Fetal programming: Masculinization increases daughter lifetime reproduction at high competition

Fetal programming: Masculinization increases daughter lifetime reproduction at high competition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fetal programming: Masculinization increases daughter lifetime reproduction at high competition Esther Carlitz, Clemens Kirschbaum, Anna K. Lindholm, Barbara König This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7435085/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract High testosterone along with masculinized traits is often considered maladaptive for female mammals, based on clinical cases of reproductive disorders and standardized animal studies. Yet, despotic species, where masculinized females dominate reproduction, challenge this view. Here, we provide support for selective benefits of female masculinization, by applying the fetal programming hypothesis. Using long-term data on free-ranging house mice exposed to naturally fluctuating densities signaling reproductive competition, we show that high-testosterone mothers produced masculinized daughters with elongated anogenital distance who achieved higher lifetime reproductive success under high-density conditions. However, population-level female testosterone and daughter masculinization declined as competition intensified. Mismatching individual fitness benefits and population trends suggest masculinization is shaped by trade-offs among maternal quality, offspring demands, and ecological and social constraints. We conclude female masculinization is not inherently maladaptive but enables competitive mothers under reproductive competition providing selective benefits for daughters – a process relevant within and likely also across species. Biological sciences/Ecology/Evolutionary ecology Biological sciences/Ecology/Ecophysiology Biological sciences/Ecology/Population dynamics Biological sciences/Zoology/Animal physiology Figures Figure 1 Figure 2 Figure 3 1. Introduction Female masculinization refers to the development of male-typical behavior, physiology, and anatomy in mammals, including humans 1 , 2 . One prominent anatomical marker of this phenomenon is the elongation of the distance between the anus and genitals (anogenital distance, AGD). Prenatal exposure to elevated testosterone levels stimulates perineal tissue growth, resulting in longer AGD after birth 3 . First mentioned in 1939 4 , the association between prenatal testosterone exposure and female masculinization has been studied in depth for more than eight decades but remains incompletely understood in terms of its underlying causes and reproductive implications. Mechanistically, the degree of masculinization is influenced by testosterone exposure during the so-called masculinization programming window 5 (gestational day 15–18 in rats, gestational week 8–14 in humans). Sources of excess testosterone include elevated maternal levels 6 or leakage from neighboring male fetuses in polytocous species (the intrauterine position phenomenon 7 ). A less understood phenomenon is that maternal stress can masculinize daughters in humans and other mammals 8 . Further, a genetic influence on the degree of female masculinization has been detected in genome-wide association studies in cows 9 . Reproductive consequences are less resolved. Several studies report that masculinized females experience a negative impact on fecundity and fertility 10 . Wild female degus, on the other hand, form social groups of similar degree of masculinization and masculinized females produce more offspring via larger litters 11 . Other findings indicate that female masculinization may provide benefits in highly competitive environments because masculinized females are more aggressive, have larger home ranges, and are superior in offspring defense 12 – 14 . It has therefore been hypothesized that masculinized females show improved fitness under conditions of high competition, such as high density 15 . This notion is supported by despotic species with pronounced reproductive skew (characterized by few or only one reproductively successful dominant female per group) where competition over reproduction is associated with highly masculinized trait expression in dominant females, including high testosterone secretion and aggression to secure reproduction 16 . The fetal programming hypothesis offers a framework for understanding divergent outcomes. This hypothesis posits that prenatal development is shaped by environmental cues to prepare offspring for the conditions they are likely to encounter after birth 17 . Following this argument, it is conceivable that highly competitive environments trigger maternal stress along with higher levels of corticosterone and testosterone, which may result in more masculinized daughters. Masculinized daughters should then show increased reproductive output in an environment of high competition. Supportive evidence includes increased daughter degree of masculinization through prenatal stress 8 , and laboratory studies showing that high population density or crowding correlates with increased levels of maternal corticosterone, aggression, testosterone, and greater masculinization of daughters 18 – 20 . In wild North American red squirrels, mothers exposed to high-density conditions or cues of it produced offspring that grew heavier during lactation, likely preparing them for competitive environments 21 . Hence, investigations under varying levels of competition may be one way forward to understand natural variation in female masculinization and its consequences for reproduction. A methodological consideration may offer another way forward to explaining divergent results on female masculinization in the literature. Anogenital distance scales proportionally with body length, as evident from repeated measures across ages in humans and cows 22 , 23 . Thus, adjusting for length is important in controlling for AGD-body length allometry. However, accurately measuring body length in many mammals poses challenges due to their flexible bodies and resistance to handling 24 , 25 . Body weight is often easier to measure and highly repeatable. While weight correlates well with body length, it is an integrated measure of lean mass and body fat mass 26 . Conditioning AGD on body weight – the traditional way of using AGD – therefore also corrects for fat accumulation. This may confound interpretations of the relationship between AGD and outcome variables because of the link between masculinization and accelerated growth 27 , 28 . Conditioning AGD on length measures may bring new insights. We use wild house mice ( Mus musculus domesticus ) in Switzerland to investigate female masculinization. Studying wild house mice in a field setting is advantageous as mice are free to perform their full behavioral repertoire, in a seasonal environment, under the influence of natural selection, in a naturally grown population with close relatives and descendents, unlike in lab mice. In Switzerland, reproductive competition experienced by female house mice differs seasonally, with breeding activity reaching a peak during spring and population density being highest in summer 29 – 31 . In addition to seasonal variation, mice experience annually changing population density and females have fewer offspring surviving during high density, indicating higher reproductive competition 32 . Varying levels of reproductive competition make this an excellent study system for investigating the fetal programming hypothesis. Using 13 years of data collected in a longitudinal study of a free-living mouse population 33 , 34 , we build a dataset of measurements of AGD, body weight, and tail length taken for 4’939 female pups at the age of 13 days, together with environmental factors including population density and season of birth. We add extensive information on adult steroid hormones, parentage, and lifetime reproductive success, resulting in an exceptional multilayer dataset across several generations. Based on assumptions of the fetal programming hypothesis, we predict that concentrations of testosterone and corticosterone in females rise with increasing reproductive competition as indicated by high population density and / or during peak breeding activity in spring (hypothesis 1 ). Consequently, the degree of female masculinization should also increase with reproductive competition in the population (hypothesis 2 ). As a mechanistic prerequisite, maternal corticosterone or testosterone needs to increase daughter degree of masculinization (hypothesis 3 ). Finally, we predict that masculinization improves female fitness, in particular under high reproductive competition (hypothesis 4 ). 2. Results General information Over a period of 13 years (January 2008 to December 2020), we measured 4’939 female pups at 13 days of age (range 12–14 days) in a population of free-ranging house mice. Population density ranged from 52 to 506 adults and increased over the years, besides annual variation over the seasons. Both mother and father could be assigned genetically at 95% confidence for 77% of these pups (n = 3’808). We further collected 762 hair samples for hormone analysis from 585 adult females during the last 4.5 years of the study period. Throughout the results, we report mean values (x̅) where variables are normally distributed and median values for other distributions (x̃). Sample sizes, descriptive statistics and variances are presented in the supplementary material (s1_Descriptive statistics). Of all female pups measured at day 13, 2’307 (47%) were recaptured at a body weight of at least 17.5 g and were thus categorized as being recruited into the adult population (the remaining 53% either died or emigrated as subadults). For 67% of recruited females (n = 1’454; corresponding to 29% of all female pups measured), we genetically identified 1 to 33 male or female offspring surviving to day 13. Reasons for unsuccessful reproduction (zero offspring surviving until day 13) may range from failure to conceive or raise offspring despite successful recruitment into the adult population, to technical problems of parental trio assignment (potentially due to errors in detection and sampling of individuals, PCR amplification, or insufficient marker variation). Female pup AGD and body weight At day 13 (range 12–14), the AGD of female pups ranged from 2 to 6 mm (x̅ = 3 mm) and their weight from 2 to 11 g (x̅ = 6 g; s1_Table1). Body weight strongly and positively correlated with tail length (r = 0.78), while AGD slightly more strongly increased with tail length (r = 0.56) than with body weight (r = 0.51, Fig. 1 A-C). Testing hypotheses Hypothesis 1 Reproductive competition increases adult female corticosterone and testosterone Contrary to our expectations, corticosterone and testosterone measured in hair of adult females decreased with increasing density (total number of adults in the population, our first measure of reproductive competition; Fig. 2 A-B, supplementary material s2_Model 1A-B). Season, our second measure of reproductive competition (with spring being characterized by highest breeding activity in the population), affected corticosterone and testosterone in different ways. Corticosterone was lowest in hair samples corresponding to the season of reduced breeding activity, winter, while testosterone did not differ significantly by season (Fig. 2 A-B, s2_Model 1A-B). Within individuals, corticosterone and testosterone nevertheless correlated positively, although weakly (Pearson r = 0.16, p < 0.001, n = 625). Hypothesis 2 Maternal exposure to reproductive competition during pregnancy increases daughter degree of masculinization We quantified masculinization of female pups at day 13 as residualized anogenital distance (AGD corrected for allometry with tail length, AGD.tail). Opposite to expectations from the fetal programming hypothesis, female pups were less masculinized (had smaller AGD.tail) at higher reproductive competition in the population (measured at daughter birth), with increasing population density and during spring (Fig. 2 C, s2_Model 2). Yet, the effects of both indices of reproductive competition together explained only 4% of the variance in masculinization. More variation was explained by sharing the same uterine environment (measured through genetic litter identity; ~30%), sharing the same mother (~ 6%) or the same father (~ 20%; Intraclass Correlation Coefficient ICC, s2_Model 2). Hypothesis 3 Maternal testosterone and corticosterone during pregnancy increases daughter degree of masculinization Mothers with higher testosterone concentrations during the period covering pregnancy produced more masculinized daughters, explaining 8% of variation in daughter masculinization, in accordance with the mechanistic assumptions of the fetal programming hypothesis (Fig. 2 D, s2_Model 3A). Maternal corticosterone levels alone did not significantly affect daughter masculinization (s2_Model 3B). Yet, the interaction between maternal testosterone and corticosterone increased the predictability of daughter masculinization to 13% (s2_Model 3C). Thus, increasing maternal testosterone induced increasing daughter masculinization only when mothers at the same time had low corticosterone. In models that included testosterone (s2_Model 3A and C), we had, by coincident, an equal number of fathers and litters. Here, the genetic litter identity explained 38% and father identity 6% of variation in daughter masculinization. Mother identity did not further explain variation in daughter masculinization. Hypothesis 4 Masculinization enhances female fitness under high reproductive competition Lifetime reproductive success Masculinization was positively associated with increasing lifetime reproductive success (Fig. 3 A, s2_Model 4A). Among the 29% of females that later reproduced, those with higher degrees of masculinization as pups had significantly more offspring surviving to day 13. While the effect was small (estimate (AGD.tail) = 0.15 more offspring with each 1 mm increase in AGD length) it was highly robust (Fig. 3 A, s2_Model 4). Notably, whether or not a female reproduced at all was not predicted by their degree of masculinization in this model. However, from this model we could not assess if increased masculinization generally led to increased reproduction, or if the advantage was linked to particular conditions of reproductive competition. Including indicators of reproductive competition (density and season) provided a more differentiated picture. Population density and season of birth were important predictors of lifetime reproductive success, although with different effects. The probability to successfully reproduce (to have at least one surviving offspring) increased for females by 6% with every 100 additional adults in the population. Among those that reproduced, however, females had 0.3 fewer surviving offspring with every 100 additional adults (s2_Model 5, negative estimates of the zero-inflation model indicate reduced probability to have zero offspring). Season of birth also significantly affected reproductive outcomes but differed from effects of density. Spring born females were about 70% less likely to reproduce than if born late in the year (autumn or winter) and, once they reproduced, had 0.2 fewer offspring surviving compared to summer and autumn born females (s2_Model 5). While both density and season were biologically meaningful predictors of lifetime reproductive success, only density moderated the effect of masculinization. The probability to have at least one surviving offspring increased for females by 20% with each 1 mm increase in AGD and every 100 adults more in the population (s2_Model 5). Among those that reproduced, females tended to have 0.09 more offspring with each 1 mm increase in AGD and every 100 adults less in the population (p = 0.09) (Fig. 3 B, s2_Model 5). Combining these two slopes in a plot, lifetime reproductive success of females decreased under high-density conditions but masculinized females (i.e., those with longer AGD) were less affected (Fig. 3 B). Recruitment into adult population Results on the likelihood of recruitment into the adult population paralleled those of the likelihood to reproduce. Recruitment probability of females generally increased by 20% with every 100 more adults in the population (s2_Model 6A). Recruitment was lower for spring and summer born females than for autumn and winter born females (Table 1, s2_Model 6A). The degree of masculinization changed female recruitment probability across density but not across season. Female recruitment probability increased by 23% with each 1 mm increase in AGD and each 100 adults more in the population (Fig. 3 C, s2_Model 6). Under low-density conditions, the degree of masculinization seemed to have no or a slightly negative influence on recruitment (Fig. 3 C). Comparing masculinization indices All models that included the masculinization index AGD corrected for tail length, AGD.tail, were repeated with an AGD corrected for body weight, AGD.weight (see supplemental material s3_Model outputs for weight-corrected AGD; s3), whereby the latter measure is the traditional way of using AGD. Both indices yielded similar results. Nevertheless, results for AGD.tail were consistently stronger than those for AGD.weight as indicated by stronger significance and lower AIC values: Maternal testosterone significantly predicted AGD.tail, and predicted AGD.weight more weakly (s2_Model 3A vs. s3_Model 3A). AGD.tail was a stronger predictor for lifetime reproductive success (s2_Model 4 and 5 vs. s3_Model 4 and 5) and recruitment into the adult population (s2_Model 6 vs. s3_Model 6). Only when predicting the degree of masculinization by density and season of birth did AGD.weight yield better results than AGD.tail (s2_Model 2 vs. s3_Model 2). This suggests that a length corrected AGD measure in pups better reflects effects driven by the degree of masculinization. 3. Discussion We studied 4’939 female pups from a free-living house mouse population monitored over 13 years to investigate female masculinization within the framework of the fetal programming hypothesis. Applied to masculinization, this hypothesis posits that reproductive competition increases female stress hormones along with testosterone, which allows mothers to increase daughter masculinization to enhance daughter competitiveness, and therefore fitness, under similar conditions of reproductive competition. In line with the fetal programming hypothesis, maternal testosterone enhanced daughter masculinization, suggesting a direct maternal effect, and those masculinized daughters had increased lifetime reproductive success as competition increased. Contrasting the hypothesis is that adult female testosterone declined population-wide as density increased, accompanied by a concurrent decline in daughter masculinization. It suggests that female masculinization through fetal programming is adaptive but only few females can afford such programming as density increases. Our first hypothesis, based on fetal programming, predicted higher adult female corticosterone and testosterone with increased reproductive competition. As expected, the two hormones correlated positively within individuals, though weakly. Contrary to predictions, both declined with population density, our proxy for reproductive competition. While few studies have measured female steroid hormones relative to group size or density, the prevailing view suggests that density elevates stress hormones, aggression, and testosterone. For example, Rhesus monkeys showed rising stress hormones and injuries at higher densities irrespective of sex 35 . Crespi et al. 2 in their recent review showed that high female testosterone enhances dominance but reduces reproductive traits, concluding that all females should increase testosterone to improve social status under competition. In line with this notion, dominant females in species with very high reproductive competition express levels of testosterone that parallel or even exceed that of males 6 . Still, we found decreasing testosterone with increasing density. An extension of the idea from Crespi et al. 2 might reconcile our findings with those from the above-mentioned studies, as masculinization affects many traits. The optimal balance of these traits shifts with environmental and social conditions. Most studies assess density effects in females confined in cages or enclosures 35 , where costs and benefits of dominance might differ from free-living individuals that can choose social partners and reproductive strategies. Our results suggest that in free-living house mice, high testosterone may disrupt group cohesion and cooperation for communal breeding, a female alternative reproductive tactic to solitary breeding 36 . Since group size and communal breeding increase with density 32 , costs of high testosterone expression or maintenance may rise under these conditions, making it less affordable. Similar testosterone patterns are observed in male mammals and birds. Group-living male African striped mice have lower testosterone than solitary males 37 , men’s testosterone decreases when they transition from a solitary lifestyle to a long-term relationship or fatherhood 38 , and colonial birds deposit less testosterone in eggs than solitary birds 39 . In female house mice, reduced corticosterone and testosterone may foster prosocial behavior and tolerance, facilitating crowding 40 as seen in some populations 41 . This hormonal decrease may also explain the observed higher female recruitment into adult populations under high density (see Hypothesis 4 ). Our second hypothesis predicted that reproductive competition during gestation increases daughter masculinization. However, like corticosterone and testosterone, daughter masculinization decreased with both indicators of reproductive competition, increasing population density and peak breeding season in spring. This again contradicts our fetal programming predictions and contrasts with guinea pigs, where daughter masculinization rises with social disruption, linked to high density 15 , 42 . Importantly, being born during high density and in spring reduced female lifetime reproductive success, confirming their biological significance as an indicator of reproductive competition. In our third hypothesis, we tested whether maternal testosterone and corticosterone positively affect daughter masculinization. We found that daughter masculinization increased with maternal testosterone, consistent with laboratory studies 43 and confirming the biological relevance of maternal testosterone in wild mammals. It suggests a direct maternal influence on daughter masculinization during the masculinization programming window, which fulfills a key mechanistic requirement of fetal programming. It also implies that reduced female testosterone at high density causally underlies the density-associated decline in masculinization. Notably, this positive effect of maternal testosterone on daughter masculinization was driven by a few mothers with exceptionally high testosterone, indicating that under high-density conditions, when hair was sampled, only some females maintain elevated levels and program their daughters accordingly. Furthermore, high maternal testosterone predicted increasing daughter masculinization in those mothers that had low levels of corticosterone. This supports predictions of the dual hormone hypothesis, claiming that testosterone only correlates with an individual’s social dominance if corticosterone is low 44 (or cortisol in other mammals). Our data suggest that, in addition, individual high testosterone together with low corticosterone jointly affect daughters and thus the next generation. Contrary to our prediction, maternal corticosterone showed no relationship with daughter masculinization, despite considerable maternal hormone variation and supporting evidence from human and lab studies 8 , 46 . This discrepancy may reflect fundamental differences between experimental or clinical settings and naturalistic settings, in which animals have more options to implement their behavioral preferences, such as ranging widely, choosing their social and sexual partners and dispersing. Daughter masculinization and female testosterone both decreased with density. From a mechanistic perspective, it is thus conceivable that density reduces females’ and thus mothers’ ability to invest into high testosterone, which reduces daughter masculinization. However, seasonal variation in female testosterone, which was lowest in summer, cannot be associated with the particularly low degree of masculinization in spring-born daughters. We thus currently miss a good mechanistic explanation for reduced masculinization in spring. Paternal testosterone decrease in spring could potentially be a driver, as we found that paternal identity significantly contributed to daughter masculinization by 20%. Yet, we lack information on male seasonal hormone patterns in house mice. In addition, lowered male testosterone during the peak breeding season would clearly contradict our expectations as well as observations in other species. On the other hand, having more receptive females available at the same time during peak breeding season might increase the possibility for low-testosterone males to reproduce, which might result in population-wide reduced daughter masculinization. Beyond parental effects, the shared uterine environment significantly shaped daughter masculinization, accounting for 30% of variation in our 13-year dataset. We do not have information on litter sex ratio at birth due to frequent infanticide before we measure pups at day 13 47,48 . The reported effect thus could either derive from a current maternal status, e.g., maternal testosterone, or from male siblings. When accounting for maternal testosterone, litter identity still accounted for 38% of variation in a subset sample. It suggests that intrauterine testosterone dynamics are at least partly driven by litter sex ratio as has been shown in wild marmots 49 . Our fourth hypothesis predicted fitness benefits of masculinization under high reproductive competition. Overall, masculinized females had higher lifetime reproductive success (offspring surviving day 13), supporting its adaptive value. Looking at specific fitness-relevant traits revealed a more variable picture. Under high-density conditions, masculinized females were more likely to be recruited into the adult population and to reproduce, thereby achieving higher lifetime reproductive success. At low densities, masculinization did not affect recruitment or reproduction probability but tended to increase lifetime offspring number among females with at least one surviving offspring. Thus, masculinization affects multiple fitness-related traits. Each specific trait may independently be modified into different directions by masculinization depending on ecological conditions, as expected by the fetal programming hypothesis. Consequently, simultaneous consideration of multiple fitness-relevant traits at varying ecological conditions seems essential to understand the adaptive value of female masculinization. Our findings that masculinized females more often remained in the population to adulthood and had more offspring when breeding during low density may parallel findings in wild degus, where females grouped by their degree of masculinization and masculinized groups had more offspring 50 . In contrast, masculinized Yellow-bellied marmots started to reproduce at older age and thus later in the year 49 , 51 , which reduced offspring survival 52 . However, none of these or other studies included measures of competition making a comparison with our results difficult (but see Monclús et al. 51 for density dependent reproductive onset). Although both population density and season of birth influenced daughter masculinization and reproductive success, only density moderated the fitness benefits of masculinization. This likely reflects that density at birth more reliably predicts future reproductive competition than season in house mice. We finally compared two indices of female masculinization, AGD corrected for tail length versus the more commonly used AGD corrected for body weight. Tail-length-corrected AGD consistently provided better model fit and stronger effect estimates (for an exception see the prediction of masculinization by indicators of competition, hypothesis 2 ). Despite this, estimates from both masculinization indices remained within a similar range, suggesting that findings based on weight-corrected AGD are not misleading, but less accurate. Correcting AGD for body weight, which includes lean as well as fat body mass, may conflate effects of prenatal testosterone exposure on AGD and body mass, as obesity and accelerated postnatal weight gain are associated with female masculinization 28 . In contrast, correcting AGD for a measure or correlate of body length accounts for allometry, independent of fat mass, and we recommend its use. In summary, our results suggest that maternal testosterone enhances daughter masculinization, which in turn increases lifetime reproductive success under high-density conditions. This supports the fetal programming hypothesis and challenges the view that female masculinization is merely a maladaptive by-product of maternal investment in sons 53 . Yet, contrary to predictions, both population-wide testosterone and daughter masculinization declined as competition intensified, suggesting that these traits are shaped by trade-offs between maternal capacity, offspring requirements, and ecological constraints. In free-living house mice, masculinized daughters gained fitness advantages through increased recruitment and reproduction, depending on density, whereas mothers may incur costs from sustaining high testosterone and raising potentially faster-growing offspring 54 . Such trade-offs, mediated by individual choice of the social environment and reproductive strategy and moderated by ecological conditions, underscore the limitations of conclusions drawn from clinical or captive studies. In conclusion, female masculinization influences multiple fitness-relevant traits, but its benefits and costs shift with ecological context. As density rises, only a subset of high-quality mothers appears able to sustain high testosterone investment, paralleling patterns in despotic species where reproduction is monopolized by few highly masculinized females. Thus, the selective advantage of female masculinization emerges along a continuum shaped by ecological pressures and social competition, with despotic species at the extreme end. This strengthens the fetal programming hypothesis for female masculinization and suggests that testosterone-associated patterns may similarly apply to females and males in allowing reproductive dominance, though on a different level. 4. Methods 2.1 Study population and data collection Study population A population of wild commensal house mice ( Mus musculus domesticus ) has been intensively studied since 2002 in a former agricultural building at the border of a forest near Zurich, Switzerland. Inside the 72 m² barn, mice are provisioned with 40 nest boxes, which mice use as protected places for resting and sleeping, and females for raising their litters. Bricks and tubes are provided as shelters as well as several structuring elements that mice can climb, so that mice can access any part of the barn. Standardized food (50:50 mix of oats and hamster food from Landi Schweiz AG, Dotzigen, Switzerland) as well as water are provided ad libitum which mimics the natural situation for commensal house mice in barns or stables in middle Europe. Mice can freely leave and enter the barn through numerous small holes or cracks that exclude predators larger than mice. Once outside, mice are subject to predation, especially from cats, foxes or owls. Mice are individually followed from the time they are pups until they leave the building (emigrate) or die, with the help of regular pup (nest) monitoring, population monitoring, RFID tracking when entering or leaving nest boxes, and through genetic identification. Below we present those methods in detail that are relevant for the present study. For more information about the setup and methods used see elsewhere 33 , 34 . Pup monitoring All nest boxes and shelters are checked for new litters at least every 13 days. Pup age is estimated according to morphological details 31 , allowing us to calculate birthdate. Each litter consisting of same-aged pups found in the same nest is assigned a unique litter identity (litter ID), and pups younger than 12 days get a litter specific tattoo in one or several paws for later recognition. When pups are about to open their eyes and become mobile at 13 days of age, we return to the nest and take standardized pup measurements, including sex, body weight (measured using digital scales to the nearest 0.1 g), anogenital distance (AGD; measured in mm with digital calipers as the distance between the center of the anus and the posterior edge of the genitals; Fig. 2 D), and tail length (measured in mm using a ruler as the distance between the center of the anus and the tip of the tail; Fig. 2 E). Additionally, a small tissue sample is taken from an ear for later genetic identification and parentage analyses (to determine the genetic father, mother and genetic siblings). To avoid too frequent disturbance of mothers and litters, we sample pups at an age range of 12–14 days. Population monitoring, RFID tagging and hair sampling The entire population is monitored at six-to-eight-week intervals by catching all mice present. Every individual is sexed and weighed, adult mice of at least 17.5 g are subcutaneously injected with a unique RFID tag (Trovan ID-100A implantable microtransponder) and a tissue sample is taken from the ear. Once tagged, mice can be permanently identified when handled or when entering or leaving nest boxes, which are equipped with automated reading devices 33 (antenna system). Mice reaching a body weight of at least 17.5 g are considered sexually mature in the study population 34 . Since December 2016, we use population monitoring events and occasionally pup monitoring events to collect adult hair samples for hormone analysis. To do so, hair is shaved with a veterinary razor from the lower back of newly tagged mice and opportunistically from tagged adults. We shave appr. 1 cm² area that corresponds to 10–15 mg hair. Shaving is avoided during colder periods to prevent additional thermoregulatory costs. During winter, we only collected hair samples from mice that were euthanized for other experiments. Parental assignment Markers at 25 polymorphic microsatellite loci are used for parentage analyses from tissue samples 55 . We assign a mother and a father to each pup using trio analysis in the program CERVUS 3.0 56 . All females and males recorded in the barn during the 30 days prior to the birth of a pup are included as potential parents, unless they are recorded as having died before the pup birthdate (for potential mothers) or the estimated conception date (potential fathers). Paternity or maternity assignments are rejected if they are assigned at less than 95% confidence. To infer a shared uterine environment during ontogeny, a genetic litter identity (genetic litter ID) is assigned based on the identity of the genetic mother and the date of birth. Pups with the same genetic litter ID can have different fathers. Life history data Adult and pup measures are connected through genetic matching between adult and pup microsatellite DNA profiles. We consider mice that were recaptured at a body weight of at least 17.5 g as being recruited into the adult population (i.e. the individual did not die or emigrate from the barn before reaching sexual maturity). Parental assignment allows us to determine individual lifetime reproductive success (total number of male and female offspring sampled at 13 days). Hormone analysis Individual hair samples are kept in an envelope at room temperature until further processing in the ‘Dresden LABservice’ ( https://dresden-labservice.com ). For preparation, hair is washed once for three minutes with 2.5 ml isopropanol before hormones are extracted from 5 ± 0.5 mg of dried whole hair (extraction for 18 h at room temperature with 1.5 ml of methanol). The extract is dried and reconstituted in 200 ml of a 50:50 mix of water and methanol. Steroid hormones (testosterone and corticosterone) are analyzed via liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS; for methodological details and details on the biological validation in house mice see elsewhere 57 , 58 . 2.2 Statistical analyses General information Data set used Our study integrates data from morphological measurements, genetics, physiology, and life history collected from female house mice sampled as pups at the age of 12 to 14 days between January 2008 and December 2020. For a subset of females, we have hormone data from adult hair sampled opportunistically between December 2016 and December 2020. Female pup AGD and calculation of derived indices We use the anogenital distance (AGD) at 12–14 days as an indicator of female degree of masculinization. To inspect the relationship between AGD, body weight (integrated measure of lean and fat body mass) and tail length (as a repeatable approximation of body length 59 ), we visualize and calculate their direct correlations. Throughout this study, we use a length corrected masculinization index, which corrects AGD for tail length (AGD.tail). More specifically, we use AGD residuals, the distance between the actual data point and the linear regression line between AGD and tail length. Positive residuals (values of AGD.tail) indicate more masculinized pups, pups with longer AGD than expected for their body length. To allow comparison of our measure of AGD.tail with the traditional way of correcting AGD by body weight (AGD.weight), we provide in the supplemental material all models investigated for AGD both corrected for tail length (AGD.tail) and corrected for body weight (AGD.weight). Since we are interested in the variation of masculinization in proportion to length and weight, and all three morphometrics of a pup (AGD, tail length, weight) are taken at the same time, we do not include the actual age at sampling (days 12–14) in our models. For simplicity, we will hereafter refer to the age at sampling as day 13. Reproductive competition among adult females is pronounced in the study population 29 , 32 , 34 . We use two measures to characterize reproductive competition. First, population density, estimated as the total number of tagged adults registered at a population monitoring event closest to the date of interest, because per capita reproductive success decreases with increasing density 32 . Second, we use season at the date of interest, because breeding activity varies with season 30 , 34 : Spring (March – May, highest breeding activity in the study population), summer (June – August), autumn (September – November), or winter (December – February, lowest breeding activity). Density and season at the date of interest refer to either date of birth (DOB) or date of hair sampling (hair), as specified. Software used : We perform all statistical analyses and create figures in R 4.3.1 with RStudio 60 using the following packages: dplyr , tidyverse and zoo for data arrangement 61 – 63 , ggplot2 , ggeffects and viridis 64 – 66 to illustrate model results and patchwork to arrange Figs. 6 7 , and pscl and DHARMa 68 , 69 for model diagnostics. Other packages are mentioned below. Testing hypotheses Hypothesis 1 Reproductive competition increases adult female corticosterone and testosterone We investigate if concentrations of corticosterone and testosterone measured in adult female hair increase with reproductive competition as specified by population density and season at sampling. Since hormones in hair are an integrated measure representing the past two to three month 29 , we refer to density and season at a time point 30 days before the hair was sampled (Density.hair, Season.hair). Testosterone and progesterone in the hair of wild derived house mice begin to differentiate between 50 to 60 days of age 57 . Since we are interested in the environmental effects on adult hormones, we only include samples from females older than 90 days. We use hormone concentrations (corticosterone or testosterone) as the response variable in a linear mixed effects model to account for repeated measures (function ‘lmer’, package lme4 70 ). Density and season as well as age at hair sampling are included as predictor variables (Density.hair, Season.hair, Age.hair), and the identity of the adult female as random factor. We summarize model outputs using the function ‘summ’ from the package jtools 71 , which provides standardized estimates, confidence intervals and the Intraclass Correlation Coefficient ‘ICC’ of our random variable female identity (‘ID’). Results from the ICC range between 0 and 1 and can be interpreted similarly to the proportion or variance explained by a random factor 72 . Hypothesis 2 Maternal exposure to reproductive competition during pregnancy increases daughter degree of masculinization To test whether reproductive competition experienced by a mother during pregnancy (specified by density and season at pup date of birth, DOB) affects daughter degree of masculinization at day 13, we use a linear mixed effects model with the response variable AGD.tail (function ‘lmer’, package lme4 70 ). Fixed predictor variables are density and season of birth (Density.DOB, Season.DOB). Random variables are genetic litter identity (to account for effects of a shared uterine environment among siblings) and mother and father identity (to control for parental effects). Hypothesis 3 Maternal testosterone and corticosterone during pregnancy increases daughter degree of masculinization Hormones in hair are an integrated measure of the past two to three months (Carlitz et al. 2022) and we here make the assumption that hair testosterone and corticosterone measures are representative of values in adult females for the previous three months. The masculinization programming window in rats, a closely related species, covers approximately 6 to 10 days before birth (embryonic day 15–18 of 24 5,73 ). We therefore only include mother – daughter pairs in this analysis when daughter birth was within 80 days before and 10 days after the mother was shaved. We use linear mixed effects models with AGD.tail as response variable (function ‘lmer’, package lme4 ; 70 . Fixed effect predictor variables are mother corticosterone (Model A) or mother testosterone (Model B) measured in hair, as well as both hormones and their interactions (Model C). Daughter genetic litter identity, and maternal and paternal identity are included as random effects predictor variable to account for repeated measures of siblings, within or across shared uterine environments. Since we are interested in potential direct effects of maternal hormones, we do not control for maternal age at hair sampling, or measures of reproductive competition (population density, season). Hypothesis 4 Masculinization enhances female fitness under high reproductive competition As a measure of fitness, we use lifetime reproductive success (number of offspring that were raised until at least day 13) in the barn. Over 70% of our study females measured as pups (n = 4’939) had zero lifetime reproductive success. Most of these females disappeared before being tagged as adults. We are not certain whether such non-recruited individuals died without recovery of the corpse, or emigrated from the barn. Dispersal in house mice, however, is considered risky and to result in low (if any) reproductive success 74 , 75 . We first examine if masculinization alone or in interaction with indices of reproductive competition (Density.DOB and Season.DOB) affects female lifetime reproductive success in the study population. To account for the highly zero-inflated data on lifetime reproductive success and a negative binomial (overdispersed) distribution among reproducing females, we apply a generalized linear effects model with number of offspring (‘offspring’) as response variable (function ‘glmmTMB’, family = truncated_nbinom2 with zero-inflation, package glmmTMB 76 . This model separately but simultaneously assesses a female’s likelihood to produce either 0 or at least 1 offspring (zero-inflation model) and estimates the number of offspring for those females that successfully reproduced (conditional model). When plotting model results, we present one regression line that combines estimates from the zero-inflation and the conditional model (function ‘ggpredict’, type = ‘zero_inflated’, packages ggeffects and ggplot 66 , 77 . In a model building approach, we test if AGD.tail alone or in interaction with Density.DOB and Season.DOB predicts lifetime reproductive success. We compare the goodness of fit for each model using the Akaike Information Criterion (AIC). Since recruitment is crucial to successful reproduction in the study population, we additionally examine the probability of recruitment of female pups in response to their degree of masculinization under varying reproductive competition. We use recruitment as a binary response variable in a generalized linear effects model (function ‘glm’, package lme4 70 . Recruitment of 0 refers to females that died or emigrated from the barn before being recaptured as adults (at a body weight of at least 17.5 g), recruitment of 1 otherwise. To explore the importance of masculinization, and of reproductive competition in moderating effects of masculinization, we add the predictor variables AGD.tail in interaction with density and season at female birth (Density.DOB, Season.DOB) in a model building approach and evaluate the goodness of model fit using the AIC. Declarations Author Contributions Conceptualization: EC and BK. Data generation: EC, AL, BK, CK. Statistical analysis: EC. Investigation: EC, AL, BK. Writing—original draft preparation, EC. Writing—review and editing, EC, BK, AL, CK. Visualization: EC. Funding acquisition: EC, BK, AL, CK. All authors have read and agreed to the published version of the manuscript. Funding The study was supported by funding from the following sources: E.C.: DFG (CA 1870/1-1) and Maria Reiche Fellowship; B.K.: SNF grant 31003A_176114, and UZH Stiftung für wissenschaftliche Forschung; A.K.L.: SNF grants 31003A_120444, 310030M_138389, Julius-Klaus and Promotor Stiftung. Declaration of competing interest We declare we have no competing interests. Acknowledgements We are grateful to all colleagues and students who contributed to the management and data collection of the long-term study on house mice in the barn since 2002. We would like to especially acknowledge the work of our barn managers Sally Steinert and Bruce Boatman. We also thank Wei Gao, Anja Schulz and the hair lab team for analyzing the hair samples and Jari Garbely for genetic laboratory work. We thank Isin Kosemen from Synosys, TU Dresden, for her interactive charts. Ethics Data were collected under the permits ZH 215/2006, ZH 51/2010, ZH 56/2013, and ZH 091/2016 from the Cantonal Veterinary Office, Kanton Zürich, Switzerland. References Vandenbergh JG, Huggett CL (1995) The anogenital distance index, a predictor of the intrauterine position effects on reproduction in female house mice. Lab Anim Sci 45:567–573 Crespi BJ, Bushell A, Dinsdale N (2024) Testosterone mediates life-history trade‐offs in female mammals. Biol Rev brv 13166. 10.1111/brv.13166 vom Saal FS, Bronson FH (1978) In Utero Proximity of Female Mouse Fetuses to Males: Effect on Reproductive Performance during Later Life. Biol Reprod 19:842–853 Greene RR, Burrill MW, Ivy AC (1939) Experimental intersexuality. The effect of antenatal androgens on sexual development of female rats. Am J Anat 65:415–469 Sharpe RM (2020) Androgens and the masculinization programming window: human-rodent differences. Biochem Soc Trans 48:1725–1735 Drea CM et al (2021) An intergenerational androgenic mechanism of female intrasexual competition in the cooperatively breeding meerkat. Nat Commun 12:7332 Clark MM, Galef BG (1995) Prenatal influences on reproductive life history strategies. Trends Ecol Evol 10:151–153 Barrett ES et al (2013) Prenatal exposure to stressful life events is associated with masculinized anogenital distance (AGD) in female infants. Physiol Behav 114–115:14–20 Gobikrushanth M et al (2019) The relationship between anogenital distance and fertility, and genome-wide associations for anogenital distance in Irish Holstein-Friesian cows. J Dairy Sci 102:1702–1711 Wainstock T, Shoham-Vardi I, Sheiner E, Walfisch A (2017) Fertility and anogenital distance in women. Reprod Toxicol 73:345–349 Correa LA et al (2025) Organizational effects of testosterone on the number of mating partners and reproductive success in females of a social rodent. Sci Rep 15:22411 Kemme K, Kaiser S, Sachser N (2007) Prenatal maternal programming determines testosterone response during social challenge. Horm Behav 51:387–394 Mann MA, Svare B (1983) Prenatal testosterone exposure elevates maternal aggression in mice. Physiol Behav 30:503–507 Zielinski WJ, vom Saal FS, Vandenbergh JG (1992) The effect of intrauterine position on the survival, reproduction and home range size of female house mice (Mus musculus). Behav Ecol Sociobiol 30:185–191 Kaiser S, Sachser N (2009) Effects of Prenatal Social Stress on Offspring Development: Pathology or Adaptation? Curr Dir Psychol Sci 18:118–121 Davies CS et al (2016) Exceptional endocrine profiles characterise the meerkat: sex, status, and reproductive patterns. Sci Rep 6:35492 Kwon EJ, Kim YJ (2017) What is fetal programming? a lifetime health is under the control of in utero health. Obstet Gynecol Sci 60:506 Chapman JC, Christian JJ, Pawlikowski MA, Michael SD (1998) Analysis of steroid hormone levels in female mice at high population density. Physiol Behav 64:529–533 Myers K, Poole W (1961) A study of the biology of the wild rabbit, Oryctolagus cuniculus (L.), in confined populations. II. The effects of season and population increase on behaviour. CSIRO Wildl Res 6:1 Zielinski WJ, Vandenbergh JG, Montano MM (1991) Effects of social stress and intrauterine position on sexual phenotype in wild-type house mice (Mus musculus). Physiol Behav 49:117–123 Dantzer B et al (2020) Maternal glucocorticoids promote offspring growth without inducing oxidative stress or shortening telomeres in wild red squirrels. J. Exp. Biol. jeb.212373 10.1242/jeb.212373 Priskorn L et al (2018) Anogenital distance as a phenotypic signature through infancy. Pediatr Res 83:573–579 Rajesh I, Gobikrushanth M, Carrelli JE, Oba M, Ambrose DJ (2022) Repeatability of anogenital distance measurements from birth to maturity and at different physiological states in female Holstein cattle. J Dairy Sci 105:2699–2707 Aplin KP (2003) Field Methods for Rodent Studies in Asia and the Indo-Pacific. Australian Centre for International Agricultural Research, Canberra Jewell PA, Fullagar PJ (1966) Body measurements of small mammals: sources of error and anatomical changes. J Zool 150:501–509 Guo J, Hall KD (2011) Predicting Changes of Body Weight, Body Fat, Energy Expenditure and Metabolic Fuel Selection in C57BL/6 Mice. PLoS ONE 6:e15961 Correa LA et al (2016) Masculinized females produce heavier offspring in a group living rodent. J Anim Ecol 85:1552–1562 Kinsley C et al (1986) Prior intrauterine position influences body weight in male and female mice. Horm Behav 20:201–211 Carlitz EHD, Lindholm AK, Gao W, Kirschbaum C, König B (2022) Steroid hormones in hair and fresh wounds reveal sex specific costs of reproductive engagement and reproductive success in wild house mice ( Mus musculus domesticus ). Horm Behav 138:105102 Evans JC, Lindholm AK, König B (2021) Long-term overlap of social and genetic structure in free-ranging house mice reveals dynamic seasonal and group size effects. Curr Zool 67:59–69 Gerber N, Auclair Y, König B, Lindholm AK (2021) Population density and temperature influence the return on maternal investment in wild house mice. Front Ecol Evol 8:602359 Ferrari M, Lindholm AK, Ozgul A, Oli MK, König B (2022) Cooperation by necessity: condition- and density-dependent reproductive tactics of female house mice. Commun Biol 5:1–10 König B et al (2015) A system for automatic recording of social behavior in a free-living wild house mouse population. Anim Biotelemetry 3:39 König B, Lindholm AK (2012) The complex social environment of female house mice ( Mus domesticus ). In: Macholan M, Baird SJE, Munclinger P (eds) Evolution of the house mouse. Cambridge University Press, Cambridge, pp 114–134. doi: 10.1017/CBO9781139044547.007 . Dettmer AM, Novak MA, Meyer JS, Suomi SJ (2014) Population density-dependent hair cortisol concentrations in rhesus monkeys ( Macaca mulatta ). Psychoneuroendocrinology 42:59–67 König B (1994) Fitness effects of communal rearing in house mice: the role of relatedness versus familiarity. Anim Behav 48:1449–1457 Schradin C, Yuen C-H (2011) Hormone levels of male African striped mice change as they switch between alternative reproductive tactics. Horm Behav 60:676–680 Roney JR, Gettler LT (2015) The role of testosterone in human romantic relationships. Curr Opin Psychol 1:81–86 Bentz AB, Becker DJ, Navara KJ (2016) Evolutionary implications of interspecific variation in a maternal effect: a meta-analysis of yolk testosterone response to competition. R Soc Open Sci 3:160499 Parmigiani S, Mainardi M, Brain PF, Haug M, Brunoni V (1989) Variation in aggressive behavior and anatomo-physiological correlates generated by crowding without physical contact in the house mouse. Aggress Behav 15:191–200 Mice Plague in Australia (1932) Nature 129:755–755 Kaiser S, Kruijver FPM, Swaab DF, Sachser N (2003) Early social stress in female guinea pigs induces a masculinization of adult behavior and corresponding changes in brain and neuroendocrine function. Behav Brain Res 144:199–210 Hotchkiss AK et al (2007) Prenatal Testosterone Exposure Permanently Masculinizes Anogenital Distance, Nipple Development, and Reproductive Tract Morphology in Female Sprague-Dawley Rats. Toxicol Sci 96:335–345 Mehta PH, Josephs RA (2010) Testosterone and cortisol jointly regulate dominance: Evidence for a dual-hormone hypothesis. Horm Behav 58:898–906 Fishman R, Kralj-Fišer S, Marglit S, Koren L, Vortman Y (2024) Fathers and sons, mothers and daughters: Sex-specific genetic architecture for fetal testosterone in a wild mammal. Horm Behav 161:105525 vom Saal FS et al (1990) Paradoxical Effects of Maternal Stress on Fetal Steroids and Postnatal Reproductive Traits in Female Mice from Different Intrauterine Positions1. Biol Reprod 43:751–761 Auclair Y, König B, Ferrari M, Perony N, Lindholm AK (2014) Nest attendance of lactating females in a wild house mouse population: benefits associated with communal nesting. Anim Behav 92:143–149 Ferrari M, Lindholm AK, König B (2019) Fitness consequences of female alternative reproductive tactics in house mice (Mus musculus domesticus) . Am Nat 193:106–124 Monclús R, Blumstein DT (2012) Litter sex composition affects life-history traits in yellow-bellied marmots. J Anim Ecol 81:80–86 Correa LA et al (2021) One for all and all for one: phenotype assortment and reproductive success in masculinized females. Behav Ecol 32:1266–1275 Monclús R, von Holst D, Blumstein DT, Rödel HG (2014) Long-term effects of litter sex ratio on female reproduction in two iteroparous mammals. Funct Ecol 28:954–962 Monclús R, Pang B, Blumstein DT (2014) Yellow-bellied marmots do not compensate for a late start: the role of maternal allocation in shaping life-history trajectories. Evol Ecol 28:721–733 Crespi B, Dinsdale NL (2023) The sexual selection of endometriosis. Evol Behav Sci 17:123–170 Helle S, Laaksonen T, Huitu O (2013) Sex-specific offspring growth according to maternal testosterone, corticosterone, and glucose levels. Behav Ecol 24:205–212 Lindholm AK, Musolf K, Weidt A, König B (2013) Mate choice for genetic compatibility in the house mouse. Ecol Evol 3:1231–1247 Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program ‘cervus’ accommodates genotyping error increases success in paternity assignment. Mol Ecol 16:1099–1106 Carlitz EHD et al (2019) Steroid hormones in hair reveal sexual maturity and competition in wild house mice ( Mus musculus domesticus ). Sci Rep 9:1–10 Gao W et al (2013) Quantitative analysis of steroid hormones in human hair using a column-switching LC–APCI–MS/MS assay. J Chromatogr B 928:1–8 Cheverud JM, Leamy LJ, Atchley WR, Rutledge JJ (1983) Quantitative genetics and the evolution of ontogeny: I. Ontogenetic changes in quantitative genetic variance components in randombred mice. Genet Res 42:65–75 R Core Team (2023) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria Wickham H (2016) tidyverse: Easily Install and Load the ‘Tidyverse’. https://doi.org/10.32614/CRAN.package.tidyverse . 2.0.0 Wickham H, François R, Henry L, Müller K, Vaughan D (2014) dplyr: A Grammar of Data Manipulation. 1.1.4 https://doi.org/10.32614/CRAN.package.dplyr Zeileis A, Grothendieck G, zoo (2005) S3 Infrastructure for Regular and Irregular Time Series. J. Stat. Softw. 14 Garnier S (2015) viridis: Colorblind-Friendly Color Maps for R. https://doi.org/10.32614/CRAN.package.viridis . 0.6.5 Lüdecke Dggeffects (2018) Tidy Data Frames of Marginal Effects from Regression Models. J Open Source Softw 3:772 Wickham H (2016) Ggplot2: Elegant Graphics for Data Analysis. Springer international publishing, Cham Pedersen TL, patchwork (2019) The Composer of Plots. https://doi.org/10.32614/CRAN.package.patchwork . 1.3.1 Hartig F, DHARMa (2016) Residual Diagnostics for Hierarchical (Multi-Level / Mixed) Regression Models. 0.4.7 https://doi.org/10.32614/CRAN.package.DHARMa Jackman Spscl (2005) Political Sci Comput Lab. https://doi.org/10.32614/CRAN.package.pscl . 1.5.9 Bates D, Maechler M, Bolker B, Walker S (2003) lme4: Linear Mixed-Effects Models using ‘Eigen’ and S4. 1.1–37 https://doi.org/10.32614/CRAN.package.lme4 Long JA (2024) jtools: Analysis and Presentation of Social ScientificData. J Open Source Softw 9:6610 Bryer J (2023) Relationship Between Intraclass Correlation (ICC) and Percent Agreement. https://irrsim.bryer.org/articles/IRRsim.html Welsh M et al (2008) Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. J Clin Invest 118:1479–1490 Bonte D et al (2012) Costs of dispersal. Biol Rev 87:290–312 Runge J-N, Lindholm AK (2018) Carrying a selfish genetic element predicts increased migration propensity in free-living wild house mice. Proc. R. Soc. B Biol. Sci. 285, 20181333 Brooks M et al (2017) glmmTMB: Generalized Linear Mixed Models using Template Model Builder. https://doi.org/10.32614/CRAN.package.glmmTMB . 1.1.11 Lüdecke Dggeffects (2017) Create Tidy Data Frames of Marginal Effects for ‘ggplot’ from Model Outputs. https://doi.org/10.32614/CRAN.package.ggeffects . 2.3.0 Additional Declarations There is NO Competing Interest. Supplementary Files s1Descriptivestatistics.docx s1_Descriptive statistics s3ModeloutputsforweightcorrectedAGD.docx s3_Model outputs for weight-corrected AGD s2ModeloutputsfortailcorrectedAGD.docx s2_Model outputs for tail-corrected AGD Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7435085","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":520088940,"identity":"2fa61da7-ca5d-428c-ab5b-145aea8e521b","order_by":0,"name":"Esther Carlitz","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-2618-5862","institution":"University of Technology Dresden","correspondingAuthor":true,"prefix":"","firstName":"Esther","middleName":"","lastName":"Carlitz","suffix":""},{"id":520088941,"identity":"5a7f3151-393f-42e4-b5e4-7b254237a03d","order_by":1,"name":"Clemens Kirschbaum","email":"","orcid":"","institution":"Dresden University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Clemens","middleName":"","lastName":"Kirschbaum","suffix":""},{"id":520088942,"identity":"b51678eb-80f6-48d4-a4d1-efbad883c489","order_by":2,"name":"Anna K. Lindholm","email":"","orcid":"https://orcid.org/0000-0001-8460-9769","institution":"University of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"K.","lastName":"Lindholm","suffix":""},{"id":520088943,"identity":"263030e2-0d7c-42a2-b6ce-169317c223a4","order_by":3,"name":"Barbara König","email":"","orcid":"https://orcid.org/0000-0001-7696-4736","institution":"University of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"König","suffix":""}],"badges":[],"createdAt":"2025-08-22 13:46:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7435085/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7435085/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92252756,"identity":"229ce74b-00d9-4e59-8c9e-8f6de0cba709","added_by":"auto","created_at":"2025-09-26 10:55:31","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1501551,"visible":true,"origin":"","legend":"","description":"","filename":"Text2025008222.docx","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/a2aa2b1e92a89ba07339ff55.docx"},{"id":92253171,"identity":"d966515e-9efe-4dad-8fa2-8af1b82955bc","added_by":"auto","created_at":"2025-09-26 11:03:31","extension":"json","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5782,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2560058.json","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/91fc5995454c659dd759df4b.json"},{"id":92252758,"identity":"29733718-6219-4831-bb6b-f5ab30ca2b17","added_by":"auto","created_at":"2025-09-26 10:55:31","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16961,"visible":true,"origin":"","legend":"","description":"","filename":"s1Descriptivestatistics.docx","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/7c5137c17404cc8abbb09e04.docx"},{"id":92251781,"identity":"b63577a4-8ff3-4a83-b594-d5972948a444","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49690,"visible":true,"origin":"","legend":"","description":"","filename":"s2ModeloutputsfortailcorrectedAGD.docx","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/3e922e29b7f2defd5784fee3.docx"},{"id":92251787,"identity":"264a7671-c836-4afe-b452-8e219cd3cf13","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44211,"visible":true,"origin":"","legend":"","description":"","filename":"s3ModeloutputsforweightcorrectedAGD.docx","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/5ccf4ddd2cc4eacad62ee9a2.docx"},{"id":92252764,"identity":"647861b1-9c3d-4ec9-9c2f-9a60f68332b7","added_by":"auto","created_at":"2025-09-26 10:55:31","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154188,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25600580enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/9f36232718557ad76f0b331b.xml"},{"id":92252761,"identity":"507003cc-677c-4d47-8a00-9ec91e3f882e","added_by":"auto","created_at":"2025-09-26 10:55:31","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":666890,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/4c2dde902de2148eb00a6c08.png"},{"id":92251786,"identity":"f8ea0f81-a306-4082-9140-fcb14de201a4","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1246267,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/98cf4c359c52cfa2ea7662cc.png"},{"id":92252762,"identity":"09d0e1f2-5cb0-4f99-828c-1fde53a58c02","added_by":"auto","created_at":"2025-09-26 10:55:31","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":853041,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/43ec924fe1960f202af66234.png"},{"id":92251789,"identity":"d88ca0f6-255c-47b3-b638-5fe4d5afc254","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":302304,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/16f741deefba89df8ba4d214.png"},{"id":92251798,"identity":"7ff97519-94c1-475b-9f7b-dec22a8d0206","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":638078,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/bc5872ff6e563a31872b5eaf.png"},{"id":92251791,"identity":"7abb0d80-f210-4394-b86d-0cf243950458","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":415378,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/3a7db4e66af3b23ba267f164.png"},{"id":92251792,"identity":"cedfc2a8-7d7c-4453-93d6-011e4024dd40","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100041,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/6e8b11ca7560fd9955404913.png"},{"id":92251800,"identity":"46835582-5c45-494c-b86d-52145f9d4417","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":198896,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/bccec32bfa4d42b3f14c56ab.png"},{"id":92251788,"identity":"4ac90ed7-d9fa-4903-9bc6-22e0b3a1533a","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111750,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/78f60db97642eb2bfa686a09.png"},{"id":92251796,"identity":"84cb5a92-a1c1-4937-abf0-b8e2159961ce","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48797,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/e211c390b1e3570099cf893e.png"},{"id":92251794,"identity":"5010156d-c4f1-4fe1-98bf-77cb4a0fb115","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106060,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/e1b26d7dbb3d8b59a3401ee5.png"},{"id":92252765,"identity":"a9fd93f8-15cf-434c-a9b7-44048da0709b","added_by":"auto","created_at":"2025-09-26 10:55:31","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":59998,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/f695b0c4226dd459372b6c5b.png"},{"id":92251801,"identity":"e274dc4d-bb51-467b-83cb-f73e3291cfb9","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152052,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25600580structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/3ee41589c5a41cf88ef8a415.xml"},{"id":92251799,"identity":"60c7f01c-8ef7-46b6-88b8-1a83443109b3","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":163824,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/43fa230037acf5dce9a13876.html"},{"id":92251778,"identity":"38f86ff2-4a79-48ea-93ca-c956c401a99e","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":666890,"visible":true,"origin":"","legend":"\u003cp\u003ePup body measures. Simple regressions between morphometrics of female pups in a population of free-ranging house mice (age range 12-14 days). (A) body weight (weight) and tail length (indicator of body length), (B) tail length and the anogenital distance, and (C) body weight and the anogenital distance. Illustration of measuring (D) the anogenital distance and (E) tail length in a 13-day-old female house mouse. Measurements are obtained from 4939 female pups living in the study population over the course of 13 years.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/bf1cfe883748061864a6d489.png"},{"id":92252757,"identity":"7852ecfd-40a4-479d-ae0e-2f596e30d25b","added_by":"auto","created_at":"2025-09-26 10:55:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1246267,"visible":true,"origin":"","legend":"\u003cp\u003eTesting preconditions of the fetal programming hypothesis. Contrasting our predictions, (A) testosterone and (B) corticosterone concentrations in hair of adult females older than 90 days decreased with increasing density (number of adults in the population). (C) Likewise, the masculinization index (AGD.tail, residualized anogenital distance corrected for tail length measured at day 13) decreased with increasing density. In line with our predictions, daughter degree of masculinization increased with (D) maternal testosterone but not with (E) corticosterone in hair. (F) Long-term data were collected from a population of free-living house mice in a former agricultural building. Hormone measures were obtained from hair samples collected over the latest 4 years of observations. To explore values of testosterone and corticosterone per individual and to split data cohorts by season, \u003ca href=\"https://synosys.github.io/fetal-programming-for-female-masculinization/\"\u003eclick here\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/228f4fe7c5a619151659b1b5.png"},{"id":92253172,"identity":"9d613833-531a-49c1-91f4-92f0491eb405","added_by":"auto","created_at":"2025-09-26 11:03:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":853041,"visible":true,"origin":"","legend":"\u003cp\u003eTesting fitness effects of female masculinization. Female lifetime reproductive success (offspring, defined as the number of offspring surviving to day 13) predicted by (A) the masculinization index alone (AGD.tail, residualized anogenital distance corrected for tail length measured at day 13) or (B) in interaction with population density as an indicator of reproductive competition (different densities indicated by different colors). Lifetime reproductive success (offspring) decreases with density but this negative effect is offset by an increasing degree of masculinization. (C) The probability for a female pup to be recruited into the adult population increases with their degree of masculinization as density increases. This is likely key to explain the positive effect of masculinization on reproductive success. Regression lines for A and B reflect the combined estimates of the conditional model (number of offspring surviving to day 13 among the 1,454 reproducing females) and the zero-inflation model (probability to reproduce among all females measured as pup). Density refers to the number of male and female adults in the population at the date of the pup’s birth. Masculinization measures are from 4,939 female 13-day-old pups living in a population of free-ranging house mice measured over the course of 13 years. To further explore the data within specific ranges of density, \u003ca href=\"https://synosys.github.io/fetal-programming-for-female-masculinization/\"\u003eclick here\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/ad3f278e0ec885d7e470c702.png"},{"id":92254017,"identity":"03c36d8f-d731-4128-aa7a-2bb0d128fd76","added_by":"auto","created_at":"2025-09-26 11:11:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3503531,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/3f63887c-d832-40e2-90a5-9c1d2b1ebeb2.pdf"},{"id":92251775,"identity":"7984ff48-2192-42aa-9a37-704a852e536b","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16961,"visible":true,"origin":"","legend":"s1_Descriptive statistics","description":"","filename":"s1Descriptivestatistics.docx","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/1c1ccba5b5d43e628dda4a61.docx"},{"id":92251780,"identity":"2c44707d-6d12-44a1-837a-e6bdd64b3bac","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":44211,"visible":true,"origin":"","legend":"s3_Model outputs for weight-corrected AGD","description":"","filename":"s3ModeloutputsforweightcorrectedAGD.docx","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/52feb58676cfd3817b94d917.docx"},{"id":92251776,"identity":"d9e50719-2a35-46cd-9912-ca2520989ef8","added_by":"auto","created_at":"2025-09-26 10:47:31","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":49690,"visible":true,"origin":"","legend":"s2_Model outputs for tail-corrected AGD","description":"","filename":"s2ModeloutputsfortailcorrectedAGD.docx","url":"https://assets-eu.researchsquare.com/files/rs-7435085/v1/ee22a9473fd20ef6379b160c.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Fetal programming: Masculinization increases daughter lifetime reproduction at high competition","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFemale masculinization refers to the development of male-typical behavior, physiology, and anatomy in mammals, including humans\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. One prominent anatomical marker of this phenomenon is the elongation of the distance between the anus and genitals (anogenital distance, AGD). Prenatal exposure to elevated testosterone levels stimulates perineal tissue growth, resulting in longer AGD after birth\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. First mentioned in 1939\u003csup\u003e4\u003c/sup\u003e, the association between prenatal testosterone exposure and female masculinization has been studied in depth for more than eight decades but remains incompletely understood in terms of its underlying causes and reproductive implications.\u003c/p\u003e\u003cp\u003eMechanistically, the degree of masculinization is influenced by testosterone exposure during the so-called masculinization programming window\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e (gestational day 15\u0026ndash;18 in rats, gestational week 8\u0026ndash;14 in humans). Sources of excess testosterone include elevated maternal levels\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e or leakage from neighboring male fetuses in polytocous species (the intrauterine position phenomenon\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e). A less understood phenomenon is that maternal stress can masculinize daughters in humans and other mammals\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Further, a genetic influence on the degree of female masculinization has been detected in genome-wide association studies in cows\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eReproductive consequences are less resolved. Several studies report that masculinized females experience a negative impact on fecundity and fertility\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Wild female degus, on the other hand, form social groups of similar degree of masculinization and masculinized females produce more offspring via larger litters\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Other findings indicate that female masculinization may provide benefits in highly competitive environments because masculinized females are more aggressive, have larger home ranges, and are superior in offspring defense\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. It has therefore been hypothesized that masculinized females show improved fitness under conditions of high competition, such as high density\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This notion is supported by despotic species with pronounced reproductive skew (characterized by few or only one reproductively successful dominant female per group) where competition over reproduction is associated with highly masculinized trait expression in dominant females, including high testosterone secretion and aggression to secure reproduction\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe fetal programming hypothesis offers a framework for understanding divergent outcomes. This hypothesis posits that prenatal development is shaped by environmental cues to prepare offspring for the conditions they are likely to encounter after birth\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Following this argument, it is conceivable that highly competitive environments trigger maternal stress along with higher levels of corticosterone and testosterone, which may result in more masculinized daughters. Masculinized daughters should then show increased reproductive output in an environment of high competition. Supportive evidence includes increased daughter degree of masculinization through prenatal stress\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and laboratory studies showing that high population density or crowding correlates with increased levels of maternal corticosterone, aggression, testosterone, and greater masculinization of daughters\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In wild North American red squirrels, mothers exposed to high-density conditions or cues of it produced offspring that grew heavier during lactation, likely preparing them for competitive environments\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Hence, investigations under varying levels of competition may be one way forward to understand natural variation in female masculinization and its consequences for reproduction.\u003c/p\u003e\u003cp\u003eA methodological consideration may offer another way forward to explaining divergent results on female masculinization in the literature. Anogenital distance scales proportionally with body length, as evident from repeated measures across ages in humans and cows \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Thus, adjusting for length is important in controlling for AGD-body length allometry. However, accurately measuring body length in many mammals poses challenges due to their flexible bodies and resistance to handling\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Body weight is often easier to measure and highly repeatable. While weight correlates well with body length, it is an integrated measure of lean mass and body fat mass\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Conditioning AGD on body weight \u0026ndash; the traditional way of using AGD \u0026ndash; therefore also corrects for fat accumulation. This may confound interpretations of the relationship between AGD and outcome variables because of the link between masculinization and accelerated growth\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Conditioning AGD on length measures may bring new insights.\u003c/p\u003e\u003cp\u003eWe use wild house mice (\u003cem\u003eMus musculus domesticus\u003c/em\u003e) in Switzerland to investigate female masculinization. Studying wild house mice in a field setting is advantageous as mice are free to perform their full behavioral repertoire, in a seasonal environment, under the influence of natural selection, in a naturally grown population with close relatives and descendents, unlike in lab mice. In Switzerland, reproductive competition experienced by female house mice differs seasonally, with breeding activity reaching a peak during spring and population density being highest in summer\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In addition to seasonal variation, mice experience annually changing population density and females have fewer offspring surviving during high density, indicating higher reproductive competition\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Varying levels of reproductive competition make this an excellent study system for investigating the fetal programming hypothesis. Using 13 years of data collected in a longitudinal study of a free-living mouse population\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, we build a dataset of measurements of AGD, body weight, and tail length taken for 4\u0026rsquo;939 female pups at the age of 13 days, together with environmental factors including population density and season of birth. We add extensive information on adult steroid hormones, parentage, and lifetime reproductive success, resulting in an exceptional multilayer dataset across several generations.\u003c/p\u003e\u003cp\u003eBased on assumptions of the fetal programming hypothesis, we predict that concentrations of testosterone and corticosterone in females rise with increasing reproductive competition as indicated by high population density and / or during peak breeding activity in spring (hypothesis \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Consequently, the degree of female masculinization should also increase with reproductive competition in the population (hypothesis \u003cspan refid=\"FPar2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As a mechanistic prerequisite, maternal corticosterone or testosterone needs to increase daughter degree of masculinization (hypothesis \u003cspan refid=\"FPar3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Finally, we predict that masculinization improves female fitness, in particular under high reproductive competition (hypothesis \u003cspan refid=\"FPar4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003eGeneral information\u003c/p\u003e\u003cp\u003eOver a period of 13 years (January 2008 to December 2020), we measured 4\u0026rsquo;939 female pups at 13 days of age (range 12\u0026ndash;14 days) in a population of free-ranging house mice. Population density ranged from 52 to 506 adults and increased over the years, besides annual variation over the seasons. Both mother and father could be assigned genetically at 95% confidence for 77% of these pups (n\u0026thinsp;=\u0026thinsp;3\u0026rsquo;808). We further collected 762 hair samples for hormone analysis from 585 adult females during the last 4.5 years of the study period.\u003c/p\u003e\u003cp\u003eThroughout the results, we report mean values (x̅) where variables are normally distributed and median values for other distributions (x̃). Sample sizes, descriptive statistics and variances are presented in the supplementary material (s1_Descriptive statistics).\u003c/p\u003e\u003cp\u003eOf all female pups measured at day 13, 2\u0026rsquo;307 (47%) were recaptured at a body weight of at least 17.5 g and were thus categorized as being recruited into the adult population (the remaining 53% either died or emigrated as subadults). For 67% of recruited females (n\u0026thinsp;=\u0026thinsp;1\u0026rsquo;454; corresponding to 29% of all female pups measured), we genetically identified 1 to 33 male or female offspring surviving to day 13. Reasons for unsuccessful reproduction (zero offspring surviving until day 13) may range from failure to conceive or raise offspring despite successful recruitment into the adult population, to technical problems of parental trio assignment (potentially due to errors in detection and sampling of individuals, PCR amplification, or insufficient marker variation).\u003c/p\u003e\u003cp\u003eFemale pup AGD and body weight\u003c/p\u003e\u003cp\u003eAt day 13 (range 12\u0026ndash;14), the AGD of female pups ranged from 2 to 6 mm (x̅ = 3 mm) and their weight from 2 to 11 g (x̅ = 6 g; s1_Table1). Body weight strongly and positively correlated with tail length (r\u0026thinsp;=\u0026thinsp;0.78), while AGD slightly more strongly increased with tail length (r\u0026thinsp;=\u0026thinsp;0.56) than with body weight (r\u0026thinsp;=\u0026thinsp;0.51, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTesting hypotheses\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHypothesis 1\u003c/strong\u003e\u003cp\u003eReproductive competition increases adult female corticosterone and testosterone\u003c/p\u003e\u003c/p\u003e\u003cp\u003eContrary to our expectations, corticosterone and testosterone measured in hair of adult females decreased with increasing density (total number of adults in the population, our first measure of reproductive competition; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, supplementary material s2_Model 1A-B). Season, our second measure of reproductive competition (with spring being characterized by highest breeding activity in the population), affected corticosterone and testosterone in different ways. Corticosterone was lowest in hair samples corresponding to the season of reduced breeding activity, winter, while testosterone did not differ significantly by season (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, s2_Model 1A-B). Within individuals, corticosterone and testosterone nevertheless correlated positively, although weakly (Pearson r\u0026thinsp;=\u0026thinsp;0.16, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;625).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHypothesis 2\u003c/strong\u003e\u003cp\u003eMaternal exposure to reproductive competition during pregnancy increases daughter degree of masculinization\u003c/p\u003e\u003c/p\u003e\u003cp\u003eWe quantified masculinization of female pups at day 13 as residualized anogenital distance (AGD corrected for allometry with tail length, AGD.tail). Opposite to expectations from the fetal programming hypothesis, female pups were less masculinized (had smaller AGD.tail) at higher reproductive competition in the population (measured at daughter birth), with increasing population density and during spring (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, s2_Model 2). Yet, the effects of both indices of reproductive competition together explained only 4% of the variance in masculinization. More variation was explained by sharing the same uterine environment (measured through genetic litter identity; ~30%), sharing the same mother (~\u0026thinsp;6%) or the same father (~\u0026thinsp;20%; Intraclass Correlation Coefficient ICC, s2_Model 2).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHypothesis 3\u003c/strong\u003e\u003cp\u003eMaternal testosterone and corticosterone during pregnancy increases daughter degree of masculinization\u003c/p\u003e\u003c/p\u003e\u003cp\u003eMothers with higher testosterone concentrations during the period covering pregnancy produced more masculinized daughters, explaining 8% of variation in daughter masculinization, in accordance with the mechanistic assumptions of the fetal programming hypothesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, s2_Model 3A). Maternal corticosterone levels alone did not significantly affect daughter masculinization (s2_Model 3B). Yet, the interaction between maternal testosterone and corticosterone increased the predictability of daughter masculinization to 13% (s2_Model 3C). Thus, increasing maternal testosterone induced increasing daughter masculinization only when mothers at the same time had low corticosterone.\u003c/p\u003e\u003cp\u003eIn models that included testosterone (s2_Model 3A and C), we had, by coincident, an equal number of fathers and litters. Here, the genetic litter identity explained 38% and father identity 6% of variation in daughter masculinization. Mother identity did not further explain variation in daughter masculinization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHypothesis 4\u003c/strong\u003e\u003cp\u003eMasculinization enhances female fitness under high reproductive competition\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLifetime reproductive success\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMasculinization was positively associated with increasing lifetime reproductive success (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, s2_Model 4A). Among the 29% of females that later reproduced, those with higher degrees of masculinization as pups had significantly more offspring surviving to day 13. While the effect was small (estimate (AGD.tail)\u0026thinsp;=\u0026thinsp;0.15 more offspring with each 1 mm increase in AGD length) it was highly robust (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, s2_Model 4). Notably, whether or not a female reproduced at all was not predicted by their degree of masculinization in this model. However, from this model we could not assess if increased masculinization generally led to increased reproduction, or if the advantage was linked to particular conditions of reproductive competition.\u003c/p\u003e\u003cp\u003eIncluding indicators of reproductive competition (density and season) provided a more differentiated picture. Population density and season of birth were important predictors of lifetime reproductive success, although with different effects. The probability to successfully reproduce (to have at least one surviving offspring) \u003cb\u003eincreased\u003c/b\u003e for females by 6% with every 100 additional adults in the population. Among those that reproduced, however, females had 0.3 \u003cb\u003efewer\u003c/b\u003e surviving offspring with every 100 additional adults (s2_Model 5, negative estimates of the zero-inflation model indicate reduced probability to have zero offspring). Season of birth also significantly affected reproductive outcomes but differed from effects of density. Spring born females were about 70% \u003cb\u003eless likely\u003c/b\u003e to reproduce than if born late in the year (autumn or winter) and, once they reproduced, had 0.2 \u003cb\u003efewer\u003c/b\u003e offspring surviving compared to summer and autumn born females (s2_Model 5).\u003c/p\u003e\u003cp\u003eWhile both density and season were biologically meaningful predictors of lifetime reproductive success, only density moderated the effect of masculinization. The probability to have at least one surviving offspring \u003cb\u003eincreased\u003c/b\u003e for females by 20% with each 1 mm increase in AGD and every 100 adults \u003cb\u003emore\u003c/b\u003e in the population (s2_Model 5). Among those that reproduced, females tended to have 0.09 \u003cb\u003emore\u003c/b\u003e offspring with each 1 mm increase in AGD and every 100 adults \u003cb\u003eless\u003c/b\u003e in the population (p\u0026thinsp;=\u0026thinsp;0.09) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, s2_Model 5). Combining these two slopes in a plot, lifetime reproductive success of females decreased under high-density conditions but masculinized females (i.e., those with longer AGD) were less affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRecruitment into adult population\u003c/b\u003e\u003c/p\u003e\u003cp\u003eResults on the likelihood of recruitment into the adult population paralleled those of the likelihood to reproduce. Recruitment probability of females generally \u003cb\u003eincreased\u003c/b\u003e by 20% with every 100 \u003cb\u003emore\u003c/b\u003e adults in the population (s2_Model 6A). Recruitment was lower for spring and summer born females than for autumn and winter born females (Table\u0026nbsp;1, s2_Model 6A). The degree of masculinization changed female recruitment probability across density but not across season. Female recruitment probability \u003cb\u003eincreased\u003c/b\u003e by 23% with each 1 mm increase in AGD and each 100 adults \u003cb\u003emore\u003c/b\u003e in the population (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, s2_Model 6). Under low-density conditions, the degree of masculinization seemed to have no or a slightly negative influence on recruitment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eComparing masculinization indices\u003c/p\u003e\u003cp\u003eAll models that included the masculinization index AGD corrected for tail length, AGD.tail, were repeated with an AGD corrected for body weight, AGD.weight (see supplemental material s3_Model outputs for weight-corrected AGD; s3), whereby the latter measure is the traditional way of using AGD. Both indices yielded similar results. Nevertheless, results for AGD.tail were consistently stronger than those for AGD.weight as indicated by stronger significance and lower AIC values: Maternal testosterone significantly predicted AGD.tail, and predicted AGD.weight more weakly (s2_Model 3A vs. s3_Model 3A). AGD.tail was a stronger predictor for lifetime reproductive success (s2_Model 4 and 5 vs. s3_Model 4 and 5) and recruitment into the adult population (s2_Model 6 vs. s3_Model 6). Only when predicting the degree of masculinization by density and season of birth did AGD.weight yield better results than AGD.tail (s2_Model 2 vs. s3_Model 2). This suggests that a length corrected AGD measure in pups better reflects effects driven by the degree of masculinization.\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eWe studied 4\u0026rsquo;939 female pups from a free-living house mouse population monitored over 13 years to investigate female masculinization within the framework of the fetal programming hypothesis. Applied to masculinization, this hypothesis posits that reproductive competition increases female stress hormones along with testosterone, which allows mothers to increase daughter masculinization to enhance daughter competitiveness, and therefore fitness, under similar conditions of reproductive competition. In line with the fetal programming hypothesis, maternal testosterone enhanced daughter masculinization, suggesting a direct maternal effect, and those masculinized daughters had increased lifetime reproductive success as competition increased. Contrasting the hypothesis is that adult female testosterone declined population-wide as density increased, accompanied by a concurrent decline in daughter masculinization. It suggests that female masculinization through fetal programming is adaptive but only few females can afford such programming as density increases.\u003c/p\u003e\u003cp\u003eOur first hypothesis, based on fetal programming, predicted higher adult female corticosterone and testosterone with increased reproductive competition. As expected, the two hormones correlated positively within individuals, though weakly. Contrary to predictions, both declined with population density, our proxy for reproductive competition. While few studies have measured female steroid hormones relative to group size or density, the prevailing view suggests that density elevates stress hormones, aggression, and testosterone. For example, Rhesus monkeys showed rising stress hormones and injuries at higher densities irrespective of sex\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Crespi et al.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e in their recent review showed that high female testosterone enhances dominance but reduces reproductive traits, concluding that all females should increase testosterone to improve social status under competition. In line with this notion, dominant females in species with very high reproductive competition express levels of testosterone that parallel or even exceed that of males\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eStill, we found decreasing testosterone with increasing density. An extension of the idea from Crespi et al.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e might reconcile our findings with those from the above-mentioned studies, as masculinization affects many traits. The optimal balance of these traits shifts with environmental and social conditions. Most studies assess density effects in females confined in cages or enclosures\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, where costs and benefits of dominance might differ from free-living individuals that can choose social partners and reproductive strategies. Our results suggest that in free-living house mice, high testosterone may disrupt group cohesion and cooperation for communal breeding, a female alternative reproductive tactic to solitary breeding\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Since group size and communal breeding increase with density\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, costs of high testosterone expression or maintenance may rise under these conditions, making it less affordable. Similar testosterone patterns are observed in male mammals and birds. Group-living male African striped mice have lower testosterone than solitary males\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, men\u0026rsquo;s testosterone decreases when they transition from a solitary lifestyle to a long-term relationship or fatherhood\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and colonial birds deposit less testosterone in eggs than solitary birds\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In female house mice, reduced corticosterone and testosterone may foster prosocial behavior and tolerance, facilitating crowding\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e as seen in some populations\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This hormonal decrease may also explain the observed higher female recruitment into adult populations under high density (see Hypothesis \u003cspan refid=\"FPar4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur second hypothesis predicted that reproductive competition during gestation increases daughter masculinization. However, like corticosterone and testosterone, daughter masculinization decreased with both indicators of reproductive competition, increasing population density and peak breeding season in spring. This again contradicts our fetal programming predictions and contrasts with guinea pigs, where daughter masculinization rises with social disruption, linked to high density\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Importantly, being born during high density and in spring reduced female lifetime reproductive success, confirming their biological significance as an indicator of reproductive competition.\u003c/p\u003e\u003cp\u003eIn our third hypothesis, we tested whether maternal testosterone and corticosterone positively affect daughter masculinization. We found that daughter masculinization increased with maternal testosterone, consistent with laboratory studies\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and confirming the biological relevance of maternal testosterone in wild mammals. It suggests a direct maternal influence on daughter masculinization during the masculinization programming window, which fulfills a key mechanistic requirement of fetal programming. It also implies that reduced female testosterone at high density causally underlies the density-associated decline in masculinization. Notably, this positive effect of maternal testosterone on daughter masculinization was driven by a few mothers with exceptionally high testosterone, indicating that under high-density conditions, when hair was sampled, only some females maintain elevated levels and program their daughters accordingly. Furthermore, high maternal testosterone predicted increasing daughter masculinization in those mothers that had low levels of corticosterone. This supports predictions of the dual hormone hypothesis, claiming that testosterone only correlates with an individual\u0026rsquo;s social dominance if corticosterone is low\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e (or cortisol in other mammals). Our data suggest that, in addition, individual high testosterone together with low corticosterone jointly affect daughters and thus the next generation.\u003c/p\u003e\u003cp\u003eContrary to our prediction, maternal corticosterone showed no relationship with daughter masculinization, despite considerable maternal hormone variation and supporting evidence from human and lab studies\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. This discrepancy may reflect fundamental differences between experimental or clinical settings and naturalistic settings, in which animals have more options to implement their behavioral preferences, such as ranging widely, choosing their social and sexual partners and dispersing.\u003c/p\u003e\u003cp\u003eDaughter masculinization and female testosterone both decreased with density. From a mechanistic perspective, it is thus conceivable that density reduces females\u0026rsquo; and thus mothers\u0026rsquo; ability to invest into high testosterone, which reduces daughter masculinization. However, seasonal variation in female testosterone, which was lowest in summer, cannot be associated with the particularly low degree of masculinization in spring-born daughters. We thus currently miss a good mechanistic explanation for reduced masculinization in spring. Paternal testosterone decrease in spring could potentially be a driver, as we found that paternal identity significantly contributed to daughter masculinization by 20%. Yet, we lack information on male seasonal hormone patterns in house mice. In addition, lowered male testosterone during the peak breeding season would clearly contradict our expectations as well as observations in other species. On the other hand, having more receptive females available at the same time during peak breeding season might increase the possibility for low-testosterone males to reproduce, which might result in population-wide reduced daughter masculinization.\u003c/p\u003e\u003cp\u003eBeyond parental effects, the shared uterine environment significantly shaped daughter masculinization, accounting for 30% of variation in our 13-year dataset. We do not have information on litter sex ratio at birth due to frequent infanticide before we measure pups at day 13\u003csup\u003e47,48\u003c/sup\u003e. The reported effect thus could either derive from a current maternal status, e.g., maternal testosterone, or from male siblings. When accounting for maternal testosterone, litter identity still accounted for 38% of variation in a subset sample. It suggests that intrauterine testosterone dynamics are at least partly driven by litter sex ratio as has been shown in wild marmots\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur fourth hypothesis predicted fitness benefits of masculinization under high reproductive competition. Overall, masculinized females had higher lifetime reproductive success (offspring surviving day 13), supporting its adaptive value. Looking at specific fitness-relevant traits revealed a more variable picture. Under high-density conditions, masculinized females were more likely to be recruited into the adult population and to reproduce, thereby achieving higher lifetime reproductive success. At low densities, masculinization did not affect recruitment or reproduction probability but tended to increase lifetime offspring number among females with at least one surviving offspring. Thus, masculinization affects multiple fitness-related traits. Each specific trait may independently be modified into different directions by masculinization depending on ecological conditions, as expected by the fetal programming hypothesis. Consequently, simultaneous consideration of multiple fitness-relevant traits at varying ecological conditions seems essential to understand the adaptive value of female masculinization.\u003c/p\u003e\u003cp\u003eOur findings that masculinized females more often remained in the population to adulthood and had more offspring when breeding during low density may parallel findings in wild degus, where females grouped by their degree of masculinization and masculinized groups had more offspring\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In contrast, masculinized Yellow-bellied marmots started to reproduce at older age and thus later in the year\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, which reduced offspring survival\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. However, none of these or other studies included measures of competition making a comparison with our results difficult (but see Moncl\u0026uacute;s et al.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e for density dependent reproductive onset).\u003c/p\u003e\u003cp\u003eAlthough both population density and season of birth influenced daughter masculinization and reproductive success, only density moderated the fitness benefits of masculinization. This likely reflects that density at birth more reliably predicts future reproductive competition than season in house mice.\u003c/p\u003e\u003cp\u003eWe finally compared two indices of female masculinization, AGD corrected for tail length versus the more commonly used AGD corrected for body weight. Tail-length-corrected AGD consistently provided better model fit and stronger effect estimates (for an exception see the prediction of masculinization by indicators of competition, hypothesis \u003cspan refid=\"FPar2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Despite this, estimates from both masculinization indices remained within a similar range, suggesting that findings based on weight-corrected AGD are not misleading, but less accurate. Correcting AGD for body weight, which includes lean as well as fat body mass, may conflate effects of prenatal testosterone exposure on AGD and body mass, as obesity and accelerated postnatal weight gain are associated with female masculinization\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In contrast, correcting AGD for a measure or correlate of body length accounts for allometry, independent of fat mass, and we recommend its use.\u003c/p\u003e\u003cp\u003eIn summary, our results suggest that maternal testosterone enhances daughter masculinization, which in turn increases lifetime reproductive success under high-density conditions. This supports the fetal programming hypothesis and challenges the view that female masculinization is merely a maladaptive by-product of maternal investment in sons\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Yet, contrary to predictions, both population-wide testosterone and daughter masculinization declined as competition intensified, suggesting that these traits are shaped by trade-offs between maternal capacity, offspring requirements, and ecological constraints. In free-living house mice, masculinized daughters gained fitness advantages through increased recruitment and reproduction, depending on density, whereas mothers may incur costs from sustaining high testosterone and raising potentially faster-growing offspring\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Such trade-offs, mediated by individual choice of the social environment and reproductive strategy and moderated by ecological conditions, underscore the limitations of conclusions drawn from clinical or captive studies.\u003c/p\u003e\u003cp\u003eIn conclusion, female masculinization influences multiple fitness-relevant traits, but its benefits and costs shift with ecological context. As density rises, only a subset of high-quality mothers appears able to sustain high testosterone investment, paralleling patterns in despotic species where reproduction is monopolized by few highly masculinized females. Thus, the selective advantage of female masculinization emerges along a continuum shaped by ecological pressures and social competition, with despotic species at the extreme end. This strengthens the fetal programming hypothesis for female masculinization and suggests that testosterone-associated patterns may similarly apply to females and males in allowing reproductive dominance, though on a different level.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Study population and data collection\u003c/h2\u003e\u003cp\u003eStudy population\u003c/p\u003e\u003cp\u003eA population of wild commensal house mice (\u003cem\u003eMus musculus domesticus\u003c/em\u003e) has been intensively studied since 2002 in a former agricultural building at the border of a forest near Zurich, Switzerland. Inside the 72 m\u0026sup2; barn, mice are provisioned with 40 nest boxes, which mice use as protected places for resting and sleeping, and females for raising their litters. Bricks and tubes are provided as shelters as well as several structuring elements that mice can climb, so that mice can access any part of the barn. Standardized food (50:50 mix of oats and hamster food from Landi Schweiz AG, Dotzigen, Switzerland) as well as water are provided \u003cem\u003ead libitum\u003c/em\u003e which mimics the natural situation for commensal house mice in barns or stables in middle Europe. Mice can freely leave and enter the barn through numerous small holes or cracks that exclude predators larger than mice. Once outside, mice are subject to predation, especially from cats, foxes or owls.\u003c/p\u003e\u003cp\u003eMice are individually followed from the time they are pups until they leave the building (emigrate) or die, with the help of regular pup (nest) monitoring, population monitoring, RFID tracking when entering or leaving nest boxes, and through genetic identification. Below we present those methods in detail that are relevant for the present study. For more information about the setup and methods used see elsewhere\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePup monitoring\u003c/p\u003e\u003cp\u003eAll nest boxes and shelters are checked for new litters at least every 13 days. Pup age is estimated according to morphological details\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, allowing us to calculate birthdate. Each litter consisting of same-aged pups found in the same nest is assigned a unique litter identity (litter ID), and pups younger than 12 days get a litter specific tattoo in one or several paws for later recognition. When pups are about to open their eyes and become mobile at 13 days of age, we return to the nest and take standardized pup measurements, including sex, body weight (measured using digital scales to the nearest 0.1 g), anogenital distance (AGD; measured in mm with digital calipers as the distance between the center of the anus and the posterior edge of the genitals; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), and tail length (measured in mm using a ruler as the distance between the center of the anus and the tip of the tail; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Additionally, a small tissue sample is taken from an ear for later genetic identification and parentage analyses (to determine the genetic father, mother and genetic siblings). To avoid too frequent disturbance of mothers and litters, we sample pups at an age range of 12\u0026ndash;14 days.\u003c/p\u003e\u003cp\u003ePopulation monitoring, RFID tagging and hair sampling\u003c/p\u003e\u003cp\u003eThe entire population is monitored at six-to-eight-week intervals by catching all mice present. Every individual is sexed and weighed, adult mice of at least 17.5 g are subcutaneously injected with a unique RFID tag (Trovan ID-100A implantable microtransponder) and a tissue sample is taken from the ear. Once tagged, mice can be permanently identified when handled or when entering or leaving nest boxes, which are equipped with automated reading devices\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e (antenna system). Mice reaching a body weight of at least 17.5 g are considered sexually mature in the study population\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSince December 2016, we use population monitoring events and occasionally pup monitoring events to collect adult hair samples for hormone analysis. To do so, hair is shaved with a veterinary razor from the lower back of newly tagged mice and opportunistically from tagged adults. We shave appr. 1 cm\u0026sup2; area that corresponds to 10\u0026ndash;15 mg hair.\u003c/p\u003e\u003cp\u003eShaving is avoided during colder periods to prevent additional thermoregulatory costs. During winter, we only collected hair samples from mice that were euthanized for other experiments.\u003c/p\u003e\u003cp\u003eParental assignment\u003c/p\u003e\u003cp\u003eMarkers at 25 polymorphic microsatellite loci are used for parentage analyses from tissue samples\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. We assign a mother and a father to each pup using trio analysis in the program CERVUS 3.0\u003csup\u003e56\u003c/sup\u003e. All females and males recorded in the barn during the 30 days prior to the birth of a pup are included as potential parents, unless they are recorded as having died before the pup birthdate (for potential mothers) or the estimated conception date (potential fathers). Paternity or maternity assignments are rejected if they are assigned at less than 95% confidence. To infer a shared uterine environment during ontogeny, a genetic litter identity (genetic litter ID) is assigned based on the identity of the genetic mother and the date of birth. Pups with the same genetic litter ID can have different fathers.\u003c/p\u003e\u003cp\u003eLife history data\u003c/p\u003e\u003cp\u003eAdult and pup measures are connected through genetic matching between adult and pup microsatellite DNA profiles. We consider mice that were recaptured at a body weight of at least 17.5 g as being recruited into the adult population (i.e. the individual did not die or emigrate from the barn before reaching sexual maturity). Parental assignment allows us to determine individual lifetime reproductive success (total number of male and female offspring sampled at 13 days).\u003c/p\u003e\u003cp\u003eHormone analysis\u003c/p\u003e\u003cp\u003eIndividual hair samples are kept in an envelope at room temperature until further processing in the \u0026lsquo;Dresden LABservice\u0026rsquo; (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dresden-labservice.com\u003c/span\u003e\u003cspan address=\"https://dresden-labservice.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For preparation, hair is washed once for three minutes with 2.5 ml isopropanol before hormones are extracted from 5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mg of dried whole hair (extraction for 18 h at room temperature with 1.5 ml of methanol). The extract is dried and reconstituted in 200 ml of a 50:50 mix of water and methanol. Steroid hormones (testosterone and corticosterone) are analyzed via liquid chromatography coupled with tandem mass spectrometry (LC\u0026ndash;MS/MS; for methodological details and details on the biological validation in house mice see elsewhere\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Statistical analyses\u003c/h2\u003e\u003cp\u003eGeneral information\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData set used\u003c/strong\u003e\u003cp\u003eOur study integrates data from morphological measurements, genetics, physiology, and life history collected from female house mice sampled as pups at the age of 12 to 14 days between January 2008 and December 2020. For a subset of females, we have hormone data from adult hair sampled opportunistically between December 2016 and December 2020.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFemale pup AGD and calculation of derived indices\u003c/strong\u003e\u003cp\u003eWe use the anogenital distance (AGD) at 12\u0026ndash;14 days as an indicator of female degree of masculinization. To inspect the relationship between AGD, body weight (integrated measure of lean and fat body mass) and tail length (as a repeatable approximation of body length\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e), we visualize and calculate their direct correlations. Throughout this study, we use a length corrected masculinization index, which corrects AGD for tail length (AGD.tail). More specifically, we use AGD residuals, the distance between the actual data point and the linear regression line between AGD and tail length. Positive residuals (values of AGD.tail) indicate more masculinized pups, pups with longer AGD than expected for their body length. To allow comparison of our measure of AGD.tail with the traditional way of correcting AGD by body weight (AGD.weight), we provide in the supplemental material all models investigated for AGD both corrected for tail length (AGD.tail) and corrected for body weight (AGD.weight). Since we are interested in the variation of masculinization in proportion to length and weight, and all three morphometrics of a pup (AGD, tail length, weight) are taken at the same time, we do not include the actual age at sampling (days 12\u0026ndash;14) in our models. For simplicity, we will hereafter refer to the age at sampling as day 13.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eReproductive competition\u003c/b\u003e among adult females is pronounced in the study population\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We use two measures to characterize reproductive competition. First, population density, estimated as the total number of tagged adults registered at a population monitoring event closest to the date of interest, because per capita reproductive success decreases with increasing density\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Second, we use season at the date of interest, because breeding activity varies with season\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e: Spring (March \u0026ndash; May, highest breeding activity in the study population), summer (June \u0026ndash; August), autumn (September \u0026ndash; November), or winter (December \u0026ndash; February, lowest breeding activity). Density and season at the date of interest refer to either date of birth (DOB) or date of hair sampling (hair), as specified.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSoftware used\u003c/b\u003e: We perform all statistical analyses and create figures in R 4.3.1 with RStudio\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e using the following packages: \u003cem\u003edplyr\u003c/em\u003e, \u003cem\u003etidyverse\u003c/em\u003e and \u003cem\u003ezoo\u003c/em\u003e for data arrangement\u003csup\u003e\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eggplot2\u003c/em\u003e, \u003cem\u003eggeffects\u003c/em\u003e and \u003cem\u003eviridis\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e to illustrate model results and \u003cem\u003epatchwork\u003c/em\u003e to arrange Figs.\u0026nbsp;6\u003csup\u003e7\u003c/sup\u003e, and \u003cem\u003epscl\u003c/em\u003e and \u003cem\u003eDHARMa\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e for model diagnostics. Other packages are mentioned below.\u003c/p\u003e\u003cp\u003eTesting hypotheses\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHypothesis 1\u003c/strong\u003e\u003cp\u003eReproductive competition increases adult female corticosterone and testosterone\u003c/p\u003e\u003c/p\u003e\u003cp\u003eWe investigate if concentrations of corticosterone and testosterone measured in adult female hair increase with reproductive competition as specified by population density and season at sampling. Since hormones in hair are an integrated measure representing the past two to three month\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, we refer to density and season at a time point 30 days before the hair was sampled (Density.hair, Season.hair). Testosterone and progesterone in the hair of wild derived house mice begin to differentiate between 50 to 60 days of age\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Since we are interested in the environmental effects on adult hormones, we only include samples from females older than 90 days.\u003c/p\u003e\u003cp\u003eWe use hormone concentrations (corticosterone or testosterone) as the response variable in a linear mixed effects model to account for repeated measures (function \u0026lsquo;lmer\u0026rsquo;, package \u003cem\u003elme4\u003c/em\u003e\u003csup\u003e70\u003c/sup\u003e). Density and season as well as age at hair sampling are included as predictor variables (Density.hair, Season.hair, Age.hair), and the identity of the adult female as random factor. We summarize model outputs using the function \u0026lsquo;summ\u0026rsquo; from the package \u003cem\u003ejtools\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e, which provides standardized estimates, confidence intervals and the Intraclass Correlation Coefficient \u0026lsquo;ICC\u0026rsquo; of our random variable female identity (\u0026lsquo;ID\u0026rsquo;). Results from the ICC range between 0 and 1 and can be interpreted similarly to the proportion or variance explained by a random factor\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHypothesis 2\u003c/strong\u003e\u003cp\u003eMaternal exposure to reproductive competition during pregnancy increases daughter degree of masculinization\u003c/p\u003e\u003c/p\u003e\u003cp\u003eTo test whether reproductive competition experienced by a mother during pregnancy (specified by density and season at pup date of birth, DOB) affects daughter degree of masculinization at day 13, we use a linear mixed effects model with the response variable AGD.tail (function \u0026lsquo;lmer\u0026rsquo;, package \u003cem\u003elme4\u003c/em\u003e\u003csup\u003e70\u003c/sup\u003e). Fixed predictor variables are density and season of birth (Density.DOB, Season.DOB). Random variables are genetic litter identity (to account for effects of a shared uterine environment among siblings) and mother and father identity (to control for parental effects).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHypothesis 3\u003c/strong\u003e\u003cp\u003eMaternal testosterone and corticosterone during pregnancy increases daughter degree of masculinization\u003c/p\u003e\u003c/p\u003e\u003cp\u003eHormones in hair are an integrated measure of the past two to three months (Carlitz et al. 2022) and we here make the assumption that hair testosterone and corticosterone measures are representative of values in adult females for the previous three months. The masculinization programming window in rats, a closely related species, covers approximately 6 to 10 days before birth (embryonic day 15\u0026ndash;18 of 24\u003csup\u003e5,73\u003c/sup\u003e). We therefore only include mother \u0026ndash; daughter pairs in this analysis when daughter birth was within 80 days before and 10 days after the mother was shaved. We use linear mixed effects models with AGD.tail as response variable (function \u0026lsquo;lmer\u0026rsquo;, package \u003cem\u003elme4\u003c/em\u003e; \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Fixed effect predictor variables are mother corticosterone (Model A) or mother testosterone (Model B) measured in hair, as well as both hormones and their interactions (Model C). Daughter genetic litter identity, and maternal and paternal identity are included as random effects predictor variable to account for repeated measures of siblings, within or across shared uterine environments.\u003c/p\u003e\u003cp\u003eSince we are interested in potential direct effects of maternal hormones, we do not control for maternal age at hair sampling, or measures of reproductive competition (population density, season).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHypothesis 4\u003c/strong\u003e\u003cp\u003eMasculinization enhances female fitness under high reproductive competition\u003c/p\u003e\u003c/p\u003e\u003cp\u003eAs a measure of fitness, we use lifetime reproductive success (number of offspring that were raised until at least day 13) in the barn. Over 70% of our study females measured as pups (n\u0026thinsp;=\u0026thinsp;4\u0026rsquo;939) had zero lifetime reproductive success. Most of these females disappeared before being tagged as adults. We are not certain whether such non-recruited individuals died without recovery of the corpse, or emigrated from the barn. Dispersal in house mice, however, is considered risky and to result in low (if any) reproductive success\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe first examine if masculinization alone or in interaction with indices of reproductive competition (Density.DOB and Season.DOB) affects female lifetime reproductive success in the study population. To account for the highly zero-inflated data on lifetime reproductive success and a negative binomial (overdispersed) distribution among reproducing females, we apply a generalized linear effects model with number of offspring (\u0026lsquo;offspring\u0026rsquo;) as response variable (function \u0026lsquo;glmmTMB\u0026rsquo;, family\u0026thinsp;=\u0026thinsp;truncated_nbinom2 with zero-inflation, package \u003cem\u003eglmmTMB\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. This model separately but simultaneously assesses a female\u0026rsquo;s likelihood to produce either 0 or at least 1 offspring (zero-inflation model) and estimates the number of offspring for those females that successfully reproduced (conditional model). When plotting model results, we present one regression line that combines estimates from the zero-inflation and the conditional model (function \u0026lsquo;ggpredict\u0026rsquo;, type = \u0026lsquo;zero_inflated\u0026rsquo;, packages \u003cem\u003eggeffects\u003c/em\u003e and \u003cem\u003eggplot\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. In a model building approach, we test if AGD.tail alone or in interaction with Density.DOB and Season.DOB predicts lifetime reproductive success. We compare the goodness of fit for each model using the Akaike Information Criterion (AIC).\u003c/p\u003e\u003cp\u003eSince recruitment is crucial to successful reproduction in the study population, we additionally examine the probability of recruitment of female pups in response to their degree of masculinization under varying reproductive competition. We use recruitment as a binary response variable in a generalized linear effects model (function \u0026lsquo;glm\u0026rsquo;, package \u003cem\u003elme4\u003c/em\u003e\u003csup\u003e70\u003c/sup\u003e. Recruitment of 0 refers to females that died or emigrated from the barn before being recaptured as adults (at a body weight of at least 17.5 g), recruitment of 1 otherwise. To explore the importance of masculinization, and of reproductive competition in moderating effects of masculinization, we add the predictor variables AGD.tail in interaction with density and season at female birth (Density.DOB, Season.DOB) in a model building approach and evaluate the goodness of model fit using the AIC.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eConceptualization: EC and BK. Data generation: EC, AL, BK, CK. Statistical analysis: EC. Investigation: EC, AL, BK. Writing\u0026mdash;original draft preparation, EC. Writing\u0026mdash;review and editing, EC, BK, AL, CK. Visualization: EC. Funding acquisition: EC, BK, AL, CK. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe study was supported by funding from the following sources: E.C.: DFG (CA 1870/1-1) and Maria Reiche Fellowship; B.K.: SNF grant 31003A_176114, and UZH Stiftung f\u0026uuml;r wissenschaftliche Forschung; A.K.L.: SNF grants 31003A_120444, 310030M_138389, Julius-Klaus and Promotor Stiftung.\u003c/p\u003e\n\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\n\u003cp\u003eWe declare we have no competing interests.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe are grateful to all colleagues and students who contributed to the management and data collection of the long-term study on house mice in the barn since 2002. We would like to especially acknowledge the work of our barn managers Sally Steinert and Bruce Boatman. We also thank Wei Gao, Anja Schulz and the hair lab team for analyzing the hair samples and Jari Garbely for genetic laboratory work. We thank Isin Kosemen from Synosys, TU Dresden, for her interactive charts.\u003c/p\u003e\n\u003ch2\u003eEthics\u003c/h2\u003e\n\u003cp\u003eData were collected under the permits ZH 215/2006, ZH 51/2010, ZH 56/2013, and ZH 091/2016 from the Cantonal Veterinary Office, Kanton Z\u0026uuml;rich, Switzerland.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVandenbergh JG, Huggett CL (1995) The anogenital distance index, a predictor of the intrauterine position effects on reproduction in female house mice. Lab Anim Sci 45:567\u0026ndash;573\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCrespi BJ, Bushell A, Dinsdale N (2024) Testosterone mediates life-history trade‐offs in female mammals. Biol Rev brv 13166. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/brv.13166\u003c/span\u003e\u003cspan address=\"10.1111/brv.13166\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evom Saal FS, Bronson FH (1978) In Utero Proximity of Female Mouse Fetuses to Males: Effect on Reproductive Performance during Later Life. Biol Reprod 19:842\u0026ndash;853\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGreene RR, Burrill MW, Ivy AC (1939) Experimental intersexuality. The effect of antenatal androgens on sexual development of female rats. Am J Anat 65:415\u0026ndash;469\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSharpe RM (2020) Androgens and the masculinization programming window: human-rodent differences. Biochem Soc Trans 48:1725\u0026ndash;1735\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDrea CM et al (2021) An intergenerational androgenic mechanism of female intrasexual competition in the cooperatively breeding meerkat. Nat Commun 12:7332\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eClark MM, Galef BG (1995) Prenatal influences on reproductive life history strategies. Trends Ecol Evol 10:151\u0026ndash;153\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarrett ES et al (2013) Prenatal exposure to stressful life events is associated with masculinized anogenital distance (AGD) in female infants. Physiol Behav 114\u0026ndash;115:14\u0026ndash;20\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGobikrushanth M et al (2019) The relationship between anogenital distance and fertility, and genome-wide associations for anogenital distance in Irish Holstein-Friesian cows. J Dairy Sci 102:1702\u0026ndash;1711\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWainstock T, Shoham-Vardi I, Sheiner E, Walfisch A (2017) Fertility and anogenital distance in women. Reprod Toxicol 73:345\u0026ndash;349\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCorrea LA et al (2025) Organizational effects of testosterone on the number of mating partners and reproductive success in females of a social rodent. Sci Rep 15:22411\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKemme K, Kaiser S, Sachser N (2007) Prenatal maternal programming determines testosterone response during social challenge. Horm Behav 51:387\u0026ndash;394\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMann MA, Svare B (1983) Prenatal testosterone exposure elevates maternal aggression in mice. Physiol Behav 30:503\u0026ndash;507\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZielinski WJ, vom Saal FS, Vandenbergh JG (1992) The effect of intrauterine position on the survival, reproduction and home range size of female house mice (Mus musculus). Behav Ecol Sociobiol 30:185\u0026ndash;191\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaiser S, Sachser N (2009) Effects of Prenatal Social Stress on Offspring Development: Pathology or Adaptation? Curr Dir Psychol Sci 18:118\u0026ndash;121\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDavies CS et al (2016) Exceptional endocrine profiles characterise the meerkat: sex, status, and reproductive patterns. Sci Rep 6:35492\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKwon EJ, Kim YJ (2017) What is fetal programming? a lifetime health is under the control of in utero health. Obstet Gynecol Sci 60:506\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChapman JC, Christian JJ, Pawlikowski MA, Michael SD (1998) Analysis of steroid hormone levels in female mice at high population density. Physiol Behav 64:529\u0026ndash;533\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMyers K, Poole W (1961) A study of the biology of the wild rabbit, Oryctolagus cuniculus (L.), in confined populations. II. The effects of season and population increase on behaviour. CSIRO Wildl Res 6:1\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZielinski WJ, Vandenbergh JG, Montano MM (1991) Effects of social stress and intrauterine position on sexual phenotype in wild-type house mice (Mus musculus). Physiol Behav 49:117\u0026ndash;123\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDantzer B et al (2020) Maternal glucocorticoids promote offspring growth without inducing oxidative stress or shortening telomeres in wild red squirrels. \u003cem\u003eJ. Exp. Biol.\u003c/em\u003e jeb.212373 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1242/jeb.212373\u003c/span\u003e\u003cspan address=\"10.1242/jeb.212373\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePriskorn L et al (2018) Anogenital distance as a phenotypic signature through infancy. Pediatr Res 83:573\u0026ndash;579\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRajesh I, Gobikrushanth M, Carrelli JE, Oba M, Ambrose DJ (2022) Repeatability of anogenital distance measurements from birth to maturity and at different physiological states in female Holstein cattle. J Dairy Sci 105:2699\u0026ndash;2707\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAplin KP (2003) Field Methods for Rodent Studies in Asia and the Indo-Pacific. Australian Centre for International Agricultural Research, Canberra\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJewell PA, Fullagar PJ (1966) Body measurements of small mammals: sources of error and anatomical changes. J Zool 150:501\u0026ndash;509\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuo J, Hall KD (2011) Predicting Changes of Body Weight, Body Fat, Energy Expenditure and Metabolic Fuel Selection in C57BL/6 Mice. PLoS ONE 6:e15961\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCorrea LA et al (2016) Masculinized females produce heavier offspring in a group living rodent. J Anim Ecol 85:1552\u0026ndash;1562\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKinsley C et al (1986) Prior intrauterine position influences body weight in male and female mice. Horm Behav 20:201\u0026ndash;211\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarlitz EHD, Lindholm AK, Gao W, Kirschbaum C, K\u0026ouml;nig B (2022) Steroid hormones in hair and fresh wounds reveal sex specific costs of reproductive engagement and reproductive success in wild house mice (\u003cem\u003eMus musculus domesticus\u003c/em\u003e). Horm Behav 138:105102\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEvans JC, Lindholm AK, K\u0026ouml;nig B (2021) Long-term overlap of social and genetic structure in free-ranging house mice reveals dynamic seasonal and group size effects. Curr Zool 67:59\u0026ndash;69\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGerber N, Auclair Y, K\u0026ouml;nig B, Lindholm AK (2021) Population density and temperature influence the return on maternal investment in wild house mice. Front Ecol Evol 8:602359\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFerrari M, Lindholm AK, Ozgul A, Oli MK, K\u0026ouml;nig B (2022) Cooperation by necessity: condition- and density-dependent reproductive tactics of female house mice. Commun Biol 5:1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eK\u0026ouml;nig B et al (2015) A system for automatic recording of social behavior in a free-living wild house mouse population. Anim Biotelemetry 3:39\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eK\u0026ouml;nig B, Lindholm AK (2012) The complex social environment of female house mice (\u003cem\u003eMus domesticus\u003c/em\u003e). In: Macholan M, Baird SJE, Munclinger P (eds) Evolution of the house mouse. Cambridge University Press, Cambridge, pp 114\u0026ndash;134. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1017/CBO9781139044547.007\u003c/span\u003e\u003cspan address=\"10.1017/CBO9781139044547.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDettmer AM, Novak MA, Meyer JS, Suomi SJ (2014) Population density-dependent hair cortisol concentrations in rhesus monkeys (\u003cem\u003eMacaca mulatta\u003c/em\u003e). Psychoneuroendocrinology 42:59\u0026ndash;67\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eK\u0026ouml;nig B (1994) Fitness effects of communal rearing in house mice: the role of relatedness versus familiarity. Anim Behav 48:1449\u0026ndash;1457\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchradin C, Yuen C-H (2011) Hormone levels of male African striped mice change as they switch between alternative reproductive tactics. Horm Behav 60:676\u0026ndash;680\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoney JR, Gettler LT (2015) The role of testosterone in human romantic relationships. Curr Opin Psychol 1:81\u0026ndash;86\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBentz AB, Becker DJ, Navara KJ (2016) Evolutionary implications of interspecific variation in a maternal effect: a meta-analysis of yolk testosterone response to competition. R Soc Open Sci 3:160499\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eParmigiani S, Mainardi M, Brain PF, Haug M, Brunoni V (1989) Variation in aggressive behavior and anatomo-physiological correlates generated by crowding without physical contact in the house mouse. Aggress Behav 15:191\u0026ndash;200\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMice Plague in Australia (1932) Nature 129:755\u0026ndash;755\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaiser S, Kruijver FPM, Swaab DF, Sachser N (2003) Early social stress in female guinea pigs induces a masculinization of adult behavior and corresponding changes in brain and neuroendocrine function. Behav Brain Res 144:199\u0026ndash;210\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHotchkiss AK et al (2007) Prenatal Testosterone Exposure Permanently Masculinizes Anogenital Distance, Nipple Development, and Reproductive Tract Morphology in Female Sprague-Dawley Rats. Toxicol Sci 96:335\u0026ndash;345\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMehta PH, Josephs RA (2010) Testosterone and cortisol jointly regulate dominance: Evidence for a dual-hormone hypothesis. Horm Behav 58:898\u0026ndash;906\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFishman R, Kralj-Fišer S, Marglit S, Koren L, Vortman Y (2024) Fathers and sons, mothers and daughters: Sex-specific genetic architecture for fetal testosterone in a wild mammal. Horm Behav 161:105525\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evom Saal FS et al (1990) Paradoxical Effects of Maternal Stress on Fetal Steroids and Postnatal Reproductive Traits in Female Mice from Different Intrauterine Positions1. Biol Reprod 43:751\u0026ndash;761\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAuclair Y, K\u0026ouml;nig B, Ferrari M, Perony N, Lindholm AK (2014) Nest attendance of lactating females in a wild house mouse population: benefits associated with communal nesting. Anim Behav 92:143\u0026ndash;149\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFerrari M, Lindholm AK, K\u0026ouml;nig B (2019) Fitness consequences of female alternative reproductive tactics in house mice \u003cem\u003e(Mus musculus domesticus)\u003c/em\u003e. Am Nat 193:106\u0026ndash;124\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoncl\u0026uacute;s R, Blumstein DT (2012) Litter sex composition affects life-history traits in yellow-bellied marmots. J Anim Ecol 81:80\u0026ndash;86\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCorrea LA et al (2021) One for all and all for one: phenotype assortment and reproductive success in masculinized females. Behav Ecol 32:1266\u0026ndash;1275\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoncl\u0026uacute;s R, von Holst D, Blumstein DT, R\u0026ouml;del HG (2014) Long-term effects of litter sex ratio on female reproduction in two iteroparous mammals. Funct Ecol 28:954\u0026ndash;962\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoncl\u0026uacute;s R, Pang B, Blumstein DT (2014) Yellow-bellied marmots do not compensate for a late start: the role of maternal allocation in shaping life-history trajectories. Evol Ecol 28:721\u0026ndash;733\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCrespi B, Dinsdale NL (2023) The sexual selection of endometriosis. Evol Behav Sci 17:123\u0026ndash;170\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHelle S, Laaksonen T, Huitu O (2013) Sex-specific offspring growth according to maternal testosterone, corticosterone, and glucose levels. Behav Ecol 24:205\u0026ndash;212\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLindholm AK, Musolf K, Weidt A, K\u0026ouml;nig B (2013) Mate choice for genetic compatibility in the house mouse. Ecol Evol 3:1231\u0026ndash;1247\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program \u0026lsquo;cervus\u0026rsquo; accommodates genotyping error increases success in paternity assignment. Mol Ecol 16:1099\u0026ndash;1106\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarlitz EHD et al (2019) Steroid hormones in hair reveal sexual maturity and competition in wild house mice (\u003cem\u003eMus musculus domesticus\u003c/em\u003e). Sci Rep 9:1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao W et al (2013) Quantitative analysis of steroid hormones in human hair using a column-switching LC\u0026ndash;APCI\u0026ndash;MS/MS assay. J Chromatogr B 928:1\u0026ndash;8\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheverud JM, Leamy LJ, Atchley WR, Rutledge JJ (1983) Quantitative genetics and the evolution of ontogeny: I. Ontogenetic changes in quantitative genetic variance components in randombred mice. Genet Res 42:65\u0026ndash;75\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eR Core Team (2023) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWickham H (2016) tidyverse: Easily Install and Load the \u0026lsquo;Tidyverse\u0026rsquo;. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32614/CRAN.package.tidyverse\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.package.tidyverse\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 2.0.0\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWickham H, Fran\u0026ccedil;ois R, Henry L, M\u0026uuml;ller K, Vaughan D (2014) dplyr: A Grammar of Data Manipulation. 1.1.4 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32614/CRAN.package.dplyr\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.package.dplyr\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeileis A, Grothendieck G, zoo (2005) \u003cem\u003eS3\u003c/em\u003e Infrastructure for Regular and Irregular Time Series. \u003cem\u003eJ. Stat. Softw.\u003c/em\u003e 14\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarnier S (2015) viridis: Colorblind-Friendly Color Maps for R. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32614/CRAN.package.viridis\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.package.viridis\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 0.6.5\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eL\u0026uuml;decke Dggeffects (2018) Tidy Data Frames of Marginal Effects from Regression Models. J Open Source Softw 3:772\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWickham H (2016) Ggplot2: Elegant Graphics for Data Analysis. Springer international publishing, Cham\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePedersen TL, patchwork (2019) The Composer of Plots. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32614/CRAN.package.patchwork\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.package.patchwork\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 1.3.1\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHartig F, DHARMa (2016) Residual Diagnostics for Hierarchical (Multi-Level / Mixed) Regression Models. 0.4.7 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32614/CRAN.package.DHARMa\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.package.DHARMa\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJackman Spscl (2005) Political Sci Comput Lab. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32614/CRAN.package.pscl\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.package.pscl\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 1.5.9\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBates D, Maechler M, Bolker B, Walker S (2003) lme4: Linear Mixed-Effects Models using \u0026lsquo;Eigen\u0026rsquo; and S4. 1.1\u0026ndash;37 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32614/CRAN.package.lme4\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.package.lme4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLong JA (2024) jtools: Analysis and Presentation of Social ScientificData. J Open Source Softw 9:6610\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBryer J (2023) Relationship Between Intraclass Correlation (ICC) and Percent Agreement. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://irrsim.bryer.org/articles/IRRsim.html\u003c/span\u003e\u003cspan address=\"https://irrsim.bryer.org/articles/IRRsim.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWelsh M et al (2008) Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. J Clin Invest 118:1479\u0026ndash;1490\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBonte D et al (2012) Costs of dispersal. Biol Rev 87:290\u0026ndash;312\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRunge J-N, Lindholm AK (2018) Carrying a selfish genetic element predicts increased migration propensity in free-living wild house mice. \u003cem\u003eProc. R. Soc. B Biol. Sci.\u003c/em\u003e 285, 20181333\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrooks M et al (2017) glmmTMB: Generalized Linear Mixed Models using Template Model Builder. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32614/CRAN.package.glmmTMB\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.package.glmmTMB\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 1.1.11\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eL\u0026uuml;decke Dggeffects (2017) Create Tidy Data Frames of Marginal Effects for \u0026lsquo;ggplot\u0026rsquo; from Model Outputs. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32614/CRAN.package.ggeffects\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.package.ggeffects\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 2.3.0\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7435085/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7435085/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHigh testosterone along with masculinized traits is often considered maladaptive for female mammals, based on clinical cases of reproductive disorders and standardized animal studies. Yet, despotic species, where masculinized females dominate reproduction, challenge this view. Here, we provide support for selective benefits of female masculinization, by applying the fetal programming hypothesis. Using long-term data on free-ranging house mice exposed to naturally fluctuating densities signaling reproductive competition, we show that high-testosterone mothers produced masculinized daughters with elongated anogenital distance who achieved higher lifetime reproductive success under high-density conditions. However, population-level female testosterone and daughter masculinization declined as competition intensified. Mismatching individual fitness benefits and population trends suggest masculinization is shaped by trade-offs among maternal quality, offspring demands, and ecological and social constraints. We conclude female masculinization is not inherently maladaptive but enables competitive mothers under reproductive competition providing selective benefits for daughters \u0026ndash; a process relevant within and likely also across species.\u003c/p\u003e","manuscriptTitle":"Fetal programming: Masculinization increases daughter lifetime reproduction at high competition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 10:47:26","doi":"10.21203/rs.3.rs-7435085/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3bba3e58-f097-4aae-a23c-b3057dd3b132","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55281937,"name":"Biological sciences/Ecology/Evolutionary ecology"},{"id":55281938,"name":"Biological sciences/Ecology/Ecophysiology"},{"id":55281939,"name":"Biological sciences/Ecology/Population dynamics"},{"id":55281940,"name":"Biological sciences/Zoology/Animal physiology"}],"tags":[],"updatedAt":"2025-11-10T00:35:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-26 10:47:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7435085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7435085","identity":"rs-7435085","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}