Defining Functional Puberty in Female C57BL/6J Mice Using Endocrine, Cytological and Morphological Markers

Defining Functional Puberty in Female C57BL/6J Mice Using Endocrine, Cytological and Morphological Markers | 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 Defining Functional Puberty in Female C57BL/6J Mice Using Endocrine, Cytological and Morphological Markers Mélanie Chester, Noalig Wyckens, Eloïse Airaud, Raphaël Corre, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8741954/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Puberty marks the transition to reproductive competence and is driven by activation of the hypothalamo/pituitary/gonadal axis, culminating in first ovulation in females. In mice, puberty onset is commonly inferred from vaginal opening and estrous cyclicity, but the precise timing of first ovulation remains unclear. Here, we aimed to define reliable parameters of functional puberty in female C57BL/6J mice by integrating external, endocrine, and morphological measures. Vaginal opening and daily vaginal cytology were combined with daily serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) measurements and ovarian histology using the Pubertal Ovarian Maturation Score (Pub-Score), which estimates ovulation based on follicular development and corpus luteum morphology. Notably, we observed substantial inter-individual variability in puberty-related events, and no link between estrous cytology and first ovulation. Pub-Score analysis indicated that first ovulation occurred between 36 and 54 days postnatal, with a mean around 44 days, with no correlation with vaginal cytology. LH surges were detected in some of the females that had ovulated and showed limited temporal correspondence with vaginal cytology or Pub-Score estimates. Together, these findings demonstrate that puberty in female mice is a gradual, asynchronous process and that external or cytological markers alone are insufficient to define functional reproductive maturity. Health sciences/Endocrinology Biological sciences/Physiology Puberty ovulation estrous cycle LH surge Pub-Score Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The concept of “puberty” has long been defined as the transitional period leading to reproductive competence, as initially described by Ojeda et al. [ 1 ]. This process involves profound neuroendocrine modifications within the hypothalamic circuitry, particularly involving glutamate, GABA, and kisspeptin (Kiss) neurons [ 2 ]. The activation of the hypothalamo-pituitary-gonadal (HPG) axis is a hallmark of puberty onset. Pulsatile secretion of gonadotropin-releasing hormone (GnRH) from hypothalamic neurons stimulates pituitary release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), thereby driving gonadal steroidogenesis and the emergence of secondary sexual characteristics. In humans, puberty typically occurs around 11–12 years of age, while in mice it takes place around 40 days postnatal (dpn) [ 3 ]. In females, the first ovulation represents a key event of this developmental stage, occurring under the control of the preovulatory GnRH-LH surge triggered by high estradiol (E2) levels acting through a positive feedback loop [ 4 ]. Defining the age at which the system becomes functional is critical in understanding key mechanisms of reproductive biology, physiology and physiopathology, and in identifying perturbations. In rodents, external indicators such as vaginal opening (VO) and subsequent stages of the estrous cycle (first estrus, E, and first diestrus 2, D2) are commonly used as indicators of puberty onset [ 5 – 8 ]. However, while vaginal opening and ovulation usually coincide in rats [ 9 ], it does not appear to be the case in mice, making the accurate identification of first ovulation challenging. To date, no reliable standardized method exists to precisely define the timing of first ovulation in intact mice. Moreover, vaginal cytology alone often fails to reflect the true onset of ovarian cyclicity. The objective of this study was therefore to determine whether the integrative analysis of different parameters could mark puberty onset and first ovulation in female mice. We used complementary approaches combining (1) vaginal opening and cytological evaluation, (2) measurement of circulating LH and FSH levels, and (3) morphological evaluation of the ovulation on ovarian histology, using the Pub-Score method described by Tena-Sempere’s group [ 9 ]. Altogether, our findings indicate that puberty onset and first ovulation in female mice cannot be reliably inferred from a single external or hormonal marker, and that commonly used criteria may lead to misinterpretation of reproductive maturity during the peripubertal window. Instead, an integrative assessment combining external, endocrine, histological, and functional reproductive readouts is required to accurately define the acquisition of ovarian cyclicity. Results Experimental design To investigate the temporal coordination of puberty onset and first ovulation in female mice of the C57BL/6J strain, which is the most widely used strain in mouse studies, three complementary experimental paradigms were implemented (Fig. 1 A). In all groups, vaginal opening (VO) was monitored daily starting at postnatal day (dpn) 26, an age at which C57BL/6J females are still sexually immature. Vaginal cytology was assessed daily, and ovaries were collected at the end of the experiment for histological dating of ovulation using the Pubertal Ovarian Maturation Score (Pub-Score) method [ 9 ]. This semi-quantitative histological method is based on ovarian morphology, integrating antral follicle development in pre-ovulatory animals and corpora lutea aging in post-ovulatory animals (Fig. 1 B). Negative scores indicate progressive pre-ovulatory follicular maturation and number of days before ovulation, score 0 corresponds to the first ovulation, and positive scores reflect the number of days since first or second ovulation. The age at first ovulation was calculated from the Pub-Score and the age at sample collection. Experiments were terminated at defined developmental milestones to capture key transitions: mice were euthanized either at the first estrus (group 1, 1st E; n = 10), at the first diestrus 2 following the first estrus (group 2, 1st D2; n = 10), or at variable time points between 9 and 16 days after VO following daily blood sampling to detect preovulatory gonadotropin surges (group 3; n = 26) (Fig. 1 A). This design enabled an integrated analysis of vaginal cytology, endocrine dynamics, and ovarian maturation across the peripubertal period. Marked inter-individual variability in estrous cycle patterns and timing of first ovulation Across the three experimental groups, vaginal opening was first observed between 27 and 38 dpn (mean ± SEM: 30.81 ± 0.28 dpn; Fig. 2 A). Subsequent estrous stages, identified by daily vaginal cytology, displayed pronounced inter-individual variability. The first estrus occurred between 29 and 48 dpn (36.9 ± 0.7 dpn), while the first diestrus 2 following first estrus occurred significantly later and was observed between 34 and 50 dpn (42.95 ± 1 dpn; Fig. 2 A). Consistent with this variability, the interval between vaginal opening and first estrus ranged from 0 to 17 days (6.5 ± 0.7 days), and the interval to first diestrus II ranged from 5 to 19 days (11.75 ± 0.98 days; Fig. 2 B). These results indicate that although vaginal opening represents an early marker of pubertal activation, it precedes the establishment of a complete estrous cycle by approximately two weeks, with substantial variability among individuals. Ovarian histology revealed that only 1 out of 10 mice studied at first estrus (10%) displayed corpora lutea, indicating that ovulation had occurred in a very small fraction of mice at this stage (Fig. 2 C). In contrast, 8 out of 10 mice (80%) euthanized at first diestrus 2 had ovulated, suggesting that most females achieved functional puberty during this interval. When we examined the estrus pattern of all mice having reached diestrus 2, we found that the mean delay between first estrus and first diestrus 2 was 4.8 days ± 0.8, ranging from 1 day up to 10 days. To further characterize ovarian maturation, ovaries from all mice (n = 46) were analyzed using the Pub-Score method, which integrates corpus luteum morphology, and follicular size. This analysis classified ovaries as Pub-Score positive (presence of corpora lutea) or negative (absence of corpora lutea). Overall, 22 out of 46 of examined mice (47.8%) exhibited histological evidence of ovulation. Based on Pub-Score estimates, first ovulation occurred between 36 and 51 dpn (mean ± SEM: 44.7 ± 0.7 dpn). In mice that had not yet ovulated at the time of sacrifice, retrospective estimation predicted first ovulation between 36 and 54 dpn (44.0 ± 1.2 dpn). When all animals were considered together, first ovulation was confined to a developmental window spanning 36–54 dpn, corresponding to 5–21 days after vaginal opening (mean ± SEM: 44.4 ± 0.7 dpn; 10.07 ± 0.75 days post-VO; Fig. 2 A). These results place the functional onset of puberty in C57BL/6J females at approximately 44 dpn, with marked inter-individual variability. Integration of endocrine, cytological, and morphological parameters To assess endocrine dynamics following vaginal opening, daily blood samples were collected in mice of group 3 at time points corresponding to the expected preovulatory LH surge, approximately one hour after lights off. Gonadotropin concentrations were measured using Luminex assays. Sampling began four days after VO and continued for 12 consecutive days. Basal LH concentrations remained below 1 ng/mL and fluctuated over time, consistent with the pulsatile release mode of this hormone (Fig. 4 ). Among the eight mice that ovulated during the sampling period, a distinct LH surge was detected in five individuals. One mouse (female 93) even exhibited two LH surges (Fig. 4 ). LH peaks occurred as early as four days after vaginal opening in two mice (females 93 and 104) and between 12 and 15 days post-VO in the remaining animals (females 91, 93, 94, and 105). FSH profiles showed substantial variability, with no consistent increase coinciding with LH surges. This may reflect the biphasic nature of FSH secretion around ovulation, with surges occurring outside the nocturnal sampling window. When gonadotropin profiles were combined with Pub-Score analysis for each individual, no clear temporal correspondence between LH surge detection and predicted ovulation timing was observed, except in one mouse (female 105; Fig. 4 ). Overall, first ovulation occurred within a 3 days range following the LH peak between 12- and 15-days post-VO, for the 5 females with a distinct LH surge, with the date of the first ovulation predicted 14 to 16 days post-VO for all the eight females. Integration with vaginal cytology indicated that LH surges predominantly occurred during estrus (Fig. 4 ). Finally, to evaluate the potential impact of repeated handling and blood sampling on pubertal timing, the estimated age of first ovulation was compared between mice subjected to blood sampling and those that were not. There was no significant effect of blood sampling when we considered age at first ovulation, but a small delay when considering the number of days after vaginal opening (Supplementary Fig. 1), suggesting that blood sampling could also interfere with puberty onset. Discussion Accurately defining the timing of puberty is key to understand the mechanisms regulating reproductive maturation and their susceptibility to genetic, metabolic, or environmental perturbations. In this study, we sought to identify parameters defining functional puberty in C57BL/6J female mice by integrating external, cytological, endocrine, and morphological readouts. By combining vaginal opening and daily vaginal cytology with gonadotropin measurements and histological ovarian assessment using the Pub-Score method, we provide a comprehensive evaluation of commonly used pubertal markers and their relationship to first ovulation. Our data demonstrate pronounced inter-individual variability across all pubertal landmarks examined, including the timing of vaginal opening, first estrus, and first diestrus 2. Histological analysis indicates that first ovulation occurs on average around 44 dpn, within a surprisingly broad developmental window spanning approximately 36 to 54 dpn. In parallel, detection of preovulatory LH surges was achieved in a subset of animals and showed limited temporal correspondence with Pub-Score-based estimates of ovulation. Together, these findings indicate that puberty in female mice is not a discrete event but rather a prolonged and asynchronous developmental process during which external, cytological, endocrine, and ovarian markers are uncoupled (Fig. 5 ). The marked variability observed between vaginal opening, estrous cyclicity, and first ovulation highlights the limitations of relying on external or cytological markers to define functional puberty. Unlike in rats, in which vaginal opening and ovulation occur in close temporal proximity [ 9 ], our data confirm that in mice vaginal opening precedes ovulation by approximately 10–15 days on average, with substantial inter-individual variation. Although the majority of mice had ovulated by the time of the first diestrus 2, only a minority had ovulated at first estrus, indicating that estrous cytology alone does not reliably reflect ovarian competence during the peripubertal period. Consistent with this, Pub-Score analysis revealed no strict association between estrous stage and ovulation, with first ovulation most frequently occurring at estrus but also detected at proestrus or diestrus I. This dissociation likely reflects the immaturity and instability of hypothalamo-pituitary-ovarian axis coordination during puberty, when estrus-like cytological patterns can emerge before the establishment of fully functional ovulatory cycles. Among the approaches tested, the Pub-Score method emerged as the most reliable indicator of first ovulation. By integrating corpus luteum morphology with follicular development, this method allows retrospective estimation of ovulation timing independently of transient endocrine or cytological fluctuations. The mean age of first ovulation identified in our study is consistent with previous reports in C57BL/6J mice and supports the notion that functional ovarian activation is substantially delayed relative to vaginal opening [ 9 ]. Notably, nearly half of the mice analyzed had not ovulated at the time of ovary collection despite exhibiting vaginal opening and, in some cases, repeated estrus-like cytology, underscoring the risk of misclassifying pubertal status when relying solely on these criteria. It would be of interest to determine whether the altered timing of puberty onset reported in numerous mouse models [ 10 – 13 ] is systematically associated with alterations in first ovulation, and thus with the functional onset of puberty. The study of LH dosage on the evening of ovulatory discharge has already been carried out in adult females with tail sampling and appears to be a good method of analysis [ 14 – 16 ]. The limited detection of LH surges in our study emphasizes the technical and biological challenges of capturing transient endocrine events during pubertal development. The brief duration of the LH surge [ 15 , 17 ], once-daily sampling, and the potential inhibitory effects of repeated handling with stress induction [ 18 ] likely contributed to the modest detection rate. Moreover, the imperfect alignment between LH surges and Pub-Score-predicted ovulation suggests that endocrine dynamics during puberty may not fully conform to the canonical adult estrous cycle on which Pub-Score staging is based. Nevertheless, the observation that LH surges were detected exclusively in animals with histological evidence of ovulation supports the conclusion that Pub-Score positivity reflects physiologically meaningful activation of the hypothalamic-pituitary-ovarian axis. Our findings are consistent with previous studies demonstrating that vaginal opening and first estrus precede functional ovarian activation in mice by a variable interval. The Pub-Score method, originally described by Gaytán et al.[ 9 ], established that neither vaginal opening nor estrous cytology can reliably define the timing of first ovulation. By integrating cytological, endocrine, and morphological readouts within the same experimental framework, our study extends these observations and highlights the magnitude of inter-individual variability even within a single inbred strain. Several limitations of our study should be acknowledged. Detection of LH surges was constrained by sampling frequency and potential stress-related suppression of gonadotropin secretion. Pub-Score analysis, while robust, is inherently cross-sectional and retrospective, preventing precise longitudinal tracking of ovulation in individual animals. In addition, this study was limited to the C57BL/6J strain, and strain-specific differences in pubertal timing and ovarian physiology may limit generalization. Taken together, our results underscore the need for integrative approaches when defining puberty in female mice. Increased sampling frequency, development of minimally invasive longitudinal methods, and incorporation of additional biomarkers of ovarian maturation may further refine the temporal resolution of pubertal staging. From a methodological standpoint, the use of Pub-Score analysis as a reference endpoint is strongly recommended when accurate identification of first ovulation is required. Importantly, experimental designs should explicitly recognize that vaginal opening and estrous cytology do not reliably predict ovulation and should avoid equating these markers with reproductive maturity. In conclusion, this study highlights the complexity and variability inherent in pubertal maturation in C57BL/6J female mice. External and cytological markers alone are insufficient to precisely determine the timing of first ovulation. Among the approaches tested, histological assessment using the Pub-Score method provides the most reliable estimate of functional puberty. These findings have important implications for experimental designs in reproductive and developmental biology and emphasize the necessity of rigorous, multi-parameter strategies when studying the onset of reproductive competence in mice. Methods Animals and treatments All experiments were conducted on C57BL/6J female mice bred in our animal facility from parents obtained from Janvier Labs (Le Genest St Isle, France). The day of birth was designated as postnatal day 0 (dpn 0), and weaning occurred at 21 dpn. Animal care and handling followed established procedures [ 19 ]. Mice were housed in groups of 3–5 per cage under a 12 h light/dark cycle, at 22 ± 1°C, with ad libitum access to standard chow and water. From 10 dpn, female mice were manipulated daily to limit the stress related to vaginal smears and tail-blood collection. Animals were randomly assigned to three experimental groups (Fig. 1 A). All sample collection and histological analyses were performed by investigators blinded to the experimental group. After VO, blood samples (20 µL) were collected daily for 12 consecutive days in one group of mice (n = 17), about one hour after lights off (8:30 PM), via tail sampling using capillaries. Prior to terminal cardiac puncture, mice were weighted and then anesthetized with ketamine (Imalgene® 1000; 100 mg/kg BW) and xylazine (Rompun® 2%; 10 mg/kg BW) injected intra-peritoneally with a 1mL sterile syringe mounted with a 26-gauge needle (Terumo). Euthanasia was performed by cervical dislocation. At the time of death, body weights were as followed (mean ± SEM): group 1, 16.2 ± 0.3 g; group 2, 17.1 ± 0.2 g; group 3, 16.6 ± 0.16 g. Serum was obtained after clotting for 15 minutes at room temperature and centrifugation at 5000 g for 5 minutes. Ovaries were collected post-mortem and fixed in Bouin’s solution (HT10132, Sigma-Aldrich) for histological processing. All procedures were approved by the Institutional Animal Care and Use Committee of Université Paris-Cité and the French Ministry of Agriculture (CEB#13-2021) and were performed in accordance with relevant guidelines and regulations. Determination of serum LH and FSH levels Serum LH and FSH concentrations were determined using the Milliplex MAP rat pituitary magnetic bead panel on 10 µL of sample (Merck-Millipore, Nottingham, UK), following the manufacturer’s instructions [ 19 ]. The assay sensitivities were 32 pg/mL for FSH and 3.2 pg/mL for LH. Inter-assay coefficients of variation were 5.4% (FSH) and 3.2% (LH), and intra-assay coefficients were 7.6% (FSH) and 5.9% (LH). Determination of estrous cyclicity Vaginal smears were collected daily following VO to monitor the onset of the estrous cycle, up to first estrus, first diestrus 2 or after 9–16 days depending on the group of mice (Fig. 1 ). Smears were obtained using 0.9% NaCl solution, stained with hematoxylin and eosin (HE), and analyzed under light microscopy. The stages of the cycle were identified based on the predominant cell types: nucleated epithelial cells (proestrus, PE), cornified cells (estrus, E), and leukocytes (diestrus 2, D2) as previously described [ 20 , 21 ]. Histological processing and Pub-Score analysis Ovaries (n = 46) were fixed in Bouin’s solution for 48 hours, rinsed in PBS, and stored in 70% ethanol. Samples were dehydrated, embedded in paraffin (HISTIM core facility, Cochin Institute, Paris), and sectioned at 5 µm. Every fifth section was mounted and stained with HE. Slides were scanned using a Pannoramic 250 Flash III system (3D HISTECH, Bicêtre Biomedical Institute). The Pub-Score method [ 9 ] was used to determine ovulation timing based on CL morphology and largest follicular size. Ovaries were classified as pre-ovulatory (no CL; largest follicle used to estimate − 1 day), ovulatory (presence of newly formed CL), or post-ovulatory (regressing CL and secondary follicular growth, + 1 day). Statistical analysis Sample sizes are provided in the text. Data were analyzed using GraphPad Prism 10. Data are shown as mean ± SEM or %, and statistical analyses were performed using one-way ANOVA or Student t-test, when appropriate. Graphical design All schematic figures were created using BioRender (BioRender.com). Declarations Competing Interests: the authors declare no competing financial interests. Funding Declaration This work was supported by Agence Nationale de la Recherche (ANR ReproFun, attributed to CJG) and Ecole Doctorale BioSPC (NW, MC). Author Contribution MC, CHP conducted vaginal smears and histological examination of the ovaries, determined the Pub-Score. CG, MC, CHP, RC and EA performed tail-blood collection. RC and MC performed histological sections. CG, MC and NW analyzed the data and prepared the figures. CG wrote the initial manuscript. CG, MC, NW and AP evaluated and discussed all the data. All authors read the manuscript. Acknowledgement The authors wish to thank the staff members of the core animal facility Buffon of Université Paris Cité (Paris, France). They acknowledge the technical assistance of M. Surenaud for gonadotropin assays (Hôpital Henri Mondor, Créteil, France) and O. Trassard for slide scanning (Cellular and tissue imaging core facility, Bicêtre Biomedical Institute). This work was supported by ANR ReproFun (CG) and ED BioSPC (MC, NW). Data Availability All data generated or analyzed during this study are included in this article, and any other data will be made available on request from the corresponding author. References Ojeda, S. R., Andrews, W. W., Advis, J. P. & White, S. S. Recent advances in the endocrinology of puberty. Endocr. Rev. 1 , 228–257 (1980). Prevot, V. Puberty in Mice and Rats. in Knobil and Neill’s Physiology of Reproduction 1395–14394th Edition, Amsterdam, The Netherlands, (2015). 10.1016/B978-0-12-397175-3.00030-2 Bell, M. R. Comparing Postnatal Development of Gonadal Hormones and Associated Social Behaviors in Rats, Mice, and Humans. Endocrinology 159 , 2596–2613 (2018). Christian, C. A. & Moenter, S. M. The Neurobiology of Preovulatory and Estradiol-Induced Gonadotropin-Releasing Hormone Surges. Endocr. Rev. 31 , 544–577 (2010). Herbison, A. E., Porteous, R., Pape, J. R., Mora, J. M. & Hurst, P. R. 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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-8741954","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":611083746,"identity":"0618a3fe-8ec3-4296-b955-f845cfb8cdc3","order_by":0,"name":"Mélanie Chester","email":"","orcid":"","institution":"Université Paris-Cité, CNRS, Inserm","correspondingAuthor":false,"prefix":"","firstName":"Mélanie","middleName":"","lastName":"Chester","suffix":""},{"id":611083747,"identity":"db321312-73cb-4a67-88f1-a249daefdc73","order_by":1,"name":"Noalig Wyckens","email":"","orcid":"","institution":"Université Paris-Cité, Inserm","correspondingAuthor":false,"prefix":"","firstName":"Noalig","middleName":"","lastName":"Wyckens","suffix":""},{"id":611083748,"identity":"f2ad77e9-5b77-408a-8bdb-27097bb7330a","order_by":2,"name":"Eloïse Airaud","email":"","orcid":"","institution":"Université Paris-Cité, CNRS, Inserm","correspondingAuthor":false,"prefix":"","firstName":"Eloïse","middleName":"","lastName":"Airaud","suffix":""},{"id":611083749,"identity":"6bbb81b6-d381-4158-ba46-762a8a4d1f2a","order_by":3,"name":"Raphaël Corre","email":"","orcid":"","institution":"Université Paris-Cité, CNRS, Inserm","correspondingAuthor":false,"prefix":"","firstName":"Raphaël","middleName":"","lastName":"Corre","suffix":""},{"id":611083750,"identity":"7875d11c-e0ea-4fe8-9bbe-19d2b2442fe6","order_by":4,"name":"Claire-Hélène Petrovic","email":"","orcid":"","institution":"Université Paris-Cité, CNRS, Inserm","correspondingAuthor":false,"prefix":"","firstName":"Claire-Hélène","middleName":"","lastName":"Petrovic","suffix":""},{"id":611083751,"identity":"f640e351-6625-4120-bdd7-3489d47d7a9e","order_by":5,"name":"Alice Pierre","email":"","orcid":"","institution":"Université Paris-Cité, Fonctions Placentaire et Reproductive, Microbiote pré et post- natal (FPRM)","correspondingAuthor":false,"prefix":"","firstName":"Alice","middleName":"","lastName":"Pierre","suffix":""},{"id":611083752,"identity":"d213bea8-c48b-4de9-a276-dec5ccea2620","order_by":6,"name":"Céline Julie Guigon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIie3OsWrCQBzH8Z9kyPJPs14R7CscZJBg0VepCHFsS5cMQs9FFx/AQukzZHKO/CEuEenWsUFwL10cSulJULJccCx43+HuOP4f7gCb7V/mqLTcD0tc3umbVg1pVEl+IkEtqZwnp6OZ+NPxmB9idJOV+759fGO03XWx3EPem4jIl4rnOQYJ01PwsmCEs6FkggyVgUjRV+xNMNBjUdNb8HOSRmDgR5pekTeFJr9H8sqQmx0OHzMT0dBEoSvZzZqe0uQjQko1ROT6Y5SJu2smJ6BsiHC+A5M0E3/K/E2j297VZlVsadRB24+cr31sJsfH+gpUDlXX2nqA+3nusM1ms11Yfwd6VIioC7wTAAAAAElFTkSuQmCC","orcid":"","institution":"Université Paris-Cité, Fonctions Placentaire et Reproductive, Microbiote pré et post- natal (FPRM)","correspondingAuthor":true,"prefix":"","firstName":"Céline","middleName":"Julie","lastName":"Guigon","suffix":""}],"badges":[],"createdAt":"2026-01-30 14:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8741954/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8741954/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105565484,"identity":"4f04b0d8-a0b6-4a57-a788-ebb478f88061","added_by":"auto","created_at":"2026-03-27 12:53:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":963808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eExperimental design used to characterize cues of puberty in C57BL/6J female mice\u003c/em\u003e\u003c/p\u003e\n\u003ch4\u003e\u003cem\u003eA-Schematic representation of the experimental design. Female C57BL/6J mice were monitored daily for vaginal opening and estrous cyclicity. In group 3, repeated blood samples were collected at a fixed nocturnal time point to detect preovulatory gonadotropin surges. Designed using BioRender (\u003c/em\u003e\u003ca href=\"https://app.biorender.com/illustrations/69455b5a781eec89c8f99f82\"\u003e\u003cem\u003ehttps://app.biorender.com/illustrations/69455b5a781eec89c8f99f82\u003c/em\u003e\u003c/a\u003e\u003cem\u003e). Ovaries were collected at the end of the experimental period for histological analysis.\u003c/em\u003e\u003c/h4\u003e\n\u003cp\u003e\u003cem\u003eB-Representative hematoxylin/eosin-stained ovarian sections from peripubertal mice with growing follicles at different stages of maturation and possibly corpora lutea indicating that ovulation had occurred. Sections show growing follicles at different stages of maturation and, in some cases, corpora lutea, indicating that ovulation had occurred. Ovarian histology was used to assign Pubertal Ovarian Maturation Scores (Pub-Scores) based on established morphological criteria, as described in Gaytan et al.[9]. Pub-Scores were classified as negative or positive according to the absence or presence of corpora lutea, respectively, and based on antral follicle size. Shown are ovaries with negative Pub-Scores, lacking corpora lutea and displaying antral follicles of 351-400 µm (Pub-Score: −2) or \u0026gt;400 µm (Pub-Score: −1). Ovaries with positive Pub-Scores exhibit corpora lutea with fully luteinized granulosa cells and antral follicles of 350-400 µm (Pub-Score: +3), or corpora lutea containing apoptotic cells with large antral follicles (\u0026gt;400 µm; Pub-Score: +4). Right panels show higher-magnification views of the boxed regions. Scale bars: 200 µm (left panels) and 50 µm (right panels).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8741954/v1/03bf27349a7ddd660a3f2dea.png"},{"id":105366888,"identity":"09ea5468-f24d-427e-9b55-f6e1782c4712","added_by":"auto","created_at":"2026-03-25 08:46:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTiming of vaginal opening, estrous cycle progression, and first ovulation in C57BL/6J female mice. A- Age at vaginal opening (VO), first estrus (1\u003c/em\u003e\u003csup\u003e\u003cem\u003est\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e E), first diestrus 2 (1\u003c/em\u003e\u003csup\u003e\u003cem\u003est\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e D2) and first ovulation. B- Interval (in days) between vaginal opening and first estrus, and between vaginal opening and first diestrus 2 or first ovulation. First ovulation was estimated by Pub-Score analysis. C-Percentage of mice having already ovulated at first estrus or at first diestrus\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e2. Data illustrate marked inter-individual variability in the timing of pubertal progression and first ovulation. Bar plots represent mean values ± SEM, with individual animals shown as dots. Data were analyzed by one-way ANOVA, with *** P\u0026lt;0.001 and ****P\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8741954/v1/6b1c207da5a77e8e6ec3bd84.png"},{"id":105366893,"identity":"103c8354-2059-4309-9aeb-77726556559d","added_by":"auto","created_at":"2026-03-25 08:46:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57899,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEstrous cycles are highly variable after vaginal opening in C57BL/6J female mice and do not show a predictive pattern of ovulation.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eShown are the vaginal smears patterns of proestrus (P), estrus (E), diestrus 1 (D1 and diestrus 2 (D2) in four female mice from vaginal opening to the first diestrus 2, with time of ovulation predicted by the Pub-Score and shown by an * on the graph.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8741954/v1/d0d8fda02bfa36e041b1a7ef.png"},{"id":105366889,"identity":"be164029-fcce-4512-9ce2-278c78566aa1","added_by":"auto","created_at":"2026-03-25 08:46:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":271127,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSerum LH and FSH profiles in peripubertal mice following vaginal opening.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDaily serum LH and FSH levels were measured from 4 to 15 days after vaginal opening in eight female mice that ovulated during this period. Hormone profiles are shown together with the corresponding estrous stage and the predicted day of first ovulation, as determined using the Pub-Score method \u003c/em\u003e[9]\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8741954/v1/ee3f8b03dd870498035e9f7c.png"},{"id":105366890,"identity":"971b9bde-372a-42af-b8ad-484f1acd00a1","added_by":"auto","created_at":"2026-03-25 08:46:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61255,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cem\u003eIntegrated markers of puberty onset and first ovulation in female C57BL/6J mice\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eSchematic summary illustrating inter-individual variability in puberty onset and the timing of first ovulation in female mice. Each horizontal line represents an individual mouse positioned along the postnatal time axis (days postnatal). Colored symbols indicate the different pubertal events assessed in this study: vaginal opening (VO), first estrus and diestrus stages identified by vaginal cytology, detection of preovulatory luteinizing hormone (LH) surges, and morphological criteria of ovulation determined by Pub-Score analysis (presence of preovulatory follicles and/or corpora lutea). The figure highlights the temporal dissociation between vaginal opening, commonly used as an external marker of puberty, and the occurrence of the first ovulation confirmed by morphological and endocrine criteria. While vaginal opening and initiation of estrous cyclicity display marked inter-individual variability, the timing of first ovulation is confined to a narrower developmental window. LH surge (when detected) occurs transiently and inconsistently relative to vaginal cytology and first ovulation.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8741954/v1/291dced5f82aba244cc7aceb.png"},{"id":105570162,"identity":"3430ae87-14d5-434c-91e0-331ef1d97204","added_by":"auto","created_at":"2026-03-27 13:15:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2144538,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8741954/v1/1585d5d1-23d0-4e94-af2f-1f46f07772c8.pdf"},{"id":105366892,"identity":"f1f5efc5-1956-4790-9319-9040e3d22e28","added_by":"auto","created_at":"2026-03-25 08:46:55","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":88201,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8741954/v1/811b89cb6730eefebf9de005.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Defining Functional Puberty in Female C57BL/6J Mice Using Endocrine, Cytological and Morphological Markers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe concept of \u0026ldquo;puberty\u0026rdquo; has long been defined as the transitional period leading to reproductive competence, as initially described by Ojeda et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This process involves profound neuroendocrine modifications within the hypothalamic circuitry, particularly involving glutamate, GABA, and kisspeptin (Kiss) neurons [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The activation of the hypothalamo-pituitary-gonadal (HPG) axis is a hallmark of puberty onset. Pulsatile secretion of gonadotropin-releasing hormone (GnRH) from hypothalamic neurons stimulates pituitary release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), thereby driving gonadal steroidogenesis and the emergence of secondary sexual characteristics.\u003c/p\u003e \u003cp\u003eIn humans, puberty typically occurs around 11\u0026ndash;12 years of age, while in mice it takes place around 40 days postnatal (dpn) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In females, the first ovulation represents a key event of this developmental stage, occurring under the control of the preovulatory GnRH-LH surge triggered by high estradiol (E2) levels acting through a positive feedback loop [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Defining the age at which the system becomes functional is critical in understanding key mechanisms of reproductive biology, physiology and physiopathology, and in identifying perturbations. In rodents, external indicators such as vaginal opening (VO) and subsequent stages of the estrous cycle (first estrus, E, and first diestrus 2, D2) are commonly used as indicators of puberty onset [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, while vaginal opening and ovulation usually coincide in rats [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], it does not appear to be the case in mice, making the accurate identification of first ovulation challenging. To date, no reliable standardized method exists to precisely define the timing of first ovulation in intact mice. Moreover, vaginal cytology alone often fails to reflect the true onset of ovarian cyclicity.\u003c/p\u003e \u003cp\u003eThe objective of this study was therefore to determine whether the integrative analysis of different parameters could mark puberty onset and first ovulation in female mice. We used complementary approaches combining (1) vaginal opening and cytological evaluation, (2) measurement of circulating LH and FSH levels, and (3) morphological evaluation of the ovulation on ovarian histology, using the Pub-Score method described by Tena-Sempere\u0026rsquo;s group [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Altogether, our findings indicate that puberty onset and first ovulation in female mice cannot be reliably inferred from a single external or hormonal marker, and that commonly used criteria may lead to misinterpretation of reproductive maturity during the peripubertal window. Instead, an integrative assessment combining external, endocrine, histological, and functional reproductive readouts is required to accurately define the acquisition of ovarian cyclicity.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eExperimental design\u003c/h2\u003e\n\u003cp\u003eTo investigate the temporal coordination of puberty onset and first ovulation in female mice of the C57BL/6J strain, which is the most widely used strain in mouse studies, three complementary experimental paradigms were implemented (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). In all groups, vaginal opening (VO) was monitored daily starting at postnatal day (dpn) 26, an age at which C57BL/6J females are still sexually immature. Vaginal cytology was assessed daily, and ovaries were collected at the end of the experiment for histological dating of ovulation using the Pubertal Ovarian Maturation Score (Pub-Score) method [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. This semi-quantitative histological method is based on ovarian morphology, integrating antral follicle development in pre-ovulatory animals and corpora lutea aging in post-ovulatory animals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Negative scores indicate progressive pre-ovulatory follicular maturation and number of days before ovulation, score 0 corresponds to the first ovulation, and positive scores reflect the number of days since first or second ovulation. The age at first ovulation was calculated from the Pub-Score and the age at sample collection.\u003c/p\u003e\n\u003cp\u003eExperiments were terminated at defined developmental milestones to capture key transitions: mice were euthanized either at the first estrus (group 1, 1st E; n\u0026thinsp;=\u0026thinsp;10), at the first diestrus 2 following the first estrus (group 2, 1st D2; n\u0026thinsp;=\u0026thinsp;10), or at variable time points between 9 and 16 days after VO following daily blood sampling to detect preovulatory gonadotropin surges (group 3; n\u0026thinsp;=\u0026thinsp;26) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). This design enabled an integrated analysis of vaginal cytology, endocrine dynamics, and ovarian maturation across the peripubertal period.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMarked inter-individual variability in estrous cycle patterns and timing of first ovulation\u003c/h3\u003e\n\u003cp\u003eAcross the three experimental groups, vaginal opening was first observed between 27 and 38 dpn (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM: 30.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 dpn; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Subsequent estrous stages, identified by daily vaginal cytology, displayed pronounced inter-individual variability. The first estrus occurred between 29 and 48 dpn (36.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 dpn), while the first diestrus 2 following first estrus occurred significantly later and was observed between 34 and 50 dpn (42.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1 dpn; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Consistent with this variability, the interval between vaginal opening and first estrus ranged from 0 to 17 days (6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 days), and the interval to first diestrus II ranged from 5 to 19 days (11.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 days; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). These results indicate that although vaginal opening represents an early marker of pubertal activation, it precedes the establishment of a complete estrous cycle by approximately two weeks, with substantial variability among individuals. Ovarian histology revealed that only 1 out of 10 mice studied at first estrus (10%) displayed corpora lutea, indicating that ovulation had occurred in a very small fraction of mice at this stage (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). In contrast, 8 out of 10 mice (80%) euthanized at first diestrus 2 had ovulated, suggesting that most females achieved functional puberty during this interval. When we examined the estrus pattern of all mice having reached diestrus 2, we found that the mean delay between first estrus and first diestrus 2 was 4.8 days\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8, ranging from 1 day up to 10 days.\u003c/p\u003e\n\u003cp\u003eTo further characterize ovarian maturation, ovaries from all mice (n\u0026thinsp;=\u0026thinsp;46) were analyzed using the Pub-Score method, which integrates corpus luteum morphology, and follicular size. This analysis classified ovaries as Pub-Score positive (presence of corpora lutea) or negative (absence of corpora lutea). Overall, 22 out of 46 of examined mice (47.8%) exhibited histological evidence of ovulation. Based on Pub-Score estimates, first ovulation occurred between 36 and 51 dpn (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM: 44.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 dpn). In mice that had not yet ovulated at the time of sacrifice, retrospective estimation predicted first ovulation between 36 and 54 dpn (44.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 dpn).\u003c/p\u003e\n\u003cp\u003eWhen all animals were considered together, first ovulation was confined to a developmental window spanning 36\u0026ndash;54 dpn, corresponding to 5\u0026ndash;21 days after vaginal opening (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM: 44.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 dpn; 10.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75 days post-VO; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). These results place the functional onset of puberty in C57BL/6J females at approximately 44 dpn, with marked inter-individual variability.\u003c/p\u003e\n\u003ch3\u003eIntegration of endocrine, cytological, and morphological parameters\u003c/h3\u003e\n\u003cp\u003eTo assess endocrine dynamics following vaginal opening, daily blood samples were collected in mice of group 3 at time points corresponding to the expected preovulatory LH surge, approximately one hour after lights off. Gonadotropin concentrations were measured using Luminex assays. Sampling began four days after VO and continued for 12 consecutive days.\u003c/p\u003e\n\u003cp\u003eBasal LH concentrations remained below 1 ng/mL and fluctuated over time, consistent with the pulsatile release mode of this hormone (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Among the eight mice that ovulated during the sampling period, a distinct LH surge was detected in five individuals. One mouse (female 93) even exhibited two LH surges (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). LH peaks occurred as early as four days after vaginal opening in two mice (females 93 and 104) and between 12 and 15 days post-VO in the remaining animals (females 91, 93, 94, and 105). FSH profiles showed substantial variability, with no consistent increase coinciding with LH surges. This may reflect the biphasic nature of FSH secretion around ovulation, with surges occurring outside the nocturnal sampling window. When gonadotropin profiles were combined with Pub-Score analysis for each individual, no clear temporal correspondence between LH surge detection and predicted ovulation timing was observed, except in one mouse (female 105; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Overall, first ovulation occurred within a 3 days range following the LH peak between 12- and 15-days post-VO, for the 5 females with a distinct LH surge, with the date of the first ovulation predicted 14 to 16 days post-VO for all the eight females. Integration with vaginal cytology indicated that LH surges predominantly occurred during estrus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Finally, to evaluate the potential impact of repeated handling and blood sampling on pubertal timing, the estimated age of first ovulation was compared between mice subjected to blood sampling and those that were not. There was no significant effect of blood sampling when we considered age at first ovulation, but a small delay when considering the number of days after vaginal opening (Supplementary Fig.\u0026nbsp;1), suggesting that blood sampling could also interfere with puberty onset.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccurately defining the timing of puberty is key to understand the mechanisms regulating reproductive maturation and their susceptibility to genetic, metabolic, or environmental perturbations. In this study, we sought to identify parameters defining functional puberty in C57BL/6J female mice by integrating external, cytological, endocrine, and morphological readouts. By combining vaginal opening and daily vaginal cytology with gonadotropin measurements and histological ovarian assessment using the Pub-Score method, we provide a comprehensive evaluation of commonly used pubertal markers and their relationship to first ovulation.\u003c/p\u003e\n\u003cp\u003eOur data demonstrate pronounced inter-individual variability across all pubertal landmarks examined, including the timing of vaginal opening, first estrus, and first diestrus 2. Histological analysis indicates that first ovulation occurs on average around 44 dpn, within a surprisingly broad developmental window spanning approximately 36 to 54 dpn. In parallel, detection of preovulatory LH surges was achieved in a subset of animals and showed limited temporal correspondence with Pub-Score-based estimates of ovulation. Together, these findings indicate that puberty in female mice is not a discrete event but rather a prolonged and asynchronous developmental process during which external, cytological, endocrine, and ovarian markers are uncoupled (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe marked variability observed between vaginal opening, estrous cyclicity, and first ovulation highlights the limitations of relying on external or cytological markers to define functional puberty. Unlike in rats, in which vaginal opening and ovulation occur in close temporal proximity [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e], our data confirm that in mice vaginal opening precedes ovulation by approximately 10\u0026ndash;15 days on average, with substantial inter-individual variation. Although the majority of mice had ovulated by the time of the first diestrus 2, only a minority had ovulated at first estrus, indicating that estrous cytology alone does not reliably reflect ovarian competence during the peripubertal period. Consistent with this, Pub-Score analysis revealed no strict association between estrous stage and ovulation, with first ovulation most frequently occurring at estrus but also detected at proestrus or diestrus I. This dissociation likely reflects the immaturity and instability of hypothalamo-pituitary-ovarian axis coordination during puberty, when estrus-like cytological patterns can emerge before the establishment of fully functional ovulatory cycles.\u003c/p\u003e\n\u003cp\u003eAmong the approaches tested, the Pub-Score method emerged as the most reliable indicator of first ovulation. By integrating corpus luteum morphology with follicular development, this method allows retrospective estimation of ovulation timing independently of transient endocrine or cytological fluctuations. The mean age of first ovulation identified in our study is consistent with previous reports in C57BL/6J mice and supports the notion that functional ovarian activation is substantially delayed relative to vaginal opening [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. Notably, nearly half of the mice analyzed had not ovulated at the time of ovary collection despite exhibiting vaginal opening and, in some cases, repeated estrus-like cytology, underscoring the risk of misclassifying pubertal status when relying solely on these criteria. It would be of interest to determine whether the altered timing of puberty onset reported in numerous mouse models [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e] is systematically associated with alterations in first ovulation, and thus with the functional onset of puberty.\u003c/p\u003e\n\u003cp\u003eThe study of LH dosage on the evening of ovulatory discharge has already been carried out in adult females with tail sampling and appears to be a good method of analysis [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. The limited detection of LH surges in our study emphasizes the technical and biological challenges of capturing transient endocrine events during pubertal development. The brief duration of the LH surge [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e], once-daily sampling, and the potential inhibitory effects of repeated handling with stress induction [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e] likely contributed to the modest detection rate. Moreover, the imperfect alignment between LH surges and Pub-Score-predicted ovulation suggests that endocrine dynamics during puberty may not fully conform to the canonical adult estrous cycle on which Pub-Score staging is based. Nevertheless, the observation that LH surges were detected exclusively in animals with histological evidence of ovulation supports the conclusion that Pub-Score positivity reflects physiologically meaningful activation of the hypothalamic-pituitary-ovarian axis.\u003c/p\u003e\n\u003cp\u003eOur findings are consistent with previous studies demonstrating that vaginal opening and first estrus precede functional ovarian activation in mice by a variable interval. The Pub-Score method, originally described by Gayt\u0026aacute;n et al.[\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e], established that neither vaginal opening nor estrous cytology can reliably define the timing of first ovulation. By integrating cytological, endocrine, and morphological readouts within the same experimental framework, our study extends these observations and highlights the magnitude of inter-individual variability even within a single inbred strain.\u003c/p\u003e\n\u003cp\u003eSeveral limitations of our study should be acknowledged. Detection of LH surges was constrained by sampling frequency and potential stress-related suppression of gonadotropin secretion. Pub-Score analysis, while robust, is inherently cross-sectional and retrospective, preventing precise longitudinal tracking of ovulation in individual animals. In addition, this study was limited to the C57BL/6J strain, and strain-specific differences in pubertal timing and ovarian physiology may limit generalization.\u003c/p\u003e\n\u003cp\u003eTaken together, our results underscore the need for integrative approaches when defining puberty in female mice. Increased sampling frequency, development of minimally invasive longitudinal methods, and incorporation of additional biomarkers of ovarian maturation may further refine the temporal resolution of pubertal staging. From a methodological standpoint, the use of Pub-Score analysis as a reference endpoint is strongly recommended when accurate identification of first ovulation is required. Importantly, experimental designs should explicitly recognize that vaginal opening and estrous cytology do not reliably predict ovulation and should avoid equating these markers with reproductive maturity.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study highlights the complexity and variability inherent in pubertal maturation in C57BL/6J female mice. External and cytological markers alone are insufficient to precisely determine the timing of first ovulation. Among the approaches tested, histological assessment using the Pub-Score method provides the most reliable estimate of functional puberty. These findings have important implications for experimental designs in reproductive and developmental biology and emphasize the necessity of rigorous, multi-parameter strategies when studying the onset of reproductive competence in mice.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eAnimals and treatments\u003c/h2\u003e\u003cp\u003eAll experiments were conducted on C57BL/6J female mice bred in our animal facility from parents obtained from Janvier Labs (Le Genest St Isle, France). The day of birth was designated as postnatal day 0 (dpn 0), and weaning occurred at 21 dpn. Animal care and handling followed established procedures [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Mice were housed in groups of 3–5 per cage under a 12 h light/dark cycle, at 22 ± 1°C, with \u003cem\u003ead libitum\u003c/em\u003e access to standard chow and water. From 10 dpn, female mice were manipulated daily to limit the stress related to vaginal smears and tail-blood collection. Animals were randomly assigned to three experimental groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). All sample collection and histological analyses were performed by investigators blinded to the experimental group. After VO, blood samples (20 µL) were collected daily for 12 consecutive days in one group of mice (n = 17), about one hour after lights off (8:30 PM), \u003cem\u003evia\u003c/em\u003e tail sampling using capillaries. Prior to terminal cardiac puncture, mice were weighted and then anesthetized with ketamine (Imalgene® 1000; 100 mg/kg BW) and xylazine (Rompun® 2%; 10 mg/kg BW) injected intra-peritoneally with a 1mL sterile syringe mounted with a 26-gauge needle (Terumo). Euthanasia was performed by cervical dislocation. At the time of death, body weights were as followed (mean ± SEM): group 1, 16.2 ± 0.3 g; group 2, 17.1 ± 0.2 g; group 3, 16.6 ± 0.16 g. Serum was obtained after clotting for 15 minutes at room temperature and centrifugation at 5000 g for 5 minutes. Ovaries were collected post-mortem and fixed in Bouin’s solution (HT10132, Sigma-Aldrich) for histological processing. All procedures were approved by the Institutional Animal Care and Use Committee of Université Paris-Cité and the French Ministry of Agriculture (CEB#13-2021) and were performed in accordance with relevant guidelines and regulations.\u003c/p\u003e\u003ch3\u003eDetermination of serum LH and FSH levels\u003c/h3\u003e\u003cp\u003eSerum LH and FSH concentrations were determined using the Milliplex MAP rat pituitary magnetic bead panel on 10 µL of sample (Merck-Millipore, Nottingham, UK), following the manufacturer’s instructions [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. The assay sensitivities were 32 pg/mL for FSH and 3.2 pg/mL for LH. Inter-assay coefficients of variation were 5.4% (FSH) and 3.2% (LH), and intra-assay coefficients were 7.6% (FSH) and 5.9% (LH).\u003c/p\u003e\u003ch2\u003eDetermination of estrous cyclicity\u003c/h2\u003e\u003cp\u003eVaginal smears were collected daily following VO to monitor the onset of the estrous cycle, up to first estrus, first diestrus 2 or after 9–16 days depending on the group of mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Smears were obtained using 0.9% NaCl solution, stained with hematoxylin and eosin (HE), and analyzed under light microscopy. The stages of the cycle were identified based on the predominant cell types: nucleated epithelial cells (proestrus, PE), cornified cells (estrus, E), and leukocytes (diestrus 2, D2) as previously described [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eHistological processing and Pub-Score analysis\u003c/h2\u003e\u003cp\u003eOvaries (n = 46) were fixed in Bouin’s solution for 48 hours, rinsed in PBS, and stored in 70% ethanol. Samples were dehydrated, embedded in paraffin (HISTIM core facility, Cochin Institute, Paris), and sectioned at 5 µm. Every fifth section was mounted and stained with HE. Slides were scanned using a Pannoramic 250 Flash III system (3D HISTECH, Bicêtre Biomedical Institute). The Pub-Score method [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e] was used to determine ovulation timing based on CL morphology and largest follicular size. Ovaries were classified as pre-ovulatory (no CL; largest follicle used to estimate − 1 day), ovulatory (presence of newly formed CL), or post-ovulatory (regressing CL and secondary follicular growth, + 1 day).\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eSample sizes are provided in the text. Data were analyzed using GraphPad Prism 10. Data are shown as mean ± SEM or %, and statistical analyses were performed using one-way ANOVA or Student t-test, when appropriate.\u003c/p\u003e\u003ch2\u003eGraphical design\u003c/h2\u003e\u003cp\u003eAll schematic figures were created using BioRender (BioRender.com).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ethe authors declare no competing financial interests.\u003c/p\u003e\n\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eThis work was supported by Agence Nationale de la Recherche (ANR ReproFun, attributed to CJG) and Ecole Doctorale BioSPC (NW, MC).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eMC, CHP conducted vaginal smears and histological examination of the ovaries, determined the Pub-Score. CG, MC, CHP, RC and EA performed tail-blood collection. RC and MC performed histological sections. CG, MC and NW analyzed the data and prepared the figures. CG wrote the initial manuscript. CG, MC, NW and AP evaluated and discussed all the data. All authors read the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors wish to thank the staff members of the core animal facility Buffon of Universit\u0026eacute; Paris Cit\u0026eacute; (Paris, France). They acknowledge the technical assistance of M. Surenaud for gonadotropin assays (H\u0026ocirc;pital Henri Mondor, Cr\u0026eacute;teil, France) and O. Trassard for slide scanning (Cellular and tissue imaging core facility, Bic\u0026ecirc;tre Biomedical Institute). This work was supported by ANR ReproFun (CG) and ED BioSPC (MC, NW).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article, and any other data will be made available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOjeda, S. R., Andrews, W. W., Advis, J. P. \u0026amp; White, S. S. Recent advances in the endocrinology of puberty. \u003cem\u003eEndocr. Rev.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, 228\u0026ndash;257 (1980).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrevot, V. 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A novel action of follicle-stimulating hormone in the ovary promotes estradiol production without inducing excessive follicular growth before puberty. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 46222 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eByers, S. L., Wiles, M. V., Dunn, S. L. \u0026amp; Taft, R. A. Mouse estrous cycle identification tool and images. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e, e35538 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePantier, L. K., Li, J. \u0026amp; Christian, C. A. Estrous Cycle Monitoring in Mice with Rapid Data Visualization and Analysis. \u003cem\u003eBio Protoc.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, e3354 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Puberty, ovulation, estrous cycle, LH surge, Pub-Score","lastPublishedDoi":"10.21203/rs.3.rs-8741954/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8741954/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePuberty marks the transition to reproductive competence and is driven by activation of the hypothalamo/pituitary/gonadal axis, culminating in first ovulation in females. In mice, puberty onset is commonly inferred from vaginal opening and estrous cyclicity, but the precise timing of first ovulation remains unclear. Here, we aimed to define reliable parameters of functional puberty in female C57BL/6J mice by integrating external, endocrine, and morphological measures. Vaginal opening and daily vaginal cytology were combined with daily serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) measurements and ovarian histology using the Pubertal Ovarian Maturation Score (Pub-Score), which estimates ovulation based on follicular development and corpus luteum morphology. Notably, we observed substantial inter-individual variability in puberty-related events, and no link between estrous cytology and first ovulation. Pub-Score analysis indicated that first ovulation occurred between 36 and 54 days postnatal, with a mean around 44 days, with no correlation with vaginal cytology. LH surges were detected in some of the females that had ovulated and showed limited temporal correspondence with vaginal cytology or Pub-Score estimates. Together, these findings demonstrate that puberty in female mice is a gradual, asynchronous process and that external or cytological markers alone are insufficient to define functional reproductive maturity.\u003c/p\u003e","manuscriptTitle":"Defining Functional Puberty in Female C57BL/6J Mice Using Endocrine, Cytological and Morphological Markers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 08:46:50","doi":"10.21203/rs.3.rs-8741954/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-23T11:14:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T20:34:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-12T11:36:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-03T08:14:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1463886894072277625366157627396291417","date":"2026-03-23T22:25:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3496583311182360103571478376215135676","date":"2026-03-23T12:15:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194866836390256852482252796076683216839","date":"2026-03-23T11:22:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176810295726136086898777743960479212555","date":"2026-03-23T05:05:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-23T04:37:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-18T04:50:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-16T09:16:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-12T10:34:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-12T10:21:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cdf08dea-a61d-4e04-886c-457712e0da5d","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65017692,"name":"Health sciences/Endocrinology"},{"id":65017693,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-05-08T10:23:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 08:46:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8741954","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8741954","identity":"rs-8741954","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}