Multigenerational Effects of an Environmentally Relevant Phthalate Mixture on Reproductive Parameters and Ovarian miRNA Expression in Female Rats

Phthalates are endocrine-disrupting chemicals used in many consumer products. Our laboratory previously developed an environmentally relevant phthalate mixture consisting of 6 phthalates and found that it disrupted female fertility in mice. However, it was unknown if maternal exposure to the mixture affects reproductive parameters and ovarian post-transcription in the F1 and F2 generation of female rats. Thus, we tested the hypothesis that maternal exposure to the phthalate mixture affects folliculogenesis, steroidogenesis, and ovarian microRNA (miRNA) in the F1 and F2 generations of female rats. Pregnant female rats were divided into 4 groups and orally dosed daily from gestational day 10 to postnatal day 21 with corn oil (control group), 20 μg/kg/day, 200 μg/kg/day, or 200 mg/kg/day of the phthalate mixture. Maternal exposure to the phthalate mixture impaired folliculogenesis in the F1 and F2 generations of female rats and affected steroidogenesis in the F1 generation of female rats compared to control. Further, the phthalate mixture altered ovarian expression of some genes related to the cell cycle and steroidogenesis compared to control in the F1 and F2 generations of female rats. The mixture also increased ovarian expression of rno-mir-184 that is involved with the oocyte maturation process. Collectively, our data show that maternal exposure to the phthalate mixture affects folliculogenesis and steroidogenesis in the F1 and F2 generations of female rats and alters ovarian miRNA expression in the F1 generation of female rats.

phthalates, ovary, steroidogenesis, endocrine-disrupting chemicals, miRNA Phthalates are chemical plasticizers that modify thermal and mechanical properties of a given polymer. These chemicals are added to polymer solutions to promote their plasticity and flexibility (Godwin, 2011). In addition, some phthalates are used to help dissolve other materials (CDC, 2021; Godwin, 2011). Phthalates are present in hundreds of products such as vinyl flooring, lubricating oils, and personal-care products (soaps, shampoos, nail polish, hair sprays). Some phthalates are in polyvinyl chloride plastics, which are used to make products such as plastic packaging, garden hoses, and medical tubing (CDC, 2021). Humans are often exposed to phthalates mainly via ingestion, but exposure also occurs by inhalation and dermal absorption. Previous studies have shown that exposure to a mixture of phthalates can cause adverse reproductive outcomes in animals. For example, exposure to a mixture of phthalates increased uterine weights, decreased anogenital distance, altered estrous cyclicity, and disrupted fertility in mice ranging from postnatal day 0 to 9 months of age in a multiple and transgenerational manner (Zhou et al., 2017a,b). Further, the same environmentally relevant mixture of phthalates accelerated biomarkers of reproductive aging in multiple generations of aging female mice (Brehm et al., 2020). Specifically, exposure to the mixture dysregulated hormone levels and increased cystic ovaries in the F1 generation, decreased the percentage of antral follicles and testosterone levels in the F2 generation, and it caused irregular cyclicity, decreased the percentage of ovarian follicles, and increased the occurrence of cysts in the F3 generation of mice (Brehm et al., 2020). In addition, studies measured several phthalate metabolites in the urine of 2636 or more people aged 6 years and older who took part in the National Health and Nutrition Examination Survey during 2003–2004 (Silva et al., 2003, 2004). These findings indicate that phthalate exposure is widespread in the U.S. population. Further, adult women have higher levels of metabolites measured in urine than do men for those phthalates that are used in soaps, body washes, shampoos, cosmetics, and similar personal care products (CDC, 2021). This is of concern because exposure to phthalates has been associated with poor reproductive outcomes in women (Cao et al., 2020; Cobellis et al., 2003; Fu et al., 2017; Lee et al., 2020; Messerlian et al., 2016; Özel et al., 2019; Pednekar et al., 2018; Reddy et al., 2006; Specht et al., 2015; Thomsen et al., 2017; Tranfo et al., 2012). Specifically, exposure to phthalates has been associated with increased risk of fibroids, leiomyoma, and endometriosis (Cobellis et al., 2003; Fu et al., 2017; Lee et al., 2020; Reddy et al., 2006). In addition, exposure to phthalates has been associated with longer time to pregnancy, earlier menopause, premature ovarian failure, and low antral follicle count (Cao et al., 2020; Messerlian et al., 2016; Özel et al., 2019; Pednekar et al., 2018; Specht et al., 2015; Thomsen et al., 2017; Tranfo et al., 2012). Further, studies have shown that exposure to phthalates can alter microRNA (miRNA) expression (Liu et al., 2018; O’Brien et al., 2018; Scarano et al., 2019; ). miRNAs are a class of noncoding small RNAs around 20–24 nucleotides in length, which are involved in the regulation of genes at both transcriptional and post-transcriptional levels (O’Brien et al., 2018). Altering miRNA expression may lead to widespread gene expression changes (O’Brien et al., 2018). For instance, phthalate exposure altered ovarian expression of miRNAs that inhibited granulosa cell proliferation and altered expression of apoptotic factors, promoting apoptosis of granulosa cells in mice (Liu et al., 2018). Another study associated higher phthalate levels in follicular fluid with extracellular vesicles of miRNAs that alter pathways linked to oocyte development, maturation, and fertilization (Barnett-Itzhaki et al., 2021). Alterations in miRNA expression could alter the regulation of genes responsible for steroidogenesis and folliculogenesis, which could eventually lead to impaired fertility. Exposure to environmental toxicants during development may result in a wide range of adverse health outcomes across the life course, and potentially be transmitted to the next generations (Woodruff et al., 2011). Thus, the objective of this study was to test the hypothesis that maternal exposure during gestation and lactation to an environmentally relevant mixture of phthalates can alter miRNA expression in the F1 and F2 generations of female rats. This study also tested the hypothesis that maternal exposure to the mixture of phthalates affects steroidogenesis and folliculogenesis in F1 and F2 generations of female rats. The novelty of this study is the evaluation of the toxic effects of the phthalate mixture during gestation and lactation periods using female rats as a model. In addition, to our knowledge, previous studies have not evaluated the effects of the phthalate mixture on ovarian miRNA expression in rats.

Chemicals Diethyl phthalate (DEP), bis(2-ethylhexyl) phthalate (DEHP), di-n-butylphthalate (DBP), diisononylphthalate (DiNP), diisobutyl phthalate (DiBP), and butylbenzylphthalate (BBzP) were purchased from Sigma-Aldrich (St Louis, Missouri). The phthalate mixture contained DEP, DEHP, DBP, DiNP, DiBP, and BzBP. The chemicals were thoroughly mixed and diluted in corn oil (vehicle control). The doses used for this study were 20 µg/kg/day, 200 µg/kg/day, and 200 mg/kg/day. This mixture has been shown to affect female reproduction, the mammary gland, and prostate in rodents (Brehm and Flaws, 2021; de Freitas et al., 2021; Scarano et al., 2019). Animals Male and female Sprague Dawley rats were supplied by the Multidisciplinary Center for Biological Research (CEMIB/UNICAMP-Brazil) and they were kept during the experimental period in the Bioterium of Small Mammals in the Department of Structural and Functional Biology, Institute of Biosciences (UNESP-Brazil). The animals were bred under controlled temperature (23°C ± 2°C) and regulated humidity conditions, with periods of 12 h light/dark cycles and food and water ad libitum. This study was carried out in accordance with the Brazilian Council on Animal Experimentation Control and the protocol was approved by the Ethical Committee on Animal Use of the Institute of Biosciences, São Paulo State University (UNESP) (Protocol: 1040/CEUA). Experimental Design Pregnant Sprague Dawley rats (n = 8/group) were exposed to the phthalate mixture by daily oral gavage. Rats were euthanized by anesthetic saturation (sodium pentobarbital) followed by cardiac puncture. Animals were divided in 4 different groups: control group (exposed to vehicle; corn oil); 20 µg of the phthalate mixture (20 µg/kg/day); 200 µg of the phthalate mixture (200 µg/kg/day); and 200 mg of the phthalate mixture (200 mg/kg/day). The phthalate mixture was diluted in tocopherol-stripped corn oil (vehicle control). The 2 lower doses were selected to mimic daily human exposure levels based on the amount of DEHP, and the highest dose was selected to compare our results with available single phthalate rodent studies (Zhou et al., 2017a). Animals from control, 20 µg/kg/day, 200 µg/kg/day, and 200 mg/kg/day of the mixture received the respective doses of the phthalate mixture in the following proportion: 21% DEHP, 35% DEP, 15% DBP, 8% DiBP, 5% BBzP, and 15% DiNP. This proportion was based on a previous study considering the proportion of phthalate metabolites detected in urine samples from pregnant women (Yazdy et al., 2018). Animals received treatment from gestational day (GD) 10 to postnatal day (PND) 21 by gavage. This is a critical period for the development of the reproductive system, especially the ovary (Scarano et al., 2019; Smith et al., 2014). Pregnant females treated with vehicle or the mixture were designated as the F0 generation (n = 8 each/group). Offspring from F0 generation were named as F1 generation. After birth, the number of F1 pups per litter was reduced to 8 (1:1 ratio between males and females whenever possible), and litters with fewer than 6 pups were euthanized. Supplementary Figures 1 and 2 indicate the specific litter sizes as well as males and females per litter prior to culling litters to 8 per treatment group. To guarantee the independence of treatments and litters, the experimental unit was the litter and not the individual animal (Brandt et al., 2014; Scarano et al., 2019). Adult females from F1 generation (n = 4–5) were used in each group to produce F2 generation. Therefore, in this study to obtain the F2 generation, F1 females were mated with nontreated adult males. The ovaries were collected and weighed at 3 different time points: F1: PND 22 (n = 8/group), PND 120 (n = 4–5); F2: PND 22 (n = 4–5/group) and designated for molecular analysis and histological evaluation. Tissues were collected from rats in diestrus. Histological Evaluation of the Ovaries Ovaries were collected, weighed, and subjected to histological evaluation (n = 4–5 ovaries per treatment group). Ovaries were fixed in Dietrich’s fixative, embedded in paraffin, and sectioned at 8 µm using a microtome. After sectioning, slides were stained with Weigert’s hematoxylin and picric acid-methyl blue. Every 10th section of the ovary was used to count the number of primordial, primary, preantral, antral, atretic, and abnormal follicles. Primordial follicles were defined as follicles with an oocyte, surrounded by a single layer of squamous granulosa cells. Primary follicles were defined as follicles that consisted of an oocyte, surrounded by a single layer of cuboidal granulosa cells. Preantral follicles were defined as follicles containing an oocyte, surrounded by multiple layers of cuboidal granulosa cells, and theca cells. Antral follicles were defined as follicles that consisted of an oocyte, surrounded by numerous layers of cuboidal granulosa cells, theca cells, and a fluid filled antrum (Sonigo et al., 2018). Atretic follicles were counted as preantral or antral follicles that contained 10% or greater number of apoptotic bodies. Abnormal follicles included follicles with double oocytes and/or fragmented nuclei. Preantral and antral follicles were required to have nuclear material present in each oocyte to avoid double counting. All sections were examined without knowledge of treatment group. Gene Expression Analysis Ovaries from females at PND 22 and PND 120 in the F1 generation and at PND 22 in the F2 generation from all treatment groups were snap frozen in liquid nitrogen, and then stored at −80°C until RNA extraction. Total RNA was isolated from the ovaries (n = 4–5 ovaries per treatment group) using the organic isolation of the RNA with TRI Reagent (Sigma-Aldrich, USA), following the instructions provided by the manufacturers using TRIZOL reagent (Ambion, USA). Total RNA concentration was determined using NanoVue (GE Healthcare, USA) and RNA purity was evaluated according to the A260nm/A280nm and A260nm/A230nm ratios (acceptable when both ratios were greater than 1.8). The quality of the RNA was obtained by the RNA integrity number (RIN) from the analysis of microfluid-based ribosomal RNAs using the 2100 system Bioanalyzer (Agilent, USA) (RIN > 7.0). MicroRNA expression miRNA cDNA was synthesized from the total RNA of 1 μg using the primers of specific miRNA acceleration and the TaqMan MicroRNA reverse transcription kit (Thermo Fisher Scientific, USA). Total RNA was reverse transcribed into cDNA using the Master RNA Mixture for high-capacity cDNA (Thermo Fisher Scientific). Analysis miRNA was performed with 10 μl reaction in duplicate (TaqMan Gene Expression Master Mix for miRNAs; Thermo Fisher Scientific) as described by the manufacturer, and performed using the QuantStudio 12K Flex System (Thermo Fisher Scientific) with the following cycle conditions: 95°C for 10 min followed by 40°C–95°C cycles for 15 s and 60°C for 1 min. Oligonucleotide TaqMan assays for miRNAs (Thermo Fisher Scientific) used were: rno-mir-184 (Assay ID 000485), rno-mir-141-3p (Assay ID 000463), and rno-mir-U6 (Assay ID 001973) in a 96-well plate according to the manufacturer’s specifications. The MammU6 was used as reference gene to normalize the data of miRNA expression. These reference genes were selected based on geNorm calculations (Vandesompele et al., 2002), and the levels of relative expression in these reference genes were similar among the control treated rno-mir-184 and rno-mir-141-3p as indicated by the Ct value reported after the analysis of the raw RT-qPCR data. The relative quantification was evaluated using real-time quantitative PCR and the 2(-Delta C(T)) method (Livak and Schmittgen, 2001; Sticht et al., 2018). Significant changes were set at p value ≤ .05. We selected mir-141 for analysis because mir-141 regulates genes involved in ovarian tumorigenesis (Mateescu et al., 2011). Further, we selected mir-184 for analysis because mir-184 can alter the levels of hormones such as progesterone, testosterone, and estradiol in human ovarian cells (Sirotkin et al., 2009). In addition, mir-184 has been shown to regulate biological events during oogenesis and early embryogenesis in Drosophila (Iovino et al., 2009). mRNA expression mRNA cDNA was synthesized from total RNA of 10 ng using High-Capacity RNA-to-cDNA master mix (Thermo Fisher Scientific, Waltham, Massachusetts). Analysis of RT-qPCR was conducted using the C and FX96 Real-Time PCR Detection System (Bio-Rad Laboratories) and accompanying CFX Manager Software according to the manufacturer’s protocol. The machine quantifies the amount of PCR product generated by measuring SsoFastEvaGreen dye (Bio-Rad Laboratories) that fluoresces when bound to double-stranded DNA. All qPCR reactions were conducted in duplicate with 2 μl (from 1.67 ng RNA), forward and reverse primers (7.5 pmol each), and SsoFastEvaGreen Supermix. Primers used included glyceraldehyde-3phosphate dehydrogenase (Gapdh), B-cell lymphoma 2 (Bcl2), Bcl2-like protein 10 (Bcl2l10), Bcl2-associated X protein (Bax), apoptosis-inducing factor 1 (Aimf1), cyclin A2 (Ccna2), cyclin B1 (Ccnb1), cyclin D2 (Ccnd2), cyclin-dependent kinase 4 (Cdk4), cyclin-dependent kinase inhibitor 1A (Cdkn1a), steroidogenic acute regulatory protein (Star), cytochrome P450 cholesterol side-chain cleavage (Cyp11a1), cytochrome P450 steroid 17-α-hydroxylase 1 (Cyp17a1), 3β-hydroxysteroid dehydrogenase 1 (Hsd3b1), and cytochrome P450 aromatase (Cyp19a1). The qPCR program consisted of an enzyme activation step (94°C for 5 min), an amplification and quantification program (36 cycles of 95°C for 10 s, 60°C for 10 s, 72°C for 10 s, and a single fluorescence reading), a melt curve (65°C–95°C heating, 0.4°C/s with continuous fluorescence readings), and a final step at 72°C for 4 min per the manufacturer’s protocol. Gapdh mRNA expression was used in qPCR analyses as a housekeeping gene. All gene expression data were normalized to the housekeeping gene Gapdh. Relative fold changes were calculated as ratios to the control group levels and were analyzed using a mathematical model for relative quantification of qPCR data (Pfaffl, 2001). Analysis of Sex Steroid Hormone Levels Blood was obtained from rats in diestrus during the collection of tissues. Plasma were subjected to enzyme-linked immunosorbent assays (ELISAs, DRG International Inc., Springfield, New Jersey) for measurement of progesterone (analytical sensitivity was 0.034 ng/ml and both intra- and interassay coefficients of variation were below 8%), testosterone (analytical sensitivity was 0.083 ng/ml and both intra- and interassay coefficients of variation were below 10%), and estradiol (analytical sensitivity was 10.6 pg/ml and both intra- and interassay coefficients of variation were below 15%). Assays were run according to the manufacturer’s instructions. Lypocheck from Bio-Rad Laboratories was used as a control with known values in all ELISAs in this study. Prediction and Functional Annotation Analysis MiRWalk (http://mirwalk.umm.uni-heidelberg.de/) was used to determine the predicted targets of the miRNAs rno-mir-184 (Rattus norvegicus). We selected the targets according to energy (more than -20) and all selected targets were in the predicted target module (Sticht et al., 2018) (Supplementary Table 1). Enrichment analysis for target mRNAs of the microRNAs were performed in KOBAs 3.0 tool (http://bioinfo.org/kobas), which gather information from different data banks. We selected data from KEGG (https://www.genome.jp/kegg) and Gene Ontology (http://geneontology.org/) databases. Pathways and ontological terms were selected by higher interest and adopting p value and adjusted p value < .05 as cut-off criterion for significance. We used package “circlize” in environment R to create the circus plot to show targets of rno-mir-184 and mRNA and pathway associations, and the SnakeyMatic (http://sankeymatic.com/) to create Alluvial diagram to represent the ontological enrichment for each mRNA. Statistical Analysis Data from follicle counting, ovarian histology, ovarian miRNA expression, and ovarian mRNA gene expression were analyzed by comparing treatment groups to control using IBM SPSS version 27 software (SPSS Inc., Chicago, Illinois). Outliers were removed by the Grubb’s test using GraphPad outlier calculator software (GraphPad Software Inc., La Jolla, California). Continuous data were assessed for normal distribution by Shapiro-Wilk analysis. If data met assumptions of normal distribution and homogeneity of variance, data were analyzed by 1-way analysis of variance (ANOVA) followed by Tukey HSD or Dunnett 2-sided post hoc comparisons. However, if data met assumptions of normal distributions, but not homogeneity of variance, data were analyzed by ANOVA followed by Games-Howell or Dunnett’s T3 post hoc comparisons. If data were presented as percentages or were not normally distributed, the independent sample Kruskal-Wallis H followed by Mann-Whitney U nonparametric tests were performed. For all comparisons, statistical significance was determined by p value ≤ .05. When p values were > .05, but < .10, data were considered to exhibit borderline significance, but were not considered statistically different. The enrichment pathways analysis was performed in KOBAS 3.0 database, using the statistical method hypergeometric test/Fisher’s exact test (p value) and FDR correction method Benjamini and Hochberg (adjusted p value).

Effects of Phthalate Mixture Exposure on Animal Body and Ovarian Weight Gestational and lactational exposure to the environmentally relevant phthalate mixture did not significantly affect body weight of F1 PND 22, F1 PND 120, or F2 PND 22 animals in any treatment group compared to control (Table 1, n = 4–8 per treatment group). However, the mixture caused a borderline increase (not statistically different) (20 μg/kg/day) and significantly increased (200 μg/kg/day) ovarian weight at PND 22 in the F1 generation compared to control (Table 1, n = 4–8 per treatment group, p = .08, p < .05). In addition, the phthalate mixture (200 mg/kg/day) caused a borderline increase (not statistically different) in ovarian weight at PND 22 in the F2 generation compared to control (Table 1, n = 4–8 per treatment group, p = .07). Table 1. | F1 PND 22 | F1 PND 120 | F2 PND 22 | ||||| |---|---|---|---|---|---|---|---| | Phthalate Mixture Groups | Sample Size | Body | Ovaries (relative to body weight) | Body | Ovaries (relative to body weight) | Body | Ovaries (relative to body weight) | | Control | 4–8 | 47.3 ± 1.35 | 0.007 ± 0.0008 | 279.5 ± 8.05 | 0.017 ± 0.001 | 40.0 ± 1.00 | 0.017 ± 0.001 | | 20 µg/kg/day | 4–8 | 48.0 ± 2.14 | 0.008 ± 0.0005^ | 272.0 ± 15.6 | 0.017 ± 0.001 | 41.5 ± 2.09 | 0.019 ± 0.002 | | 200 µg/kg/day | 4–8 | 46.7 ± 0.82 | 0.010 ± 0.0013* | 279.8 ± 10.5 | 0.016 ± 0.000 | 46.2 ± 3.20 | 0.016 ± 0.001 | | 200 mg/kg/day | 4–8 | 46.9 ± 2.76 | 0.009 ± 0.0017 | 277.0 ± 12.6 | 0.017 ± 0.001 | 35.6 ± 3.03 | 0.056 ± 0.025^ | Asterisk (*) indicates significant differences compared to control (p ≤ .05) and carets (^) indicate borderline difference (not statistically different) compared to control (.05 < p < .1). Data represent means ± standard error of the mean. Effects of Phthalate Mixture Exposure on Folliculogenesis In the F1 generation at PND 22, gestational and lactational exposure to the phthalate mixture significantly decreased the number of primary and preantral follicles in 200 mg/kg/day and 200 μg/kg/day treatment groups, respectively, compared to control (Figure 1A, n = 3–4 per treatment group, p < .05). In addition, exposure to the mixture significantly increased the number of the antral follicles at PND 22 in the 200 mg/kg/day treatment group in the F1 generation compared to control (Figure 1A, n = 3–4 per treatment group, p < .05). Further, exposure to the phthalate mixture caused a borderline increase (not statistically different) in the percentage of primary (20 μg/kg/day) and antral follicles (200 μg/kg/day) (Figure 1B, n = 3–4 per treatment group, .05 < p < .10) and significantly increased the percentage of antral follicles (200 mg/kg/day) compared to control. Exposure to the mixture caused a borderline decrease in the percentage of preantral follicles in 20 and 200 μg/kg/day treatment groups compared to control. In the F1 generation at PND 120, gestational and lactational exposure to the phthalate mixture caused a borderline decrease (not statistically different) in the number of preantral (200 μg/kg/day and 200 mg/kg/day) and antral follicles (20 μg/kg/day) compared to control (Figure 1C, n = 3–4 per treatment group, .05 < p < .10). Additionally, exposure to the mixture significantly decreased the percentage of preantral follicles in the 200 μg/kg/day treatment group compared to control (Figure 1C, n = 3–4 per treatment group, p < .05). In the F2 generation at PND 22, exposure to the phthalate mixture significantly decreased the number of primordial follicles in the 200 μg/kg/day treatment group compared to control (Figure 2A, n = 3–4 per treatment group, p < .05). Further, exposure to the mixture caused a borderline increase in the percentage of antral follicles in the 200 μg/kg/day treatment group compared to control (Figure 2B, n = 3–4 per treatment group, .05 < p < .10). Effects of Phthalate Mixture Exposure on Ovarian Gene Expression Apoptotic factors Gestational and lactational exposure to the phthalate mixture did not alter expression of the antiapoptotic factors Bcl2 and Bcl2l10 or the pro-apoptotic factors Bax and Aimf1 in any treatment group at PND 22 or 120 in the F1 generation compared to control (Figure 3, n = 3–4 per treatment group). However, exposure to the mixture significantly increased ovarian expression of Bcl2 (200 mg/kg/day) at PND 22 in the F2 generation compared to control (Figure 4A, n = 4 per treatment group, p < .05). Cell cycle regulators In the F1 generation at PND 22, gestational and lactational exposure to the phthalate mixture significantly increased (200 µg/kg/day) and decreased (200 mg/kg/day) ovarian expression of the cell cycle promoter Cdk4 compared to control (Figure 5C, n = 4 per treatment group, p < .05). Exposure to the mixture did not alter expression of the cell cycle regulators Ccna2, Ccnd2, and Cdkn1a at PND 22 in the F1 generation compared to control (Figure 5, n = 4 per treatment group). In addition, exposure to the phthalate mixture significantly decreased (200 mg/kg/day) ovarian expression of the cell cycle inhibitor Cdkn1a at PND 120 in the F1 generation compared to control (Figure 5, n = 3–4 per treatment group, p < .05). Exposure to the mixture did not alter expression of the cell cycle regulators Ccna2, Ccnd2, and Ccnb1 in the F1 PND 120 female rats compared to control (Figure 5, n = 3–4 per treatment group). In the F2 generation at PND 22, exposure to the mixture did not alter ovarian expression of any cell cycle regulator ovarian expression compared to control (Figure 6, n = 4 per treatment group). Steroidogenic factors In the F1 generation at PND 22, exposure to the phthalate mixture significantly increased ovarian expression of Star (200 µg/kg/day) compared to control (Figure 7A, n = 3–4 per treatment group, p < .05). In addition, exposure to the mixture significantly increased ovarian expression of Hsd3b1 (200 µg/kg/day and 200 mg/kg/day) compared to control (Figure 7C, n = 3–4 per treatment group, p < .05). Phthalate mixture exposure did not alter ovarian expression of Cyp11a1, Cyp17a1, or Cyp19a1 in any treatment group at PND 22 in the F1 generation compared to control (Figure 7, n = 3–4 per treatment group). Further, exposure to the mixture caused a borderline increase (not statistically different) in the expression of Cyp11a1 (20 µg/kg/day) (Figure 7F, n = 3–4 per treatment group, .05 < p < .10), but did not alter expression of Star, Hsd3b1, Cyp17a1, or Cyp19a1 in any treatment group at PND 120 in the F1 generation compared to control (Figure 7, n = 3–4 per treatment group). In the F2 generation at PND 22, exposure to the mixture significantly decreased expression (20 µg/kg/day) and borderline decreased (not statistically different) (200 mg/kg/day) Hsd3b1 expression compared to control (Figure 8C, n = 3–4 per treatment group, p < .05, p = .08). Effects of Phthalate Mixture Exposure on miRNA Expression In the F1 generation at PND 22, exposure to mixture did not alter rno-mir-141-3p or rno-mir-184 expression in any treatment group compared to control (Figs. 9A and 9B, n = 3–4 per treatment group). In the F1 generation at PND 120, exposure to the mixture increased rno-mir-184 (200 mg/kg/day) compared to control group (Figure 9D, n = 3–5 per treatment group, p < .05, p = .0483). In the F2 generation at PND 22, exposure to mixture did not alter rno-mir-141-3p or rno-mir-184 expression in any treatment group compared to control (Figs. 10A and 10B, n = 4–5 per treatment group). Functional Enrichment The rno-mir-184 has 4430 targets with 514 predicted targets of interest. Functional enrichment highlighted 37 targets associated with pathways of interest. These targets have an important influence in oocyte meiosis and progesterone-mediated maturation pathways (Figure 11 and Supplementary Table 2) and in ontological terms as oocyte maturation, differentiation and development, and hormonal response (Figure 12 and Supplementary Table 2). The number of genes and the reference to each term are shown Supplementary Table 2. Effects of Phthalate Mixture Exposure on Sex Steroid Hormone Levels Gestational and lactational exposure to the phthalate mixture (200 mg/kg/day) significantly decreased the levels of progesterone at PND 22 in the F1 generation compared with control (Figure 13A, n = 4–5 per treatment group, p < .05). In addition, exposure to the mixture significantly decreased testosterone levels (200 µg/kg/day and 200 mg/kg/day) at PND 22 in the F1 generation compared with control (Figure 13B, n = 4–5 per treatment group, p < .05). In the F1 generation at PND 120, exposure to the phthalate mixture caused a borderline increase (not statistically different) in progesterone levels and a borderline decrease (not statistically different) in testosterone levels in the 200 mg/kg/day treatment group compared with control (Figs. 13D and 13E, n = 4–5 per treatment group, .05 < p < .10). Exposure to the mixture did not alter estradiol levels at PND 22 or 120 in the F1 generation compared with control (Figs. 13C and 13F, n = 4–5 per treatment group). In the F2 generation at PND 22, exposure to the phthalate mixture did not alter the levels of sex steroid hormones in any treatment group compared with control (Figure 14, n = 4–5 per treatment group).

This study tested the hypothesis that maternal exposure during gestation and lactation to an environmentally relevant phthalate mixture affects steroidogenesis and folliculogenesis in the F1 and F2 generation of female rats. In addition, this study tested the hypothesis that maternal exposure to the mixture of phthalates impacts posttranscription in the ovaries of the F1 and F2 generations of female rats. Our results indicate that exposure to the mixture altered the number and percentage of follicles as well as ovarian expression of some genes related to apoptosis, the cell cycle, and steroidogenesis in F1 and F2 generation of female rats compared with control. Exposure to the mixture also impaired sex steroid hormone levels in the F1 generation of female rats compared with control. In addition, maternal exposure to the phthalate mixture altered ovarian miRNA expression in the F1 generation of female rats compared with control. We found that maternal exposure to the phthalate mixture borderline increased (not statistically different) (20 µg/kg/day) and significantly increased (200 µg/kg/day) the relative weight of the ovaries at PND 22 in the F1 generation of female rats compared with control. Interestingly, the phthalate mixture exposure decreased the number and percentage of follicles in the same treatment groups, but increased the number and percentage of antral follicles at the 200 mg/kg/day dose at PND 22 in the F1 generation of female rats compared with control. These results indicate that the increase in ovarian weight does not correlate with an increase in the number of follicles. We think that the mixture may be interfering with the amount of fluid or connective tissue in the ovaries, which could increase ovarian weight. In addition, the phthalate mixture decreased progesterone and testosterone levels in the F1 generation of female rats compared with control. Thus, it is not surprising that the mixture-induced decrease in the number and percentage of follicles correlates with the mixture-induced decrease in hormone levels. Maternal exposure to the phthalate mixture borderline decreased (not statistically different) the number of preantral follicles (200 µg/kg/day and 200 mg/kg/day) and antral follicles (20 µg/kg/day) at PND 120 in the F1 generation of female rats compared with control. Further, the phthalate mixture (200 µg/kg/day) significantly decreased the percentage of preantral follicles at PND 120 in the F1 generation compared with control. These results indicate that the phthalate mixture is interfering with the transition of preantral follicles to the antral follicle stage. In fact, we observed that the phthalate mixture borderline increased (not statistically different) the levels of progesterone and borderline decreased (not statistically different) the levels of testosterone in the highest concentration group, suggesting that the mixture is interfering with the ability of the follicles to produce sex steroid hormones. This is of concern because altered folliculogenesis can affect hormone production as observed here and subsequently affect fertility. A recent study demonstrated that this same phthalate mixture decreased the ability of F1 female mice to produce pups at 11 and 13 months of age, and it also decreased the ability of F1 females to carry out their pregnancy at 13 months of age (Brehm and Flaws, 2021). Future studies should investigate whether exposure to the phthalate mixture impairs fertility in the F1 generation of female rats. Similar to the F1 generation, our results determined that maternal exposure to the phthalate mixture borderline increased (not statistically different) the relative weight of the ovaries (200 mg/kg/day) at PND 22 in the F2 generation of female rats compared with control. Surprisingly, the phthalate mixture exposure decreased the number of primordial follicles (200 µg/kg/day) and increased the percentage of antral follicles (200 µg/kg/day) at PND 22 in the F2 generation compared with control. Again, the increase in ovarian weight was not associated with an increase the number of follicles. Future studies should examine if the increase in ovarian weight is due to an increase in fluid or connective tissue in the ovaries. Phthalates have been shown to disrupt the expression of proteins involved in cell cycle regulation and proliferation, affecting cell growth (Hannon et al., 2015; Rattan et al., 2019; Zhou and Flaws, 2017). Our study determined that the phthalate mixture increased ovarian expression of the cell cycle promoter Cdk4 in the 200 µg/kg/day group and decreased expression of the same gene in the 200 mg/kg/day group at PND 22 in the F1 generation of female rats. A previous study showed that exposure to a single phthalate (di(2-ethylhexyl) phthalate) did not alter ovarian expression of Cdk4; however, it affected expression of other cell cycle regulators in mice (Rattan et al., 2019). In addition, the mixture decreased expression of the cell cycle inhibitor Cdkn1a (200 mg/kg/day) at PND 120 in the F1 generation. Cell cycle regulators are heavily involved in cell proliferation and follicle growth (Robker and Richards, 1998). Therefore, alterations in the expression of these factors could affect reproductive outcomes and fertility. The ovaries play an important role in steroidogenesis. For this reason, we measured ovarian expression of factors involved in sex steroid hormone synthesis and serum hormone levels. Our results indicate that maternal exposure to the phthalate mixture significantly increased ovarian expression of Star (200 µg/kg/day) and Hsd3b1 (200 µg/kg/day and 200 mg/kg/day) at PND 22 in the F1 generation of rats. In fact, we observed a decrease in progesterone and testosterone levels in the highest concentration groups at PND 22 in the F1 generation compared with control. We speculate that the increase in expression of Star and Hsd3b1 is a form of compensation for the decreased levels of progesterone and testosterone. Further, the phthalate mixture borderline increased (not statistically different) ovarian expression of Cyp11a1 (20 µg/kg/day) and significantly decreased expression of Hsd3b1 (200 mg/kg/day) at PND 120 in the F1 generation compared with control. We also observed an increase in the levels of progesterone and a decrease in the levels of testosterone in the highest concentration groups at PND 120 in the F1 generation compared with control. In the F2 generation, maternal exposure to the phthalate mixture significantly decreased ovarian expression of Hsd3b1 (20 µg/kg/day) and borderline decreased (not statistically different) expression of the same gene (200 mg/kg/day) at PND 22 compared with control. In a different study, the effects the phthalate mixture exposure during gestation were evaluated in the F1 generation of female mice (Gill et al., 2021). Similar to our results, the phthalate mixture decreased the levels of progesterone and testosterone in the F1 generation of female mice at PND 60 compared with control (Gill et al., 2021). Interestingly, the mixture decreased the number and percentage of preantral follicles in the F1 generation at PND 60 compared with control in mice (Gill et al., 2021). Another study also determined that in utero exposure to the mixture decreased levels of testosterone in the F1 generation of female mice compared with control (Brehm et al., 2020). Contrary to our results, a previous study observed that the mixture decreased the number of primordial follicles in the F1 generation of mice (Brehm et al., 2020). However, the F1 generation data were collected at 13 months of age (Brehm et al., 2020). Part of our results differ likely because of species differences between mice and rats and the age of the animals at the time of data collection. Our results demonstrated that maternal exposure to the mixture significantly increased ovarian rno-mir-184 expression (200 mg/kg/day) at PND 120 in the F1 generation of female rats compared with control. Recently, Scarano et al. demonstrated that maternal exposure to the same mixture of phthalates altered expression of rno-mir-184 and other miRNAs in the rat prostate compared with control (Scarano et al., 2019). miRNA-184 can alter the levels of hormones such as progesterone, testosterone, and estradiol in human ovarian cells (Sirotkin et al., 2009). In addition, mir-184 has been shown to regulate biological events during oogenesis and early embryogenesis in Drosophila (Iovino et al., 2009). In fact, we observed that the phthalate mixture exposure decreased the number of primordial follicles (200 µg/kg/day) at PND 120 in the F1 generation of rats compared with control, which could be indicative of an early onset of ovarian senescence. Functional enrichment analysis revealed that rno-mir-184 is involved in the regulation of progesterone-mediated oocyte maturation and oocyte meiosis. Oocyte maturation is the reentry into meiosis that occurs just prior to ovulation and subsequent fertilization (Jamnongjit and Hammes, 2005). Oocytes are arrested in prophase I of meiosis until puberty when luteinizing hormone induces resumption of meiosis of follicle-enclosed oocytes, which then triggers the resumption of meiosis up to metaphase II (Jamnongjit and Hammes, 2005; Solc et al., 2010). Oocytes are subsequently held in meiotic arrest until fertilization when meiosis is completed (Jamnongjit and Hammes, 2005). Any interference with oocyte maturation could result in defective or low-quality germ cells, subsequently leading to adverse fertility outcomes. Future studies should investigate the effects of the phthalate mixture on oocyte quality, ovulation, and fertility in F1 and F2 generation of female rats. In general, our data showed nonmonotonic dose-responses to the phthalate mixture exposure. These results are consistent with the known effects of endocrine disrupting chemicals (EDCs). EDCs are compounds capable of interfering with the endocrine system (NIEHS, 2021). EDCs present nonmonotonic responses, in which different concentrations of an EDC show opposite effects (Vandenberg, 2014; Vandenberg et al., 2012). Several hypotheses have been proposed to explain nonmonotonic profiles of EDCs. Some hypotheses include actions at several molecular targets with differing affinities, antagonistic effects, negative feedback regulation to reduce responses, or receptor desensitization (Vandenberg et al., 2012). Phthalates are well known to be endocrine-disrupting chemicals, not only disrupting the reproductive system, but also thyroid and adrenal gland function (Derakhshan et al., 2021; Martinez-Arguelles et al., 2014). In conclusion, maternal exposure to the phthalate mixture impaired folliculogenesis in the F1 and F2 generations of female rats and affected steroidogenesis in the F1 generation of female rats compared with control. Further, the phthalate mixture altered ovarian expression of some genes related to the cell cycle and steroidogenesis in F1 and F2 generations of female rats compared with control. Finally, the phthalate mixture increased ovarian expression of rno-mir-184, which is involved in the oocyte maturation process. Future studies should investigate the effects of the phthalate mixture on ovulation, oocyte quality, and fertility parameters in female rats. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. DECLARATION OF CONFLICTING INTERESTS The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. AUTHOR CONTRIBUTIONS A.G. designed the analysis of ovarian mRNA expression, follicle counting, and hormone measurements, interpreted the data, wrote the manuscript, and revised the manuscript in response to the coauthors and reviewers. A.A. helped design and conduct the study, designed the analysis of ovarian miRNA expression, interpreted the data, helped write the manuscript, and revised the manuscript in response to the coauthors. L.G.A.C. and L.F.B. helped design and conduct the animal study and revised the manuscript in response to the coauthors. W.R.S. designed the study, obtained funding for the study, provided input on the analysis, and edited the manuscript. J.A.F. designed the study, obtained funding for the study, provided input on the analysis, and edited the manuscript. FUNDING National Institutes of Health (NIH) (R01 ES 028661, NIH R01 ES 032163); an Environmental Toxicology Fellowship; an award from the Lemann Center for Brazilian Studies; the Sao Paulo State Research Foundation FAPESP (2017/08306-2, 2018/50002-3); a Researcher Fellowship from National Council for Scientific and Technological Development, CNPq (303418/2019-2-WRS, 303751/2018-5-LFB). DATA AVAILABILITY The data underlying this article will be shared on reasonable request to the corresponding author. Supplementary Material Contributor Information Andressa V Gonsioroski, Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana, Illinois 61802, USA. Ariana M Aquino, Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP 18618-689, Brazil. Luiz G Alonso-Costa, Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP 18618-689, Brazil. Luis F Barbisan, Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP 18618-689, Brazil. Wellerson R Scarano, Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP 18618-689, Brazil. Jodi A Flaws, Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana, Illinois 61802, USA.

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