Reproductive Tract Development Research Articles

High incidence of imperforate vagina in ADGRA3-deficient mice

BackgroundTen percent of the female population suffers from congenital abnormalities of the vagina, uterus, or oviducts, with severe consequences for reproductive and psychological health. Yet, the underlying causes of most of these malformations remain largely unknown. ADGRA3 (GPR125) is involved in WNT signaling and planar cell polarity, mechanisms vital to female reproductive tract development. Although ADGRA3 is a well-established spermatogonial stem cell marker, its role within the female urogenital system remains unclear.ResultsIn this study, we found Adgra3 to be expressed throughout the murine female urogenital system, with higher expression pre-puberty than after sexual maturation. We generated a global Adgra3−/− mouse line and observed imperforate vagina in 44% of Adgra3−/− females, resulting in distension of the reproductive tract and infertility. Ovarian morphology, plasma estradiol, ovarian Cyp19a1, and vaginal estrogen receptor α (Esr1) expression were unaffected. However, compared to controls, a significantly lower bone mineral density was found in Adgra3−/− mice. Whereas vaginal opening in mice is an estrogen-dependent process, 17β-estradiol treatment failed to induce vaginal canalization in Adgra3−/− mice. Furthermore, a marked reduction in vaginal and ovarian progesterone receptor expression was observed concomitant with an upregulation of apoptotic regulators Bcl2, Bid, and Bmf in adult Adgra3−/− females with a closed vagina.ConclusionsOur collective results shed new insights into the complex mechanisms by which the adhesion receptor ADGRA3 regulates distal vaginal tissue remodeling during vaginal canalization via altered sex hormone responsiveness and balance in apoptotic regulators. This highlights the potential of ADGRA3 as a target in diagnostic screening and/or therapy for obstructive vaginal malformations in humans.

Disruption of Thyroid Hormone Receptor Thrab Leads to Female Infertility in Zebrafish.

Thyroid hormones (THs) T4 and T3 are vital for development, growth, and metabolism. Thyroid dysfunction can also cause problems in fertility, suggesting involvement of THs in reproduction. In zebrafish, there exist 2 forms of TH receptor alpha gene (thraa and thrab). Disruption of these genes by CRISPR/Cas9 showed no reproductive irregularities in the thraa mutant; however, inactivation of the thrab gene resulted in female infertility. Although young female mutants (thrabm/m) showed normal ovarian development and folliculogenesis before sexual maturation, they failed to release eggs during oviposition after sexual maturation. This spawning failure was due to oviductal blockage at the genital papilla. The obstruction of the oviduct subsequently caused an accumulation of the eggs in the ovary, resulting in severe ovarian hypertrophy, abdominal distention, and disruption of folliculogenesis. Gene expression analysis showed expression of both TH receptors and estrogen receptors in the genital papilla, suggesting a direct TH action and potential interactions between thyroid and estrogen signaling pathways in controlling genital papilla development and function. In addition to their actions in the reproductive tracts, THs may also have direct effects in the ovary, as suggested by follicle atresia and cessation of folliculogenesis in the heterozygous mutant (thrab+/m), which was normal in all aspects of female reproduction in young and sexually mature fish but exhibited premature ovarian failure in aged females. In summary, this study provides substantial evidence for roles of THs in controlling the development and functions of both reproductive tract and ovary.

Developmental programming of the ovarian reserve in livestock

Mammalian females are born with a finite number of follicles in their ovaries that is referred to as the ovarian reserve. There is a large amount of variation between females in the number of antral follicles that they are born with, but this number is positively correlated to size of the ovarian reserve, has a strong repeatability within a female, and a moderate heritability. Although the heritability is moderate, numerous external factors including health, nutrition, ambient temperature, and litter size influence the size and function of the ovarian reserve throughout life. Depletion of the ovarian reserve contributes to reproductive senescence, and genetic and epigenetic factors can lead to a more rapid decline in follicle numbers in some females than others. The relationship of the size of the ovarian reserve to development of the reproductive tract and fertility is generally positive, although some studies report antagonistic associations of these traits. It seems likely that management decisions and environmental factors that result in epigenetic modifications to the genome throughout life may cause variability in the function of ovarian genes that influence fecundity and fertility, leading to differences in reproductive longevity among females born with ovarian reserves of similar size. This review summarizes our current understanding of factors influencing size of the ovarian reserve in cattle, sheep, and pigs and the relationship of the ovarian reserve to reproductive tract development and fertility. It provides strategies to apply this knowledge to improve diagnostics for better assessment of fertility and reproductive longevity in female livestock.

MAP3K1 regulates female reproductive tract development.

Mitogen-activated protein 3 kinase 1 (MAP3K1) has a plethora of cell type-specific functions not yet fully understood. Herein, we describe a role for MAP3K1 in female reproductive tract (FRT) development. MAP3K1 kinase domain-deficient female mice exhibited an imperforate vagina, labor failure and infertility. These defects corresponded with shunted Müllerian ducts (MDs), the embryonic precursors of FRT, that manifested as a contorted caudal vagina and abrogated vaginal-urogenital sinus fusion in neonates. The MAP3K1 kinase domain is required for optimal activation of the Jun-N-terminal kinase (JNK) and cell polarity in the MD epithelium, and for upregulation of WNT signaling in the mesenchyme surrounding the caudal MD. The MAP3K1-deficient epithelial cells and MD epithelium had reduced expression of WNT7B ligands. Correspondingly, conditioned media derived from MAP3K1-competent, but not -deficient, epithelial cells activated a TCF/Lef-luciferase reporter in fibroblasts. These observations indicate that MAP3K1 regulates MD caudal elongation and FRT development, in part through the induction of paracrine factors in the epithelium that trans-activate WNT signaling in the mesenchyme.

Mammary gland development and EDC-driven cancer susceptibility in mesenchymal ERα-knockout mice.

Development of the mammary gland requires both proper hormone signaling and cross talk between the stroma and epithelium. While estrogen receptor (ERα) expression in the epithelium is essential for normal gland development, the role of this receptor in the stroma is less clear. Moreover, several lines of evidence suggest that mouse phenotypes of in utero exposure to endocrine disruption act through mesenchymal ERα in the developing fetus. We utilized a Twist2-cre mouse line to knock out mesenchymal ERα. Herein, we assessed mammary gland development in the context of mesenchymal ERα deletion. We also tested the effect of in utero bisphenol A (BPA) exposure to alter the tumor susceptibility in the mouse mammary tumor virus-neu (MMTV-neu) breast cancer mouse model. Mesenchymal ERα deletion resulted in altered reproductive tract development and atypical cytology associated with estrous cycling. The mammary gland demonstrated mature epithelial extension unlike complete ERα-knockout mice, but ductal extension was delayed and reduced compared to ERα-competent mice. Using the MMTV-Neu cancer susceptibility model, ERα-intact mice exposed to BPA had reduced tumor-free survival and overall survival compared to BPA-exposed mice having mesenchymal ERα deletion. This difference is specific for BPA exposure as vehicle-treated animals had no difference in tumor development between mice expressing and not expressing mesenchymal ERα. These data demonstrate that mesenchymal ERα expression is not required for ductal extension, nor does it influence cancer risk in this mouse model but does influence the cancer incidence associated with in utero BPA exposure.

A nonsense mutation in mouse Adamtsl2 causes uterine hypoplasia and an irregular estrous cycle

The spontaneous mutation stubby (stb) in mice causes chondrodysplasia and male infertility due to impotence through autosomal recessive inheritance. In this study, we conducted linkage analysis to localize the stb locus within a 1.6 Mb region on mouse chromosome 2 and identified a nonsense mutation in Adamtsl2 of stb/stb mice. Histological analysis revealed disturbed endochondral ossification with a reduced hypertrophic chondrocyte layer and stiff skin with a thickened dermal layer. These phenotypes are similar to those observed in humans and mice with ADAMTSL2/Adamtsl2 mutations. Moreover, stb/stb female mice exhibited severe uterine hypoplasia at 5 weeks of age and irregular estrous cycles at 10 weeks of age. In normal mice, Adamtsl2 was more highly expressed in the ovary and pituitary gland than in the uterus, and this expression was decreased in stb/stb mice. These findings suggest that Adamtsl2 may function in these organs rather than in the uterus. Thus, we analyzed Gh expression in the pituitary gland and plasma estradiol and IGF1 levels, which are required for the development of the female reproductive tract. There was no significant difference in Gh expression and estradiol levels, whereas IGF1 levels in stb/stb mice were significantly reduced to 54–59% of those in +/+ mice. We conclude that Adamtsl2 is required for the development of the uterus and regulation of the estrous cycle in female mice, and decreased IGF1 may be related to these abnormalities.

Genomic characterisation of the overlap of endometriosis with 76 comorbidities identifies pleiotropic and causal mechanisms underlying disease risk

Comorbid conditions can be driven by underlying pleiotropic and causal mechanisms that can provide insights into shared molecular and biological processes contributing to disease risk. Endometriosis is a chronic condition affecting one in nine women of reproductive age and poses many challenges including lengthy diagnostic delays and limited treatment efficacy owing to poor understanding of disease aetiology. To shed light on the underlying biological mechanisms and to identify potential risk factors, we examine the epidemiological and genomic relationship between endometriosis and its comorbidities. In the UK Biobank 292 ICD10 codes were epidemiologically correlated with endometriosis diagnosis, including gynaecological, immune, infection, pain, psychiatric, cancer, gastrointestinal, urinary, bone and cardiovascular traits. A subset of the identified comorbidities (n = 76) underwent follow-up genetic analysis. Whilst Mendelian randomisation suggested causality was not responsible for most comorbid relationships, 22 traits were genetically correlated with endometriosis, including pain, gynaecological and gastrointestinal traits, suggestive of a shared genetic background. Pleiotropic genetic variants and genes were identified using gene-based and colocalisation analysis. Shared genetic risk factors and potential target genes suggest a diverse collection of biological systems are involved in these comorbid relationships including coagulation factors, development of the female reproductive tract and cell proliferation. These findings highlight the diversity of traits with epidemiological and genomic overlap with endometriosis and implicate a key role for pleiotropy in the comorbid relationships.

P-159 Impact of the symbiotic metabolites D-amino acids on preimplantation development

Abstract Study question Are the metabolites derived from the symbiotic microbiota of reproductive tract involved in preimplantation development? Summary answer The metabolites derived from the symbiotic microbiota of reproductive tract, D-amino acid（D-AA）, contributed to preimplantation development. What is known already It has been known that Lactobacillus exist in the vagina as symbiotic bacteria. Though it was thought that Lactobacillus keep the pH environment acidic and prevent infection by pathogenic bacteria, thereby keeping the uterus and fallopian tubes sterile in mammal, we have identified Staphylococcus aureus in oviductal lavage fluid from female mice and confirmed that the antibiotics disrupt the implantation pattern. In human, the vaginal microbiota is reported to be dominated by Lactobacillus, which produces D-amino acids (D-AA). These findings suggest the involvement of bacterial metabolites D-AA in the establishment of human pregnancy. Study design, size, duration 23 women undergoing in vitro fertilization (IVF) treatment at Keio University Hospital from 2019 to 2021 were assessed for eligibility. 3 were excluded due to spontaneous pregnancy or infectious disease. Twenty subjects were allocated to the successful pregnancy group (S-group (8); mean ± standard deviation 35.5 ± 2.8 years) defined as gestational sac detected on ultrasound at 5 weeks' gestation; and to the unsuccessful pregnancy group (U- group (12); 35.5 ± 2.7 years)). Participants/materials, setting, methods Genomic DNA (gDNA) extracted from vaginal and intrauterine samples at each menstrual cycle was used for metagenome analysis at the genus and the species levels by next-generation sequencing, and by quantitative real-time PCR. Amino acids were analyzed using a two-dimensional HPLC. Immunocytochemical staining by a polyclonal antibody to D-AA oxidase (DAO), which catabolizes D-AA, was performed using B6D2F1 mice and mice with a natural point-mutation G181R in DAO gene, which abolishes DAO activity. Main results and the role of chance Both vaginal/uterine bacterial counts were higher in the luteal phase than in the follicular phase. Vaginal bacterial counts in the S-group were lower than those in the U- group. The composition of microbiota of the vaginal samples (N = 23) shifted from the follicular to the luteal phase, with Lactobacillus spp. predominating. However, there was no significant difference in the ratio of Lactobacillus spp. between the S- and U-groups. The intrauterine samples of the S-group contained a high abundance of L. crispatus, L. gasseri, and L. ultunensis, while the U-group contained a high abundance of L. jensenii. Next, we analyzed the Lactobacillus metabolite, D-AA, and detected D-Ser, D-Ala, D-Asn, and D-Pro in the vaginal, uterine, and follicular fluid of the S-group. Follicular fluid that developed into blastocysts contained more D-AA than that of arrested embryos (N = 2). To investigate the significance of D-AA in the preimplantation development, mouse embryos were cultured in amino acid-depleted medium. All of them arrested (0.0%: 0/78), whereas 41% (25/61) of the embryos with D-AA added to the amino acid-depleted medium developed into blastocysts. We confirmed the expression of DAO in oocytes and blastocysts. DAO-null mice had fewer births than wild type. Limitations, reasons for caution The main limitation is the differences of symbiotic microbiota in reproductive tract and embryonic development between mice and humans; it may be difficult to apply the results of this analysis to humans. Wider implications of the findings Our results display a correlation between species-level microbiome profiling and IVF outcomes. In addition, microbiota-derived metabolites D-AA are suggested to be crucial mediators of embryo development. These findings could give rise to the development of new diagnostic techniques and drug. Trial registration number Not applicable

Evaluation of multigenerational effects of 2-ethylhexyl 4-hydroxybenzoate in Japanese medaka.

The Japanese medaka (Oryzias latipes) extended one-generation reproduction test (MEOGRT) (Test Guideline 890.2200) is a Tier 2 test within the Endocrine Disruptor Screening Program of the US Environmental Protection Agency (US EPA). A modified MEOGRT was used to evaluate multigenerational effects of 2-ethylhexyl 4-hydroxybenzoate (2-EHHB) under flow-through conditions starting with adults (parent generation, F0) through a 3-week reproductive phase of the second generation (F2). Fish were exposed to one of five 2-EHHB test concentrations or a dechlorinated tap water control. Fecundity was affected at the lowest exposure (5.32 μg/L) and greater sensitivity occurred in the F1 and F2 generations. Percent fertility was also diminished from no effect level observed in the F0 generation to 101 and 48.8μg/L in the F1 and F2 generations, respectively. Growth indices were decreased for F0 adult females and F1 subadults and adults at 48.8μg/L 2-EHHB. Histopathologic examination of gonads, liver, kidney, and thyroid yielded possible delayed reproductive tract development in F1 subadult males, masculinization of the renal phenotype in F1 adult females (renal tubular eosinophilia) and reduced hepatic energy storage (liver glycogen vacuoles) in F1 (11.3 and 48.8μg/L) and F2 (48.8 and 101 μg/L) males and females, respectively. Endocrine-related findings included a decrease in anal fin papillae in F2 adult males at 101 μg/L. Results of this study demonstrate effects on growth, development, and reproduction that may be mediated by endocrine (weak estrogenic) and nonendocrine mechanisms. Duration of the MEOGRT should not be routinely extended beyond the OCSPP 890 guideline study design.

GWAS meta-analyses clarify the genetics of cervical phenotypes and inform risk stratification for cervical cancer.

Genome-wide association studies (GWAS) have successfully identified associations for cervical cancer, but the underlying mechanisms of cervical biology and pathology remain uncharacterised. Our GWAS meta-analyses fill this gap, as we characterise the genetic architecture of cervical phenotypes, including cervical ectropion, cervicitis, cervical dysplasia, as well as up to 9229 cases and 490 304 controls for cervical cancer from diverse ancestries. Leveraging the latest computational methods and gene expression data, we refine the association signals for cervical cancer and propose potential causal variants and genes at each locus. We prioritise PAX8/PAX8-AS1, LINC00339, CDC42, CLPTM1L, HLA-DRB1 and GSDMB as the most likely candidate genes for cervical cancer signals, providing insights into cervical cancer pathogenesis and supporting the involvement of reproductive tract development, immune response and cellular proliferation/apoptosis. We construct a genetic risk score (GRS) that is associated with cervical cancer [hazard ratios (HR)= 3.1 (1.7-5.6) for the top 15% vs lowest 15% of individuals], and with other HPV- and immune-system-related diagnoses in a phenome-wide association study analysis. Our results propose valuable leads for further functional studies and present a GRS for cervical cancer that allows additional risk stratification and could potentially be used to personalise the conventional screening strategies for groups more susceptible to cervical cancer.

Associations Between Prenatal Urinary Biomarkers of Phthalate Exposure and Preterm Birth: A Pooled Study of 16 US Cohorts

ABSTRACT Preterm birth is a leading cause of neonatal mortality. Preterm birth is a public health burden, particularly in the United States, where approximately 10% of deliveries are preterm. Although the underlying cause of most preterm births is unknown, environmental chemical exposures (such as phthalates) possibly contribute. Phthalates are common in consumer products, and exposure can occur through diet, personal care products, and even household dust. Consequently, exposure is ubiquitous for pregnant individuals. Prenatal phthalate exposure has been associated with adverse neurodevelopment in children and disordered development of the male reproductive tract. This study reviewed 16 prospective studies conducted within the United States to pool individual-level data to examine prenatal urinary biomarkers of phthalate exposure and preterm birth. In addition, potential influence of exposure to overall phthalate mixture was assessed to determine the potential impact of reduced exposure on preterm birth. Results of the study indicated that of 6045 pregnant women, 539 (9%) delivered preterm. Overall, of the entire included cohort, 802 individuals were Black (13.3%), 2576 were White (42.6%), 2323 were Latina (38.4%), and 328 had other race identity (including Native Hawaiian, Alaskan Native, or American Indian). Characteristics of participants were similar between those who delivered preterm versus term. Some 96% of urine samples displayed detectable concentrations of urinary phthalate metabolites. As demonstrated by regression analyses, higher concentrations of most phthalate metabolites bore an association of slightly higher odds (12%–16% higher) for preterm birth. In addition, the study estimated that reducing the mixture of phthalate metabolite concentrations by 10%, 30%, or 50%, respectively, could prevent 1.8, 5.9, and 11.1 preterm births per 1000 live births. The study found a relationship between higher maternal pregnancy concentrations of urinary phthalate metabolites and preterm birth. The findings of this study identify a potential benefit of phthalate exposure reduction among pregnant individuals via either regulations or behavioral interventions. Because phthalate exposure can occur through many environments and sources, the US Consumer Product Safety Commission has attempted to pinpoint major sources of phthalate exposure and has determined that the predominate exposures appear to occur through food and medications, although uncertainty in the primary source of exposure remains. Phthalate exposure also widely varies in the United States based on several factors such as whether a person is at a disadvantaged socioeconomic status, is pregnant, or is of a marginalized race or ethnicity. Targeted interventions, such as altering personal care products, is made challenging as consumers are not readily able to access accurate ingredients lists. In the United States, for example, fragrance ingredient lists are not required to list phthalates when they are included in the product. Diet interventions intending to reduce phthalate exposure have had mixed results. Although federally mandated restrictions have limited the use of phthalates in products for children, few such restrictions exist for products intended for pregnant individuals. As 28 phthalates are currently allowed as food additives or in food contact materials, they are difficult to avoid. Mitigation of population-level health effects from phthalates through regulatory means would be most effective when considering phthalates not as individual chemicals, but rather as a class. This study found that higher concentrations of several urinary phthalate metabolites in pregnancy were associated with preterm birth, a consistent finding across 16 prospective US studies. Such findings emphasize the importance of the development of policy measures and public health around phthalate exposure reduction, especially among pregnant individuals.

Fetal sex and the development of gestational diabetes mellitus in polycystic ovarian syndrome gravidae

Polycystic ovarian syndrome is characterized by elevated androgens and is a well-known risk factor for the occurrence of gestational diabetes mellitus. Androgens (particularly dehydroepiandrosterone-sulfate) are crucial for the development and characteristics of the male reproductive tract during fetal life, and fetal dehydroepiandrosterone-sulfate enters the placenta where it is metabolized and functions as an estrogen substrate. Given this unique sex-specific relationship with androgens and the association of serum dehydroepiandrosterone-sulfate concentration with insulin resistance, we hypothesized that metabolic comorbidities in pregnancy might differ by fetal sex in gravidae with polycystic ovarian syndrome, notably in those with infertility. This study aimed to evaluate the data in a large population-based database to explore if fetal sex was significantly associated with gestational diabetes mellitus in gravidae with infertility and polycystic ovarian syndrome after controlling for confounders. This study was designed to evaluate the risk for the occurrence and rates of gestational diabetes mellitus among gravidae with infertility and a history of polycystic ovarian syndrome. We used a 2-hospital, single academic institution database comprising more than 30,000 subjects enrolled from September 2011 to June 2021 to identify all gravidae with diagnoses of infertility and polycystic ovarian syndrome at the time of delivery and to compare them with gravidae who lacked these comorbidities. Data on covariates, including but not limited to maternal age, body mass index, fetal sex, race, ethnicity, presence or absence of hypertensive disease, and presence or absence of gestational diabetes were identified. Unadjusted and adjusted odds rations were calculated. We found a statistically significant association between fetal female sex and the development of gestational diabetes mellitus in gravidae with polycystic ovarian syndrome (odds ratio for female vs male, 2.13; 95% confidence interval, 1.06-4.32; P=.03). After adjusting for potential confounders identified in our univariate analyses, there continued to be a statistically significant association between female fetuses and the development of gestational diabetes mellitus (adjusted odds ratio for female vs male, 2.10; 95% confidence interval, 1.04-4.41; P=.04). In contrast, there was no significant association between fetal sex and the development of gestational diabetes mellitus in our similar analysis of gravidae without infertility and polycystic ovarian syndrome (P=.99). Although the origin of gestational diabetes mellitus is multifactorial, we found that female fetal sex is associated with gestational diabetes mellitus in gravidae with infertility and polycystic ovarian syndrome but not in their comparative controls. Further research on the molecular mechanisms driving the association between female fetuses and the development of gestational diabetes mellitus in the context of maternal polycystic ovarian syndrome is warranted.

Loss of Adgra3 causes obstructive azoospermia with high penetrance in male mice.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3-/- ), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

Mouse vaginal development with lateral enlargement at late embryonic stages and caudal elongation after birth.

Müllerian ducts give rise to the oviducts, uterus, cervix, and vagina. During female reproductive tract development in mice, the bilateral Müllerian duct epithelium grows caudally until reaching the urogenital sinus epithelium. This is followed by further caudal growth with the reduction of the urogenital sinus epithelium. Finally, the vaginal epithelium of adult mice is entirely derived from the Müllerian duct epithelium. Here, we explored the mechanisms underlying mouse vaginal development via cell proliferation, apoptosis, and lineage analyses. We found that at the late embryonic stages, apoptosis occurred at the attachment site of bilateral Müllerian duct epithelia below the cervix, resulting in bilateral lumen traffic. The Müllerian duct epithelium was enclosed by the urogenital sinus epithelium at their boundary region on embryonic day (E) 16.5, whereas the Müllerian duct epithelium encased the urogenital sinus epithelium at postnatal day (P) 0 through lateral enlargement. Lateral Müllerian duct enlargement was accompanied by focal ERK activation within the curved epithelial tips and the specific localization of mitotic nuclei on the luminal side of the Müllerian duct epithelial layer at E17.5. Descent of the Müllerian duct epithelium and shortening of the urogenital sinus epithelium occurred rapidly after birth, accompanied by cell proliferation in the Müllerian duct epithelium and its peripheral mesenchymal tissues as well as intense apoptosis in the urogenital sinus epithelium around their boundary region. Urogenital sinus epithelium was localized at the base of the vagina at P7. In conclusion, the mouse vagina develops laterally at the late embryonic stages and caudally after birth.

OR25-1 Bilateral and Unilateral Malformations of Male Reproductive Tracts in Mice with Androgen Receptor Gene Deletion in the Mesenchyme

Abstract Congenital unilateral defects of male reproductive tracts have been documented in men, suggesting the potential left-right asymmetry in male reproductive tract development. However, classic textbooks of endocrinology have been teaching us that the development of the paired male reproductive tract is governed by the universal action of the androgen receptor (Ar). During sexual differentiation, testis-derived androgens activate the Ar to promote the stabilization of the bilateral Wolffian ducts, the progenitor for the male reproductive tract. The Wolffian ducts then differentiate into the paired male reproductive tract organs, which include the epididymes, vas deferentes, and seminal vesicles. The Ar is expressed both in the epithelium and mesenchyme of the male reproductive tract during fetal development. Landmark tissue recombinant studies and the observation of normal morphology in the epithelium-specific Ar knockout mouse all support the notion that Ar action in the mesenchyme dictates the morphogenesis of the male reproductive tract. However, no genetic study has been performed to investigate the consequence of the loss of mesenchymal Ar on male reproductive tract development. To test the functional significance of the mesenchymal Ar, we designed a mesenchyme-specific Ar knockout mouse model (ARcKO) to ablate Ar specifically in the mesenchyme prior to the initiation of sexual differentiation. We performed immunohistochemistry of AR to confirm that Ar expression was absent in the mesenchyme while epithelial Ar remained intact in this ARcKO model. Based on the widely accepted notion that mesenchymal Ar regulates epithelial morphogenesis of the male reproductive tract, we expected to see abnormal patterning of the male reproductive tract. Indeed, the epididymis lost its characteristic coiling and became cystic. However, 40% of the 23 collected ARcKO males displayed these abnormalities on both horns; 43% and 17% displayed these abnormal phenotype solely on the left and right horn, respectively. This surprising observation might result from asynchronous activities of Cre on the left and right horns. However, when the Cre was crossed with a reporter (tdTomato), we observed comparable reporter expression on both horns at the onset of sexual differentiation of reproductive tracts. These observations suggest that the developmental programs in the morphogenesis of the left and right male reproductive tracts are asymmetrical and there could be a compensation mechanism for the loss of mesenchymal AR. We are currently in the process of comparing the left and the right horns in Ar knockout male mice to determine any biased signaling. Taken together, our study provides a unique model for not only studying congenital defects of male reproductive tracts but also for investigating the potential asymmetry in the masculinization program. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.

RF34 | PMON254 Why Does Androgens Fail to Promote the Survival of the Wolffian Duct in the Absence of Wnt9b?

Abstract The male reproductive tract development has been predominantly dependent upon androgen action, which is mainly mediated by the androgen receptor (AR). Under the influence of androgen actions, the primitive male reproductive tract, also known as the Wolffian duct, is first stabilized then differentiates into the epididymis, vas deference and seminal vesicle. Impairment of androgen signaling during fetal programming causes a spectrum of male reproductive disorders that manifest in adulthood. Despite the dominant roles of androgen action in the male reproductive tract development, it has been noticed that androgen's capability in promoting Wolffian duct survival and development is completely lost in the absence of Wnt9b. Wnt9b belongs to the WNT family of secreted proteins. It is specifically expressed in the Wolffian duct epithelium to regulate the surrounding mesenchyme. This project aims to investigate why the androgen action fails to maintain the male reproductive tract in the absence of Wnt9b. We first examined the expression pattern of Wnt9b during the window of sexual differentiation, which was not investigated in the previous publication. We found that during sexual differentiation, Wnt9b is still specifically expressed in the Wolffian duct epithelium in both XX and XY embryos. Androgen action exists in XY but not in the XX embryos during sexual differentiation. These observations indicate that Wnt9b does not lie downstream of the androgen signaling. We are in process of collecting Wnt9b+/+ and Wnt9b-/- male embryos to examine whether expression of AR and AR downstream genes is altered in the absence of Wnt9b. In addition, WNT9B can activate two intracellular pathways under the context of target cells: β-catenin dependent and independent pathways. Since Wolffian duct mesenchyme is the major target of epithelium-derived WNT9B, we next determined which pathways WNT9B acts through in the mesenchyme by specific deletion of β-catenin in the Wolffian duct mesenchyme. Interestingly, the loss of β-catenin in the mesenchyme led to the partial degeneration of Wolffian ducts at the caudal region and cystic formations at the cranial region, the similar phenotypes observed in mesenchymal AR knockout male embryos. Taken together, our study demonstrates that the local WNT9B/β-catenin pathway provides the permissive signaling for androgen actions in Wolffian duct development. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:12 p.m. - 1:17 p.m.

PS-6 Nutrition During Early Pregnancy Impacts Offspring Ovarian Characteristics

Abstract During early pregnancy offspring are directly exposed to nutrients consumed by their mother, and the development of their own reproductive tract is underway. The objective of this research was to determine how characteristics of offspring ovaries were affected by different maternal rates of gain during the first trimester of gestation in beef heifers. Before breeding antral follicle counts were determined via ultrasound. Beginning at breeding Angus heifers were managed to achieve one of two rates of gain: low (0.20 kg/d, n = 8; LG) or moderate (0.75 kg/d, n = 8; MG) for the first trimester of pregnancy, after which they were managed as a single group through F1 calving. Calves remained with their dams until weaning and were managed as a single group through breeding. These F1 heifer calves were then synchronized and bred to a single sire using female sexed semen. On the 84th day of gestation (approximate heifer age 17 months) ovaries were removed from the reproductive tract, weighed, and visible antral follicles were counted. Cross-sections from each ovary were post-fixed and embedded in paraffin, then each of 3 sections (5 µm, with 10 sections between to avoid counting follicles more than once) were stained with Hematoxylin and Eosin for histological evaluation. Number of primordial, primary, secondary, and antral follicles were determined for each section. Data were analyzed using the GLM procedure of SAS with significance declared at P ≤ 0.05. The CORR procedure of SAS was used to calculate correlations between pre-breeding antral follicle counts, surface follicle counts, and histological follicle counts. Though no differences were observed in number of visible follicles (P = 0.45), the corpus luteum (CL) was heavier (P = 0.03) and average ovarian length was greater (P = 0.04) in offspring from LG dams compared with those from MG dams. No differences were observed in the number of primordial, primary, secondary, or antral follicles between treatments (P ≤ 0.18). There was a correlation between the number of histological follicles and surface follicles (r = 0.73; P = 0.002) and pre-breeding antral follicles and surface follicles (r = 0.60; P = 0.014), but there was no correlation between pre-breeding antral follicles and histological follicles (P = 0.10). Heavier CLs have a positive correlation with amount of progesterone released, which is essential for pregnancy maintenance. Longer lengths of ovaries suggest more area for follicles to develop, which is indicative of future reproductive success. Data corroborates previous reports regarding positive correlations between antral follicle count determined via ultrasound and counts of surface follicles on extracted ovaries. These findings demonstrate that nutrition during the first trimester of gestation impacts offspring reproductive tract development.

Expression of CSF1, AR, and SRD5A2 during Postnatal Development of the Boar Reproductive Tract.

Simple SummaryUnderstanding the initial development of the male reproductive system, including the prostate, should provide insight into malfunctions in the adult male. Although changes in circulating androgens during development are characterized in multiple species, potential changes in the androgen receptor, in the enzyme that converts testosterone to the presumably more potent dihydrotestosterone, and in colony stimulating factor 1, a critical mediator of macrophage influence on organ development, were previously unknown and anticipated to be influenced by androgens and estrogens. Gene expression in the testis, prostate, and seminal vesicles of these three mediators of development, including responses to reduced testosterone or estrogens, were evaluated. Each of these three genes had a unique temporal pattern of expression during postnatal reproductive tract development. However, surprisingly minimal effects of altered steroid signaling were reported on the expression of these presumed pivotal genes.The male reproductive system develops from a minimally functioning gonad and nonfunctioning accessory sex glands in the neonate; sex steroids, presumed to be primary influencers of these changes, have been characterized in multiple species. This study focused on the expression of the androgen receptor as the principal mediator of androgen-induced signaling; the 5α reductase enzyme that converts testosterone to the more active dihydrotestosterone; and colony stimulating factor 1, a mediator of macrophage influence on organ development in the pig. The time points chosen to evaluate normal developmental changes during the juvenile and prepubertal intervals included the inflection time points of 6.5 weeks of age at the nadir of circulating estradiol and testosterone concentrations in juveniles, and 11 weeks of age, when these concentrations begin to increase. The role of sex steroid signaling in the regulation of gene expression was evaluated by the blockade of androgen and estrogen receptors and reduction in endogenous estrogens. Expression of colony stimulating factor 1 in the testes gradually decreased during development; developmental profiles in the prostate and seminal vesicles were clearly different. Interference with sex steroid signaling had no effect on the expression of these three genes in testicular tissue and minimal and transient effects in prostate and seminal vesicles.

Folic Acid: Sources, Chemistry, Absorption, Metabolism, Beneficial Effects on Poultry Performance and Health.

Recently, there has been an increasing interest in the study of the effects of folic acid (FA) on poultry because it was observed that FA could overcome problems in poultry health while improving its performance. FA, or folate, is a water-soluble B vitamin essential in poultry, so FA intake must be available in the feed. Sources of FA in feed come from plants or animals, and animal sources have relatively more stable FA. The ingested FA will be absorbed in the intestinal lumen and transported into the liver through the blood vessels. Therefore, FA has a positive effect on the performance and health status of poultry. The effect of FA on poultry performance is to increase reproductive tract development, FA content in eggs, hatchability, weight gain, average initial body weight, feed intake, relative growth rate, chick body weight, breast fillet percentage, and reduce FCR and white striping score. At the same time, the effect on poultry health influences antioxidant activities, thyroid hormones, blood biochemicals, anti-inflammatory gene expressions, and immune responses. The present review deals with FA sources, chemistry, absorption, metabolism, effects on performance, and poultry health, which are based on valid basic information.

Development and characterization of human fetal female reproductive tract organoids to understand Müllerian duct anomalies

Müllerian ducts are paired tubular structures that give rise to most of the female reproductive organs. Any abnormalities in the development and differentiation of these ducts lead to anatomical defects in the female reproductive tract organs categorized as Müllerian duct anomalies. Due to the limited access to fetal tissues, little is understood of human reproductive tract development and the associated anomalies. Although organoids represent a powerful model to decipher human development and disease, such organoids from fetal reproductive organs are not available. Here, we developed organoids from human fetal fallopian tubes and uteri and compared them with their adult counterparts. Our results demonstrate that human fetal reproductive tract epithelia do not express some of the typical markers of adult reproductive tract epithelia. Furthermore, fetal organoids are grossly, histologically, and proteomically different from adult organoids. While external supplementation of WNT ligands or activators in culture medium is an absolute requirement for the adult reproductive tract organoids, fetal organoids are able to grow in WNT-deficient conditions. We also developed decellularized tissue scaffolds from adult human fallopian tubes and uteri. Transplantation of fetal organoids onto these scaffolds led to the regeneration of the adult fallopian tube and uterine epithelia. Importantly, suppression of Wnt signaling, which is altered in patients with Müllerian duct anomalies, inhibits the regenerative ability of human fetal organoids and causes severe anatomical defects in the mouse reproductive tract. Thus, our fetal organoids represent an important platform to study the underlying basis of human female reproductive tract development and diseases.