Ovarian Development Research Articles

Synergistic cooperation between the β-catenin and SF1 regulates progesterone synthesis in laying hen ovarian granulosa cells

The development of ovarian follicles in poultry is a key factor affecting the performance of egg production. Ovarian follicle development is regulated via the Wnt/β-catenin signaling pathway, and β-catenin, encoded by CTNNB1, is a core component of this pathway. In this study, using ovary GCs from laying hens, we investigated the regulatory role of CTNNB1 in steroid synthesis. We found that CTNNB1 significantly regulates the expression of StAR and CYP11A1 (key genes related to progesterone synthesis) and the secretion of progesterone (P4). Furthermore, simultaneous overexpression of CTNNB1 and SF1 resulted in significantly higher levels of CYP11A1 and secretion of P4 than in cells overexpressing CTNNB1 or SF1 alone. We also found that in GCs overexpressing SF1, levels of CYP11A1 and secreted P4 were significantly greater than in controls. Silencing of CYP11A1 resulted in the inhibition of P4 secretion while overexpression of SF1 in CYP11A1-silenced cells restored P4 secretion to normal levels. Together, these results indicate that synergistic cooperation between the β-catenin and SF1 regulates progesterone synthesis in laying hen ovarian hierarchical granulosa cells to promote CYP11A1 expression.

Landscape transcriptomic analysis of bovine follicular cells during key phases of ovarian follicular development

BackgroundThere are many gaps in our understanding of the mechanisms involved in ovarian follicular development in cattle, particularly regarding follicular deviation, acquisition of ovulatory capacity, and preovulatory changes. Molecular evaluations of ovarian follicular cells during follicular development in cattle, especially serial transcriptomic analyses across key growth phases, have not been reported. This study aims to address this gap by analyzing gene expression using RNA-seq in granulosa and antral cells recovered from ovarian follicular fluid during critical phases of ovarian follicular development in Holstein cows.ResultsIntegrated analysis of gene ontology (GO), gene set enrichment (GSEA), protein–protein interaction (PPI), and gene topology identified that differentially expressed genes (DEGs) in the largest ovarian follicles at deviation (Dev) were primarily involved in FSH-negative feedback, steroidogenesis, cell proliferation, apoptosis, and the prevention of early follicle rupture. In contrast, DEGs in the second largest follicles (DevF2) were mainly related to loss of cell viability, apoptosis, and immune cell invasion. In the dominant (PostDev) and preovulatory (PreOv) follicles, DEGs were associated with vascular changes and inflammatory responses.ConclusionsThe transcriptome of ovarian follicular fluid cells had a predominance of granulosa cells in the dominant follicle at deviation, with upregulation of genes involved in cell viability, steroidogenesis, and apoptosis prevention, whereas in the non-selected follicle there was upregulation of cell death-related transcripts. Immune cell transcripts increased significantly after deviation, particularly in preovulatory follicles, indicating strong intrafollicular chemotactic activity. We inferred that immune cell invasion occurred despite an intact basal lamina, contributing to follicular maturation.Graphical

Toxic effects of microplastic and nanoplastic on the reproduction of teleost fish in aquatic environments.

Microplastics and nanoplastics are widely present in aquatic environments and attract significant scholarly attention due to their toxicity, persistence, and ability to cross biological barriers, which pose substantial risks to various fish species. Microplastics and nanoplastics can enter fish through their digestive tract, gills and skin, causing oxidative damage to the body and adversely affecting their reproductive system. Given that fish constitute a crucial source of high-quality protein for humans, it is necessary to study the impact of microplastics on fish reproduction in order to assess the impact of pollutants on ecology, biodiversity conservation, environmental sustainability, and endocrine disruption. This review explores the reproductive consequences of microplastics and nanoplastics in fish, examining aspects such as fecundity, abnormal offspring, circadian rhythm, gonad index, spermatocyte development, oocyte development, sperm quality, ovarian development, and changes at the molecular and cellular level. These investigations hold significant importance in environmental toxicology.

Trehalose metabolism mediates trade-offs between reproduction and survival in beet webworm, Loxostege sticticalis, under heat stress.

Temperature is an important determinant of developmental and reproductive rates in insects. Here, we investigated the physiological responses of adult beet webworm, Loxostege sticticalis L. (Lepidoptera: Crambidae), to three temperatures (16, 23 and 30 °C) focusing on trehalose metabolism. Exposure of moths to 30 °C accelerated eclosion and ovarian development, but shortened the oviposition period and adult longevity, whereas exposure to 16 °C had opposite effects. Transcriptome analysis revealed that vitellogenin (VG) and vitellogenin receptor (VR) genes were up-regulated at 30 °C, as were numerous genes related to energy metabolism, including those involved in the insulin signaling pathway, the tricarboxylic acid (TCA) cycle, and glycolysis. Expression of the trehalose transporter gene TRET1 was also induced at high temperature, primarily in the ovaries, where trehalose content increased, accompanied by lipid degradation in the fat body. Treatment with the trehalase inhibitor validamycin A reduced female fecundity and longevity at 23 °C, but enhanced the expression of genes related to stress resistance and reproduction, mimicking the effect of high temperature. Besides their practical utility for predicting the oviposition behavior and geographic distribution of L. sticticalis in the field, these results elucidate the various physiological roles of trehalose in L. sticticalis during exposure of moths to high temperature and may provide insights into the relationship between stress resistance and reproduction in insects more generally. © 2024 Society of Chemical Industry.

Comparative study of the reproductive activity of Calliptamus barbarus (Costa, 1839) and Calliptamus wattenwylianus (Pantel, 1836) in a subhumid bioclimatic region

The reproductive activity of Calliptamus barbarus and Calliptamus wattenwylianus was investigated to understand population dynamics and reproductive potential in a subhumid bioclimatic zone. Specimens were collected from July to October and dissected to assess ovarian development and egg-laying activity. The number of ovarioles, determined at birth, ranged from 62 to 67 for C. wattenwylianus and 48 to 58 for C. barbarus. Higher fertility was observed in C. wattenwylianus, with egg production increasing during the first oviposition for both species. The average reproductive yield was notable for C. barbarus (85.99%) and C. wattenwylianus (86.56%). Oocyte resorption occurred mainly during drought (August-September) and cold periods (October-November). C. wattenwylianus females reached maturity earlier, beginning in August, compared to C. barbarus. Egg production began in July for C. wattenwylianus and in September for C. barbarus. Oocyte retention rates were 5% for C. barbarus and 9% for C. wattenwylianus. These findings provide insights into reproductive patterns in North African Orthoptera, with implications for pest control in subhumid regions.

Energy Reserve Allocation in the Trade-Off between Migration and Reproduction in Fall Armyworm.

Striking a trade-off between migration and reproduction becomes imperative during long-range migration to ensure proper energy allocation. However, the mechanisms involved in this trade-off remain poorly understood. Here, we used a takeoff assay to distinguish migratory from non-migratory individuals in the fall armyworm, which is a major migratory insect worldwide. Migratory females displayed delayed ovarian development and flew further and faster than non-migratory females during tethered flight. Transcriptome analyses demonstrated an enrichment of fatty acid genes across successive levels of ovarian development and different migratory behaviors. Additionally, genes with roles in phototransduction and carbohydrate digestion along with absorption function were enriched in migratory females. Consistent with this, we identified increased abdominal lipids in migratory females that were mobilized to supply energy to the flight muscles in the thorax. Our study reveals that the fall armyworm faces a trade-off in allocating abdominal triglycerides between migration and reproduction during flight. The findings provide valuable insights for future research on this trade-off and highlight the key energy components involved in this strategic balance.

Reproductive performance of lumpfish (Cyclopterus lumpus, L. 1758) females: Effects of integrated photoperiod and temperature manipulations on sexual maturation and spawning.

A successful control of sexual maturation is crucial for year-round production of lumpfish juveniles destined as cleaner fish in Atlantic salmon aquaculture. This study investigated the combined effects of photoperiod and temperature manipulations on sexual maturation and spawning in lumpfish females. Lumpfish juveniles were exposed to simulated natural and nine-month compressed annual photoperiods, with subsequent temperature elevation. Body weight (BW), condition factor (K), gonadosomatic index (GSI), ovarian development, plasma levels of 17β-estradiol (E2), testosterone (T) and 11-ketotestosterone (11-KT), and spawning were assessed. Compressing the natural photoperiod caused a clear increase and decrease in GSI, T, 11-KT and E2 towards and during the spawning period. Before the temperature elevation, GSI, T, 11-KT, E2 and ovarian development were advanced in the compressed photoperiod. After the temperature elevation, GSI, T, 11-KT and E2 fluctuated more in the compressed photoperiod, while in the natural photoperiod, E2 declined, and GSI, T and 11-KT increased. Spawning was advanced by 1 month in the compressed photoperiod compared to the natural photoperiod. Temperature elevation led to higher levels, earlier peaks and declines of T, 11-KT or E2 in both photoperiods, and advanced spawning by 1.5 months in the compressed photoperiod compared to the natural photoperiod. Temperature elevation also led to increased ovulation recruitment and increased cumulative weight of spawned eggs in the natural photoperiod. Compressing the natural photoperiod and elevating temperature can thus advance sexual maturation and spawning in lumpfish females. Due to the lower amounts of spawned egg weights in the high temperature compressed photoperiod, further studies on effects of photoperiod and timing of temperature manipulations on spawning, fecundity and egg quality could optimize the photothermal manipulations on lumpfish broodstock.

The N6-methyladenosine landscape of ovarian development and aging highlights the regulation by RNA stability and chromatin state.

The versatile epigenetic modification known as N6-methyladenosine (m6A) has been demonstrated to be pivotal in numerous physiological and pathological contexts. Nonetheless, the precise regulatory mechanisms linking m6A to histone modifications and the involvement of transposable elements (TEs) in ovarian development and aging are still not completely understood. First, we discovered that m6A modifications are highly expressed during ovarian aging (OA), with significant contributions from decreased m6A demethylase FTO and overexpressed m6A methyltransferase METTL16. Then, using FTO knockout mouse model and KGN cell line, we also observed that FTO deletion and METTL16 overexpression significantly increased m6A levels. This led to the downregulation of the methyltransferase SUV39H1, resulting in reduced H3K9me3 expression. The downregulation of SUV39H1 and H3K9me3 primarily activated LTR7 and LTR12, subsequently activating ERV1. This resulted in a decrease in cell proliferation, while the levels of apoptosis, cellular aging markers, and autophagy markers significantly increased in OA. In summary, our study offers intriguing insights into the role of m6A in regulating DNA epigenetics, including H3K9me3 and TEs, as well as autophagy, thereby accelerating OA.

Nur77 ameliorates cyclophosphamide-induced ovarian insufficiency in mice by inhibiting oxidative damage and cell senescence

Premature ovarian failure (POF) is among the primary causes of ovarian dysfunction that severely affects women’s physical and mental health. The main purpose of this study was to explore the expression level of Nerve growth factor-induced protein B (Nur77/NR4A1) in cyclophosphamide (CTX)-induced POF. We then tested whether Nur77 can exert a protective effect after CTX treatment and investigated the mechanism of Nur77’s role during ovarian injury. CTX promotes follicular atresia by inducing redox imbalance, apoptosis, and senescence, thereby causing direct toxicity to gonads. Additionally, CTX decreases ovarian reserve consumption by stimulating the excessive activation of primordial follicles. Nur77 can be stimulated by oxidative stress, DNA damage, metabolism, inflammation, etc. However, its relationship with POF remains unelucidated. We here found that Nur77 is expressed at low levels in POF ovaries. Therefore, Nur77 was identified as a regulator of ovarian injury and follicular development. According to the results, Nur77 overexpression alleviated redox imbalances, reduced cell senescence and apoptosis, and improved follicular reserve. Nur77 protects ovarian function by restoring disordered sex hormone levels and estrus cycles and promoting follicle growth and development at all levels. Moreover, the rapamycin protein kinase (AKT)/mammalian target of the rapamycin (mTOR) is a crucial regulator of the primordial follicle pool and follicular development. A relationship was observed between Nur77 and AKT through string and molecular docking. Experiments confirmed the involvement of the AKT/mTOR signaling pathway in the regulatory role of Nur77 in ovarian function. Thus, Nur77 is a critical target for POF prevention and treatment.

Functional characterisation of human recessive DIS3 variants in premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is characterised by the loss or complete absence of ovarian activity in women under the age of 40. Clinical presentation of POI varies with phenotypic severity ranging from premature loss of menses to complete gonadal dysgenesis. POI is genetically heterogeneous with >100 causative gene variants identified thus far. The aetiology of POI varies from syndromic, idiopathic, monogenic to autoimmune causes the condition. Genetic diagnoses are beneficial to those impacted by POI as it allows for improved clinical management and fertility preservation. Identifying novel variants in candidate POI genes, however, is insufficient to make clinical diagnoses. The impact of missense variants can be predicted using bioinformatic algorithms but computational approaches have limitations and can generate false positive and false negative predictions. Functional characterisation of missense variants, is therefore imperative, particularly for genes lacking a well-established genotype:phenotype correlation. Here we used whole-exome sequencing (WES) to identify the first case of a homozygous missense variant in DIS3 (c.2320C > T; p.His774Tyr) a critical component of the RNA exosome in a POI patient. This adds to the previously described compound heterozygous patient. We perform the first functional characterisation of a human POI-associated DIS3 variant. A slight defect in mitotic growth was caused by the variant in a Saccharomyces cerevisiae model. Transgenic rescue of Dis3 knockdown in Drosophila melanogaster with human DIS3 carrying the patient variant led to aberrant ovarian development and egg chamber degeneration. This supports a potential deleterious impact of the human c.2320C > T; p.His774Tyr variant.

Sublethal concentration of λ-cyhalothrin inhibits insulin-like peptides and leads to reproductive toxicity in Chrysoperla sinica.

Insulin-like peptides (ILPs) act as crucial reproductive neuropeptides in insects, regulating insect reproduction through the insulin signaling pathway (ISP). Our previous studies have found that the sublethal concentrations (LC1 and LC10) of lambda-cyhalothrin (λCy) could induce severe reproductive toxicity in the lacewing, Chrysoperla sinica (Tjeder), but the toxicological mechanism remains unclear. This study discovered that λCy could inhibit CsILP transcription, leading to a decrease in insulin content and downregulation of C. sinica insulin receptor (CsInR) and C. sinica forkhead box O (CsFOXO) expression in ISP. Interference with CsILP expression resulted in downregulation of C. sinica vitellogenin (CsVg) and decreasing fecundity, while exogenous injection of bovine insulin promoted upregulation of CsVg expression and facilitated reproduction in female adults of C. sinica. Meanwhile, interference with FOXO of ILP downstream transcription factor could lead to downregulation of CsVg, hindering ovarian development and resulting in a decrease in egg production. However, exogenous injection of bovine insulin could remedy the effects caused by FOXO interference. In addition, ILP mediates juvenile hormone and 20-hydroxyecdysone biosynthesis by acting on their synthetic regulatory enzymes and influences the signal transduction of the 2 reproductive endocrine hormones, thereby regulating the reproductive endocrine environment in C. sinica. In conclusion, λCy inhibits CsILP expression, leading to disorder of ISP, leading to the reduced fecundity of C. sinica.

Genetic Diversity Losses in Mantis Shrimp Oratosquilla oratoria Offspring Created for Supportive Releases Using an Ecological Breeding in Earthen Pond Method

ABSTRACTArtificial breeding of mantis shrimp Oratosquilla oratoria, using the ecological breeding in earthen pond (EBEP) method, has been carrying out from 2019, and the produced larvae have always been used for supportive breeding programs in Bohai Bay. To assess the degree of fidelity of enhancement programs used an EBEP method to native genetic pools, genetic diversity at 10 microsatellite loci was analyzed in three hatchery offspring populations, their wild parent stocks, and their wild contemporaneous offspring. Significant decrease in the almost genetic diversity parameters and highly significant differences in shannon's information index (decreasing by 22.54%–40.97%) and allelic richness (33.80%–46.87%) were observed between in hatchery populations. This further created relatively more genetic differentiation between the hatchery and wild population, as the pairwise Fst values and genetic distances showed. The mean pairwise relatedness coefficients showed higher values within hatchery populations and lower values within wild populations. Hatchery populations contained the fewest full‐sib families and offspring were extremely distributed in 10–11 full‐sib groups in each hatchery population. The relatively small number of actual spawning females and large variance in parental contribution to the next generation possibly causes allele reduction in the hatchery population, which in turn leads to a decrease in allelic diversity and an increase in genetic similarities among individuals. Promoting synchrony of ovarian development in females, providing good conditions for incubation and larvae culture and balancing the contributions of parents will be important in future breeding propagation.

Mating behaviour and cholesterol nutritional strategies promoted ovarian development of female swimming crab (Portunus trituberculatus).

Female crabs enter a stage of rapid ovarian development after mating, and cholesterol is a substrate for steroid hormone synthesis. Therefore, in this experiment, an 8-week feeding trial was conducted to investigate the effects of mating treatments (mated crab and unmated crab) and three dietary cholesterol levels (0·09 %, 0·79 % and 1·40 %) on ovarian development, cholesterol metabolism and steroid hormones metabolism of adult female swimming crab (Portunus trituberculatus). The results indicated that crabs fed the diet with 0·79 % cholesterol significantly increased gonadosomatic index (GSI) and vitellogenin (VTG) content than other treatments in the same mating status. Moreover, mated crabs had markedly increased GSI and VTG content in the ovary and hepatopancreas than unmated crabs. The histological observation found that exogenous vitellogenic oocytes appeared in the mated crabs, while previtellogenic oocytes and endogenous vitellogenic oocytes were the primary oocytes in unmated crabs. The transmission electron microscopy analysis showed that when fed diet with 0·79 % cholesterol, the unmated crabs contained more rough endoplasmic reticulum and mated crabs had higher yolk content than other treatments. Furthermore, mating treatment and dietary 0·79 % cholesterol level both promoted cholesterol deposition by up-regulation of the mRNA and protein expression levels of class B scavenger receptors 1 (Srb1), while stimulating the secretion of steroid hormones by up-regulation of the mRNA and protein expression of steroidogenic acute regulatory protein (Star). Overall, the present results indicated that mating behaviour plays a leading role in promoting ovarian development, and dietary 0·79 % cholesterol level can further promote ovarian development after mating.

Distribution of genotypes by the growth diferential factor 9 (GDF-9) gene locus in the romanov breed sheep population

The FecG gene, also known as GDF-9 (growth factor differentiation factor 9), plays an important role in regulating reproductive function in mammals. This paper presents the results of a study of a point mutation in the differential growth factor gene in Romanov breed sheep. Mutations in exon 1 of the GDF-9 gene can lead to disturbances in ovarian development and ovulation, resulting in infertility or reduced reproductive capacity in animals. The object of the study was Romanov breed sheep in a sample of 30 individuals. For DNA extraction, venous blood samples were taken from the jugular vein. In Romanov breed sheep for the GDF-9 gene, it was found that at the G1 locus of this gene there are two alleles – G and A. Three genotypes were also identified: GG (254 bp/117 bp), AG (410bp/254/117 bp) and AA (410 bp/410 bp). The observed χ2 criterion was less than expected, therefore, the empirical and theoretical frequencies do not differ significantly, and deviations from the distribution according to the Hardy-Weinberg law are not observed. The observed heterozygosity index (Ho) was 0.27. Allele frequencies were 0,8 for G and 0,2 for A. For genotypes the following were obtained: GG – 0,66, GA – 0,27 and AA – 0,07.It can be noted that the data on the frequencies of alleles and genotypes in the population of Romanov breed sheep differ from the results for the Kulunda fine-wool, Dagestan mountain, Salsk, Lacaune and Merino breeds. The research results can be used to monitor changes in allele frequencies and genotypes in sheep during the selection process.

Role of red pigment concentrating hormone in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain

The eyestalk ganglion is a predominant neuroendocrine organ in crustaceans that synthesize crustacean hyperglycemic hormone (CHH) family participating in physiological activities such as ovarian development, glucose homeostasis, and molting. Red pigment concentrating hormone (RPCH) contributes to a diversity of physiological influences, including ovarian development; however, its role in modulating CHH-family neuropeptides in the eyestalk ganglion remains unclear. In this study, we characterized the putative RPCH receptor (RPCHR) in the mud crab Scylla paramamosain and revealed that it was expressed in a variety of tissues, including the eyestalk ganglion. In vivo administration of mature RPCH peptide significantly restrained vitellogenesis inhibiting hormone (VIH) expression and significantly enhanced RPCHR, crustacean hyperglycemic hormone (CHH) and molt inhibiting hormone (MIH) gene expression, while in contrast, in vivo injection of RPCH-dsRNA for knockdown dramatically enhanced VIH expression and decreased RPCHR, CHH and MIH expression in the eyestalk ganglion. The present study comprehensively reports that RPCH may be responsible for the regulation of ovarian maturation, glucose homeostasis, and molting in S. paramamosain through the modulation of CHH family neuropeptides in the eyestalk ganglion.

In vivo treatment with a non-aromatizable androgen rapidly alters the ovarian transcriptome of previtellogenic secondary growth coho salmon (Onchorhynchus kisutch).

Recent evidence suggests that androgens are a potent driver of growth during late the primary stage of ovarian follicle development in teleosts. We have previously shown that the non-aromatizable androgen, 11-ketotestosterone (11-KT), both advances ovarian follicle growth in vivo and dramatically alters the primary growth ovarian transcriptome in coho salmon. Many of the transcriptomic changes pointed towards 11-KT driving process associated with the transition to a secondary growth phenotype. In the current study, we implanted previtellogenic early secondary growth coho salmon with cholesterol pellets containing 11-KT and performed RNA-Seq on ovarian tissue after 3 days in order to identify alterations to the ovarian transcriptome in early secondary growth. We identified 8,707 contiguous sequences (contigs) that were differentially expressed (DE) between control and 11-KT implanted fish and were able to collapse those to 3,853 gene-level IDs, more than a 3-fold more DE contigs than at the primary growth stage we reported previously. These contigs included genes encoding proteins involved in steroidogenesis, vitellogenin and lipid uptake, follicle stimulating hormone signaling, growth factor signaling, and structural proteins, suggesting androgens continue to promote previtellogenic secondary growth.

The conserved role of miR-2 and novel miR-109 in the increase in fecundity of Diaphorina citri induced by symbiotic bacteria and pathogenic fungi.

Infection with pathogens can increase the fecundity and other fitness-related traits of insect vectors for their own advantage. Our previous research has reported that DcKr-h1 plays a critical role in the increase in fecundity of Diaphorina citri induced by the bacterium, "Candidatus Liberibacter asiaticus" (CLas) and the fungus, Cordyceps fumosorosea (Cf). However, the posttranscriptional regulation of this process remains poorly understood. Given the significance of miRNAs in gene regulation, we delved into their roles in shaping phenotypes and their underlying molecular mechanisms. Our results indicated that two miRNAs, miR-2 and novel-miR-109, jointly inhibited DcKr-h1 expression by binding to its 3' untranslated region (UTR). In both D. citri-CLas and D. citri-Cf interactions, the increased juvenile hormone (JH) titer and reduced abundance of miR-2 and novel-miR-109 ensure high levels of DcKr-h1 expression, consequently stimulating ovarian development and enhancing fecundity. These observations provide evidence that miR-2 and miR-109 are crucial players in the JH-dependent increase in fecundity in psyllids induced by infection with different pathogens.

8795 Impact of Retinoic Acid Receptor Alpha Conditional Knockout on Ovarian Follicle Development in the Mouse

Abstract Disclosure: Z. Bogin: None. A. Keisker: None. A. Urbanowski: None. C. White: None. J.L. Kipp: None. The ovaries are the primary endocrine and reproductive organs in females. The ovarian follicle is the basic structural and functional unit of the ovary. When there is abnormal development in ovarian follicles, infertility and reproductive diseases such as polycystic ovarian syndrome (PCOS), premature ovarian failure (POF), and ovarian cancers may occur. Retinoic acid (RA), a biologically active derivative of vitamin A, is a signaling molecule that plays a vital role in various physiological processes, including embryonic development, tissue differentiation, and reproduction. We have shown that RA stimulates granulosa cell proliferation through a cell signaling cascade involving Retinoic Acid Receptors (RARs), which have three isoforms alpha, beta, and gamma. In this study, we characterized a new retinoic acid receptor alpha conditional knockout (RARA cKO) mouse model with RARA deleted in granulosa cells during embryonic development. The body weight and uterus weight of the RARA cKO mice were decreased at week 7 but returned to normal at week 15 and 12 months. The animals were either sub-fertile or infertile with phenotypic evidence of POF. In addition, a variety of ovarian pathologies were observed, including the presence of hemosiderin, increased cyst formation, and abnormal follicle counts. Overall, this study provides evidence of the involvement of retinoic acid signaling, mediated by RARA, in ovarian development and fertility. This study will help to better understand the prevention and treatment of ovarian diseases. Presentation: 6/2/2024

8795 Impact of Retinoic Acid Receptor Alpha Conditional Knockout on Ovarian Follicle Development in the Mouse

Abstract Disclosure: Z. Bogin: None. A. Keisker: None. A. Urbanowski: None. C. White: None. J.L. Kipp: None. The ovaries are the primary endocrine and reproductive organs in females. The ovarian follicle is the basic structural and functional unit of the ovary. When there is abnormal development in ovarian follicles, infertility and reproductive diseases such as polycystic ovarian syndrome (PCOS), premature ovarian failure (POF), and ovarian cancers may occur. Retinoic acid (RA), a biologically active derivative of vitamin A, is a signaling molecule that plays a vital role in various physiological processes, including embryonic development, tissue differentiation, and reproduction. We have shown that RA stimulates granulosa cell proliferation through a cell signaling cascade involving Retinoic Acid Receptors (RARs), which have three isoforms alpha, beta, and gamma. In this study, we characterized a new retinoic acid receptor alpha conditional knockout (RARA cKO) mouse model with RARA deleted in granulosa cells during embryonic development. The body weight and uterus weight of the RARA cKO mice were decreased at week 7 but returned to normal at week 15 and 12 months. The animals were either sub-fertile or infertile with phenotypic evidence of POF. In addition, a variety of ovarian pathologies were observed, including the presence of hemosiderin, increased cyst formation, and abnormal follicle counts. Overall, this study provides evidence of the involvement of retinoic acid signaling, mediated by RARA, in ovarian development and fertility. This study will help to better understand the prevention and treatment of ovarian diseases. Presentation: 6/2/2024

8053 A Ligand-Based Differentiation Protocol for Human Induced Pluripotent Stem Cells Recapitulates Granulosa Cell Development and Induces Steroidogenic Pathway Genes

Abstract Disclosure: H. Kubo: None. M.M. Laronda: None. Sex determination is a developmental process that defines the gonad as a testis or ovary, and influences sex hormone production. While key signaling events are well conserved across mammals, there can be differences between species. Therefore, there is a need to develop a human model to fully understand this critical process in humans. We have developed a ligand-based differentiation protocol for human induced pluripotent stem cells (hiPSCs) to generate granulosa-like cells (GLCs). Additionally, hiPSC-derived GLCs could become the foundation for a cell-based hormone replacement therapy that recapitulates cyclicity, physiologic levels of hormones, and production of peptide hormones that are not included in currently available hormone replacement therapies. We aim to gain further understanding of the key factors and culture conditions necessary for functional GLCs that produce ovarian hormones, which includes maintained expression of FOXL2. We used our monolayer differentiation protocol that includes WNT-agonist CHIR99021 treatment for 2 days, followed by a combination treatment of BMP4, FST, and DKK1-inhibitor Gallocyanine for 3 days. We have tested our protocol on three different hiPSC lines: IISH2i-BM9s, STiPS-A3, and STiPS-A6. Expression of pluripotency marker POU5F1 is reduced during differentiation, while expression of the intermediate mesoderm markers PAX2, LHX1, and WT1 and bipotential gonad marker GATA4 peak at day 2. Peak expression of the bipotential gonad marker NR5A1 occurred at day 2 in the STiPS-A6 cells, but at day 5 in IISH2i-BM9 and STiPS-A3 cells. Interestingly, FOXL2 expression peaks at day 2 in all three lines and drops by day 5. In the IISH2i-BM9s, FOXL2 protein peaks at day 2 by Jess Western while there is the highest percentage of FOXL2-positive cells at day 5 by immunofluorescence. In murine granulosa cells, FOXL2 maintenance depends on NR5A2. NR5A2 expression drops in the GLCs, which suggests that NR5A2 loss may be preventing FOXL2 maintenance. Additionally, as FOXL2 is required for expression of aromatase and HSD17B1, we are using this model to interrogate NR5A2 upstream of FOXL2 and control hormone production. CYP19A1 and HSD17B1 along with AMH are induced during this differentiation protocol, suggesting these GLCs can be steroidogenic and produce peptide hormones. Additionally, scRNA-seq analysis of the GLCs is underway to identify types of granulosa cells produced by this protocol. Future directions include optimizing GLC culture conditions to generate hormones and to test if NR5A2 inhibitors alter FOXL2 expression. Put together, the data suggests that cells differentiated with our protocol show promise as a human model of ovarian development and a foundation for a personalized cell-based hormone replacement therapy. This work is supported by the NIH/NICHD R01HD104683 (MML), HuBMAP U01HD110336 (MML), and Gesualdo Family Research Scholar (MML). Presentation: 6/2/2024