The Orphan Nuclear Receptors Steroidogenic Factor-1 and Liver Receptor Homolog-1: Structure, Regulation, and Essential Roles in Mammalian Reproduction.

Nuclear receptors are intracellular proteins that act as transcription factors. Proteins with classic nuclear receptor domain structure lacking identified signaling ligands are designated orphan nuclear receptors. Two of these, steroidogenic factor-1 (NR5A1, also known as SF-1) and liver receptor homolog-1 (NR5A2, also known as LRH-1), bind to the same DNA sequences, with different and nonoverlapping effects on targets. Endogenous regulation of both is achieved predominantly by cofactor interactions. SF-1 is expressed primarily in steroidogenic tissues, LRH-1 in tissues of endodermal origin and the gonads. Both receptors modulate cholesterol homeostasis, steroidogenesis, tissue-specific cell proliferation, and stem cell pluripotency. LRH-1 is essential for development beyond gastrulation and SF-1 for genesis of the adrenal, sexual differentiation, and Leydig cell function. Ovary-specific depletion of SF-1 disrupts follicle development, while LRH-1 depletion prevents ovulation, cumulus expansion, and luteinization. Uterine depletion of LRH-1 compromises decidualization and pregnancy. In humans, SF-1 is present in endometriotic tissue, where it regulates estrogen synthesis. SF-1 is underexpressed in ovarian cancer cells and overexpressed in Leydig cell tumors. In breast cancer cells, proliferation, migration and invasion, and chemotherapy resistance are regulated by LRH-1. In conclusion, the NR5A orphan nuclear receptors are nonredundant factors that are crucial regulators of a panoply of biological processes, across multiple reproductive tissues.

Steroidogenic factor 1 (SF-1; Nr5a1) regulates the formation of the ovarian reserve.

The ovarian follicle reserve, formed pre- or perinatally, comprises all oocytes for lifetime reproduction. Depletion of this reserve results in infertility. Steroidogenic factor 1 (SF-1; Nr5a1) and liver receptor homolog 1 (LRH-1; Nr5a2) are two orphan nuclear receptors that regulate adult endocrine function, but their role in follicle formation is unknown. We developed models of conditional depletion of SF-1 or LRH-1 from prenatal ovaries. Depletion of SF-1, but not LRH-1, resulted in dramatically smaller ovaries and fewer primordial follicles. This was mediated by increased oocyte death, resulting from increased ovarian inflammation and increased Notch signaling. Major dysregulated genes were Iroquois homeobox 3 and 5 and their downstream targets involved in the establishment of the ovarian laminin matrix and oocyte-granulosa cell gap junctions. Disruptions of these pathways resulted in follicles with impaired basement membrane formation and compromised oocyte-granulosa communication networks, believed to render them more prone to atresia. This study identifies SF-1 as a key regulator of the formation of the ovarian reserve.

The role of steroidogenic factor 1 (SF-1) in steroidogenic cell function of the testes and ovaries of mature mice.

In brief: The nuclear receptor steroidogenic factor 1 (SF-1) is essential for mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and depletion of SF-1 in steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility. Abstract: Steroidogenic factor 1 (SF-1 or NR5A1) plays an essential role in the development of fetal gonads and regulates genes involved in steroid biosynthesis. Since SF-1 is expressed in multiple cell types in mouse gonads, we developed three novel conditional knockout (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (Nr5a1f/f) to identify its role in testes and ovaries of mature mice: Cytochrome P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f, Leydig and theca cell-specific), aromatase (Cyp19Cre/+;Nr5a1f/f, Sertoli and granulosa cell-specific), as well as a combination of both (Cyp17+Cyp19-Cre;Nr5a1f/f). Compared to control animals, Cyp19-Cre;Nr5a1f/f cKO males showed normal fertility and testicular function. The Cyp17Cre/+;Nr5a1f/f cKO males had smaller testis, with drastically reduced Leydig cell volumes and impaired steroidogenesis, though their reproductive performance remained comparable to controls. Some 50% of Cyp17Cre/++Cyp19Cre/+;Nr5a1f/f double-cKO (dKO) males were infertile, while the remaining 50% showed significantly reduced fertility. These dKO males also had smaller testis with degenerative seminiferous tubules, abnormal Leydig cell morphology and lower levels of intra-testicular testosterone. Abnormal Sertoli cell localization was noted in dKO testes, with increased Sox9, p27 and inhibin subunit ßb and decreased androgen receptor expression. Female mice from all genotypes showed normal reproductive capacity, though steroidogenic gene expression levels were significantly decreased in both Cyp17Cre/+;Nr5a1f/f cKO and dKO females. These results show the essential role of SF-1 in mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and that depletion SF-1 in all steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.

Liver receptor homolog 1 (LRH-1) regulates follicle vasculature during ovulation in mice.

In brief: It is well-established that liver receptor homolog 1 (LRH-1/NR5A2) regulates the ovarian function and is required for ovulation and luteinization in mice. In the present experiment, we showed that LRH-1 is required to control vascular changes during ovulation, a novel mechanism of action of this orphan nuclear receptor. Abstract: Liver receptor homolog 1 (LRH-1/NR5A2) is a key regulator of ovarian function, and recently, it has been suggested that it may regulate changes in follicular angiogenesis, an important event during the ovulatory process and luteal development. In the present experiment, the objective was to determine whether conditional depletion of LRH-1 in mice granulosa cells modified vascular changes during the periovulatory period and to explore the possible mechanisms of this modification. We generated mice (22- to 25-day-old) with specific depletion of LRH-1 in granulosa cells by crossing Lrh1 floxed (Lrh1 f/f) mice with mice expressing Cre-recombinase driven by the anti-Müllerian type II receptor (Amhr2-cre; conditional knockout or cKO mice). We showed that preovulatory follicles of LRH-1 cKO mice had a reduced number of endothelial cells in the theca cell layer at 8 h after human chorionic gonadotropin treatment compared with control (CON) mice. Additionally, mRNA and protein expression of leptin receptor (LEPR), a protein that stimulates angiogenesis in a vascular endothelial growth factor-A (VEGFA)-dependent manner, and teratocarcinoma-derived growth factor-1 (TDGF1), which may directly stimulate endothelial cell function, were reduced in LRH-1 cKO mice as compared to CON after the LH surge. These results showed that LRH-1 is necessary for the correct vascular changes that accompany ovulation in mice and that this effect may be regulated through VEGFA-dependent and VEGFA-independent pathways mediated by LEPR and TDGF1.

Orphan nuclear receptors in angiogenesis and follicular development.

Orphan nuclear receptors (ONRs) are a subset of the nuclear receptor family that lacks known endogenous ligands. Among 48 nuclear receptors identified in humans, 25 are classified as ONRs. They function as transcription factors and control the expression of a wide range of genes to regulate metabolism, fertility, immunity, angiogenesis, and many other functions. Angiogenic factors are essential during ovarian follicle development, including follicle growth and ovulation. The correct development of blood vessels contributes to preantral and antral follicular development, selection of the dominant follicle or follicles, follicular atresia, and ovulation. Although progress has been made in understanding the molecular mechanisms that regulate follicular angiogenesis, the role of ONRs as regulators is not clear. Based on their functions in other tissues, the ONRs NR1D1 (REV-ERBß), NR2C2 (TR4), NR2F2 (COUP-TF-II) and NR3B1, 2, and 3 (ERRα, ERRß and ERRγ) may modulate angiogenesis during antral follicle development. We hypothesize that this is achieved by effects on the expression and function of VEGFA, ANGPT1, THBS1, and soluble VEGFR1. Further, angiogenesis during ovulation is expected to be influenced by ONRs. NR5A2 (LRH-1), which is required for ovulation, regulates angiogenic genes in the ovary, including VEGFA and the upstream regulator of angiogenesis, PGE2. These angiogenic molecules may also be regulated by NR5A1 (SF-1). Evidence from outside the reproductive tract suggests that NR2F2 and NR4A1(NUR77) promote VEGFC and PGF, respectively, and NR4As (NUR77, NOR1) seem to be necessary for the angiogenic effects of VEGFA and PGE2. Together, the data suggest that ONRs are important regulators of follicular angiogenesis.

New functions for old factors: the role of polyamines during the establishment of pregnancy.

Implantation is essential for the establishment of a successful pregnancy, and the preimplantation period plays a significant role in ensuring implantation occurs in a timely and coordinated manner. This requires effective maternal-embryonic signalling, established during the preimplantation period, to synchronise development. Although multiple factors have been identified as present during this time, the exact molecular mechanisms involved are unknown. Polyamines are small cationic molecules that are ubiquitously expressed from prokaryotes to eukaryotes. Despite being first identified over 300 years ago, their essential roles in cell proliferation and growth, including cancer, have only been recently recognised, with new technologies and interest resulting in rapid expansion of the polyamine field. This review provides a summary of our current understanding of polyamine synthesis, regulation and function with a focus on recent developments demonstrating the requirements for polyamines during the establishment of pregnancy up to the implantation stage, in particular the role of polyamines in the control of embryonic diapause and the identification of an alternative pathway for their synthesis in sheep pregnancy. This, along with other novel discoveries, provides new insights into the control of the peri-implantation period in mammals and highlights the complexities that exist in regulating this critical period of pregnancy.

Transcriptome Changes in the Mink Uterus during Blastocyst Dormancy and Reactivation.

Embryo implantation in the mink follows the pattern of many carnivores, in that preimplantation embryo diapause occurs in every gestation. Details of the gene expression and regulatory networks that terminate embryo diapause remain poorly understood. Illumina RNA-Seq was used to analyze global gene expression changes in the mink uterus during embryo diapause and activation leading to implantation. More than 50 million high quality reads were generated, and assembled into 170,984 unigenes. A total of 1684 differential expressed genes (DEGs) in uteri with blastocysts in diapause were compared to the activated embryo group (p < 0.05). Among these transcripts, 1527 were annotated as known genes, including 963 up-regulated and 564 down-regulated genes. The gene ontology terms for the observed DEGs, included cellular communication, phosphatase activity, extracellular matrix and G-protein couple receptor activity. The KEGG pathways, including PI3K-Akt signaling pathway, focal adhesion and extracellular matrix (ECM)-receptor interactions were the most enriched. A protein-protein interaction (PPI) network was constructed, and hub nodes such as VEGFA, EGF, AKT, IGF1, PIK3C and CCND1 with high degrees of connectivity represent gene clusters expected to play an important role in embryo activation. These results provide novel information for understanding the molecular mechanisms of maternal regulation of embryo activation in mink.

Inhibition of polyamine synthesis causes entry of the mouse blastocyst into embryonic diapause.

Embryonic diapause is a common reproductive strategy amongst mammals, requiring an intimate cross-talk between the endometrium and the blastocyst. To date, the precise molecular signals responsible are unknown in the mouse or any other mammal. Previous studies in the mink implicate polyamines as major regulators of the control of diapause. In the mouse, inhibiting the rate-limiting enzyme of polyamine synthesis, ornithine decarboxylase (ODC1) during early pregnancy largely prevents implantation, but the fate of the nonimplanted embryos is unknown. To determine whether polyamines control mouse embryonic diapause, we treated pregnant mice with an ODC1 inhibitor from d3.5 to d6.5 postcoitum. At d7.5, 72% of females had no signs of implantation whilst the remaining females exhibited disrupted placental formation and degenerate embryos. In the females with no implantation, we obtained viable blastocysts that had attenuated cell proliferation, indicating a state of diapause. When cultured in vitro, these exhibited trophoblast outgrowth, indicative of reactivation of embryogenesis. In contrast, direct culture of d3.5 blastocysts with an ODC1 inhibitor failed to cause entry into diapause. Examination of the polyamine pathway enzymes and a number of implantation factors indicated inhibition of ODC1 resulted in a uterine phenotype that resembled diapause, with some compensatory increases in crucial genes. Thus, we conclude that an absence or paucity of polyamines induces the uterine quiescence that causes entry of the blastocyst into embryonic diapause.

Embryo arrest and reactivation: potential candidates controlling embryonic diapause in the tammar wallaby and mink†.

Embryonic diapause is a period of developmental arrest which requires coordination of a molecular cross-talk between the endometrium and blastocyst to ensure a successful reactivation, but the exact mechanisms are undefined. The objectives of this study were to screen the tammar blastocyst for potential diapause control factors and to investigate the potential for members of the epidermal growth factor (EGF) family to coordinate reactivation. A select number of factors were also examined in the mink to determine whether their expression patterns were conserved across diapause species. The full-length sequences of the tammar genes of interest were first cloned to establish their level of sequence conservation with other mammals. The uterine expression of EGF family members EGF and heparin-binding EGF (HBEGF) and their receptors (EGFR and erb-b2 receptor tyrosine kinase 4 (ERBB4)) was determined by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Both HBEGF and EGF were significantly upregulated at reactivation compared to diapause. In the blastocyst, the expression of the potential diapause factors Forkhead box class O family members (FOXO1, FOXO3, and FOXO4), tumor protein 53 (TP53), cyclin-dependent kinase inhibitor 1A (CDKN1A), and the EGF family were examined by RT-PCR and immunofluorescence. Nuclear (and hence active) FOXO expression was confirmed for the first time in a mammalian diapause blastocyst in both the tammar and the mink-CDKN1A was also expressed, but TP53 is not involved and EGFR was not detected in the blastocyst. These results indicate that the EGF family, FOXOs, and CDKN1A are promising candidates for the molecular control of embryonic diapause in mammals.

Ovary-specific depletion of the nuclear receptor Nr5a2 compromises expansion of the cumulus oophorus but not fertilization by intracytoplasmic sperm injection.

The orphan nuclear receptor, liver receptor homolog-1 (aka Nuclear receptor subfamily 5, Group A, Member 2 (Nr5a2)), is widely expressed in mammalian tissues, and its ovarian expression is restricted to granulosa cells of activated follicles. We employed the floxed Nr5a2 (Nr5a2f/f) mutant mouse line and two granulosa-specific Cre lines, Anti-Müllerian hormone receptor- 2 (Amhr2Cre) and transgenic cytochrome P450 family 19 subfamily A polypeptide 1 (tgCyp19Cre), to develop two tissue- and time-specific Nr5a2 depletion models: Nr5a2Amhr2-/- and Nr5a2Cyp19-/-. In the Nr5a2Cyp19-/- ovaries, Nr5a2 was depleted in mural granulosa, but not cumulus cells. We induced follicular development in mutant and wild-type (control, CON) mice with equine chorionic gonadotropin followed 44 h later treatment with human chorionic gonadotropin (hCG) to induce ovulation. Both Nr5a2Amhr2-/- and Nr5a2Cyp19-/- cumulus-oocyte complexes underwent a reduced degree of expansion in vitro relative to wild-type mice. We found downregulation of epiregulin (Ereg), amphiregulin (Areg), betacellulin (Btc) and tumor necrosis factor stimulated gene-6 (Tnfaip6) transcripts in Nr5a2Amhr2-/- and Nr5a2Cyp19-/- ovaries. Tnfaip6 protein abundance, by quantitative immunofluorescence, was likewise substantially reduced in the Nr5a2-depleted model. Transcript abundance for connexin 43 (Gja1) in granulosa cells was lower at 0 h and maximum at 8 h post-hCG in both Nr5a2Amhr2-/- and Nr5a2Cyp19-/- follicles, while Gja1 protein was not different prior to the ovulatory signal, but elevated at 8 h in Nr5a2Amhr2-/- and Nr5a2Cyp19-/- follicles. In both mutant genotypes, oocytes can mature in vivo and resulting embryos were capable of proceeding to blastocyst stagein vitro. We conclude that Nr5a2 is essential for cumulus expansion in granulosa cells throughout follicular development. The disruption of Nr5a2 in follicular somatic cells does not affect the capacity of the oocyte to be fertilized by intracytoplasmic sperm injection.

Polyamine-Mediated Effects of Prolactin Dictate Emergence from Mink Obligate Embryonic Diapause.

Embryonic diapause is an evolutionary strategy to ensure that offspring are born when maternal and environmental conditions are optimal for survival. In many species of carnivores, obligate embryonic diapause occurs in every gestation. Reciprocal embryo transplant studies indicate that embryo arrest during diapause is conferred by uterine conditions and is due to a lack of specific factors necessary for continued development. In previous studies, global gene expression analysis revealed reduced uterine expression during diapause of a cluster of genes in the mink that regulate the abundance of polyamines, including ornithine decarboxylase 1 (ODC1). In addition, in vivo inhibition of the conversion of ornithine to the polyamine, putrescine, induced a reversible arrest in mink embryonic development and an arrest in trophoblast cell proliferation in vitro. Previous studies have implicated prolactin as the principal endocrine signal to terminate diapause. In this study, uterine expression of both the progesterone and estrogen receptors remained low at reactivation whilst the prolactin receptor was expressed at all times. Treatment of mink uterine epithelial cells with varying doses of prolactin indicated that this hormone induces ODC1 expression in the uterus via pSTAT1 and mTOR, thereby regulating uterine polyamine levels. In addition, we performed global gene expression analysis on mink embryos to further explore dynamic changes during diapause and found 94 genes upregulated at reactivation from diapause. Three polyamine-related genes, including ODC1, were also upregulated at reactivation from diapause. To establish whether polyamines mitigate escape from embryonic diapause, we collected mink embryos in diapause and incubated them in vitro with putrescine. Increase in embryo volume, the first indication of emergence from diapause, was observed within the first 5 days of culture in all viable embryos treated with putrescine, and the duration of embryo survival was increased threefold. Concomitant increases were also observed in both the total number of cells and the proportion of dividing cells in putrescine-treated embryos whilst control embryos remained in the diapause state. In further studies, inhibition of polyamine synthesis abrogated proliferation in cells derived from the inner cell mass of the mink embryo, while putrescine induced dose-dependent increases in cell division. We conclude that supplementation of embryos in diapause with putrescine results in their escape from developmental dormancy. These results provide strong evidence that obligate diapause in vivo is caused by the paucity of polyamines necessary for activation of the embryo after prolactin-induced termination of diapause.

Relationship between adiponectin and fertility in the female pig.

Adiponectin isoforms may mediate different aspects of the pleiotropic function of the protein, including the reproductive process. We examined the pattern of circulating adiponectin and adiponectin system expression in fat and ovarian tissues of hyperfertile and subfertile sows. We demonstrated the presence of five different isoforms of adiponectin (90, 158, 180, 250 and >250kDa) in the circulation and identified a subgroup of subfertile females that displayed reduced abundance of all adiponectin isoforms as well as a lack of the 250-kDa adiponectin isoform in both serum and follicular fluid. Subfertility in these animals was associated with fewer large follicles and corpora lutea in the ovaries, as well as lower concentrations of 17ß-oestradiol in the follicular fluid of large follicles. In addition, subfertile females showed higher adiponectin mRNA in fat tissue and altered mRNA and protein expression of adiponectin and its receptors in the ovary. Changes in the abundance and pattern of circulating adiponectin isoforms have been associated with reproductive disorders in animals and humans, including polycystic ovarian syndrome (PCOS). Our findings suggest that the adiponectin system may play an important role in controlling ovarian function and influencing porcine fertility.

Dysregulation of testicular cholesterol metabolism following spontaneous mutation of the niemann-pick c1 gene in mice.

The Niemann-Pick-type C1 (Npc1) protein mobilizes LDL-derived cholesterol from lysosomes. Npc1 deficiency disease is a panethnic autosomal recessive disorder of intracellular cholesterol trafficking, leading to accumulation of cholesterol in endosomes/lysosomes. This report assesses the effects of a spontaneous inactivating mutation of the Npc1 gene on spermatogenesis and cholesterol homeostasis in mice. We quantified 1) free and esterified cholesterol levels by enzymatic analysis, 2) cholesterol enzymes and transporter protein expression by Western blotting, and 3) the number of Apostain-labeled apoptotic germ cells and apoptosis levels by ELISA in seminiferous tubule-enriched fractions. In wild-type (WT) mice, esterified cholesterol was elevated when Npc1 expression was low during puberty, while in adulthood, the levels were low (P < 0.05) when Npc1 expression was high (P < 0.01). In Npc1-/- mice, free and esterified cholesterol were significantly elevated. The abundance of cholesterol regulatory proteins, HMGR ACAT1, ACAT2, SR-BI, and ABCA1 was significantly higher in Npc1-/- than in WT mice. The level of apoptosis determined by ELISA and the number of Apostain-labeled cells/tubule were higher in Npc1-/- than in WT mice. Circulating testosterone levels in the Npc1-/- males were threefold lower than those observed in the WT. Deleting the Npc1 gene is accompanied by an increase in germ cell apoptosis and compensatory imbalances in the expression of cholesterol enzymatic and transporter factors and is associated with esterified cholesterol accumulation in seminiferous tubules.

Global gene expression in the bovine corpus luteum is altered after stimulatory and superovulatory treatments.

Equine chorionic gonadotrophin (eCG) has been widely used in superovulation and artificial insemination programmes and usually promotes an increase in corpus luteum (CL) volume and stimulates progesterone production. Therefore, to identify eCG-regulated genes in the bovine CL, the transcriptome was evaluated by microarray analysis and the expression of selected genes was validated by qPCR and western blot. Eighteen Nelore crossbred cows were divided into control (n=5), stimulated (n=6) and superovulated groups (n=7). Ovulation was synchronised using a progesterone device-based protocol. Stimulated animals received 400 IU of eCG at device removal and superovulated animals received 2000 IU of eCG 4 days prior. Corpora lutea were collected 7 days after gonadotrophin-releasing hormone administration. Overall, 242 transcripts were upregulated and 111 transcripts were downregulated in stimulated cows (P ≤ 0.05) and 111 were upregulated and 113 downregulated in superovulated cows compared to the control animals (1.5-fold, P ≤ 0.05). Among the differentially expressed genes, many were involved in lipid biosynthesis and progesterone production, such as PPARG, STAR, prolactin receptors and follistatin. In conclusion, eCG modulates gene expression differently depending on the treatment, i.e. stimulatory or superovulatory. Our data contribute to the understanding of the pathways involved in increased progesterone levels observed after eCG treatment.

Expression of steroidogenic proteins in bovine placenta during the first half of gestation.

The aim of the present study was to determine the occurrence and localisation of the principal steroidogenic proteins in bovine placenta from Day 50 to Day 120 of pregnancy. Immunohistochemistry revealed that, at all stages investigated, bovine steroidogenic acute regulatory protein (StAR), cytochrome P45011A1 and hydroxy-δ-5-steroid dehydrogenase, 3ß- and steroid δ-isomerase 1 proteins were found principally at the fetomaternal interdigitations: the chorionic villus and maternal septum. Moreover, caruncular epithelial cells and uninucleate trophoblast cells were the principal cells detected that were positive for the three markers. Western blot analysis showed that only caruncular tissue expressed all three steroidogenic markers; in contrast, cotyledons only expressed StAR and cytochrome P45011A1. Immunoblot results showed a complementary pattern of StAR and cytochrome P45011A1 expression between caruncles and cotyledons at different stages. These observations suggest that, in early pregnancy, the maternal compartment contributes significantly to bovine placental steroidogenesis, particularly for the synthesis of progesterone. Furthermore, the variation in StAR and cytochrome P45011A1 expression between caruncular and cotyledonary tissues across gestation suggests that placental steroidogenesis requires cell-to-cell communication between maternal and fetal cells.

Steroidogenic Factor 1 Regulation of the Hypothalamic-Pituitary-Ovarian Axis of Adult Female Mice.

The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable regulator of adrenal and gonadal formation, playing roles in sex determination, hypothalamic development, and pituitary function. This study aimed to identify the roles of SF-1 in postnatal female reproductive function. Using a progesterone receptor-driven Cre recombinase, we developed a novel murine model, characterized by conditional depletion of SF-1 [PR-Cre;Nr5a1f/f; conditional knockout (cKO)] in the hypothalamic-pituitary-gonadal axis. Mature female cKO were infertile due to the absence of ovulation. Reduced gonadotropin concentrations in the pituitary gland that were nevertheless sufficient to maintain regular estrous cycles were observed in mature cKO females. The cKO ovaries showed abnormal lipid accumulation in the stroma, associated with an irregular expression of cholesterol homeostatic genes such as Star, Scp2, and Acat1. The depletion of SF-1 in granulosa cells prevented appropriate cumulus oöphorus expansion, characterized by reduced expression of Areg, Ereg, and Ptgs2. Exogenous delivery of gonadotropins to cKO females to induce ovulation did not restore fertility and was associated with impaired formation and function of corpora lutea accompanied by reduced expression of the steroidogenic genes Cyp11a1 and Cyp19a1 and attenuated progesterone production. Surgical transplantation of cKO ovaries to ovariectomized control animals (Nr5a1f/f) resulted in 2 separate phenotypes, either sterility or apparently normal fertility. The deletion of SF-1 in the pituitary and in granulosa cells near the moment of ovulation demonstrated that this nuclear receptor functions across the pituitary-gonadal axis and plays essential roles in gonadotropin synthesis, cumulus expansion, and luteinization.

Uterine signaling at the emergence of the embryo from obligate diapause.

Embryonic diapause is the reversible arrest of embryo development prior to implantation under a regime of uterine control that is not well understood. Our objective was to explore uterine modifications associated with the emergence of embryonic diapause in the mink, a species in which embryonic diapause characterizes every gestation. We investigated the uterine transcriptome at reactivation using the suppressive subtractive hybridization technique. A library of 123 differentially expressed genes between uteri with blastocysts in diapause and reactivated blastocysts was generated. Among those genes, 41.5% encode for potential secreted products that are implicated in regulation of cell proliferation (14%), homeostasis (14%), protein folding (11%), electron transport chain (8%), and innate immune response (8%), therefore suggesting that these biological processes are implicated in blastocyst reactivation. Two genes, the high-mobility group nucleosome binding domain 1 (HMGN1), a chromatin remodeling factor, and the secreted protein acidic and cystein-rich (SPARC), which is implicated in extracellular cell-cell interactions, were submitted to more detailed analysis of expression patterns in the mink uterus at blastocyst reactivation. Expression of both HMGN1 and SPARC was increased significantly in the uterus at embryo reactivation compared with diapause, principally in the endometrial epithelium and subepithelial stroma. These results provide new insight into uterine signaling at the emergence of the blastocyst from diapause and highlight the factors HMGN1 and SPARC as potential inductors of uterine environment modifications underlying uterine signaling during emergence of the embryo from embryonic diapause.

Elucidating evolutionarily conserved mechanisms of diapause regulation using an in silico approach.

Embryonic diapause is an enigmatic phenomenon that appears in diverse species. Although regulatory mechanisms have been established, there is much to be discovered. Herein, we have made the first comprehensive attempt to elucidate diapause regulatory mechanisms using a computational approach. We found transcription factors unique to promoters of genes in diapause species. From pathway analysis and STRING PPI networks, the signaling pathways regulated by these unique transcription factors were identified. The pathways were then consolidated into a model to combine various known mechanisms of diapause regulation. This work also highlighted certain transcription factors that may act as 'master transcription factors' to regulate the phenomenon. Promoter analysis further suggested evidence for independent evolution for some of regulatory elements involved in diapause.

Nuclear receptors: Key regulators of somatic cell functions in the ovulatory process.

The development of the ovarian follicle to its culmination by ovulation is an essential element of fertility. The final stages of ovarian follicular growth are characterized by granulosa cell proliferation and differentiation, and steroid synthesis under the influence of follicle-stimulating hormone (FSH). The result is a population of granulosa cells poised to respond to the ovulatory surge of luteinizing hormone (LH). Members of the nuclear receptor superfamily of transcription factors play indispensable roles in the regulation of these events. The key regulators of the final stages of follicular growth that precede ovulation from this family include the estrogen receptor beta (ESR2) and the androgen receptor (AR), with additional roles for others, including steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1). Following the LH surge, the mural and cumulus granulosa cells undergo rapid changes that result in expansion of the cumulus layer, and a shift in ovarian steroid hormone biosynthesis from estradiol to progesterone production. The nuclear receptor best associated with these events is LRH-1. Inadequate cumulus expansion is also observed in the absence of AR and ESR2, but not the progesterone receptor (PGR). The terminal stages of ovulation are regulated by PGR, which increases the abundance of the proteases that are directly responsible for rupture. It further regulates the prostaglandins and cytokines associated with the inflammatory-like characteristics of ovulation. LRH-1 regulates PGR, and is also a key regulator of steroidogenesis, cellular proliferation, and cellular migration, and cytoskeletal remodeling. In summary, nuclear receptors are among the panoply of transcriptional regulators with roles in ovulation, and several are necessary for normal ovarian function.

A role for orphan nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) in primordial follicle activation.

Liver receptor homolog-1 (NR5A2) is expressed specifically in granulosa cells of developing ovarian follicles where it regulates the late stages of follicle development and ovulation. To establish its effects earlier in the trajectory of follicular development, NR5A2 was depleted from granulosa cells of murine primordial and primary follicles. Follicle populations were enumerated in neonates at postnatal day 4 (PND4) coinciding with the end of the formation of the primordial follicle pool. The frequency of primordial follicles in PND4 conditional knockout (cKO) ovaries was greater and primary follicles were substantially fewer relative to control (CON) counterparts. Ten-day in vitro culture of PND4 ovaries recapitulated in vivo findings and indicated that CON mice developed primary follicles in the ovarian medulla to a greater extent than did cKO animals. Two subsets of primordial follicles were observed in wildtype ovaries: one that expressed NR5A2 and the second in which the transcript was absent. Neither expressed the mitotic marker. KI-67, indicating their developmental quiescence. RNA sequencing on PND4 demonstrated that loss of NR5A2 induced changes in 432 transcripts, including quiescence markers, inhibitors of follicle activation, and regulators of cellular migration and epithelial-to-mesenchymal transition. These experiments suggest that NR5A2 expression poises primordial follicles for entry into the developing pool.