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.

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.

Insights from two independent transcriptomic studies of the bovine corpus luteum during pregnancy.

Several recent studies have used transcriptomics to investigate luteal changes during the maternal recognition of the pregnancy period in ruminants. Although these studies have contributed to our understanding of luteal function during early pregnancy, few attempts have been made to integrate information across these studies and distinguish key luteal transcripts or functions that are repeatably identified across multiple studies. Therefore, in this study, two independent studies of the luteal transcriptome during early pregnancy were combined and compared. In the first study, corpora lutea (CL) from day 20 of pregnancy were compared with CL collected on day 14 of pregnancy, prior to embryonic signaling. The cattle were nonlactating. In the second study, CL from day 20 of pregnancy were compared with CL collected from day 20 cyclic cattle that had been confirmed as not yet undergoing luteal regression. These were lactating cattle. Three methods were used to compare these two datasets, to identify key luteal regulators. In the first method, all transcripts with Benjamini-Hochberg-adjusted P-value (Q value) < 0.05 in both datasets were considered. This yielded 22 transcripts, including several classical interferon-stimulated genes, as well as regulators of transforming growth factor-beta (TGFB) and latent TGFB-binding proteins (LTBP)1 and 2. In the second, less conservative method, all transcripts with P < 0.01 and changed in the same direction in both datasets were considered. This yielded an additional 20 transcripts that were not identified in the first analysis, for a total of 42 common transcripts. These transcripts were regulators of functions such as inflammatory balance and matrix remodeling. In the third method, transcripts with Q < 0.10 were subject to pathway analysis, and common pathways were identified. Retinoic acid signaling and classical interferon signaling pathways were identified with this method. Finally, regulation by interferon tau (IFNT) was investigated. Among the 42 transcripts identified, 32 were regulated by IFNT in cultured luteal cells (Q < 0.05). Among those not regulated by IFNT were LTBP1 and 2, which are TGFB-binding proteins. In summary, common transcripts from two studies of the luteal transcriptome during early pregnancy were combined and shared changes were identified. This not only generated a list of potential key luteal regulators, which were mostly IFNT regulated, but also included transcripts not regulated by IFNT, including LTBP1 and 2.

Reproductive efficiency is necessary for the financial and environmental sustainability of cattle production. A critical component of this efficiency is the maintenance of pregnancy. The corpus luteum (CL) is a transient ovarian endocrine gland that produces progesterone­the hormone that maintains pregnancy in all mammals. With each new reproductive cycle, a new CL is formed from the remnant of the ovulatory follicle, and at the end of each cycle, in the absence of a pregnancy, the CL regresses in response to prostaglandin F2α from the uterus. In contrast, in the presence of a pregnancy, the CL is rescued from regression by the embryo, in a process known as maternal recognition of pregnancy. The embryo is known to alter uterine function, but its effect on the CL has remained a mystery until recent years. In this study, we compared two independent studies of global changes in the CL of pregnancy, to identify the most important luteal changes that occur during early pregnancy. We confirmed evidence for embryonic signaling to the CL and generated a list of candidate genes that are the likeliest regulators of changes to luteal function in early pregnancy. This list includes regulators of tissue integrity and inflammation.

Form of Supplemental Selenium Affects the Expression of mRNA Transcripts Encoding Selenoproteins, and Proteins Regulating Cholesterol Uptake, in the Corpus Luteum of Grazing Beef Cows.

Selenium (Se)-deficient soils necessitate supplementation of this mineral to the diet of forage-grazing cattle. Functionally, Se is incorporated into selenoproteins, some of which function as important antioxidants. We have previously shown that the source of supplemental Se; inorganic (sodium selenite or sodium selenate; ISe), organic (selenomethionine or selenocysteine; OSe) or 1:1 mix of ISe and OSe (MIX), provided to Angus-cross cows affects concentrations of progesterone (P4) during the early luteal phase of the estrous cycle. In this study, we sought to investigate (1) the effect of form of Se on the expression of mRNA encoding selenoproteins in the corpus luteum (CL), and (2) whether this previously reported MIX-induced increase in P4 is the result of increased luteal expression of key steroidogenic transcripts. Following a Se depletion and repletion regimen, 3-year-old, non-lactating, Angus- cross cows were supplemented with either ISe as the industry standard, or MIX for at least 90 days, with the CL then retrieved on Day 7 post-estrus. Half of each CL was used for analysis of targeted mRNA transcripts and the remainder was dissociated for culture with select agonists. The expression of three selenoprotein transcripts and one selenoprotein P receptor was increased (p < 0.05), with an additional five transcripts tending to be increased (p < 0.10), in cows supplemented with MIX versus ISe. In cultures of luteal cells, hCG-induced increases in P4 (p < 0.05) were observed in CL obtained from ISe-supplemented cows. The abundance of steroidogenic transcripts in the CL was not affected by the form of Se, however, the abundance of mRNA encoding 2 key transcripts regulating cholesterol availability (Ldlr and Hsl) was increased (p < 0.05) in MIX-supplemented cows. Overall, the form of Se provided to cows is reported to affect the expression of mRNA encoding several selenoproteins in the CL, and that the form of Se-induced effects on luteal production of P4 appears to be the result of changes in cholesterol availability rather than a direct effect on the expression of steroidogenic enzymes within the CL.

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.

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.

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.

Luteal Lipids Regulate Progesterone Production and May Modulate Immune Cell Function During the Estrous Cycle and Pregnancy.

Although the corpus luteum (CL) contains high concentrations of lipid in the form of steroid hormone precursors and prostaglandins, little is known about the abundance or function of other luteal lipid mediators. To address this, 79 lipid mediators were measured in bovine CL, using ultra performance liquid chromatography-tandem mass spectrometry. CL from estrous cycle days 4, 11, and 18 were compared and, separately, CL from days 18 of the estrous cycle and pregnancy were compared. Twenty-three lipids increased as the estrous cycle progressed (P < 0.05), with nine increasing between days 4 and 11 and fourteen increasing between days 4 and 18. Overall, this indicated a general upregulation of lipid mediator synthesis as the estrous cycle progressed, including increases in oxylipins and endocannabinoids. Only 15-KETE was less abundant in the CL of early pregnancy (P < 0.05), with a tendency (P < 0.10) for four others to be less abundant. Notably, 15-KETE also increased between estrous cycle days 4 and 18. Ingenuity Pathway Analysis (IPA, Qiagen) indicated that functions associated with differentially abundant lipids during the estrous cycle included leukocyte activation, cell migration, and cell proliferation. To investigate changes in CL during maternal recognition of pregnancy, this lipid dataset was integrated with a published dataset from mRNA profiling during maternal recognition of pregnancy. This analysis indicated that lipids and mRNA that changed during maternal recognition of pregnancy may regulate some of the same functions, including immune cell chemotaxis and cell-cell communication. To assess effects of these lipid mediators, luteal cells were cultured with 5-KETE or 15-KETE. One ng/mL 5-KETE reduced luteal progesterone on day 1 of culture, only in the absence of luteinizing hormone (LH), while 1 ng/mL 15-KETE induced progesterone only in the presence of LH (10 ng/mL). On day 7 of culture, 0.1 ng/mL 15-KETE reduced prostaglandin (PG)F2A-induced inhibition of LH-stimulated progesterone production, while 1 ng/mL 15-KETE did not have this effect. Overall, these data suggest a role for lipid mediators during luteal development and early pregnancy, as regulators of steroidogenesis, immune cell activation and function, intracellular signaling, and cell survival and death.

Conserved MHC gene orthologs genetically map to the turkey MHC- B.

The avian MHC-associated gene set includes orthologs to genes found throughout the human major histocompatibility complex (MHC), including some loci of the evolutionarily conserved class III region. In the turkey and other Galliformes, genes linked to the MHC have been identified because they are closely associated with class I or class II genes. This study was designed to evaluate additional class III genes for linkage to the avian MHC to further determine conservation of these loci in birds. BLAST searches were used to locate sequences in the turkey genome with similarity to genes shared between the MHC of Xenopus and humans. Primers were designed to target 25 genes, and putative orthologs were amplified by PCR and sequenced. Sequence polymorphisms were identified for 15 genes in turkey reference mapping families, and 8 genes showed significant genetic linkage to the turkey MHC-B locus. These new genetic markers and linkage relationships broaden our understanding of the composition of the avian MHC and expand the gene content for the turkey MHC-B.