Expression In Granulosa Cells Research Articles

CAMP/PKA/CREB signaling pathway-mediated effects of melatonin receptor genes on clock gene expression in Bactrian camel ovarian granulosa cells

The cAMP/PKA/CREB pathway is involved in the regulation of melatonin during important physiological activities in mammals. However, the regulation of circadian clock genes in ovarian granulosa cells remains unclear. Herein, we determined the relationship between melatonin and biological clock genes using cultured Bactrian camel ovarian granulosa cells. The enzyme-linked immunosorbent assays showed that the cAMP content was reduced when melatonin receptor (MT) genes or cryptochrome (Cry) genes were overexpressed; the quantitative polymerase chain reaction and western blot analyses revealed that the expression levels of all circadian clock genes (GNB2, PKA, CREB, Per1/2/3, and Clock) except Cry1/2 decreased significantly at 24 h. Cellular immunolocalization analysis showed that melatonin receptors were localized in the cell membrane and cytoplasm; the CRY protein was mainly localized in the nucleus. Overall, our findings indicated that the rhythmic regulation of ovarian granulosa cells was consistent with the regulatory action of the central circadian clock.

TGF-β1 stimulates aromatase expression and estradiol production through SMAD2 and ERK1/2 signaling pathways in human granulosa-lutein cells.

Estradiol (E2), one of the main steroid hormones secreted by the ovaries, plays an important rolein maintaining normal female reproductive function. Ovarian granulosa cells are the main source of E2 production because these cells express aromatase, which is encoded by the CYP19A1 gene and catalyzes the final step in E2 biosynthesis from androgens. Transforming growth factor-beta 1 (TGF-β1) and its receptors are expressed in human granulosa cells, and TGF-β1 expression can be detected in human follicular fluid. To date, TGF-β1 has been shown to regulate various ovarian functions. However, whether aromatase can be regulated by TGF-β1 in human granulosa cells has not been determined. In the present study, we demonstrate that TGF-β1 stimulates aromatase expression in primary human granulosa-lutein cells and in the human ovarian granulose-like tumor cell line, KGN. We used pharmacological inhibitors and small interfering RNA-mediated knockdown approaches to reveal that the SMAD2 and ERK1/2 signaling pathways are involved in TGF-β1-induced aromatase expression and E2 production. These results not only provide important insights into the molecular mechanisms that mediate TGF-β1-induced aromatase expression and E2 production in human granulosa cells but also increase the understanding of the normal physiological roles of TGF-β1 in the ovary.

FMR1 expression in human granulosa cells and variable ovarian response: control by epigenetic mechanisms.

In humans, FMR1 (fragile X mental retardation 1) is strongly expressed in granulosa cells (GCs) of the female germline and apparently controls efficiency of folliculogenesis. Major control mechanism(s) of the gene transcription rate seem to be based on the rate of CpG-methylation along the CpG island promoter. Conducting CpG-methylation-specific bisulfite-treated PCR assays and subsequent sequence analyses of both gene alleles, revealed three variably methylated CpG domains (FMR1-VMR (variably methylated region) 1, -2, -3) and one completely unmethylated CpG-region (FMR1-UMR) in this extended FMR1-promoter-region. FMR1-UMR in the core promoter was exclusively present only in female GCs, suggesting expression from both gene alleles, i.e., escaping the female-specific X-inactivation mechanism for the second gene allele. Screening for putative target sites of transcription factors binding with CpG methylation dependence, we identified a target site for the transcriptional activator E2F1 in FMR1-VMR3. Using specific electrophoretic mobility shift assays, we found E2F1 binding efficiency to be dependent on CpG-site methylation in its target sequence. Comparative analysis of these CpGs revealed that CpG 94-methylation in primary GCs of women with normal and reduced efficiency of folliculogenesis statistically significant differences. We therefore conclude that E2F1 binding to FMR1-VMR3 in human GCs is part of an epigenetic mechanism regulating the efficiency of human folliculogenesis. Our data indicate that epigenetic mechanisms may control GC FMR1-expression rates.

PTEN Expression in Human Granulosa Cells Is Associated with Ovarian Responses and Clinical Outcomes in IVF.

The ovarian reserve determines the success of in vitro fertilization (IVF) and embryo transfer treatment. It predicts the ovarian response in controlled ovarian hyperstimulation cycles. Apoptosis in granulosa cells surrounding oocytes is important for ovarian function and has been closely associated with follicular atresia. PTEN (encoding phosphatase and tensin homolog) is a well-known tumor suppressor gene that functions as a mediator of apoptosis and is crucial for mammal reproduction. In the present study, we analyzed the expression level of PTEN in human granulosa cells and aimed to investigate its association with the ovarian response and clinical outcomes in IVF. Apoptosis in granulosa cells were analyzed using Annexin V-Allophycocyanin staining after PTEN short hairpin RNA lentivirus transfection. Real-time fluorescent quantitative PCR analysis showed that the PTEN transcript level was significantly higher in poor responders and significantly lower in high responders, compared with that in normal responders. However, PTEN expression in the pregnancy group decreased slightly, but not significantly, compared with that in the non-pregnancy group. The apoptosis rate of granulosa cells declined significantly after 24-h transfection of the PTEN-shRNA lentivirus. These results suggest a fundamental role of PTEN in the regulation of follicular development, and that it might be involved in the pathogenesis of follicular dysplasia and ovarian dysfunction.

LH upregulates connexin 43 expression in granulosa cells by activating the Wnt/β-catenin signalling pathway.

Connexin (Cx) 43 is the most widely expressed gap junction protein in follicle granulosa cells and plays an important role in follicle development and growth. The aims of this study were to investigate the effects of LH on the expression of Cx43 and key proteins in the downstream Wnt-β/catenin signalling pathway and to explore the mechanism underlying the regulation of Cx43 expression in granulosa cells. Primary culture granulosa cells were obtained from 21-day-old Sprague-Dawley rats, and were treated with different concentrations of LH (150, 300 and 600 IU L-1). Cx43 expression in granulosa cells was detected using immunofluorescence. Western blotting was used to detect the expression of Cx43, β-catenin and Axin2 proteins (Axin2 is a protein that in humans is encoded by the AXIN2 gene, which presumably plays an important role in the regulation of the stability of β-catenin in the Wnt signaling pathway) in granulosa cells with and without FH535 treatment (a Wnt/β-catenin signalling pathway inhibitor). Cx43 expression was detected in the cytoplasm and cell membrane of granulosa cells. Treatment with a high concentration of LH (300 IU L-1) increased the expression of β-catenin and Axin2, as well as that of Cx43. FH535 treatment reduced the LH-induced increases in Cx43, β-catenin and Axin2. These results indicate that LH upregulates Cx43 expression in granular cells by activating the Wnt/β-catenin signalling pathway.

P63 expression in granulosa-luteinized cells of infertile patients with peritoneal endometriosis submitted to in vitro fertilization.

ObjectiveEndometriosis is associated with infertility, even without an anatomical abnormality. Furthermore, the peritoneal (mild) phenotype of this disease is the most prevalent and linked to infertility. The present study aimed to investigate the p63 gene and protein expression in granulosa cells from pre-ovulatory follicles in patients with endometriosis and infertility submitted to in vitro fertilization.MethodsTwenty-eight patients participated in the study and were divided into two groups according to the presence or absence of endometriosis. The p63 gene-expression levels assessment was performed by real-time PCR (qPCR) using the TaqMan assay, and we used immunofluorescence to check the p63 protein expression after IVF.ResultsThere was no significant difference between the groups regarding age, hormonal levels, oocyte standards, and p63 gene expression. The control group showed an RQ of 1.000 (0.431 to 2.323) and the study group showed an RQ of 0.725 (0.249 to 2.105), p>0.05. Both groups showed a weak expression of the p63 gene (p>0.05).ConclusionsThis study described that endometriosis may not affect the p63 gene expression. Moreover, after follicular recruitment and growth, we found a weak expression of this protein, suggesting it is not part of oocyte maturation and development control.

Fshr and lhr mRNA expression in granulosa cells of poor responder

In Vitro Fertilization (IVF) is one of the most commonly used procedures to help pregnancies in couples who have infertility problems. One of the problems of infertility is poor ovarian response and the woman who experiences it is known as poor responder. Poor responders do not have an adequate ovarian response to gonadotropin in ovarian stimulation. The success of fertilization in poor responders tends to be low due to low quantity and is generally followed by low oocyte quality. Gonadotropins consisting of FSH and LH, play a role in follicle development and ovulation. The follicle response in capturing gonadotropins depends on the exact bond between the hormone and its receptor (FSHR and LHR) in the granulosa cells surrounding the oocyte. The purpose of this research is to know the expression level of fshr and lhr mRNA in granulosa cells of poor responders through real-time PCR method which then tested statistically using t-test. Fourteen samples of each poor responders and normal women were used in this research. The results showed insignificant differences between expression level of fshr and lhr mRNA in granulosa cells of poor responders and normal women (p > 0.05).

Gonadotropin inhibitory hormone downregulates steroid hormone secretion and genes expressions in duck granulosa cells.

The mechanisms by which GnIH regulates the steroid synthesis pathway in duck granulosa cells remain poorly understood. In this study, we measured steroid hormone secretion by ELISA and reproduction-associated gene expression by quantitative real-time Polymerase Chain Reaction (qPCR) in duck granulosa cells treated with different concentrations of GnIH (0, 0.1, 1, 10, and 100 ng/mL) for 24 h. The genome-wide expression profiles of GnIH-treated cells (0 and 10 ng/mL) were evaluated by high-throughput RNA sequencing. Compared with untreated cells, the secretion of the steroid hormones E2, E1, P4, and T was downregulated, with that of E1 and P4 reaching statistical significance (P<0.05); in contrast, the secretion of ACV and INH was significantly upregulated (P<0.05) after treatment with 10 and 100 ng/mL GnIH. The expression of encoding steroidogenic proteins and enzymes genes (STAR, CYP11A1, CYP17A1, CYP19A1, and 3-β-HSD) and encoding gonadotropin receptors genes (FSHR, LHR) were significantly declined (P<0.05) in the 10 and 100 ng/mL GnIH treatments. Transcriptome sequencing identified 348 differentially expressed genes (DEGs), including 253 upregulated and 95 downregulated genes. The DEGs were mainly involved in cell growth and death, immune response, and steroid biosynthesis pathways. We identified four novel DEGs (MROH5, LOC113840576, SDR42E1, and LOC113841457) with key roles in the regulation of steroid hormone biosynthesis. Our study revealed changes in gonadal steroid hormone secretion and steroid biosynthesis pathway-related gene expression in duck granulosa cells under the inhibitory effect of GnIH. These data contribute to our understanding of the molecular and genetic mechanisms underlying reproduction in ducks.

Apoptosis-related genes expression in primary in vitro culture of human ovarian granulosa cells

Abstract Ovarian granulosa cells (GCs) play a crucial role in oocyte maturation, creating a favorable microenvironment around the oocyte. Therefore, enhanced apoptosis and GCs loss may negatively affect the intra-follicular milieu and compromise the oocyte quality, reducing pregnancy chances. Based on the RT-qPCR method, the present research revealed the differential expression of apoptosis-related genes (BCL2, BAX, p53, CASP9) during the seven days of primary in vitro culture of GCs isolated from patients undergoing in vitro fertilization (IVF) procedure. Individual gene expression changes may reflect the GCs survival and/or apoptotic status at different time points. Running title: Apoptosis-related genes expression in granulosa cells in vitro

Analysis of TGFB1, CD105 and FSP1 expression in human granulosa cells during a 7-day primary in vitro culture

Abstract The human granulosa cells (GCs) surround the oocyte and form the ovarian follicle’s proper architecture. These sub-populations include mural granulosa cells, antral granulosa cells, and cumulus granulosa cells. Their main functions are to support the oocyte’s growth (cumulus granulosa cells) and estradiol production (mural granulosa cells). After ovulation, the granulosa cells transform into the luteal cells of the corpus luteum and produce progesterone. Our study investigated the expression profile of three genes: TGFB1, CD105, and FSP1 during a 7-day in vitro culture. The analysis was conducted using the RT-qPCR technique. Changes in the expression of CD105 and FSP1 could be observed during the 7-day in vitro culture. In the case of TGFB, the expression remained at a similar level, with no statistically significant differences observed. Running title: Expression of TGFB1, CD105 and FSP1 in granulosa cells

Oral Vitamin D supplementation impacts gene expression in granulosa cells in women undergoing IVF.

Oral Vitamin D supplementation impacts gene expression in granulosa cells in women undergoing IVF.

Sustained ERK1/2 signaling is necessary for follicular rupture during ovulation in mice.

Abolition of the LH-induced ERK1/2 pathway leads to dramatic changes in gene expression in granulosa cells, subsequently abrogating ovulation. Here we explored whether sustained ERK1/2 signaling beyond immediate-early hours of the LH surge is important for ovulation in mice. First, we examined the effect of inhibition of ERK1/2 activity at 4 h after hCG stimulation on ovulation in superovulated immature mice. Treatment with the ERK1/2 pathway inhibitor PD0325901 at 4 h post-hCG disrupted follicular rupture without altering cumulus expansion, oocyte meiotic maturation and luteinization. Profiling the expression pattern of genes of the RSK family of ERK1/2 signal mediators revealed that RSK3, but not other isoforms, was induced by hCG treatment. Further, RSK3-knockout mice were sub-fertile with reduced ovulation rate and smaller litter size compared to WT mice. Given that PD0325901 inhibits all mediators of ERK1/2 signaling, we chose to evaluate the gene expression underlying deficient follicular rupture in ERK1/2 inhibited mice. We found that inhibition of ERK1/2 signaling at 4 h post-hCG resulted in an imbalance in the expression of genes involved in extracellular matrix degradation and leukocyte infiltration necessary for follicular rupture. In conclusion, our data demonstrate that sustained ERK1/2 signaling during ovulation is not required for cumulus expansion, oocyte meiotic maturation and luteinization, but is required for follicular rupture.

Expression of PGC-1ɑ in Ovarian Tissue of PCOS Rat Model

To investigate the expression of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) in the ovarian tissue of polycystic ovary syndrome (PCOS) rat model and obese PCOS rat model, and the possible mechanism of PCOS. Thirty SD rats were randomly divided into control group, PCOS rat model group and obese PCOS rat model group. DHEA dissolved in 0.2 mL soybean oil was injected daily into the rats of two PCOS groups for 21 d. Rats in obese PCOS model group were added with high-fat diet based on DHEA modeling, and each group had 10 rats. Body mass were measured before and on the 22 nd day after modeling. The serum testosterone (T) levels were measured by abdominal aortic blood, and the ovarian tissues of rats were taken for histological changes were observed by HE staining, immunohistochemical staining and Western blot to detect PGC-1ɑ protein expression. The body mass of rats in each group increased after modeling, and the body mass of rats in PCOS group and obese PCOS group increased significantly ( P<0.05). The serum T concentration of two PCOS model groups was higher than that of control group ( P<0.01). The serum T concentration in obese PCOS model group was higher than that in the PCOS group ( P<0.05). The results of HE staining of rat ovarian tissue showed that there were follicles and a small amount of corpus luteum at different developmental stages in the control group, and the granulosa cells were arranged in 4-6 layers. The number of immature small follicles in the two PCOS groups was significantly increased. The granulosa cells were arranged in 1-3 layers, relatively looser, and some follicles were atresia. In the obese PCOS group, the diameter of ovarian atretic follicles increased, the number of granulocyte layers decreased, and oocytes disappeared more obviously. Immunohistochemical staining showed that the PGC-1ɑ protein was mainly expressed in the cumulus and granulosa cells of ovarian tissue in the control group. The mean gray level of PGC-1ɑ protein expression in PCOS group (0.53±0.06) and obese PCOS group (0.36±0.03) was lower than that of the control group (0.75±0.03), with the statistical difference ( P<0.05). PGC-1ɑ expression in the obese PCOS group was lower than that in the PCOS group ( P<0.01). The results of Western blot were consistent with those of immunohistochemical staining. PGC-1ɑ is associated with damage of ovarian granulosa cells in high-fat environment. The decrease of PGC-1ɑ expression in granulosa cells of ovarian follicles may be an important cause of PCOS development.

Sirtuin-1 inhibits endothelin-2 expression in human granulosa-lutein cells via hypoxia inducible factor 1 alpha and epigenetic modifications†.

Endothelin-2 (EDN2) expression in granulosa cells was previously shown to be highly dependent on the hypoxic mediator, hypoxia inducible factor 1 alpha (HIF1A). Here, we investigated whether sirtuin-1 (SIRT1), by deacetylating HIF1A and class III histones, modulates EDN2 in human granulosa-lutein cells (hGLCs). We found that HIF1A was markedly suppressed in the presence of resveratrol or a specific SIRT1 activator, SRT2104. In turn, hypoxia reduced SIRT1 levels, implying a mutually inhibitory interaction between hypoxia (HIF1A) and SIRT1. Consistent with reduced HIF1A transcriptional activity, SIRT1 activators, resveratrol, SRT2104, and metformin, each acting via different mechanisms, significantly inhibited EDN2. In support, knockdown of SIRT1 with siRNA markedly elevated EDN2, whereas adding SRT2104 to SIRT1-silenced cells abolished the stimulatory effect of siSIRT1 on EDN2 levels further demonstrating that EDN2 is negatively correlated with SIRT1. Next, we investigated whether SIRT1 can also mediate the repression of the EDN2 promoter via histone modification. Chromatin immunoprecipitation (ChIP) analysis revealed that SIRT1 is indeed bound to the EDN2 promoter and that elevated SIRT1 induced a 40% decrease in the acetylation of histone H3, suggesting that SIRT1 inhibits EDN2 promoter activity by inducing a repressive histone configuration. Importantly, SIRT1 activation, using SRT2104 or resveratrol, decreased the viable numbers of hGLC, and silencing SIRT1 enhanced hGLC viability. This effect may be mediated by reducing HIF1A and EDN2 levels, shown to promote cell survival. Taken together, these findings propose novel, physiologically relevant roles for SIRT1 in downregulating EDN2 and survival of hGLCs.

LH-induced Transcriptional Regulation of Klf4 Expression in Granulosa Cells Occurs via the cAMP/PKA Pathway and Requires a Putative Sp1 Binding Site.

Krüppel-like factor 4 (Klf4) plays an important role in the transition from proliferation to differentiation in a wide variety of cells. Previous studies demonstrated its critical role in the luteal transition of preovulatory granulosa cells (GCs). This study used cultured rat preovulatory GCs to investigate the mechanism by which luteinizing hormone (LH) regulates Klf4 gene expression. Klf4 mRNA and protein were rapidly and transiently induced by LH treatment, reaching peak levels after 45 min and declining to basal levels by 3 h. Pretreatment with the protein synthesis inhibitor cycloheximide had no effect on LH-stimulated Klf4 expression, indicating that Klf4 is an immediate early gene in response to LH. To investigate the signaling pathway involved in LH-induced Klf4 regulation, the protein kinase A (PKA) and protein kinase C (PKC) pathways were evaluated. A-kinase agonists, but not a C-kinase agonist, mimicked LH in inducing Klf4 transcription. In addition, specific inhibitors of A-kinase abolished the stimulatory effect of LH on Klf4 expression. Truncation of a Klf4 expression construct to −715 bp (pKlf4-715/luc) had no effect on transcriptional activity, whereas deletion to −402 bp (pKlf4-402/luc) dramatically reduced it. ChIP analysis revealed in vivo binding of endogenous Sp1 to the −715/−500 bp region and maximal transcriptional responsiveness to LH required the Sp1 binding element at −698/−688 bp, which is highly conserved in mice, rats, and humans. These findings demonstrate that Klf4 is activated by LH via the cAMP/PKA pathway and a putative Sp1 binding element at −698/−688 bp is indispensable for activation and suggest that Klf4 could be a target for strategies for treating luteal phase insufficiency induced by an aberrant response to the LH surge.

Regulation of the early growth response-1 binding protein NAB2 in bovine granulosa cells and effect on connective tissue growth factor expression

Ovarian fibrosis is associated with increased expression of the transcription factor, Early growth response-1 (EGR1) and connective tissue growth factor (CCN2) in granulosa cells. The transcriptional activity of EGR1 is under negative feedback control by NAB1 and NAB2, but little is known about NAB expression in granulosa cells. Using a well-defined bovine in-vitro granulosa cell model, we show that NAB2 but not NAB1 mRNA is upregulated by fibroblast growth factor (FGF)1 and FGF2, but not by FGF4 or FGF8b. Overexpressing NAB2 abrogated the ability of FGF8b to increase EGR1 and CCN2 mRNA, as well as mRNAs encoding other FGF-target genes. Surprisingly, overexpression of NAB2 in the absence of growth factor stimulation increased abundance of mRNA encoding CCN2 and EGR1, and decreased estradiol secretion. We conclude that NAB2 is expressed in granulosa cells and plays a role in regulating EGR1-induced CCN2 expression, although cross-talk with other signaling pathways is likely occurring.

Effects of FOXO1 on the proliferation and cell cycle-, apoptosis- and steroidogenesis-related genes expression in sheep granulosa cells

Forkhead boxO (FOXO) transcription factors regulate diverse biological processes, including cellular metabolism, cell apoptosis, and the cell cycle. Results from several studies indicate FOXO1 regulates different granulosa cell (GC) pathways involved in proliferation, survival and differentiation. Functions and mechanisms of FOXO1 regulation of sheep GCs remain unclear. This study was conducted to analyze the function of FOXO1 in regulation of sheep GCs. In this study, the 1827 bp sheep FOXO1 coding sequence was cloned from sheep GCs. Multiple sequence alignment and phylogenetic analysis indicated that the FOXO1 protein sequence is highly homologous to FOXO1 protein sequences from other species. The results obtained from using CCK-8 assays indicated sheep GC proliferation increased when there was suppression of FOXO1 gene expression. When there was induced expression of the FOXO1 gene in sheep GCs, there was a resulting increased abundance of P21 and P27 mRNA transcript, whereas suppression of the FOXO1 gene expression had the opposite effect. Furthermore, the relative abundance in vitro of apoptosis-related protein mRNA transcripts (caspase3, caspase8, caspase9, Bax/Bcl-2) was markedly increased or decreased when there was induction or suppression of FOXO1 gene expression, respectively,(P < 0.05). Induction of FOXO1 gene expression resulted in an increase in abundance of steroidogenic protein mRNA transcripts (CYP11A1, 3β-HSD), while suppression of FOXO1 gene expresion resulted in a decrease abundance of the CYP11A1, STAR mRNA transcripts. Results from the present study indicated that FOXO1 inhibited the proliferation of sheep GCs and affected mRNA transcript abundance for proteins involved in regulation of apoptosis, the cell cycle and steroidogenesis.

Global analysis of FSH-regulated gene expression and histone modification in mouse granulosa cells.

Follicle-stimulating hormone (FSH) regulates ovarian follicular development through a specific gene expression program. We analyzed FSH-regulated transcriptome and histone modification in granulosa cells during follicular development. We used super-stimulated immature mice and collected granulosa cells before and 48 h after stimulation with equine chorionic gonadotropin (eCG). We profiled the transcriptome using RNA-sequencing (N = 3/time-point) and genome-wide trimethylation of lysine 4 of histone H3 (H3K4me3; an active transcription marker) using chromatin immunoprecipitation and sequencing (ChIP-Seq; N = 2/time-point). Across the mouse genome, 14,583 genes had an associated H3K4me3 peak and 63-66% of these peaks were observed within ≤1 kb promoter region. There were 72 genes with differential H3K4me3 modification at 48 h eCG (absolute log fold change > 1; false discovery rate [FDR] < 0.05) relative to 0 h eCG. Transcriptome data analysis showed 1463 differentially expressed genes at 48 h eCG (absolute log fold change > 1; FDR < 0.05). Among the 20 genes with differential expression and altered H3K4me3 modification, Lhcgr had higher H3K4me3 abundance and expression, while Nrip2 had lower H3K4me3 abundance and expression. Using ChIP-qPCR, we showed that FSH-regulated expression of Lhcgr, Cyp19a1, Nppc, and Nrip2 through regulation of H3K4me3 at their respective promoters. Transcript isoform analysis using Kallisto-Sleuth tool revealed 875 differentially expressed transcripts at 48 h eCG (b > 1; FDR < 0.05). Pathway analysis of RNA-seq data demonstrated that TGF-βsignaling and steroidogenic pathways were regulated at 48 h eCG. Thus, FSH regulates gene expression in granulosa cells through multiple mechanisms namely altered H3K4me3 modification and inducing specific transcripts. These data form the basis for further studies investigating how these specific mechanisms regulate granulosa cell functions.

Developmental and hormonal regulation of ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 gene expression in ovarian granulosa and theca cells of cattle.

Ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) is a multi-domain nuclear protein that plays an important role in epigenetics and tumorigenesis, but its role in normal ovarian follicle development remains unknown. Thus, the present study evaluated if UHRF1 mRNA abundance in bovine follicular cells is developmentally and hormonally regulated, and if changes in UHRF1 are associated with changes in DNA methylation in follicular cells. Abundance of UHRF1 mRNA was greater in granulosa cells (GC) and theca cells (TC) from small (<6 mm) than large (≥8 mm) follicles and was greater in small-follicle GC than TC. In GC and TC, fibroblast growth factor 9 (FGF9) treatment increased (P < 0.05) UHRF1 expression by 2-fold. Also, luteinizing hormone (LH) and insulin-like growth factor 1 (IGF1) increased (P < 0.05) UHRF1 expression in TC by 2-fold, and forskolin (an adenylate cyclase inducer) alone or combined with IGF1 increased (P < 0.05) UHRF1 expression by 3-fold. An E2F transcription factor inhibitor (E2Fi) decreased (P < 0.05) UHRF1 expression by 44% in TC and by 99% in GC. Estradiol, progesterone, and dibutyryl-cAMP decreased (P < 0.05) UHRF1 mRNA abundance in GC. Treatment of GC with follicle-stimulating hormone (FSH) alone had no effect but when combined with IGF1 enhanced the UHRF1 mRNA abundance by 2.7-fold. Beauvericin (a mycotoxin) completely inhibited the FSH plus IGF1-induced UHRF1 expression in small-follicle GC. Treatments that increased UHRF1 mRNA (i.e., FGF9) in GC tended to decrease (by 63%; P < 0.10) global DNA methylation, and those that decreased UHRF1 mRNA (i.e., E2Fi) in GC tended to increase (by 2.4-fold; P < 0.10) global DNA methylation. Collectively, these results suggest that UHRF1 expression in both GC and TC is developmentally and hormonally regulated, and that UHRF1 may play a role in follicular growth and development as well as be involved in ovarian epigenetic processes.

Core Binding Factors are essential for ovulation, luteinization, and female fertility in mice

Core Binding Factors (CBFs) are a small group of heterodimeric transcription factor complexes composed of DNA binding proteins, RUNXs, and a non-DNA binding protein, CBFB. The LH surge increases the expression of Runx1 and Runx2 in ovulatory follicles, while Cbfb is constitutively expressed. To investigate the physiological significance of CBFs, we generated a conditional mutant mouse model in which granulosa cell expression of Runx2 and Cbfb was deleted by the Esr2Cre. Female Cbfbflox/flox;Esr2cre/+;Runx2flox/flox mice were infertile; follicles developed to the preovulatory follicle stage but failed to ovulate. RNA-seq analysis of mutant mouse ovaries collected at 11 h post-hCG unveiled numerous CBFs-downstream genes that are associated with inflammation, matrix remodeling, wnt signaling, and steroid metabolism. Mutant mice also failed to develop corpora lutea, as evident by the lack of luteal marker gene expression, marked reduction of vascularization, and excessive apoptotic staining in unruptured poorly luteinized follicles, consistent with dramatic reduction of progesterone by 24 h after hCG administration. The present study provides in vivo evidence that CBFs act as essential transcriptional regulators of both ovulation and luteinization by regulating the expression of key genes that are involved in inflammation, matrix remodeling, cell differentiation, vascularization, and steroid metabolisms in mice.