Progesterone Production In Granulosa Cells Research Articles

MiR-21/SMAD2 Is Involved in the Decrease in Progesterone Synthesis Caused by Lipopolysaccharide Exposure in Follicular Granulosa Cells of Laying Goose.

Lipopolysaccharide (LPS) is one of the important pathogenic substances of E. coli and Salmonella, which causes injury to the reproductive system. Ovarian dysfunction due to Gram-negative bacterial infections is a major cause of reduced reproductive performance in geese. However, the specific molecular mechanisms of LPS-induced impairment of sex steroid hormone synthesis have not been determined. The regulatory mechanism of miRNA has been proposed in many physiological and pathogenic mechanisms. Therefore, the role of miRNA in breeding geese exposed to LPS during the peak laying period was investigated. In this study, twenty Yangzhou geese at peak laying period were injected with LPS for 0 h, 24 h, and 36 h. The follicular granulosa layer was taken for RNA-seq and analyzed for differentially expressed miRNAs. It was observed that LPS changed the appearance of hierarchical follicles. miRNA sequencing analysis was applied, and miR-21 and SMAD2 (SMAD family member 2) were selected from 51 differentially expressed miRNAs through bioinformatics prediction. The results showed that miR-21 down-regulated SMAD2 expression and progesterone (P4) production in LPS-treated goose granulosa cells (GCs). It also determined that overexpression of miR-21 or silence of SMAD2 suppressed the sex steroid biosynthesis pathway by decreasing STAR and CYP11A1 expression. Down-regulation of miR-21 exacerbates the LPS-induced decline in P4 synthesis and vice versa. The findings indicated that miR-21 was involved in LPS regulation of P4 synthesis in goose granulosa cells by down-regulating SMAD2. This study provides theoretical support for the prevention of LPS-induced ovarian dysfunction in geese.

Sirt3 Regulates Proliferation and Progesterone Production in Leydig Cells via Suppression of Reactive Oxygen Species.

Sirt3 is a mitochondrial protein deacetylase functioning in energy metabolism, regulation of intracellular reactive oxygen species (ROS) levels, and aging. Although Sirt3 loss has negative effects on fertility of oocytes during in vitro fertilization and on progesterone production in granulosa cells, Sirt3's function in Leydig cells remains unclear. Therefore, we investigated Sirt3 activity in Leydig cells, focusing on androgen production. To do so, we performed immunohistochemistry to confirm Sirt3 localization in gonads and observed strong Sirt3 immunostaining in Leydig cells of human testes and of Sirt3+/+ and Sirt3+/- mouse testes, while Sirt3-/- mouse testis tissue was negative. In human ovary, hilus cells were strongly Sirt3-positive, theca cells showed weak positivity, and granulosa cells showed very weak or almost no immunostaining. Next, we used the murine Leydig tumor cell line MA-10 as a model. We overexpressed Sirt3 but observed no changes in proliferation, expression of Star, Cyp11a1 (p450scc gene), and Hsd3b, or progesterone production in MA-10 cells. Sirt3 knockdown significantly reduced proliferation, suppressed expressions of steroidogenic enzymes and of transcription factors Ad4bp (Sf-1 gene) and Gata4, and decreased progesterone production. Sirt3 knockdown in MA-10 cells also increased intracellular ROS levels based on CM-H2DCFDA fluorescence dye analysis and increased the proportion of both early and late apoptotic (necrotic) cells based on Annexin V/7AAD assays. These results indicate that Sirt3 has a potential function in androgen production in Leydig cells by regulating intracellular ROS levels.

ERK1/2-SOX9/FOXL2 axis regulates ovarian steroidogenesis and favors the follicular-luteal transition.

Estradiol and progesterone are the primary sex steroids produced by the ovary. Upon luteinizing hormone surge, estradiol-producing granulosa cells convert into progesterone-producing cells and eventually become large luteal cells of the corpus luteum. Signaling pathways and transcription factors involved in the cessation of estradiol and simultaneous stimulation of progesterone production in granulosa cells are not clearly understood. Here, we decipher that phosphorylated ERK1/2 regulates granulosa cell steroidogenesis by inhibiting estradiol and inducing progesterone production. Down-regulation of transcription factor FOXL2 and up-regulation of SOX9 by ERK underpin its differential steroidogenic function. Interestingly, the incidence of SOX9 is largely uncovered in ovarian cells and is found to regulate FOXL2 along with CYP19A1 and STAR genes, encoding rate-limiting enzymes of steroidogenesis, in cultured granulosa cells. We propose that the novel ERK1/2-SOX9/FOXL2 axis in granulosa cells is a critical regulator of ovarian steroidogenesis and may be considered when addressing pathophysiologies associated with inappropriate steroid production and infertility in humans and animals.

Effect of IGF1 and FSH on the function of granulosa cells from prehierarchal follicles in chickens†.

Insulin-like growth factor 1 (IGF1) is an essential regulator of mammalian follicle development and synergizes with follicle-stimulating hormone (FSH) to amplify its effects. In avian preovulatory follicles, IGF1 increases the expression of genes involved in steroidogenesis and progesterone and inhibin A production. The role of IGF1 in prehierarchal follicles has not been well studied in chickens. The aim of this study was to investigate the role of IGF1 in granulosa cells from prehierarchal follicles and to determine whether IGF1 and FSH synergize to promote follicle development. Granulosa cells of 3-5 and 6-8mm prehierarchal follicles were cultured with IGF1 (0, 10, 100ng/mL) in the presence or absence of FSH (0, 10ng/mL). Cell proliferation, expression of genes important in follicle development (FSHR, IGF1R, AMH, STAR, CYP11A1, INHA, and INHBA), and progesterone production were evaluated following treatment. IGF1 treatment alone significantly increased STAR, CYP11A1, and INHBA mRNA expression and cell proliferation in granulosa cells of 6-8mm follicles. IGF1 and FSH synergized to increase STAR mRNA expression in 6-8mm follicles. IGF1 and FSH co-treatment were necessary to increase INHA mRNA expression in 6-8mm follicles. Although IGF1 significantly increased the expression of genes involved in steroidogenesis, progesterone production in granulosa cells of 6-8mm follicles was not affected. IGF1 did not affect AMH mRNA expression, although FSH significantly decreased AMH expression in granulosa cells of 3-5mm follicles. These results suggest that IGF1 may act with FSH to promote follicle selection at the prehierarchal follicle stage.

Corticosterone stage-dependently inhibits progesterone production presumably via impeding the cAMP-StAR cascade in granulosa cells of chicken preovulatory follicles

Stress can suppress reproduction capacity in either wild or domestic animals, but the exact mechanism behind it, especially in terms of steroidogenesis, remains under-investigated so far. Considering the important roles of progesterone in avian breeding, we investigated the modulation of corticosterone on progesterone production in cultured granulosa cells of chicken follicles at different developmental stages. Using enzyme immunoassays, our study showed that corticosterone could only inhibit progesterone synthesis in granulosa cells from F5-6, F4, and F3 follicles, but not F2 and F1 follicles. Coincidentally, both quantitative real-time PCR and western blotting revealed that corticosterone could downregulate steroidogenic acute regulatory protein (StAR) expression, suggesting the importance of StAR in corticosterone-related actions. Using the dual-luciferase reporter system, we found that corticosterone can potentially enhance, rather than inhibit, the activity of StAR promoter. Of note, combining high-throughput transcriptomic analysis and quantitative real-time PCR, phosphodiesterase 10A (PDE10A), protein kinase cAMP-dependent type II regulatory subunit alpha (PRKAR2A) and cAMP responsive element modulator (CREM) were identified to exhibit the differential expression patterns consistent with cAMP blocking in granulosa cells from F5-6, F4, and F3, but not F2 and F1 follicles. Afterward, the expression profiles of these genes in granulosa cells of distinct developmental-stage follicles were examined by quantitative real-time PCR, in which all of them expressed correspondingly with progesterone levels of granulosa cells during development. Collectively, these findings indicate that corticosterone can stage-dependently inhibit progesterone production in granulosa cells of chicken preovulatory follicles, through impeding cAMP-induced StAR activity presumptively.

TGF-β1 induces type I collagen deposition in granulosa cells via the AKT/GSK-3β signaling pathway-mediated MMP1 down-regulation.

Type I collagen is the most abundant extracellular matrix (ECM) protein in the mammalian ovary, and comprises two COL1A1 subunits and one COL1A2 subunit. Matrix metalloproteinase 1 (MMP1) is a typical collagenase of type I collagen, that can be detected in ovarian follicles and early corpus luteum. Previous studies demonstrated that MMP1-mediated degradation of type I collagen plays a functional role in regulating corpus luteum formation, and transforming growth factor β1 (TGF-β1) inhibits luteinization and progesterone production in granulosa cells (GCs). Whether TGF-β1 regulates the expression of MMP1, COL1A1, or the deposition of type I collagen during corpus luteum formation remains to be elucidated. This study aimed to investigate the molecular mechanisms through which TGF-β1 regulates MMP1 expression and type I collagen deposition in GCs. Our results show that TGF-β1 upregulates COL1A1 expressions and downregulates MMP1 expression. Inhibition approaches, including pharmacological inhibitors such as p38 inhibitor (SB203580), ERK1/2 inhibitor (U0126), AKT inhibitor (LY294002), and GSK-3β inhibitor (LiCl), as well as knockdown using siRNA specific to these genes, were used. Our results suggest that TGF-β1 decreases MMP1 production via an ALK5-mediated AKT/GSK-3β-dependent signaling pathway, and a decrease in MMP1 levels and an increase in COL1A1 levels synergistically promote type I collagen deposition in GCs. Collectively, these findings provide novel insights into the underlying molecular mechanisms by which TGF-β1 upregulates type I collagen deposition in GCs.

AREG upregulates secreted protein acidic and rich in cysteine expression in human granulosa cells

The secreted protein acidic and rich in cysteine (SPARC) is a secreted glycoprotein and the expression of ovarian SPARC peaks during ovulation and luteinization. Besides, SPARC expression was induced by human chorionic gonadotropin (hCG) in rat granulosa cells. Amphiregulin (AREG) is the most abundant epidermal growth factor receptor (EGFR) ligand expressed in human granulosa cells and follicular fluid. AREG mediates the physiological functions of luteinizing hormone (LH)/hCG in the ovary. However, to date, the biological function of SPARC in the human ovary remains undetermined, and whether AREG regulates SPARC expression in human granulosa cells is unknown. In this study, we show that AREG upregulated SPARC expression via EGFR in a human granulosa-like tumor cell line, KGN. Treatment of AREG activated ERK1/2, JNK, p38 MAPK, and PI3K/AKT signaling pathways and all of them were required for the AREG-induced SPARC expression. Using RNA-sequencing, we identified that steroidogenic acute regulatory protein (StAR) was a downstream target gene of SPARC. In addition, we demonstrated that SPARC mRNA levels were positively correlated with the levels of StAR mRNA in the primary culture of human granulosa cells. Moreover, SPARC protein levels were positively correlated with progesterone levels in follicular fluid of in vitro fertilization patients. This study provides the regulatory role of AREG on the expression of SPARC and reveals the novel function of SPARC in progesterone production in granulosa cells.

TGF-β1 suppresses de novo cholesterol biosynthesis in granulosa-lutein cells by down-regulating DHCR24 expression via the GSK-3β/EZH2/H3K27me3 signaling pathway

Cholesterol is a precursor to steroid hormones and can be obtained from serum LDL or de novo synthesis in steroidogenic cells. Before luteinizing hormone (LH) surge-induced ovulation, follicles remain avascular, and cholesterol required for progesterone production in granulosa cells (GCs) is derived from de novo biosynthesis. Previous studies have verified that the intrafollicular TGF-β1 plays inhibitory roles in GCs luteinization, vascularization, and progesterone production. Nevertheless, the regulatory function of TGF-β1 on de novo cholesterol synthesis in granulosa-lutein (GL) cells remains largely unknown. We aim to investigate this aspect in this study using in vivo cultured human GL cells. Our results suggested that TGF-β1 significantly suppresses intracellular cholesterol levels and down-regulates the expression of the final step enzyme, DHCR24, that catalyzes de novo cholesterol synthesis. We used specific inhibitors and siRNA-mediated knockdown approaches demonstrate that TGF-β1 suppression of DHCR24 expression in GL cells is mediated by the GSK-3β/EZH2/H3K27me3 signaling pathway. Further ChIP assays revealed that elevated H3K27me3 levels in the promoter region of DHCR24 play a vital role in TGF-β1-induced DHCR24 down-regulation, and RNA-sequencing results confirmed these findings. Notably, our study provides a novel insight into the molecular mechanisms by which TGF-β1 suppresses de novo cholesterol biosynthesis in GL cells.

Palmitic acid protects granulosa cells from oleic acid induced steatosis and rescues progesterone production via cAMP dependent mechanism

• Palmitate is not lipotoxic in granulosa cells. • Oleate induces steatosis and downregulates progesterone production in granulosa cells. • Palmitate prevents the oleate induced granulosa cell dysfunction. • cAMP levels regulate lipid droplet accumulation and progesterone production in granulosa cells.

Cholesterol uptake or trafficking, steroid biosynthesis, and gonadotropin responsiveness are defective in young poor responders

To investigate whether poor ovarian response in young patients undergoing invitro fertilization simply involves lesser follicle growth due to diminished ovarian reserve or whether there are intrinsic perturbations in the ovary. A translational research study. University Hospital Translational Research Center. A total of 40 patients undergoing invitro fertilization (20 normal and 20 poor responders) with ovarian stimulation using a gonadotropin-releasing hormone antagonist and recombinant follicle-stimulating hormone were included in the study. None. Luteal granulosa cells obtained during oocyte retrieval procedures were used for the experiments. Cell culture, quantitative real-time polymerase chain reaction, immunoblotting, confocal time-lapse live-cell imaging, and hormone assays were used. We tracked the steroidogenic pathway starting from the very initial step of cholesterol uptake to the final step of estradiol and progesterone production in luteal granulosa cells and identified some previously unknown intrinsic defects in the poor responders. Most notably, the expression of low-density lipoprotein receptors was significantly down-regulated and the uptake of cholesterol and its cytoplasmic accumulation and transportation to mitochondria were substantially delayed and reduced in the poor responders. Further, the expression of the steroidogenic enzymes steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, and aromatase as well as gonadotropin receptors was defective, and the response of the cells to exogenous follicle-stimulating hormone and human chorionic gonadotropin was blunted, leading to compromised basal and gonadotropin-stimulated estradiol and progesterone production in the poor responders. This study demonstrates that poor ovarian response in young individuals should not simply be regarded as lesser follicle growth due to diminished ovarian reserve because the underlying pathogenetic mechanisms appear to be much more complex.

Oleic acid reduces steroidogenesis by changing the lipid type stored in lipid droplets of ovarian granulosa cells

BackgroundOleic acid is an abundant free fatty acid present in livestock that are in a negative energy-balance state, and it may have detrimental effects on female reproduction and fertility. Oleic acid induces lipid accumulation in bovine granulosa cells, which leads to a foam cell-like morphology and reduced steroidogenesis. However, why oleic acid increases lipid accumulation but decreases steroidogenesis remains unclear. This study focused on oleic acid’s effects on lipid type and steroidogenesis.ResultsOleic acid increased the lipid accumulation in a concentration-dependent manner and mainly increased the triglyceride level and decreased the cholesterol ester level. Oleic acid also led to a decline in estradiol and progesterone production in porcine granulosa cells in vitro. In addition, oleic acid up-regulated the expression of CD36 and diacylglycerol acyltransferase 2, but down-regulated the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, scavenger receptor class B member 1 and acetyl-Coenzyme A acetyltransferase 2, as well as steroidogenesis-related genes, including cytochrome P450 family 11 subfamily A member 1, cytochrome P450 family 19 subfamily A member 1 and 3 as well as steroidogenic acute regulatory protein at the mRNA and protein levels. An oleic acid-rich diet also enhanced the triglyceride levels and reduced the cholesterol levels in ovarian tissues of female mice, which resulted in lower estradiol levels than in control-fed mice. Compared with the control, decreases in estrus days and the numbers of antral follicles and corpora lutea, as well as an increase in the numbers of the atretic follicles, were found in the oleic acid-fed female mice.ConclusionsOleic acid changed the lipid type stored in lipid droplets of ovarian granulosa cells, and led to a decrease in steroidogenesis. These results improve our understanding of fertility decline in livestock that are in a negative energy-balance state.

An integrated RNA-Seq and network study reveals that valproate inhibited progesterone production in human granulosa cells

BackgroundValproate (VPA) is an antiepileptic drug (AEDs) with an ideal effect against epilepsy as well as other neuropsychiatric diseases. There is considerable evidence that women taking VPA are prone to reproductive endocrine disorders. However, few studies have been published about VPA effects on human ovarian granulosa cells. MethodsBy treating human ovarian granulosa cell line KGN with VPA, the cell viability and progesterone production function were evaluated. RNA-sequencing was applied to uncover the global gene expression upon VPA treatment. ResultsWe revealed that VPA dose-dependently repressed the viability of KGN. VPA treatment at 600 μM inhibited the progesterone production. The mRNA and protein expression of CYP11A1 and STAR, two key enzymes in the biosynthesis of progesterone, were both suppressed. Gene set enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis of the transcriptome revealed classical functions of VPA as a neuromodulator and regulator of histone acetylation modifications. In addition to this, VPA commonly affected many steroid metabolism related genes in follicle cells, such as promoting the expression of vitamin D receptor (VDR). ConclusionOur findings suggest that VPA caused steroids metabolism pathways disturbance related with ovarian function and inhibited progesterone biosynthesis by inhibiting the expression of steroidogenesis genes. Our research may provide theoretical basis for the better use of VPA and the possible ways to counteract its side effects.

Epigallocatechin-3-gallate stimulates StAR expression and progesterone production in human granulosa cells through the 67-kDa laminin receptor-mediated CREB signaling pathway.

Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechins extracted from green tea. The health benefits of EGCG have been extendedly studied. Ovarian steroidogenesis plays a pivotal role in maintaining normal reproductive function. Granulosa cells in the ovary are essential for steroid hormone production. To date, the effect of EGCG on steroidogenesis in human granulosa cells remains unclear. In the present study, we examine the physiological concentrations of EGCG on steroidogenesis in a steroidogenic human granulosa-like tumor cell line, KGN. Our results demonstrate that treatment with EGCG upregulates steroidogenic acute regulatory protein (StAR) expression and increases progesterone (P4) production. EGCG does not affect the expression levels of other steroidogenesis-related enzymes, such as P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. In addition, we identify the expression of 67-kDa laminin receptor (67LR) in KGN cells. Moreover, EGCG-induced StAR expression and P4 production require the 67LR-mediated activation of the PKA-CREB signaling pathway. These results provide a better understanding of the function of EGCG on ovarian steroidogenesis, which may lead to the development of alternative therapeutic approaches for reproductive disorders.

Amphetamine-Decreased Progesterone and Estradiol Release in Rat Granulosa Cells: The Regulatory Role of cAMP- and Ca2+-Mediated Signaling Pathways.

The purpose of this study is to evaluate the amphetamine effects on progesterone and estradiol production in rat granulosa cells and the underlying cellular regulatory mechanisms. Freshly dispersed rat granulosa cells were cultured with various test drugs in the presence of amphetamine, and the estradiol/progesterone production and the cytosolic cAMP level were measured. Additionally, the cytosolic-free Ca2+ concentrations ([Ca2+]i) were measured to examine the role of Ca2+ influx in the presence of amphetamine. Amphetamine in vitro inhibited both basal and porcine follicle-stimulating hormone-stimulated estradiol/progesterone release, and amphetamine significantly decreased steroidogenic enzyme activities. Adding 8-Bromo-cAMP did not recover the inhibitory effects of amphetamine on progesterone and estradiol release. H89 significantly decreased progesterone and estradiol basal release but failed to enhance a further amphetamine inhibitory effect. Amphetamine was capable of further suppressing the release of estradiol release under the presence of nifedipine. Pretreatment with the amphetamine for 2 h decreased the basal [Ca2+]i and prostaglandin F2α-stimulated increase of [Ca2+]i. Amphetamine inhibits progesterone and estradiol secretion in rat granulosa cells through a mechanism involving decreased PKA-downstream steroidogenic enzyme activity and L-type Ca2+ channels. Our current findings show that it is necessary to study the possibility of amphetamine perturbing reproduction in females.

Melatonin alleviates deoxynivalenol-induced apoptosis of human granulosa cells by reducing mutually accentuated FOXO1 and ER stress‡.

Deoxynivalenol (DON) is one of the most prevalent Fusarium mycotoxins, which cause detrimental effects on human and animal reproductive systems by inducing oxidative stress. Increasing evidence has suggested the potential roles of melatonin in protecting granulosa cells from oxidative injury, but the underlying mechanisms remain largely elusive. Here, we demonstrated that suppression of FOXO1 and endoplasmic reticulum (ER) stress was engaged in melatonin-mediated protection against oxidative damage in human granulosa cells upon DON exposure in vitro. DON induced excess reactive oxygen species accumulation, cells viability loss, reduced estradiol-17β, and progesterone production in human granulosa cells, whereas melatonin ameliorated these phenotypes. Next, we found that the protective effect of melatonin against apoptosis was via reducing ER stress because the inhibition of ER stress displayed similar protective effects during DON treatment. Moreover, melatonin provided no additional protection when ER stress was inhibited. We further found that FOXO1 is a pivotal downstream effector of melatonin and ER stress in regulating DON-induced apoptosis in human granulosa cells. Blocking of FOXO1 reduced DON-induced cells death and FOXO1 activation could be suppressed by melatonin or ER stress inhibitor. However, melatonin failed to further restore cells viability in the presence of FOXO1 inhibitor. Collectively, our results reveal a new mechanism of melatonin in protecting against DON-induced apoptosis and dysfunction by suppressing ER stress and FOXO1 in human granulosa cells.

Effects of selected hormones and their combination on progesterone and estradiol production and proliferation of feline granulosa cells cultured in vitro

Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. The present study aimed to develop a hormone responsive granulosa cell culture system to measure steroidogenic and cell proliferation responses to help identify factors that might regulate ovarian function in queens. Five experiments were conducted each with 75 or more ovaries, three in spring and two in fall seasons. Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48 h after an initial 48 h plating in 10% fetal calf serum. In granulosa cells isolated from spring and fall collected feline ovaries, IGF1 alone and combined with FSH stimulated (P < 0.05) cell proliferation, whereas FSH alone had no effect (P > 0.10) on cell proliferation. Also, in granulosa cells collected in spring and fall, IGF1 alone and FSH alone increased (P < 0.05) estradiol production by severalfold, and a combination of FSH and IGF1 increased (P < 0.05) estradiol production above either FSH or IGF1 treatment alone. The FSH plus IGF1 treatment increased (P < 0.05) CYP19A1 mRNA abundance by 27-fold. In contrast, EGF decreased (P < 0.05) FSH plus IGF1-induced estradiol production by over 80% in granulosa cells of both spring and fall collected ovaries. In granulosa cells isolated from spring and fall collected ovaries, IGF1 plus FSH inhibited (P < 0.05) progesterone production. Melatonin increased (P < 0.05) FSH plus IGF1-induced cell proliferation and amplified (P < 0.05) the FSH plus IGF1-induced inhibition of progesterone production. However, melatonin and GH had no effect (P > 0.10) on estradiol production either alone or in combination with FSH plus IGF1 in both spring and fall. Prolactin, FGF9 and activin had no effect (P > 0.10) on cell proliferation or steroidogenesis. FGF2 decreased (P < 0.05) estradiol production without affecting progesterone production or cell numbers. Growth differentiation factor 9 (GDF9) increased (P < 0.05) progesterone production but had no effect (P > 0.10) on granulosa cell proliferation or estradiol production. In conclusion, the in vitro system described herewithin may be useful to assess and evaluate ovarian function in feline species and has identified EGF, FSH and IGF1 as major regulators of feline ovarian follicular function.

FSH Promotes Progesterone Synthesis by Enhancing Autophagy to Accelerate Lipid Droplet Degradation in Porcine Granulosa Cells.

Little is known about the molecular relationships among follicle stimulating hormone (FSH), lipid droplet (LD) degradation, and autophagy. In this study, we aimed to investigate the pathway by which FSH regulates autophagy and the potential role of autophagy in progesterone production. Our results revealed that FSH stimulated progesterone production in mammalian follicular granulosa cells (GCs) through a non-canonical pathway. In porcine secondary follicles cultured in vitro, FSH treatment increased the level of the autophagic marker, LC3-II, as well as increased the number of autophagic vacuoles in GCs. The underlying molecular mechanism and biological functions were then investigated in porcine GCs. Our results demonstrated that FSH could upregulate Beclin1 levels in porcine GCs; however, this effect was blocked by LY294002 (a PI3K/AKT inhibitor) and SP600125 (SAPK/JNK inhibitor). Further research confirmed that the transcriptional factor, c-Jun, was phosphorylated by FSH, then translocated into the nucleus from the cytoplasm and bound to the BECLIN1 promoter region, and that LY294002, SP600125, or c-Jun knockdown prevented the increase in Beclin1 levels induced by FSH. Interestingly, inhibition of autophagy using chloroquine or SP600125 decreased progesterone production in porcine GCs treated with FSH, although the expression of StAR and P450scc was not disturbed. Moreover, FSH treatment reduced the average number and size of LDs in porcine GCs, but these effects were eliminated by knocking down the key autophagy genes, ATG5 and BECLIN1; in addition, the effect of FSH on promoting progesterone secretion by the cells was also reduced significantly. Based on the above results, we concluded that FSH promoted progesterone production by enhancing autophagy through upregulation of Beclin1 via the PI3K/JNK/c-Jun pathway to accelerate LD degradation in porcine GCs, independent of the classical steroidogenic pathway.

Effects of the FABP4 gene on steroid hormone secretion in goose ovarian granulosa cells

ABSTRACT 1. To investigate the physiological role of FABP4 in the goose ovary, this study determined the effects of overexpressing and siRNA interfering FABP4 on progesterone (P4) and oestradiol (E2) production in granulosa cells. Measurements were made by ELISA, real-time qRT-PCR and western blotting. 2. The concentrations of P4 and E2 in the FABP4 overexpression granulosa cells were increased compared to the control group (P > 0.05 for P4; P < 0.05 for E2). Likewise, the mRNA and protein expression levels of CYP11A1 and CYP19A1 were significantly higher than in the control group (P < 0.05 or P < 0.001). Conversely, the concentrations of P4 and E2 in the FABP4 silencing granulosa cells were significantly decreased compared with the control group (P < 0.001). Likewise, the mRNA and protein expression levels of CYP11A1 and CYP19A1 were significantly lower than in the control group (P < 0.001, or P < 0.01). 3. The study indicated that the FABP4 gene may regulate steroid hormone secretion and the expression of the steroidogenic genes in geese ovarian granulosa cells. These results support the possibility that the FABP4 gene mediates ovarian steroid hormone biosynthesis function and reproduction in geese.

Differential actions of diacylglycerol acyltransferase (DGAT) 1 and 2 in regulating lipid metabolism and progesterone secretion of goose granulosa cells

Accumulating evidence shows that granulosa cells within both mammalian and avian ovaries have the ability to synthesize fatty acids through de novo lipogenesis and to accumulate triglycerides essential for oocyte and ovarian development. However, very little is known about the exact roles of key genes involved in the lipid metabolic pathway in granulosa cells. The goal of this study was to investigate the differential actions of diacylglycerol acyltransferase (DGAT) 1 and 2, which are recognized as the rate-limiting enzymes catalyzing the last step of triglyceride biosynthesis, in regulating lipid metabolism and steroidogenesis in granulosa cells of goose follicles at different developmental stages. It was observed that the mRNAs encoding DGAT1 and DGAT2 were ubiquitous in all examined granulosa cell layers but exhibited distinct expression profiles during follicle development. Notably, the mRNA levels of DGAT1, DGAT2, FSHR, LHR, STAR, CYP11A1, and 3βHSD remained almost constant in all except for 1–2 follicles within the 8−10 mm cohort, followed by an acute increase/decrease in the F5 follicles. At the cellular level, siRNA-mediated downregulation of DGAT1 or DGAT2 did not change the amount of lipids accumulated in both undifferentiated- and differentiated granulosa cells, while overexpression of DGAT2 promoted lipid accumulation and expression of lipogenic-related genes in these cells. Meanwhile, we found that interfering DGAT2 had no effect but interfering DGAT1 or overexpressing DGAT2 stimulated progesterone secretion in undifferentiated granulosa cells; in contrast, interference or overexpression of DGAT1/2 failed to change progesterone levels in differentiated granulosa cells but differently modulated expression of steroidogenic-related genes. Therefore, it could be concluded that DGAT1 is less efficient than DGAT2 in promoting lipid accumulation in both undifferentiated- and differentiated granulosa cells and that DGAT1 negatively while DGAT2 positively regulates progesterone production in undifferentiated granulosa cells.

Human BMP8A suppresses luteinization of rat granulosa cells via the SMAD1/5/8 pathway.

Bone morphogenetic proteins (BMPs) are known to play an indispensable role in preventing the precocious luteinization of granulosa cells within growing ovarian follicles. In this study, we found that the transcripts of BMP8 genes are enriched in the ovaries of humans and rodents. When analyzing transcriptomic datasets obtained from human mature granulosa cells, we further found that the BMP8 transcripts not only show the highest abundance among the searchable BMP-related ligands but also decrease significantly in women of advanced age or women with polycystic ovarian syndrome. The correlation between the BMP8 levels in granulosa cells and the decline in ovarian function in these subjects suggests that BMP8 protein may be involved in the regulation of granulosa cell function(s). Using a rat model, we demonstrated that human BMP8A protein activates the SMAD1/5/8 and the SMAD2/3 pathways simultaneously in both immature and mature granulosa cells. Furthermore, the expression of potential type I and type II receptors used by BMP8 in rat granulosa cells was characterized. We found that BMP8A treatment can significantly inhibit gonadotropin-induced progesterone production and steroidogenesis-related gene expression in granulosa cells. Pathway dissection using receptor inhibitors further revealed that such inhibitory effects occur specifically through the BMP8-activated SMAD1/5/8, but not SMAD2/3, pathway. Taken together, considering its abundance and possible functions in granulosa cells, we suggest that BMP8 may act as a novel luteinization inhibitor in growing follicles.