Response Of Granulosa Cells Research Articles

Effects of Inhibin A on Apoptosis and Proliferation of Bovine Granulosa Cells.

Simple SummaryThe ovarian granulosa cells play a crucial role in oocyte nourishing, secreting hormones that create functional bidirectional crosstalk with the oocyte. During follicle development, granulosa cells replicate, secrete hormones, and provide a critical microenvironment for follicular growth. Proliferation and differentiation of granulosa cells are essential for normal follicular growth, development of the oocyte, ovulation, and luteinization. Inhibins negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary, controlling ovarian follicle development. This study was aimed to explore the cellular and molecular adaptation of bovine granulosa cells under different concentrations of inhibin A. For instance, several physiological traits (cell viability, apoptosis, mitochondrial membrane potential, and cell proliferation) were assessed under different concentrations (0, 20, 50, and 100 ng/mL) of inhibin A. Results depicted that high doses of inhibin A boosted cell viability, mitochondrial membrane integrity, and cell proliferation, while inhibiting apoptotic rate in bovine granulosa cells.Inhibin A is well known for its inhibitory properties against follicle-stimulating hormone (FSH), released through a pituitary–gonadal negative feedback loop to regulate follicular development. Ovarian folliculogenesis, hormonal biosynthesis, and gametogenesis are dependent on inhibins, playing vital roles in promoting or inhibiting cell proliferation. The present study explored the physiological and molecular response of bovine granulosa cells (GCs) to different concentrations of inhibin A in vitro. We treated the primary GCs isolated from ovarian follicles (3–6 mm) with different levels of inhibin A (20, 50, and 100 ng/mL) along with the control (0 ng/mL) for 24 h. To evaluate the impact of inhibin A on GCs, several in vitro cellular parameters, including cell apoptosis, viability, cell cycle, and mitochondrial membrane potential (MMP) were detected. Besides, the transcriptional regulation of pro-apoptotic (BAX, Caspase-3) and cell proliferation (PCNA, CyclinB1) genes were also quantified. The results indicated a significant (p < 0.05) increase in the cell viability in a dose-dependent manner of inhibin A. Likewise, MMP was significantly (p < 0.05) enhanced when GCs were treated with high doses (50, 100 ng/mL) of inhibin A. Furthermore, inhibin A dose (100 ng/mL) markedly improved the progression of the G1 phase of the cell cycle and increased the cell number in the S phase, which was supported by the up-regulation of the proliferating cell nuclear antigen PCNA (20, 50, and 100ng/mL) and CyclinB (100 ng/mL) genes. In addition, higher doses of inhibin A (50 and 100 ng/mL) significantly (p < 0.05) decreased the apoptotic rate in GCs, which was manifested by down regulating BAX and Caspase-3 genes. Conclusively, our study presented a worthy strategy for the first time to characterize the cellular adaptation of bovine GCs under different concentrations of inhibin A. Our results conclude that inhibin A is a broad regulatory marker in GCs by regulating apoptosis and cellular progression.

MicroRNA-135a Regulates VEGFC Expression and Promotes Luteinized Granulosa Cell Apoptosis in Polycystic Ovary Syndrome.

Androgen is known to regulate microRNA-135a (miR-135a) and can be regulated by androgen, suggesting that it may contribute to polycystic ovary syndrome (PCOS) with hyperandrogenism. However, its roles and mechanisms of action in PCOS are unknown. In this study, the role and molecular mechanisms underlying miR-135a in granulosa cells (GCs) in PCOS were evaluated. miR-135a expression was upregulated in patients with PCOS and in GCs isolated from patients compared with that in the respective controls (P < 0.01), as determined by RT-qPCR. The overexpression of miR-135a inhibited GC proliferation and induced GC apoptosis, as observed by CCK-8 assay and apoptosis assay. Furthermore, miR-135a overexpression increased the expression of double-strand break maker, γH2AX, as confirmed by western blotting. Our results further suggest that these effects were mediated via downregulation of vascular endothelial growth factor C (VEGFC), which was identified as a direct target of miR-135a. Moreover, levels of VEGFC and miR-135a expression showed a negative correlation. These findings indicate that miR-135a promotes apoptosis and the DNA damage response in GCs in PCOS, likely via VEGFC signaling. This study provides novel insights into GC dysregulation in PCOS and suggests that miR-135a is a promising therapeutic target for PCOS treatment.

Effect of cryopreservation on human granulosa cell viability and responsiveness to gonadotropin.

In human, the use of freshly recovered granulosa cells for experiments remains difficult. Because of the single use of human cells, the experiments cannot be repeated, and no additional conditions can be tested afterwards with the cells of the same patient. Therefore, granulosa cell cryopreservation could be a good alternative to keep part of these cells for later controls or experiments. The aim of this study is to compare the responsiveness to FSH of fresh and frozen-thawed human primary granulosa-lutein cells (hGLC) and determine if cryopreserved granulosa cells can be used in place of fresh cells. Two cryopreservation methods were also compared: a conventional versus a simplified freezing method. This experimental study was undertaken at Igyxos S.A., Nouzilly, France. Seventy women undergoing oocyte retrieval at the IVF Unit from Bretonneau University Hospital (Tours, France) were recruited in 2016. Fresh and frozen-thawed hGLC were cultured for 7 days and then stimulated by r-FSH for 48 h. To assess r-FSH efficacy and potency, extracellular cAMP accumulated in the supernatant for each stimulation point was measured. We demonstrated that hGLC remain responsive to FSH stimulation after freezing-thawing and 7 days of pre-culture. They are able to secrete cAMP with a similar EC50 value as fresh hGLC, but FSH efficacy is lowered. As our study did not show any significant difference between the two freezing methods concerning the sensitivity of hGLC to FSH, hGLC could be cryopreserved with the simplified freezing method without taking up too much time for IVF laboratories.

Polyinosinic-polycytidylic acid induces innate immune responses via Toll-like receptor 3 in human ovarian granulosa cells.

The ovary can be infected by a variety of viruses, which may come from the female reproductive tract or the peritoneum. The innate immune responses to viral infection in the human ovary are poorly understood. The present study demonstrated that human ovarian granulosa cells had innate immune activity in response to viral RNA challenge through Toll-like receptor 3 (TLR3) activation. TLR3 was constitutively expressed in the human ovary and predominantly located in granulosa cells of developmental follicles at all stages. Polyinosinic-polycytidylic acid [poly (I:C)], a synthetic viral double-stranded RNA analog, induced innate immune responses in human ovarian granulosa cells and affected endocrine function. Poly (I:C) significantly upregulated proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1β and type I interferon (IFN-α/β), and the innate immune responses were significantly reduced by blocking TLR3 signaling. Furthermore, poly (I:C) induced antiviral genes expression, including 2'-5'-oligoadenylate synthetase, Mx GTPase 1, IFN-stimulating gene 15 and double-stranded RNA-activated protein kinase R. In contrast, the expression of P450 aromatase and inhibin was dramatically inhibited by poly (I:C). Both silencing of TLR3 and neutralizing TNF-α reversed the inhibitory effect of poly (I:C) on P450 aromatase and inhibin expression. Our study demonstrates that granulosa cells play a potential role in innate immune protection against viral infection in the normal human ovary, and the innate immune response perturbs cell endocrine function.

Development of the dominant follicle: mechanisms of selection and maintenance of oocyte quality

For a follicle to reach dominance, in mono-ovulatory species such as cattle, requires the integration of a number of processes involving both extra-ovarian signals and intra-follicular paracrine and autocrine regulators. Ovarian transplant studies in both cattle and sheep demonstrated that it takes approximately 4 months for primordial follicles to reach dominance. Gonadotrophins are not a prerequisite for the continued growth of pre-antral follicles, unlike antral follicles, but FSH does appear to stimulate development. Local growth factors, such as IGFs and BMPs, are expressed throughout follicle development and interact with gonadotrophins to stimulate development. As follicles become dominant, there is a transfer of dependency from FSH to LH. There are also differences in LH-responsiveness of theca and granulosa cells during follicular development, due to differential regulation and control by intricate local mechanisms altering LH receptor (LHR) mRNA expression. In addition, both the BMP and IGF systems can modulate the proliferative and differentiative responses of both granulosa and theca cells to gonadotrophins. There is a significant interaction between BMPs and the IGF system in regulating follicular development. A range of factors, including nutrition, will also determine the fate of the growing follicle and the quality of the oocyte. Nearly all follicles regress and apoptotic cell death throughout follicular development is an underlying mechanism of cell loss during follicular atresia. Several markers of follicular atresia have been identified including IGFBPs. There is a significant correlation between the presence of low molecular weight IGFBPs in bovine follicular fluid and caspase-3 activity of granulosa cells in individual follicles. In conclusion, it is the interaction between extra-ovarian and intra-ovarian factors that determine the fate of the follicle and the quality of the oocyte.

SFRP4 Is a Negative Regulator of Ovarian Follicle Development and Female Fertility.

WNT signaling regulates a variety of ovarian processes, including follicle development, granulosa cell (GC) proliferation and differentiation, steroidogenesis, and ovulation. The secreted frizzled-related proteins (SFRPs) comprise a family of WNT signaling antagonists. Sfrp4 expression was previously reported to be induced in ovarian GCs and cumulus cells in vivo following human chorionic gonadotropin treatment, suggesting that it may play key roles in cumulus expansion, ovulation/luteinization, and corpus luteum (CL) function. In this study, we aimed to define the physiological roles of Sfrp4 in the ovary by gene targeting. Sfrp4-null female mice were found to produce larger litters than did their wild-type littermates. Although previous studies had suggested roles of Sfrp4 in luteal cell survival, no differences in CL formation, morphology, steroidogenesis, involution, or luteal cell apoptosis were found in Sfrp4-null mice. Likewise, cumulus expansion occurred normally in Sfrp4-null mice, with minimal changes in cumulus cell gene expression. Hyperfertility in the Sfrp4-null model was ultimately attributed to decreased antral follicle atresia, leading to an enhanced ovulatory rate. Increased expression of FSH- and LH-responsive genes was found in GCs from Sfrp4-null mice, and GCs isolated from Sfrp4-null mice were found to be hyperresponsive to FSH and LH in vitro. Although Sfrp2 was found to be overexpressed in the GCs of Sfrp4-null mice (suggesting a compensatory mechanism), Sfrp2-null mice had normal fertility and ovulatory rates, and Sfrp2/4 double knockout mice did not differ from Sfrp4-null mice. Taken together, our results suggest that SFRP4 acts to attenuate GC responsiveness to gonadotropins, thereby decreasing follicle survival, ovulatory rate, and fertility.

Endogenous and Exogenous Modulation of Nrf2 Mediated Oxidative Stress Response in Bovine Granulosa Cells: Potential Implication for Ovarian Function.

Nrf2 is a redox sensitive transcription factor regulating the expression of antioxidant genes as defense mechanism against various stressors. The aim of this study is to investigate the potential role of noncoding miRNAs as endogenous and quercetin as exogenous regulators of Nrf2 pathway in bovine granulosa cells. For this cultured granulosa cells were used for modulation of miRNAs (miR-28, 153 and miR-708) targeting the bovine Nrf2 and supplementation of quercentin to investigate the regulatory mechanisms of the Nrf2 antioxidant system. Moreover, cultured cells were treated with hydrogen peroxide to induce oxidative stress in those cells. Our results showed that, oxidative stress activated the expression of Nrf2 as a defense mechanism, while suppressing the expression of those miRNAs. Overexpression of those miRNAs resulted in downregulation of Nrf2 expression resulted in higher ROS accumulation, reduced mitochondrial activity and cellular proliferation. Quercetin supplementation showed its protective role against oxidative stress induced by H2O2 by inducing the expression of antioxidant enzymes. In conclusion, this study highlighted the involvement of miR-153, miR-28 and miR-708 in regulatory network of Nrf2 mediated antioxidant system in bovine granulosa cells function. Furthermore, quercetin at a low dose played a protective role in bovine granulosa cells against oxidative stress damage.

Influence of green tea constituents on cultured porcine luteinized granulosa cell functions

The aim of the study was to examine the action of green tea polyphenols (GTPP) and their main constituent, epigallocatechin-3-gallate (EGCG), on porcine ovarian granulosa cells. For this purpose, the effect of GTPP and EGCG on cultured porcine ovarian granulosa cell functions, including proliferation, apoptosis, steroidogenesis and response to insulin-like growth factor I (IGF-I) was examined. Proliferation (the accumulation of proliferating cell nuclear antigen (PCNA) and cyclin B1), apoptosis (the accumulation of bax and caspase 3) and the release of steroid hormones (progesterone and testosterone) were evaluated by using immunocytochemistry and enzyme immunoassay. The addition of both GTPP and EGCG reduced the percentage of both PCNA- and cyclin B1-positive cells, increased the proportion of cells containing bax and caspase 3 and stimulated progesterone release. GTPP had a biphasic effect on testosterone output – stimulating at dose 1 μg/ml and inhibiting at doses 10 and 100 μg/ml, whilst EGCG did not affect testosterone secretion. IGF-I, when administered alone, promoted % of cells containing PCNA, suppressed bax accumulation, and stimulated progesterone release (only at dose 100 ng/ml). Testosterone release increased after the addition of IGF-I at 1 ng/ml, but decreased after IGF-I addition at 10 or 100 ng/ml. Both GTPP and EGCG suppressed or even reversed the effects of IGF-I on percentage of PCNA-positive cells, bax, testosterone output, and promoted IGF-I action on progesterone release. These observations suggested the inhibitory actions of green tea constituents on porcine ovarian granulosa cell functions were mediated through various regulatory mechanisms: suppression of the ovarian cell cycle in the S and G2 phases; promotion of cytoplasmic apoptosis; alteration of steroid hormone release; and, predominantly, prevention of the action of the hormonal stimulator IGF-I on ovarian cells. It can be also suggested that the major GTPP effects may result from the presence of EGCG.

Prenatal exposure to bisphenol-A altered miRNA-224 and protein expression of aromatase in ovarian granulosa cells concomitant with elevated serum estradiol levels in F1 adult offspring.

This study was aimed to predict bisphenol-A (BPA)-responsive miRNA's using an in silico approach and to study their expression in granulosa cells of animals exposed prenatally to BPA. Pregnant Wistar rats were exposed to BPA through water (25 μg/L, 250 μg/L, and 2.5 mg/L) during gestation. The expression of miRNA-133b, miRNA-378 and miRNA-224 were analyzed in ovarian granulosa cells. BPA affected the postnatal developmental landmarks such as weight of the pups at birth and reduced anogenital distance. BPA exposed animals showed elevated serum estradiol (E2) levels, while follicle-stimulating hormone levels were reduced. The expression of miRNA-224 and aromatase protein levels were found to be increased. This preliminary finding reveals the impact of early life exposure to BPA on the long-term ovarian functions that may be mediated through miRNA-based granulosa cell response. Besides, it is also a compelling indicator for the subclinical response that could have important consequences on female fertility.

The Wilms tumor gene (WT1) (+/-KTS) isoforms regulate steroidogenesis by modulating the PI3K/AKT and ERK1/2 pathways in bovine granulosa cells†.

The Wilms tumor (WT) gene WT1 encodes the splicing variants WT1(+KTS) and WT1(-KTS). Recent data suggest that WT1 plays an important role in the development of mice follicles. However, the mechanism through which WT1 influences ovarian steroidogenesis remains unknown. This study identified WT1 and evaluated the impact of splicing variants WT1(+KTS) and WT1(-KTS) on steroidogenesis using adult bovine granulosa cells (GCs). Using RT-qPCR and western blotting, we found that the ratio between WT1(+KTS) and WT1(-KTS) was stabilized. WT1 expression, however, decreased gradually in bovine GCs in response to follicle enlargement or atresia. The downregulation of WT1 increased the secretion of basal and follicle-stimulating-hormone-induced progesterone (P4), but decreased the secretion of basal-induced estradiol (E2). This was associated with an increase in the expression of 3β-HSD, and a decrease in the expression of CYP19A1. In addition, WT1(-KTS) overexpression suppresses the secretion of E2 and P4 compared with WT1(+KTS) overexpression. This was associated with a decrease in the expression of CYP19A1, CYP11A1, and 3β-HSD in cultured bovine GCs. Of note, the downregulation of WT1 suppresses the phosphorylation levels of AKT and p-ERK1/2. However, WT1(-KTS) overexpression promotes the phosphorylation levels of AKT and suppresses p-ERK1/2 levels. LY294002 (AKT inhibitor) increases MKP3 mRNA expression levels but decreases the level of p-AKT and p-ERK1/2. Collectively, WT1 significantly suppresses the mRNA expression of CYP11A1 and 3β-HSD and the secretion of P4 in bovine GCs. Moreover, it regulates CYP19A1 mRNA expression and E2 secretion with complex networks, at least in part, by modulating AKT and ERK1/2 signaling. The effect of WT1(-KTS) was more pronounced than that exerted by WT1(+KTS).

42 Nrf2 and nuclear factor kappa B cross-talk in bovine granulosa cells under lead challenge

Lead (Pb2+), one of the pervasive and protracted environmental heavy metals, is known to affect the female reproductive system by inducing oxidative stress. The Nrf2 and NF-κB are two key transcriptional factors (TF) known to regulate cellular redox status and response against stress and inflammation, respectively. Evidence showed that these TF undergo functional cross-talk. Although the properties of Pb2+ have been extensively studied, little is known about its effect on bovine granulosa cells with respect to Nrf2/NF-κB interaction. The aim of this study was to investigate the impact of Pb2+ on the response of bovine granulosa cells with regard to the cross-talk between Nrf2 and NF-κB. For this, bovine granulosa cells aspirated from small growing follicles (3-5mm of diameter) from ovaries obtained from a local slaughterhouse were in vitro cultured at 37°C and 5% CO2. Sub-confluent cells were exposed to different doses of lead acetate [Pb (C2H3O2)2] (1, 2, or 3µg mL−1) for 2h. Following this, the accumulation of intracellular reactive oxygen species was measured using the H2DCFDA fluorescent probe (Life Technologies, Darmstadt, Germany). The cell viability was assessed using Cell Counting Kit-8 (CCK-8, Dojindo, Munich, Germany) 24h after treatments. Moreover, cells were harvested and subjected to total RNA isolation using miRNeasy Mini Kit (Qiagen, Hilden, Germany) followed by cDNA synthesis using First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and the gene expression was determined using quantitative PCR. Data were analysed using comparative threshold cycle (ΔΔCT) method and normalized against the expression of GAPDH and β-actin genes. Furthermore, the protein lysate was isolated from cells to assess carbonylated protein using OxyBlot protein oxidation detection kit (abcam, Cambridge, MA, USA). Results showed that exposure of granulosa cells to lead acetate provoked intracellular reactive oxygen species accumulation and protein carbonylation. This was accompanied by cell cycle arrest at G0/G1 phase, reduction in cell viability, and decrease in the expression of cell proliferation marker genes (CCND2 and PCNA). Moreover, lead acetate elicited down-regulation of both Nrf2 and NF-κB and their downstream antioxidant genes (SOD and CAT), whereas no significant difference was shown in the level of other genes involved in both TF pathways (Keap-1, HO-1, Trx, IKK, and TNF-α). In addition, lead acetate challenge increased the expression of endoplasmic reticulum stress marker genes (GRP78 and CHOP) and the pro-apoptotic gene (Caspase-3), whereas it reduced the anti-apoptotic gene (BCL-2). Our findings suggest that Pb+2 driven-oxidative stress deregulates granulosa cell viability and proliferation, enhances endoplasmic reticulum stress, induces cell cycle arrest, and mediates apoptosis probably via disruption of Nrf2/NF-κB cross-talk.

Progesterone maintains the status of granulosa cells and slows follicle development partly through PGRMC1.

Progesterone receptor membrane component 1 (PGRMC1) mediates antimitotic and antiapoptotic actions of progesterone in granulosa cells, which indicates that PGRMC1 may play a key role in maintaining the status of granulosa cells. The current study investigated the effects of progesterone on intracellular signaling involved in differentiation, follicle development, inflammatory responses, and antioxidation, and determined the role of PGRMC1 in these processes. Our results demonstrated that progesterone slowed follicle development and inhibited p-ERK1/2, p-p38, caspase-3, p-NF-κB, and p-IκB-α signals involved in differentiation, steroidogenesis, and inflammatory responses in granulosa cells. Progesterone inhibited the steroidogenic acute regulatory protein and the cholesterol side-chain cleavage enzymeand decreased pregnenolone production. A PGRMC1 inhibitor and a PGRMC1 small interfering RNA ablated these inhibitory effects of progesterone. Interfering with PGRMC1 functions also decreased cellular antioxidative effects induced by an oxidant. These results suggest that PGRMC1 might play a critical role in maintaining the status of granulosa cells and balancing follicle numbers.

Antimicrobial peptide expression in swine granulosa cells in response to lipopolysaccharide

Antimicrobial peptides (AMP) are host defense peptides present in all species examined. The objective of the current study was to characterize the expression of a group of antimicrobial peptides in ovarian cells, and to investigate their expression response to pathogen ligands. It was found that while PG1 transcript was not detected in the ovary, the expression of BD2 is the highest in small follicle derived granulosa cells (SGC), and its expression decreases during follicular development to large follicle stage (LGC; p < 0.05). The expression of BD2 in cumulus cells also decreased from GV to MII stage of oocyte maturation. ANG4 expression increased in granulosa cells during follicular development from SGC to LGC stage (p < 0.05), although no significant difference was observed in cumulus cells from different stages of oocyte maturation. We further examined AMP expression in follicle cells treated with different toll-like receptor (TLR) ligands which mimic pathogen exposure in the ovary. Of the four TLR ligands examined, lipopolysaccharide (LPS) exposure resulted in a 11.5 fold increase of BD2 expression, and a significant decrease of LYZ in LGC. A similar response pattern in BD2 and LYZ expression was also observed in SGC. These responses of AMP expression to LPS are associated with increased TLR4 signaling pathway component in mRNA and protein level, such as MyD88 and NFkB, and pro-inflammatory cytokines/chemokines, such as IL-6, TNFα and IL-8 (p < 0.05). Our data suggest that AMPs may play a role in innate defense as well as other physiological functions during ovarian follicular development and oocyte maturation.

Histology of the Ovary of the Laying Hen (Gallus domesticus).

The laying hen (Gallus domesticus) is a robust animal model for epithelial ovarian cancer. The use of animal models is critical in identifying early disease markers and developing and testing chemotherapies. We describe the microscopic characteristics of the normally functioning laying hen ovary and proximal oviduct to establish baselines from which lesions associated with ovarian cancer can be more readily identified. Ovaries and oviducts were collected from 18-month-old laying hens (n = 18) and fixed in 10% neutral buffered formalin. Hematoxylin- and eosin-stained sections were examined by light microscopy. Both post-ovulatory follicular regression and atresia of small follicles produce remnant clusters of vacuolated cells with no histological evidence that scar tissue persists. Infiltrates of heterophils are associated with atresia of small follicles, a relationship not previously documented in laying hen ovaries. Because these tissues can be mistaken for cancerous lesions, we present a detailed histological description of remnant Wolffian tissues in the laying hen ovary. Immunohistochemical staining for pancytokeratin produced a positive response in ovarian surface epithelium and staining for vimentin produced a positive response in granulosa cells of follicles. Epithelial cells lining glands of the remnant epoöphoron had a positive response to both pancytokeratin and vimentin, a result also observed in women.

Cytosolic DNA sensor-initiated innate immune responses in mouse ovarian granulosa cells

Viral infections of the ovary may perturb ovarian functions. However, the mechanisms underlying innate immune responses in the ovary are poorly understood. The present study demonstrates that cytosolic viral DNA sensor signaling initiates the innate immune response in mouse ovarian granulosa cells and affects endocrine function. The cytosolic DNA sensors p204 and cGAS and their common signaling adaptor stimulator of interferon (IFN) genes (STING) were constitutively expressed in granulosa cells. Transfection with VACV70, a synthetic vaccinia virus (VACV) DNA analog, induced the expression of type I interferons (IFNA/B) and major inflammatory cytokines (TNFA and IL6) through IRF3 and NF-κB activation respectively. Moreover, several IFN-inducible antiviral proteins, including 2',5'-oligoadenylate synthetase, IFN-stimulating gene 15 and Mx GTPase 1, were also induced by VACV70 transfection. The innate immune responses in granulosa cells were significantly reduced by the transfection of specific small-interfering RNAs targeting p204, cGas or Sting Notably, the VACV70-triggered innate immune responses affected steroidogenesis in vivo and in vitro The data presented in this study describe the mechanism underlying ovarian immune responses to viral infection.

Apelin (APLN) regulates progesterone secretion and oocyte maturation in bovine ovarian cells.

APLN and its G-protein coupled receptor APLNR are expressed in the bovine ovary. However their role in granulosa cells and oocytes is unknown. Here, we studied their expression in bovine ovarian cells and investigated their regulation in cultured luteinizing granulosa cells in response to IGF1 and FSH. We determined the effect and the molecular mechanism of APLN (isoforms 17 and 13) on bovine granulosa cell progesterone secretion and on oocyte maturation. By RT-qPCR and immunoblot, we showed that the expression of both APLN and APLNR in granulosa and oocytes significantly increased with ovarian follicles size whereas it was similar in theca interstitial cells. In vitro, in unstimulated luteinizing bovine granulosa cells and in response to IGF1 (10-8 M) but not to FSH (10-8 M), we observed that APLN (-17 and -13) (10-9 M) increased progesterone production; this was abolished in response to the APLNR antagonist ML221. These latter effects were dependent on the MAPK ERK1/2 kinase. Furthermore, we showed that APLN (-17 and -13) (10-9 M) increased cell proliferation through AKT signaling. Conversely, the addition of APLN-13 and APLN-17 to in vitro maturation medium containing IGF1 (10-8 M) but not FSH (10-8 M) arrested most oocytes at the germinal vesicle stage, which was associated with a decrease in progesterone secretion, an inhibition in MAPK ERK1/2 phosphorylation and an increase in PRKA phosphorylation in oocytes. Thus, APLN can increase progesterone secretion and cell proliferation in bovine luteinizing granulosa cells in vitro, while it blocks meiotic progression at the germinal vesicle stage during bovine oocyte in vitro maturation.

Molecular mechanism and functional role of macrophage colony‑stimulating factor in follicular granulosa cells.

Our previous demonstrated that macrophage colony‑stimulating factor (M‑CSF) stimulated the production of estradiol (E2) and progesterone (P) in luteinized granulosa cells (GCs), and that its secretion may be regulated by follicle‑stimulating hormone (FSH). The present study aimed to examine the effect of M‑CSF alone or with Letrozole treatment on the function of non‑luteinizing granulosa cells, using the COV434cell line, and its interaction with FSH. Human luteinized granulosa cells (LGC) were isolated from the follicular fluid of superovulated infertile patients (average age, 30.8±2.1years) undergoing an intracytoplasmic sperm injection. The LGC were cultured with various concentrations of recombinant human macrophage colony stimulating factor (rhM‑CSF; 0, 10, 25, 50 or 100ng/ml), rhM‑CSF+Letrozole (10‑6mol/l), rhFSH (0, 10, 25, 50 or 100IU/ml), or rhFSH+Letrozole (10‑6 mol/l). E2 concentrations in the media were measured using ELISA. The expression levels of the FSH receptor and the M‑CSF receptor were detected via reverse transcription‑quantitative polymerase chain reaction. Following COV434cell treatment with M‑CSF, cell proliferation was quantified using the MTS assay and protein expression was detected by western blotting. It was demonstrated that M‑CSF and FSH stimulated the production of E2. The production of FSH receptors was enhanced by rhM‑CSF or rhM‑CSF+Letrozole invitro in a dose‑dependent manner. It was observed that rhFSH promoted the expression of the M‑CSF receptor, at a certain concentration. Proliferation of COV434cells increased in a dose‑dependent manner following treatment with RhM‑CSF. Furthermore, M‑CSF induced the phosphorylation of c‑Jun N‑terminal kinase (JNK) and p38; however, the level of E2 in the medium was not altered when the cells were pretreated with the JNK inhibitor SP600125 or the p38 inhibitor SB203580. The present study suggested that M‑CSF may be important in regulating the response of GCs to gonadotropin and may have a promotive effect in the early phase of follicular development. The biological effects of M‑CSF may partially be attributed to activation of the JNK and p38 signaling pathways. M‑CSF may represent a novel follicular development regulator agent in the future.

The effect of estradiol on granulosa cell responses to FSH in women with polycystic ovary syndrome

BackgroundThe influence of estradiol (E2) on granulosa cell (GC) function has not been tested clinically in women with polycystic ovary syndrome (PCOS). The objective of this study is to determine if E2 influences GC responses to FSH in women with PCOS.MethodsThis is a two phase, single cohort study conducted over a 2-year period at a single academic center. Nine women with PCOS according to NIH criteria. In Phase 1, FSH stimulation of GC responses as measured by E2 and Inhibin B (Inh B) were assessed before and at 5 and 6 weeks after GnRH agonist administration. In Phase 2, the same protocol was employed with the addition of an aromatase inhibitor (letrozole, LET) administered daily beginning at week 4 for 2 weeks.ResultsIn Phase 1, recovery of FSH, E2 and Inh B from ovarian suppression occurred at 5 and 6 weeks after GnRH agonist injection and preceded resumption of LH and androgen secretion. In Phase 2, hormone recovery after GnRH agonist was characterized by elevated FSH and suppressed E2 levels whereas recovery of LH and androgen levels were unchanged. In Phase 1, FSH stimulated E2 and Inh B responses were unaltered during recovery from ovarian suppression. In Phase 2, E2 and Inh B fold changes after FSH were significantly reduced at weeks 5 (p < 0.04) and 6 (p < 0.01), respectively.ConclusionIn anovulatory women with PCOS, chronic, unopposed E2 secretion may contribute, at least in part, to enhanced ovarian responsiveness to FSH.Trial registrationNCT02389088

Genome-wide interactions between FSH and insulin-like growth factors in the regulation of human granulosa cell differentiation.

Is the genome-wide response of human cumulus cells to FSH and insulin-like growth factors (IGFs) comparable to the response observed in undifferentiated granulosa cells (GCs)? FSH actions in human cumulus cells mimic those observed in preantral undifferentiated GCs from laboratory animals, and approximately half of the regulated genes are dependent on the simultaneous activation of the IGF1 receptor (IGF1R). Animal studies have shown that FSH and the IGFs system are required for follicle growth and maturation. In humans, IGF levels in the follicular fluid correlate with patients' responses toIVF protocols. The main targets of FSH and IGFs in the ovary are the GCs; however, the genomic mechanisms involved in the response of GCs to these hormones are unknown. Human cumulus cells isolated from IVF patients were cultured for 48 h in serum-free media in the presence of vehicle, FSH, IGF1R inhibitor or their combination. Discarded cumulus cells were donated to research by reproductive-aged women undergoing IVF due to non-ovarian etiologies of infertility at a university-affiliated clinic. The effect of FSH and/or IGF1R inhibition on cumulus cell function was evaluated using Affymetrix microarrays, quantitative PCR, western blot, promoter assays and hormone level measurements. The findings demonstrate that human cumulus cells from IVF patients respond to FSH with the expression of genes known to be markers of the preantral to preovulatory differentiation of GCs. These results also demonstrate that ~50% of FSH-regulated genes require IGF1R activity and suggest that several aspects of follicle growth are coordinately regulated by FSH and IGFs in humans. This novel approach will allow for future mechanistic and molecular studies on the regulation of human follicle maturation. Data set can be accessed at Gene Expression Omnibus number GSE86427. Experiments were performed using primary human cumulus cells. This may not represent the response of intact follicles. Understanding the mechanisms involved in the regulation of GC differentiation by FSH and IGF in humans will contribute to improving treatments for infertility. The project was financed by the National Instituted of Health grant number R56HD086054 and R01HD057110 (C.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We have no competing interests to declare.

Interference of Steroidogenesis by Gold Nanorod Core/Silver Shell Nanostructures: Implications for Reproductive Toxicity of Silver Nanomaterials.

As a widely used nanomaterial in daily life, silver nanomaterials may cause great concern to female reproductive system as they are found to penetrate the blood-placental barrier and gain access to the ovary. However, it is largely unknown about how silver nanomaterials influence ovarian physiology and functions such as hormone production. This study performs in vitro toxicology study of silver nanomaterials, focusing especially on cytotoxicity and steroidogenesis and explores their underlying mechanisms. This study exposes primary rat granulosa cells to gold nanorod core/silver shell nanostructures (Au@Ag NRs), and compares outcomes with cells exposed to gold nanorods. The Au@Ag NRs generate more reactive oxygen species and reduce mitochondrial membrane potential and less production of adenosine triphosphate. Au@Ag NRs promote steroidogenesis, including progesterone and estradiol, in a time- and dose-dependent manner. Chemical reactivity and transformation of Au@Ag NRs are then studied by electron spin resonance spectroscopy and X-ray absorption near edge structure, which analyze the generation of free radical and intracellular silver species. Results suggest that both particle-specific activity and intracellular silver ion release of Au@Ag NR contribute to the toxic response of granulosa cells.