Search

Purpose: Aromatase plays an important role in ovarian development, the normal progress of the menstrual cycle, and fertility status. Elevated aromatase activity is linked to obesity. There is a bidirectional relationship between obesity and thyroid function. Few studies have investigated the relationship between TSH and ovarian aromatase in obesity. Our aim was to investigate the effect of TSH on aromatase expression of ovarian granulosa cells in obese mice., Methods: Female mice pups were divided into an obesity group and a control group. Obese parameters and the time of pubertal onset were recorded. At the age of 5 weeks, blood and tissues were obtained. Serum aromatase and hormone concentrations were measured using ELISA. The granulosa cells were isolated and exposed to variable concentrations (0 μM, 1 μM, 10 μM, 100 μM) of TSH. The expression of CYP19A1 mRNA and protein were assessed via RT-qPCR and western blot., Results: In female mice, body weight, Lee's obesity index, and serum levels of E2, aromatase, and TSH were significantly higher in the obesity group compared to the control group, whereas the time of pubertal onset and serum T3 and T4 concentrations were significantly lower (all P < 0.001). In granulosa cells, the expression of CYP19A1 mRNA in the obesity group was lower than that in the control group at 1 μM and 100 μM concentrations of TSH (both P < 0.001). The expression of CYP19A1 protein in the obesity group was higher than that in the control group after TSH stimulation (P = 0.014, P < 0.001, and P = 0.004, respectively). With the increase of TSH concentrations, the expression of CYP19A1 mRNA and protein in the two groups significantly increased (all P < 0.001)., Conclusion: Early puberty and elevated serum aromatase and TSH levels were found in obese female mice. In the granulosa cells of obese mice, TSH directly regulates aromatase expression in a dose-dependent manner., (© 2024. The Author(s), under exclusive licence to Hellenic Endocrine Society.)

Background: The mammalian follicle is the basic functional unit of the ovary, and its normal development is required to obtaining oocytes capable of fertilization. As women get older or decline in ovarian function due to certain pathological factors, the growth and development of follicles becomes abnormal, which ultimately leads to infertility and other related female diseases. Kuntai capsules are currently used in clinical practice to improve ovarian function, and they contain the natural compound Baicalin, which is a natural compound with important biological activities. At present, the role and mechanism of Baicalin in the development of ovarian follicles is unclear., Methods: Human primary granulosa cells collected from follicular fluid, and then cultured and treated with Baicalin or its normal control, assessed for viability, subjected to RT-PCR, western blotting, flow cytometry, and hormone analyses. The estrus cycle and oocytes of CD-1 mice were studied after Baicalin administration and compared with controls. Ovaries were collected from the mice and subjected to hematoxylin-eosin staining and immunohistochemistry analysis., Results: We showed that Baicalin had a dose-dependent effect on granulosa cells cultured in vitro. A low concentration of Baicalin (for example, 10 μM) helped to maintain the viability of granulosa cells; however, at a concentration exceeding 50 μM, it exerted a toxic effect. A low concentration significantly improved the viability of granulosa cells and inhibited cell apoptosis, which may be related to the resultant upregulation of Bcl-2 expression and downregulation of Bax and Caspase 3. By constructing a hydrogen peroxide-induced cell oxidative stress damage model, we found that Baicalin reversed the cell damage caused by hydrogen peroxide. In addition, Baicalin increased the secretion of estradiol and progesterone by upregulating P450arom and stAR. The results of the in vivo experiment showed that the intragastric administration of Baicalin to aged mice improved the estrous cycle and oocyte quality. Furthermore, we observed that Baicalin enhanced the viability of granulosa cells through the mTOR pathway, which in turn improve ovarian function., Conclusion: These results indicate that Baicalin could improve the viability of ovarian granulosa cells and the secretion of steroid hormones and thus could help to improve degenerating ovarian function and delay ovarian aging., (© 2022. The Author(s).)

Melatonin, an important regulator of mammalian reproduction, is mainly produced in the pineal gland, and granulosa cells (GCs), the main mammalian ovarian secretory cells, synthesize melatonin and express melatonin receptors (MRs) MT1 and MT2. However, studies on melatonin regulation in GCs are lacking in sheep. In this study, we explored the effects of β-estradiol (E2) on melatonin production and MR expression in GCs. We cultured sheep GCs to analyze the expression of the melatonin rate-limiting enzymes AANAT and HIOMT and the effects of E2 on AANAT, HIOMT, and MR expression and melatonin synthesis. To determine whether estrogen receptors (ERs) mediated E2 action on melatonin secretion and MR expression, we assessed ERA and ERB expression in GCs and observed whether ER antagonists counterbalanced the effects of E2. GCs expressed AANAT and HIOMT mRNA, indicating that they transformed exogenous serotonin into melatonin. E2 inhibited melatonin production by downregulating AANAT, HIOMT, and MRs. GCs expressed ERA and ERB; ERA/ERB inhibitors abolished E2-mediated inhibition of melatonin secretion and MR expression. PHTPP upregulated melatonin secretion and MT1 expression in E2-treated GCs, but did not significantly affect AANAT and MT2 expression. In conclusion, melatonin secretion in GCs was inhibited by E2 through an ERA- and ERB-mediated process., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The main features of polycystic ovary syndrome (PCOS) are abnormal follicular development and ovulation dysfunction, which are caused by the excessive autophagy of ovarian granulosa cells. Acupuncture has been shown to improve ovulation dysfunction and abnormal follicular development in PCOS patients, but its mechanism is unclear. This study hypothesized that the beneficial effects of acupuncture are the result of LncMEG3-mediated effects on the PI3K/AKT/mTOR pathway. Acupuncture (CV-4, RN-3, CV-6, SP-6 and EX-CA 1) was used to treat a rat model of polycystic ovary syndrome. Hematoxylin-eosin staining was used to observe ovarian morphology and enzyme-linked immunosorbent assay, western blotting, immunohistochemistry and real-time PCR were used to detect LH, E2, FSH, T, AMH, LncMEG3, PI3K, AKT, mTOR, P62 and LC3II/I expression. The ovarian morphology of 90% of the rats in the acupuncture treatment group was significantly improved after 11 consecutive days of therapy. Acupuncture also resulted in a significant decrease in serum LH, FSH, T and AMH levels and a significant increase in E2 level (P<0.01). LncMEG3, PI3K, AKT, mTOR, P62 and LC3II/I expression was decreased in ovarian granulosa cells after acupuncture compared with PCOS and lentiviral Intervention Group (P<0.05), while the expression of follicle stimulating hormone receptor was increased (P<0.05). These results indicate that acupuncture can down-regulate the expression of LncMEG3 and thereby inhibit the PI3K/AKT/mTOR pathway, reducing granulosa cell autophagy and normalizing their proliferation. These factors ultimately remedy abnormal follicular development. These findings suggest that acupuncture has clinical potential as a safe treatment for PCOS ovulatory dysfunction., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The endocannabinoid system (ECS) plays a crucial role in human reproduction. Changes in anandamide (AEA) levels affect reproductive events and has already been suggested as biomarker of reproductive potential of male and female gametes. Although cannabinoid-receptor 1 (CB1) was already identified in human granulosa cells (hGCs) the ECS was not characterized on granulosa cells line COV434 nor the effects of AEA on GCs viability and function depicted. Therefore, the aim of this study was to characterize the ECS elements and explore the effects of AEA on both COV434 and hGCs. Our results revealed that hGCs express the full enzymatic machinery responsible for AEA metabolism as well as cannabinoid receptors. In addition, AEA induced a reduction in both COV434 and hGCs viability in a concentration and time-dependent manner. Nevertheless, the effects of AEA in cell viability was independent of either CB1 or CB2 receptors. There was no ROS release in both cell models; however, AEA induced morphological changes, presenting chromatin condensation at 72 h, and variation on mitochondrial membrane potential. Moreover, AEA induced an increase in caspase -3/-7 activities in both cell models, but in hGCs there was also an increase in caspase 8 activity. This study supports the idea that ECS balance is crucial for folliculogenesis and oocyte quality as dysregulated AEA levels may compromise female fertility., (Copyright © 2021. Published by Elsevier Ltd.)

Ovarian granulosa cells (GCs) are the most important source of estrogen. Therefore, aromatase (estrogen synthase), which is the key enzyme in estrogen synthesis, is not only an important factor of ovarian development, but also the key to estrogen secretion by GCs. Disorders of the ovarian estrogen secretion are more likely to induce female estrogen‑dependent diseases and fertility issues, such as ovarian cancer and polycystic ovary syndrome. Hence, aromatase is an important drug target; treatment with its inhibitors in estrogen‑dependent diseases has attracted increasing attention. The present review article focuses on the regulation and mechanism of the aromatase activity in the GCs, as well as the specific regulation of aromatase promoters. In GCs, follicle‑stimulating hormone (FSH) is dependent on the cyclic adenosine monophosphate (cAMP) pathway to regulate the aromatase activity, and the regulation of this enzyme is related to the activation of signaling pathways, such as phosphatidylinositol 3‑kinase (PI3K) and extracellular signal‑regulated kinase (ERK). In addition, endocrine‑disrupting substance and other related factors affect the expression of aromatase, which eventually create an imbalance in the estrogen secretion by the target tissues. The present review highlights these useful factors as potential inhibitors for target therapy.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. ZEA causes reproductive disorders, genotoxicity, and testicular toxicity in animals. However, little is known about the functions of apoptosis and autophagy after exposure to ZEA in granulosa cells. This study investigated the effects of ZEA on chicken granulosa cells. The results show that ZEA at different doses significantly inhibited the growth of chicken granulosa cells by inducing apoptosis. ZEA treatment up-regulated Bax and downregulated Bcl-2 expression, promoted cytochrome c release into the cytosol, and triggered mitochondria-mediated apoptosis. Consequently, caspase-9 and downstream effector caspase-3 were activated, resulting in chicken granulosa cells apoptosis. ZEA treatment also upregulated LC3-II and Beclin-1 expression, suggesting that ZEA induced a high level of autophagy. Pretreatment with chloroquine (an autophagy inhibitor) and rapamycin (an autophagy inducer) increased and decreased the rate of apoptosis, respectively, in contrast with other ZEA-treated groups. Autophagy delayed apoptosis in the ZEA-treated cells. Therefore, autophagy may prevent cells from undergoing apoptosis by reducing ZEA-induced cytotoxicity. In addition, our results further show that the autophagy was stimulated by ZEA through PI3K-AKT-mTOR and MAPK signaling pathways in chicken granulosa cells.

Expression levels of genes involved in the development of germ cells vary throughout the process from bipotential gonadal period to adult gonadal formation. In mice, developments of female and male reproductive system are regulated by germ cell-specific factors and hormones, and determinative days in this regulation are very important. c-Abl is a non-receptor tyrosine kinase with cellular functions including cell proliferation, growth and development. mTERT is involved in maintaining telomerase activity and proliferation of surviving cells. We suggested that c-Abl and mTERT might be important for the healthy development of prenatal and postnatal mouse ovary and testis. We aim to demonstrate localization and expressions of c-Abl and mTERT in crucial days of ovary and testis development in prenatal and postnatal period in mouse by immunofluorescence staining and qRT-PCR, respectively. The importance of c-Abl and mTERT expressions during the healthy gonadal development is indicated in the prenatal and postnatal gonadal development. Also, protein expression levels were detected by Western Blot in only postnatal ovary and testis. Determining the functions of the c-Abl and mTERT throughout the process will be important in terms of understanding the infertility cases in the female and male with future studies., Competing Interests: Declaration of Competing Interest All the authors listed have significantly contributed to he manuscript and there is no disclose any conflict of interest., (Copyright © 2020 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

In a healthy female reproductive system, a subtle hormonal and metabolic dance leads to repetitive cyclic changes in the ovaries and uterus, which make an effective ovulation and potential implantation of an embryo possible. However, that is not so in the case of polycystic ovary syndrome (PCOS), in which case the central mechanism responsible for entraining hormonal and metabolic rhythms during the menstrual cycle is notably disrupted. In this review we provide a detailed description of the possible scenario of PCOS pathogenesis. We begin from the analysis of how a set of genetic disorders related to PCOS leads to particular malfunctions at a molecular level (e.g., increased enzyme activities of cytochrome P450 (CYP) type 17A1 (17α-hydroxylase), 3β-HSD type II and CYP type 11A1 (side-chain cleavage enzyme) in theca cells, or changes in the expression of aquaporins in granulosa cells) and discuss further cellular- and tissue-level consequences (e.g., anovulation, elevated levels of the advanced glycation end products in ovaries), which in turn lead to the observed subsequent systemic symptoms. Since gene-editing therapy is currently out of reach, herein special emphasis is placed on discussing what kinds of drug targets and which potentially active substances seem promising for an effective medication, acting on the primary causes of PCOS on a molecular level.

Follicle development is the most crucial step toward female fertility and is controlled mainly by follicle-stimulating hormone (FSH). In ovarian granulosa cells (GCs), FSH activates protein kinase A by increasing 3',5'-cyclic adenosine 5'-monophosphate (cAMP). Since cAMP signaling is impinged in part by salt-inducible kinases (SIKs), we examined the role of SIKs on the regulation of FSH actions. Here, we report that SIKs are essential for normal ovarian function and female fertility. All SIK isoforms are expressed in human and rodent GCs at different levels (SIK3>SIK2>SIK1). Pharmacological inhibition of SIK activity potentiated the stimulatory effect of FSH on markers of GC differentiation in mouse, rat, and human GCs and estradiol production in rat GCs. In humans, SIK inhibition strongly enhanced FSH actions in GCs of patients with normal or abnormal ovarian function. The knockdown of SIK2, but not SIK1 or SIK3, synergized with FSH on the induction of markers of GC differentiation. SIK inhibition boosted gonadotropin-induced GC differentiation in vivo, while the genomic knockout of SIK2 led to a significant increase in the number of ovulated oocytes. Conversely, SIK3 knockout females were infertile, FSH insensitive, and had abnormal folliculogenesis. These findings reveal novel roles for SIKs in the regulation of GC differentiation and female fertility, and contribute to our understanding of the mechanisms regulated by FSH. Furthermore, these data suggest that specific pharmacological modulation of SIK2 activity could be of benefit to treat ovulatory defects in humans and to increase the propagation of endangered species and farm mammals., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Wt1 gene encodes a nuclear transcription factor which is specifically expressed in ovarian granulosa cells and testicular Sertoli cells. Our previous studies demonstrated that Wt1 is required for the lineage specification of supporting cells and inactivation of Wt1 results in Sertoli cells to Leydig-like cells transformation. To test whether Wt1 is also involved in lineage maintenance of granulosa cells during ovary development, Wt1 was specifically deleted in pre-granulosa cells using Foxl2-cre. We found that the female Wt1-/flox; Foxl2-cre mice were infertile with atrophic ovaries and no growing follicles with multiple layers of granulosa cells were observed. A large number of 3β-HSD-positive steroidogenic cells were detected in ovaries of Wt1-/flox; Foxl2-cre mice during embryonic stage and these cells were derived from Foxl2-expressing pre-granulosa cells. The quantitative results showed the expression of granulosa cell marker genes (Foxl2, Follistatin) was downregulated and steroidogenic cell marker genes (3β-HSD, Cyp11a1, Star and Sf1) was dramatically increased in Wt1-/flox; Foxl2-cre ovaries. We also found that the meiosis of germ cells in Wt1-/flox; Foxl2-cre ovaries was delayed but not arrested. This study demonstrates that Wt1 is required for lineage maintenance of granulosa cells and inactivation of Wt1 results in pre-granulosa cells to steroidogenic cells transformation which in turn causes the defect of ovary development., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sirtuins are a family of deacetylases that modify structural proteins, metabolic enzymes, and histones to change cellular protein localization and function. In mammals, there are seven sirtuins involved in processes like oxidative stress or metabolic homeostasis associated with aging, degeneration or cancer. We studied gene expression of sirtuins by qRT-PCR in human mural granulosa-lutein cells (hGL) from IVF patients in different infertility diagnostic groups and in oocyte donors (OD; control group). Study 1: sirtuins genes' expression levels and correlations with age and IVF parameters in women with no ovarian factor. We found significantly higher expression levels of SIRT1, SIRT2 and SIRT5 in patients ≥40 years old than in OD and in women between 27 and 39 years old with tubal or male factor, and no ovarian factor (NOF). Only SIRT2, SIRT5 and SIRT7 expression correlated with age. Study 2: sirtuin genes' expression in women poor responders (PR), endometriosis (EM) and polycystic ovarian syndrome. Compared to NOF controls, we found higher SIRT2 gene expression in all diagnostic groups while SIRT3, SIRT5, SIRT6 and SIRT7 expression were higher only in PR. Related to clinical parameters SIRT1, SIRT6 and SIRT7 correlate positively with FSH and LH doses administered in EM patients. The number of mature oocytes retrieved in PR is positively correlated with the expression levels of SIRT3, SIRT4 and SIRT5. These data suggest that cellular physiopathology in PR's follicle may be associated with cumulative DNA damage, indicating that further studies are necessary., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Granulosa cells play important roles in the regulation of ovarian functions. Phospholipase C is crucial in several signalling pathways and could participate in the molecular mechanisms of cell proliferation, differentiation and ageing. The objective of this study was to identify the effects of phospholipase C on the steroidogenesis of oestradiol and progesterone in porcine granulosa cells cultured in vitro. Inhibitor U73122 or activator m-3M3FBS of phospholipase C was added to the in vitro medium of porcine granulosa cells, respectively. The secretion of oestradiol decreased after 2 hr, 8 hr, 12 hr, 24 hr and 48 hr of treatment with 500 nM U73122 (p < .05) and decreased after 2 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The secretion of progesterone increased after 4 hr of treatment with 500 nM U73122 (p < .05) and increased after 2 hr and 8 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The ratio of oestradiol to progesterone decreased at each time point, except 8 hr after the addition of 500 nM U73122 (p < .05). The ratio of oestradiol to progesterone decreased after 2 hr (p < .05) of treatment with 500 nM m-3M3FBS. In genes that regulate the synthesis of oestradiol or progesterone, the mRNA expression of CYP11A1 was markedly increased (p < .05), and the mRNA expression of other genes did not change significantly in the U73122 treatment group, while the addition of m-3M3FBS did not change those genes significantly despite the contrary trend. Our results demonstrated that phospholipase C can be a potential target to stimulate the secretion of oestradiol and suppress progesterone secretion in porcine granulosa cells cultured in vitro, which shed light on a novel biological function of phospholipase C in porcine granulosa cells., (© 2019 Blackwell Verlag GmbH.)

Cyclic adenosine monophosphate (cAMP) is a ubiquitous secondary messenger that plays a central role in endocrine tissue function, particularly in the synthesis of steroid hormones. The intracellular concentration of cAMP is regulated through its synthesis by cyclases and its degradation by cyclic nucleotide phosphodiesterases (PDEs). Although the expression and activity of PDEs impact the specificity and the amplitude of the cAMP response, it is becoming increasingly clear that the sub-cellular localization of PDE emphasizes the spatial regulation of the cell signalling processes that are essential for normal cellular function. We first examined the expression of PDE8A in porcine ovarian cells. PDE8A is expressed in granulosa cells, cumulus cells and oocytes. Second, we assessed the mitochondrial sub-cellular localization of PDE8A. Using western blotting with isolated mitochondrial fractions from granulosa cells and cumulus-oocyte complexes revealed immuno-reactive bands. PDE assay of isolated mitochondrial fractions from granulosa cells measured specific PDE8 cAMP-PDE activity as PF-04957325-sensitive. The immune-reactive PDE8A signal and MitoTracker labelling co-localized supporting mitochondrial sub-cellular localization of PDE8A, which was confirmed using immuno-electron microscopy. Finally, the effect of PDE8 on progesterone production was assessed during the in-vitro maturation of cumulus-oocyte complexes. Using PF-04957325, we observed a significant increase (P < 0.05) in progesterone secretion with follicle-stimulating hormone (FSH). Active mitochondria stained with MitoTracker orange CMTMRos were also increased by the specific PDE8 inhibitor supporting its functional regulation. In conclusion, we propose the occurrence of mitochondrial sub-cellular localization of PDE8A in porcine granulosa cells and cumulus cells. This suggests that there is potential for new strategies for ovarian stimulation and artificial reproductive technologies, as well as the possibility for using new media to improve the quality of oocytes.

Heat stress can inhibit follicular development in dairy cows, and thus can affect their reproductive performance. Follicular granulosa cells can synthesize estrogen, that affects the development and differentiation of follicles by apoptosis. Heme oxygenase 1 (HO-1/heat shock protein 32) plays an antiapoptotic and cytoprotective role in various cells during stress-induced apoptosis, but little is known about its definitive function in bovine (ovarian) granulosa cells (bGCs). In our study, the roles and mechanism of HO-1 on the heat stress-induced apoptosis of bGCs were studied. Our results show that the expression of HO-1 was significantly increased under heat stress. Moreover, HO-1 silencing increased apoptosis, whereas its overexpression dampened apoptosis by regulating the expression of Bax/Bcl-2 and the levels of cleaved caspase-3. In addition, HO-1 can also play a cytoprotective role by affecting estrogen levels and decomposing heme to produce biologically active metabolite carbon monoxide (CO). Meanwhile, CO significantly increased the level of HO-1, decreased Bax/Bcl-2 levels, and inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The apoptosis of ovarian GCs can affect the secretion of estrogen and lead to disorder of the ovarian microenvironment, thus affecting the normal function of the ovary. Our results indicate that HO-1 acts as a cytoprotective enzyme and plays a protective role in heat-induced apoptosis of bGCs. In conclusion, HO-1 and its metabolite CO inhibit the apoptosis of bGCs induced by heat stress through the ERK1/2 pathway. The results of this study provide a valuable clue for improving the fertility of heat stressed cows in summer., (© 2018 Wiley Periodicals, Inc.)

Granulosa cells control oocyte maturation through paracrine signalling and changes to the microenvironment around the oocyte. Apoptosis occurs as a physiological mechanism of granulosa cell renewal, but how it relates with the ovarian response to induced ovulation is still unclear. Therefore, this study evaluated apoptosis-related gene expression levels in granulosa cells of patients undergoing controlled ovarian stimulation. We enrolled prospectively 59 consecutive IVF patients referred to a tertiary academic hospital for couple infertility treatment. Luteinized granulosa cells were isolated from follicular fluid and the RNA was extracted, reverse-transcribed and the gene expression of apoptosis inducers (caspase-3, caspase-8 and bax) and inhibitor (Bcl-2) was quantified by real-time polymerase chain reaction. Caspase-3 gene expression correlated negatively with the number of pre-ovulatory follicles (Spearman's r =  -0.308), the number of collected oocytes (r =  -0.451), the number of mature oocytes (r =  -0.526), the number of fertilized oocytes (r =  -0.439) and the number of viable embryos (r =  -0.443, all statistically significant at p < 0.02 level). No such associations were found with caspase-8, bax or bcl-2. These preliminary findings suggest that increased caspase-3 gene expression in granulosa cells is associated with a worse ovulatory response in humans.

Polycystic ovary syndrome (PCOS) is a major cause of infertility in women of reproductive age. However, its exact etiology remains unknown. In this study, we sequenced miRNAs in human follicular fluid and identified 16 downregulated and 3 upregulated miRNAs in PCOS group compared with non-PCOS group. Among the differential expressed miRNAs, miR-335-5p was verified lower abundance in PCOS than non-PCOS group using quantitative real-time PCR. Besides, miR-335-5p negatively correlated with antral follicle count, anti-Müllerian hormone and total testosterone. Bioinformatics analysis identified serum/glucocorticoid-regulated kinase family member 3 (SGK3) as a potential target gene of miR-335-5p. SGK3 is involved in protein kinase B-mammalian target of rapamycin kinase (AKT-mTOR) signaling pathway and cell proliferation. Western blotting and cell counting kit-8 assays demonstrated that miR-335-5p mimic reduced, while miR-335-5p inhibitor increased, SGK3 abundance, AKT-mTOR pathway and cell proliferation in human granulosa-like tumor KGN cells. Dual-luciferase reporter assays showed that miR-335-5p binds to the 3' untranslated region of SGK3 mRNA. Furthermore, miR-335-5p was decreased and SGK3 was elevated in human granulosa cells obtained from PCOS patients as compared with non-PCOS controls. These findings suggested that miR-335-5p is involved in granulosa cells proliferation by reducing SGK3 expression, which might provide a molecular target to improve dysfunctional granulosa cells in patients with PCOS.

MTOR (mechanistic target of rapamycin) is a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation. Here we show that conditional knockout (cKO) of Mtor in either primordial or growing oocytes caused infertility but differentially affected oocyte quality, granulosa cell fate, and follicular development. cKO of Mtor in nongrowing primordial oocytes caused defective follicular development leading to progressive degeneration of oocytes and loss of granulosa cell identity coincident with the acquisition of immature Sertoli cell-like characteristics. Although Mtor was deleted at the primordial oocyte stage, DNA damage accumulated in oocytes during their later growth, and there was a marked alteration of the transcriptome in the few oocytes that achieved the fully grown stage. Although oocyte quality and fertility were also compromised when Mtor was deleted after oocytes had begun to grow, these occurred without overtly affecting folliculogenesis or the oocyte transcriptome. Nevertheless, there was a significant change in a cohort of proteins in mature oocytes. In particular, down-regulation of PRC1 (protein regulator of cytokinesis 1) impaired completion of the first meiotic division. Therefore, MTOR-dependent pathways in primordial or growing oocytes differentially affected downstream processes including follicular development, sex-specific identity of early granulosa cells, maintenance of oocyte genome integrity, oocyte gene expression, meiosis, and preimplantation developmental competence., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Humans are exposed not only to single endocrine disruptors, but also to chemical mixtures that can adversely affect their reproductive health. Steroidogenesis in reproductive tissues is emerging as the key target of endocrine disruptor action. Here, we analyzed the effect of environmental chemical mixtures with estrogenic activity on steroidogenic processes in immature rat granulosa cells and whether the observed steroidogenic effects were mediated through estrogen receptors. Extracts from untreated wastewater were prepared by solid-phase extraction and silica gel fractionation. ER-CALUX assay showed that the polar fractions of wastewater exerted different levels of estrogenic activity. Exposure of immature granulosa cells to the polar fraction exerting 9 ng of 17β-estradiol equivalents per liter of water of estrogenic activity increased mRNA expression of the key enzymes of progesterone biosynthetic pathway Star and Hsd3b1, but did not alter the level of Cyp19a1 and Lhr. Addition of estrogen receptor inhibitor ICI 182 780 prevented the estrogenic mixture-induced increase in Hsd3b1, but not Star mRNA level in immature granulosa cells. These results indicate that the environmental chemical mixtures with estrogenic activity exert endocrine disrupting effects by augmenting the progesterone biosynthetic pathway in immature rat granulosa cells, which is an effect achieved in part through activation of the estrogen receptors., (Copyright © 2018 John Wiley & Sons, Ltd.)

Transcript analysis is usually performed by costly, time-consuming, and expertise intensive methods, like real time-PCR, microarray, etc. However, they are not much feasible in low-input laboratories. Therefore, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a means of mammalian transcript analysis. Particularly, RT-LAMP was developed for buffalo aromatase cytochrome P450 (CYP19) transcript, to study its expression in 3D-cultured buffalo granulosa cells, which were exposed to lipopolysaccharide (LPS). The CYP19-RT-LAMP assay rapidly identified the LPS-induced downregulation of the CYP19 gene within 30 min at 63°C in a water bath. The assay was visualized via unaided eye by observing the change in turbidity and fluorescence, which were decreased by increasing the LPS exposure time to granulosa cells. Overall, the developed CYP19-RT-LAMP assay provided a hope on the application of RT-LAMP for mammalian transcript analysis in low-input laboratories., (© 2017 Wiley Periodicals, Inc.)

Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder in reproductive-aged women. Hormonal abnormality caused by steroidogenesis disturbances appears to be the main culprit of the clinical picture in PCOS. Vitamin D3 could regulate steroidogenesis in granulosa cells, but the mechanism of action of vitamin D3 on steroidogenesis remains unknown. AMP-activated protein kinase (AMPK) has a modulating role in steroid hormone production. We investigated the effect of vitamin D3 on steroidogenesis in cultured granulosa cells of dehydroepiandrosterone-induced PCOS mice and studied the involvement of AMPK signalling pathway in the current process. Immunoblotting assay showed that vitamin D3 could increase phosphorylation of AMPK alpha and acetyl-CoA carboxylase, main substrate of AMPK. Vitamin D3 and 5-aminoimidazole-4-carboxamide-1-β-D-riboside or Aicar (AMPK activator) not only reduced gene expression of steroidogenic enzymes (P450scc or Cyp11a1, StAR, Cyp19a1 and 3B-HSD), but also reduced production of progesterone and 17B-estradiol assessed by radioimmunoassay. Pretreatment with compound C (AMPK inhibitor) decreased APMK phosphorylation and eliminated the effects of vitamin D3 and Aicar on steroidogenic enzymes expression and estradiol and progesterone production. This study showed that vitamin D3 has the main role in regulating of steroidogenesis in granulosa cells of mouse polycystic ovary through activation of the AMPK signalling pathway., Significance of the Study: Polycystic ovarian syndrome (PCOS) is an endocrine disorder of women in reproductive age. This disorder is partly related to disruption in steroidogenesis pathway and dysregulation of estradiol and progesterone production in granulosa cells of polycystic ovaries. Previously, we have shown that vitamin D3 could modulate steroidogenesis pathway in PCOS granulosa cells. In this study, we investigate the molecular mechanism of vitamin D3 in regulation of steroidogenesis pathway. We have shown that vitamin D3 has a modulating role in steroidogenesis pathway of granulosa cells by regulation of AMP-activated protein kinase (AMPK) as an underlying molecular mechanism in mouse polycystic ovary., (Copyright © 2018 John Wiley & Sons, Ltd.)

In mammals, preovulatory oocytes are encircled by several layers of granulosa cells (GCs) in follicular microenvironment. These follicular oocytes are arrested at diplotene arrest due to high level of cyclic nucleotides from encircling GCs. Pituitary gonadotropin acts at the level of encircling GCs and increases adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) and activates mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. The MAPK3/1 disrupts the gap junctions between encircling GCs and oocyte. The disruption of gap junctions interrupts the transfer of cyclic nucleotides to the oocyte that results a drop in intraoocyte cAMP level. A transient decrease in oocyte cAMP level triggers maturation promoting factor (MPF) destabilization. The destabilized MPF finally triggers meiotic resumption from diplotene arrest in follicular oocyte. Thus, MAPK3/1 from GCs origin plays important role in gonadotropin-mediated meiotic resumption from diplotene arrest in follicular oocyte of mammals.

Successful ovulation requires the actions of gonadotropins along with those mediated by growth factors binding to their receptor tyrosine kinases (RTKs). There are several growth factors such as epidermal growth factor family ligands and interleukins that play a role during ovulation initiated by the preovulatory surge of luteinizing hormone (LH). The aim of this project was to analyze growth factor signaling pathways induced by LH in mouse granulosa cells. Immature female mice were treated with equine chorionic gonadotropin (eCG) followed 48 hr later by human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. We performed protein array analysis where we identified higher phosphorylation of insulin-like growth factor 1 receptor (IGF1R), the fibroblast growth factor receptor 2 (FGFR2) and ephrin receptor B1 (EPHB1) in granulosa cells at 4 hr post-hCG compared to 0 hr hCG (p < 0.05). We report both a significant increase in transcript abundance (p < 0.05) and the phosphorylation level (p < 0.05) of the IGF1R in granulosa cells at hCG4h. The mRNA abundance of the Fgfr2 and Ephb1 receptors remained unaltered upon hCG treatment. Nonetheless, transcript abundance of the fibroblast growth factor 2 (Fgf2) ligand was elevated at hCG4h (p < 0.01). Based on these results we conclude that the preovulatory LH surge activates signaling pathways of IGF1R through increase in the expression of the Igf1r gene in granulosa cells of ovulating follicles in mice. The LH surge also appears to activate FGFR2 IIIc and EPHB1 signaling, although further investigation is required., (© 2018 Wiley Periodicals, Inc.)

Bisphenol A (BPA) is an endocrine disruptor used in a variety of consumer products. Exposure to BPA leads to alterations in steroidogenesis of ovarian granulosa cells. Here, we analyzed the mechanism by which BPA alters progesterone biosynthesis in immature rat granulosa cells. BPA increased expression of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase in granulosa cells; however, BPA prevented the basal and the FSH-induced progesterone production. BPA caused sequestration of cholesterol to the perinuclear area, as evident by the Filipin staining. BPA decreased mRNA expression of ATP binding cassette transporter-A1 (Abca1) and increased level of sterol regulatory element binding protein 1. Addition of exogenous cell-permeable cholesterol restored the effect of BPA on Abca1 and Star mRNA expression and partially reversed BPA's effect on progesterone production. These results indicate that exposure to BPA disrupts cholesterol homeostasis leading to decreased progesterone production in immature rat granulosa cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The majority of ovarian primordial follicles are preserved in a dormant state to maintain the female reproductive lifespan, and only a few primordial follicles are activated to enter the growing follicle pool in each wave. Recent studies have shown that primordial follicular activation depends on mammalian target of rapamycin complex 1 (mTORC1)-KIT ligand (KITL) signaling in pre-granulosa cells and its receptor (KIT)-phosphoinositol 3 kinase (PI3K) signaling in oocytes. However, the upstream regulator of mTORC1 signaling is unclear. The results of the present study showed that the phosphorylated mitogen-activated protein kinase3/1 (MAPK3/1) protein is expressed in some primordial follicles and all growing follicles. Culture of 3 days post-parturition (dpp) ovaries with the MAPK3/1 signaling inhibitor U0126 significantly reduced the number of activated follicles and was accompanied by dramatically reduced granulosa cell proliferation and increased oocyte apoptosis. Western blot and immunofluorescence analyses showed that U0126 significantly decreased the phosphorylation levels of Tsc2, S6K1, and rpS6 and the expression of KITL, indicating that U0126 inhibits mTORC1-KITL signaling. Furthermore, U0126 decreased the phosphorylation levels of Akt, resulting in a decreased number of oocytes with Foxo3 nuclear export. To further investigate MAPK3/1 signaling in primordial follicle activation, we used phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor bpV(HOpic) to promote primordial follicle activation. In this model, U0126 also inhibited the activation of primordial follicles and mTORC1 signaling. Thus, these results suggest that MAPK3/1 participates in primordial follicle activation through mTORC1-KITL signaling., (© 2017 Wiley Periodicals, Inc.)

Secretion of 17-β-estradiol (E2) by human granulosa cells can be disrupted by various environmental toxicants. In the current study, we investigated whether carbon black nanoparticles (CB NPs) affect the steroidogenic activity of cultured human granulosa cells. The human granulosa cell line KGN and granulosa cells from patients undergoing in vitro fertilization were treated with increasing concentrations of CB NPs (1 to 100 µg/mL) together or not with follicle-stimulating hormone (FSH). We observed that CB NPs are internalized in KGN cells without affecting cell viability. CB NPs could be localized in the cytoplasm, within mitochondria and in association with the outer face of the endoplasmic reticulum membrane. In both cell types, CB NPs reduced in a dose-dependent manner the activity of aromatase enzyme, as reflected by a decrease in E2 secretion. A significant decrease was observed in response to CB NPs concentrations from 25 and 50 µg/mL in KGN cell line and primary cultures, respectively. Furthermore, CB NPs decreased aromatase protein levels in both cells and reduced aromatase transcript levels in KGN cells. CB NPs rapidly activated extracellular signal-regulated kinase 1 and 2 in KGN cells and pharmacological inhibition of this signaling pathway using PD 98059 significantly attenuated the inhibitory effects of CB NPs on CYP19A1 gene expression and aromatase activity. CB NPs also inhibited the stimulatory effect of FSH on aromatase expression and activity. Altogether, our study on cultured ovarian granulosa cells reveals that CB NPs decrease estrogens production and highlights possible detrimental effect of these common NPs on female reproductive health., (Copyright © 2017 Endocrine Society.)

Follicle-stimulating hormone (FSH) promotes secretion of follicle fluid and follicle development. FSH acts via cognate FSH receptor (FSHR). It remains unknown whether the supplement of FSH-receptor binding inhibitor (FRBI) into the in vitro maturation (IVM)medium influence the estrogen receptor expression and signal pathway of oocytes in sheep. The present study aimed to investigate FRBI effects on inositol trisphosphate (IP3) of oocytes and protein kinase A (PKA) of sheep granulosa cells, further to elucidate the signal pathway of FRBI effects. Cumulus-oocyte complexes (COCs) were recovered from antral follicles. COCs were cultured for 24 h in the IVM medium supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40 μg/mL) and FSH (10IU/mL). ELISA was used to measure the concentrations of estradiol (E2) and IP3 in the IVM medium. Western blotting was utilized to detect protein expression of ERβ of COCs and protein kinase A (PKA) of granulosa cells. The results showed IP3 concentrations of FRBI-3 and FRBI-4 groups were less than that of CG and FSH groups at 22 h and 24 h (P < 0.05). PKA levels of FRBI-3 and FRBI-4 groups were significantly less than that of CG and FSH group (P < 0.05 or P < 0.01). Expression levels of ERβ mRNA and protein of FRBI-treated groups were gradually decreased in comparison to CG and FSH group. The minimum value was detected in the FRBI-4 group. ERβ protein level of the FRBI-4 group was significantly less than that of FSH group (P < 0.05). E2 concentrations of FRBI-treated groups were elevated as compared to CG, with the highest increment of FRBI-2 group (P < 0.05). Our results revealed a higher dose of FRBI reduced IP3 production. FRBI could suppress slightly expression levels of ERβ mRNA and protein of COCs and PKA of granulosa cells, additionally increased E2 production of sheep COCs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

SIRT1, a member of the sirtuin family, has recently emerged as a vital molecule in controlling ovarian function. The aims of the present study were to investigate SIRT1 expression and analyze SIRT1-mediated apoptosis in human granulosa cells (GCs). Human ovarian tissues were subjected to immunohistochemistry for localization of SIRT1 expression. SIRT1 knockdown in a human ovarian GC tumor line (COV434) was achieved by small interfering RNA, and the relationship between apoptosis and SIRT1 was assessed by quantitative reverse transcription polymerase chain reaction and western blotting. We further detected SIRT1 expression in human luteinized GCs. Associations among SIRT1 knockdown, SIRT1 stimulation (resveratrol) and expression of ERK1/2 and apoptotic regulatory proteins were analyzed in cell lines and luteinized GCs. Resveratrol downregulated the levels of nuclear factor (NF)-κB/p65, but this inhibitory effect was attenuated by suppressing SIRT1 activity. The NF-κB/p65 inhibitor pyrrolidine dithiocarbamate achieved similar anti-apoptosis effects. These results suggest that SIRT1 might play an anti-apoptotic role in apoptosis processes in GCs, possibly by sensing and regulating the ERK1/2 pathway, which has important clinical implications. Thus, our study provides a mechanistic link, whereby activation of SIRT1 function might help to sustain human reproduction by maintaining GCs as well as oocytes, offering a novel approach for developing a new class of therapeutic anti-inflammatory agents.

Epidemiological studies have shown a link between problems with offspring of couples living in a contaminated environment in comparison to those who live in an uncontaminated environment. We measured the concentrations of 16 priority polycyclic aromatic hydrocarbons (PAHs) in maternal and cord blood. To explore the mechanism of the effects of PAH mixtures on nonluteinized granulosa cells (HGrC1) and granulosa tumor cells (COV434), as well as cell proliferation and apoptosis, we investigated the effect of PAH mixtures on the expression of the aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT) and aryl hydrocarbon receptor repressor (AHRR) genes, as well as the expression and activity of target genes cytochrome P450 1A1 (CYP1A1) and catechol-O-methyltransferase (COMT). The cells were exposed to mixture 1 (M1), composed of all 16 priority PAHs, and mixture 2 (M2), composed of five PAHs which are not classified as human carcinogens, and which are observed in the highest amounts both in maternal and cord blood. All 16 priority PAHs were bioavailable in maternal and cord plasma, suggesting that perinatal exposure should be considered. In HGrC1 cells, M1 increased AHR and ARNT, but decreased AHRR expression, in parallel with increased CYP1A1 and COMT expression and activity. M2 decreased AHR and AHRR, and increased ARNT, with no effect on CYP1A1 expression and activity; however, it did increase COMT expression and activity. In tumor cells, M1 lowered AHR and up-regulated AHRR and ARNT expression, consequently decreasing CYP1A1 expression and COMT activity. M2 up-regulated AHR and ARNT, down-regulated AHRR, and had no effect on CYP1A1 and COMT expression, but decreased COMT activity. We hypothesise that, dependent on composition, mixtures of PAHs activate the AHR differently through varying transcription responses: in HGrC1, a canonical AHR mechanism of M1, with activation of CYP1A1 important for detoxication, while in COV434, a noncanonical AHR mechanism, probably by activation the nuclear factor NFkB., (Copyright © 2017. Published by Elsevier B.V.)

Purpose: Our study aimed to investigate the association of telomerase activity (TA) and telomere length (TL) in granulosa cells (GCs) with IVF outcomes of polycystic ovary syndrome (PCOS) patients, and the effects of oral contraceptive pill (OCP) pretreatment on these two parameters., Methods: One hundred sixty-three infertile women were enrolled and divided into a PCOS group (n = 65) and a non-PCOS group (n = 98). The PCOS group was further divided into an OCP pretreatment group (n = 35) and a non-OCP pretreatment group (n = 30), a TA <0.070 group (n = 34) and a TA ≥0.070 group (n = 31), and a TL <1 group (n = 41) and a TL ≥1 group (n = 24), respectively., Results: No obvious differences were observed in TA between these groups. The TL was 0.971 in PCOS group and 1.118 in non-PCOS group (P = 0.005). The patients with TL ≥1 accounted for 36.9% in PCOS group and 54.1% in non-PCOS group (P = 0.032). The average duration of infertility for PCOS patients was 5 years in TA <0.070 group and 4 years in TA ≥0.070 group (P = 0.038), and 5 years in TL <1 group and 3 years in TL ≥1 group (P = 0.006), respectively. No obvious differences were observed in IVF outcomes between these groups. No obvious differences were observed in TA, TL, or IVF outcomes between OCP pretreatment group and non-OCP pretreatment group in PCOS patients., Conclusions: Shorter TL was found in PCOS patients. The TA levels did not change significantly in PCOS patients. PCOS patients with a lower TA level and shorter telomeres had an earlier onset of infertility symptoms. No predictive value was found for TA and TL in terms of embryo quality or IVF outcomes in PCOS patients, and no effect OCP pretreatment was observed on either TA and TL.

The process of follicular development involves communications between oocyte and surrounding granulosa cells. FURIN is a member of the family of proprotein convertases that is involved in the activation of a large number of zymogens and proproteins by cleavage at its recognition motif. To investigate the functions of FURIN in female fertility, furin flox/flox (fur fl/fl ) mice were crossed with Zp3-Cre mice and Gdf9-Cre, respectively, to achieve oocyte-specific disruption of FURIN. Here we report for the first time that FURIN is dispensable for primordial follicle maintenance and activation but important for early secondary follicular development, as ablation of FURIN in oocytes caused failure of follicle development beyond the type 4 and/or 5a follicles in mutant mice, resulting in increased number of early secondary follicles and the severely decreased number of mature follicles, thus anovulation and infertility. We also found that the developmental arrest of early secondary follicles might be rooted in the loss of the mature form of ADAMTS1 (85-kDa prodomain truncated) and compromised proliferation of granulosa cells in mutant mice. Taken together, our data highlight the importance of FURIN in follicle development beyond the early secondary follicle stage and indicate that compromised FURIN function leads to follicular dysplasia and female infertility in mice.

Ornithine decarboxylase antizyme 1 (OAZ1) regulates both polyamine biosynthesis and transport and acts as a tumor suppressor because of its functions in the regulation of cell proliferation, apoptosis and growth. However, the roles of OAZ1 in the regulation of polyamine metabolism, cell proliferation, apoptosis and hormone receptor expression in granulosa cells (GCs) from geese have not been determined. To define the roles of OAZ1 in primary GCs, we constructed and transfected an Oaz1 overexpression vector with a thymidine deletion into primary GCs. Oaz1 overexpression induced 65 and 30% decreases in the putrescine and spermidine contents, respectively, 225% increase in the spermine content and up-regulated the expression levels of genes associated with polyamine metabolism (but Oaz2). The Ccnd1, Pcna, Bax and Caspase 3 mRNA expression levels in GCs overexpressing Oaz1 were significantly higher than the expression levels in the vehicle group (p < 0.05). Additionally, a slight increase in the E2 concentration in the culture medium and enhanced Er, Fshr and Lhr expression were observed in the GCs 24 h after Oaz1 overexpression (p < 0.05). These data suggested that Oaz1 up-regulated the expression of genes related to polyamine metabolic enzymes and reproductive hormone receptors, disturbed intracellular polyamine homeostasis, and affected the transcription of genes associated to cell proliferation and apoptosis in goose primary GCs. Taken together, our results indicate that modifying polyamine metabolism by Oaz1 may interfere with the expression of genes involved in cell proliferation, apoptosis and responsiveness of ovary toward hormones in goose GCs.

This study was undertaken to evaluate whether cigarette smoke is associated with changes in the expression of antioxidant enzymes in granulosa cells of women undergoing IVF treatments. For this aim, the expression of three antioxidant enzymes (SOD1, SOD2 and catalase) in non-smokers (n = 20) and smokers (n = 20) was analyzed. There was a statistically significant overexpression of SOD2 and catalase mRNA levels in smokers in comparison with non-smokers. Cigarette smoking was associated with a lower fertilization rate, implantation rate and pregnancy rate in comparison with non-smokers. There was no effect on retrieved oocytes number, metaphase II oocytes number, quality of embryos transferred and live birth rate. These findings suggest that cigarette smoke initiates oxidative stress in granulosa cells.

Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder of women of reproductive age characterized by polycystic ovarian morphology, anovulation or oligomenorrhea, and hyperandrogenism. It is shown that disruption in the steroidogenesis pathway caused by excess androgen in PCOS is a critical element of abnormal folliculogenesis and failure in dominant follicle selection. Vitamin D plays an important role in the regulation of ovulatory dysfunction and can influence genes involved in steroidogenesis in granulosa cells. In the present study, we investigated the effects of vitamin D3 on steroidogenic enzyme expression and activities in granulosa cell using a PCOS mouse model. In our study, the PCOS mouse model was developed by the injection of dehydroepiandrosterone (DHEA) for 20 days. The mRNA and protein expression levels of genes involved in steroidogenesis in granulosa cells were compared between polycystic and normal ovaries using real-time PCR and Western blotting assays. Granulosa cells of DHEA-induced PCOS mice were then cultured with and without vitamin D3 and mRNA and protein expression levels of steroidogenic enzymes and serum 17beta-estradiol and progesterone levels were investigated using qRT-PCR, western blot, and radioimmunoassay, respectively. Steroidogenic enzymes including Cyp11a1, StAR, Cyp19a1, and 3β-HSD were upregulated in granulosa cells of PCOS mice when compared to normal mice. Treatment with vitamin D3 decreased mRNA and protein expression levels of steroidogenic enzymes in cultured granulosa cells. Vitamin D3 also decreased aromatase and 3β-HSD activity that leads to decreased 17beta-estradiol and progesterone release. This study suggests that vitamin D3 could modulate the steroidogenesis pathway in granulosa cells of PCOS mice that may lead to improving follicular development and maturation. This is a step towards a possible conceivable treatment for PCOS., Abbreviations: AMHR-II: anti-müllerian hormone receptor-II; 3β-HSD: 3β-hydroxysteroid dehydrogenase; Cyp11a1: Cytochrome P450 Family 11 Subfamily A Member 1; Cyp19a1: cytochrome P450 aromatase; DHEA: dehydroepiandrosterone; FSH: follicle stimulating hormone; FSHR: follicle stimulating hormone receptor; IVF: in vitro fertilization; 25OHD: 25-hydroxy vitamin D; OHSS: ovarian hyperstimulation syndrome; PCOS: polycystic ovarian syndrome; P450scc: P450 side-chain cleavage enzyme; StAR: steroidogenic acute regulatory protein; VDRs: vitamin D receptors.

Lipids rafts are specialised membrane microdomains involved in cell signalling that can be isolated as detergent-resistant membranes (DRMs). The second messenger cyclic AMP (cAMP) has a central role in cell signalling in the ovary and its degradation is carried out by the phosphodiesterase (PDE) enzyme family. We hypothesised that PDEs could be functionally present in the lipid rafts of porcine mural granulosa cell membranes. PDE6C, PDE8A and PDE11A were detected by dot blot in the DRMs and the Triton-soluble fraction of the mural granulosa cells membrane and the cytosol. As shown by immunocytochemistry, PDEs showed clear immunostaining in mural granulosa cell membranes and the cytosol. Interestingly, cAMP-PDE activity was 18 times higher in the DRMs than in the Triton-soluble fraction of cell membranes and was 7.7 times higher in the cytosol than in the DRMs. cAMP-PDE activity in mural granulosa cells was mainly contributed by the PDE8 and PDE11 families. This study shows that PDEs from the PDE8 and PDE11 families are present in mural granulosa cells and that the cAMP-PDE activity is mainly contributed by the cytosol. In the cell membrane, the cAMP-PDE activity is mainly contributed by the DRMs. In addition, receptors for prostaglandin E2 and LH, two G-protein-coupled receptors, are present in lipid rafts and absent from the non-raft fraction of the granulosa cell membrane. These results suggest that in these cells, the lipid rafts exist as a cell-signalling platform and PDEs are one of the key enzyme families present in the raft.

The aim of these in vivo and in vitro studies was to examine the influence of caloric restriction (CR), and the administration of insulin-like growth factor (IGF-I), on rabbit fecundity and to understand the interrelationships between CR and IGF-I, as well as the endocrine and intracellular mechanisms of their effects. Female rabbits were subjected to 50% CR, injections of IGF-I (20 μg/animal/day) and a combination of the two for 10 d before and 2 d after ovulation induced by 25 IU PMSG and 0.25 IU hCG. On the day of ovulation blood samples were collected and analyzed IGF-I, leptin, progesterone (P 4 ) and estradiol (E 2 ) concentrations by RIA. Some animals from each group were killed in their periovulatory period and weighed, as were their ovaries. Granulosa cells isolated from ovaries of does subjected or not to CR were cultured for 2 d with and without IGF-I (100 ng/mL). Accumulation of markers of cell proliferation (PCNA and cyclin B1), apoptosis (bax), MAP/ERK1,2 kinase (MAPK), protein kinase A (PKA) and IGF-I were evaluated by immunocytochemistry. In addition, E 2 release by cells isolated from ovaries of animals subjected or not to CR and cultured with and without IGF-I (1, 10, 100, 1000 or 10000 ng/mL) was assessed by RIA. The remaining animals were kept until parturition, when the number of pups was recorded. CR did not affect animal and ovarian weight, but significantly increased the number of pups per litter and plasma levels of IGF-I and decreased plasma leptin and P 4 , but not E 2 concentration. Injections of IGF-I did not influence body and ovarian weights, but increased the number of pups per litter and plasma IGF-I and leptin concentration and reduced plasma E 2 but not P 4 level. IGF-I administration did not modify the main effects of CR, although it prevented the CR-induced decrease in plasma P 4 level. CR reduced accumulation of PCNA, bax, promoted accumulation of cyclin B1 but not of MAPK, PKA or IGF-I within ovarian granulosa cells. Addition of IGF-I to culture medium reduced accumulation of bax, MAPK, and IGF-I and promoted PKA accumulation and E 2 release. CR promoted the stimulatory effect of IGF-I on E 2 output. Thus, CR can increase rabbit fecundity, probably via changes in IGF-I, leptin and steroid hormones released, which in turn can affect ovarian cell cycle, apoptosis, and response to IGF-I. Furthermore, they demonstrate the stimulatory influence of IGF-I on rabbit fecundity, which was associated with changes in plasma leptin, E 2 and ovarian cell apoptosis, PKA, MAPK, IGF-I and E 2 release. The promotion of IGF-I output by CR and the ability of IGF-I to mimic/replace but not to modify CR effects on fecundity, plasma IGF-I, and ovarian cell apoptosis suggest that IGF can mediate the action of CR on these reproductive indexes. In contrast, differences in the action of CR and IGF-I on other hormones, ovarian cell proliferation, protein kinases and IGF-I suggest that CR action on these indexes is not mediated by IGF-I. We thus demonstrate that both CR and IGF-I administration can increase rabbit fecundity, and that their effects can be mediated by changes in reproductive hormones, ovarian cell proliferation, apoptosis, and the response of ovarian cells to IGF-I., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Epigenetic mechanisms play important roles in properly occurring mammalian oogenesis. One of these mechanisms is DNA methylation adding a methyl group to the fifth carbon atom of the cytosine residues using S-adenosyl-L-methionine as a methyl donor. DNA methylation generally takes place at cytosine-phosphate-guanine (CpG) dinucleotide sites and rarely occurs at cytosine-phosphate-thymine (CpT), cytosine-phosphate-adenine (CpA), or cytosine-phosphate-cytosine sites, known as non-CpG sites. Basically, two different DNA methylation processes are identified: de novo methylation and maintenance methylation. While the de novo methylation functions in methylation of unmethylated DNA strands, maintenance methylation is capable of methylating hemi-methylated DNA strands following DNA replication. Both DNA methylation processes are catalyzed by special DNA methyltransferase (DNMT) enzymes. To date, five different DNMTs have been identified: DNMT1, DNMT3A, DNMT3B, DNMT3L, and DNMT2. In this chapter, we focus particularly on temporal and spatial expression of DNMTs in mammalian oocytes and granulosa cells.

Lysyl oxidase (LOX) is the key enzyme involved in the crosslinking of collagen and elastin that is essential for the formation of extracellular matrix (ECM). LOX-mediated ECM remodeling plays a critical role in follicle development, oocyte maturation and corpus luteum formation. To date, the regulation of LOX in human ovary has never been elucidated. Activin A and its functional receptors are highly expressed in ovarian follicles from an early developmental stage. They locally regulate follicle progression. The aim of this study was to investigate the effects of activin A on the expression of LOX and its extracellular enzyme activity in primary and immortalized human granulosa-lutein cells obtained from patients undergoing an in vitro fertilization procedure. We demonstrated that activin A significantly upregulated the expression of connective tissue growth factor (CTGF) and LOX via an activin/TGF-β type I receptor mediated-signaling pathway. Using a target depletion small interfering RNA knockdown approach, we further confirmed that the upregulation of CTGF expression resulted in an activin-A-induced increases in LOX expression and activity. These findings may provide insight into the mechanisms by which intrafollicular growth factors regulate the expression of LOX for ECM formation and tissue remodeling in the human ovary., (© 2016 Society for Reproduction and Fertility.)

Aims: Relevant roles in follicular development and ovulation are played by maternal antigen that embryos require (MATER), product of a maternal effect gene, and by reactive oxygen species (ROS), indispensable for the induction of ovulatory genes. At the moment, the relationship between these two biological systems and their involvement in the ovarian aging have not been still clarified. The aim of the current experimental study was to analyse the age-related changes of the MATER and NOX proteins., Materials and Methods: MATER and ROS homeostasis was studied in granulosa cells (GCs) and cumulus cells (CCs) of infertile patients who undergone oocyte retrieval for in vitro fertilization cycles using Western blot and confocal immunofluorescence analysis. Samples were obtained from subjects with age≥40years (cases) and with age≤37years (controls)., Key Findings: The expression pattern of MATER and NOX observed in GCs was not different from that observed in CCs. High levels of both proteins were detected in the control samples. A significant lower expression of both MATER and NOX4 was observed in the case versus control samples., Significance: The expression of MATER and NOX4 proteins are closely related to the follicular development and ovulation with particular regard for ovarian aging., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Within the ovarian follicle, granulosa cells (GCs) surround and support immature oocytes. FSH promotes the differentiation and proliferation of GCs and is essential for fertility. We recently reported that ERK activation is necessary for FSH to induce key genes that define the preovulatory GC. This research focused on the phosphoregulation by FSH of ERK within GCs. FSH-stimulated ERK phosphorylation on Thr(202)/Tyr(204) was PKA-dependent, but MEK(Ser(217)/Ser(221)) phosphorylation was not regulated; rather, MEK was already active. However, treatment of GCs with the EGF receptor inhibitor AG1478, a dominant-negative RAS, an Src homology 2 domain-containing Tyr phosphatase inhibitor (NSC 87877), or the MEK inhibitor PD98059 blocked FSH-dependent ERK(Thr(202)/Tyr(204)) phosphorylation, demonstrating the requirement for upstream pathway components. We hypothesized that FSH via PKA enhances ERK phosphorylation by inhibiting the activity of a protein phosphatase that constitutively dephosphorylates ERK in the absence of FSH, allowing MEK-phosphorylated ERK to accumulate in the presence of FSH because of inactivation of the phosphatase. GCs treated with different phosphatase inhibitors permitted elimination of both Ser/Thr and Tyr phosphatases and implicated dual specificity phosphatases (DUSPs) in the dephosphorylation of ERK. Treatment with MAP kinase phosphatase (MKP3, DUSP6) inhibitors increased ERK(Thr(202)/Tyr(204)) phosphorylation in the absence of FSH to levels comparable with ERK phosphorylated in the presence of FSH. ERK co-immunoprecipitated with Myc-FLAG-tagged MKP3(DUSP6). GCs treated with MKP3(DUSP6) inhibitors blocked and PKA inhibitors enhanced dephosphorylation of recombinant ERK2-GST in an in vitro phosphatase assay. Together, these results suggest that FSH-stimulated ERK activation in GCs requires the PKA-dependent inactivation of MKP3(DUSP6)., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Oocytes play critical roles in regulating the expression of transcripts encoding the glycolytic enzymes phosphofructokinase, platelet (PFKP) and lactate dehydrogenase A (LDHA) in granulosa cells in mice, but whether this is the case in pigs or other mammals has not been adequately investigated. Therefore, the aim of this study was to determine whether porcine oocytes regulate the expression levels of these transcripts in granulosa cells in vitro. Porcine cumulus cells expressed higher levels of PFKP and LDHA transcripts than mural granulosa cells (MGCs). However, co-culturing with oocytes had no significant effect on the isolated cumulus cells. While murine oocytes promoted the expression of both Pfkp and Ldha transcripts by murine MGCs, porcine oocytes promoted the expression of only Pfkp, but not Ldha transcripts by murine MGCs. Neither murine nor porcine oocytes affected PFKP and LDHA expression by porcine MGCs. Moreover, in the presence of porcine follicular fluid, porcine oocytes maintained the expression of PFKP, but not LDHA by porcine cumulus cells. Therefore, porcine oocytes are capable of regulating the expression of PFKP but not LDHA in granulosa cells in coordination with unknown factor(s) present in the follicular fluid., (© 2015 Japanese Society of Animal Science.)

Background: d-chiroinositol (DCI) is a inositolphosphoglycan (IPG) involved in several cellular functions that control the glucose metabolism. DCI functions as second messenger in the insulin signaling pathway and it is considered an insulin sensitizer since deficiency in tissue availability of DCI were shown to cause insulin resistance (IR). Polycystic ovary syndrome (PCOS) is a pathological condition that is often accompanied with insulin resistance. DCI can positively affects several aspect of PCOS etiology decreasing the total and free testosterone, lowering blood pressure, improving the glucose metabolism and increasing the ovulation frequency. The purpose of this study was to evaluate the effects of DCI and insulin combined with gonadotrophins namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on key steroidogenic enzymes genes regulation, cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and cytochrome P450 side-chain cleavage (P450scc) in primary cultures of human granulosa cells (hGCs). We also investigated whether DCI, being an insulin-sensitizer would be able to counteract the expected stimulator activity of insulin on human granulosa cells (hGCs)., Methods: The study was conducted on primary cultures of hGCs. Gene expression was evaluated by RT-qPCR method. Statistical analysis was performed applying student t-test, as appropriate (P < 0.05) set for statistical significance., Results: DCI is able to reduce the gene expression of CYP19A1, P450scc and insulin-like growth factor 1 receptor (IGF-1R) in dose-response manner. The presence of DCI impaired the increased expression of steroidogenic enzyme genes generated by the insulin treatment in gonadotrophin-stimulated hGCs., Conclusions: Insulin acts as co-gonadotrophin increasing the expression of steroidogenic enzymes genes in gonadotrophin-stimulated granulosa cells. DCI is an insulin-sensitizer that counteracts this action by reducing the expression of the genes CYP19A1, P450scc and IGF-1R. The ability of DCI to modulate in vitro ovarian activity of insulin could in part explain its beneficial effect when used as treatment for conditions associated to insulin resistance.

ACSF2 (encoded by acyl-CoA synthetase family member 2) belongs to the acyl-CoA synthetase (ACS) family, activating fatty acids by forming a thioester bond with CoA. In our previous study, a SNP residing in the intron of ACSF2 was identified to be linked to goose egg-laying performance. But the structure of goose ACSF2 as well as its role in reproduction remains unknown. In this study, we cloned and characterized ACSF2 in Yangzhou geese. A total of four alternative splice variants, designated as ACSF2-1, ACSF2-2, ACSF2-3 and ACSF2-4 respectively, were identified in the ovary. The coding regions of the four variants are 1770, 1692, 1599 and 1917 bp in length, respectively encoding 589, 563, 532 and 638 amino acids with conserved AMP-binding sites. All ACSF2 variants were widely expressed in 11 tested tissues in geese, except that the ACSF2-2 transcript was not detected in hypothalamus, pituitary gland and granulosa cells. Subcellular localization revealed that ACSF2 is a mitochondrial matrix protein. ACSF2 mRNA level was compared between high egg production (HEP; n = 8) and low egg production (LEP; n = 10) groups and showed a lower (P < 0.05) mRNA level in the HEP group. Further experiments indicated that overexpressing ACSF2 resulted in a significant increase of caspase-3 mRNA levels and that ACSF2 knockdown triggered a decrease in the caspase-3 mRNA level in granulosa cells. Similarly, the lower caspase-3 mRNA levels were identified in ovaries of the HEP group with lower ACSF2 mRNA levels. The research showed that the ACSF2 mRNA levels had a positive correlation with caspase-3 mRNA levels in vivo (R = 0.86, P < 0.01). Our results suggest that lower ACSF2 expression promotes the laying performance of goose possibly by inhibiting granulosa cell apoptosis and facilitating follicular development., (© 2016 Stichting International Foundation for Animal Genetics.)

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that can regulate various physiological and pathological processes. The expression of S1P has been detected in human follicular fluid. In addition, two S1P receptors, S1P1 and S1P3, are expressed at a high level in human granulosa cells. Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) production plays a critical role in the regulation of ovulation. However, thus far, the effect of S1P on COX-2 expression and PGE2 production in human granulosa cells remains unknown. In the present study, our results demonstrated that treatment with S1P significantly induced COX-2, but not COX-1, expression and increased PGE2 production in human granulosa cells. The stimulatory effects of S1P on COX-2 expression and PGE2 production were attenuated by treatment with specific antagonist of S1P1 or S1P3 and siRNA-mediated knockdown of S1P1 or S1P3. In addition, the COX-2 expression was induced by S1P1 or S1P3 agonist treatment. Interestingly, treatment with S1P activated YAP signaling via S1P1 and S1P3. Moreover, knockdown of YAP partially attenuated S1P-induced COX-2 expression and PGE2 production. These results provide evidence that S1P induces COX-2 expression and PGE2 production in human granulosa cells through a S1P1/3-mediated YAP signaling pathway., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of the present study was to investigate the hormonal regulation of hyaluronan (HA) components in sheep granulosa cells. HA components are present in the reproductive tract and have a range of physical and signalling properties related to reproductive function in several species. First, abattoir-derived ovaries of sheep were used to determine the localisation of HA synthase (HAS) 1-3 and CD44 proteins in antral follicles. Staining for HAS1-3 and CD44 proteins was most intense in the granulosa layer. Accordingly, the expression of HAS2, HAS3 and CD44 mRNA was measured in cultured granulosa cells exposed to 0-50ngmL(-1) of 17β-oestradiol and different combinations of oestradiol, gonadotropins, insulin-like growth factor (IGF)-1 and insulin for 48-96h (1ngmL(-1) FSH, 10ngmL(-1) insulin, 10ngmL(-1) IGF-1, 40ngmL(-1) E2 and 25ngmL(-1) LH.). mRNA expression was quantified by real-time polymerase chain reaction using a fold induction method. The results revealed that the hormones tested generally stimulated mRNA expression of the genes of interest in cultured granulosa cells. Specifically, oestradiol, when combined with IGF-1, insulin and FSH, stimulated HAS2 mRNA expression. Oestradiol and LH had synergistic effects in increasing HAS3 mRNA expression. In conclusion, we suggest that the hormones studied differentially regulate HAS2, HAS3 and CD44 in ovine granulosa cells in vitro. Further work is needed to address the signalling pathways involved.

Background: The ovulatory LH surge rapidly alters the expression of steroidogenesis-related genes such as steroidogenic acute regulatory protein (StAR) in granulosa cells (GCs) undergoing luteinization. We recently reported that histone modifications contribute to these changes. Histone modifications are regulated by a variety of histone modification enzymes. This study investigated the changes in gene expression of histone modification enzymes in rat GCs undergoing luteinization after the induction of ovulation. The extracellular regulated kinase (ERK)-1/2 is a mediator in the intracellular signaling pathway stimulated by the ovulatory LH surge and regulates the expression of a number of genes in GCs. We further investigated whether ERK-1/2 is involved in the regulation of the histone modification at the StAR promoter region in GCs undergoing luteinization., Results: GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h) CG injection. The expressions of 84 genes regulating histone modifications or DNA methylation were measured using a PCR array. Five genes (HDAC4, HDAC10, EZH2, SETDB2, and CIITA) were identified as histone acetylation- or histone methylation-related genes, and were significantly altered after hCG injection. None of the genes were related to DNA methylation. mRNA levels of EZH2, SETDB2, HDAC4, and HDAC10 decreased and CIITA mRNA levels increased 4 or 12 h after hCG injection. GCs isolated after eCG injection were incubated with hCG for 4 h to induce luteinization. StAR mRNA levels were significantly increased by hCG accompanied by the increase in H3K4me3 of the StAR promoter region. StAR mRNA expression was inhibited by the ERK inhibitor with the significant decrease of H3K4me3. These results suggest that hCG increases StAR gene expression through the ERK-1/2-mediated signaling which is also associated with histone modification of the promoter region., Conclusions: Gene expressions of histone modification enzymes change in GCs undergoing luteinization after ovulation induction. This change may play important roles in regulating the expression of various genes during the early stage of luteinization, which may be critical for the subsequent corpus luteum formation.

Granulosa cell tumors are representative of estrogenic ovarian tumors, and some Sertoli-stromal cell tumors are also estrogenic. The exact cells that are responsible for estrogenesis, however, have yet to be identified. In the present study, 25 sex cord-stromal tumors (20 granulosa cell tumors, 4 Sertoli-Leydig cell tumors, and a Sertoli cell tumor) were immunohistochemically examined for expression of P450 aromatase, which is critical for estrogenesis. All of the tumors had been evaluated for estrogenic function, including contemporaneous endometrial hyperplasia and/or elevation of serum estradiol. Eleven of 14 estrogenic granulosa cell tumors showed sparse or aggregated immunoreactivity for aromatase, whereas 5 of 6 nonestrogenic tumors did not. Aromatase was selectively expressed by plump granulosa cells with eosinophilic or vacuolated cytoplasm, resembling luteinized granulosa cells. Such a localization of aromatase is analogous to that in normal ovaries. Aromatase expression in primary tumors was recapitulated by recurrent tumors. In Sertoli-stromal cell tumors, either undifferentiated plump cells or well-differentiated Sertoli cells expressed aromatase. In conclusion, the expression of P450 aromatase corresponds to specific cell morphology in sex cord-stromal tumors, including recurrent tumors. Aromatase status in granulosa cell tumors provides helpful information on whether serum estradiol could be a marker for recurrence.

Background: Pro-nerve growth factor must be cleaved to generate mature NGF, which was suggested to be a factor involved in ovarian physiology and pathology. Extracellular proNGF can induce cell death in many tissues. Whether extracellular proNGF exists in the ovary and may play a role in the death of follicular cells or atresia was unknown., Materials and Methods: Immunohistochemistry of human and rhesus monkey ovarian sections was performed. IVF-derived follicular fluid and human granulosa cells were studied by RT-PCR, qPCR, Western blotting, ATP- and caspase-assays., Results and Conclusion: Immunohistochemistry of ovarian sections identified proNGF in granulosa cells and Western blotting of human isolated granulosa cells confirmed the presence of proNGF. Ovarian granulosa cells thus produce proNGF. Recombinant human proNGF even at high concentrations did not affect the levels of ATP or the activity of caspase 3/7, indicating that in granulosa cells proNGF does not induce death. In contrast, mature NGF, which was detected previously in follicular fluid, may be a trophic molecule for granulosa cells with unexpected functions. We found that in contrast to proNGF, NGF increased the levels of the transcription factor early growth response 1 and of the enzyme choline acetyl-transferase. A mechanism for the generation of mature NGF from proNGF in the follicular fluid may be extracellular enzymatic cleavage. The enzyme MMP7 is known to cleave proNGF and was identified in follicular fluid and as a product of granulosa cells. Thus the generation of NGF in the ovarian follicle may depend on MMP7.

Oocyte maturation and cumulus cell expansion depend on luteinizing hormone (LH)-mediated upregulation of membrane-bound epidermal growth factor (EGF)-like ligands, including amphiregulin, epiregulin, and betacellulin. These ligands then transactivate the EGF receptor (EGFR) after release by matrix metalloproteinases (MMPs). However, direct measurement of released EGF-like ligands or MMPs from granulosa cells has not been formally evaluated, nor has direct identification of responsible MMPs. Here we address these issues by analyzing LH-induced steroidogenesis, which is also MMP and EGFR dependent, in freshly isolated mouse primary granulosa cells. We demonstrate a correlation between amphiregulin and epiregulin mRNA induction and steroid production in LH-treated granulosa cells as well as in ovaries of human chorionic gonadotropin-treated mice. In contrast, LH does not alter Mmp1, Mmp2, Mmp3, Mmp8, Mmp9, or Adam17 mRNA expression. We demonstrate that, in primary mouse granulosa cells, LH triggers release of soluble amphiregulin that correlates with steroid production, both of which are blocked by MMP2/9 inhibition, confirming that MMP2/9 likely regulates LH-induced amphiregulin release and downstream processes. Notably, LH does not alter secretion of MMP2/9 from primary granulosa cells, nor does it modulate MMP activity. These findings indicate that, in the ovary, LH dictates EGFR-mediated processes not by regulating MMPs, but instead by increasing EGF-like ligand availability. In contrast, LH stimulation of primary mouse Leydig cells does not induce EGF-like ligand expression or require MMP2/9 for steroidogenesis, confirming marked differences in LH receptor-induced processes in the testes. Our results suggest that MMP inhibition may be a means of attenuating excess ovarian steroid production in diseases like polycystic ovary syndrome., (© 2015 by the Society for the Study of Reproduction, Inc.)

Objective: To examine coenzyme Q10 (CoQ10)-dependent mitochondrial respiratory chain (MRC) activity in granulosa cells (GC) with aging and examine the effect of in vitro CoQ supplementation., Design: Experimental study., Setting: Hospital laboratory., Patient(s): Ten younger (<32 years) and 10 older (>39 years) patients undergoing in vitro fertilization (IVF) treatment., Intervention(s): Measurement of succinate-cytochrome c reductase (MRC complex II + III) activity in the presence and absence of CoQ1 (a soluble CoQ analog)., Main Outcome Measure(s): MRC enzymatic activity in human GC via complex II + III measured in GC homogenate by spectrophotometry and compared with CoQ in dependent MRC complex II and citrate synthase (CS)., Result(s): Complex II + III activity was 1.9 times higher in young patients compared with older patients (18.3 ± 5.8 and 9.6 ± 3 nmol/min/mg, respectively) whereas II and CS were not statistically significantly different. Increased II + III activity in the presence CoQ1 was observed in both groups but was statistically significantly higher in the older patients, reaching similar levels. Compared with baseline (II + III + Q/II + III), the increase was 2.47 times higher in older patients compared with young patients (6.5 ± 2.0 and 2.62 ± 0.83, respectively)., Conclusion(s): Coenzyme Q10-dependent MRC activity in GC reduces with aging. This reduction is diminished upon in vitro CoQ1 supplementation, indicating that CoQ10 deficit is the underlying cause for the mitochondrial dysfunction. The results show that functional CoQ10 status can be assessed by measuring complex II + III activity in GC and might provide a useful monitoring tool for future clinical studies of oral CoQ10 supplementation to older patients undergoing IVF treatment., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)