Human Granulosa-lutein Research Articles

High GDF-8 in follicular fluid is associated with a low pregnancy rate in IVF patients with PCOS.

Polycystic ovary syndrome (PCOS) is the most common cause of female infertility. Growth differentiation factor-8 (GDF-8) is expressed in the ovary and can be detected in human follicular fluid which provides an important microenvironment for maintaining physiological functions of the ovarian follicle. To date, the relationship between GDF-8 levels in follicular fluid and the risk of PCOS is completely unknown. In the present study, we show that during the process of the controlled ovarian hyperstimulation (COH), serum GDF-8 levels are higher on the day of gonadotropin administration and 14 days after embryo transfer in in vitro fertilization (IVF) patients with PCOS than they are in IVF patients without PCOS. Importantly, GDF-8 levels in follicular fluid at oocyte retrieval are also higher in PCOS patients than in non-PCOS patients. Treatment of primary human granulosa-lutein (hGL) cells with GDF-8 downregulates StAR protein expression and the inhibition is more pronounced in hGL cells from PCOS patients than it is in cells from non-PCOS patients. Importantly, high GDF-8 levels and low progesterone (P4) levels were associated with poor pregnancy outcomes in PCOS patients. Our results provide the first evidence that aberrant expression of GDF-8 in the follicular fluid of PCOS patients results in abnormal P4 expression, which leads to poor pregnancy outcomes.

MON-027 Activation of Endoplasmic Reticulum Stress Mediates Oxidative Stress-Induced Apoptosis of Granulosa Cells in Ovaries Affected by Endometrioma

Endometriosis exerts detrimental effects on ovarian physiology and compromises follicular health. Granulosa cells of endometriosis patients are characterized by increased apoptosis, as well as high oxidative stress. Among several pathophysiologic factors associated with endometriosis, it is expected that oxidative stress contributes to the induction of apoptosis in granulosa cells, although the underlying mechanism remains unclear. Endoplasmic reticulum (ER) stress, a local factor closely associated with oxidative stress, has emerged as a critical regulator of ovarian function. We hypothesized that ER stress is activated by high oxidative stress in granulosa cells in ovaries with endometrioma and mediates oxidative stress-induced apoptosis. Ovaries from patients with endometrioma and control were collected to determine apoptosis, oxidative stress and ER stress by TUNEL, immunohistochemical staining of 8-OHdG and ER stress sensors, respectively. Human granulosa-lutein cells (GLCs) obtained from IVF patients were cultured with H2O2 (an oxidative stress inducer) or tauroursodeoxycholic acid (TUDCA, an ER stress inhibitor in clinical use) to assess apoptosis and ER stress by quantitative PCR and FACS. Activity of pro-apoptotic factors was determined by caspase-8 activity assay and western blotting for cleaved caspase-3. Human GLCs from patients with endometrioma expressed up to two times higher level of mRNAs associated with the unfolded protein response (UPR), including ATF4, ATF6, the spliced form of XBP1, HSPA5, and CHOP. In addition, the levels of phosphorylated ER stress sensor proteins, IRE1 and PERK, were elevated. Given that ER stress results in phosphorylation of ER stress sensor proteins and induces UPR factors, these findings indicate that these cells were under ER stress. H2O2 increased expression of UPR-associated mRNAs in cultured human GLCs, and this effect was abrogated by pre-treatment with TUDCA. Treatment with H2O2 increased apoptosis and the activity of pro-apoptotic factors caspase-8 and caspase-3, both of which were attenuated by TUDCA. Our findings suggest that activated ER stress induced by high oxidative stress in granulosa cells in ovaries with endometrioma mediates apoptosis of these cells, leading to ovarian dysfunction in endometriosis patients. Targeting ER stress with currently clinically available ER stress inhibitors, or with these agents in combination with antioxidants, may serve as a novel strategy for rescuing endometriosis-associated ovarian dysfunction.

MON-033 Androgen Increases the Accumulation of Advanced Glycation End Products in Granulosa Cells by Activating ER Stress in PCOS

Polycystic ovarian syndrome (PCOS) is associated with hyperandrogenism. Previously we found that androgen activated endoplasmic reticulum (ER) stress in granulosa cells of antral follicles in PCOS, contributing to ovarian fibrosis (1) and growth arrest of antral follicles (2). In addition, recent studies demonstrated the accumulation of advanced glycation end products (AGEs) in granulosa cells from PCOS patients, which contribute to its pathology. Based on these findings, we hypothesized that androgen upregulates the expression of the receptor for AGEs (RAGE) in granulosa cells of antral follicles by activating ER stress. This in turn, increases the accumulation of AGEs in these cells. In the present study, we found that testosterone induced the expression of RAGE and accumulation of AGE in cultured human granulosa-lutein cells (GLCs). These effects were inhibited with the treatment of tauroursodeoxycholic acid (TUDCA), a clinically available ER stress inhibitor agent. Knockdown of the transcription factor C/EBP homologous protein (CHOP), an unfolded protein response (UPR) factor activated by ER stress, inhibited the testosterone-induced RAGE expression and AGE accumulation. Pretreatment with flutamide, as well as knockdown of androgen receptor decreased the testosterone-induced RAGE expression. Expression of RAGE was increased in GLCs obtained from patients with PCOS. Concomitantly, the expression of RAGE and the accumulation of AGE was increased in granulosa cells of antral follicles from PCOS patients and dehydroepiandrosterone (DHEA)-induced PCOS mice. Administration of the RAGE inhibitor, FPS-ZM1 or TUDCA to PCOS mice, reduced the expression of RAGE and the accumulation of AGE in granulosa cells of antral follicles, accompanied by a reduction of atretic follicles and improvement in the estrous cycle. In summary, our findings indicate that hyperandrogenism in PCOS increases the expression of RAGE and accumulation of AGEs in the ovary by activating ER stress. The potential therapeutic benefit of targeting the AGE-RAGE system, either with a RAGE inhibitor or an ER stress inhibitor agents, may serve as a novel approach for the treatment of PCOS. (1) Takahashi et al. Sci Rep. 2017;7(1):10824. (2) Azhary et al. Endocrinol. 2019;160(1):119–132

TGF-\u03b21 induces VEGF expression in human granulosa-lutein cells: a potential mechanism for the pathogenesis of ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome (OHSS) is one of the most serious and iatrogenic complications that can occur during in vitro fertilization treatment. Although the pathogenesis of OHSS is not fully understood, vascular endothelial growth factor (VEGF) has been recognized as an important mediator of the development of OHSS. Transforming growth factor-beta-1 (TGF-β1) is known to regulate various ovarian functions. However, whether VEGF can be regulated by TGF-β1 in human granulosa cells has not been determined. In addition, the role of TGF-β1 in the pathogenesis of OHSS remains unknown. In the present study, we demonstrate that TGF-β1 stimulates VEGF expression in and secretion from both immortalized human granulosa-lutein (hGL) cells and primary hGL cells. Our results demonstrate that the SMAD2/3, ERK1/2, and p38 MAPK signaling pathways are involved in TGF-β1-induced VEGF expression and secretion. Using a mouse OHSS model, we show that the expression levels of TGF-β1 and VEGF are increased in the ovaries of OHSS mice. Blocking TGF-β1 signaling inhibits the development of OHSS by attenuating VEGF expression. Moreover, clinical results reveal that the protein levels of TGF-β1 and VEGF are increased in the follicular fluid of patients with OHSS, and that the levels of these two proteins in the follicular fluid are positively correlated. The results of this study help to elucidate the mechanisms by which VEGF expression is regulated in hGL cells, which could lead to the development of alternative therapeutic approaches for treating OHSS.

Androgens Increase Accumulation of Advanced Glycation End Products in Granulosa Cells by Activating ER Stress in PCOS

Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism, and we previously found that androgens activate endoplasmic reticulum (ER) stress in granulosa cells from patients with PCOS. In addition, recent studies demonstrated the accumulation of advanced glycation end products (AGEs) in granulosa cells from PCOS patients, which contribute to the pathology. Therefore, we hypothesized that androgens upregulate the receptor for AGEs (RAGE) expression in granulosa cells by activating ER stress, thereby increasing the accumulation of AGEs in these cells and contributing to the pathology. In the present study, we show that testosterone increases RAGE expression and AGE accumulation in cultured human granulosa-lutein cells (GLCs), and this is reduced by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Knockdown of the transcription factor C/EBP homologous protein (CHOP), an unfolded protein response factor activated by ER stress, inhibits testosterone-induced RAGE expression and AGE accumulation. The expression of RAGE and the accumulation of AGEs are upregulated in granulosa cells from PCOS patients and dehydroepiandrosterone-induced PCOS mice. Administration of the RAGE inhibitor FPS-ZM1 or TUDCA to PCOS mice reduces RAGE expression and AGE accumulation in granulosa cells, improves their estrous cycle, and reduces the number of atretic antral follicles. In summary, our findings indicate that hyperandrogenism in PCOS increases the expression of RAGE and accumulation of AGEs in the ovary by activating ER stress, and that targeting the AGE-RAGE system, either by using a RAGE inhibitor or a clinically available ER stress inhibitor, may represent a novel approach to PCOS therapy.

ALK4-SMAD3/4 mediates the effects of activin A on the upregulation of PAI-1 in human granulosa lutein cells

In the mammalian ovary, the proteolysis of the extracellular matrix is dynamically regulated by plasminogen activator and plasminogen activator inhibitor (PAI), and it is a critical event that influences various physiological and pathological processes. Activin A is a member of the transforming growth factor-β superfamily and is expressed at a high level in human luteal cells that play an essential role in the regulation of the luteal function. At present, it is not known whether activin A can regulate the expression and production of PAI in human granulosa lutein (hGL) cells. The present study aimed to examine the effects of activin A on the expression and production of intraovarian PAI-1 and the underlying molecular mechanisms. Using primary and immortalized hGL cells as the cell model, we demonstrated that activin A upregulated the expression of PAI-1 and increased the production of PAI-1 in an autocrine/paracrine manner. Additionally, using a dual inhibition approach (molecular inhibitors and siRNA-mediated knockdown), we showed that this biological function is mediated by the ALK4-mediated SMAD3-SMAD4-dependent signaling pathway. Our findings suggest that activin A may be involved in the regulation of luteal function via the induction of PAI-1 expression and an increase in PAI-1 production.

TGF-β1 promotes vitamin D-induced prostaglandin E2 synthesis by upregulating vitamin D receptor expression in human granulosa-lutein cells.

There is increasing evidence showing the importance of vitamin D (Vit D) and its nuclear receptor, the Vit D receptor (VDR), in female reproductive health. Transforming growth factor-β1 (TGF-β1) and its functional receptors are expressed in human oocytes and granulosa cells that participate in follicular development and ovulation. Recently, Sma- and Mad-related protein 3 (SMAD3; a downstream effector of TGF-β1) has been proposed to mediate crosstalk between the Vit D and TGF-β1 signaling pathways, but this relationship has not been fully explored and has yet to be tested in human granulosa-lutein (hGL) cells. In this study, we showed that TGF-β1 significantly promoted the expression of VDR, and this stimulatory effect occurred through the activin receptor-like kinase 5 type I receptor-mediated SMAD3 and ERK1/2 signaling pathways in hGL cells. Additionally, we showed that Vit D increased the expression of cyclooxygenase 2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) in a time- and dose-dependent manner. Furthermore, we demonstrated a synergistic effect of TGF-β1 and Vit D on the expression of COX-2 and synthesis of PGE2, and this effect could be attenuated by silencing the expression of VDR. Our findings indicate that TGF-β1 upregulates the expression of VDR, which promotes Vit D-induced COX-2 expression and subsequent PGE2 production by activating the SMAD3 and ERK1/2 signaling pathways in hGL cells.

TGF-β1 Increases GDNF Production by Upregulating the Expression of GDNF and Furin in Human Granulosa-Lutein Cells.

Glial cell line-derived neurotrophic factor (GDNF) is expressed at a high level in the human ovary and GDNF signaling is involved in the direct control of follicular activation and oocyte maturation. Transforming growth factor-β1 (TGF-β1) plays an important role in the regulation of various ovarian functions. Furin is an intracellular serine endopeptidase of the subtilisin family that is closely associated with the activation of multiple protein precursors. Despite the important roles of GDNF and TGF-β1 in the regulation of follicular development, whether TGF-β is able to regulate the expression and production of GDNF in human granulosa cells remains to be determined. The aim of this study was to investigate the effect of TGF-β1 on the production of GDNF and its underlying mechanisms in human granulosa-lutein (hGL) cells. We used two types of hGL cells (primary hGL cells and an established immortalized hGL cell line, SVOG cells) as study models. Our results show that TGF-β1 significantly induced the expression of GDNF and furin, which, in turn, increased the production of mature GDNF. Using a dual inhibition approach combining RNA interference and kinase inhibitors against cell signaling components, we showed that the TβRII type II receptor and ALK5 type I receptor are the principal receptors that mediated TGF-β1-induced cellular activity in hGL cells. Additionally, Sma- and Mad-related protein (SMAD)3 and SMAD4 are the downstream signaling transducers that mediate the biological response induced by TGF-β1. Furthermore, furin is the main proprotein convertase that induces the production of GDNF. These findings provide additional regulatory mechanisms by which an intrafollicular factor influences the production of another growth factor through a paracrine or autocrine interaction in hGL cells.

Activation of endoplasmic reticulum stress mediates oxidative stress-induced apoptosis of granulosa cells in ovaries affected by endometrioma.

Endometriosis exerts detrimental effects on ovarian physiology and compromises follicular health. Granulosa cells from patients with endometriosis are characterized by increased apoptosis, as well as high oxidative stress. Endoplasmic reticulum (ER) stress, a local factor closely associated with oxidative stress, has emerged as a critical regulator of ovarian function. We hypothesized that ER stress is activated by high oxidative stress in granulosa cells in ovaries with endometrioma and that this mediates oxidative stress-induced apoptosis. Human granulosa-lutein cells (GLCs) from patients with endometrioma expressed high levels of mRNAs associated with the unfolded protein response (UPR). In addition, the levels of phosphorylated ER stress sensor proteins, inositol-requiring enzyme 1 (IRE1) and double-stranded RNA-activated protein kinase-like ER kinase (PERK), were elevated in granulosa cells from patients with endometrioma. Given that ER stress results in phosphorylation of ER stress sensor proteins and induces UPR factors, these findings indicate that these cells were under ER stress. H2O2, an inducer of oxidative stress, increased expression of UPR-associated mRNAs in cultured human GLCs, and this effect was abrogated by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Treatment with H2O2 increased apoptosis and the activity of the pro-apoptotic factors caspase-8 and caspase-3, both of which were attenuated by TUDCA. Our findings suggest that activated ER stress induced by high oxidative stress in granulosa cells in ovaries with endometrioma mediates apoptosis of these cells, leading to ovarian dysfunction in patients with endometriosis.

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.

CCN2 Mediates S1P-Induced Upregulation of COX2 Expression in Human Granulosa-Lutein Cells.

CCN1 and CCN2 are members of the CCN family and play essential roles in the regulation of multiple female reproductive functions, including ovulation. Cyclooxygenase-2 (COX2) is a critical mediator of ovulation and can be induced by sphingosine-1-phosphate (S1P) through the S1P1/3-mediated Yes-associated protein (YAP) signaling. However, it is unclear whether CCN1 or CCN2 can mediate S1P-induced upregulation of COX2 expression and increase in prostaglandin E2 (PGE2) production in human granulosa-lutein (hGL) cells. In the present study, we investigated the effects of S1P on the expressions of CCN1 and CCN2 in hGL cells. Additionally, we used a dual inhibition approach (siRNA-mediated silencing and small molecular inhibitors) to investigate the molecular mechanisms of S1P effects. Our results showed that S1P treatment significantly upregulated the expression of CCN1 and CCN2 in a concentration-dependent manner in hGL cells. Additionally, inhibition or silencing of S1P1, but not S1P3, completely abolished the S1P-induced upregulation of CCN2 expression. Furthermore, we demonstrated that S1P-induced nuclear translocation of YAP and inhibition or silencing of YAP completely abolished the S1P-induced upregulation of CCN1 and CCN2 expression. Notably, silencing of CCN2, but not CCN1, completely reversed the S1P-induced upregulation of COX2 expression and the increase in PGE2 production. Thus, CCN2 mediates the S1P-induced upregulation of COX2 expression through the S1P1-mediated signaling pathway in hGL cells. Our findings expand our understanding of the molecular mechanism underlying the S1P-mediated cellular activities in the human ovary.

Increased Expression of KISS1 and KISS1 Receptor in Human Granulosa Lutein Cells-Potential Pathogenesis of Polycystic Ovary Syndrome.

Kisspeptins are a family of neuropeptides that are essential for fertility. Recent experimental data suggest a putative role of kisspeptin signaling in the direct control of ovarian function. To explore the expression of KISS1 and KISS1 receptor (KISS1R) in human granulosa lutein cells and the potential role of KISS1/KISS1R system in the pathogenesis of polycystic ovary syndrome (PCOS), we measured the concentration of KISS1 in follicular fluid, the expression of KISS1 and KISS1R in granulosa lutein cells, and the circulating hormones. The expression levels of KISS1 and KISS1R were significantly upregulated in human granulosa lutein cells obtained from women with PCOS. The expression levels of KISS1 in human granulosa lutein cells highly correlated with those of KISS1R in non-PCOS patients, but not in patients with PCOS, most likely due to the divergent expression patterns in women with PCOS. Additionally, the expression levels of KISS1 highly correlated with the serum levels of anti-Müllerian hormone (AMH). The expression levels of KISS1 and KISS1R, as well as the follicular fluid levels of KISS1, were not significantly different between the pregnant and nonpregnant patients in both PCOS and non-PCOS groups. In conclusion, the increased expression of KISS1 and KISS1R in human granulosa lutein cells may contribute to the pathogenesis of PCOS. The expression levels of KISS1 highly correlated with the serum levels of AMH. The KISS1 and KISS1R system in the ovary may not have a remarkable role in predicting the in vitro fertilization (IVF) outcome.

Melatonin induces progesterone production in human granulosa-lutein cells through upregulation of StAR expression.

Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in ovarian steroidogenesis and progesterone (P4) synthesis. Melatonin and its receptors are expressed in human granulosa cells, and have been shown to influence basal P4 production. However, previous studies addressing the regulation of StAR expression by melatonin and its impact on P4 secretion yielded contradictory results. Here, we demonstrate that melatonin upregulates StAR expression in primary cultures of human granulosa-lutein (hGL) cells obtained from women undergoing in vitro fertilization (IVF). Using pharmacological inhibitors, we show that the stimulatory effect of melatonin on StAR expression is mediated via both MT1 and MT2 melatonin receptors. Melatonin exposure activates the PI3K/AKT signaling pathway and its inhibition attenuates the stimulatory effect of melatonin on StAR expression. Moreover, siRNA-mediated knockdown of StAR abolishes melatonin-induced P4 production. Importantly, clinical analyses demonstrate that melatonin levels in human follicular fluid are positively correlated with P4 levels in serum. By illustrating the potential physiological role of melatonin in the regulation of StAR expression and P4 production in hGL cells, our results may serve to improve current strategies used to treat clinical infertility.

Human chorionic gonadotropin-induced amphiregulin stimulates aromatase expression in human granulosa-lutein cells: a mechanism for estradiol production in the luteal phase.

Does amphiregulin (AREG), the most abundant and important epidermal growth factor receptor (EGFR) ligand in the follicular fluid, regulate aromatase expression in human granulosa-lutein (hGL) cells? AREG mediates the hCG-induced up-regulation of aromatase expression and estradiol (E2) production in hGL cells. AREG expression and secretion are rapidly induced by hCG in hGL cells and mediate physiological functions of LH/hCG in the ovary. EGFR protein is expressed in follicles not only in the pre-ovulatory phase but also throughout the luteal phase of the menstrual cycle. After the LH surge, the human corpus luteum secretes high levels of E2, which regulates various luteal cell functions. Aromatase is an enzyme responsible for a key step in the biosynthesis of E2. However, whether AREG regulates aromatase expression and E2 production in hGL cells remains unexplored. This study is an experimental study performed over a 1-year period. In vitro investigations examined the role of AREG in the regulation of aromatase expression and E2 production in primary hGL cells. Primary hGL cells were obtained from women undergoing IVF treatment in an academic research center. Aromatase mRNA and protein levels were examined after exposure of hGL cells to recombinant human AREG, hCG or LH. The EGFR tyrosine kinase inhibitor AG1478, PI3K inhibitor LY294002 and siRNAs targeting EGFR, LH receptor, StAR and AREG were used to verify the specificity of the effects and to investigate the underlying molecular mechanisms. Reverse transcription quantitative real-time PCR (RT-qPCR) and western blot were used to measure the specific mRNA and protein levels, respectively. Follicular fluid and serum were collected from 65 infertile women during IVF treatment. Pearson's correlation analysis was performed to examine the correlation coefficient between two values. Treatment of hGL cells with AREG-stimulated aromatase expression and E2 production. Using pharmacological inhibitors and specific siRNAs, we revealed that AREG-stimulated aromatase expression and E2 production via EGFR-mediated activation of the protein kinase B (AKT) signaling pathway. In addition, inhibition of EGFR activity and AREG knockdown attenuated hCG-induced up-regulation of aromatase expression and E2 production. Importantly, the protein levels of AREG in the follicular fluid were positively correlated with the E2 levels in serum after 2days of oocyte pick-up and in the follicular fluid of IVF patients. N/A. The in vitro setting of this study is a limitation that may not reflect the real intra-ovarian microenvironment. Clinical data were obtained from a small sample size. Our results provide the first evidence that hCG-induced AREG contributes to aromatase expression and E2 production in the luteal phase of the menstrual cycle. A better understanding of the hormonal regulation of female reproductive function may help to develop new strategies for the treatment of clinical infertility. This work was supported by the National Natural Science Foundation of China for Young Scientists (81601253), the specific fund of clinical medical research of Chinese Medical Association (16020160632) and the Foundation from the First Affiliated Hospital of Zhengzhou University for Young Scientists to Lanlan Fang. This work was also supported by an operating grant from the National Natural Science Foundation of China (81820108016) to Ying-Pu Sun. All authors declare no conflict of interest.

Accumulation of advanced glycation end products in follicles is associated with poor oocyte developmental competence.

Advanced glycation end products (AGEs) affect the follicular microenvironment. The close relationship between AGEs, proinflammatory cytokine production and activation of the unfolded protein response (UPR), which involves activating transcription factor 4 (ATF4), is crucial for regulation of various cellular functions. We examined whether accumulation of AGEs in follicles was associated with proinflammatory cytokine production and activation of the UPR in granulosa cells and decreased oocyte developmental competence. Concentrations of AGEs, soluble receptor for AGE (sRAGE), interleukin (IL)-6 and IL-8 in follicular fluid (FF) were examined by ELISAs in 50 follicles. mRNA expression of ATF4, IL-6 and IL-8 in cumulus cells (CCs) were examined by quantitative RT-PCR in 77 samples. Cultured human granulosa-lutein cells (GLCs) were treated with AGE-bovine serum albumin (BSA) alone or following transfection of ATF4-targeting small interfering RNA. The AGE concentration and the AGE/sRAGE ratio in FF were significantly higher in follicles containing oocytes that developed into poor-morphology embryos (group I) than those with good-morphology embryos (group II). When compared with sibling follicles from the same patients, the AGE/sRAGE and concentrations of IL-6 and IL-8 in FF, as well as ATF4, IL-6 and IL-8 mRNA expression in CCs, were significantly higher in group I follicles than group II. AGE treatment increased mRNA expression of ATF4, IL-6 and IL-8 in cultured GLCs. Knockdown of ATF4 abrogated the stimulatory effects of AGE on mRNA expression and protein secretion of IL-6 and IL-8. Our findings support the idea that accumulation of AGEs in follicles reduces oocyte competence by triggering inflammation via activation of ATF4 in the follicular microenvironment.

TGF-β1 promotes hyaluronan synthesis by upregulating hyaluronan synthase 2 expression in human granulosa-lutein cells

Hyaluronan serves as a structural component of ovarian follicles, and hyaluronan-mediated signaling cascades lead to follicular development, oocyte maturation, and ovulation. Transforming growth factor-β (TGF-β1) is highly expressed in human oocytes and granulosa cells and involved in the regulation of follicular development and ovulation. Previous studies have shown the imperative role for TGF-β signaling in the regulation of hyaluronan-mediated cumulus expansion and ovulation in human granulosa-lutein (hGL) cells. However, the detailed underlying molecular mechanisms by which TGF-β regulates the synthesis of hyaluronan in hGL cells are not fully elucidated. Using both primary and immortalized hGL cells as study models, we provide the first data showing that TGF-β1 significantly promoted the synthesis of hyaluronan by upregulating the expression of hyaluronan synthase 2 in these cells. Additionally, using dual inhibition approaches, we show that the TGF-β type II (TβRII) receptor and TGF-β type I (ALK5) receptor are functional receptors that mediate stimulatory effects in response to TGF-β1. Moreover, we found that the canonical SMAD2/SMAD3-SMAD4 signaling pathway is the principal intracellular signaling pathway that upregulates the expressionhyaluronan synthase and subsequent hyaluronan synthesis. Notably, we showed that SNAIL transcription factor is a critical molecule mediating the TGF-β signaling, which contributes to the increase in hyaluronan synthesis. These results of our in vitro studies suggest that intraovarian TGF-β1 plays a functional role in the local regulation of hyaluronan synthesis in hGL cells.

Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars.

Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f®)- with biosimilar preparations (Bemfola® and Ovaleap®)-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f® or biosimilar (1 × 10−3-1 × 103 ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ increase and β-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and β-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 ± 0.9–24 ± 1.7 ng/ml; β-arrestin 2 EC50 range = 140 ± 14.1–313 ± 18.7 ng/ml; Kruskal-Wallis test; p ≥ 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ increased upon cell treatment by 4 μg/ml Gonal-f®, while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC50s were demonstrated (Kruskal-Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.

CDK4/6 inhibitor protects chemerin-induced human granulosa-lutein cells from apoptosis by inhibiting the p53/p21 waf pathway.

Dysregulation of the cell cycle is common in human tumorigenesis. Therefore, CDK4/6 inhibitors targeting the cell cycle have been developed, and their antiapoptotic effects have been highly correlated with potential clinical therapies. The aim of this study was to identify the regulatory effect of the CDK4/6 inhibitor palbociclib on chemerin-induced apoptosis of immortalized human granulosa-lutein (hGL) cells and to elucidate its fundamental mechanism of action. Palbociclib enhanced antioxidative enzyme generation and diminished ROS generation in hGL cells. Furthermore, we found that palbociclib suppressed chemerin-induced apoptotic protein expression, reversing the Bcl-2/Bax ratio and inhibiting the p53/p21 waf pathway. Eventually, palbociclib decreased the level of cleaved caspase-3 and -9, hindering the apoptosis of hGL cells. In general, the antiapoptotic efficacy of palbociclib could be attributed in part to the modulation of the mitochondrial apoptotic pathway in hGL cells.

ID3 mediates the TGF-β1-induced suppression of matrix metalloproteinase-1 in human granulosa cells.

In the mammalian ovary, matrix metalloproteinase-1 (MMP1) is expressed in growing ovarian follicles, and MMP1-mediated extracellular matrix (ECM) remodeling plays a functional role in regulating the formation of corpus luteum. Transforming growth factor-β1 (TGF-β1) is an intraovarian growth factor that acts as a negative regulator of luteinization and progesterone production in human granulosa-lutein (hGL) cells. At present, whether TGF-β1 regulates the expression of MMP1 and thus affects ECM remodeling during corpus luteum formation remains largely unknown. The aim of this study was to investigate the effects of TGF-β1 and the molecular mechanisms by which it regulates the expression of MMP1 in immortalized human granulosa cells lines (SVOG) and primary hGL cells (obtained from consenting patients undergoing IVF treatment). We used inhibition approaches including a competitive antagonist for endogenous TGF-β type II receptor, pharmacological inhibitors (SB431542 and dorsomorphin), and specific small interfering RNA-targeted knockdown of ALK5 type I receptor and SMAD4 to demonstrate that TGF-β1 downregulates the expression and production of MMP1 via a TβRII/ALK5-mediated SMAD-dependent signaling pathway in hGL cells. Additionally, our results show that the suppressive effect of TGF-β1 on the expression of MMP1 is mediated by a transcription factor, the inhibitor of differentiation 3 (ID3) protein. Our findings provide insights into the molecular interactions and mechanisms of TGF-β1 and ID3 during the regulation of MMP1 in hGL cells.

ALK4-SMAD2/3-SMAD4 signaling mediates the activin A-induced suppression of PTX3 in human granulosa-lutein cells

As one of the members of the transforming growth factor-β (TGF-β) superfamily, activin A plays an important role in regulating follicular development and oocyte maturation. Pentraxin 3 (PTX3) is the key component that promotes the process of cumulus expansion during mammalian ovulation. At present, the regulation of PTX3 expression in human granulosa cells remains largely unknown. This study aimed to examine the effects of activin A on the expression of PTX3 in human granulosa-lutein (hGL) cells and to investigate the underlying molecular mechanisms. Using an established immortalized hGL cell line (SVOG) and primary hGL cells as study models, we demonstrated that activin A significantly increased the phosphorylation of SMAD2 and SMAD3, which suppressed the expression of PTX3 at both the mRNA and protein levels. Additionally, these effects induced by activin A were completely reversed by pretreatment with the TGF-β type I receptor inhibitor SB431542 and knockdown of ALK4. Furthermore, knockdown of SMAD2, SMAD3, or SMAD4 completely reversed the activin A-induced suppressive effects on PTX3 expression. Notably, the ChIP analyses demonstrated that phosphorylated SMADs could bind to human PTX3 promoter. Collectively, our results showed that the ALK4-SMAD2/3-SMAD4 signaling pathway most likely mediates the suppressive effect of activin A on PTX3 expression in hGL cells.