Increased Progesterone Production Research Articles

Adropin may regulate ovarian functions by improving antioxidant potential in adult mouse

The corpus luteum (CL) is a temporary endocrine gland that synthesizes progesterone. The luteal progesterone plays a central role in the regulation of the estrous cycle as well as the implantation and maintenance of pregnancy. Our previous study showed the expression of adropin and its receptor, GPR19, in the luteal cells and its significant role in luteinization. The aim of the present study was to investigate the in vitro effect of adropin on hCG-induced ovarian functions in adult mice. We also evaluated the effect of exogenous treatment with adropin on ovarian steroidogenesis and anti-oxidant parameters, with special emphasis on CL function. Our results demonstrated that adropin acts synergistically with hCG to promote ovarian steroidogenesis and survival by increasing the expression of StAR, 3β-HSD, and aromatase proteins and decreasing the BAX/BCL2 ratio. Exogenous adropin treatment increased progesterone production by increasing the expression of GPR19, StAR and 3β-HSD enzymes in the mouse ovary. Also, adropin inhibited the luteal oxidative stress by increasing nuclear translocation of NRF-2 in CL, which resulted in increased HO-1 expression and SOD, catalase activity. Decreased oxidative stress might inhibit the translocation of NF-κB into the nucleus of luteal cells, resulting into increased survival and decreased apoptosis, as evident by decreased lipid peroxidation, BAX/BCL2 ratio, caspase 3, active caspase 3 expression, and TUNEL-positive cells in adropin treated mice. Our findings suggest that adropin can be a promising candidate that can enhance the survivability of the CL.

The Role of DNMT1 and C/EBPα in the Regulation of CYP11A1 Expression During Syncytialization of Human Placental Trophoblasts.

Progesterone synthesized in the placenta is essential for pregnancy maintenance. CYP11A1 is a key enzyme in progesterone synthesis, and its expression increases greatly during trophoblast syncytialization. However, the underlying mechanism remains elusive. Here, we demonstrated that passive demethylation of CYP11A1 promoter accounted for the upregulation of CYP11A1 expression during syncytialization with the participation of the transcription factor C/EBPα. We found that the methylation rate of a CpG locus in the CYP11A1 promoter was significantly reduced along with decreased DNA methyltransferase 1 (DNMT1) expression and its enrichment at the CYP11A1 promoter during syncytialization. DNMT1 overexpression not only increased the methylation of this CpG locus in the CYP11A1 promoter, but also decreased CYP11A1 expression and progesterone production. In silico analysis disclosed multiple C/EBPα binding sites in both CYP11A1 and DNMT1 promoters. C/EBPα expression and its enrichments at both the DNMT1 and CYP11A1 promoters were significantly increased during syncytialization. Knocking-down C/EBPα expression increased DNMT1 while it decreased CYP11A1 expression during syncytialization. Conclusively, C/EBPα plays a dual role in the regulation of CYP11A1 during syncytialization. C/EBPα not only drives CYP11A1 expression directly, but also indirectly through downregulation of DNMT1, which leads to decreased methylation in the CpG locus of the CYP11A1 promoter, resulting in increased progesterone production during syncytialization.

FRI392 Functional Interrelationship Between Oxytocin And Bone Morphogenetic Proteins In Ovarian Steroidogenesis By Granulosa Cells

Abstract Disclosure: K. Yamamoto: None. Y. Nakano: None. N. Iwata: None. Y. Soejima: None. A. Suyama: None. T. Hasegawa: None. F. Otsuka: None. Oxytocin (OXT) is a neuropeptide that is produced in the hypothalamus and is secreted from the posterior pituitary. OXT has diverse physiological roles mediated by the OXT receptor (OXTR), including not only stimulation of uterine contractions during labor and peripheral functions of milk ejection during lactation but also functions in pair bonding, maternal nurturing, and social behavior related to the action of OXT on the brain. It has been shown that OXT and OXTR are expressed in ovarian granulosa cells (GCs) in several species including humans. Secretion of OXT by bovine GCs is reported to be increased after the preovulatory luteinizing hormone surge, which is coincident with an increase in progesterone secretion and a decrease in secretion of estradiol and androgen, suggesting a functional interrelationship between OXT and ovarian steroidogenesis. It has been demonstrated that application of OXT to cultures of GCs from preovulatory follicles in bovines, marmoset monkeys, and humans led to increases in progesterone production. However, the precise molecular mechanism by which OXT modulates ovarian steroidogenesis in GCs remains unknown. Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta (TGF-β) superfamily, play important roles in steroidogenesis and cellular proliferation in ovarian follicles via autocrine/paracrine fashion. BMP ligands and receptors are expressed in a cell-dependent manner in the ovary and have a pivotal role in female fertility. Throughout folliculogenesis, BMPs commonly inhibit follicle-stimulating hormone (FSH)-induced progesterone production, and oocyte-derived factors BMP-6 and BMP-15 are known to act on GCs through BMP receptors and suppress FSH-induced progesterone synthesis. To clarify the reproductive role of OXT in ovarian steroidogenesis and its functional interaction with BMPs, the effects of OXT on ovarian steroidogenesis were investigated by using primary culture of rat GCs and human granulosa KGN cells. Here we found that the OXT receptor was expressed in both rat GCs and human KGN cells and that OXT treatment significantly increased FSH- and forskolin (FSK)-induced progesterone production, but not estradiol production, by rat GCs and KGN cells, respectively. In accordance with the effects of OXT on progesterone production, OXT enhanced mRNA levels of CYP11A1 and HSD3B2 induced by FSK in KGN cells. Of note, OXT enhanced the phosphorylation of SMAD1/5/9 and the mRNA level of ID1 induced by BMP-15, but not those induced by BMP-6, in KGN cells. It was also revealed that OXT application upregulated the expression of BMPR2, a crucial type-II receptor of BMP-15, and enhanced the BMP-15-induced expression of inhibitory SMAD6 by KGN cells. Taken together, it was shown that OXT enhances ovarian progesterone production with upregulation of BMP-15 activity, leading to a fine-tuning of ovarian steroidogenesis. Presentation: Friday, June 16, 2023

Perfluorooctanoic acid (PFOA) affects steroidogenesis and antioxidant defence in granulosa cells from swine ovary

PFOA is mainly employed in products with water and oil repellent properties. Due to its persistence, bioaccumulation and critical effects on health, its use has been restricted in several countries. This research was intended to explore PFOA action on the main functions of swine ovarian granulosa cells, a valuable model for translational medicine. Moreover, since we previously demonstrated a disruptive effect on free radical generation we sought to explore PFOA effects on the main antioxidant enzymes. PFOA inhibited cell proliferation (p < 0.001), assessed by BrdU uptake. Steroidogenesis was disrupted: PFOA also stimulated 17β-estradiol production (p < 0.05), increased progesterone production (p < 0.05) at the lowest dose while it displayed an inhibitory effect at higher concentrations (p < 0.05). SOD (p < 0.001), catalase (p < 0.05) and peroxidase (p < 0.01) activities were stimulated. Therefore, our study supports a disruptive effect of PFOA in cultured swine granulosa cells.

Effects of asprosin on estradiol and progesterone secretion and proliferation of bovine granulosa cells

Asprosin is an adipokine synthesized by the white adipose tissue that regulates glucose homeostasis and that has been reported to affect bovine theca cell function and follicular growth, but its role on granulosa cell functions remains to be unveiled. Hence, the objective of this study was to investigate asprosin impacts on granulosa cell steroidogenesis. Bovine granulosa cells from small ovarian follicles were cultured in vitro to investigate the effects of asprosin on cell proliferation, production of steroids, mRNA abundance of genes that encode steroidogenic enzymes and cell cycle regulators, and protein relative abundance of steroidogenic signaling pathways. Asprosin was shown to affect granulosa cell functions in a dose-dependent manner. In the presence of FSH, asprosin enhanced estradiol production and stimulated an increase in mRNA expression of FSHR and CYP19A1 in a dose-dependent manner. In the presence of IGF1, asprosin decreased estradiol production, increased progesterone production, altered PKA relative protein expression, and tended to alter the ratio of p-ERK1/2/total ERK1/2 protein expression in a dose-dependent manner. Furthermore, asprosin increased p-53 gene expression in basal culture conditions and with or without FSH and IGF1. Taken together, findings of this study show that asprosin is a regulator of granulosa cell functions and the effects of asprosin depend on dose and cell culture conditions.

Co-cultivation of human granulosa cells with ovarian cancer cells leads to a significant increase in progesterone production

PurposeIn humans, granulosa cells (GCs) are part of the follicle and nourish the growing oocyte. GCs produce estrogen and, after ovulation, progesterone. They are embedded in a multicellular tissue structure of the ovary, which consists of a variety of different cell types that are essential for the physiological function of the ovary. However, the extent to which individual ovarian cell types contribute to overall functionality has not yet been fully elucidated. In this study, we aim to investigate the effects of co-culturing human granulosa cells with ovarian cancer cells on their progesterone and estrogen production in an in vitro model.MethodsAfter seeding, the cells were stimulated with 200 µM forskolin in DMEM for 72 h and the medium of the different cell culture experiments was collected. Subsequently, progesterone and oestradiol concentrations were determined using an Elisa assay.ResultsMorphologically, it was striking that the cells self-organize and form spatially separated areas. Compared to culturing granulosa cells alone, co-culturing human granulosa cells together with the ovarian cancer cell line OvCar-3 resulted in a significant increase in progesterone production (20.3 ng/ml versus 50.2 ng/ml; p < 0.01).ConclusionsUsing a simple in vitro model, we highlight the importance of cellular crosstalk between different ovarian cells in a complex cellular network and that it strongly influences granulosa cell hormone production. This could have potential implications for the procedure of transplanting endocrine tissues after cryopreservation, as it highlights the importance of survival of all cells for the functionality of the transplanted tissue.

Sinapic and ferulic acid phenethyl esters increase the expression of steroidogenic genes in MA-10 tumor Leydig cells

During aging, the production of androgens by the testis Leydig cells gradually decreases. Phenolic compounds can improve testosterone biosynthesis and delay the onset of hypogonadal symptoms in males. In this study, sinapic acid phenethyl ester was evaluated for its ability to regulate gene expression and steroid production in Leydig cells. Specifically, the effects of this ester on the transcriptome of MA-10 Leydig cells were investigated by RNA-Seq. To better establish a structure-function relationship of the hydroxy-methoxyphenyl moiety of sinapic and phenethyl ester, its influences on gene expression were compared to those of its ferulic acid phenethyl ester analogue. According to the transcriptomic analysis, most genes encoding enzymes related to cholesterol biosynthesis are increased in response to sinapic acid phenethyl ester treatment of MA-10 Leydig cells. Interestingly, treatments with 10 μM of ferulic acid phenethyl ester increased cAMP-dependent Star promoter activation, gene expression and protein levels. In addition, treatments of MA-10 Leydig cells with 10 μM of sinapic or ferulic acid phenethyl ester resulted in increased progesterone production. Thus, our results indicate that sinapic and ferulic acid phenethyl esters can improve cholesterol and steroid biosynthesis in testicular Leydig cells.

Light-driven progesterone production by InP–(M. neoaurum) biohybrid system

Progesterone is one of the classical hormone drugs used in medicine for maintaining pregnancy. However, its manufacturing process, coupled with organic reagents and poisonous catalysts, causes irreversible environmental pollution. Recent advances in synthetic biology have demonstrated that the microbial biosynthesis of natural products, especially difficult-to-synthesize compounds, from building blocks is a promising strategy. Herein, overcoming the heterologous cytochrome P450 enzyme interdependency in Mycolicibacterium neoaurum successfully constructed the CYP11A1 running module to realize metabolic conversion from waste phytosterols to progesterone. Subsequently, progesterone yield was improved through strategies involving electron transfer and NADPH regeneration. Mutant CYP11A1 (mCYP11A1) and adrenodoxin reductase (ADR) were connected by a flexible linker (L) to form the chimera mCYP11A1-L-ADR to enhance electron transfer. The chimera mCYP11A1-L-ADR, adrenodoxin (ADX), and ADR-related homolog ARH1 were expressed in M. neoaurum, showed positive activity and produced 45 mg/L progesterone. This electron transfer strategy increased progesterone production by 3.95-fold compared with M. neoaurum expressing mCYP11A1, ADR, and ADX. Significantly, a novel inorganic–biological hybrid system was assembled by combining engineered M. neoaurum and InP nanoparticles to regenerate NADPH, which was increased 84-fold from the initial progesterone titer to 235 ± 50 mg/L. In summary, this work highlights the green and sustainable potential of obtaining synthetic progesterone from sterols in M. neoaurum.Graphical

Comparing the effects of bisphenol A, C, and F on bovine theca cells in vitro

Endocrine disrupting chemicals (EDCs) target aspects of hormone activity. Tightly coordinated crosstalk between two somatic cells of the ovary, granulosa and theca cells, governs steroid hormone production and plays a critical role in reproduction. It is thus pertinent to understand the impact of EDCs on granulosa and theca cells. Bisphenol A (BPA), a well-known EDC, is widely used in the manufacturing of consumer products with humans routinely exposed. Strong evidence of the adverse effects of BPA on the female reproductive system has emerged and as a result, manufacturers have begun replacing BPA with other bisphenols, such as BPC and BPF. The safety of these analogs is currently unclear and should be investigated independently. Although much is known about the impact of BPA on granulosa cells, similar study of theca cells has been neglected. Further, there is a lack of studies on the impact of BPC and BPF on the female reproductive system. To fill these gaps, the present study compared the effect of BPA, BPC, and BPF on the viability and steroid production of theca cells from bovine, a clinically relevant model for human reproduction. We show that BPC is more detrimental to theca cell viability and progesterone production compared to BPA. Surprisingly, we also found that BPF induces an increase in progesterone production compared to a decrease with BPA and BPC. To determine safety for the reproductive system, we conclude that a major shift away from BPA to bisphenol analogs should be investigated more thoroughly.

EFFICACY OF A NEW DRUG BASED ON GM-CSF FOR THE TREATMENT OF COWS WITH FOLLICULAR CYSTS

The article presents the material on studying the efficacy of the new drug proovulin for the treatment of cows with follicular cysts. It was established that the double administration of proovulin, containing granulocyte-macrophage colony-stimulating factor (GM-CSF), at a dose of 10.0 ml with an interval of 4 days, is accompanied by an efficacy of 83.3% that is by 3.3-8.3% higher, compared to the dose of proovulin of 5.0 ml and surfagon, while reducing the period from calving to fertilization by 18.9-35.7 days and the fertilization rate - by 0.29-0.45. The restoration of the functional activity of the genital gonads in cows with follicular cysts when using proovulin occurs against the background of the normalization of their hormonal-cytokine profile that is manifested by an increase in progesterone production by 10.0 times, which leads to a decrease in the level of TNFα by 20.1%, as well as the concentration of IL-1β - by 18.9%.

Effects of transforming growth factor β1 on steroidogenesis of feline granulosa cells cultured

Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. To determine if transforming growth factor β1 (TGFB1), activin, epidermal growth factor (EGF), follicle stimulating hormone (FSH), luteinizing hormone (LH), melatonin, and insulin-like growth factor 1 (IGF1) regulate granulosa cell steroidogenesis and proliferation in cats, three experiments were conducted in winter season. Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48h after an initial 48h plating in 10% fetal calf serum. Treatment with IGF1 and FSH increased (P <0.05) estradiol production by 2.3- and 1.33-fold, respectively. In contrast, TGFB1 blocked (P <0.05) IGF1-induced estradiol production and inhibited FSH-induced estradiol production by 60%. Combined with FSH or FSH plus IGF1, TGFB1 inhibited (P <0.05) cell proliferation, whereas TGFB1 increased progesterone production by 2.8-fold in the presence of FSH plus IGF1. EGF decreased (P <0.05) FSH plus IGF1-induced estradiol production by 89% but did not affect progesterone production or cell numbers. Activin did not affect (P >0.10) cell numbers or steroidogenesis in the presence of FSH plus IGF1. Melatonin and LH decreased (P <0.05) estradiol production 53% and 59%, respectively, without affecting progesterone production or cell proliferation. The present study has identified TGFB1 as a major regulator of feline ovarian function, in addition to EGF, IGF1, melatonin, LH and FSH. These studies will provide useful information for future development of fertility control in feline species.

Transcriptomic analysis of MA-10 tumor Leydig cells treated with adipose derived hormones adiponectin and resistin

Obesity contributes to a decrease in testosterone production in men. Indeed, adipose tissue produces several hormones, including adiponectin and resistin, and these may influence the activity of signaling pathways responsible for regulating the expression of genes related to steroidogenesis. In this study, we wanted to identify which genes are directly regulated by these hormones using the MA-10 tumor Leydig cell model. To do this, we treated these cells with adiponectin or resistin, followed by RNA extraction and RNA-Seq transcriptome analysis. Interestingly, genes upregulated by the globular form of adiponectin (gACRP30) were associated to steroid hormones biosynthesis, whereas resistin had no effect on the transcriptome of MA-10 Leydig cells. Moreover, the expression of the Star gene, encoding the steroidogenic acute regulatory protein, was increased in response to treatments with 0.5 mM 8Br-cAMP. Such stimulation was further increased by adiponectin, resulting in increased progesterone production. However, resistin had no effect on steroid production from MA-10 tumor Leydig cells under the treatment conditions investigated. Thus, our data suggest that a direct regulation of steroidogenic genes' expressions in Leydig cells by adipose derived hormones involves cooperation between the cAMP/PKA pathway and adiponectin, but not resistin, to activate Star expression and improve progesterone synthesis.

Involvement of BMP-15 in glucocorticoid actions on ovarian steroidogenesis by rat granulosa cells

To elucidate the impact of glucocorticoids on ovarian steroidogenesis and its molecular mechanism by focusing on bone morphogenetic proteins (BMPs), we examined the effect of dexamethasone (Dex) on estradiol and progesterone synthesis by using primary culture of rat granulosa cells. It was revealed that Dex treatment dose-dependently decreased estradiol production but increased progesterone production induced by follicle-stimulating hormone (FSH) by granulosa cells. In accordance with the effects of Dex on estradiol synthesis, Dex suppressed P450arom mRNA expression and cAMP synthesis induced by FSH. Dex treatment in turn enhanced basal as well as FSH-induced levels of mRNAs encoding the enzymes for progesterone synthesis including P450scc and 3βHSD but not StAR and 20αHSD. Of note, Dex treatment significantly upregulated transcription of the BMP target gene Id-1 and Smad1/5/9 phosphorylation in the presence of BMP-15 among the key ovarian BMP ligands. It was also found that Dex treatment increased the expression level of BMP type-I receptor ALK-6 among the type-I and –II receptors for BMP-15. Inhibitory Smad6/7 expression was not affected by Dex treatment. On the other hand, BMP-15 treatment upregulated glucocorticoid receptor (GR) expression in granulosa cells. Collectively, it was revealed that glucocorticoids elicit differential effects on ovarian steroidogenesis, in which GR and BMP-15 actions are mutually enhanced in granulosa cells.

Bone morphogenetic protein 15 supplementation enhances cumulus expansion, nuclear maturation and progesterone production of in vitro-matured bovine cumulus-oocyte complexes.

In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus-oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100ng/ml), FGF16 (10ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.

Melatonergic systems of AANAT, melatonin, and its receptor MT2 in the corpus luteum are essential for reproductive success in mammals†

Corpus luteum (CL) plays a critical role in mammalian reproductive physiology. Its dysfunction will lead to infertility or habitual abortion. In the current study, by use of melatonin specific membrane receptor 2 (MT2) knocking out (KO) mice model combined with RNA-Seq, immunohistochemistry, and immunofluorescence analyses, the genes of melatonin synthetic enzyme arylalkylamine N-acetyltransferase (AANAT) and MT2 were identified to strongly express in the CL of sows and mice. KO MT2 significantly impaired the reproductive performance in mice indicated by the reduced litter sizes. Melatonin treatment elevated the progesterone production in sows suggesting the improved CL function. Mechanistic analysis showed that melatonin upregulated a set of progesterone synthesis-related genes including cytochrome P450 family 11 subfamily A member 1 (Cyp11a1), aldo-keto reductase family 1, member C18 (Akr1c18), isopentenyl-diphosphate delta isomerase 1 (Idi1), and luteinizing hormone/choriogonadotropin receptor (Lhcgr). The upregulation of these genes directly related to the increased progesterone production. The regulatory effects of melatonin on these gene expressions were mediated by MT2 and MT2KO diminished the effects of melatonin in this respect. Thus, the presence of melatonergic system of AANAT, melatonin, and its receptor MT2 in CL is essential for reproductive success in mammals.

Ovulatory signals alter granulosa cell behavior through YAP1 signaling

BackgroundThe Hippo pathway plays critical roles in regulating cell proliferation, differentiation and survival among species. Hippo pathway proteins are expressed in the ovary and are involved in ovarian function. Deletion of Lats1 causes germ cell loss, ovarian stromal tumors and reduced fertility. Ovarian fragmentation induces nuclear YAP1 accumulation and increased follicular development. At ovulation, follicular cells stop proliferating and terminally differentiate, but the mechanisms controlling this transition are not completely known. Here we explore the role of Hippo signaling in mouse granulosa cells before and during ovulation.MethodsTo assess the effect of oocytes on Hippo transcripts in cumulus cells, cumulus granulosa cells were cultured with oocytes and cumulus oocyte complexes (COCs) were cultured with a pSMAD2/3 inhibitor. Secondly, to evaluate the criticality of YAP1 on granulosa cell proliferation, mural granulosa cells were cultured with oocytes, YAP1-TEAD inhibitor verteporfin or both, followed by cell viability assay. Next, COCs were cultured with verteporfin to reveal its role during cumulus expansion. Media progesterone levels were measured using ELISA assay and Hippo transcripts and expansion signatures from COCs were assessed. Lastly, the effects of ovulatory signals (EGF in vitro and hCG in vivo) on Hippo protein levels and phosphorylation were examined. Throughout, transcripts were quantified by qRT-PCR and proteins were quantified by immunoblotting. Data were analyzed by student’s t-test or one-way ANOVA followed by Tukey’s post-hoc test or Dunnett’s post-hoc test.ResultsOur data show that before ovulation oocytes inhibit expression of Hippo transcripts and promote granulosa cell survival likely through YAP1. Moreover, the YAP1 inhibitor verteporfin, triggers premature differentiation as indicated by upregulation of expansion transcripts and increased progesterone production from COCs in vitro. In vivo, ovulatory signals cause an increase in abundance of Hippo transcripts and stimulate Hippo pathway activity as indicated by increased phosphorylation of the Hippo targets YAP1 and WWTR1 in the ovary. In vitro, EGF causes a transient increase in YAP1 phosphorylation followed by decreased YAP1 protein with only modest effects on WWTR1 in COCs.ConclusionsOur results support a YAP1-mediated mechanism that controls cell survival and differentiation of granulosa cells during ovulation.

BPA activates EGFR and ERK1/2 through PPARγ to increase expression of steroidogenic acute regulatory protein in human cumulus granulosa cells

Bisphenol A (BPA) negatively affects steroid production in human luteinized granulosa cells (GC). This study was designed to address two important questions: (1) whether BPA exerts the same disruptive effect in human cumulus granulosa cells (hCGC) and (2) to reveal the molecular mechanism underlying the BPA's action on steroidogenesis. We used cultured hCGC since these cells exert the properties of GC from early antral follicles. Results showed that BPA at 100 μM decreased estradiol level and CYP19A1 mRNA, but increased progesterone production, steroidogenic acute regulatory protein (STAR) and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression after 48 h. Shorter (6 h) exposure to BPA elevated PPARγ mRNA level in hCGC. Addition of ERK1/2 (U0126), EGFR (AG1478) and PPARγ (GW9662) inhibitors prevented the BPA-induced STAR and PPARγ mRNA expression. Western blot analysis showed that BPA induced a rapid EGFR and ERK1/2 activation. The BPA-induced EGFR phosphorylation was prevented by addition of the PPARγ inhibitor, whereas the BPA-induced ERK1/2 activation was prevented by addition of the EGFR or PPARγ inhibitor. These data show that BPA increases the progesterone and decreases the estradiol biosynthetic pathway in hCGC. Augmentation of the progesterone biosynthetic pathway is mediated through the PPARγ-dependent activation of EGFR and ERK1/2, leading to increased expression of STAR mRNA.

Co-Enzyme Q10 Supplementation Rescues Cumulus Cells Dysfunction in a Maternal Aging Model.

Over the past four decades, due to cultural and social changes, women in the developed world have significantly delayed childbirth. This trend is even worse for patients who attend infertility clinics. It is well-known that live birth rates in women older than 35 are significantly lower than in those younger, both naturally and with assisted reproduction. Fertility decline is, in part, due to an increase in oocyte aneuploidy that leads to a reduced embryo quality, as well as an increased incidence of miscarriages and birth defects. Here we show that aging-associated malfunction is not restricted to the oocyte, as cumulus granulosa cells also display a series of defects linked to mitochondrial activity. In, both, human and mouse model, a decline in cumulus cell function due to increased maternal age is accompanied by a decreased expression of enzymes responsible for Coenzyme Q (CoQ) production, particularly Pdss2 and CoQ6. In an aged mouse model supplementation with Coenzyme Q10—a potent stimulator of mitochondrial function—restored cumulus cell number, stimulated glucose uptake, and increased progesterone production. CoQ10 supplementation might, thus, improve oocyte and cumulus cells quantity and quality, by improving the mitochondrial metabolism in females of advanced maternal age.

Regulation of steroidogenic function of luteal cells by thrombospondin and insulin in water buffalo (Bubalus bubalis).

The present study examined the effect of exogenous thrombospondin 1 (TSP1) on the steroidogenic function of luteal cells cultured invitro. Furthermore, the transcriptional interaction of insulin with TSP1 and its receptor, cluster of differentiation 36 (CD36) were also investigated. At the highest dose (500ngmL-1) TSP1 significantly downregulated the expression of the angiogenic marker von Willebrand factor (vWF) and progesterone production in cultured luteal cells. Moreover, the simultaneous upregulation in the expression of caspase 3 by exogenous TSP1 was consistent with a reduction in the number of viable luteal cells as determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay after 72h of culture. However, the expression of critical enzymes in the progesterone synthetic pathway was not significantly modulated by treatment with TSP1 in cultured luteal cells. Knocking out of endogenous TSP1 with the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPRassociated protein9 (Cas9) system improved the viability of luteal cells as well as increasing progesterone production and decreasing caspase 3 activation. Insulin treatment suppressed the expression of TSP1 and CD36 in cultured luteal cells in a dose- and time-dependent manner. To conclude, TSP1 acts as a negative endogenous regulator of angiogenesis that attenuates progesterone production, possibly by reducing the number of luteal cells via apoptosis during luteal regression, whereas insulin as a luteinising signal may have inhibited the thrombospondin system for the efficient development of luteal function.

Protection of cyanidin-3-O-glucoside against acrylamide- and glycidamide-induced reproductive toxicity in leydig cells

Acrylamide (AA) occurs in many cooked carbohydrate-rich foods and has caused widespread concern as a possible carcinogen. Glycidamide (GA) is the ultimate genotoxic metabolite of AA. The present study was to investigate the protective effect of Cyanidin-3-O-glucoside (C3G) against AA- and GA-induced reproductive toxicity in R2C Leydig cells. The results demonstrated that C3G inhibited AA- and GA-induced cytotoxicity and mitochondria-mediated cell apoptosis, the effective doses of C3G were ranging from 10 to 50 μM. Besides, AA (1.925 mM) and GA (0.872 mM) exposure increased ROS level and decreased mitochondrial membrane potential, which led to a decrease in progesterone production, while C3G ranging from 10 to 50 μM reduced ROS immediately, and increased progesterone production after 24 h treatment. Furthermore, C3G up-regulated expression of Bcl-2 protein and down-regulated pro-apoptotic protein Bax and cleaved Caspase-3 after 24 h treatment in 1.925 mM AA- and 0.872 mM GA-treated R2C cells. Moreover, C3G intervention increased the protein expression of steroidogenic acute regulatory (StAR). It was concluded that C3G is effective in reducing AA- and GA-induced reproductive toxicity via inhibition of ROS generation, mitochondrial membrane depolarization and apoptosis, as well as activating steroidogenic enzymes.