Primary Cultures Of Granulosa Cells Research Articles

P-678 Cabergoline has inhibitory effects on FSH-induced cell differentiation and estrogen secretion in a primary culture system of rat ovarian granulosa cells

Abstract Study question How does cabergoline have effects on cell differentiation and hormone secretion induced by FSH stimulation in ovarian granulosa cells? Summary answer Cabergoline suppresses LH receptor expression and Estradiol (E2) secretion in primary cultures of rat ovarian granulosa cells by inhibiting cAMP production induced by FSH stimulation. What is known already Cabergoline is a dopamine receptor 2 agonist that has been used to treat hyperprolactinemia and, more recently, to prevent ovarian hyperstimulation syndrome. Although it has been previously reported that cabergoline suppresses E2 and Progesterone secretion induced by FSH stimulation in a primary culture system of rat ovarian granulosa cells, but the effect on LH receptor expression has not been investigated. It has also been reported that the activation of dopamine receptor 2 suppresses VEGF secretion and reduces the incidence of early-onset OHSS by inhibiting the VEGF/VEGFR2 pathway in research using human luteinized granulosa cells. Study design, size, duration We used a primary culture system of ovarian granulosa cells obtained from immature female rats treated with diethylstilbestrol to examine the effect of cabergoline addition on the changes in ovarian granulosa cells induced by FSH stimulation. Participants/materials, setting, methods FSH and cabergoline were added alone or co-added to a primary culture system of rat ovarian granulosa cells, and the changes induced by them were measured and compared. (1) mRNA expression of Lhcgr, Cyp19a1, and Fshr by RT-qPCR (2) E2 concentration in the culture medium (3) Measurement of hCG binding on the cell surface (4) cAMP concentration Main results and the role of chance Co-addition of cabergoline suppressed each of the changes induced by FSH stimulation as follows. (1) The mRNA expression levels of Lhcgr, Cyp19a1, and Fshr decreased in a concentration-dependent manner (0.01-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, they decreased to 14%, 9%, and 49% of FSH alone, respectively. (2) E2 concentration in the culture media also decreased in a concentration-dependent manner (1-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, it decreased to 49% of FSH alone. (3) The amount of hCG binding on the cell surface also decreased in a concentration-dependent manner (0.1-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, it decreased to 7% of FSH alone. (4) FSH-stimulated cAMP production decreased in a concentration-dependent manner (0.01-1µM) of co-added cabergoline compared to FSH alone. In the 1µM of co-added cabergoline group, it decreased to 50% of FSH alone. These results suggest that cabergoline suppresses LH receptor and aromatase expression and E2 production by inhibiting cAMP production induced by FSH stimulation. Limitations, reasons for caution Since this is an in vitro experiment using a rat primary culture system, the results of this research cannot be considered directly applicable to humans. Wider implications of the findings Our findings imply that cabergoline administration during the follicular phase may inhibit ovarian granulosa cell differentiation and estrogen secretion. In women taking relatively high doses of cabergoline, attention may need to be paid regarding the effects of cabergoline on follicular development and estrogen secretion. Trial registration number not applicable

Role of DNA damage response in cyclophosphamide-induced premature ovarian failure in mice.

To investigate the DNA damage response (DDR) in a cyclophosphamide (CTX)-induced mouse model of premature ovarian failure (POF). The POF model was established by injecting mice with CTX. The body, ovarian weights, the estrus cycle, and pathological changes of the ovaries were recorded. The serum levels of 17 β-estradiol (E2) and follicle-stimulating hormone (FSH) were measured. The expression of Ki67, β-galactosidase (β-gal), p21, p53, γH2AX, and pATM in ovarian tissues was detected by immunohistochemistry. The expression of β-gal, γH2AX, and pATM was analyzed by immunofluorescence staining of primary cultured granulosa cells (GCs). The body and ovarian weights decreased, the estrus cycles were erratic, and the FSH level increased, whereas the E2 level decreased in POF mice compared to controls. The pathological consequences of POF revealed an increase in atretic follicles, corpus luteum, and primordial follicles and a decrease in the number of primary, secondary, and tertiary follicles. Ki67 expression was reduced, β-gal, p21, p53, γH2AX, and pATM expression were elevated in the ovaries of POF mice. The expression of β-gal, γH2AX, and pATM increased in GCs with the concentration in a time-dependent manner. In total, CTX induced POF in mice, which was mediated by the DDR pathway of ATM-P53-P21.

Changes in adiponectin levels of subclinical ketosis cows and their effects on steroid hormone secretion and proliferation in follicular granulosa cells

In dairy cows, the occurrence of subclinical ketosis (SCK) is particularly high during early lactation. Previously, we documented alterations in the abundance of adiponectin (ADPN) in anestrus cows with SCK in comparison to cows in estrus. In the present study, 60 cows were divided into two groups: control (C, n = 30) and SCK (n = 30). Based on cow’s estrus situation in two group at 55–60 days postpartum, 15 anestrus SCK cows and estrus cows were designated the SCK-A group and C-E group, respectively. The SCK-A group had downregulated serum and follicular fluid ADPN levels compared with the C-E group. The serum ADPN level was positively correlated with the insulin level and follicle growth rate, and there was a positive correlation between ADPN and glucose in the follicular fluid. Primary culture of dairy cow granulosa cells (GCs) was established to observe the effect of low glucose (Glu) and/or ADPN on GCs cyclins and proteins important for steroid synthesis. The results showed that the addition of 1 µg/mL ADPN alleviated the negative effects of low Glu treatment on the proliferation of GCs and the expression of steroid secretion related protein proteins. Treatment with LY294002 (PI3K inhibitor) four experimental GCs groups: control (0 µg/mL ADPN), 1 µg/mL ADPN, LY294002 inhibitor, and 1 µg/mL ADPN+LY294002. The results showed that ADPN promotes the secretion of steroid hormones by GCs through the PI3K–AKT. In summary, ADPN plays a crucial role in ameliorating postpartum anestrus in dairy cows with SCK.

Kisspeptin stimulates oestradiol biosynthesis by upregulating steroidogenic transcripts and proliferation markers in the bubaline granulosa cells in vitro

AbstractKisspeptin (Kp), an upstream regulator of GnRH release, is essential for the development and function of reproductive axis. Previously, we demonstrated the localization of Kp and its receptor (Kiss1r) in the active follicle in the bubaline ovary. Present study aimed to determine the effect of Kp on granulosa cell (GCs) functions, especially oestradiol (E2) and progesterone (P4) production, and differential expression of genes regulating the proliferation, apoptosis and steroidogenesis in the buffalo. The ovaries with 6–10 mm size follicles obtained from the cyclic buffaloes after slaughtering were used for isolation of GCs for in vitro study. The primary GCs culture was treated with Kp (0, 10, 50 and 100 nM) and incubated for 48 h. Production of E2 and P4 was estimated in the culture supernatant by ELISA. The expression of gonadotropin receptors (FSHR and LHR), steroidogenic genes (STAR, 3β‐HSD, CYP19A1), proliferation marker (PCNA), apoptotic factors (CASP3 and BCL2) and Kp signalling molecule (extracellular signal‐regulated kinase 1/2, ERK1/2 and p‐ERK1/2) was studied in the GCs by qPCR. Significant E2 production was found in the Kp 50 and 100 nM groups (p < .05), whereas P4 production was reduced in Kp 100 nM group (p < .05). There was concomitant upregulation of FSHR, ERK1/2, STAR and CYP19A1 in the Kp 100 nM treated GCs. In addition, Kp at 100 nM stimulated the proliferation of GCs by upregulating the expression of BCL2 (5.0 fold) and PCNA (94.9 fold). Further, high immunoreactivity of p‐ERK1/2 was observed in the Kp‐treated GCs. It was concluded that Kp at 100 nM concentration stimulated E2 production by upregulating the steroidogenic pathway through ERK1/2, STAR and CYP19A1 and modulating PCNA and BCL2 expressions in the GCs. Further experiments are warranted using Kp antagonist in different combinations to establish the signalling pathway in Kp‐mediated steroidogenesis in the GCs for developing strategies to control ovarian functions.

Bioencapsulation of Oocytes and Granulosa Cells.

A protocol for the encapsulation in sodium alginate of granulosa cells in primary culture and coculture of oocyte-cumulus complexes is reported. Sodium alginate forms strong gels when jellified with barium ions, allowing the self-organization of cells into a 3D structure. This method of encapsulation is simple and cheap, allowing the culture of cells in a three-dimensional fashion.

Oral follicle-stimulating hormone receptor agonist affects granulosa cells differently than recombinant human FSH

To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing invitro fertilization. Basic science research with a preclinical allosteric FSHR agonist. University hospital. Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS. Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle. Estradiol (E2) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells. TOP5300 consistently stimulated E2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS. TOP5300 appears to stimulate E2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E2 production.

Serum granulosa cell-derived TNF-α promotes inflammation and apoptosis of renal tubular cells and PCOS-related kidney injury through NF-κB signaling.

Polycystic ovary syndrome (PCOS) is a disorder with endocrinal and metabolic problems in reproductive aged women. Evidence shows that PCOS is in a high prone trend to develop kidney diseases. In this study, we investigated the mediators responsible for PCOS-related kidney injury. We found that tumor necrosis factor (TNF-α) levels were significantly increased in serum and primary cultured granulosa cells (GCs) from PCOS patients. Serum TNF-α levels were positively correlated with serum testosterone and luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio, suggesting its positive role in the severity of PCOS. Serum TNF-α levels were also positively correlated with the levels of urinary KapU, LamU,α1-MU and β2-MU, the markers for renal tubular cell-derived proteinuria. We established a PCOS mouse model by resection of the right kidney, followed by daily administration of dihydrotestosterone (DHT, 27.5 μg, i.p.) from D7 for 90 days. We found that TNF-α levels were significantly increased in the ovary and serum of the mice, accompanied by increased renal tubular cell apoptosis, inflammation and fibrosis in kidneys. Furthermore, the receptor of TNF-α, tumor necrosis factor receptor 1 (TNFR1), was significantly upregulated in renal tubular cells. We treated human ovarian granulosa-like tumor cells (KGN) with DHT (1 μg/ml) in vitro, the conditioned medium derived from the granulosa cellculture greatly accelerated apoptotic injury in human proximal tubular epithelial cells (HKC-8), which was blocked after knockdown of TNF-α in KGN cells. Furthermore, knockdown of TNFR1 in renal tubular epithelial cells greatly ameliorated cell injury induced by granulosa cell-derived conditioned medium. These results suggest that serum TNF-α plays a key role in mediating inflammation and apoptosis in renal tubular cells associated with PCOS-related kidney injury.

Analysis of the Ovarian Marker Genes Expression Revealed the Antagonistic Effects of Serotonin and Androstenedione on the Functional State of Mouse Granulosa Cells in Primary Culture

Analysis of the Ovarian Marker Genes Expression Revealed the Antagonistic Effects of Serotonin and Androstenedione on the Functional State of Mouse Granulosa Cells in Primary Culture

Stimulatory effects of vasopressin on progesterone production and BMP signaling by ovarian granulosa cells

The aim of the present study was to clarify the effects of arginine vasopressin (AVP) on ovarian steroid production and its functional relationship to the ovarian bone morphogenetic protein (BMP) system. The results showed that AVP treatment significantly increased gonadotropin- and forskolin-induced progesterone synthesis by primary culture of rat granulosa cells and human granulosa cells, respectively. In contrast, estradiol production was not significantly affected by AVP. Treatment with AVP significantly increased forskolin-induced cAMP synthesis by human granulosa cells and mRNA levels of the progesterogenic enzymes CYP11A1 and HSD3B2 in the cells. On the other hand, AVP also enhanced BMP-15-induced phosphorylation of SMAD1/5/9 and ID1 transcription. It was further revealed that the expression levels of BMP receptors, including ALK3, ALK6 and BMPR2, were upregulated by AVP. Collectively, the results indicate that AVP stimulates progesterone production via the cAMP-PKA pathway with upregulation of BMP signaling that inhibits progesterone production, which may lead to fine adjustment of progesterone biosynthesis by granulosa cells.

Analysis of the Ovarian Marker Genes Expression Revealed the Antagonistic Effects of Serotonin and Androstenedione on the Functional State of Mouse Granulosa Cells in Primary Culture

Primary culture of granulosa cells is a prerequisite for a complete study of the normal functioning of the ovary and its pathologies. In this work, we selected the optimal protocol for obtaining a primary culture of mouse granulosa cells in the most functionally active state and revealed the effects of androstenedione and serotonin on the expression of ovarian markers that reflect the functional status of granulosa cells. The morpho-functional analysis of the ovary after PMSG stimulation revealed that 48 hours after PMSG stimulation is the optimal time for obtaining granulosa cells in the most active functional state. Using the set of 14 ovarian functional state marker genes we reveal that androstenedione inhibits cumulus and immature granulosa markers but stimulates genes characteristic of the mature state of granulosa. At the same time, granulosa cells express serotonergic receptors and transporter SERT. The ovarian marker genes expression analysis revealed that serotonin affects the expression of genes characterizing the differentiation of granulosa cells towards cumulus cells. Summarizing, we can conclude that serotonin and androstenedione have an antagonistic effect on the functional state of mouse granulosa cells in primary culture in vitro.

Oxytocin enhances progesterone production with upregulation of BMP-15 activity by granulosa cells

To elucidate the reproductive role of oxytocin (OXT) in ovarian steroidogenesis and its functional interaction with bone morphogenetic proteins (BMPs), the effects of OXT on ovarian steroidogenesis were investigated by utilizing primary culture of rat granulosa cells and human granulosa KGN cells. Here we revealed that the OXT receptor was expressed in both rat and human granulosa cells and that OXT treatment significantly increased follicle-stimulating hormone (FSH)- and forskolin (FSK)-induced progesterone production, but not estradiol production, by rat and human granulosa cells, respectively. In accordance with the effects of OXT on progesterone production, OXT enhanced mRNA expression of CYP11A1 and HSD3B2 induced by FSK in human granulosa cells. Of note, OXT enhanced the phosphorylation of SMAD1/5/9 and the transcription of ID1 induced by BMP-15, but not those induced by BMP-6, in human granulosa cells. It was also revealed that OXT treatment upregulated the expression of BMPR2, a crucial type-II receptor of BMP-15, and enhanced the BMP-15-induced expression of inhibitory SMAD6 by human granulosa cells. Collectively, it was shown that OXT accelerates ovarian progesterone synthesis with upregulation of BMP-15 activity, leading to a fine-tuning of ovarian steroidogenesis (186 words).

DEHP Decreases Steroidogenesis through the cAMP and ERK1/2 Signaling Pathways in FSH-Stimulated Human Granulosa Cells.

DEHP is an endocrine disruptor that interferes with the function of the female reproductive system. Several studies suggested that DEHP affects steroidogenesis in human and rodent granulosa cells (GC). Some studies have shown that DEHP can also affect the FSH-stimulated steroidogenesis in GC; however, the mechanism by which DEHP affects hormone-challenged steroidogenesis in human GC is not understood. Here, we analyzed the mechanism by which DEHP affects steroidogenesis in the primary culture of human cumulus granulosa cells (hCGC) stimulated with FSH. Cells were exposed to DEHP and FSH for 48 h, and steroidogenesis and the activation of cAMP and ERK1/2 were analyzed. The results show that DEHP decreases FSH-stimulated STAR and CYP19A1 expression, which is accompanied by a decrease in progesterone and estradiol production. DEHP lowers cAMP production and CREB phosphorylation in FSH but not cholera toxin- and forskolin-challenged hCGC. DEHP was not able to decrease steroidogenesis in cholera toxin- and forskolin-stimulated hCGC. Furthermore, DEHP decreases FSH-induced ERK1/2 phosphorylation. The addition of EGF rescued ERK1/2 phosphorylation in FSH- and DEHP-treated hCGC and prevented a decrease in steroidogenesis in the FSH- and DEHP-treated hCGC. These results suggest that DEHP inhibits the cAMP and ERK1/2 signaling pathways, leading to the inhibition of steroidogenesis in the FSH-stimulated hCGC.

The effects of irisin and leptin on steroidogenic enzyme gene expression in human granulosa cells: In vitro studies

Reproduction and energy metabolism are closely related, and fertility can be directly affected by either obesity or malnutrition. In this study, we investigated the in vitro effects of irisin and leptin, two hormones primarily involved in energy metabolism, on the expression of genes encoding key steroidogenic enzymes in primary cultures of human granulosa cells. Granulosa cells were purified from follicular fluid samples obtained during in vitro fertilization (IVF) procedure, cultured, and treated with irisin (125-2000 ng/ml) or leptin (25–400 ng/ml) for 1–3 days. mRNA expression levels of cytochrome P450 enzymes [CYP11A1, CYP19A1, CYP21A2], hydroxy-delta-5-steroid dehydrogenase, 3 beta and steroid delta-isomerase 1 (HSD3B1), and hydroxysteroid 17-beta dehydrogenase 3 (HSD17B3) were measured using qRT-PCR analysis. Irisin significantly upregulated CYP19A1 mRNA levels, while leptin upregulated CYP19A1 and HSD3B1 mRNA levels. These preliminary results show that irisin and leptin may directly affect the expression of the genes important for ovarian steroidogenesis and female reproduction.

Low WIP1 Expression Accelerates Ovarian Aging by Promoting Follicular Atresia and Primordial Follicle Activation

Our previous study demonstrated that ovarian wild-type P53-induced phosphatase 1 (WIP1) expression decreased with age. We hypothesized that WIP1 activity was related to ovarian aging. The role of WIP1 in regulating ovarian aging and its mechanisms remain to be elucidated. Adult female mice with or without WIP1 inhibitor (GSK2830371) treatment were divided into three groups (Veh, GSK-7.5, GSK-15) to evaluate the effect of WIP1 on ovarian endocrine and reproductive function and the ovarian reserve. In vitro follicle culture and primary granulosa cell culture were applied to explore the mechanisms of WIP1 in regulating follicular development. This study revealed that WIP1 expression in atretic follicle granulosa cells is significantly lower than that in healthy follicles. Inhibiting WIP1 phosphatase activity in mice induced irregular estrous cycles, caused fertility declines, and decreased the ovarian reserve through triggering excessive follicular atresia and primordial follicle activation. Primordial follicle depletion was accelerated via PI3K-AKT-rpS6 signaling pathway activation. In vitro follicle culture experiments revealed that inhibiting WIP1 activity impaired follicular development and oocyte quality. In vitro granulosa cell experiments further indicated that downregulating WIP1 expression promoted granulosa cell death via WIP1-p53-BAX signaling pathway-mediated apoptosis. These findings suggest that appropriate WIP1 expression is essential for healthy follicular development, and decreased WIP1 expression accelerates ovarian aging by promoting follicular atresia and primordial follicle activation.

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

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

Uptake and Metabolization of Serotonin by Granulosa Cells Form a Functional Barrier in the Mouse Ovary

Serotonin (5-HT) plays an essential role in regulating female reproductive function in many animals. 5-HT accumulates in the mammalian ovary with the involvement of membrane serotonin transporter SERT and is functionally active in the oocytes of growing follicles, but shows almost no activity in follicular cells. In this study, we clarified the interplay between 5-HT membrane transport and its degradation by monoamine oxidase (MAO) in the mammalian ovary. Using pharmacologic agents and immunohistochemical staining of the cryosections of ovaries after serotonin administration in vitro, we demonstrated the activity of transport and degradation systems in ovarian follicles. The MAO inhibitor pargyline increased serotonin accumulation in the granulosa cells of growing follicles, indicating the activity of both serotonin uptake and degradation by MAO in these cells. The activity of MAO and the specificity of the membrane transport of serotonin was confirmed in primary granulosa cell culture treated with pargyline and fluoxetine. Moreover, the accumulation of serotonin is more effective in the denuded oocytes and occurs at lower concentrations than in the oocytes within the follicles. This confirms that the activity of SERT and MAO in the granulosa cells surrounding the oocytes impedes the accumulation of serotonin in the oocytes and forms a functional barrier to serotonin.

ODP407 Effects of Leptin and Ghrelin on Ovarian Steroidogenesis and Involvement of BMP Action in Rat Granulosa Cells.

Abstract Leptin and ghrelin are the two major hormones which regulate food intake and energy balance. They have also been reported to have pleiotropic functions, including the modulation of ovarian function via the hypothalamic-pituitary-ovarian system or in a direct manner to the ovaries. Blood leptin levels increase over the menstrual cycle and peak at the time of the luteinizing hormone (LH) surge. Leptin secreted from adipocytes acts on the hypothalamus and pituitary gland to stimulate the secretion of GnRH and gonadotropins. In the ovaries, high levels of leptin inhibit follicle-stimulating hormone (FSH)-induced steroid secretion and follicular development. Ghrelin, secreted by the stomach, has been reported to modulate GnRH secretion in the hypothalamus and to affect FSH and LH secretion, as well as steroidogenesis in the ovaries. Knockout of these hormones leads to impaired fertility. However, the mechanism of the direct effects of leptin and ghrelin on the ovaries has not been well elucidated. In the present study, we investigated the effects of leptin and ghrelin on the ovarian steroidogenesis using primary culture of rat granulosa cells, by focusing on the ovarian bone morphogenetic protein (BMP) system, which plays an important role in follicular development and steroidogenesis and acts as a luteinizing inhibitor. The results showed that treatment with leptin (1 to 300 ng/mL) did not affect either estradiol (E2) or progesterone (P4) production, but enhanced FSH-induced P4 production at low concentrations (10 ng/mL). On the other hand, treatment with ghrelin (1 to 300 nM) did not affect E2 and P4 production, but enhanced FSH-induced P4 production in a concentration-responsive manner. The mRNA levels of the follicular steroid synthases (StAR, P450scc, 3βHSD, 20αHSD) were examined. Leptin treatment enhanced 3βHSD mRNA levels. Both leptin treatment and ghrelin treatment decreased FSH-induced 20αHSD mRNA levels. However, both treatments decreased FSH-induced cAMP production. In addition, leptin treatment tended to suppress the transcriptional activity of Id-1 induced by BMP-6. Collectively, these results indicate that both leptin and ghrelin enhance FSH-induced P4 production by suppressing BMP signaling in rat granulosa cells. It was thus suggested that leptin and ghrelin which regulate food intake and energy balance can also act as endogenous regulators for the modulation of P4 synthesis and luteinizing process by ovarian follicles. Presentation: No date and time listed

Optimized Primary Culture and Subculture of Granulosa Cells

Background: Primordial follicle includes an oocyte surrounded by a layer of somatic cells called Granulosa Cells (GCs). GCs, also known as nurse cells, are an important protective element for the growth and survival of oocytes. Oocytes, which lack some of the metabolic processes, require granulosa cells for their development.
 Objectives: This manuscript was provided to explain the protocol of GCs primary culture extracted from NMRI mice ovaries.
 Methods: For choosing the optimum protocol, we used two methods with different culture mediums to obtain more GCs and expedite the process. Hematoxylin and Eosin (H&E) staining and flow cytometry were used to analyze the type of extracted cells from ovaries. Besides, we evaluated the effect of crocin and DPP as two common natural products in Iran on the proliferation of these cells via MTT assay.
 Results: Second protocol method and alpha-MEM culture medium were chosen based on the results. Our findings from HE staining and flow cytometry proved the percentage of cultured GCs in the flask. Further, MTT assessment demonstrated that crocin at high doses had a toxic effect on granulosa cells, whereas date palm pollen (DPP) stimulated them to proliferation.
 Conclusion: Modifying this protocol is for the improvement of proliferation, coherence, and quality of GCs in primary culture and subculture. Regarding the effect of these two natural products on granulosa cells, we can mention the bilateral effect of crocin and DPP enhancement in proliferation.

Phthalate monoesters act through peroxisome proliferator-activated receptors in the mouse ovary

Widespread use of phthalates as solvents and plasticizers leads to everyday human exposure. The mechanisms by which phthalate metabolites act as ovarian toxicants are not fully understood. Thus, this study tested the hypothesis that the phthalate metabolites monononyl phthalate (MNP), monoisononyl phthalate (MiNP), mono(2-ethylhexyl) phthalate (MEHP), monobenzyl phthalate (MBzP), monobutyl phthalate (MBP), monoisobutyl phthalate (MiBP), and monoethyl phthalate (MEP) act through peroxisome proliferator-activated receptors (PPARs) in mouse granulosa cells. Primary granulosa cells were isolated from CD-1 mice and cultured with vehicle control (dimethyl sulfoxide) or MNP, MiNP, MEHP, MBzP, MBP, MiBP, or MEP (0.4–400 μM) for 24 h. Following culture, qPCR was performed for known PPAR targets, Fabp4 and Cd36. Treatment with the phthalate metabolites led to significant changes in Fabp4 and Cd36 expression relative to control in dose-dependent or nonmonotonic fashion. Primary granulosa cell cultures were also transfected with a DNA plasmid containing luciferase expressed under the control of a consensus PPAR response element. MNP, MiNP, MEHP, and MBzP caused dose-dependent changes in expression of luciferase, indicating the presence of functional endogenous PPAR receptors in the granulosa cells that respond to phthalate metabolites. The effects of phthalate metabolites on PPAR target genes were inhibited in most of the cultures by co-treatment with the PPAR-γ inhibitor, T0070907, or with the PPAR-α inhibitor, GW6471. Collectively, these data suggest that some phthalate metabolites may act through endogenous PPAR nuclear receptors in the ovary and that the differing structures of the phthalates result in different levels of activity.

Effects of different cyclooxygenase inhibitors on prostaglandin E2 production, steroidogenesis and ovulation of bovine preovulatory follicles.

Ovulation is an inflammation-like process, and cyclooxygenase-2 (COX-2)-dependent production of prostaglandin E2 (PGE2) is its key mediator. Balanced regulation of inflammatory processes in high-yielding dairy cows may be essential for physiological ovulation and fertility. This study aimed to elucidate the mechanisms underlying ovulation failure and cyst development after disturbing intrafollicular inflammatory cascades. Therefore, nonselective (indomethacin and flunixin-meglumine), COX-2 selective (meloxicam), and highly COX-2 selective (NS-398) inhibitors were injected into preovulatory follicles 16 h after administration of GnRH, and ovulation was monitored via ultrasound examination. Additionally, follicular fluid was collected after injection of indomethacin, meloxicam, and NS-398. Moreover, primary granulosa cell cultures from preovulatory follicles were prepared and treated with indomethacin, meloxicam, and NS-398. The concentrations of 17β-estradiol, progesterone, and prostaglandin E2 (PGE2) in the follicular fluid and cell supernatant were estimated. Indomethacin and flunixin-meglumine blocked ovulation, even at low doses, and led to ovarian cyst development. The selective and highly selective COX-2 inhibitors meloxicam and NS-398 were not effective in blocking ovulation. However, indomethacin, meloxicam, and NS-398 significantly and comparably reduced PGE2 concentration in vivo and in vitro (P < 0.05) but had no effect on estradiol or progesterone production. This may contradict the generally accepted hypothesis that PGE2 is a key mediator of ovulation and progesterone production. Our results suggest a connection between ovarian disorders and inflammatory actions in early postpartum cows.