Human Luteinized Granulosa Cells Research Articles

FSH receptor in vitro modulation by testosterone and hCG in human luteinized granulosa cells

To investigate the effect of testosterone and hCG on FSH receptor (FSHR) protein and mRNA expression in human granulosa cells (GC) in vitro. Experimental in vitro cell culture obtained from healthy women undergoing IVF/ICSI due to male factor infertility. Human follicular fluid samples were obtained and after cumulus-oocyte complexes were identified, fluids were pipetted onto Ficoll gradients and centrifuged for 15 min at 400 × g at room temperature. Cells at the interface were removed and plated in 24-well plates for 3 days in M-199 with 10% FBS. Cells were treated with different concentrations of testosterone and hCG. After purification, cells were labeled with specific antibodies and the protein expression of the FSHR was evaluated by flow cytometry in the GC population. Also, total RNA was extracted from confluent GC and the FSHR gene expression was evaluated by RT-PCR. FSHR expression was modulated by treating GC in vitro at different testosterone/hCG concentrations. When compared with untreated GC, we observed a significant effect of testosterone and hCG on the expression of the FSHR at the protein level. Time course experiments confirmed that the gene expression of the FSHR peaked at 12-24h when testosterone or hCG was used as a stimulus. Both testosterone and hCG are able to positively modulate FSHR expression at gene and protein level in human GC in vitro.

Preventing Microbial Contamination during Long-Term In Vitro Culture of Human Granulosa-Lutein Cells: An Ultrastructural Analysis

Purpose. To investigate whether the addition of antibiotic/antimycotic during human granulosa-lutein cells (GLCs) isolation and cell-plating procedures prevents microbial contamination after 144 h of culture and also evaluate the effects of contamination on GLCs ultrastructure and steroid secretion. Methods. GLCs obtained from five women submitted to assisted reproductive techniques (ARTs) were isolated with PBS supplemented with antibiotic/antimycotic or PBS nonsupplemented and cultured for 144 h. GLCs were evaluated by transmission electron microscopy (TEM), and estradiol (E2) and progesterone (P4) secretion was assayed by chemiluminescence. Results. Although no contaminating microorganisms were identified by light microscopy, TEM analyses revealed several bacterial colonies in culture dishes of GLCs isolated with only PBS. Bacterial contamination disrupted the adherence of the GLCs to the culture plate interfering with monolayer formation affecting the growth pattern of GLCs. Various cellular debris and bacteria were observed, and no organelles were found in the cytoplasm of infected cells. While bacterial contamination decreased estradiol media levels, it increased progesterone, as compared with noncontaminated group. Conclusion. Taken together, our data showed that the addition of a high dose of antibiotic/antimycotic during the isolation and cell-plating procedures prevents microbial contamination of long-term GLCs culture as its effects on cells growth and function in vitro.

Leptin suppresses anti-mullerian hormone gene expression in human luteinized granulosa cells through JAK2/STAT3 pathway

Ovarian reserve, reflected by serum anti-Mullerian hormone (AMH) levels, declines with obesity. Obesity is associated with ↑ serum and follicular fluid (FF) leptin (L) and with ↓ serum and FF adiponectin (A). We have recently shown that L treatment in culture suppresses gene expression of both AMH and its receptor (R) in granulosa cells (GCs) while A does not alter these two genes. Leptin signals through janus kinase 2 (JAK2) binding domain and a signal transducer and activator of transcription 3 (STAT3) activation domain.

Expression of lipid oxidative stress-related gene ALDH3A2 (aldehyde dehydrogenase 3 family, member A2) in human granulosa-lutein (GL) cells correlates with FSH response and pregnancy

Expression of lipid oxidative stress-related gene ALDH3A2 (aldehyde dehydrogenase 3 family, member A2) in human granulosa-lutein (GL) cells correlates with FSH response and pregnancy

Insulin-Like Growth Factor 2 (IGF2) Promoter Usage in Human Luteinized Granulosa Cells.

Insulin-Like Growth Factor 2 (IGF2) Promoter Usage in Human Luteinized Granulosa Cells.

The Human Gonadotropin-Inhibitory Hormone Ortholog RFamide-Related Peptide-3 Suppresses Gonadotropin-Induced Progesterone Production in Human Granulosa Cells

RFamide-related peptide-3 (RFRP-3), a mammalian ortholog of avian gonadotropin-inhibitory hormone, has pronounced inhibitory effects on reproduction in a number of species. RFRP-3 suppresses gonadotropin release at the hypothalamic and/or pituitary levels; however, increasing evidence also suggests putative functions within the ovary. We have now demonstrated the expression of both RFRP and its receptor (GPR147) in primary cultures of human granulosa-lutein cells. Immunohistochemical analysis of normal human ovaries from premenopausal women showed that RFRPs and GPR147 were primarily localized in the granulosa cell layer of large preovulatory follicles as well as in the corpus luteum. Treatment of human granulosa-lutein cells with RFRP-3 reduced FSH-, LH- and forskolin-stimulated progesterone production and steroidogenic acute regulatory protein expression but did not affect basal or 8-bromoadenosine 3'5'-cyclic monophosphate stimulated levels. In addition, RFRP-3 inhibited gonadotropin- and forskolin-induced intracellular cAMP accumulation, and these effects were abolished by pretreatment with an inhibitor of inhibitory G(i/o) proteins (pertussis toxin). Importantly, the effects of RFRP-3 on FSH-, LH-, and forskolin-induced cAMP and progesterone accumulation were completely eliminated by cotreatment with the bifunctional GPR147/GPR74 antagonist RF9 or by pretreatment with GPR147 small interfering RNA. These results suggest that RFRP-3 is expressed in human granulosa cells in which it acts via its receptor, GPR147, to inhibit gonadotropin signaling at the level of adenylyl cyclase via activation of a pertussis toxin-sensitive Gα(i/o) protein. This leads to reduced gonadotropin-stimulated cAMP accumulation and progesterone synthesis, likely via reduced steroidogenic acute regulatory protein expression. Thus, ovarian RFRP-3/GPR147 signaling could contribute to normal ovarian function.

Influence of vascular endothelial growth factor on the expression of insulin-like growth factor-II, insulin-like growth factor binding protein-2 and 5 in human luteinized granulosa cells

The aim of the present study was to investigate the influence of vascular endothelial growth factor (VEGF) on the expression of insulin-like growth factor (IGF)-II, insulin-like growth factor binding protein (IGFBP)-2 and in cultured human luteinized granulosa cells (LGCs). Human LGCs were obtained from the follicular fluid by transvaginal oocyte aspiration from 30 infertile patients undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF). The cells were cultured for 72 h with VEGF at concentrations of 0.1, 1.0, and 10.0 ng/ml. The cells not treated with VEGF served as controls. Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of IGF-II, IGFBP-2, and 5 mRNA. The expression of IGF-II mRNA in the 10.0 ng/ml of VEGF group was significantly higher than that in the control group. Treatment with 10.0 ng/ml of VEGF significantly increased the expression of IGFBP-5 mRNA than all other groups. There were no statistically significant differences in the expression of IGFBP-2 mRNA among all the groups. VEGF may play a regulator role in human ovarian physiology by modulating the expression of IGF-II and IGFBP-5 in LGCs.

The Effects of Epidermal Growth Factor and Transforming Growth Factor-α on Secretion of Interleukin-8 and Growth-Regulated Oncogene-α in Human Granulosa-Lutein Cells

Background: In order to investigate the roles of epidermal growth factor (EGF) and transforming growth factor (TGF)-α in ovulation, we studied the production of interleukin (IL)-8 and growth-regulated oncogene (GRO)-α in cultured human granulosa-lutein cells. Methods: Granulosa-lutein cells obtained from the follicular fluids of in vitro fertilization and embryo transfer patients were cultured and treated with EGF, TGF-α, tumor necrosis factor (TNF)-α or 12-O-tetradecanoylphorbol 13-acetate (TPA). An immortalized granulosa cell line (GC1a) was also cultured and treated with EGF, TGF-α or mitogen-activated protein kinase kinase inhibitor. The supernatants were collected, and IL-8 and GRO-α were measured by ELISA. Results: The levels of IL-8 and GRO-α were significantly increased after treatment with EGF, TGF-α, TNF-α and TPA by primary cultured granulosa-lutein cells. The levels of IL-8 and GRO-α were also significantly increased after treatment with EGF or TGF-α in a dose-dependent manner by GC1a. When GC1a was treated with EGF, TGF-α or U0126, the levels of IL-8 and GRO-α were significantly decreased. Conclusion: Our data indicate that the production of IL-8 and GRO-α is upregulated by EGF and TGF-α. It is suggested that EGF and TGF-α may play an important role in luteinization processes involving IL-8 and GRO-α production.

Resveratrol promotes expression of SIRT1 and StAR in rat ovarian granulosa cells: an implicative role of SIRT1 in the ovary

BackgroundResveratrol is a natural polyphenolic compound known for its beneficial effects on energy homeostasis, and it also has multiple properties, including anti-oxidant, anti-inflammatory, and anti-tumor activities. Recently, silent information regulator genes (Sirtuins) have been identified as targets of resveratrol. Sirtuin 1 (SIRT1), originally found as an NAD+-dependent histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. To date, the presence and physiological role of SIRT1 in the ovary are not known. Here we found that SIRT1 was localized in granulosa cells of the human ovary.MethodsThe physiological roles of resveratrol and SIRT1 in the ovary were analyzed. Immunohistochemistry was performed to localize the SIRT1 expression. SIRT1 protein expression of cultured cells and luteinized human granulosa cells was investigated by Western blot. Rat granulosa cells were obtained from diethylstilbestrol treated rats. The cells were treated with increasing doses of resveratrol, and subsequently harvested to determine mRNA levels and protein levels. Cell viability was tested by MTS assay. Cellular apoptosis was analyzed by caspase 3/7 activity test and Hoechst 33342 staining.ResultsSIRT1 protein was expressed in the human ovarian tissues and human luteinized granulosa cells. We demonstrated that resveratrol exhibited a potent concentration-dependent inhibition of rat granulosa cells viability. However, resveratrol-induced inhibition of rat granulosa cells viability is independent of apoptosis signal. Resveratrol increased mRNA levels of SIRT1, LH receptor, StAR, and P450 aromatase, while mRNA levels of FSH receptor remained unchanged. Western blot analysis was consistent with the results of quantitative real-time RT-PCR assay. In addition, progesterone secretion was induced by the treatment of resveratrol.ConclusionsThese results suggest a novel mechanism that resveratrol could enhance progesterone secretion and expression of luteinization-related genes in the ovary, and thus provide important implications to understand the mechanism of luteal phase deficiency.

Bone morphogenetic proteins (BMP)-2 and -6 are potential targets of gaba-A receptor activation in human luteinized granulosa cells (GC)

BMP's are signaling molecules in the transforming growth factor-β superfamily that play significant roles in ovarian folliculogenesis in mammals. BMP-2, BMP-6, BMP-7 and BMP-15 expression was reported in the ovary. Gamma-aminobutyric acid (GABA), its biosynthetic enzyme GAD and GABA-A receptors have been found in the oviduct and ovary. Our previous data indicate that GABA-A agonists increase estradiol (E2) secretion by human GC and alter cytosolic concentrations of Ca2+ in GC in a receptor-specific manner.

Proliferative potential and phenotypic analysis of long-term cultivated human granulosa cells initiated by addition of follicular fluid

The aim of this study was to develop and optimize a strategy for long-term cultivation of luteinizing human granulosa cells (GCs). GCs were cultivated in DMEM/F12 medium supplemented with 2% fetal calf serum. In vitro proliferation of GCs was supported by follicular fluid as well as FSH and growth factors. The cultured GCs were maintained for 45 days with a doubling time of 159 ± 24 h. GCs initiated by the addition of follicular fluid and cultivated under low serum conditions reached 10 ± 0.7 population doublings. GCs maintain the typical phenotypic expression and the telomere length according to specific culture conditions. Our present study has demonstrated that GCs can be maintained in vitro for at least 45 days and this cell model can be beneficial when studying hormonal regulation associated with follicular maturation and preparation of oocytes for fertilization.

Growth Differentiation Factor 9 (GDF9) Suppresses Follistatin and Follistatin-Like 3 Production in Human Granulosa-Lutein Cells

BackgroundWe have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β B-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.MethodologyhGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin β B-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey's test.Principal FindingsGDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin β B-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin β B-subunit mRNA and inhibin B production were attenuated by adding FST.ConclusionGDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin β B-subunit mRNA expression and inhibin B production in hGL cells.

Prostaglandin F2alpha Receptors (FP) in Human Granulosa-Lutein Cells: Acquisition of Function During Transition from Granulosa to Luteal Cell Phenotype.

Prostaglandin F2alpha (PGF2alpha) is an important physiological regulator of the corpus luteum. Data from our laboratory suggest that monkey granulosa cells expressed FP receptor protein, but granulosa cell response to the FP receptor agonist fluprostenol was not detected. In contrast, monkey luteal cells expressed functional FP receptors at mid luteal phase. The present study was performed to examine the acquisition of FP receptor function as granulosa cells differentiate into the granulosa-lutein cells of the corpus luteum. Human luteinizing granulosa cells were obtained from healthy women donating oocytes for in vitro fertilization. These cells were cultured for a total of 6 days under conditions which promote transition to the granulosa-lutein cell phenotype. Western blot and immunostaining were performed to determine FP protein levels and cellular location, respectively. Real time RT-PCR was used to quantify mRNAs, and media progesterone was measured by ELISA. FP protein levels did not change during the culture period. FP protein was distributed throughout the cytoplasm and nucleus of luteinizing granulosa cells on day 0. However, FP protein was located primarily in the nucleus of cells on day 2-6 of culture, comparable to the nuclear location of FP receptors in large, granulosa-derived cells of the monkey corpus luteum. To further examine the function of nuclear FP receptors, additional studies were conducted using human cells after 2 days of culture. Stimulation of FP by fluprostenol reduced progesterone production and early growth response factor (EGR)-1 mRNA levels. Furthermore, fluprostenol inhibited the hCG-stimulated increase in EGR-1 mRNA. These data show that FP receptor stimulation regulates progesterone and EGR-1 expression in cultured human granulosa cells in a manner similar to previous studies in luteal cells. The FP receptor is a member of the G protein-coupled receptor superfamily and interacts with Gq to activate phopholipase C (PLC)-dependent protein kinase C (PKC) in the corpus luteum. In human luteinizing granulosa cells cultured for 2 days, fluprostenol did not alter basal or hCG-stimulated cAMP. FP-mediated decrease of progesterone and EGR-1 mRNA levels was blocked by the PLC inhibitor U73122 and the selective PKC inhibitor Ro-31-8220. Treatment with the mTOR inhibitor rapamycin also prevented the FP-mediated decrease in EGR-1 mRNA. Therefore, FP receptor movement into the nucleus correlates with acquisition of FP receptor function. FP receptor likely regulates luteal functions (progesterone production and EGR-1 mRNA) via a PLC and PKC-dependent pathway in primate granulosa-lutein cells. Supported by NIH HD054691 and Schering-Plough, now Merck & Co. (poster)

Anti-Müllerian Hormone Inhibits FSH-Induced Adenylyl Cyclase Activation, Aromatase Expression, and Estradiol Production in Human Granulosa-Lutein Cells.

Anti-Müllerian Hormone Inhibits FSH-Induced Adenylyl Cyclase Activation, Aromatase Expression, and Estradiol Production in Human Granulosa-Lutein Cells.

Fractalkine is expressed in the human ovary and increases progesterone biosynthesis in human luteinised granulosa cells

BackgroundRecent evidence from rodent ovaries has demonstrated expression of fractalkine and the existence of fractalkine receptor, and showed that there is a significant increase in steroidogenesis in response to fractalkine, yet the role of fractalkine and CX3CR1 in the human ovary is still unknown. This study aimed to determine the expression levels of fractalkine and CX3CR1 in the human ovary and to investigate their roles in sexual hormone biosynthesis by human luteinising granulosa cells. This is the first detailed report of fractalkine and CX3CR1 expression and function in the human ovary.MethodsFractalkine and CX3CR1 expression levels were measured by immunohistochemistry using ovarian tissue from pathological specimens from five individuals. Granulosa cells were obtained from patients during IVF treatment. They were cultured and treated with increasing doses of hCG with or without fractalkine. Media were collected to detect estradiol and progesterone by chemiluminescence. StAR, 3-βHSD and CYP11A expression were determined in granulosa cells treated with or without fractalkine by real-time RT-PCR.ResultsFractalkine and CX3CR1 were expressed in the human ovary and in luteinising granulosa cells. However, fractalkine expression was stronger in luteinising granulosa cells. Treatment with fractalkine augmented hCG stimulation of progesterone production in a dose-dependent manner with concomitant increases in transcript levels for key steroidogenic enzymes (StAR, 3-βHSD and CYP11A) but had no effect on estradiol biosynthesis(P < 0.05).ConclusionsFractalkine and CX3CR1 were found to express in human ovary and luteinising granulosa cells. Fractalkine can increase the biosynthesis of progesterone in a dose-dependent manner by enhancing transcript levels of key steroidogenic enzymes.

Women with high telomerase activity in luteinised granulosa cells have a higher pregnancy rate during in vitro fertilisation treatment

To study the effect of telomerase activity (TA) in human luteinised granulosa cells (GCs) on the outcome of in vitro fertilisation treatment. Fifty-six women, aged 23 to 39 years, were enrolled and divided into four groups according to their levels of TA. Seventeen cases in group A exhibited nondetectable TA, 16 cases in group B expressed low levels of TA (between 0.1 and 0.65 OD × mm), 14 cases in group C expressed moderate TA levels (between 0.66 and 1.00 OD × mm) and 9 cases in group D expressed high levels of TA (more than 1.00 OD × mm). The level of total serum testosterone (T) was significantly higher in groups C and D than in group A (1.43±0.10 vs. 1.08±0.11 nmol/L, P<0.030 and 1.56±0.08 vs. 1.08±0.11 nmol/L, P<0.005, respectively). The TA level was positively correlated with T (r=0.291, P<0.011). No obvious differences were observed in rates of fertilisation, cleavage, mature oocyte formation or good-quality embryos among the groups. The patients in group D exhibited the highest rates of embryo implantation and clinical pregnancy (with rates of 52.63% and 77.78%, respectively, compared with 18.92% and 29.41% in group A, 25.71% and 37.50% in group B and 48% and 50% in group C, with P<0.018 and P=0.112, respectively). The patients in group D also had a greater likelihood of becoming pregnant than those in group A (OR: 9.703, P < 0.023), group B (OR: 14.765, P<0.009) or group C (OR: 5.560, P=0.103). Luteinised GCs have a certain potential for proliferation and TA of luteinised GCs may predict the clinical outcomes of IVF treatment. Some unknown regulatory mechanisms between TA and T should be studied in further trials.

FSH modulatory effect on human granulosa cells: a gene–protein candidate for gonadotrophin surge-attenuating factor

Previous evidence indicates a homology of gonadotrophin surge-attenuating factor (GnSAF) to the carboxyl terminal of human serum albumin (HSA) and the ability of human granulosa cells to produce mRNA transcripts corresponding to this fragment, but the underlying mechanism is still unknown. This study investigated the role of FSH in vitro in the expression of the carboxyl terminal of HSA by human luteinized granulosa cells. Cells were cultured on poly- l-lysine-coated microscope slides in the absence or presence of 10 ng/ml FSH, followed by in-situ hybridization and immunocytochemistry. In the presence of FSH, mRNA transcripts corresponding to the carboxyl terminal of the HSA gene and corresponding protein could be detected in comparable intensity to that seen by hepatic HepG2 cells (positive control). Significantly lower expression was detected in granulosa cells cultured without FSH addition ( P < 0.01), but no expression was detected in HeLa cells. These results demonstrate for the first time that FSH stimulates the expression of the carboxyl terminal fragment of the HSA gene and corresponding protein in human luteinized granulosa cells. Therefore, the carboxyl terminal of HSA has a functional role in the ovary and this further supports the notion that this HSA fragment is a GnSAF-bioactive entity. Gonadotrophin surge-attenuating factor (GnSAF) is a nonsteroidal ovarian substance which attenuates the endogenous LH surge in superovulated women. In-vivo and in-vitro evidence has shown that GnSAF is produced under the stimulation of FSH and may play a physiological role during the normal menstrual cycle. We have previously demonstrated that GnSAF has identity to the carboxyl terminal fragment of human serum albumin (HSA). In this study, we investigated the role of FSH on the expression of the carboxyl terminal fragment of HSA by human luteinizing granulosa cells in vitro. It is demonstrated for the first time that FSH stimulates the expression of this fragment of HSA gene and the corresponding protein in human luteinizing granulosa cells. This finding supports further the notion that this fragment of HSA is the GnSAF bioactive entity.

In vitro viability and secretory capacity of human luteinized granulosa cells after gonadotropin-releasing hormone agonist trigger of oocyte maturation

To evaluate viability of luteinized granulosa cells obtained from patients triggered with either GnRH agonist or hCG and to assess the secretion of steroids and vascular endothelial growth factor (VEGF) by cultured luteinized granulosa cells in the presence or absence of hCG. Prospective, randomized controlled trial. University-based fertility center. A subset of patients who underwent a randomized trial involving GnRH agonist trigger after GnRH antagonist protocol vs. hCG trigger after pituitary suppression with GnRH agonist protocol. In vitro fertilization cycles. Proportion of apoptosis; basal and hCG-induced secretion of E(2), P, and VEGF by luteinized granulosa cells; follicular-fluid VEGF and luteal-phase serum E(2), P, and plasma VEGF concentrations. There were no differences in the proportion of granulosa/luteal cell apoptosis, follicular-fluid or luteal-phase plasma VEGF concentration, or basal culture media E(2), P, and VEGF concentrations between the two groups. Addition of hCG to the culture media significantly increased the P concentration in both groups, but there were no changes in E(2) or VEGF concentrations. Serum E(2) levels were lower at 5 and 9 days after GnRH agonist compared with hCG trigger. The granulosa/luteal cells obtained on the day of oocyte retrieval after GnRH agonist trigger are still viable and have the capacity to respond to hCG by increasing the secretion of steroids.

Follicular Fluid High-Density Lipoprotein-Associated Sphingosine 1-Phosphate (S1P) Promotes Human Granulosa Lutein Cell Migration via S1P Receptor Type 3 and Small G-Protein RAC11

Coordinated migration and progesterone production by granulosa cells is critical to the development of the corpus luteum, but the underlying mechanisms remain obscure. Sphingosine 1-phosphate (S1P), which is associated with follicular fluid high-density lipoprotein (FF-HDL), was previously shown to regulate ovarian angiogenesis. We herein examined the effects of S1P and FF-HDL on the function of granulosa lutein cells. Both FF-HDL and S1P induced migration of primary human granulosa lutein cells (hGCs) and the granulosa lutein cell line HGL5. In addition, FF-HDL but not S1P promoted progesterone synthesis, and neither of the two compounds stimulated proliferation of granulosa lutein cells. Polymerase chain reaction and Western blot experiments demonstrated the expression of S1P receptor type 1 (S1PR1), S1PR2, S1PR3, and S1PR5 but not S1PR4 in hGCs and HGL5 cells. The FF-HDL- and S1P-induced granulosa lutein cell migration was emulated by FTY720, an agonist of S1PR1, S1PR3, S1PR4, and S1PR5, and by VPC24191, an agonist of S1PR1 and S1PR3, but not by SEW2871 and phytosphingosine 1-phosphate, agonists of S1PR1 and S1PR4, respectively. In addition, blockade of S1PR3 with CAY1044, suramine, or pertussis toxin inhibited hGC and HGL5 cell migration toward FF-HDL or S1P, while blockade of S1PR1 and S1PR2 with W146 and JTE013, respectively, had no effect. Both FF-HDL and S1P triggered activation of small G-protein RAC1 and actin polymerization in granulosa cells, and RAC1 inhibition with Clostridium difficile toxin B or NSC23766 abolished FF-HDL- and S1P-induced migration. The FF-HDL-associated S1P promotes granulosa lutein cell migration via S1PR3 and RAC1 activation. This may represent a novel mechanism contributing to the development of the corpus luteum.

Dynamics of intra-follicular glucose during luteinization of macaque ovarian follicles

Glucose is important to the maturation of the oocyte and development of the embryo, while hyperglycemia results in profound reproductive and developmental consequences. However, the normal physiology of glucose in the ovary remains poorly understood. The goal of this study was to determine intra-follicular glucose dynamics during the periovulatory interval in non-human primates undergoing controlled ovarian stimulation protocols. Follicular fluid and mural granulosa cells were isolated before or up to 24h after an ovulatory hCG bolus, and the human granulosa-lutein cell line hGL5 was used. Intra-follicular glucose increased 3h after hCG, and remained at that level until 12h when levels decline back to pre-hCG concentrations. Pyruvate and lactate concentrations in the follicle were not strongly altered by hCG. Mural granulosa cell expression of hexokinase 1 and 2, and glucose-6-phosphate dehydrogenase mRNA decreased following hCG, while glycogen phosphorylase (liver form) increased following hCG. Glucose uptake by hGL5 cells was delayed until 24h following stimulation. In summary, intra-follicular glucose increases following an ovulatory stimulus and mural granulosa cells do not appear able to utilize it, sparing the glucose for the cumulus-oocyte complex.