Granulosa Cell Membrane Research Articles

Interleukin-22 promotes proliferation and reverses LPS-induced apoptosis and steroidogenesis attenuation in human ovarian granulosa cells: implications for polycystic ovary syndrome pathogenesis

Objective Interleukin 22 (IL-22) plays a role in inflammatory diseases. However, whether IL-22 affects the function of ovarian granulosa cells (GCs) and its relationship with Polycystic Ovary Syndrome (PCOS)remains unclear. Methods We investigated the level of IL-22 in human follicular fluid using ELISA. The expression and localization of the IL-22 receptor 1 (IL-22R1) in GCs were investigated by RT-PCR and immunofluorescence staining, respectively. The proliferation of KGN cells (human GCs line) was assessed by CCK-8 assay and EdU assay after treatment with recombinant human IL-22 (rhIL-22) and lipopolysaccharide (LPS). Apoptosis was assessed using flow cytometry. Apoptotic proteins and steroidogenic genes were detected by western blotting. Results ELISA's results showed that compared with the control group, PCOS patients showed lower expression of IL-22 in follicular fluid. Immunofluorescence showed that IL-22R1 is expressed and localized in human granulosa cell membranes. IL-22 promoted cell proliferation and reversed LPS-induced inhibition of cell proliferation. IL-22 alone did not affect apoptotic or steroidogenic protein expression, however, it reversed LPS-induced apoptosis via downregulation of Bcl-2, upregulation of Bax and cleaved caspase-3, and restoration of LPS-downregulated StAR, CYP11A1, and CYP19A1 expression. Western blotting confirmed that IL-22 activated the JAK2/STAT3 signaling. Conclusion IL-22 promotes cell proliferation, inhibits apoptosis, and regulates KGN cell steroidogenesis confronted with LPS, and decreased IL-22 may be involved in the development of PCOS.

Effects of Dextran-Coated Superparamagnetic Iron Oxide Nanoparticles on Mouse Embryo Development, Antioxidant Enzymes and Apoptosis Genes Expression, and Ultrastructure of Sperm, Oocytes and Granulosa Cells.

BackgroundAlthough application of superparamagnetic iron oxide nanoparticles (SPIONs) in industry and medi- cine has increased, their potential toxicity in reproductive cells remains a controversial issue. This study was under- taken to address the response of sperm, oocyte, and resultant blastocyst to dextran-coated SPIONs (D-SPIONs) treat- ment during murine in vitro fertilization (IVF).Materials and Methods In this experimental study, murine mature oocytes were randomly divided into three groups: control, and low- and high-dose groups in which fertilization medium was mixed with 0, 50 and 250 µg/ml of D- SPIONs, respectively. Sperm and/or cumulus oocyte complexes (COCs) were cultured for 4 h in this medium for elec- tron microscopic analysis of sperm and COCs, and assessment of developmental competence and genes expression of Gpx1, Sod1, catalase, Bcl2l1 and Bax in the resultant blastocysts. ResultsUltrastructural study of sperm, oocyte, and granulosa showed destructed mitochondria and membranes in spermatozoa, vacuolated mitochondria and distorted cristae in oocytes, and disrupted nuclei and disorganized cell membranes in granulosa in a dose-dependent manner. Data showed that cleavage and blastocyst rates in the 250 µg/ml of D-SPIONs were significantly lower than in the control group (P<0.05). Gene expression of GPx1, Sod1, catalase, Bcl2l1 and Bax in resultant blastocysts of the high-dose group and catalase and Bax in resultant blastocysts of the low-dose group, was higher than the controls.ConclusionThere is considerable concern regarding D-SPIONs toxic effects on IVF, and mitochondrial and cell membrane damage in mouse spermatozoa and oocytes, which may be related to oxidative stress and apoptotic events.

Characterization of Membrane Microdomains on a Human Granulosa Cell Line by Atomic Force Microscopy

Signaling via the Hypothalamic‐Pituitary‐Gonadal (HPG) axis regulates fertility in both men and women. Follicle stimulating hormone (FSH), secreted from the anterior pituitary, binds to a G protein coupled receptor (GPCR) on ovarian granulosa cells. The activation of this receptor triggers a cellular signaling pathway that leads to the promotion of follicle development and estrogen secretion. Our lab has previously demonstrated that the human FSH receptor (hFSHR) shows an affinity for residing in lipid rafts of HEK293 cells which may play a pivotal role in its signaling. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids, with higher stiffnesses than bulk membrane domains. To characterize lipid rafts on the human granulosa cell line hGrC1 we used atomic force microscopy. Atomic force microscopy is a technique used to produce nanoscale resolution topographical images. It can also be used for determining mechanical characteristics, such as stiffness and strain. Here, using contact and force‐volume mode atomic force microscopy, we successfully generated topographical maps of hGrC1s and recorded membrane stiffness measurements. Areas of the granulosa cell membrane showed variable stiffness on a scale of 58.998 nm / pixel, providing the first direct evidence of lipid rafts in human granulosa cell membranes. Demonstration of lipid rafts contributes to the implications of the role of lipid rafts hFSH signalling. By understanding membrane dynamics on granulosa cells we hope to gain insight into how to manipulate hFSHR function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterisation and cryopreservation of the ovarian preantral follicle population from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831).

The aim of the present study was to characterise the ovarian preantral follicle (PF) population and to establish a solid surface vitrification (SSV) process using dimethyl sulfoxide (DMSO) as a cryoprotectant for preservation of ovarian tissue from yellow-toothed cavies (Galea spixii). Ovaries were fixed for PF population analysis or were subjected to the SSV process. The mean (± s.e.m.) PF population per ovarian pair was estimated to be 416.0±342.8. There were 140.0±56.0 (63.4%) and 125.0±58.0 (64.0%) primary follicles on the right and left ovaries, respectively. The proportion of this follicle category was significantly greater than that of other follicle categories (P<0.05). The diameter of follicles (123.7±18.3µm), oocytes (50.1±5.0µm) and nuclei (14.27±2.01µm) was larger for secondary ones when compared with other PFs categories. Most PFs were morphologically normal (94.6%), with light microscopy identifying only a few atretic follicles (5.4%). After SSV, there was a reduction in the proportion of morphologically normal PFs compared with the non-vitrified group (69.5% vs 91.2%, respectively). Transmission electron microscopy revealed preservation of oocytes and granulosa cell membranes and the morphological aspect of follicles; the primary change observed in some vitrified PFs was the presence of vacuoles in the oocytes and granulosa cells cytoplasm and turgid mitochondria. In conclusion, the present study provides an estimative and characterization for the PF population in ovaries of G. spixii. Moreover, we report its PFs cryopreservation using an SSV process.

Active 3'-5' cyclic nucleotide phosphodiesterases are present in detergent-resistant membranes of mural granulosa cells.

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

Expression of aquaporin 1, 5 and 9 in the ovarian follicles of cycling and early pregnant pigs.

Aquaporins (AQPs) are water channel proteins responsible for water homeostasis and important for proper functioning of all body systems, including reproductive structures. This study was designed to determine their localization and quantitative changes in the pig ovary during different stages of the estrous cycle and early pregnancy. The expression of AQP 1, 5 and 9 proteins was determined by immunocytochemistry and Western blot analyses. AQP1 was found in the plasma membranes of capillary endothelium, AQP5 - in the plasma membranes of granulosa cells of developing follicles and flattened follicle cells of the primordial follicles, and AQP9 - in granulosa cells of the developing follicles. In the cyclic pigs, the expression of AQP1 and 5 proteins was the highest on Days 18-20, but did not change significantly between Days 2-4, 10-12 and 14-16 of the cycle. In pregnant pigs (Days 14-16 and 30-32), the expression of AQP1 and 5 did not change and was similar to that observed during Days 10-12 and 14-16. In turn, AQP9 expression did not change between all studied periods. In conclusion, studied AQP are localized in different cells populations, the endothelial and granulosa cells, and AQP1 and 5 seem to be crucial for follicular development in pigs.

Identification and characterization of membrane androgen receptors in the ZIP9 zinc transporter subfamily: I. Discovery in female atlantic croaker and evidence ZIP9 mediates testosterone-induced apoptosis of ovarian follicle cells.

Rapid, cell surface-initiated, pregenomic androgen actions have been described in various vertebrate cells, but the receptors mediating these actions remain unidentified. We report here the cloning and expression of a cDNA from Atlantic croaker (Micropogonias undulatus) ovaries encoding a 33-kDa, seven-transmembrane protein with binding and signaling characteristics of a membrane androgen receptor that is unrelated to any previously described steroid receptor. Instead, croaker membrane androgen receptor has 81-93% amino acid sequence identity with zinc transporter ZIP9 (SLC39A9) subfamily members, indicating it is a ZIP9 protein. Croaker ZIP9 is expressed in gonadal tissues and in brain and is up-regulated in the ovary by reproductive hormones. Croaker ZIP9 protein is localized to plasma membranes of croaker granulosa cells and human breast cancer (SKBR-3) cells stably transfected with ZIP9. Recombinant croaker ZIP9 has a high affinity (dissociation constant, Kd, 12.7 nM), limited capacity (maximal binding capacity 2.8 nM/mg protein), displaceable, single binding site-specific for androgens, characteristic of steroid receptors. Testosterone activates a stimulatory G protein coupled to ZIP9, resulting in increased cAMP production. Testosterone promotes serum starvation-induced cell death and apoptosis in transfected cells and in croaker ovarian follicle cells that is associated with rapid increases in intracellular free zinc concentrations, suggesting an involvement of zinc in this nonclassical androgen action to promote apoptosis. These responses to testosterone are abrogated by treatment with ZIP9 small interfering RNA. The results provide the first evidence that zinc transporter proteins can function as specific steroid membrane receptors and indicate a previously unrecognized signaling pathway mediated by steroid receptors involving alterations in intracellular zinc.

Three-Dimensional Image Analysis to Quantify the Temporo-Spacial Expression of Cellular Receptors

� Abstract≤O varian folliculogenesis is primarily controlled by the action of gonadotropins namely follicle stimulating hormone (FSH) and luteinizing hormone (L H). Several reports indicated that the process of initial recruitment of primordial follicles to the growing follicles is not gonadotropin-dependent but Bone morphogenetic protein (B M P)-dependent. However, this has not been unequivocally confirmed. T he aim of this study was to investigate the temporo-spacial protein expression of the B M P receptors 1B (B M PR1b), FSH R and L H R in several stages of follicle development. W hile the localization of all receptors was found in granulosa cell membrane of the follicles the temporal expression was varied. B M PR1b was expressed in all follicle stages, FSH R was detected in primary follicles onward and L H R was absent in both primordial and primary follicles but appeared in later stages. Q uantitative analysis based on the intensity of fluorescent signals showed that the expression of B M PR1b, FSH R and L H R significantly (p< 0.001 p< 0.0001 p< 0.0001 respectively) increased with follicular development. W e have concluded that the combination of sensitive immunofluorescence labeling and computerized 3D image analysis proves efficient tools for in situ detection and quantification of the expression of small amount of protein in a complex tissue structure.

The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways.

BackgroundC-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles.MethodsOvaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis.ResultsThe results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src siRNA knockdown of c-src expression. Furthermore, our studies demonstrated that folliculogenesis onset was inhibited by Calphostin, PD98059 or LY294002 treatment,but none of them down-regulated c-src expression. In contrast, the expression levels of p-PKC, p-ERK1/2 and p-PI3K in the follicles were clearly decreased by c-src siRNA transfection. Correspondingly, both Calphostin and LY294002 treatment resulted in a decrease in the p-PKC level in follicles, but no change was observed in the PD98059 group. Finally, LY294002 treatment decreased the p-PI3K expression level in the follicles, but no changes were observed in the PD98059 and Calphostin groups.ConclusionsC-src plays an important role in regulating primordial follicle activation and growth via the PI3K-PKC- ERK1/2 pathway.

Effect of methanol on mitochondrial organization in zebrafish (Danio rerio) ovarian follicles

Successful cryopreservation is usually measured in terms of cell survival. However, there may also be more subtle effects within cells that survive. Previous studies on zebrafish have produced evidence of mitochondrial DNA (mtDNA) damage in cryopreserved embryonic blastomeres and, after exposure to cryoprotectants, alterations in mtDNA replication in embryos and decreased mitochondrial membrane potential, mtDNA and ATP production in ovarian follicles. This study shows that the decreased ATP levels previously observed in stage III zebrafish ovarian follicles exposed to ≥3 M methanol persisted in those follicles that subsequently developed to stage IV. However, the decreased mtDNA levels were restored in those follicles. In order to determine whether mitochondrial distribution and/or their transport network was affected by the methanol exposure, immunocytochemistry analysis of tubulin and mitochondrial cytochrome c oxidase I (COX-I) was performed, along with phalloidin staining of polymerized actin. Neat arrangements of all proteins were observed in control follicles, with COX-I and tubulin being colocalized near granulosa cell nuclei, while actin formed hexagonal and/or polygonal structures nearer granulosa cell membranes and projected into the oocyte surface. Exposure to methanol (2 to 4 M) disrupted the COX-I and tubulin arrangements and the hexagonal and/or polygonal actin distribution and actin projections into the oocyte. These effects were still observed in those follicles that developed to stage IV, although the severity was reduced. In summary, the disruption to function and distribution of mitochondria in ovarian follicles exposed to > 2 M methanol may be mediated via disruption of the mitochondrial transport system. Some recovery of this disruption may take place after methanol removal and subsequent follicle maturation.

Proteins Involved in the LH Switch Are Present in Detergent-Resistant Membranes of Porcine Granulosa Cells but Not in Cumulus-Oocyte Complexes.

Proteins Involved in the LH Switch Are Present in Detergent-Resistant Membranes of Porcine Granulosa Cells but Not in Cumulus-Oocyte Complexes.

Progestin Signaling Through mPRα in Atlantic Croaker Granulosa/Theca Cell Cocultures and Its Involvement in Progestin Inhibition of Apoptosis

Although there is substantial evidence that membrane progestin receptors (mPRs) perform a critical physiological role in meiotic maturation of fish oocytes, it is unknown whether they are also intermediaries in progestin signaling in the surrounding follicular cells. Here, we show that mPRα protein is located on the plasma membranes of both granulosa and theca cells (G/T cells) isolated from Atlantic croaker ovaries and is associated with the presence of a single high affinity, limited capacity, pertussis toxin-sensitive, specific progestin [17,20β,21-trihydroxy-4-pregnen-3-one (20β-S)] membrane binding site with the characteristics of mPRα. Treatment of G/T cells with 20β-S caused rapid G protein activation and a transient, pertussis toxin-sensitive, decrease in cAMP levels, whereas the selective nuclear progesterone receptor agonist, R5020, did not cause G protein activation, consistent with previous reports on mPRα signaling. 20β-S treatment decreased serum starvation-induced cell death in both G/T cells and in seatrout mPRα-transfected MDA-MB-231 cells, whereas R5020 was ineffective. Moreover, a selective mPRα agonist, 10-ethenyl-19-norprogesterone, mimicked the protective action of 20β-S against cell death, which was lost upon knockdown of mPRα protein but not after progesterone receptor knockdown, further demonstrating an involvement of mPRα. Signaling molecules involved in inhibition of apoptosis, Erk and serine-threonine kinase, were activated in G/T cells by 20β-S, which suggests a potential mechanism for mPRα inhibition of apoptosis. This is the first study to demonstrate endogenous mPR signaling in the ovarian follicle and to suggest a novel physiological role for mPRα in mediating the antiapoptotic actions of progestins in ovarian follicle cells.

Oogenesis defects in a mutant mouse model of oculodentodigital dysplasia

The essential role of connexin43 (Cx43) during oogenesis has been demonstrated by the severe germ cell deficiency and arrested folliculogenesis observed in Cx43 knockout mice. Recently, another mutant mouse strain became available (Gja1(Jrt)/+) that carries the dominant loss-of-function Cx43 mutation, Cx43(G60S). Gja1(Jrt)/+ mice display features of the human disease oculodentodigital dysplasia (ODDD), which is caused by mutations in the GJA1 gene. We used this new mutant strain to study how a disease-linked Cx43 mutant affects oogenesis. We found that female mutant mice are subfertile with significantly reduced mating success and small litters. The phosphorylated species of the Cx43 protein are reduced in the mutant ovaries in association with impaired trafficking and assembly of gap junctions in the membranes of granulosa cells, confirming that the mutant protein acts dominantly on its wild-type counterpart. Correspondingly, although starting with a normal abundance of germ cells, ovaries of the mutant mice contain significantly fewer pre-ovulatory follicles and do not respond to superovulation by gonadotropins, which is at least partially the result of reduced proliferation and increased apoptosis of granulosa cells. We conclude that the Gja1(Jrt) mutation has a dominant negative effect on Cx43 function in the ovary, rendering the females subfertile. Given these findings, closer examination of reproductive function in ODDD human females is warranted.

Ultrastructural features of the follicular wall in developing follicles of the sexually immature ostrich (&lt;i&gt;Struthio camelus&lt;/i&gt;)

The ultrastructure of the follicular wall in primordial, previtellogenic and vitellogenic follicles of the sexually immature ostrich is described in the present study. The follicular wall consists of a zona radiata, granulosa cell layer, basal lamina and thecal layer. Cytoplasmic processes from the plasma membranes of the granulosa cell layer and the ovocyte form the zona radiata in previtellogenic and vitellogenic follicles. The granulosa cell layer transforms from simple cuboidal epithelium in primordial follicles to simple columnar or pseudostratified columnar epithelium in previtellogenic and vitellogenic follicles. Transosomes were observed along the apical and lateral plasma membranes of granulosa cells. The thecal layer in previtellogenic and vitellogenic follicles consists of interna and externa components. The fibroblasts in the theca externa contain microfilaments, which are thought to be actin filaments. The study revealed ultrastructural features, which are associated with the transportation of yolk precursors and nutrients into the ovoplasm. In addition, the study indicates that, although the cells in the theca externa contain microfilaments, they do not possess the ultrastructural characteristics of smooth muscle cells.

Binding of progesterone to cell surfaces of human granulosa-lutein cells

Progesterone is produced by granulosa cells under the influence of luteinizing hormone. Nuclear progesterone receptors have been found in rat granulosa cells. Human granulosa-lutein cells rapidly respond to progesterone with an increase in intracellular calcium suggesting the existence of a nongenomic mechanism. This study was conducted to determine whether binding of progesterone to granulosa cells could occur at the membrane. Granulosa cells were obtained from an in vitro fertilization program and examined immunohistochemically with an antiserum to membrane progesterone receptors. Approximately 14–70% of freshly harvested or cultured granulosa cells of six patients showed a positive reaction to the antiserum, limited to the cell membrane. Western blot analysis of homogenates of granulosa cells and a granulosa cell tumour confirmed the presence of progesterone receptors A, B and C and low amounts of a putative membrane receptor. These results demonstrate that the plasma membranes of human granulosa cells possess binding components for progesterone which may be involved in its nongenomic mechanism of action.

Morphometric and ultrastructural characterization of Bos indicus preantral follicles

The aim of the present study was to characterize the ultrastructure of zebu cow preantral follicles (PAFs). Ovarian cortex samples were processed for light and transmission electron microscopy. Primordial follicles consisted of an oocyte surrounded by one layer of flattened or flattened-cuboidal granulosa cells. The oocyte contained a large and usually eccentric nucleus. Most organelles were located at the perinuclear ooplasm. Round shaped mitochondria, which contained electron-dense granules, smooth and rough endoplasma reticulum and a Golgi apparatus were also observed. Vesicles and coated pits were often observed in the cortical ooplasm. In primary follicles, the oocyte was surrounded by one layer of cuboidal granulosa cells. Short microvilli were observed on the oolema. Secondary follicles consisted of an oocyte surrounded by a variable number of layers of cuboidal granulosa cells. Small secondary follicles had an ultrastructure very similar to that observed in primary follicles. At this follicular stage, the zona pellucida was beginning to form around the oocyte. In large secondary follicles, the zona pellucida was totally developed around the oocyte. Several granulosa cell projections could be detected that were encroaching into the zona pellucida and protruding towards the oocyte, where gap junctions were observed between oocyte and granulosa cell membranes. Organelles within the oocyte were located at the periphery of the ooplasm, and clusters of cortical granules were observed. Round mitochondria were abundant in all developmental stages. In conclusion, this study described the ultrastructure of zebu cow PAFs, and some unique characteristics could be observed as compared with what has been reported for follicles of Bos taurus cattle.

Energy substrate metabolism of mouse cumulus-oocyte complexes: response to follicle-stimulating hormone is mediated by the phosphatidylinositol 3-kinase pathway and is associated with oocyte maturation.

Successful in vitro maturation (IVM) of oocytes obtained from medium-sized antral follicles could avoid the need for superovulation for in vitro fertilization. The wide range of doses of FSH used in IVM prompted us to study the effect of varying concentrations of FSH on the dynamics of nutrient uptake and production by individual maturing mouse cumulus-oocyte complexes (COCs). COCs isolated from the antral follicles of unprimed, prepubertal B6CBF(1) mice were cultured individually in increasing concentrations of FSH (0-2000 ng/ml). Following culture, pyruvate, glucose, and lactate uptake or production by individual complexes were noninvasively assessed and compared with the stage of nuclear maturation of the enclosed oocyte. FSH significantly increased oocyte maturation and produced a two- to threefold increase in glucose uptake and lactate production by COCs in which the enclosed oocyte completed maturation. In these COCs, pyruvate was taken up under control conditions but was produced in progressively higher quantities in increasing concentrations of FSH. In COCs where the oocyte failed to complete maturation, pyruvate was taken up (rather than produced) and glucose uptake and lactate production were lower and unaffected by the presence or absence of FSH. This suggests that there is dialogue between cumulus cells and the maturing oocyte that influences FSH responsiveness and substrate metabolism of the whole COC. Finally, inhibition of FSH-stimulated glucose uptake by the PI3-kinase inhibitor LY294002 and the finding of GLUT4 protein in granulosa cells suggest that FSH increases glucose uptake by PI3-kinase-mediated translocation of GLUT4 to the granulosa cell membrane.

Down-regulation of mt 1 melatonin receptors in rat ovary following estrogen exposure

In this study, we have demonstrated that 2-[ 125I]-iodomelatonin binds specifically to rat ovarian granulosa cell (GC) membranes with high affinity (K D=83 pM; B max=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt 1 melatonin receptor antibody, we have also detected mt 1 melatonin receptors in rat ovary. Because melatonin has been reported to alter the steroidogenic responses of ovarian tissues to gonadotropins, a physiological role for intra-ovarian melatonin may exist. Thus, in order to investigate a possible intra-ovarian role for melatonin, we have used both an in vivo and in vitro model of follicular development. Treatment of immature (day 21) female rats with estradiol (E; 0.2 mg/d × 3 d; subcutaneous) was used to induce follicular growth. Membranes from both untreated (U) and E-treated animals' ovaries contained high-affinity 2-[ 125I]-iodomelatonin (I-MEL) binding sites (Kd=83 and 23 pM, respectively). Estradiol treatment in vivo caused a significant decrease (P<0.05) in binding of 2-[ 125I]-iodomelatonin to ovarian membranes with untreated animals' ovaries having a B max=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a B max=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt 1 melatonin receptors in rat ovary were down-regulated (∼95%) using immunoblot analysis. Granulosa cells isolated from E-treated rats were further matured in vitro with testosterone (T) and the pituitary gonadotropin follicle-stimulating hormone (FSH). Granulosa cells were cultured with either T (10 ng/ml) or FSH (5.71 ng ovine FSH-20/ml) alone, or both FSH and T for 48 h. There was no statistically significant specific binding of 2-[ 125I]-iodomelatonin to GC membranes cultured with T or FSH alone. However, following a 48-h exposure to FSH and T in vitro specific 2-[ 125I]-iodomelatonin binding occurred with total 2-[ 125I]-iodomelatonin binding =5.5 fmol/mg protein. Therefore, the existence of hormonally-regulated expression of high-affinity melatonin binding sites suggests that melatonin may have an important intra-ovarian physiological role.

Hormone-induced receptor gene splicing: enhanced expression of the growth factor type I follicle-stimulating hormone receptor motif in the developing mouse ovary as a new paradigm in growth regulation.

The acquisition of FSH receptor(s) during follicular growth and their coupling to signaling pathways are key events in follicular development and dominance. However, little is known about the precise nature of the FSH receptor(s) involved in the growth-promoting phases of hormone action. To investigate the hormonal regulation of a newly discovered, alternatively spliced, growth factor type 1 receptor (designated FSH-R3) for the hormone, we examined expression in the adult mouse and the effect of PMSG treatment in the immature mouse ovary. Using RT-PCR and primers based on the established sheep ovarian transcript, a part of the FSH-R3 message was amplified only in wild-type (+/+), but not in the FSH-R knockout (-/-), mouse ovary. Semiquantitative RT-PCR using 3'-end primers specific for FSH-R1 (G(s)-coupled) and FSH-R3 indicated expression levels of the latter to be higher when follicular growth was induced by PMSG. Using FSH-R3-specific peptide IgG, FSH-R3 protein was detected by Western blotting in extracts of adult mouse ovary and was localized in granulosa cell membrane of mature follicles. In the immature mouse, levels of FSH-R3 protein that increased after PMSG administration in a time-dependent manner were also localized only on granulosa cell membranes of large follicles. The results reveal for the first time the expression of a different growth-promoting receptor for FSH in the developing and cycling mouse ovary. These observations introduce a new paradigm in the control of ovarian function.

Expression of regulator of G-protein signaling protein-2 gene in the rat ovary at the time of ovulation.

The ovulatory process in mammals begins when an endogenous surge in LH circulates to the ovary and couples with receptors in the plasma membranes of granulosa cells in mature ovarian follicles. This study provides evidence that the ovulatory stimulus includes induction of the gene for regulator of G-protein signaling protein-2 (RGS2). Immature Wistar rats were primed with 10 IU eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG (a homolog of LH) s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for reverse transcription-polymerase chain reaction differential display to detect gene expression in the stimulated ovarian tissue. Two of the amplified cDNAs that were upregulated within 2 h after the ovaries had been stimulated by hCG were homologous to segments of the mouse gene for RGS2. In situ hybridization indicated that the RGS2 mRNA was expressed in the granulosa layer of mature follicles. In conclusion, the gene for RGS2, which is known to regulate membrane signaling pathways, is transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin.