Protein In Granulosa Cells Research Articles

Rapid actions of progesterone on granulosa cells

Ovarian granulosa cells are responsive to progesterone but do not express the nuclear progesterone receptor. In an attempt to identify a receptor for progesterone (P4) in granulosa cells (GCs), an antibody built against the ligand binding site of the P4 receptor (i.e. C-262) was used. This antibody detected a 60 kDa protein in GCs as well as spontaneously immortalized granulosa cells (SIGCs). This C-262 detectable protein localizes to the plasma membrane and binds P4. Importantly, this C-262 detectable protein appears to be involved in mediating P4’s biological actions. This is based on the findings that C-262 1) blocks P4’s ability to inhibit mitogen-induced mitosis and apoptosis and 2) FITC-BSA-conjugated P4 binding to granulosa cells. A C-262 detectable protein was isolated using a C-262 affinity column and sequenced. This analysis identified an unnamed protein referred to as RDA288 that was found in the rat genome (Accession number: XM 216160). A nearly identical unnamed protein was found in a cDNA library of mouse lung (Accession number: AK004678). Whether RDA288 functions as a membrane receptor for P4 remains to be determined.

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.

Tumor necrosis factor-alpha induces serum amyloid A3 in mouse granulosa cells.

TNF-alpha has significant inhibitory effects on steroidogenesis and folliculogenesis and is associated with several inflammatory responses. Because ovulation is an inflammatory reaction, the effects of TNF on the family of acute-phase proteins in granulosa cells were investigated. Granulosa cells from immature mice at 28 d of age were cultured in the presence of 10 ng TNF/ml for 24 h. Serum amyloid A3 (SAA3), a main acute-phase protein, was induced by TNF in granulosa cells. The other isoforms of serum amyloid proteins SAA1, SAA2, and SAA4 were neither expressed in granulosa cells nor induced by TNF. TNF did not induce SAA3 mRNA in granulosa cells from TNF receptor type 1 (TNFR1) knockout mice, although SAA3 mRNA was induced within 3 h after TNF treatment in wild-type cells. Two SAA3 promoters, -617/+73 and -198/+73, were responsive to TNF and to p65, a component of the TNF signaling molecule nuclear factor (NF)-kappaB. The -106/+73 promoter of SAA3 lacking a NF-kappaB-like site was not responsive to TNF or p65. In granulosa cells from TNFR1 knockout mice, the SAA3 promoter (-198/+73) was responsive to transfection with the p65 component of NF-kappaB, but neither TNF treatment nor overexpression of the p50 component of NF-kappaB increased promoter activity. Similar results were observed in the murine ovarian granulosa tumor cell line (OV3121-1). Overexpression of the inhibitor of NF-kappaB (called IkappaB) blocked SAA3 promoter activity induced by TNF and by p65 in OV3121-1 cells. Closer analysis of deletion mutants of the SAA3 promoter revealed the necessity of a NF-kappaB like site for responsiveness to TNF in the OV3121-1 cells. TNF rapidly increased p65 in OV3121-1 nuclei when compared with controls not treated with TNF. TNF also increased phospho-IkB and SAA3 in whole-cell homogenates as determined by Western blots. Thus, TNF likely increased SAA3 promoter activity and protein by activating NF-kappaB signaling via TNFR1 in mouse granulosa cells. SAA3 is a novel gene in granulosa cells with yet unknown functions in the ovary.

Inhibition of small ubiquitin-related modifier-1 expression by luteinizing hormone receptor stimulation is linked to induction of progesterone receptor during ovulation in mouse granulosa cells.

The small ubiquitin-related modifier-1 (SUMO-1) is a member of a family of ubiquitin-related proteins that have effects on several important physiological functions, including reproduction. However, the regulation of SUMO-1 expression and functional distribution of SUMO-1 in vivo remain poorly understood. In the present study, we show that SUMO-1 protein is widely expressed in various tissues. In the ovary, the expression of SUMO-1 protein is suppressed around ovulation, in both the whole ovary and the granulosa cells, after gonadotropin treatment. Additionally, when the ovulatory signal, the endogenous LH surge, is blocked in vivo by pentobarbitone sodium, the expression of SUMO-1 protein in granulosa cells is increased. This effect is reversed when the missing endogenous LH surge is substituted by human chorionic gonadotropin treatment. Our findings provide the first evidence that inhibition of SUMO-1 expression is regulated by LH receptor stimulation in granulosa cells concomitant with ovulation in the mouse ovary. Furthermore, the levels of SUMO-1 protein are increased in granulosa cells treated with progesterone receptor (PR) antagonists both in vivo and in vitro, demonstrating that SUMO-1 expression is regulated by functional PR. SUMO-1 interacts with nuclear receptors in vitro, and LH receptor-mediated induction of PR is crucial for ovulation. SUMO-1 and PR are coexpressed and can be coprecipitated, providing additional evidence for a direct interaction between SUMO-1 and PR in periovulatory granulosa cells in vivo. These results suggest that a functional link between SUMO-1 and PR is of physiological importance for the local modulation of PR-mediated events in the ovary.

Differential expression of steroidogenic factor-1 and FTF/LRH-1 in the rodent ovary.

Steroidogenic factor-1 (SF-1) (NR5A1) is an orphan nuclear receptor that plays a premier role in ovarian organogenesis. Recent studies document mRNA expression of the structurally related factor NR5A2 (FTF, LRH-1, SF-2) in the adult ovary and more specifically in granulosa cells and luteal cells but not theca cells. Conversely, SF-1 was shown to be expressed at higher levels in theca/interstitial cells. These latter observations raised the possibility that FTF/LRH-1 may control target gene expression in granulosa cells of developing follicles. Using quantitative PCR our results show that FTF/LRH-1 message is expressed at higher levels in the ovary than in liver or other tissues analyzed. We show by in situ hybridization and LacZ expression in ovaries of transgenic mice bearing an FTF-promoter-LacZ fusion gene that FTF/LRH-1 is selectively expressed in granulosa cells of rat and mouse ovaries and is not present in theca cells or interstitial cells. However, by a variety of approaches, we showed that SF-1 mRNA and protein are expressed in greater amounts than FTF/LRH-1 in granulosa cells of follicles at all stages of development. Expression of SF-1 mRNA and protein in granulosa cells was verified by in situ hybridization, immunohistochemistry of ovarian sections, and immunocytochemistry of cultured rat granulosa cells. The significance of SF-1 in regulating target gene activation was supported by EMSA. An abundant granulosa cell protein binding to the SF-1-binding motif (CCAAGGTCA) present in the aromatase promoter and an FTF/LRH-1 motif (TGTCCTTGAACA) in the alpha-fetoprotein promoter was supershifted by two SF-1-specific antibodies but not by an FTF antibody. Conversely, with the same probes, a less abundant protein/DNA complex present in liver and ovarian cell extracts was shifted by an FTF antibody but not by the SF-1 antibodies. SF-1 and FTF/LRH-1 were differentially regulated in vivo by estradiol, FSH and prolactin. Collectively these data indicate that granulosa cells of small and preovulatory follicles express both SF-1 and FTF/LRH-1 and that each orphan receptor may regulate target gene expression in these cells.

Epidermal growth factor regulation of connexin 43 in cultured granulosa cells from preantral rabbit follicles.

Connexin 43 (Cx43), a gap junction protein expressed in differentiated granulosa cells, is necessary for normal follicular development. Cx43 expression and regulation by epidermal growth factor (EGF) were characterized in immature rabbit granulosa cells. Cx43 mRNA was expressed in the granulosa cells of primary follicles, but was undetectable in primordial follicles. Abundant expression of Cx43 mRNA was maintained in the granulosa cells of growing follicles through maturity. Granulosa cells were isolated from early preantral follicles and maintained in monolayer cultures for 72 hr. After the first 24 hr of culture, they were maintained for 48 hr in serum-free medium supplemented with 0, 1, 5, or 10 ng/ml of mouse EGF. Granulosa cell proteins were isolated, solubilized, and evaluated for Cx43 by Western blot analysis using antibodies to rat Cx43. Relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) were increased (P < 0.05) by EGF in a dose-dependent manner. Northern blot analysis of RNA from cultured granulosa cells demonstrated increased amounts of Cx43 mRNA in the EGF treated cultures (10 ng EGF/ml) relative to controls (P < 0.03). In summary, Cx43 gap junctions are synthesized in granulosa cells following the onset of folliculogenesis in vivo and their expression is enhanced by EGF in vitro.

Survivin as a cell cycle-related and antiapoptotic protein in granulosa cells.

Survivin is a relatively unique member of the inhibitor of apoptosis protein (IAP) family in that it contains a single baculovirus IAP repeat (BIR) domain combined with a COOH-terminal alpha-helix coiled-coil domain instead of the more common zinc-binding RING finger. Results from a variety of transformed or continuous mammalian cell lines suggest that, due to the combination of these features, Survivin is capable of regulating both cell proliferation and apoptotic cell death. However, to date there is essentially no information regarding Survivin expression, regulation or function within the ovary, or in any nonmammalian vertebrate species. In the present studies, cDNAs for chicken (ch) Survivin-142 (homologous to human Survivin-142) plus three alternatively spliced variants (ch Survivin-short, -gamma, and -delta) are described, and of these, transcripts for ch Survivin-142 and -short are expressed in granulosa cells from the hen ovary. Highest levels of Survivin mRNA during follicle development occur in mitotically active granulosa cells from undifferentiated, prehierarchal follicles. Cell cycle analysis determined that Survivin mRNA expression is elevated specifically during the G2/M phase of mitosis. Significantly, transient transfection with ch Survivin-142 in primary cultures of hen granulosa cells attenuates taxol- and N-octanoylsphingosine- (C8-ceramide-) induced caspase-3 activity, whereas overexpression of ch Survivin-short (a truncated variant that lacks much of the functional BIR domain plus the entire alpha-helix coil domain) lacks this antiapoptotic activity. Taken together, these data provide evidence for Survivin in granulosa cells acting as a bifunctional protein associated with regulation of the cell cycle and the inhibition of apoptosis.

Induced apoptosis and expression of related proteins in granulosa cells from women undergoing IVF: a preliminary study.

Because apoptosis may be involved in the outcome of IVF, the expression of pro- and anti-apoptosis proteins in a model of induced granulosa cell (GC) apoptosis was evaluated in 25 women with normal FSH levels undergoing IVF. After 1 day of culture, apoptosis was induced with interferon (IFN)-gamma (200 IU/ml), followed 24 h later by an agonistic anti-Fas antibody (0.5 microg/ml). On day 3, apoptotic GC, identified by chromatin condensation and/or nuclear fragmentation after DAPI staining, were counted among 1000 cells in randomly chosen fields under UV microscopy, and enabled allocation of women into two groups with either low (group 1) or high (group 2) percentages of apoptosis (11.6 +/- 4.8 and 59.5 +/- 14.8% respectively; P < 0.001). Immunoblotting was used to evaluate the following in proteins: poly (ADP-ribose) polymerase (PARP), caspases 8 and 3, Bcl-2, heat shock protein (HSP) 70, Bax, Bak and Stat-1 (a protein known to be inducible by IFN-gamma). Based on densitometric analysis of immunoblots, the PARP 116 kDa bands were respectively 4.3- and 33.3-fold lower for treated groups 1 and 2. Caspase 8, caspase 3 and HSP70 were expressed slightly less in treated group 2 than treated group 1. Densitometric analysis of bands corresponding to Bcl-2 showed respectively for treated groups 1 and 2, 3.2- and 2.5-fold decreases. Bak expression was similar in both control groups, and comparably lower in the two treated groups. With regard to Stat-1, densitometry showed 3.3- and 1.3-fold increases respectively in treated groups 1 and 2. These results suggested that Fas-mediated apoptosis of GC is accompanied by significant changes in proteins acting in apoptosis, and that this type of programmed cell death might play a potential prognostic role for women undergoing IVF.

Androgen receptor and follicle-stimulating hormone receptor in the pig ovary during the follicular phase of the estrous cycle.

Follicle-stimulating hormone (FSH) is an important regulator of follicular development. Some effects of FSH on ovarian follicles might be enhanced by androgens. The main objectives of the present study were to examine expression of the androgen receptor (AR) and FSH receptor (FSHR) in late developing follicles in pigs. Ovaries were collected from gilts on days 13, 15, 17, and 19 of the estrous cycle (day 0 = first day of estrus, n = 4 gilts/day), a period coincident with the follicular phase. One ovary was processed for immunohistochemistry (IHC) of AR. Samples of surface wall from the largest follicles (4-5 per gilt) were dissected from the other ovary, pooled and processed for determination of AR and FSHR mRNAs using reverse transcription-polymerase chain reaction (RT-PCR). Intense AR immunostaining was present in nuclei of granulosa cells of preantral and antral follicles. AR immunoreactivity was also present in the nuclei of oocytes. Weak staining for AR was observed in cells of the theca interna, ovarian surface epithelium, and in most cells of the ovarian stroma. Relative amounts of immunoreactive AR in granulosa cells of late developing follicles, or small antral follicles (< 2 mm), did not differ between days 13, 15, 17, and 19. However, amounts of AR in granulosa cells of small antral follicles was greater (P < 0.05) than in the largest follicles present in the same ovary. The relative amounts of AR mRNA in tissue from the largest follicles on days 13, 15, 17, and 19 did not differ; however, amounts of FSHR mRNA in the same follicles were not different between days 13, 15, and 17, but decreased (P < 0.05) by day 19. Results indicate that during the follicular phase in gilts, the AR protein is mainly present in granulosa cells. Relative amounts of AR protein in granulosa cells and mRNA in walls of late developing follicles did not significantly change from day 13 to 19; however, amounts of FSHR mRNA decreased in preovulatory follicles by day 19 of the estrous cycle.

Transforming growth factor β (TGF β) and activin stimulate nuclear translocation of Smad proteins in granulosa cells.

Objective: The TGFβ family of growth factors has long been thought to play a role in ovarian follicle development, however until recently the mechanisms of action of these growth factors have been unknown. A group of proteins that functions as intracellular mediators of TGFβ family signaling has recently been identified and named Smad. Though there are 9 Smads so far, two of the receptor-specific smads: Smad2 and Smad3 are both associated with both TGFβ and activin signaling. Receptor complexes that have bound ligand can activate receptor-specific Smads by phosphorylation. Phosphorylated Smads translocate to the nucleus where they modulate transcription. In this study we evaluated the capacity of TGFβ and activin to induce nuclear translocation of Smad2 and Smad3 in cultured granulosa cells.

Human estrogen receptor beta-specific monoclonal antibodies: characterization and use in studies of estrogen receptor beta protein expression in reproductive tissues

Investigation of the role of the second, more recently described estrogen receptor, denoted ERβ, will be critical in understanding the molecular mechanisms underlying tissue-specific gene regulation by estrogens. Expression of ERβ in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization and size of the endogenous ERβ protein, due, in part, to the limited availability of human ERβ-specific antibodies. Thus, our aim was to generate specific antibodies to human ERβ and use them to determine the tissue-specific distribution and size(s) of the ERβ protein. To this end, we have cloned three different hybridoma cell lines that produce monoclonal antibodies specific for the hormone-binding domain of human ERβ. The antibodies, made in mice against human ERβ amino acids 256–505 (hormone binding domain lacking the F domain), are designated CFK-E12 (E12), CMK-A9 (A9) and CWK-F12 (F12) and were determined to be the IgG γ1 isotype for E12, and IgG γ2b for A9 and F12. All three monoclonal antibodies could be used to detect in vitro translated, baculovirus expressed, and cell transfected and expressed ERβ protein by Western blot analyses, and all failed to detect ERα. A9 and F12 were able to immunoprecipitate efficiently the native form of ERβ protein in the presence and absence of estradiol. Epitope mapping studies indicate that the E12 and F12 antibodies recognize overlapping peptide sequences in the N-terminal region of the hormone-binding domain, a region that is highly conserved among species. Immunocytochemical studies with these antibodies reveal nuclear-specific localization of the ERβ protein in granulosa cells of the rat ovary. Nuclear ERβ is also specifically localized in epithelial and some stromal cells of mouse and rat epididymis. Western blot analysis with protein extracts from ovarian granulosa cells of human, rat, mouse, and pig showed a ca. 52 kDa and an additional ca. 62–64 kDa band in these species. These results indicate the presence of two predominant molecular size forms of the ERβ protein in ovarian granulosa cells and demonstrate the utility of these antibodies for detection of ERβ in the human and in several other mammalian species.

Characterization of Estrogen Receptor- (ER ) Messenger Ribonucleic Acid and Protein Expression in Rat Granulosa Cells

We have examined estrogen-responsiveness of ovarian granulosa cells by focusing on estrogen receptor (ER) expression. Estrogen responsiveness was determined by examining the effect of 17β-estradiol (1–10 nm) on luciferase reporter activity in rat granulosa cells transfected with an ERE-luciferase construct. The results demonstrate an estrogen-induced (approximately 3-fold) increase in luciferase reporter activity, indicating that granulosa cells contain functional estrogen response element (ERE)-binding transcriptional activators. Gel mobility shift assays in combination with ER antibodies show that ERβ is the predominant ERE-binding protein in granulosa cells. Western blotting results show that granulosa cells contain ERβ-immunoreactive protein(s) migrating at a size substantially larger than the recombinant protein generated from the originally proposed 485 amino acid open-reading frame. This size discrepancy is not due to granulosa cell expression of ERβ isoforms with insertions within the coding region because RT-PCR assays revealed products with sizes expected for ERβ, ERβB, and δ3 isoforms. This size discrepancy appears to be due to usage of a well-conserved, upstream in-frame translation initiation codon (ATG436) leading to a 530 amino acid open reading frame. ERβ messenger RNA (mRNA) characterization using 5′-rapid amplification of complementary DNA ends (5′-RACE) show the presence of two different (P1- and P2-) 5′-ends of rat ERβ mRNA encoding the full-length ERβ protein. The generation of the P2-specific exon is likely due to initiation of transcription from an alternative promoter. Both P1- and P2-specific exon-containing ERβ mRNAs are expressed in granulosa cells, and they are rapidly down-regulated by the cAMP-mediated intracellular signaling pathway in cultured granulosa cells. Taken together, our results show that rat granulosa cells produce two different 3′,5′-cAMP-regulated ERβ mRNA species and that these mRNA species are capable of encoding the full-length ERβ protein.

Characterization of estrogen receptor-beta (ERbeta) messenger ribonucleic acid and protein expression in rat granulosa cells.

We have examined estrogen-responsiveness of ovarian granulosa cells by focusing on estrogen receptor (ER) expression. Estrogen responsiveness was determined by examining the effect of 17beta-estradiol (1-10 nM) on luciferase reporter activity in rat granulosa cells transfected with an ERE-luciferase construct. The results demonstrate an estrogen-induced (approximately 3-fold) increase in luciferase reporter activity, indicating that granulosa cells contain functional estrogen response element (ERE)-binding transcriptional activators. Gel mobility shift assays in combination with ER antibodies show that ERbeta is the predominant ERE-binding protein in granulosa cells. Western blotting results show that granulosa cells contain ERbeta-immunoreactive protein(s) migrating at a size substantially larger than the recombinant protein generated from the originally proposed 485 amino acid open-reading frame. This size discrepancy is not due to granulosa cell expression of ERbeta isoforms with insertions within the coding region because RT-PCR assays revealed products with sizes expected for ERbeta, ERbetaB, and delta3 isoforms. This size discrepancy appears to be due to usage of a well-conserved, upstream in-frame translation initiation codon (ATG436) leading to a 530 amino acid open reading frame. ERbeta messenger RNA (mRNA) characterization using 5'-rapid amplification of complementary DNA ends (5'-RACE) show the presence of two different (P1- and P2-) 5'-ends of rat ERbeta mRNA encoding the full-length ERbeta protein. The generation of the P2-specific exon is likely due to initiation of transcription from an alternative promoter. Both P1- and P2-specific exon-containing ERbeta mRNAs are expressed in granulosa cells, and they are rapidly down-regulated by the cAMP-mediated intracellular signaling pathway in cultured granulosa cells. Taken together, our results show that rat granulosa cells produce two different 3',5'-cAMP-regulated ERbeta mRNA species and that these mRNA species are capable of encoding the full-length ERbeta protein.

Mechanisms of insulin-like growth factor I augmentation of follicle-stimulating hormone-induced porcine steroidogenic acute regulatory protein gene promoter activity in granulosa cells.

Insulin-like growth factor I (IGF-I) and the gonadotropin, FSH, can synergize to stimulate progesterone production in primary cultures of maturing human, rat, and pig granulosa cells. These trophic hormones act by increasing the activity and production of proteins and their gene transcripts essential to sterol uptake, delivery, and utilization in steroidogenesis. We previously observed that FSH and IGF-I interact synergistically to promote the accumulation of steroidogenic acute regulatory protein (StAR) messenger RNA and protein in granulosa cells. Here we investigate potential mechanisms of IGF-I synergy with FSH and the protein kinase A (PKA) pathway in activating the porcine StAR gene promoter. To this end, we first cloned 1423 bp of the porcine StAR promoter upstream of the transcriptional start site using PCR and created 5'-deletional constructs coupled to a cytoplasmically targeted firefly luciferase reporter gene. FSH, 8-bromo-cAMP, and transient transfection of the protein kinase A (PKA) catalytic subunit (driven by the Rous sarcoma virus promoter) were used to activate the PKA effector pathway. All three agonists alone stimulated StAR promoter-driven luciferase activity in primary cultures of granulosa cells after 4-h treatment. IGF-I significantly augmented PKA pathway agonist activation of the StAR promoter, whereas IGF-I had no effect alone. Binding experiments with 125I-labeled ovine FSH-20 in IGF-I (100 ng/ml)-treated granulosa cells showed that FSH binding affinity and receptor number were unchanged by IGF-I treatment. However, IGF-I augmented FSH-stimulated, but not forskolin-stimulated, cAMP accumulation. Analysis of 5'-deletion constructs of the StAR promoter revealed three regions of stimulatory activity within the -139-bp fragment upstream of the transcriptional start site as well as another potentially inhibitory region upstream (-1115 to 905). Elimination of the putative SF-1 site (-48 to -41) virtually abolished StAR promoter responsiveness. In summary, our data indicate that IGF-I can act via two post FSH-binding mechanisms to augment FSH/PKA pathway-mediated StAR gene promoter transactivation: at the level of cAMP accumulation and distal to cAMP production and PKA activation.

A high-molecular-weight preovulatory stage-related protein in equine follicular fluid and granulosa cells.

The high-molecular-weight proteins of equine follicular fluid were examined to determine whether some polypeptides are unique to certain physiological conditions. Fluids from ovarian follicles of various diameters and physiological stages during the follicular phase were recovered by ultrasound-guided follicular aspiration. Granulosa cells and cumulus-oocyte complexes (COC) were recovered by scraping the intrafollicular wall during puncture. Follicular fluids and corresponding serum, as well as granulosa cell lysates, were analyzed by one-dimensional SDS-PAGE and silver staining. COC morphology was assessed microscopically. A 200-kDa protein band was demonstrated in fluids from preovulatory follicles, in natural conditions or after induction of ovulation. This protein band was absent in fluids from follicles at earlier stages, subordinate follicles, and serum. The presence of this protein at the preovulatory (PO) stage was ascertained through recovery of the fluid from follicles twice during their growth. Its appearance was time dependent after induction of ovulation but was not induced by an intrafollicular injection of a physiological dose of progesterone. We also demonstrated the presence of this 200-kDa protein in granulosa cells lysates recovered from preovulatory follicles. The expression of this protein in the follicular fluid was related to the cumulus aspect and chromatin configuration of the enclosed COC. No relation was found between its presence in the follicular fluid at the PO stage and subsequent ovulation of the punctured follicle or embryo production. The identification of this molecule is approached and discussed. These results show a novel PO stage-related protein in equine follicular fluid, which may be involved in the differentiation and maturation mechanisms occurring in the follicle during the preovulatory period.

Murine oocytes suppress expression of luteinizing hormone receptor messenger ribonucleic acid by granulosa cells.

This study tested the hypothesis that murine oocytes participate in the establishment of granulosa cell phenotypic heterogeneity in preovulatory follicles. In these follicles, mural granulosa cells express LH receptors (LHR) and LHR mRNA, but expression of these molecules is low or undetectable in cumulus cells. Thus, the expression of LHR mRNA is a marker of the mural granulosa cell phenotype in preovulatory follicles. Cumulus cells expressed elevated steady-state levels of LHR mRNA when oocytes were microsurgically removed from oocyte-cumulus cell complexes, and this was prevented by paracrine factor(s) secreted by isolated oocytes. These factors also suppressed FSH-induced elevation of the level of LHR mRNA expression by mural granulosa cells isolated from small antral follicles, even when expression was augmented by culturing granulosa cells on components of basal lamina. Moreover, factor(s) secreted by oocytes suppressed the expression of LHR mRNA in mural granulosa cells isolated from preovulatory follicles already expressing elevated levels of these transcripts. The ability of oocytes to suppress the LHR mRNA expression by granulosa cells was developmentally regulated. Oocytes from preantral follicles and mature (metaphase II arrested) oocytes were less effective in suppressing expression than fully grown, germinal vesicle (GV)-stage oocytes. Furthermore, two-cell-stage embryos did not suppress LHR mRNA levels. Coculture of isolated oocytes with granulosa cells affected the synthesis of very few granulosa cell proteins detected by fluorography of two-dimensional gels after 35S-methionine labeling. Thus, oocyte suppression of FSH-induced LHR mRNA expression is specific in both the suppressing cell type and the effects on granulosa cells. It is concluded that the default pathway of granulosa cell differentiation produces the mural granulosa cell phenotype, as represented by the expression of LHR mRNA. This pathway is abrogated by oocytes. Thus, oocytes play a dominant role in establishing the fundamental heterogeneity of the granulosa cell population of preovulatory follicles.

Gonadotropins induce rapid phosphorylation of the 3',5'-cyclic adenosine monophosphate response element binding protein in ovarian granulosa cells.

The pituitary gonadotropins FSH and LH exert their effects on gonadal target cells, at least in part, through the activation of adenylyl cyclase and the production of the second messenger cAMP. To elucidate further the signal transduction pathways regulating gonadotropin-responsive genes in ovarian granulosa cells, we have investigated the expression of the cAMP response element binding protein (CREB), which mediates many of the effects of cAMP by modulating the transcription of target genes in a cAMP-dependent fashion. In situ hybridization, RNA blot analysis and RT-PCR RNA quantification demonstrated that CREB messenger RNA (mRNA) is expressed at low levels throughout the ovary, and that CREB mRNA levels do not change appreciably after gonadotropin stimulation. Similar results were obtained using immunohistochemistry and Western protein blotting to examine CREB protein in ovaries isolated from immature animals treated with gonadotropins or immunocytochemistry and Western protein blotting to examine the CREB protein in cultured granulosa cells after gonadotropin treatment. In contrast, immunocytochemistry and Western protein blotting using an antipeptide antibody specific to CREB phosphorylated at serine 133 (P-CREB), which is the activated from of the CREB protein, revealed a dramatic increase in the phosphorylated form of CREB within 20 min of gonadotropin treatment of granulosa cells that was transient and was decreased by 60 min after gonadotropin treatment. Stimulation of P-CREB was observed using granulosa cells isolated from immature animals and treated with recombinant human FSH in vitro, or using granulosa cells isolated from immature animals primed with PMSG in vivo and treated with human CG (hCG) in vitro. Stimulation of P-CREB was also observed in ovarian granulosa cells isolated from animals treated with PMSG in vivo. These results indicate that both gonadotropins can induce a rapid and transient phosphorylation of the CREB protein in granulosa cells, leading to the activation of a factor likely to play an important role in the transcription of many gonadotropin-regulated ovarian genes.

Thyroid hormone induces the synthesis of a putative protein in the rat granulosa cell which stimulates progesterone release.

125I-3,5,3'-tri-iodothyronine (T3) binds specifically to a pure nuclear preparation from rat granulosa cells. A Scatchard analysis of T3 binding showed a Kd of 0.65 x 10(-9) mol/l and a Bmax of 1.57 pmol/mg DNA. The biological relevance of T3 binding to granulosa cells was evaluated by adding T3 (20 ng/incubation) to granulosa cells (1 x 10(6) cells/incubation) which greatly stimulated progesterone release. T3-stimulated progesterone release was significantly inhibited by actinomycin-D (P < 0.01) and cycloheximide (P < 0.01). T3 caused about a twofold increase in granulosa cell protein synthesis as compared with the control which was inhibited by actinomycin-D and cycloheximide. The addition of T3 to granulosa cell incubations also resulted in a more than 2.5-fold increase in mRNA. The results indicated that T3 stimulation of progesterone release is mediated via T3-induced protein(s) or TIP. TIP was located in the soluble supernatant fraction (100,000 g supernatant; 100 k sup) from T3-incubated cells but could not be detected in the 100 k sup from the control cells or LH-incubated cells. TIP was purified based on its biological activity, i.e. its addition to granulosa cell incubations stimulated progesterone release into the medium. The 100 k sup from T3-incubated granulosa cells was subjected to Sephadex G-75 gel filtration, FPLC Mono Q and FPLC Superose 6 chromatography which resulted a 273-fold purification over the starting material and a clearly homogeneous protein was obtained. SDS-PAGE of purified TIP showed it to be a 53 kDa monomer protein. Experiments conducted with radiolabelled TIP suggested internalization of TIP into the granulosa cell. The results therefore showed that T3 induces the synthesis of mRNA and proteins in rat granulosa cells and that one of the proteins is TIP which, in turn, stimulates progesterone release from the cell, suggesting thereby that this putative protein is a novel mediator of T3 function in the granulosa cell.

G protein expression and second messenger formation in human granulosa cells.

The expression of heterotrimeric (alpha beta gamma subunits) GTP-binding regulatory proteins (G proteins) and the activation of G protein-linked receptors in human granulosa cells were investigated. The cells were obtained from stimulated follicles in women undergoing in vitro fertilization and were cultured in serum-supplemented medium. Immunoblotting with specific antibodies showed that granulosa cell membranes express alpha s, alpha i3 alpha i1,2, alpha q,11 and beta subunits. Three antibodies against alpha o failed to detect this protein. The cells responded to hCG and to prostaglandin E2 with a dose-dependent increase in cAMP formation, confirming the functional activation of G alpha s. The alpha 2 adrenoceptor agonist, clonidine, inhibited hCG-stimulated cAMP formation and this effect was blocked with pertussis toxin, thus involving a Gi-type protein, most likely G alpha i2. Oxytocin provoked an increase in formation of inositol phosphates and intracellular calcium concentration, which was partly pertussis toxin resistant, providing evidence of G alpha q,11 activation. However, a significant component of the response to oxytocin could be blocked by pertussis toxin, indicating Gi-mediated phospholipase C activation (by either alpha i or beta gamma subunits). These data demonstrate the presence of G proteins in granulosa cells and suggest a complex regulation of hormonal signalling. The concentration of cAMP in these cells depended on the balance of G alpha s:G alpha i activation, whereas activation of the inositol phospholipid pathway and rises in intracellular calcium involved both Gq,11 and Gi pathways.

Steroidogenic factor-1 binding and transcriptional activity of the cholesterol side-chain cleavage promoter in rat granulosa cells.

The cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene is expressed in rat ovarian follicles in response to gonadotropins (FSH/LH) and cAMP. To identify functional regions within the rat P450scc promoter, 894 basepairs (bp) of 5'-flanking sequence and 5'-deletions (at -379, -101, -73, and -38 bp) were linked to the human GH reporter gene and transfected into cultured rat granulosa cells. cAMP inducibility of the rat promoter was localized to a region (between -73/-38 bp) that contains one of two AGGT/CC/TA motifs, designated SCC1 (-51/-43 bp) and SCC2 (-79/-71 bp), within the rat promoter. One of the nuclear proteins in granulosa cells that binds to SCC1 was identified as the orphan receptor, steroidogenic factor-1 (SF-1). In contrast, multiple protein-DNA complexes formed with SCC2, only one of which was clearly identified as SF-1. Nuclear extract binding was sequence specific; SCC1 bound SF-1 more strongly than did SCC2. Thus, the two AGGT/CC/TA motifs of the rat promoter appear to differ structurally and functionally. Furthermore, because the expression of SF-1 mRNA precedes hormonal/cAMP induction of P450scc mRNA and is not regulated in vitro by cAMP, the functional role of SF-1 in transcriptional regulation of the P450scc gene, including its induction by cAMP, is not entirely clear and is probably dependent on other factors and/or the modification (phosphorylation?) of SF-1.