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B C Vanderhyden

Publications and source records attributed to B C Vanderhyden.

At least 19 recordsLinked to original sources
PubMed2003

Evaluation of members of the TGFbeta superfamily as candidates for the oocyte factors that control mouse cumulus expansion and steroidogenesis.

Oocytes secrete factors that control cumulus and granulosa functions, including cumulus expansion and steroid hormone production. Some members of the transforming growth factor beta (TGFbeta) superfamily influence these activities, yet it is still not determined conclusively whether any of these superfamily members are the previously reported oocyte-secreted factors. The aim of this study was to examine the effects of TGFbeta1 and growth differentiation factor 9 (GDF-9) on cumulus expansion and progesterone production by mouse oocytectomized (OOX) complexes in culture. TGFbeta1 mimics the effects of oocytes by both enabling cumulus expansion and inhibiting progesterone production; however, neutralizing antibodies to TGFbeta1 in cultures of cumulus-oocyte complexes (COCs) or in co-cultures of OOX complexes failed to inhibit the ability of oocytes to enable cumulus expansion or inhibit progesterone production. Activin A had no effect on progesterone production by OOX complexes. In experiments using oocytes obtained from mice with deficient expression of GDF-9, OOX complexes cultured in the presence of heterozygous oocytes were capable of full expansion, whereas OOX complexes cultured with oocytes from GDF-9 null mice did not expand. Similarly, GDF-9 null oocytes failed to suppress FSH-induced progesterone production by OOX complexes. These results support the hypothesis that GDF-9 is the cumulus expansion enabling factor produced by mouse oocytes and that GDF-9 also inhibits cumulus progesterone production; however, the possibility remains that loss of GDF-9 may indirectly affect the ability of oocytes to produce the factors that regulate cumulus cell activity.

Activins↗
PubMed2001

Analysis of ubiquitination in vivo using a transgenic mouse model.

The primary pathway for the proteolytic destruction of cellular proteins is through ubiquitin-mediated targeting to the proteasome. This pathway is pivotal not only in the elimination of damaged or misfolded proteins but also in the temporal, developmental, or signal-mediated destruction of normal cellular substrates. The list of known substrates of the ubiquitin/proteasome pathway is long, but most substrates have been identified in yeast or, more recently, in cultured mammalian cells. It is likely that many mammalian substrates with developmental or disease relevance have yet to be identified because their ubiquitination occurs in tissue or organ systems that cannot be adequately modeled in vitro. We have developed a transgenic mouse model that will allow the isolation and identification of these substrates. The human UbC promoter was used to drive expression of a hexahistidine-tagged version of human ubiquitin in a variety of mouse tissues from early embryonic stages, as assessed by a green fluorescent protein marker. Cleavage of the fusion protein by endogenous enzymes produced epitope-tagged ubiquitin that was detected both in monomeric form and conjugated to cellular proteins. This mouse model should facilitate in the analysis of normal and disease-related ubiquitination events in vivo.

Animals↗
PubMed2000

Lack of expression of c-KIT in ovarian cancers is associated with poor prognosis.

The c-KIT protooncogene encodes a tyrosine kinase receptor, KIT, that is expressed in many normal and cancerous tissues. In this study, we have examined the expression of c-KIT and its ligand, stem cell factor (SCF), in human epithelial ovarian tumors, in normal ovaries and in cultured ovarian surface epithelium (OSE). Cultured cells, normal tissues and tumors were analyzed by Northern and Western blot analyses, reverse transcription-polymerase chain reaction and immunohistochemistry. Normal OSE expressed SCF, but not c-KIT; however, epithelial invaginations and inclusion cysts often expressed KIT protein. Of 15 benign ovarian tumors and tumors of low malignant potential, 87% expressed c-KIT, and 92% of these co-expressed SCF, suggesting the possibility of autocrine growth regulation. Of 35 malignant ovarian cancers, 71% expressed c-KIT (92% co-expressed SCF), with a trend for decreased c-KIT expression in advanced stage disease. Of 34 patients with malignant tumors for whom follow-up information was available (median follow-up time of 24 months), 9 had tumors that did not express c-KIT, 8 (89%) of whom have died and the remaining 1 has recurrent disease. Of the 25 patients with tumors expressing c-KIT, 56% are still alive. Eight of the patients have no evidence of disease and all had KIT-expressing tumors. Statistical analysis indicated that patients whose tumors did not express c-KIT had a significantly shorter (p < 0.05) disease-free survival time than patients who had KIT-expressing tumors. Our results suggest that c-KIT may play a role in early ovarian tumorigenesis, and that loss of c-KIT expression is associated with poor prognosis.

Blotting, Northern↗
PubMed2000

Inhibition by human embryos of mouse granulosa cell progesterone production: development of a sensitive bioassay.

Reproduction technologies could be improved by the development of methods to evaluate oocyte or embryo quality in a non-invasive, quantitative manner. Since human embryos secrete a factor that inhibits granulosa cell progesterone production, an interspecies bioassay was established to investigate whether the presence of this progesterone-inhibitory factor (PIF) in human embryo-conditioned (HEC) media is related to the health and developmental capacity of the embryos. Oocytes were microsurgically removed from oocyte-cumulus complexes isolated from superovulated mouse ovaries, and the oocytectomized complexes were cultured in HEC media in the presence of follicle stimulating hormone and testosterone. Progesterone accumulation in the media was determined by radioimmunoassay. Despite the potential limitations of very small volumes of HEC media to evaluate, and the need to freeze these media at the source, the bioassay was able to detect PIF activity in HEC media. Most embryos produced PIF activity, but the degree of inhibition was not correlated with the ability of oocytes to be fertilized, nor with embryo morphology or ability to cleave and develop after transfer. These results demonstrate that secretion of PIF by human embryos can be measured by this bioassay and that human PIF can inhibit murine granulosa cell steroidogenesis; however, PIF activity is not correlated with human embryo quality or developmental competence.

Animals↗
PubMed2000

Secretion of paracrine factors enabling expansion of cumulus cells is developmentally regulated in pig oocytes.

To demonstrate secretion of cumulus expansion-enabling factor (CEEF) by porcine oocytes, we used an interspecies testing system. Porcine oocytes were used to condition culture medium, and the presence of CEEF was tested using mouse oocytectomized complexes (OOX), which require CEEF for expansion. Follicle-stimulating hormone-stimulated expansion and synthesis of hyaluronic acid (HA) by mouse OOX were assessed after 18 h of culture in media conditioned by porcine oocytes: 1) at different stages of maturation and 2) in which maturation was inhibited with a specific inhibitor of cdk-kinases, butyrolactone I. Fully grown (GV-germinal vesicle), late-diakinesis (LD), metaphase I (MI), and metaphase II (MII) oocytes were prepared by culture of oocyte-cumulus complexes (OCC) for 0, 22, 27, and 42 h, respectively. To block GV breakdown, porcine oocytes were cultured for 27 h in medium supplemented with butyrolactone I (50 microM). Medium conditioned by oocytes in GV, LD, and after butyrolactone I block allowed full expansion of >90% of mouse OOX, whereas oocytes in MI and MII caused disintegration of mouse OOX without cumulus mucification. To measure synthesis of HA by cumulus cells, 25 mouse OOX were cultured in the conditioned media in the presence of 2.5 microCi of D-[6-(3)H]glucosamine hydrochloride. After 18 h, incorporation of the [(3)H]glucosamine into HA was determined either in complexes (retained HA) or in medium plus complexes (total HA). Total HA accumulation by mouse OOX was not different from that of intact OCC. However, oocytes in GV, LD, and after butyrolactone I treatment enabled mouse OOX to retain significantly more HA within the complex than oocytes in MI and MII. The results indicate that secretion of factors that promote the retention of HA within the complex is developmentally regulated during oocyte maturation.

4-Butyrolactone↗
PubMed1999

Transforming growth factor-beta regulates Kit ligand expression in rat ovarian surface epithelial cells.

In preparation for ovulation, paracrine communication between the preovulatory follicle and overlying theca/stromal cells and ovarian surface epithelium (OSE) must take place to facilitate the degradative and apoptotic events associated with ovulation. Kit tyrosine kinase receptors and their ligand, kit ligand (KL) are expressed within ovarian follicles, and ligand-induced receptor activation appears to account for some of the cell - cell interactions important for oocyte development. We investigated the expression of Kit receptors and KL in OSE cells and the possibility that modulation of their expression could affect OSE cell activity. KL mRNA and protein were detected in the OSE cell layer of rat ovaries, and primary cultures of rat OSE as well as the immortalized rat OSE cell line, ROSE 199, expressed KL, but not Kit receptors. Both primary and immortalized OSE cells preferentially expressed KL-1, rather than KL-2, transcripts, suggesting that these cells produce predominantly the soluble form of KL. Activation of the cAMP signalling pathway using dibutyryl cAMP decreased proliferation of ROSE 199 cells and elicited a threefold increase in KL expression. TGF-beta similarly inhibited ROSE 199 cell proliferation, but strongly inhibited dibutyryl cAMP-induced KL expression, indicating that changes in KL expression were not directly associated with OSE cell proliferation. The expression of mostly soluble KL in the surface epithelium suggests that this cytokine may be acting in a paracrine fashion, perhaps interacting with nearby Kit receptor-bearing theca cells.

Animals↗
PubMed1999

Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR.

The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the amino-terminal targeted Pkr mouse, tumor necrosis factor alpha-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targeted Pkr-null cells. The observation of intact eukaryotic initiation factor-2alpha phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2alpha kinase(s).

Animals↗
PubMed1999

Oocytectomy does not influence synthesis of hyaluronic acid by pig cumulus cells: retention of hyaluronic acid after insulin-like growth factor-I treatment in serum-free medium.

Mouse oocytes secrete a factor that enables cumulus cells to undergo expansion in response to FSH (1 microg/ml), whereas expansion of the porcine cumulus oophorus has been shown to be independent of the oocyte. The aim of this study was to assess FSH-induced synthesis of hyaluronic acid (HA) by porcine cumulus cells before and after oocytectomy. In addition, we studied the effect of insulin-like growth factor-I (IGF-I) on the ability of cumulus cells to synthesize and retain HA in response to FSH in serum-free medium. Porcine oocyte-cumulus complexes and complexes from which the oocytes had been removed by oocytectomy were cultured for 24 h in the presence of 2.5 microCi of D-[6-(3)H]glucosamine hydrochloride, fetal calf serum (FCS, 5%), and FSH. After 24 h, incorporation of [(3)H]glucosamine into HA was measured either in complexes alone (retained HA) or in medium plus complexes (total HA). Specificity of incorporation of radioactivity into HA was confirmed by the sensitivity to highly specific Streptomyces hyaluronidase. Our results suggest that 1) the synthesis of HA by pig cumulus cells in vitro is stimulated by FSH and that oocytectomy does not change this synthesis; 2) oocytes do not influence retention of HA within the complex; 3) FSH-induced synthesis of HA by cumulus cells is decreased in medium with polyvinylpyrrolidone (PVP)-supplemented (total and retained HA) compared to FCS-supplemented medium; 4) IGF-I enabled cumulus cells to synthesize HA in response to FSH in PVP-supplemented medium in a manner similar to that observed when serum is present in the medium.

Animals↗
PubMed1998

Mouse oocytes regulate granulosa cell steroidogenesis throughout follicular development.

Mouse oocytes secrete a factor(s) that inhibits progesterone and enhances estradiol production by granulosa cells. This study determined the ability of mouse oocytes to secrete this steroid-regulating factor during oocyte growth and the ability of granulosa cells to respond to the factor during follicular development. Oocyte-granulosa cell complexes from preantral and antral follicles were oocytectomized (OOX; oocytes microsurgically removed) and cultured for up to 48 h with FSH (150 ng/ml) and testosterone (500 nM). At all stages of development examined, OOX complexes produced more progesterone than did intact complexes, from 1.45-fold for early growing follicles to 23-fold for complexes from antral follicles. Significant estradiol production was restricted to intact complexes from late antral follicles. Progesterone accumulation by OOX complexes cocultured with oocytes was inhibited by all stages of oocytes examined, with maximal inhibition by fully grown oocytes. Ovulated complexes produced large quantities of progesterone, even though oocytes secreted progesterone-inhibitory factor, because of a desensitization of cumulus cells to the factor during their terminal differentiation. Even in the presence of abundant pregnenolone, OOX complexes showed reduced ability to produce and/or accumulate progesterone in the presence of oocytes, suggesting that the oocyte-secreted factor, either directly or indirectly, regulates the activity of 3beta-hydroxysteroid dehydrogenase and/or progesterone metabolism. These results demonstrate that oocytes secrete a factor with steroid-regulating activity in increasing amounts and/or potency during follicular development, but responsiveness of cumulus cells to this factor declines during luteinization.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Hormonally regulated expression and alternative splicing of kit ligand may regulate kit-induced inhibition of meiosis in rat oocytes.

Mutations in the genes encoding the Kit tyrosine kinase receptor or kit ligand (KL) cause numerous phenotypic defects, including sterility. In the postnatal ovary, Kit is expressed on the oocyte surface and KL is produced by the surrounding granulosa cells, but its function in these cells is still unknown. The purpose of this study was to determine the role KL/Kit interactions play in the regulation of oocyte meiosis. Here, we demonstrate that meiotically arrested rat oocytes that are microinjected with Kit antisense oligonucleotides have decreased Kit expression. This decreased expression is associated with an increased ability of these oocytes to resume meiosis compared with those microinjected with missense oligonucleotides or buffer alone. In addition, oocytes cultured in the presence of KL were delayed in their resumption of meiosis, but KL could not enhance the meiosis inhibitory effects of dibutyryl cAMP, suggesting that KL operates through a mechanism that is independent of cAMP. Human chorionic gonadotropin-induced meiotic resumption in oocytes was accompanied by a shift in follicular granulosa cell KL expression from membrane-bound to soluble forms and a loss of expression of both forms of KL in cumulus cells. Thus, KL-activated Kit inhibits meiotic progression, and the in vivo luteinizing hormone-stimulated resumption of meiosis may negate Kit activity by a localized decrease in KL expression and by altering the form of KL produced within the follicle.

Alternative Splicing↗
PubMed1997

Male infertility caused by epididymal dysfunction in transgenic mice expressing a dominant negative mutation of retinoic acid receptor alpha 1.

Retinoids are thought to be required for the normal development and maturation of a number of tissues, including most epithelia. The action of retinoids appears to be mediated through the binding to retinoic acid receptors (RARs) in the nucleus. The activity of retinoic acid can be inhibited in cells carrying dominant negative mutations of RAR alpha. We created transgenic mice expressing a dominant negative mutant of RAR alpha driven by the murine mammary tumor virus promoter. Expression of the transgene was evident in the epididymis and vas deferens in transgenic males. These males were either infertile or had reduced fertility, and the epithelium lining the ducts of the epididymis and vas deferens had undergone squamous metaplasia. Sperm developed normally in the testis but degenerated in the epididymis and vas deferens because inspissated ductal fluid blocked the normal passage of the sperm.

Animals↗
PubMed1997

Interactions between the oocyte and cumulus cells in the ovary of the B6.Y(TIR) sex-reversed female mouse.

The XY (B6.Y(TIR)) sex-reversed female mouse is infertile, primarily because of the early death of its embryos. We have previously determined that the XY oocyte itself, not the surrounding somatic cells, is responsible for its failure in postfertilization development. In the present study, we assessed the ability of the XY oocyte to regulate granulosa cell differentiation and functions. Oocyte-cumulus complexes (OCC) were isolated from antral follicles and were cultured in the presence of FSH and testosterone. Microsurgical removal of oocytes prevented cumulus cell expansion and suppressed estradiol production while it promoted progesterone production. Coculture with denuded oocytes from either XX or XY ovaries restored cumulus expansion and the endocrine profile observed in intact OCC. Morphology of oocytes and OCC in the preantral and antral follicles in situ as well as after isolation was compared for XX and XY ovaries. The average area of XY oocytes was smaller by 20% only at the preantral stage, whereas the zona pellucida layer was thinner by 20% at all stages. Furthermore, the XY oocyte was found to be attached to fewer cumulus cells (60% of XX control) in antral follicles and isolated OCC. In conclusion, the XY oocyte develops the normal ability of regulating granulosa cell differentiation despite its inferiority with respect to some morphometric parameters when compared to the XX oocyte.

Animals↗
PubMed1997

Proviral inactivation of the Npat gene of Mpv 20 mice results in early embryonic arrest.

The Mpv 20 transgenic mouse strain was created by infection of embryos with a defective retrovirus. When Mpv 20 heterozygous animals were crossed, no homozygous neonatal mice or midgestation embryos were identified. When embryos from heterozygous crosses were cultured in vitro, approximately one quarter arrested as uncompacted eight-cell embryos, indicating that proviral insertion resulted in a recessive lethal defect whose phenotype was manifest very early in development. Molecular cloning of the Mpv 20 insertion site revealed that the provirus had disrupted the Npat gene, a gene of unknown function, resulting in the production of a truncated Npat mRNA. Expression of the closely linked Atm gene was found to be unaffected by the provirus.

Animals↗
PubMed1996

Hormonal regulation of the ligand for c-kit in the rat ovary and its effects on spontaneous oocyte meiotic maturation.

Kit ligand (KL, c-kit ligand) mRNA was detected in the ovaries of 26-day-old prepubertal rats using in situ hybridization. In antral follicles there was a gradient in the intensity of the hybridization signal across the layers of granulosa cells, with greatest intensity observed in the cumulus granulosa cells enclosing the oocyte, and less signal occurring in the granulosa cells furthest from the oocyte. In age-matched rats 40 hr after injection of pregnant mare serum gonadotropin (PMSG), the pattern of distribution of KL resembled that in the untreated ovaries, although the intensity of the hybridization signal was greater in the PMSG-primed ovaries. This morphological observation was confirmed using Northern blot analysis, which indicated that granulosa cells of PMSG-treated rats had 3.5-fold greater abundance of KL mRNA compared to untreated rats. The abundance of KL mRNA further increased to 7-fold over control levels at 6 hr after PMSG-primed rats were treated with human chorionic gonadotropin (hCG). By contrast, treatment of rats with diethylstilbestrol to stimulate follicular growth did not cause any change in the abundance of KL transcripts. To investigate a potential role for KL in oocyte meiotic maturation, fully grown oocytes were cultured for 24 hr with or without KL (50 or 500 ng/ml). The presence of KL resulted in a significant, albeit transient, delay in the progression of spontaneous meiotic maturation, using the indices of germinal vesicle breakdown and polar body formation. The inhibitory effects of KL were specifically blocked by ACK2, an antibody to the extracellular domain of the c-kit receptor. These results indicate that KL is produced in rat granulosa cells at particularly high levels in the cells closest to the oocyte and that this production may be regulated directly by gonadotropic hormones. Furthermore, KL inhibits the progression of meiosis in cultured oocytes, which suggests a possible role in the maintenance of meiotic arrest that occurs throughout oocyte growth.

3T3 Cells↗
PubMed1996

Autologous granulosa cell coculture demonstrates zygote suppression of granulosa cell steroidogenesis.

OBJECTIVE: To determine if embryos can modulate steroid hormone production by luteinized granulosa cells. DESIGN: Granulosa cells obtained from follicular aspirates were cultured alone or in the presence of a two-pronuclear zygote. The production of E2 and P by these cultures was evaluated by RIA. SETTING: In Vitro Fertilization Unit in an academic research environment. PATIENTS: Sixteen women undergoing IVF. INTERVENTIONS: Standard IVF-ET treatment cycle using leuprolide acetate for pituitary desensitization before hMG or urofollitropin for ovarian stimulation. MAIN OUTCOME MEASURES: Estradiol and P concentration in culture media of luteinized granulosa cells alone or granulosa cells cocultured with a two-pronuclear embryo. RESULTS: Both E2 and P production by luteinized granulosa cells was reduced when cultured in the presence of an embryo. CONCLUSIONS: Human embryos secrete a factor that regulates granulosa cell steroidogenesis.

Adult↗
PubMed1995

Differential regulation of progesterone and estradiol production by mouse cumulus and mural granulosa cells by A factor(s) secreted by the oocyte.

Mouse oocytes secrete a factor(s) that inhibits progesterone and enhances estradiol production by cumulus granulosa cells. The purpose of this study was to investigate the mechanisms by which the production of these steroids is modulated. Mouse oocyte-cumulus cell complexes (intact) and complexes from which the oocytes were removed microsurgically (oocytectomized; OOX) were cultured for up to 48 h in the presence of FSH (150 ng/ml) and testosterone (5 x 10(-7) M). For these experiments, all cells were obtained from antral follicles of 24- to 26-day-old mice primed with eCG. Intact complexes produced primarily estradiol, with significant accumulation occurring between 24 and 48 h. In contrast, OOX complexes produced little estradiol but, starting at 18 h of culture, released significantly more progesterone than did intact complexes. Progesterone accumulation in cocultures of denuded oocytes with either OOX complexes or monolayers of mural granulosa cells was significantly reduced compared to that with OOX complexes or mural granulosa cells cultured alone. If dibutyryl cAMP replaced FSH in the cocultures, similar results were obtained, suggesting that the oocyte-secreted steroid-regulating factor acts downstream of the generation of cAMP to inhibit progesterone production. Since estradiol can inhibit progesterone production by granulosa cells, we investigated the possibility that the increased progesterone released by OOX complexes was secondary to the lower estradiol production. Intact complexes cultured in the presence of the nonaromatizable androgen, 5 alpha-dihydrotestosterone, or steroidal (4-hydroxyandrostenedione) or non-steroidal (CGS 16949A) aromatase inhibitors produced little estradiol; however, progesterone production by these complexes was no different from that of estradiol-producing intact complexes. These results suggest that the steroid-regulating factor(s) secreted by occytes acts to regulate granulosa cell production of estradiol and progesterone by independent mechanisms.

Animals↗
PubMed1994

Purinergic receptor-mediated intracellular Ca2+ oscillations in chicken granulosa cells.

These studies were designed to investigate the effects of extracellular ATP on intracellular calcium ion concentration ([Ca2+]i) and progesterone secretion in granulosa cells obtained from the two largest preovulatory follicles (F1 and F2) of hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 99 +/- 7 nM (n = 22). There was a 5.7 +/- 0.7-fold increase in [Ca2+]i in all (n = 140) of the cells within 5 sec of adding a maximally stimulatory concentration (100 microM) of extracellular ATP. The initial spike was followed by [Ca2+]i oscillations that returned to the resting level between spikes. The frequency and amplitude of the [Ca2+]i oscillations were varied and persisted for 1-40 min. [Ca2+]i oscillations were also triggered by 100 microM UTP, UDP, GTP, GDP, ADP, and the nonhydrolyzable analog ATP gamma S. Adenosine, AMP, GMP, and UMP (all at 100 microM) were ineffective. The lowest ATP concentration to trigger a [Ca2+]i response was 1 microM. The sustained oscillatory phase of the response, but not the initial spike, was inhibited by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA. The nucleotide-triggered [Ca2+]i oscillations were not affected by adding the dihydropyridine Ca2+ channel blockers verapamil (100 microM), methoxy-verapamil (D600; 100 microM), or nifedipine (10 microM), before or during the response. However, the oscillations, but not the initial spike, were prevented by pretreating the cells with a general Ca2+ channel blocker, lanthanum (1 mM) or cobalt (5 mM). Lanthanum and cobalt also promptly stopped the [Ca2+]i oscillations when added during the oscillatory phase. The nucleotide-triggered [Ca2+]i response was also abolished by pretreating the cells with an inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM). In 3-h incubations, adenosine (100 microM) or ATP (100 microM) did not affect basal or LH (20 or 100 ng/ml)-stimulated progesterone production. These studies demonstrate that chicken granulosa cells display P2 purinergic receptors on their surfaces. Activation of these receptors triggers [Ca2+]i oscillations that follow the release of Ca2+ from internal stores and depend on Ca2+ influx through dihydropyridine-insensitive Ca2+ channels. The physiological function(s) of P2 purinergic receptors on granulosa cells is not known.

Adenosine↗