Cultures Of Bovine Granulosa Cells Research Articles

Função do fator de crescimento fibroblástico 18 na regulação da cascata de eventos pré-ovulatórios em bovinos

ABSTRACT: Suboptimal LH surges can lead to delayed or absent ovulation during reproductive management programs in cattle. Several signaling pathways may enhance the LH signal, potentially including growth factors. In the present study, we determined whether Fibroblast growth factor 18 (FGF18), which mimics some actions of the LH surge on granulosa cells, may potentiate the action of a suboptimal dose of LH using an established bovine granulosa cell (GC) culture system. Addition of recombinant FGF18 increased the concentration of progesterone (P4) in the culture medium. A combination of LH and FGF18 in the culture medium increased the abundance of mRNA encoding EREG, EGR1 and EGR3, proteins that are early modulators of the LH-induced pre-ovulatory cascade. This study demonstrated that FGF18 may be a co-factor in the regulation of the pre-ovulatory cascade in bovine ovarian granulosa cells.

Effects of bone morphogenetic protein 4, gremlin, and connective tissue growth factor on estradiol and progesterone production by bovine granulosa cells.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β family of proteins that have been implicated in the paracrine regulation of granulosa cell (GC) function, but whether responses to BMPs change with follicular size or interact with connective tissue growth factor (CTGF) or BMP antagonists (e.g., gremlin [GREM]) to directly affect GC function of cattle is unknown. Therefore, to determine the effects of BMP4 on proliferation and steroidogenesis of GCs and its interaction with GREM or CTGF, experiments were conducted using bovine GC cultures. In vitro, BMP4 (30 ng/mL) inhibited (P < 0.05) follicle-stimulating hormone (FSH) plus insulin-like growth factor 1 (IGF1)-induced progesterone and estradiol production by large- and small-follicle GCs, but the inhibitory effect of BMP4 on estradiol production was much more pronounced in large-follicle GCs. In small-follicle GCs, BMP4 had no effect (P > 0.10) on IGF1-induced proliferation, but GREM inhibited (P < 0.05) cell proliferation and estradiol and progesterone production in IGF1 plus FSH-treated GCs. In large-follicle GCs, BMP4 (10 to 30 ng/mL) increased (P < 0.05) GC numbers and GREM (100 ng/mL) blocked this effect. In large-follicle GCs, CTGF inhibited (P < 0.05) FSH plus IGF1-induced progesterone and estradiol production, and CTGF blocked the stimulatory effect of BMP4 on GC proliferation. These results indicate that BMP4, GREM, and CTGF inhibit GC aromatase activity and progesterone production. Also, the stimulatory effect of BMP4 on GC proliferation and the inhibitory effects of BMP4 on GC steroidogenesis are more pronounced in large vs. small follicles.

Lipopolysaccharide-induced mechanisms of ovarian dysfunction in cows with uterine inflammatory diseases.

Uterine inflammatory diseases commonly occur in postpartum dairy cows, resulting in reduced reproductive performance due to aberrant uterine and ovarian activity. Infection of the uterus with gram-negative bacteria results in the detection of lipopolysaccharide (LPS) in the plasma and follicular fluid of cows along with uterine inflammation. LPS acts on follicular components such as theca cells, granulosa cells, and follicle-enclosed oocytes, leading to impaired follicular activity. Follicles with a high LPS environment exhibit reduced follicular steroidogenesis due to the inhibition of steroidogenic enzyme transcription. Primary cell cultures of bovine granulosa and theca cells have shown that LPS acts on follicular cells to impair steroid production, which may disturb follicle growth and/or reduce their ability to ovulate. Even if ovulation occurs, cows with uterine inflammation are less likely to conceive because in addition to uterine damage, LPS also impairs the developmental competence of oocytes. LPS perturbs the nuclear and cytoplasmic maturation of bovine oocytes. Moreover, oocytes matured using LPS treatment are less likely to develop into the blastocyst stage. Such oocytes also have a reduced number of trophoblast cells in blastocysts. Therefore, the detrimental effects of LPS on ovarian activity may be partly responsible for infertility in cows with uterine inflammation. Novel treatment and prevention strategies for uterine inflammatory diseases can be developed by advancing our knowledge of the pathophysiology underlying ovarian dysfunction, and this can only be achieved by further research. The present review outlines the molecular pathogenesis of LPS-induced ovarian dysfunction.

Elevated free fatty acids affect bovine granulosa cell function: a molecular cue for compromised reproduction during negative energy balance.

High-yielding dairy cows postpartum face the challenge of negative energy balance leading to elevated free fatty acids levels in the serum and follicular fluid thus affecting the ovarian function. Here, we investigated effects of physiological concentrations of palmitic acid (PA), stearic acid (SA) and oleic acid (OA) on the viability, steroid production and gene expression in a bovine granulosa cell (GC) culture model. Treatment with individual and combined fatty acids increased the CD36 gene expression, while no significant apoptotic effects were observed. Both PA and SA significantly upregulated the expression of FSHR, LHCGR, CYP19A1, HSD3B1, CCND2 and increased 17β-estradiol (E2) production, while OA downregulated the expression of these genes and reduced E2. Interestingly, STAR was equally downregulated by all fatty acids and combination treatment. E2 was significantly reduced after combination treatment. To validate the effects of OA, in vivo growing dominant follicles (10–19 mm) were injected with bovine serum albumin (BSA) with/without conjugated OA. The follicular fluid was recovered 48 h post injection. As in our in vitro model, OA significantly reduced intrafollicular E2 concentrations. In addition, expression of CD36 was significantly up- and that of CYP19A1 and STAR significantly downregulated in antral GC recovered from aspirated follicles. The ovulation rates of OA-injected follicles tended to be reduced. Our results indicate that elevated free fatty acid concentrations specifically target functional key genes in GC both in vitro and in vivo. Suggestively, this could be a possible mechanism through which elevated free fatty acids affect folliculogenesis in dairy cows postpartum.

Lactate promotes specific differentiation in bovine granulosa cells depending on lactate uptake thus mimicking an early post-LH stage

BackgroundThe LH-induced folliculo-luteal transformation is connected with alterations of the gene expression profile in cells of the granulosa layer. It has been described that hypoxic conditions occur during luteinization, thus favoring the formation of L-lactate within the follicle. Despite being a product of anaerobic respiration, L-lactate has been shown to act as a signaling molecule affecting gene expression in neuronal cells. During the present study, we tested the hypothesis that L-lactate may influence differentiation of follicular granulosa cells (GC).MethodsIn a bovine granulosa cell culture model effects of L- and D-lactate, of increased glucose concentrations and of the lactate transport inhibitor UK5099 were analyzed. Steroid hormone production was analyzed by RIA and the abundance of key transcripts was determined by quantitative real-time RT-PCR.ResultsL-lactate decreased the production of estradiol and significantly affected selected genes of the folliculo-luteal transition as well as genes of the lactate metabolism. CYP19A1, FSHR, LHCGR were down-regulated, whereas RGS2, VNN2, PTX3, LDHA and lactate transporters were up-regulated. These effects could be partly or completely reversed by pre-treatment of the cells with UK5099. The non-metabolized enantiomer D-lactate had even more pronounced effects on gene expression, whereas increased glucose concentrations did not affect transcript abundance.ConclusionsIn summary, our data suggest that L-lactate specifically alters physiological and molecular characteristics of GC. These effects critically depend on L-lactate uptake, but are not triggered by increased energy supply. Further, we could show that L-lactate has a positive feedback on the lactate metabolism. Therefore, we hypothesize that L-lactate acts as a signaling molecule in bovine and possibly other monovular species supporting differentiation during the folliculo-luteal transformation.

Molecular characterization of a disintegrin and metalloprotease-17 (ADAM17) in granulosa cells of bovine preovulatory follicles

A disintegrin and metalloprotease-17 (ADAM17) is thought to play a key role in the release of soluble and active epiregulin (EREG) and amphiregulin (AREG) in ovarian follicles but its transcriptional regulation in follicular cells remains largely unknown. The objectives of this study were to characterize the regulation of ADAM17 transcripts in bovine follicles prior to ovulation and to investigate its transcriptional control in bovine granulosa cells. To study the regulation of ADAM17 transcripts, RT-PCR analyses were performed using total RNA extracted from bovine follicles collected between 0 h and 24 h post-hCG. Results showed that levels of ADAM17 mRNA were low prior to hCG (0 h), markedly and transiently increased 6–12 h post-hCG (P < 0.05), and returned to low baseline levels at 24 h post-hCG in granulosa and theca interna cells of preovulatory follicles. To determine the transcriptional control of ADAM17 expression, primary cultures of bovine granulosa cells were used. Forskolin (FSK) stimulation induced a pattern of ADAM17 mRNA up-regulation in vitro similar to that observed by hCG in vivo. 5′-Deletion mutagenesis studies identified a minimal region of the bovine ADAM17 promoter containing basal and FSK-inducible activities, which were dependent on the presence of a consensus AP1 cis-element. Electrophoretic mobility shift assays revealed an interaction between AP1 and the trans-acting factor Fra2. Chromatin immunoprecipitation assays confirmed an endogenous interaction between Fra2 and the ADAM17 promoter in granulosa cell cultures. FSK-inducible ADAM17 promoter activity and mRNA expression were suppressed by PKA and ERK1/2 inhibitors but not by a p38MAPK inhibitor, pointing to the importance of PKA and ERK1/2 signaling pathways in the up-regulation of bovine ADAM17 mRNA. Collectively, these findings describe the gonadotropin/FSK-dependent up-regulation of ADAM17 transcripts in bovine preovulatory follicles and unravel for the first time some of the molecular mechanisms involved in ADAM17 gene expression in granulosa cells of a monoovulatory species.

Formulation and testing of a non-steroidal aromatase inhibitor intravaginal device for the control of ovarian function in cattle

The study was designed to formulate intravaginal devices that provide biologically active circulating concentrations of an aromatase inhibitor for a minimum of 4 days, and to determine their physiologic effects in cattle. Three compounds with estradiol inhibitory capability (letrozole, anastrozole and fenbendazole) were tested in vitro using bovine granulosa cell culture. Letrozole was found to be the most efficient and potent inhibitor. A wax-based vehicle was selected for further development of a letrozole intravaginal device based on its steady release rate. Cycling heifers were assigned randomly to be given an intravaginal device containing wax plus gel coat (n=4), wax formulation (n=4), no formulation (blank device, control, n=4). Intravaginal devices were inserted on Day 3 (Day 0=ovulation) and kept in place for 8 days. The addition of a letrozole-containing gel coating hastened the initial increase on plasma concentrations, while the letrozole-containing wax-based vehicle maintained prolonged delivery from the intravaginal device. The dominant follicle diameter profile was larger in heifers treated with the wax plus gel coat device (P<0.04), and the interwave interval was prolonged in heifers in the letrozole-treated groups compared to controls (P<0.001). Plasma estradiol concentrations were reduced significantly in the letrozole-treated groups. Plasma progesterone concentrations were lower in the wax letrozole-treated group (P<0.02). We concluded that wax base plus gel coat intravaginal devices are suitable for the development of a letrozole-based protocol for the synchronization of ovulation in cattle. It effectively reduced estradiol production resulting in prolonged dominant follicle growth and lifespan, without adversely affecting progesterone production.

Suitable housekeeping genes for normalization of transcript abundance analysis by real-time RT-PCR in cultured bovine granulosa cells during hypoxia and differential cell plating density.

BackgroundBovine granulosa cell culture models are important to understand molecular mechanisms of ovarian function. Folliculogenesis and luteinization are associated with increasing density of cells and local hypoxic conditions. The current study identified two reliable housekeeping genes useful for gene normalization in granulosa cells under different in vitro conditions.MethodsDuring the current experiments cells were subjected to different biological and physical stimuli, follicle stimulating hormone, different initial cell plating density and hypoxia. Transcript abundance of seven housekeeping genes was quantified by real-time RT-PCR with co-amplification of the respective external standard.ResultsThree of the genes, GAPDH, HMBS, and HPRT1 were found to be regulated by initial cell plating density, five of them, GAPDH, HMBS, HPRT1, RPLP0 and RPS18 under hypoxic conditions, but none of them after FSH stimulation. In detail, GAPDH was up regulated, but HPRT1 and HMBS were down regulated at high density and under hypoxia. Expression of RPLP0 and RPS18 was inconsistent, but was significantly down-regulated in particular at high cell density combined with hypoxia. In contrast, TBP and B2M genes were neither regulated under different plating density conditions nor by hypoxia as they showed similar expression levels under all conditions analyzed.ConclusionsThe present data indicate that TBP and B2M are appropriate housekeeping genes for normalization of transcript abundance measured by real-time RT-PCR in granulosa cells subjected to different plating densities, oxygen concentrations and FSH stimulation.

Expression and regulation of regulator of G-protein signaling protein-2 (RGS2) in equine and bovine follicles prior to ovulation: molecular characterization of RGS2 transactivation in bovine granulosa cells.

The luteinizing hormone preovulatory surge stimulates several signal pathways essential for ovulation, and the regulator of G-protein signaling protein-2 (RGS2) is thought to be involved in this process. The objectives of this study were to characterize the regulation of RGS2 transcripts in equine and bovine follicles prior to ovulation and to determine its transcriptional control in bovine granulosa cells. To assess the regulation of equine RGS2 prior to ovulation, RT-PCR was performed using total RNA extracted from equine follicles collected at various times after human chorionic gonadotropin (hCG) injection. Results showed that RGS2 mRNA levels were very low at 0 h but markedly increased 12-39 h post-hCG (P < 0.05). In the bovine species, results revealed that RGS2 mRNA levels were low in small and dominant follicles and in ovulatory follicles obtained at 0 h, but markedly increased in ovulatory follicles 6-24 h post-hCG (P < 0.05). To study the molecular control of RGS2 expression, primary cultures of bovine granulosa cells were used. Stimulation with forskolin induced an up-regulation of RGS2 mRNA in vitro. Studies using 5'-deletion mutants identified a minimal region containing full-length basal and forskolin-inducible RGS2 promoter activities. Site-directed mutagenesis indicated that these activities were dependent on CRE and ETS1 cis-elements. Electrophoretic mobility shift assays confirmed the involvement of these elements and revealed their interactions with CREB1 and ETS1 proteins. Chromatin immunoprecipitation assays confirmed endogenous interactions of these proteins with the RGS2 promoter in granulosa cells. Forskolin-inducible RGS2 promoter activity and mRNA expression were markedly decreased by PKA and ERK1/2 inhibitors, and treatment with an antagonist of PGR (RU486) and inhibitors of PTGS2 (NS398) and EGFR (PD153035) blocked the forskolin-dependent RGS2 transcript expression, suggesting the importance of RGS2 in ovulation. Collectively, this study reports for the first time the gonadotropin-dependent up-regulation of RGS2 in equine and bovine preovulatory follicles and presents some of the regulatory controls involved in RGS2 gene expression in granulosa cells.

Expression and regulation of stromal cell-derived factor-1 (SDF1) and chemokine CXC motif receptor 4 (CXCR4) in equine and bovine preovulatory follicles

The interaction between stromal cell-derived factor-1 (SDF1) and chemokine CXC motif receptor 4 (CXCR4) has been implicated in leukocyte attraction, tissue remodeling and angiogenesis. The objective of the present study was to characterize the expression and regulation of SDF1 and CXCR4 in equine follicles during the ovulatory process. Equine preovulatory follicles were isolated during estrus 0–39h after hCG treatment. Follicle wall preparations (theca interna with attached granulosa cells) and isolated preparations of granulosa cells and theca interna were obtained, and total RNA extracts were analyzed by RT-PCR/Southern blot. Results showed that levels of CXCR4 transcripts were induced by hCG in follicles at 36h post-hCG (P<0.05 vs 0h), with the induction observed in both granulosa and theca cells. Immunoblotting and immunohistochemical analyses confirmed an increase in CXCR4 protein in follicles after hCG treatment. In contrast, levels of SDF1 transcripts were very low in granulosa cells but high in theca interna cells throughout most of the ovulatory period. Studies in vivo performed with bovine preovulatory follicles collected 0–24h post-hCG revealed a marked and significant up-regulation of CXCR4 transcripts after hCG (P<0.05), as observed in equine follicles. A similar pattern of CXCR4 mRNA up-regulation was observed in cultures of bovine granulosa cells treated with forskolin (P<0.05). This forskolin-dependent induction of CXCR4 mRNA was suppressed by co-treatment with inhibitors of PKA, ERK1/2 and EGFR, and by the progesterone receptor antagonist RU486 (P<0.05), underscoring the contribution of multiple signaling pathways. In complementary studies, treatment of bovine granulosa cells with EGF or the hypoxia mimetic cobalt chloride significantly increased CXCR4 transcript levels, whereas co-treatment with forskolin and a CXCR4 antagonist repressed the expression of several ovulation-related genes. Collectively, this study describes for the first time the gonadotropin-dependent up-regulation of CXCR4 transcript in ovarian follicles of large monoovulatory species, provides some insights into the regulation of CXCR4 gene expression in granulosa cells, and identifies a potential link between follicular SDF1/CXCR4 activation and the regulation of ovulation-related genes.

Comparison of Medium Supplementation on Proliferation and Hormone Production of Bovine Granulosa Cells in a Defined Culture System

Aims: The objective of this study was to develop a serum -free bovine granulosa cell (GC) culture system in which estradiol (E2) production could be maintained in a defined media, with polyvinyl alcohol (PVA), insulin and insulin

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200® implanted heifers

BackgroundHeifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments: non-implanted (n = 5), Revalor 200 for 28 d (n = 5), or Revalor 200 for 84 d (n = 6). Small follicle (1 to 5 mm) granulosa cells were isolated from each pair and incubated with phosphate buffered saline (n = 16) or 100 ng/mL follicle stimulating hormone (n = 16) for 24 h.ResultsGranulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar (P = 0.22) to non-implanted heifers, while medium estradiol concentrations were increased (P < 0.10) at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein (P = 0.05) mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 (P < 0.10) or 84 d (P < 0.05) compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase (P = 0.57) and aromatase (P = 0.23) were demonstrated in implanted or non-implanted heifers.ConclusionsThese results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.

Expression, Regulation, and Promoter Activation of Vanin-2 (VNN2) in Bovine Follicles Prior to Ovulation1

Vanin-2 (VNN2) is known to be involved in inflammation and leukocyte migration, but its regulation in follicles remains unknown. The objectives of this work were to study the regulation of VNN2 transcripts in bovine follicles prior to ovulation and to characterize the control of its expression in bovine granulosa cells. VNN2 expression was studied using total RNA extracted from granulosa cells of small follicles (2-4 mm in diameter), dominant follicles obtained on Day 5 of the estrous cycle, ovulatory follicles obtained 0-24 h after human chorionic gonadotropin (hCG), and corpora lutea on Day 5 of the cycle. The results from RT-PCR analyses showed that levels of VNN2 mRNA were high in ovulatory follicles 24 h post-hCG but low in the other tissues. In ovulatory follicles, levels of VNN2 mRNA were low at 0 h but significantly up-regulated 12-24 h post-hCG. To determine factors controlling VNN2 gene expression, established primary cultures of granulosa cells isolated from bovine dominant follicles were used. Treatment with forskolin elevated VNN2 mRNA expression as observed in vivo. Mutation studies identified the minimal region conferring basal and forskolin-stimulated VNN2 promoter activities, which were dependent on chicken ovalbumin upstream promoter-transcription factor (COUP-TF), GATA, and Ebox cis-elements. Electrophoretic mobility shift assays identified COUP-TF, GATA4, and upstream stimulating factor proteins as key factors interacting with these elements. Chromatin immunoprecipitation assays confirmed basal and forskolin-induced interactions between these proteins and the VNN2 promoter in bovine granulosa cell cultures. VNN2 promoter activity and mRNA expression were markedly stimulated by forskolin and overexpression of the catalytic subunit of PKA, but inhibited by PKA and ERK1/2 inhibitors. Collectively, the findings from this study describe for the first time the gonadotropin/forskolin-dependent up-regulation of VNN2 transcripts in granulosa cells of preovulatory follicles and provide insights into some of the molecular bases of VNN2 gene expression in follicular cells.

Molecular Characterization and Transcriptional Regulation of a Disintegrin and Metalloproteinase With Thrombospondin Motif 1 (ADAMTS1) in Bovine Preovulatory Follicles

The ovulatory process involves a complex remodeling of the extracellular matrix during which a desintegrin and metalloproteinase with thrombospondin motif 1 (ADAMTS1) is thought to play a key role, but its transcriptional regulation in bovine follicles remains largely unknown. The objectives of this study were to characterize the regulation of ADAMTS1 in bovine follicles before ovulation and to determine its transcriptional control in bovine granulosa cells. Regulation of ADAMTS1 was assessed using total RNA isolated from bovine preovulatory follicles obtained at various times after human chorionic gonadotropin treatment. Results from RT-PCR analyses showed that levels of ADAMTS1 mRNA were very low at 0 hours but increased at 6 to 24 hours after human chorionic gonadotropin in granulosa cells. To determine the regulatory mechanisms controlling ADAMTS1 gene expression in vitro, primary cultures of bovine granulosa cells were established, and treatment with forskolin up-regulated ADAMTS1 mRNA levels. Promoter activity assays, 5'-deletion, and site-directed mutagenesis identified a minimal region conferring full-length basal and forskolin-stimulated ADAMTS1 promoter activities, with both being dependent on Ebox cis-acting elements. EMSAs revealed upstream stimulating factor (USF) proteins as key trans-activating factors interacting with Ebox. Chromatin immunoprecipitation assays confirmed such interactions between USF and Ebox in vivo, and USF binding to Ebox elements was increased by forskolin treatment. ADAMTS1 promoter activity and mRNA expression were increased by forskolin and overexpression of the catalytic subunit of protein kinase A, but not by cotreatment with inhibitors of protein kinase A, ERK1/2, and epidermal growth factor receptor signaling pathways. Furthermore, treatment with a soluble epidermal growth factor induced ADAMTS1 mRNA expression in granulosa cells. Collectively, results from this study describe the gonadotropin/forskolin-dependent up-regulation of ADAMTS1 mRNA in granulosa cells of bovine preovulatory follicles in vivo and in vitro and identify for the first time some of the molecular mechanisms responsible for ADAMTS1 promoter activation in follicular cells of a large monoovulatory species.

Human chorionic gonadotropin-dependent up-regulation of epiregulin and amphiregulin in equine and bovine follicles during the ovulatory process

Little is known about the expression and regulation of epiregulin (EREG) and amphiregulin (AREG) in ovarian follicles of large monoovulatory animal species. To characterize the gonadotropin-dependent regulation of EREG and AREG mRNAs in equine follicles prior to ovulation, extracts were prepared from equine follicles collected during estrus between 0 and 39h post-hCG and corpora lutea obtained on day 8 of the estrous cycle (day 0=day of ovulation). Results from RT-PCR/Southern blot analyses showed that levels of EREG and AREG mRNAs were very low in follicles obtained at 0h but increased thereafter (P<0.05), with maximal levels observed 33–39h post-hCG. This significant increase was observed in both granulosa and theca cells. Immunohistochemistry and immunoblot analyses confirmed the hCG-dependent induction of EREG protein in both cell types. RT-PCR/Southern blot analyses of ADAM17, which encodes an enzyme that cleaves and releases soluble bioactive EREG and AREG, showed that levels of its transcript were high and remained constant throughout the period studied. Studies on the hCG-dependent regulation of EREG and AREG in bovine preovulatory follicles in vivo showed that the induction of both transcripts was transient, observed predominantly at 6h post-hCG and localized only in granulosa cells. To characterize the effect of epidermal growth factor receptor (EGFR) activation on the expression of ovulation-related genes in granulosa cells of a large monoovulatory animal species, primary cultures of bovine granulosa cells were established. Results from RT-PCR analyses revealed that EREG and AREG mRNAs were induced by forskolin treatment in vitro; but the EGFR inhibitor PD153035 suppressed the forskolin-dependent induction of several ovulation-related transcripts, including PTGS2, PTGER2, TNFAIP6, PGR, MMP1, VEGFA, and CTSL2 mRNAs. Moreover, these transcripts were induced in granulosa cell cultures by EGF, an analog of EREG and AREG. Collectively, this study identifies differences in the temporal and cellular localization of EREG and AREG expression in equine and bovine preovulatory follicles, and underscores the potential role of follicular EGFR activation in the regulation of ovulation-regulated genes in large monoovulatory species.

Association Between Leptin, LH and its Receptor and Luteinization and Progesterone Accumulation (P4) in Bovine Granulosa Cell In Vitro

A full understanding of the cellular events that occur during in vitro luteinization of bovine granulosa cells, stimulated by LH and by leptin, is a complex goal that has not been completely achieved. The aim of this work was to study the effects of leptin, LH and leptin + LH on progesterone accumulation (P4) and on the expression of LH receptors (LHR) in bovine granulosa cells in culture. The results confirm that this in vitro model is representative of functional and morphological luteinization/differentiation. The pattern of expression of LHR with time of incubation was an important marker of in vitro luteinization, with 50-90% of cells expressing LHR by 96 h in culture. Cytoplasmic lipidic droplets were highly abundant in granulosa cells, suggesting a sufficient source of precursors for steroid hormone synthesis: P4 accumulation ranged between 40 and 550 ng/ml. In addition, a positive correlation (r = 0.58, p < 0.05) between the expression of LHR and accumulation of P4 throughout the time of incubation was observed. The expression of LHR was inhibited by LH and leptin + LH treatment. In conclusion, we found an inverse modulation between the expression of LHR and the concentration of LH, and the expression of LHR could be regulated by P4 produced by the luteinized granulosa cells. These findings are contributing to elucidate further the panoply of interactions during the differentiation of granulosa cells into luteal cells in vitro.

Effect of Angiotensin II on Bovine Follicular Growth and mRNA Encoding Steroidogenic Enzymes, Gonadotrophin Receptors, and Tissue Development Genes.

Angiotensin II (AngII) has been shown to be involved in the regulatory mechanism of ovulation and oocyte nuclear and cytoplasmic maturation in cattle. In rats, AngII induces apoptosis in granulosa cells and atresia of antral follicles during follicular development. However, the angiotensin converting enzyme (ACE) mRNA expression increases at the end of follicular maturation, suggesting a possible modulatory role of AngII for ovulation in cows. In addition, the expression of AngII type 2 receptors (AGTR2) is higher in healthy than in atretic follicles and, in vitro, is stimulated by FSH in bovine granulosa cell culture. Based on this evidence, this study was carried out to establish the role of AngII during the follicular growth and the effect of AngII inhibition on steroid secretion and follicular cell gene expression profile. Cows were pre-synchronized to induce a new follicular wave and the largest follicle was injected with saralasin (AngII inhibitor; 10μM), AngII (10μM) or saline when it reached 7-8mm diameter. The intrafollicular injections were performed through a double-needle system and guided by a 7.5 MHz vaginal ultrasound probe. All follicles were monitored by ultrasonography for 96 hours after intrafollicular injection. Follicles injected with saline (control group) reached ovulatory size and ovulated after a challenge with the GnRH agonist gonadorelin, demonstrating that the intrafollicular injection did not affect follicular growth. Saralasin inhibited follicular growth in all treated cows (4/4), suggesting that AngII is essential for follicular growth in 7-8mm follicles. However, intrafollicular injection of AngII affected neither follicular growth nor the pattern of follicular dynamics, which were similar to control cows. These results imply that bovine ovarian follicles contain sufficient AngII for follicle development. In a second experiment, a single follicle of each cow was injected with saralasin or saline and cows were ovariectomized 24h later. The follicular fluid was aspirated to determine steroid concentrations, and granulosa and theca cells were recovered for gene expression measurement. Estradiol concentration in follicular fluid was determined by radioimmunoassay. Messenger RNA levels were analyzed by real-time RT-PCR and are expressed relative to cyclophilin. The inhibition of AngII action decreased abundance of mRNA encoding aromatase (CYP19), 3β-hydroxysteroid dehydrogenase (3β HSD), LH receptor, SerpinE2 and cyclinD2 in granulosa cells but not StAR, 17β HSD, FSH receptor, growth arrest and DNA damage inducible (GADD45b) or X-linked inhibitor of apoptosis protein (XIAP). In theca cells, the inhibition of AngII decreased the expression of AGTR2 but not the expression of steroidogenic enzymes genes. Taken together, these results show that AngII is essential for follicular growth, and plays important role in regulating genes involved in granulosa cell proliferation and differentiation, which are necessary for development of the dominant follicle. These data suggest that AngII signaling is involved in follicle growth and dominance in cattle. (poster)

Cocaine- and Amphetamine-Regulated Transcript Regulation of Follicle-Stimulating Hormone Signal Transduction in Bovine Granulosa Cells

Regulation of estradiol production, central to ovarian follicular development and reproductive function, is mediated by a complex interaction of pituitary gonadotropins such as FSH with locally produced regulatory molecules. We previously demonstrated a negative association of expression of cocaine-and amphetamine-regulated transcript (CART) with follicle health status and a novel local negative role for CART in regulation of basal estradiol production by bovine granulosa cells. However, effects of CART on FSH-induced estradiol production and the underlying mechanism(s) mediating the physiological actions of CART on granulosa cells are not known. Objectives of the present study were to determine effects of CART on basal and FSH-induced intracellular cAMP levels, aromatase mRNA, estradiol accumulation, calcium signaling, and the intracellular signaling pathways involved using primary cultures of bovine granulosa cells. CART treatment potently inhibits the FSH-induced rise in granulosa cell cAMP levels, estradiol accumulation, and aromatase mRNA. Furthermore, results show that calcium is essential for FSH-induced cAMP and estradiol accumulation, and CART significantly inhibits FSH-induced calcium influx. Select G protein and protein kinase inhibitors were used to elucidate pathways involved in CART actions. The inhibitory actions of CART on FSH signaling and estradiol production are mediated via a G(o/i)-dependent pathway, whereas none of the other signaling inhibitors had any effect on CART actions. Results demonstrate novel potent inhibitory effects of CART on multiple components of the FSH signaling pathway linked to estradiol production and follicular development and shed new insight into the mechanism of action of CART potentially pertinent within and beyond the reproductive system.

The effect of oxysterols, individually and as a representative mixture from food, on in vitro cultured bovine ovarian granulosa cells

The cytotoxicity of five oxysterols identified in cooked fish, 7-ketocholesterol, 7beta-hydroxycholesterol, cholesterol 5alpha,6alpha-epoxide, cholestanetriol and 4-cholesten-3-one, was investigated in primary cultures of bovine ovarian granulosa cells. Cells were exposed to the oxysterols individually and to a mixture of the same oxysterols for 24 h. Cell viability as determined by trypan blue exclusion and mitochondrial integrity (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction) was reduced by 0.5 and 2.5 microM 7beta-hydroxycholesterol, cholesterol 5alpha,6alpha-epoxide, cholestanetriol and 4-cholesten-3-one, but not by 0.5 or 2.5 microM 7-ketocholesterol under the same culture conditions. A mixture of 7-ketocholesterol, 7beta-hydroxycholesterol, cholesterol 5alpha,6alpha-epoxide, cholestanetriol and 4-cholesten-3-one 0.5 microM each; 2.5 microM oxysterol in total) did not change cell viability relative to controls. Lipid peroxidation, as determined by thiobarbituric acid reactive substances assay, was unaffected by a 24-hour exposure of granulosa cells to individual oxysterols but was increased slightly by the oxysterol mixture. The specific activities of antioxidant enzymes superoxide dismutase and catalase were increased to different extents (1.17- to 6.43-fold), relative to controls, by the administration of individual oxysterols and the oxysterol mixture. These results indicate that while some individual oxysterols can induce cytotoxic effects and defensive responses in bovine ovarian granulosa cells, administration of the same oxysterols as a mixture does not elicit the same responses. In addition, the oxysterols tested exerted a pro-apoptotic effect on granulosa cells when administered individually at concentrations of 0.5 and 2.5 microM, but not when administered as a 2.5 microM oxysterol mixture. The results suggest that major oxysterols are not universally cytotoxic, they may complete with other oxysterols for receptor sites, and that the simultaneous presence of several different oxysterol species may reduce the adverse effects of individual oxysterols.

Role of Upstream Stimulatory Factor Phosphorylation in the Regulation of the Prostaglandin G/H Synthase-2 Promoter in Granulosa Cells

To investigate the role of USF phosphorylation in the regulation of the PGHS-2 promoter in granulosa cells, promoter activity assays were performed in primary cultures of bovine granulosa cells transfected with the chimeric PGHS-2 promoter/luciferase (LUC) construct -149/-2PGHS-2.LUC. Transfections were done in the absence or presence of forskolin; the protein kinase A (PKA) inhibitor H-89; or an expression vector encoding USF1, USF2, the catalytic subunit of PKA (cPKA), or a PKA inhibitor protein (PKI). Electrophoretic mobility shift assays were performed to study USF/DNA interactions using granulosa cell nuclear extracts and a 32P-labeled proximal PGHS-2 promoter fragment containing the E-box element. The results show that forskolin stimulation and cPKA overexpression caused a marked and significant increase in USF-dependent DNA binding and PGHS-2 promoter activities (p < 0.05). In contrast, both activities were decreased by H-89 treatment or PKI overexpression. Reverse transcription-PCR analyses revealed that these treatments had similar effects on endogenous PGHS-2 mRNA levels in granulosa cells. Cotransfection studies with a USF2 mutant lacking N-terminal activation domains (U2Delta1-220) repressed forskolin-, cPKA-, and USF-dependent PGHS-2 promoter activities. Electrophoretic mobility shift assays showed that U2Delta1-220 was able to compete with full-length USF proteins and to saturate the E-box element. Immunoprecipitation/Western blot analyses revealed an increase in the levels of phosphorylated USF1 and USF2 after forskolin treatment, whereas chromatin immunoprecipitation assays showed that binding of USF proteins to the endogenous PGHS-2 promoter was stimulated by forskolin. Site-directed mutagenesis of a consensus PKA phosphorylation site within USF proteins abolished their transactivating capacity. Collectively, these results characterize the role of USF phosphorylation in PGHS-2 expression and identify the phosphorylation-dependent increase in USF binding to the E-box as a putative molecular basis for the increase in PGHS-2 promoter transactivation in granulosa cells.