In vitro effects of two environmental toxicants, beauvericin and glyphosate in Roundup, on cell numbers and steroidogenesis of bovine ovarian cells.

Beauvericin is an emerging Fusariotoxin naturally occurring in cereal grains throughout the world whereas glyphosate (N-phosphonomethyl-glycine) is a non-selective systemic herbicide used worldwide. The purpose of this study is to evaluate a newly developed ovarian cell culture system (that includes both granulosa and theca cells) as an in vitro model for toxicological studies. Specifically, the effects of beauvericin and glyphosate in formulation with Roundup on ovarian cell numbers and steroid production were evaluated. Ovaries collected from cattle without luteal structures were sliced into 30-70 pieces each, and granulosa and theca cells were collected. Harvested cells were cultured for 48 h in 10% fetal bovine serum-containing medium followed by 48 h in serum-free medium containing testosterone (500 ng/mL; as an estrogen precursor) with the following eight treatments: (1) controls, (2) FSH (30 ng/mL) alone, (3) FSH plus insulin-like growth factor-1 (IGF1; 30 ng/mL), (4) FSH plus IGF1 plus beauvericin (3 µM), (5) FSH plus IGF1 plus glyphosate in Roundup (10 µg/mL), (6) FSH plus IGF1 plus fibroblast growth factor 9 (FGF9, 30 ng/mL), (7) a negative control without added testosterone, and (8) IGF1 plus LH (30 ng/mL) with basal medium without added testosterone. In the presence of FSH, IGF1 significantly increased cell numbers, estradiol and progesterone production by severalfold. Glyphosate in Roundup formulation significantly inhibited IGF1-induced cell numbers and estradiol and progesterone production by 89-94%. Beauvericin inhibited IGF1-induced cell numbers and estradiol and progesterone by 50-97% production. LH plus IGF1 significantly increased androstenedione secretion compared with controls without added testosterone indicating the presence of theca cells. In conclusion, the present study demonstrates that toxicological effects of beauvericin and glyphosate in Roundup formulation are observed in a newly developed ovarian cell model system and further confirms that both glyphosate and beauvericin may have the potential to impair reproductive function in cattle.

Developmental and hormonal regulation of FBN1 and OR4M1 mRNA in bovine granulosa cells.

Recent studies have reported hormonal regulation of expression of fibrillin 1 (FBN1), the gene that encodes asprosin, in bovine theca cells, however, hormonal regulation of gene expression of FBN1 and the asprosin receptor, olfactory receptor 4M1 (OR4M1), has not been evaluated in granulosa cells (GC). This study was designed to characterize FBN1 and OR4M1 gene expression in GC during development of bovine dominant ovarian follicles, and to determine the hormonal regulation of FBN1 and OR4M1 mRNA expression in GC. GC FBN1 mRNA abundance was greater (P < 0.05) in medium (5.1-8 mm) estrogen inactive (EI) follicles than in large (>8.1 mm) or small (1-5 mm) EI follicles. In comparison, GC OR4M1 mRNA abundance was greater (P < 0.05) in small EI follicles than in large or medium EI follicles. Abundance of OR4M1 mRNA in GC of follicles collected on days 3 to 4 (early growth phase) and on days 5 to 6 (late growth phase) was similar, whereas FBN1 mRNA abundance was greater (P < 0.05) on days 5 to 6 vs days 3 to 4. Hormonal regulators for FBN1 mRNA abundance in cultured small-follicle GC were identified: TGFß1 causing a 2.45-fold increase, WNT3A causing a 1.45-fold increase, and IGF1 causing a 65% decrease. Steroids, leptin, insulin, growth hormone, follicle stimulating hormone, fibroblast growth factor 9 and epidermal growth factor had no effect on FBN1 mRNA abundance. Abundance of OR4M1 mRNA in GC was regulated by progesterone with 3.55-fold increase, but other hormones did not affect GC OR4M1 mRNA abundance. Findings indicate that both FBN1 and OR4M1 gene expression are hormonally and developmentally regulated in bovine follicles, and thus may affect asprosin production and its subsequent role in ovarian follicular function in cattle.

Changes in fibroblast growth factor receptors-1c, -2c, -3c, and -4 mRNA in granulosa and theca cells during ovarian follicular growth in dairy cattle.

The various fibroblast growth factors (FGF) regulate their function via binding to 4 main FGF receptor (FGFR) subtypes and their splice variants, FGFR1b, FGF1c, FGFR2b, FGFR2c and FGFR3c and FGFR4, but which of these FGFR are expressed in the granulosa (GC) and theca cells (TC), the 2 main cell layers of ovarian follicles, or change during follicular development is unknown. We hypothesized that FGFR1c, FGFR2c and FGFR3c (but not FGFR4) gene expression in GC (but not TC) would change with follicular development. Hence, the objective of this study was to determine if abundance of FGFR1c, FGFR2c, FGFR3c, and FGFR4 mRNA change according to follicular size, steroidogenic status, and days post-ovulation during growth of first-wave dominant follicles in Holstein cattle exhibiting regular estrous cycles. Estrous cycles of non-lactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n = 8) or d 5 to 6 (n = 8) post-ovulation for GC and TC RNA extraction from small (1-5 mm), medium (5.1 to 8 mm) or large (8.1-18 mm) follicles for real-time PCR analysis. In GC, FGFR1c and FGFR2c mRNA relative abundance was greater in estrogen (E2)-inactive (ie, concentrations of E2 < progesterone, P4) follicles of all sizes than in GC from large E2-active follicles (ie, E2 > P4), whereas FGFR3c and FGFR4 mRNA abundance did not significantly differ among follicle types or days post-estrus. In TC, medium E2-inactive follicles had greater FGFR1c and FGFR4 mRNA abundance than large E2-active and E2-inactive follicles on d 5 to 6 post-ovulation whereas FGFR2c and FGFR3c mRNA abundance did not significantly differ among follicle types or day post-estrus. In vitro experiments revealed that androstenedione increased abundance of FGFR1c, FGFR2c and FGFR4 mRNA in GC whereas estradiol decreased FGFR2c mRNA abundance. Neither androstenedione nor estradiol affected abundance of the various FGFR mRNAs in cultured TC. Taken together, the findings that FGFR1c and FGFR2c mRNA abundance was less in GC of E2-active follicles and FGFR1c and FGFR4 mRNA was greater in TC of medium inactive follicles at late than at early growing phase of the first dominant follicle support an anti-differentiation role for FGF and their FGFR as well as support the idea that steroid-induced changes in FGF and their receptors may regulate selection of dominant follicles in cattle.

Postpartum nutrition affects the insulin-like growth factor system in dominant follicles and plasma of anestrous beef cows.

Effects of nutrition on insulin-like growth factor-I (IGF-I), IGF binding proteins (IGFBP), and insulin in plasma and dominant follicles were evaluated at day 72 and 56 (Exp. 1, n = 12 and Exp. 2, n = 28, respectively) postpartum in anovulatory primiparous beef cows. Cows were stratified based on body condition score at calving and randomly assigned to nutritional treatments: maintain (M), 2.27 kg of a 40 % CP supplement per day and ad libitum hay; or gain (G), ad libitum access to a 50 % concentrate diet and ad libitum hay. Blood samples were collected twice weekly starting 30 days postpartum. Ovarian follicles were evaluated using ultrasonography commencing 42 (Exp. 1) or 30 (Exp. 2) days postpartum. Body weight and condition score were greater (P < 0.05) for cows of G than M groups and postpartum interval to luteal function was longer for cows of the M than G group. Insulin and IGF-I concentrations in follicular fluid (FF) and plasma were greater (P < 0.05) for cows of the G than M group at follicular aspiration. Plasma and FF IGFBP4 and IGFBP5 concentrations were greater (P <  0.05) in Exp. 2, and IGFBP5 was greater in Exp. 1 for cows of the G than M group. Treatment did not affect FF steroid concentrations or granulosal cell CYP19A1, PAPPA, IGFBP4, and IGFBP5 mRNA abundance. These results indicate concentrations of IGF-I, insulin, IGFBP4, and IGFBP5 in FF and plasma are affected by nutritional intake and may be related to follicular function.

Effects of N-carbamylglutamate and arginine on steroidogenesis and proliferation of pig granulosa cells in vitro.

Results of in vivo studies indicate dietary N-carbamylglutamate (NCG) and arginine (ARG) can enhance reproductive performance in gilts. It was hypothesized that both NCG and ARG will alter hormone-induced estradiol (E2) production by granulosa cells (GC), explaining why these compounds could improve reproductive performance in pigs. The objective of these studies, therefore, was to evaluate the direct effects of NCG and ARG on porcine GC proliferation and steroidogenesis, using an in vitro cell culture system. The GC from small (SM; 1-5 mm) and large (LG; >5 mm) pig follicles were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing 500 ng/mL of testosterone (as an E2 precursor), and NCG or ARG at various doses in the presence of either follicle-stimulating hormone (FSH; 30 ng/mL), insulin-like growth factor-1 (IGF1; 30 ng/mL), or both. Numbers of GC were determined at the end of the experiment and concentrations of progesterone (P4) and E2 in culture medium were determined. Results indicated that LG-follicle GC were more responsive to NCG and ARG than SM-follicle GC. Specifically, in LG-follicle GC, NCG inhibited (P <  0.05) basal and FSH-induced P4 and E2 production but stimulated cell numbers; whereas ARG inhibited FSH-induced E2 production and cell numbers. In SM-follicle GC, treatment with NCG and ARG decreased IGF1 plus FSH induced P4 production, but E2 production and cell proliferation were not affected. These studies indicate that NCG and ARG may directly affect follicular function in pigs.

Fusarium mycotoxins and in vitro species-specific approach with porcine intestinal and brain in vitro barriers: A review.

Fusarium mycotoxins, such as fumonisins, trichothecenes, zearalenone and emerging fusariotoxins, common contaminants of feed and food, have received increased interest, due to the possible impact on animal and human health. In this context, it is urgent to focus our attention on fusariotoxins adverse effects, considering and analysing data in relation to their species-specificity. The in vitro approach for fusariotoxins risk assessment evaluation, through porcine epithelial barriers model, allowed to collect information on their absorption profile, bioavailability and toxicity. The aim of this review is to give an overview on Fusarium mycotoxins and their interactions with porcine intestinal and brain in vitro barriers, because they represent direct target organs of toxicity and as tools to evaluate their permeability and transport.

Transcriptome profiling of bovine ovarian theca cells treated with fibroblast growth factor 9.

We reported previously that fibroblast growth factor 9 (FGF9) acts as an antidifferentiation factor, stimulating proliferation of granulosa cells (GCs) and theca cells (TCs) while suppressing hormone-induced steroidogenesis of these cells. How FGF9 acts to simultaneously suppress steroidogenesis and stimulate proliferation remains to be fully elucidated. Thus, this study was undertaken to clarify the effects of FGF9 on the TC transcriptome. Ovaries were obtained from beef heifers at a local abattoir, TCs were isolated from large antral follicles, and cultured with or without 30 ng/mL of FGF9 for 24 h in the presence of LH and IGF-1. After treatment, total RNA was extracted from TC and processed for microarray using Affymetrix GeneChip Bovine Genome Arrays (n = 4/group). Transcriptome analysis comparing FGF9-treated TC with control TC using 1.3-fold cutoff, and a P < 0.05 significance level identified 355 differentially expressed transcripts, with 164 elements upregulated and 191 elements downregulated by FGF9. The ingenuity pathway analysis (IPA) was used to investigate how FGF9 treatment affects molecular pathways, biological functions, and the connection between molecules in bovine TC. The IPA software identified 346 pathways in response to FGF9 in TC involved in several biological functions and unveiled interesting relationships among genes related to cell proliferation (eg, CCND1, FZD5, and MYB), antioxidation/cytoprotection (eg, HMOX1 and NQO1), and steroidogenesis (eg, CYP11A1 and STAR). Overall, genes, pathways, and networks identified in this study painted a picture of how FGF9 may regulate folliculogenesis, providing novel candidate genes for further investigation of FGF9 functions in ovarian follicular development.

Effects of N-carbamylglutamate and L-arginine on gonadotrophin-releasing hormone (GnRH) gene expression and secretion in GT1-7 cells.

Recent studies have shown that N-carbamylglutamate (NCG) and arginine (ARG) supplementation improves reproductive performance in livestock. The objectives of the present study were to evaluate the effects of NCG and ARG on GT1-7 cell gonadotrophin-releasing hormone (GnRH) secretion, gene expression and cell proliferation. GT1-7 cells were treated in vitro with different concentrations of NCG (0-1.0mM) or ARG (0-4.0mM) in serum-free medium for 12 or 24h. For GnRH secretion and cell proliferation, GT1-7 cells were more sensitive to NCG than ARG. NCG treatment after 12h increased cell numbers and inhibited GnRH secretion in a dose-dependent manner (P<0.05), although there was no significant effect of NCG on these parameters after 24h culture. ARG treatment decreased GnRH secretion after 24h (P<0.05), whereas it had no effect after 12h. GT1-7 cells express GnRH, Kiss-1 metastasis-suppressor (Kiss1), G-protein coupled receptor 54 (GPR54), neuronal nitric oxide synthase (nNOS) and estrogen receptor α (ERα) genes. High concentrations of NCG (1.0mM) and ARG (4.0mM) inhibited (P<0.05) GnRH and nNOS mRNA abundance in GT1-7 cells. ARG treatment decreased Kiss1 and increased ERα mRNA abundance. Thus, high concentrations of NCG (1.0mM) and ARG (4.0mM) may act both directly and indirectly to regulate GnRH neuron function by downregulating genes related to GnRH synthesis and secretion to slow GnRH production while stimulating GT1-7 cell proliferation.

Effects of N-carbamylglutamate and L-arginine on steroidogenesis and gene expression in bovine granulosa cells.

Feeding N-carbamylglutamate (NCG) and arginine (ARG) improves reproductive measures in pigs and reduces systemic steroid levels in pregnant ewes. We hypothesized that the effects of NCG and ARG on reproduction were due to direct effects on the ovary. Thus, the objectives of this study were to investigate the effects of NCG and ARG on granulosa cell (GC) steroidogenesis, gene expression, and cell proliferation in vitro. GC were collected from small (1-5mm) bovine follicles and treated in vitro with NCG or ARG in serum-free medium for 24h to 48h. Both NCG and ARG inhibited (P<0.05) IGF1- and FSH-induced GC estradiol production but only NCG inhibited (P<0.05) progesterone production. In contrast, NCG and ARG increased (P<0.05) GC numbers induced by IGF1 and FSH. NCG inhibited (P<0.05) StAR, CYP11A1 and CYP19A1 mRNA abundance in small-follicle GC, whereas ARG had no effect (P>0.10) on StAR, CYP11A1 or CYP19A1 mRNA abundance. We conclude that NCG and ARG may act directly on GC and therefore may regulate ovarian function by slowing follicular differentiation via inhibiting IGF1 action, and steroid synthesis while stimulating GC proliferation in cattle.

Effect of melatonin on bovine theca cells in vitro.

Melatonin affects granulosa cell function in several species but its function in theca cells is less clear, particularly in monotocous animals. Thus, the objectives of this study were to determine the effects of melatonin on theca cell steroidogenesis, gene expression and cell proliferation in a monotocous species, namely cattle. Ovaries were collected from a local bovine abattoir, from which theca cells were isolated from large (8-22mm) follicles and treated with various hormones in serum-free medium for 24h or 48h. Melatonin caused a dose-dependent inhibition (P<0.05) of LH+insulin-like growth factor 1 (IGF1)-induced androstenedione and progesterone production. Also, melatonin inhibited (P<0.05) LH+IGF1-induced expression of steroidogenic acute regulatory protein (StAR) mRNA (via real-time polymerase chain reaction) in theca cells, but it had no effect (P>0.10) on cytochrome P450 11A1 (CYP11A1) and cytochrome P450 17A1 (CYP17A1) mRNA abundance. In LH+IGF1-treated theca cells, melatonin decreased caspase 3 (CASP3) mRNA to levels similar to those observed in LH-treated theca cells. In contrast, melatonin increased (P<0.05) the number of bovine theca cells in both LH- and LH+IGF1-treated cultures. In conclusion, melatonin may act as an endocrine regulator of ovarian function in cattle by stimulating theca cell proliferation and inhibiting differentiation via inhibition of hormone-induced steroidogenesis.

Current status of the role of endothelins in regulating ovarian follicular function: A review.

Endothelins (EDN) are a group of vasoactive 21 amino acid peptides reported to play roles in steroidogenesis, folliculogenesis, and ovulation. EDN1, EDN2 and EDN3 have all been shown to affect granulosa cell (GC) function in a variety of mammalians species. Herewithin, the role of EDN in regulating steroidogenesis and ovarian follicular development is reviewed, focusing on the localization and function of EDN and their receptors in ovarian follicular function emphasizing species differences. For example, in single ovulating species such as humans and cattle, in the presence of trophic hormones such as FSH and IGF1, EDN1 and EDN2 significantly inhibited GC estradiol production in 2 of 4 studies, while no effect was observed for GC progesterone production in 2 of 4 studies. In contrast, EDN1 exhibited inhibitory effects on progesterone production by GC in 3 of 3 studies in pigs and 3 of 4 studies in rats. Also, EDN1 inhibited GC estradiol production in 4 of 5 studies in rats. Altogether, these results indicate that EDN are produced by ovarian follicles and are involved in the regulation of steroidogenesis of GC of several mammalian species including humans, cattle, pigs and rats, but that these effects may vary with species and culture condition.

Evidence that gene expression of ovarian follicular tight junction proteins is regulated in vivo and in vitro in cattle.

Tight junctions (TJ) are common paracellular sealing structures that control the transport of water, ions, and macromolecules across cell layers. Because the role of TJ in bovine follicular development is unknown, we investigated the developmental and hormonal regulation of the transmembrane TJ protein, occludin (OCLN), and the cytoplasmic TJ proteins, TJ protein 1 (TJP1) and cingulin (CGN) in bovine granulosa cells (GC) and theca cells (TC). For this purpose, bovine GC and TC were isolated from large (>8 mm) and/or small (1 to 5 mm) follicles and either extracted for real-time PCR (qPCR) or cultured in vitro. The abundances of both and mRNA were greater ( < 0.05) in TC than GC, whereas the mRNA abundance was greater ( < 0.05) in GC than TC. The abundance of mRNA in both GC and TC was greater ( < 0.05) in small follicles compared with large follicles, whereas the GC of large follicles had less ( < 0.05) mRNA abundance than the GC of small follicles. The abundance of mRNA in GC or TC did not differ ( > 0.10) among follicle sizes. In vitro treatment with various growth factors known to affect ovarian folliculogenesis indicated that , , and were hormonally regulated. Fibroblast growth factor 9 (FGF9) decreased ( < 0.05) the and mRNA abundances. Tumor necrosis factor α (TNFα) and vascular endothelial growth factor A (VEGFA) increased ( < 0.05) the mRNA abundance but decreased ( < 0.05) the mRNA abundance. Dexamethasone (DEX) increased ( < 0.05) and mRNA abundances. Epidermal growth factor (EGF) decreased ( < 0.05) and dihydrotestosterone (DHT) increased ( < 0.05) the abundances of , , and mRNA. We propose that the downregulation of OCLN and other TJ proteins during follicular development could reduce barrier function, thereby participating in increasing follicle size by allowing for an increase in the volume of follicular fluid as well as by allowing additional serum factors into the follicular fluid that potentially may directly impact GC functions. The results of the current study indicate the following in cattle: 1) gene expression of TJ proteins (i.e., , , and ) differs between GC and TC and changes with follicle size, and 2) autocrine, paracrine, and endocrine regulators, such as FGF9, EGF, DHT, TNFα, and glucocorticoids, modulate , , and mRNA abundance in TC in vitro.

G protein-coupled receptor 34 in ovarian granulosa cells of cattle: changes during follicular development and potential functional implications.

Abundance of G protein-coupled receptor 34 (GPR34) mRNA is greater in granulosa cells (GCs) of cystic vs normal follicles of cattle. The present experiments were designed to determine if GPR34 mRNA in granulosa cell [GC] changes during selection and growth of dominant follicles in cattle as well as to investigate the hormonal regulation of GPR34 mRNA in bovine GC in vitro. In Exp. 1, estrous cycles of nonlactating cows were synchronized and then ovariectomized on either day 3-4 or 5-6 after ovulation. GPR34 mRNA abundance in GC was 2.8- to 3.8-fold greater (P < 0.05) in small (1-5 mm) and large (≥8 mm) estrogen-inactive dominant follicles than in large estrogen-active follicles. Also, GPR34 mRNA tended to be greater (P < 0.10) in F2 than F1 follicles on day 3-4 postovulation. In Exp. 2-7, ovaries were collected at an abattoir and GC were isolated and treated in vitro. Expression of GPR34 was increased (P < 0.05) 2.2-fold by IGF1. Tumor necrosis factor (TNF)-α decreased (P < 0.05) the IGF1-induced GPR34 mRNA abundance in small-follicle GC, whereas IGF1 decreased (P < 0.05) GPR34 expression by 45% in large-follicle GC. Treatment of small-follicle GC with either IL-2, prostaglandin E2 or angiogenin decreased (P < 0.05) GPR34 expression, whereas FSH, cortisol, wingless 3A, or hedgehog proteins did not affect (P > 0.10) GPR34 expression. In Exp. 6 and 7, 2 presumed ligands of GPR34, L-a-lysophosphatidylserine (LPPS) and LPP-ethanolamine, increased (P < 0.05) GC numbers and estradiol production by 2-fold or more in small-follicle GC, and this response was only observed in IGF1-treated GC. In conclusion, GPR34 is a developmentally and hormonally regulated gene in GC, and its presumed ligands enhance IGF1-induced proliferation and steroidogenesis of bovine GC.

Effects of angiogenin on granulosa and theca cell function in cattle.

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P<0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P<0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.

Fibroblast growth factor 9 (FGF9) regulation of cyclin D1 and cyclin-dependent kinase-4 in ovarian granulosa and theca cells of cattle.

To determine the mechanism by which fibroblast growth factor 9 (FGF9) alters granulosa (GC) and theca (TC) cell proliferation, cell cycle proteins that regulate progression through G1 phase of the cell cycle, cyclin D1 (CCND1) and cyclin-dependent kinase-4 (CDK4; CCND1's catalytic partner), were evaluated. Ovaries were obtained from a local abattoir, GC were harvested from small (1-5 mm) and large (8-22 mm) follicles, and TC were harvested from large follicles. GC and TC were plated in medium containing 10% fetal calf serum followed by various treatments in serum-free medium. Treatment with 30 ng/mL of either FGF9 or IGF1 significantly increased GC numbers and when combined, synergized to further increase GC numbers by threefold. Abundance of CCND1 and CDK4 mRNA in TC and GC were quantified via real-time PCR. Alone and in combination with IGF1, FGF9 significantly increased CCND1 mRNA expression in both GC and TC. Western blotting revealed that CCND1 protein levels were increased by FGF9 in TC after 6 h and 12 h of treatment, but CDK4 protein was not affected. A mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway inhibitor, U0126, significantly reduced FGF9-induced CCND1 mRNA expression to basal levels. For the first time we show that CCND1 mRNA expression is increased by FGF9 in bovine TC and GC, and that FGF9 likely uses the MAPK pathway to induce CCND1 mRNA production in bovine TC.

Changes in fibroblast growth factor 9 mRNA in granulosa and theca cells during ovarian follicular growth in dairy cattle.

Fibroblast growth factor 9 (FGF9) has been suggested to act as an antidifferentiation factor in cattle by reducing steroidogenesis and increasing cell proliferation in granulosa (GC) and theca (TC) cells. The objective of this study was to characterize FGF9 mRNA abundance in GC and TC during development of dominant follicles in dairy cattle. Estrous cycles of nonlactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n=8) or 5 to 6 (n=8) postovulation for GC and TC RNA extraction from small (1-5mm), medium (5.1-8mm), and large (8.1-18mm) follicles for PCR analysis. The FGF9 mRNA abundance was greater in GC than in TC. In GC, FGF9 mRNA abundance was greater in small, medium, and large estrogen-inactive [i.e., concentrations of estradiol (E2)P4) follicles at both early (d 3-4) and late (d 5-6) growing phases of first dominant follicle. Abundance of FGF9 mRNA increased in medium-sized follicles from early to late growing phase of the dominant follicle. In TC, FGF9 mRNA abundance was greater in large E2-inactive follicles than in large E2-active follicles on d 3 to 4 postovulation; no significant differences in TC FGF9 mRNA existed among follicle types on d 5 to 6 postovulation. Correlations among levels of follicular fluid hormones and FGF9 mRNA levels revealed significant negative correlations between GC FGF9 mRNA abundance and follicular fluid E2 (r=-0.68), free IGF-1 (r=-0.63), and E2-to-P4 ratio (r=-0.58). In summary, abundance of FGF9 mRNA in GC and TC increases in medium-sized follicles during development of dominant follicles and is less in dominant E2-active than subordinate E2-inactive follicles, suggesting that FGF9 signaling could contribute to normal follicle development and steroidogenesis in dairy cattle.

Changes in brain ribonuclease (BRB) messenger RNA in granulosa cells (GCs) of dominant vs subordinate ovarian follicles of cattle and the regulation of BRB gene expression in bovine GCs.

Brain ribonuclease (BRB) is a member of the ribonuclease A superfamily that is constitutively expressed in a range of tissues and is the functional homolog of human ribonuclease 1. This study was designed to characterize BRB gene expression in granulosa cells (GCs) during development of bovine dominant ovarian follicles and to determine the hormonal regulation of BRB in GCs. Estrous cycles of Holstein cows (n = 18) were synchronized, and cows were ovariectomized on either day 3 to 4 or day 5 to 6 after ovulation during dominant follicle growth and selection. Ovaries were collected, follicular fluid (FFL) was aspirated, and GCs were collected for RNA isolation and quantitative polymerase chain reaction. Follicles were categorized as small (1-5 mm; pooled per ovary), medium (5-8 mm; individually collected), or large (8.1-17 mm; individually collected) based on surface diameter. Estradiol (E2) and progesterone (P4) levels were measured by radioimmunoassay (RIA) in FFL. Abundance of BRB messenger RNA (mRNA) in GCs was 8.6- to 11.8-fold greater (P < 0.05) in small (n = 31), medium (n = 66), and large (n = 33) subordinate E2-inactive (FFL E2 < P4) follicles than in large (n = 16) dominant E2-active (FFL E2 > P4) follicles. In the largest 4 follicles, GCs BRB mRNA abundance was negatively correlated (P < 0.01) with FFL E2 (r = -0.65) and E2:P4 ratio (r = -0.46). In experiment 2, GCs from large (8-22 mm diameter) and small (1-5 mm diameter) follicles were treated with insulin-like growth factor 1 (IGF1; 0 or 30 ng/mL) and/or tumor necrosis factor alpha (0 or 30 ng/mL); IGF1 increased (P < 0.05) BRB mRNA abundance, and tumor necrosis factor alpha decreased (P < 0.001) the IGF1-induced BRB mRNA abundance in large-follicle GCs. In experiment 3 to 6, E2, follicle-stimulating hormone, fibroblast growth factor 9, cortisol, wingless 3A, or sonic hedgehog did not affect (P > 0.10) abundance of BRB mRNA in GCs; thyroxine and luteinizing hormone increased (P < 0.05), whereas prostaglandin E2 (PGE2) decreased (P < 0.05) BRB mRNA abundance in small-follicle GCs. Treatment of small-follicle GCs with recombinant human RNase1 increased (P < 0.05) GCs numbers and E2 production. In conclusion, BRB is a hormonally and developmentally regulated gene in bovine GCs and may regulate E2 production during follicular growth in cattle.

Maintenance energy requirements of beef cows and relationship with cow and calf performance, metabolic hormones, and functional proteins.

Gestating Angus, nonlactating, spring-calving cows were used to determine variation in maintenance energy requirements (MR); to evaluate the relationship among MR and cow and calf performance, plasma concentrations of IGF-I, T4, glucose, insulin, and ruminal temperature; and to describe the LM proteome and evaluate protein abundance in cows with different MR. Cows (4 to 7 yr of age) with a BCS of 5.0 ± 0.2 and BW of 582 ± 37 kg in the second to third trimester of gestation were studied in 3 trials (trial 1, n = 23; trial 2, n = 32; trial 3, n = 38). Cows were individually fed a complete diet in amounts to meet predicted MR (Level 1 Model of NRC), and feed intake was adjusted weekly until constant BW was achieved for at least 21 d (maintenance). Cows were classified on the basis of MR as low (>0.5 SD less than mean, LMR), moderate (±0.5 SD of mean, MMR), or high (>0.5 SD more than mean, HMR) MR. Blood samples were taken at maintenance and at 2 mo postpartum in trial 2. Muscle biopsies were taken from LMR and HMR after cows consumed actual MR for 28 d (trial 2) or 21 d (trial 3). Proteins from LM were separated by 2-dimensional difference gel electrophoresis and were identified, and abundance was quantified and compared. The greatest differences in MR between cows were 29%, 24%, and 25% in trials 1, 2, and 3, respectively. Daily MR (NEm, kcal·BW(-0.75)·d(-1)) averaged 89.2 ± 6.3, 93.0 ± 4.9, and 90.4 ± 4.6 in trials 1, 2, and 3, respectively. Postpartum BW and BCS, calf birth and weaning weights, postpartum luteal activity, and ruminal temperature were not influenced by MR of the cows. Concentrations of IGF-I were greater (P = 0.001) in plasma of MMR compared with LMR cows consuming predicted MR diets, and MR was negatively correlated with concentrations of IGF-I in plasma (r = -0.38; P = 0.05) at 2 mo postpartum. A total of 103 proteins were isolated from LM; 52 gene products were identified. Abundance of specific proteins in the LM was not influenced (P > 0.11) by MR. Variation in MR of cows will make it possible to improve feed efficiency by selection. Identification of biomarkers for MR will allow selection of more efficient cows, which consume less feed and produce calves with similar weaning weights. Productive cows that require less feed for maintenance will improve efficiency of production and enhance sustainability of the environment.

Effects of fibroblast growth factor 9 on steroidogenesis and control of FGFR2IIIc mRNA in porcine granulosa cells.

The objectives of this study were to investigate the effects of fibroblast growth factor 9 (FGF9) on hormone-stimulated porcine granulosa cell proliferation and steroid production and to further elucidate the hormonal and developmental control of FGFR2IIIc gene expression in granulosa cells. Porcine ovaries were collected from a local slaughterhouse and granulosa cells were collected from small to medium (1 to 5 mm) follicles for 5 in vitro studies that were conducted. Cells were cultured for 48 h in 5% fetal calf serum plus 5% porcine serum and then treated with various combinations of FSH, IGF-I, FGF9, Sonic hedgehog (SHH), cortisol, PGE2, and/or wingless-type mouse mammary tumor virus integration site family member 5A (WNT5A) in serum-free medium for an additional 24 or 48 h. Medium was collected for analysis of steroid concentration via RIA, or RNA was collected for gene expression analysis of FGFR2IIIc via quantitative reverse transcription PCR. Fibroblast growth factor 9 stimulated (P < 0.05) IGF-I-induced estradiol production in the presence of FSH and testosterone. However, FGF9 had inconsistent effects on progesterone production, stimulating progesterone production in the presence of FSH and testosterone but inhibiting progesterone production in the presence of IGF-I, FSH, and testosterone. Cell numbers were increased (P < 0.05) by FGF9 in the presence of IGF-I and FSH but not in the presence of FSH and absence of IGF-I. For FGFR2IIIc mRNA studies, granulosa cells were treated with FSH, IGF-I, FGF9, SHH, cortisol, PGE2, or WNT5A. Follicle-stimulating hormone alone had no effect (P > 0.10) whereas IGF-I increased (P < 0.05) FGFR2IIIc mRNA abundance. Cortisol, PGE2, SHH, and WNT5A had no effect (P > 0.10) on FGFR2IIIc gene expression whereas FGF9 in the presence of FSH and IGF-I inhibited (P < 0.05) FGFR2IIIc gene expression. In an in vivo study, granulosa cells from large (7 to 14 mm) follicles had greater (P < 0.05) abundance of FGFR2IIIc mRNA than small (1 to 3 mm) or medium (4 to 6 mm) follicles. In conclusion, IGF-I-induced FGFR2IIIc mRNA may be a mechanism for increased responses to FGF9 in FSH plus IGF-I-treated granulosa cells. Fibroblast growth factor 9 and IGF-I may work together as amplifiers of follicular growth and granulosa cell differentiation by stimulating estradiol production and concomitantly stimulating granulosa cell growth in pigs.

Possible role of IGF2 receptors in regulating selection of 2 dominant follicles in cattle selected for twin ovulations and births.

Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E2), progesterone (P4), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E2-active (EA) and E2-inactive (EI) follicles were selected according to their E2-to-P4 ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E2 and P4 concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles.