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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. [ABSTRACT FROM AUTHOR]

• Granulosa cell fibrillin 1 mRNA abundance was increased during follicle selection. • Granulosa cell fibrillin 1 mRNA abundance was decreased by insulin-like growth factor 1. • Olfactory receptor 4M1 mRNA abundance was increased by progesterone. • Asprosin production and its receptor are hormonally regulated in 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: TGFβ1 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. [ABSTRACT FROM AUTHOR]

Abstract: Insulin-like growth factor-I (IGF-I) plays a pivotal role in cattle fertility, acting as a monitoring signal that allows reproductive events to occur when nutritional conditions for successful reproduction are reached. However, endocrine IGF-I is not a predictor of reproductive events, but rather an indirect estimator of the suitability of the animal to achieve the reproductive event in question. Although measuring circulating IGF-I concentrations might not have any clinical application in the cattle industry, endocrine IGF-I screening will continue to be important for the study of interactions between nutrition and reproduction. In addition, endocrine IGF-I screening could be used as an ancillary test for the selection of cattle for high reproductive potential, especially in herds of high genetic merit for milk production, in which a decline in fertility has been identified. [Copyright &y& Elsevier]

Abstract: The effect of plasma from cyclic versus nutritionally induced anovulatory beef heifers was evaluated on proliferation of bovine granulosa cells in vitro. Granulosa cells were obtained from small (1–5mm) follicles of cattle and cultured for 4 days. During the last 2 days of culture, cells were exposed to medium containing 0, 1 or 10% plasma from cyclic or anovulatory heifers in the presence or absence of IGF-I (100ng/ml). Cell numbers were determined. Regardless of source, increasing percentage of plasma to culture medium increased cell numbers. However, the plasma-induced increase was greater in granulosa cells exposed to cyclic heifer plasma versus anovulatory heifer plasma. In addition, concomitant treatment with IGF-I dramatically improved cell proliferation induced by anovulatory heifer plasma. These results indicate that plasma from cyclic heifers contain factors that are a greater stimulus to granulosa cell proliferation than plasma from anovulatory heifers. Systemic factors such as IGF-I may play a role in directly regulating granulosa cell proliferation in cattle. [Copyright &y& Elsevier]

Comparisons of numbers of antral ovarian follicles and corpora lutea (CL), of blood hormone concentrations, and of follicular fluid steroid concentrations and IGFBP activity were conducted between cows selected (twinner) and unselected (control) for twin births to elucidate genetic differences in the regulation of ovarian follicular development. Ovarian follicular development was synchronized among cows by a single i.m. injection of PGF2α on d 18 of the estrous cycle; six cows per population were slaughtered at 0, 24, 48, and 72 h after PGF2α. Jugular vein blood was collected from each animal at PGF2α injection and at 24-h intervals until slaughter. Ovaries of twinner cows contained more small (≤5 mm in diameter, P < 0.05), medium (5.1 to 9.9 mm, P < 0.05), and large (≥10.0 mm, P < 0.01) follicles and more (P < 0.01) CL than ovaries of controls. Follicular fluid concentrations of estradiol, androstenedione, testosterone, and progesterone reflected the stage of follicular development and were similar for twinner and control follicles at the same stage. Earlier initiation of follicular development and/or selection of twin-dominant follicles in some twinner cows resulted in greater concentrations of estradiol in plasma at 0, 24, and 48 h and of estradiol, androstenedione, and testosterone in follicular fluid of large follicles at 0 h after PGF2α for twinner vs. control cows (follicular status × time × population, P < 0.01). Binding activities of IGFBP-5 and -4 were absent or reduced (P < 0.01) in follicular fluid of developing medium and large estrogen-active (estradiol:progesterone ratio > 1) follicles but increased with atresia. Only preovulatory Graafian follicles lacked IGFBP-2 binding, suggesting a possible role for IGFBP-2 in selection of the dominant follicle. Concentrations of IGF-I were twofold greater (P < 0.01), but GH (P = 0.10) and cholesterol (P < 0.05) were less in blood of twinners. Three generations of selection of cattle for twin ovulations and births enhanced ovarian follicular development as manifested by increased numbers of follicles within a follicular wave and subsequent selection of twin dominant follicles. Because gonadotropin secretion and ovarian steroidogenesis were similar for control and twinner cattle, enhanced follicular development in twinners may result from decreased inhibition by the dominant follicle(s), increased ovarian sensitivity to gonadotropins, and/or increased intragonadal stimulation, possibly by increased IGF-I. Key Words: Cattle; Gonadotropins; Insulin-Like Growth Factor Binding Proteins; Ovarian Folliculogenesis; Steroids; Twins [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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 (1–5 mm) bovine follicles and treated in vitro with NCG or ARG in serum-free medium for 24 h to 48 h. 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. [ABSTRACT FROM AUTHOR]

• FGFR1c and FGFR2c mRNA abundance is least in granulosa cells of dominant follicles. • Estradiol decreased FGFR2c mRNA abundance in granulosa cells. • Androgen increased FGFR1c, FGFR2c and FGFR4 mRNA abundance in granulosa cells. • FGFR3c and FGFR4 mRNA abundance in granulosa cells is similar among follicle types. • FGFR2c and FGFR3c mRNA abundance in theca cells is similar among follicle types. 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. [ABSTRACT FROM AUTHOR]