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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]

• 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]

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]

Abstract: Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease. [Copyright &y& Elsevier]

Abstract: Ovarian follicular growth and dominance are controlled by a series of hormonal and intraovarian events including a decrease in intrafollicular IGF-binding proteins −2, −4 and −5 levels. Proteolytic enzymes such as pregnancy-associated plasma protein-A (PAPP-A) degrade IGFBPs and increase bioavailability of IGF-I and -II during follicular development. The objective of this study was to determine the effect of IGF-I, IGF-II, insulin (INS), LH, FSH, estradiol (E2), leptin or cortisol on ovarian PAPP-A mRNA levels. Granulosa (GC) from small (SM) (1–5mm) and large (LG) (8–22mm) follicles as well as theca cells (TC) from LG follicles were collected from bovine ovaries and cultured for 48h in medium containing 10% FCS and then treated with various hormones in serum-free medium for an additional 24h. Cells were treated with various concentrations (3–500ng/ml) and combinations of IGF-I, IGF-II, FSH, LH, E2, INS, leptin and (or) cortisol for 24h (Experiments 1–10). PAPP-A mRNA levels were measured using quantitative real-time RT-PCR. In SM-GC and LG-GC, none of the treatments significantly affected (P >0.10) PAPP-A mRNA abundance. In LG-TC, IGF-I, LH or cortisol did not affect (P >0.10) PAPP-A mRNA levels, whereas INS with or without LH decreased (P <0.05) PAPP-A mRNA. E2 alone decreased PAPP-A mRNA levels in LG-TC, and E2 amplified the insulin-induced inhibition of PAPP-A mRNA abundance in LG-TC. We conclude that control of PAPP-A mRNA abundance in granulosa and theca cells differs, and that E2 may be part of an intraovarian negative feedback system which may reduce the bioavailable IGFs in the theca layer during growth and selection of follicles. [Copyright &y& Elsevier]

The effects of estradiol, insulin, and gonadotropins on levels of insulin-like growth factor binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and theca cells were evaluated in vitro using serum-free medium containing various hormone treatments arranged in four different experiments. Amounts of IGFBP-2, -3, -4 and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. In small-follicle (1–5 mm) granulosa cells, follicle-stimulating hormone (FSH) in the presence or absence of insulin increased (P<0.05) IGFBP-3 mRNA but did not change IGFBP-2, -4, or -5 mRNA levels; estradiol was without effect on IGFBP-2, -3, -4, or -5 mRNA levels in the absence of insulin but increased (P<0.05) IGFBP-2 mRNA levels in the presence of insulin. Luteinizing hormone (LH) in the absence (but not presence) of insulin increased (P<0.05) small-follicle granulosa cell IGFBP-3 mRNA levels. In large-follicle (>7.9 mm) granulosa cells, insulin alone increased (P<0.05) IGFBP-2 gene expression while LH, FSH, and estradiol were without effect (P>0.10). Estradiol (3 and 300 ng/ml) decreased (P<0.05) IGFBP-5 mRNA levels in large-follicle granulosa cells. In theca cells, insulin decreased (P<0.05) IGFBP-4 expression, but had no effect (P>0.10) on IGFBP-2, -3, or -5 mRNA levels. Estradiol decreased (P<0.05) IGFBP-2, -3, and -4 mRNA levels but had no effect on IGFBP-5 mRNA levels in theca cells. LH had no effect on levels of IGFBP-2, -3, -4, or -5 mRNA in theca cells. These results indicate that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by estradiol, insulin and gonadotropins, therefore discretely modulating the amount of bioavailable IGFs to these cells depending upon the specific hormonal stimuli. In particular, these studies are the first in cattle to show that estradiol selectively inhibits IGFBP-2, -3, and -4 gene expression in theca cells, inhibits IGFBP-5 gene expression in large-follicle granulosa cells, and stimulates IGFBP-2 gene expression in small-follicle granulosa cells. [Copyright &y& Elsevier]

Wilms' tumor gene WT1 encodes a nuclear transcriptional factor, which has been shown to regulate granulosa cell steroidogenesis in bovine; however, it is not known whether the functions of theca cells are regulated by WT1. Here, we determined the effects of this gene on theca cell proliferation, apoptosis, and steroidogenesis in vitro. In cultured bovine theca cells, the downregulation of WT1 increased the secretion of progesterone but had no effect on proliferation and apoptosis. WT1 includes the variants WT1(+KTS) and WT1(−KTS), which differ by 3 amino acids KTS (lysine, threonine, and serine). WT1(±KTS) upregulation increased the messenger RNA (mRNA) expression of STAR and CYP17A1 and decreased the progesterone secretion and CYP11A1 mRNA expression. In contrast to WT1(+KTS), WT1(−KTS) upregulation also decreased the mRNA expression of 3β-HSD. In both variants, WT1(−KTS) has more obvious effects. In conclusion, WT1 can decrease progesterone secretion, likely due in part to the inhibition of CYP11A1 and 3β-HSD. • WT1 was found to decrease progesterone production. • The decrease of progesterone production likely due in part to the inhibition of CYP11A1 and 3β-HSD. • In both variants, WT1(−KTS) has more obvious effects. [ABSTRACT FROM AUTHOR]