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.
Endothelins are a group of vasoactive 21 amino acid peptides reported to play roles in steroidogenesis, folliculogenesis, and ovulation (Bridges et al. 2012 Life Sci. 91, 501–506). Nevertheless, the role of endothelins in regulating steroidogenesis in the bovine species requires further investigation. Thus, the objective of this study was to investigate the effects of endothelin 1 (ET-1) and endothelin 2 (ET-2) on bovine granulosa cell (GC) steroidogenesis. Bovine ovaries were obtained from a local abattoir. Follicular fluid was aspirated from small (1–5 mm) follicles and GC were isolated and exposed to various treatments (ET-1, ET-2, or ET-1 plus ET-2 with FSH and with or without insulin-like growth factor-1). In replicated experiments, culture medium was removed and analysed for steroid production via radioimmunoassay. Granulosa cells were either harvested with trypsin and counted using a Coulter Counter or collected with Trizol for RNA extraction and quantification via real-time PCR (18S rRNA was used as a housekeeping gene). Steroid production was expressed as nanograms (in the case of progesterone) and picograms (in the case of oestradiol) per 105 cells per 24 h. Relative quantity of target gene mRNA was expressed as 2–ΔΔCt using the relative comparative threshold cycle (Ct) method. Data were analysed via ANOVA and the general linear models (GLM) procedure of SAS for Windows (SAS Institute Inc., Cary, NC). If a significant main effect was identified, differences among means were determined by Fisher’s protected least significant differences test. The values were reported as least squares means ± standard error of the mean. In the presence of insulin-like growth factor-1, ET-1 significantly inhibited oestradiol production at 300 ng mL–1 (100.30 ± 11.05; P < 0.05), but not at 30 ng mL–1 (114.47 ± 11.05; P > 0.05) in comparison to the control (141.21 ± 11.05), whereas no differences were observed for progesterone production at 300 ng mL–1 (60.11 ± 7.11; P > 0.05) or at 30 ng mL–1 (64.02 ± 7.11; P > 0.05) in comparison to control (76.75 ± 7.11). ET-2 also significantly inhibited oestradiol production at 300 ng mL–1 (91.08 ± 11.87; P < 0.01), but not at 30 ng mL–1 (112.77 ± 11.87; P > 0.05) in comparison to the control in the presence of insulin-like growth factor-1. No significant effect of ET-1 and ET-2 was observed on steroidogenesis of granulosa cells cultured without insulin-like growth factor-1. Consistent with steroids production data, real-time PCR results indicated that, in the presence of IGF-1, ET-1 (5.66 ± 1.05) and ET-2 (5.65 ± 1.05) inhibited (P < 0.05) aromatase gene expression compared to controls (11.33 + 1.05), and ET-1 plus ET-2 (2.42 ± 1.05) reduced (P < 0.05) expression below that observed with either alone. No effect of ET-1 (4.38 ± 0.95; P > 0.05), ET-2 (5.94 ± 0.95; P > 0.05), or ET-1 plus ET-2 (4.57 ± 0.95; P > 0.05) was observed for side-chain cleavage enzyme (CYP11A1) in comparison to controls (4.4 ± 1.07). Altogether, these results indicate that endothelins are involved in the regulation of steroidogenesis of bovine GC.
Fibroblast growth factor 9 (FGF9) has been suggested to act as a dedifferentiation factor during bovine folliculogenesis, reducing steroidogenesis and increasing cell proliferation in granulosa (GC) and theca (TC) cells, but whether endogenous GC production of FGF9 change during bovine folliculogenesis and atresia/apoptosis is unknown. The objective of these studies was to investigate the relationship between FGF9 mRNA, follicle size, and health status of follicles. Ovaries (n = 10 cows) from a local abattoir classified visually as in midcycle phase (i.e. presence of corpus luteum and large follicles) were collected and categorized as small (1–5 mm), medium (5.1–8 mm) or large (8.1–22 mm) in size (Experiment 1). Follicular fluid (FFL) was aspirated for measurement of oestradiol (E2) and progesterone (P4) via radioimmunoassay and GC collected for RNA extraction. Abundance of mRNA for FGF9 and Caspase-3 (CASP3), an effector of apoptosis, were measured by real-time PCR (qPCR). Data were analysed via factorial ANOVA with main factors: follicle size, follicle estrogenic status, and their interaction. The abundance of GC FGF9 mRNA was greater (P < 0.05) in large E2-inactive (E2P4 concentrations) follicles (10.5 ± 22). The abundance of GC CASP3 mRNA was greater (P < 0.01) in small E2-inactive follicles than in large and medium E2-active and E2-inactive follicles. FGF9 mRNA abundance was not correlated with E2/P4 ratio in FFL, but it was positively correlated with CASP3 mRNA abundance (r = 0.35; P < 0.05). GC CASP3 mRNA abundance was negatively correlated with E2/P4 ratio (r = –0.48; P < 0.01). To investigate the relationship between FGF9 and CASP3 mRNA abundance during experimentally-induced apoptosis, GC from large and small follicles were collected (Experiment 2) and GC were plated in medium containing 10% FCS. GC (n = 3 independent pools for small and large follicles) were then treated with or without 10% FCS for an additional 24 h or 48 h followed by RNA extraction and qPCR for measurement of abundance of FGF9 and CASP-3 mRNA. Statistical analyses with ANOVA included main factors: treatment, duration of treatment, and their interaction. In small-follicle GC, FGF9 and CASP3 mRNA abundance were not correlated and were not affected by treatments. In large follicles, FGF9 mRNA abundance was greater in GC treated without FCS (27.5 ± 2.7) than in GC treated with 10% FCS (6.6 ± 2.7) and tended to differ (P < 0.08) between 24 h (22.5 ± 2.7) and 48 h (11.6 ± 2.7). CASP3 mRNA abundance was greater in GC treated without FCS (310 ± 36) than in GC treated with 10% FCS (140 ± 36) but did not differ (P > 0.10) between 24 h and 48 h. In Experiment 2, there was no significant correlation between FGF9 and CASP3 mRNA (r = 0.28; P = 0.2). These results indicate that FGF9 mRNA abundance is greater in GC from large E2-inactive than from E2-active follicles and its production may be increased in large follicles undergoing apoptosis.
Fibroblast growth factors (FGF) regulate folliculogenesis of several species, including cattle. The cellular responses to a particular FGF are influenced by the diversity of high affinity fibroblast growth factor receptors (FGFR). There are 4 distinct genes encoding FGFR in vertebrates and the occurrence of mRNA splicing in the immunoglobulin-like domain III generates a diversity of sequences, and results in various isoforms of FGFR1, FGFR2, and FGFR3 (but not of FGFR4). Because FGFR have different ligand specificities, the presence of FGFR in bovine antral follicles is of fundamental importance for the FGF to exert their effects in the ovary. Hence, the objective of this study was to determine if FGFR1c, FGFR2c, FGFR3c, and FGFR4 mRNA abundance in granulosa cells (GC) change according to follicular size, steroidogenic status, and days post-ovulation during growth of first-wave dominant follicles in cattle. Oestrous cycles of non-lactating dairy cattle were synchronized and ovaries were collected on either Day 3–4 (n = 8) or Day 5–6 (n = 8) post-ovulation (as assessed by rectal ultrasonography). Follicular fluid (FFL) was aspirated from small (1–5 mm), medium (5.1–8 mm), or large (8.1–18 mm) follicles for measurement of oestradiol (E2) and progesterone (P4) levels by radioimmunoassay, and GC were collected for mRNA extraction. Relative quantity of target gene mRNA was expressed as 2−ΔΔCt using the comparative threshold cycle (Ct) method. Data were transformed to natural log (x + 1), to correct for heterogeneity of variance, and analysed via factorial ANOVA with the general linear model procedure of SAS and are reported as least squares means ± s.e.M. Follicle group (based on steroidogenic status and size of follicles), but not days post-ovulation or their interaction, significantly affected FGFR1c, FGFR2c, and FGFR3c mRNA abundance, whereas FGFR4 mRNA abundance was not affected by follicle group or days post-ovulation. FGFR1c mRNA abundance was greater (P < 0.01) in large (44.8 ± 11.3; n = 29), medium (63.8 ± 7.6; n = 64), and small (44.6 ± 11.2; n = 29) E2-inactive (FFL E2/P4 ratio < 1) than in large E2-active (FFL E2/P4 ratio > 1) follicles (10.5 ± 15.5; n = 16) and greater (P < 0.05) in medium E2-inactive than in large and small E2-inactive follicles. FGFR2c mRNA abundance was greater (P < 0.01) in large (423.9 ± 131.9), medium (585.8 ± 97.0), and small (273.6 ± 143.2) E2-inactive than in large E2-active (56.2 ± 195.6) follicles. The FGFR3c mRNA abundance was greater (P < 0.05) in large (143.4 ± 40.2) and medium (160.2 ± 29.3) E2-inactive than in large E2-active (43.2 ± 58.6) follicles and tended to be greater (P = 0.06) in small E2-inactive (101.9 ± 42.9) than in large E2-active follicles. Taken together, the findings that FGFR1c, FGFR2c, and FGFR3c mRNA abundance is lower in GC of E2-active follicles during growth of the first dominant follicle support an anti-differentiation role for these FGFR as well as support the idea that some FGF may regulate the selection of dominant follicles in cattle.
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