Preantral follicles serve as a reservoir of female gametes that could be used in assisted reproductive technologies in humans, livestock, and endangered animals. Invitro culture of ovarian cortex is a widely used method to grow preantral follicles. Follicle-stimulating hormone (FSH) is often added to the culture medium as a folliculogenesis-promoting factor. The roles of FSH in antral follicles is well known; however, the effects of FSH in preantral follicles and indirectly in the ovarian cortical cells is largely unknown. The aim of this study was to determine the transcriptomic responses of the ovarian cortex containing preantral follicles to FSH signalling over time. In 3 biological replicates, small strips of bovine ovarian cortex (10×5mm) were dissected from the medulla and evaluated under a stereomicroscope for removal of all visible antral follicles. Resulting cortical strips were cultured in defined medium with human-recombinant FSH or vehicle for 2 or 4 days at 38.5°C and 5% CO2. The RNA was isolated and subjected to cDNA library preparation and 3′-Tag RNA sequencing. Sequencing data analysis was performed using the edgeR and maSigPro packages (Bioconductor-R). Using a time-course analysis, genes up- or downregulated 2-fold or more and associated with an FDR<0.05 were considered differentially expressed (DEG) and were further analysed with NetworkAnalyst software. We found 252 DEG over time in response to FSH. In FSH-treated samples, significantly enriched biological functions from upregulated genes were associated with glycolysis, gluconeogenesis, carbon metabolism, and biosynthesis of amino acids. In contrast, significantly enriched biological functions from downregulated genes found in FSH-treated samples included phagosome and necroptosis. The germ cell markers BMP15, DAZL, DDX4, GDF9, and ZP2/ZP3 were expressed but unchanged by FSH, suggesting the presence of similar numbers of oocytes between samples. The gene B4GALT2, previously reported as a granulosa cell marker, was upregulated in FSH-treated samples at Day 4. The follicular marker RAB23 was expressed in all samples and not changed by FSH. One interesting finding was upregulation of MAPK signalling (represented by the genes MAPKAPK3, ELK4, MKNK2, and TGFB3) in response to FSH signalling, with no change in expression of the cAMP-response element responsive genes CYP19A1 and INHA. Together, these data indicate that FSH stimulates energy metabolism in ovarian cortical cells and represses negative cell function activity. We conclude that these responses are mostly mediated by granulosa cells, because the FSH receptor is not appreciably expressed in the ovarian cortex stroma. Moreover, the data suggest that FSH may utilise alternative signalling pathways, such as MAPK, in early follicles. This information enhances our understanding of FSH signalling pathways in the ovarian cortex, mediated by preantral follicles to create a positive environment for folliculogenesis.
Instability of the Homogeneous Distribution of Chemical Waves in the Belousov–Zhabotinsky Reaction
