Mammalian ovarian follicles are formed in the female ovary either before or after birth in different species. The first type of ovarian follicles are a population of dormant primordial follicles, which contain a primary oocyte arrested at prophase I of the first meiotic division surrounded by granulosa cells. The number of primordial follicles that are formed in the ovary can be anywhere between hundreds, thousands or even millions, which differs across species. The peak number is suggested to be fixed and this population of dormant primordial follicles serve as the oocyte reservoir for future fertility. Ovarian folliculogenesis involves the activation of dormant primordial follicles and development into primary, secondary and antral follicle stages towards the ovulation of the oocyte. The regulation of ovarian folliculogenesis is crucial to female fertility. Many studies, predominantly performed in rodents, focused on elucidating the molecular mechanisms involved in ovarian follicle and oocyte development. There are therefore still major gaps in our knowledge for larger animal models. This is particularly true for early follicle development, from the primordial primary to secondary stages, for which the molecular mechanisms remain to be fully elucidated in mammals. Using the domestic cat (Felis catus) as a model organism, we hypothesised that primordial to primary and primary to secondary follicle development is regulated by specific signalling pathways and characterized by specific gene expression patterns. Through a series of experiments, we aimed to describe transcription and protein expression during early follicle development. Domestic cat ovaries were obtained from animal shelters where routine ovariectomies were performed. The mechanical dissection technique was used to isolate whole early follicles, which consist of an oocyte and the surrounding somatic cells, from domestic cat ovaries. We collected primordial, primary and secondary follicles. Prior to ribonucleic acid (RNA) isolation, follicle samples were pooled for each type to increase the number of follicles per sample. Libraries were prepared for RNA-sequencing and custom index primers allowed us to pool the libraries prior to sequencing. In our first publication, we presented the analysis of the RNA-sequencing data, which revealed genes that were significantly differentially expressed during early follicle development in this species. We identified specific molecular mechanisms which may be involved in the regulation of early folliculogenesis. For example, we found that the phosphatidylinositol-3-kinase and protein kinase B (PI3K/Akt) and the transforming growth factor beta (TGF-β) signalling pathways were involved in early follicle development in the domestic cat. Additionally, we 14 identified that the extracellular matrix (ECM) of the ovary was participating during both developmental transitions in this species too. For validation purposes, we compared the gene expression levels of the RNA-sequencing results to quantitative reverse transcription polymerase chain reaction (qRT-PCR) data for two genes: bone morphogenetic protein 15 (BMP15) and Histone 1, H1t (HISTH1T). In the RNA-sequencing data, we found that BMP15 and HISTH1T were significantly differentially expressed during early follicular development. The qRT-PCR data was mainly concordant with these results. During our analysis for the first publication we also identified genes involved in ovarian steroidogenesis during early follicle development. In our second publication, we hypothesised that gonadotropin and sex steroid signalling is involved in early folliculogenesis and that early follicles are a source of sex steroids in the domestic cat so further investigations were pursued. We immunostained ovarian tissue sections and investigated the localisation of gonadotropin receptors, sex steroid receptors and steroidogenic enzyme proteins. We found that gonadotropin and sex steroid receptor protein signals were detected in different follicular locations in early follicles and at different intensities during early follicle development in the domestic cat. In comparison to the primordial and primary stages, when no protein signals were detectable for the analysed steroidogenic enzymes, we found that protein signals for some of them were detectable by the secondary stage. Although early follicles are fully equipped at the level of gene expression to produce sex steroids, we conclude from the protein data that it is may be possible that only secondary follicles are a source of sex steroids but not the earlier stages. We measured gene expression levels for three steroidogenic enzymes, the androgen receptor, progesterone receptors and a cholesterol transporter using qRT-PCR. The expression levels were too low to make a conclusive comparison to the RNA-sequencing results and no statistical significance was estimated for the expression of these genes during early folliculogenesis studied using qRT-PCR. In the future, a larger number of follicles per sample may overcome this and provide more insight into a smaller subset of genes. In conclusion, we presented two in-depth studies which investigated in the domestic cat for the first time potential key genes, signalling pathways and molecular mechanisms that may be regulating early follicle development. We are contributing toward an improved understanding of the principles of early follicle development in mammalian species – a topic of great interest in reproduction biology. Our results may also be useful for designing in vitro experiments for the culture of early domestic cat follicles in the future.
