dc.description.abstract
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.
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