Ovarian follicles constitute the functional unit of ovaries and supports the development and maturation of oocytes. They are pivotal to estrogen production, estrus behaviour, estrous cyclicity, oocyte competency and post-ovulation corpus luteum formation (Chu et al. 2017). Follicular health comprises a major determinant of fecundity and reproductive efficiency in females of different species, especially in litter bearing domestic animals like pigs, wherein farm economics is directly proportional to high litter size (Bharati et al. 2022a). The gonadotrophin dependent growth of ovarian follicles ensues after puberty in gilts and there is a cyclic recruitment of follicles, followed by developmental transition from small follicles (SF) to large follicles (LF) during short follicular phase of 5-7 days, in a span of 18-24 days (an average of 21 days) estrous cycle in pigs (Soede et al. 2011). Follicles are a highly heterogenous tissue, comprising of theca cell layer, granulosa cells, immune cells, oocyte, follicular fluid and the micro-vasculatures (Gad et al., 2020, Yang et al., 2019). The number of granulosa cells, oocyte quality, steroid profile, vasculature pattern and volume of follicular fluid changes during the course of development from SF to LF (Costermans et al., 2020). The LF matures into pre-ovulatory follicle, which ovulates in response to pre-ovulatory LH surge from anterior pituitary leading to the transformation of a fluid filled follicle into transient endocrine gland, the corpus luteum (Bharati et al. 2021).

Follicular development is in fact a complex process involving increase in follicular size and fluid volume, oocyte maturation, change in theca and granulosa cell functionality along with the shift in immune milieu (Chu et al., 2017, LaVoie, 2017, Yaakov et al., 2023). Previous studies have established the expression of transcription factors (GATA, E2F1, FOXO3a), genes encoding growth factors (VEGF, FGF, IGF), growth differentiation factors (GDFs) and their receptors within the ovary along with extracellular matrix (ECM) and immune components, which not only modulate the action of gonadotropins on ovarian follicles but also regulate key cellular events like proliferation, differentiation, migration, cell cycle and cell fate determination (Akkoyunlu et al. 2003; Goldman and Shalev, 2004; LaVoie 2017; Mishra et al. 2017; Yang et al. 2019; Morrell et al. 2020; Zhang et al. 2020). These factors have been proposed to regulate signalling pathways associated with follicular growth and development (Li et al. 2021); however, the complete repertoire of intrafollicular intrinsic factors is still inconclusive in pigs, which can be comprehensively obtained through high throughput techniques like RNA-Seq (Gong et al. 2023).

Ovarian follicles form a major component of assisted reproductive technology (ART) based programmes in humans and animals. Follicles are either isolated form ovaries collected from slaughter houses or oocytes are retrieved in-vivo from follicles and subsequently used for in-vitro maturation (IVM), in-vitro fertilization (IVF), in-vitro embryo production (IVEP) and transfer (Taghizabet et al. 2022). Hence, knowledge on repertoire of transcriptionally active genes and their interactions during different stages of follicular development is critical to successful ART programmes. Moreover, follicular cells viz. granulosa and theca cells, are employed as in vitro cell culture model system for studying effects of various endocrine and toxicological agents on ovarian physiology (Gilchrist, 2010, Mlynarcikova and Scsukova, 2021), which necessitates information on global gene dynamics during SF and LF stage, as this may affect the outcome of in-vitro research. With the advent of high throughput RNA-sequencing techniques and varied bioinformatics analysis platforms, the exploration and functional association of multiple genes and signalling pathways with several key biological processes has essentially become critical to fundamental understanding of follicular growth (Fan et al. 2019). Studies in bovines have confirmed the distinct transcriptomic features of ovarian follicles based on size using microarray (Hatzirodros et al. 2014), nevertheless the transcriptome profiling of ovarian follicles and their in-depth study during estrous cycle are not completely understood in Ghoongroo pigs. These are the indigenous pig breed of India, with black skin, bulldog face appearance and high prolificacy under low input system. They are the lesser-known prolific pig genetic resources, whose ovarian physiology has not been explored at the molecular levels. Hence, the present study was undertaken to gain insights into the critical processes and transcriptomic signatures during key developmental stage of ovarian follicle transition from SF to LF during estrous cycle in Ghoongroo pigs.