MicroRNAs (miRNAs) are a class of small single stranded endogenously expressed RNAs, 19-23 nucleotide long that negatively modulate complementary mRNA. They are highly conserved in eukaryotes and constitute ~1% of the total genome which can target more than 30% of the transcriptome (Lewis et al, 2005). Mainly, miRNA regulates gene expression by translational suppression, mRNA cleavage and deadenylation. In mammals, miRNAs regulate numerous biological functions including spermatogenesis, ovarian function, embryonic development (Hayashi et al, 2008; Lei et al, 2010; Otsuka et al, 2011; Houbaviy et al, 2003) while their dysregulation has been associated with many diseases. Functionally, it has been demonstrated that a specific miRNA can have multiple targets and a specific mRNA can be targeted by multiple miRNAs (Kim and Nam, 2006). miRNA genes are mainly found in intergenic (between genes) or intronic (within introns) regions but some are encoded by exonic regions as well (Rodriguez et al, 2004). They are usually transcribed by RNA polymerase II as the initial primary miRNA (pri-miRNA). These pri-miRNAs are then processed into precursor miRNAs (pre-miRNAs) through either canonical or non-canonical pathway.
During canonical miRNA biogenesis pathway, a multiprotein complex known as Microprocessor processes the pri-miRNA within the nucleus into pre-miRNA. Microprocessor complex constitute the nuclear RNase III enzyme (Drosha) and DiGeorge syndrome critical region 8 (DGCR8). The pri-miRNA binds with the RNA binding protein DGCR8 and is cleaved by Drosha yielding ~ 70 nucleotide stem loop pre-miRNA (Gregory et al, 2004; Han et al, 2006; Landthaler et al, 2004). Non-canonical miRNA biogenesis varies at this phase in that pre-miRNAs are formed by mRNA splicing machinery, avoiding the need for Drosha facilitated cleavage in the nucleus (Chong et al, 2010). In both pathways, the pre-miRNAs are then transferred to the cytoplasm through the nuclear export protein exportin-5 via a Ran-GTP dependent mechanism (Lund et al. 2004). Upon export to cytoplasm, the pre-miRNA is cleaved by a second RNAse III enzyme, DICER, into a transient RNA duplex of ~19–23 nucleotides. Following the dicer processing, the duplex RNA is further cleaved into two strands, passenger and guide strands. The passenger strand is selectively being degraded while the remaining guide strand is preferentially loaded onto the RNA-induced silencing complex (RISC). The RISC consists of one of the four different Argonaute (Argo) proteins, DICER and a TARRNA-binding protein (TRBP) (Hutvagner and Simard, 2008). Once mature miRNAs are loaded into the RISC, miRNAs are able to bind and modulate the expression of their target mRNAs. The binding of target mRNA with miRNA-induced silencing complex (miRISC) is mediated by a sequence of 2–8 nucleotides (seed region), at the 5? end of the mature miRNA. If the binding exhibits perfect base-pairing, the mRNA transcript are decayed and mRNA translation does not happen. If this binding homology between miRNA seed region and target mRNA is imperfect, mRNA translation is repressed. This probably occurs through an inhibition of translation after transcriptional initiation (Jackson andStandart, 2007) or via an interaction of eukaryotic translation initiation factor 6 (EIF6) with RISC that prevents assembly of 80S ribosomes (Chendrimada et al, 2007). In mammalian cells, Pbody proteins are thought to be involved in mRNA degradation that is the dominant mode of action of miRNAs. Pbodies are enriched with proteins and enzymes involved in mRNA deterioration and sequestration from translational machinery. Pbody constituents, such as the ATP-dependent RNA helicase p54 (also known as RCK and DDX6) (Chu et al, 2006), mRNA-decapping enzyme 1 (DCP1), DCP2 (Behm-Ansmant et al, 2006) and GW182 (also known as TNRC6A) (Liu et al, 2005; Eulalio et al, 2008) have been shown to physically interact with Argonaute proteins and are necessaryfor miRNA-mediated gene suppression.
Ovarian folliculogenesis is a fundamental and necessary process to produce competent mature oocyte at ovulation. The early folliculogenesis begins with the breakdown of germ cell cluster, leading to the formation of dormant primordial follicles (Pepling and Spradling, 2001). These follicles then undergo different stages of maturation to produce a functional egg. In order to achieve the ovulatory stage, an ovarian follicle will go through the primordial (resting), primary, secondary (pre-antral), tertiary (antral) follicular, and, finally, the mature oocyte stages (Picton et al, 1998). During this journey, a follicle grows in size, granulosa cells display mitosis and stratified, progression of theca cell layer, formation of zonapellucida and antrum along with chromosomal and cytoplasmic changes in the oocyte.
Once the primordial follicle pool is formed, the follicle is headed to three fates: to stay in a quiescent stage for maintenance of ovarian reserve, to undergo atresia (follicular programmed cell death) or to become primary follicle, a process called as recruitment. Primordial follicle recruitment process is tightly regulated by multimodal signaling between the oocyte, granulosa cells, and neighboring somatic cells which ultimately will form the theca cells. This activity depends on autocrine and paracrine modulation by various intrinsic ovarian growth factors that function through various signaling pathways and is gonadotropin independent (Hirshfield, 1991; Skinner, 2005; McGee and Hsueh, 2000; Oktem and Urman, 2010). Primordial follicle activation or quiescence is maintained by developmental factors which activates or suppresses follicular development respectively. The development from primordial follicle to primary follicle is marked by changing of granulosa cell morphology from flattened to cuboidal along with the acquisition of Zona Pellucida and increased oocyte diameter (>60 ?m) (Rankin et al, 1996). Primary follicles then grow into pre-antral follicles, also known as secondary and tertiary follicles in which oocyte is encompassed by at least two tiers of cuboidal granulosa cells and two outer theca cell layers. Follicles at this phase are gonadotropin dependent due to the presence of both theca and granulosa cells and begin producing sex steroid hormones (Knight and Glister, 2006). In this phase follicle diameter increases to 120–150 ?m at the pre-antral stage from 40–70 ?m at the primary stage. With further growth, preantral follicles then develop into antral follicles by reaching a diameter of 200 ?m and are the most mature follicle type found in the ovary. In this stage, the follicle starts to exhibit several fluid-filled spaces within the cuboidal granulosa cells, which will join to make the antral cavity. These antral follicles have two outer theca cell layers with increased vasculature and displays continued expansion of granulosa and theca cells. Every fertile estrous or menstrual cycle needs the presence of a pre-existing antral follicle pool which acts to cyclic gonadotropins and releases fertilization competent egg. Hence, folliculogenesis must stay dynamic to permit for the continual generation of antral follicles to undergo cyclic recruitment for potential ovulation. As the growth of the antral follicle progresses, their receptivity towards gonadotropins increases due to the production of estradiol. The rise in the estradiol production stimulates the luteinizing hormone (LH) surge which induces physiological response leading to ovulation of one or multiple follicles depending upon the species. This response promotes meiosis, follicular development, steroidogenesis, cumulus cell expansion, and luteinization, ultimately promoting oocyte maturation (Jamnongjit et al, 2005; Gallo, 1981). The proper maintenance and development of the follicle reserve is necessary till reproductive age. Otherwise, it would lead to infertility caused by premature ovarian failure and or premature menopause (Hoyer and Sipes, 1996; Craig et al, 2011). Premature menopause is of concern since it is linked with increased risks of osteoporosis and cardiovascular disease (Sowers and La Pietra, 1994; Luborsky et al, 2003; Mondul et al, 2005). Present study will analyze the expression profile of miRNAs and mRNAs during oocyte maturation process and identify specific markers, which are associated with proper or improper maturation.DETAILS OF HYPOTHESIS /MODEL/THEORY /EXPERIMENTS
HYPOTHESIS AND OBJECTIVE:
The role of small RNA in the function of ovary came into the light when Dicer, a ribonuclease III, was knocked down in mice, which performs the processing of pre-small RNA to functional small mature RNA resulted in the defective folliculogenesis, oocyte maturation and ovulation (Lei et al, 2010; Murchison et al, 2007; Otsuka et al, 2008; Hong et al, 2008).We hypothesize that the differential expression of miRNA may play the role in folliculogenesis as protein expression is dynamic during folliculogenesis.
This study is aimed to evaluate the comparative miRNA expression profiles of Primary, pre-antral and antral follicles in addition to mature oocytes in mouse model during folliculogenesis to determine the effects of differential miRNA expression on oocyte maturation and to identify markers associated with accurate oocyte maturation.
To investigate the expression profile of miRNAs in Primary Follicle, Pre-antral Follicle, Antral follicle and mature Oocyte.
To study the spatiotemporal expression patterns of highly regulated miRNAs in the ovarian follicles and oocyte.
To quantify the differential abundance of RNA and proteins during oocyte maturation in the ovarian follicles and oocyte.
To identify and experimentally validate the genes targeted by these miRNA during oocyte maturation.
To analyze the role of important miRNAs in murine oocyte maturation.