The Role of MicroRNAs in Mammalian Fertility: From Gametogenesis to Embryo Implantation.

The genetic codes inscribed during two key developmental processes, namely gametogenesis and embryogenesis, are believed to determine subsequent development and survival of adult life. Once the embryo is formed, its further development mainly depends on its intrinsic characteristics, maternal environment (the endometrial receptivity), and the embryo-maternal interactions established during each phase of development. These developmental processes are under strict genetic regulation that could be manifested temporally and spatially depending on the physiological and developmental status of the cell. MicroRNAs (miRNAs), one of the small non-coding classes of RNAs, approximately 19-22 nucleotides in length, are one of the candidates for post-transcriptional developmental regulators. These tiny non-coding RNAs are expressed in ovarian tissue, granulosa cells, testis, oocytes, follicular fluid, and embryos and are implicated in diverse biological processes such as cell-to-cell communication. Moreover, accumulated evidences have also highlighted that miRNAs can be released into the extracellular environment through different mechanisms facilitating intercellular communication. Therefore, understanding miRNAs mediated regulatory mechanisms during gametogenesis and embryogenesis provides further insights about the molecular mechanisms underlying oocyte/sperm formation, early embryo development, and implantation. Thus, this review highlights the role of miRNAs in mammalian gametogenesis and embryogenesis and summarizes recent findings about miRNA-mediated post-transcriptional regulatory mechanisms occurring during early mammalian development.

MicroRNAs: tiny molecules with a significant role in mammalian follicular and oocyte development.

The genetic regulation of female fertility (follicular development, oocyte maturation and early preimplantation embryo development) involves the spatio-temporal regulation of those genes that play key roles in various stages of the female reproductive axis. MicroRNAs (miRNAs), a class of small non-coding RNAs, are known to regulate the expression of a large proportion of such genes. In recent decades, multiple studies have aimed to determine the roles of these non-coding RNAs in mammalian follicular development, oocyte growth and embryo development. These studies have applied a variety of approaches, including conditional knockout of miRNA biogenesis genes, high-throughput sequencing technologies for pattern recognition in miRNA expression and loss- and gain-of-function of miRNAs in various animal models. In addition to the cellular miRNAs, a large variety of RNAs are found in circulation, being coupled with extracellular vesicles, proteins and lipids. Because of their potential as diagnostic markers for abnormal physiologies, there is increasing interest in the identification of extracellular miRNAs in various biological fluids and spent in vitro culture media. This review focuses on studies addressing the expression and potential role of cellular and extracellular miRNAs in mammalian follicular cell physiology and subsequent ovarian functionality and oocyte maturation.

Extracellular vesicle mediated molecular signaling in ovarian follicle: Implication for oocyte developmental competence.

The bidirectional communication between the oocyte and the companion somatic cells in the follicular environment is known to be mediated by either a direct communication via gap junction or transzonal projections or indirectly through endocrine, paracrine and autocrine signaling factors. Extracellular vesicles (EVs), which are found in various biological fluids, including follicular fluid (FF) are known to play important roles in mediating the communication between the oocyte and the surrounding somatic cells through shuttling bioactive molecules to facilitate follicular growth and oocyte maturation. As vesicles in the extracellular space are known to reflect the physiological status of the donor or the releasing cells, molecules carried by the EVs in the follicular environment could be markers of the internal and external stressors. EVs exhibit greater degree of heterogeneity in their size, biogenesis and the bioactive molecule they carry. The process of biogenesis of EVs is known to be regulated by several proteins associated with the endosomal sorting complex required for transport (ESCRT) proteins. The type of EVs and surface proteins markers vary according to the type of protein involved in their biogenesis. EVs are recently reported to play indispensable role in promoting cell-to-cell communication during follicular growth. Recent advancements in EV research opened the possibilities to load EVs with specific molecules like miRNA, siRNA, CRISPR-cas9 complex and protein, which showed a new horizon for their application in therapeutics. The present review explores the biogenesis, the role and the future prospects of EVs with a special emphasis given to follicular growth and oocyte maturation.

Metabolism-associated genome-wide epigenetic changes in bovine oocytes during early lactation.

Dietary intake in early lactating cows is outmatched by milk production. These cows experience a negative energy balance, resulting in a distinct blood metabolism and poor reproductive function due to impaired ovulation and increased embryo loss. We hypothesize that oocytes from lactating cows undergoing transient metabolic stress exhibit a different epigenetic profile crucial for developmental competence. To investigate this, we collected oocytes from metabolically-profiled cows at early- and mid-postpartum stages and characterized their epigenetic landscape compared with control heifers using whole-genome bisulfite sequencing. Early-postpartum cows were metabolically deficient with a significantly lower energy balance and significantly higher concentrations of non-esterified fatty acids and beta-hydroxybutyrate than mid-postpartum animals and control heifers. Accordingly, 32,990 early-postpartum-specific differentially methylated regions (DMRs) were found in genes involved in metabolic pathways, carbon metabolism, and fatty acid metabolism, likely descriptive of the epigenetic regulation of metabolism in early-postpartum oocytes. DMRs found overlapping CpG islands and exons of imprinted genes such as MEST and GNAS in early-postpartum oocytes suggest that early lactation metabolic stress may affect imprint acquisition, which could explain the embryo loss. This whole-genome approach introduces potential candidate genes governing the link between metabolic stress and the reproductive outcome of oocytes.

Extracellular vesicle-coupled miRNA profiles in follicular fluid of cows with divergent post-calving metabolic status.

Most high-yielding dairy cows enter a state of negative energy balance (NEB) during early lactation. This, in turn, results in changes in the level of various metabolites in the blood and follicular fluid microenvironment which contributes to disturbed fertility. Extracellular vesicles (EVs) are evolutionarily conserved communicasomes that transport cargo of miRNA, proteins and lipids. EV-coupled miRNAs have been reported in follicular fluid. However, the association between postpartum NEB and EV-coupled miRNA signatures in follicular fluid is not yet known. Energy balance analysis in lactating cows shortly after post-calving revealed that the majority of the cows exhibited transiently negative energy balance levels, whereas the remaining cows exhibited either consistently negative or consistently positive energy levels. Metabolic status was associated with EV-coupled miRNA composition in the follicular fluid. Cows experiencing NEB showed reduced expression of a large number of miRNAs while cows with positive energy balances primarily exhibited elevated expression of EV-coupled miRNAs. The miRNAs that were suppressed under NEB were found to be involved in various metabolic pathways. This is the first study to reveal the presence of an association between EV-coupled miRNA in follicular fluid and metabolic stress in dairy cows. The involvement of differentially expressed miRNAs in various pathways associated with follicular growth and oocyte maturation suggest the potential involvement of specific follicular miRNAs in oocyte developmental competence, which may partially explain reduced fertility in cows due to post-calving metabolic stress.