BackgroundOocyte fertilization and successful embryo implantation are key events marking the onset of pregnancy. In sexually reproducing organisms, embryogenesis begins with the fusion of two haploid gametes, each of which has undergone progressive stages of maturation. In the final stages of oocyte maturation, minimal transcriptional activity is present and regulation of gene expression occurs primarily at the post-transcriptional level. MicroRNAs (miRNA) are potent effectors of post-transcriptional gene silencing and recent evidence demonstrates that the miR-34 family of miRNA are involved in both spermatogenesis and early events of embryogenesis.MethodsThe profile of miR-34 miRNAs has not been characterized in gametes or embryos of Bos taurus. We therefore used quantitative reverse transcription PCR (qRT-PCR) to examine this family of miRNAs: miR-34a, -34b and -34c as well as their precursors in bovine gametes and in vitro produced embryos. Oocytes were aspirated from antral follicles of bovine ovaries, and sperm cells were isolated from semen samples of 10 bulls with unknown fertility status. Immature and in vitro matured oocytes, as well as cleaved embryos, were collected in pools. Gametes, embryos and ovarian and testis tissues were purified for RNA.ResultsAll members of the miR-34 family are present in bovine spermatozoa, while only miR-34a and -34c are present in oocytes and cleaved (2-cell) embryos. Mir-34c demonstrates variation among different bulls and is consistently expressed throughout oocyte maturation and in the embryo. The primary transcript of the miR-34b/c bicistron is abundant in the testes and present in ovarian tissue but undetectable in oocytes and in mature spermatozoa.ConclusionsThe combination of these findings suggest that miR-34 miRNAs may be required in developing bovine gametes of both sexes, as well as in embryos, and that primary miR-34b/c processing takes place before the completion of gametogenesis. Individual variation in sperm miR-34 family abundance may offer potential as a biomarker of male bovine fertility.

Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo.

MicroRNAs are potent regulators of gene expression that have been widely implicated in reproduction and embryo development. Recent studies have demonstrated that miR-21, a microRNA extensively studied in the context of disease, is important in multiple facets of reproductive biology including folliculogenesis, ovulation, oocyte maturation and early mammalian development. Surprisingly, little is known about the mechanisms that regulate miR-21 and no studies have characterized these regulatory pathways in cumulus-oocyte complexes (COCs). We therefore investigated miR-21 in an in vitro model of bovine oocyte maturation. Levels of the primary transcript of miR-21 (pri-miR-21) and mature miR-21 increased markedly in COCs over the maturation period. Cloning of the bovine pri-miR-21 gene and promoter by 5′3′RACE (rapid amplification of cDNA ends) revealed a highly conserved region immediately upstream of the transcription start site and two alternatively-spliced variants of pri-miR-21. The promoter region contained several putative transcription factor binding sites, including two for signal transducer and activator of transcription 3 (STAT3). Mutation of these sites significantly decreased both the intrinsic activity of pri-miR-21 promoter-luciferase constructs and the response to leukemia inhibitory factor (LIF) (a STAT3 activator) in cultured MCF7 cells. In COCs, treatment with a STAT3 pathway inhibitor markedly decreased pri-miR-21 expression and prevented cumulus expansion. Pri-miR-21 expression was also inhibited by the protein synthesis inhibitor cycloheximide, suggesting that a protein ligand or signaling cofactor synthesized during maturation is necessary for transcription. Together these studies represent the first investigation of signaling pathways that directly influence miR-21 expression in bovine oocytes and cumulus cells.

The period beginning with the signal for ovulation, when a fully‐grown oocyte progresses through meiosis to become a mature egg that is fertilized and develops as a preimplantation embryo, is crucial for healthy development. The early preimplantation embryo is unusually sensitive to cell volume perturbations, with even moderate decreases in volume or dysregulation of volume‐regulatory mechanisms resulting in developmental arrest. To prevent this, early embryos possess mechanisms of cell volume control that are apparently unique to them. These rely on the accumulation of glycine and betaine (N, N, N‐trimethylglycine) as organic osmolytes—compounds that can provide intracellular osmotic support without the deleterious effects of inorganic ions. Preimplantation embryos also have the same mechanisms as somatic cells that mediate rapid responses to deviations in cell volume, which rely on inorganic ion transport. Both the unique, embryo‐specific mechanisms that use glycine and betaine and the inorganic ion‐dependent mechanisms undergo major changes during meiotic maturation and preimplantation development. The most profound changes occur immediately after ovulation is triggered. Before this, oocytes cannot regulate their volume, since they are strongly attached to their rigid extracellular matrix shell, the zona pellucida. After ovulation is triggered, the oocyte detaches from the zona pellucida and first becomes capable of independent volume regulation. A complex set of developmental changes in each cell volume‐regulatory mechanism continues through egg maturation and preimplantation development. The unique cell volume‐regulatory mechanisms in eggs and preimplantation embryos and the developmental changes they undergo appear critical for normal healthy embryo development.