Identification and Characterization of MicroRNAs in Porcine Gametes and Pre-Implantation Embryos

MicroRNAs (miRNAs) are short ribonucleic acids that ultimately affect the production of proteins. Although miRNAs are involved in nearly every biological process examined to date, little is known of the identity or function of miRNA in porcine reproductive tissues or their potential involvement in reproductive processes in pigs or other species. The objective of this dissertation research was to determine the presence of miRNAs in porcine gametes and both in vivo- and in vitro- produced pre-implantation embryos and to identify differences in miRNA expression between normal and aberrant samples. Using a heterologous RT-PCR approach, we demonstrated the presence of a total of 92 miRNAs in porcine oocytes, spermatozoa, and/ or embryos at the 4-cell, 8-cell, 16-cell, and blastocyst stages, with hundreds more predicted by miRNA microarray. Subsequent qRT-PCR analysis showed differential expression of five miRNAs, let-7a, -7d, -7e, miR-15b, and -22, between normal sperm and morphologically abnormal sperm or sperm samples exhibiting low motility. Messenger RNA targets of the differentially expressed miRNAs encode proteins important for spermatogenesis, sperm structure, and/ or sperm cell metabolism. Differential expression was also shown among embryos at various stages in development, demonstrating a temporal expression pattern of specific miRNAs in pre-implantation embryo growth. More interestingly, miR-24 was differentially expressed between in vivo- and in vitro- produced embryos at the 8-cell and blastocyst stages, supporting the need to characterize aberrant miRNA expression associated with the abnormal embryonic development correlated with assisted reproductive technologies. All of the miRNAs examined demonstrated high sequence similarity to the corresponding human miRNA sequences, indicative of high conservation among species. Understanding miRNA expression in reproductive processes is critical to comprehending the mechanistic roles miRNAs play in the regulation of all physiological processes