Profile of microRNAs present in semen and embryos and their relationship to fertility

Bulls fertility relies of many factors. In concern to sperm cells, it depends of sperm quality (SQ; morphofunctional features) and sperm molecular components. Due to the fact thatsperm quality is not always related with fertility, the effect of many molecular factors on fertility has been intensively studied. miRNAs, which have post transcriptional action, are present on sperm cells and are important to spermatogenesis, sperm maturation and embryo development. However, the mechanisms by which they regulate male fertility are not completely understood. Thus, the objective of the present studies was to investigate the role of miRNAs on fertility regulation. On the first study, frozen-thawed semen batches of high (54.3±1.0%; HF; n=3) and low fertility (41.5±2.3%; LF; n=3) Aberdeen Angus (Bos taurus) bulls were analyzed in concern to SQ; were used in embryo in vitro production to produce and collect embryos of one cell, two cells and blastocysts; and were analyzed to the profile of 380 sperm miRNAs. The target genes of the miRNAs that presented different abundance levels on sperm cells and embryos of HF and LF bulls were investigated. Blastocysts diameter, cell number and proliferation index were also evaluated. Besides, in this study, we validated a new model to prove that some miRNAs are delivered by sperm cell to embryo. Data were analyzed by analyzes of variance (ANOVA) and Chi-Square. Signicante level was 5% unless otherwise stated. Among miRNAs evaluated, miR- 216b presented less abundance on HF sperm cells (P=0.08) and HF zygotes (P<0.05). miR- 216b target gene, KRAS, that is related with cell proliferation, presented high abundance (P<0.05) on HF two cells embryos such as the first clivage rate and the number of cells on blastocysts that were greater on this group. We also showed that miR-216b is delivered by sperm cells to embryos. On the second study, we used six Nelore (Bos indicus) bulls. Three bulls were submitted to testicular heat stress (HS group). Semen was collected three days before and 21 days after heat stress. Samples were analyzed in concern to SQ and to the profile of 380 miRNAs on sperm cells and on exossomes. Data were analyzed by ANOVA. Signicante level was 5%. HS presented 21 sperm miRNAs diferently abundant, but miRNAs from exossomes were not different between the HS and control groups. miR-126-5p and -146a presented less abundance on HS sperm cells and on BF bulls sperm cells of the first study. miR-216b was not different in the second study. Thus, we can conclude that 1) miRNAs are important to fertility; 2) there are miRNAs that are important to fertility and can be altered by spermatogenesis injuries; and 3) miR-216b is delivered by sperm cells to embryos and is important to initial embryo development regulating HF and LF embryos differently; showing that sperm miRNAs are important molecules for regulation and establishment of fertility. The studies here presented generate knowledge upon sperm miRNAs importance and present novel results that confirm that bull sperm cells contribute with more than DNA to embryo development. (AU)