Abstract
Correct transmission of the genetic material to the next generation is essential for the maintenance of genomic integrity. Therefore chromosomes should be divided properly during each meiotic division. In this thesis the function of several proteins, particularly Transforming acidic coiled-coil protein 3 (TACC3), has been examined in cattle oocytes. Immunofluorescent
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staining demonstrated the presence of TACC3 around the chromosomes during the first and second meiotic divisions. The function of TACC3 was investigated directly by injecting TACC3 targeted siRNAs into oocytes and indirectly by chemical inhibition of Aurora A activity, a kinase known to phosphorylate and thereby activate TACC3. Abnormal spindle formation and impaired embryonic development was observed. It is concluded that expression of TACC3 is important for the stability of the meiotic spindle and thereby correct segregation of chromosomes. For the survival of a species, it is also important that the genome in the gametes is protected from potentially damaging elements such as transposons. Piwi proteins, together with Piwi-interacting RNAs (piRNAs), can suppress transposon activity by endonuclease activity of the Piwi protein using piRNA as a guide for target recognition. Small RNAs in ovaries and oocytes were sequenced. In cattle ovaries, the pachytene like 30 nucleotide (nt) piRNAs were detected. Using immunohistochemistry, PIWIL1 was demonstrated in the primordial follicles, suggesting that the 30 nt piRNAs in the ovaries interact with PIWIL1. In the ovaries of adult women, expression of PIWIL1 and PIWIL2 was detected, together with piRNAs of 27-28 and 30 nt, whereas in ovaries from second trimester fetuses 27 nt piRNAs were detected, as was PIWIL2 expression. It is proposed that in oocytes from fetal follicles, PIWIL2 interacts with 27 nt piRNAs, whereas in adult ovaries, and particularly the oocytes of primary follicles, PIWIL1 and PIWIL2 function with 27-28 and 30 nt piRNAs. In bovine oocytes isolated from antral follicles, unmethylated 26 nt piRNAs were detected which were still present in 2-4 cell embryos. Proteomic analysis showed relatively large amounts of PIWIL3 in these oocytes and embryos and we propose that, in oocytes from antral follicles, PIWIL3 and unmethylated 26 nt piRNAs play a role in inhibition of transposon activity. Since we were interested in the function of miRNAs in oocytes and early embryos we analyzed miRNAs that could potentially be used as references to normalize the expression of miRNAs in oocytes, cumulus cells and embryos. In cattle, miR-93 and miR-103 were the most stably expressed, whereas in pig samples miR-26a, miR-191 and miR-93 were the most suitable reference miRNAs. A better understanding of meiotic spindle dynamics in oocytes and the function of small non-coding RNAs in mammalian oocytes is not only important from a fundamental point of view, but could eventually lead to better treatments for infertility including more efficient assisted reproduction technologies.
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