Identification and characterisation of microRNAs during bovine ovarian follicular development
Sontakke, Sadanand Dewaji
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Proper understanding of ovarian follicular and luteal development is essential to improve and optimally control reproductive function in domestic animals and to unravel causes of infertility in animals and humans. MicroRNAs (miRNAs) are key post-transcriptional gene regulators in multiple processes involving tissue growth and differentiation. The studies in this thesis were carried out to identify and characterise expression profiles of miRNAs and their potential roles during ovarian follicular development in the cow. The first study aimed to identify expression profiles of miRNAs during antral follicle growth. Follicles were collected from abattoir ovaries and their status (healthy/atretic) was assessed by measuring steroid levels and aromatase expression. An heterologous microarray approach followed by RT-qPCR validation was used to identify and compare miRNA profiles between large (13–16 mm) healthy and small (4–8 mm) follicles. A unique subset of 7 miRNAs (miR-144, miR-202, miR-210, miR-451, miR-652, miR-873 and miR-876) was identified that were enriched in the Large Healthy follicles relative to small follicles and Large Atretic follicles. Further characterisation revealed that many of these miRNAs were highly expressed in the granulosa compartment of the follicle, predominantly in the mural granulosa cells, indicating their potential involvement in regulating granulosa cell function. Expression patterns of miRNAs in the ovarian tissues were generally reflected in the follicular fluid, thus follicular fluid may be used to monitor follicular health. Finally, tissue-wide screening of miRNAs identified miR-202 as a gonad-specific miRNA in the cow. The aim of the second study was to identify potential roles of the miRNAs enriched in Large Healthy follicles. Using in silico approaches, about 1700 bovine genes were identified as the predicted targets of those miRNAs. Putatively targeted signalling pathways are primarily involved in cell survival, proliferation and differentiation. Further, as expected, top predicted gene targets (SPRED1, ATG7, TGFBR2) showed an expression pattern in follicular tissues that was opposite to the patterns of the targeting miRNAs. However, expression patterns of miR-202 or miR-210 during follicular growth could not be recapitulated in gonadotrophin-stimulated cells in vitro and moreover, over-expression or inhibition of miR-202 in these cells did not result in changes in target mRNA levels. The third study investigated the expression profiles of miRNAs during follicle-luteal transition. Microarray analyses identified 9 miRNAs that were upregulated and 14 miRNAs that were downregulated between Large Healthy follicles and early corpus luteum in the cow. Predicted targets of these miRNAs were associated with signalling pathways involved in cell survival, proliferation and differentiation, indicating that these miRNAs might play significant regulatory roles during ovulation and early luteal development. Further, expression of some of these miRNAs and their putative targets during luteinisation in vivo could be recapitulated in cultured granulosa cells providing a system to study the functional roles of these miRNAs during follicle-luteal development. In summary, this is the first study identifying unique subsets of miRNAs associated with follicular development and the follicle-luteal transition in the cow which may be important for female fertility.