P REGUNTAS DE R ESPUESTA O RAL EN C OMISIÓN

After identification of the differential expressed miRNAs between SF and DF, next attempt took place to recognize the temporal dynamic miRNA enrichment and degradation in granulosa cells within SF vs SF and DF vs DF at day 3 and day 7 of

estrous cycle. Results have displayed that the temporal miRNA expression profile changes between day 3 and day 7 of the estrous was appreciable in DF groups by which the expression profile of 131 miRNAs was found to be altered between day 3 and 7 of the estrous cycle. Conversely, in SF groups, only 5 miRNAs were differentially regulated between these two time points. This may conclude that follicles (subordinate and dominant) present during the early luteal phase act like nonovulatory follicles present during the follicular phase and become atretic. This idea of regression and follicular atresia could be associated to the influence of progesterone (Taylor and Rajamahendran 1991), and the decline in FSH commencement after day 2 of the estrous cycle which brings differential alterations in FSH dependent growth factors and hormones within the cohort of preselected follicles, which simultaneously inducing growth and boosting estradiol producing capacity of the DF and atresia of subordinate follicles (Mihm et al. 1997). The granulosa cells of DF displayed noticeable miRNA transcriptional activity variation as compared to SF in the current results (Figure 4.6). This may propose the presence of dynamic molecular shift in the follicles that could progress in size and to be entitled for dominance. The biological reason for this could be the element that bovine follicles are not adequate to attain the dominant stage at day 3 unlike to day 7 when the follicle can reach dominance stage even though they are not ovulated. Furthermore, as the estrous cycle progresses from day 3 to day 7, the follicles increase in size, the production of estradiol also elevates. The upsurge in estradiol concentrations in performance with inhibin are the main endocrine signals that suppress FSH concentrations from the anterior pituitary gland via negative feedback reducing FSH to basal concentrations (Ginther 2000, Ginther et al. 2000b, Sunderland et al. 1994). However, the increment of estradiol secretion usually does not occur more than 3 or 4 days and thus due to high levels of progesterone, the DF becomes atretic and estradiol production drops on day 6 of the estrous cycle (Mihm et al. 2002). Centered on this perception, the considerations were made to feature into the miRNAs whether their shift is associated with estradiol production. In fact, during folliculogenesis, steroidogenesis is one of crucial process that happened in the granulosa cells. For this, dehydroepiandrosterone (DHEA) is produced from pregnenolone, androstenedione from progesterone by cytochrome p450 enzyme called CYP17A1 occurs in theca cells. On the other hand, the conversion of androstenedione into estrone and conversion of testosterone into estradiol occurs in granulosa cells by aromatase gene (Craig et al.

2011). Agreeing to (Xu et al. 2011), aromatase gene (CYP19A1) is a validated target of miR-378. However, this miRNA was found to be increase in granulosa cells of DF at day 7 compared to day 3 of the estrous cycle suggesting a decrease in the level of aromatase genes expression. Similarly, the miRNAs which were found to be associated with inhibitory effect on estradiol production (Sirotkin et al., 2009) namely, miR-96b, miR-146, miR-28, miR-29a, miR-184, miR-32, miR-34a, miR-129, miR-132, miR- 133a, and miR-150 were found to be increased at day 7 of the estrous cycle. Others, miR-24, miR-122, miR-145, miR-182, miR-143 and miR-150 which increased progesterone level in human granulosa cells after they were over expressed (Sirotkin et al. 2009) were found to be increased at day 7 of the estrous cycle. Other miRNAs which were dysregulated in DF between day 7 and day 3 of the estrous cycle namely miR-21, miR-132, miR-191 and miR-99b were also exhibited temporally expression alteration in mural granulosa cells collected from mice treated with eCG for 46 h followed by injection of hCG (Fiedler et al. 2008). This may suggesting some miRNAs whose expression was suppressed or enriched in granulosa cells of DF between day 3 and 7 of the estrous may involve in steroidogenesis or they may have some role in acquisition of follicular maturation. Recruitment and selection, the processes that give rise to the DF, and dominance, the physiological state of the mature DF, are important areas of basic research. Results of these basic studies on early luteal phase of follicular development need further validation to solve the problems in farm animal reproduction.

In conclusion, this thesis reported comprehensive understandings into the miRNA enrichment and degradation in the subordinate and dominant follicle during the luteal phase of the estrous cycle using high-throughput miRNA sequencing data generated from granulosa cells obtained from subordinate and dominant follicles at day 3 and 7 of the estrous cycle. In addition to novel miRNAs, the study identified large set of miRNAs which are found to be differentially expressed in granulosa cells of SF and DF and suggested to play a diverse role in various physiological processes associated with the two follicle types during the course of folliculogenesis. Further detail functional analysis of miRNAs is required to precisely determine the regulatory role of those noncoding small RNAs in bovine follicular development.