MicroRNA Expression Profile in Bovine Granulosa Cells of Preovulatory Dominant and Subordinate Follicles during the Late Follicular Phase of the Estrous Cycle

Abstract

In bovine, ovarian follicles grow in a wave-like fashion with commonly 2 or 3 follicular waves emerging per estrous cycle. The dominant follicle of the follicular wave which coincides with the LH-surge becomes ovulatory, leaving the subordinate follicles to undergo atresia. These physiological processes are controlled by timely and spatially expressed genes and gene products, which in turn are regulated by post-transcriptional regulators. MicroRNAs, a class of short non-coding RNA molecules, are one of the important posttranscriptional regulators of genes associated with various cellular processes. Here we investigated the expression pattern of miRNAs in granulosa cells of bovine preovulatory dominant and subordinate follicles during the late follicular phase of bovine estrous cycle using Illumina miRNA deep sequencing. In addition to 11 putative novel miRNAs, a total of 315 and 323 known miRNAs were detected in preovulatory dominant and subordinate follicles, respectively. Moreover, in comparison with the subordinate follicles, a total of 64 miRNAs were found to be differentially expressed in preovulatory dominant follicles, of which 34 miRNAs including the miR-132 and miR-183 clusters were significantly enriched, and 30 miRNAs including the miR-17-92 cluster, bta-miR-409a and bta-miR-378 were significantly down regulated in preovulatory dominant follicles. In-silico pathway analysis revealed that canonical pathways related to oncogenesis, cell adhesion, cell proliferation, apoptosis and metabolism were significantly enriched by the predicted target genes of differentially expressed miRNAs. Furthermore, Luciferase reporter assay analysis showed that one of the differentially regulated miRNAs, the miR-183 cluster miRNAs, were validated to target the 3´-UTR of FOXO1 gene. Moreover FOXO1 was highly enriched in granulosa cells of subordinate follicles in comparison with the preovulatory dominant follicles demonstrating reciprocal expression pattern with miR-183 cluster miRNAs. In conclusion, the presence of distinct sets of miRNAs in granulosa cells of preovulatory dominant and subordinate follicles supports the potential role of miRNAs in post-transcriptional regulation of genes involved in bovine follicular development during the late follicular phase of the estrous cycle.

Cellular and extracellular vesicular origins of miRNAs within the bovine ovarian follicle

“…Other identified differentially expressed miRNAs that exhibited increased expression levels in COCs in this study, such as miR‐342 and miR‐542‐5p , were recently identified in EVs from bovine FF and found to be more highly expressed in large than in small follicles (Navakanitworakul et al., ). Similarly, let‐7a‐3p and miR‐542‐5p, whose expression was found to be increased in COCs compared to in GCs in this study, were previously determined to be down‐ and upregulated, respectively, in granulosa cells from bovine pre‐ovulatory compared to in subordinated follicles (Gebremedhn et al., ). Together, these results suggest that let‐7a‐3p and miR‐542‐5p likely play a different role in COCs, which is independent of the follicle stage.…”

The Role of MicroRNAs in Mammalian Fertility: From Gametogenesis to Embryo Implantation

“…Moreover, looking into the miRNA expression patterns in follicles of different size (dominant and subordinate), healthy and atretic could be a step forward towards identification of miRNAs that are potentially involved in follicular development and ovulation. With this respect, previous studies have shown differential expression of miRNAs that are involved follicular cell proliferation, steroidogenesis, luteinization, and oocyte maturation between small and large follicles or between healthy and atretic bovine follicles [31][32][33]. Moreover, miRNAs that are potentially involved in Wnt signalling pathway, Transforming growth factor (TGF-beta) signalling pathway, axon guidance and apoptosis were differential expressed between small and bigger follicles at day 3 of the bovine oestrous cycle ( Figure 1) while miRNAs that were involved in metabolism of vitamins, cofactors, lipids, lipoproteins, cysteine and methionine were differentially expressed between small and bigger follicles at day 7 of the estrous cycle [31].…”

“…Apart from this, understanding miRNA expression patterns during the follicular phase of the estrous cycle would provide a better clue about miRNAs that directly involved in ovulation. With this regard, our previous study have shown miRNAs that potentially involve in cell adhesion, cell proliferation, apoptosis and metabolism were significantly different between the granulosa cells of preovulatory dominant and subordinate follicles at day 19 of the bovine oestrous cycle [32]. Similarly, in zebra fish it is believed that small early vitellogenic follicles are unable to undergo oocyte maturation whereas oocytes in mid-to late vitellogenic follicles can be induced by Luteinizing hormone (LH) to become matured.…”

“…Understanding the expression patterns of miRNAs during the different phases of reproductive cycle (luteal or follicular phase) of mammals such as cattle would provide a better insight about post-transcriptional gene regulation underlining follicular atresia and oocyte ovulation [31,32] or to identify miRNAs governing the production of steroid essential for the normal processes of folliculogenesis [35][36][37][38][39]. Nevertheless, aberrant expression of developmentally relevant miRNAs during critical period of the reproductive cycle could negatively affect follicular development resulting for anovulation or ovulation of infertile oocytes.…”

Roles of microRNAs in mammalian reproduction: from the commitment of germ cells to peri‐implantation embryos

“…This suggests that these miRNAs might regulate the phenotypic characters of different cell types by controlling the expression of important signalling molecules (Donadeu et al, ). Additionally, miR‐708, miR‐221, miR‐21‐3p, miR‐335, and miR‐214 are expressed abundantly in the follicular fluid of SFs compared to that of DFs (Gebremedhn et al, ) indicating a potential role in the selection of follicles. Hence, differential expression of miRNAs between TCs and GCs may play an important role in the selection and recruitment of DFs, as well as during the follicular wave (in different mammalian species the recruitment, growth and development of several waves of follicles is possible before follicle rupture and ovulation occur; during this process two or more follicles might develop, but only one follicle successfully releases the egg) and atresia.…”