Identification and characterization of miRNAs expressed in the bovine ovary

Abstract

BackgroundMicroRNAs are the major class of gene-regulating molecules playing diverse roles through sequence complementarity to target mRNAs at post-transcriptional level. Tightly regulated expression and interaction of a multitude of genes for ovarian folliculogenesis could be regulated by these miRNAs. Identification of them is the first step towards understanding miRNA-guided gene regulation in different biological functions. Despite increasing efforts in miRNAs identification across various species and diverse tissue types, little is known about bovine ovarian miRNAs. Here, we report the identification and characterization of miRNAs expressed in the bovine ovary through cloning, expression analysis and target prediction.ResultsThe miRNA library (5'-independent ligation cloning method), which was constructed from bovine ovary in this study, revealed cloning of 50 known and 24 novel miRNAs. Among all identified miRNAs, 38 were found to be new for bovine and were derived from 43 distinct loci showing characteristic secondary structure. While 22 miRNAs precursor loci were found to be well conserved in more than one species, 16 were found to be bovine specific. Most of the miRNAs were cloned multiple times, in which let-7a, let-7b, let-7c, miR-21, miR-23b, miR-24, miR-27a, miR-126 and miR-143 were cloned 10, 28, 13, 4, 11, 7, 6, 4 and 11 times, respectively. Expression analysis of all new and some annotated miRNAs in different intra-ovarian structures and in other multiple tissues showed that some were present ubiquitously while others were differentially expressed among different tissue types. Bta-miR-29a was localized in the follicular cells at different developmental stages in the cyclic ovary. Bio-informatics prediction, screening and Gene Ontology analysis of miRNAs targets identified several biological processes and pathways underlying the ovarian function.ConclusionResults of this study suggest the presence of miRNAs in the bovine ovary, thereby elucidate their potential role in regulating diverse molecular and physiological pathways underlying the ovarian functionality. This information will give insights into bovine ovarian miRNAs, which can be further characterized for their role in follicular development and female fertility as well.

Characterization of microRNAs differentially expressed during bovine follicle development

“…An increase in the levels of all of these miRNAs in large follicles was accounted for increases in mural granulosa cells rather than in COC; this was not surprising as the latter contains a very small fraction of the total cells in the follicle, and therefore microarray analyses of whole follicle tissues might have missed changes in miRNA levels in the COC. Indeed, miRNAs differentially expressed in bovine COCs, cumulus cells or oocytes, in addition to changes in miRNA expression during oocyte maturation, have been reported (Hossain et al 2009, Miles et al 2012, and they did not include any of the miRNAs identified as enriched in large healthy follicles in this study. Nonetheless, our results suggest a potential involvement of bta-miR-144, bta-miR-202, bta-miR-451, bta-miR-652, and bta-miR-873 in the regulation of mural granulosa cell function during maturation of the dominant follicle, as their expression in all cases increased in healthy follicles once they reached a diameter of 12-14 mm.…”

“…QPCR analyses confirmed that at least three of these miRNAs were also upregulated significantly in some or all of the large healthy follicle categories relative to large atretic follicles, thus identifying this set of miRNAs as very strong candidates with a role in the development of bovine dominant follicles. mir-144, mir-202, mir-451, and mir-652 were reported earlier to be expressed in the ovary (Hossain et al 2009, McBride et al 2012, Kitahara et al 2013, Schauer et al 2013, although their expression patterns within follicular tissues were not studied in detail. In this study, bta-miR-202 was restricted to the bovine gonad; moreover, its expression within the follicle was highest in granulosa cells while levels in ovarian stroma were negligible.…”

“…With a few exceptions (Carletti et al 2010, Kitahara et al 2013, most evidence on follicular roles of miRNAs has been obtained using cultured cells, particularly rodent cells, and in some cases actual changes in the expression of these miRNAs during follicle development have not been demonstrated. Indeed, although genome-wide miRNA analyses of whole ovarian tissues (Landgraf et al 2007, Ro et al 2007, Mishima et al 2008, Hossain et al 2009, Ahn et al 2010, Juanchich et al 2013 or follicular, or luteal tissues (Fiedler et al 2008, McBride et al 2012, Miles et al 2012, da Silveira et al 2012, Donadeu & Schauer 2013, Schauer et al 2013, Sohel et al 2013 have been reported in several species, detailed spatiotemporal profiles encompassing several follicle developmental stages have not. Identifying such profiles will be an important step toward understanding the functional involvement of miRNAs during specific stages of follicle development.…”

Involvement of miRNAs in ovarian follicular and luteal development

“…F X DONADEU and others 325 ), equine follicular fluid (da Silveira et al 2012) and bovine corpora lutea have been identified using mouse-or human-based microarray or highthroughput qPCR. Some studies reported differences in miRNA expression between different ovarian or follicular compartments, including expression of miR-503, miR-224 and miR-383 predominantly in mouse granulosa cells and oocytes of mice (Lei et al 2010, Yao et al 2010a, Yin et al 2012, as well as differential expression of several dozen miRNAs in bovine ovarian cortex, cumulus cells and corpus luteum (CL; Hossain et al 2009). Further, a correlation was reported between follicular fluid and granulosa cell levels of several miRNAs in horses (da Silveira et al 2012).…”

“…Small RNA populations have been identified by cloningbased or next-generation sequencing of normal ovarian tissues from human (Landgraf et al 2007), mice (Ro et al 2007, Mishima et al 2008, Ahn et al 2010, pigs , cattle (Hossain et al 2009, Tripurani et al 2010, Huang et al 2011, Miles et al 2012 and sheep (McBride et al 2012). Most of those studies involved analyses on whole ovaries rather than on specific ovarian tissue components, an approach that, although very useful for comprehensive identification of miRNA sequences, provides very limited insight into their functional relevance.…”