Abstract
The mammalian ovarian follicle is the complex reproductive unit comprising germ cell, somatic cells (Cumulus and Granulosa cells), and follicular fluid (FF): paracrine communication among the different cell types through FF ensures the development of a mature oocyte ready for fertilization. This paper is focused on non-coding RNAs in ovarian follicles and their predicted role in the pathways involved in oocyte growth and maturation. We determined the expression profiles of microRNAs in human oocytes and FF by high-throughput analysis and identified 267 microRNAs in FF and 176 in oocytes. Most of these were FF microRNAs, while 9 were oocyte specific. By bioinformatic analysis, independently performed on FF and oocyte microRNAs, we identified the most significant Biological Processes and the pathways regulated by their validated targets. We found many pathways shared between the two compartments and some specific for oocyte microRNAs. Moreover, we found 41 long non-coding RNAs able to interact with oocyte microRNAs and potentially involved in the regulation of folliculogenesis. These data are important in basic reproductive research and could also be useful for clinical applications. In fact, the characterization of non-coding RNAs in ovarian follicles could improve reproductive disease diagnosis, provide biomarkers of oocyte quality in Assisted Reproductive Treatment, and allow the development of therapies for infertility disorders.
Highlights
Infertility, the inability to conceive and have children, is estimated to affect as many as 186 million people worldwide, and, represents an important social and medical problem (Inhorn and Patrizio, 2015)
Significant miRNA differences were identified by Significance of Microarrays Analysis (SAM)1, applying a two-class paired test among Ct of follicular fluid (FF) and oocytes samples by using a p-value based on 100 permutations; imputation engine: K-nearest neighbors, 10 neighbors; false discovery rate < 0.15 (Ragusa et al, 2014; Battaglia et al, 2016; Fendler et al, 2017). miRNA expression changes were calculated by applying the 2− CT method and using the average Ct of each plate as endogenous controls
Using TaqMan Low Density Array (TLDA) technology, we determined the expression profile of 384 miRNAs on 6 pools of FF and 6 pools of MII oocytes from healthy women
Summary
Infertility, the inability to conceive and have children, is estimated to affect as many as 186 million people worldwide, and, represents an important social and medical problem (Inhorn and Patrizio, 2015). Most of the antral follicles undergo apoptosis, whereas only one, the dominant Graafian follicle, ovulates to release the mature egg, ready for fertilization (Sun et al, 2009; Zuccotti et al, 2011) At this stage, the follicles consist of the germ cell in the metaphase of the second meiotic division (MII oocytes), in a fluid-filled cavity called the antrum and different layers of somatic cells, the cumulus cells (CC) surrounding the oocytes and the granulosa cells (GC) as walls of follicle (Russell and Robker, 2007; Rodgers and Irving-Rodgers, 2010). The discovery of mechanisms of autocrine and paracrine communication mediated by miRNAs, inside the ovarian follicle, has revealed that these non-coding RNAs represent important regulators inside the pathways involved in folliculogenesis and oocyte maturation (Santonocito et al, 2014; Di Pietro, 2016) Their characterization could improve our knowledge about female gametogenesis, and could pinpoint new molecules involved in reproductive disorders allowing the formulation of new therapeutic approaches
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