Abstract
Members of the interleukin-6 (IL-6) family of cytokines are important for reproductive function that are mediated through changes in gene and miRNA expression. Herein, we characterized the expression of miR-21, miR-155, miR-34c and miR-146a in bovine oocytes and cumulus cells during in vitro maturation (IVM) with leukemia inhibitory factor (LIF), IL-6 and IL-11 or unsupplemented controls. LIF-exposed COCs showed higher expression of miR-21 and miR-155 in oocytes, whereas miR-146a expression was increased in oocytes matured with IL-6 and IL-11. In cumulus cells, miR-155 expression was elevated by all treatments while only LIF increased miR-21 expression. Based on these results, we next examined how LIF exposure during IVM affected oocyte competence, through IVF and the expression of specific genes in GV- and MII-oocytes, in 2- and 8-cell embryos, and in Day 8-blastocysts. LIF supplementation did not affect cleavage rate, blastocyst yield or several other developmental parameters, but did increase hatching rate. LIF suppressed DPPA3, ZAR1 and NPM2 expression in 2 cell- and/or 8-cell embryos. LIF increased the expression of KAT2A and HSPA1A in MII-oocytes, and that of HDAC1, KAT2A and HSP90AA1 and the BAX:BCL2L1 ratio in 2-cell embryos. In contrast, HDAC1, KAT2A and HSP90AA1 expression and BAX:BCL2L1 ratio was lower in 8-cell embryos derived from LIF oocytes. IVM with LIF also increased the expression of DNMT3A, HSPA1A and HSP90AA1 in blastocysts. In conclusion, supplementation with LIF during IVM was consistently associated with changes in the relative abundance of transcripts in mature bovine oocytes and in specific embryo developmental stages.
Highlights
Oocyte differentiation occurs through complex processes beginning at the embryonic life and extending until the ovulation of a metaphase II stage (MII) oocyte
In order to investigate the potential mechanisms underlying the effects of IL-6 family cytokines on cellular function in the context of cumulus cells and the oocyte, we first evaluated the levels of miRNA transcripts in cumulus cells from Cumulus-oocyte complexes (COCs) matured in the presence of leukemia inhibitory factor (LIF), IL-6, IL-11 or in non-supplemented in vitro maturation (IVM) media
Higher (P < 0.05) miR-21 transcript levels were observed in cumulus cells from COCs in vitro matured with LIF when compared to the expression levels of miR-21 in cumulus cells from GV oocytes (Fig. 1A)
Summary
Oocyte differentiation occurs through complex processes beginning at the embryonic life and extending until the ovulation of a metaphase II stage (MII) oocyte. We propose that the identification of specific differentially expressed mRNAs and miRNAs during in vitro maturation, together with the assessment of transcript levels of genes during subsequent bovine embryo development could help us understand the effect of changing the maturation milieu on oocyte quality. To this end, we first evaluated the levels of key miRNAs known to be expressed in oocytes and cumulus cells (miR-21, miR-155, miR-34c and miR-146a) after in vitro maturation with IL-6 family cytokines. Transcript abundances of maternal-effect genes [zygote arrest 1 (ZAR1), nucleoplasmin 2 (NPM2), developmental pluripotency associated 3 (DPPA3)], and genes related to epigenetics (DNMT3A, KAT2A and HDAC1), heat stress (HSPA1A, HSP90AA1) and apoptosis (BAX, BCL2L1) were determined in bovine MII oocytes and day 8 blastocysts
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