Pre-ovulatory intercellular regulation of miR-125a-3p within mouse ovarian follicles.

miR-125a-3p, a post-transcription regulator of Fyn kinase, is expressed in mouse pre-ovulatory follicles; its expression within the follicle decreases toward ovulation. Our aim was to follow the synthesis of miR-125a-3p and regulation of its expression in all follicular compartments, focusing on intercellular communication. Mural granulosa cells (GCs) or cumulus cells (CCs) were transfected with either scrambled-miR (negative control) or miR-125a-3p mimic. Freshly isolated GCs or CCs were incubated overnight in culture media conditioned by transfected cells. To examine a possible role of gap junctions in the regulation of miR-125a-3p, we incubated large antral follicles in the presence of carbenoxolone, a gap-junction inhibitor, and triggered them to mature with hGC. Levels of miR-125a family members in GCs, CCs, oocytes, and culture media were measured by qPCR. We showed that miR-125a-3p is synthesized by all follicular components, but is regulated within the follicle as a whole. It is secreted by mural-GCs and taken up by CCs, where it remains functional, and vice versa, mural-GCs can take up miR-125a-3p secreted by CCs. miR-125a-3p is transcribed and accumulated in oocytes throughout oogenesis. Transcriptionally quiescent GV oocytes utilize their accompanying follicular cells to monitor the level of miR-125a-3p within them, as indicated in an ex vivo follicle culture. Our study reveals that miR-125a-3p expression is modulated by a network of intercellular communications within pre-ovulatory follicles, thus enabling a coordinated decrease of miR-125a-3p toward ovulation.

Fyn and argonaute 2 participate in maternal-mRNA degradation during mouse oocyte maturation.

Fertilization triggers physiological degradation of maternal-mRNAs, which are then replaced by embryonic transcripts. Ample evidence suggests that Argonaut 2 (AGO2) is a possible post-fertilization regulator of maternal-mRNAs degradation; but its role in degradation of maternal-mRNAs during oocyte maturation remains obscure. Fyn, a member of the Src family kinases (SFKs), and an essential factor in oocyte maturation, was reported to inhibit AGO2 activity in oligodendrocytes. Our aim was to examine the role of Fyn and AGO2 in degradation of maternal-mRNAs during oocyte maturation by either suppressing their activity with SU6656 - an SFKs inhibitor; or by microinjecting DN-Fyn RNA for suppression of Fyn and BCl-137 for suppression of AGO2. Batches of fifteen mouse oocytes or embryos were analyzed by qPCR to measure the expression level of nine maternal-mRNAs that were selected for their known role in oocyte growth, maturation and early embryogenesis. We found that Fyn/SFKs are involved in maintaining the stability of at least four pre-transcribed mRNAs in oocytes at the germinal vesicle (GV) stage, whereas AGO2 had no role at this stage. During in-vivo oocyte maturation, eight maternal-mRNAs were significantly degraded. Inhibition of AGO2 prevented the degreadation of at least five maternal-mRNAs, whereas inhibition of Fyn/SFK prevented degradation of at least five Fyn maternal-mRNAs and two SFKs maternal-mRNAs; pointing at their role in promoting the physiological degradation which occurs during in-vivo oocyte maturation. Our findings imply the involvement of Fyn/SFKs in stabilization of maternal-mRNA at the GV stage and the involvement of Fyn, SFKs and AGO2 in degradation of maternal mRNAs during oocyte maturation.

Regulation of GVBD in mouse oocytes by miR-125a-3p and Fyn kinase through modulation of actin filaments.

Meiotically arrested oocytes are characterized by the presence of the nuclear structure known as germinal-vesicle (GV), the breakdown of which (GVBD) is associated with resumption of meiosis. Fyn is a pivotal factor in resumption of the first meiotic division; its inhibition markedly decreases the fraction of oocytes undergoing GVBD. Here, we reveal that in mouse oocytes Fyn is post-transcriptionally regulated by miR-125a-3p. We demonstrate that in oocytes resuming meiosis miR-125a-3p and Fyn exhibit a reciprocal expression pattern; miR-125a-3p decreases alongside with an increase in Fyn expression. Microinjection of miR-125a-3p inhibits GVBD, an effect that is markedly reduced by Fyn over-expression, and impairs the organization of the actin rim surrounding the nucleus. Lower rate of GVBD is also observed in oocytes exposed to cytochalasin-D or blebbistatin, which interfere with actin polymerization and contractility of actin bundles, respectively. By down-regulating Fyn in HEK-293T cells, miR-125a-3p reduces the interaction between actin and A-type lamins, which constitute the nuclear-lamina. Our findings suggest a mechanism, by which a decrease in miR-125a-3p during oocyte maturation facilitates GVBD by allowing Fyn up-regulation and the resulting stabilization of the interaction between actin and A-type lamins.