ABSTRACT
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.
ABSTRACT
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.
