Intercellular Regulation of miR-125a-3p within Mouse Pre-Ovulatory Follicles

Introduction:

MicroRNAs, which serve as intercellular signaling molecules, are present in body fluids and transferred through gap-junctions. miR-125a-3p, found in follicular-fluids, is expressed in mouse pre-ovulatory follicles. The hCG-mediated decrease in miR-125a-3p expression, in oocytes and mural-granulosa-cells, enables the pre-ovulatory molecular processes.

Aim:

Examine whether and how does the intercellular communication-net within pre-ovulatory follicles modulate the expression of intra-follicular miR-125a-3p.

Materials and methods:

Mural-granulosa-cells, cumulus-cells and oocytes were isolated from pre-ovulatory mice follicles 48 hours after PMSG administration. Granulosa-cells were lysed or transfected with scramble-miR or miR-125a-3p-mimic and incubated over-night. Freshly isolated mural-granulosa/cumulus-cells were incubated over-night in conditioned medium of transfected granulosa cells. Oocytes were lysed or exposed to hCG and incubated for four hours. Levels of pri-miR-125a, miR-125a-3p/5p were measured by real-time PCR.

Results:

Mural-granulosa-cells and cumulus-cells can transcribe miR-125a and both maintain a similar proportion of miR-125a-3p/pri-miR-125a. miR-125a-3p is secreted by mural-granulosa-cells into their culture medium and regulate miR-125a-3p expression within the cumulus-cells and vice versa. Though miR-125a-3p is transcribed and accumulated in oocytes throughout oogenesis, its expression within fully-grown GV oocytes is regulated by cumulus-cells, which modulate intra-oocyte pri-miR-125a processing via gap-junctions within the cumulus-oocyte-complexes. Finally, we found that hCG directly down-regulate miR-125a-3p expression in fully-grown oocytes, mural-granulosa and cumulus-cells.

Conclusions:

Our findings imply that miR-125a-3p is tightly regulated within the follicle. The intercellular communications within pre-ovulatory follicles is enabled by secretion and transfer of miR-125a-3p or of an unknown factor, allowing coordinated decrease of miR-125a-3p expression in mural-granulosa/cumulus-cells and oocytes towards ovulation.