Reproduction Abstracts

Introduction: MicroRNAs, transferred between cells through gap-junctions and fluids, serving as inter-cellular signaling molecules. miR-125a-3p, which can be found in follicular-fluid, is expressed and down-regulated by hCG in mural-granulosa-cells. Our preliminary results show that similar effect of hCG in oocytes enables resumption of first-meiotic division. Cumulus-cells and oocytes maintain diverged relationship that includes bi-directional transfer of RNAs and proteins. Because mural-cells transmit the LH/hCG-ovulatory stimulus to the cumulus-oocyte-complex we hypothesized that miR-125a-3p is regulated within the follicle, which serves as a unit enabling synchronization towards ovulation.

Methods: Mural and cumulus-cells were isolated from pre-ovulatory mice follicles 48 hours after PMSG administration, lysed or transfected with scramble-miR or miR-125a-3p-mimic and incubated over-night. Freshly isolated mural/cumulus-cells were seeded and incubated over-night with conditioned medium of transfected-cells. Levels of pri-miR-125a, miR-125a-3p/5p and Fyn-mRNA (downstream target of miR-125a-3p) were measured by real-time PCR.

Results and Discussion: miR-125a-3p expression and activity (indicated by Fyn-mRNA levels) in cumulus-cells cultured in conditioned medium of miR-125a-3p-transfected mural-cells, was higher than in cells cultured in conditioned medium of scramble-transfected cells suggesting that mural-cells modulate expression of miR-125a-3p in cumulus-cells through secretion of a yet unknown factor. We examined whether mural-cells are the lone source of miR-125a-3p or it is transcribed by cumulus-cells. Pri-miR-125a was detected in both cell-types, with higher level in cumulus-cells, similar to the levels of miR-125a-3p/5p. The relative percent of miR-125a-3p/5p from pri-miR-125a was equal in mural and cumulus-cells, suggesting that either they are similarly regulated or that the cells maintain a bi-directional communication enabling them to sense and adjust miR-125a-3p/5p level. Utilizing our cell-model we show that cumulus-cells can also modulate miR-125a-3p expression in mural-cells. (Our results imply that miR-125a-3p is tightly regulated within the follicle. Secretion of miR-125a-3p or of an unknown factor allows mural and cumulus-cells to co-regulate the level of miR-125a-3p expression.)