REGULATION OF CUMULUS-OOCYTE GAP-JUNCTIONAL COMMUNICATION IN PORCINE DURING IN VITRO MATURATION: GONADOTROPIN-INDEPENDENT INCREASES OF CONNEXIN 43 PROTEIN

Abstract

3′5′-Cyclic adenosine monophosphate (cAMP) is a key regulator of oocyte nuclear maturation. Current proposed models suggest that cAMP is synthesized either directly in the oocyte or synthesized in the surrounding cumulus cells and transported to the oocyte through gapjunctions. Here, we report that porcine cumulus cells actively modulate gap-junctional communication with the oocyte and expression of connexin 43 protein during in vitro oocyte maturation (IVM). Gap-junctional communication was measured by incubating cumulus-oocyte complexes (COC) in the presence of the dye calcein-AM, a permeable non-fluorescent derivative of fluorescent calcein. Calcein-AM diffuses into the COCs and is then cleaved into the AM group and the non-permeable fluorescent calcein. After dye uptake, the COCs were washed twice and then incubated for 25 minutes to allow the dye to transfer from the cumulus to the oocyte. Thereafter COCs were denuded and fluorescence emission of calcein was then quantified in individual oocytes using a fluorophotometric- inverted microscope. By measuring this transfer from the cumulus cells to the oocyte, our results showed that there was a doubling (P<0.05) in communication between cumulus and oocyte from 0-12 hours of IVM. This increase was followed by a radical decrease of gap-junctional communication between 18 and 22 hours of IVM, simultaneous with oocyte germinal vesicle breakdown. Connexin 43 is a known constituent of gap-junction channels in granulosa and cumulus cells. Therefore, to determine how connexin 43 protein varied during IVM, it was measured using Western blotting with an anti-connexin 43 antibody. Levels of connexin 43 protein were analyzed by densitometry relative to tubulin levels. Connexin 43 protein levels were increased 3.5-fold (P<0.05) in COC between 0 and 4 hours of IVM. In our culture conditions, the increase did not require gonadotropin stimulation. To verify if the increase in connexin 43 protein was due to factors in the follicular fluid supplemented to the culture medium, polyvinyl alcohol was used instead. An increase of connexin 43 protein levels was again observed in both COC culture conditions. When cultured in media containing polyvinyl alcohol, the 4h increase in connexin 43 protein levels was prevented by adding protein synthesis (cycloheximide, 2 μg/ml), RNA synthesis (alpha-amanitin, 25 μg/ml) and polyadenylation (cordycepin, 20 μM) inhibitors. To assess whether the presence of the oocyte is necessary for the increase in connexin 43 levels, oocytes were microsurgically removed from COC and cultured for 4 hours. Connexin 43 levels after 4h were indistinguishable in complexes with and without the oocyte, demonstrating that the increase is independent of the oocyte. In summary, our results show that gap junction permeability is actively regulated in porcine COC during IVM prior to nuclear maturation. They also show that connexin 43 upregulation after 4 hours of IVM was not dependant of gonadotropin, follicular fluid or oocyte, but required de novo RNA synthesis and polyadenylation and protein synthesis. These evidences further support a functional role of cumulus-oocyte gap junctional communication in the process of oocyte meiotic resumption. (platform)