Effect of cilostamide treatment on the intercellular coupling, meiotic and embryonic developmental competence in horse oocyte-cumulus cells complexes of different origins

Design of new strategies to improve in vitro embryo production (IVP) efficiency, such as pre-maturation culture with cilostamide (CILO PMC) aimed to prolong oocytes-cumulus cells gap junction-mediated communications (GJC) functionality while delaying meiotic resumption, should consider the high heterogeneity of cumulus-oocyte complexes (COCs) retrieved from ovarian follicles. In the mare, main factors contributing to this heterogeneity are: the follicle diameter, the cumulus oophorus morphology and the reproductive seasonality. Thus, the objectives of this study were: 1) to identify a population of equine oocytes that would benefit from a CILO PMC, based on the assessment the GJC functionality by Lucifer Yellow microinjection, the oocyte chromatin configuration by DNA staining, and the meiotic competence after in vitro maturation (IVM), in COCs of different origins, since these parameters are indicative of the oocyte metabolic state and 2) to assess the effect of CILO PMC, in COCs of different origins, on the GJC functionality, the meiotic and embryonic developmental competence after IVM and intracytoplasmic sperm injection (ICSI). COCs with Compact (Cp) or Expanded (Ex) cumulus oophorus were collected from <1, 1-2 and >2 cm follicles during winter, spring-summer and fall, and cultured with our standard IVM protocol (CTRL) or prematurated in the same medium with 10-4 UI r-hFSH/ml and 10 μM Cilostamide (CILO PMC) for different times. When meiotic and developmental were assessed, COCs were subjected to CILO PMC for 6 h and than to standard IVM and ICSI. Data were analyzed by Chi square test. Overall, our results suggest that COCs that would more probably benefit from CILO PMC are those with Cp cumuli from <1 and 1-2 cm follicles collected during spring-summer and fall. In fact, they have a lower meiotic competence after 24h IVM (P<0.05), with a mean MII rate of 40%, compared to all the other groups, in which MII rate is around 60%. Moreover, they show a higher % (P<0.05) of COC with open GJC (70%), when compared with both Cp COC in the winter season and Exp COC from all follicular classes in all seasons tested, in which more that 50% of COCs have closed GJC. Independently from the season, chromatin configuration analysis reveals that oocytes with Cp cumuli from 1-2 cm follicles are in a more advanced stage of differentiation since their chromatin is more condensed (P<0.05). Study on GJC indicates that functionality can be maintained up to 10h in Cp COCs from 1-2 cm using CILO PMC when compared to the CTRL group, in which a major GJC drop is detected at 10h. In fact, COCs with open GJC are 74% and 44% in CILO PMC and CTRL groups respectively at 10 h (P<0.05) On the contrary, the exact timing for the treatment of Cp COCs from <1 cm follicles remains to be determined since GJC functionality drops after 16h in the CTRL group, with only 14% of COCs with open GJC, and no differences are observed between groups. Finally, initial evaluation of the effect of CILO PMC for 6h indicates that it does not affect the rate of embryos obtained after ICSI. Evaluation of embryo quality is in progress to assess whether CILO PMC could improve IVP efficiency beyond embryo yield. In conclusion, our data could be useful in setting up IVM strategies to improve horse IVP efficiency as well as the understanding of horse oocyte biology. Funding: Grant n. 26096200 "Ex Ovo Omnia" Regione Sardegna-Lombardia and ‘Dote Ricerca’ FSE, Regione Lombardia.