184 INTERCELLULAR COUPLING AND CHROMATIN CONFIGURATION STATE IN HORSE OOCYTE–CUMULUS CELL COMPLEXES OF DIFFERENT ORIGINS

Abstract

Fewer follicles and of variable size are found at any time on the mare ovary compared with other livestock species, also influenced by seasonal variation. This is reflected on the population of cumulus–oocyte complexes (COC) collected that is characterised by a high heterogeneity. Recovered immature oocytes presumably need different culture conditions. The main factors contributing to this high heterogeneity are the follicle diameter, the status of the cumulus oophorus, and the reproductive seasonality. Therefore, the aim of the present study was to assess the nuclear chromatin configuration and the oocytes–cumulus cell gap junction-mediated communication (GJC) functionality in COC of different origins because these parameters are indicative of the oocyte’s metabolic state and should be taken into account when designing IVM strategies. The COC with compact (Cp) or expanded (Ex) cumulus oophorus were collected from follicles of different diameters (<1, 1–2, and >2 cm) in October–November, January–February, and April–May. The GCJ functionality was assessed by Lucifer Yellow microinjection and chromatin configuration was evaluated by Hoechst and Lacmoid staining, after cumulus cells removal. Data were obtained from a total of 1003 oocytes and were analysed by chi-squared test. Overall, GJC functionality was impaired in the majority of Ex COC in each follicle category, even though a certain proportion of them had open GJC (% of Ex COC with open GJC was 39.7, 29.3, and 39.3 in <1, 1–2, and >2 cm follicles, respectively). Moreover, the proportion of Ex COC with open GJC did not differ significantly between periods (% of Ex COCs with open GJC in October–November, January–February, and April–May was, respectively, 43.3, 28.6, and 41.7 in <1 cm follicles; 45.5, 19.3, and 26.47 in 1–2 cm follicles; 66.7, 50, and 16.7 in >2 follicles). On the contrary, the majority of Cp COC from follicles <1 and 1–2 cm, showed open GCJ in October–November and April–May, whereas they decreased significantly in January–February. This tendency was not maintained in Cp COC from follicles >2 cm, where GJC functionality did not differ significantly between periods (% of Cp COC with open GJC in October–November, January–February, and April–May was, respectively, 74.4, 35.7, and 75 in <1 cm follicles; 73.8, 42.1, and 67.7 in 1–2 cm follicles; 58.3, 58.3, and 68.6 in >2-cm follicles). Chromatin configuration analysis revealed that the highest proportion (23.9%) of oocytes with fibrillar chromatin was found in Cp oocytes from <1 cm follicles, whereas the proportion of oocytes with fibrillar chromatin ranged from 5.4 to 12.5% in the other groups. Moreover, the increase in follicle diameter was generally associated with an increase of chromatin condensation in Cp COC. Interestingly, the chromatin configuration distribution did not differ significantly among seasons. Our data could be useful in setting up new in vitro cultural strategies aimed to improve horse-assisted reproductive technology efficiency as well as in the understanding of horse oocyte biology.

Funding: Grant no. 26096200 ‘Ex Ovo Omnia’ from Regione Sardegna & Lombardia and ‘Dote Ricerca Applicata’ (VL and IT).