197 Influence of cumulus cell morphology on nuclear and cytoplasmic maturation markers of equine oocytes

This study aimed to characterize the effects of holding different populations of equine cumulus–oocyte complexes (COCs) at 22°C on chromatin, mitochondria, and microtubule configurations. We hypothesized that the type of cumulus cell morphology would influence the initial presentation of markers and their progression during holding and IVM, with expanded COCs presenting a higher incidence of mature configurations throughout different times. A pool of 340 slaughterhouse-derived COCs were selected and divided into two groups: expanded (Ex, n = 161) and compact (Cp, n = 179). Selected COCs in each group were randomly allotted to four sampling times: T0h (Ex, n = 26; Cp, n = 52), at collection; T18h (Ex, n = 32; Cp, n = 38), holding in a serum-free HEPES buffered medium at 22°C for 18 h; T30h (Ex, n = 55; Cp, n = 55), placed immediately into IVM for 30 h; and T48h (Ex, n = 48; Cp, n = 34), placed into IVM after holding. At different time points, oocytes were denuded and stained with MitoTracker CMTMRos, Alexa Fluor 488 anti-α tubulin antibody, and Hoechst 33342. Classification was performed with confocal microscopy. Statistical analyses were done using Chi-square test with Yate’s correction or Fisher’s exact test. Significance was set as P ≤ 0.05. At T0h, the majority of Cp and Ex were at the germinal vesicle (GV) stage. However, Ex had more condensed chromatin (P = 0.002) and an even proportion of homogeneous (HM) and heterogeneous (HT) mitochondria and microtubules. Conversely, Cp presented a higher proportion of fibrillar/intermediate chromatin (P = 0.019) and HM mitochondria (P = 0.0001) and microtubules (P = 0.0001). At T18h, viable Ex presented more chromatin configurations associated with GV breakdown (P = 0.027), while Cp presented progression toward condensed chromatin (P = 0.0003). No differences were found in the proportions of Ex and Cp with HT, HM, or abnormal mitochondria. A reduction in the proportion of Ex with normal HT microtubules was noted (P = 0.003). In addition, Cp showed no progression from HM to HT microtubules. At T30h and T48h, maturation rates were higher for Ex (P = 0.0006; P = 0.005), with no differences between times. At T30h, Ex and Cp presented a progression to HT mitochondria (P = 0.003; P = 0.005) and microtubules (P = 0.0003; P = 0.0001) in comparison with T0h. At T48h, similar findings were observed for mitochondria (P = 0.0001; P = 0.002) and microtubules (P = 0.0003; P = 0.002) of Ex and Cp, respectively. No differences between T30h and T48h were noted. No differences in degeneration rates were noted between time points. In conclusion, cumulus cell morphology influenced the initial presentation of nuclear and cytoplasmic markers, as well as chromatin progression during holding and IVM. Holding did not enable the progression of cytoplasmic markers and may have temporarily compromised microtubule dynamics. This could be related to instability of microtubules at 22°C. We suggest that this instability could be one possible mechanism underlying inhibition of GV breakdown when COCs are transported at room temperature to laboratories for in vitro production of equine embryos.