30 The importance of cumulus cells for the survival and timing of meiotic resumption of porcine oocytes vitrified at the immature stage

Abstract

Previous research revealed that vitrification at the immature (the germinal vesicle, GV) stage triggers premature meiotic resumption in cumulus-enclosed porcine oocytes and causes a damage in gap junctions (Appeltant et al. 2017 Reprod. Fertil. Dev. 29, 2419-2429). However, the correlation between the two phenomena was not investigated yet. The present research was conducted to clarify whether premature meiotic resumption is caused by gap junction disruption and to assess the importance of cumulus cells for the survival of porcine oocytes vitrified at the GV stage. Cumulus–oocyte complexes (COCs) were collected from 3- to 6-mm antral follicles of slaughtered gilts. Immediately after collection, approximately half of them were denuded mechanically (DOs). In each replicate, groups of COCs and DOs were processed without vitrification (control groups). Treatment groups of COCs and DOs were vitrified on Cryotop sheets in a combination of 17.5% propylene glycol and 17.5% ethylene glycol and warmed in 0.4M sucrose. The oocytes were then cultured for 22h in a chemically defined porcine oocyte medium (POM) supplemented with 10ngmL−1 epidermal growth factor, 10IUmL−1 equine chorionic gonadotrophin, 10IUmL−1 human chorionic gonadotrophin, and 1mM dibutyryl cAMP. After culture, COCs were denuded and oocyte survival was assessed by morphological evaluation of membrane integrity under a stereo microscope. Then, live oocytes were fixed and stained with 1% orcein and nuclear status was evaluated under a phase-contrast microscope. The experiment was replicated 5 times. Data were analysed by ANOVA followed by Tukey’s multiple comparisons test. After vitrification and culture, the survival rate in the COC group was higher (P<0.05) than that of the DO group (160/191=84.7±3.4% vs. 153/237=65.0±6.2%, respectively) but reduced (P<0.05) compared with those in the control COC and DO groups (138/143=96.6±1.0% and 152/153=99.3±0.6%, respectively). The majority of the control COCs and DOs were at the GV stage with similar percentages (95.6±2.2% and 94.0±2.2%, respectively). In contrast, the percentages of oocytes at the GV stage in the vitrified COC and DO groups were reduced (71.6±9.4% and 45.7±10.5%, respectively; P<0.05) compared with the control groups, which were associated with increased frequencies of diakinesis and MI stages. Percentages of oocytes at the GV stage in the vitrified COC and DO groups were not significantly different (P=0.23). In conclusion, cumulus cells can prevent vitrification-related membrane damage of oocytes. Furthermore, vitrification induced premature meiosis both in the cumulus-enclosed and denuded oocytes even in the presence of the meiotic inhibitor, dibutyryl cAMP. Nevertheless, cumulus removal without vitrification did not induce premature meiosis in the oocytes. Therefore, disruption in communication with cumulus cells might not be the primary reason for premature meiosis in vitrified oocytes.