Nuclear and cytoskeletal alterations of in vitro matured porcine oocytes under hyperthermia.

Abstract

The effects of heat shock (HS) on the nucleus and cytoskeleton of in vitro matured pig oocytes were examined in this study. Porcine cumulus-oocyte complexes (COCs) were aspirated from 3 to 6 mm diameter follicles and subjected to standard in vitro maturation (IVM) procedures for 42 hr. In Experiment 1, IVM-derived oocytes were then randomly allocated to different HS treatments at 41.5 degrees C for 0 (control, C0h, n = 101), 1 (HS1h, n = 113), 2 (HS2h, n = 104), and 4 hr (HS4h, n = 111), respectively. An additional control group of oocytes was cultured for 4 hr without HS (C4h, n = 93). Immunocytochemical staining was performed using anti-tubulins and FITC-conjugated mouse IgG for microtubule (MT) labeling. The chromatin and microfilaments (MFs) were stained using Hoechst 33342 and rhodamine-phalloidin, respectively. In the severe HS (4 hr), the chromosomes of the MII oocytes became an aggregated chromatin structure and separated into groups. The spindle MTs were completely depolymerized or formed MT arrays. The relative fluorescence intensity (RFI) or amount of the MF structures including transzona processes (TZPs), vitelline ring (VR), and pericytoplasmic MF, were changed in various degrees. The reversibility of these alterations in the chromatin and the cytoskeleton depended on the duration of the HS. In general, abnormalities in the chromosomes, spindle MTs and the percentages of oocytes with pericytoplasmic MTs increased with length of HS treatment. The size of the spindle and the RFI of MFs in the HS oocytes were also altered. The significant changes in the nucleus and the cytoskeleton in porcine oocytes after HS may be associated with reduced development under hyperthermia and, perhaps, with the low pregnancy rates in domestic species during hot seasons.