Oocyte Maturation/Viability after Culture of Antral Follicles Immersed in Culture Medium
All of the oocytes derived after culture of the antral follicles immersed in culture medium were maintained at GV stage, although in some of these oocytes, abnormal chromatin condensation was observed. However, on subsequent culture, none of them were able to break down GV and develop to MII (Fig. 3a). Treatment with okadaic acid did not induce GVBD in these oocytes. Trypan blue exclusion as a test of oocyte viability revealed that the majority of these oocytes had lost viability during the culture period.
Maintenance of Meiotic Arrest
The number of oocytes cultured and the percentage of GV-stage oocytes derived from each of the three culture methods are shown in Table 1. Oocytes recovered after 24-h culture of intact antral follicles maintained the highest frequency of GV arrest (96.8%). This contrasted with 62.7% of oocytes cultured within follicle hemisections (x21 = 37.1, p < 0.001), which in turn was greater than the 24.6% for oocytes attached to a portion of the follicle wall (X21 = 48.5, p < 0.001). Visual assessment of oocyte quality suggested that the oocytes derived from whole follicle culture had a normal appearance (Fig. 3b); however, the oocytes cultured within hemisections of follicle wall showed an abnormal, slightly condensed chromatin morphology (Fig. 3c). buy diabetes drugs
FIG. 1. Bovine antral follicles after dissection from the ovary. a) Range of follicle sizes. b) Good-quality follicle showing translucency and presence of blood vessels used as selection criteria (X20; reproduced at 47%).
FIG. 2. Schematic diagram of antral follicle dissection for the preparation of follicle hemisections or isolation of oocytes attached to part of the follicle wall. Isolated follicles were pierced, and collapsed follicle walls were cut in three places. Oocytes attached to hemisections of follicle wall were cultured for 24 h.
FIG. 3. GV-stage oocytes isolated after 24 h culture. a) Group of GV oocytes showing abnormal chromatin condensation isolated after 24-h culture inside culture medium. b) GV-stage oocyte after 24-h antral follicle culture showing uncondensed chromatin. c) GV-stage oocyte after 24-h culture inside hemisection of the follicle wall showing abnormal chromatin condensation in the presence of nucleus membrane. a,b X200; c X400.
TABLE 1. Number of oocytes and percentage remaining at the GV stage after 24 h culture in three different culture systems.
|Culture methodFollicle wall Hemisections Antral follicle||No. of replicates667||Total no. of oocytes735964||No. oocytes at GV stage (%)18 (24.6)37 (62.7)62 (96.8)|