186 EFFECT OF POLYVINYLPYRROLIDONE SUPPLEMENTATION IN CULTURE MEDIUM ON THE GROWTH OF MOUSE OOCYTES

Abstract

A high volume of polyvinylpyrrolidone (PVP) supplementation in culture medium has a significant impact on the growth of bovine oocytes. The objective of the present study was to determine whether or not PVP affects oocyte growth in the mouse. Oocyte–granulosa cell complexes were isolated from 11- or 12-day-old mice (ICR) by mechanical isolation of follicles, followed by a collagenase treatment (0.1%; 10 min). Twenty complexes were placed on each insert fit in the 24-well culture plate and cultured for 10 days in an atmosphere of 5% CO₂ in air at 37°C. The culture medium was a modified α-MEM supplemented with 5% fetal bovine serum and 1 ng mL^–1 FSH. The concentration of PVP (molecular weight of 360 000) was 0%, 1%, 2%, or 3% (w/v). During the first 2 days, only medium with 0% PVP was used. The oocytes recovered on Day 10 were subjected to in vitro maturation, IVF, and embryo culture. In 12 replications, the total numbers of oocytes cultured in medium with 0%, 1%, 2%, and 3% PVP were 235, 233, 233, and 231, respectively. In some additional experiments, oocytes were fixed on Day 10 and processed for transmission electron microscopy (TEM). The oocytes in medium with 0% PVP became located within an enlarged dome-like structure. In medium with 2% PVP and 3% PVP, no such domes were formed, and the oocytes within several granulosa cell layers were exposed to medium; however, the cumulus cell mass specifically became larger than that in medium with 0% PVP. The viabilities of oocytes recovered from medium with 0%, 1%, 2%, and 3% PVP were 83%, 81%, 91%, and 93%, respectively. The survival rate was significantly higher in medium with 3% PVP than in medium with 0% PVP or 1% PVP (P < 0.05). The mean oocyte diameter increased from 59 µm (Day 0) to 72, 71, 71, and 72 µm in medium with 0, 1, 2, and 3% PVP, respectively, but they continued to be smaller than in vivo grown oocytes (81.0 µm; P < 0.01). When maturation was induced, cumulus cell mucification occurred irrespective of PVP concentration during the growth. No significant differences were found between the groups in the percentage of polar body extrusion (ranging from 78 to 88%). Developmental outcomes based on oocytes used for in vitro fertilization were the following: cleavage rates were 67, 78, 74, and 76%; and blastocyst rates were 37, 44, 47, and 36% of oocytes that had been grown in medium with 0, 1, 2, and 3% PVP, respectively. The numbers of oocytes included were 60, 59, 68, and 66, respectively. The TEM observation suggests that more intimate contacts were maintained between the oocyte and cumulus cells in medium with 2% PVP than in medium with 0% PVP. Taken together, PVP supplementation in medium has a considerable influence on the morphology of mouse oocyte–granulosa cell complexes and close contacts within the complexes in the long-term culture, as having been observed with bovine oocytes.