Complete activation of porcine oocytes induced by the sulfhydryl reagent, thimerosal

Thimerosal (200 mu M) triggered Ca2+ oscillations in 56 of 56 mature porcine oocytes. The Ca2+ oscillations were blocked by the sulfhydryl-reducing agent dithiothreitol (DTT), thus supporting the hypothesis that thimerosal acts by oxidizing critical sulfhydryl groups on intracellular Ca2+-release proteins. Thimerosal treatment alone arrested the oocytes in metaphase, probably by oxidizing tubulin sulfhydryl groups and thus destroying the spindle. However, a 10-min exposure to 200 mu M thimerosal followed by a 30-min incubation in 8 mM DTT induced complete activation, as 73.8% of the oocytes formed pronuclei. The second polar body was visible in 73.3% (55 of 75) of the activated oocytes. Combined thimerosal/DTT treatment of the oocytes also induced cortical granule exocytosis, as revealed by confocal microscopy, and the subsequent hardening of the zona pellucida. After activation, some oocytes were incubated in vitro, or in vivo in a ligated porcine oviduct, for 6 days. When cultured in vitro, 42.0% (37 of 88) of the oocytes developed to the compact morula or blastocyst stage; the average number of inner cell mass (ICM) and trophectoderm (76) nuclei in the blastocysts was 8.6 +/- 0.7 and 20.1 +/- 1.3, respectively. Culture in a ligated oviduct resulted in 42.9% development to the compact morula or blastocyst stage, with the blastocysts having a mean number of 12.5 +/- 1.0 ICM and 63.6 +/- 9.2 TE nuclei.