ACTIVATION OF DOMESTIC CAT OOCYTES USING THE SULFHYDRYL REAGENT THIMEROSAL

Abstract

Domestic cats are useful as animal models in comparative medicine programs as they provide insight into disease mechanism and facilitate investigation of new therapeutic options. The application of cloning in these animals is also beneficial in terms of conserving the precious animal models. Although successful cat cloning by nuclear transfer has recently been reported, the technology is still extremely ineffective. One potential reason for the low efficiency is suboptimal embryo development after oocyte activation. During nuclear transfer, the calcium signals that are normally triggered by the fertilizing sperm must be reproduced artificially to initiate the development of the reconstructed embryos. Thimerosal is a chemical which is able to oxidize critical sulfhydryl groups on intracellular calcium release proteins and thus induce the release of calcium from the intracellular stores and trigger oocyte activation. The effects of thimerosal have previously been reported in a number of species; the objective of this study was to evaluate whether thimerosal can induce calcium release and initiate parthenogenetic embryo development in the oocyte of the domestic cat. Cat ovaries were obtained from a local veterinary clinic. Immature oocytes collected from the ovaries were matured in vitro in Feline Optimized Culture Medium (FOCM) supplemented with 0.6 mM cysteine, 0.1 mM cysteamine, 1.0 IU/ml eCG, 2.0 IU/ml hCG, 25 ng/ml EGF, for 24 hours. For intracellular calcium measurements, cat oocytes were incubated in the presence of 2 μM fura-2 AM, a calcium indicator dye and 0.02% pluronic F-127, for 40 minutes. Individual oocytes were then transferred into TL-Hepes medium containing 200 μM thimerosal. Changes in the cytoplasmic free calcium levels were monitored using the InCyt Im2 fluorescence imaging system. Preimplantation embryonic development was also evaluated; for this purpose the oocytes were incubated in TL-Hepes in the presence of 200 μM thimerosal for 30 minutes followed by a 20-min incubation in TL-Hepes with 8 mM dithiothreitol, a sulfhydryl reducing agent. Control oocytes were cultured in TL-Hepes for 50 minutes. After the treatments, the oocytes were held in 7.5 μg/ml cytochalasin B for 4 hours to stimulate the formation of diploid embryos then cultured in FOCM for 6 days. The total number of nuclei in the developing embryos was recorded by staining with Hoechst 33342. After the addition of thimerosal, a series of transient rises in the intracellular free calcium concentration were observed in 81.8% (9 out of 11) of the oocytes during the 30-min incubation period. Furthermore, thimerosal induced cleavage in 21.8% (14/64) of the activated oocytes; 4.7% (3/64) of the oocytes developed to morula or blastocyst stage. The average cell number of those morulae/blastocysts was 19.7±3.2. In the control group, no cleavage was observed (0/43). The results support the hypothesis that thimerosal can induce an oscillation in the intracytoplasmic free calcium levels in oocytes by oxidizing sulfhydryl groups on intracellular calcium release proteins. The series of calcium increases was sufficient to trigger the signal transduction pathway in cat oocytes that resulted in embryonic development up to the blastocyst stage. The results indicate the potential application of thimerosal for oocyte activation during cat nuclear transfer. Additional studies are needed for the optimization of the activation parameters. (poster)