169 FORSKOLIN INDUCED INCREASE IN LIPOLYTIC ACTIVITY I PORCINE EMBRYOS PRODUCED IN VITRO

Abstract

The sensitivity of porcine embryos to cryopreservation is largely due to their high level of intracellular lipid content (Polge and Willadsen 1978 Cryobiology 15, 370-373). Delipation through centrifugation and micromanipulation resulted in a significant proportion of porcine embryos produced in vivo being able to survive cryopreservation and produce live births (Nagashima et al. 1995 Nature 374, 416). However, due to the intense resources needed for delipation via micromanipulation, this approach has limited practical value. In this experiment, we tested the hypothesis that delipation can be achieved through chemical stimulation of intracellular lipolysis in porcine embryos produced in vitro. Day 6 porcine blastocysts cultured in the presence of 10 µM forskolin, a lipolytic agent (Ho and Shi 1982 Biochem. Biophys. Res. Commun. 107, 157-164), in a group of 101-202 blastocysts per 50 µL of NCSU-23 + 4 mg/mL BSA (Sigma-Aldrich, St. Louis, MO, USA) yielded approximately 125 000-250 000 cells/mL (Zalatan et al. 2001 Endocrinology 142, 3783-3790). Blastocysts cultured in 50 µL NCSU-23 + 4 mg/mL BSA without the supplementation of forskolin served as control. Samples (12 µL) of the media were taken from culture at 0, 3, and 6 h and frozen at -20°C for lipolytic assay. Because the major content of intracellular lipids in porcine embryos is triacylglycerol and the hydrolysis of triacylglycerol results in the production of fatty acid and glycerol, therefore, the lipolytic activity in porcine blastocysts was measured by detection of glycerol concentration in the culture media. A commercially available Free Glycerol Regent (Sigma) was used with modifications. This kit only measures free glycerol released into the media as a result of endogenous lipase activity because the kit itself doesn't contain lipase. Ten µL of sample medium was mixed with 80 µL of Free Glycerol Regent and incubated in a 37°C water bath for 5 min. The resulting samples were read using a Cary 50 UV Spectrophotometer (Varian, Inc., Palo Alto, CA, USA). The actual concentration of glycerol in the culture medium was calculated using the glycerol standard curve. The glycerol concentration at 0 h was regarded as 0 for both the treatment group and the control group. The glycerol concentrations at other time points were calculated accordingly. The measurement was conducted four times for the treatment group and three times for the control group. The data were analyzed using a Student's t-test. Glycerol concentrations in the treatment group at 3 h and 6 h were 5.99 ± 1.74 (mean ± SEM) µM, and 10.49 ± 0.81 µM, respectively, and both values were significantly different from their counterparts in the control group, which were 0.95 ± 0.62 µM and 4.43 ± 1.31 µM, respectively. These results indicate that the hydrolysis of intracellular lipids in porcine embryos can be stimulated by lipolytic agents and result in the partial reduction of the intracellular lipid content. This approach may be used for designing a better protocol for the cryopreservation of porcine embryos produced in vitro.

This project was supported by a grant from the National Institutes of Health (U42 RR 018877).