Bioassay of fish gonadotrophin by ovarian mitochondrial cholesterol depletion

Abstract

Murrel (Channa punctatus Bloch) ovarian tissues were incubated in vitro with or without piscine gonadotrophins and then subjected to subcellular fractionation followed by nonesterified cholesterol (cholesterol) assay. Gonadotrophin from salmon (SG-G100), tilapia (TL1MS), and sturgeon (S27MS) depleted cholesterol in the mitochondrial fraction, whereas cholesterol remained unchanged in other subcellular fractions. Aminoglutethimide, an inhibitor of mitochondrial cholesterol side-chain cleavage, blocked the depletion of mitochondrial cholesterol in response to SG-G100 and murrel pituitary extract. When ovarian tissue containing [4-¹⁴C]cholesterol was challenged with SG-G100 in vitro, a dose-dependent decrease of mitochondrial [4-¹⁴C]cholesterol was observed. Increasing concentrations (1, 2, 4, 6, or 8µg/incubation) of SG-G100, TL1MS, and S27MS resulted in a clear linear depletion of mitochondrial cholesterol. The slope of the dose-response curve in different individual fish was found to be distinctly uniform and parallel. The slopes of the standard curves obtained with TL1MS and S27MS were greater than that with SG-G100, indicating that tilapia and sturgeon gonadotrophins are more potent. Carp pituitary gonadotrophin content, determined by using these standard curves, showed the sensitivity and precision of this bioassay.