Subcellular fractionation of rainbow trout gonads with emphasis on microsomal enzymes involved in steroid metabolism

Abstract

Rainbow trout gonads were subfractionated by differential centrifugation with emphasis on obtaining preparations suitable for the study of steroid-metabolizing enzymes. A fractionation scheme was evaluated for the mature testis and for 3 ovarian developmental stages. The distribution of cell organelles among the fractions was determined using enzyme-markers and electron microscopy. The fractionation scheme was found to be suitable for separating mitochondria and microsomes which were recovered at similar yields to those that had been reported for other extraheptic fish tissues. Fractionation of the mature ovary was fraught with problems probably because a large yolk protein cytosole fraction interfered with the recovery of microsomes. However, no difference in the specific activity of microsomal NADPH-cytochrome c-reductase between the various organ preparations was evident. The testis microsomes contained detectable amounts of cytochrome P450, whereas its content in the various ovary microsomes was too low to be detected. Progesterone 17 alpha-hydroxylase was detected in microsomes from testes and early developing ovaries, and microsomal aromatase activity was present in microsomes from early developing, mature and postovulatory ovaries. Furthermore, the testis microsomes contained a highly active UDP glucuronosyltransferase with testosterone used as a substrate.

Enzymatic validation of species‐specific protocols for metal subcellular fractionation in freshwater animals

“…Furthermore, the proportion of debris (i.e., membranes, granules, and nuclei) recovered in the other fractions was not assessed in our work even though this fraction is a significant one with respect to mass and represents more than half of the metal accumulated in many studies (Wallace and Luoma ; Rainbow et al ; Leonard et al ). Moreover, Andersson () has already observed that DNA could accumulate in the cytosol following subcellular fractionation and our own PCR analysis (Supporting Information Fig. S1) suggested the presence of DNA in every fractions following the fractionation of D. magna .…”

Differential gene expression of HSC70/HSP70 in yellowtail cells in response to chaperone-mediated autophagy

“…Subcellular fractionation was performed by modifying a previously described method [41]. Cells were washed twice with ice‐cold NaCl/P i and homogenized in lysis buffer containing 20 m m HEPES/NaOH, pH 7.5, 250 m m sucrose, 1 m m EDTA, and 1× protease inhibitor cocktail (F. Hoffmann‐La Roche, Basel, Switzerland) by 50 strokes in a Dounce‐type homogenizer.…”

Riochemical and physiological effects in farmed Baltic salmon fed lipids containing xenobiotics extracted from Baltic herring

“…The microsomal fractions of liver, head kidney, trunk kidney and gonads were obtained by differential centrifugation according to published methods (F6rlin, 1980;Pesonen & Andersson, 1987;Andersson, 1992) except that the post-mitochondrial supernatant was frozen in liquid nitrogen before preparation of the microsomal fraction. The microsomal fractions of liver, head kidney, trunk kidney and gonads were obtained by differential centrifugation according to published methods (F6rlin, 1980;Pesonen & Andersson, 1987;Andersson, 1992) except that the post-mitochondrial supernatant was frozen in liquid nitrogen before preparation of the microsomal fraction.…”