Comp. Biochem. Physiol. Vol. 73B, No. 2, pp. 269 to 274, 1982 0305-0491/82/100269-06503.00/0 Printed in Great Britain. © 1982 Pergamon Press Ltd SEX DIFFERENCES IN LIVER NON-SPECIFIC ESTERASES OF THE GREEN FROG RANA ESCULENTA ORFEO PICARIELLO, VIRGILIO BOTTE a n d MARINA PAOLUCCI Istituto e Museo di Zoologia, Facolt/t di Scienze, Universit/t di Napoli, Via Mezzocannone, 8, 80134 Napoli, Italy (Received 20 January 1982) Abstrac~l. Liver esterases of the green frog Rana esculenta have been fractionated on disc electro- phoresis, thin-layer electrofocusing and column electrofocusing. 2. The enzyme resolves in several molecular forms some of which result sex dependent, since they disappear in castrated animals and can be induced by sex hormone administration. 3. The enzyme molecular forms which depend on female hormones might be involved in cellular modifications of hepatocytes related to yolk protein synthesis. INTRODUCTION In most organisms non-specific esterases occur with as many as 10-30 molecular forms whose number and relative activity result both tissue and species specific (Markert & Moller, 1959; Holmes & Whitt, 1970; Masters & Holmes, 1974; Shaklee & Whitt, 1977; Moss, 1979). Some molecular forms, moreover, seem to be sex dependent, since they are synthesized and/or show a different relative activity in one of the two sexes. These enzyme fractions have been demonstrated in blood and in tissues of many animal species belonging to all vertebrate classes, including man (Botte & Basile. 1975; Lancieri et al., 1980; Tegelstr6m & Rytt- man, 1981 ; Kirkeby & Blecher, 1981). In the liver of the green frog, Rana eseulenta, two out of the five esterase molecular forms, resolved on starch gel electrophoresis, are sex dependent. They, in fact, are more active in intact females than in males and can be easily activated in both castrated males and females by estradiol administration (Botte & Basile, 1975). This observation has made possible a better definition of sex dependent diversity since the esterases are mainly bound to cytoplasmic membrane system which undergoes peculiar modifications dur- ing the synthesis of vitellogenin (Brachet et al., 1971 Lotstra et al., 1971 ; Lewis et al., 1976). MATERIALS AND METHODS Animals Adult Rana esculenta (mean body weight g 46.4 + 2.4; snout-vent length mm 74 + 13) were captured around Naples in February, at the beginning of the breeding season (February-May). Many of them were gonadecto- mized and then transferred, together with intact frogs, into terraria which had ambient photothermal regimes and run- ning tap water. Food (meal worms larvae) was given ad libitum. Hormonal treatments Ten days after the operation, the frogs were gathered into three groups, each composed of eight males and as many females, and were treated as follows. Group 1. Twice a week these frogs received 50/zg of estradiol dissolved in 50/~1 of 5~o ethyl alcohol in amphib- ian saline in the dorsal lymphatic sac for three weeks (total hormone dose: 300/zg). Group 2. After five estradiol injections as in group 1, the animals received 50/zg of progesterone dissolved in 50/21 of 5% ethyl alcohol in amphibian saline. Group 3. These frogs were injected only with the solvent. Two days after the last injection the animals were sacri- ficed, livers were collected and perfused with cold amphib- ian saline to remove the blood. Enzyme solution preparation Each liver was homogenized in ice cold 0.25 M sucrose (100 mg/ml). The suspensions were then centrifuged to get the following fractions: pellets at 7000, 50000 and 100,000O; supernatant at 100,000g. Preliminary studies showed that more than 95% of enzyme activity was re- covered in the last fraction, which therefore was used for further purifications. Protein determination was carried out according to Lowry et al. (1951) using bovine serum albumin as standard. Enzyme activity determinations Gels obtained from disc electrophoresis or from isoelec- trofocusing were incubated for 30 min at 37°C in a medium formed by 8 mM Tris-HC1 buffer pH 7.4, containing 0.003% fast blue RR salt and 0.002% substratum predis- solved in a small amount of acetone. The following sub- strata were routinely used: alpha naphthyl acetate, alpha naphthyl butrirrate, alpha naphthyl caprate. After incuba- tion the gels were rinsed in running tap water and then fixed in 30% methanol in water before photographed. The biochemical demonstration of esterases was carried out following Burlina & Galzigna's method (1972). Samples (100 #1 of 6 mg/ml of enzyme solutions) were incu- bated for 15 min at 37°C in a medium formed by 3 ml of 0.i M phosphate buffer, pH 8.5, containing 0.003% alpha naphthyl acetate. The free naphthol was then evaluated by reading at 310 rim. The above reported conditions resulted the most suitable ones for demonstration of frog liver ester- ase activity. The activity was expressed as nanomoles of alpha naphthol liberated by the hydrolysis of alpha naph- thyl acetate per/Lg protein for 15 rain. Disc electrophoresis The Davis' method (1964) was followed. Samples (con- taining 300~g of proteins) were applied to 10 × 0.7 cm 269