Effect of 17 [beta]-estradiol on induction of vitellogenesis in immature female and male frogs, Hoplobatrachus rugulosus

Abstract

The objectives of the study were to elucidate the effect of 17[beta]-estradiol (E[subscript 2]) on vitellogenesis in immature frogs, Hoplobatrachus rugulosus (H. rugulosus) which reared in the rainy season (June-October) and the dry season (November-March). Female and male frogs at the age of 5 months old of each season were daily intramuscularly injected with E[subscript 2] at a dose of 0, 50, 500 or 5,000 microgram/kg body weight for five days. Liver vitellogenin (Vtg) gene expression, plasma phosphoprotein (precursor of Vtg), the number and size of oocytes, liver-somatic index (LSI), gonadal-somatic index (GIS) as well as histology of the liver and gonad were determined. Results obtained showed that primers of Vtg gene sequence from the Vtg gene sequence of the chicken, Gallus gallus were used for RT-PCR amplification of H. rugulosus liver total RNA. Sequence of the derived RT-PCR amplicons and sequence similarity suggested that it was a Vtg gene of H. rugulosus. The expression level of liver Vtg gene was determined by semi-quantitative RT-PCR. Treatment with E[subscript 2] in frogs which reared during rainy season within the dose range studied significantly decreased the Vtg gene expression levels in immature female frogs (0.65+-0.02, 0.60+-0.02 and 0.58+-0.02 vs 0.85+-0.03, respectively). E[subscript 2] at dose of 500 microgram/kg body weight significantly increased the level of Vtg gene expression (0.80+-0.06) in immature male frog compared to control group (0.61+-0.04). In contrast, female frogs reared during dry season, treated with E[subscript 2] at all doses tended to increase the Vtg gene expression levels (0.68+-0.04, 0.78+-0.05 and 0.72+-0.04 vs 0.65+-0.04). Similar results were obtained from the immature male frog (0.70+-0.06, 0.72+-0.05 and 0.73+-0.03 vs 0.68+-0.04). Phosphoprotein staining showed that E[subscript 2] could stimulate plasma phosphoprotein levels in both immature female and male frogs which reared in both seasons. There were no changes in the number and size of oocytes, LSI and GSI as well as in the histology of the liver and oocytes in the immature female frogs which reared during rainy season were observed. Results obtained might be due to the short period of treatment (5 days). When plasma E[subscript 2] levels were measured, it was found that the levels of E[subscript 2] in immature female reared in the rainy season (327.24+-188.61 pg/ml) were higher than the female frog reared in the dry season (87.33+-24.18 pg/ml) and than the E[subscript 2] levels found in immature male in both seasons (17.59+-4.47 and 68.03+-23.81 pg/ml). It is therefore concluded that the primers designed from part of cDNA sequence of vitellogenin gene of Gallus gallus was able to amplify a fragment of at least one of the Vtg genes in the frogs, H. rugulosus which confirmed that this part of the gene is conserved among vertebrates. Since E[subscript 2] inhibited Vtg gene expression in frogs reared in the rainy season when the endogenous E[subscript 2] was high, hence it is possible that frogs with high levels of endogenous E[subscript 2] may be interfered with exogenous E[subscript 2] such as xenoestrogens in the environment resulted in impairment of reproduction in the frog