| Octyphenol(OP)is one of the degradation products of alkylphenol ethoxylates(APEOs),which belongs to the environmental endocrine disruptors(EDCs)and can interfere with the reproduction and development of animals.Gonadotropin includes follicle stimulating hormone(FSH)and luteinizing hormone(LH),which is secreted by the anterior pituitary and involved in the regulation of animal germ cell development and larval gonadal differentiation.To investigate the effects of OP on the expression of gonadotropin and its receptor genes in amphibian,we cloned some fragments of FSHf?,LH?,FSHR and LHR genes of Rana chensinensis.The distribution of this four genes in brain,eye,liver,kidney,stomach,heart,testis/ovary was analyzed by semi-quantitative PCR,and the expression profile of FSH? and LH? in the head and FSHR and LHR in gonads mesonephros complex(GMC)of the tadpoles at 33,37,43 and 46 stages was detected.Subsequently,the male adult frogs were exqosed to 10-8,10-7,10-6,10-5 mol/L OP for 10,20,30 days,respectively.And then the change of gonadosomatic index of OP treated male adults was analyzed by means of weigh and calculation.The expression of both FSH? and LH? in the brain and FSHR and LHR in the testis was detected by quantitative RT-PCR.In addition,tadpoles at stage 28 were exposed at 10-8,10-7,10-6 mol/L OP for 20,30 days and the entire larval period,respectively.The effects of OP exposure on larvae sex ratio were analyzed via statistical calculation.The expression of both FSH? and LH? in the head and FSHR and LHR in the GMC was detected by real-time quantitative PCR.Ultimately,explore the effects of OP exposure on FSH?,LH?,FSHR and LHR gene expression.The main experimental results and conclusions are as follows:1.The FSHf,LH?,FSHR and LHR cDNA fragments of R chensinensis cloned in this study were 100bp,328bp,255bp and 248bp,respectively.The results of BLAST analysis convey that the similarity of these four fragments with the corresponding genes in other species of amphibians were respectively more than 90%,which suggests that these fragments cloned in our study were exactly part of FSH?,LH?,FSHR and LHR gene of R.chensinensis.2.The semi-quantitative PCR results showed that FSH? and LH? had the highest expression in the head,and the expression of FSHR and LHR were the highest in the testis.This is consistent with that of the gonadotropin which was synthesized by the adenohypophysis and was involved in the regulation to the function of testis.The expression profile presented by semi-quantitative PCR showed that the expression of FSH? and FSHR was gradually increased during larval development,which suggests that FSH and FSHR were participated in the regulation of gonadal differentiation of tadpoles.3.The gonadosomatic index of male adults was decreased after OP exposure,which indicated that OP could affect the normal development of testis.The quantitative RT-PCR analysis showed that the expression of FSH? was up-regulated while the expression of LH? was down-regulated by OP.FSHR expression was significantly down-regulated,however,LHR expression was early decreased and then increased.These revealed that OP can affect the normal function of testis by interfering with the expression of gonadotropin and its receptor genes.4.The sex ratio of female was higher than that of the male.The quantitative RT-PCR showed that OP exposure down-regulated the expression of FSH? in both sexes and both FSHR and LHR in females,while the expression of FSHR and LHR in males at stage 46 was increased.These suggested that OP can affect the sex differentiation of juvenile by interfering with the expression of gonadotropin and its receptor genes.All the aboved results suggest that OP could interfere with the expression of gonadotropin and its receptor genes in adult and juvenile R.chensinensis,and could interfere with endocrine activity via regulating the expression of related genes,which ultimately affect male spermatogenesis and juvenile gonadal differentiation. |
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