Preliminary Studies On Mechanism Of Reproductive Endocrine In Male Barfin Flounder (Verasper Moseri)

As one of new and important culture objects for sea water cultivating in factory farm, barfin flounder (Verasper moseri) has important economic value. This paper mainly study on cultured male barfin flounder, from which the testis, sexual steroid hormone of blood plasma, aromatase and P450c17 are taken and studied by the methods of histological staining, immunohistochemical techniques, radioimmunoassay and molecular methods in details. The main results and conclusions are as follows:1. Histological study on the development and annual change in the testis of male Verasper moseriLike other teleosts, barfin flounder has a pair of testical located on two sides of the abdominal cavity. The shape of testis is thin strip at early stage of development, then it be thicken and widened gradually. Testis is usually symmetrical, and testical mesentery is connected with the body cavity lining.The testical development of barfin flounder is also similar with other teleosts, and showed significant seasonal changes. Sperm experiences 5 typical steps during its life:spermatogonia, primary spermatocytes, secondary spermatocytes, spermatid and spermatozoa. The annual change of testis development showed that the testicular development is in step with seasonal reproductive activity. Germ cells are not synchronous during early stages of testis development, and germ cell development is not synchronous in the same testis lobule. Germ cells are also at different developmental stages in different spermatogenic cysts.2. Cloning of CYP19a gene and expression analysis during the reproductive cycle in males of Verasper moseriCytochrome P450 aromatase is the terminal enzyme in steroidogenic pathway that converts androgens into estrogens. In most of vertebrates, P450arom is the product of the CYP19 gene, which exists as a single copy per haploid genome. In contrast, there are two isoforms of aromatase in teleosts, which encoded by two different genes, CYP19a and CYP19b, as well as have different distribution.This work describes the molecular cloning of the cDNA encoding the barfin flounder (Verasper moseri) gonad cytochrome P450 aromatase (CYP19a) by means of reverse transcriptase polymerase chain reaction (RT-PCR), degenerate primers PCR amplification and 5'-and 3'-rapid amplification of cDNA ends (RACE) analyses. We cloned the complete cDNA sequence of CYP19a gene and the length is 2167 bp, encoding a protein of 523 amino acids. Alignment analysis using barfin flounder CYP19a gene deduced animo acid sequence and animo acid sequences of other species showed that there were transmembrane helix region, I-helix region, Ozol's peptide region, aromatase-specific conserved region and heme-binding region in the deduced animo acid sequence. Phylogenetic analysis suggested that the occurrence of a gene duplication for cytochrome P450 aromatase in the teleost lineage after its divergence from the tetrapods. Further more, the higher identity between barfin flounder P450aromA and P450aromAs of other teleosts suggested that they have the same origin. A semiquantitative PCR was developed to measure mRNA expression levels of CYP19a gene of barfin flounder. Expression analysis in different tissues demonstrated that P450aromA transcripts were highly abundant in brain, ovary and testis, less abundant in intestine, liver and kidney, showing that not only gonads can produce steroids but also other tissues have steroidogenic capability. And we analyzed the relative mRNA expression level of CYP19a in testes at different stage of reproductive cycle, there is the highest level at stage II and the lowest level at stage V. These changes in expression profiles may imply differences in the functionality of the enzyme between different stages, suggesting an important role for P450aromA in barfin flounder testes development. 3. Molecular cloning, characterization expression of P450c17-â… and P450c17-â…¡and their functions analysis during the reproductive cycle in males of Verasper moseriP450c17, a key steroidogenic enzyme, play important roles in the production of sex steroid and cortisol. In teleost, there are two types of P450c17, P450cl7-â… possessing 17a-hydroxylase and 17,20-lyase activities, and P450c17-â…¡only possessing 17a-hydroxylase activity. This work describes the molecular cloning of the cDNA encoding the barfin flounder(Verasper moseri) P450c17-â… and P450c17-â…¡by means of reverse transcriptase and RT-PCR and 5'and 3'rapid amplification of cDNA ends (RACE) analyses, and mRNA expression profiles analyzing by semi-quantitative RT-PCR. Respectively, P450c17-â… and P450c17-â…¡mRNA levels in the testes and correlated with serumal testosterone (T) level, as well as gonadosomatic index (GSI) of males during different stages of spermatogenesis. The results demonstrated that P450c17-â… and P450c17-â…¡mRNA were expressed in the testis and ovary, suggesting that both of them participate in the production of sex steroid in barfin flounder gonads. P450c17-â… mRNA was undetectable, in contrast, P450c17-â…¡mRNA was detected at the highest level in the head kidney, meaning that only P450c17-â…¡is involved in the production of cortisol in barfin flounder. In testes, mRNA levels of P450cl7-â… and P450c17-â…¡showed little stage-dependent variation, and didn't share the same variation trend with GSI and serumal T levels of males, indicating that activities of P450cl7-â… and P450c17-â…¡are not regulated at the transcriptional level, but regulated at translation or post-translation.4. Immunohistochemical study of aromatase in testisAromatase immunohistochemical study on testis of barfin flounder showed that aromatase immunopositive reaction of spermatogonia and spermatocytes weaken gradually and spermids are immunonegative to aromatase during testis development. Overall, aromatase has stronger expression at early stage of testis development. In the testis, aromatase are expressed in spermatogonia, spermatocytes, Sertoli cells and interstitial cells. Particularly, aromatase are expressed continuously in Sertoli cells and interstitial cells. Gonadal-type aromatase gene (CYP19a) analysis showed that CYP19a are expressed continuously during reproductive cycle of males. The major role of aromatase is converting androgen into estrogen in the testis, and Sertoli cells and interstitial cells are major position where sex steroids are produced. All of these make us to speculate that aromatase play an important role during germ cell development, testis development and maturation of barfin flounder.