Study On The Molecular Mechanisms Of Reproductive Endocrinology In Paralichthys Olivaceus And Paralichthys Lethostigma

Almost all fish reared in captivity exhibit some form of reproductive dysfunction. In females, there is often failure to undergo final oocyte maturation, ovulation and spawning; while in males milt production may be reduced and of low quality. This thesis mainly studied on cultured Japanese flounder (Paralichthys olivaceus) and wild southern flounder(Paralichthys lethostigma), from which the gonad, sexual steroid hormone of blood plasma, several gene related to fish reproduction were taken and studied by the methods of histological staining, immunohistochemical techniques, radioimmunoassay and molecular methods. The aim is to provide some valuable information for understanding the reproductive mechanisms in flatfish cultured and for improving strategies of reproductive manipulations in the aquaculture industry. Main contents and results are as follows: 1. P450 aromatase (P450arom, CYP19), a CYP19 gene product, is a member of the cytochrome P450 superfamily that catalyzes the formation of aromatic C18 estrogen from C19 androgen. To characterize the involvement of the gonadal P450 aromatase gene (CYP19α) during the reproduction of Japanese flounder, the expression of CYP19αmRNA was detected in different tissue of adult fish and also determined during the various developmental stages of testes. The CYP19αmRNA was highly abundant in ovary and spleen of female fish, less abundant in testis and spleen of male fish, but not present in other tissues by RT-PCR. The changes in mRNA level of CYP19αdid not share the same changes trend with serum E2 during the reproductive cycle of male fish Immunohistochemically, aromatase immunopositive reaction of spermatogonia and spermatocytes weaken gradually and spermids were immunonegative to aromatase during testis development. Overall, immunoreactive P450arom has stronger expression at early stage of testis development. These results suggest that CYP19a gene implicated in the regulation of spermatogenesis.2. Cytochrome P450c17 is the single enzyme that mediates the 17a-hydroxylase and 17,20 lyase activities during the biosynthesis of steroid hormones in the gonads and adrenal gland. In teleost, there are two types of P450c17, P450c17-Ⅰhas both these enzymatic activity and P450cl7-II possesses only 17a-hydroxylase activities. In this study, the full length cDNA of P450c17-Ⅰand P450c17-Ⅱwere cloned from the Japanese flounder, which is an annually spawning teleost. RT-PCR analysis of Japanese flounder tissue extracts showed significant expression of P450c17-ⅠmRNA in the heart, testis and ovary. And the expression of P450c17-ⅡmRNA was relatively high in brain, head kidney, kidney, testis and ovary. We further examined the pattern of expression of two genes throughout the reproductive cycle. RT-PCR revealed that P450c17-Ⅰand P450c17-ⅡmRNA were expressed in testes of different stage. And the variation trend of serumal T level and GSI were similar with the pattern of these two genes mRNA expression. The physiological relevance of these findings remains to be further elucidated.3. Estrogens play important roles in the growth and differentiation of reproductive tissues and in the maintenance of fertility by binding to a nuclear receptor protein, the estrogen receptors. The objectives of this study were to characterize polymorphisms within the coding region of estrogen receptorβ(ERβ) gene in the collected female Japanese flounder and to analyze the association of ERβpolymorphisms with reproductive indices by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Two single nucleotide polymorphisms (SNPs), SNP1 (c.577de1C) and SNP2 [c.A891T (p.Gln1 14Leu)], were identified in the ERβgene. A one-way ANOVA revealed that SNP1 was significantly associated with the gonadosomatic index (GSI) in female Japanese flounder (P<0.05). And SNP2 was significantly associated with the serum 17β-estradiol (E2) level and GSI (P<0.05). Individuals with genotype AB of SNP2 had significantly higher serum E2 level and GSI than those of genotype AA (P< 0.05).Moreover, the hepatosomatic index (HSI), a marker for genetic effects, was significantly higher for diplotype D2 compared with the other three diplotypes (P<0.05).4. FOXL2 gene plays an important role in ovarian development, granulosa cell differentiation, and thus the proper maintenance of ovarian function. The aims of this study were to characterize polymorphisms within the FOXL2 gene in the collected female Japanese flounder and analyze the association of FOXL2 polymorphisms with reproductive performance by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP). Results indicated that five Single nucleotide polymorphisms (SNPs), which were SNP1 [c.540A>C (p.Asn102His) and c.591A>G (p.Asn119Asp)], SNP2 [c.864G>A (p.Lys210Glu) and c.875G>A] and SNP3 (c.1169C>A), were identified in the FOXL2 gene. General Linear Model (GLM) analysis showed that SNP1 in the forkhead domain was significantly associated with gonadosomatic index (GSI) (P<0.05). SNP2 in the downstream of forkhead domain was significantly associated with serum 17β-estradiol (E2) level (P<0.05). And SNP3 in the 3'-UTR was significantly associated with hepatosomatic index (HSI) (P<0.05). Moreover, the evaluation of the genetic effects for both Testosterone (T) level of diplotype D3 and GSI of diplotype D5 suggested they were significantly higher than those of other four diplotypes (P<0.05), respectively.5. Recent results suggested that membrane progestin receptor alpha (mPRα) mediates nongenomic actions of progestin hormones to induce oocyte maturation and sperm hypermotility in several teleost species. The role of mPRa in gamete and gonadal physiology was further evaluated in the present study by examining gonadal expression of mPRa during the gonadal development in male Japanese flounder.We cloned the mPRa cDNA from the Japanese flounder ovary. The predicted protein showed high sequence identity with Southern flounder (99%),Seatrout(94%) and Atlantic croaker(93%) mPRα. Computer modeling predicted that the deduced protein has seven transmembrance domains.The mPRα mRNA was detected in all the tissues tested with relatively greater expression in brain, ovary, testis and kidney. The mPRa mRNA was also expressed in Japanese flounder testes throughout the gonadal cycle. And the serumal E2 and GSI level showed the similar variation trend with the pattern of mPRa mRNA expression. Taken together, these data supported the hypothesis that mPRa is the MIS receptor in Japanese flounder.6. Recently we discovered a previously uncharacterized gene with the characteristics of a progesterone and adipoQ receptor (PAQR) in a flatfish, Japanese flounder. Here, we reported the cloning and partial characteristics of this hitherto unknown gene. Phylogenetic analysis clearly indicated the PAQR-like gene clade was between the PAQR7 clade and PAQR8 clade. The predicted protein showed high sequence identity with three-spined stickleback (77%) and medaka (72%) unnamed gene. Structural analyses of the translated cDNA suggested it encoded protein with seven transmembrance domains, typical of progesterone and adipoQ receptor. RT-PCR analysis of Japanese flounder tissue extracts showed significant expression of the PAQR-like gene in ovary, head kidney, testis and kidney, and lower expression in brain, gill, intestine, stomach and pyloric caecum. The PAQR-like gene mRNA was also expressed in Japanese flounder testes throughout the gonadal cycle. And the variation trend of GSI was similar with the pattern of the PAQR-like gene mRNA expression. These characteristics suggested that the PAQR-like gene is related to reproductive physiology in this species.7. The identity of the maturation-inducing steroid (MIS) in Southern flounder, a marine teleost which is similar to Japanese flounder, is unknown. The purpose of the present study was to identify the major progestin produced during oocyte maturation (OM) in southern flounder and investigate whether its secretion increases during this process. Intact of ovarian follicles undergoing OM were incubated in vitro with tritiated [3H]pregnenolone precursor and the tritiated products were extracted, purified, and identified by TLC. We found the tritiated products were biosynthesized from [3H]pregnenolone including 20β-S, 17a,20p-DHP,11-deoxycortisol. During the oocyte maturation, there were a significant increase in the production of 20β-S, whereas the production of 11-deoxycortisol decreased. And the amount of 17α,20β-DHP remained almost the same. In vitro,20β-S and 17α,20β-DHP were most effective steroids to induce maturation of southern flounder oocytes. These results suggest that that 20β-S and 17α,20P-DHP maybe the naturally occurring MIS in the female southern flounder.