Cloning Of Reproductive Genes And Their Expressive Manipulation Analysis In Male Spotted Sea Bass(Lateolabrax Maculatus)

Spotted sea bass is an ecologically and economically important euryhaline teleost fish that lives in a wide range of salinity. Using histological, endocrinological and molecular biological methods to detect the changes of endocrine physiology of spotted sea bass cultured in Shandong coastal areas, the results are listed below:1. Cloning of reproductive gene from spotted sea bassUsing RACE method,12 full-length sequences were cloned from spotted sea bass(GTHa subunit, FSHβ subunit, LHβ subunit, StAR, AMH, FTZ-F1α, FTZ-F1b, CYP171, 3β-HSD, vasa, cGnRH-Ⅱ and PRL),6 uncomplete genes were also acquired(CYP17-Ⅱ、 FSHR、PRLR、GR、AR、SOX9). All sequences had been submitted to NCBI. Clustal W and MEGA 5.0 methods were used to analysis their homology. It clearly showed that these genes in spotted sea bass were homologous to those of other perciformes species.2. Expression of reproductive genes during reproductive cycle and in different tissuesQPCR was performed to measure the changes of GTHs subunits, StAR, AMH, FTZ-Fl, CYP171 and 3β-HSD genes expression during testicular development cycle, it was detected that these genes change significantly during this period, suggesting they may participate in spermatogenesis of spotted sea bass. Spatial analysis showed that gene transcripts tested in this study expressed highly in steroidogenesis-related tissues, such as testis, brain, head kidney and liver. Gene transcripts can also be found in other nonsteroidogenesis tissues. The location of CYP17　Ⅰ　and 3β-HSD mRNA in spotted sea bass testis were detected by in situ hybrization. Positive signals for CYP17 Ⅰ and 3β-HSD were both observed in interstitial cells of adult testis. Combinng the changes of histology and hormone levels, it provided a deep insight into the function of reproductive genes in spotted sea bass.3. Changes of histology, T level and gene expression during hCG and GnRHa treatmentThe spermatic cell development level and serum testosterone level were increased in response to in vivo administration of hCG and GnRHa. Quantification of GTHs subunits transcripts in brain, pituitary and testis, StAR, FTZ-F1、CYP171,3β-HSD in testis and AR gene in brain revealed a trend similar to their serum testosterone level, increasing significantly during hormone treatment, while a reciprocal relationship was founded between transcripts of　AMH　and AR genes in testis and serum concentration of testosterone. It suggested that hCG and GnRHa stimulations can improve the development stage of spotted sea bass testes and these genes detected are all under the control of gonadotropic hormone and gonadotropin-releasing hormone.4. Changes of histology, hormone levels and gene expression in salinity treatmentSerum osmolality was significantly reduced in both brackish water (BW) and freshwater (FW) groups during 1-8d of acclimation, these changes were consistent with plasma testosterone levels (1-6d) and testicular histology. Serum FSH and LH concentrations decreased in FW group during salinity acclimation whereas transcript levels of GTHa, FSHβ and LHβ subunits in the pituitary showed large increases during the first two days of salinity acclimation in BW and FW groups. In addition, a decreasing trend of 3β-HSD, CYP17 I and AMH mRNA transcripts was observed in testes of spotted sea bass from the BW and FW groups especially in FW group, while AR gene mRNA levels increased substantially in BW group (4-8d). These results suggested that abrupt decreases in salinity may have negative effects on testicular function in spotted sea bass.