| The presence of sex steroids in molluscs has been demonstrated, and previousstudies showed that sex steroids play potential roles in reproduction of molluscs.However, information regarding endocrine system in molluscs is still fragmentary.Moreover, endogenous origin and action mechanism of sex steroids are stillquestionable in molluscs.Zhikong scallop (Chlamys farreri), which is dioecious and sex-stable, is acommercially important bivalve in northern China. In the present study, we monthlyexamined the sex steroids (estradiol-17β, E2and testosterone, T) levels in gonads ofC. farreri during annual reproductive cycle and analyzed the potential role of sexsteroids in reproduction. Moreover, a full-length cDNA of the17β-hydroxysteroiddehydrogenase8gene (17β-HSD8) was cloned and characterized in C. farreri, and itscatalytic activity in vitro was also detected. Furthermore, we analyzed the temporaland spatial expression profile of17β-HSD8at mRNA and protein levels in gonadsduring the reproductive cycle and in embryos and larvae during early development,thus exploring its potential roles in steroids synthesis and metabolism pathway,gametogenesis and ontogenesis.To assess the potential roles of sex steroids in modulating reproductive processesof C. farreri, variations in E2and T levels in gonads were monthly examined fromJanuary to December2012by enzyme-linked immunosorbent assay (ELISA).One-year reproductive cycle of C. farreri was divided in to seven stages, includingresting stage, proliferative stage, growing stage, mature stage, first spawning stage,restoration stage and second spawning stage, based on the histological characteristicsof gametogenesis and spawning. During the annual reproductive cycle, meanconcentrations of E2and T in gonads ranged from75.07pg/g to666.24pg/g and from 91.09pg/g to506.28pg/g, respectively. Concentrations of E2were significantlyhigher in ovaries than in testes during the whole year, while T concentrations werehigher in testes than in ovaries during gametogenesis only. Concentrations of E2inovaries and T in testes increased with gonadal development and maturation, reachedthe highest value before spawning, and decreased rapidly after spawning. A positivecorrelation between E2levels and oocyte diameters (r=0.743, P<0.05, n=25) wasobserved, suggesting that E2may play a role in oogenesis. These findings indicatethat E2and T, which are highly correlated with the reproductive cycle, may playimportant roles in maintaining sexual stability, gonadal differentiation, gametogenesisand spawning in C. farreri.17β-hydroxysteroid dehydrogenases (17β-HSDs) are important enzymescatalyzing steroids biosynthesis and metabolism in vertebrates. Although studiesindicate sex steroids play potential roles in reproduction of molluscs, little is knownabout the presence and function of17β-HSDs in molluscs. In the present study, afull-length cDNA encoding17β-HSD type8(17β-HSD8) was identified in C. farreri,which is1,104bp in length with an open reading frame of759bp, encoding a proteinof252amino acids. Phylogenetic analysis revealed that the C. farreri17β-HSD8(Cf-17β-HSD8) belongs to the short chain dehydrogenase/reductase family (SDR) andshares high homology with other known17β-HSD8homologues. The recombinantCf-17β-HSD8protein was expressed in E. coli, and the soluble protein was obtainedthrough refolding protocols. Catalytic activity assay in vitro demonstrated that therefolded Cf-17β-HSD8could effectively convert estradiol-17β (E2) to estrone (E1)and weakly catalyze the conversion of testosterone (T) to androstenedione (A) in thepresence of NAD+.Results of semi-quantitative RT-PCR indicated that the Cf-17β-HSD8mRNAwas ubiquitously expressed in all tissues analyzed in C. farreri at proliferative stagewith high levels in digestive gland, kidney and ovary, and with low levels in mantle,adductor muscle, gill and testis. The expression levels of Cf-17β-HSD8mRNA andprotein detected by qRT-PCR and Western blot increased with gametogenesis in bothovary and testis, and were significantly higher in testis than in ovary at growing stage and mature stage. Moreover, results of in situ hybridization andimmunohistochemistry revealed that the mRNA and protein of Cf-17β-HSD8werelocated in follicle cells and gametes at all stages except spermatozoa, and the intensityof positive signals in gametes decreased along with gametogenesis. Our findingssuggest that Cf-17β-HSD8may play an important role in gametogenesis throughmodulating steroids levels in gonad of C. farreri.We also examined the temporal and spatial expression pattern of Cf-17β-HSD8mRNA in embryos and larvae of C. farreri. The Cf-17β-HSD8was highly expressedin unfertilized egg suggesting that it is a maternal gene. Expression levels ofCf-17β-HSD8mRNA decreased significantly in embryos at cleavage stage comparedwith fertilized eggs, then increased sharply and reached a peak in blastula, indicatingthe zygotic expression of Cf-17β-HSD8is started at this stage. After that, theexpression levels of Cf-17β-HSD8fluctuated with individual development. In situhybridization detection revealed that the positive signals of Cf-17β-HSD8mRNAwere evenly distributed in cells of embryos at various stages, implying thatCf-17β-HSD8might participate in embryogenesis of C. farreri through modulatingsex steroids levels in embryos. In the D-shaped larva, high expression level ofCf-17β-HSD8mRNA was observed, and the positive signals gathered around thevisceral mass, suggesting a potential role of Cf-17β-HSD8in initiation oforgangenesis.In conclusion, E2and T play potential roles in maintaining sexual stability,gonadal differentiation, gametogenesis and spawning in C. farreri; the Cf-17β-HSD8in gonads participates in regulating gametogenesis through modulating steroids levelsof C. farreri, and may also be involved in the embryo development and larvalorgangenesis. Data in this study provide an academic basis for further exploration ofsteroids biosynthesis and metabolism pathway and their physiological roles inreproduction and development. |
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