Studies Of Molecular Mechanisms Of Sex Pheromones In Chinese Black Sleeper (Bostrychus Sinensis)

Olfaction plays an important role in fish reproductive communication.Many fish species employ sex pheromones to improve the synchronization of gametogenesis,spawning and fertility.Among fish sex pheromones,progestins and prostaglandins are well studied.Take goldfish for example,at the time of oocyte mature,female fish release 17a,20?-dihydroxy-pregnen--one(DHP)into water acting as a sex pheromone.DHP elicits endocrine responses in male fish,including Lh surge and increase of milt production.Oocyte maturation is followed by ovulation.At this point,female release prostaglandin F2a(PGF2a)and prostaglandin E2(PGE2)into water to induce reproductive behaviors in male,resulting in a higher fertilization rate.Our previous studies clearly showed that DHP and PGE2 were two sex pheromones in the Chinese black sleeper(Bostrychus sinensis).However,one question is still unsolved "How does male B.sinensis sense sex pheromones?" In this study,the techniques of real-time PCR,in situ hybridization,immunohistology,transactivation assay and ex vivo tissue culture were used to study the molecular mechanisms of sex pheromone in male B.sinensis.The main results are as follows:1.Olfactory progestin receptors are considered as candidates to sense sex pheromone DHP.We first cloned 9 progestin receptors in B.sinensis:membrane progestin receptors(paqr5,paqr6,paqr7a;paqr7b,paqr8 and paqr9);nuclear progesterone receptor(pgr);progestin receptor membrane component(pgrmcl and pgrmc2).These progestin receptors showed different tissue specific expression patterns,and all of them were expressed in the olfactory rosette.Interestingly,the expression of paqr8 in the olfactory rosette increased with testicular development.This pattern mirrored the pattern of plasma DHP change in female.Therefore,we suppose that olfactory Paqr8 plays an important role in sensing sex pheromone DHP.2.One main function of sex pheromone DHP is to increase plasma Lh level,resulting in testis maturation of male fish.In this study,we cloned the gonadotropin(GtH)subunits in B.sinensis and analyzed the expression patterns of GtH subunits in the pituitary at different stages of testicular development.The results suggest that like other fish,B.sinensis Fsh was involved at early stages of spermatogenesis,while Lh played important roles at late stages of spermatogenesis.Unsurprisingly,after the exposure to 5nM DHP for 6hr,the expression of lh? in the pituitary was significantly increased,while that of fsh? kept constant.In addition,plasma 11-KT level also increased 24hr after exposure.These results suggest sex pheromone DHP promotes the maturation of male B.sinensis via Lh synthesis and 11-KT production Interestingly,the pituitary lh? of immature male B.sinensis did not respond to DHP exposure,suggesting that endocrine responses are influenced by reproductive status One possibility is that reproductive status modulates the expression of DHP receptors We intraperitoneally injected nearly mature male fish with HCG and LHRH-A3 to promote testicular maturation and investigated the expression of progestin receptors Among 9 progestin receptors,only the expression of paqr8 and pgrmc2 were up-regulated,and paqr8 showed a 2-folds increase.Lately,we found that positive signals of Paqr8 protein were observed in the dendritic knobs which is responsible for sensing odors.Moreover,some DHP-activated olfactory sensory neurons(OSNs)expressed Paqr8 protein,further suggesting that Paqr8 plays a vital role in sensing DHP in B.sinensis3.Our previous studied showed that the possible receptor for sensing sex pheromone PGE2 was Ep1,and mature male B.sinensis was more sensitive to PGE2 than immature fish.This study aims to clarify how the reproductive status modulates the sensitivity of male fish to PGE2.The expression of ep1 significantly increased after HCG and LHRH-A3 administration,suggesting that epl expression might be modulated by sex hormones.Thereafter,we found androgens up-regulated the olfactory expression of ep1 both ex vivo and in vivo.Moreover,Ar agonist R1881 had the same effects on epl expression,suggesting that effects of androgens on ep1 expression were mediated by classic androgen receptor Ar.Like many other fishes,two Ar subtypes were found in B.sinensis(Ara and Ar?).Because Ar? and Ar?showed different tissue specific expression patterns,different subcellular localization and distinct DNA binding domains,it is suggested that they might display different biological functions.Which Ar subtype mediate the effects of androgens on epl expression?In the olfactory rosette of mature male fish,the expression level of ara was 3 times higher than that of and the expression level of ara increased with testicular development.Activated Ara but not Ar? transactivated epl promoter.Ara mRNA was widely distributed in OSNs and some ara-positive OSNs expressed Ep1.Taken together,we suppose that effects of androgens on epl expression are mediated by Ara but not Ar? in B.sinensis.Together with previous studies,the present study further investigated the molecular mechanism of sex pheromone DHP and PGE2 in B.sinensis.Taken all together,we hypothesize that during the spawning season,female B.sineniss release sex pheromone DHP and PGE2 into water in sequence.At the time of oocyte maturation,sex pheromone DHP released by female binds to olfactory Paqr8,which stimulates pituitary Ih? expression and finally elevates plasma 11-KT level in male fish.Eleveated 11-KT level not only promotes testicular maturation but also acts on olfactory Ar to up-regulate the epl expression,resulting in higher sensitivity of male fish to sex pheromone PGE2.Thereafter ovulatory female fish release PGE2 into water to induce reproductive behaviors of male fish.