Sex steroid level and sexual dimorphism expression of genes in gonads of the great sturgeon Huso huso Linneaus, 1758 during maturity developmental stages

Little is known about molecular mechanisms involved in gonad differentiation in sturgeon species. In non‐mammalian vertebrates, sox9, dmrt1, cyp17a1 and ar are male specific genes in testicular differentiation and are highly conserved. In order to understand the mechanism underlying testicular development in sturgeon, we investigated sex steroids level of 11‐ketotestosterone (KT) and testosterone (T) and relative expression of sox9, dmrt1, cyp17a1 and ar in great sturgeon gonads during different stages of sexual maturity. The results showed all studied genes had dimorphic patterns in males. In immature gonads (stage I), only cyp17a1 expressed in male gonads, while other genes did not express, and KT and T levels were low. Dmrt1 showed dimorphic expression pattern in male gonads at maturity stage II, III and IV. Furthermore, sox9 and ar mRNA presented significant dimorphic expression pattern in male gonads only at maturity stage 4. Plasma androgens levels were significantly higher in males compared to females during maturity stages II, III and IV. The results showed that among these four genes, only cyp17a1 expressed in male gonads at maturity stage I (immature), suggesting that this gene may be applicable as a sex marker in recently differentiated male great sturgeon. Sexually dimorphic patterns in other studied genes (sox9, dmrt1 and ar) suggesting that these genes may be important for testicular development and differentiation in premature great sturgeon. The obtained results provide a foundation for further research on sex differentiation and developing strategies for the sexing of sturgeon for aquaculture.     

Morphology,sex steroid level and gene expression analysis in gonadal sex reversal of triploid female (XXX) rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In non-mammalian vertebrates, estrogens and expressions of cyp19a1 and foxl2 play critical roles in maintaining ovary differentiation and development, while dmrt1 and sox9 are male-specific genes in testicular differentiation and are highly conserved. In order to deeply understand the morphological change, sex steroids level and molecular mechanism of triploid female gonadal reversal in rainbow trout, we studied the ovary morphology, tendency of estradiol-17β (E2) and testosterone (T) levels and the relative expressions of dmrt1, cyp19a1, sox9 and foxl2 in juvenile and adult fish. Our results demonstrated that the development of triploid female gonads in rainbow trout went through arrested development, oocytes dedifferentiation, ovary reconstruction and sex reversal finally. During early gonadal development (154–334 days post-fertilization), the expressions of foxl2 and cyp19a1 increased linearly, while expressions of dmrt1 and sox9 were extremely suppressed, and E2 level was higher, while T level was lower. During the mid-to-late period of triploid female gonadal development (574–964 days post-fertilization), the expressions of dmrt1 and sox9 remained high and were very close to the quantity of diploid male genes, and T levels were even reaching diploid male plasma concentrations, while expressions of cyp19a1 and foxl2 were decreased, leading to decrease in E2 level. We realized that the development model of rainbow trout triploid female gonads was extremely rare, and the regulatory mechanism was very special. Genes involved in gonadal development and endogenous estrogens are pivotal factors in fish natural sex reversal.     

大黄鱼雌雄性腺长链非编码RNA的挖掘与差异分析

为探究长链非编码RNA在大黄鱼性腺发育与分化中的作用,从雌雄各3尾大黄鱼(Larimichthys crocea)的性腺中提取总RNA,进行去除rRNA的链特异性转录组建库和二代测序。将测序数据比对到大黄鱼参考基因组,经比对、组装共得到来自31675个基因的66088个转录本,严格筛选得到来自3984个基因位点的5162条lncRNA。进一步分析获得了在大黄鱼雌雄性腺中差异表达的mRNA9341个,lncRNA2782个,高度相关的lncRNA-mRNA对1227个;有多个lncRNA靶向已知的性别分化和发育相关基因,其中lncRNA MSTRG.24346与大黄鱼的性别决定候选基因dmrt1距离相近,且相关性极显著。该研究表明lncRNA可能在大黄鱼性别分化中起到重要作用,值得深入研究阐明机制。     

栉孔扇贝sox9基因的cDNA克隆及其在不同发育时期性腺中的表达特征

SOX9是SOX家族的SOXE亚族成员,在脊椎动物骨骼发育、胰腺发育、性别决定与分化以及肿瘤形成中发挥重要的作用。sox9在不同种类脊椎动物性腺中的表达存在差异,在无脊椎动物性腺中的表达特征尚不清楚。本研究利用RACE技术克隆了栉孔扇贝(Chlamys farreri)sox9(Cf-sox9)全长的c DNA序列,其长度为2835 bp,开放阅读框为1413 bp,编码470个氨基酸。预测的氨基酸序列具有SOX家族的HMG-box和SOXE亚族的高保守区域,但没有脊椎动物羧基端的Pro-Gln-Ser rich区域。原位杂交和免疫组织化学技术确定Cf-sox9 m RNA和Cf-SOX9蛋白均定位在栉孔扇贝精巢和卵巢的所有生殖细胞中,并且在不同发育时期性腺中呈现了类似的表达规律,即在精巢中,阳性信号在精母细胞中最强,在精子中最弱;在卵巢中,阳性信号在卵原细胞、卵母细胞以及成熟卵中呈现逐渐减弱的趋势。这一表达特征与脊椎动物性腺中多样的性别差异表达特征不同,提示sox9在贝类性腺发育和配子发生中的作用可能与大多数脊椎动物不同。     

Dimorphic expression of sex-related genes in different gonadal development stages of sterlet, <Emphasis Type="Italic">Acipenser ruthenus</Emphasis>, a primitive fish species

Molecular mechanism of sex determination and differentiation of sturgeon, a primitive fish species, is extraordinarily important due to the valuable caviar; however, it is still poorly known. The present work aimed to identify the major genes involved in regulating gonadal development of sterlet, a small species of sturgeon, from 13 candidate genes which have been shown to relate to gonadal differentiation and development in other teleost fish. The sex and gonadal development of sterlets were determined by histological observation and levels of sex steroids testosterone (T), 11-ketotestosterone (11-KT), and 17β-estradiol (E2) in serum. Sexually dimorphic gene expressions were investigated. The results revealed that gonadal development were asynchronous in 2-year-old male and female sterlets with the testes in early or mid-spermatogenesis and the ovaries in chromatin nucleolus stage or perinucleolus stage, respectively. The levels of T and E2 were not significantly different between sexes or different gonadal development stages while 11-KT had the higher level in mid-spermatogenesis testis stage. In all the investigated gonadal development stages, gene dmrt1 and hsd11b2 were expressed higher in male whereas foxl2 and cyp19a1 were expressed higher in female. Thus, these genes provided the promising markers for sex identification of sterlet. It was unexpected that dkk1 and dax1 had significantly higher expression in ovarian perinucleolus stage than in ovarian chromatin nucleolus stage and in the testis, suggesting that these two genes had more correlation with ovarian development than with the testis, contrary to the previous reports in other vertebrates. Testicular development-related genes (gsdf and amh) and estrogen receptor genes (era and erb) differentially expressed at different testis or ovary development stages, but their expressions were not absolutely significantly different in male and female, depending on the gonadal development stage. Expression of androgen receptor gene ar or rspo, which was supposed to be related to ovarian development, presented no difference between gonadal development stages investigated in this study whenever in male or female.     

圆斑星鲽sox9基因的克隆与表达

本研究筛选到圆斑星鲽(Verasper variegatus)的性别相关基因sox9,并通过RACE技术获得了全长序列,基因全长为3287 bp,包括1431 bp的ORF,编码477个氨基酸,368 bp的5¢ UTR和1488 bp的3¢ UTR。在3¢ UTR中有多聚腺苷酸尾和加尾信号AATAAA。通过荧光定量PCR测定了sox9基因在圆斑星鲽成鱼不同组织中的表达水平,发现sox9基因在圆斑星鲽的脑、眼、鳃、心、肝、胆、肠、精巢、卵巢、肾和肌肉等各个组织中都有不同程度的表达。在鳃、脑和精巢组织中检测到较高水平的sox9转录,其中精巢中的转录水平显著高于其他组织,sox9基因在性腺中的表达显示出性别两相性差异。其在精巢中的表达水平要显著高于卵巢,说明sox9基因与雄性性腺发育相关。通过测定sox9基因在圆斑星鲽幼鱼不同发育时期(20、30、40、50、60、70和80日龄)的表达水平,发现其在20~50日龄表达量逐渐下降,在60日龄时表达量上升,推测表达量上升可能与幼鱼性腺分化相关。     

中华鳖SOX9基因在胚胎期性腺中的表达模式及在性逆转中的表达变化

SOX9基因在脊椎动物性别决定和性腺分化中扮演重要的调控作用。从mRNA和蛋白水平分析中华鳖SOX9基因在不同组织中的差异性表达、在胚胎性腺和成年睾丸中的细胞定位以及在性逆转中的表达变化,研究SOX9基因在中华鳖性别分化中的调控作用。Real-time PCR结果显示,SOX9基因在中华鳖雄性性腺中特异性表达。免疫荧光染色分析显示,SOX9蛋白在雄性18期胚胎性腺中开始表达,随着性腺的发育,SOX9蛋白定位于性腺Sertoli前体细胞细胞核中;而在雌性胚胎性腺并未见其表达。此外,在雌激素诱导的雄性向雌性性逆转胚胎中,SOX9基因显著下调,而在芳香化酶抑制剂诱导的雌性向雄性性逆转胚胎中,SOX9基因表达则显著上升。研究表明,SOX9基因为中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别分化过程中起调控作用。     

Cloning and expression of prostaglandin E2 receptor subtype 1 (ep 1 ) in Bostrichthys sinensis

Our previous studies suggested that prostaglandin E₂ (PGE₂) is a putative sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. We found immunoreactivities of PGE₂ receptor subtypes (Ep_1–3) expressed in the olfactory sac, but only Ep₁ presented higher density of immunoreactivity in mature fish than that in immature fish in both sexes. To gain a better understanding of the underlying molecular mechanism for the detection of PGE₂ in the olfactory system, we cloned an ep ₁ cDNA from the adult olfactory sac. The open-reading frame of the ep ₁ consisted of 1,134-bp nucleotides that encoded a 378-amino acid-long protein with a seven-transmembrane domain, typical for the G protein-coupled receptors superfamily. Expression of ep ₁ mRNA was observed in all tissues examined, with higher levels obtained in the olfactory sacs and testes. The expression of ep ₁ mRNA in the olfactory sacs and gonads was significantly higher in both sexes of mature fish than in those of immature ones. Taken together, our results suggested that Ep₁, which is highly expressed in the olfactory sacs and gonads of mature fish, is important for the control of reproduction and may be involved in PGE₂-initiated spawning behavior in B. sinensis.     

Studies on feminization,sex determination,and differentiation of the Southern catfish, <Emphasis Type="Italic">Silurus meridionalis</Emphasis>—a review

The sex ratio of the feral Southern catfish was reported to be about 1:1, while the fish obtained by artificial fertilization were always female. Hence, we examined the possible influence of the micro-environment during artificial insemination (pH of the ovarian fluid and concentration of the semen) and early development (feed, hatching temperature, and water) on the sex ratio of Southern catfish fry. In order to examine the possibility of the occurrence of gynogenesis during artificial propagation, cytological observations on the insemination processes and the artificial induction of gynogenesis were also performed. However, no male fish were obtained even in these experiments, excluding the possibilities of these micro-environmental changes on catfish sex ratio and the occurrence of gynogenesis during artificial propagation. Female-to-male sex reversal was achieved by treatment with fadrozole (an aromatase inhibitor) and tamoxifen (an estrogen receptor antagonist). Histological analyses on the gonadal development of both female and induced male fish were subsequently performed. Moreover, several genes involved in sex differentiation, such as dmrt1, foxl2, and cyp19, and three subunits of gonadotropin (gth), i.e., gthα, lhβ, and fshβ, were isolated. Their expression patterns were studied under normal gonadal development and sex reversal conditions. The results revealed that dmrt1, foxl2, and cyp19a were closely related to catfish sex differentiation, and the gth subunits were possibly related to ovarian differentiation and oocyte development. Taken together, we hypothesized that estrogen was highly responsible for the ovarian differentiation and feminization of catfish fry under artificial propagation, although the mechanism involved remains elusive.     

Distinct expression of three estrogen receptors in response to bisphenol A and nonylphenol in male Nile tilapias (Oreochromis niloticus)

Environmental estrogens, such as bisphenol A (BisA) and nonylphenol (NP), have been shown to affect the estrogen receptor (ER) expression and induce male reproductive abnormalities. To elucidate molecular mechanisms of action of xenoestrogenic chemicals on the expression of estrogen receptors in the testes of Nile tilapia (Oreochromis niloticus), three full-length cDNAs respectively encoding ntERα, ntERβ1 and ntERβ2 were cloned from testes. The amino acid sequences of ntERα, ntERβ1 and ntERβ2 showed a high degree of similarity to the relevant fish species. Tissue-specific expression study showed that three receptors were highly expressed in pituitary, liver, testis, kidney and intestine tissues. The ntERα, ntERβ1 and ntERβ2 mRNA expressions were significantly higher at the sexual early recrudescing stage than at other recrudesced stages. After being exposed to xenoestrogens from weeks 2 to 4, the ntERα mRNA levels were increased significantly in testes after NP treatment at all sampling times or after 4 weeks of exposure to BPA. The ntERβ1 mRNA levels remained unchanged, while a significant decrease of the ntERβ2 mRNA level was observed in testes after exposure to NP and BPA. The present study demonstrates that the regulation of all three ntER subtypes in testes may act via different molecular mechanisms of exposure to NP and BPA.     

Gonadal Development and Differentiation in Cultured Juvenile Winter Flounder, Pseudopleuronectes americanus

Winter flounder, Pseudopleuronectes americanus, is currently being evaluated as a stock enhancement candidate in New Hampshire, USA; however, little is known about the gonadal development or the sex ratio of cultured juveniles. To determine the size at gonadal differentiation, 327 cultured fish ranging from <20 to 110 mm total length (TL), in 10‐mm‐TL size classes, were examined histologically. Gonads had differentiated into testes and ovaries in fish ≥41 mm TL (98%), whereas the majority of fish (81%) smaller than 40 mm TL possessed undifferentiated gonads. A total of 313 cultured fish >40 mm TL were analyzed for sex ratio. In 2003, 67 females and 164 males were identified, yielding a sex ratio that was significantly skewed toward male (χ²= 40.7, df = 1, P < 0.001). This trend held true when cultured fish were sorted by age and length, with the exception of those fish 61–70 mm TL. This aberration probably was because of a small sample size in this length category. However, in both the 2004 and the 2005 cultured populations, flounder sex did not deviate from a 1:1 ratio (2004 χ²= 0.12, df = 1, P= 0.724 and 2005 χ²= 0.02, df = 1, P= 0.881). The 2003 data suggest that environmental or genetic factors may affect winter flounder sex determination; rearing manipulation studies in the hatchery are needed to confirm this hypothesis.     

Cryopreservation and transplantation of sexually immature gonads of rainbow trout

The objective of this investigation was to cryopreserve sexually immature gonads of rainbow trout (RBT). Gonads from juvenile male or female RBT were cryopreserved and stored in liquid nitrogen. Individual testes were thawed and surgically transplanted to isogenic male trout; individual ovaries were autografted. Four weeks following transplantation, all male fish were injected with salmon pituitary extract for 8–10 weeks. The transplanted testis was present in 5 of 8 animals in replicate 1 and in 3 of 6 animals in replicate 2. Fertility of sperm obtained from individual testes in replicate 2 was evaluated by in vitro fertilization. Sperm from the intact control testes had a fertilization rate of 95±3% (mean ± SEM), and sperm from transplanted testes had a fertilization rate of 78±7%. Ovaries that were frozen in liquid nitrogen, thawed and autografted to an ectopic site were not present at five months following surgery.