Maturation and spawning of <Emphasis Type="Italic">Spratelloides gracilis</Emphasis> Clupeidae in temperate waters off Cape Shionomisaki,central Japan

ABSTRACT:   The reproductive ecology of Spratelloides gracilis was investigated in the temperate waters off Cape Shionomisaki, central Japan. Cape Shionomisaki is located at the northern margin of the distribution range of this species. Females of S. gracilis with gonadosomatic index (GSI) ≥ 4.0 were defined as mature based on the relationship between GSI and the histological maturity phases of their ovaries. More than 50% of females greater than 60 mm standard length (SL) were mature. Hatch dates of larvae and juveniles collected in the study area were determined by otolith daily ring counts and found to extend from April to November. The size at maturity of females (60 mm SL) and the spawning season of S. gracilis in the temperate waters off Cape Shionomisaki (April–November) was larger and shorter, respectively, than those in tropical waters in the western Pacific. The reproductive traits observed for S. gracilis off Cape Shionomisaki appear to be adaptive to northern temperate waters with seasonal changes in environmental conditions.     

水温对半滑舌鳎性腺组织发育的影响

探讨了不同温度(19,21,23,25和27 ℃)对半滑舌鳎的性腺组织发育的影响。在受精后39～106 d对半滑舌鳎进行温度控制处理,通过石蜡组织切片,对半滑舌鳎早期性腺分化发育进行组织学观察。发现在19 ℃饲养条件下,受精后46 d(pfd46)半滑舌鳎性腺原始生殖细胞数量增加,有分化成卵巢的倾向;在该条件下,pfd82半滑舌鳎性腺开始分化为精巢。pfd82时,27 ℃条件下的幼鱼卵巢中卵原细胞数目大量增殖;精巢中精小叶多而密,结缔组织间隔清晰。pfd129时,27 ℃处理的幼鱼卵巢发育到Ⅱ期,其他温度处理组仍处于Ⅰ期卵巢,但随着温度的提高,卵巢中卵母细胞数目增多,且由散乱分布变为有序的排列在卵巢小叶中;精巢中贮精囊数量增多,囊腔中精子增多。pfd142时,高温处理组幼鱼性腺发育最快,其Ⅱ期卵巢中卵母细胞数目最多,精巢中后期生精细胞偏多,精子几乎分布到各个精小叶中,且贮精囊结构发达,内纳高密度精子,在周围输精管中也分布着精子。结果表明,温度影响了半滑舌鳎性腺发育程度,二者呈现线性关系,在适当温度范围内,性腺发育速度随着温度升高而加快。     

Ontogeny of sexual differentiation in different strains of zebrafish (Danio rerio)

The zebrafish (ZF) undergoes juvenile hermaphroditism, where in all fish, the gonad first develops as an ovary-like structure, before the sex is fixed and the gonad then differentiates into either a testis or an ovary. Sexual differentiation is being adopted as an endpoint for assessing endocrine disrupting chemicals, yet there is only very limited information on the timing and variability in sexual differentiation process both within and between strains of ZF. In this study, using gonad histology, the ontogeny of sexual differentiation was studied in two strains of ZF, one with a high level of heterozygocity (WIK strain) and another that had been in-bred for several generations. There was a high variability in the timing of sex differentiation between individuals within a specific strain (with no obvious relationship with body mass), but there were no differences between the two strains. Transformation of the immature gonad into testes in males started in week 5 post fertilization (pf) and completion of sexual differentiation for all fish in both study populations occurred by week 11–12 pf. The size of the fish containing transforming gonads in the WIK strain and in-bred strain were similar (ranging from 12 to 23 mm, and from 13 to 22 mm total length, respectively).     

石鲽仔、幼鱼性腺发育的组织学观察

利用组织学方法对人工培育的石鲽(Kareius bicoloratusBasiewsky)仔、幼鱼性腺发育进行研究。结果表明,性腺的发育与体长密切相关。刚孵化石鲽的原生殖细胞数目为2个,孵化3 d数目增至8个,之后经过迁移,至孵化9 d到达生殖嵴。在全长为7.2～8.5 mm(孵化9～11 d)的仔鱼中,性腺原基中的体细胞迅速增殖并包围原生殖细胞,后者在全长10～15 mm(孵化后10～35 d)的仔幼鱼中增殖成为生殖干细胞。原始性腺在全长15～30 mm(孵化40～60 d)的幼鱼中逐渐发育完善,呈细线状,位于腹腔后部中肾管下方紧贴体壁。雌性性腺最早在全长32.5 mm(孵化66 d)的个体中出现分化特征,至全长89～102 mm时雌性性腺特征完全分化。雄性性腺的分化较雌性性腺晚,最早在全长为91 mm的幼鱼中开始,至全长为114～118 mm时雄性性腺分化特征已经十分明显。     

莫桑比克罗非鱼幼鱼的性腺发育与分化

本文介绍了莫桑比克罗非鱼幼鱼性腺的发育和分化的观察结果。用组织学方法观察，莫桑比克罗非鱼幼鱼的性腺发育，可以明显地区分为三个阶段，即：性腺的原始阶段，性腺分化前期阶段，性腺分化完成阶段。作者认为，对莫桑比克罗非鱼的诱变试验，应在前两阶段进行；当幼鱼体长达到15mm以后，性腺分化已基本完成，性激素的处理不可能取得性诱变的明显效果。     

Scp3 expression in relation to the ovarian differentiation in the protogynous hermaphroditic ricefield eel <Emphasis Type="Italic">Monopterus albus</Emphasis>

Synaptonemal complex protein 3 (Scp3), which is encoded by scp3, is a meiotic marker commonly used to trace the timing of gonadal differentiation in vertebrates. In the present study, the ricefield eel scp3 cDNA was cloned, and a fragment encoding amino acids 49 to 244 was overexpressed. The recombinant Scp3 polypeptide was purified and used to generate a rabbit anti-Scp3 polyclonal antiserum. In adult ricefield eels, scp3 mRNA was predominantly detected in the gonads and faintly detected in discrete brain areas. In the gonads, Scp3 immunoreactivities were shown to be localized to the germ cells, including meiotic primary growth oocytes, spermatocytes, and pre-meiotic spermatogonia. During early ovarian differentiation, immunoreactive Scp3 was not detected in the gonads of ricefield eels at 6 days post-hatching (dph) but was found to be abundantly localized in the cytoplasm of some oogonia at 7 dph, coinciding with the appearance of the ovarian cavity and ovarian differentiation. At 14 dph, strong Scp3 immunostaining was detected on one side of the nucleus with a distinct polarity in some germ cells, presumably at the leptotene stage. Consistent with these results, the expression of scp3 mRNA was faintly detected in the gonads of ricefield eels at 6 dph, increased at 8 dph, and then remained relatively high thereafter. Taken together, these results suggest that the appearance of immunoreactive Scp3 in oogonia could be a marker for early ovarian differentiation in ricefield eels. The translocation of the Scp3 protein from the cytoplasm to the nucleus in the oogonium of ricefield eels appears to be a controlled process that warrants further study.     

Effects of estradiol-17β and 17α-methyltestosterone on gonadal sex differentiation in the F2 hybrid sturgeon, the bester

The effects of giving oral estradiol-17β (E₂) and 17α-methyltestosterone (MT) on gonadal sex differentiation in the F₂ hybrid sturgeon, the bester ( Huso huso female ×  Acipenser ruthenus male), are investigated. Giving E₂ at 10 μg/g diet to fish from 14 months until 31 months of age induced incomplete feminization and resulted in approximately 40% abnormal ovary development in which oocytes were observed without ovarian lamellar structures and gonadal shape was similar to normal testis. Giving MT at 25 μg/g diet for the same duration failed to induce masculinization, and resulted in approximately 30% undeveloped gonads even at 30–37 months of age. In contrast, E₂ and MT at only 1 μg/g diet given from 3 to 18 months of age was sufficient to induce feminization and masculinization, respectively. In these fish, feminization and masculinization were observed at 9 months, when most putative ovaries and testes were histologically distinguishable by the shape of the gonadal surface. These results indicate that sex reversal can be induced in these fish by hormone treatment that is started at 3 months age, before morphological differentiation occurs on the stroma of the gonads.     

达氏鲟dmc1基因的克隆及在精子生成中的表达分析

DNA减数分裂重组酶1基因(disrupted meiotic cDNA,dmc1)是一个在减数分裂时期特异表达的基因,为减数分裂同源染色体配对所必需。为研究dmc1基因在达氏鲟(Acipenser dabryanus)不同组织的表达特征,从达氏鲟性腺转录组数据库搜索得到该基因并设计引物,克隆获得达氏鲟dmc1的编码区序列,其中开放阅读框(ORF)为1 029 bp,编码342个氨基酸。运用生物信息学方法分析达氏鲟dmc1基因编码的蛋白序列和系统进化关系,推测达氏鲟Dmc1蛋白的分子量为82.696 kDa,理论等电点(PI)为5.04;多重序列比对发现,Dmc1氨氮酸序列在进化中可能比较保守。系统进化分析显示,AdDmc1属于RecA家族Dmc1蛋白分支,且与四足动物类聚为一支。RT-PCR结果表明,dmc1基因在精巢和卵巢中特异表达。结合组织学结果通过实时荧光定量PCR研究dmc1基因在精子生成过程中的表达特征发现,dmc1基因在0~Ⅰ期精巢中不表达;在Ⅱ、Ⅲ、Ⅳ期精巢中,其表达量逐渐升高并在Ⅳ期精巢中达到最高;在Ⅴ期精巢中,其表达量显著性下降(P<0.05),因此推断达氏鲟dmc1基因可能是减数分裂时期特异表达的基因,研究结果可为后续达氏鲟初级和次级精母细胞的鉴定奠定基础。     

Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus

The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse.     

17-β雌二醇诱导鲻雌性化的机制

用原位杂交和免疫细胞化学技术,对服用17 β雌二醇实验组和对照组幼年鲻脑各部和性腺进行芳香化酶的定位。结果发现,芳香化酶转录物和特异性蛋白在幼年鲻端脑(嗅球和大脑)、间脑、中脑和小脑是丰富的。在性别未分化时,芳香化酶免疫活性细胞在幼鲻脑各部的分布密度有显著的差异。在嗅球,对照组芳香化酶免疫阳性细胞的分布密度高于实验组,而在间脑、中脑和小脑实验组免疫阳性细胞数量比对照组多1～3倍,特别是下丘脑视前区芳香化酶的免疫阳性细胞数量尤占优势,提示芳香化酶在幼年鲻性分化中可能起关键的作用。另外,在性分化后,芳香化酶免疫活性还定位在卵巢颗粒细胞和精巢间质细胞与足细胞。同时,免疫阳性物质也定位在卵巢和精巢的生殖细胞。这些结果揭示了17 β雌二醇诱发幼鲻雌性化的机制可能是通过芳香化酶的介导,本研究首次提供形态学新的证据。最后,文中还讨论了芳香化酶在鲻性腺发育中可能的生理作用。     

栉孔扇贝Dmrt1的分子鉴定及表达模式分析

DMRT1是DMRT家族重要成员之一,主要参与动物性别决定和分化调控,但在不同动物中其表达和功能调控作用存在差异。本研究采用生物信息学方法分析了栉孔扇贝（Chlamys farreri）Dmrt1的序列特征,采用半定量RT-PCR技术确定了其mRNA在成体性腺、闭壳肌、外套膜、鳃和肾等组织中的分布,定量RT-PCR和原位杂交技术揭示了Dmrt1 mRNA在性腺中的时空表达特征。结果显示：栉孔扇贝Dmrt1序列中含有DMRT家族保守的DM结构域;其mRNA仅在栉孔扇贝性腺中表达,在精巢中的表达明显高于卵巢,并且以生长期精巢的表达水平最高;原位杂交检测Dmrt1阳性信号主要定位于生殖细胞的细胞质中。研究结果表明,Dmrt1在栉孔扇贝性腺中的表达特征与大部分动物性腺中的表达特征基本一致,推测其可能参与性别分化和精巢发育的功能调节。     

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dazl基因克隆及在生殖细胞中的表达

为探索龟鳖类生殖细胞的发育分化机制,实验通过特异性引物克隆了中华鳖dazl基因的cDNA片段,长1 007 bp,其中包括5′端非编码区197 bp,3′端非编码区33 bp,开放阅读框777 bp,编码258个氨基酸。氨基酸序列比对显示其与西部锦龟Dazl同源性最高,达96%;与小鼠Dazl同源性达75%。荧光定量PCR分析结果显示,中华鳖dazl mRNA主要在精巢和卵巢中表达,在体细胞组织中仅检测到微量表达。冰冻切片原位杂交结果显示,中华鳖dazl mRNA在生殖细胞中特异表达,且在不同时期的生殖细胞中呈动态表达模式。在精巢中,中华鳖dazl mRNA在初级和次级精母细胞中表达最强,在精原干细胞中表达水平次之,在精子细胞中未检测到信号;在卵巢中,中华鳖dazl mRNA信号在初级卵母细胞胞质中均匀分布且在最早期的初级卵母细胞中信号最强,随着卵母细胞的增大,信号逐渐聚集并逐渐减弱。研究表明,dazl基因可能对中华鳖两性生殖细胞的发生具有重要的调控作用。