黄鳝性腺自然逆转过程中vasa基因的表达分析

本研究采用RNA反义探针原位杂交技术,对vasa基因在黄鳝(Monopterusalbus)性腺发育过程中的表达情况进行了分析。结果表明:vasamRNA在Ⅰ、Ⅱ、Ⅲ期卵母细胞的胞质中均匀分布,在Ⅳ、Ⅴ期卵母细胞中vasamRNA有向胞质外周皮层迁移集中的趋势,但不明显;退化的卵粒也呈现vasamRNA阳性反应;在Ⅲ、Ⅳ期卵巢的被膜中检测到带有vasa阳性信号的细胞,这些细胞可能是待向精原细胞分化、迁移到卵巢被膜上的原始生殖细胞(Primordialgermcell,PGC),在性逆转过程中这些PGC可能由卵巢被膜迁移到精小叶中并发育成精子;在成熟精巢中,vasa在精原细胞和初级精母细胞中表达。进一步采用碱性磷酸酶染色法分析黄鳝卵巢及精巢后发现:在卵巢中,除了卵母细胞外,卵巢被膜中也检测到了带有碱性磷酸酶阳性信号的细胞;在成熟精巢中,只在生殖腺囊内的雄性生殖细胞中检测到碱性磷酸酶,而精巢被膜中没有检测到带有碱性磷酸酶阳性信号的细胞。本研究结果初步表明:黄鳝的雄性生殖细胞可能起源于雌性阶段卵巢被膜中的原始生殖细胞[动物学报51(3):469-475,2005]。     

中华鳖vasa基因克隆及在卵母细胞中的表达分析

研究利用中华鳖为研究模型进行爬行类生殖细胞发育分化成熟等生物学研究,克隆了中华鳖vasa基因的cDNA序列,全长3865 bp,包括5'端非编码区90 bp,3'端非编码区1699 bp,开放阅读框长2076 bp,共编码691个氨基酸。中华鳖Vasa氨基酸序列包含DEAD-box家族蛋白8个保守保守功能域,在N末端有4个RGG重复序列和2个GG富集区,与小鼠Vasa蛋白的同源性较高（72%）。荧光定量PCR的结果表明,中华鳖vasa mRNA主要精巢和卵巢中表达,其他体组织中均难检测到表达。卵巢冰冻切片原位杂交结果显示:中华鳖vasa mRNA在生殖细胞中特异表达;在卵子发生过程中的不同发育期卵母细胞中呈现动态的变化。即vasa mRNA在初级卵母细胞及生长期卵母细胞中表达最强,且均匀分布在细胞质中,随着卵母细胞的逐渐增大,信号逐渐减弱,直至在成熟的卵母细胞中几乎检测不到表达信号,说明vasa可能在中华鳖早期卵母细胞发育中起重要作用。同时,vasa基因可作为中华鳖生殖细胞分子标记物,根据其mRNA的表达水平来鉴别不同发育时期的卵母细胞。研究结果为进一步开展中华鳖胚胎生殖细胞发育及配子生成,特别是研究中华鳖,乃至爬行类原始生殖细胞（Primordial Germ Cells,PGCs）的起源、迁移、分化等研究奠定了基础。     

黄姑鱼三倍体的诱导、鉴定及其性腺发育特征

研究采用冷休克方法诱导黄姑鱼(Nibea albiflora)三倍体并进行鉴定,进一步采用组织学和生殖相关基因表达分析比较了三倍体黄姑鱼的性腺发育特征。研究结果表明:(1)在受精后2.5min以3℃进行10min的冷休克处理,冷休克处理组的受精率和孵化率分别为(70.31±4.49)%和(21.5±6.63)%,其受精率和孵化率显著低于二倍体对照组;(2)经流式细胞仪倍性检测和染色体核型分析发现,三倍体的DNA含量为二倍体的1.5倍,染色体数目为72条,而二倍体的染色体数目为48条,三倍体的比例为100%;(3)三倍体的生殖腺指数显著低于二倍体,进一步通过组织切片观察发现三倍体的精巢和卵巢发育较二倍体滞后,在12月龄时,二倍体精巢和卵巢处于Ⅴ期,而三倍体精巢和卵巢分别处于Ⅲ期和Ⅰ期;(4)三倍体精巢的dmrt1和vasa基因及三倍体卵巢的cyp19a基因表达量均显著低于二倍体(P<0.05);三倍体卵巢的vasa基因表达量也比二倍体低(P>0.05)。综上结果表明:研究通过冷休克处理成功诱导了黄姑鱼三倍体;三倍体的性腺发育较二倍体滞后,育性明显降低。     

异源四倍体银鲫外周血和精巢的组织学特征

通过比较D 系三倍体银鲫 (Carassius auratus gibelio Bloch) 与异源四倍体银鲫, 我们发现异源四倍体的外周血与精巢组织跟三倍体银鲫存在明显差异。HE 染色结果表明, 异源四倍体银鲫外周血红细胞有明显的分裂倾向。利用流式细胞术对D 系三倍体银鲫与异源四倍体银鲫外周血的DNA 直方图进行比较, 结果表明异源四倍体外周血的DNA 直方图有两个主峰。此外, 我们观察到异源四倍体银鲫精巢的三种类型, 其中Ⅰ型精巢可以产生正常精子, Ⅱ型可观察到精小囊结构, 但不能产生精子, Ⅲ型精巢未发育出精小囊结构。进一步用银鲫Vasa 抗体对精巢切片进行组织免疫荧光共聚焦显微分析, 结果表明, Ⅰ型精巢的生殖细胞完成了减数分裂, 能观察到精原细胞、初级精母细胞、次级精母细胞, 以及大量位于精小管中间的精子细胞和精子; 而Ⅱ型精巢的生殖细胞不能完成第二次减数分裂, 精小囊中存在大量的初级和次级精母细胞, 没有精子细胞产生。研究丰富了对异源四倍体银鲫生物学性状的认识。       

尖唇散白蚁工蚁、兵蚁和繁殖蚁精子发生的比较研究

为探讨白蚁非生殖品级和生殖品级生殖细胞发育差异,采用组织学染色技术对尖唇散白蚁Reticulitermes aculabialis Tsaiet Hwang繁殖蚁、工蚁和兵蚁的精巢发育以及精子发生进行了显微观察和比较研究。结果发现3个品级间精巢发育的程度差异很大,三者精巢切面面积相对大小之比为:繁殖蚁∶工蚁=1.7∶1;繁殖蚁∶兵蚁=29.3∶1;工蚁∶兵蚁=17.1∶1。繁殖蚁和工蚁精巢管内有精子的形成,工蚁和繁殖蚁精子的发生都经历了精原细胞、初级精母细胞、次级精母细胞、精细胞和精子时期,但工蚁有大量次级精母细胞呈细胞凋亡状态。兵蚁生殖细胞发育仅有精原细胞、初级精母细胞、次级精母细胞,没有精细胞和精子产生。工蚁的生殖细胞显著小于同一时期繁殖蚁的生殖细胞,兵蚁的各时期生殖细胞均极显著小于繁殖蚁同一时期的生殖细胞。研究表明各品级之间生殖功能分化与生殖细胞发育有直接关系,工蚁有转化为补充繁殖蚁和兵蚁的能力;而兵蚁由于精巢极度退化不能产生精细胞和精子,因此是非生殖品级分化的终极形式,不具有转化成补充繁殖蚁或其它品级的能力。     

粗糙沼虾精巢发育的组织学

利用光镜技术,对粗糙沼虾精巢发育进行了研究,根据精子发生过程中每种生殖细胞所占的比例和发生的次序,并结合精巢的形态特征,把精巢发育过程分为五个时期,即精原细胞期,精母细胞期,精细胞期,成熟精子期及退化期,精原细胞期,精巢小,透明乳白色,生精小管内的生殖细胞以精原细胞为主;精母细胞期;精巢体积增大,半透明乳白色,主要由处于初级精母细胞的次级精母细胞阶段的生殖细胞组成;精细胞期,精巢体积继续增大,颜色加深,生精小管内的生殖细胞以精细胞为主;成熟精子期,精巢体积可达最大,紫红色,生精小管内充满着成熟的精子,退化期;精巢体积减小,半透明乳白色,生精小管内的成熟精子几乎排空。     

中华鳖性腺的发生与发育研究

为了揭示中华鳖性腺的发生与发育规律,在(32±0.5)℃温度下孵化鳖卵,72h时,从组织切片中可见原始生殖细胞移向中肠附近的内胚层;第5天生殖嵴形成;第13天性腺分化为皮质和髓质;第16天性腺开始分化为精巢或卵巢,至第22天时分化结束,形成精巢或卵巢.胚胎期、稚鳖和1龄鳖的性腺均呈短细管状,表面光滑,无色,胶状透明;2龄、3龄、4龄鳖的精巢呈长椭圆形.稚鳖的卵巢发育至卵原细胞期,组成精巢的曲细精管不明显,管内为精原细胞;1龄幼鳖的卵巢发育至初级卵泡期,精巢的曲细精管内精原细胞的数量增加;2龄和3龄鳖的卵巢处于生长卵泡期,3龄末的卵巢内可见到成熟卵泡,2龄鳖的曲细精管内出现初级精母细胞,3龄末的曲细精管开始出现精细胞和精子;4龄鳖的卵巢发育至成熟卵泡期,曲细精管内由管壁向管腔依次为精原细胞、初级精母细胞、次级精母细胞、精细胞和精子.结果表明,在(32±0.5)℃温度下,鳖胚发育至22d时,精巢或卵巢形成;在自然条件下,中华鳖至4龄时,性腺才完全发育成熟.     

中华鳖boule基因在生殖细胞中的表达分析

boule基因为DAZ基因家族成员之一,是动物生殖细胞特异表达基因。在哺乳动物中,boule基因的缺失会引起精子生成障碍而导致雄性不育。在无脊椎动物秀丽线虫(Caenorhabditis elegans)中,boule基因同源物的缺失会引起其卵子发生障碍而导致雌性不育。龟鳖动物是最古老的爬行类,是从无羊膜卵到羊膜卵动物飞跃的过渡物种。相比于哺乳类及一些无脊椎动物,目前关于龟鳖动物生殖细胞发育模式的研究还非常有限。因此,本文以中华鳖(Pelodiscus sinensis)为研究对象,以期揭示boule基因对龟鳖动物生殖细胞发育分化的调控作用。首先,利用特异引物克隆获得中华鳖boule基因的cDNA序列,共1 005 bp,其中,3′端非编码区57 bp,开放阅读框948 bp,共编码315个氨基酸。氨基酸序列多重比对分析显示,中华鳖与绿海龟(Chelonia mydas)同源性最高,达92%,与小鼠(Mus musculus)的同源性达83%,与果蝇(Drosophila melanogaster)的同源性达53%,与青鳉(Oryzias latipes)的同源性达42%。反转录实时定量PCR(RT-qPCR)分析结果显示,中华鳖boulem RNA主要在性腺组织精巢和卵巢中表达,而在其他体细胞组织中几乎检测不到表达。原位杂交结果显示,中华鳖boule m RNA在两性生殖细胞中特异表达,且在不同分化时期的生殖细胞中呈动态表达。在精巢中,boule m RNA在初级精母细胞中表达最强,在精原细胞和次级精母细胞中表达较弱,在精子细胞和精子中难以检测到表达信号;在卵巢中,boule mRNA在初级卵母细胞中表达信号最强且信号在初级卵母细胞胞质中均匀分布,生殖细胞发育进入卵母细胞生长期后,信号开始聚集在核周胞质,随着卵母细胞的成熟,信号逐渐变弱。本研究结果表明,boule基因可能在中华鳖两性生殖细胞的减数分裂过程中均具有重要的调控作用。     

黄体酮对成年斑马鱼下丘脑-垂体-性腺轴相关基因转录表达的干扰效应

黄体酮(P4)是一种类固醇激素。为了探究P4的内分泌干扰效应, 选择成年斑马鱼(Danio rerio)作为受试生物, 研究了P4对斑马鱼下丘脑-垂体-性腺轴(HPG轴)相关基因转录表达影响。成年斑马鱼在不同浓度P4(2、11和16 ng•L–1)下处理21 d。结果显示: 暴露于高浓度组的P4能够抑制雌鱼大脑中促性腺激素释放激素2(gnrh2)、促性腺激素释放激素3(gnrh3), 卵泡刺激素(fshb)、雌激素受体1(esr1)基因的转录表达; 然而诱导了雄鱼大脑中fshb、黄体生成素(lhb)、雄激素受体(ar)基因的转录表达, 这些转录变化暗示了P4对成年斑马鱼有潜在的弱雄激素效应。此外, P4暴露对雌鱼卵巢和雄鱼精巢类固醇合成途径中固醇激素合成急性调节蛋白(star)、细胞色素p450介导侧链裂解酶(cyp11a1)、17α羟化酶(cyp17)、卵巢细胞色素P450芳香化酶(cyp19a1a)、11β羟化酶(cyp11b)、羟基类固醇3β脱氢酶(hsd3b)、羟基类固醇20β脱氢酶(hsd20b)、羟基类固醇17β脱氢酶3(hsd17b3)、羟基类固醇11β脱氢酶2(hsd11b2)以及受体信号途径中孕激素受体(pgr)、esr1、ar基因的转录表达没有显著影响。可见, 在P4暴露下, 斑马鱼大脑比性腺更加敏感。总而言之, P4能够改变斑马鱼大脑中HPG轴相关基因的转录表达水平, 进而对斑马鱼的内分泌系统具有潜在的危险。     

银鲫dmrt2b基因的分子特征及功能分析

实验克隆了银鲫dmrt2b基因的全长cDNA序列,并对其表达图式和在胚胎发育过程中的功能做了初步研究。银鲫dmrt2b和斑马鱼dmrt2b有相似的基因组结构。在胚胎发育过程中,银鲫dmrt2b主要在体节中表达。在成体中主要分布于肌肉中。注射银鲫的dmrt2b可以挽救斑马鱼dmrt2b敲降胚的表型。上述结果表明银鲫dmrt2b基因同斑马鱼dmrt2b基因有相同的功能。       

Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus

Vas (a Drosophila vasa homologue) gene expression pattern in germ cells during oogenesis and spermatogenesis was examined using all genetic females and males of a teleost fish, tilapia. Primordial germ cells (PGC) reach the gonadal anlagen 3 days after hatching (7 days after fertilization), the time when the gonadal anlagen was first formed. Prior to meiosis, no differences in vas RNA are observed in male and female germ cells. In the ovary, vas is expressed strongly in oogonia to diplotene oocytes and becomes localized as patches in auxocytes and then strong signals are uniformly distributed in the cytoplasm of previtellogenic oocytes, followed by a decrease from vitellogenic to postvitellogenic oocytes. In the testis, vas signals are strong in spermatogonia and decrease in early primary spermatocytes. No vas RNA expression is evident in either diplotene primary spermatocytes, secondary spermatocytes, spermatids or spermatozoa. The observed differences in vas RNA expression suggest a differential function of vas in the regulation of meiotic progression of female and male germ cells.     

雌激素受体在白蚁精子发生过程中的免疫组织化学定位(简报)

长期以来,雌激素（Estrogen）被认为是雌性特有的维持雌性性征的激素。近年来人们发现雄性体内亦存在雌激素及其受体,雌激素可作用于脊椎动物下丘脑-垂体-精巢调控轴以及精巢中体细胞和生精细胞,包括Sertoli细胞、Leydig细胞、精细胞,几乎涉及所有雄激素涉及的领域。雌激素通过与其受体结合而起作用,雌激素受体（Estrogen receptor,ER）在雄性生殖中起重要作用。     

Stage-specific gene expression during fish spermatogenesis as determined by laser-capture microdissection and quantitative-PCR in sea bass (Dicentrarchus labrax) gonads

The role of genes implicated in the regulation of spermatogenesis and their patterns of expression is still poorly understood. In this study, we took advantage of the cystic arrangement of the teleost testis to set up a laser capture microdissection procedure to isolate cells from cysts containing spermatogonia, spermatocytes, spermatids, or spermatozoa. We then used quantitative PCR to determine the stage-specific expression patterns of the germ cell marker vasa; gonadal aromatase (cyp19a); estrogen receptors (ers) alpha, beta1, and beta2 (era, erb1, and erb2, respectively); 11beta-hydroxylase (cyp11b1); androgen receptor beta (arb); insulinlike growth factor 1 (igf1); and sox17. vasa had the highest mRNA levels, followed by genes involved in androgen metabolism (cyp11b1 and arb). Most genes associated with estrogen metabolism (cyp19a, era, and erb1) had a lower expression, whereas igf1 and sox17 exhibited the lowest mRNA levels. Comparison of changes in mRNA levels revealed five patterns of gene expression, in general with progressively lower expression seen as spermatogenesis advanced. igf1 and sox17 were exclusively expressed in spermatogonia-containing cysts, suggesting effects during the proliferative stage. Genes involved in androgen synthesis (cyp11b1) and action (arb) peaked during the early stages of spermatogenesis and then sharply decreased. In contrast, genes associated with estrogen action, particularly erb2 and era, showed a more gradual decrease. Together, these results demonstrate the usefulness of fish models and suggest that whereas androgens are required at high levels and may exert their major actions at the initial stages of spermatogenesis, estrogens are also essential, albeit required at lower levels, and with a more generalized influence.     

Differential Expression of Cyclins B1 and B2 during Medaka (Oryzias latipes) Spermatogenesis

The Cdc2-cyclin B complex (named the M-phase-promoting factor, MPF) is well known to be a key regulator of G2-M transition in both mitosis and meiosis. However, MPF may have functions other than the cell cycle regulation, since its activity is detectable in post-mitotic (or post-meiotic) non-dividing cells. Cyclin B comprises several subtypes, but their functional differences are still unknown. Despite the established function of MPF during oocyte maturation, its role during spermatogenesis, where spermatogenic cells undergo drastic morphological changes after meiosis, remains to be elucidated. To address these issues, we have isolated cDNA clones encoding cyclins B1 and B2 from medaka testis and raised polyclonal antibodies against their products. Using these as probes, we examined the expression patterns of cyclins B1 and B2 in medaka testis at both mRNA and protein levels. Cyclin B1 and B2 mRNAs were expressed in all stages of spermatogenic cells except for spermatozoa, although the expression levels varied according to the spermatogenic stages. Cyclin B1 protein was expressed only in spermatogonia and spermatocytes at prophase and metaphase with a transient disappearance at anaphase. On the other hand, cyclin B2 protein was continuously expressed throughout spermatogenesis, even in spermatogonia and spermatocytes at anaphase and in post-meiotic spermatids and spermatozoa. The difference in their expression patterns suggests that cyclins B1 and B2 have distinct roles in medaka spermatogenesis; i.e., cyclin B1 controls the meiotic cell cycle, whereas cyclin B2 is involved in process(es) other than meiosis.     

Lymphoid-specific helicase (HELLS) is essential for meiotic progression in mouse spermatocytes

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells(-/-) to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells(-/-), Hells(+/-), and Hells(+/+) mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells(-/-) than from Hells(+/+) mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells(-/-) mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells(-/-) spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.     

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice

Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice overexpressing EF1alpha promoter-driven UCH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.     

Testicular dmrt1 is involved in the sexual fate of the ovotestis in the protandrous black porgy

In hermaphroditic fish, the ovotestis can respond to external stimuli so that only one type of gonadal tissue (either ovarian or testicular tissue) will remain reproductively active and the other will recede to a rudimentary stage. However, the molecular mechanism for sexual fate determination is still poorly understood in hermaphroditic fish. In the present study, we examined whether sexual fate determination with respect to testis development is due to differential expression of dmrt1. Expression of dmrt1 was limited to the spermatogonia-surrounding cells (Sertoli cells) throughout testis development. Testicular dmrt1 was differentially expressed in fish (black porgy [Acanthopagrus schlegeli Bleeker]) depending on if fish were destined to be female or male. Expression of dmrt1 in Sertoli cells did not require germ cell factors with busulfan treatment. To examine the role of dmrt1, we used virus-based RNA interference. Deficiency of dmrt1 resulted in a reduced number of germ cells in the testis and stimulated a male-to-female sex change. Higher serum luteinizing hormone levels were detected in 2(+)- to 3-yr-old male fish as compared to sex-changing female fish. Furthermore, we showed that fish treated in vivo with gonadotropin-releasing hormone (Gnrh) and fish treated in vitro with gonadotropin (Gth) had higher dmrt1 expression in the testis, suggesting that these endocrine factors may affect the male-to-female sex change. Therefore, our data suggest that dmrt1 plays a key role in initial testis differentiation and in later maintenance of male development. We show, to our knowledge for the first time, the functions of dmrt1 in hermaphroditic fish, which indicate that male-phase maintenance may be regulated by the brain-pituitary-gonadal axis via the Gnrh-Gth-Dmrt1 axis.