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。     

己烯雌酚抑制斑马鱼精子发生及其可能的分子机制

为研究内分泌干扰物己烯雌酚(DES)对鱼类精巢发育和配子发生的影响, 研究用DES(0.1、1和10 g/L, 暴露20d)对内分泌干扰研究的经典模式动物斑马鱼(Danio rerio)雄性成鱼进行了处理。组织学研究结果表明, DES严重影响斑马鱼精子发生。同时, 研究克隆了斑马鱼与生殖细胞发育和减数分裂相关的vasa、dmc1的部分cDNA, 对其组织和细胞表达模式进行了研究。结果表明, vasa仅表达于精巢的精原细胞、初级精母细胞和卵巢不同时期的生殖细胞; 而dmc1则表达于精巢精母细胞和卵巢卵母细胞发育早期。半定量PCR结果表明, DES处理后vasa的表达没有明显变化; 而dmc1的表达则被明显抑制, 且呈时间依赖性和剂量依赖性效应; 而转录因子dmrt1和雄激素合成关键酶基因P450 11的表达也被显著抑制。因此本研究推测, DES可能通过抑制dmrt1和P450 11的表达诱导了斑马鱼生殖细胞凋亡; 并通过抑制dmc1的表达阻碍了减数分裂。       

金鱼配子发生中vasa基因的表达和分布特征

用原位杂交技术,以地高辛标记的反义RNA为探针,检测了金鱼(Carassiusauratus)DEAD box家族基因vasa在卵子及精子发生中的分布及表达。结果表明在金鱼卵子发生中,在各个时期的卵母细胞的胞质中均有金鱼vasaRNA的杂交信号表达。在Ⅰ、Ⅱ期卵母细胞中vasaRNA的杂交信号强烈,均匀地分布在整个胞质。随着卵母细胞的生长发育及卵黄的积累,Ⅲ、Ⅳ期卵母细胞胞质中vasaRNA的杂交信号急剧减弱,而外周皮层区域,其阳性信号仍较强。在金鱼精子发生中,在精原细胞和初级精母细胞中可检测到金鱼vasaRNA杂交信号,后者的阳性信号比前者微弱;而精子细胞中没有阳性信号。推测vasa基因在金鱼精子发生早期发挥着重要作用;而在卵子发生中主要作为遗传信息储备物质,用于调控早期胚胎发育过程中原始生殖细胞的形成与分化。     

猪原始生殖细胞生物学特性的研究

胚胎生殖细胞（embryonic germ cell,EGC）是由胎儿原始生殖细胞（primordial germ cell,PGC）经体外驯化培养获得的一种多潜能干细胞。研究猪PGC生物学特性对于建立猪EGC及了解猪生殖细胞发育机制具有重要意义。该研究以原代培养的猪PGC为对象,探讨了其生长行为特征及其重编程过程中多能性、生殖系标志基因的表达模式。结果显示,26 d胚胎生殖嵴分离的PGC呈碱性磷酸酶阳性,细胞体积及核质比较大;体外培养初期呈现出较强的增殖及迁移能力,培养第5 d细胞增殖达到平台期,此时克隆高表达Oct4、Sox2、Nanog、c-Myc、Klf4和Ifi tm3（P〈0.05）,低表达Blimp1（P〈0.05）,Nanos1和Stella的表达水平与猪胎儿成纤维细胞无差异;猪PGC形成的原代克隆已经具有多向分化潜能。     

尼罗罗非鱼性腺发育的研究

湖南地区生长于池塘环境的尼罗罗非鱼(Tilapia nilotica),性成熟日龄是110—130天,雄性比雌性普遍早熟20天。精细胞的发生能够完成由精原细胞到精子的全部发育过程,同样,卵细胞的发生也能完成由卵原细胞到卵子的全部发育过程。初级卵母细胞处于Ⅲ时相阶段时,可由包被卵周的滤泡细胞分泌产生放射膜,但放射膜不在动物性极形成受精孔,也无精孔细胞的分化,证实尼罗罗非鱼是属于非受精孔受精类型。初级卵母细胞由Ⅲ时相发育到Ⅳ时相是非同步性的,产后卵巢的组织学结构为第Ⅳ期,卵巢系数在繁殖季节可出现三次高峰,证实尼罗罗非鱼是属于多次产卵类型。通过对精巢组织学的研究,发现尼罗罗非鱼的第Ⅰ期精巢是自然两性嵌合体。       

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。     

青鱼性腺发育的研究

湖南地区生长于池塘环境的青鱼,性成熟年龄是5—6年,雄性比雌性普遍地早熟一年。卵母细胞和滤泡细胞是同源的,都来自于卵原细胞。池养青鱼的卵母细胞只能发育到初级卵母细胞阶段(Ⅳ时相),必须通过人工催情,才能进行染色体的减数分裂,使卵母细胞由第Ⅳ时相发育到第Ⅴ时相。精细胞的发生,能够完成由精原细胞到精子的全部发育过程。青鱼在第一次性周期内,雄性精巢在第5个冬季进入第Ⅳ期,雌性卵巢在第6个冬季进入第Ⅲ期,从此以后,每年冬季,雄性精巢回复到第Ⅳ期,雌性卵巢回复到第Ⅲ期,这种性腺季节周期变化的规律,为生产上选留亲鱼提供了理论依据。青鱼雌性卵母细胞由第Ⅲ时相到第Ⅳ时相是同步性的;经人工催情产卵或自然退化后,卵巢的组织学结构又回复到第Ⅱ期,证明青鱼是一次产卵类型。已经达到性成熟年龄的雌性青鱼,卵母细胞的卵黄形成有两种不同的类型。第一种类型是泡内卵黄,第二种类型是泡外卵黄。如果饲养管理工作如投饵、水质调节不适宜,卵母细胞不能正常形成卵黄,就会出现卵子的败育现象,这是生产上一个重要问题,必须进一步研究。       

青鱼性腺发育的研究

湖南地区生长于池塘环境的青鱼,性成熟年龄是5—6年,雄性比雌性普遍地早熟一年。卵母细胞和滤泡细胞是同源的,都来自于卵原细胞。池养青鱼的卵母细胞只能发育到初级卵母细胞阶段(Ⅳ时相),必须通过人工催情,才能进行染色体的减数分裂,使卵母细胞由第Ⅳ时相发育到第Ⅴ时相。精细胞的发生,能够完成由精原细胞到精子的全部发育过程。青鱼在第一次性周期内,雄性精巢在第5个冬季进入第Ⅳ期,雌性卵巢在第6个冬季进入第Ⅲ期,从此以后,每年冬季,雄性精巢回复到第Ⅳ期,雌性卵巢回复到第Ⅲ期,这种性腺季节周期变化的规律,为生产上选留亲鱼提供了理论依据。青鱼雌性卵母细胞由第Ⅲ时相到第Ⅳ时相是同步性的;经人工催情产卵或自然退化后,卵巢的组织学结构又回复到第Ⅱ期,证明青鱼是一次产卵类型。已经达到性成熟年龄的雌性青鱼,卵母细胞的卵黄形成有两种不同的类型。第一种类型是泡内卵黄,第二种类型是泡外卵黄。如果饲养管理工作如投饵、水质调节不适宜,卵母细胞不能正常形成卵黄,就会出现卵子的败育现象,这是生产上一个重要问题,必须进一步研究。     

ERK/JNK在黄鳝雌、雄发育阶段生殖腺中的表达和定位

为探讨MAPK家族中ERK和JNK两个主要亚族在黄鳝雌、雄发育阶段生殖腺中的表达状况,应用蛋白质免疫印迹杂交技术和免疫组织化学法检测了ERK、JNK在黄鳝卵巢组织和精巢组织中的表达和定位。蛋白免疫印迹杂交显示:ERK在黄鳝雌、雄性腺组织中均有强的表达;JNK在性腺中的表达总体上弱于ERK,JNK1在精巢组织中的表达比卵巢组织显著降低,但JNK2在雌、雄性腺组织中的表达无明显差异。在免疫组织化学的观察中,ERK和JNK在卵原细胞和精原细胞中均为阳性反应,且定位相似:细胞质及细胞核核质呈阳性反应,核仁阴性。随着卵母细胞生长和成熟,ERK和JNK在卵母细胞胞质中阳性反应逐渐减弱。实验结果提示,ERK和JNK在黄鳝卵巢发育、凋亡退化以及雄性发育的启动过程中可能具有重要调控作用     

黄尾密鲴性腺发育的研究

生长在湖南地区水库环境的黄尾密鲴,雌、雄鱼性成熟一般为2冬龄。在具备繁殖条件的水库,其黄尾密鲴能自行产卵、受精、繁衍后代,不具备条件的,不能自然产卵,其卵母细胞只能发育到初级卵母细胞阶段(Ⅳ时相),而精细胞则能完成由精原细胞发育到精子的全部发育过程。冬季,达性成熟的黄尾密鲴,雌性卵巢为Ⅲ期,雄性精巢为Ⅳ期。每年的4月份,黄尾密鲴开始达人性腺成熟期,5月中旬至6月初为它的生殖盛期。初级卵母细胞由Ⅲ时相发育到Ⅳ时相基本是同步性的,卵巢成熟系数在一年中只出现1次高峰,经组织学研究与生产实践证明,黄尾密鲴系一年1次产卵类型。     

Identification and migration of primordial germ cells in Atlantic salmon,Salmo salar: Characterization of Vasa,Dead End,and Lymphocyte antigen 75 genes

No information exists on the identification of primordial germ cells (PGCs) in the super‐order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full‐length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi‐quantitative RT‐PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole‐mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid‐blastula stage, vasa‐expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp‐rt‐vasa 3′‐UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.     

Directed neural differentiation of duck embryonic germ cells

Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.     

Primordial germ cell-somatic cell partnership: a balancing cell signaling act

Recent studies demonstrate that the normal progression of the germ cell lineage during gonadogenesis involves a delicate balance of primordial germ cell survival and death factors generated by surrounding somatic cells. This balance operates in a different fashion in females and males. The fine tuning primordial germ cell specification in the wall of the yolk sac, migration through the hindgut and dorsal mesentery, and colonization in the urogenital ridges involves the temporal and spatial activation of the following signaling pathways: Primordial germ cell specification involves bone morphogenetic proteins 2, 4 and 8b, and their migration is facilitated by the c-kit receptor-ligand duet. When colonization occurs: (1) neuregulin-beta ligand is expressed and binds to an ErbB2-ErbB3 receptor tyrosine kinase heterodimer on primordial germ cells; (2) Vasa, an ortholog of the Drosophila gene vasa, member of an ATP-dependent RNA helicase of the DEAD (Asp-Glu-Ala-Asp)-box family protein is also expressed by primordial germ cells; (3) Bcl-x (cell survival factor) and Bax (cell death factor) join forces to modulate the first burst of primordial germ cell apoptosis; (4) Cadherins, integrins, and disintegrins bring together primordial germ cells and somatic cells to organize testis and ovary. Information on other inducers of primordial cell survival, such as TER (teratoma) factor, is beginning to emerge.     

Germ cell nuclear antigen (GCNA1) expression does not require a gonadal environment or steroidogenic factor 1: Examination of GCNA1 in ectopic germ cells and in Ftz-f1 null mice

The germ cell lineage is first recognized as a population of mitotically proliferating primordial germ cells that migrate toward the gonadal ridge. Shortly after arriving at the gonadal ridge, the germ cells begin to initiate a commitment to gamete production in the developing gonad. The mechanisms controlling this transition are poorly understood. We recently reported that a mouse germ cell nuclear antigen 1 (GCNA1) is initially detected in both male and female germ cells as they reach the gonad at 11.5 days postcoitum (dpc). GCNA1 is continually expressed in germ cells through all stages of gametogenesis until the diplotene/dictyate stage of meiosis I. Since GCNA1 expression commences soon after primordial germ cells arrive at the gonadal ridge, we wanted to determine whether the gonadal environment was essential for induction of GCNA1 expression. By examining GCNA1 expression in germ cells that migrate ectopically into the adrenal gland, we determined that both the gonadal and adrenal gland environments allow GCNA1 expression. We also examined GCNA1 expression in Ftz-F1 null mice, which are born lacking gonads and adrenal glands. During embryonic development in the Ftz-F1 null mice, the gonad and most germ cells undergo apoptotic degeneration at about 12.5 dpc. While most of the germ cells undergo apoptosis without expressing GCNA1, a few surviving germs cells, especially outside the involuting gonad clearly express GCNA1. Thus, although the Ftz-F1 gene is essential for gonadal and adrenal development, induction of GCNA1 expression in germ cells does not require Ftz-F1 gene products. The finding that germ cell GCNA1 expression is not restricted to the gonadal environment and is not dependent on the Ftz-F1 gene products suggests that GCNA1 expression may be initiated in the germ cell lineage by autonomous means. Mol. Reprod. Dev. 48:154–158, 1997. © 1997 Wiley-Liss, Inc.     

Drosophila pericentrin‐like protein promotes the formation of primordial germ cells

Primordial germ cells (PGCs) are the precursors to the adult germline stem cells that are set aside early during embryogenesis and specified through the inheritance of the germ plasm, which contains the mRNAs and proteins that function as the germline fate determinants. In Drosophila melanogaster, formation of the PGCs requires the microtubule and actin cytoskeletal networks to actively segregate the germ plasm from the soma and physically construct the pole buds (PBs) that protrude from the posterior cortex. Of emerging importance is the central role of centrosomes in the coordination of microtubule dynamics and actin organization to promote PGC development. We previously identified a requirement for the centrosome protein Centrosomin (Cnn) in PGC formation. Cnn interacts directly with Pericentrin‐like protein (PLP) to form a centrosome scaffold structure required for pericentriolar material recruitment and organization. In this study, we identify a role for PLP at several discrete steps during PGC development. We find PLP functions in segregating the germ plasm from the soma by regulating microtubule organization and centrosome separation. These activities further contribute to promoting PB protrusion and facilitating the distribution of germ plasm in proliferating PGCs.     

小鼠原生殖细胞建系过程及其分化特性的研究

以小鼠8.5dpc、10.5dpc、12.5dpc胚胎为材料,分离其中包含PGC的胚胎组织,使其生长于饲养层细胞上,在生长因子LIF、SCF和bFGF的共同作用下存活增殖,形成PGC克隆,经过几次分散转移至新的饲养层细胞,产生稳定增殖的EG干细胞克隆,共建成5株EG细胞系,AKP染色以及oct-4基因表达产物的免疫荧光检测均显示阳性。EG1、EG2、EG3、EG4、EG5,分别来自8.5、10.5dpc的胚胎,没有得到长期培养的12.5dpc的EG细胞系。EG细胞系在有饲养层细胞或添加LIF的环境中可稳定传代,保持不分化状态,至少15代内正常核型细胞所占比例80%以上。去除抑制分化因素的前提下,悬浮培养的EG细胞形成胚体,分化出类似胚胎内胚层和外胚层的细胞结构;贴壁生长的胚体能产生不同类型的分化细胞,包括上皮细胞、成纤维细胞、神经细胞等。EG细胞在裸鼠体内形成畸胎瘤。以上结果证实我们建立的EG细胞系具发育多能性,为研究早期胚胎和生殖细胞生长分化提供了模型。     

Specification of human germ cell fate with enhanced progression capability supported by hindgut organoids

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大鼠睾丸曲细精管组织支持细胞/生殖细胞长期共培养与精子发生分析

睾丸体外生殖模型的发展为体外研究睾丸的精子发生分子机制和睾丸毒理学提供了实验工具。很多报道的模型都无法真正地模拟体内复杂的生化分子及功能性相互作用从而导致研究价值有限。该实验拟建立一个体外长期维持睾丸生殖细胞存在,并能持续产生精子细胞的支持细胞/生殖细胞共培养体系。体系中的支持细胞和生殖细胞均由曲细精管组织块迁移到培养皿上,在不添加任何生长因子的情况下维持体外精子发生至圆形精子细胞超过2个月。RT-PCR分析显示,共培养细胞稳定表达cdh1、scp3、tnp2;免疫荧光染色结果显示,CDH1、PLZF、SCP3以及SOX9阳性细胞存在。这些结果例证了体系中同时存在精原干细胞、精母细胞、精子细胞和支持细胞。简单高效的支持细胞/生殖细胞体外共培养体系可用于雄性生殖的分子机制和毒理学研究。