人原始生殖细胞的特化和表观遗传重编程研究进展

原始生殖细胞(primordial germ cells,PGCs)起源于原肠胚阶段,是生殖细胞的前体细胞,由特定细胞经过一系列分子调控特化而成。PGCs完成特化后迁移进入生殖嵴,在迁移过程中存在一系列的表观遗传修饰的动态变化,包括DNA甲基化和组蛋白修饰等。PGCs迁移的后期会发生两性分化,迁入生殖嵴的PGCs影响原始性腺的发育。有关小鼠PGCs特化、迁移/增殖和两性分化等的机制已得到了广泛研究,而在人类中则由于伦理以及材料获取困难等因素还有待更深入的研究。该文综述了人原始生殖细胞(human PGCs,h PGCs)的特化机制、表观遗传调节在其特化和迁移过程中的作用以及h PGCs对性腺形成的影响。     

中国林蛙性腺的发育及温度对其性别分化的影响

为探讨幼蛙性别分化与温度的关系,在恒温和变温条件下培养中国林蛙（Rana chensinensis)受精卵至变态完成,结果表明：（1）胚胎发育到24期时生殖嵴开始出现,25期个别原始生殖细胞（PGCs)已迁移到生殖嵴中,生殖细胞与生殖嵴共同发育成生殖腺;（2）胚胎发育到31期生殖腺出现性别分化,卵巢分化初期较易识别,而精巢分化不明显;…（3）卵巢分化完成于37期,精巢分化完成于变态之后,两侧生殖腺等大;（4）胚胎发育从30期开始,性别分化对温度较为敏感,低温利于雌性化,高温利于雄性化;（5）15－25℃为变温培养时性比发生变化的敏感温度区,缓慢升温雄性比较显著增加,缓慢降温雌性比例显著增加。     

培养原始生殖细胞的新方法

为探讨原始生殖细胞(primordial germ cells,PGCs)在体外长期增殖、生长并长期保持分化潜能的新方法,我们将PGCs分别与睾丸支持细胞(Sertoli cells,SCs)和同源生殖嵴成纤维细胞共培养。结果与SCs共培养的PGCs集落明显多于同源生殖嵴成纤维细胞共培养PGCs集落,传代次数也显著多于同源生殖嵴成纤维细胞．目前与SCs共培养的PGCs已成功传代培养至了第51代。因此我们认为PGCs与SCs共培养,可有效提高原始生殖细胞在体外的增殖能力并可长期维持干细胞的特性。     

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]。     

哺乳动物原始生殖细胞体内特化和体外培养

原始生殖细胞(primordial germ cells, PGCs)是胚胎中最先出现的生殖细胞。PGCs来源于上胚层,最早出现在后肠,随后向生殖嵴迁移。这一过程伴随一系列复杂的分子调控机制,以及DNA甲基化重编程和组蛋白修饰等表观遗传过程。PGCs经过不断的分裂、发育及分化,最终形成配子。为了更好地研究PGCs发育与分化的调控和表观遗传过程,体外培养的研究变得越来越重要。本文以小鼠和人为例,介绍了哺乳动物PGCs的特化过程、PGCs特化过程中的表观遗传过程和PGCs的体外培养研究进展。     

棘胸蛙的性腺分化及温度对其性别决定的影响

单性养殖在棘胸蛙(Quasipaa spinosa)养殖中意义显著。为了了解棘胸蛙性腺分化,并探讨在不同的培育温度条件下性腺分化的差异。通过组织切片观察了棘胸蛙原始性腺的形成和性腺分化。棘胸蛙的性腺分化有其特殊性:生殖嵴形成时,其中既有体细胞,又有原始生殖细胞(PGCs);无论原始性腺是分化成为精巢还是卵巢,其中都出现一个带有单层扁平上皮初生性腔,当单层扁平上皮逐渐消失后形成次生性腔。性腔周围的PGCs开始长大2—3倍时,性腺将分化成为卵巢;体细胞渗入性腔中,使腔隙变小直至消失,这种原始性腺分化成为精巢。棘胸蛙蝌蚪孵化后的l7—80 d(Gosner 25—26期)为性腺分化的敏感时期。实验选取同一批次刚孵出蝌蚪(Gosner 24期),分别用不同水温(16±1)℃、(27±1)℃、(31±1)℃3组实验组及自然水温(23±1)℃对照组条件下的培育蝌蚪。结果显示,对照组的雌、雄性比为26∶24,雄性率接近50%;(16±1)℃实验组的雌、雄比例为33∶17,雄性率仅34%(P0.05);从(27±1)℃实验组起,雄性率提高,(31±1)℃实验组的雄性率达70%(P0.05)。棘胸蛙的性别分化属于温度依赖型性决定(TSD)。较高的培育温度可使棘胸蛙蝌蚪性别分化趋向雄性,而较低的培育温度则使蝌蚪雌性化。     

胡子鲇Dmrt1基因全长cDNA克隆及其表达分析

以胡子鲇(Clarias fuscus)为研究对象,利用RT-PCR技术和SMART RACE技术克隆获得Dmrt1基因cDNA全长,并利用生物信息学分析其结构及功能;利用半定量RT-PCR技术检测胡子鲇性腺(精巢/卵巢)、肌肉、肠、肝脏、心脏、头肾、鳃丝、脑和眼等10种组织以及Ⅱ—Ⅴ期精巢中Dmrt1基因表达。结果表明:胡子鲇Dmrt1基因cDNA全长为1417 bp,其中5′非编码区(5′-UTR)为35 bp,3′非编码区(3′-UTR)为516 bp,开放阅读框(ORF)包含864 bp,编码287个氨基酸(aa),预测所编码DMRT1为主要位于细胞核内的不稳定性亲水蛋白。氨基酸序列比对显示,胡子鲇DMRT1与已公布的非洲胡子鲇、蟾胡子鲇、黄颡鱼等鲇形目鱼类的相似性为83.3%—96.1%。胡子鲇DMRT1中具有DMRT基因家族共有的、保守性很高的DM结构域,此结构域具有典型的"C2H2C4"锌指结构,与上述鲇形目鱼类的相似性达100%,与斑马鱼、青鳉、虹鳟等鱼类的相似性为91.9%—97.3%,而与鸡、鼠、猪人等的相似性达80%以上。组织表达显示,胡子鲇Dmrt1基因仅在精巢中表达,且Ⅱ期精巢(即精子发生期)中Dmrt1基因表达量显著高于Ⅲ、Ⅳ和Ⅴ期精巢(P<0.05),而卵巢及其他8种组织中均无表达,表明Dmrt1是胡子鲇精巢特异性表达基因,可能与胡子鲇的雄性性别决定、精子发生及精巢发育密切相关。     

培养原始生殖细胞的新方法

为探讨原始生殖细胞（primordial germ cells,PGCs）在体外长期增殖、生长并长期保持分化潜能的新方法,我们将PGCs分别与睾丸支持细胞（Sertoli cells,SCs）和同源生殖嵴成纤维细胞共培养。结果与SCs共培养的PGCs集落明显多于同源生殖嵴成纤维细胞共培养PGCs集落,传代次数也显著多于同源生殖嵴成纤维细胞,目前与SCs共培养的PGCs已成功传代培养至了第51代。因此我们认为PGCs与SCs共培养．可有效提高原始生殖细胞在体外的增殖能力并可长期维持干细胞的特性。     

泽蛙的性腺分化及温度对性别决定的影响

通过组织学方法观察了泽蛙(Rana limnocharis)原始生殖细胞(PGCs)的迁移、原始性腺的形成和性腺分化,并且探讨在不同的培育温度条件下性腺分化的差异。泽蛙的性腺分化有其特殊性:生殖嵴形成时,其中既有体细胞,又有原生殖细胞;无论原始性腺是分化成为精巢还是卵巢,其中都出现一个初生性腔。蝌蚪孵化后的17-34d(Gosner 26-38期)为性腺分化的敏感时期。在蝌蚪孵化后的第2d(Gosner 25期),分别用不同水温18℃±1℃、30℃±1℃、32℃±1℃、34℃±1℃培育蝌蚪,直至完成变态幼蛙(Gosner 46期)形成。自然水温23℃-25℃为对照。对照组的雌、雄性比接近1∶1(1∶1.06);18℃±1℃实验组的雌、雄比例为1.83∶1,雄性率仅35.1%(P<0.01);从30℃±1℃实验组起,雄性率提高,34℃±1℃实验组的雄性率达74.0%(P<0.01)。较高的培育温度可使泽蛙蝌蚪性别分化趋向雄性,而较低的培育温度则使蝌蚪雌性化。泽蛙的性别分化属于温度依赖型性决定(TSD)。当前全球性气候变暖对两栖类性比的稳定存在着威胁。     

温度对江黄颡鱼性分化的影响

通过组织学方法观察江黄颡鱼原始生殖细胞(PGCs)迁移、生殖嵴生成和性腺分化,并且探讨在不同温度培育下性腺分化的差异。实验结果显示:1日龄仔鱼PGCs位于鱼体中肠背方的脏壁中胚层中;5日龄时,PGCs迁移到背方的腹膜上皮;8日龄时,生殖嵴形成;14日龄时,原始性腺形成;23日龄时,性腺开始分化。从孵化后的第10天开始,分别用(20±0.5)、(24±1.0自然水温、对照组)、(30±0.5)和(34±0.5)℃4种水温培育仔鱼达25天。实验结束后统计结果显示:对照组和(20±0.5)℃组的雌、雄性比接近1∶1(分别为1∶1.09和1.22∶1);(30±0.5)℃组的为1∶4.89,雄性率达(83.3±0.7)%;(34±0.5)℃组的为2.85∶1,雄性率仅为(26.4±0.4)%。提示(30±0.5)℃可使幼鱼性腺发育趋向雄性,(34±0.5)℃则使幼鱼性腺发育趋向雌性。实验结果表明,江黄颡鱼的性分化是属于温度依赖型性别决定。     

Repression of primordial germ cell differentiation parallels germ line stem cell maintenance

In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Here we show that many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. We show that following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a "PGC niche".     

Primordial germ cells originate from the endodermal strand cells in the ascidian Ciona intestinalis

The origin of germ cells in the ascidian is still unknown. Previously, we cloned a vasa homologue (CiVH) of Ciona intestinalis from the cDNA library of ovarian tissue by polymerase chain reaction and showed that its expression was specific to germ cells in adult and juvenile gonads. In the present study, we prepared a monoclonal antibody against CiVH protein and traced the staining for this antibody from the middle tailbud stage to young adulthood. Results showed that positive cells are present in the endodermal strand in middle tailbud embryos and larvae. When the larval tail was absorbed into the trunk during metamorphosis, the CiVH-positive cells migrated from the debris of the tail into the developing gonad rudiment, and appeared to give rise to a primordial germ cell (PGC) in the young juvenile. The testis rudiment separated from the gonad rudiment, the remainder of which differentiated into the ovary. PGCs of the testis rudiment and the ovary rudiment differentiated into spermatogenic and oogenic cells, respectively. When the larval tail containing the antibody-positive cells was removed, the juveniles did not contain any CiVH-positive cells after metamorphosis, indicating that the PGCs in the juvenile originated from part of the larval tail. However, even in such juveniles, positive cells newly appeared in the gonad rudiment at a later stage. This observation suggests that a compensatory mechanism regulates germline formation in C. intestinalis.     

dead end,a novel vertebrate germ plasm component,is required for zebrafish primordial germ cell migration and survival

In most animals, primordial germ cell (PGC) specification and development depend on maternally provided cytoplasmic determinants that constitute the so-called germ plasm. Little is known about the role of germ plasm in vertebrate germ cell development, and its molecular mode of action remains elusive. While PGC specification in mammals occurs via different mechanisms, several germ plasm components required for early PGC development in lower organisms are expressed in mammalian germ cells after their migration to the gonad and are involved in gametogenesis. Here we show that the RNA of dead end, encoding a novel putative RNA binding protein, is a component of the germ plasm in zebrafish and is specifically expressed in PGCs throughout embryogenesis; Dead End protein is localized to perinuclear germ granules within PGCs. Knockdown of dead end blocks confinement of PGCs to the deep blastoderm shortly after their specification and results in failure of PGCs to exhibit motile behavior and to actively migrate thereafter. PGCs subsequently die, while somatic development is not effected. We have identified dead end orthologs in other vertebrates including Xenopus, mouse, and chick, where they are expressed in germ plasm and germ-line cells, suggesting a role in germ-line development in these organisms as well.     

Expression of axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching the gonad

How germ cell specification occurs remains a fundamental question in embryogenesis. The embryos of several model organisms contain germ cell determinants (germ plasm) that segregate to germ cell precursors. In other animals, including mice, germ cells form in response to regulative mechanisms during development. To investigate germ cell determination in urodeles, where germ plasm has never been conclusively identified, we cloned a DAZ-like sequence from axolotls, Axdazl. Axdazl is homologous to Xdazl, a component of Xenopus germ plasm found in the vegetal pole of oocytes and eggs. Axdazl RNA is not localized in axolotl oocytes, and, furthermore, these oocytes do not contain the mitochondrial cloud that localizes Xdazl and other germ plasm components in Xenopus. Maternal Axdazl RNA is inherited in the animal cap and equatorial region of early embryos. At gastrula, neurula, and tailbud stages, Axdazl RNA is widely distributed. Axdazl first shows cell-specific expression in primordial germ cells (PGCs) approaching the gonad at stage 40, when nuage (germ plasm) appears in PGCs. These results suggest that, in axolotls, germ plasm components are insufficient to specify germ cells.     

Sexual differentiation in Salaria (=Blennius) pavo

The sexual differentiation of Salaria (= Blennius ) pavo is described from the stage of hatching to a body length of 35 mm. At hatching, the primordial germ cells (PGCs) can be recognized clearly. At a standard body length of 5 mm, they begin to protrude into the peritoneal cavity and at 14 mm they transform to oogonia. At 17 mm length, the first oocytes can be observed. In males at a standard length of 16–17 mm, the first signs of a differentiation into a testis can be recognized. Shortly after the differentiation of the male sex, the division of the male gonad into a testis and a testicular gland can be seen. The fine structural characteristics of the PGCs and of differentiation stages are presented.     

Germ cells are not the primary factor for sexual fate determination in goldfish

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.     

Determination of the sensitive stages for gonadal sex-reversal in Xenopus laevis tadpoles

The response of developing gonads of the clawed toad Xenopus laevis tadpoles to estradiol benzoate (EB) was studied between stages 44 and 67 using high resolution techniques. In presumptive genetic males the following results were obtained: 1) 100% sex reversal was induced when EB was administered before translocation of primordial germ cells (PGCs) from the gonadal epithelium into the medullary region (stages 44-50). 2) Ambiguous gonads were formed when EB treatment was initiated at stages 51-54, when PGCs were migrating into the medullary region. 3) Finally, normal testes differentiated when EB treatment began after the primordial germ cells had completed their translocation into the medulla (stages 55-56). These results suggest that EB might induce sex-reversal in genetic males by disruption of early somatic-germ cell interactions in the medullary region of the gonad. Consequently, later morphogenetic events might be deranged, preventing differentiation of testis. We propose a hypothesis in which precocious production of estradiol (E2) by genotypic females is the mechanism for primary sex differentiation.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus

Xdazl is an RNA component of Xenopus germ plasm and encodes an RNA-binding protein that can act as a functional homologue of Drosophila boule. boule is required for entry into meiotic cell division during fly spermatogenesis. Both Xdazl and boule are related to the human DAZ and DAZL, and murine Dazl genes, which are also involved in gamete differentiation. As suggested from its germ plasm localization, we show here that Xdazl is critically involved in PGC development in Xenopus. Xdazl protein is expressed in the cytoplasm, specifically in the germ plasm, from blastula to early tailbud stages. Specific depletion of maternal Xdazl RNA results in tadpoles lacking, or severely deficient in, primordial germ cells (PGCs). In the absence of Xdazl, PGCs do not successfully migrate from the ventral to the dorsal endoderm and do not reach the dorsal mesentery. Germ plasm aggregation and intracellular movements are normal indicating that the defect occurs after PGC formation. We propose that Xdazl is required for early PGC differentiation and is indirectly necessary for the migration of PGCs through the endoderm. As an RNA-binding protein, Xdazl may regulate translation or expression of factors that mediate migration of PGCs.