原始生殖细胞增殖调控的研究进展

1 原始生殖细胞的增殖过程 原始生殖细胞(primotdial germ cells,PGCs)是形成动物配子的前体,在胚胎的发育过程中迁移到生殖嵴.在迁移的过程中或到达生殖嵴后,PGCs进行有丝分裂,数量快速增加.     

睾丸生殖细胞肿瘤类型及起源

Skakkebaek(1972)首先发现睾丸原位癌(CIS)/小管生殖细胞肿瘤未定类型(IGCNU).CIS/IGCNU是睾丸生殖细胞肿瘤(TGCT)除精母细胞精原细胞瘤以外各型共同的前驱病变,其来源很可能为原始生殖细胞(PGC)/早期生殖母细胞(gonocyte)的恶性转化.流行病学和表型免疫组化检测证据表明IGCNU是一种发生于胎儿期的先天性病变.PGCs及gonocytes在前减数分裂阶段具有胚胎干细胞样多能性分化潜能.     

人原生殖细胞的分离与培养

目的:探讨人原生殖细胞(PGC)的分离取材时机与体外培养方法,为人类胚胎生殖细胞(EG)的建系研究奠定基础。方法:取不同孕龄的人胚,分离人PGC,在不同培养体系中,观察其增殖与分化情况。结果:孕8、9周较孕7周人胚用酶机械法分离PGC后,原代克隆形成率高,以小鼠胚胎成纤维细胞或STO细胞作为饲养层,并且在培养液中加入hLIF、hbFGF\,hSCF,能较好地维持人PGC的增殖并保持未分化。结论:孕8、9周龄人胚为分离PGC的适宜材料,酶机械法分离PGC简单有效,饲养层细胞与生长因子为人PGC细胞体外培养所必需。     

SCF/C-KIT系统与生精细胞发育、增殖和分化

人原始生殖细胞起源于卵黄囊尾侧壁的内胚层细胞，以后经后肠背系膜迁移至生殖嵴内增殖形成性原细胞，出生后增殖形成精原干细胞，青春期后再增殖分化形成精子，此过程受多种因素调控，其中SCF／C-KIT系统在该过程中起着重要的作用。     

人胚胎干细胞表达生殖细胞分化相关基因的研究

目的 探讨人胚胎干细胞(hES)系H1生殖细胞分化相关基因的表达.方法 免疫荧光、碱性磷酸酶检测和核型分析等方法联合鉴定H1的基本生物学特性,RT-PCR方法检测未分化H1的生殖细胞分化相关基因的基本表达模式,分别以人正常睾丸组织和人胚胎成纤维细胞株HFF-1作为阳性和阴性对照组织.结果 H1保持正常核型并维持未分化状态.未分化H1表达减数分裂前生殖细胞标志基因STELLAR、DAZL、FRAGILIS、PUM1、PUM2和GDF3;不表达原始生殖细胞(PGCs)标志基因BLIMP-1、减数分裂特异标志基因SCP1、减数分裂启动标志基因STRA8以及生殖细胞分化后期标志基因VASA.结论 未分化Hl细胞表达生殖细胞减数分裂前标志基因,但不表达分化后期基因,BLIMP-1可能成为区分未分化hES细胞与PGCs的特异性标志基因.     

原生殖细胞的研究进展

原生殖细胞是来源于胚胎原始生殖嵴的一种具有多向分化潜能的干细胞 ,原生殖细胞的形态、标志及体内外分化潜能都类似于胚胎干细胞 ,而且也具有其种系传递能力。研究表明 ,原生殖细胞在胚胎发生中有其相对固定的迁移途径 ,细胞的迁增殖和分化受许多细胞因子的调控 ,原生殖细胞可表达某些特异的抗原 ,据此可对其进行鉴定 ,目前许多种属的原生殖细胞已建系 ,这必将有助于原生殖细胞的深入研究。原生殖细胞作为一种多能干细胞 ,对于体外研究胚胎发育、基因研究、基因组学研究、药物筛选等有重要意义     

子宫内膜基质细胞作为饲养层培养人胚原始生殖细胞

目的探讨子宫内膜基质细胞作为饲养细胞培养人胚原始生殖细胞的可能。方法从5—9周人胚胎生殖嵴、中肾嵴、肠系膜中消化分离原始生殖细胞(PGCs),种植于经丝裂霉素c处理的人子宫内膜基质细胞饲养层上培养。结果PGCs可以在体外培养3个多月,传14代。这样培养的PGCs表达胚胎干细胞的多种标志,如碱性磷酸酶染色、SSEA和TRA抗原等。另外细胞保持正常的染色体核型。结论人子宫内膜基质细胞可以作为饲养细胞在体外培养人胚原始生殖细胞,保持未分化状态。     

微模式化基底上大鼠骨髓间充质干细胞的增殖、分化和迁移

目的　利用微模式化基底研究基底几何微结构对骨髓间充质干细胞增殖、分化及迁移过程的影响。方法　设计制作微模式化基底,用以控制细胞的铺展形态和面积。比较不同模式控制下大鼠骨髓间充质干细胞的增殖、分化和迁移数据。结果　细胞铺展宽度狭小可抑制骨髓间充质干细胞的增殖,细胞铺展形状可以调节其向成骨细胞分化的进程,细胞铺展面积受限时迁移增强,其增殖迁移行为减弱与成骨细胞诱导因子地塞米松的作用有关。结论　细胞铺展的几何形状和面积是骨髓间充质干细胞增殖、分化及迁移过程中的重要调节因子。     

人原始生殖细胞的研究现状

迄今为止干细胞有三种生殖细胞来源:(一)胚胎生殖细胞(EGs):来自原始生殖细胞(PGC);(二)胚胎癌性细胞(EGCs):可由成人睾丸肿瘤得到;(三)多能生殖干细胞(GSCs):已由小鼠精原干细胞的培养得到[1]。PGC在体外分离培养,建系成功后改称为胚胎生殖细胞。类胚体实验表明,EG细胞可分化形成包括所有3个胚层的组织细胞,证明其具有多能性,与胚胎干细胞(embryonic stem cell,ES细胞)具有相似的多向分化潜力,因此ES细胞在发育生物学基础研究、动物胚胎工程和临床医学上具有重要的作用和诱人的前景。人EG细胞的研究对生命科学的发展有重要意义。…     

小鼠原始生殖细胞体外增殖与分化的研究

目的探讨小鼠原始生殖细胞（PGC）的体外增殖与分化.方法将PGC与睾丸支持细胞（SC）共培养进行PGC的体外增殖.用原代PGC悬浮培养获取类胚体（EB）,将EB贴壁培养,观察自然分化的结果.结果PGC与SC共培养可获得大量PGC集落.组织化学与免疫组织化学法显示,PGC集落碱性磷酸酶（AKP）和阶段特异性胚胎抗原-1（SSEA-1）均呈阳性表达.将PGC悬浮培养可获得EB,EB贴壁生长后,可自然分化形成上皮样细胞、成纤维样细胞和神经样细胞.结论小鼠PGC与SC共培养能在体外大量增殖.并保持干细胞特性.PGC可形成EB,EB自然分化后大多数形成神经样细胞、其次是上皮样细胞和成纤维样细胞.     

Morphological studies on germ cell development and differentiation in mammals

The process of mammalian germ cell development from the early embryo through the adult entails a sequence of cellular events, including migration, proliferation, and differentiation. This paper is briefly reviewed our recent work concerning such fields. The 4C9 carbohydrate antigen as a reliable marker to study the origin, migration and differentiation of primordial germ cells is described. Further morphological data obtained by studying ectopic germ cells outside the fetal gonad is discussed. Moreover, evidence that both proliferation of spermatogonia and growth of oocytes are essentially dependent on c-Kit is reported. Still, a great deal of work remains in order to elucidate the fundamental mechanism controlling germ cell development and differentiation.     

Germ Cell Migration,Proliferation and Differentiation during Gonadal Morphogenesis in All‐Female Japanese Flounder (Paralichthys Olivaceus)

In this study, all genetic female (XX) broods of Japanese flounder were produced artificially by mating the females with sex‐reversed males. The proliferation and migration of primordial germ cells (PGCs), formation of ovary and oogenesis were described in detail. After hatching, around 20 individual PGCs migrated from the lateral to the dorsal of trunk region. At 15 days posthatching (dph), a part of PGCs were covered by a single layer somatic cells and formed the genital ridge. By 22 dph, the elongated gonadal primordia appeared under the ventral kidney, where the PGCs were totally enclosed by somatic cells. During the process of migration, PGCs were presumed to be mitotically inactive. From 63 to 73 dph, somatic cells rearrangement resulted in the formation of a narrow crevice, which became deeper and formed ovarian lumen. However, at 52 dph, dramatic mitotic proliferation of germ cell occurred and germline nest formed before the appearance of ovarian lumen. The onset of intensive germ cell proliferation and appearance of cell nests could be accepted as a criterion of initial ovarian differentiation. Then germ cells and somatic epithelial cells were gradually delimited by basement membrane and formed the germinal epithelium. In this period, results from in situ hybridization revealed that the early forkhead box L2 (pofoxl2) was expressed in somatic cells and oocytes in primary growth, which indicated the prefollicle cells formed. Then oogonia or oocytes, follicle cells, basement membrane, and theca cells composed a follicle complex. Finally, oocytes underwent meiosis and developed into to mature eggs. Anat Rec, 301:727–741, 2018. © 2017 Wiley Periodicals, Inc.     

Isolation of primordial germ cells from differentiating human embryonic stem cells

Of all the cell types that can be obtained from the differentiation of embryonic stem cells, primordial germ cells are arguably the most fascinating, as they represent the in vitro completion of the reproductive cycle of the organism from which the embryonic stem cell line was derived. It is also possible to obtain these cells from embryos at an appropriate stage of development, but this process yields only small numbers that are not applicable to investigations of their epigenetic architecture. A considerable body of data has been generated from the differentiation of mouse embryonic stem cells to this cell type, but despite the demonstration of their presence in human embryoid bodies, there has been little progress toward methods of producing human primordial germ cells in useful numbers. We present here a robust protocol to differentiate two human embryonic stem cell lines (H9 and hES-NCL1) that maximizes the numbers of primordial germ cells that may be obtained using a simple fluorescence-activated cell sorting strategy for their isolation. These primordial germ cells demonstrate high-level expression of the germ cell-specific VASA gene and show removal of parental imprints and chromatin modification changes that support their primordial germ cell identity.     

MAGE-A1, GAGE and NY-ESO-1 cancer/testis antigen expression during human gonadal development

BACKGROUND: Cancer/testis antigens (CTAs) are expressed in several cancers and during normal adult male germ cell differentiation. Little is known about their role in fetal development of human germ cells. METHODS: We examined expression of the CTAs MAGE-A1, GAGE and NY-ESO-1 in fetal gonads by single and double immunohistochemical staining. RESULTS: We found that GAGE was expressed in the primordial germ cells of the gonadal primordium, whereas MAGE-A1 and NY-ESO-1 were first detected in germ cells of both testis and ovary after sexual differentiation was initiated. The number of positive germ cells and the staining intensity of all three CTAs peaked during the second trimester and gradually decreased towards birth in both male and female germ cells. In oocytes, MAGE-A1 expression terminated around birth, whereas NY-ESO-1 expression persisted through the neonatal stage and GAGE expression was maintained until adulthood. The population of GAGE-expressing male and female germ cells partially overlapped the population of OCT4-positive cells, whereas MAGE-A1 and NY-ESO-1 were clearly expressed only by OCT4-negative cells. CONCLUSIONS: Our results suggest that MAGE-A1 and NY-ESO-1 are associated with highly proliferating germ cells, whereas GAGE proteins have a more general function in germ cells unrelated to any specific developmental stage. The recognition of differential cellular expression of GAGE, MAGE-A1, NY-ESO-1 and OCT4 may help define biologically distinct germ cell subpopulations.     

Differentiation and development of human female germ cells during prenatal gonadogenesis: an immunohistochemical study

BACKGROUND: In the development of the human ovary, the second trimester includes the transition from oogonial replication to primordial follicle formation. The present study was carried out to assess differentiation and proliferation of germ cells in a series of female gonads from 19 fetuses from the second and third trimester, and two neonates. METHODS: Using immunohistochemistry, the following markers were studied: placental/germ-like cell alkaline phosphatases (PLAP), the marker of pluripotency OCT3/4, the proliferation marker Ki-67, beta-catenin and E-cadherin, the stem cell factor receptor c-KIT, and VASA, a protein specific for the germ cell lineage. RESULTS: PLAP and OCT3/4 were seen during oogenesis, but not in germ cells engaged in folliculogenesis. A similar pattern was observed for Ki-67. Loss of pluripotency occurs once oocytes engage in follicle formation, suggesting a role of cell-cell interactions in the process of germ cell maturation. VASA, c-KIT, beta-catenin and E-cadherin were found in germ cells at all developmental stages of oogenesis and folliculogenesis. CONCLUSIONS: Immunohistochemically, two groups of germ cells can be distinguished. Germ cells that are predominantly found in the cortical region of the ovary before weeks 22-24 of gestation, showing an immature phenotype, are mitotically active, and express OCT3/4, a marker of pluripotency. On the other hand, germ cells undergoing folliculogenesis have lost their pluripotent potential and no longer proliferate.     

人胚胎睾丸生殖细胞发育的组织学和超微结构研究

用光镜与电镜技术，对１５例７至２８周人胚胎睾丸生殖细胞的发育进行了研究。结果：（１）据时间，位置及形态结构特点，将生殖细胞的发育过程初步分为三个阶段，即原始生殖细胞阶段，生殖母细胞阶段和前的细胞阶段。其中前精原细胞阶段又经历了早，中，晚三上时期各期出现的时间有交错。（２）１４周是胚胎生殖细胞发育的一个重要时期。     

Recent advances in the derivation of germ cells from the embryonic stem cells

In recent years, considerable progress has been made in the establishment and differentiation of human embryonic stem (ES) cell lines. The primordial germ cells (PGCs) and embryonic germ (EG) cells derived from them share many of their properties with ES cells. ES cell lines have now been derived from different stages of germ cell development and they have differentiated into gametes and shown embryonic development in mice, including the production of live pups. Conversely, germ cells can also be derived from ES cells. It has been demonstrated that murine (m) ES cells can differentiate into PGCs and subsequently into early gametes (oocytes and sperms) and blastocysts. Recently, immature sperm cells derived from mES cells in culture have produced live offspring. Preliminary research has indicated that human (h) ES cells probably have the potential to differentiate into germ cells. Adult stem cells have been reported to differentiate into mature germ cells in vitro. Therefore, stem cells may offer a valuable in vitro model for the investigation of germ cell development and the early stages of human gametogenesis, including epigenetic modifications of the germ line. This review discusses recent developments in the derivation and specification of mammalian germ cells from ES cells and describes some of the mechanisms of germ cell development.     

Temporal and spatial localization of three germline-specific proteins in medaka.

We show that germline-specific proteins, olvas (vasa), nanos, and tdrd1 (tudor), alter their localization in the cytoplasm during germline development in the medaka (Oryzias latipes). By immunohistochemical analysis, these three germline-specific proteins were detectable on granule-like structures in the cytoplasm of migrating primordial germ cells. In the germ cells of the gonadal primordia, these granules formed a hollow area lacking these three protein components. During the sexual differentiation of the female gonads, the granules were found to be reduced in size in the germ cells undergoing cystic division and they showed a perinuclear localization in the oocytes. However, the germ cells in the male gonads retained their hollow granules during this early sex differentiation stage. We further demonstrate the differential localization of olvas, nanos, and tdrd1 on these granules during medaka germline development.