由不同发育时期内细胞团构建的嵌合兔

哺乳动物嵌合体越来越受到人们的注意,因为它既可用以研究发育、遗传、免疫以及肿瘤发生中一些有意义的理论问题,而且还是使体外培养的胚胎干细胞发育成个体的唯一途径。已往大量的工作都是集中在小鼠上,有关家畜嵌合体的报道为数很少。 本文简要地报道利用胚泡注射技术制作嵌合兔,对交配后96小时和120小时的内细胞团(ICM)细胞的发育能力进行了研究。本实验以青紫蓝(毛色为灰色,眼睛为灰褐色)兔(QQ)胚泡为供体,以新西兰(毛色为白色,眼睛呈红色)兔(XX)胚泡为受体,以毛色和眼睛色素细胞作为鉴定嵌合体的指     

小鼠胚胎原始外胚层细胞的发育能力(英文)

本文利用胚泡注射技术研究了小鼠胚胎原始外胚层细胞(primitive ectoderm cells)的发育能力。从交配后第五天的129/SV-ter(灰色,GPI-1~a/~a)小鼠的胚胎中分离出原始外胚层细胞并将之注射到交配后第四天的C_(57)BL/6 J(黑色,GPI-1~b/~b)小鼠的胚泡腔内。经过显微操作后的胚泡被移回昆明白假孕鼠内发育,其出生率为83.3%,毛色嵌合体(chimeras)比例为100%。这些嵌合小鼠的磷酸葡萄糖异构酶(GPI-1)分析结果表明,注射的原始外胚层细胞参与了内、中、外三个胚层所衍生的组织和器官(如脑、血液、心脏、肾脏、生殖腺、肌肉、脾、旰等)的胚胎发生。嵌合体与C_(57)BL/6 J小鼠交配后所得的结果表明,原始外胚层细胞在嵌合体内能形成有功能的配子。上述结果说明,原始外胚层细胞与内细胞团(ICM)细胞、体外培养的胚胎干细胞(embryoderivedstem cells)一样,具有发育全能性。导入胚泡后,不仅能参与嵌合体中各种体细胞的分化,并且能经历配子发生产生有功能的雌雄配子。此外,本文还对胚泡注射技术进行了改进,改进后的方法不仅比已报道的各种方法简便,并且使注射嵌合体的比例提高到35.7%。     

家兔胚泡ICM在体外培养系统中的行为

一、用显微外科方法从兔早期胚泡分离来的ICM在所用的体外培养系统中的行为,因ICM的生长状态而不同。呈带状生长的ICM从近端向远处延伸,而细胞的分化则从远端逐渐向近端进行,它具有明显的极性。细胞的分化秩序井然,层次分明。因此呈带状生长的ICM适用于进行细胞分化和细胞谱系的研究。二、呈团块状生长的ICM,没有明显的极性,细胞分化开始于团块的外表面,逐渐向中央进行。细胞分化开始稍晚一些,速度也较慢,这种生长状态的ICM适用于分离胚胎干细胞。三、兔子胚外内胚层从ICM分化来,它经历了二次分化的过程。第一次发生于培养后第三天(相当于交配后第七天),形成第一胚外内胚层-胚外体壁内胚层,它向远处迁移,远离原始外胚层。培养后四天(相当于交配后第八天),形成第二种胚外内胚层-胚外脏壁内胚层,它尾随体壁内胚层向远处迁移,其后缘的部分细胞向原始外胚层附近的滋胚层下面侵润。四、胚外体壁内胚层和脏壁内胚层的细胞是由少数决定了的和离开ICM的细胞分化和增殖而来。五、文中讨论了早期胚胎细胞分化的谱系问题。     

小鼠早期胚胎发育期间TGF-β_1免疫组织化学定位

用兔抗人血小板TGF-β_1 N末端1—29氨基酸残基人工合成多肽抗血清作探针以及免疫荧光和免疫酶染色技术,分析了1—12天小鼠早期发育期间胚胎的TGF-β_1物质分布。结果表明,着床前胚胎包括卵裂细胞,桑椹胚和胚泡的ICM及滋养外胚层等细胞均显示TGF-β_1阳性免疫荧光染色。免疫酶染色还证明,沿囊胚腔顶部单层排列的原始内胚层细胞比邻近的ICM细胞有较深的染色反应。随着胚胎着床和进一步发育,7天龄胚胎中胚层早期形成阶段,紧靠中胚层一侧的外胚层胞质中含有浓集的棕色颗粒;各胚层的部分区域也存在着染色强度上差别。8—12天龄胚胎中,体节,心壁、间质细胞和肠道以及卵黄囊的脏壁中胚层均有显著的TGF-β_1免疫酶阳性物质。这些结果表明,着床前小鼠胚胎富含TGF-β_1物质,着床后的胚外组织,例如卵黄囊也为胚胎进一步发育提供了富含TGF-β_1物质的微环境;同时也提示,小鼠早期胚胎发育期间的胚泡形成,ICM细胞分化出原始内胚层,卵柱期中胚层形成,以及以后的神经管、体节和肢芽形成阶段等一系列形态发生和器官形成过程中,TGF-β_1可能是参与重要作用的一种生长调节因子。     

小鼠胚胎干细胞(ES-8501细胞)建系过程的核型及特性分析

小鼠胚胎性癌(EC)细胞系的细胞核型大多数异常,对用于分析EC细胞与胚胎细胞之间的关系和进行嵌合体研究等都是不利的。人们都期望能有正常核型的胚胎细胞系的建立。近年来Evans和Kaufman以及Martin等人先后用不同方法直接从小鼠的内细胞团(ICM)细胞建立了多潜能的胚胎干细胞(erabryonicstem eells,简称ES细胞),也有人称之为EK     

供体细胞在鸡—麻鸭嵌合体胚胎中的发育

用微注射法将鸡的PGCs注入到麻鸭的胚盘下腔中,用鸡W染色体DNA探针通过原位杂交对供体细胞在嵌合体胚胎中的发育作了研究,54个胚胎各器官都有不同程度的嵌合,其中肝脏的嵌合率最高,性腺最低,胚盘细胞移植可制备鸡－麻鸭的体细胞和种系嵌合体。     

大鼠内细胞团和胎儿神经干细胞构建嵌合体的潜力(英文）

嵌合体大鼠是研究人类疾病的重要动物模型。用囊胚注射法研究了大鼠内细胞团（ICM）和胎儿神经干细胞（FNS）构建嵌合体的潜力。结果发现来自黑色（DA）大鼠第5天（D5）和第6天（D6）囊胚的ICM细胞注入D5 Sprague-Dawley（SD）大鼠囊胚后得到3只嵌合体大鼠;D5 SD大鼠ICM细胞注射入D5 DA囊胚后得到4只嵌合体大鼠;而体外培养的DA或SD大鼠ICM细胞注射后均未能获得嵌合体大鼠。本研究用大鼠胎儿神经干细胞（rFNS）和LacZ转染的rFNS构建嵌合体,未能获得嵌合体大鼠;但在LacZ转染的SD rFNS注射到DA大鼠囊胚后发育来的41只胎儿中,有2只胎儿其组织切片中发现少量LacZ阳性细胞。结果表明DA和SD大鼠ICM具有参与嵌合体发育的潜力,但ICM细胞经体外培养后构建嵌合体的潜力显著下降（P<0.05）;大鼠胎儿神经干细胞构建嵌合体的潜力较低,可能仅具有参与早期胚胎发育的潜力。     

2n/4n嵌合体胚胎的发育特点及其应用

2n/4n嵌合体是指用二倍体的胚胎细胞和四倍体的胚胎细胞聚合所形成的嵌合体。这种嵌合体在胚胎的发育过程中。四倍体来源的细胞在分布上具有一定的倾向性,即倾向于分布在胚外组织,如胎盘;而在胎儿本身的组织中,很少能找到四倍体细胞的存在,就2n/4n嵌合体胚胎的制作、嵌合体胚胎的发育特点及该技术的可能应用进行了综述。     

源于129S1品系的小鼠胚胎干细胞系的建立及其生物学特性分析

从129S1小鼠早期胚胎的内细胞团分离、培养类胚胎样细胞,经反复传代,成功地建立了129S1小鼠胚胎干细胞系,命名为NM-2细胞系。形态学鉴定具有胚胎干细胞的典型形态特征,正常核型率为80％;呈碱性磷酸酶阳性、表达胚胎干细胞特异性转录因子OCT-4;体内分化后可形成源于三胚层的组织结构;经囊胚腔显微注射后所获得的子代个体中79％具有毛色嵌合表型;雄性嵌合个体中31％发生生殖腺嵌合;同时,通过育种观察到所有生殖腺嵌合体的子代小鼠表型正常。以上结果证实NM-2细胞系为一株具高生殖腺嵌合能力的小鼠胚胎干细胞系。     

大鼠内细胞团和胎儿神经干细胞构建嵌合体的潜力

嵌合体大鼠是研究人类疾病的重要动物模犁.用囊胚注射法研究了大鼠内细胞团(ICM)和胎儿神经干细胞(FNS)构建嵌合体的潜力.结果发现来自黑色(DA)大鼠第5天(D5)和第6天(D6)囊胚的ICM细胞注入D5 Sprague-Dawley(SD)大鼠囊胚后得到3只嵌合体大鼠:D5 SD大鼠ICM细胞注射入D5 DA囊胚后得到4只嵌合体大鼠:而体外培养的DA或SD人鼠ICM细胞注射后均未能获得嵌合体大鼠.本研究用大鼠胎儿神经干细胞(rFNS)和LacZ转染的rFNS构建嵌介体,未能获得嵌合体人鼠:但在LacZ转染的SD rFNS注射到DA大鼠囊胚后发育来的41只胎儿中,有2只胎儿其组织切片中发现少量LacZ阳性细胞.结果表明DA和SD大鼠ICM具有参与嵌合体发育的潜力,但ICM细胞经体外培养后构建嵌合体的潜力显著F降(P＜0.05);大鼠胎儿神经干细胞构建嵌合体的潜力较低,可能仅具有参与早期胚胎发育的潜力.     

家兔早期胚胎细胞发育能力的研究

The developmental potential of rabbit embryonic cells was studied through making chimera by separate introduction of inner cell mass from 96-h-old p. c., 120-h-old p. c., and 144-h-old p. c. of grey rabbits into 96-h-old p. c. blastocysts of New Zealand white rabbits. A total of five overt chimeras were obtained including two fertile males, two fertile females and one sterile male, from the ICM cells of 96-h-old and 120-h-old embryos but none was obtained from 144-h-old cells. Histological examination of the gonad showed that the sterile chimera derived from 120-h-old ICM cells with an ovotestis on both sides. Follicles and seminiferous tubules developed in the cortex and medulla of the gonad, respectively. Neither of them developed into functional germ cells. Analysis of karyotypes of peripheral blood showed that both XX and XY coexisted in lymphocytes. These results indicated that the sterile male chimera was a XX/XY sex chimera derived from ICM cells of donor and recipients with different sex, so as to the chimera with XX and XY genotypic cells. From the results mentioned above we may conclude that the ICM cells at 120-h-old p. c. are still pluripotential, they can not only participate in development into somatic components but also develop into germ cells. The potential of 144-h-old p. c. ICM cells seems to be rather restricted.     

Sex differentiation and germ cell production in chimeric pigs produced by inner cell mass injection into blastocysts

This study aimed at collecting background knowledge for chimeric pig production. We analyzed the genetic sex of the chimeric pigs in relation to phenotypic sex as well as to functional germ cell formation. Chimeric pigs were produced by injecting Day 6 or Day 7 inner cell mass (ICM) cells into Day 6 blastocysts. Approximately 20% of the piglets born from the injected blastocysts showed overt coat color chimerism regardless of the embryonic stage of donor cells. The male:female sex ratio was 7:2 and 6:1 in the chimeras derived from Day 6 and Day 7 ICM cells, respectively, showing an obvious bias toward males. When XX donor cells were injected into XY blastocysts at the same embryonic stage, the phenotypic sex of the resulting chimera was male with no germ-line cells formed from the donor cell lineage. On the other hand, when the donor was XY and the recipient blastocyst was XX, the phenotypic sex of the chimera was male, and germ-line cells were derived only from the donor cells. The combination of XY donor cells and XY blastocysts produced some chimeras in which the donor cell lineage did not contribute to germ-line formation even when it appeared in coat color. When the embryonic stage of the donor was advanced by 1 day in the XY-XY combination, 100% of the germ-line cells of the chimeras were derived from the donor cell lineage. These data showed that characteristics of sex differentiation and germ cell formation in chimeric pigs are similar to those in chimeric mice.     

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

Male sterility caused by p6H and qk mutations is not corrected in chimeric mice

It is not known if the male sterility caused by the pleiotropic mutations p6H (pink-eyed 6H) and qk (quaking) is intrinsic or extrinsic to spermatogenic cells. This question was addressed by juxtaposing mutant and normal cells in the testes of chimeric mice and determining whether the mutant germ cells could form functional sperm. Twenty-one male chimeras consisting of normal cells and p6H/p6H or qk/qk cells were analyzed. For each, breeding productivity and testicular and sperm morphology were determined. Karyotypes and isozyme analyses were performed to identify the two cellular components of each chimera. All male chimeras that contained p6H/p6H, XY cells were sterile. Although some chimeras with a qk/qk, XY mutant component were fertile, none produced offspring from the homozygous qk component. Spermatids of the sterile chimeras showed abnormalities characteristic of the mutations. We conclude from this study that the presence of normal XY germ and somatic cells in the testis did not rescue the male sterile phenotype of homozygous p6H or qk XY germ cells. Therefore, the action of these mutant genes in causing sperm abnormalities and sterility is autonomous to the germ cells.     

FGF9 promotes survival of germ cells in the fetal testis

In addition to its role in somatic cell development in the testis, our data have revealed a role for Fgf9 in XY germ cell survival. In Fgf9-null mice, germ cells in the XY gonad decline in numbers after 11.5 days post coitum (dpc), while germ cell numbers in XX gonads are unaffected. We present evidence that germ cells resident in the XY gonad become dependent on FGF9 signaling between 10.5 dpc and 11.5 dpc, and that FGF9 directly promotes XY gonocyte survival after 11.5 dpc, independently from Sertoli cell differentiation. Furthermore, XY Fgf9-null gonads undergo true male-to-female sex reversal as they initiate but fail to maintain the male pathway and subsequently express markers of ovarian differentiation (Fst and Bmp2). By 14.5 dpc, these gonads contain germ cells that enter meiosis synchronously with ovarian gonocytes. FGF9 is necessary for 11.5 dpc XY gonocyte survival and is the earliest reported factor with a sex-specific role in regulating germ cell survival.     

Meiotic germ cells antagonize mesonephric cell migration and testis cord formation in mouse gonads

The developmental fate of primordial germ cells in the mammalian gonad depends on their environment. In the XY gonad, Sry induces a cascade of molecular and cellular events leading to the organization of testis cords. Germ cells are sequestered inside testis cords by 12.5 dpc where they arrest in mitosis. If the testis pathway is not initiated, germ cells spontaneously enter meiosis by 13.5 dpc, and the gonad follows the ovarian fate. We have previously shown that some testis-specific events, such as mesonephric cell migration, can be experimentally induced into XX gonads prior to 12.5 dpc. However, after that time, XX gonads are resistant to the induction of cell migration. In current experiments, we provide evidence that this effect is dependent on XX germ cells rather than on XX somatic cells. We show that, although mesonephric cell migration cannot be induced into normal XX gonads at 14.5 dpc, it can be induced into XX gonads depleted of germ cells. We also show that when 14.5 dpc XX somatic cells are recombined with XY somatic cells, testis cord structures form normally; however, when XX germ cells are recombined with XY somatic cells, cord structures are disrupted. Sandwich culture experiments suggest that the inhibitory effect of XX germ cells is mediated through short-range interactions rather than through a long-range diffusible factor. The developmental stage at which XX germ cells show a disruptive effect on the male pathway is the stage at which meiosis is normally initiated, based on the immunodetection of meiotic markers. We suggest that at the stage when germ cells commit to meiosis, they reinforce ovarian fate by antagonizing the testis pathway.     

Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.     

Testicular Development Induced by a Recessive Mutation during Gonadal Differentiation of Female Common Carp (Cyprinus carpio, L.)

The phenotypic effects of a new recessive mutation mas ⁻¹, which in homozygous condition induces testicular development in XX animals of common carp ( Cyprinus carpio L.), are described. Sexual differentiation of XX; mas ⁻⁺/ mas ⁻¹ and XX; mas ⁻¹/ mas ⁻¹ animals was compared with the gonad development of XX wild type females and XY males. In XX females gonadal differentiation starts with the formation of an ovarian cavity and entry into meiosis of germ cells at around 80 days post hatching (ph). Male gonads remain quiescent until 120 days ph during which period they develop a network of loose connective tissue. Spermatogenesis starts with tubule formation and the differentiation of germ cells into spermatogonia type B. Heterozygous XX; mas ⁻⁺/ mas ⁻¹ animals developed as normal females, but in homozygous XX; mas ⁻¹/ mas ⁻¹ animals two types of gonad development were observed. In the first type, germ cells did not enter meiosis until 100 days ph when they differentiated as spermatogonia. An ovarian cavity was not formed but male specific connective tissue developed instead. These gonad developed as normal testes. In the second type, germ cells differentiated at 80 days ph as either oocytes or spermatocytes, which resulted in the gonads developing as ovotestes. The formation of an ovarian cavity was in most cases incomplete. The phenotypic effects of mas ⁻¹ are interpreted as a timing mismatch between mas activation and female sex differentiation.     

Ultrastructural aspects of gonadal morphogenesis in Bufo bufo (Amphibia Anura) 1. Sex differentiation

The morphogenesis of gonads in Bufo bufo tadpoles was studied, and ultrastructural differences between sexes were identified. All specimens analyzed initially developed gonads made up of a peripheral fertile layer (cortex) surrounding a small primary cavity. Subsequently a central layer of somatic cells (medulla) developed. Both layers were separated by two uninterrupted basal laminae between which a vestige of the primary cavity persisted. During female differentiation, the peripheral layer continued to be the fertile layer. In males, the central layer blended into the peripheral layer and the basal laminae disappeared. The somatic cells of the central layer came into direct contact with the germ cells; this did not occur in females. Testicular differentiation continued with the migration of germ cells towards the center of the gonad. The somatic elements surrounding the germ cells appeared to play an active role in their transfer to the center of the gonad. The peripheral layer shrank and became sterile. Two basal laminae then re-formed to separate the fertile central layer from the peripheral sterile one. Germ cells have always been thought to perform a passive role in sex differentiation in amphibians. Following the generally accepted "symmetric model", the mechanism of gonad development is symmetrical, with cortical somatic cells determining ovarian differentiation and medullary somatic cells determining testicular differentiation. In contrast, we found that sex differentiation follows an "asymmetric" pattern in which germ cells tend primarily toward a female differentiation and male differentiation depends on a secondary interaction between germ cells and medullary somatic cells.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4+/+ and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.