A trial for induction of supernumerary primordial germ cells in Xenopus tadpoles by injecting RNA of Xenopus vasa homologue into germline cells of 32-cell embryos

Whether overexpression of Xenopus vasa homologue or Xenopus vasa-like gene 1 (XVLG1) in germline cells of Xenopus embryos can induce supernumerary primordial germ cells (PGC) at tadpole stage was investigated. XVLG1 RNA (0.1-2.0 ng) and beta-gal RNA (0.5 ng) were injected into one of, usually, four germ plasm-bearing cells (GPBC) of 32-cell embryos, with the beta-gal RNA (2.0 ng) serving as both lineage tracer and control for XVLG1 RNA. The total number of PGC, including X-gal-stained and unstained PGC of injected and uninjected GPBC origins respectively, was examined in the experimental tadpoles developed from the injected embryos. The injected RNA, XVLG1 and beta-gal RNA, were translated, resulting in a large amount of corresponding proteins in presumptive PGC (pPGC) as well as in somatic cells derived from the injected GPBC. Nevertheless, the average number of total PGC per tadpole found in the experimental tadpoles from the XVLG1 RNA-injected embryos was not significantly different from that of beta-gal RNA-injected ones, irrespective of the injected dose of XVLG1 RNA. This indicates that the extra XVLG1 protein in pPGC is not sufficient to increase the number of PGC in the tadpoles.     

Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells

In order to understand the role of the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) in germ line cells, an attempt was made to perturb the function of the protein with the anti-vasa antibody 2L-13. The 2L-13 or the control antibody was microinjected with a lineage tracer (FITC-dextran-lysine, FDL) into single vegetal blastomeres containing the germ plasm of Xenopus 32-cell embryos, the descendants of which were destined to differentiate into a small number of primordial germ cells (PGC) and a large number of somatic cells, mostly of endodermal tissues at the tadpole stage. No significant effect of the injection of the antibodies on FDL-labeled, presumptive PGC (pPGC) was observed in embryos until stage 37/38. However, FDL-labeled PGC were not observed in almost all the 2L-13 antibody-injected tadpoles, although a similar number of labeled somatic cells were always present. As 2L-13 antibody specifically reacts with XVLG1 protein in the embryos by immunoblotting, the present results suggest that the antibody perturbed the function of XVLG1 protein in the pPGC, resulting in failure of PGC differentiation at the tadpole stage.     

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.     

Delayed fertilization of anuran amphibian (Xenopus) eggs leads to reduced numbers of primordial germ cells

Several media were tested for the extent to which they promoted high fertilization efficiencies in ovulated, stripped Xenopus eggs. One medium was selected for maintaining eggs in a ‘delayed fertilization’ (DelF) condition. DelF eggs displayed several unusual characteristics, including shift of the center of gravity, prominent sperm entrance site, and occasional polyspermy. The frequency of normal pattern formation varied according to the length of time eggs were maintained in the DelF condition. Various developmental abnormalities were observed during gastrulation, neurulation, and organogenesis. Most abnormalities appeared, however, to be related to morphogenesis of the endoderm. Primordial germ cell (PGC) development was examined in DelF eggs which displayed normal external morphological features at the swimming tadpole stage. PGC counts were usually normal in short-duration (eg, 5 hr) DelF eggs, but frequently substantially reduced or completely diminished in longer-duration (eg, 25hr) tadpoles. Six spawnings were compared and shown to exhibit considerable variability in fertility, morphogenesis, and PGC development. Yolk platelet shifts and developmental parameters were examined in two additional spawnings. The subcortical cytoplasm in which the germ plasm is normally localized appeared to be disrupted in longer duration DelF eggs. That observation may account for low PGC counts in DelF tadpoles.     

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。     

Spatio-temporal distribution of the protein of Xenopus vasa homologue (Xenopus vasa-like gene 1, XVLG1) in embryos

In order to know when the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) first appears in germ line cells and whether the protein is also present in somatic cells as is vasa protein in Drosophila , the spatio-temporal distribution of the protein in Xenopus embryos was carefully investigated by fluorescent microscopy. Part of the observation was performed by whole-mount immunocytochemistry and immunoblotting. A distinct fluorescence of XVLG1 protein was first recognized in a juxta-nuclear location of germ line cells or presumptive primordial germ cells (pPGC) at stage 12 (late gastrula) and remained associated with the pPGC or primordial germ cells (PGC) throughout the following stages until stage 46 (feeding tadpole). In contrast, weak fluorescence was seen in the animal hemisphere rather than in the vegetal hemisphere of cleaving embryos and in the perinuclear region of somatic cells at stages 10–42 (early gastrula to young tadpole), respectively. Nearly the same pattern as revealed by fluorescence was seen by whole-mount immunocytochemistry, except that a small amount of XVLG1 protein seemed to be present in the germ plasm and pPGC of embryos earlier than stage 12. The presence of the protein in the somatic cells and the PGC was also shown by immunoblotting.     

哺乳动物卵泡卵母细胞发生的分子调控

哺乳动物卵泡卵母细胞发生的研究一直是发育生物学研究的重点之一。简要叙述了哺乳动物卵泡卵母细胞发生的一般过程,重点分析了原始生殖细胞向卵母细胞分化过程中gdf9、c-kti、BMP4及TGF家族关键基因的表达调控对卵母细胞发生的影响,以及卵母细胞与颗粒细胞间的相互调节作用,介绍了卵母细胞体外发生的最新研究进展及面临的难题等,为进一步研究原始生殖细胞向卵母细胞分化以及卵泡生长发育的机制提供了理论基础。