Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture

The functional gametes of all vertebrates first arise in the early embryo as a migratory population of cells, the primordial germ cells (PGCs). These migrate to, and colonise, the genital ridges (GR) during the early organogenesis period, giving rise to the complete differentiating gonad. PGCs first become visible by alkaline phosphatase staining in the root of the developing allantois at 8.5 days post coitum (dpc). At 9.5 dpc they are found in the wall of the hind-gut and, during the following three days, they migrate along the hind-gut mesentery to the dorsal body wall, and then to the genital ridges. By 12.5 dpc, the great majority of PGCs have colonised the genital ridges. During this period the number of PGCs increases from less than 100 to approximately 4000. In a previous paper (Donovan et al. 1986), we showed that 10.5 dpc PGCs can be explanted from the hind-gut mesentery, and will spread and migrate on feeder cell layers. We showed also that the intrinsic ability of PGCs to spread and migrate changes as they colonise the genital ridges. In this paper, we examine extrinsic factors that control PGC behaviour in vitro. Using PGCs taken from 8.5 dpc embryos, at the beginning of their migratory phase, we show that culture medium conditioned by 10.5 dpc genital ridges causes an increase in the number of PGCs in these cultures. We also show that PGCs migrate towards 10.5 dpc genital ridges in preference to other explanted organs. These experiments show that genital ridges exert long-range effects on the migrating population of PGCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autonomous transition into meiosis of mouse fetal germ cells in vitro and its inhibition by gp130-mediated signaling

Mouse primordial germ cells (PGCs) arrive at the urogenital ridge (UGR) at around 10.5 days postcoitum (dpc). They proliferate until around 13.5 dpc, then enter into meiosis in the female or become mitotically arrested in the male gonads. In this study, meiotic transition of mouse PGCs was examined in vitro. Female PGCs obtained from UGRs or genital ridges at 10.5-11.5 dpc began to express meiosis-specific genes, Scp3 and Dmc1, after dissociation and cultivation on feeder cells for several days. Meiotic transition into the leptotene stage was confirmed by the formation of axial cores. Male PGCs at 10.5-11.5 dpc and migratory PGCs obtained from mesenteries at 10.5 dpc also expressed Scp3 and formed axial cores after several days of culture, supporting the hypothesis that PGCs are capable of entering meiosis before arriving at the UGR. gp130-mediated signaling, known to promote survival/growth of PGCs and also to inhibit the differentiation of embryonic stem cells, suppressed the expression of Scp3 in PGCs and inhibited the following formation of axial cores in vitro. This novel activity of gp130-mediated signaling may provide some clues for the understanding of pluripotency of mammalian germ-line cells and/or the sex differentiation of fetal germ cells.     

Synthesis of glycoconjugates in mouse primordial germ cells

The synthesis of protein-bound carbohydrates has been studied in primordial germ cells (PGCs) and in somatic cells of 12.5 to 13.5-days-postcoitum (dpc) fetal mouse gonads. Both cell types were shown to synthesize asparagine-linked glycopeptides and glycosaminoglycans (GAGs). In addition, PGCs also synthesize lactosaminoglycans (LAGs) although in different proportions in female and male germ cells. Female PGCs, which at 13.5 dpc are entering meiosis, synthesize mainly LAGs, and minor amounts of hyaluronic acid (HA) and chondroitin sulfate (CS). Male germ cells, on the other hand, synthesize mainly CS. Furthermore, somatic cells of fetal gonads synthesize HA as the major class of GAGs. It is suggested that the activation of LAG synthesis in developing germ cells might be related to the beginning of meiosis. Moreover, we propose that HA synthesis might be developmentally regulated in somatic cells of the gonad, in order to regulate the establishment of specific interactions with germ cells.     

Derivation in culture of primordial germ cells from cells of the mouse epiblast: phenotypic induction and growth control by Bmp4 signalling

Primordial germ cells (PGCs) are the embryonic precursors of the gametes of the adult. PGCs derive from cells of the most proximal part of the cup-shaped epiblast corresponding to the presumptive region of the extraembryonic mesoderm. At 7.2 days post coitum (dpc) a small group of PGCs located at the base of the allantois can be recognised due to a strong alkaline phosphatase activity. Thus far, scant information was available on the mechanism(s) controlling the lineage of PGCs in the mouse embryo. However, results obtained in mice defective for bone morphogenetic protein-4 (Bmp4) secreted molecule revealed that this growth factor has important functions for the derivation of PGCs from extraembryonic mesoderm cells. In this paper, we have studied the effects in culture of Bmp4 on epiblast cells obtained from egg-cylinder stage mouse embryos (5.5-6.0 dpc) and PGCs from 11.5 dpc embryos. We found that Bmp4 treatment enables recruitment of pluripotent cells to a PGC phenotype by a multi-step process involving an initial pre-commitment of epiblast cells and a following stage of PGC phenotypic determination. We further provide evidences that Bmp4 may promote the growth of gonadal PGCs through a Smad1/4 signalling.     

Identification of side population cells in mouse primordial germ cells and prenatal testis

In mammals, the stem cells of spermatogenesis are derived from an embryonic cell population called primordial germ cells (PGCs). Spermatogonial stem cells displaying the “side population” (SP) phenotype have been identified in the immature and adult mouse testis, but noting is known about the expression of the SP phenotype during prenatal development of germ cells. The SP phenotype, defined as the ability of cells to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In the present study, we analyzed and characterized the Hoechst SP via cytofluorimetric analysis of disaggregated gonads at different time points during embryonic development in mice. To directly test the hypothesis that the SP phenotype is a feature of germ cell lineage, experiments were performed on transgenic animals expressing enhanced green fluorescent protein (EGFP) under the control of the Oct4 promoter, to identify early germ cells up to PGCs. We found that prenatal gonads contain a fraction of SP cells at each stage analyzed, and the percentage of cells in the SP fraction decreases as development proceeds. Surprisingly, more than 50% of the PGCs displayed the SP phenotype at 11.5 dpc (days post coitum). The percentage of germ cells with the SP phenotype decreased steadily with development, to less than 1% at 18.5 dpc. Cytofluorimetric analysis along with immunocytochemistry performed on sorted cells indicated that the SP fraction of prenatal gonads, as in the adult testis, was heterogeneous, being composed of both somatic and germ cells. Both cell types expressed the ABC transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These findings provide evidence that the SP phenotype is a common feature of PGCs and identifies a subpopulation of fetal testis cells including prospermatogonia whose differentiation fate remains to be investigated.     

Pin1 regulates the timing of mammalian primordial germ cell proliferation

Primordial germ cells (PGCs) give rise to male and female germ cells to transmit the genome from generation to generation. Defects in PGC development often result in infertility. In the mouse embryo, PGCs undergo proliferation and expansion during and after their migration to the gonads from 8.5 to 13.5 days post coitum (dpc). We show that a peptidyl-prolyl isomerase, Pin1, is involved in the regulation of mammalian PGC proliferation. We discovered that both the male and female Pin1(-/-) mice had profound fertility defects. Investigation of the reproductive organs revealed significantly fewer germ cells in the adult Pin1(-/-) testes and ovaries than in wild type or heterozygotes, which resulted from Pin1(-/-) males and females being born with severely reduced number of gonocytes and oocytes. Further studies in 8.5 to 13.5 dpc Pin1(-/-) embryos showed that PGCs were allocated properly at the base of the allantois, but their cell expansion was progressively impaired, resulting in a markedly reduced number of PGCs at 13.5 dpc. Analyses using markers of cell cycle parameters and apoptosis revealed that Pin1(-/-) PGCs did not undergo cell cycle arrest or apoptosis. Instead, Pin1(-/-) PGCs had a lower BrdU labeling index compared with wild-type PGCs. We conclude that PGCs have a prolonged cell cycle in the absence of Pin1, which translates into fewer cell divisions and strikingly fewer Pin1(-/-) PGCs by the end of the proliferative phase. These results indicate that Pin1 regulates the timing of PGC proliferation during mouse embryonic development.     

Pluripotential stem cells derived from migrating primordial germ cells

Pluripotent stem cells termed embryonic germ cells (EGCs) have earlier been derived from pre- and post-migrating mouse primordial germ cells (PGCs). We have recently obtained four EGC lines from migrating PGCs of 9.5 days post coitum (dpc) embryos. All lines were male with normal karyotype and showed properties that are similar to previously established EGC lines, including colony morphology, expression of alkaline phosphatase (AP), and expression of SSEA-1 antigen. The developmental potency of two of these lines was tested in vivo. They contributed to a range of tissues in fetal chimeras including heart, lung, kidney, intestine, muscle, brain and skin. We also examined the methylation status of the imprinted genes: Igf2r, p57Kip2, Lit1, H19 and Igf2. Igf2r, p57Kip2 and Lit1 were unmethylated in all analysed EGC lines, whereas H19 and Igf2 showed significant hypo-methylation in the 9.5 dpc EGC-1 line when compared to previously derived 11.5 dpc male EGC lines. This suggests that imprint erasure in the male germ line occurs prior to 9.5 dpc for all imprinted genes examined.     

Stage-specific tissue and cell interactions play key roles in mouse germ cell specification

Primordial germ cells (PGCs) in mice have been recognized histologically as alkaline phosphatase (AP) activity-positive cells at 7.2 days post coitum (dpc) in the extra-embryonic mesoderm. However, mechanisms regulating PGC formation are unknown, and an appropriate in vitro system to study the mechanisms has not been established. Therefore, we have developed a primary culture of explanted embryos at pre- and early-streak stages, and have studied roles of cell and/or tissue interactions in PGC formation. The emergence of PGCs from 5.5 dpc epiblasts was observed only when they were co-cultured with extra-embryonic ectoderm, which may induce the conditions required for PGC formation within epiblasts. From 6.0 dpc onwards, PGCs emerged from whole epiblasts as did a fragment of proximal epiblast that corresponds to the area containing presumptive PGC precursors without neighboring extra-embryonic ectoderm and visceral endoderm. Dissociated epiblasts at these stages, however, did not give rise to PGCs, indicating that interactions among a cluster of a specific number of proximal epiblast cells is needed for PGC differentiation. In contrast, we observed that dissociated epiblast cells from a 6.5-b (6.5+15-16 hours) to 6.75 dpc embryo that had undergone gastrulation gave rise to PGCs. Our results demonstrate that stage-dependent tissue and cell interactions play key roles in PGC determination.     

Isolation of mouse primordial germ cells

Primordial germ cells (PGCs) were obtained from fetal mouse gonads of both sexes days post coitum (dpc), either by collagenase treatment, or by mechanical procedures with or without prior EDTA treatment. With mechanical procedures alone, yield was relatively low and many of the cells released were dead. After EDTA treatment, both yield and viability were significantly improved. Collagenase treatment gave the best yield of cells, since the entire gonad was disaggregated, but contamination with somatic cells was substantial, and the adhesive properties of the germ cells were altered by the treatment. When cells released following EDTA treatment were fractionated on a simple Percoll gradient, several thousand viable PGCs per gonad could be obtained in 2–3 h, with not more than 10–20% somatic cell contamination.     

Serum-free culture of murine primordial germ cells and embryonic germ cells

Fetal calf serum (FCS) has usually been used for culture of embryonic stem (ES) cell as a component of the culture medium. However, FCS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. Recently, a chemically-defined serum replacement, Knockout Serum Replacement (KSR), was developed to maintain ES cells in an undifferentiated state. In this experiment, we examined the effects of KSR on the growth and differentiation of primordial germ cells (PGCs) and embryonic germ (EG) cells. PGCs were collected 8.5 days postcoitum (dpc) from B6D2F1 (C57BL/6JxDBA/2J) female mice mated with B6D2F1 males. Most of the PGCs that were cultured in FCS-supplemented medium (FCS medium) had alkaline phosphatase (AP) activity and acquired a fibroblast cell shape. In contrast, PGCs in KSR-supplemented medium (KSR medium) proliferated, maintaining round and stem cell-like morphology. In addition, EG cells were established more easily from PGCs cultured in KSR medium than from PGCs cultured in FCS medium. The percentage of undifferentiated colonies of EG cells was significantly higher in KSR medium than in FCS medium. The germ line chimera was also produced from EG cells established in KSR medium. These results suggest that KSR can be used for sustaining an undifferentiated state of PGCs and EG cells in vitro.     

Primordial germ cells in the mouse embryo during gastrulation

With the aid of a whole-mount technique, we have detected a small cluster of alkaline phosphatase (ALP)-positive cells in whole mounts of mid-primitive-streak-stage embryos, 7-7 1/4 days post coitum (dpc). Within the cluster, about 8 cells contain a small cytoplasmic spot, intensely stained for ALP activity and possibly associated with an active Golgi complex. The cluster lies just posterior to the definitive primitive streak in the extraembryonic mesoderm, separated from the embryo by the amniotic fold. Towards the end of gastrulation, the number of cells containing the ALP-positive spot rises to between 50 and 80. Thereafter the number of cells in the extraembryonic cluster declines, and similar cells start to be seen in the mesoderm of the primitive streak and then in the endoderm. At 8 dpc, about 125 ALP-stained cells are found, mainly in the hindgut endoderm and also at the base of the allantois, their appearance and location at this stage agreeing closely with previous reports on primordial germ cells (PGCs). Embryos from which the cluster area has been removed at the 7-day stage are devoid of PGCs after culture for 48 h, whereas the excised tissue is rich in PGCs. We argue that the cells in the cluster are indeed primordial germ cells, at a stage significantly earlier than any reported previously. This would indicate that the PGC lineage in the mouse is set aside at least as early as 7 dpc, possibly as one of the first 'mesodermal' cell types to emerge, and that its differentiation, as expressed by ALP activity, is gradual.     

Immunomagnetic isolation of primordial germ cells and the establishment of embryonic germ cell lines in the mouse

The stage-specific embryonic antigen 1 (SSEA-1) is a cell marker of primordial germ cells (PGCs). In the present study, it is shown that isolation and purification of PGCs from 8.5-11.5 days post coitum (dpc) embryos can be achieved by a immunomagnetic cell sorting method using SSEA-1 antibody-conjugated magnetic beads, and then the sorted PGCs can be used for long-term culture under strict culture conditions to derive embryonic germ (EG) cell lines. Five independent EG cell lines with male karyotypes have been established. They show both a strong alkaline phosphatase activity and expression of the SSEA-1 antigen, and are karyotypically stable with a modal number of chromosomes in more than 80% of the cells. One of the EG cell lines from 8.5-dpc embryos produced chimeras after injections of the cells into 8-cell host embryos. These procedures could provide a useful and simple method for isolation of undifferentiated cells from a heterogeneous cell population and for establishment of embryo-derived stem cell lines.     

In vitro adhesion of mouse fetal germ cells to extracellular matrix components

Mouse primordial germ cells (PGCs) isolated from the dorsal mesentery and gonadal ridges of 10.5–12.5 days post coitum (dpc) embryos showed a progressively increasing adhesiveness to laminin and fibronectin coated substrates, whereas type I collagen and various glycosaminoglycans (hyaluronic acid, heparin and chondroitinsulphates) were poor adhesive substrates. At later stages germ cells appeared to lose their adhesiveness to fibronectin and laminin substrates; the ability to adhere to laminin decreased very rapidly in male and slowly in female germ cells. Oocytes and prospermatogonia from 15.5 dpc fetal gonads showed poor adhesiveness to all substrates tested. PGC adhesion to laminin and fibronectin substrates did not require calcium but was markedly trypsin sensitive. Antibodies against the fibronectin receptor of CHO fibroblasts and short peptides containing the Arg-Gly-Asp sequence greatly reduced PGC adhesion to fibronectin. Following adhesion to laminin or fibronectin, most PGCs did not exhibit a morphology typical of motile cells, but remained spherical. A significant proportion (about 30%) of oocytes from 13.5–14.5 dpc embryos appeared, however, able to spread and elongate following attachment to laminin. The results support the hypothesis that mouse PGCs may utilize laminin and/or fibronectin as adhesive substrates during migration and gonad colonization, but indicate that additional factors are probably required to promote PGC motility. In addition, our data provide indirect evidence that binding sites for specific components of extracellular matrix are present in PGCs, and that their expression may be developmentally regulated.     

Isolation and identification of germ cells from fetal bovine ovaries

Gonadal cell suspensions were made from bovine fetuses of 35–55-, 56–80-, and 80–130-day age groups corresponding to the periods predominated by primordial germ cells (PGCs), oogonia, and meiotic cells, respectively. Germ cells identified on morphological criteria prior to their isolation from suspensions were compared histochemically and morphologically with cells in cryosections, impression smears, and semithin sections of similar gonads. Oocytes were distinguished by their chromosomal configurations in cell spreads. In suspensions from 35–55-day fetuses, cells considered to be PGCs stood out by their size, large nucleus, intracytoplasmic vesicles, and occasional blebbing. The somatic cells were smaller and contained little cytoplasm and few vesicles. In bovine gonads, in contrast to murine gonads, alkaline phosphatase (AP) activity was not specific enough to identify germ cells once they had entered the gonad. In ovaries from the 56–80-day age group, cells similar to PGCs, but slightly larger and with more cytoplasmic vesicles, were identified as oogonia. The cytoplasmic vesicles stained positively for lipid. In ovaries of 80–130-day fetuses, oogonia, oocytes, degenerating germ cells, and multinucleate germ cells were recognized. Degenerating germ cells exhibited a variety of morphological characteristics and were consistently positive for acid-phosphatase activity. Binucleate germ cells appeared around day 85 of gestation, while multinucleate germ cells were seen from day 95. It was concluded that bovine mitotic germ cells can be isolated from gonadal cell suspensions and that the best time to recover them is between 50 and 70 days of gestation. © 1994 Wiley-Liss, Inc.     

Male differentiation of germ cells induced by embryonic age-specific Sertoli cells in mice

Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells.     

Ultrastructural and morphometric studies related to expression of the cell adhesion molecule PECAM-1/CD31 in developing rat lung.

It has recently been postulated that platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) might play a role in vascular tube formation. To evaluate the role of PECAM-1/CD31 in the formation of the capillary network in vivo, we conducted an ultrastructural immunohistochemical evaluation of the localization of PECAM-1/CD31 and its developmentally regulated expression in the periphery of the lungs of fetal, newborn, and adult rats. PECAM-1/CD31 was present mainly on luminal surfaces and at the junctions between endothelial cells. Moreover, in fetal lung, products of the immunoreaction were also found on the abluminal surfaces of endothelial cells. To relate those findings to the developmental changes in the capillary area of the lung, we performed a morphometric study of electronmicrographs. The cross-sectional area of blood vessels at the periphery of the lungs was significantly greater in 15-19-day-old fetuses than in postpartum animals (p<0.0001). Disappearance of the expression of PECAM-1/CD31 on the abluminal endothelial surface paralleled the changes in the cross-sectional area of blood vessels that occurred during the perinatal period. (J Histochem Cytochem 48:1283-1289, 2000)     

PECAM-1/CD31 trans-homophilic binding at the intercellular junctions is independent of its cytoplasmic domain; evidence for heterophilic interaction with integrin alphavbeta3 in Cis

PECAM-1/CD31 is a cell adhesion and signaling molecule that is enriched at the endothelial cell junctions. Alternative splicing generates multiple PECAM-1 splice variants, which differ in their cytoplasmic domains. It has been suggested that the extracellular ligand-binding property, homophilic versus heterophilic, of these isoforms is controlled by their cytoplasmic tails. To determine whether the cytoplasmic domains also regulate the cell surface distribution of PECAM-1 splice variants, we examined the distribution of CD31-EGFPs (PECAM-1 isoforms tagged with the enhanced green fluorescent protein) in living Chinese hamster ovary cells and in PECAM-1-deficient endothelial cells. Our results indicate that the extracellular, rather than the cytoplasmic domain, directs PECAM-1 to the cell-cell borders. Furthermore, coculturing PECAM-1 expressing and deficient cells along with transfection of CD31-EGFP cDNAs into PECAM-1 deficient cells reveal that this PECAM-1 localization is mediated by homophilic interactions. Although the integrin alphavbeta3 has been shown to interact with PECAM-1, this trans-heterophilic interaction was not detected at the borders of endothelial cells. However, based on cocapping experiments performed on proT cells, we provide evidence that the integrin alphavbeta3 associates with PECAM-1 on the same cell surface as in a cis manner.     

Construction and expression of an enzymatically active form of PECAM-1 containing the phosphatase domain of the protein tyrosine phosphatase, SHP-2

Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is a 130-kDa transmembrane glycoprotein that is expressed on the surfaces of platelets, endothelial cells, and certain leukocyte subsets. The extracellular region of PECAM-1 contains six immunoglobulin homology domains, two of which (domains 1 and 2) mediate PECAM-1 homophilic interactions. Recent evidence suggests that a major function of the extracellular region of PECAM-1 is to determine its localization within the plane of the plasma membrane. The cytoplasmic domain of PECAM-1 contains an immunoreceptor tyrosine-based inhibitory motif that, upon tyrosine phosphorylation, supports recruitment of the Src homology 2 domain-containing protein tyrosine phosphatase, SHP-2. However, neither the targets of this PECAM-1/SHP-2 complex nor the significance of localizing SHP-2 to the borders of opposing PECAM-1-expressing cells is yet known. As a first step in addressing these issues, we designed a cDNA encoding a chimeric protein composed of the PECAM-1 extracellular domain fused to the phosphatase domain of SHP-2, which we call PECAM-1/PhD2. When immunopurified from stably transfected HEK293 cell lines expressing this recombinant protein, PECAM-1/PhD2 was found to possess constitutive enzymatic activity and appropriate border localization. This constitutively active chimeric protein will be useful in future studies designed to define the components of signal transduction pathways modulated by PECAM-1/SHP-2 signaling complexes.     

小鼠生殖细胞的胚胎发育

本文综述了近年来小鼠胚胎发育过程中生殖细胞的起源、迁移与增殖、性别分化及其基因组修饰等方面的研究进展。小鼠生殖细胞在7~7.5dpc时由原始生殖细胞（PGC）演变而来,至12.5dpc时PGC全部迁移进入生殖嵴,到13.5dpc时停止分裂。Steel/c-kit信号途径在PGC迁移过程中起重要作用。生殖细胞的性别主要是由生殖腺中体细胞的微环境决定的。Y染色体上存在精子形成所必需的基因。生殖细胞的全基因组范围的重新甲基化晚于胚胎体细胞的重新甲基化,到18.5dpc时才完成。雌性生殖细胞的X染色体重新活化在14.5~15.5dpc时完成,并且与生殖嵴的性别分化无关。 Abstract:This paper reviewed the recent progress of the origin,migration and proliferation,sex determination,and genomic modification of murine germ cells during its embryonic development. Murine germ cells originate from primordial germ cells at about 7~7.5dpc. Then PGCs migrated into germinal ridge at about 12.5dpc during which Steel/c-kit signal pathway plays important roles and stopped division at 13.5dpc. The sex of germ cells was mainly determined by the soma microenvironment in the gonad. And there are essential genes for sperm formation on the Y chromosome. The de novo methylation of murine germ cells was much later than soma cells and was completed at about 18.5dpc. The X chromosome reactivation of female germ cells was finished at about 14.5~15.5dpc which was independent of sexual differentiation of germinal ridge.     

Reactive oxygen species induce reversible PECAM-1 tyrosine phosphorylation and SHP-2 binding

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) functions to control the activation and survival of the cells on which it is expressed. Many of the regulatory functions of PECAM-1 are dependent on its tyrosine phosphorylation and subsequent recruitment of the Src homology (SH2) domain containing protein tyrosine phosphatase SHP-2. The recent demonstration that PECAM-1 tyrosine phosphorylation occurs in cells exposed to the reactive oxygen species hydrogen peroxide (H2O2) suggested that this form of oxidative stress may also support PECAM-1/SHP-2 complex formation. In the present study, we show that PECAM-1 tyrosine phosphorylation in response to exposure of cells to H2O2 is reversible, involves a shift in the balance between kinase and phosphatase activities, and supports binding of SHP-2 and recruitment of this phosphatase to cell-cell borders. We speculate, however, that the unique ability of H2O2 to reversibly oxidize the reactive site cysteine residues of protein tyrosine phosphatases may result in transient inactivation of the SHP-2 that is bound to PECAM-1 under these conditions. Finally, we provide evidence that PECAM-1 tyrosine phosphorylation and SHP-2 binding in endothelial cells requires exposure to an "oxidative burst" of H2O2, but that exposure of these cells to sufficiently high concentrations of H2O2 for a sufficiently long period of time abrogates binding of SHP-2 to tyrosine-phosphorylated PECAM-1. These findings support a role for PECAM-1 as a sensor of oxidative stress, perhaps most importantly during the process of inflammation.