Human primordial germ cell-derived progenitors give rise to neurons and glia in vivo

We derived a cell population from cultured human primordial germ cells from early human embryos. The derivates, termed embryoid body-derived (EBD) cells, displayed an extensive capacity for proliferation and expressed a panel of markers in all three germ layers. Interestingly, EBD cells were also positive for markers of neural stem/progenitor cells, such as nestin and glial fibrillary acidic protein. When these cells were transplanted into the brain cavities of fetal sheep and postnatal NOD-SCID mice or nerve-degenerated tibialis anterior muscles, they readily gave rise to neurons or glial cells. To our knowledge, our data are the first to demonstrate that EBD cells can undergo further neurogenesis under suitable environments in vivo. Hence, with the abilities of extensive expansion, self-renewal, and differentiation, EBD cells may provide a useful donor source for neural stem/progenitor cells to be used in cell-replacement therapies for diseases of the nervous system.     

Skeletal myogenesis by human primordial germ cell-derived progenitors

We have isolated and cultured human primordial germ cells (PGCs) from early embryos. The PGCs expressed embryonic germ (EG) cell-specific surface markers, including Oct4 and Nanos. We derived a cell population from these PGCs that we termed embryoid body-derived (EBD) cells. EBD cells can be extensively expanded in vitro for more than 50 passages and express lineage markers from all three primary germ layers. The myogenic potential of the EBD cells was examined both in vitro and in vivo.In vitro, the EBD cells can be induced to form multinucleated myotubes, which express late skeletal muscle-specific markers, including MHC and dystrophin, when exposed to human galectin-1. In vivo, the EBD cells gave rise to all the myogenic lineages, including the skeletal muscle stem cells known as satellite cells. Strikingly, these cells were able to partially restore degenerated muscles in the SCID/mdx mouse, an animal model of the Duchenne’s muscular dystrophy. These results indicate the EBD cells may be a promising source of myogenic stem cells for cell-based therapies for muscle degenerative disorders.     

Differentiation of murine premigratory primordial germ cells in culture.

In the mouse embryo, primordial germ cells first appear in the extraembryonic mesoderm and divide rapidly while migrating to the fetal gonad. Shortly after their arrival in the gonad, germ cells sexually differentiate as proliferation ceases. Previous studies have established that primordial germ cells proliferate and migrate in feeder layer culture. To explore cellular regulation of fetal germ cell development, we have used germ cell nuclear antigen 1 (GCNA1), a marker normally expressed only in postmigratory germ cells, to investigate the developmental potency of both pre- and postmigratory cells in this culture system. We found that explanted premigratory germ cells will initiate expression of this marker and are, therefore, capable of undertaking some aspects of gonocyte differentiation without intimate exposure to the fetal gonad. We have also tested whether postmigratory gonocytes are stable in culture. As detected by either alkaline phosphatase or GCNA1, we did not detect long-term survival of either prospermatogonia or oogonia under conditions that support the survival, proliferation, and differentiation of earlier premigratory cells. These observations are consistent with an autonomous cellular mechanism governing the initial stages of gonocyte differentiation, and suggest that differentiation towards gonocytes is accompanied by a change in requirements for cell survival.     

Cytogenetic analysis of reconstituted one-cell mouse embryos derived from nuclear transfer of fetal male germ cells.

Micromanipulation techniques were used to produce reconstituted one-cell mouse embryos after the fusion of fetal male germ cells 15.5 day post coitum with enucleated secondary oocytes. At this stage of development, male fetal germ cells are arrested at G1 of mitotic interphase. Two distinct populations of germ cells, differing in size and ploidy, were isolated from the genital ridge of a mid-term fetus. Oocytes that had received male germ cells from the population of smaller (mononuclear) germ cells developed as diploid one-cell reconstituted embryos. When the same procedures were used to produce reconstituted one-cell embryos using male fetal germ cells from a population of larger (multinucleate) cells, they exhibited ploidy of either 4x, 6x or 8x at metaphase of the first cell division. Although most reconstituted embryos (90 and 96%) developed to the two-cell stage, the proportion of embryos receiving small germ cells developed to blastocysts was much higher (62%) than that receiving large germ cells (4%). These studies indicate that not all fetal germ cells are diploid before the onset of meiosis and have identified procedures to produce reconstituted embryos from fetal germ cells that do not carry genome or chromosome anomalies.     

Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells

The development of germ cells is a highly ordered process that begins during fetal growth and is completed in the adult. Epigenetic modifications that occur in germ cells are important for germ cell function and for post-fertilization embryonic development. We have previously shown that male germ cells in the adult mouse have a highly distinct epigenetic state, as revealed by a unique genome-wide pattern of DNA methylation. Although it is known that these patterns begin to be established during fetal life, it is not known to what extent DNA methylation is modified during spermatogenesis. We have used restriction landmark genomic scanning (RLGS) and other techniques to examine DNA methylation at multiple sites across the genome during postnatal germ cell development in the mouse. Although a significant proportion of the distinct germ cell pattern is acquired prior to the type A spermatogonial stage, we find that both de novo methylation and demethylation occur during spermatogenesis, mainly in spermatogonia and spermatocytes in early meiotic prophase I. Alterations include predominantly non-CpG island sequences from both unique loci and repetitive elements. These modifications are progressive and are almost exclusively completed by the end of the pachytene spermatocyte stage. These studies better define the developmental timing of genome-wide DNA methylation pattern acquisition during male germ cell development.     

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.     

Autonomous regulation of sex-specific developmental programming in mouse fetal germ cells

In mice, unique events regulating epigenetic programming (e.g., genomic imprinting) and replication state (mitosis versus meiosis) occur during fetal germ cell development. To determine whether these processes are autonomously programmed in fetal germ cells or are dependent upon ongoing instructive interactions with surrounding gonadal somatic cells, we isolated male and female germ cells at 13.5 days postcoitum (dpc) and maintained them in culture for 6 days, either alone or in the presence of feeder cells or gonadal somatic cells. We examined allele-specific DNA methylation in the imprinted H19 and Snrpn genes, and we also determined whether these cells remained mitotic or entered meiosis. Our results show that isolated male germ cells are able to establish a characteristic "paternal" methylation pattern at imprinted genes in the absence of any support from somatic cells. On the other hand, cultured female germ cells maintain a hypomethylated status at these loci, characteristic of the normal "maternal" methylation pattern in endogenous female germ cells before birth. Further, the surviving female germ cells entered first meiotic prophase and reached the pachytene stage, whereas male germ cells entered mitotic arrest. These results indicate that mechanisms controlling both epigenetic programming and replication state are autonomously regulated in fetal germ cells that have been exposed to the genital ridge prior to 13.5 dpc.     

Continuous loss of oocytes throughout meiotic prophase in the normal mouse ovary

The number of germ cells reaches the maximum just prior to entry into meiosis, yet decreases dramatically by a few days after birth in the female mouse, rat, and human. Previous studies have reported a major loss at the pachytene stage of meiotic prophase during fetal development, leading to the hypothesis that chromosomal pairing abnormalities may be a signal for oocyte death. However, the identification as well as the quantification of germ cells in these studies have been questioned. A recent study using Mouse Vasa Homologue (MVH) as a germ cell marker reached a contradictory conclusion claiming that oocyte loss occurs in the mouse only after birth. In the present study, we established a new method to quantify murine germ cells by using Germ Cell Nuclear Antigen-1 (GCNA-1) as a germ cell marker. Comparison of GCNA-1 and MVH immunolabeling revealed that the two markers identify the same population of germ cells. However, nuclear labeling of GCNA-1 was better suited for counting germ cells in histological sections as well as for double labeling with the antibody against synaptonemal complex (SC) proteins in chromosome spreading preparations. The latter experiment demonstrated that the majority of GCNA-1-labeled cells entered and progressed through meiotic prophase during fetal development. The number of GCNA-1-positive cells in the ovary was estimated by counting the labeled cells retained in chromosome spreading preparations and also in histological sections by using the ratio estimation method. Both methods demonstrated a continuous decline in the number of GCNA-1-labeled cells during fetal development when the oocytes progress through meiotic prophase. These observations suggest that multiple causes are responsible for oocyte elimination.     

Uncovering gene regulatory networks during mouse fetal germ cell development

The commitment of germ cells to either oogenesis or spermatogenesis occurs during fetal gonad development: germ cells enter meiosis or mitotic arrest, depending on whether they reside within an ovary or a testis, respectively. Despite the critical importance of this step for sexual reproduction, gene networks underlying germ cell development have remained only partially understood. Taking advantage of the W(v) mouse model, in which gonads lack germ cells, we conducted a microarray study to identify genes expressed in fetal germ cells. In addition to distinguishing genes expressed by germ cells from those expressed by somatic cells within the developing gonads, we were able to highlight specific groups of genes expressed only in female or male germ cells. Our results provide an important resource for deciphering the molecular pathways driving proper germ cell development and sex determination and will improve our understanding of the etiology of human germ cell tumors that arise from dysregulation of germ cell differentiation.     

Immunohistochemical profiling of germ cells within the human fetal testis: identification of three subpopulations

In the human fetal testis, germ cells that have migrated to the genital ridges become enclosed within testicular cords by 8 wk of gestation. Most papers refer to all types of germ cells as being "gonocytes" or "prespermatogonia," giving the impression that they are identical. Detailed morphological studies, however, have suggested a heterogeneous population. We have used single, double, and triple immunohistochemistry to evaluate the differentiation of cells within fetal testes recovered during the first (7-9 wk) and second (14-19 wk) trimesters. In the first trimester, differentiation of Sertoli cells preceded the formation of testicular cords and the differentiation of interstitial (Leydig, peritubular myoid) cells. Immunostaining for CHK2, C-KIT, placental alkaline phosphatase, PCTAIRE-1, and MAGE-A4 revealed that the proportion of germ cells expressing each of these proteins was correlated with gestational age. Expression of the pluripotency marker OCT4 was restricted to a population of small, round germ cells. Three types of germ cell were identified, and we propose that these should be known as gonocytes (OCT4pos/C-KITpos/MAGE-A4neg), intermediate germ cells (OCT4low/neg/C-KITneg/MAGE-A4neg), and prespermatogonia (OCT4neg/C-KITneg/MAGE-A4pos). In the first trimester, most germ cells had a gonocyte phenotype; however, from 18 wk of gestation, prespermatogonia were the most abundant cell type. These data provide evidence for the functional differentiation of human testicular germ cells during the second trimester of pregnancy, and they argue against these germ cells being considered as a homogeneous population, as in rodents.     

Mouse germ cell clusters form by aggregation as well as clonal divisions

After their arrival in the fetal gonad, mammalian germ cells express E-cadherin and are found in large clusters, similar to germ cell cysts in Drosophila. In Drosophila, germ cells in cysts are connected by ring canals. Several molecular components of intercellular bridges in mammalian cells have been identified, including TEX14, a protein required for the stabilization of intercellular bridges, and several associated proteins that are components of the cytokinesis complex. This has led to the hypothesis that germ cell clusters in the mammalian gonad arise through incomplete cell divisions. We tested this hypothesis by generating chimeras between GFP-positive and GFP-negative mice. We show that germ cell clusters in the fetal gonad arise through aggregation as well as cell division. Intercellular bridges, however, are likely restricted to cells of the same genotype.     

Expression of Fas,p53 and AFP in development of human fetal germ cells in vitro

In the present study we employed a two-step culture system to study the expression of Fas, p53 and alpha-fetoprotein (AFP) in the development in vitro of human fetal germ cells. p53 mRNA was determined by Northern blotting, and Fas content was assessed by western blotting. RT-nested polymerase chain reaction (RT-nPCR) analysis was performed to determine the expression of AFP mRNA in different stages of fetal follicular development. Follicular cell apoptosis was evaluated by DNA fragmentation analyses (DNA ladder). The results showed that by day 7 of culture approximately one-sixth of fetal germ cells grew to class C oocytes (primary oocytes) from class B oocytes (primordial oocytes) or class A oocytes. On day 45 of culture, one-third of these primary follicles doubled in size. In the meantime, there was a high proportion apoptosis of follicular cells on days 35 or 45 of culture, as evident by a clear ladder pattern of DNA fragmentation upon electrophoretic analysis. Expression of Fas antigen and p53 mRNA increased in a time-dependent manner, while AFP mRNA was expressed on days 10 to 35, and disappeared on day 45. These results indicate that human fetal germ cells can develop in a two-step culture system and AFP may play an active role in the proliferation of these germ cells. At the late stage of follicular development in vitro, a number of follicular cells became apoptotic. Moreover, apoptosis may be the mechanism responsible for fetal germ cell regression and the Fas antigen and/or p53-mediated death pathway may be central in the induction of germ cell regression.     

Mitotic arrest in teratoma susceptible fetal male germ cells

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27(KIP1), p15(INK4B), activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility.     

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63γ expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.     

Effects of laminin, fibronectin and type IV collagen on liver cell cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.     

New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.     

A role for E-cadherin in mouse primordial germ cell development

In this study we show that mouse primordial germ cells and fetal germ cells at certain stages of differentiation express E-cadherin and alpha and beta catenins. Moreover, we demonstrate that the formation of germ cell aggregates that rapidly occurs when monodispersed germ cell populations are released from embryonic gonads in culture is E-cadherin mediated, developmentally regulated, and dependent on the sex of the germ cells. Immunoblotting analyses indicate that the lower ability to form aggregates of primordial germ cells in comparison to fetal germ cells is not due to gross changes in E-cadherin expression, altered association with beta catenin, or changes in beta catenin phosphorylation. Investigating possible functions of E-cadherin-mediated adhesion in primordial germ cell development, we found that E-cadherin-mediated adhesion may stimulate the motility of primordial germ cells. Moreover, treatment of primordial germ cells cultured on STO cell monolayers with an anti-E-cadherin antibody caused a significant decrease in their number and markedly reduced their ability to form colonies in vitro. The same in vitro treatment of explanted undifferentiated gonadal ridges cultured for 4 days results in decreased numbers and altered localization of the germ cell inside the gonads. Taken together these results suggest that E-cadherin plays an important role in primordial germ cell migration and homing and may act as a modulator of primordial germ cell development.