Germ cell binding to rat Sertoli cells in vitro

The interaction between male germ cells and Sertoli cells was studied in vitro by co-incubation experiments using isolated rat germ cells and primary cultures of Sertoli cells made germ cell-free by the differential sensitivity of germ cells to hypotonic shock. The germ cell/Sertoli cell interaction was examined morphologically with phase-contrast and scanning electron microscopy and then quantified by measuring radioactivity bound to Sertoli cell cultures after co-incubation with added [3H]leucine-labeled germ cells. Germ cell binding to Sertoli cell cultures was the result of specific adhesion between these two cell types, and several features of this specific adhesion were observed. First, germ cells adhered to Sertoli cell cultures under conditions during which spleen cells and red blood cells did not. Second, germ cells had a greater affinity for Sertoli cell cultures than they had for cultures of testicular peritubular cells or cerebellar astrocytes. Third, germ cells fixed with paraformaldehyde adhered to live Sertoli cultures while similarly fixed spleen cells adhered less tightly. Neither live nor paraformaldehyde-fixed germ cells adhered to fixed Sertoli cell cultures. Fourth, germ cell binding to Sertoli cell cultures was not immediate but increased steadily and approached a maximum at 4 h of co-incubation. Saturation of germ cell binding to Sertoli cell cultures occurred when more than 4200 germ cells were added per mm2 of Sertoli cell culture surface. Finally, germ cell binding to Sertoli cell cultures was eliminated when co-incubation was performed on ice. Based on these observations, we concluded that germ cell adhesion to Sertoli cells was specific, temperature-dependent, and required a viable Sertoli cell but not necessarily a viable germ cell. These results have important implications for understanding the complex interaction between Sertoli cells and germ cells within the seminiferous tubule and in the design of future experiments probing details of this interaction.     

On the formation of germ cells: The good,the bad and the ugly

Mammalian germ cells are powerful cells, the only ones that transmit information to the next generation ensuring the continuation of the species. But “with great power, comes great responsibility”, meaning that germ cells are only a few steps away from turning carcinogenic. Despite recent advances little is known about germ cell formation in mammals, predominantly because of the inaccessibility of these cells. Moreover, it is difficult to pin down what in essence is characteristic of a germ cell, as germ cells keep changing place, morphology, expression markers and epigenetic identity. Formation of (primordial) germ cells in primate ES cell cultures would therefore be helpful to identify molecular signalling pathways associated with germ cell differentiation and to study epigenetic changes in germ cells. In addition, the in vitro derivation of functional germ cells from ES cells could be used in combination with therapeutic cloning to generate patient-specific ES cell lines, and can have applications in animal breeding. In this review we present the state-of-the-art on how mouse and human germ cells are formed in vivo (the good), we discuss the link between germ cells, pluripotency and germ cell tumours (the bad) and show that despite continuous progress in trying to differentiate germ cells in vitro (the ugly) the generation of functional germ cells is still a real challenge.     

Proliferation of germ cells during gonadal sex differentiation in medaka: Insights from germ cell-depleted mutant zenzai

The proliferation of germ cells becomes sexually dimorphic during gonadal sex differentiation, although the underlying dynamics of this are not well understood in vertebrates. By tracing GFP-labeled germ cells in vivo and analyzing the germ cell-depleted mutant, zenzai, we show that the proliferation and differentiation of germ cells are regulated in a sexually dimorphic manner in the teleost fish medaka. In the undifferentiated gonads, germ cells resume proliferation by slow intermittent division (type I), producing isolated daughter cells. While germ cells in the male gonads continue this mode of proliferation, some germ cell fractions in the female gonads initiate two to four rounds of continuous division (type II), forming cysts of four, eight, or sixteen cells, which subsequently enter meiosis synchronously. Thus, female germ cells become differentiated much earlier than do male germ cells. In the zenzai mutant, a defect in slow intermittent division eventually leads to the depletion of germ cells in the adult gonads in both sexes, despite the fact that cyst-forming division is unaffected. This argues that slow intermittent division is essential for the maintenance of germ cells. The proliferation and differentiation of germ cells are thus important components of gonadal sex differentiation in vertebrates.     

Cell-autonomous and inductive signals can determine the sex of the germ line of drosophila by regulating the gene Sxl

To investigate the mechanism of sex determination in the germ line, we analyzed the fate of XY germ cells in ovaries, and the fate of XX germ cells in testes. In ovaries, germ cells developed according to their X:A ratio, i.e., XX cells underwent oogenesis, XY cells formed spermatocytes. In testes, however, XY and XX germ cells entered the spermatogenic pathway. Thus, to determine their sex, the germ cells of Drosophila have cell-autonomous genetic information, and XX cells respond to inductive signals of the soma. Results obtained with amorphic and constitutive mutations of Sxl show that both the genetic and the somatic signals act through Sxl to achieve sex determination in germ cells.     

Signaling from germ cells mediated by the rhomboid homolog stet organizes encapsulation by somatic support cells

Germ cells normally differentiate in the context of encapsulating somatic cells. However, the mechanisms that set up the special relationship between germ cells and somatic support cells and the signals that mediate the crucial communications between the two cell types are poorly understood. We show that interactions between germ cells and somatic support cells in Drosophila depend on wild-type function of the stet gene. In males, stet acts in germ cells to allow their encapsulation by somatic cyst cells and is required for germ cell differentiation. In females, stet function allows inner sheath cells to enclose early germ cells correctly at the tip of the germarium. stet encodes a homolog of rhomboid, a component of the epidermal growth factor receptor signaling pathway involved in ligand activation in the signaling cell. The stet mutant phenotype suggests that stet facilitates signaling from germ cells to the epidermal growth factor receptor on somatic cells, resulting in the encapsulation of germ cells by somatic support cells. The micro-environment provided by the surrounding somatic cells may, in turn, regulate differentiation of the germ cells they enclose.     

The distribution and behavior of extragonadal primordial germ cells in Bax mutant mice suggest a novel origin for sacrococcygeal germ cell tumors

In the mouse, germ cells that do not reach the genital ridges rapidly die by a wave of apoptosis that requires the pro-apoptotic protein Bax. In Bax-null embryos, large numbers of ectopic (extragonadal) germ cells fail to die. We have studied the fates of these, in an effort to understand the etiology of human extragonadal germ cell tumors, which are thought to arise from ectopic germ cells. We find that ectopic germ cells in which apoptosis is blocked form a heterogeneous population, which partially differentiates along the gonocyte pathway to different extents in different regions of the embryo, and in the two genders. In particular, a previously undescribed population of ectopic germ cells was identified in the tail. These germ cells retained primitive markers for longer than ectopic germ cells in other regions, and represent a possible origin for sacrococcygeal type I extragonadal germ cell tumors found in neonates and infants. This hypothesis is supported, but not proved, by the finding of cells expressing the germ cell marker Oct4 associated with a coccygeal germ cell tumor in a human infant.     

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.     

Cross talk between germ cells and gonadal somatic cells is critical for sex differentiation of the gonads in the teleost fish, medaka (Oryzias latipes)

To evaluate the possible role of germ cells on sex differentiation of the gonads in vertebrates, the teleost fish, medaka ( Oryzias latipes ), was used to generate a gonad without germ cells. The germ cell-deficient medaka reveals multiple effects of germ cells on the process of sex differentiation. The previously isolated mutant medaka, hotei , with the excessive number of germ cells may support the contention that the proliferation of germ cells is related to feminization of the gonad. Futhermore, we show that two modes of proliferation for either maintenance of germ cells or commitment to gametogenesis are important components of the sex differentiation of medaka developing gonads. An intimate cross talk between germ cells and gonadal somatic cells during the sex differentiation will be discussed.     

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.     

Intermolecular interactions of homologs of germ plasm components in mammalian germ cells

In some species such as flies, worms, frogs and fish, the key to forming and maintaining early germ cell populations is the assembly of germ plasm, microscopically distinct egg cytoplasm that is rich in RNAs, RNA-binding proteins and ribosomes. Cells which inherit germ plasm are destined for the germ cell lineage. In contrast, in mammals, germ cells are formed and maintained later in development as a result of inductive signaling from one embryonic cell type to another. Research advances, using complementary approaches, including identification of key signaling factors that act during the initial stages of germ cell development, differentiation of germ cells in vitro from mouse and human embryonic stem cells and the demonstration that homologs of germ plasm components are conserved in mammals, have shed light on key elements in the early development of mammalian germ cells. Here, we use FRET (Fluorescence Resonance Energy Transfer) to demonstrate that living mammalian germ cells possess specific RNA/protein complexes that contain germ plasm homologs, beginning in the earliest stages of development examined. Moreover, we demonstrate that, although both human and mouse germ cells and embryonic stem cells express the same proteins, germ cell-specific protein/protein interactions distinguish germ cells from precursor embryonic stem cells in vitro; interactions also determine sub-cellular localization of complex components. Finally, we suggest that assembly of similar protein complexes may be central to differentiation of diverse cell lineages and provide useful diagnostic tools for isolation of specific cell types from the assorted types differentiated from embryonic stem cells.     

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.     

Primordial germ cells of the rabbit are specifically recognized by a monoclonal antibody labelling the perimitochondrial cytoplasm

 Instrumental for studies investigating the development of germ cells, and especially the separation of the germline in the early embryo, are molecular markers which reliably label germ cells and with which regulative factors of germ cell development may be analyzed. Here, we describe the monoclonal antibody PG-2, which is highly specific for the germ cells of the rabbit embryo and labels the perimitochondrial cytoplasm, as demonstrated by immunogold-silver staining. Identical expression patterns are found in germ cells of either sex from early organogenesis at 10 days post-conception (d.p.c.), when the germ cells leave the hindgut epithelium and settle in the gonadal anlage as primordial germ cells (PGCs), until the time immediately prior to birth (30 d.p.c.), when germ cells are either in their oogonial or prospermatogonial state. The antibody is the first to recognize specifically a cytoplasmic epitope in germ cells of a higher vertebrate and may well recognize the mammalian equivalent of the germ plasm found in inverteb-rates and lower vertebrates. The antibody can be used for early identification of PGCs and may be of help in the elucidation of mammalian germ cell development towards the gonial stages of spermatogenesis and oogenesis. Accepted: 30 May 1997     

The roles of FGF signaling in germ cell migration in the mouse

Fibroblast growth factor (FGF) signaling is thought to play a role in germ cell behavior. FGF2 has been reported to be a mitogen for primordial germ cells in vitro, whilst combinations of FGF2, steel factor and LIF cause cultured germ cells to transform into permanent lines of pluripotent cells resembling ES cells. However, the actual function of FGF signaling on the migrating germ cells in vivo is unknown. We show, by RT-PCR analysis of cDNA from purified E10.5 germ cells, that germ cells express two FGF receptors: Fgfr1-IIIc and Fgfr2-IIIb. Second, we show that FGF-mediated activation of the MAP kinase pathway occurs in germ cells during their migration, and thus they are potentially direct targets of FGF signaling. Third, we use cultured embryo slices in simple gain-of-function experiments, using FGF ligands, to show that FGF2, a ligand for FGFR1-IIIc, affects motility, whereas FGF7, a ligand for FGFR2-IIIb, affects germ cell numbers. Loss of function, using a specific inhibitor of FGF signaling, causes increased apoptosis and inhibition of cell shape change in the migrating germ cells. Lastly, we confirm in vivo the effects seen in slice cultures in vitro, by examining germ cell positions and numbers in embryos carrying a loss-of-function allele of FGFR2-IIIb. In FGFR2-IIIb(-/-) embryos, germ cell migration is unaffected, but the numbers of germ cells are significantly reduced. These data show that a major role of FGF signaling through FGFR2-IIIb is to control germ cell numbers. The data do not discriminate between direct and indirect effects of FGF signaling on germ cells, and both may be involved.     

Development of the larval ovary in the moth, Plodia interpunctella

The morphogenesis of ovaries and the organization of germ cells within them were visualized during the larval stages of the moth, Plodia interpunctella. The germ cells were observed by utilizing confocal microscopy coupled with immuno-fluorescent staining for the alpha-crystallin protein 25 (alphaCP25). The alphaCP25 was previously shown to be specific to germ cells of pupae and adults, and this study shows that alphaCP25 is present in larval germ cells as well. A cluster of 28 germ cells that stain for alphaCP25 was found in the gonads of newly hatched first instar larvae. The founding germ cells became segregated into four clusters, most likely by somatic cell intrusion, around the beginning of the second instar. Division of the primary germ cells began by the end of the second instar and the formation of all cystoblasts appeared to be completed within the four ovarioles by the end of the third instar. Within the ovarioles of third instar larvae, the germ cells were organized with a distal cap of seven germ cells which was segregated from the majority of the germ cells. The main body of germ cells was arranged around a central germ cell-free core as a spiral. Divisions of the cystoblasts to form cystocyte clusters were nearly completed during the fourth (last) larval instar. These features suggest that the strategy to produce follicles in moths is fundamentally different from the fruitfly, Drosophila. It appears that during the initial stages of ovary development in P. interpunctella, the primary germ cells undergo stage-complete divisions that are completed prior to the onset of the next set of divisions, which results in a complete complement of follicles available by the time of adult eclosion, while in Drosophila the primary germ cell divisions are initiated in the adult stage, and follicles are produced individually as resources are available.     

Selective loss of Sertoli cell and germ cell function leads to a disruption in sertoli cell-germ cell communication during aging in the Brown Norway rat

We investigated the effects of aging on Sertoli cell-germ cell interactions from Brown Norway rats using the induction of four specific mRNAs as markers. The testes from aging (24 mo old) Brown Norway rats can be normal size or regressed. One marker, a von Ebner's-like protein, is expressed in coculture and "in vivo" in germ cells from normal testes of 6- and 24-mo-old rats but not in germ cells from regressed testes of 24-mo-old rats. A second germ cell marker, the Huntington disease protein, is expressed in all germ cells. Two Sertoli cell markers, a serotonin receptor and a novel gene, are induced in Sertoli cells by meiotic germ cells. The serotonin receptor mRNA is expressed in Sertoli cells from 20-day, 6-mo, and 24-mo normal testes but not in those from 24-mo regressed testes. The novel gene is induced in Sertoli cells from all testes. We conclude that Sertoli cells from aged regressed testes are unable to respond to selective signals from germ cells from young rats, and germ cells from regressed testes show a similar selective loss. Such disruptions in communication between Sertoli cells and germ cells likely contribute to germ cell loss during aging.     

Continuing primordial germ cell differentiation in the mouse embryo is a cell-intrinsic program sensitive to DNA methylation

The initial cohort of mammalian gametes is established by the proliferation of primordial germ cells in the early embryo. Primordial germ cells first appear in extraembyronic tissues and subsequently migrate to the developing gonad. Soon after they arrive in the gonad, the germ cells cease dividing and undertake sexually dimorphic patterns of development. Male germ cells arrest mitotically, while female germ cells directly enter meiotic prophase I. These sex-specific differentiation events are imposed upon a group of sex-common differentiation events that are shared by XX and XY germ cells. We have studied the appearance of GCNA1, a postmigratory sex-common germ cell marker, in cultures of premigratory germ cells to investigate how this differentiation program is regulated. Cultures in which proliferation was either inhibited or stimulated displayed a similar extent of differentiation as controls, suggesting that some differentiation events are the result of a cell-intrinsic program and are independent of cell proliferation. We also found that GCNA1 expression was accelerated by agents which promote DNA demethylation or histone acetylation. These results suggest that genomic demethylation of proliferative phase primordial germ cells is a mechanism by which germ cell maturation is coordinated.     

Regulative germ cell specification in axolotl embryos: a primitive trait conserved in the mammalian lineage

How germ cells are specified in the embryos of animals has been a mystery for decades. Unlike most developmental processes, which are highly conserved, embryos specify germ cells in very different ways. Curiously, in mouse embryos germ cells are specified by extracellular signals; they are not autonomously specified by maternal germ cell determinants (germ plasm), as are the germ cells in most animal model systems. We have developed the axolotl (Ambystoma mexicanum), a salamander, as an experimental system, because classic experiments have shown that the germ cells in this species are induced by extracellular signals in the absence of germ plasm. Here, we provide evidence that the germ cells in axolotls arise from naive mesoderm in response to simple inducing agents. In addition, by analysing the sequences of axolotl germ-cell-specific genes, we provide evidence that mice and urodele amphibians share a common mechanism of germ cell development that is ancestral to tetrapods. Our results imply that germ plasm, as found in species such as frogs and teleosts, is the result of convergent evolution. We discuss the evolutionary implications of our findings.