Migratory and postmigratory mouse primordial germ cells behave differently in culture

In all vertebrate groups, the progenitors of the germ line, the primordial germ cells (PGCs) arise extragonadally and move to the developing gonad early in embryonic development. We have examined the behavior of isolated pregonadal and gonadal PGCs in vitro on feeder layers of an embryo-derived cell line. Histochemically and serologically identified pregonadal germ cells are found to be actively motile in vitro and, furthermore, show behavior characteristic of invasive cells. PGCs isolated from the developing gonad, however, show little locomotory activity and are not invasive on the same cellular substrate. These observations suggest that PGCs undergo a major change in phenotype at the time of their entry into the gonad anlagen.     

Evolution of germ cell development in tetrapods: comparison of urodeles and amniotes

SUMMARY. The embryonic development of germ cells in tetrapods is described, focusing on groups with the inductive mode of germ cell specification. In mammals PGCs are induced early in the gastrulation process, they are internalized with future extraembryonic mesoderm in the early posterior primitive streak, and specified soon thereafter. Strong evidence indicates that a similar process occurs in turtles and some other reptiles. In amniotes, the PGCs appear well before formation of the gonad in the posterior trunk, resulting in a period in which they are located outside the embryo before their migration to the gonad. In contrast, in urodeles the PGCs appear relatively late, and throughout development maintain a position close to precursors of the somatic cells of the gonad so that migration is not required. In lampreys early development of germ cells is strikingly similar to that in urodeles, suggesting this is the primitive process. As amniotes evolved large yolky eggs and better access to nutrition, development of the posterior half of the trunk became more dependent on cell proliferation; this was followed or accompanied by a shift of early germ cell development to the equivalent of the early primitive streak. A similar process may have occurred as some basal vertebrates developed large yolky eggs.     

Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells

In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.     

Tre1, a G Protein-Coupled Receptor, Directs Transepithelial Migration of Drosophila Germ Cells

In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.     

Development of the mammalian gonad: the fate of the supporting cell lineage

Sex determination in mammals is mediated via the supporting cell lineage in the fetal gonad. In the very early stages of gonadal development, the fate of the supporting cell population is critically dependent on the expression of the male-determining gene on the Y chromosome. If this gene is absent or fails to be expressed, or is expressed too late or in too small a number of supporting cells, all supporting cells (XX or XY) differentiate as pre-follicle cells and development proceeds along the female pathway. Supporting cells in which the male-determining gene is expressed in a timely manner differentiate as pre-Sertoli cells; given sufficient such cells, testis cords form and development proceeds in a male direction. If XX supporting cells are also present, a few may be recruited into the pre-Sertoli population and participate in testis cord formation. The subsequent fate of pre-follicle cells depends critically on interaction with the germ cell population in the developing gonad: absence of germ cells may lead to partial masculinization of the gonad, and/or to disappearance of the supporting cell component.     

Development and early differentiation of gonad in the european eel (Anguilla anguilla [L.], Anguilliformes,Teleostei): A cytological and ultrastructural study

The structure of the gonad of the European eel (Anguilla anguilla [L.]), an “undifferentiated” gonochoristic teleost, was investigated by transmission electron microscopy from 6–8 cm elvers to 22 cm yellow eels with juvenile hermaphroditic gonads. The pear-shaped gonads of 6–8 cm elvers assume, in 12–15 cm eels, a lamellar shape and enlarge by migration of germ cells, which we refer to as primary primordial germ cells. In the gonads of ∼ 16 cm eels, the primary primordial germ cells multiply, giving rise to clusters of germ cells that have ultrastructural characteristics of the primary primordial germ cells but show giant mitochondria, enlarged Golgi complexes, and round bodies not limited by membranes. We refer to these as secondary primordial germ cells. In 16–18 cm eels, syncytial clones of oogonia interconnected by cytoplasmic bridges are also observed. In 18–22-cm-long eels, the gonads contain primordial germ cells, oogonial clones, early oocyte cysts, single oocytes in early growth stages, and primary spermatogonia. Such germ cells are present in the same cross section where they are either intermingled or are in areas of predominantly female germ cells close to areas with predominantly male germ cells. These gonads are juvenile hermaphroditic and should be considered ambisexual because in larger eels they differentiate either into an ovary or into a testis. Somatic cells always envelop the germ cells following their migration into the gonad. These somatic cells first show similar ultrastructural features and then differentiate either into early Sertoli cells investing spermatogonia, or into early follicular (granulosa) cells investing the early previtellogenic oocytes. In eels ∼ 14 cm long, primitive steroid-producing cells also migrate into the gonad. In the ambisexual gonad they differentiate either into immature Leydig cells in the male areas, or into early special cells of the theca in the female areas. Nerve fibers are joined to the steroid-producing cells. Gonad development and differentiation are also associated with structural changes of the connective tissue characterized by the progressive appearance and deposition of collagen fibrils first in the mesogonadium, then in the gonad vascular region, and then in the germinal region. The collagen-rich areas are massive in the male areas and reduced in the female ones. J. Morphol. 231:195–216, 1997. © 1997 Wiley-Liss, Inc.     

Germ cells are not the primary factor for sexual fate determination in goldfish

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.     

Drosophila E-cadherin is essential for proper germ cell-soma interaction during gonad morphogenesis

In most animal species, germ cells require intimate contact with specialized somatic cells in the gonad for their proper development. We have analyzed the establishment of germ cell-soma interaction during embryonic gonad formation in Drosophila melanogaster, and find that somatic cells undergo dramatic changes in cell shape and individually ensheath germ cells as the gonad coalesces. Germ cell ensheathment is independent of other aspects of gonad formation, indicating that separate morphogenic processes are at work during gonadogenesis. The cell-cell adhesion molecule Drosophila E-cadherin is essential both for germ cell ensheathment and gonad compaction, and is upregulated in the somatic gonad at the time of gonad formation. Our data indicate that differential cell adhesion contributes to cell sorting and the formation of proper gonad architecture. In addition, we find that Fear of Intimacy, a novel transmembrane protein, is also required for both germ cell ensheathment and gonad compaction. E-cadherin expression in the gonad is dramatically decreased in fear of intimacy mutants, indicating that Fear of Intimacy may be a regulator of E-cadherin expression or function.     

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.     

On the control of germ cell development in Caenorhabditis elegans

After hatching, the germ line progenitor cells in C. elegans begin to divide mitotically; later, some of the germ line cells enter meiosis and differentiate into gametes. In the adult, mitotic germ cells, or stem cells, are found at one end (the distal end) and meiotic cells occupy the rest of the elongate gonad. Removal of two somatic gonadal cells, the distal tip cells, by laser microsurgery has a dramatic effect on germ cell development. In either sex, this operation leads to the arrest of mitosis and the initiation of meiosis in germ cells. The function of the distal tip cell in the intact animal appears to be the inhibition of meiosis (or stimulation of mitosis) in nearby germ cells. During development, this permits growth and, in the adult, it maintains the germ line stem cell population. A change in the position of the distal tip cell in the gonad at an early point in development is correlated with a change in the axial polarity of the germ line tissue. This suggests that the localization of the distal tip cell's inhibitory activity at the distal end of the gonad establishes the axial polarity of the germ line tissue in the intact animal.     

Fanconi anemia complementation group C is required for proliferation of murine primordial germ cells

Fanconi anemia is a polygenic trait hypothesized to be a DNA damage repair disease. We show that all three Fanconi anemia loci that have been cloned are expressed in the embryonic gonad during the period of primordial germ cell proliferation. Mice mutant for the Fanconi anemia complementation group C locus (Fancc) have reduced germ cell numbers as early as embryonic day E12.5, suggesting the Fancc protein functions prior to meiosis in both sexes. Depletion in the mutant occurs at a time when all three loci would be expressed in a wild-type gonad, implying a function in the early germline. Determination of the mitotic index of primordial germ cells by BrdU incorporation shows that germ cells in Fancc(-/-) mice proliferate significantly more slowly than littermate controls. This study demonstrates Fancc is required for mitotic proliferation of primordial germ cells.     

P granules in the germ cells of Caenorhabditis elegans adults are associated with clusters of nuclear pores and contain RNA

The germ cells, and germ cell precursors, in the nematode Caenorhabditis elegans contain distinctive granules called P granules. During early embryogenesis, P granules are segregated asymmetrically into those blastomeres that eventually produce the germ line. Because of the correlation between P granule distribution and the development of the germ line, P granules are widely thought to function in some aspect of germ line specification or differentiation. Most of the analysis of P granule structure and localization has focused on the early embryo, when P granules are located in the cytoplasm. However, during most of development P granules are associated with germ cell nuclei. We report here an ultrastructural analysis of the nuclear-associated P granules in the germ cells of the adult hermaphrodite gonad. We show that P granules are tightly associated with nuclear pores and that the positions of certain structures within the P granules correspond to the positions of pores on the nuclear envelope. We present immunocytochemical and ultrastructural data suggesting that P granules can associate, or remain associated, with pore-like structures even after they detach from the nuclear envelope during oogenesis. Finally, we show that nuclear-associated P granules in the gonad contain RNA, complementing previous studies showing that cytoplasmic P granules in embryos contain RNA.     

From primordial germ cell to primordial follicle: mammalian female germ cell development

In mammals, the final number of oocytes available for reproduction of the next generation is defined at birth. Establishment of this oocyte pool is essential for fertility. Mammalian primordial germ cells form and migrate to the gonad during embryonic development. After arriving at the gonad, the germ cells are called oogonia and develop in clusters of cells called germ line cysts or oocyte nests. Subsequently, the oogonia enter meiosis and become oocytes. The oocyte nests break apart into individual cells and become packaged into primordial follicles. During this time, only a subset of oocytes ultimately survive and the remaining immature eggs die by programmed cell death. This phase of oocyte differentiation is poorly understood but molecules and mechanisms that regulate oocyte development are beginning to be identified. This review focuses on these early stages of female germ cell development.     

The matricellular protein SPARC is internalized in Sertoli, Leydig, and germ cells during testis differentiation

The gene encoding the matricellular protein secreted protein, acidic and rich in cysteine (SPARC) was identified in a screen for genes expressed sex-specifically during mouse gonad development, as being strongly upregulated in the male gonad from very early in testis development. We present here a detailed analysis of SPARC gene and protein expression during testis development, from 11.5 to 15.5 days post coitum (dpc). Section in situ hybridization analysis revealed that SPARC mRNA is expressed by the Sertoli cells in the testis cords and the fetal Leydig cells, found within the interstitial space between the testis cords. Immunodetection with anti-SPARC antibody showed that the protein was located inside the testis cords, within the cytoplasm of Sertoli and germ cells. In the interstitium, SPARC was present intracellularly within the Leydig cells. The internalization of SPARC in Sertoli, Leydig, and germ cells suggests that it plays an intracellular regulatory role in these cell types during fetal testis development.     

Freemartin in the amphibian Pleurodeles waltl: parabiosis between individuals from opposite sex triggers both germ and somatic cells alterations during female gonad development

Wild type embryos of the newt Pleurodeles waltl were used to realize parabiosis, a useful model to study the effect of endogenous circulating hormones on gonad development. The genotypic sex of each parabiont (ZZ male or ZW female) was determined early from the analysis of the sex chromosome borne marker peptidase-1. In ZZ/ZZ and ZW/ZW associations, gonads develop according to genetic sex. In ZZ/ZW associations, the ZZ gonads differentiate as normal testes while ZW gonads development shows numerous alterations. At the beginning of sex differentiation, these ZW gonads possess a reduced number of germ cells and a reduced expression of steroidogenic factor 1 and P450-aromatase mRNAs when compared to gonads from ZW/ZW associations. During gonad differentiation, conversely to the control situation, these germ cells do not enter meiosis as corroborated by chromatin status and absence of the meiosis entry marker DMC1; the activity of the estradiol-producing enzyme P450-aromatase is as low as in ZZ gonads. At adulthood, no germ cells are observed on histological sections, consistently with the absence of VASA expression. At this stage, the testis-specific marker DMRT1 is expressed only in ZZ gonads, suggesting that the somatic compartment of the ZW gonad is not masculinized. So, when exposed to ZZ hormones, ZW gonads reach the undifferentiated status but the ovary differentiation does not occur. This gonad is inhibited by a process affecting both somatic and germ cells. Additionally, the ZW gonad inhibition does not occur in the case of an exogenous estradiol treatment of larvae.