Somatic and germ cell interactions during histogenetic aggregation of mouse fetal testes.

In the present study we examined the capacity of somatic and germ cells dissociated from fetal mouse testes at various stages to reform seminiferous cords in culture. We found that after 12 h in culture, seminiferous cords became segregated from stromal cells. Although Sertoli cells were incorporated into seminiferous cords at all stages studied, the germ cells dramatically changed their histogenetic behavior with age. Most germ cells which had been dissociated at 12.5 days postcoitum (dpc) were incorporated into the seminiferous cords, whereas at 14.5 dpc or later the majority remained among the stromal cells or as clusters on the surface of the aggregates. We considered three possible causes for this change in behavior of germ cells: (i) Failure to deposit some extracellular matrix components in the aggregates. (ii) Decrease in adhesiveness of prospermatogonia to either extracellular matrix components or Sertoli cells. (iii) A change in adhesiveness of Sertoli cells to germ cells with age. We found that laminin and fibronectin were similarly deposited in aggregates at 12.5 and 15.5 dpc. When prospermatogonia at 15.5 dpc labeled with colloidal gold were reaggregated with somatic cells at 12.5 dpc, 50% were incorporated into seminiferous cords. Moreover, [3H]thymidine-labeled Sertoli cells at 15.5 dpc formed heterochronic seminiferous cords with Sertoli cells at 12.5 dpc. These results suggest that mouse Sertoli cells change their surface property which is essential for binding to germ cells when they enter the mitotic resting stage (T-prospermatogonia).     

TGF-beta3 induces ectopic mineralization in fetal mouse dental pulp during tooth germ development

Several members of the transforming growth factor (TGF)-beta superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF-beta1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF-beta3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF-beta3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose-dependent manner. Further, TGF-beta3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF-beta3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF-beta3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF-beta3 soaked beads in vivo. These results indicate for the first time that TGF-beta3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts.     

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.     

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.     

Mouse germ cell development during embryogenesis

The discrimination and differentiation of germ cells from somatic cells is a fundamental issue during development. The early specification of mouse primordial germ cells (PGCs) is achieved by the induction of Blimp1, a key regulator of germ cells. Nanos3 is one of the genes activated in early PGCs and prevents apoptosis during their migration stage. Once PGCs enter the embryonic gonads, they differentiate according to the somatic sex of the organism. During this process, Nanos2 plays an important role as it promotes male germ cell pathway by suppressing the female fate. In this review, the process of germ cell development in the mouse is discussed with a particular focus on the functions of the key proteins, Blimp1, Nanos, and Dead end1.     

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.     

Ultrastructure of germ cell development in the human fetal testis

Summary Electron-microscopic examination of the human fetal testis between 10 and 20 weeks gestation reveals the presence of two distinct cell types within the tubules: Sertoli cells and germ cells. The latter are distinguished by their spherical shape, smooth nuclear membranes, globular mitochondria and paucity of cytoplasmic organelles. The gonocytes, or primitive germ cells, occur as single cells in the central portions of the tubules. Their chromatin is finely granular and evenly dispersed. Nucleoli are centrally placed and of uniform electron density. Various stages in the migration of gonocytes to the tubular periphery are indicated by the extension of cytoplasmic processes toward the basal lamina. Bands of microtubules are present within the processes. Spermatogonia are arranged in pairs and groups at the tubular periphery. They lack the nucleolar and mitochondrial characteristics of adult spermatogonia. Except for slight changes in chromatin density and nucleolar structure, the fetal spermatogonia retain the ultrastructural characteristics of gonocytes. Intercellular bridges connect adjacent spermatogonia. Degeneration affecting large numbers of germ cells, but primarily gonocytes, begins with nuclear infolding and chromatin condensation and eventually involves both nuclear and cytoplasmic structures. The degenerated cells are removed by phagocytosis by adjacent Sertoli cells. Large phagosomes are present in the cytoplasm of many of the Sertoli cells.Supported by a grant from the Ford Foundation and by General Research Support Grant RR055511 from the National Institutes of Health. Technical assistance was provided by Mrs. Lucy A. Conner.     

Regulation of primordial germ cell development in the mouse

Primordial germ cells (PGCs) are the founders of the gametes. They arise at the earliest stages of embryonic development and migrate to the gonadal ridges, where they differentiate into oogonia/oocytes in the ovary, and prospermatogonia in the testis. The present article is a review of the main studies undertaken by the author with the aim of clarifying the mechanisms underlying the development of primordial germ cells. Methods for the isolation and purification of migratory and post-migratory mouse PGCs devised in the author's laboratory are first briefly reviewed. Such methods, together with the primary culture of PGCs onto suitable cell feeder layers, have allowed the analysis of important aspects of the control of their development, concerning in particular survival, proliferation and migration of mouse PGCs. Compounds and growth factors affecting PGC numbers in culture have been identified. These include survival anti-apoptotic factors (SCF, LIF) and positive regulators of proliferation (cAMP, PACAPs, RA). Evidence has been provided that the motility of migrating PGCs relies on integrated signals from extracellular matrix molecules and the surrounding somatic cells. Moreover, homotypic PGC-PGC interaction has been evidenced that might play a role in PGC migration and in regulating their development. Several molecules (i.e. integrins, specific types of oligosaccharides, E-cadherin, the tyrosine kinase receptor c-kit) have been found to be expressed on the surface of PGCs and to mediate adhesive interactions of PGCs with the extracellular matrix, somatic cells and neighbouring PGCs.     

Evolution of gene regulatory networks controlling body plan development

Evolutionary change in animal morphology results from alteration of the functional organization of the gene regulatory networks (GRNs) that control development of the body plan. A major mechanism of evolutionary change in GRN structure is alteration of cis-regulatory modules that determine regulatory gene expression. Here we consider the causes and consequences of GRN evolution. Although some GRN subcircuits are of great antiquity, other aspects are highly flexible and thus in any given genome more recent. This mosaic view of the evolution of GRN structure explains major aspects of evolutionary process, such as hierarchical phylogeny and discontinuities of paleontological change.     

Members of the ErbB receptor tyrosine kinases are involved in germ cell development in fetal mouse gonads.

To isolate the genes involved in mouse primordial germ cell (PGC) development, we carried out subtraction cDNA cloning between PGC-derived embryonic germ (EG) cells and inner cell mass-derived embryonic stem cells. Among the genes preferentially expressed in EG cells, we found a gene encoding a receptor tyrosine kinase ErbB3. By in situ hybridization and immunohistochemical staining, the expression of ErbB3 as well as that of ErbB2, a coreceptor for ErbB3, was detected in PGCs in genital ridges at 12.5 dpc (days postcoitum). The expression was, however, downregulated at 14.5 dpc when the PGCs underwent growth cessation. Neuregulin-beta, a ligand for ErbB2 and ErbB3, was also expressed in genital ridges. In addition, a recombinant Neuregulin-beta enhanced the number of PGCs in 12.5-dpc embryos in culture. Taken together, these observations suggest that ErbB signaling controls the growth or survival of PGCs in genital ridges.     

Changes in Leydig cell gene expression during development in the mouse

Developmental changes in the expression of 18 Leydig cell-specific mRNA species were measured by real-time polymerase chain reaction to partially characterize the developmental phenotype of the cells in the mouse and to identify markers of adult Leydig cell differentiation. Testicular interstitial webs were isolated from mice between birth and adulthood. Five developmental patterns of gene expression were observed. Group 1 contained mRNA species encoding P450 side chain cleavage (P450(scc)), P450(c17), relaxin-like factor (RLF), glutathione S-transferase 5-5 (GST5-5), StAR protein, LH receptor, and epoxide hydrolase (EH); group 2 contained 3beta-hydroxysteroid dehydrogenase (3beta-HSD) VI, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) III, vascular cell adhesion molecule 1, estrogen sulfotransferase, and prostaglandin D (PGD)-synthetase; group 3 contained patched and thrombospondin 2 (TSP2); group 4 contained 5alpha-reductase 1 and 3alpha-hydroxysteroid dehydrogenase; group 5 contained sulfonylurea receptor 2 and 3beta-HSD I. Group 1 contained genes that were expressed in fetal and adult Leydig cells and which increased in expression around puberty toward a maximum in the adult. Group 2 contained genes expressed only in the adult Leydig cell population. Group 3 contained genes with predominant fetal/neonatal expression in the interstitial tissue. Group 4 contained genes with a peak of expression around puberty, whereas genes in group 5 show little developmental change in expression. Highest mRNA levels in descending order were RLF, P450(c17), EH, 17beta-HSD III, PGD-synthetase, GST5-5, and P450(scc). Results identify five genes expressed in the mouse adult Leydig cell population, but not in the fetal population, and one gene (TSP2) that may be expressed only in the fetal Leydig cell population. The developmental pattern of gene expression suggests that three distinct phases of adult Leydig cell differentiation occur.     

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.     

Smad5 is required for mouse primordial germ cell development

Smad5, together with Smad1 and Smad8, have been implicated as downstream signal mediators for several bone morphogenetic proteins (BMPs). Recent studies have shown that primordial germ cells (PGCs) are absent or greatly reduced in Bmp4 or Bmp8b mutant mice. To define the role of Smad5 in PGC development, we examined PGC number in Smad5 mutant mice by Oct4 whole-mount in situ hybridization and alkaline phosphatase staining. We found ectopic PGC-like cells in the amnion of some Smad5 mutant mice, however, the total number of PGCs was greatly reduced or completely absent in Smad5 mutant embryos, similar to Bmp4 or Bmp8b mutant embryos. Therefore, Smad5 is an important factor involved in PGC generation and localization.