Steroid Signaling within Drosophila Ovarian Epithelial Cells Sex-Specifically Modulates Early Germ Cell Development and Meiotic Entry

Drosophila adult females but not males contain high levels of the steroid hormone ecdysone, however, the roles played by steroid signaling during Drosophila gametogenesis remain poorly understood. Drosophila germ cells in both sexes initially follow a similar pathway. After germline stem cells are established, their daughters form interconnected cysts surrounded by somatic escort (female) or cyst (male) cells and enter meiosis. Subsequently, female cysts acquire a new covering of somatic cells to form follicles. Knocking down expression of the heterodimeric ecdysteroid receptor (EcR/Usp) or the E75 early response gene in escort cells disrupts 16-cell cyst production, meiotic entry and follicle formation. Escort cells lose their squamous morphology and unsheath germ cells. By contrast, disrupting ecdysone signaling in males does not perturb cyst development or ensheathment. Thus, sex-specific steroid signaling is essential for female germ cell development at the time male and female pathways diverge. Our results suggest that steroid signaling plays an important sex-specific role in early germ cell development in Drosophila, a strategy that may be conserved in mammals.     

Genetic control of sex determination in the germ line of Caenorhabditis elegans

The nematode Caenorhabditis elegans normally exists as one of two sexes: self-fertilizing hermaphrodite or male. Development as hermaphrodite or male requires the differentiation of each tissue in a sex-specific way. In this review, I discuss the genetic control of sex determination in a single tissue of C. elegans: the germ line. Sex determination in the germ line depends on the action of two types of genes:--those that act globally in all tissues to direct male or female development and those that act only in the germ line to specify either spermatogenesis or oogenesis. First, I consider a tissue-specific sex-determining gene, fog-1, which promotes spermatogenesis in the germ line. Second, I consider the regulation of the hermaphrodite pattern of germ-line gametogenesis where first sperm and then oocytes are produced.     

The role of glutathione in mammalian gametes

The paper reviews a recent research on the role of glutathione (GSH) in the male and female germ cells as well as during the early stages of embryo development in mammals. In both the male and female gametes, GSH is involved in the protection of these cells against oxidative damage. Glutathione has been implicated in maintaining the meiotic spindle morphology of the oocyte. After fertilization, this thiol plays an active role in the formation of the male pronucleus, and has a beneficial effect on early embryogenesis to the blastocyst stage. GSH concentrations change within the oocytes during meiotic maturation and its synthesis is regulated by gonadotropins. Furthermore, GSH concentrations in the maturing spermatozoa gradually decline during spermatogenesis. This review also addresses the important role of cumulus cells in glutathione synthesis.     

Epigenetic transitions in germ cell development and meiosis

Germ cell development is controlled by unique gene expression programs and involves epigenetic reprogramming of histone modifications and DNA methylation. The central event is meiosis, during which homologous chromosomes pair and recombine, processes that involve histone alterations. At unpaired regions, chromatin is repressed by meiotic silencing. After meiosis, male germ cells undergo chromatin remodeling, including histone-to-protamine replacement. Male and female germ cells are also differentially marked by parental imprints, which contribute to sex determination in insects and mediate genomic imprinting in mammals. Here, we review epigenetic transitions during gametogenesis and discuss novel insights from animal and human studies.     

Reconstitution of Gametogenesis In Vitro:Meiosis Is the Biggest Obstacle

Germ-line cells are responsible for transmitting genetic and epigenetic information across generations, and ensuring the creation of new individuals from one generation to the next. Gametogenesis process requires several rigorous steps, including primordial germ cell （PGC） specification, proliferation, migration to the gonadal ridges and differentiation into mature gametes such as sperms and oocytes. But this process is not clearly explored because a small number of PGCs are deeply embedded in the developing embryo. In the attempt to establish an in vitro model for understanding gametogenesis process well, several groups have made considerable progress in differen- tiating embryonic stem cells （ESCs） and adult stem cells （ASCs） into germ-like cells over the past ten years. These stem cell-derived germ cells appear to he capable of undergoing meiosis and generating both male and female gametes. But most of gametes turn out to be not fully functional due to their abnormal meiosis process compared to endogenous germ cells. Therefore, a robust system of differentiating stem cells into germ cells would enable us to investigate the genetic, epigenetic and environmental factors associated with germ cell development. Here, we review the stem cell-derived germ cell development, and discuss the potential and challenges in the differentiation of functional germ cells from stem cells.     

Heterochromatin endoreduplication prior to gametogenesis and chromatin diminution during early embryogenesis in Mesocyclops edax (Copepoda: Crustacea)

The segregation of progenitor somatic cells from those of the primordial germ cells that sequester and retain elevated levels of DNA during subsequent developmental events, poses an interesting, alternative pathway of chromosome behavior during the reproductive cycle of certain species of cyclopoid copepods and several other organisms. Separation of maternal and paternal chromosome sets during very early cleavages (gonomery) is often a feature following marked elevations of DNA levels in germ cells for some of these species. Here, we report on the accumulation of large amounts of DNA in germ line nuclei of both female and male juveniles and adults of a freshwater copepod, Mesocyclops edax (Forbes, 1890). We also report the robust uptake of 3H-thymidine by germ cells prior to gametogenesis in this species. By using cytophotometric analysis of the DNA levels in both germ line cells and somatic cells from the same specimens we demonstrate that germ cell nuclei accumulate high levels of DNA prior to the onset of gametogenesis. These elevated amounts coincide with the levels of heterochromatic DNA discarded during chromatin diminution. A new model is proposed of major cytological events accompanying the process of chromatin diminution in M. edax.     

A germline-specific gap junction protein required for survival of differentiating early germ cells

Germ cells require intimate associations and signals from the surrounding somatic cells throughout gametogenesis. The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Animals with a null mutation in zpg are viable but sterile and have tiny gonads. Adult zpg-null gonads contain small numbers of early germ cells, resembling stem cells or early spermatogonia or oogonia, but lack later stages of germ cell differentiation. In the male, Zpg protein localizes to the surface of spermatogonia, primarily on the sides adjacent to the somatic cyst cells. In the female, Zpg protein localizes to germ cell surfaces, both those adjacent to surrounding somatic cells and those adjacent to other germ cells. We propose that Zpg-containing gap junctional hemichannels in the germ cell plasma membrane may connect with hemichannels made of other innexin isoforms on adjacent somatic cells. Gap junctional intercellular communication via these channels may mediate passage of crucial small molecules or signals between germline and somatic support cells required for survival and differentiation of early germ cells in both sexes.     

Effect of methylmethane sulfonate-sensitive mutations on nondisjunction and loss of sex chromosomes in Drosophila males

The MMS-sensitive mutants mus(1) 120M1 and mus(1) 121M1 of Drosophila melanogaster were investigated regarding their effects on spontaneous and X-ray induced (2000 R) aneuploidy in male germ cells, during different stages of spermatogenesis. In matings of males carrying mus mutation and a doubly marked Y-chromosome (BsYy+) with repair proficient y f females, the frequencies of partial loss, nondisjunctions and especially complete loss were significantly higher than in the control. Apparently, MMS-sensitive mutants deal with meiotic processes and maintenance of chromosome structural stability both in females and in males, in somatic and germ cells.     

Details of the female and male pathway of the Keimbahn determined by enzyme histochemical and autoradiographic studies

Autoradiographic studies and the use of enzyme histochemistry have revealed that early germ line cells (female and male PGC, oogonia, prediplotene oocytes and prospermatogonia) as well as the more advanced germ cells (diplotene oocytes, spermatogonia, spermatocytes and spermatids) show specific patterns of their DNA and RNA synthesis and their enzymatic equipment. The female and male germ lines show similar kinetics up to the arise of oocytes and T prospermatogonia (T for transitional), the final products of a first limited multiplication process of primitive gonia. In former studies we supposed that oocytes and T prospermatogonia are the first exponents of the female and male pathway of the germ line (Hilscher and Hilscher, 1989a). Recently, it could be shown--using the reverse PLM method in slides of plastic embedded material--that the first differences between female and male GC can already be stated at the end of the first proliferation wave of oogonia and multiplying prospermatogonia; that means even before the existence of oocytes and T prospermatogonia (Hilscher and Hilscher, 1989b). Oogonia and M prospermatogonia (M for multiplying) are equipped both with only one active X chromosome. While oocytes traverse the prediplotene stages of meiotic prophase T prospermatogonia prepare for a second extensive proliferation process: spermatogenesis. Oocytes in meiosis are provided with two active X chromosomes, T prospermatogonia possess only one, and the presence of the Y chromosome is not vital for them. However, the Y chromosome is required for the normal course of spermatogenesis characterized by a stock of stem cells, that are responsible for the continuous production of male gamets. The mammalian oocyte--similar as that of insects and amphibia but to a lower degree--acts as pre-embryo.     

Efficient GFP‐labeling and analysis of spermatogenic cells using the IRG transgene and flow cytometry

Spermatogenesis is a highly ordered developmental program that produces haploid male germ cells. The study of male germ cell development in the mouse has provided unique perspectives into the molecular mechanisms that control cell development and differentiation in mammals, including tissue‐specific gene regulatory programs. An intrinsic challenge in spermatogenesis research is the heterogeneity of germ and somatic cell types present in the testis. Techniques to separate and isolate distinct mouse spermatogenic cell types have great potential to shed light on molecular mechanisms controlling mammalian cell development, while also providing new insights into cellular events important for human reproductive health. Here, we detail a versatile strategy that combines Cre‐lox technology to fluorescently label germ cells, with flow cytometry to discriminate and isolate germ cells in different stages of development for cellular and molecular analyses.     

小鼠胚胎干细胞向生殖细胞分化的研究

小鼠胚胎干细胞(ESC)在体外可以分化为多种细胞类型,其中包括各阶段的生殖细胞,甚至精细胞和成熟卵母细胞。ESC向生殖细胞分化的效率受到包括生长因子、激素和体细胞等多种因素的影响,在体外形成的是雌性配子还是雄性配子与ESC是XX型还是XY型没有必然联系。简要综述了小鼠生殖细胞在体内外的分化发育、性别决定和增殖等,并总结和展望了ESC向生殖细胞分化研究面临的问题和应用前景。     

In vitro methods of male germ cell specification and differentiation

Germ line specification is an early cell fate decision essential for the transmission of totipotency over generations. Two types of germ line stem cells populate the male gonads in mammals. Primordial germ cells (PGCs) are the germ line founders only present during prenatal life. Spermatogonial stem cells (SSCs) appear a few days after birth and divide asymmetrically to give rise to one stem cell and one spermatogonia that initiates differentiation to produce spermatozoa. Germ cell specification and differentiation involve specific environmental stimuli and a sequential order of maturing phases required for gamete function. Spatio-temporal controls similarly dictate the erasure of somatic methylation marks and the subsequent acquisition of sex-specific marks at imprinted genes in gametes. We review here the recent advancements in male germ cell derivation from ES cells and discuss the limits of these in vitro methods in providing a kinetics and a microenvironment suitable for the programming of a proper gametic and parental identity.     

Germ cell sex and cell cycle

Germ cells are the only cells in the body capable of transferring an individual's genetic and epigenetic information to the next generation. However, the developmental processes that provide the foundation for male and female germ line development and later gamete production are complex and poorly understood. In mice the primordial germ cells enter the bipotential gonad at E10.5 and, in response to the testicular or ovarian micro-environment, commit to spermatogenesis or oogenesis. This paper reviews progress in understanding the molecular processes underlying the early stages of male and female germ line development.     

Probing spermatogenesis in Drosophila with P-element enhancer detectors.

Formation of motile sperm in Drosophila melanogaster requires the coordination of processes such as stem cell division, mitotic and meiotic control and structural reorganization of a cell. Proper execution of spermatogenesis entails the differentiation of cells derived from two distinct embryonic lineages, the germ line and the somatic mesoderm. Through an analysis of homozygous viable and fertile enhancer detector lines, we have identified molecular markers for the different cell types present in testes. Some lines label germ cells or somatic cyst cells in a stage-specific manner during their differentiation program. These expression patterns reveal transient identities for the cyst cells that had not been previously recognized by morphological criteria. A marker line labels early stages of male but not female germ cell differentiation and proves useful in the analysis of germ line sex-determination. Other lines label the hub of somatic cells around which germ line stem cells are anchored. By analyzing the fate of the somatic hub in an agametic background, we show that the germ line plays some role in directing its size and its position in the testis. We also describe how marker lines enable us to identify presumptive cells in the embryonic gonadal mesoderm before they give rise to morphologically distinct cell types. Finally, this collection of marker lines will allow the characterization of genes expressed either in the germ line or in the soma during spermatogenesis.     

Prospects for spermatogenesis in vitro

In recent years, extraordinary progress has been made in a broad range of reproductive technologies, including spermatogonial transplantation in the male. However, effective procedures for the complete recapitulation of spermatogenesis in vitro, including meiosis, have remained elusive. Such procedures have the potential to facilitate (1) mechanistic studies of spermatogenesis, (2) directed genetic modification of the male germ line, and (3) treatment of male factor infertility. Early studies demonstrated the importance of germ cell-Sertoli association for germ cell survival in vitro. Recently, evidence for male germ cell survival and progression through meiosis has been reported for the rat, mouse, and man. We demonstrated the expression of spermatid-specific genes (protamine and transition protein 1) by alginate-encapsulate neonatal bull testis cells after 10 weeks in culture, suggesting that meiosis had occurred. Although identifiable germ cells in these cultures were very sparse, some indication of acrosome development was observed. Following round spermatid injection (ROSI) with presumptive spermatids produced in vitro, 50% of blastocysts produced were diploid and 37% were Y-chromosome positive. Improved culture conditions, which promote germ cell survival, differentiation, and proliferation, are essential for in vitro spermatogenesis (IVS) to become a useful technology. Other approaches to male germ cell manipulation and spermatid production are discussed.