Identification of side population cells in mouse primordial germ cells and prenatal testis

In mammals, the stem cells of spermatogenesis are derived from an embryonic cell population called primordial germ cells (PGCs). Spermatogonial stem cells displaying the "side population" (SP) phenotype have been identified in the immature and adult mouse testis, but noting is known about the expression of the SP phenotype during prenatal development of germ cells. The SP phenotype, defined as the ability of cells to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In the present study, we analyzed and characterized the Hoechst SP via cytofluorimetric analysis of disaggregated gonads at different time points during embryonic development in mice. To directly test the hypothesis that the SP phenotype is a feature of germ cell lineage, experiments were performed on transgenic animals expressing enhanced green fluorescent protein (EGFP) under the control of the Oct4 promoter, to identify early germ cells up to PGCs. We found that prenatal gonads contain a fraction of SP cells at each stage analyzed, and the percentage of cells in the SP fraction decreases as development proceeds. Surprisingly, more than 50% of the PGCs displayed the SP phenotype at 11.5 dpc (days post coitum). The percentage of germ cells with the SP phenotype decreased steadily with development, to less than 1% at 18.5 dpc. Cytofluorimetric analysis along with immunocytochemistry performed on sorted cells indicated that the SP fraction of prenatal gonads, as in the adult testis, was heterogeneous, being composed of both somatic and germ cells. Both cell types expressed the ABC transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These findings provide evidence that the SP phenotype is a common feature of PGCs and identifies a subpopulation of fetal testis cells including prospermatogonia whose differentiation fate remains to be investigated. © 2011 UBC Press

Meiotic wave adds extra asymmetry to the development of female chicken gonads

Development of female gonads in the chicken is asymmetric. This asymmetry affects gene expression, morphology, and germ cell development; consequently only the left ovary develops into a functional organ, whereas the right ovary remains vestigial. In males, on the other hand, both gonads develop into functional testes. Here, we revisited the development of asymmetric traits in female (and male) chicken gonads between Hamburger Hamilton stage 16 (HH16) and hatching. At HH16, primordial germ cells migrated preferentially to the left gonad, accumulating in the left coelomic hinge between the gut mesentery and developing gonad in both males and females. Using the meiotic markers SYCP3 and phosphorylated H2AFX, we identified a previously undescribed, pronounced asymmetryc meiotic progression in the germ cells located in the central, lateral, and extreme cortical regions of the left female gonad from HH38 until hatching. Moreover, we observed that-in contrast to the current view-medullary germ cells are not apoptotic, but remain arrested in pre-leptotene until hatching. In addition to the systematic analysis of the asymmetric distribution of germ cells in female chicken gonads, we propose an updated model suggesting that the localization of germ cells-in the left or right gonad; in the cortex or medulla of the left gonad; and in the central part or the extremities of the left cortex-has direct consequences for their development and participation in adult reproduction

Prospects for transgenesis in the chick

Research to develop a useful method for genetic modification of the chick has been on-going since the first demonstrations in the mouse in the 1980s that genetic modification is an invaluable tool for the study of gene function. Manipulation of the chick zygote is possible but inefficient. Considerable progress has been made in developing potentially pluripotent embryo stem cells and their contribution to somatic chimeric birds well-established. Germ line transmission of gametes derived from genetically modified embryo cells has not been described. Transfer of primordial germ cells from a donor embryo to a recipient and production of functional gametes from the donor-derived cells is possible. Genetic modification of primordial germ cells before transfer and their recovery through the germ line has not been achieved. The first transgenic birds described were generated using retroviral vectors. The use of lentiviral vectors may make this approach a feasible method for transgenic production, although there are limitations to the applications of these vectors. It is likely that a method will be developed in the next few years that will enable the use of transgenesis as a tool in the study of development in the chick and for many other applications in basic research and biotechnology

X-chromosome inactivation mosaicism in the three germ layers and the germ line of the mouse embryo

Electrophoretic variant forms of the X-linked enzyme phosphoglycerate kinase (PGK-1, E.C.2, 7, 2, 3) have been used to examine X-chromosome mosaicism in tissues from 121/2-day post coitum heterozygous female mouse embryos. Samples of yolk-sac endoderm, neural ectoderm, heart (mesoderm), liver (endoderm) and germ cells were analysed from each embryo. In all tissues except yolk-sac endoderm, both PGK-1 isozymes were expressed. The extent of covariance among tissues with respect to the PGK-1 isozyme contribution is consistent with all tissues being derived from the same pool of cells after X-inactivation. The covariance among tissues gives an estimate of the size of this pool (47 cells) and places the earliest time of X-inactivation in epiblast cells between 41/2 and 51/2 days post coitum. From the independent variance among tissues within an individual, the average primordial precursor pool size for the three germ layers and the germ line itself was estimated as 193 cells

Effect of FSH on testicular morphology and spermatogenesis in gonadotrophin-deficient hypogonadal mice lacking androgen receptors

Follicle stimulating hormone (FSH) and androgen act to stimulate and maintain spermatogenesis. FSH acts directly on the Sertoli cells to stimulate germ cell number and acts indirectly to increase androgen production by the Leydig cells. In order to differentiate between the direct effects of FSH on spermatogenesis and those mediated indirectly through androgen action we have crossed hypogonadal (hpg) mice which lack gonadotrophins with mice lacking androgen receptors (AR) either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with recombinant FSH for 7 days and testicular morphology and cell numbers assessed. In untreated hpg and hpg.SCARKO mice germ cell development was limited and did not progress beyond the pachytene stage. In hpg.ARKO mice testes were smaller with fewer Sertoli cells and germ cells compared to hpg mice. Treatment with FSH had no effect on Sertoli cell number but significantly increased germ cell numbers in all groups. In hpg mice FSH increased numbers of spermatogonia and spermatocytes and induced round spermatid formation. In hpg.SCARKO and hpg.ARKO mice, in contrast, only spermatogonial and spermatocyte numbers were increased with no formation of spermatids. Leydig cell numbers were increased by FSH in hpg and hpg.SCARKO mice but not in hpg.ARKO mice. Results show that in rodents 1) FSH acts to stimulate spermatogenesis through an increase in spermatogonial number and subsequent entry of these cells into meiosis, 2) FSH has no direct effect on the completion of meiosis and 3) FSH effects on Leydig cell number are mediated through interstitial ARs

Migration of germline progenitor cells is directed by sphingosine-1-phosphate signalling in a basal chordate.

The colonial ascidian Botryllus schlosseri continuously regenerates entire bodies in an asexual budding process. The germ line of the newly developing bodies is derived from migrating germ cell precursors, but the signals governing this homing process are unknown. Here we show that germ cell precursors can be prospectively isolated based on expression of aldehyde dehydrogenase and integrin alpha-6, and that these cells express germ cell markers such as vasa, pumilio and piwi, as well as sphingosine-1-phosphate receptor. In vitro, sphingosine-1-phosphate (S1P) stimulates migration of germ cells, which depends on integrin alpha-6 activity. In vivo, S1P signalling is essential for homing of germ cells to newly developing bodies. S1P is generated by sphingosine kinase in the developing germ cell niche and degraded by lipid phosphate phosphatase in somatic tissues. These results demonstrate a previously unknown role of the S1P signalling pathway in germ cell migration in the ascidian Botryllus schlosseri

An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure.

The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis

Primate Primordial Germ Cells Acquire Transplantation Potential by Carnegie Stage 23.

Primordial germ cells (PGCs) are the earliest embryonic progenitors in the germline. Correct formation of PGCs is critical to reproductive health as an adult. Recent work has shown that primate PGCs can be differentiated from pluripotent stem cells; however, a bioassay that supports their identity as transplantable germ cells has not been reported. Here, we adopted a xenotransplantation assay by transplanting single-cell suspensions of human and nonhuman primate embryonic Macaca mulatta (rhesus macaque) testes containing PGCs into the seminiferous tubules of adult busulfan-treated nude mice. We discovered that both human and nonhuman primate embryonic testis are xenotransplantable, generating colonies while not generating tumors. Taken together, this work provides two critical references (molecular and functional) for defining transplantable primate PGCs. These results provide a blueprint for differentiating pluripotent stem cells to transplantable PGC-like cells in a species that is amenable to transplantation and fertility studies

DNA methylation and regulatory elements during chicken germline stem cell differentiation

Funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.The production of germ cells in vitro would open important new avenues for stem biology and human medicine, but the mechanisms of germ cell differentiation are not well understood. The chicken, as a great model for embryology and development, was used in this study to help us explore its regulatory mechanisms. In this study, we reported a comprehensive genome-wide DNA methylation landscape in chicken germ cells, and transcriptomic dynamics was also presented. By uncovering DNA methylation patterns on individual genes, some genes accurately modulated by DNA methylation were found to be associated with cancers and virus infection, e.g., AKT1 and CTNNB1. Chicken-unique markers were also discovered for identifying male germ cells. Importantly, integrated epigenetic mechanisms were explored during male germ cell differentiation, which provides deep insight into the epigenetic processes associated with male germ cell differentiation and possibly improves treatment options to male infertility in animals and humans

Mitochondrial fission regulates germ cell differentiation by suppressing ROS-mediated activation of Epidermal Growth Factor Signaling in the Drosophila larval testis.

Mitochondria are essential organelles that have recently emerged as hubs for several metabolic and signaling pathways in the cell. Mitochondrial morphology is regulated by constant fusion and fission events to maintain a functional mitochondrial network and to remodel the mitochondrial network in response to external stimuli. Although the role of mitochondria in later stages of spermatogenesis has been investigated in depth, the role of mitochondrial dynamics in regulating early germ cell behavior is relatively less-well understood. We previously demonstrated that mitochondrial fusion is required for germline stem cell (GSC) maintenance in the Drosophila testis. Here, we show that mitochondrial fission is also important for regulating the maintenance of early germ cells in larval testes. Inhibition of Drp1 in early germ cells resulted in the loss of GSCs and spermatogonia due to the accumulation of reactive oxygen species (ROS) and activation of the EGFR pathway in adjacent somatic cyst cells. EGFR activation contributed to premature germ cell differentiation. Our data provide insights into how mitochondrial dynamics can impact germ cell maintenance and differentiation via distinct mechanisms throughout development