Primordial Germ Cells Apoptosis Research Articles

021.Identification and study of genes important for fetal germ cell biology in mice

We are using a multi-pronged approach to discovering genes and proteins that regulate the allocation, proliferation, migration, differentiation and apoptosis of primordial germ cells (PGCs) in the developing mouse embryo. First, we are using suppression PCR and microarray screening methods to identify genes whose expression is restricted to, or enriched in, gonads of a specific sex or developmental stage. In this way several genes were identified whose expression is restricted to germ cells. Second, we are using a proteomic approach to identify important proteins and the genes that encode them. Protein expression profiles are being compared between different sexes and stages of mouse fetal gonad development. Data so far indicate that this method is a useful adjunct to transcriptional profiling, capable of identifying not only proteins that are differentially expressed, but also those that are differentially modified, for example by phosphorylation. Third, in silico screening of mouse EST databases identified 23 new candidate genes whose expression appears to be limited to pluripotent cells and the germline. Many of these genes are novel uncharacterised transcripts. Preliminary in situ expression analyses show that eight of these genes are indeed limited to the germline and to pluripotent cells. These genes may have important functions in germline specification and function. We are currently developing approaches, including inducible RNAi-based methods, for examining the function of these genes, initially in vitro but also ultimately in vivo.

Ethylnitrosourea-induced apoptosis in primordial germ cells of the rat fetus

Ethylnitrosourea (ENU) is a simple alkylating agent. It induces gene mutations in fetal primordial germ cells (PGCs), and a high incidence of congenital malformations is also found in the offspring of male mice treated with ENU at the embryonic stage. It is also reported that decreases in the fertility rate and weights of the testis and ovary were found in the offspring from dams treated with ENU. In this study, we analyzed the occurrence of apoptotic cell death and the expression of p53 protein which is thought to play an important role in the DNA damage-induced apoptosis after administration of ENU to pregnant rats on day 13 of gestation to obtain a clue for clarifying the toxic effect of ENU on PGCs. Apoptotic cells increased in PGCs in fetal gonads from 3 h after treatment. The number of apoptotic PGCs peaked at 6 h and gradually decreased towards 24 h after treatment. On the other hand, p53-positive PGCs increased from I h after treatment, prior to the induction of apoptosis. The number of p53-positive PGCs peaked at 3 h and returned to the control level at 24 h after treatment. These results suggest that ENU induces apoptosis in rat fetal PGCs immediately after its administration to dams and excess cell death by apoptosis may have a close relation to the later occurrence of decreases in the fertility rate and gonadal weight. Moreover, a possible involvement of p53 is suggested in the ENU-induced apoptosis in PGCs.

Theter/terGonadal Somatic Cells Cause Apoptosis inter/terPrimordial Germ Cells (PGCs) with Normal Survivability and Proliferation Ability in the Mouse: Evidence from PGC-Somatic Cell “Exchange-Co-Culture”

Abstract The ter (teratoma, chromosome 18) mutation causes a deficiency in primordial germ cells (PGCs) of ter/ter embryos from ter congenic mouse strains at around 8.0 days post coitum (dpc). Our previous studies indicated that in vivo, apoptosis of PGCs was caused by ter/ter gonadal somatic cells. To examine survival and proliferation in ter/ter PGCs and the deficiency caused by ter/ter gonadal somatic cells in vitro, we performed “exchange-co-culture” of PGCs and gonadal somatic cells by combining different tergenotypes, on Sl/Sl4-m220 feeder cells. The number of PGCs after 3 days culture of 9.5 dpc ter/ter PGCs with +/+ 12.5 dpc gonadal somatic cells increased similar to that of +/ter or +/+ PGCs. The numbers of PGCs after 12 hr culture of +/+ and ter/ter 11.5 dpc PGCs with 11.5 dpc ter/ter gonadal somatic cells decreased significantly when compared to those cultured with +/+ somatic cells. PGCs preferred the WT1-positive gonadal somatic cells, Sertoli cells, to the feeder cells. Both TUNEL and BrdU a...

Bcl-x and Bax regulate mouse primordial germ cell survival and apoptosis during embryogenesis.

Restricted germ cell loss through apoptosis is initiated in the fetal gonad around embryonic day 13.5 (E13.5) as part of normal germ cell development. The mechanism of this germ cell attrition is unknown. We show that Bcl-x plays a crucial role in maintaining the survival of mouse germ cells during gonadogenesis. A bcl-x hypomorphic mouse was generated through the introduction of a neomycin (neo) gene into the promoter of the bcl-x gene by homologous recombination. Mice that contained two copies of the hypomorphic allele had severe reproductive defects attributed to compromised germ cell development. Males with two mutant alleles lacked spermatogonia and were sterile; females showed a severely reduced population of primordial and primary follicles and exhibited greatly impaired fertility. Primordial germ cells (PGCs) in bcl-x hypomorph mice migrated to the genital ridge by E12.5 but were depleted by E15.5, a time when Bcl-x and Bax were present. Two additional bcl-x transcripts were identified in fetal germ cells more than 300 bp upstream of previously reported start sites. Insertion of a neo cassette led to a down-regulation of the bcl-x gene at E12.5 in the hypomorph. Bax was detected by immunohistochemistry in germ cells from bcl-x hypomorph and control testes at E12.5 and E13.5. Bcl-x function was restored, and animals of both genders were fertile after removal of the neo selection cassette using Cre-mediated recombination. Alternatively, the loss of Bcl-x function in the hypomorph was corrected by the deletion of both copies of the bax gene, resulting in a restoration of germ cell survival. These findings demonstrate that the balance of Bcl-x and Bax control PGC survival and apoptosis.

Germ line development in fishes.

Classical work on germ cells in fishes has dealt with three main issues; their embryonic origin, the proliferation, and migration pathway during embryonic and larval development. Until recently, primordial germ cells (PGCs) have been studied in a number of fishes using morphological criteria only. The identification of the Drosophila vasa homolog gene of zebrafish now allows comparison of these morphological data with vasa RNA expression patterns in zebrafish. Teleost PGCs can be distinguished from somatic cells by their distinct morphology, at the earliest during gastrulation, and in most fishes their number varies between 10 and 30 during pregonial development. Mitosis is generally not observed in PGCs at extragonadal locations, whereas they are mitotically active once at the gonadal ridges. During gastrulation, PGCs appear to translocate from the epiblast to the hypoblast and during somitogenesis they are found associated with the most peripheral yolk syncitial layer (YSL). From the peripheral YSL they migrate through the median mesoderm into the dorsal mesoderm and then to the dorsal mesentery, where they establish the gonad primordia with mesenchymal cells. Vasa RNA positive cells, the PGCs of the zebrafish conform to these general observations. Interestingly, classical descriptive and experimental data can now be reevaluated using vasa as a molecular marker of the fish germ line. The power of zebrafish genetics together with possibilities of experimental embryology should accelerate research on aspects of vertebrate germ line development such as PGC migration, division and apoptosis, as well as (in) fertility. The present review summarizes some of the classical data on germ line development in fishes in relation to recent data on vasa expression in zebrafish and compares these findings, where appropriate, with those in other model organisms. Special emphasis is placed on vasa gene expression as a potential universal germ line marker and suggestions are made for novel, zebrafish specific approaches to investigate the vertebrate germ line.

Gene Transfection of Mouse Primordial Germ Cellsin Vitroand Analysis of Their Survival and Growth Control

We evaluated electroporation, liposome-mediated transfection, and the calcium phosphate (CaPO4) coprecipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to the investigation of molecular mechanisms of the germ cell development. We found that electroporation severely damaged PGCs, and the efficiency of liposome-mediated transfection was very low. In contrast, using the CaPO4coprecipitation method, 18% of PGCs transfected with plasmid pSV-LT expressed simian virus 40 large tumor antigen (SV 40 T-Ag) transiently. However, we did not detect any effects on the proliferation and survival of PGCs obtained from the embryonic gonads at 11.5 days postcoitum (d.p.c.) during 2 days of culture after the transfection. PGCs isolated from the 11.5-d.p.c. gonads change from spread- to round-shape and exhibit growth arrest during a few days of culture, and these rounded PGCs quickly disappear from the culture. We found that the transfection and expression of Bcl-xlor adenovirus type 2 E1B 19,000-molecular-weight protein (E1B 19K) significantly promoted the survival of PGCs and retarded the disappearance of rounded PGCs from the culture system. These results suggest that the Bcl-xlor E1B 19K can prevent the apoptosis of PGCs and inhibit the cell death of the rounded PGCs in culture.

Growth factors in mouse primordial germ cell migration and proliferation

Information obtained mainly from in vitro culture studies and genetic analysis of mouse mutants White spotting and Steel indicate a pivotal role of growth factors in the development of mouse primordial germ cells (PGCs). While stem cell factor (SCF) and TGFβ1 seem to have a role in PGC migration (as an adhesion factor and a chemoattractant, respectively), the former is certainly required for PGC survival in vitro and probably in vivo as well. Recent findings suggest that the mechanism by which SCF supports PGC survival is by preventing PGC apoptosis. A similar action appears to be exerted by leukemia inhibitory factor (LIF), a further growth factor influencing PGC growth in culture. PGC proliferation seems to be mainly induced by cAMP dependent mechanisms, but futther investigations are needed to clarify the interrelationships among the different molecular pathways activated by SCF, LIF, cAMP and other putative PGC growth factors (i.e. bFGF). Stimulation of long-term proliferation of PGCs, leading to derivation of ES-like cells (embryonal germ cells) obtained by using a combination of growth factors (bFGF, SCF and LIF), opens new intriguing perspectives for such studies and transgenic technology.