Proliferation Of Primordial Germ Cells Research Articles

Spatial and temporal patterns of c-kit-expressing cells in WlacZ/+ and WlacZ/WlacZ mouse embryos.

In the mouse, the Kit receptor and its ligand, the stem cell factor (SCF), are encoded at the W/Kit and Steel loci, respectively. The Kit/SCF transduction pathway is involved in promoting cellular migration, proliferation and/or survival of melanoblasts, hematopoietic progenitors and primordial germ cells. Furthermore, a functional Kit/SCF pathway is required for the development of interstitial cells of Cajal (ICC) in the small intestine. Whereas all c-kit-expressing cells in embryogenesis were not identified, previous studies clearly demonstrated that the c-kit expression pattern extends well beyond cells known to be affected by W mutations. To investigate further Kit function, we specifically marked the c-kit-expressing cells and followed their fate during embryogenesis. A mutation was introduced by gene targeting at the W/Kit locus in mouse embryonic stem cells. The lacZ reporter gene was inserted into the first exon of c-kit, thus creating a null allele, called WlacZ. The lacZ expression reflects normal expression of the c-kit gene in WlacZ/+ embryos. The comparison of the patterns of lacZ-expressing cells between WlacZ/+ and WlacZ/WlacZ embryos allowed us to detect where and when melanoblasts, primordial germ cells and hematopoietic progenitors failed to survive in the absence of Kit. We also observed that ICC express c-kit during embryogenesis. ICC are found identically in WlacZ/+ and WlacZ/WlacZ embryos. Therefore, ICC do not depend on Kit expression during embryogenesis. These results indicate that the function of the c-kit gene is only required for the postnatal development of the ICC. Unexpected sites of c-kit expression were uncovered in embryos, including endothelial, epithelial and endocrine cells. None of these cells are dependent on Kit expression for their migration, proliferation and/or survival during embryogenesis. Nevertheless, we assume that the Kit/SCF pathway could be involved in the growth of transformed endothelial, epithelial and endocrine cells.

A combination of Buffalo rat liver cell-conditioned medium, forskolin and membrane-bound stem cell factor stimulates rapid proliferation of mouse primordial germ cells in vitro similar to that in vivo.

Recent studies have shown that stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and the enhancement of cAMP levels increase proliferation and survival of mouse primordial germ cells (PGC) in vitro. Even after the addition of these factors, however, it is still not possible to obtain proliferation of PGC at a rapid rate similar to that in vivo, suggesting the presenge of other growth factor(s) in vivo. We previously reported that tumor necrosis factor-α stimulates proliferation of PGC at earlier migration stages. We now show that the use of SI/SI4-m220 feeder cells and the addition of a medium conditioned with Buffalo rat liver cells and forskolin to the culture medium stimulate PGC obtained from 8.5 days post coitum embryos to proliferate in culture at a rate comparable to that in vivo. Under such conditions, proliferation of PGC continued several days past the timing of growth arrest in vivo; however, it did stop afterwards. Such proliferating PGC continue to express c-kit and Oct-3 proteins. The characteristics of the culture medium and the requirement of feeder cells were different from those for embryonic stem (ES) cells, suggesting that these rapidly proliferated PGC are not transformed into ES-like EG cells.

Testicular degeneration induced in rat offspring by maternal treatment with sobuzoxane

Pregnant rats were orally administered sobuzoxane (an antitumor drug) during various days of gestation and the testicular development of their offspring was examined histopathologically on day 21 after birth. When 15.6 to 250 mg/kg of sobuzoxane was given every day from day 7 to 17 of gestation, seminiferous tubules devoid of spermatogonia and/or spermatocytes were found in a dose-related manner. The critical period for testicular degeneration was identified to be day 13 and 14 of gestation by single administration experiments and single treatment with 15.6 to 250 mg/kg of sobuzoxane on day 14 of gestation gave a dose-dependent incidence of the testis abnormality. These results suggest that the testis abnormality in offspring from dams treated with sobuzoxane is due to inhibition of the proliferation of primordial germ cells during testis organogenesis.

Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells.

Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on SI/SI4-m220 cells. Interestingly, IL-11 stimulated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action of PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT-mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates adenylate cyclase and promotes proliferation of mouse primordial germ cells

During migration and for about 2 days after their arrival in the gonadal ridges, primordial germ cells (the embryonic precursors of gametes of the adult animal) proliferate actively. Certain growth factors, such as stem cell factor and leukemia inhibitory factor, seem to be essential for survival, proliferation and possibly differentiation of mouse primordial germ cell in vivo and/or in vitro. Similarly, increase in intracellular cAMP is followed by a marked enhancement of primordial germ cell proliferation, at least in culture. In the present study, we show that pituitary adenylate cyclase-activating polypeptides (PACAP-27 and PACAP-38), two neuropeptides of the secretin-glucagon-vasoactive intestinal polypeptide-GH-releasing hormone family, stimulate in vitro proliferation of mouse primordial germ cells, bind to primordial germ cells and gonadal somatic cells (possibly to type I PACAP receptor) and activate adenylate cyclase in the same cells. Moreover, PACAP-like immunoreactivity was found in gonadal ridges, mostly on germ cell surface. In conclusion, evidence is provided that PGC proliferation can be stimulated by certain bioactive polypeptides, thus suggesting a novel regulatory role for such compounds in early gonad development.

The germ cell deficient locus maps to mouse Chromosome 11A2?3

The autosomal recessive mouse mutation, germ cell deficient, gcd, manifests as infertility in both sexes owing to improper migration and/or proliferation of primordial germ cells during embryonic development. Mice harboring this mutation have been hypothesized to be animal models of the human syndromes, premature ovarian failure and Sertoli cell only syndrome. Since the gcd mutation arose from the insertion of over 100 kb of foreign DNA into the chromosome during a transgenic mouse experiment, fluorescent in situ hybridization with the transgene as a probe was used to determine the chromosomal position of the gcd locus. DAPI chromosomal banding in conjunction with double labeling with the alpha 1(I) collagen gene revealed that the gcd locus is situated on mouse Chromosome (Chr) 11A2-3. Two candidate genes, Lif and Oncostatin M, map near the gcd locus; however, Southern blot hybridization analysis revealed no gross rearrangements in these genes in gcd mice. The chromosomal position of the gcd locus will prove valuable in the search for other candidate genes as well as a landmark for positional cloning experiments.

Effects of feeder cells and growth factors on the proliferation of mouse primordial germ cells

There are various kinds of stem cell which have the ability to self-renew under appropriate microenvironments and growth factors. In vitro culture of stem cells may provide a tool for elucidating the mechanism of self-renewal of these cells. Hematopoietic B cell progenitor cell proliferation is supported on the born marrow-derived cell line, ST2. Embryonic stem (ES) cells derived from mouse blastocysts acquire their ability for self-renewal and pluripotency on mouse embryonic fibroblast cells (MEF). In the present study, we examined effects of these 2 different kinds of feeder cells, in the presence of growth factors, on the ability of mouse primordial germ cells (PGCs) collected at 8.5 d post coitum (dpc) to proliferate and acquire of pluripotency. The addition of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF), ST2, but not MEF, supported the proliferation of the PGCs for at least 5 d in culture. The presence of either LIF or bFGF renders a lower ability to support the proliferation of PGCs. Reverse transcription-PCR analysis shows that both feeder cells transcribed the soluble form of stem cell factor (SCF) but not membrane-bound SCF, which is an established factor related to the proliferation and pluripotency of PGCs. These results suggest that both hematopoietic stem cells and PGCs make use of the common growth factors for their growth in culture. In addition, the inability of MEF to affect proliferation of PGCs may suggest that the initial culture conditions for the proliferation could be different between ES cells and PGCs in the mouse.

The ter mutation first causes primordial germ cell deficiency in ter/ter mouse embryos at 8 days of gestation.

The ter (teratoma) mutation causes primordial germ cell (PGC) deficiency in ter/ter embryos at 9.5-12.5 days of post-coitum (dpc) in mouse strains 129/Sv-ter and LTXBJ-ter. To study the effects of the ter mutation on the PGC development more precisely, we examined the PGC number and distribution in 7.5-12.5 dpc embryo of ter congenic C57BL/6J-ter strain using their complete serial sections. The ter genotypes of embryos were identified by the polymerase chain reaction (PCR) polymorphisms of the microsatellite DNA of the Grl-1 locus mapped near the ter locus. Results showed that: (i) the PGC number in ter/ter embryos was similar to those of + /ter and + / + embryos at 7.5 dpc, and did not increase at 8.0-12.5 dpc, although those of normal littermates did usually; (ii) the PGC migration to genital ridges was never affected in all embryos; and (iii) the ter genotype difference in the PGC numbers was not recognized between + /ter and + / + embryos. We concluded that the ter mutation does not affect the PGC appearance around 7.5 dpc, but first causes PGC deficiency around 8.0 dpc at the beginning of their migration and proliferation, suggesting that the normal function of the ter gene may be essential for the proliferation or survival mechanisms of PGC.

Proliferation of chick primordial germ cells cultured on stroma cells from the germinal ridge.

Fluorescent reagent-labelled PGCs isolated from the blood of 2-day-old chick embryos were cultured on stroma cells derived from 5-day-old germinal ridge in Medium 199 supplemented with 10% FBS, human IGF-1, bovine FGF-b, and murine LIF. In 7 experiments, the number of MCs increased by an average of 4.8 fold in 4 days. Intrinsic PGCs in the 5-day embryonic germinal ridge were observed loosely attached to the stroma cells, and they also increased 3.8 fold during culture for 4 days. These results indicate the possibility of applying this culture method to the production of transgenic chickens.

Heterogeneity in the in vitro survival and proliferation of human seminoma cells.

The in vitro culture conditions allowing survival and initial proliferation of murine primordial germ cells from 10.5 days post coitum embryos, which include the use of a murine embryonal fibroblast (STO) feeder, were applied to 21 human seminomas, composed of tumour cells which are considered as the malignant counterparts of human primordial germ cells. Cells from 18 seminomas attached poorly to STO, and only a few survived through day 10. In contrast, three seminomas showed a higher degree of attachment. Two of them showed initial proliferation and enhanced survival: 30 days for tumour SE1 and 25 days for tumour SE3. Tumour SE1 was more extensively studied, using the culture conditions allowing the derivation of pluripotent embryonic stem cells from 8.5 days post coitum murine primordial germ cells, which include the use of STO feeder, stem cell factor, leukaemia inhibitory factor and basic fibroblast growth factor. The presence of stem cell factor was necessary and sufficient for colonies of tumour cells to form during the first 3 days of culture. While the cell number decreased after day 3 in medium without fetal calf serum, it increased until day 9 in medium containing fetal calf serum. No reprogramming of SE1 cells to pluripotent stem cells was observed. Our data indicate that seminomas form a tumour population with a heterogeneous in vitro behaviour not equivalent to that of 8.5-10.5 days post coitum murine primordial germ cells.

Stimulation of gonadal and germ cell development in larval and juvenile carp (Cyprinus carpio L.) by homologous pituitary extract

The data presented show that in larval carp, gonadal size has increased distinctly after treatment with homologous pituitary extract (PE). Moreover, the precocious onset of primordial germ cell proliferation and of sex differentiation into male and female gonads was induced. Body weight of treated and control specimens did not differ significantly from each other throughout the experiment. Treatment of juvenile carp with homologous PE led also to an increase in gonadal size without concomitant increase in body weight. Induction of precocious spermatogenesis was observed too. GTH-levels in control and PE-treated carp were monitored by means of a homologous radioimmunoassay, showing that in PE-treated carp the GTH-level was distinctly elevated. The possible role of pituitary hormones in larval and juvenile gonadal development is discussed.

Apoptosis in mouse primordial germ cells: a study by transmission and scanning electron microscope.

A detailed study of the death in vitro of mouse primordial germ cell (PGCs) by means of transmission and scanning electron microscopy is reported. The results show that after 4-5 h of culture 15-20% PGCs assume the typical morphological features of apoptotic cells, including chromatin condensation in dense masses under the nuclear membrane, compaction of the cytoplasm, crowding of organelles and surface protuberances. Cells then break up into discrete fragments (apoptotic bodies) which eventually degenerate by "secondary necrosis". It is possible that apoptosis plays a biologically useful role in avoiding uncontrolled PGC proliferation and in eliminating misplaced germ cells whose survival might be harmful to the animal.

Tumor Necrosis Factor-α (TNF-α) Stimulates Proliferation of Mouse Primordial Germ Cells in Culture

Recent studies have shown that stem cell factor, leukemia inhibitory factor, and basic fibroblast growth factor increase proliferation and survival of the mouse primordial germ cells (PGCs) in culture. We now show that addition of tumor necrosis factor-α (TNF-α) to culture medium stimulates proliferation of PGCs without transforming them into embryonic stem cells and that its effect is specific for the PGCs at younger stages before and during their migration to gonads. A previously reported finding that TNF-α is expressed at these stages in mouse embryos suggests possible involvement of TNF-α in the proliferative regulation of the PGCs in the embryo.

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.

Proliferation of mouse primordial germ cells on the bone marrow-derived stromal cell line ST2

Proliferation of mouse primordial germ cells on the bone marrow-derived stromal cell line ST2

Proliferation and migration of primordial germ cells in We/We mouse embryos.

We have examined the numbers and distribution of primordial germ cells in We/We, We/+, and +/+ mouse embryos using Southern blotting to determine embryo genotypes. At early somite stages (5-7 somites: approximately 8 1/2 days post coitum [dpc]) there are 50 to 100 germ cells in embryos of all genotypes. The number of germ cells in We/+ and +/+ embryos then begins to increase: at later somite stages (17-19 somites: approximately 9 1/2 dpc) they number about 200, and by 10 1/2 dpc there are approximately 725 We/+ and 850 +/+ germ cells. During this time, however, the number of germ cells in We/We embryos remains less than 100. At 8 1/2 dpc, the distribution of germ cells in the hindgut endoderm is the same in all genotypes. By 9 1/2 dpc, 30% of We/We germ cells are found in ectopic sites (allantois and vitelline artery); germ cell distribution along the length of the hindgut appears normal, but germ cells remain confined to the floor of the gut in We/We embryos, rather than being distributed around its circumference as in the other two genotypes. By 10 1/2 days, the migration of We/We germ cells through the dorsal mesentery lags behind that of the other genotypes, and a larger proportion remains in the gut wall.

Combined action of stem cell factor, leukemia inhibitory factor, and cAMP on in vitro proliferation of mouse primordial germ cells

In the present paper we investigated the effects of stem cell factor/mastocyte growth factor (SCF/MGF), leukemia inhibitory factor/differentiating inhibitory activity (LIF/DIA) (two growth factors known to affect primordial germ cell growth in vitro) and forskolin (FRSK) (an activator of adenylate cyclase in many cell types) alone or in combination on the survival and proliferation of primordial germ cells (PGCs) obtained from 8.5, 10.5, and 11.5 days post coitum (dpc) mouse embryos and cultured without pre-formed cell feeder layers. The results showed that both at 1 and 3 days of culture the addition of 100 ng/ml SCF, 20 microM FRSK, or in some instances 20 ng/ml LIF alone caused a significant increase of PGC number as compared with controls. The highest effects were obtained when SCF and/or LIF were used together with FRSK. Moreover, we found that FRSK elevated cAMP levels in purified 11.5 dpc PGCs and that this compound, but not SCF and LIF, stimulated PGC proliferation, as assessed by 5-bromo-2'-deoxyuridin (BrdU) incorporation. These results suggest a mechanism of combined action of cAMP with SCF and/or LIF in the control of proliferation of mouse PGCs in vitro.

Migration, proliferation and potency of primordial germ cells

In most species, the cells allocated to the germ line, the primordial germ cells (PGCs) arise very early in embryo-genesis, and migrate to join the somatic cells at the site where the gonad will form. In three widely studied animals; the mouse, the frog and Drosophila, the PGCs associate with the developing gut, from which they migrate during the period of organogenesis to the gonads. During this migration, the germ cell population increases by an amount which is more or less constant for a particular species. Genes important in the control of PGC migration and population are being identified in two ways. In invertebrates, and to a lesser extent in mice, genetic approaches have identified important loci or gene products. Culturing PGCs in a variety of conditions has been an alternative approach in mouse embryos. From these latter studies, it is now known that a number of growth factors, released from surrounding tissues, control many aspects of PGC behaviour, including their proliferation, migration, potency, and survival. Attention is also focusing on changes in PGC adhesiveness during migration.

Proliferation of Mouse Primordial Germ Cells in Vitro: A Key Role for cAMP

Two agents known to enhance the level of intracellular cAMP (dibutyryl cAMP and forskolin) markedly increase the number of 8.5, 10.5, and 11.5 days postcoitum (dpc) mouse primordial germ cells (PGCs) cultured on TM 4 cell feeder layers. Forskolin (FRSK) caused a significant increase of PGC number also in monodispersed cell suspensions obtained from PGC-containing tissues of the three embryonic ages studied and in purified 11.5 dpc PGCs cultured without feeder layers. The addition to the culture medium of adenosine-3′,5′-cyclic monophosphorothioate RP isomer (Rp-cAMPS, a competitive antagonist for cAMP-dependent protein kinases), significantly reduced the effects of FRSK. Last, FRSK stimulated PGC proliferation, as assessed by 5-bromo-2′-deoxyuridin incorporation. We conclude that cAMP-dependent mechanisms play a crucial role in the control of mitotic proliferation of mouse PGCs in culture.

Leukaemia inhibitory factor is necessary for maintenance of haematopoietic stem cells and thymocyte stimulation.

Leukaemia inhibitory factor (LIF) has a variety of effects on different cell types in vitro, inhibiting the differentiation of embryonic stem cells and promoting the survival and/or proliferation of primitive haematopoietic precursors and primordial germ cells. Here we show that LIF-deficient mice derived by gene targeting techniques have dramatically decreased numbers of stem cells in spleen and bone marrow. Injection of spleen and marrow cells from these mice promotes long-term survival of lethally irradiated wild-type animals, however, showing that the LIF- stem cells remain pluripotent. The numbers of committed progenitors are also reduced in the spleen but not the bone marrow, suggesting that stem cells interact differently with the splenic and medullary microenvironment. Heterozygous animals are intermediate in phenotype, implying that LIF has a dosage effect, and defects in stem cell number can be compensated by exogenous LIF. LIF thus appears to be required for the survival of the normal pool of stem cells, but not their terminal differentiation.