| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TISSUE-SPECIFIC STEM CELLS |
Murdoch Children's Research Institute, ARC Centre of Excellence in Biotechnology and Development, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Melbourne, Victoria, Australia
Key Words. Fetal • Germ cells • Cell cycle • Mitotic arrest • Retinoblastoma • p27Kip1
Correspondence: Correspondence: Patrick S. Western, Ph.D., Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria 3052, Australia. Telephone: 61-3-83416353; Fax: 61-3-83416429; e-mail: patrick.western{at}mcri.edu.au
Received on August 1, 2007;
accepted for publication on November 5, 2007.
First published online in STEM CELLS EXPRESS November 15, 2007.
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
Disclosure of potential conflicts of interest is found at the end of this article.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
Mitosis is characterized by the phases G1, S, G2, and M, with strict regulation of the cyclin/cyclin-dependent kinase (CDK) complexes involved in each phase. More specifically, a central function of CyclinD-cdk4/6 and CyclinE-cdk2 complexes is to hyperphosphorylate and inactivate the critical checkpoint regulator retinoblastoma protein (pRB), allowing the cell cycle to proceed. Similarly, two pRB-like proteins, p107 and p130, also play important roles in G1-S-phase checkpoint regulation [11]. In its hypophosphorylated form, pRB binds E2Fs, repressing the transcription of genes required for DNA synthesis. Hyperphosphorylation of pRB allows its dissociation from E2F containing complexes and DNA synthesis to proceed, effectively inactivating the G1-S-phase checkpoint for that cell cycle. In response to antiproliferation signals, key proteins inhibiting the G1-S-phase transition are activated [12, 13]. These include members of the INK4 and Cip/Kip cell cycle inhibitor families, which inhibit the CyclinD-cdk4/6 and CyclinE-cdk2 complexes and hence prevent phosphorylation of pRB. The resulting hypophosphorylated form of pRB forces cells into quiescence through activation of the G1-S-phase checkpoint. Therefore, regulated expression of the Cip/Kip (p21Cip1, p27Kip1, and p57Kip2) and INK4 (p15INK4b, p16INK4a, p18INK4c, and p19INK4d) cell cycle inhibitors is essential in the control of G1/G0 arrest.
Surprisingly, the G1-S-phase activators and inhibitors remain to be examined in arresting fetal germ cells. Since the timing of germ cell cycle arrest has been poorly defined, the studies performed to date have focused mainly on germ cells that have undergone arrest. Previous studies have examined germ cell proliferation from E14 until E18 [7] but have not assessed proliferation in younger germ cells. Although several reports have examined expression of isolated G1-S-phase regulators in arrested and apparently cycling germ cells [14, 15], an important limitation of these studies is the lack of a comprehensive and direct comparison of the G1-S-phase cell cycle regulators in cells shown to be undergoing mitotic arrest. Interestingly, it has been shown that common molecular abnormalities found in testis cancer include mutations in some regulators of the G1-S-phase of the cell cycle [16]. Defining the normal function of these genes in the cell cycle of developing germ cells promises to ultimately increase our understanding of testis cancer.
This study examines the timing and molecular control of mitotic arrest of male germ cells in developing mouse embryos. To clarify the timing of mitotic arrest, we developed a flow cytometric protocol to examine the progression of germ cells through mitotic arrest. By analyzing DNA content, 5-bromo-2'-deoxyuridine (BrdU) incorporation and Ki67 expression in developing fetal mouse germ cells, we show that male germ cell arrest occurs in the G0 stage of the cell cycle between E12.5 and E14.5. Furthermore, we analyzed the molecular dynamics of G1-S-phase progression in arresting male germ cells and show that this arrest phase is likely to be mediated primarily by preventing pRB protein phosphorylation through the activation of p27Kip1 and p15INK4b and by suppressing CyclinE1 and CyclinE2. For the first time, the data presented here accurately define the mitotic arrest of male germ cells by combining the analysis of cell cycle changes with the examination of functionally defined cell cycle regulators.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
Flow Cytometry
Pregnant mice were injected intraperitoneally with 100 mg/kg BrdU and euthanized 2 hours after the injection. Gonads were dissected, and single-cell suspensions were obtained by dissociation in 0.25% Trypsin/EDTA. Flow cytometry analyses were carried out using the BrdU-allophycocyanin (APC) Flow Kit (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) according to the manufacturer's instructions. Germ cells were identified by mouse vasa homolog (MVH) antibody (1/2,000) staining followed by goat anti-rabbit Alexa 488 secondary antibody (1/500; Molecular Probes, Eugene, OR, http://probes.invitrogen.com), and BrdU-positive cells with anti-BrdU-APC-conjugated antibody. Samples were resuspended in 500 µl of phosphate-buffered saline (PBS)/0.5% bovine serum albumin containing 100 µg/ml propidium iodide and 200 µg/ml RNase A. Flow cytometry was performed using an LSR II instrument (BD Biosciences, San Diego, http://www.bdbiosciences.com), and data were analyzed using FACSDiva (BD Biosciences) and Modfit LT cell cycle analysis (Verity Software House, Topsham, ME, http://www.vsh.com) software. MVH-positive and -negative populations were gated, and BrdU staining was assessed in each population. A minimum of 25,000 germ cells were analyzed for each sample. BrdU gating was set against samples obtained from mice exposed to BrdU but not to the BrdU antibody (Fig. 1A).
|
Reverse Transcription and Real-Time Polymerase Chain Reaction Analysis
One hundred nanograms of aRNA was reverse transcribed using random hexamers and Superscript III (Invitrogen) according to the manufacturer's instructions. Real-time polymerase chain reaction (PCR) was performed using the mouse Universal Probe Library (UPL) system and Faststart Taqman Probe Master mix with ROX (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). One nanogram of cDNA was subject to amplification (ABI 7900 HT instrument, Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Samples were run in triplicate, and experiments were performed twice. All samples were normalized against a combination of Hprt and MapK1 using the comparative CT method (
CT). The genes against which normalization was carried out were chosen from a panel of nine candidate genes. The transcriptional stability of these genes was assessed using reverse transcription and quantitative real time polymerase chain reaction analysis (qRTPCR) of E12.5–E15.5 germ and somatic cell aRNA and geNorm software analysis (supplemental online Fig. 2) (http://www.medgen.ugent.be/
jvdesomp/genorm/) [18]. Cycle parameters and sequences for all qRTPCR primer/UPL probe set combinations used are included in supplemental online Table 1. For all qRTPCR data presented here, no template controls exhibited no amplification. In addition, each amplification set was performed with standard curves to confirm primer/probe efficiency.
Immunoblotting
Pure germ cell and somatic cell populations were sorted from dissociated gonads as described above. Cells were collected by centrifugation and resuspended in 2 x Laemmli buffer at a concentration of 10,000 cells per microliter based on the FACS cell counts and immediately boiled for 15 minutes. Five microliters of cell lysate was loaded in each lane on 7%–15% polyacrylamide gels (as appropriate), and immunoblotting was performed according to standard protocols. Antibodies were obtained from commercial sources and identified single bands of the appropriate sizes on all immunoblots shown (Figs. 3 and 4). Antibodies used for immunoblotting and immunofluorescence are listed in supplemental online Table 2.
Fixation of Tissue and Immunofluorescence
Gonads with mesonephros were dissected and fixed in PBS containing 4% paraformaldehyde for 15–75 minutes at room temperature. Ten-micron sections were collected and immunofluorescence was performed as described previously [19]. Images were obtained using a Leica TCS SP2 SE laser scanning confocal microscope (Leica, Heerbrugg, Switzerland, http://www.leica.com). All images displayed are single optical sections. All antibodies raised in mouse were applied to sections using the Mouse on Mouse staining kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) according to the manufacturer's instructions. Finally, for all immunofluorescence experiments, control sections were incubated with secondary antibody only, to confirm that staining was due specifically to primary antibody binding (not shown).
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
To more accurately define the timing and dynamics of germ cell cycle arrest, we analyzed E11.5, E12.5, E13.5, and E14.5 developing embryos. At E11.5 and E12.5, BrdU was strongly incorporated in the DNA of both the germ and somatic cell populations isolated from male and female embryos collected from CD1 x C57Bl6 matings. Using this analysis, there were no significant differences in BrdU incorporation observed between E11.5 or E12.5 male and female germ cells, indicating that mitotic arrest is initiated at or after E12.5 in male germ cells (Fig. 1B). Interestingly, at E11.5 and E12.5, a significantly higher proportion of the germ cell population incorporated BrdU than the somatic population (at E12.5; 47.4% positive [+ve] compared with 13.4% +ve respectively) (Fig. 1C). The high level of BrdU incorporation in E11.5 and E12.5 germ cells demonstrates that at these stages, germ cells were rapidly cycling. In contrast to the high incorporation observed at E12.5, by E13.5 a significant proportion of the male germ cells had ceased to incorporate BrdU (28.8% +ve), and by E14.5 the germ cells no longer incorporated BrdU (1.7% +ve), strongly indicating that by E14.5 male germ cells had entered mitotic arrest (Fig. 1C). To determine the percentage of male germ cells in each stage of the cell cycle for each developmental stage, we used ModFit LT to analyze the proportions of the germ cell populations in G0/G1, S, and G2/M. These data revealed that 37.79%, 55.13%, and 94.97% of germ cells were in G1/G0 at E12.5, E13.5, and E14.5, respectively (Fig. 1C).
Absence of Ki67 staining is often used to distinguish quiescent cell populations arrested in G0 from those in the G1 or other phases of the cell cycle. To support the flow cytometric data and determine whether the developing germ cells were arrested in G0, we used immunofluorescence with Ki67 and germ cell nuclear antigen (GCNA; a germ cell-specific marker) antibodies to double-stain sections of developing gonads from E12.5–E15.5 male embryos. At E12.5, germ cells strongly positive for Ki67 were commonly detected. However, by E13.5, few Ki67 strongly positive germ cells were detected, and by E14.5, germ cells were Ki67-negative or very weakly positive, indicating that arrested germ cells had accumulated in G0 and were no longer cycling through G1 (supplemental online Fig. 1, E12.5 and E14.5 shown). These data, combined with the flow cytometric analysis presented here, show that male germ cell cycle arrest occurs in G0 during E12.5–E14.5.
Several Key Regulators of G1-S-Phase Are Transcriptionally Regulated During Mitotic Arrest
Having accurately determined the timing of G0/G1 arrest in developing male germ cells, we then investigated the molecular basis of germ cell cycle arrest. Initially, we used qRTPCR to quantitatively examine transcriptional changes in the key regulators of G1-S-phase. To achieve accurate quantification of mRNA levels in developing germ cells, we cleanly separated the gonad from the neighboring mesonephric tissue and purified germ cells from the surrounding gonadal somatic cells using FACS activated by Oct4-eGFP transgene expression (supplemental online Fig. 2A). Purity of the sorted cells was confirmed using several techniques. FACS analysis of the sorted germ and somatic cell populations revealed that the sorted cells were 99% pure, whereas no contaminating expression was detected in the germ cell population using qRTPCR for specific markers of germ and somatic cells (Mvh and Oct4, both specific to germ cells, and Sox9, specific to somatic cells) (supplemental online Fig. 2B). Western blotting revealed that MVH was detected only in the germ cell population and not the somatic cells (supplemental online Fig. 2C). These data confirm that both the germ cell and somatic cells isolated by FACS are of very high purity.
To ensure that an accurate quantitative real time PCR assay was used in this analysis, we used geNorm analysis to identify housekeeping genes that exhibited the highest transcriptional stability (geNorm Gene Stability Measure [M]) possible. This was achieved by analyzing the expression of nine candidate genes for constant expression in male E12.5–E15.5 germ and somatic cells. This resulted in the identification of MapK1 and Hmbs as the most transcriptionally stable genes tested in both germ and somatic cells (supplemental online Fig. 3). For the first time, this has allowed an accurate transcriptional expression analysis of genes in pure fetal germ cell RNA extracts using qRTPCR normalized against verified control genes.
qRTPCR analysis revealed striking suppression of CyclinE1 and CyclinE2 expression in E12.5–E14.5 male germ cells during the early stages of cell cycle arrest. Expression of CyclinE1 and CylcinE2 then remained low during E14.5–E15.5 (Fig. 2A). Although both CyclinE1 and CyclinE2 expression was also detected in somatic cells, the level of expression was comparatively low (4–5-fold lower than for E12.5 germ cells) and was not regulated. Analysis of CyclinD3 revealed similar levels of expression in somatic and germ cells at E12.5. However, CyclinD3 expression was then downregulated 2.5-fold between E12.5 and E13.5 in arresting germ cells, whereas its expression was maintained at similar levels in the somatic cells over the same period. By contrast, CyclinD1 and CyclinD2 expression in germ cells was 20–100-fold lower than the maximal levels detected in the somatic cells, strongly indicating that these two cyclins are not expressed at functional levels in fetal male germ cells.
|
The predominant inhibitors of CyclinD-cdk4/6 and CyclinE-cdk2 activity are the INK4 and Cip/Kip family members. Examination of Cip/Kip transcription revealed relatively strong male germ cell expression of p21Cip1 at E12.5 and E13.5, a sharp increase in germ cell expression of p27Kip1 between E12.5 and E13.5, and a 3.5-fold increase in p57Kip2 expression between E12.5–E15.5 (Fig. 2C). For p21Cip1, expression decreased during E14.5 and E15.5, whereas for p27Kip1 and p57Kip2, the levels at E15.5 were similar to those observed at E13.5. Transcription of the INK4 inhibitors varied considerably in arresting male germ cells. Both p15INK4b and p16INK4a were transcribed exclusively in germ cells, whereas p19INK4d transcription was similar in germ and somatic cells, and p18INK4c was predominantly transcribed in somatic cells (Fig. 2D). Strikingly, p16INK4a was upregulated 120-fold in E15.5 compared with E12.5 male germ cells. Although this is a huge increase in transcriptional activity, much of this upregulation occurred in the later stages of arrest (at E14.5, p16INK4a was increased 20-fold, and at E15.5, p16INK4a was increased 120-fold in germ cells), both stages in which germ cell DNA synthesis had been abolished (Figs. 1C, 2D). p15INK4b transcription also steadily increased in arresting germ cells, so that by E13.5 and E14.5 expression was twofold and fourfold higher than at E12.5, respectively. In summary the transcriptional analysis of G1-S-phase regulators indicates that arresting germ cells express the pocket protein pRB; the G1 cyclins CyclinE1, CyclinE2, and CyclinD3; the INK4 members p15INK4b and p16INK4a; and the Cip/Kip members p21Cip1, p27Kip1, and p57Kip2.
The G1-S-Phase Proteins p27Kip1 and pRB Are Strongly Regulated During the Critical Phase of Germ Cell Mitotic Arrest
Next, we examined expression of pRB, p16INK4a, p21Cip1, p27Kip1, and p57Kip2 using immunoblotting and/or immunofluorescence. Due to the lack of a suitable antibody, p15INK4b was not examined. Immunoblotting of E12.5–E15.5 pure germ cell extracts (50,000 germ cells per lane) showed that pRB was almost undetectable in somatic cells but was strongly expressed in male and female germ cells (somatic data not shown). In E12.5 male and female germ cells, pRB was detected almost exclusively in its hyperphosphorylated form (Fig. 3A). However, 1 day later, male-specific, rapid dephosphorylation of pRB had occurred to the extent that virtually all pRB was hypophosphorylated in E13.5 male germ cells. This dephosphorylation event was then followed by degradation of the pRB protein, so that by E15.5 pRB was detected only at very low levels in male germ cells (Fig. 3A). Use of immunofluorescence to examine pRB expression in E12.5–E14.5 developing gonads revealed that pRB was expressed exclusively by germ cells, and its expression was downregulated during E12.5–E14.5, supporting the result obtained using Western blotting (Fig. 3B). This striking regulation of pRB protein phosphorylation and expression level indicates that pRB mediates mitotic arrest of germ cells through its control of the G1-S-phase checkpoint and that once arrested, germ cells no longer require pRB. By contrast to males, in females, pRB was almost completely phosphorylated, showing that pRB phosphorylation was not inhibited in E12.5–E15.5 female germ cells (Fig. 3A). Interestingly, pRB protein was not degraded in female germ cells by E15.5.
|
|
Specific expression of all three Cip/Kip proteins and the INK4 inhibitor p16INK4a was detected in developing germ cells using immunofluorescent analysis of gonad sections. p27Kip1 expression was not detected at E12.5, but robust expression was detected throughout the nucleus of germ cells at E14.5. Similarly, both p21Cip1 and p57Kip2 were detected in E14.5 germ cells, but only very weakly in E12.5 male germ cells (Fig. 4B–4D). Finally, expression of p16INK4a was weak or undetected in E12.5 male germ cells but increased strongly in both the cytoplasm and nucleus of arrested germ cells at E14.5 (Fig. 4E). Interestingly, the expression domains of p27Kip1, p21Cip1, and p57Kip2 differed substantially in male germ cells. Whereas p27Kip1 was distributed in an even speckled pattern through the nucleus, p21Cip1 protein was located in several highly localized spots within the nucleus, and p57Kip2 was expressed in a perinuclear spot in all germ cells (Fig. 4F). By contrast to the germ cell staining observed for p57Kip2, highly specific nuclear staining was observed in some somatic cells of the gonad. It therefore appears that although all three Cip/Kip inhibitors are expressed during germ cell arrest their distribution, and presumably their functions in arresting germ cells are distinct.
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
To definitively examine mitotic arrest of fetal male germ cells, we established a flow cytometric protocol that had the ability to specifically and reproducibly analyze the developing germ cell population. This technology proved powerful in that it allowed the consistent cell cycle analysis of large numbers of fetal germ cells from an in vivo environment. This has never been achieved for fetal germ cells and promises to allow analysis of many aspects of germ cell development during this critical phase. In addition, we isolated germ cells undergoing mitotic arrest and directly monitored levels of protein and RNA expression in these populations using immunoblotting, immunofluorescence, and qRTPCR. This combination of approaches has provided a system in which we can accurately and directly compare the stages of mitotic arrest with protein and RNA expression profiles during fetal germ cell development in cells collected in vivo.
Flow cytometric analysis of germ cell cycle state coupled with BrdU incorporation revealed that mitotic arrest occurs rapidly between E12.5 and E14.5 in male germ cells. In addition, the decrease in BrdU incorporation between E12.5 and E14.5 (Fig. 1B) indicates that arrest of the germ cell population occurs in an unsynchronized manner. Finally, our unpublished data indicate that differences of as much as 24 hours exist in the timing of mitotic arrest in outbred CD1 and inbred C57Bl6 mouse strains. Despite this, analysis in inbred C57Bl6, outbred CD1, or a cross between these two strains shows that the process of mitotic arrest in both outbred and inbred strains is similar and occurs between E12.5 and E14.5.
An Early Response of Arresting Germ Cells Is the Upregulation of p27Kip1 and Dephosphorylation of pRB
The flow cytometric data presented here show that at E11.5 and E12.5, male and female germ cells are rapidly cycling. In support of these data, we observed extensive expression of Ki67 and a particularly high proportion of hyperphosphorylated pRB in germ cells at this time. This indicates that although the signaling mechanism initiating male commitment of the germ cells may be active at E12.5 [8], the germ cells have not yet substantially altered their cell cycle in response to that signal.
However, just 1 day later, we observed substantial molecular changes specifically in male germ cells, including strong upregulation of p27Kip1 and p15INK4b, together with dephosphorylation of pRB. Flow cytometric analysis of germ cells shows that although E12.5 germ cells are still strongly cycling, they have clearly begun arrest by E13.5 (Fig. 1B). This is reflected in a substantial decrease in BrdU incorporation and an increase in the percentage of germ cells in G1/G0. When protein expression changes were analyzed in equivalent cycling (E12.5) and arresting (E13.5) male germ cells, p27Kip1 and p15INK4b became strongly upregulated. Also, during this time, pRB changed from being almost completely hyperphosphorylated to being predominantly hypophosphorylated, and CyclinE was strongly suppressed at the transcriptional level. Therefore, in the early stages of male germ cell cycle arrest, protein expression and phosphorylation of the key functional regulators of G1-S-phase transition, p27Kip1, p15INK4b, pRB, and CyclinE, are substantially altered.
It has been established that an unknown signal produced by the somatic cells of the gonad initiates male germ cell sex determination at around E12.5 [8]. It has also been well-established that p27Kip1 and p15INK4b function to mediate cell cycle arrest by inhibiting CyclinE-cdk2 and CyclinD-cdk4/6 complexes, respectively. This prevents phosphorylation of pRB and allows hypophosphorylated pRB to block the G1-S-phase transition and to suppress CyclinE expression [13, 21, 22]. These observations combined with the current data have led us to propose a functional model for male germ cell arrest.
Germ cell-specific upregulation of p27Kip1 and p15INK4b appears to be a relatively direct response to the signaling mechanism that communicates an antiproliferative signal from the somatic cells to the germ cells. Given the established role of p27Kip1 and p15INK4b in mediating cell cycle arrest, the upregulation of p27Kip1, p15INK4b, and p16INK4a during germ cell arrest indicates that these proteins play important roles in mediating mitotic arrest in male germ cells. Coincident with the upregulation of p27Kip1 and p15INK4b, we observed a rapid loss of hyperphosphorylated pRB. We propose that in arresting male germ cells, p27Kip1 and p15INK4b respond to a somatic cell-derived antiproliferative signal and are upregulated. These proteins then inhibit CyclinE-cdk2 and CyclinD-cdk4/6, respectively, preventing hyperphosphorylation of pRB in arresting germ cells. The change in balance from predominantly hyperphosphorylated pRB (at E12.5) to the partially hyperphosphorylated form (E13.5) and finally to only the hypophosphorylated form (by E14.5) allows pRB to activate the G1-S checkpoint across the germ cell population and mediate cell cycle arrest. Once lost, pRB hyperphosphorylation would not be reestablished in male germ cells that have upregulated p27Kip1 and p15INK4b, as these proteins inhibit this phosphorylation through their action on the CyclinE-cdk2 and CyclinD-cdk4/6 complexes (Fig. 5). An important additional role of pRB in the early stages of germ cell arrest may be to stabilize p27Kip1 by initiating degradation of the p27Kip1-destabilizing protein Skp2, through the APC/CCdh1 complex [23]. In addition, since p16INK4a is also strongly upregulated at the transcriptional level in the later stages of mitotic arrest, it may also perform an important role in the inhibition of the CyclinD-cdk4/6 complex during the later stages of mitotic arrest in fetal male germ cells.
|
Since p21Cip1, p57Kip2 and p16INK4a are also upregulated in E14.5 germ cells, the expression of these proteins may support the role of p27Kip1 and p15INK4b in mitotic arrest. However, distinct distributions of the Cip/Kip proteins were observed within (p21Cip1 and p27Kip1) and next to (p57Kip2) the nucleus (Fig. 4E), suggesting that the role of each of these proteins during germ cell mitotic arrest is functionally distinct. Despite these distinct patterns, the complex regulation of several key cell cycle inhibitors (p27Kip1, p21Cip1, p57Kip2, p15INK4b, and p16INK4a) during the early (E12.5–E13.5) and later (E13.5–E14.5) stages of mitotic arrest may provide opportunities for functional backup mechanisms that ensure the arrest of germ cells in situations where the function of one of these members is lost. Redundancy in the mechanisms underlying mitotic arrest may explain the apparent lack of obvious germ cell arrest phenotypes in some knockout mouse models of G1-S-phase inhibitors. However, it is interesting to speculate that loss of pRB may result in aberrant mitotic arrest in male germ cells, as this is the single predominant RB-like protein whose expression and phosphorylation state is regulated during early germ cell arrest. This remains to be determined, as mice null for pRB exhibit a lethal phenotype at around the time of germ cell arrest [28–30].
|
CONCLUSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
|
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. Forand, P. Fouchet, J.-B. Lahaye, A. Chicheportiche, R. Habert, and J. Bernardino-Sgherri Similarities and Differences in the In Vivo Response of Mouse Neonatal Gonocytes and Spermatogonia to Genotoxic Stress Biol Reprod, May 1, 2009; 80(5): 860 - 873. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yoshioka, J. R. McCarrey, and Y. Yamazaki Dynamic Nuclear Organization of Constitutive Heterochromatin During Fetal Male Germ Cell Development in Mice Biol Reprod, April 1, 2009; 80(4): 804 - 812. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Childs, P. T.K. Saunders, and R. A. Anderson Modelling germ cell development in vitro Mol. Hum. Reprod., September 1, 2008; 14(9): 501 - 511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Daggag, T. Svingen, P. S. Western, J. A. v. d. Bergen, P. J. McClive, V. R. Harley, P. Koopman, and A. H. Sinclair The Rhox Homeobox Gene Family Shows Sexually Dimorphic and Dynamic Expression During Mouse Embryonic Gonad Development Biol Reprod, September 1, 2008; 79(3): 468 - 474. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
