1 Wolfson Institute for Biomedical Research, University College London, The Cruciform Building, Gower Street, London, WC1E 6BT, UK
2 Department of Histopathology, University College London, Rockefeller Building, University Street, London, WC1E 6JJ, UK
3 Welcome Trust Biocentre, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
4 Centre for Applied Medical Statistics and General Practice and Primary Care Research Unit, Department of Public Health and Primary Care, University of Cambridge, Forvie Site, Robinson Way, Cambridge, CB2 2SR, UK
5 Medical Research Council Biostatistics Unit, Institute of Public Health, Forvie Site, Robinson Way, Cambridge, CB2 2SR, UK
Author for correspondence (e-mail: k.stoeber{at}ucl.ac.uk)
Accepted 19 August 2004
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Summary |
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Key words: Ki67, Cdc6, Cdt1, MCM, Geminin, DNA replication licensing, Oogenesis, Spermatogenesis, Gametogenesis
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Introduction |
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Importantly, initiation of chromosomal replication also represents a final and critical step in growth control downstream of complex networks of cell signalling pathways that have evolved to specify when and where cells divide in multicellular organisms (Stoeber et al., 2001). Over the last decade studies in yeast and Xenopus have identified some of the molecular mechanisms that control this critical decision point in cell proliferation, but it remains unclear how origin licensing is regulated in human tissue development and maintenance. The major role of cell division in adult life is to maintain the number of differentiated functional cells, replacing cells lost through death or injury. Tissues in the adult with the most rapid turnover are referred to as self-renewing systems and include skin, gut, testis and the haemopoietic system. These tissues share similar hierarchies of cellular development from stem cells to terminally differentiated mature cells via transit amplifying cells (Myster and Duronio, 2000
; Watt and Hogan, 2000
). We have previously shown that the origin licensing pathway plays a critical role in coordinating growth in human tissues; specifically in self-renewing tissues the transition from progenitor cells to the non-mitotic, terminally differentiated phenotype is coupled to repression of origin licensing through downregulation of the MCM helicase and its chromatin loading factor Cdc6 (Stoeber et al., 2001
; Williams et al., 1998
). Thus differentiation and DNA replication licensing appear to be mutually exclusive processes in keeping with the concept of antagonism between cellular circuits controlling proliferation and differentiation (Olson, 1992
; Olson and Spiegelman, 1999
). In this context two intriguing questions arise; first, is the physical connection of differentiation-promoting and replication-inhibiting domains within Geminin involved in triggering the proliferation-differentiation switch in self-renewing systems? Secondly, does Geminin play a role in preventing untimely DNA synthesis during other physiological processes?
To address these important questions for tissue development and maintenance, we first investigated regulation of origin licensing factors including Geminin in cycling human cells using a novel non-chemical synchronisation methodology (Thornton et al., 2002; Helmstetter et al., 2003
). Extension of our findings in cultured cells to expression profiling of Cdc6, Cdt1, Mcm2 and Geminin in human gut epithelium and testis has revealed fundamental differences in the control of origin licensing between self-renewing systems of somatic and germ cell type. To investigate further whether Geminin blocks untimely DNA synthesis during germ cell development in higher vertebrates, we compared origin licensing in male and female human germ cells. Our findings show that human primary spermatocytes and primary oocytes prevent untimely DNA synthesis during meiosis I (MI) through repression of origin licensing by very different mechanisms. This intriguing sexual dimorphism may contribute to the striking difference in error rates during meiosis in the male and female germlines.
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Materials and Methods |
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Cell culture and synchronisation
MOLT-4 human leukemic lymphoblasts (ATCC CRL-1582; Rockville, MD, USA) were cultured at 37°C in CO2-independent Leibovitz (L-15) medium supplemented with 2 g/l dextrose, 50 units/ml penicillin G, 50 µg/ml streptomycin sulphate and 10% FCS (all from Invitrogen, Paisley, UK). Newborn (early G1 phase) cells were collected by membrane elution and followed during synchronous growth as described previously (Helmstetter et al., 2003; Thornton et al., 2002
). Samples were removed at specific time points for determination of cell concentrations, cell sizes, DNA and protein content. Cell density and size were determined using a Coulter Z2 Particle Count and Size Analyzer (Beckman-Coulter, High Wycombe, UK).
Asynchronous HL-60 cells (ECACC 8501143, Salisbury, Wiltshire, UK) were maintained between 1-5x105 cells/ml at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 2 mM glutamine and 10% FCS. HeLa S3 cells (ECACC 87110901) were cultured at 37°C and 5% CO2 in DMEM supplemented with 10% FCS and synchronised in S phase by blocking with thymidine (2.5 mM) for 25 hours and releasing for 2 hours prior to harvesting.
Rabbit polyclonal antibody generation
pET14b-Geminin (Wohlschlegel et al., 2000) was expressed in E. coli BL21(DE3) and His6-tagged Geminin protein purified by Ni-NTA metal affinity chromatography following the manufacturer's instructions (Qiagen, Crawley, UK). Recombinant Geminin was further purified using a (FPLC) Hi-load Q Sepharose 16/10 column in NaPi buffer, and eluted with increasing concentrations of NaCl. Rabbits were injected with recombinant Geminin (125 µg) and received three boost injections following a standard immunisation protocol (Eurogentech, Seraing, Belgium). Sera were affinity-purified on a CNBr column against 10 mg of recombinant Geminin, eluted with 0.1 M glycine pH 2.5, and dialysed into PBS/1% BSA/0.1% sodium azide. An equal volume of sterile glycerol was added and antibodies designated G94 and G95 were stored at 20°C. Antibody purification was quality controlled by SDS-PAGE and ELISA. Specificity of antibodies was demonstrated by immunoblotting, by quenching all immunohistochemical staining after preincubating antibodies with recombinant Geminin for 1 hour at room temperature, and by flow cytometric analyses of asynchronous MOLT-4 cells after preincubation with either antibody G95 and recombinant Geminin (1:10 ratio), or G95 alone. Rabbit polyclonal antibodies were raised against a C-terminal fragment (amino acid residues 238-546) of bacterially expressed human Cdt1 as previously described (Wohlschlegel et al., 2000
).
Flow cytometric analysis of DNA, Ki67 and Geminin content
For flow cytometric analysis of DNA content, cells were fixed in 80% ethanol and stained with propidium iodide as described previously (Helmstetter et al., 2003). For determination of Ki67 and Geminin content, cells were fixed and permeabilised with non-ionic detergent as described previously (Gong et al., 1995
). After centrifugation, cell pellets were resuspended in Dulbecco's phosphate-buffered saline (D-PBS) and either 10 µl of FITC-conjugated Ki67 mAb (clone B56) or matched non-specific staining control (FITC-conjugated IgG; PharmingenTM, Oxford, UK), or 0.1 µg of anti-Geminin antibody G95, or G95 plus recombinant Geminin (1:10 ratio). After a 2-hour incubation at 4°C, cells were washed with D-PBS/1% BSA, concentrated via centrifugation and stained with 50 µg/ml propidium iodide and 20 µg/ml RNAse A in PBS. Analyses of light scatter properties and DNA/protein content were performed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Cell doublets were excluded by gating on a dot plot of the width versus the area of DNA fluorescence intensity (Erlanson and Landberg, 1998
). In most samples 104 cells were examined and data stored/analysed using CellQuestTM software (BD Biosciences), Cylchred (V.1.0.0.1) and WinMDI (V.2.7).
Preparation of protein extracts and immunoblotting
MOLT-4 and HL-60 total cell lysates (2x106 cells) were prepared in sample buffer (3% SDS, 100 mM DTT, 60 mM Tris pH 6.8, 0.01% Bromophenol Blue, 10% glycerol) as described previously (Harlow and Lane, 1999). Lysates from asynchronous Jurkat and HeLa cells were obtained commercially (BD Biosciences, Oxford, UK) and 12.5 µg lysate was loaded per well. Lysates were separated on 10% SDS-polyacrylamide gels (Invitrogen) and immunoblotted as described previously (Stoeber et al., 2001
) using anti-Geminin antibodies G94 and G95 (and corresponding preimmune sera), anti-Mcm5 mAb (Stoeber et al., 2002
) and antibodies obtained from commercial sources against the following antigens: Cdc6 (NeoMarkers, Fremont, CA, USA; clone DCS-180.2), Mcm2 (clone 46) and PCNA (clone 24) (BD Transduction LaboratoriesTM, Lexington, KY, USA), Mcm7 (NeoMarkers; clone 47DC141), Cyclin A (Santa Cruz; CA, USA; clone C-19) and Actin (Sigma Aldrich, Gillingham, UK; clone AC-15). Western blots of human tissue lysates were obtained from Geno Technology, Inc (St Louis, MO, USA) and probed with G94, G95 and anti-Cdc6 antibody.
Immunoprecipitation
Total lysates from HeLa cells (500 µg) harvested 2 hours after release from thymidine block were prepared in RIPA buffer containing 1 mM 1,4-dithiothreitol (DTT) and 1 tablet of Complete Mini, EDTA-free protease inhibitor cocktail per 10 ml buffer (Roche Diagnostics GmbH, Mannheim, Germany). Geminin protein was immunoprecipitated with anti-Geminin antibody G94 (1.5 µg) essentially as described previously (Harlow and Lane, 1999) with final washes in lysis buffer A (140 mM NaCl, 10 mM Tris pH 8.0, 0.5% NP-40) before resuspension in Laemmli buffer. Proteins were separated by SDS-PAGE, and immunoblots were probed with anti-Geminin antibody G95.
(q)RT-PCR analysis of geminin splice variants
For conventional RT-PCR using primers spanning the coding region of geminin, 300 ng of total RNA isolated with RNeasy Mini Kit (Qiagen) was reverse transcribed for 1 hour at 42°C in a reaction mix containing 10 mM dNTPs, 0.5 µl RNasin (Promega, Southampton, UK), 80 pmol of oligo(dT) primers, AMV RT buffer and 0.6 µl AMV reverse transcriptase (both Promega). After purification with Qiaquick PCR Purification Kit (Qiagen), 4 µl cDNA was amplified for 35 cycles (95°C for 1 minute, 55°C for 1 minute and 72°C for 1 minute) in 50 µl reactions containing Pfu polymerase buffer, 1 µl Pfu polymerase (each from Stratagene, Amsterdam, the Netherlands), 10 mM dNTPs and 0.2 µg of each geminin-specific primer. Geminin primers were designed within the first and last exons (exon 7) as follows [for: 5' AGC AGG GCT TTA CTG CAG AG 3', rev: 5' AAG TCA TGG CTG ACA ACT GAG A 3'].
For real-time RT-PCR analyses of geminin isoforms, RNA from human tissues was obtained from BD Biosciences and 2 µg was reverse transcribed as described previously (Lobenhofer et al., 2002) for 10 minutes at 25°C, 30 minutes at 48°C and 5 minutes at 95°C in a reaction mix containing 1x RT buffer (Applied Biosystems, Warrington, UK), 5.5 mM MgCl2, 500 µM of each dNTP, 2.5 µM random hexamers, 0.4 units/µl RNase inhibitor and 1.25 U/µl MultiScribeTM reverse transcriptase (Applied Biosystems). Undiluted cDNA was amplified in duplicate or triplicate PCR reactions containing either (1) Geminin splice-variant-specific primers and SYBR Green PCR mastermix (QuantitectTM SYBR® Green PCR kit, Qiagen, La Jolla, CA, USA) or (2) 18s rRNA dual-labelled probes and TaqMan® Universal PCR Master Mix (each from Applied Biosystems). All reactions were performed on an ABI 7700 Sequence Detector (Applied Biosystems) following the manufacturers' recommendations with thermal cycling parameters of 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Primers for four Geminin splice variants designated GemA [for: 5' CTG TGG CCT TTT GCG AGG T 3', rev: 5' GGG TGG AGA CGC TCA ATC C 3'], GemB [for: 5' GGG CCT CCG GGA CAA 3', rev: 5' TTA TGT AGA TGG TGA AGC ACA GAA GA 3'], GemD [for: 5' AGT TAG CAG GGC TTT ACT GCA GA 3', rev: 5' AGA AGA CTA CGC TGA TCC CCA C 3'] and GemE [for: 5' CGT CTG CGT CAG TTG GTC AC 3', rev: 5' AAA GCC CTG CTA ACT CCG C 3'] were designed using GenBank and Primer Express software (V.1.0; Applied Biosystems). Primer pairs for GemB, GemD and GemE are specific as one or both primers were designed within unique exons. Using Sequence Detection Software (Applied Biosystems), the threshold cycle (Ct) for each amplification reaction was determined. A variation of n cycles between reactions or samples represents a 2n-fold difference in message levels.
Immunohistochemistry
Anti-Mcm2 mAb (clone 46) was obtained from BD Transduction LaboratoriesTM (Lexington, KY, USA), anti-human Ki67 mAb (clone Mib-1) from DAKO (Glostrup, Denmark), and anti-Cdc6 mAb (clone DCS-180) from Neomarkers. Three µm sections of formalin-fixed, paraffin-embedded tissues were cut onto DAKO TechmateTM S2024 slides, dewaxed in xylene and rehydrated through an alcohol series to water. For antigen retrieval, tissues were pressure-cooked for 2-3 minutes (Ki67, Cdc6, Cdt1, Mcm2) or microwaved for 20 minutes in 0.1 M citrate buffer pH 6.0 (Geminin). For Ki67, Mcm2 and Geminin detection, automatic immunostaining was performed on a DAKO TechMateTM 500 as described previously (Dogan et al., 2000); Cdt1 immunostaining was performed manually. After blocking endogenous peroxidase activity, slides were incubated with primary antibodies for 1 hour at room temperature using the following concentrations: (a) ovary: Ki67 (1/50), Cdt1 (1/6000), Mcm2 (1/4000), G94 (1/4000), G95 (1/2000); (b) testis: Ki67 (1/50), Cdt1 (1/7500), Mcm2 (1/4000), G94 (1/4000), G95 (1/1500); (c) colon: Ki67 (1/50), Cdt1 (1/9000), Mcm2 (1/2000), G94 (1/4000), G95 (1/2000). Antigen-bound primary Ki67, Cdt1, Mcm2 and Geminin antibodies were detected with ChemMateTM EnVisionTM Detection Kit (DAKO). For staining of Cdc6, slides were covered with 50 µl of Cdc6 antibody (1/50) for 4 hours at 37°C. To detect antigen-bound Cdc6 antibody, a labelled streptavidin-biotin visualisation system was utilised (ChemMateTM Detection Kit K5001, DAKO). Primary antibodies were omitted in negative controls and, in addition, appropriate tissue sections were used as positive and negative controls. To detect in situ expression of Geminin in ethanol-fixed MOLT-4 cells, staining was performed as described above with G95 (1/2000) and detected with the labelled streptavidin-biotin visualisation system (ChemMateTM Detection Kit K5001, DAKO).
Protein expression profile analysis
Slides were examined and images captured with an Olympus BX51 microscope/CCD camera setup using ANAlysis software (SIS, Münster, Germany). Captured images were printed and cells expressing the protein of interest counted. In colon, epithelial cells in the basal compartment (BC: lower third of colonic crypts) and luminal compartment (LC, upper two thirds of colonic crypts) were evaluated separately. In testis, spermatogonia (SG), primary spermatocytes (SC), early spermatids (EST) and late spermatids (LST) were examined separately. Different stages of spermatogenesis were identified according to established morphological criteria (Trainer, 1997). In ovary, primary oocytes located within primordial follicles of the ovarian cortex were investigated. To assess interobserver variability, 10% of all cases were recounted by an independent investigator with high agreement.
Statistical analysis
Colon and testis data were analysed using identical statistical methods. For each of these sites, five subjects each provided 100 crypts (colon) or tubules (testes) for data analysis. Each of the five markers was used to stain 20 crypts/tubules from each subject. For each crypt/tubule we formed a summary statistic (Altman, 1991), the proportion of cells stained positive by the marker used for the crypt/tubule. The labelling index (percentage of positive cells) was estimated for each marker using a linear mixed model, a form of analysis of variance that accounts for the repeated measures within subject (Laird and Ware, 1982
). For ovary, five subjects each provided five histology sections for data analysis. Each marker was used to stain one section from each subject. In each section, all primary oocytes were assessed for protein expression by calculating the proportion of cells stained positive. A minimum of 10 primary oocytes were evaluated per case for each protein (overall between 120 and 164 primary oocytes were examined for protein expression). The labelling index was estimated for each marker by averaging these proportions across the subjects. Labelling indices for different pairs of markers, or for the same marker in different compartments, were compared by calculating ratios of the indices for each subject, and averaging these across the subjects. Intervals that did not contain the value 1 indicated that the two indices were considered different at the 5% level of statistical significance. Analysis was carried out using SPSS 11.5 for Windows (SPSS Inc., Chicago, IL, USA) and S-PLUS 6 (MathSoft Inc., Seattle, WA, USA).
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Results |
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To study expression of origin licensing factors including Geminin during synchronous growth of proliferating human cells, we exploited membrane elution (Fig. 2A), a novel non-chemical synchronisation methodology (Thornton et al., 2002; Helmstetter et al., 2003
). Newborn (early G1 phase) MOLT-4 cells isolated by membrane elution progressed through a full cycle of synchronous growth as demonstrated by FACS analysis of the DNA content (Fig. 2B). In total cell lysates prepared from synchronous batch cultures (Fig. 2C), Cdc6 oscillates during the cell cycle with levels increasing during S phase before diminishing in late G2/M. Cdt1 expression peaks in early G1 with protein levels declining during S phase progression. Protein levels of the DNA helicase subunits Mcm2, Mcm5 and Mcm7 increase linearly and in parallel in early G1 up to a maximum in S phase before decreasing during G2/M. Oscillation of MCM protein levels was also observed in a second successive cycle of synchronous MOLT-4 growth (data not shown), suggesting that these changes in protein levels may have been previously obscured by metabolic perturbation through use of chemical synchronisation agents. As shown in asynchronous MOLT-4 cells, by flow cytometry (Fig. 1D), Geminin is absent during the permissive window for pre-RC assembly in G1 with levels becoming detectable at the G1/S transition and increasing linearly during S/G2 before mitotic degradation. The specificity of the anti-Geminin antibodies and the cell cycle periodicity of Geminin expression is further supported by in situ analysis of MOLT-4 cells showing expression restricted to late S/G2 phase but not in G1 phase (Fig. 2D). The periodicity in Geminin expression is similar to that seen for Cyclin A and is consistent with findings in HeLa cells (McGarry and Kirschner, 1998
; Wohlschlegel et al., 2000
). Protein levels of proliferating cell nuclear antigen (PCNA) and the cytoskeletal filamentous protein Actin were essentially unchanging during synchronous progression through the cell cycle. Moreover, flow cytometric analysis showed that the fraction of cells expressing the standard proliferation marker Ki67 during synchronous growth (85-95%) remained constant and was similar to the proportion of Ki67-positive cells in asynchronous culture.
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Origin licensing factors are downregulated during the proliferation-differentiation switch in colon but not testis
To investigate regulation of the origin licensing pathway in human somatic and germ cell self-renewing tissues, we analysed the protein expression profiles of Ki67, Cdc6, Cdt1, Mcm2 and Geminin in colon and testis. Labelling indices (percentage of positive cells) for each protein are described in Table 1, and representative immunostained tissue sections are shown in Figs 3 and 4. In colonic crypts (Fig. 3), expression of Ki67 and Mcm2 is found in a high proportion of stem-transit progenitor cells within the basal compartment (BC; 82% and 88%, respectively). A significantly lower percentage of these cells express Geminin [28% (with antibody G94); 33% (G95)] and Cdt1 (44%). Low level expression (1.6%) of Cdc6 is detected in crypt cells within the BC. In testis (Fig. 4), 24% of basal proliferative cells (spermatogonia) express Ki67, with Cdt1 and Mcm2 expression found in a significantly higher percentage of cells (35% and 45%, respectively), suggesting that a proportion of these progenitor cells may reside in a licensed but non-proliferating state (Stoeber et al., 2001). Geminin is expressed in a significantly lower percentage of spermatogonia [11% (G94); 10% (G95)] and Cdc6 in a subpopulation (0.8%).
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In contrast to the basal proliferative compartment, the Ki67 labelling index is significantly lower in the luminal compartment (82% in the BC against 12% in the LC) of colonic crypts. In the latter compartment, which contains differentiating goblet and enteroendocrine cells, the drop in Ki67 labelling index corresponds to loss of proliferative capacity as cells migrate to the surface. Triggering of the proliferation-differentiation switch also coincides with downregulation of Cdc6 (1.6% in the BC compared with 0.1% in the LC) and Cdt1 (44% in the BC against 13% in the LC). Similarly, Geminin is significantly downregulated as cells migrate from the proliferative to the differentiated compartment [28% (G94)/33% (G95) in the BC compared to 3.1% (G94)/3.7% (G95) in the LC]. Importantly, expression of Mcm2 persists at relatively high levels (40%) in early differentiation with downregulation occurring only as cells terminally differentiate. Notably, these origin factors are absent in cells at the base of colonic crypts (fourth position or below), the putative stem cell compartment (Potten, 1986). This suggests that colonic stem cells, which have a high capacity for self-renewal but divide infrequently, reside in an out-of-cycle, non-licensed quiescent state (S. R. Kingsbury, M.L., T.F., E. C. Obermann, unpublished observation).
In testis, the migration of cells into the differentiated LC, which contains germ cells in meiotic and post-meiotic phases, is also linked to downregulation of Ki67 as cells lose their ability to initiate DNA synthesis. This is reflected in the significant decrease in the Ki67 labelling index from 24% for spermatogonia to 0.6% in spermatocytes. However, in marked contrast to early differentiating colonocytes, in which downregulation of origin licensing factors occurs in parallel with Ki67, early differentiation in testis (i.e. maturation of spermatogonia into primary spermatocytes) is associated with continued presence of these replicative factors (Fig. 5). Primary spermatocytes show strikingly high levels of Cdc6, Cdt1, Mcm2 and Geminin in contrast to the marked downregulation seen in the differentiating LC of colon [Cdc6: 42% against 0.1%; Cdt1: 33% against 13%; Mcm2: 99% against 40%; Geminin: 91% (G94)/95% (G95) against 3.1% (G94)/3.7% (G95)]. Cdt1 expression levels fall in late prophase during the pachytene stage of meiosis I (MI). During the later stages of differentiation, as germ cells terminally differentiate into spermatids, there is downregulation of Cdc6, Mcm2 and Geminin.
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Untimely DNA synthesis during meiosis I is repressed via different mechanisms in the male and female germline
Like primary spermatocytes, primary oocytes (within the primordial follicles of the ovary) are arrested in prophase of MI. Representative immunostained tissue sections of human ovary are illustrated in Fig. 6. A comparison of labelling indices for Cdc6, Cdt1, Mcm2, Geminin and Ki67 in primary spermatocytes and primary oocytes is described in Fig. 7. Neither primary spermatocytes nor primary oocytes express Ki67. In contrast, the male and female germ cells each express significant levels of Cdt1 (spermatocytes: 33%, oocytes: 97%) and Mcm2 (spermatocytes: 98.9%, oocytes: 99.6%). Notably, although Cdc6 and Geminin are present at high levels in primary spermatocytes (42% and 95%, respectively), these origin licensing factors are absent in primary oocytes (Fig. 7). These data demonstrate a striking sexual dimorphism in DNA replication licensing of male and female germ cells, and implicate Geminin as a key factor in maintaining stability of the male germline genome.
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Given high Geminin levels in primary spermatocytes and unique mechanisms of gene expression in testis (Goldberg, 1996; Walker et al., 1999
), we sought to investigate whether germ cell-specific transcripts of geminin are expressed during spermatogenesis. Conventional RT-PCR analyses using primers spanning the geminin locus gave two distinct bands when total RNA from a panel of cell lines (Fig. 8A) and human tissues (Fig. 8B) (including testis) was investigated. Although geminin message levels were generally more abundant in self-renewing tissues such as testis, colon and bone marrow than in stable (liver) or permanent (heart) tissues, the two alternatively spliced forms were not equally expressed in different tissues. Sequence analysis of the isoforms revealed that the larger form (designated GemA) contained a sequence within exon 1 (132 bp), which was not found within the smaller variant (GemE). Quantitative RT-PCR was used to study these and two additional variants supported by cDNA clones within AceView (http://www.ncbi.nlm.nih.gov) in RNA from human testis, ovary, colon and cervix (Fig. 8C). Analyses revealed that each variant was present in each tissue with geminin message levels generally higher in testis (data not shown). Message levels of the endogenous control gene (18s rRNA) were virtually identical for all tissues (inset, Fig. 8C). Significantly, the GemE splice variant was considerably more abundant (
8-fold) in testis than in ovary, colon or cervix. GemE represents a mRNA that could encode 258 amino acids if translated. Analysis of additional amino acids at the N terminus suggests that the predicted protein could possess a zinc finger motif which may facilitate protein-protein or protein-DNA interactions (Sujatha and Chatterji, 1991). Interestingly, the GemE isoform also has cell cycle periodicity in MOLT-4 cells during synchronous growth with a peak in expression at G1/S (data not shown).
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At the protein level, immunoblotting of human testis and ovary lysates with anti-Geminin antibodies G94 and G95 detected the 33 kDa form of Geminin in testes but not ovary (Fig. 8D). These data are in keeping with our immunohistochemical findings (Figs 4 and 6). Interestingly, in addition to the 33 kDa form, the Geminin-specific antibodies also detected a polypeptide with a molecular mass of 20 kDa in testis lysates. Preincubation of the antibodies with recombinant Geminin blocks this interaction (Fig. 8D), suggesting that the additional polypeptide detected may constitute a testis-specific Geminin isoform. This band could of course also be a result of non-specific proteolytic cleavage after tissue lysis or merely a protein with a shared epitope. However, it is noteworthy that similar proteolytic cleavage after lysis of other tissues was not observed and that detection of the 20 kDa polypeptide with two independent polyclonal antibodies was blocked after preincubation with recombinant Geminin. As is the case for Geminin, a single protein with a molecular mass of
62 kDa consistent with the reported electrophoretic mobility of human Cdc6 was detected with an anti-Cdc6 antibody in lysates from testis but not ovary (Fig. 8E), again confirming our immunohistochemical finding that Cdc6 is absent in primary oocytes (Fig. 6). Taken together, our data suggest alternative transcripts of geminin may be involved in germ cell-specific functions. Future detailed analyses at RNA and protein level are required to characterise these isoforms.
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Discussion |
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The presence of high Geminin levels in primary spermatocytes suggests protein expression and/or prolonged half-life in these cells, which are locked in prophase of MI. At transcription level, testis is unique in that many germ cell-specific transcripts are produced during spermatogenesis (Goldberg, 1996). Our detection of a geminin mRNA isoform significantly more abundant in testis than in its female germline counterpart or in somatic tissues, suggests that GemE may indeed have testis-specific functions during spermatogenesis. Notably it has been suggested that deletions within the 5'UTR, as is the case for GemE when compared with the larger isoform, could be associated with increased mRNA expression or transcriptional modulatory function (Yu et al., 2001
). At the protein level, a prolonged half-life of Geminin protein may occur through testis-specific mechanisms that modulate anaphase promoting complex-targeted proteolysis during meiosis. This has been observed for Cyclin B1, a target for the anaphase promoting complex that is incompletely degraded at the end of MI in Xenopus and that is essential for suppression of DNA synthesis after Cdc2 inactivation at MI exit (Iwabuchi et al., 2000
; Iwabuchi et al., 2002
; Stern, 2003
).
Cell cycle kinetics in the human germline are remarkably different. Oogonia begin meiosis during foetal development but arrest part-way through prophase of MI to form primary oocytes, and do not complete the first division until ovulation. The second meiotic division (MII) is completed only if the egg is fertilised. Thus oogenesis may last for several decades. In contrast, male meiosis begins at puberty and is a continuous process with spermatocytes progressing through prophase I and through the second reduction division in around 22 days (Wolgemuth et al., 2002). Our data and those of Nishitani et al. (Nishitani et al., 2001
) suggest that all factors required for origin licensing are present within testis. This implicates Geminin as an inhibitor of origin licensing and thus a suppressor of DNA replication in primary spermatocytes during the prolonged prophase of MI. An important question is therefore whether a similar mechanism operates during female meiosis. Our studies have revealed a very different mechanism by which primary oocytes suppress chromosomal replication (Fig. 7). Although primary oocytes express the MCM helicase proteins and Cdt1, the second essential loading factor Cdc6 is absent. Interestingly, Geminin, which in contrast to primary spermatocytes would not be required to suppress origin licensing in the absence of Cdc6, is also absent in primary oocytes. A similar molecular mechanism for acquisition of DNA replication competence during oocyte maturation has been reported for Xenopus, in that competence to replicate in the unfertilised egg is conferred by Cdc6 during meiotic maturation (Lemaitre et al., 2002
; Whitmire et al., 2002
). Our finding that Geminin is absent in human primary oocytes is also in keeping with the reported absence of Geminin in stage VI (arrested) Xenopus oocytes with the synthesis of Geminin being induced during oocyte maturation (McGarry, 2002
). Thus Cdc6 is most probably the crucial regulator in humans, which ensures that eggs do not engage in untimely DNA replication during the prolonged prophase of MI which (might last several years). It is noteworthy that Cdc6 is rate-limiting for acquisition of DNA replication competence not only in female germ cells, but also in somatic cells reentering the cell division cycle from quiescence (Stoeber et al., 1998
; Lea et al., 2003
).
Sexual dimorphism has been identified at many levels in relation to meiosis and the production of haploid gametes. Mammalian males and females use different strategies, transit meiosis with different levels of success, and exit with different end products. Importantly, the quality control mechanisms that ensure genetic integrity of gametes during meiosis also show a striking sexual dimorphism (Hunt and Hassold, 2002). It has been shown that the pachytene and spindle assembly checkpoints are missing or less stringent in mammalian oogenesis than in spermatogenesis (Wolgemuth et al., 2002
; Voet et al., 2003
), and that these differences might account for the high error rates observed in oocytes. Direct studies of human gametes show chromosomal abnormalities in up to 20% of oocytes but only 3-4% of sperm (Martin and Rademaker, 1991
). It is possible that the alternative strategies for repression of origin licensing observed by us in this study of human gametogenesis could contribute to the striking difference in error rates during meiosis in the male and female germlines.
Here we demonstrate that suppression of proliferation in terminally differentiated cells of self-renewing tissues (spermatocytes and surface colonocytes) is achieved through downregulation of the Cdc6, Cdt1 and MCM origin licensing factors. Differentiated hepatocytes of adult liver, a stable tissue, and mature adult neurones and myocardiocytes, the latter examples of permanent tissues (Leblond, 1963), also suppress origin licensing through Cdc6 and MCM downregulation (Stoeber et al., 2001
). Primary oocytes, and also quiescent T cells (Lea et al., 2003
), mimic somatic cells with induction of Cdc6 rate-limiting for acquisition of replication competence. Cdc6, the loading factor for the MCM helicase, therefore functions as the `master regulator' of cell proliferation control in these tissues. In contrast to somatic and female germ cells, repression of origin licensing in primary spermatocytes appears to depend on inhibition of origin licensing by Geminin with Cdt1 only becoming rate-limiting in late prophase. Taken together our studies have revealed a remarkable heterogeneity in the regulation of origin licensing in somatic and germ cell tissues.
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Acknowledgments |
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Footnotes |
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References |
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Altman, D. G. (1991). Practical statistics for medical research. Norwell, MA: Chapman and Hall.
Bell, S. P. and Dutta, A. (2002). DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333-374.[CrossRef][Medline]
Blow, J. J. and Hodgson, B. (2002). Replication licensing defining the proliferative state? Trends Cell Biol. 12, 72-78.[CrossRef][Medline]
Brown, D. C. and Gatter, K. C. (2002). Ki67 protein: the immaculate deception? Histopathology 40, 2-11.[CrossRef][Medline]
Champion, M. D. and Hawley, R. S. (2002). Playing for half the deck: the molecular biology of meiosis. Nat. Cell Biol. 4, S50-S56.[CrossRef][Medline]
Del Bene, F., Tessmar-Raible, K. and Wittbrodt, J. (2004). Direct interaction of geminin and Six3 in eye development. Nature 427, 745-749.[CrossRef][Medline]
DePamphilis, M. L. (2003). The `ORC cycle': a novel pathway for regulating eukaryotic DNA replication. Gene 310, 1-15.[CrossRef][Medline]
Dimitrova, D. S., Prokhorova, T. A., Blow, J. J., Todorov, I. T. and Gilbert, D. M. (2002). Mammalian nuclei become licensed for DNA replication during late telophase. J. Cell Sci. 115, 51-59.
Dogan, A., Bagdi, E., Munson, P. and Isaacson, P. G. (2000). CD10 and BCL-6 expression in paraffin sections of normal lymphoid tissue and B-cell lymphomas. Am. J. Surg. Pathol. 24, 846-852.[CrossRef][Medline]
Erlanson, M. and Landberg, G. (1998). Flow cytometric quantification of cyclin E in human cell lines and hematopoietic malignancies. Cytometry 32, 214-222.[CrossRef][Medline]
Goldberg, E. (1996). Transcriptional regulatory strategies in male germ cells. J. Androl. 17, 628-632.
Gong, J., Traganos, F. and Darzynkiewicz, Z. (1995). Growth imbalance and altered expression of cyclins B1, A, E, and D3 in MOLT-4 cells synchronized in the cell cycle by inhibitors of DNA replication. Cell Growth Differ. 6, 1485-1493.[Abstract]
Gopalakrishnan, V., Simancek, P., Houchens, C., Snaith, H. A., Frattini, M. G., Sazer, S. and Kelly, T. J. (2001). Redundant control of rereplication in fission yeast. Proc. Natl. Acad. Sci. USA 98, 13114-13119.
Harlow, E. and Lane, D. P. (1999). Using antibodies; a laboratory manual, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Helmstetter, C. E., Thornton, M., Romero, A. and Eward, K. L. (2003). Synchrony in human, mouse and bacterial cell cultures a comparison. Cell Cycle 2, 42-45.[Medline]
Hunt, P. A. and Hassold, T. J. (2002). Sex matters in meiosis. Science 296, 2181-2183.
Iwabuchi, M., Ohsumi, K., Yamamoto, T. M., Sawada, W. and Kishimoto, T. (2000). Residual Cdc2 activity remaining at meiosis I exit is essential for meiotic M-M transition in Xenopus oocyte extracts. EMBO J. 19, 4513-4523.
Iwabuchi, M., Ohsumi, K., Yamamoto, T. M. and Kishimoto, T. (2002). Coordinated regulation of M phase exit and S phase entry by the Cdc2 activity level in the early embryonic cell cycle. Dev. Biol. 243, 34-43.[CrossRef][Medline]
Kroll, K. L., Salic, A. N., Evans, L. M. and Kirschner, M. W. (1998). Geminin, a neuralizing molecule that demarcates the future neural plate at the onset of gastrulation. Development 125, 3247-3258.
Labib, K. and Diffley, J. F. (2001). Is the MCM2-7 complex the eukaryotic DNA replication fork helicase? Curr. Opin. Genet. Dev. 11, 64-70.[CrossRef][Medline]
Laird, N. M. and Ware, J. H. (1982). Random-effects models for longitudinal data. Biometrics 963-974.
Lea, N. C., Orr, S. J., Stoeber, K., Williams, G. H., Lam, E. W., Ibrahim, M. A., Mufti, G. J. and Thomas, N. S. (2003). Commitment point during G0>G1 that controls entry into the cell cycle. Mol. Cell. Biol. 23, 2351-2361.
Leblond, C. P. (1963). Classification of cell populations on the basis of their proliferative behaviour. NCI Monogr. 14, 19-145.
Lei, M. and Tye, B. K. (2001). Initiating DNA synthesis: from recruiting to activating the MCM complex. J. Cell Sci. 114, 1447-1454.
Lemaitre, J. M., Bocquet, S. and Mechali, M. (2002). Competence to replicate in the unfertilized egg is conferred by Cdc6 during meiotic maturation. Nature 419, 718-722.[CrossRef][Medline]
Lobenhofer, E. K., Bennett, L., Cable, P. L., Li, L., Bushel, P. R. and Afshari, C. A. (2002). Regulation of DNA replication fork genes by 17beta-estradiol. Mol. Endocrinol. 16, 1215-1229.
Luo, L., Yang, X., Takihara, Y., Knoetgen, H. and Kessel, M. (2004). The cell-cycle regulator geminin inhibits Hox function through direct and polycomb-mediated interactions. Nature 427, 749-753.[CrossRef][Medline]
Martin, R. H., Ko, E. and Rademaker, A. (1991). Distribution of aneuploidy in human gametes: comparison between human sperm and oocytes. Am. J. Med. Genet. 39, 321-331.[Medline]
Masai, H. and Arai, K. (2002). Cdc7 kinase complex: a key regulator in the initiation of DNA replication. J. Cell Physiol. 190, 287-296.[CrossRef][Medline]
McGarry, T. J. (2002). Geminin deficiency causes a Chk1-dependent G2 arrest in Xenopus. Mol. Biol. Cell 13, 3662-3671.
McGarry, T. J. and Kirschner, M. W. (1998). Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93, 1043-1053.[Medline]
Mendez, J. and Stillman, B. (2000). Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20, 8602-8612.
Mihaylov, I. S., Kondo, T., Jones, L., Ryzhikov, S., Tanaka, J., Zheng, J., Higa, L. A., Minamino, N., Cooley, L. and Zhang, H. (2002). Control of DNA replication and chromosome ploidy by geminin and cyclin A. Mol. Cell. Biol. 22, 1868-1880.
Myster, D. L. and Duronio, R. J. (2000). To differentiate or not to differentiate? Curr. Biol. 10, 302-304.
Nishitani, H. and Lygerou, Z. (2002). Control of DNA replication licensing in a cell cycle. Genes Cells 7, 523-534.
Nishitani, H., Taraviras, S., Lygerou, Z. and Nishimoto, T. (2001). The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase. J. Biol. Chem. 276, 44905-44911.
Olson, E. N. (1992). Interplay between proliferation and differentiation within the myogenic lineage. Dev. Biol. 154, 261-272.[Medline]
Olson, E. and Spiegelman, B. M. (1999). Cell differentiation. Curr. Opin. Cell Biol. 11, 653-654.[CrossRef]
Potten, C. S. (1986). Cell cycles in cell hierarchies. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 49, 257-278.[Medline]
Potten, C. S. (1997). Stem Cells. London, UK: Academic Press.
Quinn, L. M., Herr, A., McGarry, T. J. and Richardson, H. (2001). The Drosophila Geminin homolog: roles for Geminin in limiting DNA replication, in anaphase and in neurogenesis. Genes Dev. 15, 2741-2754.
Shreeram, S., Sparks, A., Lane, D. P. and Blow, J. J. (2002). Cell type-specific responses of human cells to inhibition of replication licensing. Oncogene 21, 6624-6632.[CrossRef][Medline]
Stern, B. M. (2003). FEARless in meiosis. Mol. Cell 11, 1123-1125.[CrossRef][Medline]
Stoeber, K., Mills, A. D., Kubota, Y., Krude, T., Romanowski, P., Marheineke, K., Laskey, R. A. and Williams, G. H. (1998). Cdc6 protein causes premature entry into S phase in a mammalian cell-free system. EMBO J. 17, 7219-7229.
Stoeber, K., Tlsty, T. D., Happerfield, L., Thomas, G. A., Romanov, S., Bobrow, L., Williams, E. D. and Williams, G. H. (2001). DNA replication licensing and human cell proliferation. J. Cell Sci. 114, 2027-2041.
Stoeber, K., Swinn, R., Prevost, A. T., de Clive-Lowe, P., Halsall, I., Dilworth, S. M., Marr, J., Turner, W. H., Bullock, N., Doble, A. et al. (2002). Diagnosis of genito-urinary tract cancer by detection of minichromosome maintenance 5 protein in urine sediments. J. Natl. Cancer Inst. 94, 1071-1079.
Sujatha, S. and Chatterji, D. (1999). Detection of putative Zn(II) binding sites within Escherichia coli RNA polymerase: inconsistency between sequence-based prediction and 65Zn blotting. FEBS Lett. 454, 169-171.[CrossRef][Medline]
Tada, S., Li, A., Maiorano, D., Mechali, M. and Blow, J. J. (2001). Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nat. Cell Biol. 3, 107-113.[CrossRef][Medline]
Thepaut, M., Hoh, F., Dumas, C., Calas, B., Strub, M. P. and Padilla, A. (2002). Crystallization and preliminary X-ray crystallographic analysis of human Geminin coiled-coil domain. Biochim. Biophys. Acta 1599, 149-151.[Medline]
Thornton, M., Eward, K. L. and Helmstetter, C. E. (2002). Production of minimally disturbed synchronous cultures of hematopoietic cells. Biotechniques 32, 1098-1105.[Medline]
Trainer, T. D. (1997). Testis and Excretory Duct System. 2nd edn. Philadelphia, PA: Lippincott-Raven.
Vaziri, C., Saxena, S., Jeon, Y., Lee, C., Murata, K., Machida, Y., Wagle, N., Hwang, D. S. and Dutta, A. (2003). A p53-dependent checkpoint pathway prevents rereplication. Mol. Cell 11, 997-1008.[Medline]
Voet, T., Liebe, B., Labaere, C., Marynen, P. and Scherthan, H. (2003). Telomere-independent homologue pairing and checkpoint escape of accessory ring chromosomes in male mouse meiosis. J. Cell Biol. 162, 795-807.
Walker, W. H., Delfino, F. J. and Habener, J. F. (1999). RNA processing and the control of spermatogenesis. Front. Horm. Res. 25, 34-58.[Medline]
Watt, F. M. and Hogan, B. L. (2000). Out of Eden: stem cells and their niches. Science 287, 1427-1430.
Wharton, S. B., Hibberd, S., Eward, K. L., Crimmins, D., Jellinek, D. A., Levy, D., Stoeber, K. and Williams, G. H. (2004). DNA replication licensing and cell cycle kinetics of oligodendroglial tumours. Br. J. Cancer (in press).
Whitmire, E., Khan, B. and Coue, M. (2002). Cdc6 synthesis regulates replication competence in Xenopus oocytes. Nature 419, 722-725.[CrossRef][Medline]
Williams, G. H., Romanowski, P., Morris, L., Madine, M., Mills, A. D., Stoeber, K., Marr, J., Laskey, R. A. and Coleman, N. (1998). Improved cervical smear assessment using antibodies against proteins that regulate DNA replication. Proc. Natl. Acad. Sci. USA 95, 14932-14937.
Wohlschlegel, J. A., Dwyer, B. T., Dhar, S. K., Cvetic, C., Walter, J. C. and Dutta, A. (2000). Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Science 290, 2309-2312.
Wolgemuth, D. J., Laurion, E. and Lele, K. M. (2002). Regulation of the mitotic and meiotic cell cycles in the male germ line. Recent Prog. Horm. Res. 57, 75-101.
Wuarin, J. and Nurse, P. (1996). Regulating S phase: CDKs, licensing and proteolysis. Cell 85, 785-787.[Medline]
Yan, Z., DeGregori, J., Shohet, R., Leone, G., Stillman, B., Nevins, J. R. and Williams, R. S. (1998). Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells. Proc. Natl. Acad. Sci. USA 95, 3603-3608.
Yu, J. J., Thornton, K., Guo, Y., Kotz, H. and Reed, E. (2001). An ERCC1 splicing variant involving the 5'-UTR of the mRNA may have a transcriptional modulatory function. Oncogene 20, 7694-7698.[CrossRef][Medline]
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