p53 controls low dna damage-dependent premeiotic...

p53 Controls Low DNA Damage-dependent PremeioticCheckpoint and Facilitates DNA Repairduring Spermatogenesis1

Dov Schwartz, Naomi Goldfinger, Zvi Kam, andVarda Rotter2

Department of Molecular Cell Biology, Weizmann Institute of Science,Rehovot, Israel 76100

AbstractPreviously, it was implicated that p53 plays a role inspermatogenesis. Here we report that p53 knockoutmice exhibit significantly less mature motilespermatozoa than their p53(1/1) counterparts. Tobetter understand the role of p53 in spermatogenesis,we analyzed the response of spermatogenic cells toDNA insult during prophase. It was found that althoughlow-level g-irradiation activated a p53-dependentpremeiotic delay, higher levels of g-irradiation induceda p53-independent apoptosis during meiosis.Furthermore, p53 knockout mice exhibited reduced invivo levels of unscheduled DNA synthesis, indicative ofcompromised DNA repair. Thus, p53 provides anotherlevel of stringency in addition to other spermatogenic“quality control” mechanisms.

IntroductionSpermatogenesis involves the genetic recombination proc-ess, which takes place during the prolonged arrest at thetetraploid DNA content, termed meiotic prophase. Double-and single-strand DNA breaks, actively generated by stillunknown mechanisms during the zygotene stage of the mei-otic prophase, serve as substrates for the strand exchangeduring recombination. Strict maintenance of genomic stabil-ity and prevention of mutagenesis are essential for success-ful outcome of meiosis to assure the fidelity sufficient forproper heredity (1).

It is therefore not surprising that p53, the “guardian of thegenome” (2), seems to play a role in spermatogenesis (3–19).Indeed, p53 mRNA and protein are highly expressed duringmouse and rat spermatogenesis (4, 5, 8) and are predomi-nantly evident in the premeiotic primary spermatocytes at the

zygotene-pachytene stages, before the onset of meiotic di-vision (5, 8). Furthermore, p53 knockout mice and mice withreduced levels of p53 exhibit germ cell degeneration duringthe meiotic prophase, manifested by the appearance ofmultinucleated giant cells (3). p53 is also suggested to me-diate stress-related spermatogonial apoptosis after DNAdamage (15) as well as after overheating of the testiculartissue (13). p53 knockout mice exhibit an increased inci-dence of testicular cancer, indicating that p53 has a role inthe prevention of carcinogenesis during the mitotic stages ofspermatogenesis (6, 9, 10). The role of p53 in the spermato-gonial stress response is supported also by the extremelygood responsiveness of testicular cancer cells expressingwild-type p53 to chemo- and radiotherapy (12, 14, 16). Thiswas shown to be a result of activation of “normally latent”wild-type p53, which in turn induces extensive apoptoticresponse (11).

Several reports deal with the role of p53 protein in meioticand premeiotic stages of spermatogenesis. Recently, it wasshown that the fidelity of the meiotic crossing-over in severalgenomic loci is not severely affected in p53-knockout mice(17). Odorisio et al. (18) reported that whereas spermatogo-nial DNA-damage induced apoptosis is p53 dependent, themeiotic “quality control” chromosome-synapsis-dependentcheckpoint at meiotic metaphase I is p53 independent (18).On the other hand, it was observed that knockout mice forboth p53 and ATM genes proceed to later stages ofprophase than those knockout mice with the ATM gene only(20). Yin et al. (19) showed that p53(2/2) mice exhibit com-promised apoptosis specially in tetraploid DNA state. Theseresults suggest that the DNA damage-dependent checkpointsituated in meiotic prophase is p53 dependent.

p53 protein plays an important role in the maintenance ofgenomic stability during mitotic proliferation (21–23). Thesefunctions are carried out by composite regulation of keycellular responses to DNA damage (24). On the one hand,p53 mediates the arrest of cells at the G1-S boundary (25)and affects the G2 (26–28) and spindle (29–31) checkpoints.This permits the cells to repair the DNA damage, prior tostages of its fixation and propagation, which may lead tocarcinogenic transformation (32, 33). Moreover, p53 wasshown to facilitate general genomic repair of DNA damage(33) and to bind proteins involved in DNA repair like XPB (34),RPA (35), and rad51 (36, 37). On the other hand, in manycellular systems, p53 promotes apoptosis of cells harboring“irreparable” or high DNA damage (32).

Several lines of evidence suggest that p53 is involved inDNA recombination and rearrangement. p53 binds the mam-malian homologue of yeast rad51 protein, which is directlyinvolved in homologous recombination, and in repair of DNAdouble-strand breaks (36). Recently, it was shown that

Received 6/4/99; revised 8/20/99; accepted 8/23/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 This work was supported in part by grants from the Israel-USA Bina-tional Science Foundation, the German Israeli Foundation for ScientificResearch and Development, and the Israel Cancer Association. V. R. isthe incumbent of the Norman and Helen Asher Professional Chair inCancer Research at the Weizmann Institute.2 To whom requests for reprints should be addressed, at Department ofMolecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel76100. Phone: 972-8-9344501; Fax: 972-8-9465265; E-mail: [email protected].

665Vol. 10, 665–675, October 1999 Cell Growth & Differentiation

knockout mice with the ATM gene, which is known to regu-late the DNA damage-dependent p53 function, are infertile(20, 38–40). Moreover, p53 was shown to promote homol-ogous recombination measured in vivo (41) and to bind hol-iday junctions, which are recombination intermediates (42).Involvement of p53 in DNA rearrangement was concludedfrom the observation that p53 mediates g-irradiation-dependent partial rescue of the rearrangement intermediatesin SCID mutation (43–45). Moreover, the general rise in ille-gitimate recombination levels and aneuploidy upon p53 in-activation in cancer cells is well documented (24, 46).

In this report, we compare the performance of the sper-matogenic process in mice with a different p53 genotype. Wefound that p53(2/2) spermatocytes exhibit a deregulatedprogression toward meiosis after application of low-levelDNA damage during the meiotic prophase. In vivo analysis ofUDS,3 indicative of DNA repair, revealed that p53(2/2) miceexhibited lower levels of DNA repair compared with that ofp53(1/1) littermates.

We also found that p53(2/2) mice have significantly lessmature motile spermatozoa in comparison with theirp53(1/1) counterparts. This reduction in the amount of mo-tile sperm is, at least in part, a result of pronounced loss ofcells during meiotic divisions. The p53(2/2) spermatocytesexhibit a deregulated progression toward meiosis after ap-plication of low-level DNA damage during the meioticprophase. In vivo analysis of UDS, indicative of DNA repair,revealed that p53(2/2) mice exhibited lower levels of DNArepair compared with that of p53(1/1) littermates.

These results suggest that p53 plays a role in the low-levelDNA damage-dependent premeiotic checkpoint and con-tributes to the efficiency of DNA repair, which in turn ensureappropriate quality and quantity of mature spermatozoa.

ResultsComparison of Epididimal Sperm Motility in p53(1/1),p53(1/2), and p53(2/2) Mice. Although not well docu-mented, it is known that p53(2/2) mice suffer infertility (7).Previously, we observed that p53(2/2) mice or those withreduced levels of p53 protein exhibit a degenerative processof the spermatogenic tissue, termed the giant cell syndrome(3). It was, therefore, of importance to evaluate whether lackof p53 would significantly affect the overall yield of motilespermatozoa, a primary measure of male fertility. To thatend, we surgically removed the cauda epididimi of C57BI/6mice of various p53 genotypes. The mature spermatozoareleased from the epididimi were analyzed for motility usingthe “swim-up” assay. As shown in Fig. 1, the amount ofmotile spermatozoa of p53(1/2) or p53(2/2) mice wasabout 60% of the p53(1/1) control mice. This indicates thatindeed lack of p53 reduces the yield of “functional” sperma-tozoa.

In Vivo Analysis of Spermatogenic Progression UsingBrdUrd Pulse Labeling. To elucidate whether the reducednumbers of motile spermatozoa in p53 (1/2) and p53(2/2)mice are a result of enhanced degeneration of spermato-genic cells, we in vivo pulse labeled replicating spermatogo-nia with BrdUrd and followed the abundance and DNA con-tent distribution of the cohort of labeled cells along meiosis(see chart, Fig. 2a). The proportion of the BrdUrd-labeledcells in the total population and the DNA content distributionat various days after the in vivo BrdUrd pulse were measuredby FACS analysis. Fig. 2b depicts a typical example of sper-matogenic population progression as a function of time.Analysis of mice with various p53 genotypes indicated nosignificant difference in the percentage of mitotically dividingspermatogonia (24 h after BrdUrd pulse) and of cells at thepachytene stage of the meiotic prophase (9 days afterBrdUrd pulse; data not presented). This suggests that lack ofp53 does not affect spermatogonial proliferation or earlystages of the meiotic prophase. However, after traverse ofmeiotic divisions (14 and 15 days after BrdUrd pulse), thepercentage of haploid labeled cells in p53(2/2) or p53(1/2)mice was significantly reduced in comparison with that ofp53(1/1) controls (Fig. 2, c and d). This implies that lack ofp53 in mice may cause spermatogenic degeneration dur-ing meiosis, even without subjection to exogenous DNAdamage.

Effect of Low-Level g-Irradiation on SpermatogenicProgression in the Various Mice. To examine whether thepresence of aberrant or damaged DNA in p53 knockoutspermatocytes undergoing meiotic division is indeed theprimary cause for observed enhancement in cell degenera-tion, we analyzed the meiotic progression upon induction ofexogenous DNA damage at the pachytene and spermatogo-nial stages. For this purpose, mice with different p53 geno-types were g-irradiated (1 or 3.5 Gy) 9 days or 24 h after invivo BrdUrd pulse, and cell survival and DNA content distri-bution were analyzed by FACS. At these DNA damage timepoints, most of the BrdUrd-labeled cell cohort was situatedin the mid/late pachytene stage or in the spermatogonialstage, respectively. Analysis of the BrdUrd-labeled cells 4days after 1 Gy g-irradiation at the spermatogonial stage

3 The abbreviations used are: UDS, unscheduled DNA synthesis; BrdUrd,5-bromo-2-deoxyuridine; FACS, fluorescence-activated cell sorter; 3AB,3-aminobenzamide; AO, acridine orange; DDW, double-distilled water;Tdt, terminal deoxynucleotidyl transferase.

Fig. 1. Reduced numbers of motile epididimal spermatozoa in p53(2/2)mice. The cauda epididimi of three mice with p53(1/1), p53(1/2), andp53(2/2) genotype were surgically removed and minced. The motility ofspermatozoa was measured by the “swim-up” assay. Bars, SD.

666 p53 Facilitates DNA Repair during Spermatogenesis

revealed no significant difference between the mice (data notpresented). In contrast, the percentage of tetraploid sper-matogenic cells in p53(1/1) and p53(1/2) mice was signif-icantly increased after 1 Gy g-irradiation at the pachytenephase (Fig. 3a). The same differences were scored whenmice were exposed to 3.5 Gy of g-irradiation. Starting fromthese DNA damage levels, the premeiotic delay was accom-panied by p53-independent apoptosis of cells undergoingmeiotic divisions (data not shown).

To examine whether other types of DNA damage will alsoactivate the in vivo p53-dependent or -independent check-point controls, the BrdUrd-pulsed mice with a spermato-

genic cohort situated at the pachytene (Fig. 3b) were treatedwith cisplatin or 3AB. Cisplatin causes interstrand DNA ad-ducts, thus activating the nucleotide excision repair path-way, whereas 3AB is a potent inhibitor of DNA damage-dependent protein poly(ADP)-rybozilation, which facilitatesthe ligation of the intrinsic DNA damage. As can be seen inFig. 3b, introduction of 3AB reduced the meiotic progressionin a p53-independent manner. Introduction of cisplatincaused a p53-dependent activation of the premeiotic check-point in a manner similar to g-irradiation (data regardingcisplatin is not shown). This suggests that the kind of DNAdamage applied during the meiotic prophase will determine

Fig. 2. Reduced efficiency of meiotic progression in p53(2/2) mice. p53(1/1), p53(1/2), or p53(2/2) mice were pulse-labeled with BrdUrd. Subse-quently, the progression along meiosis of the labeled cohort was followed using composite anti-BrdUrd-propidium iodide analysis by FACS. A chart ofspermatogenic progression indicates the duration of each phase and the timing of the DNA-damaging treatments that were used in the followingexperiments (a). b, representative analysis of spermatogenic progression obtained by FACS analysis using in vivo BrdUrd pulse [the indication of the methodof gating (upper part) and presentation of the meiotic progression of the gated cells (lower part)] c, the relative distribution of DNA content of the variousBrdUrd-labeled mice 13, 14, and 15 days after BrdUrd pulse, respective to progression of meiotic divisions. d, percentage of haploid BrdUrd-labeled cellsof total population in mice containing various p53 content.

667Cell Growth & Differentiation

whether the stress response will be p53-dependent or p53-independent.

Previously, we observed an elevated expression of p53 atthe pachytene phase (5). Therefore, we decided to examinethe effect of g-irradiation on the levels of p53 protein expres-sion in the various stages of spermatogenesis. To that end,we analyzed p53 expression in well-defined, enriched sper-matogenic populations obtained from p53(1/1) mice beforeand after exposure to g-irradiation (3.5 Gy). p53 expressionwas assessed by FACS and by immunoprecipitation withmonoclonal PAb-421 anti-p53 antibody. Detection of mei-otic, mitotic, and interphase cells was based on differentialDNA denaturability assay using the AO fluorescent DNAbinding dye. The identity of each spermatogenic populationwas determined by the DNA content, DNA denaturability(respective to chromosomal condensation), and chromo-some morphology. We verified the identity of the variouspopulations appearing in adult spermatogenic cells using theAO assay by analyzing their appearance during the firstspermatogenic round. In particular, we identified a distinctpopulation of tetraploid cells possessing condensed chro-mosomes as diplotene spermatocytes, because this popu-lation first appeared in the first round just before the onset ofthe meiotic division (data not shown). To further confirmthese results, we analyzed the p53 levels using immunopre-cipitation and the AO pattern of adult spermatogenic cells,enriched for various spermatogenic stages using centrifugalelutriation. Fig. 4a shows the DNA content of the spermato-genic populations obtained by centrifugal elutriation. Analy-sis of DNA morphology and DNA denaturability indicatedthat fraction I consisted mainly (.85%) of round sperma-

tids, whereas fraction II contained early prophase andmeiotic cells. Fractions III and IV contained zygotene-earlypachytene and late pachytene cells, respectively (Fig. 4b).Analysis of p53 expression in the various isolated fractionswas carried out by immunoprecipitation and FACS analy-sis of p53 levels using p53-specific antibodies. In agree-ment with our previous report, we observed that p53 isexpressed predominantly in fractions III and IV enrichedfor the zygotene-pachytene spermatocytes (Fig. 4c; Ref.5). Interestingly, however, under the present experimentalconditions, no significant up-regulation of p53 levels wasdetected after DNA damage in p53-containing fractions.Previously, it was suggested that activation of p53 proteinin testis after DNA insults is not associated with an accu-mulation in protein levels (11, 16).

Next we determined the outcome of DNA damage on thepercentage of meiotic tetraploid cells using the AO assay(Fig. 5a). We found that g-irradiation (1 Gy) of p53(1/1) micecaused a gradual reduction in the percentage of tetraploidmeiotic cells possessing condensed chromosomes, whichwas detectable at ;2 days after treatment (Fig. 5b). This timecourse correlates with the traverse of mid/late pachytenecells through meiosis. In parallel with the reduction of themeiotic tetraploid population, the only population of tet-raploid cells detectable was the high p53 expressors (datanot shown). This indicates that DNA damage causes theaccumulation of p53-positive premeiotic cells.

In contrast, 1 Gy of g-irradiation of p53(2/2) or p53(1/2)mice did not significantly change the percentage of tetraploidmeiotic cells. Under the same conditions, a 50% decline ofthis population in p53(1/1) mice was noticed. As can be

Fig. 3. Low levels of DNA dam-age induce p53-dependent pre-meiotic delay. The experimentwas performed and analyzed asdescribed in Fig. 2, except someof the mice were g-irradiatedduring pachytene phase 9 daysafter BrdUrd pulse. a, compari-son of DNA content distributionof BrdUrd-labeled spermato-genic cells 13 days after theBrdUrd pulse, with and withoutapplication of 1 Gy g-irradiation9 days after the pulse. b, sameexperiment, performed after treat-ment with 3AB instead of g-irradi-ation. Full graph, untreated mice;empty graph, treated mice.


seen in Fig. 5a, similar patterns of spermatogenic popula-tions were observed before and after DNA insult. Quantita-tive comparison of tetraploid meiotic cells indicated similarcell numbers in the p53(2/2) and p53(1/2) mice at differenttime points after exposure to g-irradiation, thus suggestingthat knockout of p53 expression releases a restraint prevent-ing prophase spermatocytes possessing low levels of DNAdamage to progress toward chromosomal condensation, aninitial step of meiotic division. This implies that the presenceof low levels of DNA damage at the end of the meioticprophase activates a p53-dependent checkpoint.

Analysis of apoptosis in the various mice 4 days after 1 Gyof g-irradiation using Tdt nick end labeling assay on testicularsections revealed a mild increase in apoptosis only in sper-matogonial cells. However, this increment was observed ir-respective of p53 genotype. Upon application of g-irradiationhigher than 5 Gy, dose-dependent apoptosis was also ob-served in spermatocytes in a p53-independent manner. Thisindicates that p53-independent mechanisms are responsiblefor elimination of spermatogenic cells containing high levelsof DNA damage during and after meiosis.

Analysis of UDS after In Vivo DNA Damage in the Var-ious Mice. In addition to controlling cell cycle checkpoints,p53 was also suggested to play a role in DNA repair, andbecause spermatogenesis involves such pathways exten-sively, we next examined the possibility that p53 participatesin the DNA repair-related processes of spermatogenesis. Tothat end, we utilized an assay measuring the in vivo levels ofUDS in testicular tissue. It should be noted that the levels ofUDS reflect the repair rate of a variety of DNA repair mech-anisms because the de novo nucleotide incorporation is a

step shared by practically all of the DNA repair pathways.This was carried out by measurement of the in vivo BrdUrdincorporation in nonreplicating, postmitotic cells by usingmonoclonal anti-BrdUrd antibodies with preferential affinitytoward low levels of BrdUrd incorporation in DNA, typical forDNA repair (47). Mice of different p53 genotypes were im-planted s.c. with BrdUrd pills (see “Materials and Methods”)several hours prior to application of in vivo DNA damage byeither g-irradiation or injection of various doses of cisplatin.The levels of BrdUrd incorporation were measured 24 h later.As can be seen in Fig. 6a, the untreated p53(1/1) andp53(2/2) mice exhibited no significant difference in the lev-els BrdUrd incorporation into postmitotic cells. After cisplatintreatment, the p53(1/1) mice exhibited a dose-dependentincrease in BrdUrd incorporation in nonreplicating cells. Asimilar increment was measured in these mice after exposureto increasing doses of g-irradiation. However, the DNA dam-age-dependent increase in BrdUrd incorporation measuredin the p53(2/2) mice subjected to similar treatments wassignificantly less pronounced. Fig. 6b represents a quantita-tive summary of the results obtained. On the basis of theFACS analysis of BrdUrd incorporation in the various sper-matogenic cell populations, it appears that the DNA repairlevels measured here are significantly lower in p53(2/2)mice than in p53(1/1) littermates.

To confirm these results and to identify the cells engagedin DNA repair, we analyzed in parallel the levels of UDS intesticular sections from the same mice, using computerizedquantitation of BrdUrd incorporation. To that end, testicularsections obtained from mice with different p53 genotypeswere subjected to an immunohistochemical staining using

Fig. 4. p53 is expressed in tet-raploid premeiotic pachytenespermatocytes. The spermato-genic cells were separated intofour enriched fractions by centrif-ugal elutriation: I, 17–21; II, 40–43; III, 43–52; and IV, 52–58 (see“Materials and Methods”). a,analysis of DNA content of sper-matogenic fractions obtained bycentrifugal elutriation. b, AOFACS analysis of the same frac-tions. c, p53 levels analyzed byimmunoprecipitation with mono-clonal anti-p53 PAb-421 antibodyin g-irradiated (5 h after 1 Gy) andnonirradiated enriched fractions.


the monoclonal anti-BrdUrd antibodies that were further vi-sualized by a secondary FITC-conjugated antibody (see“Materials and Methods”). Sections were then analyzed un-der the same data acquisition conditions to enable semi-quantitative analysis of the data. Exposure of animals to 10Gy of g-irradiation induced a significant increase in the num-ber of the low-level BrdUrd-incorporating cells of thep53(1/1) mice. These cells were clearly situated in the re-gions of the seminipherous tubuli containing postmitoticcells, mapping internally to the peripheral, spermatogonialcells. No such increment in BrdUrd-incorporating cells wasevident in sections obtained from the p53(2/2) mice. Similarpatterns of spermatogonial cell proliferation, scored as highBrdUrd incorporators, were evident in all sections analyzed.These cells, located at the borders of the somniferous tubuli,represent the replicating spermatogonia.

Treatment of cells with cisplatin seemed to induce thesame patterns of cells incorporating BrdUrd. Again, thep53(1/1) mice exhibited increased numbers of cells ex-pressing fine grains of incorporated BrdUrd, correspondingto UDS, as compared with p53(2/2) mice. Although somedifferences in proliferating cell distribution could be seen, the

general pattern of high BrdUrd-incorporating cells inp53(1/1) and p53(2/2) mice was comparable.

These results suggest, therefore, that at least a part ofDNA repair activity measured by the UDS assay in spermat-ogenic cells seems to be p53 dependent.

DiscussionA role for p53 in spermatogenesis was suggested by theobservation that changes in p53 expression are associatedwith key phases regulating meiotic progression, peaking atthe pachytene stage (5), and that mice deficient in p53 exhibita degenerative syndrome manifested by the appearance ofgiant cells (3). Furthermore, although not well documented,p53 knockout mice seem to suffer infertility. p53 was sug-gested to take part in a number of pathways that were shownto be associated essentially with the maintenance of genomestability. p53 seems to control, among others, cell growtharrest, induction of apoptosis, cell differentiation, and DNArepair. These activities were shown to be associated with thedifferent cell cycle phases and checkpoints, most likely in acell type-specific manner (48). It is, therefore, of interest toelucidate the contribution of p53 in spermatogenesis, a

Fig. 5. p53-knockout mice do not arrest upon low-level DNA damage. p53(1/1), p53(1/2), or p53 (2/2) mice were g-irradiated (1 Gy). Subsequently, thepercentage of the meiotic and premeiotic cells was assessed using the AO FACS-based assay. a, representative analyses of spermatogenic populationsobtained 4 days after 1 Gy of g-irradiation in various mice. b, percentage of tetraploid meiotic cells in p53(1/1) mice after g-irradiation. c, percentage oftetraploid meiotic cells 4 days after 1 Gy of g-irradiation (■) compared with nonirradiated control in various mice (h). Bars: b and c, SD.


Fig. 6. Spermatocytes of p53-knockout mice exhibit less efficient in vivo DNA repair. The UDS levels after treatment with various doses of g-irradiation or cisplatinwere measured in vivo in p53(2/2) or p53(1/1) mice by FACS or by immunohistochemistry. a, representative results obtained by FACS analysis. b, statisticalanalysis of UDS levels after 10 Gy of g-irradiation and 8 mg/kg treatment with cisplatin after normalization with nonirradiated controls of p53(1/1) (■) and p53(2/2)mice (h). Bars, SD. Immunohistochemical detection of UDS after 10 Gy of g-irradiation (c), or cisplatin treatment (d), using semiquantitative computerized analysis,is shown. Green, Hoechst 33342 staining for DNA morphology; red, BrdUrd incorporation, measured indirectly with the IU-4 anti-BrdUrd antibody.


physiological process that engages many of the p53-asso-ciated activities.

Spermatogenesis in most mouse strains, carrying eitherstable chromosomal translocations or deregulated premei-otic DNA recombination and DNA repair (40, 58), arrests atthe pachytene stage (49). This implies that this stage harborsa checkpoint that is dependent on chromosomal aberrations.In some cases, elevated apoptosis during the mitotic divi-sions of spermatogonia is prevalent (18, 50). Normally, adegenerative process is known to take place during the tworeduction divisions of meiosis and at the first stages of sper-matogenesis (51). The primary cause of this degenerationwas found to be chromosomal abnormalities (50, 52). Giventhis fact as well as the fact that p53 is expressed during themeiotic prophase led us to adopt the working hypothesis thatp53 functions in the meiotic prophase in a similar manner asit does in DNA damage-dependent G2 delay during the mi-totic cell cycle.

The involvement of p53 in the DNA damage-dependentdelay at the G2 phase of the cell cycle and its contribution tothe DNA repair at this phase were alluded to in severalstudies. Recently, Bunz et al. (53) demonstrated that knock-out of either p53 or p21/waf1 gene expression in humancolorectal cancer cell lines causes premature exit of DNAdamage-dependent G2 delay accompanied by failure of cy-tokinesis, resulting in endoreduplaction of the tetraploid cellsand formation of polyploid giant cells. It appears that highlevels of p53 can modulate the arrest at the G2 phase prior tomitotic chromosomal condensation. The duration of the DNAdamage-dependent delay is also affected by the efficiency ofDNA repair at the G2 phase (26–28, 54–58).

There are several studies dealing with the possible involve-ment of p53 in spermatogenesis. The observation that nodifferences in recombination frequency in specific genomicloci were found upon comparison of p53 knockout mice withtheir intact litter mates suggested no role for p53 during andafter recombination in spermatogenesis (17). Furthermore, itwas observed that the DNA damage-dependent apoptosisoccurring during meiotic metaphase I is p53-independent(18). Others, however, suggested that the radio sensitivity(15) and (partially) the thermo sensitivity (59) of replicatingspermatogonia are p53-dependent. It should be noted thatthese studies did not assess the effect of p53 targeting onthe general quantitative aspects of spermatogenic progres-sion and maturation.

In this report, we compared the pattern of progression ofcells along the spermatogenic differentiation in mice of dif-ferent p53 genotypes. To evaluate the nature of p53 involve-ment in the regulation of spermatogenic process, we firstpulse-labeled with BrdUrd a cohort of replicating spermato-gonia and quantitated the percentage of labeled cells at keyphases of spermatogenesis, i.e., mitotic proliferation, meioticreduction divisions, and mature spermatozoa. We found thatwithout application of exogenous DNA damage, the percent-age of BrdUrd-labeled replicating spermatogonia andprophase spermatocytes is comparable between p53 knock-out and normal mice. Thus, in these conditions, there was nosignificant difference in levels of spermatogonial apoptosisor cell loss before and during prophase between these mice.

However, upon completion of meiosis, p53 knockout micehad only 60–65% of the labeled haploid cells found in nor-mal mice. This suggests that the reduction in the numbers ofmotile spermatozoa observed in p53 knockout mice may beattributed to cell degeneration at some stage of spermato-genesis.

The pronounced degenerative process during meiosis ob-served in p53-knockout mice (which is manifested by theappearance of multinucleated giant cells) can be caused byeither deficient DNA repair during the meiotic prophaseand/or by deregulation of the checkpoint that is supposed toprevent the entrance of cells possessing damaged DNA tomeiosis. To compare the response to DNA damage at themeiotic prophase, we used BrdUrd pulse labeling, as well asthe AO DNA denaturability assay, to quantify the progressionof spermatogenic cells without BrdUrd labeling. Using thesetwo methods, we found the existence of a p53-dependentcheckpoint during the pachytene stage, which regulates theprogression toward meiosis upon low levels of DNA damage.In addition, p53-knockout mice exhibited lower levels of invivo DNA repair, measured by semiquantitative evaluation ofUDS upon insult with g-irradiation or cisplatin.

Taken together, it appears that in p53-knockout mice, partof the postmeiotic spermatids possessing DNA damage pre-sumably mature to spermatozoa, which in many cases areimmotile because of the mutations that were not eliminatedduring the meiotic divisions. Some of these haploid cells maydegenerate by the p53-independent pathways, as suggestedby others (18). We suggest, therefore, that the end result ofthe p53-knockout phenotype is less efficient spermatogen-esis, characterized by lower numbers of motile “functional”spermatozoa.

It was already shown that the duration of late-pachytenephase is regulated by phosphorylation, because okadaicacid treatment is sufficient to induce transfer from pachyteneto dyplotene phases in vitro, without requirement for any denovo protein translation (60). This implies that progressiontoward premeiotic chromosome condensation is regulatedby a typical protein-phosphorylation based “checkpoint”mechanism. We propose that among other proteins, p53regulates this process in a specific set of conditions, whichare disregarded by other DNA damage-sensing mecha-nisms. Our results suggest that p53 may provide an addi-tional level of stringency, especially in low levels of DNAdamage, to the existing p53-independent “quality control”mechanisms responsible for elimination or repair of sper-matocytes with damaged DNA. Moreover, p53 contributes tothe efficiency of DNA repair during the postmitotic stages ofspermatogenesis. By these means, p53 function ensuresappropriate “quality” and “yield” of mature intact and func-tional spermatozoa in normal mice.

Materials and MethodsMouse Maintenance and Genotype Analysis. p53(2/2) mice of C57BLstrain (61) were bred by mating p53(1/2) or p53(2/2) parents. Genotypeanalysis was performed by PCR analysis, which permits the specificidentification of p53(1/1), p53(1/2), and p53(2/2) carriers. Tissue frag-ments obtained from either adult mice or embryos (tails or ears) wereanalyzed. Genomic DNA was prepared by incubating tissues in 0.5 ml ofTE containing 0.4 mg proteinase K and 0.5% SDS overnight at 37°C. The


samples were extracted three times in phenol/chloroform/isoamyl andtwice in alcohol. DNA was washed in isopropanol, centrifuged, washedtwo times in 70% ethanol, and dissolved in DDW. Each PCR reactioncontained 0.5 mg of genomic DNA. The primers used were as describedbefore (62); for the wild-type p53 sequences, we used the 595W2 primerGTC CGC GCC ATG GCC ATC TA and the 393W2 primer ATG GGA GGCTGC CAG TCC TAA CCC. For the neo p53(2/2)-specific marker, we usedthe 59 ND, CAG CCC AGT GGA GTG ACA CAC ACC T (specificallydesigned in our lab); the sequence of the 39 3N2 was TTT ACG GAG CCCTGG CGC TCG ATG T. The PCR program included 5 min at 94°C, 36cycles of 30 s at 94°C, 30 s at 69°C, 60 s at 72°C, and finally 7 min in 72°C.Typical patterns of DNA fragments were obtained for the p53(1/1) ho-mozygous (138 bp), p53(2/2) homozygous (180 bp), and p53(1/2) het-erogeneous (138 bp and 180 bp).

Spermatozoa Motility Measured by the “Swim-Up” Assay. Thecauda epididimi of mice were removed surgically after sacrifice, gently cutto small (about 1-mm size) pieces using a blade, as described previously(63). The cut tissue was resuspended in modified McCoy 5A mediumsupplemented with glucose (2 g/l), lactate (1.8 g/l), and sodium pyruvate(0.11 g/l; Sigma). The tissue was spun down at 800 3 g and incubated for1 h in 32°C. The supernatant medium containing the motile spermatozoathat swam up after centrifugation was carefully collected and counted ina hemocytometer.

Testicular Cell Suspension Preparation Using the Collagenase-Trypsin Method. Testicles were surgically removed and decapsulated.To remove the extra tubular tissue, the tubuli were incubated for 20 minunder shaking in McCoy 5A medium containing 1 mg/ml of collagenase IV(Sigma). After two washes with the McCoy 5A medium, the tubuli wereresuspended in the same medium containing 2.5 mg/ml trypsin (Sigma)and 1 mg/ml DNase I (Sigma). The tubuli were incubated for another 15min at 32°C under shaking. Then the tubuli were gently dispersed bypipettation with a Pasteur pipette. Subsequently, the cell suspension waswashed once in McCoy 5A medium supplemented with 10% FCS, 0.5%BSA, and 0.08% w/v soybean trypsin inhibitor (Sigma; type I–A) and againwith same solution except the trypsin inhibitor. Finally, the cell suspensionwas filtered through nylon mesh and washed twice with McCoy 5A me-dium.

Isolation of Enriched Spermatogenic Populations by CentrifugalElutriation. Spermatogenic cells (1–3 3 108), obtained as describedabove, were loaded into an elutriation rotor [J-6MI centrifuge equippedwith a JE-5.0 elutriation system, including a Sanderson chamber (Beck-mann Instruments Inc.) and MasterFlex (Cole-Parmer Instruments)] peri-staltic pump presterilized with 5% sodium hypochlorite. The separationwas performed at room temperature (20–22°C) using McCoy 5A mediumsupplemented with lactate and pyruvate (see above) and 2% heat-inac-tivated fetal calf serum with a constant centrifuge speed of 2500 rpm(rmax 5 ;470). The cells were loaded with a pump speed of 13 ml/min;haploid spermatids were isolated at 17–21 ml/min pump speed. In addi-tion, several tetraploid-enriched fractions at pump speeds of 40–43,43–52, and 52–58 ml/min were collected. Separated cell populations werewashed once with DPBS and analyzed by FACS and microscope forploidity and cell morphology, respectively, to confirm their identity andstage of enrichment.

Analysis of Meiotic and Mitotic Testicular Cells by FACS Using theAO DNA Denaturability Assay. Testicular cell suspensions (1 3 106

cells/ml) were fixed for 2–24 h in 70% ethanol-30% HBSS v/v at 220°C.Cells were washed once with HBSS and resuspended in HBSS with 0.5mg/ml RNase A. After 1-h incubation at 37°C, cells were washed once andresuspended in HBSS at 2 3 106 cells/ml. Cell suspensions (200 ml) wereadded to 0.5 ml of 0.1 M HCl. After 40-s incubation at room temperature,2 ml of Acridine Orange AO (Molecular Probes) staining solution of pH 2.6,containing 90% v/v 0.1 M sodium citrate, 10% v/v 0.2 M Na2HPO4, and 6mg/ml AO, were added (64). Cells were analyzed by the FACS using theFACSort flow cytometer and CellQuest list mode analysis software (Beck-ton-Dickinson; Ref. 65).

Labeling of Replicating Spermatogonia with BrdUrd and Subse-quent FACS Analysis. The protocol is essentially as described previ-ously (66–68). Mice received i.v. injections of 100 mg/kg BrdUrd (Sigma)and were sacrificed at various times later. The spermatogenic cells wereprepared (see above) and subsequently fixed in ice-cold solution contain-ing 70% v/v ethanol (Biolab), 30% HBSS and stored at 220°C untilanalysis.

The FACS assay of labeled cells was essentially according to theBeckton Dickinson protocol using the FITC-labeled anti-BrdUrd antibody.The data were acquired on a FACsort machine (Beckton Dickinson). Cells(10,000) of each mouse (in duplicates) were analyzed by a CellQuestsoftware (Beckton Dickinson).

In Vivo and In Vitro UDS Assay. Mice were sedated with pentobar-bital, and two pills containing 50 mg of BrdUrd (Boehringher Mannheim)were s.c. implanted in the nape of the neck. The time allowed for theBrdUrd concentration to reach the testis, to permit sufficient stochiomet-ric labeling of DNA replication and repair, was 7 h. This was determined byflow cytometric measurement of BrdUrd incorporation in replicating andrepairing spermatogenic cells (data not shown). To assure that the BrdUrdincorporation is stochiometric to the DNA damage dose, all experimentswere performed within a range of DNA-damaging levels. The damagingagents were introduced either by tail injection of PBS [for 3-AB andcisplatin (16)] or by whole-body g-irradiation. Twenty-four h after DNAdamage, the mice were sacrificed, and the testicles were removed andfixed in 70% ethanol or in buffered formalin for immunohistochemicalanalysis. The doses used were 5, 10, 15, and 20 Gy of g-irradiation; 4, 8,and 16 mg/kg of cisplatin; and 100, 200, and 400 mg/kg of 3AB (Sigma).

UDS Measure by FACS. For flow cytometrical analysis, the testicleswere dispersed using the collagenase-trypsin method (as describedabove) and fixed in 70% ethanol at 220°C. The protocol is essentially aspublished by Selden et al. (47). In brief, the ethanol-fixed cells were treatedwith RNaseA, resuspended in cold 0.1 N HCl 0.5% Triton X-100, andincubated on ice for 10 min. Next, the cells were resuspended in deionizedH2O and incubated for 15 min at 90°C to additionally denature the DNA.Cells were incubated for an additional 1 h at room temperature withanti-BrdUrd-specific monoclonal IU-4 antibody (CALTAG Laboratoriescode MD5010; (Ref. 47). After washing with HBSS, the cells were incu-bated with secondary FITC-conjugated anti-mouse IgG antibody,washed, and resuspended in HBSS containing 5 mg/ml propidium iodide(Sigma) and analyzed by FACS, using the FACSort (Beckton Dickinson)machine operated by the CellQuest software (Beckton Dickinson).

Computerized Measure of UDS in Testicular Sections. Testicleswere fixed in 70% ethanol (BioLab) or in formalin (Sigma) and subse-quently embedded in paraffin. The sections obtained from paraffin blockswere deparaffinized according to standard protocol and subjected to acidand heat treatment as indicated for UDS measure by FACS. The BrdUrdincorporation was determined by overnight incubation in 4°C with the IU-4monoclonal antibody. After application of secondary FITC-conjugatedanti-mouse IgG antibody (Sigma F8521), the sections were incubated with5 m/ml Hoechst 33342 DNA stain (Molecular Probes, Inc.) and analyzed byAxioplan fluorescent microscope (Zeiss) equipped with computerizedCCD camera and image processing software. Pictures were presented ingray or in false color.

In Vivo Apoptosis. Apoptosis was analyzed by the in situ Tdt nick endlabeling staining that was carried out as described before (69). Briefly,after deparaffinization of the tissue sections, they were immersed in DDW.Sections were then incubated for 15 min in 23 SSC (13 SSC is 0.15 M

sodium chloride and 15 mM sodium citrate) buffer at 60°C, washed inDDW, and incubated with 20 mg/ml of proteinase K (Boehringer Mann-heim) for 15 min at room temperature. After a wash with DDW, endoge-nous peroxidases were inactivated by incubating the sections with 2%H2O2 in PBST (PBS with 0.05% Tween 20) for 10 min at room tempera-ture. Sections were then incubated in TdT buffer (Boehringer Mannheim)for 5 min at room temperature, and then 50 ml of the reaction mixture [5 3TdT buffer, 1 ml biotin-21-dUTP (Clontech, 1 mM stock), and 8 units of TdTenzyme (Boehringer Mannheim)] were added. The reactions were carriedout at 37°C for 1.5 h in a humid chamber. The slides were washed with 23SSC, DDW, and finally with PBS and covered with 10% SM in PBST for15 min. After SM was removed, the sections were incubated with ABCsolution from the ABC kit (Vector Laboratories, Inc.) for 30 min at roomtemperature, washed with PBS, and stained using AEC procedure (Sig-ma). The slides were then washed three times in DDW and stained withhematoxylin for 30 s and mounted by Kaiser’s glycerol gelatin (Merck).

Immunoprecipitation. Spermatogenic cell suspension was obtainedby the collagenase trypsin method. The enriched populations for variousspermatogenic phases were obtained by centrifugal elutriation and incu-bated for 1.5 h in methionine-deficient medium supplemented with 50mCi/5 3 106 cells of [35S]methionine.


[35S]methionine-labeled proteins were immunoprecipitated with anti-p53-specific antibodies. The complexes generated were precipitated with30 ml of Sepharose-protein A (50%) and washed three times in PLB buffer[10 mM NaH2HPO4 (pH 7.5), 100 mM NaCl, 1% Triton X-100, 0.5% sodiumdeoxycholate, and 0.1% SDS. The immune complexes were separated onSDS-PAGE.

AcknowledgmentsWe thank Ahuva Kapon for excellent technical help, David Wiseman forthe fruitful discussion, and Vivienne Laufer for help in preparation of themanuscript.

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