Meiotic Prophase Abnormalities and Metaphase Cell Death in MLH1-Deﬁcient Mouse Spermatocytes: Insights Into Regulation of Spermatogenic Progress

Developm ental Biology 249, 85–95 (2002) doi:10.1006/dbio.2002.0708

Meiotic Prophase Abnorm alities and Metaphase Cell Death in MLH1-Deﬁcient Mouse Sperm atocytes: Insights into Regulation of Sperm atogenic Progress

Shannon Eaker,¹John Cobb,²April Pyle, and Mary Ann Handel³

Departm ent of Biochem istry and Cellular and Molecular Biology, U niversity of Tennessee, Knoxville, Tennessee 37996

The MLH1 protein is required for norm al m eiosis in m ice and its absence leads to failure in m aintenance of pairing between bivalent chrom osom es, abnorm al m eiotic division, and ensuing sterility in both sexes. In this study, we investigated whether failure to develop foci of MLH1 protein on chrom osom es in prophase would lead to elim ination of prophase sperm atocytes, and, if not, whether univalent chrom osom es could align norm ally on the m eiotic spindle and whether m etaphase sperm atocytes would be delayed and/or elim inated. In spite of the absence of MLH1 foci, no apoptosis of sperm atocytes in prophase was detected. In fact, chrom osom es of pachytene sperm atocytes from Mlh1^؊/؊m ice were com petent to condense m etaphase chrom osom es, both in vivo and in vitro. Most condensed chrom osom es were univalents with spatially distinct FISH signals. Typical m etaphase events, such as synaptonem al com plex breakdown and the phosphorylation of Ser10 on histone H3, occurred in Mlh1^؊/؊sperm atocytes, suggesting that there is no inhibition of onset of m eiotic m etaphase in the face of m assive chrom osom al abnorm alities. However, the condensed univalent chrom osom es did not align correctly onto the spindle apparatus in the m ajority of Mlh1^؊/؊sperm atocytes. Most m eiotic m etaphase sperm atocytes were characterized with bipolar spindles, but chrom osom es radiated away from the m icrotubule-organizing centers in a prom etaphase-like pattern rather than achieving a bipolar orientation. Apoptosis was not observed until after the onset of m eiotic m etaphase. Thus, sperm atocytes are not elim inated in direct response to the initial m eiotic defect, but are elim inated later. Taken together, these observations suggest that a spindle assem bly checkpoint, rather than a recom bination or chiasm ata checkpoint, m ay be activated in response to m eiotic errors, thereby ensuring elim ination of chrom osom ally abnorm al gam ete precursors. © 2002 Elsevier Science (USA)

som es and recom bination between them . Recom bination results in the form ation of physical links, chiasm ata, be-

INTRODUCTION

tween hom ologous chrom osom es. These are required for proper chrom osom e alignm ent and segregation in the ﬁrst m eiotic division (Carpenter, 1994; Koehler et al., 1996). Since recom bination is a prerequisite for faithful segregation of chrom osom es, it is im portant to understand not only the m olecular events of recom bination but also the consequences of error and whether error is m onitored in order to ensure gam ete quality. Meiotic recom bination is a com plex series of steps, m ediated by a large num ber of proteins that likely act together in com plexes (Cohen and Pollard, 2001; Sm ith and N icolas, 1998). The events of recom bination include DN A double-strand breaks, strand invasion, form ation of Holliday junctions and heteroduplex DN A, and processing of recom bination interm ediates by reactions that include DN A m ism atch repair. A signiﬁcant
Making a genetically com plete and norm al gam ete is essential for reproduction and continuity of the species. The process of m eiosis ensures that gam etes receive a haploid, 1N , com plem ent of chrom osom es and the 1C com plem ent of DN A. The stage is set for accurate segregation of chrom osom es by the events of m eiotic prophase, principally pairing and synapsis of hom ologous chrom o-

1

Present address: Am erican Red Cross, Holland Laboratory,
Rockville, MD 20855.

2

Present address: University of Geneva, Departm ent of Zoology,
Sciences III, 30 Quai Ernest-Anserm et, 1211 Geneva 4, Switzerland.

3

To whom correspondence should be addressed. Fax: (865) 974-
6306. E-m ail: m [email protected].

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FIG. 1. Chrom osom e pairing is defective in Mlh1^Ϫ/Ϫsperm atocytes as revealed by FISH analysis of MI sperm atocytes from Mlh1^ϩ/ϩ(A) and Mlh1^Ϫ/Ϫ(B) m ice with chrom osom e paint probes (for chrom osom es 2, green, and 8, red, with DAPI-stained chrom atin in blue). In (A), signals of the sam e color are juxtaposed in a single bivalent, indicating paired hom ologous chrom osom es in control sperm atocytes. In (B), univalent chrom osom es are identiﬁed by two separate signals per chrom osom e in m utant sperm atocytes. FIG. 2. Pachytene sperm atocytes from Mlh1^Ϫ/Ϫm ice are com petent to condense chrom osom es in response to OA treatm ent in vitro. (A) Control Mlh1^ϩ/ϩsperm atocytes treated with OA for 6 h. (B) Mlh1^Ϫ/Ϫsperm atocytes treated with OA for 6 h. Condensed chrom osom es are usually univalents, assessed by chrom osom e num ber, m orphology, and absence of visible chiasm ata. (C) Mlh1^Ϫ/Ϫsperm atocytes treated with OA for 6 h, showing occasional chiasm ate bivalents (arrow).

num ber of proteins have been im plicated, directly or indirectly, in the events com prising recom bination in m am - m als (Cohen and Pollard, 2001). While m uch is already known about the m olecular processes of m eiotic recom bination, virtually nothing is known about the m echanism s in m am m alian gam etogenesis that m ight m onitor the progress of m eiosis and ensure gam ete quality. In the apparent absence of m utations affecting m am m alian m eiotic checkpoint m echanism s, evidence for such processes can be gleaned from experim ental investigation of m utants with errors in m eiotic processes. N ull or knockout m utations have been particularly useful to test possible downstream effects, perhaps checkpoint-m ediated, of failure in specific m eiotic processes. However, there is an im portant caveat: failure in progress of gam etogenesis in the absence of a specific gene product can be explained in at least two ways. It could be due either to a requirem ent for the gene product in order to progress to the subsequent step in m eiosis or to checkpoint m onitoring of a failed event and subsequent elim ination by apoptosis of germ cells that are otherwise progressing in differentiation. N onetheless, m utations or conditions interfering with the events of m eiosis can be inform ative about specific requirem ents and possibly provide indirect evidence for the existence of m eiotic checkpoint m echanism s. Here, we study the effects on m eiotic progress in sperm atogenesis of absence of the MLH1 protein. The MLH1 protein prom otes crossing over in budding yeast (Hunter and Borts, 1997), and in m ice and hum ans, the MLH1 protein localizes with m eiotic crossover sites, corresponding to the num ber and distribution of chiasm ata (Anderson et al., 1999; Barlow and Hulten, 1998). Mice that are hom ozygous for a knockout of the Mlh1 gene are sterile, exhibiting a failure either to form or to m aintain chiasm ata, revealed by presence of univalent chrom osom es at m eiotic m etaphase (Baker et al., 1996; Edelm ann et al., 1996). In m utant fem ale m ice, oocytes clearly progress to m etaphase, at which tim e they exhibit abnorm alities of chrom osom e alignm ent and spindle assem bly (Woods et al.,

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FIG. 3. Events of the G ₂/M occur in norm al tem poral order in Mlh1^Ϫ/Ϫsperm atocytes. (A) Section of stage XI–XII tubule from control Mlh1^ϩ/Ϫtestis, stained with antibodies for division-phase MPM-2 epitopes (red) and phosphorylated histone H3 (green), showing MI sperm atocytes with aligned chrom osom es. (B) Section of stage XI–XII tubule from testis of an Mlh1^Ϫ/Ϫm ouse, stained as in Fig. 1A. N ote that diplotene sperm atocytes show “speckled” staining for phosphorylated histone H3 at the centrom eric heterochrom atin located near the nuclear envelope (arrow) and that MI sperm atocytes with heavily phosphorylated histone H3 do not have neatly aligned chrom osom es. (C) This im age of a surface-spread control MI sperm atocyte, stained with antibodies against SYCP3 (red) and phosphorylated histone H3 (green), shows typical residual SYCP3 staining at the paired m etaphase centrom eres (arrow). (D) This Mlh1^Ϫ/Ϫsperm atocyte, prepared as in Fig. 3C, shows that histone H3 is phosphorylated at MI, but that hom ologous centrom eres are not paired.

1999). In m utant m ale m ice, data about loss of sperm atocytes are a bit m ore am biguous, but apparently it occurs either in late m eiotic prophase (Edelm ann et al., 1996) or in the division phase (Baker et al., 1996); the difference could be due to different m utations or differing interpretation of the phenotype. We analyzed the consequences of chiasm ata failure for survival and progress of sperm atocytes, and determ ined the tim e of cell death with reference to m eiotic events. In spite of absence of MLH1 protein, sperm atocytes are not arrested and do not undergo apoptosis during the pachytene stage when MLH1 foci are ﬁrst assem bled onto chrom osom es. Instead, there is an apparently norm al transition from prophase to prom etaphase, with the m ajority of sperm atocytes dying at m etaphase. Thus, if cell death is induced by a m eiotic checkpoint, the checkpoint seem ingly detects abnorm alities at the stage of spindle assem bly and chrom osom e alignm ent, well after the m anifestation of the ﬁrst m eiotic abnorm ality in m utant sperm atocytes.

MATERIALS AND METHODS

Mice

Mice carrying the Mlh1 targeted m utation were generously provided by Sean Baker (Baker et al., 1996) and offspring were genotyped by PCR reactions for the norm al and targeted alleles of

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the Mlh1 gene from DN A obtained from tail tips. Mice were housed under 14 h light/10 h dark photoperiods at a constant tem perature (21°C), with free access to standard laboratory chow and water.

and

8

(Cam bio Inc., Cam bridge, UK) were warm ed to 37°C,

denatured at 65°C, then cooled to 37°C for 1 h. A 15-␮l aliquot of each chrom osom e paint probe was added to the slides. The slides were coverslipped, sealed, and incubated overnight at 37°C in a hum idiﬁed cham ber. After two washes at 45°C for 5 m in in 50% form am ide/2ϫ SSC, followed by two washes in 0.1ϫ SSC, detection reagents from the m anufacturer (Cam bio, Inc.) were added to each slide. The slides were then processed for ﬂuorescent visualization as described below.

Cell and Tissue Preparation

Male m ice were killed by cervical dislocation. Testes were rem oved and fixed by overnight im m ersion in cold 4% paraform aldehyde (Sigm a) at 4°C. After fixation and dehydration, testes were em bedded in paraffin and sectioned at 3 ␮m . The deparaffinized sections were m icrowaved (10 m in at power 3) to unm ask antigens before reaction with antibody. To obtain isolated germ cells, testes were detunicated, digested in 0.5 m g/m l collagenase (Sigm a) in Krebs–Ringer bicarbonate (KRB) at 32°C for 20 m in, then digested in 0.5 m g/m l trypsin (Sigm a) in KRB at 32°C for 13 m in. After filtration through 80-␮M m esh and three washes in KRB, sperm atocytes were either fixed in a fibrin clot (see below) or enriched for isolation of pachytene sperm atocytes by sedim entation on a bovine serum album in (BSA) gradient at unit gravity (Bellve´, 1993). After isolation of pachytene sperm atocytes, the cells were cultured in MEM m edium /5% fetal bovine serum (Gibco/BRL). After overnight culture at 32°C with 5% CO ₂, cells were treated for 6 h with 5 ␮M okadaic acid (OA) or the ethanol solvent (Cobb et al., 1999a). Chrom atin configurations were visualized by Giem sastaining of air-dried preparations of the treated cells (Evans et al., 1964; Wiltshire et al., 1995). Surface-spread chrom atin preparations for synaptonem al com plex visualization were perform ed as previously described (Cobb et al., 1999a). Briefly, germ cells were fixed in 2% paraform aldehyde and allowed to dry onto slides. The slides were fixed in 2% paraform aldehyde/0.03% SDS, then in 2% paraform aldehyde, then blocked in 10% goat serum /3% BSA in phosphate-buffered saline (PBS) prior to processing for im m unofluorescence.

Apoptosis Analysis

Apoptosis assays were perform ed by using the In Situ Cell Death Detection Kit (Roche Pharm aceuticals), utilizing the end-labeling TUN EL reaction on testes fixed and sectioned as described above. The TUN EL reaction was perform ed according to the m anufacturer’s protocol, with the exception of a 15-m in incubation with the enzym e on the slides. After deparaffinization in xylene and rehydration, the slides were incubated in 80 ␮l of the reaction m ix at 37°C in a hum idified cham ber. After two 5-m in washes in PBS, the slides were processed for im m unofluorescence. To determ ine the tim ing of cell death relevant to m eiotic stage, a developm ental analysis was perform ed. Apoptosis was scored in cross-sections of sem iniferous tubules from m ice 16, 18, 20, 22, and 24 days old, as well as from adults. Three control and three m utant m ice from each age were used. Tubules with m ore than three apoptotic germ cells were scored as apoptotic, consistent with previously established criteria (Kon et al., 1999); approxim ately 500 tubule crosssections per m ouse were scored.

Immunolocalization

Antisera used were polyclonal anti-SYCP3 (Eaker et al., 2001), anti-tubulin (Am ersham ), anti-phosphorylated histone H3 (Upstate Biotechnology), and anti-MPM-2 (Upstate Biotechnology). Following overnight incubation in prim ary antibody, slides were incubated with rhodam ine- or ﬂuorescein-conjugated secondary antibodies (Pierce), and m ounted with Prolong Antifade (Molecular Probes) containing DAPI (Molecular Probes) to stain DN A. Antibody localization was observed by using an Olym pus epiﬂuorescence m icroscope, and im ages were captured to Adobe PhotoShop with a Ham am atsu color CCD cam era. Confocal im ages were collected by using a Leica TC SP2 laser-scanning confocal m icroscope.
To obtain a preparation enriched in m eiotically dividing sperm atocytes, a variation of the transillum ination procedure (Parvinen et al., 1993) was used (Eaker et al., 2001). Testes from adult m ice were detunicated, and then digested with 0.5 m g/m l collage-

nase for

8

m in at 33°C. Tubule segm ents were excised and

transferred onto m icroscope slides in KRB. A coverslip was then placed on top of the segm ent, allowing the tubules to spread onto the slide. The entire slide was then frozen in liquid N ₂for 30 s, the coverslip was rem oved, and the slide was fixed in 3:1 ethanol/acetic acid. Prior to incubation with antibodies, the slide was blocked in PBS/10% goat serum for 30 m in. Sperm atocytes from germ cell preparations were em bedded in fibrin clots as previously described (Eaker et al., 2001). Germ cells were isolated as described above and brought to a concentration of 25 ϫ 10⁶cells/m l. A 3-␮l aliquot of fibrinogen (Calbiochem , 10 m g/m l fresh) and 1.5 ␮l of the cell suspension were pipetted onto a slide. Then, 2.5 ␮l of throm bin (Sigm a; 250 units) was added, and the slide was allowed to clot for 2 m in. The slide was then fixed in 4% paraform aldehyde (Sigm a) for 15 m in, washed in 0.2% Triton X-100 (Sigm a) for 5 m in, then processed for im m unofluorescence. Chrom osom e painting, using fluorescence in situ hybridization
(FISH), was perform ed as previously described (Eaker et al., 2001). Briefly, sperm atocytes were fixed in 3:1 ethanol:acetic acid, then dropped onto slides and allowed to dry. After dehydration in an increasing ethanol series, cells were denatured by incubation in 70% form am ide/2ϫ SSC at 65°C for 2 m in, followed by another dehydration series. Chrom osom e paint probes, for chrom osom es 2

RESULTS

Chromosome Univalence and Events of the G ₂/M Transition in Mlh1^؊/؊Spermatocytes

Previous findings of lack of MLH1 protein foci in Mlh1^Ϫ/Ϫsperm atocytes and m eiotic chrom osom e univalence (Baker et al., 1996; Edelm ann et al., 1996) were confirm ed. Chrom osom e behavior in Mlh1^Ϫ/Ϫsperm atocytes was studied by using FISH with chrom osom e-specific (Chrs. 2 and 8) paint probes on surface-spread chrom osom e preparations. This analysis was carried out on m etaphase I (MI) sperm atocytes retrieved from testes as well as on pachytene sperm atocytes induced to reach MI by treatm ent with the phosphatase inhibitor OA (Wiltshire et al., 1995), a protocol providing a

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larger num ber of MI sperm atocytes for statistical purposes. Appropriate chrom osom e pairing, indicated by juxtaposed FISH signals, was observed for Chrs. 2 and 8 in control (Mlh1^ϩ/ϩ) sperm atocytes, with 0% Ϯ 0.00 m ispairing both in vivo as well as in vitro after treatm ent with OA (Fig. 1A). Am ong Mlh1^Ϫ/Ϫsperm atocytes, the m ajority (90% Ϯ 0.58) of Chrs. 2 and 8 were neither hom ologously paired nor in physical proxim ity; that is, they were separated by m ore than one FISH signal dom ain (Fig. 1B). Since surface-spread chrom osom es were scored, it is not known whether hom ologous chrom osom es could be in closer physical proxim ity in vivo. N onetheless, this analysis reveals a clear difference in proxim ity of hom ologs when m utant and control sperm atocytes were com pared. tubules from Mlh1^Ϫ/Ϫm ice, this was considered to be an artifact deriving from the absence of postm eiotic stages of sperm atid differentiation. The fact that Mlh1^Ϫ/Ϫsperm atocytes go through an apparently norm al G ₂/M transition is supported by observations of OA-treated sperm atocytes, where, as in controls, all sperm atocytes with condensed chrom osom es show disassem bly of the synaptonem al com - plex and phosphorylation of histone H3 throughout the chrom atin (Figs. 3C and 3D). These data show that, although univalent chrom osom es are form ed, the entry into m etaphase in Mlh1^Ϫ/Ϫsperm atocytes is sim ilar to that of Mlh1^ϩ/ϩsperm atocytes, im plying that although the MLH1 protein m ay be required for chiasm ata form ation or m aintenance, chiasm ata are not part of the signal m achinery enabling either the norm al or precocious, OA-induced, G ₂/M transition.
Incubation of pachytene sperm atocytes with OA is an assay that allowed assessm ent of com petence of the MLH1- deﬁcient sperm atocytes to undergo various events of the G ₂/M transition (Cobb et al., 1999a). In these analyses, characteristic processes of the G ₂/M were m onitored. These included disassem bly of the axes of the synaptonem al com plex recognized by the antibody to m ouse SYCP3, condensation and individualization of chrom osom es, the phosphorylation of histone H3, a characteristic m arker of the transition into m etaphase in both m itotic and m eiotic cells (Cobb et al., 1999b), and presence of division-phase phosphorylated epitopes recognized by the MPM-2 antiserum . In both control and m utant Mlh1^Ϫ/Ϫsperm atocytes, treatm ent with OA led to chrom osom e condensation and other events of the G ₂/M transition, revealing com petence of m utant sperm atocytes to undergo events of the G ₂/M transition (Figs. 2 and 3). Analysis of MI chrom osom es in standard Giem sa-stained chrom osom e preparations (Fig. 2) revealed severe reduction in num ber of chiasm ata in m utant Mlh1^Ϫ/Ϫsperm atocytes (Fig. 2B), although about 25% of the sperm atocytes had one or two chiasm ate bivalents (Fig. 2C). This analysis also showed com petence of Mlh1^Ϫ/Ϫsperm atocytes to condense chrom osom es in response to OA treatm ent (Fig. 2B). In both control and m utant sperm atocytes, all stages of condensation were observed. Likewise, as previously reported (Wiltshire et al., 1995; Cobb et al., 1999a,b), not all sperm atocytes exhibit chrom osom e condensation after OA treatm ent, and interm ediate stages are seen. N onetheless, over a series of four experim ents, the frequency of Mlh1^Ϫ/Ϫsperm atocytes with condensed chrom osom es was equal to or slightly greater than the frequency am ong control sperm atocytes in the sam e experim ent. Both histone H3 phosphorylation and disassem bly of the synaptonem al com plex (SC), events consistent with m etaphase entry, were also observed in Mlh1^Ϫ/Ϫsperm atocytes (Fig. 3). These events occur in a tem porally norm al m anner in MLH1-deﬁcient sperm atocytes (Fig. 3B), with histone H3 phosphorylation originating in the centrom eric heterochrom atin in diplotene sperm atocytes and pervasive throughout the chrom atin by MI (Figs. 3A and 3B). In both control and m utant stage XII tubule sections, robust num - bers of diplotene and MI sperm atocytes were seen. Although there appeared to be m ore such cells in stage XII