The Mouse Meiotic Mutation Mei1 Disrupts Chromosome Synapsis with Sexually Dimorphic Consequences for Meiotic Progression

Developmental Biology 242, 174–187 (2002) doi:10.1006/dbio.2001.0535, available online at http://www.idealibrary.com on The Mouse Meiotic Mutation mei1 Disrupts Chromosome Synapsis with Sexually Dimorphic Consequences for Meiotic Progression

Brian J. Libby,* Rabindranath De La Fuente,* Marilyn J. O’Brien,* Karen Wigglesworth,* John Cobb,† Amy Inselman,† Shannon Eaker,† Mary Ann Handel,† John J. Eppig,* and John C. Schimenti*,1 *The Jackson Laboratory, Bar Harbor, Maine 04609; and †Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840

mei1 (meiosis defective 1) is the first meiotic mutation in mice derived by phenotype-driven mutagenesis. It was isolated by using a novel technology in which embryonic stem (ES) cells were chemically mutagenized and used to generate families of mice that were screened for infertility. We report here that mei1/mei1 spermatocytes arrest at the zygotene stage of meiosis I, exhibiting failure of homologous chromosomes to properly synapse. Notably, RAD51 failed to associate with meiotic chromosomes in mutant spermatocytes, despite evidence for the presence of chromosomal breaks. Transcription of genes that are markers for the leptotene and zygotene stages, but not genes that are markers for the pachytene stage, was observed. mei1/mei1 females are sterile, and their oocytes also show severe synapsis defects. Nevertheless, unlike arrested spermatocytes, a small number of mutant oocytes proved capable of progressing to metaphase I and attempting the first meiotic division. However, their chromosomes were unpaired and were not organized properly at the metaphase plate or along the spindle fibers during segregation. mei1 was genetically mapped to chromosome (Chr) 15 in an interval that is syntenic to human Chr 22q13. This region, which has been completely sequenced, contains no known homologs of genes specifically required for meiosis in model organisms. Thus, mei1 may be a novel meiotic gene. © 2002 Elsevier Science (USA) Key Words: meiosis; mouse; synaptonemal complex; Rad51; sexual dimorphism; infertility.

INTRODUCTION maintenance since unproductive matings are unremark- able. Mouse mutants with meiotic defects have therefore The fundamental purpose of meiosis is to generate hap- been derived primarily by ablating orthologs of yeast meio- loid cells from diploid cells. In the ﬁrst meiotic prophase, sis genes by homologous recombination in embryonic stem homologous chromosomes align, synapse, and undergo re- (ES) cells. Examples of genes evaluated by this approach combination. A reductional division then occurs, in which include Dmc1, Msh4, Msh5, and Spo11 (Baudat et al., 2000; homologous chromosomes segregate to form haploid prod- Edelmann et al., 1999; Kneitz et al., 2000; Pittman et al., ucts, followed by an equational division that segregates 1998; Romanienko and Camerini-Otero, 2000; Yoshida et sister chromatids. The genetic control of these processes is al., 1998). Knockouts of genes encoding mammalian pro- best understood in Saccharomyces cerevisiae, an organism teins that associate with meiotic chromosomes, including well-suited to the isolation and characterization of muta- Sycp3 and Hsp70–2, have also proven fruitful (Dix et al., tions that disrupt meiosis. 1996; Yuan et al., 2000). Finally, knockouts made in genes The genetic control of mammalian meiosis is less well for reasons other than the study of meiosis sometimes have understood, in part due to the relative paucity of mutations meiotic phenotypes. Examples include the DNA repair and affecting meiosis. Spontaneous mutations resulting in ste- oncogenes Atm (Xu et al., 1996), Mlh1 (Baker et al., 1996), rility are difﬁcult to detect in the course of routine colony Pms2 (Baker et al., 1995), A-myb (Toscani et al., 1997), and Bax (Knudson et al., 1995). 1 To whom correspondence should be addressed. Fax: (207) 288- While exploitation of cross-species homology to identify 6082. E-mail: [email protected]. and mutate mouse genes involved in meiosis has been

productive, there are pitfalls and shortcomings to this since this completely sequenced region of HSU22 does not approach. First, there are genes important for meiosis in contain any clear homologs of genes known to be required yeast that either do not exist or have not been identified in for meiosis in other model organisms. mammals. These include genes encoding synaptonemal complex proteins, e.g., Zip1 (Sym and Roeder, 1994), Zip3 (Agarwal and Roeder, 2000), and Red1 (Rockmill and Roe- MATERIALS AND METHODS der, 1988), checkpoint control molecules (San-Segundo and Genetic Mapping Roeder, 1999), and transcriptional regulators (Chu and Her- skowitz, 1998). Second, knockouts of mouse genes can The mei1 mutation was induced by treating CJ7 ES cells, which produce phenotypes that are vastly different from the mei- are of the 129S1/Sv strain (129), with EMS. Chimeras were out- otic defects caused by deficiency of the yeast orthologs. For crossed to C57BL/6J (B6) to produce first generation (G1) offspring, example, mutations in some members of the Rad52 epista- which were in turn crossed to B6 partners to produce second sis group have subtle or no effects on meiosis (Rad52 and generation (G2) progeny. The G2 mice were mated to G1 parents to generate a G3 screening class (it was in the G3 class in which the Rad54) (Essers et al., 1997; Rijkers et al., 1998), while null mei1 mutant phenotype was first observed). The genetic mapping mutations in the RecA-related members of this epistasis strategy was to find concordance between the mei1 phenotype and group (Rad51, Rad51b, and Rad51d) cause embryonic le- homozygosity of 129 sequences in G3 animals. To expand the thality (Pittman and Schimenti, 2000; Shu et al., 1999; number of utilizable animals for linkage analysis, G3 heterozygotes Tsuzuki et al., 1996), precluding analysis of their potential were identified by backcrossing to proven G2 heterozygotes and roles in meiosis (although the construction of conditional determining whether any sterile males resulted. That mei1 was at alleles might address the problem). Finally, it is likely that the root of infertility of such animals was verified by the presence there are many genes that are required for proper meiosis in of small testes upon dissection. mammals that are not present in yeast, due in part to the A genome scan using approximately 60 MIT microsatellite complexity and sexual dimorphism of gametogenesis in markers polymorphic between B6 and 129, representing every autosome (at least three markers per autosome roughly evenly mammals. Such genes encode growth factors, hormones, spaced), was used to analyze the genomes of an initial group of nine signaling molecules, and factors involved in the somatic mutants. This indicated linkage to Chr 15. As indicated in the text, cell/germ cell interactions that are critical to successful tight linkage to Dmc1 was also implied by analysis of these gametogenesis. These issues can be solved, at least in part, animals. Once linkage to Chr 15 was observed and refined to an by a phenotype-driven approach to identify genes that affect interval between D15Mit156 and D15Mit33, all subsequent ani- meiosis in a mammalian model. Not only does this allow mals were typed only for these (plus some flanking markers), and identification of new genes that affect meiosis, but it also recombinants were examined for the mei1 phenotype. facilitates the recovery of hypomorphic alleles that would otherwise be lethal as nulls. Histology We previously reported the identification of a meiotic Testes were fixed in Bouin’s solution Ͼ24 h before embedding in mei1 mutant in mice, , in a novel ES cell-based chemical paraffin. Ovaries were taken from 8-week-old females and similarly mutagenesis screen (Munroe et al., 2000). It was induced in processed. Sections (5 ␮M) were cut and stained with hematoxylin 129S1/Sv ES cells by ethylmethanesulfonate (EMS) and and eosin. isolated in a three-generation, genome-wide recessive screen. Male homozygotes were sterile, displaying arrest in Genotyping the first meiotic prophase at what appeared to be the zygotene or pachytene stage. In this report, the genetic Approximately 2-mm tail tips were lysed by incubation at 55°C ␮ defects in mei1 mutant mice are investigated in molecular in 100 l of buffer PBND [50 mM KCl; 10 mM Tris, pH 8.3; 2.5 mM and biological detail. Mutant spermatocytes show defects MgCl2; 0.1 mg/ml gelatin; 0.45% (v:v) NP40; 0.45% (v:v) Tween 20]. The lysate (1 ␮l) was directly used as template in subsequent in meiotic chromosome synapsis, fail to assemble RAD51 standard PCRs. PCR products were electrophoresed on a horizontal onto meiotic chromosomes, and arrest in meiotic prophase 3.5% MetaPhor (FMC) gel. before entering pachynema. While homozygous mutant females also are sterile and display defective synapsis, there is sexual dimorphism in the arrest phenotype. Mutant Northern Analysis ovaries display reduced numbers of early- to late-stage Total RNA from adult testes was prepared by using an RNeasy follicles, containing oocytes that have progressed past the kit (Qiagen) following manufacturer’s protocols. RNA (10 ␮g) was zygotene-to-pachytene stage arrest characteristic of sper- electrophoresed on formaldehyde agarose gels by using standard matocytes. Mutant oocytes that were matured in vitro procedures and blotted onto a nylon membrane (MSI). The blot was condense chromosomes in an abnormal metaphase I, in hybridized with the Tcp10 probe, then stripped and rehybridized with the ␤-actin probe. which homologous chromosomes remain unpaired, do not congress to a well-defined spindle equator and migrate to opposite poles in a disorganized manner. mei1 was mapped RT-PCR to a Ͻ1-cM interval on chromosome (Chr) 15 that corre- Wild-type and mutant cDNA were generated from 0.5 ␮g total sponds to human Chr 22q13. mei1 may be a novel gene RNA (obtained from adult testes as described above) by using the

Superscript Preamplification kit (Gibco/BRL) according to the Oocytes were exposed to 3.8 ␮g/ml of a mouse monoclonal manufacturer’s protocol. Reverse transcription product (1 ␮l) was anti-␤-tubulin antibody (IgG; Sigma) for1hat37°C, followed by included in a standard 50-␮l PCR incubated for 35 cycles of 94°C two washes (30 min each) in PBS with 10% fetal calf serum at 37°C. for 30 s; 55°C for 30 s (Dmc1 and ␤-actin; 46°C for 30 s for Xmr); Immunocytochemical detection of spindle microtubules was car- and 72°C for 45 s. Either 5 (Dmc1 and ␤-actin) or 10 ␮l(Xmr)of ried out by exposure to 1.3 ␮g/ml of a fluorescein isothiocyanate product was run per lane on a horizontal 1% agarose gel. (FITC)-conjugated sheep anti-mouse IgG (H ϩ L) F(ab)2 fragment (Jackson ImmunoResearch, West Grove, PA) at 37°C for 1 h followed by incubation with PBS with 10% fetal calf serum for 1 h Electron Microscopy of Spermatocyte Nuclei at 37°C. Chromosomes were counterstained with 10 ␮g/ml pro- Spermatocytes from 8-week-old mice and oocytes from E17 pidium iodide (Sigma) for 10 min. Oocytes were then mounted on females were surface spread and stained essentially as described poly-L-lysine-coated slides (Sigma) in 4 ␮l of Vectashield antifading (Dresser and Moses, 1979). Briefly, testicular cells were harvested medium (Vector Laboratories, Burlingame, CA). Analyses of mei- from adult testes and spread in 0.5% NaCl onto the surface of otic configuration and spindle assembly were performed by laser Formvar-coated slides. Slides were fixed by submersing in 4% scanning confocal microscopy on a Leica TCS-NT confocal micro- paraformaldehyde with 0.03% SDS (followed by 4% paraformalde- scope equipped with an air-cooled argon ion laser (568 nm excita- hyde without SDS) and rinsed twice in 0.4% Photoflo (Kodak) tion) system (Leica Microsystems). before a final rinse in dH2O. Slides were then air-dried prior to staining of chromosomes with 50% silver nitrate solution. RESULTS Immunohistochemistry Genetic and Physical Map of the mei1 Critical Immunofluorescence analysis of surface spread gonocytes was Region and Identification of Candidate Genes performed as described (Pittman et al., 1998). For tail fibroblasts, cultures were first established by removing the outer skin from The mei1 mutation was identified in a genome-wide approximately 1 cm of tail biopsies and allowing fibroblasts to grow screen for recessive male sterility mutations, using a novel out in Dulbecco’s Modified Eagle’s media ϩ 10% FBS. Following mutagenesis technology. ES cells treated with the chemical two or three passages to establish short-term culture, subsets of EMS were used to establish pedigrees segregating highly 125 cells were exposed to 400–900 RADs of radiation from a Cs mutagenized genomes (Munroe et al., 2000). Preliminary source and were either immediately fixed to slides as described histological examination of mutant testes suggested that above for electron microscopy or were incubated at 37°C for3hto spermatogenesis in affected males was arrested in the first recover prior to surface spreading. The anti-RAD51 antibody was obtained from Oncogene Research Products (Cambridge, MA), meiotic prophase. anti-phosphorylated H2AX was from Upstate Biotechnology (Lake To simplify the identification of affected animals for the Placid, NY), and the antiserum to SYCP3 was previously described biological analyses described below (and for eventual posi- (Eaker et al., 2001). tional cloning), the mutation was genetically mapped. Het- erozygotes (identified by progeny testing) were intercrossed, Oocyte Culture and Assessment of Meiotic and a genome-wide scan of sterile offspring was performed Progression with microsatellite markers polymorphic between 129S1/ Sv (129; the strain in which the mutation was induced) and Ovaries were dissected 44 h after treatment with equine chori- C57BL/6J (B6; the strain to which the mutation was out- onic gonadotropin and cultured as described to test for spontaneous crossed in the three-generation breeding scheme used to resumption of meiosis (Eppig, 1999; Eppig and Telfer, 1993). The detect recessive mutations). Also, given the similarity of oocytes were examined by using a stereomicroscope after 15 h of the meiotic arrest phenotype to that of DMC1-null mice culture and assessed for germinal vesicle breakdown indicative of the resumption of meiosis and the presence of a polar body, which (Pittman et al., 1998), possible linkage to the Dmc1 locus is usually characteristic of progression to metaphase II. on Chr 15 was evaluated. In the first 20 animals tested, 19 affected were homozygous for the 129 allele of Dmc1, and linkage to microsatellite (Mit) markers on Chr 15 was Analysis of Chromosome Preparations in Oocytes indicated. After genotyping and phenotyping 409 animals Fully grown oocytes were collected after 14 h of in vitro representing 818 meioses, 2 recombinants between Dmc1 maturation and transferred to a hypotonic solution of 1% sodium and mei1 were found. Furthermore, Dmc1 transcripts were citrate for 10 min before processing for chromosome analysis. present at normal levels (see below), and the sequence of Air-dried chromosome spreads were prepared essentially as de- Dmc1 transcripts from mei1 homozygotes was wild-type scribed (Tarkowsky, 1966) and stained with a solution of 4% (data not shown). Finally, six Dmc1/ϩ, mei1/ϩ double Giemsa. Chromosomes were analyzed by using a DMRX/E micro- scope (Leica Microsystems, Exton, PA) under a 100ϫ objective. heterozygotes (four males/two females) were fertile, which indicates complementation between mei1 and Dmc1. After determining that mei1 is not allelic to Dmc1, the Immunofluorescent Detection of Meiotic Spindle critical region was narrowed by using additional markers in Assembly the region that are polymorphic between B6 and 129. These After in vitro maturation, oocytes were immediately fixed in a efforts place mei1 in an interval of about 0.38 cM (5 4% paraformaldehyde–PBS solution for 18 h at room temperature. recombinants out of 1320 meioses) between D15Mit69 and

FIG. 1. Genetic and physical map of mei1 region on mouse Chr 15. Shown in (A) is a diagram of mouse Chr 15 with microsatellite loci (abbreviated by substituting “M” for “D15Mit”) and a gene (Dmc1) mapped near mei1. The number of meioses scored for crossovers in particular intervals are indicated both in raw numbers (# recombinants/# meioses) and recombination fractions. The data placed mei1 in a 0.38-cM region between markers D15Mit69 and D15Mit189. (B) An exploded view near the critical region, showing certain BAC and YAC (dashed line) clones mentioned in the text. (C) The region of human Chr 22 corresponding to the mouse mei1 critical region is shown, with all known genes organized in linear order but not to scale.

D15Mit189 (Fig. 1). The mei1 mutation could therefore be quence from the BAC containing D15Mit69 was found to be followed in matings by using these two loci as surrogate homologous to the novel gene dJ377F16.1 in humans, markers. which maps 2 Mb centromeric to CYP2D. Thus, if synteny To begin generating a physical contig of the mei1 critical is preserved between species, the human MEI1 ortholog region, YAC and BAC clones were isolated by using the should be contained in this interval. However, this region markers flanking mei1, D15Mit69, and D15Mit189. Se- contains no annotated homologs of genes known to affect quences of BAC ends were determined, along with some meiosis specifically in model organisms. It is possible that internal sequence. End sequence from a BAC (170K19) mei1 is expressed in multiple tissues, including germ cells, containing D15Mit189 revealed a 189-bp stretch that is but that this particular allele impacts exclusively on meio- approximately 95% identical to the first exons of three sis. Examples of candidate genes from this region that may testosterone 16␣-hydroxylase gene family members fall into this category include G22P1, which encodes Ku70, (Cyp2d9, Cyp2d10, and Cyp2d11) that had been previously a protein essential for nonhomologous end joining of DSBs; described and mapped to central Chr 15 (Wong et al., 1989). Tob2, which inhibits cell cycle progression from G0/G1 to The mei1 region of mouse Chr 15 exhibits conserved S and is highly expressed in oocytes (Ikematsu et al., 1999); synteny with human 22q13, the sequence of which has and NHP2L1, the product of which interacts with the DNA been completely determined (Dunham et al., 1999). Se- damage checkpoint molecule RAD17 (Chang et al., 1999).

FIG. 2. Histology of mei1 mutant gonads. Shown are paraffin sections of testes and oocytes stained with hematoxylin and eosin. The genotypes of the samples are indicated. (A, B) Testis cross-sections through a seminiferous tubule. (C, D) Sections of heterozygote and mutant ovaries, with nearly the entire organ shown. (E) Higher magnification image of a severely affected mutant ovary. (F) A late antral follicle with an essentially normal structure. (G) A mutant ovary with blood-filled follicles. Cl, corpus luteum. Arrows point to follicles.

FIG. 3. Analysis of metaphase chromosomes in mei1/mei1 oocytes. Laser scanning confocal micrographs of single oocytes from females heterozygous (A, B) or homozygous (C, D) for the mei1 mutation. The heterozygote samples were prepared after 7–8 h of in vitro maturation, the time of normal prometaphase and metaphase. Samples of mutant oocytes were ﬁxed at 15 h of maturation to illustrate abnormal progression of meiosis. Immunoﬂuorescence staining of spindle microtubules was performed by using an antibody to ␤-tubulin

Sterility Phenotype of mei1/mei1 Mutants TABLE 1 In Vitro Maturation of mei1 Mutant Oocytes In homozygous males, histological examination of sec- tioned testes revealed that spermatogenesis was severely Oocyte stage ϩ/ϪϪ/Ϫ P value disrupted. The seminiferous tubules were completely devoid of postmeiotic cells, and the most advanced tubules GV 28 (6%) 21 (6%) ϽϽ contained cells that appeared to be arrested at or before the MI 205 (47%) 300 (88%) P 0.0001 MII 207 (47%) 20 (6%) P ϽϽ 0.0001 pachytene stage of meiosis I (Munroe et al., 2000; Figs. 2A Total 440 341 and 2B). To investigate effects on oogenesis, females generated from intercrosses of heterozygotes were tested for Note. Determination of the progress of meiosis was made after fertility by mating to wild-type males. Homozygous fe- 15 h of maturation for all groups. P values are from Fisher’s Exact males failed to produce litters. Histological preparations of test. GV, germinal vesicle stage; MI and MII, metaphase I and II. these females revealed marked ovary defects. The most severely affected adult ovaries were small, disorganized, and devoid of normal follicles (Fig. 2E). However, most ovaries did have follicles, albeit greatly reduced in number Examination of chromosome behavior and spindle struc- (Fig. 2D). The follicles ranged from the primordial to the ture in mei1/mei1 oocytes by confocal microscopy and in antral stages of development. The latter contained germinal spread preparations of metaphase chromosomes revealed vesicle-stage oocytes that appeared morphologically normal significant aberrations (Fig. 3). Prometaphase nuclei of (Fig. 2F). Some follicles exhibited blood-filled antral cavities oocytes from mei1/mei1 females were unusual in that the (Fig. 2G). Corpora lutea were present in the ultrastructur- chromosomes were present in an atypical rosette forma- ally intact mutant ovaries, suggesting that ovulation, or tion. Instead of being uniformly organized within the mi- significant follicular development, may have taken place crotubule network (Fig. 3A), chromosomes from mutant (Fig. 2D). oocytes were present at the periphery (Fig. 3C). In most mutant oocytes, the chromosomes did not congress properly to the metaphase spindle equator and were found Defective Meiosis and Aberrant First Meiotic chaotically distributed along the spindle fibers (compare Division in mei1/mei1 Oocytes heterozygote to homozygote in Figs. 3B and 3D, respec- To better characterize the developmental potential of tively). Metaphase spreads of chromosomes revealed that, mei1/mei1 oocytes, they were matured in vitro. When instead of the normal configuration of 20 bivalents with removed from the follicular environment, in which granu- chiasmata (Fig. 3E), mutant oocytes contained 40 unpaired losa cells arrest meiotic progression at the diplotene stage, univalents (Fig. 3F). Thus, as in the example of Fig. 3D, it wild-type oocytes will efficiently resume the first meiotic appears that karyokinesis may have been attempted, but division and finally arrest at metaphase II. Accordingly, anaphase I was highly disorganized with asynchronous and mei1/mei1 females were treated with equine chorionic unequal segregation of univalents. gonadotropin to promote follicle development, and 44 h later their ovaries were removed and fully grown oocytes at MEI1 Function Is Autonomous to Oocytes the germinal vesicle (GV) stage were isolated and placed in culture. Their ability to undergo spontaneous resumption Since development of germ cells is controlled in part by of meiosis, and the degree to which they progressed, was somatic cells, either cellular compartment may be the monitored. Only a small percentage (6%, compared with primary site of mei1 activity, though the observed mutant 47% in heterozygote controls) of mei1/mei1oocytes ap- phenotype is manifested in germ cells. To determine peared to complete the first meiotic division and produce a whether oocyte development failure in mutants is a conse- polar body (Table 1). Thus, fully grown mei1/mei1 oocytes quence of defects intrinsic to the oocyte or in the follicular can resume meiosis, but progression to metaphase II is somatic cells, reaggregated chimeric ovaries were con- severely impaired. structed (Eppig and Wigglesworth, 2000). This was done by

(green). Chromosomes were counterstained with propidium iodide (red). (A) Prometaphase-stage normal oocyte showing bivalent chromosomes uniformly distributed in a rosette configuration within the microtubule network. (B) Metaphase I-stage normal oocyte showing a typical barrel shaped spindle with chromosomes aligned at the spindle equatorial region. (C) Prometaphase-stage mutant oocyte showing an abnormal rosette configuration with the majority of univalent chromosomes loosely attached to the peripheral region of the microtubule network. (D) Abnormal anaphase-like configuration in a mutant oocyte showing failure to segregate univalent chromosomes. (E) Chromosome preparations from oocytes obtained from wild-type (ϩ/ϩ) females showing pairing of homologous chromosomes in a typical bivalent configuration at the metaphase I stage. Note chiasmata. (F) Chromosome configuration observed in oocytes from mei1/mei1 females at the metaphase I stage. Note that chromosomes are present as unpaired univalents.

completely exchanging the oocyte and somatic cell components of ovaries between wild-type and mei1/mei1 newborn mice. The reaggregated ovaries were grafted beneath the renal capsules of SCID mice for 21 days to allow oocyte and follicular development. Wild-type oocytes from recon- structed ovaries matured normally, showing nearly 50% polar body production, regardless of whether their development was in the presence of wild-type or mutant somatic cells. Thus, the presence of mutant mei1/mei1 somatic cells does not cause the mei1 phenotype in oocytes. In contrast, the reaggregated ovaries created with mei1/mei1 oocytes did not develop, whether constructed with mutant or normal somatic cells. Thus, mutant oocytes did not survive the reaggregation protocols. Taken together, these results suggest that MEI1 function is oocyte-autonomous.

Synapsis Is Defective in mei1/mei1 Gonocytes To determine whether the meiotic progression defects of mei1 mutant gonocytes stem from aberrant meiotic chromosome behavior during early prophase, progression of chromosome pairing and synaptonemal complex formation were examined in spermatocytes from sexually mature males and in oocytes obtained from ϳ17-day fetal ovaries. As assessed by electron microscopy, mutant oocytes and spermatocytes both had clear defects in chromosome synapsis. In contrast to the complete autosomal synapsis observed in wild-type spermatocytes (Fig. 4A) and oocytes (not shown), a complete set of synapsed chromosomes was not found in any nuclei from mutant gonocytes. However, some nuclei contained a few chromosome pairs that appeared partially synapsed (Figs. 4B and 4C), and in rare cases, a fully paired or synapsed pair was observed (Fig. 4C, inset). It could not determined whether pairing was between homologous chromosomes. Chromosome structure was further investigated by immunohistochemical analyses of molecules known to associate with meiotic chromosomes in characteristic patterns at various stages of meiotic prophase: SYCP3 and RAD51. SYCP3 is a component of axial elements of unsynapsed chromosomes, which becomes the lateral element of synapsed chromosomes. Meiotic chromosomes from mei1/ mei1 spermatocytes and fetal oocytes (not shown) exhibited staining of axial elements with antiserum to SYCP3 (Fig. 5B), conﬁrming that mutant cells progress at least to early meiotic prophase. Partial synapsis of chromosomes was observed in many mutant nuclei, and uniform anti-SYCP3 staining along the lengths of chromosomes in these nuclei suggests that these cells reached a zygotene-like developmental stage.

FIG. 4. Synapsis defects in spermatocytes. (A–C) Electron micrographs of silver nitrate-stained spermatocyte (A, B) and oocyte (C) Occasionally, full synapsed chromosome pairs are seen (arrow- chromosomes. The wild-type chromosomes are fully synapsed (A), head). Chromosomes in mutant oocyte nuclei are also predomi- whereas most chromosomes in the mei1/mei1 spermatocytes (B) nantly unsynapsed (C), although occasional regions of pairing are either not synapsed (asterisk) or partially synapsed (arrow). and/or synapsis can be seen (C, inset).

The RecA homolog RAD51, which is important for lated with specific stages of meiosis. These genes can be meiotic recombination in yeast, can be detected in discrete used as markers of meiotic progress in the context of foci along chromosome cores in normal meioses (Ashley et mutations that perturb meiosis. Transcription of three al., 1995; Barlow et al., 1997; Moens et al., 1997; Plug et al., genes that are expressed at the zygotene (Dmc1, Xmr) and 1996). RAD51 catalyzes strand invasion during homologous pachytene (Tcp10) stages of meiosis (Calenda et al., 1994; recombination and is thought to localize to single-stranded Ewulonu et al., 1993; Habu et al., 1996; Vidal et al., 1998) DNA at the sites of DSBs in meiotic chromosomes (Shino- was examined by RT-PCR (Dmc1 and Xmr) and Northern hara et al., 1997; Sung, 1994). While RAD51 foci were blot (Tcp10) analysis of whole testis RNA. Whereas Dmc1 observed in mei1/ϩ spermatocytes (Fig. 5C), none were transcripts were present at approximately normal levels, detected on chromosome cores or synapsed chromosomal Xmr was not detectable in mei1/mei1 RNA (Fig. 7). Tcp10 regions in mei1/mei1 spermatocytes (Fig. 5D). Nonetheless, transcripts could not be detected by Northern blotting (Fig. normal or elevated levels of RAD51 protein could be 7), although low levels were detected by RT-PCR (not detected in Western blots of mei1/mei1 testes (not shown). shown). Thus, from the standpoint of the meiotic transcrip- Thus, it appears that the protein product of the mei1 gene is tional program, mutant spermatocytes do not progress to a required, directly or indirectly, for association of antigeni- normal pachytene stage, confirming results from cytologi- cally recognizable association of RAD51 with meiotic chro- cal analyses. Given the zygotene-like appearance of meiotic mosomes. The absence of this association implies that chromosomes in mutant spermatocytes, in which some recombination in mei1/mei1 spermatocytes may not occur synapsis is seen, the absence of Xmr transcription was normally or at all. surprising, since this protein is a marker of all stages of Since RAD51 associates with sites of DSBs in spermato- meiosis I prophase in spermatocytes (Mahadevaiah et al., cytes, its absence in mei1/mei1 spermatocytes could reflect 2001). either a lack of DSBs or failure to recruit RAD51 to the breaks. Phosphorylation of histone H2AX in chromatin is another marker of DNA DSBs. The staining patterns of the DISCUSSION phosphorylated form (␥-H2AX) have been described in spermatocytes and have been shown to depend on the presence Phenotype-driven mutagenesis is a powerful approach to of the SPO11 protein that is required for DSB formation identify genes required for meiosis and, importantly, does (Mahadevaiah et al., 2001). To test for the presence of DSBs not depend on preexisting knowledge of the molecular in mutant spermatocytes, anti-␥-H2AX antibody was used nature of such genes. mei1 was isolated in a screen for as a probe. Positive staining of mutant spermatocytes was recessive mutations causing male infertility by using a observed (Figs. 5E and 5F), consistent with the existence of novel mutagenesis methodology. Rather than treating DSBs. The data suggest that DSBs occur in mei1/mei1 whole animals with a mutagen such as ethylnitrosourea spermatocytes but that RAD51 does not effectively associ- (Russell et al., 1979), ES cells were mutagenized with EMS. ate with them. These cells were used to generate germline chimeras, which Since RAD51 protein is expressed in all tissues and is in turn founded families to conduct three-generation reces- presumed to be involved in recombinational DSB repair sive mutation screens (Munroe et al., 2000). To our knowl- (Lim and Hasty, 1996; Sharan et al., 1997), a possible role for edge, mei1 is the first meiosis-specific mutation isolated by mei1 in somatic cells was explored. First, primary fibroblast random mutagenesis in mice. cultures from mei1/mei1 mice were tested for radiation ES cell mutagenesis provides several unique advantages sensitivity. No difference in survival of mei1/mei1 vs. over whole animal mutagenesis, including the ability to mei1/ϩ fibroblasts across a range (50–400 RAD) of ionizing modulate and premeasure the mutation rate, use different radiation exposure was observed (not shown). Second, mutagens, and screen for mutations in culture before the mei1/mei1 fibroblasts were exposed to high levels of ioniz- generation of animals. At the Hprt locus of EMS-treated ES ing radiation (900 RADs) and probed with anti-RAD51 cells, the predominant classes of mutation were G 3 Aor antibody. Cells treated in such a fashion typically display C 3 T transitions (Munroe et al., 2000). While intragenic numerous nuclear RAD51 foci that presumably correspond point changes can result in hypomorphic mutations, which to sites of DSB repair (Haaf et al., 1995). RAD51 foci were can yield valuable insight into protein function, we cannot observed in both normal and mutant cells after irradiation yet conclude whether the mei1 allele described here is null (Figs. 6A and 6B). These results suggest that the defect in (see below). Ultimately, a complementation test with a mei1 mutants with respect to RAD51 chromatin binding is targeted null allele of the mei1 gene will address this issue meiosis-specific. and rule out the remote possibility that the phenotype is attributable to mutations in more than one gene. Other than the germ cell defects reported here, no other Transcription of Meiotic Marker Genes in mei1/ obvious problems were observed in mei1/mei1 animals. mei1 Mice There were no indications of a possible role for MEI1 in Certain genes expressed during spermatogenesis display repair of double-strand breaks in somatic cells, based on the highly regulated patterns of expression that can be corre- observation that MEI1-deficient fibroblasts were not radia-

FIG. 5. Immunohistochemical analysis of meiocytes. (A) Normal and (B) mutant spermatocyte nuclei stained with an antiserum directed against SYCP3, an axial element component. In the mei1/mei1 nucleus, disorganized and partial pairing of chromosomes is seen. (C, D) Spermatocyte nuclei costained with anti-SYCP3 (red) and anti-RAD51 (green), where yellow spots are points of colocalization. Numerous foci are present along wild-type zygotene-stage chromosomes (C), but no foci are seen in the mutant (D). (E) Normal and (F) mutant spermatocyte nuclei costained with an antiserum directed against phosphorylated histone H2AX (green), which is believed to be an indicator of DSBs, and anti-SYCP3 (red).

FIG. 6. Anti-RAD51 staining of tail ﬁbroblasts treated with gamma irradiation. Both wild-type (A) and mutant (B) show a normal pattern of RAD51 foci 3 h after treatment.

tion sensitive and RAD51 foci formed normally on irradi- tion, such as the cohesin cores, which have been shown to ated fibroblasts from mutant mice. Mutant animals have recruit the recombination protein DMC1 to meiotic chro- been maintained for up to 16 months without any obvious mosomes (Pelttari et al., 2001). Assembly of mature SCs detrimental effects. In addition, experiments using chi- requires interaction of several proteins, and it is likely that meric reaggregated ovaries indicate that the defect in the not all of these have been identified. progression of meiosis is intrinsic to the oocyte and not a Phenotypically, the mei1 mutation shows striking simi- consequence of deficits in the companion somatic cells. larities to SPO11-deficient mice, which fail to induce DSBs Thus, evidence is that MEI1 is required specifically by germ in meiotic chromosomes (Baudat et al., 2000; Romanienko cells. and Camerini-Otero, 2000). Both show partial chromosome The most notable basis for meiotic failure in mei1/mei1 synapsis, lack of RAD51 binding to meiotic chromosomes, meiocytes is a defect in chromosome synapsis, found both and greatly reduced (but present) ovarian follicular develop- in this study and by Romanienko and Camerini-Otero ment. However, mei1/mei1 spermatocytes, unlike Spo11 (2000). This synaptic failure may be related to the absence mutants (Mahadevaiah et al., 2001), appear to contain DSBs of RAD51 protein foci along meiotic chromosome cores, as assessed by anti-␥-H2AX binding. Thus, the similarity in despite the presence of RAD51 protein in mutant spermato- phenotype may not have a common underlying basis. cytes. In wild-type spermatocytes, RAD51 foci are present Nevertheless, it is possible that the presence of ␥-H2AX in from leptonema through pachynema. It is thought that mei1/mei1 spermatocytes reflects chromatin modifications these foci represent sites where genetically induced DSBs other than those due to meiotically induced DSBs. are repaired by recombination and that RAD51 is a critical mei1/mei1 oocytes also share a remarkable similarity constituent of a recombination complex that conducts the with MLH1-deficient oocytes. In both cases, MI metaphase repair. The absence of RAD51 binding suggests that recom- chromosomes in mutant oocytes are present as univalents, bination is defective in mei1/mei1 nuclei. Since mutant loosely organized along the metaphase plate. Disorganized nuclei were stained by anti-␥-H2AX, a marker of chromatin segregation of chromosomes at anaphase then occurs, and associated with DSBs, it is possible that recombinational entry to MII is largely absent (Woods et al., 1999). This repair of DSBs may not occur. The failure to repair DSBs, or phenomenon may be due to the absence of tension which the absence of recombination itself, may be responsible for normally exists between paired homologs, as a result of the observed defects in synapsis. Mice lacking the RecA equal pull by spindle fibers from opposite poles. An impor- homolog DMC1 that have a presumed deficiency in meiotic tant difference between these two mutations, however, is DSB repair, present a similar complete meiotic arrest in that Mlh1Ϫ/Ϫ oocytes probably undergo synapsis, even spermatocytes (Pittman et al., 1998; Yoshida et al., 1998). though the mutation presumably prevents normal recom- However, unlike mei1/mei1 oocytes, there is no progres- bination (Baker et al., 1996). Thus the phenotypic resem- sion past pachynema in DMC1-deficient oocytes. Alterna- blance of MI in mei1/mei1 and Mlh1Ϫ/Ϫ oocytes may be a tively, synaptic failure in mei1/mei1 meiocytes could be secondary consequence of the mutation (univalent chromo- explained if MEI1 were required for assembly of other somes at MI) rather than a similarity between MEI1 and structures ultimately required for synapsis and recombina- MLH1 biochemical functions.

for particular protein products at particular meiotic stages. It has been suggested that oocytes have a less rigorous or nonexistent checkpoint system that allows meiosis to pro- ceed through pachynema without fully synapsed chromosomes (Burgoyne and Baker, 1985; Odorisio et al., 1998). Given the current data, there remain several possibilities for the function of MEI1. It may facilitate assembly of multiprotein complexes. It could enable RAD51 to bind meiotic chromatin breaks in a way the causes immunohis- tochemically detectable foci. It could be a synaptonemal complex protein that must be present for RAD51 binding. It could be a protein required for other proteins to recognize DSBs. It also must be considered that this mei1 allele may not be a null mutation, since it was induced by a point mutagen. The fact that some synapsis occurs in mutant spermatocytes is a feature that may be consistent with a hypomorphic allele; however, it remains to be demon- strated whether the synapsis that occurs is homologous. The region of mouse Chr 15 to which mei1 was mapped corresponds to a roughly 2-Mb segment of human 22q13, which has been completely sequenced. The overall structure of this interval appears highly conserved between the species, based on the facts that the mouse orthologs of several human genes, namely G22P1, RANGAP1, TEF, FIG. 7. Northern blot and RT-PCR analysis of mutant and control testis RNAs with probes for meiotically expressed genes. (A) ADSL, NAGA, and SREBF2, are known to reside on central Northern blot showing that pachytene-expressed Tcp10 is absent in MMU15 (Mouse Genome Database: www.informatics.jax. mei1/mei1 testes. (B) RT-PCR analysis of Dmc1 and Xmr expres- org), and that the Celera mouse genome sequence indicates sion. The zygotene-expressed Dmc1 shows normal transcript levels sequence colinearity (the authors’ observations). Based on in mutant testes, whereas Xmr transcripts are undetectable in the surveys of currently available annotations of the human same testes. and mouse sequences (from publicly available sources and also the Celera database), this region does not contain any clear homologs of genes known to cause meiosis-speciﬁc defects in mammals or other organisms. Thus, mei1 may be Many aspects of mammalian meiosis show sexual dimor- a novel meiotic gene, highlighting the value of forward phism (Handel and Eppig, 1998), and there are several genetic approaches for gene discovery in the ﬁeld of mam- mouse mutants in which meiosis is differentially impacted malian gametogenesis and meiosis. in oocytes and spermatocytes. Those with defects directly affecting synapsis tend to have sexually dimorphic phenotypes. For example, translocations that prevent abnormal ACKNOWLEDGMENTS synapsis of single chromosomes cause pachynema arrest in males but not females (Burgoyne and Baker, 1985; Odorisio We thank the histology group at The Jackson Laboratory for preparing tissue slides; E. Akeson for gonocyte spreading protocols et al., 1998). Mutation of the SC protein SYCP3 in mice and technical advice; P. Finger for assisting with electron micros- causes early meiotic prophase arrest in males, but females copy; G. Martin for capturing the images of histological sections; R. are actually fertile (although it is not known whether Bergstrom for colony maintenance; and Drs. Barbara Knowles and synapsis is normal) (Yuan et al., 2000). Mice homozygous Wes Beamer for critical reading of the manuscript. This work was for a gene-targeted Spo11 mutation have a dimorphic arrest supported by NIH Grants GM45415, HD33816, and HD23839 to phenotype similar to mei1, in that some oocytes are made. J.C.S., M.A.H., and J.E., respectively. A Cancer Center Grant These mice also exhibit synapsis failure, presumably be- (CA34196) to The Jackson Laboratory supported core facilities used cause they fail to induce genetically programmed DSBs in this work. during leptonema (Baudat et al., 2000; Romanienko and Camerini-Otero, 2000), but SPO11 may also have a role in REFERENCES the SC maintenance (Romanienko and Camerini-Otero, 2000). Agarwal, S., and Roeder, G. S. (2000). Zip3 provides a link between The ability of such mutant oocytes, but not spermato- recombination enzymes and synaptonemal complex proteins. cytes, to progress through pachynema and to reach Cell 102, 245–255. metaphase/anaphase I may be a consequence of either sex Ashley, T., Plug, A. W., Xu, J., Solari, A. J., Reddy, G., Golub, E. I., differences in meiotic checkpoint control or in requirement and Ward, D. C. (1995). Dynamic changes in Rad51 distribution

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