REPRODUCTIONRESEARCH

K K Gene expression profiles of Spo11 / mouse testes with spermatocytes arrested in meiotic prophase I

Natalya A Smirnova, Peter J Romanienko, Pavel P Khil and R Daniel Camerini-Otero Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, 5 Memorial Drive, Bethesda, Maryland 20892, USA Correspondence should be addressed to R D Camerini-Otero; Email: [email protected]

P J Romanienko is now at Developmental Biology Program, Mouse Genetics Core, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York 10021, USA

Abstract Spo11, a meiosis-specific protein, introduces double-strand breaks on chromosomal DNA and initiates meiotic recombination in a wide variety of organisms. Mouse null Spo11 spermatocytes fail to synapse chromosomes and progress beyond the zygotene stage of K K meiosis. We analyzed gene expression profiles in Spo11 / adult and juvenile wild-type testis to describe genes expressed before and after the meiotic arrest resulting from the knocking out of Spo11. These genes were characterized using the Gene Ontology data K K base. To focus on genes involved in meiosis, we performed comparative gene expression analysis of Spo11 / and wild-type testes from 15-day mice, when spermatocytes have just entered pachytene. We found that the knockout of Spo11 causes dramatic changes in the level of expression of genes that participate in meiotic recombination (Hop2, Brca2, Mnd1, FancG) and in the meiotic checkpoint (cyclin B2, Cks2), but does not affect genes encoding protein components of the synaptonemal complex. Finally, we discovered unknown genes that are affected by the disruption of the Spo11 gene and therefore may be specifically involved in meiosis and spermatogenesis. Reproduction (2006) 132 67–77

Introduction Villeneuve & Hillers 2001, Bannister & Schimenti 2004). In addition to mitotic DNA repair proteins, meiotic Spo11 protein, a type II like topoisomerase, generates recombination requires several meiosis-specific double-strand breaks (DSBs) during meiosis. It is proteins, such as Dmc1 and the relatively recently structurally and functionally conserved in a wide variety described Hop2 and Mnd1 proteins (Bishop et al. 1992, of organisms such as yeast, insects, worms, plants, mice Leu et al. 1998, Pittman et al. 1998, Yoshida et al. 1998, and humans (Keeney et al. 1997, Dernburg et al. 1998, McKim & Hayashi-Hagihara 1998, Romanienko & Rabitsch et al. 2001, Tsubouchi & Roeder 2002, Camerini-Otero 1999, 2000, Baudat et al. 2000, Grelon Petukhova et al. 2003, 2005). Nevertheless, many gene et al. 2001, Storlazzi et al. 2003). The wide distribution products specifically involved in meiotic recombination and the high degree of sequence similarity of Spo11 are still unknown. homologs suggest that the introduction of DSBs is an Apart from its catalytic function, Spo11 may play a essential function in meiosis (Keeney 2001). Generated structural role in chromosome pairing (Romanienko & by Spo11, DSBs are used for initiation of meiotic Camerini-Otero 2000, Prieler et al. 2005). Pairing of recombination and promote pairing and synaptonemal homologous chromosomes, but not synapsis, was complex (SC) formation between homologous chromo- observed in yeast with a catalytically inactive Spo11 somes in many organisms (Romanienko & Camerini- mutant, while complete deletion of the Spo11 gene Otero 2000, Storlazzi et al. 2003, Henderson & Keeney eliminates this homolog pairing (Cha et al. 2000). 2004). Synapsis facilitates the completion of recombina- Furthermore, in yeast, Spo11 foci were abundant in tion that leads to the correct segregation of chromosomes some pachytene cells where formation of DSBs are during meiosis I (Lichten 2001). Meiotic DSBs recruit a completed and during pachytene, most Spo11 foci number of conserved enzymes including Mre11, Nbs1, touched or overlapped with Zip1 – the central element Rpa, Rad50, Rad51 and Rad54 that also participate in component of the SC (Prieler et al. 2005). Spo11 may DNA repair in mitotic cells (Baarends et al. 2001, also negatively regulate exit from the bouquet stage,

q 2006 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00997 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access 68 N A Smirnova and others a cell-wide regulatory transition accompanying global Materials and Methods chromosome movements, via non-catalytic function Animals in Sordaria macrospora at zygotene/pachytene stage (Storlazzi et al. 2003). In mouse, Spo11 localizes to All animal use procedures were performed according to discrete foci early in meiosis, which is consistent with the NIH Guide for the Care and Use of Laboratory K K catalytic function of Spo11 in leptotene, and later to the Animals. Spo11 / knockout mice were generated as regions of homologous chromosome synapsis that described previously (Romanienko & Camerini-Otero K K suggested an additional structural role for Spo11 2000). Wild-type mice were siblings of Spo11 / mice. (Romanienko & Camerini-Otero 2000). Taken together, these findings indicate that Spo11 may have a role in the Total RNA isolation progression of meiotic prophase independent of DSBs. Testes were surgically removed from mice and stored at Knockout of the mouse Spo11 prevents formation of K80 8C until total RNA was isolated using a Trizol DSBs and SC during meiosis and leads to the meiotic arrest of spermatocytes at zygotene (Baudat et al. 2000, solution (Life Technologies). Total RNA then was treated Romanienko & Camerini-Otero 2000). The arrested with RNase-free DNase (Promega) at room temperature K K cells undergo apoptosis. As a result, Spo11 / for 15 min. RNA concentration was determined by UV homozygous male mice are sterile and have small, spectrophotometry and RNA integrity was confirmed m underdeveloped testes. A growing body of information using agarose gel electrophoresis. Total RNAs (10 g) indicates that mouse knockouts of genes with many were used as templates in reverse transcription (RT) different functions in meiosis, such as Msh4 (DNA reactions. mismatch repair protein), Dmc1 (meiotic recombina- tion and repair protein), Hop2 (proper homologous Microarray procedures chromosome pairing protein), Sycp3 (structural com- Amino-allyl modified cDNA was synthesized using ponent of the axial/lateral element of SC) and Mei1 (a SuperScript II RNase H-RT (Invitrogen) oligo (dT) (Life possible partner of Spo11) have a similarly arrested Technologies) and amino-allyl modified dUTP (Sigma), meiotic phenotype (de Rooij & de Boer 2003, followed by labeling with fluorescent dyes Cy3 or Cy5 Petukhova et al. 2003). These mutant mice have (Amersham). Labeled products were purified with spermatocytes that do not develop beyond zygotene QIAquick PCR Purification Kit (Qiagen). The hybrid- and are sterile in the homozygous state (de Rooij & de izations were performed on glass slide microarrays Boer 2003, Petukhova et al. 2003). A common feature Mouse NIA 15K (Keck Biotechnology Resource Lab, for all these mutant spermatocytes is the failure to Yale University, New Haven, CT, USA) containing synapse homologous chromosomes (Pittman et al. 15 000 mouse genes according to the manufacturer’s 1998, Yoshida et al. 1998, Kneitz et al. 2000, Yuan protocol. Microarray experiments were performed in et al. 2000, Libby et al. 2002, 2003, Petukhova et al. quadruplicate with different mouse pairs and dye- 2003). It is not clear how this failure in chromosome reversed hybridizations. The chips were scanned using synapsis leads to the arrest in meiosis and subsequent the GenePix 4000A scanner (Axon Instruments, Union apoptosis. It might be the result of the action of a City, CA, USA) and primary data were analyzed using the pachytene checkpoint (Roeder & Bailis 2000) as has Genepix 3.0 software (Axon Instruments). Microarray been observed for budding yeast or the transcriptional data are available under the GEO accession numbers silencing imposed at pachytene on unsynapsed meiotic GSE1138 and GSE3436. chromosomes (Turner et al. 2005). The latter might be sufficient in its own. In other words, the transcriptional Data analysis inactivation of genes essential for meiotic progression may precipitate meiotic arrest before pachytene in the Primary data were flagged using four default parameters absence of a checkpoint as such. A comparative set in the Genepix 3.0 program. For further analysis, the analysis of gene expression in mutant and wild-type data were imported into Excel (Microsoft) and mouse testes may yield new information about the normalized by the Median Centering Method. We general mechanisms leading to meiotic arrest and performed the statistical analysis using a modified t-test uncover new genes, which participate in meiosis, implemented in SAM software (http://www-stat.stanford. meiotic checkpoint and spermatogenesis. edu/~tibs/SAM/)(Tusher et al. 2001). We defined We used cDNA microarray analysis to measure gene differentially expressed genes at a 1% false discovery K K expression levels in adult and juvenile Spo11 / testes. rate confidence level and a cutoff for differential K K Differentially expressed genes were further charac- expression equal to 1.5 for juvenile Spo11 / and 2.0 K K terized by their functional classification and by the for adult Spo11 / , juvenile wild-type microarray tissue specificity of their expression. Analysis of gene experiments and for analysis of testis specificity. Gene K K expression of juvenile Spo11 / testes has allowed us to clustering was performed with the K-means method and recognize genes whose expression depends on Spo11. a hierarchical clustering package based on the Pearson

Reproduction (2006) 132 67–77 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access K K Gene expression profiles of Spo11 / 69 correlation coefficient, using the average linkage of the Results and discussion log ratio (Eisen et al. 1998). K K 2 Expression profile of adult Spo11 / mouse testes To functionally characterize genes, expressed early and late in spermatogenesis, we used the gene ontology To investigate the effect of knocking out Spo11 on the mining tool eGOn (www.egon.com), a web-based tool expression of genes in testis, we performed a competi- for mapping microarray data onto the gene ontology tive microarray hybridization of total RNA samples from K K structure. We performed the Target–Master test, where a Spo11 / and wild-type mouse testis. Disruption of given gene set (the target gene list) was compared with Spo11 gene results in a gross change of the gene the master list, contained all the genes printed on the expression profile from whole testis (Fig. 1). We found slide. The total number of genes in the target list divided that about 4000 genes changed their expression in K K by the total number of genes in the master list was called Spo11 / testes more than twofold (Fig. 1a). To validate the overall proportion. The Target–Master test the microarray data, we performed quantitative RT-PCR determined gene ontology (GO) classes where the for selected genes (Fig. 1c). Differential expression proportion of the number of genes in a target list divided ratios obtained by microarray analysis and quantita- by the number of genes in the master list is different from tive RT-PCR were in qualitative agreement for all the overall proportion with P!0.05. A two-sided one genes tested, although we found some quantitative sample binomial test was implemented. differences. The obvious dramatic changes in expression patterns K K between Spo11 / and wild-type testis are due to Quantitative RT-PCR K/K differences in the cell composition of Spo11 and For the 3 0-most 500 bp of the full-length mRNA wild-type testis. Wild-type testes of adult mice are sequence primer pairs were designed using the web- heterogeneous and contain more than 10 cell types based program Primer version 3.0 (Rozen & Skaletsky (Russell et al. 1990). The development of testis in K K 2000) to yield a short (120–150 bp) PCR fragment with a Spo11 / mice is abnormal, because of the early melting temperature of about 60 8C. For each reaction, meiotic arrest. Consequently, these testes are devoid of 1 ml of diluted cDNA (4 mg/ml) was mixed with 300 pmol all the cell types past the arrest point including of each primer in 25 ml (final volume) of SYBR Green pachytene and secondary spermatocytes, spermatids Master Mix (Applied Biosystems, Foster City, CA, USA). and spermatozoa. However, they are relatively enriched RT-PCR was performed in an ABI 7000 (Applied in pre arrest germ cells (primordial germ cells, Biosystems). All amplifications were run in triplicate. spermatogonia, early meiotic prophase spermatocytes)

Figure 1 Scatter plots of gene expression values in testis of adult (a) and juvenile (15 days) (b) K K Spo11 / vs wild-type mice. Each gene is represented by a dot on scatter plots and the axes are the associated intensity levels of gene K K expression in Spo11 / and wild-type testis. Genes whose expression level was at least two K K times greater in Spo11 / testis are shown in yellow and genes whose expression level was at least two times greater in wild-type testis are shown in blue (c). The bar graph shows ratios of mRNA levels of selected genes in adult K K Spo11 / versus wild-type testis as determined by microarray analysis and quan- titative RT-PCR. www.reproduction-online.org Reproduction (2006) 132 67–77

Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access 70 N A Smirnova and others

Table 1 Average expression in spermatogonia, pachytene spermatocytes, and rounded spermatids. dpp; days post partum.

Average signal ratio K K K K Adult wild-type 15 dpp wild-type Adult Spo11 / 15 dpp Spo11 / Type vs reference RNA vs adult wild-type vs adult wild-type vs 15 dpp wild-type Spermatogonia 0.79 1.10 1.35 0.96 Pachytene spermatocytes 2.37 0.53 0.45 0.75 Rounded spermatids 2.32 0.40 0.43 0.97 and somatic Leydig and Sertoli cells (de Rooij & de Boer testis on average. On the other hand, spermatogonia- 2003, Khil et al. 2004). expressed genes are slightly enriched. K K These developmental abnormalities of Spo11 / Spermatogenesis involves the development of sperma- testes can be verified by analyzing the expression levels togonia into mature spermatozoa. In the mouse, this of cell type-specific genes. Recently, Shima et al. (2004) process starts at birth and is continuously re-initiated defined genes that are expressed predominantly in thereafter (Russell etal. 1990). Meiotic progression reaches K K spermatogonia, pachytene spermatocytes and rounded approximately the stage of the block in Spo11 / mice at spermatids on a genome-wide scale. We calculated the 15 days of age. In agreement with this developmental average expression levels of genes from each cell type in similarity, we found that the gene expression profiles of K K our experiments (Table 1) and found that genes 15-day wild type and Spo11 / adult testis are very similar expressed in spermatocytes and spermatids have more (Fig. 2, lanes S and W). The majority of the genes for those K K K K than twofold lower expression level in Spo11 / adult transcripts depleted in Spo11 / adult testis are also

K K Figure 2 Comparison of gene expression profiles in Spo11 / adult testes and 15 day juvenile wild-type testes with the spermatogenesis time course (Schultz et al. 2003). First, we normalized gene expression for every time point to gene expression on day 1 after birth. Then, we performed K (KZ9) means clustering of the normalized gene expression in the spermatogenesis time course and a hierarchical clustering of gene expression for K K every nine gene clusters including gene expression data for Spo11 / adult testis (lane S) and 15 day juvenile wild-type testes (lane W) vs adult wild- type testes. Testis-specific genes were determined from microarray experiments where the gene expression profile in adult wild-type testes is compared to reference RNA (lane T). We calculated the median of the ratio of the expression of every gene to reference RNA for four microarray experiments and consider a gene testis-specific, if the median of this ratio is equal or greater than 2. On the right, the biological processes over-represented for the class of testis-specific genes are listed. The evaluation of P value is given in Materials and Methods.

Reproduction (2006) 132 67–77 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access K K Gene expression profiles of Spo11 / 71 depleted in juvenile testis, and similarly enriched tran- Functional characterization of ‘early’ and ‘late’ genes K K K K scripts in Spo11 / are also enriched in juvenile testis. A in the expression profile of Spo11 / adult and cumulative analysis of genes expressed in individual cell wild-type juvenile testis populations of testis shows the same trends as for K/K To characterize those genes expressed early or late in Spo11 testis (Table 1). The average level of transcripts spermatogenesis and to integrate expression specificity of genes expressed in pachytene spermatocytes and with functional annotation, we performed gene ontology rounded spermatids was at least twofold less in K/K mining using the web-based tool eGOn (www.egon.com). Spo11 and juvenile testis when compared to adult Differentially expressed genes were divided into six wild-type testis (Table 1). Despite the almost identical clusters based on their regulation pattern in either K/K K K expression pattern of transcripts in Spo11 and juvenile Spo11 / or juvenile testes (Fig. 3). Cluster 1 includes K K testis, we found some interesting differences. These genes, whose transcripts are enriched only in Spo11 / differences are discussed in detail later. testes; cluster 2 – common early genes; cluster 3 – early Independent evidence for the conclusion that the genes only in 15 day wild-type testes; cluster 4 – genes, K K development of testis reaches the stage of the block in whose transcripts are depleted only in Spo11 / testes; Spo11 mice at approximately 15 days after birth, comes cluster 5 – common late genes and cluster 6 – genes from an analysis of the time course of spermatogenesis expressed late only in juvenile testes (see Fig. 3). Although K K (Schultz et al. 2003)(Fig. 2). We divided genes into nine gene expression patterns in adult Spo11 / and juvenile clusters based on the similarity of their expression wild-type testis have high similarity, there is a profound profiles as a function of time using K-means clustering. difference observed in the expression level of early genes. We found that transcripts that are more abundant in This difference is due to the apoptosis of arrested K/K K K Spo11 testis are associated with clusters of genes that spermatocytes in Spo11 / testes and the enrichment in K K reach their maximum of expression in testis before Spo11 / testes of Leydig cells that are the primary 14 days after birth (clusters 6, 7 and 8, see Fig. 2). The producers of steroid hormones in males. Therefore, the K/K majority of transcripts depleted in Spo11 testis largest cluster for early genes (genes, whose transcripts are K K increase their expression in testis on or after day 14 enriched only in Spo11 / testis) is over-represented with after birth (e.g. cluster 3). GO classes, such as steroid biosynthesis, fatty acid Therefore, in our microarray analysis, we can consider metabolism, proteolysis and peptidolysis, response to K K genes whose transcripts are enriched in Spo11 / testes external stimuli, immune response and cytolysis (Table 2, as genes expressed in spermatogonia and early sperma- Table 4 which can be viewed at: http://www.reproduction- tocytes, while genes, whose transcripts are depleted in online.org/supplemental/). The first cluster contains main K K Spo11 / testis are expressed later in meiosis and intracellular effectors of apoptosis: caspases 9 and 6, spermatogenesis. We call the first group of genes, ‘early’ Gadd45 g (growth arrest and DNA-damage-inducible genes and the second group ‘late’ genes. 45 gamma) and the members of the Bcl-2 family of

Figure 3 Heat map of genes, whose transcripts are K K depleted or enriched in adult Spo11 / and juvenile wild-type testis. We selected genes whose transcripts are K K depleted or enriched twofold either in adult Spo11 / (lane S) or juvenile wild-type testis (lane W) and divided them into six clusters: (1) transcripts enriched only in Spo11 mutant, (2) common early genes in Spo11 mutant and juvenile wild-type testis, (3) early genes in juvenile wild-type testis, (4) transcripts depleted only in Spo11 mutant, (5) common late genes in Spo11 mutant and juvenile wild-type testis, (6) late genes only in juvenile wild-type testis. On the right, selected genes from each cluster are listed. www.reproduction-online.org Reproduction (2006) 132 67–77

Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access 72 N A Smirnova and others

Table 2 Statistically significant biological processes associated with early and late genes in spermatogenesis.

GO Name of GO class P value K K Cluster 1.‘Early ’ genes for Spo11 / testes GO:0030003 cation homeostasis 0.002 GO:0007031 peroxisome organization and biogenesis 0.013 GO:0006694 steroid biosynthesis 0.001 GO:0006631 fatty acid metabolism 0.001 GO:0019752 carboxylic acid metabolism 0.012 GO:0018108 peptidyl-tyrosine phosphorylation 0.026 GO:0009605 response to external stimulus 0.001 GO:0006955 immune response 0.001 GO:0006508 proteolysis and peptidolysis 0.046 GO:0006731 coenzymes and prosthetic group metabolism 0.01 K K Cluster 2. Common ‘Early’ genes for 15 dpp wild-type and Spo11 / testes GO:0007154 cell communication 0.001 GO:0008283 cell proliferation 0.02 GO:0007049 cell cycle 0.006 GO:0000074 regulation of cell cycle 0.02 GO:0007143 female meiosis 0.001 GO:0015671 oxygen transport 0.001 GO:0006839 mitochondrial transport 0.001 GO:0006405 RNA-nucleus export 0.001 GO:0006817 phosphate transport 0.001 GO:0006928 cell motility 0.001 GO:0007275 development 0.011 GO:0009408 response to heat 0.001 Cluster 3. ‘Early’ genes for 15 dpp wild-type testes GO:0007126 meiosis 0.023 GO:0008283 cell proliferation 0.023 GO:0007049 cell cycle 0.012 GO:0000074 regulation of cell cycle 0.049 GO:0000280 nuclear division 0.021 GO:0015671 oxygen transport 0.002 K K Cluster 4. ‘Late’ genes for Spo11 / testes GO:0000278 mitotic cell cycle 0.004 GO:0000087 M phase of mitotic cell cycle 0.019 GO:0016569 covalent chromatin modification 0.019 GO:0006352 transcription initiation 0.003 GO:0006366 transcription from Pol II promoter 0.009 GO:0016481 negative regulation of transcription 0.042 GO:0006446 regulation of translational initiation 0.029 GO:0009314 response to radiation 0.047 GO:0009408 response to heat 0.001 K K Cluster 5. Common ‘Late’ genes for 15 dpp wild-type and Spo11 / testes GO:0000067 DNA replication and chromosome cycle 0.038 GO:0007059 chromosome segregation 0.022 GO:0000280 nuclear division 0.007 GO:0007017 microtubule-based process 0.006 GO:0006338 chromatin remodeling 0.017 GO:0000278 mitotic cell cycle 0.019 GO:0000279 M phase 0.004 GO:0007067 mitosis 0.008 GO:0007126 meiosis 0.019 GO:0007276 gametogenesis 0.012 GO:0006066 alcohol metabolism 0.001 GO:0006096 glycolysis 0.001 GO:0006457 protein folding 0.027 GO:0006464 protein modification 0.001 GO:0006470 protein amino acid dephosphorylation 0.001 GO:0006512 ubiquitin cycle 0.018 Cluster 6. ‘Late’ genes for 15 dpp wild-type testes only GO:0007283 spermatogenesis 0.034 GO:0016601 RAC protein signal transduction 0.023 GO:0007266 Rho protein signal transduction 0.035 GO:0030003 cation homeostasis 0.027 GO:0000082 G1/S transition of mitotic cell cycle 0.021 GO:0006694 steroid biosynthesis 0.044 GO:0006629 lipid metabolism 0.009

Cluster order correspond to cluster number from Fig. 3. GO; gene ontology, dpp; days post partum.

Reproduction (2006) 132 67–77 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access K K Gene expression profiles of Spo11 / 73

Table 3 Microarray data for genes, implicated in meiosis.

Average signal ratio

15 dpp wild-type vs K K K K ID Name adult wild-type adult Spo11 / vs adult wild-type 15 dpp Spo11 / vs 15 dpp wild-type BG085454 Mlh3 3.33 1.53 0.84 BG066554 Mre11 0.65 0.44 0.98 BG086280 Msh6 1.51 1.86 1.10 AU042878 Hop2 0.35 0.17 0.42 BG072904 Rad51 1.99 1.53 0.97 BG068566 Msh4 0.91 0.75 0.93 BG070803 Mnd1 0.93 5.92 4.57 BG087717 Pms2 1.51 1.64 1.02 AU043450 Msh2 3.39 4.06 0.85 BG085912 Rad50 1.32 1.33 0.88 BG068496 RAD6 0.60 1.25 1.40 BG068820 Brca2 0.69 0.44 0.60 BG065309 RAD54 0.51 0.37 0.91 BG077083 RAD1 0.58 0.56 0.63 BG064423 ATM 1.39 1.37 1.07 BG075806 Ku70 0.49 0.66 0.86 BG074715 SMC1b 0.99 0.66 1.05 BG085432 SMC3 0.22 0.18 0.67 BG076157 Scp1 2.11 1.14 0.70 BG075601 Stag3 0.42 0.39 0.79 BG076124 Scp3 1.43 1.80 1.09 BG072786 Rec8 0.70 0.73 0.97 BG081004 RPA 0.32 0.42 1.32 BG088069 Fkbp6 1.54 1.61 0.86 BG074262 Terf2 1.78 2.62 1.15 AU022550 Suv39h2 2.19 3.67 0.79 BG087679 Suv39h1 1.22 1.19 1.01 BG073019 Cdk2 1.32 1.34 0.83 BG070939 Chek1 1.84 2.78 1.02 BG085472 p18 3.39 3.97 0.95 BG083522 Cks2 0.99 0.66 0.54 BG071915 cyclin B2 0.27 0.12 0.46 BG083482 Chek2/CHK2 1.48 0.54 1.00 BG066279 CDC25A 0.49 0.36 1.02 AW544792 cyclin E2 1.50 0.91 BG077073 cyclin H 1.13 0.51 0.75 dpp; days post partum. proteins that promote the mitochondria-dependent DNA repair and apoptosis, and corresponding genes are apoptotic pathway (Green & Kroemer 2004)(Fig. 3, induced before or in early stage of meiosis, we find in Table 2, Table 4, which can be viewed at: http://www. cluster 2 Msh2 (DNA mismatch repair protein), Rad21 reproduction-online.org/supplemental/). The appearance (DBSs repair protein), Casp3 (apoptosis related cysteine of cytolysis and immune response among over-represented protease or caspase 3) and Pdcd8 (programmed cell death GO classes reflects the intensive apoptosis, which occurs 8- apoptosis-inducing factor). K K in adult Spo11 / testes. Cluster 3 (early genes in 15 day wild-type testes only) K K Both juvenile wild-type and adult Spo11 / testes are show as a positive association with such GO classes as also enriched with spermatogonia cells that undergo meiosis, cell proliferation, cell cycle and nuclear several mitotic divisions before entering meiosis. There- division. Some of the genes found in this cluster include fore, among common early genes (cluster 2), the Mlh3 (establishing and/or maintaining crossovers), Sycp1 following GO classes are over-represented: cell prolifer- (major structural protein of the central element in SC), ation, cell cycle, regulation of cell cycle, cell motility and Rad51 (eukaryotic RecA homolog); E2f6 (transcriptional development (Table 2, Table 4, which can be viewed at: regulation of various genes implicated in chromatin http://www.reproduction-online.org/supplemental/). remodeling), Anapc1 (anaphase promoting complex Smc1l1 (structural maintenance of chromosomes 1-like subunit 1) and Dazl (translation regulator). The obser- 1) and cell-cycle regulators such as Cdk5 (cyclin- vation that these genes are highly expressed only in dependent kinase 5), Ccna2 (cyclin A2) and Anapc4, 7 juvenile wild-type testis is consistent with the normal (anaphase promoting complex subunit 4 and 7) are in this development of meiosis in juvenile wild-type testis (Table cluster. Consistent with the fact that the normal 4, which can be viewed at: http://www.reproduction- development of spermatogenesis is accompanied by online.org/supplemental/). It is interesting to note that www.reproduction-online.org Reproduction (2006) 132 67–77

Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access 74 N A Smirnova and others in contrast to Anapc4 and Anapc7, the Anapc1 (meiotic means clustering of microarray data obtained from a K K check point regulator) is induced only in 15-day wild- comparison between Spo11 / and wild-type testes, type testis and might be critical for the zygotene/ and wild-type adult testes and reference RNA showed pachytene transition. that 90% of testis-specific genes mapped to the cluster of K K The difference between late genes in Spo11 / and genes expressed in the late stages of meiosis and juvenile testis is not as profound as the difference spermatogenesis (Fig. 2, lanes S and T). This conclusion between early genes (Fig. 3). The majority of genes was also confirmed if we used the other criteria of testis K K whose transcripts are depleted in both Spo11 / and specificity based on the abundance of clones in Unigene juvenile testis belong to cluster 5 (common late genes). cDNA libraries and publicly available Affymetrix data of As expected, we found a positive association of genes mouse gene expression in different tissues (Khil et al. from cluster 5 with processes that occur late in male 2004). We found only 10% of testis-specific genes in the meiosis and spermatogenesis. These GO classes ’early’ cluster of gene expression in Spo11 testis. In include gametogenesis (Rad23b, Mea1, Zfp35, Tce1), addition, GO analysis of testis-specific genes determined meiosis (Tsga2, Hop2, Rpa1, Stag3), chromatin remo- from a comparison of wild-type testes and reference deling (Morf4l1, Brd6) and chromosome segregation RNA showed a statistically significant association with (Ccnb2, Chc1, Cdc25A, Cdc25C) (Table 2, Table 4, the same GO classes that were observed in cluster 5 K K which can be viewed at: http://www.reproduction- (genes transcripts, depleted in both Spo11 / adult and online.org/supplemental/). 15 days testis) (Fig. 2, Table 2). The relatively small cluster 4 (gene transcripts K/K depleted only in adult Spo11 testis) is enriched with genes involved in mitotic cell cycle, covalent chromatin modification, transcription initiation and regulation of The effect of Spo11 knockout on gene expression in the translational initiation (Table 2). We can speculate that testes of juvenile mice the lack of expression of these genes was the con- Previously, we found that the major effect of Spo11 sequence of the meiotic arrest and induction of apoptosis knockout is the disruption of the normal development of K K in Spo11 / testis. Interestingly, in this cluster, we find testes. To focus on genes involved in meiosis I, we K K important meiotic recombination proteins involved in compared gene expression profiles of Spo11 / and the processing of DSBs, such as Mre11 (a major DSB wild-type testes from 12- and 15-days mice. On day 12, repair protein) and Brca2 (breast cancer 2). In a mutant most of wild-type spermatocytes (about 85%) are in K K Brca2 / mouse carrying a human BRCA2 transgene leptotone/zygotene stages and at 15 days, they are (the Brca2 null mutation is embryonic lethal for mice) entering pachytene (Goetz et al. 1984). Comparing K K spermatocytes undergo normal DSBs formation but fail Spo11 / and wild-type mice at this age may allow us to to complete recombination and do not progress beyond focus on genes expressed in early meiotic prophase I that zygotene (Sharan et al. 2004). In our study, the observed are more clearly dependent on the formation of DSBs. depletion of Brca2 could be a consequence of the These genes include those involved in meiotic recombi- absence of DSBs in Spo11 knockout testis. nation, homologous pairing, SC formation and meiotic The last cluster, cluster 6 contains late genes, whose checkpoints. The use of this relatively early time point transcripts are depleted only in 15-day wild-type testis helps to eliminate from the analysis, genes expressed in and these genes are implicated in spermatogenesis, later stages of spermatogenesis and apoptotic genes that steroid biosynthesis and lipid metabolism. The presence are induced in response to the meiotic arrest of K K of these last two categories reflects the incomplete Spo11 / spermatocytes. establishment of hormone production in juvenile testis. Apart from a few genes, including Mnd1, the microarray analysis did not reveal any significant differences in gene expression between Spo11 KO and wild-type testis at 12 day of age (Table 5, which can The majority of testis-specific genes are expressed in be viewed at: http://www.reproduction-online.org/ late stages of meiosis and spermatogenesis supplemental/). On day 15, we found that only 250 To further characterize genes differentially expressed in genes have changed their expression level more than 1.5- K K K K Spo11 / adult testes, we determined the distribution of fold in Spo11 / compared to wild-type testis (Fig. 1b, mouse testis-specific genes among early and late genes Table 5 available online). First of all, this selection K K in the gene expression profiles of Spo11 / testes. We includes some genes critical for meiotic recombination performed cDNA microarray analysis, where we (Table 3, Table 5 available online). Among them are compared gene expression in adult mouse testes to Hop2, Brca2, Mnd1, FancG (Fanconi anemia (group G) reference RNA. Reference RNA included samples of (Yang et al. 2001, Petukhova et al. 2003, 2005, Chen et al. total RNA from 11 different mouse tissues. We classified 2004, Gudmundsdottir & Ashworth 2004). According to a genes as testis-specific, if their expression level in testes current model, Brca2 is involved in delivering Rad51 and is at least twofold more than in reference RNA. K (KZ9) Dmc1 to repair DSBs by initiating strand exchange

Reproduction (2006) 132 67–77 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access K K Gene expression profiles of Spo11 / 75

K K (Gudmundsdottir & Ashworth 2004). In Spo11 / components of the machinery that controls meiotic spermatocytes, programmed DSBs are not formed and progression from zygotene to pachytene. Although cyclin this function of Brca2 would not be required. Indeed, we B1 can substitute cyclin B2 and the expression level of found a 1.5-fold depletion of Brca2 transcripts in juvenile cyclin B1 did not change in 15-day Spo11 testes, still K K Spo11 / testis. Furthermore, we observed a twofold Ccnb1/Cdk1 complex can be compromised by decrease in the level of expression FancG transcripts in downregulation of the expression of some genes. Among K K Spo11 / spermatocytes. FancG protein is capable of them, we found calmodulin, a gene that can affect binding to two separate sites in the Brca2 protein (Hussain cyclinB1/Cdk1 (also known as Cdc2) through its partner, et al. 2003). It is co-immunoprecipitated with Brca2 from the testis-specific heat-shock protein Hsp70-2 (Zhu et al. human cells and co-localized in nuclear foci with both 1997, Moriya et al. 2004). Hsp-70-2 deficient mice exhibit Brca2 and Rad51 following DNA damage with mitomy- failed meiosis (Dix et al. 1996) and it has been suggested cin C (Hussain et al. 2003). Without doubt, FancG protein that Hsp-70-2 functions as a molecular chaperone for the K/K also participates in meiosis because the FancG mice Ccnb1/Cdk1 (Cdc2) complex (Zhu et al. 1997). have germ cell defects and decreased fertility (Yan g et al. Among other genes that might play an essential role in 2001). One of the most interesting observations is a meiotic progression, we can emphasize Ddx4, the twofold decrease in the transcription level of Hop2, mouse homologue of the Drosophila gene Vasa, that another abundant meiotic protein closely associated with controls the initiation of translation (Carrera et al. 2000) K K the Dmc1 and Rad51 in meiosis (Petukhova et al. 2003, and those expression is a twofold depleted in Spo11 / 2005, Chen et al. 2004). It has been shown in yeast and juvenile testes (Table 5, which can be viewed at: http:// mice that the Mnd1/Hop2 heterodimeric protein www.reproduction-online.org/supplemental/). The complex is required for the homologous pairing of mouse Vasa transcript is bound by Dazl (Reynolds et chromosomes during meiosis and that these proteins act al. 2005), and knockouts of either Dazl or Ddx4 lead to by promoting Rad51 or Dmc1 activity during the meiotic arrest of mouse spermatocytes at a zygotene-like K K formation of the first recombination intermediates in stage similar to that found in the Spo11 / knockout meiosis (Petukhova et al. 2003, 2005, Chen et al. 2004). (Tanaka et al. 2000, Saunders et al. 2003). The dramatic decrease in the expression of Hop2 in K/K Other promising candidates potentially involved in Spo11 juvenile testes may be the reflection of the meiotic checkpoint, meiotic recombination and SC absence of DSBs and the lack of the need for Hop2 under formation may be found among the 53 genes with these circumstances. We could not find a satisfactory unknown function differentially expressed in juvenile explanation for increased transcription of Mnd1 in K K K K / Spo11 / juvenile testes because the function of Spo11 testis. Based on the expression data obtained Mnd1, besides the role in forming a complex with Hop2 from the GEO profile database (http://www.ncbi.nlm. nih.gov/geo/), we determined that the majority of that stimulates Rad51 and Dmc1, is unclear. K K K K / Although Spo11 / spermatocytes fail to form SC unknown genes downregulated in young Spo11 between homologous chromosome (Baudat et al. 2000, testis increase their expression on day 14 after birth Romanienko & Camerini-Otero 2000), we did not observe (Table 6 which can be viewed online at: http://www. significant changes in the expression of genes encoding reproduction-online.org/supplemental/). We found that proteins that are part of the lateral and central elements in some unknown proteins contain conserved domains SC such as Smc1beta, Rec8, Stag3, Smc3, Fkbp6, Sycp1 involved in DNA recombination, microtubule-based and Sycp3 (Table 3). Therefore, our gene expression data process and regulation of developmental pathways support the conclusion of previous study that cohesin cores (Table 6 available online). can form independently of DSB formation and homo- In summary, microarray analysis of gene expression in K/K logous pairing in mice (James et al. 2002). adult Spo11 testes reveals that the Spo11 mutation K K Spo11 / spermatocytes are arrested at zygotene in causes dramatic changes in the gene expression pattern of K/K meiotic prophase I. Little is known about the mechanisms Spo11 mouse testis. Spo11 knockout activates of cell cycle control and checkpoint prior to pachytene. It is meiotic arrest in zygotene spermatocytes and, as a consequence, the disruption of spermatogenesis and interesting in this regard that we found that the meiosis- K K specific Ccnb2 (cyclin B2) and the Cks2 (CDC28 protein normal testis development. Spo11 / testes are enriched kinase regulatory subunit 2) are twofold downregulated in with spermatogonia and spermatocytes in early stages of K K K K 15-day Spo11 / testis. The Cks2 modulates substrate meiosis. Therefore, Spo11 / testes are a useful model recognition by a Cdk1 that is part of the meiotic cell cycle for studies of gene expression in spermatogonia and early regulatory complex Ccnb2/Cdk1 (Patra & Dunphy 1998, spermatocytes. Using the GO mining tool eGOn, we Spruck et al. 2003, Murray 2004). Although in Cks2- determined the relevant biological processes for genes deficient mice, meiosis arrests at metaphase I (Spruck et al. expressed early and late in spermatogenesis. A compara- K K K K 2003) and Ccnb2 / mice are fertile (the function of tive analysis of gene expression in juvenile Spo11 / cyclin B2 is probably rescued by cyclin B1 (Ccnb1) mouse testes showed a dramatic decrease in the (Brandeis et al. 1998), the cyclin B2 and Cks2 may be key expression level of genes involved in meiotic www.reproduction-online.org Reproduction (2006) 132 67–77

Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access 76 N A Smirnova and others recombination, meiotic checkpoints, but an absence of Gudmundsdottir K & Ashworth A 2004 BRCA2 in meiosis: turning over significant changes in the expression of genes encoding a new leaf. Trends in Cell Biology 14 401–404. proteins for SC. This analysis also revealed about 50 genes Henderson KA & Keeney S 2004 Tying synaptonemal complex initiation to the formation and programmed repair of DNA double- of unknown function specifically induced or repressed in strand breaks. PNAS 101 4519–4524. K/K Spo11 testes. Such genes are very likely to be Hussain S, Witt E, Huber PA, Medhurst AL, Ashworth A & specifically involved in meiosis and spermatogenesis Mathew CG 2003 Direct interaction of the Fanconi anaemia and to be affected by disruption of the Spo11 gene. protein FANCG with BRCA2/FANCD1. Human Molecular Studies of these novel proteins may shed a new light on Genetics 12 2503–2510. our understanding of the function of Spo11 protein in James RD, Schmiesing JA, Peters AH, Yokomori K & Disteche CM 2002 Differential association of SMC1alpha and SMC3 proteins with meiosis and may help to find more partners of Spo11 in meiotic chromosomes in wild-type and SPO11-deficient male mice. mammals. Chromosome Research 10 549–560. Keeney S 2001 Mechanism and control of meiotic recombination initiation. Current Topics in Developmental Biology 52 1–53. Keeney S, Giroux CN & Kleckner N 1997 Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely References conserved protein family. Cell 88 375–384. Khil PP, Smirnova NA, Romanienko PJ & Camerini-Otero RD 2004 The Baarends WM, van der Laan R & Grootegoed JA 2001 DNA repair mouse X chromosome is enriched for sex-biased genes not subject to mechanisms and gametogenesis. Reproduction 121 31–39. selection by meiotic sex chromosome inactivation. Nature Genetics Bannister LA & Schimenti JC 2004 Homologous recombinational 36 642–646. repair proteins in mouse meiosis. Cytogenetic and Genome Research 107 191–200. Kneitz B, Cohen PE, Avdievich E, Zhu L, Kane MF, Hou H Jr, Kolodner Baudat F, Manova K, Yuen JP, Jasin M & Keeney S 2000 Chromosome RD, Kucherlapati R, Pollard JW & Edelmann W 2000 MutS homolog synapsis defects and sexually dimorphic meiotic progression in mice 4 localization to meiotic chromosomes is required for chromosome lacking Spo11. Molecular Cell 6 989–998. pairing during meiosis in male and female mice. Genes & Bishop DK, Park D, Xu L & Kleckner N 1992 DMC1: a meiosis-specific Development 14 1085–1097. yeast homolog of E. coli recA required for recombination, Leu JY, Chua PR & Roeder GS 1998 The meiosis-specific Hop2 protein synaptonemal complex formation, and cell cycle progression. Cell of S. cerevisiae ensures synapsis between homologous chromo- 69 439–456. somes. Cell 94 375–386. Brandeis M, Rosewell I, Carrington M, Crompton T, Jacobs MA, Kirk J, Libby BJ, De La Fuente R, O’Brien MJ, Wigglesworth K, Cobb J, Gannon J & Hunt T 1998 Cyclin B2-null mice develop normally Inselman A, Eaker S, Handel MA, Eppig JJ & Schimenti JC 2002 The and are fertile whereas cyclin B1-null mice die in utero. PNAS 95 mouse meiotic mutation mei1 disrupts chromosome synapsis with 4344–4349. sexually dimorphic consequences for meiotic progression. Develop- Carrera P, Johnstone O, Nakamura A, Casanova J, Jackle H & Lasko P mental Biology 242 174–187. 2000 VASA mediates translation through interaction with a Libby BJ, Reinholdt LG & Schimenti JC 2003 Positional cloning Drosophila yIF2 homolog. Molecular Cell 5 181–187. and characterization of Mei1, a vertebrate-specific gene required Cha RS, Weiner BM, Keeney S, Dekker J & Kleckner N 2000 for normal meiotic chromosome synapsis in mice. PNAS 100 Progression of meiotic DNA replication is modulated by inter- 15706–15711. chromosomal interaction proteins, negatively by Spo11p and Lichten M 2001 Meiotic recombination: breaking the genome to save positively by Rec8p. Genes and Development 14 493–503. it. Current Biology 11 R253–R256. Chen YK, Leng CH, Olivares H, Lee MH, Chang YC, Kung WM, Ti McKim KS & Hayashi-Hagihara A 1998 mei-W68 in Drosophila SC, Lo YH, Wang AH, Chang CS et al. 2004 Heterodimeric melanogaster encodes a Spo11 homolog: evidence that the complexes of Hop2 and Mnd1 function with Dmc1 to promote mechanism for initiating meiotic recombination is conserved. meiotic homolog juxtaposition and strand assimilation. PNAS Genes & Development 12 2932–2942. 101 10572–10577. Moriya M, Ochiai M, Yuasa HJ, Suzuki N & Yazawa M 2004 de Rooij DG & de Boer P 2003 Specific arrests of spermatogenesis in Identification of Ca2C-dependent calmodulin-binding proteins in genetically modified and mutant mice. Cytogenetic and Genome rat spermatogenic cells as complexes of the heat-shock proteins. Research 103 267–276. Molecular Reproduction and Development 69 316–324. Dernburg AF, McDonald K, Moulder G, Barstead R, Dresser M & Murray AW 2004 Recycling the cell cycle: cyclins revisited. Cell 116 Villeneuve AM 1998 Meiotic recombination in C. elegans initiates 221–234. by a conserved mechanism and is dispensable for homologous Patra D & Dunphy WG 1998 Xe-p9, a Xenopus Suc1/Cks protein, is chromosome synapsis. Cell 94 387–398. essential for the Cdc2-dependent phosphorylation of the Dix DJ, Allen JW, Collins BW, Mori C, Nakamura N, Poorman-Allen P, anaphase- promoting complex at mitosis. Genes and Development Goulding EH & Eddy EM 1996 Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. 12 2549–2559. PNAS 93 3264–3268. Petukhova GV, Romanienko PJ & Camerini-Otero RD 2003 The Hop2 Eisen MB, Spellman PT, Brown PO & Botstein D 1998 Cluster protein has a direct role in promoting interhomolog interactions analysis and display of genome-wide expression patterns. PNAS 95 during mouse meiosis. Developmental Cell 5 927–936. 14863–14868. Petukhova GV, Pezza RJ, Vanevski F, Ploquin M, Masson JY & Goetz P, Chandley A & Speed R 1984 Morphological and temporal Camerini-Otero RD 2005 The Hop2 and Mnd1 proteins act in sequence of meiotic prophase development at puberty in the male concert with Rad51 and Dmc1 in meiotic recombination. Nature mouse. Journal of Cell Science 65 249–263. Structural & Molecular Biology . Green DR & Kroemer G 2004 The pathophysiology of mitochondrial Pittman DL, Cobb J, Schimenti KJ, Wilson LA, Cooper DM, cell death. Science 305 626–629. Brignull E, Handel MA & Schimenti JC 1998 Meiotic prophase Grelon M, Vezon D, Gendrot G & Pelletier G 2001 AtSPO11-1 is arrest with failure of chromosome synapsis in mice deficient for necessary for efficient meiotic recombination in plants. EMBO Dmc1, a germline-specific RecA homolog. Molecular Cell 1 Journal 20 589–600. 697–705.

Reproduction (2006) 132 67–77 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access K K Gene expression profiles of Spo11 / 77

Prieler S, Penkner A, Borde V & Klein F 2005 The control of Spo11’s Storlazzi A, Tesse S, Gargano S, James F, Kleckner N & Zickler D 2003 interaction with meiotic recombination hotspots. Genes and Meiotic double-strand breaks at the interface of chromosome Development 19 255–269. movement, chromosome remodeling, and reductional division. Rabitsch KP, Toth A, Galova M, Schleiffer A, Schaffner G, Aigner Genes & Development 17 2675–2687. E, Rupp C, Penkner AM, Moreno-Borchart AC, Primig M et al. Tanaka SS, Toyooka Y, Akasu R, Katoh-Fukui Y, Nakahara Y, Suzuki R, 2001 A screen for genes required for meiosis and spore Yokoyama M & Noce T 2000 The mouse homolog of Drosophila formation based on whole-genome expression. Current Biology Vasa is required for the development of male germ cells. Genes and 11 1001–1009. Development 14 841–853. Reynolds N, Collier B, Maratou K, Bingham V, Speed RM, Taggart M, Tsubouchi H & Roeder GS 2002 The Mnd1 protein forms a complex Semple CA, Gray NK & Cooke HJ 2005 Dazl binds in vivo to specific with hop2 to promote homologous chromosome pairing and meiotic transcripts and can regulate the pre-meiotic translation of Mvh in double-strand break repair. Molecular and Cellular Biology 22 germ cells. Human Molecular Genetics 14 3899–3909. 3078–3088. Roeder GS & Bailis JM 2000 The pachytene checkpoint. Trends in Turner JM, Mahadevaiah SK, Fernandez-Capetillo O, Nussenz- Genetics 16 395–403. weig A, Xu X, Deng CX & Burgoyne PS 2005 Silencing of Romanienko PJ & Camerini-Otero RD 1999 Cloning, character- unsynapsed meiotic chromosomes in the mouse. Nature ization, and localization of mouse and human SPO11. Genomics Genetics 37 41–47. 61 156–169. Tusher VG, Tibshirani R & Chu G 2001 Significance analysis of Romanienko PJ & Camerini-Otero RD 2000 The mouse Spo11 gene is required for meiotic chromosome synapsis. Molecular and Cellular microarrays applied to the ionizing radiation response. PNAS 98 Proteomics 6 975–987. 5116–5121. Rozen S & Skaletsky HJ 2000 Primer 3 on the WWW for general users Villeneuve AM & Hillers KJ 2001 Whence meiosis? Cell 106 and for biologist programmers. In Bioinformatics Methods and 647–650. Protocols: Methods in Molecular Biology. pp 365–386. Ed, S Yang Y, Kuang Y, De Oca RM, Hays T, Moreau L, Lu N, Seed B & Krawetz & S Misner. Totowa, NJ, USA: Humana Press. D’Andrea AD 2001 Targeted disruption of the murine Fanconi Russell LD, Ettlin RA, Sinha Hikim AP & Clegg ED 1990 Histological anemia gene, Fancg/Xrcc9. Blood 98 3435–3440. and Histopathological Evaluation of the Testis, edn 1 pp 62–194. Yoshida K, Kondoh G, Matsuda Y, Habu T, Nishimune Y & Morita Clearwater, FL, USA:Cache River Press. T 1998 The mouse RecA-like gene Dmc1 is required for Saunders PT, Turner JM, Ruggiu M, Taggart M, Burgoyne PS, Elliott D & homologous chromosome synapsis during meiosis? Molecular Cooke HJ 2003 Absence of mDazl produces a final block on germ Cell 1 707–718. cell development at meiosis. Reproduction 126 589–597. Yuan L, Liu JG, Zhao J, Brundell E, Daneholt B & Hoog C 2000 Schultz N, Hamra FK & Garbers DL 2003 A multitude of genes The murine SCP3 gene is required for synaptonemal complex expressed solely in meiotic or postmeiotic spermatogenic cells offers assembly, chromosome synapsis, and male fertility. Molecular a myriad of contraceptive targets. PNAS 100 12201–12206. Cell 5 73–83. Sharan SK, Pyle A, Coppola V, Babus J, Swaminathan S, Benedict J, Zhu D, Dix DJ & Eddy EM 1997 HSP70-2 is required for CDC2 kinase Swing D, Martin BK, Tessarollo L, Evans JP et al. 2004 BRCA2 activity in meiosis I of mouse spermatocytes. Development 124 deficiency in mice leads to meiotic impairment and infertility. 3007–3014. Development 131 131–142. Shima JE, McLean DJ, McCarrey JR & Griswold MD 2004 The murine testicular transcriptome: characterizing gene expression in the testis during the progression of spermatogenesis. Biology of Reproduction 71 319–330. Received 6 October 2005 Spruck CH, de Miguel MP, Smith AP, Ryan A, Stein P, Schultz RM, First decision 14 December 2005 Lincoln AJ, Donovan PJ & Reed SI 2003 Requirement of Cks2 for the first metaphase/anaphase transition of mammalian meiosis. Science Revised manuscript received 16 March 2006 300 647–650. Accepted 5 May 2006

www.reproduction-online.org Reproduction (2006) 132 67–77

Downloaded from Bioscientifica.com at 09/24/2021 07:01:38PM via free access