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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