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 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 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.
Introduction
Spo11 protein, a type II like topoisomerase, generates double-strand breaks (DSBs) during meiosis. It is structurally and functionally conserved in a wide variety of organisms such as yeast, insects, worms, plants, mice and humans (Keeney et al. 1997, Dernburg et al. 1998, McKim & Hayashi-Hagihara 1998, Romanienko & Camerini-Otero 1999, 2000, Baudat et al. 2000, Grelon et al. 2001, Storlazzi et al. 2003). The wide distribution and the high degree of sequence similarity of Spo11 homologs suggest that the introduction of DSBs is an essential function in meiosis (Keeney 2001). Generated by Spo11, DSBs are used for initiation of meiotic recombination and promote pairing and synaptonemal complex (SC) formation between homologous chromosomes in many organisms (Romanienko & Camerini-Otero 2000, Storlazzi et al. 2003, Henderson & Keeney 2004). Synapsis facilitates the completion of recombination that leads to the correct segregation of chromosomes during meiosis I (Lichten 2001). Meiotic DSBs recruit a number of conserved enzymes including Mre11, Nbs1, Rpa, Rad50, Rad51 and Rad54 that also participate in DNA repair in mitotic cells (Baarends et al. 2001, Villeneuve & Hillers 2001, Bannister & Schimenti 2004). In addition to mitotic DNA repair proteins, meiotic recombination requires several meiosis-specific proteins, such as Dmc1 and the relatively recently described Hop2 and Mnd1 proteins (Bishop et al. 1992, Leu et al. 1998, Pittman et al. 1998, Yoshida et al. 1998, Rabitsch et al. 2001, Tsubouchi & Roeder 2002, Petukhova et al. 2003, 2005). Nevertheless, many gene products specifically involved in meiotic recombination are still unknown.
Apart from its catalytic function, Spo11 may play a structural role in chromosome pairing (Romanienko & Camerini-Otero 2000, Prieler et al. 2005). Pairing of homologous chromosomes, but not synapsis, was observed in yeast with a catalytically inactive Spo11 mutant, while complete deletion of the Spo11 gene eliminates this homolog pairing (Cha et al. 2000). Furthermore, in yeast, Spo11 foci were abundant in some pachytene cells where formation of DSBs are completed and during pachytene, most Spo11 foci touched or overlapped with Zip1 – the central element component of the SC (Prieler et al. 2005). Spo11 may also negatively regulate exit from the bouquet stage, a cell-wide regulatory transition accompanying global chromosome movements, via non-catalytic function in Sordaria macrospora at zygotene/pachytene stage (Storlazzi et al. 2003). In mouse, Spo11 localizes to discrete foci early in meiosis, which is consistent with catalytic function of Spo11 in leptotene, and later to the regions of homologous chromosome synapsis that suggested an additional structural role for Spo11 (Romanienko & Camerini-Otero 2000). Taken together, these findings indicate that Spo11 may have a role in the progression of meiotic prophase independent of DSBs.
Knockout of the mouse Spo11 prevents formation of DSBs and SC during meiosis and leads to the meiotic arrest of spermatocytes at zygotene (Baudat et al. 2000, Romanienko & Camerini-Otero 2000). The arrested cells undergo apoptosis. As a result, Spo11−/ −homozygous male mice are sterile and have small, underdeveloped testes. A growing body of information indicates that mouse knockouts of genes with many different functions in meiosis, such as Msh4 (DNA mismatch repair protein), Dmc1 (meiotic recombination and repair protein), Hop2 (proper homologous chromosome pairing protein), Sycp3 (structural component of the axial/lateral element of SC) and Mei1 (a possible partner of Spo11) have a similarly arrested meiotic phenotype (de Rooij & de Boer 2003, Petukhova et al. 2003). These mutant mice have spermatocytes that do not develop beyond zygotene and are sterile in the homozygous state (de Rooij & de Boer 2003, Petukhova et al. 2003). A common feature for all these mutant spermatocytes is the failure to synapse homologous chromosomes (Pittman et al. 1998, Yoshida et al. 1998, Kneitz et al. 2000, Yuan et al. 2000, Libby et al. 2002, 2003, Petukhova et al. 2003). It is not clear how this failure in chromosome synapsis leads to the arrest in meiosis and subsequent apoptosis. It might be the result of the action of a pachytene checkpoint (Roeder & Bailis 2000) as has been observed for budding yeast or the transcriptional silencing imposed at pachytene on unsynapsed meiotic chromosomes (Turner et al. 2005). The latter might be sufficient in its own. In other words, the transcriptional inactivation of genes essential for meiotic progression may precipitate meiotic arrest before pachytene in the absence of a checkpoint as such. A comparative analysis of gene expression in mutant and wild-type mouse testes may yield new information about the general mechanisms leading to meiotic arrest and uncover new genes, which participate in meiosis, meiotic checkpoint and spermatogenesis.
We used cDNA microarray analysis to measure gene expression levels in adult and juvenile Spo11−/ −testes. Differentially expressed genes were further characterized by their functional classification and by the tissue specificity of their expression. Analysis of gene expression of juvenile Spo11−/ −testes has allowed us to recognize genes whose expression depends on Spo11.
Materials and Methods
Animals
All animal use procedures were performed according to the NIH Guide for the Care and Use of Laboratory Animals. Spo11−/ −knockout mice were generated as described previously (Romanienko & Camerini-Otero 2000). Wild-type mice were siblings of Spo11−/ −mice.
Total RNA isolation
Testes were surgically removed from mice and stored at −80 °C until total RNA was isolated using a Trizol solution (Life Technologies). Total RNA then was treated with RNase-free DNase (Promega) at room temperature for 15 min. RNA concentration was determined by UV spectrophotometry and RNA integrity was confirmed using agarose gel electrophoresis. Total RNAs (10 μg) were used as templates in reverse transcription (RT) reactions.
Microarray procedures
Amino-allyl modified cDNA was synthesized using SuperScript II RNase H-RT (Invitrogen) oligo (dT) (Life Technologies) and amino-allyl modified dUTP (Sigma), followed by labeling with fluorescent dyes Cy3 or Cy5 (Amersham). Labeled products were purified with QIAquick PCR Purification Kit (Qiagen). The hybridizations were performed on glass slide microarrays Mouse NIA 15K (Keck Biotechnology Resource Lab, Yale University, New Haven, CT, USA) containing 15 000 mouse genes according to the manufacturer’s protocol. Microarray experiments were performed in quadruplicate with different mouse pairs and dye-reversed hybridizations. The chips were scanned using the GenePix 4000A scanner (Axon Instruments, Union City, CA, USA) and primary data were analyzed using the Genepix 3.0 software (Axon Instruments). Microarray data are available under the GEO accession numbers GSE1138 and GSE3436.
Data analysis
Primary data were flagged using four default parameters set in the Genepix 3.0 program. For further analysis, the data were imported into Excel (Microsoft) and normalized by the Median Centering Method. We performed the statistical analysis using a modified t-test implemented in SAM software (http://www-stat.stanford.edu/~tibs/SAM/) (Tusher et al. 2001). We defined differentially expressed genes at a 1% false discovery rate confidence level and a cutoff for differential expression equal to 1.5 for juvenile Spo11−/ −and 2.0 for adult Spo11−/ −, juvenile wild-type microarray experiments and for analysis of testis specificity. Gene clustering was performed with the K-means method and a hierarchical clustering package based on the Pearson correlation coefficient, using the average linkage of the log2 ratio (Eisen et al. 1998).
To functionally characterize genes, expressed early and late in spermatogenesis, we used the gene ontology mining tool eGOn (www.egon.com), a web-based tool for mapping microarray data onto the gene ontology structure. We performed the Target–Master test, where a given gene set (the target gene list) was compared with the master list, contained all the genes printed on the slide. The total number of genes in the target list divided by the total number of genes in the master list was called the overall proportion. The Target–Master test determined gene ontology (GO) classes where the proportion of the number of genes in a target list divided by the number of genes in the master list is different from the overall proportion with P<0.05. A two-sided one sample binomial test was implemented.
Quantitative RT-PCR
For the 3′-most 500 bp of the full-length mRNA sequence primer pairs were designed using the web-based program Primer version 3.0 (Rozen & Skaletsky 2000) to yield a short (120–150 bp) PCR fragment with a melting temperature of about 60 °C. For each reaction, 1 μl of diluted cDNA (4 μg/ml) was mixed with 300 pmol of each primer in 25 μl (final volume) of SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). RT-PCR was performed in an ABI 7000 (Applied Biosystems). All amplifications were run in triplicate.
Results and discussion
Expression profile of adult Spo11−/ −mouse testes
To investigate the effect of knocking out Spo11 on the expression of genes in testis, we performed a competitive microarray hybridization of total RNA samples from Spo11−/ −and wild-type mouse testis. Disruption of Spo11 gene results in a gross change of the gene expression profile from whole testis (Fig. 1). We found that about 4000 genes changed their expression in Spo11−/ −testes more than twofold (Fig. 1a). To validate the microarray data, we performed quantitative RT-PCR for selected genes (Fig. 1c). Differential expression ratios obtained by microarray analysis and quantitative RT-PCR were in qualitative agreement for all genes tested, although we found some quantitative differences.
The obvious dramatic changes in expression patterns between Spo11−/−and wild-type testis are due to differences in the cell composition of Spo11−/ −and wild-type testis. Wild-type testes of adult mice are heterogeneous and contain more than 10 cell types (Russell et al. 1990). The development of testis in Spo11−/ −mice is abnormal, because of the early meiotic arrest. Consequently, these testes are devoid of all the cell types past the arrest point including pachytene and secondary spermatocytes, spermatids and spermatozoa. However, they are relatively enriched in pre arrest germ cells (primordial germ cells, spermatogonia, early meiotic prophase spermatocytes) and somatic Leydig and Sertoli cells (de Rooij & de Boer 2003, Khil et al. 2004).
These developmental abnormalities of Spo11−/ −testes can be verified by analyzing the expression levels of cell type-specific genes. Recently, Shima et al.(2004) defined genes that are expressed predominantly in spermatogonia, pachytene spermatocytes and rounded spermatids on a genome-wide scale. We calculated the average expression levels of genes from each cell type in our experiments (Table 1) and found that genes expressed in spermatocytes and spermatids have more than twofold lower expression level in Spo11−/ −adult testis on average. On the other hand, spermatogonia-expressed genes are slightly enriched.
Spermatogenesis involves the development of spermatogonia into mature spermatozoa. In the mouse, this process starts at birth and is continuously re-initiated thereafter (Russell et al. 1990). Meiotic progression reaches approximately the stage of the block in Spo11−/ −mice at 15 days of age. In agreement with this developmental similarity, we found that the gene expression profiles of 15-day wild type and Spo11−/ −adult testis are very similar (Fig. 2, lanes S and W). The majority of the genes for those transcripts depleted in Spo11−/ −adult testis are also depleted in juvenile testis, and similarly enriched transcripts in Spo11−/ −are also enriched in juvenile testis. A cumulative analysis of genes expressed in individual cell populations of testis shows the same trends as for Spo11−/ −testis (Table 1). The average level of transcripts of genes expressed in pachytene spermatocytes and rounded spermatids was at least twofold less in Spo11−/ −and juvenile testis when compared to adult wild-type testis (Table 1). Despite the almost identical expression pattern of transcripts in Spo11−/ −and juvenile testis, we found some interesting differences. These differences are discussed in detail later.
Independent evidence for the conclusion that the development of testis reaches the stage of the block in Spo11 mice at approximately 15 days after birth, comes from an analysis of the time course of spermatogenesis (Schultz et al. 2003) (Fig. 2). We divided genes into nine clusters based on the similarity of their expression profiles as a function of time using K-means clustering. We found that transcripts that are more abundant in Spo11−/ −testis are associated with clusters of genes that reach their maximum of expression in testis before 14 days after birth (clusters 6, 7 and 8, see Fig. 2). The majority of transcripts depleted in Spo11−/ −testis increase their expression in testis on or after day 14 after birth (e.g. cluster 3).
Therefore, in our microarray analysis, we can consider genes whose transcripts are enriched in Spo11−/ −testes as genes expressed in spermatogonia and early spermatocytes, while genes, whose transcripts are depleted in Spo11−/ −testis are expressed later in meiosis and spermatogenesis. We call the first group of genes, ‘early’ genes and the second group ‘late’ genes.
Functional characterization of ‘early’ and ‘late’ genes in the expression profile of Spo11−/ −adult and wild-type juvenile testis
To characterize those genes expressed early or late in spermatogenesis and to integrate expression specificity with functional annotation, we performed gene ontology mining using the web-based tool eGOn (www.egon.com). Differentially expressed genes were divided into six clusters based on their regulation pattern in either Spo11−/ −or juvenile testes (Fig. 3). Cluster 1 includes genes, whose transcripts are enriched only in Spo11−/ −testes; cluster 2 – common early genes; cluster 3 – early genes only in 15 day wild-type testes; cluster 4 – genes, whose transcripts are depleted only in Spo11−/ −testes; cluster 5 – common late genes and cluster 6 – genes expressed late only in juvenile testes (see Fig. 3). Although gene expression patterns in adult Spo11−/ −and juvenile wild-type testis have high similarity, there is a profound difference observed in the expression level of early genes. This difference is due to the apoptosis of arrested spermatocytes in Spo11−/ −testes and the enrichment in Spo11−/ −testes of Leydig cells that are the primary producers of steroid hormones in males. Therefore, the largest cluster for early genes (genes, whose transcripts are enriched only in Spo11−/ −testis) is over-represented with GO classes, such as steroid biosynthesis, fatty acid metabolism, proteolysis and peptidolysis, response to external stimuli, immune response and cytolysis (Table 2, Table 4 which can be viewed at: http://www.reproduction-online.org/supplemental/). The first cluster contains main intracellular effectors of apoptosis: caspases 9 and 6, Gadd45 g (growth arrest and DNA-damage-inducible 45 gamma) and the members of the Bcl-2 family of proteins that promote the mitochondria-dependent apoptotic pathway (Green & Kroemer 2004) (Fig. 3, Table 2, Table 4, which can be viewed at: http://www.reproduction-online.org/supplemental/). The appearance of cytolysis and immune response among over-represented GO classes reflects the intensive apoptosis, which occurs in adult Spo11−/ −testes.
Both juvenile wild-type and adult Spo11−/ −testes are also enriched with spermatogonia cells that undergo several mitotic divisions before entering meiosis. Therefore, among common early genes (cluster 2), the following GO classes are over-represented: cell proliferation, cell cycle, regulation of cell cycle, cell motility and development (Table 2, Table 4, which can be viewed at: http://www.reproduction-online.org/supplemental/). Smc1l1 (structural maintenance of chromosomes 1-like 1) and cell-cycle regulators such as Cdk5 (cyclin-dependent kinase 5), Ccna2 (cyclin A2) and Anapc4, 7 (anaphase promoting complex subunit 4 and 7) are in this cluster. Consistent with the fact that the normal development of spermatogenesis is accompanied by DNA repair and apoptosis, and corresponding genes are induced before or in early stage of meiosis, we find in cluster 2 Msh2 (DNA mismatch repair protein), Rad21 (DBSs repair protein), Casp3 (apoptosis related cysteine protease or caspase 3) and Pdcd8 (programmed cell death 8- apoptosis-inducing factor).
Cluster 3 (early genes in 15 day wild-type testes only) show as a positive association with such GO classes as meiosis, cell proliferation, cell cycle and nuclear division. Some of the genes found in this cluster include Mlh3 (establishing and/or maintaining crossovers), Sycp1 (major structural protein of the central element in SC), Rad51 (eukaryotic RecA homolog); E2f6 (transcriptional regulation of various genes implicated in chromatin remodeling), Anapc1 (anaphase promoting complex subunit 1) and Dazl (translation regulator). The observation that these genes are highly expressed only in juvenile wild-type testis is consistent with the normal development of meiosis in juvenile wild-type testis (Table 4, which can be viewed at: http://www.reproduction-online.org/supplemental/). It is interesting to note that in contrast to Anapc4 and Anapc7, the Anapc1 (meiotic check point regulator) is induced only in 15-day wild-type testis and might be critical for the zygotene/ pachytene transition.
The difference between late genes in Spo11−/ −and juvenile testis is not as profound as the difference between early genes (Fig. 3). The majority of genes whose transcripts are depleted in both Spo11−/ −and juvenile testis belong to cluster 5 (common late genes). As expected, we found a positive association of genes from cluster 5 with processes that occur late in male meiosis and spermatogenesis. These GO classes include gametogenesis (Rad23b, Mea1, Zfp35, Tce1), meiosis (Tsga2, Hop2, Rpa1, Stag3), chromatin remodeling (Morf4l1, Brd6) and chromosome segregation (Ccnb2, Chc1, Cdc25A, Cdc25C) (Table 2, Table 4, which can be viewed at: http://www.reproduction-online.org/supplemental/).
The relatively small cluster 4 (gene transcripts depleted only in adult Spo11−/ −testis) is enriched with genes involved in mitotic cell cycle, covalent chromatin modification, transcription initiation and regulation of translational initiation (Table 2). We can speculate that the lack of expression of these genes was the consequence of the meiotic arrest and induction of apoptosis in Spo11−/ −testis. Interestingly, in this cluster, we find important meiotic recombination proteins involved in the processing of DSBs, such as Mre11 (a major DSB repair protein) and Brca2 (breast cancer 2). In a mutant Brca2−/ −mouse carrying a human BRCA2 transgene (the Brca2 null mutation is embryonic lethal for mice) spermatocytes undergo normal DSBs formation but fail to complete recombination and do not progress beyond zygotene (Sharan et al. 2004). In our study, the observed depletion of Brca2 could be a consequence of the absence of DSBs in Spo11 knockout testis.
The last cluster, cluster 6 contains late genes, whose transcripts are depleted only in 15-day wild-type testis and these genes are implicated in spermatogenesis, steroid biosynthesis and lipid metabolism. The presence of these last two categories reflects the incomplete establishment of hormone production in juvenile testis.
The majority of testis-specific genes are expressed in late stages of meiosis and spermatogenesis
To further characterize genes differentially expressed in Spo11−/ −adult testes, we determined the distribution of mouse testis-specific genes among early and late genes in the gene expression profiles of Spo11−/ −testes. We performed cDNA microarray analysis, where we compared gene expression in adult mouse testes to reference RNA. Reference RNA included samples of total RNA from 11 different mouse tissues. We classified genes as testis-specific, if their expression level in testes is at least twofold more than in reference RNA. K (K=9) means clustering of microarray data obtained from a comparison between Spo11−/ −and wild-type testes, and wild-type adult testes and reference RNA showed that 90% of testis-specific genes mapped to the cluster of genes expressed in the late stages of meiosis and spermatogenesis (Fig. 2, lanes S and T). This conclusion was also confirmed if we used the other criteria of testis specificity based on the abundance of clones in Unigene cDNA libraries and publicly available Affymetrix data of mouse gene expression in different tissues (Khil et al. 2004). We found only 10% of testis-specific genes in the ’early’ cluster of gene expression in Spo11 testis. In addition, GO analysis of testis-specific genes determined from a comparison of wild-type testes and reference RNA showed a statistically significant association with the same GO classes that were observed in cluster 5 (genes transcripts, depleted in both Spo11 −/ −adult and 15 days testis) (Fig. 2, Table 2).
The effect of Spo11 knockout on gene expression in the testes of juvenile mice
Previously, we found that the major effect of Spo11 knockout is the disruption of the normal development of testes. To focus on genes involved in meiosis I, we compared gene expression profiles of Spo11 −/ −and wild-type testes from 12- and 15-days mice. On day 12, most of wild-type spermatocytes (about 85%) are in leptotone/zygotene stages and at 15 days, they are entering pachytene (Goetz et al. 1984). Comparing Spo11−/ −and wild-type mice at this age may allow us to focus on genes expressed in early meiotic prophase I that are more clearly dependent on the formation of DSBs. These genes include those involved in meiotic recombination, homologous pairing, SC formation and meiotic checkpoints. The use of this relatively early time point helps to eliminate from the analysis, genes expressed in later stages of spermatogenesis and apoptotic genes that are induced in response to the meiotic arrest of Spo11−/ −spermatocytes.
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 be viewed at: http://www.reproduction-online.org/supplemental/). On day 15, we found that only 250 genes have changed their expression level more than 1.5-fold in Spo11−/ −compared to wild-type testis (Fig. 1b, Table 5 available online). First of all, this selection includes some genes critical for meiotic recombination (Table 3, Table 5 available online). Among them are Hop2, Brca2, Mnd1, FancG (Fanconi anemia (group G) (Yang et al. 2001, Petukhova et al. 2003, 2005, Chen et al. 2004, Gudmundsdottir & Ashworth 2004). According to a current model, Brca2 is involved in delivering Rad51 and Dmc1 to repair DSBs by initiating strand exchange (Gudmundsdottir & Ashworth 2004). In Spo11−/ −spermatocytes, programmed DSBs are not formed and this function of Brca2 would not be required. Indeed, we found a 1.5-fold depletion of Brca2 transcripts in juvenile Spo11−/ −testis. Furthermore, we observed a twofold decrease in the level of expression FancG transcripts in Spo11 −/ −spermatocytes. FancG protein is capable of binding to two separate sites in the Brca2 protein (Hussain et al. 2003). It is co-immunoprecipitated with Brca2 from human cells and co-localized in nuclear foci with both Brca2 and Rad51 following DNA damage with mitomycin C (Hussain et al. 2003). Without doubt, FancG protein also participates in meiosis because the FancG−/ −mice have germ cell defects and decreased fertility (Yang et al. 2001). One of the most interesting observations is a twofold decrease in the transcription level of Hop2, another abundant meiotic protein closely associated with the Dmc1 and Rad51 in meiosis (Petukhova et al. 2003, 2005, Chen et al. 2004). It has been shown in yeast and mice that the Mnd1/Hop2 heterodimeric protein complex is required for the homologous pairing of chromosomes during meiosis and that these proteins act by promoting Rad51 or Dmc1 activity during the formation of the first recombination intermediates in meiosis (Petukhova et al. 2003, 2005, Chen et al. 2004). The dramatic decrease in the expression of Hop2 in Spo11−/ −juvenile testes may be the reflection of the absence of DSBs and the lack of the need for Hop2 under these circumstances. We could not find a satisfactory explanation for increased transcription of Mnd1 in Spo11−/ −juvenile testes because the function of Mnd1, besides the role in forming a complex with Hop2 that stimulates Rad51 and Dmc1, is unclear.
Although Spo11−/ −spermatocytes fail to form SC between homologous chromosome (Baudat et al. 2000, Romanienko & Camerini-Otero 2000), we did not observe significant changes in the expression of genes encoding proteins that are part of the lateral and central elements in SC such as Smc1beta, Rec8, Stag3, Smc3, Fkbp6, Sycp1 and Sycp3 (Table 3). Therefore, our gene expression data support the conclusion of previous study that cohesin cores can form independently of DSB formation and homologous pairing in mice (James et al. 2002).
Spo11−/ −spermatocytes are arrested at zygotene in meiotic prophase I. Little is known about the mechanisms of cell cycle control and checkpoint prior to pachytene. It is interesting in this regard that we found that the meiosis-specific Ccnb2 (cyclin B2) and the Cks2 (CDC28 protein kinase regulatory subunit 2) are twofold downregulated in 15-day Spo11−/ −testis. The Cks2 modulates substrate recognition by a Cdk1 that is part of the meiotic cell cycle regulatory complex Ccnb2/Cdk1 (Patra & Dunphy 1998, Spruck et al. 2003, Murray 2004). Although in Cks2-deficient mice, meiosis arrests at metaphase I (Spruck et al. 2003) and Ccnb2−/ −mice are fertile (the function of cyclin B2 is probably rescued by cyclin B1 (Ccnb1) (Brandeis et al. 1998), the cyclin B2 and Cks2 may be key components of the machinery that controls meiotic progression from zygotene to pachytene. Although cyclin B1 can substitute cyclin B2 and the expression level of cyclin B1 did not change in 15-day Spo11 testes, still Ccnb1/Cdk1 complex can be compromised by downregulation of the expression of some genes. Among them, we found calmodulin, a gene that can affect cyclinB1/Cdk1 (also known as Cdc2) through its partner, the testis-specific heat-shock protein Hsp70-2 (Zhu et al. 1997, Moriya et al. 2004). Hsp-70-2 deficient mice exhibit failed meiosis (Dix et al. 1996) and it has been suggested that Hsp-70-2 functions as a molecular chaperone for the Ccnb1/Cdk1 (Cdc2) complex (Zhu et al. 1997).
Among other genes that might play an essential role in meiotic progression, we can emphasize Ddx4, the mouse homologue of the Drosophila gene Vasa, that controls the initiation of translation (Carrera et al. 2000) and those expression is a twofold depleted in Spo11−/ −juvenile testes (Table 5, which can be viewed at: http://www.reproduction-online.org/supplemental/). The mouse Vasa transcript is bound by Dazl (Reynolds et al. 2005), and knockouts of either Dazl or Ddx4 lead to meiotic arrest of mouse spermatocytes at a zygotene-like stage similar to that found in the Spo11−/ −knockout (Tanaka et al. 2000, Saunders et al. 2003).
Other promising candidates potentially involved in meiotic checkpoint, meiotic recombination and SC formation may be found among the 53 genes with unknown function differentially expressed in juvenile Spo11−/ −testis. Based on the expression data obtained from the GEO profile database (http://www.ncbi.nlm.nih.gov/geo/), we determined that the majority of unknown genes downregulated in young Spo11−/ −testis increase their expression on day 14 after birth (Table 6 which can be viewed online at: http://www.reproduction-online.org/supplemental/). We found that some unknown proteins contain conserved domains involved in DNA recombination, microtubule-based process and regulation of developmental pathways (Table 6 available online).
In summary, microarray analysis of gene expression in adult Spo11−/ −testes reveals that the Spo11 mutation causes dramatic changes in the gene expression pattern of Spo11−/ −mouse testis. Spo11 knockout activates meiotic arrest in zygotene spermatocytes and, as a consequence, the disruption of spermatogenesis and normal testis development. Spo11−/ −testes are enriched with spermatogonia and spermatocytes in early stages of meiosis. Therefore, Spo11−/ −testes are a useful model for studies of gene expression in spermatogonia and early spermatocytes. Using the GO mining tool eGOn, we determined the relevant biological processes for genes expressed early and late in spermatogenesis. A comparative analysis of gene expression in juvenile Spo11−/ −mouse testes showed a dramatic decrease in the expression level of genes involved in meiotic recombination, meiotic checkpoints, but an absence of significant changes in the expression of genes encoding proteins for SC. This analysis also revealed about 50 genes of unknown function specifically induced or repressed in Spo11−/ −testes. Such genes are very likely to be specifically involved in meiosis and spermatogenesis and to be affected by disruption of the Spo11 gene. Studies of these novel proteins may shed a new light on our understanding of the function of Spo11 protein in meiosis and may help to find more partners of Spo11 in mammals.
Average expression in spermatogonia, pachytene spermatocytes, and rounded spermatids. dpp; days post partum.
| Average signal ratio | ||||
|---|---|---|---|---|
| Type | Adult wild-type vs reference RNA | 15 dpp wild-type vs adult wild-type | Adult Spo11−/− vs adult wild-type | 15 dpp Spo11−/− 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 |
Statistically significant biological processes associated with early and late genes in spermatogenesis.
Microarray data for genes, implicated in meiosis.
| Average signal ratio | ||||
|---|---|---|---|---|
| ID | Name | 15 dpp wild-type vs adult wild-type | adult Spo11−/ −vs adult wild-type | 15 dpp Spo11−/ −vs 15 dpp wild-type |
| dpp; days post partum. | ||||
| 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 | SMC1β; | 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 |
Scatter plots of gene expression values in testis of adult (a) and juvenile (15 days) (b) 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 expression in Spo11−/ −and wild-type testis. Genes whose expression level was at least two 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 Spo11−/ −versus wild-type testis as determined by microarray analysis and quantitative RT-PCR.
Citation: Reproduction 132, 1; 10.1530/rep.1.00997
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 (K=9) means clustering of the normalized gene expression in the spermatogenesis time course and a hierarchical clustering of gene expression for 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.
Citation: Reproduction 132, 1; 10.1530/rep.1.00997
Heat map of genes, whose transcripts are depleted or enriched in adult Spo11−/ −and juvenile wild-type testis. We selected genes whose transcripts are 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.
Citation: Reproduction 132, 1; 10.1530/rep.1.00997
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