Proteomic Analysis of Proteins Involved in ... - ACS Publications

Jan 25, 2010 - (3) These processes are all important to enable mature sperm to fertilize an egg ..... of the acrosome involves many membrane-associate...
2 downloads 0 Views 5MB Size
Download PDF
Proteomic Analysis of Proteins Involved in Spermiogenesis in Mouse Xuejiang Guo,†,# Jian Shen,†,# Zhengrong Xia,† Rui Zhang,‡ Ping Zhang,† Chun Zhao,† Jun Xing,† Ling Chen,† Wen Chen,† Min Lin,† Ran Huo,† Bing Su,*,‡ Zuomin Zhou,*,† and Jiahao Sha† Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China, and State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming 650223, Yunnan, China Received August 17, 2009

Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testisexpressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility. Keywords: spermiogenesis • proteome • acrosome biogenesis • translational regulation

Introduction The development of mammalian sperm in the testis from spermatids to spermatozoa is called spermiogenesis. Once spermatocytes have completed meiosis, the haploid round spermatids undergo spermiogenesis to form spermatozoa. Spermiogenesis is a unique process in the mammalian body and involves formation of the acrosome with a large vesicle structure,1 condensation of the nucleus,2 generation of the sperm tail and removal of most of the cytoplasm as a residual body.3 These processes are all important to enable mature sperm to fertilize an egg outside the male body. The condensed nucleus can protect the paternal genome from nucleases or mutagens that are potentially present in the internal or external media. In addition, together with removal of the residual body, nuclear condensation can provide a more compact and hydrodynamic nucleus, which allows spermatozoa to move faster. * To whom correspondence should be addressed: Prof. Zuomin Zhou, Lab of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, China. E-mail: [email protected]; Tel & Fax: 86-25-86862908; Prof. Bing Su, State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming 650223, Yunnan, China. E-mail [email protected]; Tel & fax: 86-871-5193137. † Nanjing Medical University. # These authors contributed equally to the work. ‡ Kunming Institute of Zoology and Kunming Primate Research Center.

1246 Journal of Proteome Research 2010, 9, 1246–1256 Published on Web 01/25/2010

The spermatozoa with the most hydrodynamic nuclei would move fastest, and would be able to fertilize the oocyte first.4 The tail provides spermatozoa the force required to move forward, and the acrosome, an acidic secretory vesicle that contains hydrolytic enzymes, provides the enzymes required for spermatozoa to penetrate the zona pellucida around the oocyte.5 Although many studies of spermiogenesis have been performed, the detailed mechanisms of these processes are still unknown. In this regard, the profiling of proteins involved in spermiogenesis would be particularly useful for advanced studies. Although the protein profiles of spermatogonia, sperm, and different developmental stages of the testis have been studied in different mammalian species,6-12 the protein profile involving spermiogenesis is still not well-characterized. Using flow cytometry, we purified haploid male germ cells that covered the entire process of spermiogenesis from adult mouse testes, and constructed a large-scale mouse haploid proteome of 2116 proteins that were involved in spermiogenesis.

Materials and Methods Sorting and Preparation of Haploid Germ Cells. Briefly, the testes from adult ICR mice were decapsulated, and then disaggregated for 1 min into a single cell suspension using a Medimachine (BD Bioscience, San Jose, CA). The suspension 10.1021/pr900735k

 2010 American Chemical Society

Proteomic Analysis of Spermiogenesis Proteins in Mouse of testicular cells was filtered (300 µm pore size) and incubated with Hoechst H33342 (Sigma Chemical Co., St. Louis, MO) according to manufacturer’s instructions. The haploid germ cells were sorted and collected using a FACSVantageSE flow cytometry system (BD Biosciences). Sample Preparation. After the haploid germ cells had been sorted, protein lysates were prepared with a lysis buffer that contained 7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 65 mM dithiothreitol (DTT), and 1% (v/v) protease inhibitor cocktail. The extracted protein (∼300 µg) was separated by SDSPAGE and digested in-gel according to a previously published procedure.13 The only modification was that the gel was cut into 31 slices before cutting into gel particles measuring 1 mm3 for in-gel digestion. Capillary Reverse-Phase Liquid Chromatography (LC) and Mass Spectrometric Analysis. The trypsin-digested fractions were loaded sequentially onto a Michrom peptide CapTrap (MW 0.5-50 kDa, 0.5 × 2 mm; Michrom BioResources, Inc., Auburn, CA) at a flow rate of 60 µL/min with buffer A (see below). The trap column effluent was then applied to a reversephase microcapillary column (0.1 × 150 mm, packed with 5 µm 100 Å Magic C18 resin; Michrom Bioresources). The reversephase separation of peptides was performed using the following buffers: 5% acetonitrile (ACN) with 0.1% formic acid (buffer A) and 95% ACN with 0.1% formic acid (buffer B) using a 73-min gradient (5-45% B for 55 min, 90% B for 5 min, and 5% B for 13 min). Peptide analysis was performed using a hybrid linear ion trap (LTQ) Orbitrap mass spectrometer (ThermoFinnigan, San Jose, CA) coupled directly to an LC column and the same parameters as in the published procedure.13 Settings for Peak List File Generation and Database Searches. The SEQUEST analysis software (Bioworks version 3.3; ThermoFinnigan) was used to identify the peptide sequences in a mouse protein database that best matched the observed tandem mass spectrometry (MS/MS) spectra.14 Peak lists in DTA files were generated for each MS/MS spectrum with a minimum ion count of 8 from the raw data using default parameters (Bioworks version 3.3) for the peptide mass range of 400-8,000. The International Protein Index (IPI) mouse database (ipi.MOUSE.v3.31; Jul 14, 2007, downloaded from ftp.ebi.ac.uk/pub/databases/IPI), which contained 56 555 entries, was used for SEQUEST searching. Peptide (parent ion) tolerance of 10 ppm, fragment ion tolerance of 1 Da and 2 missed cleavages for trypsin were allowed, and carbamidomethylation on Cys (+57 Da) was set as a fixed modification and oxidation on Met (+16 Da) as differential modification. The following criteria were used for filtering peptides with low confidence scores: cross-correlation values (Xcorr) greater than 2.49 and 3.0 were used for doubly charged ions and triply- or higher charged ions, respectively; ∆Cn values (difference in Xcorr with the next highest value) of less than 0.1 were removed from the matched sequences. The false-positive rate (FPR) was calculated by analyzing all files by the same method, but against a “sequence-reversed” IPI Mouse database. Annotation of Proteins in the Haploid Proteome. For bioinformatics analysis, each IPI accession number was converted to an Entrez Gene ID according to the IPI Protein CrossReferences file from ftp://ftp.ebi.ac.uk/pub/databases/IPI. Only proteins that mapped to one unique Entrez Gene ID were used for further bioinformatics analysis. All Entrez Gene IDs were loaded onto the Database for Annotation, Visualization and Integrated Discovery (DAVID)15 to identify the enriched bio-

research articles logical themes, including Gene Ontology terms, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. To find the relationships between proteins and cellular processes, the Entrez Gene IDs were imported into Pathway Studio 5.0 (Ariadne Genomics, Inc., Rockville, MD) using the ResNet Mammalian Database. A gene that encoded an identified protein was considered to be a retrogene if it satisfied the published criteria.16 Proteins were considered to function in cilia if they had orthologs in human cilia (default E-value, reciprocal best hits) according to Ciliaproteome Database 2.0 (http://www.ciliaproteome. org/).17 Mouse knockout data were downloaded from the Mouse Genome Informatics (MGI) database (MGI_4.1, http:// www.informatics.jax.org/). Chromosome Distribution Analysis of the Haploid Proteome. The chromosomal distribution of genes that encoded the identified proteins in the mouse haploid proteome was compared with the known distribution of genes on mouse chromosomes using a standard χ2 contingency table, according to a previously published method.18 Paralogs were batchdownloaded from Ensembl 48 using BioMart (http://www. ensembl.org/biomart/martview/). Gene Expression Data Sets. The data for mRNA expression across tissues were obtained from data compiled by Smith et al.19 or Unigene’s EST expression profiles from the UniGene’s EST ProfileViewer (http://www.ncbi.nlm.nih.gov/UniGene). Mouse genes that are differentially expressed in testis (designated as DET in the BioMart query form) according to Chalmel et al.’s transcriptome data for mouse gametogenesis were downloaded from BioMart of GermOnline. Genes highly expressed in spermatogonia, spermatocytes, and spermatids were from the mitotic cluster, meiotic cluster, and postmeiotic cluster of DET genes, respectively.20 Indirect Immunofluorescence. Testis tissue was embedded in optimal cutting temperature (OCT) compound, cryosectioned into 5-µm thick sections using a Leica Model CM1900 cryostat (Leica Microsystems, Wetzlar, Germany), mounted on slides, and left to dry before fixation. Single testicular cell suspensions prepared with the Medimachine (BD Bioscience) or sperm from the cauda epididymis were air-dried onto slides. The prepared slides were fixed with 4% formaldehyde/ phosphate buffered saline (PBS) for 30 min, washed three times with PBS for 5 min, and then blocked with horse serum (Beijing ZhongShan Biotechnology Co., Beijing, China) for 2 h at room temperature. Following incubation with primary antibodies overnight at 4 °C, the cells were incubated with secondary antibody labeled with fluorescein isothiocyanate (FITC; Beijing ZhongShan Biotechnology Co.) at a dilution of 1:200 for 1 h at room temperature. The negative controls were incubated without the primary antibodies, but otherwise the same. The primary antibodies used were anti-VAMP4 (Abcam, Cambridge, U.K.), anti-PRDX6 (Novus Biologicals, Littleton, CO), anti-XPO1 (Santa Cruz Biotechnology), anti-KIF3A (Abcam), and antiDDX4 (Abcam). The nuclei were stained with 5 µg/mL Hoechst H33342 (Sigma) for 30 s. For acrosomal staining, the fixed sperm were incubated in 30 µg/mL of FITC-labeled Pisum sativum agglutinin (PSA) (Sigma) for 1 h21 and then the indirect immunofluorescence was performed as described above. Western Blot Analysis. Samples that contained 100 µg of protein from adult mouse testes were electrophoresed and transferred to a nitrocellulose membrane (Amersham Biosciences, Uppsala, Sweden). The membranes were blocked and then incubated overnight with antibodies against VAMP4, Journal of Proteome Research • Vol. 9, No. 3, 2010 1247

research articles

Guo et al.

Figure 1. Immunofluorescence of purified mouse haploid germ cells. Haploid germ cells purified by flow cytometry were stained with Hoechst (blue) only (A), or with both Hoechst (blue) and anti-DDX4 antibody (green) (B). DDX4 is a germ cell-specific protein and is only localized in the cytoplasm of spermatocytes and spermatids in testis. Strong staining with anti-DDX4 antibody was seen in almost all cells except some elongated spermatids with little cytoplasm. All cells observed were small in size and were haploid germ cells.

PRDX6, XPO1, KIF3A. They were washed and then incubated for 1 h with horseradish peroxidase (HRP)-conjugated antirabbit IgG (diluted 1:2000; Beijing ZhongShan Biotechnology Co.). Specific proteins were detected using an ECL kit and AlphaImager (Amersham). Intratesticular Injection of Vamp4 Small Interfering RNA (siRNA). siRNAs against Vamp4 mRNA (Invitrogen, Carlsbad, CA; Catalog No. MSS244064, MSS244065, and MSS244066) were purchased, diluted to a final concentration of 20 µM, and stored at -20 °C. The efficacies of the three siRNAs were verified using the GC2 cell line (ATCC catalog number CRL-2196) as per the manufacturer’s instructions, and the one with the highest efficacy (MSS244065) was used for in vivo studies. Approximately 3-5 µL of individual siRNA was injected into the seminiferous tubules of one testis of a 3-week-old ICR mouse using the injection procedure described previously.22 Trypan blue (0.4%) was used as an indicator to ensure that the microinjection was successful. The other testis of the same mouse was injected with Neg Control siRNA (Invitrogen; Catalog No. 12935-400). Transmission Electron Microscopy. For ultrastructural examination, tissue blocks of testis or cauda epididymis, or spermatozoa from the cauda epididymis were postfixed with 2% OsO4 and embedded in Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate and inspected using an electron microscope (JEM.1010; JEOL, Tokyo, Japan).

Results Peptide and Protein Identification. After the mouse testis cells had been sorted by flow cytometry, we confirmed that the purity of sorted haploid germ cells was over 99% by counting 500 sorted cells under the microscope (Figure 1). We extracted and separated the protein by SDS-PAGE (Additional data file 1 in Supporting Information), digested it with trypsin and, following capillary reverse-phase liquid chromatography, obtained 131 145 MS/MS spectra from the tryptic peptides using a Finnigan LTQ Orbitrap hybrid mass spectrometer. For reliable proteomic identification, we selected the high-score peptide sequences with an FPR of 1%, and considered only proteins with a confidence score of not less than 95%, as calculated by the ProteinProphet algorithm using the Scaffold software (v01_07_00; Proteome Software, Portland, OR).23 Redundant proteins or protein isoforms that could not be differentiated from each other on the basis of MS/MS data were presented as a unique protein group. As a result, 2116 unique 1248

Journal of Proteome Research • Vol. 9, No. 3, 2010

proteins were identified successfully. Of the identified proteins, 1926 could be mapped to unique genes. Two genes have more than one protein isoforms unambiguously identified, and finally, 1924 unique genes were identified. The list of 2116 proteins identified in the present study, with confidence scores, is presented in Additional data file 2 in Supporting Information and the detailed sequence information of the peptides acquired by MS/MS is presented in Additional data file 3. Single peptidebased identification data are presented in Additional data files 4-7. All the mass spectrometry data for identified proteins were deposited in the PRoteomics IDEntifications database24 (PRIDE; http://www.ebi.ac.uk/pride/). Pathway and Gene Ontology Annotation of the Haploid Germ Cell Proteome. To characterize the proteome, we first analyzed the KEGG pathway annotation of the proteins using DAVID,15 and found that the haploid proteome contained all the 31 proteins in the 26S proteasome pathway in mouse. Pathway annotation of the haploid proteome by Pathway Studio revealed that 84 proteins were annotated to vesiclerelated events (Figure 2A). Each identified protein was assigned a subcellular localization by DAVID.15 When an individual protein was known to be localized in more than one cellular compartment, all of the localizations were counted nonexclusively. Figure 2B shows the cellular distribution of the identified proteins. As shown in the figure, the largest proportion of proteins identified was annotated as cytoplasm (982), followed by nucleus (515), membrane (467) and unknown localization (459). Some proteins were localized in subcellular structures such as the mitochondrion (227), endoplasmic reticulum (114), Golgi apparatus (84), and lysosome (20; Additional data file 8 in Supporting Information). Biological process analysis of the proteome using DAVID showed that many processes relevant to spermiogenesis were overrepresented, some of which are translation (95), nucleocytoplasmic transport (39) and chromatin assembly or disassembly (22). In addition, it was unsurprising that there was a significant enrichment of proteins involved in the process of male gamete generation (48) (see Figure 2C and Additional data file 8). Comparison with the Human Cilia Proteome. According to Ciliaproteome Database 2.0 (http://www.ciliaproteome. org/), 231 proteins from our proteome have orthologs in human cilia17 (Additional data file 9 in Supporting Information). The MGI database contains information on knockouts for 46 of

Proteomic Analysis of Spermiogenesis Proteins in Mouse

research articles

Figure 2. Functional annotations of the mouse haploid proteome. The vesicle-related processes regulated by mouse haploid proteins (A) were generated by Pathway Studio software. Regulatory events are denoted by arrows and documented by literature citations (see Additional data file 11 in Supporting Information). The pie chart of subcellular localizations (B) and the bar graph of enriched biological processes (C) were based on gene ontology annotations by DAVID.

these. Among the 12 genes not annotated as causing embryonic, perinatal or postnatal lethality when knocked out, 4 genes (33%) can affect sperm flagella and influence male reproduction: Tekt2, Spag6, Spag16 and Ppp1cc. These ciliary proteins may also have important functions in the formation and/or function of the sperm tail, given the similar structure of the cilium and the sperm flagellum, which both have a typical “9 + 2” array of microtubules. Testis Specific Genes. Smith et al.19 found that 1 119 genes (of the 32 496 genes in the mouse genome in total according to the NCBI Entrez Gene database) showed evidence of specificity in the mouse testis. We identified 299 testis-specific genes (among the total of 1924 uniquely identified genes) in our haploid proteome (Additional data file 10 in Supporting Information). Statistical analysis showed a very significant enrichment of testis-specific genes in the proteome compared with the whole genome distribution (p ) 2.3 × 10-148 for χ2 test). Immunolocalization of Proteins from the Haploid Proteome. To better characterize the proteins annotated to spermiogenesis, we studied the localizations of a protein annotated to vesicle-related events, VAMP4. VAMP4 is a soluble Nethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, which can mediate membrane interactions.

In elongated spermatids, VAMP4 was localized in the acrosomal region around the nucleus,, whereas in the mature spermatozoa, it was localized mainly in the acrosome region with a faint signal in the tail region (Figure 3A). In addition, PRDX6, a lysosomal protein, was also shown to be localized in the acrosomal region around the nucleus of elongated spermatids and in the acrosome of the mature spermatozoa (Figure 3B). This is consistent with the hypothesis that there is a molecular link between the machinery involved in lysosome and acrosome biogenesis because many of the enzymes in the acrosome are associated with the lysosome in somatic cells.1 Immunofluorescence studies also showed that the ciliary protein, KIF3A, was localized to the developing tail of spermatids, and was located finally in the principle segment of the tail in mature spermatozoa (Figure 3C). This implied a possible function for KIF3A in sperm tail formation and/or function. In addition, XPO1, a protein annotated by gene ontology to function in nucleocytoplasmic transport, was shown to undergo nucleocytoplasmic translocalization during spermiogenesis (Figure 3D). The expression of XPO1 decreased during spermiogenesis and it was undetectable in spermatozoa. In Vivo Study of the Function of VAMP4 in Spermiogenesis by Intratestiscular Injection of siRNA against Vamp4. To further investigate the role of vesicle-related proteins in sperJournal of Proteome Research • Vol. 9, No. 3, 2010 1249

research articles

Guo et al.

Figure 3. Changes of localization of proteins during spermiogenesis by immunofluorescence. The localization of VAMP4 (A), PRDX6 (B), KIF3A (C), and XPO1 (D) during different stages of spermiogenesis in the mouse testis was visualized by immunofluorescence using specific FITC-labeled antibodies (green). Immunofluorescence with secondary antibody only was used as control (E). The nuclei were stained with Hoechst (blue), and schematic representations of the developing haploid germ cells during the stages of spermiogenesis are shown on the left. The specificity of the antibodies used was demonstrated by Western blotting using mouse testis protein (F).

miogenesis, mice seminiferous tubules were injected with siRNA against Vamp4, which introduced siRNA into approximately 30-40% of the seminiferous tubules (Figure 4A) without causing testis tissue damage. Double-stranded Stealth siRNAs against Vamp4 were purchased from Invitrogen. The siRNA that gave the highest level of protein inhibition in the GC2 cell line after 72 h was used for subsequent analysis. siRNAs has been shown to be effective for in vivo studies without the use of electroporation or liposome.25,26 After intratestiscular injection of siRNA against Vamp4, the VAMP4 protein was observed to be suppressed significantly in the testis as early as 24 h after injection, and it was consistently suppressed at 48 and 72 h after injection (Figure 4B,C). Immunofluorescence also showed that seminiferous tubules injected with siRNA (traced by trypan blue) had low levels of 1250

Journal of Proteome Research • Vol. 9, No. 3, 2010

VAMP4 in spermatids, whereas tubules in the same testis that did not contain siRNA had high levels of VAMP4 (Figure 4D-K). Equal amount of siRNA against Vamp4 and control siRNA were separately injected into each of the paired testes from each mouse under the same conditions. And the siRNA can only be introduced into 30-40% of testicular tubules without causing testis tissue damage. Abnormalities in sperm from the cauda epididymis were counted 3 weeks after the injection; the proportion of head abnormalities was increased markedly to 23.7% in these sperm, which was significantly higher than that in the negative control siRNA group (10.6%) (p < 0.01) (Figure 5A-D). Immunofluorescence studies of the abnormal sperm from the cauda epididymis showed various head abnormalities. In the sperm with abnormal heads, the acrosome was also shown to be abnormal (Figure 5F-J). In some sperm, the

Proteomic Analysis of Spermiogenesis Proteins in Mouse

research articles

Figure 4. RNAi-mediated knockdown of VAMP4 mRNA. The testes of 3-week-old mice were injected with siRNA against Vamp4 and trypan blue (red) was used to trace the seminiferous tubules into which the siRNA was injected (A). As indicated in the figure, 30-40% of the seminiferous tubules was injected. The abundance of VAMP4 protein was assayed by Western blotting (B), the data represent an average of triplicate experiments, and values are expressed as fold changes relative to the negative control (mean ( standard deviation) (C) at 24, 48, or 72 h after injection. The tubulin was used as internal control. Immunofluorescence studies were performed on the testes 2 days after injection. The seminiferous tubules that had been injected with Vamp4 or negative control siRNA were traced by trypan blue (red). In the tubule injected with siRNA against Vamp4, the level of VAMP4 protein in spermatids was significantly decreased compared with that without siRNA in the same testis (D-G). However, in the testis injected with negative control siRNA, the VAMP4 signal remained strong in spermatids in injected tubules (H-K). And the VAMP4 signal in spermatids in tubules injected with siRNA against Vamp4 (shown by dotted circle) was significantly lower than in those treated by control siRNA (shown by dotted circle). The seminiferous tubules with spermatids are marked with asterisks.

acrosome was even located in the rear region of the sperm head (Figure 5H). Transmission electron microscopy studies performed 2 weeks after siRNA treatment showed abnormal acrosome formation of acrosomes in the round spermatid with discrete unfused small acrosomal vesicles around the nucleus and accumulation of many vesicles in the cytoplasm (Figure 6B). During elongation, abnormal acrosomes were usually observed in the spermatids together with misshapen heads (Figure 6C-F). In the cauda epididymis, normal sperm have an elongated condensed nucleus and one intact acrosome that covers the nucleus. However, in the cauda epididymis that corresponded to the testis treated with siRNA against Vamp4, sperm with misshapen nucleus with an abnormal acrosome, fragmented discrete acrosome vesicles, or no acrosome were observed (Figure 6H-L). Many small vesicles could be observed in sperm heads that did not contain an acrosome (Figure 6I). The acrosomal abnormalities imply that of the vesicles do not fuse successfully during acrosome formation. Chromosome Distribution Analysis. We mapped all 1924 uniquely identified genes onto the polytene chromosomes and

observed the following distribution: X chromosome, 30 genes; chromosome 11, 190 genes; chromosome 7, 142 genes; chromosome 4, 133 genes. χ2 analysis of this distribution versus that of all chromosomal genes revealed significant deviations (p ) 8.49 × 10-17) (Figure 7). The number of genes identified on the X chromosome deviated most from the expected number. In addition, a significant difference was observed when we compared the proportion of X-to-autosomal haploid genes with the genomic ratio (p ) 4.21 × 10-14). We removed the genes on the X chromosome from the analysis and compared the distributions among the autosomes. We observed a nonrandom distribution of genes in the haploid proteome across the autosomes (p ) 1.29 × 10-6); the number of genes on chromosome 11 deviated most from the expected number. A significant difference was observed when we compared the proportion of haploid genes on chromosome 11 with that on all autosomes (p ) 1.33 × 10-7). Therefore, haploid genes were overrepresented on chromosome 11. By iteration, we also found that haploid genes were underrepresented on chromosomes 6 and 7. The distribution of haploid proteome Journal of Proteome Research • Vol. 9, No. 3, 2010 1251

research articles

Figure 5. Effect of in vivo RNAi against Vamp4 on sperm morphology in the cauda epididymis. Paired testes from the same 3-week-old mouse were injected with siRNA against Vamp4 and negative control siRNA, respectively. Different types of abnormality of sperm in the cauda epididymis were counted 3 weeks later under the microscope and compared (A). The ratios were subjected to arcsine square root transformation prior to Student’s t test. Significant differences are indicated by * (p < 0.05) or ** (p < 0.01). Data are expressed as the mean ( SD. The shapes of sperm from the cauda epididymis are shown for mouse testes that were untreated (B), treated with negative control siRNA (C), and treated with siRNA against Vamp4 (D). Sperm with head abnormalities are indicated with arrows. Fluorescent staining of the acrosome with PSA (green) and nucleus with Hoechst (blue) is shown for normal sperm (E) and sperm with abnormal heads (F-J). The sperm with abnormal heads contained an abnormal acrosome.

across chromosomes was only similar to the genomic distribution after the genes on these four chromosomes had been removed from the analysis (p ) 0.09). Chromosome 11 is the only chromosome on which mouse haploid genes are overrepresented. To identify the reason, we divided the genes into two groups, paralogs and nonparalogs, and found no significant difference for haploid nonparalogs between chromosome 11 and the other chromosomes when compared with the total genomic distribution (p > 0.05). However, when the paralogs were compared, the difference was highly significant (p ) 2.88 × 10-5). Therefore, the overrepresentation of the haploid proteome on chromosome 11 is caused primarily by an overrepresentation of testis-expressed paralogs which are formed by gene duplications on chromosome 11. Retrogene Analysis. Retrogenes are a class of functional genes generated by retrotransposition of the parental genes. Using the same criteria as Shiao et al.’s approach,16 we identified five retrogenes in our haploid proteome (Table 1), and demonstrated that they were well-differentiated from their 1252

Journal of Proteome Research • Vol. 9, No. 3, 2010

Guo et al. parental genes by identifying their unique peptides. All five genes were testis-specific according to Unigene’s EST expression profiles, and all were located on the autosomes. Three of them (67%) were derived from parental genes on X chromosome. None of their parental genes were testis-specific. Comparison with Gene Expression Data. To determine whether the haploid proteome had a high coverage of genes that are expressed highly in haploid germ cells, we compared the proteome data with microarray data. Among genes in the mitotic cluster (spermatogonia; 2585 Ensembl Genes), meiotic cluster (spermatocytes; 1583 Ensembl Genes), and postmeiotic cluster (spermatids; 1348 Ensembl Genes) identified by Chalmelet al.’s analysis of the transcriptome during mouse gametogenesis, we identified the proteins encoded by 307 (11.9%), 356 (22.5%) and 160 (11.9%) of the genes, respectively (Figure 8). To our surprise, the proportion of genes from the haploid proteome in postmeiotic cluster was significantly less than that in the meiotic cluster (p ) 5.34 × 10-14, χ2 test), and similar to that in the mitotic cluster (p ) 0.995, χ2 test). To test the possibility that genes in the postmeiotic cluster are expressed at a lower absolute level in the haploid germ cells than that of genes from the other two clusters, we compared their absolute levels of mRNA expression in spermatids..We found that genes from the postmeiotic cluster were expressed at a significantly higher level when compared with those from the mitotic cluster and meiotic clusters (p ) 5.6e-16 and p , 1 × 10-16, respectively; Wilcoxon-Mann-Whitney statistical test). In addition, the mean level of expression of genes in the postmeiotic cluster was 16 times greater than that for the mitotic cluster. The low coverage of genes from the postmeiotic cluster in the haploid proteome is not consistent with the assumption that a gene is highly expressed during the stage that it functions. This assumption is the basis of prediction of gene function by microarray analysis.

Discussion To help characterize the process of spermiogenesis, we constructed a large-scale proteome of 2116 proteins for spermiogenesis, of which 299 were testis-specific. In addition, we identified all 31 proteins from the 26S proteasome pathway annotated in KEGG database and provided the evidence of the full functional 26S proteasome pathway during spermioenesis. This large-scale proteome could provide a rich resource for the study of spermiogenesis. During spermiogenesis, biogenesis of the acrosome involves many membrane-associated activities such as the generation of proacrosomal granules (PAGs) from trans-Golgi stacks and fusion of these PAGs to form a single large acrosome that associates with the nuclear envelope.27 Although the morphologic changes of these vesicle events have been studied in detail, the molecular basis is still largely unknown as yet.28 The fusion of individual PAGs to one acrosome vesicle may use the common cytosolic fusion machinery, which involves the formation of complexes of v- and t-SNAREs.1,28 Of the 84 proteins involved in vesicle-related events in the haploid proteome, a new v-SNARE protein, VAMP4, was identified. In somatic cells, VAMP4 is a v-SNARE protein associated with the trans-Golgi network.29,30 PAGs were generated from trans-Golgi network during spermiogenesis. So VAMP4 may play an important function in the generation of acrosome. Our immunolocalization studies of VAMP4 showed that it was localized in the acrosomal region of elongated spermatids and sperm. Using an in vivo RNAi-mediated knockdown mouse

Proteomic Analysis of Spermiogenesis Proteins in Mouse

research articles

Figure 6. Transmission electron micrographs of spermatids from testis and sperm from the cauda epididymis. Transmission electron micrographs of spermatids from mouse testis 2 weeks after siRNA injection (B-F). Round spermatid with a flat, cap-like acrosome in normal testis (A). Discrete proacrosomic vesicles (arrow heads) were seen around the nuclear envelope and many vesicles were observed in the cytoplasm of round spermatid (B). Misshapen heads were observed in spermatids from the testes treated with siRNA against Vamp4 (C-F). Transmission electron micrographs of sperm from the cauda epididymis 3 weeks after injection with siRNA (G-L). Normal sperm have an elongated condensed head. However, abnormally shaped sperm heads were observed in mice treated with siRNA against Vamp4. Nuclei without a surrounding acrosome (H) or with many small vesicles (arrowhead) (I) were seen in the sperm. In addition, sperm with several discrete acrosomal vesicles were also observed (J, K). Even in sperm with only one acrosomal vesicle, the acrosome sometimes had a nearly ovoid structure, rather than its normal cap-like shape (L). Abbreviations are as follows: Nu, nucleus; Ac, acrosome.

model, we found the first direct evidence for the important role of SNARE proteins in acrosome formation. In mice with suppressed VAMP4 protein, abnormalities of acrosomal vesicle fusion and of the nucleus were seen in spermatids and sperm. Microscopically, there was evidence of increased levels of abnormality of sperm heads. The malformed acrosomes may fail to correctly withstand the exogenous morphogenic clutch force exerted through the apical ectoplasmic specializations and thus contribute to the abnormal nuclear morphogenesis.31 Indepth study of the 84 proteins annotated to vesicle-related

events may help us to elucidate the molecular pathway of acrosome biogenesis, and to characterize the cause of globozoospermia, a severe disease that involves abnormal acrosome biogenesis and causes male infertility. The compact nucleus of sperm is formed during spermiogenesis by chromatin remodeling and nuclear condensation. In the haploid proteome, 22 proteins were annotated to function in chromatin assembly or disassembly and may be involved in chromatin remodeling. In addition, 39 proteins were annotated to be involved in nucleocytoplasmic transport. Journal of Proteome Research • Vol. 9, No. 3, 2010 1253

research articles

Guo et al.

Figure 7. Observed and expected number of genes in the haploid proteome for each mouse chromosome. The chromosome annotations of the mouse haploid proteome were from NCBI Build 37.1 (Sep 12, 2007). The two chromosomes that showed the most deviation from the expected values were the X chromosome and chromosome 11.

During nuclear condensation, the nucleus shrinks in size, and many proteins are “squeezed” out of the nucleus. For example, Deleted in Azoospermia Associated Protein 1 (DAZAP1) translocates from the nucleus to the cytoplasm during spermatid elongation.32 Immunofluorescence studies showed that XPO1, an essential mediator of the nuclear export signal-dependent nuclear export of proteins in eukaryotic cells,33 actively moved out of the nucleus during nucleus condensation and may help export proteins into the cytoplasm to shrink the nucleus in volume. The complex processes of nuclear condensation and chromatin remodeling are important for spermiogenesis, and disruption of these processes may generate abnormal sperm, such as macrocephalous sperm without a condensed nucleus. The flagella of sperm have a similar structure to cilia. A total of 231 proteins in the haploid proteome were found expressed in human cilia. Of the proteins that did not cause lethality before sexual maturation when knocked out, 33% could affect sperm flagella and influence male reproduction. In addition, Kif3a, a subunit of the Kinesin II motor complex required for intraflagellar transport and the formation of cilia,34 was immunolocalized to the developing tail and the principle segment of the mature sperm tail. Detailed studies of these ciliary proteins will improve our understanding of the biogenesis and function of the sperm tail and enable the mechanisms of asthenozoospermia to be characterized. Although many proteins in the haploid proteome were of known functions, there were also many novel proteins. With respect to the genes that correspond to the identified proteins, 135 were identified by Riken Clone Identifier such as “1700029i08rik”, which shows that they correspond to the only sequencing information available, and 20 have gene symbols that begin with “LOC”, which shows that they were discovered by prediction. These novel proteins could fulfill as yet uncharacter-

Figure 8. Comparison of the mouse haploid proteome with transcriptional data for gametogenesis. The boxplot of mRNA expression values (A) in spermatids shows mouse genes in the mitotic, meiotic, and postmeiotic clusters described by Chalmel et al.20 The corresponding percentages of genes identified in the mouse haploid proteome are shown in panel B.

ized functions in the testis and study of these genes could improve our understanding of the mechanisms of spermiogenesis. Analysis of chromosomal distribution of the haploid proteome showed a nonrandom distribution across the chromosomes. The two chromosomes that deviated most significantly from the expected distribution were the X chromosome and chromosome 11. Chromosome 11 is the only chromosome on which haploid genes are overrepresented. A previous large-scale study of mouse mutations, using the point mutagen N-ethylN-nitrosourea (ENU), showed that many genes important for male fertility are located on chromosome 11. In that study, mutations in 6 out of 33 genes (18%) on chromosome 11, which cause phenotypes but are not lethal, can cause male infertility.35 By classifying genes as paralogs and nonparalogs, we found that overrepresentation of haploid genes on chromosome 11 was primarily due to the overrepresentation of paralogs on this chromosome. Paralogs are genes formed by duplication during evolution. It seems that on chromosome 11, testis-expressed genes families have expanded by this process. Mutations on chromosome 11 may be prone to cause male fertility problems. The X chromosome is inactivated during meiosis due to meiotic sex chromosome inactivation, it retains a repressed state during spermiogenesis,36 and it was shown to repress most of X-linked genes on mRNA levels in mammals.37 Our study of the functional forms of genes, proteins, showed that the effect also existed on the protein level in an omics scale in mammals. And it provides the advanced evidence for the tendency of some genes to escape from X chromosome to autosomes as retrogenes to maintain their function during spermiogenesis in spite of X chromosome repression.16 In the haploid proteome, three retrogenes were identified whose parental genes were located on the X chromosome. The retrogenes were all located on autosomes and expressed in a testis-specific manner, whereas the products of the three

Table 1. Proteins Encoded by Retrogenes Identified in the Mouse Haploid Proteomea IPI accession

retrogene name

retrogene Ensembl ID

chr

parental gene Ensembl ID

parental gene name

chr

IPI00121288 IPI00555060 IPI00133708 IPI00116929 IPI00403905

4930521a18rik pgk2 d1pas1 Pabpc3 kif2b

ENSMUSG00000026063 ENSMUSG00000031233 ENSMUSG00000039224 ENSMUSG00000046173 ENSMUSG00000046755

1 17 1 17 11

ENSMUSG00000042433 ENSMUSG00000062070 ENSMUSG00000000787 ENSMUSG00000022283 ENSMUSG00000021693

E230019M04Rik Pgk1 Ddx3x Pabpc1 Kif2a

X X X 15 13

a Proteins were separated by one-dimensional gel electrophoresis and identified by LC-MS/MS using mouse male haploid germ cells purified by flow cytometry.

1254

Journal of Proteome Research • Vol. 9, No. 3, 2010

research articles

Proteomic Analysis of Spermiogenesis Proteins in Mouse parental X-linked genes were not detected in the proteome. Pgk2 is such a retrogene. Its parental gene, Pgk1, encodes an enzyme of the glycolytic pathway, and therefore is required in all metabolically active cell types for the production of ATP. Pgk1 transcription is repressed after X-chromosome inactivation during spermatogenesis, whereas the testis-specific autosomal retrogene Pgk2 is expressed to replace the function of its parental gene.38 The protein level study of spermatogenesis has been less explored compared with transcriptional level ones.20,39 To determine the degree of consistency between transcriptome and the proteome, we compared the proteomics data with microarray data from enriched germ cells,20 and found significant inconsistencies. Genes that have been shown to be highly expressed in haploid germ cells by microarray studies were identified to a lesser degree in the haploid proteome. Although the comparison between protein and mRNA levels was based on different genes, the inconsistency between protein and mRNA levels may still reveal significant translational regulation of these genes. In the testis, some proteins have already been reported to be translationally regulated during spermatogenesis. For example, Mst87F is first transcribed in primary spermatocytes in Drosophila, but translation is not initiated until at least 3 days later when the spermatids have fully elongated.40 In addition, the premature translation of protamine mRNA can lead to failed spermatogenesis.41 However, these studies of translational regulation by the comparison of protein and mRNA levels were only performed on individual genes. The construction of large-scale haploid proteome allowed revealing translational regulation during spermiogenesis in an omics scale. Translational regulation may be required to timely control the complex morphogenesis of spermiogenesis. Therefore, to study the gene functions in testis more effectively, it is important to study the protein directly, and not only limit the analysis to transcriptional regulation.

Conclusions By characterizing the proteins that are involved in spermiogenesis, we found many proteins that possibly function in unique processes during spermiogenesis. The comparison of data on the proteome and transcriptome showed that significant translational regulation occurs during spermiogenesis, and emphasized the importance of studying spermatogenesis at the protein level. This large-scale proteome should provide a rich resource for the study of spermiogenesis, improve our understanding of the mechanisms that cause male infertility such as teratozoaspermia and asthenospermia, which are two major types of male infertility, and provide candidates for drug targets for male contraception and male infertility.

Acknowledgment. This study was supported by grants from the 973 program (2009CB941703, 2006CB504002), the Chinese Natural Science Funds (30630030), and the Program of Changjiang Scholars and Innovative Research Team in University (IRT0631). Supporting Information Available: Additional data file 1 shows the SDS-PAGE separation of mouse male haploid germ cell protein. Additional data file 2 is a table that shows the protein identification of the haploid proteome with confidence scores. Additional data file 3 is a table that shows all the peptide identifications for the haploid proteome. Additional data file 4 is a table that shows the single peptide-based protein identi-

fications. Additional data files 5-7 correspond to MS/MS spectra and fragment assignments of the single peptide-based identifications. Additional data file 8 is a table that shows the gene ontology annotation of proteins in the mouse haploid proteome. Additional data file 9 is a table of the mouse haploid proteins with orthologs in human cilia. Additional data file 10 is a table of the testis-specific proteins in the mouse haploid proteome. Additional data file 11 contains literature citations for vesicle-related processes regulated by mouse haploid proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Moreno, R. D.; Alvarado, C. P. The mammalian acrosome as a secretory lysosome: new and old evidence. Mol. Reprod. Dev. 2006, 73 (11), 1430–4143. (2) Toshimori, K.; Ito, C. Formation and organization of the mammalian sperm head. Arch. Histol. Cytol. 2003, 66 (5), 383–396. (3) Escalier, D. Knockout mouse models of sperm flagellum anomalies. Hum. Reprod. Update 2006, 12 (4), 449–461. (4) Oliva, R. Protamines and male infertility. Hum. Reprod. Update 2006, 12 (4), 417–435. (5) Abou-Haila, A.; Tulsiani, D. R. Mammalian sperm acrosome: formation, contents, and function. Arch. Biochem. Biophys. 2000, 379 (2), 173–182. (6) Guillaume, E.; Dupaix, A.; E, M.; Courtens, J. L.; Je´gou, B.; Pineau, C. Proteome analysis of spermatogonia: identification of a first set of 53 proteins. Proteome 2000, 1, 1–20. (7) Com, E.; Evrard, B.; Roepstorff, P.; Aubry, F.; Pineau, C. New insights into the rat spermatogonial proteome: identification of 156 additional proteins. Mol. Cell. Proteomics 2003, 2 (4), 248– 261. (8) Martinez-Heredia, J.; Estanyol, J. M.; Ballesca, J. L.; Oliva, R. Proteomic identification of human sperm proteins. Proteomics 2006, 6 (15), 4356–4369. (9) Baker, M. A.; Hetherington, L.; Reeves, G. M.; Aitken, R. J. The mouse sperm proteome characterized via IPG strip prefractionation and LC-MS/MS identification. Proteomics 2008, 8 (8), 1720– 1730. (10) Baker, M. A.; Hetherington, L.; Reeves, G.; Muller, J.; Aitken, R. J. The rat sperm proteome characterized via IPG strip prefractionation and LC-MS/MS identification. Proteomics 2008, 8 (11), 2312– 2321. (11) Huang, X. Y.; Guo, X. J.; Shen, J.; Wang, Y. F.; Chen, L.; Xie, J.; Wang, N. L.; Wang, F. Q.; Zhao, C.; Huo, R.; Lin, M.; Wang, X.; Zhou, Z. M.; Sha, J. H. Construction of a proteome profile and functional analysis of the proteins involved in the initiation of mouse spermatogenesis. J. Proteome Res. 2008, 7 (8), 3435–3446. (12) Cao, W.; Gerton, G. L.; Moss, S. B. Proteomic profiling of accessory structures from the mouse sperm flagellum. Mol. Cell. Proteomics 2006, 5 (5), 801–810. (13) Guo, X. J.; Zhang, P.; Huo, R.; Zhou, Z. M.; Sha, J. H. Analysis of the human testis proteome by mass spectrometry and bioinformatics. Proteomics: Clin. Appl. 2008, 2 (12), 1651–1657. (14) Eng, J. K.; Mccormack, A. L.; Yates, J. R. An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database. J. Am. Soc. Mass Spectrom. 1994, 5, 976– 989. (15) Dennis, G., Jr.; Sherman, B. T.; Hosack, D. A.; Yang, J.; Gao, W.; Lane, H. C.; Lempicki, R. A. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003, 4 (5), P3. (16) Shiao, M. S.; Khil, P.; Camerini-Otero, R. D.; Shiroishi, T.; Moriwaki, K.; Yu, H. T.; Long, M. Origins of new male germ-line functions from X-derived autosomal retrogenes in the mouse. Mol. Biol. Evol. 2007, 24 (10), 2242–2253. (17) Gherman, A.; Davis, E. E.; Katsanis, N. The ciliary proteome database: an integrated community resource for the genetic and functional dissection of cilia. Nat. Genet. 2006, 38 (9), 961–962. (18) Dorus, S.; Busby, S. A.; Gerike, U.; Shabanowitz, J.; Hunt, D. F.; Karr, T. L. Genomic and functional evolution of the Drosophila melanogaster sperm proteome. Nat. Genet. 2006, 38 (12), 1440– 1445. (19) Smith, A. D.; Sumazin, P.; Zhang, M. Q. Tissue-specific regulatory elements in mammalian promoters. Mol. Syst. Biol. 2007, 3, 73. (20) Chalmel, F.; Rolland, A. D.; Niederhauser-Wiederkehr, C.; Chung, S. S.; Demougin, P.; Gattiker, A.; Moore, J.; Patard, J. J.; Wolgemuth,

Journal of Proteome Research • Vol. 9, No. 3, 2010 1255

research articles

(21) (22)

(23) (24)

(25) (26) (27)

(28) (29)

(30)

1256

D. J.; Jegou, B.; Primig, M. The conserved transcriptome in human and rodent male gametogenesis. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (20), 8346–8351. Zhang, J.; Wu, J.; Huo, R.; Mao, Y.; Lu, Y.; Guo, X.; Liu, J.; Zhou, Z.; Huang, X.; Sha, J. ERp57 is a potential biomarker for human fertilization capability. Mol. Hum. Reprod. 2007, 13 (9), 633–639. Lu, L.; Lin, M.; Xu, M.; Zhou, Z. M.; Sha, J. H. Gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells. Asian J. Androl. 2006, 8 (1), 53–59. Nesvizhskii, A. I.; Keller, A.; Kolker, E.; Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 2003, 75 (17), 4646–4658. Jones, P.; Cote, R. G.; Cho, S. Y.; Klie, S.; Martens, L.; Quinn, A. F.; Thorneycroft, D.; Hermjakob, H. PRIDE: new developments and new datasets. Nucleic Acids Res. 2008, 36 (Database issue), D878– 883. Gonzalez-Gonzalez, E.; Lopez-Casas, P. P.; Del Mazo, J. Gene silencing by RNAi in mouse Sertoli cells. Reprod. Biol. Endocrinol. 2008, 6, 29. Aigner, A. Gene silencing through RNA interference (RNAi) in vivo: strategies based on the direct application of siRNAs. J. Biotechnol. 2006, 124 (1), 12–25. Yao, R.; Ito, C.; Natsume, Y.; Sugitani, Y.; Yamanaka, H.; Kuretake, S.; Yanagida, K.; Sato, A.; Toshimori, K.; Noda, T. Lack of acrosome formation in mice lacking a Golgi protein, GOPC. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (17), 11211–11216. Ramalho-Santos, J.; Schatten, G.; Moreno, R. D. Control of membrane fusion during spermiogenesis and the acrosome reaction. Biol. Reprod. 2002, 67 (4), 1043–1051. Zeng, Q.; Tran, T. T.; Tan, H. X.; Hong, W. The cytoplasmic domain of Vamp4 and Vamp5 is responsible for their correct subcellular targeting: the N-terminal extenSion of VAMP4 contains a dominant autonomous targeting signal for the trans-Golgi network. J. Biol. Chem. 2003, 278 (25), 23046–23054. Steegmaier, M.; Klumperman, J.; Foletti, D. L.; Yoo, J. S.; Scheller, R. H. Vesicle-associated membrane protein 4 is implicated in transGolgi network vesicle trafficking. Mol. Biol. Cell 1999, 10 (6), 1957– 1972.

Journal of Proteome Research • Vol. 9, No. 3, 2010

Guo et al. (31) Kierszenbaum, A. L.; Tres, L. L. The acrosome-acroplaxomemanchette complex and the shaping of the spermatid head. Arch. Histol. Cytol. 2004, 67 (4), 271–284. (32) Lin, Y. T.; Yen, P. H. A novel nucleocytoplasmic shuttling sequence of DAZAP1, a testis-abundant RNA-binding protein. RNA 2006, 12 (8), 1486–1493. (33) Fukuda, M.; Asano, S.; Nakamura, T.; Adachi, M.; Yoshida, M.; Yanagida, M.; Nishida, E. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 1997, 390 (6657), 308–311. (34) Hirokawa, N. Stirring up development with the heterotrimeric kinesin KIF3. Traffic 2000, 1 (1), 29–34. (35) Kile, B. T.; Hentges, K. E.; Clark, A. T.; Nakamura, H.; Salinger, A. P.; Liu, B.; Box, N.; Stockton, D. W.; Johnson, R. L.; Behringer, R. R.; Bradley, A.; Justice, M. J. Functional genetic analysis of mouse chromosome 11. Nature 2003, 425 (6953), 81–86. (36) Turner, J. M.; Mahadevaiah, S. K.; Ellis, P. J.; Mitchell, M. J.; Burgoyne, P. S. Pachytene asynapsis drives meiotic sex chromosome inactivation and leads to substantial postmeiotic repression in spermatids. Dev. Cell 2006, 10 (4), 521–529. (37) Namekawa, S. H.; Park, P. J.; Zhang, L. F.; Shima, J. E.; McCarrey, J. R.; Griswold, M. D.; Lee, J. T. Postmeiotic sex chromatin in the male germline of mice. Curr. Biol. 2006, 16 (7), 660–667. (38) McCarrey, J. R.; Kumari, M.; Aivaliotis, M. J.; Wang, Z.; Zhang, P.; Marshall, F.; Vandeberg, J. L. Analysis of the cDNA and encoded protein of the human testis-specific PGK-2 gene. Dev. Genet. 1996, 19 (4), 321–332. (39) Wrobel, G.; Primig, M. Mammalian male germ cells are fertile ground for expression profiling of sexual reproduction. Reproduction 2005, 129 (1), 1–7. (40) Schafer, M.; Kuhn, R.; Bosse, F.; Schafer, U. A conserved element in the leader mediates post-meiotic translation as well as cytoplasmic polyadenylation of a Drosophila spermatocyte mRNA. EMBO J. 1990, 9 (13), 4519–4525. (41) Lee, K.; Haugen, H. S.; Clegg, C. H.; Braun, R. E. Premature translation of protamine 1 mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice. Proc. Natl. Acad. Sci. U.S.A. 1995, 92 (26), 12451–12455.

PR900735K