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Developmental Expression of Src-Related Tyrosine Kinases in the Mouse Testis

SERGE GOUPIL^*,

SOPHIE LA SALLE^,

JACQUETTA M. TRASLER

LOUIS-JEAN BORDELEAU^*,

PIERRE LECLERC^*,

From the ^* Ontogenie et Reproduction, Centre de Recherche du Centre Hospitalier Universitaire de Québec-Centre Hospitalier de l'Université Laval, Ste-Foy, Québec, Canada; the Département d'Obstétrique/Gynécologie, Centre de Recherche en Biologie de la Reproduction, Université Laval, Ste-Foy, Québec, Canada; and the Departments of Pediatrics, Human Genetics, and Pharmacology and Therapeutics, McGill University, and the Research Institute at the Montreal Children's Hospital of the McGill University Health Centre, Montreal, Canada. Present address: The Jackson Laboratory, 600 Main St, Bar Harbor, ME 04609.

Correspondence to: Pierre Leclerc, Ontogenie et Reproduction, Centre de Recherche du CHUQ-CHUL, 2705 Boul. Laurier, Ste-Foy, QC, Canada G1V 4G2 (e-mail: pierre.leclerc{at}crchul.ulaval.ca).

Received for publication March 22, 2010; accepted for publication August 13, 2010.

Abstract

An increase in protein tyrosine phosphorylation occurs during sperm capacitation in numerous species. The involvement of Src-related tyrosine kinases in this phenomenon has been demonstrated using different inhibitors specifically targeting this family of enzymes. In mammals, this group of nonreceptor tyrosine kinases is made up of 8 members with similar SRC homology domain 3 (SH3) and SH2 domains. Although some members of this group of enzymes can compensate for one another, showing some redundancy, each is unique and may perform specific functions during male germ cell development. To further characterize the importance of Src-related tyrosine kinases in the events leading to proper sperm formation, and because no inhibitor affecting a single gene product exists, expression of Src, Yes1, Fyn, Lyn, Lck, Hck, Blk, and Fgr was assessed by real-time polymerase chain reaction in developing mouse testes and in enriched populations of mouse spermatogenic cells, revealing distinct expression profiles for each kinase during testis development and in isolated male germ cells. Immunolocalization of SRC, LYN, and HCK in adult mouse testes as well as in mature spermatozoa further confirmed differential localization of these kinases during spermatogenesis. Although mRNA levels of these latter kinases were higher in spermatogonia and spermatocytes than in spermatids, protein levels were highest in spermatids, suggesting delayed transcript translation. Taken together, these results clearly show an uneven expression of each kinase in different spermatogenic cells, indicating that each member may play a different role during spermatogenesis, in addition to highlighting the complexity of Src-related kinase expression regulation in male germ cells. Furthermore, differential localization of these tyrosine kinases in mature spermatozoa also suggests a specific role for each member in sperm function and integrity.

     Key words: Reproductive tract, sperm, spermatogenesis, tyrosine kinase

In male germ cells, it is during sperm capacitation that protein tyrosine phosphorylation has been investigated the most. An increase in tyrosine phosphorylation of specific proteins has been demonstrated during sperm capacitation in many species (Aitken et al, 1995; Visconti et al, 1995a,b; Leclerc et al, 1996, 1997, 1998; Galantino-Homer et al, 1997). The mechanisms involved in the control of sperm protein phosphotyrosine content during the process of capacitation result from the interactions between cAMP, oxygen derivatives, and Ca²⁺-mediated signaling pathways (Aitken et al, 1998; Leclerc et al, 1998; Dorval et al, 2002). These signaling reactions possibly occur in a cascade of events triggered by membrane cholesterol efflux (Visconti et al, 1999). Although several of the sperm phosphotyrosine-containing proteins affected by capacitation have been identified (Ficarro et al, 2003; Bailey et al, 2005; Baker et al, 2006), there is little information on the enzymes, kinases, or phosphatases involved in this increase in protein phosphotyrosine content. Because basal and capacitation-associated protein tyrosine phosphorylation is abolished by different kinase inhibitors targeting more specifically Src-related kinases (Dorval et al, 2002, 2003; Baker et al, 2006; Lawson et al, 2008; Mitchell et al, 2008), this family of protein tyrosine kinases was suspected to play an important role in sperm. Although the presence of YES1, SRC, LYN, and other Src-related tyrosine kinases has recently been demonstrated in mature spermatozoa (Leclerc and Goupil, 2002; Baker et al, 2006; Lalancette et al, 2006; Lawson et al, 2008; Mitchell et al, 2008), their precise role in motility, capacitation, acrosome reaction, or interaction with the egg still needs further investigation.

Interactions between germ and Sertoli cells are crucial to the formation of functional spermatozoa during spermatogenesis. As germ cells proliferate and differentiate, extensive remodeling of the seminiferous epithelium must take place to allow for movement of germ cells across the blood-testis barrier toward the lumen of the seminiferous tubules (O'Donnell et al, 2006). Communication between germ and Sertoli cells is also required for the timely release of spermatozoa from the seminiferous epithelia via the process of spermiation (O'Donnell et al, 2006). Although the involvement of Src-related tyrosine kinases in proliferation, differentiation, and cytoskeletal reorganization has been demonstrated, and is recognized, in somatic cells (Thomas and Brugge, 1997), the role of these kinases in the nuclear and cytoplasmic events occurring during spermatogenesis remains elusive. Therefore, the goal of this study was to describe the expression of the 8 members of the Src-related kinase family during spermatogenesis to further evaluate their specific involvement in the cellular and nuclear reorganization characterizing male germ cell development and leading to the formation of functional spermatozoa.

Materials and Methods

Germ Cell Isolation and RNA Extraction

Purified populations of germ cells were obtained from the testes of 8-, 17-, and 70-day-old CD-1 mice (Charles Rivers, St-Constant, Canada) according to the sedimentation velocity cell separation method on a 2% to 4% bovine serum albumin gradient as previously described (Bellvé et al, 1977; La Salle and Trasler, 2006). Animals were housed and treated according to the guidelines of the Canadian Council on Animal Care and approved by the McGill University and Laval University animal care committees. Cells were identified on the basis of morphologic criteria and size. Populations of type A spermatogonia (average purity, 86%) and type B spermatogonia (average purity, 83%) were obtained from the testes of 8-day-old mice (n = 2 cell separations). Preleptotene spermatocytes (average purity, 85%), leptotene/zygotene spermatocytes (average purity, 87%), and prepubertal (early) pachytene spermatocytes (average purity, 80%) were obtained from the testes of 17-day-old mice (n = 2 cell separations). Pachytene spermatocytes (average purity, 81%), round spermatids (average purity, 88%), and elongating spermatids mixed with residual bodies (average purity, 86%) were obtained from 70-day-old mice (n = 2 cell separations). Total RNA was extracted from snap-frozen pellets of isolated germ cells using the RNeasy Mini kit with DNaseI treatment according to the manufacturer's protocol (Qiagen Inc, Mississauga, Canada).

As an alternative, enriched populations of premeiotic and postmeiotic spermatogenic cells were separated according to their DNA content using fluorescence-activated cell sorting. Because the genome of spermatocytes in prophase I of meiosis is diploid (2n), but each chromosome is made up of 2 sister chromatids (4c), their DNA content can be distinguished from that of spermatids, which have undergone 2 meiotic divisions and are haploid (1c, 1n). Germ cell isolation was performed with adult (90-day-old) mice according to the procedure reported by Meistrich (1972), with some modifications. The tunica albuginea was removed from the testes, then the seminiferous tubules were minced in small fragments (<2 mm), placed in 1 mL of Dulbecco phosphate-buffered saline containing 1 mg/mL glucose (D-PBS) in a 35-mm diameter Petri dish, and agitated for 10 minutes at 31°C. Trypsin (Gibco BRL/Life Technologies, Burlington, Canada) was added to a final concentration of 0.1%, and the mixture was incubated for another 10 minutes without agitation. MgCl₂ (10 mM, final concentration) and bovine pancreas DNase I (2 µg/mL, final concentration; Sigma, St Louis, Missouri) were added, and the mixture was incubated at 31°C with agitation for 10 minutes. Cell detachment from the tubules was facilitated by pipetting, then fetal bovine serum was added to a final concentration of 8% to quench trypsin activity. Next, the sample was filtered through a 50-µm pore screen and centrifuged (500 x g) at room temperature. The cells were washed again with D-PBS and prepared for cell sorting as described previously (Mays-Hoopes et al, 1995). Briefly, cells were fixed in 0.4 M sodium citrate (pH 2.35) made in 0.1% diethylpyrocarbonate-treated H₂O for 1 day at room temperature, then in the same solution at pH 4.5 for 3 to 7 days at 4°C. One day prior to the sort, cells were centrifuged and resuspended in HIB medium (10 mM Hepes [pH 7], 8.6 g/L NaCl, 0.38 g/L KCl, 0.4 g/L EDTA, 0.2 g/L phenoxyethanol, and 0.1% bovine serum albumin in 0.1% diethylpyrocarbonate-treated H₂O; Mays-Hoopes et al, 1995), added with 0.1 mg/mL propidium iodide to stain DNA, and kept in the dark at 4°C until the next day. At the time of the sort, the cell suspension was allowed to equilibrate to room temperature, and cells were isolated by fluorescence-activated cell sorting according to their nucleic acid content as described previously (Mays-Hoopes et al, 1995). The spermatocyte (4c, 2n) and spermatid (1c, 1n) fractions were collected, and RNA was isolated as described previously (Chomczynski and Sacchi, 1987). Contaminating genomic DNA was removed by a 30-minute treatment with RNase-free DNase I performed at 37°C (Promega, Madison, Wisconsin).

Total RNA was also isolated from 7-, 14-, 21-, and (adult) 90-day-old CD-1 mouse testes. During the first wave of spermatogenesis, seminiferous tubules from 7-day-old mice contain spermatogonia and Sertoli cells, whereas pachytene spermatocytes and haploid spermatids are first detected in 14- and 21-day-old animals, respectively. In 90-day-old animals, spermatogenesis is ongoing, and all spermatogenic cell types are present within the seminiferous tubules (Bellvé et al, 1977). The tunica albuginea was removed, and the testes were snap frozen on dry ice and kept at –80°C until use. Total RNA was isolated from these testes using Trizol (Gibco) according to the manufacturer's instructions.

Reverse Transcription Polymerase Chain Reaction Amplification of Src-Related Kinase mRNA

DNase I-treated mRNA isolated from whole testes or germ cells was reverse transcribed according to the manufacturer's instructions using 1 µg of poly d(T)_12–18 (Pharmacia Biotech, Dorval, Canada), 200 units of SuperScript III reverse transcriptase (Gibco BRL), 500 µM each dinucleotide triphosphate, 5 mM dithiothreitol, and 40 units of RNasin (Promega) in the provided first-strand reaction buffer. Complementary DNA encoding the Src-related kinases was next amplified using degenerate primers (forward: 5'-TNAARCAY TAYAARATYMG-3', and reverse: 5'-GTSAYRATRWAR ATGGGYTC-3'; where N stands for A, T, G, or C; R stands for A or G; Y stands for C or T; M stands for A or C; S stands for C or G; and W stands for A or T) designed within the highly conserved amino acids KHYKIR and EPIYIV from the SRC homology domain 2 (SH2) and kinase domains of Src-related kinases, respectively. Polymerase chain reaction (PCR) was performed using 10 µL of cDNA, 2.5 units of Taq DNA polymerase (Qiagen), 200 µM each dinucleotide triphosphate, and 1 µg each of the forward and reverse primers in a total volume of 100 µL of PCR Buffer (Qiagen). Cycling conditions were as follows: 1.5-minute denaturation at 94°C, 2-minute annealing at 50°C, and 3-minute extension at 72°C for 40 cycles; a final extension period of 10 minutes was added at the end. The PCR products were separated by electrophoresis through a 0.8% agarose gel containing ethidium bromide.

Quantitative PCR

PCR amplification was performed on testis mRNA isolated from adult mice using primer sets specific for each of the Src-related kinases (see the Table for primer sequences). For each kinase, the primers used for amplification were designed within different exons to span at least 1 intron, except for Blk, for which they were designed within the same exon. The amplified products were electrophoresed on an agarose gel, cloned using pGEM-T easy vectors (Promega), and sequenced at the research center sequencing core facility (Centre de Recherche du Centre Hospitalier Universitaire de Québec-Centre Hospitalier de l'Université Laval Research Center, Ste-Foy, Canada). The plasmids containing the cloned PCR fragments were serially diluted and used as templates to establish standard curves in quantitative PCR. Quantitative PCR was performed using a LightCycler (Roche Diagnostics, Laval, Canada) in a 20-µL reaction mix that contained 5 µL of either testis or germ cell cDNA or plasmid cDNA at known concentrations, 0.6 µM each primer, 2 mM MgCl₂, and 2 µL of FastStart Master SYBRGreen I mix (Roche Diagnostics). The volume was completed with nuclease-free water (Promega). The amplification procedure was preceded by a 10-minute denaturation period at 95°C followed by 50 cycles of 10-second denaturation at 95°C, 5-second annealing (from 58°C to 68°C, according to the set of primers; see the Table), and 20-second extension at 72°C, with single acquisition of fluorescence at the end of the extension step. At the end, the samples were slowly heated from 74°Cto96°C with continuous reading of fluorescence to obtain a melting curve. A single peak was obtained for each gene product. The relative expression of each gene was calculated in relation to the appropriate standard curve and normalized to the expression of the housekeeping gene Gapdh (Table). Prior to undertaking these experiments, the choice of Gapdh to normalize gene expression was validated by comparing the expression profiles obtained using Gapdh to the ones obtained after normalization to total RNA content (OD₂₆₀) measured before reverse transcription or to 18S RNA levels (after reverse transcription PCR using random hexamers); similar expression patterns were obtained using all 3 approaches.

Immunoblotting Analysis of Src-Related Tyrosine Kinases

The presence of Src-related tyrosine kinases was assessed in total protein extracts prepared from mouse spermatozoa. Cauda epididymal spermatozoa were resuspended in D-PBS (6.8 mM CaCl₂, 2.68 mM KCl, 1.46 mM KH₂PO₄, 0.492 mM MgCl₂, 136.9 mM NaCl, and 8.06 mM Na₂HPO₄), washed by centrifugation (150 x g, 20 minutes) at room temperature, and resuspended in the same buffer. Proteins were extracted by the addition of 5x concentrated solubilization buffer (1x final concentrations: 2% sodium dodecyl sulfate, 10% glycerol, 5% β-mercaptoethanol, and 62.5 mM Tris-HCl [pH 6.8]) and heated at 100°C for 5 minutes. Proteins were separated by electrophoresis on 7.5% sodium dodecyl sulfate–polyacrylamide gels (Laemmli, 1970) and electrotransferred (Towbin et al, 1979) to nitrocellulose (0.22-µm pore size; MSI Inc, Westborough, Massachusetts). Nonspecific sites on the membranes were blocked in 5% (wt/vol) dry skimmed milk in Trisbuffered saline (0.9% NaCl and 20 mM Tris-HCl [pH 7.8]) supplemented with 0.1% Tween-20 (TTBS). The membranes were incubated for 1 hour at room temperature with one of the following antibodies diluted in TTBS: monoclonal anti-YES (Transduction Laboratories, Lexington, Kentucky), or polyclonal anti-SRC, anti-LYN, or anti-HCK antibodies (Santa Cruz Biotechnology, Santa Cruz, California). Membranes were extensively washed in TTBS and incubated with either goat anti-mouse or goat anti-rabbit immunoglobulin G (IgG) conjugated to horseradish peroxidase (Jackson Immunoresearch, West Grove, Pennsylvania) in TTBS for 45 minutes at room temperature. Finally, the membranes were extensively washed, and positive immunoreactive bands were revealed by chemiluminescence using the ECL Western Blotting detection kit (GE Healthcare, Baie d'Urfé, Canada) on x-ray films according to the manufacturer's instructions.

Immunodetection of Src-related kinases was also performed on protein extracts prepared from the testes of 7-, 14-, 21-, and 90-day-old mice. Detunicated testes were homogenized (Polytron; Kinematica Inc, Bohemia, New York) in solubilization buffer devoid of reducing agent and then centrifuged at 10 000 x g for 10 minutes. The supernatant was added with β-mercaptoethanol to a final concentration of 5%, heated at 100°C for 5 minutes, and kept frozen until use. Protein concentration was determined using the Micro BCA Protein Assay kit (Pierce Biotechnology Inc, Rockford, Illinois) after precipitation by trichloroacetic acid to get rid of detergents and reducing agents. Immunoblotting was performed as described above on equal protein load.

Indirect Immunofluorescence

Washed spermatozoa were placed on poly-L-lysine–coated coverslips, fixed for 15 minutes in 3.7% formaldehyde in PBS (1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄, 137 mM NaCl, and 2.7 mM KCl [pH 7.4]), rinsed with PBS, permeabilized for 10 minutes in 0.2% Triton X-100 in PBS, and rinsed again with PBS. For SRC immunodetection, cells were further incubated at –20°C for 10 minutes with methanol. Nonspecific sites were blocked with PBS supplemented with 1% bovine serum albumin. Samples were incubated overnight at 4°C with any of the tyrosine kinase antibodies described above, or with commercial nonimmune rabbit IgG as negative controls, diluted in the blocking solution. Cells were next rinsed with PBS and incubated with goat anti-rabbit IgG conjugated to fluorescein (Jackson Immunoresearch) for 1 hour at room temperature. After extensive washes in PBS, 25 µL of the antibleaching agent diazabicyclo[2,2,2]octane (1.5% made in 90% glycerol) was deposited on slides that were next covered with the coverslip. Immunofluorescence was detected by epifluorescence microscopy with an ultraviolet light.

Immunohistochemistry

For immunohistochemical investigations, testes from adult CD-1 or C57BL/6 (Charles River) mice were collected, and fixative solution (4% formaldehyde in 50 mM PBS [pH 7.4]) was injected directly into the testes. They were next immersed in fixative for 24 hours, then dehydrated in ethanol and embedded in paraffin. Sections (6 µm thick) were deparaffinized in toluene and rehydrated; endogenous peroxidase activity was eliminated by preincubation with 3% H₂O₂ in methanol for 30 minutes. Antigen retrieval was achieved by incubation of the slides for 10 minutes in a boiling bath containing 10 mM sodium citrate (pH 6.0). After cooling, the slides were blocked with 1% bovine serum albumin in PBS for 1 hour and incubated overnight at 4°C with the primary antibodies diluted in the blocking solution. After being washed 5 times with PBST (PBS supplemented with 0.05% Tween-20), the sections were incubated with a biotinylated donkey anti-rabbit IgG (Jackson Immunoresearch) in the blocking solution for 1 hour at room temperature, washed again in PBST, and incubated with horseradish peroxidase–conjugated streptavidin (Jackson Immunoresearch) for 45 minutes at room temperature. The slides were extensively washed in PBST, and the immune complex was revealed with 3,3'-diaminobenzidine (Sigma-Aldrich, Oakville, Canada) and counterstained with hematoxylin. At last, the tissue sections were mounted in Mowiol (Calbiochem; EMD Biosciences Inc, San Diego, California) and observed by light microscopy (Zeiss Axioskop 2). Testis sections incubated with the same concentration of commercial nonimmune rabbit IgG (Sigma) or primary antibodies preadsorbed with an excess of their immunizing peptide were used as negative control.

Results

Expression Dynamics of Src-Related Tyrosine Kinases in the Developing Testis

To assess the overall presence of Src-related kinases in the testis, PCR amplification using degenerate primers was first performed to establish whether the region encoding a highly conserved domain among the 8 Src-related kinases could be detected in adult testis cDNA. Amplification with this set of degenerate primers resulted in a 415- to 418-bp product (Figure 1), which was in complete agreement with the expected size range according to the nucleotide sequence of the different Src-related kinase genes. However, because the testis is composed of both somatic cells, such as Sertoli or Leydig cells, and spermatogenic cells, the same set of primers was used next to amplify cDNA made from RNA isolated from fluorescence-activated cell–sorted spermatocytes (4c, 2n) and spermatids (1c, 1n) to determine whether Src-related kinases were specifically expressed in germ cells. Again, as for whole-testis cDNA, a product of 415 to 418 bp was amplified in both cell populations (Figure 1), suggesting that Src-related kinases are specifically expressed in certain types of male germ cells.

View larger version (41K): [in this window] [in a new window]   Figure 1. Polymerase chain reaction amplification of Src-related kinase RNA in testis, spermatocytes, and spermatids. Total RNA was isolated from whole testes denuded from their tunica albuginea or from diploid spermatocytes (2n, 4c) and haploid spermatids (1n, 1c) separated by flow cytometry according to their DNA content. Upon reverse transcription, the cDNA was amplified using degenerate primers designed to amplify the region encoding a portion of the SRC homology domain 2 (SH2) and kinase domains, which are highly conserved among the 8 members of the Src family. A single band of 418 bp was observed.

View larger version (34K): [in this window] [in a new window]   Figure 2. Testicular expression of RNA encoding Src-related tyrosine kinases in 7-, 14-, 21-, and 90-day-old mice. RNA was isolated from whole testes denuded from their tunica albuginea, reverse transcribed using poly-d(T)12–18, and amplified by real-time polymerase chain reaction. For each tyrosine kinase, relative expression was normalized to that of Gapdh. Data are presented as means ± standard error of the mean (n = 5 animals at each age). Bars with different letters are significantly different (P < .05) using Bonferroni's multiple comparison test after analysis of variance.

Although the expression analysis conducted using testes of 7-, 14-, and 21-day-old mice gave an indication of the cell types that could be expressing the various Src-related kinases, it did not allow for precise determination of cell-specific expression. Therefore, the expression of the 8 Src-related tyrosine kinases was evaluated in enriched spermatogenic cell populations. The tyrosine kinase Src, which showed the strongest expression in the testes of 7-day-old mice, was expressed in a constant manner in type A and type B spermatogonia, as well as in preleptotene, leptotene/zygotene, early pachytene, and late pachytene spermatocytes (Figure 3). In round spermatids, Src expression levels were about one-third of those measured in spermatogonia and spermatocytes, and further decreased in elongated spermatids/residual bodies, where the expression level was only 11% of that observed in spermatogonia and spermatocytes.

View larger version (34K): [in this window] [in a new window]   Figure 3. Expression of RNA encoding Src-related tyrosine kinases in an enriched population of spermatogenic cells. RNA was isolated from enriched populations of male germ cells, reverse transcribed using poly-d(T)12–18, and amplified by real-time polymerase chain reaction. For each cell preparation and each tyrosine kinase, relative expression was normalized to that of Gapdh, and the values were expressed as percentages of the sum of all the spermatogenic cells. Data from each of the 2 cell preparations are shown. A and B indicate type A and type B spermatogonia, respectively; PL, LZ, EP, and P, preleptotene, leptotene/zygotene, early pachytene, and pachytene spermatocytes, respectively; RS and ES, round and elongating spermatids, respectively. For pachytene spermatocytes, expression levels of each tyrosine kinase relative to Gapdh were: 2.99 x 10–4 (Src), 1.95 x 10–3 (Yes1), 7.57 x 10–3 (Fyn), 3.74 x 10–3 (Lyn), 5.21 x 10–3 (Lck), 8.21 x 10–6 (Hck), 2.47 x 10–5 (Blk), and 4.39 x 10–5 (Fgr).

View larger version (146K): [in this window] [in a new window]   Figure 4. Developmental expression of testicular SRC. The expression of c-SRC was detected by immunohistochemistry on testis sections from adult mice. The positive signal was revealed by diaminobenzidine and appears as a brownish precipitate. Panels A, B, and C show sections of seminiferous tubules at different stages of spermatogenesis. No signal was obtained when commercial rabbit immunoglobulin G was used as a negative control instead of the primary antibody (Panel D).

Immunodetection of Src-Related Kinases in the Testis and in Mature Spermatozoa

Having established expression patterns for the 8 members of the Src-related tyrosine kinase family at the mRNA level in enriched populations of male germ cells, we further investigated the expression of the encoded tyrosine kinases by immunohistochemistry in the adult mouse testis. Germ cell expression of SRC was dependent on cell type. No staining was detected in the interstitial tissue, including the Leydig cells. Within the seminiferous tubules, spermatogonia and Sertoli cells were negative for SRC, whereas a weak positive signal was observed in spermatocytes (Figure 4). A strong positive signal was detected in round and elongated spermatids. The tyrosine kinase SRC was mostly associated with the cell cytoplasm. As for SRC, no LYN staining was detected in Leydig cells, Sertoli cells, or spermatogonia (Figure 5). However, both spermatocytes and spermatids expressed the tyrosine kinase LYN. Signal intensity peaked in round spermatids and then decreased as these cells progressed through spermiogenesis. HCK staining was detected in spermatocytes and spermatids, whereas spermatogonia and Sertoli cells remained unstained (Figure 6). The strongest signal was found in round spermatids, and a distinctively stained body could be observed in the nucleus of pachytene spermatocytes (Figure 6A). Although no labeling was obtained by immunohistochemical methods with the anti-YES antibody used, immunoblot analysis showed that the testicular levels of this tyrosine kinase increased between days 7 and 14, where it reached maximal levels (Figure 7), suggesting that expression of YES begins at the early pachytene spermatocyte stage.

View larger version (150K): [in this window] [in a new window]   Figure 5. Developmental expression of testicular LYN. The expression of LYN was detected by immunohistochemistry on testis sections from adult mice. The positive signal was revealed by diaminobenzidine and appears as a brownish precipitate. Panels A, B, and C show sections of seminiferous tubules at different stages of spermatogenesis. No signal was obtained when commercial rabbit immunoglobulin G was used as a negative control instead of the primary antibody (Panel D).

View larger version (155K): [in this window] [in a new window]   Figure 6. Developmental expression of testicular HCK. The expression of HCK was detected by immunohistochemistry on testis sections from adult mice. The positive signal was revealed by diaminobenzidine and appears as a brownish precipitate. Panels A, B, C, and D show sections of seminiferous tubules at different stages of spermatogenesis. No signal was obtained when the anti-HCK antibody was preadsorbed with the immunizing peptide (Panel E) or when commercial rabbit immunoglobulin G was used as a negative control instead of the primary antibody (Panel F).

View larger version (33K): [in this window] [in a new window]   Figure 7. Developmental expression of testicular YES. (A) The expression of c-YES was detected by Western blot analysis on testicular protein extracts prepared from 7-, 14-, 21-, and 90-day-old mice. (B) Quantification of the c-YES–positive band shown in Panel A. The data represent means ± standard error of the mean (n = 4 for day 7 [D-7]; n = 5 for D-14; and n = 6 for D-21 and D-90, respectively) and are shown as arbitrary units. *Significantly different (P < .05) from the expression measured at D-7.

The next step of our study was to determine, using indirect immunofluorescence, whether the Src-related tyrosine kinases expressed in testicular germ cells were also present in mature spermatozoa. As previously reported (Baker et al, 2006), SRC was detected in the entire flagellum of mouse spermatozoa isolated from cauda epididymis (Figure 8). A strong positive signal was also detected in the acrosomal region of the sperm head. This distribution pattern differed from the one of the tyrosine kinases, LYN, which displayed a strong signal in the acrosomal area (Figure 9). In the flagellum, LYN was detected in the principal and end pieces but was absent from the midpiece. Positive signal was also observed associated with the cytoplasmic droplet. Unlike SRC and LYN, HCK was not detected in the head but was restricted to the principal piece of the flagellum (Figure 10). Interestingly, the intensity of the HCK signal was maximal in the anterior portion, immediately next to the midpiece, and decreased toward the middle of the principal piece, where it became barely detectable. For these 3 kinases, the expression pattern observed in mature sperm is in agreement with their distribution in the seminiferous epithelium of adult mice.

View larger version (67K): [in this window] [in a new window]   Figure 8. Localization of SRC in mature spermatozoa. (A) The presence of the tyrosine kinase SRC was assessed by indirect immunofluorescence on cauda epididymal sperm. (B) As a negative control, fixed permeabilized cells were incubated with the same concentration of commercial rabbit immunoglobulin G. Panels C and D are phase-contrast images of the same fields presented in Panels A and B, respectively.

View larger version (94K): [in this window] [in a new window]   Figure 9. Localization of LYN in mature spermatozoa. (A) The presence of the tyrosine kinase LYN was assessed by indirect immunofluorescence on cauda epididymal sperm. (B) As a negative control, fixed permeabilized cells were incubated with the same concentration of commercial rabbit immunoglobulin G. Panels C and D are phase-contrast images of the same fields presented in Panels A and B, respectively.

View larger version (85K): [in this window] [in a new window]   Figure 10. Localization of HCK in mature spermatozoa. (A) The presence of the tyrosine kinase HCK was assessed by indirect immunofluorescence on cauda epididymal sperm. (B) As a negative control, fixed permeabilized cells were incubated with the same concentration of commercial rabbit immunoglobulin G. Panels C and D are phase-contrast images of the same fields presented in Panels A and B, respectively.

In the present study, we clearly demonstrated that spermatogenic germ cells express protein tyrosine kinases from the Src family. Several experimental approaches were used to establish the presence of Src-related tyrosine kinases within seminiferous tubules. PCR amplification was carried out using degenerate primers designed to detect the region encoding for part of the SH2 and catalytic domains of these kinases, which are 2 highly conserved regions among the different members of the Src family. Using oligonucleotide primers specific to each of the 8 Src-related kinases, the expression pattern of these kinases was revealed by quantitative PCR in testes from 7-, 14-, 21-, and 90-day-old mice, as well as in enriched populations of spermatogenic cells. Expression of specific members of the Src family was also assessed by immunodetection using specific antibodies.

This study is the first to show that expression of the specific transcripts encoding each of the Src-related tyrosine kinases varies according to the developmental stage of the cells undergoing spermatogenesis. The mRNA encoding Src is present at high levels in 7-day-old mouse testis and decreases afterward. This suggests that this transcript is present mostly in spermatogonia and/or Sertoli cells as well as in interstitial cells, such as Leydig cells. In adult rat testis, SRC has been shown primarily within the cytoplasm of the Sertoli cells (Wang et al, 2000), which differs from our immunohistochemical results showing no specific labeling in Sertoli or Leydig cells (Figure 4). In our study, however, where the transcript levels were assessed in isolated populations of testicular germ cells, the mRNA encoding Src was at high levels from the type A spermatogonia stage to the late pachytene spermatocyte stage, whereas protein expression started at the late pachytene spermatocyte stage up to the late spermatid stage. When assessed in mature spermatozoa, this kinase was detected in the acrosomal region as well as in the principal piece of the flagellum. The flagellar localization of SRC in mature epididymal sperm reflects perfectly the protein distribution in elongating and elongated spermatids in testis seminiferous tubules. The presence of SRC in the anterior portion of the head as well as in the tail is also in agreement with the reported presence of SRC in human sperm (Lawson et al, 2008), but differs with what was previously reported in mature mouse sperm, where it was found exclusively in the flagellum (Baker et al, 2006). Whether this difference depends on the source of the antibody used or the immunodetection procedure remains elusive.

As for Src, mRNA expression of Fyn decreased in the testes from 7- to 90-day-old mice. When assessed in enriched spermatogenic cells, the RNA levels of this kinase were at their highest in type B spermatogonia. Although this kinase was not investigated at the protein level in this study, FYN has been reported in mouse testis, mostly in Sertoli cells, in association with cytoskeletal elements (Maekawa et al, 2002). Recently, a truncated form of FYN, devoid of the kinase domain, was described in rat spermatids, whereas only the complete protein was detected in mature spermatozoa (Kierszenbaum et al, 2009). According to the oligonucleotide primers used, only the full-length sequence of Fyn was amplified in the present study. Similarly, the mRNA levels of Hck decreased in the testis from days 7 to 90; as for Fyn, levels were highest in type B spermatogonia. Using a set of degenerate primers, Hck-encoding mRNA has also been detected by reverse transcription PCR in a mixed population of mouse germ cells (Visconti et al, 2001). Furthermore, the mRNA encoding the entire sequence of Hck has been detected by PCR amplification in bovine haploid testicular germ cells, and the kinase was successfully detected in a protein extract from bull testis (Bordeleau and Leclerc, 2008). In the present study, it is clearly shown that HCK is present in spermatocytes, but more so in spermatids. In mature sperm, it was localized predominantly in the anterior portion of the principal piece.

The expression pattern of Yes mRNA during the development of the mouse testis is different. The highest levels are measured in 21-day-old mice, which suggest that spermatocytes or spermatids could be the cells responsible for this elevated level. When Yes mRNA levels were measured in isolated germ cells, the highest expression was observed in spermatocytes. This latter result agrees with the expression of the kinase, the levels of which are at their highest in the testes of 14-day-old mice, as shown by immunoblotting. The kinase c-YES was also detected in adult mouse testis, in agreement with the previously reported presence of YES in rat testis (Zhao et al, 1990). Furthermore, Yes, as well as Hck and Fgr, were the Src-related kinases with the lowest mRNA levels in postmeiotic germ cells. Unlike what was reported for rat epididymal (Zhao et al, 1990) and human ejaculated (Leclerc and Goupil, 2002) spermatozoa, we were unable to detect the kinase YES in mouse epididymal sperm (data not shown). Taken together, these data suggest species-specific differences in the expression of these kinases, which could reflect differences in the regulation or the role of these kinases in the testis.

As shown in this study, levels of Src-encoding mRNA are elevated in spermatogonia and primary spermatocytes and low in spermatids, which differs completely from the protein expression levels revealed by immunohistochemistry on testis sections; SRC is strongly present in spermatids and almost absent from the spermatogonia. The observed discrepancies between RNA and protein expression in different germ cells during spermatogenesis can occur through different mechanisms. It has been known for a long time that high levels of RNA synthesis occur during meiosis (reviewed by Eddy and O'Brien, 1998), and although translation of some of these newly produced RNAs proceeds at this stage, some can also be stored and their translation postponed until after completion of meiosis, during spermatid differentiation. Accumulation of polyadenylated RNAs in the nucleus of spermatocytes (Morales and Hecht, 1994) and mRNA storage in spermatocyte and in the chromatoid body of spermatids (Saunders et al, 1992) have been reported. RNA helicases regulating transcript translation and transport from the nucleus to the cytoplasm are also present in the chromatoid body (Sato et al, 2010). It is not known, however, whether mRNAs encoding Src-related tyrosine kinases are among the mRNAs stored within these structures.

Differences between RNA and protein expression can also result from the difference in the age of the animals used to perform these various analyses. In this study, localization of Src-related kinases was investigated strictly in the testes of adult males, where spermatogenesis is a continuous process, whereas levels of their encoding mRNAs were measured in cells obtained, in part, from prepubertal animals undergoing the first wave of spermatogenesis. Differences may occur in the intracellular regulation of an initiation compared with a maintenance process. It would be interesting to investigate the testicular distribution of Src-related tyrosine kinases in juvenile males to determine whether protein expression correlates with transcript levels. Alternatively, mRNA levels could be assessed in spermatogonia, spermatocytes, and spermatids from adult males, but it would be highly difficult to obtain "pure" enriched germ cell populations.

There is an increasing amount of evidence that Src-related tyrosine kinases are present within the seminiferous epithelium and that they play a role in the process of spermatogenesis. The involvement of this family of tyrosine kinases has recently been demonstrated in the renewal and proliferation of spermatogonial stem cells, an important feature of spermatogenesis (Braydich-Stolle et al, 2007; Oatley et al, 2007). Among the 8 mammalian Src-related tyrosine kinases, Src, Yes, Fyn, Lyn, and Hck were detected by reverse transcription PCR in spermatogonial stem cells (Oatley et al, 2007), and, using an siRNA approach, it was shown that the first 4 kinases listed above mediate GDNF-induced spermatogonial stem cell proliferation (Braydich-Stolle et al, 2007). On the other hand, the mechanisms by which these stem cells differentiate into type A spermatogonia remain elusive. In the present study, our enriched populations of type A and type B spermatogonia were prepared using 8-day-old mice and could have contained spermatogonial stem cells. In fact, for spermatogonial stem cell transplantations, spermatogonial stem cells are usually isolated from mice of the same age (Oatley and Brinster, 2006). A strong increase in Fyn, Yes, Hck, Blk, and Fgr mRNA expression was measured in type B spermatogonia compared with type A spermatogonia, which is similar to a previously reported increase in Fyn expression in type B compared with type A spermatogonia (Yu et al, 2003). It is unknown whether similar results would have been obtained if type A and type B spermatogonia were isolated from adult animals with well-established spermatogenesis. Notwithstanding, type B spermatogonia are the last cells that proliferate by mitosis as they prepare to enter meiosis and differentiate into preleptotene spermatocytes. There are numerous genes known to be either up- or down-regulated during the transition from mitosis to meiosis (Yu et al, 2003; Rossi et al, 2008). Within seminiferous tubules, DNA methyltransferase 3a (DNMT3a) has recently been shown to be expressed predominantly in type B spermatogonia (La Salle and Trasler, 2006). In transfected MCF-7 cells, induced expression of DNMT3a is mediated by members of the Src-related tyrosine kinase family, mainly by SRC (Shafiei et al, 2008). In the present study, expression of Src was elevated up to the pachytene spermatocyte stage, and whether it is involved in the expression of DNMT3a or other DNA methyltransferases remains to be established.

During prophase of the first meiotic division, which is characterized by its long duration, cells undergo massive DNA synthesis in the absence of cell division, and homologous chromosomes pair, synapse, and undergo meiotic recombination (Kerr et al, 2006). The X and Y sex chromosomes, which are largely nonhomologous in contrast to autosomal chromosomes, form a transcriptionally inactive and heterochromatic territory known as the XY body in pachytene spermatocytes (Handel, 2004). Interestingly, we detected HCK labeling in pachytene spermatocytes in a structure reminiscent of the XY body, but localization to this subnuclear territory still needs to be confirmed. Once cells have completed prophase I of meiosis, they quickly go through the first meiotic division to form secondary spermatocytes, and then the second meiotic division occurs to produce haploid spermatids. The cues controlling the transition from prophase I to metaphase I (G₂/MI) are still unclear. Neither cyclin-dependent kinases nor aurora kinases appear to be responsible for desynapsis when the G₂/MI transition is induced by okadaic acid in mouse pachytene spermatocytes (Sun and Handel, 2008). Involvement of Src-related tyrosine kinases during meiosis in the male still has to be elucidated, although the involvement of members of this tyrosine kinase family has been demonstrated during meiosis resumption in the egg (Sette et al, 2002; Talmor-Cohen et al, 2004a,b; Tomashov-Matar et al, 2007, 2008). During egg activation, FYN and/or YES1 would act downstream from the Ca²⁺ increase to promote meiotic resumption. When transposed to male meiosis, exit from the second metaphase (MII) would take place in secondary spermatocytes, the lifespan of which is very short, and therefore highly difficult to study.

Haploid spermatids go through numerous cellular modifications, collectively referred to as spermiogenesis, to become spermatozoa. These changes result in formation of an acrosome and a flagellum, along with reorganization of the nucleus and other cellular organelles. Although the involvement of Src-related kinases in such processes is well recognized in somatic cells, there is no indication thus far whether these kinases play a role in spermiogenesis. It has been demonstrated that the testicular variant of the tyrosine kinase FER is associated with acrosome development and remodeling during spermatid head shaping (Kierszenbaum et al, 2008). In the present study, the relative RNA expression of Lck, Blk, and, to a lower extent, Hck was increased in round spermatids, which suggests a role in the cellular transformations occurring in postmeiotic cells. This increase in Lck mRNA is in perfect agreement with the increase in mRNA expression measured in adult testes compared with 21-day-old mice, suggestive of an involvement in elongating spermatids. Therefore, contribution of Src-related kinases to the final steps of spermiogenesis cannot be neglected.

The results presented in this study clearly show that Src-related tyrosine kinases are expressed in a defined, spatiotemporal manner during testis development. Testicular levels of specific transcripts vary according to the age of the mice and are in close agreement with the profiles obtained in isolated spermatogenic cells. To our surprise, Src, Lyn, and Hck transcript levels and expression differed from their protein expression. Their mRNA levels were high in spermatogonia and spermatocytes, whereas detection of these kinases started in late pachytene spermatocytes. Storage and delayed translation of specific gene products expressed in haploid testicular germ cell is a phenomenon that has been known for many years, but the exact mechanisms explaining the discrepancies observed here in the case of Src-related kinases remain to be further investigated. Our data support the idea that each Src-related kinase is expressed in a specific manner at different times during spermatogenesis, which implies specific and potentially nonredundant roles for each of these kinases. On the other hand, no male infertility has been reported in mice with targeted disruption for a single gene of this tyrosine kinase family (reviewed in Lowell and Soriano, 1996), suggesting that they can compensate for each other. Further studies are required to clearly identify the specific processes affected by these tyrosine kinases during the formation of fully differentiated and functional male gametes. Furthermore, because specific Src-related tyrosine kinases are detected in epididymal spermatozoa and found at different localizations, they may be involved in various sperm functions, such as activation of motility, hyperactivation, and capacitation, as well as other events occurring at fertilization.

Acknowledgments

We are thankful to C. Lalancette and C. Lachance for their critical comments on this manuscript. We also want to acknowledge Dr M. Dufour from the Cytometry Core Facility of Centre de Recherche du Centre Hospitalier Universitaire de Québec-Centre Hospitalier de l'Université Laval.

Footnotes

Supported by grants from the Canadian Institutes of Health Research (P.L. and J.M.T.) and the Natural Science and Engineering Research Council of Canada (P.L.). S.L. is the recipient of fellowships from the Fonds de la Recherche en Santé du Québec and the Canadian Institute of Health Research. J.M.T. is a James McGill Professor of McGill University. The McGill University Health Centre Research Institute and the Centre de Recherche du Centre Hospitalier Universitaire de Québec-Centre Hospitalier de l'Université Laval receive support from the Fonds de la Recherche en Santé du Québec.

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