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Testis |
Department of Biomedical Science,2 Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan
Department of Degenerative Neurological Disease,3 National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, 187-8502, Japan
Laboratory of Pathophysiology,4 Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, 812-8582, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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male reproductive tract, meiosis, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies show that there are at least four mammalian UCH isozymes, among which the residues surrounding the active site share a high degree of homology [8, 9]. Mouse Uchl1 and Uchl3 encode proteins of similar size that share 52% amino acid sequence identity [8, 10]. However, the distribution of these isozymes is quite distinct; UCH-L1 mRNA is selectively and highly expressed in the testis/ovary and neuronal cells [10–12], whereas UCH-L3 mRNA is expressed in all tissues, including the testis/ovary and brain [13, 14]. UCH-L1-specific antibodies stain the testis, especially spermatogonia and Sertoli cells [15–18]. In addition, UCH-L1-deficient gracile axonal dystrophy (gad) mutant mice exhibit pathological changes, such as progressively decreasing spermatogonial stem cell proliferation [18]. These results led us to postulate that UCH isozymes in the testis function in the development of spermatogonia into mature sperm. Spermatogenesis is a complex, highly organized process that is divided into three phases: proliferation, meiosis, and spermiogenesis [19], each of which may require the activity of specific UCH isozymes. However, our understanding of the functional roles and the localization of UCH isozymes during spermatogenesis is limited.
In the present study, we generated peptide-specific antibodies against sequences within UCH-L1 or UCH-L3 and purified developing sperm cells at each stage from testes using immunomagnetic beads followed by discontinuous Percoll gradient centrifugation [20–23]. We found that the expression level of each UCH isozyme increased during the first round of spermatogenesis, and that the isozymes exhibited differential expression in the mouse testis [24]. Our results suggest that UCH isozymes play an important role in the regulation of spermatogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The gad [11] and Uchl3 knockout [13] mice were used as controls. The gad mouse is an autosomal recessive mutant that was obtained from the cross between CBA and RFM mice. The mutant line was maintained by intercross for more than 20 generations. The Uchl3 knockout mouse was generated by the standard method using homologously recombinant ES cells from 129SV mice. The knockout line was back-crossed several times to C57BL/6J mice. Male Balb/c mice were purchased from Nihon CLEA, Inc. (Tokyo, Japan), and all animals were maintained at the National Institute of Neuroscience, National Center of Neurology and Psychiatry (Japan).
All mouse experiments were performed in accordance with the institution's regulations for animal care and with the approval of the Animal Investigation Committee.
Isolation of Type A Spermatogonia and Sertoli Cells
Sequential enzymatic digestion of testicular tubules was performed as previously described [21]. After treatment with erythrocyte lysing buffer, testes from ten 2-wk-old and two 8-wk-old Balb/c mice were incubated twice for 5 min at 34°C with medium containing 0.5 mg/ml collagenase IV-S (Sigma-Aldrich, St. Louis, MO) and digested for 10 min at 34°C with medium containing 1 mg/ml trypsin (Sigma-Aldrich).
After the sequential enzymatic digestion, type A spermatogonia were isolated using immunomagnetic beads. The cells were incubated at room temperature for 15 min with biotin-conjugated rat anti-mouse CD117 (1 µg/106 cells), which recognizes the extracellular domain of the c-kit receptor (clone 2B8; Pharmingen, San Diego, CA). The cell suspension was then centrifuged at 300 x g for 5 min and washed with Dulbecco modified Eagle medium to remove excess antibody. The cell pellet was then resuspended in 80 µl buffer and incubated with 20 µl MACS antibiotin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) per 107 total cells at 6–12°C for 15 min. The cell suspension was then washed carefully and resuspended in 500 µl buffer per 108 total cells. The c-kit-positive cells (type A spermatogonia) were separated with a MACS separator (Miltenyi Biotec) and collected. The suspension containing Sertoli cells was resuspended at a concentration of 1 x 106 cells/ml in tissue culture medium containing 10% fetal calf serum.
Sertoli Cell Culture and Purification
The suspension containing Sertoli cells obtained from testes of ten 2-wk-old mice was plated on lectin- (Datura stramonium agglutinin; Sigma, St. Louis, MO) coated dishes as described [25, 26] and incubated for 3 days at 37°C. Alkaline phosphatase activity was visualized according to the procedure of Cox and Singer [27]. Cultured cells were incubated in reaction buffer (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5) containing 0.17 mg/ml 5-bromo-4-chloro-3-indolyl phosphate and 0.33 mg/ml nitro blue tetrazolium chloride.
Isolation of Spermatocytes and Spermatids
After isolating type A spermatogonia from testes of 8-wk-old mice using immunomagnetic beads, the testicular cell suspension was separated by discontinuous Percoll (Amersham Biosciences, Piscataway, NJ) gradient centrifugation [20]. The recovered cell populations were analyzed by phase-contrast microscopy [28], fluorescence-activated cell sorting (FACS) [29], and reverse transcription-polymerase chain reaction (RT-PCR).
RT-PCR Analysis of Isolated Cells Expressing Specific Marker Genes
From each population of spermatogonia, spermatocytes, spermatids, and Sertoli cells isolated from testicular cell suspensions, mRNAs were extracted using the QuickPrep micro mRNA purification kit (Amersham Biosciences) and subjected to reverse transcription with a first-strand cDNA synthesis kit (Amersham Biosciences) according to the manufacturer's instructions. PCR was performed with the following primers; c-kit [30], 5'-AAGATTTGCGATTTCGGGC-3' and 5'-CTGAAAATGCTCTCTGGTGCC-3'; Histone H1t [7], 5'-GTCCAGCTCTTGACCATGTCG-3' and 5'-GCTTTTTCCCTCGCCTTTTAG-3'; SP-10 [31], 5'-TTTATCTGCTTGGATCTGCCC-3', and 5'-GCTTGAAAGTTGCTGAACCG-3'; stem cell factor (SCF) [32], 5'-ATAGGAAAGCCGCAAAGGC-3' and 5'-TTACAAGCGAAATGAGAGCCG-3'; and glyceraldehyde-3-phosphate dehydrogenase [33]. Each sample was amplified using the AmpliTaq Gold GeneAmp system (Applied Biosystems, Foster City, CA).
FACS Analysis of Isolated Cells
FACS analysis was performed as described by Malkove et al. [29, 34]. Briefly, isolated spermatogenic cells were fixed in 70% ethanol overnight at 4°C and then incubated in propidium iodide staining solution (50 µg/ ml and 100 U/ml RNase A in PBS) for 30 min at room temperature. Within 2 h poststaining, the isolated spermatogenic cells were analyzed by FACS (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ). Excitation was at 488 nm and emission was at 
600 nm.
Quantitative mRNA Analysis of UCH Isozyme Genes by Real-Time PCR
SYBR Green-based real-time quantitative RT-PCR (ABI PRISM 7700 Sequence detection system, Columbia, MD) was performed [33, 35] in SYBR Green Master mix using the following primers; UCH-L1, 5'-TTCTGTTCAACAACGTGGACG-3' and 5'-TCACTGGAAAGGGCATTCG-3'; UCH-L3, 5'-TGAAGGTCAGACTGAGGCACC-3' and 5'-AATTGGAAATGGTTTCCGTCC-3'; UCH-L4, 5'-AAACAAACCATCAGCAATGCC-3' and 5'-GACCCTGATTCAAAGTGCACC-3'; UCH-L5, 5'-TTTTCTTTTCAAGTGGCAGCC-3' and 5'-GATAGCCTGAGTGGCACAAGC-3'; and ß-actin, 5'-CGTGCGTGACATCAAAGAGAA-3' and 5'-CAATAGTGATGACCTGGCCGT-3'. For comparing relative UCH isozyme gene expression in isolated germ cells and Sertoli cells, the formula 2–ddCt was used to calculate relative expression levels compared with spermatogonia of two-week-old mice. For comparing the expression level of UCH isozyme genes in the time course of testicular maturation, the formula 2–ddCt was used to calculate relative expression levels compared with the testes of 5-day-old mice.
Western Blotting of UCH-L1 and UCH-L3 from Isolated Cells
Total protein was extracted [24] from isolated testicular cells or whole testes of 5-, 7-, 15-, 19-, 21-, 23-, 26-, and 33-day-old Balb/c mice. Control extracts were obtained from testes of gad and Uchl3 knockout mice. For preparation of antibodies, we designed two specific peptides, AQHENFRKKQIEELKGQEVSPK (R891A, GenBank no. NP_057932, residues 57–78) and EKYEVFRTEEEEKIKSQGQDVTSS (R837A, GenBank no. NP_035800, residues 60–83) corresponding to mouse UCH-L1 and UCH-L3, respectively. Polyclonal antibodies against R891A and R837A were raised in rabbits and the IgG fraction was isolated (Tana Laboratories, L.C., Houston, TX). Each sample was adjusted to 5 µg protein/10 µl and subjected to SDS-PAGE (15% acrylamide; XV Pantera gel; DRC, Tama, Japan). After transferring the proteins to a nitrocellulose membrane and blocking with 5% skim milk, the membranes were incubated at 4°C overnight with the primary antibody to UCH-L1 (1:1000) or UCH-L3 (1:400). The membranes were then incubated with peroxidase-conjugated goat anti-rabbit IgG (H+L) (1:10 000; Pierce, Rockford, IL) for 60 min at room temperature. Immunoreactivity was visualized using the SuperSignal detection kit (Pierce) and analyzed with a ChemiImager (Alpha Innotech, San Leandro, CA).
Immunohistochemistry of UCH-L1 and UCH-L3
Tissues were fixed in vivo with 4% paraformaldehyde in phosphate-buffered saline (PBS) and embedded in paraffin, and sections (4 µm thickness) were treated with absolute methanol containing 3% H2O2 for 30 min to block endogenous peroxidase activity. After blocking with 10% goat serum for 1 h at room temperature, the sections were incubated at 4°C overnight with antibodies to UCH-L1 (1:500) or UCH-L3 (1:200) diluted in PBS containing 1% BSA. The sections were then incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:200, Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature and examined by confocal laser scanning microscopy (Olympus, Tokyo, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using the c-kit antibody, we isolated an average of 17 x 105 type A spermatogonia from ten 2-wk-old juvenile mice and an average of 3.5 x 105 type A spermatogonia from two 8-wk-old adult mice. The purity of each isolated population was <84.5% (Fig. 1A and Table 1). Using phase-contrast microscopy, we determined that these cells were 14–16 µm in diameter. Following elimination of the remaining spermatogonia using magnetic microbeads, the remaining cells were separated into two principal visible bands by centrifugation through a discontinuous Percoll gradient. Phase-contrast microscopy revealed that these two bands consisted primarily of pachytene spermatocytes and round spermatids (Fig. 1, B and C). We isolated an average of 3 x 105 spermatocytes and 200 x 105 spermatids from two 8-wk-old adult mice. We also isolated an average 20 x 105 Sertoli cells from ten 2-wk-old juvenile mice (Fig. 1D). Peritubular myoid cells were identified by alkaline phosphatase staining. Although >95% of these cells were unstained, 5% of the Sertoli cells showed considerable alkaline phosphatase activity (Fig. 1E).
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RT-PCR Analysis of Isolated Cells Expressing Specific Marker Genes
RT-PCR using extracts of isolated cells confirmed the expression of mRNAs from c-kit, Histone H1t, SP-10, and SCF. Each gene was expressed in only one cell type, as follows: c-kit in spermatogonia, the Histone H1t gene in spermatocytes, SP-10 in spermatids, and SCF in Sertoli cells (Fig. 2).
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Characterization of Isolated Cells by FACS
To determine the purity of isolated germ cells, we used FACS analysis to follow the differentiation-dependent acquisition of stage-specific patterns during spermatogenesis and monitored the percentage of cells with differing DNA content. The histogram in Figure 3 presents the number of cells at each fluorescence level (FL2-A). Diploid (2n DNA) cells were observed in 84.5% of the isolated spermatogonia, tetraploid (4n DNA) cells were observed in 79.6% of the isolated spermatocytes, and haploid (1n DNA) cells were observed in 85.3% of the isolated spermatids (Table 1). These results demonstrate that the majority of cells in each population exhibited the expected ploidy.
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Expression of UCH Isozymes During Spermatogenesis
We characterized the expression pattern of each UCH gene in isolated testicular cell populations during spermatogenesis using SYBR Green-based real-time quantitative RT-PCR (Fig. 4). The 2–ddCt values indicate the relative mRNA expression levels compared with spermatogonia from 2-wk-old mice (Sg2). The genes encoding UCH-L1 and UCH-L4 were expressed mainly in spermatogonia (Fig. 4, A and C). UCH-L1 was also expressed significantly in the Sertoli population, and UCH-L4 was expressed to a lesser degree in spermatocytes and spermatids. UCH-L3 and UCH-L5 genes were expressed primarily in the spermatid population and to a much lesser extent in spermatocytes (Fig. 4, B and D). We further examined the expression of UCH mRNAs during testicular maturation (Fig. 5A) in whole testes from 5-, 7-, 15-, 19-, 21-, 23-, 26-, and 33-day-old mice. The UCH-L1 and UCH-L4 mRNAs were expressed similarly during development and likewise for the UCH-L3 and UCH-L5 genes. The expression of UCH-L1 mRNA appears relatively high on Postnatal Day 15. However, the percentage of spermatogonia and Sertoli cells would have become diluted by meiotic and postmeiotic germ cells after Day 15, thereby accounting for the relatively lower levels of UCH-L1 at subsequent time points. RT-PCR data suggest that UCH-L1 mRNA expression in spermatogonia and Sertoli cells increased continuously even after Postnatal Day 15 (Fig. 5B). We also analyzed the protein expression patterns of UCH-L1 and UCH-L3 in the mouse testis using peptide-specific antibodies that recognize regions in mouse UCH-L1 or UCH-L3. Western blot analysis detected UCH-L1 in spermatogonia and Sertoli cells, and UCH-L3 was detected primarily in spermatocytes and spermatids, as expected, although it was also found to a lesser extent in spermatogonia (Fig. 6A). Also, UCH-L3 expression increased in a differentiation-dependent manner during juvenile spermatogenesis (Fig. 5C). Immunohistochemistry detected homogeneous UCH-L1 expression in both spermatogonia and Sertoli cells from wild-type (Balb/ c) and Uchl3 knockout mice, whereas UCH-L3 was detected mainly in spermatocytes and round spermatids of wild-type (Balb/c) and gad mice (Fig. 6, B and C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ubiquitin C-terminal hydrolases catalyze the hydrolysis of C-terminal esters and amides of ubiquitin. These enzymes are believed to play a key role in processing polyubiquitin and ubiquitylated proteolytic peptides [8]. The genes for at least four UCHs, UCH-L1 and UCH-L3–5, have been identified in the mouse. Although the specificity and function of these isozymes in spermatogenesis remains elusive, each of these enzymes contains conserved residues that are critical for enzymatic activity [8–10, 44, 45]. The predominant mouse isozymes, UCH-L1 and UCH-L3, share 52% amino acid sequence identity [10, 13, 45]. However, UCH-L1 mRNA is selectively expressed in the mouse testis and nervous systems [11], whereas UCH-L3 mRNA is expressed in nearly every tissue tested, with high levels in the testis [13]. Intracellular localization of UCH-L1 was reported to be closely associated with the proliferative activity of spermatogonia and Sertoli cells [15–18]. In contrast, the expression of UCH-L3 has not been examined in the testis. To address this question, we first generated polyclonal antibodies that specifically react with mouse UCH-L3. Using RT-PCR and western blotting, we detected high levels of UCH-L3 mRNA and protein in meiotic pachytene spermatocytes and postmeiotic spermatids during spermatogenesis (Figs. 4B and 6A). These results suggest that UCH-L1 and UCH-L3 may play distinct roles in spermatogenesis, in that UCH-L1 may function in mitotic proliferation, whereas UCH-L3 may function in the meiotic differentiation of spermatocytes into spermatids.
Around Postnatal Day 15, the higher expression of UCH-L1 mRNA suggests that UCH-L1 might have certain functions during testicular maturation. Furthermore, the increased expression of UCH-L1 after Postnatal Day 15 suggests that it might play an active role in mitotic proliferation. In addition to the Uchl1 genes, we also analyzed transcription from two other UCH isozyme genes, Uchl3 and Uchl5, in isolated testicular cells. UCH-L3 and UCH-L5 mRNAs were found in meiotic pachytene spermatocytes and postmeiotic spermatids (Fig. 4, B and D) and showed similar expression patterns during the course of testicular maturation (Fig. 5B). These results suggest that the Uchl3 and Uchl5 genes may have overlapping functions during spermatogenesis. The ubiquitin pathway is very active during the postmitotic phase of spermatogenesis [3, 36]. Thus, UCH-L3 may function to regulate the cell cycle and chromatin structure during the meiotic phase but may facilitate the en masse degradation of cytoplasmic proteins as well as organelle/nuclear remolding during the postmeiotic phase. The present study demonstrates for the first time that UCH-L3 is expressed mainly in meiotic spermatocytes and postmeiotic spermatids during spermatogenesis. Although UCH-L3 shares considerable sequence homology with UCH-L1, the hydrolytic activity of UCH-L3 in vitro differs substantially from that of UCH-L1. The rate of UCH-L3-mediated catalysis (Kcat) is more than 200 times greater than UCH-L1 using ubiquitin amide as a substrate [46]. This relatively high activity of UCH-L3 is consistent with its expression during the postmitotic phase of spermatogenesis, in that maturation from spermatocytes to spermatids may be critically dependent on the ubiquitin pathway despite the fact that Uchl3 knockout mice exhibit normal fertility [13].
In conclusion, our results demonstrate that the expression of UCH isozymes is differentially and developmentally regulated during spermatogenesis and that UCH-L1 and UCH-L3 likely have distinct functions during different developmental phases. These results enhance our understanding of how the ubiquitin pathway is regulated by UCH isozymes during spermatogenesis. Moreover, isolation of mouse germ cells and Sertoli cells from testes may afford the opportunity to assess UCH isozyme function during spermatogenesis in vitro. UCH-L1 has been suggested to associate with monoubiquitin and thereby increase the half-life of ubiquitin in neurons [47]. In addition, a UCH-L1 ubiquityl ligase-like activity has also been proposed [46]. Further biochemical and genetic analyses of UCH family members will help elucidate the role of UCHs in the complex molecular mechanisms involved in spermatogenesis.
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FOOTNOTES |
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Received: 20 January 2004.
First decision: 5 February 2004.
Accepted: 19 March 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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