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Testis |
Department of Obstetrics and Gynaecology,2
Department of Pathology,3
Department of Anatomy,4
Department of Surgery,5 The University of Hong Kong, Pokfulam Road, Hong Kong, Special Administrative Region of the People's Republic of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Each of the AZF regions contains many genes that have not been fully characterized. These genes may be the potential candidates for AZF. So far, only the RNA-binding motif (RBM) gene in the AZFb region and the deleted-in-azoospermia (DAZ) gene in the AZFc region have been extensively studied, and they are believed to play important roles in normal spermatogenesis. The RBM gene is expressed specifically in the nuclei of testicular germ cells [7, 8] and belongs to a large family of genes spreading over the Y chromosome. The DAZ gene is also expressed specifically in the testis and bears an RNA recognition motif [9]. These two genes are members of Y-encoded gene families with autosomal homologues. The cell biology of RBM is complex and suggests a role in pre-mRNA splicing [10]. A role for the DAZ family in the regulation of mRNA translation is supported by several lines of circumstantial evidence, including the association of DAZ-like protein with polyribosomes [11].
The VCY2 (variable charge, Y chromosome, 2; alias BPY2) gene, identified by Lahn and Page [12], locates in the most frequently deleted AZFc region in infertile men [3, 5]. VCY2 is composed of eight exons spanning 21 kilobases. Only five exons (exons 4–8) are translated into amino acids. Fluorescence in situ hybridization on interphase chromatin fibers (fiber-FISH) on relaxed chromatin located a functional copy of VCY2 within the DAZ gene cluster and multiple partial nonfunctional copies consisting of 5'-untranslated exons scattered along chromosome Yq [13].
Our recent study indicated that ubiquitination may be required for VCY2 function through the specific interaction with ubiquitin protein ligase (UBE3A) [14]. Furthermore, both VCY2 and UBE3A mRNAs have been localized to ejaculated human spermatozoa, suggesting that they may have important roles in spermatogenesis. To further confirm the involvement of VCY2 in spermatogenesis, the present study aimed to examine the expression of VCY2 in human testis with normal spermatogenesis. Testicular biopsy specimens from infertile men with different testicular histology were also included for immunohistochemical analysis to determine the possible relationship of VCY2 in the pathogenesis of male infertility.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the expression and the localization pattern of VCY2 in human testicular tissues with normal spermatogenesis, fresh testicular tissues were obtained from a total of three unrelated men undergoing orchiectomy as hormonal treatment for prostate cancer. The average patient age was 68 yr (range, 63–76 yr). The operations were performed during the year 2002 at the Department of Surgery, The University of Hong Kong. This study protocol was approved by the Ethics Committee of The University of Hong Kong. Histological examination of testicular sections was performed by a pathologist after hematoxylin-and-eosin staining. All testicular tissues showed morphologically normal spermatogenesis with normal tubule cellularity and late spermatids in all tubular cross-sections. These tissue specimens were divided into two pieces: One was used for protein lysate preparation for Western blot analysis, and one was fixed overnight in 10% neutral buffered formalin, washed in 70% ethanol, and paraffin embedded for immunohistochemical analysis.
Testicular Biopsy Specimens from Infertile Patients
Fresh testicular biopsy specimens from infertile patients were not available from our laboratory. To determine the possible relationship of VCY2 in the pathogenesis of male infertility, VCY2 expression was examined in archival, formalin-fixed, paraffin-embedded biopsy specimens from infertile patients. Such biopsy specimens from 16 infertile men were obtained from the Department of Pathology, The University of Hong Kong. The average patient age was 39 yr (range, 33–44 yr). These biopsy specimens were taken by open surgery on men being assessed for infertility. Four of these infertile patients had been assessed previously for Y-chromosome microdeletion and VCY2 gene deletion [3]. These included two AZFc-deleted patients (one with hypospermatogenesis and one with Sertoli cell-only syndrome) and two non-AZF-deleted patients (one with arrested spermatogenesis and one with Sertoli cell-only syndrome). These testicular sections were used to compare the expression pattern of VCY2 with the same testicular histology. If VCY2 immunoreactivities were not detected in testicular sections from non-AZFc-deleted patients, this result would indicate that the absence of VCY2 expression was not caused by AZFc or VCY2 deletion. Spermatogenesis in these infertile patients was classified as Sertoli cell-only syndrome (n = 6), maturation arrest (n = 4), and hypospermatogenesis (n = 6) during histological examination by a pathologist. In histological evaluation, Sertoli cell-only syndrome was defined as a biopsy specimen showing seminiferous tubules with Sertoli cells only and no spermatogenesis. If the tubules consisted of all stages of spermatogenesis but with reduced numbers, the patient was classified as having hypospermatogenesis. The cessation of spermatogenesis at a specific stage of spermatogenesis was defined as maturation arrest.
Preparation of Human Ejaculated Spermatozoa
To determine the VCY2 expression in human ejaculated spermatozoa at the ultrastructural level, we used immunogold electron microscopic analysis. Semen specimens from three unrelated, normal, healthy donors (20–50 x 106 spermatozoa/ml) were obtained from patients attending the in vitro fertilization clinic of Queen Mary Hospital, Hong Kong. The average patient age was 36 yr (range, 35–37 yr). Semen parameters were assessed using the criteria for normal concentration and motility according to the World Health Organization (WHO) guidelines. Semen samples with normal semen parameters refer to fertile sperm according to the WHO criteria for fertility, but these sperm can only be regarded as fertile if they have produced offspring. One to 2 ml of each sample were layered on top of a discontinuous 45%/90% Percoll gradient and centrifuged at 300 x g for 20 min. Sperm were washed twice by centrifugation at 550 x g in wash buffer (Ham F-10 medium containing 3% sucrose [w/v]). Subsequently, these sperm samples were fixed and used for Immunogold electron microscopic analysis.
Preparation of Protein Lysates from Testis for Western Blot Analysis
Testicular tissues with normal spermatogenesis from three unrelated patients with prostate cancer undergoing orchiectomy were used for Western blot analyses as described above. The following steps were performed at 0–4°C unless otherwise stated: Tissue specimens were homogenized in double-strength Laemmli lysis buffer [15] containing 0.125 M Tris-HCl (pH 6.8), 2% (w/v) SDS, 20% (v/v) glycerol, and 130 mM dithiothreitol. The samples were then placed on ice and sonicated vigorously for 20 sec. Supernatants of the tissue extract were subjected to protein analyses using the Bradford assay. The protein extracts were stored at -70°C before Western blot analysis.
Generation of Antibody Against VCY2
Antisera were raised in rabbits against a peptide consisting of amino acid residues 7–21 (RARTRAGQDHYSHPC) in the N-terminal of the VCY2 protein (GenBank accession no. AF000980). Peptide synthesis and antibody production were purchased from Alpha Diagnostic International (San Antonio, TX). Rabbit antisera were subsequently peptide affinity-purified using SulfoLink (Pierce, Rockville, IL) coupling gel to remove nonspecific binding activities according to the manufacturer's instructions. The specificity of the antibody was tested by Western blot analysis.
Western Blot Analysis
The expression of VCY2 in human testicular tissues with normal spermatogenesis was examined by Western blot analysis. Testicular biopsy specimens from infertile patients were not available for Western blot analysis. Approximately 200 µg of total protein extracts per lane were separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA) using a Bio-Rad transfer system. Nonspecific sites were blocked by incubation of the membrane with PBS containing 0.1% (v/v) Tween 20 at 4°C overnight. The membrane was then incubated with rabbit anti-VCY2 antibody (diluted 1:1000) or monoclonal antiactin antibody (diluted 1:2500; Sigma-Aldrich Corporation, St. Louis, MO) for 2 h at room temperature. After washing with PBS, horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin (Ig) G (diluted 1:2000) or HRP-conjugated sheep anti-mouse IgG (diluted 1:2000; Amersham Biosciences, Buckinghamshire, U.K.) was added and incubated for 1 h at room temperature. The enhanced chemiluminescence Western blotting detection kit (Amersham Biosciences) was used to visualize the antigen/antibody HRP complex, and autoradiography of the membrane was made on Kodak Biomax films (Eastman Kodak, Rochester, NY). Specificity of the VCY2 antibody was verified by preabsorbing VCY2 antibody with VCY2 immunizing peptide. Preabsorbed VCY2 antibody at a dilution similar to that of VCY2 antisera was used for Western blot analysis on the same membrane.
Immunohistochemical Analysis
All sections were cut at a thickness of 5 µm, mounted, and deparaffinized in xylene. Endogenous peroxidase activity of the sections was removed by incubation with 3% hydrogen peroxide in absolute methanol for 10 min. After washing with water, the sections were rehydrated and permeabilized using a microwave at high power for 10 min in citrate buffer (10 mmol/L citrate, pH 6.0). After cooling, the sections were washed twice with PBS for 5 min each time. Immunostaining was carried out with a nonbiotin amplification (NBA) kit as recommended by the manufacturer (Zymed Laboratories, Inc., San Francisco, CA). The sections were incubated first in the blocking solution as provided in the NBA kit and subsequently with the primary antibody (diluted 1:100 in blocking solution) at 4°C overnight in a humidified chamber. Following the washing steps with PBS, all sections were incubated first with secondary antibody and subsequently with HRP-conjugated tertiary antibody. The presence of peroxidase was then revealed by addition of diaminobenzidine (DAB) substrate and H2O2. Sections were then lightly counterstained with hematoxylin, dehydrated, and mounted with Permount (Fisher, Fair Lawn, NJ). The negative-control sections were incubated in parallel with blocking solution or with preabsorbed VCY2 antibody. Preabsorbed VCY2 antibody at a dilution similar to that of the VCY2 antisera was used for immunostaining.
Immunogold Electron Microscopic Analysis
Human ejaculated spermatozoa were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in PBS for 15 min at room temperature. The fixatives were removed by washing three times in wash buffer, and the spermatozoa were dehydrated by passage through a series of graded ethanol ranging from 50% to 100%. Spermatozoa were infiltrated with and embedded in LR White resin (London Resin Company Ltd., Reading, U.K.). The blocks were polymerized overnight at room temperature, and ultrathin sections (thickness, 100 nm) were cut and mounted on 200-mesh, uncoated gold grids. To stain the sections, the sections were first blocked in undiluted normal goat serum (NGS) for 15 min at room temperature. They were then incubated for 16 h at 4°C with either undiluted normal goat serum (NGS) or undiluted VCY2 antibodies containing incubation medium (1% NGS, 1% BSA, and 0.1% Tween 20). After the sections were washed four times in wash buffer, they were incubated with gold (10 nm)-conjugated secondary antibody, goat anti-rabbit IgG (Amersham Biosciences) diluted 1:35 in incubation medium. The sections were subsequently washed in distilled water and stained with uranyl acetate before examination with a Philip CM100 BioTwin Transmission Electron Microscope (FEI Company Electron Optics, Eindhoven, The Netherlands). Digital images were captured by MegaView digital camera (Soft Imaging System GmbH, Münster, Germany).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western blot analysis utilizing an antibody generated against the N-terminal of human VCY2 detected a single specific band of approximately 14 kDa in size, which corresponds to the expected size of VCY2, in human testicular tissues with normal spermatogenesis from three unrelated men (Fig. 1). VCY2 expression was detected in all three testicular tissues with similar intensity. The same protein loading was shown in these samples on the same blot when probed with antiactin antibody (Fig. 1). The specificity of the VCY2 antibodies was shown by the absence of the band in human lymphocytes (Fig. 1) and by the absence of signals on the same membrane when exposed to preabsorbed VCY2 antibody (data not shown).
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Immunostaining
In the testicular tissue sections with normal spermatogenesis, VCY2 was detected in the nuclei of spermatogonia, early and late spermatocytes, and round spermatids with variable degrees of expression, but it was not detected in elongated spermatids (Fig. 2, A and B). VCY2 expression appears to vary neither in a germ cell-specific manner (i.e., spermatogonia vs. spermatocytes vs. spermatids) nor in a stage-specific manner. No immunoreactivities were found in endothelial cells and myoid cells. However, some immunoreactivities were detected in the Leydig cell cytoplasm (Fig. 2A). Sertoli cell nucleoli possessed some immunoreactivity, which was in fact the result of nonspecific staining with the secondary antibody, because similar immunoreactivity was also present in the negative-control sections, in which primary antibody was omitted (Fig. 2C). No immunoreactivity was detected in germ cell nuclei in the negative-control sections when primary antibody was omitted (Fig. 2C). The specificity of VCY2 antisera was demonstrated by the absence of nuclear staining in germ cells when VCY2 antisera was preabsorbed with the VCY2 immunizing peptide (Fig. 2D).
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We next sought to determine the expression of VCY2 in testicular tissues with impaired spermatogenesis. When spermatogenesis was arrested at the spermatocyte stage, VCY2 immunoreactivity was only weakly detected at the spermatogonial level. No immunoreactivities were found in spermatocytes (Fig. 2E). An identical result was also observed in the maturation-arrest biopsy specimens without AZFc deletion. In all cases of hypospermatogenesis, weak immunoreactivity of VCY2 was detected at the spermatogonial level in seminiferous tubules with spermatogenesis progressing to the spermatid stage. No VCY2 immunoreactivity was detected in spermatocytes and spermatids (Fig. 2F). In the patient with hypospermatogenesis and AZFc deletion encompassing the regions of DAZ and VCY2 genes, no immunoreactivities were detected in germ cells at any state of development.
In all six Sertoli cell-only syndrome biopsy specimens, VCY2 immunoreactivities were detected in the cytoplasm and in the Sertoli cell nucleoli within the seminiferous tubules (Fig. 2G). The immunoreactivity in the Sertoli cell nucleoli was in fact the result of nonspecific staining with the secondary antibody, because immunopositive staining in Sertoli cell nucleoli was also detected in the negative-control sections (Fig. 2C) and in the patient with Sertoli cell-only syndrome and AZFc deletion.
Immunogold Labeling
Immunogold labeling with anti-VCY2 antibodies was performed to localize VCY2 in human ejaculated spermatozoa at the ultrastructural level. Gold particles were most prominent in the nucleus of all spermatozoa that we examined in the section (Fig. 3, A–C). The gold particles were randomly distributed within the whole nucleus. Occasionally, a few gold particles were detected within the acrosomal membrane. Gold particles were not observed in the basal plate, axoneme, mitochondrial sheath, and fibrous sheath. In the negative-control section when primary antibody was omitted, gold particles were not detected within the nucleus of spermatozoa (Fig. 3D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The VCY2 antibody used in the present study was raised against a 15-amino-acid synthetic peptide corresponding to a part of the N-terminal region of VCY2. The sequence was unique to VCY2 in BLAST analysis in the GenBank nucleotide database. Using Western blot analysis, the specificity of the affinity-purified VCY2 antibody was shown by the presence of a single specific band with the expected size of VCY2 in all the testicular biopsy specimens with normal spermatogenesis that were examined. VCY2 expression was also detected in human ejaculated spermatozoa by Western blot analysis (data not shown), and this result was confirmed by our Immunogold electron microscopic analysis.
The localization of VCY2 in human male germ cells was investigated by immunostaining and Immunogold electron microscopy. Immunohistochemical analyses demonstrated the expression of VCY2 in all germ cell nuclei, except elongated spermatids, in testicular biopsy specimens with normal spermatogenesis. The expression of VCY2 in the nucleus of human ejaculated spermatozoa at the ultrastructural level suggests that the absence of VCY2 expression in elongated spermatids is caused by the inaccessibility of the antibody in these germ cells with condensed nuclei. Although the exact function of VCY2 is not known, variable degrees of expression intensity of VCY2 in germ cell nuclei suggest that VCY2 is present during mitosis and meiosis and is controlled temporally during spermatogenesis.
We demonstrated different patterns of expression in testicular biopsy specimens with spermatogenic disorders when compared with testicular tissues having normal spermatogenesis. Both maturation-arrest and hypospermatogenesis biopsy specimens demonstrated weak VCY2 expression in the nuclei of spermatogonia and no expression in spermatocytes and spermatids. Identical results were also observed in the maturation-arrest tissue without AZFc or VCY2 deletion. The absence of VCY2 expression in tissues without Y-chromosome microdeletion could be attributed to the lack of activating transcription factors or to epigenetic modifications, such as DNA methylation of the promoter region. In the hypospermatogenesis tissue with AZFc and VCY2 deletions, VCY2 immunoreactivities were absent in all spermatogenic cells. The impaired expression of VCY2 suggests its involvement in the pathogenesis of male infertility, and it implies that VCY2 may be important for the production of mature spermatids.
Our recent study showed that ubiquitination may be required for VCY2 function through the specific interaction with UBE3A [14]. Different phases of mammalian spermatogenesis probably require different specialized activities of the ubiquitin system. Similar to VCY2, ubiquitin protein ligase was localized to male germ cells, and its inactivation also could lead to spermatogenetic arrest and male sterility [17, 18]. In spermatocytes, an intricate link exists among DNA repair, the ubiquitin system, and regulation of meiotic chromatin structure, as indicated by the colocalization of proteins involved in these processes on meiotic recombination complexes [19]. Taken together, these data have implicated the requirement of VCY2 and ubiquitin protein ligase for the normal germ cell development.
It has been thought that the main function of the AZFc genes in the Y chromosome is associated with spermatogenesis. However, the presence of VCY2 in human ejaculated spermatozoa may suggest a role of VCY2 in fertilization. This hypothesis is supported by one previous study [20] that showed ICSI with ejaculated spermatozoa from men with a microdeletion in the AZFc region of the Y chromosome resulted in a reduced fertilization rate and embryo score.
Although the exact function of VCY2 is not yet known, the specific localization of the protein in germ cell nuclei indicates that VCY2 is likely to function in the nucleus during spermatogenesis. The loss of VCY2 by deletion or some other dysfunction in infertile men is associated with the pathogenesis of male infertility. We suggest that the absence of VCY2 gene or protein together with other genes within the AZFc region on the Y chromosome may greatly affect both the quantity and quality of sperm production. Further analysis of the gene and protein function is necessary to elucidate the mechanism of spermatogenetic regulation by VCY2.
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FOOTNOTES |
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Received: 27 January 2003.
First decision: 17 February 2003.
Accepted: 17 April 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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