Molecular Human Reproduction, Vol. 5, No. 12, 1122-1126,
December 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular aspects of spermatogenesis |
Specific expression of heat shock protein HspA2 in human male germ cells
1 Center for Reproduction and Genetics, 2 Department of Obstetrics and Gynecology, 3 Department of Urology of Pundang Je-Saeng General Hospital, Kyungki-do, and 4 Department of Life Science, Sogang University, Seoul, Korea
Abstract
In the mouse, the heat shock protein 70-2 (Hsp70-2) has been found to play a critical role in spermatogenesis. The HspA2 gene is the human homologue of the murine Hsp70-2 gene with 91.7% identity in the nucleotide coding sequence. We examined the expression of HspA2 in human tissues. To detect HspA2 expression, antiserum 2A that was raised against mouse Hsp70-2 and that cross-reacted with human HspA2 protein expressed in Escherichia coli was used. The results of Western blotting indicate that significant HspA2 expression occurs in testes with normal spermatogenesis, whereas only a low amount of HspA2 was expressed in testis with Sertoli cell-only syndrome. Only a small amount of HspA2 was detected in breast, stomach, prostate, colon, liver, ovary, and epididymis. Immunoreactivity to HspA2 was present in spermatocytes and spermatids in the testes with normal spermatogenesis, while immunoreactivity to HspA2 in testis with Sertoli cell-only syndrome was remarkably decreased or inconspicuous over the entire cell. These results demonstrate that the HspA2 protein is highly expressed in human male specific germ cells, suggesting that HspA2 protein may play a specific role during meiosis in human testes as found in the murine model.
heat shock proteins/HspA2/male germ cells/spermatogenesis/testis
Introduction
Spermatogenesis is a developmental process occurring in three distinct phases, an initial mitotic phase, meiosis, and a post-meiotic phase characterized by extensive remodelling of haploid germ cells to produce spermatozoa. A selective set of genes is expressed in each phase of spermatogenesis and produce a selective set of proteins with restricted patterns of expression (Eddy et al., 1991
). However, the mechanism of spermatogenesis remains elusive.
The heat shock proteins (HSP) are a group of homologous proteins encoded by a multigene family. Their function as chaperones, in modulating the conformation of newly synthesized polypeptides, appears to have been conserved throughout evolution (Georgopoulos and Welch, 1993; Welch, 1993). Their expression can be constitutive and/or increased when cells are subjected to heat shock or metabolic stress (Miller et al., 1991). However, two mouse Hsp genes, Hsp70-2 and Hsc70t are apparently expressed only in the male germ cell line at specific limited stages of spermatogenesis (Allen et al., 1988; Zakeri et al., 1988; Maekawa et al., 1989; Matsumoto and Fujimoto, 1990). The level of expression of Hsp70-2 is not responsive to heat shock in mouse germ cells, suggesting a restricted role such as modulating maturation of the male germ line cells (Allen et al., 1988; Zakeri et al., 1988; Matsumoto and Fujimoto, 1990).
HspA2 is the human homologue of the murine Hsp70-2 gene with a 91.7% identity in the nucleotide coding sequence and 98.2% in the corresponding amino acid sequence (Bonnycastle et al., 1994). HspA2 has <90% amino acid homology to other members of the human Hsp70 gene family, 83.3% to the heat-inducible Hsp70-1 gene and 86.1% with the human heat shock cognate gene Hsc70 (Bonnycastle et al., 1994). Therefore, we postulate that human HspA2 may have important function in spermatogenesis of human testis. However, nothing is known about the expression of HspA2 protein in human testes and other tissues. The objectives of the present study were to determine the expression of HspA2 protein in human tissues.
Materials and methods
Tissue preparation
Human testis tissues were obtained from patients who had given their consent and were undergoing pathological evaluation. After biopsy, testis tissue was fixed immediately in Bouin's solution and then embedded in paraffin, sectioned, and stained with haematoxylin and eosin in the standard fashion for pathological evaluation. Immunohistochemistry was also performed using the sectioned slides. After pathological evaluation, the tissue of testes with normal spermatogenesis and Sertoli cell-only syndrome was used in this study. Fresh testis tissue and fresh human tissues were stored in liquid nitrogen for the Western blot analysis.
Expression of His6-tagged HspA2 protein
The open reading frame (ORF) of the HspA2 gene was amplified from genomic DNA by PCR with specific primers to give a 1930 bp EcoRI/HindIII fragment (Figure 1A). PCR amplification was for 35 cycles (94°C, 1 min; 62°C, 1 min; 72°C, 2 min). The PCR amplicons were cloned into pBS vector (Amersham, Arlington, IL, USA), and the direction and sequence of inserted cDNA was confirmed by sequence analyses. For the preparation of the expression vector for HspA2, cloned HspA2 ORF was cut by EcoRI/HindIII digestion after amplification in Escherichia coli. The DNA fragments were ligated in expression vector pTrcHisB (Invitrogen, San Diego, CA, USA) and introduced into the E.coli strain Top10 (Figure 1B). HspA2 protein expression was induced by 1 mmol/l isopropylthio-ß-D-galactoside (IPTG) and the cross-reactivity was examined with rabbit polyclonal antiserum 2A (generous gift from E.M.Eddy, NIH; Rosario et al., 1992) raised against mouse Hsp70-2 protein by Western blot analysis.
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Preparation of protein extracts from human tissues
Protein extracts were prepared from each tissue. Total lysates were prepared by homogenization of tissues in fresh ice-cold protein lysis buffer [50 mmol/l Tris pH 7.5, 150 mmol/l NaCl, 0.1% NP-40, 50 mmol/l NaF, 1 mmol/l dithiothreitol (DTT), 1 mmol/l phenylmethylsulphonyl fluoride (PMSF), 10 µg/ml soybean trypsine inhibitor, 10 µg/ml leupeptine, 10 µg/ml aprotinin]. The extract was centrifuged at 12 000 rpm for 10 min at 4°C, and the supernatant was collected for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting. The protein concentration of each tissue extract was determined by micro BCA assay and the same amounts of total proteins were used for each sample.
Western blot analysis
Total protein (20 µg) extracts from each tissue were re-suspended in 10 µl of 1x SDS protein sample buffer (62.5 mmol/l Tris pH 6.8, 40 mmol/l DTT, 2% SDS, 0.025% Bromophenol Blue, 10% glycerol), boiled for 5 min and subjected to one-dimensional PAGE on 9% polyacrylamide gels containing SDS, followed by transfer to nitrocellulose membranes. The blots were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h at room temperature. The blots were then reacted with a 1:2000 dilution of antiserum 2A for 1 h at room temperature. The proteins were detected using the 4-chloro-1-naphtol.
Immunohistochemistry
Immunohistochemical staining was performed using an avidin–biotin immunoperoxidase technique (Dako, Carpinteria, CA, USA). A rabbit polyclonal antiserum2A was used as diluted 1:200. The second incubation with biotinylated polyvalent antibody and the third incubation with avidin–horseradish peroxidase followed the first incubation with primary antibody. The chromogenic reaction was developed by incubation with a solution of AEC+ substrate-chromogen (DAKO, USA). Negative controls were incubated with pre-immune serum. All sections were counterstained with haematoxylin and mounted in an aqueous medium.
Results
Human Hsp A2 protein can be detected by antiserum 2A
The human HspA2 gene is highly homologous to the mouse Hsp70-2 gene (Bonnycastle et al., 1994). Antiserum 2A contains polyclonal antibodies to mouse Hsp70-2 protein. To examine the cross-reactivity of the antiserum 2A with human HspA2 protein, Western blotting was performed with human HspA2 protein expressed in E.coli system. Recombinant HspA2 fusion protein was induced by supplementation of 1 mmol/l IPTG in E.coli culture media and the production of HspA2 was analysed by Western blotting with antiserum 2A. (Figure 2). The amount of expressed HspA2 was not different after 2–4 h of induction. (Figure 2A lanes 2 and 3). Expressed HspA2 fusion protein was detected by Western blot analysis with antiserum 2A (Figure 2B, lane 2). The antiserum 2A does not cross-react with human Hsp70/Hsc70 proteins (data not shown).
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Expression of hspA2 protein in human tissues
Expression of HspA2 in a variety of human tissues was examined by Western blot analysis with short termination of colour reaction (Figure 3
). HspA2 70 kDa protein was only detected in the testis with normal spermatogenesis (Figure 3A, lane 1). Almost no HspA2 protein was detected in testis with Sertoli cell-only syndrome (Figure 3A, lane 2). However, a very low amount of HspA2 protein could be detected in testis with Sertoli cell-only syndrome with a longer colouring reaction (data not shown). Very low amounts of HspA2 (similar to those in Sertoli cell-only testis) were also detected in breast, stomach, prostate, colon, liver, ovary, and epididymis (Figure 3B). Therefore, HspA2 protein was expressed specifically in testis with normal spermatogenesis. To browse the pattern of HspA2 expression and its cellular specificity in testicular epithelium, immunohistochemistry was performed. Figure 4 shows haematoxylin and eosin staining of the testis with normal spermatogenesis and Sertoli cell-only syndrome (Figure 4A,B). When these testes were stained with antiserum 2A, positive immunoreactivities of HspA2 were specifically localized on the spermatocytes and spermatids of the normal spermatogenic testes (Figure 4C). Even in the normal testis, the spermatogonia in the basal layer, Leydig cells, Sertoli cells, and somatic cells were HspA2 negative (Figure 4C). In addition, the immunoreactivities of HspA2 in testis with Sertoli cell-only syndrome were remarkably decreased or inconspicuous in all cell types (Figure 4D).
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Discussion
This study demonstrates that the HspA2 protein is specifically expressed in human male germ cells. These results suggest that HspA2 protein may play a specific role during meiosis in the human testis.
A protein that was continuously expressed without heat shock stress during the murine spermatogenesis was isolated and named as Hsp70-2; it had previously been referred to as P70 (Allen et al., 1988). These authors also reported that the Hsp70-2 expression is stage-specific with the highest expression in the pachytene stage of the spermatocyte. The Hsp70-2 protein has been confirmed as the product of the Hsp70-2 gene by Western blot analysis using antiserum 2A raised against Hsp70-2, and the antiserum does not cross-react with other heat shock proteins (Rosario et al., 1992). Recently, direct evidence (Dix et al., 1996) that Hsp70-2 has an important role in murine spermatogenesis, has indicated that knockout of the Hsp70-2 gene leads to spermatogenic arrest and infertility in the male mice, but does not produce any problems in female mice.
The human HspA2 gene is highly homologous to the mouse Hsp70-2 gene and the rat testis-specific Hsp gene (Zakeri et al., 1988; Wisniewski et al., 1993). The rat and mouse gene products were 98.4 and 98.2% identical in amino acid sequence to the HspA2 gene products respectively (Bonnycastle et al., 1994). Therefore, human HspA2 may have an important function in spermatogenesis of human testes.
In order to investigate HspA2 distribution in normal and Sertoli cell-only testes, we confirmed that the antiserum 2A reacted with human HspA2. The antiserum 2A is a polyclonal antibody to mouse Hsp70-2 peptide (amino acids 611–628). The antigenic peptide sequence is NH2-SKLYQGGPGGGGSSGGPT-COOH. In this region, the sequence of HspA2 protein is NH2-SKLYQGGPGGGSGGGGSGASGGPT-COOH. HspA2 gene has 6 additional amino acids near the 3' end of the human sequence (amino acids 622–627), which is the region of the largest difference between the human and the rodent sequences. Therefore, we expressed HspA2 protein in E.coli to examine whether the antiserum 2A had cross-reacted with human HspA2. As shown in Figure 2
, we demonstrated that antiserum 2A antibody could detect the expressed HspA2 protein of MW 73 kDa. Histidine and additional amino acids caused the increase of the size to 73 kDa (Figure 2A and B).
It is known that spermatogenesis is initiated by endocrine cues transmitted indirectly through surrounding somatic cells, by growth factors, or by short-loop paracrine and autocrine signals (Jégou and Sharpe, 1993). However, how these extrinsic cues influence germ cell development is not understood precisely. For the precise pattern of germ cell development, the specific gene products required, and the conserved nature of the process implies that the primary regulator of spermatogenesis might be an intrinsic genetic programme. The intrinsic regulation of gene expression in spermatogenesis occurs at three levels: transcription, translation and post-translation. Transcriptional regulation is the primary determinant of gene expression in most cells. However, translational regulation probably has a greater role in germ cells than in other cell types (Eddy, 1998). It has been reported that HspA2 mRNA is constitutively expressed in most tissues and a very high level of message is made in testis (Bonnycastle et al., 1994). However, the presence of HspA2 protein in various tissues has not been reported. The amount of HspA2 protein was significantly higher in testis with normal spermatogenesis than that of other tissues. Also, a very low amount of HspA2 protein was detected in ovary and testis with Sertoli cell-only syndrome. The HspA2 protein expression is in agreement with the studies of Bonnycastle et al. (1994) implying that HspA2 expression is regulated mostly at the transcriptional level. Also, HspA2 protein may be expressed specifically in male germ cells.
The immunohistochemical results indicate that HspA2 protein was mainly detected in germ cells, especially pachytene spermatocyte stage. Therefore, it could be speculated that HspA2 protein might play a specific role during spermatogenesis in man.
Although the roles of Hsp70-2 remains to be defined in mouse, it was suggested that Hsp70-2 is required for the heterodimer formation of cdc2 with cyclin B1 (Zhu et al., 1997), and for desynapsis of synaptonemal complex during meiotic prophase (Allen et al., 1996; Dix et al., 1997). So, it might be informative to investigate the cdc2/cyclin B1 heterodimer formation in human testes with or without HspA2, and this is under investigation. Further molecular analysis of the gene and protein function is necessary in order to elucidate the mechanism of spermatogenesis regulation by HspA2.
Acknowledgments
We are very grateful to Ri-Cheng Chian, Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada H3A, 1A1, for critical review of this manuscript. The authors also wish to thank Hyun-Ju Kim for his helpful suggestions and discussions.
Notes
5 To whom correspondence should be addressed at: Center for Reproduction and Genetics, Pundang Je-Saeng General Hospital, Kyungki-do 463–050, Korea 
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Submitted on June 7, 1999; accepted on August 16, 1999.
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