Repression of hspa2 messenger RNA in human testes with abnormal spermatogenesis

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1 FERTILITY AND STERILITY VOL. 73, NO. 6, JUNE 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Repression of hspa2 messenger RNA in human testes with abnormal spermatogenesis Weon-Young Son, M.Sc., a,b Ching-Tack Han, Ph.D., b Suh-Ha Hwang, M.Sc., b Jae-Ho Lee, M.Sc., a Seokjoong Kim, M.D., a,c and Young Chan Kim, M.D. a,d Pundang Je-Saeng General Hospital, Dae-jin Medical Center, Kyungki-do; and Sogang University, Seoul, Korea Received August 18, 1999; revised and accepted December 7, Presented at the 94th Annual Meeting of the American Urological Association, Dallas, Texas, May 1 6, Reprint requests: Weon- Young Son, M.Sc., Center for Reproduction and Genetics, Pundang Je- Saeng General Hospital, Dae-jin Medical Center, Kyungki-do , Korea (FAX: ; sonyoung@dmc.or.kr). a Center for Reproduction and Genetics, Pundang Je-Saeng General Hospital. b Department of Obstetrics and Gynecology, Pundang Je-Saeng General Hospital. c Department of Urology, Pundang Je-Saeng General Hospital. d Department of Life Science, Sogang University /00/$20.00 PII S (00) Objective: To evaluate the messenger RNA (mrna) expression of hspa2 in testes of infertile men with azoospermia. Design: Prospective study. Setting: Center for Reproduction and Genetics, Pundang Je-Saeng General Hospital, Dae-Jin Medical Center, Korea. Patient(s): Azoospermic patients (n 15) undergoing testicular biopsy for pathologic evaluation were selected. Intervention(s): After pathologic evaluation, testicular biopsy specimens were subdivided into three groups: group 1, normal spermatogenesis (n 5); group 2, spermatocyte arrest (n 5); and group 3, Sertoli cell only syndrome (n 5). The levels of hspa2 mrna expression were compared in testes of group 1, group 2, and group 3 with the use of a competitive reverse transcription polymerase chain reaction (RT-PCR) technique. Main Outcome Measure(s): Comparison of hspa2 mrna levels in testes. Result(s): On competitive RT-PCR analyses for hspa2 mrna, significant hspa2 expression was observed in group 1, whereas a very low level of hspa2 was expressed in groups 2 and 3. Conclusion(s): This study demonstrates that hspa2 gene expression is down-regulated in human testes with abnormal spermatogenesis, which in turn suggests that the hspa2 gene might play a specific role during meiosis in human testes. (Fertil Steril 2000;73: by American Society for Reproductive Medicine.) Key Words: Spermatogenic arrest, human testes, hspa2 mrna, Sertoli-cell only syndrome, competitive RT-PCR Spermatogenesis is a unique process of cellular differentiation in which diploid testicular stem cells differentiate into haploid spermatozoa (1, 2). During meiosis and spermiogenesis, male germ cells undergo major morphologic and biochemical changes. These changes are regulated by the stage-specific activation of a large number of genes under temporal and cellspecific regulation (3). Recently, some genes expressed during spermatogenesis have been identified as causes of abnormal spermatogenesis in murine models. About 10% 20% of testicular biopsy specimens obtained from azoospermic men demonstrate spermatogenic arrest, which may be associated with hormonal disturbances and/or chromosomal abnormalities (4 8). Although spermatogenic arrest may occur at any number of stages, an in-depth understanding of its biochemical regulation remains elusive (9, 10). Members of the 70-kd heat shock protein (hsp70) family, which assist in the folding, transport, and assembly of proteins in the cytoplasm, are associated with the development of male germ cells (11). It has been demonstrated that two unique members of the hsp70 family, hsp70-2 and hsc70t, are expressed during murine spermatogenesis (12 15). Dix et al. (16) demonstrated that knockout of the hsp70-2 gene in mice leads to arrest in maturation of the primary spermatocytes at stage 1 of meiosis, whereas the homozygous mutant (hsp70-2 / ) male mice become infertile. These observations indicate that the hsp70-2 gene has a 1138

2 unique function, which is required for murine spermatogenesis. The hspa2 gene is the human homolog of the murine hsp70-2 gene, with 91.7% identity in the nucleotide coding sequence (17). To investigate the biologic significance of the hspa2 gene in human spermatogenesis, we evaluated the mrna expression of the hspa2 gene in the testis biopsy specimens of infertile men with azoospermia. MATERIALS AND METHODS Tissue Preparation Before beginning the study, we obtained approval from the Institutional Review Board of the Pundang Je-Saeng General Hospital. Testicular biopsies were performed in infertile men with azoospermia in a standard clinical fashion. The patients had no other significant pathologies. After biopsy, the testicular tissue was divided into two parts. One part was stored immediately in liquid nitrogen for reverse transcription polymerase chain reaction (RT-PCR). Another portion was fixed in Bouin s solution and later embedded in paraffin, sectioned, and stained with hematoxylin and eosin in a standard fashion. In addition, immunohistochemistry to cyclic adenosine monophosphate (camp)-responsive element modulator was performed in sectioned testis for the evaluation of spermatocyte arrest. After pathologic and immunohistochemical evaluation, the specimens stored in liquid nitrogen were subdivided into three groups on the basis of the results: group 1, normal spermatogenesis (n 5); group 2, spermatocyte arrest (spermatids not present) (n 5); and group 3, Sertoli cell only syndrome (n 5). Immunohistochemistry Immunohistochemical staining was performed with use of an avidin-biotin immunoperoxidase technique (DAKO, Cambridge, UK). Rabbit polyclonal antibodies (Calbiochem, San Diego, CA) against camp-responsive element modulator protein were used as primary antibodies with a dilution of 1:50. After incubation with the primary antibody, the second incubation was with biotinylated polyvalent antibody and the third incubation was with avidin-horseradish peroxidase. The chromogenic reaction was developed by incubation with a solution of substrate-chromogen (AEC ; DAKO). Negative controls were incubated with preimmune serum. All sections were counterstained with hematoxylin and mounted in an aqueous medium. Total RNA Extractions Total RNA was obtained from each testis according to a procedure developed by Gibco BRL (Grand Island, NY). The testicular specimens were placed in a glass tissue grinder in 1 ml of TriZol solution (Gibco BRL) and homogenized until an even suspension was obtained. The homogenate was transferred to a microcentrifuge tube and incubated for 5 minutes. Chloroform was added to the tube, and the samples were centrifuged at 12,000 g for 20 minutes at 4 C. The aqueous phase was transferred to a fresh tube and isopropanol (1 vol) was added. After incubation for 10 minutes, the RNA was pelleted by centrifugation at 12,000 g for 10 minutes at 4 C. The pellet was washed with 75% ethanol and dried briefly. The RNA pellet was resuspended in 20 L of diethyl pyrocarbonate treated sterile water. The contaminated DNA was removed by DNase I (Gibco BRL) digestion. To measure the concentration, we diluted 2 L of the RNA suspension with 600 L of diethyl pyrocarbonate treated sterile water and the optical density at 260 nm was read on the spectrophotometer. The remaining RNA was stored at 85 C for later use. Construction of the Competitor for Competitive RT-PCR The sequences of oligonucleotide primers for hspa2 were determined with a 343-bp fragment from the 3 -untranslated portion of the cdna ( ), which does not show sequence homology with other human hsp70 genes. The synthetic oligonucleotide primers that were used in RT-PCR analysis of hspa2 were: sense, 5 -TTGTTGGAAGTCCTTG GTATA-3 ; and antisense, 5 -CATTTGCATTTATGCATT TGT-3. Template 343-bp cdnas for hspa2 were obtained by RT-PCR amplification of total testis RNA. The RT-PCR amplicons were cloned into pmosblue vector (Amersham, Arlington, IL), and the direction and sequence specificity of inserted cdna were confirmed by sequence analysis. Deletion mutant cdna for hspa2 was constructed as follows. Digestion of RT-PCR amplicons with MseI produced several fragments. MseI-digested cdna fragments were religated without further purification. Ligates were cloned into pmosblue vector, and bacterial clones containing mutant cdna were selected by PCR amplification of plasmid DNAs. The direction and sequence specificity of inserted competitor were confirmed by sequence analyses. A deletion mutant cdna of hspa2 was 200 bp in size. Reverse Transcription Polymerase Chain Reaction Reverse transcription was performed by priming with antisense primers for hspa2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH was used as an endogenous internal control to which other PCR amplification products were normalized. The sequences for GAPDH were as described by Wong et al. (18). Total RNA (5 g) was denatured in the presence of 2 pmol of antisense primers for 10 minutes at 65 C, followed by quick chilling in ice, and 7 L of master mix (10 RT buffer, 1 L dntp mix [2.5 mm each], 50 mm MgCl 2, 200 mm dithiothreitol) was added to the RNA. The RNA mixture was then prewarmed for 5 minutes at 42 C. Reverse transcriptase (AMVe; Promega, Madison, WI) was added and incubated for 50 minutes at 42 C. The temperature was raised to 65 C for 15 minutes to terminate the reaction. FERTILITY & STERILITY 1139

3 For competitive PCR, sequential dilutions of mutant cdnas were added into the RT product before PCR. PCR amplification was performed with 2 L of RT reaction mixture in 50 L of reaction mixture containing 5 L of 10 PCR buffer (supplied by Takara, Tokyo, Japan), 2.5 L of dntp mix (2.5 mm each), and 0.5 U of Ex-Taq polymerase (Takara). In each reaction, 10 pmol of PCR primers was added. The samples were then subjected to amplification on the PCR cycler. PCR was performed under the following conditions: 94 C for 30 seconds, 58 C for 30 seconds, and 72 C for 30 seconds. After the last cycle, incubation at 72 C was extended for 10 minutes. The RT-PCR products were separated by electrophoresis in a 2% agarose gel. The ethidium bromide stained gels were visualized with an ultraviolet light source. The gels were photographed on a gel video imager (Gel Documentation system 1000; Bio-Rad Laboratories, Hercules, CA). The integral values of the peak areas for each band were quantified with the use of ImageMaster VDS software (Pharmacia Biotech, Uppsala, Sweden). RESULTS In normal spermatogenesis, the seminiferous tubules contained spermatogenic cells in mitotic (spermatogonia), meiotic (spermatocytes), and postmeiotic (spermatids) phases of development (Fig. 1A). However, patients with spermatocyte arrest were deficient in postmeiotic spermatids (Fig. 1B), and the seminiferous tubules contained spermatocytes with condensed nuclei. The seminiferous tubules of Sertoli cell only syndrome were completely deficient in meiotic germ cells (Fig. 1C). Spermatocyte arrest was also confirmed by immunohistochemistry to camp-responsive element modulator, which is expressed in round spermatid cells with specificity (19) (Fig. 1). As expected, camp-responsive element modulator was not detected in testes with spermatocyte arrest and Sertoli cell only syndrome (Figs. 1E, F). However, we observed a typical staining pattern in round spermatid cells with normal spermatogenesis (Fig. 1D; arrowheads). The pathologic and immunohistochemical stains in the three groups are shown in Figure 1. For a quantitative comparison of hspa2 mrna between testes with normal spermatogenesis (group 1) and abnormal testes (groups 2 and 3), competitive RT-PCR analysis was performed (Fig. 2). Significant hspa2 mrna expression was observed in all testes of group 1 (Fig. 2B). However, the groups with spermatocyte arrest (group 2) and Sertoli cell only syndrome (group 3) showed a constitutively low level of hspa2 mrna expression (Figs. 2C, D). In contrast, the conspicuous pattern of GAPDH mrna, as the internal control, was uniform among the groups (Fig. 2A). The relative levels of hspa2 mrna expression in testes of all groups used for these studies are shown in Figure 3. DISCUSSION This study demonstrates that hspa2 mrna expression is repressed in human testes with abnormal spermatogenesis. These findings provide a critical clue that the role of hspa2 in human spermatogenesis might be same as the role of hsp70-2 in mouse spermatogenesis. The role of hsp70-2 was proved by the fact that knockout of hsp70-2 in the seminiferous epithelium arrested spermatogenesis. Genetically engineered hsp70-2 deficient mice with a normal phenotype except the seminiferous epithelium were completely devoid of spermatids and spermatozoa (16). Because the hspa2 gene is the human homolog of the murine hsp70-2 gene, we hypothesized that hspa2 gene expression may be disrupted in human testes with abnormal spermatogenesis. To test this hypothesis, we examined the expression of hspa2 mrna in infertile men with azoospermia. The results from spermatocyte maturation arrest in infertile men were consistent with hspa2 mrna deficiency. The spermatocyte arrest was evaluated by histology and immunohistochemistry. We characterized the testes of men with azoospermia due to gonadal failure in terms of the presence of spermatids. In 1998, Lin et al. (19) suggested that camp-responsive element modulator has an important role in spermatid development and is a stage-specific regulator of human spermatogenesis. In that study, although campresponsive element modulator was present in the round spermatid stage of normal or abnormal spermatogenesis (spermatid maturation arrest and hypospermatogenesis), the complete absence of expression of camp-responsive element modulator was confirmed in the specimens from patients with Sertoli cell only syndrome and spermatocyte maturation arrest. Therefore, we used the expression of camp-responsive element modulator as a stage-specific marker of human spermatogenesis. On the basis of this expression and the histologic findings, the samples were classified into three groups and the hspa2 expression was quantified by RT-PCR. In quantitative RT-PCR, there are some important limitations for mrna quantification (18). For example, PCR amplifications do not reside in the exponential phase indefinitely; beyond a defined point in the reaction, the product levels no longer accumulate in proportion to the starting materials. A second problem is the variability in product output from one tube to another. This variability in amplification efficiency is largely uncontrollable and is likely due to a number of unrelated factors. Because of these problems in the RT-PCR reaction, very small differences in the amplification efficiency of patient samples can have profound effects on the analysis of the final product. To compensate for these variations in amplification and reverse transcription efficiencies, we performed the PCR reaction by coamplification of target templates together with 1140 Son et al. Expression of hspa2 mrna in human testes Vol. 73, No. 6, June 2000

4 FIGURE 1 Hematoxylin and eosin stains and immunohistochemistry (magnification, 200). For hematoxylin and eosin stains, (A) shows testis of normal spermatogenesis, group 1; (B) shows testis of spermatocyte arrest, group 2; (C) shows testis of Sertoli cell only syndrome, group 3. For immunohistochemistry of camp-responsive element modulator protein, (D) shows testis of normal spermatogenesis; (E) shows testis of spermatocyte arrest; (F) shows testis of Sertoli cell only syndrome. Positive campresponsive element modulator immunoreactivity is indicated by the arrows. Son. Repression of hspa2 mrna. Fertil Steril FERTILITY & STERILITY 1141

5 FIGURE 2 Competitive RT-PCR analysis for hspa2 transcripts. (A), For internal control, GAPDH was used; lane M molecular-weight markers; lane 1 normal spermatogenesis; lane 2 spermatogenic arrest; lane 3 Sertoli cell only syndrome; lane 4 negative control. For competitive RT-PCR, (B) shows normal spermatogenesis, group 1; (C) shows spermatogenic arrest, group 2; (D) shows Sertoli cell only syndrome, group 3. Son. Repression of hspa2 mrna. Fertil Steril exogenous competitor plasmid. The results from our competitive RT-PCR assays provide relative RNA data, which in many cases are sufficient for studies of mrna expression kinetics and other comparative experiments. The expression of hspa2 mrna in testes with spermatogenic arrest was significantly lower than that in testes with normal spermatogenesis, implying that the hspa2 gene is repressed in testes with spermatocyte arrest (Fig. 3). The 1142 Son et al. Expression of hspa2 mrna in human testes Vol. 73, No. 6, June 2000

6 FIGURE 3 Relative levels of hspa2 mrna expression in testes in the three groups. Group 1 normal spermatogenesis; group 2 spermatogenic arrest; group 3 Sertoli cell only syndrome. studies, implying that hspa2 expression is regulated mostly at the transcriptional level. In conclusion, it is strongly suggested that the hspa2 gene may play a specific role during human spermatogenesis. Repression of the hspa2 gene may be a cause of testicular failure in various types of male infertility. This finding has important implications in the clinical management of infertility in men in the era of rapidly advancing assisted reproductive technology. Further molecular studies at the DNA and protein levels are necessary to elucidate the mechanism of regulation of spermatogenesis by the hspa2 gene. Son. Repression of hspa2 mrna. Fertil Steril very low level of hspa2 mrna expression also was observed in testes with Sertoli cell only syndrome, showing that hspa2 mrna is expressed basically in mitotic cells. It seems that the hspa2 mrna was constitutively expressed in most human tissues (17). The clinical features associated with the histologic diagnosis of human spermatogenesis are extremely variable (20, 21). Biopsy evidence of bilateral testicular epithelium does not indicate total phenomena of spermatogenesis in the testes (21). However, we analyzed hspa2 expression by competitive RT-PCR in same biopsy specimens as for the histologic and immunohistochemical analyses. Therefore, the patterns of spermatogenesis were not variable, at least in the biopsied samples in our study. In general, the regulation of gene expression in cells occurs at three levels: transcription, translation, and posttranslation. 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 (3, 22). The human hspa2 gene has a single open reading frame of 1,917 bp with a predicted molecular weight of 70,030 d (17). The hspa2 gene product was 98.2% identical in amino acid sequence to the mouse hsp70-2 gene product (17). We previously demonstrated that antiserum 2A (23) raised against mouse hsp70-2 protein cross-reacted with human hspa2 protein (24). In that study, we reported that hspa2 protein is highly expressed in germ cells of human testis. However, the hspa2 protein expression in testes with Sertoli cell only syndrome was very low. The hspa2 mrna expression is in agreement with the protein Acknowledgments: The authors thank Ri-Cheng Chian, Ph.D. (Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada), for critical review of this manuscript. They also thank Edward D. Kim, M.D., Ph.D. (Department of Surgery/Urology, University of Tennessee Medical Center, Knoxville, Tennessee), for his helpful suggestions and discussions. References 1. de Kretser DM, Kerr JB. The cytology of the testis. In: Knobil E, Neill J, editors. The physiology of reproduction. New York: Raven Press, 1988: Eddy EM, O Brien DA, Welch JE. Mammalian sperm development in vivo and in vitro. In: Wassarman PM, editor. Elements of mammalian fertilization. Boca Raton (FL): CRC Press, 1991: Eddy EM. Regulation of gene expression during spermatogenesis. Semin Cell Dev Biol 1998;9: Meyer JM, Roos M, Rumpler Y. Statistical study of a semiquantitative evaluation of testicular biopsies. Arch Androl 1988;10: Cantu JM, Rivas F, Hernandez-Jauregui P, Diaz M, Cortes-Gallegos V, Vaca G, et al. Meiotic arrest at first spermatocyte level: a new inherited infertility disorder. Hum Genet 1981;59: Kretser DM, de Burger HG, Hudson B. 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7 expression by polymerase chain reaction: the primer-dropping method. Anal Biochem 1994;223: Lin WW, Lamb DJ, Lipshultz LI, Kim ED. Absence of cyclic adenosine 3 :5 monophosphate responsive element modulator expression at the spermatocyte arrest stage. Fertil Steril 1998;69: Silber SJ, Nagy A, Devroey P, Tournaye H, Van Steirteghem AC. Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure. Hum Reprod 1997;12: Tournaye H, Verheyen G, Nagy P, Ubaldi F, Goossens A, Silber S, et al. Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Hum Reprod 1997;12: Kleene KC. Patterns of translational regulation in the mammalian testis. Mol Reprod Dev 1996;43: Rosario MO, Perkins SL, O Brien DA, Allen RL, Eddy EM. Identification of the gene for the developmentally expressed 70 kda heat-shock protein (P70) of mouse spermatogenic cells. Dev Biol 1992;150: Son WY, Hwang SH, Han CT, Lee JH, Kim S, Kim YC. Specific expression of heat shock protein HspA2 in human male germ cells. Mol Hum Reprod 1999;12: Son et al. Expression of hspa2 mrna in human testes Vol. 73, No. 6, June 2000

EXPRESSION PROFILING OF CREM GENE IN TESTIS WITH NORMAL AND IMPAIRED SPERMATOGENESIS IN EGYPTIAN MALES

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