- Email: [email protected]
Androgen receptor is expressed in both X- and Y-carrier human spermatozoa
Androgen receptor is expressed in both X- and Y-carrier human spermatozoa Daniela Zuccarello, Ph.D., Andrea Garolla, Ph.D., Alberto Ferlin, Ph.D., Massimo Menegazzo, B.Sc., Luca De Toni, B.Sc., Marina Carraro, B.Sc., Caterina Veronese, Ph.D., and Carlo Foresta, Ph.D. Department of Histology, Microbiology and Medical Biotechnologies, Centre for Male Gamete Cryopreservation, University of Padova, Padova, Italy
Objective: To quantify the amount of AR messenger RNA (mRNA) and to analyze the presence and functionality of the AR protein in X- and Y-carrier human spermatozoa. Design: A molecular and cellular research study. Setting: Academic research clinic and laboratories. Patient(s): Ten normozoospermic volunteers. Intervention(s): Sperm were analyzed for expression of AR mRNA and protein. The functionality of AR protein was assessed after incubation with 1 nM of synthetic androgen. Main Outcome Measure(s): Reverse-transcription polymerase chain reaction (PCR), real-time PCR, Western immunoblotting, confocal immunofluorescence, and fluorescence in situ hybridization analysis were performed. Result(s): A significant amount of AR mRNA (around 30% of that found in the testis) was found in sperm. Androgen receptor protein was found in both X- and Y-carrier spermatozoa and translocates into the nucleus in the presence of synthetic androgen. Conclusion(s): A functional AR is expressed in human sperm. In view of the fact that AR protein is found in both X- and Y-carrier spermatozoa, it most probably is translated in earlier steps of spermatogenesis and moves to Y-carrier spermatids through the cytoplasmic bridges. (Fertil Steril 2009;91:193–200. 2009 by American Society for Reproductive Medicine.) Key Words: Androgen receptor, sperm, RNA, non-genomic effect, X-carrier Y-carrier sperm
Androgens, principally T and 5-dihydrotestosterone, are steroid hormones that mediate a wide range of physiological responses and developmental processes, involving both reproductive and nonreproductive systems in the male (1). Many physiological actions of androgens are mediated by the androgen receptor (AR), whose function is essential in males for proper sexual differentiation, pubertal development, and regulation of normal spermatogenesis (2). Androgen receptor activity is regulated by the steroid ligand T and its metabolite 5-dihydrotestosterone, the binding of which on the receptor initiates nuclear translocation and the transcriptional regulatory function of AR (3). Recently, in addition to this genomic mode of action by steroids, an increasing body of evidence suggests that androgens can exert a rapid, nongenomic effect, mediated through a membrane AR or through c-Src kinase–AR complex (4). The central role of androgens in the male reproductive system has prompted investigation into the presence and distribution of AR in male reproductive tissues (5). The site of action of androgens in the testis has been studied extensively, and for a long time the expression of AR has been restricted exclusively to Sertoli cells, Leydig cells, and peritubular or myoid cells in the testis (6–9), suggesting that the role of Received October 18, 2007; revised and accepted November 14, 2007. Reprint requests: Carlo Foresta, Ph.D., Department of Histology, Microbiology, and Medical Biotechnologies, Centre for Male Gamete Cryopreservation, University of Padova, Via Gabelli 63, 35121 Padova, Italy (FAX: 39-049-8218520; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2007.11.040
androgens in spermatogenesis is mediated by these cells (10). The recent generation and characterization of male AR knockout mice and Sertoli cell–selective AR knockout mice confirmed the essential role of AR signaling for both external and internal male phenotype development (11, 12). Recently, the presence of AR in human sperm was demonstrated by Solakidi et al. (13) and Aquila et al. (14), suggesting a constitutive functional role of AR in these cells. Because the ejaculated spermatozoa are considered transcriptionally silent cells, a nongenomic action was assumed, as suggested by a large series of experiments highlighting the ability of AR to modulate the phosphatidylinositol-3-OH/alpha-protein kinase B pathway. Besides, Solakidi et al. (13) used immunofluorescence to localize the AR in the sperm midpiece region, whereas Aquila et al. (14) localized AR in the sperm head, imputing the discrepancy to the different methods used to process samples. The conflicting findings concerning the expression pattern of AR in sperm cells have motivated us to better explore the distribution of this steroid hormone receptor in human sperm. Moreover, the origin of AR protein has not been sufficiently explained, because it may derive from translation in the early steps of spermatogenesis or during spermatid development (spermiogenesis). Therefore, in this study, we quantified the amount of AR mRNA in human sperm, confirmed the presence of AR protein, showed the functional integrity of the AR protein, and examined the presence of the AR protein in X- and Y-carrier spermatozoa.
Fertility and Sterility Vol. 91, No. 1, January 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
193
MATERIALS AND METHODS Semen Sample Collection and Processing Human semen samples were obtained by masturbation after 2– 5 days of sexual abstinence, from 10 normozoospermic volunteers who had provided informed consent. Samples were allowed to liquefy for 30 minutes and were examined for seminal parameters according to the World Health Organization (15) criteria. Samples with abnormal viscosity, presence of leukocytes, or immature germ cells were not included in this study. The samples were washed once using sperm washing medium (Irvine Scientific, Santa Ana, CA). Motile spermatozoa were selected by using a routine swim-up procedure, and cell viability was evaluated by eosin test. In all cases, the percentage of living cells was >90%. In stimulation experiments, spermatozoa were suspended in sperm washing medium at a concentration of 2 106 cells/mL and were incubated overnight with 1 nM of synthetic androgen R1881 (methyltrienolone) at 37 C; in the negative control, the hormone was omitted. Each experiment was performed four times, and in at least three of those, normal samples were pooled. Synthesis of Complementary DNA and ReverseTranscription Polymerase Chain Reaction Analysis Ribonucleic acid was extracted from human sperm and testis (testicular biopsy from a man with obstructive azoospermia) by using the RNeasy Micro Kit (Qiagen, Milan, Italy), which includes DNase treatment. Reverse-transcription polymerase chain reaction (RT-PCR; complementary DNA [cDNA] synthesis) was performed by using total RNA (50 ng) and the QuantiTect RT Kit (Qiagen), with random hexamers. The quality of obtained RNA and cDNAwas tested by spectrophotometer measurement (NanoDrop; Celbio, Milan, Italy). Total cDNAwas amplified by PCR using specific exon 1 primers for human AR (forward, 50 -TAGCCCCCTACGGCTACA-30 ; reverse, 50 -TTCCGAAGACGACAAGATGGAC-30 ; 322 bp), as described elsewhere (1). As a negative control, the cDNA was omitted, whereas for RT control, a template-free RT enzyme was used. Primers for CD4 (forward, 50 -CAGGGAAAGAAAGTGGTGC T-30 ; reverse, 50 - TTCTGGTCCTCCACTTCACA-30 ; 275 bp; exon spanning) and acrosin (forward, 50 -AACTCTGCGACA GAGGGAAA-30 ; reverse, 50 -CACACATTGGTTGGCTGA AC-30 ; 272 bp; exon spanning) were used to exclude the presence of leukocyte contamination and to verify the presence of sperm cDNA, respectively. The melting temperature was 60 C for both primers’ pairs. The RT-PCR products were electrophoretically analyzed through 1% agarose gel, were visualized by SYBR-safe DNA gel staining (Invitrogen, Milan, Italy), and were confirmed by direct sequencing on an ABI Prism sequencer (Applied Biosystems, Monza, Italy). Real-Time PCR For real-time PCR, we used the ABI PRISM 7900 HT (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The amplification reactions were performed in a 25-mL final volume containing 12.5 194
Zuccarello et al.
AR in human spermatozoa
mL of 2 TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 2.5 mL of AR TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA), and 10 mL (20 ng) of cDNA obtained from ejaculated sperm and testis biopsy. The assay’s probes span an exon junction (exon boundary, 4–5 for AR and 1–2 for b-actin) and do not detect genomic DNA. Amplification was performed for 40 cycles. After an initial hot start for 10 minutes, each cycle consisted of 15 seconds of denaturation, 30 seconds of annealing at 60 C, and 30 seconds of extension at 60 C. To normalize the amount of expressed AR mRNAs, the internal housekeeping gene b-actin was used, and each cDNA product was tested in triplicate. To calculate our data, we used the comparative Ct method for relative quantification (the DDCt method), which describes the change in expression of the target gene in the tested sample relative to a calibrator sample and provides an accurate comparison between samples of the initial level of template in each. As a calibrator sample, we used RNA extracted by skin fibroblasts, which was processed in the same way as the tested sample. Data were analyzed by using the Sequence Detector System software (version 2.1; Applied Biosystems, Foster City, CA). Western Immunoblotting Sperm lysates were prepared from sperm by using the Bioplex Cell Lysis Kit (BioRad, Milan, Italy). Total protein content was quantified by using the Bradford Protein Assay Kit (BioRad), and protein samples (30 mg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 7% gel and blotted onto polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Milan, Italy). The membrane was probed with the mouse monoclonal antibody against human AR, which recognizes amino acid residues 301–320, corresponding to the N-terminal domain, followed by incubation with the goat anti-mouse immunoglobins–horseradish peroxidase (BioRad). Immunoreactive proteins were visualized by using the enhanced chemiluminescence system detection kit (ECL; PerkinElmer, Monza, Italy) and by using autoradiography with radiography film. A human testis protein library (Celbio) was used as positive control. The negative control was obtained by omitting the primary antibody. Immunofluorescence Sperm samples were washed and suspended in phosphatebuffered saline at a concentration of 2 106 cells per microliter. One hundred microliters of that cell suspension were spotted on Polysine Microscope Slides (Menzel GmbH & Co, Braunschweig, Germany) by means of Cytospin 3 (Thermo Electron Corporation, Waltham, MA) at 1,500 rpm for 2 minutes. Spermatozoa samples were allowed to air dry, fixed in phosphate-buffered saline–paraformaldehyde 4% for 15 minutes, and permeabilized with phosphate-buffered saline–Triton X-100 (3%) for 5 minutes (Sigma Aldrich, St. Louis, MO), all at room temperature. Samples then were treated with phosphate-buffered saline–normal goat serum Vol. 91, No. 1, January 2009
15% (Vector Labs, Burlingame, CA) at room temperature and were incubated overnight with primary antibodies at 4 C. For double immunostaining, samples were simultaneously incubated with rabbit polyclonal antibody to AR (1:1,000) and mouse monoclonal anti-b-actin antibody (1:100; Abcam, Cambridge, UK). Primary immunoreactions then were detected by incubation with rabbit F(ab0 ) R-phycoerythrin–conjugated anti-mouse immunoglobulins and swine F(ab0 ) fluorescein isothiocyanate–conjugated anti-rabbit immunoglobulins (both, 1:100; Dako Cytomation, Glostrup, Denmark) for 1 hour at room temperature. Finally, slides were counterstained with 6-diamino-2-phenylindole and were mounted with antifade buffer and a 24 24-mm coverslip. The immunostaining was evaluated by using a laser confocal microscope (UltraView; PerkinElmer, Monza, Italy). For each subject, R100 cells were evaluated. Negative controls were performed by omitting the primary antibody. Sperm Chromosome Analysis The study of sperm chromosomes was performed by using multicolor fluorescence in situ hybridization, as reported elsewhere (16). Hybridization of DNA was performed by using human a-satellite probes that were specific for chromosomes X, Y, and 18 (Kreatech Biotechnology B.V., Amsterdam, the Netherlands) that were directly labeled by using fluorophores PlatinumBright495 (chromosome X, green) and PlatinumBright550 (chromosome Y, red); for the detection of chromosome 18, a mixture (1:1) of PlatinumBright495 and PlatinumBright550 directly labeled specific probes was used, resulting in a yellow signal. Deoxyribonucleic acid denaturation of sperm and probes, incubation, and posthybridization washing were performed according to the Kreatech Biotechnology protocol. After preparation, slides were stored (1–4 d, at 4 C) or immediately were observed by using an Eclipse E600 Fluorescence Microscope (Nikon, Melville, NY) that was fitted with a 100-W mercury lamp and a triple band-pass filter that was suitable for the fluorochromes in use. We performed the fluorescence in situ hybridization analysis on previously AR-immunohybridized, nonstimulated sperm, as already described earlier in this subsection. Single spots were evaluated as reported elsewhere (17, 18). For each subject, R200 cells were scored. RESULTS Analysis of RNA and cDNA Reverse-transcription PCR amplification resulted in the expected PCR product size of 322 bp in sperm and testis (Fig. 1a). Real-time PCR assay was used to quantify the amount of mRNA for AR in sperm, compared with testis. By fixing the amount of AR mRNA expressed in testis to 100, we estimated the total amount of AR mRNA in sperm to be 27.7% (relative quantification for testis, 9.803, with SD of 0.611; and relative quantification for sperm, 2.718, with SD of 0.815), as shown in Figure 1b. By evaluation of b-actin internal housekeeping gene expression, we observed that the amount of total mRNA present in ejaculated sperm Fertility and Sterility
was globally lower (mean b-actin Ct in testis, 24.4271; vs. in sperm, 30.47002) than that in testis. Western Immunoblotting The presence of AR protein in sperm was investigated by Western blot by using a monoclonal antibody recognizing amino acid residues 301–320, corresponding to the N-terminal domain. The antibody showed the presence in sperm of a prominent 110-kDa band, corresponding to the full AR protein, and a very light 87-kDa band, which probably represents an AR cleavage product (Fig. 1c). The same pattern of bands, with a similar intensity, was found in human testis protein extract, which was used as positive control. Immunofluorescence Immunolocalization of androgen receptor in nonstimulated sperm demonstrated a diffuse light staining of the sperm tail and an intensification of the signal in the postacrosomal region and initial region of the midpiece (Fig. 2a–g), as shown in the overlapping image of b-actin (green, Fig. 2a) and AR (red, Fig. 2b), resulting in a yellow color (Fig. 2f). The merged picture (Fig. 2g: red for AR, blue for DAPI, and green for b-actin) clearly showed the acrosomal region in green, the nucleus in blue, and the postacrosomal region and initial region of the midpiece in yellow. No purple color (overlapping of nuclear blue and red AR) was shown in this basal-condition experiment. After overnight androgen stimulation, the signal in the postacrosomal region and midpiece disappeared, whereas it was present strongly in the sperm head (Fig. 3b) and weakly in the sperm tail, as shown in the overlapping image of AR (red) and nucleus signals (blue), resulting in a purple color (Fig. 3e), and in the overlapping of AR (red) and b-actin (green; Fig. 3f). The merged picture (Fig. 3g) showed a clear superimposition of AR (red) and nuclear signal (blue DAPI), without any overflow in the acrosomal region. Negative controls (not pictured in the figures) showed no signal. Fluorescence In Situ Hybridization Analysis By using a fluorescence in situ hybridization technique for recognizing the X, Y, and 18 chromosomes on previously AR-immunohybridized, nonstimulated sperm, we found that both X- and Y-carrier spermatozoa expressed AR (Fig. 4a and b). The AR signal remained localized in the midpiece of spermatozoa, as we observed in fresh ejaculated sperm that were not subjected to fluorescence in situ hybridization treatment. However, clear AR immunolocalization within this picture was not possible, because the cells underwent strong chemical and physical treatments for fluorescence in situ hybridization analysis. The percentage of X-carrier and Y-carrier sperm was around 1:1 (53% vs. 47%, respectively). DISCUSSION Androgens, by signaling through the androgen receptor, mediate a wide range of physiological responses and 195
FIGURE 1
FIGURE 1 Continued Expression of AR in human-ejaculated spermatozoa. (A) Reverse-transcription PCR analysis of the human AR gene. The expected 322-bp band is displayed both in testis and sperm samples. Primers for CD4 and acrosin were used, respectively, to exclude the presence of leukocyte contamination and to verify the presence of sperm cDNA. In the RT lanes, a template-free RT enzyme was used. In the RTþ lanes, a template plus RT enzyme was used. In the negative lane, the cDNA was omitted. (B) Real-time PCR of AR gene. The amount of AR mRNA in sperm (white rectangle) is 27.7% of the AR mRNA expressed in testis (gray rectangle). (C) Western blotting of AR protein by using a monoclonal antibody raised against the N-terminal domain, showing a two-band pattern (110 and 87 kDa), both in sperm and testis. Negative control was obtained by omitting the primary antibody. All the experiments were repeated at least three times on 10 different samples, and the results of one representative experiment are depicted. No significant differences were detected among the samples.
suggesting a functional role of AR in these cells. However, some questions have remained open, in particular regarding the precise sperm cell localization of the AR (head or midpiece), its functional integrity, and the origin of AR protein.
Zuccarello. AR in human spermatozoa. Fertil Steril 2009.
developmental processes, including the correct development of functional male gametes. Indeed, expression of AR has been demonstrated clearly in Sertoli, Leydig, peritubular myoid, and vascular smooth muscle cells of the testis (6, 19–26), and it initially was thought that AR was not expressed in germ cells (19, 20, 27). However, more recent evidence has suggested expression of AR in spermatogonia (21, 24), spermatocytes (21), and elongated spermatids at spermatogenic stage XI (23), in both rat and human beings. Recently, the presence of the AR in human sperm was demonstrated by Solakidi et al. (13) and Aquila et al. (14), 196
Zuccarello et al.
AR in human spermatozoa
In this study, we confirmed the presence of AR in human spermatozoa at the RNA and protein level, including a relative quantification of the total amount of AR mRNA present in these cells. Our results showed that the sperm content of AR mRNA is around 30% of that found in the testis. Moreover, in evaluating the b-actin gene expression, we observed that the amount of total RNA present in ejaculated sperm is very low, confirming reports elsewhere (28) that indicated low RNA quantity in these transcriptionally dormant cells. On the basis of the general assumption that ejaculated spermatozoa do not synthesize RNA and proteins (29), the presence of transcripts in these cells traditionally has been attributed to the presence of residual cytoplasmic droplets that escape absorption by the Sertoli cells or to the presence of immature spermatids or somatic cells in the ejaculates from which spermatozoa are traditionally obtained. Nevertheless, in the last 15 years, a large number of investigators have demonstrated the presence of numerous mRNA in spermatozoa (29), and recently, Ostermeier et al. (30) reported the simultaneous detection by using DNA arrays of R3,500 unique mRNA in the ejaculate sperm. These RNA are thought to originate in early steps of spermatogenesis or during spermatid development and may diffuse among spermatids, thanks to the cytoplasmic sharing between connected spermatids. Interestingly, although it now has been confirmed that human sperm do not transcribe novel RNA (31) because Vol. 91, No. 1, January 2009
FIGURE 2 Immunolocalization of AR in human ejaculated spermatozoa in basal condition (fresh ejaculate), as evaluated by laser confocal microscope. (A) Green, b-actin; (B) red, AR; (C) blue, DAPI; (D) green þ blue; (E) red þ blue; (F) green þ red, resulting in yellow; and (G) merged image (green þ red þ blue).
Zuccarello. AR in human spermatozoa. Fertil Steril 2009.
of the shutdown of nuclear transcription that precedes the package of spermatozoal DNA, some studies have indicated the presence of a latent translational capacity in sperm, on the basis of a measured response to cycloheximide (32) and as also suggested by the finding that mitochondrial ribosomes are actively involved in protein translation in spermatozoa (33). However, the real function of these RNA in sperm remains unclear, even if the most likely conjectures suppose a role in the selective nuclear DNA repackaging mediated by protamines, in paternal imprinting mediation and in the male-gamete contribution to early embryogenesis (29). By Western blot analysis, we found both the 110- and the 87-kDa bands both in testis and sperm. The 110-kDa AR form universally is considered to correspond to the full-length AR, whereas the 87-kDa form is considered to be the result of in vitro proteolysis, as extensively demonstrated by Gregory et al. (34). This consideration is consistent with our experimental series, in which we observed the 87-kDa band more or less intensely in different instances, depending on the number of cycles of freezing and thawing (personal data). Fertility and Sterility
By immunofluorescence and confocal microscope observation, we have demonstrated the presence of AR protein in the sperm postacrosomal region and initial part of the midpiece in the basal condition, followed by nuclear (head) translocation of the receptor after overnight androgen stimulation. In agreement with our observations, Solakidi et al. (13) reported an intense staining of the sperm midpiece, corresponding to the site of concentration of the mitochondria, in addition to a diffuse reactivity in the tail region. On the contrary, Aquila et al. (14) detected a prevalent signal localization in the sperm head and ascribed this apparent discrepancy to the different methods used to process samples. In particular, it is not clear in the article by Aquila et al. (14) whether the spermatozoa used to perform immunofluorescence assays were at the basal condition or were being used after 30 minutes of androgen stimulation or incubation in capacitating medium, making difficult a direct comparison of their results with ours. One of the most important findings of our study is that the AR protein is fully functional, because it translocates into the 197
FIGURE 3 Immunolocalization of AR in human-ejaculated spermatozoa after overnight stimulation by R1881. (A) Green, b-actin; (B) red, AR; (C) blue, DAPI; (D) green þ blue; (E) red þ blue; (F) green þ red resulted in yellow; and (G) merged image (green þ red þ blue).
Zuccarello. AR in human spermatozoa. Fertil Steril 2009.
nucleus after androgen stimulation. Therefore, our findings may suggest that androgens exert their effect directly on sperm, through a classical AR genomic pathway. However, spermatozoa are considered transcriptionally inactive, and therefore much evidence has concentrated on the possible rapid, nongenomic effects of androgens, as mediated by membrane-bound AR. In fact, it has been demonstrated that androgens in sperm lead in a dose-dependent manner to the rapid phosphorylation of specific AR residues, which leads to the start of the phosphatidylinositol-3-OH/alpha¼protein kinase B pathway, which is involved in activation of caspases 3, 8, and 9 (14). Moreover, in osteoblasts and osteocytes (35), the non-genotropic signaling of the androgens through the AR also has been described, with complete dissociation from transcriptional activity mediated by the receptor. In these cells, the nongenomic effects of AR involve the MAPK pathway, interacting with the SH3 domain of c-Src. The significance of the nuclear translocation of the AR after stimulation with androgens in sperm therefore remains unclear. 198
Zuccarello et al.
AR in human spermatozoa
On the basis of AR sperm midpiece localization in the basal condition, it has been supposed that AR is involved in mitochondrial function in a way that is related to the energy requirements of these cells (13). In fact, Solakidi et al. (13) showed that AR internal sequences are similar to those observed in some mitochondrial inner membrane proteins, assuming a possible role of AR in sperm motility regulation. Besides, it is possible that intact AR protein, located in the sperm nucleus after androgen stimulation, plays a role in the first steps of embryonic development, on the basis of the well-known anabolic action of androgens (36). The second most important finding of our study is the presence of AR protein in both X- and Y-carrier spermatozoa, which gives a clear indication regarding the origin of this protein. In accordance with the current opinion that mature spermatozoa are transcriptionally silent, it may be assumed that the AR gene is transcribed and/or translated during the early steps of spermatogenesis by XY diploid spermatocytes, or Vol. 91, No. 1, January 2009
FIGURE 4 Fluorescence in situ hybridization analysis of 18,X (a) and Y (b) chromosomes in human sperm, evaluated by fluorescence microscope. After AR immunohybridization of fresh ejaculated sperm, the fluorescence in situ hybridization analysis showed that both X- and Y-carrier spermatozoa express AR in the midpiece. Arrows show the specific chromosome signals (yellow, 18; red, Y; green, X). White ring indicates AR immunolocalization.
Zuccarello. AR in human spermatozoa. Fertil Steril 2009.
during spermiogenesis by X-carrier haploid spermatids. The AR protein then could move to Y-carrier spermatids through the cytoplasmic bridges that connect the spermatids (37). Indeed, there is evidence in the literature that other gene products transit the cytoplasmic bridges of the elongated spermatids (38). In conclusion, we showed that the localization of AR in sperm is dependent on the hormonal-stimulation status of the sperm. We showed the functional integrity of AR protein, its translocation into the nucleus after androgen stimulation, and the presence of AR in both X- and Y-carrier spermatozoa, suggesting a possible role for this receptor in the final step of spermatogenesis and/or sperm function. Acknowledgments: The authors thank Denis Bison, B.Sc. (University of Padova, Padova, Italy), for technical assistance with confocal microscopy, Elisa Prana, B.Sc. for technical assistance with RT-PCR, and Albert O. Brinkmann (Erasmus University, Rotterdam, the Netherlands) for kindly providing both the mouse monoclonal and rabbit polyclonal AR antibodies. They also thank all the staff of the Centre for Male Gamete Cryopreservation for helpful discussion.
REFERENCES 1. Ferlin A, Vinanzi C, Garolla A, Selice R, Zuccarello D, Cazzadore C, et al. Male infertility and androgen receptor gene mutations: clinical features and identification of seven novel mutations. Clin Endocrinol 2006;65:606–10.
Fertility and Sterility
2. Weinbauer GF, Nieschlag E. Hormonal control of spermatogenesis. In: de Kretser DM, ed. Molecular biology of the male reproductive system. San Diego, CA: Academic Press, 1993:99–142. 3. Lindzey J, Kumar MV, Grossman M, Young C, Tindall DJ. Molecular mechanisms of androgen action. Vitam Horm 1994;49:383–432. 4. Heinlein CA, Chang C. The roles of androgen receptors and androgenbinding proteins in nongenomic androgen actions. Mol Endocrinol 2002;16:2181–7. 5. McLachlan RI, O’Donnell L, Stanton PG, Balourdos G, Frydenberg M, de Kretser DM, et al. Effects of testosterone plus medroxyprogesterone acetate on semen quality, reproductive hormones, and germ cell populations in normal young men. J Clin Endocrinol Metab 2002;87:546–56. 6. Ruizeveld de Winter JA, Trapman J, Vermey M, Mulder E, Zegers ND, van der Kwast TH. Androgen receptor expression in human tissues: an immunohistochemical study. J Histochem Cytochem 1991;39:927–36. 7. Iwamura M, Abrahamsson PA, Benning CM, Cockett AT, Di Sant’Agnese PA. Androgen receptor immunostaining and its tissue distribution in formalin fixed, paraffin-embedded sections after microwave treatment. J Histochem Cytochem 1994;42:783–8. 8. Goyal HO, Bartol FF, Wiley AA, Neff CW. Immunolocalization of receptors for androgen and estrogen in male caprine reproductive tissues: unique distribution of estrogen receptors in efferent ductule epithelium. Biol Reprod 1996;56:90–101. 9. Suarez-Quian CA, Martinez-Garcia F, Nistal M, Regadera J. Androgen receptor distribution in adult human testis. J Clin Endocrinol Metab 1999;84:350–8. 10. McLachlan RI, O’Donnell L, Meachem SJ, Stanton PG, de Kretser DM, Pratis K, et al. Hormonal regulation of spermatogenesis in primates and man: insights for development of the male hormonal contraceptive. J Androl 2002;23:149–62. 11. Yeh S, Tsai MY, Xu Q, Mu XM, Lardy H, Huang KE, et al. Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues. Proc Natl Acad Sci USA 2002;99:13498–503. 12. Tan KA, De Gendt K, Atanassova N, Walker M, Sharpe RM, Saunders PT, et al. The role of androgens in sertoli cell proliferation and functional maturation: studies in mice with total or Sertoli cell-selective ablation of the androgen receptor. Endocrinology 2005;146: 2674–83. 13. Solakidi S, Psarra AM, Nikolaropoulos S, Sekeris CE. Estrogen receptors alpha and beta (ERalpha and ERbeta) and androgen receptor (AR) in human sperm: localization of ERbeta and AR in mitochondria of the midpiece. Hum Reprod 2005;20:3481–7. 14. Aquila S, Middea E, Catalano S, Marsico S, Lanzino M, Casaburi I, et al. Human sperm express a functional androgen receptor: effects on PI3K/ AKT pathway. Hum Reprod 2007;22:2594–605. 15. World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge, United Kingdom: Cambridge University Press, 1999. 16. Foresta C, Galeazzi C, Bettella A, Stella M, Scandellari C. High incidence of sperm sex chromosomes aneuploidies in two patients with Klinefelter’s syndrome. J Clin Endocrinol Metab 1998;83:203–5. 17. Robbins WA, Baulch JE, Moore D, Weier HU, Blakey D, Wyrobek AJ. Three-probe fluorescence in situ hybridization to assess chromosome X, Y, and 8 aneuploidy in sperm of 14 men from two healthy groups: evidence for a paternal age effect on sperm aneuploidy. Reprod Fertil Dev 1995;7:799–809. 18. Guttenbach M, Martinez-Exposito MJ, Michelmann HW, Engel W, Schmid M. Incidence of diploid and disomic sperm nuclei in 45 infertile men. Hum Reprod 1997;12:468–73. 19. Anthony CT, Kovacs WJ, Skinner MK. Analysis of the androgen receptor in isolated testicular cell types with a microassay that uses an affinity ligand. Endocrinology 1989;125:2628–35. 20. Sar M, Lubahn DB, French FS, Wilson EM. Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 1990;127:3180–6. 21. Kimura N, Mizokami A, Oonuma T, Sasano H, Nagura H. Immunocytochemical localization of androgen receptor with polyclonal antibody in
199
22.
23.
24.
25.
26.
27.
28.
paraffin-embedded human tissues. J Histochem Cytochem 1993;41: 671–8. Bremner WJ, Millar MR, Sharpe RM, Saunders PT. Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology 1994;135:1227–34. Vornberger W, Prins G, Musto NA, Suarez-Quian CA. Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis. Endocrinology 1994;134:2307–16. Zhou X, Kudo A, Kawakami H, Hirano H. Immunohistochemical localization of androgen receptor in mouse testicular germ cells during fetal and postnatal development. Anat Rec 1996;245:509–18. Pelletier G, Labrie C, Labrie F. Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs. J Endocrinol 2000;165:359–70. Zhou Q, Nie R, Prins GS, Saunders PT, Katzenellenbogen BS, Hess RA. Localization of androgen and estrogen receptors in adult male mouse reproductive tract. J Androl 2002;23:870–81. Grootegoed JA, Peters MJ, Mulder E, Rommerts FF, Van der Molen HJ. Absence of a nuclear androgen receptor in isolated germinal cells of rat testis. Mol Cell Endocrinol 1977;9:159–67. Zhao Y, Li Q, Yao C, Wang Z, Zhou Y, Wang Y, et al. Characterization and quantification of mRNA transcripts in ejaculated spermatozoa of fertile men by serial analysis of gene expression. Hum Reprod 2006;21: 1583–90.
200
Zuccarello et al.
AR in human spermatozoa
29. Miller D, Ostermeier GC, Krawetz SA. The controversy, potential and roles of spermatozoal RNA. Trends Mol Med 2005;11:156–63. 30. Ostermeier GC, Dix DJ, Miller D, Khatri P, Krawetz SA. Spermatozoal RNA profiles of normal fertile men. Lancet 2002;360:772–7. 31. Grunewald S, Paasch U, Glander HJ, Anderegg U. Mature human spermatozoa do not transcribe novel RNA. Andrologia 2005;37:69–71. 32. Naz RK. Effect of actinomycin D and cycloheximide on human sperm function. Arch Androl 1998;41:135–42. 33. Gur Y, Breitbart H. Mammalian sperm translate nuclear-encoded proteins by mitochondrial-type ribosomes. Genes Dev 2006;20:411–6. 34. Gregory CW, He B, Wilson EM. The putative androgen receptor-A form results from in vitro proteolysis. J Mol Endocrinol 2001;27:309–19. 35. Kousteni S, Bellido T, Plotkin LI, O’Brien CA, Bodenner DL, Han L, et al. Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 2001;104:719–30. 36. Bhasin S, Woodhouse L, Casaburi R, Singh AB, Mac RP, Lee M, et al. Older men are as responsive as young men to the anabolic effects of graded doses of testosterone on skeletal muscle. J Clin Endocrinol Metab 2005;90:678–88. 37. Dym M, Fawcett DW. Further observations of spermatogonia, spermatocytes, and spermatids connected by intercellular bridges in the mammalian testis. Biol Reprod 1971;4:195–215. 38. Braun RE, Behringer RR, Peschon JJ, Brinster RL, Palmiter RD. Genetically haploid spermatids are phenotypically diploid. Nature 1989;337: 373–6.
Vol. 91, No. 1, January 2009
Objective: To quantify the amount of AR messenger RNA (mRNA) and to analyze the presence and functionality of the AR protein in X- and Y-carrier human spermatozoa. Design: A molecular and cellular research study. Setting: Academic research clinic and laboratories. Patient(s): Ten normozoospermic volunteers. Intervention(s): Sperm were analyzed for expression of AR mRNA and protein. The functionality of AR protein was assessed after incubation with 1 nM of synthetic androgen. Main Outcome Measure(s): Reverse-transcription polymerase chain reaction (PCR), real-time PCR, Western immunoblotting, confocal immunofluorescence, and fluorescence in situ hybridization analysis were performed. Result(s): A significant amount of AR mRNA (around 30% of that found in the testis) was found in sperm. Androgen receptor protein was found in both X- and Y-carrier spermatozoa and translocates into the nucleus in the presence of synthetic androgen. Conclusion(s): A functional AR is expressed in human sperm. In view of the fact that AR protein is found in both X- and Y-carrier spermatozoa, it most probably is translated in earlier steps of spermatogenesis and moves to Y-carrier spermatids through the cytoplasmic bridges. (Fertil Steril 2009;91:193–200. 2009 by American Society for Reproductive Medicine.) Key Words: Androgen receptor, sperm, RNA, non-genomic effect, X-carrier Y-carrier sperm
Androgens, principally T and 5-dihydrotestosterone, are steroid hormones that mediate a wide range of physiological responses and developmental processes, involving both reproductive and nonreproductive systems in the male (1). Many physiological actions of androgens are mediated by the androgen receptor (AR), whose function is essential in males for proper sexual differentiation, pubertal development, and regulation of normal spermatogenesis (2). Androgen receptor activity is regulated by the steroid ligand T and its metabolite 5-dihydrotestosterone, the binding of which on the receptor initiates nuclear translocation and the transcriptional regulatory function of AR (3). Recently, in addition to this genomic mode of action by steroids, an increasing body of evidence suggests that androgens can exert a rapid, nongenomic effect, mediated through a membrane AR or through c-Src kinase–AR complex (4). The central role of androgens in the male reproductive system has prompted investigation into the presence and distribution of AR in male reproductive tissues (5). The site of action of androgens in the testis has been studied extensively, and for a long time the expression of AR has been restricted exclusively to Sertoli cells, Leydig cells, and peritubular or myoid cells in the testis (6–9), suggesting that the role of Received October 18, 2007; revised and accepted November 14, 2007. Reprint requests: Carlo Foresta, Ph.D., Department of Histology, Microbiology, and Medical Biotechnologies, Centre for Male Gamete Cryopreservation, University of Padova, Via Gabelli 63, 35121 Padova, Italy (FAX: 39-049-8218520; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2007.11.040
androgens in spermatogenesis is mediated by these cells (10). The recent generation and characterization of male AR knockout mice and Sertoli cell–selective AR knockout mice confirmed the essential role of AR signaling for both external and internal male phenotype development (11, 12). Recently, the presence of AR in human sperm was demonstrated by Solakidi et al. (13) and Aquila et al. (14), suggesting a constitutive functional role of AR in these cells. Because the ejaculated spermatozoa are considered transcriptionally silent cells, a nongenomic action was assumed, as suggested by a large series of experiments highlighting the ability of AR to modulate the phosphatidylinositol-3-OH/alpha-protein kinase B pathway. Besides, Solakidi et al. (13) used immunofluorescence to localize the AR in the sperm midpiece region, whereas Aquila et al. (14) localized AR in the sperm head, imputing the discrepancy to the different methods used to process samples. The conflicting findings concerning the expression pattern of AR in sperm cells have motivated us to better explore the distribution of this steroid hormone receptor in human sperm. Moreover, the origin of AR protein has not been sufficiently explained, because it may derive from translation in the early steps of spermatogenesis or during spermatid development (spermiogenesis). Therefore, in this study, we quantified the amount of AR mRNA in human sperm, confirmed the presence of AR protein, showed the functional integrity of the AR protein, and examined the presence of the AR protein in X- and Y-carrier spermatozoa.
Fertility and Sterility Vol. 91, No. 1, January 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
193
MATERIALS AND METHODS Semen Sample Collection and Processing Human semen samples were obtained by masturbation after 2– 5 days of sexual abstinence, from 10 normozoospermic volunteers who had provided informed consent. Samples were allowed to liquefy for 30 minutes and were examined for seminal parameters according to the World Health Organization (15) criteria. Samples with abnormal viscosity, presence of leukocytes, or immature germ cells were not included in this study. The samples were washed once using sperm washing medium (Irvine Scientific, Santa Ana, CA). Motile spermatozoa were selected by using a routine swim-up procedure, and cell viability was evaluated by eosin test. In all cases, the percentage of living cells was >90%. In stimulation experiments, spermatozoa were suspended in sperm washing medium at a concentration of 2 106 cells/mL and were incubated overnight with 1 nM of synthetic androgen R1881 (methyltrienolone) at 37 C; in the negative control, the hormone was omitted. Each experiment was performed four times, and in at least three of those, normal samples were pooled. Synthesis of Complementary DNA and ReverseTranscription Polymerase Chain Reaction Analysis Ribonucleic acid was extracted from human sperm and testis (testicular biopsy from a man with obstructive azoospermia) by using the RNeasy Micro Kit (Qiagen, Milan, Italy), which includes DNase treatment. Reverse-transcription polymerase chain reaction (RT-PCR; complementary DNA [cDNA] synthesis) was performed by using total RNA (50 ng) and the QuantiTect RT Kit (Qiagen), with random hexamers. The quality of obtained RNA and cDNAwas tested by spectrophotometer measurement (NanoDrop; Celbio, Milan, Italy). Total cDNAwas amplified by PCR using specific exon 1 primers for human AR (forward, 50 -TAGCCCCCTACGGCTACA-30 ; reverse, 50 -TTCCGAAGACGACAAGATGGAC-30 ; 322 bp), as described elsewhere (1). As a negative control, the cDNA was omitted, whereas for RT control, a template-free RT enzyme was used. Primers for CD4 (forward, 50 -CAGGGAAAGAAAGTGGTGC T-30 ; reverse, 50 - TTCTGGTCCTCCACTTCACA-30 ; 275 bp; exon spanning) and acrosin (forward, 50 -AACTCTGCGACA GAGGGAAA-30 ; reverse, 50 -CACACATTGGTTGGCTGA AC-30 ; 272 bp; exon spanning) were used to exclude the presence of leukocyte contamination and to verify the presence of sperm cDNA, respectively. The melting temperature was 60 C for both primers’ pairs. The RT-PCR products were electrophoretically analyzed through 1% agarose gel, were visualized by SYBR-safe DNA gel staining (Invitrogen, Milan, Italy), and were confirmed by direct sequencing on an ABI Prism sequencer (Applied Biosystems, Monza, Italy). Real-Time PCR For real-time PCR, we used the ABI PRISM 7900 HT (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The amplification reactions were performed in a 25-mL final volume containing 12.5 194
Zuccarello et al.
AR in human spermatozoa
mL of 2 TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 2.5 mL of AR TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA), and 10 mL (20 ng) of cDNA obtained from ejaculated sperm and testis biopsy. The assay’s probes span an exon junction (exon boundary, 4–5 for AR and 1–2 for b-actin) and do not detect genomic DNA. Amplification was performed for 40 cycles. After an initial hot start for 10 minutes, each cycle consisted of 15 seconds of denaturation, 30 seconds of annealing at 60 C, and 30 seconds of extension at 60 C. To normalize the amount of expressed AR mRNAs, the internal housekeeping gene b-actin was used, and each cDNA product was tested in triplicate. To calculate our data, we used the comparative Ct method for relative quantification (the DDCt method), which describes the change in expression of the target gene in the tested sample relative to a calibrator sample and provides an accurate comparison between samples of the initial level of template in each. As a calibrator sample, we used RNA extracted by skin fibroblasts, which was processed in the same way as the tested sample. Data were analyzed by using the Sequence Detector System software (version 2.1; Applied Biosystems, Foster City, CA). Western Immunoblotting Sperm lysates were prepared from sperm by using the Bioplex Cell Lysis Kit (BioRad, Milan, Italy). Total protein content was quantified by using the Bradford Protein Assay Kit (BioRad), and protein samples (30 mg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 7% gel and blotted onto polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Milan, Italy). The membrane was probed with the mouse monoclonal antibody against human AR, which recognizes amino acid residues 301–320, corresponding to the N-terminal domain, followed by incubation with the goat anti-mouse immunoglobins–horseradish peroxidase (BioRad). Immunoreactive proteins were visualized by using the enhanced chemiluminescence system detection kit (ECL; PerkinElmer, Monza, Italy) and by using autoradiography with radiography film. A human testis protein library (Celbio) was used as positive control. The negative control was obtained by omitting the primary antibody. Immunofluorescence Sperm samples were washed and suspended in phosphatebuffered saline at a concentration of 2 106 cells per microliter. One hundred microliters of that cell suspension were spotted on Polysine Microscope Slides (Menzel GmbH & Co, Braunschweig, Germany) by means of Cytospin 3 (Thermo Electron Corporation, Waltham, MA) at 1,500 rpm for 2 minutes. Spermatozoa samples were allowed to air dry, fixed in phosphate-buffered saline–paraformaldehyde 4% for 15 minutes, and permeabilized with phosphate-buffered saline–Triton X-100 (3%) for 5 minutes (Sigma Aldrich, St. Louis, MO), all at room temperature. Samples then were treated with phosphate-buffered saline–normal goat serum Vol. 91, No. 1, January 2009
15% (Vector Labs, Burlingame, CA) at room temperature and were incubated overnight with primary antibodies at 4 C. For double immunostaining, samples were simultaneously incubated with rabbit polyclonal antibody to AR (1:1,000) and mouse monoclonal anti-b-actin antibody (1:100; Abcam, Cambridge, UK). Primary immunoreactions then were detected by incubation with rabbit F(ab0 ) R-phycoerythrin–conjugated anti-mouse immunoglobulins and swine F(ab0 ) fluorescein isothiocyanate–conjugated anti-rabbit immunoglobulins (both, 1:100; Dako Cytomation, Glostrup, Denmark) for 1 hour at room temperature. Finally, slides were counterstained with 6-diamino-2-phenylindole and were mounted with antifade buffer and a 24 24-mm coverslip. The immunostaining was evaluated by using a laser confocal microscope (UltraView; PerkinElmer, Monza, Italy). For each subject, R100 cells were evaluated. Negative controls were performed by omitting the primary antibody. Sperm Chromosome Analysis The study of sperm chromosomes was performed by using multicolor fluorescence in situ hybridization, as reported elsewhere (16). Hybridization of DNA was performed by using human a-satellite probes that were specific for chromosomes X, Y, and 18 (Kreatech Biotechnology B.V., Amsterdam, the Netherlands) that were directly labeled by using fluorophores PlatinumBright495 (chromosome X, green) and PlatinumBright550 (chromosome Y, red); for the detection of chromosome 18, a mixture (1:1) of PlatinumBright495 and PlatinumBright550 directly labeled specific probes was used, resulting in a yellow signal. Deoxyribonucleic acid denaturation of sperm and probes, incubation, and posthybridization washing were performed according to the Kreatech Biotechnology protocol. After preparation, slides were stored (1–4 d, at 4 C) or immediately were observed by using an Eclipse E600 Fluorescence Microscope (Nikon, Melville, NY) that was fitted with a 100-W mercury lamp and a triple band-pass filter that was suitable for the fluorochromes in use. We performed the fluorescence in situ hybridization analysis on previously AR-immunohybridized, nonstimulated sperm, as already described earlier in this subsection. Single spots were evaluated as reported elsewhere (17, 18). For each subject, R200 cells were scored. RESULTS Analysis of RNA and cDNA Reverse-transcription PCR amplification resulted in the expected PCR product size of 322 bp in sperm and testis (Fig. 1a). Real-time PCR assay was used to quantify the amount of mRNA for AR in sperm, compared with testis. By fixing the amount of AR mRNA expressed in testis to 100, we estimated the total amount of AR mRNA in sperm to be 27.7% (relative quantification for testis, 9.803, with SD of 0.611; and relative quantification for sperm, 2.718, with SD of 0.815), as shown in Figure 1b. By evaluation of b-actin internal housekeeping gene expression, we observed that the amount of total mRNA present in ejaculated sperm Fertility and Sterility
was globally lower (mean b-actin Ct in testis, 24.4271; vs. in sperm, 30.47002) than that in testis. Western Immunoblotting The presence of AR protein in sperm was investigated by Western blot by using a monoclonal antibody recognizing amino acid residues 301–320, corresponding to the N-terminal domain. The antibody showed the presence in sperm of a prominent 110-kDa band, corresponding to the full AR protein, and a very light 87-kDa band, which probably represents an AR cleavage product (Fig. 1c). The same pattern of bands, with a similar intensity, was found in human testis protein extract, which was used as positive control. Immunofluorescence Immunolocalization of androgen receptor in nonstimulated sperm demonstrated a diffuse light staining of the sperm tail and an intensification of the signal in the postacrosomal region and initial region of the midpiece (Fig. 2a–g), as shown in the overlapping image of b-actin (green, Fig. 2a) and AR (red, Fig. 2b), resulting in a yellow color (Fig. 2f). The merged picture (Fig. 2g: red for AR, blue for DAPI, and green for b-actin) clearly showed the acrosomal region in green, the nucleus in blue, and the postacrosomal region and initial region of the midpiece in yellow. No purple color (overlapping of nuclear blue and red AR) was shown in this basal-condition experiment. After overnight androgen stimulation, the signal in the postacrosomal region and midpiece disappeared, whereas it was present strongly in the sperm head (Fig. 3b) and weakly in the sperm tail, as shown in the overlapping image of AR (red) and nucleus signals (blue), resulting in a purple color (Fig. 3e), and in the overlapping of AR (red) and b-actin (green; Fig. 3f). The merged picture (Fig. 3g) showed a clear superimposition of AR (red) and nuclear signal (blue DAPI), without any overflow in the acrosomal region. Negative controls (not pictured in the figures) showed no signal. Fluorescence In Situ Hybridization Analysis By using a fluorescence in situ hybridization technique for recognizing the X, Y, and 18 chromosomes on previously AR-immunohybridized, nonstimulated sperm, we found that both X- and Y-carrier spermatozoa expressed AR (Fig. 4a and b). The AR signal remained localized in the midpiece of spermatozoa, as we observed in fresh ejaculated sperm that were not subjected to fluorescence in situ hybridization treatment. However, clear AR immunolocalization within this picture was not possible, because the cells underwent strong chemical and physical treatments for fluorescence in situ hybridization analysis. The percentage of X-carrier and Y-carrier sperm was around 1:1 (53% vs. 47%, respectively). DISCUSSION Androgens, by signaling through the androgen receptor, mediate a wide range of physiological responses and 195
FIGURE 1
FIGURE 1 Continued Expression of AR in human-ejaculated spermatozoa. (A) Reverse-transcription PCR analysis of the human AR gene. The expected 322-bp band is displayed both in testis and sperm samples. Primers for CD4 and acrosin were used, respectively, to exclude the presence of leukocyte contamination and to verify the presence of sperm cDNA. In the RT lanes, a template-free RT enzyme was used. In the RTþ lanes, a template plus RT enzyme was used. In the negative lane, the cDNA was omitted. (B) Real-time PCR of AR gene. The amount of AR mRNA in sperm (white rectangle) is 27.7% of the AR mRNA expressed in testis (gray rectangle). (C) Western blotting of AR protein by using a monoclonal antibody raised against the N-terminal domain, showing a two-band pattern (110 and 87 kDa), both in sperm and testis. Negative control was obtained by omitting the primary antibody. All the experiments were repeated at least three times on 10 different samples, and the results of one representative experiment are depicted. No significant differences were detected among the samples.
suggesting a functional role of AR in these cells. However, some questions have remained open, in particular regarding the precise sperm cell localization of the AR (head or midpiece), its functional integrity, and the origin of AR protein.
Zuccarello. AR in human spermatozoa. Fertil Steril 2009.
developmental processes, including the correct development of functional male gametes. Indeed, expression of AR has been demonstrated clearly in Sertoli, Leydig, peritubular myoid, and vascular smooth muscle cells of the testis (6, 19–26), and it initially was thought that AR was not expressed in germ cells (19, 20, 27). However, more recent evidence has suggested expression of AR in spermatogonia (21, 24), spermatocytes (21), and elongated spermatids at spermatogenic stage XI (23), in both rat and human beings. Recently, the presence of the AR in human sperm was demonstrated by Solakidi et al. (13) and Aquila et al. (14), 196
Zuccarello et al.
AR in human spermatozoa
In this study, we confirmed the presence of AR in human spermatozoa at the RNA and protein level, including a relative quantification of the total amount of AR mRNA present in these cells. Our results showed that the sperm content of AR mRNA is around 30% of that found in the testis. Moreover, in evaluating the b-actin gene expression, we observed that the amount of total RNA present in ejaculated sperm is very low, confirming reports elsewhere (28) that indicated low RNA quantity in these transcriptionally dormant cells. On the basis of the general assumption that ejaculated spermatozoa do not synthesize RNA and proteins (29), the presence of transcripts in these cells traditionally has been attributed to the presence of residual cytoplasmic droplets that escape absorption by the Sertoli cells or to the presence of immature spermatids or somatic cells in the ejaculates from which spermatozoa are traditionally obtained. Nevertheless, in the last 15 years, a large number of investigators have demonstrated the presence of numerous mRNA in spermatozoa (29), and recently, Ostermeier et al. (30) reported the simultaneous detection by using DNA arrays of R3,500 unique mRNA in the ejaculate sperm. These RNA are thought to originate in early steps of spermatogenesis or during spermatid development and may diffuse among spermatids, thanks to the cytoplasmic sharing between connected spermatids. Interestingly, although it now has been confirmed that human sperm do not transcribe novel RNA (31) because Vol. 91, No. 1, January 2009
FIGURE 2 Immunolocalization of AR in human ejaculated spermatozoa in basal condition (fresh ejaculate), as evaluated by laser confocal microscope. (A) Green, b-actin; (B) red, AR; (C) blue, DAPI; (D) green þ blue; (E) red þ blue; (F) green þ red, resulting in yellow; and (G) merged image (green þ red þ blue).
Zuccarello. AR in human spermatozoa. Fertil Steril 2009.
of the shutdown of nuclear transcription that precedes the package of spermatozoal DNA, some studies have indicated the presence of a latent translational capacity in sperm, on the basis of a measured response to cycloheximide (32) and as also suggested by the finding that mitochondrial ribosomes are actively involved in protein translation in spermatozoa (33). However, the real function of these RNA in sperm remains unclear, even if the most likely conjectures suppose a role in the selective nuclear DNA repackaging mediated by protamines, in paternal imprinting mediation and in the male-gamete contribution to early embryogenesis (29). By Western blot analysis, we found both the 110- and the 87-kDa bands both in testis and sperm. The 110-kDa AR form universally is considered to correspond to the full-length AR, whereas the 87-kDa form is considered to be the result of in vitro proteolysis, as extensively demonstrated by Gregory et al. (34). This consideration is consistent with our experimental series, in which we observed the 87-kDa band more or less intensely in different instances, depending on the number of cycles of freezing and thawing (personal data). Fertility and Sterility
By immunofluorescence and confocal microscope observation, we have demonstrated the presence of AR protein in the sperm postacrosomal region and initial part of the midpiece in the basal condition, followed by nuclear (head) translocation of the receptor after overnight androgen stimulation. In agreement with our observations, Solakidi et al. (13) reported an intense staining of the sperm midpiece, corresponding to the site of concentration of the mitochondria, in addition to a diffuse reactivity in the tail region. On the contrary, Aquila et al. (14) detected a prevalent signal localization in the sperm head and ascribed this apparent discrepancy to the different methods used to process samples. In particular, it is not clear in the article by Aquila et al. (14) whether the spermatozoa used to perform immunofluorescence assays were at the basal condition or were being used after 30 minutes of androgen stimulation or incubation in capacitating medium, making difficult a direct comparison of their results with ours. One of the most important findings of our study is that the AR protein is fully functional, because it translocates into the 197
FIGURE 3 Immunolocalization of AR in human-ejaculated spermatozoa after overnight stimulation by R1881. (A) Green, b-actin; (B) red, AR; (C) blue, DAPI; (D) green þ blue; (E) red þ blue; (F) green þ red resulted in yellow; and (G) merged image (green þ red þ blue).
Zuccarello. AR in human spermatozoa. Fertil Steril 2009.
nucleus after androgen stimulation. Therefore, our findings may suggest that androgens exert their effect directly on sperm, through a classical AR genomic pathway. However, spermatozoa are considered transcriptionally inactive, and therefore much evidence has concentrated on the possible rapid, nongenomic effects of androgens, as mediated by membrane-bound AR. In fact, it has been demonstrated that androgens in sperm lead in a dose-dependent manner to the rapid phosphorylation of specific AR residues, which leads to the start of the phosphatidylinositol-3-OH/alpha¼protein kinase B pathway, which is involved in activation of caspases 3, 8, and 9 (14). Moreover, in osteoblasts and osteocytes (35), the non-genotropic signaling of the androgens through the AR also has been described, with complete dissociation from transcriptional activity mediated by the receptor. In these cells, the nongenomic effects of AR involve the MAPK pathway, interacting with the SH3 domain of c-Src. The significance of the nuclear translocation of the AR after stimulation with androgens in sperm therefore remains unclear. 198
Zuccarello et al.
AR in human spermatozoa
On the basis of AR sperm midpiece localization in the basal condition, it has been supposed that AR is involved in mitochondrial function in a way that is related to the energy requirements of these cells (13). In fact, Solakidi et al. (13) showed that AR internal sequences are similar to those observed in some mitochondrial inner membrane proteins, assuming a possible role of AR in sperm motility regulation. Besides, it is possible that intact AR protein, located in the sperm nucleus after androgen stimulation, plays a role in the first steps of embryonic development, on the basis of the well-known anabolic action of androgens (36). The second most important finding of our study is the presence of AR protein in both X- and Y-carrier spermatozoa, which gives a clear indication regarding the origin of this protein. In accordance with the current opinion that mature spermatozoa are transcriptionally silent, it may be assumed that the AR gene is transcribed and/or translated during the early steps of spermatogenesis by XY diploid spermatocytes, or Vol. 91, No. 1, January 2009
FIGURE 4 Fluorescence in situ hybridization analysis of 18,X (a) and Y (b) chromosomes in human sperm, evaluated by fluorescence microscope. After AR immunohybridization of fresh ejaculated sperm, the fluorescence in situ hybridization analysis showed that both X- and Y-carrier spermatozoa express AR in the midpiece. Arrows show the specific chromosome signals (yellow, 18; red, Y; green, X). White ring indicates AR immunolocalization.
Zuccarello. AR in human spermatozoa. Fertil Steril 2009.
during spermiogenesis by X-carrier haploid spermatids. The AR protein then could move to Y-carrier spermatids through the cytoplasmic bridges that connect the spermatids (37). Indeed, there is evidence in the literature that other gene products transit the cytoplasmic bridges of the elongated spermatids (38). In conclusion, we showed that the localization of AR in sperm is dependent on the hormonal-stimulation status of the sperm. We showed the functional integrity of AR protein, its translocation into the nucleus after androgen stimulation, and the presence of AR in both X- and Y-carrier spermatozoa, suggesting a possible role for this receptor in the final step of spermatogenesis and/or sperm function. Acknowledgments: The authors thank Denis Bison, B.Sc. (University of Padova, Padova, Italy), for technical assistance with confocal microscopy, Elisa Prana, B.Sc. for technical assistance with RT-PCR, and Albert O. Brinkmann (Erasmus University, Rotterdam, the Netherlands) for kindly providing both the mouse monoclonal and rabbit polyclonal AR antibodies. They also thank all the staff of the Centre for Male Gamete Cryopreservation for helpful discussion.
REFERENCES 1. Ferlin A, Vinanzi C, Garolla A, Selice R, Zuccarello D, Cazzadore C, et al. Male infertility and androgen receptor gene mutations: clinical features and identification of seven novel mutations. Clin Endocrinol 2006;65:606–10.
Fertility and Sterility
2. Weinbauer GF, Nieschlag E. Hormonal control of spermatogenesis. In: de Kretser DM, ed. Molecular biology of the male reproductive system. San Diego, CA: Academic Press, 1993:99–142. 3. Lindzey J, Kumar MV, Grossman M, Young C, Tindall DJ. Molecular mechanisms of androgen action. Vitam Horm 1994;49:383–432. 4. Heinlein CA, Chang C. The roles of androgen receptors and androgenbinding proteins in nongenomic androgen actions. Mol Endocrinol 2002;16:2181–7. 5. McLachlan RI, O’Donnell L, Stanton PG, Balourdos G, Frydenberg M, de Kretser DM, et al. Effects of testosterone plus medroxyprogesterone acetate on semen quality, reproductive hormones, and germ cell populations in normal young men. J Clin Endocrinol Metab 2002;87:546–56. 6. Ruizeveld de Winter JA, Trapman J, Vermey M, Mulder E, Zegers ND, van der Kwast TH. Androgen receptor expression in human tissues: an immunohistochemical study. J Histochem Cytochem 1991;39:927–36. 7. Iwamura M, Abrahamsson PA, Benning CM, Cockett AT, Di Sant’Agnese PA. Androgen receptor immunostaining and its tissue distribution in formalin fixed, paraffin-embedded sections after microwave treatment. J Histochem Cytochem 1994;42:783–8. 8. Goyal HO, Bartol FF, Wiley AA, Neff CW. Immunolocalization of receptors for androgen and estrogen in male caprine reproductive tissues: unique distribution of estrogen receptors in efferent ductule epithelium. Biol Reprod 1996;56:90–101. 9. Suarez-Quian CA, Martinez-Garcia F, Nistal M, Regadera J. Androgen receptor distribution in adult human testis. J Clin Endocrinol Metab 1999;84:350–8. 10. McLachlan RI, O’Donnell L, Meachem SJ, Stanton PG, de Kretser DM, Pratis K, et al. Hormonal regulation of spermatogenesis in primates and man: insights for development of the male hormonal contraceptive. J Androl 2002;23:149–62. 11. Yeh S, Tsai MY, Xu Q, Mu XM, Lardy H, Huang KE, et al. Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues. Proc Natl Acad Sci USA 2002;99:13498–503. 12. Tan KA, De Gendt K, Atanassova N, Walker M, Sharpe RM, Saunders PT, et al. The role of androgens in sertoli cell proliferation and functional maturation: studies in mice with total or Sertoli cell-selective ablation of the androgen receptor. Endocrinology 2005;146: 2674–83. 13. Solakidi S, Psarra AM, Nikolaropoulos S, Sekeris CE. Estrogen receptors alpha and beta (ERalpha and ERbeta) and androgen receptor (AR) in human sperm: localization of ERbeta and AR in mitochondria of the midpiece. Hum Reprod 2005;20:3481–7. 14. Aquila S, Middea E, Catalano S, Marsico S, Lanzino M, Casaburi I, et al. Human sperm express a functional androgen receptor: effects on PI3K/ AKT pathway. Hum Reprod 2007;22:2594–605. 15. World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge, United Kingdom: Cambridge University Press, 1999. 16. Foresta C, Galeazzi C, Bettella A, Stella M, Scandellari C. High incidence of sperm sex chromosomes aneuploidies in two patients with Klinefelter’s syndrome. J Clin Endocrinol Metab 1998;83:203–5. 17. Robbins WA, Baulch JE, Moore D, Weier HU, Blakey D, Wyrobek AJ. Three-probe fluorescence in situ hybridization to assess chromosome X, Y, and 8 aneuploidy in sperm of 14 men from two healthy groups: evidence for a paternal age effect on sperm aneuploidy. Reprod Fertil Dev 1995;7:799–809. 18. Guttenbach M, Martinez-Exposito MJ, Michelmann HW, Engel W, Schmid M. Incidence of diploid and disomic sperm nuclei in 45 infertile men. Hum Reprod 1997;12:468–73. 19. Anthony CT, Kovacs WJ, Skinner MK. Analysis of the androgen receptor in isolated testicular cell types with a microassay that uses an affinity ligand. Endocrinology 1989;125:2628–35. 20. Sar M, Lubahn DB, French FS, Wilson EM. Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 1990;127:3180–6. 21. Kimura N, Mizokami A, Oonuma T, Sasano H, Nagura H. Immunocytochemical localization of androgen receptor with polyclonal antibody in
199
22.
23.
24.
25.
26.
27.
28.
paraffin-embedded human tissues. J Histochem Cytochem 1993;41: 671–8. Bremner WJ, Millar MR, Sharpe RM, Saunders PT. Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology 1994;135:1227–34. Vornberger W, Prins G, Musto NA, Suarez-Quian CA. Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis. Endocrinology 1994;134:2307–16. Zhou X, Kudo A, Kawakami H, Hirano H. Immunohistochemical localization of androgen receptor in mouse testicular germ cells during fetal and postnatal development. Anat Rec 1996;245:509–18. Pelletier G, Labrie C, Labrie F. Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs. J Endocrinol 2000;165:359–70. Zhou Q, Nie R, Prins GS, Saunders PT, Katzenellenbogen BS, Hess RA. Localization of androgen and estrogen receptors in adult male mouse reproductive tract. J Androl 2002;23:870–81. Grootegoed JA, Peters MJ, Mulder E, Rommerts FF, Van der Molen HJ. Absence of a nuclear androgen receptor in isolated germinal cells of rat testis. Mol Cell Endocrinol 1977;9:159–67. Zhao Y, Li Q, Yao C, Wang Z, Zhou Y, Wang Y, et al. Characterization and quantification of mRNA transcripts in ejaculated spermatozoa of fertile men by serial analysis of gene expression. Hum Reprod 2006;21: 1583–90.
200
Zuccarello et al.
AR in human spermatozoa
29. Miller D, Ostermeier GC, Krawetz SA. The controversy, potential and roles of spermatozoal RNA. Trends Mol Med 2005;11:156–63. 30. Ostermeier GC, Dix DJ, Miller D, Khatri P, Krawetz SA. Spermatozoal RNA profiles of normal fertile men. Lancet 2002;360:772–7. 31. Grunewald S, Paasch U, Glander HJ, Anderegg U. Mature human spermatozoa do not transcribe novel RNA. Andrologia 2005;37:69–71. 32. Naz RK. Effect of actinomycin D and cycloheximide on human sperm function. Arch Androl 1998;41:135–42. 33. Gur Y, Breitbart H. Mammalian sperm translate nuclear-encoded proteins by mitochondrial-type ribosomes. Genes Dev 2006;20:411–6. 34. Gregory CW, He B, Wilson EM. The putative androgen receptor-A form results from in vitro proteolysis. J Mol Endocrinol 2001;27:309–19. 35. Kousteni S, Bellido T, Plotkin LI, O’Brien CA, Bodenner DL, Han L, et al. Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 2001;104:719–30. 36. Bhasin S, Woodhouse L, Casaburi R, Singh AB, Mac RP, Lee M, et al. Older men are as responsive as young men to the anabolic effects of graded doses of testosterone on skeletal muscle. J Clin Endocrinol Metab 2005;90:678–88. 37. Dym M, Fawcett DW. Further observations of spermatogonia, spermatocytes, and spermatids connected by intercellular bridges in the mammalian testis. Biol Reprod 1971;4:195–215. 38. Braun RE, Behringer RR, Peschon JJ, Brinster RL, Palmiter RD. Genetically haploid spermatids are phenotypically diploid. Nature 1989;337: 373–6.
Vol. 91, No. 1, January 2009