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Regular Article |
a Howard Hughes Medical Institute, Whitehead Institute, and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142
b Department of Obstetrics, Gynecology and Reproductive Sciences, and Departments of Physiology and Urology, University of California, San Francisco, California 94143-0720
c Department of Pathology and
d Department of Urology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
e MRC Human Genetics Unit, Edinburgh, Scotland EH4 2XU, United Kingdom
ABSTRACT
The human DAZ gene family is expressed in germ cells and consists of a cluster of nearly identical DAZ (deleted in azoospermia) genes on the Y chromosome and an autosomal homolog, DAZL (DAZ-like). Only the autosomal gene is found in mice. Y-chromosome deletions that encompass the DAZ genes are a common cause of spermatogenic failure in men, and autosomal homologs of DAZ are essential for testicular germ cell development in mice and Drosophila. Previous studies have reported that mouse DAZL protein is strictly cytoplasmic and that human DAZ protein is restricted to postmeiotic cells. By contrast, we report here that human DAZ and human and mouse DAZL proteins are present in both the nuclei and cytoplasm of fetal gonocytes and in spermatogonial nuclei. The proteins relocate to the cytoplasm during male meiosis. Further observations using human tissues indicate that, unlike DAZ, human DAZL protein persists in spermatids and even spermatozoa. These results, combined with findings in diverse species, suggest that DAZ family proteins function in multiple cellular compartments at multiple points in male germ cell development. They may act during meiosis and much earlier, when spermatogonial stem cell populations are established.
sperm, spermatogenesis
INTRODUCTION
Infertility affects 10–15% of couples in industrialized countries, and nearly half of all cases are traced to the male [1]. Y-chromosome deletions encompassing the DAZ (deleted in azoospermia) gene cluster comprise the most common molecularly defined cause of spermatogenic failure in infertile men [2–14]. In both Drosophila and mice, disruption of the autosomal DAZ homolog results in male infertility and, in mice, female infertility as well [15, 16].
The human Y-linked DAZ gene cluster arose during primate evolution by transposition and amplification of the still-extant autosomal gene, DAZL (DAZ-like, formerly known as DAZLA, DAZH, or SPGYLA), which is widely conserved among vertebrates and invertebrates [15, 17–25]. In all species examined, expression of DAZ, DAZL, and their homologs is reported only in germline cells [18, 19, 24–27]. The presence of RNA recognition motifs suggests that DAZ family proteins bind RNA [28], but little is known of their cellular functions.
Previous studies of the cellular localization of DAZ family proteins in testes of various species have reported surprisingly different results. In Drosophila, the DAZ-homologous protein Boule was found in the nuclei of premeiotic cells, moving to the cytoplasm at the onset of meiosis [29]. In mice, however, the DAZL protein was reported to be strictly cytoplasmic [16]. In humans, DAZ protein was stated to be restricted to postmeiotic cells [30].
Using independent antisera, we re-examined how DAZ family proteins were distributed in testicular germ cells of humans and mice. We wished to determine whether the DAZL and DAZ proteins are present in fetal as well as adult testes, whether they exist in both premeiotic and postmeiotic cells, and whether they are found in the nucleus or cytoplasm.
MATERIALS AND METHODS
Preparation of Antisera
Polyclonal antibodies were raised by injecting synthetic oligopeptides coupled to a multiple-antigen peptide resin into rabbits (Research Genetics, Inc., Huntsville, AL). Twelve to 20 wk after initial injection of peptides, serum antibodies were purified on Protein A columns according to the manufacturer's instructions (Hi-Trap; Pharmacia, Inc., Uppsala, Sweden) and preabsorbed on mouse liver acetone powder to remove nonspecific binding activities.
Western Blotting
Mouse or human testis samples or semen were lysed by heating to 95°C for 5 min in 10 volumes of 0.1 M Tris-Cl (pH 6.8), 200 mM dithiothreitol (DTT), 4% SDS, 0.2% bromophenol blue, and 20% glycerol. The samples were placed on ice and sonicated vigorously to shear DNA. Twenty micrograms of protein extract were run in each lane of a 12% polyacrylamide gel and then electrophoretically transferred to nitrocellulose filters according to the manufacturer's instructions (BioRad, Inc., Hercules, CA). The nitrocellulose filters underwent the following series of room-temperature incubations in solutions containing 5% milk powder, 0.1 M Tris-HCl (pH 7.5), 0.9% NaCl, and 0.2% Nonidet 40 (NP40): 1) preincubation for 1 h, 2) incubation with a 1:1000 dilution of antisera 149, 150, or 133 for 1 h, 3) three rinses of 5 min each, 4) incubation with a 1:10 000 dilution of secondary antibody, anti-rabbit IgG/peroxidase conjugate (Sigma Chemical Co., St. Louis, MO), for 1 h, and 5) two rinses of 5 min each. There were three more room-temperature rinses of 5 min each: one rinse in 5% milk powder, 0.1 M Tris-HCl (pH 7.5), 0.9% NaCl, and 0.1% Tween 20; and two rinses in 0.1 M Tris-HCl (pH 7.5), 0.9% NaCl. Peroxidase reactions were visualized using the electrochemiluminescence system according to the manufacturer's instructions (Amersham, Arlington Heights, IL).
Immunohistochemistry
Human testis sections were obtained from a 20–21-wk fetus and from a 38-yr-old man who presented with a seminoma; testes sections were obtained from the Department of Pathology, Brigham and Women's Hospital, Boston, following institutional review board approval. Mouse testis sections were obtained from embryos 18 days postcoitum and from 60-day-old adult mice; an Institutional Animal Care and Use Committee approved the use of the mice.
Formalin-fixed, paraffin-embedded human tissues were sectioned at 5 µm. After baking at 60°C for 1 h, sections were deparaffinized and rehydrated (100% xylene, four times, 3 min each; 100% ethanol, twice, 3 min each; 95% ethanol, twice, 3 min each; water, 5 min). Slides were blocked by treating with 3% hydrogen peroxide in methanol for 5 min at room temperature, then washed in water for 5 min, and microwave treated (800 W, General Electric) at 199°F for 30 min in 10 mM citrate (pH 6.0). Slides were cooled for 15 min, transferred to PBS, 0.1% Tween 20, and preincubated for 15 min at room temperature in 1.5% goat serum. Slides were incubated for 1 h at room temperature with antisera 149 (at 1:4000 dilution), antisera 150 (1:1000), or antisera 133 (1:200) in 2% goat serum. Slides were washed three times (5 min each) in PBS, 0.1% Tween 20, and then incubated for 30 min with biotinylated horse anti-rabbit IgG (Vector Laboratories, Burlingame, CA). After three washes (5 min each) in PBS, 0.1% Tween 20, slides were incubated for 40 min at room temperature with avidin:biotinylated-peroxidase complex (Vector), followed by reaction with 3,3'-diaminobenzidine tetrachloride:hydrogen peroxide. Sections were subsequently stained with 2% Gill hematoxylin. Column-fractionated preimmune sera corresponding to fractions containing the specific antibodies were used as negative controls.
Mouse tissues were treated in a similar manner except that tissues were fixed in Bouin fixative rather than formalin, the microwave treatment was eliminated, and goat anti-rabbit horseradish peroxidase-conjugated antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) replaced the biotin:avidin system described above.
Cell types were determined by enlarging the images and examining the position, size, and morphology of cells that were immunopositive. The criteria to assign cell types were those previously reported [31–33].
RESULTS
Three peptides against which polyclonal antibodies were raised and the corresponding portions of the human DAZ (Y-encoded), human DAZL (autosomal), and mouse DAZL (autosomal) proteins are shown in Figure 1a. We anticipated that antisera 149 would recognize all three proteins, that antisera 150 would recognize the autosomally encoded (human and mouse) DAZL proteins, and that antisera 133 would recognize the human Y-encoded DAZ protein. Indeed, on Western blots of adult mouse tissues, a testis-specific protein with a molecular weight of 33 kDa, as predicted for mouse DAZL, was detected by antisera 149 and 150 (Fig. 1b). These two antisera also recognized the human DAZL protein, again with a molecular weight of 33 kDa, on Western blots of human adult testes. As anticipated, a human testis protein of higher molecular weight (46 kDa), DAZ, was detected by antisera 149 and 133 (Fig. 1c). (Competition with the immunizing peptides abolished all of these signals; not shown.) Antisera 133, raised against a peptide present in human Y-encoded DAZ but absent in the autosomal proteins, did not recognize any proteins in mice (Fig. 1, b and d, left-hand lane), where the Y chromosome does not carry any DAZ genes. However, mice carrying a DAZ transgene express a DAZ protein as a fusion with 14 amino acids from herpes simplex virus thymidine kinase protein that is recognized by antisera 133 (Fig. 1d, right-hand lane; transgene construction is as reported in Slee et al. [34]). This indicates that antibody 133 is specific to the DAZ protein encoded by the human Y chromosome. The size of the DAZ protein in human testes in these studies corresponds to a protein with an estimated tandem array of roughly nine DAZ repeats; no other forms of DAZ were detected. Another report, in which a single antibody was used and specificity was not confirmed with transgenic mice, suggested that the predominant form of DAZ protein expressed was 66 kDa [30]. This suggests the possibility that there may be several different DAZ proteins expressed in human testis. Although we have not observed any additional DAZ proteins on multiple blots, this may be expected because there is heterogeneity in the number of repeats in different gene copies in different men [35]. In any case, confidence that the 46-kDa protein represents authentic DAZ is based on the observations that this protein is absent from wild-type mouse testis extracts, present in human testis extracts, and present in transgenic mice expressing DAZ protein from a Y-chromosome yeast artificial chromosome transgene.
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We next used these antisera to explore the distribution of DAZ and DAZL proteins in human testis sections from a 20- to 21-wk fetus and from an adult. (The adult had presented with a seminoma; the sections we studied were not adjacent to the tumor and were comprised of histologically normal seminiferous tubules with a full range of spermatogenic cell types.) We found that, in humans, the DAZ and DAZL proteins are present in male germ cells at many stages in their development, both prenatally and during spermatogenesis. Antisera 149 that recognizes both DAZ and DAZL clearly stained the gonocytes of the fetus and the spermatogonia and spermatocytes of the adult (Fig. 2); we also observed weaker staining of round and elongating spermatids. A similar pattern was obtained with antisera 150 that is specific for DAZL, although here the staining of spermatids was more pronounced (Fig. 2). Staining with antisera 133, which is specific for (Y-encoded) DAZ, was largely restricted to the less mature germ cells: gonocytes, spermatogonia, to a lesser extent, spermatocytes, and only occasionally round spermatids (Fig. 2). These results are confirmed by examination of multiple tubules in testicular biopsies (Fig. 3). Our impression that DAZL, but not DAZ, was present in spermatids throughout their differentiation was further explored by Western blotting of protein extracts from a normal semen sample. We observed the 33-kDa DAZL protein readily in these preparations, while we did not detect DAZ in mature spermatids (Fig. 4). We conclude that both the autosomally encoded DAZL and Y-encoded DAZ proteins are present in human male germ cells through much of their development, both prenatally and during spermatogenesis, but that only DAZL is detected in postmeiotic, mature spermatids.
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By studying the immunohistochemically stained sections of human fetal and adult testes, we found that the intracellular locations of the DAZ and DAZL proteins change during the progression from fetal gonocytes to spermatogonia to spermatocytes to spermatids. In gonocytes, DAZ and DAZL were present in both the nucleus and the cytoplasm (Fig. 2). In spermatogonia, DAZ and DAZL were most abundant in the nucleus but could also be detected in the cytoplasm (Fig. 2; for example see upper right panel and cells marked with an asterisk). In spermatocytes, the proteins appear to be restricted to the cytoplasm (see especially antisera 149, adult testis in Fig. 2; cells indicated by an asterisk). Finally, as the spermatogenic cells mature into round spermatids and begin to elongate, the pattern of DAZL staining is consistent with cytoplasmic localization (see especially antisera 150, adult testis in Fig. 2; cell marked by an asterisk). Thus, our results are consistent with the hypothesis that DAZ and DAZL are predominantly nuclear in spermatogonia, but at meiosis the proteins are largely located in the cytoplasm, where DAZL persists in differentiating spermatids.
We next sought to determine whether DAZL protein is present in the germ cells of mice at the same developmental stages and in the same cellular compartments as in humans. (As mentioned earlier, there are no Y-linked DAZ genes in mice.) In mice, as in humans, DAZL protein is present in testicular germ cells in both fetuses and adults, as revealed by staining with antisera 149 and 150 (Fig. 5). As expected, no staining is observed with antiserum 133 that specifically recognizes human Y-chromosome-encoded DAZ protein (Fig. 5). In addition, there appears to be no staining of Sertoli cells or other somatic cells, in agreement with previous Northern blotting and in situ hybridization studies [18, 19, 27]. In mouse fetal gonocytes, DAZL appears to be present in both the nucleus and the cytoplasm (Fig. 5). In sections of adult mouse testis counterstained conventionally with hematoxylin and eosin, DAZL staining is most evident in the cytoplasm of meiotic cells (spermatocytes; Fig. 5). To ensure that no DAZL staining was obscured, we also examined adult testicular sections that were not counterstained. With no counterstaining, the localization of DAZL to spermatocyte cytoplasm was confirmed, and a second localization—to the nuclei of spermatogonia—was revealed (Fig. 6). We conclude that during spermatogenesis in mice, as in humans, DAZL shifts from a predominantly nuclear to a predominantly cytoplasmic location at meiosis.
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These findings with mouse DAZL conflict with a recent report indicating that the protein is strictly cytoplasmic in murine male germ cells [16]. As in the present study, Ruggiu et al. [16] reported strong staining for DAZL in spermatocytes, where the protein is cytoplasmic. They did not observe staining in spermatogonial nuclei despite using fluorescently labeled antibodies. As the two antibodies are raised against different parts of the DAZL protein, it is possible that different epitopes have different availabilities in different cell types.
DISCUSSION
Our immunohistochemical studies in human and mouse, together with mutation and expression analysis of DAZ homologs in diverse organisms, provide evidence that mammalian DAZ family proteins may function at multiple points in male germ cell development. Specifically, we hypothesize that the mammalian DAZ family proteins act both during meiosis and much earlier, during the establishment of spermatogonial stem cell populations. As demonstrated here in both humans and mice, the DAZ family proteins are expressed in gonocytes of fetal testes, long before the onset of spermatogenesis and meiosis (at puberty). In mutant male mice lacking Dazl function, germ cells are lost between Days 15 and 19 of embryonic development [16], also long before spermatogenesis begins. Taken together, these results suggest that DAZ family proteins in both humans and mice play a critical role in early male germ cell development, when spermatogonial stem cell precursors must emerge and be maintained. The putative RNA-binding proteins of the DAZ family are largely nuclear during these early, premeiotic phases of male germ cell development, leading us to speculate that the early functions of the mammalian DAZ family proteins are directed at RNA processing or storage in the nucleus [28].
Genetic analysis of diverse species demonstrates the importance of DAZ family proteins in meiosis, in one or both sexes, and our protein localization studies hint at a similar meiotic role in male mammals. In male flies lacking boule function, meiotic divisions do not occur [15]. The Xenopus DAZ homolog Xdazl, when expressed in Drosophila, rescues the boule meiotic entry defect [25]. Also, loss of Dazl function in mutant female mice or suppression of DAZ-homolog function in female nematodes result in germ cell loss at meiosis [16, 24]. (In Dazl-mutant male mice, germ cells are lost during fetal development [16], precluding any manifestation of the male meiotic role that we postulate here.) As we report, the mammalian DAZ family proteins appear to move from a predominantly nuclear to a predominantly cytoplasmic location at male meiosis. The protein product of the Drosophila boule gene, homologous to mammalian DAZ and DAZL, undergoes a similar redistribution from nucleus to cytoplasm when male meiosis is initiated [29]. The late functions of the mammalian DAZ family proteins may begin at the onset of meiosis.
Previous in situ hybridization studies had revealed that, in the human adult testis, DAZ/DAZL transcripts are most abundant in spermatogonia and primary spermatocytes [26]. For DAZ, these in situ hybridization data are in close accord with the present immunohistochemical findings. In contrast, DAZL protein appears to persist long after transcript levels have peaked. The DAZL protein observed in spermatozoa and their (postmeiotic) spermatid precursors may have been synthesized many days or even weeks earlier, during or prior to meiosis. Our findings with DAZ are at odds with the report of Habermann et al. [30] that this Y-encoded human protein is found only in late stage germ cells, spermatids, and spermatozoa. Spermatids and spermatozoa are the only male germ cells in which we do not detect the Y-chromosome-encoded DAZ protein (Figs. 2–4). Habermann et al. [30] employed a single antiserum that detected many protein species on Western blots of human testis extracts. This antiserum may not have been specific for DAZ. In the present study, three different antisera were used and results were confirmed via several different methods.
The expression patterns of the human DAZ and DAZL proteins overlap but are not identical, suggesting that the two human proteins may contribute differentially to the postulated early and late DAZ family functions. As described earlier, the human Y-encoded DAZ protein is most prominently observed in less mature germ cells, especially gonocytes and spermatogonia, while the human autosomally encoded DAZL protein is clearly present after meiosis, persisting even in mature spermatozoa. Thus, the Y-borne DAZ gene cluster, which arose by transposition and amplification of the autosomal DAZL gene during primate evolution [17], may have evolved toward specialization in early functions; the human DAZL gene may have evolved toward specialization in late (meiotic or postmeiotic) functions. This would fit well with previous evidence that men who are deleted for the Y-borne DAZ gene cluster are infertile because of a problem in the generation or maintenance of spermatogonial stem cell populations (an early function) [2, 3] and not a defect in the differentiation pathway of spermatogenesis itself (a late function). Thus, infertility in DAZ-deleted men may have its origins long before puberty and perhaps even during fetal development.
ACKNOWLEDGMENTS
We thank L. Brown and J. Cook-Chrysos for graphic art; and A. Bortvin, L. Brown, B. Lahn, D. Menke, R. Saxena, C. Sun, P.J. Turek, and J. Wang for comments on the manuscript.
FOOTNOTES
First decision: 10 November 1999.
1 Supported by the National Institutes of Health. 
2 Correspondence: Renee A. Reijo, Dept. of Ob/Gyn & RS, 513 Parnassus, Box 0720, HSW1480, University of California, San Francisco, CA 94143-0720. FAX: 415 476 6145; reijo{at}itsa.ucsf.edu
Accepted: July 12, 2000.
Received: October 14, 1999.
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