BIOLOGY OF REPRODUCTION 68, 1490-1496 (2000) DAZ Fal ly Proteins Exist Throughout Male Germ Cell Development and Transi from Nucleus to Cytoplasm at Meiosis in Humans and Mice! Renee A. Rei Howard Cooke,’ and David C. Page? = David M. Dorfman,* Roger Slee,’ Andrew A. Renshaw,* Kevin R. Loughlin,* Howard Hughes Medical Institute,’ Whitehead Institute, and Department of Biology, Massachuseus institute (fF Technology, Cambridge, Massachusetts 02142 Deparment of Obstetrics, Gynecology and Reproductive Sciences,’ and Departments of Physiology and Urology University of California, 5 in Francisco, California 94143-0720 Department of Pathology” and Department of Urology." Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusets 02115 MRC Human Genetics Unit” ABSTRACT ‘he human DAZ gene family is expresed in germ cll ane consis of cluster of nearly identical DAZ (deleted 2200" Spermia genes on the Y chromosome and an autosomal hom log, DAZE DAZ ke) Only the autosomal gene found in mie. ¥Ekeomocome deletions ha encompass the DAZ gener are 4 Common caine of spermatagene faire i men, and autosomal ttomotogs of DAZ are eral for testiciar germ call devel pment in mice and ja Previous sues have reported that mouse DAZL protein strictly eytoplasmic and that Raman DAZ protein i resticted to postmette cell, By contac, we feport here that human DAZ and human and mouse DAZL pro {chm are present in both the nuclel and eytopasn of fetal gon: tyler and io spermatogonial nucle The proteins rlocae to the cytoplasm during male melons Further obserwatons using thiman tases date that, ue DAZ, human DAZL protein sss in spermatids and even spermatoroa These results, com: Fined with findings in diverse species, suggest that DAZ family protein funtion im multiple cellar compartments at maple points n male germ cel development. hey may act during me this and mucheafr, when spermatogonial stem cell popula tions are extablihed. sperm, spermatogenesis INTRODUCTION Infertility affects 10-15% of couples in industrialized countries, and nearly hall ofall cases tne traced to the male [IL Y-cheomosome deletions encompassing the DAZ (de: Jeted in azoospermia) gene cluster comprise the most eom- ‘mon molecularly defined cause of spermatagenie failure in infertile men (2-14), In both Drosophila and mice, distup- tion of the autosomal D4Z homolog results ia maie inker- tility and, in mice, female infertility as well (15, 16) ‘The human Y-linked D4Z gene cluster arose during pri tc evolution by transposition and amplification of the siill-extant. autosomal. gene, DAZL (DAZ-like, formerly known as DAZE, DAZH, or SPGYLA), which is widely ‘Sappond bythe National ott of Heath ‘Corespundence: Renee A. Re, Dep of ObGyn & RS, 513 Pass, Ho o7b HOW 40, Univ of Calon Sa raeleo, CA B48 (720, FAK 415 476 6145, oma rejowien vest ee Received 14 October 199, Fist decision: 10 Nevers 199, Pecepted 12 ay 2000 Sno by he Society or he Study of Repaction, tn ISSN 00063353 psn hiepocong, Edinburgh, Scotland EH4 2XU, United Kingdom 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 litle is known of their cellular finetions Previous studies of the cellular localization of DAZ. fam. ily proteins in tesies of various species have reported sur prisingly different results. In Drosophila, the DAZ-bomol- ‘ogous protein Boule was found in tho nucle of premeiotie cells, moving to the cytoptasm atthe onset of mess [29] In mice, however, the DAZL protein was reported to be sicictly eytoplasmic [16]. In humans, DAZ protein was stal- ced to be restricted 10 posimeiotic cells (30) Using independent antisera, we re-examined how DAZ family proteins were distributed in testicular germ cells of hhumans 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 post- ieiotic celis, and whether they are found in the nucleus or cytoplasm, MATERIALS AND METHODS Proparation of Antisera Polyclonal antibodies were raised by injecting synthetic oligopeptides coupled to a multiple-antigen peptide resin ino rabbits. (Research Genetics, Tnc., Huntsville, AL) ‘Twelve to 20 wk after initial injection ‘of peptides, serum antibodies were purified on Protein A columns according lo the manufacturer's insisuctions (HicTrap: Pharmacia, Inc., Uppsala, Sweden) and preabsorbed on’ mouse liver acetone povwder 10 remove nonspecific binding activities Western Bloting ‘Mouse o- human testis samples or semen were lysed by heating 10 95°C for 5 min in 1D 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 exiraet were run in each lane ‘of a 12% polyaerylamide gel and then eleetrophoretically iwansferred to nitrocellulose filers according to the manu: facturer’s instructions (BioRad, Ine., Hercules, CA). The nitrocellulose filters underwent the following series of room-iemperature incubations in solutions containing 5% milk powder, 0.1 M Tris-HCI (pil 7-5), 0.9% NaCl, and 1490)DAZ FAMILY PROTEINS IN MALE GERM CELL DEVELOPMENT. 1491 0.2% Nonidet 40 (P40): 1) preincubation for 1 h, 2) ine ccubation with a 1:1000 dilution of antisera 149, 150, or 133 for 1 h, 3) three rinses of $ min each, 4) incubation with a 1110000 dilution of secondary aatibody, anti-rabbit IgG, peroxidase conjugate (Sigma Chemical Ca., St. Louis, Mo), for 1 and 5) two rinses of 5 min eaeh There were three more room-temperature rinses of $ min each: one rinse in 5% mill powder, 0.1 M Tris-HICI (pIl 7.5), 0.9% NaCl, and 0.1% Tween 20; and two rinses in 0.1 M Tis HCI (pH 7.5), 09% NaCl. Peroxidase reactions were vie sualized using the electrochemilumineseence system ac- cording to the manufacturer's instructions (Amersham, Ar Tingion Heights, IL), Immunohistochemistry Human tests sections were oblained from a 20-21-wk fews and from a 38-yrold nan who presented witha sem inoma, testes seeions were olained from the Department Gt Pathology, Brigham and Women’s Hospital, Boston, f= Towing insttutional review boar! approval, Mouse test's sections were obined from embryos 18 days posteoitam and fiom 6b-day-old adult mice, an Tnstuional Anita Cave and Use Commitee approved the use of the mice omalinfixed, parfin-embedded buman tissues were sectioned at © um, Aller baking at 60°C for 1h, sections Ivete depatainized and rehydrated (100% xylene, four Times, 3 min each 100% ethane, bvie, 3 min each, 95% cthanol, twice, 3 min each; water, 3 tin), Slides we blocked by treating with 3% hydrogen peroxide in metha- nol for 8 min at room temperature, then washed in water for 4 min, and miowave tesled (800 W. General Electr) 20 199°F far 30 min in 10 mM eta (pT 6.0). Slides were fooled for 15min, transferred to PBS, 0% Tween 20, and preincubated fer TS in al room temperatre in 159% goal Serum, Sides were incubated for I hal room leriperaure ‘with antisera 149 (at 1-000 dition), antisera 130 (1 TOA), or antisera 133 (1:200) in-2%% goat scrum, Slides were Washed thre times (S min exch) in PBS, 0.19% Tween 20, and then incubated for 30 min with biotinyated horse antrabbit IgG (Veetor Laboratories, Burlingame, CA). AE ter three washes {5 min each) in PBS, 0.1% Tween 20, Slides were incubated for 40 mua at room tempersture with avidinbitinylated peroxidase complex (Vector, followed by reaction wi 3.3°-dlaminobenzidinetetrachoride!hydro= gen peroxide, Seelions were subsequently stained with 2% Gil hematorylin, Colume-factonated. preimmune sera corresponding to fractions containing te specific aibod- {es were sed as negative controls ‘Mouse tissues were Weated in a similar manner except that tssies were fixed in Bouin fixative rather than for Inalin, the microwave treatment was eliminaied, and goat Entiaibblt oreradish peturidace conjugated anibonies Gackson tmmunoResearh Laboratories; West Grove, PA) feplaced the biotinavidin system described above Cell typos were determined by enlarging the images and examining the positon, siz, and tnorphology of cals that ‘vere immunopesitve, The etna fo assign ell iypes were those previously reported [31-33] RESULTS ‘Three peptides against which polyclonal antibodies were raised and the corresponding portions of the human DAZ (Yeencaded), human DAZL (autosomal), and mouse DAZ (autosomal) proteins are shown in Figure Ta, We anticipa ced that antisera 149 would recognize all three proteins, that [ fe — +50 FG. 1A NZL sel DAZ pon a he ses api “chan nee oped pape 15; pena bo eet Oe Sen stmoraiy ecoes EL pope und only DRE: ad ci 19nd ey n OA Ain sides te core {eg human A, hman DAP nd wer AM ee 2 aera Date geet Mont aaron pate {tometer ego al nn acs sg we STheu'nth pefnmore soon eri tere 155, bel apis mmamtenendd D4? ep ssctn mice ees tg a nate Sota on be ppt sa weg rhe De pon SEs lopmentnly a6 Beye ee Ot Sin 9 ron ‘arse ne on ema fh We og Tota A sig heed whe mee "Ss whos BA? arene) when sn igs ae ped th SMESY BS ha‘sipettshonanlvomosona! DAZ ple Ho ‘Sac nage eran wat pred ne al a Ps ‘Siow of sear tse om mc of humors wee ppd a SNe isis aeeod. antisera 150 would recognize the autosomal encoded (hu ‘man and mouse) DAZL proieins, and that antisera 133 ‘would recognize the human Y-encoded DAZ protein. [n- deed, on Wester blots of adult mouse tissues, a testis-spe- Gif protein with a molecular weight of 33 kDa, as pre- dictod for mouse DAZL, was detected by antisera 149 and 150 (Fig. 1b). These two antisera also recognized the hu- man DAZL protein, again with a molecular weight of 33, Da, on Westera blots of human adult testes, AS anticipat- ced, human testis protein of higher molecular weight (46 KDa), DAZ, was detected by antisera 149 and 133 (Fig. To). (Competition with the immunizing peptides abolished all ofthese signals; not shown.) Amtisera 133, raised against peplide present in human Y-encoded DAZ but absent in the autosomal proteins, did nat recognize any proteins in1492 Antisera 149 133 Invmunpevoidave sing of human tts eins fetal week 20-21 and adult Ante REUO ET AL. 49 ecoppizes bs DAZL and DAZ. Anti recogsizs DAZ” Anisra (33 recognizes DAZ iy fel section” srows ptf representative gcytes.Albrevitons in ad sete Sp, Spematogonom; pe, spomatacte Spt spermati SC, Sel cal Note hat nig with antes 11 sometime ete depont cf brn des invegans of tubule mens where no cel ae ound, Sections were coumtrstalned wth 2% il horatonyin, mice (Fig. 1, b and d, left-hand lane), where the Y chro- mosome does not carry any DAZ yenes. However, mice carrying a DAZ transgene express a DAZ protein as a fi sion with 14 amino acids from herpes simplex virus thy. ‘midine kinase protein that is recognized by antisera 133 (Fig. 1d, nght-hand lane; transgene construction is as re- ported in Siee ct al. [34)). This indicates that antibody 133 ts specific tthe DAZ protein encoded by the human Y chromosome, The size of the DAZ. provein 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, ia 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 proieins on muluiple blots, this may be expected because there is heterogeneity in the number of repeats in different gene copies in different men [35]. Ia any ease, confidence that the 46-kDa protein represents au- thealic DAZ is based on the observations that this protein is absent from wild-type mouse testis extracts, present in hhuman testis extracts, and present in tansgenic: mice «x- pressing DAZ protin fom a Y-chromosome yeast ail chromosome transgene Wo next used these antisera to explore the distribution of DAZ and DAZE proteins in human testis seetions from 20 to 2l-wk fetus and fom an adult. (The adult had presented with a seminoma; the sections we studied were hot adjacent to the tumor and were comprised of histolog- ically normal seminiferous tubules with a full range of sper- ‘matogenic cell types.) We found that, in humans, the DAZ. ark DAZL proteins are present in mate germ cells at many sages in their development, both prenatally and during spermatogenesis, Antisera 149 uhat recognizes both DAZ and DAZL clearly stained the gonocyles of the fetus and the spermatogonia and spermatocytes of the adult (Fis, 2); wwe 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-cneoded) 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 nomal semen sample. We observed the 33-kDa DAZL. protein readily in these preparations, while we didDAZ FAMILY PROTEINS IN MALE GERM CELL DEVELOPMENT. Antisera 149 eo e4 150 ag 38 Rea Le 133 FG, 3, Immunoperonidase taining of human tests sections ram aus Inccatingvtning potere in mule tubules Ames 169 recognizes thos DAZL and OAZ. Antisera 150 recognizes OAZL. Antseta 3 te gnats DAZ Nove tht sang wih atsra 133 sorties esa a Sk dat gee ue en when a not detect DAZ in mature spermatids (Fig. 4), We conclude that both the autosomally encaded! 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 post ‘meiotic, mature spermatic. ‘By studying the immunohistochemically stained scetions oC human fetal and adult testes, we found thatthe intacel- Tutar locations of the DAZ and DAZL proteins change d ing the progression from fetal gonoeytes fo spermatogonia to spermatocytes to spermatids. In gonoeytes, DAZ and DAZL were present in both the nucleus and the cytoplasm (Fig. 2). In spermatogonia, DAZ ancl DAZ were most abundant ia the nucleus but could also be detected in the feyloplasm (Fig. 2; for example see upper right pane! and cells marked with an asterisk). In spermatocytes, the pro- teins appear fo be restricted tothe eytoplasm (see especially antisera 149, adult testis in Fig. 2; cells indicated by an asterisk). Finally, as the spermatouenic cells mature” in round spermatids and begin to elongate, the patiem of DAZL staining is consistent with cytoplasmic Ioealization Gee expecially antisera 150, adult testis in Fig. 25 c ‘marked by an asterisk). Thus, our results are consistent wi 1493 FG. 4. Wesem blating of prtcng in mature human spermatoros. ‘Western bling as one ising antsera 133. Prin exacts of pera from 3 normal semen sample wee prepared ae described i Maer tnd Meth the hypothesis that DAZ and DAZL are predominantly nu clear in spermatogonia, but at meiosis the proteins are largely locaied in the eytopiasm, where DAZL persists in differentiating spermatids We next sought to determine whether DAZL protein is, present inthe germ cells of mice atthe same developmental ‘tages and in the same cellular compartments asin humans. (As mentioned earlier, there ars 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 130 (Fig. 5). As expected, ro staining is observed with antiserum 183 that speeifically recognizes human Y-chromosome-eneaded DAZ. protein (Fig. 5). In addition, there appears 10 be no staining of Sertoli cells or other somatic cells, in agreement with pre= ‘vious Northem bloting and in situ hybridization studies [18, 19, 27]. In mouse feral gonoeytes, DAZL appears 20 bee present in both the nucleus and the eytoplasm (Fig. 5) Ih Seotions of adult mouse testis countersiained conven: Uionally 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 net coun- tersiained, With no counterstaining, the localization of DAZL to spermatocyte eytoplasm was confirmed, and a second localization—to the nuclei of spermatogonia—was revealed (Fig. 6). We conclude that during spermatogencsis jn mice, as in humans, DAZL shifls [rom a predominantly nuclear 16 a predominantly cytoplasmic location at meiosis. “These findings with mouse DAZL conflict with a recent report indicating that the protein is strictly eytoplasmic in ‘murine male germ cells [16], As inthe present sudy, Rug~ iu et al. [16] reported strong staining for DAZL in, sper Iatocytes, where the protcin is cytoplasmic. They didnot ‘observe staining in spermatogonial nuclei despite using fiu- forescently labeled antibodies. As the two antibodies are1494 REUO ET AL. Invmnwptronidie ang of mwse tests sets with avers 149,150, and 155: al (18 days poston and al 60 days ate bi. Ahtvevation inl echons pq spermatogeniams Sp spermato: KS mu spat FS, elongating spermatel St, Seno ell Av expecta Sizes 13, specie to encoded DAZ, dif na stan ny spermatngenic cel monte ei section. neal acne, arows point representative onecyes ried ayaiot different parts of the DAZE prose, i pesuble tha diferent epiops have dierent availabiin In dtorat cll Spe DISCUSSION ‘Our immunohistochemical studies in human and mouse, together with mutation and expression analysis of DAZ hor FIG. 6. Adult mouse tsi sane! with anise 149 but with 0 he Istoxjlinenunteraning, _mologs in diverse organisms, provide evidence that mam- ‘malian DAZ family. proteins may function at anultiple points in male germ eell development. Specifically, we hy- ppothesize that dhe mammalian DAZ family proteins vet beth during meiosis and much earlier, during the establishment ‘of spermatogonial stem cell populations. As demonstrated hhere in both humans and mice, die DAZ. family proteins are expressed in gonoeyies of fetal testes, long before the ‘onset of spermatogenesis and meiosis (at puberty). In mu: tant male mice lacking Daz! function, germ cells are lost beiween Days 15 and 19 of embryonic development [16], also long before spermatogenesis begins. Taken together these results suggest that DAZ family proteins in both hue ‘mans and mice play a ertieal 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 carly, premciotic phases of male gcim ecll development, leading us to speculate thatthe early functions of the mam: ‘malian DAZ family proteins are diteeted at RNA. process: ing or storage in the nucleus [28] Genetic analysis of diverse species demonstrates the im- portance of DAZ family proteins in meiosis, in one oF both sexes, and our protein localization studies hint at a similar meiotic role in male mammals. In male ies lacking boule function, meiotic divisions da not accur [15]. The Xenopus DAZ homolog Xdazl, when expressed in Drosophila, res- ‘cuss the boule meiotic enlry detect [25]. Also, [oss of Das function in mutant female mice or suppression of Ds homolog function in female nematodes result in germ cell loss at meiosis (16, 24]. (In Dazi-mutant male mice, germ cells are lost during fetal development [16], precluding anyDAZ FAMILY PROTEINS IN MALE GERM CELL DEVELOPMENT. 1s manifesation of the male meiotic role that we postulate here.) As we report, the mammalian DAZ family proteins appear to move from a predominantly nuclear o a predom- inanily eytoplasmie location at male meiosis, The protein product of the Drosophila houle gene, homologous 10 ‘mammalian DAZ and DAZL, undergoes a similar redistri- bution fiom nucieus to eyioplasm when male meiosis is initiated [29]. The late fanctons of the mammalian DAZ family proieins may begin at the onset of meiosis Previous in situ hybridization studies had revealed that, in the human adult testis, D4Z/DAZL transeripts ate most abundant in spermatogonia. and primary. spermatocytes [26}, For DAZ, these in sity hybridization data are in close Accord with the present immunohistoclemical findings. In contrast, DAZL procein appears to persist long after ran- seript levels have peaked. The DAZL protein observed in spermatozoa and their (postmeioti) spermatid precursors may have been synthesized many days or even weeks ear lice ducing or prior to meiosis, Our findings with DAZ are at ods with the report of Habermann et al. [30] that this Y¥-eneaded human protcin is found only in late stage germ cells, spermatids, and spermatozoa. Spermatids and sper- ‘maiozoa are the only male germ eells in which we do not detect the Y-chromosome-cncoded DAZ. protein (Figs. 2 44), Habermann etal. [30] employed a single antiserum that dewected 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 sed and ests Were confine vi evra erent meth ‘The expression patterns of the human DAZ and DAZL. proteins exeriap but are not identical, suggesting that the {wo human proteins may contribute difleretialy to the postulated ettly and late DAZ funily functions. AS de- scribed carlier, the human Y-encoded DAZ protein is most prominently observed in less mature germ cells, especially Bonoeytes and spermatogonia, while the human autoso- mally encoded DAZL protein is clearly present after mei- ‘sis, persisting even in mature spermatozoa. Thus, the Y- ome DAZ gene cluster, which arose by transposition and amplification of the autosomal DAZL gone during primate evolution [17], may have evolved toward specialization in carly finetions; the human DAZL gene may have evolved toward specialization in late (meiotic or postmeiotic) ane lions. This would ft well with previous evidence at men who are deleted far the Ycbore DAZ gene clusier are in fertile because of a problem in the generation or mainte hance of spermatogonial stem cell populations (an early function) (2, 3} and not a defect in the differentiation path ‘way of spermatogenesis itself (a late fumetion). Thus, in- ferilty in D4Z-deleted men may have its origins long be- fore puberty and perhaps even during fetal development ACKNOWLEDGMENTS. ‘We than L. Seow and J Coak-Chrysos fr grap st an A. Bet vin t-Bu Lahn, D. 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