Characterization of D1Pas1, a mouse autosomal homologue of the human AZFa region DBY, as a nuclear protein in spermatogenic cells

FERTILITY AND STERILITY威 VOL. 76, NO. 4, OCTOBER 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

Characterization of D1Pas1, a mouse autosomal homologue of the human AZFa region DBY, as a nuclear protein in spermatogenic cells Donna R. Session, M.D.,a,b Grace S. Lee, B.A.,c and Debra J. Wolgemuth, Ph.D.a,c,d Columbia University College of Physicians and Surgeons, New York, New York

Received January 3, 2001; revised and accepted April 23, 2001. Supported by National Institute of Child Health and Development Physician Scientist Awards K11 HD00974 (D.R.S.) and RO1 HD18122 (D.J.W.). Presented at the Society of Gynecologic Investigation Annual Meeting, San Diego, California, March 19 –22, 1997. Reprint requests: Donna R. Session, M.D., Mayo Clinic, Department of Obstetrics and Gynecology, 200 First Street SW, Rochester, Minnesota 55905. (FAX: 507-284-1774; E-mail: [email protected]). a Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, New York, New York. b Present address: Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, Minnesota. c Genetics and Development, Columbia University College of Physicians and Surgeons, New York, New York. d The Center for Reproductive Sciences, Columbia University College of Physicians and Surgeons, New York, New York. 0015-0282/01/$20.00 PII S0015-0282(01)01996-3

804

Objective: To gain insight into the function of D1Pas1 in spermatogenesis. Design: The cellular and subcellular distribution of D1Pas1 protein were examined. Setting: Academic research laboratory. Animals: Swiss Webster and C57B1/6J mice. Intervention(s): Antibodies were generated against a D1Pas1 fusion protein. Immunoblot analysis was performed on lysates of testicular cells separated into enriched populations of spermatogenic cells and fractionated into nuclear and cytoplasmic compartments. Immunohistochemistry was performed on histological sections of testis from adult and postnatal day 17 mice. Main Outcome Measure(s): D1Pas1 protein distribution. Result(s): D1Pas1 was expressed in germ cells, and its expression was developmentally regulated because it was detected specifically in the meiotic and postmeiotic haploid stages of spermatogenesis. D1Pas1 protein was predominantly localized in the nucleus, with weak cytoplasmic staining. Conclusion(s): Nuclear localization of D1Pas1 in the testis and its sequence homology to putative RNA helicases suggests a role of D1Pas1 in pre-mRNA processing during spermatogenesis. Germ cell expression of D1Pas1 and homology to the Y chromosome gene DBY, which is located in an area deleted in azoospermia, suggests a potential role for an autosomal gene in the regulation of spermatogenesis. (Fertil Steril威 2001;76: 804 –11. ©2001 by American Society for Reproductive Medicine.) Key Words: AZF, DBY, DEAD box, D1Pas1, RNA helicase, spermatogenesis, PL10

The genetic program that regulates mammalian gametogenesis is poorly understood. One strategy to determine the regulation of spermatogenesis is to identify genes that are expressed in testicular cells. The association of azoospermia and deletions of the Y chromosome (1) suggested evidence for the presence of testisspecific genes located on the Y chromosome. To this end, a human Y-chromosome– derived genomic probe designated 12f3 that detects testis-specific transcripts in humans was used to screen a mouse testis cDNA library (2). In the mouse, 12f3 hybridized weakly to D1Pas1 (formerly designated PL10) (3). Murine D1Pas1 is an autosomal gene that maps to chromosome 1 (4) and is thought to be a retroposon of the X chromosome dbx gene (5). There are several other examples of functional

retroposons, which are believed to originate from duplication of RNA intermediates (6 –11). Interestingly, several of the functional retroposons demonstrate germ-cell–specific expression (6 –9). Although the usual fate of a retroposon is degeneration of the sequence into a processed pseudogene, a functional autosomal retroposon locus in spermatogenic cells may have been maintained by selective pressure during evolution. It has been suggested that the transcriptional inactivation of X and Y chromosomes during spermatogenesis or the absence of the X or Y chromosome in haploid cells would explain why the cells may need a second copy of an X or Y gene on an autosome if it is needed during spermatogenesis. In addition, the expression of homologues of Ychromosome genes may explain variations in spermatogenesis observed in families of patients with Y-chromosome deletions.

The D1Pas1 cDNA is highly homologous to the Y-chromosome gene DBY in both mouse and human (5, 12). DBY is located in the euchromatic part of the long Y-chromosome arm (Yq11), and deletions of this region have been associated with azoospermia and/or severe oligospermia (13). DBY shares conserved sequences with D1Pas1 that are suggestive of RNA helicase activity. The protein deduced from the coding region of the D1Pas1 cDNA shares sequence homology with several other proteins, which are members of the DEAD box RNA helicase gene family (14). The DEAD box proteins are members of a family of prokaryotic, eukaryotic, and viral NTPases. This family of proteins has been implicated in cellular processes involving the alteration of RNA secondary structure, because RNA helicases catalyze the separation of basepaired regions. RNA secondary structure is essential for RNA function in pre-mRNA splicing, mRNA translation, ribosome assembly, and RNA stability. The involvement of RNA helicases in the regulation of these processes has been demonstrated in translation initiation (15) and mRNA splicing (16). This evidence and the fact that D1Pas1 mRNA expression is developmentally regulated with the highest levels of transcripts present during the meiotic and haploid stages of spermatogenesis (3) support a regulatory rather than structural role for D1Pas1 in the testis. This study was performed to further characterize the possible function of the D1Pas1 gene by producing a fusion protein, which was used to generate antibodies for use in immunoblot and immunohistochemical analysis to identify the cellular and subcellular distribution of the protein during spermatogenesis.

MATERIALS AND METHODS Source of Tissues Swiss Webster mice obtained from Charles River (Wilmington, DE) or C57B1/6J mice obtained from Jackson Laboratory (Bar Harbor, ME) were used as the source of testes. Testes were obtained from mice older than 35 days and from mice at postnatal day 17. Dissected tissues were fixed in 4% paraformaldehyde in 1⫻ phosphate-buffered saline (PBS; 130 mM NaCl, 7 mM Na2PO4, and 3 mM Na2PO4) overnight at 4°C. The paraformaldehyde was replaced by 1⫻ PBS, followed by washes of 0.85% saline, 0.85% saline/ethanol (1:1), and 70% ethanol. Tissues were dehydrated by transfer through graded ethanols, followed by xylene, and were embedded in paraffin wax (Tissue Tek embedding medium, Fisher Scientific, Pittsburgh, PA). Sections were cut at 5 ␮m, collected onto pretreated slides (Fisher Scientific), and stored at 4°C until needed. Investigations were performed after approval by the institutional review board.

Enriched populations of pachytene spermatocytes, round spermatids, and residual bodies and cytoplasmic fragments were obtained, and lysates were prepared for use in immunoblot analysis.

Tissue Preparation and Protein Quantitation Fresh or frozen testes were homogenized in 2⫻ sample buffer (18) and denatured for 5 minutes at 100°C, and insoluble components were removed by centrifugation. Homogenates were either used immediately or stored at ⫺80°C. Protein quantitation was determined by the Bradford dye assay (Bio-Rad Laboratories, Hercules, CA) (19).

In Vitro Transcription and Translation of D1Pas1 Synthetic messenger RNA was prepared according to the manufacturer’s protocol (Promega Corporation, Madison, WI) using the 3.2-kb D1Pas1 cDNA (3). The control used the same vector with an untranslatable cDNA fragment. The reaction contained 1 ␮g of linearized vector DNA, 2 mM DTT, 50 units RNasin, 500 ␮M of each ribonucleoside triphosphate, and 15 units of RNA polymerase in a volume of 100 ␮L of transcription buffer (Promega). After transcription for 1 hour at 37°C, the DNA template was removed by incubation with 1 unit per microgram of DNase for 30 minutes at 37°C. RNA was recovered by ethanol precipitation and dissolved at a final concentration of 0.5 ␮g/mL. In vitro translations were carried out in rabbit reticulocyte lysate in a final volume of 50 ␮L according to the protocol provided by the manufacturer (Promega). The reaction was performed in nuclease-treated lysate containing 50 units of RNasin, 1 ␮L of 1 mM amino acid mixture (minus methionine), 40 uCi of [35S] methionine, and 1 ␮g RNA and was incubated for 1 hour at 30°C. Aliquots of the sample were mixed with sample buffer and resolved by using sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) electrophoresis. The gel was dried and exposed to X-ray film.

Production of the D1Pas1 Fusion Protein A XmnI to NheI fragment (1695 bp) of the 3.2-kb D1Pas1 cDNA (3) was subcloned into the pET21B-derived expression vector (Novagen, Madison, WI) (20). The DNA at the subcloning sites was sequenced to confirm that no cloning artifacts had occurred. DNA sequencing was performed on an applied Biosystems Model 373A DNA sequencer (Applied Biosystems, Foster City, CA) and analyzed using IBI Pustell sequence analysis software (21). The expression vector was used to transform Escherichia coli strain BL21 (DE3) LysS. Induction of expression, cell harvest, and protein purification by metal chelation chromatography using His-Bind resin under denaturing conditions were carried out under conditions suggested by the manufacturer (Novagen).

Separation of Testicular Cells

Production and Purification and D1Pas1 Antibodies

Testicular cells were separated on a 2%– 4% BSA gradient at unit gravity, as described by Wolgemuth et al. (17).

The protein was coupled to protein A to enhance antigenicity. The protein was emulsified in complete Freund’s

FERTILITY & STERILITY威

805

adjuvant and injected into rabbits (Pocono Rabbit Farm Laboratory, Canadenesis, PA). Individual rabbits were immunized by subcutaneous injections. Animals were boosted monthly and bled 10 days after each immunization. Antibodies were affinity purified against the fusion protein on nitrocellulose strips (22). The eluted antibody was concentrated by centrifugation (micron 30, Amicon, Bedford, MA), and the final concentration was estimated via absorption at OD280. The purification was confirmed via immunoblotting.

Kit, Vector Laboratories, Burlingame, CA). The sections were incubated in ABC reagent for 2 hours at room temperature, followed by three 10-minute washes with 1⫻ PBS containing 0.1% Triton X-100, equilibrated with 0.1 M Tris, pH 7.2, for 5 minutes. Detection was accomplished with 0.2 mg/mL diaminobenzidine and 0.01% hydrogen peroxide in 0.1 M Tris, pH 7.2. The sections were counterstained with hematoxylin, and coverslips were applied using Protexx mounting media (Baxter Diagnostics, McGaw Park, IL).

Immunoblot Analysis

Subcellular Fractionation

Lysates from total testis or enriched populations of germ cells were run on 7%–10% SDS–polyacrylamide gels and blotted onto supported nitrocellulose (Schleicher & Schuell, Keene, NH) using a Hoefer tank transfer apparatus (Hoefer Scientific Instruments, San Francisco, CA). Blots were blocked for 3 hours in Blotto (6% nonfat dry milk in Trisbuffered saline [1⫻ TBS: 20mM Tris, pH 7.5, 150mM NaCl]) at room temperature. Blots were incubated with primary antibodies at a concentration of 1:1,000 for whole serum and 1 ␮g per milliliter for affinity-purified antibody in Blotto for 2 hours at room temperature. The filters were then washed three times for 10 minutes in 1⫻ TBS. The filters were then incubated with the secondary antibody, goat antirabbit IgG (Sigma Chemical Company, St. Louis, MO) conjugated to horseradish peroxidase (1:3,000), for 1 hour at room temperature, followed by three 5-minute rinses in 1⫻ TBS. An Enhanced Chemiluminescence Kit (ECL Kit, Amersham, Chicago, IL) followed by autoradiography was used to detect immune complexes.

Nuclear and cytoplasmic subcellular compartments were obtained from a total testicular homogenate, which was fractionated by differential centrifugation on a sucrose gradient (24). Briefly, testes of five Swiss Webster mice were decapsulated and suspended in 1⫻ PBS. The tubules were then treated with collagenase to remove interstitial cells and allowed to settle. They were then washed three times with 1⫻ PBS before treatment with trypsin and DNase to disrupt the tubules (17). The tubules were filtered through a nylon mesh and concentrated by centrifugation. The pellet was washed three times with 1⫻ PBS and resuspended in TMK (20 mM Tris, pH 7.7, 40 mM KCl, 10 mM MgCl2), centrifuged at 225 ⫻ g for 5 minutes, and resuspended in 0.32 M sucrose, 1% Triton X-100, and observed for cell lysis. One milliliter was overlaid on a 1.1 M sucrose solution and centrifuged at 500 ⫻ g for 10 minutes; the pellet was resuspended in 0.32 M sucrose and centrifuged at 135 ⫻ g for 5 minutes. Lysates were prepared and the proteins fractionated by SDS-PAG electrophoresis according to our standard procedures described above.

Immunohistochemistry The sections were deparaffinized by two 10-minute incubations in Histoclear (National Diagnostics, Atlanta, GA). Tissues were rehydrated through graded ethanol and washed in 1⫻ PBS containing 0.1% Triton X-100. The sections were microwaved twice for 5 minutes each in 0.1 M citrate buffer (23). Endogenous peroxidase activity was abolished by incubating sections in methanol containing 0.3% hydrogen peroxide for 20 minutes. Sections were then rehydrated by two 10-minute washes in 1⫻ PBS containing 0.1% Triton X-100. All of the subsequent incubations were performed in a humidified atmosphere. Sections were preblocked with 1⫻ PBS containing 0.1% Triton X-100, 1% normal goat serum for 1 hour at 4°C. The primary antibody was a 1:500 dilution (in 1⫻ PBS containing 0.1% Triton X-100, 1% normal goat serum) of whole serum from rabbits immunized with the D1Pas1 protein and preimmune serum from the same rabbits overnight at 4°C. In the preblocked controls, the dilution of whole serum was incubated with D1Pas1 fusion protein (10 ␮g) for 1 hour prior at room temperature on a rocking platform. This was followed by three 10-minute washes with 1⫻ PBS containing 0.1% Triton X-100. Localization of antibody was detected by biotinylated secondary goat anti-rabbit antibodies and stained with immunoperoxidase (Vectastain ABC 806

Session et al.

D1Pas1 and spermatogenesis

Sequence Analysis Homology searches and pairwise computer alignment were performed using the software package of the Genetics Computer Group (version 9.0 –9.1; Genetics Computer Group, Madison, WI) for nucleic and amino acid sequence comparison.

RESULTS Polyacrylamide Gel Electrophoresis Analysis of D1Pas1 in the Testis In vitro transcription and translation of D1Pas1 protein yields a single protein migrating at the relative molecular weight predicted from the D1Pas1 cDNA. There were no detectable bands in the control lane in which the reaction lacked a translatable cDNA template (Fig. 1). A portion of this cDNA was used to produce D1Pas1 in E. coli. To determine whether the anti-D1Pas1 fusion protein antibodies could recognize native D1Pas1, protein lysates of murine testis and the fusion protein were immunoblotted (Fig. 2). The antibodies detected a band at the appropriate size for native D1Pas1 as well as the truncated fusion protein. The expression of D1Pas1 in cells from different stages of spermatogenesis was examined by immunoblot analysis of Vol. 76, No. 4, October 2001

FIGURE 1 In vitro transcription/translation of D1Pas1. The 35S-labeled in vitro translation product was run on a 10% SDS-PAGE and the autoradiograph exposed overnight. A single band of the predicted size is detected in the samples of the protein derived from the 3.2-kb D1Pas1 cDNA (3). A band was not detected in the control lane, which included linearized vector with an untranslatable cDNA fragment. The relative molecular weight of the protein is indicated.

FIGURE 2 Detection of D1Pas1 native and fusion protein. Lysates of the fusion protein (F) and total testis (T) of adult Swiss Webster mice were immunoblotted with 1 ␮g/mL of affinity-purified antibody. The molecular weights of the markers are indicated, and the entire blot is represented. Sixty micrograms of protein was loaded per lane.

Session. D1Pas1 and spermatogenesis. Fertil Steril 2001.

lysates from adult testes separated on an albumin gradient. Testicular cells were separated into three enriched populations: pachytene spermatocytes, round spermatids, and residual bodies and cytoplasmic fragments. Residual bodies are the cytoplasm that is lost from elongating spermatids during spermatogenesis. Cytoplasmic fragments arise as artifacts of the cell fractionation technique and are predominantly from elongating spermatids. A band migrating at 70 kd was detected in pachytene spermatocytes, round spermatids, and cytoplasmic fragments and residual bodies (Fig. 3A). Immunoblotting, therefore, suggests the presence of D1Pas1 in several stages of spermatogenic cells. The presence of D1Pas1 in cytoplasmic fragments and residual bodies likely represents contamination; typically, this procedure yields fractions with approximately 5% contaminating cells (17). The subcellular distribution D1Pas1 was examined by immunoblot analysis performed on testicular cells that had been fractionated into nuclear and cytoplasmic compartments (Fig. 3B). The anti-D1Pas1 antibodies identify a band of the appropriate size in the nuclear fraction that was not detected in the cytoplasmic fraction. A lysate from total testis was included as a positive control.

Immunohistochemical Localization of D1Pas1 in the Testis Immunohistochemistry was performed to localize D1Pas1 more precisely to specific cell types in the testis to determine its expression pattern in the specific stages of meiosis and to confirm its apparent nuclear localization. In the testis, D1Pas1 was expressed predominantly in the nuclei of germ cells that were undergoing meiosis (Fig. 4A and B). There was no detectable staining in the interstitial Leydig cells (Fig. 4D and E). The controls preblocked with the immunizFERTILITY & STERILITY威

Session. D1Pas1 and spermatogenesis. Fertil Steril 2001.

ing protein and preimmune serum (Fig. 4C) revealed no significant staining. Localization of D1Pas1 to specific stages of spermatogenesis was determined by the staging of the seminiferous tubules according to the method described by Oakberg (25) and Russell et al. (26). D1Pas1 protein was expressed from zygotene to diplotene of meiotic prophase. D1Pas1 was abundantly expressed in round spermatids. As spermatids became fully elongated, no nuclear D1Pas1 protein was detected. The highest level of detection occurred in pachytene spermatocytes. The expression of D1Pas1 was confirmed using testes from day 17 mice (Fig. 4B), which are enriched in pachytene spermatocytes.

Sequence Homology with DBX/DBY Alignment searches revealed significant identity of the mouse D1Pas1 protein with human DBX/DBY (12). D1Pas1 807

FIGURE 3 Subcellular expression of D1Pas1 in the testis. The relative molecular weights of the proteins are indicated. (A), Detection of D1Pas1 protein in separated testicular cells. Testicular cells were separated into purified pools of pachytene spermatocytes (P), round spermatids (RS), and cytoplasmic fragments and residual bodies (CF/RB). Lysates from the pooled cells were analyzed for the presence of D1Pas1 protein by immunoblotting with 1 ␮g/mL of purified antibody. Two hundred micrograms of total protein was loaded per lane. (B), Detection of D1Pas1 protein in a testicular cell fractionation. Sixty micrograms of protein was loaded per lane. Immunoblotting was performed using a 1:1,000 dilution of whole serum. Lane N ⫽ nuclear fraction; T ⫽ total testis; C ⫽ cytoplasmic fraction.

sociation within the seminiferous tubules. Our immunohistochemistry studies demonstrated that D1Pas1 is most highly expressed in the nuclei of spermatocytes from pachytene to diplotene of meiosis I. Immunohistochemistry performed on day 17 testis confirms the expression of D1Pas1 during meiosis. In testis of day 17 mice, the germ cells are predominantly in the pachytene stage (30). The lack of staining in the sections preincubated with the immunizing peptide and in sections incubated with preimmune serum suggests specificity for the anti-D1Pas1 antibodies. In addition, D1Pas1 protein expression closely correlates with the previously determined pattern of expression of D1Pas1 transcripts (3). The immunohistochemical analysis and immunoblot assessment of subcellular fractions revealed the localization of D1Pas1 in the nuclei of cells undergoing spermatogenesis. This nuclear localization, along with D1Pas1’s homology to yeast genes known to be involved in splicing, suggested that the D1Pas1 protein may have an analogous function in mammals. D1Pas1 exhibits a relatively high degree of homology (53% amino acid identity) to the yeast protein DED1 (31). Mutants of DED1 are defective in the initiation of translation, and D1Pas1 cDNA has been shown to complement yeast DED1 mutants (31). Genetic studies have also shown that DED1 can act as a suppressor for the splicing defect in PRP8 mutants (32). Moreover, D1Pas1 contains a C-terminal motif (designated RS/GF) that is enriched in Arg, Ser, Gly, and Phe. The RS/GF domain is reminiscent of the basic RS domains found in several splicing factors, including snRNP (33) and the non-snRNP proteins (34). However, D1Pas1 does not possess the prototypical RS domain.

Session. D1Pas1 and spermatogenesis. Fertil Steril 2001.

revealed 95% and 88% identity with DBX (X-chromosome homologue of DBY) at the amino acid and nucleic acid levels, respectively. D1Pas1 revealed 89% and 81% identity with the Y-chromosome gene DBY at the amino acid and nucleic acid levels, respectively. In comparing the three sequences, significant stretches of absolute identity and conservative substitutions were seen distributed over the entire alignment (Fig. 5). All three proteins shared the highly conserved segments already described in RNA helicases, indicated by the shaded areas (Fig. 5) (27, 28). The extensive homology suggested that D1Pas1 is a mouse homologue of the human DBY/DBX gene.

DISCUSSION During spermatogenesis in mice, the appearance of spermatocytes occurs in a defined sequence of differentiation (29). It is possible to determine the stage of meiosis based on morphological criteria and the characteristic patterns of as808

Session et al.

D1Pas1 and spermatogenesis

Alternatively, D1Pas1 could be involved in nuclear processes such as transcription or nucleolar assembly, similar to that proposed for the human nuclear protein p68 (35). Human nuclear protein p68 has demonstrated RNA-dependent ATPase activity (36) and ATP-dependent RNA helicase activity in vitro (37). The p68 protein undergoes dramatic changes in nuclear localization during the cell cycle. It is localized in the nucleoplasm during interphase and translocates to the nucleoli during telophase, suggesting a role in nucleolar assembly (38). D1Pas1 has the highest degree of homology to DBX, also denoted DDX3 and CAP-Rf, in the human (39 – 41). DBX has been characterized through its interaction with the hepatitis C virus (39, 40). Immunofluorescent staining of HeLa cells showed that DBX is predominantly expressed in nuclear speckles and at low levels in the cytoplasm, consistent with the expression pattern of D1Pas1 in the mouse testis (40). In vitro, DBX has ATPase activity, similar to the case with other RNA helicases, which is enhanced by the hepatitis C virus core protein (39). DBX leads to an enhancement of reporter plasmid activity using a luciferase assay, indicating that DBX is involved in the regulation of gene expression (39). Although the homology to RNA helicase proteins is Vol. 76, No. 4, October 2001

FIGURE 4 Immunohistochemical localization of D1Pas1 in the testis. Immunohistochemistry was performed on paraffin-embedded Swiss Webster testis sections using a 1:500 dilution of whole serum on adult testis (A), day 17 testis (B) at ⫻100 magnification, and preimmune serum on day 17 testis (C) at ⫻40 magnification; and a 1:500 dilution of whole serum on adult testis (D) and day 17 (E) testis at ⫻40 magnification. The sections were stained brown with diaminobenzidine and counterstained with hematoxylin, which stains nuclei blue.

Session. D1Pas1 and spermatogenesis. Fertil Steril 2001.

high, the biochemical function of the mammalian D1Pas1 during spermatogenesis remains to be determined. The Y chromosome has been shown to be essential for male fertility. Recently, additional candidate genes for Y male fertility genes have been identified (12). Most of the candidate genes have been mapped to one of three regions in the euchromatic part of the Y chromosome, designated AZF regions a,b,c (13). These genes have been sorted into two classes: Y genes with a homologous gene copy on the X chromosome (DBY, DFFRY, E1F1AY, UTY) and Y-specific repetitive gene families expressed solely in the testis (BPY1, BPY2, CDY, PRY, TTY1, TTY2, XKRY, TSPY, RBM, DAZ). Genes of the first group have several shared features. Each gene has a homologue on the X chromosome encoding a similar but not identical protein. Each exists in a single copy and is expressed in a wide variety of tissues. Some AZF genes express testis-specific transcripts (DBY, E1F1AY). The D1Pas1 cDNA shares sequence homology with the human AZFa gene, DBY. D1Pas1 is believed to be a retroposon of the X homologue of mouse gene Dby, Dbx (5). The tight linkage and order of mouse Dffry-Dby-Uty was shown FERTILITY & STERILITY威

to be conserved in the human Y chromosome and is the first example of syntenic homology between Y chromosomes from two distinct mammalian orders (5). This syntenic homology between mouse and human may have functional significance, as deletions affecting the Dffry-Dby-Uty block are associated with severe early blockages in spermatogenesis (42). Although a single phenotypic correlation does not exist, microdeletions restricted to the AZFa region usually result in patients with Sertoli cell– only syndrome or severe oligospermia (42). Genes associated with Sertoli cell– only syndrome would be expected to be expressed in the early stages of spermatogenesis. The expression of D1Pas1 in early meiotic stages of spermatogenesis would be consistent with severe oligospermia if D1Pas1 and its homologue on the Y chromosome are essential for spermatogenesis. In summary, the D1Pas1 protein product was shown to be expressed in the germ cells of the testis and its expression developmentally regulated. The protein was localized predominantly in the nuclei of the cells, which would suggest a role for D1Pas1 in the regulation of mRNA processing rather than translation initiation. Functional implications derived 809

FIGURE 5 Amino acid alignment of mouse and human genes. Amino acids identical in D1Pas1, DBX, and DBY are shown in upper-case letters. Dots indicate sequence breaks that are included to give optimal alignments. Shaded boxes correspond to the consensus motifs I–IX in proteins identified as DEAD box RNA helicases (27, 28).

Session. D1Pas1 and spermatogenesis. Fertil Steril 2001.

from structural similarities are of course speculative. However, it seems that the DEAD box family shares a common ATP-dependent helicase function. The putative helicase structure of D1Pas1 suggests that it is not a structural pro810

Session et al.

D1Pas1 and spermatogenesis

tein. The cellular and temporal specificity of the expression in the germ line strongly suggest a specific regulatory role for the D1Pas1 protein during spermatogenesis. References 1. Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet 1976;34:119 –24. 2. Leroy P, Seboun E, Mattei MG, Fellous M, Bishop CE. Testis-specific transcripts detected by a human Y-DNA-derived probe. Development 1987;101:177– 83. 3. Leroy P, Alzari P, Sassoon D, Wolgemuth D, Fellous M. The protein encoded by a murine male germ cell-specific transcript is a putative ATP-dependent RNA helicase. Cell 1989;57:549 –59. 4. Kingsmore SF, Vik DP, Kurtz CB, Leroy P, Tack BF, Weis JH, et al. Genetic organization of complement receptor-related genes in the mouse. J Exp Med 1989;169:1479 – 84. 5. Mazeyrat S, Saut N, Sargent CA, Grimmond S, Longepied G, Ehrmann IE, et al. The mouse Y chromosome interval necessary for spermatogonial proliferation is gene dense with syntenic homology to the human AZFa region. Hum Mol Genet 1998;7:1713–24. 6. Dahl HHM, Brown RM, Hutchison WM, Maragos C, Brown GK. A testis-specific form of the human pyruvate dehydrogenase E1␣ subunit is coded for by an intronless gene on chromosome 4. Genomics 1990; 8:225–32. 7. McCarrey JR, Thomas K. Human testis-specific PGK gene lacks introns and possesses characteristics of a processed gene. Nature 1987; 326:501–5. 8. Hendriksen PJM, Hoogerbrugge JW, Baarends WM, de Boer P, Vreeburg JTM, Vos EA, et al. Testis-specific expression of a functional retroposon encoding glucose-6-phosphate dehydrogenase in the mouse. Genomics 1997;41:350 –9. 9. Boer PH, Adra CN, Lau Y-F, McBurney MW. The testis-specific phosphoglycerate kinase gene pgk-2 is a recruited retroposon. Mol Cell Biol 1987;7:3107–12. 10. Reinton N, Haugen TB, Orstavik S, Skalhegg BS, Hansson V, Jahnsen T, et al. The gene encoding the C ␥ catalytic subunit of cAMP-dependent protein kinase is a transcribed retroposon. Genomics 1998;49:290–7. 11. Ehrmann IE, Ellis PS, Mazeyrat S, Duthie S, Brockdorff N, Mattei MG, et al. Characterization of genes encoding translation initiation factor eIF-2␥ in mouse and human: sex chromosome localization, escape from X-inactivation and evolution. Hum Mol Genet 1998;7:1725–37. 12. Lahn BT, Page DC. Functional coherence of the human Y chromosome. Science 1997;278:675– 80. 13. Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, et al. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 1996;5:933– 43. 14. Schmid SR, Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol 1992;6:283–92. 15. Nielsen PJ, McMaster GK, Trachsel H. Cloning of eukaryotic protein synthesis initiation factor genes: isolation and characterization of cDNA clones encoding factor eIF-4A. Nucleic Acids Res 1985;13: 6867– 80. 16. Strauss EJ, Guthrie C. PRP28, a “DEAD-box” protein, is required for the first step of mRNA splicing in vitro. Nucleic Acids Res 1994;22: 3187–93. 17. Wolgemuth DJ, Gizang-Ginsberg E, Engelmyer E, Gavin BJ, Ponzetto C. Separation of mouse testis cells on a Celsep apparatus and their usefulness as a source of high molecular weight DNA or RNA. Gamete Res 1985;12:1–10. 18. Laemmli UK. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 1970;227:680 –5. 19. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 –54. 20. Rosenberg AH, Lade BN, Chui D-S, Lin S-W, Dunn JJ, Studier FW. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 1987;56:125–35. 21. Pustell JM. Interactive molecular biology computing. Nucleic Acids Res 1988;16:1813–20. 22. Lillie SH, Brown SS. Artifactual immunofluorescent labeling in yeast, demonstrated by affinity purification of antibody. Yeast 1987;3:63–70. 23. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991;39:741– 8. 24. Bellve AR. Purification, culture, and fractionation of spermatogenic cells. Methods Enzymol 1993;225:84 –113. 25. Oakberg EF. A description of spermiogenesis in the mouse and its use

Vol. 76, No. 4, October 2001

26. 27. 28.

29. 30. 31. 32. 33.

in the analysis of the cell cycle of the seminiferous epithelium and germ cell renewal. Am J Anat 1956;99:391– 409. Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED. Histological and histopathological evaluation of the testis. Clearwater, FL: Cache River Press, 1990. Linder P, Lasko PF, Ashburner M, Leroy P, Nielsen PJ, Nishi K, et al. Birth of the D-E-A-D box [letter]. Nature 1989;337:121–2. Pause A, Methot N, Sonenberg N. The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis. Mol Cell Biol 1993; 13:6789 –98. Nebel BR, Amarose AP, Hackett EM. Calendar of gametogenic development in the prepubertal mouse. Science 1961;134:832–3. Bellve AR, Cavicchia JC, Millette CF, O’Brien DA, Bhatnager YM, Dym M. Spermatogenic cells of the prepubertal mouse. Isolation and morphological characterization. J Cell Biol 1977;74:68 – 85. Chuang R-Y, Weaver PL, Liu Z, Chang T-H. Requirement of the DEAD-Box protein Ded1p for messenger RNA translation. Science 1997;275:1468 –71. Jamieson DJ, Rahe B, Pringle J, Beggs JD. A suppressor of a yeast splicing mutation (prp8-1) encodes a putative ATP-dependent RNA helicase. Nature 1991;349:715–7. Theissen H, Etzerodt M, Reuter R, Schneider C, Lottspeich F, Argos P, et al. Cloning of the human cDNA for the U1 RNA-associated 70K protein. EMBO J 1986;5:3209 –17.

FERTILITY & STERILITY威

34. Fu X-D, Maniatis T. Isolation of a complementary DNA that encodes the mammalian splicing factor SC35. Science 1992;256:535– 8. 35. Ford MJ, Anton IA, Lane DP. Nuclear protein with sequence homology to translation initiation factor eIF-4A. Nature 1988;332:736 – 8. 36. Iggo RD, Lane DP. Nuclear protein p68 is an RNA-dependent ATPase. EMBO J 1989;8:1827–31. 37. Hirling H, Scheffner M, Restle T, Stahl H. RNA helicase activity associated with the human p68 protein. Nature 1989;339:562– 4. 38. Iggo RD, Jamieson DJ, MacNeill SA, Southgate J, McPheat J, Lane DP. p68 RNA helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts. Mol Cell Biol 1991;11:1326 –33. 39. You L-R, Chen C-M, Yeh T-S, Tsai T-Y, Mai R-T, Lin C-H, et al. Hepatitis C virus core protein interacts with cellular putative RNA helicase. J Virol 1999;73:2841–53. 40. Owsianka AM, Patel AH. Hepatitis C virus core protein interacts with a human DEAD Box Protein DDX3. Virology 1999;257:330 – 40. 41. Park SH, Lee S-G, Kim Y, Song K. Assignment of a human putative RNA helicase gene DDX3, to human X chromosome bands p11.3p11.23. Cytogenet Cell Genet 1998;81:178 –9. 42. Kent-First M, Muallem A, Shultz J, Pryor J, Roberts K, Nolten W, et al. Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection. Mol Reprod Dev 1999;53:27– 41.

811