Expression patterns of the DAZ-associated protein DAZAP1 in rat and human ovaries

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1 Expression patterns of the DAZ-associated protein DAZAP1 in rat and human ovaries Hsien-An Pan, M.D., a,b Yue-Shan Lin, M.D., c,d Ko-Hung Lee, M.D., a Jin-Ru Huang, M.Sc., e Ying-Hui Lin, M.D., a and Pao-Lin Kuo, M.D. a a Department of Obstetrics and Gynecology, b Institute of Clinical Medicine; c Department of Obstetrics and Gynecology, Chi-Mei Hospital; d Center of General Education, National Chung Cheng University, Kaohsiung, Taiwan; and e Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Kaohsiung, Taiwan Objective: To evaluate the expression of DAZAP1 (deleted in azoospermia associated protein 1) in rat and human ovaries. Design: Experimental study. Setting: University hospital. Patient(s): Twelve corpus luteum (CL) specimens were collected during operation, either by laparoscopic surgery for CL rupture or by laparotomy for benign gynecologic conditions. Intervention(s): Surgical excision of 12 human CL. Main Outcome Measure(s): Proteins analyzed by immunohistochemical staining, Western blotting, and coimmunoprecipitation experiments. Result(s): DAZAP1 is expressed in rat and human luteal cells. Expression of DAZAP1 decreases with advancing stages of CL. Co-immunoprecipitation experiments show in vivo interaction of DAZ-like (DAZL) protein with DAZAP1 in the ovarian tissues. Conclusion(s): The expression patterns of DAZAP1 and DAZL are identical within rat and human ovaries. In mammalian species, DAZAP1 may be involved in diverse reproductive functions, ranging from cell cycle regulation and maturation of oocytes to differentiation of luteal cells. (Fertil Steril 2005;84(Suppl 2): by American Society for Reproductive Medicine.) Key Words: Corpus luteum, DAZAP1, DAZL Proteins that bind RNA play important roles in the posttranscriptional regulation of gene expression. They also participate in the processing, transport, localization, and translational control of messenger RNA (1, 2). Ribonucleic acids that localize to the vegetal cortex of Xenopus oocytes are involved in early embryonic patterning and cell fate specification. Vg1 is a maternal mrna that becomes localized to the vegetal cortex of the mature oocyte and is required for generating dorsal mesoderm at the blastula stage of Xenopus embryogenesis (3, 4). Proline-rich RNA-binding protein (Prrp) binds to Vg1 mrna and VegT mrna, which are essential for the proper localization of Vg1 mrna to the vegetal cortex of the Xenopus oocytes (5). The ortholog of Prrp in the mammalian species is DAZAP1 (deleted in azoospermia associated protein 1). DAZAP1 shares 89% similarity and 81% identity in amino acid sequences with Xenopus Prrp (6). DAZAP1 also is an RNA-binding protein, identified through its interaction with putative male infertility factors, DAZ and DAZ-like (DAZL), in a yeast twohybrid system (7). DAZAP1 contains two RNA-binding Received November 2, 2004; revised and accepted March 13, Supported by research grants from the National Science Council, Taiwan (NSC B and NSC B ). Reprint requests: Pao-Lin Kuo, M.D., Department of Obstetrics and Gynecology, National Cheng-Kung University Medical Center, 138 Sheng-Li Road, Tainan Taiwan (FAX: ; paolink@mail.ncku.edu.tw). domains in the N-terminal portion and a proline-rich domain in the C-terminal portion and is expressed most abundantly in the testis (8). The DAZ gene family consists of three members in the human being: DAZ on Yq11.2, DAZL on 3p25, and BOULE on 2q33. The DAZ gene is present only on the Y chromosomes of great apes and Old World monkeys (9, 10). The autosomal DAZLgene is present in all vertebrates studied (10 13), and BOULE orthologs have been isolated from Drosophila and Caenorhabditis elegans in addition to mice and humans (14 16). Both DAZ and DAZL are thought to have evolved from the same ancestral BOULE gene, and they share common characteristics (16). They both encode RNA-binding proteins that are expressed mainly in germ cells. The DAZ gene encode several isoforms with a polymorphic DAZ repeat region containing 8 24 copies of a 24 amino acid DAZ repeat, whereas the DAZL protein contains a single DAZ repeat unit (9 11, 17). Deletion of the DAZ repeat region dramatically reduced the binding of DAZ/DAZL to DAZAP1 (7). DAZLis unique in terms of its tissue distribution patterns among DAZ gene family members, considering its expression not only in germ cells but also in somatic cells. In the male reproductive system, the DAZL protein is expressed in multiple stages of germ cell development, including gonocyte, spermatogonia, spermatocyte, spermatid, and spermatozoa (18 20). In the female reproductive system, DAZL is expressed in the fetal oogonium, adult oocytes, /05/$30.00 Fertility and Sterility Vol. 84, Suppl 2, October 2005 doi: /j.fertnstert Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. 1089

2 granulosa cells, and theca interna (21 23). In a report elsewhere, we showed evidence that DAZL protein is expressed in granulosa-lutein cells from different phases of the corpus luteum (CL) (24). Like DAZ/DAZL, DAZAP1 also binds to RNA homopolymers, with a preference for polyuridylic acid and polyguanylic acid (7). In vitro, DAZAP1 binds to both the Y chromosome encoded DAZ and an autosome-encoded DAZL protein. However, the in vivo interaction of DAZ gene family members with DAZAP1 has not been demonstrated. Most proteins function through their interaction with other proteins. In this study, we showed expression patterns of DAZAP1 in rat and human ovaries. We also demonstrated the in vivo interaction of DAZL with DAZAP1. MATERIALS AND METHODS Collection of Human Ovarian Tissues This study was approved by the institutional review board of National Cheng Kung University Hospital. We collected 12 specimens of CL: 4 in the early luteal phase, 4 in the mid-luteal phase, and 4 in the late luteal phase. The specimens were taken during operation, either by laparoscopic surgery for CL rupture or by laparotomy for benign gynecologic conditions. Each patient had regular menstrual periods, and the dating had been confirmed by the histology of the endometrium. The luteal phase was subdivided into an early luteal phase (days of the cycle, or young CL, 1 5 days of age), mid-luteal phase (days of the cycle, or mature CL, 6 10 days of age), and late luteal phase (days 25 27, or old CL, days of age). Cycle day 14 was considered to be CL day 0. Human luteinized granulosa cells were obtained from four women undergoing IVF at our medical center. Granulosa cells were isolated, as described elsewhere (25), from aspirated follicular fluid after ovum retrieval. After centrifugation, the GLC layer on the top of a red blood cell pellet was resuspended in 1X phosphate buffer saline (PBS) containing 100 g/ml DNase I (Roche Molecular Biochemicals, Mannheim, Germany) and 2mg/mL type IV collagenase (Gibco, Grand Island, NY). The cell suspension was incubated at 37 C for 20 minutes and centrifuged at 300 Xg for 10 minutes at room temperature. The pellet was resuspended in lysis buffer (120mM NaCl, 10mM Tris-HCl (ph 6.8), 1% Noidet P-40, 0.1% SDS and 1% deoxycholate) with protease inhibitor cocktail [2mM 4-(2-aminoethyl)benzenesulfony fluoride (AEBSF), 1mM EDTA, 130 M bestatin, 14 M E-64, 1mM leupetin and 0.3 M aprotinin] (Sigma, St. Louis, MO) for protein extraction. Generation of Antibodies, Co-Immunoprecipitation, and Western Blotting A goat anti-dazapl antibody was generated against the last 19 aa residues of the mouse DAZAP1 protein (26). A rabbit anti-dazl antibody was generated against the 272nd to 290th amino acids of the DAZL protein (19, 24). Antibodies then were affinity purified on protein A columns (ImmunoPure- Plus Immobilized Protein A IgG Purification Kit; Pierce, Rockford, IL). Western blotting of rat and human testis and ovary extracts detected a band of 50 kda for DAZAP1 and a band of 33 kda for DAZL (19, 24, 26). For co-ip experiments, the tissues were homogenized and mixed with twice the volume of lysis buffer containing protease inhibitor (T-PER Tissue Protein Extraction Reagent; Pierce). After complete mixing, the samples were repetitively frozen thawed in a liquid-nitrogen tank and a 37 C water bath several times to dissociate the chromosomal DNA and proteins. Approximately 500 g of protein was added to 50 L of protein A Sepharose (Amersham Bioscience, Uppsala, Sweden), gently mixed for at least 1 hour, kept at 4 C overnight, and processed by centrifuge at 12,000 g for 20 seconds. Five microliters of polyclonal antibodies to DAZL or DAZAP1 and 50 L of protein A Sepharose were added to the supernatant, gently mixed for 1 hour at room temperature, and kept at 4 C overnight. After centrifugation, the pellets were washed three times with lysis buffer and fractionated on a 12% sodium dodecyl sulfate polyacrylamide gel, and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA) by using a BioRad transfer system at 110 V for 1 hour. The membranes were washed with Tris-buffered saline containing 0.5% of Tween 20 and then incubated in a blocking solution (5% milk powder in the wash buffer) for 1 hour. The membranes then were incubated in a 1:100 to 1:2,500 dilution of a primary antiserum overnight and washed three times with the wash buffer, followed by incubation with a 1:10,000 dilution of the secondary antibody (goat anti-rabbit immunoglobulin G, peroxidase conjugated; Pierce) in the wash buffer. The filters were then washed several times, and the peroxidase activities were visualized by using SuperSignal substrate, following the manufacturer s instructions (Pierce). Immunohistochemical Detection of DAZAP1 and DAZL in Human CL and Dazap1 and Dazl in the Rat Ovary Specimens were dehydrated, embedded in paraffin, and sectioned at 5 m. Immunostaining of DAZAP1 or DAZL was carried out as described elsewhere (19, 24, 26). Briefly, sections were deparaffinized with 100% xylene and rehydrated with 100%, 95%, and 70% ethanol. The slides then were blocked with 3% hydrogen peroxide in absolute methanol for 5 minutes, washed with water for 5 minutes, and heated at 90 C for 5 minutes in preheated citrate acid buffer. After cooling, the slides were washed twice with Tris-buffered saline for 5 minutes each. The slides were incubated with primary antibody (1:1,000) for 60 minutes at room temperature. Binding of primary antibodies to tissue sections was detected by using a goat ABC staining system (Santa Cruz Biotechnology, Santa Cruz, CA). After the washing steps with Tris-buffered saline, sections were incubated with biotinated mouse anti-rabbit immunoglobulin G antibody (Dako, Carpinteria, CA) for 30 minutes at room temperature, washed with Tris-buffered saline, then incubated with avidin-biotinylated peroxidase complex for 30 minutes at room temperature, followed by 1090 Pan et al. Expression patterns of DAZAP1 in ovaries Vol. 84, Suppl 2, October 2005

3 FIGURE 1 Immunohistochemical staining with anti-dazap1 antibodies shows expression of DAZAP1 in the cytoplasm of granulosa luteal (GL) cells from the early stage (A, 200), mid stage (B, 200), and late stage (C, 200) of CL. Early stage of a CL tissue section stained with preimmune serum shows no signals (D, 200). reaction with 3,3=-diaminobenzidine (DAB) chromogen in hydrogen peroxide. Sections were subsequently counterstained with hematoxylin, dehydrated, and mounted. RESULTS Localization of DAZAP1 in the Ovarian Tissue Immunohistochemical staining with antiserum to DAZAP1 was applied to the human CL. Homogeneous staining was observed in the cytoplasm of granulosa luteal cells. The staining was strongest in the early luteal phase (Fig. 1A), less intensive in the midluteal phase (Fig. 1B), and weakest in the late luteal phase (Fig. 1C). Control experiments using preimmune serum did not show any signal in consecutive sections of early (Fig. 1D), mid, and late CL (data not shown). The expression pattern of DAZAP1 in different stages of CL is reminiscent of the DAZL expression pattern reported in our previous study (24). In the rat ovary, immunohistochemical staining showed intense staining of Dazap1 in the cytoplasm of oocytes and theca interna cells. Some positive scattering staining also was noted in the cytoplasm of granulosa cells of the preovulatory follicles (Fig. 2A). Consecutive section stained with Dazl antibody showed a similar expression pattern in the rat ovary (Fig. 2B). Control experiments with the preimmune serum showed no signals in consecutive sections of rat ovary (Fig. 2C). Homogeneous Dazap1 staining also was observed in luteal cells of the rat (Fig. 2D). Consecutive section staining with Dazl antibody shows a similar staining pattern in LC (Fig. 2E). A control experiment with pre-immune serum shows no signals (Fig. 2F). In Vivo Interaction of DAZAP1 With DAZL To confirm whether the two proteins (DAZL and DAZAP1 in the human, Dazl and Dazap1 in the rat) interact in vivo, we performed co-immunoprecipitation (IP) studies in the rat testes, rat ovary, human testis, human CL, and human luteinized granulosa cells. In rat, immunoblot with antiserum to Dazap1 showed expression of Dazap1 protein in rat testis and ovary (Fig. 3A, lanes 1 and 2). Immunoprecipitation with the antiserum to Dazl resulted in pull-down of a 50- kda Dazap1 protein, indicating positive interaction of Dazap1 with Dazl (Fig. 3A, lanes 3 and 4). Likewise, co-ip experiments using human testis, CL, and luteinized granulosa cells showed the same results, indicating in vivo interaction between DAZL and DAZAP1 (Fig. 3B). Control experiments using preimmune serum instead of specific antiserum to DAZL during the IP experiments did not show Fertility and Sterility 1091

4 FIGURE 2 In the rat ovary, immunohistochemical staining shows intense staining of Dazap1 in the cytoplasm of the oocytes (O), theca interna cells (T), and scattered signals in granulosa cells (G) of preovulatory follicles (A, 100). Immunohistochemical staining of consecutive section with Dazl antibody shows similar staining pattern in rat ovary (B, 100). Control experiment with preimmune serum shows no signals (C, 100). There is homogeneous staining of Dazap1 in luteal cells (LC; D, 100). Consecutive section staining with Dazl antibody shows a similar staining pattern in LC (E, 100). A control experiment with preimmune serum shows no signals (F, 100). any band (data now shown). Pull-down of DAZL by antiserum to DAZAP1 again showed interaction of DAZAP1 with DAZL in human (Fig. 3C) and rat tissues (data now shown). Decreased Expression of DAZAP1 Protein With Advancing Stages of CL Western blot of the early, mid, and late stages of the CL showed the absence of DAZAP1 protein expression in the late luteal phase (Fig. 4). Normalization with -actin showed decreasing amounts of DAZAP1 with advancing stages of CL. The ratio of DAZAP1 -actin in early CL ranged between 1.03 and 1.95, whereas the ratio ranged between 0.98 and 0.64 for the midstage CL. DAZAP1 was undetectable in the late-stage CL. DISCUSSION In the mouse, Dazap1 protein was present most abundantly in the testis and, to a lesser degree, in the ovary, spleen, liver, lung, and brain (6). In the testes, Dazap1 was expressed in the spermatogonia, pachytene spermatocyte, round spermatid, and elongated spermatid (26). Although the expression of Dazap1 during germ cell development paralleled that of Dazl, the temporal and spatial expression of Dazap1 in mouse testes only partially overlapped that of Dazl. It was suggested that the interaction between Dazap1 and Dazl may be transient (8). In the present study, we showed expression of Dazap1 protein in cytoplasm of the oocyte, theca interna, and luteal cells of the rat ovary. We also showed expression of DAZAP1 protein in human luteinized granulosa cells, as well as luteal cells. It appears that the expression patterns of 1092 Pan et al. Expression patterns of DAZAP1 in ovaries Vol. 84, Suppl 2, October 2005

5 FIGURE 3 Protein extract from rat testis and ovary was incubated with Dazl antibody, and immunoprecipitated proteins were analyzed by Western blot using Dazap1 antibody. Immunoblot analyses of rat testicular and ovarian tissues by using Dazapl antibody were used as positive controls (A). Protein extracts from human testis, CL, and luteinized granulosa cells were incubated with DAZL antibody, and immunoprecipitated proteins were analyzed by Western blot using DAZAP1 antibody. Immunoblot analysis of human testicular tissues by using DAZAP1 antibody was used as a positive control (B). Protein extracts from human testis, CL, and luteinized granulosa cells were incubated with DAZAP1 antibody, and immunoprecipitated proteins were analyzed by Western blot by using DAZL antibody. Immunoblot analysis of human testicular tissues by using DAZL antibody was used as a positive control (B). IB immunoblot. DAZ family in the regulation of mrna translation is supported by the association of DAZL with polyribosomes (8). It was also shown that zebrafish DAZL protein controls translation via the sequence GUUC (27). However, sucrose gradient analyses of the postmitochondrial fraction showed that, unlike Dazl, Dazap1 did not co-sediment with polyribosomes. The absence of Dazap1 from polyribosomes indicates that it is not directly involved in protein synthesis (6). Although Dazap1 protein is present most abundantly in the testis, it also is expressed in nonreproductive organs: the spleen, liver, lung, and brain. It is possible that in reproductive organs, DAZAP1 interacts with the DAZ gene family to facilitate the translation of their target mrnas. On the other hand, DAZAP1 may act as a repressor by competing with other translational factors for the binding to DAZL or DAZ. When in nonreproductive organs, DAZAP1 is involved in functions unrelated to the DAZ gene family. DAZAP1 is an evolutionarily conserved RNA-binding protein with unknown functions. Its predominant expression in testes suggests a role in spermatogenesis. In the testes, DAZAP1 most likely concerts with DAZ gene families to regulate the cell cycle progression of germ cells. In Xenopus oocytes, Prrp appears to interact with multiple mrnas and proteins. It binds to Vg1 mrna and VegT mrna and concentrated in the vegetal cortex of stage III/IV oocytes. It also is associated with two microfilament- associated proteins, profilin and Mena, through its proline-rich domain. Therefore, Prrp also may facilitate local actin polymerization in Xenopus oocytes (5). Prrp also can bind to a Xenopus homolog of the human transcription factor, FUSE-binding protein (EBP2), which is present in the nucleus and throughout the cytoplasm at all stages of oogenesis. This association does not require the presence of Vg1 mrna (28). Recently, Prrp was found to bind to a novel vegetally localized RNA that encodes a protein with a functionally uncharacterized NIF-motif in Xenopus oocytes (29). In the ovary, it is possible that DAZAP1 is involved in diverse functions, ranging from cell cycle regulation or maturation of oocytes to the differentiation of somatic cells (granulosa cells and luteal cells). Several RNA substrates for DAZ/DAZL have been identified DAZAP1 and DAZL are identical within ovaries. Like DAZL, expression of DAZAP1 also decreases with advancing stages of CL (24). Previous attempts to co-immunoprecipitate Dazap1 and Dazl with antibodies against Dazl or Dazap1 have not been successful (7). In the present study, we confirmed the in vivo interaction of DAZL and DAZAP1. Although DAZAP1 interacts with DAZL in both ovarian and testicular tissues, their roles may not be completely identical. A role for the FIGURE 4 Western blot shows decreasing amount of DAZAP1 protein with advancing stages of human CL. Fertility and Sterility 1093

6 (30, 31). Because DAZAP1 also is an RNA-binding protein, it would be interesting to observe whether DAZAP1 and DAZ/ DAZL share some of the substrates. It also would be interesting to investigate whether human DAZAP1 interacts with similar sets of proteins or mrnas, as does Prrp in Xenopus oocytes. Collectively, the DAZAP1 expression has been identified in human testes, CL, and luteinized granulosa cells. In the rat ovary, Dazap1 is expressed in the cytoplasm of the oocytes, theca interna, and luteal cells. Co-IP experimentation has shown the interaction of DAZL and DAZAP1 proteins. These two proteins may act together to facilitate the expression of a set of genes in both germ cells and somatic cells, given the similar expression pattern in ovaries. Acknowledgment: The authors thank Pauline Yen (Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan) for the anti-dazap1 antibody. REFERENCES 1. Burd CG, Dreyfus G. 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