Molecular Human Reproduction, Vol. 5, No. 6, 495-497,
June 1999
© 1999 European Society of Human Reproduction and Embryology
Existence of human DAZLA protein in the cytoplasm of human oocytes
1 Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, Sapporo, and 2 Department of Biochemistry, Hokkaido University School of Medicine, Sapporo, Japan
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Abstract |
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The human deleted in azoospermia-like autosomal (DAZLA) gene is thought to be a candidate gene for azoospermia. cDNA encoding the C-terminal 94 amino acid residues of human DAZLA was used to express a bacterial fusion protein which was then used to raise polyclonal antibodies in rabbits. Immunohistochemical analyses with the human DAZLA antiserum showed that the DAZLA protein is expressed at a cytoplasmic location in female germ cells. Available evidence suggests that the DAZLA gene is a participant in human oogenesis.
DAZLA/immunohistochemistry/oocyte/oogenesis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Deletion of the azoospermia factor (AZF) region of the human Y chromosome results in spermatogenic failure. The DAZ (deleted in azoospermia) gene, which is located on human chromosome Yq11, is regarded as a candidate gene of azoospermia (Reijo et al., 1995
). A recent study demonstrated that the mouse homologue of human DAZ is autosomal, and this gene has been referred to as Dazla (DAZ-like autosomal) (Cooke et al., 1996; Reijo et al., 1996a). In contrast to male-specific expression of human DAZ, mouse Dazla is expressed exclusively in the testis and the ovary (Cooke et al., 1996). Furthermore, sequence analysis has revealed relatively low homology between human DAZ and mouse Dazla. We previously identified human DAZLA on chromosome 3p25 (Seboun et al., 1997). In the present study, we examined immunohistochemically the localization of human DAZLA protein in the human ovary to confirm its participation in human oogenesis. |
Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Preparation of specific antiserum against human DAZLA protein
cDNA encoding the C-terminal 94 amino acid residues of human DAZLA protein was cloned into a pMAL-c2 vector (New England Biolabs Inc, Beverly, MA, USA). After transformation into Escherichia coli strain TB1, expression of the fusion protein was induced by isopropyl-1-thio-ß-D-galactoside according to the manufacturer's instructions. The fusion protein was purified by an amylose resin column, emulsified in Freund's adjuvant, and injected intradermally into rabbits to raise antisera. Immunoglobulin (Ig)G fraction was purified from the rabbit antisera by ammonium sulphate precipitation, followed by DEAE-cellulose column chromatography (Livingston, 1997).
Western blot analysis
The Western blot analysis of human tissues, including testis extracts, was performed using the polyclonal IgG produced by immunizing rabbits with the fusion protein. Tissues were homogenized in 5 vol (w/w) of ice-cold 0.25 M sucrose containing 2 mM EDTA, 10 mM Tris–HCl (pH 7.2), 4 mg/ml of aprotinin, and 1 mM phenyl-methanesulphonyl fluoride. The tissue lysates were centrifuged at 1500 g for 10 min at 4°C. After adding sodium dodecyl sulphate (SDS) at a final concentration of 1%, the supernatant was centrifuged at 10 000 g for 10 min at 4°C. SDS–polyacrylamide gel electrophoresis (PAGE) was performed under reducing conditions according to the standard method of Laemmli (1970). Western blotting of protein bands to nitrocellulose membrane (BA-S NC) (Schleicher & Schuell, Dassel, Germany) was carried out as described earlier (Bjerrum et al., 1986) using the rabbit IgG to human DAZLA (1:5000 dilution) as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10 000 dilution) as the second antibody. Detection was performed using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Tokyo, Japan).
Application of antiserum to immunohistochemistry
A total of 10 ovary specimens were obtained at either surgery or autopsy from women aged 14–49 years (Table I). In all cases, the Helsinki Declaration regarding the use of human tissues was strictly observed. For light microscopical analysis, the tissues were fixed in cold Bouin's solution overnight (cases 1–3) or in 10% neutral-buffered formalin for 72 h (cases 4–10), dehydrated, embedded in paraffin, and sectioned serially at 6 µm thickness. Tissue sections were incubated with the rabbit anti-DAZLA IgG diluted from 1:50 to 1:500. Immunostaining was performed by the strepto–avidin–biotin–complex method with rabbit IgG to the antiserum for DAZLA protein using a Histofine SAB-PO(R) kit (Nichirei Corporation, Tokyo, Japan), and counterstained with haematoxylin or Methyl Green.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Western blot analysis was performed using the polyclonal IgG produced by immunizing rabbits with the DAZLA fusion protein. Under non-reducing conditions, we were able to detect a band representing a protein of ~33 kDa, in human testis extracts exclusively (Figure 1
). No bands were detected in any tissues when the preimmune rabbit serum was used as the primary antibody (data not shown).
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Immunohistochemical analysis was performed to determine the localization of DAZLA protein in the ovary (Figure 2A–C
). Human ovary sections stained for DAZLA showed intense staining in all germ cells from all patients. In oocytes, the staining was exclusively in the cytoplasm. DAZLA was also present in the theca folliculi (theca interna) of the maturing follicles (Figure 2C). In contrast, DAZLA immunostaining was not detected in the ovary of the menopausal woman (case 3). No staining was observed in the tissues when primary antibody was withheld and preimmune antisera used as a negative control (data not shown). Thus, the staining in the oocytes was specific to DAZLA.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, we expressed the human DAZLA protein in bacteria, raised polyclonal antibody in rabbits, and performed Western blot analyses of human testis extracts. However, extracts of ovaries were not examined, since mouse Dazla transcripts were detectable in the ovary only by reverse transcription–polymerase chain reaction (RT–PCR) (Cooke et al., 1996
), and thus Dazla transcripts are presumably not abundant in the ovaries. DAZLA is expressed in the testes of all mammals examined to date, although DAZ is Y-linked only in highly evolved primates (Cooke et al., 1996; Seboun et al., 1997). Therefore, DAZLA is considered to be a strong candidate gene associated with spermatogenesis, particularly in species lacking DAZ. A recent study shows that disruption of the mouse Dazla gene leads to complete absence of gamete production in both male and female mice, and thus that mouse Dazla is essential for gametogenesis (Ruggiu et al., 1997). In the present study, we initially attempted to demonstrate the existence and localization of human DAZLA in testis. To investigate the function of DAZLA in human gametogenesis, it is crucial to determine whether DAZLA, an autosomal gene unlike DAZ, is also expressed in human ovaries. In the mouse, Dazla is present in maturing follicles, but exists in the cytoplasm of oocytes in the embryonic and prepubertal ovaries. In humans, it is very difficult to investigate the fetal and prepubertal ovary. However, the present results indicate that the DAZLA protein is located in the cytoplasm of human oocytes but not in the menopausal ovaries, suggesting that DAZLA plays an important role in human oogenesis, much like that of mouse Dazla. However, it remains to be seen whether or not ovarian DAZLA plays a role in ovary maturation.
Although DAZLA is expressed in adult testis, we do not know whether it is expressed in somatic tissue or in the germ cell compartment. In the mouse, Dazla transcripts cannot be detected in testes that lack germ cells (Reijo et al., 1996b). Because both DAZLA and DAZ are members of the RNA-binding protein family (Reijo et al., 1995, 1996a; Cooke et al., 1996; Seboun et al., 1997), and human DAZLA protein is located in the cytoplasm of oocytes, as is mouse Dazla protein, we conclude that DAZLA is expressed in germ cells during meiosis and may participate in translational control or transportation of mRNA.
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Acknowledgments |
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We thank Yumi Gokan, Hiromi Shimakage, and Miyuki Yasui for their skilful technical assistance. This study was supported in part by Grants-in-Aid for Scientific Research (Nos. 08671858 and 10671509) from the Ministry of Education, Science, Sports and Culture of Japan (to N.H.).
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Notes |
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3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, N15 W7, Kita-ku, Sapporo 060–8638, Japan
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References |
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Bjerrum, O.J. and Schafer-Nielsen, C. (1986) Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus. In Dunn, M.J. (ed.), Electrophoresis `86. VCH Publisheers, Deerfield Beach, FL, USA, pp. 315–327.
Cooke, H.J., Lee, M., Kerr, S. and Ruggiu, M. (1996) A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum. Mol. Genet., 5, 513–516.
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Submitted on September 9, 1998; accepted on March 2, 1999.
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