- Email: [email protected]
The expression of DAZL1 in the ovary of the human female fetus
FERTILITY AND STERILITY威 VOL. 73, NO. 3, MARCH 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
The expression of DAZL1 in the ovary of the human female fetus Meng-Yin Tsai, M.D., M.Sc., Shiuh-Young Chang, M.D., Hsin-Yi Lo, M.Sc. I-Hsuan Chen, M.Sc. Fu-Jeng Huang, M.D. Fu-Tsai Kung, M.D. Ying-Jen Lu, M.D. Kaohsiung Chang-Gung Memorial Hospital, Kaohsiung County, Taiwan and Chang-Gung University, Taoyuan County, Taiwan
Objective: To determine whether DAZL1 is expressed in human fetal ovarian tissue. Design: The presence of DAZL1 expression was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Setting: Academic tertiary care medical center and research unit of university. Patient(s): Five female abortuses between the 19th and 22nd week of gestational age. Intervention(s): Fetal ovarian tissues were collected immediately after the cessation of the heart beat. Main Outcome Measure(s): The product of RT-PCR. Result(s): DAZL1 expression was detected in all five samples. Conclusion(s): DAZL1 is not only expressed in human testes but also in ovaries. It may play a role in germ cell survival and gonad development in both sexes. (Fertil Steril威 2000;73:627–30. American Society for Reproductive Medicine.) Key Words: DAZL1, fetus, ovary, reverse transcriptase polymerase chain reaction (RT-PCR)
Received June 1, 1999; revised and accepted September 17, 1999. Reprint requests: Meng-Yin Tsai, M.D., Department of Obstetrics and Gynecology, Kaohsiung Chang-Gung Memorial Hospital, No. 123, Tapei Road, Niao-Sung Hsian Kaohsiung County 83305, Taiwan FAX: 886-7-7322915; E-mail: [email protected]). Department of Obstetrics and Gynecology, Kaohsiung Chang-Gung Memorial Hospital. Department of Obstetrics and Gynecology, College of Medicine, Chang-Gung University. 0015-0282/99/$20.00 PII S0015-0282(99)00544-0
Germ cell proliferation is a complex process involving a series of signal transductions that are regulated by stringent stage-specific gene expression (1–3). Although knowledge concerning germ cell proliferation is accumulating rapidly, the process of regulation remains poorly understood. One of the most well-known regions responsible for gametogenesis is the azoospermic factor (AZF) region located on chromosome Yq11. Patients with deletions in this region are azoospermic and infertile (4). A candidate gene, called DAZ (deletion in azoospermia) was recently mapped to this region (5) and has been found to be deleted in 3%– 13% of azoospermic patients (5– 8). Although DAZ is on the Y chromosome of humans, the only homologous gene identified in mice is not on the Y chromosome, but on chromosome 17 (9, 10). This homologous gene has been called dazl (9, 10). In addition to its homology with DAZ, dazl has other homology in humans (11–14). It was mapped to chromosome 3 and called DAZL1 (11–14). Sequencing analysis has revealed that the nucleotides of DAZL1 are 83% homologous to DAZ (15).
The function of DAZL1 has not yet been clarified, and most of the information concerning its function has come from animal studies of the homologous gene, dazl. With use of a combination of genetic and biochemical approaches, the product of dazl has been shown to be a germ cell-specific cytoplasmic protein expressed in the diploid stage of spermatocytes and the pachytene stage of oocytes (16). Unlike the homologous, boule gene in drosophila, which is only important for spermatogenesis (17), both male and female dazl knockout mice appear to be infertile (16). This finding suggests that dazl gene expression is necessary for germ cell survival and gonad development in mice of both sexes (16). In humans, DAZL1 is specifically expressed in adult testes producing germ cells (9, 12–14) but not expressed in some idiopathic azoospermic men (13, 18). Whether DAZL1 is expressed in women is unclear. Previous Northern blot analysis of DAZL1 expression in adult female ovaries has unanimously displayed negative findings (11–14). However, the results of reverse transcriptase polymerase chain reaction 627
tions. The extracted RNA was quantified with a spectrophotometer (DU640, Beckman, Fullerton, CA).
TABLE 1 Demographic and clinical characteristics of the five abortuses supplying ovarian tissues.
Patient no. 1 2 3 4 5
Gestational age (week)
Birth weight (g)
Reason for termination
21 22 21 19 20
400 420 480 160 470
Chromosome abnormality, 47,XXX PPROM Victim of rape Achondrogenesis PPROM
Note: PPROM ⫽ preterm premature rupture of membrane. Tsai. Expression of DAZL1. Fertil Steril 2000.
(RT-PCR) have been inconsistent. Saxena et al. (12) reported a PCR product, suggesting the positive expression of DAZL1 in the adult ovary, but Shan et al. (11) could not achieve the same result. In female mice, the most abundant dazl expression was reported to occur in the fetal stage (16). As the homologous gene in humans, DAZL1 may also be most abundantly expressed in the fetal stage. Randomized analysis of ovarian tissues at different ages could yield divalent results. In this study, we sampled and analyzed fetal ovarian tissues from five abortuses to evaluate whether DAZL1 is expressed in the human female fetus.
MATERIALS AND METHODS Samples Permission was obtained from the Medical Ethics and the Human Clinical Trials Committee of Chang-Gung Memorial Hospital to collect fetal tissue for the purposes of this study. The fetal ovarian tissues were collected from five female abortuses that were aborted because of various reasons between the 19th and 22nd week of gestation (Table 1). The ovarian tissues were obtained immediately after the cessation of fetal heart beat. Testicular tissues collected from two azoospermic men, one who had secretory-type lesions and the other who had obstructive-type lesions, were used as positive controls. DAZL1 had been proved to be expressed in the tissues in the preliminary work. Endometrial tissue obtained from a 35-year-old woman was used as a negative control. All of the collected tissues were frozen at ⫺196°C immediately and remained frozen at this temperature until used in RNA extraction procedures.
RNA Isolation The RNA of the samples was extracted by the guaniethidium isothiocyanate method (Trizol, GIBCO BRL, Gaithersburg, MD). The details of the extraction procedures were performed according to the manufacturer’s instruc628
Tsai et al.
DAZL1 in human fetal ovary
RT-PCR of DAZL1 A pair of DAZL1-specific primers that yielded a 128-bp product from the RNA extracts were used for RT-PCR reaction. The nucleotide sequence of the primers were as follows: Forward: 5⬘-GGAGCTATGTTGTACCTCC-3⬘ Reverse: 5⬘-GTGGGCCATTTCCAGAGGG-3⬘ They were located on exon 8 and exon 9 of the DAZL1 gene, respectively (12). RT-PCR was performed with use of a commercial kit (Promega, Madison, WI). The reactions were done in 50-l volumes of 1.5 mM of MgCl2, 5 mM NH4Cl, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 100 M deoxynucleotide triphosphate (dNTPs), with 1 U of AMV reverse transcriptase, 1 U Tfl DNA polymerase, 100 ng of RNA, and 1 M of each primer. The reaction conditions were as follows: reverse transcription at 48°C for 45 minutes, denaturation at 94°C for 3 minutes, 35 cycles of 1 minute at 94°C, 1.5 minute at 56°C, and 1 minute at 72°C. The final extension was 5 minutes at 72°C. A pair of -actinspecific primers that yielded a 430-bp product were also amplified. This product was used as an internal control to ensure the stability of the extracted mRNA. The PCR products were separated on a 3% agarose gel.
Southern Blot Hybridization The specificity of the PCR product was confirmed by Southern blot hybridization performed by a standard procedure. Briefly, the product on agarose gel was transferred to a nylon transfer membrane (Hybond-N⫹, Amersham Life Science, Chalford, Buckinghamshire, United Kingdom). A 40bp DAZL1-specific antisense sequence spanning between the two primers was used as a probe. The nucleotide sequence of the probe was as follows: 5⬘-TAGTTAACAGCTGAATAAGCCGGAGGTACAACATAGCTCC-3⬘ The probe was first labeled with digoxigenin-conjugateddideoxy-uracil triphosphate (dd-UTP) at the 3⬘-end by terminal transferase (Boehringer Mannheim, Mannheim, Germany). The membrane and probe were hybridized at 54°C overnight and stepwise washed with 2⫻ SSC, 1⫻ SSC, and 0.1⫻ SSC. The signal was finally developed by incubating the membrane in alkaline phosphatase-conjugated antidigoxigenin antibody solution (Boehringer Mannheim) and the chromogen, Nitroblue tetrazolium (NBT) solution (Boehringer Mannheim), consecutively.
RESULTS The results of RT-PCR are shown in Figure 1. The DAZL1-specific primers yielded a 128-bp RT-PCR product in all five fetal ovarian tissues and two of the positive Vol. 73, No. 3, March 2000
FIGURE 1
FIGURE 2
Results of RT-PCR. Left (arrowhead): A 128-bp RT-PCR product that was amplified by DAZL1-specific primers was demonstrated in five patients and two positive controls, but not in negative control. Right (arrow): A 430-bp product that was amplified by -actin-specific primers was demonstrated in all patients, including positive and negative controls. m ⫽ DNA marker with 100-bp ladder; s and o ⫽ positive controls, testicular tissues from patients with secretory-type and obstructive-type azoospermia, respectively; n ⫽ negative control, endometrial tissue from a 35-year-old woman. 1,2,3,4,5 ⫽ the ovarian tissues from the five abortuses aligned in consecutive sequence as in Table 1.
Results of Southern blot hybridization. A 40-bp antisenseoligonucleotide probe spanning between the sequences of the two primers was used as a probe for Southern blot hybridization. The specificity of the RT-PCR product shown in Figure 1 was confirmed. s and o ⫽ positive controls, testicular tissues from patients with secretory-type and obstructive-type azoospermia, respectively; n ⫽ negative control, endometrial tissue from a 35-year-old woman. 1,2,3,4,5 ⫽ the ovarian tissues from the five abortuses aligned in consecutive sequence as in Table 1.
Tsai. Expression of DAZL1. Fertil Steril 2000. Tsai. Expression of DAZL1. Fertil Steril 2000.
controls, but not in the negative control. The -actin-specific primers yielded a 430-bp product in all samples. The specificity of the DAZL1 product was confirmed by Southern blot hybridization, and the results are shown in Figure 2.
DISCUSSION The most abundant expression of dazl in female mice has been reported to occur in the pachytene cells of fetal ovarian tissues that have just finished the process of proliferation and entered prophase I (16). In humans, pachytene cells exist during the midtrimester of gestation. They pass through the pachytene stage and stop at the dictyotene stage late in the third trimester (19, 20). During the process of meiosis, most of the oocytes simultaneously undergo regression and apoptosis. The number of oocytes diminishes from 7,000,000 at the 20th week of gestation to 2,000,000 in the newborn period and 300,000 at puberty (19, 20). Therefore, if DAZL1 is also expressed in pachytene cells, the optimal samples for study should be fetal ovarian tissues obtained around the 20th week of gestation. Our samples were collected at this gestational age. We found that DAZL1 was obviously expressed in human fetal ovaries. Although Saxena et al. (12) suggested that DAZL1 could be expressed at a low level in the adult ovary, FERTILITY & STERILITY威
the results of the present study provide the first and the strongest evidence supporting DAZL1 expression in the fetal ovary. This suggests that the function of DAZL1 in humans could be similar to the function of dazl in mice and that it is therefore likely to be important for gonad development and germ cell survival in both sexes. We did not measure DAZL1 expression levels in the present study; therefore, it remains unclear whether the DAZL1 expression level is related to the number of pachytene cells. Comparison of expression levels would require a large number of fetal ovaries from a wide range of gestational ages. Legal and ethical considerations limit the availability of such samples. Multicenter cooperation and the establishment of a nationwide tissue committee are needed to enable such studies to be performed in the future. In conclusion, we have demonstrated the expression of DAZL1 in human fetal ovary. This new evidence suggests that DAZL1 is not only essential for spermatogenesis but is also important for oogenesis. References 1. Page AW, Orr-Weaver TL. Stopping and starting the meiotic cell cycle. Curr Opin Genet 1997;7:23–31. 2. Sassone-Corsi P. Transcriptional checkpoints determining the fate of male germ cells. Cell 1997;88:163– 6. 3. Sagata N. What does Mos do in oocytes and somatic cells? Bioassays 1997;19:13–21. 4. Tiepolo L, Zuffardi O. Localization of factors controlling spermatogen-
629
5.
6.
7.
8. 9. 10.
11.
esis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet 1976;34:119 –24. Reijo R, Lee TY, Salo P, Alagappan R, Brown LG, Rosenberg M, et al. Diverse spermatogenic defects in human caused by Y chromosome deletions encompassing a novel RNA binding protein gene. Nature Genet 1995;10:383–92. Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, et al. Human Y chromosome azoospermia factor mapped to different subregions in Yq11. Hum Mol Genet 1996;5:933– 43. Simoni M, Gromoll J, Dworniczak B, Rolf C, Abshagen K, Kamischke A, et al. Screening for deletions of the Y chromosome involving the DAZ (Deleted in AZoospermia) gene in azoospermia and oligozoospermia. Fertil Steril 1997;67:542–7. Chang SY, Tsai MY. Deletion of azoospermic factor genes in Chinese azoospermic and severe oligozoospermic patients. J Assist Reprod Genet 1999;16:237– 40. Cooke HJ, Lee M, Kerr S, Ruggiu M. A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum Mol Genet 1996;5:513– 6. Reijo R, Seligman J, Dinulos MB, Jaffe T, Brown LG, Disteche CM, et al. Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans, is expressed in male germ cells before and after puberty. Genomics 1996;35:346 –52. Shan Z, Hirschmann P, Seebacher T, Edelmann A, Jauch A, Morell J, et al. A SPGY copy homologous to the mouse gene dazla and drosophila gene boule is autosomal and expressed only in the human male gonad. Hum Mol Genet 1996;5:2005–11.
630
Tsai et al.
DAZL1 in human fetal ovary
12. Saxena R, Brown LG, Hawkins T, Alagappan RK, Skaletsky H, Reeve MP, et al. The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned. Nature Genet 1996;14:292–9. 13. Yen PH, Chai NN, Salido EC. The human autosomal gene DAZLA: testis specificity and a candidate for male infertility. Hum Mol Genet 1996;5:2013–7. 14. Seboun E, Barbaux S, Bourgeron T, Nishi S, Algonik A, Egashira M, et al. Gene sequence, localization, and evolution of DAZLA, a candidate male sterility gene. Genomics 1997;41:227–35. 15. Chai NN, Phillips A, Fernandez A, Yen PH. A putative human infertility gene DAZLA: genomic structure and methylation status. Mol Hum Reprod 1997;3:705– 8. 16. Ruggiu M, Speed R, Taggart M, McKay SJ, Kilanoski F, Saunders P, et al. The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 1997;389:73–7. 17. Eberhart CG, Maines JZ, Wasserman SA. Meiotic cell cycle requirement for a fly homologue of human Deleted In Azoospermia. Nature 1996;381:783–5. 18. Lee JH, Lee DR, Yoon SJ, Chai YG, Roh SI, Yoon HS. Expression of DAZ (deleted in azoospermia), DAZL1 (DAZ-like) and protamine-2 in testis and its application for diagnosis of spermatogenesis in nonobstructive azoospermia. Mol Hum Reprod 1998;4:827–34. 19. Himelstein-Braw R, Byskov AG, Peters H, Faber M. Follicular atresia in the infant human ovary. J Reprod Fertil 1976;46:55–9. 20. Adashi EY, Rock JA, Rosenwaks Z. Reproductive endocrinology, surgery, and technology. Philadelphia: Lippincott-Raven Publishers, 1995:19.
Vol. 73, No. 3, March 2000
The expression of DAZL1 in the ovary of the human female fetus Meng-Yin Tsai, M.D., M.Sc., Shiuh-Young Chang, M.D., Hsin-Yi Lo, M.Sc. I-Hsuan Chen, M.Sc. Fu-Jeng Huang, M.D. Fu-Tsai Kung, M.D. Ying-Jen Lu, M.D. Kaohsiung Chang-Gung Memorial Hospital, Kaohsiung County, Taiwan and Chang-Gung University, Taoyuan County, Taiwan
Objective: To determine whether DAZL1 is expressed in human fetal ovarian tissue. Design: The presence of DAZL1 expression was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Setting: Academic tertiary care medical center and research unit of university. Patient(s): Five female abortuses between the 19th and 22nd week of gestational age. Intervention(s): Fetal ovarian tissues were collected immediately after the cessation of the heart beat. Main Outcome Measure(s): The product of RT-PCR. Result(s): DAZL1 expression was detected in all five samples. Conclusion(s): DAZL1 is not only expressed in human testes but also in ovaries. It may play a role in germ cell survival and gonad development in both sexes. (Fertil Steril威 2000;73:627–30. American Society for Reproductive Medicine.) Key Words: DAZL1, fetus, ovary, reverse transcriptase polymerase chain reaction (RT-PCR)
Received June 1, 1999; revised and accepted September 17, 1999. Reprint requests: Meng-Yin Tsai, M.D., Department of Obstetrics and Gynecology, Kaohsiung Chang-Gung Memorial Hospital, No. 123, Tapei Road, Niao-Sung Hsian Kaohsiung County 83305, Taiwan FAX: 886-7-7322915; E-mail: [email protected]). Department of Obstetrics and Gynecology, Kaohsiung Chang-Gung Memorial Hospital. Department of Obstetrics and Gynecology, College of Medicine, Chang-Gung University. 0015-0282/99/$20.00 PII S0015-0282(99)00544-0
Germ cell proliferation is a complex process involving a series of signal transductions that are regulated by stringent stage-specific gene expression (1–3). Although knowledge concerning germ cell proliferation is accumulating rapidly, the process of regulation remains poorly understood. One of the most well-known regions responsible for gametogenesis is the azoospermic factor (AZF) region located on chromosome Yq11. Patients with deletions in this region are azoospermic and infertile (4). A candidate gene, called DAZ (deletion in azoospermia) was recently mapped to this region (5) and has been found to be deleted in 3%– 13% of azoospermic patients (5– 8). Although DAZ is on the Y chromosome of humans, the only homologous gene identified in mice is not on the Y chromosome, but on chromosome 17 (9, 10). This homologous gene has been called dazl (9, 10). In addition to its homology with DAZ, dazl has other homology in humans (11–14). It was mapped to chromosome 3 and called DAZL1 (11–14). Sequencing analysis has revealed that the nucleotides of DAZL1 are 83% homologous to DAZ (15).
The function of DAZL1 has not yet been clarified, and most of the information concerning its function has come from animal studies of the homologous gene, dazl. With use of a combination of genetic and biochemical approaches, the product of dazl has been shown to be a germ cell-specific cytoplasmic protein expressed in the diploid stage of spermatocytes and the pachytene stage of oocytes (16). Unlike the homologous, boule gene in drosophila, which is only important for spermatogenesis (17), both male and female dazl knockout mice appear to be infertile (16). This finding suggests that dazl gene expression is necessary for germ cell survival and gonad development in mice of both sexes (16). In humans, DAZL1 is specifically expressed in adult testes producing germ cells (9, 12–14) but not expressed in some idiopathic azoospermic men (13, 18). Whether DAZL1 is expressed in women is unclear. Previous Northern blot analysis of DAZL1 expression in adult female ovaries has unanimously displayed negative findings (11–14). However, the results of reverse transcriptase polymerase chain reaction 627
tions. The extracted RNA was quantified with a spectrophotometer (DU640, Beckman, Fullerton, CA).
TABLE 1 Demographic and clinical characteristics of the five abortuses supplying ovarian tissues.
Patient no. 1 2 3 4 5
Gestational age (week)
Birth weight (g)
Reason for termination
21 22 21 19 20
400 420 480 160 470
Chromosome abnormality, 47,XXX PPROM Victim of rape Achondrogenesis PPROM
Note: PPROM ⫽ preterm premature rupture of membrane. Tsai. Expression of DAZL1. Fertil Steril 2000.
(RT-PCR) have been inconsistent. Saxena et al. (12) reported a PCR product, suggesting the positive expression of DAZL1 in the adult ovary, but Shan et al. (11) could not achieve the same result. In female mice, the most abundant dazl expression was reported to occur in the fetal stage (16). As the homologous gene in humans, DAZL1 may also be most abundantly expressed in the fetal stage. Randomized analysis of ovarian tissues at different ages could yield divalent results. In this study, we sampled and analyzed fetal ovarian tissues from five abortuses to evaluate whether DAZL1 is expressed in the human female fetus.
MATERIALS AND METHODS Samples Permission was obtained from the Medical Ethics and the Human Clinical Trials Committee of Chang-Gung Memorial Hospital to collect fetal tissue for the purposes of this study. The fetal ovarian tissues were collected from five female abortuses that were aborted because of various reasons between the 19th and 22nd week of gestation (Table 1). The ovarian tissues were obtained immediately after the cessation of fetal heart beat. Testicular tissues collected from two azoospermic men, one who had secretory-type lesions and the other who had obstructive-type lesions, were used as positive controls. DAZL1 had been proved to be expressed in the tissues in the preliminary work. Endometrial tissue obtained from a 35-year-old woman was used as a negative control. All of the collected tissues were frozen at ⫺196°C immediately and remained frozen at this temperature until used in RNA extraction procedures.
RNA Isolation The RNA of the samples was extracted by the guaniethidium isothiocyanate method (Trizol, GIBCO BRL, Gaithersburg, MD). The details of the extraction procedures were performed according to the manufacturer’s instruc628
Tsai et al.
DAZL1 in human fetal ovary
RT-PCR of DAZL1 A pair of DAZL1-specific primers that yielded a 128-bp product from the RNA extracts were used for RT-PCR reaction. The nucleotide sequence of the primers were as follows: Forward: 5⬘-GGAGCTATGTTGTACCTCC-3⬘ Reverse: 5⬘-GTGGGCCATTTCCAGAGGG-3⬘ They were located on exon 8 and exon 9 of the DAZL1 gene, respectively (12). RT-PCR was performed with use of a commercial kit (Promega, Madison, WI). The reactions were done in 50-l volumes of 1.5 mM of MgCl2, 5 mM NH4Cl, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 100 M deoxynucleotide triphosphate (dNTPs), with 1 U of AMV reverse transcriptase, 1 U Tfl DNA polymerase, 100 ng of RNA, and 1 M of each primer. The reaction conditions were as follows: reverse transcription at 48°C for 45 minutes, denaturation at 94°C for 3 minutes, 35 cycles of 1 minute at 94°C, 1.5 minute at 56°C, and 1 minute at 72°C. The final extension was 5 minutes at 72°C. A pair of -actinspecific primers that yielded a 430-bp product were also amplified. This product was used as an internal control to ensure the stability of the extracted mRNA. The PCR products were separated on a 3% agarose gel.
Southern Blot Hybridization The specificity of the PCR product was confirmed by Southern blot hybridization performed by a standard procedure. Briefly, the product on agarose gel was transferred to a nylon transfer membrane (Hybond-N⫹, Amersham Life Science, Chalford, Buckinghamshire, United Kingdom). A 40bp DAZL1-specific antisense sequence spanning between the two primers was used as a probe. The nucleotide sequence of the probe was as follows: 5⬘-TAGTTAACAGCTGAATAAGCCGGAGGTACAACATAGCTCC-3⬘ The probe was first labeled with digoxigenin-conjugateddideoxy-uracil triphosphate (dd-UTP) at the 3⬘-end by terminal transferase (Boehringer Mannheim, Mannheim, Germany). The membrane and probe were hybridized at 54°C overnight and stepwise washed with 2⫻ SSC, 1⫻ SSC, and 0.1⫻ SSC. The signal was finally developed by incubating the membrane in alkaline phosphatase-conjugated antidigoxigenin antibody solution (Boehringer Mannheim) and the chromogen, Nitroblue tetrazolium (NBT) solution (Boehringer Mannheim), consecutively.
RESULTS The results of RT-PCR are shown in Figure 1. The DAZL1-specific primers yielded a 128-bp RT-PCR product in all five fetal ovarian tissues and two of the positive Vol. 73, No. 3, March 2000
FIGURE 1
FIGURE 2
Results of RT-PCR. Left (arrowhead): A 128-bp RT-PCR product that was amplified by DAZL1-specific primers was demonstrated in five patients and two positive controls, but not in negative control. Right (arrow): A 430-bp product that was amplified by -actin-specific primers was demonstrated in all patients, including positive and negative controls. m ⫽ DNA marker with 100-bp ladder; s and o ⫽ positive controls, testicular tissues from patients with secretory-type and obstructive-type azoospermia, respectively; n ⫽ negative control, endometrial tissue from a 35-year-old woman. 1,2,3,4,5 ⫽ the ovarian tissues from the five abortuses aligned in consecutive sequence as in Table 1.
Results of Southern blot hybridization. A 40-bp antisenseoligonucleotide probe spanning between the sequences of the two primers was used as a probe for Southern blot hybridization. The specificity of the RT-PCR product shown in Figure 1 was confirmed. s and o ⫽ positive controls, testicular tissues from patients with secretory-type and obstructive-type azoospermia, respectively; n ⫽ negative control, endometrial tissue from a 35-year-old woman. 1,2,3,4,5 ⫽ the ovarian tissues from the five abortuses aligned in consecutive sequence as in Table 1.
Tsai. Expression of DAZL1. Fertil Steril 2000. Tsai. Expression of DAZL1. Fertil Steril 2000.
controls, but not in the negative control. The -actin-specific primers yielded a 430-bp product in all samples. The specificity of the DAZL1 product was confirmed by Southern blot hybridization, and the results are shown in Figure 2.
DISCUSSION The most abundant expression of dazl in female mice has been reported to occur in the pachytene cells of fetal ovarian tissues that have just finished the process of proliferation and entered prophase I (16). In humans, pachytene cells exist during the midtrimester of gestation. They pass through the pachytene stage and stop at the dictyotene stage late in the third trimester (19, 20). During the process of meiosis, most of the oocytes simultaneously undergo regression and apoptosis. The number of oocytes diminishes from 7,000,000 at the 20th week of gestation to 2,000,000 in the newborn period and 300,000 at puberty (19, 20). Therefore, if DAZL1 is also expressed in pachytene cells, the optimal samples for study should be fetal ovarian tissues obtained around the 20th week of gestation. Our samples were collected at this gestational age. We found that DAZL1 was obviously expressed in human fetal ovaries. Although Saxena et al. (12) suggested that DAZL1 could be expressed at a low level in the adult ovary, FERTILITY & STERILITY威
the results of the present study provide the first and the strongest evidence supporting DAZL1 expression in the fetal ovary. This suggests that the function of DAZL1 in humans could be similar to the function of dazl in mice and that it is therefore likely to be important for gonad development and germ cell survival in both sexes. We did not measure DAZL1 expression levels in the present study; therefore, it remains unclear whether the DAZL1 expression level is related to the number of pachytene cells. Comparison of expression levels would require a large number of fetal ovaries from a wide range of gestational ages. Legal and ethical considerations limit the availability of such samples. Multicenter cooperation and the establishment of a nationwide tissue committee are needed to enable such studies to be performed in the future. In conclusion, we have demonstrated the expression of DAZL1 in human fetal ovary. This new evidence suggests that DAZL1 is not only essential for spermatogenesis but is also important for oogenesis. References 1. Page AW, Orr-Weaver TL. Stopping and starting the meiotic cell cycle. Curr Opin Genet 1997;7:23–31. 2. Sassone-Corsi P. Transcriptional checkpoints determining the fate of male germ cells. Cell 1997;88:163– 6. 3. Sagata N. What does Mos do in oocytes and somatic cells? Bioassays 1997;19:13–21. 4. Tiepolo L, Zuffardi O. Localization of factors controlling spermatogen-
629
5.
6.
7.
8. 9. 10.
11.
esis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet 1976;34:119 –24. Reijo R, Lee TY, Salo P, Alagappan R, Brown LG, Rosenberg M, et al. Diverse spermatogenic defects in human caused by Y chromosome deletions encompassing a novel RNA binding protein gene. Nature Genet 1995;10:383–92. Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, et al. Human Y chromosome azoospermia factor mapped to different subregions in Yq11. Hum Mol Genet 1996;5:933– 43. Simoni M, Gromoll J, Dworniczak B, Rolf C, Abshagen K, Kamischke A, et al. Screening for deletions of the Y chromosome involving the DAZ (Deleted in AZoospermia) gene in azoospermia and oligozoospermia. Fertil Steril 1997;67:542–7. Chang SY, Tsai MY. Deletion of azoospermic factor genes in Chinese azoospermic and severe oligozoospermic patients. J Assist Reprod Genet 1999;16:237– 40. Cooke HJ, Lee M, Kerr S, Ruggiu M. A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum Mol Genet 1996;5:513– 6. Reijo R, Seligman J, Dinulos MB, Jaffe T, Brown LG, Disteche CM, et al. Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans, is expressed in male germ cells before and after puberty. Genomics 1996;35:346 –52. Shan Z, Hirschmann P, Seebacher T, Edelmann A, Jauch A, Morell J, et al. A SPGY copy homologous to the mouse gene dazla and drosophila gene boule is autosomal and expressed only in the human male gonad. Hum Mol Genet 1996;5:2005–11.
630
Tsai et al.
DAZL1 in human fetal ovary
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Vol. 73, No. 3, March 2000