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doi: 10.1016/S0015-0282(99)00544-0

The expression of DAZL1 in the ovary of the human female fetus

Keywords

DAZL1. fetus. ovary. reverse transcriptase polymerase chain reaction (RT-PCR).

Resumen

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.

Artículo

Germ cell proliferation is a complex process involving a series of signal transductions that are regulated by stringent stage-specific gene expression . 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 . A candidate gene, called DAZ (deletion in azoospermia) was recently mapped to this region and has been found to be deleted in 3%–13% of azoospermic patients .

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 . This homologous gene has been called dazl . In addition to its homology with DAZ, dazl has other homology in humans . It was mapped to chromosome 3 and called DAZL1 . Sequencing analysis has revealed that the nucleotides of DAZL1 are 83% homologous to DAZ .

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 . Unlike the homologous, boule gene in drosophila, which is only important for spermatogenesis , both male and female dazl knockout mice appear to be infertile . This finding suggests that dazl gene expression is necessary for germ cell survival and gonad development in mice of both sexes .

In humans, DAZL1 is specifically expressed in adult testes producing germ cells but not expressed in some idiopathic azoospermic men . Whether DAZL1 is expressed in women is unclear. Previous Northern blot analysis of DAZL1 expression in adult female ovaries has unanimously displayed negative findings . However, the results of reverse transcriptase polymerase chain reaction (RT-PCR) have been inconsistent. Saxena et al. reported a PCR product, suggesting the positive expression of DAZL1 in the adult ovary, but Shan et al. could not achieve the same result.

In female mice, the most abundant dazl expression was reported to occur in the fetal stage . 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 . 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 instructions. The extracted RNA was quantified with a spectrophotometer (DU640, Beckman, Fullerton, CA).

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 . RT-PCR was performed with use of a commercial kit (Promega, Madison, WI). The reactions were done in 50-μl volumes of 1.5 mM of MgCl2, 5 mM NH4Cl, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 100 μM deoxynucleotide triphosphate (dNTPs), with 1 U of AMV reverse transcriptase, 1 U Tfl DNA polymerase, 100 ng of RNA, and 1 μM 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 β-actin-specific 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 40-bp 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-conjugated-dideoxy-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 . The DAZL1-specific primers yielded a 128-bp RT-PCR product in all five fetal ovarian tissues and two of the positive controls, but not in the negative control. The β-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 .

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 . 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 . 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 . 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. suggested that DAZL1 could be expressed at a low level in the adult ovary, 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.

Received 1 June 1999
Received 17 September 1999
Accepted 17 September 1999

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