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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 11 4379-4386
Copyright © 2000 by The Endocrine Society

Original Studies

Expression of Bcl-2 and Bax in the Human Corpus Luteum during the Menstrual Cycle and in Early Pregnancy: Regulation by Human Chorionic Gonadotropin1

Norihiro Sugino, Takashi Suzuki, Shiro Kashida, Ayako Karube, Shuji Takiguchi and Hiroshi Kato

Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine (N.S., S.K., A.K., S.T., H.K.), Minamikogushi 1-1-1, Ube 755-8505, Japan; and Department of Pathology, Tohoku University School of Medicine (T.S.), 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

Address all correspondence and requests for reprints to: Norihiro Sugino, M.D., Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Minamikogushi 1-1-1, Ube 755-8505, Japan. E-mail: obgyn{at}po.cc.yamaguchi-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate the relationship between apoptosis and the Bcl-2/Bax system in the human corpus luteum (CL), the frequency of apoptosis and expression of Bcl-2 and Bax were examined in the CL during the menstrual cycle and in early pregnancy. In situ analysis of DNA fragmentation showed that the number of apoptotic cells was much greater in the regressing CL than that in the midluteal phase CL, whereas there were almost no apoptotic cells in the CL of early pregnancy. Immunohistochemistry revealed that Bcl-2 expression was observed in the luteal cells in the midluteal phase and early pregnancy, but not in the regressing CL. In contrast, Bax immunostaining was observed in the regressing CL, but not in the midluteal phase and early pregnancy. bcl-2 messenger ribonucleic acid (mRNA) levels in the CL during the menstrual cycle were highest in the midluteal phase and lowest in the regressing CL. In the CL of early pregnancy, bcl-2 mRNA levels were significantly higher than those in the midluteal phase. In contrast, bax mRNA levels were highest in the regressing CL and remarkably low in the CL of early pregnancy. Western blot analyses revealed that Bcl-2 expression was significantly lower in the regressing CL than in the midluteal phase and early pregnancy, and that Bax expression was, in contrast, significantly higher in the regressing CL than in the midluteal phase and was remarkably low in the CL of early pregnancy. When corpora lutea of the midluteal phase were incubated with hCG, hCG significantly increased the mRNA and protein levels of Bcl-2 and significantly decreased those of Bax. In conclusion, Bcl-2 and Bax may play important roles in the regulation of the life span of the human CL by controlling the rate of apoptosis. hCG may act to prolong the life span of the CL by increasing Bcl-2 expression and decreasing Bax expression when pregnancy occurs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EPHEMERALITY AND prolongation of corpus luteum (CL) function have been a matter of concern for many years. In humans, the life span of the CL is 14 days after ovulation if pregnancy does not occur, but the CL can be further maintained if pregnancy occurs. The regression of the CL is necessary for follicular development during the next reproductive cycle, whereas the rescue of the CL is essential for the maintenance of pregnancy. However, the mechanism regulating the life span of the CL is poorly understood. Recent evidence has shown that apoptosis plays an important role in CL regression in several species (1, 2, 3, 4, 5, 6, 7). In humans, apoptosis was detected in the regressing CL, but not in the CL of pregnancy, suggesting that apoptosis may be involved in regulation of the life span of the CL (3). Apoptosis is controlled by the expression of a number of regulatory genes, for example the Bcl-2 family and Fas (8, 9, 10, 11, 12, 13). In particular, it has been suggested that the Bcl-2 family may be of importance in controlling the rate of apoptosis. Bcl-2 is known to protect cells from apoptosis, and it is now apparent that the activity of Bcl-2 is determined by the interaction with Bax that has a degree of homology to Bcl-2. Bax forms homodimers and induces apoptosis. If Bcl-2 forms heterodimers with Bax, then Bcl-2 is protecting the cells by inhibiting the formation of Bax homodimers. Thus, it is thought that the ratio of Bcl-2 to Bax expression is the critical determinant of cell fate, such that elevated Bcl-2 favors extended survival of cells, whereas increasing levels of Bax expression accelerate cell death (14, 15, 16). Recently, Bcl-2 and Bax have been found in the human CL (17, 18). However, little is known regarding the change in expression of Bcl-2 and Bax in the human CL during the menstrual cycle and in early pregnancy. In the present study to investigate the possible role of Bcl-2 and Bax in the regulation of the life span of the human CL, the frequency of apoptosis and the change in expression of messenger ribonucleic acid (mRNA) and protein of Bcl-2 and Bax were examined in the human CL throughout the menstrual cycle and in early pregnancy. We further examined the role of hCG in the regulation of Bcl-2 and Bax expression in the human CL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This project was reviewed and approved by the committee on investigations involving human subjects of Yamaguchi University School of Medicine. Informed consent from the patient was obtained before the collection of any tissue samples for this study.

Materials

RPMI 1640 medium was purchased from Flow Laboratories, Inc. (McLean, VA). Streptomycin, penicillin, deoxynucleotide triphosphates, and Moloney murine leukemia virus reverse transcriptase were obtained from Life Technologies, Inc. (Grand Island, NY). hCG was purchased from Sigma (St. Louis, MO). Random hexamer and Taq DNA polymerase were obtained from Perkin-Elmer Corp. (Foster City, CA). [{alpha}-32P]Deoxy-CTP was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Isogen was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Tissue samples

CL were obtained from patients with normal menstrual cycles, aged 39–49 yr, undergoing hysterectomy for myoma uteri or cervical cancer. The menstrual history and endometrial histology were used to determine the age of the CL. CL from the menstrual cycle were classified into four different groups according to age: the early luteal phase (days 1–5 of luteal phase, with day 1 being the day of ovulation), the midluteal phase (days 6–11), the late luteal phase (days 12–15), and the regression phase (after the onset of menstruation, days 3–7 of the follicular phase). CL of early pregnancy (6–8 weeks of pregnancy) were obtained from patients, aged 24–30 yr, with ectopic pregnancy. Tissue samples were washed with saline to remove blood, immediately frozen in liquid nitrogen, and stored at -80 C until isolation of RNA for RT-PCR and isolation of protein for Western blot analysis. In some patients, blood samples were obtained during surgery for determination of serum progesterone concentrations. Serum progesterone concentrations (mean ± SEM) were significantly higher in the midluteal phase (13.1 ± 1.8 ng/mL; n = 5) than in the early luteal phase (4.8 ± 1.5 ng/mL; n = 4) and the late luteal phase (3.2 ± 0.6 ng/mL; n = 5), whereas those of all patients in the regression phase were less than 1.0 ng/mL.

Apoptosis detection by in situ analysis

DNA fragmentation was detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method using an apoptosis in situ detection kit (Wako Pure Chemical Industries Ltd., Osaka, Japan) in 3 tissue samples from the midluteal phase, regression phase, and early pregnancy. CL were fixed in Carnoy solution, embedded in paraffin, and sectioned (4 µm thick). The tissue sections were deparaffinized in xylene, dehydrated in a graded series of ethanol, and immersed with 100 µL TdT for 10 min at 37 C after treatment with a protein digestion enzyme for 5 min at 37 C. After 3 washes with phosphate-buffered saline and inhibition of endogenous peroxidase activity with 3% H2O2 for 5 min at room temperature, tissue sections were incubated with a peroxidase-conjugated antibody for 10 min at 37 C. Peroxidase activity was visualized by incubating the sections with diaminobenzidine solution for 2–2.5 min. Counterstaining was performed with methyl green. For the positive control, tissue sections were treated with deoxyribonuclease I solution for 15 min at 37 C before TdT treatment. For the negative control, tissue sections were incubated with a TdT buffer that does not contain the enzyme. The number of apoptotic cells was counted within a grid area of 0.125 mm2 at x200 in the tissue sections from 3 samples obtained from the midluteal phase, regression phase, and early pregnancy. Counting was performed on 10 randomly chosen areas in each sample by 3 independent observers. An observer-related mean was calculated for each sample, and the mean of the 3 observer-related means was used as a single observation.

Immunohistochemistry

The immunohistochemical staining was performed on tissue samples from the midluteal phase, regression phase, and early pregnancy taken from three different patients. CL were fixed in Carnoy solution, embedded in paraffin, and sectioned (3 µm thick). The tissue sections were deparaffinized in xylene and dehydrated in a graded series of ethanol. Then, the slides were heated in an autoclave at 120 C for 5 min in a citric acid buffer (2 mmol/L citric acid and 9 mmol/L trisodium citrate dehydrate, pH 6.0). Immunohistochemistry for Bcl-2 and Bax was performed with the streptavidin-biotin amplification method using a Histofine Kit (Nichirei Co. Ltd., Tokyo, Japan) as reported previously (19). The dilution of primary antibodies used in this study was 1:40 in phosphate-buffered saline-BSA (1%) for Bcl-2 (monoclonal antibody; clone 124, DAKO Corp. Japan, Tokyo, Japan) and 1:200 for Bax (polyclonal antibody; DAKO Corp. Japan). The antigen-antibody complex was visualized by incubating the sections with 3,3'-diaminobenzidine·4HCl (Nacalai Tesque Co. Ltd., Tokyo, Japan) in 0.05 mol/L Tris-HCl buffer (pH 7.6) containing 0.01% H2O2 for 2–3 min. For the negative control, normal mouse or rabbit serum was used instead of the primary antibodies, and no specific immunoreactivity was detected in these sections. Counterstaining was performed with Meyer’s hematoxylin.

Incubation of CL

CL obtained from the midluteal phase were sliced into small pieces and pooled, and then one or two pieces were randomly collected and incubated in serum-free RPMI 1640 medium (35–60 mg wet weight/mL·tube) at 37 C for 1 h under an atmosphere of 95% O2-5% CO2 in a shaking water bath. The medium was then changed to the test medium containing hCG (1 and 10 IU/mL), and the incubation was continued for 6 h under the same atmosphere as that described above. After incubation, the CL tissue was immediately frozen in liquid nitrogen and stored at -80 C until isolation of RNA for RT-PCR and isolation of protein for Western blot analysis. The incubation was performed in triplicate. It was confirmed in this incubation system that progesterone concentrations in the medium were significantly increased by hCG [control, 3.0 ± 0.2 ng/mg wet wt; hCG (1 IU/mL), 5.7 ± 0.7 ng/mg wet wt; hCG (10 IU/mL), 7.9 ± 1.3 ng/mg wet wt; mean ± SEM; n = 3].

RT-PCR

Total RNA was isolated from the CL with Isogen using the method provided by the manufacturer. For mRNA analysis, RT-PCR was performed as reported previously (20) with the oligonucleotide primers for Bcl-2 (5'-GACTTCGCCGAGATGTCCAG-3' and 5'-TCACTTGTGGCTCAGATAGG-3') and Bax (5'-GGTTTCATCCAGGATCGAGACGG-3' and 5'-ACAAAGATGGTCACGGTCTGCC-3'), designed by Hori et al. (21) and Kugu et al. (22), respectively. Direct sequence analyses of the PCR products were performed for sequence verification. Two oligonucleotide primers (5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-GGACAGAGTCTTGATGATCTC-3') were also used to amplify ribosomal protein L19 as an internal control (23). In brief, 3 µg total RNA were reverse transcribed at 42 C in a reaction mixture (single strength PCR buffer, 2.5 mmol/L deoxynucleotide triphosphates, 5 µmol/L random hexamer primer, 1.5 mmol/L MgCl2, and 200 U Moloney murine leukemia virus reverse transcriptase). The RT product was divided into equal aliquots and placed into two tubes with Bcl-2 (or Bax) primers and L19 primers, and PCR was performed. For PCR amplification, a mixture containing the oligonucleotide primers (50 pmol), [{alpha}-32P]deoxy-CTP (2 µCi at 3000 Ci/mmol), and Taq DNA polymerase (2.5 U) was added to each reaction. Amplification was carried out for 30 cycles consisting of 94 C for 1 min, 60 C for 1 min, and 72 C for 1.5 min for Bcl-2, and 27 cycles consisting of 94 C for 1 min, 50 C for 1 min, and 72 C for 2 min for Bax, followed by 10 min of final extension at 72 C in a programmed temperature control system (PC-800, ASTEC, Fukuoka, Japan). The predicted sizes of the PCR-amplified products were 390 bp for Bcl-2, 445 bp for Bax, and 194 bp for L19. A linear curve was plotted using the number of cycles of amplification vs. densitometric values of the PCR products, measured with a BAS2000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). The optimal number of cycles for amplification that fit within the linear range was chosen for each set of primers of Bcl-2, Bax, and L19 (data not shown). To separate the band of Bcl-2/Bax and L19, reaction products were electrophoresed on an 8% polyacrylamide nondenaturing gel under 200 V for 2 h. After autoradiography, band intensities were analyzed using a bioimaging analyzer BAS2000. For quantification, the densities of Bcl-2 and Bax were normalized to that of the internal control L19.

Western blot analysis

CL were homogenized with distilled water and centrifuged at 800 x g for 10 min at 4 C. The supernatant was used for Western blot analysis. Eighty micrograms of protein, determined by the Lowry method (24), for Bcl-2 and Bax were separated by SDS-PAGE in 15% gels under reduced conditions. The proteins in the gel were electrophoretically transferred to nitrocellulose membranes and reacted with the rabbit antihuman Bcl-2 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit antihuman Bax polyclonal antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:50 with 0.5% skimmed milk in Tris-buffered saline (pH 7.5). The membranes were then immersed in the reaction buffer containing peroxidase-conjugated swine antirabbit Ig. The reacted band was developed with an ECL kit (Amersham Pharmacia Biotech, Aylesbury, UK). Reacted bands of Bcl-2 and Bax were scanned, and their optical densities were measured by NIH Image.

Progesterone assay

Progesterone concentrations in the serum and medium were determined by a specific RIA as reported previously (25). The sensitivity of the assay was 100 pg/mL, and the intra- and interassay coefficients of variation were 7.0% and 14.4%, respectively.

Statistical analysis

Data were examined by ANOVA and Duncan’s new multiple range test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In situ analysis of DNA fragmentation by the TUNEL method showed that the positively stained cells were observed in the CL of the midluteal phase (Fig. 1AGo) and the regression phase (Fig. 1BGo). The number of positive cells in the CL was much greater in the regression phase than in the midluteal phase (Fig. 1EGo), whereas there were almost no positive cells in the CL of early pregnancy (Fig. 1CGo).



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  		Figure 1. In situ analysis of DNA fragmentation in the CL from the midluteal phase (A), the regression phase (B), and early pregnancy (C). In situ analysis of DNA fragmentation was analyzed by the TUNEL method. D, Negative control. Original magnification, x400. E, A quantitative analysis of apoptotic cells in the CL from the midluteal phase, the regression phase, and early pregnancy. The number of TUNEL-positive cells (brown staining), indicated by arrows, was counted within a grid area of 0.125 mm2. Values are the mean ± SEM of different three samples. a, P < 0.01 vs. the other two groups.

 
Immunostaining of Bcl-2 was observed in the luteal cells of the midluteal phase and early pregnancy (Fig. 2Go, A and C), whereas luteal cells of the regressing CL showed no immunostaining (Fig. 2BGo). In contrast, immunostaining of Bax was observed in the luteal cells of the regressing CL (Fig. 2FGo), but not in the luteal cells of the midluteal phase and early pregnancy (Fig. 2Go, E and G).



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  		Figure 2. Immunohistochemical staining for Bcl-2 (A–D) and Bax (E–H) in the human CL from the midluteal phase (A and E), the regression phase (B and F), and early pregnancy (C and G). D (from early pregnancy) and H (from the regression phase), Negative controls. Original magnification, x236.

 
Figure 3Go shows the mRNA levels of bcl-2 and bax in the CL obtained from different stages of the menstrual cycle and early pregnancy. bcl-2 mRNA levels in the CL during the menstrual cycle were highest in the midluteal phase, decreased toward the regression phase, and were lowest in the regressing CL (Fig. 3AGo). In the CL of early pregnancy, bcl-2 mRNA levels were significantly higher than those in the midluteal phase (Fig. 3AGo). In contrast, bax mRNA levels were significantly higher in the late luteal phase than in the early and midluteal phases and were highest in the regressing CL (Fig. 3BGo). In the CL of early pregnancy, bax mRNA levels were remarkably low (Fig. 3BGo). It has been reported that the ratio of Bcl-2 to Bax expression is the critical determinant of cell fate, such that elevated Bcl-2 favors extended survival of cells, whereas increasing levels of Bax expression accelerate cell death (14, 15, 16). As shown in Fig. 4Go, the ratio of bcl-2 to bax mRNA was the lowest in the regressing CL, and there was a significant difference between the midluteal phase and the regression phase, whereas the ratio of bcl-2 to bax mRNA was remarkably high in the CL of early pregnancy.



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  		Figure 3. Changes in bcl-2 (A) and bax (B) mRNA levels in the human CL during the menstrual cycle and in early pregnancy. Samples were obtained from the early luteal phase (days 1–5 of the luteal phase, with day 1 being the day of ovulation; n = 3), the midluteal phase (days 6–11; n = 4), the late luteal phase (days 12–15; n = 3), the regression phase (after the onset of menstruation, days 3–7 of the follicular phase; n = 5), and early pregnancy (6–8 weeks of pregnancy; n = 5). Total RNA was isolated and subjected to RT-PCR. The intensity of the signals of Bcl-2 or Bax was normalized to that of the internal control L19. The quantification data (the ratio of Bcl-2 or Bax to L19) represent the mean ± SEM. A: a, P < 0.05 vs. early luteal phase; b, P < 0.01 vs. mid and late luteal phases and pregnancy; c, P < 0.05 vs. midluteal phase. B: a, P < 0.05 vs. early luteal phase; b, P < 0.01 vs. midluteal phase; c, P < 0.01 vs. the other groups.

 


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  		Figure 4. Changes in the ratio of bcl-2 to bax mRNA in the human CL during the menstrual cycle and in early pregnancy. The ratio of bcl-2/bax was calculated from each sample in Fig. 3Go. Values are the mean ± SEM. a, P < 0.05 vs. early and midluteal phases; b, P < 0.01 vs. the other groups.

 
Western blot analyses revealed that protein expression of Bcl-2 in the CL was significantly lower in the regression phase than in the midluteal phase and early pregnancy (Fig. 5AGo). Protein expression of Bcl-2 in the CL of early pregnancy tended to be higher than that in the midluteal phase; however, there was no significant difference between these values (Fig. 5AGo). In contrast, protein expression of Bax in the CL was significantly higher in the regression phase than in the midluteal phase and was remarkably low in early pregnancy (Fig. 5BGo).



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  		Figure 5. Western blot analyses for Bcl-2 (A) and Bax (B) in the CL obtained from the midluteal phase, the regression phase, and early pregnancy. Samples were obtained from the midluteal phase (n = 4), the regression phase (n = 4), and early pregnancy (n = 5). The density of the band was measured by NIH Image, and the data were expressed as a percentage of the midluteal phase value. Values are the mean ± SEM. a, P < 0.01 vs. the other two groups.

 
To study whether hCG is involved in the change in the expression of Bcl-2 and Bax, CL of the midluteal phase were incubated with hCG. hCG significantly increased the mRNA levels of bcl-2 and significantly decreased the mRNA levels of bax (Fig. 6Go). In addition, hCG significantly increased protein expression of Bcl-2 and significantly decreased protein expression of Bax in the Western blot analyses (Fig. 7Go).



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  		Figure 6. Effects of hCG on bcl-2 (A) and bax (B) mRNA levels in the CL. CL obtained from the midluteal phase were incubated with hCG (1 and 10 IU/mL) for 6 h. Total RNA was isolated and subjected to RT-PCR. The intensity of the signals of Bcl-2 or Bax was normalized to that of the internal control L19. The quantification data (the ratio of Bcl-2 or Bax to L19) represent the mean ± SEM of three different experiments. a, P < 0.01; b, P < 0.05 (vs. control).

 


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  		Figure 7. Effects of hCG on Bcl-2 (A) and Bax (B) protein levels in the CL. CL obtained from the midluteal phase were incubated with hCG (1 and 10 IU/mL) for 6 h. Western blot analysis was performed for the determination of protein levels. The density of the band in the Western blot analyses was measured by NIH Image, and the data were expressed as a percentage of the control value. Values are the mean ± SEM of three different experiments. a, P < 0.01; b, P < 0.05 (vs. control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study showed that the frequency of apoptosis was remarkably high in the regressing CL, whereas there were almost no apoptotic cells in the CL of early pregnancy. This finding is in agreement with the previous reports (3, 5) and suggests that apoptosis may be involved in human luteal regression. However, little is known regarding the factors controlling apoptosis in the human CL. It has been proposed that the ratio of Bcl-2 to Bax is the critical determinant of cell fate, such that elevated Bcl-2 favors extended survival of cells, whereas increasing levels of Bax expression accelerate cell death (14, 15, 16). The present study showed that high Bcl-2 and low Bax expression were observed in the CL in the midluteal phase and early pregnancy, whereas low Bcl-2 and high Bax expression were found in the regressing CL, and the change in the ratio of bcl-2 to bax mRNA was parallel to luteal function and inversely related to the frequency of apoptosis in the CL. Therefore, Bcl-2 and Bax may play important roles in the regulation of the life span of the human CL, probably by controlling the rate of apoptosis. Of course, we cannot neglect the presence of some factors, such as p53, that may act upstream of Bcl-2 family members via transcriptional regulation of the bcl-2 and bax genes (13, 22).

Regarding the mechanism controlling the prolongation of the luteal life span when pregnancy occurs, several factors, including hCG, have been reported to be involved in the rescue of the CL (26, 27, 28, 29, 30, 31, 32, 33). The present study showed the remarkably high ratio of Bcl-2 to Bax in the CL of early pregnancy. In addition, the present study demonstrated in vitro that hCG increased Bcl-2 expression and decreased Bax expression in the midluteal phase CL. Therefore, the high ratio of Bcl-2 to Bax seen in the CL of early pregnancy may be due to the effect of hCG. Tilly et al. (34, 35) reported that gonadotropins prevented follicular apoptosis, which was related to the increase in the ratio of Bcl-2 to Bax. Dharamarjan et al. (36) also reported that hCG-mediated inhibition of apoptosis involved down-regulated Bax expression in rabbit luteal cells. Thus, these findings may suggest that hCG rescues the human CL by increasing the ratio of Bcl-2 to Bax when pregnancy occurs.

Recently, Rodger et al. (17, 18) reported that immunohistochemical staining for Bcl-2 and Bax was found in the CL obtained from the early, mid, and late luteal phases and in the CL rescued by hCG administration, but there was no difference in the intensity of immunostaining. They also showed by immunoblotting analyses that Bcl-2 and Bax expression were detected at constant levels in the CL throughout the luteal phase and in the CL rescued by hCG (17, 18). Compared with our data, the change in Bax expression seems to be in a discrepancy. It is hard to clearly explain the difference; however, one explanation may be the difference in experimental techniques, sample preparation, and antibodies used. Also, it should be taken into consideration that they did not examine the CL of early pregnancy.

In conclusion, the present study suggests that Bcl-2 and Bax play important roles in the regulation of the life span of the human CL by controlling the rate of apoptosis. hCG may act to prolong the life span of the CL by increasing Bcl-2 expression and decreasing Bax expression when pregnancy occurs.


   Footnotes
 
1 This work was supported in part by a grant from the UBE Foundation and Grant-in-Aid 11671623 from the Ministry of Education, Science, and Culture, Japan. Back

Received March 8, 2000.

Revised May 31, 2000.

Revised July 25, 2000.

Accepted July 28, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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