: Kor. J. Fertil. Steril., Vol. 31, No. 2, 2004, 6 GnRH-agonist PBR 1, 2, Apoptosis and Peripheral Benzodiazepin Receptor

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1 : Kor. J. Fertil. Steril., Vol. 31, No. 2, 2004, 6 GnRH-agonist PBR 1, 2, Apoptosis and Peripheral Benzodiazepin Receptor (PBR) Expression in Human Granulosa-Luteal Cells by GnRH-agonist Sei Kwang Kim 1, Yun-Hee Youm 2, Jeong-Mi Yoon 1, Sang Wook Bai 1, Hyunwon Yang 3, Dong Jae Cho 1, Yong-Dal Yoon 2, Chan Ho Song 1 1 Department of Obstetrics and Gynecology, College of Medicine, Yonsei University, 2 Department of Life Sciences, College of Natural Sciences, Hanyang University Life Science Institute, 3 Eulji University School of Medicine Objective: To investigate whether GnRH-agonist (GnRH-Ag) using in IVF-ET affects apoptosis of human granulosa-luteal cells and expression of peripheral benzodiazepine receptor (PBR) protein involved in the apoptosis of the cells. Methods: Granulosa-luteal cells obtained during oocyte retrieval were cultured and treated with 10 5 M GnRH-Ag. Apoptosis of the cells by the treatment was confirmed using DNA fragmentation analysis 24 h after culture. The presence of PBR protein within the cells was examined by immunofluorescence staining and the expression of the protein was analyzed by Western blotting. In addition, it was measured for progesterone and nitric oxide (NO) produced by granulosa-luteal cells after GnRH-Ag treatment. To evaluate the relationship between NO production and PBR expression, sodium nitroprusside (SNP) as a NO donor was added in media and investigated the expression of PBR protein by Western blotting. Results: Apoptosis increased in the granulosa-luteal cells 24 h after GnRH-Ag treatment, whereas the expression of PBR protein significantly decreased. Furthermore, the production of progesterone and nitric oxide (NO) by the cells significantly fell from 12 h after the treatment. In the results of Western blotting after SNP treatment, the expression of PBR protein increased in the treatment with SNP alone to the granulosa-luteal cells, but was suppressed in the treatment with GnRH-Ag and SNP. Additionally, the staining result of PBR protein in the cells showed the even distribution of it through the cell. Conclusion: These results demonstrate that GnRH-Ag treatment induces apoptosis, decreasing expression of PBR protein and NO production in human granulosa-luteal cells. The present study suggests that one of the apoptosis mechanism of human granulosa-luteal cells by GnRH-Ag might be a :, ) , Tel: (02) , Fax: (02) , skkim@yumc.yonsei.ac.kr *

2 signal transduction pathway via NO and PBR. Key Words: Apoptosis, GnRH-agonist, Granulosa-luteal cells, Nitric Oxide (NO), Peripheral benzodiazepine receptor (PBR) (GnRH). 11 GnRH-Ag 10 peripheral benzodiazepine receptor (PBR) steroidogenic acute FSH LH,. 1 GnRH GnRH GnRH NO nitric oxide synthase GnRH (NOS), 16, 17 18,, 19, 20., GnRH cyclic nucleotide 21 LH prolactin FSH, LH, IGF-1 estradiol progesterone progesterone, androgen estro-. 19,22 gen. 5 NO GnRH, 6 inhibin. 7 GnRH GnRH regulatory (StAR). 12 PBR StAR. 13 PBR. 2 GnRH, PBR GnRH GnRH Nitric oxide (NO). NO. 23 NO, NO. 24 NO. 8,9 GnRH IGFBP-4 GnRH, (atresia) GnRH-Ag,. 10. GnRH Bax GnRH-Ag NO NO Bcl-xL, PBR

3 GnRH. 70%. 1 ml 100, well culture plate (Nunc, Denmark) 1 well ( 1 ml) (IVF-ET) 100,000, 37 95%. 5% CO 2 100% hmg (Meri-. Dulbecco's Modified onal; IBSA, Swiss) hfsh (Metrodin; Serono, Swiss) Eagle Medium (dmem; GIBCO BRL) 10% fetal bo-. vine serum (FBS; GIBCO BRL) 2 mm L-glutamine (step-down fashion) (GIBCO BRL), 100 U/ml penicillin (GIBCO BRL),, hmg hfsh µg/ml streptomycin (GIBCO BRL) (Medison 128; Medison Co., Korea), hmg. 18 mm 17 mm 2 hcg (Profasi; Serono, Swiss) 10,000 IU. hcg NO PBR. St. Louis, MO) GnRH-Ag Hemonglobin (Sigma, St. Louis, MO). 24 PBR NO. 1 ml 40% percoll 3 ml 300 xg ml % collagenase (Sigma, St. Louis, MO) G heamocytometer. trypan blue, chloroform: isoamyl alco- hol (24:1, V:V). 1.5 ml well culture plate 24 plate M GnRH-Ag (Sigma, St. Louis, MO) NO SNP (Sigma, 12.5 µl 10% SDS µl 8 M potassium acetate 60 4, 5000 xg ml phenol: chloroform: iso- amyl alcohol (25:24:1, V:V:V) DNA

4 , , protease inhibitors (0.1 mm phenyl methyl-sulfonyl- 100% ethanol, -70 fluoride, 5 g/ml aprotinin, and 5 g/ml leupeptin) 1. 4 homogenization buffer 12,000 xg 14,000 xg 30 DNA 30. DC 50 µl 1X TE buffer (10 mm Tris-HCl, 1 protein assay kit (Bio-Rad Laboratories, Inc., Hercules, mm EDTA, ph 8.0) 1 µl DNasefree RNase (500 µg/ml; Boehringer-Mannheim, IN) DNA phenol : chloroform : isoamyl alcohol, chloroform: isoamyl alcohol M sodium acetate % ethanol DNA ,000 xg 30, ml 80% ethanol. 25 µl, 260 nm DNA -20. DNA lane 5 µg 1.5% agarose logy, Santa Cruz, CA) TTBS 1:1,000 gel loading, running buffer TBE 40., 50 V 3 membrane chemiluminescence (ECL kit; ethidium bromide tran- Amersham Life Science, Buckinghamshire, U.K.) silluminator. Science, Buckinghamshire, U.K.). NO nitrate/nitrite colorimetric assay kit (Alexis PBR Biochemicals, San Diego, CA), 1%. 10 kda molecular mass cut-off filter (Amicon, Millipore Co., Bedford, MA). goat nor-, nitrate reductase enzyme cofactor 3. sulfanila- rabbit polyclonal mide N-[1-naphthyl] ethylenediamine 1. PBS 10 microplate reader (Spectra Max 250, Molecular Devices, Sunnyvale, CA) anti-rabbit IgG (Jackson Immuno Research laboratories, 540 nm.. Propidium Iodide. 50 mm Tris-base PBR (Ni- (ph 7.4), 150 mm NaCl, 10 mm EDTA, 0.1% Tween- kon, Tokyo, Japan) CA, U.S.A.) 10% SDS-PAGE. blotting nitro-cellulose membrane, 5% non-fat dry milk Tris-buffered saline (TTBS; 10 mm Tris (ph 7.6), 150 mm NaCl, 0.1% Tween-20) 1 blocking. Blocking membrane rabbit polyclonal anti-human PBR (Trevigen, Gai- thersbug, MD) 1:1,000 TTBS 1. TTBS 1 3 anti-rabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechno- X-ray film (Hyperfilm, Amersham Life paraformaldehyde PBS mal serum 30. anti-human PBR, 1:100 fluorescein isothiocyanate (FITC) West Grove, PA) 30

one-way ANOVA stu- 4h 24 h Marker Saline GnRH Saline GnRH dent's t-test p 0.05. GnRH-Ag DNA. 4 GnRH-Ag DNA. 24 DNA GnRH-Ag (Figure 1). A 4 h 12 h 24 h Saline GnRH Saline GnRH Saline GnRH PBR Figure 1.

The cells cultured for 24 h showed an increase in DNA fragmentation in GnRH-Ag treatment compared with those in the saline. β-actin B Figure 2.

5 one-way ANOVA stu- 4h 24 h Marker Saline GnRH Saline GnRH dent's t-test p GnRH-Ag DNA. 4 GnRH-Ag DNA. 24 DNA GnRH-Ag (Figure 1). A 4 h 12 h 24 h Saline GnRH Saline GnRH Saline GnRH PBR Figure 1. Effects of GnRH-Ag treatment on DNA fragmentation in the cultured human granulosa-luteal cells. The cells cultured for 24 h showed an increase in DNA fragmentation in GnRH-Ag treatment compared with those in the saline. β-actin B Figure 2. Nitric oxide levels in the medium (mean EM; n = 5) at points timed after initiation of the treatment. *p<0.05 compared with corresponding saline controls. Figure 3. Western blot analysis (mean EM; n = 5) of PBR proteins in the granulosa-luteal cells at points timed after the commencement of treatment. *p<0.05 compared with corresponding saline controls

12 GnRH-Ag 3.33 0.66 µm/ml 6.21 1.52 µm/ml Figure 2 GnRH-Ag NO. NO (p<0.05), 24 2.35 0.91 µg/ml 4.04 1.23 µg/ml A SNP (mm) Saline 0.01 0.

Western blot analysis (mean EM; n = 5) of PBR protein in the granulosa-luteal cells (A & B) and nitric oxide levels (mean EM; n = 5) in the medium 24 h after sodium nitroprusside (SNP) treatment in a

6 12 GnRH-Ag µm/ml µm/ml Figure 2 GnRH-Ag NO. NO (p<0.05), µg/ml µg/ml A SNP (mm) Saline A Saline SNP SNP+ SNP Hb GnRH Hb +GnRH PBR PBR β-actin β-actin B B C C Figure 4. Western blot analysis (mean EM; n = 5) of PBR protein in the granulosa-luteal cells (A & B) and nitric oxide levels (mean EM; n = 5) in the medium 24 h after sodium nitroprusside (SNP) treatment in a dose-dependent manner. *p<0.05 compared with corresponding saline controls. Figure 5. Western blot analysis (mean EM; n = 5) of PBR protein in the granulosa-luteal cells (A & B) and nitric oxide levels (mean EM; n = 5) in the medium 24 h after treatment with GnRH-Ag, sodium nitroprusside (SNP), or hemoglobin (Hb). *p<0.05 compared with corresponding saline controls

(p<0.05). PBR SNP (239.11 23.12) Hb (178.56 13.32) PBR. GnRH-Ag, 4 (123.98 21.87) SNP PBR, 12 GnRH-Ag (71.34 10.21) (48.67 6.98) (p<0.05). 24 GnRH- Ag (72.78 12.32) (43.34 5.22) (p<0.05) (Figure 3).

7 (p<0.05). PBR SNP ( ) Hb ( ) PBR. GnRH-Ag, 4 ( ) SNP PBR, 12 GnRH-Ag ( ) ( ) (p<0.05). 24 GnRH- Ag ( ) ( ) (p<0.05) (Figure 3). Hb ( µg/ml). GnRH-Ag NO SNP PBR, PBR 0.01 mm SNP (62.48 Hb ( µg/ml) GnRH-Ag ( µg/ml) 9.20) 0.1 mm SNP ( ) PBR 1 mm SNP ( ) (5.66± 1.24 µg/ml) (Figure 4B). NO (Figure 5C). SNP (Figure 4C). Figure mm SNP, Hb GnRH- Ag PBR PBR NO.. Hb ( ) GnRH-Ag ( ) PBR ( ) (Figure 5B). NO SNP ( µg/ml), NO SNP µg/ml. A B Figure 6. Localization of PBR protein in the cultured human granulosa-luteal cells. Green spots displaying PBR expression localize on mitochondria in even throughout the cell. A; Fluorescent and B; Light microphotographs. Original magnification X

8 , Bax Bcl-xL (Figure 6).. 11,14 GnRH GnRH-Ag. 28 GnRH, GnRH. GnRH-Ag NO. 25 FSH GnRH-Ag. 29, GnRH-Ag. 26 GnRH, GnRH-Ag. GnRH-Ag GnRH-Ag NO PBR. GnRH- GnRH-Ag. GnRH-Ag GnRH-Ag DNA. 24 DNA GnRH-Ag. GnRH-Ag DNA,,, GnRH-Ag. GnRH-Ag, 27 GnRH-Ag. NO PBR GnRH-Ag NO. 30 NO,. 31 NO. GnRH-Ag NO Ag NO. GnRH-Ag NO NOS, GnRH-Ag NO NOS. 32 NO S-nitroso-N-acetyl-DL-penicillamine, 33 SNP FSH. 34 GnRH-Ag NO PBR

9 NO potential SNP PBR (proapoptotic). PBR, NO PBR. 1 MPT mm SNP NO, PBR potential. NO. NO PBR NO SNP NO Hb PBR. SNP 4864 PK PBR, Hb. 40 PBR. GnRH-Ag SNP PBR. SNP NO Hb. NO. GnRH-Ag - PBR 18 kda. 35 PBR Ro isoquinoline carboxamide benzodiazepine voltagedependent anion channels 1. Barbieri RL, Hornstein MD. Assisted reproduction-in. 36 oxidative pho- vitro fertilization success is improved by ovarian sphorylation, stimulation with exogenous gonadotropins and pi- tuitary suppression with gonadotropin-releasing hor-. 13 mone analogues. Endocr Rev 1999; 20: PBR 2. Beckers NG, Macklon NS, Eijkemans MJ, Ludwig. 32 M, Felberbaum RE, Diedrich K, et al. Nonsupple- mented luteal phase characteristics after the admi-. 38 PBR nistration of recombinant human chorionic gonado- tropin, recombinant luteinizing hormone, or gona-. dotropin-releasing hormone (GnRH) agonist to in- (mitochondrial permeability transition pore; MPT) duce final oocyte maturation in in vitro fertilization PBR ligand., leukae- mic cells (HL 60) PBR ligand PK 11195, Ro5-4864, pyrrolo-1,5-benzoxazepines. 39 Ro5- NO. GnRH-Ag PBR. NO GnRH-Ag PBR PBR NO SNP. NO GnRH, NO PBR

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歯 PDF * * The Effects of Oocyte Oocytes Intracytoplasmic ICSI) Kee TaekHoo Hai * SikChun Department of Obstetrics and Gynecology, School of Medicine, Kyungpook Taegu, *Department of Animal Science, Taegu Kyungbuk,