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Molecular events in decidualization: cDNAsubtraction to detect progesterone-induced genes in human endometrial stromal cells
A.IO MOLECULAR EVENTS IN DECIDUALIZATION: eDNA subtraction to detect progesterone-induced genes in human endometrial stromal cells. Hideharu Kanzaki*, Mariko Fujimoto**, Toshihiro Higuchi**, Hirohiko Watanabe**, Jun Fujita*** and Takahide Mori**, *Department of Obstetrics & Gynec.ology, Kansai Medical University, Osaka Japan; and Department of **Gynecology & Obstetrics and ***Clinical Molecular Biology, Faculty of Medicine, Kyoto University, Kyoto, Japan. Little is known as to the molecular events involved in the process of decidual transformation of endometrial stromal cells induced by progesterone (P). In order to identify genes induced by P, human endometrial stromal cells were cultured with or without P for 12 days, and poly(A) RNAs were isolated from the cultured ceils. After synthesis of double strmd cDNA from poly(A) RNA, eDNA was enzymatieally digested, ligated with oligonucleotide adaptor, and amplified by PCR. Target eDNA obtained from decidualized cells was hybridized with excess amount of biotinylated competitor eDNA obtained from fibroblastic stromal ceils, and biotinylated hybrids were subtracted by streptavidin incubation. After 4 rounds of subtraetive hybridization, the subtracted eDNAs were finally cloned in to a pBlueseript SK(-) plasmid, and each clone was analyzed by Northern blotting. After analyzing 2,000 clones, 12 clones were predominantly P-dependent, and a clone which was induced within 6 hours after P-addition, was revealed to be tissue transglutaminase (TGase). Since both an inhibitor for TGase (monodansyleadaverin) and oligodeoxynucleotide complementary to the TGase mRNA inhibit the in vitro P-induced decidual transformation. TGase is considered to be an indispensable factor in the decidual transformation of human endometrial stromal ceil. Further analysis on the other P-induced genes is now in progress.
RELATIONSHIPS BETWEEN CLINICAL / LABORATORY FINDINGS AND HISTOPATHOLOGICAL FEATURES OF P R E E C L A M P T I C P L A C E N T A E . N o b u h i s a N a g a i t, K o h W a t a n a b e ~, S a t o s h i H a y a k a w a ), K a z u o Satoh t, N o r i m i c h i Nemoto-' and I s a m u S a k u r a i 2 , 1 ; D e p t . Obstet./Grynaecol., 2;2nd Dept. Pathol. Nihon University School of Medicine, Itabashi, Tokyo, Japan Objective ; Our purpose was to compare h i s t o p a t h o l o g i c a l f i n d i n g s of p r e e c l a m p t i c p l a c e n t a e and clinical or laboratory f i n d i n g s in order to clarify p a t h o g e n e s i s and p a t h o p h y s i o l o g y of p r e e c l a m p s i a . Study Design; Review of singleton live-borne 97 cases of preeclampsia with their clinical courses and routine laboratory data. Routine H-E stain, elastic fiber stain and T U N E L s t a i n for d e t e c t i o n of a p o p t o s i s were p e r f o r m e d . R e s u l t s ; M o r e f r e q u e n t l y in e a r l y o n s e t p r e e c l a m p s i a b e f o r e 2 8 w k of g e s t a t i o n v e r s u s late o n s e t g r o u p , we o b s e r v e d h e m o r r h a g i c i n f a r c t , deciduitis, vasculitis, repair hyperplasia and increased apoptosis. In patients with positive auto-antibodies or d e c r e a s e d c o m p l e m e n t t i t e r s , we o b s e r v e d m o r e evident inflammatory changes. Conclusion ; Histopathological findings reflect clinical and laboratory findings of preeclampsia
Placenta (1996), Vol. 17 PEROXISOME P R O L I F E R A T O R REGULATES JEG-3 CHORIOCARSINOMA CELL FUNCTION. I-L MATSUO and J. F. STRAUSS HI *, Department of Obstetrics and Gynecology, Faculty of Medicine, ~ University,Kobe.Japan, * I ~ ~ Ob6"telricsand ~logy, Fal:ultyofMedicine,Pennsylvat~Ur,lvetsity,Pl'fladdphia.
USA It has recently become evident that effects of fatty acids or peroxisome pmliferators may be mediated by pemxisome pmlifleralor-activated receptors(PPARs), newly described members of the nuclear receptor s t e a m i l y at tbe level of gene ~ ' n o . To examino the h ~ that nutritional signals regulate trophoblast cell function. JEG-3 chorioearcinoma cells were teated with cl~ibric acid that stimulates PPARs.TI~ donal tran.~ormedtroph~last cell llne d i ~ y s a n ~ of the functional features of syncytiotrophoblast. We used Northern h y b f i ~ o n analysisas the primaly~ for as~ssingreslXmSebecanse the effects on multiple genes could be examined by sequenlial probing. Clofibdc add, known peroxisome prniiferator, suppressed JEO-3 cell growth in associatienwith increases in the tumor suppremor p53 protein and its mRNA. It r e ~ c ~ chi~onic gonedonq:in (CG) secredon and CG and CO/~ mR.NAin growing cells without effecting aetin mRNA. However, clofibric acid did not in&me pero~some la'oftleretion in the JEG-3 cells by alteringlevels of mRNAs for ~ proteinssuch as sterol carrier protein 2 and aeyl-CoA oxidase, and mitochondrial cholester~ side-ehein cleavage enzyme, c ~ P450see. AU-trans refinoicacid and 9-cisrelindc add irmeased CG secretionand CG c~ and CGfl mRNAs, but dofibde add blunted these sfirr~atory, effects. We condude l)thatJEG-3 cells expressPPARs md are subjectedto regulation by their stimulation; 2)that PPAR stimulation in JEG-3 cells does not promote ~ proliferation; md 3)that pemxisonneprolgeratino and retinoid differentially regulate JE.G-3 ~11 endocrine activities. It is suggested from the-~efindings that JEG-3 cells possess mechanisms to reSlX~ to r~rient cues,
THE EFFECT OF IMMORTALIZED T R O P H O B L A S T C E L L ON PGE2 S Y N T H E S I S B Y I M M O R T A L I Z E D D E C I D U A L CELL. Hiroyuki Seki, Satoru T a k e d a , K i k u m i M a t s u o k a , Y o u k o H a g a and K a t s u y u k i K i n o s h i t a , D e p a r t m e n t of O b s t e t r i c s and Gynecology, Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama, Japan P u r p o s e : The interaction of t r o p h o b l a s t and decidual c e l l s is s u p p o s e d to h a v e an i m p o r t a n t r o l e f o r i m p l a n t a t i o n a n d m a i n t e n a n c e of p r e g n a n c y . T h e e f f e c t of t r o p h o b l a s t c e l l o n PGE2 s y n t h e s i s b y d e c i d u a l c e l l w a s i n v e s t i g a t e d to e l u c i d a t e t h i s interaction. Methods : Immortalized trophoblast ( T C L ) a n d d e c i d u a l cell l i n e ( D E L ) w h i c h w e r e established by transfecting SV40 large T antigen, were u s e d in t h e f o l l o w i n g e x p e r i m e n t s , (1) D E L w a s cultured with the s u p e r n a t a n t from T C L for 24 hours. Then, PGE2 s y n t h e s i s by DEL and the a m o u n t of D N A of DEL were measured. (2) The co-culture of T C L and DEL, the cultures of T C L alone and DEL, alone were d o n e for 24 h o u r s , r e s p e c t i v e l y . T h e n , e a c h PGE2 s y n t h e s i s and the a m o u n t of D N A w a s m e a s u r e d and compared. Results : (1)The supernatant from TCL decreased PGE2 s y n t h e s i s of DEL in a dose d e p e n d e n t m a n n e r . P G E 2 s y n t h e s i s in c o - c u l t u r e of T C L a n d D E L is less than that in c u l t u r e of D E L alone. T C L had no effects on the a m o u n t of D N A of DEL in each experiment. Conclusion : The compound released from T C L d e c r e a s e d PGE2 s y n t h e s i s by DEL w i t h o u t any effect on D N A content of D E L .
RELATIONSHIPS BETWEEN CLINICAL / LABORATORY FINDINGS AND HISTOPATHOLOGICAL FEATURES OF P R E E C L A M P T I C P L A C E N T A E . N o b u h i s a N a g a i t, K o h W a t a n a b e ~, S a t o s h i H a y a k a w a ), K a z u o Satoh t, N o r i m i c h i Nemoto-' and I s a m u S a k u r a i 2 , 1 ; D e p t . Obstet./Grynaecol., 2;2nd Dept. Pathol. Nihon University School of Medicine, Itabashi, Tokyo, Japan Objective ; Our purpose was to compare h i s t o p a t h o l o g i c a l f i n d i n g s of p r e e c l a m p t i c p l a c e n t a e and clinical or laboratory f i n d i n g s in order to clarify p a t h o g e n e s i s and p a t h o p h y s i o l o g y of p r e e c l a m p s i a . Study Design; Review of singleton live-borne 97 cases of preeclampsia with their clinical courses and routine laboratory data. Routine H-E stain, elastic fiber stain and T U N E L s t a i n for d e t e c t i o n of a p o p t o s i s were p e r f o r m e d . R e s u l t s ; M o r e f r e q u e n t l y in e a r l y o n s e t p r e e c l a m p s i a b e f o r e 2 8 w k of g e s t a t i o n v e r s u s late o n s e t g r o u p , we o b s e r v e d h e m o r r h a g i c i n f a r c t , deciduitis, vasculitis, repair hyperplasia and increased apoptosis. In patients with positive auto-antibodies or d e c r e a s e d c o m p l e m e n t t i t e r s , we o b s e r v e d m o r e evident inflammatory changes. Conclusion ; Histopathological findings reflect clinical and laboratory findings of preeclampsia
Placenta (1996), Vol. 17 PEROXISOME P R O L I F E R A T O R REGULATES JEG-3 CHORIOCARSINOMA CELL FUNCTION. I-L MATSUO and J. F. STRAUSS HI *, Department of Obstetrics and Gynecology, Faculty of Medicine, ~ University,Kobe.Japan, * I ~ ~ Ob6"telricsand ~logy, Fal:ultyofMedicine,Pennsylvat~Ur,lvetsity,Pl'fladdphia.
USA It has recently become evident that effects of fatty acids or peroxisome pmliferators may be mediated by pemxisome pmlifleralor-activated receptors(PPARs), newly described members of the nuclear receptor s t e a m i l y at tbe level of gene ~ ' n o . To examino the h ~ that nutritional signals regulate trophoblast cell function. JEG-3 chorioearcinoma cells were teated with cl~ibric acid that stimulates PPARs.TI~ donal tran.~ormedtroph~last cell llne d i ~ y s a n ~ of the functional features of syncytiotrophoblast. We used Northern h y b f i ~ o n analysisas the primaly~ for as~ssingreslXmSebecanse the effects on multiple genes could be examined by sequenlial probing. Clofibdc add, known peroxisome prniiferator, suppressed JEO-3 cell growth in associatienwith increases in the tumor suppremor p53 protein and its mRNA. It r e ~ c ~ chi~onic gonedonq:in (CG) secredon and CG and CO/~ mR.NAin growing cells without effecting aetin mRNA. However, clofibric acid did not in&me pero~some la'oftleretion in the JEG-3 cells by alteringlevels of mRNAs for ~ proteinssuch as sterol carrier protein 2 and aeyl-CoA oxidase, and mitochondrial cholester~ side-ehein cleavage enzyme, c ~ P450see. AU-trans refinoicacid and 9-cisrelindc add irmeased CG secretionand CG c~ and CGfl mRNAs, but dofibde add blunted these sfirr~atory, effects. We condude l)thatJEG-3 cells expressPPARs md are subjectedto regulation by their stimulation; 2)that PPAR stimulation in JEG-3 cells does not promote ~ proliferation; md 3)that pemxisonneprolgeratino and retinoid differentially regulate JE.G-3 ~11 endocrine activities. It is suggested from the-~efindings that JEG-3 cells possess mechanisms to reSlX~ to r~rient cues,
THE EFFECT OF IMMORTALIZED T R O P H O B L A S T C E L L ON PGE2 S Y N T H E S I S B Y I M M O R T A L I Z E D D E C I D U A L CELL. Hiroyuki Seki, Satoru T a k e d a , K i k u m i M a t s u o k a , Y o u k o H a g a and K a t s u y u k i K i n o s h i t a , D e p a r t m e n t of O b s t e t r i c s and Gynecology, Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama, Japan P u r p o s e : The interaction of t r o p h o b l a s t and decidual c e l l s is s u p p o s e d to h a v e an i m p o r t a n t r o l e f o r i m p l a n t a t i o n a n d m a i n t e n a n c e of p r e g n a n c y . T h e e f f e c t of t r o p h o b l a s t c e l l o n PGE2 s y n t h e s i s b y d e c i d u a l c e l l w a s i n v e s t i g a t e d to e l u c i d a t e t h i s interaction. Methods : Immortalized trophoblast ( T C L ) a n d d e c i d u a l cell l i n e ( D E L ) w h i c h w e r e established by transfecting SV40 large T antigen, were u s e d in t h e f o l l o w i n g e x p e r i m e n t s , (1) D E L w a s cultured with the s u p e r n a t a n t from T C L for 24 hours. Then, PGE2 s y n t h e s i s by DEL and the a m o u n t of D N A of DEL were measured. (2) The co-culture of T C L and DEL, the cultures of T C L alone and DEL, alone were d o n e for 24 h o u r s , r e s p e c t i v e l y . T h e n , e a c h PGE2 s y n t h e s i s and the a m o u n t of D N A w a s m e a s u r e d and compared. Results : (1)The supernatant from TCL decreased PGE2 s y n t h e s i s of DEL in a dose d e p e n d e n t m a n n e r . P G E 2 s y n t h e s i s in c o - c u l t u r e of T C L a n d D E L is less than that in c u l t u r e of D E L alone. T C L had no effects on the a m o u n t of D N A of DEL in each experiment. Conclusion : The compound released from T C L d e c r e a s e d PGE2 s y n t h e s i s by DEL w i t h o u t any effect on D N A content of D E L .