Life Sciences, Vol. Printed in the U S A

51, pp. 817-822

Pergamon Press

HUMAN

CHORIONIC GONADOTROPIN CAUSES AN ESTROGEN-MEDIATED INDUCTION OF RAT OVARIAN CANBONYL REDUCTASE

Norlhlsa I n a ~ u 1, N i r o I n a b a 2, T e t s u o S a t o h U, Tomoko F u J i i I Department of Pharmacology, Teikyo University School of Medicine. Department of Pharmacology a n d T o x i c o l o g y ~, T o k y o C o l l e g e o f P h a r m a c y . Laboratory of_Biochemical Pharmacology and Blotoxicology a, Faculty of Pharmaceutical Sciences, Chlba University. (Received in final form June 30, 1992)

SUMMARy

We e a r l i e r reported t h a t human c h o r l o n l c gonadotropin (hCG) s t i m u l a t e s rat ovarian carbonyl reductase (CR) a c t i v i t y and content, and that estrogen enhances the stimulatory effect. The p r e s e n t s t u d y was p e r f o r m e d t o d e t e r m i n e t h e mode o f a c t i o n of the gonadotropln. Cyclohexlmlde (CHX) a n d a c t l n o m y c l n D (AD) w e r e g i v e n t o e s t r a d l o l - p r e t r e a t e d i m m a t u r e r a t s 6 h b e f o r e hCG t r e a t m e n t . The e n z y m e activity was measured with three substrates, and the enzyme content was determined by the method of Westernblot analysis using anti-rat o v a r i a n CR a n t i serum as the first antibody. Both protein lnhibitors significantly prevented hCG f r o m i n c r e a s i n g the enzyme activity and content in estradiol-pretreated ovary. These results indicate t h a t r a t o v a r i a n CR i s i n d u c e d b y LH v i a t h e a c t i o n of estrogen. Rat ovaries contain t w o l s o e n z y m e s (CR1, CR2) of carbonyl reductase (CR, EC 1 . 1 . 1 . 1 8 4 ) and their enzymologlcal and immunochemlcal properties have been reported [1,2]. Both isoenzymes catalyze the conversion of 13,14-dihydro-15ketoprostaglandln F2a (15KD-PGF2a), a physiologically inactive PG, to the bloactlve 13,14-dlhydro-prostaglandln F2a (13,14H2PGF2a) [3]. The CR a c t i v i t y in rat ovary was significantly increased before ovulation, following the surge of lutelnlzlng hormone (LH) and constantly decreased during pregnancy and pseudopregnancy [4,5]. Furthermore, in immature rats the activity and content of CR w a s m a r k e d l y i n c r e a s e d by human chorlonic gonadotropln (hCG), and estradiol-178 (E 2) potentiated the stimulatory effect o f hCG [ 6 , 7 ] . Some a n t l e s t r o g e n s , such as tamoxlfen, clomlfen and nafoxidlne, inhibited not only ovarian CR 1Address: Medicine,

Dept. Pharmacol., Teikyo 2-11-1 Kaga, Itabashl-ku,

Copyright

Univ. School of Tokyo 173, Japan.

0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights

reserved.

818

Regulation of Ovarian Carbonyl Reductase

Vol. 51, No. Ii, 1992

activity but also ovulation in adult female rats, and those inhibitions were restored by treatment w l t h hCG a n d lutelnizing hormone releasing h o r m o n e (LH-RH) [8,9]. These antlestrogens completely antagonized the action of E2 on ovarian CR I n i m m a t u r e rats [7]. These findings indicate that ovarian CR m ay be regulated via the hypothalamo-pltultary-ovarlan axis, especially b y LH a n d E 2 , a n d i n t i m a t e l y involved In ovarian function. However, It remains unclear that the stlmulatory effect of hCG on ovarian CR i n E 2 - p r e t r e a t e d rats ls due to the enzyme protein synthesis, a s we h a v e n o t c o n f i r m e d it by using protein synthesis inhlbltors. The present study was undertaken by using cyclohexlmlde (CHX), a cytoplasmic protein synthesis inhibitor, and actlnomycin D ( A D ) , a D N A - p r l m e d RNA p o l y m e r a s e inhibitor, in attempt to establish the effect o f hCG o n o v a r l a n CR.

MATERIALS

AND METHODS

Animals. Immature female Wlstar-KY strain rats (20 days of age) were purchased f r o m SLC ( S h l z u o k a , Japan) and housed In group cages (4-5 rats/cage) under controlled conditions of light (12 h o n , 12 h o f f ) and temperature (23 °C). Food and water were always available. Hormone and drug treatments were carried out from 26 days of age. Chemicals. Cyclohexlmide (CHX), 4-benzoylpyridlne (4BP) and menadione were obtained f r o m Wako P u r e C h e m i c a l Industries Co. (Osaka, Japan), and nlcotinamlde adenine dlnucleotlde phosphate (reduced f o r m , NADPH) w a s f r o m O r i e n t a l Y e a s t Co. ( O s a k a , Japan). Actinomycln D (AD) a n d E 2 w e r e p u r c h a s e d from Sigma Chemical Co. (MO, USA). Tritlated 15KD-PGF2a (80 C1/mmol) was obtained from Amersham Int. plc (Buckinghamshire, UK) a n d a u t h e n t i c 15KD-PGF2a was from Upjohn Pharmaceuticals Ltd. (MI, USA). Authentic 13,14H 2 -PGF2a was kindly provided b y Ono Pharmaceutical Co. (Osaka, Japan). HCG w a s purchased from Telkoku Hormone Mfg (Tokyo, Japan). Other chemicals of reagent grade were obtained from Wako Pure Chemlcal Industries and Blo-Rad (Tokyo, Japan). Drug treatments. E2 was dissolved in sesame oil and l0 ug/rat were subcutaneously administered to Immature animals once a day for 3 days starting o n 26 d a y s o f a g e . CHX ( 3 0 0 u g / r a t ) and AD (100 ug/rat), respectively, were dissolved In 0.9% saline and subcutaneously administered to the animals a t 0 9 0 0 h o n 28 days of age. HCG (10 IU/rat) was dissolved in 0.9% saline and subcutaneously administered a t 1 5 0 0 h o n 28 d a y s o f a g e . Control rats were given vehicle alone. All immature rats were sacrificed at 0900 h o n 29 d a y s o f a g e a n d t h e o v a r i e s of each rat were immediately isolated and weighed. Enzyme assay. The ovaries were homogenized I n 10 mM p h o s p h a t e buffer (pH 6 . 5 ) containing 1 mM d l t h l o t h r e l t o l , 0 . 5 mM EDTA a n d 0 . 1 5 4 M KC1, a n d t h e h o m o g e n a t e was centrifuged a t 9 0 0 0 x g f o r 20 min at 4 °C. The 9000 xg supernatant was used as source o f CR activity. CR activity was measured by uslng three substrates (15KD-PGF2., 4BP a n d m e n a d i o n e ) as previously described [6,7]. The reduction o f 4BP (1 mM) a n d m e n a d l o n e ( 0 . 2 mM) w a s d e t e r m i n e d spectrophotometrlcally in a total v o l u m e o f 1 ml c o n t a i n i n g 100 mM p h o s p h a t e buffer (pH 6 . 5 ) , 0 . 1 mM NADPH a n d 0 . 3 m l o f 9 0 0 0 xg

Vol. 51, No. ii, 1992

Regulation of Ovarian Carbonyl Reductase

819

supernatant a ~ the enzyme activity was expressed as units/mg protein x 10 ~ ° o f NADPH o x i d i z e d . The reduction of 15KD-PGF 2(283 pmoles/0.05 uCi) was measured radiochemlcally in a tota~ volume o f 0 . 5 ml c o n t a i n i n g 1 0 0 mM p h o s p h a t e buffer (pH 7 . 0 ) , 1 mM NADPH and 0.1 m l o f 9O00 x g supernatant and the enzyme activity was expressed as pmoles/mg protein/15 mln of 13,142 PGF2a formed. The protein concentration in the supernatant was determined by the method of Lowry et al. [10], uslng bovine serum albumin as a standard. D e t e r m i n a t i o n o f enzyme c o n t e n t . The c o n t e n t o f CR In t h e o v a r i a n s u p e r n a t a n t was d e t e r m i n e d by Westernblot-PAP a n a l y s i s a f t e r SDSPAGE (10% p o l y a c r y l a m l d e g e l ) u s i n g p u r i f i e d r a t o v a r i a n CR as a standard [ 7 ] . The amount o f t h e enzyme p r o t e i n on t h e blot was measured by d e n s i t o m e t r y , and t h e s t a n d a r d c u r v e was p r e p a r e d in t h e range from 4.46 t o 17.85 ng. Statistical a n a l y s i s . S t a t i s t i c a l s i g n i f i c a n c e of the data d e t e r m i n e d by t h e S t u d e n t t t e s t and a n a l y s i s o f v a r i a n c e a t 95% c o n f i d e n c e l e v e l by t h e D u n n e t t ' s m u l t l p l e range t e s t .

was the

RESULTS

Table I shows changes in the ovarian and uterine weights, and in the content of ovarlan CR a f t e r treatment wlth the protein synthesis inhibitors CHX a n d AD. The ovarian weight was significantly increased to 1.8-fold of control levels by coadministration of E 2 a n d hCG, b u t t h e s t l m u l a t o r y effect was significantly i n h i b i t e d by both CHX and AD, 68 and 65%, r e s p e c t i v e l y . Both inhlbltors also negatively affected the o v a r i a n weight gain of r a t s t r e a t e d with E2 o r hCG a l o n e . Although both CHX and AD produced a f a l l i n the u t e r i n e w e i g h t i n the a b s e n c e o f hCG and E2, no s i g n i f i c a n t i n h i b i t i o n by CHX was o b s e r v e d i n E 2 - t r e a t e d r a t s , whereas t h e u t e r i n e w e i g h t g a l n by E2 was p a r t i a l l y a b o l i s h e d by AD t r e a t m e n t . The content of o v a r i a n CR from E2-, hCG- and E 2 - h C G - t r e a t e d r a t s was greatly (7.5-, 9 . 4 - and 1 4 . 5 - f o l d , r e s p e c t i v e l y ) i n c r e a s e d r e l a t i v e to c o n t r o l l e v e l s , and both p r o t e i n s y n t h e s i s i n h l b l t o r s p a r t i a l l y prevented the i n c r e a s e i n t h e enzyme by E2-hCG treatment,CHX b e i n g more p o t e n t . The r e s u l t s i n d i c a t e t h a t the hCG-induced increase of CR i n r a t o v a r y p r e t r e a t e d w l t h E2 I s mediated by protein synthesls. Changes in ovarian CR a c t i v i t y using three substrates are shown in FIG. 1 . We h a v e previously reported that estrogen potentiates the stimulatory effect o f hCG o n o v a r i a n CR a c t i v i t y and content in immature rats [6,7]. As e x p e c t e d , CHX a n d AD significantly inhibited thls potentiation. The decrease in ovarian CR a c t i v i t y by these two protein synthesis tnhibitors was correlated well wlth that in ovarian CR c o n t e n t . Especially, the reduction o f 1 5 K D - P G F 2 a a n d 4BP a f t e r treatment wlth E2-hCG was significantly decreased t o m o r e t h a n 50% b y b o t h CHX a n d AD.

DISCUSSION

We

have a l r e a d y d e m o n s t r a t e d t h a t e s t r o g e n p o t e n t i a t e s

the

820

Regulation of Ovarian Carbonyl Reductase

TABLE

Vol. 51, No. ii, 1992

I

Changes in ovarian and uterine weights, and ovarian carbonyl reductase c o n t e n t in i m m a t u r e r a t s a f t e r treatment with protein synthesis inhibitors Tissue weight(mg) Enzyme content Ovary Uterus (ug/mg prot.) None(Control) CHX AD

14.0±2.19 13.0±0.71 11.0±0.71

100.4±17.95 52.8±3.97 a 49.8±3.52 a

E2 +CHX +AD

19.6±0.75 a 11.3±1.44 f 12.5±0.50 f

152.7±4.80 a 141.3±22.29 I03.3±3.94 f

hCG +CHX +AD

22.0±0.68 a 15.5±0.89 f 13.3±1.11 f

E2+hCG +CHX +AD

25.6±1.72 c 17.4±0.90 e 16.6±0.81 f

64.8±2.66 61.8±2.59 45.8±4.75a, e 141.0±4.87 124.7±4.00 d I06.6±5.56 f

2.94±0.455 2.34±0.523 0.55±0.378 a 22.08±4.833 b 4.17±0.86 e 1 . 2 6 ± 0 . 2 2 7 b,e 27.52±4.260 c 4.98±2.210 f 17.45±4.463 a 42.66±1.492 c 9 . 8 2 ± 1 . 4 9 8 a,f 21.36±9.034

~ ( 1 0a g e .p g /Cr aHt X) ( 3 0 0 wpags / r ga ti )v e n

s.c. f o r 3 d a y s f r o m 26 days and AD(IO0 ug/rat), respectively, were given s.c. a t 0 9 0 0 h o f 28 d a y s o f a g e a n d h C G ( 1 0 I U / rat) was given s.c. at 1500 h o f 28 d a y s o f a g e . The ovaries and uteri w e r e r e m o v e d a t 0 9 0 0 h o f 29 d a y s o f age. Each. value shows the meanZSE of 4-8 immature rats. ap

Human chorionic gonadotropin causes an estrogen-mediated induction of rat ovarian carbonyl reductase.

We earlier reported that human chorionic gonadotropin (hCG) stimulates rat ovarian carbonyl reductase (CR) activity and content, and that estrogen enh...
343KB Sizes 0 Downloads 0 Views