PROSTAGLANDINS

P K O S T A G L A N D I N F AND P R O G E S T E R O N E S E C R E T I O N BY P O R C I N E E N D O M E T R I U M AND C O R P U S L U T E U M IN V I T R O

Charles E. Patek and John Watson* D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of S t r a t h c l y d e , Glasgow C_r4 0NR, Scotland

Abstract S l i c e s of p o r c i n e e n d o m e t r i u r n a n d c o r p u s l u t e u m t i s s u e o b t a i n e d f r o m m a t u r e s o w s t h r o u g h o u t the l u t e a l p h a s e of t h e o e s t r o u s c y c l e w e r e i n c u b a t e d in c u l t u r e m e d i u m which was a n a l y s e d at r e g u l a r i n t e r v a l s o v e r a p e r i o d of 8 h o u r s f o r p r o s t a g l a n d i n F a n d p r o g e s t e r o n e . Prostaglandin F secretion was greatest by endometrium obtained during the m i d III to l a t e I l u t e a l s t a g e of t h e c y c l e a n d the i n c r e a s e d l e v e l s s e c r e t e d b y t h i s t i s s u e w e r e p a r a l l e l e d b y h i g h l e v e l s of s e c r e t i o n from corpus luteum tissue. The a d d i t i o n of i n d o m e t h a c i n (10 fLg/rnl) to the c u l t u r e m e d i u m c o m p l e t e l y a b o l i s h e d p r o s t a g l a n d i n F s e c r e t i o n b y b o t h e n d o m e t r i u m a n d l u t e a l t i s s u e i n d i c a t i n g t h a t the h i g h l e v e l s of the p r o s t a g l a n d i n w e r e due t o s y n t h e s i s . P r o g e s t e r o n e s e c r e t i o n b y the corpus luteum was maximal from early luteal tissue and had declined to c o n s i d e r a b l y l o w e r l e v e l s b y late stage t i s s u e when p r o s t a g l a n d i n s e c r e t i o n was g r e a t e s t . T h e p o s s i b l e p h y s i o l o g i c a l s i g n i f i c a n c e of luteal prostaglandin F secretion is discussed.

Acknowledgement s T h e a u t h o r s w i s h to t h a n k D r . J . E . P i k e of the U p j o h n C o m p a n y for the supply of P G F z u and Professor E . W . H o r t o n of the Department of Pharmacology, Edinburgh University for the supply of antibody to PGFza. This w o r k w a s supported by grants f r o m the Medical Research Council and Science Research Council ( C E P is the recipient of a C A S E student ship in conjunction with ICI Ltd. ) for which we are extremely grateful. * To whom correspondence should be addressed.

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Introduction Exogenously administered prostaglandin Fza (PGFza) has been shown to be lute olytic thereby causing the cessation of progesterone s e c r e t i o n a n d s t r u c t u r a l r e g r e s s i o n of t h e c o r p u s l u t e u r n in s e v e r a l s p e c i e s i n c l u d i n g t h e cow, g u i n e a p i g , h a m s t e r , p i g , r a b b i t , r a t a n d i n p a r t i c u l a r t h e e w e in w h i c h s p e c i e s i t i s a l s o t h e n a t u r a l l u t e o l y s i n s e c r e t e d b y t h e u t e r u s ( 1 ) . H y s t e r e c t o m y w i l l p r o l o n g t h e l i f e s p a n of the corpus luteum in those species in which PGFZa is luteolytic thereby i m p l i c a t i n g t h e u t e r u s a s t h e m a j o r s o u r c e of P G F 2 a , a n d i n f a c t i n t h e Sheep it has been demonstrated that uterine PGF2a is transferred from the uterine vein to the ovarian artery by a countercurrent mechanism (2). Humans and primates however have a different utero-ovarian v a s c u l a t u r e f r o m m o s t a n i m a l s a n d h y s t e r e c t o m y doe s n o t p r o l o n g t h e l i f e s p a n of t h e c o r p u s l u t e u m (1) n o r d o e s e x o g e n o u s P G F z a c a u s e luteolysis. POFza of ovarian origin may play an important role in luteolysis in humans and primates as it has been demonstrated that the c o r p o r a l u t e a of b o t h s p e c i e s c o n t a i n c o n s i d e r a b l e a m o u n t s of P G F z a (1) a n d it h a s r e c e n t l y b e e n d e m o n s t r a t e d t h a t l u t e o l y s i s c a n b e i n d u c e d in h u m a n s b y t h e d i r e c t i n j e c t i o n of P G F Z a i n t o t h e c o r p u s l u t e u m (3). The present study was undertaken to determine whether the s e c r e t i o n of P G F f r o m t h e p o r c i n e e n d o m e t r i u m i n v i t r o w a s s i m i l a r t o t h e k n o w n p a t t e r n of u t e r o - o v a r i a n v e n o u s p l a s m a l e v e l s of P G F in v i v o t h r o u g h o u t t h e l u t e a l p h a s e of t h e o e s t r o u s c y c l e (4) a n d f u r t h e r to determine whether any such changes were accompanied by similar c h a n g e s in t h e s e c r e t i o n of P G 1v f r o m t h e c o r p u s l u t e u m i n v i t r o . Materials and Methods

M a t e r i a l s : A l l c h e m i c a l s w e r e of A R g r a d e , a n d s o l v e n t s w e r e f u r t h e r p u r i f i e d b y d i s t i l l a t i o n i n a n a l l g l a s s s y s t e m . [ 1, 2, 6, 7 - 3H] P r e g n 4 - e r i e - 3 , 2 0 - d i o n e ( p r o g e s t e r o n e 87 C i / m M o l e ) a n d [5, 6, 8, l l , 12, 14, 15 (n) - 3H] P r o s t a g l a n d i n - l P 2 a (175 C i / m M o l e ) w e r e o b t a i n e d f r o m t h e Radiochemical Centre, Amersham. M e d i u m 199 ( t y p e T C 45) w a s obtained from Wellcome Reagents Ltd., Beckenham, Kent. Buffer for radioimmunoassays ( G P B S ) c o n s i s t e d of p h o s p h a t e b u f f e r ( 0 . 1 M, p H 7 . 4 ) c o n t a i n i n g s o d i u m c h l o r i d e ( 0 . 1 5 M) a n d g e l a t i n ( 0 . 1 ~o). Scintillator for radioimmunoassays c o n s i s t e d of P P O (2 g) a n d P O P O P ( 0 . 1 g, K o c h - L i g h t L a b o r a t o r i e s L t d . ) d i s s o l v e d i n t o l u e n e (800 m l ) a n d T r i t o n X - 1 0 0 (200 m l , H o p k i n s & W i l l i a m s L t d . ) . Radioactive s a m p l e s w e r e c o u n t e d u s i n g a N u c l e a r C h i c a g o I s o c a p 300 L i q u i d S c i n t illation counter. Porcine endometrium and luteal tissue: Intact ovaries plus uterus obtained from mature sows immediately after slaughter in the local abattoir were chilled and transported to the laboratory. The tissue

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w a s o b t a i n e d f r o m f o u r s e p a r a t e g r o u p s ( A B C D ) of a n i m a l s s u c h t h a t t h e r e w a s t i s s u e f r o m a s w i d e a s p r e a d of the l u t e a l p h a s e o f t h e c y c l e a s p o s s i b l e in e a c h e x p e r i m e n t a l g r o u p . In e x p e r i m e n t s B a n d C, corpora lutea and uteri were obtained from the same animals. The c o r p o r a lutea w e r e c a r e f u l l y e x c i s e d f r o m the o v a r y , t r i m m e d f r e e of c o n n e c t i v e t i s s u e , w e i g h e d , dated as d e s c r i b e d b e l o w and f i n a l l y s l i c e d to a t h i c k n e s s of 0 . 4 rnm u s i n g a hand m i c r o t o m e , E n d o m e t r i u m c u r r e t i n g s f r o m t h e m i d h o r n r e g i o n of t h e u t e r u s w e r e obtained from the myometriurn. T h e s t a g e of t h e o e s t r o u s c y c l e w a s a s s e s s e d b y c a r e f u l e x a m i n a t i o n of t h e c o r p o r a l u t e a , a n d t h e s e w e r e d i v i d e d i n t o e a r l y ( d a y s 3 - 6 ) m i d ( d a y s 7 - 1 5 ) a n d l a t e ( d a y 16+) c o r p o r a l u t e a on t h e b a s i s of v i s i b l e a n d h i s t o l o g i c a l e x a m i n a t i o n a s d e s c r i b e d p r e v i o u s l y (5). It w a s f u r t h e r p o s s i b l e to subdivide both the e a r l y and m i d c o r p o r a l u t e a into e a r l y I, I I , I l l o r m i d I, It, III t i s s u e . In t h e c a s e of t h e e a r l y l u t e a l t i s s u e t h i s w a s a s s e s s e d on t h e s i z e of t h e c e n t r a l b l o o d c l o t , w h i l e i n t h e c a s e of t h e m i d l u t e a l t i s s u e t h i s w a s a s s e s s e d on t h e b a s i s of t h e v a s c u l a r i t y of t h e t i s s u e a n d t h e i n c r e a s i n g n u m b e r of p y c n o t i c n u c l e i in the a g e i n g lutein c e l l . L a t e c o r p o r a lutea w e r e subdivided into two s t a g e s , l a t e I o r II on t h e b a s i s of t h e i r d e c r e a s i n g s i z e a n d t e n d e n c y t o t u r n y e l l o w / w h i t e clue t o i n c r e a s i n g a m o u n t s of u n u s e d c h o l e s t e r o l
esters.

T i s s u e i n c u b a t i o n : T i s s u e s a m p l e s (50 rag) i n q u a d r u p l i c a t e w e r e i n c u b a t e d i n s i l a n i s e d g l a s s c o n i c a l f l a s k s (25 m l ) c o n t a i n i n g m e d i u m 199 (2 m l ) p l u s t h e a n t i b i o t i c s p e n i c i l l i n (6 m g / m l ) a n d s t r e p t o m y c i n s u l p h a t e (10 m g / m l ) . I n c u b a t i o n s w e r e c a r r i e d out a t 3 7 ° C w i t h s h a k i n g , a n d t h e m e d i u m w a s g a s s e d f o r 10 s e c o n d s e v e r y h o u r w i t h o x y g e n / C O 2 ( 9 5 : 5 ) . T h e i n c u b a t i o n s w e r e c a r r i e d out f o r a t o t a l o f 8 h o u r s du~ing which the m e d i u m was r e n e w e d e v e r y two h o u r s , and m e d i u m f r o m the i n c u b a t i o n s was s t o r e d at - Z 0 ° C . E x t r a c t i o n s with a p p r o p r i a t e s o l v e n t s ( d e s c r i b e d below) for p r o g e s t e r o n e and p r o s t a g l a n d i n F w e r e p e r f o r m e d w i t h i n 24 h o u r s , a n d t h e e x t r a c t s w e r e then s t o r e d at -Z0°C until a n a l y s e d by r a d i o i m r n u n o a s s a y . In t h e e x p e r i m e n t w h e r e i n d o m e t h a c i n w a s u s e d , t h i s w a s p1~esent i n t h e i n c u b a t i o n m e d i u m a t a c o n c e n t r a t i o n of 10 ~ g / m l . P r o g e s t e r o n e a s s a y : P r o g e s t e r o n e w a s m e a s u r e d b y a c o m b i n a t i o n of a n e x t r a c t i o n t e c h n i q u e (5) w h i c h d e p e n d s on t h e s e l e c t i v e e x t r a c t i o n o f p r o g e s t e r o n e w i t h s p e c i f i c b a t c h e s of l i g h t p e t r o l e u m ( b . p . 4 0 - 5 0 °C) a n d t h e r a d i o i m m u n o a s s a y m e t h o d of T h o r n e y c r o f t a n d S t o n e (6). T h e a n t i b o d y which was h i g h l y s p e c i f i c for p r o g e s t e r o n e was r a i s e d in r a b b i t s a g a i n st 11 a - h y d r o x y p r o g e s t e r o n e - 11 - h e m i s u c c i n a t e - b o v i n e serum albumin.

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T h e p r o g e ste r o n e c o n c e n t r a t i o n s we r e e s t i m a t e d b y m e a n s of a s t a n d a r d c u r v e and c o r r e c t e d for w a t e r b l a n k s ( c o n s i s t e n t l y l e s s than 5 pg) and e x t r a c t i o n l o s s e s . T h e c o e f f i c i e n t of v a r i a t i o n f o r a c o n s t a n t a m o u n t of progesterone in a standard sample w a s 8 . 2 % b e t w e e n assays (reproducibility) and 6 . 1 % within assays (precision). P r o s t a g l a n d i n F a s s a . y : I n c u b a t i o n s a m p l e s (0.5 m l ) in t r i p l i c a t e w e r e e x t r a c t e d w i t h p e t r o l e u m e t h e r (5 m l ; b . p . 4 0 - 5 0 ° C ) t o r e m o v e non p o l a r l i p i d s , and the r e m a i n i n g a q u e o u s solution c o n t a i n i n g p r o s t a g l a n d i n s w a s a c i d i f i e d t o pH 3 . 0 w i t h o x a l i c a c i d . T h i s w a s t h e n e x t r a c t e d w i t h 3 x 3 m l of e t h y l a c e t a t e , t h e e x t r a c t s p o o l e d , t h e s o l v e n t r e m o v e d in v a c u o a n d t h e r e s i d u e d i s s o l v e d i n e t h a n o l (2 m l ) . Prostaglandin recovery from this extraction procedure was consistently of t h e o r d e r of 9 5 % . A l i q u o t s of t h e e t h a n o l s o l u t i o n w e r e a s s a y e d f o r p r o s t a g l a n d i n le by r a d i o i m m u n o a s s a y using an antibody raised against P G F 2 a in rabbit, kindly supplied by Professor E° W. Horton, and exhibiting negligible cross-reaction against other prostaglandins except with which it cross reacts 100%.

PGFIa

S t a n d a r d s of P G F z a in t r i p l i c a t e a n d u n k n o w n s in e t h a n o l w e r e a d d e d to a p p r o p r i a t e a s s a y t u b e s a n d t h e s o l v e n t r e m o v e d i_~nv a c u o . A solution of a n t i b o d y (100 ~1; d i l u t e d 1:2700) in G P B S b u f f e r w a s a d d e d to e a c h t u b e a l o n g w i t h a s o l u t i o n of t r i t i a t e d P G l e 2 a ( 0 . 0 2 ~Ci i n 100 ~1 G P B S b u f f e r ) a n d 500 ~tl of G P B S b u f f e r . The tubes were i n c u b a t e d f o r 1 . 5 h o u r s i n an i c e b a t h a n d a s o l u t i o n (100 ~1) of g e l a t i n (0.4%) i n G P B S b u f f e r t h e n a d d e d . F r e e p r o s t a g l a n d i n w a s separated f r o m a n t i b o d y b o u n d m a t e r i a l by t h e a d d i t i o n of d e x t r a n / c h a r c o a l (500 ~I of G P B S buffer containing 4 m g / m l Norit A charcoal and O. 25 m g / m l dextran T. 70) followed by vigorous mixing, incubation for 10 rain at 4 ° C and finally centrifugation at 3000 rprn for 3 minutes at 4°C. A n aliquot of the supernatant (500 ~i) containing the antibody bound prostaglandin w a s immediately r e m o v e d and added to a counting vial containing 10 m l scintillator. Radioactivity w a s m e a s u r e d in the scintillation counter, an assay curve constructed f r o m the P G F z a standards, and the concentration of P G F in the u n k n o w n s calculated f r o m this. T h e P G F concentrations w e r e corrected f r o m m e d i u m blanks (consistenly less than i0 pg) and extraction losses. T h e coefficient of variation for a constant a m o u n t of P G F 2 a in a standard sample w a s 4 . 0 % between a s s a y s ( r e p r o d u c i b i l i t y ) a n d 5. 1% w i t h i n a s s a y s ( p r e c i s i o n ) . Results T a b l e I s h o w s t h e t e m p o r a l s e c r e t i o n of P G F ( n g / 1 0 0 m g t i s s u e ) a t t w o h o u r l y i n t e r v a l s o v e r a p e r i o d of 8 h o u r s b y p o r c i n e e n d o m e t r i u m i n v i t r o f r o m t h r e e s e p a r a t e g r o u p s (B, C , D ) of s o w s . T o t a l p r o d u c t i o n of P O l e p e r 100 m g t i s s u e / 8 h o u r s i s s h o w n i n t h e h i s t o g r a m s in F i g . 1. 100 J U L Y 1976 V O L . 12 N O . 1

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R e l a t i v e l y l o w l e v e l s of P G F w e r e p r o d u c e d b y t h e e n d o m e t r i u m up to t h e m i d II l u t e a l p h a s e of t h e o e s t r o u s c y c l e , b u t b o t h m i d III a n d l a t e I l u t e a l t i s s u e s e c r e t e d h i g h l e v e l s of p r o s t a g l a n d i n s w h i c h h a d d r o p p e d c o n s i d e r a b l y b y t h e l a t e II l u t e a l s t a g e .
P G F secretion (ng/lO0 m g tissue) by porcine corpus luteum tissue in vitro under the s a m e conditions as above with two of the s a m e groups (B, C) of s o w s and one other (group A) is s h o w n in Table II. Total PGF production (ng/100 mg tissue/8hr) i s s h o w n in F i g . Z, a l o n g with the e s t i m a t e d p r o d u c t i o n p e r o v a r y ( r i g / o v a r y / 8 h r ) . T h i s l a t t e r f i g u r e w a s c a l c u l a t e d by a s s e s s i n g the a v e r a g e w e i g h t of e a c h c o r p u s i u t e u m i n t h e o v a r y a n d m u l t i p l y i n g t h i s b y t h e n u m b e r of c o r p o r a l u t e a . It w a s f e l t w o r t h w h i l e to i n c l u d e t h i s e s t i m a t e d f i g u r e to g i v e s o m e i d e a of t o t a l o v a r i a n p r o d u c t i o n a l t h o u g h i t i s o b v i o u s l y p r o n e to s o m e e r r o r . It s h o u l d a l s o be b o r n e in m i n d t h a t t i s s u e s a m p l e s w e r e f r o m d i f f e r e n t a n i m a l s w i t h v a r y i n g n u m b e r s of c o r p o r a l u t e a a n d t h a t t h e c o r p o r a l u t e a t e n d e d to b e s m a l l e r i n t h e l a t e s t a g e of t h e c y c l e . T h i s c o u l d p o s s i b l y a c c o u n t f o r t h e a p p a r e n t l a c k of c o r r e l a t i o n b e t w e e n t h e s e c r e t i o n / 1 0 0 m g t i s s u e a n d s e c r e t i o n / o v a r y d u r i n g t h e l a t e I s t a g e of t h e c y c l e i n e x p e r i m e n t s A a n d B. A p a r t f r o m the r e l a t i v e l y h i g h P G F s e c r e t i o n f r o m t h e e a r l y II l u t e a l t i s s u e i n e x p e r i m e n t s A a n d C, the p a t t e r n of s e c r e t i o n of P G F v e r y c l o s e l y p a r a l l e l e d t h a t f r o m the e n d o m e t r i u m i n t h a t m a x i m u m s e c r e t i o n of P G F o c c u r r e d f r o m t h e c o r p u s l u t e u m d u r i n g t h e m i d III t o l a t e I l u t e a l p h a s e of t h e o e s t r o u s cycle. T h e h i g h l e v e l of P G F s e c r e t i o n b y t h e e a r l y l I l u t e a l t i s s u e c o u l d w e l l be a s s o c i a t e d w i t h t h e c e n t r a l b l o o d c l o t i n t h e l u t e a l t i s s u e w h i c h is difficult to r e m o v e .

P r o g e s t e r o n e s e c r e t i o n by t h e c o r p u s l u t e u m of t h e s o w s i n g r o u p s B a n d C i s s h o w n in T a b l e III ( n g / 1 0 0 m g t i s s u e / Z h r ) a n d F i g . S (~tg/100 m g t i s s u e / 8 h r ) . T h e l a t t e r a l s o s h o w s a n e s t i m a t e of t h e t o t a l p r o d u c tionper ovary. This shows that progesterone production is greatestby t h e e a r l y l u t e a l t i s s u e , w h e n P G F p r o d u c t i o n i s l e a s t , a n d at i t s l o w e s t in the late l u t e a l t i s s u e w h e n P G F p r o d u c t i o n is at a m a x i m u m . T a b l e IV s h o w s t h e e f f e c t of i n d o m e t h a c i n on P G F p r o d u c t i o n by b o t h e n d o m e t r i u m a n d c o r p u s l u t e u m t i s s u e . In b o t h c a s e s t h i s i n h i b i t e d P G F p r o d u c t i o n b y o v e r 95% a n d c l e a r l y d e m o n s t r a t e s t h a t t h e p r o s t a g l a n d i n i n a l l t h e e x p e r i m e n t s r e p o r t e d a b o v e i s d u e to de n o v o s y n t h e s i s of t h e c o m p o u n d a n d n o t s i m p l y f l u s h i n g o u t of p o s s i b l e endogenous material.

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t-~

Table

I

by porcine

endometrium

(mid horn)

in vitro

during

Prostaglandin F secretion (ng/100 mg tissue) the luteal phase of the oestrous cycle.

©

Experiment 0 - 2 2 - 4

Corpus luteum state

Time

(hours) 4 - 6

6 - 8

B

earlyll midII midIII latel
_ + +

7.3~. + 74.7 ~_ 57.1 ~. 76.4 0.9 I0.3 15.8 9

1 .9 a II 9.2 5.6

7.3~. + 41.6 ~_ 80.3 ~_ 179 -

7.1 y~ 0.6 + 12.1 ~_ 2 37.8_~ 4.7 125 - 26+5

5.8~_ + 13.6 T. 47.3 ~ 143 -

0.4 1.9 14.7 12

C

e a r l y II early III m i d II m i d III l a t e II 13.6 + 1 3 8 . 8 + 3.6 76.4+-23.2 287 -+23.5 7 9 . 2 + 55 9.2~ 1.2 31 6 6.3 67.6 +- 7.8 + 79.2_T_ 2 3 . 1 194 - 48.5

8.8~_ 50 60.8 + 5 0 . 4 ~76.3 +

0.8 9.5 5.6 9.5 15.2

9.2 39.2 60.4 73.6 112

1.2 5.4 3. 1 17.1 25

D

©

mid mid late late

lI III I II

51.3 185.3 265 89.5

~_ + 9.2 b ~ 32.7 ~ 52 - 14.7

39.5 + 8 . 3 I 0 3 . 4 ~ 20 1 78 ~. 26.5 52.5 - 7.1

20. I ~+ 4 . 2 69.5 % 9.7 92.5 ~_ 1 7.4 38.7 - 4 . 8

15.1 + 37.6 i 43.4~ 12.1 -

4.6 7.7 9.1 1.3

b~

a

mean

+ - SEM,

n = 4

b

mean

+ - SEM,

n = 5

T a b l e II

Prostaglandin

F secretion (ng/100 mg tissue) by porcine corpus luteum tissue ~ vitro.

Experiment
0 7 Z 4 4 6

Time (hours)
6 8

~O

Corpus luteum state

+
5.7 ~ 0.7 a Z . 5 ~ 0.4 14.8 - 0.6 4.5 ~_ 0.7 g.?~0.Z 7.4 - I.Z

+

+
3.Z _~ 0.6 2.9-0.1 3.8 + 0.7

+
2.3 ~. 0.3 3 . g ~ 0.3 Z.9 - 0.4

A

early II m i d II late I

B I 31.9 - 8.5

e a r l y III m i d II

+ 4 . 3 Yc 0 . 5 1 9 . 5 Yr 4 . 9
19.Z - 3.5

+ 3.4~_ 0 . 9 2 0 . 3 ~. 6 . 1

3 . 1 -+ 0 . 4 9.6 + 1.4
Z 0 . 5 +- 5.4

+ 4.1 ~ 0.6 9.8 ~ 1.8
2 3 . 3 - 0.3

late

0.6

1.0
I.i

C

©
OO

early II m i d II m i d III late II

6.6 5.7 ZI. 1 4.4

0.4 0.6 I.Z 0. Z

4.5 Z.Z 7.2 1.6

0.Z 0.2 0.3

2.1 0.4

0.5 0.7 0.3 0.Z

1.2 1.5 2.0 0.03

0.3 0.3 0.3 O.OZ

~.~

a

mean-

+

SEM,

n

= 4

Table

III

Progesterone

s e c r e t i o n ( n g / 1 0 0 m g t i s s u e ) by p o r c i n e c o r p u s l u t e u m t i s s u e i n v i t r o .

©
in

>

Experiment
0
2 Z 6

Time (hours)
4 4
6 -

Corpus luteum state * 1140
79.3~ 9.6 391 Z66 130 8.1 + ~ ~ ~ 62 33 3Z 0.4 6.5 1.0

8

a 1118
56.5~ 0.2 + 349 ~ 201 ~ 92 ~ 9.5-

÷ ~ lZ7

÷ ~ IZZ
5.3 0.2 66 21.6 Z2.5 1.2

1101

÷ ~ 110
43.5 ~ 0.3+ 365 ~ 195 ~ 76 ; 12.37.6 0.3 89 22 21.5 1.2

B

early III m i d II late I 482 393 85 8.6 67 30 17.8 I.I

1135 65 84.9 Z.9 24.9 - 7.3

early II m i d II m i d III late II

©

L,~

a

mean

+ - SF.M,

n

= 4.

PROSTAGLANDINS

600 400i

B

I

~

2W'

.~.
C
600 = 4N

t
I~e

200
m

C
L_

n.

600 400 200

D

--~
I I
I

]Z
F i g u r e 1.

]~

late Z

late I

I

P r o s t a g l a n d i n F s e c r e t i o n by p i g + e n d o m e t r i u m in vitro. Each point r e p r e s e n t s the m e a n ° SEM f r o m four incubations.

JULY 1976

VOL. 12 NO. 1

105

m

©

0 G~t Q

I-I Prostaglandin F (ng/lOOmg ti,ssue/8h)
o o 6 o 0

N

~i~

o

o

I~ < o ~L%"

W
I:I

3>

q

Q

a

o

Di

O

©

M o

E

t

i

|

~tut
8h )

q

im

l!

Prostaglondin F ( n g / o v a r y /

0

[]
--" I~ ~ 4b Ut

Progesterone

( pg/lOOmg t i s s u e / 8 h )

<
0

I=

0

~1~

m

t~

p

It•

('1
m

in l

m

©

g

Prog aste rone

( J~g/ovary / 8h )
C~

PROSTAGLANDINS

Table IV

E f f e c t of i n d o m e t h a c i n o n p r o s t a g l a n d i n F s e c r e t i o n (ng/100 mg tissue) by porcine endometrium and corpus l u t e u m t i s s u e i n v i t r o d u r i n g t h e m i d tI l u t e a l p h a s e of the cycle.

Tissue

Experiment
0-4 Control 3.9

Time (hours) 4-8
+ - 0.45

Total/8h
+ - 0.3

+ - 0.3 a 4.4

8.5

Corpus l u t e u m indomethacin (I0 ~g/ml m e d i u m ) Control 0.2Z + 0.05 0.1Z +- 0.05 0.34 + 0.I

12.7+4.9

10.8 -+ 1.8

Z3.5 -+ 5.5 0.ZZ + 0.06

endometrium
indomethacin (I0 ~g/ml m e d i u m )
a +

0.l -+ 0.04 0.0Z -+ 0.02

mean-

SEM,

n

= 4.

Discussion The above results clearly demonstrate that the porcine endometrium i n v i t r o h a s t h e c a p a c i t y to s y n t h e s i s e h i g h l e v e l s of P G F d u r i n g t h e m i d l I I to l a t e I l u t e a l p h a s e of t h e o e s t r o u s c y c l e . T h i s o b s e r v a t i o n i s i n a g r e e m e n t w i t h p r e v i o u s i n v i v o s t u d i e s w h e r e t h e c o n c e n t r a t i o n of P G F i n t h e u t e r o - o v a r i a n v e n o u s p l a s m a of s o w s w a s a t i t s h i g h e s t a r o u n d d a y 16 of t h e c y c l e (4) w h i c h c o r r e s p o n d s w i t h t h e m i d / l a t e l u t e a l p h a s e of t h e c y c l e . T h i s e v i d e n c e f u r t h e r s t r e n g t h e n s t h e a s s o c i a t i o n b e t w e e n l u t e o l y s i s a n d t h e e n d o m e t r i a [ s e c r e t i o n of P G F i m p l i c a t e d b y t h e f a c t s t h a t (a) h y s t e r e c t o m y p r o l o n g s t h e l i f e s p a n of t h e c o r p u s l u t e u r n i n s o w s (7), (b) e n d o m e t r i a l f l u s h i n g s f r o m s o w s i n t h e l a t e r p a r t of t h e o e s t r o u s c y c l e c a u s e s l u t e o l y s i s i n t h e p i g (8, 9) a n d (c) P G F z a i n d u c e d r e g r e s s i o n of p o r c i n e c o r p o r a l u t e a m a i n t a i n e d b y o e s t r o g e n (10).
T h e finding that the pig corpus luteum also produced considerable amounts of P G F during the m i d Ill "to late I luteal phase of the cycle in concert with the endometrial secretion is of particular interest and places s o m e doubt on the simplified uterine luteolysin concept. Whether the observed changes in luteal prostaglandin activity are a consequence of luteolysis or play an active role in effecting luteolysis could not be ascertained f r o m the present study. T h e dating of the oestrous cycles of t h e a n i m a l s u s e d w a s n o t s u f f i c i e n t l y s p e c i f i c to d e t e r m i n e w h e t h e r

108

JULY 1976

VOL, 12 NO. 1

PROSTAGLANDINS

t h e e n d o m e t r i a l a n d l u t e a l s e c r e t i o n s of P G F a r e c o i n c i d e n t o r w h i c h event occurs first. Further studies with endometrium and corpus l u t e u m ~ s a m p l e s f r o m p i g s a t precisely k n o w n s t a g e s i n t h e l u t e a l p h a s e of t h e c y c l e a r e r e q u i r e d to e s t a b l i s h t h i s . In e i t h e r e v e n t h o w e v e r t h e a b i l i t y of t h e c o r p u s l u t e u m to s y n t h e s i s e P G F a t p e a k l e v e l s i n t h e l a t e r s t a g e s of t h e c y c l e r a i s e s i n t e r e s t i n g p o s s i b i l i t i e s f o r t h e r e g u l a t i o n of l u t e a l f u n c t i o n t h r o u g h control of intrinsic P G F synthesis. T h e relatively high levels of P G F s y n t h e s i s e d b y t h e e a r l y II l u t e a l t i s s u e w e r e n o t c o n s i d e r e d s i g n i f i c a n t a s t h e s e w e r e m o s t l i k e l y a s s o c i a t e d w i t h t h e c e n t r a l b l o o d c l o t of the c o r p u s l u t e u m , a n d it h a s b e e n d e m o n s t r a t e d t h a t b l o o d p l a t e l e t s r e l e a s e l a r g e q u a n t i t i e s of P G F z u . (11) I t i s p o s s i b l e t h a t i n t h e p i g a l t h o u g h t h e l i f e s p a n of t h e c o r p u s l u t e u m i s u n d e r u t e r i n e r e g u l a t i o n (8, 9) t h e u t e r i n e P G F m a y n o t b e t h e u l t i m a t e e f f e c t o r of l u t e o l y s i s , b u t r a t h e r a c t s a s a t r i g g e r f o r t h i s event in which luteal P G F could t h e n play an i m p o r t a n t r o l e . Indeed this mechanism could be important in other animals where luteolysis i s k n o w n to b e u n d e r u t e r i n e c o n t r o l (1) a n d m a y b e of p r i m e i m p o r t a n c e in primates and humans where there is no apparent uterine control (12, 13). T h e a b i l i t y o£ r a t c o r p o r a l u t e a to s y n t h e s i s e P G F 2 u h a s b e e n d e m o n s t r a t e d (14) a n d t h e b o v i n e o v a r y i n v i v o h a s b e e n s h o w n t o h a v e t h e c a p a c i t y t o c o n v e r t a r a c h i d o n i c a c i d t o P G F (15). In t h i s l a t t e r study it w a s suggested that since partial luteal regression occurred arachidonic and could be the luteolysin although it is equally possible that the increased level of P G F w a s the trigger for luteolysis. Both p r i m a t e a n d h u m a n c o r p o r a l u t e a h a v e b e e n s h o w n to c o n t a i n s u b s t a n t i a l a m o u n t s of P G F Z ~ (1, 16), a n d a l t h o u g h l i t t l e v a r i a t i o n i n l u t e a l l e v e l s h a s b e e n d e m o n s t r a t e d d u r i n g t h e m e n s t r u a l c y c l e (17), i t h a s b e e n shown recently that direct injection of P G F Z a into the h u m a n corpus luteum induces luteolysis (3). H e n d e r s o n ~ M c N a t t y (1) r e c e n t l y p r o p o s e d a b i o c h e m i c a l h y p o t h e s i s to e x p l a i n t h e m e c h a n i s m of l u t e a l r e g r e s s i o n w h e r e b y t h e u t e r u s o r o v a r y s e c r e t e d i n c r e a s e d a m o u n t s of P G F 2 a u n d e r t h e i n f l u e n c e of progesterone. The present demonstration that both the ovary and ~terus h a v e t h e c a p a c i t y to s y n t h e s i s e l a r g e a m o u n t s of P G F a r o u n d d a y s 1 4 - 1 7 of t h e e s t r o u s c y c l e i s e n t i r e l y c o n s i s t e n t w i t h t h i s h y p o t h e s i s a s m a x i m u m p r o g e s t e r o n e l e v e l s in vivo h a v e b e e n shown to o c c u r a r o u n d d a y s 8 - 1 3 o f t h e c y c l e (18). T h e o b s e r v a t i o n t h a t l u t e a l P G F s e c r e t i o n a t t a i n s a m a x i m u m at about the s a m e t i m e as e n d o m e t r i a l PGF a l l o w s s p e c u l a t i o n on t h e p o s s i b l e r o l e of t h i s m a t e r i a l i n l u t e o l y s i s , p a r t i c u l a r ly in the p r i m a t e and h u m a n .

JULY

1976

V O L . 12 N O . 1

109

PROSTAGLANDINS

References 1. Henderson, K.M. and McNatty, K.P. : A biochemical hypothesis t o e x p l a i n t h e m e c h a n i s m of l u t e a l r e g r e s s i o n . P r o s t a g l a n d i n s 9 : 7 7 9 - 7 9 7 , 1975. McCracken, J.A., Carlson, J.C., Glew, M.E., Goding, J.K. and Baird, D.T. : Prostaglandin F2~ identified as a luteolytic h o r m o n e i n s h e e p . N a t u r e ( N e w Biol) 238 : 1 2 9 - 1 3 4 , 1972.

2.

3.

Korda, A.K., Shutt, D.A., Smith, I.D., Shearman, K.P. and
L y n e h a m , R . C . : A s s e s s m e n t of a p o s s i b l e l u t e o l y t i c e f f e c t of i n t r a - o v a r i a n i n j e c t i o n of p r o s t a g l a n d i n F 2 a i n the h u m a n . P r o s t a g l a n d i n s 9 : 4 4 3 - 4 4 9 , 1975.

4.

G l e e s o n , A . K . , T h o r b u r n , G . D . a n d Cox, K . I . : P r o s t a g l a n d i n F c o n c e n t r a t i o n s i n t h e u t e r o - o v a r i a n v e n o u s p l a s m a of t h e s o w d u r i n g t h e l a t e l u t e a l p h a s e of t h e o e s t r o u s c y c l e . P r o s t a g l a n d i n s 5 : 5 2 1 - 5 2 9 , 1974. Watson, J. and Leask, J.T.S. of o v a r i a n s t e r o i d o g e n e s i s . : S u p e r f u s i o n i n v i t r o i n the s t u d y J . E n d o c r i n o l . 64 : 163~173, 1975.

5.

6.

T h o r n e y c r o f t , I . H . a n d S t o n e , S . C . : K a d i o i m m u n o a s s a y of s e r u m progesterone in women receiving oral contraceptive steroids. C o n t r a c e p t i o n 5 : 1 2 9 - 1 4 6 , 1972. Anderson, L.L., Butcher, K.L. and Melampy, R.M. :Uterus a n d t h e o c c u r r e n c e of o e s t r u s i n p i g s . N a t u r e 198 : 3 1 1 - 3 1 2 , 1963. S c h o m b e r g , D . W . : A d e m o n s t r a t i o n i_n v i t r o of l u t e o l y t i c a c t i v i t y i n pig u t e r i n e f l u s h i n g s . J . E n d o c r . 3 8 : 3 5 9 - 3 6 0 , 1967. C h r i s t e n s o n , K . K . a n d D a y , B . N . : L u t e o l y t i c e f f e c t s of e n d o m e t r i a l e x t r a c t s i n t h e p i g . J . A n i t a . S c i . 34 : 620, 1971. Kraeling, R.R., Barb, C.R. and Davis, B.J. : Prostaglandin i n d u c e d r e g r e s s i o n of p o r c i n e c o r p o r a l u t e a m a i n t a i n e d b y o e s t r o g e n . P r o s t a g l a n d i n s 9 : 4 5 9 - 4 6 2 , 1975. S i l v e r , M . J . , S m i t h , J . B . , I n g e r m a n , C. a n d K a s i s , J . J . Human blood prostaglandins : formation during clotting. Prostaglandins 1:429-436, 1972.
:

7.

8.

9.

10.

11.

110

JULY 1976

VOL. 12 NO. 1

PROSTAGLANDINS

12.

Neill, J.D., Johansson, E.D.G. and Knobil, E. :Failure h y s t e r e c t o m y to i n f l u e n c e t h e n o r m a l p a t t e r n of c y c l i c progesterone secretion in the rhesus monkey. E n d o c r i n o l o g y 84 ; 4 6 4 - 4 6 5 , 1969.

of

13.

Fraser, J.S., Baird, D.T., Hobson, B.M., Michie, E.A. Hunter W.M. : Cyclical ovarian function in women with c o n g e n i t a l a b s e n c e of u t e r u s a n d v a g i n a . J. Clin. Endocr. Metab. 36:634-637, 1973.

and

14.

Demers, L.M., Behrman, H.R. and Greep, R.O. : E f f e c t s of p r o s t a g l a n d i n s a n d g o n a d o t r o p h i n s on l u t e a l p r o s t a g l a n d i n a n d steroid biosynthesis. Advances in the Biosciences 9 : 701-707, 1973. S h e m e s h , M. a n d H a n s e l , W. : A r a c h i d o n i c a c i d a n d b o v i n e c o r p u s l u t e u m f u n c t i o n . P r o c . Soc. E x p . B i o l . M e d . 148 : 2 4 3 - 2 4 6 , 1975. Wilks, J.W., Forbes, K.K. and Norland, J.F. :in The Prostaglandins - Clinical applications in human reproduction. ( E . M . S o u t h e r n , E d i t o r ) p . 4 7 , 1972. Challis, J . R . G . , Calder, A.A., Dilley, S., Forster, C.S., Hillier, K., Hunter, D.3. S., Mackenzie, I.Z. and T h o r b u r n , G . D . : P r o d u c t i o n of p r o s t a g l a n d i n s E a n d F a b y corpora lutea, corpora albicantes and stroma from the human ovary. J. Endocr. 68:401-408, 1976. Henricks, D.M., Guthrie, H.D. and Handlin, D.L. :Plasma estrogen, progesterone and luteinizing hormone levels during t h e e s t r o u s c y c l e i n p i g s . B i o l . K e p r o d . 6 : 2 1 0 - 2 1 8 , 1972.

15.

16.

17.

18.

JULY

1976

VOL.

12 N O . 1

111

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