The release of pituitary gonadotrophins by luteinizing hormone releasing hormone in the intact, castrated and aspermatogenic rat

Llfe Sciences, Vol. 31, pp. 2717-2721 Printed in the U.S.A.

Pergamon Press

THE RELEASE OF PITUITARY GONADOTROPHINSBY LUTEINIZING HORMONE RELEASING HORMONE IN THE INTACT, CASTRATED AND ASPERMATOGENIC RAT I.D. Morris and S. Azmatullah Department of Pharmacology, Materia Hedica and Therapeutics, U n i v e r s i t y of Hanchester, Stopford B u i l d i n g , Oxford Road, Manchester M13 9PT, U.K. (Received in final form September 7, 1982) Summary The change in serum gonadotrophin c o n c e n t r a t i o n in response to s y n t h e t i c L u t e i n i z i n g Hormone Releasing Hormone (LHRH - 400 ng i . v . ) was i n v e s t i g a t e d under b a r b i t u r a t e anaesthesia in a d u l t male rats e i t h e r c h r o n i c a l l y c a s t r a t e d , rendered aspermatogenic by the a d m i n i s t r a t i o n o f ~ - c h l o r o h y d r i n 12-16 weeks p r e v i o u s l y ( t o remove i n h i b i n ) , or t r e a t e d w i t h v e h i c l e . A s i n g l e i n j e c t i o n o f LHRH i n creased serum LH and FSH c o n c e n t r a t i o n s s i m i l a r l y in both i n t a c t and aspermatogenic r a t s . In c a s t r a t e d rats the amount o f LH released was much g r e a t e r and the FSH s e c r e t i o n sustained. A second i n j e c t i o n produced a s i m i l a r increase although a second peak o f FSH could not be detected in c a s t r a t e d rats as the FSH level was s t i l l e l e v a t e d . The increase in LH l e v e l s was two to three times l a r g e r in response to the second i n j e c t i o n o f LHRH than to the f i r s t in a l l groups. The r e s u l t s do not support the hypothesis t h a t the enhanced gonadotropin response to c a s t r a t i o n in the aspermatogenic r a t is due to increased p i t u i t a r y s e n s i t i v i t y to LHRH. I t is well e s t a b l i s h e d t h a t in the female r at the ovary can modulate the p i t u i t a r y s e n s i t i v i t y to endogenous and exogenous l u t e i n i z i n g hormone r e l e a s i n g hormone (LHRH). C a s t r a t i o n decreases the p i t u i t a r y s e n s i t i v i t y to LHRH which can be restored by the a d m i n i s t r a t i o n o f the o v a r i a n s t e r o i d s . Oestrogen and progesterone have a l s o been shown to be p a r t i a l l y r e s pons ib le f o r the increased p i t u i t a r y s e n s i t i v i t y to LHRH found p r i o r to o v u l a t i o n in the pr o- oes t r u s r a t (2, 3, 4). In the male r a t the f a c t o r s c o n t r o l l i n g p i t u i t a r y responses to LHRH are less well d e f i n e d . I t is g e n e r a l l y agreed t h a t the testes generate two feedback s i g n a l s both o f which can p o t e n t i a l l y modify p i t u i t a r y responses to LHRH (5). The f i r s t o f these is androgen secreted by the i n t e r s t i t i a l cells, t e s t o s t e r o n e and i t s met a b o l i t e s decrease the response o f the p i t u i t a r y to LHRH (4, 6, 7). The second, but less w e l l d e f i ned s i g n a l , i n h i b i n is thought to a r i s e from the seminiferous t u b u l e . The evidence f o r i t s e x i s t e n c e is l a r g e l y c i r c u m s t a n t i a l and comes mainly from o b s e r v a t i o n s t h a t FSH is p r e f e r e n t i a l l y elevated when the seminiferous e p i t h e l i u m is damaged or absent and the i n t e r s t i t i u m is s t i l l s e c r e t i n g androgen (5). I t has r e c e n t l y been demonstrated t h a t in the aspermatogenic r a t , in which i n h i b i n is presumably absent and the i n t e r s t i t i u m f u n c t i o n i n g n o r m a l l y , the increase in gonadotrophin in response to removal o f the t e s t i c u l a r androgen feedback signal by c a s t r a t i o n is more rapid than in the i n t a c t animal (8). This o b s e r v a t i o n r a i s e s the p o s s i b i l i t y t h a t in the i n t a c t r a t i n h i b i n i n h i b i t s the response o f the p i t u i t a r y to endogenous LHRH. This hypothesis has been examined by comparing the changes in gonadotrophin in response to s y n t h e t i c LHRH in c h r o n i c a l l y c a s t r a t e d and aspermatogenic rats w i t h those in i n t a c t r a t . 0024-3205/82/242717-05503.00/0 Copyright (c) 1982 Pergamon Press Ltd.

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Gonadotrophlns After LHRH in Male Rats

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M a t e r i a l s and Methods A d u l t male Wistar rats weighing 240-260 g i n i t i a l l y were maintained under c o n t r o l l e d l i g h t i n g (on 05.00-19.00 hr) and allowed f r e e access to food and ~ t e r . The rats were randomly d i v i d e d i n t o three groups. Those in the f i r s t group were b l l a t e r a l l y c a s t r a t e d under et h e r anaesthesia, those in the second group received 0.9% NaCI 2ml/kg i . p . and those in the t h i r d group were i n j e c t e d w i t h ( d . l . ) ~ - c h l o r o h y d r i n (Koch L i g h t , Colnbrook, England) 100 mg/kg in 0.9~ NaCI 2 ml/kg i . p . In our l a b o r a t o r y t h i s dose o f ~ - c h l o r o h y d r i n has p r e v i o u s l y been shown to render a l l t r e a t e d rats aspermatogenic i n d i r e c t l y by producing cysts in the epididymis which r e s u l t s in back pressure necrosis o f the seminal e p i t h e l i u m (9). Twelve to s i x t e e n weeks l a t e r the rats were randomly assigned to experimental groups. (Body weight 400-500 g) Rats were anaesthetised w i t h a n a e s t h e t i c et her (May & Baker) and the l e f t femoral vein cannulated. The ether was then withdrawn and anaesthesia m a i n t a i n ed f o r the rest o f the experiment w i t h sodium p e n t o b a r b i t o n e (Sagatal, May & Baker) i n j e c t e d i n t r a v e n o u s l y . The mean t o t a l doses o f a n a e s t h e t i c administered in d i v i d e d doses throughout the experiment were 37±11, 40±6 and 46±14 mg/kg (Mean±S.E.M., n=6-12) f o r the c a s t r a t e d , s a l i n e t r e a t e d and ~ - c h l o r o h y d r i n t r e a t e d groups r e s p e c t i v e l y . The d i f f e r e n c e s between groups were not s i g n i f i cant. Heparin 2000 iu/kg ( P u l a r i n , Evans Medical) was given i . v . 40 min a f t e r the b a r b i t u r a t e . Ten minutes a f t e r t h i s (T=O) and subsequently every 20 min f o r 120 min blood samples (0.5 ml) were withdrawn and the volume replaced w i t h s a l i n e In t e s t animals 400 ng o f s y n t h e t i c LHRH ( I . C . I . Ltd) was included in the r e p l a cement s a l i n e at T=O and T=60 min. Plasma was prepared and stored at -20 oc unt i l assayed f o r gonadotrophins. When the l a s t sample had been obtained the a n i mals were k i l l e d and the testes and seminal v e s i c l e s removed and weighed. Plasma samples (2 x 50 ~ l ) were assayed f o r FSH and LH in a s i n g l e r a d i o immunoassay using k i t s k i n d l y provided by N.I.A.M.D. Rat P i t u i t a r y d i s t r i b u t i o n program, U.S.A. w i t h minor m o d i f i c a t i o n s as o u t l i n e d elsewhere (10). The w i t h in assay c o e f f i c i e n t o f v a r i a t i o n f o r LH and FSH r e s p e c t i v e l y were 10% and 12%. The lowest c o n c e n t r a t i o n s d e t e c t a b l e by the assay were 20 ng/mI o f LH and 90 ng/ml o f FSH. In some animals plasma LH and FSH was not d e t e c t a b l e t h e r e f o r e values o f 20 ng/ml f o r LH and 90 ng/ml f o r FSH were taken f o r the c o n s t r u c t i o n o f the graphs. In the graphs values o f means ± s.e. means are shown which, although i ~ a p p r o p r i a t e s t a t i s t i c s f o r non-parametric comparisons, serve to i l l u s t r a t e the magnitude o f the changes. Tests o f s i g n i f i c a n c e were made using the Mann-Whitney U t e s t . For the gonadotrophins, comparisons o f the l e v e l s at each time are made w i t h the corresponding l e v e l a t T = O f o r each treatment and not w i t h the concurrent v e h i c l e t r e a t e d c o n t r o l because o f the unexpected and unexplained d i f f e r e n c e between the l e v e l s at T = 0 (not s i g n i f i c a n t ) . Comparisons o f t i s s u e weights were made using Student's t t e s t (two t a i l e d ) . Results The weights o f the testes in the ~ - c h l o r o h y d r i n t r e a t e d rats (2.15 ± 0.1 g) were s i g n i f i c a n t l y d i f f e r e n t from those o f s a l i n e t r e a t e d rats (3.7 ± 0.08 g) (P
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Gonadotrophins After LHRB in Male Rats

These r e s u l t s a r e s i m i l a r t o more d e t a i l e d o b s e r v a t i o n s o f o u r l a b o r a t o r y and c o n f i r m t h a t t h e ~ - c h l o r o h y d r i n has r e n d e r e d t h e r a t s a s p e r m a t o g e n i c .

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(9)

In c a s t r a t e d r a t s , LHRH caused an immediate r i s e in plasma LH w h i c h was maximal a f t e r 40 mln and w h i c h began t o f a l l a t 60 min. A f u r t h e r i n j e c t i o n o f LHRH a t T = 60 produced a second r i s e in plasma LH ( F i g . 1). In i n t a c t and a s p e r m a t o g e n i c r a t s a l m o s t i d e n t i c a l changes in LH were seen; a f t e r LHRH t h e plasma LH c o n c e n t r a t i o n r o s e , r e a c h i n g a p l a t e a u a f t e r 20-60 min. A second i n j e c t i o n o f LHRH a g a i n i n c r e a s e d the plasma LH. The r a t i o o f the mean i n c r e a s e in LH a f t e r the second dose t o t h a t a f t e r the f i r s t dose o f LHRH was 2 . 7 , 2 . 6 and 2.1 f o r t h e i n t a c t , a s p e r m a t o g e n i c and t h e c a s t r a t e r a t s r e s p e c t i v e l y .

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FIG. I A and B. Mean (± SEM; n = 3=7) plasma LH concentrations (ng/ml) in b a r b i t u r a t e anaesthetised male rats a f t e r intravenous LHRH (h00 ng: closed symbols) o r vehi c l e (open symbols) given irnmediateIy a f t e r withdrawal o f the blood samples a t T = 0 and 60 min. m ~ - c h l o r o h y d r i n t r e a t e d aspermatogenic r a t ; • i n t a c t cont r o l r a t ; • castrated r a t . Time 0 v 40 *P<0.05. Time 60 v I00 + P
Changes in plasma FSH d i f f e r e d from those o f LH and between treatment groups. In castrated rats there was an i n i t i a l r i s e in response to LHRH, but

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Gonadotrophins After LHRH in Male Rats

t h e l e v e l s d i d not f a l l iable further rise. In caused a r i s e in plasma the second i n j e c t i o n o f

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again. The second i n j e c t i o n o f LHRH caused no a p p r e c b o t h t h e a s p e r m a t o g e n i c and t h e i n t a c t r a t s , LHRH FSH. The l e v e l s then f e l l b u t rose a g a i n In response t o LHRH.

LHRH v e h i c l e d i d n o t cause any s i g n i f i c a n t any o f the t r e a t m e n t g r o u p s .

changes in e i t h e r

LH o r FSH in

Discussion

The i n c r e a s e s in plasma LH a f t e r LHRH in i n t a c t and a s p e r m a t o g e n i c r a t s were s i m i l a r . In c a s t r a t e d r a t s the amount o f r a d i o - i m m u n o a s s a y a b l e LH r e l e a s e d was much g r e a t e r than in t h e o t h e r g r o u p s . However, in r e l a t i v e terms the i n c r e a s e s were s i m i l a r in a l l groups - a p p r o x i m a t e l y 10O~. T h i s s u g g e s t s t h a t t h e response r e f l e c t s the s e c r e t i o n o f a f i x e d f r a c t i o n o f a r e a d i l y r e l e a s a b l e pituitary pool o f LH as p r e v i o u s l y s u g g e s t e d by o t h e r s ( 4 , 11, 12). The second dose o f LHRH was a d m i n i s t e r e d t o t e s t w h e t h e r t h e s e l f - p r i m i n g activity o f LHRH was a l t e r e d by t h e t r e a t m e n t s . A p r i m i n g e f f e c t was indeed d e m o n s t r a t e d , t h e second LHRH i n j e c t i o n c a u s i n g the r e l e a s e o f a p p r o x i m a t e l y t w i c e as much as t h e F i r s t dose in a l l t h r e e t r e a t m e n t g r o u p s . Other r e p o r t s (12, 14) c o u l d n o t d e m o n s t r a t e a p r i m i n g e f f e c t o f LHRH in the i n t a c t male r a t s . A i y e r e t al (2) r e p o r t e d t h a t in o e s t r o g e n t r e a t e d c a s t r a t e d ~ l e rats, priming by LHRH c o u l d be d e m o n s t r a t e d w h i c h was s i m i l a r in m a g n i t u d e t o t h a t r e p o r t e d here b u t no d a t a were p r e s e n t e d f o r i n t a c t o r c a s t r a t e d male r a t s . In a n o t h e r s t u d y , LHRH f a i l e d t o i n c r e a s e plasma LH in i n t a c t r a t s and p r i m i n g a c t i v i t y was a b s e n t in a c u t e l y c a s t r a t e d male r a t s ( 1 5 ) . However, a low dose o f LHRH (50 ng) was u t i l i s e d w h i c h may e x p l a i n the d i s c r e p a n c y . The i n c r e a s e s in plasma FSH in response t o LHRH were not so c l e a r l y d e f i n e ~ b o t h i n j e c t i o n s o f LHRH i n c r e a s i n g plasma FSH by s i m i l a r amounts in a s p e r m a t o g e n i c and i n t a c t r a t s . However, no c l e a r p r i m i n g a c t i v i t y was seen. In c a s t r a ~ ed n ~ l e r a t s i t d i d n o t p r o v e p o s s i b l e t o e l i c i t a second i n c r e a s e in FSH w i t h LHRH; the i n c r e a s e in FSH in response t o the f i r s t dose o f LHRH was g r a d u a l and c o n t i n u e d t h r o u g h o u t t h e e x p e r i m e n t . The reasons f o r t h i s a r e n o t c l e a r , b u t may r e f l e c t the p l e o m o r p h i s m o f FSH under d i f f e r e n t e n d o c r i n o l o g i c a l s t a t e s r e s u l t i n g in d i f f f e r e n t r a t e s o f c l e a r a n c e o f t h e FSH m o l e c u l e ( 1 6 ) . These e x p e r i m e n t s s u g g e s t t h a t an i n c r e a s e in p i t u i t a r y responsiveness to endogenous LHRH does not e x p l a i n the enhanced f e e d b a c k response seen in t h e aspermatogenic rat. I f i n h i b i n is a b s e n t in the a s p e r m a t o g e n i c r a t , t h e d a t a would i n d i c a t e t h a t i n h i b i n does n o t m o d u l a t e t h e p i t u i t a r y response t o LHRH and t h e r e f o r e o t h e r e x p l a n a t i o n s must be s o u g h t . However, o t h e r d a t a a r e a v a i l a b l e t o suggest t h a t i n h i b i n decreases t h e p i t u i t a r y response t o LHRH. In v i t r o i n h i b i n p r e p a r a t i o n s d e c r e a s e the LHRH induced r e l e a s e o f LH and FSH by p i t u i t a r y c e l l c u l t u r e s (17, 18, 19). As a r e s u l t o f e x p e r i m e n t s in v i v o , Franchimont Chari & Demoulin (20) f i r s t suggested t h a t a p K o t e i n a c e o u s f r a c t i o n o f seminal f l u i d , presumably c o n t a i n i n g i n h i b i n , c o u l d d e c r e a s e the s e c r e t i o n o f p i t u i t a r y g o n a d o t r o p h i n s t i m u l a t e d by exogenous LHRH. More r e c e n t l y t h e a c u t e e f f e c t s o f a c r u d e i n h i b i n c o n t a i n i n g p r e p a r a t i o n upon L H R H s t i m u l a t e d g o n a d o t r o p h i n secr e t i o n in b a r b i t u r a t e a n a e s t h e t i s e d and c o n s c i o u s a d u l t male r a t s have been examined ( 2 1 ) . This p r e p a r a t i o n suppressed the LHRH induced r i s e in FSH b u t d i d not a l t e r the r i s e in LH. When they i n c r e a s e d t h e dose o f LHRH t o 500 ng the i n h i b i n c o n t a i n i n g p r e p a r a t i o n was i n e f f e c t i v e . These d a t a suggest t h a t the dose o f LHRH used in t h e p r e s e n t s t u d y may have been t o o l a r g e , a l t h o u g h o t h e r s have shown t h a t 500 ng is sub-maximal ( 6 ) . The e x p e r i m e n t s d e s c r i b e d here and those o f H o r r l s & Jackson ( 9 ) . a r e not d i r e c t l y c o m p a r a b l e w i t h those o f Rush & L i p n e r ( 2 1 ) , as in the f o r m e r case the f e e d b a c k s i g n a l s had been removed whereas in the l a t t e r the f e e d b a c k s i g n a l s were s t i l l o p e r a t i n g and the e f f e c t s o f the

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Gonadotrophins After LHRH in Male Rats

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crude inhibin preparation superimposed upon this system. This could account for the discrepancies. It would be expected that relatively small changes in gonadotrophins would occur in response to exogenous LHRH were endogenous inhibin exerting a near maximal feedback inhibition itself. By using models in which the inhibin feedback was absent, it was hoped that there would be greater scope for changes in the release gonadotrophin in response to LHRH, as suggested by the previous castration experiments, an expectation which seems not to have been met. Further understanding of the testicular feedback upon gonadotropin secretion will have to await the development of assays for plasma inhibin. Such assays will establish whether in vivo models do indeed remove inhibin from the circulation and also whether the administration of inhibin extracts result in physiological or pharmacological plasma concentrations. REFERENCES I) Acknowledgements: We are grateful to Dr H. Gregory, I.C.I. for the supply of synthetic LHRH, and to the Rat Pituitary Hormone Program, N.I.A.M.D., U.S.A. for the radioimmunoassay kits. 2) M.S. Aiyer, M.C. G o o d & K.Brown-Grant. J.Endocr., 6._99,255-262 (1976). 3) C.A.Blake. Endocrinology, 9~8, 451-460 (1976). 4) G.Fink & S.R.Henderscn. J.Endocr., 73, 157-164 (1977). 5) S.J. Main, R.V. Davies& B.P.Setchell. J.Reprod. Fertil., Supplement 26, 3-14 (1979). 6) H.L.Verjans & K.B.Eik-Nes. Acta Endocr(Kbh), 8~3, 493-505 (1976). 7) D.D.Nansel, M.S.Aiyer, W.H.Meinzer & E.M.Bogdanove. Endocrinology, 104, 524-531 (1979). 8) I.D.Morris & C.M.Jackson. Acta Endocr(Kbh), 88, 38-47 (1978). 9) I.D.Morris & C.M.Jackson. Int.J.Androl., I, 8-6"/-95(1978). 10) I.D.Morris. J.Reprod. Fertil., 57, 469-475--(1979). 11) M.C.Martinez. J.Reprod. Fertil.-~--48, 195-197 (1976). 12) T.M.Badger, C.E.Wilcox, E.R.Meyer, R.D. B e l l & T.J.Cicero. Endocrinology, 102, 136-141 (1978). 13) H.R.Grotjan Jr. & D. £. Johnson. Acta Endocr(Kbh), 84.4, 254-267 (1977). 14) A.E.Miller & G.D. Riegle. Proc. Soc. Exp. B i o l . & Pied., 157, 494-499 (1978). 15) M.K.Vaughan, C.Trakulrungsi, L.H.Petterborg, L.Y.Johnson, D.E.Blask, W. Trakulrungsi & R.J.Reiter. Mol.Cell.Endocr., I__44,59-71 (1979). 16) E.M.Bogdanove, G.T. Campbell, E.D.Blair, M.E.Mula, A.E.Miller & G.J.Grossman. Endocrinology, 95, 219-228.(1974). 17) A.Steinberger. J.Reprod. Fertil., Supplement 2_66, 31-45 (1979). 18) F.H.De Jong, R.Welschen, W.P.Hermans, S.D.Smith & H.J.Van der Molen. J. Reprod. Fertil., Supplement 26, 47-59 (1979). 19) R.V.Davies, S.J.Main & B.P.S~tchell. Int.J.Androl., Supplement ~, 102-113 (1978). 20) P. Franchimont, S.Chari & A. Demoulin. J.Reprod.F e r t i l . , 44, 335-350 (1975). 21) M.E.Rush & H.Lipner. Endocrinology, ]05, 187-193 (1979).--