15.10.1975
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1237
1
Fig. 1. Control 1-day-old rat testis. The seminiferous tubules show gonocytes and supporting cells. Occasional Leydig ceils. HE, • 320. Fig. 2. LH-RH treated, l-day-old rat testis. Similar cell types. HE, • 320. Fig. 3. Control ll-day-old rat testis. The tubular diameter and the nmnber of supporting cells has increased. Primitive type A spermatogortia are seen. He, • 320.
Fig. 4. LH-RH treated l l-day-old rat testis. Similar to the Figure 3. HE, x320. Fig. 5. Control 13-day-old rat testis. Tubular diameter increased. Adult type spermatogonia and primary spermatocyt@s are present in the germinal epithelium. HE, • 320. Fig. 6. LH-RH treated 13-day-old-rat. Testes similar to the control. HE, • 320. r e p o r t e d o n l y slight s t i m u l a t i o n of t h e testis. RAMIREZ a n d MCCANN 9 h a v e s u g g e s t e d t h a t t h e o n s e t of ' p u b e r t y ' in t h e r a t is d e t e r m i n e d b y a c h a n g e in t h e h y p o p h y s i s s e n s i t i v i t y to releasing h o r m o n e s r a t h e r t h a n b y c h a n g e s of t h e g o n a d o t r o p i n c o n t e n t of t h e glands. I n h u m a n , ROTH et al.~ s t a t e d t h a t p u b e r t a l a n d a d u l t t e s t e s a p p e a r s to be m o r e r e s p o n s i v e t h a n t h e i m m a t u r e testis in releasing t e s t o s t e r o n e after t h e a d m i n i s t r a t i o n of L H R H . Also SANDOW a n d BABEJ 10 s p e c u l a t e d t h a t chronic L H - R H t r e a t m e n t m a y h a v e a n e g a t i v e f e e d - b a c k effect on p i t u i t a r y s e n s i t i v i t y t o t h e g o n a d o t r o p i n releasing hormones. O u r results showed t h a t a d m i n i s t e r e d s y n t h e t i c L H R H is n e i t h e r able to i n i t i a t e per se t h e d e v e l o p m e n t of s p e r m a t o g e n e s i s a n d m a t u r a t i o n of L e y d i g cells, n o r to accelerate t h e n o r m a l r h y t h m of t h e n a t u r a l e v o l u t i o n of t h e gonads. I t is q u i t e possible t h a t o t h e r m e c h a n i s m s m i g h t be i n v o l v e d in t h e o n s e t of p u b e r t y . On t h e o t h e r h a n d , we h a d no e v i d e n c e of a n e g a t i v e f e e d - b a c k i n d u c e d b y t h e i n j e c t e d L H - R H , since b o t h t r e a t e d a n d u n t r e a t e d a n i m a l s s h o w e d n e i t h e r q u a l i t a t i v e nor q u a n t i t a t i v e histological differences in t h e testis. The significance of t h e h i g h e r n u m b e r of s p e r m a t o g o n i a l cells in u n t r e a t e d 14-day-old r a t s is o p e n to q u e s t i o n .
Resumen. R a t a s m a c h o s i n m a d u r o s t r a t a d o s cr6nic a m e n t e con f a c t o r l i b e r a d o r sintdtico de L H d e s d e el p r i m e r o h a s t a el 15 dia de e d a d a l a s ddsis de 5 ~xg subcutAneos c u a t r o veces al dia c a d s c u a t r o h o r a s p o r animal, fueron i n c a p a c e s de iniciar o acelerar el proceso de m a d u racidn t e s t i c u l a r e s t u d i a n d o el proceso e s p e r m a t o g 6 n i c o por c o n t a g e s eelulares diferenciales en secciones t r a n s versales y el desarrollo de las c61ulas de L e y d i g m a d u r a s . M. A. GARCIA-HJARLES 11 a n d O. VILAR 12
Centro de Investigaciones en Reproducci6n, 2a Cdtedra de Histologla, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 10 ~ piso, Bzwnos Aires (Rep~blica Argentina), 15 A p r i l 1975. D. V. RAMIREZand S. M. M~CCANN,Endocrinology 72, 452 (1963). 10 j. SANDOWand 1Vf.BABEL Acta endocr., Copenh. Suppl. 177, 297 (1973). 11 Post-Graduate fellow of the Latin American Training Course on Reproductive Biology (W.H.0.). Inst. de Invest. de la Altura, Apt. No. 6083, Lima, Peru. 12 Established Investigator, Consejo Nacional de Investigaciones Cientlficas y Tdcnicas. R6publica Argentina.
Effect of C a s t r a t i o n o n t h e R a t P i n e a l G l a n d : a F l u o r e s c e n c e E a r l i e r i n v e s t i g a t i o n s 1 a d e m o n s t r a t e d t h a t some p i n e a l o c y t e s of t h e r a b b i t , rat, h e d g e h o g a n d mole c o n t a i n a yellow a u t o f l u o r e s c e n t s u b s t a n c e . I n r a b b i t a n d r a t pinealocytes, t h i s m a t e r i a l a p p e a r e d t o be a t r y p t o p h a n - r i c h p r o t e i n 1, 2 t h e f u n c t i o n of w h i c h is still u n k n o w n . i t m i g h t p o s s i b l y be a carrier p r o t e i n of pineal h o r m o n e s 2. Considering t h i s h y p o t h e s i s , it s e e m e d of i n t e r e s t to
Histochemical
and Biochemical
Study
d e t e r m i n e , in t h e r a t pineal gland, t h e q u a n t i t y of cells c o n t a i n i n g yellow a u t o f l u o r e s c e n t m a t e r i a l u n d e r v a r y i n g 1 A. R. SMITH, J. F. JONGKIND and J. ARIJ~NS KAPPERS, Gen. cotnp. Endoer. 78, 364 (1972). 2 A. R. SmTu, Thesis (Amsterdam 1972). a p. PEVET, not published.
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Table I. Number of autofluorescent cells in the pineal gland after
castration (cells/mm 2) 4 days Controls
Castrated
158 i 15 147 ~ 23
1 week Controls
Castrated
3 weeks Controls
137 ~ 15
216 -~ 44
160 -4- 37 287 :k 40
Castrated
e x p e r i m e n t a l c o n d i t i o n s r e l a t e d t o t h e f u n c t i o n of t h e e p i p h y s i s in t h e r e g u l a t i o n of t h e h y p o t h a l a m o - h y p o p h y s e o - g o n a d a l s y s t e m . B e c a u s e S~ITH 2 d e m o n s t r a t e d an inverse r e l a t i o n in d i f f e r e n t c i r c u m s t a n c e s b e t w e e n t h e a m o u n t of a u t o f l u o r e s c e n t m a t e r i a l a n d t h a t of s e r o t o n i n in t h e r a t pineal, it was n e c e s s a r y t o s t u d y t h e s e r o t o n i n c o n c e n t r a t i o n u n d e r t h e s a m e e x p e r i m e n t a l conditions. The present investigation concerns the determination of t h e n u m b e r of cells c o n t a i n i n g yellow a u t o f l u o r e s c e n t m a t e r i a l a n d t h e q u a n t i t y of pineal serotonin, respectively, in a d u l t c a s t r a t e d male r a t s as c o m p a r e d t o t h o s e in c o n t r o l s over a p e r i o d of 3 w e e k s a f t e r c a s t r a t i o n . Material and methods. 84 m a l e w i s t a r r a t s (weight 200 g) were used for t h i s s t u d y (30 for t h e fluorescence histoc h e m i c a l s t u d y , 54 for t h e b i o c h e m i c a l s t u d y ) . T h e y were k e p t in cages a t 25 ~ a n d c o n s t a n t h u m i d i t y . T h e a n i m a l s were e x p o s e d to 12 h light daily (from 07.00 h t o 19.00 h). T h e y r e c e i v e d t a p w a t e r a n d s t a n d a r d r a t pellets ad libitum. F o u r t y - t w o r a t s were c a s t r a t e d b y t h e a b d o m i n a l route, t h e o t h e r s were s h a m - o p e r a t e d . 28 r a t s (14 c a s t r a t e d - 5 for fluorescence h i s t o c h e m i c a l s t u d y , 9 for b i o c h e m i c a l s t u d y - a n d 14 s h a m - o p e r a t e d ) w e r e killed 4 d a y s a f t e r c a s t r a t i o n a n d t h e 56 others, r e s p e c t i v e l y 1 a n d 3 w e e k s after the operation. Fluorescence histochemical study. A f t e r d e c a p i t a t i o n ( w i t h o u t a n a e s t h e s i a , b e t w e e n 09.00 a n d 10.00 h), t h e pineal was r e m o v e d w i t h i n 1 m i n a n d frozen ill freon-12, cooled t o - - 1 5 0 ~ b y liquid nitrogen. Sections of 15 ~xm w e r e c u t on a c r y o s t a t a t --18~ F r o m e a c h pineal gland, a p p r o x i m a t e l y 20 f r o n t a l sections were c u t as d e s c r i b e d above. 10 a l t e r n a t i n g sections were used for t h e s t u d y of a u t o f l u o r e s c e n t material. A f t e r c u t t i n g , t h e sections were t r a n s p o r t e d to a freeze-dryer. I n a v a c u u m c h a m b e r , t h e c r y o s t a t sections were f r o z e n - d r i e d a t - - 3 0 ~ for 45 min. D u r i n g t h i s period, t h e p r e s s u r e dec r e a s e d t o 0.01 Tor. T h e n t h e sections w e r e b r o u g h t w i t h i n 10 m i n t o r o o m t e m p e r a t u r e in vacuo. F o r 2 h,
EXPERIENTIA 31/10
Table II. Quantity of serotonin in the pineM gland of rat after castration (y/g of pineal tissue) 4 days Controls
Castrated
I week Controls
Castrated
3 weeks Controls Castrated
47.8 ~= 2.3 43.9 • 3.6 45.1 =k 4.6 46.8 ~ 5.4 46.4 • 4 45;3 ~ 4
t h e f r o z e n - d r i e d sections w e r e placed a t 80 ~ ( t e c h n i q u e of FALCK e t al. 4, as m o d i f i e d b y HEENE 5, w i t h o u t f o r m a l d e h y d e v a p o u r t r e a t m e n t . F o r m o r e details see SMITH 2, ~). F o r t h e d e m o n s t r a t i o n of autofluorescence, a Leitz fluorescence microscope e q u i p p e d w i t h a m e r c u r y l a m p (Hb0200) was used, T h e q u a n t i f i c a t i o n of yellow a u t o f l u o r e s c e n t cells was d e t e r m i n e d b y counting, in t h e d i f f e r e n t sections, t h e n u m b e r of t h e s e ceils p r e s e n t in a d e t e r m i n e d area (0.03 mm~). Countings were m a d e in 10 to 15 areas p e r section, t h e n u m b e r of areas s t u d i e d d e p e n d i n g o n t h e size of t h e section. T h e s t a n d a r d error w a s c a l c u l a t e d in t h e results o b t a i n e d in all r a t s of t h e g r o u p s t u d i e d (5 r a t s p e r group). Biochemical study. F o r t h e d e t e r m i n a t i o n of t h e q u a n t i t y of s e r o t o n i n in t h e pineal gland, pools were m a d e . F o r e a c h g r o u p (4 days, 1 a n d 3 w e e k s a f t e r c a s t r a t i o n ; 4 d a y s , 1 a n d 3 weeks a f t e r s h a m - o p e r a t i o n ) 3 d e t e r m i n a t i o n s were m a d e , a n d for 1 d e t e r m i n a t i o n 3 pineal glands were pooled. All r a t s were sacrificed b e t w e e n 09.15 h a n d 10.30 h. As this p e r i o d was r e l a t i v e l y long, c a s t r a t e d a n d c o n t r o l r a t s were sacrificed a l t e r n a t i v e l y . If t h e r e is an effect of t h e circadian r h y t h m on t h e s e r o t o n i n c o n c e n t r a t i o n of t h e pineal g l a n d d u r i n g t h i s t i m e lapse, t h e effect is i d e n t i c a l in t h e t w o groups, so t h a t it is possible t o c o m p a r e t h e s e t w o groups. I s o l a t i o n of s e r o t o n i n (5-HT) is b a s e d on A m b e r l i t e CG50 c o l u m n c h r o m a t o g r a p h y . P i n e a l glands were r e m o v e d , pooled a n d e x t r a c t e d in ice-cold 10 ml of 0.4 M perchloric acid, c o n t a i n i n g 0.1 m l ascorbic acid (2%) a n d 0.2 m l of 10% E D T A . T h e e x t r a c t s were a d j u s t e d to a p H of 5.6 w i t h 5 N K~Co 3, c e n t r i f u g e d a n d t h e supern a t a n t s were p u t on A m b e r l i t e CG50 c o l u m n s (4.5 • 50 mn)
4 B. FALCK,N. A. HILLARP,G. THIEME and A. ToRP, J. Histochem. 10, 348 (1962). R. HENNE, Histochemie 74, 324 (1968). 8 A. R. SMITH and J. A~Ns KAPPERS,Brain Res. 86, 353 (1975).
E ~ Controls 50
Castrated __ [
l
T
150
Fig. 1. Number of autofluorescent cells in the pineal gland after castration (cells/mm 2)
: I
Fig. 2. Quantity of serotonin in the pineal gland after castration (y/g of pineal tissue)
15.10. 1975
Specialia
a t a r a t e n o t e x c e e d i n g 0.5 m l / m n . T h e n t h e c o l u m n s w e r e w a s h e d w i t h 10 m l 0.02 M p h o s p h a t e b u f f e r (pH 6.5) c o n t a i n i n g 0.2% E D T A . E l u t i o n was p e r f o r m e d w i t h 1 N HC1. The first 6 m l of t h e elution c o n t a i n e d 5-HT. S e r o t o n i n was d e t e r m i n e d f l u o r i m e t r i c a l l y L The results were e x p r e s s e d in 7/g of pineal tissue, a n d t h e s t a n d a r d error is m e a s u r e d for each 3 d e t e r m i n a t i o n s . Results. 4 d a y s a f t e r t h e operation, no s t a t i s t i c a l l y s i g n i f i c a n t differences in t h e q u a n t i t y of a u t o f l u o r e s c e n t cells (Figure 1 a n d T a b l e I) a n d t h e q u a n t i t y of s e r o t o n i n (Figure 2 a n d Table II) b e t w e e n t h e c a s t r a t e d a n d s h a m o p e r a t e d r a t pineal glands were observed. One w e e k a f t e r t h e c a s t r a t i o n , t h e q u a n t i t y of a u t o f l u o r e s c e n t cells in t h e pineal g l a n d of t h e o p e r a t e d r a t s was a b o u t 37% increased in r e s p e c t to t h e s h a m - o p e r a t e d r a t g r o u p (Figure 1, T a b l e I). No s t a t i s t i c a l l y significant differences h a v e b e e n o b s e r v e d in t h e s e r o t o n i n c o n c e n t r a t i o n in t h e c a s t r a t e d a n d s h a m - o p e r a t e d r a t pineal g l a n d s (Figure 2, Table II). T h r e e -weeks a f t e r t h e o p e r a t i o n , t h e q u a n t i t y of a u t o f l u o r e s c e n t cells in t h e pineal gland of t h e c a s t r a t e d r a t s was increased, t h i s increase being larger t h a n a f t e r 1 w e e k (about 660/o in r e s p e c t t o t h e s h a m - o p e r a t e d group) (Figure 1, Table I). H o w e v e r , no s t a t i s t i c a l l y significant differences were o b s e r v e d b e t w e e n t h e s e r o t o n i n concent r a t i o n of t h e c a s t r a t e d a n d s h a m - o p e r a t e d r a t pineal g l a n d (Figure 2, T a b l e II). Discussion. P i n e a l r e s p o n s e to c a s t r a t i o n a p p e a r e d to be v e r y slow. 4 d a y s a f t e r t h e o p e r a t i o n we h a v e n o t o b s e r v e d a n y effect on t h e n u m b e r of a u t o f l u o r e s c e n t cells a n d t h e 5-HT levels. A f t e r 1 week, a small increase of t h e q u a n t i t y of a u t o f l u o r e s c e n t cells was observed, while a f t e r 3 weeks t h i s increase is larger. This slowness of t h e pineal r e s p o n s e is p o s s i b l y due to t h e p a r a m e t e r s chosen. I n d e e d , in o t h e r e x p e r i m e n t s using d i f f e r e n t p a r a m e t e r s (metabolic c o m p o u n d s S), we h a v e o b s e r v e d a q u i c k r e s p o n s e of t h e pineal g l a n d to c a s t r a t i o n . The f a c t t h a t t h e r e was no m o d i f i c a t i o n in s e r o t o n i n levels in t h e pineal g l a n d of o r c h i d e c t o m i z e d a d u l t r a t s is i n t e r e s t i n g b u t difficult t o i n t e r p r e t e . I t is possible t h a t t h e c a s t r a t i o n does n o t effect t h e p r o d u c t i o n of pineal indole d e r i v a t i v e s b u t it is also possible t h a t , after castration, t h e 5-HT t u r n o v e r changes w i t h o u t a n y c h a n g e in t h e 5HT levels in tissue. The increase of q u a n t i t y of cells c o n t a i n i n g autofluor e s c e n t m a t e r i a l d e m o n s t r a t e d t h a t o r c h i d e c t o m y h a s an effect on t h e pineal gland. J u s t a f t e r c a s t r a t i o n , an a b r u p t
1239
d r o p of a n d r o g e n p l a s m a levels occurs. This causes a r a p i d increase in p l a s m a g o n a d o t r o p i n ~-n. A t t h a t m o m e n t it is n o t possible to d e t e r m i n e w h e t h e r t h e effect of c a s t r a t i o n , d e s c r i b e d in t h e p r e s e n t paper, is due to t h e decreasing p l a s m a a n d r o g e n level or to t h e increase of F S H a n d L H levels. P e r h a p s b o t h m a y h a v e a n effect on t h e pineal gland. I t is i n t e r e s t i n g t h a t , some t i m e a f t e r c a s t r a t i o n , a n o t a b l e increase was o b s e r v e d of p i n e a l p r o t e i n s y n t h e s i s , i.e., increase of t h e q u a n t i t y of a u t o f l u o r e s c e n t material. I n t e n s i f i e d pineal p r o t e i n s y n t h e s i s is also k n o w n to occur in e s t r o g e n - t r e a t e d i m m a t u r e female rats. This is caused b y increased L H s e c r e t i o n b y t h e a d e n o h y p o p h y s i s 12. As c a s t r a t i o n also p r o v o k e s a n increase of L H s e c r e t i o n 9-~1, it m a y t h e r e f o r e be t h a t t h e increase of a u t o f l u o r e s c e n t p i n e a l o c y t e s a f t e r c a s t r a t i o n is caused b y i n c r e a s e d L H secretion. I t is possible t h a t t h e yellow a u t o f l u o r e s c e n t m a t e r i a l r e p r e s e n t s a n a c t i v e p r o t e i n c o m p o u n d (pineal h o r m o n e ?) d i f f e r e n t t o p i n e a l indole d e r i v a t i v e s . U n d e r t h e s e e x p e r i m e n t a l c o n d i t i o n s , c a s t r a t i o n m a y be r e s t r i c t e d to t h e s y n t h e s i s of t h e s e a c t i v e p r o t e i n c o m p o u n d s ~a.
Summary. The o r c h i d e c t o m y of t h e a d u l t r a t i n d u c e s a n increased q u a n t i t y of cells c o n t a i n i n g a u t o f l u o r e s c e n t m a t e r i a l ( p r o t e i n a c e o u s m a t e r i a l 1, 2).
P. PEVET, A. R. SMITH,X. VAN DE I(AR a n d H. VAN BRONSWIJK 11
Netherlands Central Institute for Brain Research, Ijdijk 28, Hmsterdam-O (The Netherlands), 28 February 1975.
7 N. E. A~DEN and iF. MAGNUSSON, Acta physiol, scand. 69, 89 (1967). 8 l). PEVI,;T,in preparation. 9 V. L. GAY and A. R. MID(;LEY, Endocrinology 8d, 1359 (1969). 10 M. YAMA:,IOTO,N. 1). I)I~-'BICLand E. M. BOGDANOW:,Endocrinology 86, 1102 (1970). 11 R. 15. BLACKWELLand M. S. AMOSS,Expl. Biol. Med. 136, 11 (1971). 12 I. NIR, N. KAISER,N. ttlRSCItMANN and F. G. SULMAN,Life Sci. 9, 851 (1970). ~s R. J. WURT.~tA~, H. M. SnEIN, J. Axelrod and F. LARIN, Proe. natn. Acad. Sci., USA 62, 749 (1969). 14 Acknowledgments. The authors are much indebted to Mr. S. 1.n,:M and I. OBERINKfor technical assistance and Miss P. RORING for typing the manuscript.
Suppression of Pupal Esterase Activity in Aedes aegypti (Diptera: Culicidae) by an Insect Growth Regulator Considerable i n t e r e s t has b e e n g e n e r a t e d b y t h e suggestion t h a t insect p o p u l a t i o n s m a y be c o n t r o l l e d b y use of analogues a n d m i m i c s of n a t u r a l juvenile h o r m o n e 1, 2. These insect g r o w t h r e g u l a t o r s (IGR) are believed to alter t h e n o r m a l h o r m o n a l b a l a n c e a n d t h u s interfere w i t h p o s t - e m b r y o n i c d e v e l o p m e n t ; h o w e v e r , t h e precise m o d e of action h a s n o t y e t b e e n resolved. SLADE a n d WlLI~INSON 3 h a v e i n d i c a t e d t h a t , because m a n y I G R ' s are s t r u c t u r a l l y dissimilar to t h e n a t u r a l h o r m o n e , it is unlikely t h a t t h e i r effect is m e d i a t e d d i r e c t l y t h r o u g h a n i n t e r a c t i o n w i t h t h e n a t u r a l h o r m o n e receptor. T h e y p r o p o s e t h a t t h e I G R ' s stabilize t h e n a t u r a l h o r m o n e b y i n h i b i t i n g t h e n o r m a l d e g r a d a t i o n p a t h w a y s . One e n z y m e r e s p o n s i b l e for c a t a b o l i s m of e n d o g e n o u s juvenile h o r m o n e is a c a r b o x y e s t e r a s e w h i c h is i n d u c e d w i t h i n 30
m i n of t h e a p p e a r a n c e of j u v e n i l e h o r m o n e 4. T h e p r e s e n t s t u d y was u n d e r t a k e n to d e t e r m i n e t h e effect of one I G R (isopropyl 11-methoxy-3, 7, 1 1 - t r i m e t h y l - d o d e c a - 2 , 4- dienoate, Altosid | (ZR 515) o n t h e esterases of t h e m o s q u i t o , Aedes aegypti (L.), an insect species w h i c h is p a r t i c u l a r l y sensitive to t h e effects of I G R ' s ~.
1 Z. SLAMA, A. Rev. Biochem. dO, 1079 (1971). 2 j. j. MEN~; and M. BEROZA, Insect ,Juvenile Hormones: Chemistry and Action (Academic Press, New York 1972). 3 M. SLADEand C. F. WILKINSON,Science 781, 672 (1973). 4 D. WHITMORE JR., ]~. WHITMORE and L. I. GILBERT, Proc. natn. Acad. Sci., USA 69, 1592 (1972). 5 A. SPIELMANand C. M. ~VILLIAMS, Science 154, 1043 (1966).
Speeialia
1237
1
Fig. 1. Control 1-day-old rat testis. The seminiferous tubules show gonocytes and supporting cells. Occasional Leydig ceils. HE, • 320. Fig. 2. LH-RH treated, l-day-old rat testis. Similar cell types. HE, • 320. Fig. 3. Control ll-day-old rat testis. The tubular diameter and the nmnber of supporting cells has increased. Primitive type A spermatogortia are seen. He, • 320.
Fig. 4. LH-RH treated l l-day-old rat testis. Similar to the Figure 3. HE, x320. Fig. 5. Control 13-day-old rat testis. Tubular diameter increased. Adult type spermatogonia and primary spermatocyt@s are present in the germinal epithelium. HE, • 320. Fig. 6. LH-RH treated 13-day-old-rat. Testes similar to the control. HE, • 320. r e p o r t e d o n l y slight s t i m u l a t i o n of t h e testis. RAMIREZ a n d MCCANN 9 h a v e s u g g e s t e d t h a t t h e o n s e t of ' p u b e r t y ' in t h e r a t is d e t e r m i n e d b y a c h a n g e in t h e h y p o p h y s i s s e n s i t i v i t y to releasing h o r m o n e s r a t h e r t h a n b y c h a n g e s of t h e g o n a d o t r o p i n c o n t e n t of t h e glands. I n h u m a n , ROTH et al.~ s t a t e d t h a t p u b e r t a l a n d a d u l t t e s t e s a p p e a r s to be m o r e r e s p o n s i v e t h a n t h e i m m a t u r e testis in releasing t e s t o s t e r o n e after t h e a d m i n i s t r a t i o n of L H R H . Also SANDOW a n d BABEJ 10 s p e c u l a t e d t h a t chronic L H - R H t r e a t m e n t m a y h a v e a n e g a t i v e f e e d - b a c k effect on p i t u i t a r y s e n s i t i v i t y t o t h e g o n a d o t r o p i n releasing hormones. O u r results showed t h a t a d m i n i s t e r e d s y n t h e t i c L H R H is n e i t h e r able to i n i t i a t e per se t h e d e v e l o p m e n t of s p e r m a t o g e n e s i s a n d m a t u r a t i o n of L e y d i g cells, n o r to accelerate t h e n o r m a l r h y t h m of t h e n a t u r a l e v o l u t i o n of t h e gonads. I t is q u i t e possible t h a t o t h e r m e c h a n i s m s m i g h t be i n v o l v e d in t h e o n s e t of p u b e r t y . On t h e o t h e r h a n d , we h a d no e v i d e n c e of a n e g a t i v e f e e d - b a c k i n d u c e d b y t h e i n j e c t e d L H - R H , since b o t h t r e a t e d a n d u n t r e a t e d a n i m a l s s h o w e d n e i t h e r q u a l i t a t i v e nor q u a n t i t a t i v e histological differences in t h e testis. The significance of t h e h i g h e r n u m b e r of s p e r m a t o g o n i a l cells in u n t r e a t e d 14-day-old r a t s is o p e n to q u e s t i o n .
Resumen. R a t a s m a c h o s i n m a d u r o s t r a t a d o s cr6nic a m e n t e con f a c t o r l i b e r a d o r sintdtico de L H d e s d e el p r i m e r o h a s t a el 15 dia de e d a d a l a s ddsis de 5 ~xg subcutAneos c u a t r o veces al dia c a d s c u a t r o h o r a s p o r animal, fueron i n c a p a c e s de iniciar o acelerar el proceso de m a d u racidn t e s t i c u l a r e s t u d i a n d o el proceso e s p e r m a t o g 6 n i c o por c o n t a g e s eelulares diferenciales en secciones t r a n s versales y el desarrollo de las c61ulas de L e y d i g m a d u r a s . M. A. GARCIA-HJARLES 11 a n d O. VILAR 12
Centro de Investigaciones en Reproducci6n, 2a Cdtedra de Histologla, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 10 ~ piso, Bzwnos Aires (Rep~blica Argentina), 15 A p r i l 1975. D. V. RAMIREZand S. M. M~CCANN,Endocrinology 72, 452 (1963). 10 j. SANDOWand 1Vf.BABEL Acta endocr., Copenh. Suppl. 177, 297 (1973). 11 Post-Graduate fellow of the Latin American Training Course on Reproductive Biology (W.H.0.). Inst. de Invest. de la Altura, Apt. No. 6083, Lima, Peru. 12 Established Investigator, Consejo Nacional de Investigaciones Cientlficas y Tdcnicas. R6publica Argentina.
Effect of C a s t r a t i o n o n t h e R a t P i n e a l G l a n d : a F l u o r e s c e n c e E a r l i e r i n v e s t i g a t i o n s 1 a d e m o n s t r a t e d t h a t some p i n e a l o c y t e s of t h e r a b b i t , rat, h e d g e h o g a n d mole c o n t a i n a yellow a u t o f l u o r e s c e n t s u b s t a n c e . I n r a b b i t a n d r a t pinealocytes, t h i s m a t e r i a l a p p e a r e d t o be a t r y p t o p h a n - r i c h p r o t e i n 1, 2 t h e f u n c t i o n of w h i c h is still u n k n o w n . i t m i g h t p o s s i b l y be a carrier p r o t e i n of pineal h o r m o n e s 2. Considering t h i s h y p o t h e s i s , it s e e m e d of i n t e r e s t to
Histochemical
and Biochemical
Study
d e t e r m i n e , in t h e r a t pineal gland, t h e q u a n t i t y of cells c o n t a i n i n g yellow a u t o f l u o r e s c e n t m a t e r i a l u n d e r v a r y i n g 1 A. R. SMITH, J. F. JONGKIND and J. ARIJ~NS KAPPERS, Gen. cotnp. Endoer. 78, 364 (1972). 2 A. R. SmTu, Thesis (Amsterdam 1972). a p. PEVET, not published.
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Specialia
Table I. Number of autofluorescent cells in the pineal gland after
castration (cells/mm 2) 4 days Controls
Castrated
158 i 15 147 ~ 23
1 week Controls
Castrated
3 weeks Controls
137 ~ 15
216 -~ 44
160 -4- 37 287 :k 40
Castrated
e x p e r i m e n t a l c o n d i t i o n s r e l a t e d t o t h e f u n c t i o n of t h e e p i p h y s i s in t h e r e g u l a t i o n of t h e h y p o t h a l a m o - h y p o p h y s e o - g o n a d a l s y s t e m . B e c a u s e S~ITH 2 d e m o n s t r a t e d an inverse r e l a t i o n in d i f f e r e n t c i r c u m s t a n c e s b e t w e e n t h e a m o u n t of a u t o f l u o r e s c e n t m a t e r i a l a n d t h a t of s e r o t o n i n in t h e r a t pineal, it was n e c e s s a r y t o s t u d y t h e s e r o t o n i n c o n c e n t r a t i o n u n d e r t h e s a m e e x p e r i m e n t a l conditions. The present investigation concerns the determination of t h e n u m b e r of cells c o n t a i n i n g yellow a u t o f l u o r e s c e n t m a t e r i a l a n d t h e q u a n t i t y of pineal serotonin, respectively, in a d u l t c a s t r a t e d male r a t s as c o m p a r e d t o t h o s e in c o n t r o l s over a p e r i o d of 3 w e e k s a f t e r c a s t r a t i o n . Material and methods. 84 m a l e w i s t a r r a t s (weight 200 g) were used for t h i s s t u d y (30 for t h e fluorescence histoc h e m i c a l s t u d y , 54 for t h e b i o c h e m i c a l s t u d y ) . T h e y were k e p t in cages a t 25 ~ a n d c o n s t a n t h u m i d i t y . T h e a n i m a l s were e x p o s e d to 12 h light daily (from 07.00 h t o 19.00 h). T h e y r e c e i v e d t a p w a t e r a n d s t a n d a r d r a t pellets ad libitum. F o u r t y - t w o r a t s were c a s t r a t e d b y t h e a b d o m i n a l route, t h e o t h e r s were s h a m - o p e r a t e d . 28 r a t s (14 c a s t r a t e d - 5 for fluorescence h i s t o c h e m i c a l s t u d y , 9 for b i o c h e m i c a l s t u d y - a n d 14 s h a m - o p e r a t e d ) w e r e killed 4 d a y s a f t e r c a s t r a t i o n a n d t h e 56 others, r e s p e c t i v e l y 1 a n d 3 w e e k s after the operation. Fluorescence histochemical study. A f t e r d e c a p i t a t i o n ( w i t h o u t a n a e s t h e s i a , b e t w e e n 09.00 a n d 10.00 h), t h e pineal was r e m o v e d w i t h i n 1 m i n a n d frozen ill freon-12, cooled t o - - 1 5 0 ~ b y liquid nitrogen. Sections of 15 ~xm w e r e c u t on a c r y o s t a t a t --18~ F r o m e a c h pineal gland, a p p r o x i m a t e l y 20 f r o n t a l sections were c u t as d e s c r i b e d above. 10 a l t e r n a t i n g sections were used for t h e s t u d y of a u t o f l u o r e s c e n t material. A f t e r c u t t i n g , t h e sections were t r a n s p o r t e d to a freeze-dryer. I n a v a c u u m c h a m b e r , t h e c r y o s t a t sections were f r o z e n - d r i e d a t - - 3 0 ~ for 45 min. D u r i n g t h i s period, t h e p r e s s u r e dec r e a s e d t o 0.01 Tor. T h e n t h e sections w e r e b r o u g h t w i t h i n 10 m i n t o r o o m t e m p e r a t u r e in vacuo. F o r 2 h,
EXPERIENTIA 31/10
Table II. Quantity of serotonin in the pineM gland of rat after castration (y/g of pineal tissue) 4 days Controls
Castrated
I week Controls
Castrated
3 weeks Controls Castrated
47.8 ~= 2.3 43.9 • 3.6 45.1 =k 4.6 46.8 ~ 5.4 46.4 • 4 45;3 ~ 4
t h e f r o z e n - d r i e d sections w e r e placed a t 80 ~ ( t e c h n i q u e of FALCK e t al. 4, as m o d i f i e d b y HEENE 5, w i t h o u t f o r m a l d e h y d e v a p o u r t r e a t m e n t . F o r m o r e details see SMITH 2, ~). F o r t h e d e m o n s t r a t i o n of autofluorescence, a Leitz fluorescence microscope e q u i p p e d w i t h a m e r c u r y l a m p (Hb0200) was used, T h e q u a n t i f i c a t i o n of yellow a u t o f l u o r e s c e n t cells was d e t e r m i n e d b y counting, in t h e d i f f e r e n t sections, t h e n u m b e r of t h e s e ceils p r e s e n t in a d e t e r m i n e d area (0.03 mm~). Countings were m a d e in 10 to 15 areas p e r section, t h e n u m b e r of areas s t u d i e d d e p e n d i n g o n t h e size of t h e section. T h e s t a n d a r d error w a s c a l c u l a t e d in t h e results o b t a i n e d in all r a t s of t h e g r o u p s t u d i e d (5 r a t s p e r group). Biochemical study. F o r t h e d e t e r m i n a t i o n of t h e q u a n t i t y of s e r o t o n i n in t h e pineal gland, pools were m a d e . F o r e a c h g r o u p (4 days, 1 a n d 3 w e e k s a f t e r c a s t r a t i o n ; 4 d a y s , 1 a n d 3 weeks a f t e r s h a m - o p e r a t i o n ) 3 d e t e r m i n a t i o n s were m a d e , a n d for 1 d e t e r m i n a t i o n 3 pineal glands were pooled. All r a t s were sacrificed b e t w e e n 09.15 h a n d 10.30 h. As this p e r i o d was r e l a t i v e l y long, c a s t r a t e d a n d c o n t r o l r a t s were sacrificed a l t e r n a t i v e l y . If t h e r e is an effect of t h e circadian r h y t h m on t h e s e r o t o n i n c o n c e n t r a t i o n of t h e pineal g l a n d d u r i n g t h i s t i m e lapse, t h e effect is i d e n t i c a l in t h e t w o groups, so t h a t it is possible t o c o m p a r e t h e s e t w o groups. I s o l a t i o n of s e r o t o n i n (5-HT) is b a s e d on A m b e r l i t e CG50 c o l u m n c h r o m a t o g r a p h y . P i n e a l glands were r e m o v e d , pooled a n d e x t r a c t e d in ice-cold 10 ml of 0.4 M perchloric acid, c o n t a i n i n g 0.1 m l ascorbic acid (2%) a n d 0.2 m l of 10% E D T A . T h e e x t r a c t s were a d j u s t e d to a p H of 5.6 w i t h 5 N K~Co 3, c e n t r i f u g e d a n d t h e supern a t a n t s were p u t on A m b e r l i t e CG50 c o l u m n s (4.5 • 50 mn)
4 B. FALCK,N. A. HILLARP,G. THIEME and A. ToRP, J. Histochem. 10, 348 (1962). R. HENNE, Histochemie 74, 324 (1968). 8 A. R. SMITH and J. A~Ns KAPPERS,Brain Res. 86, 353 (1975).
E ~ Controls 50
Castrated __ [
l
T
150
Fig. 1. Number of autofluorescent cells in the pineal gland after castration (cells/mm 2)
: I
Fig. 2. Quantity of serotonin in the pineal gland after castration (y/g of pineal tissue)
15.10. 1975
Specialia
a t a r a t e n o t e x c e e d i n g 0.5 m l / m n . T h e n t h e c o l u m n s w e r e w a s h e d w i t h 10 m l 0.02 M p h o s p h a t e b u f f e r (pH 6.5) c o n t a i n i n g 0.2% E D T A . E l u t i o n was p e r f o r m e d w i t h 1 N HC1. The first 6 m l of t h e elution c o n t a i n e d 5-HT. S e r o t o n i n was d e t e r m i n e d f l u o r i m e t r i c a l l y L The results were e x p r e s s e d in 7/g of pineal tissue, a n d t h e s t a n d a r d error is m e a s u r e d for each 3 d e t e r m i n a t i o n s . Results. 4 d a y s a f t e r t h e operation, no s t a t i s t i c a l l y s i g n i f i c a n t differences in t h e q u a n t i t y of a u t o f l u o r e s c e n t cells (Figure 1 a n d T a b l e I) a n d t h e q u a n t i t y of s e r o t o n i n (Figure 2 a n d Table II) b e t w e e n t h e c a s t r a t e d a n d s h a m o p e r a t e d r a t pineal glands were observed. One w e e k a f t e r t h e c a s t r a t i o n , t h e q u a n t i t y of a u t o f l u o r e s c e n t cells in t h e pineal g l a n d of t h e o p e r a t e d r a t s was a b o u t 37% increased in r e s p e c t to t h e s h a m - o p e r a t e d r a t g r o u p (Figure 1, T a b l e I). No s t a t i s t i c a l l y significant differences h a v e b e e n o b s e r v e d in t h e s e r o t o n i n c o n c e n t r a t i o n in t h e c a s t r a t e d a n d s h a m - o p e r a t e d r a t pineal g l a n d s (Figure 2, Table II). T h r e e -weeks a f t e r t h e o p e r a t i o n , t h e q u a n t i t y of a u t o f l u o r e s c e n t cells in t h e pineal gland of t h e c a s t r a t e d r a t s was increased, t h i s increase being larger t h a n a f t e r 1 w e e k (about 660/o in r e s p e c t t o t h e s h a m - o p e r a t e d group) (Figure 1, Table I). H o w e v e r , no s t a t i s t i c a l l y significant differences were o b s e r v e d b e t w e e n t h e s e r o t o n i n concent r a t i o n of t h e c a s t r a t e d a n d s h a m - o p e r a t e d r a t pineal g l a n d (Figure 2, T a b l e II). Discussion. P i n e a l r e s p o n s e to c a s t r a t i o n a p p e a r e d to be v e r y slow. 4 d a y s a f t e r t h e o p e r a t i o n we h a v e n o t o b s e r v e d a n y effect on t h e n u m b e r of a u t o f l u o r e s c e n t cells a n d t h e 5-HT levels. A f t e r 1 week, a small increase of t h e q u a n t i t y of a u t o f l u o r e s c e n t cells was observed, while a f t e r 3 weeks t h i s increase is larger. This slowness of t h e pineal r e s p o n s e is p o s s i b l y due to t h e p a r a m e t e r s chosen. I n d e e d , in o t h e r e x p e r i m e n t s using d i f f e r e n t p a r a m e t e r s (metabolic c o m p o u n d s S), we h a v e o b s e r v e d a q u i c k r e s p o n s e of t h e pineal g l a n d to c a s t r a t i o n . The f a c t t h a t t h e r e was no m o d i f i c a t i o n in s e r o t o n i n levels in t h e pineal g l a n d of o r c h i d e c t o m i z e d a d u l t r a t s is i n t e r e s t i n g b u t difficult t o i n t e r p r e t e . I t is possible t h a t t h e c a s t r a t i o n does n o t effect t h e p r o d u c t i o n of pineal indole d e r i v a t i v e s b u t it is also possible t h a t , after castration, t h e 5-HT t u r n o v e r changes w i t h o u t a n y c h a n g e in t h e 5HT levels in tissue. The increase of q u a n t i t y of cells c o n t a i n i n g autofluor e s c e n t m a t e r i a l d e m o n s t r a t e d t h a t o r c h i d e c t o m y h a s an effect on t h e pineal gland. J u s t a f t e r c a s t r a t i o n , an a b r u p t
1239
d r o p of a n d r o g e n p l a s m a levels occurs. This causes a r a p i d increase in p l a s m a g o n a d o t r o p i n ~-n. A t t h a t m o m e n t it is n o t possible to d e t e r m i n e w h e t h e r t h e effect of c a s t r a t i o n , d e s c r i b e d in t h e p r e s e n t paper, is due to t h e decreasing p l a s m a a n d r o g e n level or to t h e increase of F S H a n d L H levels. P e r h a p s b o t h m a y h a v e a n effect on t h e pineal gland. I t is i n t e r e s t i n g t h a t , some t i m e a f t e r c a s t r a t i o n , a n o t a b l e increase was o b s e r v e d of p i n e a l p r o t e i n s y n t h e s i s , i.e., increase of t h e q u a n t i t y of a u t o f l u o r e s c e n t material. I n t e n s i f i e d pineal p r o t e i n s y n t h e s i s is also k n o w n to occur in e s t r o g e n - t r e a t e d i m m a t u r e female rats. This is caused b y increased L H s e c r e t i o n b y t h e a d e n o h y p o p h y s i s 12. As c a s t r a t i o n also p r o v o k e s a n increase of L H s e c r e t i o n 9-~1, it m a y t h e r e f o r e be t h a t t h e increase of a u t o f l u o r e s c e n t p i n e a l o c y t e s a f t e r c a s t r a t i o n is caused b y i n c r e a s e d L H secretion. I t is possible t h a t t h e yellow a u t o f l u o r e s c e n t m a t e r i a l r e p r e s e n t s a n a c t i v e p r o t e i n c o m p o u n d (pineal h o r m o n e ?) d i f f e r e n t t o p i n e a l indole d e r i v a t i v e s . U n d e r t h e s e e x p e r i m e n t a l c o n d i t i o n s , c a s t r a t i o n m a y be r e s t r i c t e d to t h e s y n t h e s i s of t h e s e a c t i v e p r o t e i n c o m p o u n d s ~a.
Summary. The o r c h i d e c t o m y of t h e a d u l t r a t i n d u c e s a n increased q u a n t i t y of cells c o n t a i n i n g a u t o f l u o r e s c e n t m a t e r i a l ( p r o t e i n a c e o u s m a t e r i a l 1, 2).
P. PEVET, A. R. SMITH,X. VAN DE I(AR a n d H. VAN BRONSWIJK 11
Netherlands Central Institute for Brain Research, Ijdijk 28, Hmsterdam-O (The Netherlands), 28 February 1975.
7 N. E. A~DEN and iF. MAGNUSSON, Acta physiol, scand. 69, 89 (1967). 8 l). PEVI,;T,in preparation. 9 V. L. GAY and A. R. MID(;LEY, Endocrinology 8d, 1359 (1969). 10 M. YAMA:,IOTO,N. 1). I)I~-'BICLand E. M. BOGDANOW:,Endocrinology 86, 1102 (1970). 11 R. 15. BLACKWELLand M. S. AMOSS,Expl. Biol. Med. 136, 11 (1971). 12 I. NIR, N. KAISER,N. ttlRSCItMANN and F. G. SULMAN,Life Sci. 9, 851 (1970). ~s R. J. WURT.~tA~, H. M. SnEIN, J. Axelrod and F. LARIN, Proe. natn. Acad. Sci., USA 62, 749 (1969). 14 Acknowledgments. The authors are much indebted to Mr. S. 1.n,:M and I. OBERINKfor technical assistance and Miss P. RORING for typing the manuscript.
Suppression of Pupal Esterase Activity in Aedes aegypti (Diptera: Culicidae) by an Insect Growth Regulator Considerable i n t e r e s t has b e e n g e n e r a t e d b y t h e suggestion t h a t insect p o p u l a t i o n s m a y be c o n t r o l l e d b y use of analogues a n d m i m i c s of n a t u r a l juvenile h o r m o n e 1, 2. These insect g r o w t h r e g u l a t o r s (IGR) are believed to alter t h e n o r m a l h o r m o n a l b a l a n c e a n d t h u s interfere w i t h p o s t - e m b r y o n i c d e v e l o p m e n t ; h o w e v e r , t h e precise m o d e of action h a s n o t y e t b e e n resolved. SLADE a n d WlLI~INSON 3 h a v e i n d i c a t e d t h a t , because m a n y I G R ' s are s t r u c t u r a l l y dissimilar to t h e n a t u r a l h o r m o n e , it is unlikely t h a t t h e i r effect is m e d i a t e d d i r e c t l y t h r o u g h a n i n t e r a c t i o n w i t h t h e n a t u r a l h o r m o n e receptor. T h e y p r o p o s e t h a t t h e I G R ' s stabilize t h e n a t u r a l h o r m o n e b y i n h i b i t i n g t h e n o r m a l d e g r a d a t i o n p a t h w a y s . One e n z y m e r e s p o n s i b l e for c a t a b o l i s m of e n d o g e n o u s juvenile h o r m o n e is a c a r b o x y e s t e r a s e w h i c h is i n d u c e d w i t h i n 30
m i n of t h e a p p e a r a n c e of j u v e n i l e h o r m o n e 4. T h e p r e s e n t s t u d y was u n d e r t a k e n to d e t e r m i n e t h e effect of one I G R (isopropyl 11-methoxy-3, 7, 1 1 - t r i m e t h y l - d o d e c a - 2 , 4- dienoate, Altosid | (ZR 515) o n t h e esterases of t h e m o s q u i t o , Aedes aegypti (L.), an insect species w h i c h is p a r t i c u l a r l y sensitive to t h e effects of I G R ' s ~.
1 Z. SLAMA, A. Rev. Biochem. dO, 1079 (1971). 2 j. j. MEN~; and M. BEROZA, Insect ,Juvenile Hormones: Chemistry and Action (Academic Press, New York 1972). 3 M. SLADEand C. F. WILKINSON,Science 781, 672 (1973). 4 D. WHITMORE JR., ]~. WHITMORE and L. I. GILBERT, Proc. natn. Acad. Sci., USA 69, 1592 (1972). 5 A. SPIELMANand C. M. ~VILLIAMS, Science 154, 1043 (1966).
