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Inhibition of in vitro human chorionic gonadotropin-stimulated testosterone production in testis and of ovulation in the rat by charcoal-treated rat testicular extract
Life Sciences, Vol. 34, pp. 273-280 Printed in the U.S.A.
Pergamon Press
INHIBITION OF IN VITRO HUMAN CHORIONIC GONADOTROPIN-STIMULATED TESTOSTERONE PRODUCTION IN TESTIS AND OF OVULATION IN THE RAT BY CHARCOAL-TREATED RAT TESTICULAR EXTRACT* Gabriela A. de Bellabarba 1, Walter Bishop 1 and Francisco 7. Rojas 2 1Departamento de Fisiopatologia, Facultad de Medicina, Universidad de Los Andes, M6rida, Venezuela and 2Department of O b s t e t r i c s and Gynecology, The U n i v e r s i t y of T e x a s Health Science Center at San Antonio, Texas 78284 (Received in final form November i, 1983) SUMMARY
P r e v i o u s l y , we d e s c r i b e d the p r e s e n c e of a factor obtained from a 105,000 x g s u p e r n a t a n t of r a t testis that was found to inhibit human chorionic gonadotropin (hCG) binding to gonadal receptors. In the p r e s e n t s t u d y , similarly p r e p a r e d testicular e x t r a c t was t e s t e d for its effects on in vitro hCG-stimulated t e s t o s t e r o n e production by isolated testis ] h t e r ~ i a l cells and for its effect on s p o n t a n e o u s ovulation in the r a t . Incubation of interstitial cells with c h a r c o a l - t r e a t e d e x t r a c t significantly inhibited the steroidogenic r e s p o n s e to hCG in a d o s e - r e l a t e d manner. This inhibition was also a p p a r e n t a f t e r heating the e x t r a c t for 10 rain at 100°C. Preincubation of the cells with c h a r c o a l - t r e a t e d e x t r a c t r e s u l t e d in an inhibitory effect that was not readily r e v e r s e d b y s u b s e q u e n t addition of hCG, revealing an element of i r r e v e r s i b i l i t y in the mechanism of inhibition. A single i . p . injection of testicular e x t r a c t given between 1430-1630 h of p r o e s t r u s inhibited s p o n t a n e ous ovulation in the r a t . This effect was also o b s e r v e d a f t e r heating the e x t r a c t for 10 min at 100°C; in c o n t r a s t , no significant effect was obtained with the injection of a similar dose of liver extract. Administration of 5 IU hCG a f t e r p r e t r e a t m e n t with the testicular e x t r a c t did not r e v e r s e the inhibitory effect on ovulation, indicating t h a t this effect was p r o b a b l y not e x e r t e d at the h y p o t h a l a m u s - p i t u i t a r y level. It is concluded that the aqueous testicular e x t r a c t contains a factor able to antagonize the physiological e v e n t s mediated b y luteinizing hormone ( L H ) / h C G , and t h a t this factor is consistent with the p r e s e n c e of an L H / h C G - b i n d i n g inhibitory activity in r a t testis.
*Supported by an Institutional Research Grant (S07-RR05654) from The University of Texas Health Science Center at San Antonio and by the Consejo de DesarroIlo Cient[fico y Humanfstico de la Universidad de Los Andes. This work was presented in part at the 16th Annual Meeting of the Society for the Study of Reproduction, August 8-10, 1983, Cleveland, Ohio. Address requests for reprints to: Dr. Francisco J. Rojas, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284. 0024-3205/8& $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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Recently, we d e s c r i b e d the p r e s e n c e of a factor having the ability to inhibit luteinizing hormone gonadotropin (hCG) binding to gonadal r e c e p t o r s (1). heat stable, not steroid in n a t u r e and p r e s e n t e d Binding studies and inhibition kinetics indicated t h a t hCG binding mainly b y i n t e r f e r i n g with the formation complex and t h a t it competes with the hormone for C o n s e q u e n t l y , it was d e m o n s t r a t e d t h a t the inhibitor of the 9onadotropin and its r e c e p t o r s in t e s t i c u l a r and
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obtained from r a t testis ( L H ) / h u m a n chorionic This factor was partially a low molecular weight. the inhibitor affects LH/ of the h o r m o n e - r e c e p t o r the same binding sites. p r e v e n t s the interaction ovarian t i s s u e s .
In the p r e s e n t r e p o r t , we extend our p r e v i o u s findings and demonstrate that charcoal-treated testicular extract interferes with hCG-stimulated t e s t o s t e r o n e production b y e n z y m e - d i s p e r s e d t e s t i c u l a r interstitial cells in vitro and t h a t its administration d u r i n g p r o e s t r u s inhibits ovulation in tKe r a t . These r e s u l t s are c o n s i s t e n t with the p r e s e n c e of an LH/hCG binding inhibitor in r a t t e s t i s , s u g g e s t i n g t h a t this factor is not only able to inhibit the binding of the hormone to the r e c e p t o r s , b u t also to act as an a n t a g o n i s t of the physiological functions mediated b y LH/hCG. Materials and Methods P r e p a r a t i o n s of a 105,000 x 9 s u p e r n a t a n t of r a t liver and of r a t testis containing the LH/hCG binding inhibitor were obtained in either p h o s p h a t e b u f f e r (0.01 M sodium p h o s p h a t e b u f f e r , pH 7.5) or in Medium 199 (Gibco L a b o r a t o r i e s ) as d e s c r i b e d elsewhere (1). Under these conditions, the r e s u l t ing aqueous e x t r a c t s contained 100 m9 tissue equivalent p e r 100 pl of e x t r a c t . Charcoal t r e a t m e n t involved addition of 200 mg charcoal (Norit A) p e r 2 ml t e s t i c u l a r e x t r a c t i n c u b a t e d for 30 min at 4°C. Pilot experiments using [3H] t e s t o s t e r o n e showed t h a t charcoal removed more t h a n 98% of the added steroid. For heating s t u d i e s , c h a r c o a l - t r e a t e d e x t r a c t s were boiled in a water bath for 10 min followed b y c e n t r i f u g a t i o n at 10,000 x 9 for 10 rain. The r e s u l t i n g clear s u p e r n a t a n t was t h e n u s e d for inhibitory activities. In g e n e r a l , heating of the e x t r a c t s was p e r f o r m e d just before t h e i r use. P r e p a r a t i o n of d i s p e r s e d interstitial cells was c a r r i e d out b y collagenase digestion of d e c a p s u l a t e d r a t testis according to the method d e s c r i b e d b y Dufau et al. (2). Final r e s u s p e n s i o n of the cells was in Medium 199 at a ratio of 100 mg tissue p e r 200 pl of cell s u s p e n s i o n . The general p r o c e d u r e s outlined b y C i g o r r a g a et al. (3) were employed for cell incubations. Two h u n d r e d microliters of cell sfi-spension were incubated at 34°C u n d e r an atmosphere of 95% 0 2:5% CO 2 in a final volume of I ml of Medium 199. At the end of the incubation period, the mixture was boiled in a water b a t h for 5 min, centrif u g e d at 10,000 x g for 10 min and the s u p e r n a t a n t stored at -30°C for s u b s e q u e n t determination of total (medium plus t i s s u e ) t e s t o s t e r o n e production b y radioimmunoassay. In the p r e s e n c e of an excess of hCG (5 IU; A y e r s t L a b s , New Y o r k ) , the total t e s t o s t e r o n e p r o d u c t i o n was linear for at least 3 hours. The a n t i s e r u m to t e s t o s t e r o n e , obtained from Radioassay System L a b o r a t o r i e s ( C a r s o n , CA), had high specificity for t e s t o s t e r o n e with relatively low c r o s s - r e a c t i v i t y for d i h y d r o t e s t o s t e r o n e (6.6%), h 4 - a n d r o s t e n e d i o n e (0.9%) and 5 a - a n d r o s t a n e - 3 t L 17~-diol (2.19%). Radioimmunoassays were p e r f o r m e d directly on samples as outlined b y Dufau et al. (4). The i n t e r a s s a y and i n t r a - a s s a y coefficients of variation were 12.5°~-an~ 10.1%, r e s p e c t i v e l y . Rats of the S p r a g u e - D a w l e y s t r a i n were u s e d . The animals were housed in a controlled e n v i r o n m e n t , 12 L:12 D, with r a t chow and tap w a t e r available ad libitum, and were sacrificed by decapitation immediately p r i o r to use. For ~-u l¢ti-e~ on inhibition of ovulation, r a t s exhibiting at least two consecutive
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Antigonadotropin Activity in Rat Testis
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4 - d a y cycles were selected; the LH s u r g e was o b s e r v e d between 1600 h and 1800 h of p r o e s t r u s . The influence of c h a r c o a l - t r e a t e d testicular and liver e x t r a c t s , or charcoal- and h e a t - t r e a t e d testicular e x t r a c t s was a s s e s s e d in normal p r o e s t r o u s r a t s r e c e i v i n g a single i . p . injection of 3.5 g tissue equivalent/BW at various times between 1130 h and 1630 h. The r a t s were sacrificed on the following day (1000 h of e s t r u s ) and the number of ova in the oviducts counted u n d e r a dissecting microscope. Data were analyzed using S t u d e n t ' s t t e s t . Results Effects of c h a r c o a l - t r e a t e d testicular e x t r a c t upon hCG-stimulated steroidogenesis ~ y d i s p e r s e d testicular l~ff~-rstitial cells in vitro. Incubation of interstitial cells with 5 IU of hCG in the p r e s e n c e of t e s t i c u l a r e x t r a c t s which had been t r e a t e d with either charcoal or with charcoal followed by heating for 10 min at 100°C r e s u l t e d in a significant decrease in the ability of the cells to p r o d u c e t e s t o s t e r o n e (Fig. 1). T h e r e was a doser e s p o n s e relationship between the q u a n t i t y of testicular e x t r a c t and the d e g r e e of inhibition of s t e r o i d o g e n e s i s , with 400 pl of c h a r c o a l - t r e a t e d e x t r a c t r e s u l t i n g in a d e c r e a s e of approximately 85% in the net gonadotropin-stimulated p r o d u c t i o n of t e s t o s t e r o n e . Inhibition in the p r e s e n c e of 400 ~l of the charcoal- and h e a t - t r e a t e d e x t r a c t was about 40%. This ability to retain some inhibitory a c t i v i t y a f t e r heating at 100°C a g r e e d with previous data showing t h a t the hCG/LH binding inhibitor is partially heat stable with r e s p e c t to its ability to i n t e r f e r e with the binding of the hormone (1). In c o n t r a s t to the t e s t i c u l a r e x t r a c t , addition of 400 ~l of c h a r c o a l - t r e a t e d liver e x t r a c t heated at 100°C for 10 min failed to show detectable inhibitory activity on the hCG-stimulated s t e r o i d o g e n e s i s of the cells (Fig. 1). In a n o t h e r set of e x p e r i m e n t s , interstitial cells were f i r s t incubated with c h a r c o a l - t r e a t e d t e s t i c u l a r e x t r a c t for 30 min at 34°C. The inhibitor was then removed and the cells r e c o n s t i t u t e d with medium and incubated with 5 IU of hCG for 2 h. Results indicated t h a t preincubation with the testicular factor diminished the s u b s e q u e n t stimulation of t e s t o s t e r o n e production b y the g o n a d o t r o p i n added d u r i n g the second incubation (Table I ) . This effect may be a t t r i b u t e d to the ability of the inhibitor to p r e v e n t the formation of the h o r m o n e - r e c e p t o r complex as p r e v i o u s l y d e s c r i b e d (1). F u r t h e r m o r e , the o b s e r v a t i o n t h a t inhibition of steroidogenesis in interstitial cells was not r e v e r s e d a f t e r addition of hCG f u r t h e r s u p p o r t s the concept t h a t binding of the inhibitor to LH/hCG r e c e p t o r s may proceed in an i r r e v e r s i b l e manner (1). Effects of a single injection of c h a r c o a l - t r e a t e d s p o n t a n e o u s ovulation in normal cycling r a t s .
testicular e x t r a c t on the
Because the LH/hCG binding inhibitor p r e v e n t s the interaction of hCG and its r e c e p t o r s in testis and o v a r y (1), an attempt was made to see w h e t h e r the t e s t i c u l a r factor might have an inhibitory effect on LH/hCG binding in vivo. In preliminary s t u d i e s , we o b s e r v e d t h a t the administration of t e s t i c u l a r e x t r a c t to immature r a t s was able to alter the ovulation induced b y an s . c . injection of 5 IU hCG, as determined b y the number of ova p e r ovulating r a t 72 h a f t e r the hormone t r e a t m e n t (data not shown). This effect was most a p p a r e n t when the testicular e x t r a c t was injected in single doses of 3 to 5 g tissue e q u i v a l e n t / r a t , 54-55 h following hCG administration. Lower doses had no detectable e f f e c t s . Under the conditions of this experimental model, hCG alone may induce ovulation in a b o u t 75% of immature r a t s and it is t h o u g h t t h a t the g o n a d o t r o p i n elicits, between 54 and 57 h a f t e r the injection, an endogenous s u r g e of LH which is p r e s u m a b l y r e s p o n s i b l e for t r i g g e r i n g ovulation (5). Although our initial r e s u l t s were e n c o u r a g i n g , t h e y were highly
276
Antigonadotropin
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Vol. 34, No. 3, 1984
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FIG. I Effects of the testicular inhibitor on hCG-stimulated dispersed testicular interstitial cells in_vitro.
400uI,Heated Liver Extract
testosterone production by
Cell preparations were incubated with .% IU of h C G in the presence of either charcoal-treated testicular extract, or testicular extract which has been treated with charcoal followed by heating for 10 min at I00°C. The incubations were carried out in Medium 199 at 34°C for 3 h under an atmosphere of 95% 02:5% C O 2. At the end of the incubation total (medium plus tissue) testosterone production was determined as described under Materials and Methods. Last column at right shows incubation with charcoal-treated liver extract heated for 10 rain at I00°C. All tissue extracts were prepared in Medium 199 at a ratio of 100 mg tissue equivalent per 100 pl extract. Each value represents the mean + S E M of three separate determinations. *, P <0.005; e, P <0.01 compared to the group having h C G alone.
v a r i a b l e and failed to r e s p o n s e to the e x t r a c t .
show
a
consistent
pattern
of
ovulation-inhibitory
B e t t e r and more r e p r o d u c i b l e r e s u l t s were obtained using normal cycling r a t s which r e c e i v e d an i . p . injection of 3.5 g tissue equivalent/BW of c h a r c o a l - t r e a t e d t e s t i c u l a r e x t r a c t at d i f f e r e n t times during p r o e s t r u s . The single dose selected was b a s e d on the p r e v i o u s o b s e r v a t i o n s using the model of ovulation induction in immature r a t s d e s c r i b e d above. Table II shows t h a t injection of t e s t i c u l a r e x t r a c t between 1430-1630 h of p r o e s t r u s significantly inhibited s p o n t a n e o u s ovulation, as evidenced b y the number of animals in which ova were found. In c o n t r a s t , r a t s receiving a similar dose of liver e x t r a c t ovulated normally. As e x p e c t e d , in view of the partial heat stability of LH/hCG binding inhibitor, administration of a single dose of charcoal- and h e a t - t r e a t e d e x t r a c t also r e s u l t e d in a significant inhibition of ovulation, although to a l e s s e r e x t e n t t h a n the nonheated e x t r a c t . Administration of the t e s t i c u l a r e x t r a c t at 1130 h was not i n h i b i t o r y , p r e c l u d i n g the likelihood t h a t
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TABLE I Effects of Preincubation of Dispersed T e s t i c u l a r I n t e r s t i t i a l Cells w i t h T e s t i c u l a r I n h i b i t o r on in v i t r o hCG Stimulation of Testosterone Production
Second incubation
Testosterone produced (ng/g tissue)
Decrease of hCG-stimulated testosterone production (~)
Assay
First incubation a
1
I n t e r s t i t i a l cells
hCG
102.0 + 4.7
2
I n t e r s t i t i a l cells + 200 lal e x t r a c t
hCG
74.5 + 4.2
27 b
3
I n t e r s t i t i a l cells + 400 pl e x t r a c t
hCG
59.3 + 7.4
42 b
control
aCells were f i r s t incubated at 34°C f o r 30 min with e i t h e r c h a r c o a l - t r e a t e d t e s t i c u l a r e x t r a c t s or w i t h Medium 199 alone ( c o n t r o l ) . After centrifugation and w a s h i n g , the cells were resuspended and incubated a second time at 34°C f o r 2 h in the presence of 5 IU hCG. Total p r o d u c t i o n of testosterone was determined as described u n d e r Materials and Methods. Data are the mean (-+ SEM) of two separate sets of e x p e r i m e n t s . bp <0.005 vs. c o n t r o l
T A B L E II I n h i b i t o r y A c t i v i t y of T e s t i c u l a r E x t r a c t upon Spontaneous Ovulation in Normal C y c l i n g Rats a
Extact b
Time of Injection c
Control
--
8/8
Testicular extract Charcoal-treated Charcoal-treated Charcoal-treated Charcoal-treated C h a r c o a l - t r e a t e d and heated
1130 1430 1530 1630 1530
Liver extract,
1530 h
heated
No. No. ova (+ SEM) ovulating per o v u l a t i n g rat rat
h h h h h
5/5 6/11 5/13 5/5 4/4 4/4
pd
11.2 + 0.9 9.6 6.6 2.0 3.0 5.3
+ -+ t + -+
2.1 0.7 0.3 0.7 1.1
10.7 + 0.8
NS <0.01 <0.001 <0.001 <0.01 NS
aNormal p r o e s t r o u s rats received a single dose of tissue e x t r a c t s and were sacrificed at 1000 h the n e x t day f o r c o u n t i n g the number of ova in the oviducts. bwhen heated, 10 rain.
the c h a r c o a l - t r e a t e d
extracts
were boiled in a water bath f o r
CTime of p r o e s t r u s at w h i c h injection was g i v e n dp vs. control
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Antigonadotropin
Activity in Rat Testis
Vol.
34, No.
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the effects d e s c r i b e d in Table II were a t t r i b u t a b l e to a nonspecific deleterious effect upon the o v a r y . In o r d e r to i n v e s t i g a t e f u r t h e r the n a t u r e of the inhibition of ovulation, we f i r s t injected the r a t s with the inhibitor at 1530 h of p r o e s t r u s , followed by an injection of hCG 1 h later. Figure 2 shows that the administration of 5 IU of hCG was unable to r e v e r s e the inhibitory effect induced by the single dose of c h a r c o a l - t r e a t e d e x t r a c t . T h e s e r e s u l t s s t r o n g l y s u g g e s t t h a t inhibition may be due to a direct i n t e r f e r e n c e at the level of the o v a r y . F u r t h e r m o r e , the inhibitory effect induced with the charcoal- and h e a t - t r e a t e d e x t r a c t was p a r tially r e v e r s e d b y the s u b s e q u e n t administration of hCG, a r e s u l t which is in agreement with the partial retention of activity following heating of the inhibit o r , and which indicates t h a t the ability to inhibit ovulation was d o s e - r e l a t e d . Discussion The p r e s e n t r e s u l t s d e m o n s t r a t e t h a t the aqueous e x t r a c t p r e p a r e d from r a t testis contains a factor able to p r e v e n t hCG-stimulated t e s t o s t e r o n e production by testicular interstitial cells in vitro and to inhibit ovulation in the cycling r a t . These findings were c o n s i s t e n t ~ t h the p r e s e n c e of an LH/hCG binding inhibitor in the t e s t i c u l a r e x t r a c t which has been r e p o r t e d to be partially heat stable, n o n - e x t r a c t a b l e by charcoal, and able to compete with LH/hCG for the same binding sites in testicular and ovarian tissues (1). The in v i t r o studies showed t h a t p r e i n c u b a t i o n with the testicular factor significantly m-iS-~Dited the s u b s e q u e n t stimulation of steroidogenesis by hCG compared to controls in which the f i r s t incubation of the interstitial cells was in medium alone. T h u s , the i r r e v e r s i b i l i t y in the mechanism of action of the inhibitor o b s e r v e d in binding e x p e r i m e n t s (1) was also evident in studies concerning steroidogenesis in vitro (Table I ) . A similar o b s e r v a t i o n has been r e p o r t e d by Kumari et alT. ~ using an LH r e c e p t o r binding inhibitor (LHRBI) obtained from e x t r a c t s of porcine c o r p u s luteum. These i n v e s t i g a t o r s found t h a t the ability of the LHRBI to inhibit p r o g e s t e r o n e secretion in c u l t u r e d g r a n u l o s a cells is not r e v e r s e d a f t e r addition of LH. It is a p p a r e n t , t h e r e f o r e , t h a t an element of i r r e v e r s i b i l i t y may be a common f e a t u r e in the mode of action of other gonadotropin binding inhibitors. The inhibitory effects of the testicular factor upon t e s t o s t e r o n e p r o d u c t i o n by interstitial cells in r e s p o n s e to hCG c o n t r a s t e d g r e a t l y with thoa~ o b s e r v e d with LHRH b y Yang and c o - w o r k e r s (7). Studies from these a u t h o r s show t h a t the LHRBI obtained from the ovaries of p s e u d o p r e g n a n t r a t s inhibits in vitro LH-stimulated p r o g e s t e r o n e s y n t h e s i s b y ovarian tissue and t h a t , in t e ~ a r t i s s u e , the LHRBI p r e p a r a t i o n not only enhances the in v i t r o LH stimulation of t e s t o s t e r o n e , b u t also, b y itself, stimulates the t e s t o s t e r o n e production (7). These o b s e r v a t i o n s might s u g g e s t the existence of a dichotomy of b e h a v i o r between LH r e c e p t o r s from ovaries as compared with the r e c e p t o r s from t e s t i s . In c o n t r a s t , our data demonstrating t h a t the t e s t i c u l a r factor is an a n t a g o n i s t of LH/hCG functioning at the gonadal level in both the male and the female do not s u p p o r t such a concept. Furthermore, the evidence that the testicular factor inhibits hCG binding to testicular and ovarian LH/hCG r e c e p t o r s (1) d i s a g r e e s with the s u g g e s t i o n of Yang et al. (7,8) t h a t ovarian LH r e c e p t o r s are s t r u c t u r a l l y d i f f e r e n t . The p r e s e n t r e s u l t s , t h e r e f o r e , s u p p o r t the t h o u g h t t h a t LHRBI obtained from ovaries of p s e u d o p r e g n a n t r a t s and the LH/hCG binding inhibitor obtained from r a t testis may be distinct molecular entities with d i f f e r e n t modes of inhibition. The p r e c i s e mechanism of action of the ovulation-inhibiting activity d e m o n s t r a t e d in testicular e x t r a c t is not known, b u t the fact t h a t s u p p r e s s i o n of ovulation o c c u r r e d a f t e r administration of hCG s u g g e s t s t h a t the effect was
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Antigonadotropin Activity in Rat Testis
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FIG. 2 pre-treated
with t e s t i c u l a r i n h i b i t o r d u r i n g
Rats were f i r s t given a single i . p . injection of either phosphate b u f f e r (0.01 M, pH 7 . 5 ) , charcoal-treated, or charcoal- and heat-treated t e s t i c u l a r e x t r a c t s at 1530 h of proestrus. Following this p r e - t r e a t m e n t , a single i . p . injection of 5 IU hCG was given at 1630 h of proestrus. The animals were then sacrificed at 1000 h next day and the number of ova in the oviduct counted. T e s t i c u l a r extracts were prepared in phosphate b u f f e r and the dose injected was of 3.6 g tissue equivalent/BW. *, P <0.001; 0, P <0.01. Statistical analyses refers to a comparison between the experimental and control ( b u f f e r - injected ) g rou ps.
p r o b a b l y not at the h y p o t h a l a m i c - p i t u i t a r y level, b u t , r a t h e r , due to i n t e r f e r ence in the binding of the gonadotropin to its ovarian r e c e p t o r s . Recently, the inhibitory action of a c r u d e c h a r c o a l - t r e a t e d aqueous e x t r a c t of porcine c o r p u s luteum upon ovulation in the r a b b i t was r e p o r t e d (9). This effect was also a t t r i b u t e d to a direct inhibitory action on ovarian LH binding, although an effect on h y p o t h a l a m i c - p i t u i t a r y function was not ruled out (9). Whether the inhibitory s u b s t a n c e p r e s e n t in the porcine c o r p u s luteum e x t r a c t is identical to the L H / h C G - b i n d i n g factor e x t r a c t e d from r a t testis remains to be elucidated. The existence d e s c r i b e d h e r e of a t e s t i c u l a r factor h a v i n g ovulationinhibiting a c t i v i t y s u g g e s t s its potential use as a c o n t r a c e p t i v e acting at the ovarian level. Since the p r e p a r a t i o n of large quantities of the inhibitor is
280
Antigonadotropin Activity in Rat Testis
n e c e s s a r y for more e x t e n s i v e and complete s t u d i e s , awaits its purification and chemical c h a r a c t e r i z a t i o n . t h a t efforts to d e s c r i b e the various activities of the and fluids capable of affecting normal physiological worthwhile.
Vol. 34, No. 3, 1984
a definitive conclusion In the meantime, we feel gonadal tissue e x t r a c t s p r o c e s s e s are certainly
Acknowledgments
We wish to t h a n k Ms. Gretta Small for her a s s i s t a n c e in p r e p a r i n g this manuscript. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
F . J . ROJAS, I. MORETTI-ROJAS, G. DE BELLABARBA and W. BISHOP, Biol. Reprod. 25:519-529 (1981). M.L. D U F A U , C . R . MENDELSON and K . J . CATT, J. Clin. Endocrinol. Metab. 39:610-613 (1974). S . G . CI--GORRAGA, M.L. DUFAU and K . J . CATT, J. Biol. Chem. 253:4297-4304 (1978). M.L. DUFAU, K.J. CATT and T. TSURUHARA, Endocrinology 90:1032-1040 (1972). S~K. KOSTYK, E . J . DROPCHO, H. MOLTZ and J . R . SWARTWOUT, Biol. R e p r o d . 19:1102-1107 (1978). G.L. KU-MARI, S. TUCKER and C . P . CHANNING, Biol. Reprod. 21:1043-1050 (1979). K--IP. YANG, N.A. SAMAAN and D.N. WARD, Endocrinology 104:552-558 (1979). K-P. YANG, N.A. SAMAAN and D.N. WARD, Proc. Soc. Exp. Biol. Med, 152:606-609 (1976). C.P. CHANNING, S.K. BATTA and I-H. BAg, Proc. Soc. Exp. Biol. Med. 166:479-483 (1981).
Pergamon Press
INHIBITION OF IN VITRO HUMAN CHORIONIC GONADOTROPIN-STIMULATED TESTOSTERONE PRODUCTION IN TESTIS AND OF OVULATION IN THE RAT BY CHARCOAL-TREATED RAT TESTICULAR EXTRACT* Gabriela A. de Bellabarba 1, Walter Bishop 1 and Francisco 7. Rojas 2 1Departamento de Fisiopatologia, Facultad de Medicina, Universidad de Los Andes, M6rida, Venezuela and 2Department of O b s t e t r i c s and Gynecology, The U n i v e r s i t y of T e x a s Health Science Center at San Antonio, Texas 78284 (Received in final form November i, 1983) SUMMARY
P r e v i o u s l y , we d e s c r i b e d the p r e s e n c e of a factor obtained from a 105,000 x g s u p e r n a t a n t of r a t testis that was found to inhibit human chorionic gonadotropin (hCG) binding to gonadal receptors. In the p r e s e n t s t u d y , similarly p r e p a r e d testicular e x t r a c t was t e s t e d for its effects on in vitro hCG-stimulated t e s t o s t e r o n e production by isolated testis ] h t e r ~ i a l cells and for its effect on s p o n t a n e o u s ovulation in the r a t . Incubation of interstitial cells with c h a r c o a l - t r e a t e d e x t r a c t significantly inhibited the steroidogenic r e s p o n s e to hCG in a d o s e - r e l a t e d manner. This inhibition was also a p p a r e n t a f t e r heating the e x t r a c t for 10 rain at 100°C. Preincubation of the cells with c h a r c o a l - t r e a t e d e x t r a c t r e s u l t e d in an inhibitory effect that was not readily r e v e r s e d b y s u b s e q u e n t addition of hCG, revealing an element of i r r e v e r s i b i l i t y in the mechanism of inhibition. A single i . p . injection of testicular e x t r a c t given between 1430-1630 h of p r o e s t r u s inhibited s p o n t a n e ous ovulation in the r a t . This effect was also o b s e r v e d a f t e r heating the e x t r a c t for 10 min at 100°C; in c o n t r a s t , no significant effect was obtained with the injection of a similar dose of liver extract. Administration of 5 IU hCG a f t e r p r e t r e a t m e n t with the testicular e x t r a c t did not r e v e r s e the inhibitory effect on ovulation, indicating t h a t this effect was p r o b a b l y not e x e r t e d at the h y p o t h a l a m u s - p i t u i t a r y level. It is concluded that the aqueous testicular e x t r a c t contains a factor able to antagonize the physiological e v e n t s mediated b y luteinizing hormone ( L H ) / h C G , and t h a t this factor is consistent with the p r e s e n c e of an L H / h C G - b i n d i n g inhibitory activity in r a t testis.
*Supported by an Institutional Research Grant (S07-RR05654) from The University of Texas Health Science Center at San Antonio and by the Consejo de DesarroIlo Cient[fico y Humanfstico de la Universidad de Los Andes. This work was presented in part at the 16th Annual Meeting of the Society for the Study of Reproduction, August 8-10, 1983, Cleveland, Ohio. Address requests for reprints to: Dr. Francisco J. Rojas, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284. 0024-3205/8& $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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Recently, we d e s c r i b e d the p r e s e n c e of a factor having the ability to inhibit luteinizing hormone gonadotropin (hCG) binding to gonadal r e c e p t o r s (1). heat stable, not steroid in n a t u r e and p r e s e n t e d Binding studies and inhibition kinetics indicated t h a t hCG binding mainly b y i n t e r f e r i n g with the formation complex and t h a t it competes with the hormone for C o n s e q u e n t l y , it was d e m o n s t r a t e d t h a t the inhibitor of the 9onadotropin and its r e c e p t o r s in t e s t i c u l a r and
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obtained from r a t testis ( L H ) / h u m a n chorionic This factor was partially a low molecular weight. the inhibitor affects LH/ of the h o r m o n e - r e c e p t o r the same binding sites. p r e v e n t s the interaction ovarian t i s s u e s .
In the p r e s e n t r e p o r t , we extend our p r e v i o u s findings and demonstrate that charcoal-treated testicular extract interferes with hCG-stimulated t e s t o s t e r o n e production b y e n z y m e - d i s p e r s e d t e s t i c u l a r interstitial cells in vitro and t h a t its administration d u r i n g p r o e s t r u s inhibits ovulation in tKe r a t . These r e s u l t s are c o n s i s t e n t with the p r e s e n c e of an LH/hCG binding inhibitor in r a t t e s t i s , s u g g e s t i n g t h a t this factor is not only able to inhibit the binding of the hormone to the r e c e p t o r s , b u t also to act as an a n t a g o n i s t of the physiological functions mediated b y LH/hCG. Materials and Methods P r e p a r a t i o n s of a 105,000 x 9 s u p e r n a t a n t of r a t liver and of r a t testis containing the LH/hCG binding inhibitor were obtained in either p h o s p h a t e b u f f e r (0.01 M sodium p h o s p h a t e b u f f e r , pH 7.5) or in Medium 199 (Gibco L a b o r a t o r i e s ) as d e s c r i b e d elsewhere (1). Under these conditions, the r e s u l t ing aqueous e x t r a c t s contained 100 m9 tissue equivalent p e r 100 pl of e x t r a c t . Charcoal t r e a t m e n t involved addition of 200 mg charcoal (Norit A) p e r 2 ml t e s t i c u l a r e x t r a c t i n c u b a t e d for 30 min at 4°C. Pilot experiments using [3H] t e s t o s t e r o n e showed t h a t charcoal removed more t h a n 98% of the added steroid. For heating s t u d i e s , c h a r c o a l - t r e a t e d e x t r a c t s were boiled in a water bath for 10 min followed b y c e n t r i f u g a t i o n at 10,000 x 9 for 10 rain. The r e s u l t i n g clear s u p e r n a t a n t was t h e n u s e d for inhibitory activities. In g e n e r a l , heating of the e x t r a c t s was p e r f o r m e d just before t h e i r use. P r e p a r a t i o n of d i s p e r s e d interstitial cells was c a r r i e d out b y collagenase digestion of d e c a p s u l a t e d r a t testis according to the method d e s c r i b e d b y Dufau et al. (2). Final r e s u s p e n s i o n of the cells was in Medium 199 at a ratio of 100 mg tissue p e r 200 pl of cell s u s p e n s i o n . The general p r o c e d u r e s outlined b y C i g o r r a g a et al. (3) were employed for cell incubations. Two h u n d r e d microliters of cell sfi-spension were incubated at 34°C u n d e r an atmosphere of 95% 0 2:5% CO 2 in a final volume of I ml of Medium 199. At the end of the incubation period, the mixture was boiled in a water b a t h for 5 min, centrif u g e d at 10,000 x g for 10 min and the s u p e r n a t a n t stored at -30°C for s u b s e q u e n t determination of total (medium plus t i s s u e ) t e s t o s t e r o n e production b y radioimmunoassay. In the p r e s e n c e of an excess of hCG (5 IU; A y e r s t L a b s , New Y o r k ) , the total t e s t o s t e r o n e p r o d u c t i o n was linear for at least 3 hours. The a n t i s e r u m to t e s t o s t e r o n e , obtained from Radioassay System L a b o r a t o r i e s ( C a r s o n , CA), had high specificity for t e s t o s t e r o n e with relatively low c r o s s - r e a c t i v i t y for d i h y d r o t e s t o s t e r o n e (6.6%), h 4 - a n d r o s t e n e d i o n e (0.9%) and 5 a - a n d r o s t a n e - 3 t L 17~-diol (2.19%). Radioimmunoassays were p e r f o r m e d directly on samples as outlined b y Dufau et al. (4). The i n t e r a s s a y and i n t r a - a s s a y coefficients of variation were 12.5°~-an~ 10.1%, r e s p e c t i v e l y . Rats of the S p r a g u e - D a w l e y s t r a i n were u s e d . The animals were housed in a controlled e n v i r o n m e n t , 12 L:12 D, with r a t chow and tap w a t e r available ad libitum, and were sacrificed by decapitation immediately p r i o r to use. For ~-u l¢ti-e~ on inhibition of ovulation, r a t s exhibiting at least two consecutive
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4 - d a y cycles were selected; the LH s u r g e was o b s e r v e d between 1600 h and 1800 h of p r o e s t r u s . The influence of c h a r c o a l - t r e a t e d testicular and liver e x t r a c t s , or charcoal- and h e a t - t r e a t e d testicular e x t r a c t s was a s s e s s e d in normal p r o e s t r o u s r a t s r e c e i v i n g a single i . p . injection of 3.5 g tissue equivalent/BW at various times between 1130 h and 1630 h. The r a t s were sacrificed on the following day (1000 h of e s t r u s ) and the number of ova in the oviducts counted u n d e r a dissecting microscope. Data were analyzed using S t u d e n t ' s t t e s t . Results Effects of c h a r c o a l - t r e a t e d testicular e x t r a c t upon hCG-stimulated steroidogenesis ~ y d i s p e r s e d testicular l~ff~-rstitial cells in vitro. Incubation of interstitial cells with 5 IU of hCG in the p r e s e n c e of t e s t i c u l a r e x t r a c t s which had been t r e a t e d with either charcoal or with charcoal followed by heating for 10 min at 100°C r e s u l t e d in a significant decrease in the ability of the cells to p r o d u c e t e s t o s t e r o n e (Fig. 1). T h e r e was a doser e s p o n s e relationship between the q u a n t i t y of testicular e x t r a c t and the d e g r e e of inhibition of s t e r o i d o g e n e s i s , with 400 pl of c h a r c o a l - t r e a t e d e x t r a c t r e s u l t i n g in a d e c r e a s e of approximately 85% in the net gonadotropin-stimulated p r o d u c t i o n of t e s t o s t e r o n e . Inhibition in the p r e s e n c e of 400 ~l of the charcoal- and h e a t - t r e a t e d e x t r a c t was about 40%. This ability to retain some inhibitory a c t i v i t y a f t e r heating at 100°C a g r e e d with previous data showing t h a t the hCG/LH binding inhibitor is partially heat stable with r e s p e c t to its ability to i n t e r f e r e with the binding of the hormone (1). In c o n t r a s t to the t e s t i c u l a r e x t r a c t , addition of 400 ~l of c h a r c o a l - t r e a t e d liver e x t r a c t heated at 100°C for 10 min failed to show detectable inhibitory activity on the hCG-stimulated s t e r o i d o g e n e s i s of the cells (Fig. 1). In a n o t h e r set of e x p e r i m e n t s , interstitial cells were f i r s t incubated with c h a r c o a l - t r e a t e d t e s t i c u l a r e x t r a c t for 30 min at 34°C. The inhibitor was then removed and the cells r e c o n s t i t u t e d with medium and incubated with 5 IU of hCG for 2 h. Results indicated t h a t preincubation with the testicular factor diminished the s u b s e q u e n t stimulation of t e s t o s t e r o n e production b y the g o n a d o t r o p i n added d u r i n g the second incubation (Table I ) . This effect may be a t t r i b u t e d to the ability of the inhibitor to p r e v e n t the formation of the h o r m o n e - r e c e p t o r complex as p r e v i o u s l y d e s c r i b e d (1). F u r t h e r m o r e , the o b s e r v a t i o n t h a t inhibition of steroidogenesis in interstitial cells was not r e v e r s e d a f t e r addition of hCG f u r t h e r s u p p o r t s the concept t h a t binding of the inhibitor to LH/hCG r e c e p t o r s may proceed in an i r r e v e r s i b l e manner (1). Effects of a single injection of c h a r c o a l - t r e a t e d s p o n t a n e o u s ovulation in normal cycling r a t s .
testicular e x t r a c t on the
Because the LH/hCG binding inhibitor p r e v e n t s the interaction of hCG and its r e c e p t o r s in testis and o v a r y (1), an attempt was made to see w h e t h e r the t e s t i c u l a r factor might have an inhibitory effect on LH/hCG binding in vivo. In preliminary s t u d i e s , we o b s e r v e d t h a t the administration of t e s t i c u l a r e x t r a c t to immature r a t s was able to alter the ovulation induced b y an s . c . injection of 5 IU hCG, as determined b y the number of ova p e r ovulating r a t 72 h a f t e r the hormone t r e a t m e n t (data not shown). This effect was most a p p a r e n t when the testicular e x t r a c t was injected in single doses of 3 to 5 g tissue e q u i v a l e n t / r a t , 54-55 h following hCG administration. Lower doses had no detectable e f f e c t s . Under the conditions of this experimental model, hCG alone may induce ovulation in a b o u t 75% of immature r a t s and it is t h o u g h t t h a t the g o n a d o t r o p i n elicits, between 54 and 57 h a f t e r the injection, an endogenous s u r g e of LH which is p r e s u m a b l y r e s p o n s i b l e for t r i g g e r i n g ovulation (5). Although our initial r e s u l t s were e n c o u r a g i n g , t h e y were highly
276
Antigonadotropin
Activity in Rat Testis
Vol. 34, No. 3, 1984
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FIG. I Effects of the testicular inhibitor on hCG-stimulated dispersed testicular interstitial cells in_vitro.
400uI,Heated Liver Extract
testosterone production by
Cell preparations were incubated with .% IU of h C G in the presence of either charcoal-treated testicular extract, or testicular extract which has been treated with charcoal followed by heating for 10 min at I00°C. The incubations were carried out in Medium 199 at 34°C for 3 h under an atmosphere of 95% 02:5% C O 2. At the end of the incubation total (medium plus tissue) testosterone production was determined as described under Materials and Methods. Last column at right shows incubation with charcoal-treated liver extract heated for 10 rain at I00°C. All tissue extracts were prepared in Medium 199 at a ratio of 100 mg tissue equivalent per 100 pl extract. Each value represents the mean + S E M of three separate determinations. *, P <0.005; e, P <0.01 compared to the group having h C G alone.
v a r i a b l e and failed to r e s p o n s e to the e x t r a c t .
show
a
consistent
pattern
of
ovulation-inhibitory
B e t t e r and more r e p r o d u c i b l e r e s u l t s were obtained using normal cycling r a t s which r e c e i v e d an i . p . injection of 3.5 g tissue equivalent/BW of c h a r c o a l - t r e a t e d t e s t i c u l a r e x t r a c t at d i f f e r e n t times during p r o e s t r u s . The single dose selected was b a s e d on the p r e v i o u s o b s e r v a t i o n s using the model of ovulation induction in immature r a t s d e s c r i b e d above. Table II shows t h a t injection of t e s t i c u l a r e x t r a c t between 1430-1630 h of p r o e s t r u s significantly inhibited s p o n t a n e o u s ovulation, as evidenced b y the number of animals in which ova were found. In c o n t r a s t , r a t s receiving a similar dose of liver e x t r a c t ovulated normally. As e x p e c t e d , in view of the partial heat stability of LH/hCG binding inhibitor, administration of a single dose of charcoal- and h e a t - t r e a t e d e x t r a c t also r e s u l t e d in a significant inhibition of ovulation, although to a l e s s e r e x t e n t t h a n the nonheated e x t r a c t . Administration of the t e s t i c u l a r e x t r a c t at 1130 h was not i n h i b i t o r y , p r e c l u d i n g the likelihood t h a t
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Antigonadotropin Activity in Rat Testis
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TABLE I Effects of Preincubation of Dispersed T e s t i c u l a r I n t e r s t i t i a l Cells w i t h T e s t i c u l a r I n h i b i t o r on in v i t r o hCG Stimulation of Testosterone Production
Second incubation
Testosterone produced (ng/g tissue)
Decrease of hCG-stimulated testosterone production (~)
Assay
First incubation a
1
I n t e r s t i t i a l cells
hCG
102.0 + 4.7
2
I n t e r s t i t i a l cells + 200 lal e x t r a c t
hCG
74.5 + 4.2
27 b
3
I n t e r s t i t i a l cells + 400 pl e x t r a c t
hCG
59.3 + 7.4
42 b
control
aCells were f i r s t incubated at 34°C f o r 30 min with e i t h e r c h a r c o a l - t r e a t e d t e s t i c u l a r e x t r a c t s or w i t h Medium 199 alone ( c o n t r o l ) . After centrifugation and w a s h i n g , the cells were resuspended and incubated a second time at 34°C f o r 2 h in the presence of 5 IU hCG. Total p r o d u c t i o n of testosterone was determined as described u n d e r Materials and Methods. Data are the mean (-+ SEM) of two separate sets of e x p e r i m e n t s . bp <0.005 vs. c o n t r o l
T A B L E II I n h i b i t o r y A c t i v i t y of T e s t i c u l a r E x t r a c t upon Spontaneous Ovulation in Normal C y c l i n g Rats a
Extact b
Time of Injection c
Control
--
8/8
Testicular extract Charcoal-treated Charcoal-treated Charcoal-treated Charcoal-treated C h a r c o a l - t r e a t e d and heated
1130 1430 1530 1630 1530
Liver extract,
1530 h
heated
No. No. ova (+ SEM) ovulating per o v u l a t i n g rat rat
h h h h h
5/5 6/11 5/13 5/5 4/4 4/4
pd
11.2 + 0.9 9.6 6.6 2.0 3.0 5.3
+ -+ t + -+
2.1 0.7 0.3 0.7 1.1
10.7 + 0.8
NS <0.01 <0.001 <0.001 <0.01 NS
aNormal p r o e s t r o u s rats received a single dose of tissue e x t r a c t s and were sacrificed at 1000 h the n e x t day f o r c o u n t i n g the number of ova in the oviducts. bwhen heated, 10 rain.
the c h a r c o a l - t r e a t e d
extracts
were boiled in a water bath f o r
CTime of p r o e s t r u s at w h i c h injection was g i v e n dp vs. control
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Activity in Rat Testis
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34, No.
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the effects d e s c r i b e d in Table II were a t t r i b u t a b l e to a nonspecific deleterious effect upon the o v a r y . In o r d e r to i n v e s t i g a t e f u r t h e r the n a t u r e of the inhibition of ovulation, we f i r s t injected the r a t s with the inhibitor at 1530 h of p r o e s t r u s , followed by an injection of hCG 1 h later. Figure 2 shows that the administration of 5 IU of hCG was unable to r e v e r s e the inhibitory effect induced by the single dose of c h a r c o a l - t r e a t e d e x t r a c t . T h e s e r e s u l t s s t r o n g l y s u g g e s t t h a t inhibition may be due to a direct i n t e r f e r e n c e at the level of the o v a r y . F u r t h e r m o r e , the inhibitory effect induced with the charcoal- and h e a t - t r e a t e d e x t r a c t was p a r tially r e v e r s e d b y the s u b s e q u e n t administration of hCG, a r e s u l t which is in agreement with the partial retention of activity following heating of the inhibit o r , and which indicates t h a t the ability to inhibit ovulation was d o s e - r e l a t e d . Discussion The p r e s e n t r e s u l t s d e m o n s t r a t e t h a t the aqueous e x t r a c t p r e p a r e d from r a t testis contains a factor able to p r e v e n t hCG-stimulated t e s t o s t e r o n e production by testicular interstitial cells in vitro and to inhibit ovulation in the cycling r a t . These findings were c o n s i s t e n t ~ t h the p r e s e n c e of an LH/hCG binding inhibitor in the t e s t i c u l a r e x t r a c t which has been r e p o r t e d to be partially heat stable, n o n - e x t r a c t a b l e by charcoal, and able to compete with LH/hCG for the same binding sites in testicular and ovarian tissues (1). The in v i t r o studies showed t h a t p r e i n c u b a t i o n with the testicular factor significantly m-iS-~Dited the s u b s e q u e n t stimulation of steroidogenesis by hCG compared to controls in which the f i r s t incubation of the interstitial cells was in medium alone. T h u s , the i r r e v e r s i b i l i t y in the mechanism of action of the inhibitor o b s e r v e d in binding e x p e r i m e n t s (1) was also evident in studies concerning steroidogenesis in vitro (Table I ) . A similar o b s e r v a t i o n has been r e p o r t e d by Kumari et alT. ~ using an LH r e c e p t o r binding inhibitor (LHRBI) obtained from e x t r a c t s of porcine c o r p u s luteum. These i n v e s t i g a t o r s found t h a t the ability of the LHRBI to inhibit p r o g e s t e r o n e secretion in c u l t u r e d g r a n u l o s a cells is not r e v e r s e d a f t e r addition of LH. It is a p p a r e n t , t h e r e f o r e , t h a t an element of i r r e v e r s i b i l i t y may be a common f e a t u r e in the mode of action of other gonadotropin binding inhibitors. The inhibitory effects of the testicular factor upon t e s t o s t e r o n e p r o d u c t i o n by interstitial cells in r e s p o n s e to hCG c o n t r a s t e d g r e a t l y with thoa~ o b s e r v e d with LHRH b y Yang and c o - w o r k e r s (7). Studies from these a u t h o r s show t h a t the LHRBI obtained from the ovaries of p s e u d o p r e g n a n t r a t s inhibits in vitro LH-stimulated p r o g e s t e r o n e s y n t h e s i s b y ovarian tissue and t h a t , in t e ~ a r t i s s u e , the LHRBI p r e p a r a t i o n not only enhances the in v i t r o LH stimulation of t e s t o s t e r o n e , b u t also, b y itself, stimulates the t e s t o s t e r o n e production (7). These o b s e r v a t i o n s might s u g g e s t the existence of a dichotomy of b e h a v i o r between LH r e c e p t o r s from ovaries as compared with the r e c e p t o r s from t e s t i s . In c o n t r a s t , our data demonstrating t h a t the t e s t i c u l a r factor is an a n t a g o n i s t of LH/hCG functioning at the gonadal level in both the male and the female do not s u p p o r t such a concept. Furthermore, the evidence that the testicular factor inhibits hCG binding to testicular and ovarian LH/hCG r e c e p t o r s (1) d i s a g r e e s with the s u g g e s t i o n of Yang et al. (7,8) t h a t ovarian LH r e c e p t o r s are s t r u c t u r a l l y d i f f e r e n t . The p r e s e n t r e s u l t s , t h e r e f o r e , s u p p o r t the t h o u g h t t h a t LHRBI obtained from ovaries of p s e u d o p r e g n a n t r a t s and the LH/hCG binding inhibitor obtained from r a t testis may be distinct molecular entities with d i f f e r e n t modes of inhibition. The p r e c i s e mechanism of action of the ovulation-inhibiting activity d e m o n s t r a t e d in testicular e x t r a c t is not known, b u t the fact t h a t s u p p r e s s i o n of ovulation o c c u r r e d a f t e r administration of hCG s u g g e s t s t h a t the effect was
Vol. 34, No. 3, 1984
Antigonadotropin Activity in Rat Testis
279
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8 o
o
Q
±
6
o d)
E z
2 I !
I
0 First Injection:
Buffer
Second Injection: 5 I U
Charcoal Ext.
Charcoal Ext. ~ Heated
51U
5IU
(hCG) Administration proestrus.
of
hCG
to
rats
FIG. 2 pre-treated
with t e s t i c u l a r i n h i b i t o r d u r i n g
Rats were f i r s t given a single i . p . injection of either phosphate b u f f e r (0.01 M, pH 7 . 5 ) , charcoal-treated, or charcoal- and heat-treated t e s t i c u l a r e x t r a c t s at 1530 h of proestrus. Following this p r e - t r e a t m e n t , a single i . p . injection of 5 IU hCG was given at 1630 h of proestrus. The animals were then sacrificed at 1000 h next day and the number of ova in the oviduct counted. T e s t i c u l a r extracts were prepared in phosphate b u f f e r and the dose injected was of 3.6 g tissue equivalent/BW. *, P <0.001; 0, P <0.01. Statistical analyses refers to a comparison between the experimental and control ( b u f f e r - injected ) g rou ps.
p r o b a b l y not at the h y p o t h a l a m i c - p i t u i t a r y level, b u t , r a t h e r , due to i n t e r f e r ence in the binding of the gonadotropin to its ovarian r e c e p t o r s . Recently, the inhibitory action of a c r u d e c h a r c o a l - t r e a t e d aqueous e x t r a c t of porcine c o r p u s luteum upon ovulation in the r a b b i t was r e p o r t e d (9). This effect was also a t t r i b u t e d to a direct inhibitory action on ovarian LH binding, although an effect on h y p o t h a l a m i c - p i t u i t a r y function was not ruled out (9). Whether the inhibitory s u b s t a n c e p r e s e n t in the porcine c o r p u s luteum e x t r a c t is identical to the L H / h C G - b i n d i n g factor e x t r a c t e d from r a t testis remains to be elucidated. The existence d e s c r i b e d h e r e of a t e s t i c u l a r factor h a v i n g ovulationinhibiting a c t i v i t y s u g g e s t s its potential use as a c o n t r a c e p t i v e acting at the ovarian level. Since the p r e p a r a t i o n of large quantities of the inhibitor is
280
Antigonadotropin Activity in Rat Testis
n e c e s s a r y for more e x t e n s i v e and complete s t u d i e s , awaits its purification and chemical c h a r a c t e r i z a t i o n . t h a t efforts to d e s c r i b e the various activities of the and fluids capable of affecting normal physiological worthwhile.
Vol. 34, No. 3, 1984
a definitive conclusion In the meantime, we feel gonadal tissue e x t r a c t s p r o c e s s e s are certainly
Acknowledgments
We wish to t h a n k Ms. Gretta Small for her a s s i s t a n c e in p r e p a r i n g this manuscript. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
F . J . ROJAS, I. MORETTI-ROJAS, G. DE BELLABARBA and W. BISHOP, Biol. Reprod. 25:519-529 (1981). M.L. D U F A U , C . R . MENDELSON and K . J . CATT, J. Clin. Endocrinol. Metab. 39:610-613 (1974). S . G . CI--GORRAGA, M.L. DUFAU and K . J . CATT, J. Biol. Chem. 253:4297-4304 (1978). M.L. DUFAU, K.J. CATT and T. TSURUHARA, Endocrinology 90:1032-1040 (1972). S~K. KOSTYK, E . J . DROPCHO, H. MOLTZ and J . R . SWARTWOUT, Biol. R e p r o d . 19:1102-1107 (1978). G.L. KU-MARI, S. TUCKER and C . P . CHANNING, Biol. Reprod. 21:1043-1050 (1979). K--IP. YANG, N.A. SAMAAN and D.N. WARD, Endocrinology 104:552-558 (1979). K-P. YANG, N.A. SAMAAN and D.N. WARD, Proc. Soc. Exp. Biol. Med, 152:606-609 (1976). C.P. CHANNING, S.K. BATTA and I-H. BAg, Proc. Soc. Exp. Biol. Med. 166:479-483 (1981).