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Lordosis inhibiting effects of endogenous progesterone in the male rat primed with estrogen
Physiology& Behavior,Vol. 45, pp. 1007-1010. ©Pergamon Press plc., 1989. Printed in the U.S.A.
0031-9384/89 $3.00 + .00
Lordosis Inhibiting Effects of Endogenous Progesterone in the Male Rat Primed with Estrogen A. C H A B L I , CH. S C H A E F F E R A N D CL. A R O N 1
Institute o f Histology, Faculty o f Medicine and URA 552 CNRS, 67000 Strasbourg, France R e c e i v e d 2 January 1989
CHABLI, A., CH. SCHAEFFER AND CL. ARON. Lordosis inhibiting effects of endogenousprogesterone in the male rat primed with estrogen. PHYSIOL BEHAV 45(5) 1007-1010, 1989.--The aim of this study was to investigate the mechanisms involved in the inhibitory action of progesterone on estrogen-induced facilitatory effects of estradiol benzoate on lordosis behavior in the male rat. Intact adult male rats were given 1) 25 p.g estradiol benzoate (EB) and 100 I~g progesterone (P) at an interval of 42 hr. EB injected animals served as controls 2) EB followed by 3 doses of 400 ~g dexamethasone (DEXA) and P as above. EB + DEXA injected animals served as controls. Testing for lordosis behavior was performed by 50 -+hr after EB injection. A significant decrease in the number of the males displaying lordosis in response to the mounts of stimulus males resulted from P injection following EB treatment as compared to EB controls. DEXA treatment significantly reduced the number of EB animals showing lordosis responses but completely prevented the inhibitory effects of exogenous P to occur. Blood P values appeared to be significantly lower in EB + DEXA males than in their EB counterparts. The results provide evidence that endogenous P is involved in the display of lordosis behavior by EB-treated intact males. They mainly suggest that the effects of exogenous P on estrogen-induced lordosis behavior in the intact male rat result from sequential inhibitory mechanisms involving exposure of the animals to the successive action of endogenous and exogenous P. Male rat
Lordosis behavior
Estrogen
Endogenous progesterone
IT is now well established that male rats castrated as adults may be mounted by other males and display lordosis responses following treatment with successive doses (2, 10, 11) or a single dose (14, 26, 28) of estradiol benzoate (EB). This effect of estrogen in castrated males was shown to be facilitated by exogenous progesterone (P) (9,28) and by endogenous P of adrenal origin (22). The male rat thus behaves like the female in which P is known to facilitate the effects of EB on the display of lordosis behavior in ovariectomized (18,27) and in cyclic (17,25) animals. However if it is widely accepted that P can act in synergy with estrogen to facilitate behavioral events, it may be also effective as a blocker of estrogen-facilitated sexual behavior in the females of various rodent species [for review, (16)]. Regarding the female rat it was reported (6) that a sequential inhibition occurred after the display of an estrogen-progesterone induced period of lordosis behavior when a second P treatment was given some hours after the animals were receptive. The aim of this investigation was therefore to examine the possibility of such a sequential inhibition in intact male rats since preliminary unpublished observations in our laboratory showed that exogenous P was capable of inhibiting the facilitatory effects of EB on the display of lordosis behavior in intact animals. This paper describes an experiment in which the role played by P of
adrenal origin in the development of a sequential inhibition of lordosis behavior by exogenous P was studied. METHOD
Animals Sexually unexperienced adult male rats (strain WI) raised in our colony were used when 2.5 to 3.5 months old and weighing 250-300 g. They were kept under conditions of controlled temperature (22-24°C) and lighting (lights on 0100-1500 hr). They had free access to a commercial laboratory food (UAR) and tap water ad lib.
Procedure Experiment 1. The aim of this experiment was to determine whether the blockade of the secretion of endogenous P by the adrenals would affect lordosis behavior in intact male rats primed with estradiol benzoate (EB) associated or not with progesterone (P). Dexamethasone (DEXA) which is known (12) to exert suppressive effects on pituitary adrenal activity was used because previous experiments in our laboratory (22) clearly demonstrated that this drug sharply depressed the adrenocortical secretion of P in castrated male rats primed with EB.
~Requests for reprints should be addressed to Pr. C1. Aron, Institute of Histology, Faculty of Medicine, 4 Rue Kirschleger, 67000 Strasbourg, France.
1007
1008
CHABLI, SCHAEFFER AND ARON
Progesterone ( ng/ml ) 2,5
TABLE 1 DISPLAY OF LORDOSIS BEHAVIOR IN INTACT MALE RATS GIVEN ESTRADIOL BENZOATE (EB) AND PROGESTERONE (P). EFFECTS OF DEXAMETHASONE (DEXA) TREATMENT
2,0
1,5
Treatment*
1,0
0,5 T
Non injected controls
EB
J
EB t DEXA
FIG. 1. Blood progesterone concentration in intact male rats given a single dose of 25 p~g estradiol benzoate (EB). Trunk blood collection at 1300 hr by 47 hr after EB. Three successive doses of Dexarnethasone (DEXA) were given at 0800 hr and 2000 hr on the day after EB and at 0800 hr on the following day (10 animals in each group). Values are mean ± SEM. *p<0.05 compared to EB +DEXA and controls (Scheffe's test).
Sixty four animals were divided into 4 groups of 16 rats each that were assigned to the following hormonal treatment: 25 p,g EB (SC) at 1400 hr; 25 p,g EB + 100 p,g P (SC) 42 hr later; 25 p,g EB + 3 × 400 p,g dexamethasone (DEXA) (SC) (at 0800 hr and 2000 hr on the day after EB injection and at 0800 hr on the following day, i.e., by 18 hr, 30 hr and 42 hr after EB injection respectively); 25 Ixg EB + 3 × 400 Ixg DEXA + 100 p,g P as in the preceding groups. The animals were tested for lordosis behavior at the beginning of the dark period under a dim light by 50 +- 1 hr after EB injection. Experiment 2. Experiment 2 determined the changes in blood progesterone concentration induced by DEXA in intact male rats primed with a single dose of 25 p,g EB (SC). Three groups of 10 animals each were used. The first one consisted of rats given EB only. The second one was composed of rats given EB and 3 doses of 400 p,g of DEXA as in Experiment 1. A third group of oil injected rats served as controls. All the animals were decapitated at 1300 hr by 47 hr after EB injection and trunk blood was collected for P measurement and stored at - 20°C until further use.
Behavioral Testing The tests for lordosis behavior were carried out in a 60 cm diameter Plexiglas mating arena during a behavioral session lasting 10 min. Previous work (9,21) showed that over such a time period ORCH estrogenized rats were mounted on an average of at least ten times by sexually vigorous males. Stimulus males were selected that were shown to be fully sexually active when tested at the beginning of the dark period with ovariectomized estrogenprogesterone treated female rats. A 5 rain period of adaptation of the stimulus males in the mating arena was allowed prior to the introduction of the experimental animals. As in our previous behavioral studies (1,21) we determined 1) the proportion of animals in each group that showed willingness to mate and therefore to display one or more lordosis in response to male mounts; 2) the lordosis quotient (LQ) in each male by dividing the
25 p,g EB 25 p,g EB + 100 i.LgPt 25 txg EB + DEXA:~ 25 p,g EB + DEXA + 100 p,g P
Proportion of Animals Displaying Lordosis Responses
Mean Lordosis Quotient +_ SEM in Rats Displaying Lordosis Responses
Mean Number of Mounts --- SEM Displayed by the Stimulus Males
15/16§ 9/16
38.5 ± 7.27¶ 78.4 ± 5.40
14.9 + 1.43§ 10.7 --- 1.72
6/16#
40.8 _+ 5.83
14.3 ± 1.87
53.8 --- 8.29
13.8 --- 1.81
14/16
*Testing with a vigorous male was performed between 1500 and 1700 hr by 50--- 1 hr after EB injection (lights on 0100-1500 hr). ~P at 0800 hr by 42 hr after EB and 8 - 1 hr before sexual behavioral testing. ~:400 p,g DEXA at 0800 and 2000 hr on the day after EB injection and at 0800 hr on the day of sexual behavioral testing.. §p<0.05 vs. EB + P, ¶p<0.0t vs. EB + P #p<0.001 vs. EB + DEXA + P.
number of lordosis by the number of mounts, then, multiplying by 100. Lordosis behavior in male rats strictly resembles that displayed by the estrous female. It consisted in arching the back, extending the neck and deviating the tail to one side to expose the genital region. Only deep lordosis in which both head and tall were noticeably raised were taken into consideration. A mean LQ was calculated in each group of animals for the rats showing lordosis and served as measurement of the mating performance.
P Radioimmunoassay (RIA) P was estimated after hexane extraction by RIA method using an antibody of characteristic specificity for 11 ahydroxyprogesterone hemisuccinate bovine serumalbumine (RIA-kit Biomerieux). P measurement was made without column separation because previous assays (19) using Sephadex LH 20 microcolumns as described by Youssefnejadian et al. (29) showed that P values did not differ using either procedure. Intraassay and interassay coefficients were 10% and 8% respectively. Plasma concentration were expressed in ng/ml.
Statistics The Chi square method with or without the continuity correction of Yates was used to compare the proportion of animals showing lordosis responses in the different experimental groups• Variance analysis was used to study LQ values (after angular transformation of the data) and the number of mounts and P values (after logarithmic transformation of the data). Newman-Keuls method for localization of differences was used when necessary. RESULTS
Effects of Endogenous and Exogenous Progesterone on the Display of Lordosis Behavior in Intact Male Rats Primed With Estradiol Benzoate Table 1 shows that the animals given 23 p,g EB and 100 p,g P
LORDOSIS INHIBITION BY PROGESTERONE
1009
successively displayed less frequent lordosis responses to male mounts that those given EB only (9/16 vs. 15/16; )(2 Yates =4.16; p<0.05). DEXA treatment appeared to sharply decrease the number of animals which displayed lordosis behavior and this effect was completely reversed by administration of P (6/16 vs. 14/16; ×2=6.0; p<0.01). The LQ values appeared significantly higher in EB + P animals than in their EB counterparts (78.4 - 5.40 vs. 38.5 - 7 . 2 7 ; p<0.01). As to the mounts, EB + P animals were mounted less frequently than EB animals (10.7--. 1.72 vs. 14.9±
1.43; p<0.05). Changes in Blood Progesterone Concentration Following Estradiol Benzoate and Dexamethasone Treatment As shown in Fig. 1, blood P values appeared to be higher in EB-treated animals than in the two other groups (F(2,27)= 9.18, p<0.001) and P values in DEXA animals and in controls were significantly lower than those found in EB-treated animals
(p<0.05). DISCUSSION In this study we have shown that P was capable of inhibiting the facilitatory effects of EB on the display of lordosis behavior in intact male rats. We observed that 100 p,g P given 42 hours after 25 p,g EB significantly reduced the number of animals displaying lordosis in response to male mounts. At first sight, such a result was unexpected since lordosis behavior has been shown to be facilitated by exogenous P in the castrated male rat (9,28) and also in the intact male rat (28). Moreover previous findings in our laboratory (22) have provided evidence that EB was capable of increasing P concentration in castrated male rats and that endogenous P might thus facilitate the induction of lordosis behavior by estrogen. It then might be hypothesized that EB caused an increase in blood P concentration in our experimental model and rendered the animals sensitive to the inhibitory effects of a subsequent exposure to exogenous P. This assumption, which is based on the well known sequential inhibitory effects of P which occur in ovariectomized rats when they are submitted to two successive injections of P (14-16), is fully supported by the present study. Reduced lordosis response in intact male rats following removal of endogenous P by DEXA treatment confirms (22) that P of adrenal origin--which is secreted under the influence of estrogen--may exert facilitatory effects on the display of lordosis behavior. Furthermore lordosis behavior appeared fully restored by exogenous P in EB + DEXA-treated animals which appeared deprived of endogenous P. In this group exogenous P likely exerted facilitatory effects on lordosis response, instead of its inhibitory action in nonDEXA-treated animals, because endogenous P was suppressed. This clearly demonstrates the sequential nature of P
inhibition in animals given EB and exogenous P successively. It is worth noting that DEXA nonspecific debilitating effects were not responsible for the decrease in lordosis responses in the DEXAtreated males. Changes in motor activity were not observed following DEXA treatment. However we must explain some apparent discrepancy between our results and those of Van de Poll and Van Dis (28) who showed that P could enhance the effects of estrogen on the display of lordosis behavior in intact male rats. This could be accounted for by the fact that estrogen can prevent the appearance of sequential inhibitory effects of P on lordotic responsiveness depending on the priming dose administered (6,24). The shorter interval (36 hr) between estrogen and progesterone injection in Van de Poll and Van Dis's study may also explain the discrepancy between their results and those of our study. Moreover P in high doses may also prevent a desensitization effect on lordosis behavior (3). Unpublished data in our laboratory also show that high doses of EB prevent the inhibitory effects of P to occur in intact male rats. In fact, Van de Poll and Van Dis (28) used higher doses of EB and P than those used in the present work. Two last points deserve attention. Despite the sequential inhibitory effects of P, the copulatory performance as measured by the LQ appeared to be higher in EB + P animals than in those given EB only. Experiments are in progress in our laboratory which suggest that different neuroendocdne mechanisms are involved in the control of the willingness to mate (number of males in a given group which display lordosis responses to male mounts) and the copulatory performance (LQ values). This high degree of female-like "receptivity" in the males given EB + P probably explains that a reduced number of mounts by the stimulus males was sufficient to induce lordosis behavior. Estrogen are known to induce P receptors in the hypothalamus of female (4, 5, 23) and male (8, 13, 20) rodents. But P administration in the female is also known to lead to a loss of loosely bound nuclear P receptors in the brain (5, 7, 23). This accounts for the development of the sequential inhibitory effect of P on the display of lordosis behavior in the estrogen primed ovariectomized female rat (3). The question then may be raised whether this mechanism is involved in the inhibitory effects of P in our experimental model. Of course, further work is required to validate this assumption. In conclusion, the results presented here demonstrate that the intact male rat constitutes a useful tool for a better understanding of the hormonal mechanisms of bisexuality in rodents. ACKNOWLEDGEMENTS We are grateful to Dr. N. Boehm for her assistance in radioimmunoassay and to Mrs. Machart and Ms. Ganter for their technical assistance. We would like to thank Mr. Dejol for the figures and Ms. Gangloff for typing the manuscript. We are thankful to Dr. M. Roos for the statistical analysis of the data and to Mr. G. Weisman for his linguistic assistance.
REFERENCES 1. Al Sattli, M.; Aron, C1. Influence of olfactory bulb removal on sexual receptivity in the rat. Psychoneuroendocrinology 2:399-407; 1977. 2. Ar~n-Engelbrektsson, B.; Larsson, K.; S6dersten, P.; Wilhelmsson, M. The female lordosis pattern induced in male rats by estrogen. Horm. Behav. 1:181-188; 1970. 3. Blanstein, J. D. Progesterone in high doses may overcome progesterone desensitization effect on lordosis by translocation of hypothalamic receptors. Horm. Behav. 16:175-190; 1982. 4. Blanstein, J. D.; Feder, H. H. Cytoplasmic progesterone receptors in guinea pig brain: characteristics and relationship to the induction of sexual behavior. Brain Res. 169:481-497; 1979. 5. Blaustein, J. D.; Feder, H. H. Nuclear progestin receptors in guinea pig brain measured by an in vitro exchange assay after hormonal treatments that affect lordosis. Endocrinology 106:1061-1069; 1980.
6. Blaustein, J. D.; Wade, G. N. Sequential inhibition of sexual behavior by progesterone in female rats: comparison with a synthetic antiestrogen. J. Comp. Physiol. Psychol. 91:752-760; 1977. 7. Blaustein, J. D.; Wade, G. N. Progestin binding by brain and pituitary cell nuclei and female rat sexual behavior. Brain Res. 140:360-367; 1978. 8. Blaustein, J. D.; Ryer, H. I.; Feder, H. H. A sex difference in the progesterone receptor system of guinea-pig brain. Neuroendocrinology 31:403-409; 1980. 9. Chabli, A.; Schaeffer, Ch.; Samama, B.; Aron, C1. Hormonal control of the perception of the olfactory signals which facilitate lordosis behavior in the male rat. Physiol. Behav. 35:729-734; 1985. 10. Davidson, J. M. Effects of estrogen on the sexual behavior of male rat. Endocrinology 84:1365-1372; 1969.
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11. Davidson, J. M.; Levine, S. Progesterone and beterotypic sexual behaviour in male rats. J. Endocrinol. 44:129-130; 1969. 12. De Kloet, E. R.; Van der Vies, J.; Wied, D. The site of the suppressive action of dexamethasone on pituitary adrenal activity. Endocrinology 94:61-73; 1974. 13. Fraile, I. G.; Pfaff, D. W.; McEwen, B. S. Progestin receptors with and without induction in male and female hamster brain. Neuroendocrinology 45:487-491; 1987. 14. Hansen, S.; Stdersten, P. Reversal of progesterone inhibition on sexual behaviour in ovariectomized rats by high doses of progesterone. J. Endocfinol. 80:381-388; 1979. 15. Marrone, B. L.; Rodriguez-Sierra, F. J.; Feder, H. H. Lordosis inhibiting effects of progesterone in the female rat. Horm. Behav. 8:391--402; 1977. 16. Morin, L. P. Progesterone: inhibition of rodent sexual behavior. Physiol. Behav. 18:701-715; 1977. 17. Plas-Roser, S.; Aron, C1. New data concerning the control by the adrenals of sexual receptivity in the rat. Physiol. Behav. 19:57--60; t977. 18. Powers, J. B. Hormonal control of sexual receptivity during the estrous cycle of the rat. Physiol. Behav. 5:831-835; 1970. 19. Roser, S. Facteurs hormonanx de la regulation du rythme oestral et du comportement sexuel chez la ratte. Ph. D. Thesis, Universit¢ Louis Pasteur, Strasbourg, France, 1974:203. 20. Samama, B.; Aron, CI. Changes in estrogen receptors in the mediobasal hypothalamus mediate the facilitory effects exerted by the male's olfactory cues and progesterone on feminine behavior in the male rat. J. Steroid Biochem.; in press.
CHABLI, SCHAEFFER AND ARON
21. Schaeffer, Ch.; Aron, C1. Studies on feminine behavior in the male rat: influence of olfactory stimuli. Horm. Behav. 15:377-385; 1981. 22. Schaeffer, Ch.; Chabli, A.; Aron, C1. Endogenous progesterone and lordosis behavior in male rats given estrogen alone. J. Steroid Biocbem. 25:99-102; 1986. 23. Schwartz, S. M.; Blanstein, J. D.; Wade, G. N. Inhibition of estrous behavior by progesterone in rats: role of neural estrogen and progesterone receptors. Endocrinology 105:1078-1082; 1979. 24. Shivers, B. D.; Harlan, R. E.; Parker, C. R.; Moss, R. L. Sequential inhibitory effect of progesterone on lordotic responsiveness in rats: time course, estrogenic nullification and actinomycin-D insensitivity. Biol. Reprod. 23:963-973; 1980. 25. SOdersten, P.; Hansen, S. Effects of oestradiol and progesterone on the induction and duration of sexual receptivity in cyclic female rats. J. Endocrinol. 74:477-485; 1977. 26. Stdersten, P.; Larsson, K. Lordosis behavior in castrated male rats treated with estradiol benzoate or testosterone in combination with an estrogen antagonist MER 25 and in intact male rats. Horm. Behav. 5:13-18; 1974. 27. Stidersten, P.; Eneroth, P.; Hansen, S. Induction of sexual receptivity in ovariectomized rats by pulse administration of oestradiol-1713. J. Endocrinol. 89:55-62; 1981. 28. Van de Poll, N. E.; Van Dis, H. Hormone induced lordosis and its relation to masculine sexual activity in male rats. Horm. Behav. 8:1-7; 1977. 29. Youssefnejadian, E.; Florensa, E.; Collins, W. P.; Sommerville, I. F. Radioimmunoassay of plasma progesterone. J. Steroid Biocbem. 3:893-901; 1972.
0031-9384/89 $3.00 + .00
Lordosis Inhibiting Effects of Endogenous Progesterone in the Male Rat Primed with Estrogen A. C H A B L I , CH. S C H A E F F E R A N D CL. A R O N 1
Institute o f Histology, Faculty o f Medicine and URA 552 CNRS, 67000 Strasbourg, France R e c e i v e d 2 January 1989
CHABLI, A., CH. SCHAEFFER AND CL. ARON. Lordosis inhibiting effects of endogenousprogesterone in the male rat primed with estrogen. PHYSIOL BEHAV 45(5) 1007-1010, 1989.--The aim of this study was to investigate the mechanisms involved in the inhibitory action of progesterone on estrogen-induced facilitatory effects of estradiol benzoate on lordosis behavior in the male rat. Intact adult male rats were given 1) 25 p.g estradiol benzoate (EB) and 100 I~g progesterone (P) at an interval of 42 hr. EB injected animals served as controls 2) EB followed by 3 doses of 400 ~g dexamethasone (DEXA) and P as above. EB + DEXA injected animals served as controls. Testing for lordosis behavior was performed by 50 -+hr after EB injection. A significant decrease in the number of the males displaying lordosis in response to the mounts of stimulus males resulted from P injection following EB treatment as compared to EB controls. DEXA treatment significantly reduced the number of EB animals showing lordosis responses but completely prevented the inhibitory effects of exogenous P to occur. Blood P values appeared to be significantly lower in EB + DEXA males than in their EB counterparts. The results provide evidence that endogenous P is involved in the display of lordosis behavior by EB-treated intact males. They mainly suggest that the effects of exogenous P on estrogen-induced lordosis behavior in the intact male rat result from sequential inhibitory mechanisms involving exposure of the animals to the successive action of endogenous and exogenous P. Male rat
Lordosis behavior
Estrogen
Endogenous progesterone
IT is now well established that male rats castrated as adults may be mounted by other males and display lordosis responses following treatment with successive doses (2, 10, 11) or a single dose (14, 26, 28) of estradiol benzoate (EB). This effect of estrogen in castrated males was shown to be facilitated by exogenous progesterone (P) (9,28) and by endogenous P of adrenal origin (22). The male rat thus behaves like the female in which P is known to facilitate the effects of EB on the display of lordosis behavior in ovariectomized (18,27) and in cyclic (17,25) animals. However if it is widely accepted that P can act in synergy with estrogen to facilitate behavioral events, it may be also effective as a blocker of estrogen-facilitated sexual behavior in the females of various rodent species [for review, (16)]. Regarding the female rat it was reported (6) that a sequential inhibition occurred after the display of an estrogen-progesterone induced period of lordosis behavior when a second P treatment was given some hours after the animals were receptive. The aim of this investigation was therefore to examine the possibility of such a sequential inhibition in intact male rats since preliminary unpublished observations in our laboratory showed that exogenous P was capable of inhibiting the facilitatory effects of EB on the display of lordosis behavior in intact animals. This paper describes an experiment in which the role played by P of
adrenal origin in the development of a sequential inhibition of lordosis behavior by exogenous P was studied. METHOD
Animals Sexually unexperienced adult male rats (strain WI) raised in our colony were used when 2.5 to 3.5 months old and weighing 250-300 g. They were kept under conditions of controlled temperature (22-24°C) and lighting (lights on 0100-1500 hr). They had free access to a commercial laboratory food (UAR) and tap water ad lib.
Procedure Experiment 1. The aim of this experiment was to determine whether the blockade of the secretion of endogenous P by the adrenals would affect lordosis behavior in intact male rats primed with estradiol benzoate (EB) associated or not with progesterone (P). Dexamethasone (DEXA) which is known (12) to exert suppressive effects on pituitary adrenal activity was used because previous experiments in our laboratory (22) clearly demonstrated that this drug sharply depressed the adrenocortical secretion of P in castrated male rats primed with EB.
~Requests for reprints should be addressed to Pr. C1. Aron, Institute of Histology, Faculty of Medicine, 4 Rue Kirschleger, 67000 Strasbourg, France.
1007
1008
CHABLI, SCHAEFFER AND ARON
Progesterone ( ng/ml ) 2,5
TABLE 1 DISPLAY OF LORDOSIS BEHAVIOR IN INTACT MALE RATS GIVEN ESTRADIOL BENZOATE (EB) AND PROGESTERONE (P). EFFECTS OF DEXAMETHASONE (DEXA) TREATMENT
2,0
1,5
Treatment*
1,0
0,5 T
Non injected controls
EB
J
EB t DEXA
FIG. 1. Blood progesterone concentration in intact male rats given a single dose of 25 p~g estradiol benzoate (EB). Trunk blood collection at 1300 hr by 47 hr after EB. Three successive doses of Dexarnethasone (DEXA) were given at 0800 hr and 2000 hr on the day after EB and at 0800 hr on the following day (10 animals in each group). Values are mean ± SEM. *p<0.05 compared to EB +DEXA and controls (Scheffe's test).
Sixty four animals were divided into 4 groups of 16 rats each that were assigned to the following hormonal treatment: 25 p,g EB (SC) at 1400 hr; 25 p,g EB + 100 p,g P (SC) 42 hr later; 25 p,g EB + 3 × 400 p,g dexamethasone (DEXA) (SC) (at 0800 hr and 2000 hr on the day after EB injection and at 0800 hr on the following day, i.e., by 18 hr, 30 hr and 42 hr after EB injection respectively); 25 Ixg EB + 3 × 400 Ixg DEXA + 100 p,g P as in the preceding groups. The animals were tested for lordosis behavior at the beginning of the dark period under a dim light by 50 +- 1 hr after EB injection. Experiment 2. Experiment 2 determined the changes in blood progesterone concentration induced by DEXA in intact male rats primed with a single dose of 25 p,g EB (SC). Three groups of 10 animals each were used. The first one consisted of rats given EB only. The second one was composed of rats given EB and 3 doses of 400 p,g of DEXA as in Experiment 1. A third group of oil injected rats served as controls. All the animals were decapitated at 1300 hr by 47 hr after EB injection and trunk blood was collected for P measurement and stored at - 20°C until further use.
Behavioral Testing The tests for lordosis behavior were carried out in a 60 cm diameter Plexiglas mating arena during a behavioral session lasting 10 min. Previous work (9,21) showed that over such a time period ORCH estrogenized rats were mounted on an average of at least ten times by sexually vigorous males. Stimulus males were selected that were shown to be fully sexually active when tested at the beginning of the dark period with ovariectomized estrogenprogesterone treated female rats. A 5 rain period of adaptation of the stimulus males in the mating arena was allowed prior to the introduction of the experimental animals. As in our previous behavioral studies (1,21) we determined 1) the proportion of animals in each group that showed willingness to mate and therefore to display one or more lordosis in response to male mounts; 2) the lordosis quotient (LQ) in each male by dividing the
25 p,g EB 25 p,g EB + 100 i.LgPt 25 txg EB + DEXA:~ 25 p,g EB + DEXA + 100 p,g P
Proportion of Animals Displaying Lordosis Responses
Mean Lordosis Quotient +_ SEM in Rats Displaying Lordosis Responses
Mean Number of Mounts --- SEM Displayed by the Stimulus Males
15/16§ 9/16
38.5 ± 7.27¶ 78.4 ± 5.40
14.9 + 1.43§ 10.7 --- 1.72
6/16#
40.8 _+ 5.83
14.3 ± 1.87
53.8 --- 8.29
13.8 --- 1.81
14/16
*Testing with a vigorous male was performed between 1500 and 1700 hr by 50--- 1 hr after EB injection (lights on 0100-1500 hr). ~P at 0800 hr by 42 hr after EB and 8 - 1 hr before sexual behavioral testing. ~:400 p,g DEXA at 0800 and 2000 hr on the day after EB injection and at 0800 hr on the day of sexual behavioral testing.. §p<0.05 vs. EB + P, ¶p<0.0t vs. EB + P #p<0.001 vs. EB + DEXA + P.
number of lordosis by the number of mounts, then, multiplying by 100. Lordosis behavior in male rats strictly resembles that displayed by the estrous female. It consisted in arching the back, extending the neck and deviating the tail to one side to expose the genital region. Only deep lordosis in which both head and tall were noticeably raised were taken into consideration. A mean LQ was calculated in each group of animals for the rats showing lordosis and served as measurement of the mating performance.
P Radioimmunoassay (RIA) P was estimated after hexane extraction by RIA method using an antibody of characteristic specificity for 11 ahydroxyprogesterone hemisuccinate bovine serumalbumine (RIA-kit Biomerieux). P measurement was made without column separation because previous assays (19) using Sephadex LH 20 microcolumns as described by Youssefnejadian et al. (29) showed that P values did not differ using either procedure. Intraassay and interassay coefficients were 10% and 8% respectively. Plasma concentration were expressed in ng/ml.
Statistics The Chi square method with or without the continuity correction of Yates was used to compare the proportion of animals showing lordosis responses in the different experimental groups• Variance analysis was used to study LQ values (after angular transformation of the data) and the number of mounts and P values (after logarithmic transformation of the data). Newman-Keuls method for localization of differences was used when necessary. RESULTS
Effects of Endogenous and Exogenous Progesterone on the Display of Lordosis Behavior in Intact Male Rats Primed With Estradiol Benzoate Table 1 shows that the animals given 23 p,g EB and 100 p,g P
LORDOSIS INHIBITION BY PROGESTERONE
1009
successively displayed less frequent lordosis responses to male mounts that those given EB only (9/16 vs. 15/16; )(2 Yates =4.16; p<0.05). DEXA treatment appeared to sharply decrease the number of animals which displayed lordosis behavior and this effect was completely reversed by administration of P (6/16 vs. 14/16; ×2=6.0; p<0.01). The LQ values appeared significantly higher in EB + P animals than in their EB counterparts (78.4 - 5.40 vs. 38.5 - 7 . 2 7 ; p<0.01). As to the mounts, EB + P animals were mounted less frequently than EB animals (10.7--. 1.72 vs. 14.9±
1.43; p<0.05). Changes in Blood Progesterone Concentration Following Estradiol Benzoate and Dexamethasone Treatment As shown in Fig. 1, blood P values appeared to be higher in EB-treated animals than in the two other groups (F(2,27)= 9.18, p<0.001) and P values in DEXA animals and in controls were significantly lower than those found in EB-treated animals
(p<0.05). DISCUSSION In this study we have shown that P was capable of inhibiting the facilitatory effects of EB on the display of lordosis behavior in intact male rats. We observed that 100 p,g P given 42 hours after 25 p,g EB significantly reduced the number of animals displaying lordosis in response to male mounts. At first sight, such a result was unexpected since lordosis behavior has been shown to be facilitated by exogenous P in the castrated male rat (9,28) and also in the intact male rat (28). Moreover previous findings in our laboratory (22) have provided evidence that EB was capable of increasing P concentration in castrated male rats and that endogenous P might thus facilitate the induction of lordosis behavior by estrogen. It then might be hypothesized that EB caused an increase in blood P concentration in our experimental model and rendered the animals sensitive to the inhibitory effects of a subsequent exposure to exogenous P. This assumption, which is based on the well known sequential inhibitory effects of P which occur in ovariectomized rats when they are submitted to two successive injections of P (14-16), is fully supported by the present study. Reduced lordosis response in intact male rats following removal of endogenous P by DEXA treatment confirms (22) that P of adrenal origin--which is secreted under the influence of estrogen--may exert facilitatory effects on the display of lordosis behavior. Furthermore lordosis behavior appeared fully restored by exogenous P in EB + DEXA-treated animals which appeared deprived of endogenous P. In this group exogenous P likely exerted facilitatory effects on lordosis response, instead of its inhibitory action in nonDEXA-treated animals, because endogenous P was suppressed. This clearly demonstrates the sequential nature of P
inhibition in animals given EB and exogenous P successively. It is worth noting that DEXA nonspecific debilitating effects were not responsible for the decrease in lordosis responses in the DEXAtreated males. Changes in motor activity were not observed following DEXA treatment. However we must explain some apparent discrepancy between our results and those of Van de Poll and Van Dis (28) who showed that P could enhance the effects of estrogen on the display of lordosis behavior in intact male rats. This could be accounted for by the fact that estrogen can prevent the appearance of sequential inhibitory effects of P on lordotic responsiveness depending on the priming dose administered (6,24). The shorter interval (36 hr) between estrogen and progesterone injection in Van de Poll and Van Dis's study may also explain the discrepancy between their results and those of our study. Moreover P in high doses may also prevent a desensitization effect on lordosis behavior (3). Unpublished data in our laboratory also show that high doses of EB prevent the inhibitory effects of P to occur in intact male rats. In fact, Van de Poll and Van Dis (28) used higher doses of EB and P than those used in the present work. Two last points deserve attention. Despite the sequential inhibitory effects of P, the copulatory performance as measured by the LQ appeared to be higher in EB + P animals than in those given EB only. Experiments are in progress in our laboratory which suggest that different neuroendocdne mechanisms are involved in the control of the willingness to mate (number of males in a given group which display lordosis responses to male mounts) and the copulatory performance (LQ values). This high degree of female-like "receptivity" in the males given EB + P probably explains that a reduced number of mounts by the stimulus males was sufficient to induce lordosis behavior. Estrogen are known to induce P receptors in the hypothalamus of female (4, 5, 23) and male (8, 13, 20) rodents. But P administration in the female is also known to lead to a loss of loosely bound nuclear P receptors in the brain (5, 7, 23). This accounts for the development of the sequential inhibitory effect of P on the display of lordosis behavior in the estrogen primed ovariectomized female rat (3). The question then may be raised whether this mechanism is involved in the inhibitory effects of P in our experimental model. Of course, further work is required to validate this assumption. In conclusion, the results presented here demonstrate that the intact male rat constitutes a useful tool for a better understanding of the hormonal mechanisms of bisexuality in rodents. ACKNOWLEDGEMENTS We are grateful to Dr. N. Boehm for her assistance in radioimmunoassay and to Mrs. Machart and Ms. Ganter for their technical assistance. We would like to thank Mr. Dejol for the figures and Ms. Gangloff for typing the manuscript. We are thankful to Dr. M. Roos for the statistical analysis of the data and to Mr. G. Weisman for his linguistic assistance.
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