Attractivity of male rats induced by estradiol and progesterone

Physiology &Behavior,Vol. 40, pp. 737-740. Copyright©Pergamon Journals Ltd., 1987. Printed in the U.S.A.

0031-9384/87 $3.00 + .00

Attractivity of Male Rats Induced by Estradiol and Progesterone J. M E R K X , A . K . S L O B A N D J. J. V A N D E R W E R F F T E N B O S C H

Department of Endocrinology, Growth and Reproduction, Faculty of Medicine Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands R e c e i v e d 18 J u n e 1987 MERKX, J., A. K. SLOB AND J. J. VAN DER WERFF TEN BOSCH. Attractivityof male rats inducedby estradioland progesterone. PHYSIOL BEHAV 40(6) 737-740, 1987.--From the present study it appears that castrated male rats can be made attractive to intact males by estradiol benzoate plus progesterone (EB + P). For the determination of attractivity a residential plus-maze was used in which the resident male could choose between the company oftbe experimental male and that of an ovariectomized female with or without EB + P. In one experiment the males were castrated in adulthood; these animals were less attractive than the ovariectomized females with a similar hormone treatment. In a second experiment it was found that males that had been castrated at birth were as attractive as the ovariectomized females, after treatment with EB + P. These findings indicate that the attractivity that can be induced by EP + P in gonadectomized adult rats is somewhat but not wholly reduced if androgen is present during the neonatal period. Attractivity

Neonatal castration

Estradiol

Progesterone

specific (in this case the intact male rat) towards the female incentive [3], they concluded that neonatally androgenized female rats were less attractive than neonatally untreated female rats. The present study was designed to show: (1) if castrated male rats could be made attractive by EB and P, and (2) if neonatal castration would affect hormonally induced attractivity in adulthood. Two experiments were carried out. In both experiments a residential plus-maze was used, in which a male rat (the resident rat) was housed during several weeks. The time this resident spent in the neighbourbood of a conspecific was used as a measure o f the latter's attractivity. In an earlier study [13] it had been demonstrated that when this conspecific was an intact female, the resident male would spend nearly all of his time in her vicinity during the night of her oestrus, while on other days he was less interested in her.

ATTRACTIVITY, proceptivity and receptivity are three terms to describe characteristics of a female rat during phases of sexual interaction [3]. Attractivity can be defined as the fentale's stimulus value; proceptivity connotes her appetitive reactions towards the male and receptivity comprises those responses of the female which make an intravaginal ejaculation by the male possible. It has long been known that mating behavior of the female rat is dependent on ovarian hormones for expression [1]. After ovariectomy, administration of estradiol benzoate (EB) in combination with progesterone (P) will induce the characteristic behaviors of the three phases in mating behavior (e.g., [2, 7, 9, 13]). It has been known for some time that occasionally male rats castrated in adult life also exhibit receptive behavior (lordosis), following treatment with EB and P [16,17]. The activating effects of these hormones on lordosis behavior are facilitated by the absence of androgen during the neonatal period, as in the normal female rat and in the male rat after neonatal castration [4, 6, 8, 21]. Other studies have shown that neonatal castration of male rats also allows facilitating effects on proceptive behaviors to be induced by estradiol (benzoate) and progesterone [6, 14, 20]. F a d e m and Barfield [6] showed that the presence of androgen during the neonatal period blocked both proceptive (i.e., ear-wiggiing) and receptive (lordosis) behaviors in adult rats. The effects of androgen in the neonatal period on oestrogen induced attractivity have been investigated recently. De Jonge, Meyerson and van de Poll [11], using an open field test situation, showed that intact adult male rats preferred the neighbourhood of a normal female rat over the company of a neonatally androgertized female rat, when both females were ovariectomized and given repeated injections with EB. Since attractivity is inferred from the behavior of a con-

METHOD

General Locally bred R × U Wistar rats were used, which were housed in isosexual groups after weaning at the age of 3 weeks. Female rats were ovariectomized at the age of 3 months. At the beginning of each experiment the animals were about 4 months old and none of them had heterosexual mating experience. In both experiments male rats (the resident rats) were housed individually in a residential plus-maze, which has been described in earlier publications [10,13]. A plus-maze consists o f a central box and four arms extending from it to four peripheral boxes. In small cages on top of two opposite peripheral boxes stimulus animals can be housed in such a way that the resident male can see, hear and smell the

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adulthood (hatched columns) and with an ovariectomized female rat (open columns). EB and P indicate the nights following injection of estradiol benzoate and progesterone into one of the stimulus rats. n =number of resident males from which data could be collected.

stimulus animal through a wire-mesh. The residential rat had free access to the four peripheral boxes 24 hr a day. His whereabouts were monitored by two infra-red light detectors in each arm. In this way the duration of the visits to each of the four peripheral boxes were registered (for more details see [ 18]). Since earlier studies have revealed that the resident rat is most active during the dark period (21.00-07.00 hr) only the data o f this period were analyzed [13]. Both the resident rats and the stimulus rats were adapted to the maze during several days before data collection was started. For analysis of results non-parametric statistics were used [5,19], since data were assumed not to be distributed homogeneously. However, in Figs. 1 and 2 parametric results, i.e., m e a n s + S E M , are shown. The test of Friedman was used for the analysis of the time male rats spent during different nights of each treatment period of five days in the vicinity of one particular stimulus rat, i.e., in the peripheral box below the latter animal. The Wilcoxon matched-pairs signed-ranks test was used for analyzing the difference in time the resident male rats spent in the neighbourhood of either of the two stimulus animals.

circumstances [13]. The castrated male rat was treated in the first and third week, the ovariectomized female rat in the second and fourth week (see Fig. 1).

Experiment 2 Twelve male rats were castrated within 24 hr after birth. When they were about 4 months old they were put in one of the two top-cages. In the opposite top-cage an ovariectomized female rat was housed. The dose and timing of hormone administration were the same as in experiment 1. There were 4 treatment periods of one week each. During the first period the ovariectomized female rat and during the second period the neonatally castrated male rat was treated. During the third period both stimulus animals were treated simultaneously. This was also done in the fourth period, but during the night following the injection of P both stimulus animals were anaesthetized with phenobarbital (150 mg/kg body-weight IP), a dose sufficient for anaesthesia for about 6 hr. RESULTS

Experiment 1

Experiment I

At the age of 2 months 6 male rats were castrated. Two months later they were placed in one of the two cages on top of the terminal boxes, while in each opposite top-cage an ovariectomized female rat was housed. These two stimulus animals in each plus-maze were treated alternately with an SC injection of EB (2.5 ~g) followed 44 hr later by an SC injection of P (0.5 rag). This latter injection was administered at 17.00 hr, i.e., 4 hr before lights went off. This regime of hormone treatment has been shown to induce attractivity in ovarieetomized female rats of this strain under these test

The time that the resident males spent in the neighbourhood of the stimulus animals during the different nights in the four treatment periods is shown in Fig. 1. Due to failure of two light detectors only the data of 4 plus-mazes could be used from treatment period II onward. Figure 1 clearly shows that during treatment periods I and III the resident male spent more time near the castrated male after the latter animal was injected with P than during other nights (Friedman K1=138, p<0.05; K3=115, p<0.01). The results of treatment periods II and IV are in agreement with earlier

HORMONES AND ATTRACTIVITY OF MALE RATS

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FIG. 2. Mean number of min per 10 hr night spent by intact male rats with a neonatally castrated male rat (hatched columns) and with an ovariectomized female rat (open columns). EB and P indicate the nights followinginjection of estradiol benzoate and progesterone into one or both of the stimulus rats. n=number of resident males from which data could be collected.

experiments: during the night following the injection of P into the ovariectomized female, the resident male rat spent more time in her company than during other nights (Friedman Ks= 140, p<0.01; K4 = 134, p<0.01). During all nights of the four treatment periods, except the night after P injection to the castrated male rats in periods I and III, most (in I, 4 out of 6; and 3 out of 4 in II, III and IV) of the resident males spent more time near the ovariectomized female than near the castrated male. The time that the resident males spent near the ovariectomized female rat in the two nights after P injection (mean_+SEM: 509.1_+15.12 min/night) was significantly longer than the time they spent near the castrated male rat in both nights after P treatment (mean_+SEM: 244.5_+77.88 min/night; Wilcoxon V=21, p<0.05).

Experiment 2 Figure 2 shows the results of this experiment. During the first treatment period the resident males spent more time near the ovariectomized females during the night after P injection than during other nights (Friedman K1=678, p<0.01) and also spent more time near the females than near the neonatally castrated males (Wilcoxon V=64, p<0.01). During the second treatment period, when the neonatally castrated males were treated with EB + P, the resident males preferred the company of these rats during the night following P treatment as compared to other nights of this treatment period (Friedman K~=550, p<0.01). During this particular night they also spent more time with the neonatally castrated male than with the ovariectomized female (Wilcoxon V =45, p <0.05). In the third treatment period both the ovariectomized and

the neonatally castrated male rat were treated simultaneously with hormones. In the night following P injection the resident males spent more time near the ovariectomized female than during other nights of this treatment period (Friedman Ka= 330, p<0.01). During that particular night 6 out of 10 resident males spent more time with her than with the neonatally castrated male (Wilcoxon V=30, ns). There were no differences between the times they spent during different nights near the neonatally castrated male (Friedman Ks= 164, ns). In the last treatment period both stimulus animals were treated with hormones and in the night following P injection they were anaesthetized. During this night 6 out of 10 resident males spent more time near the ovariectomized female than near the neonatally castrated male (Wilcoxon V=32, ns). As during treatment period III the resident males stayed longer near the P treated ovariectomized females than during other nights (Friedman K4=264, p <0.05), while there was no such difference in the time spent near the neonatally castrated male rats during different nights (Friedman K4= 105, ns).

DISCUSSION

It has recently been found that in the plus-maze a female rat is more attractive to an intact heterosexually naive male during the night of oestrus than during other nights. Such preference was displayed irrespective whether oestrus occurred naturally or was induced by exogenous hormones (oestrogen plus P) [13]. From the first experiment of the present study it appears that treatment with EB and P also causes a long-term castrated male rat to become more attractive to a resident male. Since it is known that the hor-

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MERKX, SLOB AND VAN DER WERFF TEN BOSCH

mones used will also induce receptive behaviors in castrated male rats [16,17], it can be c o n c l u d e d that both phases of 'feminine' sexual b e h a v i o r are determined by the presence of adequate h o r m o n a l stimulation in adulthood. Despite the similar treatment of g o n a d e c t o m y and h o r m o n e administration in adulthood (experiment 1), the males seem less attractive than the females since the resident males spent twice as m u c h time with the latter animals as with the former, after EB and P treatment. Males castrated at birth are equally attractive as females o v a r i e c t o m i z e d in adulthood when both are treated with E B and P (experiment 2). It thus appears that the p r e s e n c e of androgen during the neonatal period is an important factor in determining the degree of E B + P induced attractivity in adulthood. De Jonge, M e y e r s o n and van de Poll [11] have s h o w n that intact male rats were more attracted to a neonatally untreated female rat than to a female rat which had been treated neonatally with a single injection of testosterone propionate. T o g e t h e r with the experiments o f F a d e m and Barfield [6] who showed that neonatally castrated male rats displayed p r o c e p t i v e and receptive behaviors in an equal amount as do females, the present experiments support the hypothesis that all three aspects of feminine sexual b e h a v i o r in rats are strongly influenced by the presence of androgen during the neonatal period. The results of the fourth treatment period of the second

experiment indicate that anaesthetized oestrous females are still attractive to male rats, a finding which is corroborated by ongoing research in our laboratory. These results indicate that non-behavioral cues determine to a great extent the attractivity of oestrous female rats. Although it has been shown for the hamster that during oestrus the vaginal discharge contains chemical substances which attract male hamsters and stimulate their mating b e h a v i o r [151], the rat findings appear to need a different explanation. In the last two treatment periods of experiment 2 male rats (which had no vagina) were as attractive to male rats following E B + P treatment as o v a r i e c t o m i z e d females. This leads to the conclusion that in the rat the vaginal discharge does not necessarily play an important role in the emission of so-called sex attractants. At least it is not a 'conditio sine qua non.' Lucas, D o n o h o e and T h o d y [12] have shown that the preputial glands play an important role in the production of sex attractants. E x p e r i m e n t s are going on to determine to what extent the preputial glands contribute to female attractivity as measured in the plus-maze situation.

ACKNOWLEDGEMENTS The authors are indebted to Frits Vels, Pieter van der Schoot and Paula van der Vaart for valuable assistance.

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