Activation of Estrus by Pheromones in a Marsupial: Stimulus Control and Endocrine Factors

Transcription

1 BIOLOGY OF REPRODUCTION 36, (1987) Activation of Estrus by Pheromones in a Marsupial: Stimulus Control and Endocrine Factors BARBARA H. FADEM Department of Psychiatry and Mental Health Science University of Medicine and Dentistry of New Jersey New Jersey Medical School Newark, New Jersey ABSTRACT Little is known about the effects of social factors or about the hormonal control of social factors on reproduction in marsupials. In previous studies on the gray opossum, a small, Brazilian didelphid, the presence of males was found to activate reproduction in females. In this study, the effects of pheromones produced by intact males, castrated males, and intact females on estrus in female gray opossums was examined. It was found that females housed alone rarely showed spontaneous periods of estrus, that exposure to pheromonal cues provided by intact males reliably induced estrus in % of the experimental females, and that pheromonal cues provided by castrated males or intact, stimulus females induced estrus in 25% of the experimentalfemales. These findings are discussed with respect to the ecology, social behavior, and reproductive characteristics of the didelphid marsupials. INTRODUCTION In eutherian mammals, social cues can coordinate reproductive events. While visual, tactile, auditory, or other exteroceptive factors may be involved (Eleftheriou et al., 1973), pheromones are often utilized in such coordination (Whitten, 1966). In mice, pheromones produced by intact males activate estrus in females (Marsden and Bronson, 1964). Since the ability to activate estrus disappears after a male is castrated, returns when he is treated with androgen, and is present in ovariectomized females that are given androgen (Bronson and Whitten, 1968), production of the estrus-activating pheromone in mice is androgen-dependent. Little is known about the effects of social cues or the relationship between hormonal condition and social cues on reproduction in marsupials. The only reports of such effects were the observations that Virginia opossum (Dideiphis virginiana) females from the same litter came into estrus at the same time (Reynolds, 1952), that reproduction in subordinate Accepted August 28, Received May 27, This study was supported by NIH grant HD (NICHHD). male sugar gliders (Petaurus breviceps) was inhibited by odors produced by the dominant male (Schultz- Westrum, 1969) (in these two studies, no data were presented), and that the presence of females increased production of testosterone in male tammer wallabys (Catling and Sutherland, 1980). Studies of the effects of social and hormonal factors on reproduction in marsupials have not been done largely because it has been difficult to maintain and breed marsupials in the laboratory (Farris, 1950; Barnes, 1968). Recently, the gray opossum (Monodeiphis domestica), a small, South American member of the Didelphidae, the same family as the Virginia opossum, has become a practical laboratory species (Fadem et a!., 1982; VandeBerg, 1983). A recent study demonstrated activation of estrus in female gray opossums by exposure to males (Fadem, 1985). In that study, tactile, auditory, visual, olfactory, and gustatory contact between male and female were permitted while mating was prevented by means of a wire mesh. Thus, the means by which males induced estrus in females could not be identified. The present study was designed to examine the hypothesis that, as in eutherians, the social cue for estrus induction in gray opossums is pheromonal in nature, and further, that this pheromone is androgen-dependent. To do this, intact 328

2 ESTRUS INDUCTION BY PHEROMONES IN A MARSUPIAL 329 female gray opossums were exposed to pheromonal cues provided by intact males, but not to the males themselves. The effects on estrus of pheromonal cues provided by castrated males and by other intact females was also examined. Animals MATERIALS AND METHODS The animals used in the present study were part of a breeding colony of gray opossums at the University of Medicine and Dentistry of New Jersey, New Jersey \ tedical School, Newark, NJ. Adult female gray opossums weigh g, and adult males, g. Sexual maturity is reached at 4-5 mo; breeding has occurred up to age 28 mo in females and 39 mo in males, pregnancy is days and breeding occurs throughout the year (Fadem et a!., 1982; VandeBerg, 1983). The estrous cycle is approximately 32 days, although short, possibly anovulatory cycles of approximately 14 days are also seen in females housed singly in a room containing both males and females (Fadem and Rayve, 1985). For this experiment, eight females aged 7-17 mo served as subjects. Sixteen males (two groups of eight males each) and eight females aged 9-18 mo served as stimulus animals. Maintenance All female subjects were housed in one room (3.4 X 6.1 m) containing no other animals for a minimum of 1 mo prior to the start of the study. Female stimulus animals were housed in a room (1.6 X 3.4 m) containing only other females for a minimum of 1 mo prior to their use in the study. Male stimulus animals were housed in a 3.4 X 6.1 m room Containing both males and females for a minimum of 4 mo prior to the start of the study. A reversed lighting schedule was used in all three rooms with white fluorescent light for 14 h ( h)/24 h, and temperature was maintained at 24#{176}C.The ventilation systems in the three rooms were independent. Water and food (fox food, Milk Products, Inc., New Holstein, WI) were available ad libitum, the water in a standard water bottle with sipper tube and the food in a metal food hopper. Individual animals were housed in polyethylene rat cages (46 X 23 X 19 cm) with wire tops. The bottom of each cage was covered with a 1-cm layer of pine shavings. Except as noted below, cages were washed and shavings were changed weekly. Vaginal Smears Vaginal smears were obtained from the subjects every Monday, Wednesday, and Friday for 42 of the 48 wk of the study. Smears were not taken during one 6-wk period in late July and August. Smears were taken between 0900 and 1100 h, before any cage changes or switching took place (see below). To obtain the smear, a saline-soaked, cotton-tipped swab was inserted approximately 5 mm into the urogenital sinus, aimed slightly downward, rotated once, and removed. The material on the swab was spread on an albumin-coated slide, dried in a warming oven at 30#{176}Cfor 10 mm, placed in a solution of methylene blue for 1 mm, and washed twice in distilled water. The criteria for identifying periods of estrus in this species appear elsewhere (Fadem and Rayve, 1985). Cage-Switch Procedure To present only pheromonal cues provided by the stimulus animal to the experimental female without the confounding physical presence of the stimulus animal, a cage-switch procedure was devised. To conduct a cage switch, the experimental female was put into the cage of the stimulus animal and the stimulus animal was put into the experimental female s cage. Two days later, cages of the experimental female and the stimulus animal were again switched, and two days later, switched again. The experimental female was housed in this cage for an additional 9 days after which she was placed into a clean cage. Thus, the total period of exposure of the experimental female to the odor of the stimulus animal (the experimental period) was 14 days. The cage of the experimental female was always switched with the cage of the same stimulus animal during an experimental period. All cage switching was done after vaginal smearing for that day, outside of the room housing the experimental females. During a cage switch, the stimulus animal was placed in a neutral cage, the experimental female was placed in the stimulus animal s cage, and then the stimulus animal was placed in the experimental female s cage. Experimental females never left their room and were never in visual or auditory contact with the stimulus animals during the cage switch. Cages of animals of both groups were not cleaned for one wk prior to the cage switch, during the week of the cage switch, and for one wk thereafter. At all other times, cages were cleaned weekly as noted above.

3 330 FADEM Experimental Procedure To start the study, vaginal smears were taken from the 8 experimental females for a period of 4 wk. Over the following 44 weeks, the cage-switch procedure was employed with 5 different stimulus animal groups. The stimulus animal groups were (in order of presentation) 1) intact males, 2) the same males 17 days after castration, 3) the same males 129 days after castration, and 4) intact females. To control for the effects of age or multiple exposures to the odors of other animals, experimental females were exposed to the cages of a new group of intact males (Group 5) at the conclusion of the study. To avoid possible cyclicity engendered by presentation of stimulus animal groups at set intervals, time intervals between presentations of these groups were varied randomly. Castration was accomplished under anesthesia induced by methoxyflurane (Metofane; Pitman-Moore, Inc., Washington Crossing, NJ) inhalation. Vaginal smearing continued during and between cage-switch procedures, as above. Data Analysis The truncated binomial distribution (Johnson and Kotz, 1969) was used to compare the number of females coming into estrus during each 14-day period after the cages of each stimulus animal group were switched (experimental period) to the number of females coming into estrus in the period preceding each presentation of the cages of a stimulus animal group when the females were in clean cages (control period). The number of days (N) in each control period were as follows for each stimulus animal group: 1) 28 days, 2)35 days, 3)56 days, 4) 63 days, and 5) 35 days. The probability (p) of estrus not occurring for the N days in each experimental or control period was modeled as pn. The Neyman-Pearson likelihood ratio test (Kendall and Stewart, 1967) was used to test the hypothesis of no difference in the probability of estrus occurring between experimental and control periods for each stimulus animal group and between experimental periods for the five stimulus animal groups. RESULTS A total of twenty-seven periods of estrus were seen in the eight experimental females in the 42 wk that vaginal smears were taken in this study. Twenty-three of these periods occurred in the 2 wk after exposure to the cages of other animals, while only four occurred spontaneously in the 32 control wk of the study. Each of the four spontaneous estrous periods was exhibited by a different experimental female. These results are summarized in Figure 1. All eight experimental females came into estrus 4-11 days after their first exposure to the cages of intact males. Six of these females again came into estrus after their second exposure to the cages of a different group of intact males at the end of the study 42 wk later. The probability of estrus occurring after each of the two exposures to the cages of intact males was significantly greater than in each control period (Xl = 24.5, p<0.0005; Xl 21.4, p<0.0005, INTACT CASTRATED CASTRATED INTACT INTACT MALES I MALES MALES FEMALES MALES I I SHORT LONG TERM (/) L _, TERM (I) w I w sf I - 124< l I I li2- -ll2 IOI I. -IOl l I - IO5 li6 - - ll6 l13 _. U >< 113 I I - U j_., SWITCH CAGES END OF EXPERIMENT FIG. 1. Effects of pheromones produced by intact males, castrated males, and stimulus females on estrus in female gray opossums. Each bar represents a period of estrus. The first vertical dotted line for each stimulus condition is the first day that the experimental females were exposed to pheromones produced by stimulus animals. The second vertical line for each stimulus condition is the day that the experimental females were placed in clean cages.

4 ESTRUS INDUCTION BY PHEROMONES IN A MARSUPIAL 331 respectively), and there was no significant difference in the probability of estrus occurring under the two intact stimulus male conditions, one at the start and one at the end of the study. Five of the eight experimental females came into estrus after exposure to males that had been castrated for 2.5 wk, whereas only two females came into estrus after exposure to these same males 16 wk later. The probability of estrus occurring after exposure to the cages of castrated males was significantly higher than in the control periods in both cases (Xl = 12.0, p<0.001 and Xl = 6.4, p<0.o25) and significantly lower, in the case of males castrated for 18.5 wk, than after exposure to the cages of intact males at the end of the study (Xl = 5.1,p<0.025). One experimental female (#123) came into estrus spontaneously on the morning before the cage switch with intact females and was thus considered to be responding in the control period. Somewhat surprisingly, estrus was activated in two of the remaining experimental females after exposure to the cages of intact stimulus females. The probability of estrus occurring after exposure to the cages of intact stimulus females was significantly higher than in the control period (Xl = 7.15, p<0.ool). Routine weekly cage changes had no effect on the occurrence of estrus. The latency of experimental females to display estrus was different between groups of stimulus animals. While estrus occurred in four females within 4 days after exposure to the cages of intact males, the latency to display estrus was at least 7 days after exposure to the cages of castrated males or intact females. DISCUSSION The results of this study indicate that in gray opossums, 1) females rarely exhibit spontaneous periods of estrus when there is no direct contact with pheromonal cues from conspecifics; 2) pheromonal cues provided by intact males, in the absence of any other exteroceptive stimuli, are sufficient to induce estrus reliably in % of experimental females; and 3) at least partial androgen dependence of pheromone production is likely since pheromonal cues provided by males castrated for an extended period or other intact females induce estrus in only 25% of the experimental females. This study is the first to show that pheromones may be a mechanism by which reproduction is regulated in a female marsupial. Although its speciesspecificity remains to be examined, this mechanism could allow the female gray opossum to take advantage of the presence of a male by entering reproductive condition quickly after exposure to a pheromone that he produces. While the estrus-inducing pheromone in gray opossums is at least partially androgen-dependent, its source remains to be identified. Urine may be the source, as in a number of eutherians (Marsden and Bronson, 1964; Carter et a!., 1980). Also, since male but not female gray opossums possess a suprasternal gland (Fadem and Schwartz, 1985) used in scent marking (Fadem and Cole, 1985), which disappears on castration (Fadem, 1986), secretions of this androgen-dependent structure could serve a reproductive function, such as estrous induction, in this species. Future studies will examine the source of the estrusinducing pheromone in gray opossums. Although an explanation for the ability of castrated males or other females to induce estrus in some female gray opossums is not readily apparent, a number of possibilities exist. First, if a specific primer pheromone is involved in estrus induction in gray opossums, production of this pheromone may not be totally androgen-dependent. Second, as in some eutherian mammals in which estrus may be induced by bedding changes (Hughes, 1964) or by stress (Terman, 1973), pheromones provided by another animal could have caused a nonspecific neuroendocrine response (Bronson, 1985), resulting in estrus in some experimental females. Both specific and nonspecific responses may have been involved in estrus induction in gray opossums. Whatever the mechanism responsible for the results obtained here, this study has shown that reproductive condition in female gray opossums is extremely sensitive to social/pheromonal cues. Physiological responsiveness to social cues is closely related to the ecology of a species (Conaway, 1971). Although little is known about the South American marsupials (Hunsaker, 1977), the findings obtained here can help to generate some hypotheses about the ecology and social behavior of these South American forms. Like the North American opossum, South American didelphids are thought to be nomadic and solitary and to show little social behavior (Hunsaker, 1977; Charles-Dominique, 1983; Streilein, 1982; Lee and Cockburn, 1985). It has been suggested that the activation of estrus by close contact with males would not be of advantage to solitary species in which the

5 332 FADEM physical dispersion of males and females is often great (Whitten and Bronson, 1970; Stoddart, 1980). Thus, although characterized as socially simple and solitary, estrus induction by males seen here, as well as the extensive scent marking seen in a previous study (Fadem and Cole, 1985), indicate that gray opossums may not be as socially simple and solitary as previously described. Rather, it is likely that male and female gray opossums maintain an important communication network via chemosignals. ACKNOWLE DGME NTS I thank the veterinarians and staff of animal caretakers at the UMDNJ-New Jersey Medical School for help with the care and maintenance of animals; Lavy Abramovitch of the Department of Statistics, Rutgers University, for conducting the statistical analyses; and Doris Kraus and Sean Dorrell for technical assistance. REFERENCES Barnes RD, Marmosa mitis, a small marsupial for experimental biology. In: Animal Models for Biomedical Research. Washington, DC: National Academy of Sciences, pp Bronson FH, Mammalian reproduction: an ecological perspective. Biol Reprod 32:1-26 Bronson FH, Whitten WK, Oestrus-accelerating pheromones of mice. Assay, androgen dependency and presence in bladder urine. J Reprod Fertil 15: Carter CS, Getz LL, Gavish L, McDermott JL, Arnold P, Malerelated pheromones and the activation of reproduction in the prairie vole (Microtus ochrogaster). Biol Reprod 2 3: Catling PC, Sutherland RL, Effect of gonadectomy, season and the presence of female rammer wallabys (Macropus euqenii) on concentrations of testosterone, luteinizing hormone and follicle stimulating hormone in the plasma of male tammar wallabys. J Endocrinol 86:25-33 Charles-Dominque P, Ecology and social adaptations in didelphid marsupials: comparison with eutherians of similar ecology. In: Eisenberg J, Kleiman D (eds.), Advances in the Study of Mammalian Behavior Special Publication No. 7. The American Society of Mammalogists, pp Conaway CH, Ecological adaptation and mammalian reproduction. Biol Reprod 4: Eleftheriou BE, Christenson CM, Zarrow MV, The influence of exteroceptive stimuli and pheromonal facilitation of ovulation in different strains of mice. Endocrinology 5 7: Fadem BH, Evidence for the activation of female reproduction by males in a marsupial, the gray short-tailed opossum (Mon 0- delphis domestica). Biol Reprod 33: Fadem BH, Chemical communication in gray short-tailed opossums (Monodelphis dornestica) with comparisons to other marsupials and with reference to monotremes. In: Duvall D, Muller- Schwarze D, Silverstein RM (eds.), Chemical Signals in Vertebrates IV: Ecology, Evolution and Comparative Biology. New York: Plenum Publishing Corp. pp Fadem BH, Cole EA, Scent-marking in the grey short-tailed opossum (Monodelphis domestica). Anim Behav 3 3: Fadem BH, Rayve RS, Characteristics of the oestrous cycle and influence of social factors in grey short-tailed opossums (Monodelphis doniestica). J Reprod Fertil 73: Fadem BH, Schwartz RA, A sexually dimorphic suprasternal scent gland in gray short-tailed opossums (Monodeiphis domestica). J Mammal 67: Fadem BH, Trupin GL, VandeBerg JL, Maliniak E, Hayssen V, Care and breeding of the gray short-tailed opossum (Monodelphis domestica). Lab Anim Sci 23: Farris EJ, The opossum. In: Farris EJ (ed.), Care and Breeding of Laboratory Animals. New York: Wiley, pp Hughes RL, Effect of changing cages, introduction of the male, and other procedures on the oestrous cycle of the rat. CSIRO Wild Res 9: Hunsaker D, Ecology of new world marsupials. In: Hunsaker D (ed.), Biology of Marsupials. New York: Academic Press, pp Johnson NL, Kotz S, Discrete Distributions. Boston: Houghton Mifflin Co. Kendall MG, Stewart A, The Advanced Theory of Statistics, Vol. 2. New York: Hafner Lee AK, Cockburn A, Evolutionary Ecology of Marsupials. Cambridge, England: Cambridge University Press Marsden HM, Bronson FH, Estrous synchrony in mice: alteration by exposure to male urine. Science 144:1469 Reynolds HC, Studies on reproduction in the opossum (Dideiphis vi,iniana virginiana). Univ Calif PubI Zool 52: Schultz-Westrum TG, Social communication by chemicals in flying phalangers. In: Pfaffman C (ed.). Olfaction and Taste: Proceedings of the Third International Symposium. New York: Rockefeller University Press, pp Stoddart DM, The Ecology of Vertebrate Olfaction. New York: Chapman and Hall Streilein KE, Behavior, ecology and distribution of South American marsupials. In: Mores MA, Genoways HH (eds.), Mammalian Biology in South America. Linesville, PA: Pymatuning Laboratory of Ecology: Special Publication, pp Terman CR, Reproductive inhibition in asymptotic populations of prairie deer mice. J Reprod Fertil (Suppl.) 19: VandeBerg JL, The gray short-tailed opossum: a new laboratory animal. ILAR News 26(3):9-12 Whitten WK, Pheromones and mammalian reproduction. Adv Reprod Physiol 1: Whitten WK, Bronson FH, The role of pheromones in mammalian reproduction. In: Johnston JW Jr, Moulton DC, Turk A (eds.), Advances in Chemoreception, Vol 1. Englewood Cliffs, NJ: Appleton Century Crofts-Prentiss Hall, pp