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Ovarian hormones influence odor stimulated flank marking behavior in the hamster (Mesocricetus auratus)
Physiology&Behavior,Vol. 45, pp. 113-117. Copyright© Pergamon Press pie, 1989. Printed in the U.S.A.
0031-9384/89 $3.00 + .00
Ovarian Hormones Influence Odor Stimulated Flank Marking Behavior in the Hamster (Mesocricetus auratus) 1 H. ELLIOTT ALBERS
AND CEYLON
M. R O W L A N D
Laboratory of Neuroendocrinology and Behavior Departments of Biology and Psychology, Georgia State University, Atlanta, GA 30303 R e c e i v e d 11 J u l y 1988 ALBERS, H. E. AND C. M. ROWLAND. Ovarian hormones influence odor stimulatedflank marking behavior in the hamster (Mesocricetus auratus). PHYSIOL BEHAV 45(1) 113-117, 1989.--The effect of ovarian hormones on the frequency of female flank marking in response to the odors of male hamsters was examined. The amount of flank marking differed significantly (p<0.01) among the days of the estrous cycle, with the lowest levels of flank marking occurring on estrus. Ovariectomy (OVX) eliminated the 4 day rhythm, and significantly (p<0.01) reduced the total amount of flank marking. Implantation of Silastic capsules containing estradiol benzoate (EB) in OVX hamsters increased flank marking by approximately two-fold when compared to hamsters implanted with blank Silastic capsules. In a second series of experiments, OVX hamsters given EB flank marked significantly (p<0.01) more than those with blank capsules. Subcutaneous injection of progesterone (P) into OVX-EB treated hamsters 6 hr prior to testing significantly reduced flank marking when compared to OVX-EB treated hamsters given oil. No differences were observed in flank marking between groups of OVX hamsters with blank capsules and injected with P or oil. In summary, these data indicate that the four day rhythm of odor stimulated flank marking in intact female hamsters may be the result of the 4 day fluctuations of estrogen and progesterone that occur during the estrous cycle. Estrogen
Progesterone
Scent marking
M A N Y mammalian species have developed specialized scent glands and scent marking behaviors that allow individuals to communicate by depositing odors in the environment [for reviews see (11, 14, 30)]. Scent marking occurs in a variety of situations and appears to communicate socially important information, such as reproductive state and dominance status. As a result, it is not surprising that gonadal hormones have a major role in the control of scent marking behavior. In male mammals, castration reduces the frequency of scent marking and testosterone replacement therapy restores precastration levels of the behavior (30). In female mammals, the role o f gonadal hormones in the control o f scent marking is less clear-cut. F o r example, the frequency of urine scent marking in mice is not altered by ovariectomy and does not correlate with the changing levels of gonadal hormones during the ovulatory cycle (17). In female gerbils, ovarian hormones do appear to influence the frquency o f scent marking, at least in females that exhibit high levels of marking behavior (19, 27, 29). One form of scent marking observed in golden hamsters (Mesocricetus auratus) is flank marking. Flank marking appears to be involved in communication o f dominance status (9,14) and female mate choice (10). Since solitary hamsters flank mark infrequently in their home cage, flank marking
has been studied primarily using two experimental paradigms. One approach has been to study odor stimulated flank marking which occurs when a hamster is placed in the recently vacated cage of another hamster. A second approach has been to study socially stimulated flank marking, which occurs when two or more hamsters are placed in the same arena. In males, odor stimulated flank marking appears to be testosterone dependent (12), however flank marking stimulated during social encounters can occur at high levels in castrate hamsters (28). In females, the data from several studies suggest that the frequency of flank marking during social encounters is influenced by ovarian hormones. Ovariectomy reduces the frequency of flank marking in hamsters paired with other females (22) and hormone replacement can restore precastration levels of flank marking (26). The only data available on whether ovarian hormones influence the frequency of odor stimulated flank marking have reported a tendency for females to flank mark less on the day of estrus than on other days of the estrous cycle in response to odors of male hamsters (13). The purpose of the present study was to investigate the role of reproductive state in the control of odor stimulated flank marking in female hamsters. Experiment 1 determined whether the frequency of odor stimulated flank
1This work was supported by NSF Grant BNS-8711373.
113
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DAY OF ESTROUS CYCLE FIG. l. Effect of ovariectomy (black bars) or sham ovariectomy (white bars) on the four day rhythm in odor stimulated flank marking approximately two weeks following surgery. The day of the estrus cycle was determined by monitoring vaginal discharge in the sham ovariectomized group and predicted from the monitoring of vaginal discharge before surgery in the ovariectomized group. The numbers of hamsters are indicated in each bar. Abbreviations: diestrus 1 (D1), diestrus 2 (D2), proestrus (P) and estrus (E).
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marking varies over the estrous cycle, and Experiment 2 examined how estrogen and progesterone influence flank marking in response to male odors. METHOD
Adult male and female hamsters obtained from Harlan Sprague Dawley, Inc. were housed under a reverse lightdark cycle containing 14 hr light and 10 hr dark (lights off at 0900) and provided free access to food and water throughout all experiments. The estrous cycle was monitored in intact hamsters using the method of Orsini (18). All hamsters were anesthetized with sodium pentobarbital prior to ovariectomy or sham ovariectomy. An incision was made in the skin and muscle wall directly above the ovary. The ovary was removed and the muscle and skin sutured. Sham ovariectomies were produced using the same method, except the ovary was not removed. Silastic capsules were implanted subcutaneously near the back o f the neck following anesthetization with ether. All Silastic capsules (0.062 in. i.d., 0.125 o.d,; No. 602-285, Dow Coming Corp., Midland, MI) were packed with 5 mm of crystalline 17B-estradiol-3-benzoate (EB) (Sigma, St. Louis) and sealed with Medical Adhesive Silicone Type A (Dow Coming Corp.). EB capsules made in this manner have been reported to produce blood levels of 128-+ 10 pg/ml (15), which is within the physiological range of estradiol values seen during the hamster estrous cycle (3,4). Blank Silastic capsules were also 5 mm in length. Progesterone (Sigma) was injected in a dose o f 0.5 mg in 0.1 ml of corn oil. This dose of progesterone has been reported to produce blood levels of 6.7-+2.6 ng/ml (15), and to be sufficient for the induction of behavioral heat (5). Control injections contained 0.1 ml of corn oil. Adult male hamsters were individually housed in cages ( 1 0 x l 8 x 8 in.) containing bed-o-cobs laboratory animal bedding (The Andersons, Maumee, OH) for 10 days, without cage cleaning. Immediately before odor stimulated tests of female flank marking, the male resident of the cage was removed. The female was placed in the cage for 10 min and the
FIG. 2. Reduction in odor stimulated flank marking following ovariectomy (OVX), but not sham ovariectomy (SHAM-OVX). The left side of the figure indicates the amount of flank marking observed for each group on three consecutive tests conducted at two week intervals. Surgery was performed immediately following the flank marking tests conducted on week 5. The right side of the figure indicates the amount of flank marking observed in each group following surgery. The number of hamsters in each group is indicated in the bars.
number of flank marks recorded. A flank mark was scored each time the hamster pressed its flank gland against the side of the cage and moved forward. Flank marking tests were conducted on four consecutive days at two week intervals. The same male's cage was used for all tests of individual females. Testing was done during the dark phase o f the light-dark cycle under dim red illumination. In Experiment 1, tests for odor stimulated flank marking were conducted on each of the four consecutive days of the estrous cycle. Each test consisted o f a 10 mm exposure to the recently vacated cage of a male hamster every two weeks for six weeks. Statistical differences between groups were analyzed with the one-way or Subjects/Groups x Trials classifications of the analysis of variance (21). RESULTS
Experiment 1 The frequency of flank marking was found to differ significantly (p <0.01) among the days of the estrous cycle. The amount o f flank marking on the first three days o f t b e estrous cycle was similar; diestrus 1:14.64+2.1/10 min; diestrus 2: 16.97+ 1.44; proestrus: 15.71_+ 1.97. In contrast, the number of flank marks observed on estrus were 2-3 times lower than on any other day o f the cycle, i.e., 5.77_+1.47/10 min. To determine whether the 4 day cycle in the frequency of flank marking was induced by ovarian hormones, hamsters were either ovariectomized (OVX) ( N = l l ) or shamovariectomized (SHAM-OVX) (N=5). Tests o f odor sfimu-
OVARIAN HORMONES AND FLANK MARKING
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WEEKS FIG. 3. The effects of implantation of empty Silastic capsules (OVX+BLANK) or Silastic capsules containing estradiol benzoate (OVX+E) on odor stimulated flank marking. Prior to capsule implantation, both groups, which had been ovariectomized for at least seven weeks, were tested for odor stimulated flank marking (C). The higher levels of flank marking observed in the OVX+E group approached, but did not reach statistical significance (O<0.1). The number of hamsters in each group is indicated in the bars.
lated flank marking were again conducted at two week intervals for six consecutive weeks. As can be seen in Fig. 1, a 4 day rhythm in the frequency of flank marking did not persist in OVX hamsters, however in SHAM-OVX the lowest levels o f flank marking continued to occur on estrus. In addition to eliminating the 4 day rhythm in flank marking, OVX significantly (p<0.01) reduced the total amount of flank marking observed in tests conducted four and six weeks postsurgery (Fig. 2). Interestingly, the OVX induced reduction in flank marking was not observed two weeks following surgery. In fact, in eight o f the 11 hamsters higher levels of flank marking was observed two weeks following ovariectomy than during the test immediately preceding the surgery. No statistically significant alterations were observed in the amount of flank marking of the S H A M - O V X group. To investigate the effects of estrogen on odor stimulated flank marking, Silastic capsules containing estradiol benzoate (EB) (N=6) or blank capsules (N=5) were implanted in hamsters that had been ovariectomized for approximately seven weeks. As can be seen in Fig. 3, prior to implantation of Silastic capsules both groups flank marked similar amounts in response to the odors of male hamsters. In flank marking tests conducted three and five weeks following implantation of Silastic capsules, flank marking levels had nearly doubled in the group receiving estrogen, whereas the amount of flank marking observed in hamsters receiving blank capsules remained at about the same levels as before capsule implantation. These between group differences approached, but did not reach statistical significance (p<0.1).
Experiment 2 To more clearly define the role o f ovarian hormones in the control of odor stimulated flank marking a second series of experiments was conducted. Hamsters were ovariectomized and then implanted with blank Silastic capsules or Silastic capsules containing EB one week later. In flank marking
OVX+B
OVXeE +OIL
OVX÷E + PROGI
OVX÷B +OIL
OVX44J +PROG
FIG. 4. The effects of estrogen and progesterone on odor stimulated flank marking. (A) Ovariectomized hamsters implanted with blank Silastic capsules (OVX+B) flank marked significantly 09<0.01) less than hamsters implanted with Silastic capsules containing estradiol benzoate (OVX+E). (B) OVX+E treated hamsters receiving subcutaneous injections of progesterone (PROG) 6 hr before testing were found to flank mark significantly (o<0.05) less than OVX+E hamsters given control injections of oil. (C) No significant (o >0.05) differences in the amount of flank marking were found between OVX+B treated hamsters injected with PROG 6 hr before testing and OVX+B treated hamsters given control injections of oil. The number of hamsters in each group is indicated in the bar.
tests conducted one month later hamsters receiving EB (N = 10) were found to flank mark twice as much as hamsters receiving blank capsules (N=9). This between group difference was statistically significant (p<0.01). To determine whether progesterone might influence the stimulatory effects o f estrogen on flank marking, OVX hamsters were implanted with Silastic capsules containing EB (N=5) or blank Silastic capsules (N=5), and then injected with progesterone or oil 6 hr before tests of odor stimulated flank marking. In OVX hamsters given oil, flank marking levels were similiar to those observed previously, i.e., 13.6_+2.2/10 min. In contrast, injection of progesterone into OVX hamsters given EB significantly (/9<0.05) depressed the frequency of flank marking by over 50%. The effects of progesterone on flank marking appeared to require the presence of estrogen, since no differences in the amount of flank marking was observed between groups of OVX hamsters implanted with blank Silastic capsules and injected with progesterone (N=5) or oil (N=5) 6 hr before testing. DISCUSSION The present study demonstrates that ovarian hormones significantly influence the frequency of hamster flank marking behavior stimulated by the odors of male hamsters. In intact hamsters, the amount of flank marking observed was related to the stage of the estrous cycle. During the first three days of the cycle the amount of flank marking observed was similar, however on estrus the frequency o f flank marking was considerably reduced. Correlation o f flank marking with the known fluctuations o f ovarian hormones during the estrous cycle indicates that the greatest amount of flank marking occurs when estrogen levels are elevated (4). The dramatic reduction in the frequency of flank marking on estrus is preceded by a surge in progesterone levels on the afternoon of proestrus (20). The effects of estrogen and progesterone on the fre-
116
A L B E R S AND R O W L A N D
quency o f flank marking observed in the present study are consistent with the possibility that the fluctuations in estrogen and progesterone during the estrous cycle are responsible for the four day cycle in the frequency of flank marking. Ovariectomy reduced flank marking behavior and administration of physiological levels of estrogen restored precastration levels of flank marking. Administration of progesterone to estrogen primed ovariectomized hamsters resulted in levels of flank marking similar to those observed in intact hamsters on estrus. Progesterone appears to influence flank marking behavior by acting in conjunction with estrogen, since progesterone was found to be without effect unless estrogen was present. The medial preoptic-anterior hypothalamus (MPOA-AH) appears to be involved in mediating the effects of gonadal hormones on a number of behaviors (16). Recently, arginine vasopressin (AVP) within the M P O A - A H has been implicated in the control of hamster flank marking behavior. Microinjection of A V P into the M P O A - A H stimulates intense bouts of flank marking in both male and female hamsters (6,8), and AVP antagonists microinjected into the MPOAA H inhibit flank marking, whether the marking was stimulated by AVP injection, odors of conspecifics or social interactions (2,7). One mechanism which may underlie the effects of testosterone on male flank marking has been identiffed (1). Castration, which reduces odor stimulated flank marking (12), also reduces the induction of flank marking by A V P microinjected into the MPOA-AH. Administration of physiological levels of testosterone restores precastration levels of odor stimulated flank marking in response to AVP. These data suggest that one way testosterone may influence the frequency of flank marking is by altering the response of the M P O A - A H to AVP. The site(s) at which ovarian hormones influence flank marking behavior are not known. Although AVP can stimulate female flank marking when microinjected into the MPOA-AH, the relationship between this finding and the site(s) at which ovarian hormones act awaits further investi-
gation. Several studies have examined the effects of the implantation of estrogen within specific hypothalamic regions on female flank marking. In these studies females were adapted to large arenas and then tested for flank marking stimulated during one hour social interactions with male hamsters (23-25). No significant differences were observed in the amount of flank marking which occurred following the local implantation of estrogen into a variety of hypothalamic sites, including the medial preoptic and anterior hypothalamus. One possible explanation for finding no stimulation of flank marking by estrogen regardless of the hypothalamic site of administration might be provided by the findings of the present study. In the present study approximately three weeks was required for the full expression of the reduction in flank marking produced by ovariectomy, and for the full restoration of precastration levels of flank marking by estrogen. These observations suggest that flank marking may represent an exception to the nearly immediate behavioral effect of ovariectomy normally observed. As a result, the time interval of less than one week between ovariectomy and hypothalamic implant of estrogen used in these studies (23-25) may not have been sufficient to observe the effects of the estrogen implants on flank marking. Data from males suggest that differences may also exist in the endocrine bases of odor stimulated and socially stimulated flank marking. Although odor stimulated flank marking is testosterone dependent (12), flank marking stimulated by social encounters can occur at high levels in castrate males (28). The studies which have investigated the effects of hypothalamic implantation of estrogen examined flank marking stimulated by social encounters with males in large arenas (23-25). In this type of testing arrangement female flank marking occurs at much lower levels than in the tests used for odor stimulated marking in the present study. It will be important to determine whether differences exist in the neuroendocrine control of flank marking induced by odors and social encounters.
REFERENCES
1. Albers, H. E.; Liou, S. Y.; Ferris, C. F. Testosterone alters the behavioral response of the medial preoptic-anterior hypothalamus to microinjection of arginine vasopressin in the hamster. Brain Res. 456:382-386; 1988. 2. Albers, H. E.; Pollock, J.; Simmons, W. H.; Ferris, C. F. A Vl-like receptor mediates vasopressin induced flank marking within the hamster hypothalamus. J. Neurosci. 6:2085-2089; 1986. 3. Albers, H. E.; Moline, M. L.; Moore-Ede, M. C. Sex differences in circadian control of LH secretion. J. Endocrinol. 100:101-105; 1984. 4. Baranczuk, R.; Greenwald, G. S. Peripheral levels of estrogen in the cyclic hamster. Endocrinology 92:805-812; 1973. 5. Carter, C. S.; Clemens, L. G.; Hoekema, D. J. Neonatal androgen and adult sexual behavior in the golden hamster. Physiol. Behav. 9:89-95; 1972. 6. Fen'is, C. F.; Albers, H. E.; Wesolowski, S. M.; Goldman, B. D.; Leeman, S. E. Vasopressin injected into the hypothalamus triggers a complex stereotypic behavior in golden hamsters. Science 224:521-523; 1984. 7. Ferris, C. F.; Pollock, J.; Albers, H. E.; Leeman, S. E. Inhibition of flank marking behavior in golden hamsters by microinjection of a vasopressin antagonist into the hypothalamus. Neurosci. Lett. 55:163-168; 1985.
8. Ferris, C. F.; Meenan, D. M.; Albers, H. E. Microinjection of kainic acid into the hypothalamus of golden hamsters prevents vasopressin-dependent flank marking behavior. Neuroendocrinology 44:112-116; 1986. 9. Ferris, C. F.; Axelson, J. F.; Shinto, L. H.; Albers, H. E. Scent marking and the maintenance of dominant/subordinate status in male golden hamsters. Physiol. Behav. 40:661-664; 1987. 10. Huck, U. W.; Lisk, R. D.; Gore, A. C. Scent marking and mate choice in the golden hamster. Physiol. Behav. 35:389-393; 1985. 11. Johnson, R. P. Scent marking in mammals. Anita. Behav. 21:521-535; 1973. 12. Johnston, R. E. Testosterone dependence of scent marking by male hamsters (Mesocricetus auratus). Behav. Neural Biol. 31:96-99; 1981. 13. Johnston, R. E. The causation of two scent-marking behaviour patterns in female hamsters (Mesocrieetus auratus). Anita. Behav. 25:317-327; 1977. 14. Johnston, R. E. Communication. In: Siegel, H. I., ed. The hamster. New York: Plenum Press; 1985:121-154. 15. Jorgenson, K. L.; Schwartz, N. B. Effect of steroid treatment on tonic and surge secretion of LH and FSH in the female golden hamster: effect of photoperiod. Neuroendocrinology 39:549-554; 1984.
OVARIAN HORMONES AND FLANK MARKING 16. Malsbury, C. W.; Miceli, M. O.; Scouten, C. W. Neural basis of reproductive behavior. In: Siegel, H. I., ed. The hamster. New York: Plenum Press; 1985:229-259. 17. Maruniak, J. A.; Owen, K.; Bronson, F. H.; Desjardins, C. Urinary marking in female house mouse: effects of ovarian steroids, sex experience and type of stimulus. Behav. Biol. 13:211-217; 1975. 18. Orsini, M. W. The external vaginal phenomenon characterizing the stages of the estrous cycle, pregnancy, pseudopregnancy, lactation and the anestrous hamster, (Mesocricetus auratus) Waterhouse. Proc. Anim. Care Panel. 11:193-206; 1961. 19. Owen, K. ; Thiessen, D. D. Estrogen and progesterone interaction in the regulation of scent marking in the female Mongolian gerbil (Meriones unguiculatus). Physiol. Behav. 12:351-355; 1974. 20. Ridley, K.; Greenwald, G. S. Progesterone levels measured every two hours in the cyclic hamster. Proc. Soc. Exp. Biol. Med. 149:10-12; 1975. 21. Steinmetz, J. E.; Roman, A. G.; Patterson, M. M. Statistical programs for the Apple II microcomputer. Behav. Res. Methods Instrnm. 13:702; 1981. 22. Takahashi, L. K.; Lisk, R. D. Intrasexual interactions among female golden hamsters (Mesocricetus auratus) over the estrous cycle. J. Comp. Psychol. 98:267-275; 1984. 23. Takahashi, L. K.; Lisk, R. D. Estrogen action in anterior and ventromedial hypothalamus and the modulation of heterosexual behavior in female golden hamsters. Physiol. Behav. 34:233239; 1985.
117 24. Takahashi, L. K.; Lisk, R. D.; Burnett, A. L., II. Dual estradiol action in diencephalon and the regulation of sociosexual behavior in female golden hamsters. Brain Res. 359:194--207; 1985. 25. Takahashi, L. K.; Lisk, R. D. Diencephalic organization of estradiol sensitive sites regulating sociosexual behavior in female golden hamsters: contralateral versus ipsalateral activation. Brain Res. 425:337-345; 1987. 26. Vandenbergh, J. G. The effects of gonadal hormones on the aggressive behaviour of adult golden hamsters (Mesocricetus auratus). Anim. Behav. 19:589-594; 1971. 27. Wallace, P. K.; Owen, K.; Theissen, D. D. The control and function of maternal scent marking in the Mongolian gerbil (Meriones unguiculatus). Physiol. Behav. 10:463-466; 1973. 28. Whitsett, J. M. The development of agressive and marking behavior in intact and castrated male hamsters. Horm. Behav. 6:47-57; 1975. 29. Yahr, P.; Theissen, D. D. Steroid regulation of territorial scent marking in the Mongolian gerbil (Meriones unguiculatus). Horm. Behav. 3:359-368; 1972. 30. Yahr, P. Hormonal influence on territorial marking behavior. In: Svare, B., ed. Hormones and aggressive behavior. New York: Plenum Press; 1983:145-175.
0031-9384/89 $3.00 + .00
Ovarian Hormones Influence Odor Stimulated Flank Marking Behavior in the Hamster (Mesocricetus auratus) 1 H. ELLIOTT ALBERS
AND CEYLON
M. R O W L A N D
Laboratory of Neuroendocrinology and Behavior Departments of Biology and Psychology, Georgia State University, Atlanta, GA 30303 R e c e i v e d 11 J u l y 1988 ALBERS, H. E. AND C. M. ROWLAND. Ovarian hormones influence odor stimulatedflank marking behavior in the hamster (Mesocricetus auratus). PHYSIOL BEHAV 45(1) 113-117, 1989.--The effect of ovarian hormones on the frequency of female flank marking in response to the odors of male hamsters was examined. The amount of flank marking differed significantly (p<0.01) among the days of the estrous cycle, with the lowest levels of flank marking occurring on estrus. Ovariectomy (OVX) eliminated the 4 day rhythm, and significantly (p<0.01) reduced the total amount of flank marking. Implantation of Silastic capsules containing estradiol benzoate (EB) in OVX hamsters increased flank marking by approximately two-fold when compared to hamsters implanted with blank Silastic capsules. In a second series of experiments, OVX hamsters given EB flank marked significantly (p<0.01) more than those with blank capsules. Subcutaneous injection of progesterone (P) into OVX-EB treated hamsters 6 hr prior to testing significantly reduced flank marking when compared to OVX-EB treated hamsters given oil. No differences were observed in flank marking between groups of OVX hamsters with blank capsules and injected with P or oil. In summary, these data indicate that the four day rhythm of odor stimulated flank marking in intact female hamsters may be the result of the 4 day fluctuations of estrogen and progesterone that occur during the estrous cycle. Estrogen
Progesterone
Scent marking
M A N Y mammalian species have developed specialized scent glands and scent marking behaviors that allow individuals to communicate by depositing odors in the environment [for reviews see (11, 14, 30)]. Scent marking occurs in a variety of situations and appears to communicate socially important information, such as reproductive state and dominance status. As a result, it is not surprising that gonadal hormones have a major role in the control of scent marking behavior. In male mammals, castration reduces the frequency of scent marking and testosterone replacement therapy restores precastration levels of the behavior (30). In female mammals, the role o f gonadal hormones in the control o f scent marking is less clear-cut. F o r example, the frequency of urine scent marking in mice is not altered by ovariectomy and does not correlate with the changing levels of gonadal hormones during the ovulatory cycle (17). In female gerbils, ovarian hormones do appear to influence the frquency o f scent marking, at least in females that exhibit high levels of marking behavior (19, 27, 29). One form of scent marking observed in golden hamsters (Mesocricetus auratus) is flank marking. Flank marking appears to be involved in communication o f dominance status (9,14) and female mate choice (10). Since solitary hamsters flank mark infrequently in their home cage, flank marking
has been studied primarily using two experimental paradigms. One approach has been to study odor stimulated flank marking which occurs when a hamster is placed in the recently vacated cage of another hamster. A second approach has been to study socially stimulated flank marking, which occurs when two or more hamsters are placed in the same arena. In males, odor stimulated flank marking appears to be testosterone dependent (12), however flank marking stimulated during social encounters can occur at high levels in castrate hamsters (28). In females, the data from several studies suggest that the frequency of flank marking during social encounters is influenced by ovarian hormones. Ovariectomy reduces the frequency of flank marking in hamsters paired with other females (22) and hormone replacement can restore precastration levels of flank marking (26). The only data available on whether ovarian hormones influence the frequency of odor stimulated flank marking have reported a tendency for females to flank mark less on the day of estrus than on other days of the estrous cycle in response to odors of male hamsters (13). The purpose of the present study was to investigate the role of reproductive state in the control of odor stimulated flank marking in female hamsters. Experiment 1 determined whether the frequency of odor stimulated flank
1This work was supported by NSF Grant BNS-8711373.
113
114
ALBERS AND ROWLAND
INTACT
OVX
20
z
'111, I .2kllA D1
10. C) o
D2
P
E
DAY OF ESTROUS CYCLE FIG. l. Effect of ovariectomy (black bars) or sham ovariectomy (white bars) on the four day rhythm in odor stimulated flank marking approximately two weeks following surgery. The day of the estrus cycle was determined by monitoring vaginal discharge in the sham ovariectomized group and predicted from the monitoring of vaginal discharge before surgery in the ovariectomized group. The numbers of hamsters are indicated in each bar. Abbreviations: diestrus 1 (D1), diestrus 2 (D2), proestrus (P) and estrus (E).
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marking varies over the estrous cycle, and Experiment 2 examined how estrogen and progesterone influence flank marking in response to male odors. METHOD
Adult male and female hamsters obtained from Harlan Sprague Dawley, Inc. were housed under a reverse lightdark cycle containing 14 hr light and 10 hr dark (lights off at 0900) and provided free access to food and water throughout all experiments. The estrous cycle was monitored in intact hamsters using the method of Orsini (18). All hamsters were anesthetized with sodium pentobarbital prior to ovariectomy or sham ovariectomy. An incision was made in the skin and muscle wall directly above the ovary. The ovary was removed and the muscle and skin sutured. Sham ovariectomies were produced using the same method, except the ovary was not removed. Silastic capsules were implanted subcutaneously near the back o f the neck following anesthetization with ether. All Silastic capsules (0.062 in. i.d., 0.125 o.d,; No. 602-285, Dow Coming Corp., Midland, MI) were packed with 5 mm of crystalline 17B-estradiol-3-benzoate (EB) (Sigma, St. Louis) and sealed with Medical Adhesive Silicone Type A (Dow Coming Corp.). EB capsules made in this manner have been reported to produce blood levels of 128-+ 10 pg/ml (15), which is within the physiological range of estradiol values seen during the hamster estrous cycle (3,4). Blank Silastic capsules were also 5 mm in length. Progesterone (Sigma) was injected in a dose o f 0.5 mg in 0.1 ml of corn oil. This dose of progesterone has been reported to produce blood levels of 6.7-+2.6 ng/ml (15), and to be sufficient for the induction of behavioral heat (5). Control injections contained 0.1 ml of corn oil. Adult male hamsters were individually housed in cages ( 1 0 x l 8 x 8 in.) containing bed-o-cobs laboratory animal bedding (The Andersons, Maumee, OH) for 10 days, without cage cleaning. Immediately before odor stimulated tests of female flank marking, the male resident of the cage was removed. The female was placed in the cage for 10 min and the
FIG. 2. Reduction in odor stimulated flank marking following ovariectomy (OVX), but not sham ovariectomy (SHAM-OVX). The left side of the figure indicates the amount of flank marking observed for each group on three consecutive tests conducted at two week intervals. Surgery was performed immediately following the flank marking tests conducted on week 5. The right side of the figure indicates the amount of flank marking observed in each group following surgery. The number of hamsters in each group is indicated in the bars.
number of flank marks recorded. A flank mark was scored each time the hamster pressed its flank gland against the side of the cage and moved forward. Flank marking tests were conducted on four consecutive days at two week intervals. The same male's cage was used for all tests of individual females. Testing was done during the dark phase o f the light-dark cycle under dim red illumination. In Experiment 1, tests for odor stimulated flank marking were conducted on each of the four consecutive days of the estrous cycle. Each test consisted o f a 10 mm exposure to the recently vacated cage of a male hamster every two weeks for six weeks. Statistical differences between groups were analyzed with the one-way or Subjects/Groups x Trials classifications of the analysis of variance (21). RESULTS
Experiment 1 The frequency of flank marking was found to differ significantly (p <0.01) among the days of the estrous cycle. The amount o f flank marking on the first three days o f t b e estrous cycle was similar; diestrus 1:14.64+2.1/10 min; diestrus 2: 16.97+ 1.44; proestrus: 15.71_+ 1.97. In contrast, the number of flank marks observed on estrus were 2-3 times lower than on any other day o f the cycle, i.e., 5.77_+1.47/10 min. To determine whether the 4 day cycle in the frequency of flank marking was induced by ovarian hormones, hamsters were either ovariectomized (OVX) ( N = l l ) or shamovariectomized (SHAM-OVX) (N=5). Tests o f odor sfimu-
OVARIAN HORMONES AND FLANK MARKING
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WEEKS FIG. 3. The effects of implantation of empty Silastic capsules (OVX+BLANK) or Silastic capsules containing estradiol benzoate (OVX+E) on odor stimulated flank marking. Prior to capsule implantation, both groups, which had been ovariectomized for at least seven weeks, were tested for odor stimulated flank marking (C). The higher levels of flank marking observed in the OVX+E group approached, but did not reach statistical significance (O<0.1). The number of hamsters in each group is indicated in the bars.
lated flank marking were again conducted at two week intervals for six consecutive weeks. As can be seen in Fig. 1, a 4 day rhythm in the frequency of flank marking did not persist in OVX hamsters, however in SHAM-OVX the lowest levels o f flank marking continued to occur on estrus. In addition to eliminating the 4 day rhythm in flank marking, OVX significantly (p<0.01) reduced the total amount of flank marking observed in tests conducted four and six weeks postsurgery (Fig. 2). Interestingly, the OVX induced reduction in flank marking was not observed two weeks following surgery. In fact, in eight o f the 11 hamsters higher levels of flank marking was observed two weeks following ovariectomy than during the test immediately preceding the surgery. No statistically significant alterations were observed in the amount of flank marking of the S H A M - O V X group. To investigate the effects of estrogen on odor stimulated flank marking, Silastic capsules containing estradiol benzoate (EB) (N=6) or blank capsules (N=5) were implanted in hamsters that had been ovariectomized for approximately seven weeks. As can be seen in Fig. 3, prior to implantation of Silastic capsules both groups flank marked similar amounts in response to the odors of male hamsters. In flank marking tests conducted three and five weeks following implantation of Silastic capsules, flank marking levels had nearly doubled in the group receiving estrogen, whereas the amount of flank marking observed in hamsters receiving blank capsules remained at about the same levels as before capsule implantation. These between group differences approached, but did not reach statistical significance (p<0.1).
Experiment 2 To more clearly define the role o f ovarian hormones in the control of odor stimulated flank marking a second series of experiments was conducted. Hamsters were ovariectomized and then implanted with blank Silastic capsules or Silastic capsules containing EB one week later. In flank marking
OVX+B
OVXeE +OIL
OVX÷E + PROGI
OVX÷B +OIL
OVX44J +PROG
FIG. 4. The effects of estrogen and progesterone on odor stimulated flank marking. (A) Ovariectomized hamsters implanted with blank Silastic capsules (OVX+B) flank marked significantly 09<0.01) less than hamsters implanted with Silastic capsules containing estradiol benzoate (OVX+E). (B) OVX+E treated hamsters receiving subcutaneous injections of progesterone (PROG) 6 hr before testing were found to flank mark significantly (o<0.05) less than OVX+E hamsters given control injections of oil. (C) No significant (o >0.05) differences in the amount of flank marking were found between OVX+B treated hamsters injected with PROG 6 hr before testing and OVX+B treated hamsters given control injections of oil. The number of hamsters in each group is indicated in the bar.
tests conducted one month later hamsters receiving EB (N = 10) were found to flank mark twice as much as hamsters receiving blank capsules (N=9). This between group difference was statistically significant (p<0.01). To determine whether progesterone might influence the stimulatory effects o f estrogen on flank marking, OVX hamsters were implanted with Silastic capsules containing EB (N=5) or blank Silastic capsules (N=5), and then injected with progesterone or oil 6 hr before tests of odor stimulated flank marking. In OVX hamsters given oil, flank marking levels were similiar to those observed previously, i.e., 13.6_+2.2/10 min. In contrast, injection of progesterone into OVX hamsters given EB significantly (/9<0.05) depressed the frequency of flank marking by over 50%. The effects of progesterone on flank marking appeared to require the presence of estrogen, since no differences in the amount of flank marking was observed between groups of OVX hamsters implanted with blank Silastic capsules and injected with progesterone (N=5) or oil (N=5) 6 hr before testing. DISCUSSION The present study demonstrates that ovarian hormones significantly influence the frequency of hamster flank marking behavior stimulated by the odors of male hamsters. In intact hamsters, the amount of flank marking observed was related to the stage of the estrous cycle. During the first three days of the cycle the amount of flank marking observed was similar, however on estrus the frequency o f flank marking was considerably reduced. Correlation o f flank marking with the known fluctuations o f ovarian hormones during the estrous cycle indicates that the greatest amount of flank marking occurs when estrogen levels are elevated (4). The dramatic reduction in the frequency of flank marking on estrus is preceded by a surge in progesterone levels on the afternoon of proestrus (20). The effects of estrogen and progesterone on the fre-
116
A L B E R S AND R O W L A N D
quency o f flank marking observed in the present study are consistent with the possibility that the fluctuations in estrogen and progesterone during the estrous cycle are responsible for the four day cycle in the frequency of flank marking. Ovariectomy reduced flank marking behavior and administration of physiological levels of estrogen restored precastration levels of flank marking. Administration of progesterone to estrogen primed ovariectomized hamsters resulted in levels of flank marking similar to those observed in intact hamsters on estrus. Progesterone appears to influence flank marking behavior by acting in conjunction with estrogen, since progesterone was found to be without effect unless estrogen was present. The medial preoptic-anterior hypothalamus (MPOA-AH) appears to be involved in mediating the effects of gonadal hormones on a number of behaviors (16). Recently, arginine vasopressin (AVP) within the M P O A - A H has been implicated in the control of hamster flank marking behavior. Microinjection of A V P into the M P O A - A H stimulates intense bouts of flank marking in both male and female hamsters (6,8), and AVP antagonists microinjected into the MPOAA H inhibit flank marking, whether the marking was stimulated by AVP injection, odors of conspecifics or social interactions (2,7). One mechanism which may underlie the effects of testosterone on male flank marking has been identiffed (1). Castration, which reduces odor stimulated flank marking (12), also reduces the induction of flank marking by A V P microinjected into the MPOA-AH. Administration of physiological levels of testosterone restores precastration levels of odor stimulated flank marking in response to AVP. These data suggest that one way testosterone may influence the frequency of flank marking is by altering the response of the M P O A - A H to AVP. The site(s) at which ovarian hormones influence flank marking behavior are not known. Although AVP can stimulate female flank marking when microinjected into the MPOA-AH, the relationship between this finding and the site(s) at which ovarian hormones act awaits further investi-
gation. Several studies have examined the effects of the implantation of estrogen within specific hypothalamic regions on female flank marking. In these studies females were adapted to large arenas and then tested for flank marking stimulated during one hour social interactions with male hamsters (23-25). No significant differences were observed in the amount of flank marking which occurred following the local implantation of estrogen into a variety of hypothalamic sites, including the medial preoptic and anterior hypothalamus. One possible explanation for finding no stimulation of flank marking by estrogen regardless of the hypothalamic site of administration might be provided by the findings of the present study. In the present study approximately three weeks was required for the full expression of the reduction in flank marking produced by ovariectomy, and for the full restoration of precastration levels of flank marking by estrogen. These observations suggest that flank marking may represent an exception to the nearly immediate behavioral effect of ovariectomy normally observed. As a result, the time interval of less than one week between ovariectomy and hypothalamic implant of estrogen used in these studies (23-25) may not have been sufficient to observe the effects of the estrogen implants on flank marking. Data from males suggest that differences may also exist in the endocrine bases of odor stimulated and socially stimulated flank marking. Although odor stimulated flank marking is testosterone dependent (12), flank marking stimulated by social encounters can occur at high levels in castrate males (28). The studies which have investigated the effects of hypothalamic implantation of estrogen examined flank marking stimulated by social encounters with males in large arenas (23-25). In this type of testing arrangement female flank marking occurs at much lower levels than in the tests used for odor stimulated marking in the present study. It will be important to determine whether differences exist in the neuroendocrine control of flank marking induced by odors and social encounters.
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