Progesterone can either increase or decrease weight gain and adiposity in ovariectomized syrian hamsters

Physiology & Behavior, Vol. 46. pp. 273-278. ©Pergamon Press plc, 1989. Printed in the U.S.A.

0031-9384/89 $3.00 + .00

Progesterone Can Either Increase or Decrease Weight Gain and Adiposity in Ovariectomized Syrian Hamsters A N I T A J. B H A T I A 1 A N D G E O R G E N. W A D E

Neuroscience and Behavior Program and Department o f Psychology University o f Massachusetts, Amherst, M A 01003 R e c e i v e d 9 February 1989

BHATIA, A. J. AND G. N. WADE. Progesterone can either increase or decrease weight gain and adiposity in ovariectomized Syrian hamsters. PHYSIOL BEHAV 46(2) 273-278, 1989.--We examined the effects of estradiol and progesterone on weight gain, food intake, and carcass composition in Syrian hamsters (Mesocricetusauratus). In ovariectomized (OVX) hamsters injections of 5 p.g/day estradiol benzoate (EB) alone decreased weight gain and adiposity, whereas treatment with progesterone alone (1 or 5 rag/day) resulted in increased weight gain and adiposity. However, concurrent treatment with progesterone and EB reduced body fat content to levels significantly below those of hamsters treated with EB alone. In a second experiment, 1713-estradioland progesterone were administered via subcutaneous Silastic capsules in doses which produce physiological levels of steroids. Implants of estradiol significantly decreased body weight gain and fat content. As in the first experiment, these effects of estradiol were exaggerated by concurrent progesterone administration. Implants of progesterone alone did not affect body weight or fatness in OVX hamsters. These data indicate that estradiol and progesterone interact to decrease body lipid stores in hamsters, whereas in rats progesterone reverses the adiposityreducing actions of estradiol. This species difference in responses to progesterone could help to explain why rats increase, whereas hamsters decrease, their body lipid stores during pregnancy, when circulating progesterone levels are elevated. Estradiol

Pregnancy

Body weight

DURING pregnancy rats exhibit substantial weight gains in excess of the conceptus mass, and these weight gains are accompanied by a substantial hyperphagia. Carcass analyses have shown that much of this maternal weight gain is due to increased deposition of lipid in white adipose tissue (20). These lipid stores are then mobilized during lactation (19). Syrian hamsters differ from rats in their body weight and fat content responses to pregnancy. Syrian hamsters do not alter food intake during pregnancy and actually lose up to 40% of their body lipid prior to parturition (3, 4, 24). It is possible that differences in responses to progesterone could underlie the species difference in energy balance and fat storage during pregnancy. During pregnancy, circulating levels of progesterone are greatly elevated in both species (8, 10, 12). Both rats and Syrian hamsters gain weight and fatten following ovariectorny, and estradiol treatment alone reverses or prevents these weight gains (2, 14, 23). However, the available data suggest that the two species differ in their responses to progesterone. In the presence of estradiol [in gonadally-intact rats or estradiol-primed ovariectomized (OVX) rats] treatment with exogenous progesterone mimics the effects of pregnancy and causes increases in food intake, body weight, and carcass fat content. Progesterone has no effect on body weight or food intake in OVX rats (i.e., in the

absence of estradiol) (5, 7, 9, 13, 15-17). Unlike rats, OVX Syrian hamsters gain weight during treatment with large doses of progesterone alone (5 mg/day). These progesterone-induced weight gains are evident up to 6 weeks following the cessation of hormone treatment in OVX hamsters (21,25), suggesting that they are due to overall growth, although carcass analyses were not done in these studies. Treatment with a combination of estradiol and progesterone has been reported to both decrease (6) or increase body weight (18) in OVX hamsters. In that latter study the weight gain was attributed to overall growth, rather than an increase in any one carcass component (18). In the following experiments we examined the effects of progesterone and estradiol on food intake, body weight, and fat stores in Syrian hamsters. Our aims were to: a) clarify the nature of estradiol-progesterone interactions in the control of energy balance in hamsters, and b) determine whether increased progesterone levels (12) could contribute to the loss of body fat (24) in pregnant hamsters. GENERALMETHOD

Animals and Housing Adult, female Syrian hamsters (initial body weights 90-100 g)

~Requests for reprints should be addressed to Anita J. Bhatia, Department of Psychology, University of Massachusetts, Amherst, MA 01003.

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of the Lak:LVG strain were obtained from Charles River Breeding Laboratories, Wilmington, MA. All hamsters were housed individually in wire-bottomed stainless-steel cages for at least one week prior to the start of any experiments. Tap water and Purina Laboratory Rodent chow (No. 5001) were available ad lib. A 16:8 hr light:dark schedule with lights on at 0700 hr was maintained. Room temperature was controlled at 22---2°C.

Anesthesia and Surgery The anesthesia used throughout these experiments was methoxyflurane (Metofane, Pitman-Moore) unless otherwise stated. Ovariectomies were done via bilateral, dorsolateral incisions irrespective of estrous cycle day. In Experiment 2 implants containing crystalline 1713-estradiol (Sigma) in 1.0 cm lengths of Silastic tubing (No. 602-235, 0.058" i.d., 0.077" o.d., 0.009" wall thickness, Dow Coming Co.), crystalline progesterone (Sigma) in 2.5 cm lengths of Silastic tubing, and/or 1.0 cm and/or 2.5 cm empty blanks were placed subcutaneously in the intrascapular region via a single dorsorostral incision. Hormone implants were made as outlined by Takeda and Leavitt (22). All implants were placed in 0.9% saline at room temperature with 17% benzalkonium chloride (Zephrin Chloride) in a 1:750 dilution for 4-10 hr prior to insertion in order to sterilize all implants and prime those implants containing hormone.

Carcass Analyses At the end of each experiment all animals were anesthetized and decapitated. In Experiment 1 the retroperitoneal (RWAT), parametrial (PWAT), and inguinal (IWAT) white adipose tissues were dissected bilaterally and weighed to the nearest I mg. In Experiment 2 the RWAT and IWAT were dissected and weighed. In both experiments uteri were dissected and weighed. All dissected tissues were returned to the carcass for subsequent measurement of terminal carcass composition. In Experiment 1 terminal carcass composition was measured using a modification (7) of the method of Leshner et al. (11). Shaved, eviscerated hamsters were dried to a constant weight at 70-75°C to determine carcass water content. Lipid content was measured by grinding the dehydrated carcass in a blender and extracting a homogeneous sample ( - 5 0 0 mg) to a constant weight with petroleum ether. The weight of the remaining tissue represented the fat-free dry weight. All samples were extracted in duplicate. In Experiment 2 eviscerated hamsters were dried to a constant weight, and carcass lipid was estimated from carcass water content. In hamsters percent carcass water and percent lipid are highly correlated (r=.98; % l i p i d = - 1 . 3 2 x % w a t e r + 96.54; N = 2 8 9 ; Wade, unpublished data). Fat-free dry weight was calculated as the total eviscerated carcass weight less water and estimated lipid contents.

Statistical Analyses All data are presented as mean--SE. The data were analyzed using a mixed model or one-way ANOVA (two-tailed) as appropriate with hormone treatment as a between groups variable and days as a within subjects variable. Post hoc analyses using Newman-Keuls multiple comparison, rank order tests (p<0.05) were done only if the relevant main or interaction statistic was significant at p<0.05. Post hoc statistical analyses of betweengroup differences always compared data for individual days; data were never averaged across days.

BHATIA AND WADE

Daily food intakes were summed over four days (Experiment 1) and cumulative body weight gain calculated (Experiments 1 and 2) for the analyses. EXPERIMENT 1

Procedure Nine weeks after ovariectomy, hamsters were divided into 6 groups matched for daily food intake (spillage and pouching accounted for) and body weight. Hamsters received daily injections of either sesame oil vehicle (Oil, n =9), 5 ~g estradiol3-benzoate (EB, n = 10), 1 mg progesterone (P1, n = 10), or 5 mg progesterone (P5, n = 5) in 0.1 ml/day total volume or 5 ~g EB in combination with P1 (EB+P1, n = 10) or P5 (EB+P5, n = 10) in two separate injections with a total volume of 0.2 ml/day. The EB was dissolved in sesame oil. Progesterone was dissolved in sesame oil containing 4.57% benzyl benzoate and 11.41% benzyl alcohol by volume. Body weight was measured daily, and food intake was measured daily for the first 16 days and then every other day for days 17-44 following the start of injections. After 44 45 days of injections, the animals were anesthetized with chloropentobarbital (Chloropent), measured for nasoanal length to the nearest 1 mm, and decapitated between 0800 and 1300.

Results Significant changes were seen over time in weight gain [days x hormone interaction: F(250,2350)= 13.0, p<0.001] and food intake [days x hormone interaction: F(55,528)= 1.6, p<0.05] (Fig. 1). Weight gains and food intakes were similar among groups across the baseline period. By day 6 of injections, progesterone treatment (5 mg/day), whether administered alone or in combination with estradiol, resulted in significant weight gains. The weight gains of the P5 group exceeded those of the Oil group from day 6 until the end of the experiment. The EB + P5 hamsters had larger weight gains compared to the Oil hamsters on days 6--12. Following this period of weight gain, EB+P5 hamsters began to lose weight, and at 30--44 days following the start of injections the weight changes of the EB+P5 group were significantly lower than the P1 and P5 groups. The weight gains of the P1 hamsters exceeded those of the Oil group on days 30--44. Treatment with EB or EB+P1 caused significant decreases in body weight (days 35--44). Progesterone treatment at both the 1 mg and 5 mg doses elevated food intake, and the 5 mg dose was more effective than the 1 mg dose (data not shown). Food intakes of the P5 group exceeded those of the Oil group on days 9-16 and 29-32, and the food intakes of the P1 group exceeded those of the Oil group on days 17-20 and 29-32. None of the other hormone treatments affected food intake. Carcass lipid content was affected significantly by hormone treatment, F(5,48)=9.5, p<0.001 (Fig. 2). Treatment with EB decreased carcass lipid content, whereas the high dose of progesterone alone (P5, but not P1) increased body fat levels. Progesterone plus EB (EB+P1 and EB+P5) was significantly more effective than EB alone in decreasing carcass fat content. There was a significant effect of hormone treatment on carcass fat-free dry weight, F(5,48)=2.7, p < 0 . 0 3 (Fig. 2). Post hoc analyses showed that only the P5 hamsters had a higher carcass fat-free dry weight compared with the EB hamsters. Although there was an overall effect for body water, F(5,48) = 2.6, p<0.05, post hoc analysis revealed no significant differences among groups (Fig. 2). The effects of the hormone treatments on carcass adiposity

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were mirrored in the weights of all the fat pads examined [PWAT: F(5,48) = 13.9, p<0.001; RWAT: F(5,48)= 20.1, p<0.001, and IWAT: F(5,58)=24.8, p<0.001] (Fig. 3). Estradiol alone and with progesterone (both the 1 and 5 mg doses) reduced RWAT, IWAT, and PWAT weights compared with oil vehicle or progesterone alone. Estradiol and progesterone in combination (both the 1 and 5 mg doses) decreased IWAT weight more than treatment

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Procedure

Forty-three female hamsters were ovariectomized. Two weeks following surgery baseline food intake and body weight were measured every 4 days for 16 days. These animals were divided into 7 groups matched for average baseline food intake and body weight and then given two subcutaneous Silastic implants. Twenty-three hamsters received a 1.0 cm blank and a 2.5 cm blank, 8 hamsters a E2 and a 2.5 cm blank [E2 (implanted)], 4 hamsters a P capsule and a 1.0 cm blank [P (implanted)], and 8 hamsters a E2 and a P capsule [E2+P (implanted)]. These hormone implants induce plasma estradiol levels of 98---5 pg/ml and progesterone levels of 10 - 2 ng/ml (22) which are in the physiological range for estrous cycling Syrian hamsters on the second day of diestrus and early proestrus, respectively (1, 10, 12). All animals were then injected for 39--40 days; of the 23 hamsters with two blank capsules 5 were injected dally with sesame oil (Oil), 5 with 2.5 Ixg unesterified E2 [E2 (injected)], 5 with 1.0 mg P[P (injected)], and 8 with 2.5 v,g E2 plus 1.0 mg P [E2+P (injected)]. Hamsters with hormone-containing implants received sesame oil injections. At the end of this period all animals were anesthetized and decapitated between 0800 and 1300.

There was no significant effect of any hormone treatment on terminal nasoanal lengths, F(5,48) = 0.9, p>0.48 (data not shown). Discussion

Rats and hamsters differ in their responses to progesterone both in the presence and absence of estradiol. In rats, progesterone reverses the weight-reducing actions of estradiol (7,23). However, in this experiment progesterone synergized with estradiol to further reduce body weight gain and fat content in hamsters. Progesterone alone at 1 mg and 5 mg/day increased weight gain in hamsters compared with OVX controls. In contrast, treatment with progesterone alone has no effects on the body weight or carcass composition of OVX rats (9, 16, 17). However, only the 5 mg dose increased carcass lipid content. Therefore, some of the progesterone-induced weight gain may be due to nonsignificant increases in all body components, although skeletal length does not appear to be affected as indicted by nasoanal length. The EB+P5 hamsters also exhibited increased weight gain during the first 6-18 days of hormone treatment, but this may be due to increases in carcass water content. Schneider et al. (18) found that following 16 days of estradiol benzoate (2.5 t~g/day) and progesterone (5 mg/day) administration, OVX hamsters exhibit increased body weight compared to oil-treated OVX controls, but the only

Results

Hormone treatments significantly affected body weight gain [days x hormone interaction, F(246,1476)=10.3, p<0.001] (Fig. 4). Weight gains were similar across the baseline period. Progesterone injections (1 mg), but not implants, significantly increased body weight from day 8 until the end of the experiment.

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DAYS OF INJECTIONS FIG. 4. Cumulative weight gain of ovariectomized hamsters which received daily injections of sesame oil vehicle (Oil), 2.5 I.Lg estradiol [E2 (inj)], estradiol plus 1 mg progesterone [E2+P (inj)], l mg progesterone [P (inj)], or had subcutaneous implants of estradiol [E 2 (imp)], estradiol plus progesterone [E2+P (imp)], or progesterone [P (imp)].

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Unesterified estradiol injected at a dose of 2.5 Ixg did not affect weight gain, but when administered with progesterone (1 mg) weight gain was significantly reduced. Estradiol implants both alone and with progesterone implants decreased weight gain. While there was no interaction effect with time, there was a main effect of hormone treatment on food intake, F(6,37)= 4.3, p<0.005 (data not shown). Post hoc analysis of the data showed that the food intakes of the P (injected) hamsters exceeded those of the oil-treated controls and those of both groups treated with estradiol and progesterone. The only carcass component that was significantly affected by hormone treatments was lipid content, F(6,36)= l l. 1, p<0.001 (Fig. 5). Progesterone injections (1 rag), but not implants, increased carcass lipid. Unesterified estradiol decreased body fat levels when given in implants, but not when injected at a dose of 2.5 fl,g/day. When progesterone was given to estradiol-treated hamsters it produced further significant reductions in carcass lipid content, whether injected or administered in implants. The effects of the different hormone treatments on RWAT weight, F(6,36)= 18.4, p<0.001, and IWAT weight, F(6,36)= 15.0, p<0.001, paralleled those of total carcass lipid (Fig. 6). Estradiol plus progesterone (injected or implanted) reduced RWAT and IWAT weights. Estradiol when injected (2.5 Ixg) had no effect on RWAT or IWAT weights, but in implants estradiol reduced

both RWAT and IWAT weights. Progesterone (injected but not implanted) increased RWAT and IWAT weights. Uterine weights varied significantly with hormone treatment, F(6,36) = 36.3, p<0.001 (Table 1). As in Experiment 1, animals receiving both estradiol and progesterone had the largest uteri; uteri of animals receiving estradiol alone were intermediate; and animals treated with oil or progesterone alone had the lightest uteri.

Discussion Unesterified estradiol at 2.5 Ixg/day did not affect body weight gain or fatness in OVX Syrian hamsters in contrast to the higher 5 I~g dose of estradiol benzoate used in Experiment 1. However, 2.5 ~.g E 2 did prime animals for the weight- and adiposity-reducing effects of progesterone. Progesterone with estradiol (implants) decreased body fat content compared with estradiol alone (implants), although it did not affect body weight gain. This finding points out that body weight alone cannot be used as a measure of adiposity. GENERALDISCUSSION These experiments show that in OVX Syrian hamsters, unlike

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BHATIA AND WADE

OVX rats, progesterone can either decrease or increase body weight, adiposity, and food intake. In the presence of estradiol, progesterone decreases body weight and adiposity whether administered by injection or implants. In addition, even though injections of unesterified estradiol (2.5 p~g/day) did not affect body weight or fat content, this dose of estradiol did induce a responsiveness to progesterone so that the two steroids together decreased body fat stores (Experiment 2). High doses of progesterone (1 mg and 5 mg/day) in the absence of estradiol increase weight gain. While weight gains induced by progesterone alone were accompanied by some increases in food intake, treatment with progesterone in combination with estradiol did not significantly affect food intake. Similar to what occurs in OVX rats, injections of estradiol benzoate (5 ixg/day) or subcutaneous implants of unesterified estradiol decreased weight gain primarily through reductions in adiposity.

This species difference in the effects of progesterone on body weight and lipid content may account for the difference in the effects of pregnancy on these endpoints in the two species. In both species treatment with estradiol and progesterone together mimic the effects of pregnancy on body weight and fat stores. During pregnancy rats increase their food intake and fatten (20), just as estradiol-primed rats do during treatment with progesterone (5, 7, 9, 23). Conversely, in hamsters, progesterone treatment exaggerates estradiol-induced weight losses without affecting food intake, just as pregnant hamsters lose - 4 0 % of their body lipid stores without undereating (3, 4, 24). ACKNOWLEDGEMENTS Supported by NIH Research Grants NS 10873 and DK 32976 and by NIMH Research Scientist Development Award MH 00321. We are grateful to Jay Alexander for expert technical assistance and to Jill Schneider and Jeff Blaustein for helpful advice and suggestions.

REFERENCES 1. Baranczuk, R.; Greenwald, G. S. Peripheral levels of estrogen in the cyclic hamster. Endocrinology 92:805-812; 1973. 2. Edens, N. K.; Wade, G. N. Effects of estradiol on tissue distribution of newly synthesized fatty acids in rats and hamsters. Physiol. Behav. 31:703-709; 1983. 3. Fleming, A. S. Food intake and body weight regulation during the reproductive cycle of the golden hamster (Mesocricetus auratus). Behav. Biol. 24:291-306; 1978. 4. Fleming, A. S.; Miceli, M. Effect of diet on feeding and body weight regulation during pregnancy and lactation in the golden hamster (Mesocricetus auratus). Behav. Neurosci. 97:246-254; 1983. 5. GaUeti, F.; Klopper, A. The effect of progesterone on the quantity and distribution of body fat in the female rat. Acta Endocrinol. Copenh. 46:379-386; 1964. 6. Gerall, A. A.; Thiel, A. R. Effects of perinatal gonadal secretions on parameters of receptivity and weight gain in hamsters. J. Comp. Physiol. Psychol. 89:580-589; 1975. 7. Gray, J. M.; Wade, G. N. Food intake, body weight, and adiposity in female rats: Actions and interactions of progestins and antiestrogens. Am. J. Physiol. 240:E474-E481; 1981. 8. Grota, L. J.; Eik-Nes, K. B. Plasma progesterone concentrations during pregnancy and lactation in the rat. J. Reprod. Fertil. 13:83-91; 1967. 9. Hervey, E.; Hervey, G. R. The relationship between the effects of ovariectomy and of progesterone treatment on body weight and composition of the female rat. J. Physiol. 87:44P-45P; 1966. 10. Leavitt, W. W.; Blaha, G. C. Circulating progesterone levels in the golden hamster during the estrous cycle, pregnancy and lactation. Biol. Reprod. 3:353-361; 1970. 11. Leshner, A. I.; Litwin, V. A.; Squibb, R. L. A simple method for carcass analysis. Physiol. Behav. 9:281-282; 1972. 12. Lukaszewska, J. H.; Greenwald, G. S. Progesterone levels in the cyclic and pregnant hamster. Endocrinology 86:1-9; 1970. 13. Mook, D. G.; Kenney, N. J.; Roberts, S.; Nussbaum, A. I.; Rodier, W. I., III. Ovarian-adrenal interactions in regulation of body weight

by female rats. J. Comp. Physiol. Psychol. 81:198-211; 1972. 14. Morin, L. P.; Fleming, A. S. Variation of food intake and body weight with estrous cycle, ovariectomy, and estradiol benzoate treatment in hamsters (Mesocrietus auratus). J. Comp. Physiol. Psychol. 92:1-6; 1976. 15. Roberts, S.; Kenney, N. J.; Mook, D. G. Overeating induced by progesterone in the ovariectomized, adrenalectomized rat. Horm. Behav. 3:267-276; 1972. 16. Rodier, W. I., III. Progesterone-estrogen interactions in the control of activity wheel running in the female rat. J. Comp. Physiol. Psychol. 74:365-373; 1971. 17. Ross, G. E.; Zucker, I. Progesterone and the ovarian-adrenal modulation of energy balance in rats. Horm. Behav. 5:43-62; 1974. 18. Schneider, J. E.; Palmer, L. A.; Wade, G. N. Effects of estrous cycles and ovarian steroids on body weight and energy expenditure in Syrian hamsters. Physiol. Behav. 38:119-126; 1986. 19. Smith, R. W.; Walsh, A. Effect of lactation on lipolysis in rat adipose tissue. Lipids 11:418-420; 1976. 20. Steingrimsdottir, L.; Greenwood, M. R. C.; Brasel, J. A. Effect of pregnancy, lactation and high fat diet on adipose tissue in OsborneMendel rats. J. Nutr. 110:600--609; 1980. 21. Swanson, H. H. Effect of progesterone on the body weight of hamsters. J. Endocrinol. 41:xiii; 1968. 22. Takeda, A.; Leavitt, W. W. Temporal effects of progesterone domination on estrogen and oxytocin receptors in hamster uterus. J. Steroid Biochem. 25:219-224; 1986. 23. Wade, G. N. Some effects of ovarian hormones on food intake and body weight in female rats. J. Comp. Physiol. Psychol. 88:183-193; 1975. 24. Wade, G. N.; Jennings, G.; Trayhuru, P. Energy balance and brown adipose tissue thermogenesis during pregnancy in Syrian hamsters. Am. J. Physiol. 250:R845-R850; 1986. 25. Zucker, I.; Wade, G. N.; Zeigler, R. Sexual and hormonal influences on eating, taste preferences, and body weight of hamsters. Physiol. Behav. 8:101-11 l; 1972.