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Feminine saccharin preference in the genetically androgen insensitive male rat pseudohermaphrodite
HORMONES
ANI)
Feminine
HFHAVIOR,
4: 371-375
Saccharin Insensitive
BERNARD
(1973)
Preference in the Genetically Male Rat Pseudohermaphrodite
H. SHAPIKO
and ALLEN
Androgen
S. GULUMAN
7’l?c C’hildren’s IIospital of Philadelphia, Division of Exprrimen~ul Putl~olo~gv, I 740 Hainhridge Skeet, Philudelphia, Perlnsylvmiu I9 146 und Department of Pediatrics. University of Petvnylvania School of Medicine, Philadelphia, Pcrmsylvuniu I9 IO4
Saccharin prefcrencc in rhe StanlcyCumbreck male rat pseudohermaphrodite is characteristically feminine. The pseudohermaphrodite conbumes significantly greater amounts of 0.25, 0.5, and 1.W saccharin solutions than mnle littermares. The enhanced saccharin preference of pseudohcrmaphrodites suggest incomplete or absent masculine imprinting of’ brain centers regulating fluid consumption. Saccharin preference was unaffected by gonadectomy in pseudohermaphrodites \vith previous experience in selecting saccharin.
The maie rat pseutlohermaphrodile(Stanley and Gumbreck, 1964) has a female phenotype with an XY karyotype and a chromatin negative nuclear sex. The affected animals have a blind vagina and nipples, but no masculine rcproductivc tract other than bilateral inguinal testes. Target organs from the pseudohermaphroditeare generally unresponsive to physiologic dosesof testosterone or dihydrotestosterone (Bardin, Bullock, secretion af Sherins, Mowszowicz, and Blackburn, 1973) and testicular testosterone is subnormal (Bardin, Allison, Stanley, and Gumbreck, 1969). In addition. the male pseudohermaphroditc has a fcmalc pattcm of steroid excretion (Gustafsson and Goldman, 1972). Bardin et al. (1973) have concluded that the primary genetic defect of the male rat pseudohermaphrodite is the inherited end organ insensitivity to androgens reflected at the cellular level by deficient target organ antlrc,gerl-birldingproteins. In the rat, sexual behavior is “programmed” during late fetal and neonatal life (Harris, lY64; Whalen, 1968). ‘I’hus, adult behavioral patterns can reflect abnormalities occurring during masculine or feminine differentiation. Antlrogen dependent masculinereproductive behavior cannot be studied in the male rat pseudopermaphrodite because of the animals unrcsponsivcncssto androgens. Preference for sweet solutions is one of the few nonreproductive behaviors for which reliable sex differences exist among rodents (Valensteiq 371 Copyright All rights
@ 1973 by Academic Press, Inc. of reproduction in any form reserved.
372
SIIAPIRO
AND
GOLDMAN
Kakolewski, and Cox, 1967; Wade and Zucker, 1969a). Furthermore, the appearance and maintenance of saccharin preference is relatively independent of androgen action (Zucker, 1969). With these facts in mind, our purpose was to study, indirectly, sexual differentiation in the male rat pseudohermaphrodite, by measuring the saccharin preference of the rat and determining the influence of the gonads.
METHODS
AND MATERIALS
Groups of five or six experimentally naive male, female, and pseudohermaphroditic King-X Holtzman rats were housed individually in animal quarters maintained on. a 14 hr light/10 hr dark lighting schedule. Each rat had continuous access to dry lab chow and to solutions available from two 100 ml Richter tubes mounted on the front of each cage. Each day the 24 hr fluid consumption was measured, bottles were washed, and fresh solutions were provided. Spilled fluid was accounted for after collecting in plastic cups mounted under each drinking tube. Following an initial adaptation period rats were offered a choice between tap water and various concentrations of sodium saccharin solutions (Sweeta, Squibb). At the conclusion of the testing period, the animals were bilaterally gonadectomized and fed dry lab chow and tap water only, ad lib., for the next 4 wk. Following the above procedures, saccharin preference was measured in the castrated rats. Body weights were recorded at the beginning and the conclusion of each testing period, and from these two values an average was calculated. Statistical significance was determined by Students t.
RESULTS AND DISCUSSION The total amounts of water and saccharin consumed by male rat pseudohermaphrodites and females were almost identical, but control males drank significantly less saccharin, at all concentrations tested, than pseudohermaphrodites or females, and when high concentrations of saccharin were offered, males consumed more water (Fig. 1). Although body weights of female rats were significantly lower than males and pseudohermaphrodites (Table l), when saccharin consumption is calculated on a relative basis (ml consumed/l00 g body weight) the taste preference of pseudohermaphrodites remains characteristically feminine. Our results agree with previous reports that have demonstrated a greater preference for sweet solutions among adult female rats than adult males (Valenstein ef al., 1967; Wade and Zucker, 1969a). Furthermore, with the exception of bilateral inguinal testes, the male
SACCIIARIN
PREFERENCE TABLE
Body
_Male Female Pseudohermaphrodite
.---
Weights
.~...._
of Male,
Female,
No. of rats 5 5 6
IS THE
373
PSEUDO
1 and Pseudohermaphrodite
Rats
Body --weights -_-----_---.(g)b Postcastration Prccastration 318.4 283.6 352.2
f 21.4+ + 12.8; + 25.1
354.8 291.0 351.0
f 44.2 + 32.1; 2 54.5
‘Mean f standard deviation. bRoman letters represent a significant difference between males and females, greek letters demonstrate a statistical difference between pseudohermaphrodites males or fcmalcs. (1 or A: p < .OOl; p or B: p < .Ol; y or C: p < .05.
and and
Fig. 1. Mean daily consumption of water (shaded bars) and various concentrations of saccharin (opened bars) by males, females, and pseudohermaphrodites before and after gonadectomy. Roman letters represent a significant difference between males and females, and Greek letters demonstrate a statistical difference between pscudohcrmaphrodites and males or females. A or a: p < .OO I ; B or p: p < .Ol ; C or y: p < .05.
rat pseudohermaphrodite is anatomically a phenotypic female (Stanley and Gumbreck, 1964) and could be expected to have a feminine saccharin preference. The lower saccharin preference of male rats is probably a result of the organizing action of androgens during neural differentiation (Wade and
374
SHAPIRO
AND
GOLDMAN
Zucker, 1969b). In adults, on the other hand, testicular secretions exert at most a weak suppressive effect on saccharin preference (Zuckcr, 1969). The greater saccharin consumption of the pseudohermaphrodite may be due to incomplete masculinization of the brain. In vitro, testicular steroidogenesis in the prepubertal pseudohermaphrodite is similar to littermate males (Coffey, Aronin, French, and Nayfeh, 1972) which implies that steroid secretions in the pseudohermaphrodite are normal during sexual differentiation. IIowcver, developing brain centers which influence taste prcfcrence may be insensitive to the organizing action of androgens, as target organs in the adult pseudohermaphrodite are unresponsive to testosterone (Rardin et al., 1973). Feminine organogenesis in mammals is independent of ovarian control (Jost, 1970), and it is possible that feminization of brain centers, controlling taste preference, can also develop without ovarian influence. IIowever, neonatal hormone secretions have been detected in the female rat (Reiter, Goldenberg, Vaitukaitis, and Ross, 1972) and an injection of estradiol benzoatc on Day 5 after birth increases saccharin consumption in adult females (Wade and Zucker, 1969b). In adult rats ovariectomy results in diminished saccharin preference and daily injections of estradiol and progesterone can elevate saccharin consumption in spayed females (Zucker, 1969). Thus, ovarian hormones are probably required for the development and/or maintenance of the enhanced saccharin preference in females. Although the pseudohermaphrodite also possesses this enhanced taste preference, the animal has testes which produce, in vitro, mostly ring A reduced androgens (Coffey et al., 1972). Recently, in our laboratory, we observed that vaginal estrous smears in pseudohermaphrodites are abolished following castration (in preparation). This suggests that the testes of the pseudohermaphrodite may secrete enough estrogens to maintain the rat’s enhanced saccharin preference and vaginal cornification. In an attempt to determine the role of the gonads on saccharin consumption, male, female, and pseudohermaphrodites were castrated following 6 days of saccharin testing and retested 4 wk later. Saccharin preference in females and pseudohermaphrodites was unaffected by gonadectomy, while males tended to consume larger quantities of 0.5% and 1% saccharin solutions after castration (Fig. 1). Females and pseudohermaphrodites drank similar quantities of saccharin. and water pre- and postoperatively and continued to consume more saccharin at all concentrations tested, than males. It has been reported that ovariectomized rats have a much lower saccharin preference than intact females, while the saccharin preference vf orchiectomized rats is only slightly greater than those of intact males (Zucker, 1969). Ilowever, prior experience with sweet solutions is an important variable in the extent to which ovarian hormones regulate taste preference. For rats having 95 days experience in the selection of saccharin, saccharin consumption was unaltered by ovariectomy while among inexperienced rats, saccharin preference was
SACCHARIS
PREFERENCE
IN THE
PSEUDO
37.5
much greater for intact than for ovariectomized females (Wade and Zucker, 1969b). Thus, in our experiment it is possible that the 6 days of testing with saccharin solutions prior to castration was sufficient time to acquire a learned preference for saccharin and overcome the hormonal influences.
ACKNOWLEDGMENTS A. S. Goldman is a recipient of a Career Development Award (IID-13,628) from the U.S.P.H.S. Research was supported by grants from the National Foundation “March of Dimes,” from the U.S.P.H.S. (HD-4683), and from the Rockefeller Foundation.
REFERENCES Bardin,
W. C., Allison, J. E., Stanley, A. J., and Gumbreck, L. G. (1969). Secretion of testosterone by the pseudohermaphroditc rat. Endocrinology 84, 435-436. Bardin, C. W., Bullock, L. P., Sherins, R. J., Mowszowicz, I., and Blackburn, W. R. (1973). Androgcn metabolism and mechanism of action in male pscudohermaphroditism: A study of testicular feminization. Rec. f’rog. Hor. Res., 29, 65-109. Coffey, J. C., Aronin, P. A., French, F. S., and Nayfeh, S. N. (1972). Steroid metabolism by testicular homogenates of the StanlcyCumbreck pseudohermaphrodite male rat. I. Increased formation of androsterone and androstanediol. Steroids 19, 433454. Gustafsson, J.-A., and Goldman, A. S. (1972). Steroid monosulfates in urine from male rat pseudohermaphrodites. Endocrinology 90, 931-935. Harris, G. W. (1964). Sex hormones, brain development and brain function. Endocrinology 78, 627-648. Jest, A. (1970). Hormonal factors in the sex differentiation of the mammalian foetus. Philos. Trans. Roy. Sot. London B 259, 119-l 30. Keiter, E. O., Goldenberg, R. L., Vaitukaitis, J. L. and Ross, G. ‘1’. (1972). A role for endogenous estrogen in normal ovarian dcvclopment in the neonatal rat. Endocrinology 91, 1537-1539. Stanley, A. J., and Gumbreck, L. G. (1964). Male pseudohermaphroditism with feminizing testis in the male rat: a sex-linked recessive character. 46th Meeting, Endocrine Sot. San Francisco, 40. Valenstein, E. S., Kakolewski, J. W., and Cox, V. C. (1967). Sex differences in taste preference for glucose and saccharin solutions. Science 156, 942-943. Wade, G. N., and Zucker, I. (1969a). Hormonal and dcvclopmcntal influences on rat saccharin preferences. J. Conrp. Physiol. Psychol. 69, 291-300. Wade, C. N., and Zucker, I. (1969b). Taste preferences of female rats: Modification by neonatal hormones, food deprivation and prior expericncc. Physiol. Behuv. 4, 935-943. Whalen, R. C. (1968). Diffcrcntiation of the neural mechanisms which control gonadotrophin secretion and sexual behavior. In M. Diamond (Ed.), Perspectives in Reproduction 4nd Sexual Behavior, pp. 303-340, Indiana University Press, Bloomington, IN. Zucker, I. (1969). Hormonal determinants of sex differences in saccharin preference, food intake and body weight. Physiol. Behav. 4, 595602.
ANI)
Feminine
HFHAVIOR,
4: 371-375
Saccharin Insensitive
BERNARD
(1973)
Preference in the Genetically Male Rat Pseudohermaphrodite
H. SHAPIKO
and ALLEN
Androgen
S. GULUMAN
7’l?c C’hildren’s IIospital of Philadelphia, Division of Exprrimen~ul Putl~olo~gv, I 740 Hainhridge Skeet, Philudelphia, Perlnsylvmiu I9 146 und Department of Pediatrics. University of Petvnylvania School of Medicine, Philadelphia, Pcrmsylvuniu I9 IO4
Saccharin prefcrencc in rhe StanlcyCumbreck male rat pseudohermaphrodite is characteristically feminine. The pseudohermaphrodite conbumes significantly greater amounts of 0.25, 0.5, and 1.W saccharin solutions than mnle littermares. The enhanced saccharin preference of pseudohcrmaphrodites suggest incomplete or absent masculine imprinting of’ brain centers regulating fluid consumption. Saccharin preference was unaffected by gonadectomy in pseudohermaphrodites \vith previous experience in selecting saccharin.
The maie rat pseutlohermaphrodile(Stanley and Gumbreck, 1964) has a female phenotype with an XY karyotype and a chromatin negative nuclear sex. The affected animals have a blind vagina and nipples, but no masculine rcproductivc tract other than bilateral inguinal testes. Target organs from the pseudohermaphroditeare generally unresponsive to physiologic dosesof testosterone or dihydrotestosterone (Bardin, Bullock, secretion af Sherins, Mowszowicz, and Blackburn, 1973) and testicular testosterone is subnormal (Bardin, Allison, Stanley, and Gumbreck, 1969). In addition. the male pseudohermaphroditc has a fcmalc pattcm of steroid excretion (Gustafsson and Goldman, 1972). Bardin et al. (1973) have concluded that the primary genetic defect of the male rat pseudohermaphrodite is the inherited end organ insensitivity to androgens reflected at the cellular level by deficient target organ antlrc,gerl-birldingproteins. In the rat, sexual behavior is “programmed” during late fetal and neonatal life (Harris, lY64; Whalen, 1968). ‘I’hus, adult behavioral patterns can reflect abnormalities occurring during masculine or feminine differentiation. Antlrogen dependent masculinereproductive behavior cannot be studied in the male rat pseudopermaphrodite because of the animals unrcsponsivcncssto androgens. Preference for sweet solutions is one of the few nonreproductive behaviors for which reliable sex differences exist among rodents (Valensteiq 371 Copyright All rights
@ 1973 by Academic Press, Inc. of reproduction in any form reserved.
372
SIIAPIRO
AND
GOLDMAN
Kakolewski, and Cox, 1967; Wade and Zucker, 1969a). Furthermore, the appearance and maintenance of saccharin preference is relatively independent of androgen action (Zucker, 1969). With these facts in mind, our purpose was to study, indirectly, sexual differentiation in the male rat pseudohermaphrodite, by measuring the saccharin preference of the rat and determining the influence of the gonads.
METHODS
AND MATERIALS
Groups of five or six experimentally naive male, female, and pseudohermaphroditic King-X Holtzman rats were housed individually in animal quarters maintained on. a 14 hr light/10 hr dark lighting schedule. Each rat had continuous access to dry lab chow and to solutions available from two 100 ml Richter tubes mounted on the front of each cage. Each day the 24 hr fluid consumption was measured, bottles were washed, and fresh solutions were provided. Spilled fluid was accounted for after collecting in plastic cups mounted under each drinking tube. Following an initial adaptation period rats were offered a choice between tap water and various concentrations of sodium saccharin solutions (Sweeta, Squibb). At the conclusion of the testing period, the animals were bilaterally gonadectomized and fed dry lab chow and tap water only, ad lib., for the next 4 wk. Following the above procedures, saccharin preference was measured in the castrated rats. Body weights were recorded at the beginning and the conclusion of each testing period, and from these two values an average was calculated. Statistical significance was determined by Students t.
RESULTS AND DISCUSSION The total amounts of water and saccharin consumed by male rat pseudohermaphrodites and females were almost identical, but control males drank significantly less saccharin, at all concentrations tested, than pseudohermaphrodites or females, and when high concentrations of saccharin were offered, males consumed more water (Fig. 1). Although body weights of female rats were significantly lower than males and pseudohermaphrodites (Table l), when saccharin consumption is calculated on a relative basis (ml consumed/l00 g body weight) the taste preference of pseudohermaphrodites remains characteristically feminine. Our results agree with previous reports that have demonstrated a greater preference for sweet solutions among adult female rats than adult males (Valenstein ef al., 1967; Wade and Zucker, 1969a). Furthermore, with the exception of bilateral inguinal testes, the male
SACCIIARIN
PREFERENCE TABLE
Body
_Male Female Pseudohermaphrodite
.---
Weights
.~...._
of Male,
Female,
No. of rats 5 5 6
IS THE
373
PSEUDO
1 and Pseudohermaphrodite
Rats
Body --weights -_-----_---.(g)b Postcastration Prccastration 318.4 283.6 352.2
f 21.4+ + 12.8; + 25.1
354.8 291.0 351.0
f 44.2 + 32.1; 2 54.5
‘Mean f standard deviation. bRoman letters represent a significant difference between males and females, greek letters demonstrate a statistical difference between pseudohermaphrodites males or fcmalcs. (1 or A: p < .OOl; p or B: p < .Ol; y or C: p < .05.
and and
Fig. 1. Mean daily consumption of water (shaded bars) and various concentrations of saccharin (opened bars) by males, females, and pseudohermaphrodites before and after gonadectomy. Roman letters represent a significant difference between males and females, and Greek letters demonstrate a statistical difference between pscudohcrmaphrodites and males or females. A or a: p < .OO I ; B or p: p < .Ol ; C or y: p < .05.
rat pseudohermaphrodite is anatomically a phenotypic female (Stanley and Gumbreck, 1964) and could be expected to have a feminine saccharin preference. The lower saccharin preference of male rats is probably a result of the organizing action of androgens during neural differentiation (Wade and
374
SHAPIRO
AND
GOLDMAN
Zucker, 1969b). In adults, on the other hand, testicular secretions exert at most a weak suppressive effect on saccharin preference (Zuckcr, 1969). The greater saccharin consumption of the pseudohermaphrodite may be due to incomplete masculinization of the brain. In vitro, testicular steroidogenesis in the prepubertal pseudohermaphrodite is similar to littermate males (Coffey, Aronin, French, and Nayfeh, 1972) which implies that steroid secretions in the pseudohermaphrodite are normal during sexual differentiation. IIowcver, developing brain centers which influence taste prcfcrence may be insensitive to the organizing action of androgens, as target organs in the adult pseudohermaphrodite are unresponsive to testosterone (Rardin et al., 1973). Feminine organogenesis in mammals is independent of ovarian control (Jost, 1970), and it is possible that feminization of brain centers, controlling taste preference, can also develop without ovarian influence. IIowever, neonatal hormone secretions have been detected in the female rat (Reiter, Goldenberg, Vaitukaitis, and Ross, 1972) and an injection of estradiol benzoatc on Day 5 after birth increases saccharin consumption in adult females (Wade and Zucker, 1969b). In adult rats ovariectomy results in diminished saccharin preference and daily injections of estradiol and progesterone can elevate saccharin consumption in spayed females (Zucker, 1969). Thus, ovarian hormones are probably required for the development and/or maintenance of the enhanced saccharin preference in females. Although the pseudohermaphrodite also possesses this enhanced taste preference, the animal has testes which produce, in vitro, mostly ring A reduced androgens (Coffey et al., 1972). Recently, in our laboratory, we observed that vaginal estrous smears in pseudohermaphrodites are abolished following castration (in preparation). This suggests that the testes of the pseudohermaphrodite may secrete enough estrogens to maintain the rat’s enhanced saccharin preference and vaginal cornification. In an attempt to determine the role of the gonads on saccharin consumption, male, female, and pseudohermaphrodites were castrated following 6 days of saccharin testing and retested 4 wk later. Saccharin preference in females and pseudohermaphrodites was unaffected by gonadectomy, while males tended to consume larger quantities of 0.5% and 1% saccharin solutions after castration (Fig. 1). Females and pseudohermaphrodites drank similar quantities of saccharin. and water pre- and postoperatively and continued to consume more saccharin at all concentrations tested, than males. It has been reported that ovariectomized rats have a much lower saccharin preference than intact females, while the saccharin preference vf orchiectomized rats is only slightly greater than those of intact males (Zucker, 1969). Ilowever, prior experience with sweet solutions is an important variable in the extent to which ovarian hormones regulate taste preference. For rats having 95 days experience in the selection of saccharin, saccharin consumption was unaltered by ovariectomy while among inexperienced rats, saccharin preference was
SACCHARIS
PREFERENCE
IN THE
PSEUDO
37.5
much greater for intact than for ovariectomized females (Wade and Zucker, 1969b). Thus, in our experiment it is possible that the 6 days of testing with saccharin solutions prior to castration was sufficient time to acquire a learned preference for saccharin and overcome the hormonal influences.
ACKNOWLEDGMENTS A. S. Goldman is a recipient of a Career Development Award (IID-13,628) from the U.S.P.H.S. Research was supported by grants from the National Foundation “March of Dimes,” from the U.S.P.H.S. (HD-4683), and from the Rockefeller Foundation.
REFERENCES Bardin,
W. C., Allison, J. E., Stanley, A. J., and Gumbreck, L. G. (1969). Secretion of testosterone by the pseudohermaphroditc rat. Endocrinology 84, 435-436. Bardin, C. W., Bullock, L. P., Sherins, R. J., Mowszowicz, I., and Blackburn, W. R. (1973). Androgcn metabolism and mechanism of action in male pscudohermaphroditism: A study of testicular feminization. Rec. f’rog. Hor. Res., 29, 65-109. Coffey, J. C., Aronin, P. A., French, F. S., and Nayfeh, S. N. (1972). Steroid metabolism by testicular homogenates of the StanlcyCumbreck pseudohermaphrodite male rat. I. Increased formation of androsterone and androstanediol. Steroids 19, 433454. Gustafsson, J.-A., and Goldman, A. S. (1972). Steroid monosulfates in urine from male rat pseudohermaphrodites. Endocrinology 90, 931-935. Harris, G. W. (1964). Sex hormones, brain development and brain function. Endocrinology 78, 627-648. Jest, A. (1970). Hormonal factors in the sex differentiation of the mammalian foetus. Philos. Trans. Roy. Sot. London B 259, 119-l 30. Keiter, E. O., Goldenberg, R. L., Vaitukaitis, J. L. and Ross, G. ‘1’. (1972). A role for endogenous estrogen in normal ovarian dcvclopment in the neonatal rat. Endocrinology 91, 1537-1539. Stanley, A. J., and Gumbreck, L. G. (1964). Male pseudohermaphroditism with feminizing testis in the male rat: a sex-linked recessive character. 46th Meeting, Endocrine Sot. San Francisco, 40. Valenstein, E. S., Kakolewski, J. W., and Cox, V. C. (1967). Sex differences in taste preference for glucose and saccharin solutions. Science 156, 942-943. Wade, G. N., and Zucker, I. (1969a). Hormonal and dcvclopmcntal influences on rat saccharin preferences. J. Conrp. Physiol. Psychol. 69, 291-300. Wade, C. N., and Zucker, I. (1969b). Taste preferences of female rats: Modification by neonatal hormones, food deprivation and prior expericncc. Physiol. Behuv. 4, 935-943. Whalen, R. C. (1968). Diffcrcntiation of the neural mechanisms which control gonadotrophin secretion and sexual behavior. In M. Diamond (Ed.), Perspectives in Reproduction 4nd Sexual Behavior, pp. 303-340, Indiana University Press, Bloomington, IN. Zucker, I. (1969). Hormonal determinants of sex differences in saccharin preference, food intake and body weight. Physiol. Behav. 4, 595602.