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Estradiol contributes to the postnatal demasculinization of female Japanese quail (Coturnix coturnix japonica)
HORMON!ZS
AND
18, 287-297 (1984)
BEHAVIOR
Estradiol Contributes to the Postnatal Demasculinitation of Female Japanese Quail (Coturnix coturnix japonica) J. BALTHAZART' AND M. SCHUMACHER Labomtoire
de Biochimie
GnPrule
et ComparPe, CJniversit6 de LiPge, 17 place Delcour, B-4020 LiPge, Belgilrm
Two experiments were performed to characterize the process of postnatal demasculinization in Japanese quail. In the first experiment. it was shown that estradiol (E2) can complete female demasculinization during the first 4 weeks of life. By contrast, E2 did not demasculinize sexual behavior and cloaca1 gland in neonatally castrated males. Neonatally gonadectomized females preferentially performed mount attempts when tested in their home cage by comparison to a test arena. In Experiment 2, E2 Silastic implants (40-mm) maintained full copulatory behavior in castrated males but not in females. This large dose of E2 did not demasculinize adult sexually active birds (males or females) even if treatment lasted for 1 month. It is concluded that E2 can demasculinize sexual behavior only in females and only if treatment is performed in very young birds.
The permanent organizational actions of gonadal hormones are thought to be limited to a short critical period in early development. This critical period of sexual ditferentiation would take place at the end of the embryonic life and during the first days after birth in the rat (McEwen, 1978). In quail, it would end around Day 12 of incubation (Adkins, 1979). Several studies, however, suggest that the permanent actions of steroids are not restricted to such a short period. If female rats are lightly androgenized after birth, they will display normal estrous cycles when they reach puberty. However, this capacity will be lost at an early age and they will become anovulatory (Gorski, 1968; Harlan and Gorski, 1977). Postpubertal ovarian secretions (estrogens and/or androgens) are responsible for the appearance of this delayed anovulatory syndrome (DAS; Harlan and Gorski, 1978) which is a good model of permanent changes induced by steroids during the postpubertal period (Harlan, Gordon, and Gorski, 1979). The present data support the conclusion that the competent (“critical”) period during which gonadal steroids may exert permanent effects is not I To whom all correspondence and reprint requests should be addressed. 287 0018-506X/84 s I .50 Copyright f 1YX4 by Academic Press. Inc. All rights of reproduction in any rorm xscrvcd.
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limited to a concise perinatal period of development. Examples are available which show the following: -The competent period is not the same for different responses. In rats, the differentiation of the lordosis response and pituitary function is essentially completed 4-5 days after birth while estrogens still have permanent feminizing effects on open-field behavior later in the development (Steward and Cygan, 1980). -Different aspects of a response (e.g., sexual behavior) can differentiate independently. In rats, defeminization (decreasedcapacity to show lordosis) occurs mainly during the first 4-5 days after birth, whereas masculinization (increased capacity to show male-like mounting) may begin before birth (McEwen, 1978). -For a given response, the competent period is to some extent dose dependent. Androgenization of female rats (suppression of ovulatory cycles by neonatal testosterone treatment) is possible up to Day 5 of life with low doses of testosterone but can be obtained until Day 11 if higher doses are given (Lob1 and Gorski, 1974; Harlan et al, 1979). -The observed effects of neonatal treatment could depend on the test situation or hormonal stimuli used to elicit behavioral responses in adulthood (Feder and Goy, 1983; Clemens, Hiroi, and Gorski, 1969). The concepts of a critical period for sexual differentiation thus “retain importance but must be reshaped to fit more recent evidence” (Harlan et al, 1979). In Japanese quail, sexual differentiation proceeds mainly through demasculinization of the females (Adkins and Adler, 1972; Adkins, 1975; Balthazart and Schumacher, 1983).It was thought that this process occurred before Day 12 of incubation (Adkins, 1976, 1979) but we showed recently that the female demasculinization is not completed at hatching and that full demasculinization of female behavior and morphology is achieved under the influence of ovarian secretion during the first 2-4 weeks of posthatching life (Schumacher and Balthazart, 1983a, 1983b, 1984). Postnatal testicular secretions, by contrast, seem to have no permanent effects in males except on the control mechanisms of gonadotropin secretion (Schumacher and Balthazart, 1984). The present experiments were designed to determine the nature of the ovarian secretion responsible for the postnatal demasculinization in female quail. Considering that estradiol demasculinizes the behavior of males when injected in embryos (Adkins, 1975, 1979; Whitsett, Irwin, Edens, and Thaxton, 1977). we have tested the effect of this steroid in neonatally gonadectomized males and females. GENERAL METHODS
Neonatal gonadectomy and hormonal treatments with Silastic implants were performed as described previously (Schumacher and Balthazart,
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1984). During behavioral tests, the sexual behavior of the birds was recorded during a 5-min presentation to a receptive female either in the home cages or in a test arena. Frequency and latency of occurrence of the following behavior patterns were recorded: neck grab (NG), mount attempt (MA), mount (M), and cloaca1 contact movement [CCM; see Adkins and Adler (1972) and Hutchison (1978) for description]. Cloaca1 gland areas were measured to the nearest square millimeter (Sachs, 1%7) and at sacrifice, stemotracheal (syringeal) muscles (androgendependent structures) were weighed to the nearest milligram [see Schumacher and Balthazart (1984) for more detail]. EXPERIMENT 1 Material
and Methods
Male and female quail came from three different hatches in nearly equal numbers. Birds from Groups 1 and 3 were hatched in our laboratory while those from Group 2 were bought from a local dealer (origin of our colony) at the age of 1 day. Birds from the different hatches were distributed in a random order between the different experimental groups so that birds from each hatch were present in each group in similar numbers. At the age of 4 days, males and females were gonadectomized and 2 days later were implanted with IO-mm Silastic implants filled with estradiol (E2) or left empty as control (C). Two weeks later, the length of the implants was doubled (another lo-mm capsule was implanted). Indirect estimations based on the plasma levels of steroid induced in quail by similar testosterone implants (Desjardins and Turek. 1977):on the relative release rates of E2 and T from Silastic capsules (Dziuk and Cook. 1966; Kincl, Benagiano, and Angee, 1968; Wada, 1982), and on data showing E2 plasma levels in rats bearing E2 Silastic capsules (Brawer, Schipper, and Robaire, 1983; Sodersten, Pettersson, and Eneroth, 1983) suggest that the E2 implants used here produced plasma levels of steroid in the range 100 to 1500 pg/ml which compares reasonably with levels seen in developing chicken (Tanabe, Nakamura, Fujiokd, and Doi, 1979) or in adult quail (Doi, Takai, Nakamura, and Tanabe, 1980). This dose was also known to have behavioral effects in quail not associated with obvious toxic effects (Balthazart, Malacarne, and Schumacher, unpublished data). Treatment was maintained for I month as the postnatal differentiation of behavior in quail is completed during that period (Schumacher and Balthazart, 1984). Ali birds (I 1 E2 males, 13 C males, 26 E2 females, and 21 C females) were raised in heterosexual groups until the age of 34 days. At that time (4 weeks after the implantation), the implants were removed and quail were isolated in individual home cages for 2 weeks and left without hormone treatment. They were then implanted with 60-mm (3 x 20) Silastic capsules filled with crystalline T and then starting 2 weeks later
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they were tested for sexual behavior five times in their home cage and five times in the arena. Tests were performed in a random order (arena versus home cage) at the rate of two tests per week. At the end of the experiment, quail were 102 days old. Sexually inactive birds were killed and checked for castration and presence of implants. Sexually active birds were introduced in Experiment 2 (see below). At the end of Experiment 1, cloaca1 gland area was measured in each bird and syringeal muscles of the sacrificed birds were weighed. Body weight and plumage type were also recorded when birds were 34 (end of postnatal E2 treatment), 48 (start of T treatment), and 102 (end of experiment) days old. The type of plumage was quantified on a nonparametric scale ranging from 1 to 4 with I = rufous-colored male-type breast feathers, 2 = rufous-colored breast feathers with a few brown spots, 3 = rufous-colored breast feathers with large number of brown spots, and 4 = pale-colored female-type breast feathers with brown spots. In quail, E2 induces a female-type plumage in both sexes and the male plumage is neutral or anhormonal (Hutchison, 1978). Results Se-xual behaviclr.During the home-cage tests, nearly all males attempted to mount the stimulus females (Fig. 1). Females were less active than a--me--+-----,,
E2d
cpzr--
FIG. 1. Cumulative percentage of males and females which received empty (C birds) or EZ-filled (E2 birds) Silastic implants during their first 4 weeks of life and showed mount attempts (MA) when tested in their home cage. The x axis indicates time since T implantation. Above: results for the 3 hatches of birds. Below: results for Hatches 1 and 3 only (Hatch 2 from another origin excluded; see text). *** = P < 0.001, ** = P < 0.01, and * = P < 0.05 by Fisher exact probability tests when compared to C females.
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males. During the first and third tests, more C than E2 females showed sexual behavior. This difference was still present in the other tests but did not reach significance. However, when results from Hatches 1 and 3 were analyzed separately (birds from Hatch 2 which were not hatched in the laboratory being dropped from the data), C females were then sexually more active than E2 females during each of the tests (Fig. 1, below). Females from Hatch 2 were not demasculinized at all by postnatal estrogens (6/7 C females and 9112E2 females showed MA at least once) while this effect was clearly present in the other females. As shown previously (Schumacher and Balthazart, 1984) females preferentially showed male sexual behavior when tested in their home cage by comparison with the arena (Fig. 2). The percentage of females which showed sexual behavior was thus higher in the C than in the E2 group only for tests performed in the home cages but not for those in the arena. A small percentage of females only performed cloaca1 contact movements in the arena (6/47) or in the home cages (13/47). By contrast, nearly all males showed full copulatory behavior in both test situations. There was no difference in latency or frequency for the different behavior patterns between C and E2 males whatever the test situation. Morphology. On Day 102 after nearly 8 weeks of T treatment, males had larger cloaca1 glands than females. In addition, C females had larger glands than E2 females (Fig. 3). Syringeal muscles had similar weights in the 4 experimental groups. At 34 and 48 days of age, C males were characterized by a male-type plumage whereas E2 males and E2 females had a completely female-type plumage. The plumage of C females was intermediate with a median score of 2. At the end of the experiment after the T treatment, C birds of both sexes had a male-type plumage whereas all E2 birds had a female like plumage (Fig. 3). On Day 48, E2 males were lighter than E2 females and C males (P < 0.01 and P < 0.05, respectively, by one-way analysis of variance followed by NewmanHome Cq I
C- Males EZ-Males
I
C-Females
m
&-Females
FIG. 2. Sexual behavior (percentage showing mount attempts, MA) of neonatally gonadectomized quail treated with E2 or empty (C) silastic implants and tested in their home cage or in the arena. * = P < 0.05 and 000 = P < 0.001, 00 = P < 0.01, and 0 = P < 0.05 by Fisher exact probability tests for comparisons to corresponding group of males (0) or to C females (*).
292
BALTHAZART
iZ3
EZ-Males
AND SCHUMACHER
IT3
E2-Females
FIG. 3. Cloaca1 gland area at 102 days (left) and plumage type at 34, 48, and 102 days of life in neonatally gonadectomized quail treated with E2 or kept as control(C). Cloaca1 gland areas were analyzed by one-way analysis of variance followed by Newman-Keuls tests, plumage type by the.Kruskall-Wallis analysis of variance followed by Mann-Whitney U tests. *** or 000 = P < 0.001, **or00 = P
Keuls tests), At the end of the experiment, E2 males were still lighter than E2 females (P < 0.05, same test). EXPERIMENT
2
Having shown that E2 can demasculinize female behavior during the first month after hatching, we decided to see whether the same effect could be obtained in adult birds. We also tried to see whether E2 Silastic implants would maintain full copulatory behavior in neonatally gonadectomized males but not females as suggested by a previous experiment (Schumacher and Balthazart, 1984). Material
and Methods
T implants of the sexually active males and females from previous experiment were removed and birds were implanted on the same day (Day 102 of life) with 40 mm of empty of EZfilled Silastic capsules, Due to the small number of active females, no C females were retained for this experiment. Results were not influenced by the postnatal hormone treatment (C or E2) and the present results are thus only presented as a function of the adult hormone treatment [control adult (CA): 8 males, and estradiol adult (E2A): 8 males and 19 females including 8 with neonatal E2 and 11 neonatal C]. These implants were left in place during 46 days during which birds were tested for sexual behavior 11 times in their home cages and at the end twice in the arena. Tests were performed at the rate of two tests per week. Fifty-one days after the implantation, capsules were removed and birds
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were reimplanted with 60-mm (3 x 20) Silastic T capsules. After 2 weeks of T treatment, sexual behavior was tested once in the home cage and once in the arena. On Day 168, cloaca1 glands were measured, and birds were sacrificed by decapitation and checked for castration and presence of implants. Results During the estrudiol treatment. Estradiol maintained full copulatory behavior in nearly all males but not in females throughout the implantation period (home-cage tests; Fig. 4). Surprisingly, two CA males still performed cloacal contact movements during the 46 days without hormonal treatment. Six out of the eight E2A males attempted to mount the stimulus female and five performed cloaca1 contact movements during the last two tests in the arena. E2A females were sexually inactive in this test situation. During the testosferone treatment. All males performed cloaca1 contact movements in response to T when tested in the arena as well as in the home cages. Only 13 of the 19 females which were previously ac?ive before the E2 treatment attempted to mount the stimulus female during the single test in the home cage. However, this small decrease in activity
TIME
(DAYS1
FK. 4. Percentage of birds showing mount attempt (MA) and cloaca1 contact movement (CCM) when treated with EZ-filled or empty (C, males only) silastic implants. All these birds were sexually active during the previous phase of the experiment when they were treated with testosterone. Behavioral tests were performed in the home cages. The x axis indicates time since removal of T implants and placement of E2 or C implants. * = P < 0.05 compared to C males. 000 or n n n = P < 0.001, and 0 = P < 0.05 compared to E2 females. All comparisons by the Fisher exact probability test. Most intermediate tests are omitted for clarity.
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SCHUMACHER
only reflected the fact that birds were tested five times before and only once after being exposed to the adult E2 treatment. If this difference in test frequency is taken into account by comparing for example the numbers of females which showed mount attempts during at least three out of five tests before E2 treatment to those which showed the behavior after, this difference vanishes (before: 14/19; after: 13/19). Similarly, the E2 treatment in adulthood did not alter the response of the cloacal gland to T. The average gland sizes before and after E2 were not different (before: 191 + 21 mm*; after: 184 -t 43 mm’). GENERAL DISCUSSION We showed previously that female quail demasculinization is a continuous process extending into posthatching life which is not completed on Day 12 of incubation as was suggested by earlier studies. Although neonatally ovariectomized females are less sensitive than males to the activating effects of testosterone, demasculinization is completed only after 2-3 weeks of posthatching life under the influence of ovarian secretions (Schumacher and Balthazart, 1984). The present experiments suggest that estradiol can induce the postnatal demasculinization in female quail. Small E2 implants placed in neonatally ovariectomized females for the first month of life inhibited almost completely the ability of the females to respond to T by showing male-type sexual behavior. Neonatal E2 treatment also demasculinized the cloaca1 gland of females but did not affect their syringeal muscles. A previous experiment suggested that the demasculinization of these muscles is only completed 2 weeks after hatching (Schumacher and Balthazart, 1984). Why E2 did not demasculinize the muscles in the present experiment is unclear and could be related to inadequate dosage or to the nature of the hormone used (different ovarian hormones could differentiate the different aspects of the sexual phenotype). In rats for example, it is known that estrogens can defeminize sexual behavior [for review: McEwen (1978) and Darner (1980)] but do not control the differentiation of the spinal nucleus of the bulbocavernosus which is induced by androgens exclusively (Breedlove and Arnold, 1980). Similarly, in quail testicular hormones are responsible for the late differentiation of the gonadotrophin secreting system (Schumacher and Balthazart, 1984) while sexual behavior would rather differentiate under the influence of the ovary. In the first experiment, we were able to demasculinize sexual behavior of gonadectomized females but not males. The postnatal reactivity to the organizing effects of E2 on sexual behavior is thus sexually differentiated before hatching. Females are presensitized to the organizing action of E2 and it seems likely that embryonic E2 is itself responsible for this effect. It is known that the in virro production of estrogens in organotypic culture is higher for the left ovary than for the testes in quail on Days
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10 and 14 of incubation (Guichard, Scheib, Haffen, Mignot, and Cedard, 1980). In chicken, female embryos also have higher E2 and estrone plasma levels than males between Day 7 of incubation and hatching (Woods and Et-ton, 1978; Woods and Brazzil, 1981). These data thus support the idea that sexual differentiation is biphasic. Embryonic estrogens would sensitize the undifferentiated target tissues during a critical period of embryonic development which would permit the more progressive organizing action of the same hormone during 2-3 weeks after hatching. This timing could be slightly variable as suggested by the fact that sensitivity to the organizing action of E2 was aparently lost already at 4 days of age in one batch of quail in Experiment 1. These birds were from a different origin (see Methods) and had thus been incubated in slightly different conditions which possibly changed their rate of maturation and consequently the exact timing of their competent periods [see Schumacher and Balthazart (1984) for a more detailed discussion]. Neonatally ovariectomized females had a plumage showing both male and female characteristics during the first 7 weeks of life. As the femaletype plumage is induced by estrogens in quail (Kannankeril and Domm, 1%8; Hutchison, 1978), this suggests that these females including those in the control group (without E2 implants) were still under the influence of estrogens. These estrogens, which are probably secreted by the adrenals [see Tanabe et al. (1979) for a study in chicken] might have attenuated the differences between females with and without exogenous E2 and thus have decreased the magnitude of the effects observed here. However, concentrations of circulating estrogens in young ovariectomized females were probably very low because estrogens induce female-type plumage at low concentrations which ate not sufficient to suppress LH secretion (Gibson, Follett, and Gledhill, 1975). In Experiment 2, E2 maintained full copulatory behavior in males but not in females. This suggests that one aspect of the sexual differentiation is the loss in females of the sensitivity to the activating effects of E2 on male-type sexual behavior. This experiment also showed that high doses of E2 (40 mm) given for more than a month in adult quail have no permanent demasculinizing effects in males as well as in females. As lo- to 20-mm E2 implants given during a comparable period to neonatal quail are sufficient to demasculinize sexual behavior and morphology of females, this suggests that the competent period for female demasculinization is terminated before birds become adult. This conclusion must, however, be moderated by the fact that behavior had been activated by T before the E2 treatment was applied and sequential effects of the hormone treatments are quite possible. In conclusion these experiments demonstrate that sexual differentiation is completed only during the first few weeks of life in female quail under the influence of ovarian secretions among which estradiol could play a
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critical role. The sensitivity to these progressive organizing effects of estradiol is, however, lost before hatching in the males and before puberty in females, ACKNOWLEDGMENTS We are indebted to Professor E. Schoffeniels for his continued interest in our research. This work was supported by a grant from the Belgian FNRS (credit aux chercheurs) to J. Balthazart and Grant 2.451880 from the FRFC to Professor E. Schoffeniels. M. Schumacher is supported by a grant from the IRSIA.
REFERENCES Adkins, E. K. (1975). Hormonal basis of sexual differentiation in the Japanese quail. J. Comp. Physiol. Psychol. 89, 61-71. Adkins, E. K. (1976). Embryonic exposure to an antiestrogen masculinizes behavior of female quail. Physiol. Behav. 17, 357-359. Adkins, E. K. (1979). Effect of embryonic treatment with estradiol or testosterone on sexual differentiation of the quail brain. Neuroendocrinology 29, 178-185. Adkins, E. K., and Adler, N. T. (1972). Hormonal control of behavior in the Japanese quail. J. Comp. Physiol. Psychol. 81, 27-36. Balthazart, J., and Schumacher, .M. (1983).Testosterone metabolism and sexual differentiation in quail. In J. Bahhazart, E. Prove, and R. Gilles (Eds.), Hormones and Behavior in Higher Vertebrates, pp.237-260. Springer-Verlag, Berlin. Brawer, J., Schipper, H., and Robaire, B. (1983). Effects of long term androgen and estradiol exposure on the hypothalamus. Endocrinology 112, 194-199. Breedlove, M. S., and Arnold, A. P. (1980). Hormone accumulation in a sexually dimorphic motor nucleus of the rat spinal cord. Science 210, X54-566. Clemens, L. G., Hiroi, M., and Gorski, R. A. (1969). Induction and facilitation of female mating behavior in rats treated neonatally with low?doses of testosterone propionatc. Endocrinology 84, 1430-1438. Desjardins, C., and Turek, F. W. (1977). Effects of testosterone on spermatogenesis and luteinizing hormone release in Japanese quail. Gen. Comp. Endocrinol. 33, 293-303. Doi, 0.. Takai, T., Nakamurd, T., and Tanabe, Y. (1980). Changes in pituitary and plasma LH, plasma and follicular progesterone and estradiol, and plasma testosterone and estrone concentrations during the ovulatory cycle of the quail (Coturnix coturnix juponica).
Gen. Comp. Endocrinol.
41, 1X-163.
Darner, G. (1980). Sexual differentiation of the brain. Vitam. Horm. 38, 325-381. Dziuk, P. J., and Cook, B. (1966). Passageof steroids through silicone rubber. Endocrin0log.y 78, 208-2 I 1. Feder, H. H., and Goy, R. W. (1983). Effects of neonatal estrogen treatment of female guinea pigs on mounting behavior in adulthood. Horm. Behav. 17, 284-291. Gibson, W. R., Follett, B. K., and Gledhill, B. (1975). Plasma levels of luteinizing hormone in gonadectomized Japanese quail exposed to short or long day length. J. Endocrinol. 64, 87-101. Gorski, R. A. (1968). Influence of age on the response to paranatal administration of a low dose of androgen. Endocrinology 82, 1001-1004. Guichard, A., Scheib, D., Haffen, K., Mignot, Th.lM., and Cedard, L. (1980). Comparative study in steroidogenesis by quail and chick embryonic gonads in organ culture. J. Steroid
biochem.
12, 83-87.
Harlan, R. E., and Gorski, R. A. (1977). Steroid regulation of luteinizing hormone secretion in normal and androgenizcd rats at different ages. Endocrinology 101, 741-749.
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Harlan, R. E.. and Gorski, R. .4. (1978). Effects of postpubertal ovarian steroids on reproductive function and sexual differentiation of lightly androgenized rats. Endocrinology
102, 17 16- 1724.
Harlan, R. E., Gordon, J. H., and Gorski, R. A. (1979). Sexual differentiation of the brain: Implications for neuroscience. In D. M. Schneider (Ed.), Reviews of Neuro.vciencr Vol. 4, pp.31-69. Raven Press, New York. Hutchison, R. E. (1978). Hormonal differentiation of sexual behavior in Japanese quail. Horm.
Behv.
11, 363-387.
Kannankeril, J. V., and Domm, L. V. (1968). The influence of gonadectomy on sexual characters in the Japanese quail. J. &forphol. 126, 395-412. Kincl, F. A., Benagiano, G., and Angee, I. (1968). Sustained release hormonal preparations. 1. Diffusion of various steroids through polymer membranes. Skroids 11, 673-680. Lobl, R. T., and Gorski, R. A. (1974). Neonatal intrahypothalamic androgen administration: The influence of dose and age of androgenization of female rdts. Endocrinology 94. 1325-1330. McEwen, B. S. (1978). Sexual maturation and differentiation: The role of gonadal steroids. Prog. Brain Res. 48, 291-307. Sachs. B. D. (1967). Photoperiodic control of the cloaca1 gland of the Japanese quail. Science 157, 201-203.
Schumacher, M., and Balthazart, J. (1983a). Effects of castration on postnatal differentiation in the Japanese quail (Coturnix coturnix juponica). IRCS Med. Sci. Lihr. Compend. 11, 102-103.
Schumacher, M., and Balthazart, J. (1983b). The post-natal differentiation of sexual behavior in the Japanese quail (Coturnix cofurnix juponicu). Behav. Processes 8, 189-195. Schumacher, IM., and Balthazart, J. (1984). The postnatal demasculinization of sexual behavior in the Japanese quail (Cofurnti coturnix juponicu). Horm. Behav. 18, 298312. SGdersten, P., Pettersson, A., and Eneroth, P. (1983). Pulse administration of estradioll7B cancels sex differences in behavioral estrogen sensitivity. Endocrinology 112. 1883-1885. Steward, J.. and Cygan, D. (1980). Ovarian hormones act early in development to feminize adult open-field behavior in the rat. Harm. Behav. 14, 20-32. Tanabe, Y., h’akamura, T., Fujioka. K., and Doi, 0. (1979). Production and secretion of sex steroid hormones by the testes, the ovary and the adrenal glands of embryonic and young chickens (Gallus domesticus). Gen. Comp. Endocrinol. 39, 26-33. Wada, M. (1982). Effects of sex steroids on calling, locomotor activity, and sexual behavior in castrated male Japanese quail. How. Behuv. 16, 147-157. Whitsett, J. M., Irwin, E. W., Edens, F. W., and Thaxton, J. P. (1977). Demasculinization of male Japanese quail by prenatal estrogen treatment. Harm. Behav. 8, 254-263. Woods, J. E., and Erton, L. H. (1978). The synthesis of estrogens in the gonads of the chick embryo. Gen. Comp. Endocrinol. 36, 360-378. Woods, J. E., and Brazzil, D. M. (1981). Plasma 17fi-estrddiol levels in the chick embryo. Gen. Camp. Endocrinol.
49, 37-43.
AND
18, 287-297 (1984)
BEHAVIOR
Estradiol Contributes to the Postnatal Demasculinitation of Female Japanese Quail (Coturnix coturnix japonica) J. BALTHAZART' AND M. SCHUMACHER Labomtoire
de Biochimie
GnPrule
et ComparPe, CJniversit6 de LiPge, 17 place Delcour, B-4020 LiPge, Belgilrm
Two experiments were performed to characterize the process of postnatal demasculinization in Japanese quail. In the first experiment. it was shown that estradiol (E2) can complete female demasculinization during the first 4 weeks of life. By contrast, E2 did not demasculinize sexual behavior and cloaca1 gland in neonatally castrated males. Neonatally gonadectomized females preferentially performed mount attempts when tested in their home cage by comparison to a test arena. In Experiment 2, E2 Silastic implants (40-mm) maintained full copulatory behavior in castrated males but not in females. This large dose of E2 did not demasculinize adult sexually active birds (males or females) even if treatment lasted for 1 month. It is concluded that E2 can demasculinize sexual behavior only in females and only if treatment is performed in very young birds.
The permanent organizational actions of gonadal hormones are thought to be limited to a short critical period in early development. This critical period of sexual ditferentiation would take place at the end of the embryonic life and during the first days after birth in the rat (McEwen, 1978). In quail, it would end around Day 12 of incubation (Adkins, 1979). Several studies, however, suggest that the permanent actions of steroids are not restricted to such a short period. If female rats are lightly androgenized after birth, they will display normal estrous cycles when they reach puberty. However, this capacity will be lost at an early age and they will become anovulatory (Gorski, 1968; Harlan and Gorski, 1977). Postpubertal ovarian secretions (estrogens and/or androgens) are responsible for the appearance of this delayed anovulatory syndrome (DAS; Harlan and Gorski, 1978) which is a good model of permanent changes induced by steroids during the postpubertal period (Harlan, Gordon, and Gorski, 1979). The present data support the conclusion that the competent (“critical”) period during which gonadal steroids may exert permanent effects is not I To whom all correspondence and reprint requests should be addressed. 287 0018-506X/84 s I .50 Copyright f 1YX4 by Academic Press. Inc. All rights of reproduction in any rorm xscrvcd.
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limited to a concise perinatal period of development. Examples are available which show the following: -The competent period is not the same for different responses. In rats, the differentiation of the lordosis response and pituitary function is essentially completed 4-5 days after birth while estrogens still have permanent feminizing effects on open-field behavior later in the development (Steward and Cygan, 1980). -Different aspects of a response (e.g., sexual behavior) can differentiate independently. In rats, defeminization (decreasedcapacity to show lordosis) occurs mainly during the first 4-5 days after birth, whereas masculinization (increased capacity to show male-like mounting) may begin before birth (McEwen, 1978). -For a given response, the competent period is to some extent dose dependent. Androgenization of female rats (suppression of ovulatory cycles by neonatal testosterone treatment) is possible up to Day 5 of life with low doses of testosterone but can be obtained until Day 11 if higher doses are given (Lob1 and Gorski, 1974; Harlan et al, 1979). -The observed effects of neonatal treatment could depend on the test situation or hormonal stimuli used to elicit behavioral responses in adulthood (Feder and Goy, 1983; Clemens, Hiroi, and Gorski, 1969). The concepts of a critical period for sexual differentiation thus “retain importance but must be reshaped to fit more recent evidence” (Harlan et al, 1979). In Japanese quail, sexual differentiation proceeds mainly through demasculinization of the females (Adkins and Adler, 1972; Adkins, 1975; Balthazart and Schumacher, 1983).It was thought that this process occurred before Day 12 of incubation (Adkins, 1976, 1979) but we showed recently that the female demasculinization is not completed at hatching and that full demasculinization of female behavior and morphology is achieved under the influence of ovarian secretion during the first 2-4 weeks of posthatching life (Schumacher and Balthazart, 1983a, 1983b, 1984). Postnatal testicular secretions, by contrast, seem to have no permanent effects in males except on the control mechanisms of gonadotropin secretion (Schumacher and Balthazart, 1984). The present experiments were designed to determine the nature of the ovarian secretion responsible for the postnatal demasculinization in female quail. Considering that estradiol demasculinizes the behavior of males when injected in embryos (Adkins, 1975, 1979; Whitsett, Irwin, Edens, and Thaxton, 1977). we have tested the effect of this steroid in neonatally gonadectomized males and females. GENERAL METHODS
Neonatal gonadectomy and hormonal treatments with Silastic implants were performed as described previously (Schumacher and Balthazart,
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1984). During behavioral tests, the sexual behavior of the birds was recorded during a 5-min presentation to a receptive female either in the home cages or in a test arena. Frequency and latency of occurrence of the following behavior patterns were recorded: neck grab (NG), mount attempt (MA), mount (M), and cloaca1 contact movement [CCM; see Adkins and Adler (1972) and Hutchison (1978) for description]. Cloaca1 gland areas were measured to the nearest square millimeter (Sachs, 1%7) and at sacrifice, stemotracheal (syringeal) muscles (androgendependent structures) were weighed to the nearest milligram [see Schumacher and Balthazart (1984) for more detail]. EXPERIMENT 1 Material
and Methods
Male and female quail came from three different hatches in nearly equal numbers. Birds from Groups 1 and 3 were hatched in our laboratory while those from Group 2 were bought from a local dealer (origin of our colony) at the age of 1 day. Birds from the different hatches were distributed in a random order between the different experimental groups so that birds from each hatch were present in each group in similar numbers. At the age of 4 days, males and females were gonadectomized and 2 days later were implanted with IO-mm Silastic implants filled with estradiol (E2) or left empty as control (C). Two weeks later, the length of the implants was doubled (another lo-mm capsule was implanted). Indirect estimations based on the plasma levels of steroid induced in quail by similar testosterone implants (Desjardins and Turek. 1977):on the relative release rates of E2 and T from Silastic capsules (Dziuk and Cook. 1966; Kincl, Benagiano, and Angee, 1968; Wada, 1982), and on data showing E2 plasma levels in rats bearing E2 Silastic capsules (Brawer, Schipper, and Robaire, 1983; Sodersten, Pettersson, and Eneroth, 1983) suggest that the E2 implants used here produced plasma levels of steroid in the range 100 to 1500 pg/ml which compares reasonably with levels seen in developing chicken (Tanabe, Nakamura, Fujiokd, and Doi, 1979) or in adult quail (Doi, Takai, Nakamura, and Tanabe, 1980). This dose was also known to have behavioral effects in quail not associated with obvious toxic effects (Balthazart, Malacarne, and Schumacher, unpublished data). Treatment was maintained for I month as the postnatal differentiation of behavior in quail is completed during that period (Schumacher and Balthazart, 1984). Ali birds (I 1 E2 males, 13 C males, 26 E2 females, and 21 C females) were raised in heterosexual groups until the age of 34 days. At that time (4 weeks after the implantation), the implants were removed and quail were isolated in individual home cages for 2 weeks and left without hormone treatment. They were then implanted with 60-mm (3 x 20) Silastic capsules filled with crystalline T and then starting 2 weeks later
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they were tested for sexual behavior five times in their home cage and five times in the arena. Tests were performed in a random order (arena versus home cage) at the rate of two tests per week. At the end of the experiment, quail were 102 days old. Sexually inactive birds were killed and checked for castration and presence of implants. Sexually active birds were introduced in Experiment 2 (see below). At the end of Experiment 1, cloaca1 gland area was measured in each bird and syringeal muscles of the sacrificed birds were weighed. Body weight and plumage type were also recorded when birds were 34 (end of postnatal E2 treatment), 48 (start of T treatment), and 102 (end of experiment) days old. The type of plumage was quantified on a nonparametric scale ranging from 1 to 4 with I = rufous-colored male-type breast feathers, 2 = rufous-colored breast feathers with a few brown spots, 3 = rufous-colored breast feathers with large number of brown spots, and 4 = pale-colored female-type breast feathers with brown spots. In quail, E2 induces a female-type plumage in both sexes and the male plumage is neutral or anhormonal (Hutchison, 1978). Results Se-xual behaviclr.During the home-cage tests, nearly all males attempted to mount the stimulus females (Fig. 1). Females were less active than a--me--+-----,,
E2d
cpzr--
FIG. 1. Cumulative percentage of males and females which received empty (C birds) or EZ-filled (E2 birds) Silastic implants during their first 4 weeks of life and showed mount attempts (MA) when tested in their home cage. The x axis indicates time since T implantation. Above: results for the 3 hatches of birds. Below: results for Hatches 1 and 3 only (Hatch 2 from another origin excluded; see text). *** = P < 0.001, ** = P < 0.01, and * = P < 0.05 by Fisher exact probability tests when compared to C females.
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males. During the first and third tests, more C than E2 females showed sexual behavior. This difference was still present in the other tests but did not reach significance. However, when results from Hatches 1 and 3 were analyzed separately (birds from Hatch 2 which were not hatched in the laboratory being dropped from the data), C females were then sexually more active than E2 females during each of the tests (Fig. 1, below). Females from Hatch 2 were not demasculinized at all by postnatal estrogens (6/7 C females and 9112E2 females showed MA at least once) while this effect was clearly present in the other females. As shown previously (Schumacher and Balthazart, 1984) females preferentially showed male sexual behavior when tested in their home cage by comparison with the arena (Fig. 2). The percentage of females which showed sexual behavior was thus higher in the C than in the E2 group only for tests performed in the home cages but not for those in the arena. A small percentage of females only performed cloaca1 contact movements in the arena (6/47) or in the home cages (13/47). By contrast, nearly all males showed full copulatory behavior in both test situations. There was no difference in latency or frequency for the different behavior patterns between C and E2 males whatever the test situation. Morphology. On Day 102 after nearly 8 weeks of T treatment, males had larger cloaca1 glands than females. In addition, C females had larger glands than E2 females (Fig. 3). Syringeal muscles had similar weights in the 4 experimental groups. At 34 and 48 days of age, C males were characterized by a male-type plumage whereas E2 males and E2 females had a completely female-type plumage. The plumage of C females was intermediate with a median score of 2. At the end of the experiment after the T treatment, C birds of both sexes had a male-type plumage whereas all E2 birds had a female like plumage (Fig. 3). On Day 48, E2 males were lighter than E2 females and C males (P < 0.01 and P < 0.05, respectively, by one-way analysis of variance followed by NewmanHome Cq I
C- Males EZ-Males
I
C-Females
m
&-Females
FIG. 2. Sexual behavior (percentage showing mount attempts, MA) of neonatally gonadectomized quail treated with E2 or empty (C) silastic implants and tested in their home cage or in the arena. * = P < 0.05 and 000 = P < 0.001, 00 = P < 0.01, and 0 = P < 0.05 by Fisher exact probability tests for comparisons to corresponding group of males (0) or to C females (*).
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iZ3
EZ-Males
AND SCHUMACHER
IT3
E2-Females
FIG. 3. Cloaca1 gland area at 102 days (left) and plumage type at 34, 48, and 102 days of life in neonatally gonadectomized quail treated with E2 or kept as control(C). Cloaca1 gland areas were analyzed by one-way analysis of variance followed by Newman-Keuls tests, plumage type by the.Kruskall-Wallis analysis of variance followed by Mann-Whitney U tests. *** or 000 = P < 0.001, **or00 = P
Keuls tests), At the end of the experiment, E2 males were still lighter than E2 females (P < 0.05, same test). EXPERIMENT
2
Having shown that E2 can demasculinize female behavior during the first month after hatching, we decided to see whether the same effect could be obtained in adult birds. We also tried to see whether E2 Silastic implants would maintain full copulatory behavior in neonatally gonadectomized males but not females as suggested by a previous experiment (Schumacher and Balthazart, 1984). Material
and Methods
T implants of the sexually active males and females from previous experiment were removed and birds were implanted on the same day (Day 102 of life) with 40 mm of empty of EZfilled Silastic capsules, Due to the small number of active females, no C females were retained for this experiment. Results were not influenced by the postnatal hormone treatment (C or E2) and the present results are thus only presented as a function of the adult hormone treatment [control adult (CA): 8 males, and estradiol adult (E2A): 8 males and 19 females including 8 with neonatal E2 and 11 neonatal C]. These implants were left in place during 46 days during which birds were tested for sexual behavior 11 times in their home cages and at the end twice in the arena. Tests were performed at the rate of two tests per week. Fifty-one days after the implantation, capsules were removed and birds
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were reimplanted with 60-mm (3 x 20) Silastic T capsules. After 2 weeks of T treatment, sexual behavior was tested once in the home cage and once in the arena. On Day 168, cloaca1 glands were measured, and birds were sacrificed by decapitation and checked for castration and presence of implants. Results During the estrudiol treatment. Estradiol maintained full copulatory behavior in nearly all males but not in females throughout the implantation period (home-cage tests; Fig. 4). Surprisingly, two CA males still performed cloacal contact movements during the 46 days without hormonal treatment. Six out of the eight E2A males attempted to mount the stimulus female and five performed cloaca1 contact movements during the last two tests in the arena. E2A females were sexually inactive in this test situation. During the testosferone treatment. All males performed cloaca1 contact movements in response to T when tested in the arena as well as in the home cages. Only 13 of the 19 females which were previously ac?ive before the E2 treatment attempted to mount the stimulus female during the single test in the home cage. However, this small decrease in activity
TIME
(DAYS1
FK. 4. Percentage of birds showing mount attempt (MA) and cloaca1 contact movement (CCM) when treated with EZ-filled or empty (C, males only) silastic implants. All these birds were sexually active during the previous phase of the experiment when they were treated with testosterone. Behavioral tests were performed in the home cages. The x axis indicates time since removal of T implants and placement of E2 or C implants. * = P < 0.05 compared to C males. 000 or n n n = P < 0.001, and 0 = P < 0.05 compared to E2 females. All comparisons by the Fisher exact probability test. Most intermediate tests are omitted for clarity.
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only reflected the fact that birds were tested five times before and only once after being exposed to the adult E2 treatment. If this difference in test frequency is taken into account by comparing for example the numbers of females which showed mount attempts during at least three out of five tests before E2 treatment to those which showed the behavior after, this difference vanishes (before: 14/19; after: 13/19). Similarly, the E2 treatment in adulthood did not alter the response of the cloacal gland to T. The average gland sizes before and after E2 were not different (before: 191 + 21 mm*; after: 184 -t 43 mm’). GENERAL DISCUSSION We showed previously that female quail demasculinization is a continuous process extending into posthatching life which is not completed on Day 12 of incubation as was suggested by earlier studies. Although neonatally ovariectomized females are less sensitive than males to the activating effects of testosterone, demasculinization is completed only after 2-3 weeks of posthatching life under the influence of ovarian secretions (Schumacher and Balthazart, 1984). The present experiments suggest that estradiol can induce the postnatal demasculinization in female quail. Small E2 implants placed in neonatally ovariectomized females for the first month of life inhibited almost completely the ability of the females to respond to T by showing male-type sexual behavior. Neonatal E2 treatment also demasculinized the cloaca1 gland of females but did not affect their syringeal muscles. A previous experiment suggested that the demasculinization of these muscles is only completed 2 weeks after hatching (Schumacher and Balthazart, 1984). Why E2 did not demasculinize the muscles in the present experiment is unclear and could be related to inadequate dosage or to the nature of the hormone used (different ovarian hormones could differentiate the different aspects of the sexual phenotype). In rats for example, it is known that estrogens can defeminize sexual behavior [for review: McEwen (1978) and Darner (1980)] but do not control the differentiation of the spinal nucleus of the bulbocavernosus which is induced by androgens exclusively (Breedlove and Arnold, 1980). Similarly, in quail testicular hormones are responsible for the late differentiation of the gonadotrophin secreting system (Schumacher and Balthazart, 1984) while sexual behavior would rather differentiate under the influence of the ovary. In the first experiment, we were able to demasculinize sexual behavior of gonadectomized females but not males. The postnatal reactivity to the organizing effects of E2 on sexual behavior is thus sexually differentiated before hatching. Females are presensitized to the organizing action of E2 and it seems likely that embryonic E2 is itself responsible for this effect. It is known that the in virro production of estrogens in organotypic culture is higher for the left ovary than for the testes in quail on Days
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10 and 14 of incubation (Guichard, Scheib, Haffen, Mignot, and Cedard, 1980). In chicken, female embryos also have higher E2 and estrone plasma levels than males between Day 7 of incubation and hatching (Woods and Et-ton, 1978; Woods and Brazzil, 1981). These data thus support the idea that sexual differentiation is biphasic. Embryonic estrogens would sensitize the undifferentiated target tissues during a critical period of embryonic development which would permit the more progressive organizing action of the same hormone during 2-3 weeks after hatching. This timing could be slightly variable as suggested by the fact that sensitivity to the organizing action of E2 was aparently lost already at 4 days of age in one batch of quail in Experiment 1. These birds were from a different origin (see Methods) and had thus been incubated in slightly different conditions which possibly changed their rate of maturation and consequently the exact timing of their competent periods [see Schumacher and Balthazart (1984) for a more detailed discussion]. Neonatally ovariectomized females had a plumage showing both male and female characteristics during the first 7 weeks of life. As the femaletype plumage is induced by estrogens in quail (Kannankeril and Domm, 1%8; Hutchison, 1978), this suggests that these females including those in the control group (without E2 implants) were still under the influence of estrogens. These estrogens, which are probably secreted by the adrenals [see Tanabe et al. (1979) for a study in chicken] might have attenuated the differences between females with and without exogenous E2 and thus have decreased the magnitude of the effects observed here. However, concentrations of circulating estrogens in young ovariectomized females were probably very low because estrogens induce female-type plumage at low concentrations which ate not sufficient to suppress LH secretion (Gibson, Follett, and Gledhill, 1975). In Experiment 2, E2 maintained full copulatory behavior in males but not in females. This suggests that one aspect of the sexual differentiation is the loss in females of the sensitivity to the activating effects of E2 on male-type sexual behavior. This experiment also showed that high doses of E2 (40 mm) given for more than a month in adult quail have no permanent demasculinizing effects in males as well as in females. As lo- to 20-mm E2 implants given during a comparable period to neonatal quail are sufficient to demasculinize sexual behavior and morphology of females, this suggests that the competent period for female demasculinization is terminated before birds become adult. This conclusion must, however, be moderated by the fact that behavior had been activated by T before the E2 treatment was applied and sequential effects of the hormone treatments are quite possible. In conclusion these experiments demonstrate that sexual differentiation is completed only during the first few weeks of life in female quail under the influence of ovarian secretions among which estradiol could play a
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critical role. The sensitivity to these progressive organizing effects of estradiol is, however, lost before hatching in the males and before puberty in females, ACKNOWLEDGMENTS We are indebted to Professor E. Schoffeniels for his continued interest in our research. This work was supported by a grant from the Belgian FNRS (credit aux chercheurs) to J. Balthazart and Grant 2.451880 from the FRFC to Professor E. Schoffeniels. M. Schumacher is supported by a grant from the IRSIA.
REFERENCES Adkins, E. K. (1975). Hormonal basis of sexual differentiation in the Japanese quail. J. Comp. Physiol. Psychol. 89, 61-71. Adkins, E. K. (1976). Embryonic exposure to an antiestrogen masculinizes behavior of female quail. Physiol. Behav. 17, 357-359. Adkins, E. K. (1979). Effect of embryonic treatment with estradiol or testosterone on sexual differentiation of the quail brain. Neuroendocrinology 29, 178-185. Adkins, E. K., and Adler, N. T. (1972). Hormonal control of behavior in the Japanese quail. J. Comp. Physiol. Psychol. 81, 27-36. Balthazart, J., and Schumacher, .M. (1983).Testosterone metabolism and sexual differentiation in quail. In J. Bahhazart, E. Prove, and R. Gilles (Eds.), Hormones and Behavior in Higher Vertebrates, pp.237-260. Springer-Verlag, Berlin. Brawer, J., Schipper, H., and Robaire, B. (1983). Effects of long term androgen and estradiol exposure on the hypothalamus. Endocrinology 112, 194-199. Breedlove, M. S., and Arnold, A. P. (1980). Hormone accumulation in a sexually dimorphic motor nucleus of the rat spinal cord. Science 210, X54-566. Clemens, L. G., Hiroi, M., and Gorski, R. A. (1969). Induction and facilitation of female mating behavior in rats treated neonatally with low?doses of testosterone propionatc. Endocrinology 84, 1430-1438. Desjardins, C., and Turek, F. W. (1977). Effects of testosterone on spermatogenesis and luteinizing hormone release in Japanese quail. Gen. Comp. Endocrinol. 33, 293-303. Doi, 0.. Takai, T., Nakamurd, T., and Tanabe, Y. (1980). Changes in pituitary and plasma LH, plasma and follicular progesterone and estradiol, and plasma testosterone and estrone concentrations during the ovulatory cycle of the quail (Coturnix coturnix juponica).
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Darner, G. (1980). Sexual differentiation of the brain. Vitam. Horm. 38, 325-381. Dziuk, P. J., and Cook, B. (1966). Passageof steroids through silicone rubber. Endocrin0log.y 78, 208-2 I 1. Feder, H. H., and Goy, R. W. (1983). Effects of neonatal estrogen treatment of female guinea pigs on mounting behavior in adulthood. Horm. Behav. 17, 284-291. Gibson, W. R., Follett, B. K., and Gledhill, B. (1975). Plasma levels of luteinizing hormone in gonadectomized Japanese quail exposed to short or long day length. J. Endocrinol. 64, 87-101. Gorski, R. A. (1968). Influence of age on the response to paranatal administration of a low dose of androgen. Endocrinology 82, 1001-1004. Guichard, A., Scheib, D., Haffen, K., Mignot, Th.lM., and Cedard, L. (1980). Comparative study in steroidogenesis by quail and chick embryonic gonads in organ culture. J. Steroid
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Harlan, R. E., and Gorski, R. A. (1977). Steroid regulation of luteinizing hormone secretion in normal and androgenizcd rats at different ages. Endocrinology 101, 741-749.
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Harlan, R. E.. and Gorski, R. .4. (1978). Effects of postpubertal ovarian steroids on reproductive function and sexual differentiation of lightly androgenized rats. Endocrinology
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Harlan, R. E., Gordon, J. H., and Gorski, R. A. (1979). Sexual differentiation of the brain: Implications for neuroscience. In D. M. Schneider (Ed.), Reviews of Neuro.vciencr Vol. 4, pp.31-69. Raven Press, New York. Hutchison, R. E. (1978). Hormonal differentiation of sexual behavior in Japanese quail. Horm.
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Kannankeril, J. V., and Domm, L. V. (1968). The influence of gonadectomy on sexual characters in the Japanese quail. J. &forphol. 126, 395-412. Kincl, F. A., Benagiano, G., and Angee, I. (1968). Sustained release hormonal preparations. 1. Diffusion of various steroids through polymer membranes. Skroids 11, 673-680. Lobl, R. T., and Gorski, R. A. (1974). Neonatal intrahypothalamic androgen administration: The influence of dose and age of androgenization of female rdts. Endocrinology 94. 1325-1330. McEwen, B. S. (1978). Sexual maturation and differentiation: The role of gonadal steroids. Prog. Brain Res. 48, 291-307. Sachs. B. D. (1967). Photoperiodic control of the cloaca1 gland of the Japanese quail. Science 157, 201-203.
Schumacher, M., and Balthazart, J. (1983a). Effects of castration on postnatal differentiation in the Japanese quail (Coturnix coturnix juponica). IRCS Med. Sci. Lihr. Compend. 11, 102-103.
Schumacher, M., and Balthazart, J. (1983b). The post-natal differentiation of sexual behavior in the Japanese quail (Coturnix cofurnix juponicu). Behav. Processes 8, 189-195. Schumacher, IM., and Balthazart, J. (1984). The postnatal demasculinization of sexual behavior in the Japanese quail (Cofurnti coturnix juponicu). Horm. Behav. 18, 298312. SGdersten, P., Pettersson, A., and Eneroth, P. (1983). Pulse administration of estradioll7B cancels sex differences in behavioral estrogen sensitivity. Endocrinology 112. 1883-1885. Steward, J.. and Cygan, D. (1980). Ovarian hormones act early in development to feminize adult open-field behavior in the rat. Harm. Behav. 14, 20-32. Tanabe, Y., h’akamura, T., Fujioka. K., and Doi, 0. (1979). Production and secretion of sex steroid hormones by the testes, the ovary and the adrenal glands of embryonic and young chickens (Gallus domesticus). Gen. Comp. Endocrinol. 39, 26-33. Wada, M. (1982). Effects of sex steroids on calling, locomotor activity, and sexual behavior in castrated male Japanese quail. How. Behuv. 16, 147-157. Whitsett, J. M., Irwin, E. W., Edens, F. W., and Thaxton, J. P. (1977). Demasculinization of male Japanese quail by prenatal estrogen treatment. Harm. Behav. 8, 254-263. Woods, J. E., and Erton, L. H. (1978). The synthesis of estrogens in the gonads of the chick embryo. Gen. Comp. Endocrinol. 36, 360-378. Woods, J. E., and Brazzil, D. M. (1981). Plasma 17fi-estrddiol levels in the chick embryo. Gen. Camp. Endocrinol.
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