The role of endogenous hormones in differentiating the sexes is an area of continuing research. The bulk of findings in this field support the notion that mammalian sexual differentiation is primarily mediated by androgens of testicular origin and that the presence of these androgens in early life produces a "male" brain. In contrast, the female brain is thought to develop via a hormonal default mechanism, in the absence of androgen. Findings are reviewed which show that ovarian hormones also play a significant role in sexual differentiation, and that the process of ovarian feminization has a considerably later sensitive period than androgen-mediated masculinization.
1. Historically, reviews concerning the role of hormones in sexual differentiation of mammals have focused on males and the effects of neonatal exposure to testicular androgens (e.g., testosterone, the predominant androgen). Emphasis on androgen stems from the preponderance of data demonstrating that neonatal castration of the male prevents or interferes with the masculinization process, whereas early exposure of females to high doses of androgens results in male-like patterns in adulthood (see Breedlove, 1992; or Toran-Allerand, 1986 for review). Such observations range from reports of cognitive differences among human populations with abnormal androgenic exposure (Masica, Money, Ehrhardt & Lewis, 1969; Resnick & Berenbaum, 1982) to alterations in hypothalamic morphology following perinatal androgenic manipulations in rats (Gorski, 1984).
2. The role of ovarian hormones has not been as extensively investigated. When effects are found, they are generally subtle as compared to the marked effects of androgens. Indeed, some researchers have reported that ovarian manipulations are without effect (Lisk & Suydam, 1967; Whalen & Edwards, 1967). Yet, over the past twenty years a number of papers have reported significant behavioral and neuromorphological effects (1) after removal of the ovaries, or (2) after low-dose estrogen exposure following removal of the testes. In view of such findings, some researchers have suggested that estrogen may play an active role in differentiation of the female brain (Dohler, Hancke, Srivastava, Hofmann, Shryne & Gorski, 1984a; Dohler, 1991; Hendricks, 1992; Toran-Allerand, 1992). However, these data have not been assimilated into a widely accepted model of developmental ovarian effects.
3. It is the purpose of this paper to review findings, including data from our laboratory, in support of the thesis that exposure to ovarian hormones (primarily estrogen) during development is necessary for differentiation of the female brain.
4. Mechanisms of hormone action have traditionally been divided into effects which occur early in development and are permanent ("organizational") versus those which occur later in development and are transitory since they depend upon the presence of circulating hormones ("activational"). Within this framework, sex differences in neuromorphological structure were assumed to reflect the permanent effects of organizing steroids. Some behavioral effects were interpreted as organizational (e.g., reduced rough and tumble play following neonatal castration of the male), whereas other behaviors, particularly those which could be mimicked by experimental manipulation of circulating steroids (e.g., the "priming" of female rodents for sexual receptivity via exogenous estrogen and progesterone), were considered activational.
5. It has been recently acknowledged, however, that the organizational/activational dichotomy is not absolute. Several reviews exploring the changing definitions of these terms have been written (Arnold & Breedlove, 1985; Stewart, 1988; Williams, 1986). Specifically, the designations of organizational effects as physiological or structural characteristics set in the brain during a sensitive period of early development, and activational effects as occurring primarily in adulthood, are no longer strictly held. Instead, the primary distinction now depends upon whether induced changes represent permanent or transient effects, whenever in life they occur.
6. Recent research has also demonstrated activational changes, traditionally characterized by fluctuations in behavior, in cellular and/or neurochemical parameters. These findings muddy the traditional definition of sexual differentiation, which has always included early organizational effects but excluded activational effects. Yet, the cyclic nature of the female brain has its roots in the way the brain was organized initially. Thus, cyclicity per se is a permanent (organizational) feature of the female brain. In as much as cyclic hormonal changes induce specific neural alterations, such effects characterize the female brain and thus would appear to fall under the rubric of sexual differentiation. We cite below several findings demonstrating activational ovarian effects on neural parameters.
III.1 OVARIAN HORMONES AND REPRODUCTIVE BEHAVIOR
7. Blizard and Denef (1973) reported that the inhibitory effects of neonatal testosterone exposure on female rat sexual behavior (lordosis, or receptive posturing) were suppressed if the ovaries were present during development. Thus, treated subjects showed a greater lordosis response if the ovaries had been present neonatally. These findings are consistent with the results of Sodersten (1976), who found that postpubertally ovariectomized female rats showed more lordosis as adults (after priming) than neonatally ovariectomized females. Thus, subjects with the ovaries retained neonatally again showed more lordosis as adults. Sodersten concluded that "Although the nature and mechanism of the action of these ovarian secretions remain to be determined we feel that the fact should be recognized that they do exert a modifying influence on psychosexual differentiation" (p. 419).
8. Other researchers have found that when proceptive or soliciting components of female rat sexual behavior - hopping, darting and ear-wiggling - are examined, ovarian effects become even more evident. Gerall and his colleagues (Gerall, Dunlap & Hendricks, 1973) reported higher proceptive behavior in estrogen-primed female rats ovariectomized postpubertally compared to those ovariectomized neonatally. Similarly, neonatally gonadectomized males who received prepubertal ovarian transplants or low-dose estrogen treatment were more proceptive in adulthood than males who were gonadectomized only. Interestingly, the former males exhibited as much proceptive darting behavior as normal females, suggesting that exposure to physiological levels of ovarian estrogen may be particularly important in the organization of proceptivity.
9. The findings summarized above suggest that lordosis is under different hormonal control than proceptive behavior, and that the latter behavior may be particularly sensitive to ovarian effects. This thesis is supported by Ward's (1983) finding that prenatally stressed male rats exhibited lower mounting and increased lordosis in adulthood, but did not exhibit any of the proceptive components of female sexual behavior.
III.2 OVARIAN HORMONES AND NON-REPRODUCTIVE BEHAVIOR
10. OPEN-FIELD BEHAVIOR. Female rats are normally more active in the open field than males. Ovariectomy on Day 1 or 8 of life reduced open-field behavior in adult female rats to male levels (Denti & Negroni, 1975; Stewart & Cygan, 1980). This finding is consistent with the report by Blizard and Denef (1973) that the presence of ovaries during development suppressed the masculinizing effects of neonatal testosterone treatment on open field behavior. Stewart and Cygan (1980) also studied the effect of estrogen on open field behavior in ovariectomized female rats and concluded that "estrogens given during the period prior to weaning can have a feminizing effect on adult open-field behavior, and that the sex difference normally observed in adult rats is dependent in part on the presence of the ovaries during a period after birth" (p. 20).
11. PLUS MAZE. Recently, Zimmerberg and Farley (1993) found that intact adult female rats spent significantly more time in the open arms of a plus maze than males. When females were either exposed neonatally to the estrogen receptor-blocker tamoxifen, or ovariectomized at puberty, they spent less time in the open arms as adults. Females who received both treatments spent the least time in the open. In contrast, neonatal and pubertal manipulations of androgens in males (via administration of the androgen receptor-blocker flutamide and castration) had little effect. The authors concluded "These experiments indicate that female gonadal hormones play an important role both organizationally and activationally in plus maze behavior" (p. 1119). This assertion was further confirmed by Leret, Molina-Holgado, and Gonzales (1994), who also reported that neonatally ovariectomized female rats behaved similarly to males when tested in a plus maze paradigm.
12. MAZE LEARNING. Krasnoff and Weston (1976) found that sex differences in maze learning emerged around the time of puberty in rats, with females making more errors after puberty and male behavior remaining essentially unchanged. Other findings showed that intact or neonatally castrated male rats, given low-doses of estrogen neonatally, had lower spatial learning scores in adulthood than untreated males (Dawson, Cheung & Lau, 1975). In humans, sex differences in spatial ability have also been reported to appear at puberty (see Halpern, 1992 for review), although the hormonal basis for this effect is not fully understood.
13. AVOIDANCE LEARNING. Sex differences in two-way shuttlebox learning do not appear until after puberty in rats (Bauer, 1978) yet are influenced by ovarian hormones at various times in development. Denti and Negroni (1975) found that neonatal ovariectomy decreased avoidance learning of adult female rats to male-typical levels. In contrast, Diaz-Veliz, Soto, Dussaubat, and Mora (1989) reported that postpubertal ovariectomy led to a significant decrease in errors made in a shuttlebox paradigm in rats, an effect which was reversed with three days of estrogen replacement. Diaz-Veliz and her colleagues also reported that avoidance learning varied across the estrous cycle, with high estrogen levels associated with increased errors. The latter findings are likely to reflect activational hormonal effects (given their transient nature), while the neonatal ovariectomy appears to have had a permanent (organizational) effect upon this behavior. It is noteworthy that, in the above example, the activational effects of estrogen on avoidance behavior were opposite to developmental (organizational) estrogen effects.
14. ROTATION BEHAVIOR. Camp, Robinson, and Becker (1984) have demonstrated sexual dimorphism of the nigrostriatal system in rats as measured by various tests of amphetamine-induced rotation (with females showing more asymmetry on some tasks and less on others). In addition, Becker and colleagues (Becker & Cha, 1989; Castner & Becker, 1990) reported that endogenous, or exogenously administered, pulsatile estrogen potentiated the dopaminergic and behavioral response to amphetamine in female, but not male, rats. Ovariectomy depressed striatal dopaminergic release and turnover in females, while physiological concentrations of estrogen stimulated dopaminergic release. Castration or estrogen exposure had no similar effect on male striatal tissue. This sex difference appeared to emerge at puberty through changes in the response of female striatal tissue to estrogen.
III.3 ESTROUS EFFECTS
15. As an aside, it should be noted that not all experiments regarding ovarian effects on behavior have controlled for the possibility of adult estrous effects. Where neonatally ovariectomized females are compared to intact females as adults, behavioral differences may reflect the consequence of activational rather than organizational ovarian influence. However, findings such as significant behavioral differences between neonatal and pubertal ovariectomy (Gerall et al., 1973), potentiation of the effects of pubertal ovariectomy by neonatal treatment with tamoxifen (Zimmerberg & Farley, 1993), and differential effects of neonatal and pubertal ovariectomy (Denti & Negroni, 1975; Diaz-Veliz et al., 1989), give clear evidence of developmental rather than activational effects.
III.4 OVARIAN HORMONES AND SUBCORTICAL MORPHOLOGY
16. Dohler and colleagues (Dohler et al., 1984a, 1984b) found that neonatal administration of the estrogen antagonist, tamoxifen, to female rats decreased the size of the sexually dimorphic nucleus of the preoptic area (SDN-POA), which is normally significantly larger in male as compared to female rats, in adulthood. They proposed that "feminization" of this structure may require some low level of estrogen. This thesis is supported by more recent evidence that neonatal treatment with estrogen mRNA antisense also significantly reduced the size of the SDN-POA in intact adult female rats (McCarthy, Schlenker & Pfaff, 1993).
17. In a related study, Bloch and Gorski (1987; 1988) reported that the hypothalamic anteroventral preoptic nucleus (AVPv) is significantly larger in female rats than in males, and that postpubertal castration of males followed by treatment with low doses of estrogen and progesterone significantly enlarged this structure. These males also had smaller SDN-POA as compared to control males. Similar effects were not observed in males that were gonadectomized only. More recent work has shown that sex differences in the AVPv emerge at puberty, and are the consequence of increases in AVPv size in females (Davis, Elihu, Shryne & Gorski, 1993).
18. Frankfurt, Gould, Woolley, and McEwen (1990) reported that dendritic spine density of ventromedial hypothalamic neurons varied across the estrous cycle in rats. They found that adult ovariectomy brought about a reduction in density and that estrogen or estrogen plus progesterone replacement increased density. Although such effects are properly described as activational, they demonstrate that circulating hormones can temporarily change neural structure. Thus, transient fluctuations in adult physiology and behavior may also be accompanied by alterations in neuroanatomy despite the traditional view that structural changes are permanent and restricted to the early organizational period.
III.5 OVARIAN HORMONES AND CORTICAL/HIPPOCAMPAL MORPHOLOGY
19. CORTICAL THICKNESS. Neonatal ovariectomy increased the cortical thickness of 90-day old female rats as compared to sham operated littermate controls (Diamond, Johnson & Ehlert, 1979). Other females were ovariectomized at either 90 or 300 days while littermate controls received sham surgery. Ninety days later (i.e., at 180 or 390 days of age, respectively) cortical thickness measurements were obtained. No differences were found between either group as compared to shams, thereby implying that ovarian hormones had acted during early development to affect the thickness of the cerebral cortex.
20. Pappas, Diamond, and Johnson (1979) sought to determine the relative contributions of estrogen and progesterone to cortical thickness. In their first experiment they replicated the neonatal ovariectomy effect of an increase in cortical thickness at 90 days. In a second experiment one group of females were ovariectomized on Day 1 while female littermate controls received sham surgery. From 40 to 90 days of age, the experimental females received daily injections of ethinylestradiol while the controls received an equal volume of sesame oil. Measurements at 90 days found that the ovariectomy-plus-estrogen group had significantly thinner cortices than controls. A third experiment followed the same procedure as experiment 2 except that ovariectomized females received daily injections of progesterone from Days 40-90 while littermate controls received oil. The progesterone-treated animals had significantly thicker cortices. These findings suggest that estrogen and progesterone act reciprocally to affect cortical thickness.
21. In later studies Diamond and her colleagues found the right cerebral cortex of the male rat to be significantly thicker than the left throughout life; whereas the cerebral cortex of the female showed no significant right-left differences (but a left thicker than right trend; Diamond, Johnson, Young & Singh, 1983). To investigate the role of ovarian hormones in this sexual dimorphism, a group of females were ovariectomized on Day 1 while female littermate controls received sham surgery. Cortical thickness measurements at 90 days showed that the ovariectomized females exhibited a male pattern of right-greater-than-left in all areas measured, significantly so in the visual cortex (Diamond, Dowling & Johnson, 1981), while controls again showed no significant right-left differences.
22. Stewart and Kolb (1988) attempted to replicate this latter finding. Although they found the described sex difference, they did not find that ovariectomy reversed the cortical asymmetry pattern in females.
23. DENDRITIC SPINE DENSITY. The development of dendritic spines in the visual cortex occurs later in intact male rats than in intact females (Munoz-Cueto, Garcia-Segura & Ruiz-Marcos, 1990). After Day 20, dendritic spine numbers continued to increase for males, whereas females showed a significant decrease. The female-typical loss of dendritic spines was prevented by ovariectomy on Day 30, leading to a higher number of cortical dendritic spines among ovariectomized females as compared to intact females on Day 60. Munoz-Cueto et al. postulated that estrogen exerted inhibitory effects on cortical dendritic spine growth in both sexes, with aromatized testosterone delaying development in males during the early period, and ovarian hormones promoting a loss of spines during the later time period (Days 20 to 60).
24. Finally, Gould, Woolley, Frankfurt, and McEwen (1990) reported that ovariectomy in adulthood decreased dendritic spine density on CA1 pyramidal cells in the hippocampus of female rats, and that this effect was blocked by the concurrent administration of estrogen and progesterone. They suggested that variations in the density of these spines may accompany the estrous cycle. This was confirmed by Woolley, Gould, Frankfurt, and McEwen (1990). Miyakawa and Arai (1987) also found that post-pubertal estrogen treatment increased the number of axodendritic synapses in the lateral septum of intact female rats, but not intact males. As discussed earlier, such findings suggest that morphological changes may underlie some of the behavioral effects of circulating ovarian hormones. It is also possible that some behavioral effects of ovarian hormones observed in humans [e.g., variations in spatial ability across the menstrual cycle (Hampson, 1990; Hampson & Kimura, 1988)] may be associated with similar morphological changes.
III.6 OVARIAN HORMONES AND DEVELOPMENTAL PERIODS
25. The concept of critical, or sensitive, periods in development has played a central role in theories of sexual differentiation. Thus, it has been generally agreed that testicular androgens such as testosterone exert masculinizing effects on the CNS of male rats during the period between about gestational day 17 and postnatal days 8-10 (Rhees, Shryne & Gorski, 1990a; Rhees, Shryne & Gorski, 1990b; see also Gorski, 1984). This perinatal period of sensitivity to the masculinizing effects of testosterone appears to be similar in mice (e.g., see Wagner & Clemens, 1989). Testosterone presumably modifies neuromorphological and neurochemical systems to bring about permanent structural changes. After puberty, circulating levels of testosterone activate the male-organized brain and induce temporary functional changes in behavior.
26. A different set of temporal parameters appears to apply to female brain development. The sensitive period for permanent structural and behavioral ovarian effects ("feminization") does not end by day 10, as in the male, but extends quite late in life. For example, the critical period for feminization of sexual behavior has been shown to extend up to puberty in female rats (Gerall et al., 1973), and exposure to low doses of estrogen as late as Day 30 to 40 resulted in feminized open-field behavior in previously ovariectomized rats (Stewart & Cygan, 1980). With regard to neuroanatomy, ethinylestradiol exposure from Days 40 to 90 led to a thinner cortex in ovariectomized female rats (Pappas et al., 1979). Post-pubertal castration of male rats followed by low-dose estrogen and progesterone treatment increased the size of the AVPv, and decreased the size of other sexually dimorphic nuclei (Bloch & Gorski, 1988). Finally, ovariectomy of female rats on Day 30 prevented the female-typical decrease in cortical pyramidal dendritic spines (Munoz-Cueto et al., 1990). These findings are in contrast to the relatively early (< Day 10) sensitive period for masculinizing effects of androgen on neuromorphology and behavior in rats.
27. Recent evidence also demonstrates that onset of sensitivity to activational effects may be different in males and females. In an important study, Williams (1986) showed activational effects of estrogen on lordosis in female rat pups as young as 6 days of age. Although such early activational effects of masculinizing androgens may exist, none that we are aware of has yet been reported.
28. The work reviewed provides a strong basis for the assertion that ovarian hormones influence development of the female brain, a process which seems appropriately termed "feminization." The findings discussed here do not refute or contradict the profound evidence of androgen-mediated masculinization, but suggest that ovarian hormones may exert parallel influences on the development of brain and behavior in the female. This, in turn, compels us to broaden the concept of sexual differentiation by recognizing that both testicular and ovarian hormones are active participants. This idea was anticipated more than 10 years ago by Stewart and Cygan who wrote in 1980 that "while both testicular and ovarian hormones contribute to normal male and female behavioral development, their actions are not merely reciprocal and probably occur at different times in development" (p. 24).
29. We next review data showing that ovarian and testicular hormones each play a critical role in the growth and development of the corpus callosum of the rat.
IV.1 THE ORIGINAL FINDINGS
30. We have systematically investigated the role of neonatal gonadal hormones on callosal development in the rat, prompted by the finding that the corpus callosum is significantly larger in adult male than female Purdue-Wistar rats (Berrebi, Fitch, Ralphe, Denenberg, Friedrich & Denenberg, 1988).
31. In our initial study, entire litters of male and female pups received handling stimulation between birth and weaning, or were nonhandled controls. Handling was included because of prior data showing that this procedure affects the development of cerebral laterality and may influence callosal size as well (Denenberg, 1981). Handling consisted of removing the newborn pups from the maternity cage, leaving the mother in the cage, placing each pup into a 1 gallon can containing shavings, leaving them for 3 minutes, and returning them to the home cage (Denenberg 1977). This was done daily from Day 1 through 20, with weaning on Day 21. Subjects were then group housed with same-sexed littermates. At 110 days they were perfused, the brains were removed, and a mid-sagittal section of the callosum was obtained. Using a projection microscope the callosum was magnified and drawn. Computerized analysis of callosal size demonstrated that males had a larger absolute cross-sectional callosal area than females, particularly among handled subjects.
32. In all of our experiments we have measured and analyzed callosal area, perimeter, length, and seven regional width factors derived from prior factor analysis (Denenberg, Berrebi, & Fitch, 1989). Typically we have found that when a manipulation alters callosal size there are significant effects on callosal area, one or both of the two anterior width factors, and one or both of the two posterior width factors. For purposes of this review only the callosal area data are reported since, with one exception to be discussed below, these accurately reflect the findings from the complete data set. It is also important to note that brain weight was uncorrelated with corpus callosum (CC) area in the original study, or any study we have conducted since, allowing us to conclude that cross-sectional callosal area can be evaluated in absolute terms and does not need to be corrected for brain weight.
33. The Berrebi et al. findings demonstrated a clear sexual dimorphism in callosal size in the rat, whether handled in infancy or not. This result has since been independently replicated in conventionally reared Long-Evan rats (Zimmerberg & Scalzi, 1989; Zimmerberg & Mickus, 1990). Because it is relatively rare to find sex differences in cortex, we set out to determine whether gonadal hormones influenced the sexually dimorphic CC. Unless otherwise noted, all experiments described below were conducted with animals handled in infancy, since we wished to maximize the baseline sex differences.
IV.2 TESTOSTERONE AND CALLOSAL MASCULINIZATION
34. TESTOSTERONE ADMINISTRATION TO FEMALES. In our first hormone study we found that a single injection of 1 mg testosterone propionate (TP) administered to handled 4-day old female pups was sufficient to significantly increase their adult CC area, compared to oil-treated female littermates (Fitch, Berrebi, Cowell, Schrott & Denenberg, 1990a). Indeed, the increase was so large that the TP female CC values did not differ significantly from those of male littermates.
35. We repeated the Fitch et al. experiment with nonhandled (NH) rats, fully expecting to obtain the same effects (Denenberg, Fitch, Schrott, Cowell & Waters, 1991a). To our surprise NH females given TP on Day 4 did not have an enlarged callosum. We then did a second experiment in which we had both handled (H) and NH animals. We replicated the finding that H females given TP had an enlarged callosum, and we also replicated the finding that NH females given TP did not have an enlarged callosum (Denenberg et al., 1991a). For the two studies using handled animals, the average increase in callosal area as a consequence of the testosterone treatment on Day 4 was 6.41%. We interpreted these data as suggesting a synergy between the presence of testosterone and the effects of handling on adrenal corticosteroids (Denenberg, Brumaghim, Haltmeyer & Zarrow, 1967; Meaney, Aitken, Bhatnagar, Van Berkel & Sapolsky, 1988). The finding that TP treatment must be associated with handling in order to significantly enlarge the female's callosum suggests that the mechanism underlying this effect is more complex than simple exposure of the female to androgen, and clearly requires further experimental investigation.
36. DEVELOPMENTAL EFFECTS OF TP TREATMENT. Handled female rats were given 1 mg TP or oil on Day 4, and littermates from each condition (as well as male controls) were sacrificed at 30, 55, or 90 days (Fitch, Cowell, Schrott & Denenberg, 1990b). The callosal size of males and TP treated females was significantly greater than oil females at 30 days, and there were no significant differences between TP females and males. This pattern continued through days 55 and 90.
37. CASTRATION OF MALES. To investigate the role of endogenous testicular androgens in callosal development, we castrated handled male pups on Day 1 of life (Fitch et al., 1990a). Contrary to our expectations, the callosal size of these males was not affected in adulthood. Denenberg et al. (1991a) also failed, in two experiments, to see an effect of Day 1 castration on CC size.
38. These findings suggested that testosterone exposure in the prenatal and early (<24 hour) postnatal period had already exerted organizing effects in males. Therefore, we conducted another experiment in which an androgen receptor blocker, flutamide, was administered to pregnant dams (Fitch, Cowell, Schrott & Denenberg, 1991a). Prenatal flutamide exposure was followed by neonatal castration of male pups. All other pups (flutamide treated females and controls of both sexes) received sham surgery. In this study, unlike those described above, non-handled animals were used. Under these conditions callosal area was significantly smaller among treated males compared to control males, and did not differ from that of female littermates (Fitch et al., 1991a). These results have since been replicated with handled animals (Fitch, 1990; Fitch, Cowell, Schrott & Denenberg, in prep).
39. Zimmerberg and Scalzi (1989) have provided independent evidence that the organizing effects of testosterone on the callosum occur quite early in development. They found a sexual dimorphism in callosal size for 3-day old pups. In addition, they also found that the callosal sex difference in 3-day old pups is eliminated by prenatal exposure to alcohol, a process thought to suppress the prenatal surge of testosterone in the male fetus (McGivern, Clancy, Hill & Noble, 1988).
40. THE SENSITIVE PERIOD FOR TESTOSTERONE EFFECTS. Based on the above findings, gestational Day 17 appears to define the beginning of the sensitive period for the effect of testosterone upon cortical organization in the rat. The end of the sensitive period falls somewhere between postnatal Day 4 and Day 8, since TP administered to females on Day 4 increased callosal size (Fitch et al., 1990a), but TP administration on Day 8, 12, or 16 did not (Fitch, Cowell, Schrott & Denenberg, 1991b). This sensitive period is consistent with the reports of others. Breedlove and Arnold (1983) found that castration on Day 1 did not demasculinize the size of the bulbocavernosus spinal nucleus in male rats, whereas prenatal treatment with flutamide, combined with castration, did. Wagner and Clemens (1989) found that TP treatment to female mice on postnatal Days 1, 3, and 5, but not 7, 9, and 11, significantly increased the size of this nucleus to that of males.
IV.3 OVARIAN HORMONES AND CALLOSAL FEMINIZATION
41. TAMOXIFEN EFFECTS. The first hint that callosal size is affected by ovarian hormones was from a study in which the estrogen receptor blocker tamoxifen (TX) was given to 4-day old male and female pups. TX resulted in a near-significant (p<.06) increase in callosal area in females, but did not affect callosal size in males (Fitch et al., 1990a). In the same experiment the synthetic estrogen diethylstilbestrol (DES) was administered to males and females on Day 4. DES did not significantly affect callosal area in either sex (despite significant effects on body weight), suggesting that tamoxifen was not acting as an estrogenic agent.
42. OVARIECTOMY EFFECTS. The findings described above suggested (but did not prove) that the increase in the size of the female callosum was a consequence of temporary estrogen "removal" during development. One way to test this hypothesis was to remove estrogen via ovariectomy, and measure callosal size in adulthood. We did this experiment, and found that when neonatally handled females were ovariectomized on Day 8, 12, or 16, all three castrated groups had significantly larger callosa than sham-operated female littermates in adulthood (Fitch et al., 1991b). Further, the three groups did not differ among themselves for callosal width or area. These results suggest that the sensitive period for ovarian manipulations extends to at least Day 16, much later than for TP which was effective at Day 4 but not 8.
43. The above results have been replicated for both handled and non-handled females (Mack, Fitch, Cowell, Schrott & Denenberg, 1993; Mack, Cowell & Denenberg, 1992). For the three ovariectomy studies, the average increase in callosal area was 6.68%. Apparently, ovariectomy altered callosal size regardless of the presence or absence of handling, whereas TP effects on the female callosum were observed only in combination with handling. This distinction may relate to the later sensitive period for ovarian as compared to TP manipulations. Handling occurred from Days 1-21, which encompasses the sensitive period for testosterone action, but which may have preceded the sensitive period for the action of ovarian hormones.
44. EFFECTS OF OVARIECTOMY AND ESTROGEN REPLACEMENT. Since ovariectomy deprives the female of both estrogen and progesterone, it was not possible, based on the findings reviewed above, to determine which hormone was the effective agent in altering callosal size. We had previously found that TX resulted in an enlarged callosum, arguing for estrogen as the causal agent. We recently confirmed this interpretation by ovariectomizing females on Day 12 and giving half of them a low-dose silastic estrogen implant on Day 25 (Mack et al., 1993). In adulthood, those ovariectomized females with the implant had significantly smaller callosa than littermates who received ovariectomy only. Indeed they were smaller than intact female littermates as well. Our results are consistent with those of Pappas et al. (1979), who found that ethinylestradiol given on Days 40 to 90 had an inhibitory effect on the development of cortical thickness in Ovx females. These findings emphasize that the sensitive period for ovarian effects extends much later than that of testosterone, in this case as late as Day 25.
45. DEVELOPMENTAL EFFECTS OF OVARIAN HORMONES. In a related study on callosal development in intact and hormonal manipulated animals, we found that the effects of TP on callosal size were significant by 30 days of age (paralleling early sex effects), whereas the effects of ovariectomy were not evident until 90 days of age (Fitch et al., 1990b).
46. ELIMINATION OF ADRENAL ANDROGEN EFFECTS. To further support the assertion that ovarian hormones have direct effects on cortical differentiation, it was necessary to demonstrate that the removal of the ovaries did not lead to an increase in adrenal androgen output and hence indirect "masculinization." In fact, the opposite effect was found. Ovariectomized females secreted half as much basal androstenedione, the primary adrenal androgen, as sham-operated female littermates (Fitch, McGivern, Redei, Schrott, Cowell & Denenberg, 1992). Furthermore, ovariectomized females did not show the stress-mediated rise in androstenedione observed for intact females following exposure to a novel environment.
47. EVIDENCE THAT ESTROGEN EXERTS ORGANIZING EFFECTS. A key question is whether morphological changes in the callosum reflect permanent organizational steroid effects, or activational effects as a function of estrogen levels at the time of sacrifice. Three sets of data support the organizational hypothesis. First, the fact that effects following ovariectomy at Day 12 were not observed at 30 and 55 days of age argues against an activational mechanism, although one might counter that activational effects are often dependent on puberty, and therefore early manipulations of estrogen level may not be "activationally" expressed until postpuberty. Second, we examined intact adult female rats for phase of estrous at the time of sacrifice, and then measured ovarian weight and uterine weight in addition to our standard measures of callosal size. Although a significant effect of estrous on uterine weight was observed, no relationship between callosal size and estrous was found (Mack, Fitch & Denenberg, submitted). Third, we ovariectomized a group of females at 78 days, well after puberty, and did sham surgery on female littermate controls. At 110 days we found no significant difference between the CC of the two groups (F<1.0) (Mack, Fitch & Denenberg, submitted). These three sets of findings, in combination with the data we have reviewed, strongly favor the hypothesis that ovarian hormones, primarily estrogen, exert permanent morphological (organizational) effects upon the callosum.
48. SUMMARY. These combined findings establish that, in rats: (1) the removal of ovarian hormones in early life leads to callosal enlargement; (2) these effects can be countered by the administration of estrogen; (3) the sensitive period for this phenomenon extends at least through Day 25 of life, considerably later than for testosterone effects; (4) these findings do not reflect secondary effects on adrenal androgen output; (5) ovarian effects on the callosum do not interact with handling in the same manner as androgenic manipulations; (6) developmentally, the expression of this effect begins considerably later than that of testosterone; and (7) these are permanent (organizational) effects, not transitory (activational) ones.
49. In the human literature there have been reports of sex differences in cerebral lateralization (Kimura, 1987; Kimura & Harshman, 1984; McGlone, 1980), but the primary finding of structural sexual dimorphism in human cerebral cortex has been for the corpus callosum. The initial paper by DeLacoste-Utamsing and Holloway (1982), that the splenium of the callosum is larger in women than men, has been contradicted a number of times (e.g., Bell & Variend, 1985; Bleier, Houston & Byne, 1986; Byne, Bleier & Houston, 1986; Demeter, Ringo & Doty, 1985; Kertesz, Polk, Howell & Black, 1987; Nasrallah, Andreasen, Coffman, Olson, Dunn, Ehrhardt & Chapman, 1986; Oppenheim, Lee, Nass & Gazzaniga, 1987), although one report (Allen, Richey, Chai & Gorski, 1991) recently found the splenium to be larger in women. Alternatively, several recent and comprehensive studies found the callosum to be larger, overall, in men (Clarke, Kraftsik, Van der Loos & Innocenti, 1989; Witelson, 1989; 1991). A major reason for inconsistent reports of sex differences is that callosal region and subject age are key parameters which interact with sex to affect CC measurements (Cowell, Allen, Zalatimo, and Denenberg, 1992).
50. The finding of interactions between sex, age and callosal region is not surprising since regional interactions with some variables have also been found in the rat CC. However, in the rat CC studies reviewed here, both sex and hormone manipulations exerted such profound effects on callosal size (evident in both anterior and posterior callosal regions) that these effects were apparent by measuring overall callosal area. This allowed us to simplify our presentation by using CC area as the only dependent variable for the purpose of documenting the role of ovarian hormones upon brain development. It should be noted, however, that more subtle regional differences do exist in the rat callosum. Some are based upon anterior-posterior differences in the emergence of callosal sex differences, and others are based upon environment-hormone interactions involving early experience variables such as handling and environmental enrichment (Denenberg et al., 1991a; Fitch et al., 1990a, 1990b; Juraska & Kopcik, 1988; Mack et al., 1992 & 1993). Such regional effects are perhaps to be expected given that the callosum is a highly heterogeneous structure, encompassing fibers of passage from widely divergent areas of cortex.
51. Similarly, data obtained in the human callosal literature emphasizes the importance of regional analyses of the CC. One region in which gender effects have been reliably found is the isthmus. Witelson (1989) showed this region to be sensitive to gender when consistency of hand usage was also taken into consideration. Specifically, she found the isthmus portion of the callosum to be significantly larger in non-consistent right-handed men. This finding has recently been confirmed in two independent studies (Habib, Gayraud, Olivia, Regis, Salamon, & Khalil, 1991; Denenberg, Kertesz, & Cowell, 1991b).
52. Another approach to the study of the CC is via correlational methodology. Witelson (1991) has found structural-functional correlations for callosal measures in males but not in females, and proposed that males and females are "dichotomous," or qualitatively distinct, populations with respect to cortical organization and lateralization. Further, she suggested that the mechanisms underlying the development of cerebral laterality may be entirely different for males and females (see also McCormick, Witelson & Kingstone, 1990).
53. In summary, several key parameters have been found which interact with sex differences in the human CC: age, CC region, degree of hand preference, and direction of hand preference. Although our studies with rats have not directly addressed these same variables or their equivalents (e.g., age, paw preference, etc.), limiting our ability to draw generalizations from one dataset to the other, it is important to note that there is good evidence of sexual dimorphism in the human, as well as the rat CC. Thus it is quite likely that future studies will demonstrate hormonal effects on human CC development, as we have shown in the rat.
54. The results reviewed here prompt the consideration of feminization as a process which functions parallel to masculinization. The two processes are qualitatively different and operate during different developmental periods. In order for the brain to become sexually differentiated, males need exposure to testicular androgens during the perinatal period (roughly from embryonic day 17 through postnatal day 10 in rats), and females need exposure to ovarian secretions including, but not necessarily limited to, estrogen, during a later period that may extend to or even beyond puberty.
55. Given the presence of two processes, one must consider the extent to which they interact in vivo, particularly in an experimental condition where an intact female is treated with androgen. For example, testosterone exposure in infancy combined with handling stimulation is sufficient to enlarge the female callosum to the size of a male, yet to what extent TP affects or redirects the feminization process that normally occurs in the female is unclear. Some findings suggest that the presence of the ovaries may modify the developmental actions of androgens (e.g., Blizard & Denef, 1973). Alternately, exposure to androgens may alter the activity of the ovaries themselves, greatly confounding the interpretation of endogenous processes (e.g., Barraclough, 1961). Consequently, one must consider the implications of these findings for the common research practice of using intact female "controls" in hormonal manipulation experiments. Although we originally based our conclusions about the actions of androgen on the sexual differentiation of the callosum on comparisons between TP-treated intact and oil-treated intact females, it is possible that this comparison has limited validity. It is unclear to what extent endogenous ovarian hormones interact with exogenous hormonal manipulations. Certainly in future research ovariectomized females should be used as a baseline control to assess developmental hormonal effects, as gonadectomized males are the common control for hormonal manipulations in the male.
56. The data presented here support the concept that ovarian hormones play an important role in the development of the female brain and that the temporal parameters and mechanisms of "ovarian feminization" are markedly different from those of androgenic masculinization. Such findings speak to the need to complement our current model of androgen-mediated sexual differentiation of the brain with what is now known about the parallel role of the ovaries.
ACKNOWLEDGMENTS: The authors would like to express thanks to Patty Cowell, Lisa Schrott, and Christine Mack, whose contributions to the projects described here are innumerable.
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