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The prenatal stress syndrome: Current status
Psyclloneuroendocrinology, Vol. 9,
No. 1, pp. 3 - 1 I, 1984.
0306 - 4530/84 $3.00 + 0.00 © 1984 PergamonPressLid.
Printed in Great Britain.
THE
PRENATAL
STRESS
SYNDROME:
CURRENT
STATUS*
INGEBORG L. WARD Department of Psychology,Villanova University, Villanova, PA 19085, U.S.A. (Received 27 July 1983)
SUMMARY Exposure of female rats to stressors during the last week of pregnancy results in a selective feminization and demasculinization of adult sexual behaviors in the male offspring. No behavioral abnormalities are detectable in the female offspring, and reproductive morphological structures appear normal in both sexes. Existing data suggest that the mechanism mediating the so called Prenatal Stress Syndromein male rats is an alteration in fetal testicular enzyme,activity. This, in turn, leads to abnormal levels of testosterone, the hormone believed to masculinize sexual behavior potentials at critical stages of perinatal development. Specifically, the activity of the steroidogenic enzymeAS-3lS-hydroxysteroiddehydrogenasein fetal Leydigcells and plasma titers of testosterone are low in prenatally stressed males on days 18 and 19 of gestation, a time when both of these substances reach maximal levels in control males. The implications of this model for sexual behavior differentiation in higher organisms is explored. IT HAS been shown in several mammalian species that the differentiation o f normal male sexual behaviors requires exposure to adequate amounts o f androgen during very specific stages o f perinatal development (see reviews by Baum, 1979; Goy & McEwen, 1980; Ward & Ward, 1984). This principle has been demonstrated in laboratory animals by surgical or chemical castration o f fetal or, in some species, neonatal males. As adults, such males show a feminization and demasculinization in reproductive morphology and behavior which is directly proportional to the timing and extent to which androgen was withheld during perinatal life. The degree to which such hormonal mechanisms generated on lower species generalize to explain intersexed human behavior has been the subject of considerable speculation, particularly in recent years. In all likelihood this problem will remain in the realm of speculation for the forseeable future, since controlled experiments comparable to those done with animals do not exist for humans and, from a practical and ethical point o f view, are unlikely to be generated. The present paper makes no attempt to resolve this fundamental problem, but it does describe yet another animal model which may or may not turn out to have clinical significance. The unique contribution of this rat model lies in its demonstration that maternal, stress during pregnancy can have profound effects on the sexual behavior o f the male offspring, while leaving gross reproductive morphology unaltered. In 1972 it was discovered that the male offspring o f rats stressed during pregnancy have a reduced potential for the male ejaculatory pattern, while at the same time showing abnormally high levels of female lordotic behavior (Ward, 1972). These behavioral alterations have collectively been termed the Prenatal Stress Syndrome. What follows is a *Presented in part at the XIV International Congress of the International Society of Psychoneuroendocrinology, New York City, 12- 16 June 1983.
4
IN(;EB()R(; l,. WAR[)
brief description of some of the sexually dimorphic behaviors which characterize prenatally stressed males and of what is known about the physiological mechanism that may underlie the etiology of this syndrome. The standard stress treatment utilized in my laboratory consists of a combination of restraint and light. Beginning on day 14 of gestation and continuing through day 21, pregnant rats are placed 3 times daily for 45 min into Plexiglas restrainers illuminated by floodlights delivering approximately 200 foot-candles of light. To verify that this treatment is stressful, plasma levels of corticosterone were measured in unstressed mothers and their male and female fetuses, as well as in mothers and fetuses killed in the middle of a stress session on days 18 and 21 of gestation (Ward & Weisz, 1984). As seen in Fig. 1, both mothers and their fetuses, when killed in the middle of a treatment session, have markedly elevated corticosterone levels compared to unstressed controls. Thus this treatment, imposed on the pregnant rat, stresses not only her but is somehow transmitted to her fetuses. CONTROL
sTRE. RRLE 480
C 0 R T I C 0 S T E R
440-
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]
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HOTHER
!--1
400-
360-
0 N E
320-
N 6 /
Z80-
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240-
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FEHRLE
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L 200" R S m n
160-
IB
21
O~Y OF G E S T B T I O N FiG. 1. Plasma corticosterone levels of 18 and 21 day pregnant rats and their male and female fetuses. The bloods were taken from untreated animals (control) and from animals sacrificed in the middle of a stress session (stress). Adapted from Ward & Weisz (in press).
The lasting consequences of this maternal treatment on the development o f the fetuses are most discernible when the latter reach adulthood. When tested, it becomes immediately obvious that few of the mate offspring of females stressed during pregnancy
PRENATAL STRESSSYNDROME
5
are able to display normal copulatory behavior. Not only is there a marked reduction in the percentage of males that ejaculate, but most fail even to initiate copulation, even though given repeated access to estrous females. If these same animals are gonadectomized and injected with dosages of estrogen and progesterone sufficient to activate estrous behavior in female rats, they show much higher levels of the female lordosis posture than do normal males (Ward, 1972; 1977). Figure 2 depicts the characteristic lordotic pattern exhibited by prenatally stressed males when mounted by a vigorous stud male. If the stress treatment is delayed until birth, the males are behaviorally normal. Neonatal male rats exposed to a variety of stressors including handling, cold, or vibration copulate normally and show no enhanced bisexual potentials. Variations of this basic observation have now been documented in at least seven laboratories, and the types of treatments shown to induce components of the prenatal stress syndrome have been extended to exposure of pregnant mothers to social crowding, malnutrition, or a conditioned emotional response, as well as the standard restraint and light procedure already described (Ward, 1972; 1977; Herrenkohl & Whitney, 1976; Masterpasqua et al., 1976; DahlOf et al., 1977; Dunlap et al., 1978; GOtz & DOrner, 1980; Rhees & Fleming, 1981).
FXG.2. Femalelordosisposture shownby a prenatallystressedmalerat followingcastrationand treatmentwith estrogen and progesterone. The female littermates of these males also have been studied at great length. In our hands, prenatally stressed females are normal with regard to sexual behavior, ability to reproduce, levels of maternal behavior, and their overall success in rearing young (Ward, 1974; Meisel, 1980; Beckhardt & Ward, 1983). Their male siblings of course are profoundly affected.
6
|NGEBORGL. WARD
Since the original study, we have tried several different therapies to determine whether the impairment in ejaculatory behavior found in prenatally stressed males could be overcome. In one attempt, prenatally stressed males that failed to copulate spontaneously were injected daily with testosterone propionate (TP) (1 mg) for six weeks (Ward, 1977). This treatment activated ejaculatory behavior in approximately 23°70 of these previous noncopulators. Nevertheless, even after such extensive androgen treatment, significantly fewer stressed (46070) than control (77070) males ejaculated. Paradoxically, if the animals were tested with a vigorous stud male while receiving testosterone therapy, 62070 of the prenatally stressed males responded with the female lordosis pattern. No control males emitted this behavior under identical testing conditions. Closer inspection revealed that a larger percentage of stressed males showed lordosis following treatment with TP than showed ejaculatory behavior. The androgen treatment divided the prenatally treated males into four distinct subgroups, the relative incidences of which are shown in Fig. 3. A few males were normal in that they spontaneously ejaculated and showed no lordosis behavior. A larger proportion evidenced lordosis when treated with gonadal hormones but did not ejaculate. A very small percentage seemed to be totally asexual, showing neither male nor female behavior. Finally, some animals were potentially bisexual and eventually evidenced male behavior following prolonged androgen treatment. However, this category of prenatally stressed males was also fully capable of showing lordosis. Following activation of the bisexual potential by TP injections, these males would alter the behavioral pattern they were emitting to be appropriate for the sexual partner with whom they were paired. They mounted a receptive female but, within minutes, lordosed if placed with a vigorous stud male. The direction of the behavior emitted by the bipotential animals was entirely determined by the stimulus animal. Along with this description of the dimensions in which prenatally stressed males deviate from controls, it becomes important to point out that in other ways they appear to be I00.
Control
Stress 80.
P" Z 60W 40"
20" 0
Mole Only
Bisexuot F e m a l e Only
Asexuol
BEHAVIORAL POTENTIAL
FIG. 3. The relativepercentageof control and prenatallystressedmale rats showingonlyejaculatorybehavior (maleonly),onlylordosisbehavior(femaleonly),both patterns(bisexual),or neitherpattern( ~ u a l ) f o l ~ n g prolonged treatmentwith testosteronepropionate, n = 13 per group. Adapted from Ward(1977).
PRENATAL STRESS SYNDROME
7
quite normal. Typically in experimental studies in which androgen levels are manipulated during fetal life, alterations in reproductive morphology as well as behavior result. It thus seemed likely that the feminization and demasculinization of behavior found in prenatally stressed male rats would be accompanied by concomitant changes in standard measures of androgen-dependent morphology. Thus far, this has not turned out to be the case (Ward, 1977). Body and testis weight and ano-genital distance typically are reduced if measured at birth (Dahl6f et al., 1978; B. Ward, unpublished observations), but if the measures are repeated in adulthood, stressed and control males do not differ. Furthermore, stressed and control males are not distinguishable with regard to the size of such androgen dependent structures as the penis, testes or epididymi. Furthermore, histology of the adult testes reveals that prenatally stressed males produce sperm in adulthood. Thus stressed males cannot be distinguished from normal males on the basis of morphology. Exposure to stress during fetal life seems to alter certain sexually dimorphic behaviors while leaving morphology unaffected. However, not all sexually dimorphic behaviors are feminized. A variety of behaviors other than those involved in copulation have been identified as differing in amount and intensity between male and female rats (see review by Beatty, 1979). These include levels of aggression, patterns of juvenile play and performance in an open field. The one most thoroughly investigated in stressed males is active avoidance responding, a task in which female rats excel compared to males (Meisel et al., 1979). Stressed and control males and females were trained in a standard two-way shuttle box. If the animal moved from one compartment to the other within five seconds of the onset of dark (CS) in the occupied compartment it avoided foot shock (UCS). The performance of the four groups on 150 trials distributed evenly across six consecutive days indicated that prenatal stress did not feminize this behavior. While both female groups learned and performed the task at a higher rate than did males, prenatally stressed males were not different from control males. To summarize, the male offspring of female rats stressed during the last week of pregnancy generally are unable to ejaculate spontaneously. A few will copulate in response to various therapies, such as prolonged androgen treatment or cohabitation with a female for several weeks, but regardless of their eventual ability to ejaculate, most show high levels of lordotic behavior. However, the quality of this pattern is not identical to that of an estrous female. As seen in Fig. 2, prenatally stressed males lordose when mounted by a vigorous male, and some will even briefly hold the lordosis once the stimulus animal has dismounted. However, while estrous female rats hop, dart, and earwiggle to promote mounting by a vigorous male, stressed males lack the ability to display any of the soliciting components of the female rat's estrous behavior pattern. Thus, as their performance in the active avoidance task also indicated, the feminization of behavioral potentials is only partial. Our working hypothesis about the etiology of the prenatal stress syndrome is that it stems from the same hormonal mechanism underlying sexual behavior differentiation in both normal males and females. As mentioned earlier, the normal masculinization and defeminization of sexually dimorphic behaviors requires exposure to adequate amounts of androgenic steroids during specific stages of perinatal development. To determine whether induction of the Prenatal Stress Syndrome involved abnormalities in testicular
8
INGEBORG L. WARD
hormones, we measured plasma testosterone levels in fetal males and females taken from control and stressed mothers on the days of gestation during which sexual differentiation is thought to occur in the rat (Ward & Weisz, 1980; Weisz & Ward, 1980; Ward & Weisz, 1984). This revealed a number of interesting features. To begin with, plasma testosterone titers in normal male and female rat fetuses are significantly different only after day 17 of gestation. Between days 17 and 18 postconception, males, but not females, experience a sharp surge in testosterone which lasts for two days. This is followed by a drop to stable levels, which are only slightly higher than those of female siblings. This characteristic pattern was totally altered by prenatal stress. Stressed males had prominently elevated levels of testosterone on day 17 of gestation, a time when control males were not different from control females. This was followed by a sharp decline on days 18 and 19, which contrasted with the surge in testosterone shown by control males. After that time, the two groups were not different. We have examined several other physiological parameters whose patterns of functioning during perinatal life have been implicated in the process of sexual behavior differentiation. One of these is the activity of key steroidogenic enzymes utilized by fetal Leydig cells in the synthesis of testosterone. Using quantitative cytochemistry, we have characterized the ontogenic pattern of one of these, AL313-hydroxysteroid dehydrogenase (Orth et ai., 1983). As can be seen in Fig. 4, the difference between stressed and control males in the pattern of this enzyme is very similar to that described for blood levels of testosterone. This suggests that the abnormal titers of testosterone in the circulation are the direct consequence of alterations in the steroidogenic activity of the fetal gonads. Finally, it is becoming increasingly more likely that the aromatization of androgen to estrogen may be required for proper masculinization of the nervous system, at least in the rat. Figure 5 demonstrates that prenatally stressed rats have significantly lower levels of brain aromatase activity on days 18, 19, and 20 of gestation than do control animals (Weisz e t a / . , 1982). The possible contribution this abnormality makes to the behavioral syndrome shown by the males remains to be elucidated. 55 50 ¸
S
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DAYS POST-CONCEPTION
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FIG. 4. The activity of AL3l~-hydroxysteroid dehydrogenase in individual Leydig cells of rat fetuses f r o m 16 to 21 days of gestation. Each point represents the mean for a single litter. The total number of fetuses used in each group is given in parentheses. From Orth e t aL (1983).
PRENATAL STRESS SYNDROME m | !
9
(47)
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~CONTROL 1
,,o
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711'
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(27) FIG. 5. Mean (_+S.E.M.) steroid aromatase activity of combined hypothalamic-amygdaloid tissue fractions taken from control and stressed rat fetuses betweendays 17 and 21 of gestation. The number of subjects in each group is given in parentheses. From Weisz et al. (1982). Taken together, these data provide compelling evidence that the partial modification of sexually dimorphic behaviors seen in prenatally stressed male rats results from a failure to experience the normal surge in testosterone which should take place on days 18 and 19 of gestation. If CNS components critical to adult sexual behavior begin to differentiate on these days, then in females and stressed males they do so without benefit of the testosterone surge characterizing control males. The Prenatal Stress Syndrome has provided a very useful tool for studying the mechanisms by which normal sexual differentiation are achieved. Further, it has revealed a variety of ways in which environmental stressors acting on pregnant dams can alter the hormonal milieu in which their fetuses differentiate, thereby affecting the reproductive behavior potentials o f the next generation of male rodents. Whether or not this model holds as one moves up the phylogenetic scale to primates remains to be determined. The optimistic conclusions o f D0rner et al. that this syndrome provides a direct explanation o f homosexuality in human males (D0rner, 1980; 1981) should be greeted with some caution. Not only is it a fundamental precept in science to demand conclusive data rather than to accept untested generalizations, but there also are known differences among various animal species in the mechanisms underlying sexual behavior differentiation, particularly between primates and rodents. For example, exposure of neonatal female rats to the nonaromatizable androgen dihydrotestosterone (DHT) results in the masculinization of reproductive morphology, but sexually dimorphic behaviors differentiate as in normal females (Luttge & Whalen, 1970; McDonald & Doughty, 1974; Korenbrot et al., 1975). Comparable treatment o f fetal female rhesus monkeys causes severe masculinization of
10
INGEBORG L. WARD
both morphology and various behaviors, including copulation and a wide array of social patterns (Goy, 1981; G o y & R o b i n s o n , 1982; T h o r n t o n & Goy, unpublished o b s e r v a t i o n s ) . T h i s h a s b e e n t a k e n as o n e l i n e o f e v i d e n c e i n d i c a t i n g t h a t in r a t s t e s t o s t e r o n e f u n c t i o n s a s a p r o h o r m o n e a n d t h a t it is its a r o m a t i z a t i o n p r o d u c t , e s t r a d i o l , that actively mediates the masculinization and defeminization of behavioral potentials d u r i n g p e r i n a t a l d e v e l o p m e n t . I n p r i m a t e s , t h e a r o m a t i z a t i o n s t e p is n o t r e q u i r e d . T h u s , a t t h i s p o i n t in t i m e , t h e P r e n a t a l S t r e s s S y n d r o m e is i n t e r e s t i n g a n d s u g g e s t i v e , b u t it r e m a i n s t o b e d e m o n s t r a t e d w h e t h e r it h a s a n y c l i n i c a l s i g n i f i c a n c e . The research reported in this paper has been supported by Grant HD04688 from the NICHHD and by NIMH Research Scientist Development Award II l-K2-MH00049 (to I. L. W.) and by Grant HD009542 to Dr. Judith Weisz of the Hershey Medical Center.
REFERENCES
BAUM,M. J. (1979) Differentiation of coital behavior in mammals: a comparative analysis. Neurosci. Biobehav. Rev. 3, 2 6 5 - 284. BEATTY, W. W. (1979) Gonadal hormones and sex differences in nonreproductive behaviors in rodents: organizational and activational influences. Horm. Behav. 12, 112- 163. BECKHARDT, S. & WARD, I. L. (1983) Reproductive functioning in the prenatally stressed female rat. Devl. PsychobioL 16, 111 - 118. DAHLOF, L. G., I-I~RD, E. & LARSSON, K. (1977) Influence of maternal stress on offspring sexual behavior. Anim. Behav. 25, 193- 195. DAHLOF, L. G., H)iRD, E. & LARSSON, K. (1978) Influence of maternal stress on the development of the fetal genital system. Physiol. Behav. 20, 193 - 195. DORNER, G. (1980) Sexual differentiation of the brain. Vitamins Horm. 38, 3 2 5 - 381. DORNER, G. (1981) Sex hormones and neurotransmitters as mediators for sexual differentiation of the brain. Endokrinologie 78, 129- 138. DUNLAP, J: L., ZADINA, J. E. & GOUGIS, G. (1978) Prenatal stress interacts with prepubertal social isolation to reduce male copulatory behavior. Physiol. Behav. 21,873 -875. GOTZ, F. & DONNER, G. (1980) Homosexual behaviour in prenatally stressed male rats after castration and oestrogen treatment in adulthood. Endokrinologie 76, 115 - ! 17. Gov, R. W. & MCEWEN, B. S. (1980) Sexual Differentiation of the Brain. MIT Press, Cambridge. Gov, R. W. (1981) Differentiation of male social traits in female rhesus macaques by prenatal treatment with androgens: variation in type of androgen, duration, and timing of treatment. In Fetal Endocrinology, M. J. Novy and J. A. Resko (Eds.), pp. 319-339. Academic Press, New York. CoY, R. W. & ROBINSON, J. A. (1982) Prenatal exposure of rhesus monkeys to potent androgens: morphological, behavioral and physiological consequences. Banbury Report I1. Environmental Factors in Human Growth and Development. Cold Spring Harbor Laboratory, pp. 355 - 378. HERRENKOHL,L. R. & WHITNEY,J. B. (1976) Effects of prepartal stress on postpartai nursing behavior, litter development and adult sexual behavior. Physiol. Behav. 17, 1019- 1021. KORENBROT, C. C., PAUP, D. C. & CoRSKI, R. A. (1975) Effects of testosterone propionate or dihydrotestosterone propionate on plasma FSH and LH levels in neonatal rats and on sexual differentiation of the brain. Endocrinology 97, 7 0 9 - 717. LUTTGE, W. G. & WHALEN, R. E. (1970) Dihydrotestosterone, androstenedione, testosterone: comparative effectiveness in masculinizing and defeminizing reproductive systems in male and female rats. Horm. Behav. l, 265 - 281. MASTERPASQUA,F., CHAPMAN, R. H. & LORE, R. K. (1976) The effects of prenatal psychological stress on the sexual behavior and reactivity of male rats. DevL Psychobiol. 9, 403 - 41 I. MCDONALD, P. G. & DOUGHTY,C. (1974) Effects of neonatal administration of different androgens in the female rat: correlation between aromatization and the induction of sterilization. J. Endocr. 61, 9 5 - 103. MEISEL, R. L., DOHANICH, G. P. & WARD, I. L. (1979) Effects of prenatal stress on avoidance acquisition, open-field performance and lordotic behavior in male rats. Physiol. Behav. 22, 527 - 530. MEISEL, R. L. (1980) Effects of prenatal stress and uterine position on the sexual behavior and morphological differentiation of male and female rats. M.S. thesis, Villanova University, Villanova, PA.
PRENATAL STRESS SYNDROME
11
ORTH, J. M., WEISZ, J., WARD, O. B. & WARD, I. L. (1983) Environmental stress alters the developmental pattern of A~-3[3-hydroxysteroid dehydrogenase activity in Leydig cells of fetal rats: a quantitative cytochemical study. Biol. Reprod. 28, 6 2 5 - 631. RHEES, R. W. & FLEMING, D. E. (1981) Effects of malnutrition, maternal stress, or ACTH injections during pregnancy on sexual behavior of male offspring. Physiol. Behav. 27, 879 - 882. WARD, 1. L. (1972) Prenatal stress feminizes and demasculinizes the behavior of males. Science |7S, 82 - 84. WARD, I. L. (19"/4) Sexual behavior differentiation: prenatal hormonal and environmental control. In Sex Differences in Behavior, R. C. Friedman, R. M. Richart and R. L. Vande Wiele (Eds.), pp. 3 - 17. John Wiley, New York. WARD, I. L. (1977) Exogenous androgen activates female sexual behavior in noncopulating prenatally stressed males. J. comp. physiol. Psychol. 9 1 , 4 6 5 - 4 7 1 . WARD, I. L. & WARD, O. B. (1984) Sexual behavior differentiation: effects of prenatal manipulations in rats. Handbook of Behavioral Neurobiology, Vol. 8, Neurobiology of Reproduction, N. Adler and D. Pfaff (Eds.). Plenum Press, New York (in press). WARD, I. L. & WEISZ, J. (1980) Maternal stress alters plasma testosterone in fetal males. Science 207, 328 - 329. WARD, I. L. & WEISZ, J. (1984) Differential effects of maternal stress on circulating levels of corticosterone, progesterone, and testosterone in male and female rat fetuses and their mother. Endocrinology (in press). WEISZ, J. & WARD, 1. L. (1980) Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology 106, 3 0 6 - 316. WEISZ, J., BROW~, B. L. & WARD, I. L. (1982) Maternal stress decreases steroid aromatase activity in brains of male and female rat fetuses. Neuroendocrinology 35, 374 - 379.
No. 1, pp. 3 - 1 I, 1984.
0306 - 4530/84 $3.00 + 0.00 © 1984 PergamonPressLid.
Printed in Great Britain.
THE
PRENATAL
STRESS
SYNDROME:
CURRENT
STATUS*
INGEBORG L. WARD Department of Psychology,Villanova University, Villanova, PA 19085, U.S.A. (Received 27 July 1983)
SUMMARY Exposure of female rats to stressors during the last week of pregnancy results in a selective feminization and demasculinization of adult sexual behaviors in the male offspring. No behavioral abnormalities are detectable in the female offspring, and reproductive morphological structures appear normal in both sexes. Existing data suggest that the mechanism mediating the so called Prenatal Stress Syndromein male rats is an alteration in fetal testicular enzyme,activity. This, in turn, leads to abnormal levels of testosterone, the hormone believed to masculinize sexual behavior potentials at critical stages of perinatal development. Specifically, the activity of the steroidogenic enzymeAS-3lS-hydroxysteroiddehydrogenasein fetal Leydigcells and plasma titers of testosterone are low in prenatally stressed males on days 18 and 19 of gestation, a time when both of these substances reach maximal levels in control males. The implications of this model for sexual behavior differentiation in higher organisms is explored. IT HAS been shown in several mammalian species that the differentiation o f normal male sexual behaviors requires exposure to adequate amounts o f androgen during very specific stages o f perinatal development (see reviews by Baum, 1979; Goy & McEwen, 1980; Ward & Ward, 1984). This principle has been demonstrated in laboratory animals by surgical or chemical castration o f fetal or, in some species, neonatal males. As adults, such males show a feminization and demasculinization in reproductive morphology and behavior which is directly proportional to the timing and extent to which androgen was withheld during perinatal life. The degree to which such hormonal mechanisms generated on lower species generalize to explain intersexed human behavior has been the subject of considerable speculation, particularly in recent years. In all likelihood this problem will remain in the realm of speculation for the forseeable future, since controlled experiments comparable to those done with animals do not exist for humans and, from a practical and ethical point o f view, are unlikely to be generated. The present paper makes no attempt to resolve this fundamental problem, but it does describe yet another animal model which may or may not turn out to have clinical significance. The unique contribution of this rat model lies in its demonstration that maternal, stress during pregnancy can have profound effects on the sexual behavior o f the male offspring, while leaving gross reproductive morphology unaltered. In 1972 it was discovered that the male offspring o f rats stressed during pregnancy have a reduced potential for the male ejaculatory pattern, while at the same time showing abnormally high levels of female lordotic behavior (Ward, 1972). These behavioral alterations have collectively been termed the Prenatal Stress Syndrome. What follows is a *Presented in part at the XIV International Congress of the International Society of Psychoneuroendocrinology, New York City, 12- 16 June 1983.
4
IN(;EB()R(; l,. WAR[)
brief description of some of the sexually dimorphic behaviors which characterize prenatally stressed males and of what is known about the physiological mechanism that may underlie the etiology of this syndrome. The standard stress treatment utilized in my laboratory consists of a combination of restraint and light. Beginning on day 14 of gestation and continuing through day 21, pregnant rats are placed 3 times daily for 45 min into Plexiglas restrainers illuminated by floodlights delivering approximately 200 foot-candles of light. To verify that this treatment is stressful, plasma levels of corticosterone were measured in unstressed mothers and their male and female fetuses, as well as in mothers and fetuses killed in the middle of a stress session on days 18 and 21 of gestation (Ward & Weisz, 1984). As seen in Fig. 1, both mothers and their fetuses, when killed in the middle of a treatment session, have markedly elevated corticosterone levels compared to unstressed controls. Thus this treatment, imposed on the pregnant rat, stresses not only her but is somehow transmitted to her fetuses. CONTROL
sTRE. RRLE 480
C 0 R T I C 0 S T E R
440-
FEHflLE
I
]
HOTNER
HOTHER
!--1
400-
360-
0 N E
320-
N 6 /
Z80-
N L
240-
HRLE -I
FEHRLE
P
L 200" R S m n
160-
IB
21
O~Y OF G E S T B T I O N FiG. 1. Plasma corticosterone levels of 18 and 21 day pregnant rats and their male and female fetuses. The bloods were taken from untreated animals (control) and from animals sacrificed in the middle of a stress session (stress). Adapted from Ward & Weisz (in press).
The lasting consequences of this maternal treatment on the development o f the fetuses are most discernible when the latter reach adulthood. When tested, it becomes immediately obvious that few of the mate offspring of females stressed during pregnancy
PRENATAL STRESSSYNDROME
5
are able to display normal copulatory behavior. Not only is there a marked reduction in the percentage of males that ejaculate, but most fail even to initiate copulation, even though given repeated access to estrous females. If these same animals are gonadectomized and injected with dosages of estrogen and progesterone sufficient to activate estrous behavior in female rats, they show much higher levels of the female lordosis posture than do normal males (Ward, 1972; 1977). Figure 2 depicts the characteristic lordotic pattern exhibited by prenatally stressed males when mounted by a vigorous stud male. If the stress treatment is delayed until birth, the males are behaviorally normal. Neonatal male rats exposed to a variety of stressors including handling, cold, or vibration copulate normally and show no enhanced bisexual potentials. Variations of this basic observation have now been documented in at least seven laboratories, and the types of treatments shown to induce components of the prenatal stress syndrome have been extended to exposure of pregnant mothers to social crowding, malnutrition, or a conditioned emotional response, as well as the standard restraint and light procedure already described (Ward, 1972; 1977; Herrenkohl & Whitney, 1976; Masterpasqua et al., 1976; DahlOf et al., 1977; Dunlap et al., 1978; GOtz & DOrner, 1980; Rhees & Fleming, 1981).
FXG.2. Femalelordosisposture shownby a prenatallystressedmalerat followingcastrationand treatmentwith estrogen and progesterone. The female littermates of these males also have been studied at great length. In our hands, prenatally stressed females are normal with regard to sexual behavior, ability to reproduce, levels of maternal behavior, and their overall success in rearing young (Ward, 1974; Meisel, 1980; Beckhardt & Ward, 1983). Their male siblings of course are profoundly affected.
6
|NGEBORGL. WARD
Since the original study, we have tried several different therapies to determine whether the impairment in ejaculatory behavior found in prenatally stressed males could be overcome. In one attempt, prenatally stressed males that failed to copulate spontaneously were injected daily with testosterone propionate (TP) (1 mg) for six weeks (Ward, 1977). This treatment activated ejaculatory behavior in approximately 23°70 of these previous noncopulators. Nevertheless, even after such extensive androgen treatment, significantly fewer stressed (46070) than control (77070) males ejaculated. Paradoxically, if the animals were tested with a vigorous stud male while receiving testosterone therapy, 62070 of the prenatally stressed males responded with the female lordosis pattern. No control males emitted this behavior under identical testing conditions. Closer inspection revealed that a larger percentage of stressed males showed lordosis following treatment with TP than showed ejaculatory behavior. The androgen treatment divided the prenatally treated males into four distinct subgroups, the relative incidences of which are shown in Fig. 3. A few males were normal in that they spontaneously ejaculated and showed no lordosis behavior. A larger proportion evidenced lordosis when treated with gonadal hormones but did not ejaculate. A very small percentage seemed to be totally asexual, showing neither male nor female behavior. Finally, some animals were potentially bisexual and eventually evidenced male behavior following prolonged androgen treatment. However, this category of prenatally stressed males was also fully capable of showing lordosis. Following activation of the bisexual potential by TP injections, these males would alter the behavioral pattern they were emitting to be appropriate for the sexual partner with whom they were paired. They mounted a receptive female but, within minutes, lordosed if placed with a vigorous stud male. The direction of the behavior emitted by the bipotential animals was entirely determined by the stimulus animal. Along with this description of the dimensions in which prenatally stressed males deviate from controls, it becomes important to point out that in other ways they appear to be I00.
Control
Stress 80.
P" Z 60W 40"
20" 0
Mole Only
Bisexuot F e m a l e Only
Asexuol
BEHAVIORAL POTENTIAL
FIG. 3. The relativepercentageof control and prenatallystressedmale rats showingonlyejaculatorybehavior (maleonly),onlylordosisbehavior(femaleonly),both patterns(bisexual),or neitherpattern( ~ u a l ) f o l ~ n g prolonged treatmentwith testosteronepropionate, n = 13 per group. Adapted from Ward(1977).
PRENATAL STRESS SYNDROME
7
quite normal. Typically in experimental studies in which androgen levels are manipulated during fetal life, alterations in reproductive morphology as well as behavior result. It thus seemed likely that the feminization and demasculinization of behavior found in prenatally stressed male rats would be accompanied by concomitant changes in standard measures of androgen-dependent morphology. Thus far, this has not turned out to be the case (Ward, 1977). Body and testis weight and ano-genital distance typically are reduced if measured at birth (Dahl6f et al., 1978; B. Ward, unpublished observations), but if the measures are repeated in adulthood, stressed and control males do not differ. Furthermore, stressed and control males are not distinguishable with regard to the size of such androgen dependent structures as the penis, testes or epididymi. Furthermore, histology of the adult testes reveals that prenatally stressed males produce sperm in adulthood. Thus stressed males cannot be distinguished from normal males on the basis of morphology. Exposure to stress during fetal life seems to alter certain sexually dimorphic behaviors while leaving morphology unaffected. However, not all sexually dimorphic behaviors are feminized. A variety of behaviors other than those involved in copulation have been identified as differing in amount and intensity between male and female rats (see review by Beatty, 1979). These include levels of aggression, patterns of juvenile play and performance in an open field. The one most thoroughly investigated in stressed males is active avoidance responding, a task in which female rats excel compared to males (Meisel et al., 1979). Stressed and control males and females were trained in a standard two-way shuttle box. If the animal moved from one compartment to the other within five seconds of the onset of dark (CS) in the occupied compartment it avoided foot shock (UCS). The performance of the four groups on 150 trials distributed evenly across six consecutive days indicated that prenatal stress did not feminize this behavior. While both female groups learned and performed the task at a higher rate than did males, prenatally stressed males were not different from control males. To summarize, the male offspring of female rats stressed during the last week of pregnancy generally are unable to ejaculate spontaneously. A few will copulate in response to various therapies, such as prolonged androgen treatment or cohabitation with a female for several weeks, but regardless of their eventual ability to ejaculate, most show high levels of lordotic behavior. However, the quality of this pattern is not identical to that of an estrous female. As seen in Fig. 2, prenatally stressed males lordose when mounted by a vigorous male, and some will even briefly hold the lordosis once the stimulus animal has dismounted. However, while estrous female rats hop, dart, and earwiggle to promote mounting by a vigorous male, stressed males lack the ability to display any of the soliciting components of the female rat's estrous behavior pattern. Thus, as their performance in the active avoidance task also indicated, the feminization of behavioral potentials is only partial. Our working hypothesis about the etiology of the prenatal stress syndrome is that it stems from the same hormonal mechanism underlying sexual behavior differentiation in both normal males and females. As mentioned earlier, the normal masculinization and defeminization of sexually dimorphic behaviors requires exposure to adequate amounts of androgenic steroids during specific stages of perinatal development. To determine whether induction of the Prenatal Stress Syndrome involved abnormalities in testicular
8
INGEBORG L. WARD
hormones, we measured plasma testosterone levels in fetal males and females taken from control and stressed mothers on the days of gestation during which sexual differentiation is thought to occur in the rat (Ward & Weisz, 1980; Weisz & Ward, 1980; Ward & Weisz, 1984). This revealed a number of interesting features. To begin with, plasma testosterone titers in normal male and female rat fetuses are significantly different only after day 17 of gestation. Between days 17 and 18 postconception, males, but not females, experience a sharp surge in testosterone which lasts for two days. This is followed by a drop to stable levels, which are only slightly higher than those of female siblings. This characteristic pattern was totally altered by prenatal stress. Stressed males had prominently elevated levels of testosterone on day 17 of gestation, a time when control males were not different from control females. This was followed by a sharp decline on days 18 and 19, which contrasted with the surge in testosterone shown by control males. After that time, the two groups were not different. We have examined several other physiological parameters whose patterns of functioning during perinatal life have been implicated in the process of sexual behavior differentiation. One of these is the activity of key steroidogenic enzymes utilized by fetal Leydig cells in the synthesis of testosterone. Using quantitative cytochemistry, we have characterized the ontogenic pattern of one of these, AL313-hydroxysteroid dehydrogenase (Orth et ai., 1983). As can be seen in Fig. 4, the difference between stressed and control males in the pattern of this enzyme is very similar to that described for blood levels of testosterone. This suggests that the abnormal titers of testosterone in the circulation are the direct consequence of alterations in the steroidogenic activity of the fetal gonads. Finally, it is becoming increasingly more likely that the aromatization of androgen to estrogen may be required for proper masculinization of the nervous system, at least in the rat. Figure 5 demonstrates that prenatally stressed rats have significantly lower levels of brain aromatase activity on days 18, 19, and 20 of gestation than do control animals (Weisz e t a / . , 1982). The possible contribution this abnormality makes to the behavioral syndrome shown by the males remains to be elucidated. 55 50 ¸
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FIG. 4. The activity of AL3l~-hydroxysteroid dehydrogenase in individual Leydig cells of rat fetuses f r o m 16 to 21 days of gestation. Each point represents the mean for a single litter. The total number of fetuses used in each group is given in parentheses. From Orth e t aL (1983).
PRENATAL STRESS SYNDROME m | !
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(27) FIG. 5. Mean (_+S.E.M.) steroid aromatase activity of combined hypothalamic-amygdaloid tissue fractions taken from control and stressed rat fetuses betweendays 17 and 21 of gestation. The number of subjects in each group is given in parentheses. From Weisz et al. (1982). Taken together, these data provide compelling evidence that the partial modification of sexually dimorphic behaviors seen in prenatally stressed male rats results from a failure to experience the normal surge in testosterone which should take place on days 18 and 19 of gestation. If CNS components critical to adult sexual behavior begin to differentiate on these days, then in females and stressed males they do so without benefit of the testosterone surge characterizing control males. The Prenatal Stress Syndrome has provided a very useful tool for studying the mechanisms by which normal sexual differentiation are achieved. Further, it has revealed a variety of ways in which environmental stressors acting on pregnant dams can alter the hormonal milieu in which their fetuses differentiate, thereby affecting the reproductive behavior potentials o f the next generation of male rodents. Whether or not this model holds as one moves up the phylogenetic scale to primates remains to be determined. The optimistic conclusions o f D0rner et al. that this syndrome provides a direct explanation o f homosexuality in human males (D0rner, 1980; 1981) should be greeted with some caution. Not only is it a fundamental precept in science to demand conclusive data rather than to accept untested generalizations, but there also are known differences among various animal species in the mechanisms underlying sexual behavior differentiation, particularly between primates and rodents. For example, exposure of neonatal female rats to the nonaromatizable androgen dihydrotestosterone (DHT) results in the masculinization of reproductive morphology, but sexually dimorphic behaviors differentiate as in normal females (Luttge & Whalen, 1970; McDonald & Doughty, 1974; Korenbrot et al., 1975). Comparable treatment o f fetal female rhesus monkeys causes severe masculinization of
10
INGEBORG L. WARD
both morphology and various behaviors, including copulation and a wide array of social patterns (Goy, 1981; G o y & R o b i n s o n , 1982; T h o r n t o n & Goy, unpublished o b s e r v a t i o n s ) . T h i s h a s b e e n t a k e n as o n e l i n e o f e v i d e n c e i n d i c a t i n g t h a t in r a t s t e s t o s t e r o n e f u n c t i o n s a s a p r o h o r m o n e a n d t h a t it is its a r o m a t i z a t i o n p r o d u c t , e s t r a d i o l , that actively mediates the masculinization and defeminization of behavioral potentials d u r i n g p e r i n a t a l d e v e l o p m e n t . I n p r i m a t e s , t h e a r o m a t i z a t i o n s t e p is n o t r e q u i r e d . T h u s , a t t h i s p o i n t in t i m e , t h e P r e n a t a l S t r e s s S y n d r o m e is i n t e r e s t i n g a n d s u g g e s t i v e , b u t it r e m a i n s t o b e d e m o n s t r a t e d w h e t h e r it h a s a n y c l i n i c a l s i g n i f i c a n c e . The research reported in this paper has been supported by Grant HD04688 from the NICHHD and by NIMH Research Scientist Development Award II l-K2-MH00049 (to I. L. W.) and by Grant HD009542 to Dr. Judith Weisz of the Hershey Medical Center.
REFERENCES
BAUM,M. J. (1979) Differentiation of coital behavior in mammals: a comparative analysis. Neurosci. Biobehav. Rev. 3, 2 6 5 - 284. BEATTY, W. W. (1979) Gonadal hormones and sex differences in nonreproductive behaviors in rodents: organizational and activational influences. Horm. Behav. 12, 112- 163. BECKHARDT, S. & WARD, I. L. (1983) Reproductive functioning in the prenatally stressed female rat. Devl. PsychobioL 16, 111 - 118. DAHLOF, L. G., I-I~RD, E. & LARSSON, K. (1977) Influence of maternal stress on offspring sexual behavior. Anim. Behav. 25, 193- 195. DAHLOF, L. G., H)iRD, E. & LARSSON, K. (1978) Influence of maternal stress on the development of the fetal genital system. Physiol. Behav. 20, 193 - 195. DORNER, G. (1980) Sexual differentiation of the brain. Vitamins Horm. 38, 3 2 5 - 381. DORNER, G. (1981) Sex hormones and neurotransmitters as mediators for sexual differentiation of the brain. Endokrinologie 78, 129- 138. DUNLAP, J: L., ZADINA, J. E. & GOUGIS, G. (1978) Prenatal stress interacts with prepubertal social isolation to reduce male copulatory behavior. Physiol. Behav. 21,873 -875. GOTZ, F. & DONNER, G. (1980) Homosexual behaviour in prenatally stressed male rats after castration and oestrogen treatment in adulthood. Endokrinologie 76, 115 - ! 17. Gov, R. W. & MCEWEN, B. S. (1980) Sexual Differentiation of the Brain. MIT Press, Cambridge. Gov, R. W. (1981) Differentiation of male social traits in female rhesus macaques by prenatal treatment with androgens: variation in type of androgen, duration, and timing of treatment. In Fetal Endocrinology, M. J. Novy and J. A. Resko (Eds.), pp. 319-339. Academic Press, New York. CoY, R. W. & ROBINSON, J. A. (1982) Prenatal exposure of rhesus monkeys to potent androgens: morphological, behavioral and physiological consequences. Banbury Report I1. Environmental Factors in Human Growth and Development. Cold Spring Harbor Laboratory, pp. 355 - 378. HERRENKOHL,L. R. & WHITNEY,J. B. (1976) Effects of prepartal stress on postpartai nursing behavior, litter development and adult sexual behavior. Physiol. Behav. 17, 1019- 1021. KORENBROT, C. C., PAUP, D. C. & CoRSKI, R. A. (1975) Effects of testosterone propionate or dihydrotestosterone propionate on plasma FSH and LH levels in neonatal rats and on sexual differentiation of the brain. Endocrinology 97, 7 0 9 - 717. LUTTGE, W. G. & WHALEN, R. E. (1970) Dihydrotestosterone, androstenedione, testosterone: comparative effectiveness in masculinizing and defeminizing reproductive systems in male and female rats. Horm. Behav. l, 265 - 281. MASTERPASQUA,F., CHAPMAN, R. H. & LORE, R. K. (1976) The effects of prenatal psychological stress on the sexual behavior and reactivity of male rats. DevL Psychobiol. 9, 403 - 41 I. MCDONALD, P. G. & DOUGHTY,C. (1974) Effects of neonatal administration of different androgens in the female rat: correlation between aromatization and the induction of sterilization. J. Endocr. 61, 9 5 - 103. MEISEL, R. L., DOHANICH, G. P. & WARD, I. L. (1979) Effects of prenatal stress on avoidance acquisition, open-field performance and lordotic behavior in male rats. Physiol. Behav. 22, 527 - 530. MEISEL, R. L. (1980) Effects of prenatal stress and uterine position on the sexual behavior and morphological differentiation of male and female rats. M.S. thesis, Villanova University, Villanova, PA.
PRENATAL STRESS SYNDROME
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ORTH, J. M., WEISZ, J., WARD, O. B. & WARD, I. L. (1983) Environmental stress alters the developmental pattern of A~-3[3-hydroxysteroid dehydrogenase activity in Leydig cells of fetal rats: a quantitative cytochemical study. Biol. Reprod. 28, 6 2 5 - 631. RHEES, R. W. & FLEMING, D. E. (1981) Effects of malnutrition, maternal stress, or ACTH injections during pregnancy on sexual behavior of male offspring. Physiol. Behav. 27, 879 - 882. WARD, 1. L. (1972) Prenatal stress feminizes and demasculinizes the behavior of males. Science |7S, 82 - 84. WARD, I. L. (19"/4) Sexual behavior differentiation: prenatal hormonal and environmental control. In Sex Differences in Behavior, R. C. Friedman, R. M. Richart and R. L. Vande Wiele (Eds.), pp. 3 - 17. John Wiley, New York. WARD, I. L. (1977) Exogenous androgen activates female sexual behavior in noncopulating prenatally stressed males. J. comp. physiol. Psychol. 9 1 , 4 6 5 - 4 7 1 . WARD, I. L. & WARD, O. B. (1984) Sexual behavior differentiation: effects of prenatal manipulations in rats. Handbook of Behavioral Neurobiology, Vol. 8, Neurobiology of Reproduction, N. Adler and D. Pfaff (Eds.). Plenum Press, New York (in press). WARD, I. L. & WEISZ, J. (1980) Maternal stress alters plasma testosterone in fetal males. Science 207, 328 - 329. WARD, I. L. & WEISZ, J. (1984) Differential effects of maternal stress on circulating levels of corticosterone, progesterone, and testosterone in male and female rat fetuses and their mother. Endocrinology (in press). WEISZ, J. & WARD, 1. L. (1980) Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology 106, 3 0 6 - 316. WEISZ, J., BROW~, B. L. & WARD, I. L. (1982) Maternal stress decreases steroid aromatase activity in brains of male and female rat fetuses. Neuroendocrinology 35, 374 - 379.