Comparison of the adverse effects of adrenal and ovarian steroids on early pregnancy in mice

Psychoneuroendocrinology,Vol. 16, No. 6, pp. 525-536, 1991

0306-4530191 $3.00+0.00 ©1992 Pergamon Press pie

Printed in Great Britain

COMPARISON OF THE ADVERSE EFFECTS OF ADRENAL AND OVARIAN STEROIDS ON EARLY PREGNANCY IN MICE DENYS DE CATANZARO, ELAINE MACNIVEN, a n d FLORA RICCIUTI Department of Psychology, McMaster University, Hamilton, Ontario, Canada (Received I August 1990; in finalform 31 January 1991)

SUMMARY In one experiment, female C57- and HS-strain mice were inseminated according to standard procedures and randomly assigned to conditions involving administration of various adrenal and ovarian steroids in the first trimester of pregnancy. The pattern of effects was similar in the two strains, although generally C57 females produced fewer and smaller litters than did HS females. Oestradiol benzoate injections completely blocked pregnancy at doses far less than those effective for other steroids. Corticosterone injections did not produce any significant pregnancy block. Androstenedione injections produced a pregnancy block at 500 gtg per day. Dehydroepiandrosterone produced a mild pregnancy block at both 500 gtg and 100 gtg per day. In a second experiment, the dose-response curve for oestradiol was examined. Daily doses of 0.333 I.tg and greater completely blocked pregnancy, a dose of 0.111 I.tg did so in the majority of females, and smaller doses had little effect. These results, taken together with other data, suggest that oestrogenic action may mediate the stress-induced block of pregnancy. INTRODUCTION WHETHER OR NOT FERTILIZATIONis followed by completion of fetal development and parturition depends to a large extent upon environmental and hormonal variations during pregnancy. Evidence suggests that psychological factors during early pregnancy can impede intrauterine implantation of fertilized ova or produce fetal abortion or resorption. In laboratory rodents, diverse stressors, including human handling (Runner, 1959), exposure to predators (de Catanzaro, 1988), exposure to strange males and their odors (Bruce, 1959; 1960; 1963; de Catanzaro & Storey, 1989), chronic restraint (Euker & Riegle, 1973; Wiebold et al., 1986; MacNiven & de Catanzaro, 1990a), and environmental and social changes (Bronson et al., 1964) can all disrupt pregnancy. In agricultural studies, exposure of pregnant ewes and cattle to environmental changes and transport have increased the rate of embryonic loss (Doney et al., 1976). Although there are methodological concerns, some human case studies similarly suggest that anxiety and life stress correlate with spontaneous abortion (see McDonald, 1968). T h e p h y s i o l o g i c a l e v e n t s u n d e r l y i n g this e f f e c t h a v e not y e t b e e n d e t e r m i n e d . Psychologically or physiologically demanding stimuli can cause stereotyped activation of the hypothalamo-pituitary-adrenal axis, resulting in secretion of corticosteroids (Selye, 1956). Some studies (Robson & Sharaf, 1952; Velardo, 1957; Yang et al., 1969; Kittinger et al., 1980; Address correspondence and reprint requests to: Dr. Denys de Catanzaro, Department of Psychology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, CANADA. 525

526

D. DECArANZAROet al.

de Catanzaro et al., 1986) have indicated that administration of adrenocorticotropic hormone (ACTH) during pregnancy can result in inhibition of intrauterine implantation of fertilized ova, reduced litter size, spontaneous abortion, and fetal resorption. Administration of glucocorticoids also can inhibit uterine growth and preparation for implantation (Szego, 1952; Velardo et al., 1956; Bitman & Cecil, 1967). Nevertheless, it has not been established whether major glucocorticoids like cortisol and corticosterone directly inhibit pregnancy, or whether any adrenal activity directly mediates the block of pregnancy induced by psychological variations. Of two studies examining the effects of adrenalectomy upon strange-male-induced pregnancy blocks, Snyder and Taggert (1967) reported that adrenalectomy diminished such a block, while Sahu and Dominic (1981) reported a failure of this surgery to affect the block. Androstenedione and dehydroepiandrosterone, which are both released from the adrenal during stress (Fuller et al., 1984; Fenske, 1986), have been reported to cause an expulsion of fertilized ova from the reproductive tract (Harper, 1967a; 1967b; 1969). However, sample sizes in these studies were small, limiting statistical power, and no replications have been reported. There is also much evidence suggesting that excessive oestrogenic activity can be harmful to early pregnancy in rats (e.g., Dreisbach, 1959; Edgren & Shipley, 1961; Saunders, 1965) and mice (e.g., Greenwald, 1965; Emmens et al., 1967; Smith, 1968; Smith & Biggers, 1968). Treatment with various synthetic oestrogens has detrimental effects upon migration of fertilized ova through the fallopian tubes and intrauterine implantation (Whitney & Burdick, 1936; Dreisbach, 1959; Davis, 1963; Chang & Yanagimachi, 1965; Greenwald, 1965; Harper, 1968). However, there is little comparable evidence for more natural oestrogens. Given that exogenous ACTH can result in increased oestrogen levels (Arai et al., 1972; Strott et al., 1975) and that endogenous oestrogen may rise in early pregnancy in response to stress (MacNiven & de Catanzaro, 1990b), this may be important for understanding physiological mediation of the stress-induced block of pregnancy. If there is a common hormonal mediator of the adverse influences of psychological and physiological stress on early pregnancy, it has not yet been clearly identified. The present series of experiments was designed to determine whether adrenal hormones have any impact upon the course of pregnancy after insemination in mice. The influence of corticosterone, androstenedione, dehydroepiandosterone, and oestradiol was compared. In order to shed further light on the role of endogenous oestrogen in early pregnancy, dose-response data were gathered for oestradiol. EXPERIMENT 1

This experiment was designed to compare the effect of four steroids upon early pregnancy. Corticosterone is the major glucocorticoid secreted by the mouse adrenal gland in response to stress (Barlow et al., 1975), but there are no published studies on its effects on early pregnancy. Oestradiol-1713, a principal oestrogen, was also examined because of evidence that oestrogens block early pregnancy (Whitney & Burdick, 1936; Chang & Yanagimachi, 1965; Greenwald, 1965). Androstenedione and dehydroepiandrosterone were examined because of a need to confirm previous reports (Harper, 1967a; 1967b; 1969) of their adverse effect on pregnancy. MATERIALSAND METHODS Animals

Female C57-strain mice were obtained from Canadian BreedingFarms, La Prairie, Qudbec, at 60 days of

STEROIDS AND PREGNANCY DISRUPTION

527

age. Female HS- (heterogeneous) strain mice were bred in our laboratory from stock acquired from the University of Toronto. Prior to insemination, females were housed in groups of four in standard polypropylene cages measuring 28x 16xll cm with wire grid tops allowing continuous access to food and water. The colony room was maintained under reversed 12:12-hr dark:light cycle and at 21+ I ° C. Insemination

When females were between 80 and 100 days of age, they were each housed alone at the start of the dark phase of the light cycle with one male of the same strain in a clean cage like that described above. Males were sexually experienced and preselected for sexual vigour, and had been deprived of access to a female for at least 7 days. Each pair was observed at approximately 3-hr intervals for mating behaviour, vaginal redness, and the presence or absence of a sperm plug until the end of the dark phase. The moment of detection of the sperm plug was taken as the inception of pregnancy (day 0). Once a sperm plug was detected, the female was left with the male for 24 hr. The female then was weighed on a triple beam balance and housed individually, with the injections of the various steroids beginning at this time. The procedures were repeated daily until a sufficient number of subjects was obtained. The subjects were assigned randomly to the conditions, with conditions counterbalanced across days of insemination. Experimental conditions

In the initial study, there were nine treatments, examined in each of the two strains, for a total of 18 conditions. A vehicle injection control group in each strain involved daily sc injections of 0.05 cc peanut oil, delivered about 8 hr after commencement of the dark phase of the lighting cycle. These injections began the day after detection of the sperm plug and continued through the 6th day after detection of the plug. The eight other treatments involved two doses of each of the steroids oestradiol-17[~ benzoate, corticosterone, dehydroepiandrosterone, and androstenedione. Doses of 100 I.tg and 500 Ixg were employed, except in the case of oestradiol, in which 10 I~g and 50 I.tg were employed, based on previous evidence of exceptional potency of synthetic oestrogens in blocking pregnancy. All injections were given SC in 0.05 cc peanut oil, except for corticosterone, which due to poor solubility in oil was given in 0.05 cc propylene glycol. Outcome measures

Upon completion of the injections, females remained housed individuallyand were left undisturbed. On the 18th day after insemination they were weighed, as an index of pregnancy prior to parturition, and thereafter inspected visually on two occasions each day for the occurrence of birth. The number of pups born, number of stillborn pups, and the number of cannibalized pups were recorded. Since cannibalization and neonatal death usually occur within 2 days of birth, additional measures of the number of pups surviving to 3 days of age and total surviving litter weight 3 days after birth were recorded for each female. A weight of 0.0 g was assigned to each female with no litter. A blunt wooden prod was used to move the pups gently for counting without leaving human odours. Stillborn or partially cannibalized pups were removed by hand while wearing a surgical glove. Additional studies

In a separate small experiment, we also administered corticosterone to both strains in a daily 500 ~tg dose (n = 12 per strain), suspended in peanut oil that was heated briefly and agitated immediately before injection. This was due to concerns about comparability of vehicles. We also examined oestradiol benzoate at a dally 500 ktg dose (n =9 per strain), to provide direct comparability of doses. RESULTS Figure 1 gives the m e a n n u m b e r of pups b o m in each c o n d i t i o n in the first experiment. Table I provides measures of maternal weight on day 18, n u m b e r of pups on day 3 after birth, and the litter weight of pups on day 3 after birth in each condition. Table II gives information on sample size and stillbirths and cannibalizations. Oestradiol blocked p r e g n a n c y entirely in both strains at both doses. Corticosterone failed to produce a p r e g n a n c y block at either dose

528

D. DECATANZAROet al.

TABLE I. MEANS ( + S E M ) MATERNAL WEIGHT ON DAY 18 OF PREGNANCYs NUMBER OF PUPS SURVIVING TO DAY 3 AFTER BIRTH, AND LrITER WEIGHT ON DAY 3 AFTER BIRTH FOR ALL TREATMENTS IN EXPERIMENT 1

Treatment

Dose

Maternal Weight (g)

Pups Surviving

Litter Weight (g)

C57 Strain Vehicle Oestradiol-17~ Benzoate

10 }~g 50 ~g

33.1 + 2.5

3.4 + 1.1

7.2 + 2.4

24.4+ 0.6 24.6+ 0.3

0.0 + 0.0 0.0+ 0.0

0.0+ 0.0 0.0+ 0.0

Corticosterone

100 ~g 500 ~g

33.0 + 2.4 30.6+ 2.9

4.7 + 1.0 3.1 + 1.1

8.9 + 2.0 6.3+ 2.3

Androstenedione

100 p.g 500 ~g

28.9 + 2.8 26.8+ 2.1

2.2 + 1.1 0.9+ 0.7

4.1 + 2.1 2.3+ 1.6

Dehydroepiandrosterone

100 ~tg 500 ~g

27.6 + 2.3 29.4+ 2.8

1.6 + 0.9 1.7+ 1.0

3.0 + 1.6 3.8+ 2.2

HS Strain Vehicle

40.1 + 3.3

6.1 + 1.3

13.5+ 2.8

10 ~g 50 ~tg

27.1 + 0.9 28.3+ 0.8

0.0 + 0.0 0.0+ 0.0

0.0 + 0.0 0.0+ 0.0

Corticosterone

100 ].tg 500 ~g

32.0 + 2.0 40.3+ 3.1

3.7 + 1.4 5.8+ 1.5

8.2 + 3.0 14.3+ 3.7

Androstenedione

100 500 100 500

40.0 + 31.2+ 32.1 + 32.4+

6.2 + 1.4+ 1.6 + 1.8+

14.9 + 3.6+ 4.3 + 4.5+

Oestradiol-17~ Benzoate

Dehydroepiandrosterone

~g ~g ].tg ~g

3.5 2.7 2.4 2.0

1.5 1.0 0.9 1.0

[]

HS Strain

•

C57 Strain

3.8 2.4 2.4 2.3

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100 500 Corticosterone

100 500 Anclrostenedione

100 500 p.g Dehydroepiandrostemne

Fla. I: Mean (+ SEM) number of pups born after various hormonal treatments in the first trimester of pregnancy in Experiment 1.

STEROIDSANDPREGNANCYDISRUPTION

529

TABLE II. S A M P L E SIZES,,PERCENT OF FEMALES BEARING LrI'rER$,,PERCENT OF PUPS CANNIBALIZED~, AND PERCENT OF PUPS STILLBORN IN EXPERIMENT 1

Treatment

C57 Strain Vehicle Oestradiol-171~ Benzoate Corticosterone Androstenedione Dehydroepiandrosterone HS Strain Vehicle Oestradiol-171~ Benzoate Corticosterone Androstenedione Dehydroepiandrosterone

Dose

n

% Females Bearing Litters

% Pups Cannibalized

% Pups Stillborn

10 lag 50 lag 100 I~g 500 ~tg 100 lag 500 lag 100 lag 500 ktg

13 12 12 12 11 11 12 12 11

54 0 0 67 45 27 17 25 27

4 0 0 0 0 0 10

6 0 5 8 8 14 0

10 Ixg 50 lag 100 lag 500 ~tg 100 lag 500 lag 100 Ixg 500 lag

11 12 12 11 11 11 11 12 11

73 0 0 45 64 64 18 25 36

0 0 0 0 0 0 5

7 11 2 0 0 0 0

level in either strain. Androstenedione clearly reduced the number of pregnancies in both strains at the higher dose. Dehydroepiandrosterone produced an equivalent partial block at both doses. A 2 x 9 analysis of variance was conducted for each of the four measures: matemal weight on day 18 of pregnancy, number of pups born, number of living pups on day 3 after birth, and surviving litter weight on day 3 after birth. For maternal weight on day 18, there was significant effect of strain [F(1,190)= 20.63, p =0.0001] and of treatment [F(8,190)= 5.50, p < 0 . 0 0 0 1 ] , but no significant interaction. For the number of pups born, strain, [F(1,190)= 4.38, p = 0 . 0 4 ] and treatment [F(8,190)=8.31, p < 0 . 0 0 0 1 were significant, but the interaction was not. Similarly, strain [F(1,190) = 4.85, p = 0.03] and treatment [F(8,190) = 8.18, p <0.0001] were significant for number of pups on day 3, and strain [F(1,190)= 8.88, p = 0.005] and treatment [F(8,190)= 7.55, p < 0.0001] were. significant for mean litter weight on day 3, but neither interaction was significant. Newman-Keuls multiple comparisons (p < 0.05) were conducted for each of these four measures, with the nine treatments being compared with strain collapsed. For both the number of pups b o m and the number alive on day 3, the treatments fell clearly into two distinct groups, with the vehicle control, the 100 lag and 500 I.tg corticosterone, and the 100 lag androstenedione treatments differing significantly from the 100 lag and 500 lag dehydroepiandrosterone, the 500 lag androstenedione, and the I0 lag and 50 lag oestradiol treatments. For maternal weight on day 18, there were significant differences, with the vehicle control differing from the 100 lag dehydroepiandrosterone, the 500 lag androstenedione, and both oestradiol groups. The 500 lag corticosterone and 100 lag androstenedione treatments each

530

D. DECATANZAROet al.

differed from both oestradiol treatments, while the 100 lag corticosterone treatment differed from only the 10 lag oestradiol condition. For litter weight on day 3, the vehicle, 500 l,tg c o r t i c o s t e r o n e , and 100 lag a n d r o s t e n e d i o n e t r e a t m e n t s all d i f f e r e d f r o m b o t h dehydroepiandrosterone, both oestradiol, and the 500 lag androstenedione treatments, while the 100 lag corticosterone treatment differed for the 500 lag dehydroepiandrosterone and both oestradiol treatments. Although statistical analyses were not conducted for the number of cannibalizations and stillbirths, there were no obvious differences in these measures, with only a small number of occurrences in either condition, as indicated in Table I. In the supplementary study, females given daily injections of 500 lag corticosterone suspended in oil produced an average of 4.5 + 1,1 pups for the HS strain and 3.0 + 0.9 for the C57 strain, which is consistent with results for the same steroid in propylene glycol. No pups were produced by females of either strain given 500 lag oestradiol benzoate. EXPERIMENT 2 The first experiment indicated that oestradiol was far more potent in inhibiting pregnancy than were any of the other steroids examined. Previous studies of oestrogenic inhibition of early pregnancy have used synthetic compounds, whereas information is needed on more natural substances if we are to understand the hormonal mediation of stress-induced pregnancy block. Since oestrogens can be produced by the adrenal glands, and some evidence indicates that exogenous ACTH stimulates oestrogen secretion (Arai et al., 1972; Strott et al., 1975), it is conceivable that a small increase in endogenous oestrogens might mediate the block of pregnancy. The second study was designed to develop a thorough dose-response curve for oestradiol, in order to provide a basis for confirming or rejecting this hypothesis. This experiment was first conducted with oestradiol-1713 benzoate. Because it remained possible that the potency observed was due in part to the biochemical properties of the benzoate form, we subsequently replicated the study using an unmodified form of oestrogen, oestradiol-1713. MATERIALS AND METHODS Female HS mice were prepared and inseminated in the same way as in Experiment 1. The only exception was that upon detection of a sperm plug, females were immediately removed, weighed, and assigned to groups. Nine groups of subjects were prepared. The groups consisted of an untreated control group, a vehicle injection control group, and seven doses of oestradiol-17[~: 0.004, 0.013, 0.037, 0.111, 0.333, 1.000, and 3.000 ~tg per injection, each dissolved in 0.05 cc peanut oil. All injections were administered daily about 5 hr after commencement of the dark phase of the lighting cycle. The injections began on day 1 of pregnancy and continued through day 6. Outcome measures were identical to those of Experiment 1. As mentioned above, the experiment was replicated with oestradiol-17l~ in the unmodified form. Doses and procedures were identical to those described above, except that an additional group with a dose of 0.0013 ~tg was included, and there was no replication of the untreated control (no injections). Outcome measures also were the same, except that maternal weight on day 18 was not recorded. RESULTS Figure 2 gives the results in the number of pups born for the initial study with oestradiol1713 benzoate. Table III gives the number of pups surviving to day 3, maternal weight on day 18, the total litter weight on day 3 after birth, number of pups per litter, number of stillbirths, and number of cannibalizations. Table IV gives the percentages giving birth, stillbom,

531

STEROIDS AND PREGNANCYDISRUPTION

and cannibalized. It is clear that pregnancy was completely blocked at daily doses of 0.333 Ixg and greater. There were only two completed pregnancies at a daily dosage of 0.111 pg. Most females produced litters of normal sizes at the 0.037 pg dosage, while females receiving lower doses were indistinguishable from controls. Analyses of variance showed significant effects of condition for n u m b e r of pups born [F(8,79) = 22.50, p < 0.0001], n u m b e r o f pups alive on day 3 [F(8,79) -- 22.57, p < 0.0001], and total litter weight on day 3 [F(8,79) -- 22.77, p < 0.0001]. For each of these measures, N e w m a n - K e u l s comparisons showed that doses of 0.111 pg and greater differed significantly from all lower doses. Table V presents the results from the replication with u n m o d i f i e d oestradiol-1713. These results conformed to those with the benzoate form of the hormone, except that the unmodified h o r m o n e a p p e a r e d to be slightly m o r e potent, as p r e g n a n c y b l o c k b e g a n at a l o w e r dose

TABLE III. MEANS (+SEM) MATERNALWEIGHTON DAY 18 OF PREGNANCY,NUMBEROF PUPS SURVIVING TO DAY 3 AFTER BIRTHt AND LITTER WEIGHT AT DAY 3 AFTER BIRTH AFTEREXPOSURETO VARIOUSDOSESOF OESTRADIOL-17~BENZOATEIN EXPERIMENT2 Dose

Maternal Weight (g)

Pups Surviving

Litter Weight (g)

48.0 + 9.6 a 42.6+ 13.2 46.1 + 10.3 a 49.2+ 6.6 40.9+ 9.7 29.3 + 5.1a 27.1 + 1.6 27.0+ 2.0 26.5 + 0.8

9.9 + 1.6 6.8+ 1.5 9.1 + 1.3 10.9+ 0.4 7.1 + 1.6 0.4 + 0.3 0.0+ 0.0 0.0+ 0.0 0.0 + 0.0

23.5 + 3.6 16.5+ 3.6 22.1 + 3.7 23.9+ 1.1 16.4+ 3.7 1.1 + 0.9 0.0+ 0.0 0.0+ 0.0 0.0 + 0.0

Control 0.000 btg 0.004 ~g 0.013 pg 0.037 pg 0.111 pg 0.333 ~tg 1.000 ~tg 3.000 pg

a Based on one dam less than the full sample size due to parturition on day 18 of pregnancy.

TABLE IV. SAMPLE SIZES, PERCENT OF FEMALES BEARING LITFERS~PERCENT OF PUPS CANNIBALIZED, AND PERCENT OF PUPS STILLBORN AFTER EXPOSURETO VARIOUSDOSES OF OESTRADIOL-17~BENZOATEIN EXPERIMENT2 Dose

n

% Females Bearing Litters

% Pups Cannibalized

% Pups Stillborn

Control 0.000 ~tg 0.004 ~g 0.013 ~g 0.037 pg 0.111 pg 0.333 ~g 1.000 ~tg 3.000 btg

8 10 10 10 10 10 10 10 10

87 70 90 100 70 20 0 0 0

2 0 0 0 0 17 -

2 7 2 2 0 17 -

532

D. DE CATANZAROet al.

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1.000

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II II

Control

Oestradiol - 17~ Dosage (gg) l~c,. 2: Mean (± SEM) n u m b e r of p u p s b o r n after various daffy d o s e s of oestradiol- 17!3 benzoate in the first trimester of pregnancy in Experiment 2.

(0.037 lag/day). Analysis of variance was significant for number of pups bom [F(8,47) = 9.83, p < 0.0001], number of pups alive on day 3 [F(8,47)= 9.82, p <0.0001], and litter weight on day 3 [F(8,47) = 8.76, p < 0.0001]. DISCUSSION Oestradiol-1713 had the greatest adverse effect on early pregnancy, producing a total block in all inseminated females at doses that were far lower than those examined for the other steroids. Dehydroepiandrosterone was second in potency for pregnancy disruption, preventing pregnancy in some but not all inseminated females at both doses in both strains. Androstenedione ranked third in pregnancy-blocking capacity, disrupting pregnancy in some but not all females from both strains, but only at the higher dose. In contrast, the major adrenal glucocorticoid, corticosterone, in no case produced a significant decrement in pregnancy relative to vehicletreated control females. This is despite evidence that corticosterone has adverse influences on other female reproductive parameters, behavioural oestrus in particular (de Catanzaro et al., 1981; 1985; de Catanzaro, 1987). Overall, oestradiol-171~ produced a pregnancy block at one thousandth of the lowest dose of dehydroepiandrosterone examined and one five-thousandth of the minimal effective dose of androstenedione. Taken with other findings from our laboratory, the present results suggest that oestrogens may mediate the stress-induced block of pregnancy. We have assayed endogenous corticosterone, progesterone, and oestradiol-17[~ in recently inseminated female rats with and without chronic restraint stress (MacNiven & de Catanzaro, 1990b). Such restraint stress is well-known to block pregnancy (Euker & Riegle, 1973; Wiebold et al., 1986; MacNiven & de Catanzaro, 1990a). All three of these steroids were significantly elevated by chronic stress around the time of intrauterine implantation of fertilized ova. We also have found that concurrent injections of oestrogen antibodies partially prevents the pregnancy-blocking effects of restraint-stress in inseminated mice (MacNiven et al., 1990). The present results with oestradiol-1713 comple-

533

STEROIDSAND PREGNANCY DISRUPTION TABLE V. S A M P L E SIZES A N D M E A N S ('4"SEM) N U M B E R OF PUPS BORN, N U M B E R OF PUPS SURVIVING TO DAY 3 AFTER BIRTHs A N D LITTER WEIGHT AT DAY 3s AFTER EXPOSURE TO VARIOUS DOSES OF UNMODIFIED OESTRADIOL-17fi IN EXPERIMENT 2

Dose

n

Pups Born

Pups Surviving

Litter Weight (g)

0.000 I.tg 0.0013 ~tg 0.004 ~tg 0.013 ~tg 0.037 ~tg 0.111 ~tg 0.333 Ixg 1.000 ~tg 3.000 ~tg

7 6 6 5 6 6 6 7 7

8.0+ 1.6 9.0+ 1.9 6.8+ 2.2 4.8+ 2.0 0.0 + 0.0 0.0+ 0.0 0.5+ 0.5 0.0+ 0.0 0.0+ 0.0

7.9+ 1.6 9.0+ 1.9 6.8+ 2.2 4.8+ 2.0 0.0 + 0.0 0.0+ 0.0 0.5+ 0.5 0.0+ 0.0 0.0+ 0.0

16.7+ 3.27 17.6+ 3.56 16.3+ 5.46 12.4+ 5.15 0.0+ 0.06 0.0+ 0.06 1.7+ 1.76 0.0+ 0.07 0.0+ 0.07

ment these findings and suggest that oestrogens might constitute a mediating factor in the stress-induced block of pregnancy. Relative to previous studies, Experiment 2 provides a more comprehensive dose-response curve for the effects of oestrogen administration on early pregnancy. Most of the earlier studies utilized synthetic oestrogens (e.g., Emmens et al., 1967; Greenwald, 1965), and earlier doseresponse data address the oestrogen requirement for implantation (Smith & Biggers, 1968; Smith, 1968). Our effective doses are largely consistent with the effective doses in previous studies, although differences in species, form of oestrogen, parameters of administration, and dependent measures complicate such comparisons. Greenwald (1965) examined the state of the corpora lutea in hamsters treated with a single injection of 1, 25, 50, 100, or 250 ~tg of oestradiol cyclopentylpropionate on the day of insemination. Despite a small sample size, the results indicated that the first two doses had no apparent effect, while the others resulted in the regression of the corpora lutea in all subjects. Greenwald (1965) suggested that these effects may be indirect, being mediated by prevention of release of pituitary luteotrophic hormones. Whitney and Burdick (1936) and Burdick and Whitney (1937) examined the influences of three synthetic forms of oestrogen upon the rate of passage of ova through the fallopian tubes, finding a locking of fertilized ova in the fallopian tubes with lower doses and acceleration of rate of passage through the tubes with higher doses. Unfortunately, the synthetic nature of their compounds prevents direct comparison of their results with those of other studies. Chang and Yanagimachi (1965) examined the influence of repeated oral administration of natural and synthetic oestrogens during the first few days after insemination in female rabbits and hamsters and found greater degeneration of ova with higher doses, a greater influence of the natural form of oestrogen, and greater sensitivity in rabbits. Harper (1968) demonstrated that synthetic oestrogens can be as potent as the naturally occurring compounds. Our results also confirm reports by Harper (1967a; 1967b; 1969) that the lesser known steroids, dehydroepiandrosterone and androstenedione, have detrimental effects on pregnancy. The large sample of animals in the present study allows statistical confirmation of Harper's findings, and they extend the species generality by demonstrating effects in mice. Since these two steroids can be released by the adrenal gland under stressful stimulation (Fenske, 1986), they might also play a role in psychogenic pregnancy blocks. Harper (1969) suggested that conversion of androstenedione and dehydroepiandrosterone to oestrogens may be responsible

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for their pregnancy blocking effects. Only a small fraction of these steroids would need to be transformed to oestrogens to account for the effects observed in this study. It also is possible that the mechanism involving enhanced oestrogen under stress m a y involve the hypothalamicpituitary axis, and g o n a d o t r o p i n s m a y play a role (Chatterjee & Harper, 1970); this idea demands further research. However, corticosterone is an unlikely mediator of pregnancy block, unless it can be shown to have effects well outside the dose range of the present study. Unlike androstenedione and dehydroepiandrosterone, corticosterone is not a precursor o f oestrogens (Temple & Liddle, 1970). Ineffectiveness of corticosterone may be viewed as inconsistent with reports (Szego, 1952; Velardo et al., 1956; Bitman & Cecil, 1967; Schlough, 1971) that administration of glucocorticoids inhibits uterine growth and physiological preparation for implantation, although such studies did not examine pregnancy outcome in intact animals. The exceptional potency of oestrogen as an inhibitor o f early pregnancy is worthy of much additional attention. Since oestrogens have a dramatically adverse effect on early pregnancy at very low exogenous doses, it is conceivable that only a small enhancement o f their natural levels by psychological and physiological stressors could account for the negative influences of such stressors on gestation. REFERENCES Arai K, Kuwabara Y, Okinaga S (1972) The effect of adrenocorticotropic hormone and dexarnethasone, administered to the fetus in utero, upon maternal and fetal estrogens. Am J Obstet Gynecol 113: 316-322. Barlow SM, Morrison PJ, Sullivan FM (1975) Effects of acute and chronic stress on plasma corticosterone levels in the pregnant and non-pregnant mouse. J Endocrino166: 93-99. Bitman J, Cecil HC (1967) Differential inhibition by cortisol of estrogen-stimulated uterine responses. Endocrinology 80: 423-429. Bronson FH, Eleftheriou BE, Garick E1 (1964) Effects of intra- and inter-specific social stimulation on implantation in deer mice. J Reprod Fert 8: 23-27. Bruce HM (1959) An exteroceptive block to pregnancy in the mouse. Nature 184: 105. Bruce HM (1960) A block to pregnancy caused by the proximity of strange males. J Reprod Fert 1: 96-103. Bruce HM (1963) Olfactory block to pregnancy among grouped mice. JReprod Fert 6: 451-460. Burdick HO, Whitney R (1937) Acceleration of the rate of passage of fertilized ova through the fallopian tubes of mice by massive injections of an estrogenic substance. Endocrinology 21: 637-643. Chang MC, Yanagimachi R (1965) Effect of estrogens and other compounds as oral antifertility agents on the development of rabbit ova and hamster embryo. Fertil Steri116: 281-291. Chatterjee A, Harper MJK (1970) Interruption of implantation and gestation in rats by reserpine, chlorpromazine and ACTH: possible mode of action. Endocrinology 87: 966-969. Davis, BK (1963) Studies on the termination of pregnancy with norethynodrel. J Endocrino127: 99-106. de Catanzaro D (1987) Alteration of estrogen-induced lordosis through central administration of corticosterone in adrenalectomized-ovariectomized rats. Neuroendocrinology 46: 468-474. de Catanzaro D (1988) Effect of predator exposure upon early pregnancy in mice. Physiol Behav 43: 691-696. de Catanzaro D, Knipping RP, Gorzalka BB (1981) Antagonism of estrogen-induced lordosis by corticosterone in adrenalectomized-ovariectomized rats. Pharm Biochem Behav 15: 761-766. de Catanzaro D, Lee PCS, Kerr TH (1985) Facilitation of sexual receptivity in female mice through blockade of adrenal 11~-hydroxylase. Horm Behav 19: 77- 85. de Catanzaro D, Maerz M, Heaven RKB, Wilson W (1986) Repeated failure of ACTH administration to alter masculine behavior in mice. Dev Psychobio119: 501-510. de Catanzaro D, Storey AE (1989) Partial mediation of strange-male-induced pregnancy blocks by sexual activity in mice (Mus musculus). J Comp Psychol 103: 381-388. Doney JM, Smith WF, Gunn RG (1976) Effects of post-mating environmental stress or administration of ACTH on early embryonic loss in sheep. JAgric Sci 87: 133-136. Dreisbach RH (1959) The effects of steroid sex hormones on pregnant rats. J Endocrinol 18: 271-277.

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