Behauioural Processes, 2 (1977) o Elsevier Scientific Publishing

41-73 Company,

41 Amsterdam

-

Printed

in The Netherlands

INFLUENCE OF ENVIRONMENTAL DISTURBANCES ON UTERINE MOTILITY DURING PREGNANCY AND PARTURITION IN RABBIT AND SHEEP

ELISABETH

H.M. BONTEKOE*,

S.J. DIELEMAN**

J.F. BLACQUIERE*,

C. NAAKTGEBOREN*,

and P.P.M. WILLEMS*

Research group for Comparative Obstetrics *Department of Obstetrics and Gynaecology, Wilhelmina Gasthuis, le Helmersstraat 104, Amsterdam (The Netherlands) **Department of Veterinary Obstetrics, State University Utrecht, Yalelaan 7, Uithof, Utrecht (The Netherlands) (Received

25 March 1976;

revised 10 February

1977)

ABSTRACT Bontekoe, E.H.M., Blacquiere, J.F., Naaktgeboren, C., Dieleman, S.J. and Willems, P.P.M., 1977. Influence of environmental disturbances on uterine motility during pregnancy and parturition in rabbit and sheep. Behav. Processes, 2: 41-73. The effect of stressful stimuli on uterine motility during pregnancy and parturition was studied in sheep and rabbits. The effects of epinephrine and various alpha- and betaadrenergic blocking agents were also investigated. By comparing the results of these experiments, the authors conclude that the increase in epinephrine level (stress response of the organism) is the direct cause of the observed changes in uterine motility. Both stress and epinephrine caused either an activation or an inhibition of uterine motility. The direction of the effect depended on the ratio of sex-steroid concentrations in the plasma. Blood plasma levels of oestradiol-17p and of progesterone were determined in the sheep. Both stress and epinephrine inhibited uterine motility only when plasma levels of oestradiol17p were very high (oestrogen-domination). The biological relevance and clinical implications are discussed. INTRODUCTION

In many mammalian species environmental disturbances causing fright or anxiety may lead to a prolongation of labour, varying from a slight delay to a complete inhibition of the birth process. Moreover, longlasting stressful events may cause an abortion or premature labour (Naaktgeboren and Slijper, 1970; Naaktgeboren and Bontekoe, 1976) in rabbits, non-human primates and several other mammals. It seemed important to study the effects of stress upon uterine motility during pregnancy and parturition. In former experiments it was observed that in the cyclic ewe, stress can have two different effects upon uterine motility, either inhibitory or excitatory. Stress caused an excitation of uterine motility during the luteal phase

42

of the cycle, but an inhibition during oestrus. It was supposed that these different effects are dependent on the levels of circulating sex-steroid hormones (Naaktgeboren and Bontekoe, 1975). Uterine atony in the rabbit was suggested to be caused by inhibition of oxytocin release due to stress (Porter and Schofield, 1966). This explanation does not seem adequate, since the occurrence of the stress-induced increase in uterine motility in this hypothesis remains unclarified. Stress causes an arousal of the sympathetic nervous system, leading to a release of epinephrine and other catecholamines (Selye, 1956). From in vitro studies it became clear that epinephrine is able to either excite or to inhibit uterine activity (Riisse, 1965). We have suggested that the effects of stress upon uterine motility are mediated by an extra output of catecholamines (Naaktgeboren and Bontekoe, 1975). In order to test this hypothesis, we investigated the effects of epinephrine on uterine motility in sheep and rabbits. These two species have been used for studies of normal uterine activity during pregnancy and parturition (Naaktgeboren et al., 1975a,b). Thus the present paper represents a comparative approach, which may enable generalizations to be made beyond &he physiological adaptations of one species. MATERIAL

AND METHODS

Animals The influence of stress and of epinephrine upon uterine motility was studied in fifteen sheep (Texel breed). Nine of the ewes had also been used in experiments on normal uterine activity during pregnancy and parturition (Naaktgeboren et al., 197515). Forty-four rabbits of several different breeds were used. Fourteen does were used merely to study the effects of stress and epinephrine on uterine activity. The remaining thirty were used as controls. The does were operated at day 22 or 23 of pregnancy. Uterine activity was recorded every day for at least one hour, starting from the second day after operation. Some animals aborted. The results from these animals have been treated separately. During all experiments the animal’s behaviour was noted carefully. In addition, a closed-circuit television unit was used to observe the ewe’s behaviour. Recording

technique

Uterine activity was recorded by the electrophysiological method as described by Naaktgeboren (1974). Four pairs of silver electrodes were attached to the myometrium of each animal under NembutaI@ (pentobarbital) anaesthesia [ 0.5 ml/kg, i.e. 30 mg/kg bodyweight] (rabbit) or fluothane/N,O inhalation anaesthesia (sheep). Each pair of electrodes was connected by silastic-coated cables to a contact set, implanted in the animal’s neck (rabbit) or in the abdominal wall (sheep). In order to take blood samples or to inject drugs

43

without causing stress all animals were fitted with a jugular cannula. For each trial the animal was connected to the preamplifiers of the recorder (Elema Schonander Minograf 81 and 42B) by plugging a cable into the contact set. Uterine activity was recorded at a paper speed of 2.5 mm/s, and the amplifiers were set at 30 Hz with a time constant of 0.03 s. Uterine activity was quantified by measuring the duration of the recorded electrical activity. In the graphs electrical activity is expressed in s per unit of time (2 min for rabbit experiments; 12 min for ewes). Stress stimuli Although the term “stress” has a variety of meanings in the literature, we use this short term because it is more convenient than a long description. We considered any environmental disturbance which caused behavioural arousal in the experimental animal to be a stressful stimulus. Control experiments have shown that these same stimuli do not evoke a reaction in the anaesthetised animal. Thus it may be stated that the stimuli under study are elaborated psychologically and that they do not directly influence uterine activity by physiological pathways. We frightened the animals by various kinds of environmental disturbances (stress stimuli). The variety in stimuli to which the animals were exposed was necessary in order to avoid adaptation. Since it is probable that the various stimuli are not equivalent, we have presented the results of each stimulus separately. For the rabbit, the following stimuli were under study. The introduction of much cigarette smoke into the cage was the stress stimulus most frequently used. It caused an immobility reflex. The effects of a cat placed into the doe’s cage, a dog barking near the cage, or the giving of a sham intramuscular injection were also studied. Furthermore, a doe was removed from her nest during labour and the effects of this very disturbing event on the labour contractions were studied. An observer entering the animal’s room during a recording session, may disturb the animal (“presence of an observer”). During reattachment of the recording cable, the animal has to be handled; this almost always led to behavioural arousal (“cable attachment”). In the ewe the effects of “presence of an observer” and of “cable attachment”, as defined above, were studied. Since the ewes were housed isolated and observed by closed-circuit television, environmental influences were introduced only by the investigators. In addition, the effects were studied of sudden unpleasant noise (produced by explosion of small bombs, loud whistling which hurt the ears, and blowing a trumpet); pain induced by giving a sham intramuscular injection; occlusion of the animal’s nostrils by way of laying a hand on them; a dog barking near the cage and later inside the cage; a plastic bag rustled in front of the animal; and finally, removal of the newborn lamb(s) from the ewe.

44

Drugs The dosage of epinephrine used for the rabbits was 0.05 mg or 0.125 mg per animal, administered via the implanted jugular catheter. In sheep, either a single dose of 0.2 mg or an infusion of 0.1 mg/min was given. We worked on a limited scale with noradrenaline, phentolamine (Regitine@), proprano101 (Inderal’) and with combinations of these agents. In rabbits and sheep 21 and 47 of these experiments were carried out, respectively. Dosages and other details are given in the text. Estimation

of sex-steroid

hormones

Peripheral blood samples were taken via an implanted jugular catheter twice a day and more frequently during periods of stress, catecholamine-infusions and parturition. Heparinized blood was immediately centrifuged at +4”C and plasma was stored at -25°C until assay. Progesterone levels were determined by radioimmunoassay similar to the method of De Jong et al. (1974). Antiserum’ produced in rabbits against progesterone 11 (uBSA conjugates was used with (1,2,6,7-3H) progesterone (84 Ci/mmol) as the tracer. Dextran-coated charcoal was used for separation of bound and free fractions. The radioimmunoassay was checked by GLC with FID as earlier described (Brand et al., 1976). Oestradiol-170 levels were determined by radioimmunoassay similar to the above progesterone procedure, after extraction of the plasma with freshly distilled peroxide-free diethyl ether. Antiserum’ produced in sheep against oestradiol-17/I---CG-keto-oxime BSA conjugates was used with (1,2,6,7,-3H) oestradiol-170 (86 Ci/mmol) as the tracer. Dextrancoated charcoal in gelatine PBS-buffer was used for separation of antibodybound and free oestradiol fractions. Cross-reaction of the oestradiol antisera with oestrone and with oestradiol-17awas minimal (3% and 1.2% respectively). The other steroids tested cross-reacted less than 0.1%. RESULTS

Stress and epinephrine resulted in four types of effect on uterine motility: (a) Activation and restoration of the original level of activity. The activation was either an increase in the number or in the duration of the contractions (Fig.1 and 10). Both will be called the positive (+) effect. (b) Activation followed by a decrease to less than the original level and thereafter a restoration of the initial pattern of motility; (+ -) effect (Fig.5). (c) Inhibition of the activity, either completely or as a decrease in the number and/or duration of the contractions; (-) effect (Fig.4). ’ The antiserum 5RlPR4 was generously supplied by Dr. Van der Molen, Erasmus University, Rotterdam, The Netherlands. 2The antiserum OR-580 was generously supplied by Dr. R. Scaramuzzi, MRC Unit for Reproductive Biology, Edinburgh, Scotland.

0

0

0

N stress

N intravenous epinephrine

2

3

(-)

0

0

(-)

0

2

~~

1

0

(+-)

0

1

0

0

(0)

0

1

(+)

0

0

(0)

0

1

(-)

1

3

(-)

6

13

(+-)

0

0

(+-) 0

(0)

3

4

(+)

0

0

(0)

4-l days p.ab.

see text

1

(+)

0

0

(-)

3

(-)

3

2

(+-)

0

(+-)

Less than 24 h. p.p.

p.ab. = post abortum

2 and 3 days p.ab.

(+)

Parturition

in rabbits

(+)

results

N = Number of observations; p.p. = post partum; (+) = Increase in uterine activity - (-) = Decrease in uterine activity (+ -) = Initial increase followed by a decrease (0) = No effect. For explanation see text.

0

(0)

(+)

Effect on uterine motility

I

10

Less than 24 h. p.ab.

20

intravenous epinephrine

N

29

N stress

(0)

Pregnancy

of the experimental

(+)

I

Effect on uterine motility

Summary

TABLE

0

0

(-)

3

5

(+)

0

0

(0)

2

4

t---j

Total p.ab. (Columns 5+6+7)

0

0

5

0

(0)

(+)

10

15

(+-)

1

1

(+--)

More than 24 h. p.p.

f

Fig.la. Electrical activity, recorded at two sites of the uterus in a 25.day experimental room, at II the cat is placed next to the rabbit. This results

200 pv

MIN.

^A

,.

“~ pregnant rabbit. At I a cat is brought in an increase in uterine activity.

t

j

into the

47

(d) No changes in the pattern of uterine activity; (0) effect. In the rabbit all four effects were found, whereas in the sheep the (+ -) effect has not been recorded.

The effect of stress and of catecholamines

on uterine activity

in the rabbit

The data from all experiments are given in Table I. Since the effects depend on the functional state of the uterus, the data will be discussed separately.

Pregnancy During pregnancy the uterus is normally quiescent until immediately before parturition (Naaktgeboren et al., 1975a). Stressing the does during pregnancy either resulted in an increase in uterine activity or had no effect. Fig.la shows the increase in uterine activity when a cat was placed next to a 25-day pregnant rabbit. At arrow I the cat was brought into the room, at arrow II the cat was placed into the doe’s cage. The doe showed signs of severe anxiety, i.e., she assumed a freezing position. At the same time an increase in the uterine activity was recorded (Fig.lb). Uterine activity was greatly increased (up to 45 s/2 min) during the time the cat was next to the doe. Only when the cat was removed (IV) did uterine activity fall to the pre-stress level of about 10s of activity per 2 minutes. Likewise, the administration of 0.05 mg epinephrinercaused either an activation of the uterine motility or had no effect. We were also able to block the (+) t

30

\/ ** I

* II

III

\ i

*

IV

‘\i

i *

:i_, TIME

(-___-.~.___

10

MIN.)

Fig.lb. Uterine activity as shown in Figure lA, quantified and expressed in seconds of activity per 2 min. At I the cat is brought into the room, at II the cat is placed next to the rabbit, at III the cat is put closer to the rabbit, at IV the cat is removed.

48

effect of both stress and of epinephrine with a dose of 1 mg of the alphablocker Regitine@ (ohentolamine), administered intravenously. Table I shows the number of times we observed a (+) effect, due to stress, compared to the number of times that epinenhrine-treatment caused a (+) effect. On the whole, the effects of stress and of epinephrine during pregnancy were quite comparable. Parturition Stress during labour caused a diminution or a complete inhibition of uterine activity. The effect is quantitatively represented in Fig.2. The cross-hatched area represents the variations in normal activity during parturition in a large series of control observations in non-stressed animals (Naaktgeboren et al., 1975a). The uterine activity of the disturbed animals was far less than was expected at this stage of labour. In rabbit XX the only disturbances which caused response in uterine motility were the connection of the cable and the presence of an observer. The other animal (rabbit XXIX) had to be removed from its cage to establish th> cable connection. Removal of an animal from its nest during labour is known to delay parturition (Naaktgeboren and Bontekoe, 1976). From behavioural observations (nestbuilding and plucking behaviour), delivery in these two rabbits was expected earlier than it actually occurred. Thus we concluded that parturition was delayed by stress. HOWACT.IN 500

SEC,%,

MIN

:/::,: .. _--

CONTROLES RABBIT

XXIX

RABBIT

XX

30

MIN.

Fig.2. Uterine activity in seconds per 10 min around parturition in the rabbit. In the crosshatched area the measured values of uterine activity of a large series of non-stressed control rabbits are situated (after Naaktgeboren et al., 1975a). The solid and the dotted lines represent uterine activity of two disturbed animals. P is the birth of the first young. Further explanation in text.

49

ever, the duration of the delay could not be calculated. We therefore plotted the time of birth of the first young in the control animals and in the stressed does at the same spot on the curve (P). It appears that the activity of the uterus was lower at the time just before delivery of the first pup in the stressed animals. The uterine activity of rabbit XXIX, taken from her nest during parturition, is also represented in Fig.3A. The data suggest that uterine activity is completely absent for about half an hour after the start of the recording session. Moreover, not only did the number of contractions diminish due to stress, but the propagation of the contractions along the uterine horn was also impaired (Fig.3B). After half an hour of complete inactivity, activity reappears, but the contractions are still poorly propagated. Not until two hours after the disturbing event was propagation fully reestablished, and half an hour later the expulsion phase began. Administration of epinephrine during the onset of labour (nest building) resulted in a short period of labour-like activity, immediately followed by a decrease towards the original level of uterine activity. In fact, uterine activity increased so strongly during the epinephrine treatment, that two young were expelled. In contrast, epinephrine administration during the expulsion phase or immediately after the birth of A

ACT in sec/iomin

Number of contractions

(pro 6 min)

B

Fig.3. A. Quantitative representation of uterine activity of rabbit XXIX (the same as from Fig.2) as recorded at three sites of the uterus. The positions of the electrodes on the myometrium is represented schematically. B. Partogram of rabbit XXIX. Solid lines: homogeneous contractions, dotted lines: feeble contractions.

50

the last young resulted in a marked decrease of uterine activity. In one case five young were expelled when 0.03 mg of epinephrine was given; labour contractions disappeared completely and did not reappear until 20 minutes later. Then two other young were expelled, and although they were fully developed, they were stillborn, possibly due to the long delay in the expulsion phase. Post par-turn phase Immediately post par-turn a very regular pattern of uterine activity was recorded in the rabbits. When a doe was stressed less than 24 hours post parturn the most common result was an inhibition of uterine activity as well as impairment of the propagation of the contractions (Fig.4). The top part of Fig.4 represents a portion of the record of the electrical activity of the uterus of a rabbit six hours post par-turn; the lower portion represents the frequency of the contractions. The uterus displayed a regular activity until the rabbit was frightened by a barking dog (arrow), causing complete inhibition of the uterine activity for 30 minutes. In one case, stress caused an activation on the first day post partum. More than 24 hours post partum, stress most often resulted in an increase in uterine activity. An inhibitory effect was not ob-

. .,,

Fig.4. Top: part of the record of uterine activity of a 6-hours post partum rabbit. At the arrow the rabbit is frightened by a barking dog; this leads to inhibition of uterine activity. Bottom: the same record, represented as a partogram, in which the number and the quality of the contractions are shown. The position of the electrodes on the myometrium is represented schematically. At the arrow the rabbit is frightened.

I’

.

. j/

j

..::‘i*$&~::.+_

,&{q,

_ .-

,

,~““,‘,,.

>A

,

.i.., %$J,:;.:y””

.i*-,;,~l;r*,

._,.

:...

.~

-----

_.

h

.,’

,.

1._

.,,

.,

_,.? I”

i ,.

..I..

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ij ,,..(,,;,“‘.?-

,

1~

.

.;.,%,:..‘J

. . _--

-

mopv

I

I_,^

;_

.~

*.q __.

eig.5. Electrical uterine activity of a 1.5-day post abortum rabbit. Position of the electrodes as in Fig.4. This is a very good example of the so-called biphasic effect of stress. When the rabbit is frightened the uterus first displays one long contraction, followed by a period of complete arrest of all electrical activity.

_._~~

..,>

;..*

.

52

served later than 24 hours post par-turn (Table I). Epinephrine administered less than 24 hours post partum resulted in a very clear biphasic effect, namely an initial increase, followed by a long lasting inhibition. Since this experiment was carried out after an induced labour, this was not recorded in Table I. The inhibitory component of the reaction could be blocked by the administration of 1 mg of the beta-blocking agent InderaI@ (propranolol) intravenously.

Post abortum phase Less than 24 hours post abortum,

stress resulted in decreased uterine activity. However, two and three days post abortum, stress caused a biphasic response (Fig.5 and 6). The lower part of Fig.6 represents the uterine activity in one rabbit three days post abortum. On the upper portion variations in heart rate are expressed, At 1 (arrow) a sham injection of physiological saline solution caused a sharp rise in uterine activity, followed by a period of inhibition of about 10 minutes. When the observers left the room (2) a restoration of the normal 3 days post abortum uterine activity occurred (20s activity per 2 minutes). Following the sham injection the heart rate increased and remained high, even during the time when the uterus was quiescent. Heart rate decreased to normal levels only after the observers had left the room. More than four days post abortum the result of stress was either an increase in uterine activity or the (+ -) effect (Table I). The (-) effect was most frequently observed during the first day post abortum, whereas the (+) effect was most often observed 4 and more days post abortum. The (+ -) effect occurred most frequently at 2 and 3 days post abortum. Epinephrine administered less than 24 hours post abortum usually resulted in inhibition of uterine activity. However 2 and 3 days post abortum, epinephrine usually caused a (+ -) effect. More than 4 days post abortum uterine activity either increased or a biphasic reaction was observed after the administration of epinephrine. The excitatory component of the biphasic reaction to stress and to epinephrine could be blocked by the alpha-blocking agents Dibenyline@ (ph enoxybenzamine) and Regitine@(phentolamine) in doses of 0.5 mg and 1 mg respectively. The effects of stress and of epinephrine were strikingly similar (Fig.7a). At A, a normal uterine activity in a rabbit 3 days post abortum is represented. The uterus is now quite quiescent. When a large quantity of cigarette smoke was blown into the cage (B), uterine activity increased. A new pattern of uterine activity was observed: a long burst of electrical activity, followed by a large number of trains. Gradually the intervals between the trains increased in length, until the effect was over. Epinephrine (C) caused a reaction similar to that caused by the cigarette smoke. The effects of stress and of epinephrine could be blocked by the adninistration of 1 mg of the alpha-blocking agent Regitine@ (Fig.7b). The same animal was exposed to smoke on the same day as in Eig.7a. However, before stressing the doe we had administered 1 mg of Regitine@. The effects of stress and epinephrine were both reduced. Only a very small contraction was observed after smoke (A) or epinephrine (B). The effect of norepinephrine was one

53

HRin 340

260

bpm

1

k

160

1

100

+ ? 4

ACT. I” sec./ 2 min

75

50

25

1

Fig.6. Uterine activity expressed in seconds of activity per 2 min of a 3-days post abortum rabbit. At 1 (arrow) the rabbit receives a sham injection with physiological saline solution, at 2 the observers leave the experimental room, so providing some rest for the experimental animal. The stress of the injection clearly results in the so-called biphasic uterine response. Above: the heart rate of the same rabbit is represented in beats per minute. The stress of the injection causes the heart rate to rise. Heart rate and uterine activity become normal after the observers leave the room.

contraction (J?ig.7b, C), which occurred simultaneously at all recorded sites of the uterus. The reaction was quite different from the reaction provoked by epinephrine and could be blocked by Regitine@ (D). After the adminis-

54

.

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A: ”

* I,

e *, . =. _-* _

.*-.__

.“b_

_r_-_ -__

__-._A”

.

. X,“__._

_ I ~ ^X_ - _-.“_

__:,__*_

I “._x

-._

200&w

“^~

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*

_ _______,

1 MIN

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L _

.,

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56

tration of 1 mg Regitine@ there was no uterine activity when the doe received 0.003 mg norepinephrine. The effects of epinephrine, stress, alphaadrenergic blockade and norepinephrine are quantitatively represented in Fig.8. The doe was three days post abortum. At C the extension of the cannula was connected to the implanted jugular catheter. At S cigarette smoke was blown into the animal’s cage. Uterine activity increased up to 80 s per 2 minutes. A similar increase was observed after the administration of 0.05 mg epinephrine. However, norepinephrine caused only one small contraction. After the administration of the alpha-adrenergic blocker Regitine@ (Ph) the effects not only of stress, but also of epinephrine or norepinephrine were partially if not completely blocked. Usually, epinephrine administered less than 24 hours post abortum resulted in an inhibition of the very regular uterine activity. We were able to blo k this inhibitory effect of epinephrine with the beta-blocking agent Inderal fi . Uterine inertia occurred in a few cases

60 .

40

. l9

*

. ~ *

,..

. b.

. l .d

.

.

.

.

5

C

.

,Ld i.i

L.c..4

.

4*

*

c

4

E

*

*

**

NE

NE

PHS

* E

& l * . c-4 e_) F NE

ilME

+ 10 _~MIN_+ Fig.8. The effects on uterine motility of connecting the animal to the recorder and inserting the cannula into the jugular catheter (C); smoke (S); readjustment of the cannula (C); as ministration of 0.05 mg epinephrine (E); of 0.003 mg norepinephrine (NE); of Regitine (PH); and the effects of smoke (S), of epinephrine (E) and norepinephrine (NE) after alpha-adrenergic blockade with phentolamine (Regitine@) in a 3-day post abortum rabbit. (Activity expressed in seconds per 2 min.) It is clear that after alpha-adrenergic blockade the effects of stress and of epinephrine on uterine motility are reduced, and that norepinephrine has no effect after blocking uterine alpha-adrenergic receptors (cf. Fig.7a and 7b).

_ 21

--

D

I= yaAW+S

444

4

4

OSOmg INDERAL

time

I 1Omin

+-+ - -3 NUMBERof TRAINS -t-.-ACTIVITYsec/2min

Fig.9. The effect of propranolol and stress on uterine motility, expressed in number of trains and in seconds of activity per 2 min in a P-day Post aborturn rabbit. The animal is very anxious (at D: freezing position). F: flush with physiological saline solution. S: the rabbit is frightened by a dog. F’ropranolol restores normal uterine activity and prevents the effect of stress (S).

2_

4_

N’ 6_

AC 4c:.

58

following cable connection. However, after treatment with 1 mg Inderal@ on such an occasion, uterine activity was restored to the normal levels. When 0.05 mg of epinephrine was given, uterine activity increased, followed by inhibition. However, after the administration of 1 mg Inderal@, epinephrine was no longer able to inhibit uterine activity. After a third treatment with Inderal@ an increase in uterine activity was noticed. Fig.9 represents uterine activity in a doe two days post abortum. The doe sat constantly immobilized (“freezing position”). Uterine activity was minimal. The stress-induced uterine inertia could be counteracted by the administration of 0.25 mg Inderal@. The effect of Inderal@ lasted about 10 minutes. Thereafter the uterus again became quiescent until another dose of 0.25 mg Inderal@ was given. Again we observe a restoration of uterine activity to a normal level for two days post abortum (40 s/2 minutes). Concluding remarks Our results in rabbits can be summarized as follows: Most of the time stress and epinephrine during pregnancy caused an increase in uterine motility. The excitatory effects could be blocked by the alphablocking agents Dibenyline@ and Regitine@. At parturition a decrease or total inhibition of uterine activity occurred when the doe was stressed. Epinephrine administration during the period preceding parturition resulted in the premature birth of two young, followed by a period of rest? During the expulsion phase epinephrine caused a complete inhibition of labour activity. Until 24 hours post partum, stress usually reduced uterine activity, whereas epinephrine administration resulted in one contraction followed by inhibition. The inhibitory effect of epinephrine could be blocked by the beta-blocking agent Inderal@. More than 24 hours post partum, stress resulted in an activation in most cases, whereas epinephrine still caused the biphasic reaction. Less than 24 hours post abortum, stress always caused an inhibition of uterine activity, while epinephrine did so in most cases. Two or three days post abortum, the effect of stress was a biphasic reaction. The same reaction was caused by the administration of epinephrine. The excitatory component of the response to stress and to epinephrine could be blocked by the alpha-blocking agents Dibenyline@ and Regitine @ and the inhibitory component by the beta-blocking agent Inderal@. More than four days post abortum, stress again caused only an increase in uterine motility. The effect of epinephrine was either an increase in uterine motility or an increase followed by an inhibition. A real (-) effect as a reaction to stress was found ten times (Table I). Nine observations were made during parturition or within 24 hours of labour or abortion. Three times a (-) effect was seen after epinephrine. Once this was during parturition and two times less than 24 hours after abortion.

59

The effects of stress and of catecholamines

on uterine activity

in the ewe

Pregnancy Spontaneous uterine motility during pregnancy and parturition in the sheep was described in detail by Naaktgeboren et al. (1975b). They distinguished seven patterns in uterine activity in the pregnant and parturient ewe. Until one month before parturition the uterus was very quiescent. However, the uterus gradually became more active, with periods of rest alternating with periods or bursts of activity. We studied pregnant ewes only after day 120 of pregnancy. Normally the uterus displayed the activity pattern as mentioned above at this time. When the pregnant ewe was stressed, uterine activity increased or did not respond at all. When there was an increase in activity, a variety of responses (one extra burst, one small contraction or only some isolated peaks) were observed. Generally quantification was difficult. It was only possihle to induce a burst of activity when it was due to appear, i.e. about 30 minutes after a spontaneous burst of activity. In that case, stress caused a new burst of activity (Fig.10). At 1 the observers entered the cage, at 2 the ewe was frightened by noise, at 3 the nostrils of the ewe were covered by a hand. Immediately thereafter a burst of activity began. Immediately after it had disappeared the same procedure was repeated (at 4 and 5), but now only a small contraction occurred. Sometimes stress had no effect. The number of observations is given in Table II. Frequently a burst of activity was observed at the beginning of the recording session (these bursts are not included in Table II). Particularly when the animal was not used to the recording equipment, as a rule during the first experiments, or if the ewe was a very nervous animal, connection of the recording equipment was experienced as a stressful event, which induced TABLE Summary

II of the experimental

results in sheep Effect on uterine motility Number of observations

_

__~ Day 120 of pregnancy 1 day before birth

(+)

(0)

(-)

26 9

0 0

until

Stress Epinephrine

68 11

24 Hours before birth until 20 hours after birth

Stress Epinephrine

0 0

09 0

5

21 Hours after birth until 5 days post partum

Stress Epinephrine

7 2

40 0

0

(+) = Increase in uterine activity (0) = No effect (-) = Decrease in uterine activity

60

i

>,

-w

61

.

62

a burst of activity. Whether this is a coincidence or a real induction of bursts of activity is unknown, due to the method we used. It was impossible to check whether a burst would have occurred if the animal had not been connected to the recorder. However, records were of a long duration (up to 4 hours) and the bursts of activity appeared regularly and were of about the same duration (Lt 7 minutes). From control observations we were able to calculate the prohability that at an arbitrary time a burst of activity would appear (P = 0.02). Since the mean duration of a burst of activity was 7 minutes, the chance that at the time recording was initiated, a burst of activity would occur is 0.14. In this case, one record in seven would begin during a burst of activity. However, one in three recordings started with a burst of activity. This is more than can be expected by chance. During pregnancy we administered epinephrine, either as a single intravenous administration of 0.2 mg or as a continuous infusion of 0.1 mg per minute. Epinephrine caused either an increase in uterine activity or had no effect (Table II). The pattern of the increase in uterine motility was similar to that observed after stress stimuli. A continuous epinephrine infusion caused the appearance of uterine activity only at the start of the infusion; then uterine activity declined rapidly (Fig.11). We were able to block the epinephrine-induced increase in uterine motility by administration of 50 mg Dibenyline @. Isoprenaline-treatment during pregnancy in a dose of 0.5 mg did not affect uterine motility. The and Propranolol@) did not block the spontanebloc king agents (Dibenyline@ ous pattern of activity.

Parturition The duration of either the dilatation phase or of the afterbirth stage cannot be estimated accurately. We therefore included the day before and the day after parturition within observations on the day of parturition. From 24 hours before until 20 hours after birth of the lamb, stress resulted in a decrease or a total inhibition of uterine motility. Thus the effect of stress changed from activation during pregnancy to inhibition near parturition. This change in the effect of stress may be due to the shift in the oestrogen/ progesterone ratio (Table III). Whether stress caused a short-term decrease in uterine activity or a severe disturbance of labour activity depended on individual differences between animals. Once we observed short-term inhibition of uterine activity when we connected the recorder to a ewe in labour. In a very anxious animal however, a similar event led to severe disturbance of labour activity (Fig.12). In normal, undisturbed labour, uterine activity was maximal during the expulsion phase (Fig.l2a, ewe 24). Uterine activity just before and after the actual expulsion of the lamb were of the same magnitude. However, the dilatation phase was characterized by short contractions occurring more frequently than during the post par-turn phase (cf. Fig. 12b and c, ewe 24). Data recorded from the very nervous ewe 2 are also plotted on these graphs. The

63

TABLE

III

Blood plasma levels of oestradiol-17@

(in pglml) as a function

Data from 4 ewes with a single lamb

Data from 2 ewes with twin lambs Number of samples

Min.

Max

Mean

25

46

100

74

5

24

9

71

153

115

5

161

124

9

183

335

210

4

Min.

Max.

Mean

~__.__ 121 h-48 h before birth

0

31

10

48 h-24 h before birth

0

55

24 h before5 h after birth 32

Period

of time before and after birth

Number of samples

5 h-20 h after birth

7

30

19

2

74

100

87

3

20 h-48 h after birth -~

0

22

8

5

0

94

30

8

completion of the expulsion of the lamb for both ewes is indicated by arrow III. At I ewe 2 was connected because she appeared to be in labour. However, the expected uterine activity was not recorded. Thus the quiescence could not be anything but a psychogenic uterine atony. After half an hour, uterine activity reappeared and reached the expected level (Fig.1 2a); however, the pattern remained aberrant, because more trains occurred (Fig.12b) with a shorter duration (Fig.12~). At the moment the uterine activity resembled the normal situation, one of the observers entered the cage (at arrow II), resulting in total inhibition of labour activity. Therefore, at that point, recording was terminated and within one hour the lamb was delivered. At arrow IV the ewe was reconnected to record post partum activity, which also showed very suppressed values. We removed the lambs from their mother six hours after parturition. The ewe, bleating vigorously, showed signs of excitation. Uterine activity immediately decreased (Fig.13). Electrode 1 displayed only some irregular groups of spikes and electrode 2 displayed no activity at all. At 1 the lambs were removed, at 2 the lambs bleated vigorously, and at 3 the ewe could see the lambs which were returned at 4 and placed next to the ewe (5). Uterine activity increased and contractions reappeared. The lambs did not suckle, so the reappearance of the contractions was not caused by an oxytocin release. The inhibition of uterine activity by removal of the lambs from the ewe could be blocked by the administration of 1 mg Inderal@ intravenously. The effects of stress and of epinephrine administration were comparable (Table II, Fig.14). At S (Fig.14) the lamb was removed from the ewe, resulting in a total inhibition of all electrical activity. After restoration of uterine activity, epinephrine treatment also caused inhibition

Fig.12. Data from two labouring ewes as described in the text. Ewe 24; control, two sites on the uterus, indicated with triangles. Ewe 2: an anxious, easily disturbed animal, two sites on the uterus, indicated ith circles. (a) Above: activity in sec/l2 min. (b) Below: number of trains. (c) Facing page: mean duration of trains (in seconds). I: Connection of ewe 2 to the recorder. II: It is decided not to record any longer, while this procedure leads to too much disturbance for ewe 2. III: Birth of the lambs for both ewes. IV: Reconnection of ewe 2 to the recorder to record post partum activity.

65

of the electrical activity. Therefore epinephrine administered during parturition and immediately after birth of the lamb caused a decrease or a total inhibition of uterine activity, as did stress.

Postpartum

period

Removal of the lambs on the second post partum day resulted in an increase in uterine activity (Table II). Again we observed a change in the direction of the effect of stress, from an inhibition during parturition and immediately thereafter to an activation when stimulation occurred more than 20 hours post partum. The peak in circulating oestrogens observed immediately before and during parturition did not decline until about 5 hours post partum (Table III). This change in the direction of the effect of stress and of epinephrine on uterine motility, observed on the second post partum day, was concomitant with changes in the balance of sex-steroids.

Relationship between the oestrogenlprogesterone sponse to stress and to epinephrine

ratio and the uterine re-

Plasma progesterone level was high (5-12 ng/ml from day 120 of pregnancy), when we began our experiments, until one or two days before parturition. During the same time however, plasma levels of oestradiol-170 were very low. Progesterone levels in periphere blood plasma decreased approximately 1 week before parturition. Mean values of progesterone in single pregnancies were 8.1 ng/ml from day -21 till day -7. Between day -7 and 48 hours before parturition mean values declined from 8.1 ng/ml to 4.3 ng/ml. Near the onset of delivery a further decrease from 4.3 ng/ml to 0.5 ng/ml occurred. After the birth of the lamb the mean serum progesterone value was 0.3 ng/ml. In ewes carrying twins, higher values were observed: from day -21 till day -7 16.5 ng/ml; from day -7 till 48 hours before parturition a de-

Fig. 13. Electrical activity at two electrodes of a ewe 6 hours postpartum, and the influence thereupon of removing the newborn lamb. The uterus displays very regular contractions at both recorded sites. At 1 the lambs are removed from the ewe. The ewe is very restless, while the lambs bleat vigorously (2). At 3 the lambs become visible to the ewe, at 4 the lambs are back, and at 5 they lie down quietly next to the ewe. It is clear that removing the lambs causes an inhibition of uterine activity. However, at the very moment the lambs become visible to the ewe, the uterus again starts to display regular contractions.

67

cline from 24.5 ng/ml to 8.5 ng/ml; from 48 hours before parturition till delivery a decline from 8.5 ng/ml to 1.4 ng/ml; and after the birth of the lambs 0.2 ng/ml. Stabenfeldt et al. (1972) and Charnley et al. (1973) observed a similar decrease. In contrast, Thompson and Wagner (1974) reported a decline in jugular progesterone levels at day -1 and day 0, while progesterone levels in uterine venous blood declined from approximately day -7. The concentration of peripheral plasma progesterone reported in this paper agrees with the data reported by Stabenfeldt et al. (1972) and Charnley et al. (1973). The oestradiol-170 levels in the peripheral blood plasma reached values of 100-200 pg/ml in ewes which delivered a single lamb. Oestradiol-17P levels in two ewes with twins increased to values of 265 pg/ml and 335 pg/ml respectively (Table III). These values are in agreement with other reports. Charnley et al. (1973) and Robertson and Smeaton (1973) reported oestradiol170 concentrations in the jugular vein of 100-150 pg/ml for ewes which delivered a single lamb. The latter authors reported oestradiol-17/I levels of 200-400 pg/ml for ewes which delivered twins. After parturition, plasma concentrations of both oestradiol and progesterone decreased very rapidly. More than one day after parturition the levels

40

20

+ s

I

\ 4

0.2 mg EPINEPHRINE

I

’ 10 Ylll )

Fig.14. Uterine activity of a ewe, 18 hours post partum (seconds of activity per 2 minutes). In this figure the effect of removing the lamb and of the administration of 0.2 mg epinephrine intravenously are compared. The stress of being separated from the lamb leads to an inhibition of uterine activity. The administration of 0.2 mg epinephrine also leads to inhibition of uterine activity at this time post partum.

68

EWE 1-2

-

PROGESTERONE

- - b-

OESTRADIOL-170

birth

180

I

E20XlOIP4RATlO

: 160

I

-1

-6

-j

-i

-3

time I” days (pg/ml) and progesterone (ngiml) Fig.15. Plasma levels of estradiol-170 tween these two hormones (EJP,) X 10 nging mass, in the ewe during parturition.

and the ratio bethe days around

of both the steroids had declined to very low values (progesterone below 0.5 ng/ml; oestradiol-17p below 25 pg/ml). During the time plasma progesterone levels were high and oestradiol-170 levels were low, uterine motility displayed the pattern described by Naaktgeboren et al. (1975b), i.e. bursts of activity or bursts of activity with trains. Twenty-four hours before parturition the uterine motility pattern gradually changed to one of periodically regular activity, which means an increase in total activity. The increase in uterine activity was concomitant with the rise in plasma levels of oestradiol-170 and the fall in plasma concentrations of progesterone. The relationship between oestradiol and progesterone is expressed as a ratio (E,/P) X 10 ng/ng mass (Fig.15). During the expulsion phase, the uterus was very regularly active and very high levels of plasma oestradiol-17P were detectable. When the plasma values of oestradiol-17fl decreased after parturition, uterine activity decreased and became irregular. More than one day post partum the uterus was quiescent and plasma sex-steroid levels had declined to the low levels as mentioned above (Fig.15). Either stress stimuli or epinephrine caused an increase in uterine activity from day 120 of pregnancy until one day post partum. During this period, plasma oestradiol-17/I levels were low. More than one day post partum the response to stress or to epinephrine was likewise an increase in uterine activity when the oestradiol-17P values were very low. Only when the oestradiol170 values were high, i.e. during the day preceding the birth of the lamb and shortly after its birth, did stress and epinephrine always cause an inhibition of uterine activity (cf. Tables II and III).

69

In conclusion, the concentration of progesterone and the oestradiol-17fl in the peripheral blood may determine not only the pattern of uterine activity but also the direction of the effect of stress and epinephrine on myometrial motility. DISCUSSION

From the facts that (1) both the uterus and the heart react to environmental disturbances (stress stimuli), (2) the effects can be mimicked by epinephrine, and (3) that they can be blocked with selective alpha- and beta-blocking drugs, we speculate that the effects of stress on uterine motility may be mediated by a stress-induced activation of the sympathetic nervous system, leading to a release mainly of epinephrine from the adrenal medulla into the bloodstream. It should be noted however, that for many species the uterine autonomic innervation is not yet thoroughly investigated, and that many contradictory reports on this topic exist in the literature. In the rabbit it appears from the work of Owman and Sjijberg (1966) that there exists a well developed autonomic uterine innervation, which is most prominent in the outer muscular layer. Rosengren and Sjoberg (1968) found that in the first half of pregnancy a marked increase in neuronal noradrenaline occurs, and that this increase is followed by a dramatic decrease in total noradrenaline during the latter half of pregnancy. At the end of pregnancy the uterus is completely devoid of noradrenaline-containing sympathetic nerves. We believe that from their data it may be concluded that there is no evidence that uterine motility can be influenced by neuronal pathways at the end of pregnancy. Since we worked on rabbits at the end of pregnancy (day -25 and later), we may conclude that our findings can only be explained by assuming that epinephrine is the catecholamine responsible. It is a well known fact that one of the organism’s reactions to stressful stimuli is an extra release of catecholamines by the adrenals (Selye, 1956). The investigations of Frankenhguser (1971) are interesting in this respect. She was able to show that in humans epinephrine was mainly released in those stress situations that were characterised by ambiguity and the impossibility of actively coping with the stressful situation. In cases where the subjects had a certain situational control, although a certain degree of tension was still present, norepinephrine was released, but epinephrine release seemed to be counteracted when subjects had control of their situation. The same phenomenon was reported by Brady (1970) for monkeys. The stress stimuli we applied in our investigation were randomly scheduled, and therefore rather ambiguous; our animals did not have the opportunity to flee or to avoid being stressed. We presume that in most of the stress stimuli which we created, epinephrine was the most important hormone released, although itwas not measured. This phenomenon could be basic to the success of psychological education for childbirth: teaching the expectant mother how to cope with her situation, thus reducing uncertainty by informing her about what is going to happen. This might re-

70

duce the release of epinephrine from the adrenals in cases where the mother is still tense and nervous and norepinephrine is still being released. From our pilot experiments with norepinephrine, it is evident that this reduction of epinephrine-release thus might prevent severe labour-inertia. Friedman (1975) reported that in the human the stress of leaving home and going to the hospital for delivery (the “transposition-stage”) may lead to inhibition of uterine contractions. Friedman (1974) states that uterine function is related to the attitudes and motivation of the parturient woman. His findings support the hypothesis outlined above. In contrast, the hypothesis that stress may cause a disturbed release of oxytocin, leading to labour-inertia (Porter and Schofield, 1966), cannot explain the occurrence of the stress-induced activation during pregnancy. Epinephrine is, to a certain degree, antagonistic with oxytocin and impairs the regularity of the labour contractions, even when the uterus is first stimulated by oxytocin. We were able to block labour-inertia in the rabbit with the betareceptor blocking agent Inderal@ as Zacutti and Brugnoli (1970) were able to do for women in labour. This indicates the importance of epinephrine as the effective hormone for mediating the changes in uterine motility during stressful events. The blocking agents themselves do not affect uterine motility. The oestrogen/progesterone ratio may be a determining factor for both the pattern of the uterine activity and the direction of the effect of both stress and epinephrine. A quiescent uterus, predominantly under the influence of progesterone (an ET/P ratio in favour of progesterone) was activated both by stressful stimuli and by epinephrine. When the uterus was active (i.e. when the EJP ratio was in favour of the oestrogens) stress and epinephrine both caused an inhibition of uterine motility. Although the oestradiol-170 levels in peripheral plasma were low as early as 5 hours after birth, the inhibiting effect of stress and of epinephrine lasted until about 20 hours after birth. The uterus displayed the effects of oestrogenic domination not only during the time that the peak of oestradiol-17P was detectable in peripheral blood plasma, but also up to 15 hours thereafter. This was true for the contraction patterns as well as for the response to stress and to epinephrine in sheep. Stress may lead to an abortion or to premature labour in the rabbit. A doe, in which epinephrine caused the expulsion of two young, had not yet fully shown the well known signs of parturition such as plucking. In contrast, the does used in the stress experiments and in the epinephrine experiment, had shown plucking. Therefore it is probable that all does were not in the same stage of parturition. Possibly the response to stress and to epinephrine is not the same in the dilatation phase and during the expulsion phase. The partus praematurus in the rabbit even has a function in regulating population-density (Mykytowycz and Fullagar, 1973). Since parturition in the rabbit may take place before the establishment of oestrogen-domination is complete (Naaktgeboren et al., 1975a), the fact that premature labour can be induced by epinephrine is understandable. This may offer an explanation for the findings of Mykytowycz and Fullagar (1973). Stress and epinephrine sometimes

have no effect on uterine motility. This may be due to the high level of the monoaminoxydase-enzyme detectable during pregnancy. Rapid catecholamine depletion could protect pregnancy in cases of short-term stress. However, longlasting and severe stress situations may lead to a disturbance of pregnancy in some species. This is also known from several species of monkeys (Naaktgeboren and Bontekoe, 1976). Inhibition of labour contractions in a stressful situation offers the mother the opportunity to move to a more favourable environment, but after premature labour or abortion in a stressful situation, the mother has more chance to survive and is able to engage in reproductive activity next season. The effects of stress on uterine motility could be regarded as very important adaptations with biological relevance. The influence of the E2/P ratio on uterine motility patterns and on the direction of the effect of various stressors and of epinephrine on uterine motility that changes with a changing E2/P ratio cannot simply be regarded as a shift in alpha- and beta-receptor sensitivity. A study of the influence of various alpha- and beta-sympathicometics on uterine motility during pregnancy and parturition appears to give support to the concept of Langley (1906) who included the direction of the effect of adrenergic drugs in defining alpha- and beta-receptors. In the literature this concept is usually disregarded, whereas that of Ahlquist (1948), who spoke of the relative sensitivity of an organ to adrenergic drugs in defining the alpha- and beta-receptor, paying no attention to the direction of the effect, is accepted. Nevertheless, some authors define the inhibitory action of epinephrine as a beta-effect and the excitatory action as an alpha-effect. Others term the inhibitory action of progesterone on the myometrium a beta-effect and the excitatory action of oestrogens on the myometrium an alpha-effect (Cieciorowska and Telko, 1961) and then speak of a shift in alpha- and betareceptors at the end of pregnancy. This interpretation of the alpha-beta-concept is not consistent with the original concept, since the terms alpha- and beta-receptors have not been defined on the basis of the action of steroids, but on the action of various adrenergic drugs. These inconsistencies result in confusion in obstetrical literature. Each time a pregnant doe was stressed or given epinephrme, uterine motility increased in contrast to the uterus of the normally quiescent pregnant rabbit. However, when the uterus becomes active, as was the case during parturition and immediately post partum, stress or epinephrine lead to uterine inhibition and sometimes to a (+ -) effect. The possibility that these two reactions are not so different as they seem, should not be excluded, but it might be that only the component of the reaction becomes clear, which is most deviant from the original pattern. However, this is a problem which remains to be clarified.

72

ACKNOWLEDGEMENTS

The authors are most grateful for the technical assistance and surgical assistance and animal care by Prof. Dr. P. Klopper, Dr. P. Fontyne, Miss D.M. Blankenstein, Miss H.A. Van Oord, D. Van Bruggen, J.K. Van Dijk, J. Lubberink, A.V.P. Van de Poll and P.J. Timmerman, and to Mrs.P. Noden for correcting the manuscript. REFERENCES Ahlquist, R.P., 1948. A study of the adrenotropic receptors. Am. J. Physiol., 153: 586600. Brady, J.V., 1970. Endocrine and autonomic correlates of emotional behaviour. In: P. Black (Editor), Physiological Correlates of Emotion. Academic Press, New York and London, pp. 95-125. Brand, A., Taverne, M.A.M., Van der Weyden, G.C., Aarts, M.H., Dieleman, S.J., Fontijne, P., Drost, M. and De Bois, C.H.W., 1976. Non-surgical embryo transfer in cattle. I. Myometrial activity as a possible cause of embryo expulsion. In: L.E.A. Rowson (Editor), Egg Transfer in Cattle. Commission of the European Communities, Agricultural Research Seminar (EUR 5491), pp. 41--56. Charnley, W.A., Buckmaster, J.M., Cerini, M.E., Cumming, I.A., Goding, J.R., Obst, J.M., Williams, A. and Winfield, C., 1973. Changes in levels of progesterone, corticosteroids, estrone, estradiol-170, luteinizing hormone and prolactin in the peripheral plasma of the ewe during late pregnancy and at parturition. Biol. Reprod., 9: 30-35. Cieciorowska, A. and Telko, M., 1961. Die Wirkung des Adrenalin und des Noradrenalin auf die Kontraktionen des menschlichen Uterus und ein Erkllrungsversuch dieser Wirkung. Gynaekologia, 152: 39-49. De Jong, F.H., Baird, D.I. and Van der Molen, H.J., 1974. Ovarian secretion rates of oestrogens, androgens and progesterone in normal women and in women with persistent ovarian follicles. Acta Endocrinol., 77: 575-587. Frankenhauser,M.,1971. Experimental approaches to the study of human behaviour as related to neuroendocrine functions. In: L. Levi (Editor), Society, Stress and Disease. I. The Psychosocial Environment and Psychosomatic Diseases. Proc. Int. Interdisciplinary Symp. Stockholm, April 1970. Oxford University Press, London, New York, Toronto, pp. 22-35. Friedman, D.D., 1974. Parturiphobia. Am. J. Obstet. Gynecol., 118: 13+135. Friedman, D.D., 1975. Conflict behaviour in the parturient. In: H. Hirsch (Editor), The Family. 4th Int. Congr. Psychosom. Obstet. Gynecol., Tel Aviv, 1974. Karger Verlag, Base], pp. 373-375. Langley, J.N., 1906. On the reaction of cells of nerve endings to certain poisons chiefly as regards the reaction of striated muscle to nicotine and to curare. J. Physiol. (London), 33: 374. Mykytowycz, R. and Fullagar, R.P., 1973. Effect of social environment on reproduction in the rabbit, Oryctolagus cuniculus. J. Reprod. Fertil., Suppl., 19: 503-522. Naaktgeboren, C., 1974. Myometriai activity and its exploration by electromyography of uterine smooth muscle. Z. Tierz. Zuchtungsbiol., 91: 278-296. Naaktgeboren, C. and Bontekoe, F.H.M.. 1975. The uterus, a mirror of the soul. In: H. Hirsch (Editor), The Family. 4th Int. Congr. Psychosom. Obstet. Gynecol., Tel Aviv, 1974. Karger Verlag, Basel, pp. 511-519.

73

Naaktgeboren, C. and Bontekoe, E.H.M., 1976. Vergleichend geburtkundliche Betrachtungen und experimentelle Untersuchungen iiber psychosomatischen StSrungen der Schwangerschaft und des Geburtsablaufes. Z. Tierz. Ziichtungsbiol., 93: 264-320. Naaktgeboren, C. and Slijper, E.J., 1970. Biologie der Geburt. Eine Einfiihrung in vergleichende Geburtskunde. Paul Parey, Hamburg, Berlin, 225 pp. Naaktgeboren, C., Kroon, C.H. and Schoof, A.G., 1975a. Uber die Wehentltigkeit des Kaninchens. Eine elektrophysiologische Studie der Uterusaktivitat und eine Theorie iiber die Auslijsung des Geburtbeginnes. Z. Saugetierk., 40: 150-172. Naaktgeboren, C., Pool, C., Van der Weyden, G.C., Taverne, M.A.M., Schoof, A.G. and Kroon, C.H., 1975b. Elektrophysiologische Untersuchungen iiber die Uteruskontraktionen des Schafes wahrend der Trachtigkeit und der Geburt. Z. Tierz. Ziichtungsbiol., 92: 220-243. Owman, C. and Sjiiberg, N.O., 1966. Adrenergic nerves in the female genital tract of the rabbit. With remarks on cholinesterase-containing structures. Z. Zellforsch., 74: 182-197. Porter, D.G. and Schofield, M.J., 1966. Intrauterine pressure changes during pregnancy and parturition in rabbits. Endocrinology, 36: 291-299. Robertson, H.A. and Smeaton, T.C., 1973. The concentration of unconjugated oestrone, oestradiol-17P and oestradiol-17a in the maternal plasma of the pregnant ewe in relation to the initiation of parturition and lactation. J. Reprod. Fertil., 35: 461-468. Rosengren, E. and Sjoberg, N.-O., 1968. Changes in the amount of adrenergic transmitter in the female genital tract of rabbit during pregnancy. Acta Physiol. Stand., 72: 412424. Riisse, M., 1965. Geburtsablauf beim Rind. Eine Betrachtung des funktionelles Geschehens bei der Normalgeburt. Arch. J. Exp. Vet. Ned., 19: 763 -870. Selye, H., 1956. The Stress of Life. 4th Edition, New York, 340 pp. Stabenfeldt, G.H., Drost, M. and Franti, C.E., 1972. Peripheral plasma progesterone levels in the ewe during pregnancy and parturition. Endocrinology, 90: 144-150. Thompson, F.N. and Wagner, W.C., 1974. Plasma progesterone and oestrogens in sheep during late pregnancy: contribution of the maternal adrenal and ovary. J. Reprod. Fertil., 41: 57-66. Zacutti, A. and Brugnoli, C.A., 1970. Influenza delle catecholamine sull’ attivitl contrattile in travaglio di parto. Minerva Ginecol., 22: 303-306.