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The induction of pseudopregnancy and pregnancy by mating in albino rats exposed to constant light
HORMONES
AND
BEHAVIOR
(1977)
8,62-76
The Induction of Pseudopregnancy Mating in Albino Rats Exposed
and Pregnancy by to Constant Light
K. BROWN-GRANT’ Department of Human Anatomy, University of Oxford, and the A.R.C. Institute of Animal Physiology, Babraham, Cambridge, England
Oxford,
Adult female albino rats anovulatory as a result of exposure to constant light (CL rats) were shown to ovulate in response to limited coital stimulation without necessarily becoming either pseudopregnant or pregnant. A higher level of coital stimulation would induce pseudopregnancy provided that intromission was not prevented. The occurrence of an ejaculation was not essential for the induction of pseudopregnancy but the incidence of pseudopregnancy was higher when a series of intromissions included one or more intromissions associated with ejaculation than when it did not. Similar findings regarding the induction of pseudopregnancy were obtained in experiments on female rats maintained under normal light-dark conditions (LD rats). Increasing the interval between intromissions from less than I min to as much as 20 min did not reduce the incidence of pseudopregnancy in CL rats. Splitting a set number of intromissions into two groups separated by up to 180 min did not reduce the incidence of pseudopregnancy in CL or LD rats. Conditions for the induction of pregnancy were more critical than for the induction of pseudopregnancy. CL rats showed only a low incidence of pregnancy and, if pregnant, either had small litters or did not deliver living pups even when a high level of coital stimulation from several males, including multiple ejaculations, occurred over a limited period of time. A high success rate was only observed when CL rats were mated with a single male overnight. LD rats showed a higher incidence of pregnancy than CL rats in response to a restricted amount of coital stimulation over a short time period, especially when an ejaculation was the terminal event in the mating sequence. This dependence on the occurrence of an ejaculation as the terminal event was not observed in CL rats, probably because, unlike LD rats, their uteri were not distended with fluid at the time of mating.
Adult female rats exposed to constant light cease to ovulate and show persistent vaginal cornification. Such animals also show persistent behavioral estrus (Hardy, 1970), but ovulation can be induced by mating or, less consistently, by the injection of progesterone or nonspecific stress, probably as a consequence of the secretion of progesterone by the adrenal cortex (Brown-Grant, Davidson, and Greig, 1973). Dempsey and Searles (1943) showed that at least some female rats exposed to constant light became pseudopregnant after mating. Separate neural mechanisms have been suggested to be involved in the control of reflex ovulation and ’ Medical Research Council Medicine, Memorial University,
External Scientific Staff. St. John’s, Newfoundland,
Present address: Faculty Canada AlC 5S7.
of
62 Copyright All rights
@ 1977 by Academic Press. Inc. of reproductmn in any form reserved.
ISSN 0018-506X
PREGNANCY IN PERSISTENT ESTROUS RATS
63
induction of pseudopregnancy in the rat (Everett, 1968; Everett and Quinn, 1966). If this were so, then mating-induced ovulation in the persistent estrous rat might occur without a subsequent pseudopregnancy or pregnancy. This possibility and the requirements for the induction of pseudopregnancy and pregnancy by mating in rats exposed to constant light have been investigated and compared with the findings in females of the same strain maintained under normal light-dark conditions and mated at proestrus. METHODS
The rats were adults of the albino Wistar strain obtained from Charles River, England, and they were housed six to eight to a cage. Three types of animals were used. Female rats were kept under constant light after their arrival at the laboratory and were used after 8 to 12 weeks of exposure. They are referred to as CL rats (constant light-induced persistent estrous rats). Other females were kept under normal light-dark conditions (lights on at 0500 hr and off at 1900 hr), and daily vaginal smears were obtained for 10 to 20 days; only rats that had shown at least two consecutive 4-day cycles were used. These animals are referred to as LD (light-dark) rats. The males used as stimulus animals were kept under reversed light conditions (lights off at 1100 hr and on at 2300 hr), and tests were carried out during the later part of their dark period. For mating, the females were brought from their home cages to the room housing the males which was dimly lit by low-power red bulbs in the cages where mating took place. Thus, CL rats had up to a 4-hr period of much lower intensity light than usual during the matings and they were returned to cages in constant light after mating. This procedure, without contact with males, did not induce ovulation. The LD rats, depending on the time they were mated, had a period of exposure to light of a reduced intensity either late in their normal lights-on phase or early in their usual dark period before returning to their usual lighting conditions. These procedures, without contact with males, did not induce pseudopregnancy. Matings were generally carried out with two or three males and a single female in the cage, though, in some experiments, a one-to-one arrangement was used as described under Results. The occurrence of mounts, mounts resulting in a lordosis response, lordosis responses associated with intromissions, and intromissions associated with ejaculations were recorded. In addition, the occurrence of incidents of rejection behavior (Hardy, 1972) on the part of the females in response to an attempt of the male to mount was noted. The lordosis quotient [LQ = (number of lordoses/number of mounts) x 1001was calculated for each test, as was the rejection quotient [RQ = (number of episodes of rejection behavior/ number of mounts or attempts to mount) x 1001.The period over which the coital stimulus was received varied from experiment to experiment as
64
K. BROWN-GRANT
the required level of stimulation varied, but the period of contact with the males under observation was generally between 2 and 20 min. Further details of the lighting conditions, the effects on the females, and the performance of the mating tests can be found in Brown-Grant (1974, 1975) and Brown-Grant et al. (1973). In a few preliminary experiments on CL rats, the occurrence of pseudopregnancy was assessed by the examination of daily vaginal smears beginning on the morning of the day after mating (Day 0 of pseudopregnancy or pregnancy), but, in all later experiments, smears were not taken, and the animals were tested for a decidual reaction on Day 4 as will be described. In experiments on LD rats, daily vaginal smears were continued after mating and, if a cornified smear was observed on Day 4, smearing was continued to confirm that the animal was not pseudopregnant and that it continued to show regular 4-day cycles. If the smear on Day 4 was leucocytic, the rats were anesthetized with Avertin (tribromoethanol plus amylene hydrate) and one uterine horn was traumatized along the anti-mesometrial surface of the uterine lumen as described by De Feo (1963). The animals were killed 5 days later, and the weight of the traumatized horn was expressed as a percentage of the control horn. The responses obtained were, in fact, always clearly positive or negative, the weight of the traumatized horn being either 800 to 120% or within + 20% of the weight of the control. The incidence of pregnancy was assessed by allowing the animals to survive to term and noting the number of pups born, or by killing the animals a few days after the expected day of delivery and recording the number of placental scars or fetuses at autopsy. In other experiments, the uterus was examined for implantation sites at autopsy on Day 9 when the decidual response was assessed. Values given in the text and tables are group means t SEM. The t-test or the appropriate nonparametric test (Siegel, 1956) was used to compare groups of animals. A value of P < 0.05 was regarded as significant. RESULTS Induction
of Pseudopregnancy
Preliminary experiments. Twenty-one CL rats were mated; 19 were responsive (LQ > 50) and each received twenty intromissions plus one or two ejaculations. Vaginal smears were taken up to Day 13 and the rats were killed after they had littered or on Day 24. Seventeen of the rats had ovulated and had leucocytic smears up to Day 13 and one set of large corpora lutea in their ovaries when they were killed. Only two rats delivered live litters (five and ten pups), and none of the others had placental scars or any evidence of implantation sites. The incidence of pseudopregnancy appeared to be 10%. An additional 23 CL rats were mated; 21 were responsive and received twenty intromissions plus one or
PREGNANCY
IN PERSISTENT
ESTROLJS RATS
65
two ejaculations. Their uteri were traumatized on Days 3, 4, or 5, and they were killed 5 days later. Sixteen rats had ovulated, and all showed a marked decidual response. A single implantation site was seen in one animal at autopsy but none of the others had any signs of implantation. The weight of the traumatized horn (as a percentage of the control) was 333 2 83% for five rats traumatized on Day 3, 1089 + 113% for six traumatized on Day 4, and 586 + 120% for five traumatized on Day 5. The magnitude of the responses on these 3 days was very close to the values of 207, 1118, and 546% respectively obtained in pseudopregnant LD rats of this strain (Brown-Grant and Raisman, 1972), indicating that the time course of the changes in uterine sensitivity in early pseudopregnancy was not affected by previous or continuing exposure to constant light. As a control procedure, the uteri of five CL rats that had mated but had not ovulated were traumatized on Day 4. When they were killed on Day 9, the traumatized horns were 109 ? 9% of the weight of the control horns. Ovulation without pseudopregnancy. The possibility that matinginduced ovulation was invariably associated with a subsequent pseudopregnancy was investigated in experiments in which CL rats received varying amounts of coital stimulation. Before placing the females with the males the vagina was occluded with surgical tape (Brown-Grant et al., 1973; Hardy, 1970, 1972) so that intromissions could not occur on the initial mounts (taped rats). Each female was mounted 20 to 25 times in all, and, as the LQ was high (90-lOO%), most mounts induced a lordosis response. In some cases, the plaster was not removed until the end of the test (mounts only or zero intromission group). In others, the plaster was removed after 20, 15, 10, or 5 mounts, and mating continued until the appropriate number of intromissions with or without ejaculation occurred (5, 10, 15, or 20, respectively). In this way, the total number of mounts and the duration of exposure to males was equal for each group. All CL rats were traumatized on Day 4 and killed on Day 9; the results are given in Table 1. It is clear that ovulation, based on finding fresh corpora lutea in the ovaries on Day 4, can occur in response to mating without the CL rat becoming pseudopregnant. Two further experiments were carried out. Twelve rats were given an intramuscular injection of 2.5 mg of progesterone in 0.1 ml of oil at 1200 hr; 5 days later, the ovaries were examined for recent corpora lutea. Eight rats had ovulated and in these animals one uterine horn was traumatized. When they were killed 5 days later, the weight of the traumatized horn was 105 + 5% of that of the control horn. Six further CL rats received a subcutaneous injection of 50 pg of ovine LH (NIH-LH-Sl 1) in 0.1 ml of saline. All ovulated, but none showed a decidual response though the traumatized horns were slightly heavier than the controls, 129 2 5%, due possibly to inflammatory changes. Normal LD rats were mated in the same way at proestrus. Failure to
66
K. BROWN-GRANT TABLE I Incidence of Pseudopregnancy after Varying Amounts of Coital Stimulation in Rats Exposed to Constant Light (CL) and in Normal Cyclic Rats (LD)” Number of intromissions
Treatment CL rats Ovulation Pseudopregnant LD rats Pseudopregnant
0
5
10
15
20
911lb
@2Y
10111 (91)
14114 (100)
14114 (100)
II/l1 (100)
019 (0)
O/IO (0)
6114 (43)
12114
(86)
lOill (91)
lill (9)
011 (0)
Iill (9)
5112 (42)
819 (89)
a Rats mated between 1600 and 1800 hr of proestrus. * Number of rats responding. c Number within parentheses is percentage of rats responding.
become pseudopregnant was established by the continuation of a regular 4-day cycle of vaginal smears. If the vaginal smear on Day 4 was leucocytic, the occurrence of pseudopregnancy was confirmed by a positive decidual response to uterine traumatization. The incidence of pseudopregnancy was slightly higher in the CL rats in the 10 and 15 intromissions groups, but the differences were not statistically significant (Table 1). With no or a low number of intromissions pseudopregnancy was rarely induced in either CL or LD rats, but 20 intromissions were generally effective. Variation in the timing of the coital stimulus. CL rats received 20 mounts while taped and then five intromissions within 1 or 2 min or spaced 5, 10,20, or 40 min apart. In a second experiment, each received 15 mounts while taped and then 10 intromissions in 3 to 4 min or spaced 5, 10, or 20 min apart. The results are given in Table 2. Spacing 5 or 10 intromissions in this way did not significantly affect the incidence of pseudopregnancy though there was some indication that the 5-min interval might be the most effective. CL rats received 15 or 10 mounts while taped followed immediately by 10 or 15 intromissions (zero interval group of Table 3) or mounts followed immediately by five, seven, or eight intromissions. After an interval of 30 to 180 min, the latter animals received further intromissions to bring the total up to 10 or 15. A similar experiment with fifteen intromissions per rat was carried out on LD rats. The results are given in Table 3. Though there was some indication that with a 180-min gap the incidence of ovulation in CL rats might be reduced, neither these nor any other
PREGNANCY
Incidence
1N PERSISTENT
ESTROUS
TABLE 2 of Pseudopregnancy in CL Rats in Response to Five or Ten Spaced Intromissions Time interval
Number of intromissions 5
10
67
RATS
(mitt)
5
10
20
40
019"
w
216 (331
016 (0)
015 (0)
016 (0)
6114
9112
6112
8112
-
(431
(751
(50)
(67)
‘I Number of rats responding. h Number within parentheses is percentage
of rats responding.
differences within treatment groups were significant in the Fisher exact probability or the Chi-square test. Sensitirvity at different times during the receptive period at proestrus. The previous series of experiments had involved mating LD rats as much as 3 hr apart in time. The possibility of a systematic variation in sensitivity to the induction of pseudopregnancy by coital stimulation during the receptive period of the normal cycle was investigated both to see whether the rather minor changes seen in the LD rats in the previous experiment (Table 3) might be due to such a variation, and also to see whether valid comparisons could be made between the sensitivity of the LD rats of the strain used and that reported by other workers for different strains, who Incidence
Type
TABLE 3 of Pseudopregnancy in Response to Ten or Fifteen Given as One Group or Split into Two Groups at Different Times Apart
of rat
Number of intromissions
CL
10
0 Ovulation Pseudopregnant
CL
1.5
Ovulation Pseudopregnant
15
Pseudopregnant
60
120
180
14114” ( 1OO)b 6114 (431
12112 (100) 7112
12112 (100) 8112
(58)
IO/l0 (100) 7110 (701
12116 (751 6112 (501
14114 (100) 12114
12112 (100) 12112 (100)
14114 (1W 11114 (79)
14115 (931 II/l4 (79)
3110 (301
5110 (501
319 (331
5112
(42) ’ Number of rats responding. ’ Number within parenthesis is percentage
(min)
30
(86) LD
Interval
--___
Intromissions
of rats responding.
(67)
13115 (871 11/13
(83 l/5
(20)
68
K. BROWN-GRANT
have used mainly rats held under reversed light conditions and tested them near the middle of the dark period. LD rats were tested between 20 min to and 20 min past the hour at the times indicated on the day of proestrus. They received either five mounts while taped and twenty intromissions with or without ejaculations or 10 mounts while taped followed by fifteen intromissions. The LQ and RQ were calculated for each animal and combined for all animals tested at a particular time. No animal was tested more than once on any one day. The results obtained are shown in Table 4. There was a rapid increase in receptivity during the afternoon of proestrus; the LQ value for the group as a whole followed a similar pattern. The RQ for the group as a whole fell as the LQ rose but lagged somewhat behind. RQ values for nonresponders were still increasing at a time when the RQ of responsive rats was falling rapidly. There was no evidence of a systematic change in the incidence of pseudopregnancy in response to a given level of coital stimulation over the period studied, implying that stimulation is as effective in a receptive female with a moderate LQ and a high RQ at, say 1700 hr, as it is in a highly receptive female with a low RQ at, say 2100 hr. Importance of ejaculations for the induction of pseudopregnancy. A retrospective study was made of the relative effectiveness in inducing pseudopregnancy of a given number of intromissions in a particular temporal pattern or at a particular time in proestrus when the series included one or more ejaculations or no ejaculations. The results are given in Table 5. The results for the CL rats suggest and the results for the LD rats show conclusively that the probability of inducing a pseudopregnancy with any particular pattern of coital stimulation or at any time during the period of natural receptivity is much higher when the series of intromissions includes one or more ejaculations than when it does not. The results also demonstrate that the occurrence of an ejaculation, however, is not essential for the induction of pseudopregnancy nor is it, by itself, sufficient. The results of the studies on the effects of increasing levels of stimulation and on spacing intromissions or splitting them into two groups and the study on the effect of stimulation at different times in proestus were all analyzed retrospectively to see whether within each experiment there was any significant variation in the proportion of series with and those without an ejaculation between the different experimental groups. No significant differences were found. Induction
of Pregnancy
Preliminary experiments. In the initial experiment on the induction of pseudopregnancy in CL rats, the animals received twenty intromissions plus one or more ejaculations and were killed after the expected date of delivery. Only two out of seventeen rats had litters (five and ten pups), and none of the others had any signs of implantation sites at autopsy. The
0 Day of estrus. b Number within ’ Nonresponsive
rats
80 _t 5
-
I4
84%
3/16 (19) 17 -t 9 65 i 6 62 -c 8
-
O/6 (0) L 0 53 + 14 53 2 14
parentheses indicates percentage. = LQ s 20; responsive = LQ > 20
of responsive
LQ. all rats RQ. all rats RQ. nonrerponsive~ rats LQ, responsive’ rats RQ, responsive rats Pseudopregnanr rats (20 intromissions) Pseudopregnant rats (IS intromissions)
Number
I5w
819 (89) l/IO
13/14 (93) i/IO (10)
8113 (62) 5112
(42)
4/lO (40)
(IO)
23 f 5
95 2 2
23129 (79) 90?5 26 f 5 382 I5
22 f 3
97 2 I
2413 I (77) 79 2 7 32 f 5 75 + 17
39 2 3
89 t 3
52179 (661 59 2- 5 50 25 3 74 z 7
1700
51 25
85 r 4
20154 (37) 33 2 6 56 z 4 60 + 6
1600
Time tested
(hrj
(40)
4/10
2Oca
(36)
11115 (73) 4111
9+3
96 ? I
(100) 96 2 I 923
25125
2100
Z/IO
2-z
IO0
10110 (loo) 100 212
22cRl
2300
6110
I,1
94 -c I
9110 (90) 89i IO 726 61
TABLE 4 Incidence of Responsivity (LQ > 20), LQ and RQ Values, and Incidence of Pseudopregnancy in LD Rats Tested and Mated at Different Times During the Afternoon and Evening of Proestrus
(88)
718
3~2
919 IIW loo 3~2
0300”
70
K. BROWN-GRANT TABLE 5 Incidence of Pseudopregnancy in CL and LD Rats Following Different Levels of Coital Stimulation Incidence of pseudopregnancy
Level of stimulation CL rats 5 to 20 10
With ejaculation(s) 19125
UW’ 10 I spaced’
12118
(67) 10 I split’
17127
(6% 15 I split’
34141
(83 LD rats 5 to 20 I 15 I split’
20 1 r. timeg
18133 (55) 14131 (45) 15132 (47) 20/28 (71)
30135
8136
(86)
(2-a
1.5125
3121 (14) 4141 (10) 8/16 (50)
(60) 15 I V. timeg
Without ejaculation
12134 (35) 3013.5
(86)
Pa
NS’ NS NS NS
a Probability in the Chi-square test. * I indicates intromissions. c NS indicates P > 0.05. d Percentage within parentheses. e See Table 2. f See Table 3. Q See Table 4.
possibility that the level of coital stimulation might have been inadequate was tested. A group of 15 CL rats were mated with several males successively; each female received 55 to 75 intromissions and 7 to 14 ejaculations. Only one rat failed to ovulate; the other 14 had mucus-containing smears and, at laparotomy on Day 7, they had large corpora lutea in their ovaries and small uteri without fluid but no implantation sites at that time or when they were killed on Day 24. The possibility that eggs shed at the first ovulation after a prolonged period of anovulation were defective was investigated. CL rats were mated (20 intromissions plus one or two ejaculations), and the occurrence of pseudopregnancy was confirmed by vaginal smears. When their smears became proestrous or estrous in character the females were placed with males and, if receptive, they were mated a second time at the same level
PREGNANCY
IN PERSISTENT
ESTROUS
RATS
71
of coital stimulation as before. Of the 28 rats that mated at the end of the first pseudopregnancy, three failed to ovulate, and nine ovulated but did not become pseudopregnant or pregnant. Of the remaining 16 rats, ten were pseudopregnant and six pregnant. Only three had live litters (six, eight, and ten pups), and three produced dead pups some days after the expected date of delivery. LD rats were mated between 1700 and 1900 hr on the day of proestrus at the same level of coital stimulation; of the 17 animals mated, three continued to cycle, seven became pseudopregnant, and seven pregnant. All pregnant rats delivered live litters (mean pup number, 6.0 + 1S). The incidence of pregnancy was low in LD rats as well as in CL rats with this pattern of stimulation. The results for LD rats differed from those for CL rats at the mating that induced the first ovulation (P < 0.02 in the Chisquare test), but the results for the CL rats mated a second time were not significantly different from the LD rats. Influence of the rate of stimulation and of the occurrence of a terminal ejuculation. Two possibilities to explain the low incidence of pregnancy were considered. The matings were carried out with two or more males in the cage and they were replaced at once if they ejaculated. Thus, the pacing of the intromissions was very different from that obtained in the one-to-one situation and ejaculation could occur at any time during the test rather than as the terminal event in a normal ejaculatory series (ES). Both factors might have had an adverse effect on the incidence of pregnancy (Diamond, 1970; Adler and Zoloth, 1970; see also Discussion). CL rats and LD rats between 1700 and 1900 hr of proestrus were each mated with a single fresh male and mating was allowed to continue until the male ejaculated after which he was removed. At this stage, either two or three fresh males were placed in the cage and mating was allowed to continue until eight intromissions without an ejaculation had occurred or a single fresh male was introduced and allowed to mate until he too ejaculated. The matings were concluded by allowing a single male to mate until he ejaculated with females that had already received the series of eight intromissions (ES rats) and by allowing several males to mate with the females that had already had two ES until eight further intromissions had occurred (I rats). Thus, the total level of coital stimulation was approximately the same for the two groups but one had a normal ES with a terminal ejaculation as the last event and the other had eight intromissions. The incidence of pseudopregnancy was determined from the vaginal smears and pregnant animals were allowed to litter or they were killed on Day 24 if they had not littered by then. The results are given in Table 6. For the CL rats, the incidence of pregnancy was not different for the two types of mating, nor did the proportion of pregnant rats littering differ but litter size was significantly lower in the I rats (P < 0.05 in the z-test). The LD rats in the ES group where the terminal event was an
12
K. BROWN-GRANT TABLE 6 Incidence of Pseudopregnancy, Pregnancy, and Litter size in CL Rats and in LD Rats Mated at Proestrus” CL Rats
Number mated Ovulating Pseudopregnant Pregnant
ES
I
ES
I
14 13114 (93Y 4113 (31) 9113
13 12113
11 -
13 -
2111 (18) 9111
9112 (75) 3112 (25) 213
(6% Littering Litter size
LD Rats
419 (44) 6.5 ‘- 1.0
(92) 6112 (50) 6112 (50) 316 (50) 2.7 2 0.3
(821 s/9 (89) 8.4 2 0.9
(67) 4.0 2 2.0
a The ES rats had an ES, eight intromissions, and a second ES; the 1 rats had two ES followed by eight intromissions after the second ejaculation. b Number within parentheses is percentage.
ejaculation had a significantly higher incidence of pregnancies and of live births (P < 0.01 in each case in the Chi-square test). The incidence of live births in pregnant LD rats was significantly higher than in CL rats when both treatment groups were combined (P < 0.05) or for the ES rats alone (P < 0.05). In a final experiment, CL and LD rats at proestrus were mated under observation with several males until 20 intromissions and one or two ejaculations had occurred and then were left overnight in a cage with a single rested male. The cage floors below the wide mesh grill were covered with paper and the number of copulation plugs was counted the next morning when the female was removed and returned to her usual cage in light-dark or constant light, Nine CL rats mated (plug number 6.4 + 1.8), one became pseudopregnant, eight became pregnant, and all delivered live litters (pup number 8.9 r+ 0.6). The incidence of mating, pregnancy, and delivery of a live litter was 100% for the group of seven LD rats; plug number was 5.3 5 0.7 and pup number 9.9 + 0.8 A retrospective analysis of the other experimental records was made for both CL and LD rats, and all instances where a series of intromissions including at least one ejaculation had induced pseudopregnancy and the coexistence of pregnancy could have been verified, if it had occurred, either by birth of a litter or by inspection of the uterus for implantation sites, fetuses, or placental scars were noted and classified on the basis of whether an ejaculation was the terminal event in the mating sequence (TE group) or was followed by one or more intromissions (NTE group). The incidence of pregnancy was significantly higher in the TE groups of LD
PREGNANCY
IN PERSISTENT
ESTROUS
RATS
73
rats (16 out of 25 rats) than in the NTE group (13 out of 54 rats), P < 0.001, but did not differ significantly between the TE and NTE groups of CL rats in the Chi-square test (9 out of 26 rats and 15 out of 84 rats respectively). DISCUSSION
In female rats anovulatory as a result of exposure to constant light, the coital stimulation necessary for the induction of ovulation was qualitatively and quantitatively different from that necessary for the induction of pseudopregnancy. Mounts without intromissions or a small number of intromissions induced ovulation, but did not induce pseudopregnancy (Table 1). Ovulation without a subsequent pseudopregnancy or pregnancy occurred following as many as 15 or more intromissions and one or more ejaculations. Clearly, in CL rats, ovulation induced by mating is not necessarily associated with pseudopregnancy. Essentially the same findings have been reported for the vole (Microtus agrestis), a reflex ovulator (Milligan, 1975). The necessary coital stimulation for the induction of pseudopregnancy in CL rats and in rats of the same strain kept in normal light-dark conditions and mated at proestrous was quite similar. Mounts without intromissions were ineffective in both. The incidence of pseudopregnancy increased with increasing numbers of intromissions in both CL and LD rats, generally reaching 90% or more in rats receiving 20 or more intromissions. When the males were manipulated so that a given number of intromissions occurred at different intervals or a given number of intromissions occurred in two groups different times apart, the effectiveness of the stimulation in inducing pseudopregnancy was not reduced with spacings of from less than 1 to as much as 20 or 40 min in CL rats (Table 2) or with separation of groups of intromissions by up to 180 min in CL or LD rats (Table 3). These results are comparable to those obtained by Edmonds, Zoloth, and Adler (1972) for cyclic rats and suggest that the CL rat can also “store copulatory stimulation” with respect to the maintenance of luteal function, though perhaps not with respect to the induction of ovulation (Table 3). The occurrence of an ejaculation is neither necessary nor in itself sufficient for the induction of pseudopregnancy in either CL or LD rats (Table 5). That an ejaculation was not necessary in cyclic rats was previously demonstrated by Adler (1969), Chester and Zucker (1970) and Edmonds et al. (1972). This finding is in contrast to the situation in the mouse where McGill and Coughlin (1970) have shown that the occurrence of the ejaculatory reflex in the male is essential for the coital induction of persistent luteal activity. In the present experiments, a retrospective analysis of the data showed that the incidence of pseudopregnancy in response to a particular pattern of coital stimulation was higher if one or
74
K. BROWN-GRANT
more ejaculations had occurred than if only intromissions had occurred (Table 5). This finding is not in agreement with the statement of Chester and Zucker (1970) about their own results with cyclic rats or their report of Adler’s conclusions (Adler, 1969). In fact, the results in these two papers do support the view that, though an ejaculation is neither necessary nor sufficient in itself to induce pseudopregnancy, the occurrence of an ejaculation increases the chance of pseudopregnancy occurring. For Chester and Zucker (1970), if the results given in their Tables 1 and 2 are divided into groups receiving one to five, six to ten, or more than ten intromissions, and the incidence of pseudopregnancy in the “with ejaculation” and “without ejaculation” groups are compared, the incidence in the “with ejaculation” group is significantly higher for the groups receiving a small number of intromissions. Similarly, the data of Figs. 1 and 2 of Adler (1969) show highly significant differences when analyzed in this way. The incidence of pregnancy as opposed to pseudopregnancy shows some interesting differences between CL and LD rats. Both types of rats had a low incidence of pregnancy when the level of coital stimulation was 20 intromissions plus one or two ejaculations. Increasing the level to 55 to 75 intromissions and seven to fourteen ejaculations did not increase the incidence of pregnancy in CL rats. When a single male was allowed to mate to satiety overnight with either a CL or an LD female, the incidence of pregnancy was high for both types of females and the incidence of live births and litter size were also both high and not statistically different. In the case of the CL rats, in particular, where a comparable level of stimulation delivered by several males over a short time period was quite ineffective, these results suggest that the normal timing of the coital stimulation was the reason for its greater effectiveness. It seems likely that factors related to sperm deposition and transport or possibly to egg transport may also be involved in these results for CL rats as a relatively high incidence of pseudopregnancy was obtained even with the abnormal pattern of coital stimulation. The state of the genital tract in the CL rat in persistent estrus after 2 to 3 months of exposure to constant light is very different from that of the LD female at proestrus. There is very rarely any detectable luminal fluid in the uterus of the CL rat unless there has been a recent “spontaneous” or induced ovulation as evidenced by the presence of macroscopically obvious corpora lutea in the ovaries (unpublished observation). In contrast, the normal cyclic female at proestrus has a uterus that is ballooned with luminal fluid. It seems likely that the requirements for and the mechanisms of sperm deposition in the uterus and transport to the oviduct will be quite different in these CL animals than those in the normal cyclic rat mated at proestrus (Blandau, 1945; Chester and Zucker, 1970). The critical role of the copulation plug in preventing leakage of ejaculate diluted by luminal fluid in the normal
PREGNANCY
IN PERSISTENT
ESTROUS
RATS
75
female does not seem to be of comparable importance in the CL rat. The findings of Adler and 2010th (1970) were confirmed by the results for LD rats where both experiments specifically designed to test the hypothesis and retrospective analysis of the records of other experiments showed that coital stimulation was far more effective in inducing pregnancy when the terminal event was an ejaculation than when the stimulation ended with one or more intromissions. It seems probable, as suggested by Dziuk (1971), that displacement of the copulation plug which, if in situ, prevents the normal hormonally controlled discharge of luminal fluid through the cervices between proestrus and estrus (Armstrong, 1968), may be the basis of this effect. In unpublished experiments, LD rats were given the same coital stimulation at proestrus as for the I group of Table 6 and were killed the next morning (expected day of estrus, Day 0 of pregnancy). In six out of ten rats, the uterus contained no macroscopically obvious fluid in contrast to the persistent ballooning with turbid fluid normally seen on the morning of Day 0 of pregnancy. The CL rat, whose uterus contains no fluid, did not show dependence on the occurrence of a terminal ejaculation for successful impregnation to any significant degree. A study of the hormonal changes that occur in the CL rat when pregnancy or pseudopregnancy is induced by mating would be of considerable interest. In rats under normal light-dark conditions there are two relatively brief daily spikes of prolactin secretion, a diurnal surge at 1500 to 2100 hr and also a nocturnal surge at 0300 to 0500 hr (Butcher, Fugo, and Collins, 1972). Whether the pseudopregnant or pregnant rat in constant light has a continuously elevated plasma concentration or a random pattern of spikes in prolactin secretion remains to be established. The results may be of value in investigating the mechanism of control of this hormone. ACKNOWLEDGMENTS I am grateful to Mr. M. R. Sherwood for his help in carrying out these experiments. work was supported by grants from the Medical Research Council (U.K.).
The
REFERENCES Adler, N. T. (1969). Effects of the male’s copulatory behavior on successful pregnancy of the female rat. J. Camp. Physiol. Psychoi. 69, 613-622. Adler, N. T., and Zoloth, S. R. (1970). Copulatory behavior can inhibit pregnancy in female rats. Science 168, 1480-1482. Armstrong, D. T. (1968). Hormonal control of uterine lumen fluid retention in the rat. Amer. J. Physiol. 213, 764-771. Blandau, R. J. (1945). On the factors involved in sperm transport through the cervix uteri of the albino rat. Amer. J. Ancrr. 77, 253-272. Brown-Grant, K. (1974). The role of the retina in the failure of ovulation in female rats exposed to constant light. Nrrr,orndocn’noloR!: 16, 243-254.
76
K. BROWN-GRANT
Brown-Grant, K. (1975). A re-examination of the lordosis response in female rats given high doses of testosterone propionate or estradiol benzoate in the neonatal period. Horm. Behav. 6, 351-378. Brown-Grant, K., Davidson, J. M., and Greig, F. (1973). Induced ovulation in albino rats exposed to constant light. J. Endocrinol. 57, 7-22. Brown-Grant, K., and Raisman, G. (1972). Reproductive function in the rat following selective destruction of afferent fibres to the hypothalamus fom the limbic system. Brain Res. 46, 23-42.
Butcher, R. L., Fugo, N. W., and Collins, W. E. (1972). Semicircadian rhythm in plasma levels of prolactin during early gestation in the rat. Endocrinology 90, 1125-l 127. Chester, R. V., and Zucker, 1. (1970). Influence of male copulatory behavior on sperm transport, pregnancy and pseudopregnancy in female rats. Physiol. Behav. 5, 35-43. De Feo, V. J. (1963). Temporal aspect of uterine sensitivity in the pseudopregnant rat. Endocrinology
72, 305-3 16.
Dempsey, E. W., and Searles, H. F. (1943). Environmental modification of certain endocrine phenomena. Endocrinology 32, 119-128. Diamond, M. (1970). Intromission pattern and species vaginal code in relation to induction of pseudopregnancy. Science 169, 995-997. Dziuk, P. (197 I). Copulation and the inhibition of pregnancy in rats. Science 171, 3 Il. Edmonds, S., Zoloth, S. R., and Adler, N. T. (1972). Storage of copulatory stimulation in the female rat. Physiol. Behav. 8, 161-164. Everett, J. W. (1968). “Delayed pseudopregnancy” in the rat, a tool for the study of central neural mechanisms in reproduction. In M. Diamond (Ed.), Perspectives in Reproduclion and Sexual Behavior, Chap. 4, pp. 25-3 I. Indiana University Press, Bloomington. Everett, J. W., and Quinn, D. L. (1966). Differential hypothalamic mechanisms inciting 78, 141-150. ovulation and pseudopregnancy in the rat. Endocrinology Hardy, D. F. (1970). The effect of constant light on the estrous cycle and behavior of the female rat. Physiol. Behav. 5, 421-425. Hardy, D. F. (1972). Sexual behavior in continuously cycling rats. Behaviour 41,288-297. McGill, T. E., and Coughlin, R. C. (1970). Ejaculatory reflex and luteal activity induction in Mus musculus. J. Reprod. Fertil. 21, 215-220. Milligan, S. R. (1975). Mating, ovulation, and corpus luteum function in the vole, Microfus agrestis.
J. Reprod.
Fertil.
Siegel, S. (1956). Nonpuramerric York.
42, 35-44. Statisfics,for
the Behavioral
Sciences. McGraw-Hill,
New
AND
BEHAVIOR
(1977)
8,62-76
The Induction of Pseudopregnancy Mating in Albino Rats Exposed
and Pregnancy by to Constant Light
K. BROWN-GRANT’ Department of Human Anatomy, University of Oxford, and the A.R.C. Institute of Animal Physiology, Babraham, Cambridge, England
Oxford,
Adult female albino rats anovulatory as a result of exposure to constant light (CL rats) were shown to ovulate in response to limited coital stimulation without necessarily becoming either pseudopregnant or pregnant. A higher level of coital stimulation would induce pseudopregnancy provided that intromission was not prevented. The occurrence of an ejaculation was not essential for the induction of pseudopregnancy but the incidence of pseudopregnancy was higher when a series of intromissions included one or more intromissions associated with ejaculation than when it did not. Similar findings regarding the induction of pseudopregnancy were obtained in experiments on female rats maintained under normal light-dark conditions (LD rats). Increasing the interval between intromissions from less than I min to as much as 20 min did not reduce the incidence of pseudopregnancy in CL rats. Splitting a set number of intromissions into two groups separated by up to 180 min did not reduce the incidence of pseudopregnancy in CL or LD rats. Conditions for the induction of pregnancy were more critical than for the induction of pseudopregnancy. CL rats showed only a low incidence of pregnancy and, if pregnant, either had small litters or did not deliver living pups even when a high level of coital stimulation from several males, including multiple ejaculations, occurred over a limited period of time. A high success rate was only observed when CL rats were mated with a single male overnight. LD rats showed a higher incidence of pregnancy than CL rats in response to a restricted amount of coital stimulation over a short time period, especially when an ejaculation was the terminal event in the mating sequence. This dependence on the occurrence of an ejaculation as the terminal event was not observed in CL rats, probably because, unlike LD rats, their uteri were not distended with fluid at the time of mating.
Adult female rats exposed to constant light cease to ovulate and show persistent vaginal cornification. Such animals also show persistent behavioral estrus (Hardy, 1970), but ovulation can be induced by mating or, less consistently, by the injection of progesterone or nonspecific stress, probably as a consequence of the secretion of progesterone by the adrenal cortex (Brown-Grant, Davidson, and Greig, 1973). Dempsey and Searles (1943) showed that at least some female rats exposed to constant light became pseudopregnant after mating. Separate neural mechanisms have been suggested to be involved in the control of reflex ovulation and ’ Medical Research Council Medicine, Memorial University,
External Scientific Staff. St. John’s, Newfoundland,
Present address: Faculty Canada AlC 5S7.
of
62 Copyright All rights
@ 1977 by Academic Press. Inc. of reproductmn in any form reserved.
ISSN 0018-506X
PREGNANCY IN PERSISTENT ESTROUS RATS
63
induction of pseudopregnancy in the rat (Everett, 1968; Everett and Quinn, 1966). If this were so, then mating-induced ovulation in the persistent estrous rat might occur without a subsequent pseudopregnancy or pregnancy. This possibility and the requirements for the induction of pseudopregnancy and pregnancy by mating in rats exposed to constant light have been investigated and compared with the findings in females of the same strain maintained under normal light-dark conditions and mated at proestrus. METHODS
The rats were adults of the albino Wistar strain obtained from Charles River, England, and they were housed six to eight to a cage. Three types of animals were used. Female rats were kept under constant light after their arrival at the laboratory and were used after 8 to 12 weeks of exposure. They are referred to as CL rats (constant light-induced persistent estrous rats). Other females were kept under normal light-dark conditions (lights on at 0500 hr and off at 1900 hr), and daily vaginal smears were obtained for 10 to 20 days; only rats that had shown at least two consecutive 4-day cycles were used. These animals are referred to as LD (light-dark) rats. The males used as stimulus animals were kept under reversed light conditions (lights off at 1100 hr and on at 2300 hr), and tests were carried out during the later part of their dark period. For mating, the females were brought from their home cages to the room housing the males which was dimly lit by low-power red bulbs in the cages where mating took place. Thus, CL rats had up to a 4-hr period of much lower intensity light than usual during the matings and they were returned to cages in constant light after mating. This procedure, without contact with males, did not induce ovulation. The LD rats, depending on the time they were mated, had a period of exposure to light of a reduced intensity either late in their normal lights-on phase or early in their usual dark period before returning to their usual lighting conditions. These procedures, without contact with males, did not induce pseudopregnancy. Matings were generally carried out with two or three males and a single female in the cage, though, in some experiments, a one-to-one arrangement was used as described under Results. The occurrence of mounts, mounts resulting in a lordosis response, lordosis responses associated with intromissions, and intromissions associated with ejaculations were recorded. In addition, the occurrence of incidents of rejection behavior (Hardy, 1972) on the part of the females in response to an attempt of the male to mount was noted. The lordosis quotient [LQ = (number of lordoses/number of mounts) x 1001was calculated for each test, as was the rejection quotient [RQ = (number of episodes of rejection behavior/ number of mounts or attempts to mount) x 1001.The period over which the coital stimulus was received varied from experiment to experiment as
64
K. BROWN-GRANT
the required level of stimulation varied, but the period of contact with the males under observation was generally between 2 and 20 min. Further details of the lighting conditions, the effects on the females, and the performance of the mating tests can be found in Brown-Grant (1974, 1975) and Brown-Grant et al. (1973). In a few preliminary experiments on CL rats, the occurrence of pseudopregnancy was assessed by the examination of daily vaginal smears beginning on the morning of the day after mating (Day 0 of pseudopregnancy or pregnancy), but, in all later experiments, smears were not taken, and the animals were tested for a decidual reaction on Day 4 as will be described. In experiments on LD rats, daily vaginal smears were continued after mating and, if a cornified smear was observed on Day 4, smearing was continued to confirm that the animal was not pseudopregnant and that it continued to show regular 4-day cycles. If the smear on Day 4 was leucocytic, the rats were anesthetized with Avertin (tribromoethanol plus amylene hydrate) and one uterine horn was traumatized along the anti-mesometrial surface of the uterine lumen as described by De Feo (1963). The animals were killed 5 days later, and the weight of the traumatized horn was expressed as a percentage of the control horn. The responses obtained were, in fact, always clearly positive or negative, the weight of the traumatized horn being either 800 to 120% or within + 20% of the weight of the control. The incidence of pregnancy was assessed by allowing the animals to survive to term and noting the number of pups born, or by killing the animals a few days after the expected day of delivery and recording the number of placental scars or fetuses at autopsy. In other experiments, the uterus was examined for implantation sites at autopsy on Day 9 when the decidual response was assessed. Values given in the text and tables are group means t SEM. The t-test or the appropriate nonparametric test (Siegel, 1956) was used to compare groups of animals. A value of P < 0.05 was regarded as significant. RESULTS Induction
of Pseudopregnancy
Preliminary experiments. Twenty-one CL rats were mated; 19 were responsive (LQ > 50) and each received twenty intromissions plus one or two ejaculations. Vaginal smears were taken up to Day 13 and the rats were killed after they had littered or on Day 24. Seventeen of the rats had ovulated and had leucocytic smears up to Day 13 and one set of large corpora lutea in their ovaries when they were killed. Only two rats delivered live litters (five and ten pups), and none of the others had placental scars or any evidence of implantation sites. The incidence of pseudopregnancy appeared to be 10%. An additional 23 CL rats were mated; 21 were responsive and received twenty intromissions plus one or
PREGNANCY
IN PERSISTENT
ESTROLJS RATS
65
two ejaculations. Their uteri were traumatized on Days 3, 4, or 5, and they were killed 5 days later. Sixteen rats had ovulated, and all showed a marked decidual response. A single implantation site was seen in one animal at autopsy but none of the others had any signs of implantation. The weight of the traumatized horn (as a percentage of the control) was 333 2 83% for five rats traumatized on Day 3, 1089 + 113% for six traumatized on Day 4, and 586 + 120% for five traumatized on Day 5. The magnitude of the responses on these 3 days was very close to the values of 207, 1118, and 546% respectively obtained in pseudopregnant LD rats of this strain (Brown-Grant and Raisman, 1972), indicating that the time course of the changes in uterine sensitivity in early pseudopregnancy was not affected by previous or continuing exposure to constant light. As a control procedure, the uteri of five CL rats that had mated but had not ovulated were traumatized on Day 4. When they were killed on Day 9, the traumatized horns were 109 ? 9% of the weight of the control horns. Ovulation without pseudopregnancy. The possibility that matinginduced ovulation was invariably associated with a subsequent pseudopregnancy was investigated in experiments in which CL rats received varying amounts of coital stimulation. Before placing the females with the males the vagina was occluded with surgical tape (Brown-Grant et al., 1973; Hardy, 1970, 1972) so that intromissions could not occur on the initial mounts (taped rats). Each female was mounted 20 to 25 times in all, and, as the LQ was high (90-lOO%), most mounts induced a lordosis response. In some cases, the plaster was not removed until the end of the test (mounts only or zero intromission group). In others, the plaster was removed after 20, 15, 10, or 5 mounts, and mating continued until the appropriate number of intromissions with or without ejaculation occurred (5, 10, 15, or 20, respectively). In this way, the total number of mounts and the duration of exposure to males was equal for each group. All CL rats were traumatized on Day 4 and killed on Day 9; the results are given in Table 1. It is clear that ovulation, based on finding fresh corpora lutea in the ovaries on Day 4, can occur in response to mating without the CL rat becoming pseudopregnant. Two further experiments were carried out. Twelve rats were given an intramuscular injection of 2.5 mg of progesterone in 0.1 ml of oil at 1200 hr; 5 days later, the ovaries were examined for recent corpora lutea. Eight rats had ovulated and in these animals one uterine horn was traumatized. When they were killed 5 days later, the weight of the traumatized horn was 105 + 5% of that of the control horn. Six further CL rats received a subcutaneous injection of 50 pg of ovine LH (NIH-LH-Sl 1) in 0.1 ml of saline. All ovulated, but none showed a decidual response though the traumatized horns were slightly heavier than the controls, 129 2 5%, due possibly to inflammatory changes. Normal LD rats were mated in the same way at proestrus. Failure to
66
K. BROWN-GRANT TABLE I Incidence of Pseudopregnancy after Varying Amounts of Coital Stimulation in Rats Exposed to Constant Light (CL) and in Normal Cyclic Rats (LD)” Number of intromissions
Treatment CL rats Ovulation Pseudopregnant LD rats Pseudopregnant
0
5
10
15
20
911lb
@2Y
10111 (91)
14114 (100)
14114 (100)
II/l1 (100)
019 (0)
O/IO (0)
6114 (43)
12114
(86)
lOill (91)
lill (9)
011 (0)
Iill (9)
5112 (42)
819 (89)
a Rats mated between 1600 and 1800 hr of proestrus. * Number of rats responding. c Number within parentheses is percentage of rats responding.
become pseudopregnant was established by the continuation of a regular 4-day cycle of vaginal smears. If the vaginal smear on Day 4 was leucocytic, the occurrence of pseudopregnancy was confirmed by a positive decidual response to uterine traumatization. The incidence of pseudopregnancy was slightly higher in the CL rats in the 10 and 15 intromissions groups, but the differences were not statistically significant (Table 1). With no or a low number of intromissions pseudopregnancy was rarely induced in either CL or LD rats, but 20 intromissions were generally effective. Variation in the timing of the coital stimulus. CL rats received 20 mounts while taped and then five intromissions within 1 or 2 min or spaced 5, 10,20, or 40 min apart. In a second experiment, each received 15 mounts while taped and then 10 intromissions in 3 to 4 min or spaced 5, 10, or 20 min apart. The results are given in Table 2. Spacing 5 or 10 intromissions in this way did not significantly affect the incidence of pseudopregnancy though there was some indication that the 5-min interval might be the most effective. CL rats received 15 or 10 mounts while taped followed immediately by 10 or 15 intromissions (zero interval group of Table 3) or mounts followed immediately by five, seven, or eight intromissions. After an interval of 30 to 180 min, the latter animals received further intromissions to bring the total up to 10 or 15. A similar experiment with fifteen intromissions per rat was carried out on LD rats. The results are given in Table 3. Though there was some indication that with a 180-min gap the incidence of ovulation in CL rats might be reduced, neither these nor any other
PREGNANCY
Incidence
1N PERSISTENT
ESTROUS
TABLE 2 of Pseudopregnancy in CL Rats in Response to Five or Ten Spaced Intromissions Time interval
Number of intromissions 5
10
67
RATS
(mitt)
5
10
20
40
019"
w
216 (331
016 (0)
015 (0)
016 (0)
6114
9112
6112
8112
-
(431
(751
(50)
(67)
‘I Number of rats responding. h Number within parentheses is percentage
of rats responding.
differences within treatment groups were significant in the Fisher exact probability or the Chi-square test. Sensitirvity at different times during the receptive period at proestrus. The previous series of experiments had involved mating LD rats as much as 3 hr apart in time. The possibility of a systematic variation in sensitivity to the induction of pseudopregnancy by coital stimulation during the receptive period of the normal cycle was investigated both to see whether the rather minor changes seen in the LD rats in the previous experiment (Table 3) might be due to such a variation, and also to see whether valid comparisons could be made between the sensitivity of the LD rats of the strain used and that reported by other workers for different strains, who Incidence
Type
TABLE 3 of Pseudopregnancy in Response to Ten or Fifteen Given as One Group or Split into Two Groups at Different Times Apart
of rat
Number of intromissions
CL
10
0 Ovulation Pseudopregnant
CL
1.5
Ovulation Pseudopregnant
15
Pseudopregnant
60
120
180
14114” ( 1OO)b 6114 (431
12112 (100) 7112
12112 (100) 8112
(58)
IO/l0 (100) 7110 (701
12116 (751 6112 (501
14114 (100) 12114
12112 (100) 12112 (100)
14114 (1W 11114 (79)
14115 (931 II/l4 (79)
3110 (301
5110 (501
319 (331
5112
(42) ’ Number of rats responding. ’ Number within parenthesis is percentage
(min)
30
(86) LD
Interval
--___
Intromissions
of rats responding.
(67)
13115 (871 11/13
(83 l/5
(20)
68
K. BROWN-GRANT
have used mainly rats held under reversed light conditions and tested them near the middle of the dark period. LD rats were tested between 20 min to and 20 min past the hour at the times indicated on the day of proestrus. They received either five mounts while taped and twenty intromissions with or without ejaculations or 10 mounts while taped followed by fifteen intromissions. The LQ and RQ were calculated for each animal and combined for all animals tested at a particular time. No animal was tested more than once on any one day. The results obtained are shown in Table 4. There was a rapid increase in receptivity during the afternoon of proestrus; the LQ value for the group as a whole followed a similar pattern. The RQ for the group as a whole fell as the LQ rose but lagged somewhat behind. RQ values for nonresponders were still increasing at a time when the RQ of responsive rats was falling rapidly. There was no evidence of a systematic change in the incidence of pseudopregnancy in response to a given level of coital stimulation over the period studied, implying that stimulation is as effective in a receptive female with a moderate LQ and a high RQ at, say 1700 hr, as it is in a highly receptive female with a low RQ at, say 2100 hr. Importance of ejaculations for the induction of pseudopregnancy. A retrospective study was made of the relative effectiveness in inducing pseudopregnancy of a given number of intromissions in a particular temporal pattern or at a particular time in proestrus when the series included one or more ejaculations or no ejaculations. The results are given in Table 5. The results for the CL rats suggest and the results for the LD rats show conclusively that the probability of inducing a pseudopregnancy with any particular pattern of coital stimulation or at any time during the period of natural receptivity is much higher when the series of intromissions includes one or more ejaculations than when it does not. The results also demonstrate that the occurrence of an ejaculation, however, is not essential for the induction of pseudopregnancy nor is it, by itself, sufficient. The results of the studies on the effects of increasing levels of stimulation and on spacing intromissions or splitting them into two groups and the study on the effect of stimulation at different times in proestus were all analyzed retrospectively to see whether within each experiment there was any significant variation in the proportion of series with and those without an ejaculation between the different experimental groups. No significant differences were found. Induction
of Pregnancy
Preliminary experiments. In the initial experiment on the induction of pseudopregnancy in CL rats, the animals received twenty intromissions plus one or more ejaculations and were killed after the expected date of delivery. Only two out of seventeen rats had litters (five and ten pups), and none of the others had any signs of implantation sites at autopsy. The
0 Day of estrus. b Number within ’ Nonresponsive
rats
80 _t 5
-
I4
84%
3/16 (19) 17 -t 9 65 i 6 62 -c 8
-
O/6 (0) L 0 53 + 14 53 2 14
parentheses indicates percentage. = LQ s 20; responsive = LQ > 20
of responsive
LQ. all rats RQ. all rats RQ. nonrerponsive~ rats LQ, responsive’ rats RQ, responsive rats Pseudopregnanr rats (20 intromissions) Pseudopregnant rats (IS intromissions)
Number
I5w
819 (89) l/IO
13/14 (93) i/IO (10)
8113 (62) 5112
(42)
4/lO (40)
(IO)
23 f 5
95 2 2
23129 (79) 90?5 26 f 5 382 I5
22 f 3
97 2 I
2413 I (77) 79 2 7 32 f 5 75 + 17
39 2 3
89 t 3
52179 (661 59 2- 5 50 25 3 74 z 7
1700
51 25
85 r 4
20154 (37) 33 2 6 56 z 4 60 + 6
1600
Time tested
(hrj
(40)
4/10
2Oca
(36)
11115 (73) 4111
9+3
96 ? I
(100) 96 2 I 923
25125
2100
Z/IO
2-z
IO0
10110 (loo) 100 212
22cRl
2300
6110
I,1
94 -c I
9110 (90) 89i IO 726 61
TABLE 4 Incidence of Responsivity (LQ > 20), LQ and RQ Values, and Incidence of Pseudopregnancy in LD Rats Tested and Mated at Different Times During the Afternoon and Evening of Proestrus
(88)
718
3~2
919 IIW loo 3~2
0300”
70
K. BROWN-GRANT TABLE 5 Incidence of Pseudopregnancy in CL and LD Rats Following Different Levels of Coital Stimulation Incidence of pseudopregnancy
Level of stimulation CL rats 5 to 20 10
With ejaculation(s) 19125
UW’ 10 I spaced’
12118
(67) 10 I split’
17127
(6% 15 I split’
34141
(83 LD rats 5 to 20 I 15 I split’
20 1 r. timeg
18133 (55) 14131 (45) 15132 (47) 20/28 (71)
30135
8136
(86)
(2-a
1.5125
3121 (14) 4141 (10) 8/16 (50)
(60) 15 I V. timeg
Without ejaculation
12134 (35) 3013.5
(86)
Pa
NS’ NS NS NS
a Probability in the Chi-square test. * I indicates intromissions. c NS indicates P > 0.05. d Percentage within parentheses. e See Table 2. f See Table 3. Q See Table 4.
possibility that the level of coital stimulation might have been inadequate was tested. A group of 15 CL rats were mated with several males successively; each female received 55 to 75 intromissions and 7 to 14 ejaculations. Only one rat failed to ovulate; the other 14 had mucus-containing smears and, at laparotomy on Day 7, they had large corpora lutea in their ovaries and small uteri without fluid but no implantation sites at that time or when they were killed on Day 24. The possibility that eggs shed at the first ovulation after a prolonged period of anovulation were defective was investigated. CL rats were mated (20 intromissions plus one or two ejaculations), and the occurrence of pseudopregnancy was confirmed by vaginal smears. When their smears became proestrous or estrous in character the females were placed with males and, if receptive, they were mated a second time at the same level
PREGNANCY
IN PERSISTENT
ESTROUS
RATS
71
of coital stimulation as before. Of the 28 rats that mated at the end of the first pseudopregnancy, three failed to ovulate, and nine ovulated but did not become pseudopregnant or pregnant. Of the remaining 16 rats, ten were pseudopregnant and six pregnant. Only three had live litters (six, eight, and ten pups), and three produced dead pups some days after the expected date of delivery. LD rats were mated between 1700 and 1900 hr on the day of proestrus at the same level of coital stimulation; of the 17 animals mated, three continued to cycle, seven became pseudopregnant, and seven pregnant. All pregnant rats delivered live litters (mean pup number, 6.0 + 1S). The incidence of pregnancy was low in LD rats as well as in CL rats with this pattern of stimulation. The results for LD rats differed from those for CL rats at the mating that induced the first ovulation (P < 0.02 in the Chisquare test), but the results for the CL rats mated a second time were not significantly different from the LD rats. Influence of the rate of stimulation and of the occurrence of a terminal ejuculation. Two possibilities to explain the low incidence of pregnancy were considered. The matings were carried out with two or more males in the cage and they were replaced at once if they ejaculated. Thus, the pacing of the intromissions was very different from that obtained in the one-to-one situation and ejaculation could occur at any time during the test rather than as the terminal event in a normal ejaculatory series (ES). Both factors might have had an adverse effect on the incidence of pregnancy (Diamond, 1970; Adler and Zoloth, 1970; see also Discussion). CL rats and LD rats between 1700 and 1900 hr of proestrus were each mated with a single fresh male and mating was allowed to continue until the male ejaculated after which he was removed. At this stage, either two or three fresh males were placed in the cage and mating was allowed to continue until eight intromissions without an ejaculation had occurred or a single fresh male was introduced and allowed to mate until he too ejaculated. The matings were concluded by allowing a single male to mate until he ejaculated with females that had already received the series of eight intromissions (ES rats) and by allowing several males to mate with the females that had already had two ES until eight further intromissions had occurred (I rats). Thus, the total level of coital stimulation was approximately the same for the two groups but one had a normal ES with a terminal ejaculation as the last event and the other had eight intromissions. The incidence of pseudopregnancy was determined from the vaginal smears and pregnant animals were allowed to litter or they were killed on Day 24 if they had not littered by then. The results are given in Table 6. For the CL rats, the incidence of pregnancy was not different for the two types of mating, nor did the proportion of pregnant rats littering differ but litter size was significantly lower in the I rats (P < 0.05 in the z-test). The LD rats in the ES group where the terminal event was an
12
K. BROWN-GRANT TABLE 6 Incidence of Pseudopregnancy, Pregnancy, and Litter size in CL Rats and in LD Rats Mated at Proestrus” CL Rats
Number mated Ovulating Pseudopregnant Pregnant
ES
I
ES
I
14 13114 (93Y 4113 (31) 9113
13 12113
11 -
13 -
2111 (18) 9111
9112 (75) 3112 (25) 213
(6% Littering Litter size
LD Rats
419 (44) 6.5 ‘- 1.0
(92) 6112 (50) 6112 (50) 316 (50) 2.7 2 0.3
(821 s/9 (89) 8.4 2 0.9
(67) 4.0 2 2.0
a The ES rats had an ES, eight intromissions, and a second ES; the 1 rats had two ES followed by eight intromissions after the second ejaculation. b Number within parentheses is percentage.
ejaculation had a significantly higher incidence of pregnancies and of live births (P < 0.01 in each case in the Chi-square test). The incidence of live births in pregnant LD rats was significantly higher than in CL rats when both treatment groups were combined (P < 0.05) or for the ES rats alone (P < 0.05). In a final experiment, CL and LD rats at proestrus were mated under observation with several males until 20 intromissions and one or two ejaculations had occurred and then were left overnight in a cage with a single rested male. The cage floors below the wide mesh grill were covered with paper and the number of copulation plugs was counted the next morning when the female was removed and returned to her usual cage in light-dark or constant light, Nine CL rats mated (plug number 6.4 + 1.8), one became pseudopregnant, eight became pregnant, and all delivered live litters (pup number 8.9 r+ 0.6). The incidence of mating, pregnancy, and delivery of a live litter was 100% for the group of seven LD rats; plug number was 5.3 5 0.7 and pup number 9.9 + 0.8 A retrospective analysis of the other experimental records was made for both CL and LD rats, and all instances where a series of intromissions including at least one ejaculation had induced pseudopregnancy and the coexistence of pregnancy could have been verified, if it had occurred, either by birth of a litter or by inspection of the uterus for implantation sites, fetuses, or placental scars were noted and classified on the basis of whether an ejaculation was the terminal event in the mating sequence (TE group) or was followed by one or more intromissions (NTE group). The incidence of pregnancy was significantly higher in the TE groups of LD
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rats (16 out of 25 rats) than in the NTE group (13 out of 54 rats), P < 0.001, but did not differ significantly between the TE and NTE groups of CL rats in the Chi-square test (9 out of 26 rats and 15 out of 84 rats respectively). DISCUSSION
In female rats anovulatory as a result of exposure to constant light, the coital stimulation necessary for the induction of ovulation was qualitatively and quantitatively different from that necessary for the induction of pseudopregnancy. Mounts without intromissions or a small number of intromissions induced ovulation, but did not induce pseudopregnancy (Table 1). Ovulation without a subsequent pseudopregnancy or pregnancy occurred following as many as 15 or more intromissions and one or more ejaculations. Clearly, in CL rats, ovulation induced by mating is not necessarily associated with pseudopregnancy. Essentially the same findings have been reported for the vole (Microtus agrestis), a reflex ovulator (Milligan, 1975). The necessary coital stimulation for the induction of pseudopregnancy in CL rats and in rats of the same strain kept in normal light-dark conditions and mated at proestrous was quite similar. Mounts without intromissions were ineffective in both. The incidence of pseudopregnancy increased with increasing numbers of intromissions in both CL and LD rats, generally reaching 90% or more in rats receiving 20 or more intromissions. When the males were manipulated so that a given number of intromissions occurred at different intervals or a given number of intromissions occurred in two groups different times apart, the effectiveness of the stimulation in inducing pseudopregnancy was not reduced with spacings of from less than 1 to as much as 20 or 40 min in CL rats (Table 2) or with separation of groups of intromissions by up to 180 min in CL or LD rats (Table 3). These results are comparable to those obtained by Edmonds, Zoloth, and Adler (1972) for cyclic rats and suggest that the CL rat can also “store copulatory stimulation” with respect to the maintenance of luteal function, though perhaps not with respect to the induction of ovulation (Table 3). The occurrence of an ejaculation is neither necessary nor in itself sufficient for the induction of pseudopregnancy in either CL or LD rats (Table 5). That an ejaculation was not necessary in cyclic rats was previously demonstrated by Adler (1969), Chester and Zucker (1970) and Edmonds et al. (1972). This finding is in contrast to the situation in the mouse where McGill and Coughlin (1970) have shown that the occurrence of the ejaculatory reflex in the male is essential for the coital induction of persistent luteal activity. In the present experiments, a retrospective analysis of the data showed that the incidence of pseudopregnancy in response to a particular pattern of coital stimulation was higher if one or
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more ejaculations had occurred than if only intromissions had occurred (Table 5). This finding is not in agreement with the statement of Chester and Zucker (1970) about their own results with cyclic rats or their report of Adler’s conclusions (Adler, 1969). In fact, the results in these two papers do support the view that, though an ejaculation is neither necessary nor sufficient in itself to induce pseudopregnancy, the occurrence of an ejaculation increases the chance of pseudopregnancy occurring. For Chester and Zucker (1970), if the results given in their Tables 1 and 2 are divided into groups receiving one to five, six to ten, or more than ten intromissions, and the incidence of pseudopregnancy in the “with ejaculation” and “without ejaculation” groups are compared, the incidence in the “with ejaculation” group is significantly higher for the groups receiving a small number of intromissions. Similarly, the data of Figs. 1 and 2 of Adler (1969) show highly significant differences when analyzed in this way. The incidence of pregnancy as opposed to pseudopregnancy shows some interesting differences between CL and LD rats. Both types of rats had a low incidence of pregnancy when the level of coital stimulation was 20 intromissions plus one or two ejaculations. Increasing the level to 55 to 75 intromissions and seven to fourteen ejaculations did not increase the incidence of pregnancy in CL rats. When a single male was allowed to mate to satiety overnight with either a CL or an LD female, the incidence of pregnancy was high for both types of females and the incidence of live births and litter size were also both high and not statistically different. In the case of the CL rats, in particular, where a comparable level of stimulation delivered by several males over a short time period was quite ineffective, these results suggest that the normal timing of the coital stimulation was the reason for its greater effectiveness. It seems likely that factors related to sperm deposition and transport or possibly to egg transport may also be involved in these results for CL rats as a relatively high incidence of pseudopregnancy was obtained even with the abnormal pattern of coital stimulation. The state of the genital tract in the CL rat in persistent estrus after 2 to 3 months of exposure to constant light is very different from that of the LD female at proestrus. There is very rarely any detectable luminal fluid in the uterus of the CL rat unless there has been a recent “spontaneous” or induced ovulation as evidenced by the presence of macroscopically obvious corpora lutea in the ovaries (unpublished observation). In contrast, the normal cyclic female at proestrus has a uterus that is ballooned with luminal fluid. It seems likely that the requirements for and the mechanisms of sperm deposition in the uterus and transport to the oviduct will be quite different in these CL animals than those in the normal cyclic rat mated at proestrus (Blandau, 1945; Chester and Zucker, 1970). The critical role of the copulation plug in preventing leakage of ejaculate diluted by luminal fluid in the normal
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female does not seem to be of comparable importance in the CL rat. The findings of Adler and 2010th (1970) were confirmed by the results for LD rats where both experiments specifically designed to test the hypothesis and retrospective analysis of the records of other experiments showed that coital stimulation was far more effective in inducing pregnancy when the terminal event was an ejaculation than when the stimulation ended with one or more intromissions. It seems probable, as suggested by Dziuk (1971), that displacement of the copulation plug which, if in situ, prevents the normal hormonally controlled discharge of luminal fluid through the cervices between proestrus and estrus (Armstrong, 1968), may be the basis of this effect. In unpublished experiments, LD rats were given the same coital stimulation at proestrus as for the I group of Table 6 and were killed the next morning (expected day of estrus, Day 0 of pregnancy). In six out of ten rats, the uterus contained no macroscopically obvious fluid in contrast to the persistent ballooning with turbid fluid normally seen on the morning of Day 0 of pregnancy. The CL rat, whose uterus contains no fluid, did not show dependence on the occurrence of a terminal ejaculation for successful impregnation to any significant degree. A study of the hormonal changes that occur in the CL rat when pregnancy or pseudopregnancy is induced by mating would be of considerable interest. In rats under normal light-dark conditions there are two relatively brief daily spikes of prolactin secretion, a diurnal surge at 1500 to 2100 hr and also a nocturnal surge at 0300 to 0500 hr (Butcher, Fugo, and Collins, 1972). Whether the pseudopregnant or pregnant rat in constant light has a continuously elevated plasma concentration or a random pattern of spikes in prolactin secretion remains to be established. The results may be of value in investigating the mechanism of control of this hormone. ACKNOWLEDGMENTS I am grateful to Mr. M. R. Sherwood for his help in carrying out these experiments. work was supported by grants from the Medical Research Council (U.K.).
The
REFERENCES Adler, N. T. (1969). Effects of the male’s copulatory behavior on successful pregnancy of the female rat. J. Camp. Physiol. Psychoi. 69, 613-622. Adler, N. T., and Zoloth, S. R. (1970). Copulatory behavior can inhibit pregnancy in female rats. Science 168, 1480-1482. Armstrong, D. T. (1968). Hormonal control of uterine lumen fluid retention in the rat. Amer. J. Physiol. 213, 764-771. Blandau, R. J. (1945). On the factors involved in sperm transport through the cervix uteri of the albino rat. Amer. J. Ancrr. 77, 253-272. Brown-Grant, K. (1974). The role of the retina in the failure of ovulation in female rats exposed to constant light. Nrrr,orndocn’noloR!: 16, 243-254.
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Brown-Grant, K. (1975). A re-examination of the lordosis response in female rats given high doses of testosterone propionate or estradiol benzoate in the neonatal period. Horm. Behav. 6, 351-378. Brown-Grant, K., Davidson, J. M., and Greig, F. (1973). Induced ovulation in albino rats exposed to constant light. J. Endocrinol. 57, 7-22. Brown-Grant, K., and Raisman, G. (1972). Reproductive function in the rat following selective destruction of afferent fibres to the hypothalamus fom the limbic system. Brain Res. 46, 23-42.
Butcher, R. L., Fugo, N. W., and Collins, W. E. (1972). Semicircadian rhythm in plasma levels of prolactin during early gestation in the rat. Endocrinology 90, 1125-l 127. Chester, R. V., and Zucker, 1. (1970). Influence of male copulatory behavior on sperm transport, pregnancy and pseudopregnancy in female rats. Physiol. Behav. 5, 35-43. De Feo, V. J. (1963). Temporal aspect of uterine sensitivity in the pseudopregnant rat. Endocrinology
72, 305-3 16.
Dempsey, E. W., and Searles, H. F. (1943). Environmental modification of certain endocrine phenomena. Endocrinology 32, 119-128. Diamond, M. (1970). Intromission pattern and species vaginal code in relation to induction of pseudopregnancy. Science 169, 995-997. Dziuk, P. (197 I). Copulation and the inhibition of pregnancy in rats. Science 171, 3 Il. Edmonds, S., Zoloth, S. R., and Adler, N. T. (1972). Storage of copulatory stimulation in the female rat. Physiol. Behav. 8, 161-164. Everett, J. W. (1968). “Delayed pseudopregnancy” in the rat, a tool for the study of central neural mechanisms in reproduction. In M. Diamond (Ed.), Perspectives in Reproduclion and Sexual Behavior, Chap. 4, pp. 25-3 I. Indiana University Press, Bloomington. Everett, J. W., and Quinn, D. L. (1966). Differential hypothalamic mechanisms inciting 78, 141-150. ovulation and pseudopregnancy in the rat. Endocrinology Hardy, D. F. (1970). The effect of constant light on the estrous cycle and behavior of the female rat. Physiol. Behav. 5, 421-425. Hardy, D. F. (1972). Sexual behavior in continuously cycling rats. Behaviour 41,288-297. McGill, T. E., and Coughlin, R. C. (1970). Ejaculatory reflex and luteal activity induction in Mus musculus. J. Reprod. Fertil. 21, 215-220. Milligan, S. R. (1975). Mating, ovulation, and corpus luteum function in the vole, Microfus agrestis.
J. Reprod.
Fertil.
Siegel, S. (1956). Nonpuramerric York.
42, 35-44. Statisfics,for
the Behavioral
Sciences. McGraw-Hill,
New