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Effect of Estrogens and Other Compounds as Oral Antifertility Agents on the Development of Rabbit Ova and Hamster Embryos
Effect of Estrogens and Other Compounds as Oral Antifertility Agents on the Development of Rabbit Ova and Hamster Embryos M. C. CHANG, Ph.D., and R. YANAGIMACHI, Ph.D."
in rabbits can be interrupted by the injection of estrogens. 6 , 14, 16 The interruption is due to degeneration or loss of ova before implantation. 7 , 10, 11 Greenwald reported 13 that a single injection of 0.1 mg. estradiol cyclopentylpropionate (ECP) immediately after coitus caused a total death of rabbit embryos 8 days after mating. Feeding 0.5 mg.jkg. daily for 10 days of a derivative of diphenyl-dihydronaphthalane (U-H100A) to 5 rabbits that had just mated, Duncan et aP observed a complete inhibition of pregnancy. The effectiveness and the mechanism of action of several antifertility agents have been repOlted." This paper reports the relative effectiveness of H241, (A-nor androstane-2a-17a-diethynyl-2,B, 17,B-diol), ECP, U-1HOOA, and estrone in producing degeneration of rabbit ova and hamster embryos when orally administered before or after insemination.
PREGNANCY
METHODS
ch
ds
pu Imouse of nt tuof a
ndita-
with a
c, en-
QUES:T.
e (17
tranol L-"e'9
SEY
The methods employed upon the rabbits were the same as those reported previously.5 A group of 6-8 adult female rabbits were inseminated with semen collected from 2-3 fertile bucks, and intravenously injected with LH for the induction of ovulation. Usually, 3 oral doses of the test substance were administered 1, 2, and 3 days after insemination. ECP was dissolved in cottonseed oil, U1HOOA in a diluent, t and H241 in a steroid-suspending From the 'Vorcester Foundation for Experimental Biology, Shrewsbury, Mass., and The Department of Biology, Boston University, Boston, Mass. This work was supported by a grant from the Population Council, Inc., and Grant GM-08167 and Research Career Award 5-K6-HD-18,334 from the U. S. Public Health Service. Sincere thanks are due Miss Dorothy M. Hunt for assistance throughout this study. Thanks are also due Dr. D. J. Patanelli of the Merck Institute and Dr. G. W. Duncan of The Upjohn Co., respectively, for the supplies of H241 and of U-lllOOA used in this study. *Present address: The Zoological Institute, Faculty of Science, Hokkaido University, Sapporo, Japan. tDiluent 122 from the Upjohn Company, Kalamazoo, Mich.
281
TABLE 1. Development of Rabbit Ova After Administration of Antifertility Agents Ova recovered Dose Agent
H241
ECP
V-I1100A
Estrone
Control
mg./mbbit
2 1 0.5 0.2 0.1 1 0.5 0.25 0.2 0.1 20 10 5 2.5 1 1 0.5 0.2 0.1
mg./kg.
0.45 -0.49 0.23 -0.26 0.12 -0.14 0.049-0.057 0.023-0.028 0.22 -0.36 0.10 -0.13 0.054-0.061 0.047-0.059 0.021-0.027 4.25 -5.88 2.15 -2.75 1.16 -1.42 0.60 -0.69 0.24 -0.28 0.23 -0.26 0.12 -0.13 0.05 -0.064 0.026-0.028
No. of rabbits
4 4 6 6 6 7 6 8 6 6 7 6 6 6 6 6 6 6 6 12
Total No. O.L.
43 60 71 69 76 78 63 89 63 63 74 55 65 78 61 68 74 70 71 141
Rabbits were fed on Days 1, 2, and 3 and examined on Day 6. *Calculated according to the total number of corpora lutea.
From tubes (deg.)
Deg.
19 4 3
6 2
9 21 12 23 6 1 15 10 11 17 4 9 3
From uterus
2 3 9 2 9 5 1 8 2 6 1 4 4 5 6
Small
2 3 4 6 1 3 2
1 4 4 9 16 6 6
Normal
7 15 39 13 2 5 37 41 15 10 2 11 21
From vagina (deg.)
3 4 1 4
1
5 1 6 16 106
2
Ova ('10)* Normal
Recov.
10 22 51 17 3 6 59 64 20 18 3 14 34 0 1.4 8.6 23 75
49 22 27 30 55 49 30 46 71 79 30 20 40 35 51 38 51 29 42 87
VOL.
16, No.3, 1965
ESTROGEN ANTIFERTILITY
283
vehicle, * and estrone in cottonseed oil. Six days after insemination, the animals were killed and their fallopian tubes, uterine horns, and vaginas were Hushed for the recovery of eggs. Normal, small, and degenerated blastocysts 5 recovered from the different parts of the tract were recorded and compared with the number of corpora lutea. If there was any evidence that the recovered ova might be unfertilized these ova were mounted and stained by the method of Chang. 3 Animals in which unfertilized ova were found were excluded from the study. This occurred, however, in only a few cases. In other series of experiments, the test substances were fed only once after insemination, 3 times before insemination, or 3 times at the time of implantation, and the ova, blastocysts, or fetuses were examined accordingly. With hamsters, a group of females weighing 130--180 gm. were mated with fertile males in the evening. They were fed with an agent 1, 2, and 3 days or 4, 5, and 6 days after mating, and killed 12 or 13 days after mating. The number of corpora lutea, implantation sites, resorption, and normal fetuses were recorded. RESULTS Rabbit Ova 6 Days After Insemination
The results of this study on rabbits are summarized in Table 1. Fifteen animals were inseminated at different times and served as controls. When examined, 6 days after insemination, no ova were recovered from 1 animal, and 2 of them had 2 crops of corpora lutea and no fertilized ova. The other 12 animals yielded a total of 141 corpora lutea and 123 ova. Of these 123 ova, 3 degenerated ova were recovered from the tube, 2 degenerated ova from the vagina, and 6 small blastocysts (less than 1.5 mm.) and 106 normal blastocysts were recovered from the uterus. The mean size of normal blastocysts from each animal ranged from 2.02 ± 0.66 to 3.77 -+- 0.29 mm. From Table 1 it can be seen that estrone, which is a natural estrogen, is the most effective of the test substances in causing degeneration of rabbit ova when orally administered for 3 days. It caused almost complete degeneration of ova at a dose of 0.5--1 mg. per rabbit. Even at a dose of 0.1 mg., 77% of ova were degenerated. H241 was effective at a dose of 1-2 mg., but about 50% of the ova had degenerated following the feeding of 0.1 mg. per rabbit. Feeding of ECP at 0.25--1 mg. per rabbit caused about 83-97% ova degeneration, although the dose and effect were rather variable. The results of feeding U-ll100A, however, were most inconsistent, degeneration *Developed by the National Institutes of Health.
TABLE 2. Variation of ECP Feedings Ova recovered
C.L.
Prom tubes (deg.)
Deg.
Small
Normal
Normal
Recov.
Dose No. of feedings
3 (Day 1, 2, 3) 1 (Day 1) 1 (Day 3)
Total
Ova (%)*
Prom uteru8
lIIg./rabbit
Mg./kg.
No. of rabbits
0.5
0.10-0.13
6
63
12
3
2
2
3
30
0.5
0.11-0.14
7
69
3
9
7
30
43
71
0.5
0.12-0.14
7
85
0
2
4
42
49
60
Each dose: 0.5 mg./rabbit. *Calculated according to the total number of corpora lutea.
VOL. 16, No.3, 1965
ESTROGEN ANTIFERTILITY
285
of 86--97% of ova occurring at a dose of 2.5-5 mg./rabbit, but less at a dose of 20 mg. or 1 mg./rabbit. As it is known" that only normal blastocysts (as so assessed on Day 6) would implant and develop into normal fetuses, we are confident that the effect of these agents, determined at 6 days after insemination, would offer a good assay for their antifertility activity. The retention of ova in the fallopian tubes 6 days after insemination, when they were expected to be in the uterus, was more obvious following the feeding of ECP (17%) than other agents (7.8-11%, based on the number of corpora lutea). The higher the dose, the more ova tended to remain in the tube, except, perhaps, in the case of U-11100A. The expulsion of ova from the uterus to the vagina was observed when H241, ECP, or U-11100A was fed. No definite conclusion on this point is possible, however, because of the difficulty in recovering ova and the fast degeneration of ova in the vagina. The rate of recovery (the number of ova recovered on' the basis of the total number of corpora lutea) was much decreased following the feeding of any of the agents, but its relation to dosage cannot be established from our limited data, although there were indications that the lower rate of recovery was correlated with an effective dosage. Variation of ECP Feedings
Table 2 presents the results when ECP (0.5 mg. per rabbit) was fed for 3 days or only on Day 1 or Day 3. From the results, it seems obvious that feeding on Day 1 or on Day 3 was much less effective as compared with 3 feedings on Days 1, 2, and 3. Although the other agents were not tested in this fashion, it can be assumed that 3 feedings of these agents would be more effective than 1 feeding. Oral Administration During Implantation
Table 3 presents the results when an agent was fed on Days 6, 7, and 8 after insemination, i.e., at the time of implantation. When examined 20-25 days after insemination, 31-89% implantation sites and 14-77% normalfetuses (in terms of the number of corpora lutea) were found. From the results with this limited number of animals, it appears that oral administration of these agents at the time of implantation has no marked adverse effect on implantation or the development of the embryo. Only in the cases of feeding of estrone or ECP were total implantations reduced. Considering, however, that at this dose level, feeding on Days 1,2, and 3 induced a 80-100% ova-degeneration 6 days after insemination, while 31-89% implantation sites were observed when fed at the time of implantation, it appears that the antifertility action occurred while the ova were in the tube rather than while they were in the uterus at implantation. Furthermore, it should be stressed
286
CHANG &
Y AN AGIMACHI
FERTILITY & STERILITY
TABLE 3. Development of Rabbit Fetuses After Administration of Antifertility Agents Placental degeneration Normal fetuses*
Fetal
Maternal
Implantation
No.
0/0
No.
%
Dose (mg./kg.)
No. of rabbits
Total
C.L.
No.
0/0
No.
0.23-0.29
4t
53
41
77
4
8
2
4
47
89
(1 mg·frab.)
0.23-0.30
4
64
16
25
4
6
8
13
28
44
U-I1100A (20 mg.frab.)
4.1 -5.2
4
60
27
45
6
10
3
5
36
60
0.21-0.28
4 10
51 115
7 74
14 64
8 2
16
1
2
16 76
31 66
Agent
H241 (1 mg.frab.)
0/0
ECP
Estrone
(1 mg.frab.) Controlt
Rabbits were fed on Days 6, 7, and 8 and examined 20-25 days after insemination. *Macerated fetuses: with H241, 2; with ECP, 1; with estrone, 1. tOne animal died and one aborted 19 days after insemination. :tData from Chang and Hunt, 1963.
here that unless the blastocysts are in good physiological condition, the termination of pregnancy cannot be entirely attributed to a disturbance of implantation. Oral Administration Before Insemination
In order to determine whether ovulation and fertilization can be inhibited by these compounds, rabbits were fed 3 times: 2 days, 1 day, and immediately before insemination. Table 4 presents the findings when the animals were examined 2 days after insemination. When H241 and Ull100A were orally administered, the rate of ova recovery was rather low: 36 and 59%, respectively; when ECP and estrone were given, there was no significant effect on the rate of recovery: 93 and 79%, respectively. Among 30 ova, 8 were found unfertilized in the 4 animals treated with estrone, as were 3 of 17 ova from 3 animals treated with U-ll100A, but all the other animals yielded fertilized ova, although 1 degenerated ovum was recovered from the uterus of 1 animal treated with U-ll100A. Except in the animals treated with ECP (93%), the percentage of normal or fertilized ova in terms of the number of corpora lutea was low (36-58%), but it was high (73100%) in terms of the total number of ova recovered. Thus, it appears that these agents have no significant effect on ovulation and fertilization of ova when administered before insemination.
VOL. 16, No.3, 1965 TABLE
287
ESTROGEN ANTIFERTILITY
4. Ovulation and Fertilization When Agents Administered Before Insemination Normal ('70) U'lHt re('(}t~(! rl,tl
Agent
H241 (1 mg.frab.)
No. of rabbits
Total C.L. Total
From tube
From uterus
From vagina
B(UJed onNo. ofC.L.
Based on No.of Recovery ova rec. (0/0 )
3
25
9
6
3
0
36
100
36
3
27
25
25
0
0
93
100
93
3
29
17
16
1"
0
45
77
59
4
38
30
24
4
2
58
73
79
ECP
(1 mg.frab.) U-lllOOA
(20mg·frab.) Estrone
(1 mg.frab.)
In the U-lllOOA group, 3 unfertilized ova were recovered from the tube; in the estrone group, 5 unfertilized ova were recovered from the tube, 1 from the uterus, and 2 from the vagina. Rabbits were fed 2 days, 1 day, and immediately before insemination, and examined 2 days later. *Degenerated.
The fast transportation of ova from the tube to the uterus was shown, however, by the recovery of a few ova from the uterus 2 days after insemination. Since the majority of the ova were normal and still in the fallopian tube 2 days after insemination, treatment before insemination, therefore, does not appear to be effective for antifertility purposes. Examination 2 Days After Insemination
From Table 5 it can be seen that the proportion of ova recovered varied with the different agents, from 46--70%. The percentage of normal ova (at a stage of 12-16 cells), in terms of the total number of ova recovered is quite high-84-100%. The percentages of ova recovered from the uterus were 72, 42, 17, and 14%, respectively, when H241, ECP, U-11100A, and estrone were administered. A few ova were recovered from the vagina. It is obvious that the fast transportation of ova from the tubes to the uterus is the major effect of these agents 5 and is the cause of ova-degeneration. 2 When the ova recovered from the tubes and from the uterus were cultured in rabbit serum and saline (1: 1) for 2 days, 77% of 31 ova recovered from the tubes and 58% of 19 ova recovered from the uterus developed into early blastocysts, indicating that the agents administered were not necessarily toxic to the ova, and that those ova recovered from the uterus were probably in poor physiological condition.
288
CHANG & Y AN AGIMACHI
FERTILITY & STERILITY
TABLE 5. Examination 2 Days After Insemination N ormnl «1<,) (hI"
Agent
H241 (1 mg.Jrab.) ECP (1 mg./rab.) U-ll100A (20 mg./rab.) Estrone (1 mg./rab.)
No. of 7'otal rabbits O.L. Total
rt'rfli'e rt'd
Prom tube
Ra .• ('(l
n".'t,,z
Prom uterll .•
t'ff.t/il1lt
No. ofC.L.
J?rOllt
011
0'" No. of Becoveru OVct rel'. (%)
3
39
18
3
14
1
39
84
46
3
27
19
11
8
0
70
100
70
3
27
18
15
3
0
67
100
67
3
32
22
17
3
2
63
91
69
In the H241 group, 1 ovum recovered from the tube and 1 from the uterus were unfertilized; the ovum recovered from the vagina was degenerated. Rabbits were fed on Day 1 and examined on Day 2.
Study of Hamsters
Adult hamsters were fed with these agents, either 1, 2, and 3 days after mating or at the time of implantation. ECP and estrone were dissolved in 0.5 ml. of cottonseed oil, H241 in 0.1 ml. of the steroid-suspending vehicle and V-ll100A in 0.1 ml. of the diluent mentioned above. The solution was fed to hamsters through a blunt needle (15-gauge) inserted into the back of the mouth or food pouch. From Table 6, it can be seen that at a dose level of 1 mg. per hamster, estrone and ECP were more effective in interTABLE 6. Embryonic Development of Hamsters After Administration of Antifertility Agents
Agent
H241 E.C.P. U-ll100A Estrone Control
Dosel hamster (mg.)
1 0.2 1 1 1 10 1 1
t
Days given
1,2,3 1,2,3 1,2,3 4,5,6 1,2,3 1,2,3 1,2,3 4,5,6 1,2,3
No. mated
No. pregnant
12
4 11
31 153
1 9 8 9 7 8
11
12 10 10 12 10 12 8
*Including all uterine swellings with or without fetuses. tColltrol animals were given 0.5 ml. of cottonseed oil.
Oorpora lutea
Implant. sites*
Normal fetuses
22 107
145 120 121
30 137 3 132 115 113
93 110
91 106
55 104
80 88 91
----
VOL.
16, No.3, 1965
ESTROGEN ANTIFERTILITY
289
rupting pregnancy (100 and 92% of the females failed to become pregnant) than H241 and U-lllOOA (67 and 20% failed). Females with at least one implantation site were considered to be pregnant, and only corpora lutea of pregnant females were counted, not including cyclic corpora lutea. Further, the implantation sites were 0,0.25,2.5, and 11.5jfemale when estrone, ECP, H241, and U-l1100A, respectively were fed on Days 1, 2, and 3. Feeding of 1 mg. of ECP at the time of implantation, on Days 4, 5, and 6, or at a dose of 0.2 mg.jhamster on Days 1,2, and 3, however, were not effective. From this account, it can be said that with the possible exception of U-lllOOA, these agents' antifertility activity was demonstrated in the hamster as it was in the rabbit. Since, however, a hamster weighs only about 150 gm. while a rabbit weighs about 4 kg., it is obvious that a much higher dose is required for effectiveness in the hamster. DISCUSSION
Antifertility activity of a nonsteroid, MER-25, was first reported by Segal and Nelson,17 and then confirmed by Chang. 4 These workers concluded that effects of MER-25 appear to be exerted prior to the exit of ova from the oviduct, and involve the degeneration of ova. A derivative of MER-25, clomiphene, was later examined by Segal and Nelson/ 8 Segal and Davidson, and Chang. 5 Other compounds, such as U-1l555A, a derivative of 2,3,diphenylindene, and U-lllOOA, were also found to be effective when administered at the period of tubal transport of the zygotes. 5 • 8. 9 U-1l555A, however, does not "exhibit significant progestational, antiprogestational, antigonadotropic, androgenic, or estrogenic activities."8 The interruption of pregnancy or other antifertility effects of several estrogens administered by injection was recently investigated by Greenwald whose observations were very similar to those described above. Other steroids that have similar effects have been reported by Banik and Pincus, and Pincus. These workers, however, considered that these compounds are anti-progestins and affect the implantation of fertilized eggs. Comparison of several antifertility agents 5 demonstrated clearly that these agents all had the ability to cause the retention of ova in the tube, the expulsion of ova from the tube or from the uterus, and also the degeneration of ova if they reached the uterus too early, as shown by Chang? The present investigation demonstrates that a natural estrogen, estrone, when administered orally, also can be used effectively as an antifeTtility agent. In view of these findings, it appears that whether the agent is a phenolic steroid, such as estrone, or an A-norsteroid, such as H241, or nonsteroidal, such as U-l1100A or clomiphene, its activity is similar (depending on dosage administered) in affecting tubal ova. Since this activity is also manifested by oral adminis-
290
CHANG
&
YANAGIMACHI
FERTILITY
& STERILITY
tration of a natural steroid, estrone, one is forced to conclude that any such agents, whether or not they have estrogenic activity in other bioassays, certainly have one common estrogenic activity, as reported here. Thus, one may deduce that any compound with estrogenic activity may have antifertility activity if taken at a particular time soon after mating. SUMMARY
A-norsteroid (H241), estradiol cyclopentylpropionate (ECP), a derivative of diphenyl-dihydronaphthalene (U-11100A) and estrone were fed to rabbits 1, 2, and 3 days after insemination in order to test antifertility activities. When examined 6 days after mating, estrone, H241, and ECP in the dose of 0.5 or 1 mg. per rabbit, induced 98-100,90-100, and 83-97% degeneration of ova, respectively. The results of administration of U-11100A were inconsistent; 5 or 10 mg. per rabbit induced 82-97% degeneration of ova. Feeding of ECP, 0.5 mg. per rabbit once on Day 1 or 3 was less effective than feeding 3 times, on Day 1, 2, and 3. Except for estrone and ECP, feeding these compounds on Days 6, 7, and 8, at the time of implantation, had no effect on implantation. Feeding these compounds 3 times before insemination did not inhibit ovulation or fertilization, and did not indicate their antifertility activity. When the animals were fed on Day 1 and examined on Day 2, a certain percentage of ova was found in the uterus sooner than expected, although the morphology (not necessarily the physiology) of the ova recovered from the tube or the uterus was normal. When hamsters were fed with these compounds (with the exception of U-11100A) 3 times on Days 1, 2, and 3 after mating, and examined on Day 12-13, a high percentage of embryonic degeneration was observed at a dose level of 1 mg. per hamster, indicating that a much higher dose level is required for the hamsters than for the rabbits. It is concluded that natural estrogen or any other compounds that have the activity of causing retention of ova in the tube, expulsion of ova, and fast transportation of ova into the uterus, would be effective as an antifertility agent if administered soon after mating. Worcester Foundation Shrewsbury, Mass.
ADDENDUM Groups of 6 rabbits were fed with estradiol on Days 1, 2, and 3 and examined on Day 6. At a dose level of 0.5, 0.2, and 0.1 mg. per rabbit, 3.5% (37% recovery), 7.5% (36% recovery), and 5.2% (43% recovery) of normal blastocysts, respectively, were observed. When stilbestrol, at a dose of 1, 0.5, and 0.1 mg. per rabbit, was similarly fed, 0% (25% recovery), 9.4% (11% recovery), and 62% (69% recovery) of normal blastocysts, respectively, were obtained. As expected, estradiol appears to be more potent than estrone in this respect. After this manuscript was submitted, the action of estrogens on the early pregnancy
VOL. 16, No.3, 1965
ESTROGEN ANTIFERTILITY
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of mice and rats (STONE, G. M., and EMME:-.rS, C. W.,]. Endocrinol. 29:137,147, 1964), and on the transportation of rat eggs (BANIK, U., and PINCUS, G., Proc. Soc. Exp. BioI. & Med. 116:1032, 1964) was published, which also points to the same effect as reported here in the rabbit and hamster.
REFERENCES 1. BANIK, U. K., and PINCUS, G. Effect of steroids anti-progestins on implantation of fertilized eggs of rats and mice. Proc. Soc. Exp. BioI. & Med. 111:595, 1962. 2. CHANG, M. C. Development and fate of transferred rabbit ova or blastocysts in relation to the ovulation time of recipients. J. Exp. Zool. 114:197, 1950. 3. CHANG, M. C. Fertilizability of rabbit ova and the effects of temperature in vitro on their subsequent fertilization and activation in vivo. J. Exp. Zool. 121:351, 1952. 4. CHANG, M. C. Degeneration of ova in the rat and rabbit following oral administration of 1- (P-2-diethylaminoethoxyphenyl) -1-phenyl-2-P-anisylethanol. Endocrinology 65:339, 1959. 5. CHANG, M. C. Effects of certain antifertility agents on the development of rabbit ova. Fertil. & Steril. 15:97, 1964. 6. COURRIER, R, and RAYNAUD, R. Etude quantitative de l'avortment folliculinique chez la lapine, par l'hormone cristalisee. realisation d' un avortment partie!. Compt. rend. Soc. BioI. 116:1073, 1934. 7. CSAPO, A. "The Mechanism of Myometrial Function and its Disorders." In Modern Trends in Obstetrics and Gynecology, ED. by Rowes, K. Butterworth, London, 1955, pp. 20-48. 8. DUNCAN, G. W., STUCKI, J. C., LYSTER, S. C~, and LEDNICER, D. An orally effective mammalian antifertility agent. Proc. Soc. Exp. BioI. & Med. 109:163, 1962. 9. DUNCAN, G. W., LYSTER, S. C., CLARK, J. J., and LEDNICER, D. Antifertility activities of two Diphenyl-Dihydronaphthalene derivatives. Proc. Soc. Exp. BioI. & Med. 112:439, 1963. 10. GREENWALD, G. S. Interruption of pregnancy in the rabbit by the administration of estogen. J. Exp. Zool. 135:461, 1957. 11. GREENWALD, G. S. The comparative effectiveness of estrogens in interrupting pregnancy in the rabbit. Fertil. & Steril. 10:155, 1959. 12. GREENWALD, G. S. The antifertility effects in pregnant rats of a single injection of estradiol cyclopentylpropionate. Endocrinology 69: 1068, 1961. 13. GREENWALD, G. S. Interruption of early pregnancy in the rabbit by a single injection of oestradiol cyclopentylpropionate. J. Endocrinol. 26:133, 1963. 14. HECKEL, G. P., and ALLEN, W. M. Maintenance of the corpus luteum and inhibition of parturition in the rabbit by injection of estrogenic hormone. Endocrinology 24:137, 1939. 15. PINCUS, G. "Frontiers in Methods of Fertility Control." In Human Fertility and Population Problems, ED. by Greep, R O. Schenkman Publishing Co., Cambridge, Mass. 1963, 177-203. 16. PINCUS, G., and KIRSCH, R E. The sterility in rabbits produced by injections of oestrone and related compounds. Am. J. Physiol. 115:219, 1936. 17. SEGAL, S. J., and NELSON, W. O. An orally active compound with antifertility effects in rats. Proc. Soc. Exp. BioI. & Med. 98:431, 1958. 18. SEGAL, S. J., and NELSON, W. O. Antifertility action of chloromiphene (Abstract). Anat. Rec. 139:273, 1961. 19. SEGAL, S. J., and DAVIDSON, O. Prolonged antifertility action of chlomiphene in delayed implantation (Abstract). Anat. Rec. 142:278, 1962.
in rabbits can be interrupted by the injection of estrogens. 6 , 14, 16 The interruption is due to degeneration or loss of ova before implantation. 7 , 10, 11 Greenwald reported 13 that a single injection of 0.1 mg. estradiol cyclopentylpropionate (ECP) immediately after coitus caused a total death of rabbit embryos 8 days after mating. Feeding 0.5 mg.jkg. daily for 10 days of a derivative of diphenyl-dihydronaphthalane (U-H100A) to 5 rabbits that had just mated, Duncan et aP observed a complete inhibition of pregnancy. The effectiveness and the mechanism of action of several antifertility agents have been repOlted." This paper reports the relative effectiveness of H241, (A-nor androstane-2a-17a-diethynyl-2,B, 17,B-diol), ECP, U-1HOOA, and estrone in producing degeneration of rabbit ova and hamster embryos when orally administered before or after insemination.
PREGNANCY
METHODS
ch
ds
pu Imouse of nt tuof a
ndita-
with a
c, en-
QUES:T.
e (17
tranol L-"e'9
SEY
The methods employed upon the rabbits were the same as those reported previously.5 A group of 6-8 adult female rabbits were inseminated with semen collected from 2-3 fertile bucks, and intravenously injected with LH for the induction of ovulation. Usually, 3 oral doses of the test substance were administered 1, 2, and 3 days after insemination. ECP was dissolved in cottonseed oil, U1HOOA in a diluent, t and H241 in a steroid-suspending From the 'Vorcester Foundation for Experimental Biology, Shrewsbury, Mass., and The Department of Biology, Boston University, Boston, Mass. This work was supported by a grant from the Population Council, Inc., and Grant GM-08167 and Research Career Award 5-K6-HD-18,334 from the U. S. Public Health Service. Sincere thanks are due Miss Dorothy M. Hunt for assistance throughout this study. Thanks are also due Dr. D. J. Patanelli of the Merck Institute and Dr. G. W. Duncan of The Upjohn Co., respectively, for the supplies of H241 and of U-lllOOA used in this study. *Present address: The Zoological Institute, Faculty of Science, Hokkaido University, Sapporo, Japan. tDiluent 122 from the Upjohn Company, Kalamazoo, Mich.
281
TABLE 1. Development of Rabbit Ova After Administration of Antifertility Agents Ova recovered Dose Agent
H241
ECP
V-I1100A
Estrone
Control
mg./mbbit
2 1 0.5 0.2 0.1 1 0.5 0.25 0.2 0.1 20 10 5 2.5 1 1 0.5 0.2 0.1
mg./kg.
0.45 -0.49 0.23 -0.26 0.12 -0.14 0.049-0.057 0.023-0.028 0.22 -0.36 0.10 -0.13 0.054-0.061 0.047-0.059 0.021-0.027 4.25 -5.88 2.15 -2.75 1.16 -1.42 0.60 -0.69 0.24 -0.28 0.23 -0.26 0.12 -0.13 0.05 -0.064 0.026-0.028
No. of rabbits
4 4 6 6 6 7 6 8 6 6 7 6 6 6 6 6 6 6 6 12
Total No. O.L.
43 60 71 69 76 78 63 89 63 63 74 55 65 78 61 68 74 70 71 141
Rabbits were fed on Days 1, 2, and 3 and examined on Day 6. *Calculated according to the total number of corpora lutea.
From tubes (deg.)
Deg.
19 4 3
6 2
9 21 12 23 6 1 15 10 11 17 4 9 3
From uterus
2 3 9 2 9 5 1 8 2 6 1 4 4 5 6
Small
2 3 4 6 1 3 2
1 4 4 9 16 6 6
Normal
7 15 39 13 2 5 37 41 15 10 2 11 21
From vagina (deg.)
3 4 1 4
1
5 1 6 16 106
2
Ova ('10)* Normal
Recov.
10 22 51 17 3 6 59 64 20 18 3 14 34 0 1.4 8.6 23 75
49 22 27 30 55 49 30 46 71 79 30 20 40 35 51 38 51 29 42 87
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ESTROGEN ANTIFERTILITY
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vehicle, * and estrone in cottonseed oil. Six days after insemination, the animals were killed and their fallopian tubes, uterine horns, and vaginas were Hushed for the recovery of eggs. Normal, small, and degenerated blastocysts 5 recovered from the different parts of the tract were recorded and compared with the number of corpora lutea. If there was any evidence that the recovered ova might be unfertilized these ova were mounted and stained by the method of Chang. 3 Animals in which unfertilized ova were found were excluded from the study. This occurred, however, in only a few cases. In other series of experiments, the test substances were fed only once after insemination, 3 times before insemination, or 3 times at the time of implantation, and the ova, blastocysts, or fetuses were examined accordingly. With hamsters, a group of females weighing 130--180 gm. were mated with fertile males in the evening. They were fed with an agent 1, 2, and 3 days or 4, 5, and 6 days after mating, and killed 12 or 13 days after mating. The number of corpora lutea, implantation sites, resorption, and normal fetuses were recorded. RESULTS Rabbit Ova 6 Days After Insemination
The results of this study on rabbits are summarized in Table 1. Fifteen animals were inseminated at different times and served as controls. When examined, 6 days after insemination, no ova were recovered from 1 animal, and 2 of them had 2 crops of corpora lutea and no fertilized ova. The other 12 animals yielded a total of 141 corpora lutea and 123 ova. Of these 123 ova, 3 degenerated ova were recovered from the tube, 2 degenerated ova from the vagina, and 6 small blastocysts (less than 1.5 mm.) and 106 normal blastocysts were recovered from the uterus. The mean size of normal blastocysts from each animal ranged from 2.02 ± 0.66 to 3.77 -+- 0.29 mm. From Table 1 it can be seen that estrone, which is a natural estrogen, is the most effective of the test substances in causing degeneration of rabbit ova when orally administered for 3 days. It caused almost complete degeneration of ova at a dose of 0.5--1 mg. per rabbit. Even at a dose of 0.1 mg., 77% of ova were degenerated. H241 was effective at a dose of 1-2 mg., but about 50% of the ova had degenerated following the feeding of 0.1 mg. per rabbit. Feeding of ECP at 0.25--1 mg. per rabbit caused about 83-97% ova degeneration, although the dose and effect were rather variable. The results of feeding U-ll100A, however, were most inconsistent, degeneration *Developed by the National Institutes of Health.
TABLE 2. Variation of ECP Feedings Ova recovered
C.L.
Prom tubes (deg.)
Deg.
Small
Normal
Normal
Recov.
Dose No. of feedings
3 (Day 1, 2, 3) 1 (Day 1) 1 (Day 3)
Total
Ova (%)*
Prom uteru8
lIIg./rabbit
Mg./kg.
No. of rabbits
0.5
0.10-0.13
6
63
12
3
2
2
3
30
0.5
0.11-0.14
7
69
3
9
7
30
43
71
0.5
0.12-0.14
7
85
0
2
4
42
49
60
Each dose: 0.5 mg./rabbit. *Calculated according to the total number of corpora lutea.
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ESTROGEN ANTIFERTILITY
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of 86--97% of ova occurring at a dose of 2.5-5 mg./rabbit, but less at a dose of 20 mg. or 1 mg./rabbit. As it is known" that only normal blastocysts (as so assessed on Day 6) would implant and develop into normal fetuses, we are confident that the effect of these agents, determined at 6 days after insemination, would offer a good assay for their antifertility activity. The retention of ova in the fallopian tubes 6 days after insemination, when they were expected to be in the uterus, was more obvious following the feeding of ECP (17%) than other agents (7.8-11%, based on the number of corpora lutea). The higher the dose, the more ova tended to remain in the tube, except, perhaps, in the case of U-11100A. The expulsion of ova from the uterus to the vagina was observed when H241, ECP, or U-11100A was fed. No definite conclusion on this point is possible, however, because of the difficulty in recovering ova and the fast degeneration of ova in the vagina. The rate of recovery (the number of ova recovered on' the basis of the total number of corpora lutea) was much decreased following the feeding of any of the agents, but its relation to dosage cannot be established from our limited data, although there were indications that the lower rate of recovery was correlated with an effective dosage. Variation of ECP Feedings
Table 2 presents the results when ECP (0.5 mg. per rabbit) was fed for 3 days or only on Day 1 or Day 3. From the results, it seems obvious that feeding on Day 1 or on Day 3 was much less effective as compared with 3 feedings on Days 1, 2, and 3. Although the other agents were not tested in this fashion, it can be assumed that 3 feedings of these agents would be more effective than 1 feeding. Oral Administration During Implantation
Table 3 presents the results when an agent was fed on Days 6, 7, and 8 after insemination, i.e., at the time of implantation. When examined 20-25 days after insemination, 31-89% implantation sites and 14-77% normalfetuses (in terms of the number of corpora lutea) were found. From the results with this limited number of animals, it appears that oral administration of these agents at the time of implantation has no marked adverse effect on implantation or the development of the embryo. Only in the cases of feeding of estrone or ECP were total implantations reduced. Considering, however, that at this dose level, feeding on Days 1,2, and 3 induced a 80-100% ova-degeneration 6 days after insemination, while 31-89% implantation sites were observed when fed at the time of implantation, it appears that the antifertility action occurred while the ova were in the tube rather than while they were in the uterus at implantation. Furthermore, it should be stressed
286
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TABLE 3. Development of Rabbit Fetuses After Administration of Antifertility Agents Placental degeneration Normal fetuses*
Fetal
Maternal
Implantation
No.
0/0
No.
%
Dose (mg./kg.)
No. of rabbits
Total
C.L.
No.
0/0
No.
0.23-0.29
4t
53
41
77
4
8
2
4
47
89
(1 mg·frab.)
0.23-0.30
4
64
16
25
4
6
8
13
28
44
U-I1100A (20 mg.frab.)
4.1 -5.2
4
60
27
45
6
10
3
5
36
60
0.21-0.28
4 10
51 115
7 74
14 64
8 2
16
1
2
16 76
31 66
Agent
H241 (1 mg.frab.)
0/0
ECP
Estrone
(1 mg.frab.) Controlt
Rabbits were fed on Days 6, 7, and 8 and examined 20-25 days after insemination. *Macerated fetuses: with H241, 2; with ECP, 1; with estrone, 1. tOne animal died and one aborted 19 days after insemination. :tData from Chang and Hunt, 1963.
here that unless the blastocysts are in good physiological condition, the termination of pregnancy cannot be entirely attributed to a disturbance of implantation. Oral Administration Before Insemination
In order to determine whether ovulation and fertilization can be inhibited by these compounds, rabbits were fed 3 times: 2 days, 1 day, and immediately before insemination. Table 4 presents the findings when the animals were examined 2 days after insemination. When H241 and Ull100A were orally administered, the rate of ova recovery was rather low: 36 and 59%, respectively; when ECP and estrone were given, there was no significant effect on the rate of recovery: 93 and 79%, respectively. Among 30 ova, 8 were found unfertilized in the 4 animals treated with estrone, as were 3 of 17 ova from 3 animals treated with U-ll100A, but all the other animals yielded fertilized ova, although 1 degenerated ovum was recovered from the uterus of 1 animal treated with U-ll100A. Except in the animals treated with ECP (93%), the percentage of normal or fertilized ova in terms of the number of corpora lutea was low (36-58%), but it was high (73100%) in terms of the total number of ova recovered. Thus, it appears that these agents have no significant effect on ovulation and fertilization of ova when administered before insemination.
VOL. 16, No.3, 1965 TABLE
287
ESTROGEN ANTIFERTILITY
4. Ovulation and Fertilization When Agents Administered Before Insemination Normal ('70) U'lHt re('(}t~(! rl,tl
Agent
H241 (1 mg.frab.)
No. of rabbits
Total C.L. Total
From tube
From uterus
From vagina
B(UJed onNo. ofC.L.
Based on No.of Recovery ova rec. (0/0 )
3
25
9
6
3
0
36
100
36
3
27
25
25
0
0
93
100
93
3
29
17
16
1"
0
45
77
59
4
38
30
24
4
2
58
73
79
ECP
(1 mg.frab.) U-lllOOA
(20mg·frab.) Estrone
(1 mg.frab.)
In the U-lllOOA group, 3 unfertilized ova were recovered from the tube; in the estrone group, 5 unfertilized ova were recovered from the tube, 1 from the uterus, and 2 from the vagina. Rabbits were fed 2 days, 1 day, and immediately before insemination, and examined 2 days later. *Degenerated.
The fast transportation of ova from the tube to the uterus was shown, however, by the recovery of a few ova from the uterus 2 days after insemination. Since the majority of the ova were normal and still in the fallopian tube 2 days after insemination, treatment before insemination, therefore, does not appear to be effective for antifertility purposes. Examination 2 Days After Insemination
From Table 5 it can be seen that the proportion of ova recovered varied with the different agents, from 46--70%. The percentage of normal ova (at a stage of 12-16 cells), in terms of the total number of ova recovered is quite high-84-100%. The percentages of ova recovered from the uterus were 72, 42, 17, and 14%, respectively, when H241, ECP, U-11100A, and estrone were administered. A few ova were recovered from the vagina. It is obvious that the fast transportation of ova from the tubes to the uterus is the major effect of these agents 5 and is the cause of ova-degeneration. 2 When the ova recovered from the tubes and from the uterus were cultured in rabbit serum and saline (1: 1) for 2 days, 77% of 31 ova recovered from the tubes and 58% of 19 ova recovered from the uterus developed into early blastocysts, indicating that the agents administered were not necessarily toxic to the ova, and that those ova recovered from the uterus were probably in poor physiological condition.
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TABLE 5. Examination 2 Days After Insemination N ormnl «1<,) (hI"
Agent
H241 (1 mg.Jrab.) ECP (1 mg./rab.) U-ll100A (20 mg./rab.) Estrone (1 mg./rab.)
No. of 7'otal rabbits O.L. Total
rt'rfli'e rt'd
Prom tube
Ra .• ('(l
n".'t,,z
Prom uterll .•
t'ff.t/il1lt
No. ofC.L.
J?rOllt
011
0'" No. of Becoveru OVct rel'. (%)
3
39
18
3
14
1
39
84
46
3
27
19
11
8
0
70
100
70
3
27
18
15
3
0
67
100
67
3
32
22
17
3
2
63
91
69
In the H241 group, 1 ovum recovered from the tube and 1 from the uterus were unfertilized; the ovum recovered from the vagina was degenerated. Rabbits were fed on Day 1 and examined on Day 2.
Study of Hamsters
Adult hamsters were fed with these agents, either 1, 2, and 3 days after mating or at the time of implantation. ECP and estrone were dissolved in 0.5 ml. of cottonseed oil, H241 in 0.1 ml. of the steroid-suspending vehicle and V-ll100A in 0.1 ml. of the diluent mentioned above. The solution was fed to hamsters through a blunt needle (15-gauge) inserted into the back of the mouth or food pouch. From Table 6, it can be seen that at a dose level of 1 mg. per hamster, estrone and ECP were more effective in interTABLE 6. Embryonic Development of Hamsters After Administration of Antifertility Agents
Agent
H241 E.C.P. U-ll100A Estrone Control
Dosel hamster (mg.)
1 0.2 1 1 1 10 1 1
t
Days given
1,2,3 1,2,3 1,2,3 4,5,6 1,2,3 1,2,3 1,2,3 4,5,6 1,2,3
No. mated
No. pregnant
12
4 11
31 153
1 9 8 9 7 8
11
12 10 10 12 10 12 8
*Including all uterine swellings with or without fetuses. tColltrol animals were given 0.5 ml. of cottonseed oil.
Oorpora lutea
Implant. sites*
Normal fetuses
22 107
145 120 121
30 137 3 132 115 113
93 110
91 106
55 104
80 88 91
----
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ESTROGEN ANTIFERTILITY
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rupting pregnancy (100 and 92% of the females failed to become pregnant) than H241 and U-lllOOA (67 and 20% failed). Females with at least one implantation site were considered to be pregnant, and only corpora lutea of pregnant females were counted, not including cyclic corpora lutea. Further, the implantation sites were 0,0.25,2.5, and 11.5jfemale when estrone, ECP, H241, and U-l1100A, respectively were fed on Days 1, 2, and 3. Feeding of 1 mg. of ECP at the time of implantation, on Days 4, 5, and 6, or at a dose of 0.2 mg.jhamster on Days 1,2, and 3, however, were not effective. From this account, it can be said that with the possible exception of U-lllOOA, these agents' antifertility activity was demonstrated in the hamster as it was in the rabbit. Since, however, a hamster weighs only about 150 gm. while a rabbit weighs about 4 kg., it is obvious that a much higher dose is required for effectiveness in the hamster. DISCUSSION
Antifertility activity of a nonsteroid, MER-25, was first reported by Segal and Nelson,17 and then confirmed by Chang. 4 These workers concluded that effects of MER-25 appear to be exerted prior to the exit of ova from the oviduct, and involve the degeneration of ova. A derivative of MER-25, clomiphene, was later examined by Segal and Nelson/ 8 Segal and Davidson, and Chang. 5 Other compounds, such as U-1l555A, a derivative of 2,3,diphenylindene, and U-lllOOA, were also found to be effective when administered at the period of tubal transport of the zygotes. 5 • 8. 9 U-1l555A, however, does not "exhibit significant progestational, antiprogestational, antigonadotropic, androgenic, or estrogenic activities."8 The interruption of pregnancy or other antifertility effects of several estrogens administered by injection was recently investigated by Greenwald whose observations were very similar to those described above. Other steroids that have similar effects have been reported by Banik and Pincus, and Pincus. These workers, however, considered that these compounds are anti-progestins and affect the implantation of fertilized eggs. Comparison of several antifertility agents 5 demonstrated clearly that these agents all had the ability to cause the retention of ova in the tube, the expulsion of ova from the tube or from the uterus, and also the degeneration of ova if they reached the uterus too early, as shown by Chang? The present investigation demonstrates that a natural estrogen, estrone, when administered orally, also can be used effectively as an antifeTtility agent. In view of these findings, it appears that whether the agent is a phenolic steroid, such as estrone, or an A-norsteroid, such as H241, or nonsteroidal, such as U-l1100A or clomiphene, its activity is similar (depending on dosage administered) in affecting tubal ova. Since this activity is also manifested by oral adminis-
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&
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FERTILITY
& STERILITY
tration of a natural steroid, estrone, one is forced to conclude that any such agents, whether or not they have estrogenic activity in other bioassays, certainly have one common estrogenic activity, as reported here. Thus, one may deduce that any compound with estrogenic activity may have antifertility activity if taken at a particular time soon after mating. SUMMARY
A-norsteroid (H241), estradiol cyclopentylpropionate (ECP), a derivative of diphenyl-dihydronaphthalene (U-11100A) and estrone were fed to rabbits 1, 2, and 3 days after insemination in order to test antifertility activities. When examined 6 days after mating, estrone, H241, and ECP in the dose of 0.5 or 1 mg. per rabbit, induced 98-100,90-100, and 83-97% degeneration of ova, respectively. The results of administration of U-11100A were inconsistent; 5 or 10 mg. per rabbit induced 82-97% degeneration of ova. Feeding of ECP, 0.5 mg. per rabbit once on Day 1 or 3 was less effective than feeding 3 times, on Day 1, 2, and 3. Except for estrone and ECP, feeding these compounds on Days 6, 7, and 8, at the time of implantation, had no effect on implantation. Feeding these compounds 3 times before insemination did not inhibit ovulation or fertilization, and did not indicate their antifertility activity. When the animals were fed on Day 1 and examined on Day 2, a certain percentage of ova was found in the uterus sooner than expected, although the morphology (not necessarily the physiology) of the ova recovered from the tube or the uterus was normal. When hamsters were fed with these compounds (with the exception of U-11100A) 3 times on Days 1, 2, and 3 after mating, and examined on Day 12-13, a high percentage of embryonic degeneration was observed at a dose level of 1 mg. per hamster, indicating that a much higher dose level is required for the hamsters than for the rabbits. It is concluded that natural estrogen or any other compounds that have the activity of causing retention of ova in the tube, expulsion of ova, and fast transportation of ova into the uterus, would be effective as an antifertility agent if administered soon after mating. Worcester Foundation Shrewsbury, Mass.
ADDENDUM Groups of 6 rabbits were fed with estradiol on Days 1, 2, and 3 and examined on Day 6. At a dose level of 0.5, 0.2, and 0.1 mg. per rabbit, 3.5% (37% recovery), 7.5% (36% recovery), and 5.2% (43% recovery) of normal blastocysts, respectively, were observed. When stilbestrol, at a dose of 1, 0.5, and 0.1 mg. per rabbit, was similarly fed, 0% (25% recovery), 9.4% (11% recovery), and 62% (69% recovery) of normal blastocysts, respectively, were obtained. As expected, estradiol appears to be more potent than estrone in this respect. After this manuscript was submitted, the action of estrogens on the early pregnancy
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of mice and rats (STONE, G. M., and EMME:-.rS, C. W.,]. Endocrinol. 29:137,147, 1964), and on the transportation of rat eggs (BANIK, U., and PINCUS, G., Proc. Soc. Exp. BioI. & Med. 116:1032, 1964) was published, which also points to the same effect as reported here in the rabbit and hamster.
REFERENCES 1. BANIK, U. K., and PINCUS, G. Effect of steroids anti-progestins on implantation of fertilized eggs of rats and mice. Proc. Soc. Exp. BioI. & Med. 111:595, 1962. 2. CHANG, M. C. Development and fate of transferred rabbit ova or blastocysts in relation to the ovulation time of recipients. J. Exp. Zool. 114:197, 1950. 3. CHANG, M. C. Fertilizability of rabbit ova and the effects of temperature in vitro on their subsequent fertilization and activation in vivo. J. Exp. Zool. 121:351, 1952. 4. CHANG, M. C. Degeneration of ova in the rat and rabbit following oral administration of 1- (P-2-diethylaminoethoxyphenyl) -1-phenyl-2-P-anisylethanol. Endocrinology 65:339, 1959. 5. CHANG, M. C. Effects of certain antifertility agents on the development of rabbit ova. Fertil. & Steril. 15:97, 1964. 6. COURRIER, R, and RAYNAUD, R. Etude quantitative de l'avortment folliculinique chez la lapine, par l'hormone cristalisee. realisation d' un avortment partie!. Compt. rend. Soc. BioI. 116:1073, 1934. 7. CSAPO, A. "The Mechanism of Myometrial Function and its Disorders." In Modern Trends in Obstetrics and Gynecology, ED. by Rowes, K. Butterworth, London, 1955, pp. 20-48. 8. DUNCAN, G. W., STUCKI, J. C., LYSTER, S. C~, and LEDNICER, D. An orally effective mammalian antifertility agent. Proc. Soc. Exp. BioI. & Med. 109:163, 1962. 9. DUNCAN, G. W., LYSTER, S. C., CLARK, J. J., and LEDNICER, D. Antifertility activities of two Diphenyl-Dihydronaphthalene derivatives. Proc. Soc. Exp. BioI. & Med. 112:439, 1963. 10. GREENWALD, G. S. Interruption of pregnancy in the rabbit by the administration of estogen. J. Exp. Zool. 135:461, 1957. 11. GREENWALD, G. S. The comparative effectiveness of estrogens in interrupting pregnancy in the rabbit. Fertil. & Steril. 10:155, 1959. 12. GREENWALD, G. S. The antifertility effects in pregnant rats of a single injection of estradiol cyclopentylpropionate. Endocrinology 69: 1068, 1961. 13. GREENWALD, G. S. Interruption of early pregnancy in the rabbit by a single injection of oestradiol cyclopentylpropionate. J. Endocrinol. 26:133, 1963. 14. HECKEL, G. P., and ALLEN, W. M. Maintenance of the corpus luteum and inhibition of parturition in the rabbit by injection of estrogenic hormone. Endocrinology 24:137, 1939. 15. PINCUS, G. "Frontiers in Methods of Fertility Control." In Human Fertility and Population Problems, ED. by Greep, R O. Schenkman Publishing Co., Cambridge, Mass. 1963, 177-203. 16. PINCUS, G., and KIRSCH, R E. The sterility in rabbits produced by injections of oestrone and related compounds. Am. J. Physiol. 115:219, 1936. 17. SEGAL, S. J., and NELSON, W. O. An orally active compound with antifertility effects in rats. Proc. Soc. Exp. BioI. & Med. 98:431, 1958. 18. SEGAL, S. J., and NELSON, W. O. Antifertility action of chloromiphene (Abstract). Anat. Rec. 139:273, 1961. 19. SEGAL, S. J., and DAVIDSON, O. Prolonged antifertility action of chlomiphene in delayed implantation (Abstract). Anat. Rec. 142:278, 1962.