- Email: [email protected]
Reproductive function in rats after mifepristone-induced termination of pregnancy
ELSEVIER
Reproductive Function in Rats After Mifepristone-Induced Termination of Pregnancy Carlos M. Telleria and Ricardo
P. Deis
The effect of mifepristone, a potent progesterone receptor antagonist, on the reproductive function during early pregnancy in rats was examined. A single dose of this drug (10 mglkg) WAS injected S.C. at 1200 h on day 4 (Group I), day 7 (Group II) or day 10 (Group III) of pregnancy. Gestation WAS interrupted and the vaginal cytology showed A typical proestrus condition two days after mifepristone treatment in all the groups. When compared with cycling proestrus rats, serum LH concentrations at 1800 h in the mifepristone-induced proestrus were lower in Group I, similar in Group II and higher in Group III, and serum prolactin (PRL) values were lower in Group I, but not different in Groups II and III. Serum progesterone levels were higher in the three experimental groups when compared with cycling proestrus rats, and similar to that of pregnant rats. Rats in Group I showed A significantly lower sexual receptivity and ovulation rate when compared with Groups II and III or cycling proestrus rants.Most of the mifepristone-treated rats that copulated during the night of the induced proestrus did not become pregnant and showed a delayed pseudopregnancy-like condition. These results indicate that mifepristone administered in A single dose to early pregnant rats terminates pregnancy and induces A proestrus condition two days after treatment followed by successful postovulatory contraception. The mifepristone-induced proestrus is characterized by a differential pattern of serum LH, PRL and progesterone concentrations, mating behavior and ovulation rates, depending on the day of pregnancy when mifepristone is administered. CONTRACEPTION 1996; 53:185-190 KEY WORDS:
mifepristone,
RU486,
pregnancy, rat
Laboratorio de Reproducci6n y Lactancia. LARLAC-CONICET, Casil a de Correo 855. 5500 Mendoza, Argentina Carlos M. Telleria, Ph.D., DepartName and address for corr spondence: Avenue, Uniment of Physiology and Bioph sits, M/C 901, 835 South Wolcott icago, IL 60612. Tel: (312) 996.7688; Fax: (312) versity of Illinois at Chicago, Cf 996-1414 Submitted for publication N ~vember 28, 1995 Accepted for publication D d cember 18, 1995
0 1996 Elsevier Science Inc. 655 Avenue of the Americas,
II rights reserved. ew York, NY 10010
Introduction rogesterone plays a crucial role in mammalian reproduction and it is essential for the initiation P and maintenance of pregnancy. This hormone regulates the function of female reproductive tissues112 through an intracellular receptor that is a hormonally regulated transcription factor.‘14 Therefore, the discovery of the progesterone receptor antagonist, mifepristone (see review, Refs. 5,6) allowed its use as a potent contraceptive agent to terminate early or advanced gestation in several species,7,8 thereby supporting the importance of progesterone in the maintenance of pregnancy. Several studies in pregnant rats have focused on the anti-implantation effects,‘j9-” abortifacient actions,7’12-14 or induction of preterm delivery15-l 7 by mifepristone. However, no attempts have been made to evaluate the female fertility immediately after termination of pregnancy induced by mifepristone. A preliminary study from our laboratory reported that early pregnant rats receiving an abortifacient dose of mifepristone showed a vaginal smear corresponding to a proestrus condition two days after treatment, which was designated mifepristone-induced proestrus.‘* Consequently, the present experiments were undertaken to investigate in detail the reproductive function of the mifepristoneinduced proestrus during early pregnancy in rats.
Materials
and Methods
Animals
Virgin female rats bred in our laboratory (originally Wistar strain) and weighing 180-220 g at the start of the experiments were used. They were kept under controlled conditions of light [lights on 0600-2000 h) and temperature (22-24°C); standard rat chow (Nutric, Cordoba, Argentina) and water were available adlibitum. To induce pregnancy, female rats were caged individually with fertile males beginning on the afternoon of proestrus. Positive matings were verified on the following morning by identifying sperm or copulation plug in the vagina. This day was desigISSN 0010.7824/96/$15.00 PII SOOlO-7824(96)00009-O
186
Telleria
and Deis
nated as day 1 of pregnancy. In our laboratory, usually give birth on day 23.
Contraception 199653: 185-l 90
rats
Injections of Mifepristone The progesterone antagonist mifepristone (17Bhydroxy- 11 B-[4-dimethyl-aminophenyll17c~-[ lpropynyll-estra-4,9-diene-3-one; RU- 486), kindly provided by Roussel-Uclaf, Romainville, France, was dissolved in sunflower seed oil at 10 mg/ml and injected S.C. at a single dose of 10 mgfkg. Control rats were injected with the vehicle alone. Experimental Groups In the first experiment, rats were injected with mifepristone at 1200 h on days 4 (Group I), 7 (Group II) or 10 (Group III) of pregnancy. Vaginal smears were taken daily until the animals were killed. Since a vaginal cytology corresponding to proestrus was observed in all mifepristone-treated pregnant rats two days after treatment, blood samples were obtained 54 h after mifepristone or vehicle administration [i.e., at 1800 h of the induced proestrus). The rats treated with mifepristone were killed on the morning following sampling, and the Fallopian tubes were examined for the presence of eggs. Vehicle-treated cycling rats were bled at 1800 h on the proestrus, sacrificed the following morning and used as controls. In the second experiment, groups of pregnant rats treated with mifepristone were caged individually with sexually active males overnight on the day of the induced proestrus and the presence of spermatozoa was checked in the vaginal smear the following morning and after that, vaginal smears were recorded daily. These groups were compared with cycling rats treated with vehicle and caged with males on proestrus. Blood Sampling Blood samples were obtained by cardiac puncture of the conscious animals. Under our conditions, cardiac puncture caused no significant stress to the rats and the serum PRL values obtained in these rats were not different from those obtained in decapitated rats from the same experimental gr0ups.l’ Sera were separated and stored frozen at -20°C until assayed for LH, PRL and progesterone. Decidualization The stimulus for deciduoma induction was provided to the right horn of the uterus by scratching with a hooked needle, as described by De Feo,20 under ether anaesthesia on diestrous day fourth. The left horn served as control and was not scratched. Twelve days after surgery, animals were killed and both horns
were excised and weighed. The uterine weight was used as an index of the responsiveness of the uterus to deciduoma formation. Cervico-vaginal Stimulation Stimulation of the uterine cervix was performed with a wood rod for 30 set at 1300 h on the estrus. This mating stimulus to the uterine cervix induces a state of pseudopregnancy characterized by the induction of a twice-daily pattern of PRL release that ultimately results in an increased and sustained secretion of progesterone.21,22 RIAs Progesterone was assayed using a highly specific antiserum developed in our laboratory.23 The specificity, validity, and reliability of this RIA has been previously reported.23,24 PRL and LH were measured by using materials kindly provided by the NIADDK, Bethesda, MD, USA, as previously described.25 Statistics Data were expressed as means + SEM of groups of six to ten animals. Comparisons between means of two groups were done by Student’s t-test. One-way analysis of variance (ANOVA I) followed by the Tukey’s test was used for multiple comparisons. When variances were not homogeneous, logarithmic transformation of data was applied. When appropriate, the nonparametric Fisher’s exact probability test was used. A value of P>O.O5 was judged not significant.
Results Effect of Mifepristone Administration on Days 4 (Group I), 7 (Group II) or 20 (Group III) of Pregnancy on Serum LH, PRL and Progesterone Levels 54 h After Treatment Pregnancy was interrupted by mifepristone treatment irrespective of the day of administration, and a vaginal proestrus condition was observed two days after treatment in all groups. In control animals, pregnancy was not affected by vehicle administration. LH values were not detectable in serum of pregnant rats treated with vehicle, but increased significantly in the mifepristone-induced proestrus (Figure 1A). When compared with normal cycling rats at 1800 h on proestrus, serum LH concentrations were lower in Group I, similar in Group II and higher in Group III. Serum PRL concentration increased in all the groups of pregnant rats receiving mifepristone with respect to control pregnant rats (Figure 1B). However,
Reproductive
Contraception 1996;53: 185-I 90
2
25
32
20
1
15 E 2 d
Pregnancy
187
10 5
c
After Mifepristone-Terminated
tered on day 4 of pregnancy (i.e., Group I), serum progesterone was not different from control pregnant rats (Figure 1C). When compared with cycling proestrus rats, serum progesterone concentrations in the afternoon of the mifepristone-induced proestrus were higher in the three experimental groups. Interestingly, this increase was more pronounced in Group I.
35 (A) 30
Function
“5
Ovulatory and Mating Responses in the Mifepristone-induced Proestrus in Rats During Early Pregnancy
1 (B)
I
II
Groups
III
Figure 1. Serum concentrations of (A) LH, (B) PRL and (C) progesteroneat 1800 h in the proestrus induced in pregnant rats by treatment with mifepristone (solid bars, 10 mg/kg, s.c.) at 1200 h on day 4 (Group I), day 7 (Group II) or day 10 (Group III) of pregnancy. Control values were obtained from normal pregnant rats (iopenbars) on days 6, 9 or 12 of pregnancy, respectively, treated with vehicle two days before. Data are means f SEM of groups of six to ten animals. The shaded area represents values + SEM at 1800 of a group of eight cyclic rats on proestrus day used ascontrols. ‘P
when compared with cycling proestrus rats, serum PRL levels at 1800 of the mifepristone-induced proestrus were lower : ,n Group I, but not different in Groups II and III. Circulating proge decreased significantly in corresponding to en compared with control pregwas adminis-
The mating behavior and ovulation rates are summarized in Table 1. Ovulation was induced in all the rats treated with mifepristone on day 10 of pregnancy (i.e., Group III) and in 9 out of 12 rats treated on day 7 (i.e., Group II). A significant decrease in the number of ovulating animals occurred in the group receiving mifepristone on day 4 of pregnancy (i.e., Group I) when compared with normal proestrus rats and Groups II and III. On the other hand, the number of ova per ovulating rat was similar to control in Groups II and III, but showed a significant reduction in Group I. Rats treated with mifepristone and mated on the night of the induced proestrus showed a different sexual receptivity to the male, depending on the day of drug administration. Thus, the number of animals showing sperm in the vagina was significantly lower in Group I with respect to control cycling rats caged with males on proestrus. Conversely, no significant differences were observed after treatment with mifepristone in Groups II and III. Only 3 out of 17 animals treated with mifepristone and showing spermatozoa after mating became pregnant. However, each rat delivered only two pups on day 24 of pregnancy.
Induction of a Pseudopregnancy-like Rats Treated with Mifepristone
Condition
in
Most of the mifepristone-treated rats that copulated during the induced proestrus and did not become pregnant showed four days of a particular estrous cycle with a vaginal smear representative of estrus on days 1, 3 and 4, and diestrus on day 2. After this, a period of constant diestrus of 11.O f 0.3 days (n=19) like a pseudopregnancy state was attained irrespective of the day of treatment with mifepristone (Table 1). The remaining rats did not become pregnant and did not show a pseudopregnancy-like period, but had two or three irregular cycles before cycling regularly. To establish that the pseudopregnancy-like condition is the consequence of a coital stimulus which occurred in the ovulatory proestrus induced by mifepristone, we replace the stimulus of the coitus by a mechanical cervico-vaginal stimulation. This was achieved on the estrus following the mifepristone-
188
Telleria
and Deis
Contraception 1996:53:185-190
Table 1. Ovulation rate and mating behavior in the proestrus induced in pregnant rats after treatment with RU486 (10 mgkg-‘) on Days 4 (Group I), 7 (Group II) or 10 (Group III) of pregnancy; proestrus rats with four-day cycles were used as control
Groups
Number of Ovulating Rats
Number of Ova per Ovulating Rat
Sexual Receptivity (Number of Rats Showing Spermatozoa)
Number of Pregnant Rats
Rats Showing a Pseudopregnancy like Period
I II III Control
11/17* 9112 919 11/12
5.8 + 0.7* * 8.0 * 0.8 9.3 + 0.8 10.2 + 0.5
2/9* 6111 9/l 1 11/12
119 l/11 l/11 11/12
319 8/11 8/11 -
Values are mean ?r SEM. ‘Simificantlv (ANOVA followed by Tukey’s multipie
different from the control range test).
enxm IP < 0.05; Fisher’s
induced proestrus in rats treated on day 7 of pregnancy that have a normal ovulation rate. After 3 days with an irregular cycle with most rats showing a vaginal estrus condition, a state of constant diestrus occurred. The length of this period (10.1 * 0.34 days; n=14) was not different from that observed in the mifepristone-treated groups caged with potent males on the night of the induced proestrus. To determine the existence of functional corpora lutea in the pseudopregnancy-like condition observed after mifepristone treatment, a deciduoma response was induced. The ability to achieve a deciduoma response was taken as sufficient evidence of a progestational endometrium.“j The deciduoma was induced by scratching the endometrium on the fourth day of diestrus in a group of rats receiving mifepristone on day 7 of pregnancy. The mean weight of the scratched horns twelve days later was significantly higher (P~0.01) compared with the contralateral horns used as control (830 f 190 mg vs. 222 f 21.5 mg; n=lO).
Discussion Results of this investigation have shown that a single dose of 10 mg mifepristone administration to rats during early pregnancy, interrupts gestation and induces a state of proestrus two days after treatment, followed by successful postovulatory contraception. The proestrus induction by mifepristone is characterized by a differential pattern of serum LH, PRL and progesterone concentrations, mating behavior and ovulation rates, dependent on the day of pregnancy when mifepristone is administered. The possible mechanism by which mifepristone induce modifications in the LH surge seems to be due to a blockade of an inhibitory or a stimulatory effect of progesterone on LH secretion27’28 according to the day when pregnancy is interrupted. Short-term studies show that mifepristone inhibits pituitary gonadotropin secretion, but long-term treatment induces an increase in the mean LH and LH pulse amplitude.’ Antagonizing diestrous progesterone actions in cy-
exact probability
test). *‘P < 0.01 compared
with
control
group
cling rats by mifepristone from metestrus through proestrus, increases basal LH concentrations, but when the mifepristone is given only on the day of proestrus, the LH surge is reduced.29,30 The steroidal milieu in rats treated with mifepristone is characterized by the presence of moderately high levels of estradio131 and abnormally high levels of testosterone, with an inappropriate feedback system that results in a dysregulation of the pituitary LHRH secretion.3283 After withdrawal of the central progesterone action, the hormonal milieu of the hypothalamus-hypophysial axis in the mifepristone-induced proestrus may change according to the day of pregnancy the drug is administered, inducing a different type of LH surge. It is interesting to analyze the high serum progesterone levels observed in the afternoon of the mifepristone-induced proestrus in all the experimental groups, which are almost similar to serum progesterone concentrations in the corresponding days of pregnancy, and significantly higher when compared with levels in control proestrus rats. This high progesterone production may indicate that the corpora lutea of the interrupted pregnancy are still active at the time of the induced proestrus. It is known that during the first week of pregnancy, PRL is released from the pituitary in the form of two large daily surges34,35 that are essential for maintaining basal progesterone production in rats.36 Thus, the two daily surges of PRL in early pregnancy together with the high serum PRL obtained in the induced proestrus may have preserved and activated the corpora lutea in spite of the interrupted pregnancy. High doses of mifepristone in early pregnant rats induce changes characteristic of luteof corpora olysis 10,14~37with a temporary impairment lutea but with full recovery of the function by day 6.l’ It seems that the single dose of mifepristone used in the present study does not induce luteolysis since the serum progesterone levels continue to rise. A similar
result was obtained by Vanage et a13’ after administration of 1 mg/kg of mifepristone on days 7,
Contraception 1996;53:185-190
Reproductive
8 and 9 of pregnancy. It was recently observed that mifepristone induces an increase in serum progesterone concentration when administered directly into the ovarian bursa in proestrus and early pregnat rats25 or in late pregnancy.“’ Thus, other possible explanation for the high levels in serum progesterone observed after mifepristone treatment is to consider a direct ovarian stimullatory effect of mifepristone on progesterone production. Only 2 out of 31 pregnant rats treated with mifepristone started a new pregnancy after mating in the induced proestrus. However, this pregnancy was not normal because rats delivered only two pups on day 24, that is, a day after the control pregnant rats usually give birth. This effect is probably due to a blockade of implantation by the antiprogesterone.‘-l ‘f3s We have to consider that resorption of fetuses from the last pregnancy could be incomplete and the uterus may be incapable of receiving and holding fertilized eggs recently after abortion. However, the animals receiving mifepristone on day 4 of pregnancy, i.e., before implantation, and showing spermatozoa in the vagina the day after the induced proestrus, did not become pregnant. Thus, we consider that a longlasting antiprogestational effect of mifepristone at the uterine level may be the major cause of the postovulatory contraception observed immediately after abortion performed on early pregnancy. Supporting this hypothesis, most animals showed a period of four days with a clear predominance of vaginal cornification after the proestrus-induced mifepristone treatment, due most probably to a local action of the antagonist as indicated by others.29,39 On the other hand, the presence af distended uteri (ballooning) in the first estrus following the induced proestrus (results not shown), is an evidence of a long-lasting antiprogestational action of mifepristone allowing estrogen to act in the oviduct and endometrium without being counteracted by progesterone.40 After a typical four-day cycle showing vaginal cornification, most of the animals caged with fertile males on the night of the mifepristone-induced proestrus showed a pseudopregnant-like condition. Most probably that is the consequence of the copulatory stimuli without having implatation of the fertilized eggs. The clear deciduoma response obtained after scratching the endornetrium on the fourth day of diestrus could be taken as sufficient evidence of a progestational endomet+.rm and a true pseudopregnancy stage.
supported by a grant (PID No. Consejo National de Investiga-
Function
After Mifepristone-Terminated
Pregnancy
189
ciones Cientificas y Tecnicas (CONICET), Argentina, and from the Programa Latinoamericano de Capacitacion e Investigation en Reproduction Humana (PLACIRH) (PLI-014/90). The authors are indebted to the Hormone Distribution Program, NIADDK, for kindly providing the materials for the PRL and LH RIAs, to Roussel-Uclaf (Romainville, France) for providing RU38486 and to Dr. R.K. Srivastava for the careful revision of the manuscript.
References 1. Bardin W, Milgrom E, Mauvis-Jarvis P, eds. Progester-
one and progestins. New York:Raven Press, 1983. 2. Clark JH, Mani SK. Actions of ovarian steroid hormones. In: Knobil E, Neil1 JD, eds. The Physiology of Reproduction, 2nd ed. New York:Raven Press Ltd, 1994: 101l-59. 3. Evans RM. The steroid and thyroid hormone receptor superfamily. Science 1988;240:889-95. 4. O’Malley B. The steroid receptor superfamily: more excitement predicted for the future. Mol Endocrinol 1990; 4:363-9. 5. Mao J, RegelsonW, Kalimi M. Molecular mechanism of RU486 action: a review. Mol Cell Biochem 1992;109: l-8.
6. Spitz IM, Bardin CW. Clinical pharmacology of RU486-an antiprogestin and antiglucocorticoid. Contraception 1993;-48r403-44. 7. Philibert D, Moguilewsky M, Mary I, et al. Pharmacological profile of RU486 in animals. In: The antiprogesterone steroid RU486 and human fertility control. Baulieu EE, Segal SJ, eds. New York:Plenum Press, 1985;49-68. 8. Baulieu EE. RU486: an antiprogestin with contragestive activity in women. In: The antiprogesterone steroid RU486 and human fertility control. Baulieu EE, Segal SJ, eds. New York:Plenum Press, 1985;1-25. 9. Psychoyos A, Prapas I. Inhibition of egg development and implantation in rats after post-coital administration of the progesterone antagonist RU486. J Reprod Fertil 1987;80:487-91. 10. Kawano T, Okamura H, Tajima C, Fukuma K, Katabuchi H. Effect of RU486 on luteal function in the early pregnant rat. J Reprod Fertil 1988;83:279-85. 11. Sarantis L, Roche D, Psychoyos A. Displacement of receptivity for nidation in the rat by the progesterone antagonist RU486: a scanning electron microscopy study. Hum Reprod 1988;3:251-55. 12. Beier S, Kaufmann J, Neef G. Antifertily effects of antigestagens (AG) at various stagesof pregnancy in rats. Acta Endocrinol, Abstract 9, 1986jSuppl 274:8-9. 13. Garfield RE, Gase JM,Baulieu EE. Effect of the antiprogesterone RU486 on preterm birth in the rat. Am J Obstet Gynecol 1987;157:1281-5. 14. Singh G, Singh MM, Maitra SC, et al. Luteolytic action of two antiprogestational agents (RU-38486 and ZK98734) in the rat. J Reprod Fertil 1988;83:73-83. 15. Bose MJ, Germain G, Nicolle A, Mouren M, Philibert D, Baulieu EE. Control of birth in rats by RU486, an antiprogesterone compound. J Reprod Fertil 1987379: l-8. 16. Cabrol D, Carbonne B, Bienkiewicz A, Dallot E, Alj AE, Cedard L. Induction of labor and cervical maturation using mifepristone (RU486) in the late pregnant rat. In-
190
17.
18.
19. 20. 21.
22. 23. 24.
25.
26. 27. 28.
Telleria
and Deis
fluence of a cyclooxygenase inhibitor (Diclofenac). Prostaglandins 1991;42:71-9. Telleria CM, Stocco CO, Deis RP. Luteolytic action of RU486: modulation of luteal 3Bhydroxysteroid dehydrogenase and 20a-hydroxysteroid dehydrogenase activities in late pregnant rats. J Steroid Biochem Mol Biol 1995;52:567-73. Telleria CM, Deis RP. Respuesta diferencial de1 eje hipotalamo-hipfisis-ovario a la action de1 antiprogesterona RU486 (Mifepristona) en ratas preriadas. Proceedings of the IX Annual Meeting of the Society of Biology of Cuyo, Argentina, Abstract 33, 199 1. Vermouth NT, Deis RP. Prolactin release and lactogenesis after ovariectomy in pregnant rats: effect of ovarian hormones. J Endocrinol 1974;63:13-20. De Feo VJ. Temporal aspects of uterine sensitivity in the pseudopregnant or pregnant rats. Endocrinology 1963;72:305-16. Castro-Vazquez A, Luque EH, Carrerio NB. Modulation of sensitivity to cervicovaginal stimulation during the estrous cycle: evidence for an extrapituitary action of LH-RH. Brain Res 1984;305:231-7. Gunnet JW, Freeman ME. The mating-induced release of prolactin: a unique neuroendocrine response. Endocrine Rev 198334:44-61. Bussmann LE, Deis RP. Studies concerning the hormonal induction of lactogenesis by prostaglandin F2a in pregnant rats. J Steroid Biochem 1979;11:1485-9. Telleria CM, Carrizo DG, Deis RP. Levonorgestrel inhibits luteinizing hormone-stimulated progesterone production in rat luteal cells. J Steroid Biochem Mol Biol 1994;50:161-6. Telleria CM, Deis RP. Effect of RU486 on ovarian progesterone production at proestrus and during pregnancy: a possible dual regulation of the biosynthesis of progesterone. J Reprod Fertil 1994;102:379-84. De Feo VJ. Decidualization. In: Cellular biology of the uterus. Wynn R, ed. New York:Plenum Press, 1967j191-290. Caligaris L, Astrada JJ, Taleisnik S. Biphasic effect of progesterone on the release of gonadotropin in rats. Endocrinology 1971;89:331-7. Ortman 0, Emons G, Knuppen R, Catt KL. Inhibitory effects of the antiprogestin RU486 on progesterone actions and luteinizing hormone secretion in pituitary gonadotrophs. J Steroid Biochem 1989;32:291-7.
Contraception 199653: 185-I 90
JE, Bellido C, Galiot F, Lopez FJ, Gay29. Sanchez-Criado tan F. A possible dual mechanism of the anovulatory action of antiprogesterone RU486 in the rat. Biol Reprod 1990;42:877-86. 30. Rao IM, Mahesh VB. Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the pregnant mare’s serum gonadotropinprimed immature rat and the adult rat. Biol Reprod 1986;35:1154-61. 31. Sanchez-Criado JE, Galiot F, Bellido C, Gonzalez D, Tebar M. Hypothalamus-pituitary-ovarian axis in cyclic rats lacking progesterone actions. Biol Reprod 1993j48: 916-25. 32. Schuiling GA, Moes H, Koiter TR. Interdependence of oestradiol and 5a-dihydrotestosterone in modulating LH and FSH secretion in rats. J Endocrinol 1989j123: 257-62. in go33. Carriere PO, Brawer JR, Farookhi R. Alterations nadotropin-releasing hormone-dependent gonadotropin secretion in rats with polycystic ovaries. Biol Reprod 1991345:685-90. RL, Fugo NW, Collins WE. Semicircadian 34. Butcher rhythm in plasma levels of prolactin during early gestation in the rat. Endocrinology 1972;90:1125-7. 35. Freeman ME, Smith MS, Nazian SJ, Neil1 JD. Ovarian and hypothalamic control of the daily surges of prolactin secretion during pseudopregnancy in the rat. Endocrinology 1974;94:875. 36. Gibori G, Keyes PL. The role of estradiol in early pregnancy in the rat. Endocrinology 198Oj106:1584-89. 37. Vanage G, Kadam PA, Bardin CW, Koide SS. Potentiating effect of epostane on pregnancy-terminating activity of RU486 in the rat. Contraception 1993;47:499506. 38. Roblero LS, Croxatto HB. Effect of RU486 on development and implantation of rat embryos. Mol Reprod Develop 1991;29:342-6. 39. van der Schoot P, Bakker GH, Klijn JGM. Effects of the progesterone antagonist RU486 on ovarian activity in the rat. Endocrinology 1987;121:1375-82. 40. Slayden OVD, Hirst JJ, Brenner RM. Estrogen action in the reproductive tract of rhesus monkeys during antiprogestin treatment. Endocrinology 1993; 132: 1845-56.
Reproductive Function in Rats After Mifepristone-Induced Termination of Pregnancy Carlos M. Telleria and Ricardo
P. Deis
The effect of mifepristone, a potent progesterone receptor antagonist, on the reproductive function during early pregnancy in rats was examined. A single dose of this drug (10 mglkg) WAS injected S.C. at 1200 h on day 4 (Group I), day 7 (Group II) or day 10 (Group III) of pregnancy. Gestation WAS interrupted and the vaginal cytology showed A typical proestrus condition two days after mifepristone treatment in all the groups. When compared with cycling proestrus rats, serum LH concentrations at 1800 h in the mifepristone-induced proestrus were lower in Group I, similar in Group II and higher in Group III, and serum prolactin (PRL) values were lower in Group I, but not different in Groups II and III. Serum progesterone levels were higher in the three experimental groups when compared with cycling proestrus rats, and similar to that of pregnant rats. Rats in Group I showed A significantly lower sexual receptivity and ovulation rate when compared with Groups II and III or cycling proestrus rants.Most of the mifepristone-treated rats that copulated during the night of the induced proestrus did not become pregnant and showed a delayed pseudopregnancy-like condition. These results indicate that mifepristone administered in A single dose to early pregnant rats terminates pregnancy and induces A proestrus condition two days after treatment followed by successful postovulatory contraception. The mifepristone-induced proestrus is characterized by a differential pattern of serum LH, PRL and progesterone concentrations, mating behavior and ovulation rates, depending on the day of pregnancy when mifepristone is administered. CONTRACEPTION 1996; 53:185-190 KEY WORDS:
mifepristone,
RU486,
pregnancy, rat
Laboratorio de Reproducci6n y Lactancia. LARLAC-CONICET, Casil a de Correo 855. 5500 Mendoza, Argentina Carlos M. Telleria, Ph.D., DepartName and address for corr spondence: Avenue, Uniment of Physiology and Bioph sits, M/C 901, 835 South Wolcott icago, IL 60612. Tel: (312) 996.7688; Fax: (312) versity of Illinois at Chicago, Cf 996-1414 Submitted for publication N ~vember 28, 1995 Accepted for publication D d cember 18, 1995
0 1996 Elsevier Science Inc. 655 Avenue of the Americas,
II rights reserved. ew York, NY 10010
Introduction rogesterone plays a crucial role in mammalian reproduction and it is essential for the initiation P and maintenance of pregnancy. This hormone regulates the function of female reproductive tissues112 through an intracellular receptor that is a hormonally regulated transcription factor.‘14 Therefore, the discovery of the progesterone receptor antagonist, mifepristone (see review, Refs. 5,6) allowed its use as a potent contraceptive agent to terminate early or advanced gestation in several species,7,8 thereby supporting the importance of progesterone in the maintenance of pregnancy. Several studies in pregnant rats have focused on the anti-implantation effects,‘j9-” abortifacient actions,7’12-14 or induction of preterm delivery15-l 7 by mifepristone. However, no attempts have been made to evaluate the female fertility immediately after termination of pregnancy induced by mifepristone. A preliminary study from our laboratory reported that early pregnant rats receiving an abortifacient dose of mifepristone showed a vaginal smear corresponding to a proestrus condition two days after treatment, which was designated mifepristone-induced proestrus.‘* Consequently, the present experiments were undertaken to investigate in detail the reproductive function of the mifepristoneinduced proestrus during early pregnancy in rats.
Materials
and Methods
Animals
Virgin female rats bred in our laboratory (originally Wistar strain) and weighing 180-220 g at the start of the experiments were used. They were kept under controlled conditions of light [lights on 0600-2000 h) and temperature (22-24°C); standard rat chow (Nutric, Cordoba, Argentina) and water were available adlibitum. To induce pregnancy, female rats were caged individually with fertile males beginning on the afternoon of proestrus. Positive matings were verified on the following morning by identifying sperm or copulation plug in the vagina. This day was desigISSN 0010.7824/96/$15.00 PII SOOlO-7824(96)00009-O
186
Telleria
and Deis
nated as day 1 of pregnancy. In our laboratory, usually give birth on day 23.
Contraception 199653: 185-l 90
rats
Injections of Mifepristone The progesterone antagonist mifepristone (17Bhydroxy- 11 B-[4-dimethyl-aminophenyll17c~-[ lpropynyll-estra-4,9-diene-3-one; RU- 486), kindly provided by Roussel-Uclaf, Romainville, France, was dissolved in sunflower seed oil at 10 mg/ml and injected S.C. at a single dose of 10 mgfkg. Control rats were injected with the vehicle alone. Experimental Groups In the first experiment, rats were injected with mifepristone at 1200 h on days 4 (Group I), 7 (Group II) or 10 (Group III) of pregnancy. Vaginal smears were taken daily until the animals were killed. Since a vaginal cytology corresponding to proestrus was observed in all mifepristone-treated pregnant rats two days after treatment, blood samples were obtained 54 h after mifepristone or vehicle administration [i.e., at 1800 h of the induced proestrus). The rats treated with mifepristone were killed on the morning following sampling, and the Fallopian tubes were examined for the presence of eggs. Vehicle-treated cycling rats were bled at 1800 h on the proestrus, sacrificed the following morning and used as controls. In the second experiment, groups of pregnant rats treated with mifepristone were caged individually with sexually active males overnight on the day of the induced proestrus and the presence of spermatozoa was checked in the vaginal smear the following morning and after that, vaginal smears were recorded daily. These groups were compared with cycling rats treated with vehicle and caged with males on proestrus. Blood Sampling Blood samples were obtained by cardiac puncture of the conscious animals. Under our conditions, cardiac puncture caused no significant stress to the rats and the serum PRL values obtained in these rats were not different from those obtained in decapitated rats from the same experimental gr0ups.l’ Sera were separated and stored frozen at -20°C until assayed for LH, PRL and progesterone. Decidualization The stimulus for deciduoma induction was provided to the right horn of the uterus by scratching with a hooked needle, as described by De Feo,20 under ether anaesthesia on diestrous day fourth. The left horn served as control and was not scratched. Twelve days after surgery, animals were killed and both horns
were excised and weighed. The uterine weight was used as an index of the responsiveness of the uterus to deciduoma formation. Cervico-vaginal Stimulation Stimulation of the uterine cervix was performed with a wood rod for 30 set at 1300 h on the estrus. This mating stimulus to the uterine cervix induces a state of pseudopregnancy characterized by the induction of a twice-daily pattern of PRL release that ultimately results in an increased and sustained secretion of progesterone.21,22 RIAs Progesterone was assayed using a highly specific antiserum developed in our laboratory.23 The specificity, validity, and reliability of this RIA has been previously reported.23,24 PRL and LH were measured by using materials kindly provided by the NIADDK, Bethesda, MD, USA, as previously described.25 Statistics Data were expressed as means + SEM of groups of six to ten animals. Comparisons between means of two groups were done by Student’s t-test. One-way analysis of variance (ANOVA I) followed by the Tukey’s test was used for multiple comparisons. When variances were not homogeneous, logarithmic transformation of data was applied. When appropriate, the nonparametric Fisher’s exact probability test was used. A value of P>O.O5 was judged not significant.
Results Effect of Mifepristone Administration on Days 4 (Group I), 7 (Group II) or 20 (Group III) of Pregnancy on Serum LH, PRL and Progesterone Levels 54 h After Treatment Pregnancy was interrupted by mifepristone treatment irrespective of the day of administration, and a vaginal proestrus condition was observed two days after treatment in all groups. In control animals, pregnancy was not affected by vehicle administration. LH values were not detectable in serum of pregnant rats treated with vehicle, but increased significantly in the mifepristone-induced proestrus (Figure 1A). When compared with normal cycling rats at 1800 h on proestrus, serum LH concentrations were lower in Group I, similar in Group II and higher in Group III. Serum PRL concentration increased in all the groups of pregnant rats receiving mifepristone with respect to control pregnant rats (Figure 1B). However,
Reproductive
Contraception 1996;53: 185-I 90
2
25
32
20
1
15 E 2 d
Pregnancy
187
10 5
c
After Mifepristone-Terminated
tered on day 4 of pregnancy (i.e., Group I), serum progesterone was not different from control pregnant rats (Figure 1C). When compared with cycling proestrus rats, serum progesterone concentrations in the afternoon of the mifepristone-induced proestrus were higher in the three experimental groups. Interestingly, this increase was more pronounced in Group I.
35 (A) 30
Function
“5
Ovulatory and Mating Responses in the Mifepristone-induced Proestrus in Rats During Early Pregnancy
1 (B)
I
II
Groups
III
Figure 1. Serum concentrations of (A) LH, (B) PRL and (C) progesteroneat 1800 h in the proestrus induced in pregnant rats by treatment with mifepristone (solid bars, 10 mg/kg, s.c.) at 1200 h on day 4 (Group I), day 7 (Group II) or day 10 (Group III) of pregnancy. Control values were obtained from normal pregnant rats (iopenbars) on days 6, 9 or 12 of pregnancy, respectively, treated with vehicle two days before. Data are means f SEM of groups of six to ten animals. The shaded area represents values + SEM at 1800 of a group of eight cyclic rats on proestrus day used ascontrols. ‘P
when compared with cycling proestrus rats, serum PRL levels at 1800 of the mifepristone-induced proestrus were lower : ,n Group I, but not different in Groups II and III. Circulating proge decreased significantly in corresponding to en compared with control pregwas adminis-
The mating behavior and ovulation rates are summarized in Table 1. Ovulation was induced in all the rats treated with mifepristone on day 10 of pregnancy (i.e., Group III) and in 9 out of 12 rats treated on day 7 (i.e., Group II). A significant decrease in the number of ovulating animals occurred in the group receiving mifepristone on day 4 of pregnancy (i.e., Group I) when compared with normal proestrus rats and Groups II and III. On the other hand, the number of ova per ovulating rat was similar to control in Groups II and III, but showed a significant reduction in Group I. Rats treated with mifepristone and mated on the night of the induced proestrus showed a different sexual receptivity to the male, depending on the day of drug administration. Thus, the number of animals showing sperm in the vagina was significantly lower in Group I with respect to control cycling rats caged with males on proestrus. Conversely, no significant differences were observed after treatment with mifepristone in Groups II and III. Only 3 out of 17 animals treated with mifepristone and showing spermatozoa after mating became pregnant. However, each rat delivered only two pups on day 24 of pregnancy.
Induction of a Pseudopregnancy-like Rats Treated with Mifepristone
Condition
in
Most of the mifepristone-treated rats that copulated during the induced proestrus and did not become pregnant showed four days of a particular estrous cycle with a vaginal smear representative of estrus on days 1, 3 and 4, and diestrus on day 2. After this, a period of constant diestrus of 11.O f 0.3 days (n=19) like a pseudopregnancy state was attained irrespective of the day of treatment with mifepristone (Table 1). The remaining rats did not become pregnant and did not show a pseudopregnancy-like period, but had two or three irregular cycles before cycling regularly. To establish that the pseudopregnancy-like condition is the consequence of a coital stimulus which occurred in the ovulatory proestrus induced by mifepristone, we replace the stimulus of the coitus by a mechanical cervico-vaginal stimulation. This was achieved on the estrus following the mifepristone-
188
Telleria
and Deis
Contraception 1996:53:185-190
Table 1. Ovulation rate and mating behavior in the proestrus induced in pregnant rats after treatment with RU486 (10 mgkg-‘) on Days 4 (Group I), 7 (Group II) or 10 (Group III) of pregnancy; proestrus rats with four-day cycles were used as control
Groups
Number of Ovulating Rats
Number of Ova per Ovulating Rat
Sexual Receptivity (Number of Rats Showing Spermatozoa)
Number of Pregnant Rats
Rats Showing a Pseudopregnancy like Period
I II III Control
11/17* 9112 919 11/12
5.8 + 0.7* * 8.0 * 0.8 9.3 + 0.8 10.2 + 0.5
2/9* 6111 9/l 1 11/12
119 l/11 l/11 11/12
319 8/11 8/11 -
Values are mean ?r SEM. ‘Simificantlv (ANOVA followed by Tukey’s multipie
different from the control range test).
enxm IP < 0.05; Fisher’s
induced proestrus in rats treated on day 7 of pregnancy that have a normal ovulation rate. After 3 days with an irregular cycle with most rats showing a vaginal estrus condition, a state of constant diestrus occurred. The length of this period (10.1 * 0.34 days; n=14) was not different from that observed in the mifepristone-treated groups caged with potent males on the night of the induced proestrus. To determine the existence of functional corpora lutea in the pseudopregnancy-like condition observed after mifepristone treatment, a deciduoma response was induced. The ability to achieve a deciduoma response was taken as sufficient evidence of a progestational endometrium.“j The deciduoma was induced by scratching the endometrium on the fourth day of diestrus in a group of rats receiving mifepristone on day 7 of pregnancy. The mean weight of the scratched horns twelve days later was significantly higher (P~0.01) compared with the contralateral horns used as control (830 f 190 mg vs. 222 f 21.5 mg; n=lO).
Discussion Results of this investigation have shown that a single dose of 10 mg mifepristone administration to rats during early pregnancy, interrupts gestation and induces a state of proestrus two days after treatment, followed by successful postovulatory contraception. The proestrus induction by mifepristone is characterized by a differential pattern of serum LH, PRL and progesterone concentrations, mating behavior and ovulation rates, dependent on the day of pregnancy when mifepristone is administered. The possible mechanism by which mifepristone induce modifications in the LH surge seems to be due to a blockade of an inhibitory or a stimulatory effect of progesterone on LH secretion27’28 according to the day when pregnancy is interrupted. Short-term studies show that mifepristone inhibits pituitary gonadotropin secretion, but long-term treatment induces an increase in the mean LH and LH pulse amplitude.’ Antagonizing diestrous progesterone actions in cy-
exact probability
test). *‘P < 0.01 compared
with
control
group
cling rats by mifepristone from metestrus through proestrus, increases basal LH concentrations, but when the mifepristone is given only on the day of proestrus, the LH surge is reduced.29,30 The steroidal milieu in rats treated with mifepristone is characterized by the presence of moderately high levels of estradio131 and abnormally high levels of testosterone, with an inappropriate feedback system that results in a dysregulation of the pituitary LHRH secretion.3283 After withdrawal of the central progesterone action, the hormonal milieu of the hypothalamus-hypophysial axis in the mifepristone-induced proestrus may change according to the day of pregnancy the drug is administered, inducing a different type of LH surge. It is interesting to analyze the high serum progesterone levels observed in the afternoon of the mifepristone-induced proestrus in all the experimental groups, which are almost similar to serum progesterone concentrations in the corresponding days of pregnancy, and significantly higher when compared with levels in control proestrus rats. This high progesterone production may indicate that the corpora lutea of the interrupted pregnancy are still active at the time of the induced proestrus. It is known that during the first week of pregnancy, PRL is released from the pituitary in the form of two large daily surges34,35 that are essential for maintaining basal progesterone production in rats.36 Thus, the two daily surges of PRL in early pregnancy together with the high serum PRL obtained in the induced proestrus may have preserved and activated the corpora lutea in spite of the interrupted pregnancy. High doses of mifepristone in early pregnant rats induce changes characteristic of luteof corpora olysis 10,14~37with a temporary impairment lutea but with full recovery of the function by day 6.l’ It seems that the single dose of mifepristone used in the present study does not induce luteolysis since the serum progesterone levels continue to rise. A similar
result was obtained by Vanage et a13’ after administration of 1 mg/kg of mifepristone on days 7,
Contraception 1996;53:185-190
Reproductive
8 and 9 of pregnancy. It was recently observed that mifepristone induces an increase in serum progesterone concentration when administered directly into the ovarian bursa in proestrus and early pregnat rats25 or in late pregnancy.“’ Thus, other possible explanation for the high levels in serum progesterone observed after mifepristone treatment is to consider a direct ovarian stimullatory effect of mifepristone on progesterone production. Only 2 out of 31 pregnant rats treated with mifepristone started a new pregnancy after mating in the induced proestrus. However, this pregnancy was not normal because rats delivered only two pups on day 24, that is, a day after the control pregnant rats usually give birth. This effect is probably due to a blockade of implantation by the antiprogesterone.‘-l ‘f3s We have to consider that resorption of fetuses from the last pregnancy could be incomplete and the uterus may be incapable of receiving and holding fertilized eggs recently after abortion. However, the animals receiving mifepristone on day 4 of pregnancy, i.e., before implantation, and showing spermatozoa in the vagina the day after the induced proestrus, did not become pregnant. Thus, we consider that a longlasting antiprogestational effect of mifepristone at the uterine level may be the major cause of the postovulatory contraception observed immediately after abortion performed on early pregnancy. Supporting this hypothesis, most animals showed a period of four days with a clear predominance of vaginal cornification after the proestrus-induced mifepristone treatment, due most probably to a local action of the antagonist as indicated by others.29,39 On the other hand, the presence af distended uteri (ballooning) in the first estrus following the induced proestrus (results not shown), is an evidence of a long-lasting antiprogestational action of mifepristone allowing estrogen to act in the oviduct and endometrium without being counteracted by progesterone.40 After a typical four-day cycle showing vaginal cornification, most of the animals caged with fertile males on the night of the mifepristone-induced proestrus showed a pseudopregnant-like condition. Most probably that is the consequence of the copulatory stimuli without having implatation of the fertilized eggs. The clear deciduoma response obtained after scratching the endornetrium on the fourth day of diestrus could be taken as sufficient evidence of a progestational endomet+.rm and a true pseudopregnancy stage.
supported by a grant (PID No. Consejo National de Investiga-
Function
After Mifepristone-Terminated
Pregnancy
189
ciones Cientificas y Tecnicas (CONICET), Argentina, and from the Programa Latinoamericano de Capacitacion e Investigation en Reproduction Humana (PLACIRH) (PLI-014/90). The authors are indebted to the Hormone Distribution Program, NIADDK, for kindly providing the materials for the PRL and LH RIAs, to Roussel-Uclaf (Romainville, France) for providing RU38486 and to Dr. R.K. Srivastava for the careful revision of the manuscript.
References 1. Bardin W, Milgrom E, Mauvis-Jarvis P, eds. Progester-
one and progestins. New York:Raven Press, 1983. 2. Clark JH, Mani SK. Actions of ovarian steroid hormones. In: Knobil E, Neil1 JD, eds. The Physiology of Reproduction, 2nd ed. New York:Raven Press Ltd, 1994: 101l-59. 3. Evans RM. The steroid and thyroid hormone receptor superfamily. Science 1988;240:889-95. 4. O’Malley B. The steroid receptor superfamily: more excitement predicted for the future. Mol Endocrinol 1990; 4:363-9. 5. Mao J, RegelsonW, Kalimi M. Molecular mechanism of RU486 action: a review. Mol Cell Biochem 1992;109: l-8.
6. Spitz IM, Bardin CW. Clinical pharmacology of RU486-an antiprogestin and antiglucocorticoid. Contraception 1993;-48r403-44. 7. Philibert D, Moguilewsky M, Mary I, et al. Pharmacological profile of RU486 in animals. In: The antiprogesterone steroid RU486 and human fertility control. Baulieu EE, Segal SJ, eds. New York:Plenum Press, 1985;49-68. 8. Baulieu EE. RU486: an antiprogestin with contragestive activity in women. In: The antiprogesterone steroid RU486 and human fertility control. Baulieu EE, Segal SJ, eds. New York:Plenum Press, 1985;1-25. 9. Psychoyos A, Prapas I. Inhibition of egg development and implantation in rats after post-coital administration of the progesterone antagonist RU486. J Reprod Fertil 1987;80:487-91. 10. Kawano T, Okamura H, Tajima C, Fukuma K, Katabuchi H. Effect of RU486 on luteal function in the early pregnant rat. J Reprod Fertil 1988;83:279-85. 11. Sarantis L, Roche D, Psychoyos A. Displacement of receptivity for nidation in the rat by the progesterone antagonist RU486: a scanning electron microscopy study. Hum Reprod 1988;3:251-55. 12. Beier S, Kaufmann J, Neef G. Antifertily effects of antigestagens (AG) at various stagesof pregnancy in rats. Acta Endocrinol, Abstract 9, 1986jSuppl 274:8-9. 13. Garfield RE, Gase JM,Baulieu EE. Effect of the antiprogesterone RU486 on preterm birth in the rat. Am J Obstet Gynecol 1987;157:1281-5. 14. Singh G, Singh MM, Maitra SC, et al. Luteolytic action of two antiprogestational agents (RU-38486 and ZK98734) in the rat. J Reprod Fertil 1988;83:73-83. 15. Bose MJ, Germain G, Nicolle A, Mouren M, Philibert D, Baulieu EE. Control of birth in rats by RU486, an antiprogesterone compound. J Reprod Fertil 1987379: l-8. 16. Cabrol D, Carbonne B, Bienkiewicz A, Dallot E, Alj AE, Cedard L. Induction of labor and cervical maturation using mifepristone (RU486) in the late pregnant rat. In-
190
17.
18.
19. 20. 21.
22. 23. 24.
25.
26. 27. 28.
Telleria
and Deis
fluence of a cyclooxygenase inhibitor (Diclofenac). Prostaglandins 1991;42:71-9. Telleria CM, Stocco CO, Deis RP. Luteolytic action of RU486: modulation of luteal 3Bhydroxysteroid dehydrogenase and 20a-hydroxysteroid dehydrogenase activities in late pregnant rats. J Steroid Biochem Mol Biol 1995;52:567-73. Telleria CM, Deis RP. Respuesta diferencial de1 eje hipotalamo-hipfisis-ovario a la action de1 antiprogesterona RU486 (Mifepristona) en ratas preriadas. Proceedings of the IX Annual Meeting of the Society of Biology of Cuyo, Argentina, Abstract 33, 199 1. Vermouth NT, Deis RP. Prolactin release and lactogenesis after ovariectomy in pregnant rats: effect of ovarian hormones. J Endocrinol 1974;63:13-20. De Feo VJ. Temporal aspects of uterine sensitivity in the pseudopregnant or pregnant rats. Endocrinology 1963;72:305-16. Castro-Vazquez A, Luque EH, Carrerio NB. Modulation of sensitivity to cervicovaginal stimulation during the estrous cycle: evidence for an extrapituitary action of LH-RH. Brain Res 1984;305:231-7. Gunnet JW, Freeman ME. The mating-induced release of prolactin: a unique neuroendocrine response. Endocrine Rev 198334:44-61. Bussmann LE, Deis RP. Studies concerning the hormonal induction of lactogenesis by prostaglandin F2a in pregnant rats. J Steroid Biochem 1979;11:1485-9. Telleria CM, Carrizo DG, Deis RP. Levonorgestrel inhibits luteinizing hormone-stimulated progesterone production in rat luteal cells. J Steroid Biochem Mol Biol 1994;50:161-6. Telleria CM, Deis RP. Effect of RU486 on ovarian progesterone production at proestrus and during pregnancy: a possible dual regulation of the biosynthesis of progesterone. J Reprod Fertil 1994;102:379-84. De Feo VJ. Decidualization. In: Cellular biology of the uterus. Wynn R, ed. New York:Plenum Press, 1967j191-290. Caligaris L, Astrada JJ, Taleisnik S. Biphasic effect of progesterone on the release of gonadotropin in rats. Endocrinology 1971;89:331-7. Ortman 0, Emons G, Knuppen R, Catt KL. Inhibitory effects of the antiprogestin RU486 on progesterone actions and luteinizing hormone secretion in pituitary gonadotrophs. J Steroid Biochem 1989;32:291-7.
Contraception 199653: 185-I 90
JE, Bellido C, Galiot F, Lopez FJ, Gay29. Sanchez-Criado tan F. A possible dual mechanism of the anovulatory action of antiprogesterone RU486 in the rat. Biol Reprod 1990;42:877-86. 30. Rao IM, Mahesh VB. Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the pregnant mare’s serum gonadotropinprimed immature rat and the adult rat. Biol Reprod 1986;35:1154-61. 31. Sanchez-Criado JE, Galiot F, Bellido C, Gonzalez D, Tebar M. Hypothalamus-pituitary-ovarian axis in cyclic rats lacking progesterone actions. Biol Reprod 1993j48: 916-25. 32. Schuiling GA, Moes H, Koiter TR. Interdependence of oestradiol and 5a-dihydrotestosterone in modulating LH and FSH secretion in rats. J Endocrinol 1989j123: 257-62. in go33. Carriere PO, Brawer JR, Farookhi R. Alterations nadotropin-releasing hormone-dependent gonadotropin secretion in rats with polycystic ovaries. Biol Reprod 1991345:685-90. RL, Fugo NW, Collins WE. Semicircadian 34. Butcher rhythm in plasma levels of prolactin during early gestation in the rat. Endocrinology 1972;90:1125-7. 35. Freeman ME, Smith MS, Nazian SJ, Neil1 JD. Ovarian and hypothalamic control of the daily surges of prolactin secretion during pseudopregnancy in the rat. Endocrinology 1974;94:875. 36. Gibori G, Keyes PL. The role of estradiol in early pregnancy in the rat. Endocrinology 198Oj106:1584-89. 37. Vanage G, Kadam PA, Bardin CW, Koide SS. Potentiating effect of epostane on pregnancy-terminating activity of RU486 in the rat. Contraception 1993;47:499506. 38. Roblero LS, Croxatto HB. Effect of RU486 on development and implantation of rat embryos. Mol Reprod Develop 1991;29:342-6. 39. van der Schoot P, Bakker GH, Klijn JGM. Effects of the progesterone antagonist RU486 on ovarian activity in the rat. Endocrinology 1987;121:1375-82. 40. Slayden OVD, Hirst JJ, Brenner RM. Estrogen action in the reproductive tract of rhesus monkeys during antiprogestin treatment. Endocrinology 1993; 132: 1845-56.