MOLECULAR REPRODUCTION AND DEVELOPMENT 29:342-346 (1991)
Effect of RU486 on Development and Implantation of Rat Embryos LUIS S. ROBLERO AND HORACIO B. CROXATTO lnstituto Chileno de Medicina Reproductiva and Unidad de Reproduceion y Desarrollo, Universidad Catolica de Chile, Santiago, Chile This study evaluated the effectsof ABSTRACT postcoital treatment with the antiprogestin RU486 on transport, development and implantation of rat embryos. Doses of 0.1, 0.5, 1.0,2.0, or 3.0 mghat of RU486 were injected subcutaneously on days 1, 1 +2, or 4 of pregnancy. Autopsies were carried out on days 5 or 12 of pregnancy. RU486 provoked a significant dose-related reduction in the number of recovered embryos and inhibited their development (day 5) and decreased the number and size of implanted embryos (day 12).Treatment on day 4 was the least effective. Blastocysts recovered from RU486-treated rats exhibited comparable rate of trophoblastic outgrowth in vitro as the controls. Blastocysts transferred from RU486-treated rats to synchronous untreated pseudopregnant recipients yielded implanted embryos 12 days later in all recipients, although at a significantly lower rate than the controls. Blastocysts transferred from control pregnant rats to RU486-treated pseudopregnant recipients failed to implant completely when the dose was 21.0 mg. The interceptive mechanism of postcoital treatment with RU486 in the rat involves loss of embryos from the reproductive tract and altered development prior to implantation. Endometrial receptivity or the ability of the uterus to retain the embryos until the time of implantation are also impaired by RU486. The embryos that survive these effects may experience delayed implantation in their mothers.
Key Words: RU486, Embryo development, Rat embryo transfer, Oviductal transport
several steps of the reproductive process. Given continuously for the first four days, it shortens by 12% the sojourn of the embryos in the oviduct (Fuentealba et al., 1987). Treatment restricted to the first 2 days of pregnancy also accelerates tuba1 transport, it interferes with the development of morulae into blastocysts, and it can delay implantation or suppress it (Psychoyosand Prapas, 1987). The aims of the present work, in the rat, were (1)to assess time- and dose-related effects of postcoital treatment with RU486 on the proportion of embryos reaching the blastocyst stage on day 5 and their mean size on day 12 of pregnancy; and (2) to assess the relative importance of embryo damage versus impairment of the genital tract milieu in implantation failure caused by RU486. The latter was attempted in two ways: (1)by testing trophoblast outgrowth in vitro and viability in vivo, in antiprogestin-free environment, of embryos grown to blastocyst stage in their treated mothers; and (2) by testing the rate of implantation of normal blastocysts transferred to the uterus of treated pseudopregnant recipients.
MATERIALS AND METHODS Mature female Sprague-Dawley rats (190-210 g) in proestrus, were mated overnight with fertile males. The next day was designated day 1 of pregnancy if spermatozoa were detected in the vaginal smear. The following experiments were carried out with these pregnant females. Effect of Different Doses of RU486 on Development and Implantation of Rat Embryos These experiments were designed to define time- and dose-related effects of postcoital treatment with RU486 on development and implantation. A total of 250 pregnant females were divided into groups of 10 and injected sc with 0.1, 1.0, 2.0 or 3.0 mg of RU486 per animal or with vehicle on day 1,1+2 or 4 of pregnancy. RU486 was diluted in propylene glycol, and all concentrations were injected sc in 0.1 ml of the vehicle. Groups of 10 females injected on day 1 or 1+ 2 were
INTRODUCTION Previous work in the mouse demonstrated that progesterone produced after fertilization is essential for normal transport and development of embryos in the oviduct and to sustain their viability and implantation in the uterus. The effects of ovariectomy on these processes were reversed by replacement therapy with physiological doses of progesterone (Roblero and Garavagno, 1979)and were mimicked by neutralization of endogenous progesterone with antiprogesterone antibodies (Wang et al., 1984) or the antiprogestin RU486 (Roblero et al., 1987). June 29, 1990; accepted February 26, 1991. In the rat, the administration of the antiprogestin Received Address reprint requests to Dr. Luis S. Roblero, Clinica Las Condes, RU486 (Philibert et al., 19851 after fertilization alters Lo Fontecilla 441, Santiago, Chile.
0 1991 WILEY-LISS, INC.
RU486 EFFECTS ON RAT EMBRYOS killed on day 5 a t 1O:OO h to determine the number, location and development of embryos. Additional groups of 10 females each, injected with RU486 on day 1, 1 + 2 or 4 of pregnancy were killed on day 12 of pregnancy to determine the number of implanted embryos. Embryos were recovered by flushing the oviducts and uteri with a buffered salt solution (PBS). The embryos from day 5 of pregnancy were classified according to their development as morula, blastocyst, or arrested. To determine the number of implanted embryos on day 12, uterine swellings, produced by the implanted embryos were counted. The size was determined by measuring the crown-rump axis of dissected embryos using a Vernier caliper accurate to 0.10 mm.
Effect of RU486 on In Vitro Trophoblast Outgrowth of Rat Blastocysts These experiments were designed to clarify whether treatment with RU486 in vivo interferes subsequently with trophoblast outgrowth in vitro (Jenkinson, 1978) in an antiprogestin-free medium. For this purpose, donor rats were injected sc with 0.1 or 1.0 mg of RU486 or vehicle on day 1 of pregnancy. Blastocysts were recovered aseptically from the uterine horns of donor rats in the morning of day 5 of pregnancy. Blastocysts were cultured in 100 pl of Ham-F10 culture medium (Sigma Chemical Co., St. Louis, MO) under mineral oil in Falcon petri dishes (Falcon 3037, Falcon Plastics, Oxnard, CA) a t 37"C, with 90% humidity, and 5% C 0 2 in air. Ham-F10 was supplemented with fetal bovine serum (FBS)(Sigma Chemical Co., St. Louis, MO) (10% viv). After 72-h incubation, embryonic development and trophoblastic outgrowth were evaluated using an inverted microscope. Effect of Exposing Embryos to RU486 In Vivo on Their Subsequent Development in Untreated Foster Mothers and Uterine Receptivity of Treated Pseudopregnant Foster Mothers to Normal Blastocysts These experiments were intended to determine to which extent the alteration of embryonic development induced by RU486 can be reversed in the normal uterine environment (not exposed to the drug) and whether or not control blastocysts can implant in the uteri of pseudopregnant rats exposed to RU486. In the first experiment, donor rats injected on day 1 or 1+ 2 of pregnancy with 0.1 of 1.0 mg of RU486 or vehicle were killed on day 5 of pregnancy. Because no embryos were recovered after treatment with 1.0 mg RU486, a dose of 0.5 mg was used in a separate experiment to improve the yield. Blastocysts were transferred to the uteri of untreated, synchronous pseudopregnant rats obtained by mating with vasectomized males (McLaren, 1956). In the second experiment, blastocysts recovered on day 5 of pregnancy from untreated rats were transferred to the uteri of synchronous pseudopregnant rats
343
treated on day 1or 1+ 2 with 0.1, 1.0, 2.0, or 3.0 mg of RU486. Embryo transfers were done by puncturing the oviductal end of the uterus with a calibrated glass needle. Blastocysts were delivered into one uterine horn in 1.0 pl of culture medium. The contralateral horn was left undisturbed. On day 8 after transfer, pseudopregnant foster mothers were killed by cervical dislocation, and the number of implanted embryos and/or resorption sites was determined. For statistical purposes, a t-test was used to compare the mean number of implanted embryos and the test of significance for differences of proportions was used for comparing the percentages of pregnant females, with significance set at P S 0.05.
RESULTS Effect of Different Doses of RU486 on Development and Implantation of Rat Embryos The effect of treatment with different doses of RU486 on day 1 or 1+ 2 of pregnancy on the number and type of embryos recovered on day 5 is shown in Table 1. Treatment with RU486 provoked a significant 'I( < 0.01) dose-related reduction in the number of recovered embryos. All embryos recovered were in the uterus. Treatment on day 1with 0.1, 1.0,2.0, or 3.0 mg reduced the number of recovered embryos to 81.8%, 23.6%, 3.6%, and 0%, respectively, of control values. Treatment on day 1+ 2 had no effect at the 0.1 mg dose but caused total loss of embryos at higher doses. The proportion of morulae and blastocysts in the 0.1 mg dose was normal but in animals treated with higher doses all embryos recovered were arrested. The effect of treatment with RU486 on day 1,1+2, or 4 of pregnancy on the number and mean size of implanted embryos on day 12 is shown in Table 2. RU486 provoked a dose-related decrease in the number of implantations. The most and the least severe effects were observed with treatments given on days 1+ 2 and 4, respectively. Reduction in the number of implanted embryos was the most sensitive parameter followed by the proportion of rats with embryos and the size of the embryos. Only treatment with 1.0 mg/rat on day 1 reduced significantly the proportion of rats with implantations, as well as the size of implanted embryos. Effect of RU486 on In Vitro Trophoblast Outgrowth of Rat Blastocysts In vitro trophoblast outgrowth of rat blastocysts recovered from experimental and control rats is shown in Table 3. Trophoblast outgrowth was observed in only 45% of control blastocysts and no significant differences were found between blastocysts from rats treated with 0.1 mg of RU486 or with vehicle. Few'blastocysts were obtained from rats injected with 1.0 mg of RU486, and the lower percentage of in vitro trophoblast outgrowth in this group with respect to the control group was close to, but did not reach, statistical significance.
344
L.S. ROBLERO AND H.B. CROXATTO
+
TABLE 1. Effect of RU486 Injected SC on Day 1 or 1 2 on Embryos Recovered on Day 5 of Pregnancy No. of No. of No. of RU486 Rats w/embryos egbryos Eorulae bla_stocysts (%) X +SE X +SE X +SE mg/rat Day 1 Control 0.1 1.0 2.0 3.0 Day 1 2 Control 0.1 1, 2, or 3.0
+
+
+ 0.2
+ 0.2
+
0.0 0.0 0.0
10.4 1.0 8.6 f 0.5 0.0 0.0 0.0
+
0.0 0.0 0.0
10.2 0.6 9.0 0.9 0.0
100 100 80 20 0
11.0 f 1.0 9.0 0.4 2.6 f 1.2* 0.4 0.4* 0.0
100 100 0
10.2 0.6 9.0 f 0.9 0.0
0.4 0.2
+
+ +
* P vs. control
Effect of RU486 on Development and Implantation of Rat Embryos LUIS S. ROBLERO AND HORACIO B. CROXATTO lnstituto Chileno de Medicina Reproductiva and Unidad de Reproduceion y Desarrollo, Universidad Catolica de Chile, Santiago, Chile This study evaluated the effectsof ABSTRACT postcoital treatment with the antiprogestin RU486 on transport, development and implantation of rat embryos. Doses of 0.1, 0.5, 1.0,2.0, or 3.0 mghat of RU486 were injected subcutaneously on days 1, 1 +2, or 4 of pregnancy. Autopsies were carried out on days 5 or 12 of pregnancy. RU486 provoked a significant dose-related reduction in the number of recovered embryos and inhibited their development (day 5) and decreased the number and size of implanted embryos (day 12).Treatment on day 4 was the least effective. Blastocysts recovered from RU486-treated rats exhibited comparable rate of trophoblastic outgrowth in vitro as the controls. Blastocysts transferred from RU486-treated rats to synchronous untreated pseudopregnant recipients yielded implanted embryos 12 days later in all recipients, although at a significantly lower rate than the controls. Blastocysts transferred from control pregnant rats to RU486-treated pseudopregnant recipients failed to implant completely when the dose was 21.0 mg. The interceptive mechanism of postcoital treatment with RU486 in the rat involves loss of embryos from the reproductive tract and altered development prior to implantation. Endometrial receptivity or the ability of the uterus to retain the embryos until the time of implantation are also impaired by RU486. The embryos that survive these effects may experience delayed implantation in their mothers.
Key Words: RU486, Embryo development, Rat embryo transfer, Oviductal transport
several steps of the reproductive process. Given continuously for the first four days, it shortens by 12% the sojourn of the embryos in the oviduct (Fuentealba et al., 1987). Treatment restricted to the first 2 days of pregnancy also accelerates tuba1 transport, it interferes with the development of morulae into blastocysts, and it can delay implantation or suppress it (Psychoyosand Prapas, 1987). The aims of the present work, in the rat, were (1)to assess time- and dose-related effects of postcoital treatment with RU486 on the proportion of embryos reaching the blastocyst stage on day 5 and their mean size on day 12 of pregnancy; and (2) to assess the relative importance of embryo damage versus impairment of the genital tract milieu in implantation failure caused by RU486. The latter was attempted in two ways: (1)by testing trophoblast outgrowth in vitro and viability in vivo, in antiprogestin-free environment, of embryos grown to blastocyst stage in their treated mothers; and (2) by testing the rate of implantation of normal blastocysts transferred to the uterus of treated pseudopregnant recipients.
MATERIALS AND METHODS Mature female Sprague-Dawley rats (190-210 g) in proestrus, were mated overnight with fertile males. The next day was designated day 1 of pregnancy if spermatozoa were detected in the vaginal smear. The following experiments were carried out with these pregnant females. Effect of Different Doses of RU486 on Development and Implantation of Rat Embryos These experiments were designed to define time- and dose-related effects of postcoital treatment with RU486 on development and implantation. A total of 250 pregnant females were divided into groups of 10 and injected sc with 0.1, 1.0, 2.0 or 3.0 mg of RU486 per animal or with vehicle on day 1,1+2 or 4 of pregnancy. RU486 was diluted in propylene glycol, and all concentrations were injected sc in 0.1 ml of the vehicle. Groups of 10 females injected on day 1 or 1+ 2 were
INTRODUCTION Previous work in the mouse demonstrated that progesterone produced after fertilization is essential for normal transport and development of embryos in the oviduct and to sustain their viability and implantation in the uterus. The effects of ovariectomy on these processes were reversed by replacement therapy with physiological doses of progesterone (Roblero and Garavagno, 1979)and were mimicked by neutralization of endogenous progesterone with antiprogesterone antibodies (Wang et al., 1984) or the antiprogestin RU486 (Roblero et al., 1987). June 29, 1990; accepted February 26, 1991. In the rat, the administration of the antiprogestin Received Address reprint requests to Dr. Luis S. Roblero, Clinica Las Condes, RU486 (Philibert et al., 19851 after fertilization alters Lo Fontecilla 441, Santiago, Chile.
0 1991 WILEY-LISS, INC.
RU486 EFFECTS ON RAT EMBRYOS killed on day 5 a t 1O:OO h to determine the number, location and development of embryos. Additional groups of 10 females each, injected with RU486 on day 1, 1 + 2 or 4 of pregnancy were killed on day 12 of pregnancy to determine the number of implanted embryos. Embryos were recovered by flushing the oviducts and uteri with a buffered salt solution (PBS). The embryos from day 5 of pregnancy were classified according to their development as morula, blastocyst, or arrested. To determine the number of implanted embryos on day 12, uterine swellings, produced by the implanted embryos were counted. The size was determined by measuring the crown-rump axis of dissected embryos using a Vernier caliper accurate to 0.10 mm.
Effect of RU486 on In Vitro Trophoblast Outgrowth of Rat Blastocysts These experiments were designed to clarify whether treatment with RU486 in vivo interferes subsequently with trophoblast outgrowth in vitro (Jenkinson, 1978) in an antiprogestin-free medium. For this purpose, donor rats were injected sc with 0.1 or 1.0 mg of RU486 or vehicle on day 1 of pregnancy. Blastocysts were recovered aseptically from the uterine horns of donor rats in the morning of day 5 of pregnancy. Blastocysts were cultured in 100 pl of Ham-F10 culture medium (Sigma Chemical Co., St. Louis, MO) under mineral oil in Falcon petri dishes (Falcon 3037, Falcon Plastics, Oxnard, CA) a t 37"C, with 90% humidity, and 5% C 0 2 in air. Ham-F10 was supplemented with fetal bovine serum (FBS)(Sigma Chemical Co., St. Louis, MO) (10% viv). After 72-h incubation, embryonic development and trophoblastic outgrowth were evaluated using an inverted microscope. Effect of Exposing Embryos to RU486 In Vivo on Their Subsequent Development in Untreated Foster Mothers and Uterine Receptivity of Treated Pseudopregnant Foster Mothers to Normal Blastocysts These experiments were intended to determine to which extent the alteration of embryonic development induced by RU486 can be reversed in the normal uterine environment (not exposed to the drug) and whether or not control blastocysts can implant in the uteri of pseudopregnant rats exposed to RU486. In the first experiment, donor rats injected on day 1 or 1+ 2 of pregnancy with 0.1 of 1.0 mg of RU486 or vehicle were killed on day 5 of pregnancy. Because no embryos were recovered after treatment with 1.0 mg RU486, a dose of 0.5 mg was used in a separate experiment to improve the yield. Blastocysts were transferred to the uteri of untreated, synchronous pseudopregnant rats obtained by mating with vasectomized males (McLaren, 1956). In the second experiment, blastocysts recovered on day 5 of pregnancy from untreated rats were transferred to the uteri of synchronous pseudopregnant rats
343
treated on day 1or 1+ 2 with 0.1, 1.0, 2.0, or 3.0 mg of RU486. Embryo transfers were done by puncturing the oviductal end of the uterus with a calibrated glass needle. Blastocysts were delivered into one uterine horn in 1.0 pl of culture medium. The contralateral horn was left undisturbed. On day 8 after transfer, pseudopregnant foster mothers were killed by cervical dislocation, and the number of implanted embryos and/or resorption sites was determined. For statistical purposes, a t-test was used to compare the mean number of implanted embryos and the test of significance for differences of proportions was used for comparing the percentages of pregnant females, with significance set at P S 0.05.
RESULTS Effect of Different Doses of RU486 on Development and Implantation of Rat Embryos The effect of treatment with different doses of RU486 on day 1 or 1+ 2 of pregnancy on the number and type of embryos recovered on day 5 is shown in Table 1. Treatment with RU486 provoked a significant 'I( < 0.01) dose-related reduction in the number of recovered embryos. All embryos recovered were in the uterus. Treatment on day 1with 0.1, 1.0,2.0, or 3.0 mg reduced the number of recovered embryos to 81.8%, 23.6%, 3.6%, and 0%, respectively, of control values. Treatment on day 1+ 2 had no effect at the 0.1 mg dose but caused total loss of embryos at higher doses. The proportion of morulae and blastocysts in the 0.1 mg dose was normal but in animals treated with higher doses all embryos recovered were arrested. The effect of treatment with RU486 on day 1,1+2, or 4 of pregnancy on the number and mean size of implanted embryos on day 12 is shown in Table 2. RU486 provoked a dose-related decrease in the number of implantations. The most and the least severe effects were observed with treatments given on days 1+ 2 and 4, respectively. Reduction in the number of implanted embryos was the most sensitive parameter followed by the proportion of rats with embryos and the size of the embryos. Only treatment with 1.0 mg/rat on day 1 reduced significantly the proportion of rats with implantations, as well as the size of implanted embryos. Effect of RU486 on In Vitro Trophoblast Outgrowth of Rat Blastocysts In vitro trophoblast outgrowth of rat blastocysts recovered from experimental and control rats is shown in Table 3. Trophoblast outgrowth was observed in only 45% of control blastocysts and no significant differences were found between blastocysts from rats treated with 0.1 mg of RU486 or with vehicle. Few'blastocysts were obtained from rats injected with 1.0 mg of RU486, and the lower percentage of in vitro trophoblast outgrowth in this group with respect to the control group was close to, but did not reach, statistical significance.
344
L.S. ROBLERO AND H.B. CROXATTO
+
TABLE 1. Effect of RU486 Injected SC on Day 1 or 1 2 on Embryos Recovered on Day 5 of Pregnancy No. of No. of No. of RU486 Rats w/embryos egbryos Eorulae bla_stocysts (%) X +SE X +SE X +SE mg/rat Day 1 Control 0.1 1.0 2.0 3.0 Day 1 2 Control 0.1 1, 2, or 3.0
+
+
+ 0.2
+ 0.2
+
0.0 0.0 0.0
10.4 1.0 8.6 f 0.5 0.0 0.0 0.0
+
0.0 0.0 0.0
10.2 0.6 9.0 0.9 0.0
100 100 80 20 0
11.0 f 1.0 9.0 0.4 2.6 f 1.2* 0.4 0.4* 0.0
100 100 0
10.2 0.6 9.0 f 0.9 0.0
0.4 0.2
+
+ +
* P vs. control
