Journal of Assisted Repn~duction and Genetics, Vol. 13, No. 4, 1996
ANIMAL EXPERIMENTATION
ANIMAL EXPERIMENTATION
Effect of Anordrin on the Development of Mouse Preimplantation Embryos in Vitro S U B H A S H C. JUNEJA z'3 and R. STAN WILLIAMS-"
Submitted: July 15, 1995 Accepted: October 17, 1995
txg/ml fi~r 80 hr caused significant inhibition of the formation of hatching blastocysts in a dose-dependent manner. Conclusion: Anordrin and its metabolite anordiol inhibit the development of two-cell embryos in vitro.
Purpose: The in vitro effect of anordrin and anordiol on the development of mouse two-cell embryos was studied. Method: Female mice were primed with gonadotropins for superovulation and caged with male mice. Preimplantation embr?,'os, at the two-cell stage, were recovered from the oviducts at 40 hr post-hCG, hi the first experiment, twocell embryos were exposed to culture medium containing different concentrations of anordrin for 3, 12, 24, and 80 hr and then grown in the anordrin-free culture medium and assessed for the formation of total and hatching blastocysts at 80 hr. In the second experiment, two-cell embryos were grown in culture medium containing different concentrations of anordiot and assessed for the formation of total and hatching blastocysts at 80 hr in vitro. Results: Exposure of two-cell embryos to anordrin concentrations of 2.5-Z5 txg/ml for I2 hr, 2.5-5.0 ~g/ml for 24 hr, and 2.5 I~glml for 80 hr caused significant inhibition of the formation of total blastocysts and to 2.5-Z5 p,g/ml for 12 hr, 1.0-2.5 ~glml for 24 hi;, and 1.0 p.g/ml for 80 hr caused significant inhibition o f the formation of hatching blastocysts, in a exposure time-dependent and dose-dependent manner. Exposure of two-cell embryos to anordiol concentrations of 15-25 txgtmt for 80 hr caused significant inhibition of the formation of total blastocysts and to 15-20
KEY WORDS: mouse embryos; anordrin; anordiol; contraceptive.
INTRODUCTION Antifertility activity of A-nor steroids was first reported in 1962 (1). The most widely studied A-nor steroid, used in China as a contraceptive, is anordrin (2c~, 17a-diethynyl-A-nor-5a-androstane-2[3, 1713-diol diproprionate). Rapid hydrolysis of propionate esters of anordrin (AF-53) takes place in vivo, leading to a biologically active diol form called anordiol or AF-45 (2a, 17a-diethynyl-A-nor-5a-androstane-2[3, 1713-diol) (2). The chemical structure of anordrin and anordiol is shown in Fig. 1. Since the early 1970s, anordrin has been used as a "vacation pill" in China. A 7.5-mg tablet is taken orally daily or on alternate days starting 1 to 5 days prior to ovulation or one 7.5-mg tablet is taken postcoitum (pc) orally by women. The contraceptive efficacy of anordrin has been reported to be 99.5% in more than 6000 cycles studied (3, 4). Anordrin has been shown to have estrogenic, antiestrogenic, antiprogestational, or antiandrogenic properties depending upon the species, stage of the cycle of treatment in female, and reproductive function studied (5-7). Anordrin increases cycle length and delays ovulation in women (6, 8), inhibits follicular development in monkeys (9), and causes luteolysis and depression of progesterone in women and baboons (4, 10, 11).
Presented orally at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5-10, 1994, Abstract No. O- 176. "~Department of Obstetrics and Gynecology,College of Medicine, University of Florida, Gainesville. Florida 32610. 3To whom correspondence should be addressed at Laboratory of Molecular Embryology,Division of Molecular Genetics, Department of Zoology,Universityof Western Ontario, London,Ontario, Canada N6A 5B7. Fax: 519-661-2014. 1058.046819610400.O356$(FL50/O ©
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trolled illumination (t4-hr light and 10-hr darkness). Water and pelleted food were supplied ad libitum. Female mice (age, 3.5-4.5 weeks; body weight, 10-15 g) were superovulated with 7 IU pregnant mare's serum gonadotropin (PMSG; Sigma Chemical Co., St. Louis, MO) administered intraperitoneally (ip), followed 48 hr later by 7 IU human chorionic gonadotropin (hCG: Sigma) ip. Immediately after hCG injection, mice were caged with fertile males (age, 10--14 weeks) individually and checked for a seminal plug in the vagina on the following morning (day of positive plug = day t of pregnancy). Mice were sacrificed by cervical dislocation. The embryos at the two-cell stage were recovered at 40 hr post-hCG from the oviducts. The oviducts were flushed through tubal ostium with the help of a 30-gauge needle fitted with a 1-ml syringe and a pair of fine forceps. The embryos were washed thrice in culture medium and transferred to the experimental culture dishes containing treatment solutions.
Culture Medium and Anordrin Solution
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Anordioi Fig. 1. Chemical structureof anordrin and anordiol. Anordiol delayed ovulation and increased cycle length in rat (12, 13). Complete pregnancy was inhibited when hamsters were fed with anordrin on days I-4 pc (14). Anordrin or anordiol administered on day 1 of pregnancy inhibited implantation in the rat (15). The mechanism of postcoital action of anordrin or anordiol, in inhibiting early pregnancy, is not well understood. Since early embryo development is one of the several steps toward successful pregnancy (16), the current study was designed to study the in vitro effect of anordrin and anordiol on the development of preimplantation embryos as assessed by their blastocyst formation and hatching.
MATERIALS AND METHODS
Embryo culture medium and flushing medium, used in the experiment, were Ham FI0 nutrient mixture supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (242 mg/L), penicillin G (37.5 IU/ml), and streptomycin sulfate (25 i~g/ml). Cell culture-quality water with low endotoxin levels (Irvine Scientific, Santa Ana, CA) was used to prepare the medium. The osmolality of the medium was adjusted to 279-281 mosmol/kg H20. Medium was supplemented with 3 mg bovine serum albumin (fraction V) per ml and was filtered through a 0.2-p.m filter (Millipore Products Division, Bedford, MA). All the chemicals were purchased from Sigma Chemical Company. Culture studies were conducted in center-well organ culture dishes (60 × 15 mm, Falcon, Becton Dickinson Labware, Lincoln Park, N J) at 5% CO2 in air in a humidified incubator at 37°C. Culture medium was equilibrated in the CO,_ incubator for 24 hr before the embryos were added, following the addition of anordrin or anordiol or vehicle. Anordrin or anordiol was dissolved in ethanol as a stock solution. The stock solution was added to embryo culture media to get the desired concentration of the drug. The final ethanol concentration in culture medium was 0.10% in the anordrin experiment (experiment 1) and 0.25% in the anordiol experiment (experiment 2). Ethanol served as the vehicle.
Embryo Recovery
Embryo Culture
Male and female B6D.,FI mice (Jackson Laboratory, Bar Harbor, ME) were housed at 23-25°C under con-
In experiment 1, the two-cell embryos recovered from different mice were pooled and then redistributed
Journal of Assisted Reproduction and Genetics. Vol. 13. No. 4. 1996
358
randomly at 6-13 embryos per dish containing different concentrations of anordrin (1.0, 2.5, 5.0, 7.5 p-g/ ml) in 2 ml of culture medium. The control medium contained the vehicle. The embryos were cultured for 3, 12, 24, and 80 hr in the above treatment groups. At the end of each incubation, the embryos were washed three times in the anordrin-free culture medium and then transferred to dishes containing vehicle only. Embryos were assessed for the formation of total and hatching blastocysts at 80 hr. In the case of the 80-hr treatment group, the embryos were assessed immediately. The experiment was conducted in five replicates and each replicate contained duplicate dishes of each treatment. In experiment 2, the two-cell embryos recovered from different mice were pooled and then redistributed randomly at 10-13 embryos per dish containing different concentrations of anordiol (10, 15, 20, and 25 p-g/ ml) in 2 ml of culture medium. The control medium contained the vehicle. The embryos were cultured for 80 hr in this experiment. The embryos were assessed for the formation of total and hatching blastocysts at the end of the experiment. The experiment was conducted in three replicates and each replicate contained duplicate dishes of each treatment.
Statistical Analysis The data from each treatment from all the replicates of each experiment were pooled and analyzed by the 2 × 2 chi-square method using contingency tables with Yates correction. RESULTS
Experiment 1 Results are shown in Figs. 2A--C. Anordrin exposure to two-cell embryos, at concentrations of 2.5-7.5 ixg/ ml for 12 hr and 2.5-5.0 ~g/ml for 24 hr, caused significant inhibition of the formation of total blastocysts in a dose-dependent manner. Exposure at 2.5-7.5 txg/ml for 12 hr and 1.0-2.5 p-g/ml for 24 hr caused significant inhibition of the formation of hatching blastocysts, in a dose-dependent manner. Exposure at 7.5 p-g/ml for 12 hr, 5.0 p-g/ml for 24 hr, and 2.5 p-g/ ml for 80 hr resulted in the formation of 0.0% total blastocysts. Exposure at 5.0 p-g/ml for 12 hr, 2.5 p.g/ ml for 24 hr, and 1.0 p-g/ml for 80 hr caused the formation of 0.0% hatching blastocysts. Anordrin exposure to two-cell embryos for 3 hr at all concentrations (1.0-7.5 p-g/ml), did not affect early embryo
JUNEYA AND WILLIAMS
development in vitro (data not shown). A photomicrograph of 24-hr-treated embryos, with a 5 p-g/ml anordrin concentration, at 80 hr is shown in Fig. 3A. All embryos degenerated at the two- to four-cell stage are shown. Embryos developed to blastocysts and hatching blastocysts are shown in the control group (Fig. 3B).
Experiment 2 Anordiol exposure of two-cell embryos at concentrations of 15-25 p-g/ml for 80 hr caused significant inhibition of the formation of total blastocysts, and exposure at 15-20 I,zg/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts in a dose-dependent manner (Fig. 4). Exposure at 25 and 20 p-g/ml resulted in the formation of 0.0% total blastocysts and hatching blastocysts, respectively (Fig. 4). Anordiot exposure to two-cell embryos, at a 10 p-g/ml concentration, did not affect the development of preimplantation embryo development in vitro (Fig. 4). Photomicrographs of the embryos developed from two-cell embryos at 80 hr, treated with 20 and 25 p-g/ ml anordiol, and from the control group are shown in Figs. 5A, B, and C, respectively. Some of the embryos reached the blastocyst stage in the 20 p-g/ml treatment group (Fig. 5A) and all the embryos degenerated or remained at the two- to eight-cell stage in the 25 p-g/ ml treatment group (Fig. 5B). A mixture of blastocysts, hatching blastocysts, and hatched blastocysts is shown in the control group (Fig. 5C). DISCUSSION There is a growing interest in contraceptives of natural or synthetic origin. The possible mechanism and side effects of each drug are determined before it can be prescribed to human beings. Anordrin is a drug of Chinese origin. In China, it has been shown to be a safe contraceptive. Although it has been shown that anordrin and its metabolite, anordiol, prevent implantation and inhibit early embryo development in vivo (3, 4, 14, 15, 17, 18), the mechanism of their action in preventing female fertility functions is not well unde~tood. The present study showed a direct effect of anordrin and anordiol on early embryo development in vitro. Anordrin caused inhibition of the formation of total and hatching blastocysts from two-cell embryos in a dose- and time-dependent manner. The effective anordrin concentration in inhibiting blastocyst formation was in the range of 2.5-7.5 p-g/ml when the embryos were exposed to the drug tbr 12-80 hr. The effective anordrin concentration in inhibiting the hatching of Journal of Assisted Reproduction and Genetics, Vol. 13, No. 4, 1996
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Fig. 2. In vitro formation of total and hatching blastocysts at 80 hr of culture from two cell embryos exposed to different concentrations of anordrin for 12 hr (A), 24 hr (B), and 80 hr (C); 0.0% anordrin represents the control. (*) P < 0.0001 and (**) P < 0.05; P value differs from its respective control value, n represents the number of embryos.
blastocysts was in the range of 1.0-2.5 p.g/ml when the embryos were exposed to the drug for 24-80 hr. The effect of anordiol on two-cell embryos was studied only for the 80-hr treatment, and the embryos were assessed after treatment without further culture. The study showed that anordiol is inhibitory to the formation and hatching of blastocysts at concentrations of 15 i,zg/ml and higher. The study showed that both anordrin and anordiol are inhibitory to preimplantation embryo development in vitro. Various studies have shown that anordrin or anordiol inhibits pregnancy in women and rodents when treated in early pregnancy. In women, one tablet of anordrin (7.5 Journal of Assisted Reproduction and Genetics. Vol. 13, No. 4, 1996
mg) inhibits pregnancy when given pc (3, 4). Anordrin showed an antifertility effect in hamsters treated with a dose of 10 mg/kg/day for 3--4 days after mating (14). In hamsters fed with anordrin from day 1 to day 3 after mating, underdeveloped eggs and degenerated preimplantation embryos were recovered (14). Anordrin or anordiol at a dose of 2 mg/kg completely inhibited implantation in the rat (15). In mice treated ip with anordrin (3 mg/kg), decidual cells and blastocysts cells showed degeneration and necrosis (17, 18) Since early embryo development is one of the several steps toward successful pregnancy (16), the drugs can inhibit early pregnancy by disrupting the functions
360
JUNEYA AND WILLIAMS
Fig. 3. Photomicrographs showing development of two-cell embryos at 80 hr. (A) After 24-hr treatment with 5.0 l.~g/ml anordrin; all the embryos degenerated with granular cytoplasm at the two- to four-cell stage. (B) Control, expanded blastocysts (small arrows) and hatching blastocysts (large arrows) are shown.
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of the uterus or early embryos or both. Synchronization of the uterine system and early embryonic development is mandatory for successful implantation. Underdevelopment of early embryos or failure of uterus preparation for implantation can inhibit implantation. The current study showed that anordrin and anordiol inhibit early embryo development in vitro. Anordrin or anordiol has been shown to disrupt the uterine system by various mechanisms. Anordrin showed weak estrogenicity by increasing the uterine wet weight and protein content (5). Anordrin inhibited progesterone-induced DCR when it was administered to mice in combination with progesterone, indicating inhibition of progesterone-induced DCR by anordrin and showing its antiprogestational property of anordrin (5). Anordiol showed weak antiestrogeniciy when estradiol-1713 and anordiol were given in a combination dose; the increment in the uterine weight caused by estradiol-17~3 was reduced by anordiol (12). In immature rats, anordiol showed estrogenicity. When Journal of Assisted Reproduction and Genetics. Vol, 13, No, 4, 1996
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~'ig. 5, Pho|omicrographs showing development of two-cell embryos at 80 hr, (A) Anordiol, 20 l.Lg/ml+ treatment; a mixture of underdeveloped embryos (small arrows) and blastocysts (large Lrrows) is shown, tB) Anordiol, 25 l.~g/ml, treatment; all the embryos degenerated with granular cytoplasm at the two- to eight+cell stage. (C) Control, expanded blastocyst (small arrow) and latching blastocysts (large arrows) are shown,
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anordiol was administered on days 25 to 29 of age, organ weights of the uterus and the serum LH concentration increased as measured on day 32 of age (7). In vivo anordiol has been shown to be more effective than anordrin in inhibiting pregnancy (19), whereas slightly opposite results were seen in vitro. The dose difference of the in vivo vs vitro action is a question of further investigation. However, it could be that, in vivo, the drugs might be inhibiting pregnancy by a double-fold function, altering uterine function as well as inhibiting early embryo development. The current study showed that anordrin and anordiol have pharmacological deleterious effects on the development of two-cell embryos in vitro. Various studies have shown that anordrin or anordiol is more effective in terminating pregnancy when combined with RU486 during the preimplantation period ( 19, 20) and during the postimplantation period (21-23). The purpose of the present study was to study the exclusive effect of anordrin and anordiol on early embryo development in vitro. However, our earlier study showed that RU486 inhibits early embryo development in vitro (24). In conclusion, anordrin and anordiol inhibit preimplantation embryo development in vitro.
JUNEYA AND WILLIAMS
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ACKNOWLEDGMENTS We thank Dr. B. J. Masterson, Professor and Chairman of the Department of Obstetrics and Gynecology, for providing funding for the research. We appreciate and thank Drs. C. Wayne Bardin and Samuel S. Koide, Population Council, New York, for providing us with the generous gift of anordrin and anordiol.
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Journal of Assisted Reproduction and Genetics, Vol. 13, No, 4, 1996