Effect of RU486 on different stages of mouse preimplantation embryos in vitroj SUBHASH C. JUNEJA~AND MELVING. D O D S ~ N ~ DepaHment of Obstetrics am? Gynecology5 East Tennessee State- Uaiversiog,,College of Medicirse, Joh~asonCiQ9 774 376d49 U.S.A.
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/09/14 For personal use only.
Received April 3, 1990
YUNEJA, S . C., and BOBSON, M. Go 1980. Effect sf RU486 on different stages sf mouse preimplaenhtion embryos in a~itra acol. 68: 1457- 1460. Can. $. Physiol. Ph 190-Hydroxy-Hlfi-(4-dime&ylamin%ophenyI)-17a-(I-propynyl)estra-49-dien-3-one(RU486) inhibited the in virro development sf different stages sf mouse preimplan&tiow embryos under study. Two-celled embryos, m o d a e , and early blastocysts were obtained from B,D,F, mice. The embryos were grown in Ham F-10 nutrient mixture (with gluhmine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 rng/L), and bovine serum albumin (fraction V, 3 rng/ml) at 37°C in a humidified incubator supplied with 5 9% CO, in air. WU486 was added to the culture medium at concentrations of 1, 5 , 18, and 20 yglrnL. Cultare medium with 0.05% ethanol sewed as the control. In vitro growth of embryos was assessed by the fdlowirmg criteria: (i) two-celled stage embryo development to blastoeyst stage after 72 h, (ii) momla stage grown to blastocyst stage after 24 $, and (iii) early blastocyst stage development ts hatching blastscyst after 12 h, in culture. WU486 inhibited the in p.'itro development of two-celled embryos, momlae, and early blastocysts at concentrations s f 5, 10, and 20 pg/mE cuHture medium ( p < 0.001). The irhibitory effect sf RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU484 did not affect embryo development at E pglml culture medium. The study indicates the direct adverse effect of RU486 at 5 pglmL and higher concentrations in culture medium on the development of mouse preimplantatiomn embryos in visro, and it encourages its further investigation as a postcoit.ea8 conbaceptive in animal models and humans. Key words : WU486, mouse, preimpIantatisn embryos, embryo culture, postcoital csntraceptbve.
BUNEJA,
S . C., et DODSON,M. G, %990.Effect of WU486 s n different stages sf mouse preimplenhbon embryos in vitro. Can. J. Physiol. Phamacol. 68 : 1457- 1468. Ee 176-hydroxy-1l~-(4-dimC~ylaminophCnyl)-17a-(l-pr~grrayl)estra49B-dien-3-one (WU486)a inhibe, chez la souris, les divers sbdes de dkveloppment ka vitro des embqons de p&implantatiora h l'dade. Des embwows B deux cellules, rnomlas et blastoeystes prCcoces, ont 6t6 obtewus de ssueis B,B,F,. Ees ernbrgrons ont 6tC d6veloppCs dans un mC%angenatrieif Ham F-10 (avee glubmine) additionnb Be bicarbonate de sodium (2, B g/E), de lactate Qecalcium (282 rng/L) et de skwm albumine de bsvln (fraction V, 3 ra~g/mE),B 37"C, dans un incubateur humidifid avw 5 % de C0, dans l'air. Le RU486 a 6tC ajoant.6 au mileu de culture B des concentrations de 1, 5, 18 et 20 pg/mL. Le milieu de culture contemnt QO,O%%dd'6thanaola servi de milieu tCmoin. La croissawce in vitro des embryom a &tCCvaluC selsa Bes crit&l-essuivrants: (i) dkvdoppement du stade embryon B deux celEules en stade blastocyste aprbs 72 %a, (di) passage du stade momla en shde blastscyste apr&s224 h et (idi) dCveloppmertnt du stade blastocyste pr6coce en blastoeyste d'Cclosion aprks 12 h, en culture. Le RU486 a inhibe le dkveloppement in v i ~ m&s embq-ans B deux cellu!es, momlas et blastocystes prCcwes, B des concentrations de 5, l0 et 20 p g / d Bans le milieu de culture ( p < 8,801). A ces concentrations, 19effetinhibiteur du WU486 snr le dkveloppement de tous Tes shdes embqonnaires B I96tude a Ct6 irrkversible. Toutefois, le RU486 n'a pas influewe6 le dkveloppemeant embvonnaire B nne concentration de I pglmE Bans le milieu de culture. Ces observations dCmontrent qa'B une concentration de plus de 5 pg/mE dans le milieu de culture, ie WU484 a un effet inhibiteur direct sue le dCveloppemenrt in vikro des embqprsns de souris en pCride de prCirnplantation; il serait intCressant.de 196tudieren tapat que eontraceptif post-coXhl chez des msdkles anirnaux et chez l'humaiw. Mats clds : RU486, souris, ernbqsns de prkimplantatiow, culture d'embryons, contraceptif pest-coital. [T~aduitpar la revue]
Introduction E 70-Wydrgsxy- B 10-(4-dimethylaminopheny1)-17a-(I -propynyl)estra4,9-dien-3-one (RU486) is an antiprgsgestin steroid and has been used as an abflifacient in humans (Baulieu 1989). At the molecular level, RU486 blocks the progesterone action at the receptor level maulieu 1988). The drug enhances cervix ripening and facili&tes the delivery s f dead fetuses (Wolf et id. 1989). In vitm RU486 causes a reduction in the synthesis sf human placenM hormones by syncytiotrsphoblast cells (Das and Catt 1987). Recently, the dmg has been shown IBart s f this work was presented at 45th American Fertility Sociew meeting at San Francisco, CA, October 1989, abstract p 1 10. 2Present and correspondence address: Reproductive Biology Unit, o ~ / ~ - wwa y p ~ civic , H ~ ~W ~ w~a , M on&rio5 , cam&~ 1 4y~ 9 . %Presentaddress: OB/GYN, Miami Vdley Hospital, Wright State University, Bayton? OH 45409, U.S.A. Printed in Canada
i
Imprirnk au Canada
to inhibit fertilization in v i m (Suneja and Bsdson 1990). RU486 intempts pregnancy when administered postccitally through an intravaginal t a p a n beginning 3 days after potential fertilization in monkeys (Hsdgen 1985). WU486 interrupts early pregnancy by delaying and or inhibiting embryo implanbtiona in many laboratory species including mice and rats (Psychoyas and Prapas 1987; Roblers et id. 1987). Underdeveloped and degenerated embryos were rewvered from RU486-treated early pregnant rats (Psychoyss and Prapas 1987). In other studies, RU486 blocks uterine cell proliferation in mice in vim (Caallingfsrd and Pollard 1988). All these foregoing reports on e a r ~ ~ - ~ r e ~ n conclude a n c y that RU486 causes the uterus to be wonreceptive for the preirnplantation embryo, which in turn results in the inhibition of 6 ' s ~ " h r o n ~ 9between q the and the growing embryo by inhibiting the uterus proliferation. The current 8bd$1Was designed to observe whether RU486 has any direct effect on preimplantatioa embryo development in I&-0.
CAN.J. E3mEYSIBL. PHARMACBL. VOL.
1458
100
68, 1990
astocyst Formation
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/09/14 For personal use only.
Hatching B aslocyst
9pg per rnL medium Fpg per mb. medium
lC#g P~T mL medim 2Cpg pm m k medim
Early B l a s t o ~ y s t
n Vitro Treatment of Embryos with RU486 PIG. 1. h z vitro effect of RU486 on different stages of mouse preimplanQtiion embryos; percent blastocyst foebrmaticn from two-celled emb~yss at 7%h; percent blastocyst formation from momla stage embryos at 224 h; percent hatching blastscyst formation from early blastscyst at
12 h in culture.
Methods and materials Male and fernale B,D,F, mice (Charles Rivers Labs, Raleigh, NC) were b u s e d at 24-25°C under controlled illumination (14 BB tight and 10 h darkness). Water and geIleted foe$ were supplied ad 1Qituln. Female mice (body weight, 28-25 g ; age, 7-8 weeks) were superovuhted with 4 EU of pregnant mare's semm gsna$otropimn (PMSG; Sigma Chemical Co., St. Louis, MO) ip followed 48 h later by 7 IU of human chorionic gonadotropin (RCG; Sigma). FemHe mice were caged individually with males (age, 10- 12 weeks) after h@G injection and smeared for positive seminal plug in the vagina next morning. Preimpkan~tionembryos at different stages of development were recovered by sacrificing the positive-smeared mice at different intervals post-hCG injection. Oviducts and (or) uteri were excised and transferred to sterile culture dishes containing culture medium. Using a fine needle (30 GI and fine forceps, embryos were flushed from the oviducts and uteri under a dissecting micrsscope. Two-celled embryos from oviducts, momlae from oviducts and uteri, and early blastocysts from uteri were flushed from positive-smeared mice sacrificed at 40, 72, and 84 h post-hC6. Each oviduct was flushed with 0.2 mL culture. medium and each uterus was flushed with 8.5 mL of culture medium. MoqhologicaHIy good-Isoking embryos at either s&ge of development, rec~veredfmm different mice, were washed, pooled, and then distributed randomly into humidified centre-well culture dishes (diameter, 35 mm: Falcon, Lincoln Park, NJ) at 15-20 ernbryos/dish containing 2 cullare medium at 3'9°C at 5 % CO, in air.
RU486 was obtained from RousseS-Uclaf Co., France, as a gift. The crystalline powder of the compound was dissolved in ethanol as the stock solution at concentrations of 2, 10, 20, and $0 rng/rnL ethanol. With the kelp nf sterile Hamilton syringes (Hamilton Go., Reno, NV)' I pL of the different stock solutions was added very gently to 2 rnk culture medium in culture dishes to get the Gnal WU486 concentrations at 1, 5, 18, and 28 gkg/mL culture medium, respeetivelg~.The precipitation of RU486 was avoided by simultaneous shaking of the culture dish while adding the stock solutions. Any dish showing precipitates observed under the dissecting microscope was not included in the experiment. The ethanol c~ncentrationin treatment groups as well as in control groups was maintained at 0.05 % . Embryos were cultured in the medium containing RU486 at 1, 5, 10, and 20 pg/mL culture medium. The effect s f RU486 an the different stages of preimplantation embryos was assessed by the folHswing criteria: (i) tws-celled stage embryo development to blastscyst stage after 72 12; (ik) momla stage embryo development to blastocyst after 24 h; ( k i k ) early blastocyst development to hatching blastscyst after 12 h in culture. The normality of the embryos at the beginning and end of the experiment mias assessed by their rnc~rphslogical appearance under the micwscope at a magnification of 408 x . Culture medium used in the experiment was Ham F-SO nutrient mixture (with glutamine, Sigma Chemical Cooproduct No. N-4635) supplemented with sodium bicarbonate (2.1 g/%), calcium kamte (282 mg/L). and bovine semm albumin (fraction V, 3 mg/m&). All the components sf the culture medium were purchased from Sigma Chemical Co. St. Louis, MO. The components sf the medium were dissolved in ultrapure water (mycoplasma screened;
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/09/14 For personal use only.
Received April 3, 1990
YUNEJA, S . C., and BOBSON, M. Go 1980. Effect sf RU486 on different stages sf mouse preimplaenhtion embryos in a~itra acol. 68: 1457- 1460. Can. $. Physiol. Ph 190-Hydroxy-Hlfi-(4-dime&ylamin%ophenyI)-17a-(I-propynyl)estra-49-dien-3-one(RU486) inhibited the in virro development sf different stages sf mouse preimplan&tiow embryos under study. Two-celled embryos, m o d a e , and early blastocysts were obtained from B,D,F, mice. The embryos were grown in Ham F-10 nutrient mixture (with gluhmine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 rng/L), and bovine serum albumin (fraction V, 3 rng/ml) at 37°C in a humidified incubator supplied with 5 9% CO, in air. WU486 was added to the culture medium at concentrations of 1, 5 , 18, and 20 yglrnL. Cultare medium with 0.05% ethanol sewed as the control. In vitro growth of embryos was assessed by the fdlowirmg criteria: (i) two-celled stage embryo development to blastoeyst stage after 72 h, (ii) momla stage grown to blastocyst stage after 24 $, and (iii) early blastocyst stage development ts hatching blastscyst after 12 h, in culture. WU486 inhibited the in p.'itro development of two-celled embryos, momlae, and early blastocysts at concentrations s f 5, 10, and 20 pg/mE cuHture medium ( p < 0.001). The irhibitory effect sf RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU484 did not affect embryo development at E pglml culture medium. The study indicates the direct adverse effect of RU486 at 5 pglmL and higher concentrations in culture medium on the development of mouse preimplantatiomn embryos in visro, and it encourages its further investigation as a postcoit.ea8 conbaceptive in animal models and humans. Key words : WU486, mouse, preimpIantatisn embryos, embryo culture, postcoital csntraceptbve.
BUNEJA,
S . C., et DODSON,M. G, %990.Effect of WU486 s n different stages sf mouse preimplenhbon embryos in vitro. Can. J. Physiol. Phamacol. 68 : 1457- 1468. Ee 176-hydroxy-1l~-(4-dimC~ylaminophCnyl)-17a-(l-pr~grrayl)estra49B-dien-3-one (WU486)a inhibe, chez la souris, les divers sbdes de dkveloppment ka vitro des embqons de p&implantatiora h l'dade. Des embwows B deux cellules, rnomlas et blastoeystes prCcoces, ont 6t6 obtewus de ssueis B,B,F,. Ees ernbrgrons ont 6tC d6veloppCs dans un mC%angenatrieif Ham F-10 (avee glubmine) additionnb Be bicarbonate de sodium (2, B g/E), de lactate Qecalcium (282 rng/L) et de skwm albumine de bsvln (fraction V, 3 ra~g/mE),B 37"C, dans un incubateur humidifid avw 5 % de C0, dans l'air. Le RU486 a 6tC ajoant.6 au mileu de culture B des concentrations de 1, 5, 18 et 20 pg/mL. Le milieu de culture contemnt QO,O%%dd'6thanaola servi de milieu tCmoin. La croissawce in vitro des embryom a &tCCvaluC selsa Bes crit&l-essuivrants: (i) dkvdoppement du stade embryon B deux celEules en stade blastocyste aprbs 72 %a, (di) passage du stade momla en shde blastscyste apr&s224 h et (idi) dCveloppmertnt du stade blastocyste pr6coce en blastoeyste d'Cclosion aprks 12 h, en culture. Le RU486 a inhibe le dkveloppement in v i ~ m&s embq-ans B deux cellu!es, momlas et blastocystes prCcwes, B des concentrations de 5, l0 et 20 p g / d Bans le milieu de culture ( p < 8,801). A ces concentrations, 19effetinhibiteur du WU486 snr le dkveloppement de tous Tes shdes embqonnaires B I96tude a Ct6 irrkversible. Toutefois, le RU486 n'a pas influewe6 le dkveloppemeant embvonnaire B nne concentration de I pglmE Bans le milieu de culture. Ces observations dCmontrent qa'B une concentration de plus de 5 pg/mE dans le milieu de culture, ie WU484 a un effet inhibiteur direct sue le dCveloppemenrt in vikro des embqprsns de souris en pCride de prCirnplantation; il serait intCressant.de 196tudieren tapat que eontraceptif post-coXhl chez des msdkles anirnaux et chez l'humaiw. Mats clds : RU486, souris, ernbqsns de prkimplantatiow, culture d'embryons, contraceptif pest-coital. [T~aduitpar la revue]
Introduction E 70-Wydrgsxy- B 10-(4-dimethylaminopheny1)-17a-(I -propynyl)estra4,9-dien-3-one (RU486) is an antiprgsgestin steroid and has been used as an abflifacient in humans (Baulieu 1989). At the molecular level, RU486 blocks the progesterone action at the receptor level maulieu 1988). The drug enhances cervix ripening and facili&tes the delivery s f dead fetuses (Wolf et id. 1989). In vitm RU486 causes a reduction in the synthesis sf human placenM hormones by syncytiotrsphoblast cells (Das and Catt 1987). Recently, the dmg has been shown IBart s f this work was presented at 45th American Fertility Sociew meeting at San Francisco, CA, October 1989, abstract p 1 10. 2Present and correspondence address: Reproductive Biology Unit, o ~ / ~ - wwa y p ~ civic , H ~ ~W ~ w~a , M on&rio5 , cam&~ 1 4y~ 9 . %Presentaddress: OB/GYN, Miami Vdley Hospital, Wright State University, Bayton? OH 45409, U.S.A. Printed in Canada
i
Imprirnk au Canada
to inhibit fertilization in v i m (Suneja and Bsdson 1990). RU486 intempts pregnancy when administered postccitally through an intravaginal t a p a n beginning 3 days after potential fertilization in monkeys (Hsdgen 1985). WU486 interrupts early pregnancy by delaying and or inhibiting embryo implanbtiona in many laboratory species including mice and rats (Psychoyas and Prapas 1987; Roblers et id. 1987). Underdeveloped and degenerated embryos were rewvered from RU486-treated early pregnant rats (Psychoyss and Prapas 1987). In other studies, RU486 blocks uterine cell proliferation in mice in vim (Caallingfsrd and Pollard 1988). All these foregoing reports on e a r ~ ~ - ~ r e ~ n conclude a n c y that RU486 causes the uterus to be wonreceptive for the preirnplantation embryo, which in turn results in the inhibition of 6 ' s ~ " h r o n ~ 9between q the and the growing embryo by inhibiting the uterus proliferation. The current 8bd$1Was designed to observe whether RU486 has any direct effect on preimplantatioa embryo development in I&-0.
CAN.J. E3mEYSIBL. PHARMACBL. VOL.
1458
100
68, 1990
astocyst Formation
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/09/14 For personal use only.
Hatching B aslocyst
9pg per rnL medium Fpg per mb. medium
lC#g P~T mL medim 2Cpg pm m k medim
Early B l a s t o ~ y s t
n Vitro Treatment of Embryos with RU486 PIG. 1. h z vitro effect of RU486 on different stages of mouse preimplanQtiion embryos; percent blastocyst foebrmaticn from two-celled emb~yss at 7%h; percent blastocyst formation from momla stage embryos at 224 h; percent hatching blastscyst formation from early blastscyst at
12 h in culture.
Methods and materials Male and fernale B,D,F, mice (Charles Rivers Labs, Raleigh, NC) were b u s e d at 24-25°C under controlled illumination (14 BB tight and 10 h darkness). Water and geIleted foe$ were supplied ad 1Qituln. Female mice (body weight, 28-25 g ; age, 7-8 weeks) were superovuhted with 4 EU of pregnant mare's semm gsna$otropimn (PMSG; Sigma Chemical Co., St. Louis, MO) ip followed 48 h later by 7 IU of human chorionic gonadotropin (RCG; Sigma). FemHe mice were caged individually with males (age, 10- 12 weeks) after h@G injection and smeared for positive seminal plug in the vagina next morning. Preimpkan~tionembryos at different stages of development were recovered by sacrificing the positive-smeared mice at different intervals post-hCG injection. Oviducts and (or) uteri were excised and transferred to sterile culture dishes containing culture medium. Using a fine needle (30 GI and fine forceps, embryos were flushed from the oviducts and uteri under a dissecting micrsscope. Two-celled embryos from oviducts, momlae from oviducts and uteri, and early blastocysts from uteri were flushed from positive-smeared mice sacrificed at 40, 72, and 84 h post-hC6. Each oviduct was flushed with 0.2 mL culture. medium and each uterus was flushed with 8.5 mL of culture medium. MoqhologicaHIy good-Isoking embryos at either s&ge of development, rec~veredfmm different mice, were washed, pooled, and then distributed randomly into humidified centre-well culture dishes (diameter, 35 mm: Falcon, Lincoln Park, NJ) at 15-20 ernbryos/dish containing 2 cullare medium at 3'9°C at 5 % CO, in air.
RU486 was obtained from RousseS-Uclaf Co., France, as a gift. The crystalline powder of the compound was dissolved in ethanol as the stock solution at concentrations of 2, 10, 20, and $0 rng/rnL ethanol. With the kelp nf sterile Hamilton syringes (Hamilton Go., Reno, NV)' I pL of the different stock solutions was added very gently to 2 rnk culture medium in culture dishes to get the Gnal WU486 concentrations at 1, 5, 18, and 28 gkg/mL culture medium, respeetivelg~.The precipitation of RU486 was avoided by simultaneous shaking of the culture dish while adding the stock solutions. Any dish showing precipitates observed under the dissecting microscope was not included in the experiment. The ethanol c~ncentrationin treatment groups as well as in control groups was maintained at 0.05 % . Embryos were cultured in the medium containing RU486 at 1, 5, 10, and 20 pg/mL culture medium. The effect s f RU486 an the different stages of preimplantation embryos was assessed by the folHswing criteria: (i) tws-celled stage embryo development to blastscyst stage after 72 12; (ik) momla stage embryo development to blastocyst after 24 h; ( k i k ) early blastocyst development to hatching blastscyst after 12 h in culture. The normality of the embryos at the beginning and end of the experiment mias assessed by their rnc~rphslogical appearance under the micwscope at a magnification of 408 x . Culture medium used in the experiment was Ham F-SO nutrient mixture (with glutamine, Sigma Chemical Cooproduct No. N-4635) supplemented with sodium bicarbonate (2.1 g/%), calcium kamte (282 mg/L). and bovine semm albumin (fraction V, 3 mg/m&). All the components sf the culture medium were purchased from Sigma Chemical Co. St. Louis, MO. The components sf the medium were dissolved in ultrapure water (mycoplasma screened;
