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Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice
Mutation Research,
147 (1985) 189-195
189
Elsevier MTR 04047
Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice L. Elbling and M. Colot Institute for Applied and Experimental Oncology, Vienna University, Borschkegasse 8a, A - 1090 Vienna (Austria)
(Received30 October 1984) (Revisionreceived27 December1984) (Accepted17 January1985)
Summary Both sister-chromatid exchange (SCE) response and embryonic development and transport in preimplantation embryos were evaluated on day 3 of gestation (vaginal plug = 1) of superovulated Swiss mice. Superovulation was found to have significant effects on number of preimplantation embryos (increase), embryo localization (accelerated transport), cleavage rate (advanced development) and abnormality rate (misshaped, fragmented, dead embryos). Superovulated 4- and 8-cell embryos collected from oviducts and uteri and incubated in vitro with 5-bromodeoxyuridine (BrdU) displayed up to 4 times higher SCE frequency than spontaneously ovulated embryos. This increase is independent of stage of development and location at the time of embryo collection. The results indicate that superovulated embryos may have induced DNA lesions.
Since the introduction of hormones (gonadotropins and sex steroid hormones) in medical practice and animal research, there have been controversies about the possible harmful effects of such agents. Gonadotropic hormone treatment for ovulation induction may lead to a hyperstimulation syndrome with multiple pregnancies, abortions and a discrepancy between ovulation and pregnancy rates (Schenker et al., 1981). There are conflicting reports as to whether or not specific malformations are induced by gonadotropic and sex hormones (Schardein, 1980). Animal experiments have not been helpful in the interpretation of findings in man. Studies reporting the association of increased malformation rates and ovulation-inducing hormones (Nishimura and Shikata, 1958; Elbling, 1973, 1975a, b) are contradicted by Smith and Chrisman (1975). Several studies on mutagenicity of gonadotropins and sex hormones
have been performed. Sex steroids have no mutagenic effect in the Ames test (Lang and Redmann, 1979). However, both positive and negative chromosomal aberration effects have been reported in various organisms (cited in Fraser, 1977; in Wallace et al., 1979; in Czeizel, 1980). Of particular interest is the finding that chromosomal aberrations were increased in superovulated early embryos (Fujimoto et al., 1974; Takagi and Sasaki, 1976; Maudlin and Fraser, 1977). Since the introduction of the SCE assay in mutagenicity testing (Perry and Evans, 1975) as a high-resolution technique for measuring subtle alterations in genetic material, there have been few studies employing this system with reproductive hormones. One negative finding (Husum et al., 1982) is contrasted by reports of increased SCE (Murthy and Prema, 1979, 1983; Dutkowski et al., 1983) in users of contraceptives. No SCE analysis has been con-
0165-1161/85/$03.30 © 1985 ElsevierSciencePublishersB.V.(BiomedicalDivision)
190 ducted after hormone-induced ovulation. It is recognized that the interrelationship between ovarian steroid secretions and their feedback effect on gonadotropins is an important component in the control of normal ovulatory function. Parameters used to assess follicular development include both steroid and gonadotropin content of follicular fluid. In most cases, superovulation regimens are non-physiological and may result in an abnormal follicular steroid milieu (Miller and Armstrong, 1981, rat; Pittaway and Wentz, 1983, man). The in vivo exposure (superovulation)-in vitro SCE assay in preimplantation embryos (Elbling and Colot, 1985) may be useful for the detection of mutagenic effects. We report on both SCE analysis of preimplantation embryos derived from hyperstimulated cycles and indicators (embryonic development and transport) of this hyperstimulation following gonadotropin-induced ovulation in random-bred Swiss mice. Materials and methods
Mice and hormone-induced ovulation Random-bred, 2 months old, female Swiss mice and males of the same strain and age were used. Ovulation was induced by intraperitoneai injections of 5 IU PMS (Gestyl, Organon) followed 48 h later by 5 IU H C G (Pregnyl, Organon). At the expected time of ovulation injected females were placed with fertile males for 2 - 4 h and then checked for vaginal plugs ( = day 1). Spontaneously ovulating (non-hormone-treated) females synchronized with males 2 days before mating (Marsden and Bronson, 1964) were caged together with males overnight and examined for the presence of a vaginal plug the following morning. S C E analysis Mated females were killed on day 3 of gestation. Preimplantation embryos were flushed in phosphate-buffered saline with 2% bovine serum albumin from the reproductive tract (oviduct and uterus were flushed separately). Embryos were collected into standard medium (Brinster, 1963) keeping track of their derivation and were classified according to the stages of development and to morphological aberrations by microscopic examination. Embryos found arrested at the 1- or
2-cell stage, or having undergone degeneration or with cytoplasmic granulation at 4- and 8-cell stages were discarded. Thus, only normal-appearing 4and 8-cell embryos were incubated in BrdU for DNA labelling and differential chromatid staining. Groups of thirty 4- or 8-cell embryos were pulsed with 10 - 6 M BrdU (Sigma Chemical Co.) in standard medium for 24 h. The BrdU medium was replaced with fresh every 6 h. The experimental procedure employed for chromosome preparation and sister-chromatid differentiation has been described previously (Elbling and Colot, 1985). Homogeneity of SCE data, which was determined by variance analysis, allowed the pooled presentation of several experiments. Results and discussion
Under superovulation conditions 40-50% of the females ovulated and mated successfully with non-treated males. About 30% of spontaneously ovulating females became pregnant. After superovulation, most of the females produced 31 73 embryos (controls 14-20) with a range of 17-93 embryos (versus a control range of 10-23). Only a small proportion (7%) of hormone-induced females ovulated within the normal range. Details of the preimplantation pregnancy are given in Tables 1 and 2. The individual experiments are listed separately to illustrate the reproducibility of the results. All experiments were performed within a 1-month period in order to exclude seasonal variations in response (Elbling, 1975a). Totals of 3027 and 892 embryos were obtained from 57 superovulated females and from 55 controls, respectively. An unusually high proportion (40%) of superovulated embryos was recovered from the uterus. 2 days after fertilization, the great majority of cleavage embryos from uninduced females were located in the oviduct (Table 1). This accelerated transport of the embryos, which is consistent with other reports (Harrington, 1965; Beaumont and Smith, 1975; Fiser and Macpherson, 1982), may be attributed to an excessive estrogen production by the ovaries after hyperstimulation (Harrington, 1965; Dukelow and Riegle, 1974; Miller and Armstrong, 1981). Abnormality rates of embryos, calculated either for tubal location only or as a total, were significantly increased in hormone-
191 TABLE 1 LOCAL DISTRIBUTION, ABNORMALITY a AND WASTAGE b OF PREIMPLANTATION EMBRYOS ON DAY 3 OF GESTATION (PLUG = l) FOLLOWING SPONTANEOUS OVULATION (K5, K7, K9) AND SUPEROVULATION ($5, $7, $9) OF SWA MICE Expt. c groups
Number of embryos Total Average
K5 K7 K9
163 144 585
18.1 +1.2 d 16.0_+1.1 15.8_+0.5
(9) e (9) (37)
Total
892
16.6
Total distribution (~)
Abnormal a embryos/mouse (~)
Oviduct
Oviduct
Uterus
Total abnormal ~ (%)
Total wastage h (~)
Uterus
100 95.5 99.0
0 4.9 1.0
3.7_+1.1 1.7_+0.5 3.1_+0.5
0 0.3 0
19.7+6.1 12.8_+3.5 19.2_+3.1
23.8_+5.7 16.8+6.3 21.3_+3.3
(55)
98.2
2.0
2.8
0.1
17.2
20.6
$5 $7 $9
744 1212 1071
49.6_+4.9***.f 52.7_+3.3 *** 56.4_+5.3***
(15) (23) (19)
56.3 52.8 70.0
43.7 47.2 30.0
5.9_+ 1.4 * 12.1 _+2.3 *** 13.2_+2.8**
7.4_+2.6 7.2+1.8 6.2+2.0
36.2_+3.4 * 35.5-+3.7*** 34.0-+3.5**
61.4-+7.9 *** 67.9-+7.9 *** 59.2_+5.9 ***
Total
3 027
52.9
(57)
59.7
40.3
10.4
6.9
35.2
62.8
a Morphologically abnormal 1-8-cell stages and empty zonae. b Percentage of early wastage included morphological abnormalities (misshaped, fragmented, dead embryos) in unfertilized or cleavage-blocked embryos (1-cell and 2-cell embryos), empty zonae and 4- and 8-cell embryos recovered from the uterus. ¢ Control (K) and superovulated (S) (Nos. 5, 7 and 9 indicate experiments run at the same time). Females were killed on day 3 of gestation. d Mean + S.E.M. e Number in parentheses: number of pregnant females. f p values: * < 0.05, ** < 0.01, *** < 0.001 in Fisher-Behrens test (N.S., not significant).
treated females ( a b o u t 10 s u p e r o v u l a t e d e m b r y o s vs. 3 control e m b r y o s in the oviduct a n d 35% vs. 17% total a b n o r m a l i t i e s , T a b l e 1). There is no difference in the a b n o r m a l i t y rates between those in the oviducts a n d those in the uteri. T h e high m o r t a l i t y rate, however, increased b y 3 times when e m b r y o s arriving p r e m a t u r e l y in the uterus were i n c l u d e d in the c a l c u l a t i o n of e m b r y o n i c wastage. T h e e x t r e m e l y high loss (60%, T a b l e 1) of the e m b r y o s observed, which c o r r e s p o n d s with o t h e r r e p o r t s (Allen a n d M c L a r e n , 1971; B e a u m o n t a n d Smith, 1975), can be e x p l a i n e d b y the deleterious, p r e m a t u r e e x p o s u r e of tubal e m b r y o s to uterine e n v i r o n m e n t ( D o y l e et al., 1963). Thus our qualitative a n d q u a n t i t a t i v e analyses indicate that total e m b r y o n i c wastage in s u p e r o v u l a t e d females is the c o n s e q u e n c e of lethality at a r o u n d the time of fertilization (significantly m o r e 1-cell a n d fragm e n t e d e m b r y o s in T a b l e 2 i n d i c a t e a fertilization o r cleavage b l o c k of egg o r zygote) a n d of lethality caused b y localized factors such as p r e m a t u r e arrival in the uterus. The n u m b e r of d e a d e m b r y o s
( g r a n u l a t i o n a n d vacuolization of c y t o p l a s m , syncytial a p p e a r a n c e , b l a s t o m e r e s lysed or of different size), taking into account o n l y 4- a n d 8-cell stages, seems not to be h o m o g e n e o u s a m o n g the 3 e x p e r i m e n t s p e r f o r m e d . O n l y groups 4 a n d 7 show a significantly increased p r o p o r t i o n of a b n o r m a l ity in these e m b r y o s . Therefore, taking m o r t a l i t y d u r i n g p r e i m p l a n t a t i o n d e v e l o p m e n t as an indic a t o r for i m p a i r e d d e v e l o p m e n t might lead to err o n e o u s interpretations. T h e most i m p o r t a n t question r e m a i n s the genetic n o r m a l i t y of non-lethally affected e m b r y o s of either n o r m a l or accelerated t r a n s p o r t a n d development. A c c e l e r a t e d d e v e l o p m e n t , also rep o r t e d elsewhere ( D u k e l o w a n d Riegle, 1974), is i n d i c a t e d b y a significantly increased n u m b e r of 8-cell e m b r y o s (Table 2). C y t o g e n e t i c studies have b e e n limited to possible clastogenic effects, although the i n d u c t i o n of SCEs as an i n d i c a t o r of m u t a g e n i c i t y is generally c o n s i d e r e d to be a m o r e sensitive p a r a m e t e r for genetic d a m a g e than visible c h r o m o s o m a l a b e r r a t i o n s (Perry a n d Wolff, 1975;
(55)
(15)
(23)
(19)
(57)
Total
$5
$7
$9
Total
(42.7)
(33.4)
(43.1)
(51.6)
(0.8)
(0) (0) (2.5)
28.9
37.7_+4.6 ***
26.8_+3.4***
27.3+-4.8 ***
67.9
65.5_+8.7 76.3+-9.4 62.0+_4.2
(42.6)
(31.6)
(48.2)
(47.9)
(2.4)
(3.5) (3.5) (0.9)
(%)
3.2
2.3+0.8 (N.S.) 2.7+-1.0 (N.S.) 4.5+ 1.7 (N.S.)
2.8
4.1_+3.6 0 4.4+_1.2
2-cell
(0)
(0) (0) (0)
(%)
(57.5)
(30.3)
(57.1)
(57.9)
~' Mean + S.E.M. b Percentage of embryos recovered from the uterus. c Number in parentheses: number of pregnant females. d p values: * < 0.05, ** < 0.01, *** < 0.001 in Fisher-Behrens test (N.S., not significant).
43.4
35.2_+5.0 *
46.7_+4.0**
48.7+5.4 ***.d
16.0
14.6_+6.7 11.5+-8.1 21.9+_3.4
(9)" (9) (37)
K5 K7 K9
4-cell
8-cell
(%) b
Stages of development at recovery (%) ~
Expt.
groups
6.5
7.7+- 1.7 *
5.8_+1.2"**
6.0+- 1.6 *
1.5
1.2+_0.7 0.6+-0.6 2.8+_0.6
1-cell
(40.0)
(30.3)
(32.3)
(47.6)
(2.1)
(0) (0) (6.3)
(%)
16.7
18.5+_2.7 ***
15.1 +-3.0 (N.S.) 16.5+3.0"
7.0
7.8_+2.7 6.8+_2.9 6.4_+1.5
Fragmented
(41.2)
(39.7)
(31.3)
(52.7)
(0)
(0) (0) (0)
(%)
3.0
6.4+-0.8 (N.S.) 1.7_+0.6 (N.S.) 0.9+_0.6 (N.S.)
4.6
6.7_+3.5 4.7+1.9 2.5_+0.5
Zonae
(77.0)
(70.0)
(60.9)
(100.0)
(0)
(0) (0) (0)
(%)
EMBRYONIC STAGES AND SITE OF RECOVERY ON DAY 3 OF GESTATION (PLUG = l) F O L L O W I N G SPONTANEOUS O V U L A T I O N (K5, K7, K9) A N D SUPEROVULATION ($5, $7. $9) OF SWA MICE
TABLE 2
193 TABLE 3 SCE F R E Q U E N C I E S a IN 4- A N D 8-CELL EMBRYOS Stage
4-celt 8-cell
Spontaneously ovulated
Superovulated
Oviduct
Oviduct
3.5+0.2 b 2.9+0.3
11.6+0.8 12.0+__0.4
t test d Uterus N.S. c N.S.
10.6+0.4 9.6+1.5
p < 0.001 p < 0.001
a Calculation based on a m i n i m u m of 20 second replication metaphases for each stage and each localization per experiment (data pooled). b Mean + S.E.M. c N.S. (not significant), localization without influence on SEC frequencies. d Superovulated significantly higher than control SCEs.
a
Fig. 1. Metaphase chromosomes with differentially stained sister chromatids from preimplantation embryos after in vitro incubation in BrdU (10 -6 M) for 2 replication cycles. (a) Preparation from a spontaneous ovulation-derived morula showing background SCE frequency (average level about 2.9/diploid blastomere); arrows point at 2 SCEs; magnification 140:1. (b, c) Multiple SCEs (average range from 9.6 to 12.0/blastomere) after superovulation; arrows indicate 16 and 14 SCEs for 8-cell (b) and morula (c) stages, respectively; magnification 1120:1. Following superovulation, background SCE increases up to about 4 times in preimplantation embryos.
Popescu et al., 1977; Carrano and Thompson, 1978; Latt et al., 1981). We report here that superovulation induces a significant increase in SCEs among preimplantation embryos (Table 3, Fig. 1). Tubal and uterine 4- and 8-cell embryos were handled separately in order to verify possible differences between stages and localization. Only morphologically normal embryos that appeared to cleave regularly to morula stage after BrdU incubation were used for chromosome spreading. No difference in the frequency of SCEs was observed between the differently staged and localized embryos (Table 3). We think that controversy about adverse effects of superovulation can be explained by the individualized response to gonadotropins which is linked to endogenous and/or exogenous factors. Exogenous hormones may induce hormone levels within the normal range, thus having no negative effect. However, induction of ovulation with gonadotropic hormones often results in abnormally elevated levels of steroids. In our experiments, certain tendencies such as increased embryo number, abnormality rate, higher cleavage rate and accelerated tubal transport are indications of hyperstimulation and excessive steroid level. It has been suggested that the rise of estrogens reflects the quality of follicular maturation and subsequent conception and pregnancy (Pittaway and Wentz, 1983). Any disturbance of the proper preovulatory or oviductal milieu, caused by exogenous hormones or by an abnormal endogenous hormone level, might be deleterious to the egg and/or preimplantation embryo. The consistently higher frequency of SCEs in
194 superovulated embryos requires further investigat i o n s to d e t e r m i n e w h e t h e r the i n c r e a s e in S C E s is a d i r e c t e f f e c t o f e x o g e n o u s g o n a d o t r o p i n s o r increased endogenous
sex s t e r o i d s o n
m a t e r i a l o f t h e m e i o t i c egg a n d / o r embryo.
Regarding
additional
the genetic
early cleavage indirect
conse-
q u e n c e s o f s u p e r o v u l a t i o n - - s u c h as a l o n g - t e r m alteration of the h o r m o n e tract --
m i l i e u in t h e g e n i t a l
w e c o u l d n o t d e t e c t a n y d i f f e r e n c e s in
S C E level a n d a b n o r m a l i t y r a t e b e t w e e n t u b a l a n d uterine-located embryos.
This
indicates
no
ad-
ditive effect of the hostile uterine milieu on the p r e i m p l a n t a t i o n e m b r y o s at t h e t i m e o f i n v e s t i g a tion but rather p o i n t s to d a m a g e during meiosis and/or
t h e first d i v i s i o n s .
T h e s e r e s u l t s s u g g e s t a p o t e n t i a l risk for t h e conceptus caused by superovulatory hormone doses.
Acknowledgements The authors thank Mrs. Elisabeth Weidinger f o r h e r skillful t e c h n i c a l a s s i s t a n c e . T h e y a r e a l s o i n d e b t e d to t h e F o n d s O s t e r r . K r e b s f o r s c h u n g s i n s t i t u t for generous support.
References Allen, J., and A. McLaren (1971) Cleavage rate of mouse eggs from induced and spontaneous ovulation, J. Reprod. Fertil., 27, 137-140. Beaumont, H.M., and A.F. Smith (1975) Embryonic mortality during the pre- and post-implantation periods of pregnancy in mature mice after superovulation, J. Reprod. Fertil., 45, 437-448. Brinster, R.L. (1963) A method for in vitro cultivation of mouse ova from two-cell to blastocyst, Exp. Cell. Res., 32, 205 208. Carrano, A.L., L.H. Thompson, D.G. Stetka, J.L. Minkler, J. Mazrimas and S. Framy (1974) DNA cross-linking, sister chromatid exchange and specific locus mutations, Mutation Res., 63, 175-188. Czeizel, A. (1980) Are contraceptive pills teratogenic?, Acta Morphol. Acad. Sci. Hung., 28, 177-188. Doyle, L.L., A.H. Gates and R.W. Noyes (1963) Asynchronous transfer of mouse ova, Fertil. Steril., 14, 215-225. Dukelow, W.R., and G.D. Riegle (1974) Transport of gametes and survival of the ovum as a function of the oviduct, in: A.D. Johnson and C.W. Foley (Eds.), Oviduct and its Function, Academic Press, New York, pp. 193-220. Dutkowski, R.T., M.J. Kevin and E.C. Jenkins (1983) The effect of oral contraceptives on sister chromatid exchange, blast transformation, mitotic index and micronuclei formation, Exp. Cell. Biol., 51, 115-120.
Elbling, L. (1973) Does gonadotropin-induced ovulation in mice cause malformations in the offspring?, Nature (London), 246, 37 39. Elbling, L. (1975a) Congenital malformations in mice after gonadotropin induced ovulation, Proc. Soc. Exp. Biol. Med., 149, 349-379. Elbling, L. (1975b) Malformations induced by hormones in mice and their transmission to the offspring, Exp. Pathol.. 11, 115-122. Elbling, L., and M. Color (1985) A method for analysing sister chromatid exchange in mouse preimplantation embryos, Mutation Res., 147, 23 28. Fiser, P.S., and J.W. Macpherson (1982) Survival of preimplanration embryos in the uteri of mice induced to superovulate and subsequently ovariectomized, J. Reprod. Fertil.. 64, 33 36. Fraser, LR. (1977) Fertilization and preimplantation development in vitro of mouse eggs obtained following stimulation with different doses of pregnant mare serum, Differentiation, 9, 29 35. Fujimoto, S.. N. Pahlavan and W.R. Dukelow (1974) Chromosomal abnormalities in rabbit preimplantation blastocysts induced by superovulation, J. Reprod. Fertil.. 4(/, 177-181. Harrington, F.E. (1965) Transportation of ova and zygotes through the genital tract of immature mice treated with gonadotropins, Endocrinology, 77, 635-640. Husum, B., H.C. Wolf and E. Niebuhr (1982) Normal sister chromatid exchanges in oral contraceptive users, Mutation Res., 103, 161 164. Lang, R., and U. Redmann (1979) Non-mutagenicity of some sex-hormones in the Ames Salmonella/microsome mutagenicity test, Mutation Res., 67, 361 365. Latt, S.A., J. Allen, S.E. Bloom, A. Carrano, A. Falke, D. Kram, E. Schneider, R. Schreck, R. Tice, B. Whitfield and S. Wolff (1981) Sister chromatid exchanges: A report of GENE-TOX program, Mutation Res., 87, 17-62. Marsden, H.M., and E.H. Bronson (1964) Estrous synchrony in mice: alteration by exposure to male urine, Science, 144, 1469. Maudlin, l., and L.R. Fraser (1977) The effect of PMSG dose on the incidence of chromosomal anomalies in mouse embryos fertilized in vitro, J. Reprod. Fertil., 50, 275-280. Miller, B.G., and D.T. Armstrong (1981) Effects of a superovulatory dose of pregnant mare serum gonadotropin on ovarian function, serum estradiol, and progesterone levels and early embryo development in immature rats. Biol. Repro&, 25, 261 -.271. Murthy, P.B.K., and K. Prema (1979) Sister-chromatid exchanges in oral contraceptive users, Mutation Res., 68, 149-152. Murthy, P.B.K., and K. Prema (1983) Further studies on sister-chromatid exchange frequency in users of hormonal contraceptives, Mutation Res., 119, 351 354. Nishimura, H., and A. Shikata (1958) The maldevelopment of the fetuses of mice treated with gonadotropic hormone before conception, Arch. Pathol., 31, 195-202. Perry, P., and H.J. Evans (1975) Cytological detection of
195 mutagen-carcinogen exposure by sister chromatid exchange, Nature (London), 258, 121-125. Pittaway, D.E., and A.C. Wentz (1983) Evaluation of the exponential rise of serum estradiol concentrations in human menopausal gonadotropin-induced cycles, Fertil. Steril., 40, 763-767. Popescu, N.C., D. Turnbull and J.A. DiPaolo (1977) Sister chromatid exchange and chromosome aberration analysis with the use of several carcinogens and noncarcinogens: brief comm., J. Natl. Cancer Inst., 59, 289-293. Schardein, J. (1980) Congenital abnormalities and hormones during pregnancy: a clinical review, Teratology, 22, 251-270.
Schenker, J.G., S. Yarkoni and M. Granat (1981) Multiple pregnancies following induction of ovulation, Fertil. Steril., 35, 105-123. Smith, C.M., and C.L. Chrisman (1975) Failure of exogenous gonadotropin controlled ovulation to cause digit abnormalities in mice, Nature (London), 253, 631. Takagi, N., and M. Sasaki (1976) Digynic triploidy after superovulation in mice, Nature (London), 264, 278-281. Wallace, M.E., F.M. Badr and R.S. Badr (1979) Studies in mice on the mutagenicity of two contraceptive drugs, J. Med. Genet., 16, 206-209.
147 (1985) 189-195
189
Elsevier MTR 04047
Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice L. Elbling and M. Colot Institute for Applied and Experimental Oncology, Vienna University, Borschkegasse 8a, A - 1090 Vienna (Austria)
(Received30 October 1984) (Revisionreceived27 December1984) (Accepted17 January1985)
Summary Both sister-chromatid exchange (SCE) response and embryonic development and transport in preimplantation embryos were evaluated on day 3 of gestation (vaginal plug = 1) of superovulated Swiss mice. Superovulation was found to have significant effects on number of preimplantation embryos (increase), embryo localization (accelerated transport), cleavage rate (advanced development) and abnormality rate (misshaped, fragmented, dead embryos). Superovulated 4- and 8-cell embryos collected from oviducts and uteri and incubated in vitro with 5-bromodeoxyuridine (BrdU) displayed up to 4 times higher SCE frequency than spontaneously ovulated embryos. This increase is independent of stage of development and location at the time of embryo collection. The results indicate that superovulated embryos may have induced DNA lesions.
Since the introduction of hormones (gonadotropins and sex steroid hormones) in medical practice and animal research, there have been controversies about the possible harmful effects of such agents. Gonadotropic hormone treatment for ovulation induction may lead to a hyperstimulation syndrome with multiple pregnancies, abortions and a discrepancy between ovulation and pregnancy rates (Schenker et al., 1981). There are conflicting reports as to whether or not specific malformations are induced by gonadotropic and sex hormones (Schardein, 1980). Animal experiments have not been helpful in the interpretation of findings in man. Studies reporting the association of increased malformation rates and ovulation-inducing hormones (Nishimura and Shikata, 1958; Elbling, 1973, 1975a, b) are contradicted by Smith and Chrisman (1975). Several studies on mutagenicity of gonadotropins and sex hormones
have been performed. Sex steroids have no mutagenic effect in the Ames test (Lang and Redmann, 1979). However, both positive and negative chromosomal aberration effects have been reported in various organisms (cited in Fraser, 1977; in Wallace et al., 1979; in Czeizel, 1980). Of particular interest is the finding that chromosomal aberrations were increased in superovulated early embryos (Fujimoto et al., 1974; Takagi and Sasaki, 1976; Maudlin and Fraser, 1977). Since the introduction of the SCE assay in mutagenicity testing (Perry and Evans, 1975) as a high-resolution technique for measuring subtle alterations in genetic material, there have been few studies employing this system with reproductive hormones. One negative finding (Husum et al., 1982) is contrasted by reports of increased SCE (Murthy and Prema, 1979, 1983; Dutkowski et al., 1983) in users of contraceptives. No SCE analysis has been con-
0165-1161/85/$03.30 © 1985 ElsevierSciencePublishersB.V.(BiomedicalDivision)
190 ducted after hormone-induced ovulation. It is recognized that the interrelationship between ovarian steroid secretions and their feedback effect on gonadotropins is an important component in the control of normal ovulatory function. Parameters used to assess follicular development include both steroid and gonadotropin content of follicular fluid. In most cases, superovulation regimens are non-physiological and may result in an abnormal follicular steroid milieu (Miller and Armstrong, 1981, rat; Pittaway and Wentz, 1983, man). The in vivo exposure (superovulation)-in vitro SCE assay in preimplantation embryos (Elbling and Colot, 1985) may be useful for the detection of mutagenic effects. We report on both SCE analysis of preimplantation embryos derived from hyperstimulated cycles and indicators (embryonic development and transport) of this hyperstimulation following gonadotropin-induced ovulation in random-bred Swiss mice. Materials and methods
Mice and hormone-induced ovulation Random-bred, 2 months old, female Swiss mice and males of the same strain and age were used. Ovulation was induced by intraperitoneai injections of 5 IU PMS (Gestyl, Organon) followed 48 h later by 5 IU H C G (Pregnyl, Organon). At the expected time of ovulation injected females were placed with fertile males for 2 - 4 h and then checked for vaginal plugs ( = day 1). Spontaneously ovulating (non-hormone-treated) females synchronized with males 2 days before mating (Marsden and Bronson, 1964) were caged together with males overnight and examined for the presence of a vaginal plug the following morning. S C E analysis Mated females were killed on day 3 of gestation. Preimplantation embryos were flushed in phosphate-buffered saline with 2% bovine serum albumin from the reproductive tract (oviduct and uterus were flushed separately). Embryos were collected into standard medium (Brinster, 1963) keeping track of their derivation and were classified according to the stages of development and to morphological aberrations by microscopic examination. Embryos found arrested at the 1- or
2-cell stage, or having undergone degeneration or with cytoplasmic granulation at 4- and 8-cell stages were discarded. Thus, only normal-appearing 4and 8-cell embryos were incubated in BrdU for DNA labelling and differential chromatid staining. Groups of thirty 4- or 8-cell embryos were pulsed with 10 - 6 M BrdU (Sigma Chemical Co.) in standard medium for 24 h. The BrdU medium was replaced with fresh every 6 h. The experimental procedure employed for chromosome preparation and sister-chromatid differentiation has been described previously (Elbling and Colot, 1985). Homogeneity of SCE data, which was determined by variance analysis, allowed the pooled presentation of several experiments. Results and discussion
Under superovulation conditions 40-50% of the females ovulated and mated successfully with non-treated males. About 30% of spontaneously ovulating females became pregnant. After superovulation, most of the females produced 31 73 embryos (controls 14-20) with a range of 17-93 embryos (versus a control range of 10-23). Only a small proportion (7%) of hormone-induced females ovulated within the normal range. Details of the preimplantation pregnancy are given in Tables 1 and 2. The individual experiments are listed separately to illustrate the reproducibility of the results. All experiments were performed within a 1-month period in order to exclude seasonal variations in response (Elbling, 1975a). Totals of 3027 and 892 embryos were obtained from 57 superovulated females and from 55 controls, respectively. An unusually high proportion (40%) of superovulated embryos was recovered from the uterus. 2 days after fertilization, the great majority of cleavage embryos from uninduced females were located in the oviduct (Table 1). This accelerated transport of the embryos, which is consistent with other reports (Harrington, 1965; Beaumont and Smith, 1975; Fiser and Macpherson, 1982), may be attributed to an excessive estrogen production by the ovaries after hyperstimulation (Harrington, 1965; Dukelow and Riegle, 1974; Miller and Armstrong, 1981). Abnormality rates of embryos, calculated either for tubal location only or as a total, were significantly increased in hormone-
191 TABLE 1 LOCAL DISTRIBUTION, ABNORMALITY a AND WASTAGE b OF PREIMPLANTATION EMBRYOS ON DAY 3 OF GESTATION (PLUG = l) FOLLOWING SPONTANEOUS OVULATION (K5, K7, K9) AND SUPEROVULATION ($5, $7, $9) OF SWA MICE Expt. c groups
Number of embryos Total Average
K5 K7 K9
163 144 585
18.1 +1.2 d 16.0_+1.1 15.8_+0.5
(9) e (9) (37)
Total
892
16.6
Total distribution (~)
Abnormal a embryos/mouse (~)
Oviduct
Oviduct
Uterus
Total abnormal ~ (%)
Total wastage h (~)
Uterus
100 95.5 99.0
0 4.9 1.0
3.7_+1.1 1.7_+0.5 3.1_+0.5
0 0.3 0
19.7+6.1 12.8_+3.5 19.2_+3.1
23.8_+5.7 16.8+6.3 21.3_+3.3
(55)
98.2
2.0
2.8
0.1
17.2
20.6
$5 $7 $9
744 1212 1071
49.6_+4.9***.f 52.7_+3.3 *** 56.4_+5.3***
(15) (23) (19)
56.3 52.8 70.0
43.7 47.2 30.0
5.9_+ 1.4 * 12.1 _+2.3 *** 13.2_+2.8**
7.4_+2.6 7.2+1.8 6.2+2.0
36.2_+3.4 * 35.5-+3.7*** 34.0-+3.5**
61.4-+7.9 *** 67.9-+7.9 *** 59.2_+5.9 ***
Total
3 027
52.9
(57)
59.7
40.3
10.4
6.9
35.2
62.8
a Morphologically abnormal 1-8-cell stages and empty zonae. b Percentage of early wastage included morphological abnormalities (misshaped, fragmented, dead embryos) in unfertilized or cleavage-blocked embryos (1-cell and 2-cell embryos), empty zonae and 4- and 8-cell embryos recovered from the uterus. ¢ Control (K) and superovulated (S) (Nos. 5, 7 and 9 indicate experiments run at the same time). Females were killed on day 3 of gestation. d Mean + S.E.M. e Number in parentheses: number of pregnant females. f p values: * < 0.05, ** < 0.01, *** < 0.001 in Fisher-Behrens test (N.S., not significant).
treated females ( a b o u t 10 s u p e r o v u l a t e d e m b r y o s vs. 3 control e m b r y o s in the oviduct a n d 35% vs. 17% total a b n o r m a l i t i e s , T a b l e 1). There is no difference in the a b n o r m a l i t y rates between those in the oviducts a n d those in the uteri. T h e high m o r t a l i t y rate, however, increased b y 3 times when e m b r y o s arriving p r e m a t u r e l y in the uterus were i n c l u d e d in the c a l c u l a t i o n of e m b r y o n i c wastage. T h e e x t r e m e l y high loss (60%, T a b l e 1) of the e m b r y o s observed, which c o r r e s p o n d s with o t h e r r e p o r t s (Allen a n d M c L a r e n , 1971; B e a u m o n t a n d Smith, 1975), can be e x p l a i n e d b y the deleterious, p r e m a t u r e e x p o s u r e of tubal e m b r y o s to uterine e n v i r o n m e n t ( D o y l e et al., 1963). Thus our qualitative a n d q u a n t i t a t i v e analyses indicate that total e m b r y o n i c wastage in s u p e r o v u l a t e d females is the c o n s e q u e n c e of lethality at a r o u n d the time of fertilization (significantly m o r e 1-cell a n d fragm e n t e d e m b r y o s in T a b l e 2 i n d i c a t e a fertilization o r cleavage b l o c k of egg o r zygote) a n d of lethality caused b y localized factors such as p r e m a t u r e arrival in the uterus. The n u m b e r of d e a d e m b r y o s
( g r a n u l a t i o n a n d vacuolization of c y t o p l a s m , syncytial a p p e a r a n c e , b l a s t o m e r e s lysed or of different size), taking into account o n l y 4- a n d 8-cell stages, seems not to be h o m o g e n e o u s a m o n g the 3 e x p e r i m e n t s p e r f o r m e d . O n l y groups 4 a n d 7 show a significantly increased p r o p o r t i o n of a b n o r m a l ity in these e m b r y o s . Therefore, taking m o r t a l i t y d u r i n g p r e i m p l a n t a t i o n d e v e l o p m e n t as an indic a t o r for i m p a i r e d d e v e l o p m e n t might lead to err o n e o u s interpretations. T h e most i m p o r t a n t question r e m a i n s the genetic n o r m a l i t y of non-lethally affected e m b r y o s of either n o r m a l or accelerated t r a n s p o r t a n d development. A c c e l e r a t e d d e v e l o p m e n t , also rep o r t e d elsewhere ( D u k e l o w a n d Riegle, 1974), is i n d i c a t e d b y a significantly increased n u m b e r of 8-cell e m b r y o s (Table 2). C y t o g e n e t i c studies have b e e n limited to possible clastogenic effects, although the i n d u c t i o n of SCEs as an i n d i c a t o r of m u t a g e n i c i t y is generally c o n s i d e r e d to be a m o r e sensitive p a r a m e t e r for genetic d a m a g e than visible c h r o m o s o m a l a b e r r a t i o n s (Perry a n d Wolff, 1975;
(55)
(15)
(23)
(19)
(57)
Total
$5
$7
$9
Total
(42.7)
(33.4)
(43.1)
(51.6)
(0.8)
(0) (0) (2.5)
28.9
37.7_+4.6 ***
26.8_+3.4***
27.3+-4.8 ***
67.9
65.5_+8.7 76.3+-9.4 62.0+_4.2
(42.6)
(31.6)
(48.2)
(47.9)
(2.4)
(3.5) (3.5) (0.9)
(%)
3.2
2.3+0.8 (N.S.) 2.7+-1.0 (N.S.) 4.5+ 1.7 (N.S.)
2.8
4.1_+3.6 0 4.4+_1.2
2-cell
(0)
(0) (0) (0)
(%)
(57.5)
(30.3)
(57.1)
(57.9)
~' Mean + S.E.M. b Percentage of embryos recovered from the uterus. c Number in parentheses: number of pregnant females. d p values: * < 0.05, ** < 0.01, *** < 0.001 in Fisher-Behrens test (N.S., not significant).
43.4
35.2_+5.0 *
46.7_+4.0**
48.7+5.4 ***.d
16.0
14.6_+6.7 11.5+-8.1 21.9+_3.4
(9)" (9) (37)
K5 K7 K9
4-cell
8-cell
(%) b
Stages of development at recovery (%) ~
Expt.
groups
6.5
7.7+- 1.7 *
5.8_+1.2"**
6.0+- 1.6 *
1.5
1.2+_0.7 0.6+-0.6 2.8+_0.6
1-cell
(40.0)
(30.3)
(32.3)
(47.6)
(2.1)
(0) (0) (6.3)
(%)
16.7
18.5+_2.7 ***
15.1 +-3.0 (N.S.) 16.5+3.0"
7.0
7.8_+2.7 6.8+_2.9 6.4_+1.5
Fragmented
(41.2)
(39.7)
(31.3)
(52.7)
(0)
(0) (0) (0)
(%)
3.0
6.4+-0.8 (N.S.) 1.7_+0.6 (N.S.) 0.9+_0.6 (N.S.)
4.6
6.7_+3.5 4.7+1.9 2.5_+0.5
Zonae
(77.0)
(70.0)
(60.9)
(100.0)
(0)
(0) (0) (0)
(%)
EMBRYONIC STAGES AND SITE OF RECOVERY ON DAY 3 OF GESTATION (PLUG = l) F O L L O W I N G SPONTANEOUS O V U L A T I O N (K5, K7, K9) A N D SUPEROVULATION ($5, $7. $9) OF SWA MICE
TABLE 2
193 TABLE 3 SCE F R E Q U E N C I E S a IN 4- A N D 8-CELL EMBRYOS Stage
4-celt 8-cell
Spontaneously ovulated
Superovulated
Oviduct
Oviduct
3.5+0.2 b 2.9+0.3
11.6+0.8 12.0+__0.4
t test d Uterus N.S. c N.S.
10.6+0.4 9.6+1.5
p < 0.001 p < 0.001
a Calculation based on a m i n i m u m of 20 second replication metaphases for each stage and each localization per experiment (data pooled). b Mean + S.E.M. c N.S. (not significant), localization without influence on SEC frequencies. d Superovulated significantly higher than control SCEs.
a
Fig. 1. Metaphase chromosomes with differentially stained sister chromatids from preimplantation embryos after in vitro incubation in BrdU (10 -6 M) for 2 replication cycles. (a) Preparation from a spontaneous ovulation-derived morula showing background SCE frequency (average level about 2.9/diploid blastomere); arrows point at 2 SCEs; magnification 140:1. (b, c) Multiple SCEs (average range from 9.6 to 12.0/blastomere) after superovulation; arrows indicate 16 and 14 SCEs for 8-cell (b) and morula (c) stages, respectively; magnification 1120:1. Following superovulation, background SCE increases up to about 4 times in preimplantation embryos.
Popescu et al., 1977; Carrano and Thompson, 1978; Latt et al., 1981). We report here that superovulation induces a significant increase in SCEs among preimplantation embryos (Table 3, Fig. 1). Tubal and uterine 4- and 8-cell embryos were handled separately in order to verify possible differences between stages and localization. Only morphologically normal embryos that appeared to cleave regularly to morula stage after BrdU incubation were used for chromosome spreading. No difference in the frequency of SCEs was observed between the differently staged and localized embryos (Table 3). We think that controversy about adverse effects of superovulation can be explained by the individualized response to gonadotropins which is linked to endogenous and/or exogenous factors. Exogenous hormones may induce hormone levels within the normal range, thus having no negative effect. However, induction of ovulation with gonadotropic hormones often results in abnormally elevated levels of steroids. In our experiments, certain tendencies such as increased embryo number, abnormality rate, higher cleavage rate and accelerated tubal transport are indications of hyperstimulation and excessive steroid level. It has been suggested that the rise of estrogens reflects the quality of follicular maturation and subsequent conception and pregnancy (Pittaway and Wentz, 1983). Any disturbance of the proper preovulatory or oviductal milieu, caused by exogenous hormones or by an abnormal endogenous hormone level, might be deleterious to the egg and/or preimplantation embryo. The consistently higher frequency of SCEs in
194 superovulated embryos requires further investigat i o n s to d e t e r m i n e w h e t h e r the i n c r e a s e in S C E s is a d i r e c t e f f e c t o f e x o g e n o u s g o n a d o t r o p i n s o r increased endogenous
sex s t e r o i d s o n
m a t e r i a l o f t h e m e i o t i c egg a n d / o r embryo.
Regarding
additional
the genetic
early cleavage indirect
conse-
q u e n c e s o f s u p e r o v u l a t i o n - - s u c h as a l o n g - t e r m alteration of the h o r m o n e tract --
m i l i e u in t h e g e n i t a l
w e c o u l d n o t d e t e c t a n y d i f f e r e n c e s in
S C E level a n d a b n o r m a l i t y r a t e b e t w e e n t u b a l a n d uterine-located embryos.
This
indicates
no
ad-
ditive effect of the hostile uterine milieu on the p r e i m p l a n t a t i o n e m b r y o s at t h e t i m e o f i n v e s t i g a tion but rather p o i n t s to d a m a g e during meiosis and/or
t h e first d i v i s i o n s .
T h e s e r e s u l t s s u g g e s t a p o t e n t i a l risk for t h e conceptus caused by superovulatory hormone doses.
Acknowledgements The authors thank Mrs. Elisabeth Weidinger f o r h e r skillful t e c h n i c a l a s s i s t a n c e . T h e y a r e a l s o i n d e b t e d to t h e F o n d s O s t e r r . K r e b s f o r s c h u n g s i n s t i t u t for generous support.
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Elbling, L. (1973) Does gonadotropin-induced ovulation in mice cause malformations in the offspring?, Nature (London), 246, 37 39. Elbling, L. (1975a) Congenital malformations in mice after gonadotropin induced ovulation, Proc. Soc. Exp. Biol. Med., 149, 349-379. Elbling, L. (1975b) Malformations induced by hormones in mice and their transmission to the offspring, Exp. Pathol.. 11, 115-122. Elbling, L., and M. Color (1985) A method for analysing sister chromatid exchange in mouse preimplantation embryos, Mutation Res., 147, 23 28. Fiser, P.S., and J.W. Macpherson (1982) Survival of preimplanration embryos in the uteri of mice induced to superovulate and subsequently ovariectomized, J. Reprod. Fertil.. 64, 33 36. Fraser, LR. (1977) Fertilization and preimplantation development in vitro of mouse eggs obtained following stimulation with different doses of pregnant mare serum, Differentiation, 9, 29 35. Fujimoto, S.. N. Pahlavan and W.R. Dukelow (1974) Chromosomal abnormalities in rabbit preimplantation blastocysts induced by superovulation, J. Reprod. Fertil.. 4(/, 177-181. Harrington, F.E. (1965) Transportation of ova and zygotes through the genital tract of immature mice treated with gonadotropins, Endocrinology, 77, 635-640. Husum, B., H.C. Wolf and E. Niebuhr (1982) Normal sister chromatid exchanges in oral contraceptive users, Mutation Res., 103, 161 164. Lang, R., and U. Redmann (1979) Non-mutagenicity of some sex-hormones in the Ames Salmonella/microsome mutagenicity test, Mutation Res., 67, 361 365. Latt, S.A., J. Allen, S.E. Bloom, A. Carrano, A. Falke, D. Kram, E. Schneider, R. Schreck, R. Tice, B. Whitfield and S. Wolff (1981) Sister chromatid exchanges: A report of GENE-TOX program, Mutation Res., 87, 17-62. Marsden, H.M., and E.H. Bronson (1964) Estrous synchrony in mice: alteration by exposure to male urine, Science, 144, 1469. Maudlin, l., and L.R. Fraser (1977) The effect of PMSG dose on the incidence of chromosomal anomalies in mouse embryos fertilized in vitro, J. Reprod. Fertil., 50, 275-280. Miller, B.G., and D.T. Armstrong (1981) Effects of a superovulatory dose of pregnant mare serum gonadotropin on ovarian function, serum estradiol, and progesterone levels and early embryo development in immature rats. Biol. Repro&, 25, 261 -.271. Murthy, P.B.K., and K. Prema (1979) Sister-chromatid exchanges in oral contraceptive users, Mutation Res., 68, 149-152. Murthy, P.B.K., and K. Prema (1983) Further studies on sister-chromatid exchange frequency in users of hormonal contraceptives, Mutation Res., 119, 351 354. Nishimura, H., and A. Shikata (1958) The maldevelopment of the fetuses of mice treated with gonadotropic hormone before conception, Arch. Pathol., 31, 195-202. Perry, P., and H.J. Evans (1975) Cytological detection of
195 mutagen-carcinogen exposure by sister chromatid exchange, Nature (London), 258, 121-125. Pittaway, D.E., and A.C. Wentz (1983) Evaluation of the exponential rise of serum estradiol concentrations in human menopausal gonadotropin-induced cycles, Fertil. Steril., 40, 763-767. Popescu, N.C., D. Turnbull and J.A. DiPaolo (1977) Sister chromatid exchange and chromosome aberration analysis with the use of several carcinogens and noncarcinogens: brief comm., J. Natl. Cancer Inst., 59, 289-293. Schardein, J. (1980) Congenital abnormalities and hormones during pregnancy: a clinical review, Teratology, 22, 251-270.
Schenker, J.G., S. Yarkoni and M. Granat (1981) Multiple pregnancies following induction of ovulation, Fertil. Steril., 35, 105-123. Smith, C.M., and C.L. Chrisman (1975) Failure of exogenous gonadotropin controlled ovulation to cause digit abnormalities in mice, Nature (London), 253, 631. Takagi, N., and M. Sasaki (1976) Digynic triploidy after superovulation in mice, Nature (London), 264, 278-281. Wallace, M.E., F.M. Badr and R.S. Badr (1979) Studies in mice on the mutagenicity of two contraceptive drugs, J. Med. Genet., 16, 206-209.