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Ova and embryos for quality control in reproductive biology
THERIOGENOLOGY OVA ANU EMBRYOS FOR QUALITY CONTROL
IN REPRODUCTIVE BIOlO6Y
S. P. Leibo Departn\ent_ofObsfetrfcs_and $ynecology Scott Uepartment of urology Bay~~~s~~~le e of Redicine , aexas 77030 ABSTRACT The ever increasing application of methods of reproductive biology to human and veterinary medicine and to agriculture carries serious risks as well as benefits. The manipulation of animal and human ova and embryos to achieve these benefits simultaneously exposes the ova and embryos to potentially deleterious changes in their biological, chemical., and physical environment. One way to minimize or at least identify such changes is to adhere to stringent methods of quality control. Rodent embr OS especially have been used in many disciplines as "surrogates" fy or both human and domestic animal ova and embryos. Keywords:
Quality control, ova, embryos, embryo culture. INTRODUCTION
The last ten years has witnessed a remarkable increase in the application of reproductive biology to the fields of medicine and agriculture. There are numerous examples. The transfer of bovine embryos has become common lace as a method to increase the reduction of calves from valuable t! am-sire matings; frozen bovine em !ryos have become a form of international currency in agricultural commerce. The in vitro fertilization of human ova and the transfer of resultant embryos has become an accepted form of medically assisted conception to alleviate human infertility. Zona-free hamster ova are used routinely in the so-called Sperm Penetration Assay (SPA} to measure the fertilizing capacity of human sperm. They are also being used with increasing frequency to assess the functional capacity of sperm not on1 of do~stic species, such as cattle, goats and pigs, but also 0 fy such non-do~stic species as dolphins and ti ers. The production of transgenic mice by the microinjection o 9 DNA into fertilized ova has become a routine procedure. These and other examples demonstrate that the emerging understanding of maturation, fertilization, and development of the mammalian ovum, all performed in vitro under controlled conditions, now permits its routine use in many applications other than in purely research contexts. One of these has been the use of rodent ova and embryos for quality control of many other procedures in reproductive biology. Rodents, as multiparous species, respond to exogenous hormones by Because of this and because of large numbers of oocytes. their re9 atively low maintenance costs, rodents have been usedMyus; plentiful source of ova and embryos for quality control.
ovulatin
JANUARYWDVOL.33NO.l
67
THERIOGENOLOGY embryos, especially because of the availability and homogeneity of genetical1.y in-bred.strains, have been extensively used in this way. n bred mice and their embryos tend to respond in a far more reproducible fashion than do out-bred animals. One example is the rather reliable response of in-bred mice to superovulatory hormones in contrast to the highly variable response of cows, even of the same breed, to analogous hormones. Most of the ap lied procedures of reproductive biolog provide many opportunities Por treated ova and embryos to be exposedydirectly or indirectly to chemical and/or biolo ical contamination and to other adverse conditions. Mouse ova anc embryos have been used as "surrogates" to screen media., instruments, glass and plastic wares used for media preparation, incubators, gases, and other aspects of the procedure being used.
reproductive biology. APPLICATIONS OF REPRODUCTIVE BIOLOGY First demonstrated as an experimental procedure more than thirty years ago., the transfer of bovine embryos from superovulated donors to recipients to increase the production of superior calves has become a routine procedure (1, 2, 3). Tens of thousands of calves have been produced b this method. Yet, because this method requires the collection, han& ling, and transfer of usually six- to eight-day old embryos, its success rate can vary enormously. Pregnancy rates have been reported to range from about 40% to more than 80% after the transfer of large numbers of embryos (4, 5 . In addition to the biolo ical and veterinary as ects, part o c’ this variability can undou% tedly be attributed to tR e many procedural variables that must be controlled if an embryo, collected on the sixth day of its gestation, is to develop normally into a full-term calf. These variables include the media used to flush the embryo out of the donor's uterus, the devices used for collection, chemicals, instruments and materials used to prepare the media, devices used to handle the embryo itself, and the materials and methods used to transfer the embryo into the uterus of the recipient foster mother. Each of these is subject to error, contamination, and mishandling. Nevertheless, many practitioners have become adept and extremely skilled at performing all of the ste s of bovine embryo transfer reliabl and successfully. Because o P the large costs required to produce I omestic animal embryos and the relatively limited supply of them, many
88
JANUARYWBDVOL.33NO.l
THERIOGENOLOGY workers have substituted mouse embryos to develop their skills and to evaluate the media, instruments and materials used in their work. Progress in the in vitro fertilization and transfer (IVF-ET of human embryos has been even more remarkable than that of bovine embryo transfer. The first children resulting from this procedure were born little more than ten years ago (6). Since then, thousands more have been born at clinics all over the world (see reviews in 7-10). Because this procedure involves human embryos, with the attendant ethical and legal ramifications in addition to the strictly
experimentation. Mouse embryos, which can be produced in large numbers from superovulated females and at relatively low cost, rovide the only justifiable and reasonable alternative. Thus, they ave been extensively used as a form of "quality control" of most of the procedures used in human IVF-ET.
R
Another application of reproductive biology for routine use has been the Sperm Penetration Assay to measure functional properties of human sperm. This method, first described in 1976 (II), de ends upon the fact that the ovum of the Syrian golden hamster denuBed of its zona is penetrated by sperm of a very wide variety of mammalian Since then, the SPA has become an accepted clinical species (12). method to evaluate the fertilizing capacity of human sperm (13, 14). There are divergent opinions as to the predictive value of the SPA; some authors have concluded that it has excellent prognostic value of a man's fertility (14-16), whereas others have concluded the opposite (17, 18). Nevertheless, because the SPA does measure the binding of sperm to an ovum, it is extremely sensitive to very slight alterations of the assay conditions. It thus becomes another indicator of changes in the chemical and/or physical pro erties of the materials and methods used in applied reproductive biofogy. Yet another applied use of early embryos has been to measure the toxicity of certain chemicals, environmental pollutants and carcinoBecause of their distinctive development in culture, e.g. a !!enscell embryo developing into a blastocyst, their fastidiousness with respect to culture conditions, and the ease with which large numbers of mouse embryos can be collected and used, they have been extensively used for toxicological studies. They also can serve as what might be referred to as "the ultimate assay" of toxic compounds. That is, treated mouse embryos can be transferred into recipients to determine the number capable of developing into normal live young, the number exhibiting teratogenic defects, and the number of genetic mutants produced by the treatment.
JANUARY19BOVOL.33NO.1
THERIOGENOLOGY QUALITY COKTROL WITH MUSE
EMBRYOS
Evidence from Bovine Embryo Transfer Rethods Although the impetus for them has been quite variable, numerous ations have demonstrated the efficacy of using mouse embryos Consider two ity control in a plied reproductive biolog from the field oP bovine embryo transfer. J' otential sources of embryotoxic compounds may arise even as a result of procedures considered to be good laboratory ractice. For example, sterilization of media and instruments wou fd seem essential to prevent deleterious microbial contamination. Recently, the in vitro culture of mouse embryos helped to identify the presence of embryotoxic compounds as a result of sterilization methods. It is common practice to sterilize rotein- containing solutions by filtering them from a plastic sten +Pe syringe throu h a microporosity filter. Takeda and Hasler (19) found that the ru% ber plun er tips of certain brands of syringes, when used for filter stern-9. lzation, leached an unknown toxic substance into the media that would kill 100% of mouse embryos exposed to it for 3 hours at room temperature. Glass syringes used in the same way caused no dama e to the embryos. The deleterious effect of this toxic compoun8 on bovine embryos might remain undetected for many months, since the pregnancy diagnosis after embryo transfer is often not performed for three months or more. The use of mouse embryos by Takeda and Hasler identified the problem efficiently and quickly. An analo ous "syringe toxicity", as Burles et al (20) termed it, had previous 91y been discovered in cultures of amniotic cells. Another example of toxicity resulting from a practice intended to insure sterility was recently identified by Schiewe et al (21). Plastic straws used for th_e_non-surgical transfer of bovine embryos ;;itoften purchased unsterilized in large quantities to reduce their It is common practice to sterilize them with gaseous ethylene oxide (ETO). Prompted by their own previous studies of ET0 contamination of plastic culture dishes, Schiewe et al (21) studied the consequences of holding mouse embryos in plastic straws previously sterilized by ETO. It is known that the gas gradually defuses out of plasticware. They placed mouse embryos into ETO-sterilized straws that had been aerated for 0. 24. and 72 hours after sterilization. and exposed the embryos for varying times up to 9 hours. As shown by their results in Fig. 1, there was an interaction between the aeration time of the straws (indicated by the labels) and the duration of exposure of the embryos within the straws. All of the embryos incubated for 3 hours or more in freshly sterilized straws were killed. Even after 72 hours of aeration, there was still enough residual ET0 in the plastic straws to kill 2W of the embryos after 6 hours of exposure, and 80% of the embryos after 9 hours. Again mouse embr OS .T ly provided an efficient and inexpensive means.to identify a potentia &;f;;rious result from a seemingly beneficial practice of stenli-
.
70
JANUARY188OVOL.33NO.l
THERIOGENOLOGY
EFFECT OF ETHYLENE OXIDE ON MOUSE EMBRYOS
Figure 1. The combined influence on mouse embryo development of aeration intervals of O_, 24, or 72 hours after sterilization of plastic straws with ethylene oxide and the duration of exposure of the embryos to the straws. The data are those of Schwiewe et al, 1988. Evidence from Human IVF-ET It is in the field of human IVF-ET that the use of mouse embryos has been an especial1 powerful tool for quality control to identify toxic effects and sK ortcomings of culture media and components, instruments, and laboratory practices. First proposed in 1982 (22), the mouse embryo culture system is now used by most IVF-ET clinics. It is not, of course, a panacea for all potential problems that may arise in a human or a bovine clinic. Nevertheless, as its value has been recognized, mouse embryo culture has helped to identify both positive and negative features of procedures used in human IVF-ET. Initially, the culture of two-cell or even eight-cell mouse embryos was used to monitor laboratory procedures. For example, Hillier et al (23) reported that the "embryo hatch rate" of two-cell embryos decreased progressively during a three-week period in which the hospital corridors in the vicinity of the embryo laboratory were being repainted. They concluded that "This disaster coincided wit.!,; particularly unsuccessful phase of clinical IVF activity". culture of two-cell embryos was also sensitive enough to detect an embryotoxic effect of sera collected into standard clinical blood collection tubes; sera collected from the same individuals into
JANUARYlWOVOL.33NO.l
71
THERIOGENOLOGY plastic centrifuge tubes was not embryotoxic (24). A cautionary note the use of the two-cell mouse culture system has been
As early as 1984, @inn et al 427) sug ested that the mouse zygote is a more sensitive indicator for quas*ity control than the two-cell embryo, and urged that its develop~nt to the fully expanded blastocyst stage be used to screen media, instruments, and procedures. They attributed more consistent human pregnancy rates to the quality control instituted by the use of fertilized mouse ova. Recently, Davidson et al (28) compared the sensitivity of one-cell vs. two-cell mouse embryos to detect suboptimal changes in pH and osmolality of culture media. The observed that the development rate of one-cell embryos to the b! astocyst stage was significantly impaired even under conditions in which two-cell stage embryos In addition, when cultured in continued their normal develop~nt. the presence of various concentrations of an embryotoxic compound Cidex, one-cell embryos were damaged by much lower concentrations of Cidex than were two-cell embryos. They concluded that the one-cell embryo culture system is a much more sensitive screening method to optimize media and rocedures than the two-cell system. An even more recent study by %abon et al (29) found that one-cell embryos developed less well when cultured or even exposed briefly to 2Cl% oxygen, but developed normally in the presence of 5% oxygen. They also concluded that mouse zygotes are a more sensitive system to detect suboptimal conditions than are two-cell stage embryos. Regardless of the specific details, it is clear that the culture of mouse embryos has proved to be a sensitive and efficient method to optimize the solutions, instruments and practices of human IVF-ET. Evidence from Toxicological Studies Mouse embryos have also been used in a totally different discipline to analyze the toxic effects of environmental pollutants, mutagens and carcinogens. These studies also illustrate the fact that mouse embryos can be used to detect chemical contamination that might be encountered in other studies of reproductive biology. Even late ;;;vage-stage embryos can be a sensltlve monitor of such contaminaFor example,. potassium dichromate is often used to clean glassware for tissue culture use. The necessity to rinse the glassware extensively to remove traces of dichromate has been demonstrated by the work of Iijima et al.(30). The percentage of embryos that in the presence of low concentrations (2 ;at;;_y ;;te; FygI;u)tured Molar) of dichromate was the same as the .
72
JANUARY199DVOL.33NO.l
THERIOGENOLOGY controls. But the development of inner cell mass outgrowths from the embryos y s significantly reduced even at a concentration as low as 2.5 x lo- Molar. There were other equally significant developmental anomalies caused by very low concentrations of dichromate in the culture media. An even more dramatic effect of the noxious effects of nickel contamination on embryonic development was reported b Storeng and Jonsen (31) They found that exposure of eight-eelT mouse embryos to 4 x low4 Molar NiC12 for as little as 5 minutes was sufficient to reduce blastocyst formation to 60% of the controls. Longer exposures caused further reduction in embryonic development. Remarkably,. however, the percentage of nickel-treated embryos that developed in vivo to full-term fetuses was only slightly lower than that of conii Another example of the sensitivity of mouse embryos to an environmental pollutant was reported by Matsumoto and Spindle (32). ;ie{ fxour;_&hatexposure of embryos at various stages of.development Molar methylmercuric chloride caused significant reduction in blastocyst formation. Interestingly, the sensitivity of the embryos to mercury varied with developmental stage, early blastocysts bein most sensitive, followed by four-cell, and then by two-cell. Moru9 ae were relatively resistant to mercury poisoning. These stage-dependent effects illustrate the subtleties by which certain compounds may affect preimplantation embryos. One final example emphasizes the efficacy of rodent embryos for quality control. Methylnitrosourea (MNU) is a potent mutagen. Nevertheless, morula-stage embryos exposed to low concentrations of MNU develop to the blastocyst stage at about the same rate as When MNU-treated embryos are transferred into controls (33). recipients, they develop into live young. However, Iannaccone (33) found that the perinatal mortality of live-young is significantly and dramatically lower than that of the controls. This is particularly significant because it demonstrates that the deleterious consequences of exposing preimplantation embryos to toxic substances may not appear until long after birth. It is commonly believed that the response of preimplantation embryos to deleterious effects is "all or none". That is, it has usually been assumed that if treated embryos survived and developed to term, then the resulting offspring would be normal. The findings of Iannaccone refute that view. Thus, the risks of exposing preimplantation embryos, human or otherwise, to potentially deleterious compounds may be even greater than previously thought, especially if the effects of the compounds are not manifested until after birth. The need for stringent adherence to procedures of quality control in applied reproductive biology, such as in human IVF-ET, cannot be overemphasized.
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REFERENCES Seidel, G.E., Jr, Science =:351-358
Superovulation and embryo transfer in cattle. (1981).
Mapletoft, R-3, Embr o transfer technolog animal reproduction, $ioTechnology 2:149-l& Sreenan, J.M. Embryo transfer: Vet. Record =:624-629 (1988).
for the enhancement of (1984).
Its uses and recent developments.
Non-surgical embryo transfer in cattle: Wright, J.M. recipient interactions. Theri~enology _@:43-56 (I98I)+
embryo-
Hasler, J.F., McCauley, A.&, Lathrop, W.F., and Foote* R.H. Effect of donor-embr o-recipient interactions on pregnancy rate in a large-scale ovine embryo transfer program. Theriogeno?ogy i: a:I39-168 (1987)" Steptoe, P.C. and Edwards, R.G. Birth after reimplantation of a human embryo. Lancet 2,366 (1978).
Wood, C. and Trownson,A.
Clinical In Vitro Fertilization. Springer-Verlag, London, 1989.
2nd ed.,
Jones, H.W., Jr,, Jones* 8.5, Hodqen, G.D., and Rosenwaks, Z, (eds.). fn Vitro Fertilization, Williams & Wilkins, Baltimore, 1986a Fishel, S. and 9 onds, E.M. feds,]. Press, Oxford, 196 6.
In Vitro Fertilisation.
Edwards, R.G., Purdy, J-M,, and Steptoe, P.C, eds.). of the Human Embryo. hcademic Press, London, 19Q 5.
IRL
Implantation
Yanagimachi, R., Yanagiaachi, H., and Rogers, B.J. The use of zona-free animal *ova as a test-system for the assessment of the fo;o;t)irrngcapacity of human spermatozoa. Blol. Reprod. s:47I-476
. Yanaqimachi, R. Mona-free hamster eggs: Their use in assessing fertilizing capacity and examining chro~so~s of human spermatozoa. Gamete Res. @187-232 (1984). The sperm penetration assay: Rogers, B.J, reevaluated. fert. Serif. a:821-840 (1985).
its
usefulness
Lamb, D.J., Johnson, A.R., Bassham, E-L., and Lipshultz, L-1. How the s rm penetration assay diagnoses infertility. Contemp. OBfGyn _22:18&4 (1989). Ausmanas, M., Tureck, R-W,, Blasco, L., Kopf, G.S., Ribas, J., and Mastroianni, t? Jr., The zona-free hamster eg penetration assay as a prognostic indicator in a human in vitro Pertilization program. Fert. Steril. 43:933-437 (1985).
THERIOGENOLOGY figalioth, E.J., Feinmesser,‘M., Navot, D., Mordel, N., and Bronson, The long-term predlcttve value of the zona-free hamster ova sperm penetration assay. Fert. Steril. Z:490-494 (1989). Foreman, R., Cohen, J., Fehilly, C.B. Fishel, S.B., and Edwards, R.G. The application of the zona-free hamster egg test for the prognosis of human in vitro fertilization. J. in Vitro Fertil. and Emb.
Transf.
1:166-171 (1984).
Kuzan, F.B., Muller, Cd., Zarutskie, P.M., Dixon, L.L., and Soules, M.R. Human sperm penetration assay as an indicator of sperm function in human in vitro fertilization. Fert. Steril. %:282-286 (1987). Takeda, T. and Hasler, J.F. Effect of plastic disposable syrin es on development of mouse embryos in culture. Theriogenology 5:205 9 (I986). Burles, J.V., Huxley, M.P., and Kennedy, T.S. Syringe toxicity in amniotic fluid cultures. Lancet, June 11, 1983, pp. 1336-1337. Schiewe, M.D., Schmidt, P.M., Pontbriand, D., and Wildt, D.E. Toxicity potential of residual ethylene oxide on fresh or frozen embryos maintained in plastic straws. Gam. Res. B:31-39 (1988). Wood, C. and Trounson, A. In vitro fertilization and embryo transfer. _&: Bonnar, J. (ed) Recent Advances in Obstetrics and Gynaecology. Churchill Livingstone, Edinburgh, 1982, pp. 259-282. Hillier, S.G., Dawson, K.J., Afnan, M., Margara, R-A., Ryder, T.A., Wickings, E.J., and Winston, R.M.L. Embryo culture: Quality control in clinical in vitro fertilisation. In: Thompson, W., Joyce, D.N., and Newton, J.R. (eds) In Vitro Fertilisation and Donor Insemination. Royal College of Obstet. and Gynaecol., London, 1985, p. 125. Haimovici, F., Hill, J.A., and Anderson, D.J. Variables affecting toxicity of human sera in mouse embryo cultures. J. in Vitro Fertil. and Embryo Trans. 8:202-206 (1988). Pratt, H.P.M., and Braude, P.R. The use of mouse embryos for quality control of culture reagents in in vitro fertilization programs: a cautionary note. Fert. Steril. u:858-860 (1987). Amy,
M.,
Nachti all,
L.,
and
Quagliarello, J.
The
effect
of
’ preimplantation cu4 ture conditions on murine embryo implantation and fetal development.
Fert.
Steril. @:861-865
(1987).
Quinn, P., Warnes, G.M., Kerin, J-F., and Kirby, C. Culture factors in relation to the success of human in vitro fertilization and embryo transfer. Fert. Steril. u:202-209 (1984). Davidson, A., Vermesh, M., Lobo, R.A., and Paulson, R.J. Mouse embryo culture as quality control for human in vitro fertilization: the one-cell versus the two-cell model. Fert. Steril. @:516-521 (1988).
JANUARY1990
VOL.33NO.l
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THERIOGENOLOGY Pabon, J.E., Jr., Findley, W.E., and Gibbons, W.E. The toxic effect of short exposures to the atmospheric o gen concentration on early mouse embryonic development. Fert. SternY . =:896-899 (1989). Iijima, S., Spindle, A., and. Pederson, R.A. Developmental and ;$;genetic effects of potassium dichromate on mouse embryos in . Teratology a:109-115 (1983). Recovery of mouse embryofox;;;Tr R. and Jonsen, J. short-term in vitro exposure to toxic nickel chloride. Lett. a:85-91 (1984).
Storeng,
Matsumoto, N. and Spindle, A. Sensitivity of early mouse embryos to methylmercury toxicity. Toxicol. Appl. Pharm. a:108-117 (1982). Iannaccone, P.M. Long-term effects of exposure to methylnitro;;;;;; on blastocysts following transfer to surrogate female mice. Res. fi:2785-2789 (1984).
76
JANUARY1990VOL.33NO.l
IN REPRODUCTIVE BIOlO6Y
S. P. Leibo Departn\ent_ofObsfetrfcs_and $ynecology Scott Uepartment of urology Bay~~~s~~~le e of Redicine , aexas 77030 ABSTRACT The ever increasing application of methods of reproductive biology to human and veterinary medicine and to agriculture carries serious risks as well as benefits. The manipulation of animal and human ova and embryos to achieve these benefits simultaneously exposes the ova and embryos to potentially deleterious changes in their biological, chemical., and physical environment. One way to minimize or at least identify such changes is to adhere to stringent methods of quality control. Rodent embr OS especially have been used in many disciplines as "surrogates" fy or both human and domestic animal ova and embryos. Keywords:
Quality control, ova, embryos, embryo culture. INTRODUCTION
The last ten years has witnessed a remarkable increase in the application of reproductive biology to the fields of medicine and agriculture. There are numerous examples. The transfer of bovine embryos has become common lace as a method to increase the reduction of calves from valuable t! am-sire matings; frozen bovine em !ryos have become a form of international currency in agricultural commerce. The in vitro fertilization of human ova and the transfer of resultant embryos has become an accepted form of medically assisted conception to alleviate human infertility. Zona-free hamster ova are used routinely in the so-called Sperm Penetration Assay (SPA} to measure the fertilizing capacity of human sperm. They are also being used with increasing frequency to assess the functional capacity of sperm not on1 of do~stic species, such as cattle, goats and pigs, but also 0 fy such non-do~stic species as dolphins and ti ers. The production of transgenic mice by the microinjection o 9 DNA into fertilized ova has become a routine procedure. These and other examples demonstrate that the emerging understanding of maturation, fertilization, and development of the mammalian ovum, all performed in vitro under controlled conditions, now permits its routine use in many applications other than in purely research contexts. One of these has been the use of rodent ova and embryos for quality control of many other procedures in reproductive biology. Rodents, as multiparous species, respond to exogenous hormones by Because of this and because of large numbers of oocytes. their re9 atively low maintenance costs, rodents have been usedMyus; plentiful source of ova and embryos for quality control.
ovulatin
JANUARYWDVOL.33NO.l
67
THERIOGENOLOGY embryos, especially because of the availability and homogeneity of genetical1.y in-bred.strains, have been extensively used in this way. n bred mice and their embryos tend to respond in a far more reproducible fashion than do out-bred animals. One example is the rather reliable response of in-bred mice to superovulatory hormones in contrast to the highly variable response of cows, even of the same breed, to analogous hormones. Most of the ap lied procedures of reproductive biolog provide many opportunities Por treated ova and embryos to be exposedydirectly or indirectly to chemical and/or biolo ical contamination and to other adverse conditions. Mouse ova anc embryos have been used as "surrogates" to screen media., instruments, glass and plastic wares used for media preparation, incubators, gases, and other aspects of the procedure being used.
reproductive biology. APPLICATIONS OF REPRODUCTIVE BIOLOGY First demonstrated as an experimental procedure more than thirty years ago., the transfer of bovine embryos from superovulated donors to recipients to increase the production of superior calves has become a routine procedure (1, 2, 3). Tens of thousands of calves have been produced b this method. Yet, because this method requires the collection, han& ling, and transfer of usually six- to eight-day old embryos, its success rate can vary enormously. Pregnancy rates have been reported to range from about 40% to more than 80% after the transfer of large numbers of embryos (4, 5 . In addition to the biolo ical and veterinary as ects, part o c’ this variability can undou% tedly be attributed to tR e many procedural variables that must be controlled if an embryo, collected on the sixth day of its gestation, is to develop normally into a full-term calf. These variables include the media used to flush the embryo out of the donor's uterus, the devices used for collection, chemicals, instruments and materials used to prepare the media, devices used to handle the embryo itself, and the materials and methods used to transfer the embryo into the uterus of the recipient foster mother. Each of these is subject to error, contamination, and mishandling. Nevertheless, many practitioners have become adept and extremely skilled at performing all of the ste s of bovine embryo transfer reliabl and successfully. Because o P the large costs required to produce I omestic animal embryos and the relatively limited supply of them, many
88
JANUARYWBDVOL.33NO.l
THERIOGENOLOGY workers have substituted mouse embryos to develop their skills and to evaluate the media, instruments and materials used in their work. Progress in the in vitro fertilization and transfer (IVF-ET of human embryos has been even more remarkable than that of bovine embryo transfer. The first children resulting from this procedure were born little more than ten years ago (6). Since then, thousands more have been born at clinics all over the world (see reviews in 7-10). Because this procedure involves human embryos, with the attendant ethical and legal ramifications in addition to the strictly
experimentation. Mouse embryos, which can be produced in large numbers from superovulated females and at relatively low cost, rovide the only justifiable and reasonable alternative. Thus, they ave been extensively used as a form of "quality control" of most of the procedures used in human IVF-ET.
R
Another application of reproductive biology for routine use has been the Sperm Penetration Assay to measure functional properties of human sperm. This method, first described in 1976 (II), de ends upon the fact that the ovum of the Syrian golden hamster denuBed of its zona is penetrated by sperm of a very wide variety of mammalian Since then, the SPA has become an accepted clinical species (12). method to evaluate the fertilizing capacity of human sperm (13, 14). There are divergent opinions as to the predictive value of the SPA; some authors have concluded that it has excellent prognostic value of a man's fertility (14-16), whereas others have concluded the opposite (17, 18). Nevertheless, because the SPA does measure the binding of sperm to an ovum, it is extremely sensitive to very slight alterations of the assay conditions. It thus becomes another indicator of changes in the chemical and/or physical pro erties of the materials and methods used in applied reproductive biofogy. Yet another applied use of early embryos has been to measure the toxicity of certain chemicals, environmental pollutants and carcinoBecause of their distinctive development in culture, e.g. a !!enscell embryo developing into a blastocyst, their fastidiousness with respect to culture conditions, and the ease with which large numbers of mouse embryos can be collected and used, they have been extensively used for toxicological studies. They also can serve as what might be referred to as "the ultimate assay" of toxic compounds. That is, treated mouse embryos can be transferred into recipients to determine the number capable of developing into normal live young, the number exhibiting teratogenic defects, and the number of genetic mutants produced by the treatment.
JANUARY19BOVOL.33NO.1
THERIOGENOLOGY QUALITY COKTROL WITH MUSE
EMBRYOS
Evidence from Bovine Embryo Transfer Rethods Although the impetus for them has been quite variable, numerous ations have demonstrated the efficacy of using mouse embryos Consider two ity control in a plied reproductive biolog from the field oP bovine embryo transfer. J' otential sources of embryotoxic compounds may arise even as a result of procedures considered to be good laboratory ractice. For example, sterilization of media and instruments wou fd seem essential to prevent deleterious microbial contamination. Recently, the in vitro culture of mouse embryos helped to identify the presence of embryotoxic compounds as a result of sterilization methods. It is common practice to sterilize rotein- containing solutions by filtering them from a plastic sten +Pe syringe throu h a microporosity filter. Takeda and Hasler (19) found that the ru% ber plun er tips of certain brands of syringes, when used for filter stern-9. lzation, leached an unknown toxic substance into the media that would kill 100% of mouse embryos exposed to it for 3 hours at room temperature. Glass syringes used in the same way caused no dama e to the embryos. The deleterious effect of this toxic compoun8 on bovine embryos might remain undetected for many months, since the pregnancy diagnosis after embryo transfer is often not performed for three months or more. The use of mouse embryos by Takeda and Hasler identified the problem efficiently and quickly. An analo ous "syringe toxicity", as Burles et al (20) termed it, had previous 91y been discovered in cultures of amniotic cells. Another example of toxicity resulting from a practice intended to insure sterility was recently identified by Schiewe et al (21). Plastic straws used for th_e_non-surgical transfer of bovine embryos ;;itoften purchased unsterilized in large quantities to reduce their It is common practice to sterilize them with gaseous ethylene oxide (ETO). Prompted by their own previous studies of ET0 contamination of plastic culture dishes, Schiewe et al (21) studied the consequences of holding mouse embryos in plastic straws previously sterilized by ETO. It is known that the gas gradually defuses out of plasticware. They placed mouse embryos into ETO-sterilized straws that had been aerated for 0. 24. and 72 hours after sterilization. and exposed the embryos for varying times up to 9 hours. As shown by their results in Fig. 1, there was an interaction between the aeration time of the straws (indicated by the labels) and the duration of exposure of the embryos within the straws. All of the embryos incubated for 3 hours or more in freshly sterilized straws were killed. Even after 72 hours of aeration, there was still enough residual ET0 in the plastic straws to kill 2W of the embryos after 6 hours of exposure, and 80% of the embryos after 9 hours. Again mouse embr OS .T ly provided an efficient and inexpensive means.to identify a potentia &;f;;rious result from a seemingly beneficial practice of stenli-
.
70
JANUARY188OVOL.33NO.l
THERIOGENOLOGY
EFFECT OF ETHYLENE OXIDE ON MOUSE EMBRYOS
Figure 1. The combined influence on mouse embryo development of aeration intervals of O_, 24, or 72 hours after sterilization of plastic straws with ethylene oxide and the duration of exposure of the embryos to the straws. The data are those of Schwiewe et al, 1988. Evidence from Human IVF-ET It is in the field of human IVF-ET that the use of mouse embryos has been an especial1 powerful tool for quality control to identify toxic effects and sK ortcomings of culture media and components, instruments, and laboratory practices. First proposed in 1982 (22), the mouse embryo culture system is now used by most IVF-ET clinics. It is not, of course, a panacea for all potential problems that may arise in a human or a bovine clinic. Nevertheless, as its value has been recognized, mouse embryo culture has helped to identify both positive and negative features of procedures used in human IVF-ET. Initially, the culture of two-cell or even eight-cell mouse embryos was used to monitor laboratory procedures. For example, Hillier et al (23) reported that the "embryo hatch rate" of two-cell embryos decreased progressively during a three-week period in which the hospital corridors in the vicinity of the embryo laboratory were being repainted. They concluded that "This disaster coincided wit.!,; particularly unsuccessful phase of clinical IVF activity". culture of two-cell embryos was also sensitive enough to detect an embryotoxic effect of sera collected into standard clinical blood collection tubes; sera collected from the same individuals into
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THERIOGENOLOGY plastic centrifuge tubes was not embryotoxic (24). A cautionary note the use of the two-cell mouse culture system has been
As early as 1984, @inn et al 427) sug ested that the mouse zygote is a more sensitive indicator for quas*ity control than the two-cell embryo, and urged that its develop~nt to the fully expanded blastocyst stage be used to screen media, instruments, and procedures. They attributed more consistent human pregnancy rates to the quality control instituted by the use of fertilized mouse ova. Recently, Davidson et al (28) compared the sensitivity of one-cell vs. two-cell mouse embryos to detect suboptimal changes in pH and osmolality of culture media. The observed that the development rate of one-cell embryos to the b! astocyst stage was significantly impaired even under conditions in which two-cell stage embryos In addition, when cultured in continued their normal develop~nt. the presence of various concentrations of an embryotoxic compound Cidex, one-cell embryos were damaged by much lower concentrations of Cidex than were two-cell embryos. They concluded that the one-cell embryo culture system is a much more sensitive screening method to optimize media and rocedures than the two-cell system. An even more recent study by %abon et al (29) found that one-cell embryos developed less well when cultured or even exposed briefly to 2Cl% oxygen, but developed normally in the presence of 5% oxygen. They also concluded that mouse zygotes are a more sensitive system to detect suboptimal conditions than are two-cell stage embryos. Regardless of the specific details, it is clear that the culture of mouse embryos has proved to be a sensitive and efficient method to optimize the solutions, instruments and practices of human IVF-ET. Evidence from Toxicological Studies Mouse embryos have also been used in a totally different discipline to analyze the toxic effects of environmental pollutants, mutagens and carcinogens. These studies also illustrate the fact that mouse embryos can be used to detect chemical contamination that might be encountered in other studies of reproductive biology. Even late ;;;vage-stage embryos can be a sensltlve monitor of such contaminaFor example,. potassium dichromate is often used to clean glassware for tissue culture use. The necessity to rinse the glassware extensively to remove traces of dichromate has been demonstrated by the work of Iijima et al.(30). The percentage of embryos that in the presence of low concentrations (2 ;at;;_y ;;te; FygI;u)tured Molar) of dichromate was the same as the .
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THERIOGENOLOGY controls. But the development of inner cell mass outgrowths from the embryos y s significantly reduced even at a concentration as low as 2.5 x lo- Molar. There were other equally significant developmental anomalies caused by very low concentrations of dichromate in the culture media. An even more dramatic effect of the noxious effects of nickel contamination on embryonic development was reported b Storeng and Jonsen (31) They found that exposure of eight-eelT mouse embryos to 4 x low4 Molar NiC12 for as little as 5 minutes was sufficient to reduce blastocyst formation to 60% of the controls. Longer exposures caused further reduction in embryonic development. Remarkably,. however, the percentage of nickel-treated embryos that developed in vivo to full-term fetuses was only slightly lower than that of conii Another example of the sensitivity of mouse embryos to an environmental pollutant was reported by Matsumoto and Spindle (32). ;ie{ fxour;_&hatexposure of embryos at various stages of.development Molar methylmercuric chloride caused significant reduction in blastocyst formation. Interestingly, the sensitivity of the embryos to mercury varied with developmental stage, early blastocysts bein most sensitive, followed by four-cell, and then by two-cell. Moru9 ae were relatively resistant to mercury poisoning. These stage-dependent effects illustrate the subtleties by which certain compounds may affect preimplantation embryos. One final example emphasizes the efficacy of rodent embryos for quality control. Methylnitrosourea (MNU) is a potent mutagen. Nevertheless, morula-stage embryos exposed to low concentrations of MNU develop to the blastocyst stage at about the same rate as When MNU-treated embryos are transferred into controls (33). recipients, they develop into live young. However, Iannaccone (33) found that the perinatal mortality of live-young is significantly and dramatically lower than that of the controls. This is particularly significant because it demonstrates that the deleterious consequences of exposing preimplantation embryos to toxic substances may not appear until long after birth. It is commonly believed that the response of preimplantation embryos to deleterious effects is "all or none". That is, it has usually been assumed that if treated embryos survived and developed to term, then the resulting offspring would be normal. The findings of Iannaccone refute that view. Thus, the risks of exposing preimplantation embryos, human or otherwise, to potentially deleterious compounds may be even greater than previously thought, especially if the effects of the compounds are not manifested until after birth. The need for stringent adherence to procedures of quality control in applied reproductive biology, such as in human IVF-ET, cannot be overemphasized.
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THERIOGENOLOGY Pabon, J.E., Jr., Findley, W.E., and Gibbons, W.E. The toxic effect of short exposures to the atmospheric o gen concentration on early mouse embryonic development. Fert. SternY . =:896-899 (1989). Iijima, S., Spindle, A., and. Pederson, R.A. Developmental and ;$;genetic effects of potassium dichromate on mouse embryos in . Teratology a:109-115 (1983). Recovery of mouse embryofox;;;Tr R. and Jonsen, J. short-term in vitro exposure to toxic nickel chloride. Lett. a:85-91 (1984).
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Matsumoto, N. and Spindle, A. Sensitivity of early mouse embryos to methylmercury toxicity. Toxicol. Appl. Pharm. a:108-117 (1982). Iannaccone, P.M. Long-term effects of exposure to methylnitro;;;;;; on blastocysts following transfer to surrogate female mice. Res. fi:2785-2789 (1984).
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