Intrinsic embryonic factors that may affect survival after transfer

THERIOGENOLOGY INTRINSICNMRRYONIC FACTORS THAT NAY AFFECT SURVIVAL AFTER TRANSFER W.A. King Centre de Recherche en ReproductionAnimale Faculte de M6decine Vethrinaire,Universitede Montreal C.P. 5000, St-Hyacinthe,Quebec, Canada, J2S 7~6

Introduction The expressionof phenotypic traits has genetic and environmental components which may be thought of as intrinsic and extrinsic. If embryo viability or survival is regarded as a phenotypic trait, it follows that embryo loss may result from faults in either, or both, of these genetic and environmentalcomponents, or from the interplay between the two. The extent of embryo loss under various conditionshas been well studied. As new techniquessuch as AI (with fresh or frozen semen), embryo transfer (first by surgical then by non-surgical methods), vitro fertilization, and "gene freezing, micromanipulation, g injection"are incorporatedinto livestockproduction,pregnancy rates are used to evaluate the embryo loss associatedwith each technique. These studies are importantand, understandably,numerous. They have revealed three general trends. The first is that a certain level of embryonic loss always occurs, no matter what the conditions. The second is that the more an embryo is handled, the greater the incidenThe third is that pregnancy rates usually ce of embryo loss. increase as the practice of any new technique improves, suggesting that some of the deleteriousextrinsic factors can be overcome. However, the underlyingcauses of these embryonic losses have received less attention,are poorly understood,and are obviously of considerable relevance to the embryo transfer industry. Among the intrinsic factors that are known to adversely affect the embryo are chromosomeabnormalitiesand single gene mutations: chromosome abnormalitiesin man are consideredto be one of the major causes of fetal death (1); single mutant genes which are lethal and begin to affect embryonic developmentas early as the first cleavage division (2,3) have been identifiedin mice. However, another class of intrinsic factors, mutagen-inducedirreversiblemodifications of the DNA, may also be a source of embryonic loss. The importanceof such factors is hinted at by extrapolationfrom studies of cultured cells and embryos. These have demonstratedlong-lastingdeleterious effects on cells exposed to many of the same insults that embryos may suffer during transfer. While the initial defect under such Acknowledgments The author is grateful to his colleaguesat the CRRA and to Lena Dahlstrom and Gail Jeffcoate for helping prepare this manuscript; to Helen Rheaume for patiently typing it; and to NSERC for financial support. JANUARY 1985 VOL. 23 NO. 1

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conditions is triggered by exposure to a temporary extrinsic factor, the permanent result certainly becomes intrinsic and will be considered as such in this paper. If one is to consider eliminating, or even reducing, embryo loss due to intrinsic factors, it is prudent to determine their'extent as well as their origin and time of action. The aim of this paper is 1) to review what is known of obviously adverse intrinsic factors, such as chromosome abnormalities, in embryos and; 2) by extrapolation from other research models, to suggest some less obvious intrinsic factors which may also contribute to embryonic loss in domestic animals.

Chromosome Anomalies Chromosome abnormalities are inherited or arise de novo gametogenesis, fertilization and early cleavage stagesT4).

during

During gametogenesis, abnormal meiosis can produce gametes with an unbalanced chromosomal composition. These most often include duplication or deletion of segments of a chromosome, the duplication or deletion of whole chromosomes or the failure of the reductional Such gametes are capable of participating in fertilization division. and result itiembryos Mith ohromosome abnormalities (5). Chromosome abnormalities which arise at the time of fertilization These are usually those which include whole sets of chromosomes. result in either polyploid (typically 3n) or fertilization-derived These result from polyspermic chimeric embryos (2n/ln, 2n/3n). fertilization, ,the failure to extrude one or both polar bodies, or fertilization of both the ovum and the polar body. Abnormalities which might arise during the first few cleavage non-disjunction leading to the loss of one or divisions include: more chromosomes from a cell with the possible gain of these chromosomes by its sister cell; the failure of mitosis resulting in polyploid nuclei; -orthe failure of cytokinesis resulting in polynuclear cells. The cytogenetic studies carried out on the early embryos of domestic animals can be principally divided into two groups: those in which one or both parents are heterozygous for a particular chromosome abnormality, and those in which the parents were chromosomally normal. Aside from the presence or absence of parental chromosomal abnormalities, other factors have been taken into account such as the type of ovulation (superovulation vs natural) age of donors (cows vs heifers) These studies are or site of fertilization (in -- vitro vs in viva). The abnormalities have been distrisummarized in Tables I and II. buted according to their most likely origin.

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TABLE I.

Cytogenetic analysis of embryos produced by which one parent was heterozygous for a balanced abnormality1

Species

Stage (Day)

Embryos Analysed

Meiosis

l-3' 7* 13 l-56

16 22 52 13 75 113 69 27 27

6.2 4.5 3.9 15.4 0 10.6 28.8 40.7 22.2

Cattle

Sheep Pigs

10-18 10-88 1-16 g-10 21

Abnormalities (%I Fertilization Cleavage

12.4 0 0 0 0 0 0 0 0

matings in chromosome

Reference

6 6 7 8 9 10 11 12 13

1 The data summarized here have been regrouped according to possible origin of chromosome abnormality When more than one origin was suggested, the one felt to be the most likely has been chosen. * PMSG stimulated

TABLE II.

Cytogenetic analysis of embryos produced by or natural ovulation'

superovulation

Abnormalities (%) Species Ovula- Stage Embryos Reference (Day) Analysed Meiosis Fertilization Cleavage tion

Sheep

Cattle

Pig

Nat PMSG PMSG FSH FSH PMSG FSH Nat PMSG FSH PMSG Nat Nat

2-3 2-3 20 2-3 7* 7* 5-7 12-16 12-18 11" IO 10 25

80 ::, 13 61 17

24 12 159 15 169 88 76

5.0 4.4 0

0 1.6 0 0 0 0 6.6 0 1.1 l-3

1.3 16.5 0 30.7

0 11.8 16.6 0 1.9 20.0 0 5.7 0

1.3 1.1 1.2 0 11.4 23.5 0

0 0 0 0 0 0

14 15 15 16 17 17

18 19 20 21 22 23 24

1 The data summarized here have been regrouped according to When more than one origin possible origin of chromosome abnormality. was suggested the one felt to be the most likely has been chosen. * Fertilized in vitro Type C (poor) embryos l* PMSG: Pregnant mares serum gonadotropin Nat: Natural ovulation FSH: Follicle stimulating hormone

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THERIOGENOLOGY The frequency of chromosome anomalies during meiosis is considerably higher in embryos produced by heterozygotes for chromosome abnormalities than in embryos from normal animals (0 to 40% vs 0 to 5%, see Tables I and II). This disruption of meiosis is to be anticipated in the carriers of abnormalities, and emphasises the need for selection of sires and embryo donors free of chromosome abnormalities. The investigation of embryos from unselected animals has shown that the incidence of fertilization abnormalities ranged from 0 to 31% and was influenced by the type of ovulation, the site of fertilization, the species under investigation and age of donor. Williams and Long (15) have clearly shown that embryos from superovulated sheep (1000 iu PMSG) had a higher frequency of abnormalities occurring at the time of fertilization than did those produced by natural ovulation In pigs, superovulation (800 iu PMSC) was not asso(17% vs 1%). ciated with an increase in chromosome anomalies detected at day 10 In contrast, 12% (2/16) fertilization-associated anomalies (22). (5n, 2n/ln) were observed in day-l to -3 cattle embryos produced by PMSG (2000 iu) stimulation (6). Abnormalities associated with fertilization (2n/ln chimeras and 3n) were observed in 31% (4/13) of cattle embryos produced by superovulation (FSH 33 mg) and examined at the lto a-cell stage. Furthermore, three unfertilized oocytes recovered on day-3 had only recently reached MI1 stage and probably reflected either an immaturity of the oocyte at ovulation or a late ovulation It must be emphasized that these observations in cattle are on (16). a very small number of zygotes and that suitable non-superovulated control studies do not exist. Susceptibility of superovulated oocytes to polyspermic fertilization and/or mitotic activity of the polar body is strongly suggested by studies in sheep (15) mice (25) and cattle (16). Perhaps up to one third of zygotes produced in this manner have fertilization-associated cytogenetically detectable abnormalities. Amongst bovine oocytes produced by gonadotrophin stimulation (FSH 33 mg) but fertilized in vitro (26, 21) and examined before cleavage, 27% (4/15) exhibited abnormalities arising during fertilization (3/15 polyspermic) or meiosis (l/l5 aneuploid). Polyspermic fertilization and formation of multiple pronuclei may occur in up to 30% of human ova fertilized -in vitro. These abnormalities have been attributed to the type of stimulation of oocyte donors, the length of time in culture before fertilization, or the type of infertility being treated (27, 28, 29). Karyotyping of three human embryos resulting from fertilization -in vitro revealed two errors of meiosis (30) and, in a second study of multipronuclear ova, errors of meiosis in five of the 29 pronuclei (29). In mice, aneuploidy was higher in ova fertilized in vitro than -in vivo (1.9% and 0.7%, respectively; 25). -Abnormalities arising during early cleavage are not well documented. Certain mosaics in live-born domestic animals have been reported in conjunction with congenital malformation possibly arising from errors of cleavage (31, 32). The study of degenerating Day-7 embryos produced by superovulation (FSH or PMSG) in cattle has revealed the presence of mixoploid embryos (embryos with diploid and polyploid cells) prior to hatching (17). While this is a normal phenomenon in the bovine trophoblast from day 12 onwards , it has not yet been observed in over 100 morphologically normal (Type A) blastocysts examined

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THERIOGENOLOGY It also appears that the incidence of chromosome on Day 7 (36). abnormalities in degenerating day-7 embryos was higher when donors were stimulated with PmG (2000 iu) than with FSH (33 mg), 35% (6117) vs 13% (6/81). Gayerie de Abrea et al. (33) have reported that embryos produced by cows had marginally more abnormal karyotypes than did embryos from heifers (9% vs 6%). However, the results were not presented in sufficient detail to distinguish between abnormalities incurred during fertilization and those produced during early cleavage.

Effect of Chromosome Anomalies -Unbalanced chromosome anomalies in humans are most often identified as a cause of spontaneous abortions or congenital malformations In domestic animals they are recognized as a source of reduced (1). Very few live-born litter size and return to service (11, 12, 13). animals with chromosome anomalies have been recorded and those obserThe ved usually manifest debilitating congenital malformation (34). study of embryos of different ages produced under various conditions has shown that certain types of anomalies are associated with certain stages of development with a general trend towards progressive elimination as gestation progresses. Abnormalities incurred during meiosis are mostly eliminated during the first month (e.g. pigs, 11, 13, 24) with some even being eliminated as early as the 2-cell stage (e.g. The chimeras (2n/ln) observed both in 2-cell Chinese hamster, 35). (16) and day-5 to -7 bovine embryos (18) have not been observed in newborns. Since these embryos are chimeric in that they arise from at least three gametes, similar blood chimerism would be expected. However, this has not been observed in embryo transfer calves which have been blood-typed (G.J. Kraay, Agriculture Canada, personal communication) suggesting that these abnormal embryos are either eliminated or that the haploid cells have no importance in the further In sheep, such chimeras appear to be development of the embryo. Pure triploid (3n) cattle eliminated during the first 20 days (15). embryos have only been observed at the 2-cell stage and at days 12-15 (19, 36). The embryos hatches (37). live- or

diploid/polyploid mosaics observed in the degenerating day-7 are probably eliminated since this type of embryo rarely in vitro and produces a low pregnancy rate when transferred Furthermore, this type of mosaic has not been reported in stillborn domestic animals.

Single Gene Loci Single genes that affect early development in domestic animals have not been identified even though many are known that lead to congenital malformation and fetal death (for review see 38). However, in the mouse, several gene loci that affect early embryonic development have been reported (Table III). These mutations appear to have specific actions which can be associated with defined stages of

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THERIOGENOLOGY Table III.

Mutations affecting early development in from Magnuson and Epstein 1981)

Designation

mice

(adapted

Expression

Stage of development

T12, ~~32

Day 3

Failure of blastulation (homozygous lethal)

TW73

Day 5-6

Abnormal trophoblast formation

To

Day 5-7

Defective extra embryonic ectoderm

Tw5

Day 7

Death of embryonic ectoderm

w9

Day

Tw'

Day 9

Defect in differentiation of neural tube and brain

PED (fast/slow)

1st cleavage

Influences rate of cleavage (fast dominant allele)

Yellow (Ay / Ay)

Day 5+

Defect of trophectoderm (homozygous lethal)

Oligosyndactylism (OS/OS)

Day 4

Mitotic arrest (homozygous lethal)

Albino C25H

2-6 cell

Failure of cleavage (homozygous lethal)

Tailshort (Ts)

Morula

Abnormal blastulation (homozygous lethal)

7-a

Failure of mesoderm and notochord formation

development. The identification of these mutations has been faciliSuch tated by the study of inbred and congenic strains of mice. levels of homozygosity may never occur in domestic animal models and therefore identification of single lethal gene mutations will be difficult. However, if one extrapolates from the observations of such mutations in mice, certain genes, gene products or stages of development might be of particular relevance to the -in utero development of domestic animals. One example is the gene known as PED (preimplantation embryo development), recognized as having two functional alleles, one fast (dominant) and one slow, and which affects the rate of early cleavage. This gene is associated with the major histocompatibility complex in mice and is being pursued as one which might have a counterpart in domestic pigs (2).

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A great deal of fundamental research is needed before the activation of the embryonic genome is understood or the impact of single One approach to mutant genes on embryo survival can be assessed. understanding these problems is to study the transgenic strains of mice in which known gene sequences have been incorporated by way of Since these viruses may be incorporated into several retroviruses. different regions of the host genome, their presence may serve to activate or disrupt nearby genes (39). Such disturbance can also lead to a mutant phenotype after spontaneous viral integration, as is the case with the lethal yellow (Ay) coat colour in mice (40); or can be induced by experimental incorporation, as is the case' with the recessive lethal MOV-13 locus which blocks the expression of the alpha 1 (I) collagen gene and eventually results in embryonic death (41).

Acquired Intrinsic Factors As discussed elsewhere in this symposium, the handling of embryos during embryo transfer increases embryonic loss for largely unknown reasons. Such losses may be brought about immediately or through more gradual processes. For example, a frozen embryo may be recognized as degenerate upon thawing, or may look viable, prolong the cycle of the recipient, but be lost subsequently. It seems reasonable to suspect that deferred loss might result permanent alterations from (mutations) in the DNA of the embryo brought about by exposure to mutagens in vitro before transfer. Potential mutagens include factors encountered during culture with heterologdus serum, exposure to UV radiation, DNA binding dyes, radioactivity, cryoprotectants and Information on the potential mutagenicity ethylene oxide residues. and/or toxicity of these treatments can be obtained from several sources. One useful test is to determine the level of treatment which inhibits development of at least 50% of embryos (median lethal dose, LD50). Another is the study of sister chromatid exchange (SCE), perhaps one of the most sensitive cytological measures of chromosome damage available today. When cytogenetic techniques for demonstrating SCEs are used, chromosome breaks and the interchange of DNA molecules at homologous loci on sister chromatids of the same chromosome are Such alterations would not be detected by other staining detected. in utero or in vitro, the In cultured cells and embryos -techniques. frequency of SCEs has been shown to increase after exposure to various toxic substances and environmental mutagens (for examples see 42). Baseline SCE frequencies -in vivo are typically lower than in vitro (43) emphasizing the effect of exposure to exogenous factors which would not occur under normal circumstances. The induction of SCEs has been shown to persist for at least three cell cycles after DNA damage The severity of the effect of DNA damage depends on the (44). function of the DNA sequences involved and the efficiency of the appropriate DNA repair system in removing the lesion (45, 46). The following summary of selected literature concerns the toxic or mutagenic effect of various extrinsic factors to which embryos are exposed during production or transfer. This summary is not intended to be complete but merely to draw attention to potential and, in some cases, avoidable sources of embryo loss.

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THERIOGENOLOGY i) PMSC: Aside from the increase in polyspermic fertilization observed in sheep (15) and mice (25) and an increase in the incidence of mixoploidy in cattle embryos (17) attributable to PMSC stimulation, a direct toxic effect on mouse embryo development has been observed. A significant increase in fragmented embryos and SCE rate was observed in groups of mouse embryos produced after superovulation with 3.0 iu and 10.0 iu PMSG compared with control and 1.5 iu groups (47). ii) Media and serum: The types of culture media used have been shown to have a small but significant effect on baseline SCE rates in cultured lymphocytes (48). Growth of embryos cultured with sera from different donors varied significantly more than did that of embryos cultured with serum from a single donor (49). Media containing sera from different sources led to different rates of SCE but heatinactivation reduced this effect. Apparently not all batches of serum contain SC&inducing factors; this is thought to result from the variability of processing procedures (48, 49, 50). iii) UV and DNA-binding Dyes: W-light is a known potent inducer of SCEs in mammalian cells. The lesions induced by exposure to UV are not necessarily repaired in a single cell cycle but may persist for several generations (44). Near UV light (300-360 nm wavelength) can induce SCEs if appropriate photosensitizing agents such as pH indicators or antibiotics are present in the culture medium (51). Various DNA binding dyes (e.g. acridine orange and Hoechst 33258) have been shown to increase SCE rates (52). However, the potential toxicity of the fluorescent vital dye, fluoroscein diacetate has been recently tested on bovine and hamster embryos and similar pregnancy rates were obtained after transfer of treated and untreated embryos in both species (53). iv) Radioactivity: Several kinds of ionizing radiation are known to be effective in damaging DNA and promoting chemical modification of DNA or chromosomal rearrangement (44, 541, although high energy irradiation (e.g. X-rays) is not an effective inducer of SCE (44). Culture of embryos with tritiated amino acids (beta-irradiation) have revealed LD5Os which vary from 0.03 to 0.13 uCi/mL depending on the amino acid used (55). The LD50 of tritiated-thymidine was approximately 0.08 uCi/mL (55) but quantities as low as 0.1 nCi/mL were shown to decrease the number of embryos that could form inner cell masses In general, preimplantation mammalian embryos are apparently (56). extremely sensitive to beta radiation from tritiated compounds of synthetic importance (55). Ethylene oxide: The toxicity of ethylene oxide residues (a known v) mutagen, 57) in plastic culture vessels used for embryo culture has been tested on mouse embryos. It was shown that exposure of culture dishes to ethylene oxide for 24 h, followed by aeration of the containers for 36 h or less, led to the formation of residues which adversely affected embryo development and quality in vitro (58). vi) Viral genes: Viral transformation of human fibroblasts is associated with increased SCEs (59) while integration of retrovirus sequences has not been shown to be associated with similar increases (60). 168

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THERIOGENOLOGY Summary and Conclusions More than half of the embryos from superovulatedcattle undergo abnormal developmentby day 8 (61). Even when only morphologically normal embryos are transferred, substantialadditional losses are incurred subsequently. Chromosomeabnormalitiesand mutant gene(s) are among the intrinsic factors which no doubt account for a significant portion of these losses. Chromosomal anomalies incurred during meiosis are typicallymore frequentwhen either the sire or the dam are heterozygousfor a balanced chromosomeabnormality. Selection of embryo donors and semen can reduce this risk and, as Moor et al. (62) suggested,II...new work on the nature of the administered hormones and their mode of application" may lead to improved viability of the superovulated embryo. This might be brought about by reducing the susceptibilityof the superovulated oocyte to polyspermic fertilization, and/or the mitotic activity of the polar body and cleavage failure. The impact of even minor changes in DNA is emphasized by the finding that a single mutant gene can lead to timed embryonic death. The fact that specificgene function can be blocked by the incorporation of segments of viral genome (41) lend credance to the suggestion that exposure to mutagens can have the same effect. An awareness of the type of mutagen likely to be encounteredand the degree to which they affect embryo survival might also contributeto a reduction in embryonic loss due to the interplaybetween extrinsic and intrinsic factors.

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