CONGENITAL MALFORMATIONS CAUSED BY DAMAGE TO SPERM
Studies by Nomura at Osaka Univerity summarised in Figures 2.5 and 2.6 showed that treatment of mouse sperm with radiation or chemicals can cause congenital malformations and cancer including leukaemia in offspring and some of the mutations are transmitted to the second generation (Nomura, 1975, 1978, 1982). Mary Lyon and her colleagues confirmed that irradiation of male or female mice before mating can lead to congenital malformations in offspring (Kirk & Lyon, 1982, 1984). The same team showed that, while some mutations can be passed on to the second generation by male offspring of irradiated parents, the major proportion of the malformations are eliminated in the first generation (Lyon & Renshaw, 1986, 1988). Adams et al. (1981) and Trasler et al. (1985) described foetal malformations and behavioural abnormalities following exposur.e of male mice before mating to cyclophosphamide. Nagao (1987) reported dose-dependent defects in offspring from treatment of male mice with MNU (methylnitrosourea). Russell and Hunsicker (1983) reported that exposure of sperm to MNU can produce specific locus mutations. Generoso et al. (1984) reported that exposure of sperm to MNU can produce heritable translocations. Mutation in male germ cells and the resulting abnormalities of offspring was the subject of a 144 page special issue of Mutation Research entitled "Male-mediated F1 abnormalities" (April 1990, Vol. 229, No 2) There is no evidence that male germ cells are any better protected than female germ cells from the ravages of mutagens.
It was suggested that the capacity of a substance to produce congenital defects in offspring by exposure of male sperm in animals should be used as a test of mutagenicity (Knudsen et al., 1977). Not all the mutations produced in this way can be identified but those which can are generally found to be genetically dominant and autosomal. The consequences of such dominant mutations are diverse and difficult to identify. Testing has inevitably been generally restricted to the identification of dominant lethal mutations (Green et al., 1985, Brusick, 1980; Burger et al., 1989). Many hundreds of mutagens have been identified using the valuable dominant lethal test in which treated males are mated with untreated females and the resulting fertilised ova are examined for lethal defects. This test does not, however, identify many non-lethal dominant mutations such as those initiating viable malformations and tumour development. The first initiating mutation has been reported to be mainly of parental origin in cases of inherited retinoblastoma and sporadic osteosarcoma (Ejima et al., 1988; Schroeder er al., 1987; Togucida et al., 1989). A Medical Research Council team has suggested that leukaemia in children living near nuclear plants may be caused by their fathers' exposure to radiation resulting in germ cell mutations (Gardner et al., 1990).
Abnormal sperm can cause miscarriage. Figure 3.1 shows an increasing risk of miscarriage with increasing sperm abnormality assessed using a phase contrast microscope: 200 cells were examined from specimens of semen from 317 men who were either husbands of women who had had miscarriages or were under investigation for infertility at hospitals in Stockholm (Furuhjelm et at., 1962). Probably about half the miscarriages were attributable to the husband but the men in this study were in no sense representative. The same Swedish study showed that a low sperm concentration was also associated with an increased risk of miscarriage as illustrated in Figure 3.2.
Low sperm concentration has been shown in other studies to be highly correlated with low percentages of morphologically normal sperm (Jouannet et al., 1981). Volume of ejaculate is not reported to be associated with sperm quality so that sperm concentration may be more indicative than total sperm count. A slow-down in DNA synthesis and consequential reduction in the rate of cell replication could explain the copying errors associated with reduced rates of DNA synthesis. The increase in miscarriage rates is prima facie evidence that sperm abnormality may be associated with a raised mutation rate.
Research at the Ontario Cancer Institute showed that mutagens do indeed cause such sperm abnormalities visable under the microscope in animals (Wyrobek & Bruce, 1975). By 1982 tests had been done on 9 species of mammal including primates and 160 different chemicals (Wyrobek, 1982). The most sensitive visible indication of exposure to a mutagen is change in sperm shape. The head of the sperm is normally oval but may be too small, too large, round, double, narrow at the base, pear-shaped or the sperm may have an abnormal tail or two tails instead of one. In order to produce abnormalities of shape a mutagen must either damage the genes that determine shape or must affect the expression of these genes. Figure 3.3 shows the percentage of abnormal sperm produced by exposure to a mutagen at different times before ejaculation in rabbits (Fox et al., 1963). It is seen that sperm shape is much more easily made abnormal by a mutagen before meiosis I and II. The sperm immediately after meiosis I and II is a spherical cell and sperm structure only develops subsequently at the spermatid stage. All the information necessary for programming sperm morphology is therefore carried by genes until the beginning of the spermatid stage. It is a reasonable inference that abnormal sperm mor phology originating before meiosis I and II is a consequence of dominant gene mutations. This conclusion is supported by the long list of chemicals, known to be mutagens from other tests, that cause morphological sperm abnormality if fed to male animals. Morphological sperm abnormality has been shown not to be associated with chromosomal abnormality, further emphasizing that the morphological abnormality caused by mutagens must have its origin before the spermatid stage and before meiosis I and II as the result of dominant mutation of genes rather than chromosomal mutation (Martin & Rademaker, 1988).
In Figure 3.3 it is seen that the curve showing the percentage of abnormal sperm continues backwards in time until 70 days before ejaculation. In these particular experiments with rabbits exposure to the mutagen 70 days before ejaculation stopped spermatogenesis. Figure 3.4 shows that 70 days before ejaculation was the point of maximum susceptibility, if measured in numbers of sperm per ejaculate. Comparing Figures 3.3 and 3.4 it is seen that the number of sperm declined as the percentage of abnormal sperm increased. At times longer that 70 days susceptibility to the mutagen dose declined and recovery from temporary infertility approached completion. The period of highest susceptibility in rabbits is seen to be well before meiosis I and II when the highest rates of DNA synthesis and cell replication take place. During this period germ cell numbers increase about 100 times. Spermatogonial replication ends when the germ cells are renamed spermatocytes about 35 days before ejaculation in mice, 44 days in rabbits and 58 days average in men as shown in Figure 3.5 in which the time-table of spermatogenesis is shown diagram matically.
Spermatogenesis in all male mammals has a great power of recovery from temporary exposure to a mutagenic influence, but recovery takes time. In Figure 3.4 the 91 days for a rabbit and the 120 days for a man are indications of the minimum recovery times. There are many case histories showing times for recovery of sperm 0count following severe exposure to radiation or chemicals ranging up to 6 years, but there is generally no good information about the extent of the exposure. There may be no recovery of sperm count from very severe exposure (Whorton & Milby, 1980). Such severe exposure is, however, very uncommon. Temporary infertility resulting from interference with the replication of spermatogonia 2 or 3 months before attempts at conception are much commoner. The resistance of spermatogonial stem cells to mutagens continues to increase as the time interval to ejaculation increases beyond 120 days. This is apparent from case histories describing recovery after much longer intervals following exposure to radiation and chemicals. The existence of what have been called "reserve stem cells" or "spermatogonia Ao" that are isolated and rarely divide have been described in bulls, mice, monkeys and rats (Clermont & Bustos-Obregon, 1968; Clermont & Hermo, 1975). The existence of such resistant dormant male germ cells obviously had survival value like the resistant, dormant female germ cells more than a few months before ovulation.
THE TIME-TABLE OF SPERM SUSCEPTIBILITY
The longer the lapse of time between exposure to a mutagen and fertilization the greater is the opportunity for elimination or repair of a damaged germ cell. Figure 3.5 shows 58 + or - 13 days from the end of spermatogonial cell replication until ejaculation. As less than 1 sperm in l09 ever fertilize an ovum the selection of less damaged sperm can offset effects of exposure to mutagens 2 or 3 months beforehand. The elimination of germ cells at every stage is selective not random. Sperm with a very wide range of characteristics reducing viability fail to get through. Adler of the West German Institute of Genetics writes (Adler, 1983b): selective not random. Sperm with a very wide range of characteristics reducing viability fail to get through. Adler of the West German Institute of Genetics writes (Adler, 1983b):
"Chemically induced chromosomal changes in spermatogonia are eliminated by germinal selection. In contrast gene mutations are transmitted from stem-cell spermatogonia through meiosis to the offspring."
This is illustrated in Figure 3.5. The dominant lethal test depends mainly on chromosomal changes. Such testing provides no information about the effect of mutagens on particular genes, which requires very large numbers of animals. An important study at the US Oak Ridge National Laboratory needed 304,479 mice offspring to study the mutation of only 7 genes (Russell, 1977). Such studies are costly and can never be done on men or women.
Gene mutations are thought to produce many more cases of congenital disorder than chromosomal mutations, but the evidence about causes is largely inferred from animal experiments and even then indirectly. It is inferred from animal experiments that mutagens can cause gene mutations that are responsible for morphological abnormality of sperm. Because gene mutations may have an immense variety of consequences which are impossible to identify in routine testing there is a danger that the length of the period of heightened susceptibility may be underestimated. The chromosomal abnormalities produced by the drug mitomycin C in mice is illustrated in Figure 3.6. It is seen that no chromosomal abnormalities were recorded in the mice as resulting from a mutagen dose more than 27 days before mating (Ehling, 1971).
This limitation to 27 days is seen in Figure 3.7 to be the result of failure to survive of any sperm produced more than 31 days before mating. Figure 3.8 shows another susceptibility-time curve based on studies at the US Oakridge National Laboratory using a chemical (MMS) that affects primarily the spermatid stage (Brewen et al., 1975).
Such susceptibility time-curves are only available for a few drugs and chemicals, are unpredictable and expensive to produce. The practical conclusion is that when such information is not available any drug or chemical should be assumed to be able to produce chromosomal abnormalities in offspring from early in the spermatocyte stage but not longer, that is up to 58b1 13 days before ejaculation in men or not longer than 12 weeks.
Ebling and Neuhiiuser-Klaus (1988) of the West German Institute for Mam malian Genetics compared the data for cyclophosphamide for dominant lethal mutations and specific-locus gene mutations in experiments involving an examination of a total of 248,413 mouse pups. The susceptibility-time curves for dominant-lethal mutations were not very different from Figure 3.8 for MMS. Moreover susceptibility to gene mutations at 7 specific loci was highest during the 21 days before mating and after meiosis. When, however, the effect 968; of cyclophosphamide was enhanced with X-rays the period of heightened susceptibility increased to about 42 days before mating to include spermatocytes and differentiating spermatogonia, and there were a few mutations at even longer intervals. The period of enhanced susceptibility to gene mutations may be at least twice as long as for dominant lethal mutations and chromosomal abnormalities as suggested in Figure 3.5. and begins well before the first cell divisions of spermatogenesis.
The experiments of Nomura (1982) described in Chapter Two and illustrated in Figures 2.5 and 2.6 showed no significant increase in dominant lethal mutations following exposure of spermatogonia to either radiation or urethane. But such exposure resulted in an increase in malformations and tumours in surviving pups not caused, in Nomura's words, by "gross chromosomal changes" but by dominant gene mutations. Nomura pointed to greater susceptibility of the postmeiotic stages, but also said that the spermatocytes were uch most susceptible to radiation "22 to 42 days before mating" without however giving the data.
The time-table of human sperm development summarized in Figure 3.5 shows the length of the period of heightened susceptibility to gene mutations as 102 days. This is a conservative figure based on the studies of Heller and Clermont (1964) in the U.S.A. and Canada. They estimated the duration of nay sperm development at 90 days, to which a variable period of storage of the finished spermatozoa has to be added. An average period of 12 days has been ned estimated (Harper, 1982). The length of storage falls as sexual activity in- ro- creases and may be as short as 3 days (Mann & Lutwak-Maun, 1981). The viability of the spermatozoa within the epididymis probably lasts for at least 3 weeks. The storage period of the spermatozoa may therefore be said to be 12 days with a tolerance of + or - days. viability of the spermatozoa within the epididymis probably lasts for at least 3 weeks. The storage period of the spermatozoa may therefore be said to be 12 days with a tolerance of b19 days. viability of the spermatozoa within the epididymis probably lasts for at least 3 weeks. The storage period of the spermatozoa may therefore be said to be 12 days with a tolerance of + or - 9 days.
The number of male germ cells, and the amount of new DNA, reach a maximum at the beginning of the spermatocyte stage, 58 + or - 13 days before ejaculation when all replication stops. The tolerances shown in Figure 3.5 mainly reflect the variable storage time in the epididymis. The peak of susceptibility for gene mutations may be around 80 to 90 days before ejaculation. The decline in susceptibility to mutagens going backwards in time to 120 days before ejaculation is substantial. Four months is a conservative estimate of the time needed for male reproductive capacity to recover from a not very potent dose of mutagen. Because the susceptible periods for mutagens vary widely, and are generally unpredictable, longer rather than shorter periods have been chosen when in doubt so that Figure 3.5 should cover most causes of mutation. Russell, who was quoted in Chapter Two as emphasizing the importance of the great increase in susceptibility in the female during ovulatory maturation, has also emphasized the susceptibility of the male to chemical mutagens during spermatogenesis (Russell, 1986). Mary Lyon said when discussing chemical mutagens (Lyon, 1988):
"The major part of the data available concerns males, and the majority of the chemicals tested so far fall in the category of those that have little effect on the spermatogonial stem cells. Thus, for these it is only any dose received in a few weeks or months before conception that need be considered."
Spermatogonial stem cells may be damaged by radiation and by some chemicals but a high proportion of all mutations in men happen later, during the susceptible periods before conception.
HEIGHTENED SUSCEPTIBILITY IMMEDIATELY BEFORE CONCEPTIONThe effects of exposure to mutagens immediately before mating have been studied. The mutagenic effect of a drug, fosfestrol, on male mice, as indicated by the fertilized ova found to be lethally damaged, is shown in Figure 3.9 from Ehling (1979).
Batches of 40 male mice were mated at different time intervals between dosing and mating to healthy undosed females. Drugs given to male animals immediately before mating can cause congenital malformations, reduced birthweight, small litters, stillbirths and reduced neonatal survival. This effect is not necessarily a result of the reaction of the drug with the male germ cell, as it has been shown by Lutwak-Mann (1964) in research at Cambridge that drugs can be carried by sperm into the female tract to the point of fertilization where they can cause congenital malformations. Exposure of sperm to thalidomide has been shown to cause congenital malformations in this way (Lutwak-Mann, 1964; Lutwak-Mann et al., 1967).
The interest in these experiments is not in the drug but in the effects on offspring of drugging sperm. The effects of methadone on sperm have been studied at the University of Vermont (Soyka & Joffe, 1980). Methadone given to male rats during the 24 hours before mating caused an increase in the percentage of pups born dead from 13 to 54 per cent as shown in Figure 3.10.
The pups born alive following drugging of the male sperm also had reduced viability as shown in Figure 3.11 which also shows that morphine had a similar effect. It is seen in Figure 3.11 that nearly 100 per cent of control pups sur vived, but only 65 per cent of pups from males dosed with morphine, and 28 Chromosomat abnormaUties produced in mature sperm by per cent of pups from males dosed with methadone survived the first 20 days of life. Both stillbirths and neonatal deaths followed the drugging of spenn. These drugs must either have damaged the male genetic code shortly before fertilization or must have caused genetic damage around the time of fertilization or shortly afterwards. Both methadone and morphine have been shown in other studies to be mutagenic and Figures 3.10 and 3.11 may be regarded as further evidence of their mutagenicity (Badr & Rabouh, 1983; Badr et al., 1979). The common analgesic codeine, found in many non-prescription painkillers, is a derivative of morphine and morphine is one of the metabolic products of codeine which must therefore also be assumed to be mutagenic. Codeine has been reported in other experiments to cause congenital malformations (Zellers & Gautieri, 1977).
THE COMPARATIVE SUSCEPTIBILITY OF MEN AND WOMEN
It has been suggested by a number of writers that new mutations in surviving children may be more frequently of male than female origin, because the male germ cell undergoes many more cell divisions than the female germ cell and most new mutations are thought to be the result of copying errors during division (Vogel, 1984). The larger number of male germ cells facilitates more effective germinal selection which eliminates chromosomal abnormalities produced during the replication of the spermatogonia at the beginning of spermatogenesis. Germinal selection is not, however, so effective in eliminating dominant gene mutations. 1,443 (+ 1,114 not fully validated) dominant genetic disorders which are distinctive have so far been listed and the commoner disorders which reduce fertility have their population prevalence maintained by new mutations (McKusick, 1988). Dominant autosomal mutations are discussed again in Chapter Eight in the context of ageing.
There is a longer period in the man of 34 b1 12 days from mciosis I and II to fertilization than in a woman which may explain, for example, the 80 per cent maternal and 20 per cent patemal origin of Down's syndrome. However in a study of triploidy, a major cause of miscarriage, 72 per cent of cases were reported to be of paternal origin (Sperling, 1984). In Klinefelter's syndrome, which seriously affects male sexual development, there is an extra X chromosome which was reported to have its origin at meiosis I in the father in about one third of a series of cases (Sanger et al., 1977). A study from the University of Oregon (1980) of 42 other chromosomal rearrangements found that 22 were of maternal and 19 of paternal origin (Chamberlin & Magenis, 1980; Mattei et al., 1982); the chromosomal abnormalities originating in the father were mostly a consequence of chromosomal breakage and rejoining at the wrong place.
It is currently a cautious and wise assumption that men and women have germ cells equally vulnerable. This is the conclusion of several studies that have emphasized the difficulty of any comparison of the responsibilities of mothers and fathers for an unfavourable pregnancy outcome caused by damage to germ cells (Adler, 1982a, 1982b, 1982c). The male time-table of susceptibility differs from that of the female and is more complicated and varies from one mutagen to the next making comparisons difficult. Some one-generation genetic disease is more paternal and some more maternal in origin. The International Commission of the Mutagen Societies (1983) discussing short-term exposure of men and women concluded that:
It may be particularly important to know the risk to maturing germ cells (in men and women), since it may be possible to avoid the genetic harm done to these stages if the accidentally exposed individuals refrain from conception for 3 months".
Where possible exposure to X-rays or drugs should be avoided during the 3 or 4 months before conception. Risk is, however, a product of susceptibility, enhanced during this period of the maturing cell, and the exposure to mutagenic influences of all kinds. The literature assumes that everyone has a general mutation rate affecting both somatic and germ cells. The concept of a mutation rate is not without difficulties but is useful. The reproductive risk is then the product of this mutation rate and an enhancement factor depending upon time of exposure. The reproductive risk is obviously zero for someone beyond reproductive age, but greatly enhanced for men and women around the time of conception.
Chapters Two and Three have discussed this enhancement of susceptibility and have only discussed factors that increase mutation such as X-rays or drugs in order to illustrate this enhancement more particularly during the period immediately before conception. The rest of this book is primarily concerned with the ever increasing number of factors that are known both to increase and also to reduce human mutation rates.