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Non-disjunction in aging female mice Martin, Renée Halo 1975

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NON-DISJUNCTION IN AGING FEMALE MICE by RENEE HALO MARTIN B .Sc , University of Brit ish Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Field of Genetics We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1975 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thesis fo r f i nanc ia l gain sha l l not be allowed without my written permission. Department of M E_^ > \ C.A L. G - E . M & T I C S The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 pate S E P T . 2 ^ m s i i ABSTRACT Classical studies have shown that reproductive performance declines with maternal age in humans and other mammals. There is an increase of trisomic offspring with maternal age in humans and an increase of trisomic embryos and fetuses with maternal age in mice. It has been sug-gested that this increase in non-disjunction is due to the decrease in chiasma frequency arid increase in univalents observed in the oocytes of old mice. This study was under-taken to determine the effects of maternal age on non-dis-junction in the oocytes of CBA mice. Oocytes from CBA mice varying in age from two to eleven months were cultured to the metaphase II stage of meiosis and the chromosomes were analysed. The oocytes from three maternal age groups were compared with respect to the mean number of oocytes obtained per mouse, the f re-quency of maturation to metaphase II, and the frequency of numerical chromosome abnormalities. Both the mean number of oocytes obtained per mouse and the frequency of matura-tion decreased markedly with maternal age. The frequency of chromosome abnormalities in the oocytes increased with i i i maternal age from the young to the middle aged mice but dropped off in the oldest maternal age group. No hyper-ploid (n+1) oocytes were observed in the young or old group of mice but 4.9% hyperploidy occurred in the middle age group. It is suggested that the lack of hyperploid oocytes in the old CBA females might be due to a threshold effect in which oocytes which are damaged by the number or type of univalents become atret ic and do not progress to metaphase II. The frequency of diploid (2n) oocytes was 1.7% and was not maternal age dependent. An overall theory of maternal age related non-disjunction is proposed in which the various environmental and genetic factors known to affect non-disjunction are linked to an underlying mechanism of univalent production in oocytes. iv TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF APPENDICES < ix ACKNOWLEDGMENTS x I. INTRODUCTION 1 Reproductive performance in mammals 1 Theories of maternal age related non-disjunction 5 A. Theories of non-disjunction due to aging of maternal systems 6 1. Intrafol1icular aging of ova 6 2. Tubal aging of ova 9 3. pH disturbances 11 4. Autoimmune disease 12 B. Theories of non-disjunction due to aging of the ovum during the dictyo-tene stage of meiosis 13 1. Sate l l i te associations of chromo-somes 13 2. X irradiation 17 V Page 3. Production of univalents in oocytes 18 II. MATERIALS'AND METHODS 22 Source and maintenance of mice 22 Procedure for obtaining oocytes 22 Procedure for culturing oocytes 23 Procedure for oocyte fixation and chromosome preparation 24 Procedure for analysing oocytes 26 III. RESULTS 28 Collection and in vitro maturation of oocytes 28 Chromosomal analysis of oocytes 29 IV. DISCUSSION 47 In vitro oocyte maturation 47 Normality of oocyte development in vitro 48 Comparison of results to other studies 49 1. Diploid.oocytes 49 2. Aneuploid oocytes 50 Characteristics of CBA mice 53 Possible explanations for the lack of hyperploidy in the oocytes from old CBA mice 54 vi Page Relation of chromosomal abnormalities to the decline in f e r t i l i t y in old female mammals 56 Discussion of results in relation to the theories of non-disjunction 57 A unified theory of maternal age related non-disjunction 58 Models for the decrease in the frequency of chiasmata with maternal age 64 V. CONCLUSIONS 67 REFERENCES 70 APPENDIX 82 v i i LIST OF TABLES Effect of maternal age on the mean number of oocytes per mouse and on in vitro oocyte maturation Chromosomal analysis of 473 metaphase II oocytes from mice of different ages vi i i LIST OF FIGURES Figure Page 1. Second meiotic metaphase showing a normal complement of 20 chromosomes 38 2. Second meiotic metaphase with 19 chromo-somes 39 3. Second meiotic metaphase with 21 chromo-somes 40 4. Second meiotic metaphase with 21 chromo-somes. The axes of the chromatids are drawn for c l a r i f i ca t ion . 41 5. Second meiotic metaphase with 40 chromo-somes 42 6. Second meiotic metaphase with 40 chromo-somes 43 7. Second meiotic metaphase with i ts polar body 44 8. Second meiotic metaphase which could not be analysed 45 9. Second meiotic metaphase which could not be analysed 46 ix LIST OF APPENDICES Appendix Page I. Constituents of Krebs-Ringer medium for oocyte culture 82 X ACKNOWLEDGMENTS I would l ike to express my sincere thanks to the members of my thesis committee at the University of Br it ish Columbia for their support, guidance, and friendship through-out this project: Dr. J . R. M i l ler , Department of Medical Genetics (Chairman); Dr. F. J . D i l l , Department of Medical Genetics (thesis supervisor); Dr. B. J . Poland, Department of Obstetrics and Gynecology; Dr. C. W. Roberts, Department, of Poultry Science; Dr. D. G. Holm, Department of Zoology; Dr. C. 0. Person, Department of Botany. I am especially grateful to Dr. F. J . Di l l for his generous and constant assistance with the day-to-day crises that kept cropping up. I am very grateful to Dr. R. P. Donahue, Depart-ment of Obstetrics and Gynecology, University of Washington, Seattle, for his knowledgeable suggestions at the outset of this project and for his generous assistance in establishing the techniques for oocyte culture. The help of Mrs. D. Smith who assisted with many technical problems is also greatly appreciated. I would l ike to thank Dr. B. K. Trimble, Depart-ment of Medical Genetics, U.B.C., for his assistance with xi the s tat i s t ica l analysis and for making computer time ava i l -able. The help of Mr. T. Balabanov for assistance with the computer f a c i l i t y is gratefully acknowledged. I acknowledge the support of Medical Research Coun-c i l Grant No. MT-1062 to Dr. J . R. Mi l ler and the assistance of a Medical Research Council Studentship from 1971-1975. Final ly, I would l ike to express my warmest perso-nal regards and sincere thanks to Dr. Mi l ler and to a l l the graduate students, members of staff and faculty in the Department of Medical Genetics for providing a warm, and s t i -mulating atmosphere during the course of this study. 1 I. INTRODUCTION Reproductive performance in mammals Advanced maternal age in mammals is associated with reduced f e r t i l i t y . In humans, the frequency of o f f -spring with chromosome abnormalities, particularly trisom-ies, increases with maternal age (Penrose 1933; Court Brown et a l . , 1969; Polani, 1969). Penrose (1933) f i r s t d i s -covered that the mean maternal age for mothers of children with Down's syndrome (trisomy 21) was 35 years compared to a mean maternal age of 28 years for the general population. Collman and Stol ler (1962) noted that the incidence of Down's syndrome increased dramatically with maternal age: from .043% in mothers between 15 and 19 years to 2.2% in mothers 45 years and older. Lenz, et a l . (1966) and Magenis, et a l . (1968) have shown that the distribution of maternal age in trisomy D and trisomy E shows a s imi lar ity to that for Down's syndrome. There is also evidence for an i n -creased mean maternal age for spontaneous abortions where the abortus has been shown to be an autosomal trisomic, i n -cluding types of trisomy not yet found in post-natal l i f e (Kerr and Rashad, 1966; Carr, 1967a). To date, studies in other mammals have not shown an increase in the frequency of chromosomally abnormal offspring with maternal age. However, most mammals spend a consider-able portion of their lifespan in a post-reproductive condi-tion and a marked reduction in l i t t e r size with increasing maternal age has been documented in several laboratory ani-mals (Talbert, 1968). Biggers et a l . (1962) and Harman and Talbert (1967) have shown that the reduction in l i t t e r size in old female mice is not accompanied by a lower ovulation rate. Ingram, Mandl, and Zuckerman (1958) have shown that l i t t e r size de-clines with maternal age in rats but the number of corpora lutea remains constant. Perry (1954) found no reduction in the number of corpora lutea in old pigs that were discarded for low f e r t i l i t y . Similarly, several investigators (Par-kening and Soderwall, 1973; Connors et a l . , 1972; Thorney-croft and Soderwall, 1969) have shown that the ovulation rate is the same in young and old hamsters, although l i t t e r size declines with age. But Blaha (1964) found a decrease in the number of corpora lutea in old hamsters. Since the vast majority of studies indicate that the decrease in l i t t e r size is not accompanied by a decrease in the number of oocytes ovulated, many embryos of older female mammals must be dying before b i r th. Conners et a l . (1972) have shown a highly s i g n i f i -cant increase in preimplantation and postimplantation death of embryos in old female hamsters. Many of the surviving embryos were retarded in development in the old females. Thorneycroft and Soderwall (1969) established that old female hamsters have a seven-fold increase in preimplantation death and a two-fold increase in resorption of established implan-tation sites by the eighth day of pregnancy. Embryonic mor-ta l i t y is also higher in old mice: Gosden (1974a) determined that 91% of oocytes from young mice survived to the blasto-cyst stage whereas only 63% of oocytes from old mice were alive at this stage. Gropp (1973) produced aneuploid embryos by using parental translocation strains and found that no aneuploid fetuses survived to term: monosomic embryos died in preimplantation stages and trisomies survived to become fetuses but died before b ir th. While some of the prenatal death that occurs in old females may be due to chromosome abnormalities, the decreased survival of embryos and fetuses in old females may also be due to uterine factors. Gosden (1974a) and Talbert and Krohn (1966) used the technique of ovum transplantation to determine whether pre-natal death in old female mice is due to uterine factors or defects in the ova. Blastocysts, recovered from young and old mice, were transferred into the uteri of young mice which had been made pseudopregnant by mating with a vasectomized male. Blastocysts from young and old donors survived equally well in the young recipients. When blastocysts from young and old donors were transferred to old recipients, survival was reduced. Therefore, both studies implicated uterine fac-tors as an important cause of the decline in reproduction. However, the number of blastocysts recovered from old mice was s ignif icant ly lower and the embryonic loss before the blastocyst stage might be due to chromosomal errors. Blaha (1964) carried out a similar study in hamsters, but in con-trast to the results obtained with the mice, 49% of blastocysts recovered from young donors developed into normal term fetuses in young hosts but only 4.5% of blastocysts from old donors developed successfully. This indicates that the blastocysts from old hamsters may have been in t r ins ica l l y defective. There is no definite explanation for the apparent great d i f -ference in the v i ab i l i t y of embryos recovered from old -5-hamsters and old mice. A species variation is possible, but there were also technical differences in timing and handling of the ova which may have influenced the results. Therefore the relat ive contributions of ovarian and uterine factors toward pre-natal loss in old females is s t i l l unclear. Two recent studies of chromosomal abnormalities in embryos (Gosden, 1973) and fetuses (Yamamoto et a l . , 1973a) from female mice of various ages demonstrate a s ignif icant increase of aneuploidy with maternal age in mice as well as in humans, although in mice autosomal trisomic embryos do not survive to term (Goodlin, 1965). Therefore, mice can be used as an experimental model to elucidate the cause of maternal age related non-disjunction leading to aneuploid embryos. Theories of maternal age related non-disjunction Several theories have been proposed to explain the effect that maternal age could have on non-disjunction. These theories can be divided into those that depend on the aging of various maternal systems and those that depend on the aging of the ovum due to the prolonged dictyotene stage of meiosis in mammals. Theories of non-disjunction due to aging of mater-nal systems are: in t ra fo l l i cu la r and tubal aging of eggs, pH disturbances in the female reproductive tract, and auto-immune disease. Theories of non-disjunction due to aging of the ovum during the dictyotene stage are: sa te l l i te association of chromosomes, X i rradiat ion, and production of univalents in oocytes. A) Theories of non-disjunction due to aging of maternal systems Intrafo l l icular aging of ova In 1922, Witschi f i r s t suggested that delayed ovulation could lead to in t ra fo l l i cu la r aging of the ovum and subsequent abnormalities in the embryo. In the A f r i -can clawed toad, Xenopus, i f the sexes are separated, the female retains her eggs and wil l ovulate only after an in -jection of gonadotropic hormones. In a series of experi-ments, Witschi (1969) determined that Xenopus eggs aged in this manner had a greatly increased incidence of mor-ta l i ty and embryonic abnormality. Witschi hypothesized that delayed ovulation in humans could lead to chromosomal abnormalities as well as malformations. Delayed ovulation could be caused by hormonal i rregular i t ies which are most often seen at the extremes of maternal a g e — i n very young women whose cycles are just getting established and in -7-older women approaching the menopause. There have been some studies in humans which lend support to this theory. In 1955, Hendricks deter-mined that, according to the 1953 Ohio records, women less that 15 years old or greater than 35 years old had a higher incidence of offspring with congenital malfor-. mations (2%) than women between the ages of 15 and 35 (1%). Carr (1971a) determined that there was an increase of chromosomal abnormalities in spontaneous abortions from young and old women. Women younger than 17 years had 40% heteroploid abortions; women older than 40 had 33% hetero-ploid abortions. The general frequency for heteroploid abortions in women of a l l ages was only 22%. However, these were both retrospective studies and the data was not analysed s ta t i s t i ca l l y . Furthermore, the increase of ab-normalities in the young and old females was not necessar-i l y due to hormone i rregular i t ies or delayed ovulation; they could have been caused by other factors. Hertig (1967) related the frequency of abnormal human embryos to the time of conception. He determined that women who ovulate regu-lar ly have a 92% chance of producing normal offspring i f conception occurs on day 14 of the menstrual cycle. If conception occurs on day 15 or later, the poss ib i l i ty of a normal conceptus drops to 42%. Therefore he fe l t that a delay in ovulation could cause abnormalities. This study was also retrospective and depended on estimates of the timing of ovulation which is d i f f i c u l t to determine exactly. Only one experimental mammal, the rat, has been used to study this hypothesis. The reproductive cycle in female rats is normally 4 days; however, old rats have a 6 day cycle (Fugo and Butcher, 1970). Therefore ovulation may be delayed in these old rats. In a series of experi-ments Butcher and Fugo (1966, 1967, 1969a) used nembutal to cause a two-day delay in ovulation in young rats. The rats treated with nembutal had a decreased fe r t i l i za t i on and implantation rate, an increased frequency of abnormal embryos, and a three-fold increase in chromosomally abnor-mal embryos. The majority of the chromosomally abnormal embryos were mosaics but there was also a small increase in the number of aneuploids. However, one cannot be certain whether the increase was due to the delay in ovulation or some other effect of the nembutal. No studies of chromo-some abnormalities in untreated old rats have been reported to date. Tubal aging of ova German (1968) proposed that the increase of t r i -somy with maternal age could be explained by tubal aging of the egg due to delayed f e r t i l i z a t i on . He reasoned that older women had less frequent intercourse and this caused a delay in f e r t i l i z a t i on . The human studies by Hendricks (1955) and Carr (1971a) demonstrating an increase of abnor-mal offspring and embryos in young and old mothers could be interpreted as supporting a delayed fe r t i l i z a t i on hypothesis rather than delayed ovulation. Very young women, as well as older women, could have infrequent intercourse. Hertig's (1967) results could also be explained by delayed f e r t i l i z a -t ion. He found that embryonic abnormality increased i f con-ception occurred on day 15 or later in the cycle and this could be due to a delay in the sperm reaching the egg rather than to delayed ovulation. However, a l l three studies were retrospective and the increase of abnormalities could s t i l l be caused by something other than delayed ovulation or fer -t i l i z a t i on . Several people have c r i t i c i zed German's hypothesis on s tat i s t ica l grounds. Matsunaga and Maruyama (1969) believe -lO-th at the age dependency for the frequency of -intercourse is not suff ic ient to account for the great increase in Down's syndrome with maternal age. There had been studies on the effect of delayed fe r t i l i za t i on in many experimental animals-—guinea pigs (Blandau and Young, 1939); rats (Braden, 1959); rabbits (Shaver and Carr, 1969); mice (Vickers, 1969); hamsters (Yamamoto and Ingalls, 1972); pigs (Hunter, 1967). In a l l these studies, there was an increase in embryonic abnormal-i t ies and death, but the commonest effect of the delayed fe r t i l i z a t i on was t r ip lo idy. After delaying f e r t i l i z a t i on for seven hours in mice, Vickers (1969) got a s l ight increase in trisomy and a nine-fold increase in t r ip lo idy. Shaver and Carr (1969) found that tr ip loidy increased from 1.4% to 13% after delaying fe r t i l i za t i on for 6-10 hours in rabbits. This dramatic increase in the frequency of t r ip lo id embryos may be due to a defect in the spindle fibre struc-ture in aging eggs. Szollosi (1971) has determined that the spindle in aging eggs often rotates and migrates to the centre of the ovum. This would inhibit the second meiotic division and a diploid egg would result. The t r ip lo id embryo could also be caused by fai lure of the aging oocyte to prevent more than one sperm entering the egg. Therefore, tubal aging of the egg due to delayed fer t i l i za t ion does cause embryonic abnormalities, particu-lar ly t r ip lo idy, but i t does not seem to be a cause of aneuploidy. pH disturbances Ingalls and Shimada (1974) have recently proposed that aneuploid offspring may result from pH disturbances in the fallopian tubes of older women. They have determined that a pH of 6.7 to 6.9 causes a broad spectrum of chromo-somal anomalies in cultured lymphocytes from adult blood. However, no studies have been published on the pH levels in fallopian tubes or on the effect of pH disturbances on gametes. If pH changes in the fallopian tube did cause aneuploidy, non-disjunction would occur at the second meio-t i c division since the oocyte is ovulated at metaphase II (Zuckerman, 1962). But non-disjunction generally occurs at the f i r s t meiotic division. Using fluorescent markers, Sas-aki and Hara (1973) demonstrated that seven out of eight cases of trisomy 21 occurred at the f i r s t meiotic division -12-of the oocyte. Therefore i t seems unlikely that pH distur-bances are a s ignif icant cause of non-disjunction. Autoimmune disease In 1963, Engel and Forbes f i r s t determined that chromosome non-disjunction occurs more frequently in fami-l ies with a genetic predisposition to autoimmunity. A high prevalence of thyroid autoantibodies have been demonstrated in children with Down's syndrome (Engel, 1967) and their mothers (Fialkow, 1964). Since the prevalence of autoim-mune disease increases with age, Burch (1969) proposed that non-disjunction during meiosis was caused by an autoagressive attack on the germ ce l l s . This autoagressive attack would occur more frequently in older women. This hypothesis could account for the cases of familial clustering of chromosomal abnormalities since a predisposition to some autoimmune d i -seases is inherited (Hecht et al., 1964). But there is no experimental evidence to support this theory and the correla-tions between non-disjunction and autoimmunity are present chiefly in young, not old, mothers. B) Theories of non-disjunction due to aging of the ovum during the dictyotene stage of meiosis In mammals, meiosis begins in the fetal ovary and progresses to diplotene. At this stage, meiosis is arrested and does not resume until shortly before ovulation in the adult (Zuckerman, 1965a). This extended diplotene, termed dictyotene, can persist in some oocytes until the end of the reproductive l i f e of the female; that i s , up to two years in the mouse and f i f t y years in man. Since the frequency of aneuploid offspring and embryos increases with maternal age, several people have suggested that changes in the oocyte during the prolonged dictyotene increase the frequency of non-disjunction. The following theories suggest the various mechanisms which might cause non-disjunction. Satel l i te associations of chromosomes Sate l l i te associations of the acrocentric D and G chromosomes are often present in mitotic cel ls (Heneen and Nichols, 1966). The sate l l i tes of the acrocentric chromo-somes have nucleolar organizers (Ferguson-Smith, 1964) and their association is generally regarded as a holdover from -14-their proximity to the nucleolus. Since the D, E, and 6 chromosomes are most frequently involved in non-disjunction in live-born children, fai lure of the nucleolus to disperse has been proposed as a cause of non-disjunction. Polani et a l . (1960) suggested that the duration of the dictyate cycle might cause a slower dispersion of the nucleolus in older oocytes, interfering with chromosome pairing and chiasma formation in D and G chromosomes. But this hypothesis is inval id, since chromosome pairing and chiasma formation occur during prophase in the fetal ovary before dictyotene. A modification of this theory has been proposed by Evans (1967). He believes that the nucleolus may fa i l to disperse and persist into metaphase I, preventing separation of the bivalents associated with i t . Many studies have been done on the frequency and type of nucleolar associations in different populations. If sa te l l i te associations are causing non-disjunction in man, then in the D group, chromosome 13 should be present in associations more frequently than chromosome 14 or 15, and in the G group, chromosome 21 more often than chromosome -15-22 because chromosome 13 and 21 are more frequently i n -volved in non-disjunction. Cooke (1972) has determined that chromosome 13 is present in nucleolar associations more frequently than chromosome 14 or 15. However, Naka-gome (1973) has shown that sa te l l i te associations do not occur more frequently in chromosome 21 than in chromosome 22. Moreover, the parents of children with Down's syndrome do not have more chromosome 21 associations than chromosome 22 (Curtis, 1974). It is important to note that the above reasoning is based on the assumption that chromosomes 13 and 21 are also the most frequent trisomies in pre-natal stages and recent evidence from spontaneous abortions demon-strates that this may not be true. In any case, a l l of these studies have been performed in mitotic tissue, and the results may be quite different in meiotic tissue. To date, there have not been any reports on associations between the sa te l l i te chromosomes in the oocytes of humans or experimen-tal mammals. Because of reports of clustering of Down's syndrome, some people have suggested that viruses can cause non-disjunc-tion (Stol ler and Collman, 1965; Robinson et a l . , 1969). Evans (1967) believes that viruses may reduce the capacity -16-for dissolution of the nucleolus because DNA viruses r e p l i -cate within the nucleus and an increase in the size of the nucleolus is often seen with virus infection. If the nucleolus is involved in causing non-dis-junction, i t would explain why the D and G chromosomes occur so frequently in trisomic conditions. But i t would not explain non-disjunction of the E chromosomes. Yunis (1965) suggested that the D, E, and G chromosomes are most common in trisomic conditions because they are largely genetically inert, since large portions of these chromosomes replicate late in the cel l cycle. Franceschini et a l . (1973) pointed out that chromosomes 13, 18, and 21 have much larger areas of fluorescence after treatment with quinacrine than the other members of the D, E, and G groups of chromosomes. Furthermore, since Sanchez and Yunis (1974) have recently shown that Q bands due to quinacrine fluorescence, are sites of repetitive DNA, this implicates the genetic inert-ness of the specif ic chromosomes most common in trisomic conditions. Therefore, a loss or gain of these chromosomes would be less deleterious than the loss of other chromo-somes and trisomic embryos involving these chromosomes would be more l ike ly to survive to term. -17-Therefore the evidence supporting the theory of nucleolar association of sa te l l i te chromosomes is real ly restricted to mitotic ce l l s . Further studies in oocytes are needed to determine i f nucleolar associations persist into metaphase I. X irradiation There have been several studies showing a possible association between maternal irradiat ion and the incidence of aneuploid offspring (Uchida and Curtis, 1961; Alberman et^  a l . , 1972). But they have generally been retrospective studies complicated by ascertainment bias. In 1968, Uchida et a l . reported the results of a prospective study on chromosomal anomalies among the children of irradiated mothers. They found that women exposed to abdominal irradiation ran an increased risk of producing aneuploid children, especially late in reproductive l i f e . Advanced maternal age seemed to increase the ab i l i ty of X rays to cause non-disjunction. Uchida and Lee (1974) studied the effect of low-dose X irradiation on the oocytes of young female mice. They found a small increase in the frequency of non-disjunc-tion during the f i r s t meiotic division in the oocytes. -18-Yamamoto et a l . (1973b) studied the effects of low-dose X irradiation on the production of heteroploid fetuses in young and old female mice. They found a spon-taneous rate of aneuploid fetuses in both young and old mothers and the frequency of aneuploid fetuses was s i g n i f i -cantly higher in older female mice. Radiation increased the frequency of aneuploid fetuses but only in old female mice. Radiation did not cause a s ignif icant increase of aneuploidy in young mice. Hence, there must be some under-lying mechanism, other than radiation, which causes an in -crease of non-disjunction with maternal age. Once this process has affected the rate of non-disjunction in older females, radiation can then cause a further increase. 3.. Production of univalents in oocytes Mather (1938) and others have pointed out that a decrease-in the frequency of crossing-over and chiasmata is associated with an increase in non-disjunction of chromosomes. A minimum of one chiasma per bivalent is necessary to ensure that chromosomes remain paired and segre-gate normally at anaphase I of meiosis (Maguire, 1974). Loss of chiasmata has been shown to result in precocious -19-univalent production in plants (Thomas and Rajhathy, 1966), insects (Shaw, 1971), mice (Purnell, 1973), and man (Pear-son et a l . , 1970). The univalents have then led to anomal-ous segregation of chromosomes and to aneuploid gametes (Carpenter and Sandler, 1973; Sharma and Reinbergs, 1974). Slizynski (1960) f i r s t suggested that the increase of aneu-ploid offspring with maternal age in humans might be ex-plained by a loss of chiasmata during the long dictyotene stage of meiosis. Henderson and Edwards (1968) found a s ignif icant decrease in the chiasma frequency in the oocytes of old female mice of three different s t ra ins—CBA, CBA/T6T6, and C57BL. The location of the chiasmata was more terminal and an increase in the frequency of univalents was also ob-served in older females. In male mice, a comparable de-cline in chiasma frequency or increase in univalents was not seen. Henderson and Edwards proposed that univalents were produced in the older females during the prolonged dictyotene stage of meiosis and that none were seen in older males because spermatogenesis is a continuous process. Henderson and Edwards also observed univalents in some human oocytes but the numbers v/ere too small to be -20-stat i s t ica l ly s ignif icant. However, they suggested that a similar reduction in chiasma frequency with subsequent uni-valent production could explain the increased incidence of trisomy in older women. In 1973, Luthardt et a l . confirmed these results in two different strains of mice, C57 BL/6J and ICR. They demonstrated a signif icant decrease in chiasma frequency and an increase in univalents with age. They also discovered that univalent production was nonrandom and involved small chromosomes predominantly. This is not surprising since small chromosomes have fewer chiasmata (Sl izynski, 1960) and a progressive loss of chiasmata would result in univa-lent formation f i r s t in the small chromosomes. It is inter-esting to note that the three types of trisomy seen in humans al l involve small chromosomes and that the incidence of trisomy 13, 18, and 21 varies inversely with the mean chiasma frequency. Trisomy 21, Down's syndrome, has the lowest chiasma frequency and is the most common trisomy (Lange et a l . , 1975). Henderson and Edwards (1968) and Luthardt et a l . (1973) proposed that the univalents seen at metaphase I in the old female mice led to non-disjunction and production -21-of aneuploid gametes. But the univalents could be subject to any one of several modes of behaviour during the ensuing anaphase: random segregation, fa i lure to migrate and ex-clusion from the main nucleus by micronuclei formation, misdivision of the centromere resulting in isochromosome formation, or normal segregation (John and Lewis, 1965). Detailed chromosome counts of metaphase II oocytes would be required to determine the fate of the univalents. Since this theory of maternal age related non-disjunction seemed the most plausible and the most amen-able to experimentation, the present study was undertaken to determine i f the univalents observed at metaphase I do result in non-disjunction in older female mice. -22-II. MATERIALS AND METHODS Source and maintenance of mice Virgin female CBA mice, 4 - 5 weeks old were ob-tained from the Jackson Laboratory (Bar Harbor, Maine). The animals were aged in the Zoology Vivarium at the Uni-versity of Brit ish Columbia and were housed 4 mice per cage. The animal rooms were kept on a 12 hour l ight (8 a.m. to 8 p.m.) and dark cycle. The mice were allowed to eat Purina lab chow and drink water ad l i b . Procedure for obtaining oocytes Oocytes were obtained without the use of gonado-tropic hormones to induce superovulation since recent reports have indicated that these hormones can increase the frequency of chromosome abnormalities (Fujimoto, Pah-lavan, and Dukelow, 1974; Boue and Boue, 1973). The mice were k i l led by cervical dislocation. The ovaries were dissected out and placed into an embryological watchglass containing 2 ml. of oocyte culture medium under 2 ml. of equilibrated paraffin o i l . Subsequent manipulations were performed under a Zeiss stereodissecting microscope -23-at a magnification of 12 times for work with the ovaries and 30 times for work with the oocytes. The ovaries were cleaned of any adhering fallopian tubes or fat and trans-ferred to new media. The oocytes were then liberated by puncturing the follicles with a 25-gauge sterile hypodermic needle. All liberated oocytes were washed twice by trans-ferring them via a mouth-controlled Pasteur micropipette to two other embryological watch glasses each containing 2 ml. of medium under paraffin o i l . Any adhering fo l l i c l e ce l l s were removed by sucking the oocyte in and out of the micropipette. Any undamaged oocytes possessing an intact germinal vesicle were selected for culturing. When the oocytes were not being directly manipulated, they were kept at 37°C in a 5% atmosphere. The maximum time span from the death of the mouse to the placement of the oocytes in the final culture medium was one hour. Procedure for culturing oocytes The medium used to culture the oocytes was a chemically defined Krebs-Ringer salt solution containing sodium pyruvate, albumin and antibiotics as described by Donahue (1968) (cf. Appendix I). The medium was steri l -ized by positive pressure millipore filtration. The -24-culture system consisted of microdroplets of medium under s t e r i l e , equil ibrated paraffin o i l in a 35 x 10 mm. Falcon p last ic tissue culture dish (Brinster, 1963). The paraffin o i l was equil ibrated with the culture medium. Between 5 and 10 oocytes were injected into each microdroplet. The oocytes were cultured in a humidified 5% CX^ incubator at 376C . Procedure for oocyte f ixation and chromosome preparation After 17 - 18 hours in culture, the oocytes were transferred to an embryological watch glass containing hypotonic solution (1% sodium citrate) for 10 minutes. The number of oocytes with a polar body was recorded. The oocytes were placed in the centre of a clean s l ide and the excess hypotonic solution was drawn off. Four drops (each 20X) of a 3:1 mixture of ethanol-glacial acetic acid were applied to the s l ide. The sl ides were then gently blown dry (Tarkowski, 1966). A maximum of 5 oocytes were placed on each s l ide. The preparations were stained for centro-meric bands using the basic method of Arrighi and Hsu (1971) with a few modifications. The procedure was as fol1ows: -25-1. .2 N HC1 at room temperature for 30 minutes. 2. Three rinses in d i s t i l l ed water. Air dry. 3. Pancreatic RNase (100 ug ml in 2 x SSC, pH 7.25) at 37°C in a moist chamber for 45 minutes. 4. Two rinses in each of 2 x SSC (pH 7) 70% ethanol, 95% ethanol. Air dry. 5. .07 N NaOH at room temperature for 50 seconds. 6. Two rinses each in 70% ethanol and 95% ethanol. Air dry. The NaOH must be removed as rapidly as possible to prevent further denaturation. The alcohol wash must be kept separate and not be reused in other parts of the staining procedure. 7. 2 x SSC (pH 7) at 65°C for 16 hours. 8. Two rinses each in 70% and 95% ethanol. Air dry. 9. Stain for 15 - 30 minutes in a Giemsa solution pre-pared by the following formula: 50 ml. d i s t i l l ed water, 1.5 ml. . 1M c i t r i c acid, adjust pH with . 2M Na2HP0^ to pH 7, 1.5 ml. pure methanol, 5 ml. Giemsa stock solution (Gurr). This solution must be prepared immediately before use as i t forms a preci -pitate after a few hours. 10. Two rinses in d i s t i l l ed water. Air dry. Mount in permount. The chromosomes of some oocytes (about 10%) required re-staining for 3 hours to achieve the desired intensity. -26-Several other simpler and faster staining procedures were attempted but no other technique produced good centromeric bands consistently. Procedure for analysing oocytes The slides were coded so that maternal age was not known at the time of analysis. The slides were anal-ysed in groups of approximately 20, containing oocytes from mice of different ages. The number of oocytes on each slide was recorded so that all oocytes or remnants of oocytes were accounted for during the analysis. The chromosome number was determined using the oil immersion lens of a Zeiss photomicroscope. Oocytes were not in-cluded in the analysis i f the chromosomes were spread more than one microscopic f ield. All oocytes with an abnormal number of chromosomes were photographed. Only oocytes with an unambiguous number of chromosomes were used for calculations of non-disjunction frequencies; therefore, many oocytes were discarded because the chromo-somes were overlapped, too contracted, too spread apart, or the C bands were not clear enough to distinguish between chromatids and chromosomes. However, -27-a l l oocytes in which a diploid or haploid number (+2 chromosomes) could be discerned were used to calculate the incidence of oocytes with 40 chromosomes. Fisher's exact test was used for the s ta t i s t i ca l analysis. -28-I I I . RESULTS Collection and in vitro maturation of oocytes A total of 1402 oocytes obtained from 174 CBA females were set up in culture. After 1 7 - 1 8 hours, an average of 80.5% of the oocytes had progressed to meta-phase II. The mice were grouped into three different mater-nal age classes based on l i t t e r size data in CBA females. The l i t t e r size of CBA mice increases and reaches a maxi-mum at 150 days, then decreases until 240 days, and after this fa l l s to a very low level until a l l oocytes are de-pleted at approximately 330 days. (Jones and Krohn, 1961). As shown in Table I (p. 36), the maternal age had a str iking effect on the mean number of oocytes obtained per mouse and on the percentage of oocytes maturing in v itro. It was very d i f f i c u l t to obtain oocytes from mice in the oldest age group. The ovaries contained very few oocytes and many were atret ic. As shown in Table I, the oldest maternal age group required almost three times the number of mice used in the youngest age group since the mean number of oocytes obtained per mouse decreased from -29-14.7 in young females (60-150 days) to 9.6 in middle-aged fema'ies (151-240 days) to 4.4 in old females (241-330 days). The percentage of oocytes maturing in vitro also decreased with maternal age. Eighty-seven per cent of oocytes from young mice progressed to metaphase II. How-ever, only 80.7% of the oocytes from the middle aged group matured in v i tro. This was s ignif icant ly different from the young mice ,(p = .01, Fisher's exact test). The f re -quency of maturation decreased further to 71.9% in the old maternal age group. This was s ignif icantly different from the middle aged mice (p = .004) and the drop from 87% in the young mice to 71.9% in the old was highly s ignif icant (p^.000001 ). Therefore, even at this early stage of develop-ment, oocytes from older females are less viable than those from younger females. This must occur because of a defect in the oocyte i t s e l f since al l oocytes were handled in the same manner and cultured in the same medium. Chromosomal analysis of oocytes Chromosome preparations which yielded exact chromo-some counts were obtained in 473 oocytes (33.7% of the total 1402). These oocytes were used in calculations of aneuploid -30-frequencies. Chromosome preparations which could be counted to the diploid or haploid number! 2 chromosomes were used to calculate the frequency of diploid oocytes. This was poss-ible in 877 oocytes (62.6% of the tota l ) . Failure of oocyte maturation, loss of oocytes during fixation and poor chromo-some quality accounted for the remaining oocytes. Examples of analysed oocytes as well as discarded oocytes are shown in Figures 1 - 9 (p. 38 - 46). The centromeric banding was very useful in deter-mining the number of chromosomes, part icular ly when the chromosomes were crowded as in Figure 4. However, i t was not possible to karyotype the metaphase II chromosomes and thereby identify a missing or extra chromosome. Mouse mito-t i c chromosomes are very d i f f i cu l t to karyotype because they are a l l telomeric and there is very l i t t l e difference in size. Meiotic chromosomes pose added problems because they vary greatly in morphology and size and the chromatids are convoluted and spread far apart. The chromosomal analysis of the oocytes is shown in Table II (p. 37). A very small number of oocytes had less than 18 chromosomes. These were assumed to be due to a r t i f i c i a l loss and were discarded from the analysis. No -31-oocytes were found with more than 21 chromosomes (other than the diploid oocytes). Eleven oocytes (2.3%) lost two chromosomes. There was no signif icant difference among the maternal age groups and these oocytes probably resulted from a r t i f i c i a l chromosome loss during oocyte f ixat ion. Thirty-four oocytes (7.2%) lost one chromosome. An example of a hypoploid oocyte is shown in Figure 2. The middle maternal age group had 10.7% oocytes with 19 chromosomes and this was s igni f icant ly different (p = .04) when compared to the old maternal age group which had only 3.8% oocytes with 19 chromosomes. Neither the youngest nor the oldest mice had any oocytes with 21 chromosomes. But the middle maternal age group had six (4.9%) hyperploid oocytes; this was s i g n i f i -cantly different when compared to the young (p = .006) and the old (p = .01) maternal age group. These six hyper-ploid oocytes were obtained from six different mice and they were not confined to one certain time or season or group of mice. (The mice were ordered from the Jackson laboratory in three groups). It is highly unlikely that oocytes with 21 chromosomes resulted from the addition of -32-one "wandering" chromosome from another oocyte. Chromo-somes from different oocytes varied greatly in s ize, shape, and centromeric banding, and since only five oocytes were placed on each s l ide, they were generally well separated from each other. Therefore, the six hyperploid oocytes probably resulted from non-disjunction during the f i r s t meiotic div is ion. Examples of hyperploid oocytes are shown in Figures 3 and 4. Theoretically, non-disjunction should produce an equal number of oocytes with 19 and 21 chromosomes. From Table II, i t is obvious that the frequency of oocytes with 19 chromosomes exceeds the frequency of oocytes with 21 chromosomes. The preponderance of hypoploid oocytes may be due to different causes: 1. Hyperploid nuclei may be preferential ly emitted into the f i r s t polar body (and therefore not be detected at metaphase II). 2. One chromosome may be lost due to blowing during oocyte f ixat ion. This last reason seems the most plausible since oocytes with less than 19 chromosomes were found but none -33-were found with more than 21. Other investigators have also observed an excess of hypoploid oocytes (Rohrborn, 1972; Hansmann, 1974; Uchida and Lee, 1974). The frequency of oocytes with 19 chromosomes is s ignif icantly higher in the middle maternal age group. Presumably this increase occurs because this class is composed of hypoploid oocytes resulting from non-disjunction (the reciprocal event of the hyperploid oocytes in the same age group) as well as chromo-some loss. Since i t is not possible to get an accurate e s t i -mate of the frequency of hypoploid oocytes due to non-dis-junction, a conservative estimate of the non-disjunction rate can be obtained by doubling the hyperploid rate. Thus the overall rate of non-disjunction was 2.6% but i t was restricted to the middle maternal age group with a non-dis-junction rate of 9.8%. The overall frequency of oocytes with 40 chromo-somes was 1.7%. Examples of diploid oocytes are shown in Figures 5 and 6. There was no signif icant difference in the frequency of diploid oocytes among the different mater-nal age groups. These diploid oocytes probably resulted from fai lure of f i r s t polar body formation. Other possible -34-interpretat ions include: 1) Two normal oocytes which are in close proximity dur-ing chromosome preparation. But the chromosomes of the two oocytes would need to have exactly the same s i z e , shape, and C banding. Since chromosomes from d i f fe rent oocytes are so var iab le , th i s i s un l i ke l y . Also, i f th i s occurred, two polar bodies would be expected in the same area and no polar bodies were ever found near the d i p l o i d oocytes. 2) Proximity of the chromosomes of the oocyte and the extruded f i r s t polar body. However, the chromosomes of the f i r s t polar body are generally not d i s t i n c t ; they are usually represented by condensed degenerate chromatin. An example of an oocyte with a typ ica l polar body is shown in Figure 7. The f i f t h column of Table II shows the f r e -quency of normal oocytes in the d i f f e ren t maternal age groups. There is no s i g n i f i c an t difference be-tween the young (86.9%) and the old maternal age group (91.3%). However, the middle age group has a s i g n i f i c an t l y Tower frequency of normal oocytes (77.9%) than e i ther the young group (p = .05) or the old group -35-(p = .003). Thus, although the oldest age group has least number of oocytes per mouse and the poorest rate of in vitro maturation (Table I )» the oocytes that do reach metaphase II have the highest f re -quency of a normal chromosome complement. -36-TABLE I Effect of maternal age on the mean number of oocytes obtained per mouse and on in vitro oocyte maturation. MATERNAL MEAN NO. % OOCYTES AGE IN NUMBER OOCTYES* PROGRESSING DAYS OF MICE OBTAINED/MOUSE TO MET II Young „ • n , * 60-150 151-240 Old 241-330 32 14.7 87.0 Middle c n n r + 59 9.6 T80.7 83 4.4 71.9 TOTAL 174 8.1 80.5 * Signif icantly different when compared to middle maternal age group (p = .01) and to old maternal age group (p<.000001 ). t S ignif icantly different when compared to old maternal age group (p = .004). % Undamaged oocytes possessing a germinal vesicle. -37-TABLE II Chromosomal analysis of 473 metaphase II oocytes from mice of di fferent ages. MATERNAL TOTAL NO. AGE IN OOCYTES 13 DAYS ANALYSED (n-2) 19 (n-1) TOTAL NO. DIPLOID & 20 21 40 HAPLOID (n) (n+1) (2n) ( ± 2)00CYTES Young 60-150 191 3.1% (6) 7.9% (15) 86.9% 1.1% (4) 353 Middle 151-240 122 1.6% (2) *10.7% (13) +77.9% *4.9% (6) 2.1% (6) 280 Old 241-330 160 1.9% (3) 3.8% (6) 91.3% 2.0% (5) 244 TOTAL 473 2.3% (11) 7.3% (34) 86.0% 1.3% (6) 1.7% (15) 877 Signif icantly different when compared to old maternal age group (p = .04), Fisher's exact test. + S ignif icantly different when compared to young maternal age group (p - .05) and to old maternal age group (p = .003). t Signif icantly different when compared to young maternal age group (p = .006) and to old maternal age group (p = .01). © Used for calculation of the frequency of 2n (40) oocytes, (cf. text p. 30) -38-Second meiotic metaphase showing a normal complement of 20 chromosomes. Reproduced at 3100 X. -39-Figure 2. Second meiotic metaphase with 19 chromo-somes. Reproduced at 3200 X. -40-Figure 3. Second meiotic metaphase with 21 chromosomes. Reproduced at 2400 X. i -41-Figure 4. Second meiotic metaphase with 21 chromosomes. The axes of the chromatids are drawn fo r c l a r i f i c a t i o n . The chromosomes are crowded and fuzzy, but they can s t i l l be counted u t i l i z i n g C bands. Reproduced at 3000 X. -42-Figure 5. Second meiotic metaphase with 40 chromosomes. Reproduced at 2200 X. -43-Figure 6. Second meiotic metaphase with 40 chromosomes. Reproduced at 2800 X. -44-Figure 7. Second meiotic metaphase with its polar body. Reproduced at 780 X. -45-Figure 8. Second meiotic metaphase which could not be analysed because the chromosomes are too crowded together and the C bands are not clear enough. Included in calcula-tions of diploid oocyte frequency. Reproduced at 2900 X. -46-Figure 9. Second meiotic metaphase which could not be analysed because of the presence of some chromatids and debris. Included in calcu-lations of diploid oocyte frequency. Reproduced at 3400 X. -47-IV. DISCUSSION In vitro oocyte maturation Pincus and Enzmann (1935) or ig inal ly observed that the mechanical release of rabbit oocytes from the ovarian f o l l i c l e s into various culture media resulted in spontaneous breakdown of the germinal vesicle and the continuation of meiosis to the metaphase II stage. Spontaneous maturation of oocytes has subsequently been described in numerous mam-malian species (Donahue, 1972a) and occurs in chemically defined as well as in biological media (Biggers, 1972). There have been several attempts to explain this phenomenon but very l i t t l e is known about oocyte maturation in vivo or in v itro. Fo l l i c le cel ls have been implicated in the inh ib i -tion of oocyte maturation in rabbits (Foote and Thibault, 1969) and in pigs (Tsafr ir i and Channing, 1975). This hypo-thesis is attractive because i t could explain the arrest of meiosis in vivo and the resumption of meiosis as soon as the oocyte is free of the f o l l i c l e ce l l s , either in vivo or in v i tro. However, oocytes with investing f o l l i c l e cel ls have matured successfully in vitro in several species -48-(Kennedy and Donahue, 1969; Robertson and Baker, 1969) and Cross and Brinster (1970) have shown that mouse oocytes cu l -tured with f o l l i c l e cel ls have an increased frequency of maturation. Fo l l i c le cel ls are capable of converting var-ious substrates to pyruvate, which the oocyte requires as an energy source (Donahue and Stern, 1968). Since oocytes of several species have been cultured with f o l l i c l e ce l l s , they cannot be inhibit ing maturation. Therefore the mechan-ism which causes oocytes to mature in v i tro remains unknown. Normality of oocyte development in vitro Several studies have been done to determine i f oocyte maturation in vitro is comparable to development in  vivo and i f normal embryonic development can proceed. Th i -bault (1973) demonstrated that rabbit oocytes matured in  v itro progress to metaphase II in exactly the same length of time as control oocytes matured in vivo after coitus. Calarco et a l . (1972) found no s ignif icant ultrastructural differences between mouse oocytes maturing in vivo and in  v itro. Early embryonic development is also normal in rabbit and mouse embryos f e r t i l i zed and grown in vitro (van Bler-kom and Manes, 1974; Cross and Brinster, 1970); in addition -49-normal adult rabbits and 15 day-old mouse fetuses have been produced from oocytes matured and fe r t i l i zed in vitro and transferred to pseudopregnant foster mothers (Thibault, 1973; Cross and Brinster, 1970). Thus, experiments to date show that in vitro oocyte maturation is similar to in vivo maturation and does not cause an increase in abnormal de-velopment. « Comparison of results to other studies 1. Diploid oocytes The overall incidence of oocytes with 40 chromo-somes in this study was 1.7% and this was not maternal age dependent. If a diploid oocyte were fe r t i l i zed by a nor-mal sperm, a t r ip lo id embryo would result. Other studies of mouse embryos have also shown that tr ip lo idy is not dependent on maternal age (Yamamoto et a l . , 1973a; Gosden, 1973). Carr (1971b) has shown that tr ip loidy does not in -crease with maternal age in humans. Donahue (1970) found an incidence of only .02% diploid oocytes in CF-1 mice. But he found that 1.2% of f i r s t cleavage divis ion mouse embryos were t r i p l o id ; these were caused equally by dis-permy and digyny (Donahue, 1972b). Yamamoto et a l . (1973a) -50-found an overall incidence of 1% tr ip lo idy in 10.5 day CF-1 mouse fetuses. Gosden (1973) found 4.2% tr iploidy in 3.5 day CBA/HT6 mouse embryos. Presumably, these differences ref lect the different gestational stages examined and the different mouse strains used. It is d i f f i c u l t to estimate the frequency of t r ip lo id embryos in humans since they can-not be studied unless the embryos are aborted. Carr (1967b) has determined that tr ip lo idy occurs in 4% of spontaneous abortuses and in 19% of abortuses with chromosome abnormal-i t i e s . However, the frequency of tr ip lo idy in humans is probably higher in ear l ier stages of gestation since the frequency of chromosome abberations decreases with gesta-tional age (Boue, 1972). Tr iploid embryos are generally aborted and only a few human tr ip lo ids have survived to term (Niebuhr, 1974). In the mouse, t r ip lo id embryos are eliminated during the f i r s t few days after implantation (Wroblewska, 1971). 2. Aneuploid oocytes Several investigators have studied the frequency of aneuploidy during meiosis and early development in young mice. Using a hybrid s t ra in, Uchida and Lee (1974) found no spontaneous aneuploidy in metaphase II oocytes. Dona-hue (1972b) found no hyperploidy during the f i r s t cleavage division of CF-1 embryos. Similarly, Gosden (1973) and Yamamoto et a l . (1973a) did not find any trisomy in CBA/HT6 embryos or CF-1 fetuses from young female mice. The pre-sent study agrees with these observations; no oocytes were found with 21 chromosomes in the young CBA mice. However, Hansmann (1974) has found that 2.4% of metaphase II oocytes from young C3H female mice have 21 chromosomes; and Phi l l ips and Kaufman (1974) have observed 2.9% hyperdiploidy in oocytes from young (C3H x 101)F-j mice. This higher f re -quency of hyperdiploidy may ref lect a higher spontaneous rate of non-disjunction in C3H mice. Genetic differences can alter the frequency of aneuploidy. Fishberg and Beatty (1951) discovered that the mutant si Tver (si) in mice causes a high spontaneous frequency of heteroploid embryos. At 4^ days gestation 14.3% of embryos are heteroploid i f the mother is homozygous for the s i l ver mutation. If both parents are s i l ver , the frequency of heteroploid embryos is higher. Phi l l ips and Kaufman (1974) have found a sex-linked mutant (FxO) which causes non-disjunction of the X chromosome in approximately one-third of oocytes. Human - 5 2 -families with a high frequency of aneuploid offspring have also been recorded (Hecht et a l . , 1964). Since very few human oocytes or early embryos have been studied, i t is d i f f i c u l t to estimate the frequency of aneuploidy at a comparable stage in humans. However the frequency of aneu-ploidy is def in ite ly higher in humans than in mice since 35% of spontaneous abortions are heteroploid and 66% of the heteroploidy is due to aneuploidy (Carr, 1971a). The effect of age on non-disjunction in mice has been analysed in only two studies. Gosden (1973) studied 3.5 day embryos from C8A/HT6 mice and found 5.2% trisomic embryos in the old females. Yamamoto et a l . (1973a) deter-mined that 1.9% of 10.5 day fetuses from old CF-1 females were trisomic. Both studies demonstrated a s ignif icant increase of non-disjunction with maternal age. In this study the frequency of oocytes with 21 chromosomes (which would lead to trisomy i f f e r t i l i zed by a normal sperm) was 4.9% in the middle aged female mice. But there was a com-plete lack of hyperploid oocytes in old females. This may be explained by the pecul iar it ies of the mouse strain used. -53-Characteristics of CBA mice CBA mice were chosen for this study because of their short reproductive lifespan (10 - 11 months) (Jones and Krohn, 1961) and because of the high frequency of uni-valents in the oocytes from old females (Henderson and Edwards, 1968). Henderson and Edwards found that 75% of the oocytes from old CBA females had univalents. There-fore, i t was f e l t that i f non-disjunction resulted from the univalents, i t would be most evident in oocytes from old CBA females. But the CBA strain is very peculiar. Jones and Krohn (1961) studied four different strains of mice and found that CBA females have the smallest endow-ment of oocytes at b i r th; the most rapid depletion of oocytes; and they are the only strain in which the ovary is completely depleted of oocytes long before death. .The smaller endowment of oocytes makes i t much more d i f f i -cult to culture large numbers of oocytes; even in young females, a maximum of only 20 oocytes per mouse can be collected. The number of oocytes decreases with age and in old CBA females very few oocytes can be obtained (cf. Table I). The small number of oocytes is not because -54-virgin mice were used in this study since Jones and Krohn (1961) have demonstrated that there is no s ignif icant d i f -ference in the rate of depletion of oocytes in virgin or multiparous mice. Possible explanations for the lack of hyperploidy in the  oocytes from old CBA mice It may be possible that only normal oocytes are being selected for culture from old CBA females. But i f this is true, one would have to postulate that oocytes which have many univalents, or are damaged in some other way, preferential ly become atret ic. Since 87% of oocytes from young females mature in culture compared to only 71.9% in old females, a higher frequency of univalents may prevent defective eggs from reaching metaphase II. If this is the case, a threshold effect must be postulated i .e . oocytes from young females have no univalents and no aneuploidy; oocytes from middle aged females have some univalents and these result in non-disjunction lead-ing to 4.9% hyperploidy; oocytes from old females may reach a threshold in which some oocytes have so many uni-valents that they either become atretic or f a i l to progress -55-to metaphase II. Alternatively, the fa i lure of maturation may not be due to the higher number of univalents but to the type of univalents seen in old CBA females. Henderson and Edwards (1968) c lass i f ied univalents as loosely-associated, tightly-associated, and true univalents•(no association). Although the frequency of loose and tight univalents in-creased with maternal age, true univalents were only seen in the oldest maternal age group. Therefore, loosely or tightly associated univalents may cause incorrect attach-ment of spindle fibres resulting in non-disjunction. Spindle fibres may not be able to attach to true univalents and meiosis might then be arrested in the oocytes from old CBA female mice. Evidence that a reduced chiasma frequency and in -creased univalent frequency can lead to meiotic arrest has been presented in one male mouse (Purnell, 1973) and ten men (Hulten et a l . , 1974). In every case the males had a high frequency of univalents and were s ter i le because of a block in spermatogenesis. Hulten et a l . (1974) investigated one patient in detail using electron microscopy and found that cel ls at diakinesis showed signs of arrest: -56-centrioles were located at the centre of the group of chromosomes and no microtubular bundles were found either around the centrioles or the centromeres; a large number of vacuoles and swelling mitochondria were also observed in these ce l l s . Therefore, abnormal oocytes from older females'may be eliminated before metaphase II, when they are normally ovulated, so that only normal oocytes are seen at this stage. Thung (1961) has observed that in old female mice oocytes are often retained in the f o l l i c l e and not ovulated so that corpora lutea containing oocytes are formed. Anovulatory cycles are also common in women approaching the menopause (Riley, 1964). Therefore, there may be some mechanism which detects abnormal oocytes, or oocytes which have been arrested at some stage before metaphase II, and prevents the ovulation of these oocytes. Relation of chromosomal abnormalities to the decline in  f e r t i l i t y in old female mammals It is obvious that although there is an increase in the frequency of chromosome abnormalities with maternal age, this increase is not great enough to entirely account -57-for the large drop in l i t t e r size in older mammals. In this study, the age group with the highest frequency of chromosome abnormalities had an estimated frequency of 9.8% aneuploidy (2 x 4.9%) and 2.1% tr iploidy or approximately 12% of oocytes with numerical chromosome anomalies. Gos-den (1973) found that 12.1% of 3.5 day embryos from old female mice had abnormal chromosome constitutions. Since l i t t e r size can drop by 50% in old mice (Jones and Krohn, 1961), there must be other factors responsible for the decrease. The retention of oocytes in their f o l l i c l e s in old mice may also contribute to the decline in l i t t e r size and hormonal function may be impaired in the old mice (Gosden, 1974b). Discussion of results in relation to the theories of non- disjunction The results presented in this paper do not d i r -ectly confirm or discredit any of the theories of non-disjunction presented in the introduction. However, since the mice were not exposed to radiation and the oocytes were not aged by delayed ovulation or f e r t i l i z a t i o n , i t is not l ike ly that these causes are responsible for the - 5 8 -non-disjunction observed in the middle age group of mice. The increase of meiosis I non-disjunction with maternal age observed in metaphase II oocytes in this study does not correspond directly to the increase of univalents with age seen in the studies of Luthardt et a l . (1973) or Henderson and Edwards (1968). If an increase in uni-valents with maternal age is causing non-disjunction, then a threshold effect must be postulated to explain the lack of hyperploidy in metaphase II oocytes of old CBA female mice. The evidence that a threshold for the num-ber or type of univalents may exist is presented above. Since CBA mice are peculiar both in their reproductive pattern and their high frequency of univalents in old females, i t would be very interesting to determine the frequency of non-disjunction with maternal age in meta-phase II oocytes in other strains of mice. To date, no one has published any data on the effect of maternal age on non-disjunction in oocytes. A unified theory of maternal age related non-disjunction The univalent theory of non-disjunction is attractive because i t provides a mechanism by which -59-non-disjunction can occur; that i s , i f bivalents become unpaired or only loosely paired, spindle fibres may not be able to attach correctly and non-disjunction may result. Since univalents have been shown to result in aneuploid gametes in other organisms (Carpenter and Sandler, 1973; Sharma and Reinbergs, 1974) i t seems l ikely that the univalents seen in older mice could ex-plain the increase of aneuploidy. Other theories lack an actual mechanism which could cause non-disjunction. For example, theories of tubal or in t ra fo l l i cu la r aging of the ova or radiation damage do not real ly explain how these factors could affect the chromosomes and thereby cause non-disjunction. However, there is some evidence that various factors such as temperature and radiation can cause an increase in the frequency of non-disjunction. These other factors may be affecting the non-disjunction rate by the mechanism of univalent production. Univalents are produced with advancing maternal age in mice as the frequency of chiasmata decreases. The spontaneous reduc-tion of chiasmata and increase of univalents with maternal age could be further accelerated by environmental factors. - 6 0 -Radiation has been shown to cause a terminali-zation of chiasmata, a decrease in chiasma frequency, and an increase in univalents in plants (Ramulu, 1973). Since radiation has also been shown to increase the frequency of non-disjunction in mice (Yamamoto et a l . , 1973b; Uchida and Lee, 1974), the mechanism may be to further decrease the chiasma frequency occurring spontaneously and acceler-ate univalent production. It may be necessary for preco-cious terminalization of chiasmata to begin before radia-tion can further affect the process since Yamamoto et a l . (1973b) found that radiation could only increase the rate of non-disjunction in old female mice not in young mice. An increase in temperature in Schistocera greg- aria also causes a terminalization of chiasmata, a decrease in chiasma frequency and an increase in univalents (Hender-son, 1962).. Grell (1971) has determined that elevated temperature causes an increase in non-disjunction of the sex chromosomes as well as an increase in noncrossover X tetrads in Drosophila melanogaster. Therefore, i t again seems l ike ly that any heat induced non-disjunction would occur by the mechanism of univalent production. No exper-iments have been performed on the effect of increased -61-temperature on non-disjunction in mammals since i t would be d i f f i c u l t to increase the temperature of a warm-blooded animal. However, the finding that heat can induce non-disjunction in lower organisms raises the poss ib i l i ty that physiological changes in temperature, perhaps incident to disease, may contribute to various types of aneuploidy by increasing the number of univalent chromosomes. The sea-sonal clustering of mongolism and other types of trisomy reported in the l i terature (Stoller and Collman, 1965; Nielsen and Friedrich, 1969) may be due to infections causing an increase in body temperature. The s l ight increase of non-disjunction reported with aging of ova (Butcher and Fugo, 1967; Vickers, 1969) may also be due to univalent formation. Rodman (1971) de-termined that in some aging mouse oocytes, s ister chroma-tids had disjoined prematurely; and Yamamoto and Ingalls (1972) found chromatid separation in aged hamster oocytes. Therefore any aging of the oocyte may further increase the frequency of univalents leading to non-disjunction. Thus environmental factors known to affect non-dis-junction frequencies may al l be operating by their effect on univalent production increasing the number of univalents -62-formed due to advancing maternal age. Variation in chiasma frequency and univalent production can be due to genetic as well as environmental factors. Selection for genetic strains with high or low mean chiasma frequencies has been accomplished in Droso- phila melanogaster (Chinnici, 1971) and Schistocerca  gregaris (Shaw, 1974). Shaw (1974) determined that 60% of the total variance in chiasma frequency in Schistocerca is due to genetic factors. Genetic mutants which f a i l to form chiasmata have also been isolated in plants (Sharma and Reinbergs, 1974), mice (Purnell, 1973) and humans (Pearson et a l . , 1970). A genetic variation in chiasma frequency could explain the clustering of aneuploidy in some families (Hecht et a l . , 1964). A genetically deter-mined low frequency of chiasmata would cause a greater susceptibi l i ty to non-disjunction since fewer environmental factors and less aging would be required to reduce the num-ber of chiasma to the extent that some chromosomes lost a l l chiasmata and became unpaired. An inherent low frequency of chiasmata might be part of the cause of Down's syndrome babies born to young mothers. A large number of studies have shown that the -63-mean age of mothers of Down's syndrome children is 6 to 8 years higher than the population mean (Penrose and Smith, 1966). But each study shows two bumps in the curve one close to the population mean and a larger one in the mid-thirt ies. These bumps are usually large enough to make the curve bimodal. Therefore Down's syndrome has been stated to be caused by A, a maternal age-independent class and by B, a maternal age-dependent class. Since class A has the same mean as the population, Penrose and Smith (1966) have suggested that Down's syndrome in class A is due to translocations, mosaicism, carr ier parents, secondary non-disjunction, and speci f ic genes inducing non-disjunction. The speci f ic genes could be lowering the chiasma frequency, increasing the formation of univa-lents and thereby causing non-disjunction. An increase in maternal age would not be expected in any of these causes. Class B, the maternal age-dependent group, would then be caused by aging of the oocyte during the prolonged dictyotene stage. Therefore although several environmental, gene-t i c , and aging factors can affect the frequency of non-disjunction by varying degrees, the mechanism of action -64-may be due to a single cause, namely univalent production and subsequent unbalanced segregation of the chromosomes. Models for the decrease in the frequency of chiasmata  with maternal age Henderson and Edwards (1968) suggested that de-creasing chiasma frequency with age could be explained either by chiasma terminalization or by a "production l ine" hypothesis. Terminalization or movement of chiasmata to the distal ends of chromosomes has been observed in plants and insects (Mather, 1938). If terminalization occurred during dictyotene, chiasmata could be progressively lost leading to the formation of univalents, especially in the smaller chromosomes which have fewer chiasmata (Sl izynski, 1960). The fact that chiasmata become more terminal in older female mice supports this view. Also, heat and radiation have been shown to cause non-disjunction in lower organisms and both also result in terminalization of chiasmata (Henderson, 1962; Yamamoto et a l . , 1973b). The main objection to the view that terminalization causes the observed decrease of chiasmata is that i t is d i f f i c u l t to envisage a molecular mechanism accounting for movement -65-of chiasmata to the chromosome ends. So Henderson and Edwards (1968) proposed that the decrease in chiasma f re -quency in old females was due to differences in chiasma frequencies in the fetal ovary. To explain this a "pro-duction l ine" in the ovary would be necessary. That i s , oocytes formed early during oogenesis would possess higher chiasma frequencies than those formed later. The oocytes would subsequently be ovulated in the same sequence account-ing for the reduction in chiasma frequency in older mice. Oocytes are formed over a period of weeks in humans (Ohno et a l . , 1962) and 4 - 5 days in mice (Borum, 1961), allow-ing suff ic ient time for gradients in chiasma frequencies to develop. Gradients in nutritional or developmental factors are known to affect chiasma frequencies in plants (Couzin and Fox, 1974; Rees and Naylor, 1960). The decline in the recombination frequency between the genes pal l i d and fidget with maternal age in mice (Bodmer, 1961) was interpreted by Henderson and Edwards as being favour-able to the "production l ine" hypothesis since recombina-tion occurs during prophase in the fetal ovary, i . e . before dictyotene. Therefore, i f gradients causing a decrease in chiasma frequency occurred in the fetal ovary, -66-a decrease in recombination would be expected in old female mice. However this decrease in recombination frequency with maternal age in mice has only been shown for these two genes. Reid and Parsons (1963) did not find a s ta t i s -t i c a l l y s ignif icant decrease in recombination frequency between the genes leaden and fuzzy with maternal age. This hypothesis of sequential oocyte development in mice could be tested by label l ing the DNA while oocytes are being formed in the fetal ovary and determining i f those oocytes formed last are conserved into old age. No formal proof exists for the "production l ine" model but neither is there any direct evidence for the loss of chiasmata by terminalization during the por-longed dictyotene stage. Therefore, the cause for the reduction in chiasma frequency and increase in univalents with maternal age in mice is not known. -67-V. CONCLUSIONS The frequency of numerical chromosome abnormal-i t ies in metaphase II oocytes from aging CBA mice was de-termined. The mice were divided into three different age groups, based on l i t t e r size data in CBA females. The frequency of diploid oocytes (which would result in t r i -ploid embryos) was 1.7% and did not vary with maternal age. Other studies have reported rates of tr ip loidy varying from .02% to 4.2% depending on the mouse strain and stage of development. There has not been a reported increase of tr iploidy with maternal age in other studies. Oocytes with 21 chromosomes were used as an index of non-disjunc-tion since chromosomes may be lost during sl ide prepara-t ion, leading to an elevated frequency of oocytes with 19 chromosomes. The overall frequency of oocytes with 21 chromosomes was 1.3% but these v/ere al l concentrated in the middle age group of mice with 4.9% hyperploidy. There have been no other published studies of meiosis I non-disjunction observed at metaphase II in aging female mice which could be compared to these results. Two studies-of aneuploidy in mouse embryos and fetuses have shown an increase with maternal age, but no -68-comparable drop in the frequency of aneuploidy in the o ld -est age group. The lack of hyperploidy in the oocytes from old CBA females may be due to pecul iar i t ies of CBA mice. Therefore similar studies in other strains of mice are required to determine i f this lack of hyperploidy in old females is strain spec i f ic . However, i t would be necessary to study much older female mice than in this project since CBA Ii i ce have an extremely short reproductive l i fespan. Several theories of non-disjunction have been discussed, part icular ly Henderson and Edward's theory of univalent formation. The results of this study do not d i rect ly confirm any of the theories unless a threshold effect is proposed to explain the lack of hyperploidy in the oocytes of old female mice. Evidence from this study and also other studies is presented to show that a thres-hold for the type or number of univalents may exist. Once the threshold level has been surpassed, meiosis may break down so that oocytes do not reach metaphase II. This would lead to a lower maturation rate of oocytes, but a higher frequency of chromosomal normality in the old females. A unif ied model of non-disjunction is presented in which the various environmental, genetic, and aging -69-factors known to affect the frequency of non-disjunction, act by an underlying mechanism of univalent formation. Evidence supporting this view is drawn from lower organ-isms as well as mammals. 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Eng. 37:133. -82-APPENDIX I Krebs-Ringer Medium for Oocyte Culture Constituents 6ram/Litre NaCI 6.96 Na Pyruvate .028 KC1 .356 CaCl 2 .189 KH2P04 .162 MgS04 * 7H20 .294 NaHC03 2.106 Pen/Strep 100U/ml/50ugm/ml Bovine Serum Albumin 1.00 Phenol Red .01 Medium for equil ibrating paraffin oi l is made as above but without pyruvate or albumin. 


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