Long-term effects of delayed motherhood in mice on

Human Reproduction Vol.18, No.8 pp. 1580±1587, 2003

DOI: 10.1093/humrep/deg349

Long-term effects of delayed motherhood in mice on postnatal development and behavioural traits of offspring Juan J.TarõÂn1,6, Vanessa GoÂmez-Piquer1, Carmen Manzanedo2, JoseÂ MinÄarro3, Carlos Hermenegildo4 and Antonio Cano5 1

Department of Functional Biology and Physical Anthropology, Faculty of Biological Sciences, University of Valencia, Burjassot, 46100 Valencia, 2Department of Psychobiology, Faculty of Psychology, Complutense University of Madrid, 28223 Madrid, 3 Area of Psychobiology, Faculty of Psychology, University of Valencia, 46071 Valencia, 4Research Unit, Hospital ClõÂnico de Valencia and Department of Physiology, University of Valencia, 46010 Valencia, and 5Department of Pediatrics, Obstetrics and Gynecology, Faculty of Medicine, University of Valencia, 46010 Valencia, Spain 6

To whom correspondence should be addressed at: Department of Pediatrics, Obstetrics and Gynecology, Faculty of Medicine, University of Valencia, Avda. Blasco IbanÄez 17, 46010 Valencia, Spain. E-mail: [email protected]

Key words: ageing/behaviour/gamete biology/mouse model/ovum

Introduction Although some authors claim that the risks of delayed motherhood are overstated (Ales et al., 1990; Berkowitz et al., 1990; Antinori et al., 1995; Bowman et al., 1995) or even that women who live to >100 years are four times more likely to have had children while in their forties than women who survive only to age 73 years (Perls et al., 1997), overwhelming evidence shows that delayed motherhood is not only associated with infertility and obstetrical problems but also with fetal and perinatal morbidity and mortality (TarõÂn et al., 1998; Tough et al., 2001, 2002; Abel et al., 2002; Alonzo, 2002; Astol® and Zonta, 2002; de La Rochebrochard and Thonneau, 2002; Seoud et al., 2002). In contrast, information about the potential negative long-term effects of delayed motherhood on offspring is scarce or fragmentary. In human beings, it has been reported that advanced maternal age at conception is associated with decreased percentage of male offspring and higher probability of offspring suffering from trisomy, infertility (TarõÂn et al., 2000) and mitochondrial DNA 1580

diseases including congenital sensorial hearing loss, cerebellar ataxia, type I (insulin-dependent) diabetes mellitus and Alzheimer's disease (TarõÂn et al., 1998). On the other hand, whereas epidemiological evidence suggests that children born to older parents (both mothers and fathers) have lower intellectual development and intellectual maturity than children whose parents are younger (Roberts and Engel, 1974) and exhibit minor neurodevelopmental disorders including ®ne-motor problems, visual±perceptual dysfunction and attentional de®cit signs (Gillberg, 1982), other epidemiological studies (Auroux et al., 1989) show that advanced maternal age, unlike the male counterpart, is not associated with decreased mental function of progeny. The lack of effect of maternal age at conception on cerebral performance of offspring has also been observed in rats during early adulthood (Vorhees, 1988). However, during the preweaning period, rats from old dams appear to move more effectively and orient towards their home cage scent slightly better than offspring from younger dams (Vorhees, 1988). ã European Society of Human Reproduction and Embryology

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BACKGROUND: Some epidemiological evidence tentatively suggests that children born to older parents may have lower intellectual development and maturity than children whose parents are younger. This study aims to analyse the long-term effects of delayed motherhood in mice on postnatal development and behavioural traits later in life. METHODS: Hybrid females, either at the age of 10 weeks or 51 weeks, were individually housed with a randomly selected 12±14 week old hybrid male. After a postweaning resting period of 1 week, dams were caged again with a new randomly selected 12±14 week old male. This sequence of events was repeated until old females reached the end of their reproductive life. RESULTS: Delayed motherhood in mice not only had negative effects on reproductive potential but also on preweaning development of offspring as evidenced by higher mortality, retarded sensorimotor integration and lower body weights as well as on behavioural traits of young adult offspring including decreased spontaneous motor activity, lower step-through latencies in the retention trial of a passive avoidance behaviour test, and no changes in escape latencies throughout ®ve daily sessions in a Morris water maze test. CONCLUSION: Advanced maternal age at conception may in¯uence preweaning development and learning capacity of offspring in the mouse model.

Long-term effects of maternal age on offspring

The purpose of the present study is to analyse, in the mouse, the long-term effects of delayed motherhood on postnatal development and behavioural traits of young adult offspring including spontaneous motor activity and learning capacity, tested in a Morris water maze, a Y-maze and a passive avoidance behaviour test. Materials and methods

Housing and birth of offspring As soon as F0 females displayed visual signals of pregnancy, females were examined once a day until parturition. Within the ®rst 24 h after parturition, litter size and sex of pups were recorded. Pups were sexed by means of the ano-genital distance, which is longer in males; this was con®rmed in later examinations during preweaning development. On postnatal day 3, litter size and sex ratio of pups from young females were matched with litter size and sex ratio of pups from aged females. Whenever possible, young mother litters were matched with those old mother litters exhibiting similar litter size and sex ratio of pups. If a particular young mother litter had surplus pups, selection of pups for matching was randomly performed. The surplus young mother pups were killed by decapitation. First generation (F1) mice were weighed within the ®rst 24 h after parturition and on postnatal days 3, 10 and 21 (at weaning). Furthermore, they were weighed just before the onset of each one of the developmental and behaviour tests applied (see below). Each animal was marked by labelling its skin with a silver nitrate±diamant

Preweaning development of offspring The righting re¯ex test was performed on postnatal days 3 to 12 between 0900 and 1030 h in all F1 pups. The righting response was de®ned as the time it took a pup that had been placed on its back to turn over and place the four paws on a solid surface. An upper limit of 180 s was set for this test. This test of sensorimotor integration was performed daily until pups righted themselves immediately (although mice took 0.05) (marginal means 6 SEM).

in litter 3), sex of offspring (P < 0.008; 34.1 6 5.0% in females versus 38.3 6 5.8% in males), interaction between litter number and the co-variate postnatal day of attaining immediate righting (P < 0.019) and interaction between sex of offspring and the co-variate body weight of offspring just before being tested in the Morris water maze (P < 0.021) had a signi®cant effect on percentage of occasions that offspring reached the hidden platform. Figure 2B shows the escape latencies, i.e. the times required for mice to ®nd and climb onto the platform, of only those young and old mother offspring that found and climbed onto the platform (young mother group: 19 males and 17 females, 22 males and 19 females, 24 males and 23 females, 26 males and 23 females, and 20 males and 27 females on days 1, 2, 3, 4 and 5 respectively; old mother group: 18 males and 15 females, 21 males and 21 females, 24 males and 20 females, 28 males and 25 females, and 25 males and 26 females in day 1, 2, 3, 4 and 5 respectively). Age group had a signi®cant (P < 0.002) effect on escape latencies in that mice from the young mother group exhibited a decrease in escape latencies during the 5 consecutive days of testing (slope 6 SEM: ±2.343 6 0.508, P < 0.019), whereas old mother offspring did not display any reduction in 1584

Simple discrimination learning test Figure 3 shows the percentage of occasions that offspring from reproductively young and old dams entered the correct choice arm of a wooden black Y-maze when tested during ®ve daily sessions of 10 trials each. The probability of entering into the correct arm during the ®ve training sessions was not signi®cantly different between young and old mother offspring. The only signi®cant factors were litter number (P < 0.020; 55.3 6 1.8% in litter 1, 57.0 6 2.0% in litter 2 and 51.0 6 3.3% in litter 3), sex of offspring (P < 0.005; 46.9 6 2.2% in females versus 62.0 6 2.7% in males), interaction between litter number and sex of offspring (P < 0.003), interaction between litter number and the co-variate body weight of offspring just before being put on diet (P < 0.029) and interaction between sex of offspring and the co-variate postnatal day of attaining immediate righting (P < 0.018). Passive avoidance behaviour test Delayed motherhood had no signi®cant effect on percentage of offspring that remembered the electric discharge given in the acquisition trial and did not enter the dark compartment during the retention trial (42.4% in the young mother group versus 53.7% in the old mother group). However, litter number (P < 0.0005; 67.2% in litter 1, 31.5% in litter 2 and 38.1% in litter 3) and sex of offspring (P < 0.001; 62.3% in females versus 32.8% in males) were signi®cant factors. As the percentage of offspring that remembered the punishment in¯icted in the acquisition trial and did not enter the dark compartment during the retention trial was not affected by age group, a further statistical analysis was performed using only those mice that entered the dark compartment in the retention trial (24 males and 14 females in the young mother group, and 19 males and 12 females in the old mother group). The analysis


escape latencies during the same period of time (slope 6 SEM: 0.022 6 0.865, P > 0.05). Other signi®cant factors were litter number (P < 0.0005; 22.4 6 0.5 s in litter 1, 24.4 6 0.5 s in litter 2 and 24.5 6 0.5 s in litter 3) and interaction between age group and litter number (P < 0.030).

Long-term effects of maternal age on offspring

Figure 4. Effect of age group on counts of spontaneous motor activity of offspring during a session of 40 min divided into eight blocks of 5 min each (marginal means 6 SEM)

Spontaneous motor activity of offspring Figure 4 exhibits the spontaneous motor activity, measured in an actimeter, of young and old mother offspring. Old mother offspring displayed a signi®cantly (P < 0.047) lower motor activity than the young mother counterparts. Litter number (P < 0.002; 350.3 6 14.1 counts in litter 1, 420.3 6 15.6 counts in litter 2 and 387.3 6 25.5 counts in litter 3) and interaction between litter number and body weight of offspring just before being tested in the actimeter (P < 0.001) were also signi®cant factors. No signi®cant effect of age group on number of pellets defecated (11.8 6 0.7 pellets in the old mother group versus 10.4 6 0.7 pellets in the young mother group) was found. In contrast, litter number (P < 0.03; 10.4 6 0.7 in litter 1, 12.8 6 0.7 in litter 2 and 10.1 6 1.2 in litter 3) and the co-variate body weight of offspring just before being tested in the actimeter (P < 0.001; slope 6 SEM: 20.146 6 5.787, P < 0.001) were signi®cant factors. Discussion The present study shows that delayed motherhood in the mouse not only has negative effects on female reproductive performance but also affects: (i) postnatal development of offspring as

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showed that time to enter the dark compartment was signi®cantly (P < 0.011) lower in the old mother group than that exhibited by the young mother counterparts (estimated marginal means 6 SEM: 45.1 6 1.1 s versus 162.4 6 0.6 s). Likewise, litter number (P < 0.008; estimated marginal means 6 SEM: 130.8 6 0.7 s in litter 1, 76.4 6 0.2 s in litter 2 and 81.3 6 1.2 s in litter 3), sex of offspring (P < 0.026; estimated marginal means 6 SEM: 104.8 6 0.2 s in females versus 85.1 6 0.2 s in males), the co-variate litter size at birth (P < 0.009; slopes 6 SEM: ±0.998 6 0.350, P < 0.009), interaction between litter number and the co-variate postnatal day of attaining immediate righting (P < 0.022), and interaction between sex of offspring and the co-variate time of day at which the passive avoidance test was performed (P < 0.008) were signi®cant factors.

evidenced by higher mortality, retarded sensorimotor integration and lower body weights during the preweaning period; and (ii) behavioural traits of offspring including decreased spontaneous motor activity and lower step-through latencies in the retention trial of a passive avoidance behaviour test, while there were no changes in escape latencies throughout ®ve daily sessions in a Morris water maze test. These results agree with previous studies in rats (Vorhees, 1988), mice (Albert et al., 1965) and human beings (TarõÂn et al., 1998; Alonzo, 2002; de La Rochebrochard and Thonneau, 2002) showing that advanced maternal age at conception increases perinatal and preweaning mortality of offspring. Likewise, our data agree with other studies in mice showing decreased body weight of offspring (Albert et al., 1965; Wang and vom Saal, 2000) although Vorhees (1988) found in the rat that advanced maternal age at conception is associated with increased body weight of progeny during preweaning development. Although downstream oocyte effects could be the primary cause, it appears that the environment provided by the uterus of reproductively old females is a major factor in this event. In fact, it has been reported (Wang and vom Saal, 2000) that grown offspring of middle-aged CF-1 female mice have lower body weights than those produced by young adult dams despite pups being reared by young adult foster mothers. We cannot discard, however, possible differences in feeding and nutrition of pups between young and aged female mice. The negative effects of delayed motherhood on behavioural traits of young adult offspring observed in the present study support previous epidemiological evidence suggesting that children born to older parents (both mothers and fathers) have lower intellectual development and intellectual maturity (Roberts and Engel, 1974) and exhibit minor neurodevelopmental disorders including ®ne-motor problems, visual± perceptual dysfunction and attentional de®cit signs (Gillberg, 1982). However, they disagree with the lack of effect of maternal age at conception on mental function reported by both Auroux et al. (1989) in human beings and by Vorhees (1988) in rats. We have to bear in mind, however, that the differences between young and old mother offspring in behaviour and cognitive function found in the present study are very subtle, and they may have not been detected if appropriate variables had not been analysed. For instance, preliminary analysis of our data showed no signi®cant effects of maternal age at conception on offspring's probability of: (i) reaching the platform of a Morris water maze; (ii) entering into the correct arm of a Y-maze; and (iii) entering into the dark compartment during the retention trial of a passive avoidance behaviour test. These results may have led us, therefore, to conclude that advanced maternal age at conception has no effect on learning capacity of offspring. However, further analysis of data showed that old mother offspring exhibited no changes in escape latencies throughout ®ve daily sessions in a Morris water maze test and lower step-through latencies in the retention trial of a passive avoidance behaviour test. Although the outcome obtained in the Morris water maze may be a random effect induced by the fact that mice did not perform any pretraining to be familiarized with the maze before starting the ®ve training sessions (note that the

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`imprint' the function of fetal cells in reproductive organs, the brain and many other tissues (Wang and vom Saal, 2000). Alternatively, although maternal ageing primarily induces numerical chromosomal aberrations in the female gamete, differences in behavioural traits between young and old mother offspring may be induced by age-associated accumulation of mutations in nuclear DNA of oocytes while arrested in meiosis I after meiotic DNA replication. Several experimental studies support this notion. For instance, it has been shown in transgenic mice that advanced maternal age is associated with an increase in frequency and magnitude of intergenerational instability of nucleotide triplet repeats in nuclear DNA (Kaytor et al., 1997). Furthermore, there is compelling evidence supporting a role for DNA mutations in mental function. For instance, if experimental mutations in the male rat are induced by using mutagenic antimitotics such as cyclophosphamide, the offspring derived from these rats display diminished learning capacity and spontaneous activity (Auroux et al., 1990b) associated with a degradation of some biochemical substrates of memory (Auroux et al., 1990a). Acknowledgement This study was supported by grant FIS 01/0138 from `Instituto de Salud Carlos III, Fondo de InvestigacioÂn Sanitaria, Ministerio de Sanidad y Consumo'.

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performance of old mother offspring was much better on the ®rst day of training when compared with the young mother group), results from the passive avoidance behaviour test suggest that advanced maternal age at conception may be associated with decreased learning capacity of offspring. If the potential negative long-term effects of delayed motherhood on offspring have not been investigated very often, studies aimed to ascertain the molecular, biochemical and cellular mechanisms involved in age effects are still scarcer. We (TarõÂn, 1995; 1996; TarõÂn et al., 1998) and another group (Arbuzova, 1995; Arbuzova et al., 2002) have proposed independently a mechanism based on the `the oxygen radical± mitochondrial injury hypothesis of ageing' (Miquel et al., 1980) to explain the negative long-term effects of advanced maternal age on offspring. Although one research laboratory (Brenner et al., 1998; Barritt et al., 1999) has not found a correlation between woman's age and mtDNA rearrangements in oocytes, our hypothesis is supported by the fact that several independent laboratories have reported an increased rate of mtDNA deletions in ovarian tissue (Kitagawa et al., 1993), oocytes (Keefe et al., 1995) and luteinized granulosa cells from older and/or postmenopausal women when compared with ovaries/oocytes from younger women. In addition, preovulatory oocytes from middle-aged women show increased mitochondrial number, mitochondrial volume ratio and mitochondrial pro®le area suggesting, therefore, subtle but generalized changes in the oxidative phosphorylation capacity (MuÈller-HoÈcker et al., 1996). These changes have been recently con®rmed by Wilding et al. (2001), which detected the presence of a negative correlation between the activity of mitochondria in fresh human metaphase II oocytes and maternal age. We should note, however, that the occurrence of maternal age-induced oxidative damage to oocyte mtDNA is not the only mechanism capable of explaining the present results. Ageing of females before conception may affect many molecular, biochemical and cellular pathways in oocytes that may jeopardize not only pre- and postimplantation embryo/ fetus development but also later life of offspring. It is known, for instance, that maternal ageing in mammals, in addition to inducing an increase in incidence of oocytes displaying a reduction of pole-to-pole distance of the metaphase II spindle, c-meiosis (colchicine-meiosis: anomalies in chromosomal distribution similar to those induced by treatment with colchicine) and aneuploidy (TarõÂn et al., 2001, 2002; for review, see TarõÂn, 1996), it is associated with shortened prophase I stage, decreased ability of oocytes to mature and extrude a polar body both in vivo and in vitro (Peluso et al., 1980; Hewitt and England, 1998; Wu et al., 2000; for reviews, see TarõÂn, 1996; Eichenlaub-Ritter et al., 1998), decreased glucose-6-phosphate dehydrogenase activity (de Schepper et al., 1987) and presence of apoptotic traits including DNA fragmentation (Fujino et al., 1996; Lopes et al., 1998; Wu et al., 2000), mitochondrial aggregation, degeneration of the entire granulosa wall leaving the oocyte completely denuded of cumulus cells and shrinkage of the oocyte (TarõÂn et al., 2001; 2002). Likewise, maternal age-related changes in testosterone and estradiol levels in the mouse uterus may permanently

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