1 Department of Functional Biology and Physical Anthropology, Faculty of Biological Sciences, University of Valencia, Burjassot, 46100 Valencia, 2 Department of Psychobiology, Faculty of Psychology, Complutense University of Madrid, 28223 Madrid, 3 Area of Psychobiology, Faculty of Psychology, University of Valencia, 46071 Valencia, 4 Research Unit, Hospital Clínico de Valencia and Department of Physiology, University of Valencia, 46010 Valencia, and 5 Department 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 Ibañez 17, 46010 Valencia, Spain. e-mail: tarinjj{at}uv.es
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Abstract |
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Key words: ageing/behaviour/gamete biology/mouse model/ovum
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Introduction |
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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.
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Materials and methods |
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Females were housed with a 1214 week old male (only one female per male) using an intermittent schedule. In particular, from day 10 after adding the male to the cage, females were examined once a day for physical evidence of pregnancy, i.e. the presence of a distended abdomen. When the researcher (V.G.P.) was assured a female was pregnant, the male was removed and the female allowed to give birth and breast-feed her pups until weaning. When the first litter was weaned, females were left alone in the cage for another week. After this resting period, females were caged again with a new randomly selected 1214 week old hybrid male. This sequence of events was repeated until females from the old mother group reached the end of their reproductive life (3 months of infertile co-habitation). At this time, each co-habitating male was housed with a young female of 1012 weeks of age to confirm his fertile condition.
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 first 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 confirmed 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 first 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 nitratediamant fuchsin stain and waterproof felt-tipped pen before weaning and by ear punching/cutting after weaning. At weaning, male and female offspring were separated and housed in groups of 10 in 35.5x23.5x 18.5 cm plastic cages. Offspring were fed the same diet and housed under the same light:dark cycle and temperature conditions as their parents.
Preweaning development of offspring
The righting reflex test was performed on postnatal days 3 to 12 between 0900 and 1030 h in all F1 pups. The righting response was defined 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 <1 s to right themselves; righting latencies in these cases were recorded as zero) when placed on their backs (Tarín et al., 1999).
Learning ability of offspring
Spatial learning test
At the age of 1214 weeks, one randomly selected F1 male mouse and female mouse (if available) from the first (14 males and 16 females from each group), second (15 males and 13 females from each group) and third (5 males and 6 females from each group) litters were tested between 1130 and 1500 h in a Morris water maze, following a modified method previously published (Xu et al., 2001). Briefly, the maze consisted of a plastic circular pool (104 cm in diameter and 30 cm in height) partially filled with water (2325°C) with black walls and white floor. The pool was divided into four quadrants called northeast (NE), northwest (NW), southeast (SE) and southwest (SW). A clear plastic escape platform (6x6 cm side to side and 13 cm in height) was submerged 2 cm below the surface and placed in the centre of the NW quadrant. Distal visual cues were kept unchanged throughout the testing period. Mice were given four trials per day to find the hidden platform for 5 consecutive days. Mice were gently placed into the water with the nose pointing toward the wall at one of the four quadrants, which varied from trial to trial. Thereafter, the escape latency, i.e. the time required for the mouse to find and climb onto the platform, was recorded for up to 60 s. Each mouse was allowed to remain on the platform for 30 s, and then removed from the maze to its home cage. If the mouse did not find the platform within 60 s, the mouse was manually placed on the platform and returned to its home cage after a period of 30 s. An inter-trial interval of 30 s was used.
Simple discrimination learning test
At the age of 2628 weeks, one randomly selected F1 male mouse and one female mouse from the first (14 males and 16 females from each group), second (14 males and 13 females from each group) and third (5 males and 6 females mice from each group) litters (the same mice that were used in the Morris water maze test) were tested between 1100 and 1425 h in a wooden black Y-maze, which had three arms of equal size (60 cm long, 11.5 cm wide and 25 cm high) as previously described (Aguilar et al., 2000). The arm where the mice were placed at the beginning of each trial was considered the starting arm. The other arms, each of which had a food cup located at the end, were considered the choice arms. Pretraining was carried out for 3 days to familiarize the mice with the maze. During these pretraining days, mice were individually placed for 5 min in the maze with 45 mg food pellets (Dustless Precision Pellets; BIO-SERV, USA) scattered throughout the three arms (on day 1), at the end of both choice arms (on day 2), and throughout one of the two choice arms (on day 3). This last arm was the arm in which the food pellets were always placed within the food cup during the following training sessions performed on 5 consecutive days. Four days before starting the pretraining sessions, animals were dieted so that their body weight during the pretraining and training sessions was
80% (82.6 ± 0.3%) of their free-feeding body weight that they displayed just before being put on diet. This was done to increase the appetite of mice and improve their discrimination learning capacity. During the training sessions, the reward for the correct response, i.e. the mouse entered the arm with food pellets within the food cup, consisted of one food pellet. If the mouse made an incorrect response, i.e. entered the choice arm lacking food pellets, it was allowed to go to the empty food cup at the end of the arm, but it was removed from the Y-maze 3 s after reaching the empty food cup. Mice were trained for 10 trials per day, staggered with inter-trial intervals of 30 s in their home cage.
Passive avoidance behaviour test
At the age of 3537 weeks, one randomly selected F1 male mouse and one female mouse from the first (13 males and 16 females from each group), second (14 males and 12 females in the young mother group and 15 males, and 13 females in the old mother group) and third (5 males and 6 females in the young mother group, and 5 males and 6 females in the old mother group) litters (the same mice that were used in the Morris water maze and simple discrimination learning tests) were tested for passive avoidance behaviour as previously described (Tarín et al., 1999). The experimental apparatus used on this occasion, however, was a two section box in which the walls of one section were black and those of the other section white and illuminated with a lamp (60 W). The two sections were separated by an automatic door. In the acquisition trial, each mouse was placed in the illuminated compartment, facing the dark section with the door closed. After 60 s the door automatically opened and the time for a mouse to enter the dark compartment was registered. As soon as the mouse entered the dark compartment, the door automatically closed and an electrical foot-shock (0.3 mA) was delivered during a period of 5 s. Immediately after this shock, the animal was returned to its home cage. In the retention trial, which was performed exactly 24 h after the acquisition trial (between 1030 and 1130 h), the mouse was again placed in the illuminated compartment, but no electrical shock was administered if it entered the dark section. In both the acquisition and the retention trial, the time to enter the dark compartment was recorded as step-through latency. The maximum step-through latency allowed when the mouse did not enter the dark compartment in the retention trial was 300 s.
Spontaneous motor activity of offspring
At the age of 2426 weeks, just 2 weeks before performing the simple discrimination learning test, the spontaneous motor activity of one F1 male mouse and one female mouse from the first (14 males and 16 females from each group), second (14 males and 13 females in the young mother group, and 15 males and 13 females in the old mother group) and third (5 males and 6 females from each group) litters (the same mice that were used in the Morris water maze test) was measured in a computer-controlled actimeter (Actisystem II, Panlab S.L., Spain). The actimeter consisted of four 35x35 cm sensory plates, which registered any activity of the animals through an electromagnetic system, an interface, and a computer that allowed the acquisition and storage of data from the sensory plates. Mice underwent a single motor-activity session between 0815 and 0915 h. Each session began with an adaptation period of 20 min followed by a monitoring period of 40 min. During this period of time, any motility of animals, resulting or not in a displacement, was registered at intervals of 5 min. After each session, the actimeter was cleaned with water and the number of defaecations scored (Tarín et al., 1999).
Statistical analysis
Fixed-effects (models with only fixed effects, co-variate and the residual term) designs of analysis of variance (ANOVA), mixed-effects (some effects are random and some are fixed) nested designs of ANOVA, and repeated measures nested designs of ANOVA with two-way interactions between variables were applied for comparisons of means. Nested designs were applied to control the potential correlation between observations within a particular litter (littermates) and avoid spurious inflation of the sample size (Wainwright, 1997). KolmogorovSmirnov one-sample test was used to check whether variables were normally distributed. If the normality assumption was violated, logarithmic or square root transformation of the variable was applied to induce normality. The effect of age group on percentage of litters with at least one pup cannibalized at birth and percentage of preweaning deaths was determined using hierarchical log-linear models with backward step-wise variable selection. Differences in sex ratios (% males) at birth between groups were tested using logistic regression analysis with forward step-wise variable selection. One-sample binomial test was utilized to test the null hypothesis that the probability of being male in each group was 0.5. Logistic regression analysis with forward step-wise variable selection was also used to ascertain the effect of delayed motherhood on percentage of offspring that did not enter the dark compartment in the retention trial of the passive avoidance behaviour test. Significance was defined as P < 0.05. The statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS Inc., USA). Whenever possible, for brevity, only significant findings are described in the results section.
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Results |
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On the other hand, body weight of pups on days 10 and 21 was significantly (P < 0.006) lower in the old mother group when compared with the young mother counterparts (Figure 1). In addition, body weight of pups on days 1, 10 and 21 was affected by litter number (P < 0.0005; 4.3 ± 0.5 g in litter 1, 4.3 ± 0.5 g in litter 2 and 4.7 ± 0.5 g in litter 3), sex of offspring (P < 0.0005; 4.3 ± 0.5 in females versus 4.4 ± 0.5 in males), interaction between age group and litter number (P < 0.0005), and interaction between age group and the co-variate litter size at birth (P < 0.0005).
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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 five daily sessions of 10 trials each. The probability of entering into the correct arm during the five training sessions was not significantly different between young and old mother offspring. The only significant factors were litter number (P < 0.020; 55.3 ± 1.8% in litter 1, 57.0 ± 2.0% in litter 2 and 51.0 ± 3.3% in litter 3), sex of offspring (P < 0.005; 46.9 ± 2.2% in females versus 62.0 ± 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).
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As the percentage of offspring that remembered the punishment inflicted 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 showed that time to enter the dark compartment was significantly (P < 0.011) lower in the old mother group than that exhibited by the young mother counterparts (estimated marginal means ± SEM: 45.1 ± 1.1 s versus 162.4 ± 0.6 s). Likewise, litter number (P < 0.008; estimated marginal means ± SEM: 130.8 ± 0.7 s in litter 1, 76.4 ± 0.2 s in litter 2 and 81.3 ± 1.2 s in litter 3), sex of offspring (P < 0.026; estimated marginal means ± SEM: 104.8 ± 0.2 s in females versus 85.1 ± 0.2 s in males), the co-variate litter size at birth (P < 0.009; slopes ± SEM: 0.998 ± 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 significant factors.
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 significantly (P < 0.047) lower motor activity than the young mother counterparts. Litter number (P < 0.002; 350.3 ± 14.1 counts in litter 1, 420.3 ± 15.6 counts in litter 2 and 387.3 ± 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 significant factors.
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Discussion |
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These results agree with previous studies in rats (Vorhees, 1988), mice (Albert et al., 1965
) and human beings (Tarín 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 fine-motor problems, visualperceptual dysfunction and attentional deficit 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 significant effects of maternal age at conception on offsprings 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 five 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 five training sessions (note that the performance of old mother offspring was much better on the first 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 (Tarín, 1995; 1996; Tarín et al., 1998
) and another group (Arbuzova, 1995
; Arbuzova et al., 2002
) have proposed independently a mechanism based on the the oxygen radicalmitochondrial 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 womans 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 profile area suggesting, therefore, subtle but generalized changes in the oxidative phosphorylation capacity (Müller-Höcker et al., 1996
). These changes have been recently confirmed 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 (Tarín et al., 2001, 2002; for review, see Tarín, 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 Tarín, 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 (Tarín et al., 2001
; 2002). Likewise, maternal age-related changes in testosterone and estradiol levels in the mouse uterus may permanently 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
).
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Acknowledgement |
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Submitted on March 11, 2003; accepted on May 7, 2003.