Saturday, October 18, 2008

Long-term effects of delayed fatherhood in mice on postnatal development and behavioral traits of offspring1

Long-term effects of delayed fatherhood in mice on postnatal
development and behavioral traits of offspring1
Short title: Long-term effects of paternal age on offspring
Summary sentence: Delayed fatherhood has long-term effects on preweaning development,
spontaneous motor activity and passive-avoidance learning capacity of offspring in the mouse
Key words: delayed fatherhood long-term effects offspring
Silvia García-Palomares3, José F. Pertusa3, José Miñarro4, Miguel A. García-Pérez5,6,
Carlos Hermenegildo5,7, Francisco Rausell3, Antonio Cano8 and Juan J. Tarín2,3
1Supported by grant BFI2003-04761 from “Ministerio de Ciencia y Tecnología”, cofinanced by
the “Fondo Europeo de Desarrollo Regional (FEDER); grant ISCIII2006-PI0405 from “Instituto
de Salud Carlos III, Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo”,
cofinanced by the FEDER; and grants GV2004-B-206 and AE/2007/001 from “Generalitat
Valenciana, Conselleria d’Émpresa, Universitat i Ciencia”.
2Correspondence: Juan J. Tarín, Department of Functional Biology and Physical Anthropology,
Faculty of Biological Sciences, University of Valencia, Dr. Moliner 50, 46100 Burjassot,
Valencia, Spain; Tel. 34-96-354 3221; E-mail:
3Department of Functional Biology and Physical Anthropology, Faculty of Biological Sciences,
University of Valencia, Burjassot, 46100 Valencia, Spain
BOR Papers in Press. Published on October 15, 2008 as DOI:10.1095/biolreprod.108.072066
Copyright 2008 by The Society for the Study of Reproduction.
García-Palomares et al.
4Department of Psychobiology, Faculty of Psychology, University of Valencia, 46071 Valencia,
5Research Unit, Hospital Clínico de Valencia, 46010 Valencia, Spain
6Department of Genetics, University of Valencia, 46100 Valencia, Spain
7Department of Physiology, University of Valencia, 46010 Valencia, Spain
8Department of Pediatrics, Obstetrics and Gynecology, Faculty of Medicine, University of
Valencia, 46010 Valencia, Spain
García-Palomares et al.
This study aims to analyze, in mice, the long-term effects of delayed fatherhood on
postnatal development, spontaneous motor activity and learning 5 capacity of offspring. Hybrid
parental-generation (F0) males, at the age of 12, 70, 100 and 120 weeks, were individually housed
with a randomly-selected 12 week-old hybrid female. The resulting first-generation (F1)
offspring were tested for several developmental and behavioral variables. Cumulative percentage
of F1 pups that attained immediate righting in the 120-week group was lower than that found in
10 the 12-, 70- and 100-week groups. Furthermore, the postnatal day of attaining immediate
righting was higher in pups from the 120-week group when compared to pups from the other age
groups. At the age of 20 weeks, F1 offspring from the 120-week group displayed lower counts of
motor activity than offspring from the 12-, 70- and 100-week groups. One week later, a higher
percentage of offspring from the 100- and 120- groups entered the dark compartment during the
15 retention trial of the passive avoidance test when compared to offspring from the 12-week group.
Offspring from the 120-week group exhibited also lower step-through latency in the retention
trial than offspring from the 12-, 70- and 100-week groups. These results show that advanced
paternal age at conception has long-term effects on preweaning development, spontaneous motor
activity and reduced passive-avoidance learning capacity of mouse offspring.
García-Palomares et al.
The present trend of couples from most Western countries to pursue educational and
professional goals before conceiving is increasingly forcing them 25 to postpone parenthood until
the mid-thirties or even beyond [1], i.e. the latter part of a woman’s childbearing years.
Furthermore, the wish of parenthood in a new partnership is increasing due to the changing
patterns of marriage and divorce that are taking place in the Western society. Although male
reproductive functions do not cease at middle age such as it occurs in women, it is known that
30 men > 40 years contribute to reduced fertility and fecundity of a couple [2]. In addition, delayed
fatherhood is associated with increased risks of conceiving a child suffering from dominant or Xlinked
recessive diseases, non-cytogenetic congenital defects, athetoid/dystonic cerebral palsy,
cogenital hemiplegia, multiple sclerosis [3], schizophrenia [4-5], autism [6], epilepsy [7],
decreased learning capacity [8] and/or mental retardation of unknown etiology [2, 9-12].
35 Advanced paternal age has been also related with increased risks of spontaneous abortion [13-
14], fetal loss [13-15], preeclampsia [16], stillbirth [17], neonatal mortality [15] and childhood
cancers [10, 14, 18-19]. In contrast, there is no clear evidence for the existence of a negative
paternal effect on preterm delivery and incidence of low birth weight [2, 14].
The purpose of the present study is to analyze, in the mouse, the long-term effects of
40 delayed fatherhood on postnatal development and behavioral traits of offspring including
spontaneous motor activity and learning capacity using a passive avoidance behavior test.
García-Palomares et al.
Mouse strain, housing and pairings of parental generation (F0) males
All the animal experiments performed in this study 45 were conducted in accordance with
the National Research Council’s (NRC) publication Guide for the Care and Use of Laboratory
Animals [20]. On postnatal day 21 (at weaning), 4 cohorts of 20, 20, 44 and 70 hybrid
(C57BL/6JIco female X CBA/JIco male) virgin F0 males (Criffa, Barcelona, Spain) were
randomly selected for the study. These mice were housed in groups of 10 in 35.5 x 23.5 x 18.5
50 cm plastic cages, fed a standard laboratory diet and tap water ad libitum and maintained on a
14L:10D photoperiod (lights-on at 0800 h) in a temperature-controlled room at 21 to 23ºC until
the age of 12, 70, 100 or 120 weeks. At this time, those males that were still alive (20, 20, 30 and
8, respectively) were individually housed in 26.5 x 20.5 x 13.5 cm plastic cages with a randomlyselected
12-week-old hybrid F0 female to produce a single litter of first-generation (F1) mice.
Birth and housing of F1 offspring
From day 10 after adding the F0 female 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
(SGP) was assured a female was pregnant (20, 20, 20 and 5 females in the 12-, 70-, 100- and 120-
60 week group, respectively), the male was removed and the female allowed to give birth and breastfeed
her pups until weaning. Within the first 24 h after parturition, litter size and gender of F1
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. Pups were
weighed within the first 24 h after parturition, on postnatal days 3, 10 and 21 (at weaning) and
65 just before the onset of the developmental and behavior tests applied (see below). Each animal
was marked by labeling its skin with a silver nitrate-diamant fuchsin stain and water-proof feltGarcía-
Palomares et al.
tipped pen before weaning and by ear punching/cutting after weaning. At weaning, male and
female F1 offspring were separated and housed in groups of 10 in 35.5 x 23.5 x 18.5 cm plastic
cages with wood shavings as bedding. Bedding was changed weekly. Offspring were fed the
same diet and housed under the same light:dark cycle and temperature 70 conditions as their parents.
Righting reflex of F1 offspring
The righting reflex test was performed on postnatal days 3 to 10 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
75 on its back to turn over to restore their normal prone position. An upper limit of 180 sec was set
for this test. This test of sensorimotor integration was performed daily until pups righted
themselves immediately (although mice took < 1 sec to right themselves, righting latencies in
these cases were recorded as zero) when placed on the supine position [21].
80 Spontaneous motor activity of F1 offspring
At the age of 20 weeks (just 1 week before performing the passive avoidance behavior
test), the spontaneous motor activity of F1 offspring was measured in a computer-controlled
actimetre (Actisystem II, Panlab S.L., Barcelona, Spain). The actimetre consisted of four 35 x 35
cm sensory plates, which registered any activity of the animals through an electromagnetic
85 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 0700 and 0900 h. In
each session, any motility of animals, resulting or not in a displacement was registered at
intervals of 5 min during a period of 60 min. After each session, the actimetre was cleaned with
water and the number of defecations scored as a measure of emotionality [21].
García-Palomares et al.
Passive avoidance behavior of F1 offspring
At the age of 21 weeks, F1 offspring were tested for passive avoidance behavior as
previously described [21]. This test was chosen because it fits the learning abilities of F1
offspring better than other tests such as the spatial-learning Morris water maze and the simplediscrimination-
learning Y-maze [21]. The experimental apparatus used 95 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 sec the door automatically opened and the time for a mouse to enter the
100 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
sec. 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 0700 and 0900 h), the
mouse was again placed in the illuminated compartment, but no electrical shock was
105 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
110 Statistical analysis
Fixed-effects (models with only fixed effects, covariate and the residual term) designs of
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
García-Palomares et al.
potential correlation among observations within a particular litter 115 (littermates) and avoid spurious
inflation of the sample size [22]. Several covariates were introduced in the statistical analyses to
control for possible confounders not included in the study design, e.g. day 3-10 body weight
(mean body weight of measures taken on postnatal days 3 and 10) and time at which mice
underwent the spontaneous motor activity session or the passive avoidance behavior test.
120 Kolmogorov-Smirnov one-sample test was used to check whether variables were normally
distributed. If the normality assumption was violated, logarithmic or square root (if variables
were metric), or arcsine square root (if data were percentages) transformation of the variable was
applied to induce normality. Bonferroni test (when the variances were assumed to be equal) or
Dunnett’s T3 test (when the variances were assumed to be unequal) were applied to perform post
125 hoc pairwise multiple comparisons between groups. Levene test was used to test the
homogeneity of variance for each dependent variable across all level combinations of the
between-subjects factors. Automated binomial logistic regression analysis with forward stepwise
variable selection was used to ascertain the effect of paternal



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