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Endocrinology Vol. 147, No. 4 1916-1923
Copyright © 2006 by The Endocrine Society

Circadian Clock Mutation in Dams Disrupts Nursing Behavior and Growth of Pups

Kyoko Hoshino, Yukako Wakatsuki, Masayuki Iigo and Shigenobu Shibata

Department of Physiology and Pharmacology, Waseda University School of Science and Engineering (K.H., Y.W., S.S.), Nishitokyo 202-0021, Japan; and Department of Applied Biochemistry, Faculty of Agriculture, Utsunomiya University (M.I.), Tochigi 321-8505, Japan

Address all correspondence and requests for reprints to: Dr. Shigenobu Shibata, Department of Physiology and Pharmacology, Waseda University School of Science and Engineering, Higashifushimi 2-7-5, Nishitokyo 202-0021, Japan. E-mail: shibatas{at}waseda.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To understand the role of the circadian molecular clock in mouse reproduction, we investigated the daily rhythms associated with nursing and pup growth in Clock-mutant mice maintained under light-dark housing conditions. The daily rhythm associated with the maternal behavior of crouching had a strong diurnal peak and two weak nocturnal peaks in wild-type dams, whereas homozygotes (Clock/Clock) exhibited no significant peaks in activity. Wild-type, but not Clock-mutant, dams showed high, rhythmic levels of prolactin content in serum that corresponded with crouching. Pup body weight increased at a significantly slower rate in Clock-mutant dams compared with wild-type dams under all experimental conditions when the pups ranged from 10–15 in number. Heterozygote dams equally bred wild-type, heterozygote, or homozygote pups. The amount of milk secreted from dams, as calculated by the increase in pup body weight through suckling, was lower in Clock-mutant mothers vs. wild-type mice. When Clock-mutant dams gave birth to more than 10 pups, survival was poor for offspring until the time of weaning. The present results demonstrate that Clock mutation disrupts daily maternal behavior and the growth and survival rate of pups, especially with the breeding of more than 10 pups.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, THE MASTER circadian pacemaker (or circadian clock) is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and coordinates circadian physiological and behavioral rhythms. Circadian output from the SCN plays a major role in the regulation of reproduction. SCN ablation or the severing of neuronal pathways between the SCN and the preoptic hypothalamic area results in estrous acyclicity and infertility (1, 2). Molecular clock components such as Clock (3), Bmal1 (4), Per1 and Per2 (5, 6), and Cry1 and Cry2 (7, 8) participate in circadian oscillation. Recent reports suggest that circadian Clock mutation disrupts estrous cyclicity, interferes with pregnancy (9), and results in perinatal and postpartum problems and poor pup survival until weaning (10). Clock-mutant female mice also show an unexpected decline in progesterone levels at midpregnancy and a shortened duration of pseudopregnancy, suggesting that maternal prolactin release may be abnormal (9). We conducted the present experiments to elucidate the role of Clock mutation in association with maternal behavior during lactation.

Prolactin plays multiple roles in reproduction, including lactation, luteal function, and reproductive behavior (reviewed in Ref.11). The varied effects of prolactin on the mammary gland include the growth and development of the mammary gland, milk synthesis, and the maintenance of milk secretion (11). Probably the best-characterized prolactin-driven reproductive behaviors are seen in the parents. Maternal behaviors such as nest building, gathering, grouping, cleaning, and crouching behavior have been most extensively studied in rats (12, 13) and less extensively in mice (14). Mice lacking a functional prolactin receptor exhibit profound deficits in the expression of maternal cues (14), clearly demonstrating that prolactin functions in mediating this behavior. Consistent with what is known about the neuronal circuitry regulating maternal behavior, it appears that the action of prolactin to stimulate maternal behavior (15) is exerted in the medial preoptic nucleus of the hypothalamus in rats (16).

There is ample chronobiological evidence that the temporal organization of prolactin secretion in rats is controlled by circadian input (17, 18). For example, the rhythm of prolactin release is maintained in constant conditions and abolished by SCN lesion in rats (17, 19). The best-known physiological stimulus affecting prolactin secretion is suckling of the nipple by neonates (20), which involves a suckling-induced decrease in dopamine neuronal activity in the arcuate nucleus (21). The prolactin secretory response to nursing is reported to be superimposed by the daily rhythm of prolactin secretion (22, 23).

In taking all previous findings into consideration, mutation of the circadian clock may affect reproductive behavior not only during pregnancy, but also during lactation. In this study we examined daily rhythms associated with the maternal behavior of crouching as well as the prolactin level and the growth and survival rates of pups from Clock-mutant dams.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and housing
Clock-mutant mice were purchased from The Jackson Laboratory (stock no. 002923; Bar Harbor, ME) and backcrossed to ICR strains more than eight times. Animals were maintained on a 12-h light, 12-h dark cycle with lights on at 0800 h (room temperature, 23 ± 2 C) and allowed access to commercial chow (Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum. During the light period, light intensity was 100–150 lux at cage level. ZT0 was defined as the lights-on time, and ZT12 as the lights-off time. Female wild-type and Clock-mutant mice were mostly mated with male wild-type and Clock-mutant mice, respectively, but in some experiments, they were mated with opposite genotype male mice (wild-type female x Clock-mutant male; Clock-mutant female x wild-type male). To examine the effect of Clock mutation on pup growth minus the effects of the Clock mutation in dams, heterozygote females and males were mated. Wild, heterozygote, and homozygote pups were bred by heterozygote dams. In this experiment, pups ears were punched for individual identification until postpartum d 7, and the genotypes of the pups were determined after weaning.

Genotypes were determined using a PCR mutagenesis method that introduces a restriction site that allows for direct detection of the Clock mutation (24). The mutant allele has an A to T transversion in the third nucleotide of the intron splice donor site (from CAGgtaac to CAGgttac, where lower case represents the intronic sequence), resulting in the skipping of exon 19. The 5' primer (63 nucleotides; 5'-GCAAGAAGAACTAAGGAAAATTCAAGAGCAACTTCAGATGGTCCATGGTCAAGGGCTACAGTT-3') is based on the sequence of exon 18 and includes the first two nucleotides of the intron, but terminates with CAGtt (rather than CAGgt). The 3' primer (5'-TAGTGCCCTAGATGGCCCTGTTGG-3') is the reverse complement of the intronic sequence terminating 398 bp 3' of the mutation. PCR amplification results in the introduction of a HincII site (Gttaac) in the wild-type allele, whereas the corresponding section of the mutant allele (Gtttac) is not cut by HincII. Mouse genomic DNA was extracted from tail biopsies by proteinase K digestion and isopropanol precipitation. Genomic DNA was subjected to PCR amplification using a hot start protocol and Taq DNA polymerase. Cycling parameters were 94 C for 3 min; 30 cycles of 94 C for 1 min, 60 C for 2 min, and 72 C for 3 min; followed by a final extension at 72 C for 10 min. Amplification products were digested with HincII (TakaraBio, Osaka, Japan) without extraction and were separated by 1.5% agarose gel electrophoresis. The Clock allele produces an amplified product of 460 bp that is unaffected by HincII. The wild-type allele is sensitive to HincII; cleavage of the 5' primer results in a 398-bp band.

To reduce the gender effects in experiments on growth and survival rates, crouching behavior, milk secretion, and prolactin content, the male and female ratio between wild and mutant pups was adjusted. Some dams were killed to measure prolactin in serum. Experimental animal care was conducted under the permission of the experimental animal welfare committee of the School of Science and Engineering at Waseda University).

Video observation of crouching behavior
Crouching, which has previously been reported in rats (25) and mice (26), is a maternal behavior associated with nursing. In this experiment, crouching behavior, including arched back nursing, was observed in 12 pups from both wild-type and Clock-mutant mice and was recorded by video for evaluation later. Crouching for over 5 min was counted as one event, and the duration was marked on an event sheet. Behavioral rhythmicity was assessed for approximately 4 wk after pups were born using a {chi}2 periodogram (27) in the range of 20–28 h. Crouching behavior was analyzed from delivery day to weaning day by an experimenter who was blind to the animals’ genotypes.

Measurement of milk secretion
On postpartum d 3 or 7, the dams were separated from their pups for 8 h from ZT2 to ZT10. After an 8-h separation, the pups were returned to the dams. We then measured the increase in pup body weight 1 h after reunion. Because the amount of prolactin released is related to stimulus intensity and is somewhat commensurate with the number of pups nursing (28), the pup number was set at 12.

Nest-building behavior
To evaluate nest-building behavior, we measured the height from the base to the top of the nest. If the height was more than 4.5 cm and included a ridge formation, we designated this nest completed. The nest was defined as incomplete if it did not meet these conditions. A researcher who was blind to the animals’ genotypes measured each nest, and the percentage of complete or incomplete nests was calculated.

Exchange of pups among wild-type and Clock-mutant dams
Half the litters were cross-fostered on postpartum d 3 or 4. In this experiment, we only used litters with more than 12 pups. Total pup number was adjusted to 12, and the gender and genotype of pups were also adjusted to equal numbers. Some dams were killed to measure prolactin in the serum.

Sample preparation
Mice were deeply anesthetized with ether, and blood was intracardially collected. Serum was separated and stored at –80 C until it determination of the prolactin level.

Measurement of prolactin levels in serum
In this experiment, we measured prolactin secretion during crouching. To ensure controlled conditions, blood samples were collected during times of stimulation by pup suckling. Dams were killed when they demonstrated an arched back or blanket nursing behavior toward pups for 20–30 min during video surveillance. A previous study (29) showed that in rats, blood prolactin concentrations begin to rise within 3 min of nursing initiation, peak within 10 min, and reach a constant level with continuous nursing. Although we do not know whether the same situation occurs in mice, with these experimental conditions we could measure prolactin secretion under suckling-stimulation conditions. Because a significant delay in pup growth was observed when pup number ranged from 10–15, we consistently examined wild-type and Clock-mutant dams who were nursing 10–15 pups. We also measured the prolactin secretion of both wild-type and Clock-mutant dams under basal conditions in the absence of nursing.

Prolactin levels in serum were determined using a newly developed time-resolved fluoroimmunoassay (TR-FIA) for mouse prolactin. The mouse prolactin RIA kit (National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases) was provided by Dr. Masanobu Yamada (Gunma University, Maebashi, Japan). Highly purified mouse prolactin for iodination (AFP-10777D; 2 µg) was labeled with europium using the DELFIA Eu-N1 ITC labeling kit (PerkinElmer Japan, Tokyo, Japan) at 22 C for 48 h according to the manufacturer’s instructions. Free europium and europium-labeled prolactin were then separated by PD-10 (Amersham Biosciences, Tokyo, Japan).

A second antibody (antirabbit IgG; 20 µg/ml in 50 mM K2HPO4 containing 0.9% NaCl and 0.5% NaN3, 200 µl; Shibayagi, Shibukawa, Japan) was immobilized to the surface of the microtiter plate (no. 437958; Nunc, Rosklide, Denmark) at 4 C for 18 h. After three washes with the washing solution (150 mM NaCl containing 0.02% Tween 20 and 0.05% NaN3), the plate was blocked with 0.1% BSA (Sigma-Aldrich Corp., St. Louis, MO) in 50 mM Na2HPO4 and 0.05% NaN3 (300 µl) at room temperature for 1 h. The plate was washed three times, and 100 µl antimouse prolactin (AFP 131078, x400,000 in DELFIA Assay Buffer, PerkinElmer Japan) was dispensed into the wells. After incubation at 4 C for 18 h, the plate was washed three times, and 50 µl mouse prolactin reference preparation for RIA (AFP-6476C; 0.078–40 ng/ml) or mouse serum samples diluted with the assay buffer were dispensed into the wells. Usually, 5-µl serum samples were used. After incubation at room temperature for 1 h, europium-labeled mouse prolactin (50 µl, ~10,000 counts per second) was dispensed into the wells and further incubated at 4 C for 18 h. After three washes, DELFIA Enhancement Solution (100 µl; PerkinElmer Japan) was added to dissociate europium from the antibody-antigen complex on the surface of the plate, and the plates were shaken for 5 min at room temperature. Time-resolve fluorescence was measured using an ARVO-Sx (PerkinElmer Japan). Prolactin levels in serum were calculated using PRISM (version 2.0; GraphPad, Inc., San Diego, CA).

To validate TR-FIA, the parallelism of the inhibition curves for a serial 2-fold dilution of the mouse serum and the mouse prolactin standard was tested using a parallel line assay (2 x 3 points). The inhibition curve for the serial 2-fold dilution of the serum was parallel to the curve for the standard (data not shown). Intra- and interassay coefficients of variation of the TR-FIA were 7.4% (n = 3) and 9.1% (n = 4) at the 0.06 ng/tube level, respectively. The minimum detectable level, defined as 2 SD from the buffer controls, was approximately 5 pg/tube.

Statistical analysis
The data are expressed as the mean ± SEM. For statistical analysis, one- or two-way ANOVA was applied, followed by the Bonferroni-Dunn or Student’s t test. In some experiments, we used the nonparametric Mann-Whitney U test for statistical analysis. The Fisher exact probability test was applied for incidence analysis. StatView for Windows (version 5.0; SAS Institute, Cary, NC) statistical analysis software was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight increase, survival rate, and number of pups in wild-type and Clock-mutant mice
We obtained a total of 746 pups from 65 wild-type pregnancies and 741 pups from 72 Clock-mutant pregnancies. There were significant differences in the number of pups born to wild-type (11.5 ± 0.28) and Clock-mutant mothers (10.3 ± 0.32; P < 0.01, by Student’s t test; Fig. 1AGo). Although the body weight of pups born to Clock-mutant mice tended to be slightly lower, there were no significant differences between the two genotypes (P > 0.05, by Student’s t test; Fig. 1BGo). When we observed the developmental pattern of pups obtained from 55 wild-type and 64 Clock-mutant dams, interrupted nursing behavior occurred in 13 of 64 Clock-mutant dams (P < 0.05 vs. wild-type, by Fisher probability test) and in three of 55 wild-type dams (Fig. 1CGo), and all pups died within 1 wk of birth. A reduction in the number of pups was observed in 13 of 64 Clock-mutant dams (P < 0.05 vs. wild-type, by Fisher probability test) and five of 55 wild-type dams up until the day of weaning (postpartum d 28; Fig. 1CGo).


Figure 1
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  		FIG. 1. Number of pups birthed by wild-type and Clock-mutant dams, their body weights at birth, and weight development. A, Distribution of dams (percentage of total dams) compared with the number of pups from each mother. Data are based on 65 (total number of pups, 746) wild-type and 72 (total number of pups, 741) Clock-mutant mothers. **, P < 0.01 between mean number of pups (11.5 ± 0.28 for wild-type and 10.3 ± 0.32 for Clock-mutant mice; by Student’s t test). {circ}, Wild-type mice; bullet, Clock-mutant mice. B, Birth weights of pups born to wild-type (n = 65) and Clock-mutant (n = 72) dams. {square}, Wild-type mice; {blacksquare}, Clock-mutant mice. C, Percentage of dams showing abnormal behavior during nursing. {square}, Normal dams; Figure 1, dams that had a reduction in the number of nursing pups; {blacksquare}, dams that completely interrupted nursing within 1 wk after delivery. Numbers in parentheses refer to the number of dams. *, P < 0.05 vs. wild-type mice (by Fisher exact probability test). D, Survival rate of pups. bullet and {circ}, Survival rate of pups numbering between 10–15 (n = 19 for wild-type; n = 22 for Clock-mutant mothers); {blacksquare} and {square}, dams with fewer than nine pups (n = 4 for wild-type; n = 6 for mutant mothers); {circ} and {square}, wild-type mice; {blacksquare} and bullet, Clock-mutant mice. #, P < 0.05; ##, P < 0.01 (vs. postpartum d 0, by Bonferroni/Dunn test). *, P < 0.05 (vs. wild-type mice, by Student’s t test). E, Weight gain of pups numbering 10–15 (bullet and {circ}) or less than nine ({blacksquare} and {square}) from wild-type ({circ} and {square}) or Clock-mutant (bullet and {blacksquare}) dams. Data came from seven wild-type and Clock-mutant mothers that bred 10–15 pups and five wild-type and mutant mothers that bred fewer than nine pups. *, P < 0.05 (vs. wild-type mice, by Student’s t test).

 
Nursing dams were divided into two groups depending on pup number (less than nine or more than 10), because mothers with less than nine pups did not show a reduced pup number until weaning. Data from dams with interrupted nursing were excluded from the following results. Under these criteria, dams with more than nine pups had no significant differences in pup survival between wild-type (12.2 ± 0.24; n = 52) and Clock-mutant (11.8 ± 0.23; n = 47; P > 0.05, by Student’s t test) dams. Clock-mutant dams exhibited a significant decrease in pup survival rate compared with wild-type dams (P < 0.05, by Student’s t test) only when they bred 10–15 pups (Fig. 1DGo). Two-way ANOVA revealed that weight gain corresponding to postnatal week was significantly slower in Clock-mutant pups vs. wild-type (df = 4,60; F = 3.4; P < 0.05, genotype x week), when pups numbered between 10 and 15 at birth (Fig. 1EGo). If pups numbered from six to nine, both wild-type and Clock-mutant pups grew faster, and there were no significant differences between the two groups (Fig. 1EGo).

Daily rhythm of crouching behavior in wild-type and Clock-mutant dams
The daily bouts of crouching were recorded and then evaluated at a later time. If dams showed arched back nursing or blanket nursing for at least 5 min, we measured the event duration time. Daily crouching patterns of the representative mice were plotted as an actogram (Fig. 2AGo), and the averaged daily pattern of crouching events during 30-min epochs from birth until weaning was plotted (Fig. 2Go, A and B). Wild-type dams exhibited a daily pattern of events with a major peak between 0900 and 1500 h and two minor peaks between 2100 and 0000 h and between 0200 and 0500 h. Clock-mutant dams did not display clear rhythmicity. Periodogram analysis revealed significant 24-h and weak 12-h rhythmicity in wild-type mothers, but not in Clock-mutant mothers (Fig. 2DGo). The average of daily crouching episodes was calculated for three wild-type and three Clock-mutant mice (Fig. 2CGo), and two-way ANOVA demonstrated a significant difference in the daily pattern of crouching events between the two groups (df = 47, 192; F = 2.2; P < 0.01, genotype x clock time; Fig. 2CGo). The duration of crouching per event (Fig. 2EGo, left) and the total length per day (Fig. 2EGo, middle) were significantly longer (P < 0.05, by Student’s t test) in wild-type mice vs. Clock mutants, and event number per day was slightly lower in wild-type mice (Fig. 2EGo, right).


Figure 2
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  		FIG. 2. Daily rhythm of crouching behavior in wild-type and Clock-mutant mice. A and B, Upper panel, Representative actogram of crouching activity in wild-type (A) and Clock-mutant dams (B) maintained under a light-dark cycle. Lower panel, Crouching as a percentage of events from 30-min intervals taken from delivery to weaning. C, Percentage of crouching episodes in three wild-type and three Clock-mutant mice. Shaded areas under the horizontal clock timeline show a significant change between wild-type and mutant mice (P < 0.05, by Student’s t test). D, Left and right periodograms were obtained from wild-type (A) and Clock-mutant (B) dams, respectively. E, Left, Duration of crouching per event until d 10 after delivery; middle, number of crouching events per day; right, total duration of crouching events per day. {circ}, Wild-type mice; bullet, Clock-mutant mice. *, P < 0.05 (vs. wild-type mice, by Student’s t test).

 
Daily rhythms of prolactin content and milk secretion in wild-type and Clock-mutant dams
Prolactin content was higher in nursing mothers than in virgin female mice (Fig 3AGo). As we did not control the suckling duration for each dam, prolactin content fell over a wide range. Therefore, we statistically analyzed prolactin content in serum using the nonparametrical Mann-Whitney U test and found that wild-type mice exhibited a clear rhythmic daily change (P < 0.05, ZT6 and ZT18 vs. ZT12), whereas Clock-mutant mice did not (P > 0.05; Fig. 3AGo). The mean daily prolactin content was slightly higher in wild-type mice vs. Clock mutants (227 ng/ml for wild-type mice; 198 ng/ml for Clock-mutant mice), but this was not a significant difference (P > 0.05, by Student’s t test). Interestingly the prolactin content in wild-type and Clock-mutant dams without pups suckling for 2 d was almost the same (Fig. 3AGo).


Figure 3
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  		FIG. 3. Daily profile of prolactin content in serum and milk secretion of wild-type and Clock-mutant dams. A, Prolactin content in serum of wild-type and Clock-mutant dams nursing 10–15 pups on d 6 after delivery. Each point consists of three to eight wild-type and three to eight Clock-mutant dams. *, P < 0.05 (by Mann-Whitney U test).V, Virgin female killed at ZT8. B, Basal level of prolactin content in dams not nursing. B, Estimated milk secretion according to weight gain. After starvation from ZT2 to ZT10, average body weight increase in 12 pups was measured for the next 1 h of suckling. **, P < 0.01 (vs. wild-type mice, by Student’s t test); ##, P < 0.01 (vs. d 3, by Student’s t test).

 
Milk secretion was estimated by the weight gain of 12 suckling pups. The increase in body weight after suckling was significantly lower (P < 0.01, by Student’s t test) in Clock-mutant pups vs. wild-type pups (Fig. 3BGo). Wild-type (P < 0.01, by Student’s t test), but not Clock-mutant (P = 0.06, by Student’s t test), pups showed significantly increased body weight from d 3 to 7. When there were fewer than nine pups, no difference in milk secretion was observed (data not shown).

Nest building in wild-type and Clock-mutant mice
Because the Clock-mutant mouse exhibited a deficit in crouching behavior and prolactin release in the serum after pup delivery, we investigated whether maternal behavior was also disturbed before delivery. Up until the time of delivery, many of the Clock-mutant mice made incomplete nests compared with wild-type mice, with significant differences between the two groups (P < 0.01 and P < 0.05, by Fisher’s probability test; Fig. 4AGo). However, there were no significant differences in weight gain during pregnancy between wild-type and Clock-mutant females (Fig. 4BGo).


Figure 4
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  		FIG. 4. Nest-building behavior and body weight change in pregnant wild-type and Clock-mutant mice. A, Percentage of complete ({square}) or incomplete ({blacksquare}) nests built by pregnant wild-type (n = 15) and Clock-mutant (n = 22) dams. *, P < 0.05; **, P < 0.01 (vs. wild-type mice, by Fisher’s exact probability test). B, Change in body weight of pregnant mice in the latter half of pregnancy. {square}, Wild-type mice; {blacksquare}, Clock-mutant mouse.

 
Slow increase in body weight related to Clock-mutant dams, but not to pups
Mating of heterozygote males and females produced wild-type, heterozygote, and homozygote pups. Heterozygote mothers bred 10–15 pups of all genotypes equally, and there were no significant differences in the body weight increase among the pups (Fig. 5AGo). In a second experiment, wild-type and Clock-mutant females were mated with Clock-mutant and wild-type males, respectively. Wild-type and Clock-mutant dams bred heterozygous pups only. The body weight gain in pups bred by wild-type dams was faster than that in pups bred by Clock-mutant dams when pups numbered between 10 and 15 (Fig. 5BGo). When there were less than nine pups, there was no difference in body weight gain (data not shown).


Figure 5
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  		FIG. 5. Slow increase in body weight related to clock mutation of dams and their pups. A, Four heterozygous dams bred 10–15 pups that were homozygous ({blacksquare}), heterozygous (Figure 5), or wild type ({square}). B, Wild-type (n = 5) and Clock-mutant homozygous (n = 5) mice bred heterozygous pups only. When the number of pups ranged from 10–14, a slow increase in body weight was seen in pups bred by Clock-mutant dams. *, P < 0.05 (vs. wild-type mice, by Student’s t test).

 
Clock-mutant foster mother and nursing behavior
To confirm that Clock-mutant dams have a deficit in nursing behavior, we conducted cross-fostering experiments. In this case, half the litters were cross-fostered on postpartum d 3 or 4. The body weight increase in pups bred by Clock-mutant dams was slow compared with that in pups of wild-type dams, even when pups were wild type or mutant (Fig. 6AGo). Although the number of pups was adjusted to 12 on the day of exchange, the pup survival rate significantly decreased in Clock-mutant dams vs. wild-type dams (Fig. 6BGo). A decreased prolactin content in the serum was more evident in the Clock-mutant foster dams than in wild-type foster dams (Fig. 6CGo), but this difference was not significant (P > 0.05, by Student’s t test).


Figure 6
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  		FIG. 6. Effect of cross-fostering pups between wild-type (W) and Clock-mutant (M) mothers on nursing behavior. A, Wild-type (n = 7) and Clock-mutant (n = 7) dams bred their 12 pups (six were from a true or foster mother). *, P < 0.05; **, P < 0.01 (vs. wild-type mice, by Student’s t test). B, Survival rate of pups. {circ}, Wild-type mice (n = 7); bullet, Clock-mutant mice (n = 7). *, P < 0.05 (vs. wild-type mice, by Student’s t test). C, Half the litters were cross-fostered, and the prolactin content of dams with their own and foster pups was measured. Numbers in parentheses represent the number of mothers.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we examined whether Clock mutation affected the maternal behavior of crouching during nursing, prolactin release, and weight gain in pups. A clear daily rhythm in the occurrence of crouching was present in wild-type dams, whereas Clock-mutant dams exhibited a loss of daily rhythm. Clock-mutant dams also demonstrated shorter durations of crouching per event and a low total time spent crouching per day. In rodents, the prolactin secretory response to nursing is superimposed by the daily rhythm of prolactin secretion; that is, the same intensity of suckling stimulus can elevate prolactin levels more effectively at certain times of the day when the daily input enhances the suckling stimulus-evoked secretory response (22, 23). In the present experiment, Clock mutation did not seem to enhance the suckling stimulus-evoked prolactin secretion on a daily basis. Based on all findings, we suggest that longer durations of crouching, which occur most often during the day in wild-type mothers, may be vital to normal pup growth. Alternately, disruption of the daily feeding cycle in Clock-mutant mice (30) could then be associated with altered behavior toward the pups.

Turek et al. (30) recently reported that Clock-mutant mouse pups showed an initial body weight gain similar to wild-type mice, but then became obese after weaning. In the present experiment, there were no significant differences in the weight gain and survival rate of pups born to wild-type and Clock-mutant mothers when the number of pups was less than nine. Thus, Clock mutation of dams did not affect pup growth when the number of pups was small; however, it severely disrupted pup growth when the number of pups ranged from 10–15. Thus, we believe that lower milk production can sustain only a certain number of pups, beyond which it becomes more difficult regardless of whether Clock-mutant dams carry the C57black or ICR strains.

Because a larger number of pups was born to dams carrying the ICR strain than to those carrying the C57black strain, Turek et al. (30) may not have found a difference in weight gain due to the small number of pups born to Clock-mutant mice with a C57black background gene. Our Clock-mutant pups gained weight after weaning and caught up with wild-type mice within 5–6 wk after birth (data not shown). The ICR mouse strain generally grows larger in size than the C57black strain; therefore, we believe there were no significant differences in body weight between wild-type and Clock-mutant ICR mice in the adult stage (data not shown).

Miller et al. (9) reported that circadian Clock mutation both disrupts estrous cyclicity and interferes with the ability of mice to carry a pregnancy to full-term. Clock-mutant C57black strain females exhibit increased fetal reabsorption during pregnancy and have a high rate of full-term pregnancy failure. Interestingly, a recent report demonstrated that Clock-mutant mice carrying the C57black strain also show more perinatal and postpartum problems and very poor offspring survival until weaning (10). Findings from our present experiment indicated that Clock mutation in ICR mice had a significant, but weak, effect on reproductive performance. Recently, Kennaway et al. (31) reported that Clock-mutant C57black strain mice backcrossed to CBA strain mice showed a redundancy of genes in the part of the circadian system that allows for reproductive cyclicity. Considering all findings, we suggest that the C57black strain carrying the Clock mutation exhibits a more severe, abnormal pattern of reproduction performance than the CBA and ICR strains also carrying the Clock mutation.

The live birth of pups has never resulted from homozygous pairings of Bmal1-null mutants (32). Nevertheless, Bmal1 females ovulate, mate, and fertilize ova after mating with wild-type males, but either delayed embryo development or early embryo loss prevents full-term pregnancies. Thus, normal clock function seems necessary to maintain normal reproduction.

In this study, Clock-mutant dams exhibited a disrupted daily rhythm of prolactin release that corresponded with the disrupted daily rhythm of crouching. Disrupted crouching rhythms may affect suckling behavior, resulting in the disrupted rhythm of prolactin release. Abnormal prolactin release may, in turn, affect not only milk production and secretion, which would then affect pup growth, but also other various maternal behaviors. Prolactin has actually been found to modulate maternal behaviors through the prolactin receptor located in the medial preoptic nucleus (20). We saw in this investigation that a smaller amount of milk was secreted in Clock-mutant dams vs. wild-type dams. However, the present results simply show a correspondence between two phenotypes, such as prolactin secretion and milk production. The direction of causality remains unclear.

There are no clear answers to the questions of how and where the Clock mutation alters maternal behavior and breeding success. It is possible that the absence of the Clock gene causes errors in prolactin production/secretion by virtue of disrupted circadian rhythm regulation of prolactin synthesis/release. There is important literature on the necessity of circadian clock for maintenance of luteal function during pregnancy (9). Taken together, these findings suggest that at least some of the Clock-mutant pregnancy abnormalities and nursing abnormalities are probably due to abnormal daily control of maternal prolactin release. Our hypothesis is consistent with the findings of other groups that have described a link between the circadian system and prolactin secretion (33). However, we still do not know whether the present findings are the result of a defective circadian clock in the SCN or the absence of normal clock gene function at one or more loci outside the SCN. Regarding to this hypothesis, a previous paper suggested that Clock-mutant females display irregular estrous cycles and failed to have a coordinated LH surge due to a disruption of the daily timing signal from the SCN to the GnRH neurons, rather than as a result of pituitary defects or inappropriate feedback from ovarian hormones (9). The daily prolactin surge induced by continuous exposure of ovariectomized rats to high estradiol levels is eliminated by SCN lesions (34, 35). Vasopressin (33) and vasoactive intestinal polypeptide (36) outputs from the rat SCN have been reported to be neurotransmitters for the regulation of this daily prolactin surge. Although a direct effect of the Clock mutation on prolactin synthesis/release of lactotrophs in vivo remains to be examined, we hypothesize that Clock-mutant dams failed to have daily prolactin release because the SCN in mutant animals does not provide a coordinated time of day signal to prolactin surges.

In contrast, the Clock genes may directly regulate prolactin synthesis through the control of prolactin promoter activity in MMQ prolactin cells (37). Imaging studies of isolated brain regions from transgenic rats using a luciferase reporter driven by the mPer1 promoter established that average Per1-luciferase expression is rhythmic in the arcuate nucleus (38). Furthermore, a circadian rhythm associated with the Per1:green fluorescence protein reporter signal was observed in tyrosine hydroxylase-positive neurons in the arcuate nucleus (39). Thus, we could not rule out the possibility that maternal behavior is disrupted by the deficits of Clock gene function at one or more loci outside the SCN. Additional experiments are necessary to elucidate the detailed mechanism of deficits of maternal behavior observed in Clock-mutant mice.

Weight gain was similar in wild-type, heterozygous, and homozygous pups bred by heterozygous dams, whereas heterozygous pups bred by Clock-mutant mothers exhibited a slow increase in body weight compared with heterozygous pups bred by wild-type dams. In addition, the cross-fostering experiment showed disrupted weight gain in pups bred by Clock-mutant mothers. Thus, we suggest that the slow increase in pup body weight relates not to a Clock mutation in pups, but to that in dams.

It is known that Clock mutation disrupts the reproductive process of estrous cyclicity during ovulation and the ability to carry a pregnancy to full term (9). We found in the present study that Clock mutation disrupted maternal behavior and interfered with weight gain in pups, thereby reducing the survival rate of offspring until the time of weaning.


   Acknowledgments
 
We thank Dr. A. F. Parlow, a member of the National Hormone and Pituitary Program and National Institute of Diabetes and Digestive and Kidney Diseases, for his help; and Dr. Masanobu Yamada (Gunma University, Maebashi, Japan) for providing us with the mouse prolactin RIA kit.


   Footnotes
 
This work was partially supported by grants (to S.S.) from the Japanese Ministry of Education, Science, Sports, and Culture (13470016) and Waseda University (2005-96).

K.H., Y.W., M.I., and S.S. have nothing to declare.

First Published Online January 26, 2006

Abbreviations: SCN, Suprachiasmatic nuclei; TR-FIA, time-resolved fluoroimmunoassay; ZT0, lights-on time; ZT12, lights-off time.

Received October 24, 2005.

Accepted for publication January 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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