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Comparative Study
. 2004 Aug 10;14(15):1367-73.
doi: 10.1016/j.cub.2004.07.055.

Circadian clock mutation disrupts estrous cyclicity and maintenance of pregnancy

Brooke H Miller  1 , Susan Losee Olson, Fred W Turek, Jon E Levine, Teresa H Horton, Joseph S Takahashi
Affiliations
Comparative Study

Circadian clock mutation disrupts estrous cyclicity and maintenance of pregnancy

Brooke H Miller et al. Curr Biol. .

Abstract

Classic experiments have shown that ovulation and estrous cyclicity are under circadian control and that surgical ablation of the suprachiasmatic nuclei (SCN) results in estrous acyclicity in rats. Here, we characterized reproductive function in the circadian Clock mutant mouse and found that the circadian Clock mutation both disrupts estrous cyclicity and interferes with the maintenance of pregnancy. Clock mutant females have extended, irregular estrous cycles, lack a coordinated luteinizing hormone (LH) surge on the day of proestrus, exhibit increased fetal reabsorption during pregnancy, and have a high rate of full-term pregnancy failure. Clock mutants also show an unexpected decline in progesterone levels at midpregnancy and a shortened duration of pseudopregnancy, suggesting that maternal prolactin release may be abnormal. In a second set of experiments, we interrogated the function of each level of the hypothalamic-pituitary-gonadal (HPG) axis in order to determine how the Clock mutation disrupts estrous cyclicity. We report that Clock mutants fail to show an LH surge following estradiol priming in spite of the fact that hypothalamic levels of gonadotropin-releasing hormone (GnRH), pituitary release of LH, and serum levels of estradiol and progesterone are all normal in Clock/Clock females. These data suggest that Clock mutants lack an appropriate circadian daily-timing signal required to coordinate hypothalamic hormone secretion. Defining the mechanisms by which the Clock mutation disrupts reproductive function offers a model for understanding how circadian genes affect complex physiological systems.

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Figures

Figure 1
Figure 1. Clock/Clock females display lengthened and irregular estrous cycles
A, Representative estrous cycles as measured by vaginal cytology in wildtype (top), Clock/+ (middle), and Clock/Clock (bottom) females. C = cornified, N = nucleated, L = leukocytic. B, Clock/Clock females have significantly more days of cornified smears compared to wildtype females, as determined by an unpaired t-test for the number of consecutive days cornified (* indicates p < 0.05, ** indicates p < 0.01).
Figure 2
Figure 2. Clock/Clock mutants fail to have a coordinated LH surge on the day of proestrus
A, Individual LH traces from all wildtype (open circles) and Clock/Clock (closed circles) females sampled from either ZT5-16 or ZT9-21. B, Individual peak LH values in serum obtained from serially sampled mice. Peak Clock/Clock LH values were compared using an unpaired t-test and found to be significantly lower than peak wildtype values (** indicates p < 0.01). Due to the limited sample volume collected, samples with LH values exceeding the range of the RIA could not be re-assayed at lower sample concentrations, preventing absolute measurements of serum concentrations above 10 ng/ml. Therefore, for analysis and presentation purposes, these values were set at 10 ng/ml, and we defined the minimum “surge” value as 14% of the maximum, or 1.4 ng/ml, as previous studies in rats have shown that 14 percent of the peak surge is the minimum value of LH required for ovulation [38]. Using these criteria, only one Clock mutant reached the minimum surge level at any time point.
Figure 3
Figure 3. Clock/Clock pregnancies are characterized by increased rates of fetal reabsorption, reduced serum levels of estradiol and progesterone, and possible dysregulation of prolactin release
A, The percentage of Clock mutant dam pregnancies showing any signs of fetal reabsorption is significantly greater than wildtype at dpc 14 (** indicates p < 0.01) and full-term (* indicates p ≤ 0.05), as determined by chi square analysis. In many cases, both wildtype and Clock pregnancies showed some signs of reabsorption by full-term, but reabsorbing fetuses occurred more frequently in Clock pregnancies. While all wildtype pregnancies delivered normally, 40% of Clock mutant dams carried to full-term but failed to deliver. Importantly, Clock mutant females exhibited copulatory plugs as frequently as wildtype females, indicating that mating behavior was intact in the mutants. Additionally, the average number of fetuses per genotype at dpc 11 was almost identical between genotypes (7.6 fetuses per wildtype dam, 7.75 fetuses per Clock/Clock dam), indicating that the early stages of pregnancy, including ovulation, fertilization, implantation, and early fetal development, occur normally in Clock mutants. WT = wildtype, CL = Clock/Clock, DPC = days post copulation. B, Estradiol levels are significantly reduced in Clock/Clock (closed circles) mice compared to wildtypes (open circles) (p < 0.001). Estradiol in both genotypes is elevated in late pregnancy (dpc 17, 19) compared to mid-pregnancy (dpc 11, 14) (p < 0.01). C, Progesterone is significantly lower in Clock/Clock females compared to wildtypes (p < 0.05) due to very low progesterone levels in Clock/Clock females at dpc 11. In both genotypes, progesterone levels at dpc 14 are elevated compared to dpc 11 and dpc 19 values (p ≤ 0.01). For both B and C, * indicates p < 0.05. Prior to testing the hypotheses, data were checked to verify that they met the assumptions of normality and equality of variance required for analysis of variance. Because the data violated one or more of the assumptions, data were transformed using logarithms and statistical analysis (two-way ANOVA and Fisher’s LSD) was performed on the log transformed data. D, The duration of pseudopregnancy is significantly shorter in Clock/Clock females compared to wildtype controls as determined by t-test (** indicates p ≤ 0.001). Wildtype (n = 10) and Clock/Clock (n = 26) females were mated with vasectomized CD-1 males and the length of pseudopregnancy, as indicated by leukocytic vaginal cytology, was measured by vaginal lavage. The presence of a copulatory plug was designated as dpc 1, and the final day of leukocytic smears was designated as the last day of pseudopregnancy.
Figure 4
Figure 4. Hypothalamic-pituitary gonadal axis function in Clock mutants
A, Estradiol benzoate treatment resulted in a significant elevation in serum LH in wildtype ovariectomized females (* indicates p < 0.01), but did not produce elevated LH levels in Clock/Clock females. NS = not significant. Two-way ANOVA. Treatment (F = 8.9, df (1,24) p < 0.01); Genotype (F = 11.8, df (1,24), p < 0.01). Mice were ovariectomized and implanted with estradiol capsules. Six days following implantation, mice received an injection of either estradiol benzoate or sesame oil at 0800h, and samples were collected the following evening at ZT13. B, There is no difference in hypothalamic GnRH peptide content among wildtype, Clock/+, and Clock/Clock females on the afternoon of proestrus. One-way ANOVA (F = 0.1, df (2,11), p = 0.9). C, GnRH treatment (400 ng/kg, sc) resulted in significantly increased serum LH compared to control levels in both wildtype and Clock/Clock females that had been hormone primed as described above (** indicates p < 0.001).
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