Long days restore regular oestrous cyclicity in mice lacking circadian rhythms
Description
Many female mammals have recurring cycles of ovulation and sexual behaviours that are regulated by reproductive hormones and confer reproductive success. In addition to sexual behaviours, circadian behaviour rhythms of locomotor activity also fluctuate across the oestrous cycle in rodents. Moreover, there is a bidirectional relationship between circadian rhythms and oestrous cyclicity since mice with disrupted circadian rhythms also have compromised oestrous cycles resulting in fewer pregnancies. In the present study, we assessed whether extending day length, which alters circadian rhythms, normalises oestrous cyclicity in mice. We found that Period (Per) 1/2/3 triple knockout (KO) mice, that have disabled canonical molecular circadian clocks, have markedly disrupted oestrous cycles. Surprisingly, extending the day length by only 2 hours per day restored regular 4- or 5-day oestrous cycles to Per1/2/3 KO mice. Longer days have also induced consistent 4-day, rather than 5-day, oestrous cycles in C57BL/6J mice. These data demonstrate that extending daytime light exposure could be used for enhancing reproductive success.
Files
Steps to reproduce
Materials and Methods (a) Measuring wheel-running activity Each mouse was individually housed in a cage (183 × 340 × 148 mm; CL-0135, CLEA Japan, Tokyo Japan) equipped with a running wheel (12 cm diameter, Sanko, Osaka, Japan). The cages were placed in light-tight, ventilated boxes, and the light intensity at the bottom of the cage was 200 – 300 lx (white light LED LDA2L-GAG5, Ohm Electric Inc., Saitama, Japan). The number of wheel revolutions was counted by a magnetic sensor-activated signal (59070-010, Littelfuse, Inc., Chicago, IL, USA) and recorded by a computer every minute. The data was analysed by the Chronobiology Kit (Stanford Software Systems, Naalehu, HI, USA) and ClockLab software (Actimetrics, Wilmette, IL). The actograms were plotted in the scaled format. (b) Determination of oestrous cycle length The stages of the oestrous cycle were determined by the pattern of daily total activity changes as previously described [10-12]. Briefly, proestrus/oestrus was defined as the day when there was a peak in total wheel revolutions that was preceded and succeeded by at least 2-3 days of lower activity. We defined this peak as "oestrus" since previous studies demonstrated that wheel-running activity is synchronised with the ovulatory cycle and animals run more on the nights of proestrus than other dioestrus nights [13-18]. Occasionally activity levels peaked for 2 days before declining to a trough. In this case, the 2nd day of peak activity was defined as oestrus. Adult C57BL/6J mice have a 4-day or 5-day oestrus cycle [11-12]. Therefore, we defined 4-day or 5-day oestrus cycles as regular oestrus cycles. If oestrus appeared shorter than 4 days or longer than 5 days, this was classified as other (irregular). Among the 28 days of recording, the percentage of days in each cycle duration (4-day, 5-day, or other) were determined for each mouse between the first day oestrus was observed and the last day oestrus was observed. Then the percentage of days spent in each group (4-day, 5-day, or other) were compiled for all mice of each genotype in 12L:12D and 14L:10D (Fig. 1E, 1F). (c) Evaluation of ovulated eggs and implantation The stage of the oestrous cycle was determined using vaginal cytology as previously described [19]. Eggs were collected from the oviductal ampulla of female mice staged at oestrus to analyse natural ovulation. The total number of eggs that were ovulated at oestrus were counted for each mouse. Female mice staged at proestrus were housed with age-matched C57BL/6J male mice. Female mice with vaginal plugs the next morning were determined to be pregnant, and decidua was observed at the implant site 1 week after copulation. (f) Statistics Results were considered significant at P<0.05. Pearson’s χ2 test was used to compare the oestrous cycle duration between the two groups. For other data, Student’s t-tests were used to examine the differences between the two groups.