Neuroendocrine effects of the duper mutation in Syrian hamsters: a role for Cryptochrome 1

Abstract

Molecular and physiological determinants of the timing of reproductive events, including the pre-ovulatory LH surge and seasonal fluctuations in fertility, are incompletely understood. We used the Cryptochrome 1-deficient duper mutant to examine the role of this core circadian clock gene in Syrian hamsters. We find that the phase of the LH surge and its stability upon shifts of the light: dark cycle are altered in duper mutants. The intensity of immunoreactive PER1 in GnRH cells of the preoptic area peaks earlier in the day in duper than wild type hamsters. We note that GnRH fibers coursing through the suprachiasmatic nucleus (SCN) contact vasopressin- and VIP-immunoreactive cells, suggesting a possible locus of circadian control of the LH surge. Unlike wild types, duper hamsters do not regress their gonads within 8 weeks of constant darkness, despite evidence of melatonin secretion during the subjective night. In light of the finding that the duper allele is a stop codon in Cryptochrome 1, our results suggest important neuroendocrine functions of this core circadian clock gene.

Keywords: Cryptochrome 1; LH surge; circadian rhythms; duper; melatonin; photoperiod.

FIGURE 1

(A) Design of experiment 2. Female hamsters housed in 14L:10D cycle were subjected to an acute phase advance on proestrus, by way of shortening dark phase. Starting on the third day after phase advance, animals were sacrificed at time points (ZT) spanning the next anticipated proestrus, as indicated by arrows. The light and dark phases are indicated by the white and black background, respectively. Abbreviation: P: proestrus. (B) Double-plotted actograms of representative female wild type (left) and duper (right) hamsters subjected to an acute 8 h phase advance on day 5. Yellow shading indicates light phase. Note that duper hamster entrains with positive phase angle (activity begins 2-3 h before lights out), and re-gains the new phase more rapidly than wt upon shift of the LD cycle.

FIGURE 2

The LH surge occurs earlier in duper than in wt hamsters entrained to 14L:10D. Symbols represent mean (+SEM) concentrations in serum of 5-6 animals collected at each zeitgeber time (ZT). *, statistically significant difference (p < 0.001) in LH concentrations between genotypes at the indicated ZT.

FIGURE 3

BMAL1 and PER1 proteins are co-localized with GnRH in a time-of- day dependent manner in both wild type and duper Syrian hamsters. Representative triple-label immunocytochemical staining for GnRH, PER1, and BMAL1 are shown in 40 μm sections of preoptic area from wild-type and duper hamsters. Arrows mark BMAL1-positive (blue) GnRH cells at ZT 4 and PER1-positive (green) GnRH cells at ZT 16. Scale bar = 50 μm.

FIGURE 4

Quantification of immunofluorescence for BMAL1 and PER1 in GnRH neurons on proestrus in duper and wild-type hamsters reveals earlier onset of PERI in duper hamsters. (A) Proportion of BMAL1-positive GnRH cells for wild-type and duper hamsters across zeitgeber sampling times (black bars and gray bars, respectively. (B) Mean BMAL1 intensity in GnRH cells for wild-type and duper hamsters. (C) Proportion of PERI- positive GnRH cells for wild-type and duper hamsters. (D) Mean PER1 intensity in GnRH cells for wild-type and duper hamsters. Bar graphs represent mean ± SEM. *, p < 0.05.

FIGURE 5

GnRH fibers traverse the SCN of both male and female hamsters. (A) SCN of female hamster sacrificed on proestrus at ZT8.5. (B) SCN of male hamster sacrificed at ZT8.5. 650x confocal z-stack images for both sexes show sub 1 micron appositions between GnRH fibers containing GnRH varicosities (Green) and SCN structures. GnRH (Green), VIP (Red), AVP (Blue) at 200x and 650x.

FIGURE 6

GnRH fibers are most extensive in rostral SCN of proestrous hamsters. Micrographs showing rostral (A) and caudal (B) SCN sections stained for GnRH (green), VIP (red), and AVP (blue). Higher magnification image at right illustrates appositions. GnRH fibers are more extensive in rostral than caudal SCN.

FIGURE 7

GnRH fiber projections in the SCN region are not restricted to proestrus and differ between hamsters and mice. (A) Female Syrian hamster sacrificed on metestrus at ZT8.5. GnRH fibers (Green), VIP (Red), AVP (Blue). GnRH fibers overlap with SCN structures. (B) Proestrous female C57bl/6 mouse; a few GnRH fibers are detected within the SCN, but more travel in the midline between the two SCN nuclei and appear as dots in the coronal plane. Left to right, rostral to caudal sequence.

FIGURE 8

Pre-ovulatory LH surge in ovary intact duper hamsters shifts rapidly in response to an 8 hour phase advance compared to wild-types. Design of experiment 2 and actograms of representative wild type and mutant hamsters are shown in Figure 1. Mean (+/− SEM) serum LH concentrations of duper and wt hamsters are shown in blue and red, respectively. Values differ at Zeitgeber Time (ZT) 5 (p < 0.05) and ZT 13 (p < 0.01).

FIGURE 9

Maintenance in 8 weeks of constant darkness in Experiment 3 arrested reproducitve function in wild type, but not duper mutant hamsters. Top: Double plotted actograms of wild type (A) and duper mutant (B) hamsters subjected to an 8 h phase advance and released into DD 10 days later. Yellow shading indicates light phase. Note that wild type hamsters are slow to re-entrain after the shift. In DD, duper female hamster shows scalloping, indicating persistence of the estrous cycle. Wild type hamsters exhibit progressive disorganization of the free running behavioral rhythm, but dupers do not. Bottom: Mean (+SEM) uterine (C) and paired testis (D) weights at time of sacrifice after 8 weeks of DD.