Circadian Function in Multiple Cell Types Is Necessary for Proper Timing of the Preovulatory LH Surge

Abstract

The timing of the preovulatory surge of luteinizing hormone (LH), which occurs on the evening of proestrus in female mice, is determined by the circadian system. The identity of cells that control the phase of the LH surge is unclear: evidence supports a role of arginine vasopressin (AVP) cells of the suprachiasmatic nucleus (SCN), but it is not known whether vasopressinergic neurons are necessary or sufficient to account for circadian control of ovulation. Among other cell types, evidence also suggests important roles of circadian function of kisspeptin cells of the anteroventral periventricular nucleus (AvPV) and gonadotropin-releasing hormone (GnRH) neurons of the preoptic area (POA), whose discharge is immediately responsible for the discharge of LH from the anterior pituitary. The present studies used an ovariectomized, estradiol-treated preparation to determine critical cell types whose clock function is critical to the timing of LH secretion. As expected, the LH surge occurred at or shortly after ZT12 in control mice. In further confirmation of circadian control, the surge was advanced by 2 h in tau mutant animals. The timing of the surge was altered to varying degrees by conditional deletion of Bmal1 in AVP^Cre, Kiss^CreBAC, and GnRH^CreBAC mice. Excision of the mutant Cnsk1e (tau) allele in AVP neurons resulted in a reversion of the surge to the ZT12. Conditional deletion of Bmal1 in Kiss1 or GnRH neurons had no noticeable effect on locomotor rhythms, but targeting of AVP neurons produced variable effects on circadian period that did not always correspond to changes in the phase of LH secretion. The results indicate that circadian function in multiple cell types is necessary for proper timing of the LH surge.

Keywords: Bmal1; GnRH; circadian; kisspeptin; luteinizing hormone; vasopressin.

Figure 1.

A, representative double plotted actogram of a wild type mouse that was initially maintained in 12L:12D (gray shading indicates lights off) before release into constant darkness (DD). B., Actogram of a representative AVP^Cre,Bmal1^fl/fl mouse under the same conditions. C, mean (+SEM) free running period (τ_DD) of 14 mice studied in this experiment (* indicates p < 0.05).

Figure 2.

Actograms of AVP^Cre/+, Cnsk1e^tau/tau mice that were maintained in 12L:12D (gray shading indicates dark phase) and then released into DD. In some cases (A-C), free running rhythms were similar to mice not bearing a Cre allele (τ_DD approximately 20h). (D, E), Other AVP^Cre mice showed a partial reversion to a longer period, although none of these mice free ran with as long a period as WT mice. Several mice (F, G) showed nocturnal activity in 12L:12D but unstable patterns of running wheel activity in DD.

Figure 3.

A, LH concentrations (ng/ml) in individual ovariectomized control mice given priming followed by surge-inducing doses of estradiol benzoate. B, C, D., LH values in similarly treated AVP^Cre/+,Bmal1^fl/fl, Kiss^CreBAC,Bmal^fl/fl, and GnRH^CreBAC,Bmal^fl/fl mice, respectively.

Figure 4.

A, Circulating LH (ng/ml) in individual CnSK1e^tau ovariectomized control mice given priming followed by surge-inducing doses of estradiol benzoate. B, LH concentrations in identically treated AVP^Cre/+,Cnsk1e^tau/tau mice. C. LH concentrations in identically treated Kiss^CreBAC,CnSK1e^tau/tau mice.