Puberty onset is initiated by activation of neurons that secrete gonadotropin-releasing hormone (GnRH). The timing and progression of puberty may depend upon temporal coordination of two opposing central mechanisms-a restraint of GnRH secretion before puberty onset, followed by enhanced stimulation of GnRH release to complete reproductive maturation during puberty. Neuronal estrogen receptor α (ERα) has been implicated in both controls; however, the underlying neural circuits are not well understood. Here we test whether these mechanisms are mediated by neurons that express kisspeptin, a neuropeptide that modulates GnRH neurosecretion. Strikingly, conditional ablation of ERα in kisspeptin neurons results in a dramatic advancement of puberty onset in female mice. Furthermore, subsequent pubertal maturation is arrested in these animals, as they fail to acquire normal ovulatory cyclicity. We show that the temporal coordination of juvenile restraint and subsequent pubertal activation is likely mediated by ERα in two separate kisspeptin neuronal populations in the hypothalamus.

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.

No abstract

The push-pull perfusion technique was used in combination with a sequential bleeding schedule to estimate simultaneously the release patterns of LHRH and LH in unanesthetized ovariectomized sheep and to determine the temporal relationship between the release of these two hormones. Ovariectomized (greater than 30 days) ewes received unilateral push-pull cannula (PPC) implants (od, 0.85 mm) into the median eminence. After at least 6 days of recovery, each ewe was fitted with an indwelling jugular catheter. For push-pull perfusion, a stylette was removed from the outer PPC, and an inner cannula assembly (od, 0.40 mm) was inserted. Artificial cerebrospinal fluid was pushed through the inner cannula and pulled up between the cannulae at 20 microliters/min. Continuous 10-min perfusate fractions were collected, acidified, and stored at -20 C for LHRH RIA. Blood samples were obtained every 10 min via the jugular catheter, each being drawn 5 min after the start of a perfusate collection interval. Plasma LH levels were determined by RIA. The duration of the sampling was 3-7 h. LHRH output was distinctly pulsatile, occurring at a frequency of approximately one pulse every 30-40 min (n = 5 sheep). LHRH pulse amplitude and frequency remained relatively constant throughout each perfusion. Plasma LH values also were pulsatile, and all LH peaks occurred either during the same interval or during the interval after a LHRH pulse. LH pulses always were accompanied or directly preceded by LHRH pulses, but LHRH pulses were not always followed by LH pulses. The amplitudes of LH pulses and corresponding LHRH pulses were highly correlated (r = 0.81; P less than 0.01). Histological examination revealed that detection of LHRH in perfusates depended upon the placement of the PPC tip into either the zona externa of the central median eminence or adjacent areas. These experiments demonstrate that 1) hypothalamic LHRH release in the Ovx ewe occurs in discrete pulses, with a mean interpulse interval of 38.7 +/- 1.5 min, 2) LH pulses invariably are preceded or accompanied by LHRH pulses, and 3) LH pulse amplitude is highly correlated with LHRH pulse amplitude.