Teresa H. Horton

Circadian Clock Mutation Disrupts Estrous Cyclicity and Maintenance of Pregnancy

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.

Aging Alters Circadian and Light-Induced Expression of Clock Genes in Golden Hamsters

Aging alters numerous aspects of circadian biology, including the amplitude of rhythms generated by the suprachiasmatic nuclei (SCN) of the hypothalamus, the site of the central circadian pacemaker in mammals, and the response of the pacemaker to environmental stimuli such as light. Although previous studies have described molecular correlates of these behavioral changes, to date only 1 study in rats has attempted to determine if there are age-related changes in the expression of genes that comprise the circadian clock itself. We used in situ hybridization to examine the effects of age on the circadian pattern of expression of a subset of the genes that comprise the molecular machinery of the circadian clock in golden hamsters. Here we report that age alters the 24-h expression profile of Clock and its binding partner Bmal1 in the hamster SCN. There is no effect of age on the 24-h profile of either Per1 or Per2 when hamsters are housed in constant darkness. We also found that light pulses, which induce smaller phase shifts in old animals than in young, lead to decreased induction of Per1, but not of Per2, in the SCN of old hamsters.

Circadian rhythms and depression: effects of exercise in an animal model

There is a clear link between altered circadian rhythms and depressive disorders, although the nature of this relationship is unknown. In addition, exercise affects both mood and alters clock function. To investigate the relationship between circadian rhythms, depression, and exercise, 3-wk-old mice housed on a 12:12-h light-dark cycle were exposed to chronic stress (CS) for 6 wk before being placed into constant darkness (DD). One-half of both the control and stressed mice were given access to a running wheel. Stressed mice consumed significantly less of a 2% sucrose solution during CS and exhibited a significant increase in immobility in the forced swim test 3 wk after the termination of stress relative to control mice. These effects were more pronounced in mice without running wheels. Stressed mice also exhibited altered percent distribution of total activity and increased fragmentation of daily activity rhythms during CS relative to control mice. Alterations in percent distribution were more pronounced in animals without running wheels. No activity rhythm changes were seen in DD, and there were no differences in light-induced phase shifts between stressed and control mice. These results suggest that CS causes long-term depressive-like symptoms but does not have long-lasting effects on activity rhythms. These changes were more pronounced in mice without running wheels, suggesting that exercise may protect against the harmful effects of stress.

Progesterone receptors mediate male aggression toward infants

Neuroendocrine mechanisms that mediate male aggression toward infants are poorly understood. Although testosterone is known to enhance aggression in other social contexts, evidence that it modulates aggression toward infants is equivocal. We have found that male progesterone receptor knockout (PRKO) mice exhibit no infanticidal behavior and little aggression toward young. Male PRKO mice also display significantly enhanced parental behaviors. In wild-type mice, blockade of PR induces a behavioral phenotype similar to that of the PRKO males, whereas progesterone exacerbates aggressive tendencies toward infants. Aggressive behaviors directed toward adult males, by contrast, are unaffected by progesterone, PR antagonism, or PR gene deletion. Previously thought to be of diminished importance in male animals, PRs play a critical and specific role in modulating infant-directed behaviors in male mice.

Progesterone Receptor A (PRA) and PRB-Independent Effects of Progesterone on Gonadotropin-Releasing Hormone Release

Progesterones (P4) negative feedback actions in the female reproductive axis are exerted in part by suppression of hypothalamic GnRH release. Here we show that P4 can inhibit GnRH release by a mechanism independent of a nuclear P4 receptor (PR(A/B)). Injections of P4, but not vehicle, allopregnanolone, or dexamethasone, acutely suppressed LH levels in both wild-type and P4 receptor knockout ovariectomized mice; pituitary responsiveness to GnRH was retained during P4 treatment, indicating a hypothalamic action. Superfusion of GnRH-producing GT1-7 cells with medium containing 10(-7) m P4 produced a rapid reduction in GnRH release. Incubation with P4 (10(-9) to 10(-7) M) inhibited forskolin-stimulated cAMP accumulation; cotreatment with pertussis toxin prevented this effect. Treatment of GT1-7 cell membranes with P4 caused activation of an inhibitory G protein (G(i)), as shown by immunoprecipitation with a G(i) antibody of most of the increase in membrane-bound [(35)S]GTPgamma-S. Saturation binding analyses demonstrated the presence of a high affinity (K(d) 5.85 nM), limited capacity (Bmax 62.2 nM) binding site for P4. RT-PCR analysis revealed the presence of mRNAs encoding both isoforms of the membrane P4 receptors, mPRalpha and mPRbeta. Western blotting, immunocytochemistry, and flow cytometry experiments similarly revealed expression of mPR proteins in the plasma membranes of GT1-7 cells. Treatment with mPRalpha siRNA attenuated specific P4 binding to GT1-7 cell membranes and reversed the P4 inhibition of cAMP accumulation. Taken together, our results suggest that negative feedback actions of P4 include rapid PR(A/B)-independent effects on GnRH release that may in part be mediated by mPRs.

Vasopressin regulation of the proestrous luteinizing hormone surge in wild-type and Clock mutant mice.

Abstract In the female mouse, ovulation and estrous cyclicity are under both hormonal and circadian control. We have shown that mice with a mutation in the core circadian gene Clock have abnormal estrous cycles and do not have a luteinizing hormone (LH) surge on the afternoon of proestrus due to a defect at the hypothalamic level. In the present study, we tested the hypotheses that vasopressin (AVP) can act as a circadian signal to regulate the proestrous release of LH, and that this signal is deficient in the Clock mutant. We found that Avp expression in the suprachiasmatic nucleus (SCN) and AVP 1a receptor (Avpr1a) expression in the hypothalamus is reduced in Clock mutant mice compared to wild-type mice. Intracerebroventricular (i.c.v.) injection of AVP on the afternoon of proestrus is sufficient to induce LH secretion, which reaches surge levels in 50% of Clock mutant mice. The effect of AVP on the Clock mutant LH surge is mediated by AVPR1A, as co-infusion of AVP and an AVPR1A-specific antagonist prevents AVP induction of LH release, although infusion of an AVPR1A antagonist into wild-type mice failed to prevent a proestrous LH surge. These results suggest that reduced hypothalamic AVP signaling plays a role in the absence of the proestrous LH surge in Clock mutant mice. The results also support the hypothesis that AVP produced by the SCN may be a circadian signal that regulates LH release.

Neuroendocrine Consequences of Androgen Excess in Female Rodents

Androgens exert significant organizational and activational effects on the nervous system and behavior. Despite the fact that female mammals generally produce low levels of androgens, relative to the male of the same species, increasing evidence suggests that androgens can exert profound effects on the normal physiology and behavior of females during fetal, neonatal, and adult stages of life. This review examines the effects of exposure to androgens at three stages of development--as an adult, during early postnatal life and as a fetus, on reproductive hormone secretions in female rats. We examine the effects of androgen exposure both as a model of neuroendocrine sexual differentiation and with respect to the role androgens play in the normal female. We then discuss the hypothesis that androgens may cause epigenetic modification of estrogen target genes in the brain. Finally we consider the clinical consequences of excess androgen exposure in women.

Estrogen receptors in neuropeptide Y neurons: at the crossroads of feeding and reproduction

Hypothalamic neuropeptide Y (NPY) neurons function as physiological integrators in at least two different neuroendocrine systems - one governing feeding and the other controlling reproduction. Estrogen might modulate both systems by regulating NPY gene expression; it might reduce food intake by suppressing NPY expression, and evoke reproductive hormone surges by stimulating it. How can estrogen exert opposing effects in an ostensibly homogeneous NPY neuronal population? Recent work with immortalized NPY-producing cells suggests that the ratio of estrogen receptor alpha:estrogen receptor beta can determine the direction and temporal pattern of transcriptional responses to estrogen. Because this ratio might itself be physiologically regulated, these findings provide one explanation for multiple neuropeptidergic responses to a single steroid hormone.

Twenty-four-hour profiles of serum leptin in siberian and golden hamsters: photoperiodic and diurnal variations.

Serum leptin concentrations were obtained from male Siberian hamsters (Phodopus sungorus) and golden hamsters (a.k.a. Syrian, Mesocricetus auratus) housed on long [light:dark (LD) 16:8] and short (LD 6:18) photoperiods for 10-11 weeks. Blood samples were collected at 45-min intervals for 24 h from individual animals using an in-dwelling atrial catheter. In Siberian hamsters, exposure to short photoperiods as compared to long photoperiods reduced body weight (32.5 +/- 1.5 vs 47.7 +/- 1.1 g) and leptin (24-h mean: 5.3 +/- 0.4 ng/ml vs 18.6 +/- 2.1 ng/ml). Although photoperiod influenced the temporal distribution of leptin in golden hamsters, the main effect of photoperiod on leptin levels in golden hamsters did not reach significance (24-h mean: 7.1 +/- 1.0 ng/ml vs 5.1 +/- 0.8 ng/ml.). Body weights of golden hamsters did not vary significantly following exposure to short photoperiod for 11 weeks (178.3 +/- 3.6 g in LD 6:18 vs 177.8 +/- 7.3 g in LD 16:8). There was no nocturnal increase in serum leptin in either species. Marked interindividual differences were apparent in individual leptin profiles. Periodogram analysis revealed that only a few animals exhibited 24-h periodicities; the presence of a significant 24-h periodicity was more common in hamsters exposed to short days. Photoperiod-associated differences in the 24-hour profile of leptin secretion may be the result of photoperiod-associated changes in feeding behavior or metabolism. A full understanding of the regulation of leptin secretion in multiple time domains may enhance our understanding of the function of leptin.

Rapid photoperiod-induced increase in detectable GnRH mRNA-containing cells in Siberian hamster

To determine whether changes in gonadotropin-releasing hormone (GnRH) neurons are early indicators of photostimulation, Siberian hamsters were placed in short days (6:18-h light-dark) at 3 (experiment 1) or 6 (experiment 2) wk of age where they were held for 3 (experiment 1) or 4 (experiment 2) wk. Hamsters were then moved to long photoperiod (16:8-h light-dark). In experiment 1, brains were collected 1-21 days after transfer from short to long days. In experiment 2, brains were collected only on the second morning of long day exposure. Long and short day controls were included in both experiments. Cells containing GnRH mRNA, as visualized by in situ hybridization, were counted. As expected, there were no differences in the number of detectable GnRH mRNA-containing cells among animals chronically exposed to long or short photoperiods. However, on the second morning after transfer from short to long photoperiod, a positive shift in the distribution of GnRH mRNA-containing cells occurred relative to the respective controls in the two experiments. Increases in follicle-stimulating hormone secretion and gonadal growth occurred days later. In conclusion, a rapid but transient increase in the distribution of detectable GnRH mRNA-containing cells is an early step in the photostimulation of the hypothalamic-pituitary-gonadal axis.