Seasonal Variation of Melatonin Concentration and mRNA Expression of Melatonin-Related Genes in Developing Ovarian Follicles of Mares Kept under Natural Photoperiods in the Southern Hemisphere.

This study investigated the seasonal variations in mRNA expression of FSH (Fshr), LH (Lhr) receptors, melatonin (Mt1 and Mt2) receptors, melatonin-synthetizing enzymes (Asmt and Aanat) and melatonin concentration in developing follicles from mares raised in natural photoperiods. For one year, ultrasonographic follicular aspiration procedures were performed monthly, and small (<20 mm), medium (20 to 35 mm) and large (>35 mm) follicles were recovered from five mares. One day before monthly sample collections, an exploratory ultrasonography conducted to record the number and the size of all follicles larger than 15 mm. The total number of large follicles were higher during the spring/summer (8.2 ± 1.9) than during autumn/winter (3.0 ± 0.5). Compared to autumn/winter seasons, there was an increase of Fshr and Aanat mRNA expressions in small, medium and large follicles, an increase of Lhr and Asmt mRNA expressions in medium and large follicles and an increase of Mt1 and Mt2 mRNA expressions in small and large follicles during spring/summer. The melatonin levels in follicular fluid were also higher during the spring/summer seasons. The present data show that melatonin locally upregulates the mRNA expression of Mt1 and Mt2 receptors and melatonin-forming enzymes in mare developing follicles during reproductive seasons.

Arginine-vasopressin-expressing neurons in the murine suprachiasmatic nucleus exhibit a circadian rhythm in network coherence in vivo.

The suprachiasmatic nucleus (SCN) is composed of functionally distinct subpopulations of GABAergic neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons, and which aspects result from neuronal interaction within a network. Here, we utilize in vivo miniaturized microscopy to measure fluorescent GCaMP-reported calcium dynamics in arginine vasopressin (AVP)-expressing neurons in the intact SCN of awake, behaving mice. We report that SCN AVP neurons exhibit periodic, slow calcium waves which we demonstrate, using in vivo electrical recordings, likely reflect burst firing. Further, we observe substantial heterogeneity of function in that AVP neurons exhibit unstable rhythms, and relatively weak rhythmicity at the population level. Network analysis reveals that correlated cellular behavior, or coherence, among neuron pairs also exhibited stochastic rhythms with about 33% of pairs rhythmic at any time. Unlike single-cell variables, coherence exhibited a strong rhythm at the population level with time of maximal coherence among AVP neuronal pairs at CT/ZT 6 and 9, coinciding with the timing of maximal neuronal activity for the SCN as a whole. These results demonstrate robust circadian variation in the coordination between stochastically rhythmic neurons and that interactions between AVP neurons in the SCN may be more influential than single-cell activity in the regulation of circadian rhythms. Furthermore, they demonstrate that cells in this circuit, like those in many other circuits, exhibit profound heterogenicity of function over time and space.

Identification of insulin-regulated aminopeptidase (IRAP) in the rat pineal gland and the modulation of melatonin synthesis by angiotensin IV

A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-ß-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.

Identification of insulin-regulated aminopeptidase (IRAP) in the rat pineal gland and the modulation of melatonin synthesis by angiotensin IV

A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-ß-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.

Chronic treatment with dexamethasone alters clock gene expression and melatonin synthesis in rat pineal gland at night.

BACKGROUND: Melatonin is a neuroendocrine hormone that regulates many functions involving energy metabolism and behavior in mammals throughout the light/dark cycle. It is considered an output signal of the central circadian clock, located in the suprachiasmatic nucleus of the hypothalamus. Melatonin synthesis can be influenced by other hormones, such as insulin and glucocorticoids in pathological conditions or during stress. Furthermore, glucocorticoids appear to modulate circadian clock genes in peripheral tissues and are associated with the onset of metabolic diseases. In the pineal gland, the modulation of melatonin synthesis by clock genes has already been demonstrated. However, few studies have shown the effects of glucocorticoids on clock genes expression in the pineal gland. RESULTS: We verified that rats treated with dexamethasone (2 mg/kg body weight, intraperitoneal) for 10 consecutive days, showed hyperglycemia and pronounced hyperinsulinemia during the dark phase. Insulin sensitivity, glucose tolerance, melatonin synthesis, and enzymatic activity of arylalkylamine N-acetyltransferase, the key enzyme of melatonin synthesis, were reduced. Furthermore, we observed an increase in the expression of Bmal1, Per1, Per2, Cry1, and Cry2 in pineal glands of rats treated with dexamethasone. CONCLUSION: These results show that chronic treatment with dexamethasone can modulate both melatonin synthesis and circadian clock expression during the dark phase.

A Short-Day Photoperiod Delays the Timing of Puberty in Female Mice via Changes in the Kisspeptin System.

The reproduction of seasonal breeders is modulated by exposure to light in an interval of 24 h defined as photoperiod. The interruption of reproductive functions in seasonally breeding rodents is accompanied by the suppression of the Kiss1 gene expression, which is known to be essential for reproduction. In non-seasonal male rodents, such as rats and mice, short-day photoperiod (SP) conditions or exogenous melatonin treatment also have anti-gonadotropic effects; however, whether photoperiod is able to modulate the puberty onset or Kiss1 gene expression in mice is unknown. In the present study, we investigated whether photoperiodism influences the sexual maturation of female mice via changes in the kisspeptin system. We observed that SP condition delayed the timing of puberty in female mice, decreased the hypothalamic expression of genes related to the reproductive axis and reduced the number of Kiss1-expressing neurons in the rostral hypothalamus. However, SP also reduced the body weight gain during development and affected the expression of neuropeptides involved in the energy balance regulation. When body weight was recovered via a reduction in litter size, the timing of puberty in mice born and raised in SP was advanced and the effects in hypothalamic mRNA expression were reverted. These results suggest that the SP delays the timing of puberty in female mice via changes in the kisspeptin system, although the effects on hypothalamic-pituitary-gonadal axis are likely secondary to changes in body weight gain.