Luis Valladares

Melatonin in the rat testis: Evidence for local synthesis

The vertebrate pineal gland rhythmically produces melatonin, a hormone involved in regulation of several physiological and behavioral processes. Melatonin is synthesized from serotonin essentially by two enzymatic steps involving N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase activities. We have previously demonstrated the presence of melatonin binding sites in the rat testes, and an inhibitory effect of melatonin on testicular gonadotrophin-stimulated androgen production. It is unknown whether these effects are mediated by melatonin synthesized locally or by melatonin from pineal origin. To assess the potential capacity of melatonin production by the testis, we used radiolabeled precursors to measure the activities of N-acetyltransferase and hydroxyindole-O-methyltransferase. The production of N-acetylserotonin was time-dependent during over 10 min of incubation. Melatonin had a linear increase throughout the 30 min incubation period with S-adenosyl-L-[methyl-14C]methionine. Identities of melatonin and N-acetylserotonin were confirmed by thin-layer chromatography. The ability of the testis to synthesize melatonin during sexual maturation was also analyzed. When activity of NAT was expressed per mg of protein, the maximal activity was observed on day 40. In contrast, when activity of NAT is expressed by the testis, the amount of NAT increased to peak on day 40 and remained elevated through day 70. We determined that both activities were predominantly localized in interstitial cells. NAT activity in seminiferous tubules was substantially decreased, representing 6.4% of NAT activity in interstitial cells. We concluded that rat testes are capable of synthesizing melatonin due to the presence of the enzymes necessary for the transformation of serotonin to melatonin.

Epigenetic and phenotypic changes result from a continuous pre and post natal dietary exposure to phytoestrogens in an experimental population of mice

BackgroundDevelopmental effects of exposure to endocrine disruptors can influence adult characters in mammals, but could also have evolutionary consequences. The aim of this study was to simulate an environmental exposure of an experimental population of mice to high amounts of nutritional phytoestrogens and to evaluate parameters of relevance for evolutionary change in the offspring. The effect of a continuous pre- and post-natal exposure to high levels of dietary isoflavones was evaluated on sexual maturity, morphometric parameters and DNA methylation status in mice. Adult mice male/female couples were fed ad libitum either with control diet (standard laboratory chow) or ISF diet (control diet plus a soy isoflavone extract at 2% (w/w) that contained the phytoestrogens genistein and daidzein). In the offspring we measured: i) the onset of vaginal opening (sexual maturation) in females, ii) weight and size in all pups at 7, 14, 21 and 42 days post-natal (dpn) and iii) DNA methylation patterns in skeletal α-actin (Acta1), estrogen receptor-α and c-fos in adults (42 dpn).ResultsVaginal opening was advanced in female pups in the ISF group, from 31.6 ± 0.75 dpn to 25.7 ± 0.48. No differences in size or weight at ages 7, 14 or 21 dpn were detected between experimental groups. Nevertheless, at age 42 dpn reduced size and weight were observed in ISF pups, in addition to suppression of normal gender differences in weight seen in the control group (males heavier that females). Also, natural differences seen in DNA methylation at Acta1 promoter in the offspring originated in the control group were suppressed in the ISF group. Acta1 is known to be developmentally regulated and related to morphomotric features.ConclusionThis study demonstrates in mammals that individuals from a population subjected to a high consumption of isoflavones can show alterations in characters that may be of importance from an evolutionary perspective, such as epigenetic and morphometric characters or sexual maturation, a life history character.

Environmental signaling and evolutionary change: can exposure of pregnant mammals to environmental estrogens lead to epigenetically induced evolutionary changes in embryos?

Summary DNA methylation is one of the epigenetic and hereditary mechanisms regulating genetic expression in mammalian cells. In this review, we propose how certain natural agents, through their dietary consumption, could induce changes in physiological aspects in mammalian mothers, leading to alterations in DNA methylation patterns of the developing fetus and to the emergence of new phenotypes and evolutionary change. Nevertheless, we hypothesize that this process would require (i) certain key periods in the ontogeny of the organism where the environmental stimuli could produce effects, (ii) particular environmental agents as such stimuli, and (iii) that a genomic persistent change be consequently produced in a population. Depending on the persistence of the environmental stimuli and on whether the affected genes are imprinted genes, induced changes in DNA methylation patterns could become persistent. Moreover, some fragments could be more frequently methylated than others over several generations, leading to biased base change and evolutionary consequences.

Effect of melatonin on rat spinal cord nociceptive transmission.

Melatonin has been shown to exert potent antinociception but the sites and mechanisms of action underlying this effect have not yet been clarified. The effect of melatonin on spinal cord nociceptive transmission was studied in rats by assessing wid-up activity in a C-fiber reflex responses paradigm evoked by repetitive (0.6 Hz) electric stimulation. Intraperitoneal administration of 1.25, 2.5, 5.0 and 10.0 mg/kg melatonin induced a dose-dependent inhibition of spinal wind-up activity, the higher dose of the drug used being able to depress completely the C reflex gain. Results indicate that melatonin markedly depresses spinal wind-up in rats, probably through hyperpolarization of dorsal horn neurons consecutive to melatonin binding to membrane receptors, and/or via intracellular interference with a NMDA receptor-dependent nitric oxide generating pathway.

Mild prenatal protein malnutrition increases α2C‐adrenoceptor density in the cerebral cortex during postnatal life and impairs neocortical long‐term potentiation and visuo‐spatial performance in rats

Mild reduction in the protein content of the mothers diet from 25 to 8% casein, calorically compensated by carbohydrates, does not alter body and brain weights of rat pups at birth, but leads to significant enhancements in the concentration and release of cortical noradrenaline during early postnatal life. Since central noradrenaline and some of its receptors are critically involved in long‐term potentiation (LTP) and memory formation, this study evaluated the effect of mild prenatal protein malnutrition on the α2C‐adrenoceptor density in the frontal and occipital cortices, induction of LTP in the same cortical regions and the visuo‐spatial memory. Pups born from rats fed a 25% casein diet throughout pregnancy served as controls. At day 8 of postnatal age, prenatally malnourished rats showed a threefold increase in neocortical α2C‐adrenoceptor density. At 60 days‐of‐age, α2C‐adrenoceptor density was still elevated in the neocortex, and the animals were unable to maintain neocortical LTP and presented lower visuo‐spatial memory performance. Results suggest that overexpression of neocortical α2C‐adrenoceptors during postnatal life, subsequent to mild prenatal protein malnutrition, could functionally affect the synaptic networks subserving neocortical LTP and visuo‐spatial memory formation.

Melatonin-induced inhibition of spinal cord synaptic potentiation in rats is MT2 receptor-dependent.

Systemically administered melatonin has been reported to produce antinociception and to inhibit spinal nociceptive transmission in rats. The present study was designed to investigate in anesthetized rats (i) whether intrathecally administered melatonin can depress synaptic potentiation (wind-up) in the spinal cord, and (ii) whether this effect is prevented by intrathecal (i.t.) administration of the MT2 receptor antagonist luzindole. Results showed that melatonin i.t. (10, 30 and 90 microg) induced dose-dependent inhibition of wind-up activity (ED50=52.06 microg i.t.), an effect that was prevented by 100 microg i.t. of luzindole. Since wind-up is dependent on NMDA receptor activation, the results suggest that melatonin can interfere with the NMDA-mediated glutamatergic component of pain transmission in rat spinal cord by acting on MT2 receptors.

α2-Adrenoceptor modulation of long-term potentiation elicited in vivo in rat occipital cortex

Pretreatment with the alpha(2)-adrenoceptor agonist clonidine (31.25, 62.5, or 125 microg/kg, i.p.) dose-dependently reduced long-term potentiation (LTP) elicited in vivo in the occipital cortex of anesthetized rats, whereas pretreatment with the alpha(2)-adrenoceptor antagonist yohimbine (0.133, 0.4, or 1.2 mg/kg, i.p.) increased neocortical LTP in a dose-dependent fashion. These effects could be related to the reported disruptive and facilitatory actions induced on memory formation by pretreatment with alpha(2)-adrenoceptor agonists and antagonists, respectively.

Fetal undernutrition induces overexpression of CRH mRNA and CRH protein in hypothalamus and increases CRH and corticosterone in plasma during postnatal life in the rat

Prenatal undernutrition induces a variety of cardiovascular alterations in mammals when adults, including hypertension and hypercortisolism, which are thought to be caused by decreased glucocorticoid feedback control of the hypothalamus-pituitary-adrenal (HPA) axis programmed during fetal life. Hypothalamic CRH seems to be involved in blood pressure elevation of spontaneously hypertensive rats and in primary hypertension of humans, but the influence of prenatal undernutrition on CRH expression has deserved little attention. Here, we studied the expression of both CRH mRNA and CRH protein in the hypothalamus of neonatal and juvenile offspring of rats undernourished during fetal life, as well as the plasma levels of CRH and corticosterone. Prenatal undernutrition of pups was induced by submitting pregnant rats to diet restriction (10g daily of 21% protein standard laboratory diet). Pups born from dams with free access to the standard laboratory diet served as controls. At day 2 of postnatal age, undernourished pups showed lower body and brain weights, but higher plasma CRH and corticosterone than normal pups. At day 40 of age, brain weight was significantly decreased in the undernourished rats, while plasma corticosterone, plasma CRH and systolic pressure were significantly increased in these animals. At days 2 and 40 of postnatal age, increased CRH mRNA expression and CRH concentration were found in the hypothalamus of undernourished rats. Results indicate that, in the rat, prenatal undernutrition led to fetal programming of CRH overexpression, a neuropeptide serving as activating signal to the HPA axis and/or to extrahypothalamic brain regions concerned with cardiovascular regulation.

Perinatal neuroendocrine regulation. Development of the circadian time-keeping system

During gestation, the perinatal neuroendocrine axis keeps clock time. In primates, the suprachiasmatic nucleus (biological clock in mammals), shows oscillatory function by midgestation. There is evidence in rodents that the mother, during pregnancy, entrains the fetal suprachiasmatic nucleus (SCN) and newborn circadian rhythms. We are investigating the role of maternal melatonin as an entraining signal for the newborn circadian time-keeping system in the Cebus apella (New World non-human primate). Twenty-four hour rhythms of temperature and cortisol are present in the 4 days old C. apella newborn. Preliminary data suggests that inhibition of maternal melatonin by exposing pregnant females to constant light alters these rhythms. We have found binding sites for melatonin and expression of mRNA for Mel 1A receptor in hypothalamus, kidney and testis. These preliminary results suggest that maternal melatonin may play a role in relating the perinatal circadian time-keeping system to environmental signals.

GAMETOGENESIS AND SEX STEROID PROFILES IN CULTURED COHO SALMON (ONCORHYNCHUS KISUTCH, WALBAUM)

The gametogenesis of a 2-year-old coho salmon broodstock population cultured in a fish farm in southern Chile was studied. Gonadosomatic index (GSI), microscopic gonadal traits, and serum levels of estradiol-17β(E2), testosterone, and 17α,20β-dihydroxy-4-pregnene-3-one (17,20P) in both sexes were recorded beginning 9 months before spawning in bimonthly samplings. Maximum GSI means were reached during May, the month of spawning, with 16.8 ± 4.1% for females and 8.4 ± 0.8% for males, both values within the range described in the literature. GSI in males, however, was triple that of females during January, showing a faster rate of gonadal growth in males in early summer. Gonadal microscopy for both sexes showed stages corresponding to those described by different authors for other salmonids such as rainbow trout. The secondary vitellogenesis period was 4 to 5 months and corresponded with the short vitellogenesis model described in rainbow trout for broodstocks maturing at 2 years of age. The serum profiles of sex steroids in both sexes are consistent with those described in coho salmon and other salmonid species. In females, E2 and 17,20P show opposite profiles, reaching their maximum levels (E2: 45.13 ± 11.3 ng/ml; 17,20P: 24.47 ± 7.34 ng/ml) during vitellogenesis (March) and ovulation (May), respectively. In both sexes, testosterone concentration shows maximum levels in May (females: 61.68 ± 15.75 ng/ml; males: 107.8 ± 11.6 ng/ml), suggesting the physiological importance of this hormone during maturation, either directly or as a substrate for the synthesis of other hormones. In males, the maximum level of 17,20P (22.33 ± 4.5 ng/ml) also occurs in May during total spermiation, which confirms its role in semen production and semen fluid regulation as described in the literature about this hormone in salmonid males. On the basis of the data obtained, a reproductive pattern is proposed for 2-year-old salmon cultured in southern Chile. J. Exp. Zool. 280:429–438, 1998.