Melatonin, Its Beneficial Effects on Embryogenesis from Mitigating Oxidative Stress to Regulating Gene Expression.

Embryogenesis is a complex multi-stage process regulated by various signaling molecules including pineal and extrapineal melatonin (MT). Extrapineal MT is found in the placenta and ovaries, where it carries out local hormonal regulation. MT is necessary for normal development of oocytes, fertilization and subsequent development of human, animal and avian embryos. This review discusses the role of MT as a regulator of preimplantation development of the embryo and its implantation into endometrial tissue, followed by histo-, morpho- and organogenesis. MT possesses pronounced antioxidant properties and helps to protect the embryo from oxidative stress by regulating the expression of the NFE2L2, SOD1, and GPX1 genes. MT activates the expression of the ErbB1, ErbB4, GJA1, POU5F1, and Nanog genes which are necessary for embryo implantation and blastocyst growth. MT induces the expression of vascular endothelial growth factor (VEGF) and its type 1 receptor (VEGF-R1) in the ovaries, activating angiogenesis. Given the increased difficulties in successful fertilization and embryogenesis with age, it is of note that MT slows down ovarian aging by increasing the transcription of sirtuins. MT administration to patients suffering from infertility demonstrates an increase in the effectiveness of in vitro fertilization. Thus, MT may be viewed as a key factor in embryogenesis regulation, including having utility in the management of infertility.

The Lhx4 homeobox transcript in the rat pineal gland: Adrenergic regulation and impact on transcripts encoding melatonin-synthesizing enzymes.

Homeobox genes generally encode transcription factors involved in regulating developmental processes. In the pineal gland, a brain structure devoted to nocturnal melatonin synthesis, a number of homeobox genes are also expressed postnatally; among these is the LIM homeobox 4 gene (Lhx4). We here report that Lhx4 is specifically expressed in the postnatal pineal gland of rats and humans. Circadian analyses revealed a fourfold rhythm in Lhx4 expression in the rat pineal gland, with rhythmic expression detectable from postnatal day 10. Pineal Lhx4 expression was confirmed to be positively driven by adrenergic signaling, as evidenced by in vivo modulation of Lhx4 expression by pharmacological (isoprenaline injection) and surgical (superior cervical ganglionectomy) interventions. In cultured pinealocytes, Lhx4 expression was upregulated by cyclic AMP, a second messenger of norepinephrine. By use of RNAscope technology, Lhx4 transcripts were found to be exclusively localized in melatonin-synthesizing pinealocytes. This prompted us to investigate the possible role of Lhx4 in regulation of melatonin-producing enzymes. By use of siRNA technology, we knocked down Lhx4 by 95% in cultured pinealocytes; this caused a reduction in transcripts encoding the melatonin-producing enzyme arylalkylamine N-acetyl transferase (Aanat). Screening the transcriptome of siRNA-treated pinealocytes by RNAseq revealed a significant impact of Lhx4 on the phototransduction pathway and on transcripts involved in development of the nervous system and photoreceptors. These data suggest that rhythmic expression of Lhx4 in the pineal gland is controlled via an adrenergic-cyclic AMP mechanism and that Lhx4 acts to promote nocturnal melatonin synthesis.

Melatonin and male reproduction.

Melatonin is a neurohormone secreted by the pineal gland whose concentrations in the body are regulated by both the dark-light and seasonal cycles. The reproductive function of seasonal breeding animals is clearly influenced by the circadian variation in melatonin levels. Moreover, a growing body of evidence indicates that melatonin has important effects in the reproduction of some non-seasonal breeding animals. In males, melatonin affects reproductive regulation in three main ways. First, it regulates the secretion of two key neurohormones, GnRH and LH. Second, it regulates testosterone synthesis and testicular maturation. Third, as a potent free radical scavenger that is both lipophilic and hydrophilic, it prevents testicular damage caused by environmental toxins or inflammation. This review summarizes the existing data on the possible biological roles of melatonin in male reproduction. Overall, the literature data indicate that melatonin affects the secretion of both gonadotropins and testosterone while also improving sperm quality. This implies that it has important effects on the regulation of testicular development and male reproduction.

Role of melatonin in embryo fetal development.

Melatonin is an indoleamine produced by the pineal gland and secreted in a circadian manner. In the past few decades, research over this topic has been enhanced. Melatonin has many important roles in the human physiology: regulator of the circadian rhythms, sleep inducer, antioxidant, anticarcinogenic. This paper reviews the involvement of melatonin in embryo fetal development. The pineal gland develops completely postpartum, so both the embryo and the fetus are dependent on the maternal melatonin provided transplacentally. Melatonin appears to be involved in the normal outcome of pregnancy beginning with the oocyte quality and finishing with the parturition. Its pregnancy night-time concentrations increase after 24 weeks of gestation, with significantly high levels after 32 weeks. Melatonin receptors are widespread in the embryo and fetus since early stages. There is solid evidence that melatonin is neuroprotective and has a positive effect on the outcome of the compromised pregnancies. In addition, chronodisruption leads to a reproductive dysfunction. Thus, the influence of melatonin on the developing human fetus may not be limited to the entertaining of circadian rhythmicity, but further studies are needed.

Sleep and hormonal changes in aging.

Age-related sleep and endocrinometabolic alterations frequently interact with each other. For many hormones, sleep curtailment in young healthy subjects results in alterations strikingly similar to those observed in healthy old subjects not submitted to sleep restriction. Thus, recurrent sleep restriction, which is currently experienced by a substantial and rapidly growing proportion of children and young adults, might contribute to accelerate the senescence of endocrine and metabolic function. The mechanisms of sleep-hormonal interactions, and therefore the endocrinometabolic consequences of age-related sleep alterations, which markedly differ from one hormone to another, are reviewed in this article.

[Age-dependent morphology of human pineal gland: supravital study].

On the base of analysis of 5784 events of diagnostic magnetic-resonance tomography studies of the head of patients in radio diagnosis departments the database is formed. Only events (n=411) without cerebral, oncology, endocrine and other pathology are taken in database. The material was grouped to time and date of the study, sex and age in accordance with generally accepted categorization. Maximum linear sizes of pineal gland and hypophysis cerebri in sagittal, axial and coronar projection were measured in all events; volumes of the organs were calculated on the formula of a ball. It is defined that the volume of pineal gland increases from birth till 17-21 year age, gradually falls till the second mature age and is getting stable in old age. The normative factors of the volume of pineal gland and hypophysis cerebri for 8 age groups are determined. "Brain sand" and false cysts in pineal gland can be observed in all age groups. The petrification degree of pineal gland, as of computer tomography, varies from 30 to 277 ed. HV. For the factor of pineal gland volume and factor of cysts frequency in pineal gland a puberty "collapse" is typical, mainly in men.

Metallothionein-I-II expression in young and adult bovine pineal gland.

Aging is characterized, among other features, by an increased concentration of metal ions in the brain that may contribute to a greater increase in free radicals production. The present paper reports data regarding the concentration of some relevant metal ions (Cu, Fe, Mn, Zn), as well as the immunopositivity of metallothionein-I-II and GFAP in the bovine pineal gland with respect to animal aging. The pineal gland of young bovines displays several immunoreactive metallothionein-I-II positive elements in the parenchyma, whose number decreases with age. We also report that a well defined group of neurons bordering the third ventricle and located close to the subcommissural organ shows an intense metallothionein-I-II immunopositivity. The presence of metallothionein-I-II was confirmed by means of liquid chromatography coupled to tandem mass spectrometry. In particular, it proved possible to identify the amino acid sequences of the unique tryptic peptide not containing cysteine and two pepsin fragments containing cysteines. In conclusion, our data suggest the presence of a metallothionein-I-II expressing system in the pineal gland and ventricle-adjacent areas of the bovine epithalamus might possibly be related to the anti-aging effects of melatonin.

Influência da melatonina sobre o desenvolvimento corporal e ósseo de ratos / Ineluence of melatonin on body and bone development of rats

Objetivo: A glândula pineal produz o hormônio melatonina (MLT), que apresenta efeitos demonstrados sobre diversas células, entre elas os adipócitos e osteoblastos. Este trabalho objetivou verificar a influência da MLT no crescimento e desenvolvimento corporal e ósseo em ratos. Método e Resultados: Ratos Wistar machos foram submetidos a pinealectomia (PINX) ou cirurgia fictícia (CONTR) aos 5 dias do nascimento e estudados quanto ao peso e comprimento corporal; comprimento, espessura da tábua óssea e do disco epifisário da tíbia após 10, 20, 30, 60, 90 e 120 dias da operação. O peso dos animais PINX foi maior que o dos CONTR aos 100 e 120 dias. Os animais PINX tiveram ainda menor espessura da tábua óssea aos 60, 90 e 120 dias e menor espessura do disco epifisário, aos 90 e 120 dias após o procedimento cirúrgico, comparados aos animais CONTR. Conclusões: A falta de MLT resultou em aumento do peso corporal, provavelmente pela maior captação de ácidos graxos pelos adipócitos e redução da termogênese: menor espessura da tábua óssea e disco epifisario da tibia pela redução da atividade de condrócitos e osteoblastos. Estes efeitos poderiam ser diretos e/ou indiretos, e seu esclarecimento demanda mais estudos.

Five days old Wistar male rats were either pinealectomized or submitted to a sham operation. The animals were followed up to 120 days after surgery, being weighed and measured every 10 days. Groups of animals were then sacrificed at 10, 20, 30, 60, 90, and 120 days after the surgery. Thicknesses of the bone board and the epiphyseal plate of the tibia were measured. Results: (1) The average weight of the pinealectomized animals was greater than that of the control animals between 100 and 120 days after surgery; (2) There was no difference between the groups in the lengths of the body and of the back paw of the animals; (3) The length of the tibia was the same in both groups but the thickness of bone board after 60, 90, and 120 days of the surgery and the thickness of the epiphysial plate after 90 and 120 days of the surgery were smaller in the pinealectomized animals when compared with the control groups. Conclusion: the pineal gland, through the secretion of melatonin, participates in the bone development in rats by unknown mechanisms.

Ciliary neurotrophic factor inhibits differentiation of photoreceptor-like cells in rat pineal glands in vitro.

Ciliary neurotrophic factor (CNTF) is a unique member of the interleukin-6 (IL-6) family, whose receptor subunit for ligand binding is exclusively expressed in the nervous system and muscle. The role of CNTF in mammalian development remains unknown. We recently reported the specific expression of CNTF in the pineal gland and eyes. To further examine the expression pattern and role of CNTF in development, we prepared a polyclonal antibody against rat CNTF, performed western blotting with this antibody, and confirmed a strong and specific expression of the CNTF protein in pineal glands and a moderate expression in the eyes among the various tissues examined in newborn rats. In pineal organ cultures of newborn rats, exogenously added recombinant rat CNTF potently inhibited the differentiation of photoreceptor-like cells in a dose-dependent manner, while CNTF did not influence the survival of pineal cells. Among several cell growth factors known to have a similar effect in retinal cultures examined, strong inhibitory effects were seen only with CNTF and the leukemia inhibitory factor (LIF), both of which belong to the IL-6 cytokine family. This inhibitory effect was the strongest during three to 6 days of culture when CNTF was added to these cultures. These results suggest that CNTF plays an inhibitory role in the development of photoreceptor-like cells in early postnatal rat pineal glands.

Maternal photoperiodic exposures alter the neonatal growth, pineal functions and sexual development of the Indian palm squirrel F. pennanti. Short communication.

The phenomenon of maternal transfer of photic information to their young ones is still an enigma. Existing reports in some rodents of temperate zone suggest that photoperiodic condition experienced by mother during their gestation period influences the pineal physiology of fetus, but nothing has been reported about the growth and sexual development of pups. Present experiment for the first time explains the effect of gestational photoperiod on the growth and sexual development of pups from a seasonally breeding tropical rodent F. pennanti. The results suggest that, constant light (LL; 24L: OD) and long day length (LDL; 14L: 10D) experiencing mother conveyed the photic information to young ones to inhibit the pineal function, while short day length (SDL; 10L: 14D) stimulated the pineal function in pups. Altered pineal functions of pups ultimately interfered with their growth and sexual maturation. Most interestingly, the pups delivered by DD experiencing mothers and then reared under same condition, at the age of 40 days attained a level of growth and sexual maturity equivalent to the growth and sexual maturation of 60 days old pups under natural day length (NDL) condition. Therefore, we may suggest that the photic information perceived by the mother alter her normal melatonin level, hence, passing through placenta melatonin influences the growth and sexual maturation of the young ones.

Rhythmic variation in beta1-adrenergic receptor mRNA levels in the rat pineal gland: circadian and developmental regulation.

In the rat pineal gland noradrenaline is released in large quantities from sympathetic nerve endings at the onset of darkness, thereby driving rhythmic melatonin synthesis with elevated levels at night-time. Upon release, noradrenaline interacts with postsynaptic beta1-adrenergic receptors to activate the cyclic AMP signalling pathway. Well characterized third messengers of this signalling cascade affect cyclic AMP-inducible genes that are crucially involved in initiation, maintenance and termination of hormone production. Among these third messengers are CREB (cyclic AMP responsive element binding protein) as an activating and ICER (inducible cyclic AMP early repressor) as an inhibitory transcription factor. Because a cyclic AMP-inducible promoter element is present on the beta1-adrenergic receptor gene, the expression of the receptor itself may be under control of the cyclic AMP-signalling pathway. By in situ hybridization, Northern blot analysis and RT-PCR we demonstrate a day/night rhythm in beta1-adrenergic receptor mRNA in the rat pineal gland with elevated levels during the dark period. As this rhythm persists, under constant darkness but is abolished upon removal of the sympathetic innervation, it is truly circadian. A marked day/night difference in the levels of beta1-adrenergic receptor mRNA becomes evident only after postnatal day 10, coinciding with the appearance of a functional cyclic AMP signalling pathway in the rat pineal gland. Furthermore, targeting ICER expression by transfection of pinealocytes with an antisense ICER construct, clearly indicates that the levels of the beta1-adrenergic receptor mRNA are regulated by the cyclic AMP-signalling pathway in a feedback mechanism.

Morphology of the brain and sense organs in the snailfish Paraliparis devriesi: neural convergence and sensory compensation on the Antarctic shelf.

The Antarctic snailfish Paraliparis devriesi (Liparidae) is an epibenthic species, inhabiting depths of 500-650 m in McMurdo Sound. Liparids are the most speciose fish family in the Antarctic Region. We examine the gross morphology and histology of the sense organs and brain of P. devriesi and provide a phyletic perspective by comparing this morphology to that of four scorpaeniforms and of sympatric perciform notothenioids. The brain has numerous derived features, including well-developed olfactory lamellae with thick epithelia, large olfactory nerves and bulbs, and large telencephalic lobes. The retina contains only rods and exhibits a high convergence ratio (82:1). Optic nerves are small and nonpleated. The tectum is small. The corpus of the cerebellum is large, whereas the valvula is vestigial. The rhombencephalon and bulbospinal junction are extended and feature expanded vagal and spinal sensory lobes as well as hypertrophied dorsal horns and funiculi in the rostral spinal cord. The lower lobes of the pectoral fins have taste buds and expanded somatosensory innervation. Although the cephalic lateral line and anterior lateral line nerve are well developed, the trunk lateral line and posterior lateral line nerve are reduced. Near-field mechanoreception by trunk neuromasts may have been compromised by the watery, gelatinous subdermal extracellular matrix employed as a buoyancy mechanism. The expanded somatosensory input to the pectoral fin may compensate for the reduction in the trunk lateral line. The brains of P. devriesi and sympatric notothenioids share well-developed olfactory systems, an enlarged preoptic-hypophyseal axis, and subependymal expansions. Although the functional significance is unknown, the latter two features are correlated with habitation of the deep subzero waters of the Antarctic shelf.

Reactive oxygen intermediates, molecular damage, and aging. Relation to melatonin.

Melatonin, the chief secretory product of the pineal gland, is a direct free radical scavenger and indirect antioxidant. In terms of its scavenging activity, melatonin has been shown to quench the hydroxyl radical, superoxide anion radical, singlet oxygen, peroxyl radical, and the peroxynitrite anion. Additionally, melatonin's antioxidant actions probably derive from its stimulatory effect on superoxide dismutase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase and its inhibitory action on nitric oxide synthase. Finally, melatonin acts to stabilize cell membranes, thereby making them more resistant to oxidative attack. Melatonin is devoid of prooxidant actions. In models of oxidative stress, melatonin has been shown to resist lipid peroxidation induced by paraquat, lipopolysaccharide, ischemia-reperfusion, L-cysteine, potassium cyanide, cadmium chloride, glutathione depletion, alloxan, and alcohol ingestion. Likewise, free radical damage to DNA induced by ionizing radiation, the chemical carcinogen safrole, lipopolysaccharide, and kainic acid are inhibited by melatonin. These findings illustrate that melatonin, due to its high lipid solubility and modest aqueous solubility, is able to protect macromolecules in all parts of the cell from oxidative damage. Melatonin also prevents the inhibitory action of ruthenium red at the level of the mitochondria, thereby promoting ATP production. In humans, the total antioxidative capacity of serum is related to melatonin levels. Thus, the reduction in melatonin with age may be a factor in increased oxidative damage in the elderly.

Developmental expression pattern of phototransduction components in mammalian pineal implies a light-sensing function.

Whereas the pineal organs of lower vertebrates have been shown to be photosensitive, photic regulation of pineal function in adult mammals is thought be mediated entirely by retinal photoreceptors. Extraretinal regulation of pineal function has been reported in neonatal rodents, although both the site and molecular basis of extraretinal photoreception have remained obscure. In this study we examine the developmental expression pattern of all of the principal components of retinal phototransduction in rat pineal via cRNA in situ hybridization. All of the components needed to reconstitute a functional phototransduction pathway are expressed in the majority of neonatal pinealocytes, although the expression levels of many of these genes decline dramatically during development. These findings strongly support the theory that the neonatal rat pineal itself is photosensitive. In addition, we observe in neonatal pinealocytes the expression of both rod-specific and cone-specific phototransduction components, implying the existence of functionally different subtypes of pinealocytes that express varying combinations of phototransduction enzymes.

Expression of brain nitric oxide synthase in the developing rat pineal gland coincides with the appearance of the adrenergic cyclic guanosine 3',5'-monophosphate response.

In the developing rat pineal gland, norepinephrine (NE)-stimulated cyclic guanosine 3',5'-monophosphate (cGMP) formation appears 2 weeks later than that of cyclic adenosine 3',5'-monophosphate (cAMP) formation. Since NE-stimulated cGMP formation requires formation of nitric oxide (NO) in the adult rat pineal, we investigated the developmental appearance of the NO-forming enzyme brain NO-synthase (NOS) to find out whether the delayed cGMP response results from the late appearance of NOS. In this study, we investigated pineal glands of rats aged 8-21 days and 8 weeks. Western blot analysis revealed that NOS-immunoreactivity (NOS-IR) was barely detectable until day 12. On day 15 NOS-IR exhibited the same intensity as in adult rats. Expression of NOS at this postnatal stage appears to be responsible for the developmental appearance of the adrenergic cGMP response.

Development of the pineal gland: measurement with MR.

PURPOSE: To use MR imaging in the analysis of the size of the normal pineal gland in infants, children, and adolescents. METHODS: We retrospectively analyzed the size of the pineal gland in 249 patients (129 male and 120 female) aged 2 weeks to 20 years old. The maximum length (L), height (H), and width (W) of the gland were determined from a combination of sagittal, coronal, and axial MR images obtained on a 1.5-T scanner. The volume was calculated by using the formula 1/2 x L x H x W. RESULTS: The size of the pineal gland was significantly smaller in patients younger than 2 years old than in older patients. The size of the pineal gland increased until 2 years of age and remained stationary between the ages of 2 and 20 years. We found a large variation in size among all age groups. No difference in size was noted between males and females. CONCLUSION: This study establishes norms for pineal gland size in infants younger than 2 years old and in children and adolescents 2 to 20 years old as detected with MR imaging. Knowledge of the size of the normal pineal gland is important in the detection of abnormalities of the pineal gland, particularly neoplasms.

Developmental maturation of pineal gland function: synchronized CREM inducibility and adrenergic stimulation.

The cAMP response element modulator (CREM) gene encodes multiple activators and repressors of cAMP-responsive transcription. Differential splicing generates a developmental switch in CREM function during spermatogenesis, while the use of an alternative promoter is responsible for the production of a cAMP-inducible transcriptional repressor, ICER (inducible cAMP early repressor). The ICER promoter is strongly inducible by cAMP because of the presence of four tandemly repeated cAMP response elements. Furthermore, ICER negatively autoregulates the ICER promoter activity, thus generating a feedback loop. CREM constitutes an early response gene of the cAMP pathway in several neuroendocrine cells. We have previously shown that CREM is highly expressed in the adult rat pineal gland at nighttime. Here, we show that the only additional site of rhythmic ICER expression within the photoneuroendocrine system is the lamina intercalaris. Ontogenetically, the ICER day-night switch and cAMP inducibility mature in the pineal gland at the end of the first postnatal week. Importantly, this correlates with the onset of melatonin synthesis and the establishment of functional adrenergic innervation. At this developmental phase we document a significant increase in protein kinase A levels, thus suggesting that ICER inducibility reflects a complete maturation of the cAMP-dependent signaling pathway at the nuclear level.

Lack of pineal growth during childhood.

During childhood, serum melatonin concentrations drop by approximately 80%, but the 24-h melatonin excretion is stable. Arrest of pineal growth after the end of infancy has been proposed as one possible mechanism underlying that phenomenon. To test this hypothesis, we reviewed 332 magnetic resonance imaging brain studies, classified as normal, of endocrine-normal children, aged 1 day to 15 yr, and estimated the pineal and pituitary sizes. The pineal was identified in 277 of 332 magnetic resonance imaging studies (83%). The average size (mean +/- SEM) of the pineal gland (transaxial diameter, 5.6 +/- 2.1; midsagittal diameter, 5.0 +/- 2.4; planimetric area, 28.5 +/- 17.8) did not differ with age. A total of 74 of 277 pineals with cysts (26.7%) were found. The occurrence of pineal cysts was equally distributed among the different age groups (chi 2 = 11.6; df = 14; P = 0.7). Ten pineals showed more than 1 cyst (3.6%). The pituitary was identified in 325 of 332 brain images (97.9%). The average pituitary size increased by some 100% from 1 to 15 yr of age [transaxial diameter: F = 2.2; P = 0.005 (by two-way analysis of variance); midsagittal diameter: F = 3.7; P = 0.0001; planimetric area: F = 7.1; P = 0.0001]. The pituitary was slightly larger in females than in males [midsagittal diameter: F = 8.8; P = 0.003 (by two-way analysis of variance); planimetric area: F = 7.9, P = 0.005]. The data presented indicate a lack of a discernible pineal growth after age 1 yr, which contrasts with pituitary development in the same individuals. The data are in agreement with a hypothesis suggesting a growth arrest of the pineal after infancy.

Calbindin-D28k, calretinin, and recoverin immunoreactivities in developing chick pineal gland.

Calbindin-D28k, calretinin, and recoverin, three intracellular calcium-binding proteins belonging to the troponin C/calmodulin superfamily, were immunohistochemically localized in chick pineal during development [from embryonic day 16 (E16) to postnatal day 14 (P14)]. At E18, only calretinin immunoreactivity could be detected in nuclei from follicular pinealocytes. With development, calretinin immunoreactivity expanded from nucleus to cytoplasm, and calretinin immuno-positive cell number increased. At P14 almost al pinealocytes were calretinin positive. Calbindin-D28k immunoreactivity was not detected before E20. During development, many follicular and parafollicular pinealocytes became strongly calbindin-D28k positive, reaching a peak both in intensity and in number at P7; thereafter their number decreased. In addition to pinealocytes, neuron-like cells appeared calbindin-D28k positive at E20 and calretinin positive at P7. Recoverin, a myristoylated protein isolated from vertebrate photoreceptor and which might participate in the inactivation of the phototransduction cascade, was transiently expressed in follicular and parafollicular pinealocytes from P1 to P14 with a maximal expression at P7. This transitory expression may coincide with a transitory light sensitivity period in chick pinealocytes, before complete maturity of the pineal gland.

Effect of photoperiod on the pineal melatonin rhythm in neonatal rats.

Photoperiod already modulates pineal melatonin rhythm in neonatal rats. Pineal melatonin content was about 500 fmol during day and increased up to 2000 and 3000 fmol at night in 8- and 12-day-old rats, respectively. On long photoperiods (LD 14:10) melatonin was increased above 1000 fmol for about 8 h while on short photoperiods (LD 8:16) for 12 to 14 h. Melatonin pattern may thus transduce photoperiodic effects in neonatal rats. However, no differences in plasma LH were found in the rats kept on long and short photoperiods.