Presence Of Melatonin Research Articles

Reprint of “pH tuning of Nafion ® for selective detection of tryptophan”

Selective and sensitive detection of the amino acid tryptophan is of importance in food processing, pharmaceutical formulations and in biological fluids. Electrochemical methods of detection of tryptophan are hampered by sluggish electron transfer kinetics and in complex matrices through overlapping peaks from interferents. This study examines the potential of the cation exchange membrane Nafion ® to enhance selectivity and sensitivity of this analyte through a seldom explored feature of this membrane: pH manipulation. A detailed examination of the effect of pH on the selectivity afforded by Nafion ® as a function of the analyte charge is presented. Selective detection of tryptophan and significant increases in sensitivity of its detection was observed in the presence of melatonin, dopamine and other interferents present in a pharmaceutical formulation through manipulation of the pH of the solution. At pH 3.0 at a Nafion ®-modified electrode, changes in the protonation of melatonin and tryptophan lowered the anodic potential of the analytes in a non-uniform manner increasing the peak resolution and permitting analyses with detection limits of 1.6 ± 0.1 nM and 1.6 ± 0.2 nM, respectively.

Amyloid‐β neurotoxicity in organotypic culture is attenuated by melatonin: involvement of GSK‐3β, tau and neuroinflammation

Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by accumulation of extracellular deposits of amyloid-beta (Abeta) peptide in brain regions that are important for memory and cognition. The buildup of Abeta aggregates in the AD is followed by the formation of intracellular neurofibrillary tangles and activation of neuroinflammatory reactions. The present study investigated whether melatonin possesses a neuroprotective effect against Abeta-induced toxicity. For this purpose, organotypic hippocampal slices were cultured and exposed to 25 microm of Abeta(25-35) in the absence or in the presence of melatonin (25, 50, or 100 microm). In addition, the authors have investigated the involvement of GSK-3beta, tau protein, astroglial, and microglial activation, and cytokine levels in the melatonin protection against Abeta-induced neurotoxicity. Melatonin prevented the cell damage in hippocampus induced by the exposure to Abeta(25-35). In addition, melatonin significantly reduced the activation of GSK-3beta, the phosphorylation of tau protein, the glial activation and the Abeta-induced increase of TNF-alpha and IL-6 levels. On the basis of these findings, we speculate that melatonin may provide an effective therapeutic strategy for AD, by attenuating Abeta-induced phosphorylation of tau protein, and preventing GSK-3beta activation and neuroinflammation.

Melatonin as a potential tool against oxidative damage and apoptosis in ejaculated human spermatozoa

It is assumed somatic cells can die in the apoptotic, the autophagic, or the necrotic way; however, the mechanisms of sperm death are not clear. Here, ejaculated human spermatozoa were evaluated for apoptosis and reactive oxygen species production in the absence or presence of melatonin, and we concluded that melatonin reverses sperm apoptosis due to its free radical scavenging actions.

Melatonin protects N2a against ischemia/reperfusion injury through autophagy enhancement

Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury. Although melatonin is known as an effective antioxidant, the mechanism of the protection cannot be explained merely by antioxidation. This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy, since autophagy has been frequently suggested to play a crucial role in neuron survival. To find it out, an ischemia/reperfusion model in N2a cells was established for examinations. The results showed that autophagy was significantly enhanced in N2a cells treated with melatonin at reperfusion onset following ischemia and greatly promoted cell survival, while autophagy blockage by 3-MA led to the shortened N2a cell survival as assessed by MTT, transmission electron microscopy, and laser confocal scanning microscopy. Besides, the protein levels of LC3II and Beclin1 were remarkably increased in ischemia/reperfusion-injured N2a in the presence of melatonin, whereas the expression of p-PKB, key kinase in PI3K/PKB signaling pathway, showed a decrease when compared with untreated subjects as accessed by immunoblotting. Taken together these data suggest that autophagy is possibly one of the mechanisms underlying neuroprotection of melatonin.

In vitro methodology, hormonal and nutritional effects and fibre production in isolated ovine and caprine anagen hair follicles

Mammalian hair follicles are complex multicellular structures in the skin, which produce hair fibre under the influence of locally produced and systemic signalling systems. Investigation to determine mechanisms of regulation, follicular responses and the importance of nutritional supply have utilised a number of in vivo and in vitro approaches. Included in these are studies on isolated intact anagen secondary follicles singly or in groups with incubation in culture medium. These utilise techniques developed for investigation of follicles from human skin. Results from selected studies reviewed here demonstrate differences in capacity for hair growth and protein synthesis between secondary follicles from Angora and cashmere-bearing goats. Mohair follicles were shown to exhibit faster hair shaft elongation both in vivo and in vitro, to have greater DNA content per follicle and to deposit significantly more protein per follicle and per unit of DNA. Incubation of anagen mohair and cashmere follicles in the presence of melatonin or prolactin showed positive responses in hair shaft growth and protein synthesis to both signalling molecules. This result indicated directly acting effects on the follicle in addition to any indirect effects arising at a whole animal level in response to, for example, variation in photoperiod. Similarly, epidermal growth factor was shown to alter elongation and protein synthesis in mohair follicles and to produce, at higher concentration, club hair structures similar to effects observed in other species. The vitamin biotin was shown to be important in maintaining viability of isolated sheep secondary hair follicles where supplementation increased the proportion continuing to grow. Effects on growth and apparent protein synthesis suggested comparatively lesser effects on follicles, which remained viable. Histology on follicles indicated effects of biotin deficiency in reducing proliferation of basal keratinocytes. The final study, included in this review, demonstrated that supply of the essential sulphur-containing amino acid l-methionine was necessary to maintain the viability and growth of mohair follicles. l-cysteine was not required in the presence of l-methionine, although there was evidence of an optimisation when both amino acids were present in adequate concentrations. Consideration is given to the importance of transport mechanisms and capacity to utilise absorbed nutrients when considering optimising nutritional supply to individual follicles. These may then provide targets for attainment in applied nutrition of animals in vivo.

Melatonin‐induced estrogen receptor α‐mediated calbindin‐D9k expression plays a role in H2O2‐mediated cell death in rat pituitary GH3 cells

Calbindin-D9k (CaBP-9k) is a 9-kDa polypeptide possessing two calcium-binding sites that is expressed in the mammalian intestine, uterus, and pituitary gland. The factors regulating the expression of the estrogen receptor (ER) and CaBP-9k in the pituitary gland are currently unknown. In this study, we investigated whether the ER and CaBP-9k expression are regulated by melatonin during H(2)O(2)-induced cell death in rat pituitary GH3 cells. Cell survival increased by approximately 27-36% in H(2)O(2) plus melatonin compared to H(2)O(2) alone, and CaBP-9k expression was augmented by treatment with H(2)O(2) plus melatonin. These results suggest that the increase in cell survival and the melatonin-induced CaBP-9k expression may play a role in protecting cells against H(2)O(2)-mediated cell death. This result is also consistent with the increase in CaBP-9k expression leading to rises in p-ERK and p-Bad (S112). Over-expression of CaBP-9k caused an increase in p-ERK. ERalpha expression was higher in H(2)O(2) plus melatonin-treated cells compared to those treated with H(2)O(2) alone, while ERbeta expression was not. Also, ERalpha in the nuclear fraction increased in the presence of melatonin and decreased in the presence of ICI 182 780 or ICI 182 780 plus melatonin. The relative binding affinity of ERalpha for melatonin was higher than that of ERbeta, suggesting that melatonin has the potential to preferentially bind ERalpha. In conclusion, these results indicate that melatonin may increase CaBP-9k expression through ERalpha.

Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBPβ transcriptional activity

Considering that melatonin has been implicated in body weight control, this work investigated whether this effect involves the regulation of adipogenesis. 3T3-L1 preadipocytes were induced to differentiate in the absence or presence of melatonin (10(-3) m). Swiss-3T3 cells ectopically and conditionally (Tet-off system) over-expressing the 34 kDa C/EBPbeta isoform (Swiss-LAP cells) were employed as a tool to assess the mechanisms of action at the molecular level. Protein markers of the adipogenic phenotype were analyzed by Western blot. At 36 hr of differentiation of 3T3-L1 preadipocytes, a reduction of PPARgamma expression was detected followed by a further reduction, at day 4, of perilipin, aP2 and adiponectin protein expression in melatonin-treated cells. Real-time PCR analysis also showed a decrease of PPARgamma (60%), C/EBPalpha (75%), adiponectin (30%) and aP2 (40%) mRNA expression. Finally, we transfected Swiss LAP cells with a C/EBPalpha gene promoter/reporter construct in which luciferase expression is enhanced in response to C/EBPbeta activity. Culture of such transfected cells in the absence of tetracycline led to a 2.5-fold activation of the C/EBPalpha promoter. However, when treated with melatonin, the level of C/EBPalpha promoter activation by C/EBPbeta was reduced by 50% (P = 0.05, n = 6). In addition, this inhibitory effect of melatonin was also reflected in the phenotype of the cells, since their capacity to accumulate lipids droplets was reduced as confirmed by the poor staining with Oil Red O. In conclusion, melatonin at a concentration of 10(-3 ) m works as a negative regulator of adipogenesis acting in part by inhibiting the activity of a critical adipogenic transcription factor, C/EBPbeta.

Effects of the number of ring substituents of cobalt carboxyphthalocyanines on the electrocatalytic detection of nitrite, cysteine and melatonin

Cobalt phthalocyanine (CoPc), cobalt tetracarboxy phthalocyanine (CoTCPc) and cobalt octacarboxy phthalocyanine (CoOCPc), adsorbed onto glassy carbon electrodes, have been used for the electrocatalytic detection of nitrite, L-cysteine and melatonin. The modified electrodes electrocatalytically detected nitrite around 800 mV vs. Ag|AgCl , a value less positive compared to that of an unmodified glassy carbon electrode (at 950 mV vs. Ag|AgCl ) and also gave detection limits in the 10-7 M range for nitrite detection. L-cysteine was detected by the modified electrodes at potentials between 0.50 to 0.65 V vs. Ag|AgCl , with L-cysteine detection limits also in the 10-7 M range. The detection limits for melatonin ranged from 10-7 to 10-6 M. CoPc-modified electrodes displayed good separation of interferents (tryptophan and ascorbic acid) in the presence of melatonin. Analyses of commercial melatonin tablets using modified electrodes gave excellent agreement with manufacturer's value for all modified electrodes of this work.

Chemical stress by different agents affects the melatonin content of barley roots

The presence of melatonin (N-acetyl-5-methoxytryptamine) in plants has been clearly demonstrated. However, while this indoleamine has been intensively studied in animals, especially in mammals, the same is not true in the case of plants, where one of the most interesting aspects is its possible role as antioxidative molecule in physiological processes. Some data reflect the possible protective role that melatonin may exert in some stress situations such as ultraviolet (UV)-radiation, induced senescence and copper stress. The present work was designed to establish how the melatonin content changes in plants as a result of chemically induced stress. For this, barley plants were exposed in different treatments to the chemical-stress agents: sodium chloride, zinc sulphate or hydrogen peroxide. After different times, the content of melatonin in treated roots and control roots were determined using liquid chromatography (LC) with time-of-flight/mass spectrometry and LC with fluorescence detection for identification and quantification, respectively. The data show that the melatonin content in roots increased due to stress, reaching up to six times the melatonin content of control roots. Induction was time dependent, while hydrogen peroxide (10 mm) and zinc sulphate (1 mm) were the most effective inducers. The capacity of roots to absorb melatonin from soil was also studied. The data establish, for first time, that the chemical-stress agents assayed can induce the biosynthesis of melatonin in barley roots and produce a significant increase in their melatonin content. Such an increase in melatonin probably plays an important antioxidative role in the defense against chemically induced stress and other abiotic/biotic stresses.

PH tuning of Nafion ® for selective detection of tryptophan

Selective and sensitive detection of the amino acid tryptophan is of importance in food processing, pharmaceutical formulations and in biological fluids. Electrochemical methods of detection of tryptophan are hampered by sluggish electron transfer kinetics and in complex matrices through overlapping peaks from interferents. This study examines the potential of the cation exchange membrane Nafion ® to enhance selectivity and sensitivity of this analyte through a seldom explored feature of this membrane: pH manipulation. A detailed examination of the effect of pH on the selectivity afforded by Nafion ® as a function of the analyte charge is presented. Selective detection of tryptophan and significant increases in sensitivity of its detection was observed in the presence of melatonin, dopamine and other interferents present in a pharmaceutical formulation through manipulation of the pH of the solution. At pH 3.0 at a Nafion ®-modified electrode, changes in the protonation of melatonin and tryptophan lowered the anodic potential of the analytes in a non-uniform manner increasing the peak resolution and permitting analyses with detection limits of 1.6 ± 0.1 nM and 1.6 ± 0.2 nM, respectively.

Common variant in MTNR1B associated with increased risk of type 2 diabetes and impaired early insulin secretion

Genome-wide association studies have shown that variation in MTNR1B (melatonin receptor 1B) is associated with insulin and glucose concentrations. Here we show that the risk genotype of this SNP predicts future type 2 diabetes (T2D) in two large prospective studies. Specifically, the risk genotype was associated with impairment of early insulin response to both oral and intravenous glucose and with faster deterioration of insulin secretion over time. We also show that the MTNR1B mRNA is expressed in human islets, and immunocytochemistry confirms that it is primarily localized in beta cells in islets. Nondiabetic individuals carrying the risk allele and individuals with T2D showed increased expression of the receptor in islets. Insulin release from clonal beta cells in response to glucose was inhibited in the presence of melatonin. These data suggest that the circulating hormone melatonin, which is predominantly released from the pineal gland in the brain, is involved in the pathogenesis of T2D. Given the increased expression of MTNR1B in individuals at risk of T2D, the pathogenic effects are likely exerted via a direct inhibitory effect on beta cells. In view of these results, blocking the melatonin ligand-receptor system could be a therapeutic avenue in T2D.

Melatonin prevents the injury‐induced decline of Akt/forkhead transcription factors phosphorylation

Melatonin plays a neuroprotective role against brain injury through the activation of Akt and the inhibition of apoptotic cell death. This study investigated whether melatonin modulates the anti-apoptotic signal through the activation of Akt and its downstream targets, FKHR, AFX, and 14-3-3. Adult male rats were treated with melatonin (5 mg/kg) prior to middle cerebral artery occlusion (MCAO) and brain tissues were collected at 24 hr after MCAO. This study confirmed that melatonin significantly reduces infarct volume and decreases the number of TUNEL-positive cells in the cerebral cortex. Potential activation was measured by phosphorylation of PDK1 at Ser(241), Akt at Ser(473), FKHR at Ser(256), and AFX at Ser(193) using Western blot analysis. Melatonin prevented the injury-induced reduction of pPDK1, pAkt, pFKHR, and pAFX. However, melatonin did not affect the level of 14-3-3, which acts as an anti-apoptotic factor through interaction of pFKHR. Further, in the presence of melatonin, the interaction of pFKHR and 14-3-3 increased, compared with that of control animals. This study suggests that melatonin plays a potent protective role against brain injury and that Akt activation and FKHR phosphorylation by melatonin mediated these protective effects.

Critical time periods of melatonin exposure enhance osteoblast differentiation from human mesenchymal stem cells

We have previously shown that melatonin enhances bone stem cell differentiation (hAMSCs) into osteoblasts following 10 days melatonin exposure, that is dependent upon MT2 melatonin receptors (MT2R), internalization and MEK(1/2). We extended the exposure to 21 days as calcium deposition, an essential component of bone differentiation process, takes place between 14 and 21 days in culture. The objective of this study was to determine the critical time periods of melatonin exposure during a 21 day exposure to osteogenic medium. HAMSCs were cultured in osteogenic medium for 21 days and then exposed to melatonin for 2, 5, 10, 14 and 21 days. Melatonin increased alkaline phosphatase (ALP) activity, a differentiation marker, when cells were exposed to melatonin for 2 days or 21 days. Calcium deposition was increased by about 900% in presence of melatonin, but this was seen only when the cells were co‐exposed to melatonin for 21 days. Melatonin's effects on ALP activity at days 2 and 21 were dependent upon MT2R, as these effects were blocked with 4P‐PDOT, an MT2R antagonist. RT‐PCR studies show an increase in early, intermediate and late genes involved in osteoblast differentiation, which is dependent upon the duration of melatonin exposure. These studies indicate that melatonin acting through MT2R can drive the differentiation of hAMSCs into osteoblasts, perhaps by activating different genes at critical time periods.

Effects of melatonin on the proliferation and differentiation of rat adipose-derived stem cells.

Osteogenesis driven by adipose-derived stem cells (ADSCs) is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM) could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs. ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM). After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and flow cytometry, respectively. The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level) also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased. These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.

Effects of melatonin on the proliferation and differentiation of rat adipose-derived stem cells

Background:Osteogenesis driven by adipose-derived stem cells (ADSCs) is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM) could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs.Materials and Methods:ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM). After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and flow cytometry, respectively.Results:The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level) also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased.Conclusion:These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.

Interactions between Dopamine and Melatonin Organize Circadian Rhythmicity in the Retina of the Green Iguana

Circadian physiology in the vertebrate retina is regulated by several neurotransmitters. In the lateral eyes of the green iguana the circadian rhythm of melatonin content peaks during the night while the rhythm of dopamine peaks during the day. In the present work, the authors explore the interaction of these 2 neurotransmitters during the circadian cycle. They depleted retinal dopamine with intravitreal injections of 6-hydroxydopamine (6-OHDA) and measured ocular melatonin content in vivo throughout 1 circadian cycle. The circadian rhythm of ocular melatonin not only persisted but increased 10-fold in amplitude. This increase was substantially reduced by the intraocular administration of dopamine. 6-OHDA-treated retinas, unlike those from untreated animals, did not express a circadian rhythm of melatonin synthesis in vitro. To deplete retinal melatonin, the authors pinealectomized iguanas and blocked retinal melatonin synthesis by depleting serotonin with intraocular injections of 5,6-dihydroxytryptamine. In animals so treated, they found that the circadian rhythm of retinal dopamine content was abolished, the levels of dopamine were lowered, and the levels of dopamine metabolites were greatly increased. The data suggest that in iguanas, the amplitude of the circadian rhythm of melatonin synthesis in the eye is suppressed by dopamine while the rhythm of dopamine depends, at least in part, on the presence of melatonin.

MT1 melatonin receptor internalization underlies melatonin‐induced morphologic changes in Chinese hamster ovary cells and these processes are dependent on Gi proteins, MEK 1/2 and microtubule modulation

Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell-type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1-Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1-CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2-dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin-mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Gi(alpha) 2,3, Gq(alpha), beta-arrestin-2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin-mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin-induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with beta-arrestin-2/MEK 1/2 and ERK 1/2. The MT1-CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.

Melatonin attenuates the focal cerebral ischemic injury by inhibiting the dissociation of pBad from 14‐3‐3

It has recently been reported that melatonin protects neuronal cells from damage by enhancing Akt activation, thus mediating antiapoptosis signals. However, there is little information regarding the effects of melatonin on the activation of genes further downstream in the Akt signaling pathway in ischemic brain injury. This study investigated whether melatonin modulates the antiapoptotic signal through Akt and its downstream targets, Bad and 14-3-3. Adult male rats were treated with melatonin (5 mg/kg) prior to middle cerebral artery occlusion (MCAO). Brains were collected at 24 hr after MCAO and infarct volumes were analyzed. Our results confirm that melatonin significantly reduces infarct volume and decreases the positive reaction of TUNEL staining in the cerebral cortex. Signal pathway activation was measured by phosphorylation of Akt at Ser(473) and Bad at Ser(136) using Western blot analysis. Melatonin prevented the injury-induced decrease of pAkt and pBad levels. However, melatonin did not affect the expression of 14-3-3, which acts as an antiapoptotic factor through interaction with Bad. Immunoprecipitation analysis showed that the interaction between pBad and 14-3-3 increased in the presence of melatonin, compared to that of control animals. Our findings suggest that melatonin prevents cell death because of brain injury and that these protective effects are mediated through maintaining the interaction between pBad and 14-3-3, thus blocking activation of the apoptotic pathway.

Melatonin increases cleavage rate of porcine preimplantation embryos in vitro

Melatonin has been used to promote in vitro embryo development in different species. This study determined the effects of melatonin on in vitro porcine embryo development; in particular, cleavage rate, blastocyst rate, and blastocyst cell number. Starting 5 hr after insemination, porcine zygotes were cultured in porcine zygote medium 3 (PZM-3) culture medium supplemented with melatonin at increasing concentrations (10(-12) M, 10(-9) M, 10(-6) M, 10(-3) M). Melatonin at a concentration of 10(-9) M had a positive effect on cleavage rates, while the highest concentration of melatonin (10(-3) M) significantly decreased cleavage rates. Although blastocyst rates were not increased by 10(-9) M melatonin, blastocyst cell numbers were significantly higher for embryos subjected to 10(-9) M melatonin. The expression levels of the pro-apoptotic gene BAX and anti-apoptotic gene BCL2L1 in blastocysts were not affected by the presence of melatonin in the culture medium. To further study the protective properties of 10(-9) M melatonin against stressful conditions, hydrogen peroxide (0.01 mm) and heat (40 degrees C) were used during embryo culture. The addition of melatonin to embryos subjected to 40 degrees C for 3 hr increased cleavage rates, but had no protective effect for embryos subjected to 0.01 mm H(2)O(2), probably because the physiological levels of melatonin could not counteract the pharmacological levels of H(2)O(2). Our data indicate that 10(-9) M melatonin has a positive effect on porcine embryo cleavage rates and blastocyst total cell numbers and it might have a protective effect against heat stress.

INFLUENCE OF OXIDATIVE STRESS ON OOCYTE QUALITY AND PROTECTIVE ROLE OF MELATONIN AS AN ANTIOXIDANT

Poor oocyte quality is a critical factor of female infertility. Reactive oxygen species are produced within the follicle especially during the ovulatory process. It has been reported that oxidative stress may be a cause of poor oocyte quality. Melatonin, a pineal hormone, is detected in the human follicular fluid. Recent reports have shown that melatonin works as a potent radical scavenger. This study was undertaken to examine a relationship between oxidative stress and poor oocyte quality and whether melatonin improves oocyte quality as an antioxidant. To investigate the effect of oxidative stress and melatonin on oocyte maturation, oocytes were recovered from preovulatory follicles 48 h after injection of 10 IU of equine chorionic gonadotropin (eCG) in immature ICR mice. Oocytes were then incubated in the presence or absence of hydrogen peroxide (H2O2; 1, 10, 100, 1000 μM). After 12 h incubation, oocytes with a first polar body were defined as mature oocytes. The percentage of the mature oocytes was significantly decreased (0%) by addition of H202 (≥10 μM) compared with control (62%). When oocytes were incubated with H202 (10 μM) in the presence of melatonin (1, 10 ng/ml), the decrease in the percentage of mature oocytes caused by H202 was blocked by melatonin (10 ng/ml) addition. It is, therefore, suggested that oxidative stress inhibits oocytes maturation and melatonin showed a protective activity against oxidative stress. Follicular fluid was sampled at the oocyte retrieval in IVF-ET patients. Intrafollicular concentrations of 8-hydroxy-2′-deoxyguanosine (8-OHdG) or Hexanoyl-lysine adduct (HEL) were determined by ELISA as an oxidative stress marker. 8-OHdG concentrations in women with high rates (≥30%) of degenerate oocytes were significantly higher than those in women with <30% degenerate oocytes, suggesting a close relationship between oxidative stress and poor oocyte quality. In order to reduce oxidative stress in the follicles, melatonin (3 mg/day, at 22:00) was orally given to IVF-ET patients (n=56), who failed to achieve a fertilization rate of at least 50% in the previous IVF-ET cycle, from the fifth day of the previous menstrual cycle to the day of oocyte retrieval of the IVF-ET cycle. Fifty-nine women underwent IVF-ET without melatonin treatment. The fertilization rate was improved by melatonin treatment compared with the previous IVFET cycle (20.0±19.0 % vs. 50.0±38 %), and 11 women (19.6%) conceived. On the other hand, without melatonin treatment, the fertilization rate was not changed (20.9±16.5 % vs. 22.8±19.0 %) and 6 women (10.2%) conceived. Intrafollicular melatonin concentrations were significantly increased (112±51 vs. 432±260 pg/ml) and intrafollicular concentrations of 8-OHdG and HEL were significantly decreased by melatonin treatment (17.4±8.8 vs. 11.6±0.7 ng/ml, 25.0±7.2 vs. 19.2±2.5 nmol/L, respectively). In conclusions, oxidative stress causes toxic effect on oocyte maturation and melatonin protects oocytes from oxidative stress. Melatonin administration is likely to improve oocyte quality. (platform)