Importance of Melatonin in Assisted Reproductive Technology and Ovarian Aging.

Melatonin is probably produced in all cells but is only secreted by the pineal gland. The pineal secretion of melatonin is determined by the light-dark cycle, and it is only released at night. Melatonin regulates biological rhythms via its receptors located in the suprachiasmatic nuclei of the hypothalamus. Melatonin also has strong antioxidant activities to scavenge free radicals such as reactive oxygen species (ROS). The direct free radical scavenging actions are receptor independent. ROS play an important role in reproductive function including in the ovulatory process. However, excessive ROS can also have an adverse effect on oocytes because of oxidative stress, thereby causing infertility. It is becoming clear that melatonin is located in the ovarian follicular fluid and in the oocytes themselves, which protects these cells from oxidative damage as well as having other beneficial actions in oocyte maturation, fertilization, and embryo development. Trials on humans have investigated the improvement of outcomes of assisted reproductive technology (ART), such as in vitro fertilization and embryo transfer (IVF-ET), by way of administering melatonin to patients suffering from infertility. In addition, clinical research has examined melatonin as an anti-aging molecule via its antioxidative actions, and its relationship with the aging diseases, e.g., Alzheimer's and Parkinson's disease, is also underway. Melatonin may also reduce ovarian aging, which is a major issue in assisted reproductive technology. This review explains the relationship between melatonin and human reproductive function, as well as the clinical applications expected to improve the outcomes of assisted reproductive technology such as IVF, while also discussing possibilities for melatonin in preventing ovarian aging.

Usefulness of intermittent clomiphene citrate treatment for women with polycystic ovarian syndrome that is resistant to standard clomiphene citrate treatment.

PURPOSE: Clomiphene citrate (CC) has been used as a first-line treatment for anovulatory polycystic ovary syndrome (PCOS). However, some patients with PCOS are resistant to standard CC treatment. In this study, a new CC treatment protocol was developed, named "intermittent CC treatment" (ICT) and its efficacy was investigated on the induction of follicular growth in patients with PCOS who were resistant to standard CC treatment. METHODS: Of the 42 patients with PCOS who were resistant to standard CC treatment (50 mg/day, 5 days), 26 underwent ICT. They were given 100 mg/day of CC for 5 days from the next menstrual cycle day (MCD) 5 (first CC). If follicular growth was not observed on MCD 14, they were given 100 mg/day of CC for 5 days (MCD 14-MCD 18) (second CC). If follicular growth still was not observed on MCD 23, they were treated with CC again in the same way (third CC). RESULTS: The first CC, second CC, and third CC were effective for 3/26 (11.5%) patients, 12/23 (52.2%) patients, and 6/11 (54.5%) patients, respectively. In total, ICT was effective for 21/26 (80.8%) patients with CC-resistant PCOS. CONCLUSION: Thus, ICT is a useful treatment and could be an alternative to gonadotropin therapy for patients with CC-resistant PCOS.

Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei, mitochondria, and plasma membranes in mice.

Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 µg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylation (γH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle.

Melatonin and female reproduction.

Melatonin (N-acetyl-5-methoxytryptamine) is secreted during the dark hours at night by the pineal gland. After entering the circulation, melatonin acts as an endocrine factor and a chemical messenger of light and darkness. It regulates a variety of important central and peripheral actions related to circadian rhythms and reproduction. It also affects the brain, immune, gastrointestinal, cardiovascular, renal, bone and endocrine functions and acts as an oncostatic and anti-aging molecule. Many of melatonin's actions are mediated through interactions with specific membrane-bound receptors expressed not only in the central nervous system, but also in peripheral tissues. Melatonin also acts through non-receptor-mediated mechanisms, for example serving as a scavenger for reactive oxygen species and reactive nitrogen species. At both physiological and pharmacological concentrations, melatonin attenuates and counteracts oxidative stress and regulates cellular metabolism. Growing scientific evidence of reproductive physiology supports the role of melatonin in human reproduction. This review was conducted to investigate the effects of melatonin on female reproduction and to summarize our findings in this field.

Importance of C/EBPß binding and histone acetylation status in the promoter regions for induction of IGFBP-1, PRL, and Mn-SOD by cAMP in human endometrial stromal cells.

Dynamic changes of gene expressions occur in human endometrial stromal cells (ESCs) during decidualization. CCAAT/enhancer-binding proteinß (C/EBPß) regulates the expression of a number of decidualization-related genes. In addition to transcription factors, it is important to know the role of epigenetic mechanisms, such as histone modifications in the regulation of decidualization-related genes. This study investigated the molecular and epigenetic mechanisms by which cAMP up-regulates the expression of IGF-binding protein-1 (IGFBP-1), prolactin (PRL), and manganese superoxide dismutase (Mn-SOD) in ESC. ESCs isolated from proliferative phase endometrium were incubated with cAMP to induce decidualization. IGFBP-1, PRL, and Mn-SOD mRNA expressions were determined by real-time RT-PCR. The C/EBPß binding and histone modification status (acetylation of histone-H3 lysine-27 [H3K27ac]) in the promoter were examined by chromatin immunoprecipitation assay. Knockdowns of C/EBPß were performed using the small interfering RNA method. cAMP induced mRNA expressions of IGFBP-1 and PRL accompanied by the increases in both C/EBPß binding activities and H3K27ac levels in the promoters. The stimulatory effects of cAMP on mRNA levels and H3K27ac levels were completely abolished by C/EBPß knockdown. cAMP increased Mn-SOD mRNA levels and C/EBPß binding activities in the enhancer region. C/EBPß knockdown inhibited Mn-SOD mRNA levels. The H3K27ac levels in the enhancer were high before cAMP stimulus but were not further increased by cAMP and were not inhibited by C/EBPß knockdown. These results show that C/EBPß regulates the expression of IGFBP-1 and PRL by altering the histone acetylation status of their promoters but differently regulates Mn-SOD gene expression in human ESC during decidualization.

Melatonin as a free radical scavenger in the ovarian follicle.

This review summarizes new findings related to beneficial effects of melatonin (N-acetyl-5-methoxytryptamine) on reproductive physiology. Recently many researchers have begun to study the local role of melatonin as an antioxidant. We focused on intra-follicular role of melatonin in the ovary. Melatonin, secreted by the pineal gland, is taken up into the follicular fluid from the blood. Reactive oxygen species (ROS) are produced within the follicles, during the ovulatory process. Melatonin reduces oxidative stress as an antioxidant, and contribute to oocyte maturation, embryo development and luteinization of granulosa cells. Our clinical study demonstrated that melatonin treatment for infertile women increases intra-follicular melatonin concentrations, reduces intra-follicular oxidative damage, and elevates fertilization and pregnancy rates. Melatonin treatment also improves progesterone production by corpus luteum in infertile women with luteal phase defect. Melatonin treatment could become a new cure for improving oocyte quality and luteal function in infertile women.

Induction of IGFBP-1 expression by cAMP is associated with histone acetylation status of the promoter region in human endometrial stromal cells.

Many genes are up- or down-regulated in human endometrial stromal cells (ESCs) undergoing decidualization. IGF-binding protein-1 (IGFBP-1) and prolactin (PRL) are preferentially expressed during decidualization and are recognized as specific markers of decidualization. This study investigated the involvement of epigenetic mechanisms in the regulation of IGFBP-1 and PRL induction by decidualization in ESCs. ESCs isolated from the proliferative phase endometrium were incubated with cAMP to induce decidualization. Human dermal fibroblasts (HDFs) were used as a nonendometrial control. cAMP induced the expressions of both genes in ESCs but induced the expression of only PRL in HDFs. Histone acetylation levels of the IGFBP-1 promoter region evaluated by chromatin immunoprecipitation assay were higher in ESCs than in HDFs. The IGFBP-1 promoter regions in the two cell types showed similar levels of DNA hypomethylation. The histone acetylation levels and DNA methylation status of the PRL promoter and enhancer regions were similar in the two cell types. cAMP had no significant effects on the histone acetylation levels and DNA methylation status of the IGFBP-1 promoter and the PRL promoter and enhancer regions in ESCs. Cotreatment of HDF with cAMP and histone deacetylase inhibitors induced IGFBP-1 expression, which was accompanied by an increased histone acetylation level and recruitment of CCAAT/enhancer-binding protein-ß to the promoter region. These results show that, during decidualization in ESCs, high histone acetylation status of the promoter regions of IGFBP-1 and PRL is associated with the induction of the IGFBP-1 and PRL genes by making the promoter regions accessible to transcriptional factors.

The role of melatonin as an antioxidant in the follicle.

Melatonin (N-acetyl-5-methoxytryptamine) is secreted during the dark hours at night by pineal gland, and it regulates a variety of important central and peripheral actions related to circadian rhythms and reproduction. It has been believed that melatonin regulates ovarian function by the regulation of gonadotropin release in the hypothalamus-pituitary gland axis via its specific receptors. In addition to the receptor mediated action, the discovery of melatonin as a direct free radical scavenger has greatly broadened the understanding of melatonin's mechanisms which benefit reproductive physiology. Higher concentrations of melatonin have been found in human preovulatory follicular fluid compared to serum, and there is growing evidence of the direct effects of melatonin on ovarian function especially oocyte maturation and embryo development. Many scientists have focused on the direct role of melatonin on oocyte maturation and embryo development as an anti-oxidant to reduce oxidative stress induced by reactive oxygen species, which are produced during ovulation process. The beneficial effects of melatonin administration on oocyte maturation and embryo development have been confirmed by in vitro and in vivo experiments in animals. This review also discusses the first application of melatonin to the clinical treatment of infertile women and confirms that melatonin administration reduces intrafollicular oxidative damage and increase fertilization rates. This review summarizes our recent works and new findings related to the reported beneficial effects of melatonin on reproductive physiology in its role as a reducer of oxidative stress, especially on oocyte maturation and embryo development.

Simple method for isolation of glyceraldehyde 3-phosphate dehydrogenase and the improvement of myofibril gel properties.

Porcine glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (G3PD) was prepared effectively by a combination of ethylene diamine tetra-acetate (EDTA) pretreatment and affinity purification. After salting out of porcine sarcoplasmic proteins (SP) with ammonium sulfate at 75% saturation, the obtained supernatant (SP-f3) was treated with EDTA, leaving G3PD in the supernatant (G3PD-E) and most other SPs in the precipitate. At that time, the separation of G3PD-E required more than 20 mmol/L EDTA. G3PD-E was then subjected to affinity purification by batchwise method using blue-sepharose CL-6B, and purified G3PD (G3PD-AP) was obtained using 2 mol/L potassium chloride (KCl) as an eluent. Texture analysis showed that the hardness, adhesiveness and gumminess of the myofibril gel at 0.2-mol/L NaCl increased with the addition of G3PD-AP. Scanning electron microscopy revealed that the G3PD-AP reinforced the gel network of the myofibril. However, scanning electron micrograph analysis showed that the network-structure of the gel by the addition of G3PD-AP developed in a different manner from that by adding 0.6 mol/L NaCl. These results showed that glycolytic enzyme, G3PD, contributes to the improvement of the rheological properties of meat products.

Miogadial and miogatrial with alpha,beta-unsaturated 1,4-dialdehyde moieties--novel and potent TRPA1 agonists.

AIMS: Most of the terpenoids with an alpha,beta-unsaturated 1,4-dialdehyde moiety, which are found in plants, fungi, and insects, have a pungent taste. However, the neural receptors responsible for the pungency of these terpenoids have not been identified yet. The transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1), which are expressed in the nociceptive neurons, induce a sensation of heat on activation by some pungent ingredients in food. In this study, we selected miogadial (MD), miogatrial (MT), and polygodial (PG) from the terpenoids with an alpha,beta-unsaturated 1,4-dialdehyde moiety and examined the effects of these 3 terpenoids on TRPA1 or TRPV1. MAIN METHODS: TRPV1 and TRPA1 activity by 3 terpenoids were evaluated using Ca(2+) imaging and patch-clamp methods in mammalian cells that express TRP heterologously and mouse sensory neurons. KEY FINDINGS: The 3 terpenoids activated TRPA1 that was heterologously expressed in HEK293 or CHO cells. The potencies of activation by the 3 terpenoids were equal and almost 10 times stronger than that of allyl isothiocyanate (AITC), which is known as the most potent TRPA1 agonist among all natural products. Moreover, these 3 terpenoids exhibited increased intracellular Ca(2+) concentration in mouse sensory neuron cells compared to AITC. High concentrations of the 3 terpenoids also activated TRPV1 that was heterologously expressed in HEK293 cells. SIGNIFICANCE: These results indicated that MD, MT, and PG were more potent in activating TRPA1 than TRPV1, and suggested that they primarily activate TRPA1 to induce pungency.

TRPA1 agonists--allyl isothiocyanate and cinnamaldehyde--induce adrenaline secretion.

Thermosensitive transient receptor potential (TRP) channels, especially TRPV1 and TRPA1, are activated by the pungent compounds present in spices. TRPV1 activation by the intake of capsaicin, the irritant in hot pepper, induces adrenaline secretion and increases energy consumption. TRPV1 is mainly expressed in the sensory neurons and coexpressed with TRPA1 at a high frequency. However, the mechanism underlying adrenaline secretion by TRPA1 agonists such as allyl isothiocyanate (AITC) and cinnamaldehyde (CNA), the pungent ingredients in mustard and cinnamon, is not known. We examined whether AITC and CNA could induce adrenaline secretion in anesthetized rats. An intravenous injection of AITC or CNA (10 mg/kg) increased adrenaline secretion. These responses disappeared completely in capsaicin-treated rats with an impaired sensory nerve function. Moreover, pretreatment with cholinergic blockers (hexamethonium and atropine) attenuated the AITC- or CNA-induced adrenaline secretion. These results suggest that TRPA1 agonists activate the sensory nerves and induce adrenaline secretion via the central nervous system.

Monoacylglycerols activate capsaicin receptor, TRPV1.

Transient receptor potential vanilloid subtype 1 (TRPV1) is known as capsaicin (CAP) receptor and activated by CAP. Activation of TRPV1 by CAP increases energy expenditure and thermogenesis in rodents or human. Therefore, TRPV1 may be target for energy expenditure enhancement and thermogenesis. To search for novel TRPV1 agonist, we screened 19 types of foods by using TRPV1-expressing HEK293 cells. TRPV1 was activated by hexane extract of wheat flour, and its functional compounds were 1-monoacylglycerols containing oleic, linoleic, and alpha-linolenic acids. Their potencies (EC50) were about 50 times larger than that of CAP and their efficacies (maximal response) were about half of that of CAP. TRPV1 was activated by 1-monoacylglycerols (MGs) having C18 and C20 unsaturated and C8-C12 saturated fatty acid (FA). Moreover, 2-MGs having C18 and C20 unsaturated FA acted on TRPV1 with the same potency. On the other hand, no activation of TRPV1 was induced by MGs having C16 and C18 saturated FA, di- or triacylglycerols of C18:1 FA. Pain-relating aversive responses were induced when TRPV1-activating 1-monoacylglycerols (50 mM) was administered subcutaneously into rat hind paw. These effects were inhibited by the co-injection of capsazepine (10 mM) which is a TRPV1 competitive antagonist. These results suggested that these 1-monoacylglycerols activate TRPV1 in vitro and in vivo.

Synthesis of the epimer of pericosine B from (-)-quinic Acid.

Synthesis of the epimer of pericosine B from (-)-quinic acid was achieved. This synthesis involves some regioselective and stereoselective processes. The desired product showed lower cytotoxic activity in comparison with natural pericosine B against the P388 leukemia cell line. The result implies that the stereocenter of C-6 in natural pericosine B plays an important role in this activity.