Lifa Lee

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 melatonins 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.

DNA methyltransferase expression in the human endometrium: down-regulation by progesterone and estrogen

BACKGROUND Epigenetic regulation may be involved in modulation of gene expression during the normal cyclic changes of the human endometrium. We investigated expression of DNA methyltransferases (DNMTs) in endometrium during the menstrual cycle and the influence of sex steroid hormones on DNMT in endometrial stromal cells (ESC) in culture. METHODS Expression of DNMT1, DNMT3a and DNMT3b was assessed by immunohistochemistry and real-time RT-PCR in endometrial tissue (n = 42 women). ESC (n = 3 women) were cultured with estradiol and medroxyprogesterone acetate (E + MPA) for 17 days, and DNMT mRNA levels were measured by real-time RT-PCR. RESULTS Nuclei of both epithelial and stromal cells immunostained for DNMT1, DNMT3a and DNMT3b during each phase of the menstrual cycle. Tissue levels of DNMT1 and DNMT3a mRNA were significantly lower in the mid-secretory phase than in the proliferative phase (P < 0.01). For DNMT3b, the change in mRNA levels showed a similar trend to that for DNMT3a. In ESC culture, DNMT3a and DNMT3b mRNA levels were significantly decreased by E + MPA treatment (P < 0.01 and P < 0.05, respectively) at Day 8 and Day 17. CONCLUSIONS DNMT mRNAs declined in the human endometrium during the secretory phase, and E + MPA down-regulated DNMT3a and DNMT3b mRNAs in ESC in culture. These results suggest that DNMTs have regulatory functions in gene expression that is associated with decidualization.

Protective role of melatonin in progesterone production by human luteal cells.

Abstract:  This study investigated whether melatonin protects luteinized granulosa cells from reactive oxygen species (ROS) as an antioxidant to enhance progesterone production in the follicle during ovulation. Follicular fluid was sampled at the time of oocyte retrieval in women undergoing in vitro fertilization and embryo transfer (IVF‐ET). Melatonin concentrations in the follicular fluid were positively correlated with progesterone concentrations (r = 0.342, P < 0.05) and negatively correlated with the concentration of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), an oxidative stress marker (r = −0.342, P < 0.05). The progesterone and 8‐OHdG concentrations were negatively correlated (r = −0.246, P < 0.05). Luteinized granulosa cells were obtained at the time of oocyte retrieval in women undergoing IVF‐ET. Cells were incubated with H2O2 (30, 50, 100 μm) in the presence or absence of melatonin (1, 10, 100 μg/mL). Progesterone production by luteinized granulosa cells was significantly inhibited by H2O2. Melatonin treatment overcame the inhibitory effect of H2O2. Twenty‐five patients who had luteal phase defect (serum progesterone concentrations <10 ng/mL during the mid‐luteal phase) were divided into two groups during the next treatment cycle: 14 women were given melatonin (3 mg/day at 22:00 hr) throughout the luteal phase and 11 women were given no medication as a control. Melatonin treatment improved serum progesterone concentrations (>10 ng/mL during the mid‐luteal phase) in nine of 14 women (64.3%), whereas only two of 11 women (18.1%) showed normal serum progesterone levels in the control group. In conclusion, melatonin protects granulosa cells undergoing luteinization from ROS in the follicle and contributes to luteinization for progesterone production during ovulation.

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 melatonins 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.

Genome-Wide DNA Methylation Analysis Reveals a Potential Mechanism for the Pathogenesis and Development of Uterine Leiomyomas

Background The pathogenesis of uterine leiomyomas, the most common benign tumor in women, remains unclear. Since acquired factors such as obesity, hypertension and early menarche place women at greater risk for uterine leiomyomas, uterine leiomyomas may be associated with epigenetic abnormalities that are caused by unfavorable environmental exposures. Principal Findings Profiles of genome-wide DNA methylation and mRNA expression were investigated in leiomyomas and in myometrium with and without leiomyomas. Profiles of DNA methylation and mRNA expression in the myometrium with and without leiomyomas were quite similar while those in leiomyomas were distinct. We identified 120 genes whose DNA methylation and mRNA expression patterns differed between leiomyomas and the adjacent myometrium. The biological relevance of the aberrantly methylated and expressed genes was cancer process, including IRS1 that is related to transformation, and collagen-related genes such as COL4A1, COL4A2 and COL6A3. We also detected 22 target genes of estrogen receptor (ER) alpha, including apoptosis-related genes, that have aberrant DNA methylation in the promoter, suggesting that the aberrant epigenetic regulation of ER alpha-target genes contributes to the aberrant response to estrogen. Conclusions Aberrant DNA methylation and its related transcriptional aberration were associated with cancer processes, which may represent a critical initial mechanism that triggers transformation of a single tumor stem cell that will eventually develop into a monoclonal leiomyoma tumor. The aberrant epigenetic regulation of ER alpha-target genes also may contribute to the aberrant response to estrogen, which is involved in the development of uterine leiomyomas after menarche.

Progesterone Increases Manganese Superoxide Dismutase Expression via a cAMP-Dependent Signaling Mediated by Noncanonical Wnt5a Pathway in Human Endometrial Stromal Cells

CONTEXT Manganese superoxide dismutase (Mn-SOD), an antioxidant enzyme in the mitochondria, protects cells by scavenging superoxide radicals in human endometrial stromal cells (ESCs). Mn-SOD increases in ESCs during decidualization induced by progesterone. OBJECTIVE The present study investigated the molecular mechanism for Mn-SOD expression induced by progesterone in human ESCs. METHODS ESCs were incubated with medroxyprogesterone acetate (MPA; 10(-6) m) or dibutyryl-cAMP (0.5 mm) for 17 d. To determine whether a cAMP-dependent signaling pathway is involved in the MPA-induced Mn-SOD expression, ESCs were treated with H89, an inhibitor of cAMP-dependent protein kinase A. A chromatin immunoprecipitation assay was performed to examine the binding of cAMP-binding protein to the cAMP-response element on the Mn-SOD gene promoter. To examine the involvement of Wnt5a signaling, anti-Wnt5a antibodies were used to neutralize the Wnt5a activities. RESULTS Mn-SOD and Wnt5a mRNA levels and intracellular cAMP concentrations were significantly increased by MPA. These increases were accompanied by an increase in the mRNA expression of IGF-binding protein-1, a marker of decidualization. The increase in Mn-SOD mRNA levels by MPA or dibutyryl-cAMP was completely inhibited by H89. The chromatin immunoprecipitation assay revealed that MPA induced cAMP-binding protein binding with cAMP-response element on the Mn-SOD gene promoter. The increase in intracellular cAMP concentrations by MPA was completely inhibited by treatment with anti-Wnt5a antibodies. MPA treatment had no effects on β-catenin expression. CONCLUSIONS Progesterone increased Mn-SOD expression via a cAMP-dependent pathway in ESCs during decidualization. cAMP-dependent signaling stimulated by progesterone is mediated by noncanonical Wnt5a pathways that signal independently of β-catenin.

Changes in Histone Modification and DNA Methylation of the StAR and Cyp19a1 Promoter Regions in Granulosa Cells Undergoing Luteinization during Ovulation In Rats

The ovulatory LH surge induces rapid up-regulation of steroidogenic acute regulatory (StAR) protein and rapid down-regulation of aromatase (Cyp19a1) in granulosa cells (GCs) undergoing luteinization during ovulation. This study investigated in vivo whether epigenetic mechanisms including histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression. GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h)CG injection. StAR mRNA levels rapidly increased after hCG injection, reached a peak at 4 h, and then remained higher compared with 0 h until 12 h. Cyp19a1 mRNA levels gradually decreased after hCG injection and reached their lowest level at 12 h. A chromatin immunoprecipitation assay revealed that levels of histone-H4 acetylation (Ac-H4) and trimethylation of histone-H3 lysine-4 (H3K4me3) increased whereas H3K9me3 and H3K27me3 decreased in the StAR promoter after hCG injection. On the other hand, the levels of Ac-H3 and -H4 and H3K4me3 decreased, and H3K27me3 increased in the Cyp19a1 promoter after hCG injection. Chromatin condensation, which was analyzed using deoxyribonuclease I, decreased in the StAR promoter and increased in the Cyp19a1 promoter after hCG injection. A chromatin immunoprecipitation assay also showed that binding activities of CAATT/enhancer-binding protein β to the StAR promoter increased and binding activities of phosphorylated-cAMP response element binding protein to the Cyp19a1 promoter decreased after hCG injection. These results provide in vivo evidence that histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression by altering chromatin structure of the promoters in GCs undergoing luteinization during ovulation.

Importance of C/EBPβ 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 proteinβ (C/EBPβ) 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/EBPβ binding and histone modification status (acetylation of histone-H3 lysine-27 [H3K27ac]) in the promoter were examined by chromatin immunoprecipitation assay. Knockdowns of C/EBPβ were performed using the small interfering RNA method. cAMP induced mRNA expressions of IGFBP-1 and PRL accompanied by the increases in both C/EBPβ binding activities and H3K27ac levels in the promoters. The stimulatory effects of cAMP on mRNA levels and H3K27ac levels were completely abolished by C/EBPβ knockdown. cAMP increased Mn-SOD mRNA levels and C/EBPβ binding activities in the enhancer region. C/EBPβ 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/EBPβ knockdown. These results show that C/EBPβ 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 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.

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-β 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.