Melatonin protects against visible light-induced oxidative stress and promotes the implantation potential of mouse blastocyst in vitro.

Improvement of embryo culture media using antioxidant agents could help to improve embryo quality against environmental factors such as visible light and could overcome implantation failures. The usefulness of the melatonin against the effect of light on the expression of the primary implantation receptors, ErbB1 and ErbB4 on pre-implantation mouse embryo was investigated. Two-cell mouse embryos were exposed to the 1600 LUX light for 30 min then randomly divided into 3 groups including: Melatonin-Treated; Luzindole Treated and Simple media as a Control group. After 72-96  The expanded blastocysts were examined for morphological quality of the embryos by Hoechst and propidium iodide staining and for the expression of ErbB1 and ErbB4 by Real-time PCR and immunocytochemistry. The expression of the Sirt3 gene was also assayed. Furthermore, intracellular reactive oxygen species (ROS) levels and the total antioxidant capacity (TAC) were examined by DCFH-DA fluorescence intensity and radical cation respectively. The number of cells in the inner cell mass (ICM) and outer cell mass (OCM) were elevated significantly in the Melatonin-treated group suggesting increased viability and proliferation. Furthermore, we found that melatonin significantly increased the expression levels of ErbB1, ErbB4, and Sirt3 genes, and the protein expression of ErbB1, ErbB4 correlated with intracellular ROS levels and TAC significantly increased after melatonin treatment. Together, these results demonstrate that melatonin could be helpful to improve preimplantation embryos through its effects in decreasing ROS levels and increasing expression of implantation-related genes.

Augmenting Peripheral Nerve Regeneration Using Hair Follicle Stem Cells in Rats.

Introduction: Cell therapy is the most advanced treatment of peripheral nerve injury. This study aimed to determine the effects of transplantation of hair follicle stem cells on the regeneration of the sciatic nerve injury in rats. Methods: The bulge region of the rat whisker were isolated and cultured. Morphological and biological features of the cultured bulge cells were observed by light microscopy and immunocytochemistry methods. Percentages of CD34, K15, and nestin cell markers expression were demonstrated by flow cytometry. Rats were randomly divided into 3 groups of injury, epineurium, and epineurium with cells in which rat Hair Follicular Stem Cells (rHFSCs) were injected into the site of the nerve cut. HFSCs were labeled with Bromodeoxyuridine (BrdU), and double-labeling immunofluorescence was performed to study the survival and differentiation of the grafted cells. After 8 weeks, electrophysiological, histological, and immunocytochemical analysis assessments were performed. Results: Rat hair follicle stem cells are suitable for cell culture, proliferation, and differentiation. The results suggest that transplantation of rat hair follicle stem cells can regenerate sciatic nerve injury; moreover, electrophysiology and histology examinations show that sciatic nerve repair was more effective in the epineurium with cell group than in the other experimental group (P<0.05). Conclusion: The achieved results propose that hair follicle stem cells improve axonal growth and functional recovery after peripheral nerve injury. Highlights: This study showed that rat hair follicle stem cells are suitable for cell culture, proliferation and differentiationThe results suggested that transplantation of rat hair follicle stem cells had the potential capability of regenerating sciatic nerve injuryEvidence of electrophysiology and histology showed Concomitant use of epineurium with hair follicle stem cell was more effective repairment. Plain Language Summary: Although repairing damaged peripheral nerves has always been a medical challenge, but peripheral nerve injury has been successfully repaired using various procedures such as nerve auto-graft or stem cell therapy. The functional reconstruction is the most important after therapy because of that primary nerve repair or use of nerve autograft, are still accepted as golden standard methods for treatment. Considerable recent interest has been focused on adult stem cells for both research and clinical applications. A highly promising source of relatively abundant and accessible, active, multipotent adult stem cells are obtained from hair follicles. In research the hair follicle stem cells implanted into the gap region of a severed sciatic nerve injury greatly enhanced the rate of nerve regeneration and the restoration of nerve function. Time is one of the several aspects require specific attention in the clinical treatment of peripheral nerve injury. Because delay of nerve injury treatment may cause neurobiological alterations in neurons and Schwann cells, impairing nerve functional recovery and affect neuron survival. In this study, concluded that stem cell injection 2 weeks after injury in the damaged nerve epineurium repairs nerve fibers, while electrophysiology of the leg muscles showed that muscle function was significantly improved. It indicates the repair of muscular innervation and nerve repair. The results pave the way for further research on this topic.

The Effect of Melatonin on Mitochondrial Function and Autophagy in In Vitro Matured Oocytes of Aged Mice.

OBJECTIVE: This study examined the in vitro effect of melatonin on the protein synthesis of mitochondria, as well as autophagy in matured oocytes of aged mice. MATERIALS AND METHODS: In this experimental study, germinal vesicles (GV) oocytes were collected from aged (with the age of six-months-old) and young mice (with age range of 6-8 weeks old) and then cultured in the in vitro culture medium (IVM) for 24 hours to each metaphase II (MII) oocytes and then supplemented with melatonin at a concentration of 10 µM. The culture medium of MII oocytes was devoid of melatonin. Afterward, the expression of the SIRT-1 and LC3 was assessed by immunocytochemistry. ATP-dependent luciferin-luciferase bioluminescence assay was employed for the measurement of the ATP contents. Intracellular reactive oxygen specious (ROS) was detected by DCFH-DA, and the total antioxidant capacity (TAC) level was determined by TAC assay. RESULTS: The expression of SIRT-1 and LC3, as well as the measurement of the ATP content, was significantly increased in oocytes treated with melatonin compared with the oocytes receiving no treatment. Moreover, TAC was considerably higher in melatonin-treated oocytes than oocytes receiving no treatment. On the other hand, the level of ROS was significantly decreased in oocytes treated with melatonin in comparison with the untreated oocytes. The results indicated that melatonin considerably improved the development of oocytes as well. CONCLUSION: According to the data, melatonin increased mitochondrial function and autophagy via an increase in the expression of SIRT1 and LC3, as well as the ATP contents while it decreased the levels of ROS and increased TAC in oocytes derived from aged mice.

Melatonin Pretreated Blastocysts along with Calcitonin Administration Improved Implantation by Upregulation of Heparin Binding-Epidermal Growth Factor Expression in Murine Endometrium.

OBJECTIVES: Implantation failure is an obstacle in assisted reproduction techniques (ART). Calcitonin is a molecules involved in uterine receptivity and embryo implantation. Melatonin can promote embryo quality and improve implantation. This study examines the effect of pretreatment of blastocysts with melatonin and calcitonin on heparin binding-epidermal growth factor (HB-EGF) expression in murine endometrium. MATERIALS AND METHODS: In this experimental study, we collected 2-cell embryos from the oviducts of 1.5 day pregnant NMRI mice. Embryos were cultured to the blastocyst in GTM medium with or without 10-9 M melatonin. Pregnant and pseudo-pregnant mice received intraperitoneal (IP) injections of 2 IU calcitonin. After 24 hours, we transferred the cultured blastocysts into the uteri of pseudo-pregnant mice. Two days later, implantation sites were counted and we assessed the levels of HB-EGF mRNA and protein in the uteri of naturally pregnant and pseudo-pregnant mice by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Statistical analysis was performed with one-way ANOVA followed by the Tukey post hoc test. P<0.05 was considered statistically significant. RESULTS: Melatonin pretreatment of blastocysts along with calcitonin administration significantly increased HB-EGF mRNA and protein (P<0.001) in the endometrium of pseudo-pregnant mice. Administration of calcitonin in naturally pregnant mice significantly increased HB-EGF mRNA and protein levels (P<0.001). Compared with the control group (2.6 ± 0.5), the average number of implantation sites in the melatonin group (4.6 ± 0.5, P<0.05) and calcitonin group (7 ± 1, P<0.001) significantly increased. There was a significant increase in implantation sites in the combined melatonin and calcitonin group (8.6 ± 0.5, P<0.001). Calcitonin significantly enhanced calcitonin receptor mRNA (P<0.001) and protein (P<0.05) in the uteri of naturally pregnant and pseudo-pregnant mice. CONCLUSIONS: Melatonin pretreated blastocysts along with calcitonin increased HB-EGF expression in the uteri of pseudopregnant mice. Calcitonin administration upregulated HB-EGF in uteri of naturally pregnant mice.

Melatonin upregulates ErbB1 and ErbB4, two primary implantation receptors, in pre-implantation mouse embryos.

OBJECTIVES: To evaluated the effects of melatonin on early embryo competence and the expression rate of the primary implantation receptors (ErbB1 and ErbB4). MATERIALS AND METHODS: Two-cell mouse embryos were cultured in 3 groups: simple media, melatonin-treated (10-9 M melatonin) and Luzindole-treated (10-9 M luzindole). Then, the rate of ErbB1 and ErbB4 gene and protein expression, the level of intracellular ROS, antioxidant capacity, and also the number of cells were evaluated and compared with the fourth group in vivo developed blastocysts (control group). RESULTS: We concluded that melatonin significantly up-regulated the ErbB1 and ErbB4 gene and protein expression, decreased intracellular ROS, increased the total antioxidant capacity, and also elevated the cell numbers in the melatonin-treated group compared with the other groups (P≤ 0.05). CONCLUSION: The use of melatonin may be a helpful factor in improving the embryo quality and enhancing the expression of ErbB1 and ErbB4, two important implantation-related genes and proteins.

In vitro induction effect of 1,25(OH)2D3 on differentiation of hair follicle stem cell into keratinocyte.

BACKGROUND: Stem cells are characterized by self-renewal and differentiation capabilities. The bulge hair follicle stem cells (HFSCs) are able to convert to epithelial components. The active metabolite of vitamin D, 1,25(OH)2D3, plays important roles in this differentiation process. In the present study has found that 1,25(OH)2D3 induces the HFSCs differentiation into keratinocyte. METHODS: HFSCs are isolated from rat whiskers and cultivated in DMEM medium. To isolate bulge stem cell population, flow cytometry and immunocytochemistry using K15, CD34 and nestin biomarkers were performed. In order to accelerate the HFSCs differentiation into eratinocyte, HFSCs were treated with 10-12 M, 1,25(OH)2D3 every 48 h for a week. RESULTS: Immunocytochemistry results showed that bulge stem cells are nestin and CD34 positive but K15 negative before differentiation. Subsequently flow cytometry results, showed that the expression of nestin, CD34 and K15 were 70.96%, 93.03% and 6.88% respectively. After differentiation, the immunocytochemical and flow cytometry results indicated that differentiated cells have positive reaction to K15 with 68.94% expression level. CONCLUSION: It was concluded that 10-12 M, 1,25(OH)2D3 could induce the HFSCs differentiation into keratinocytes.