Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold.

Electrospun nanofiber matrices sufficiently mimic the structural morphology of natural extracellular matrix. In this study, we aimed to examine the effects of agar/polyvinyl alcohol nanofiber (PVA) scaffold on the proliferation efficiency and differentiation potential of neonate mouse spermatogonial stem cells (SCCs). Testicular cells were isolated from testes of 40 mouse pups and were seeded in: 1) 2D cell culture plates in the absence (2D/-GF) or presence (2D/+GF) of growth factors and 2) onto agar/PVA scaffold in the absence (3D/-GF) or presence (3D/+GF) of growth factors. The cells were subsequently cultured for 4 weeks. First 2 weeks were dedicated to proliferative phase, whereas the next 2 weeks emphasized the differentiation phase. The identity of the SCCs was investigated at different time-points by flow cytometry and quantitative reverse transcription PCR (qRT-PCR) analyses against the germ cell markers, including PLZF, Id-4, Gfrα-1, Tekt-1, and Sycp-3. After 2 weeks of culture, the 3D/+GF group showed the highest percentage of PLZF-positive cells among culture systems (P < 0.05). The expression levels of pre-meiotic markers (Id-4 and Gfrα-1) decreased significantly in all groups, particularly in 3D/+GF group after 28 days of culture. Additionally, the cells in the 3D/+GF group displayed the highest expression of meiotic (Sycp-3) and post-meiotic markers (Tekt-1) 14 days after differentiation induction. Seemingly, the combination of the agar/PVA scaffold and growth factor-supplemented medium synergistically increased the differentiation rate of mouse SSCs into meiotic and post-meiotic cells. Thus, agar/PVA nanofiber scaffolds may have the potential for applications in the restoration of infertility, especially in azoospermic males. ABBREVIATIONS: 2D: two dimentional; 3D: three dimentional; bFGF: basic fibroblast growth factor; BMP-4: bone morphogenetic protein 4; DMEM: Dulbecco's modified Eagle's medium; ECM: extracellular matrix; FCS: fetal calf serum; FTIR: Fourier-transform infrared spectroscopy; GDNF: glial cell line-derived neurotrophic factor; GF: growth factors; Gfrα-1, GDNF family co-receptor α1; Id-4, Inhibitor of DNA Binding 4; MTT: methylthiazoltetrazolium; PLZF: promyelocytic leukemia zinc finger; PVA: polyvinyl alcohol; qRT-PCR: quantitative reverse transcription PCR; RA: retinoic acid; SACS: soft agar culture system; SD: standard deviation; SEM: scanning electron microscope; SSCs: spermatogonial stem cells; Sycp-3, Synaptonemal complex protein 3; Tekt-1, Tektin 1.

Different effects of olive leaf extract on antioxidant enzyme activities in midbrain and dopaminergic neurons of Substantia Nigra in young and old rats.

OBJECTIVES: Study of the effects of olive leaf extract on antioxidant enzyme activities in midbrain and dopaminergic neurons of Substantia Nigra in young and old rats. METHODS: Male wistar rats age 4 and 18 months were randomized into control and experimental groups. A single daily dose of 50 mg/kg of olive leaf extract was administered orally by gavage to each rat for 6 months. The control group received only distilled water. All rats were sacrificed 2 hours after the last gavage and their midbrains were separated for Malondialdehyde (MDA) and antioxidant enzyme activitiy analysis. TUNEL assay and immunohistochemical (IHC) staining were used for evaluation of the number of neurons in the Substantia Nigra. RESULTS: The level of Catalase, Glutathione Peroxidase and Superoxide Dismutase enzyme activity were significantly increased in experimental young and old groups compared to their control groups. However the level of Superoxide Dismutase enzyme activity was significantly increased in experimental old group when compared to control group (P< 0.05), the level of Superoxide Dismutase enzyme activity was not significantly changed in young groups. MDA level was decreased significantly in experimental young and old rats compared to their control groups. Histological analysis demonstrated that the number of neurons in Substantia Nigra of experimental old group was more than the control group (P<0.05). The number of apoptotic cells was significantly decreased in experimental old group compared to the corresponding control group (P<0.05). In IHC and TUNEL assay, no change was observed in the number of neurons between experimental and control young groups. CONCLUSION: Long term treatment with olive leaf extract increases antioxidant enzyme activity and protects the neurons in Substantia Nigra against oxidative stress.

Evaluation of effect of oleuropein on skin wound healing in aged male BALB/c mice.

OBJECTIVE: Olive oil and olive leaf extract are used for treatment of skin diseases and wounds in Iran. The main component of olive leaf extract is Oleuropein. This research is focused on the effects of Oleuropein on skin wound healing in aged male Balb/c mice. MATERIALS AND METHODS: In this experimental study, Oleuropein was provided by Razi Herbal Medicine Institute, Lorestan, Iran.Twenty four male Balb/c mice, 16 months of age, were divided equally into control and experimental groups.Under ether anesthesia, the hairs on the back of neck of all groups were shaved and a 1 cm long full-thickness incision was made.The incision was then left un-sutured. The experimental group received intradermal injections with a daily single dose of 50 mg/kg Oleuropein for a total period of 7 days.The control group received only distilled water. On days 3 and 7 after making the incision and injections, mice were sacrificed, and the skin around incision area was dissected and stained by hematoxylin and eosin (H&E) and Van Gieson's methods for tissue analysis.In addition, western blot analysis was carried out to evaluate the level of vascular endothelial growth factor (VEGF) protein expression. The statistical analysis was performed using SPSS (SPSS Inc., Chicago, USA). The t test was applied to assess the significance of changes between control and experimental groups. RESULTS: Oleuropein not only reduced cell infiltration in the wound site on days 3 and 7 post incision, but also a significant increase in collagen fiber deposition and more advanced re- epithelialization were observed (p<0.05) in the experimental group as compared to the control group. The difference of hair follicles was not significant between the two groups at the same period of time. Furthermore, western blot analysis showed an increased in VEGF protein level from samples collected on days 3 and 7 post-incision of experimental group as compared to the control group (p<0.05). CONCLUSION: These results suggest that Oleuropein accelerates skin wound healing in aged male Balb/c mice. These findings can be useful for clinical application of Oleuropein in expediting wound healing after surgery.