In vitro maturation medium supplementation: utilization of repaglinide, L-carnitine, and mesenchymal stem cell-conditioned medium to improve developmental competence of oocytes derived from endometriosis mouse models.

Endometriosis (EMS) is one of the most prevalent causes for female infertility. Herein, we investigated the effect of the repaglinide (RG), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) supplementation during in vitro maturation (IVM) on the quality, maturation, and fertilization rates, as well as embryonic quality and development of oocytes derived from normal and EMS mouse model. Immature oocytes were collected from two groups of normal and EMS-induced female NMRI mice at 6-8 weeks of age. Oocytes were cultured in IVM medium unsupplemented (control group), or supplemented with 1 M RG, 0.3 and 0.6 mg/mL LC, and 25 and 50% BMSC-CM. After 24 h of oocyte incubation, IVM rate and antioxidant status were assessed. Subsequently, the rates of fertilization, cleavage, blastulation, and embryonic development were assessed. Our results demonstrated that supplementation of IVM medium with LC and BMSC-CM, especially 50% BMSC-CM, significantly enhanced IVM and fertilization rates, and markedly improved blastocyst development and total blastocyst cell numbers in EMS-induced mice compared to the control group (53.28±0.24 vs 18.09±0.10%). Additionally, LC and BMSC-CM were able to significantly modulate EMS-induced nitro-oxidative stress by boosting total antioxidant capacity (TAC) and mitigating nitric oxide (NO) levels. Collectively, LC and BMSC-CM supplementation improved oocyte quality and IVM rates, pre-implantation developmental competence of oocytes after in vitro fertilization, and enhanced total blastocyst cell numbers probably by attenuating nitro-oxidative stress and accelerating nuclear maturation of oocytes. These outcomes may provide novel approaches to refining the IVM conditions that can advance the efficiency of assisted reproductive technologies in infertile couples.

In vitro maturation medium supplementation: utilization of repaglinide, L-carnitine, and mesenchymal stem cell-conditioned medium to improve developmental competence of oocytes derived from endometriosis mouse models

Endometriosis (EMS) is one of the most prevalent causes for female infertility. Herein, we investigated the effect of the repaglinide (RG), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) supplementation during in vitro maturation (IVM) on the quality, maturation, and fertilization rates, as well as embryonic quality and development of oocytes derived from normal and EMS mouse model. Immature oocytes were collected from two groups of normal and EMS-induced female NMRI mice at 6-8 weeks of age. Oocytes were cultured in IVM medium unsupplemented (control group), or supplemented with 1 M RG, 0.3 and 0.6 mg/mL LC, and 25 and 50% BMSC-CM. After 24 h of oocyte incubation, IVM rate and antioxidant status were assessed. Subsequently, the rates of fertilization, cleavage, blastulation, and embryonic development were assessed. Our results demonstrated that supplementation of IVM medium with LC and BMSC-CM, especially 50% BMSC-CM, significantly enhanced IVM and fertilization rates, and markedly improved blastocyst development and total blastocyst cell numbers in EMS-induced mice compared to the control group (53.28±0.24 vs 18.09±0.10%). Additionally, LC and BMSC-CM were able to significantly modulate EMS-induced nitro-oxidative stress by boosting total antioxidant capacity (TAC) and mitigating nitric oxide (NO) levels. Collectively, LC and BMSC-CM supplementation improved oocyte quality and IVM rates, pre-implantation developmental competence of oocytes after in vitro fertilization, and enhanced total blastocyst cell numbers probably by attenuating nitro-oxidative stress and accelerating nuclear maturation of oocytes. These outcomes may provide novel approaches to refining the IVM conditions that can advance the efficiency of assisted reproductive technologies in infertile couples.

Follicular fluid supplemented with copper and zinc increase the maturation rate of mouse vitrified-warmed oocytes.

BACKGROUND: Immature oocyte cryopreservation is a therapeutic option in assisted reproductive technology. OBJECTIVE: To evaluate the effects of supplementing oocyte maturation medium with human follicular fluid (hFF), zinc and copper. MATERIALS AND METHODS: Four different maturation media supplemented with 10% follicular fluid, 4 µg/mL copper and 1 µg/mL zinc were used for vitrified-warmed oocytes of mouse. RESULTS: Maturation rate was the highest (63.3%) in the presence of zinc. Cleavage and blastocyst rates in groups with copper and zinc were significantly higher than the control group (39.9% and 46.4% vs. 28.8%), without any significant difference between Zn and Cu. CONCLUSION: Our findings indicate the high importance of using hFF as a natural medium, and also zinc and copper as two efficient trace elements in the maturation medium for vitrified-warmed oocytes.

Supplementation of Vitrification Medium with Vanadium: Evaluation of Histological Change and Follicle Growth.

BACKGROUND: Cryopreservation of ovary is an effective option for treatment of infertility and much effort has been made to improve the ovarian cryopreservation. OBJECTIVE: The study was to investigate the effect of vanadium in the vitrification medium on histology, viability rate and follicle growth. MATERIALS AND METHODS: Mouse ovaries were vitrified in the presence of 0, 10, 100 and 250 µM vanadium (assigned as Group V0, V1, V2 and V3). After thawing, the ovaries were fixed for histological studies, mechanically isolated follicles were cultured, and then viability and growth rate of follicles were assessed. RESULTS: Vanadium supplementation to the vitrification medium significantly increased the number of morphologically normal follicles. In vitro growth and viability rate of follicles was higher in the V2 than V0 group (P < 0.05). CONCLUSION: Vanadium can improve in vitro growth and viability rate of isolated follicles from the vitrified ovaries.

The presence of zinc in the mouse ovary vitrification medium: histological evaluation and follicle growth.

BACKGROUND: Cryopreservation of ovary is a relevant option for preserving fertility of cancer patients undergoing chemotherapy. OBJECTIVE: To evaluate the effect of zinc in the vitrification medium on histology and follicle growth. MATERIALS AND METHODS: Mouse ovaries were vitrified in vitrification medium supplemented with 0, 100, 150 or 200 µg/dl zinc, identified as V0, V1, V2 and V3 groups, respectively. Histological evaluation of ovaries was carried out. The isolated pre-antral follicles were cultured. The size and growth of follicles were assessed. RESULTS: The percentage of morphologically normal follicles increased with increasing zinc concentration in the vitrification medium (P < 0.05). Follicle viability was higher in the V3 than V0 group at the beginning and end of culture (P < 0.05). The highest follicle diameter was obtained in the V3 group after 6 days of culture (P < 0.05). CONCLUSION: Supplementation of the vitrification medium with zinc can improve follicle viability and growth.

Phosphoinositid signal pathway mediate neurite outgrowth in PC12 cells by staurosporine.

OBJECTIVES: In previous studies, we showed that staurosporine uses intracellular calcium ions to affect cell death in PC12 cells. The bulk release of intracellular excessive Ca(2+) from intracellular sources into cytosol contributes to neuronal apoptotic events, which in turn results in neuronal cell death. However, the mechanisms of Ca(2+)-induced neuronal cell death or neurite elongation is still unclear. Therefore, we investigated the relation between phosphoinositid signal pathway, intracellular calcium, and reactive oxygen species on one hand, with staurosporine-induced neurite outgrowth in PC12 cells on the other. RESULTS: The inhibition of phospholipase C or IP3 receptor antagonist or phosphoinositid signal transduction antagonist produced cell death and suppressed neurite outgrowth by staurosporine in PC12 cells. The inhibition of these enzymes and pathway results in an increase in intracellular Ca(2+) although subsequent hydroxyl radical (•OH) production began after inhibitors exposure. •OH production was significantly attenuated in inhibitor supplemented medium treatment, and it was dependent on the intracellular Ca(2+) concentration. These data indicate that staurosporine activates phosphoinositid signal pathway while endoplasmic Ca(2+), and subsequent •OH production are critical events in staurosporine-induced neurite outgrowth in PC12 cells. CONCLUSION: We conclude that the fact that staurosporine mobilizes Ca2+, probably via activating the subcellular compartment, is responsible for staurosporine-induced (Ca2+]i increase during neurite outgrowth in PC12 cells (Fig. 7, Ref. 30).

Diospyros lotus L. fruit extract protects G6PD-deficient erythrocytes from hemolytic injury in vitro and in vivo: prevention of favism disorder.

OBJECTIVE: The aim of this study was to evaluate the protective effect of Diospyros lotus L. fruit extract against the hemolytic damage induced by Vicia faba beans extract in both G6PD enzyme-deficient human and rat erythrocyte in vitro and in vivo. MATERIALS AND METHODS: In the former model, venous blood samples were obtained from five subjects with known G6PD deficiency and erythrocyte hemolysis induced by Vicia faba L. bean extract was asessed spectrophotometrically in the presence and absence of Diospyros lotus L. fruits extract. In the in vivo model, G6PD-deficient rats (induced by intraperitoneal injection of dehydroepiandrosterone for 35 days) pre-treated with different doses of Diospyros lotus L. (500, 750, 1000, and 1500 mg/kg, p.o for 7 days) were challenged with Vicia faba beans extract and the protective effect of the fruit extract against hemolysis was evaluated as above. RESULTS AND CONCLUSIONS: The results have shown that Diospyros lotus L. fruits extract has antioxidant activity that may protect against hemolytic damage induced by Vicia faba bean extract in both G6PD-deficient human and rat erythrocytes. The study gives a scientific basis for the efficacy of the fruit extract as used in Iran. The fact that this was shown in human erythrocytes in vitro is significant and provides a rationale for further testing in vivo in G6PD-deficient human populations.

Cytotoxicity of hydro-alcoholic extracts of Cucurbitapepo and Solanum nigrum on HepG2 and CT26 cancer cell lines.

Plants are used worldwide for the treatment of diseases, and novel drugs continue to be developed through research from plants. There are more than 20,000 species of plants used in traditional medicines, and these are all potential reservoirs for new drugs. Cucurbitapepo has been used in traditional folk medicine to treat cold and alleviate ache. Previous pharmacological tests have shown that it possesses antiviral, anti-inflammatory, and analgesic effects. Also, Solanum nigrum has been used as a diuretic and an antipyretic agent and it has also been used to cure inflammation, edema, mastitis and hepatic cancer. In this investigation, cytotoxicity of specific concentrations of hydro-alcoholic extracts of C. pepo and S. nigrum was studied on normal [Chinese hamster ovarian cells (CHO) and rat fibroblast] and cancer (HepG2 and CT26) cell lines. The cytotoxic effects and IC(50) of the extracts on the selected cell lines were studied followed by colonogenic assay method. The results showed that IC(50) of S. nigrum extract was significantly lower than that of the C. pepo extract on all four cell lines (P < 0.05). On the other hand, IC(50) of S. nigrum extract was significantly higher than the extract of Taxus baccata and Cisplatin, herbal and chemical control positive anticancer compounds, respectively, on all four cell lines (P < 0.05). As a result, it is concluded that the extract of S. nigrum has almost similar cytotoxicity to the extract of T. baccata on cancer cells.

Comparison of the cytotoxic effects of Juniperus sabina and Zataria multiflora extracts with Taxus baccata extract and Cisplatin on normal and cancer cell lines.

Isolation and identification of some potent anti-tumor compounds from medicinal plants has motivated researchers to screen different parts of plant species for the determination of anti-tumor effects. In this study, cytotoxic effects and IC(50) of specific concentrations of hydro-alcoholic extracts of fruits of Juniperus sabina and leaves of Zataria multiflora were compared with hydro-alcoholic extract of bark of Taxus baccata and Cisplatin, well-known anticancer compounds, on normal (CHO and rat fibroblast) and cancer (HepG2 and SKOV3) cell lines. The hydro-alcoholic extracts of the plants were prepared by percolation. The cytotoxic effects and IC(50) of the extracts on the cell lines were studied followed by colonogenic assay after 72 h incubation. The results showed that the extract of Juniperus sabina possesses lower IC(50) in comparison with Zataria multiflora extract on all 4 normal and cancer cell lines (P<0.05); but, IC(50) of the Juniperus sabina extract was significantly higher than the Taxus baccata extract and Cisplatin on all 4 normal and cancer cell lines (P<0.05). As a result, it is concluded that the extract of J. sabina has almost similar cytotoxicity with the extract of Taxus baccata on cancer cells.

Prevention of selenite-induced cataractogenesis by Origanum vulgare extract.

The present study sought to assess antioxidant effect of Origanum vulgare extract in preventing selenite-induced cataractogenesis. This study was performed on Young white rats received sodium selenite (30 nmol g(-1) birth weight) subcutaneously on day 13 post partum during two months in 2009. Cataract formation and intensity was detected and measured by slit-lamp. Origanum vulgare (Ov) extract (2 g kg(-1)) was given (1-2 times) intraperitoneal at different times with respect to the selenite administration lens opacification was analyzed in selenite, selenite-Ov, Ov and control groups on day 7 after selenite administration. Ov extract have revealed a significant protective effect against selenite induced cataract when injected 1 and 2 day (2 times) before selenite injection. There is a protective effect of Ov against selenite induced cataract formation. It is supposed that the anticataract effect of Ov extract could be based on direct or indirect antioxidant mechanisms.

Evaluation of the immunomodulatory effects of five herbal plants.

A group of medicinal plants including, Silybum marianum, Matricaria chamomilla, Calendula officinalis, Cichorium intybus and Dracocephalum kotschyi which grow in Iran, were extracted with ethanol 70% and the mitogenic activity was examined both on human peripheral blood lymphocytes and thymocytes. Effect of these extracts on proliferative responsiveness of human lymphocytes to phytohemagglutinin (PHA) and on the mixed lymphocyte reaction (MLR) was also investigated. The results obtained indicated that none of the extracts had a direct mitogenic effect on human lymphocytes or thymocytes (stimulation index, SI<0.07). Among the plants studied, C. intybus and C. officinalis showed a complete inhibitory effect on the proliferation of lymphocytes in the presence of PHA (SI range 0.01-0.49). A dose dependent inhibitory effect was obtained in the case of D. kotschyi. Extract of M. chamomilla showed almost no stimulatory effect. A significant decrease in proliferation assay due to 0.1-10 microg/ml of S. marianum was observed (SI<0.46, P<0.05). In MLR, a markedly stimulatory effect with some lower concentrations of all the extracts except Dracocephalum was detected. The highest stimulatory effect was due to 100 microg/ml of S. marianum (SI 2.82). Treatment of mixed lymphocytes with 0.1-10 microg/ml of C. officinalis (SI range 1.34-1.80) and 10 microg/ml of M. chamomilla and C. intybus (SI 2.18 and 1.70, respectively) strongly increased the cell proliferation. In conclusion, this in vitro study revealed the capacity of all the extracts except Dracocephalum to enhance the proliferation of lymphocytes after stimulation with the allogenic cells.