Effects of in vitro repaglinide supplementation on improving sperm motility parameters, viability, and DNA integrity in normozoospermic and asthenozoospermic men.

OBJECTIVE: Human sperm motility and hyperactivation (HA) are induced by different factors such as intracellular calcium concentration. Repaglinide is an antidiabetic drug that, via the blocking of ATP-sensitive potassium channels (K-ATP channels), depolarization of the ß-cell membrane, and opening of the voltage-gated calcium channels leads to an increase in intracellular calcium. The present study aimed to examine the effects of repaglinide on in vitro sperm motility parameters, viability, and DNA integrity in normozoospermic and asthenozoospermic men. METHODS: Semen samples were collected from two groups of normozoospermic donors and asthenozoospermic patients. The samples were washed free of seminal plasma and then treated with medium alone (control) or with 100 nM and 1µM concentrations of repaglinide. After 1 h of incubation, percent sperm motility and hyperactivation were assessed; after 2 h of incubation, sperm viability and DNA fragmentation rate were evaluated by the Eosin-Y and acridine orange staining, respectively. RESULTS: In both groups, repaglinide at a concentration of 100 nM and 1µM significantly improved percent sperm motility, hyperactivation, and vital sperms with normal DNA; in specimens from normozoospermic men, the 1µM concentration had a noticeable effect on progressive motility; in samples from asthenozoospermic men, the highest hyperactivation rate was seen at a concentration of 100 nM as compared with the 1µM concentration and controls (p<0.05). CONCLUSIONS: Our results suggest that repaglinide can improve sperm motility, hyperactivity, viability, and DNA integrity in both normozoospermic and asthenozoospermic men.

Therapeutic effects of L-arginine, L-carnitine, and mesenchymal stem cell-conditioned medium on endometriosis-induced oocyte poor quality in an experimental mouse model.

AIM: The present study aimed to explore the potential ameliorative effects of L-arginine (LA), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on endometriosis (EMS) model in vivo and in vitro. METHODS: The animals were divided into two main groups, normal and EMS-induced mice. Normal and EMS-induced groups were injected with or without LA (250 mg/kg), LC (250 mg/kg), and BMSC-CM (a final volume of 100 µL of CM/mouse). At the end of the study, the level of total antioxidant capacity (TAC), nitric oxide (NO), and total oxidative status (TOS) were measured in plasma. Furthermore, immature oocytes were collected from two groups and cultured in a maturation medium. Subsequently, the rates of in vitro maturation, in vitro fertilization (IVF), and in vitro embryonic development were evaluated. RESULTS: The results revealed that administration of LA, LC, and BMSC-CM ameliorated the oxidative status through maintaining TAC and alleviating TOS and NO levels. More importantly, the maturation and fertilization rates, blastocyst development, and total blastocyst cell numbers significantly increased in LA, LC, and BMSC-CM-administrated groups compared to the control group. In both the normal and EMS groups, the highest IVF, cleavage, and blastocyst percentages were associated with BMSC-CM treatment (p < 0.05). CONCLUSION: Altogether, LA, LC, and BMSC-CM have therapeutic effects on impaired oocyte quality and promote subsequent development in vitro, probably through normalization of nitro-oxidative stress, thus offering potential alternatives to conventional therapies during assisted reproductive technologies for patients with EMS-associated sub/infertility.

Therapeutic effects of silver nanoparticle and L-carnitine on aerobic vaginitis in mice: an experimental study.

Introduction: Aerobic vaginitis (AV) is a type of vaginal infection that occurs at the reproductive age of women. In this study, we aimed to study the possible anti-AV therapeutic effects of silver nanoparticles (AgNPs) and L-carnitine (LC) in the mouse model. Methods: AV model was established by intra-vaginal inoculation of 108 CFU/mL Staphylococcus aureus and Escherichia coli (1:1) in adult NMRI mice. Susceptibilities of the bacteria were examined against AgNPs by inhibitory concentration (IC-50 and IC-90) and minimum biofilm inhibitory concentration (MBIC- 90) methods. The regimens therapy was intra-vaginal inoculation of AgNPs at MBIC- 90 and a daily injection of 250 mg/kg LC for two weeks. Mice were classified into healthy (control) and AV groups and then treated by LC, AgNPs, and AgNPs + LC. The vaginal smears were taken daily and tissue sections were prepared using the hematoxylin and eosin (H & E) method. Results: Minimum inhibitory concentrations (MICs) of AgNPs for E. coli, S. aureus, and their mixture were 250, 125, and 500 ppm, and their MBIC-90% were 500, 250, and 1000 ppm, respectively. The estrus cycle of mice treated with co-administration of AgNPs and LC was similar to the control group (P < 0.05). The results of histology also showed that infected mice were treated with AgNPs and LC, simultaneously. Conclusion: Single bacteria are more sensitive than their mixed model to these NPs. Co-administration of AgNPs as an antibacterial agent and LC as an antioxidant agent can treat AV in the infected mice.

Effect of Silver Nanoparticles and L-Carnitine Supplement on Mixed Vaginitis Caused by Candida albicans/ Staphylococcus aureus in Mouse Models: An Experimental Study.

The evolution of antimicrobial-resistant pathogens is a global health and development threat. Nanomedicine is rapidly becoming the main driving force behind ongoing changes in antimicrobial studies. Among nanoparticles, silver (AgNPs) have attracted attention due to their versatile properties. The study aimed to investigate the effects of AgNPs and L-carnitine (LC) on mixed Candida albicans and Staphylococcus aureus in the mice vaginitis model. Study of antimicrobial activity of AgNPs evaluated by Minimum Inhibitory Concentration (MIC) and Minimum Biocidal Concentration (MBC) assays. AgNPs inhibited biofilm formation of microbial strains, which was tested by using crystal violet staining. In the current study, we evaluated the effects of AgNPs and LC in NMRI mice infected intravaginally with C. albicans/ S. aureus for two weeks. The proportion of mice in each stage of the estrous cycle (proestrus, estrus, metestrus, and diestrus) was examined. Histological properties were assessed by hematoxylin/ eosin (H&E) staining of formalin-fixed, paraffin-embedded vaginal tissue sections. Based on the results, MICs of AgNPs against S. aureus, C. albicans, and their combination were 252.3, 124.8, and 501.8 ppm, and their minimum biofilm inhibitory concentration (MBIC) was 500, 250, and 1000 ppm, respectively. The estrous cycle in the treated group was similar to the control. Vaginal histology and cytology showed that LC can improve tissue damages caused by vaginitis and AgNPs. This study demonstrates the promising use of AgNPs as antimicrobial agents and the combination of AgNPs/ LC could be a great future alternative in the control of vaginitis.

Effect of Pentoxifylline on Staurosporine-Induced Neurite Elongation in PC12 Cells.

Objective: Pentoxifylline enhances neurite elongation in PC12 cells. This study investigated the effects of pentoxifylline on staurosporine-induced neurite elongation in PC12 cells. Materials and Methods: There were five treatment groups, including treatment group I (1 nM), treatment group II (10 nM), treatment group III (100 nM), treatment group IV (1uM), and treatment group V (10 mM of pentoxifylline), together with 214 nM staurosporine for a range of time (6, 12 and 24 hours). Cells only treated with staurosporine at a concentration of 214 nM were used as the control group. Cell proliferation, cell death, immunocytochemistry assay, and Total Neurite Length were assessed. Results: The results showed that pentoxifylline increased cell viability (p<0.05) in a dose- and time-dependent manner, and cell death assay showed that cell death decreased in a dose- and time-dependent manner (p<0.05). TNL increased significantly compared with control cells (p<0.05). Immunocytochemistry assay showed that pentoxifylline at low and high concentrations enhanced ß-tubulin III and GFAP protein expression compared with control cells. Conclusion: It can be concluded that pentoxifylline has positive effects on the staurosporine-induced neurite outgrowth process in PC12 cells.

The protective effects of curcumin on cytotoxic and teratogenic activity of retinoic acid in mouse embryonic liver.

BACKGROUND: Retinoic acid (RA) is a synthetic vitamin derivative. It exerts toxic and teratogenic effects on the development of embryonic organs in dose- and time-dependent manners in mice. Curcumin is a compound obtained from rhizomes of turmeric (Curcuma longa) and has protective effects on teratogenic agents. The current study examined the effects of curcumin on embryos treated with RA. METHODS: A total of 24 female NMRI mice (8-week-old pregnant mice) were investigated in the current study. All of them were treated for 10 days during days 15 to 50 of pregnancy. In the first group, the animals were fed with normal diets (control); in the second group, with 60 mg/kg all- trans RA; in the third group, with 10 mg/kg curcumin; and in the fourth group, with RA and curcumin in their diets. The animals were killed by cervical dislocation at the 18th day of pregnancy and embryos were separated from the uteruses. The embryo weight and crown rump (CR) length were measured, and the SPSS software was used to analyze data. RESULTS: There was a significant increase in the lengths of CR and weights of embryos after using curcumin, but RA had no effect on the length of CR and weight of embryos at a dose of 60 mg/kg. Morphometric assay of liver tissue was performed, and data analysis indicated that there were significant differences between groups in terms of morphometric parameters of liver tissue. Therefore, RA increased the cell number and sinusoid diameter and decreased the cell areas in the embryonic liver tissue. However, curcumin decreased these side effects of RA on the embryonic liver tissue. CONCLUSION: The results indicated that curcumin could decrease the toxic and teratogenic effects of RA in mouse embryos.

First comprehensive report of bacteria spp. associated with cloaca of Laudakia nupta (Sauria: Agamidae) in Iran using molecular studies.

Iran bears a remarkable variety of reptiles. One of the lizard families occurring in Iran is the Family Agamidae which is widely are distributed throughout the old world. The large-scaled rock agamid, Laudakia nupta, is one of the well-known agamid. There are few reports of cloacal microbial on reptiles hence their function in cloacae remains unknown. Laudakia nupta usually live in rural and urban areas and close vicinity to man, they are likely to play an important role in the spread of disease that may be caused by these microorganisms and their transmission to man. Therefore, the aim of this study was to identify the bacterial flora colonizing the cloacal region of Laudakia nupta using molecular studies. The cloacal fluids were directly placed on nutrient agar (NA) plates and incubated at 25 ± 2 °C for 48 h. The resulting bacterial colonies were transferred to fresh nutrient agar (NA) plates for molecular studies. Twelve isolates were obtained from 17 specimens of Laudakia nupta. All bacteria isolates were identified as Bacillus subtillis (5), Bacillus cereus (4), Bacillus sp. (1), Pseudomonas putida (1), and Pseudomonas sp. (1) based on partial sequences of the 16 s rRNA gene. This is the first comprehensive report of bacteria spp. associated with cloaca of Laudakia nupta using molecular studies. In this research, we found that Laudakia nupta can be a carrier of bacteria which can transfer microorganisms to hosts.

L-Carnitine improves endocrine function and folliculogenesis by reducing inflammation, oxidative stress and apoptosis in mice following induction of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is related to low levels of serum l-carnitine, which has antioxidant, anti-inflammatory and antiapoptotic properties. The aim of this study was to investigate the effect of l-carnitine on folliculogenesis in mice following induction of PCOS. PCOS was induced by daily injections of testosterone enanthate (1mg per 100g, s.c., for 35 days). NMRI mice (21 days old) were divided into four groups (n=6 per group): Control, Control+l-carnitine, PCOS and PCOS+l-carnitine. Mice were treated with 500mgkg-1, i.p., l-carnitine every second day for 28 days. Ovaries were studied stereologically and serum concentrations of FSH, LH, testosterone, interleukin (IL)-6 and tumour necrosis factor (TNF)-α were determined using ELISA kits. Serum concentrations of malondialdehyde (MDA) and the ferric ion reducing antioxidant power (FRAP) were also analysed. Apoptosis of follicles was evaluated by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL). CD31 was assessed immunohistochemically. Data were analysed using one-way analysis of variance (ANOVA) and Tukey's test, differences considered significant at P<0.05.The total volume of the ovary, cortex volume, oocyte volume, zona pellucida thickness and the number of antral follicles increased significantly, whereas the number of primary and preantral follicles decreased significantly, in the PCOS+l-carnitine versus PCOS group. In the PCOS+l-carnitine group, serum concentrations of FSH and FRAP increased significantly, whereas there were significant decreases in serum concentrations of testosterone, LH, MDA, IL-6 and TNF-α, as well as in the percentage of TUNEL-positive apoptotic cells, compared with the PCOS group. l-Carnitine improves folliculogenesis and is therefore suggested as a therapeutic supplement in the treatment of PCOS.

Regulatory networks upon neurogenesis induction in PC12 cell line by small molecules.

Alteration in the normal regulatory pathway of differentiation can lead to the induction of programmed cell death. Accordingly, some chemicals like staurosporine, nerve growth factor, pituitary adenylate cyclase activating peptide, and trimethyltin are shown to be able to induce differentiation in vitro, via different mechanisms in the PC12 cell line. Hence, understanding the details of the molecular mechanisms of differentiation induction by these small molecules are important for further application of these molecules in neurogenesis. Therefore, we sought to determine these signaling pathways, using gene regulatory networks analysis. Then, we have conducted a comparative analysis of the alterations in the gene expression pattern of the PC12 cell lines in response to these chemicals at the early stages. Based on the comparative analysis and previous knowledge, we have proposed the affected pathways during differentiation and apoptosis. Our findings could be useful in the development of protocols to reprogramming of neurons by such small molecules with high efficiency.

L-arginine/5-fluorouracil combination treatment approaches cells selectively: Rescuing endothelial cells while killing MDA-MB-468 breast cancer cells.

Reducing the adverse effects of chemotherapy on normal cells such as endothelial cells is a determinant factor of treatment success especially in pregnant women. In this regard, modulatory effect of L-arginine on various cancers is still a controversial topic in cancer therapy. So, this study aimed to compare the effect of L-arginine treatment alone and in combination with 5-fluorouracil (5-FU) on the survival and angiogenesis of primary human umbilical vein endothelial cells (HUVECs) and the breast cancer cell line of MDA-MB-468. Combinations of L-arginine and 5-FU increased cell survival in HUVECs but induced cell death in MDA-MB-468 cells. Nitric oxide assay showed an increase of this molecule in both cell lines. Assessments of metabolic changes as well as molecular docking indicated a decrease in glycolytic activity of cancer cells but not normal cells. Angiogenesis induction in HUVECs was confirmed through VEGF and MMP-2,9 up-regulated gene expressions. However, a down-regulation of the above-mentioned genes expression was observed in MDA-MB-468. Furthermore, an in vivo increased angiogenesis and decreased embryo toxicity was observed in combination treatment. Altogether, these findings clearly suggest that L-arginine inhibits cell death induced by 5-FU in HUVECs through attenuating the adverse effects of 5-FU, while it does not do so in breast cancer cells.

Protective Effect of Rosa Canina Extract Against Doxorubicin-induced Testicular Toxicity in Mice

Abstract The present study was aimed to investigate the in vivo effects of Rosa canina extract on doxorubicin-induced testicular toxicity in mice for the first time. Male NMRI mice were randomly divided into six treatment groups (10=per group) as follows: (I) vehicles, (II) doxorubicin alone (3 mg/kg, i.p. on days 7, 14 and 21), (III and IV): Rosa canina extract alone (100 mg/kg and 200 mg/kg per day, i.p. for 28 days), (V and VI) Rosa canina extract plus doxorubicin (each dose given 1 h post Rosa canina). Doxorubicin-treated mice displayed smaller body and testicular weights, decreased serum levels of testosterone, loss in the number of germ cells and Sertoli cells, and reduced sperm count, viability, morphology and motility. Doxorubicin treatment significantly decreased the mean testis diameter, seminiferous tubular diameter and seminiferous epithelial height and increased seminiferous luminal diameter. However, Rosa canina pretreatment could effectively improve all of these abnormalities in doxorubicin- treated mice. The treatment with a higher dose of the extract (200 mg/kg) was more effective compared to doxorubicin and the lower dose of the extract. These findings suggested that the Rosa canina extract has protective effects against doxorubicin-induced reproductive toxicity.

Improvement of the folliculogenesis by transplantation of bone marrow mesenchymal stromal cells in mice with induced polycystic ovary syndrome.

BACKGROUND: Many studies have reported that inflammation and oxidative stress are involved in the pathogenesis of polycystic ovary syndrome (PCOS). Bone marrow mesenchymal stromal cells (BM-MSCs) have anti-oxidant and anti-inflammation properties. In this study, we investigate the beneficial effect of stem cell therapy on folliculogenesis in mice with induced PCOS METHODS: Mouse model of PCOS was performed through daily injection of testosterone enanthate (1 mg/100 g/body weight subcutaneous (s.c).) for a period of 5 weeks. Naval Medical Research Institute (NMRI) mice (21 days old) were divided into three groups: control, PCOS and PCOS + BM-MSCs. BM-MSCs were labeled with Hoechst 33342 (0.5 µg/mL) and then injected into the mice (106/animal, via the tail vein) at 1 and 14 days after PCOS confirmation. Mice were humanely killed at 2 weeks after last transplantation. Ovarian stereological studies were done. Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), testosterone, interleukin (IL)-6 and tumor necrosis factor (TNF)-α serum levels were measured. The levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) in serum were analyzed. Apoptotic index for ovarian follicles was assessed using Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). CD31 expression in ovarian vessels was assessed with the immunohistochemistry. RESULTS: There was a significant increase in the total volume of ovary, cortex, number of antral follicles, volume of oocyte and zona pellucida thickness, and there was a significant decrease in the primary and preantral follicles number in the PCOS + BM-MSCs group compared with the PCOS group. There was a significant increase in the serum level of FSH and TAC and a significant decrease in the serum level of testosterone, LH, MDA and percentage of TUNEL-positive apoptotic cells in the PCOS + BM-MSCs group in comparison with the PCOS group. DISCUSSION: BM-MSC transplantation improves folliculogenesis in mice with induced PCOS. BM-MSC therapy can be an operative treatment for PCOS via anti-inflammatory, anti-oxidant and anti-apoptotic properties.

Ovary stereological features and serum biochemical factors following induction of polycystic ovary syndrome with testosterone enanthate in mice: An experimental study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine disorder featured by insulin resistance and hyperandrogenism. Testosterone enanthate can induce PCOS in mice models. OBJECTIVE: We investigated the ovary stereological features along with the oxidative stress and inflammatory factors in mice following PCOS induction using testosterone enanthate. MATERIALS AND METHODS: Twelve female NMRI mice (3 wk old) were divided into 2 groups (n=6/each): Control and PCOS. PCOS was induced through daily injections of testosterone enanthate (1 mg/100g subcutaneous s.c for 5 wk). Finally, ovaries were studied stereologically. The serum levels of the follicle-stimulating hormone, luteinizing hormone, testosterone, interleukin-6, and tumor necrosis factor-α were measured using ELISA kit. Serum levels of Malondialdehyde and the antioxidant capacity were measured relatively using thiobarbituric acid and ferric reducing antioxidant power assay. RESULTS: The mean total volume of ovary and the mean volume of cortex (p<0.001), volume of oocyte in the preantral (p=0.011) and antral follicle (p=0.015), thickness of zona pellucida (p=0.016), the number of antral follicles (p=0.012), the serum levels of follicle-stimulating hormone (p<0.001) and the antioxidant capacity (p=0.020) reduced significantly in the PCOS group compared to the control. The number of primary (p=0.017) and preantral (p=0.006) follicles and the serum levels of testosterone (p<0.001), Luteinizing hormone (p=0.002), Malondialdehyde, Interleukin 6 and Tumor necrosis factor-α (p<0.001) showed a significant increase in the PCOS group compared to the control. CONCLUSION: Testosterone enanthate induced PCOS causes stereological features in the ovary, increases the oxidative stress and inflammatory markers in mice.

The beneficial effect of repaglinide on in vitro maturation and development ability of immature mouse oocytes.

Repaglinide is a hypoglycemic drug, causing depolarization of the cell membrane, opening the voltage-gated calcium channels, and then increasing intracellular calcium in the pancreatic B cells by inhibition of the K-ATP-sensitive channels. Oocyte in vitro maturation (IVM) is influenced by different factors such as calcium signaling. In this study, we examined the effects of repaglinide on in vitro maturation and fertilization ability of mouse oocyte. Immature oocytes were isolated from female Naval Medical Research Institute mice which are 6-8 wk old mechanically and then cultured in 30 µl droplets of T6 medium with different concentrations of repaglinide. The control group did not receive repaglinide (R0). Treatment groups received different concentrations (5, 10, and 100 nM and 1 and 10 µM) of repaglinide (R1, R2, R3, R4, and R5, respectively). Oocyte in vitro maturation rate was assessed after 24 h. In vitro fertilization was performed using metaphase II oocytes obtained from R0 and R4 treatments. Embryo cleavage rate was calculated at 48 h post-IVF. Chi-square test was used for evaluating difference between control and treatment groups (p < 0.05). Oocyte maturation rate after 24 h in treatment groups R2, R3, R4, and R5 was significantly higher than that in the control (p < 0.05). Supplementation of medium with 1 µM of repaglinide (R4) during IVM significantly improved outcome of embryo cleavage rate than control at 48 h post-IVF (p < 0.05). In conclusion, repaglinide can be considered as an effective agent for in vitro oocyte maturation and embryo cleavage.

Impact of prolonged oocyte incubation time before vitrification on oocyte survival, embryo formation, and embryo quality in mice.

Oocyte incubation time before freezing is one of the factors affecting oocyte vitrification. In the assisted reproductive technology (ART) clinics, it is sometimes decided to perform oocyte vitrification after a long period of incubation time due to various conditions, such as inability to collect semen samples, unsuccessful urological interventions (PESA, TESE, etc.), or unexpected conditions. A time factor of up to 6 h has been studied in the available reports. Therefore, this study was designed to evaluate oocyte incubation time before freezing at 0, 6, 12, 18, and 24 h after retrieval. Metaphase II (MII) oocytes were obtained from NMRI female mice after being randomly divided into the five groups of 0, 6, 12, 18, and 24 h of freezing via hormonal stimulation following retrieval and entered into the vitrification-warming process. The thawed oocytes were evaluated according to the survival criteria and then inseminated with the sperms of male mice for in vitro fertilization. The next day, the embryo formation rate and embryo quality were assessed. Our results demonstrated that even after 24 h of incubation, the survival rate of oocytes was 51.35% with the embryo formation rate of 73.21%. However, the survival and embryo formation rates significantly decreased within 12, 18, and 24 h after retrieval compared to the groups vitrified at 0 h. The embryo quality was significantly reduced by vitrification at 0 to 24 h after retrieval. According to our data, although a prolonged incubation time before freezing reduced the survival rate, there was still a chance for oocytes to stay alive with acceptable embryo formation and quality rates after vitrification warming of oocytes.

l-arginine alters the effect of 5-fluorouracil on breast cancer cells in favor of apoptosis.

Chemoresistance in breast cancer is a major obstacle, especially in p53 mutation types. The aim of this study was to evaluate if a combination therapy of l-arginine with 5-fluorouracil (5-FU) can alter the effect of this chemotherapy drug on breast cancer cells. The study was performed on BT-20 and MCF-7 cell lines. The effects of l-arginine alone and in combination with 5-FU were investigated on cell viability, apoptosis and nitric oxide (NO) production. Drugs effects on the cellular energetic metabolism were investigated through the lactate production and glucose-6-phosphate dehydrogenase (G6PD) activity assay. Migration and invasion of treated cells were assessed. Real- time PCR was used for analyzing the changes in the expression level of CXCL12 and CXCR4 as two important genes involved in migration and metastasis of breast cancer cells. l-arginine increased 5-FU effect on BT-20 and MCF-7 cell lines by reducing cell viability and increasing apoptosis and NO production. Lactate production and G6PD activity assays showed that cellular energetic metabolism of both cells was altered in favor of cell death. Moreover, l-arginine decreased the metastatic activity of both cells which was confirmed through migration, invasion and gene expression results performed for both cell lines. However, drugs effect on MCF-7 (p53 wild-type) was greater than that of BT-20 (p53 mutation) in all sets of experiments. Our findings indicated that l-arginine increased the anticancer effect of 5-FU in BT-20 and MCF-7 cell lines. So, combination therapy with l-arginine and 5-FU could be considered as an effective strategy in breast cancer therapy.

Zinc supplementation of vitrification medium improves in vitro maturation and fertilization of oocytes derived from vitrified-warmed mouse ovaries.

Oocyte cryopreservation is an approach for fertility preservation for normal women and cancer patients facing chemo and radiotherapy. The present study evaluated the effect of adding zinc chloride to the vitrification medium used for whole mouse ovaries and then assessing the in vitro maturation and fertilization of oocytes when they were subsequently extracted from these vitrified ovarian tissues. Four vitrification solutions with 0, 100,150 and 200 µg/dl zinc (V0, V1, V2 and V3 respectively) were compared. The viability of oocytes isolated from ovaries vitrified-warmed in the highest concentration of zinc (V3) was significantly higher after 24 than in the control V0 group (72.99 vs 85.97). Progression to the MII stage, fertilization and cleavage by 48 h was also higher in the V3 than V0 control group (35.55 vs 44.73), (47.67 vs 63.74), (28.72 vs 43.03) (P < 0.05) respectively. These results indicate that supplementation of vitrification medium for intact ovaries with zinc can improve the oocyte viability and in vitro maturation-fertilization rate.

Isolation of breast cancer stem cell from MDA-MB231 cell line using vincristine / Aislamiento de células madre de cáncer de mama de la línea celular MDA-MB231 utilizando vincristina

Cancer has been considered as a stem cell disease. Suspension culture combined with anti-cancer drugs has recently been proposed for isolation of cancer stem cells (CSCs). In the current study, Vincristine as an anti-cancer drug combined with suspension culture was used for isolation and purification of CSCs from human breast cancer cell line (MDA-MB231). The cells were treated with different concentrations of vincristine (0, 2, 4, 6 and 8 ng/ml). Stem cells were identified with the expression of OCT4, nanog, SOX2 and nucleostemin genes by RT-PCR. Mammosphere forming unit was measured upon suspension culture containing EGF, bFGF, LIF, B27, insulin and BSA. The isolated mammospheres were investigated for CD44 expression. Results showed that 4 ng/ml of vincristine for 72 hours could be utilized as the best and most reliable dose which eliminates around 80 % of non-cancer stem cells with no destructive effect on CSCs' viability (P> 0.05). RT-PCR demonstrated that drug treated cells expressed OCT4, nanog, SOX2 and nucleostemin. Mammosphere formation unit of cells pretreated with vincristine was significantly higher than unpretreated ones (P>0.05). Immunofluorescence staining for CD44 depicted high expression of CSC marker among the isolated mammospheres. Vincristine combined with suspension culture can be considered as an appropriate method to isolate CSC.

El cáncer ha sido considerado como una enfermedad de células madre. Recientemente se ha propuesto cultivo en suspensión en combinación con medicamentos contra el cáncer para aislamiento de las células madre del cáncer (CMC). En este estudio se utilizó la vincristina como fármaco anticanceroso combinado con cultivo en suspensión para el aislamiento y purificación de las células madre cancerosas, de la línea celular de cáncer de mama humano (MDA-MB231). Las células se trataron con diferentes concentraciones de vincristina (0, 2, 4, 6 y 8 ng/ml). Las células madre se identificaron mediante la expresión de los genes OCT4, Nanog, SOX2 y nucleostemin por RT-PCR. La unidad de formación mammosphere se midió a través de cultivo en suspensión que contenía EGF, bFGF, LIF, B27, insulina y BSA. Los mammospheres aislados fueron estudiados para la expresión de CD44. Los resultados mostraron que 4 ng/ml de vincristina durante 72 horas podrían ser utilizados como la mejor y más fiable dosis que permite eliminar alrededor del 80 % de las células madre no cancerosas, sin causar un efecto destructivo sobre la viabilidad de las CMC (P> 0,05). La RT-PCR mostró que en las células tratadas con él fármaco hubo expresión de los genes OCT4, Nanog, SOX2 y nucleostemin. La unidad de formación de las células pretratadas con vincristina fue significativamente más alta que las unidades sin tratamiento previo (P>0,05). La inmunofluorescencia para CD44 muestró una alta expresión del marcador de CMC entre mammospheres aisladas. La vincristina en combinación con el cultivo en suspensión puede ser considerado como un método apropiado para aislar CMC.

Production of hepatocyte-like cells from human umbilical vein mesenchymal stem cells.

The human umbilical vein, as a readily available stem cell source, is a good alternative to harvest mesenchymal stem cells. Human umbilical cord vein mesenchymal stem cells have recently been isolated and have demonstrated the ability to differentiate into various cell types such as fat, bone, cartilage and neuronal cells. In this study, we have investigated whether human umbilical cord vein mesenchymal stem cells are also able to differentiate into hepatocyte-like cells. Hepatic differentiation was performed with a 2-step protocol and the use of hepatocyte growth factor and oncostatin M for cell culture. During four weeks of induction, most cells displayed a cuboidal morphology. Immunological analysis indicated that umbilical cord vein mesenchymal stem cells-derived hepatocyte-like cells expressed liver-specific protein markers such as albumin and cytokeratin-18. The hepatocyte-like cells also displayed several characteristics of hepatocytes, including expression of transthyretin, glucose 6-phosphatase, cytokeratin-8,18, alpha-fetoprotein, hepatocyte nuclear factor-3ß and albumin. The result of indocyanine green cell uptake, as a test substance to evaluate hepatocyte-like cell function, was positive for differentiated cells. Glycogen storage was examined by periodic acid-Schiff staining. Accumulation of intracellular glycogen was detected in the hepatocyte-like cells. Based on these observations, we have concluded that umbilical cord vein mesenchymal stem cells are endowed with hepatogenic potential and may provide a stem cell source to be used as cell therapy for liver diseases.

Hydrostatic pressure affects in vitro maturation of oocytes and follicles and increases granulosa cell death.

OBJECTIVE: This study examines the effects of hydrostatic pressure on in vitro maturation (IVM) of oocytes derived from in vitro grown follicles. MATERIALS AND METHODS: In this experimental study, preantral follicles were isolated from 12-day-old female NMRI mice. Each follicle was cultured individually in Alpha Minimal Essential Medium (α-MEM) under mineral oil for 12 days. Then, follicles were induced for IVM and divided into two groups, control and experiment. In the experiment group follicles were subjected to 20 mmHg pressure for 30 minutes and cultured for 24-48 hours. We assessed for viability and IVM of the oocytes. The percentage of apoptosis in cumulus cells was determined by the TUNEL assay. A comparison between groups was made using the student's t test. RESULTS: The percentage of metaphase II oocytes (MII) increased in hydrostatic pressuretreated follicles compared to controls (p<0.05). Cumulus cell viability reduced in hydrostatic pressure-treated follicles compared to controls (p<0.05). Exposure of follicles to pressure increased apoptosis in cumulus cells compared to controls (p<0.05). CONCLUSION: Hydrostatic pressure, by inducing apoptosis in cumulus cells, participates in the cumulus oocyte coupled relationship with oocyte maturation.