Investigating the effect of Nigella sativa on the testicular function of first-generation offspring of mice treated with titanium oxide nanoparticles.

Objective: Nanoparticles include primary particles with at least one of their dimensions being less than 100 nm. The goal of this research was to determine the possible protective role of Nigella sativa (NS) against toxic effects mediated by titanium oxide nanoparticle (TNP). Materials and Methods: 30 adult mice (10 males and 20 females) were used. After mating, the pregnant female mice were randomly divided into 4 study groups (n=5 mice in each group). From the 13th day of gestation until delivery, the mice were given TNP and NS. After delivery, 10 newborn male mice were selected from each group and kept under standard conditions until puberty according to the previous grouping (4 groups). The epididymis of each mouse was removed and the sperm was collected for the evaluation of in vitro fertilization and testis for histopathology and spermatogenesis of in vitro fertilization of first-generation mice. Results: No significant difference was observed between the NS group and the control group (p>0.05). In the TNP, a degree of epithelial lysis and a significant decrease in sperm motility was observed (p<0.05) compared with the control group. In the TNP and NS group, NS had an ameliorating effect on TNP-induced testicular germ cell damage (p<0.05). Conclusion: In the present study, it was found that NS had no destructive effect on the germinal epithelium. However, NS had an ameliorating effect on TNP-induced testicular germ cell damage in mice.

Evaluation of the Effect of Vitamin E on Reproductive Parameters in Morphine-Treated Male Mice.

Background: Morphine is a narcotic pain reliever that is prescribed to reduce postoperative pain and can produce reactive oxygen species (ROS). Therefore, it can have negative effects on spermatogenesis and male fertility. Vitamin E is an effective antioxidant which plays an important role in membrane lipid peroxidation due to increased ROS. The present study aimed to evaluate the effects of vitamin E and morphine on sperm parameters, level of malondialdehyde (MDA), and diameter of seminiferous tubules in morphine-treated mice. Methods: In this experimental study, 80 mice were divided into ten groups (n=8) including control, normal saline, vehicle, morphine, various doses of vitamin E (100, 200, 300 mg/kg), and morphine plus vitamin E (100, 200, 300 mg/kg) groups. The groups were followed up for 30 consecutive days. Sperm parameters, testis weight, the diameter of seminiferous tubules, and the level of MDA were analyzed and compared. Findings: Data analysis showed seminal parameters decreased significantly (excluding sperm count) and there was an increase in the level of MDA in morphine-treated mice compared with the normal saline group (P<0.05). Administration of E100 to morphinetreated mice did not show a significant difference in the evaluated parameters compared with the morphine group. However, E200 and E300 significantly reduced MDA and improved sperm parameters (P≤0.05). Conclusion: The results showed co-administration of vitamin E in high doses (200 & 300) could prevent the deleterious effects of morphine on some reproductive parameters and decrease the level of MDA in morphine-treated mice.

Ameliorative effect of Stevia rebaudiana Bertoni on sperm parameters, in vitro fertilization, and early embryo development in a streptozotocin-induced mouse model of diabetes.

Diabetes mellitus (DM) is a common metabolic disease characterized by high blood sugar levels. It is well known that men with diabetes frequently experience reproductive disorders and sexual dysfunction. In fact, sperm quality has a significant effect on fertilization success and embryo development. The current study aimed to investigate the effect of Stevia rebaudiana hydroalcoholic extract on serum testosterone levels, sperm parameters, in vitro fertilization (IVF) success, and in vitro embryonic developmental potential to reach the blastocyst stage in a streptozotocin (STZ)-induced mouse model of diabetes. In this research, 30 male mice were distributed randomly into control, diabetic (streptozotocin 150 mg/kg) and diabetic + Stevia (400 mg/kg) groups. The results revealed a decrease in body and testis weight and elevated blood fasting blood sugar (FBS) levels in the diabetic group, compared with the control. However, Stevia treatment significantly increased body and testis weight, while serum FBS levels were decreased compared with the diabetic group. In addition, Stevia significantly increased blood testosterone levels compared with the diabetic group. Moreover, sperm parameters were improved considerably by Stevia treatment compared with the diabetic group. Furthermore, Stevia administration significantly promoted IVF success rate and in vitro development of fertilized oocytes compared with the diabetic group. In summary, our data indicated that Stevia enhanced sperm parameters, IVF success, and in vitro embryonic developmental competency in diabetic mice, probably because of its antioxidant effects. Therefore, Stevia could ameliorate sperm parameters that, in turn, increase fertilization outcomes in experimental-induced diabetes.

Testicular Tissue Vitrification: a Promising Strategy for Male Fertility Preservation.

Destruction of spermatogonial stem cells in juvenile men survivors of pediatric cancers leads to infertility as a side effect of gonadotoxic therapies. Sperm freezing before cancer treatment is commonly used in the clinic for fertility preservation, but this method is not applicable for prepubertal boys due to the lack of mature sperm. In these cases, cryopreservation of testicular tissues is the only option for fertility preservation. Although controlled slow freezing (CSF) is the most common procedure for testicular tissue cryopreservation, vitrification can be used as an alternative method. Controlled vitrification has prevented cell damage and formation of ice crystals. Procedures were done easily and quickly with a brief exposure time to high concentration of cryoprotectants without expensive equipment. Different studies used vitrification of testicular tissues and they assessed the morphology of seminiferous tubules, apoptosis, and viability of spermatogonial cells. Transplantation of vitrified testicular tissue into infertile recipient mice as well as in vitro culture of vitrified tissues was done in previous studies and their findings showed complete spermatogenesis and production of mature sperm. Review articles usually have compared controlled slow freezing with vitrification. In this review, we focused only on the vitrification method and its results. Despite promising results, many studies have been done for finding an optimal cryopreservation protocol in order to successfully preserve fertility in prepubertal boys.

Feeding role of mouse embryonic fibroblast cells is influenced by genetic background, cell passage and day of isolation.

Mouse embryonic fibroblast (MEF) cells are commonly used as feeder cells to maintain the pluripotent state of stem cells. MEFs produce growth factors and provide adhesion molecules and extracellular matrix (ECM) compounds for cellular binding. In the present study, we compared the expression levels of Fgf2, Bmp4, ActivinA, Lif and Tgfb1 genes at the mRNA level and the level of Fgf2 protein secretion and Lif cytokine secretion at passages one, three and five of MEFs isolated from 13.5-day-old and 15.5-day-old embryos of NMRI and C57BL/6 mice using real-time PCR and enzyme-linked immunosorbent assay. We observed differences in the expression levels of the studied genes and secretion of the two growth factors in the three passages of MEFs isolated from 13.5-day-old and 15.5-day-old embryos, respectively. These differences were also observed between the NMRI and C57BL/6 strains. The results of this study suggested that researchers should use mice embryos that have different genetic backgrounds and ages, in addition to different MEF passages, when producing MEFs based on the application and type of their study.

The effect of adding Rosmarinic and Ascorbic acids to vitrification media on fertilization rate of the mice oocyte: An experimental study.

BACKGROUND: Oocytes vitrification is a pivotal step for the widespread and safekeeping of animal genetic resources. Oocytes endure notable morphological and functional damage during cryopreservation. Oxidative stress is one of the adverse effects that vitrification imparts on oocytes. OBJECTIVE: In the present study, we investigated the antioxidant effect of Rosmarinic and Ascorbic acids on the quality and fertilizing ability of frozen-thawed mice oocyte. MATERIALS AND METHODS: In this experimental study, germinal vesicle oocytes obtained from two-months-old (30-40gr) NMRI mice were randomly divided into four groups. The basic cryoprotectants were 7.5% (v/v) ethylene glycol+7.5% (v/v) Propanediol as an equilibration media. Vitrification medium contained 15% (v/v) ethylene glycol+15% (v/v) propanediol, and 0.5 M sucrose. In the first group (Control), nothing was added to vitrification mediums, whereas, in the second and third groups, 0.5 mmol/L of Ascorbic acid and 105 µmol/L of Rosmarinic acid were added into vitrification medium, respectively. The cumulative concentration of Rosmarinic and Ascorbic acids were added to group 4. Mouse oocytes were vitrified and preserved for one month. The thawed oocytes were transferred into the α-MEM medium (Alpha Minimum Essential Medium) and maintained in this medium for 24 hr, to be matured and reach the metaphase II stage. RESULTS: The addition of Rosmarinic and Ascorbic acids to the vitrification solution improved the survival, maturation of Germinal vesicles, fertilization rate, and finally development to 4-cell stage. Maturation rates to 4-cell stage for Ascorbic acid, Rosmarinic acid, and both of them together were 80%, 80.76%, and 86.61%, respectively. CONCLUSION: These results indicate that the addition of a cumulative concentration of 0.5 mmol/L Ascorbic acid and 105 µmol/L of Rosmarinic acid to the cryopreservation solution for the mouse immature oocytes would be of significant value (p < 0.01).

The effects of human menstrual blood stem cells-derived granulosa cells on ovarian follicle formation in a rat model of premature ovarian failure.

Many studies have reported that human endometrial mesenchymal stem cells (HuMenSCs) are capable of repairing damaged tissues. The aim of the present study was to investigate the effects of HuMenSCs transplantation as a treatment modality in premature ovarian failure (POF) associated with chemotherapy-induced ovarian damage. HuMenSCs were isolated from menstrual blood samples of five women. After the in vitro culture of HuMenSCs, purity of the cells was assessed by cytometry using CD44, CD90, CD34, and CD45 FITC conjugate antibody. Twenty-four female Wistar rats were randomly divided into four groups: negative control, positive control, sham, and treatment groups. The rat models of POF used in our study were established by injecting busulfan intraperitoneally into the rats during the first estrus cycle. HuMenSCs were transplanted by injection via the tail vein into the POF-induced rats. Four weeks after POF induction, ovaries were collected and the levels of Amh, Fst, and Fshr expression in the granulosa cell (GC) layer, as well as plasma estradiol (E2) and progesterone (P4) levels were evaluated. Moreover, migration and localization of DiI-labeled HuMenSCs were detected, and the labeled cells were found to be localized in GCs layer of immature follicles. In addition to DiI-labelled HuMenSCs tracking, increased levels of expression of Amh and Fshr and Fst, and the high plasma levels of E2 and P4 confirmed that HuMenSC transplantation had a significant effect on follicle formation and ovulation in the treatment group compared with the negative control (POF) group.

Combined Effect of Retinoic Acid and Basic Fibroblast Growth Factor on Maturation of Mouse Oocyte and Subsequent Fertilization and Development.

BACKGROUND: In this experimental study, germinal vesicle (GV) oocytes obtained from two-months-old NMRI mice were randomly divided into control, sham and three experimental groups. The basic culture medium was α-MEM supplemented with 10% fetal bovine serum (FBS), 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ ml epidermal growth factors. Each of the experimental groups received one of the following treatments: RA (2 µM), bFGF (20 ng/ml) or combination of RA and bFGF with the indicated concentrations. After 24 hours, capacitated spermatozoa were added to in vitro matured oocytes. Five hours later, the oocytes were cultured in fresh droplets of M2 medium for 24 hours and assessed for cleavage to the two-cells stage. MATERIALS AND METHODS: In this experimental study, germinal vesicle (GV) oocytes obtained from two-months-old NMRI mice were randomly divided into control, sham and three experimental groups. The basic culture medium was α-MEM supplemented with 10% fetal bovine serum (FBS), 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ ml epidermal growth factors. Each of the experimental groups received one of the following treatments: RA (2 µM), bFGF (20 ng/ml) or combination of RA and bFGF with the indicated concentrations. After 24 hours, capacitated spermatozoa were added to in vitro matured oocytes. Five hours later, the oocytes were cultured in fresh droplets of M2 medium for 24 hours and assessed for cleavage to the two-cells stage. RESULTS: As compared with the control group, the rate of maturation was significantly increased in the RA (P<0.001) and bFGF+RA (P<0.02) groups with 58 ± 10 and 57 ± 3.46, respectively. The rate of maturation was significant in the RA (P<0.02) and bFGF+RA (P<0.03) groups, in comparison with the bFGF group. The bFGF+RA group had higher rate (83 ± 1.52) of two-cells development, than control (33 ± 1, P<0.001). CONCLUSION: Our findings showed beneficial effects of 2 µM RA and 20 ng/ml bFGF combination on mouse oocyte IVM.

Post-treatment of melatonin with CCl4 better reduces fibrogenic and oxidative changes in liver than melatonin co-treatment.

Therapeutic effects of melatonin (MEL) in targeting CCl4 -induced liver fibrosis has been widely known, but there is no study comparing oxidative and fibrogenic changes in co- and post-treatment of MEL with CCl4 , which was further aimed in this experiment. Male SD rats were injected with CCl4 (1 mL/kg/i.p./daily) dissolved 1:1 in olive oil for 1 month. Some animals received MEL (20 mg/kg/i.p./daily) diluted in 1 mL PBS in combination with CCl4 (co-treatment), and some rats were treated with MEL, beginning with injection of the last dose of CCl4 for one month (post-treatment). The groups were control, CCl4 , CCl4 -co vehicle, CCl4 -post vehicle, post-CCl4 , MEL co-treatment, and MEL post-treatment. MEL post-treatment group showed significantly lower lipid deposition, serum malondialdehyde (MDA), serum alanine aminotransferase (ALT), and liver hydroxyproline. This group also had low expressions of Bax and transforming growth factor-ß1 (TGF-ß1). MEL post-treatment group revealed higher sera levels of albumin, superoxide dismutase (SOD) and glutathione peroxidase (GPx). Expression levels of metalloproteinase-13 (MMP-13) and Bcl2 was also higher in this group (P ≤ 0.05 vs co-treatment). Results of the present study indicated that MEL post-treatment is more powerful in reduction of CCl4 -induced liver fibrosis through reduction of oxidative stress and maintenance of matrix balance.

The effect of the ovarian varicose vein on the DNA methylation in the rat's oocyte.

OBJECTIVES: We intended to determine whether the ovarian varicose which is one of the common etiologies of the pelvic congestion syndrome, has the ability to interfere with the DNA methylation reprogramming in the oocyte and thereby affect the oocyte quality or not. MATERIALS AND METHODS: Varicose model was induced according to the Turner's method in the rats. Briefly, a 20-gauge needle was placed on the left renal vein and a thread was tied over both the needle and the renal vein medial to the insertion of the ovarian vein, and then the needle was removed. Evaluation of prooxidant-antioxidant balance (PAB) was assessed using specific kits and the expression level of the DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3L was assessed by Real-time PCR. Immunofluorescent staining for 5-methylcytosine in the oocytes evaluated the global DNA methylation. RESULTS: A significant PAB increase in the ovaries from varicose group was seen. Real-time PCR demonstrated a remarkable decrease in the expression of the Dnmt3a and Dnmt3L which are responsible for de novo DNA methylation in the oocytes. Immunofluorescent staining for 5-mC showed a reduction in the fluorescence intensity in the oocytes collected from the varicose group. CONCLUSION: Our findings from Real-time PCR and immunocytochemistry suggest that the epigenetic parameters in the oocyte could be affected by varicose induction and these epigenetic alteration has the potential to affect the oocyte quality. We suggest that the epigenetic changes could happen in the oocytes after the induction of ovarian varicose and lead to the oocyte quality reduction or even infertility.

The effects of an experimentally induced unilateral varicose ovarian vein on the activities of anti-oxidant enzymes in an adult rat ovary / Efectos de la inducción experimental unilateral de una vena ovárica varicosa sobre las actividades de las enzimas anti-oxidantes en ovarios de ratas adultas

In this study, we aim to examine effects of an experimentally induced unilateral varicose ovarian vein on the activities of anti-oxidant enzymes in an adult rat ovary. In this experimental study, a total of 30 adult female Wistar albino rats were divided into three groups. 10 rats in group 1 as the varicocele group, 10 rats in group 2 as the control group and 10 rats in group 3 as the sham group, that underwent a sham operation and. Anti-oxidant assays were assessed via specific assay kits. Statistical analysis was performed using the one way ANOVA and Tukey's tests were used for post hoc multiple comparisons, P<0.05 was considered statistically significant. The effects of the unilateral varicosity was more evident on the left side when compared to the right side as all activities of the anti-oxidant assayed were significantly reduced, P 0.05 when compared to the right side. Also, in this present study, the effect of the unilateral varicose vein was bilateral as there were no significant differences recorded between the two sides. Finally the result of this study shows that varicocele may lead to female infertility through various factors that includes reduction in the activities of anti-oxidant enzymes.

En este estudio, nuestro objetivo fue examinar los efectos de la inducción experimental unilateral de una vena ovárica varicosa en la actividad de enzimas antioxidantes en un ovario de rata adulta. Un total de 30 ratas albinas Wistar, hembras adultas, se dividieron en tres grupos. Diez ratas en el grupo 1 (grupo varicocele), diez ratas en el grupo 2 (grupo de control) y diez ratas en el grupo 3 (grupo de tratamiento simulado), que se sometió a una operación simulada. Ensayos con anti-oxidantes se evaluaron a través de kits de ensayo específicos. El análisis estadístico se realizó mediante ANOVA de una vía y las pruebas de Tukey fueron utilizadas para comparaciones múltiples Post Hoc, siendo el P<0,05 considerado como estadísticamente significativo. Los efectos de la varicosidad unilateral fue más evidente en el lado izquierdo cuando fue comparada con el lado derecho en todas las actividades del ensayo con anti-oxidante que se redujeron significativamente, el P 0,05 cuando se compara con el lado derecho. Asimismo, en el presente estudio, el efecto de la vena varicosa unilateral fue bilateral ya que no hubo diferencias significativas registradas entre las dos partes. Por último, el resultado de este estudio muestra que el varicocele puede conducir a la infertilidad femenina a través de diversos factores que incluye la reducción en la actividad de las enzimas antioxidantes.

The effects of unilateral varicose ovarian vein on antioxidant capacity and oocyte quality in rat ovary.

OBJECTIVES: Several researchers have reported the relationship between infertility in male and varicocele for so many years but the implication of varicocele in female patients is remains elusive. Here, we aim to examine the effects of unilateral varicose ovarian vein on antioxidant capacity and oocyte quality of rat ovary after the experimental creation of varicocele in female rats. MATERIALS AND METHODS: In this study, thirty adult female albino rats were divided into three equal groups: Group 1 as the control group has 10 rats, Group 2 as the sham group has 10 rats and they underwent a sham operation and finally Group 3 has the varicocele group has 10 rats. Antioxidant assays for superoxide dismutase, glutathione peroxidase and catalase were performed using specific assay kits and gene expression for Bax, Bmp-15, Hsp-27 and Gdf-9 was done via real time PCR. RESULTS: The adverse effects of the experimentally induced varicocele were reported and recorded on the left ovary compared to the right sided ovary (no varicocele induction) in the varicocele group. Real time PCR data shows that the expression of Gdf-9, Hsp-27 and Bmp-15 genes were all significantly reduced at p≤ 0.05. CONCLUSION: The results of this study show that reduced gene expression of Bmp-15, Gdf-9 and Hsp-27, increased gene expression of bax and an imbalance between pro-oxidant/antioxidant ratio are few of the several mechanisms by which varicocele may lead to infertility in female.

The effect of aminoguanidine on sperm motility and mitochondrial membrane potential in varicocelized rats.

OBJECTIVES: Increased levels of nitric oxide (NO) in the testicular veins of people suffering from varicocele have already been reported. However, the role of NO-synthase (NOS) isozymes and their inhibitors have not been extensively studied. We aimed to evaluate the inhibitory effects of aminoguanidine (AG), on sperm motility, vitality, and mitochondrial membrane potential (MMP) in varicocelized rats. MATERIALS AND METHODS: Twenty fore male Wister rats were divided into control, sham, varicocele, and treatment groups. Varicocele and treatment groups underwent partial ligation of left renal vein. Rats in the sham group underwent the same procedures as the varicocele group with the exception of vein ligation. 10 weeks after varicocele induction, sperm parameters were evaluated in all groups. The treatment group received 50 mg/kg AG injection daily for 10 weeks after which they were sacrificed prior to assessment of the parameters. Sperm viability and MMP were assessed by flow cytometry using propidium iodide (PI) and rhodamine 123 (Rh123), respectively. RESULTS: The results of this study show a decrease in sperm viability, motility and MMP in the varicocele group compared with the other groups. After AG injection, we observed that all the parameters were significantly enhanced in the treatment group compared with the other groups. Rh123 staining revealed a positive relation between MMP and sperm motility, whereas PI staining showed a positive relation between sperm motility and viability. CONCLUSION: The findings of our study show that AG improves sperm motility and MMP, and thus, might be useful in the management of varicocele-related infertility.

Effect of beta-carotene on titanium oxide nanoparticles-induced testicular toxicity in mice.

OBJECTIVE: This study evaluated the protective effect of beta-carotene (BC) on titanium oxide nanoparticle (TNP) induced spermatogenesis defects in mice. MATERIALS AND METHODS: Thirty-two NMRI mice were randomly divided into four groups. BC group received 10 mg/kg of BC for 35 days. TNP group received 300 mg/kg TNP for 35 days. TNP+BC group initially received 10 mg/kg BC for 10 days and was followed by concomitant administration of 300 mg/kg TNP for 35 days. Control group received only normal saline for 35 days. Epididymal sperm parameters, testicular histopathology, spermatogenesis assessments and testosterone assay were performed for evaluation of the TNP and BC effects on testis. RESULTS: Serum testosterone levels were markedly decreased in TNP-intoxicated mice. Epididymal sperm parameters including sperm number, motility and percentage of abnormality were significantly changed in TNP-intoxicated mice (p < 0.01). Histopathological criteria such as epithelial vacuolization, sloughing of germ cells and detachment were significantly increased in TNP-intoxicated mice (p < 0.001). BC+TNP treatment significantly prevented these changes (p < 0.05). BC also significantly elevates testosterone levels in BC+TNP group compared to TNP-treated mice (p < 0.01). DISCUSSION AND CONCLUSION: The results of this study demonstrated that BC improved the spermatogenesis defects in TNP-treated mice. BC had a potent protective effect against the testicular toxicity and might be clinically useful.