Thymoquinone effects on autophagy, apoptosis, and oxidative stress in cisplatin-induced testicular damage in mice.

PURPOSE: In this study, the effect of thymoquinone (TQ) on CP-induced spermatogenesis defects in mice has been investigated. METHODS: Sperm parameters, serum testosterone concentration, histology, Bax/Bcl-2 ratio, and expression of autophagy-related biomarkers have been assessed. Total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI) in testicular tissue were examined for the evaluation of oxidative stress levels. RESULTS: CP has induced histological changes and significantly increased the Bax/Bcl-2 ratio, decreased testosterone concentration, testicular weight, and sperm quality. CP induced oxidative stress by elevating OSI in the testicular tissue (p < 0.05). Expression of the autophagy-inducer genes (ATG7, ATG5, and Beclin-1) and ratio of LC3B/LC3A proteins were significantly decreased, while mTOR expression was increased in the CP group. TQ pretreatment dose-dependently decreased the Bax/Bcl-2 ratio and mTOR gene expression while increasing the expression of ATG5 and ATG7 genes, LC3B/LC3A ratio, and Beclin-1 proteins. TQ could also dose-dependently reverse the histology, testosterone level, and sperm quality of the CP-intoxicated mice. CONCLUSIONS: These findings show that TQ pretreatment can enhance sperm production by inducing autophagy and reducing apoptosis and oxidative stress in the CP-intoxicated mouse testicles.

Evaluation of the expression level of microRNA-21, microRNA-15a, microRNA-372 in human follicular fluid stem cells-derived oocyte-like cells (OLCs).

OBJECTIVE: Today, researchers have succeeded in achieving oocyte-like cells through the in vitro differentiation of stem cells. MicroRNAs are key regulators of oocyte development. In this study we decided to evaluate the expression pattern of microRNA-21, microRNA-15a, and microRNA-372 in oocyte-like cells, to determine the maturation stage of oocyte-like cells. METHODS: Human follicular fluid samples were collected and centrifuged, and their cells were divided into 3 groups; day 7 as control group, days 14 and 21. During this period, the cells were evaluated for their morphological appearance and viability by inverted microscopy. RNA isolation was performed and cDNA was reversely transcribed by specific stem-loop RT primers. Real-time RT-PCR was used to detect microRNA expression. RESULTS: The relative expression of microRNA-21 and microRNA-15a on day 21 was significantly down-regulated compared to the control group (day 7), but microRNA-372 did not show a significant difference. Also, on day 14 compared to the control group (day 7), microRNA-21 did not show a significant difference; but microRNA-15a and microRNA-372 were significantly down-regulated. MicroRNA-21 and microRNA-15a on day 21 compared to day 14 revealed down-regulated levels, but microRNA-372 revealed up-regulated levels. CONCLUSIONS: Our results showed significant decreases in the expression of microRNA-21 and microRNA-15a in oocyte-like cells, as well as in oocytes, which may lead to cytoplasmic maturation, germinal vesicle break down and the completion of meiosis І. In addition, down-regulation expression of microRNA-372 maybe a confirmation that mesenchymal stem cells have differentiated into germ cells, and these cells were differentiated into oocyte-like cells.

Adipose-Derived Stem Cells' Secretome Attenuates Lesion Size and Parasite Loading in Leishmaniasis Caused by Leishmania Major in Mice.

Background: Stem cell-derived secretome (SE) released into the extracellular space contributes to tissue repair. The present study aimed to investigate the impact of isolated secretome (SE) from adipose-derived mesenchymal stem cells (ASCs) on Leishmania major (L. major) lesions in BALB/c mice. Methods: This experimental study was conducted at Ahvaz University of Medical Sciences (Ahvaz, Iran) in 2021. Forty female BALB/c mice were infected with stationary phase promastigotes through intradermal injection in the bottom of their tail and randomly divided into four groups (n=10 per group). The mice were given SE (20 mg/mL), either alone or in combination with Glucantime (GC, 20 mg/mL/Kg), meglumine antimoniate (20 mg/mL/Kg) for the GC group, and phosphate-buffered saline (PBS) for the control group. After eight weeks, the lesion size, histopathology, the levels of Interleukin 10 (IL-10), and Interleukin 12 (IL-12) were assessed. For the comparison of values between groups, the parametric one-way ANOVA was used to assess statistical significance. Results: At the end of the experiment, the mice that received SE had smaller lesions (4.56±0.83 mm versus 3.62±0.59 mm, P=0.092), lower levels of IL-10 (66.5±9.7 pg/mL versus 285.4±25.2 pg/mL, P<0.001), and higher levels of IL-12 (152.2±14.2 pg/mL versus 24.2±4.4 pg/mL, P<0.001) than the control. Histopathology findings revealed that mice treated with SE had a lower parasite burden in lesions and spleen than the control group. Conclusion: The current study demonstrated that ADSC-derived SE could protect mice infected with L. major against leishmaniasis.

Irradiation and conditioned media from human umbilical cord stem cells suppress epithelial-mesenchymal transition biomarkers in breast cancer cells.

Objectives: Breast cancer cells developing radioresistance during radiation may result in cancer recurrence and poor survival. One of the main reasons for this problem is the changes in the regulation of genes that have a key role in the epithelial-mesenchymal transition (EMT). Utilizing mesenchymal stem cells can be an effective approach to overcome therapeutic resistance. In this study, we investigated the possibility of combining mesenchymal medium with cancer cell medium in sensitizing breast carcinoma cells to radiation. Materials and Methods: In this experimental study, the cells were irradiated at a dose of 4 Gy alone and in combination with stem cells and cancer cells media. Apoptosis, cell cycle, Western blotting, and real-time PCR assays evaluated the therapeutic effects. Results: We found that the CSCM could decrease the expression of several EMT markers (CD133, CD44, Vimentin, Nanog, Snail, and Twist), resulting in increased cell distribution in the G1 and G2/M phases, apoptosis rate, and protein levels of p-Chk2 and cyclin D1; furthermore, it exhibits synergetic effects with radiation treatment in vitro. Conclusion: These findings show that CSCM inhibits the expansion of breast cancer cells and makes them more susceptible to radiotherapy, offering a unique approach to treating breast cancer by overcoming radioresistance.

Oocyte quality and aging.

It is well known that female reproduction ability decreases during the forth decade of life due to age-related changes in oocyte quality and quantity; although the number of women trying to conceive has today increased remarkably between the ages of 36 to 44. The causes of reproductive aging and physiological aspects of this phenomenon are still elusive. With increase in the women's age, during Assisted Reproductive Technologies (ART) we have perceived a significant decline in the number and quality of retrieved oocytes, as well as in ovarian follicle reserves. This is because of increased aneuploidy due to factors such as spindle apparatus disruption; oxidative stress and mitochondrial damage. The aim of this review paper is to study data on the potential role of the aging process impacting oocyte quality and female reproductive ability. We present the current evidence that show the decreased oocyte quality with age, related to reductions in female reproductive outcome. The aging process is complicated and it is caused by many factors that control cellular and organism life span. Although the factors responsible for reduced oocyte quality remain unknown, the present review focuses on the potential role of ovarian follicle environment, oocyte structure and its organelles. To find a way to optimize oocyte quality and ameliorate clinical outcomes for women with aging-related causes of infertility.

Use of alginate hydrogel to improve long-term 3D culture of spermatogonial stem cells: stemness gene expression and structural features.

The quality and quantity of a spermatogonial stem-cell (SSC) culture can be measured in less time using a 3D culture in a scaffold. The present study investigated stemness gene expression and the morphological and structural characterization of SSCs encapsulated in alginate. SSCs were harvested from BALB/c neonatal mice testes through two-step mechanical and enzymatic digestion. The spermatogonial populations were separated using magnetic-activated cell sorting (MACS) using an anti-Thy1 antibody and c-Kit. The SSCs then were encapsulated in alginate hydrogel. After 2 months of SSC culturing, the alginate microbeads were extracted and stained to evaluate their histological properties. Real-time polymerase chain reaction (PCR) was performed to determine the stemness gene expression. Scanning electron microscopy (SEM) was performed to evaluate the SSC morphology, density and scaffold structure. The results showed that encapsulated SSCs had decreased expression of Oct4, Sox2 and Nanos2 genes, but the expression of Nanog, Bcl6b and Plzf genes was not significantly altered. Histological examination showed that SSCs with pale nuclei and numerous nucleolus formed colonies. SEM evaluation revealed that the alginate scaffold structure preserved the SSC morphology and density for more than 60 days. Cultivation of SSCs on alginate hydrogel can affect Oct4, Sox2 and Nanos2 expression.

Taurine ameliorates cytotoxic effects of Di(2-ethylhexyl) phthalate on Leydig cells.

It has been revealed that di(2-ethylhexyl)phthalate (DEHP) has toxic impacts on the male reproductive system. Taurine (TAU) is an amino acid with antioxidant property and beneficial impacts on the male reproductive system. In this study, protective impacts of Taurine (TAU) on DEHP-induced Leydig TM3 cell toxicity were investigated. The cells exposed to DEHP (0.8 µmol) or TAU (100 mg/ml) for 24 hr. Cell viability (MTT assay), apoptosis, oxidative stress and testosterone level were examined. DEHP could significantly decrease the cell viability percentage, reduce testosterone level, increase apoptosis, elevate Bax/ Bcl-2 ratio and enhance caspase-3 and -9 activity in the TM3 cells. Additionally, DEHP significantly elevated malondialdehyde contents and reactive oxygen species levels. It also augmented superoxide dismutase and catalase activity in the Leydig cells. Co-treatment of DEHP with TAU increased viability and testosterone level, while oxidative stress and apoptosis significantly reduced. TAU could decrease Bax/Bcl-2 ratio and caspase-3 and -9 activity in the DEHP-intoxicated cells. Our results have clearly shown that TAU protects TM3 cells against oxidative stress and apoptosis induced by DEHP.

Therapeutic effects of Wharton's jelly-derived Mesenchymal Stromal Cells on behaviors, EEG changes and NGF-1 in rat model of the Parkinson's disease.

Human Wharton's jelly-derived Mesenchymal Stromal Cells (hWJ-MSCs) have shown beneficial effects in improving the dopaminergic cells in the Parkinson's disease (PD). In the present study, the effects of hWJ-MSCs on hyperalgesia, anxiety deficiency and Pallidal local electroencephalogram (EEG) impairment, alone and combined with L-dopa, were examined in a rat model of PD. Adult male Wistar rats were divided into five groups: 1) sham, 2) PD, 3) PD + C (Cell therapy), 4) PD + C+D (Drug), and 5) PD + D. PD was induced by injection of 6-OHDA (16 µg/2 µl into medial forebrain bundle (MFB)). PD + C group received hWJ-MSCs (1 × 106 cells, intravenous (i.v.)) twice post PD induction. PD + C+D groups received hWJ-MSCs combined with L-Dopa/Carbidopa, (10/30 mg/kg, intraperitoneally (i.p.)). PD + D group received L-Dopa/Carbidopa alone. Four months later, analgesia, anxiety-like behaviors, were evaluated and Pallidal local EEG was recorded. Level of insulin-like growth factor 1 (IGF-1) was measured in the striatum and dopaminergic neurons were counted in substantia nigra (SNc). According to data, MFB-lesioned rats showed hyperalgesia in tail flick, anxiety-like symptoms in cognitive tests, impairment of electrical power of pallidal local EEG as field potential, count of dopaminergic neurons in SNc and level of IGF-1 in striatum. These complications restored significantly by MSCs treatment (p < 0.001). Our findings confirm that chronic treatment with hWJ-MSC, alone and in combination with L-Dopa, improved nociception and cognitive deficit in PD rats which may be the result of increasing IGF-1 and protect the viability of dopaminergic neurons.

Neuroprotective effects of Wharton's jelly-derived mesenchymal stem cells on motor deficits due to Parkinson's disease.

OBJECTIVES: Human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) have been recognized as a potential tool to replace damaged cells by improving the survival of the dopaminergic cells in Parkinson's disease (PD). In this study, we examined the effects of hWJ-MSCs and associated with L-dopa/carbidopa on motor disturbances in the PD model. MATERIALS AND METHODS: PD was induced by injection of 6-hydroxydopamine (6-OHDA) (16 µg/2 µl into medial forebrain bundle (MFB)). Sham group received a vehicle instead of 6-OHDA. PD+C group received hWJ-MSCs twice on the 14th and 28th days post PD induction. PD+C+D group received hWJ-MSCs and also L-dopa/carbidopa (10/30 mg/kg). PD+D group received L-dopa/carbidopa alone. Four months later, motor activities (the parameters of locomotor and muscle stiffness) were evaluated, dopaminergic neurons were counted in substantia nigra pars compacta (SNc), the level of dopamine (DA), and tyrosine hydroxylase (TH) were measured in the striatum. RESULTS: Data indicated that motor activities, the number of dopaminergic neurons, and levels of DA and TH activities were significantly reduced in PD rats as compared to the sham group (P<0.001). However, the same parameters were improved in the treated groups when compared with the PD group (P<0.001 and P<0.01, respectively). CONCLUSION: The chronic treatment of PD rats with hWJ-MSCs and L-dopa/carbidopa, improved motor activity, which may be the result of increased TH activity and due to released DA from dopaminergic neurons.

Differentiation potential of adipose tissue-derived mesenchymal stem cells into germ cells with and without growth factors.

This study aimed to culture the adipose tissue-derived mesenchymal stem cells (AT-MSCs) with and without leukaemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), epidermal growth factor (EGF) and retinoic acid (RA), and investigate their impact on the differentiation of these cells into germ cells. MSCs were separated from adipose tissue of mice, and the nature of these cells is confirmed by flow cytometry. The cells were cultured in different conditions, including MSCs grown in the presence of the growth factors, MSCs without the growth factors, MSCs cultured with combined growth factors and RA, and MSCs cultured with RA. After 2 weeks, the gene expression of c-Kit, Gcnf, Mvh and Scp3 and the protein expression of c-Kit and Gcnf were assessed by real-time PCR and Western blot, respectively. Scp3 was overexpressed in the groups supplemented with RA (p < .01). The expression of c-Kit and Mvh in the growth factor-supplemented groups was increased (p < .01). Western blot analysis confirmed the real-time PCR results. The use of the growth factors for the long-term culture of stem cells can be beneficial. However, to promote germ cell differentiation, the growth factors might be used by other meiosis inducer factors, such as RA.

Bone morphogenetic protein 15 induces differentiation of mesenchymal stem cells derived from human follicular fluid to oocyte-like cell.

Follicular fluid (FF) is essential for developing ovarian follicles. Besides the oocytes, FF has abundant undifferentiated somatic cells containing stem cell properties, which are discarded in daily medical procedures. Earlier studies have shown that FF cells could differentiate into primordial germ cells via forming embryoid bodies, which produced oocyte-like cells (OLC). This study aimed at isolating mesenchymal stem cells (MSC) from FF and evaluating the impacts of bone morphogenetic protein 15 (BMP15) on the differentiation of these cells into OLCs. Human FF-derived cells were collected from 78 women in the assisted fertilization program and cultured in human recombinant BMP15 medium for 21 days. Real-time polymerase chain reaction and immunocytochemistry staining characterized MSCs and OLCs. MSCs expressed germline stem cell (GSC) markers, such as OCT4 and Nanog. In the control group, after 15 days, OLCs were formed and expressed zona pellucida markers (ZP2 and ZP3), and reached 20-30 µm in diameter. Ten days after induction with BMP15, round cells developed, and the size of OLCs reached 115 µm. A decrease ranged from 0.04 to 4.5 in the expression of pluripotency and oocyte-specific markers observed in the cells cultured in a BMP15-supplemented medium. FF-derived MSCs have an innate potency to differentiate into OLCs, and BMP15 is effective in promoting the differentiation of these cells, which may give an in vitro model to examine germ cell development.

Curcumin as an indirect methylation inhibitor modulates the effects of Toxoplasma gondii on genes involved in male fertility.

Toxoplasma gondii is a common protozoan parasite, which infects warm-blooded mammals, including mice and humans, throughout the world. The negative effects of T. gondii infection on the human reproductive system have been documented, especially in females. However, only few studies have examined the effects of T. gondii infection on the male reproductive system. Previous research shows that T. gondii can induce DNA methylation in some gene promoters, which are key regulators of spermatogenesis. Therefore, this study aimed to evaluate the effects of curcumin on the activity of DNA methyltransferases (DNMTs), as well as selected genes, involved in spermatogenesis in spermatogenic cells. In the spermatogenic cells exposed to T. gondii, there was a significant increase in DNMT1 and DNMT3A gene expression and a significant reduction in HSPA1A, MTHR, and DAZL gene expression, compared to the controls. The present results showed that curcumin could regulate changes in T. gondii-mediated gene expression. The effect of T. gondii on DNMT activity was also investigated in this study. A 40 % increase in DNMT activity was observed due to T. gondii infection. However, DNMT activity was restored by treatment with 20 µM curcumin for eight hours. The results revealed that T. gondii increases the NF-κB activity, compared to the control group. The increase in NF-κB activity, induced by T. gondii, was inhibited by curcumin. In conclusion, T. gondii, by increasing DNMT expression and activity, leads to an increase in NF-κB activity in cells. On the other hand, curcumin reduced DNA methylation, induced by T. gondii, owing to its NF-κB-inhibiting properties. Therefore, curcumin, as a hypomethylating agent, can be potentially used to alleviate the negative effects of T. gondii on the male reproductive system.

Transplanted Wharton's jelly mesenchymal stem cells improve memory and brain hippocampal electrophysiology in rat model of Parkinson's disease.

BACKGROUND: Experimental findings have shown that stem cell transplantation is a therapeutic procedure for Parkinson's disease (PD). In this study, effects of human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs), alone and combined with l-dopa, were examined for repairing memory impairment in a rat model of PD. METHODS: Fifty adult male Wistar rats were randomly divided into five groups: 1) sham, 2) PD, 3) PD + C, 4) PD + C+D, and 5) PD + D. PD was induced by 6-OHDA injection (16 µg/2 µl) into medial forebrain bundle (MFB) and was confirmed 14 days later by contralateral rotation using apomorphine injection. The rats received hWJ-MSCs (1 × 106 cells, i.v.) twice on the 14th and 28th days post PD induction. Treated PD rats received hWJ-MSCs alone or combined with l-Dopa and Carbidopa (10/30 mg/kg, i.p.). Four months later, memory, hippocampal long-term potentiation (hLTP), histological changes, and the levels of BDNF and NGF in striatum were evaluated. RESULTS: PD caused both cell loss with small dark stained nuclei in granular zone as well as significant decrement of BDNF and NGF (P < 0.001) in striatum. These pathological alterations were associated with memory and hLTP deficits (P < 0.001 respectively). Treating PD rats with hWJ-MSCs, alone (P < 0.05 and P < 0.001) and combined with l-Dopa (P < 0.001), significantly restored the levels of both of the neurotrophins followed by improving cognition and hLTP (P < 0.001). CONCLUSION: Current findings showed that chronic treatment of PD rats with hWJ-MSCs, alone and in combination with l-Dopa, could restore memory and hLTP by reconstructing dopaminergic neurons and elevating the BDNF and NGF factors.

Spermatogonia apoptosis induction as a possible mechanism of Toxoplasma gondii-induced male infertility.

OBJECTIVES: The protozoan Toxoplasma gondii as an intracellular protozoan is widely prevalent in humans and animals. Infection generally occurs through consuming food contaminated with oocysts and tissue cysts from undercooked meat. The parasite is carried in sexual fluids like semen but there is little information about the effect of T. gondii on the male reproductive system. In this study, we examined the effect of T. gondii tachyzoites on apoptosis induction in type B spermatogonia (GC-1) cells. MATERIALS AND METHODS: Fresh tachyzoites taken of infected BALB/c mice, GC-1 spg cells were infected with increasing concentrations of tachyzoites of T. gondii, then apoptotic cells were identified and quantified by flow cytometry. The genes associated with apoptosis were evaluated by RT2 Profiler PCR Array. RESULTS: PCR array analysis of 84 apoptosis-related genes demonstrated that 12 genes were up-regulated at least 4-fold and that one gene was down-regulated at least 2-fold in the T. gondii infection group compared with levels in the control group. The number of genes whose expression had increased during the period of infection with T. gondii was significantly higher than those whose expressions had decreased (18 versus 1) and Tnfrsf11b had the highest rate of gene expression. CONCLUSION: T. gondii induce in vitro apoptosis of GC-1 spg cells. This effect shows a trend of concentration-dependent increase so that with an increase in the ratio of parasite burden to spermatogonial cells, in addition to an increase in the number of genes whose expression has changed, the fold of these changes has increased as well.

Taurine induces autophagy and inhibits oxidative stress in mice Leydig cells.

OBJECTIVES: This study evaluated taurine (TAU) effects on autophagy, apoptosis and oxidative stress in mice Leydig TM3 cells. METHODS: We treated TM3 cells with TAU (100 µg/mL) or 3-Methyladenine (3-MA, an autophagy inhibitor) for 24 h, and assessed cell viability, testosterone level, oxidative stress, apoptosis, and autophagy. RESULTS: The results showed that TAU markedly increased cell viability, testosterone levels, expression of autophagy-related genes and percentage of LC3-II-positive cells. TAU significantly reduced malondialdehyde (MDA) contents and reactive oxygen species (ROS) levels and increased the activities of SOD (superoxide dismutase) and CAT (Catalase) enzymes in the TM3 cells. TAU in the presence of autophagy inhibitor (3-MA) increased oxidative stress and decreased testosterone levels. CONCLUSION: The results showed that autophagy might be involved in TAU-increased testosterone levels in mice Leydig TM3 cells.

Utilizing Calcium Alginate for the Assessment of Bone Morphogenetic Protein 15 Induction Effect on the Differentiation of Mesenchymal Stem Cell Derived from Human Follicular Fluid to Oocyte-Like Structure.

BACKGROUND: Follicular fluid (FF)-derived mesenchymal stem cells (MSCs) are possible new source of cells in the study of oogenesis and regenerative medicine. Several biomaterials have been used as scaffolds to mimic ovarian tissue stroma. Using good matrix is essential for increasing the cell survival rate, proliferation, and differentiation. However, no study has been performed to investigate the effects of BMP15 and calcium alginate hydrogel on the differentiation potential of FF-derived MSCs to oocyte-like structures (OLSs). MATERIALS AND METHODS: In this work, FF MSCs, which were collected from women in routine in vitro fertilization procedure, were capsulated with 0.5% calcium alginate, and then the encapsulated cells were cultured in medium containing BMP15 for 2 weeks. Trypan blue staining was carried out to determine cell viability. Real-time polymerase chain reaction (PCR) and immunofluorescence (ICC) staining method were performed to characterize the expression of OCT4, Nanog, ZP2, and ZP3 genes and protein. The encapsulation process did not change the morphology and viability of the encapsulated cells. RESULTS: Reverse-transcription-PCR and ICC showed that MSCs expressed germ line stem cell markers such as OCT4 and Nanog. After 4 days of culture, OLSs formed and expressed zona pellucida markers. OLSs at least reached 180-230 µm in diameter in the control and BMP15-treated groups. Finally, a reduction in the expression pattern of pluripotency and ZP markers was detected in the encapsulated cells cultured in the BMP15-supplemented medium. CONCLUSION: The three-dimensional alginate culture system seems to be a promising method of getting in vitro differentiation and development of ovarian cells, which could mimic the native ovarian condition.

Spermatogenesis Recovery Potentials after Transplantation of Adipose Tissue-Derived Mesenchymal Stem Cells Cultured with Growth Factors in Experimental Azoospermic Mouse Models.

OBJECTIVE: Approximately 1% of the male population suffers from obstructive or non-obstructive azoospermia. Previous in vitro studies have successfully differentiated mesenchymal stem cells (MSCs) into germ cells. Because of immunemodulating features, safety, and simple isolation, adipose tissue-derived MSCs (AT-MSCs) are good candidates for such studies. However, low availability is the main limitation in using these cells. Different growth factors have been investigated to overcome this issue. In the present study, we aimed to comparatively assess the performance of AT-MSCs cultured under the presence or absence of three different growth factors, epidermal growth factor (EGF), leukemia inhibitory factor (LIF) and glial cell line-derived neurotrophic factor (GDNF), following transplantation in testicular torsion-detorsion mice. MATERIALS AND METHODS: This was an experimental study in which AT-MSCs were first isolated from male Naval Medical Research Institute (NMRI) mice. Then, the mice underwent testicular torsion-detorsion surgery and received bromodeoxyuridine (BrdU)-labeled AT-MSCs into the lumen of seminiferous tubules. The transplanted cells had been cultured in different conditioned media, containing the three growth factors and without them. The expression of germ cell-specific markers was evaluated with real-time polymerase chain reaction (PCR) and western-blot. Moreover, immunohistochemical staining was used to trace the labeled cells. RESULTS: The number of transplanted AT-MSCs resided in the basement membrane of seminiferous tubules significantly increased after 8 weeks. The expression levels of Gcnf and Mvh genes in the transplanted testicles by AT-MSCs cultured in the growth factors-supplemented medium was greater than those in the control group (P<0.001 and P<0.05, respectively). The expression levels of the c-Kit and Scp3 genes did not significantly differ from the control group. CONCLUSION: Our findings showed that the use of EGF, LIF and GDNF to culture AT-MSCs can be very helpful in terms of MSC survival and localization.

Effect of vitamin D on apoptotic marker, reactive oxygen species and human sperm parameters during the process of cryopreservation.

OBJECTIVES: Sperm cryopreservation plays an undeniable role in assisted reproductive technology. However, this process significantly reduces the motility, viability, morphology and nuclear integrity of sperm. Reasons of these changes were oxidative stress and apoptosis. The aim of this study was to evaluate the influence of vitamin D on the survival and integrity of fertile sperm after cryopreservation. MATERIALS AND METHODS: Semen sample of 18 males with normal parameters was used. After swimming up, each sample was divided into two parts. 20 µmol vitamin D was added to one part as experimental group and the other part was left untreated as control group. The samples in all groups were frozen for 14 days. Post-thawing, the groups were evaluated for sperm motility, and viability using eosin staining, morphology using the Diff-Quick staining and apoptosis by TUNEL, Annexin-V and caspase-3 activity assay. By using nitrobluetetraxolium test and thiobarbituric acid, the reactive oxygen species (ROS) and lipid peroxidation of sperms were measured, respectively. RESULTS: In comparison with control groups, motile and viable sperm concentration was substantially higher in treated groups (P-value<0.05); however, morphological analysis did not show any remarkable changes. Also, ROS and lipid peroxidation values were dramatically reduced by vitamin D (P-value<0.05). TUNEL and Annexin assay for apoptosis were considerably lower in treated groups (P-value<0.05), but caspase activity assay revealed no significant difference between groups. CONCLUSION: The results have shown that the addition of vitamin D to a freezing medium leads to higher quality and function of human sperm.

Combination of Epigallocatechin-3-gallate and Silibinin: A Novel Approach for Targeting Both Tumor and Endothelial Cells.

Despite promising benefits, anti-angiogenic strategies have revealed several drawbacks, which necessitate development of novel approaches in cancer therapy strategies including non-small-cell lung cancer, as one of the leading causes of cancer death, all over the world. Combination of flavonoids could be a safe and effective option to synergize their impact on mechanisms controlling tumor angiogenesis. In this study, we have investigated the plausible synergism of epigallocatechin-3-gallate (EGCG) and silibinin on endothelial cells, for the first time. Cell viability and migration were evaluated by survival and wound healing assays, respectively. Then, we assessed the expression of VEGF, VEGFR2, and miR-17-92 cluster using real-time polymerase chain reaction in endothelial-tumor cell and endothelial-fibroblast coculture models. EGCG ± silibinin suppressed endothelial and lung tumor cell migration in lower than 50% toxic doses. VEGF, VEGFR2, and pro-angiogenic members of the miR-17-92 cluster were downregulated upon treatments. Specifically, the combination treatment upregulated an anti-angiogenic member of the cluster, miR-19b. Our data provides evidence to utilize the EGCG and silibinin combination as a novel approach to target tumor angiogenesis in the future.

A Case of Mayer-Rokitansky-Küster-Hauser Syndrome with a Fused Pancake-shaped Pelvic Kidney.

Mayer-Rokitansky-Küster-Hauser (MRKH) Syndrome is a female reproductive system disorder. It is characterized by a defect in the Müllerian ducts development, and it causes the absence of the uterus in variable degrees in upper vaginal hypoplasia. In addition, it is often associated with the unilateral renal dysplasia. Müllerian agenesis affects 1 in 4500 newborn girls and is considered as a sporadic anomaly. Women with MRKH Syndrome have a normal female chromosome pattern 46, XX with normal ovarian function. The presence of bilateral kidney agenesis with a pelvic pancake-shaped kidney is a rare condition, and a few cases have been reported in medical journals. This case study focuses on a case of MRKH Syndrome with bilateral renal agenesis and a pancake-shaped kidney.