Role of nuclear factor erythroid 2-related factor 2 (Nrf2) in female and male fertility.

Oxidative stress refers to a condition where there is an imbalance between the production of reactive oxygen species and their removal by antioxidants. While the function of reactive oxygen species as specific second messengers under physiological conditions is necessary, their overproduction can lead to numerous instances of cell and tissue damage. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of many cytoprotective genes that respond to redox stresses. Nrf2 is regularly degraded by kelch-like ECH-associated protein 1 through the ubiquitin-proteasome pathway. The kelch-like ECH-associated protein 1 and Nrf2 complex have attracted attention in both basic and clinical infertility research fields. Oxidative stress is implicated in the pathogenesis of female infertility, including primary ovarian insufficiency, polycystic ovarian syndrome, and endometriosis, as well as male infertility, namely varicocele, cryptorchidism, spermatic cord torsion, and orchitis. Most scientists believe that Nrf2 is a potential therapeutic method in female and male infertility disorders due to its antioxidant effect. Here, the potential roles of oxidative stress and Nrf2 in female and male infertility disorders are reviewed. Moreover, the key role of Nrf2 in the inhibition or induction of these diseases is discussed.

Sertoli Cell-Conditioned Medium Induces Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells to Male Germ-Like Cells in Busulfan-Induced Azoospermic Mouse Model.

Non-obstructive azoospermia is a severe form of male infertility, with limited effective treatments. Bone marrow mesenchymal stem cells (BMSCs) can differentiate to different cell lines; therefore, transplantation of these cells is used for treatment of several diseases. Since these cells require induction factors to differentiate into germ cells, we co-transplanted bone marrow stem cells (BMSCs) with Sertoli cell-conditioned medium (SCCM) into the testis of azoospermic mice. This study was carried out in two sections, in vitro and in vivo. For in vitro study, differentiating factors (c-kit and ID4) were examined after 15 days of co-culture of bone marrow cells with Sertoli cell-conditioned medium, while for in vivo study, the azoospermia model was first created by intraperitoneal administration of a single-dose busulfan (40 mg/kg) followed by single-dose CdCl2 (2 mg/kg) after 4 weeks. Mice were divided into 4 groups including control (azoospermia), BMSC, SCCM, and BMSC + SCCM. Eight weeks after transplantation, samples were assessed for proliferation and differentiation via the expression level of MVH, ID4, SCP3, Tp1, Tp2, and Prm1 differentiation markers. The results showed that BMSC co-cultured with SCCM in vitro differentiated BMSC to germ-like cells. Similarly, in vivo studies revealed a higher level of BMSC differentiation into germ-like cells with significant higher expression of differentiation markers in transplanted groups compared to the control. This study confirmed the role of SCCM as an inductive factor for BMSC differentiation to germ cells both in vivo and in vitro conditions.

Recovering Spermatogenesis By Protected Cryopreservation Using Metformin and Transplanting Spermatogonial Stem Cells Into Testis in an Azoospermia Mouse Model.

Cryopreservation and transplantation of spermatogonial stem cells (SSCs) may serve as a new method to restore male fertility in patients undergoing chemotherapy or radiotherapy. However, SSCs may be damaged during cryopreservation due to the production of reactive oxygen species (ROS). Therefore, different antioxidants have been used as protective agents. Studies have shown that metformin (MET) has antioxidant activity. The aim of this study was to assess the antioxidant and antiapoptotic effects of MET in frozen-thawed SSCs. In addition, the effect of MET on the proliferation and differentiation of SSCs was evaluated. To this end, SSCs were isolated from mouse pups aged 3-6 days old, cultured, identified by flow cytometry (ID4, INTEGRIN ß1+), and finally evaluated for survival and ROS rate. SSCs were transplanted after busulfan and cadmium treatment. Cryopreserved SSCs with and without MET were transplanted after 1 month of cryopreservation. Eight weeks after transplantation, the recipient testes were evaluated for the expression of apoptosis (BAX, BCL2), proliferation (PLZF), and differentiation (SCP3, TP1, TP2, PRM1) markers using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction. The findings revealed that the survival rate of SSCs was higher in the 500 µm/mL MET group compared to the other groups (50 and 5000 µm/mL). MET significantly decreased the intracellular ROS production. Transplantation of SSCs increased the expression level of proliferation (PLZF) and differentiation (SCP3, TP1, TP2, PRM1) markers compared to azoospermia group, and their levels were significantly higher in the MET group compared to the cryopreservation group containing basic freezing medium (p < 0.05). MET increased the survival rate of SSCs, proliferation, and differentiation and decreased the ROS production and the apoptosis rate. Cryopreservation by MET seems to be effective in treating infertility.

A systematic method introduced a common lncRNA-miRNA-mRNA network in the different stages of prostate cancer.

Introduction: The present study aimed to investigate the interaction of the common lncRNA-miRNA-mRNA network involved in signaling pathways in different stages of prostate cancer (PCa) by using bioinformatics and experimental methods. Methods: Seventy subjects included sixty PCa patients in Local, Locally Advanced, Biochemical Relapse, Metastatic, and Benign stages, and ten healthy subjects were entered into the current study. The mRNAs with significant expression differences were first found using the GEO database. The candidate hub genes were then identified by analyzing Cytohubba and MCODE software. Cytoscape, GO Term, and KEGG software determined hub genes and critical pathways. The expression of candidate lncRNAs, miRNAs, and mRNAs was then assessed using Real-Time PCR and ELISA techniques. Results: 4 lncRNAs, 5 miRNAs, and 15 common target genes were detected in PCa patients compared with the healthy group. Unlike the tumor suppressors, the expression levels of common onco-lncRNAs, oncomiRNAs, and oncogenes showed a considerable increase in patients with advanced stages; Biochemical Relapse and Metastatic, in comparison to the primary stages; Local and Locally Advanced. Additionally, their expression levels significantly increased with a higher Gleason score than a lower one. Conclusion: Identifying a common lncRNA-miRNA-mRNA network associated with prostate cancer may be clinically valuable as potential predictive biomarkers. They can also serve as novel therapeutic targets for PCa patients.

COVID-19 vaccine side effects on menstrual disturbances among Iranian women.

Background: Many studies reported of menstrual disturbances as possible side effects of COVID-19 vaccination. Our objective was to evaluate the association between vaccination and the occurrence of menstrual disturbances among Iranian women. Methods: We used to google form questionnaires to collect reports of menstrual disturbances from 455 women aged 15-55 years in Iran. We estimated the relative risk of menstrual disturbances according to vaccination in a self-controlled case-series design after vaccination. We examined the occurrence of such disorders after the first, second and third doses of vaccine. Results: Findings The prevalence of menstrual disturbance was more in latency and heavy bleeding than others disorder after vaccination, although Ö® %50 of women doesn't have any disturbance. We observed increased risks after vaccination also for other menstrual disturbances, in menopausal women too (>10%). Discussion: Menstrual disturbances were generally common regardless of vaccination. We found a significant increase in menstrual disturbances after vaccination, particularly for latency and heavier bleeding than usual, longer duration and for short interval between menstruations. Mechanisms underlying these findings may involve bleeding disturbances in general, as well as endocrine alterations of immune system stimulation and relation to hormonal secretion.

Pentoxifylline promotes spermatogenesis via upregulation of the Nrf2-ARE signalling pathway in a mouse model of germ-cell apoptosis induced by testicular torsion-detorsion.

CONTEXT: Testicular torsion-detorsion results in loss of germ cells and infertility. Pentoxifylline has been shown to prevent tissue damage. AIMS: To determine the effect of pentoxifylline on germ cell survival in torsion-detorsion induced apoptosis Methods: Twenty male mice were divided into four groups of five animals each: Control, T1 (Torsion-detorsion+single dose 100mg/kg Pentoxifylline/IP), T2 (Torsion-detorsion+daily 20mg/kg pentoxifylline/IP for 2weeks, and T/D (Torsion-detorsion only). 35thday after torsion-detorsion, the left testes of all the animals were harvested for histological and biochemical analysis. KEY RESULTS: Histomorpholoical analysis showed significant increase (P <0.05) in seminiferous tubule diameter, Johnsen's score and germ cells of Control and T1 compared to T2 and T/D, with no significant difference (P >0.05) in testis weight, sertoli, leydig and myoid cells. Tunnel assay showed significant increase (P <0.05) in apoptotic cells of T/D and T2 animals compared to Control and T1. RT-PCR analysis showed significant high (P <0.01) mRNA expression of Bax gene in T/D compared to T1 and T2 and significant increase (P <0.05) of Bcl2 in Control, T1, T2 compared to T/D. Nrf2-ARE transcripts revealed significant increase (P <0.05) in Control and T1 compared to T2 and T/D. Western blot showed significantly increased (P <0.05) caspase-3 in T/D compared to Control, T1 and T2. CONCLUSION: Pentoxifylline promotes spermatogenesis and suppressed apoptosis induced by testicular torsion-detorsion. IMPLICATION: Pentoxifylline could serve as adjunct therapy to surgery in the treatment of torsion-detorsion induced germ cell apoptosis.

Protective effects of human amniotic membrane derived mesenchymal stem cells (hAMSCs) secreted factors on mouse spermatogenesis and sperm chromatin condensation following unilateral testicular torsion.

Testicular torsion is considered a urological disorder that requires immediate detorsion surgery. Ischemia/reperfusion (I/R) injury after testicular torsion detorsion causes of drastic impairment of spermatogenesis and infertility. Cell-free-based approaches seem to be a promising strategy to prevent I/R injury, they have more stable biological properties, and they contain paracrine factors of mesenchymal stem cells. The purpose of this study was to evaluate the protective effects of human amniotic membrane derived mesenchymal stem cells (hAMSCs) secreted factors on mouse sperm chromatin condensation and spermatogenesis improvement after I/R injury. hAMSCs were isolated and characterized by RT- PCR and flow cytometry, preparation of hAMSCs secreted factors was performed. Forty male mice were randomly divided into 4 groups: sham-operated, torsion detorsion, torsion detorsion+ intratesticular injection of DMEM/F-12, and torsion detorsion+ intratesticular injection of hAMSCs secreted factors. After one cycle of spermatogenesis, the mean number of germ cells, Sertoli, Leydig, myoid as well as tubular parameters, Johnson score, and spermatogenesis indexes were evaluated by H& E and PAS stainings. Sperm chromatin condensation and relative expression of c-kit and prm 1 genes were assessed by aniline blue staining and real-time PCR, respectively. The mean number of spermatogenic cells, Leydig, myoid, Sertoli, spermatogenesis parameters, Johnson score, as well as germinal epithelial height and diameters of seminiferous tubules decreased significantly after I/R injury. The thickness of basement membrane and percentage of sperm with excessive histone significantly increased, while the relative expression of c-kit and prm 1 significantly decreased in torsion detorsion group (p 0.001). hAMSCs secreted factors remarkably restored normal sperm chromatin condensation, spermatogenesis parameters and histomorphometric organization of seminiferous tubules via intratesticular injection (p 0.001). Thus, hAMSCs secreted factors may potentially salvage torsion-detorsion-induced infertility.

Evaluation of Nrf2/ARE Signaling Pathway in the Presence of Pentoxifylline as a Cryoprotectant in Mouse Spermatogonial Stem Cells.

The process of spermatogonial stem cell cryopreservation (SSCs) in young male cancer survivors is associated with increased reactive oxygen species (ROS), DNA fragmentation, apoptosis, decreased cell activity, and finally reduced fertility of SSCs. Therefore, it is necessary to add cryoprotectants to the freezing medium to minimize the injuries associated with cryopreservation. In addition, the Nrf2/ARE pathway is a main cellular pathway that regulates the antioxidant defense system. The purpose of this study was to evaluate the cryoprotective effect of pentoxifylline (PTX) on SSCs after freezing-thawing through the Nrf2/ARE pathway. SSCs extracted from neonatal mice testes were isolated and their purity was measured by flow cytometry with GDNF family receptor alpha-1 (GFRα1) and inhibitor of differentiation 4 (ID4). After culturing, the cells were frozen in different groups for 1 month. After freezing-thawing, cell viability, colonization rate, and intracellular ROS, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were evaluated. Quantitative real-time polymerase chain reaction and western blotting were done to assess the expression levels of Nrf2, Keap-1, PI3K, and AKT genes and proteins. The survival and colonization rates of SSCs, SOD, and CAT levels, and Nrf2, PI3K, and AKT expression levels were significantly higher in the PTX group compared with the other cryopreservation groups. The Keap-1 expression level and the ROS and MDA production levels also decreased significantly in the PTX group (p-value <0.05). According to our findings, PTX can activate the antioxidant defense through the Nrf2/ARE signaling pathway; therefore, it could be a suitable cryoprotectant candidate for freezing and long-term storage of SSCs in the clinical setting.

Cryoprotective Effect of Pentoxifylline on Spermatogonial Stem Cell During Transplantation into Azoospermic Torsion Mouse Model.

Preserving the spermatogonial stem cells (SSCs) in long periods of time during the treatment of male infertility using stem cell banking systems and transplantation is an important issue. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using 10 mM pentoxifylline (PTX) as an antioxidant in basal freezing medium. Testicular torsion-a mouse model for long-term infertility-was used to transplant fresh SSCs (n = 6), fresh SSCs treated with PTX (n = 6), cryopreserved SSCs with basal freezing medium (n = 6), and cryopreserved SSCs treated with PTX (n = 6). Eight weeks after germ cell transplantation, samples were assessed for proliferation, through evaluation of Ddx4 and Id4 markers, and differentiation via evaluation of C-Kit and Sycp3, Tnp1, Tnp2, and Prm1 markers. According to morphological and flow cytometry results, SSCs are able to form colonies and express Gfra1, Id4, α6-integrin, and ß1-integrin markers. We found positive influence from PTX on proliferative and differentiative markers in SSCs transplanted to azoospermic mice. In the recipient testis, donor SSCs formed spermatogenic colonies and sperm. Respecting these data, adding pentoxifylline is a practical way to precisely cryopreserve germ cells enriched for SSCs in cryopreservation, and this procedure could become an efficient method to restore fertility in a clinical setup. However, more studies are needed to ensure its safety in the long term.

Pentoxifylline improves the survival of spermatogenic cells via oxidative stress suppression and upregulation of PI3K/AKT pathway in mouse model of testicular torsion-detorsion.

Testicular torsion-detorsion results in enhanced formation of free radicals which contribute to the pathophysiology of testicular tissue damage. Recent reports have identified protective role of pentoxifylline (PTX) against free radicals. Thus, we determined the protective effect of pentoxifylline against testicular damage in mouse model of testicular torsion-detorsion. Twenty (6 weeks old) male mice were divided into 4 groups of 5 animals each namely: Control (sham operated group), T1 (Torsion-detosion + single dose 100 mg/kg PTX, T2 (torsion-detorsion + 20 mg/kg PTX for 2 weeks and T/D (torsion-detorsion only). Animals in T1, T2 and T/D groups underwent 2 h of testicular torsion with the left testes rotated 720° (clockwisely) followed by 30 min of detorsion. After detorsion, drug administration was done intraperitoneally. The left testes of all the animals were excised on the 35th day after torsion-detortion for histopathological and biochemical assay. Histomorphological analysis of the seminiferous tubules showed that there were significant increase (P < 0.01 or 0.05) in the mean seminiferous tubule diameter, Johnson score and germ cells of animals in Control and T1 compared to T2 and T/D with no significant difference (P > 0.05) in testes weight, sertoli, leydig and myoid cells in all groups. IHC results showed significant increase (P < 0.01 or 0.05) in id4 and scp3 protein markers in Control, T1 and T2 compared to T/D. Oxidative stress analysis revealed that Pentoxifylline significantly increased (P < 0.01 or 0.05) the level of SOD, catalase, mRNA expression of akt and pi3k genes but significantly suppress (P < 0.01 or 0.05) MDA and Caspase-3 level in Control, T1 and T2 compared to T/D. Pentoxifylline could be used as an adjunct therapy to surgery in the treatment of torsion-detorsion related testicular injury, However, Further studies are needed to evaluate the effects of pentoxifylline on testicular torsion.

Effect of a Freezing Medium Containing Melatonin on Markers of Pre-meiotic and Post-meiotic Spermatogonial Stem Cells (SSCs) After Transplantation in an Azoospermia Mouse Model Due to Testicular Torsion.

Spermatogonial stem cells (SSCs) are essential to the initiation of spermatogenesis. Cryopreservation, long-term maintenance, and auto-transplantation of SSCs could be a new treatment for infertility. The aim of this study was to add melatonin to the basic freezing medium and to evaluate its effect on the efficiency of the thawed SSCs after transplantation into the testicles of azoospermic mice. SSCs were isolated from newborn NMRI mice, and the cells were enriched to assess morphological features. The thawed SSCs were evaluated for survival, apoptosis, and ROS level before transplantation, and the proliferation (MVH and ID4) and differentiation (c-Kit, SCP3, TP1, TP2, and Prm1) markers of SSCs were examined using immunofluorescence, western blot, and quantitative real-time polymerase chain reaction (PCR) after transplantation. It was found that the survival rate of SSCs after thawing was significantly higher in the melatonin group compared with the cryopreservation group containing basic freezing medium, and the rate of apoptosis and level of ROS production also decreased significantly in the cryopreservation group with melatonin (p < 0.05). The expression of proliferation and differentiation markers after transplantation was significantly higher in the cryopreservation group with melatonin compared to the cryopreservation group (p < 0.05). The results suggest that adding melatonin to the basic freezing medium can effectively protect the SSCs by increasing the viability and reducing the ROS production and apoptosis and improve the transplantation efficiency of SSCs after cryopreservation, which will provide a significant suggestion for fertility protection in the clinic.

Promotion of Burn Wound Healing by Local Application of Adipose-Derived Mesenchymal Stem Cells: An Experimental Study.

Background:The burn wound is one of the health problems in the world that affects physical and mental health. Today, adipose-derived mesenchymal stem cells (ADSCs) have received medical attention for their accessibility and the ability to reproduce and repair. The present study was designed to investigate the effect of ADSCs on burn wound healing. Methods : The present experimental study was performed on 36 male Wistar rats divided into 1 control group and 3 experimental groups. The second-degree burns with a radius of 10 mm were induced after anesthesia. ADSCs and Dulbecco's Modified Eagle Medium (DMEM) were injected into the dermis around the burn area in the ADSCs and DMEM groups, respectively. Silver sulfadiazine (SSD) ointment was applied topically once daily as the SSD group. The control group did not receive any treatment. The rats were evaluated for 21 days. Wound healing rate, histopathological parameters, and the number of fibroblasts were evaluated by the immunofluorescence technique and vascular endothelial growth factor and transforming growth factor ß (TGF-ß) gene expression by reverse transcription-polymerase chain reaction. The results were entered into SPSS software (SPSS Inc) and analyzed with 1-way analysis of variance and repeated measures analysis. Results: The number of fibroblasts, the number of vessels, TGF-ß, and VEGF gene expression in the burn area were significantly higher in the ADSCs group than in the SSD, DMEM, and control groups. The results also showed that the amount of inflammation was significantly lower in the ADSCs group compared with the control group (p<0.001). Moreover, the percentage of wound recovery was significantly higher in the ADSCs group compared with other groups (p<0.001). Conclusion: ADSCs accelerate and improve burn wound healing by affecting fibroblasts, keratinocytes, and inflammatory cells as well as increasing the expression of the TGF-ß and VEGF genes, and thus increase in angiogenesis.

Comparison of the Effect of Adipocyte-derived Stem Cells and Curcumin Nanoliposomes with Phenytoin on Open Cutaneous Wound Healing in Rats.

BACKGROUND: Reducing the healing time of wounds can decrease the patient's immobility time and their medical costs, leading a faster return of the patients to daily work. OBJECTIVE: The aim of the present study is to compare the effect of adipose-derived stem cells and curcumin- containing liposomal nanoparticles with phenytoin on wound healing. METHODS: After anesthesia of the rats, open skin ulcers were made by a bistoury blade. Subsequently, stem cells were removed from the adipose tissue of the upper border of the epididymis. The originality of stem cells was then confirmed by the flow cytometry. The fusion method was used to prepare the liposome; and also, nanoliposomal particles were confirmed by using the DLS microscope. The percentage of recovery and the cell count was measured with IMAGEJ. The expression of genes was assessed by PCR. The number of fibroblasts was counted by immunohistochemistry techniques. The amount of collagen was determined by Tri-chromosome staining, and the number of capillaries was enumerated by H & E staining. RESULTS: The expression of the TGF-β1 gene, vascular number, wound healing rate and the number of fibroblasts increased significantly in adipose tissue-derived stem cells and curcumin nanoliposome groups (p<0.05); the wound surface was also decreased significantly (p<0.05). CONCLUSION: Based on the results of our research, adipose tissue-derived stem cells and curcumin nanoliposomes can heal wounds efficiently.

Comparison of Nasal Index Between Northwestern Nigeria and Northern Iranian Populations: An Anthropometric Study.

PURPOSE: The nasal index has a great value in anthropological studies, because it is one of the anthropometric indices acknowledged in nasal surgery as well as management. Anthropometric studies are very important area for craniofacial surgery and syndromology. The aim of this research was to compare the nasal characteristics between northwestern Nigerian and Iranian populations and compare them with other studies. METHODS: The nasal breadths and heights were measured from 400 individuals with 200 participants from Hausa ethnic group of northwestern Nigeria and 200 participants from Northern Tehran, Iran. Nasal index (NI) was calculated and analyzed statistically. RESULTS: There were significant difference in the nasal breadth (P = 0.0001), height (P = 0.0001) and NI (P = 0.0001) of sex groups in both Iranian and Nigeria population. The distribution of the nasal shapes for Iranian population is 127 leptorrhine (31.9%), 62 mesorrhine (15.6%) and nine platyrrhine (2.3%), while Nigeria population has 120 mesorrhine (30.2%), 75 leptorrhine (18.8%) and five platyrrhine (1.3%). This shows that Nigeria Hausa population has predominantly mesorrhine nose shape, while Northern Iranians are leptorrhine. CONCLUSION: The NI of males is higher than females in both population and this study can be of clinical and surgical interest in Rhinology. We recommend further studies to compare the NI of Nigeria and Iranian population of different ethnic groups and with other countries.

Three-dimensional co-culture of human spermatogonial stem cells with Sertoli cells in soft agar culture system supplemented by growth factors and Laminin.

Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than non-supplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis.

Reproductive toxicity of manganese dioxide in forms of micro- and nanoparticles in male rats.

BACKGROUND: Manganese Dioxide (MnO 2 ) has long been used in industry, and its application has recently been increasing in the form of nanoparticle. Objective: The present study was an attempt to assess the effects of MnO 2 nanoparticles on spermatogenesis in male rats. MATERIALS AND METHODS: Micro- and nanoparticles of MnO 2 were injected (100 mg/kg) subcutaneously to male Wistar rats (150 ± 20 gr) once a week for a period of 4 weeks, and the vehicle group received only normal saline (each group included 8 rats). The effect of these particles on the bodyweight, number of sperms, spermatogonia, spermatocytes, diameter of seminiferous tubes, testosterone, estrogen, follicle stimulating factor, and the motility of sperms were evaluated and then compared among the control and vehicle groups as the criteria for spermatogenesis. RESULTS: The results showed that a chronic injection of MnO 2 nanoparticles caused a significant decrease in the number of sperms, spermatogonia, spermatocytes, diameter of seminiferous tubes (p < 0.001) and in the motility of sperms. However, no significant difference was observed in the weight of prostate, epididymis, left testicle, estradiol (p = 0.8) and testosterone hormone (p = 0.2). CONCLUSION: It seems that the high oxidative power of both particles was the main reason for the disturbances in the function of the testis. It is also concluded that these particles may have a potential reproductive toxicity in adult male rats. Further studies are thus needed to determine its mechanism of action upon spermatogenesis.

Roles for Kisspeptin in proliferation and differentiation of spermatogonial cells isolated from mice offspring when the cells are cocultured with somatic cells.

Kisspeptin (Kp) expression in testis has caused most of the recent research surveying its functional role in this organ. This peptide influences spermatogenesis and sperm capacitation, so it is considered as a regulator of reproduction. Kp roles exert through hypothalamic/pituitary/gonadal axis. We aimed to evaluate direct roles for Kp on proliferation and differentiation of spermatogonial cells (SCs) when the cells are cocultured with somatic cells. Somatic cells and SCs were isolated from adult azoospermic and newborn mice and then enriched using a differential attachment technique. After the evaluation of identity and colonization for SCs, the cells were cocultured with somatic cells, and three doses of Kp (10-8 -10-6 M) was assessed on proliferation (through evaluation of MVH and ID4 markers) and differentiation (via evaluation of c-Kit and SCP3 , TP1, TP2 , and, Prm1 markers) of the coculture system. Investigations were continued for four succeeding weeks. At the end of each level of testosterone in the culture media was also evaluated. We found positive influence from Kp on proliferative and differentiative markers in SCs cocultured with somatic cells. These effects were dose-dependent. There was no effect for Kp on testosterone level. From our findings, we simply conclude that Kp as a neuropeptide for influencing central part of reproductive axis could also positively affect peripheral processes related to spermatogenesis without having an effect on steroidogenesis.

Roles for osteocalcin in proliferation and differentiation of spermatogonial cells cocultured with somatic cells.

Spermatogonial cells (SCs) are key cells for spermatogenesis. These cells are affected by paracrine signals originated from nearby somatic cells, among them Leydig cells have receptors for osteocalcin, a hormone known for exerting positive roles in the promotion of spermatogenesis. The aim of this study was to evaluate roles for osteocalcin on SCs proliferative and differentiation features after coculture with Leydig cells. SCs and Leydig cells were isolated from neonate NMRI offspring mice and adult NMRI mice, respectively. SCs population were then enriched in a differential attachment technique and assessed for morphological features and identity. Then, SCs were cocultured with Leydig cells and incubated with osteocalcin for 4 weeks. Evaluation of proliferation and differentiation-related factors were surveyed using immunocytochemistry (ICC), Western blot, and quantitative real-time polymerase chain reaction (PCR). Finally, the rate of testosterone release to the culture media was measured at the end of 4th week. Morphological and flow cytometry results showed that the SCs were the population of cells able to form colonies and to express ID4, α6-, and ß1-integrin markers, respectively. Leydig cells were also able to express Gprc6α as a specific marker for the cells. Incubation of SCs/Leydig coculture with osteocalcin has resulted in an increase in the rate of expressions for differentiation-related markers. Levels of testosterone in the culture media of SCs/Leydig was positively influenced by osteocalcin. It could be concluded that osteocalcin acts as a positive inducer of SCs in coculture with Leydig cells probably through stimulation of testosterone release from Leydig cells and associated signaling.

Effects of Viola tricolor Flower Hydroethanolic Extract on Lung Inflammation in a Mouse Model of Chronic Asthma.

Asthma is a chronic inflammatory disease of the lungs driven by T cell activation. Viola tricolor L. as a traditional medical herb could suppress activated T lymphocytes and has been used empirically for asthma remedy. In the present study, we investigated the anti-inflammatory effect of Viola tricolor and its underlying mechanism on asthma characteristics induced by ovalbumin (OVA) in mice. BALB/c mice were randomly divided into six groups: normal control, Ovalbumin (OVA) control, OVA mice treated with Viola tricolor (50, 100 and 200 mg/kg) and dexamethasone (3 mg/kg). All mice except normal controls were sensitized and challenged with OVA. Asthmatic mice were treated orally in the last 7 days of OVA challenge. The total and differential leukocyte counts, Interleukin (IL)-4 and interferon (IFN)-γ levels in bronchoalveolar lavage fluid (BALF) were determined. H and E staining for lung inflammation was performed. Viola tricolor treatment at 200 mg/kg significantly decreased IL-4 level but did not considerably affect the IFN-γ level. Therefore, it effectively reduced asthma characteristics including infiltration of leukocytes particularly eosinophil and peribronchial inflammation as compared to dexamethasone. However, Viola tricolor at 100 mg/kg had the most prominent inhibitory effect on the IL-4 level and also markedly increased IFN-γ level. As result, it prevented further reduction of inflammatory parameters in this group compared to the Viola tricolor-treated group at 200 mg/kg. Our study demonstrated that Viola tricolor has anti-inflammatory effects via inhibition of T-helper type 2 (Th2) cytokine production and validated its empirical usage in traditional medicine.

Safety Assessment of Mentha mozaffarianii Essential Oil: Acute and Repeated Toxicity Studies.

BACKGROUND: Mentha mozaffarianii, an endemic species from the Labiatae family, is used in Iranian traditional medicine. This study evaluated the acute and repeated oral toxicity of the Mentha mozaffarianii essential oil (MMEO) in rats and mice. METHODS: To assess the toxicity profile of the MMEO, we administered the essential oil to 48 rats and mice of both sexes by gavage in acute and repeated models. In acute toxicity, the animals were administered the MMEO (2000 mg/kg) and were monitored for 14 days. In the repeated toxicity, the MMEO was administered (100 mg/kg) daily for 4 weeks. On the 28th day, all the animals were scarified and blood and tissue samples were prepared. All the clinical, biochemical, and histopathological changes were assessed and compared with those in the controls. Statistical significance was determined by one- and two-way analyses of variance, followed by the Tukey test using GraphPad Prism 6. RESULTS: In the acute test, there was no mortality; therefore, the oral LD50 value determined in the mice and rats of both sexes was greater than 2000 mg/kg. In the repeated test, the animals received the MMEO and there was no mortality. In the biochemical analysis, there were significant increases in blood glucose, cholesterol, ALT, AST, ALP, and TSH in the female rats and also in BUN in the male rats. The histopathological studies revealed evidence of microscopic lesions in the liver, kidney, stomach, and small intestine tissues of the MMEO group. CONCLUSION: The results indicated that the acute toxicity of the MMEO in the mice and rats was of a low order and it revealed slight tissue damage to several organs when given subchronically at a dose of 100 mg/kg.