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Introduction: According to highlighted role of men in family planning, the use of reversible methods of contraception with no adverse effects on androgen-dependent metabolic reactions is very important. in order to achieve a conductor combination in the designing contraceptive drugs, in the study, the effect of two: Benzyl Ester and Cyclohexyl on histological parameters of seminiferous tubules was investigated. Materials and Methods: This study was conducted on the 36 rat Sprogue-Dawley with weighing 250-300 g and least 100 days. Then they were divided into two groups containing 13 mice and one group containing 10 mice and were Received respectively 15 mg/kg of Cyclohexyl and Benzyl Ester and Normal Saline as subcutaneous injection daily for 60 days. Next, samples of testicular fixative were stained by the usual method (H&E) and Histological parameters of seminiferous tubules were measured through Stereometry and Morphometry methods. Results: Reduce the Perimeter of seminiferous tubules in the group of Benzyl Ester had significant difference with the two other groups (p<0.001), While the differences diameter of the seminiferous tubules in the two groups was significant compared with the control group (p<0.05) and there was not noticeable difference among the three studied groups in terms of seminiferous tubules number. Conclusion: Benzyl ester was more effective on Histological parameters of seminiferous tubules. Thus with eliminating the cardiovascular effects and Conservation the contraceptives properties, It can be used as a guide combination. [ABSTRACT FROM AUTHOR]

Introduction: Nitric Oxide (NO)- as one of the smallest active products, involved in control of a number of physiological processes including cell growth, apoptosis, regulation of blood pressure, defense mechanism and especially in the reproductive process. Materials and Methods: We used 40 Wistar pregnant rats between 200 and 250 grams in weight and aged eight weeks. Based on observation of vaginal plug, pregnant mice were divided into five groups. The first group was received 2 mg/kg normal saline and the others were received respectively 200 mg/kg LArginine, 20 mg/kg L-NAME and a mixture of the same doses of L-Arginine and L-NAME on 3, 4 and 5th gestational days via intraperitoneal. The control group did not received any injection. Ovaries were removed on 18th gestational days, and after fixation and tissue preparation via staining by the routine H&E method, studied by Light microscopy. Results: Comparing the groups using ANOVA, there was a significant difference in reducing the number of primary follicles and increasing atretic follicles in the LArginine group in comparision with the other groups. The histological changes were also observed in the LArginine group. Conclusion: The study results showed that nitric oxide during pregnancy has damaging effects on ovaries and is recommended to be used with caution during pregnancy. [ABSTRACT FROM AUTHOR]

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 CdCl 2 (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., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

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.

Cryopreservation of spermatogonial stem cells (SSCs) is an important method to restore and maintain fertility in preadolescent children suffering from cancer. For protection of SSCs from cryoinjury, various antioxidant agents have been used. The aim of this study was to assess the antiapoptotic and antioxidant effects of melatonin in frozen-thawed SSCs. SSCs were isolated from testes of neonatal mice (3-6 days old) and their purities were measured by flow cytometry with promyelocytic leukemia zinc finger protein. After culturing, the cells were frozen in two groups (1) control and (2) melatonin (100 μM) and stored for 1 month. Finally, the cell viability, colonization rate, expression of Bcl-2 and BAX gene, and intracellular reactive oxygen species (ROS) were evaluated after freezing-thawing. Melatonin increased the viability and colonization of SSCs and Bcl-2 gene expression. It also diminished BAX gene expression and intracellular ROS. The results of this study show that melatonin with antioxidant and antiapoptotic effects can be used as an additive for freezing and long-term storage of cells and infertility treatment in the clinic.

Background: Cryostorage of spermatogonial stem cells (SSCs) is an appropriate procedure for long-term storage of SSCs for fertility preservation. However, it causes damage to cellular structures through overproduction of ROS and oxidative stress. In this study, we examined the protective effect of melatonin as a potent antioxidant in the basic freezing medium to establish an optimal cryopreservation method for SSCs., Methods: SSCs were obtained from the testes of neonatal male mice aged 3-6 days. Then, 100 μM melatonin was added to the basic freezing medium containing DMSO for cryopreservation of SSCs. Viability, apoptosis-related markers (BAX and BCL2), and intracellular ROS generation level were measured in frozen-thawed SSCs before transplantation using the MTT assay, immunocytochemistry, and flow cytometry, respectively. In addition, Western blotting and immunofluorescence were used to evaluate the expression of proliferation (PLZF and GFRα1) and differentiation (Stra8 and SCP3) proteins in frozen-thawed SSCs after transplantation into recipient testes., Results: The data showed that adding melatonin to the cryopreservation medium markedly increased the viability and reduced intracellular ROS generation and apoptosis (by decreasing BAX and increasing BCL2) in the frozen-thawed SSCs (p < 0.05). The expression levels of proliferation (PLZF and GFRα1) and differentiation (Stra8 and SCP3) proteins and resumption of spermatogenesis from frozen-thawed SSCs followed the same pattern after transplantation., Conclusions: The results of this study revealed that adding melatonin as an antioxidant to the cryopreservation medium containing DMSO could be a promising strategy for cryopreservation of SSCs to maintain fertility in prepubertal male children who suffer from cancer., (© 2022. The Author(s).)

Cryopreservation of spermatogonial stem cells (SSCs) is a useful method for fertility preservation in preadolescent children suffering from cancer. However, SSCs may become damaged during cryopreservation due to the generation of reactive oxygen species (ROS). For this reason, various antioxidant agents have been used to protect SSCs from cryopreservation-induced damages. Recently, it has been reported that miR-30a-5p has antiapoptotic and antioxidant activity. The aim of this study was to assess the antiapoptotic and antioxidant effects of miR-30a-5p mimics in frozen-thawed SSCs. To this end, SSCs were isolated from male BALB/C mice (3-6 days old) and cultivated for 14 days. After the detection of optimum concentration, a miR-30a-5p mimic or miR-30a-5p inhibitor with Lipofectamine was transfected into SSCs and, finally, the cell groups were frozen for 1 week. After thawing, different properties, including cell viability (using MTT), colonization of SSCs (number and diameter of colonies), ROS generation (using DCFH-DA assay), levels of malondialdehyde (MDA) and superoxide dismutase (SOD), and gene expression of Bcl-2 and BAXBax (using quantitative real-time PCR), were investigated. The transfection of SSCs with miR-30a-5p mimics before the freezing-thawing process significantly increased the viability, number, and diameter of SSCs colonies. Also, the miR-30a-5p mimic decreased the levels of ROS production and MDA, but it increased the SOD levels. Moreover, the miR-30a-5p mimic decreased BAX and increased Bcl-2 expression in frozen-thawed SSCs. The transfection of SSCs with the miR-30a-5p mimic can increase cell viability and antioxidant defense, and it can decrease apoptosis during the freezing-thawing process. If SSC is able to produce spermatozoa after the transfection of miR-30a-5p and the freezing-thawing process, it can be suggested as a promising strategy for the cryopreservation of SSCs in prepubertal boys suffering from cancer.

Background: The importance of spermatogonial stem cells (SSCs) in spermatogenesis is crucial and intrinsic factors and extrinsic signals mediate fate decisions of SSCs. Among endogenous regulators, microRNAs (miRNAs) play critical role in spermatogenesis. However, the mechanisms which individual miRNAs regulate self- renewal and differentiation of SSCs are unknown. The aim of this study was to investigate effects of miRNA-30a-5p inhibitor on fate determinations of SSCs., Methods: SSCs were isolated from testes of neonate mice (3-6 days old) and their purities were performed by flow cytometry with ID4 and Thy1 markers. Cultured cells were transfected with miRNA- 30a-5p inhibitor. Evaluation of the proliferation (GFRA1, PLZF and ID4) and differentiation (C-Kit & STRA8) markers of SSCs were accomplished by immunocytochemistry and western blot 48 h after transfection., Results: Based on the results of flow cytometry with ID4 and Thy1 markers, percentage of purity of SSCs was about 84.3 and 97.4 % respectively. It was found that expression of differentiation markers after transfection was significantly higher in miRNA-30a- 5p inhibitor group compared to other groups. The results of proliferation markers evaluation also showed decrease of GFRA1, PLZF and ID4 protein in SSCs transfected with miRNA-30a-5p inhibitor compared to the other groups., Conclusions: It can be concluded that inhibition of miRNA-30a-5p by overexpression of differentiation markers promotes differentiation of Spermatogonial Stem Cells.

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.

Background: Spermatogenesis is a complex process that is controlled by interactions between germ cells and somatic cells. The commitment of undifferentiated spermatogonia to differentiating spermatogonia and normal spermatogenesis requires the action of gonadotropins. Additionally, numerous studies revealed the role of retinoic acid signaling in induction of germ cell differentiation and meiosis entry., Main Text: Recent studies have shown that expression of several RA signaling molecules including Rdh10, Aldh1a2, Crabp1/2 are influenced by changes in gonadotropin levels. Components of signaling pathways that are regulated by FSH signaling such as GDNF, Sohlh1/2, c-Kit, DMRT, BMP4 and NRGs along with transcription factors that are important for proliferation and differentiation of spermatogonia are also affected by retinoic acid signaling., Conclusion: According to all studies that demonstrate the interface between FSH and RA signaling, we suggest that RA may trigger spermatogonia differentiation and initiation of meiosis through regulation by FSH signaling in testis. Therefore, to the best of our knowledge, this is the first time that the correlation between FSH and RA signaling in spermatogenesis is highlighted.

Human sperm cryopreservation is a common technique which is used in assisted reproductive technologies. Despite the existence of evidence supporting the production of ROS and DNA fragmentation during sperm cryopreservation, there is little and equivocal information about the cryopreservation effects on methylation of imprinted genes and imprinting control regions. In this study, we have investigated the effects of cryopreservation on DNA methylation in promoter regions of SNURF-SNRPN and UBE3A imprinted genes, PWS-ICR and AS-ICR in the chromosome 15q11-q13 region. Semen samples from 10 healthy normozoospermic men were collected and each sample was divided into four equal aliquots: fresh, cryoprotectant, cryopreservation, and H 2 O 2 . We measured the ROS levels and DNA fragmentation using DCFH-DA and TUNEL assay respectively by flow cytometry. DNA methylation in promoter regions of SNURF-SNRPN and UBE3A imprinted genes, PWS-ICR and AS-ICR in the chromosome 15q11-q13 region were evaluated by quantitative methylation-specific PCR technique. Intracellular levels of ROS and percentage of TUNEL-positive spermatozoa significantly increased in cryopreservation group compared to fresh group. Exposure to cryoprotectant had no significant effect on ROS levels and DNA fragmentation. Neither cryopreservation nor exposure to cryoprotectant significantly affected DNA methylation of the selected gene regions. However, DNA fragmentation had positive correlation with DNA methylation of AS-ICR. In conclusion, based on our study, clinical use of sperm cryopreservation for fertility treatments appear to be safe in regard to DNA methylation in the chromosome 15q11-q13 region.

Background: Spermatogonial stem cells (SSCs) are considered as special stem cells since they have the ability of self-renewal, differentiation, and transferring genetic information to the next generation. Also, they considered as vital players in initiating and preserving spermatogenesis. The fate decisions of SSCs are mediated by intrinsic and extrinsic factors, among which microRNAs (miRNAs) are one of the most essential factors in spermatogenesis among endogenous regulators. However, the mechanisms by which individual miRNAs regulate self-renewal and differentiation of SSCs are unclear. The present study aimed to evaluate the impact of miRNA-30 mimic on fate determinations of SSCs., Materials and Methods: The obtained SSCs from neonatal mice (3-6 days old) were purified by MACS and flow cytometry with a promyelocytic leukemia zinc-finger marker. Then, the cultured cells were transfected with miRNA- 30 mimic, and finally, the changes in expressing ID4 and c-kit proteins were assessed by western blot analysis., Results: According to flow cytometry findings, the percentage of SSC purity was about 98.32. The expression of ID4 protein and colonization increased significantly through the transfection of miRNA-30 mimic (P<0.05)., Conclusion: The miRNA-30 controls spermatogonial stem cell self-renewal and differentiation, which may have significant implications for treating male infertility., (Copyright© 2020, Galen Medical Journal.)

Chronic demyelination in the central nervous system (CNS) is accompanied by an increase in the number of reactive astrocytes and astrogliosis. There are controversial issues regarding astrocytes and their roles in demyelinating diseases in particular for multiple sclerosis (MS). We aimed to evaluate possible roles for pharmacologic astrocyte ablation strategy using La-aminoadipate (L-AAA) on remyelination in a cuprizone model of demyelination. Male C57BL/6 mice were fed with 0.2% cuprizone for 12 weeks followed by 2-week administration of L-AAA through a cannula inserted 1 mm above the corpus callosum. Rotarod test showed a significant decrease in the range of motor coordination deficits after ablation of astrocytes in mice receiving cuprizone. Results of Luxol fast blue (LFB) and transmission electron microscopy (TEM) for evaluation of myelin content within the corpus callosum revealed a noticeable rise in the percentage of myelinated areas and in the number of myelinated fibers after L-AAA administration in the animals. Astrocyte ablation reduced protein expressions for GFAP (an astrocyte marker) and Iba-1 (a microglial marker), but increased expression of Olig2 (an oligodendrocyte marker) assessed by immunofluorescence. Finally, expression of genes related to recruitment of microglia (astrocyte chemokines CXCL10 and CXCL12) and suppression of oligodendrocyte progenitor cell (OPC) differentiation (astrocyte peptides ET-1 and EDNRB) showed a considerable decrease after administration of L-AAA (for all p < 0.05). These results are indicative of improved remyelination after ablation of astrocytes possibly through hampering microgliosis and astrogliosis and a further rise in the number of matured Olig2 + cells.

In Wistar rats, reproductive behavior is controlled in a neural circuit of ventral forebrain including the medial amygdala (Me), bed nucleus of the stria terminalis (BNST) and medial preoptic area (MPOA) via perception of social odors. Diabetes Mellitus (DM) is a widespread metabolic disease that affects many organs in a variety of levels. DM can cause central neuropathies such as neuronal apoptosis, dendritic atrophy, neurochemical alterations and also causes reproductive dysfunctions. So we hypothesized damage to the nuclei of this circuit can cause reproductive dysfunctions. Therefore in this project we assessed diabetic effect on these nuclei. For this purpose neuron tracing technique and TUNEL assay were used. We injected HRP in the MPOA and counted labeled cells in the Me and BNST to evaluate the reduction of neurons in diabetic animals. Also, coronal sections were analyzed with the TMB histochemistry method. Animals in this study were adult male Wistar rats (230 ± 8g) divided to control and 10-week streptozotocin-induced diabetic groups. After data analysis by SPSS 16 software, a significant reduction of HRP-labeled neurons was shown in both Me and BNST nuclei in the diabetic group. Moreover, apoptotic cells were significantly observed in diabetic animals in contrast to control the group. In conclusion, these alterations of the circuit as a result of diabetes might be one of the reasons for reproductive dysfunctions.

BACKGROUND Early diagnosis and endoscopic resection of adenomatous polyps is the main approach for screening and prevention of colorectal cancer (CRC). We aimed to assess polyp detection rate (PDR) and to characterize demographic, clinical, and pathological features of colorectal polyps in an Iranian population. METHODS We retrospectively analyzed the data from 5427 colonoscopies performed during 2007-2012 at Masoud Clinic, the main endoscopy center associated with Sasan Alborz Biomedical Research Center, in Tehran, Iran. RESULTS Our sample included 2928 (54%) women and 2499 (46%) men, with the mean age of 48.3 years (SD=16.1). The most common reasons for colonoscopy included screening in 25.0%, and gastrointestinal bleeding in 15.2%. Cecal intubation was successful in 86% of patients. The quality of bowel preparation was fair to excellent in 78.1% (n=4235) of colonoscopies. Overall PDR was 42.0% (95% CI: 40.6-43.3). The PDR in men (51.1%, 95% CI: 49.1-53.1) was significantly higher than women (34.2%, 95% CI: 32.4-35.9, p<0.001). Polyps were more frequently observed in patients after the 6(th) decade of life (F=3.2; p=0.004). CRC was detected in 2.9% (73/2499) of men and 1.9% (57/2928) of women (p=0.02). The mean age for patients with cancer was significantly higher than that for individuals with polyps, 60.9 (SD=13.4) year vs. 56.9 (SD=13.7) year, respectively (p=0.001). Almost 82.8% of the lesions were precancerous with tubular type predominance (62.3%) followed by tubulo-villous (10.3%), villous (6.6%), and serrated (3.6%). Hyperplastic/inflammatory polyps comprised 17.2% of lesions. CONCLUSION Distal colon was more prone to develop polyps and cancer than proximal colon in our series. These findings provide a great infrastructure for next preventive programs and have implications for colorectal cancer screening at population-level.