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Infertility is intricately linked with alterations in circadian rhythms along with physiological decline and stem cell senescence. Yet, the direct involvement of circadian mechanisms in nicotine-induced injury to the testes, especially the senescence of spermatogonia stem cells (SSCs), is not well comprehended. This study revealed that nicotine exposure induced testis injury by triggering SSCs senescence along with the upregulation of senescence marker genes and senescence-associated secretory phenotype components. Moreover, nicotine treatment caused mitochondrial hyper-fusion, increased oxidative stress, and DNA damage. Exposure to nicotine was found to suppress the expression of sirtuin 6 (SIRT6), which accelerated the senescence of spermatogonia stem cells (SSCs). This acceleration led to increased acetylation of brain and muscle ARNT-like protein (Bmal1), consequently reducing the expression of Bmal1 protein. Conversely, the overexpression of Bmal1 alleviated mitochondrial hyper-fusion and senescence phenotypes induced by nicotine. Overall, this study unveiled a novel molecular mechanism behind nicotine-induced disorders in spermatogenesis and highlighted the SIRT6/Bmal1 regulatory pathway as a potential therapeutic target for combating nicotine-associated infertility., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organization that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitle., (Copyright © 2024. Published by Elsevier Inc.)

The classical traditional Chinese medicine formula Huangqi-Guizhi-Wuwutang (HGW) has been shown to enhance sperm production. However, the bioactive components and comprehensive mechanisms underlying the therapeutic effects remain unclear. The present study investigates the potential active ingredients and underlying mechanisms of HGW against spermatogenesis dysfunction. The chemical components of HGW were analyzed by mass spectrometry. And then the "components-targets-pathway-disease" network was constructed using network pharmacology research methods, which aimed to identify the key active components and potential targets of HGW in treating oligospermia. Experimental validation was finally conducted in animal model. The male-specific pathogen-free Kunming mice were divided into five groups: Sham group, Model group, and HGW groups (8, 16, and 32 g/kg of HGW by gavage for 35 days). Chemical profile and network pharmacology results revealed that potential bioactive compounds were dihydrocinnacasside, isomucronulatol, and 6-gingerol, and the mechanism of which was enriched in regulating spermatogenic stem cells (SSCs), endocrine function, and apoptosis. The administration of HGW significantly improved oligospermia in mice. HGW significantly upregulated the expression of marker proteins in SSCs and the potential targets within the testis simultaneously. Our data indicates that HGW enhances the proliferation of SSCs, and HGW can be a promising therapeutic candidate for oligospermia., (© 2024 John Wiley & Sons Ltd.)

Purpose: The use of 3D (3-Dimensional) culture systems supported cell-to-cell and cell-to-extracellular matrix (ECM) interactions, proliferation, and differentiation of SSCs (Spermatogonial stem cells). The potential advantages of ECM-based scaffolds for in vitro spermatogenesis have been indicated in human and animal experiments. Furthermore, the strong antioxidant and anti-inflammatory activities of melatonin have improved in vitro manipulation of human SSCs in culture conditions., Materials and Methods: SSCs were isolated from the testis of three dead-brain patients and then propagated for four weeks. The characterization of SSC colonies was done using real-time PCR (Polymerase chain reaction), ICC (Immunocytochemistry), and xenotransplantation to mice model. Decellularization of the human testis was performed using 0.3% sodium dodecyl sulfate (SDS) solution and 1% Triton X-100. Also, various characterizations of DTM (Decellularized testicular matrix ) were carried out using histological staining and DNA content analysis. The optimum dose of melatonin was selected by MTT (Methyl thiazol tetrazolium). SSCs were cultured in 4 groups: control, melatonin, ECM, and ECM-melatonin in a differentiation medium for four weeks. The expression of differentiation genes was evaluated by real-time polymerase chain reaction. In addition, the viability of cultured cells was assessed by MTT assay., Results: The results of ICC and real-time PCR showed the expression of undifferentiated SSC markers (PLZF and GRFA1) in SSC colonies following the 2D culture of isolated SSCs. The presence of testicular ECM components after different staining methods; and the reduction of DNA content confirmed the proper decellularization process. Germ cell apoptosis significantly decreased in melatonin and ECM groups, and the higher viability of SSCs was seen in the ECM-melatonin group. The relative expression of GFRA1 and PRM2 decreased and increased in ECM and ECM-melatonin groups, respectively., Conclusion: Our study showed that the addition of melatonin to the human naturally-derived ECM scaffold could provide a suitable platform for inducing the differentiation and preserving the viability of SSCs.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. Systematically exploring the critical factors associated with the formation of SSCs will provide new insight to improve the formation efficiency, and their practical application. Here we explore the regulatory mechanism of the ECM-receptor interaction signaling pathway and related genes during differentiation of SSCs in chicken. Firstly, the positive cell rate of SSCs protein marker was detected by immunofluorescence and flow cytometry and qRT-PCR was used to identify, the expression of related marker genes after 10 days of RA-induction. Secondly, the ESCs on 0d/ 4d /10d after RA- induction/self-differentiation were collected, and the total RNA was then extracted from cells. Finally, high-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. After PCA analysis of the RNA-seq data, Venny analysis, GO and KEGG enrichment were further used to find the key signaling pathways and genes in the RA-induction process. The results showed that on day 10 of RA-induction, grape cluster growth cells expressed integrinβ1, the specific marker protein of SSCs cells, and the integrinβ1 positive rate was 35.1%. Also, SSCs marker genes CVH, Integrinβ1, Integrinα6 were significantly up-regulated during RA-induction. Moreover, the significantly enriched pathway, ECM-receptor interaction signaling, in current study may play a crucial role in RA-induction. Then, JASPAR was used to predict the differential gene transcription factors in the signaling pathway, finding that RA receptor was a transcription factor of COL5A1, COL5A2 and COL3A1. The qRT-PCR results showed that the expression levels of RA receptors (RXRA, RARA and RXRG) and the predicted genes (COL5A1, COL5A2 and COL3A1) were both significantly increased during RA-induction. Also, dual-luciferase reporter assay showed that RA could affect the luciferin activities of COL5A1, COL5A2 and COL3A1. These results suggest that RA plays a crucial role in the formation of chicken spermatogonial stem cells via the transcription levels of COL5A1, COL5A2 and COL3A1 to regulate the ECM-receptor interaction signaling pathway. Additionally, knockdown of COL5A1/COL5A2/COL3A1 could effectively reduce the formation efficiency of SSCs. This indicated that the interference of RA receptor binding genes in the ECM-receptor interaction signaling pathway could decrease the efficiency of RA induced SSCs formation. Therefore, this study concludes that RA promotes formation of chicken spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 β1-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., (© 2021. Society for Reproductive Investigation.)

Background: Cryopreservation can expand the usefulness of spermatogonial stem cells (SSCs) in various fields. However, previous investigations that have attempted to modulate cryoinjury-induced mechanisms to increase cryoprotective efficiency have mainly focused on apoptosis and necrosis., Objectives: This study aimed to establish an effective molecular-based cryoprotectant for SSC cryopreservation via autophagy modulation., Materials and Methods: To determine the efficacy of autophagy modulation, we assessed the recovery rate and relative proliferation rate and performed western blotting for the determination of autophagy flux, immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization, and spermatogonial transplantation for in vivo SSC functional activity., Results: The results showed that a basal level of autophagy caused a higher relative proliferation rate (pifithrin-μ 0.01 μM, 184.2 ± 11.2%; 3-methyladenine 0.01 μM, 175.3 ± 10.3%; pifithrin-μ 0.01 μM + 3-methyladenine 0.01 μM, P3, 224.6 ± 22.3%) than the DMSO control (100 ± 6.2%). All treatment groups exhibited normal characteristics, suggesting that these modulators could be used as effective cryoprotectants without changing the properties of the undifferentiated germ cells. According to the results of the in vivo spermatogonial transplantation assay, the colonies per total number of cultured SSCs was significantly higher in the pifithrin-μ 0.01 μM (1596.7 ± 172.5 colonies), 3-methyladenine 0.01 μM (1522.1 ± 179.2 colonies), and P3 (1727.5 ± 196.5 colonies) treatment groups than in the DMSO control (842.8 ± 110.08 colonies), which was comparable to that of the fresh control (1882.1 ± 132.1 colonies)., Discussion: A basal level of autophagy is more essential for resilience in frozen SSCs after thawing, rather than the excessive activation or inhibition of autophagy., Conclusion: A basal level of autophagy plays a critical role in the pro-survival response of frozen SSCs after thawing; herein, a new approach by which SSC cryoprotective efficiency can be improved was identified., (© 2021 American Society of Andrology and European Academy of Andrology.)

Many child cancer patients endure anticancer therapy containing alkylating agents before sexual maturity. Busulfan (BU), as an alkylating agent, is a chemotherapy drug, causing DNA damage and cytotoxicity in germ cells. In the present study, we aimed to investigate the protective effect of astaxanthin (AST), as a potent antioxidant and powerful reactive oxygen species (ROS) scavenger, on BU-induced toxicity in human spermatogonial stem cells. For this purpose, testes were obtained from four brain-dead donors. After tissue enzymatic digestions, testicular cells were cultured for 3 weeks for spermatogonial stem cell (SSC) isolation and purification. K562 cell line was cultured to survey the effect of AST on cancer treatment. The cultured SSCs and K562 cell line were finally treated with AST (10μM), BU (0.1nM), and AST+BU. The expression of NRF-2, HO-1, SOD2, SOD3, TP53, and apoptotic genes, including CASP9, CASP3, BCL2, and BAX, were assayed using real-time PCR. Moreover, ROS level in different groups and malondialdehyde level and total antioxidant capacity in cell contraction of SSCs were measured using ELISA. Data showed that AST significantly upregulated the expression of NRF-2 gene (P<0.001) and protein (P<0.005) and also significantly decreased the production of BU-induced ROS (P<0.001). AST activated the NRF-2/HO-1 pathway that could remarkably restrain BU-induced apoptosis in SSCs. Interestingly, AST upregulated the expression level of apoptosis genes in the K562 cell line. The results of this study indicated that AST reduces the side effects of BU on SSCs without interference with its chemotherapy effect on cancerous cells through modulation of the NRF-2/HO-1 and mitochondria-mediated apoptosis pathways., (© 2021. Society for Reproductive Investigation.)

Perinatal exposures to endocrine disrupting chemicals (EDCs) alter the male reproductive system. Infants are exposed to genistein (GEN) through soy-based formula, and to Mono(2-ethylhexyl) Phthalate (MEHP), metabolite of the plasticizer DEHP. Spermatogonial stem cells (SSCs) are formed in infancy and their integrity is essential for spermatogenesis. Thus, understanding the impact of EDCs on SSCs is critical. Prostaglandins (PGs) are inflammatory mediators synthesized via the eicosanoid pathway starting with cyclooxygenases (Coxs), that regulate physiological and pathological processes. Our goal was to study the eicosanoid pathway in SSCs and examine whether it was disrupted by GEN and MEHP, potentially contributing to their adverse effects. The mouse C18-4 cell line used as SSC model expressed high levels of Cox1 and Cox2 genes and proteins, and eicosanoid pathway genes similarly to levels measured in primary rat spermatogonia. Treatments with GEN and MEHP at 10 and 100 μM decreased Cox1 gene and protein expression, whereas Cox2, phospholipase A2, prostaglandin synthases transcripts, PGE2, PGF2a and PGD2 were upregulated. Simultaneously, the transcript levels of spermatogonia progenitor markers Foxo1 and Mcam and differentiated spermatogonial markers cKit and Stra8 were increased. Foxo1 was also increased by EDCs in primary rat spermatogonia. This study shows that the eicosanoid pathway is altered during SSC differentiation and that exposure to GEN and MEHP disrupts this process, mainly driven by GEN effects on Cox2 pathway, while MEHP acts through an alternative mechanism. Thus, understanding the role of Cox enzymes in SSCs and how GEN and MEHP exposures alter their differentiation warrants further studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mouse CD90 + SSCs were enriched using the MACS technique and incubated with different doses of estradiol, ranging from 0.01 ng/mL to 500 μg/mL, for 7 days. The viability of SSCs was determined using an MTT assay. The combined effects of estradiol plus Sertoli cell differentiation medium on the orientation of SSCs toward Sertoli-like cells were also assessed. Using immunofluorescence imaging, we monitored protein levels of Oct3/4 after being exposed to estradiol. In addition, protein levels of testosterone, TF, and ABP were measured using ELISA. The expression of Sertoli cell-specific genes such as SOX9, GATA4, FSHR, TF, and ESR-1 and -2 was monitored using real-time PCR assay, and the effects of 14-day injection of estradiol on sperm parameters and Oct3/4 positive progenitor cells in a model of mouse were determined. Data showed that estradiol increased the viability of mouse SSCs in a dose-dependent manner compared to the control (p < 0.05). Along with these changes, cells displayed morphological changes and reduced Oct3/4 transcription factor levels compared to the control SSCs. 7-day incubation of SSCs with estradiol led to the up-regulation of SOX9, GATA4, FSHR, TF, and ESR-1 and -2, and levels of testosterone, TF, and ABP were increased compared to the control group (p < 0.05). The in-vivo examination noted that estradiol reduced sperm parameters coincided with morphological abnormalities (p < 0.05). Histological examination revealed pathological changes in seminiferous tubules and reduction of testicular Oct3/4 + progenitor cells. In conclusion, estradiol treatment probably can induce Sertoli cell differentiation of SSCs while exogenous administration leads to testicular progenitor cell depletion and infertility in long term., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Initiation of spermatogonial differentiation in the mouse testis begins with the response to retinoic acid (RA) characterized by activation of KIT and STRA8 expression. In the adult, spermatogonial differentiation is spatiotemporally coordinated by a pulse of RA every 8.6 days that is localized to stages VII-VIII of the seminiferous epithelial cycle. Dogmatically, progenitor spermatogonia that express retinoic acid receptor gamma (RARG) at these stages will differentiate in response to RA, but this has yet to be tested functionally. Previous single-cell RNA-seq data identified phenotypically and functionally distinct subsets of spermatogonial stem cells (SSCs) and progenitor spermatogonia, where late progenitor spermatogonia were defined by expression of RARG and Dppa3. Here, we found late progenitor spermatogonia (RARGhigh KIT-) were further divisible into two subpopulations based on Dppa3 reporter expression (Dppa3-ECFP or Dppa3-EGFP) and were observed across all stages of the seminiferous epithelial cycle. However, nearly all Dppa3+ spermatogonia were differentiating (KIT+) late in the seminiferous epithelial cycle (stages X-XII), while Dppa3- late progenitors remained abundant, suggesting that Dppa3+ and Dppa3- late progenitors differentially responded to RA. Following acute RA treatment (2-4 h), significantly more Dppa3+ late progenitors induced KIT, including at the midpoint of the cycle (stages VI-IX), than Dppa3- late progenitors. Subsequently, single-cell analyses indicated a subset of Dppa3+ late progenitors expressed higher levels of Rxra, which we confirmed by RXRA whole-mount immunostaining. Together, these results indicate RARG alone is insufficient to initiate a spermatogonial response to RA in the adult mouse testis and suggest differential RXRA expression may discriminate responding cells.

Orchitis is one of the leading causes of male animal infertility and is associated with inflammatory reactions caused by the bacterium. It has been reported that there is a mutual coupling effect between endoplasmic reticulum stress (ERS) and inflammatory response. Our studies showed that lipopolysaccharide (LPS) could cause testicular damages, apoptosis, ERS, and inflammatory responses in spermatogonial stem cells (SSCs); ERS-related apoptosis proteins were activated and the expression of ERS genes was significantly upregulated; meanwhile, the expression of Toll-like receptor 4 and inflammation factors was apparently increased with LPS treatment. Moreover, melatonin (MEL) could rescue testicular damage, and significantly inhibited the expression of ERS-related apoptosis genes, ERS markers, and inflammatory factors in SSCs and MEL played repairing and anti-infection roles in LPS-induced testicular damage. Therefore, MEL may be used as a drug to prevent and control bacterial infections in male reproductive systems. However, the specific molecular mechanism of MEL to resist ERS and inflammatory response remains to be further studied., (© 2020 Wiley Periodicals LLC.)

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.

Several lines of evidence strongly suggest that retinoic acid (RA) and stem cell factor (SCF)/c-Kit signal transduction pathways are involved in the differentiation of spermatogonial stem cells (SSCs). This study was aimed to investigate the effect of RA and SCF on in vitro differentiation of SSCs via evaluation of the mRNA expression of meiosis-specific genes in cultured testicular tissues. Testicular tissue samples were obtained from bilaterally vasectomized rats and also healthy adult rats and then were cultured for 25, 30, and 35 days on different conditions. The cultured testicular pieces were sectioned and stained with PAS to histological analysis. The total RNA was extracted from cultured testicular samples, and the expression of ACR, PRTM1, SYCP3, STRA8, c-KIT, PIWIL2, and OCT4 genes at mRNA level was quantified using real-time polymerase chain reaction (qPCR) procedure. After 1-month surgery, bilateral testicular weight showed a significant decrease in vasectomized adult rats compared with healthy adult rats (P < 0.05). Reduction in the diameter of the seminiferous tubules and depletion of advanced germinal elements in vasectomized rats compared with healthy adult rats were also observed. Our findings also demonstrated that the mRNA expression level of PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 genes in cultured testicular tissues significantly up-regulated in experimental group II compared with the control group (P < 0.001). Our findings lead us to conclude that SCF improves in vitro differentiation of SSCs in the OA rats, at least partially, by transcriptionally upregulating PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 genes.

Humanin (HN) is a mitochondrial-derived peptide that protects many cells/tissues from damage. We previously demonstrated that HN reduces stress-induced male germ cell apoptosis in rodents. HN action in neuronal cells is mediated through its binding to a trimeric cell membrane receptor composed of glycoprotein 130 (gp130), IL-27 receptor subunit (IL-27R, also known as WSX-1/TCCR), and ciliary neurotrophic factor receptor subunit (CNTFR). The mechanisms of HN action in testis remain unclear. We demonstrated in ex-vivo seminiferous tubules culture that HN prevented heat-induced germ cell apoptosis was blocked by specific anti-IL-27R, anti-gp130, and anti-EBI-3, but not by anti-CNTFR antibodies significantly. The cytoprotective action of HN was studied by using groups of il-27r-/- or ebi-3-/- mice administered the following treatment: (1) vehicle; (2) a single intraperitoneal (IP) injection of HN peptide; (3) testicular hyperthermia; and (4) testicular hyperthermia plus HN. We demonstrated that HN inhibited heat-induced germ cell apoptosis in wildtype but not in il-27r-/- or ebi-3-/- mice. HN restored heat-suppressed STAT3 phosphorylation in wildtype but not il-27r-/- or ebi-3-/- mice. Dot blot analyses showed the direct interaction of HN with IL-27R or EBI-3 peptide. Immunofluorescence staining showed the co-localization of IL-27R with HN and gp130 in Leydig cells and germ cells. We conclude that the anti-apoptotic effects of HN in mouse testes are mediated through interaction with EBI-3, IL-27R, and activation of gp130, whereas the role of CNTFR needs further studies. This suggests a multicomponent tissue-specific receptor for HN in the testis and links HN action with the IL-12/IL-27 family of cytokines., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cryopreservation as a method that enables long-term storage of biological material has long been used for the conservation of valuable zebrafish genetic resources. However, currently, only spermatozoa of zebrafish can be successfully cryopreserved, while protocols for cryopreservation of eggs and embryos have not yet been fully developed. Transplantation of germline stem cells (GSCs) has risen as a favorable method that can bypass the current problem in cryopreservation of female genetic resources and can lead to reconstitution of fish species and lines through surrogate production. Here, we describe essential steps needed for the cryopreservation of spermatogonial stem cells (SSCs) and their utilization in the conservation of zebrafish genetic resources through SSC transplantation and surrogate production.

Improvements in survival rates with gonad-sparing protocols for childhood and adolescence cancer have increased the optimism of survivors to become parents after treatment. Findings in rodents indicate that chromosomal aberrations can be induced in male germ cells by genotoxic exposures and transmitted to offspring and future generations with effects on development, fertility and health. Thus, there is a need for effective technologies to identify human sperm carrying chromosomal aberrations to assess the germ-line risks, especially for cancer survivors who have received genotoxic therapies. The time-dependent changes in the burden of sperm carrying structural chromosomal aberrations were assessed for the first time in a cancer setting, using the AM8 sperm FISH protocol which simultaneously detects abnormalities in chromosomal structure and number in sperm. Nine Hodgkin lymphoma (HL) patients provided 20 semen samples before, during, and after NOVP therapy (Novantrone, Oncovin, Velban and Prednisone) and radiation therapy that produced scattered gonadal doses from <0.05 to 0.6 Gy. Late meiosis was found to be the most sensitive to NOVP treatment for the production of sperm with chromosomal abnormalities, both in structure and number. Earlier stages of spermatogenesis were less sensitive and there was no evidence that therapy-exposed stem cells resulted in increased frequencies of sperm with abnormalities in chromosomal structure or number. This indicates that NOVP therapy may increase the risks for paternal transmission of chromosomal structural aberrations for sperm produced 32 to 45 days after a treatment with these drugs and implies that there are no excess risks for pregnancies conceived more than 6 months after this therapy. This clinical evaluation of the AM8 sperm FISH protocol indicates that it is a promising tool for assessing an individual's burden of sperm carrying chromosomal structural aberrations as well as aneuploidies after cancer therapy, with broad applications in other clinical and environmental situations that may pose aneugenic or clastogenic risks to human spermatogenesis., Competing Interests: The authors have declared that no competing interests exist.

Background: In infants, fever is often treated with acetaminophen or ibuprofen, two antipyretic and analgesic drugs inhibiting cyclooxygenases (COXs), enzymes catalyzing prostaglandin production. Infancy represents a critical developmental period when neonatal germ cells/gonocytes differentiate to spermatogonial stem cells required for spermatogenesis., Objectives: (a) Determine the expression of Cox2 and associated genes in postnatal day (PND)3 rat gonocytes compared to spermatogonia. (b) Examine whether acetaminophen or ibuprofen disrupts neonatal gonocyte functions. (c) Determine whether neonatal gonocytes produce prostaglandins and whether this process is altered by acetaminophen and ibuprofen., Materials and Methods: The expression of Cox2 and related genes was determined by gene arrays and qPCR. Cox2 protein levels were determined by immunocyto/histochemistry and immunoblots. The effects of acetaminophen and ibuprofen on PND3 gonocyte viability, apoptosis, proliferation, and differentiation were examined alone and with a proliferation cocktail or differentiation factor. Prostaglandins were examined by immunocyto/histochemistry and LC-MS., Results: Cox2 and related genes are highly expressed in gonocytes and spermatogonia. Acetaminophen and ibuprofen did not affect gonocyte survival or apoptosis, but they increased gonocyte proliferation. Ibuprofen significantly reduced RA-induced Stra8 expression, indicating an inhibitory effect on differentiation. Ibuprofen combined with RA decreased Cox2 mRNA and protein expression. PGE2 and PGF2α were produced by neonatal gonocytes and decreased by acetaminophen and ibuprofen., Discussion: The concomitant decrease of Stra8 expression, Cox2 expression, and PGE2 and PGF2a production in gonocytes co-treated with RA suggests that Cox2 plays a role in PND3 gonocyte differentiation. The effects of acetaminophen and ibuprofen on proliferation suggest a negative relationship between Cox2 and proliferation. Treating neonates with acetaminophen or ibuprofen could disrupt gonocyte development, leading to adverse reproductive effects., Conclusion: Understanding COX2 role in neonatal gonocytes and the potential risk of acetaminophen and ibuprofen treatment of infants may help prevent male reproductive pathologies., (© 2019 American Society of Andrology and European Academy of Andrology.)

The objective of this study was to investigate effects of selenium (Se) on proliferation and apoptosis of sheep spermatogonial stem cells (SSC) in vitro. The SSC were assigned to five treatment groups (0, 2.0, 4.0, 8.0 and 16.0 μmol/L Se). After treatment with Se for 96 h, cell proliferation and apoptosis were evaluated. The relative abundance of P53 mRNA transcript and protein, cell cycle and apoptosis-related genes were detected using real-time PCR and Western blot quantifications, respectively. The results indicate there were the least cell proliferation rates in the Se 16.0 group. Treatments with relatively greater Se concentrations (8.0 and 16.0 μmol/L) resulted in a greater percentage of apoptotic cells, which was consistent with the relative abundances of P53, P21, P27 and pro-apoptosis mRNA transcripts. There were relatively greater ROS concentrations in the control, Se 8.0 and Se 16.0 groups. Compared with the control group, treatment with the Se concentration of 16.0 μmol/L resulted in an increased abundance of P53, P21, P27 and BAX proteins. Treatment with Pifithrin-α suppressed the increase in abundance of P53 and P21 proteins induced by the relatively greater concentration of Se (16.0 μmol/L), however, did not result in a change in abundances of P27 and BAX proteins. These results indicate the regulatory functions of Se on proliferation and apoptosis of sheep SSC is associated with the P21-mediated P53 signalling pathway. The P27 and BAX proteins have limited functions during the apoptotic process of SSC induced by the relatively greater concentrations of Se., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the effects of different oxygen (O 2 ) concentrations on the growth of mouse spermatogonial stem cells (SSCs) and the possible mechanisms of cell proliferation in vitro. The SSCs from testicular cells were cultured in various O 2 concentrations (1%, 2.5%, 5%, and 20% O 2 ) for 7 days. Colonies of SSCs were identified morphologically and by immunofluorescence. The number of mouse SSC colonies and the area covered by them were measured. Cell cycle progression of the SSCs was analyzed to identify the state of cell proliferation. The effects of O 2 concentrations on the levels of intracellular reactive oxygen species (ROS) and expression of ATP binding cassette subfamily G member 2 (ABCG2) were also analyzed in the SSCs. Following culturing for 7 days, the SSCs were treated with Ko143 (a specific inhibitor of ABCG2) for 1 h, and the ROS level and expression of bcl-2, bax, and p53 were analyzed. The results showed that mouse SSCs formed compact colonies and had unclear borders in different O 2 concentrations for 7 days, and there were no major morphologic differences between the O 2 treatment groups. The expression of the SSC marker, GFR α1 was studied in each O 2 treatment group. The number and area of SSC colonies, and the number of GFR α1 positive cells were the highest in the 2.5% O 2 treatment group. Compared with other O 2 concentrations, the number of cells in G 0 cycle was significantly higher, while the level of intracellular ROS was lower at 1% O 2 . Moreover, the intracellular ROS levels gradually increased with increasing O 2 concentration from 1% to 20%. The expression of ABCG2 in the SSCs cultured at 2.5% O 2 was higher than in the other O 2 groups. Inhibition of ABCG2 increased intracellular ROS generation, and the expression of the pro-apoptotic genes bax and p53, and decreased the expression of the anti-apoptotic gene bcl-2. In conclusion, moderate to low O 2 tension increases ABCG2 expression to maintain mild ROS levels, triggers the expression of the anti-apoptotic genes, suppresses the proapoptotic gene pathway, and further promotes the proliferation of mouse SSCs in vitro., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1., (© 2019 Blackwell Verlag GmbH.)

Since proliferation and differentiation of spermatogonial stem cells (SSCs) in culture system provide successful transplantation in this study, culture of human SSCs was compared to SACS (soft agar culture system), gelatin and control groups. The cells were isolated from seminiferous tubules of non-azoospermia patients (NOA) and cultured in DMEM for 3 weeks. The presence of SSCs in culture system was confirmed by immunocytochemistry of GFR-α1 and ITGα6 antibodies. The proliferated cells were cultured in three mentioned groups in the presence of retinoic acid and Sertoli cells conditioned medium for another 2 weeks. The number of colonies in the SACS group was significantly higher than two other groups. Before 2 weeks of culture, only Oct4 expression was observed in testicular cells (2.32 ± 0.25). After 2 weeks, the expression of Oct4 in the gelatin group was higher than that of the SACS group on day 7. The maximum expression of Stra8 was observed in SACS and gelatin groups after 7 days, but its expression was significantly decreased after 14 days of culture ( p  < .05). The expression of Scp3 and Acrosin genes were higher after 14 days in the SACS group compared to other groups. SACS has positive effects on proliferation and differentiation of hSSCs.

Postnatal female germline stem cells (FGSCs) are a type of germline stem cell with self-renewal ability and the capacity of differentiation toward oocyte. The proliferation, differentiation, and apoptosis of FGSCs have been researched in recent years, but autophagy in FGSCs has not been explored. This study investigated the effects of the small-molecule compound 89 (C89) on FGSCs and the underlying molecular mechanism in vitro. Cytometry, Cell Counting Kit-8 (CCK8), and 5-ethynyl-2'-deoxyuridine (EdU) assay showed that the number, viability, and proliferation of FGSCs were significantly reduced in C89-treated groups (0.5, 1, and 2 µM) compared with controls. C89 had no impact on FGSC apoptosis or differentiation. However, C89 treatment induced the expression of light chain 3 beta II (LC3BII) and reduced the expression of sequestosome-1 (SQSTM1) in FGSCs, indicating that C89 induced FGSC autophagy. To investigate the mechanism of C89-induced FGSC autophagy, RNA-seq technology was used to compare the transcriptome differences between C89-treated FGSCs and controls. Bioinformatics analysis of the sequencing data indicated a potential involvement of the phosphatidylinositol 3 kinase and kinase Akt (PI3K-Akt) pathway in the effects of C89's induction of autophagy in FGSCs. Western blot confirmed that levels of p-PI3K and p-Akt were significantly reduced in the C89- or LY294002 (PI3K inhibitor)-treated groups compared with controls. Moreover, we found cooperative functions of C89 and LY294002 in inducing FGSC autophagy through suppressing the PI3K-Akt pathway. Taken together, this research demonstrates that C89 can reduce the number, viability, and proliferation of FGSCs by inducing autophagy. Furthermore, C89 induced FGSC autophagy by inhibiting the activity of PI3K and Akt. The PI3K-Akt pathway may be a target to regulate FGSC proliferation and death.

The transcription factor positioning in promoter regions relate to gene regulation, and the level of DNA methylation and histone acetylation also impact the promoter activity. In this study, we tested and verified the core promoter region and key transcription factor of Nanos2 which is a male-critical gene in the differentiation of embryonic stem cells to male germ cells, meanwhile, epigenetic effects by mean of 5-Aza-2'-deoxycytidine (5-Azadc) and Trichostin A (TSA) on the activity of Nanos2 promoter were detected. The results reveal that key transcription factor Foxd3 is a negative regulator of Nanos2, which suggests that loss-of-function of Foxd3 causes strong expression of Nanos2 responsive to large amounts of primordial germ cells and spermatogonial stem cells,whereas its overexpression causes the opposite effect. Furthermore, both 5-Azadc and TSA can provoke responses of Nanos2, but the combination effect of the two is better., (© 2018 Wiley Periodicals, Inc.)

Male infertility is due to genetics, hormonal or environmental causes, or is idiopathic. Azoospermia is linked to local testicular microenvironment deregulation, with inflammatory cells present in the 15% of testicular biopsies of infertile patients. As widely reported, spermatogenesis and steroidogenesis are controlled by local immunoregulatory agents produced by immune and nonimmune cells. Moreover IL-6R, TNFR1, Fas and IL-1R are expressed on germ cells, indicating a direct action of pro-inflammatory agents on these cells. Beyond the known function of cytokines and nitric oxide on testicular function at the stable levels present in the normal testis, this review focalises on the effect of pro-inflammatory factors on germ cell survival and death when inflammatory conditions are established in the testis. As no cure for male infertility has been found up to the present, intracytoplasmic sperm injection is the therapeutic option for azoospermic patients who wish to achieve genetic parenthood. Therapies with antioxidant and anti-inflammatory agents in experimental models of testicular damage have been successful. However, clinical implementation is uncertain in cases with a prolonged inflammatory state of the testis. Therapies offering multiple approaches to treat infertility by restoring the spermatogonial stem cell niche and protecting germ cells from apoptosis should be considered., (© 2018 Blackwell Verlag GmbH.)

Diabetes mellitus affects a large number of men of reproductive age and it usually leads to serious reproductive disorders. However, the underlying mechanisms and specific therapies still remain largely unknown. We observed Leydig cell loss in the testes of diabetic mice. Continuous high glycemic status of testes stimulated expression of Caspase12, Grp78, and Chop, the three ERS response factors; this might induce cell cycle arrest and apoptosis of Leydig cells in response to ERS. In these diabetic mouse models, melatonin alleviated apoptosis of testicular stromal cell induced by ERS, and promoted SSCs self-renewal by recovering Leydig cells secretion of CSF1 after 8 weeks of treatment. To explore the relationship between CSF-1 and ERS in Leydig cells, we treated Leydig tumor cell line with an activator Tuniamycin and an inhibitor 4-Phenylbutyrate of ERS. Our data showed that the CSF-1 expression in mouse Leydig cell lines decreased six-fold while reversely increasing five-fold in the 4-Phenylbutyrate-treated group. Thus, melatonin likely alleviates the loss of Leydig cells in diabetic testes and provides a healthier niche for SSCs to self-renew and continually provide healthy sperm for male fertility.

Many of the testicular cancer-survived patients, treated with chemotherapeutic drugs, show infertility, pre and postimplantation loss, and germ cell abnormality. Studies examining the negative effects of chemotherapeutic drugs on testicular germ cells are ongoing; however, information on the stemness properties and proteomic profiles of these cells are lacking. This study investigated the effects of chemotherapeutic drugs etoposide, cisplatin, bleomycin, and their combination (BEP) on the physiology and stem cell activity of mouse germ cells in vitro. Our results showed that treatment with the abovementioned drugs affected germ cell viability and decreased the number of proliferating germ cells significantly at specific concentrations (0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP), which maintained a survival rate of >90%. We also observed a significantly higher percentage of apoptotic cells and alterations in the expression of undifferentiated and differentiated spermatogonia-related genes and marker proteins in germ cells exposed to abovementioned concentrations of the drugs. Next, we performed germ cell transplantation into recipient mice and observed a remarkable reduction in stemness properties of spermatogonial stem cells at these concentrations. Based on these results, we assessed the levels of differentially expressed proteins by performing proteomic analysis. We found that treatment with the abovementioned drugs induced cell damage, oxidative stress, metabolic disruption, and immune deficiency which may promote tumor regeneration, cytotoxicity, infertility, and transgenerational cellular function transmission. Thus, this study provides information about the chemotherapy-induced recurrent destruction and thereby can lead possible changes in medication.

The protein encoded by double sex and mab-3 related transcription factor 1 (Dmrt1) gene contains a double sex/mab-3 domain, which was considered as one of the most conservative structures in sex determination. However, its effect on spermatogenesis of dairy goat spermatogonial stem cells (SSCs) remains to be clarified. For the first time, the roles of Dmrt1 in spermatogenesis of livestock are highlighted. Here, we investigated the expression pattern of Dmrt1 in the testes of dairy goats. Dmrt1 primarily located in undifferentiated SSCs. Moreover, Dmrt1 enhanced differentiation and proliferation of mGSCs. On the contrary, the level of meiosis was down-regulated, as Dmrt1 determines whether SSCs undergo mitosis and spermatogonial differentiation or meiosis. In the busulfan-treated mice testes, Dmrt1 repair germ cell damage was emphasized as well. Our results exposed that Dmrt1 maintenance mGSCs in two ways: facilitating proliferation and self-renewal of SSCs; and reducing the inflammatory response caused by reproductive injury. These findings identify a central role for Dmrt1 in controlling population stability and injury restoring of SSCs., (© 2017 Wiley Periodicals, Inc.)

Spermatogenesis requires retinoic acid (RA) induction of the undifferentiated to differentiating transition in transit amplifying (TA) progenitor spermatogonia, whereas continuity of the spermatogenic lineage relies on the RA response being suppressed in spermatogonial stem cells (SSCs). Here, we discovered that, in mouse testes, both spermatogonial populations possess intrinsic RA-response machinery and exhibit hallmarks of the differentiating transition following direct exposure to RA, including loss of SSC regenerative capacity. We determined that SSCs are only resistant to RA-driven differentiation when situated in the normal topological organization of the testis. Furthermore, we show that the soma is instrumental in "priming" TA progenitors for RA-induced differentiation through elevated RA receptor expression. Collectively, these findings indicate that SSCs and TA progenitor spermatogonia inhabit disparate niche microenvironments within seminiferous tubules that are critical for mediating extrinsic cues that drive fate decisions., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Given the potential biological functions of spermatogonial stem cells (SSCs) in spermatogenesis and in delivering parental genetic information to the next generation, how these cells respond to environmental toxins and carcinogens should be investigated. We examined the toxic effect of hexavalent chromium (Cr(VI)) on global histone modifications and apoptotic signaling pathways in SSCs. We determined the effect of melatonin, one of the most powerful endogenous free radical scavengers and wide-spectrum antioxidants, in protecting SSCs from Cr(VI)-induced apoptosis and global histone modification by Western blot analysis. In addition, we examined the in vivo effect of melatonin on Cr(VI)-induced histological changes of seminiferous tubules in mouse testes. We also evaluated the fertility of male mice by monitoring litter size following intraperitoneal injection of these chemicals. Our study demonstrated the Cr(VI)-induced global increases in H3K9me3 and H3K27me3 and activated the apoptotic signaling pathway. Pretreatment of SSCs with melatonin alleviated Cr(VI)-induced apoptosis and the global increase of H3K9me3. Exposure to melatonin also attenuated the Cr(VI)-induced increase of the abundance of histone methyltransferase ESET. Furthermore, exogenous administration of melatonin protected mice against Cr(VI)-induced changes in testicular histology and germ cell apoptosis, which helped maintain normal spermatogenesis and male fertility. Our study revealed a potential new therapeutic approach for male reproductive injury caused by Cr(VI)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Melatonin is a pleiotropic hormone with powerful antioxidant activity both in vivo and in vitro. The present study aimed to investigate the effects of melatonin on the proliferation efficiency of neonatal mouse spermatogonial stem cells (SSCs) using a three-dimensional soft agar culture system (SACS) which has the capacity to induce development of SSCs similar to in vivo conditions., Methods: SSCs were isolated from testes of neonate mice and their purities were assessed by flow cytometry using PLZF antibody. Isolated testicular cells were cultured in the upper layer of the SACS in αMEM medium in the absence or presence of melatonin extract for 4 weeks., Results: The identity of colonies was confirmed by alkaline phosphatase staining and immunocytochemistry using PLZF and α6 integrin antibodies. The number and diameter of colonies of SSCs in the upper layer were evaluated at days 14 and 28 of culture. The number and diameter of colonies of SSCs were significantly higher in the melatonin group compared with the control group. The levels of expression of ID-4 and Plzf, unlike c-kit, were significantly higher in the melatonin group than in the control group., Conclusions: Results of the present study show that supplementation of the culture medium (SACS) with 100 μM melatonin significantly decreased reactive oxygen species (ROS) production in the treated group compared with the control group, and increased SSC proliferation.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, which is dependent on the ability to self-renew and differentiation. Controlling self-renewal and differentiation of SSCs could apply to treatment of disease such as male infertility. Recently, in the field of stem cell research, it was demonstrated that effective increase in stem cell activity can be achieved by using growth factors derived from plant extracts. In this study, our aim is to investigate components from natural plant to improve the self-renewal of SSCs. To find the components, germ cells were cultured with comprehensive natural plant extracts, and then the more pure fraction, and finally single compound at different concentrations. As a result, we found 5H-purin-6-amine at 1 µg/mL, originated from Sedum sarmentosum, was a very effective compound induced SSCs proliferation. Our data showed that germ cells cultured with 5H-purin-6-amine could maintain their stable characteristics. Furthermore, transplantation results demonstrated that 5H-purin-6-amine at 1 µg/mL increased the activity of SSCs, indicating the compound could increase true SSC concentration within germ cells to 1.96-fold. These findings would be contributed to improve further reproductive research and treat male infertility by using natural plant extracts.

Destruction of spermatogonial stem cells (SSCs) along the chemotherapy and radiotherapy is one of the side effects of cancer treatments that lead to infertility. In vitro propagation of hSSCs is necessary to obtain an adequate number of cells for successful transplantation. In this study, hSSCs were isolated from testis biopsies of the patients with maturation arrest and proliferated in DMEM in the presence of LIF and bFGF for 5 weeks. The various types of human spermatogonia were identified in culture system and compared with testis tissue using morphological criteria at the ultrastructural level. The results showed that although many various types of spermatogonia were identified, but no remarkable difference was observed between spermatogonial cells in culture system and testis tissue. Electron and light microscopic studies of hSSC colonies did not show differentiated SSCs in the culture system. The results also showed that probably the suitable time for transplanting of SSCs in recipient testis is 2-3 weeks after culture. Because apoptosis which may affect the development of germ cells has not started in colony cells at this time and the population of apoptotic cells are low., (© 2016 Blackwell Verlag GmbH.)

The third-generation aromatase inhibitors (AIs), anastrozole, letrozole and exemestane, are highly effective for the treatment of estrogen receptor-positive breast cancer in postmenopausal women. AIs inhibit the aromatase (CYP19A1)-mediated production of estrogens. Most patients taking AIs achieve undetectable blood estrogen concentrations resulting in drug efficacy with tolerable side effects. However, some patients have suboptimal outcomes, which may be due, in part, to inherited germline genetic variants. This review summarizes published germline genetic associations with AI treatment outcomes including systemic AI concentrations, estrogenic response to AIs, AI treatment efficacy and AI treatment toxicities. Significant associations are highlighted with commentary about prioritization for future validation to identify pharmacogenetic predictors of AI treatment outcomes that can be used to inform personalized treatment decisions in patients with estrogen receptor-positive breast cancer.

Aim: This systematic review provides an overview of publications concerning pharmacogenetic research in pediatric patients with medulloblastoma and low-grade glioma., Materials & Methods: Three electronic databases searches including a manual search were performed to identify studies investigating potential interactions between germline variants and chemotherapy efficacy and toxicity., Results: Out of 3570 citations, 21 studies were included. Outcomes include overall survival, progression-free survival and treatment-related adverse events (n = 5), cisplatin-induced ototoxicity (n = 13) and vincristine-induced neurotoxicity (n = 3)., Conclusion: This review shows that the number of pharmacogenetic studies in well-defined pediatric brain tumor cohorts is poor and studies often report conflicting results. Large-scale international collaborations allowing analysis of sufficiently sized cohorts are therefore very important for the future of personalized medicine in brain tumors.

Restricted availability of retinoic acid (RA) in the testicular milieu regulates transcriptional activity of c-kit (KIT, CD117), which aids in the determination of spermatogonial stem-cell differentiation. The effect of RA on c-kit has been reported previously, but its mode of genomic action remains unresolved. We studied the molecular machinery guiding RA responsiveness to the c-kit gene using spermatogonial stem-cell line C18-4 and primary spermatogonial cells. A novel retinoic acid response element (RARE) positioned at -989 nucleotides upstream of the transcription start site (TSS) was identified, providing a binding site for a dimeric RA receptor (i.e. retinoic acid receptor gamma (RARγ) and retinoic X receptor). RA treatment influenced c-kit promoter activity, along with endogenous c-kit expression in C18-4 cells. A comprehensive promoter deletion assay using the pGL3B reporter system characterised the region spanning -271bp and -1011bp upstream of the TSS, which function as minimal promoter and maximal promoter, respectively. In silico analysis predicted that the region -1011 to +58bp comprised the distal enhancer RARE and activators such as spleen focus forming virus proviral integration oncogene (SPFI1) (PU.1), specificity protein 1 (SP1) and four E26 transformation-specific (ETS) tandem binding sites at the proximal region. Gel retardation and chromatin immunoprecipitation (ChIP) assays showed binding for RARγ, PU.1 and SP1 to the predicted consensus binding sequences, whereas GABPα occupied only two out of four ETS binding sites within the c-kit promoter region. We propose that for RA response, an enhanceosome is orchestrated through scaffolding of a CREB-binding protein (CBP)/p300 molecule between RARE and elements in the proximal promoter region, controlling germ-line expression of the c-kit gene. This study outlines the fundamental role played by RARγ, along with other non-RAR transcription factors (PU.1, SP1 and GABPα), in the regulation of c-kit expression in spermatogonial stem cells in response to RA.

The male germinal lineage, which is defined as unipotent, produces sperm through spermatogenesis. However, embryonic primordial germ cells and postnatal spermatogonial stem cells (SSCs) can change their fate and convert to pluripotency in culture when they are not controlled by the testicular microenvironment. The mechanisms underlying these reprogramming processes are poorly understood. Testicular germ cell tumors, including teratoma, share some molecular characteristics with pluripotent cells, suggesting that cancer could result from an abnormal differentiation of primordial germ cells or from an abnormal conversion of SCCs to pluripotency in the testis. Here, we investigated whether the somatic reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM) could play a role in SSCs reprogramming and induce pluripotency using a doxycycline-inducible transgenic Col1a1-4F2A-OSKM mouse model. We showed that, in contrast to somatic cells, SSCs from adult mice are resistant to this reprogramming strategy, even in combination with small molecules, hypoxia, or p53 deficiency, which were previously described to favour the conversion of somatic cells to pluripotency. This finding suggests that adult SSCs have developed specific mechanisms to repress reprogramming by OSKM factors, contributing to circumvent testicular cancer initiation events.

The culture of spermatogonial cells for future transplantation, based on the specific biology of these cells is important and necessary. Recently, the use of scaffolds especially alginate for culturing stem cells has been the focus of many researchers. The aim of this study was to evaluate the cytotoxicity of alginate hydrogels to cultures of type A spermatogonial stem cells. Spermatogonial stem cells of 6day-old immature mice were isolated by surgery; thereafter, the cells were purified by MACS using antibodies against thy-1 and C-kit and cultured on a layer of laminin. After purification, spermatogonial stem cells were encapsulated in alginate hydrogels. After one month of encapsulation and culture in DMEM culture medium containing 10ng/ml GDNF, cells were removed from hydrogel and were examined for viability, cell morphology and structure, cytotoxicity and expression of apoptosis genes Fas, P53, Bax, Bcl2, Caspase3 by staining with trypan blue, scanning electron microscopy, LDH test, and Real time PCR, respectively. The encapsulation did not change the morphology and viability of spermatogonial stem cells. Investigations showed that spermatogonial stem cells preserve by the high viability (74.08%) and cytotoxicity of alginate hydrogel was estimated to be 5%. Expression of Fas gene increased in main group compared with the control group, and expression of Bax and P53 was reduced in main group compared with the control group. Expression of Bcl2 and Caspase3 genes did not show any significant difference between the main group and the control group. Considering the lack of cytotoxicity and antioxidant properties of alginate hydrogel scaffold and high viability of cells, this three-dimensional scaffold is applicable for culturing and encapsulation of spermatogonial stem cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The decline of quantity and quality of sperm are correlated with the increasing age and some anti-cancer compounds such as busulfan. Previous studies have shown that Resveratrol (Res) inhibits tumorigenesis and metastasis of many cancers including mammary tumor, prostate and pancreatic cancers. It acts as anti-age in mouse and human, however, little is known about its protective effect on aged spermatogonial stem cells (SSCs). Here, we investigated the effects of Res in vitro on SSCs using C18-4 cells and in vivo in busulfan-induced azoospermia mice model. The results showed that Res at different concentrations had different effects on C18-4 cells. Treatment with 2 μM of Res promotes cell proliferation and inhibits apoptosis, but stimulates apoptosis with a higher concentration (20 μM) in C18-4 cells. Using busulfan-induced infertility mice model, we demonstrated that Res (30 mg/kg/d and 100 mg/kg/d) clearly ameliorated SSCs loss to recover the spermatogenesis. Taken together, our data suggest that Res might be an approach for therapeutic intervention to promote SSC proliferation and cease SSCs loss in azoospermia mice model induced by busulfan.

The present study analyzed the dose-dependent cyto- and genotoxicity of graphene oxide and reduced graphene oxide on spermatogonial stem cells (SSCs) for the first time. The results showed that graphene oxide significantly increased oxidative stress at concentrations of 100 and 400μg/ml, while low concentrations did not have a significant effect. In addition, according to the MTT assay, the cell number decreased in high-concentration (100 and 400μg/ml) graphene oxide-treated samples compared to untreated cells. However, a reduced graphene-treated sample demonstrated a significant increase in cell number. Moreover, microscopic analysis found high concentrations of graphene nanosheets in cell culture medium that reduced the number of colonies and colony forming cells. We conclude that a high concentration of graphene can be toxic to SSCs. However, such toxicity can be reduced by the surface modification of graphene nanomaterials., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Several different strategies have been adopted in attempt to recover from chemotherapy-damaged spermatogenesis that is often seen in oncologic patients. In this study, we have evaluated the impact of short period of exposure to busulphan on the haemogram and seminiferous epithelium of adult rats, focusing on spermatogonial depletion and Sertoli cell (SC) integrity. We then examined whether vitamin B 12 supplementation improves the haematological parameters and spermatogonia number. The animals received 10 mg/kg of busulphan (BuG) or busulfan+vitamin B 12 (Bu/B 12 G) on the first and fourth days of treatment. In H.E.-stained testicular sections, the areas of the seminiferous tubule (ST) and seminiferous epithelium were measured. The number of spermatogonia in H.E-stained and PCNA-immunolabelled testicular sections was quantified. The frequency of tubules with abnormal SC nuclei or TUNEL-positive SC was evaluated. Vimentin immunofluorescence in ST was also evaluated. In BuG and Bu/B 12 G, the animals showed leukopenia and thrombocytopenia, but the body weight reduced only in BuG. The areas of ST and seminiferous epithelium decreased in Bu/B 12 G and BuG. In BuG, the number of H.E.-stained and PCNA-immunolabelled spermatogonia reduced significantly. The frequency of tubules containing abnormal SC nuclei and TUNEL-positive SC increased and the vimentin immunoexpression pattern changed. In Bu/B 12 G, the number of H.E.-stained or PCNA-immunolabelled spermatogonia increased fourfold in comparison with BuG. The structural changes in ST after 6 days of busulphan exposure may be associated with the potential effect of this anti-neoplastic agent on SC. The increased number of spermatogonia in the busulphan-treated animals receiving vitamin B 12 indicates that this vitamin can be an adjuvant therapy to improve the fertility in male cancer patients., (© 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology.)

Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is one of the major helpful chemotherapeutic agents for treatment of this cancer. In addition, exposure of testes cancer cells to cisplatin could potentially eliminate tumour cells from germ cells in patients. The aim of this study was to evaluate the effect of cisplatin on viability of mouse acute lymphoblastic leukaemia cell line (EL-4) and neonatal mouse spermatogonial cells in vitro. In this study, the isolated spermatogonial stem cells (SSC) and EL-4 were divided into six groups including control (received medium), sham (received DMSO in medium) and experimental groups which received different doses of cisplatin (0.5, 5, 10 and 15 μg ml(-1) ). Cells viability was evaluated with MTT assay. The identity of the cultured cells was confirmed by the expression of specific markers. Our finding showed that viability of both SSC and EL-4 cells was reduced with the dose of 15 μg/ml when compared to the control group (P ≤ 0.05). Also, the differences between the IC50 in doses 10 and 15 μg/ml at different time were significant (P ≤ 0.05). The number of TUNEL-positive cells was increased, and the BAX and caspase-3 expressions were upregulated in EL4 cells for group that received an effective dose of cisplatin). In conclusion, despite the dramatic effects of cisplatin on both cells, spermatogonial stem cells could form colony in culture., (© 2015 Blackwell Verlag GmbH.)

Tri-ortho-cresyl phosphate (TOCP) has been widely used as plasticizers, plastic softeners, and flame-retardants in industry, which can be metabolized to High-toxic saligenin cyclic-o-tolyl phosphate (SCOTP). Our previous results found that TOCP could disrupt the seminiferous epithelium in the testis and induce autophagy of rat spermatogonial stem cells. Little is known about the toxic effect of SCOTP on rat spermatogonial stem cells. The present study showed that SCOTP decreased viability of rat spermatogonial stem cells in a dose-dependent manner. Both LC3-II and the ratio of LC3-II/LC3-I were significantly increased; autophagy proteins atg5 and Beclin 1 were also markedly increased after treatment with SCOTP, indicating SCOTP could induce autophagy of the cells. Ultrastructural observation under the transmission electron microscopy (TEM) indicated that there were autophagic vacuoles in the cytoplasm in the SCOTP-treated cells. However, cell cycle arrest was not observed by flow cytometry; and the mRNA levels of p21, p27, p53 and cyclin D1 in the cells were also not affected by SCOTP. Meanwhile, SCOTP didn't induce apoptosis of the cells. In summary, we showed that SCOTP could induce autophagy of rat spermatogonial stem cells, without affecting cell cycle and apoptosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To investigate the effects of collagen and growth factors on in vitro proliferation of human spermatogonial stem cells obtained from patients with non-obstructive azoospermia., Methods: The experimental cross-sectional study was conducted from February 2013 to April 2015 after obtaining approval from the ethics committee of Ahvaz Jundishapur University of Medical Sciences, Iran. Testicular sperm extractions of non-obstructive azoospermic patients were obtained from the Clinical Urology and Embryology, In Vitro Fertilization Department of Imam Khomeini Hospital. Spermatogonial stem cells and Sertoli cells, obtained from human testis biopsies by a two-step enzymatic digestion method, were purified using fluorescence- activated cell-sorting and daturastramonium-lectin, and were cultured separately. To investigate a more direct influential factor on colony formation, one control and two experimental groups were formed. Group 1 acted as the control in which spermatogonial stem cells were co-cultured with Sertoli cells alone. In group 2 they were co-cultured with Sertoli cells and growth factors such as leukaemia inhibitory factor, epidermal growth factor and glial cell-derived neurotrophic factor, and in group 3 with Sertoli cells along with growth factors in the presence of collagen-coated dishes. Number and diameter of the colonies were evaluated after 7 weeks., Results: Specimens obtained related to 21 patients. Number and diameter of the colonies in group 3 (18±2.6 and 276.6±45.5) were significantly more than both groups 1 (3.5±1 and D1:81.6±12) and group 2(11±2.2 and 165.2±32.5) (p<0.05 each). Also, the number and diameter of colony in group 2 were significantly better than the control group (p<0.05).Expression profile of the VASA, promyelocytic leukaemia zinc-finger (PLZF), Octamer-binding transcription factor 4 (OCT4) and integrin a6 (INTGa6) were detected in all groups. Based on cytochemical findings, OCT4 was expressed in the colonies of all three groups., Conclusions: According to positive effects of collagen and growth factors on the colonisation of spermatogonial stem cells, it seems that using the cells may lead to better colonisation of this type of stem cells.