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Serum is a common supplement that is widely used to protect various cells and tissues from cryopreservation because it provides the necessary active components for cell growth and maintenance. In this study, we compared the effects of newborn calf serum (NCS) and fetal bovine serum (FBS) on the cryopreservation of mouse spermatogonial stem cells (SSCs). The isolated SSCs were cryopreserved in two groups: freezing medium that contained 10% DMSO (dimethyl sulfoxide) and 10% FBS in DMEM (Dulbecco's Modified Eagle's Medium) (group 1) and freezing medium that contained 10% DMSO and 10% NCS in DMEM (group 2). Real-time PCR was performed for stemness gene expression. The SSCs' viability was performed by trypan blue. We observed that the SSCs had increased viability in the NCS-freeze/thaw group (87.82%) compared to the FBS-freeze/thaw group (79.83%), but this increase was not statistically significant (P < 0.105). Promyelocytic leukemia zinc finger (Plzf) and Lin28 gene expression levels in the NCS-frozen/thawed SSCs were not significantly different compared to the FBS-frozen/thawed SSCs; however, Nanog gene expression increased considerably, and Dazl gene expression decreased significantly. The results in this study demonstrated that the presence of NCS in a solution of cryopreserved SSCs increased their viability after freeze/thawing and might promote the proliferation of cultivated SSCs in vitro by increasing the relative expression of Nanog.

In testis, a rare undifferentiated germ cell population with the capacity to regenerate robustly and support spermatogenesis, is defined as spermatogonial progenitor cells (SPCs) population. As a widely used drug for tumor therapy or bone marrow transplantation, busulfan has a severe side effect on SPCs population and causes a consequent infertility. Recently, accumulating evidence revealed the protective role of autophagy in stem cell maintenance under exogenous stress. To better understand the role of autophagy in SPCs fates, we investigated the potential function of autophagy in SPCs under busulfan stress, and found that treatment of busulfan induced the formation of autophagic vesicles and autophagosomes in mouse SPCs. Subsequently, a connection of autophagy and SPCs maintenance and survival was demonstrated in a dose-dependent manner. Moreover, mTOR was identified as an essential factor for autophagy in SPCs with a complicated mechanism: (1) mTOR is phosphorylated by AKT to activate its target genes, p70s6 kinase, resulting in the inhibition of autophagy during short-term busulfan treatment. (2) mTOR mediates autophagy with p53 together, to regulate the fate of SPCs. Collectively, observations from this study indicate that moderate autophagy effectively protects SPCs from the stress of chemotherapy, which may provide an important hint for fertility protection in clinic.

Sericin is a natural protein component of silks of silkworm and has potential utility in multiple areas such as pharmacological, cosmetics, and biotechnological industries. However, the understanding of its toxicological safety is still limited. This study evaluated the safety of water-extract sericin from silkworm ( Bombyx mori ) cocoons using different model approaches, including three genotoxicity studies (the bacterial reverse mutation test, the mammalian erythrocyte micronucleus test, and the mouse spermatogonia chromosomal aberration test) and a 90-day subchronic toxicity study in Sprague-Dawley (SD) rats. The results of this study showed that water-extract sericin was nonmutagenic and nongenotoxic both in vitro and in vivo . Sericin did not induce significant changes in the body and organ weight, food intake, blood hematology and serum biochemistry, urine index, and histopathology in rats. The NOAEL of sericin was determined to be 1 g/kg/day for male and female rats. These results indicated that water-extract sericin was of low toxicity in the experimental conditions of the current study and had the potential for application in food-related products., Competing Interests: No potential conflict of interest was reported by the authors., (Copyright © 2020 Huiyan Qin et al.)

Sirolimus, also known as rapamycin, and its closely related rapamycin analog (rapalog) Everolimus inhibit "mammalian target of rapamycin complex 1" (mTORC1), whose activity is required for spermatogenesis. Everolimus is Food and Drug Administration approved for treating human patients to slow growth of aggressive cancers and preventing organ transplant rejection. Here, we test the hypothesis that rapalog inhibition of mTORC1 activity has a negative, but reversible, impact upon spermatogenesis. Juvenile (P20) or adult (P>60) mice received daily injections of sirolimus or Everolimus for 30 days, and tissues were examined at completion of treatment or following a recovery period. Rapalog treatments reduced body and testis weights, testis weight/body weight ratios, cauda epididymal sperm counts, and seminal vesicle weights in animals of both ages. Following rapalog treatment, numbers of differentiating spermatogonia were reduced, with concomitant increases in the ratio of undifferentiated spermatogonia to total number of remaining germ cells. To determine if even low doses of Everolimus can inhibit spermatogenesis, an additional group of adult mice received a dose of Everolimus ∼6-fold lower than a human clinical dose used to treat cancer. In these animals, only testis weights, testis weight/body weight ratios, and tubule diameters were reduced. Return to control values following a recovery period was variable for each of the measured parameters and was duration and dose dependent. Together, these data indicate rapalogs exerted a dose-dependent restriction on overall growth of juvenile and adult mice and negative impact upon spermatogenesis that were largely reversed; following treatment cessation, males from all treatment groups were able to sire offspring., (© 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.)

Extensive application of zinc oxide (ZnO) nanoparticles (NPs) in everyday life results in increased exposure to these NPs. Spermatogonial stem cells (SSCs) guarantee sperm production throughout the male reproductive life by providing a balance between self-renewal and differentiation. We used an in vitro platform to investigate the ZnO NPs effects on SSCs. We successfully synthesized ZnO NPs. In order to investigate these NPs, we isolated SSCs from mouse testes and cultured them in vitro. Our results confirmed the uptake of ZnO NPs by the cultured SSCs. We observed a dose- and time-dependent decrease in SSC viability. Both spherical and nanosheet ZnO NPs had the same cytotoxic effects on the SSCs, irrespective of their shapes. Moreover, we have shown that short time (one day) exposure of SSCs to a low concentration of ZnO NPs (10 μg/mL) promoted expressions of specific genes (Plzf, Gfr α1 and Bcl6b) for SSC self-renewal and differentiation genes (Vasa, Dazl, C-kit and Sycp3) expressed by spermatogonia during spermatogenesis. Our study provides the first insight into ZnO NPs function in SSCs and suggests a new function for ZnO NPs in the male reproductive system. We demonstrated that ZnO NPs might promote spermatogenesis via upregulation of gene expression related to SSC self-renewal and differentiation at low concentrations. Additional research should clarify the possible effect of ZnO NPs on the SSC genome and its effects on human SSCs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Copper is a widespread heavy metal in environment and has toxic effects when exposed. However, study of copper-induced male reproductive toxicity is still insufficient to report, and the underlying mechanisms are unknown. Keeping in view, RNA-Seq and metabolomic were performed to identify metabolic pathways that were distressed in mouse spermatogonia with the effect of copper sulfate, and the integrated analysis of the mechanism of copper administered GC-1 cells from metabolomic and transcriptomic data. Our results demonstrated that many genes and metabolites were regulated in the copper sulfate-treated cells. The differential metabolites analysis showed that 49 and 127 metabolites were significantly different in ESI+ and ESI- mode, respectively. Meanwhile, a total of 2813 genes were up-regulated and 2488 genes were down-regulated in the treatment groups compared to those in the control groups. Interestingly, ophthalmic acid and gamma glutamylleucine were markedly increased by copper treatment in two modes. By integrating with transcriptomic and metabolomic data, we revealed that 37 and 22 most related pathways were over-enriched in ESI+ and ESI- mode, respectively. Whereas, amino acid biosynthesis and metabolism play essential role in the potential relationship between DEGs and metabolites, which suggests that amino acid biosynthesis and metabolism may be the major metabolic pathways disturbed by copper in GC-1 cells. This study provides important clues and evidence for understanding the mechanisms responsible for copper-induced male spermatogenesis toxicity, and useful biomarkers indicative of copper exposure could be discovered from present study., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Previous studies have demonstrated that Zearalenone (ZEA) affects not only maternal reproductive function but also that of the offspring. However, the transgenerational toxic effects of ZEA on the spermatogonia of male F1 mice are not clear. The present study was thus designed to determine whether the fertility of male F1 mice was affected following exposure of F0 pregnant mice to ZEA. In present study, 32 pregnant female mice were divided into 4 groups and exposed to ZEA of 0, 2.5 and 5.0 mg/kg, respectively, and the testis development and reproductive performance of 96 male F1 mice were analyzed. The results demonstrated that the F0 pregnant mice treated with ZEA resulted in increased anogenital distances in the newborn male F1 mice. Moreover, ZEA caused abnormal vacuole structures and loose connections in the testes of male F1 offspring, compared with the controls. Further ultramicrostructural analysis showed that the mitochondria appeared to be vacuolated with ablated membranes and cristae, and this was accompanied by the presence of large lipid droplets in the spermatogonia. Further, the semen quality and sperm counts declined significantly, and increased malformation rates and decreased testosterone levels were observed in the male F1 offspring from experimental groups. Our results reveal the toxic effects of ZEA on F0 pregnant mice is transgenerational, and affects the fertility of male F1 mice by damaging the spermatogonial cells. This offers a new viewpoint of ZEA-induced reproductive toxicity in male animals and provides a new potential direction for the treatment and prevention of ZEA-induced cytotoxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Inc.)

Bisphenol-A (BPA) exposure in an adult male can affect the reproductive system, which may also adversely affect the next generation. However, there is a lack of comprehensive data on the BPA-induced disruption of the association and functional characteristics of the testicular germ cells, which the present study sought to investigate. Adult male mice were administered BPA doses by gavage for six consecutive weeks and allowed to breed, producing generations F1-F4. Testis samples from each generation were evaluated for several parameters, including abnormal structure, alterations in germ cell proportions, apoptosis, and loss of functional properties of spermatogonial stem cells (SSCs). We observed that at the lowest-observed-adverse-effect level (LOAEL) dose, the testicular abnormalities and alterations in seminiferous epithelium staging persisted in F0-F2 generations, although a reduced total spermatogonia count was found only in F0. However, abnormalities in the proportions of germ cells were observed until F2. Exposure of the male mice (F0) to BPA alters the morphology of the testis along with the association of germ cells and stemness properties of SSCs, with the effects persisting up to F2. Therefore, we conclude that BPA induces physiological and functional disruption in male germ cells, which may lead to reproductive health issues in the next generation.

As the male reproductive organ, the main task of the testis is the production of fertile, haploid spermatozoa. This process, named spermatogenesis, starts with spermatogonial stem cells, which undergo a species-specific number of mitotic divisions until starting meiosis and further morphological maturation. The pituitary gonadotropins, luteinizing hormone, and follicle stimulating hormone, are indispensable for vertebrate spermatogenesis, but we are still far from fully understanding the complex regulatory networks involved in this process. Therefore, we developed an ex vivo testis cultivation system which allows evaluating the occurring changes in histology and gene expression. The experimental circulatory flow-through setup described in this work provides the possibility to study the function of the male tilapia gonads on a cellular and transcriptional level for at least 7 days. After 1 week of culture, tilapia testis slices kept their structure and all stages of spermatogenesis could be detected histologically. Without pituitary extract (tilPE) however, fibrotic structures appeared, whereas addition of tilPE preserved spermatogenic cysts and somatic interstitium completely. We could show that tilPE has a stimulatory effect on spermatogonia proliferation in our culture system. In the presence of tilPE or hCG, the gene expression of steroidogenesis related genes ( cyp11b2 and stAR2 ) were notably increased. Other testicular genes like piwil1, amh , or dmrt1 were not expressed differentially in the presence or absence of gonadotropins or gonadotropin containing tilPE. We established a suitable system for studying tilapia spermatogenesis ex vivo with promise for future applications., (Copyright © 2020 Thönnes, Vogt, Steinborn, Hausken, Levavi-Sivan, Froschauer and Pfennig.)

Cryopreservation of spermatogonial stem cells (SSCs) is essential for preservation of valuable livestock and clinical applications. Although optimal equilibration of cryoprotectants has emerged as a promising approach to improve the cryopreservation efficiency, standard equilibration protocols have not yet been considered in cryopreservation of SSCs. This study aimed to establish a standard equilibration protocol to improve the cryopreservation efficiency of murine germ cells enriched for SSCs. After time- and temperature-dependent equilibration, the germ cells were cryopreserved with 10% dimethyl sulfoxide (DMSO) and 200 mM trehalose. To investigate cryopreservation efficiency at different equilibration conditions, the survival and proliferation rates were assessed after thawing, and then, cytotoxicity and intracellular trehalose quantification were analyzed. Protein (PLZF, GFRα1, VASA, and c-Kit) and gene ( Bcl6b , Erm , Dazl , and Sycp1 ) expression was determined using immunofluorescence and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. The proliferation rate increased significantly following equilibration for 20 minutes at room temperature (RT; 163.7% ± 24.6%) or 4°C (269.0% ± 18.2%). Cytotoxicity was reduced in 10% DMSO with 200 mM trehalose compared with that of 10% DMSO alone. Also, intracellular trehalose was observed after equilibration. The immunofluorescence and RT-qPCR data revealed that the murine germ cells enriched for SSCs retained their self-renewal ability after cryopreservation following equilibration. The most effective protocol was equilibration with 10% DMSO and 200 mM trehalose for 20 minutes at RT or 4°C, which is due to synergistic effects of intracellular and extracellular trehalose. This improved methodology will contribute toward the development of a standardized freezing protocol for murine germ cells enriched for SSCs and thereby expand their application in various fields.

The aim of this study was to investigate carnitine action against negative effects of etoposide on stem/progenitor spermatogonia and on sperm production. Carnitine (250 mg/kg body weight/day) and etoposide (5 mg/kg body weight/day) were administered from 25-days postpartum to 32-days postpartum. Testes were collected at 32-days postpartum, 64-days postpartum, and 127-days postpartum, and submitted to the immuno-labeling of UTF1, SOX2, and PLZF proteins to identify undifferentiated spermatogonia populations. At 127-days postpartum, sperm were collected for analysis. Carnitine+etoposide group showed a higher numerical density of spermatogonia labeled for all studied proteins at 64-days postpartum (critical age) compared to the etoposide group. Moreover, there was an improvement of spermatic parameters and sperm DNA integrity in rats of the carnitine+etoposide group in comparison with rats of the etoposide group. The results suggest that carnitine improves the self-renewal of undifferentiated spermatogonia and promotes a partial protection on them, alleviating the etoposide harmful late effects and leading to an enhancement of the sperm parameters in adulthood.

Cell differentiation during spermatogenesis requires a proper actin dynamic, regulated by several proteins, including formins. Disheveled-Associated-Activator of Morphogenesis1 (DAAM1) belongs to the formins and promotes actin polymerization. Our results showed that oral D-Aspartate (D-Asp) administration, an excitatory amino acid, increased DAAM1 protein levels in germ cells cytoplasm of rat testis. Interestingly, after the treatment, DAAM1 also localized in rat spermatogonia (SPG) and mouse GC-1 cells nuclei. We provided bioinformatic evidence that DAAM1 sequence has two predicted NLS, supporting its nuclear localization. The data also suggested a role of D-Asp in promoting DAAM1 shuttling to the nuclear compartment of those proliferative cells. In addition, the proliferative action induced by D-Asp is confirmed by the increased levels of PCNA, a protein expressed in the nucleus of cells in the S phase and p-H3, a histone crucial for chromatin condensation during mitosis and meiosis. In conclusion, we demonstrated, for the first time, an increased DAAM1 protein levels following D-Asp treatment in rat testis and also its localization in the nucleus of rat SPG and in mouse GC-1 cells. Our results suggest an assumed role for this formin as a regulator of actin dynamics in both cytoplasm and nuclei of the germ cells.

Zinc Oxide Nanoparticles (ZnO NPs) are a type of metal oxide nanoparticle with an extensive use in biomedicine. Several studies have focused on the biosafety of ZnO NPs, since their size and surface area favor entrance and accumulation in the body, which can induce toxic effects. In previous studies, ZnO NPs have been identified as a dose- and time-dependent cytotoxic inducer in testis and male germ cells. However, the consequences for the first cell stage of spermatogenesis, spermatogonia, have never been evaluated. Therefore, the aim of the present work is to evaluate in vitro the cytotoxic effects of ZnO NPs in spermatogonia cells, focusing on changes in cytoskeleton and nucleoskeleton. For that purpose, GC-1 cell line derived from mouse testes was selected as a model of spermatogenesis. These cells were treated with different doses of ZnO NPs for 6 h and 12 h. The impact of GC-1 cells exposure to ZnO NPs on cell viability, cell damage, and cytoskeleton and nucleoskeleton dynamics was assessed. Our results clearly indicate that higher concentrations of ZnO NPs have a cytotoxic effect in GC-1 cells, leading to an increase of intracellular Reactive Oxygen Species (ROS) levels, DNA damage, cytoskeleton and nucleoskeleton dynamics alterations, and consequently cell death. In conclusion, it is here reported for the first time that ZnO NPs induce cytotoxic effects, including changes in cytoskeleton and nucleoskeleton in mouse spermatogonia cells, which may compromise the progression of spermatogenesis in a time- and dose-dependent manner., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Fertility preservation is a common concern for male cancer survivors of reproductive age. However, except for testicular tissue cryopreservation, which is not very effective, there is no feasible and precise therapy capable of protecting spermatogenesis for prepubertal boys before or during gonadotoxic treatment. This study aims to investigate the effects of inhibiting necroptosis of spermatogonial stem cell (SSC) in fertility preservation. Male mice 12 weeks of age were used to establish gonadotoxicity with two intraperitoneal injections of busulfan at a total dose of 40 mg kg -1 . The mouse model and the primary cultured mouse SSCs were used to characterize the relationship between necroptosis of SSC and gonadotoxicity. Meanwhile, the effects of an inhibitor of necroptosis pathway, RIPA-56, were observed on day 36 in the mouse model of busulfan-induced gonadotoxicity. We found that the number of SSCs was decreased, but the level of necroptosis was upregulated on day 18 after busulfan treatment in testes from gonadotoxic mice. Further experiments in primary cultured cells showed that the necroptosis caused cell death in busulfan-treated SSCs and could be inhibited by RIPA-56. After suppressing the necroptosis of SSCs, the busulfan-induced mice had a decreased loss of spermatogenic cells as shown by histology and an increased Johnsen's score. Moreover, the quantities of SSCs and epididymal spermatozoa were restored after intervention with RIPA-56, indicating a series of beneficial effects by targeting the necroptosis of SSCs in mice undergoing busulfan treatment. In conclusion, our findings reveal that the necroptosis of SSCs plays a critical role in busulfan-induced gonadotoxicity and may be a potential target for male fertility preservation.

This study investigated the effects of rutin against reproductive damage caused by toxic mercury in male rats. Thirty-five Sprague Dawley rats were used. Control group was injected with saline for 7 days. The rutin-100 group received 100 mg/kg/b.w. rutin for 7 days. Mercuric chloride (HgCl 2 ) group received 1.23 mg/kg/b.w. of HgCl 2 for 7 days. Mercury chloride + rutin-50 group received 50 mg/kg/b.w. rutin and HgCl 2 1.23 mg/kg/b.w. for 7 days. HgCl 2  + rutin-100 group received 100 mg/kg/b.w. rutin and HgCl 2 1.23 mg/kg/b.w. for 7 days. It was detected that HgCl 2 treatment increased malondialdehyde (MDA) levels, tumour necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) expressions, necrosis and degeneration of spermatogonium, dead and abnormal sperm percentages; tubular walls thinning; and decreased antioxidant enzyme activities and sperm motility. It was determined that rutin application reduced testicular damage caused by HgCl 2 . In conclusion, rutin administration may treat HgCl 2 toxicity in testes., (© 2020 Blackwell Verlag GmbH.)

Background: Curcumin has shown many pharmacological activities in both preclinical and clinical studies. Many technologies have been developed and applied to improve the solubility and bioavailability of curcumin, especially the nanotechnology-based delivery systems. However, there has been evidence that certain nanoparticles have potential reproductive toxicity in practice., Methods: Curcumin-poly (lactic-co-glycolic acid) (PLGA)-PEG nanoparticles (Cur-PLGA-NPs for short) were prepared. The Cur-PLGA-NPs were evaluated with its effect on the proliferation of mouse testicular cell lines in vitro and spermatogenesis in vivo, while PLGA-NPs were used as control. For animal experiments, male BALB/c mice were treated with 20 mg/kg of Cur-PLGA-NPs for continuous 10 days via tail vein injection., Results: We found the curcumin nanoparticles suppressed the proliferation of testicular cell lines in vitro. Furthermore, a short-term intravenous delivery of curcumin-loaded nanoparticles could be harmful to the differentiation of spermatogonia, the elongation of spermatids, as well as the motility of mature sperms., Conclusion: In the present study, we disclosed the acute damage on mouse spermatogenesis and sperm parameters by curcumin-loaded nanoparticles. Our results suggested that the reproductive toxicity of nanoformulated curcumin needs to be prudently evaluated before its application., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Xia et al.)

An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg -1 . In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg -1 ) on day 36 or a dose of 40 mg kg -1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg -1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg -1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity., Competing Interests: None

Arsenic is a toxic metalloid that can cause male reproductive malfunctions and is widely distributed in the environment. The aim of this study was to investigate the cytotoxicity of arsenic trioxide (ATO) induced GC-1 spermatogonial (spg) cells. Our results found that ATO increased the levels of catalase (CAT) and malonaldehyde (MDA) and reactive oxygen species (ROS), while decreasing glutathione (GSH) and the total antioxidant capacity (T-AOC). Therefore, ATO triggered oxidative stress in GC-1 spg cells. In addition, ATO also caused severe mitochondrial dysfunction that included an increase in residual oxygen consumption (ROX), and decreased the routine respiration, maximal and ATP-linked respiration (ATP-L-R), as well as spare respiratory capacity (SRC), and respiratory control rate (RCR); ATO also damaged the mitochondrial structure, including mitochondrial cristae disordered and dissolved, mitochondrial vacuolar degeneration. Moreover, degradation of p62, LC3 conversion, increasing the number of acidic vesicle organelles (AVOs) and autophagosomes and autolysosomes are demonstrated that the cytotoxicity of ATO may be associated with autophagy. Meanwhile, the metabolomics analysis results showed that 20 metabolites (10 increased and 10 decreased) were significantly altered with the ATO exposure, suggesting that maybe there are the perturbations in amino acid metabolism, lipid metabolism, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins. We concluded that ATO was toxic to GC-1 spg cells via inducing oxidative stress, mitochondrial dysfunction and autophagy as well as the disruption of normal metabolism. This study will aid our understanding of the mechanisms behind ATO-induced spermatogenic toxicity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Optimization of tissue culture systems able to complete male germ cell maturation to post-meiotic stages is considered as an important matter in reproductive biology. Considering that hypoxia is one of the factors limiting the efficiency of organ culture, the aim of this study was to use isolated seminiferous tubules (STs), having more surface and less thickness, in an organotypic culture system in order to improve oxygen diffusion and reduce hypoxia. The mechanically separated STs embedded in agarose or alginate and 1-3-mm 3 testicular tissue fragments of 3 adult mice were separately placed on the flat surface of agarose gel that was half-soaked in the medium. Survival and differentiation of germ cells using PLZF and SCP3 markers, identity of Sertoli cell using GATA4, cell proliferation with the Ki67 marker, and ST integrity using a ST scoring were evaluated up to 36 d at different culture times, each corresponding to the duration of one spermatogenic cycle. We observed a significantly reduced ST integrity in STs embedded in agarose or alginate on day 9 (versus tissue fragments p ≤ 0.05). There was no difference in the number of PLZF-positive cells between groups, but the number of SCP3 (in all-time points) and GATA4-positive cells was significantly higher in the culture of embedded STs. Although embedding STs can be useful for the progress of in vitro spermatogenesis, it makes them sensitive to degeneration. Further improvements are required to modify the air-liquid interface method to maintain ST integrity.

Atrazine (ATZ), a widely used agricultural pesticide and benzo[a]pyrene (BaP), a ubiquitous environmental human carcinogen can induce alterations of spermatogenesis. In the present study, we showed first that our seminiferous tubule culture model, in bicameral chambers, allowed the settlement of the blood-testis barrier (BTB) in 8-day-old male rat cultures and the differentiation of spermatogonia into round spermatids.The effect of a mixture of 1 μg/L of ATZ and 1 μg/L of BaP was then investigated either during or after the establishment of the BTB by using 8- or 20-22-day-old rats. Cultures were performed over a 3-week period. Our results show that claudin-11 and connexin 43 two proteins of the BTB, were impaired by the mixture which also reduced the number of round spermatids (the direct precursors of spermatozoa), by targeting the middle to late pachytene spermatocytes. These effects were observed in 8- and 20-22-day -old rat seminiferous tubule cultures. However, the decrease of the number of round spermatids was faster and more marked in the 8-day- than in the 20-22-day -old rat seminiferous tubule cultures. Our study emphasizes the possible influence of the age of an individual on the effect of (a) toxicant(s) on spermatogenesis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As a widely used plasticizer in industry, di-2-ethylhexylphthalate (DEHP) can cause testicular toxicity, yet little is known about the potential mechanism. In this study, DEHP exposure dramatically inhibited cellviability and induced apoptosis of mouse GC-1 spg cells. Furthermore, DEHP significantly increased the levels of autophagy proteins LC3-II, Beclin1 and Atg5, as well as the ratio ofLC3-II/LC3-I. Transmission electron microscopy (TEM) further confirmed that DEHP induced autophagy of mouse GC-1 spg cells. DEHP was also shown to induceoxidative stress; while inhibition of oxidative stress with NAC could increase cell viability and inhibit DEHP-induced apoptosis and autophagy. These results suggested that DEHP induced apoptosis and autophagy of mouse GC-1 spg cells via oxidative stress. 3-MA, an inhibitor of autophagy, could rescue DEHP-induced apoptosis. In summary, DEHP induced apoptosis and autophagy of mouse GC-1 spg cells via oxidative stress, and autophagy might exert a cytotoxic effect on DEHP-induced apoptosis., (© 2019 Wiley Periodicals, Inc.)

Emerging data indicate that structural analogs of bisphenol A (BPA) such as bisphenol S (BPS), tetrabromobisphenol A (TBBPA), and bisphenol AF (BPAF) have been introduced into the market as substitutes for BPA. Our previous study compared in vitro testicular toxicity using murine C18-4 spermatogonial cells and found that BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS. Recently, we developed a novel in vitro three-dimensional (3D) testicular cell co-culture model, enabling the classification of reproductive toxic substances. In this study, we applied the testicular cell co-culture model and employed a high-content image (HCA)-based single-cell analysis to further compare the testicular toxicities of BPA and its analogs. We also developed a machine learning (ML)-based HCA pipeline to examine the complex phenotypic changes associated with testicular toxicities. We found dose- and time-dependent changes in a wide spectrum of adverse endpoints, including nuclear morphology, DNA synthesis, DNA damage, and cytoskeletal structure in a single-cell-based analysis. The co-cultured testicular cells were more sensitive than the C18 spermatogonial cells in response to BPA and its analogs. Unlike conventional population-averaged assays, single-cell-based assays not only showed the levels of the averaged population, but also revealed changes in the sub-population. Machine learning-based phenotypic analysis revealed that treatment of BPA and its analogs resulted in the loss of spatial cytoskeletal structure, and an accumulation of M phase cells in a dose- and time-dependent manner. Furthermore, treatment of BPAF-induced multinucleated cells, which were associated with altered DNA damage response and impaired cellular F-actin filaments. Overall, we demonstrated a new and effective means to evaluate multiple toxic endpoints in the testicular co-culture model through the combination of ML and high-content image-based single-cell analysis. This approach provided an in-depth analysis of the multi-dimensional HCA data and provided an unbiased quantitative analysis of the phenotypes of interest., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Resmethrin is a widely used pyrethroid insecticide, which causes low toxicity in mammals. However, its toxicity in testes has not been fully investigated. Therefore, we evaluated the toxicity of resmethrin in mouse testes using an in vitro organ culture. Mouse testicular fragments (MTFs) derived from neonates were cultured in medium containing resmethrin for 30 days. Effects on spermatogenesis in the cultured testes were investigated as functions of both time and dose. Resmethrin significantly downregulated the transcription levels of marker genes for spermatogonia and the number of spermatogenic germ cells relative to those of the controls, according to quantitative PCR and immunostaining. In addition, spermatocyte was observed in the control, but not in 50 μM resmethrin-exposed cultures. Levels of the SYCP3 meiotic marker and phosphorylated H2AX decreased by resmethrin treatment, as observed by Western blotting. Toxic or apoptotic effects of resmethrin in Sertoli and Leydig cells from MTFs were not observed by immunostaining and Tunnel assay. No changes in the expression of steroidogenic enzymes were noted. Apoptosis was only detected in the germ cells of resmethrin-treated MTFs. Thus, the highest dose of resmethrin tested (50 μM) completely inhibited spermatogenesis, because of apoptosis of germ cells and spermatocytes. Although the in vivo toxicity of resmethrin has not yet been studied in detail, significant evidence for cytotoxicity was observed in our organ cultures. This methodological approach is useful for the study of reproductive toxicity before proceeding to animal models, as it greatly reduces the use of laboratory animals., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study was designed to explore the protective effects of methanol (Meth, 200 mg kg -1 body wt) and aqueous ethanol (Eth-OH, 200 mg kg -1 body wt) extracts of Anthocleista djalonensis roots on testicular inflammation induced by lipopolysaccharide (LPS, 5 mg/kg body wt) and depletion of tubular germ cells induced by busulfan (15 mg/kg body wt) in rats after 60 days of oral administration. As expected, LPS stimulation of the animals significantly increased serum and intra-testicular interleukin-6 and serum nitrite levels which were significantly inhibited in the Eth-OH + LPS and Meth + LPS animals. The increase in testicular and not serum myeloperoxidase activity that was induced by LPS treatment was synergistically increased in the Eth-OH + LPS animals, whereas it was inhibited in the Meth + LPS animals compared to LPS-treated animals. Furthermore, the administration of the Eth-OH or Meth extracts protected against busulfan-induced depletion of tubular germ cells and promotes the re-population of the seminiferous tubules with germ cells (spermatogonia, spermatocytes and round spermatids) at different stages of development. The extracts were found to contain 7'-oxaspiro [cyclopropane-1,4'-tricyclo [3.3.1.0 (6,8)] nonan-2'-one], cis,cis-7,10-hexadecadienal, hexadecanoic acid, methyl ester, hexadecanoic acid, ethyl ester, 9,12-octadecadienoic acid, methyl ester, and 9,12-octadecadienoic acid (Z,Z)-) which may partly explain the observed anti-inflammatory effects. In conclusion, Meth extracts of A. djanonesis have better anti-inflammatory effects than the Eth-OH extract for the management of impaired testicular function due to inflammation. However both extracts exhibited protective effect on the histology of the testis allowing for the recovery of spermatogenesis.

The mitochondrial proteins involved in spermatogenic cells apoptosis in zebrafish after carbon ion radiation (CIR) were screened. The relative biological effectiveness (RBE) of CIR in zebrafish testes was investigated. Apoptosis of testicular cells was measured within 24 hr following 1 and 4 Gy CIR. Immunoblotting was used to assess the levels of mitochondrial apoptotic proteins in testes, and proliferative and apoptotic spermatogenic cells were detected by immunofluorescence after CIR. Label-free quantitative (LFQ) and parallel reaction monitoring-based target proteomics (PRM) were combined to screen and validate differential mitochondrial proteins in testes between 4 Gy and control groups at 24 hr after CIR. The RBE of CIR in zebrafish testes was 1.48 ± 0.04, and induction of apoptosis by CIR was higher than that of X-rays in testicular cells. Mitochondrial apoptotic pathways play a crucial role in spermatogenic cells apoptosis after CIR, with 60 differential mitochondrial proteins identified. Among 20 target proteins, 12 were significantly upregulated, 2 were significantly downregulated in the 4 Gy CIR group. The results of PRM were consistent with label-free analysis. This is the first study to screen the differential mitochondrial proteins and provide useful information to understand the underlying mechanisms of spermatogenic cell apoptosis in zebrafish following CIR., (© 2019 Wiley Periodicals, Inc.)

Study Question: Could small molecules (SM) which target (or modify) signaling pathways lead to increased proliferation of undifferentiated spermatogonia following chemotherapy?, Summary Answer: Inhibition of transforming growth factor-beta (TGFb) signaling by SM can enhance the proliferation of undifferentiated spermatogonia and spermatogenesis recovery following chemotherapy., What Is Known Already: Spermatogonial stem cells (SSCs) hold great promise for fertility preservation in prepubertal boys diagnosed with cancer. However, the low number of SSCs limits their clinical applications. SM are chemically synthesized molecules that diffuse across the cell membrane to specifically target proteins involved in signaling pathways, and studies have reported their ability to increase the proliferation or differentiation of germ cells., Study Design, Size, Duration: In our experimental study, spermatogonia were collected from four brain-dead individuals and used for SM screening in vitro. For in vivo assessments, busulfan-treated mice were treated with the selected SM (or vehicle, the control) and assayed after 2 (three mice per group) and 5 weeks (two mice per group)., Participants/materials, Setting, Methods: We investigated the effect of six SM on the proliferation of human undifferentiated spermatogonia in vitro using a top-bottom approach for screening. We used histological, hormonal and gene-expression analyses to assess the effect of selected SM on mouse spermatogenesis. All experiments were performed at least in triplicate and were statistically evaluated by Student's t-test and/or one-way ANOVA followed by Scheffe's or Tukey's post-hoc., Main Results and the Role of Chance: We found that administration of SB431542, as a specific inhibitor of the TGFb1 receptor (TGFbR1), leads to a two-fold increase in mouse and human undifferentiated spermatogonia proliferation. Furthermore, injection of SB to busulfan-treated mice accelerated spermatogenesis recovery as revealed by increased testicular size, weight and serum level of inhibin B. Moreover, SB administration accelerated both the onset and completion of spermatogenesis. We demonstrated that SB promotes proliferation in testicular tissue by regulating the cyclin-dependent kinase (CDK) inhibitors 4Ebp1 and P57 (proliferation inhibitor genes) and up-regulating Cdc25a and Cdk4 (cell cycle promoting genes)., Limitations, Reasons for Caution: The availability of human testis was the main limitation in this study., Wider Implications of the Findings: This is the first study to report acceleration of spermatogenesis recovery following chemotherapy by administration of a single SM. Our findings suggest that SB is a promising SM and should be assessed in future clinical trials for preservation of fertility in men diagnosed with cancer or in certain infertility cases (e.g. oligospermia)., Study Funding/competing Interest(s): This study was supported by Royan Institute and National Institute for Medical Research Development (NIMAD, grant no 963337) granted to H.B. The authors have no conflict of interest to report., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

DNA methylation have been suggested as possible mediators of long-term health effects of environmental stressors. This study aimed to evaluate the potential therapy of methylation of S-adenosyl-l-methionine (SAM) on PFOS induced trangeneral reproductive toxicity. In this study, postnatal 5d Sprague Dawley rats were randomly divided into four groups: control, PFOS, PFOS + SAM, and PFOS + Decitabine (DAC). The F0 rats were exposed to 5 mg/kg PFOS and SAM or DAC until PND60. The development of the offsprings were monitored without PFOS exposure. The fertility in F0, F1 rats, and change in F1 testes were observed. The results were as follows. The significant increase in F0 pregnancy rate, and survival rate in F1 offspring in PFOS + SAM relative to PFOS group were observed. Changes of birth weights and physical development in F1 offspring with SAM were approached as a corresponding variation of the control after the deparation period. No pregnant in F1 maternal rats in the PFOS and DAC groups were found, but pregnant in the SAM group. Significantly decrease in the percentage of abnormal seminiferous tubules and increase in expression of promyelocytic leukemia zinc finger (PLZF+) spermatogonial stem cells in F1 testis compared with the PFOS group. Taken together, Methyl donor SAM improve PLZF + spermatogonia stem cell proliferation, attenuate damage in testicular tissue structure, which subsequently improve the transgenerational growth retard and infertility induced by PFOS chronic stress., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fertility preservation for prepubertal boys relies exclusively on cryopreservation of immature testicular tissue (ITT) containing spermatogonia as the only cells with reproductive potential. Preclinical studies that used a nude mice model to evaluate the development of human transplanted ITT were characterized by important spermatogonial loss. We hypothesized that the encapsulation of testicular tissue in an alginate matrix supplemented with nanoparticles containing a necrosis inhibitor (NECINH-NPS) would improve tissue integrity and germ cells' survival in grafts. We performed orthotopic autotransplantation of 1 mm³ testicular tissue fragments recovered form mice (aged 4-5 weeks). Fragments were either non-encapsulated, encapsulated in an alginate matrix, or encapsulated in an alginate matrix containing NECINH-NPs. Grafts were recovered 5- and 21-days post-transplantation. We evaluated tissue integrity (hematoxylin-eosin staining), germ cells survival (immunohistochemistry for promyelocytic leukemia zinc-finger, VASA, and protein-boule-like), apoptosis (immunohistochemistry for active-caspase 3), and lipid peroxidation (immunohistochemistry for malondialdehyde). NECINH-NPs significantly improved testicular tissue integrity and germ cells' survival after 21 days. Oxidative stress was reduced after 5 days, regardless of nanoparticle incorporation. No effect on caspase-dependent apoptosis was observed. In conclusion, NECINH-NPs in an alginate matrix significantly improved tissue integrity and germ cells' survival in grafts with the perspective of higher reproductive outcomes.

Male fertility depends on the regulatory balance between germ cell self-renewal and differentiation, and the spatial and temporal patterns of this balance must be maintained throughout the life cycle. Retinoic acid and its receptors are important factors in spermatogenesis. Spermatogonia cells can self-proliferate and differentiate and have unique meiotic capabilities; they halve their genetic material and produce monomorphic sperm to pass genetic material to the next generation. A number of studies have found that the spermatogenesis process is halted in animals with vitamin A deficiency and that most germ cells are degraded, but they tend to recover after treatment with RA or vitamin A. This literature review discusses our understanding of how RA regulates sperm cell differentiation and meiosis and also reviews the functional information and details of RA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Male germ cell development is a critical period during which epigenetic patterns are established and maintained. The progression from diploid spermatogonia to haploid spermatozoa involves the incorporation of testis-specific histone variants, mitotic and meiotic divisions, haploid gene expression, histone-protamine transitions and massive epigenetic reprogramming. Understanding the protein players and the epigenetic mark network involved in the setting of the epigenetic programme in spermatogenesis is an exciting new clue in the field of reproductive biology with translational outcomes. As information in the existing literature regarding cross-talk between DNA methylation and histone hyperacetylation in the advanced stages of murine spermatogenesis is still scarce and controversial we have investigated the effect of a DNA-methyltransferase inhibitor, 5-aza-2'-deoxycytidine, at the cytological and molecular level (by transmission electron microscopy, immunocytochemistry and immunoprecipitation methods). Our results revealed an important role for regulation of DNA methylation in controlling histone hyperacetylation and chromatin remodelling during spermatogenesis.

The aims of the present research are to further validate the application of the improved three-dimensional (3 D) rat testicular cell co-culture model to evaluate the effects of various reprotoxic chemicals on the function of the main somatic cells, as well as on spermatogonial cell differentiation and even spermatogenesis, and to investigate the specific toxicant mechanisms in testes treated with HZ1006, a hydroxamate-based a hydroxamate-based histone deacetylase inhibitor (HDACI). Based on the characteristics of HZ1006, the appropriate exposure duration (8, 16, or 24 days), dosage (0, 3.125, 6.25, 12.5, or 25 μM) and toxic endpoints suitable for detection were selected in the experiments. The results showed inhibition of cell proliferation, reduced testosterone levels, and decreased spermatogonial cell meiosis-specific gene expression, as well as decreased protein levels of androgen receptor (AR) and decreased expression of the AR target gene PSA, accompanied by inhibition of Hdac6 expression after HZ1006 exposure in the 3 D rat testicular cell co-culture model. These findings indicate that the improved 3 D rat testicular cell co-culture model we have established has the potential to become a new testicular toxicity test system that can be used to test toxic characteristics and mechanisms of new compounds and has good application prospects, although more research on the model is required.

The increased incidence of testicular disorders in young men and the possible influence of environmental chemicals, such as dibutyl phthalate (DBP) and acrylamide (AA), requires experimental models for identifying modes of action. Most published reproductive toxicologic studies use RNA samples from the total testis to evaluate testicular gene expression; however, analyses of isolated cell types could provide a more specific tool. Among testicular germ cells, spermatogonia are critical since they represent the onset of spermatogenesis. This study aimed, (1) to establish a technique for spermatogonia isolation; (2) to apply this isolation technique to verify possible gene expression alterations (Pou5f1, Kitlg, Mki-67, Bak1 and Spry4) in prepubertal post-natal day, (PND24) and pubertal (PND45) testes after in utero and postnatal exposure to DBP or AA. The technique was efficient for isolation of a majority of spermatogonia. In utero DBP exposure led to reduced litter body weight at birth, reduced anogenital distance of male pups on PND4, and increased frequency of male nipple retention on PND14 compared to controls. DBP-exposed relative testes weights were reduced only at PND24 compared to control but they did not differ at PND45. DBP-exposed animals showed reduced expression levels of Pou5f1 and Mki67 on PND24, and reduced expression of Pou5f1 and Spry4 on PND45. AA exposure reduced expression of Pou5f1, Mki67, and Spry4 at PND45 although not significantly. Our results suggest that DBP acts by reducing cell proliferation and impairing differentiation in prepubertal and pubertal testes.

Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Pig is wildly used as a model animal for understanding reproduction mechanisms of human being. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male infertility. Here, we established an in-vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of Stra8 but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, transition protein 1 (Tnp1) and protamine 1 (Prm1) were upregulated in the presence of RA. The addition of an RA receptor (RAR) inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein through RAR and promoted the formation of round spermatids. We established an efficient culture system for in-vitro differentiation of pig SSCs. Our study represents a model for human testis disease and toxicology screening. Molecular regulators of SSC differentiation revealed in this study might provide a therapeutic strategy for male infertility., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study mainly focuses on the cytogenetic toxicity induction by zoledronic acid (ZA), a nitrogen containing bisphosphonate (N-BPs) in the male germline cells of Swiss albino mice. A single intraperitoneal exposure with three different doses of ZA (2, 4, and 8 mg/kg body weight), toxicity was assessed by analyzing spermatogonial metaphase chromosome aberrations at 24 h, aberrant primary spermatocytes at week 4, and abnormal spermatozoa at week 8 posttreatment. Cyclophosphamide (40 mg/kg) and 0.9% NaCl were used as positive and vehicle controls respectively in the study. The results showed that there was a significant induction in the number of chromosomal aberrations especially at two doses of ZA (4 and 8 mg/kg) after 24 h in the spermatogonial cells (p < 0.001) compared to vehicle control. The transmission genetic damages were noticed as aberrant spermatocytes with atypical bivalents (X-Y/autosomal asynapsis) at 4 mg/kg of ZA (p < 0.01) and at 8 mg/kg of ZA (p < 0.001) at week 4 posttreatment. A statistically significant higher number of abnormal spermatozoa (sperm) were also noticed at week 8 posttreatment of both at 4 and 8 mg/kg of ZA (p < 0.001). Hence, from these genotoxicity studies, it can be concluded that ZA is genotoxic in male germline cells and has the potential of transmitting the genotoxic effects from spermatogonial cells to sperm in male Swiss mice.

The mechanism of testicular toxicity of simultaneous multiple exposures to metals is poorly understood. Previous studies reported that the toxic effect of cadmium (Cd) is modified by tissue concentration of iron (Fe). Using the mice (Mus musculus) model in the present study, we demonstrated that combined Cd (25 mg kg -1 bw) and Fe (100 mg kg -1 bw) treatment increased both Cd and Fe testicular concentrations much more than separate exposures to either of the metals. Intratesticular Cd and Fe concentrations were inversely correlated (r = - 0.731, p < 0.05) on administration of Fe but not on combined exposure to both metals when they were positively correlated (versus Cd; r = 0.793, versus Fe; r = 0.779, p < 0.05). Additionally, Cd + Fe treatment increased testicular lipid peroxidation and depleted intratestesticular testosterone, cholesterol and glutathione concentrations much more than their separate treatment. This was also associated with decreased activity of the germ cell marker, testicular lactate dehydrogenase, and increased testicular myeloperoxidase activity. These changes resulted in decreased seminiferous epithelial height, tubular diameter, germ cell (spermatogonia, spermatocytes, and spermatids) numbers, and severe tissue damage. In conclusion, Cd + Fe intake have synergistic toxic effects on testicular steroid formation and spermatogenesis due to the high testicular concentrations of both metals.

Research Question: Does recombinant human vascular endothelial growth factor (VEGF-165) improve the efficiency of human immature testis tissue (ITT) xenotransplantation?, Design: ITT fragments from three prepubertal boys were cultured for 5 days with VEGF-165 or without (control) before xenotransplantation into the testes of immunodeficient mice. Xenotransplants were recovered at 4 and 9 months post-transplantation and vascularization, seminiferous tubule integrity, number of spermatogonia and germ cell differentiation were evaluated by histology and immunohistochemistry., Results: Transplants from donor 1 and donor 2 treated with VEGF demonstrated higher vascular surface (P = 0.004) and vessel density (P = 0.011) overall and contained more intact seminiferous tubules (P = 0.039) with time, compared with controls. The number of spermatogonia was increased over time (P < 0.001) irrespective of treatment and donor, whereas, for the VEGF-treated transplants, the increase was even higher over time (P = 0.020). At 9 months, spermatocytes were present in the xenotransplants, irrespective of treatment. No transplants could be recovered from donor 3, who had already received treatment with cyclosporine for aplastic anaemia before biopsy., Conclusions: In-vitro pre-treatment of human prepubertal testis tissue with VEGF improved transplant vascularization in two out of three cases, resulting in improved seminiferous tubule integrity and spermatogonial survival during xenotransplantation. Although further studies are warranted, we suggest VEGF to be considered as a factor for improving the efficiency of immature testis tissue transplantation in the future., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. This requires alterations in the germline (sperm or egg) epigenome. Previously, the agricultural fungicide vinclozolin was found to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs) termed epimutations that help mediate this epigenetic inheritance. The current study was designed to investigate the developmental origins of the transgenerational DMRs during gametogenesis. Male control and vinclozolin lineage F3 generation rats were used as a source of embryonic day 13 (E13) primordial germ cells, embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus vinclozolin lineage samples were determined for each developmental stage. The top 100 statistically significant DMRs for each stage were compared. The developmental origins of the caudal epididymal sperm DMRs were assessed. The chromosomal locations and genomic features of the different stage DMRs were investigated. In addition, the DMR associated genes were identified. Previous studies have demonstrated alterations in the DMRs of primordial germ cells (PGCs). Interestingly, the majority of the DMRs identified in the current study for the caudal sperm originated during the spermatogenic process in the testis. A cascade of epigenetic alterations initiated in the PGCs appears to be required to alter the epigenetic programming during spermatogenesis to modify the sperm epigenome involved in the transgenerational epigenetic inheritance phenomenon.

As the main toxic component of aristolochic acid, aristolochic acid I (AAI) is primarily found in Aristolochiaceae plants such as Aristolochia, Aristolochia fangchi and Caulis aristolochiae manshuriensis. AAI has been proven to be carcinogenic, mutagenic and nephrotoxic. Although the role of AAI in testicular toxicity has been reported, its mechanism of action is unknown. Using metabonomics and molecular biology techniques, we tried to identify the differential endogenous metabolites of AAI that may affect the changes in testicular function in mice, map the network of metabolic pathways, and systematically reveal the molecular mechanism of AAI-induced testicular toxicity. We found that AAI inhibited amino acid metabolism in mouse testicular cells, impeded the uptake and oxidative decomposition of fatty acids, prevented normal glucose uptake by testicular cells, which inhibited glycolysis and gluconeogenesis, affected the mitochondrial tricarboxylic acid (TCA) cycle, which impaired the ATP energy supply, decreased the number of spermatogenic cells and sperm in the testes, induced changes in the mitochondrial state of spermatogonial cells, and ultimately led to physiological and pathological changes in the testes. AAI also regulated the testicular physiological activity by regulating the androgen receptor and hormone levels. This study used metabonomics and other methods to elucidate the mechanism of AAI-induced testicular toxicity from a new angle., (Copyright © 2019 Elsevier Inc. All rights reserved.)

It is now established that diethylstilbestrol (DES) has damaging effects on the male reproductive system. However, to date there have been no studies morphological analysis of adult rat testes upon treatment with DES. Here, we examined whether DES has any significant morphological effect on steroidogenesis and spermatogenesis. DES was injected subcutaneously at 3 μg/day and 30 μg/day in adult male Sprague-Dawley (SD) rats for two different treatment lengths (1 or 3 weeks), after which rats were necropsied. TUNEL labeling, cell counting, and morphological analysis were used to evaluate the effects of DES. A high dose of DES and longer exposure severely affected the cellular development of the testis. Specifically, DES treatment disrupted both steroidogenesis and spermatogenesis by decreasing the number of spermatogonia, Sertoli cells, and Leydig cells in a dose- and time-dependent manner. Thus, DES may account for decreases in the number of spermatogenic cells, Sertoli cells and Leydig cells, which in turn may lead to reduced fertility in males., (Copyright © 2019. Published by Elsevier Ltd.)

Three-dimensional (3D) organoids can serve as an in vitro platform to study cell-cell interactions, tissue development, and toxicology. Development of organoids with tissue architecture similar to testis in vivo has remained a challenge. Here, we present a microwell aggregation approach to establish multicellular 3D testicular organoids from pig, mouse, macaque, and human. The organoids consist of germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells forming a distinct seminiferous epithelium and interstitial compartment separated by a basement membrane. Sertoli cells in the organoids express tight junction proteins claudin 11 and occludin. Germ cells in organoids showed an attenuated response to retinoic acid compared to germ cells in 2D culture indicating that the tissue architecture of the organoid modulates response to retinoic acid similar to in vivo. Germ cells maintaining physiological cell-cell interactions in organoids also had lower levels of autophagy indicating lower levels of cellular stress. When organoids were treated with mono(2-ethylhexyl) phthalate (MEHP), levels of germ cell autophagy increased in a dose-dependent manner, indicating the utility of the organoids for toxicity screening. Ablation of primary cilia on testicular somatic cells inhibited the formation of organoids demonstrating an application to screen for factors affecting testicular morphogenesis. Organoids can be generated from cryopreserved testis cells and preserved by vitrification. Taken together, the testicular organoid system recapitulates the 3D organization of the mammalian testis and provides an in vitro platform for studying germ cell function, testicular development, and drug toxicity in a cellular context representative of the testis in vivo., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Busulfan is a widely used chemotherapeutic drug for chronic myelogenous leukemia and bone marrow transplantation. As a cell cycle nonspecific alkylation agent, busulfan has a severe side effect on germ cells, especially on spermatogonia before meiosis. Studies have revealed that busulfan causes DNA strand crosslinks in spermatogonia and induces apoptosis, and many corresponding strategies have been developed to ameliorate the side effects. However, fertility maintenance after busulfan treatment is still a challenging project in the clinic. Here, we demonstrated that continuous injection of melatonin effectively alleviated germline cytotoxicity both in recipient mice and cultured spermatogonia, and busulfan/melatonin recipient mice produced normal litters. We further revealed that melatonin rescues spermatogonia from apoptosis by neutralizing reactive oxidative species (ROS) induced by busulfan and recovered the phosphorylation of ATM and p53 to normal levels, and as a result apoptosis in spermatogonial progenitor cells was avoided. This study reports that pineal gland hormone melatonin effectively protects spermatogonia from the stress of chemotherapy and oxidation and reveals the underlying molecular mechanisms, which will provide an important hint for fertility protection in clinic., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Androgen plays a pivotal role in spermatogenesis, accompanying a question how androgen acts on germ cells in testis since germ cells lack of androgen receptors (AR). Promyelocytic leukemia zinc-finger (PLZF) is essential for maintenance of undifferentiated spermatogonia population which is terminologically called spermatogonia progenitor cells (SPCs)., Aims: We aim to figure out the molecular connections between androgen and fates of PLZF + SPCs population., Method: Immunohistochemistry was conducted to confirm that postnatal testicular germ cells lacked endogenous AR. Subsequently, total cells were isolated from 5 dpp (day post partum) mouse testes, and dihydrotestosterone (DHT) and/or bicalutamide treatment manifested that Plzf was indirectly regulated by androgen. Then, Sertoli cells were purified to screen downstream targets of AR using ChIP-seq, and gene silence and overexpression were used to attest these interactions in Sertoli cells or SPCs-Sertoli cells co-culture system. Finally, these connections were further verified in vivo using androgen pharmacological deprivation mouse model., Results: Gata2 is identified as a target of AR, and β1-integrin is a target of Wilms' tumor 1 (WT1) in Sertoli cells. Androgen signal negatively regulate β1-integrin on Sertoli cells via Gata2 and WT1, and β1-integrin on Sertoli cells interacts with E-cadherin on SPCs to regulate SPCs fates., Conclusion: Androgen promotes differentiation of PLZF + spermatogonia pool via indirect regulatory pattern.

Ensuring robust gamete production even in the face of environmental stress is of utmost importance for species survival, especially in mammals that have low reproductive rates. Here, we describe a family of genes called melanoma antigens (MAGEs) that evolved in eutherian mammals and are normally restricted to expression in the testis (http://MAGE.stjude.org) but are often aberrantly activated in cancer. Depletion of Mage-a genes disrupts spermatogonial stem cell maintenance and impairs repopulation efficiency in vivo. Exposure of Mage-a knockout mice to genotoxic stress or long-term starvation that mimics famine in nature causes defects in spermatogenesis, decreased testis weights, diminished sperm production, and reduced fertility. Last, human MAGE-As are activated in many cancers where they promote fuel switching and growth of cells. These results suggest that mammalian-specific MAGE genes have evolved to protect the male germline against environmental stress, ensure reproductive success under non-optimal conditions, and are hijacked by cancer cells.

Glyphosate is the most widely used herbicide in the world. Several studies have investigated the effects of glyphosate and glyphosate-based herbicides (GBHs) on male reproduction, but there is still little and conflicting evidence for its toxicity. In this study, we analyzed the effects of glyphosate, alone or in formula, on the male reproductive system. Pregnant mice were treated from E10.5 to 20 days postpartum by adding glyphosate or a GBH (Roundup 3 Plus) to their drinking water at 0.5 (the acceptable daily intake, ADI dose), 5 and 50 mg/kg/day. Male offspring derived from treated mice were sacrificed at 5, 20, and 35 days old (d.o.) and 8 months old (m.o.) for analysis. Our result showed that exposure to glyphosate, but not GBH, affects testis morphology in 20 d.o. and decrease serum testosterone concentrations in 35 d.o. males. We identified that the spermatozoa number decreased by 89% and 84% in 0.5 and 5 mg/kg/day of GBH and glyphosate groups, respectively. Moreover, the undifferentiated spermatogonia numbers were decreased by 60% in 5 mg/kg/day glyphosate group, which could be due to the alterations in the expression of genes involved in germ cell differentiation such as Sall4 and Nano3 and apoptosis as Bax and Bcl2. In 8 m.o. animals, a decreased testosterone level was observed in GBH groups. Our data demonstrate that glyphosate and GBHs could cause endocrine-disrupting effects on male reproduction at low doses. As glyphosate has effects at the ADI level, our data suggest that the current ADI for glyphosate could be overestimated., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Kisspeptin (Kp) expression in testis has caused most of the recent research surveying its functional role in this organ. This peptide influences spermatogenesis and sperm capacitation, so it is considered as a regulator of reproduction. Kp roles exert through hypothalamic/pituitary/gonadal axis. We aimed to evaluate direct roles for Kp on proliferation and differentiation of spermatogonial cells (SCs) when the cells are cocultured with somatic cells. Somatic cells and SCs were isolated from adult azoospermic and newborn mice and then enriched using a differential attachment technique. After the evaluation of identity and colonization for SCs, the cells were cocultured with somatic cells, and three doses of Kp (10 -8 -10 -6  M) was assessed on proliferation (through evaluation of MVH and ID4 markers) and differentiation (via evaluation of c-Kit and SCP 3 , TP 1, TP 2 , and, Prm 1 markers) of the coculture system. Investigations were continued for four succeeding weeks. At the end of each level of testosterone in the culture media was also evaluated. We found positive influence from Kp on proliferative and differentiative markers in SCs cocultured with somatic cells. These effects were dose-dependent. There was no effect for Kp on testosterone level. From our findings, we simply conclude that Kp as a neuropeptide for influencing central part of reproductive axis could also positively affect peripheral processes related to spermatogenesis without having an effect on steroidogenesis., (© 2018 Wiley Periodicals, Inc.)

Complete spermatogenesis has been achieved in vitro in mouse testicular explants with resulting sperm used to produce pups after Intra Cytoplasm Sperm Injection and Embryo Transfer. In the present study, we evaluated the influence of sphingosine-1-phosphate (S1P) on spermatogenesis of frozen-thawed lamb testis explants in vitro. Thawed testicular pieces were cultured for 12 d on agarose blocks in serum-free growth medium containing 0, 2, 5 or 10 μM S1P. At the end of D6 and D12, some pieces were fixed and processed for histology. Other pieces were processed for RNA isolation and quantitation of proliferation (PCNA, Ki67) and differentiation (PLZF) markers and genes involved in S1P signaling (S1PR1, SGPL1, SGPP1, AKT1 and NFKBIA) by qPCR. Histology revealed an increase (P < 0.05) in seminiferous cord (SC) diameter under all culture conditions, except 5 and 10 μM S1P by D6. In the presence of 5 μM S1P, percentage of gonocytes decreased (P < 0.05) by D6 (control, 24.9% vs. S1P, 10.3%) with a concomitant increase (P < 0.05) in spermatogonia formation (control, 74.4% vs. S1P, 88.1%). S1P induced PCNA or Ki67 expression by D6, whereas PLZF was up-regulated (P < 0.05) by D6 in 2 μM S1P and D12 in 5 & 10 μM S1P. Expression of SGPL1 and SGPP1 increased 4-12-fold in tissues cultured in 10 μM S1P by D12 compared to D12 control. AKT1 and NFKBIA mRNA expression was low (P < 0.05) in 5 and or 10 μM S1P treatments on D6. These results demonstrate that S1P promotes germ cell proliferation during first week of culture and may exert an anti-apoptotic influence on the seminiferous cord in sheep testicular explants in vitro., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The object of this study was to investigate if testis germ cell transplantation (TGCT) into a heterologous recipient would result in donor-origin spermatogenesis in the dromedary camel. First, we investigated a workable protocol for TGCT in camels, including donor cell isolation, enrichment by density gradient centrifugation (Percoll and Bovicoll), rete testis injection and microsatellite detection of donor and recipient genotypes. Second, the effects of three doses of Dolichos biflorus agglutinin (DBA), a glycoprotein that specifically binds to gonocytes or Type A spermatogonia, on testis germ cell depletion were investigated by direct injection into the rete testis of a male camel. Seven recipients were prepared with DBA treatment, two males were castrated at 4 weeks for depletion assessment and the remaining five received donor cells 4-6 weeks after treatment. On average, ~17 million cells were isolated per gram of testis tissue, with 19.5±1.9% DBA-positive (DBA+) cells. Percoll centrifugation yielded a 1.5-fold increase in DBA+ cells while Bovicoll centrifugation produced a 2.5-fold increase from the input cells of 18.6±2.1% DBA+ cells. Semen was collected from the recipients 13-20 weeks after transfer and the presence of donor DNA in the samples was determined using microsatellite markers. In two of the five recipients, all semen samples were shown to be positive for donor-derived cells. These results demonstrate for the first time that: (1) heterologous testicular germ cell transplantation in camels is feasible and the recipients are able to produce spermatozoa of donor origin and (2) DBA can be used effectively to deplete endogenous stem cells.

Despite the essential role of the active metabolite of vitamin A, all-trans retinoic acid (atRA) in spermatogenesis, the enzymes, and cellular populations responsible for its synthesis in the postnatal testis remain largely unknown. The aldehyde dehydrogenase 1A (ALDH1A) family of enzymes residing within Sertoli cells is responsible for the synthesis of atRA, driving the first round of spermatogenesis. Those studies also revealed that the atRA required to drive subsequent rounds of spermatogenesis is possibly derived from the ALDH1A enzymes residing within the meiotic and post-meiotic germ cells. Three ALDH1A isozymes (ALDH1A1, ALDH1A2, and ALDH1A3) are present in the testis. Although, ALDH1A1 is expressed in adult Sertoli cells and is suggested to contribute to the atRA required for the pre-meiotic transitions, ALDH1A2 is proposed to be the essential isomer involved in testicular atRA biosynthesis. In this report, we first examine the requirement for ALDH1A2 via the generation and analysis of a conditional Aldh1a2 germ cell knockout and a tamoxifen-induced Aldh1a2 knockout model. We then utilized the pan-ALDH1A inhibitor (WIN 18446) to test the collective contribution of the ALDH1A enzymes to atRA biosynthesis following the first round of spermatogenesis. Collectively, our data provide the first in vivo evidence demonstrating that animals severely deficient in ALDH1A2 postnatally proceed normally through spermatogenesis. Our studies with a pan-ALDH1A inhibitor (WIN 18446) also suggest that an alternative source of atRA biosynthesis independent of the ALDH1A enzymes becomes available to maintain atRA levels for several spermatogenic cycles following an initial atRA injection., (© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Testicular impairment is a common complication of Diabetes mellitus (DM). Zinc Oxide Nanoparticles (ZnO NPs) are a novel agent for Zn delivery with antidiabetic and antioxidant activities. However, few reports were recorded on it. The current study aimed to investigate the possible ameliorating effect of ZnO NPs treatment on testicular tissues alterations in streptozotocin (STZ)-induced diabetic rats. Therefore, thirty mature male Wistar rats were divided into three main groups: Control group (n = 18) was subdivided equally into three subgroups (negative control, vehicle and ZnO NPs), Diabetic group (n = 6) and ZnO NPs-treated diabetic group (n = 6). Induction of diabetes was done by a single intraperitoneal injection of STZ (60 mg/kg bw). The rats were orally treated by ZnO NPs (10 mg/kg bw) for 30 constitutive days. At the end of the experiment, blood glucose and serum testosterone levels were measured. Also, testicular tissues were obtained for histopathological investigations and immunohistochemical staining with anti-PCNA (proliferating cell marker), anti-ssDNA (apoptotic cell marker), anti-SOX9 (Sertoli cell marker), anti-Stella (spermatogonia marker), anti-STRA8 (preleptotene and early-leptotene spermatocytes marker), anti-DMC1 (leptotene and zygotene spermatocytes marker), anti-Dnmt3a (a marker for cells under DNA methylation) and anti-α-SMA (peritubular myoid cell marker). The biochemical analysis revealed that diabetes resulted in a significant elevation in blood glucose level and a reduction in serum testosterone level. Moreover, histopathological investigations revealed disorganized seminiferous epithelium and sever hyalinization with vacuolization of the testicular interstitium containing Leydig cells. The immunohistochemical findings support spermatogenesis impairment in the diabetic group. However, ZnO NPs treatment restores architecture of seminiferous epithelium and Leydig cells. Furthermore, more PCNA, SOX9, Stella, STRA8, DMC1 and Dnmt3a immunopositive cells with an improvement of peritubular α-SMA immunopositive expression, as well as few ssDNA-immunopositive cells were detected in the seminiferous epithelium. This study suggested the possible protective role of orally administered ZnO NPs on testicular alterations in the STZ-induced diabetic group via steroidogenesis and spermatogenesis enhancement. In addition, further researches are acquired for evaluation mechanism of ZnO NPs treatment via oral or parenteral routes in a dose-dependent manner to identify the more effective route and dose in the treatment of testicular diabetic complications., (Copyright © 2018 Elsevier GmbH. All rights reserved.)