Survival Of Spermatogonial Stem Cells Research Articles

Bisphenol Analogs Downregulate the Self-Renewal Potential of Spermatogonial Stem Cells.

In this study, we investigated the effect of bisphenol-A (BPA) and its major analogs, bisphenol-F (BPF), and bisphenol-S (BPS), on spermatogonial stem cells (SSCs) populations using in vitro SSC culture and in vivo transplantation models. SSCs enriched from 6- to 8-day-old C57BL/6-eGFP+ male mice testes were treated with varying concentrations of bisphenols for 7 days to examine bisphenol-derived cytotoxicity and changes in SSC characteristics. We utilized flow cytometry, immunocytochemistry, real-time quantitative reverse transcription-PCR, and western blot analysis. The functional alteration of SSCs was further investigated by examining donor SSC-derived spermatogenesis evaluation through in vivo transplantation and subsequent testis analysis. BPF exhibited a similar inhibitory effect on SSCs as BPA, demonstrating a significant decrease in SSC survival, inhibition of proliferation, and induction of apoptosis. On the other hand, while BPS was comparatively weaker than BPA and BPF, it still showed significant SSC cytotoxicity. Importantly, SSCs exposed to BPA, BPF, and BPS exhibited a significant reduction in donor SSC-derived germ cell colonies per total number of cultured cells, indicating that, like BPA, BPF, and BPS can induce a comparable reduction in functional SSCs in the recipient animals. However, the progress of spermatogenesis, as evidenced by histochemistry and the expressions of PCNA and SSC specific markers, collectively indicates that BPA, BPF, and BPS may not adversely affect the spermatogenesis. Our findings indicate that the major BPA substitutes, BPF and BPS, have significant cytotoxic effects on SSCs, similar to BPA. These effects may lead to a reduction in the functional self-renewal stem cell population and potential impacts on male fertility.

Notch pathway is required for protection against heat stress in spermatogonial stem cells in medaka.

Gamete production is a fundamental process for reproduction; however, exposure to stress, such as increased environmental temperature, can decrease or even interrupt this process, affecting fertility. Thus, the survival of spermatogonial stem cells (SSCs) is crucial for the recovery of spermatogenesis upon stressful situations. Here, we show that the Notch pathway is implicated in such survival, by protecting the SSCs against thermal stress. First, we corroborated the impairment of spermatogenesis under heat stress in medaka, observing an arrest in metaphase I at 10days of heat treatment, an increase in the number of spermatocytes, and downregulation of ndrg1b and sycp3. In addition, at 30days of treatment, an interruption of spermatogenesis was observed with a strong loss of spermatocytes and spermatids. Then, the exposure of adult males to thermal stress condition induced apoptosis mainly in spermatogenic and supporting somatic cells, with the exception of the germinal region, where SSCs are located. Concomitantly, the Notch pathway-related genes were upregulated, including the ligands (dll4, jag1-2) and receptors (notch1a-3). Moreover, during thermal stress presenilin enhancer-2 (pen-2), the catalytic subunit of γ-secretase complex of the Notch pathway was restricted to the germinal region of the medaka testis, observed in somatic cells surrounding type A spermatogonia (SGa). The importance of Notch pathway was further supported by an ex vivo approach, in which the inhibition of this pathway activity induced a loss of SSCs. Overall, this study supports the importance of Notch pathways for the protection of SSCs under chronic thermal stress.

Glutamine protects mouse spermatogonial stem cells against NOX1-derived ROS for sustaining self-renewal division in vitro.

Reactive oxygen species (ROS) are generated from NADPH oxidases and mitochondria; they are generally harmful for stem cells. Spermatogonial stem cells (SSCs) are unique among tissue-stem cells because they undergo ROS-dependent self-renewal via NOX1 activation. However, the mechanism by which SSCs are protected from ROS remains unknown. Here, we demonstrate a crucial role for Gln in ROS protection using cultured SSCs derived from immature testes. Measurements of amino acids required for SSC cultures revealed the indispensable role of Gln in SSC survival. Gln induced Myc expression to drive SSC self-renewal in vitro, whereas Gln deprivation triggered Trp53-dependent apoptosis and impaired SSC activity. However, apoptosis was attenuated in cultured SSCs that lacked NOX1. In contrast, cultured SSCs lacking Top1mt mitochondria-specific topoisomerase exhibited poor mitochondrial ROS production and underwent apoptosis. Gln deprivation reduced glutathione production; supra-molar Asn supplementation allowed offspring production from SSCs cultured without Gln. Therefore, Gln ensures ROS-dependent SSC-self-renewal by providing protection against NOX1 and inducing Myc.

CHD4 acts as a critical regulator in the survival of spermatogonial stem cells in mice†.

Spermatogenesis is sustained by homeostatic balance between the self-renewal and differentiation of spermatogonial stem cells, which is dependent on the strict regulation of transcription factor and chromatin modulator gene expression. Chromodomain helicase DNA-binding protein 4 is highly expressed in spermatogonial stem cells but roles in mouse spermatogenesis are not fully understood. Here, we report that the germ-cell-specific deletion of chromodomain helicase DNA-binding protein 4 resulted in complete infertility in male mice, with rapid loss of spermatogonial stem cells and excessive germ cell apoptosis. Chromodomain helicase DNA-binding protein 4-knockdown in cultured spermatogonial stem cells also promoted the expression of apoptosis-related genes and thereby activated the tumor necrosis factor signaling pathway. Mechanistically, chromodomain helicase DNA-binding protein 4 occupies the genomic regulatory region of key apoptosis-related genes, including Jun and Nfkb1. Together, our findings reveal the determinant role of chromodomain helicase DNA-binding protein 4 in spermatogonial stem cells survival in vivo, which will offer insight into the pathogenesis of male sterility and potential novel therapeutic targets.

Establishment of a controlled slow freezing-based approach for experimental clinical cryopreservation of human prepubertal testicular tissues

ObjectiveTo develop an efficient, clinical-grade, freezing protocol toward experimental clinical cryopreservation of testicular tissues in prepubertal boys suffering from cancer.DesignExperimental cryopreservation of testicular tissue.SettingUniversity Medical Center.Patient(s)Adult patients undergoing orchiectomy for various tumors and prepubertal boys scheduled for gonadotoxic treatment.Intervention(s)None.Main Outcome Measure(s)Histopathological analysis of tissue architecture, structural integrity, and cellular morphology was performed for control and frozen-thawed cryopreserved tissues.The number of seminiferous tubules per testicular section was calculated. The survival of spermatogonial stem cells (SSCs) and Sertoli cells of the control and frozen-thawed cryopreserved tissues was analyzed by immunofluorescence staining.Result(s)Uncontrolled Slow Freezing, Controlled slow freezing, and vitrification similarly preserved the integrity of the adult testicular tissues and the survival of SSCs and Sertoli cells. Controlled slow freezing of prepubertal testicular tissues effectively preserved their architecture, the number of tubules, SSCs, and Sertoli cells. In addition, we observed SSC loss after chemotherapy in prepubertal boys, reemphasizing the importance of fertility preservation before gonadotoxic treatment.Conclusion(s)Future fertility restoration for male survivors of pediatric cancers depends on the development of an optimal prepubertal testicular tissue cryopreservation method. Our findings demonstrate the effectiveness of controlled slow freezing for cryopreservation of human prepubertal testicular tissues and may contribute to more effective banking of these tissues and potential fertility restoration.Clinical Trial Registration NumberNIH research clinical trials number: NCT02529826.

The role of quercetin in primary culture of ovine spermatogonial stem cells

The aim of the present study was to examine the effect of quercetin on the survival and primary culture of ovine spermatogonial stem cells (SSCs). The two-time enzymatic digestion process was employed to obtain SSCs from lamb testes. In the next step, the use of filtration and differential plating methods caused an increase in the number of SSCs in the cell suspension resulting from enzymatic and mechanical digestions. Mitomycin-C-treated Sertoli cells were used to prepare the feeder layer. The stem cells were then cultured on the Sertoli cell feeder layer. The identification of the colonies was done through alkaline phosphatase staining methods and specific gene expression of ram’s SSCs (nanog and Plzf). The results of methylthiazolyldiphenyl-tetrazolium bromide assay on SSCs 72 h after culture with different treatments of quercetin demonstrated that the highest percentage of survival was for 5 μM and 10 μM concentrations, respectively; however, compared to the control, no significant difference was observed. In comparison with the control, the concentration equal to and greater than 20 μM quercetin caused a significant decrease in the survival of SSCs (P < 0.05). Seven days after culture, 40 μM quercetin caused a substantial reduction in the mean number of colonies, compared to the control (P < 0.05). The results demonstrated that compared to the control, 5 μM to 40 μM of quercetin significantly reduced Plzf gene expression. Furthermore, the concentration equal to and higher than 10 μM quercetin significantly decreased bcl-2 gene expression in the cells under study (P < 0.05). Based on the findings of the present study, the use of quercetin for the primary culture of ovine SSCs is not recommended. It is suggested that the function of this antioxidant should be investigated on the differentiation of SSCs.

The Role of MicroRNA 143 and MicroRNA 206 in The Regulation of Apoptosis in Mouse Lukemia Cancer Cells and Spermatogonial Cells.

Objective In cancer treatments, smart gene delivery via nanoparticles (NPs) can be targeted for cancer cells, while concurrently minimizing damage to healthy cells. This study assessed the efficiency of poly lactic-co-glycolic acid (PLGA)-miR 143/206 transfection on apoptosis in mouse leukemia cancer cells (El4) and spermatogonial stem cells (SSCs). Materials and Methods In this experimental study, neonatal mouse spermatogonia cells and EL4 cancer cell lines were used. MicroRNA-PLGA NPs were prepared, characterized, and targeted with folate. Several doses were evaluated to obtain a suitable miR dose that can induce appropriate apoptosis in EL4 cells, while not harming SSCs. Cells were treated separately at 3 doses of each miR (for miR 143, doses of 25, 50 and 75 nmol and for miR 206, doses of 50, 100 and 150 nmol). The experiments were performed at 24, 48 and 72 hours. Viability and apoptosis were investigated by MTT and Annexin Kits. Results Based on MTT assay results, the optimal dose of miR 143 was 75 nmol (59.87 ± 2.85 % SSC and 35.3 ± 0.78 % EL4) (P≤0.05), and for miR 206, the optimal dose was 150 nmol (54.82 ± 6.7 % SSC and 33.92 ± 3.01% EL4) (P≤0.05). The optimal time was 48 hours. At these doses, the survival rate of the EL4 cells was below the half maximal inhibitory concentration (IC50) and SSC survival was above 50%. Annexin V staining also confirmed the selected doses (for miR 143 total apoptosis was 6.62% ± 1.8 SSC and 37.4% ± 4.2 EL4 (P≤0.05), and miR 206 was (10.98% ± 1.5 SSC and 36.4% ± 3.7 EL4, P≤0.05). ConclusionUsing intelligent transfection by NPs, we were able to induce apoptosis on EL4 cells and maintain acceptable SSC survival rates.

The Histone Methyltransferase SETDB1 Modulates Survival of Spermatogonial Stem/Progenitor Cells Through NADPH Oxidase.

SETDB1, a histone H3 lysine 9 (H3K9) methyltransferase, is crucial in meiosis and embryo development. This study aimed to investigate whether SETDB1 was associated with spermatogonial stem cells (SSC) homeostasis. We found that knockdown of Setdb1 impaired cell proliferation, led to an increase in reactive oxygen species (ROS) level through NADPH oxidase, and Setdb1 deficiency activated ROS downstream signaling pathways, including JNK and p38 MAPK, which possibly contributed to SSC apoptosis. Melatonin scavenged ROS and rescued the phenotype of Setdb1 KD. In addition, we demonstrated that SETDB1 regulated NADPH oxidase 4 (Nox4) and E2F1. Therefore, this study uncovers the new roles of SETDB1 in mediating intracellular ROS homeostasis for the survival of SSC.

Spermatogonial stem cell survival in ram lambs following busulfan treatment.

To clarify the effect of busulfan on the depletion of spermatogonial stem cells (SSCs) from shal rams testis, in the first experiment, lambs were treated by intraperitoneal injection with 4 mg/kg busulfan. In the second experiment, different concentrations of busulfan (1, 2 and 4 mg/kg) were injected directly into both sides of the left testis. The testes of 8 lambs were collected by standard castration procedure for histological analysis five weeks after the treatments and the left testis of remaining lambs were collected after eight weeks and a two-time enzymatic digestion process was used to isolate SSCs. The results showed that all rams that had received intraperitoneal injections of busulfan died. But by testicular injecting of same dose of the drug, 40% of the animals died. The testicular injection of rams with 1, 2 and 4 mg/kg of busulfan resulted in a dose dependent decrease in testis size and also spermatocytes population after 5 weeks of treatments. From the results of colony formation 8 weeks after treatment with busulfan, it can be concluded that only in 1 and 2 mg/kg of busulfan, recovery of endogenous germ cells was performed. In conclusion, the results demonstrated that intra-testicular injections of busulfan (2 mg/kg) reduced spermatocytes population in ram testis within 5 weeks of treatments, and this effect was reversible within 8 weeks of injection. However, it was not recommended to inject 4 mg/kg busulfan into the peritoneal cavity or testis of lambs based on its side effects.

Fibroblast growth factor-5 promotes spermatogonial stem cell proliferation via ERK and AKT activation

BackgroundSertoli cells are the most important somatic cells contributing to the microenvironment (named niche) for spermatogonial stem cells (SSCs). They produce amounts of crucial growth factors and structure proteins that play essential roles in the complex processes of male SSCs survival, proliferation, and differentiation. It has been suggested that Sertoli cell abnormalities could result in spermatogenesis failure, eventually causing azoospermia in humans. However, to the end, the gene expression characteristics and protein functions of human Sertoli cells remained unknown. In this study, we aimed to evaluate the effect of fibroblast growth factor-5 (FGF5), a novel growth factor downregulated in Sertoli cells from Sertoli cell-only syndrome (SCOS) patients compared to Sertoli cells from obstructive azoospermia (OA) patients, on SSCs.MethodsWe compared the transcriptome between Sertoli cell from SCOS and OA patients. Then, we evaluated the expression of FGF5, a growth factor which is downregulated in SCOS Sertoli cells, in human primary cultured Sertoli cells and testicular tissue. Also, the proliferation effect of FGF5 in mice SSCs was detected using EDU assay and CCK-8 assay. To investigate the mechanism of FGF5, Phospho Explorer Array was performed. And the results were verified using Western blot assay.ResultsUsing RNA-Seq, we found 308 differentially expressed genes (DEGs) between Sertoli cells from SCOS and OA patients. We noted and verified that the expression of fibroblast growth factor-5 (FGF5) was higher in Sertoli cells of OA patients than that of SCOS patients at both transcriptional and translational levels. Proliferation assays showed that rFGF5 enhanced the proliferation of mouse SSCs line C18-4 in a time- and dose-dependent manner. Moreover, we demonstrated that ERK and AKT were activated and the expression of Cyclin A2 and Cyclin E1 was enhanced by rFGF5.ConclusionThe distinct RNA profiles between Sertoli cells from SCOS and OA patients were identified using RNA-Seq. Also, FGF5, a growth factor that downregulated in SCOS Sertoli cells, could promote SSCs proliferation via ERK and AKT activation.

Histone methyltransferase SETDB1 maintains survival of mouse spermatogonial stem/progenitor cells via PTEN/AKT/FOXO1 pathway

Spermatogonial stem cells (SSCs) possess the capacity of self-renewal and differentiation, which are the basis of spermatogenesis. In maintenance of SSC homeostasis, intrinsic/extrinsic factors and various signaling pathways tightly control the fate of SSCs. Methyltransferase SETDB1 (Set domain, bifurcated 1) catalyzes histone H3 lysine 9 (H3K9) trimethylation and represses gene expression. SETDB1 is required for maintaining the survival of spermatogonial stem cells in mice. However, the underlying molecular mechanism remains unclear. In the present study, we found that Setdb1 regulates PTEN/AKT/FOXO1 pathway to inhibit SSC apoptosis. Co-immunoprecipitation and reporter gene assay revealed that SETDB1 interacted and coordinated with AKT to regulate FOXO1 activity and expression of the downstream target genes Bim and Puma. Among the SETDB1-bound genes, the H3K9me3 levels on the promoter regions of Bim and Pten decreased in Setdb1-KD group; in contrast, H3K9me3 status on promoters of Bax and Puma remained unchanged. Therefore, SETDB1 was responsible for regulating the transcription activity of genes in the apoptotic pathway at least in part through modulating H3K9me3. This study replenishes the research on the epigenetic regulation of SSC survival, and provides a new insight for the future study of epigenetic regulation of spermatogenesis.

Yangjing Capsule extract promotes proliferation of GC-1 spg cells via up-regulated POU3F1 pathway.

As is similar to glial cell line-derived neurotrophic factor (GDNF), the Yangjing Capsule (YC) extract could also lead to proliferation of spermatogonial stem cells (SSCs) by stimulating the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway; however, the regulatory effect of YC extract on the expression of POU3F1 still remains unknown. The objective of this study is to determine whether the transcription factor POU3F1 is up-regulated by YC extract through the PI3K/AKT signaling pathway to regulate SSCs survival and proliferation. Cultured GC-1 spermatogonial (spg) cells were treated with 0.01, 0.1, and 1 mg/mL YC extract for 48 h. Cell viability was analyzed using MTT assay, while POU3F1 expression was quantitatively detected using real time-polymerase chain reaction and Western blot analysis. POU3F1, GDNF family receptor alpha1 (GFRα1) short interfering ribonucleic acid (siRNA), and LY294002 (PI3K inhibitor) were applied as blockers to explore the underlying pathway. After 48 h treatment with YC extract, GC-1 spg cells proliferated and POU3F1 expression was significantly increased in a dose-dependent manner. POU3F1 siRNA partially blocked those effects of YC extract. Both GFRα1 siRNA and LY294002, as upstream blockers, reduced POU3F1 expression induced by YC extract. The conclusion is that YC extract promotes proliferation of GC-1 spg cells via up-regulation of POU3F1. The potential mechanism is that YC extract triggers the activation of the PI3K/AKT pathway and then up-regulates POU3F1 expression.

Long-term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring.

Tree shrews have a close relationship to primates and have many advantages over rodents in biomedical research. However, the lack of gene manipulation methods has hindered the wider use of this animal. Spermatogonial stem cells (SSCs) have been successfully expanded in culture to permit sophisticated gene editing in the mouse and rat. Here, we describe a culture system for the long-term expansion of tree shrew SSCs without the loss of stem cell properties. In our study, thymus cell antigen 1 was used to enrich tree shrew SSCs. RNA-sequencing analysis revealed that the Wnt/β-catenin signaling pathway was active in undifferentiated SSCs, but was downregulated upon the initiation of SSC differentiation. Exposure of tree shrew primary SSCs to recombinant Wnt3a protein during the initial passages of culture enhanced the survival of SSCs. Use of tree shrew Sertoli cells, but not mouse embryonic fibroblasts, as feeder was found to be necessary for tree shrew SSC proliferation, leading to a robust cell expansion and long-term culture. The expanded tree shrew SSCs were transfected with enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors. After transplantation into sterilized adult male tree shrew's testes, the EGFP-tagged SSCs were able to restore spermatogenesis and successfully generate transgenic offspring. Moreover, these SSCs were suitable for the CRISPR/Cas9-mediated gene modification. The development of a culture system to expand tree shrew SSCs in combination with a gene editing approach paves the way for precise genome manipulation using the tree shrew.

A long non-coding RNA interacts with Gfra1 and maintains survival of mouse spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are unique male germline stem cells that support spermatogenesis and male fertility. Long non-coding RNAs (lncRNA) have been identified as key regulators of stem cell fate; however, their role in SSCs has not been explored. Here, we report that a novel spermatogonia-specific lncRNA (lncRNA033862) is essential for the survival of murine SSCs. LncRNA033862 is expressed in early spermatogonia including SSC and was among 805 lncRNAs identified by global expression profiling as responsive to glial cell-derived neurotrophic factor (GDNF), a growth factor required for SSC self-renewal and survival. LncRNA033862 is an antisense transcript of the GDNF receptor alpha1 (Gfra1) that lacks protein coding potential and regulates Gfra1 expression levels by interacting with Gfra1 chromatin. Importantly, lncRNA033862 knockdown severely impairs SSC survival and their capacity to repopulate recipient testes in a transplantation assay. Collectively, our data provide the first evidence that long non-coding RNAs (lncRNAs) regulate SSC fate.

Henna, An Antimicrobial Herbal Medicine With Less Negative Effect on Spermatogonial Stem Cell Culture

Background: Herbal extracts have recently received the greatest attention in the path of finding naturally occurring chemicals with antibacterial and therapeutic value; however, each type of herbal remedy may have its own side effects. Objectives: The aim of the current experiment was to study the antibacterial effect of myrtle, parsley, mint, henna and chamomile extracts on Escherichia coli and their effects on colony formation and survival of spermatogonial stem cells (SSCs). Materials and Methods: Spermatogonial stem cells were isolated by two-time enzymatic digestion from slaughterhouse origin ovine testis and plant extraction by deionized water. Comparisons between different treatments were performed using analysis of variance (ANOVA) followed by Duncan’s multiple range tests. Results: The results showed that there was no significant difference between mint, henna and penicillin, on inhibition of Escherichia coli growth, however parsley, myrtle and chamomile were significantly different from penicillin (P < 0.05). The formation of SSC colonies was not affected by different herbal medicines. Myrtle and chamomile resulted in death of SSC colonies compared with the control, and the survival of spermatogonial and Sertoli cells were significantly affected by myrtle, mint, chamomile and parsley (P < 0.05). Conclusions: The results of these experiments provide evidence that henna by antibacterial activity had no detrimental affect on SSC and Sertoli cells and is a good candidate for substitution of antibiotics.

Identification of CHD1L as an Important Regulator for Spermatogonial Stem Cell Survival and Self-Renewal

Chromodomain helicase/ATPase DNA binding protein 1-like gene (Chd1l) participates in chromatin-dependent processes, including transcriptional activation and DNA repair. In this study, we have found for the first time that Chd1l is mainly expressed in the testicular tissues of prepubertal and adult mice and colocalized with PLZF, OCT4, and GFRα1 in the neonatal mouse testis and THY1+ undifferentiated spermatogonia or spermatogonial stem cells (SSCs). Knockdown of endogenous Chd1l in cultured mouse undifferentiated SSCs inhibited the expression levels of Oct4, Plzf, Gfrα1, and Pcna genes, suppressed SSC colony formation, and reduced BrdU incorporation, while increasing SSC apoptosis. Moreover, the Chd1l gene expression is activated by GDNF in the cultured mouse SSCs, and the GDNF signaling pathway was modulated by endogenous levels of Chd1l; as demonstrated by the gene expression levels of GDNF, inducible transcripts Etv5, Bcl6b, Pou3f, and Lhx1, but not that of GDNF-independent gene, Taf4b, were significantly downregulated by Chd1l knockdown in mouse SSCs. Taken together, this study provides the first evidence to support the notion that Chd1l is an intrinsic and novel regulator for SSC survival and self-renewal, and it exerts such regulation at least partially through a GDNF signaling pathway.

Left-Biased Spermatogenic Failure in 129/SvJ Dnd1Ter/+ Mice Correlates with Differences in Vascular Architecture, Oxygen Availability, and Metabolites.

Homozygosity for the Ter mutation in the RNA-binding protein Dead end 1 (Dnd1(Ter/Ter)) sensitizes germ cells to degeneration in all mouse strains. In 129/SvJ mice, approximately 10% of Dnd1(Ter/+) heterozygotes develop spermatogenic failure, and 95% of unilateral cases occur in the left testis. The first differences between right and left testes were detected at Postnatal Day 15 when many more spermatogonial stem cells (SSCs) were undergoing apoptosis in the left testis compared to the right. As we detected no significant left/right differences in the molecular pathway associated with body axis asymmetry or in the expression of signals known to promote proliferation, differentiation, and survival of germ cells, we investigated whether physiological differences might account for asymmetry of the degeneration phenotype. We show that left/right differences in vascular architecture are associated with a decrease in hemoglobin saturation and increased levels of HIF-1alpha in the left testis compared to the right. In Dnd1 heterozygotes, lower oxygen availability was associated with metabolic differences, including lower levels of ATP and NADH in the left testis. These experiments suggest a dependence on oxygen availability and metabolic substrates for SSC survival and suggest that Dnd1(Ter/+) SSCs may act as efficient sensors to detect subtle environmental changes that alter SSC fate.

Persistent DNA Damage in Spermatogonial Stem Cells After Fractionated Low-Dose Irradiation of Testicular Tissue

Testicular spermatogenesis is extremely sensitive to radiation-induced damage, and even low scattered doses to testis from radiation therapy may pose reproductive risks with potential treatment-related infertility. Radiation-induced DNA double-strand breaks (DSBs) represent the greatest threat to the genomic integrity of spermatogonial stem cells (SSCs), which are essential to maintain spermatogenesis and prevent reproduction failure. During daily low-dose radiation with 100mGy or 10mGy, radiation-induced DSBs were monitored in mouse testis by quantifying 53 binding protein 1 (53BP-1) foci in SSCs within their stem cell niche. The accumulation of DSBs was correlated with proliferation, differentiation, and apoptosis of testicular germ cell populations. Even very low doses of ionizing radiation arrested spermatogenesis, primarily by inducing apoptosis in spermatogonia. Eventual recovery of spermatogenesis depended on the survival of SSCs and their functional ability to proliferate and differentiate to provide adequate numbers of differentiating spermatogonia. Importantly, apoptosis-resistant SSCs resulted in increased 53BP-1 foci levels during, and even several months after, fractionated low-dose radiation, suggesting that surviving SSCs have accumulated an increased load of DNA damage. SSCs revealed elevated levels of DSBs for weeks after radiation, and if these DSBs persist through differentiation to spermatozoa, this may have severe consequences for the genomic integrity of the fertilizing sperm.

The Trp53-Trp53inp1-Tnfrsf10b Pathway Regulates the Radiation Response of Mouse Spermatogonial Stem Cells

SummaryGerm cells are thought to exhibit a unique DNA damage response that differs from that of somatic stem cells, and previous studies suggested that Trp53 is not involved in the survival of spermatogonial stem cells (SSCs) after irradiation. Here, we report a critical role for the Trp53-Trp53inp1-Tnfrsf10b pathway during radiation-induced SSC apoptosis. Spermatogonial transplantation revealed that Trp53 deficiency increased the survival of SSCs after irradiation. Although Bbc3, a member of the intrinsic apoptotic pathway, was implicated in apoptosis of germ and somatic stem cells, Bbc3 depletion inhibited apoptosis in committed spermatogonia, but not in SSCs. In contrast, inhibition of Tnfrsf10b, an extrinsic apoptosis regulator, rescued SSCs. Tnfrsf10b, whose deficiency protected SSCs, was upregulated by Trp53inp1 upon irradiation. These results suggest that the Trp53-Trp53inp1-Tnfrsf10b pathway responds to genotoxic damage in SSCs and that stem and progenitor cells exhibit distinct DNA damage responses in self-renewing tissue.

Vitrification preserves proliferation capacity in human spermatogonia

Does vitrification of human immature testicular tissue (ITT) have potential benefits for future fertility preservation? Does vitrification of human ITT have potential benefits in an in vivo murine xenotransplantation model? Vitrification is able to maintain proliferation capacity in spermatogonial cells after 6 months of xenografting. Controlled slow-freezing is the procedure currently applied for ITT cryobanking in clinical practice. Vitrification has been proposed as a promising technique for long-term storage of ITT, with a view to preserving spermatogonial stem cells (SSCs) for future fertility restoration in young boys suffering from cancer. After vitrification of ITT, in vitro survival of SSCs was demonstrated, but their functionality was not evaluated. Ten ITT pieces issuing from 10 patients aged 2-12 years were used. Fragments of fresh tissue (serving as controls) and fresh, frozen-thawed and vitrified-warmed testicular pieces xenografted to the scrotum of nude mice for 6 months were compared. Upon graft removal, histological and immunohistochemical analyses were performed to evaluate spermatogonia (SG) (MAGE-A4), intratubular proliferation (Ki67), proliferating SG and Leydig cells (3β-HSD). The entire piece of grafted tissue was assessed in each case. Seminiferous tubules showed good integrity after cryopreservation and xenografting for 6 months in all three groups. Survival of SG and their ability to proliferate was observed by immunohistochemistry in all grafted groups. SG were able to initiate spermatogenesis, but blockage at the pachytene stage was observed. The recovery rate of SG was 3.4 ± 3.8, 4.1 ± 7.3 and 7.3 ± 6.3%, respectively, for fresh, slow-frozen and vitrified-warmed tissue after 6 months of xenografting. The study is limited by the low availability of ITT samples of human origin. The mouse xenotransplantation model needs to be refined to study human spermatogenesis. The findings of the present study have potential implications for cryobanking of ITT and fertility preservation. Spermatogonial loss recorded after fresh ITT transplantation indicates that the avascular grafting technique needs to be optimized. There are so far no convincing data justifying modification of current clinical practice for ITT storage with slow-freezing, but this study demonstrates that it is worth pursuing optimization of ITT vitrification as an alternative for preservation of SSCs. The present study was supported by a grant from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie N° 7. 4.572.09.F). The authors declare that there is no conflict of interest.