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Alkylating agents and irradiation induce testicular damage, which results in prolonged azoospermia. Even very low doses of radiation can significantly impair testis function. However, re-irradiation is an effective strategy for locally targeted treatments and the pain response and has seen important advances in the field of radiation oncology. At present, little is known about the relationship between the harmful effects and accumulated dose of irradiation derived from continuous low-dose radiation exposure. In this study, we examined the levels of mRNA transcripts encoding markers of 13 markers of germ cell differentiation and 28 Sertoli cell-specific products in single- and re-irradiated mice. Our results demonstrated that re-irradiation induced significantly decreased testicular weights with a significant decrease in germ cell differentiation mRNA species ( Spo11 , Tnp1 , Gfra1, Oct4, Sycp3, Ddx4 , Boll, Crem, Prm1, and Acrosin ). In the 13 Sertoli cell-specific mRNA species decreased upon irradiation, six mRNA species ( Claudin-11, Espn, Fshr, GATA1, Inhbb , and Wt1 ) showed significant differences between single- and re-irradiation. At the same time, different decreases in Sertoli cell-specific mRNA species were found in single-irradiation ( Aqp8 , Clu , Cst12 , and Wnt5a ) and re-irradiation ( Tjp1, occludin, ZO -1, and ZO -2) mice. These results indicate that long-term aspermatogenesis may differ after single- and re-irradiated treatment.

Rationale: Radiotherapy has become a mainstay for tumor management, and more than 50% of patients with thoracic tumor need to be treated with radiotherapy. However, the potential adverse effects of thoracic radiotherapy on the reproductive system remain elusive. Methods: Western blot analysis, immunofluorescence assay and transmission electron microscopy (TEM) analysis were performed to investigate the integrity of blood-testis barrier (BTB) in male mice after hypofractionated irradiation (IR) on the right thorax. RNA sequencing, co-immunoprecipitation (IP), Duolink PLA and inhibitor experiments were carried out to demonstrate the molecular mechanisms of the BTB dynamics changes and the subsequent reproductive effect. Results: It was found that the hypofractionated IR on right thorax evoked ultrastructural destruction in distant testes, and thus caused radiation-induced abscopal reproductive effect (RIARE) in male mice. Mechanistically, thoracic IR induced significant nuclear translocation of Rac Family Small GTPase 1 (Rac1) in abscopal Sertoli cells, which closely correlated with the activation of TNF-α/p38 mitogen activated protein kinase (MAPK) pathway. Of note, YWHAZ, a critical polarity protein, was found to be co-localized with Rac1 in Sertoli cells, and this interaction was indispensable for thoracic IR-induced Rac1 nuclear translocation and subsequent degradation of BTB-associated proteins. Conclusions: Our findings imply for the first time that YWHAZ-mediated Rac1 nuclear translocation plays central roles in RIARE, and TNF-α/p38 MAPK/Rac1 axis can be employed as a therapeutic target against RIARE for young male patients receiving hypofractionated radiotherapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The aim of this study was to evaluate the effect of photobiomodulation (PBM) on testicular tissues and fresh sperm analysis factors in streptozotocin (STZ)-induced type one diabetes mellitus (T1DM) mice. T1DM was induced in 15 male Syrian mice by injection of 200 mg/kg STZ. After one month, mice were divided randomly into three groups, harboring 5 mice each: 1, control group; 2, first laser group (890 nm, 80 Hz, 0.03 J/cm 2 ) and 3, second laser group (0.2 J/cm 2 ). Then the mice were euthanized and testicles were dissected for stereological studies, and both epididymis and vas deferens were removed for fresh sperm analysis. Data were analyzed by statistical methods. A significant increase was observed in the Sertoli cell count in both PBM groups, compared to the control group. In addition, the second PBM group shows a significant increase in the Sertoli cell count, compared to the first PBM group. Both PBM groups show significant increase in the Leydig cell count, compared to the control group. There were significant increases of the length in the seminiferous tubules in both PBM groups, compared to the control group. In addition, the second PBM group showed a significant increase of the length in the seminiferous tubules, compared to the first PBM group. The second PBM group showed a significant increase in the sperm count, compared to the control, and first PBM groups. The first PBM group showed a significant increase in sperm count, compared to the control group. The sperm motility and count were significantly increased in the second PBM group, compared to the control and first PBM groups. The sperm motility was significantly increased in the first PBM group, compared to the control group. PBM with 0.2 J/cm 2 and 0.03 J/cm 2 energy densities significantly improved the stereological parameters and fresh sperm analysis factors, compared to the control group in STZ-induced T1DM in mice. Moreover, the PBM with 0.2 J/cm 2 energy density was statistically more effective, compared to the 0.03 J/cm 2 ., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: it has been demonstrated in various experimental studies that radiation exposure produces a negative impact on the processes of spermatogenesis associated with the disturbances of the microcirculation processes in the testes and the development of cellular and intracellular disintegration expressed as destructive changes in the cells leading to their death., Aim: The objective of the present study was to detect the ultrastructural abnormalities in the cells of Sertoli and spermatogonia under conditions of their exposure to radiation and to identify the peculiarities of their regeneration under the influence of the therapeutic and prophylactic application of low-intensity ultra-high frequency (UHF) electromagnetic radiation (EMR) and low-intensity low-frequency magnetic field (MF)., Material and Methods: The experiments were carried out on 28 non-pedigree mature male rats with the body weight 180-220 g that were divided into four groups. The first study group was comprised of the animals exposed to radiation followed by the application of low-intensity ultra-high frequency UHF electromagnetic radiation EMR. The rats in the second study group experienced effects of radiation and low-intensity low-frequency MF. The animals of the third (control) group were exposed to radiation alone, and those comprising the fourth group 1 (only radiation exposure) were considered to be intact., Results: The studies with the use of electron microscopy showed that the therapeutic and prophylactic application of low-intensity ultra-high frequency (UHF) electromagnetic radiation and low-intensity low-frequency magnetic field caused the decrease in the number and the severity of post-radiation defects in the treated cells together with the increase of the number and size of mitochondria as well as hyperplasia of ribosomes; moreover, it promoted cellular and intracellular regeneration. UHF electromagnetic radiation had a more pronounced stimulating effect on the regeneration processes as compared with low-frequency MF. Particularly active processes of intracellular regeneration evolved in Sertoli cells; they were manifested as the increase in the number and size of mitochondria, enhanced hyperplasia of ribosomes, and formation of polysomes and new membranes of the granular endoplasmic reticulum. In spermatogonia, intracellular regeneration was less pronounced than in the Sertoli cells but was accompanied by enhanced cell regeneration and a greater number of reserve stem/progenitor cells., Conclusions: The results of the present study provide a rationale for the possibility of the application of a low-frequency magnetic field and especially UHF electromagnetic radiation for the further development of the promising therapeutic and preventive technologies with a view to their introduction into routine clinical practice dealing with radiation-induced pathology.

Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G 1 -like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid . Applying the same dose of IR to Prdkc -/- and Ku -/- mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc -/- cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku -/- MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.

Study Question: Does irradiation evoke adverse effects in germ and somatic cells in testis xenografts from prepubertal monkeys?, Summary Answer: In addition to the expected depletion of germ cells, a dose-dependent effect of irradiation was observed at the mRNA and protein level in Sertoli and peritubular myoid cells., What Is Known Already: Testicular irradiation studies in monkeys have focused on the dose-dependent effects on germ cells. Previous studies using intact animals or xenografts reported that germ cells are highly sensitive to irradiation. Their depletion was demonstrated by morphometric and histological analyses. The effect of irradiation on expression of Sertoli and peritubular myoid cell markers, however, has not yet been described., Study Design, Size, Duration: The testes of two prepubertal macaques (Macaca fascicularis) were dissected into testicular fragments. Fragments were randomly exposed in vitro to one of the following three doses of irradiation: 0 Gy, n = 60; 1 Gy, n = 54; 4 Gy, n = 72. Non-irradiated control fragments (0 Gy) were placed into the Faxitron for 6.6 min without irradiation. For 1 Gy and 4 Gy irradiation was applied for 1.7 and 6.6 min, respectively. Grafts were then either immediately analyzed or subcutaneously implanted under the back skin of 39 nude mice and analyzed after 6.5 months., Participants/materials Setting Methods: Post grafting, 133 testicular xenografts were retrieved. The body weight, serum testosterone level and seminal vesical weight of the host mice as well as the number and weight of retrieved grafts were determined. Larger grafts were used to evaluate both mRNA expression profiles and protein expression patterns. In total, 71 testicular fragments were used for morphometric and histological analysis while 68 fragments were analyzed for gene expression. For PCR arrays, M. fascicularis-specific primer sequences were employed. Irradiation-induced changes in the transcript levels of 34 marker genes were determined for each testicular graft. The effects of irradiation on peritubular myoid cells and Sertoli cells were confirmed by immunohistochemical analysis of chemokine (C-X-C motif) ligand type 11 (CXCL11), alpha smooth muscle actin (SMA) and chemokine (C-X-C motif) ligand type 12 (CXCL12)., Main Results and the Role of Chance: The four testes gave rise to 106 xenografts, which were individually analyzed, limiting the role of chance despite using only two monkeys in the study. Prior to grafting, the two donors displayed spermatogonia as the most advanced germ cell type in 95% and 70% of seminiferous tubules, respectively, while remaining tubules contained SCO. No spermatocytes were encountered prior to grafting in either monkey. After 6.5 months, non-irradiated grafts displayed spermatocytes in 15.4% and 1.8% of seminiferous tubules indicating an induction of meiosis. Irradiation resulted in a complete absence of spermatocytes. The percentage of seminiferous tubules containing spermatogonia declined in a dose-dependent manner. In non-irradiated xenografts, ~40% of tubules contained spermatogonia. This proportion was reduced to 3.4% and 4.3% in the 1 Gy treated group and to 1.3% and 0.2% in 4 Gy irradiated grafts. A dose-dependent decline in mRNA levels of selected germ cell marker genes supported the morphologically detected loss of germ cells. Irradiation had no effect on CXCL12 transcript levels. At the protein level, CXCL12-positive Sertoli cells were most abundant in the 1 Gy group compared to the 4 Gy group (P < 0.05), indicating a potential role of CXCL12 during recovery of primate spermatogenesis. The most prominent radiation-evoked changes were for CXCL11, which was localized to smooth muscle cells of blood vessels and seminiferous tubules. Transcript levels declined in a dose-dependent manner in grafts from both monkeys (MM687: P < 0.01 (0 Gy versus 4 Gy), MM627: P < 0.05 (0 Gy versus 4 Gy), P < 0.001 (1 Gy versus 4 Gy)). CXCL11 patterns of protein expression revealed irradiation-dependent changes as well. That peritubular cells are affected by X-irradiation was substantiated by changes at the transcript level between 1 and 4 Gy exposed groups (P < 0.01) and at the protein level of SMA (P < 0.05, 0 Gy versus 4 Gy)., Large Scale Data: n/a., Limitations, Reasons for Caution: The spermatogonial stem cell system in primates is remarkably different from rodents. Therefore, data from a non-human primate may be more relevant to man. However, species-specific differences amongst primates cannot be fully excluded and the use of only two donors may raise concerns toward the generalization of the findings. There may also be important differences across the prepubertal period (e.g. infancy, early childhood) that are not represented by the ages included in the present study., Wider Implications of the Findings: This study is the first to indicate relevant testicular somatic cell responses following irradiation of prepubertal primate tissue. In addition to the well-known depletion of germ cells, the changes in Sertoli, and in particular peritubular myoid, cells may have important consequences for spermatogenic recovery. These novel findings should be taken into consideration when irradiation effects are assessed in tumor survivors., Study Funding and Competing Interest(s): Interdisciplinary Center for Clinical Research (IZKF) Münster (Schl2/001/13) and the Excellence Cluster 'Cells in Motion' at the University Münster. There are no conflicts of interest to declare., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Objective: To investigate the influence of cellphone electromagnetic radiation (CER) on the testicular ultrastructure and the apoptosis of spermatogenic cells in male rats.atability, feasibility, applicability, and controllability in the construction of experimental animal models, we compared the major anatomic features of the penis of 20 adult beagle dogs with those of 10 adult men. Using microsurgical techniques, we performed cross-transplantation of the penis in the 20 (10 pairs) beagle dogs and observed the survival rate of the transplanted penises by FK506+MMF+MP immune induction. We compared the relevant indexes with those of the 10 cases of microsurgical replantation of the amputated penis., Methods: Thirty adult male SD rats were equally randomized into a 2 h CER, a 4 h CER, and a normal control group, the former two groups exposed to 30 days of 900 MHz CER for 2 and 4 hours a day, respectively, while the latter left untreated. Then the changes in the ultrastructure of the testis tissue were observed under the transmission electron microscope and the apoptosis of the spermatogenic cells was determined by TUNEL., Results: Compared with the normal controls, the rats of the 2 h CER group showed swollen basement membrane of seminiferous tubules, separated tight junction of Sertoli cells, increased cell intervals, apparent vacuoles and medullization in some mitochondria, and increased apoptosis of spermatogenic cells, mainly the apoptosis of primary spermatocytes (P<0.05 ). In comparison with the 2 h CER group, the animals of the 4 h CER group exhibited swollen basement membrane of seminiferous tubules, more separated tight junction of Sertoli cells, wider cell intervals, incomplete membrane of spermatogonial cells, fragments of cytoplasm, nuclear pyknosis and notch, slight dilation of perinuclear space, abnormalities of intracellular mitochondria with vacuoles, fuzzy structure, and fusion or disappearance of some cristae, and increased damage of mitochondria and apoptosis of spermatogenic cells, including the apoptosis of spermatogonial cells, primary spermatocytes, and secondary spermatocytes (P<0.05 )., Conclusions: CER can damage the testicular ultrastructure and increase the apoptosis of spermatogenic cells of the male rat in a time-dependent manner, and the apoptosis of spermatogenic cells may be associated with the damage to mitochondria.

Testicular development and function is the culmination of a complex process of autocrine, paracrine and endocrine interactions between multiple cell types. Dissecting this has classically involved the use of systemic treatments to perturb endocrine function, or more recently, transgenic models to knockout individual genes. However, targeting genes one at a time does not capture the more wide-ranging role of each cell type in its entirety. An often overlooked, but extremely powerful approach to elucidate cellular function is the use of cell ablation strategies, specifically removing one cellular population and examining the resultant impacts on development and function. Cell ablation studies reveal a more holistic overview of cell-cell interactions. This not only identifies important roles for the ablated cell type, which warrant further downstream study, but also, and importantly, reveals functions within the tissue that occur completely independently of the ablated cell type. To date, cell ablation studies in the testis have specifically removed germ cells, Leydig cells, macrophages and recently Sertoli cells. These studies have provided great leaps in understanding not possible via other approaches; as such, cell ablation represents an essential component in the researchers' tool-kit, and should be viewed as a complement to the more mainstream approaches to advancing our understanding of testis biology. In this review, we summarise the cell ablation models used in the testis, and discuss what each of these have taught us about testis development and function., (© 2015 The Authors. Andrology published by John Wiley & Sons Ltd on behalf of American Society of Andrology and European Academy of Andrology.)

As an efficient route to control pet overpopulation and develop neutered experimental animals, male sterilization via surgical techniques, chemical injections, and antifertility vaccines has brought particular attention recently. However, these traditional ways usually induce long-term adverse reactions, immune suppression, and serious infection and pain. To overcome the above limitations, we developed a platform in the present study by using plasmonic copper sulfide nanocrystals (Cu2-xS NCs) as intelligent light-driven sterilants with ideal outcomes. Upon NIR laser irradiation, these well-prepared Cu2-xS NCs can possess NIR-induced hyperthermia and generate high levels of reactive oxygen species (ROS). Due to the cooperation of photothermal and photodynamic effects, these nanocrystals exhibited NIR-mediated toxicity toward Sertoli cells both in vitro and in vivo in a mild manner. We attribute the potential mechanism of cellular injury to the apoptosis-related death and denaturation of protein in the testicles. Furthermore, the possible metabolism route and long-term toxicity of these nanocrystals after testicular injection indicate their high biocompatibility. Taking together, our study on the NIR-induced toxicity of Cu2-xS NCs provides keen insights for the usage of plasmonic nanomaterials in biomedicine.

The experiments on the outbred female rats using the electron microscopic technique have demonstrated that the application of ultrahigh frequency low-intensity electromagnetic radiation (LIEMR) with a flux density below 1 mCW/Cm2 and a frequency of approximately 1,000 MHz in the regime of primary prophylaxis and therapeutic-preventive action suppressed the development of the post-stress pathological ultrastructural changes and increased the activity of the regenerative processes in the Sertoli cells. It was shown that the developing adaptive and compensatory changes in the Sertoli cells most frequently involve the energy-producing structures (mitochondria) that undergo the enlargement of their average and total dimensions. Simultaneously, the amount of granular endoplasmic reticulum and the number of ribosomes increased while the intracellular links between the organelles strengthened and the reserve potential of the cells improved. It is concluded that the observed effects may be due to the action of both local and systemic regulation mechanisms.

Objective: To determine the effects of two different radiation doses on sperm parameters and the role of testosterone treatment on rat spermatogenesis., Methods: The experimental animal study was conducted at Marmara University, Istanbul, Turkey, from September 2012 to January 2013. Male Sprague Dawley 4-6 months old rats weighing 300-350g were randomely divided into 5 equal groups as control, low dose irradiation, testosterone administration following low dose irradiation, high dose irradiation, and testosterone administration following high dose irradiation. The animals were kept at a constant temperature in a room with 12h light and dark cycles. After the group-wise intervention, sperm concentration, testicular size, and histopathological examination of seminiferous tubules were noted. SPSS 10 was used for statistical analysis., Results: The 40 rats in the study were divided in 5 groups of 8(20%) each. In low dose radiation, adverse effects were only temporarily observed with the return of almost normal testicular function at the end of two months with or without testosterone supplementation. In contrast, in high dose radiation, hormonal treatment effect was controversial., Conclusions: Testosterone treatment had no significant effect upon recovery after irradiation. In order to prevent the untoward effects of radiation, shielding of the remaining testis in a proper manner is crucial to avoid the harmful effects of the scattered radiation.

Ionizing radiation has been shown to arrest spermatogenesis despite the presence of surviving stem spermatogonia, by blocking their differentiation. This block is a result of damage to the somatic environment and is reversed when gonadotropins and testosterone are suppressed, but the mechanisms are still unknown. We examined spermatogonial differentiation and Sertoli cell factors that regulate spermatogonia after irradiation, during hormone suppression, and after hormone suppression combined with Leydig cell elimination with ethane dimethane sulfonate. These results showed that the numbers and cytoplasmic structure of Sertoli cells are unaffected by irradiation, only a few type A undifferentiated (Aund) spermatogonia and even fewer type A1 spermatogonia remained, and immunohistochemical analysis showed that Sertoli cells still produced KIT ligand (KITLG) and glial cell line-derived neurotrophic factor (GDNF). Some of these cells expressed KIT receptor, demonstrating that the failure of differentiation was not a result of the absence of the KIT system. Hormone suppression resulted in an increase in Aund spermatogonia within 3 days, a gradual increase in KIT-positive spermatogonia, and differentiation mainly to A3 spermatogonia after 2 weeks. KITL (KITLG) protein expression did not change after hormone suppression, indicating that it is not a factor in the stimulation. However, GDNF increased steadily after hormone suppression, which was unexpected since GDNF is supposed to promote stem spermatogonial self-renewal and not differentiation. We conclude that the primary cause of the block in spermatogonial development is not due to Sertoli cell factors such (KITL\GDNF) or the KIT receptor. As elimination of Leydig cells in addition to hormone suppression resulted in differentiation to the A3 stage within 1 week, Leydig cell factors were not necessary for spermatogonial differentiation.

Background: Radiotherapy is used routinely to treat testicular cancer. Testicular cells vary in radio-sensitivity and the aim of this study was to investigate cellular and molecular changes caused by low dose irradiation of mice testis and to identify transcripts from different cell types in the adult testis., Methods: Transcriptome profiling was performed on total RNA from testes sampled at various time points (n = 17) after 1 Gy of irradiation. Transcripts displaying large overall expression changes during the time series, but small expression changes between neighbouring time points were selected for further analysis. These transcripts were separated into clusters and their cellular origin was determined. Immunohistochemistry and in silico quantification was further used to study cellular changes post-irradiation (pi)., Results: We identified a subset of transcripts (n = 988) where changes in expression pi can be explained by changes in cellularity. We separated the transcripts into five unique clusters that we associated with spermatogonia, spermatocytes, early spermatids, late spermatids and somatic cells, respectively. Transcripts in the somatic cell cluster showed large changes in expression pi, mainly caused by changes in cellularity. Further investigations revealed that the low dose irradiation seemed to cause Leydig cell hyperplasia, which contributed to the detected expression changes in the somatic cell cluster., Conclusions: The five clusters represent gene expression in distinct cell types of the adult testis. We observed large expression changes in the somatic cell profile, which mainly could be attributed to changes in cellularity, but hyperplasia of Leydig cells may also play a role. We speculate that the possible hyperplasia may be caused by lower testosterone production and inadequate inhibin signalling due to missing germ cells.

In utero irradiation (IR) and postnatal hyperthermia (HT) exposure cause infertility by decreasing spermatogenic colony growth and the number of sperm in rats. Four groups were used: (i) Control group, (ii) HT group (rats exposed to hyperthermia on the 10th postnatal day), (iii) IR group (rats exposed to IR on the 17th gestational day) and (iv) IR + HT group. Three and six months after the procedures testes were examined by light and electron microscopy. Some degenerated tubules in the HT group, many vacuoles in spermatogenic cells and degenerated tight junctions in the IR group, atrophic tubules and severe degeneration of tight junctions in the IR + HT group were observed. ZO-1 and occludin immunoreactivity were decreased and disorganized in the HT and IR groups and absent in the IR + HT group. The increase in the number of apoptotic cells was accompanied by a time-dependent decrease in haploid, diploid and tetraploid cells in all groups. Degenerative findings were severe after 6 months in all groups. The double-hit model may represent a Sertoli cell only model of infertility due to a decrease in spermatogenic cell and alterated blood-testis barrier proteins in rat., (© 2012 The Authors. International Journal of Experimental Pathology © 2012 International Journal of Experimental Pathology.)

Objective: To study the protective effects of puerarin on 60Co-y-induced acute injury of sertoli cells of testis in mice and the mechanisms for its radiation protection., Methods: Forty male Wistar mice were randomly divided into four groups, i. e., the normal group, the model group, the low dose puerarin group, and the high dose puerarin group, respectively, 10 in each. The lower abdomen and scrotal area of mice were irradiated with a single dose of 8 Gy 60Co-gamma ray for 517 s in the model and two puerarin groups. Twenty-four h after modeling, different concentrations of puerarin (at the daily dose of 230 and 450 mg/kg respectively) were given to mice in the low and high dose groups by gastrogavage, once daily for two successive weeks. Equal volume of physiological saline was given to mice in the normal group and the model group, once daily for two successive weeks. The morphology of the testicular tissues was observed. The levels of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and inhibin beta were determined by ELISA. The relative expressions of Inhibin beta mRNA in the testicular tissues were determined by Real-time quantitative PCR., Results: Sertoli cells and spermatogenic cells were injured at different degrees in the model group. They were improved in the low and high dose puerarin groups, more obviously in the high dose puerarin group. Compared with the normal group, the level of serum FSH in the model group was significantly improved, while the serum level of Inhibin beta and the expression of Inhibin beta mRNA obviously decreased, showing statistical difference (P<0.05). Compared with the model group, the levels of serum FSH, Inhibin beta, and the expression of Inhibin beta mRNA were significantly improved in the low and high dose puerarin groups (P<0.05), more obviously in the high dose puerarin group (P<0.01, P<0.05)., Conclusion: Chinese medicine monomer of puerarin had certain protective effects on 60Co-gamma-induced acute injury of Sertoli cells in mice.

Objective: To investigate whether the low-intensity electromagnetic waves transmitted by cell phones cause histopathological or ultrastructural changes in the testes of rats., Materials and Methods: Wistar-Kyoto male rats were placed into either a control group or a group that was exposed to an electromagnetic field (EMF). Two cell phones with Specific Absorbation Rate values of 1.58 were placed and left off in cages that housed 15 rats included in the control group, and four cell phones were placed and left on in cages that housed 30 rats included in the experimental group. After 3 months, weights, seminiferous tubule diameters, and spermatogenic cell conditions of all testes of the rats were evaluated. One half of each testis was examined also under an electron microscope., Results: No significant differences were observed between the testis weights, seminiferous tubule diameters, and histopathological evaluations between rats that had and had not been exposed to EMF. Electron microscope analysis revealed that the membrana propria thickness and the collagen fiber contents were increased and the capillary veins extended in the experimental group. Common vacuolization in the cytoplasm of the Sertoli cells, growth of electron-dense structures, and existence of large lipid droplets were noted as the remarkable findings of this study., Conclusion: Although the cells that had been exposed to long-term, low-dose EMF did not present any findings that were contrary to the control conditions, the changes observed during ultrastructural examination gave the impression that significant changes may occur if the study period were to be extended. Longer studies are needed to better understand the effects of EMFs on testis tissue., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In the testis, developing germ cells are dependent on supportive physical and paracrine interactions with Sertoli cells. The intimate nature of this relationship is demonstrated by the fact that a toxic insult compromising the stability of Sertoli cells will have deleterious effects on the associated germ cells. 2,5-Hexanedione (HD) and x-radiation (x-ray) are testicular toxicants, each with a unique cellular target. HD exposure disrupts microtubule function in Sertoli cells, and x-ray exposure causes double-strand breaks in the DNA of germ cells. Despite their differing modes of action, exposure to either toxicant has the similar ultimate effect of increased germ cell apoptosis. In this study, adult male F344 rats were exposed to 1% HD in the drinking water for 18 days with or without coexposure to 2 or 5 Gy x-ray 12 h prior to necropsy. Incidence of retained spermatid heads was increased in the HD and coexposure groups. Germ cell apoptosis was significantly increased in the x-ray and coexposure groups. There was a striking stage-dependent attenuation of apoptosis with coexposure compared with x-ray alone. Detailed histopathological analysis revealed a significant suppression of x-ray-induced germ cell apoptosis by HD pretreatment in stages I-VI of the seminiferous cycle, most noticeably at stages II/III. We hypothesize either that subacute HD pretreatment compromises the ability of the Sertoli cells to eliminate x-ray-damaged germ cells or that germ cells are more resistant to x-ray-induced damage, having adapted to a less supportive environment.

When a single dose of X-rays is applied to the adult rat testis, stem spermatogonia are damaged, and spermatogenesis is interrupted. Supported by Sertoli cells, spermatogenic cells that endure irradiation complete their differentiation and gradually leave the testis as spermatozoa. In this study, the in vivo changes taking place a number of weeks after irradiation revealed cell-specific features of testicular lipid classes. A linear drop, taking about six weeks, in testis weight, nonlipid materials, free cholesterol, and 22:5n-6-rich glycerophospholipids took place with germ cell depletion. Sphingomyelins and ceramides with nonhydroxy very long-chain polyenoic fatty acids (n-VLCPUFA) disappeared in four weeks, together with the last spermatocytes, whereas species with 2-hydroxy VLCPUFA lasted for six weeks, disappearing with the last spermatids and spermatozoa. The amount per testis of 22:5n-6-rich triacylglycerols, unchanged for four weeks, fell between weeks 4 and 6, associating these lipids with spermatids and their residual bodies, detected as small, bright lipid droplets. In contrast, 22:5n-6-rich species of cholesterol esters and large lipid droplets increased in seminiferous tubules up to week 6, revealing they are Sertoli cell products. At week 30, the lipid and fatty acid profiles reflected the resulting permanent testicular involution. Our data highlight the importance of Sertoli cells in maintaining lipid homeostasis during normal spermatogenesis.

Objective: To explore whether microwave radiation may cause injury of primary cultured Sertoli cells., Methods: The model of primary cultured Sertoli cells in vitro was established, which was radiated by microwave with average power density 0, 30 and 100 mW/cm(2) for five minutes. The changes of cell cycle, apoptosis and death, and intracellular Ca2+ concentration in the Sertoli cells were measured at sixth hours through Annexin V-PI double labeling and Fluo-3-AM labeling, flow cytometry combined with laser scanning confocal microscopy after microwave exposure., Results: The numbers of Sertoli cells were obviously reduced in G0-G1 and G2-M phase (62.57% +/- 3.22% and 8.25% +/- 1.75%) and increased in S phase (29.17% +/- 4.87%) compared with the control groups (79.18% +/- 0.24%, 11.17% +/- 0.50% and 9.64% +/- 0.62%) (P < 0.05 or P < 0.01), but the changes of rate of apoptosis and death and intracellular Ca2+ concentration showed no difference at 6 h after exposure to 30 mW/cm(2) microwave. There was a significant increase in the Sertoli cell counts of G0-G1 phase (87.69% +/- 1.32%), and decrease in the Sertoli cell counts of G2-M and S phase (7.41% +/- 0.60% and 4.87% +/- 0.91%) (P < 0.01). There was also a significant increase in intracellular Ca2+ concentration and rate of apoptosis and death (P < 0.05 or P < 0.01) at 6 h after exposure to 100 mW/cm(2) microwave., Conclusion: 100 mW/cm(2) microwave radiation may cause growth inhibition and increase of apoptosis and death in the primary cultured Sertoli cells. The increase of intracellular Ca2+ concentration is one of the injury mechanisms.

Infertility is a serious late effect in childhood cancer survivors. Little is known about acute irradiation effects in immature primate testis. Radiation defects have previously only been studied in postpubertal primates. Here we use the juvenile rhesus monkey as a preclinical model. We expose fragments of testicular tissue to 0, 0.5, 1.0, and 4.0 Gy irradiation in vitro. We then maintain the fragments in organ culture for 24-48 h or xenograft the fragments into nude mice for 4 months. Histological endpoints were determined to explore the cellular responses to the irradiation. At the highest dose, irradiation provoked an acute depletion of A-spermatogonia and a rise of apoptotic germ and Sertoli cells in organ culture. A dose-dependent decrease in the number of seminiferous tubules containing type A dark and type A pale spermatogonia was observed in irradiated xenografts. The number of Sertoli-cell only tubules increased respectively. Outgrowth of grafts was affected by the 4-Gy dose. Our observations reveal that irradiation evoked an immediate and sustained depletion of A-spermatogonia. We conclude that spermatogonia in the juvenile primate testis are highly sensitive to irradiation and that spermatogonial depletion and cessation of proliferation is an acute response. In contrast to adult testes, where such damage is immediately visible, this damage in immature testes becomes apparent only when spermatogonial insufficiency leads to spermatogenic failure, and thus infertility, at the onset of puberty. Our methods are applicable to immature human testis and might serve as powerful tool to study irradiation toxicity in the juvenile human testis.

Context: Germ cells formed during human fetal life are essential for fertility of the adult, and several studies have described an increasing frequency of male reproductive disorders, which may have a common origin in fetal life and which are hypothesized to be caused by endocrine disruptors. However, factors inducing a genotoxic stress may also be implicated., Objectives: We investigated the effect of gamma-irradiation on the functions of human fetal testis during the first trimester of gestation by using an organ culture system. Then we focused on the role of the p53 pathway in the observed effects., Results: Germ cells were highly sensitive to irradiation even at doses as low as 0.1 and 0.2 Gy. Indeed, for these doses, one third of germ cells died by apoptosis. Other germ cells were blocked in their cycle, but no repair seemed to occur, and longer culture with the highest dose used showed that they were destined to die. Sertoli cells were less affected, although their proliferation and the level of anti-Müllerian hormone were reduced. Irradiation had no effect on testosterone secretion or on the expression of steroidogenic enzymes by Leydig cells. After irradiation, p53 phosphorylated on serine 15 was detected from 1-24 h in all cell types. This activation of p53 was accompanied by an increase in mRNA levels of proapoptotic factors Bax and Puma, whereas that of antiapoptotic Bcl-2 remained unchanged. P21, which is responsible for cell cycle arrest, was also up-regulated 6, 30, and 72 h after irradiation. Finally, when we added pifithrin-alpha, a specific inhibitor of p53 functions, a significant decrease in irradiation-induced apoptosis in both germ and Sertoli cells was observed, indicating the involvement of the p53 pathway in irradiation-induced apoptosis., Conclusions: This study demonstrated here for the first time the great sensitivity of human fetal germ cells to genotoxic stress caused by ionizing radiation.

In reproductive toxicity assessment, in vitro systems can be used to determine mechanisms of action of toxicants. However, they generally investigate the immediate effects of toxicants, on isolated germ cells or spermatozoa. We report here the usefulness of in vitro cultures of rat spermatocytes and Sertoli cells, in conjunction with the Comet Assay to analyze the evolution of DNA strand-breaks and thus to determine DNA damage in germ cells. We compared cultures of normal and gamma-irradiated germ cells. In non-irradiated spermatocytes, the Comet Assay revealed the presence of DNA strand-breaks, which numbers decreased with the duration of the culture, suggesting the involvement of DNA repair mechanisms related to the meiotic recombination. In irradiated cells, the evolution of DNA strand-breaks was strongly modified. Thus our model is able to detect genotoxic lesions and/or DNA repair impairment in cultured spermatocytes. We propose this model as an in vitro tool for the study of genotoxic injuries on spermatocytes.

Experiments on male rats have established that preventive use of low-intensive impulse laser radiation (200-400 Hz) on the adrenals prevented and limited development of postradiation disorders of spermatogenesis in irradiated male rats and their progeny.

Retinoid supplementation has been therapeutically used against various human disorders. We and others have demonstrated that retinol treatment causes free radical generation and increased iron uptake, iron storage and oxidative damage, both in vitro and in vivo. Here, we investigate the possible synergistic effect of retinol on UV-mediated free radical generation, oxidative damage to biomolecules and decreased cellular viability in primary cultured mammalian cells. Retinol treatment (7 microM) resulted in a threefold increase in UV-mediated free radical generation and a 40%, increase in lipoperoxidation. DNA fragmentation and mitochondrial oxidative damage also increased significantly in retinol-supplemented UV-irradiated cultured cells as compared to UV-irradiated control cells, which were only treated with the solvent used to deliver the retinol (0.1% ethanol). All measurements were restored to control values when an iron chelator, 1,10-phenanthroline (100 microM), or an OH* scavenger, mannitol (1 mM), was co-administrated. Rather than protecting against free radical generation, retinol seems to enhance UV-mediated oxidative damage and decreases cellular viability in cultured cells. We suggest that retinol-enhanced iron uptake and storage and increased reactive oxygen species generated by the Fenton reaction may act synergistically with UV-irradiation in causing oxidative damage to cells.

Whole body gamma-ray irradiation of rats with caesium-137 (137Cs) at embryonic day 20 induced marked reduction of the weight of the testis. Body weight and other tissues, however, seemed to remain normal. By light microscopy, complete loss of germ cells was observed in the testis. Other components, such as Sertoli cells and interstitial cells, seemed to be normal. The testes from day 8 postpartum rats contained very few spermatogonia compared with newborn rats, indicating loss of germ cells between days 0 and 8. In the adult, 137Cs-irradiated testes showed two conspicuous features other than the loss of germ cells: empty vacuolar spaces between Sertoli cells and multilayered seminiferous tubule basal laminae (lamina densa). The junctional structures (ectoplasmic specializations) between Sertoli cells, however, seemed normal. The thickness of each layer of multilayered basal laminae was the same as that of normal rats and electron-lucent layers similar to lamina lucida were interposed between them. Of the empty vacuolar spaces between Sertoli cells, basal laminae bridge the gap. The basal laminae contained laminin, type IV collagen and heparan sulphate proteoglycan evenly distributed among layers, suggesting a normal composition. Rough estimation of the amount of basal laminae deposited in 137Cs-irradiated rats indicates that it is within a range similar to that in normal testis. These features imply that Sertoli cells are, in part, determined perinatally to produce basal laminae for germ-line cells.

Today, many patients, who are often young, undergo total body irradiation (TBI) followed by bone marrow transplantation. This procedure can have serious consequences for fertility, but the long-term intratesticular effects of this treatment in primates have not yet been studied. Testes and epididymides of rhesus monkeys that received doses of 4-8.5 Gy of TBI at 2-4 yr of age were studied 3-8 yr after irradiation. In all irradiated monkeys, at least some seminiferous tubule cross-sections lacked germ cells, indicating extensive stem cell killing that was not completely repaired by enhanced stem cell renewal, even after many years. Testes totally devoid of germ cells were only found in monkeys receiving doses of 8 Gy or higher and in both monkeys that received two fractions of 6 Gy each. By correlating the percentage of repopulated tubules (repopulation index) with testicular weight, it could be deduced that considerable numbers of proliferating immature Sertoli cells were killed by the irradiation. Because of their finite period of proliferation, Sertoli cell numbers did not recover, and potential adult testis size decreased from approximately 23 to 13 g. Most testes showed some dilated seminiferous tubules, indicating obstructed flow of the tubular fluid at some time after irradiation. Also, in 8 of the 29 irradiated monkeys, aberrant, densely packed Sertoli cells were found. The irradiation did not induce stable chromosomal translocations in spermatogonial stem cells. No apparent changes were seen in the epididymides of the irradiated monkeys, and the size of the epididymis adjusted itself to the size of the testis. In the irradiated monkeys, testosterone and estradiol levels were normal, whereas FSH levels were higher and inhibin levels lower when testicular weight and spermatogenic repopulation were low. It is concluded that irradiation before adulthood has considerable long-term effects on the testis. Potential testis size is reduced, repopulation of the seminiferous epithelium is generally not complete, and aberrant Sertoli cells and dilated tubules are formed. The latter two phenomena may have further consequences at still longer intervals after irradiation.

Background: The overproduction of ROS by ionizing irradiation induces cellular damage which can be reduced by specific molecules such as GSH and cytokines. The aim of this study was to determine the relationship between the well known radioresistance of Sertoli cells and astrocytes in vitro and the GSH level and IL-6 production after irradiation., Materials and Methods: Cell viability, GSH content and IL-6 production were assessed at different times after irradiation and for different doses, on rat Sertoli cells and astrocytes., Results: After irradiation we observed a dose-dependent increase in the intracellular total GSH level and IL-6 production as compared to the controls., Discussion: These results strongly suggest the key role of GSH and IL-6 in the mechanisms of response of radioresistant cells to gamma irradiation. One hypothesis is that the increase of GSH level and IL-6 production after irradiation contributes to the adaptative response to oxidative stress generated by gamma irradiation.

Interleukin 1(IL-1) and IL-6 are cytokines involved in the response to radiation and are known for their radioprotective properties with respect to total-body irradiation. We previously showed that after gamma irradiation of Sertoli cells (SC), we observed an increase in the activity of IL-6 but not of IL-1. The aim of this study was to see whether this response is a function of the differentiation of SC, to analyse the mechanisms responsible for this induction, and to test whether this cytokine has a radioprotective role on germ cells. Unlike IL-1, a dose-dependent increase of IL-6 activity in SC following gamma irradiation at high doses was observed at all ages studied. On the other hand, radio-induction observed at low doses (<1Gy) was dose-independent. IL-6 up-regulation resulted from transcriptional activation as shown by the use of specific inhibitors. The injection of IL-1 and IL-6 in mice prior to whole-body irradiation resulted in an increased survival rate. Moreover, cytokines protected DNA from remaining cells following irradiation as shown by comet assay on germ cells. In conclusion, IL-6 seems to constitute a good marker of exposure to gamma irradiation, both at low and high doses. In addition, we showed that IL-1 and IL-6 have a radioprotective effect at testicular level., (Copyright 2001 Academic Press.)

Purpose: By using as an experimental model the male mouse gonad, which contains both radiosensitive (germ) and radioresistant (somatic) cells, we have studied the growth factor (and/or receptor) expression of transforming growth factor-beta receptor (TGFbeta RI), stem cell factor (SCF), c-kit, Fas-L, Fas, tumor necrosis factor receptor (TNF R55), and leukemia inhibiting factor receptor (LIF-R) after local irradiation., Methods and Materials: Adult male mice were locally irradiated on the testes. Induction of apoptosis in the different testicular cell types following X-ray radiation was identified by the TdT-mediated dUTP Nick End Labeling (TUNEL) approach. Growth factor expression was evidenced by semiquantitative RT-PCR and Western blot analyses., Results: Apoptosis, identified through the TUNEL approach, occurred in radiosensitive testicular (premeotic) germ cells with the following kinetics: the number of apoptotic cells increased after 24 h (p < 0.001) and was maximal 48 h after a 2-Gy ionizing radiation (p < 0.001). Apoptotic cells were no longer observed 72 h after a 2-Gy irradiation. The number of apoptotic cells increased with the dose of irradiation (1-4 Gy). In the seminiferous tubules, the growth factor expression in premeiotic radiosensitive germ cells was modulated by irradiation. Indeed Fas, c-kit, and LIF-R expression, which occurs in (radiosensitive) germ cells, decreased 24 h after a 2-Gy irradiation, and the maximal decrease was observed with a 4-Gy irradiation. The decrease in Stra8 expression occurred earlier, at 4 h after a 2-Gy irradiation. In addition, a significant (p < 0.03) decrease in Stra8 mRNA levels was observed at the lowest dose used (0.5 Gy, 48 h). Moreover, concerning a growth factor receptor, such as TGFbeta RI, which is expressed both in radiosensitive and radioresistant cells, we observed a differential expression depending on the cell radiosensitivity after irradiation. Indeed, TGFbeta RI expression was increased after irradiation in interstitial radioresistant testicular cells in a dose- and time-dependent manner, while it decreased in seminiferous radiosensitive (germ cells) testicular cells. Such a differential expression between radioresistant and radiosensitive cells in TGFbeta RI levels was observed in terms of both mRNA and protein. In contrast, the growth factors specifically expressed in the somatic radioresistant (Sertoli) cells in the seminiferous tubules (SCF, Fas-L, TNF R55) were not affected by ionizing radiation (up to 4 Gy, 72 h)., Conclusion: Growth factor expression decreased in the radiosensitive testicular cells after irradiation. Such a decrease occurred before the detection of apoptosis using the TUNEL approach. TGFbeta RI mRNA levels decreased in the radiosensitive cells, whereas it increased in the radioresistant cells, suggesting that TGFbeta RI may represent a biomarker of the intrinsic radiosensitivity of cells.

Purpose: To investigate the response of germ and Sertoli cells to gamma-irradiation at two distinct periods of testicular development in rat foetuses., Materials and Methods: Pregnant rats were exposed to 60Co gamma-rays at days 15, 19 or 21 post-coitum (p.c.), at doses ranging from 0.1 to 1.5Gy, and at different dose-rates. Testicular weight, seminiferous tubule condition and the number of germ and Sertoli cells were measured at early and late times after irradiation. Apoptosis was studied by the ISEL method and p53 expression was studied by immunohistochemistry., Results: At high dose-rates (> or = 3.3 Gy min(-1)), 1.5 Gy radiation at day 15 p.c. had a short-term effect on germ cell survival. A large proportion of these cells rapidly underwent p53-independent apoptosis. Apoptotic cells were strongly clustered. The remaining germ cells divided and differentiated normally leading to a majority of normal tubules in the adult testis. However, at low dose-rate (0.6mGy min(-1)), much greater depopulation of the seminiferous tubules occurred. When irradiation was given at day 19 p.c., the same dose had a delayed effect on germ cells, leading to sterility. Sertoli cells had a normal survival for irradiation at day 15 p.c. Their proliferation became higher in prepubescent testis compared with controls, when irradiation occurred at day 19 p.c., Conclusion: The position of gonocytes in the cell cycle at the time of irradiation seems to be a determining parameter for inducing gonocyte apoptosis. The strong effect of irradiation on germ cells at very low dose-rate and the appearance of clusters of apoptotic gonocytes may be a consequence of the syncitial organization of germ cells, favouring their cell synchronisation or the transmission of death signalling when they are in a radiosensitive period.

How radiation blocks spermatogenesis in certain strains of rats, such as LBNF(1), is not known. Because the block depends on androgen, we propose that androgen affects Sertoli cell function in irradiated LBNF(1) rats, resulting in the failure of spermatogonial differentiation. To begin to identify genes that may participate in this irradiation-induced blockade of spermatogenesis, we investigated the expression of several Sertoli genes in response to irradiation. The expression of the PEM: homeobox gene from its androgen-dependent Sertoli-specific proximal promoter (Pp) was dramatically reduced more than 100-fold in response to irradiation. In contrast, most other genes and gene products reported to be localized to the Sertoli cell, including FSH receptor (FSHR), androgen receptor (AR), SGP1, and the transcription factor CREB, did not exhibit significant changes in expression, whereas transferrin messenger RNA (mRNA) expression dramatically increased in response to irradiation. Irradiation also decreased Pp-driven PEM: mRNA levels in mouse testes (approximately 10-fold), although higher doses of irradiation than in rats were required to inhibit PEM: gene expression in testes of mice, consistent with their greater radioresistance. The decrease in Pem gene expression in mouse testis was also selective, as the expression of CREB, GATA-1, and SGP1 were little affected by irradiation. We conclude that the dramatic irradiation-triggered reduction of Pem expression in Sertoli cells is a conserved response that may be a marker for functional changes in response to irradiation.

While germ cell regulation of Sertoli cells has been extensively explored in adult rats in vivo, in contrast, very little is known about germ cell influence on Sertoli cell function at the time when spermatogenesis begins and develops. In the present study various Sertoli cell parameters (number, testicular androgen binding protein (ABP) and testin, serum inhibin-B and, indirectly, follicle-stimulating hormone (FSH)) were investigated after the exposure of 19-day-old rats to a low dose of 3 Grays of gamma-rays. Differentiated spermatogonia were the primary testicular targets of the gamma-rays, which resulted in progressive maturation depletion, sequentially and reversibly affecting all germ cell classes. Testicular weight declined to a nadir when pachytene spermatocytes and spermatids were depleted from the seminiferous epithelium and complete or near complete recovery of spermatogenesis and testicular weight was observed at the end of the experiment. Blood levels of FSH and ABP were normal during the first 11 days after irradiation, when spermatogonia and early spermatocytes were depleted. While the number of Sertoli cells was not significantly affected by the irradiation, from days 11-66 after gamma-irradiation, ABP production declined and FSH levels increased when pachytene spermatocytes and spermatids were depleted and the recovery of these parameters was only observed when spermatogenesis was fully restored. Comparison of the pattern of change in serum levels of inhibin-B and testicular levels of testin and of germ cell numbers strongly suggest a relationship between the disappearance of spermatocytes and spermatids from the seminiferous epithelium and the decrease in levels of inhibin-B and increase in levels of testin from 7 to 36 days post-irradiation. Levels of testin and inhibin-B were restored before spermatogenesis had totally returned to normal. In conclusion, this in vivo study shows that pre-pubertal Sertoli cell function is under the complex control of various germ cell classes. This control presents clear differences when compared with that previously observed in adult animals and depends on the Sertoli cell parameter of interest, as well as on the germ cell type.

Purpose: To investigate the response of Sertoli cell function to 60Co gamma-rays., Materials and Methods: Rat Sertoli cells were exposed in vitro and in vivo to 60Co gamma-rays in the dose range 3 Gy to 48 Gy and at 3 Gy and 6 Gy respectively. Cell viability and transferrin and IL-6 production were measured at different times following irradiation., Results: This study confirms the resistance of in vitro irradiated rat Sertoli cells in the dose range 3 Gy to 48 Gy in terms of cell number. Radiation had no effect on the IL-1 activity of Sertoli cells. However, the experiments show that despite the absence of a macroscopic effect, Sertoli cells respond to ionizing radiation by increasing transferrin secretion, transferrin response to (Bu)2cAMP stimulation and IL-6 activity., Conclusions: Transferrin is involved in the transport of iron into germ cells and in cell differentiation. IL-6 is a potent inhibitor of meiotic DNA synthesis. Radio-induced transferrin and IL-6 could play a role in the protection of germ cells and could explain, in part, the resistance of Sertoli cells to radiation.

Sertoli cells, the supportive cells in the seminiferous epithelium, orchestrate spermatogenesis by providing structural and nutritional support to germ cells. In the rat, physiological apoptosis occurs continuously to limit the size of the germ cell population to numbers that can be adequately supported. This form of germ cell death is exaggerated after testicular insults such as toxicant treatment, radiation, and heat exposure. The Fas system has been proposed as a key regulator of the activation of germ cell apoptosis. According to this model, Fas ligand (FasL) and Fas, expressed by Sertoli cells and germ cells, respectively, respond to environmental conditions and initiate germ cell death. To assess the role of the Fas system in various testicular injury models, a semiquantitative RT-PCR technique was used to evaluate the expression kinetics of both FasL and Fas after induction of massive germ cell death. Radiation exposure, which targets actively dividing germ cells, produced an up-regulation of Fas gene expression, but not FasL gene expression. However, administration of mono-(2-ethylhexyl)phthalate and 2,5-hexanedione, two widely studied Sertoli cell toxicants, resulted in up-regulated expression of both FasL and Fas. These data support the following hypotheses: 1) up-regulation of Fas is a common and critical step for initiating germ cell death in vivo; and 2) if Sertoli cells are injured, Sertoli cells up-regulate FasL to eliminate Fas-positive germ cells, which cannot be supported adequately.

The effect of the p53 gene on the survival of mouse testicular cells was evaluated by analysis of degenerating and terminal transferase-mediated end labeling (TUNEL)-positive cells and the subsequent production of further differentiated progeny. In p53 null mice, in contrast to wild-type mice, radiation induced negligible levels of degenerating or TUNEL-positive differentiating spermatogonia within 24 h. This was correlated with higher production of differentiated progeny of the differentiating spermatogonia in p53 null mice. Contrary to the differentiating spermatogonia, the stem spermatogonia of p53 null mice produced fewer differentiated progeny after irradiation than did the stem cells of wild-type mice. We conclude that, because the degeneration and TUNEL positivity of the differentiating spermatogonia in mice of different genotypes were correlated with each other and were dependent on p53, this process is indeed apoptosis. In the differentiating spermatogonia, p53-dependent apoptosis accounted for the bulk of the loss of their progeny after irradiation. Furthermore, whereas the differentiating spermatogonia died by apoptosis that was dependent on p53, the stem spermatogonia, which are more radioresistant, did not.

Radiation-induced DNA single-strand break (ssb) induction and rejoining were measured in murine testicular cells using the alkaline comet assay. Individual cells in different stages of differentiation were identified on the basis of DNA content. As expected, induction of DNA ssb in testis cells irradiated on ice was independent of ploidy, and the extent of damage was similar to that produced in cells from other normal tissues. However, in vivo irradiation of air-breathing mice produced more ssb in haploid than tetraploid germ cells, although their rates of rejoining were similar and comparable to repair rates of cells from other normal tissues. In addition, irradiation of testis in situ produced only half as much damage as irradiation in vitro, and this could be explained only in part by the rapid ssb rejoining occurring during irradiation and cell isolation. A lower cellular oxygenation was postulated to account for the apparent resistance of testis cells to induction of breaks and the difference in induction in relation to DNA content. This was confirmed when carbogen inhalation and treatment with nicotinamide not only increased the overall degree of ssb induction in all these cells, but also reduced differences between cells of different ploidies. Results using the hypoxic cell cytotoxin RSU 1069 confirmed that the extent of hypoxia was not as severe in the testis as in the SCCVII murine tumour. It can be concluded from these data that the oxygenation of all testis cells is low enough to confer radioresistance, and that haploid testis cells are less hypoxic than tetraploid spermatocytes.

Dose-dependent characteristics of rat testicular cytosol androgen reception at varied terms after prolonged whole-body gamma-irradiation (0.1, 0.25 and 0.5 Gy at dose rate of 5.83 x 10(-7) Gy/s) coupled with an electron microscopy morphological characteristics of Sertoli cells. Leydig's cells and other populations of testicular cells, were examined. Analysis of the data obtained suggests the existence of gamma-sensor in brain of mammals, that involved on hypothalamic-pituitary-testicular levels in realisation of radiation stress suppression of Sertoli cell functions at a relatively "low" (0.1-0.5 Gy) doses by means of hypothalamic releasing factors and, therefore pituitary gonadotropins hypersecretion.

Unlabelled: In an attempt to explain the deleterious effects of gonadal radionuclide localization, we examined the role of transferrin in testicular radionuclide uptake., Methods: In vivo testicular uptake and retention of the transferrin binding radionuclides 114mIn-citrate and 59Fe-citrate were compared with that of the nontransferrin binding isotopes 137Cs-citrate and Na125I for 63 days postinjection. Isotope uptake mechanisms were investigated in vitro using isolated seminiferous tubules and Sertoli cell monolayers grown in bicameral culture chambers., Results: Indium-114m, 59Fe and 137Cs were localized in the testis by 24 hr postinjection, but accumulation of 125I was minimal. Although testicular 114mIn remained constant, 59Fe declined slowly over the following 63 days and 137Cs fell very rapidly. When 114mIn- or 59Fe-loaded testes were fractionated, and markedly more 114mIn was associated with the seminiferous tubules than 59Fe, suggesting that 114mIn may be retained. In vitro uptake of 59Fe, 67Ga and 114mIn by isolated seminiferous tubules was inhibited by transferrin, but uptake of 137Cs and 125I was unaffected. Iron-59, 67Ga and 114mIn were retained by isolated tubules in contrast to 137Cs and 125I. Whereas 137Cs, 59Fe and 114mIn all crossed Sertoli cell monolayers, the rate of transcellular transport of 137Cs was faster than that of 59Fe or 114mIn, suggesting differences in the intracellular processing of transferrin binding and nontransferrin binding radionuclides., Conclusion: These data suggest that some radionuclides may access the seminiferous epithelium through receptor-mediated endocytosis of transferrin. Such radionuclide localization could lead to continuous irradiation of the testes, resulting in mutagenic damage to spermatogenic cells.

The effects of prenatal X irradiation on postnatal development of the CBA/P mouse testis was studied. At days 14, 15 and 18 post coitus pregnant female mice were exposed to single doses of X rays ranging from 0.25-1.5 Gy. Higher doses resulted in extensive loss of fetal mice. In the male offspring, at days 3 and 31 post partum, the numbers of gonocytes, type A spermatogonia and Sertoli cells per testis were determined using the disector method. Furthermore, after irradiation at day 15 post coitus, the numbers of Leydig cells, mesenchymal cells, macrophages, myoid cells, lymphatic endothelial cells, endothelial cells and perivascular cells per testis were also determined at days 3 and 31 post partum. At day 3 post partum, the number of germ cells was decreased after irradiation at days 14 and 15 post coitus. A D0 value of 0.7 Gy was determined for the radiosensitivity of the gonocytes at day 14 post coitus. A D0 value of 0.8 Gy was determined for the gonocytes at day 15 post coitus which, however, seems to be less accurate. No accurate D0 value could be determined for the gonocytes at day 18 post coitus. At day 31 post partum, the repopulation of the seminiferous epithelium as well as testis weights and tubular diameters were more affected by irradiation with increasing age of the mice at the time of irradiation. The percentage of tubular cross sections showing spermatids decreased with increasing dose after irradiation at days 15 and 18 post coitus, but not after irradiation at day 14 post coitus. Furthermore, in tubular cross sections showing spermatids, exposure of testes to 1.25 and 1.5 Gy at day 18 post coitus resulted in significantly lower numbers of spermatids per cross section when compared to those testes exposed to the same doses at day 15 post coitus. This indicates that the radiosensitivity of the gonocytes increases with fetal age. Prenatal irradiation did not cause significant changes in the numbers per testis of the Sertoli cells or the interstitial cell types. The present results indicate that, in the fetal mouse testis, the spermatogonial stem cells are more sensitive to X irradiation than in the adult testis, while Sertoli cells and interstitial cells are relatively resistant.

The effects of total-body X irradiation on the prepubertal testis of the CBA/P mouse have been studied. At either day 14 or day 29 post partum male mice were exposed to single doses of X rays ranging from 1.5-6.0 Gy. At 1 week after irradiation the repopulation index method was used to study the radiosensitivity of the spermatogonial stem cells. A D0 value of 1.8 Gy was determined for the stem cells at day 14 post partum as well as for the stem cells at day 29 post partum, indicating that the radiosensitivity of the spermatogonial stem cells in the prepubertal mouse testis is already comparable to that observed in the adult mouse. One, 2 or 3 weeks after irradiation total cell numbers per testis of Sertoli cells, Leydig cells, mesenchymal cells, macrophages, myoid cells, lymphatic endothelial cells, endothelium and perivascular cells were determined using the disector method. The Sertoli cells and interstitial cell types appeared to be relatively radioresistant during the prepubertal period. No significant changes in plasma testosterone levels were found, indicating that there is no Leydig cell dysfunction after exposure to doses up to 6 Gy during the prepubertal period. Taken together, the radioresponse of the prepubertal mouse testis is comparable to that of the adult mouse testis.

Magnetic resonance (MR) spectroscopy of the cryptorchid rat testis was used to test whether changes in the MR spectra of the rat testis might be a more sensitive indicator of changes in the metabolic status of germ cells in the testis rather than simply the cell types present. Testes of adult Wistar rats before and during 42 days of experimental cryptorchidism were investigated by in-vivo 31P MR spectroscopy. Results were compared to MR studies of the synchronized developing testis. The testicular phosphomonoester/ATP (PM/ATP) ratio was dependent only on the cell types present, and showed the same characteristics for each cell type present in the degenerating testis as in the developing testis. The testicular phosphodiester/ATP (PD/ATP) ratio decreased rapidly when the number of round and elongated spermatids was reduced. Similar effects, although less pronounced, were seen in the developing testis. The pH decreased rapidly after cryptorchidism, and was related inversely to the PM/ATP ratio, which was also observed in the developing testis. This study demonstrates that MR spectroscopy monitors the cell types present in the rat testis rather than its metabolic status.

Recent studies have shown that germ cells can regulate testins, two newly identified Sertoli cell proteins that are associated with junctional complexes. To investigate this possibility, several parameters of Sertoli cell function were investigated over 2-120 days post exposure of the rat testes to x-rays (3 Grays). The irradiation-induced loss of spermatogonia resulted in a maturation-depletion process progressively affecting all germ cell classes. Testis weight began to decrease when the most numerous germ cell type (spermatids) began to decline. A complete or near complete recovery of spermatogenesis and of the testis weight had occurred by day 120 post irradiation. There was no significant change in FSH, epididymal androgen-binding protein, and tubule fluid levels during the first weeks after irradiation, when the seminiferious epithelium was depleted of spermatogonia and germ cells up to early spermatids. In contrast, when the number of the more mature forms of spermatids declined (between day 21 and 54), FSH rose and androgen-binding protein as well as fluid production declined. The subsequent recovery of these parameters was also highly correlated with the number of late spermatids. By contrast, testicular testin contents reacted to the depletion of germ cells with a biphasic increase; a doubling occurred when spermatogonia, spermatocytes, and early spermatids were absent (days 4-28), and a 7-fold rise occurred by day 37 when the number of late spermatids had decreased by 50%. By day 54, when the sperm counts had reached a nadir, testin contents had returned to levels corresponding to about four times the control levels; they progressively recovered thereafter. These observations support the postulate that germ cells negatively regulate testins. This possibility was investigated with in vitro experiments showing that addition of germ cell-conditioned medium to Sertoli cell monolayers inhibited testin secretion in a dose-dependent manner. In conclusion this study; 1) highlights the complex interplay between the various germ cell classes in the control of the Sertoli cell function in the adult testis; 2) establishes that germ cell effects may be opposite on different Sertoli cell products; 3) demonstrates that several classes of germ cells negatively control testicular testin contents; and 4) emphasizes the particular role of late spermatids in Sertoli cell regulation.

The Sertoli cell extends from the basement membrane of the seminiferous tubule towards its lumen; it sends cytoplasmic processes which envelop different generations of germ cells. The use of Sertoli cell culture began to develop in 1975. To reduce germ cell contamination immature animals are generally used as Sertoli cell donors. Sertoli cell mitosis essentially occurs in sexually immature testes in mammals; mitosis of these cells is observed in vitro during a limited period of time. Sertoli cells in vivo perform an impressive range of functions: structural support of the seminiferous epithelium, displacement of germ cells and release of sperm; formation of the Sertoli cell blood-testis barrier; secretion of factors and nutrition of germ cells; phagocytosis of degenerating germ cells and of germ cell materials. Some of the Sertoli cell functions can be studied in vitro. The recent development of Sertoli cell culture on permeable supports (with or without extracellular matrix) has resulted in progress in understanding the vectorial secretion of several Sertoli cell markers. In addition to FSH and testosterone, several other humoral factors are known to influence Sertoli cell function. Furthermore, myoid cells bordering the tubules as well as germ cells are capable of regulating Sertoli cell activity. Sertoli cells are the most widely used testicular cells for in vitro toxicology. The testis is highly vulnerable to xenobiotics and radiations, yet the number of studies undertaken in this field is insufficient and should be drastically increased.

During normal sexual maturation of the male rat there is a progressive change in the route of secretion of inhibin by the Sertoli cell, from a predominantly basal route of secretion in prepuberty to a predominantly apical route of secretion in adulthood. This change may be monitored by comparing the levels of inhibin in testicular (TV), spermatic and peripheral (PV) venous blood and the levels in testicular interstitial fluid (IF). This study has assessed the role of germ cells in effecting this change by assessing (a) the effect of total germ cell depletion by X-irradiation of the males in utero, and (b) the effect of selective germ cell depletion in adulthood using the testicular toxicant, methoxyacetic acid (MAA). Female rats were X-irradiated on day 20 of gestation to produce male offspring whose testes were germ-cell deficient. Blood and IF samples were collected from groups of these offspring and age-matched controls at 35 and 100 days of age. In blood and IF samples, inhibin concentrations were significantly higher at 35 days of age than at 100 days. The absence of germ cells in X-irradiated animals did not affect the age-related fall in inhibin levels, nor the change in the predominant route of secretion of inhibin from the testis into blood. Testosterone was almost undetectable in 35-day-old controls, but was raised significantly by 100 days of age. In X-irradiated animals, testosterone levels were increased significantly at 35 days of age, and the levels in most samples were increased even more substantially by 100 days of age. However, PV levels of testosterone in 100-day-old X-irradiated animals were significantly lower than in controls. LH and FSH levels were raised in X-irradiated animals compared with their age-matched controls, but FSH levels in X-irradiated animals still fell with age, as in the controls. The role of specific germ cell types in regulating the route of secretion of inhibin from the normal adult testis was studied after depletion (80-100%) of pachytene and later spermatocytes by a single oral administration of MAA (650 mg/kg) to adult rats. At 3 days after MAA treatment, coincident with the loss of pachytene spermatocytes, plasma inhibin levels were increased significantly in blood and IF samples, and this was associated with a dramatic change in the route of secretion of inhibin from the testis, with increased secretion of this peptide via the base of the Sertoli cell into IF and TV blood.(ABSTRACT TRUNCATED AT 400 WORDS)

Spermatogonial cell loss has been observed in rats flown on Space Lab 3, Cosmos 1887, Cosmos 2044 and in mice following irradiation with X-ray or with high energy (HZE) particle beams. Spermatogonial loss is determined by cell counting in maturation stage 6 seminiferous [correction of seminferous] tubules. With the exception of Iron, laboratory irradiation experiments (with mice) revealed a similar pattern of spermatogonial loss proportional to the radiation dose at levels less than 0.1 Gy. Helium and Argon irradiation resulted in a 5% loss of spermatogonia after only 0.01 Gy exposure. However, significant spermatogonial loss (45%) occured at this radiation level with Iron particle beams. The loss of spermatogonia during each space flight was less than 10% when compared to control (non-flight) animals. This loss, although small, was significant. Although radiation may be a contributing factor in the loss of spermatogonia during space flight, exposure levels, as determined by dosimetry, were not significant to account for the total cell loss observed.

Newly developed techniques in high-resolution scanning electron microscopy (SEM) and for tissue-processing procedures have been applied to an investigation of structures of various cells in rat testes at different stages of gonadal maturation. A series of high-resolution SEM micrographs are presented which survey the surfaces of different types of testis cells during normal development, and which also illustrate ultrastructural features of some of their intracellular organelles. In addition, a series of high-resolution SEM micrographs are presented which compare the structural features of Sertoli cells in normal testes with those in germ-cell-depleted testes obtained from rats killed at varying times after having been irradiated in utero. We describe our observations on the structural properties of surfaces and intracellular organelles in Sertoli cells, Leydig cells, peritubular myoid cells, and some classes of germinal cells. We also consider the possible role of Sertoli cell apical cytoplasmic processes in lumen formation. Similarities are pointed out between the structure of germ-cell-depleted testes, resulting from irradiation in utero, and the structure of germ-cell-depleted testes in seasonal breeders during periods of involution. Finally, we discuss advantages and disadvantages of methods employed to reveal the fine structure of intracellular organelles in cells of the testis.

The regulation of spermatogenesis seems to involve complex cell interactions in the testis. Little is known about these cellular communication events. Advances in molecular technology and cell or cell group separation methods have made it possible to analyze function of defined spermatogenic and Sertoli cells, thereby giving some insights into the paracrine regulation of spermatogenesis. In this review we will describe how seminiferous tubule segments with distinct cell associations can be rapidly isolated and how the cell composition can be modified by high-energy X-irradiation. Results of the recent studies performed using these techniques will be briefly summarized. Spermatogenic cells at defined stages of their development can be isolated in living condition for morphological and biochemical studies by the transillumination technique. For accurate identification of the stages of the seminiferous epithelial cycle, phase contrast microscopy of live cell squashes has been used. The criteria described by Leblond and Clermont (Am. NY Acad. Sci., 55:548-573, 1952) can be used for accurate recognition of most of the stages of the cycle. However, stages I and II and substages of VII that are important in several studies are difficult to distinguish. Therefore, in addition to the morphology of early spermatids, development of the flagella at step 16 of spermiogenesis and the changing morphology of the cytoplasmic lobes (residual bodies) at stage VII of the cycle were used as criteria for rapid identification and isolation (preparative) of the seminiferous tubule segments. Expression of nucleoprotein and heat shock protein 70-related protein genes was analyzed with Northern blot, slot blot, and in situ hybridization techniques in accurately staged seminiferous tubules. Accurate stage-dependent timing of the onset of transcription, followed by storage and disappearance of the messages was demonstrated. The chromatoid body (cb) has been proposed to have a specific function in storage of the long-lived mRNAs in the spermatids. It is an actively moving cytoplasmic organelle that interacts with Golgi complex during formation of the acrosomic system. The chromatoid body is apparently also dependent on cytoplasmic microtubules, since its movements are inhibited and its structure becomes abnormal in the presence of vincristin, an inhibitor of tubulin polymerization. Follicle-stimulating hormone (FSH) is an important regulator of Sertoli cell function. Since both basal and FSH-dependent cyclic AMP (cAMP) production by seminiferous tubules showed marked stage dependency, Sertoli cells are apparently influenced by spermatogenic cells. Thus, Sertoli cell function varies cyclically depending on the stage of the seminiferous epithelial cycle to provide an optimal microenvironment for spermatogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

The immediate effect of IR laser radiation on rat germ cells was studied by cytophotometric quantification of the nuclear DNA content in testicular sections. Two different levels of radiation were studied: one according to clinical application (28.05 J/cm2) and another known to increase the germ cell number (46.80 J/cm2). The laser beam induced changes in the germ cell DNA content depending on the cell type, the cell cycle phase and the doses of radiation energy applied. Following irradiation at both doses the percentage of spermatogonia showing a 4c DNA content was increased, while the percentage of these with a 2c DNA content was decreased. Likewise, the percentages of primary spermatocytes with a DNA content equal to 4c (at 28.05 J/cm2), between 2c and 4c (at 46.80 J/cm2) and higher than 4c (at both doses) were increased. No change in the mean spermatid DNA content was observed. Nevertheless, at 46.80 J/cm2 the percentages of elongated spermatids with a c or 2c DNA content differed from the controls. Data show that, even at laser radiation doses used in therapy, the germ cell DNA content is increased shortly after IR laser radiation.

Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.