Changes in Expression of Specific mRNA Transcripts after Single- or Re-Irradiation in Mouse Testes.

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.

Radiation-induced abscopal reproductive effect is driven by TNF-α/p38 MAPK/Rac1 axis in Sertoli cells.

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.

[The ultrastructure of Sertoli cells and spermatogonia in the rats exposed to radiation under conditions of therapeutic and prophylactic application of low-intensity electromagnetic emission].

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.

Irradiation affects germ and somatic cells in prepubertal monkey testis xenografts.

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.

[Cellphone electromagnetic radiation damages the testicular ultrastructure of male rats].

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.

Cell-specific ablation in the testis: what have we learned?

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.

Using Plasmonic Copper Sulfide Nanocrystals as Smart Light-Driven Sterilants.

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 ultrastructural manifestations of the regenerative processes in the Sertoli cells under the action of low-intensity electromagnetic radiation in the rats subjected to stress].

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.

Effects of testosterone treatment on recovery of rat spermatogenesis after irradiation.

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.

Spermatogonial behavior in rats during radiation-induced arrest and recovery after hormone suppression.

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.

Transcriptome profiling of mice testes following low dose irradiation.

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.

Distribution of Zonula Occludens-1 and Occludin and alterations of testicular morphology after in utero radiation and postnatal hyperthermia in rats.

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.

Suppression of radiation-induced testicular germ cell apoptosis by 2,5-hexanedione pretreatment. I. Histopathological analysis reveals stage dependence of attenuated apoptosis.

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.

[Effect of microwave radiation on primary cultured Sertoli cells].

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.

Irradiation causes acute and long-term spermatogonial depletion in cultured and xenotransplanted testicular tissue from juvenile nonhuman primates.

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.

High radiosensitivity of germ cells in human male fetus.

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.

[Laser irradiation as a factor of primary prophylaxis of postradiation disorders of spermatogenesis in rats and their progeny].

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.

GSH level and IL-6 production increased in Sertoli cells and astrocytes after gamma 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.

Long-term effects of irradiation before adulthood on reproductive function in the male rhesus monkey.

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.