Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells.

The purpose of this study was to verify whether the nanosecond pulsed electric field, not eliciting thermal effects, permanently changes the molecular processes and gene expression of Leydig TM3 cells. The cells were exposed to a moderate electric field (80 quasi-rectangular shape pulses, 60 ns pulse width, and an electric field of 14 kV/cm). The putative disturbances were recorded over 24 h. After exposure to the nanosecond pulsed electric field, a 19% increase in cell diameter, a loss of microvilli, and a 70% reduction in cell adhesion were observed. Some cells showed the nonapoptotic externalization of phosphatidylserine through the pores in the plasma membrane. The cell proportion in the subG1 phase increased by 8% at the expense of the S and G2/M phases, and the DNA was fragmented in a small proportion of the cells. The membrane mitochondrial potential and superoxide content decreased by 37% and 23%, respectively. Microarray's transcriptome analysis demonstrated a negative transient effect on the expression of genes involved in oxidative phosphorylation, DNA repair, cell proliferation, and the overexpression of plasma membrane proteins. We conclude that nanosecond pulsed electric field affected the physiology and gene expression of TM3 cells transiently, with a noticeable heterogeneity of cellular responses.

Alteration of testicular regulatory and functional molecules following long-time exposure to 900 MHz RFW emitted from BTS.

The aim of this investigation was to evaluate changes in testosterone and some of the functional and regulatory molecules of testis such as P450scc, steroidogenic acute regulatory protein (StAR), tumour necrosis factor-α (TNF-α), interleukin-1α (IL-1α), interleukin-1ß (IL-1ß) and nerve growth factor (NGF) following exposure to 900 MHz radio frequency (RF). Thirty adult male Sprague Dawley rats (190 ± 20 g BW) were randomly classified in three equal groups, control (sham, without any exposure), short-time exposure (2 hr) (STE) and long-time exposure (4 hr) (LTE). The exposure was performed for 30 consecutive days. The testosterone level in both exposed groups was significantly less than control (p < .05). Level of TNF-α in both exposed groups was significantly greater than control (p < .05). IL-1α and NGF levels in LTE were significantly higher than the STE and control groups (p < .05). Level of IL-1ß in LTE was significantly higher than control (p < .05). Expression of both P450scc and StAR mRNA was significantly down-regulated in both exposed groups compared to control (p < .05). Our results showed that RFW can affect testis and reproductive function through changes in factors, which are important during steroidogenesis, and also through changes in inflammatory factors, which regulate Leydig cell functions.

[The ultrastructure of Leydig cells under the influence of drinking mineral water and electromagnetic radiation under the stress conditions in the rats]. / Ul'trastruktura kletok Leidiga pri deistvii mineral'noi vody i nizkointensivnogo elektromagnitnogo izlucheniya v usloviyakh stressa u krys.

The objective of the present study was elucidate the peculiar features of low-intensity electromagnetic radiation (LI EMR) and mineral water (MW) on the ultrastructure of rat Leydig cells under conditions of immobilization stress. The experiments were carried out on outbred male rats with the use of electron microscopy. It has been demonstrated that the prophylactic consumption of drinking sulfate-containing mineral water and the application low-intensity electromagnetic radiation (with the flow power density of 1 mcW/cm2 and frequency around 1,000 Hz) or the combination of these two modalities under conditions of immobilization stress reduced the degree of ultrastructural derangement in the rat Leydig cells and stimulated the development of regenerative processes. In the cases of the single-factor impact, drinking mineral water exerted more pronounced action than low-intensity electromagnetic radiation on mitochondrial regeneration. In case of the simultaneous application of the two factors their protective action on the Leydig cells was much more conspicuous than that of either of them applied alone. It is concluded that drinking sulfate-containing mineral water in combination with the application of low-intensity electromagnetic radiation enhances resistance of the rat Leydig cells to stress.

Prospective phase I study on testicular castration induced by radiation treatment.

INTRODUCTION: Surgery and luteinizing hormone-releasing hormone (LHRH) analogs are standard androgen deprivation treatments (ADT) for patients with advanced prostate cancer. We performed a phase I study to explore if irradiation to the testes could be used as an alternative to ADT. MATERIALS AND METHODS: All patients had advanced prostate cancer with normal testosterone levels before treatment and indication for long term castration. Treatment started with one LHRH analog injection (to induce a fast drop of testosterone) followed by irradiation to the whole scrotum. Two fractionation regimens were tested: 17 Gy in 2 fractions, and 24 Gy in 3 fractions. Hormonal blood evaluation was performed before and every 3 months after radiotherapy. Toxicity was evaluated at each visit by the CTCv3 scoring system. This was an IRB approved prospective study. RESULTS: The first three patients received 17 Gy in 2 fractions. None of them developed acute or late skin toxicity and none became castrated, keeping normal levels of testosterone during the time they were followed at 11, 24 and 36 months post-radiotherapy. Another four patients received 24 Gy in 3 fractions. Two developed grade 1 temporary acute dermatitis and, again, none of them became castrated during follow up of 11-36 months. CONCLUSIONS: Prospective studies assessing the effect of the Leydig cells after direct irradiation to the testes are rare. The two radiotherapy regimens used in this study were well tolerated, but not capable of causing castration after 11-36 months of follow up. It is not yet clear whether radiation treatment can effectively induce castration in men.

[Advanced glycation end products inhibit testosterone production in rat Leydig cells].

OBJECTIVE: To study the expression of the receptor for advanced glycation end products (RAGE) and the inhibitory effect of advanced glycation end products (AGEs) on testosterone production in rat Leydig cells. METHODS: Rat Leydig cells were primarily cultured and the expression of RAGE in the Leydig cells was detected by RT-PCR and immunofluorescence staining. The Leydig cells were treated with AGEs at the concentrations of 25, 50, 100 and 200 microg/ml, respectively, and the testosterone content was determined by ELISA. RESULTS: RT-PCR and immunofluorescence staining exhibited the expression of RAGE in the rat Leydig cells. AGEs remarkably suppressed hCG-induced testosterone production in the Leydig cells in a concentration-dependent manner in the 50, 100 and 200 microg/ml groups as compared with the control (P < 0.01). CONCLUSION: RAGE exists in rat Leydig cells and AGEs can significantly inhibit the secretion of testosterone in primarily cultured rat Leydig cells.

Radio frequency electromagnetic radiations interfere with the Leydig cell functions in-vitro.

A growing threat to male infertility has become a major concern for the human population due to the advent of modern technologies as a source of radiofrequency radiation (RFR). Since these technologies have become an integral part of our daily lives, thus, it becomes necessary to know the impression of such radiations on human health. In view of this, the current study aims to focus on the biological effects of radiofrequency electromagnetic radiations on mouse Leydig cell line (TM3) in a time-dependent manner. TM3 cells were exposed to RFR emitted from 4G cell phone and also exposed to a particular frequency of 1800 MHz and 2450 MHz from RFR exposure system. The cells were then evaluated for different parameters such as cell viability, cell proliferation, testosterone production, and ROS generation. A considerable reduction in the testosterone levels and proliferation rate of TM3 cells were observed at 120 min of exposure as compared to the control group in all exposure settings. Conversely, the intracellular ROS levels showed a significant rise at 60, 90 and 120 min of exposure in both mobile phone and 2450 MHz exposure groups. However, RFR treatment for different time durations (15, 30, 45, 60, 90, and 120 min) did not have significant effect on cell viability at any of the exposure condition (2450 MHz, 1800 MHz, and mobile phone radiation). Therefore, our findings concluded with the negative impact of radiofrequency electromagnetic radiations on Leydig cell's physiological functions, which could be a serious concern for male infertility. However, additional studies are required to determine the specific mechanism of RFR action as well as its long-term consequences.

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.

Testicular apoptosis and histopathological changes induced by a 2.45 GHz electromagnetic field.

There is a growing public concern about the potential human health hazard caused by exposure to electromagnetic radiation (EMR). The objective of this study is to investigate the effects of 2450 mhz electromagnetic field on apoptosis and histopathological changes on rat testis tissue. Twelve-week-old male Wistar Albino rats were used in this study. Eighteen rats equally divided into three different groups which were named group I, II and III. Cage control (group I), sham control (group II) and 2.45 GHz EMR (group III) groups were formed. Group III were exposed to 2.45 GHz EMR, at 3.21 W/kg specific absorption rate for 60 minutes/ day for 28 days. There was no difference among the groups for the diameter of the seminiferous tubules, pyknotic, karyolectic and karyotic cells. However, the number of Leydig cells of testis tissue of the rats in group III was significantly reduced comparing with the group I (p < 0.05). Estimation of spermatogenesis using the Johnsen testicular biopsy score revealed that the difference between groups is statistically significant. The level of TNF-α, Caspase-3 and Bcl-2 were compared, and no significant difference was found between the groups. When Bax apoptosis genes and Caspase-8 apoptosis enzyme were compared, there were significant differences between the groups (p < 0.05). Electromagnetic field affects spermatogenesis and causes to apoptosis due to the heat and other stress-related events in testis tissue.

Testosterone synthesis in testicular Leydig cells after long-term exposure to a static electric field (SEF).

China's clean energy and resources are mainly located in the west and north while electric load center is concentrated in the middle and east. Thus, these resources and energy need to be converted into electrical energy in situ and transported to electric load center through ultra-high voltage direct current (UHVDC) transmissions. China has built 25,000 km UHVDC transmission lines of 800 kV and 1100 kV, near which the impact of electric field on health has attracted public attention. Previous studies showed that time-varying electromagnetic field exposure could disturb testosterone secretion. To study the effect of non-time-varying electric field caused by direct current transmission lines on testosterone synthesis, male ICR mice were continually (24 h/d) exposed to static electric field of 56.3 ± 1.4 kV/m. Results showed that on the 3rd day of exposure and on the 7th day after ceasing the exposure of 28 d, serum testosterone level and testicular oxidative stress indicators didn't change significantly. On the 28th day of exposure, serum testosterone levels, testicular glutathione peroxidase (GSH-Px) activity, the mRNA and protein levels of testicular StAR, PBR, CYP11A1 decreased significantly, and testicular malondialdehyde (MDA) content increased significantly. Meanwhile, electron-dense edges and vacuolation appeared in lipid droplets of Leydig cells. The gap between inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) enlarged, which would cause the swelling of mitochondria, the rupture and deficiency of mitochondrial membranes. Analysis showed that testicular oxidative stress could induce the damage of mitochondrial structure in Leydig cells, which would decrease the rate of cholesterol transport from cytoplasm to mitochondria. Since cholesterol is the necessary precursor of testosterone synthesis, testosterone synthesis was inhibited. The decrease of the mRNA and protein expression levels of StAR and PBR in testes could diminish the cholesterol transported from OMM to IMM. The decrease of the mRNA and protein expression levels of CYP11A1 could reduce the pregnenolone required in testosterone synthesis and inhibit testosterone synthesis consequently.

Radiation effects on male fertility.

BACKGROUND: Spermatogenesis is a process of dynamic cell differentiation. Ionizing radiation impairs spermatogenesis, and spermatogonia are more radiosensitive than spermatocytes or spermatids. Consistent with this assumption and due to improvement in tumor curability, nowadays, fertility preservation represents a public health need. OBJECTIVES: To discuss radiotherapy-induced risk to male fertility and raise oncologic awareness of male fertility in daily clinical practice. MATERIALS AND METHODS: PubMed and Clinicaltrials.gov databases were searched for papers in English. RESULTS: We provide an overview of clinical landscape. Four main issues were proposed: (i) spermatogenesis and radiobiological general concepts; (ii) impairment of spermatogenesis; (iii) impairment of testosterone-producing Leydig cells; (iv) clinical radiotherapy evidence in oncology. CONCLUSION: This review can be useful in daily clinical work and offer some directions for future research.

[Gonadal function after cancer treatment in adult men]. / Gonadal funksjon etter kreftbehandling hos voksne menn.

BACKGROUND: Surgery, chemotherapy and radiation, but also long-term hormonal treatment may cause reduced gonadal function in male cancer patients. The germinal epithelium is more vulnerable to cytotoxic damage than the Leydig cells, which implies that subfertility/infertility is a more frequent side effect of cancer treatment than endocrine hypogonadism. MATERIAL AND METHODS: This review article is based on clinical experience and literature retrieved from PubMed. RESULTS AND INTERPRETATION: The degree of gonadal damage depends on the type of chemotherapy and the cumulative dose; with alkylating substances and procarbazine being the most gonadotoxic agents. The testicle is one of the most radiosensitive organs, and the damage depends on the radiation dose. Testicle damage may be caused by direct radiation of the testicles or scattered radiation from radiated adjacent tissue. Freezing of semen should be discussed with and offered to all men below 55 years that are about to undergo cancer treatment that may cause reduced fertility. Men surviving cancer who experience unwanted childlessness should be offered examination, advice and possibly assisted fertilization as part of the rehabilitation. Especially after intensive chemotherapy and high dose cranial radiation therapy the patient should be examined regularly throughout life with respect to premature endocrine hypogonadism.

1800 MHz radiofrequency fields inhibits testosterone production via CaMKI /RORα pathway.

Exposure to radiofrequency fields (RF) has been reported to induce adverse effects on testosterone production and its daily rhythm. However, the mechanisms underneath this effect remain unknown. In this study, male mice were exposed to 1800 MHz radiofrequency fields (RF, 40 µW/cm2 power intensity and 0.0553 W/Kg SAR) 2 h per day for 32 days. The data suggested that RF exposure: (i) significantly reduced testosterone levels, (ii) altered the expression of genes involved in its synthesis (Star, P450scc, P450c17 and 3ß-Hsd) in testicular tissue, (iii) significantly reduced regulatory protein CaMKI/RORα. Similar observations were also made in cultured primary Leydig cells exposed in vitro to RF. However, all of these observations were blocked by CaMK inhibitor, KN-93, and ionomycin reversed the down-regulation effects on intracellular [Ca2+]i and CaMKI/RORα expression induced by RF exposure. Thus, the data provided the evidence that RF-induced inhibition of testosterone synthesis might be mediated through CaMKI/RORα signaling pathway. Capsule: CaMKI/RORα signaling pathway was involved in the inhibition of testosterone synthesis induced by RF exposure.

Arecoline cannot alter testicular dysfunction and pineal activation caused by noise in wistar rat.

Millions of people consume betel nut for increased capacity to work and for stress reduction. The nut contains arecoline, which has multiple side effects on endocrine functions. Objective of the work is to investigate pineal-testicular responses to noise and after arecoline treatment in noise in rats. Noise exposure (100 dB, 6 h daily, 10 days) caused pineal stimulation ultrastructurally and at indoleamines level. Leydig cell dysfunction with fall of testosterone level and suppression of sex accessories were noticed. In contrast, pineal activity was inhibited and reproductive functions were stimulated after arecoline administration, confirmed from reversed changes to those of noise. Arecoline treatment in noise exposure showed same results as in noise both in pineal and in reproductive functions. It is concluded that noise causes testicular dysfunction probably by gonadotropin suppression induced by pineal melatonin in noise. Furthermore, arecoline cannot prevent it in noise in rats.

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.

1950MHz Radio Frequency Electromagnetic Radiation Inhibits Testosterone Secretion of Mouse Leydig Cells.

More studies that are focused on the bioeffects of radio-frequency (RF) electromagnetic radiation that is generated from the communication devices, but there were few reports with confirmed results about the bioeffects of RF radiation on reproductive cells. To explore the effects of 1950 MHz RF electromagnetic radiation (EMR) on mouse Leydig (TM3) cells. TM3 cells were irradiated or sham-irradiated continuously for 24 h by the specific absorption rate (SAR) 3 W/kg radiation. At 0, 1, 2, 3, 4, and 5 days after irradiation, cell proliferation was detected by cell counting kit-8 (CCK-8) method, cell cycle distribution, percentage of apoptosis, and cellular reactive oxygen species (ROS) were examined by flow cytometry, Testosterone level was measured using enzyme-linked immunosorbent assay (ELISA) assay, messenger ribonucleic acid (mRNA) expression level of steroidogenic acute regulatory protein (StAR) and P450scc in TM3 cells was detected by real-time polymerase chain reaction (PCR). After being irradiated for 24 h, cell proliferation obviously decreased and cell cycle distribution, secretion capacity of Testosterone, and P450scc mRNA level were reduced. While cell apoptosis, ROS, and StAR mRNA level did not change significantly. The current results indicated that 24 h of exposure at 1950 MHz 3 W/kg radiation could cause some adverse effects on TM3 cells proliferation and Testosterone secretion, further studies about the biological effects in the reproductive system that are induced by RF radiation are also needed.

Radiation exposure impairs luteinizing hormone signal transduction and steroidogenesis in cultured human leydig cells.

Therapeutic, accidental, and experimental radiation exposures decreased serum testosterone in males, leading to various sexual problems. Since testicular Leydig cells are the predominant source of circulating testosterone, findings on the direct effects of radiation on Leydig cell steroidogenesis and the mechanism behind such effects would be of greater importance to the use of safer radiation doses in cancer therapy and to adopt preventive or therapeutic measures to alleviate postirradiation lesions, respectively. Therefore, this study was undertaken to explore the same using cultured human Leydig cells. Testicles removed from advanced prostatic carcinoma patients were used for isolation and purification of Leydig cells. Purified Leydig cells were cultured and then exposed to different doses (2, 4, 6, 8, and 10 Gy) of fractioned gamma radiation. Normal and irradiated cells were used for luteinizing hormone (LH) receptor quantification or total RNA isolation to study LH receptor mRNA expression or LH/cyclic AMP (cAMP) stimulation test. While LH-stimulated cells were used for cAMP assay, LH- and cAMP-stimulated cells were used for the estimation of steroidogenic enzymes, testosterone and estradiol production. Radiation exposure caused adverse effects on Leydig cell steroidogenesis in a dose-dependent manner. While lower doses (2 and 4 Gy) were ineffective, higher doses (6 Gy and above) drastically decreased LH receptor, basal and LH-stimulated cAMP generation, and basal, LH-, and cAMP-stimulated steroidogenesis. While 2 Gy of radiation exposure increased the LH receptor mRNA level, other doses did not induce any significant change. Therefore, it is concluded that higher doses of radiation impair Leydig cell steroidogenesis by affecting LH signal transduction at the level of both pre- and post-cAMP generation. Decreased level of LH receptors following higher doses of radiation exposure is not coupled with impaired expression of its mRNA.

Effect of whole-body 1800MHz GSM-like microwave exposure on testicular steroidogenesis and histology in mice.

The aim of our study was to evaluate the possible effects of whole-body 1800 MHz GSM-like microwave exposure on male reproduction. After repeated exposure of mice to microwaves at 0.018-0.023 W/kg whole-body specific energy absorption rate (SAR) an elevated serum testosterone level was measured, but no microwave exposure related histopathological alteration could be detected in the reproductive organs. The in vitro steroidogenic response of 48 h Leydig cell cultures obtained from exposed animals did not differ from the controls, suggesting that Leydig cells were not the primary targets of the applied microwave exposure or direct action of microwaves on Leydig cells was temporary only. In exposed animals the red blood cell count and volume of packed red cells were also increased. Further investigations are required to clarify the mechanism of action of the applied microwave exposure on male mice, as well as to establish the biological significance of the observed phenomena.

Leydig cells contribute to the inhibition of spermatogonial differentiation after irradiation of the rat.

Irradiation with 6 Gy produces a complete block of spermatogonial differentiation in LBNF1 rats that would be permanent without treatment. Subsequent suppression of gonadotropins and testosterone (T) restores differentiation to the spermatocyte stage; however, this process requires 6 weeks. We evaluated the role of Leydig cells (LCs) in maintenance of the block in spermatogonial differentiation after exposure to radiation by specifically eliminating functional LCs with ethane dimethane sulfonate (EDS). EDS (but not another alkylating agent), given at 10 weeks after irradiation, induced spermatogonial differentiation in 24% of seminiferous tubules 2 weeks later. However, differentiation became blocked again at 4 weeks as LCs recovered. When EDS was followed by treatment with GnRH antagonist and flutamide, sustained spermatogonial differentiation was induced in >70% of tubules within 2 weeks. When EDS was followed by GnRH antagonist plus exogenous T, which also inhibits LC recovery but restores follicle stimulating hormone (FSH) levels, the spermatogonial differentiation was again rapid but transient. These results confirm that the factors that block spermatogonial differentiation are indirectly regulated by T, and probably FSH, and that adult and possibly immature LCs contribute to the production of such inhibitory factors. We tested whether insulin-like 3 (INSL3), a LC-produced protein whose expression correlated with the block in spermatogonial differentiation, was indeed responsible for the block by injecting synthetic INSL3 into the testes and knocking down its expression in vivo with siRNA. Neither treatment had any effect on spermatogonial differentiation. The Leydig cell products that contribute to the inhibition of spermatogonial differentiation in irradiated rats remain to be elucidated.

Effect of graded testicular doses of radiotherapy in patients treated for carcinoma-in-situ in the testis.

PURPOSE: To determine the effect of radiotherapy in doses 14 to 20 Gy on eradication of carcinoma-in-situ (CIS) testis and on the Leydig cell function. PATIENTS AND METHODS: Forty-eight patients presented with unilateral testicular germ cell cancer and CIS of the contralateral testis. The CIS-bearing testis was treated with daily irradiation doses of 2 Gy, 5 days a week, to a cumulative dose of 20 Gy (21 patients), 18 Gy (three patients), 16 Gy (10 patients), and 14 Gy (14 patients). RESULTS: All patients treated at dose levels 20 Gy to 16 Gy achieved histologically verified complete remission without signs of recurrence of CIS after an observation period of more than 5 years. One of 14 patients treated at dose level 14 Gy had a relapse of CIS 20 months after irradiation. Leydig cell function was examined before and regularly after radiotherapy in 44 of 48 patients. The levels of testosterone were lower after radiotherapy than before. Testosterone showed a stable decrease for more than 5 years after treatment (3.6% per year) without dose dependency. The levels of luteinizing hormone and follicle-stimulating hormone were increased after radiotherapy. The need of androgen substitution therapy was similar at all dose levels. CONCLUSION: Testicular irradiation is a safe treatment at dose level 20 Gy (10 x 2 Gy). Decrease of dose to 14 Gy (7 x 2 Gy) might lead to risk of relapse of CIS. Impairment of hormone production without clinically significant dose dependency is seen in the dose range 14 to 20 Gy.