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Regular Article |
a Department of Cell Biology, Medical School, Utrecht University, 3584 CX Utrecht, The Netherlands
b Department of Hematology, Erasmus University, 3015 GE Rotterdam, The Netherlands
c MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, 2333 AL Leiden, The Netherlands
d IRS, J.A. Cohen Institute, 2333 AL Leiden, The Netherlands
e Department of Internal Medicine, Erasmus University, 3015 GE Rotterdam, The Netherlands
f Department of Clinical Oncology, University Hospital Leiden, 2300 RC Leiden, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, epididymis, Sertoli cells, spermatogenesis, testis, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the organs whose function is at risk after TBI is the testis. Many reports have described deleterious effects of irradiation and chemotherapy on the human testis [3–12]. However, in addition to the establishment of infertility, testis size, and changes in hormone levels in these patients, the underlying causes within the testis cannot be studied in the human. In this respect, radiation experiments on nonhuman primates are relevant, because the response of the monkey to radiation does not seem to be significantly different from that of the human. This has been demonstrated for a number of acute effects, including those on the hemopoietic and intestinal system, and also for some late effects, such as tumor induction [13–16]. Furthermore, spermatogenesis in both the human and the monkey has been shown to be rather radiosensitive [3, 17]. The induction of genetic damage, such as stable chromosomal aberrations, seems to be very low in the rhesus monkey, probably due to the extensive spermatogonial stem cell killing by the irradiation [18].
Studies regarding the long-term effects of irradiation on the testis have also been carried out in rodents. In the mouse, an ongoing outgrowth of spermatogenic colonies, formed by surviving stem cells, during the first 4–6 mo after irradiation was found, which leveled off thereafter [19, 20]. These colonies generally showed full spermatogenesis, although after higher doses, cell production in some colonies was poor [21]. In contrast, in LBNF1 rats, spermatogenesis at first starts to recover but then deteriorates again, and no recovery was seen within 60 wk [22]. Administration of gonadotropin-releasing hormone antagonist to these rats initiated recovery [23], and it was later shown that the high testosterone levels in the testes of these rats inhibited differentiation of spermatogonia [24].
We now have studied testicular histology, the epididymides, and hormone levels in adult rhesus monkeys (Macaca mulatta) that received high doses of total-body x-irradiation before adulthood. The results indicate extensive killing of spermatogonial stem cells and of Sertoli cells, leading to permanent damage to the seminiferous epithelium. To our knowledge, this is the first account of long-term intratesticular changes in the primate after TBI before adulthood.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testicular and epididymal sections from 29 prepubertally irradiated and 6 control monkeys were studied. In the Biomedical Primate Research Center at Rijswijk (BPRC), The Netherlands, these monkeys had been used in experiments to study the feasibility of ways to improve bone marrow transplantation. Ultimately, these monkeys were killed to study the long-term effects of irradiation on a number of organs, including the testis. The testes and epididymides were taken out and weighed separately. Subsequently, one quarter of each testis and all epididymides were fixed in Bouin fluid for histological examination. Another quarter of the testis was used to make air-dried meiotic chromosome preparations [25] and was analyzed for abnormal pairing configurations indicative for the presence of reciprocal translocations [26].
All animals in the present study were bred within the BPRC colony and kept under identical housing conditions. They were fed commercial food pellets (Hope Farms, Woerden, The Netherlands) and a diet of fresh fruit and vegetables. The animals were procured, maintained, and used in accordance with Dutch law and regulations. The Animal Care and Use Committee and the Animal Ethical Committee approved all experiments.
Irradiation Procedures and Additional Treatments
The majority of the irradiations was performed with x-rays from a Philips-Müller generator (Philips, Hamburg, Germany) (300 kV; 10 mA; half value layer, 3 mm Cu) at a mean dose rate of 0.3 Gy min-1. During the irradiation procedure, the animals were conscious and placed in a cylindrical polycarbonate cage. The cage was rotated slowly along its vertical axis to improve homogeneity of the irradiation. Details on the dose distribution over the animals have been described elsewhere [27]. Some animals were irradiated with 6 MV x-rays produced with a Mullard accelerator (Philips, Surrey, U.K.) at a similar dose rate as that applied for the orthovolt x-rays. The relative biological effectiveness of 6 MV x-rays, as measured for hematopoietic stem cells, is 0.9 [28]. The irradiated monkeys received supportive care or, for doses of 7 Gy or higher and two fractions of 6 Gy each, an additional treatment. Additional treatment consisted of either cytokines-only or bone marrow transplantation or both cytokines and bone marrow transplantation. Cytokines used were human granulocyte-macrophage colony-stimulating factor, rhesus monkey interleukin (IL) 3, or rhesus monkey IL-6 for approximately 14 days.
Available Material
Although the original experiments were not done to study long-term effects on the testis, the irradiated monkeys were divided in three groups. A control group was also included and consisted of six monkeys ranging from 7 to 35 yr of age (mean age, 21 yr).
First, a group of 10 monkeys received doses ranging from 4 to 8.5 Gy of 6 MV x-rays (one monkey, 4 Gy; two monkeys, 5 Gy; three monkeys, 6 Gy; one monkey, 7 Gy; two monkeys, 8 Gy; and one monkey, 8.5 Gy), which was called the dose-response group. The ages of the animals at the time of irradiation were 28 (n = 1), 29 (n = 3), 30 (n = 1), 31 (n = 1), 35 (n = 1), 39 (n = 1), 42 (n = 1), and 46 (n = 1) mo (mean age, 34 mo). The animals were killed at intervals of 77–92 mo (mean interval, 84 mo) after irradiation. The age at death in this group varied between approximately 9 and 11 yr. This group was used to study the dose-response relationship for killing of spermatogonial stem cells in the prepubertal monkey.
Second, an interval group of 13 monkeys received 5 Gy of 300 kV x-rays. The ages of the animals at the time of irradiation were 17 (n = 1), 25 (n = 1), 30 (n = 1), 36 (n = 1), 38 (n = 3), 39 (n = 4), 41 (n = 1), and 47 (n = 1) mo (mean age, 36 mo). The animals were killed at intervals of 38–79 mo after irradiation. The age at death in this group varied between approximately 6 and 10 yr. This group was used to study the capacity of the seminiferous epithelium to recover from the irradiation by outgrowth of repopulating colonies founded by surviving spermatogonial stem cells.
Third, a group of six monkeys received various irradiation treatments. Two monkeys (at 30 and 33 mo of age) received 4 Gy and one monkey (at 33 mo) 8.5 Gy of 300 kV x-rays as described above. One monkey (at 53 mo) received 8.5 Gy of 6 MV x-rays. Furthermore, two monkeys (at 17 and 50 mo) received two doses of 6 Gy of 6 MV x-rays given 24 h apart. The age at death in this group varied between approximately 7 and 12 yr.
To study translocation induction by irradiation, a selection was made of all irradiated monkeys (see below).
Hormone Assays
Serum hormone levels were estimated by radioimmunoassay using the method described by Verjans et al. [29] for testosterone, the coat-a-count method purchased from Diagnostic Products Corporation (Los Angeles, CA) for estradiol as described earlier [30], and the method described by Welschen et al. [31] for FSH. Levels of inhibin B were estimated using the immune-enzymometric assay from Serotec (Oxford, U.K.). The use of this assay in rhesus monkeys was described earlier by Ramaswamy et al. [32].
Histological Examination of the Testis
In both the right and the left testis of each monkey, at least 1000 tubular cross-sections were examined for the presence of germ cells, and the repopulation index (RI) was calculated as the percentage of tubular cross-sections containing germ cells.
Furthermore, in each testis, the tubular diameter was measured in 30 tubular cross-sections using an ocular grid. In many monkeys, some of the tubules were grossly enlarged; these tubules were not taken into account when measuring tubular diameters. Also, in those testes in which the majority of the tubules was enlarged, this aspect was not studied.
Statistics
To evaluate the significance of the correlation between sets of data, regression statistics were calculated and ANOVA analysis carried out using the Analysis ToolPak of the Microsoft Excel 97 program (Microsoft, Redmond, WA). Data are presented as mean ± SEM.
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RESULTS |
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In all irradiated monkeys, tubular cross-sections were observed that did not contain any germ cells (Fig. 1A). Only 4 of the 29 irradiated monkeys had no repopulation at all (RI = 0%). This was found in one of the two monkeys that received a dose of 8 Gy, in one of the three that received 8.5 Gy, and in both that received two fractions of 6 Gy each with an interval of 24 h. For monkeys in which repopulation did take place, the seminiferous epithelium was usually normal and complete in those tubular cross-sections in which germ cells were present, with all generations of spermatogenic cells being present. Comparing the diameter of the normal (see below) tubular cross-sections with full spermatogenesis in irradiated and in control monkeys, no difference was found (169 ± 4 µm [n = 29] vs. 169 ± 3 µm [n = 10], respectively).
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In 25 of the 29 irradiated monkeys, seminiferous tubules with a dilated lumen were seen in one or both testes (Fig. 1, A and B). Dilation of tubules with and without spermatogenesis was observed. In 25 of the 58 irradiated testes, the extent of the tubular widening was such that it was not possible to measure the tubular diameter of normal tubules. Tubular widening might occur because of obstructed flow of the tubular fluid secreted by Sertoli cells. Therefore, serial sections were made to see whether occlusions were in the lumen of the widened tubules. No such occlusions were found. Also, dilated tubules were seen ending on the rete testis (Fig. 1B), suggesting an obstruction in the rete testis or the epididymis. However, serial sections of these organs also did not reveal any evidence of obstruction.
In five of the six control monkeys, spermatogenesis was normal, even in the two monkeys that were older than 30 yr. In the sixth monkey, which was 18 yr of age, spermatogenesis was poor for no apparent reason. This last monkey was not taken into account in all comparisons between normal and irradiated monkeys.
In the seminiferous epithelium of 8 of the 29 irradiated monkeys, patches or whole tubular cross-sections with aberrant Sertoli cells were found. These abnormal Sertoli cells were densely packed, with no germ cells in between these cells (Fig. 1, C–E). One of the most striking examples is shown in Figure 1, C and D, in which it can be seen that such a thick layer of Sertoli cells is still compatible with normal spermatogenesis. Aberrant Sertoli cells were not observed in the control monkeys.
Dose-Response Studies
In the dose-response group, the extent of repopulation of the seminiferous epithelium by surviving spermatogonial stem cells was studied by determining the percentage of tubular cross-sections showing germ cells (i.e., the RI) in each monkey (Fig. 2A). These results appeared to be rather variable. The correlation between the RI and the dose was only just significant (P = 0.036), and in view of this, no D0 value (i.e., the dose killing 63% of the stem cells) as a measure of x-ray-induced spermatogonial stem cell death was calculated. A more reliable dose-effect relationship was found when the irradiation dose was compared to testicular weight (P = 0.003) (Fig. 2B).
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Effect of the Interval after Irradiation on Spermatogenic Recovery
In the interval group, which received a dose of 5 Gy, whether the percentage of tubular cross-sections showing repopulation kept increasing between 38 and 79 mo after irradiation was tested. However, when the interval after irradiation was correlated to the RI (Fig. 3), no significant correlation was found.
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Relationship Between Spermatogenic Recovery and Testicular Weight
Testicular weights of irradiated monkeys in all groups were correlated to the RI (Fig. 4). A highly significant correlation was found (P = 1.3 x 10-6). By extrapolation of the graph to a full recovery of spermatogenesis (RI = 100%), it was calculated that the weight of a fully recovered testis would be approximately 13.1 g. However, the average testicular weight of the five control monkeys, which had normal spermatogenesis, was much higher (23.3 ± 1.9 g).
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Radiation Effects on Epididymis
Epididymal sections did not reveal any apparent radiation effects (Fig. 1F). A highly significant correlation was found between testis and epididymal weight (P = 1.8 x 10-15) (Fig. 5).
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Hormone Levels
The levels of FSH, testosterone, estradiol, and inhibin B were measured in each monkey.
Comparing the irradiated monkeys to the control monkeys, the average levels of FSH were significantly higher (6.1 ± 1.2 vs. 2.1 ± 0.1 ng/ml; P = 0.002) and of inhibin B significantly lower (691 ± 72 vs. 1227 ± 165 IU/ml; P = 0.02). Levels of testosterone as well as of estradiol were not significantly different (5.3 ± 0.8 vs. 7.6 ± 1.3 nmol/L and 16.5 ± 1.5 vs. 29.2 ± 6.7 pmol/L, respectively).
In the irradiated monkeys, FSH levels showed a significant negative relationship with testicular weight (P = 0.0025) and RI (P = 2.2 x 10-5) (Fig. 6, A and B). Highly significant positive correlations were found between the inhibin B level and both testicular weight (P = 8.3 x 10-6) and RI (P = 7.4 x 10-5) (Fig. 6, C and D).
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Translocation Induction
Of all monkeys and testes studied, only those presented in Table 1 showed enough recovery of the germinal epithelium to provide sufficient numbers of spermatocytes to be analyzed. The results of this multivalent analysis of spermatocytes at the diakinesis-metaphase I stage of meiosis are summarized in Table 1. Very few translocation configurations were present, and no induction by irradiation took place.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The age at which the monkeys were irradiated varied between 17 and 53 mo. The onset of puberty in the rhesus monkey has been studied by Plant [33]. Both FSH and LH levels were found to increase from approximately 30 mo of age onward, whereas testosterone levels started to increase at 36 mo, reaching adult levels at approximately 42 mo of age. However, how the changes in hormone levels relate to the start of spermatogenesis and Sertoli cell proliferation and differentiation is not clear. In rhesus monkeys, Sertoli cell numbers were found to increase sixfold from 15–17 to 60 mo of age [34]. In cynomolgus monkeys, which start puberty at approximately the same time as rhesus monkeys [35], very few spermatocytes were present at 42 mo of age, and at this age, Sertoli cell proliferation still took place [36]. From this, it can be concluded that in most, if not all, of our monkeys, the testis was still developing at the time of irradiation.
In most of the irradiated monkeys at the time they were killed, full spermatogenesis was present in at least some tubules, indicating that these animals were potentially fertile. However, with the lowest dose given (4 Gy), in none of the monkeys was the repopulation complete. Apparently, with doses of 4 Gy and higher, all stem cells were always killed in one or more seminiferous tubules, preventing complete repopulation. Complete sterility was seen in two of the five animals that received doses of 8 Gy or higher as well as in both monkeys that received two fractions of 6 Gy each separated by 24 h. The latter indicates that, in the monkey as in the mouse [37, 38], fractionation of the irradiation does not have a sparing effect on spermatogonial stem cells. Importantly, when tubules had become repopulated, their diameter was similar to that in the control testes, indicating that the surviving stem cells were able to give rise to a normal seminiferous epithelium.
In the dose-response group, only a poor correlation was found between the percentage of repopulated tubule cross-sections, RI, and dose of irradiation. Although a better correlation was found between testicular weight and dose of irradiation, neither the RI nor the testicular weight could be used to calculate a meaningful D0 value for spermatogonial stem cell killing by x-rays in the young monkey. The explanation for this lack of correlation became apparent from the correlation between RI and testicular weight. Clearly, testicular weight depended highly on the amount of seminiferous epithelium that was present. Interestingly, extrapolation of the graph to a fully recovered seminiferous epithelium (RI = 100%) indicated that, on average, the fully recovered testis would weigh approximately 13 g. This was considerably less than the testicular weight of approximately 23 g in adult control monkeys. The discrepancy between these two weights can be explained by radiation-induced cell death of Sertoli cells in the young monkeys. In the adult testis, the Sertoli cells are terminally differentiated, nondividing cells, but at the time of irradiation, these cells were still proliferating and, hence, radiosensitive [34, 36]. In recent years, it has become clear that the number of Sertoli cells determines the amount of seminiferous epithelium and, thereby, the potential testicular weight [39–42]. Apparently, no mechanism regulates the numbers of Sertoli cells by enhanced or prolonged proliferation when their numbers are low or by less proliferation or apoptosis when their numbers are high. Hence, irradiation before terminal differentiation of the Sertoli cells in the monkey, as in the rat [43], causes loss of Sertoli cells and, thereby, decreases the potential adult testicular weight. From this, it follows that, when testes are irradiated before adulthood, the killing of spermatogonial stem cells cannot be studied by establishing the degree of repopulation or testicular weight. The RI will be relatively too high, because the surviving stem cells will sooner repopulate the shorter tubules that are present, and testicular weight will be too low because of Sertoli cell loss.
In most testes of irradiated monkeys, varying numbers of tubules were found that were dilated. Serial sections were made of some testes in which these dilated tubules were common to see whether these tubules were obstructed at some point. This was never found to be the case, and several of these tubules were observed to connect to the rete testis while still dilated, suggesting that the obstruction was present in the rete testis or further up, in the efferent ducts or the epididymis. However, serial sections also did not reveal an obstruction or other abnormalities in these tissues. Probably, at some earlier time after irradiation, a transient problem with drainage of the tubular fluid from the testis took place, causing swelling and irreversible damage in some tubules. In this respect, an interesting comparison can be made with the morphology of the testes of estrogen receptor 
-deficient mice, in which a mixture of relatively normal and dilated seminiferous tubules was also seen, caused by inappropriate fluid resorption in the efferent ductules [44, 45].
Intriguingly, effects of irradiation on the morphology of Sertoli cells were found in eight monkeys. Possibly, irradiation at the time these cells were not yet terminally differentiated and still proliferating, and/or the abnormal hormone levels induced by the disappearance of most of the germ cells, altered some of the Sertoli cells. The appearance of these aberrant cells was hyperplastic-like, but no evidence for tumor formation was seen.
In rodent studies, no effects of irradiation on tubular width and Sertoli cell morphology have been reported. Unlike in the LBNF1 rat [22], no arrest of spermatogenesis was seen in the monkey, because the repopulated tubules generally showed full spermatogenesis. However, the present study was carried out at a very long time after irradiation, and a transient disturbance in spermatogenesis at an earlier interval after irradiation may have taken place.
The nonaffected concentrations of testosterone indicate that Leydig cell function in the monkeys was not influenced by these doses of irradiation, as was reported earlier for adult rats that had been irradiated pre- or neonatally [46] or when they were adults [47]. Because the larger part of circulating estradiol is formed peripherally from testosterone in the male monkey [48], that estradiol levels also did not change is not surprising. The lack of effect of the dose range studied is consistent with data in the human, indicating that, in boys, only doses of 20 Gy or more affect Leydig cell function [49–55].
The correlation between levels of inhibin and FSH in male mammals has been studied before. Reports in which the presently used sandwich assay with antibodies against both the inhibin and ß subunits was used showed a negative correlation between levels of inhibin B and FSH in normal [56] and infertile men [57, 58]. In the latter two publications, levels of inhibin B were directly related to quantitative aspects of spermatogenesis, as observed in testicular biopsies. The significant inverse relationship between inhibin and FSH levels in the monkeys indicates that the inhibin B immunoassay can be used to assess the concentration of biologically active inhibin in this species. Furthermore, inhibin levels provide a good indication of the quantity of spermatogenesis, as in the human.
The results of the analysis of spermatocytes for translocation induction in spermatogonial stem cells further confirm previous, preliminary data suggesting an insensitivity of the rhesus monkey to this type of genetic change. The earlier data obtained on prepubertally irradiated monkeys were rather limited (i.e., seven individuals in total [18, 59, 60]), but the present, more extensive results point in the same direction: that no clear differences in chromosomal radiosensitivity exist between monkeys irradiated before or after the onset of puberty. Similar observations have been made for the mouse [61]. As discussed before [18], the low recovery of radiation-induced reciprocal translocations in rhesus monkey stem cell spermatogonia itself is mainly due to postirradiation proliferation-differentiation patterns of surviving spermatogonia. That no induction was found at all in the present study is probably due to a combination of two other factors. The first is the relatively high doses of irradiation used, which kill all stem cells sensitive to translocation induction and leave the resistant ones. The existence of sensitive and resistant stem cells leads to a so-called humped dose-response relationship, which has been observed in all mammals studied so far [26], and, in the rhesus monkey, to no induction at all in the higher dose range [18]. A second factor might be the long recovery periods after irradiation in the present study, causing the elimination of stem cell clones carrying aberrations [62].
In conclusion, TBI in young monkeys has long-lasting effects within the testis, including incomplete germ cell repopulation, permanently lowered numbers of Sertoli cells, changes in Sertoli cell behavior, and damage to tubule structure. However, complete sterility only occurs after high or fractionated doses. These effects on the testis further complete the discovery of problems that may arise at long intervals after TBI. In the same group of monkeys, effects on body fat and thyroid gland [63] as well as gastrointestinal function [64], ophthalmological complications [65], and cardiovascular damage [66] have already been described.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from The Netherlands Organization for Scientific Research (NWO). 
2 Correspondence: Dirk G. de Rooij, Department of Cell Biology, University Medical Center Utrecht, AZU-RM G02.525, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. FAX: 31 302541797; d.g.derooij{at}med.uu.nl
Accepted: September 27, 2001.
Received: May 14, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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