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Department of Experimental Radiation Oncology (G.S., G.W., M.L.M.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; Department of Physiology (I.H.), University of Turku, 20520 Turku, Finland; and Center for Biomedical Research (M.P.H., E.N.), The Population Council, New York, New York 10021
Address all correspondence and requests for reprints to: Gunapala Shetty, Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030. E-mail: gshetty{at}audumla.mdacc.tmc.edu.
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-dihydrotestosterone, 7-methyl-19-nortestosterone, methyltrienolone, or E2. The percentage of tubules showing differentiation (tubule differentiation index) was increased to 68% by the GnRH antagonist, from a value of 0.1% in irradiated-only rats at 13 wk after irradiation. All of the added androgens inhibited spermatogenic recovery, lowering the tubule differentiation index to between 0.4–36%, but no inhibition was observed with the addition of E2. Of all the androgen treatments tested, T (given as daily injections of T propionate) minimally inhibited spermatogenic recovery while maintaining androgen-responsive tissue weights, and might be most useful in clinical studies. Hormonal measurements in androgen-treated rats were most consistent with the androgen inhibition of spermatogenic recovery in irradiated rats being a combined result of a direct inhibitory effect of all androgens on the testis and an indirect effect through the pituitary by raising levels of FSH, which seems to add to the inhibition of spermatogenic recovery. |
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Because an inhibitory effect of T on spermatogonial differentiation was contrary to the usual action of this hormone in the testis, we believe that it was essential to determine whether this unusual effect was indeed caused by T, a specific metabolite of T, or a general property of androgens in this system. There is extensive evidence on estrogens (and now, some accumulating evidence on androgens) that different ligands for the receptor can induce different responses. For example, there is differential transactivation by T and 5-dihydrotestosterone (DHT) of genes with different androgen response element sequences (8). Although T is considered to be the major androgen regulating normal spermatogenesis because of its high intratesticular concentration, it is possible that a particular metabolite of T regulates spermatogonial differentiation in the irradiated rats.
One of the ways to reconcile the inhibitory effect of T with previous dogma would be if irradiation altered androgen metabolism, resulting in disproportionate changes, which could be reversed by GnRH-antagonist treatment, in the testicular levels of this particular metabolite. Therefore, in the present study, we correlated the intratesticular levels of several metabolites of T in testes of unirradiated, irradiated, and irradiated hormone-treated rats with the ability of the spermatogonia to differentiate.
A second way to explain the inhibitory action of T, irrespective of any alteration in steroid metabolism, is that irradiation could induce a change in AR coactivators or corepressors, such that now T and/or one or more of its metabolites act in an inhibitory (rather than a stimulatory) mode. For example, specific AR coactivators can confer androgenic activity to estradiol-17ß (E2) or hydroxyflutamide (9). Hence, it is possible that, in the irradiated testis, inhibitory activity could be conferred on only a subset of the metabolites of T. To directly test this possibility, we measured the spermatogenic inhibitory action after irradiation of different androgens and E2 by providing them exogenously.
We tested E2, which is formed from T by aromatase. Under normal conditions, E2 does not act via AR, but there are estrogen receptors in the testis; and, with certain coactivators (9), E2 can act via the AR. The androgens tested, in addition to T, were DHT (a 5-reduced metabolite of T), 7-methyl-19-nortestosterone (MENT, a non-5-reducible, but aromatizable, androgen), and methyltrienolone (R1881, a nonmetabolizable androgen) (Fig. 1
). DHT is regarded as a more potent androgen than T because it has a higher affinity for AR (10) and can stimulate and maintain spermatogenesis more efficiently than T (11, 12, 13). MENT, a synthetic 19-norandrogen derivative, cannot be 5-reduced (14); and hence, in contrast to T, its biological activity is not differentially amplified in male accessory sex organs, compared with the testis (15), which has less 5-reductase activity. MENT is known to have an increased affinity for AR and enhanced nuclear retention, compared with T (16). R1881 is a nonmetabolizable synthetic androgen, which also binds to AR with greater affinity than T (17) but has very little affinity for androgen-binding protein (18, 19). Because R1881 is not 5-reduced, it shows weaker effects, compared with T, on the accessory sex organs at doses that produce equivalent stimulatory effects on the testis (20).
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(R93-2705), both donated by Organon (Oss, The Netherlands). T-antiserum-coated tubes were obtained from Diagnostic Systems Laboratories, Inc. (Webster, TX). Adult LBNF1 male rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and housed in animal facilities approved by the American Association for Accreditation of Laboratory Animal Care, in accordance with current regulations and standards of The United States Department of Agriculture and the Department of Health and Human Services, NIH. They were maintained on a 12-h light, 12-h dark cycle and were allowed food and water ad libitum. All rats were acclimatized for at least 10 d before the initiation of experiments, at which time they were 9–12 wk of age.
Irradiation
Rats were anesthetized with an im injection of 0.72 mg ketamine/kg body weight and 0.22 mg acepromazine/kg body weight. The rats were placed on their backs, and 5 mm of tissue-equivalent bolus material (Superflab; Mick Radio-Nuclear Instruments Inc., Bronx, NY) was placed over the scrotum to provide a build-up layer. The lower part of the body, with the anterior edge of the irradiation field positioned about 6 cm above the base of the scrotum, was irradiated using a 60Co 
-ray unit (Eldorado 8; Atomic Energy of Canada Ltd., Ottawa, Ontario, Canada). A single dose of 5 Gy was administered at a dose rate of 0.96 Gy/min. Control animals underwent anesthesia and sham irradiation.
Hormone treatment
At 3 wk after irradiation, the rats were treated with a GnRH antagonist, Cetrorelix, as described earlier (7). Cetrorelix acetate was used at a dose of 1.5 mg/kg to bring about an immediate effect, and a dose of Cetrorelix pamoate of 1.5 mg/kg was given to have a prolonged effect. This treatment was shown, in irradiated rats, to depress LH, FSH, and intratesticular T (ITT) levels within a day; suppression was maintained for approximately 3–4 wk and reached a maximum at 2 wk (7, 22).
The spermatogenic and/or androgenic effects of DHT, MENT, R1881, or E2 (relative to T) were tested as outlined in Fig. 2. The unirradiated, irradiated-only, and GnRH-antagonist-only-treated irradiated groups each consisted of a minimum of 10 rats. Each treatment group that received one of the androgens or E2 in addition to the GnRH antagonist consisted of 4 or 5 rats. Three weeks after irradiation, groups of rats irradiated with 5 Gy received sc injections of Cetrorelix. Some groups of these treated rats also received an androgen in two doses, differing by a factor of 3, or comparable doses of T starting at 3 wk after irradiation and continuing for a period of 4 wk. In each case, the other androgen was delivered in a mode as previously published (see below), and T was given in the same manner. Two groups of these GnRH-antagonist-treated irradiated rats also received E2 or T delivered in a comparable manner.
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Some rats were killed at 5 wk after irradiation, after 2 wk of hormone treatment, which is the midpoint of the treatment of the other rats, for hormone measurements. In the other rats, the treatment was continued for up to 7 wk after irradiation, at which time the implants were removed or the injections were stopped, and the rats were killed at 13 wk after irradiation to measure spermatogenic recovery.
Hormone and androgen response measurements
At the time the rats were killed, blood was collected by cardiac puncture, under ketamine-acepromazine anesthesia. The serum was separated and stored at -80 C. In all rats, the right testis was freed of the tunica, weighed, collected on ice, and homogenized in a known amount of cold water. In some cases, an aliquot was removed and the sperm heads counted; the remainder of all samples was stored at -80 C for ITT analysis. In addition, in androgen-treated rats, three androgen-responsive tissues (the seminal vesicle with its fluid, ventral prostate, and the bulbocavernosus muscle) were also removed, cleared of extraneous material, and weighed.
Serum levels of FSH and LH were measured using immunofluorometric assays (Delfia; Wallac, Inc. OY, Turku, Finland) as previously described (28, 29). The minimum levels of detection of LH and FSH by this method are 0.04 ng/ml and 0.1 ng/ml, respectively.
ITT was assayed, in samples from experiments involving exogenous steroid hormone treatment, by using T-antiserum-coated tubes. The assay was done directly in the testicular homogenates, which were not centrifuged, using T standards prepared in 0.1% gelatin in PBS (7). ITT was expressed as the amount per gram testis, to reflect the actual concentration of T to which the testicular cells are exposed. In the normal testis, in which the concentration of T is much higher than the concentration of AR (30), the T concentration determines the occupancy of the AR, which should be nearly 100% because the dissociation constant is low (31). The amount of AR in irradiated rats has not been quantified, but immunohistochemistry indicates that, in the AR-positive somatic cells, the average levels are similar to unirradiated rats (32). Although the decrease in testicular volume results in a higher concentration of AR, the concentration of T also increases and still seems to be much higher than that of the AR. However, in the irradiated rats treated with GnRH antagonists, it is possible [depending on the effects of T deprivation on the AR levels in testis, which are still not clear (30)] that the concentration of T is less than that of AR. In this case, provided the amount of AR per AR-positive somatic cell remains constant, that the receptor occupancy will be determined by the total amount of T per testis, because the number of AR-positive cells per testis remains unchanged. Hence, we will also express ITT as the amount per testis.
The cross-reactivities of DHT, R1881, and TP in the T assay were 6.5%, 0.9%, and 0.08%, respectively. Because MENT treatment did not elevate the measured ITT concentrations, its cross-reactivity in the T assay should be minimal.
In addition, studies were performed to determine the content of androgenic metabolites, along with that of T, in the testes of a subset of the hormone-treated irradiated rats, to test whether the spermatogenic inhibition was caused by altered androgen metabolism along the pathways shown in Fig. 1
. Of the rats used for this purpose, the unirradiated, irradiated-only, and irradiated GnRH-antagonist-treated rats, with and without T, were selected from a different experiment (n = 4 in each group), and the GnRH-antagonist+DHT-treated irradiated rats were those in comparison 2.
Serum T and DHT were analyzed, in these samples, by RIA without extraction, as previously described (33), with the following modification. Before measurement of DHT, T was oxidized using a commercial kit (Biotrak T/DHT-[3H]; Amersham Pharmacia Biotech, Piscataway, NJ) to avoid cross-reaction with the antibody. The interassay coefficients of variation of the RIAs for each steroid were all less than 15%. The values obtained from the measurements of androgen-free serum, prepared by stripping serum from androgen-suppressed male rats with charcoal, were subtracted from the RIA measurements of the experimental samples, to produce the corrected steroid concentrations.
Before the RIA of testicular homogenates for androgenic metabolites and T, 1000 cpm tritiated T, androsterone, androstenedione, androstanediol, or DHT were added to aliquots of the homogenates. The homogenates were then extracted two times with ether and dried under nitrogen gas. The dried extracts were resuspended in water. A portion of the extracted samples was removed for scintillation counting, and the counts were used to calculate the recovery of each steroid from the ether extractions. The remaining portion was defatted by elution through a C18 column (Bond Elut, catalog no. 1210-2028; Varian, Harbor City, CA) using methanol. The eluate was then dried under nitrogen. The defatted extracts were resuspended in Tris-buffered saline with 0.1% gelatin. A portion of the resuspended sample was removed for scintillation counting to correct for recovery from the elution step. The remaining portion of the sample was then assayed for T, androsterone, androstenedione, androstanediol, and DHT as previously described (33). Measurements from charcoal-stripped samples of testicular homogenate were subtracted to produce the final steroid concentrations. The amounts of all these androgens in testis were expressed both per gram testis and per testis.
Evaluation of spermatogenesis
Spermatogenesis was evaluated in rats killed at wk 13 after irradiation. An aliquot from the right testicular homogenate was sonicated at 4 C for 4 min, as described earlier (34). The sonication-resistant sperm heads, representing nuclei of 12–19 spermatids, were counted in a hemocytometer.
For histological analysis, the left testis was fixed in Bouin’s fluid and embedded in paraffin or plastic (JB4; Polysciences, Warrington, PA), and 4-µm sections were cut and stained with hematoxylin. To evaluate spermatogenesis after hormone treatment, 200 seminiferous tubules in one section from each animal were scored for the most advanced germ-cell stage present in each tubule. A tubule was scored as differentiating if it contained 3 or more cells that had reached the type B spermatogonia stage or later (34). The tubule differentiation index (TDI), which is the percentage of tubules showing differentiation, was then computed.
Statistical analysis
For sperm counts, LH, serum androgen, and intratesticular steroid measurements (the averages and SEM) were calculated on log-transformed data. The organ weights, TDI, and FSH were represented as arithmetic mean ± SEM. The differences between the treatment groups were analyzed first by one-way ANOVA. If the difference was significant (P < 0.05), a Dunnett’s test was performed to determine the significance of the difference between the treated groups and a selected control group (irradiated-only or irradiated-and-treated-with-GnRH-antagonist-alone). To compare the difference among specific groups in GnRH-antagonist+steroid-hormone-treated irradiated rats, a Student’s t test was performed. A computer-assisted statistics program (SPSS, Inc., Chicago, IL) was used for all analyses.
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, A–E). Thus, spermatogenic inhibition cannot be attributed specifically to either the absence or inhibitory action of any one of these androgenic metabolites. The fact that spermatogenic inhibition was associated with greater elevations of ITT, compared with other androgenic metabolites tested, implies that none of these androgens can be more inhibitory than T.
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, A–E). Thus, the stimulation of recovery of spermatogonial differentiation produced by GnRH-antagonist treatment cannot be attributed to a requirement for any of these androgens. Because T was the most effectively suppressed androgen by the GnRH antagonist, T must, if androgens are inhibitory, be the most inhibitory of all the androgens tested, at least based on changes in their endogenous levels.
We next examined the intratesticular levels of androgens in GnRH-antagonist-treated irradiated rats given additional treatment with T, which suppresses recovery of spermatogenesis. The addition of 6 cm T slightly increased the intratesticular concentrations of T in GnRH-antagonist-treated irradiated rats (Fig. 3G). Although the increase was not significant in this experiment, significant increases were observed in other experiments. The amounts of ITT per testis were increased with both lengths of T implants, with the increase being significant after the 6-cm implant (Fig. 4G). In contrast, intratesticular concentrations of androstenedione, DHT, androstanediol, and androsterone were generally suppressed after the addition of T to GnRH antagonist (Fig. 3, F and H–J), but their amounts per testis were generally unchanged (Fig. 4, F and H–J). These results further support our hypothesis that T itself, and not an androgenic metabolite, is the major factor inhibiting recovery of spermatogenesis under these conditions.
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) and sperm head count being close to the limit of detection by our counting method (Table 1). Treatment with the GnRH antagonist, after irradiation, restored the ability of the testis to support stem cell differentiation, raising the TDI, at 13 wk post irradiation, to 68%. The continued progression of reinitiated spermatogenesis, even after discontinuing the treatment, was exemplified by a 1000-fold increase in the sperm head counts in these GnRH-antagonist-treated irradiated rats, compared with irradiated-only rats.
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-reduced metabolites of T were capable of inhibiting spermatogonial differentiation, we compared the effects of DHT (relative to T) on GnRH-antagonist-stimulated spermatogonial recovery. Addition of T in 2-cm and 6-cm SILASTIC capsules suppressed the GnRH-antagonist-induced tubule differentiation by 90% and 95%, respectively (Fig. 5B). DHT in 5-cm and 15-cm SILASTIC capsules similarly suppressed the GnRH-antagonist-stimulated tubule differentiation by 93% and 95%, respectively. The sperm head counts in these rats were not different from those in the irradiated-only rats (Table 1). Because DHT cannot be converted back to T, 5-reduced androgens and/or their metabolites can inhibit spermatogonial differentiation in the irradiated rat.
Because it was possible that the inhibitory effect of T was only through conversion to 5-reduced forms, we next determined whether MENT, a non-5-reducible but aromatizable 19-nortestosterone, also inhibited spermatogonial differentiation after irradiation. Continuous delivery of T in osmotic pumps, at doses of 60 µg and 180 µg/d, reduced the GnRH-antagonist-stimulated tubule differentiation by 89% and 93%, respectively (Fig. 5C). A similar mode of treatment with MENT, at doses of 10 µg and 30 µg/d, more severely suppressed the GnRH-antagonist-stimulated TDI by at least 99% and suppressed sperm head counts to undetectable levels (Table 1).
After observing that both DHT and MENT treatments inhibited spermatogenic recovery after irradiation, we wanted to rule out the possibility the inhibitors were not these androgens but rather their metabolites. To this end, we tested a nonmetabolizable androgen, R1881. For comparison, T was given in the form of daily injections of TP. Supplementation with 100 µg and 300 µg TP/d dose-dependently suppressed the GnRH-antagonist-induced differentiation of tubules only by 47% and 57%, respectively (Fig. 5D). However, R1881 was more effective in suppressing spermatogenic recovery, given that daily injections of 300 µg and 900 µg R1881 suppressed the GnRH-antagonist-stimulated tubule differentiation by 78% and 94%, respectively. Concurrent with the reduced tubule differentiation, the sperm head counts were also dose-dependently reduced when these two androgens were used in addition to the GnRH-antagonist treatment, but the reduction produced by a lower dose of TP was less than that observed with the comparable dose of R1881 (Table 1).
Inhibition of spermatogenic recovery after irradiation by T and MENT, which are aromatizable, led us to test the effect of E2 on such recovery. Though a 6-cm T capsule inhibited the GnRH-antagonist-stimulated TDI by 95%, as observed earlier, E2 in a 0.5-cm capsule did not significantly alter the GnRH-antagonist-induced spermatogenic recovery, as observed by the TDI (Fig. 5E) and sperm head counts (Table 1).
Hormone levels during GnRH-antagonist and androgen treatment
Five weeks after irradiation, serum FSH levels were significantly elevated, but LH levels were not altered (Fig. 5, F and K). Although the increase in ITT concentrations at 5 wk after irradiation was not significant in these rats (Fig. 5P), irradiation of another group (Fig. 3B) did produce elevated concentrations of ITT at this time after irradiation. Failure to consistently detect the increases in LH and ITT that had been observed previously (6, 22, 35) may be attributable to the fact that, in all the previous experiments, the rats were killed at 10 or more weeks after irradiation, whereas here they were killed at only 5 wk.
Treatment with the GnRH antagonist reduced the FSH, LH, and ITT concentrations to 15%, 14%, and 6% of the values observed in irradiated-only rats (Fig. 5, F, K, and P). These measurements were taken 2 wk after GnRH-antagonist treatment, which is considered to be the midpoint of the treatment time.
T, DHT, MENT, and R1881, and E2 administered by different means significantly reversed the GnRH-antagonist-induced suppression of FSH (Fig. 5, G–J); only with the lower dose of TP was this increase in FSH levels not significant. The LH levels remained suppressed and not significantly different from the GnRH-antagonist-only-treated irradiated rats in all the steroid treated groups. In the group receiving GnRH+antagonist+5 cm DHT (Fig. 5L), one rat had an exceptionally high LH value, resulting in a high variation in the LH levels. The T treatment, especially the higher doses, increased the ITT concentrations and total amounts per testis in GnRH-treated irradiated rats in all four groups (Fig. 5, Q–T and V–Y). The ITT levels in rats treated with different androgens were not as high as those treated with T, but the androgen administered would be expected to exert the primary direct inhibitory effect.
Treatment with exogenous DHT did not seem to increase its intratesticular concentration in GnRH-antagonist-treated irradiated rats (Fig. 3H), but it did show a trend toward increasing total DHT levels per testis (Fig. 4H). Whereas DHT treatment (particularly the lower dose) significantly reduced the intratesticular concentrations of androstenedione, T, and androstanediol (Fig. 3, F, G, and I), it had little effect on their total amounts per testis (Fig. 4, F, G, and I). These results provide only equivocal evidence that DHT may inhibit spermatogenic recovery by direct action on the testis. In contrast, DHT treatment increased the serum DHT levels in the GnRH-antagonist-treated rats by several orders of magnitude (Fig. 6), suggesting that the serum levels of the androgens may contribute significantly to inhibition of spermatogenic recovery.
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, T and Y). We propose that this suppression of T levels contributed to the maintenance of spermatogenic recovery by E2, even though it produced an elevation of FSH (Fig 5J), which we presume to be inhibitory.
Weights of androgen-responsive organs
The weights of the accessory sex glands, bulbocavernosus muscle, and testis were used as measures of androgenicity of the different androgenic steroids. These androgenic responses were compared with the associated TDI, which is a measure of spermatogonial differentiation.
Depletion of germ cells by irradiation reduced the weight of the testis to 38% of the unirradiated control levels at 5 wk after irradiation (Fig. 7A). Treatment with GnRH antagonist, for 2 wk, further reduced the testis weight, to 19% of the unirradiated control, because of the withdrawal of gonadotropin and androgen support. This decline most likely reflects reduction in Sertoli cell secretions and shrinkage of the Sertoli and Leydig cells (36) and is not necessarily directly related to the subsequent spermatogonial recovery. Support for this was obtained by the observation that testis weights were increased during the androgen treatments (Fig. 7, B–D), whereas the TDI was decreased (Fig. 5, B–D). In contrast to the testis, the weights of seminal vesicle, ventral prostate, and bulbocavernosus muscle were not altered by irradiation. However, all were significantly reduced during treatment with GnRH antagonist (Fig. 7, E, I, and M).
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, B–D). DHT, MENT, and R1881 increased testis weights to levels equal to or more than those produced by relative doses of T. All the androgens tested significantly increased the weights of seminal vesicle, prostate, and bulbocavernosus muscle in these GnRH-antagonist-treated irradiated rats (Fig. 7, F–H, J–L, and N–P). All three additional androgens had greater stimulatory effect than did T on testis weight, compared with accessory sex organ weights (figure not shown), with the differential effect being most significant for R1881 (compare Fig. 7, D with H and L).
The weights of these androgen-responsive tissues were correlated with the TDIs obtained after the androgen treatments (Fig. 8). As indicated by the arrows, a shift in the lines upward and to the right corresponds to less spermatogonial inhibition and greater androgenicity; whereas a shift downward and to the left corresponds to greater spermatogonial inhibition and less androgenicity. The responses for all androgens except R1881 overlapped or paralleled each other, which is consistent with the different androgens acting dose-responsively through the same pathway. None of the additional androgens used were better than T in maintaining the weights of the testis, seminal vesicle, ventral prostate, and muscle tissue, relative to their inhibition of GnRH-antagonist-stimulated spermatogenic recovery (Fig. 8). In fact, MENT was the poorest of all, given that it inhibited spermatogonial differentiation at doses required for the maintenance of androgen-responsive tissue weights more than did T. Of all androgen treatments tested, T (given in the form of TP as daily injections) was the best in meeting the criterion of maximizing androgenicity and minimizing spermatogonial inhibition.
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The present study demonstrates that the spermatogenic inhibitory property in irradiated rats is not limited to any particular androgen. Four androgens (T, R1881, DHT, and MENT), all having different metabolic properties, inhibited spermatogenic recovery. The relatively unchanged intratesticular concentrations of androgenic metabolites of T, androstanediol, androstenedione, DHT, and androsterone, after irradiation, rules out the possibility that the adverse effect of irradiation on spermatogenesis is caused by altered production of these androgens. Furthermore, the levels of these androgenic metabolites changed less after stimulation of spermatogonial differentiation with GnRH-antagonist treatment than did T, indicating that none of these metabolites had more potential to inhibit spermatogenic recovery than did T.
Although the previous study indicated that T was likely exerting its inhibitory effect directly on the testis (7), extratesticular sites of action could not be ruled out. The ITT levels in the GnRH-antagonist-treated rats, with or without additional doses of T, showed a fairly good inverse correlation with spermatogenic recovery as observed earlier (7). Nevertheless, the significant suppression of spermatogenic recovery, which was not associated with significant increases in ITT and in testicular DHT concentrations, by a low dose of TP or DHT, respectively, suggest that the greatly increased serum levels of these androgens acting on an extratesticular target may contribute to the spermatogenic suppression. The androgens may act on the pituitary to alter serum gonadotropin levels to affect spermatogenesis. As observed earlier with T, the other androgens did not have any effect on LH levels; and furthermore, the serum LH levels did not correlate with spermatogenic recovery after irradiation (7). However, they did reverse the GnRH-antagonist-induced reduction of FSH levels in these rats, presumably by direct up-regulation of FSHß-gene transcription in the pituitary (42); the resulting increase in FSH levels may contribute to the inhibition of spermatogonial differentiation.
When we compared the FSH levels with spermatogenic recovery in irradiated rats that received various androgens, a negative correlation was found between TDI and FSH (r = 0.94) (Fig. 9), which extends our earlier observations (7). Only the GnRH-antagonist-treated irradiated rats that were given the higher doses of TP by injection showed a deviation in this regard. Thus, although this study was designed to test the inhibitory properties of the androgens, we also obtained additional evidence that the increases in FSH produced by the androgen treatments may inhibit spermatogenic recovery.
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The suggestion that androgens and FSH have additive effects on inhibiting spermatogenesis in irradiated rats has precedence in the additive effects of these hormones in supporting spermatogenesis. In unirradiated rats with suppressed gonadotropin or T levels, exogenous T, DHT, and R1881 had similar effects in supporting the later steps of spermatogenesis. In these cases, FSH also supports the later stages of differentiation. In normal rats, spermatogonial survival and differentiation are qualitatively independent of T and FSH (44, 45, 46, 47). The numbers of spermatogonia and spermatocytes produced can be modulated by, at most, a factor of two by FSH levels in both rodents (48) and primates (49). The inhibition of the differentiation of A spermatogonia in irradiated rats by moderate levels of ITT and other androgens and/or serum FSH indicates that these hormones are acting, in an inhibitory manner, on a stage of spermatogenesis that is normally only weakly dependent on hormone stimulation. The molecular basis of the negative effects of these two hormones on spermatogenesis is, as yet, not known.
Similar to androgens, exogenous E2 also increased the FSH levels in GnRH-antagonist-treated irradiated rats but, unlike the situation with androgens E2, did not inhibit GnRH-antagonist-stimulated spermatogenic recovery. Thus, the FSH level for the respective TDI in GnRH-antagonist+E2-treated irradiated rats does not fall on the correlation curve fitted for the FSH levels vs. TDI in GnRH-antagonist+androgen-treated irradiated rats (Fig. 9). However, further suppression of ITT levels by E2 in these GnRH-antagonist-treated rats suggests that the possible spermatogenic inhibitory effect of FSH may be countered, in this case, by the more effective suppression of ITT levels and perhaps also by a direct stimulatory action of E2. We are further investigating the mechanism of E2 action on spermatogonial differentiation in irradiated rats.
The maintenance of testis weight by hormones in GnRH-antagonist-treated irradiated rats should be related to the maintenance of spermatogenesis in unirradiated rats. The increase in testis weight at 5 wk after irradiation, 2 wk after treatment, likely measures support of metabolic and secretory activity of Sertoli cells by androgens and FSH. There were differences in the relative abilities of different androgenic treatments to enhance testis weight vs. inhibit spermatogonial development. MENT decreased the TDI more than T and DHT at doses that produced comparable increases in testis weights. Likewise, TP injections diminished the TDI less than did other treatments that produced similar increases in testis weights (Fig. 8D). Thus, the inhibition of spermatogenic recovery by different androgens does not overlap the increase in the testis weight, suggesting that the mechanisms of the androgen-mediated processes in these two cases are different.
Finally, the clinical application of prolonged GnRH analog-treatment would be more practical if androgen supplementation could be given. The effectiveness of the various androgens in supporting seminal vesicle function and muscle mass was compared with their ability to inhibit spermatogonial differentiation (Fig. 8). MENT inhibited spermatogonial differentiation more than T, while similarly maintaining accessory sex organ weights. The dose responsiveness of R1881 inhibition of spermatogonial development did not parallel those of other androgens, because there was a proportionately greater decrease in the TDI and less increase in tissue weight with increasing R1881 dose; the reason for this is not known. However, the high dose point overlaps the dose-response line for T. The relative abilities of DHT to stimulate seminal vesicle and muscle weight, compared with its suppression of spermatogonial development, were identical to those of T. Thus, none of the androgens that were given exogenously were better than T in maintaining the androgenic tissue response, relative to their inhibition of GnRH-antagonist-stimulated spermatogenic recovery.
In contrast, administration of T in its propionate form, given as daily injections, less severely inhibited spermatogenic recovery, relative to its stimulation of androgenic tissues, than when it is given as T by continuous administration in osmotic pumps or SILASTIC capsules. This might be a result of fluctuations in serum and testicular T levels that occur with daily injection of TP, although the reported half-life of TP (3–4 d) should result in only small fluctuations (50). It was of particular practical interest that TP inhibited spermatogonial differentiation less than did T at doses that produced equivalent increases in muscle and seminal vesicle weight.
The present results may be useful in designing clinical protocols for recovery of fertility, with treatment schedules for maintaining muscle mass and libido during GnRH-analog therapy, in patients who have undergone radiation therapy. Of all the androgens tested, T inhibited the spermatogenetic recovery the least, while maintaining androgenicity. However, intermittent T treatment may be better in achieving that goal than would be maintaining T at constant levels. Another possibility is to explore reducing the levels and/or action of FSH directly, without reducing T, and determining whether such treatments can maintain or stimulate spermatogonial differentiation.
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Abbreviations: AR, Androgen receptor; DHT, 5-dihydrotestosterone; E2, estradiol-17ß; ITT, intratesticular T; jsd, juvenile spermatogonial depletion; MENT, 7-methyl-19-nortestosterone; T, testosterone; TDI, tubule differentiation index; TP, T propionate.
Received February 20, 2002.
Accepted for publication May 7, 2002.
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References |
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  M. L Meistrich and G. Shetty Hormonal suppression for fertility preservation in males and females Reproduction, December 1, 2008; 136(6): 691 - 701. [Abstract] [Full Text] [PDF]
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 M. M. N. Nguyen, K. L. K. Tamashiro, S. J. Melhorn, L. Y. Ma, S. R. Gardner, and R. R. Sakai Androgenic Influences on Behavior, Body Weight, and Body Composition in a Model of Chronic Social Stress Endocrinology, December 1, 2007; 148(12): 6145 - 6156. [Abstract] [Full Text] [PDF]
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 Y.-C. Chen, R. K Cochrum, M. T Tseng, D. T Ghooray, J. P Moore, S. J Winters, and B. J Clark Effects of CDB-4022 on Leydig Cell Function in Adult Male Rats Biol Reprod, December 1, 2007; 77(6): 1017 - 1026. [Abstract] [Full Text] [PDF]
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 S. Aquila, E. Middea, S. Catalano, S. Marsico, M. Lanzino, I. Casaburi, I. Barone, R. Bruno, S. Zupo, and S. Ando Human sperm express a functional androgen receptor: effects on PI3K/AKT pathway Hum. Reprod., October 1, 2007; 22(10): 2594 - 2605. [Abstract] [Full Text] [PDF]
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G. Shetty, C. C. Y. Weng, K. L. Porter, Z. Zhang, P. Pakarinen, T. R. Kumar, and M. L. Meistrich Spermatogonial Differentiation in Juvenile Spermatogonial Depletion (jsd) Mice with Androgen Receptor or Follicle-Stimulating Hormone Mutations Endocrinology, July 1, 2006; 147(7): 3563 - 3570. [Abstract] [Full Text] [PDF]
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 G. Grafstrom, B.-A. Jonsson, A. M. El Hassan, J. Tennvall, and S.-E. Strand Rat testis as a radiobiological in vivo model for radionuclides Radiat Prot Dosimetry, April 1, 2006; 118(1): 32 - 42. [Abstract] [Full Text] [PDF]
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K. L. Porter, G. Shetty, and M. L. Meistrich Testicular Edema Is Associated with Spermatogonial Arrest in Irradiated Rats Endocrinology, March 1, 2006; 147(3): 1297 - 1305. [Abstract] [Full Text] [PDF]
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G. Shetty, C. C. Y. Weng, S. J. Meachem, O. U. Bolden-Tiller, Z. Zhang, P. Pakarinen, I. Huhtaniemi, and M. L. Meistrich Both Testosterone and Follicle-Stimulating Hormone Independently Inhibit Spermatogonial Differentiation in Irradiated Rats Endocrinology, January 1, 2006; 147(1): 472 - 482. [Abstract] [Full Text] [PDF]
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 H. Jimenez-Severiano, M. L. Mussard, L. A. Fitzpatrick, M. J. D'Occhio, J. J. Ford, D. D. Lunstra, and J. E. Kinder Testicular development of Zebu bulls after chronic treatment with a gonadotropin-releasing hormone agonist J Anim Sci, September 1, 2005; 83(9): 2111 - 2122. [Abstract] [Full Text] [PDF]
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 K. Boekelheide, H. A. Schoenfeld, S. J. Hall, C. C. Weng, G. Shetty, J. Leith, J. Harper, M. Sigman, D. L. Hess, and M. L. Meistrich Gonadotropin-Releasing Hormone Antagonist (Cetrorelix) Therapy Fails to Protect Nonhuman Primates (Macaca arctoides) From Radiation-Induced Spermatogenic Failure J Androl, March 1, 2005; 26(2): 222 - 234. [Abstract] [Full Text] [PDF]
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 G. Shetty and M. L. Meistrich Hormonal Approaches to Preservation and Restoration of Male Fertility After Cancer Treatment J Natl Cancer Inst Monographs, March 1, 2005; 2005(34): 36 - 39. [Abstract] [Full Text] [PDF]
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G. Shetty, C. C. Y. Weng, O. U. Bolden-Tiller, I. Huhtaniemi, D. J. Handelsman, and M. L. Meistrich Effects of Medroxyprogesterone and Estradiol on the Recovery of Spermatogenesis in Irradiated Rats Endocrinology, October 1, 2004; 145(10): 4461 - 4469. [Abstract] [Full Text] [PDF]
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S. A. Hild, B. J. Attardi, and J. R. Reel The Ability of a Gonadotropin-Releasing Hormone Antagonist, Acyline, to Prevent Irreversible Infertility Induced by the Indenopyridine, CDB-4022, in Adult Male Rats: The Role of Testosterone Biol Reprod, July 1, 2004; 71(1): 348 - 358. [Abstract] [Full Text] [PDF]
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Z. Zhang, R.V. Short, T. Meehan, D.M. de Kretser, M.B. Renfree, and K.L. Loveland Functional Analysis of the Cooled Rat Testis J Androl, January 1, 2004; 25(1): 57 - 68. [Abstract] [Full Text] [PDF]
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G. Shetty and C. C. Y. Weng Cryptorchidism Rescues Spermatogonial Differentiation in Juvenile Spermatogonial Depletion (Jsd) Mice Endocrinology, January 1, 2004; 145(1): 126 - 133. [Abstract] [Full Text] [PDF]
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 M. L. Meistrich, G. Wilson, K. L. Porter, I. Huhtaniemi, G. Shetty, and G. A. Shuttlesworth Restoration of Spermatogenesis in Dibromochloropropane (DBCP)-Treated Rats by Hormone Suppression Toxicol. Sci., December 1, 2003; 76(2): 418 - 426. [Abstract] [Full Text] [PDF]
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M. L. Meistrich and G. Shetty Inhibition of Spermatogonial Differentiation by Testosterone J Androl, March 1, 2003; 24(2): 135 - 148. [Full Text] [PDF]
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 M. K. Rao, C. M. Wayne, M. L. Meistrich, and M. F. Wilkinson Pem Homeobox Gene Promoter Sequences that Direct Transcription in a Sertoli Cell-Specific, Stage-Specific, and Androgen-Dependent Manner in the Testis in Vivo Mol. Endocrinol., February 1, 2003; 17(2): 223 - 233. [Abstract] [Full Text] [PDF]
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, P < 0.5; 
, P < 0.05; 
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—
) represents the direction of a positive androgenic tissue response and less inhibition of TDI, and the arrow with a thin head (
). The fit was done using a sigmoid curve and includes all the points except the one for GnRH-antagonist+E2.