THE MAMMALIAN TESTIS is susceptible to a variety of

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Endocrinology 145(10):4461– 4469 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2004-0440


Author Iris Andrews

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Endocrinology 145(10):4461– 4469 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2004-0440

Effects of Medroxyprogesterone and Estradiol on the Recovery of Spermatogenesis in Irradiated Rats GUNAPALA SHETTY, CONNIE C. Y. WENG, OLGA U. BOLDEN-TILLER, ILPO HUHTANIEMI, DAVID J. HANDELSMAN, AND MARVIN L. MEISTRICH

Suppression of intratesticular testosterone (ITT) levels is required for spermatogenic recovery in rats after irradiation, but maintenance of peripheral testosterone (T) levels is important for many male functions. Considering the preservation of peripheral T while suppressing ITT, we tested the effects of a combination of a progestin, medroxyprogesterone acetate (MPA), plus T on spermatogenic recovery after irradiation, and compared its effects to those of T alone or T combined with estradiol (E2). Rats were given testicular irradiation (6 Gy) and treated during wk 3–7 after irradiation with MPA ⴙ T, or the individual steroids with or without GnRH antagonist (GnRH-ant), or GnRH-ant alone, or T ⴙ E2. Whereas GnRH-ant alone stimulated differentiation in 55% of tubules 13 wk after irradiation compared with 0% in irradiated-only rats, the addition of MPA reduced the percentage of tubules showing differentiation to 18%. However, T or MPA


HE MAMMALIAN TESTIS is susceptible to a variety of gonadotoxins, including the anticancer agents radiation and procarbazine, which often deplete germ cells and so cause prolonged azoospermia in many species of rodents (1, 2), monkeys (3), and presumably humans (4). In the rat there is clear evidence that these agents can cause azoospermia by disrupting the differentiation of surviving type A spermatogonia (5). In humans, there is some histological evidence that radiation and cancer chemotherapeutic drugs can also block spermatogonial differentiation (4, 6) and the recovery of spermatogenesis after prolonged azoospermia demonstrates that surviving stem cells were blocked from completing differentiation (7). The effective suppression of gonadotropins and intratesticular testosterone (ITT) with GnRH analogs restores spermatogonial differentiation and progression of spermatogenesis in rats exposed to moderate doses of radiation or procarbazine (8, 9). We demonstrated that it is actually testosterone (T), acting through the androgen receptor that inhibits spermatogonial differentiation in irradiated rats (10). In fact, all agents with androgenic activity inhibited spermatogonial development (11). In the present study, a progestin, medroxyprogesterone Abbreviations: E2, Estradiol; GnRH-ant, GnRH antagonist; ITT, intratesticular testosterone; MPA, medroxyprogesterone acetate; T, testosterone; TDI, tubule differentiation index. Endocrinology is published monthly by The Endocrine Society (http://, the foremost professional society serving the endocrine community.

alone or the combination of the two induced germ cell differentiation in only 2– 4% of tubules. In contrast, E2 stimulated differentiation in 88% of tubules, and T combined with E2 still resulted in differentiation in 30% of tubules. Although both MPA and E2 suppressed ITT levels to approximately 2% of control (2 ng/g testis), MPA was a less effective stimulator of spermatogenic recovery than E2 or GnRH-ant alone. MPA’s function as a weak androgen was likely responsible for inhibiting spermatogenic recovery, as was the case for all other tested androgens. Thus, for clinical protection or restoration of spermatogenesis after radiation or chemotherapy by suppressing T production, MPA, at least in the doses used in the present study, is suboptimal. The combination of an estrogen with T appears to be most effective for stimulating such recovery. (Endocrinology 145: 4461– 4469, 2004)

acetate (MPA), was tested to determine whether it could stimulate spermatogenic recovery after irradiation. MPA effectively suppresses T and gonadotropin production (12) and thereby suppresses spermatogenesis in rodents (12, 13). Based on the observation that the hormonal regimens that suppress T production also stimulate spermatogonial differentiation in toxicant-treated rat models, we hypothesized that MPA given alone or in combination with T would stimulate this process in irradiated rats and facilitate spermatogenic recovery. Because MPA is being used with T in the clinical trials as a male contraceptive regimen to suppress spermatogenesis (14, 15), if our hypothesis is correct, the combination could also be used clinically to protect or restimulate spermatogenesis. It is important to include T in such a clinical regimen to maintain normal peripheral androgenic effects, such as bone and muscle mass and libido. However, even physiological serum levels of T can inhibit GnRH-ant-induced spermatogenic recovery. We therefore compared the effectiveness of MPA ⫹ T combination at stimulating recovery of spermatogenesis in irradiated rats with that of low doses of T alone, GnRH-ant ⫹ T, and estradiol (E2) ⫹ T. E2 was used because it is also known to inhibit normal spermatogenesis by suppressing gonadotropins (16) and T synthesis (17). Materials and Methods Materials E2, T, dextran-coated charcoal, MPA powder, benzyl alcohol, and olive oil were obtained from Sigma (St. Louis, MO). MPA pellets were


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Department of Experimental Radiation Oncology (G.S., C.C.Y.W., O.U.B.-T., 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 Department of Andrology, ANZAC Research Institute (D.J.H.), Concord Hospital and University of Sydney, Sydney, New South Wales 2139, Australia


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Irradiation Rats were anesthetized and their testes irradiated following the procedure described in detail earlier (11). 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. A single dose of 6 Gy was administered at a dose rate of 0.96 Gy/min.

Hormone measurements In some cases, blood was collected from the rats by sequential bleeding. Rats were restrained by hand, and the collection area was shaved and smeared with Vaseline; the saphenous or epigastric vein on the thigh was nicked using a no. 11 surgical blade and the blood was collected into a centrifuge tube. Alternate legs were used for repeated bleedings. When 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. To assess the systemic androgen response of the different treatments, seminal vesicles were freed of adhering tissues and weighed without expressing the fluid. Serum levels of FSH and LH were measured using immunofluorometric assays (Delfia, Wallac OY, Turku, Finland) as previously described (11, 18, 19). The standards used for LH and FSH were NIDDKrLH-RP-3 and NIDDK-rFSH-RP-2 (AFP 4621B), respectively. Using 25 ␮l

and 150 ␮l of serum samples, the minimum levels of detection of LH and FSH by this method are 0.04 ng/ml and 0.1 ng/ml, respectively. Serum T and ITT were assayed in samples from experiments involving exogenous steroid hormone treatment by using T-antiserum-coated tubes (Diagnostic Systems Laboratories, Webster, TX) as previously described in detail (10). The minimum T detection level was 0.04 ng/ml. To reflect the actual concentration of T to which the testicular cells are exposed, ITT was expressed as the amount per gram of testis. Serum levels of MPA were assayed by Immunometrics (London, UK) Ltd. following a RIA procedure slightly modified from that originally designed to estimate MPA in human plasma extracts (20). The samples were extracted in diethyl ether and reconstituted in the required amount of PBS containing 0.01% Thimerosol and 0.1% gelatin. The samples were then incubated overnight at 4 C with MPA antiserum (raised in goat), and tritiated MPA tracer (obtained from NEN Life Science Products, Wellesley, MA). The mixture was incubated with the charcoal reagent at 4 C for 30 min, and the free hormone was precipitated. The bound hormone in the supernatant was then counted. Standards were prepared in dextran-coated charcoal-stripped rat serum. The assay was validated by assaying rat serum samples with known amounts of MPA added. The minimum level of detection of MPA by this assay was 58 pg/ml.

Hormone treatments The schedules of hormone treatments after irradiation are given in Fig. 1. All the treatments were given for a period of 4 wk from wk 3–7 after irradiation. Experiments 1a and 1b differed by the delivery methods for MPA, resulting in different doses. The dose of MPA was selected to be between 8 and 10 mg/kg䡠d based on previous studies using injections (13, 21). In experiment 1a, we implanted in each rat one 75-mg sc MPA pellet, which nominally was supposed to release 2.7 mg/d, equivalent to 9 mg/kg䡠d, for 28 d with the rats we used. In experiment 1b, we gave MPA injections of 10 mg/kg䡠d in a solvent of 95% olive oil and 5% benzyl alcohol for 2 wk (wk 3 and 4 after irradiation), followed by 5 mg/kg injections once every 2 d for 1 wk (wk 5). Because of depot accumulation of MPA, this was sufficient to keep T levels suppressed to less than 10% of control until wk 7. The GnRH-ant Acyline was dissolved in sterile water and given as weekly sc injections of 1.5 mg/kg on wk 3–5 after irradiation followed by a reduced dose of 0.7 mg/kg at wk 6. We have determined that a dose of Acyline of 1.5 mg/kg suppresses serum T ⬍ 0.1 ng/ml within 3 d of the first injection and maintains suppression for 7–10 d (Porter, K. L., G. Shetty and M. L. Meistrich, manuscript in preparation). The dose of

FIG. 1. Schematic of the experimental protocol (A). Hormone combinations in the different groups and the mode of delivery of MPA for each of the experiments (B). SQ, Subcutaneous; N.A., not applicable.

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obtained from Innovative Research of America (Sarasota, FL). SILASTIC brand silicon tubing (catalog no. 602-305) was purchased from Dow Corning (Midland, MI). The GnRH-ant, Acyline, was obtained from the Contraceptive Development Branch of National Institute of Child Health and Human Development (North Bethesda, MD). Adult LBNF1 (F1 hybrids of Lewis and Brown-Norway) male rats were obtained from Harlan Sprague Dawley (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, National Institutes of Health. They were maintained on a 12-h light, 2-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. All the animal procedures were approved by the Institutional Animal Care and Use Committee.

Shetty et al. • Failure of Spermatogenic Recovery

Shetty et al. • Failure of Spermatogenic Recovery

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Evaluation of spermatogenesis An aliquot from the right testicular homogenate was sonicated at 4 C for 4 min as described earlier (24). The sonication-resistant sperm heads, representing nuclei of steps 12–19 spermatids, were counted in a hemocytometer. The detection limit of this assay is 3 ⫻ 103 sperm heads per testis. The left testis was fixed in Bouin’s fluid and embedded in paraffin or plastic (JB4, Polysciences, Warrington, PA). Then 4-␮m sections were cut and stained with hematoxylin. To evaluate recovery of spermatogenesis after irradiation and hormone treatment, we scored 200 seminiferous tubules in one section from each animal. A tubule was scored as differentiating if it contained three or more cells that had reached the type B spermatogonia stage or later (25). The tubule differentiation index (TDI), which is the percentage of tubules showing differentiation, was then computed.

Statistical analysis For sperm counts, LH, serum T, and ITT measurements, the averages and sem were calculated on log-transformed data. The organ weights, TDI, FSH, and MPA were represented as arithmetic mean ⫾ sem. The differences between the treatment groups were analyzed first by oneway ANOVA. If the difference was significant (P ⬍ 0.05), a t test with a Bonferroni correction 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-ant alone). To compare the differences between specific groups of GnRH-ant and steroid-hormone-treated irradiated rats, a t test was performed. All analyses were performed with the SPSS (version 11.5) statistical package.

Results MPA treatment regimens

Initial studies using MPA pellets, each designed to release 9 mg/kg䡠d, failed to suppress hormone levels and spermat-

ogenesis as much as expected for this dose of MPA (12, 13). Treatment of nonirradiated rats with MPA pellets for 4 wk did not significantly change serum T and late spermatid counts, although ITT was slightly reduced from 169 ⫾ 23 to 102 ⫾ 10 ng/g testis (data not shown). Two-week treatment of irradiated rats with MPA pellets also failed to significantly decrease LH, FSH, serum T, and ITT levels (data not shown). We therefore tested daily injections of MPA at 10 mg/kg. Four weeks of this treatment reduced both serum and intratesticular T levels to less than 1% of control and late spermatid counts to 10% of control in nonirradiated rats (Table 1). Higher doses of MPA (20 and 40 mg/kg) produced no significant further reductions in serum T, ITT, or spermatid counts. In irradiated rats, daily injections of MPA for 2 wk, unlike pellets, produced marked declines in LH, FSH, serum T, and ITT. To determine the basis for this difference in biological response to the two modes of delivery, we measured serum MPA levels for each method. Consistent with the responses, implantation of single pellets produced serum levels of 7.7 ⫾ 1.6 ng/ml in irradiated rats after 2 wk, whereas daily injections produced about 200 ng/ml (Table 1). Because delivery of MPA via injections increased serum MPA levels and reduced T and gonadotropin levels and also reduced spermatogenesis in nonirradiated rats, we used this mode of delivery in the remaining studies. Although the 10-mg/kg dose by daily injections effectively decreased the levels of T, MPA’s depot effect made the duration of its activity uncertain. It was therefore necessary to find a regimen that would limit the T depression to 4 wk. In a preliminary study, two groups of nonirradiated rats were given daily injections of MPA at 10 mg/kg for 3 wk. One received the same dose from wk 3 to 4 and the other was given a reduced dose of 5 mg/kg䡠d during this period. MPA injections suppressed serum T levels below the limits of detection within 2 wk (Fig. 2A). With both injection regimens complete suppression continued for another 2 wk after stopping the treatment. Based on these studies, the doses of MPA used in experiment 1b were further reduced: daily injections of 10 mg/kg was given for 2 wk (wk 3–5 after irradiation), 5 mg/kg injections were given every other day for 1 wk (wk 5– 6). Because of the depot effect, this dosage of MPA kept serum T suppressed ⬍ 0.2 ng/ml at wk 7 after irradiation (Fig. 2B). A similar suppression was observed in rats receiving GnRHant alone. In those receiving a combination of GnRH-ant and MPA, serum T levels were even more suppressed at wk 7. But by wk 8, serum T in all these rats reached values that were not significantly different from control levels except for those

TABLE 1. Efficacy of MPA on the suppression of sex hormone levels and spermatogenesis. MPA was given by injection of 10 mg/kg body weight/d Nonirradiated (treatment: 4 wk) Hormone treatment

None MPA injections a b c

Serum T (ng/ml)a

ITT (ng/g-testis)a

6.4 ⫾ 1.5 0.045 ⫾ 0.009c

221 ⫾ 60 1.4 ⫾ 0.2c

Sperm head count/testisa

Irradiated-6 Gy (treatment: wk 3–5 after irradiation) Serum T (ng/ml)a

230 ⫻ 106 ⫾ 13 ⫻ 106 2.2 ⫾ 0.5 22 ⫻ 106 ⫾ 1.7 ⫻ 106c 0.041 ⫾ 0.00c

ITT (ng/g-testis)a

208 ⫾ 12 2.3 ⫾ 0.3c

Mean calculated from log transformed data (⫾SEM). Mean ⫾ SEM. P ⬍ 0.001. Significance of difference between respective control and MPA-treated groups.

Serum LH (ng/ml)a

Serum FSH (ng/ml)b

Serum MPA (ng/ml)b

0.34 ⫾ .08 6.8 ⫾ 0.3 0.10 ⫾ 0.00 0.05 ⫾ 0.01c 3.86 ⫾ 0.38c 212 ⫾ 19c

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Acyline given on wk 6 was reduced to 0.7 mg/kg to maintain the T suppression only until wk 7. In addition, some groups of irradiated rats were also given T, administered in 2-cm SILASTIC brand capsules as described previously (16, 22). In rats implanted with 2-cm T capsules, with or without other suppressive treatments, serum T levels were not significantly different from average control physiological T levels (data not shown). In the second experiment, groups of rats were given sc implants of 0.5-cm SILASTIC brand capsules filled with E2, 2-cm T capsules, or both at 3 wk after irradiation. This dose of E2 was shown to have effects on spermatogenesis (22), elevate serum E2 levels, and suppress T production (23). In both experiments, some rats were killed for hormone measurements, 5 wk after irradiation, after 2 wk of hormone treatment, which is the midpoint of the treatment of the other rats. In the other rats, the treatment was continued so that the presence of the required amount of the administered hormone in rats was calculated to end at wk 7, by stopping the injections at the appropriate time, removal of the implants, and the expected degradation of the pellets. The rats were killed 13 wk after irradiation to measure spermatogenic recovery. A minimum of four rats in each group were used for hormone analysis, and a minimum of seven rats in each of the steroid-treated groups were used for the analysis of spermatogenic recovery.


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FIG. 3. Tubule differentiation indices (A, C, and E) and sperm head counts (B, D, and F) in irradiated-only (6 Gy) rats and irradiated rats treated during wk 3–7 after irradiation as follows: injections of MPA, T implants, or a combination of both (A and B), GnRH-ant with or without injections of MPA or T implants (C and D) (experiment 1b); E2 or T alone or a combination of both (E and F) (experiment 2). The rats were killed 13 wk after irradiation for the analysis of spermatogenic recovery. The numbers in parentheses on top of each column represent the number of rats in each experimental group. Significance of the difference from irradiated-only rats (for irradiated rats receiving additional treatments): *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001. Significance of difference from GnRH-ant alone-treated irradiated rats (for irradiated GnRH-ant-treated rats receiving additional treatments): ‡‡, P ⬍ 0.01; ‡‡‡, P ⬍ 0.001. Significance of difference between two groups indicated in brackets: †, P ⬍ 0.05; ††, P ⬍ 0.01; †††, P ⬍ 0.001.

treated with T ⫹ MPA (which was reduced to 0.16 ng/ml, not shown in figure). This could be due to a more highly effective suppression of T production by the combination of MPA and T that continued for a week even after the removal of the T implants. Effects of steroid treatments on spermatogonial differentiation

Irradiation with 6 Gy prevented the differentiation of surviving spermatogenic cells, the TDI being 0% at wk 13 (Fig. 3) and sperm head count falling below the limit of detection. We next evaluated the efficacy of MPA or T alone or the

combination of both to facilitate recovery of spermatogenesis. As expected, due to inefficient hormone suppression, treatment with MPA alone in pellet form in experiment 1a did not significantly stimulate differentiation and did not alter the TDI produced by treatment with 2-cm T or GnRH antagonist (data not shown). When MPA was given via injections in experiment 1b, it produced a TDI of 2% (Fig. 3A), a value that was not significantly altered by the addition of T. Steroid hormone treatments had no significant effects on sperm head counts, as they were less than 104 per testis (Fig. 3B). To determine whether MPA would have an effect on GnRH-ant-stimulated recovery of spermatogenesis, MPA

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FIG. 2. Serum T levels measured in serial serum samples from nonirradiated (A) and irradiated (B) rats treated with injections of MPA with or without other hormones. A, Two groups of rats (n ⫽ 4 each) were given daily injections of MPA at 10 mg/kg for 3 wk, one of which received 5 mg/kg䡠d of MPA from wk 3– 4 (filled triangle) and the other continued with 10 mg/kg䡠d during this period (filled square). The control rats (filled circle) (n ⫽ 2) received only the diluent for MPA. B, Irradiated rats were treated with GnRH-ant (open diamond) (n ⫽ 8), daily injections of MPA (open triangle) (n ⫽ 9), or a combination of both (open hexagon) (n ⫽ 10) according to the dosing regimens described for experiment 1b in Materials and Methods. Irradiated-only rats (open circle) (n ⫽ 4 except for 6-wk point, which only had two rats) received the diluent for MPA during this period. The hormone treatments were started 3 wk after irradiation. The rats were bled at times indicated on the graph. Significance of difference between treated rats and untreated rats at respective time points: ***, P ⬍ 0.001. ***, Applies to all three treatment groups for wk 2– 6 and wk 5–7 in panels A and B, respectively.

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Hormone levels during steroid treatment

We had previously shown that intratesticular levels of T or other androgens were mainly responsible for the inhibition of spermatogonial differentiation in irradiated rats. In the absence of other androgens the extent of recovery directly correlated with the suppression of ITT. Similar to previous observations, 5 wk after irradiation serum FSH levels and the concentration of ITT were significantly elevated, but the serum T levels were not significantly altered (Fig. 4, I, J, K, and L). Daily injections of MPA suppressed serum FSH, serum LH, and ITT levels to 56%, 15%, and 1% of the irradiated control levels, respectively (Table 1; and Fig. 4, A–C). Exogenous T or the combination of MPA ⫹ T produced a similar suppression of FSH and LH. Whereas MPA treatment was very effective at suppressing ITT to 2 ng/g testis, exogenous T treatment with or without MPA produced ITT levels of about 23 ng/g testis (Fig. 4C), which is intermediate between irradiated-only and MPA-treated rats. GnRH-ant was more effective in suppressing FSH than was MPA, but it was not effective at suppressing ITT, which was 15 ng/g testis in this group. The addition of T to the GnRH-ant treatment regimen partially reversed the suppression of FSH to levels that were observed in rats treated with T or MPA alone (Fig. 4, A and E). It should be noted that the degree of reversal of GnRHant-induced FSH suppression by MPA was similar to that caused by T, suggesting MPA has an androgenic effect. Measurements of the effects of MPA and T on the GnRH-antinduced suppression of LH were not informative because the values were already at the limit of detection of assay (Fig. 4F). MPA did further suppress the ITT levels in the GnRH-anttreated irradiated rats, bringing the levels down to those observed in irradiated rats given MPA alone (Fig. 4, C and G). E2 treatment (experiment 2) suppressed serum FSH, LH and ITT levels to 34%, 21%, and 0.9%, respectively, of the irradiated-only levels (Fig. 4, I–K), which was slightly greater suppression than that produced by MPA. E2 treatment also markedly reduced the serum T levels to 0.08 ng/ml com-

pared with about 2–3 ng/ml in control and irradiated-only rats (Fig. 4L). The addition of T to E2 treatment, as was the case when it was added to GnRH-ant treatment, significantly elevated serum FSH to the levels observed with T alone. We could not determine whether the addition of T affected LH levels, because they were already at the limit of detection of the assay. E2 was highly effective in suppression of ITT levels to 2 ng/g testis. In addition, the rats that received the combination of E2 ⫹ T treatment showed lower ITT concentrations (11 ng/g testis) than those receiving T alone (20 ng/g testis), suggesting that E2 further reduced testicular T production (Fig. 4K). The data from the various treatments were combined to evaluate the relationship between TDI and ITT concentrations. A general inverse correlation between TDI and ITT was observed for all treatment groups except those receiving MPA (Fig. 5A). The points (filled symbols) for MPA alone and GnRH-ant ⫹ MPA showed low ITT and low TDI, indicating that MPA inhibited spermatogenic recovery by a mechanism that did not involve T levels, further suggesting that MPA has an androgenic effect. The point for E2 ⫹ T (open square) was below the fitted curve, indicating that in this protocol E2 did not stimulate spermatogenic recovery by other means than its suppression of T production. The TDI value for GnRH-ant alone (open downward triangle), which strongly suppressed FSH, was above the curve, and the value for E2 ⫹ T, which only moderately suppressed FSH, was below the curve, suggesting that FSH might also have an inhibitory role. This possibility is supported by a replot of TDI as a linear function of both the ITT and FSH concentrations, which gives an even better fit for all the data points without MPA (Fig. 5B) than did using ITT alone. The combination used [T] ⫹ 2 ⫻ [FSH] was chosen among various linear functions to optimize the fit. As a measure of the androgenic effects of the different treatments, we also weighed the seminal vesicles of the irradiated rats in experiment 1b, killed during the treatments at wk 5 (Fig. 6). There were effects of MPA treatment on seminal vesicle weight that deviated from the serum T levels. For example, MPA significantly increased the seminal vesicle weights in the GnRH-ant-treated irradiated rats (Fig. 6B), despite maximum suppression of serum T levels and T production in this treatment group (Fig. 4, G and H). This result indicates some weak androgenic activity of MPA. In addition MPA significantly reduced the seminal vesicle weights in the T-treated rats (Fig. 6A) despite equivalent serum levels of T (2.9 and 3.5 ng/ml, respectively, in T and MPA ⫹ T-treated rats, Fig. 4D). This decrease suggests that high levels of MPA may bind to the androgen receptor and displace some of the more biologically active androgens, such as T or dihydrotestosterone. Discussion

Our principal goal was to study the efficacy of a currently used contraceptive regimen for stimulating the recovery of spermatogenesis in irradiated LBNF1 rats while maintaining serum T levels. In the course of these studies, we used two different dosing regimens for MPA that produced quite different serum MPA levels. However, neither level of MPA

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was combined with GnRH-ant treatment. As observed previously, 2-cm T implants decreased GnRH-ant-stimulated recovery of spermatogenesis from about 55% recovering tubules to 2%. MPA administered by injections significantly suppressed the GnRH-ant-stimulated TDI to about 18% and produced a 20-fold decline in sperm head counts (Fig. 3, C and D). Note that the combinations of GnRH-ant with T and MPA with T produced similar levels of tubule differentiation and late spermatid production. In experiment 2 we evaluated the effects of E2 on spermatogonial differentiation in irradiated rats. E2 stimulated differentiation in 88% of tubules and raised sperm counts to 7 ⫻ 107 per testis, as opposed to 55% and 4 ⫻ 106 per testis for rats treated with GnRH-ant (Fig. 3, E and F vs. C and D). When E2 and T were given in combination, the TDI was reduced, but only to an intermediate value of 30%. The sperm head counts for the irradiated rats treated with T and E2 were reduced to 1.3 ⫻ 105, which was significantly higher than the undetectable levels in irradiated-only rats (⬍ 3 ⫻ 103 per testis).

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Shetty et al. • Failure of Spermatogenic Recovery

combined with T proved effective at enhancing or producing a good level of recovery of spermatogenesis. The low serum levels of MPA (about 8 ng/ml) produced by the pellet regimen did not significantly change gonadotropin or androgen levels nor depress normal sperm production, or modulate the effects of T. In contrast, the high serum levels of MPA (about 200 ng/ml) produced by daily injections significantly suppressed serum T, ITT, and sperm production in nonirradiated rats and also suppress serum gonadotropins, serum T, and ITT levels in irradiated rats. During contraceptive trials in men, in which 300-mg (once in

3 months) depots of MPA enhanced the suppression of LH, FSH, and sperm production produced by T alone (15), serum MPA levels varied according to time since the last dose between 3.5 and 40 ng/ml (Handelsman, D. J., unpublished data). During contraceptive trials in women, the median serum level of MPA was 0.88 ng/ml toward the end of each 3-month treatment cycle, 11–14 wk after injection of 150 mg of depot MPA (20). These effective serum levels achieved in humans appear to be closer to the doses produced by the pellets indicating that spermatogenesis in the rat may be less sensitive to low serum levels of MPA that suppress repro-

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FIG. 4. Serum FSH (A, E, and I), serum LH (B, F, and J), ITT (C, G, and K), and serum T (D, H, and L) levels in irradiated (6 Gy) rats and irradiated rats treated with injections of MPA, T implants, or a combination of both (A–D); irradiated rats treated with GnRH-ant with or without injections of MPA or T implants (E–H) (experiment 2); nonirradiated rats, irradiated rats, and irradiated rats treated with E2 or T alone or a combination of both (I–L) (experiment 3). The hormone treatments were started at 3 wk after irradiation and the rats were killed at 5 wk after irradiation. The numbers in parentheses on top of each column represent the number of rats in each experimental group. Significance of the difference from irradiated-only rats (for irradiated rats receiving different treatments): *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001; Significance of difference from GnRH-ant alone-treated irradiated rats (for irradiated GnRH-ant-treated rats receiving additional treatments): ‡‡‡, P ⬍ 0.001. Significance of difference between two groups indicated in brackets: †, P ⬍ 0.05; †††, P ⬍ 0.001.

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FIG. 6. Seminal vesicle weights in irradiated-only and irradiated rats treated with injections of MPA or T implants or a combination of both (A) and in irradiated rats treated with GnRH-ant with or without injections of MPA or T implants (B). The hormone treatments were started 3 wk after irradiation, and the rats were killed at wk 5 after irradiation. The numbers in parentheses on top of each column represent the numbers of rats in each experimental group. Significance of the difference from irradiated-only rats (for irradiated rats receiving different treatments): ***, P ⬍ 0.001. Significance of difference from GnRH-ant alone-treated irradiated rats (for irradiated GnRH-ant-treated rats receiving additional treatments): ‡‡, P ⬍ 0.01; ‡‡‡, P ⬍ 0.001. Significance of difference between two indicated groups of rats receiving different steroids: †, P ⬍ 0.05; †††, P ⬍ 0.001.

ductive function in humans. However, in this study we also used high dose levels of MPA in rats that have gonadotropin and spermatogenic suppressive effects comparable with those observed in humans. Despite the suppressive effects of the high dose of MPA on hormone levels in the rat, it did not produce any marked change in tubule differentiation. MPA alone did induce spermatogonial differentiation in 2% of tubules, which was comparable with that observed with T alone; the combination was not significantly different. The paucity of stimulation was surprising in view of the high suppression of ITT observed with MPA. The inability of MPA to stimulate recovery might be due to its reported androgenic activity (26). Evidence for its androgenicity appeared in our own study: MPA treatment increased seminal vesicle weight in GnRH-anttreated irradiated rats even though it further suppressed ITT levels. Further evidence that MPA acts as an androgen is its

inhibition of GnRH-ant stimulated spermatogenic recovery, a property shared by all the androgens tested (11). Because serum MPA levels were about 200 ng/ml (about 600 nm) in the high-dose MPA group and we assume that intratesticular levels of MPA would be similar, and because ITT levels were only 2 ng/g testis (about 7 nm), and MPA does binds to androgen receptor with a binding affinity (Kd) of 1.7–3.6 nm vs. the 0.2– 0.5 nm value for T (27), there is appreciable binding of MPA to the androgen receptor in the testis. The inhibition of spermatogenic recovery implies that MPA bound to the androgen receptor in the testis acts as an androgen agonist in this system. Additional evidence for MPA-androgen receptor binding is the reduction in seminal vesicle weight when we added MPA to the T-treatment regimen. In that case, serum T levels were about 2 ng/ml (about 7 nm) and serum MPA levels were about 600 nm. These results can thus be explained if, in the seminal vesicle, MPA is a less

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FIG. 5. Correlations between tubule differentiation indices and ITT concentrations (A) and a linear combination of ITT and FSH concentrations [ITT] ⫹ 2⫻ [FSH] (B) during the treatment period in irradiated only rats and irradiated rats that received various hormone treatments: irradiated-only (open circle), irradiated rats treated with MPA (filled triangle), T (open diamond), MPA ⫹ T (filled circle), GnRH-ant (open downward triangle), GnRH-ant ⫹ MPA (filled square), GnRH-ant ⫹ T (open upward triangle), E2 (open hexagon) or E2 ⫹ T (open square). Data from experiments 1b and 2. The remaining points, after excluding those for animals treated with MPA, either alone or in combination with other hormones (all filled symbols), were fitted with sigmoidal curves.


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(36). The antiandrogenic progestin should be very effective at reducing gonadotropin levels and would also inhibit some of the residual testosterone action in the testis, stimulating spermatogenic recovery much like flutamide does (10). Alternatively, an estrogenic (37), less androgenic (38) progestin like norethisterone, which has also been shown to have contraceptive potential in men, may be considered (39). In contrast to the results with MPA, E2, which suppressed serum LH, FSH, T, and ITT levels to the same extent as MPA, produced a dramatic recovery of spermatogenesis. Furthermore, although recovery was reduced after the addition of T to E2, it was still sustained (TDI ⫽ 30%). In fact, this recovery was much greater than that observed with GnRH-ant ⫹ T (3%). This could be explained by the lower ITT levels, which were 11 ng/g testis with E2 ⫹ T compared with 27 ng/g testis with GnRH-ant ⫹ T combination. These results are consistent with a previous study showing that addition of E2 to T, given before procarbazine treatment, improved the recovery of spermatogenesis after procarbazine treatment (22, 23). Because E2 has been shown to be useful in enhancing the T-induced suppression of gonadotropins and likely ITT as indicated by suppression of sperm production in normal men (40), estrogens or selective estrogen receptor modulators might also be useful in stimulating recovery of spermatogenesis after cytotoxic therapies. Acknowledgments We are thankful to Mr. Kuriakose Abraham for the histological preparations and Mr. Walter Pagel for editorial advice. We also thank Ms. Tarja Laiho and Dr. Pirjo Pakarinen for the skillful assistance in performing gonadotropin assays and Dr. Hyun K. Kim for providing the Acyline. Received April 6, 2004. Accepted June 11, 2004. 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: [email protected] This work was supported by Research Grants R01 ES-08075 from National Institutes of Health (NIH)/National Institute of Environmental Health Sciences (to M.L.M.), Core Grant CA 16672 from the NIH, and a grant from the Lalor Foundation (to G.S.).

References 1. Kangasniemi M, Huhtaniemi I, Meistrich ML 1996 Failure of spermatogenesis to recover despite the presence of A spermatogonia in the irradiated LBNF1 rat. Biol Reprod 54:1200 –1208 2. Delic JI, Hendry JH, Morris ID, Shalet SM 1986 Dose and time relationships in the endocrine response of the irradiated adult rat testis. J Androl 7:32– 41 3. van Alphen MMA, van de Kant HJG, de Rooij DG 1988 Depletion of the spermatogonia from the seminiferous epithelium of the rhesus monkey after X irradiation. Radiat Res 113:473– 486 4. Meistrich ML, van Beek MEAB 1990 Radiation sensitivity of the human testis. Adv Radiat Biol 14:227–268 5. Meistrich ML, Shetty G 2003 Inhibition of spermatogonial differentiation by testosterone. J Androl 24:135–148 6. Kreuser ED, Kurrle E, Hetzel WD, Heymer B, Porzsolt F, Hautmann R, Gaus W, Schlipf U, Pfeiffer EF, Heimpel H 1989 Reversible germ cell toxicity after aggressive chemotherapy in patients with testiclular cancer: results of a prospective study. Klin Wochenschr 67:367–378 7. Clifton DK, Bremner WJ 1983 The effect of testicular X-irradiation on spermatogenesis in man. A comparison with the mouse. J Androl 4:387–392 8. Meistrich ML, Kangasniemi M 1997 Hormone treatment after irradiation stimulates recovery of rat spermatogenesis from surviving spermatogonia. J Androl 18:80 – 87 9. Meistrich ML, Wilson G, Shuttlesworth G, Huhtaniemi I, Reissmann T 2001 GnRH agonists and antagonists stimulate recovery of fertility in irradiated LBNF1 rats. J Androl 22:809 – 817

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effective androgen receptor agonist than is T at maintaining tissue weight. Other explanations are also possible. For example because MPA reduces testicular androgen binding protein levels but increases serum androgen binding protein levels (12), the ability of T to inhibit of spermatogonial differentiation in the testis could be enhanced but the T-stimulated rise in seminal vesicle weight decreased. Alternatively, MPA could act directly on the testis by an androgen receptor-independent mechanism. Preliminary evidence for the presence of progesterone receptors in testis (28) and the direct action of the progestin levonorgestrel on the seminiferous epithelium (29) could provide such a mechanism. MPA could also inhibit spermatogenic recovery by altering Leydig cell functions because high levels of progesterone inhibit murine Leydig cell functions in culture (30, 31). There have been contradictory reports on the effects of MPA ⫹ T treatment, given before (or during) cytotoxic treatment, on later spermatogenic recovery. One study showed that MPA ⫹ T potentiates radiation damage to spermatogenesis in Sprague Dawley rats (32). Others (21, 33) reported that MPA ⫹ T treatment produces greater recovery of spermatogenic function after the radiation or procarbazine treatment of Sprague Dawley rats. Neither of the two studies showing that MPA ⫹ T had beneficial effects compared that treatment regimen with any others, even with T alone. In the current study, we also observed a modest increase in the number of tubules with differentiating germ cells in rats given MPA ⫹ T; the small magnitude of this response is in part a result of our use of a more radiation-sensitive rat strain, LBNF1 (22), a higher radiation dose (6 Gy vs. 3 Gy), treatment after rather than before radiation, and a shorter MPA ⫹ T treatment time (28 vs. 55 d). However, the most important point from the current study is that the stimulation of recovery by MPA ⫹ T treatment is much less than after GnRHant, E2, or E2 ⫹ T treatment. Although combining MPA with T enhanced the contraceptive potential of the treatment in human clinical trials (15, 34), the addition of MPA in two different doses to a T treatment regimen did not enhance the restoration of spermatogenesis after cytotoxic treatment in rats. Although it is possible that an intermediate dose of MPA would effectively suppresses T production but may not have sufficient androgenic action to inhibit spermatogenic recovery, there appear to be better potential treatment options. Our studies are consistent with a recent report that MPA ⫹ T failed to stimulate recovery of spermatogenesis in men who had been exposed to high doses of radiation or chemotherapy for treatment of childhood cancers (35). The present study shows that T ⫹ MPA slightly stimulated spermatogenic recovery in the rat model, but other treatments were more potent. Thus, the ineffectiveness of a combination of MPA and T in restoring fertility in cancer patients (35) does not negate the possibility that a more effective hormonal treatment could protect or restore spermatogenesis and fertility after cytotoxic therapy for cancer. If the weak effect of MPA on spermatogenic recovery is caused by its androgenicity, other progestins, which lack androgenic properties, may be more effective. An antiandrogenic progestin, such as cyproterone acetate, along with T may be considered, provided doses of the two agents can be adjusted to maintain the peripheral effects of the androgen

Shetty et al. • Failure of Spermatogenic Recovery

Shetty et al. • Failure of Spermatogenic Recovery


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