Both Testosterone and Follicle-Stimulating Hormone Independently Inhibit Spermatogonial Differentiation in Irradiated Rats
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《内分泌学杂志》
Department of Experimental Radiation Oncology (G.S., C.C.Y.W., O.U.B.-T., Z.Z., M.L.M.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Prince Henry’s Institute of Medical Research (S.J.M.), Clayton, Victoria 3168, Australia
Department of Physiology (P.P., I.H.), University of Turku, 20520 Turku, Finland
Institute of Reproductive and Developmental Biology (I.H.), Imperial College London, London W12 ONN, United Kingdom
Abstract
Simultaneous suppression of both testosterone and FSH with GnRH antagonists (GnRH-ant) reverses the radiation-induced block in spermatogonial differentiation in F1 hybrids of Lewis and Brown-Norway rats. Although addition of exogenous testosterone restores the block, it also raises FSH, and hence it had not been possible to conclusively determine which hormone was inhibiting spermatogonial differentiation. In the present study, we establish the relative roles of testosterone and FSH in this inhibition using three different approaches. The first approach involved the treatment of irradiated rats, in which differentiation was stimulated by GnRH-ant plus flutamide, with FSH for 2 wk; the FSH reduced the percentage of tubules that were differentiated (TDI) by about 2-fold, indicating that FSH does have an inhibitory role. The second approach involved treatment of irradiated, hypophysectomized rats with exogenous testosterone for 10 wk; testosterone also reduced the TDI, demonstrating that testosterone had a definite inhibitory effect, independent of pituitary hormones. Furthermore, in this protocol we showed that TDI in the hypophysectomized testosterone-treated group, which had higher intratesticular testosterone levels but lacked FSH, was slightly higher than the TDI in a GnRH-antagonist-testosterone-treated group of irradiated rats, which had normal physiological levels of FSH; this result supports a role for endogenous FSH in suppressing spermatogonial differentiation in the latter group. The third approach involved injection of an active anti-FSH antibody for 10 d in untreated, GnRH-ant plus flutamide-treated, or GnRH-ant plus testosterone-treated irradiated rats. This was not sufficient to increase the TDI. However, flutamide given in a similar treatment schedule did increase the TDI in GnRH-ant plus testosterone-treated rats. We conclude that both testosterone and FSH individually inhibit spermatogonial differentiation after irradiation, but testosterone is a more highly potent inhibitor than is FSH.
Introduction
RADIATION AND CANCER chemotherapy deplete germ cells, causing prolonged azoospermia in rodents (1, 2, 3), monkeys (4), and humans (5). In the rat these agents cause azoospermia by disrupting the differentiation of surviving type A spermatogonia (6). In humans there is some histological evidence that radiation and cancer chemotherapeutic drugs can also block spermatogonial differentiation (5, 7), and the recovery of spermatogenesis after prolonged azoospermia also shows that surviving stem cells can be blocked from completing differentiation (8). However, in rats exposed to moderate doses of radiation or procarbazine, spermatogonial differentiation and progression of spermatogenesis can be restored by suppression of gonadotropins and intratesticular testosterone (ITT) using GnRH analogs (9, 10).
Because both ITT and FSH are elevated after irradiation (9, 11), both might have inhibitory effects. We initially showed that administration of exogenous testosterone (T) strongly inhibited the GnRH-analog-induced stimulation of spermatogonial recovery in a dose-dependent manner and that the effect of T was almost completely reversed if the androgen receptor antagonist flutamide was given (11). That and subsequent studies (6, 11, 12) showed excellent inverse correlations between the ITT levels and spermatogenic recovery. However, when irradiated rats treated with GnRH antagonist (GnRH-ant) were also treated with T, FSH levels were elevated in a dose-responsive manner by the action of T on the pituitary, and when these rats were also given flutamide, there was some reduction in FSH (6, 11). Thus, there also was a good inverse correlation between FSH levels and recovery of spermatogenesis. When we examined the effects of four different androgens, we found that they all suppressed spermatogenic recovery (13), but we could not determine whether suppression was a direct effect of androgen at the testicular level or a result of the elevation of FSH levels that they produced. Overall we found a better inverse correlation of spermatogenic recovery with ITT levels than FSH levels, suggesting a more dominant role of androgens than FSH in the inhibitory process. However, when the combined effects of ITT and FSH were considered, the correlations were further improved (11, 12). Because of the parallel effects of the treatments used on ITT and FSH, none of the previous studies could prove that either ITT or FSH was individually inhibitory to spermatogonial differentiation.
To show that both T and FSH have unequivocal direct effects on the inhibition of spermatogonial differentiation and to determine the role of FSH relative to T in inhibiting this process, we modulated FSH and, in some cases, T levels by three different approaches and assessed spermatogenic recovery. We first tested the effect of exogenous FSH treatment in GnRH-ant-treated, irradiated rats. Second, we compared GnRH-ant plus T treatment, which incompletely suppresses FSH, with that of hypophysectomy plus T, which produces complete FSH suppression. Third, we used an FSH antibody to reduce active serum FSH levels.
Materials and Methods
Materials
T and dextran-coated charcoal were obtained from Sigma (St. Louis, MO). Flutamide pellets were obtained from Innovative Research of America (Sarasota, FL). SILASTIC brand tubing (catalog no. 602-305) was purchased from Dow Corning (Midland, MI). Alzet miniosmotic pumps (model 2001) were obtained from Alza Corp. (now Diuret) (Palo Alto, CA). Two GnRH-ants were used in this study: cetrorelix was provided by Dr. Thomas Reissmann (ASTA Medica, Frankfurt, Germany), and Acyline was obtained from the Contraceptive Development Branch of National Institute of Child Health and Human Development (North Bethesda, MD). Two different GnRH-ants were used because there was a change in the formulation of cetrorelix pamoate, which was initially used, and the original formulation was no longer available. Hence, acyline was used in later experiments. Recombinant human FSH was a gift from Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA).
Polyclonal ovine antiserum raised against rat FSH (FSHAb) and control IgG preparations were obtained from immunized and nonimmunized sheep serum as described previously (14). The FSHAb is active in an in vitro bioassay, and in vivo a dose of 2 mg/kg FSHAb by daily sc injection neutralizes at least 90% of the circulating FSH in a normal rat (14, 15).
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 U.S. Department of Agriculture and the Department of Health and Human Services, National Institutes of Health (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 3 d before the initiation of irradiation or other treatments. They were 7 wk of age at the start of experiment 2 or 9–12 wk of age at the start of experiments 1 and 3 (Fig. 1). All the animal procedures were approved by the Institutional Animal Care and Use Committee.
Irradiation
Rats were anesthetized and their testes irradiated using procedures described in detail earlier (13). The lower part of the body was irradiated with a single dose of 5 Gy (experiments 1B, 1C, 2, and 3) (11) or 6 Gy (experiment 1A) (16).
Hypophysectomy
Rats were hypophysectomized and/or given hormone treatments within a week after irradiation. Hypophysectomy was performed by making a 1-cm incision on the ventral midline of the neck up to the mandible. The salivary glands, muscles, and trachea were retracted, and the cranium was scraped clean to see the horizontal blue suture line. Trephines were used to remove a circular piece of bone on the center of the blue line. The hypophysis was visually inspected and aspirated by inserting a cannula attached to a vacuum pump. For sham hypophysectomy, the same procedure was performed except that the pituitary was not aspirated. The incision was closed and analgesia was provided as needed based on clinical observations of pain or distress. The hypophysectomized rats were maintained on 5% sucrose solution for a week after surgery followed by drinking water supplemented with 3 mg cortisone succinate, 0.3 mg deoxycorticosterone acetate, and 0.17 mg L-thyroxine per liter of water. These hormones were given to minimize the effects due to the loss of ACTH and TSH. The hypophysectomized and sham-hypophysectomized rats were bled for hormone analysis 4 d after surgery to evaluate the completeness of the surgery.
Hormone modulation
The schedules of treatments administered after irradiation to modulate hormone levels are given in Fig. 1. The end of the treatment time was determined by stopping the injections at the appropriate time, removal of the implants or pumps, and the expected degradation of the pellets.
The effect of recombinant human (rh) FSH treatment was tested in irradiated rats in experiments 1A and B, giving the treatment either after (1A) or during (1B) the depletion of the differentiated germ cells by doses of radiation used in previous studies (11, 16). The activity of the rhFSH preparation was listed as 5 IU/μg. The dose of rhFSH (4 IU/d) was selected based on previous studies (17) and corresponds to 0.8 μg per rat per day. In experiment 1A, rats were given two weekly sc injections of the GnRH-ant acyline at a dose of 1.5 mg/kg in sterile water starting 15 wk after irradiation, which maintains T suppression for 2 wk (18). To further suppress the androgen action, the rats were also given the antiandrogen flutamide by sc implanting 20-cm-long flutamide filled SILASTIC brand implants (Porter, K. L., G. Shetty, G. A. Shuttlesworth, I. Huhtaniemi, and M. L. Meistrich, manuscript in preparation). One group of these GnRH-ant+flutamide-treated irradiated rats received 0.8 μg rhFSH in PBS per day for 2 wk from sc implanted miniosmotic pumps, which were replaced at weekly intervals. Appropriate, sham injections, placebo pellets, or Teflon bars were used for rats not receiving all treatments. In experiment 1B, groups of irradiated rats were given single sc injections of the GnRH-ant cetrorelix acetate and cetrorelix pamoate at doses of 1.5 and 0.75 mg/kg, respectively, which is expected to suppress the gonadotropin and T levels for 2 wk (11). Flutamide was given by sc implanting an 84-mg pellet calculated to deliver a dose of 20 mg/kg body weight per day for 2 wk; the residual pellet was left in place after 2 wk (11). The rhFSH was given as in experiment 1A. In experiment 1C, designed to compare the spermatogenic inhibitory effect of T with that of FSH in experiment 1B, the treatment schedule, doses, and delivery of GnRH-ant and flutamide were the same, with the exception that the residual flutamide pellet was surgically removed at the end of 2 wk. T was given to some of the rats either in 2- or 6-cm SILASTIC brand capsules sc implanted (19).
The second experiment was designed to test the role of T in the absence of FSH and compare the effects of hypophysectomy with GnRH-ant. Groups of sham-hypophysectomized rats were given weekly sc injections of acyline at a dose of 1.5 mg/kg in sterile water starting 4 d after surgery. T was provided to two groups of rats in 6-cm SILASTIC brand capsules and left in place until they were killed.
Experiment 3 was designed to compare the effects of suppressing FSH by passive immunization with FSHAb with blocking T action with flutamide. Irradiated-only rats, irradiated rats treated with GnRH-ant+flutamide, and irradiated rats treated with GnRH-ant+T were used, with and without FSHAb treatment. The GnRH-ant acyline was given by a single sc injection of 1.5 mg/kg, which had been previously shown to suppress T for 10 d in unirradiated LBNF1 rats (18), some of the rats in the current experiment had begun to show recovery of T levels at d 10. Flutamide was given by sc implanting 60-mg pellets that release the drug at a calculated dose of 20-mg/kg body weight per day for 10 d. Testosterone was administered in 2-cm SILASTIC brand capsules. The rats in these three treatment groups were treated with either FSHAb for 10 d or control IgG. All treatments were started 27 d after irradiation, and the duration of treatment was limited to 10 d because longer treatments can result in the development of antibodies to the neutralizing antisera. The doses of FSHAb given in irradiated-only rats, irradiated rats treated with GnRH-ant+flutamide, and irradiated rats treated with GnRH-ant+T were 3.62, 0.9, and 1.74 mg/kg·d, respectively, based on the dose (2 mg/kg·d) required to neutralize more than 90% of FSH in rats with a mean FSH value of 5.9 ng/ml (14) and the expected FSH levels in these rats. To directly compare the relative spermatogenic inhibitory effects of FSH and T, one group of GnRH-ant+T-treated rats was given flutamide for 10 d in 60-mg pellets.
To confirm the activity of FSHAb in blocking FSH in LBNF1 rats, a separate experiment was performed. The spermatogenic inhibitory activity of the FSHAb was tested in GnRH-ant+T-treated unirradiated rats, using a protocol slightly modified from one previously employed in Sprague Dawley rats (14). Rats were given six weekly injections of acyline at 1.5 mg/kg to suppress gonadotropin and androgen levels. At the beginning of the seventh week they were implanted with 6-cm SILASTIC brand implants containing T to partially restore ITT and FSH levels and spermatogenesis, and given daily injections of either FSHAb or control IgG at 1.74 mg/kg for 7 d. The quantitative changes in the degree of spermatogenesis in these groups of rats was assessed by counting germ cell types in 25-μm-thick testicular sections embedded in methacrylate (JB4, Polysciences, Warrington, PA) by stereology.
Stereological analysis
The optical dissector method (20) was used to determine the total number of cells per testis. All estimates were performed using a x100 objective on a DML microscope (Leica Microsystems, Wetzlar, Germany) with a motorized stage controller and joystick. A software package, Stereo Investigator (MicroBrightField, Colchester, VT), was used to superimpose an unbiased counting frame on the video image. Fields were selected by a systematic uniform random sampling scheme as described previously (20).
A counting frame with an area of 686 μm2 was used to count Sertoli cells, type A spermatogonia/intermediate spermatogonia, B spermatogonia/preleptotene spermatocytes, leptotene/zygotene/pachytene spermatocytes, and round spermatids. We counted 40–200 of each cell type in each animal. Two sections at different regions of the each testis, selected by systematic random sampling, were analyzed.
Hormone measurements
We analyzed hormone levels in a minimum of five rats in each hormone- or hormone suppressor-treated group. In some cases blood was collected from the rats by sequential bleeding as described earlier (12). When the rats were killed, blood was collected by cardiac puncture under ketamine-acepromazine anesthesia. The serum was separated and stored at –20 C. In all rats the right testis was freed of the tunica, weighed, collected on ice, and homogenized. In some cases, an aliquot was removed and the sperm heads counted; the remainder of all samples was stored at –20 C for ITT analysis.
Serum levels of FSH and LH were measured using immunofluorometric assays (Delfia, Wallac Finland Oy, Turku, Finland) as previously described (21, 22). The standards used for rat LH and FSH were NIDDK-rLH-RP-3 and NIDDK-rFSH-RP-2 (AFP 4621B), respectively. For 25- and 30-μl samples, the minimum detection levels for LH and FSH by this method are 0.04 and 0.1 ng/ml, respectively. The standard used for human (h) FSH assay was calibrated against the second international reference preparation of human pituitary FSH/LH for bioassay. The minimum level of detection for human FSH was 1 U/liter. No detectable cross-reaction of rat FSH was observed in the human FSH assay for the range of FSH levels present in the experimental rats. However, there was cross-reactivity of the human FSH in the rat assay, which was quantified by running the same samples of rhFSH in a rat FSH assay (using rat FSH as standard for analysis) and a human FSH assay (using hFSH as standard). The cross-reactivity of human FSH in the rat assay run at a series of dilutions showed that 1 U/liter of rhFSH in the hFSH assay would give a reading of 0.54 ng/ml in the rat assay. In exogenous rhFSH-treated rats, the actual rat FSH values were calculated by subtracting the amount contributed by hFSH using this relationship. To compare the concentration of the human FSH in the rat serum with endogenous rat FSH levels, it was also necessary to determine the relationship between units per liter as measured by the immunofluorometric assay for human FSH and nanograms per milliliter of the rhFSH. A series of dilutions in buffer showed that 1 U/liter in the hFSH assay represents 4.94 ng/ml of rhFSH.
Serum T and ITT were assayed by using T-antiserum-coated tubes (Diagnostic Systems Laboratories, Webster, TX) as previously described in detail (11). The minimum T detection level was 0.04 ng/ml. To reflect the actual concentration of T to which the testicular cells were exposed, ITT was expressed as the amount per gram of testis.
Evaluation of spermatogenesis
To assess spermatogenic recovery, a minimum of five rats in each of the hormone-treated groups were killed at the times indicated in Fig. 1.
An aliquot from the right testicular homogenate was sonicated at 4 C for 4 min as described earlier (23). The sonication-resistant sperm heads, representing late spermatid nuclei, were counted in a hemocytometer. The detection limit of this assay is 3 x 103 sperm heads per testis.
The left testis was fixed in Bouin’s fluid and embedded in paraffin or methacrylate. Then 4-μm sections were cut and stained with hematoxylin. To evaluate recovery of spermatogenesis after irradiation and hormone treatment, the tubule differentiation index (TDI), which is the percentage of tubules showing differentiated germ cells, was computed in one section from each animal as described previously (13, 23).
Statistical analysis
The organ weights, TDI, and FSH were represented as arithmetic mean ± SEM. For sperm counts, LH, serum T, and ITT measurements, the averages and SEM were calculated on log-transformed data. The differences between the treatment groups were analyzed first by one-way ANOVA. If the difference was significant (P < 0.05), a Dunnett’s post hoc test was performed to determine the significance of the difference between the treated groups and a selected control group. When comparing multiple treatment groups simultaneously a Student’s t test with Bonferroni correction was applied; when only two groups were compared, the Student’s t test was performed without correction. In cases in which the data deviated from a normal distribution due to outliers, nonparametric statistics were applied. All analyses were performed with the SPSS (version 11.5, SPSS Inc., Chicago, IL) statistical package.
Results
Exogenous FSH treatment
To determine whether there is an inhibitory effect of FSH on spermatogonial differentiation, in experiment 1A, rhFSH was given to rats from wk 15 to 17 after the complete depletion of differentiating germ cells by radiation, whereas the levels and actions of androgens were suppressed with GnRH-ant and flutamide. The results showed that the GnRH-ant+flutamide stimulation of spermatogonial differentiation observed at wk 17 and 19 were reduced by rhFSH (Table 1). To confirm that FSH also inhibited the restoration of spermatogonial differentiation during the postirradiation depletion phase, rhFSH was given along with GnRH-ant+flutamide during wk 3–5 after irradiation. Treatment with GnRH-ant plus flutamide dramatically stimulated spermatogonial differentiation observed at wk 13 above that observed in irradiated-only rats (Fig. 2, A and B). Addition of rhFSH to this regimen significantly reduced the TDI and sperm head count, respectively, to 48 and 17% of the values observed with GnRH-ant plus flutamide alone.
We analyzed hormone levels in the rats in experiment 1B to confirm that the inhibitory effect was actually due to FSH. Treatment with a combination of GnRH-ant and flutamide, with and without additional rhFSH treatment, suppressed the serum levels of rat FSH to about 20% of irradiation-only levels (Fig. 2C), but the treatment with rhFSH produced hFSH levels of 10.9 U/liter (Fig. 2D). Based on the assay of known amounts of rhFSH in buffer, we estimated the concentration of rhFSH in treated rat serum to be 54 ng/ml, and the value might be higher if there were matrix effects of rat serum in the human FSH assay.
The LH levels were reduced by treatment with GnRH-ant+flutamide with or without rhFSH, but they were variable and the differences were not significant (data not shown). Nevertheless, the serum T levels were reduced to the limit of detection in these groups (Fig. 2F). GnRH-ant+flutamide treatment, without and with rhFSH, reduced the ITT concentration, respectively, to 3 and 4% (not significantly different) of the irradiated-only levels (Fig. 2E). Thus, the inhibition of spermatogenic recovery caused by exogenous rhFSH was unlikely to be due to a change in ITT levels.
To determine the effectiveness of T, compared with FSH, at inhibiting spermatogonial differentiation, rats were given GnRH-ant during wk 3–5 after irradiation with flutamide, no other treatment, or exogenous T (experiment 1C). GnRH-ant-only treatment for such a short period without blocking the action of the remaining low levels of T reduced the stimulation to only 10% of that observed in GnRH-ant+flutamide-treated irradiated rats (Fig. 3A). Moreover, addition of T in 2- and 6-cm capsules to GnRH-ant treatment further reduced the TDI to 5 and 1%, respectively, of that observed in GnRH-ant+flutamide-treated rats, with a concurrent reduction in sperm head counts (Fig. 3B). Such a severe inhibition by T, compared with only about 50% inhibition of TDI by rhFSH (Fig. 2A), suggests that these levels of T are more strongly inhibitory than the FSH treatment.
To confirm that the dose of rhFSH used was biologically active in LBNF1 rats, two groups of unirradiated LBNF1 rats were given GnRH-ant+flutamide for 2 wk, and one of these groups also received rhFSH at 0.8 μg/d. The GnRH-ant+flutamide treatment reduced the sperm head count and testis weights, but the addition of rhFSH produced significant increases in both end points (Fig. 4), consistent with its expected biological activity (24).
Hypophysectomy experiments
To confirm the independent spermatogenic inhibitory role of T after irradiation and to further examine whether pituitary hormones, especially FSH, are also involved in such inhibition, we analyzed the effects of T supplementation on spermatogenic recovery after irradiation in hypophysectomized and GnRH-ant-treated rats. Successful hypophysectomy was confirmed by the lack of body weight gain and assays of T levels 4 d after surgery (data not shown). No differences were observed between irradiated-only rats and sham-hypophysectomized irradiated rats in serum T at 4 d after surgery (not shown) or in ITT, serum T, LH, and FSH levels at the end of the experiment (Fig. 5, B–E).
Irradiated-only and sham-hypophysectomized irradiated rats showed negligible spermatogonial differentiation at 11 wk after irradiation (Fig. 5A). Hypophysectomy within a week after irradiation restored spermatogonial and spermatocyte differentiation in all the tubules. The histology of the testis was qualitatively similar to that observed in unirradiated rats after hypophysectomy (data not shown). Treatment of sham hypophysectomized, irradiated rats with GnRH-ant for 10 wk started at the time of hypophysectomy of the other group also stimulated differentiation in all tubules. When the hypophysectomized, irradiated rats were also treated with T in 6-cm SILASTIC brand capsules, starting immediately after hypophysectomy, the TDI was reduced to 56 ± 4%, whereas administration of 6 cm T to the GnRH-ant-treated rats inhibited the TDI at least as much to 47 ± 5%.
Hypophysectomy or GnRH-ant-treatment reduced the LH and serum T levels to below the limits of detection (Fig. 5, B and D). However, it was noted that the ITT levels in GnRH-suppressed, irradiated rats were significantly lower than those of hypophysectomized, irradiated rats (Fig. 5C). The more profound suppression of ITT by the GnRH-ant than hypophysectomy, despite undetectable levels of LH in both cases, suggests that GnRH-ant directly inhibits T production in Leydig cells of irradiated rats, possibly through GnRH receptors (25), as had been observed previously in other systems (26, 27).
Addition of 6 cm T increased the ITT concentrations to 19.5 ± 0.3 ng/g testis in hypophysectomized, irradiated rats but only to 11.0 ± 0.8 ng/g testis in GnRH-ant-treated irradiated rats (Fig. 5C). Although T treatment increased serum T levels in both hypophysectomized and GnRH-ant-treated rats, the levels were significantly higher in the hypophysectomized group (Fig. 5D). It should be noted that in the hypophysectomized rats the volume of blood was about half of the volume in nonhypophysectomized rats, which might have affected serum hormone concentrations.
Prolonged GnRH-ant treatment reduced serum FSH levels below the limit of detection, similar to that observed in hypophysectomized rats (Fig. 5E). The reduction of FSH to undetectable levels is attributed to the 10-wk treatment time because in a previous study, when the acyline was given for only 2 wk, FSH levels were still detectable (12). Although FSH levels were still below the limit of detection in T-treated, hypophysectomized irradiated rats, the addition of T partially restored the FSH levels in GnRH-ant-treated irradiated rats to 41% of those in irradiated-only rats.
To determine whether a relationship exists between the hormone levels and TDI, the TDIs were plotted against relevant hormone levels. The relationships between mean values of TDI and those of the ITT concentrations for intact irradiated and hypophysectomized irradiated rats, with and without additional treatments, is shown in Fig. 6A. The reduction of the TDI on addition of T to hypophysectomized rats, in the absence of FSH, demonstrates that T alone can inhibit spermatogonial differentiation in irradiated rats. However, an obvious shift in the curve to the left is observed for the intact irradiated rats, compared with the hypophysectomized irradiated rats. This suggests that the ITT concentration required to bring about a specific level of spermatogenic inhibition in irradiated rats with an intact pituitary is lower than that required in hypophysectomized irradiated rats. For example, to reduce the TDI to 56%, only about 10 ng T per gram testis were required in intact irradiated rats, compared with 20 ng T per gram testis in hypophysectomized-irradiated rats. Thus, a factor from the pituitary adds to the effect of ITT to facilitate inhibition of spermatogonial differentiation.
To model the possible additive inhibitory effect of FSH on spermatogonial differentiation in the irradiated rats, we plotted TDI against a linear combination of ITT concentration and 2 x FSH levels (Fig. 6B), which was the relationship we previously derived using irradiated rats treated with different combinations of GnRH-ant and other hormones (12). The curve relating TDI and [ITT + (2 x FSH)] for irradiated GnRH-ant-treated and irradiated hypophysectomized rats were very close, suggesting that this relationship approximates the additive effect of FSH to T in the inhibition of spermatogenic recovery in irradiated rats. In this study the best fit was obtained with the linear combination, ITT + (3.4 x FSH) (Fig. 6C).
FSH passive immunization
To further quantify the spermatogenic inhibitory activity of FSH in irradiated rats, circulating FSH was inactivated with FSHAb in experiment 3. We performed the treatment under three conditions: 1) in irradiated-only rats, which have high FSH and ITT; 2) in irradiated, GnRH-ant+flutamide-treated rats, which have low FSH and ITT and the action of T is blocked; and 3) in irradiated GnRH-ant+T-treated rats with intermediate levels of FSH and ITT. To compare the relative inhibitory effects of T and FSH, we also gave flutamide instead of FSHAb to an additional group of GnRH-ant+T-treated rats.
In irradiated-only rats the 10-d treatment with FSHAb did not increase the TDI or sperm head counts (Fig. 7, A and B), both of which were essentially zero. When gonadotropins and androgenic action were suppressed in the irradiated rats during this time period using GnRH-ant+flutamide, the TDI and sperm head counts significantly increased. However, the FSHAb treatment also did not alter the TDI or sperm head counts in these gonadotropin and androgen-suppressed rats. Third, the addition of T to the GnRH treatment, which increased ITT and FSH levels, significantly decreased the TDI and sperm head counts, but no significant increases in these measures were noted after FSHAb treatment. Serum levels of T were not altered by FSHAb treatment in any of these groups (Fig. 7C), indicating that changes in T were unlikely to have any effect on the outcome of spermatogenic recovery after FSHAb treatment.
Comparing the two groups treated with GnRH-ant and FSHAb, one treated with flutamide and one treated with T, shows T significantly reduces the TDI and sperm head counts. Because the FSH should be neutralized by the antibody, this result further demonstrates the inhibitory effects of T. Moreover, addition of flutamide, unlike FSHAb, to the GnRH-ant+T-treated rats reversed the spermatogenic inhibition, highlighting the greater inhibitory effect of endogenous levels of T than endogenous levels of FSH on spermatogonial differentiation.
To prove that the FSH antibody was indeed biologically active in LBNF1 rats, we examined its spermatogenic inhibitory activity in long-term GnRH-suppressed unirradiated rats that had been given T for 1 wk (14). Testosterone treatment increased the serum T levels to similar values of 7.6 ± 0.5 or 8.2 ± 0.4 ng/ml without or with daily injections of FSHAb, respectively, and raises FSH levels (14). The daily injections of FSHAb significantly (P < 0.001) suppressed the testis weight from 0.27 ± 0.01 to 0.20 ± 0.01 g and produced significant reductions in all the germ cell types analyzed in these rats (Fig. 8), demonstrating the biological activity of the FSHAb in blocking FSH action on the testis.
Discussion
The androgen and FSH inhibition of spermatogenic recovery after irradiation seems to contradict their well-studied roles in stimulating and maintaining spermatogenesis (14, 28). Although FSH and T have overlapping and synergistic effects on spermatogenesis (17), the role of T is more pronounced in supporting the differentiation of spermatids past step 7 (29), and the role of FSH is more important in supporting spermatogonial numbers and differentiation. Nevertheless, in normal rats spermatogonial differentiation is still qualitatively independent of both ITT and FSH (30). However, in irradiated rats we have now shown that the survival and differentiation of A spermatogonia are inhibited by moderate levels of ITT or high levels of FSH.
Through the use of hypophysectomy, this study unequivocally demonstrated for the first time that T alone can inhibit spermatogenic recovery in irradiated rats independently of the pituitary hormones. Although our previous studies showed that spermatogenic inhibition by exogenous androgens occurred through the androgen receptor, FSH mediation of such inhibition was not ruled out due to the parallel effects of exogenous androgens and antiandrogen on testicular androgen levels or activity and serum FSH levels (11, 13).
In the present study, we investigated the role of FSH in the inhibition of spermatogonial differentiation after irradiation using three different protocols. The first protocol, in which high levels of FSH were maintained by continuous administration of rhFSH for 2 wk, significantly inhibited spermatogenic recovery in androgen-suppressed irradiated rats. The second protocol, comparing hypophysectomized with GnRH-ant-suppressed rats, showed that a pituitary factor was involved in the inhibition of spermatogonial differentiation, and calculations showed that the results were consistent with this factor being FSH. In contrast, the third protocol, involving a reduction of endogenous FSH levels for 10 d under three different circumstances, failed to stimulate recovery of spermatogonial differentiation.
A quantitative comparison of the results of the three protocols is given in Table 2. Because we believe that the rhFSH is highly active in rats, the 54 ng/ml of rhFSH in the serum of rhFSH-treated rats in experiment 1B, which is 8 times that of rat FSH in irradiated-only rats, represents a supraphysiological level. The inhibitory effect of high FSH levels is observed with a 2-wk treatment under conditions in which the inhibition by T was minimized by the low ITT levels and the flutamide treatment.
In experiment 2 we were able to demonstrate an inhibitory effect of 10-wk exposure to endogenous FSH levels of 3 ng/ml in the intact rats treated with GnRH-ant and T, compared with the hypophysectomized rats treated with T in which there was no FSH present (Table 2). Despite the fact that the hypophysectomized, T-treated rats had higher ITT levels than the irradiated GnRH-ant+T-treated rats, they had slightly higher TDI values. Because we know that ITT is inhibitory, the slight increase in TDI (or at least the lack of any decrease) in the hypophysectomized, T-treated rats is most likely due to the absence of an inhibitory pituitary factor. The pituitary is responsible for the production of a variety of hormones, including the gonadotropins (LH and FSH), ACTH, TSH, GH, MSH, and prolactin. All the rats in this experiment received corticosteroids and T4, which should minimize the effects of loss of ACTH and TSH. Even so, corticosteroids act only on Leydig cells to inhibit T production (31), and this would be accounted for in our measurements of ITT. Thyroid hormones act only on immature, not adult, Sertoli cells, and their effect on Leydig cells is also primarily on steroidogenesis (32). Although thyroid hormone receptors are present on germ cells, the action of thyroid hormones does not appear to be critical for spermatogenesis in the adult. The effect of LH, which was suppressed to below the limit of detection in all hypophysectomized and GnRH-ant-treated groups, has been demonstrated to act only on spermatogenesis through T production (33). Whereas GH (34) and prolactin (35) have been reported to have stimulatory effects on spermatogenesis, their effects are mostly through steroidogenesis, and these effects are not as dramatic as the gonadotropins. Although we cannot rule out the possibility that a pituitary hormone besides FSH may have inhibitory effect on spermatogonial differentiation, we have shown that the outcomes can be explained by changes in FSH levels. The differences in the curves relating TDI to the ITT in intact and hypophysectomized rats (Fig. 6) could be nearly eliminated by replotting the data according to a previously developed linear relationship involving ITT and FSH (12).
In contrast, in experiment 3 no stimulation of spermatogenesis was observed by inhibiting FSH by passive immunization with an FSHAb preparation that we demonstrated to be active in unirradiated LBNF1 rats (Fig. 8). We believe that the failure of stimulation of spermatogonial differentiation in any of the three arms of this experiment could be attributed to stronger inhibition by high ITT levels, small changes in FSH levels, and the short time of the suppressive treatment (Table 2). In the irradiated-only rats, which had FSH levels of 6.8 ng/ml, the high levels of ITT exerted such a strong inhibitory effect that no recovery could be observed even with more than 90% suppression of FSH. In rats that received treatment with GnRH-ant+flutamide, the lack of enhancement of the percentage of tubules that differentiated by FSH immunoneutralization was attributed to the low levels of endogenous FSH in these animals. In the rats that received GnRH-ant+T, the inhibition of 3.3 ng/ml of FSH by FSHAb did not increase the TDI. The different result in this experiment from the enhancement of recovery in the hypophysectomized T-treated rats, compared with the GnRH-ant+T-treated rats, which also was a result of elimination of 3 ng/ml of FSH, is attributed to the short 10-d (experiment 3) vs. the long 10-wk (experiment 2) durations of FSH suppression. In addition, the greater inhibitory effects of the higher levels of ITT in this group of experiment 3 would further reduce detection of any effect of FSH modulation. Whereas the inhibitory effects of supraphysiological levels of FSH can be detected even after short exposure periods, exposure times must be long to detect a significant inhibitory effect of physiological or lower levels of FSH. However, the demonstration that flutamide but not FSHAb could stimulate the TDI in irradiated rats treated with GnRH-ant+T, even during this short treatment period (Fig 7) shows that even subphysiological levels of ITT can be more strongly inhibitory than physiological levels of FSH (Table 2).
The greater inhibitory effects of T caused by exogenous T (experiment 1C) compared with less inhibition caused by high levels of FSH (experiment 1B) further confirms the relative inhibitory action of the two hormones. The linear relationship derived from Fig. 6C can be used to quantify the relative effects of ITT and FSH on inhibition of spermatogonial differentiation in irradiated rats. The relationship shows that the inhibitory effect of 1 ng/ml FSH was equivalent to that of 3.4 ng T per gram testis. Because the level of ITT in the irradiated rats is 195 ng/g testis and FSH level was 6.8 ng/ml, we calculated that 90% of the inhibition of spermatogonial differentiation is a result of the ITT and only 10% is a result of the FSH.
Because androgen and FSH receptors are generally believed to be absent from germ cells including spermatogonia, T and FSH must produce their effects by acting on somatic cells. The actual physiological and molecular mechanisms of the T and FSH inhibition of spermatogonial differentiation after irradiation is under investigation.
T could be acting on multiple cell types that have androgen receptors (36). The action on the vasculature or other interstitial cells that affect fluid levels or composition deserves particular attention. This is supported by a strong correlation between the quantity of interstitial fluid per testis, which varies in response to ITT levels, and the inhibition of spermatogonial differentiation (18).
FSH can exert its direct inhibitory action only on Sertoli cells because they are the only ones with the receptor, and hence a reasonable approach is to examine FSH- and T-regulated Sertoli cell genes. Inhibin and activin are important Sertoli cell genes because activin stimulates spermatogonial proliferation in vitro (37) and while inhibin inhibits spermatogonial proliferation in vivo (38). Microarray analysis of total testicular RNA samples from experiment 1A (Bolden-Tiller, O. U., D. N. Stivers, C. C. Weng, and M. L. Meistrich, unpublished data) indicated that T/FSH suppression slightly (23%) but significantly (P < 0.001) up-regulated inhibin-, although inhibin-A was not expressed and inhibin-B was not identified as present on chips. In addition, activin inhibitors, follistatin, and bone morphogenetic protein and activin membrane-bound inhibitor, were, respectively, unchanged or slightly up-regulated (40%, P < 10–5) by T/FSH suppression. These data, which indicate that FSH would stimulate spermatogonial proliferation by reducing levels of inhibin and bone morphogenetic protein and activin membrane-bound inhibitor (39), suggest that these molecules are not involved in the inhibition of spermatogonial function by FSH and T.
Further research is required to resolve questions that remain about the application of these findings to other species. For example, although T also inhibits spermatogonial differentiation in jsd mice (40), FSH does not have any inhibitory role (41). Also, because it has recently been shown that T and FSH suppression was unsuccessful in stimulating spermatogenic recovery in irradiated nonhuman primates (42, 43), the focus of future research in this area should be on understanding the molecular mechanisms of T and FSH inhibition of spermatogonial differentiation through Sertoli cells in the rat. Although these hormonal treatments per se might not be clinically applicable, modulation of the downstream factors that they regulate could be useful in restoring fertility in patients who have undergone cancer therapy.
Acknowledgments
We thank Mr. Kuriakose Abraham for the histological preparations and Mr. Walter Pagel for editorial advice. We also thank Ms. Tarja Laiho for the skillful assistance in performing gonadotropin assays. We sincerely thank Drs. R. P. Blye and Hyun K. Kim (National Institute of Child Health and Human Development) for providing the acyline and Dr. A. F. Parlow (National Hormone and Peptide Program) for providing rhFSH. Hypophysectomy was performed by Ms. Kathy Rozek (Charles River Laboratories). We thank Dr. David Robertson for assistance in the generation of the sheep antisera raised against rat FSH.
Footnotes
This work was supported by Research Grant R01 ES-08075 from the National Institutes of Health (NIH)/National Institute of Environmental Health Sciences (to M.L.M.), Cancer Center Support Grant CA 16672 from the NIH, and a grant from the Lalor Foundation (to G.S.).
First Published Online October 6, 2005
Abbreviations: FSHAb, Antiserum raised against FSH; GnRH-ant, GnRH antagonist; h, human; ITT, intratesticular testosterone; LBNF1, F1 hybrids of Lewis and Brown-Norway; rh, recombinant human; T, testosterone; TDI, tubule differentiation index.
Accepted for publication September 28, 2005.
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Prince Henry’s Institute of Medical Research (S.J.M.), Clayton, Victoria 3168, Australia
Department of Physiology (P.P., I.H.), University of Turku, 20520 Turku, Finland
Institute of Reproductive and Developmental Biology (I.H.), Imperial College London, London W12 ONN, United Kingdom
Abstract
Simultaneous suppression of both testosterone and FSH with GnRH antagonists (GnRH-ant) reverses the radiation-induced block in spermatogonial differentiation in F1 hybrids of Lewis and Brown-Norway rats. Although addition of exogenous testosterone restores the block, it also raises FSH, and hence it had not been possible to conclusively determine which hormone was inhibiting spermatogonial differentiation. In the present study, we establish the relative roles of testosterone and FSH in this inhibition using three different approaches. The first approach involved the treatment of irradiated rats, in which differentiation was stimulated by GnRH-ant plus flutamide, with FSH for 2 wk; the FSH reduced the percentage of tubules that were differentiated (TDI) by about 2-fold, indicating that FSH does have an inhibitory role. The second approach involved treatment of irradiated, hypophysectomized rats with exogenous testosterone for 10 wk; testosterone also reduced the TDI, demonstrating that testosterone had a definite inhibitory effect, independent of pituitary hormones. Furthermore, in this protocol we showed that TDI in the hypophysectomized testosterone-treated group, which had higher intratesticular testosterone levels but lacked FSH, was slightly higher than the TDI in a GnRH-antagonist-testosterone-treated group of irradiated rats, which had normal physiological levels of FSH; this result supports a role for endogenous FSH in suppressing spermatogonial differentiation in the latter group. The third approach involved injection of an active anti-FSH antibody for 10 d in untreated, GnRH-ant plus flutamide-treated, or GnRH-ant plus testosterone-treated irradiated rats. This was not sufficient to increase the TDI. However, flutamide given in a similar treatment schedule did increase the TDI in GnRH-ant plus testosterone-treated rats. We conclude that both testosterone and FSH individually inhibit spermatogonial differentiation after irradiation, but testosterone is a more highly potent inhibitor than is FSH.
Introduction
RADIATION AND CANCER chemotherapy deplete germ cells, causing prolonged azoospermia in rodents (1, 2, 3), monkeys (4), and humans (5). In the rat these agents cause azoospermia by disrupting the differentiation of surviving type A spermatogonia (6). In humans there is some histological evidence that radiation and cancer chemotherapeutic drugs can also block spermatogonial differentiation (5, 7), and the recovery of spermatogenesis after prolonged azoospermia also shows that surviving stem cells can be blocked from completing differentiation (8). However, in rats exposed to moderate doses of radiation or procarbazine, spermatogonial differentiation and progression of spermatogenesis can be restored by suppression of gonadotropins and intratesticular testosterone (ITT) using GnRH analogs (9, 10).
Because both ITT and FSH are elevated after irradiation (9, 11), both might have inhibitory effects. We initially showed that administration of exogenous testosterone (T) strongly inhibited the GnRH-analog-induced stimulation of spermatogonial recovery in a dose-dependent manner and that the effect of T was almost completely reversed if the androgen receptor antagonist flutamide was given (11). That and subsequent studies (6, 11, 12) showed excellent inverse correlations between the ITT levels and spermatogenic recovery. However, when irradiated rats treated with GnRH antagonist (GnRH-ant) were also treated with T, FSH levels were elevated in a dose-responsive manner by the action of T on the pituitary, and when these rats were also given flutamide, there was some reduction in FSH (6, 11). Thus, there also was a good inverse correlation between FSH levels and recovery of spermatogenesis. When we examined the effects of four different androgens, we found that they all suppressed spermatogenic recovery (13), but we could not determine whether suppression was a direct effect of androgen at the testicular level or a result of the elevation of FSH levels that they produced. Overall we found a better inverse correlation of spermatogenic recovery with ITT levels than FSH levels, suggesting a more dominant role of androgens than FSH in the inhibitory process. However, when the combined effects of ITT and FSH were considered, the correlations were further improved (11, 12). Because of the parallel effects of the treatments used on ITT and FSH, none of the previous studies could prove that either ITT or FSH was individually inhibitory to spermatogonial differentiation.
To show that both T and FSH have unequivocal direct effects on the inhibition of spermatogonial differentiation and to determine the role of FSH relative to T in inhibiting this process, we modulated FSH and, in some cases, T levels by three different approaches and assessed spermatogenic recovery. We first tested the effect of exogenous FSH treatment in GnRH-ant-treated, irradiated rats. Second, we compared GnRH-ant plus T treatment, which incompletely suppresses FSH, with that of hypophysectomy plus T, which produces complete FSH suppression. Third, we used an FSH antibody to reduce active serum FSH levels.
Materials and Methods
Materials
T and dextran-coated charcoal were obtained from Sigma (St. Louis, MO). Flutamide pellets were obtained from Innovative Research of America (Sarasota, FL). SILASTIC brand tubing (catalog no. 602-305) was purchased from Dow Corning (Midland, MI). Alzet miniosmotic pumps (model 2001) were obtained from Alza Corp. (now Diuret) (Palo Alto, CA). Two GnRH-ants were used in this study: cetrorelix was provided by Dr. Thomas Reissmann (ASTA Medica, Frankfurt, Germany), and Acyline was obtained from the Contraceptive Development Branch of National Institute of Child Health and Human Development (North Bethesda, MD). Two different GnRH-ants were used because there was a change in the formulation of cetrorelix pamoate, which was initially used, and the original formulation was no longer available. Hence, acyline was used in later experiments. Recombinant human FSH was a gift from Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA).
Polyclonal ovine antiserum raised against rat FSH (FSHAb) and control IgG preparations were obtained from immunized and nonimmunized sheep serum as described previously (14). The FSHAb is active in an in vitro bioassay, and in vivo a dose of 2 mg/kg FSHAb by daily sc injection neutralizes at least 90% of the circulating FSH in a normal rat (14, 15).
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 U.S. Department of Agriculture and the Department of Health and Human Services, National Institutes of Health (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 3 d before the initiation of irradiation or other treatments. They were 7 wk of age at the start of experiment 2 or 9–12 wk of age at the start of experiments 1 and 3 (Fig. 1). All the animal procedures were approved by the Institutional Animal Care and Use Committee.
Irradiation
Rats were anesthetized and their testes irradiated using procedures described in detail earlier (13). The lower part of the body was irradiated with a single dose of 5 Gy (experiments 1B, 1C, 2, and 3) (11) or 6 Gy (experiment 1A) (16).
Hypophysectomy
Rats were hypophysectomized and/or given hormone treatments within a week after irradiation. Hypophysectomy was performed by making a 1-cm incision on the ventral midline of the neck up to the mandible. The salivary glands, muscles, and trachea were retracted, and the cranium was scraped clean to see the horizontal blue suture line. Trephines were used to remove a circular piece of bone on the center of the blue line. The hypophysis was visually inspected and aspirated by inserting a cannula attached to a vacuum pump. For sham hypophysectomy, the same procedure was performed except that the pituitary was not aspirated. The incision was closed and analgesia was provided as needed based on clinical observations of pain or distress. The hypophysectomized rats were maintained on 5% sucrose solution for a week after surgery followed by drinking water supplemented with 3 mg cortisone succinate, 0.3 mg deoxycorticosterone acetate, and 0.17 mg L-thyroxine per liter of water. These hormones were given to minimize the effects due to the loss of ACTH and TSH. The hypophysectomized and sham-hypophysectomized rats were bled for hormone analysis 4 d after surgery to evaluate the completeness of the surgery.
Hormone modulation
The schedules of treatments administered after irradiation to modulate hormone levels are given in Fig. 1. The end of the treatment time was determined by stopping the injections at the appropriate time, removal of the implants or pumps, and the expected degradation of the pellets.
The effect of recombinant human (rh) FSH treatment was tested in irradiated rats in experiments 1A and B, giving the treatment either after (1A) or during (1B) the depletion of the differentiated germ cells by doses of radiation used in previous studies (11, 16). The activity of the rhFSH preparation was listed as 5 IU/μg. The dose of rhFSH (4 IU/d) was selected based on previous studies (17) and corresponds to 0.8 μg per rat per day. In experiment 1A, rats were given two weekly sc injections of the GnRH-ant acyline at a dose of 1.5 mg/kg in sterile water starting 15 wk after irradiation, which maintains T suppression for 2 wk (18). To further suppress the androgen action, the rats were also given the antiandrogen flutamide by sc implanting 20-cm-long flutamide filled SILASTIC brand implants (Porter, K. L., G. Shetty, G. A. Shuttlesworth, I. Huhtaniemi, and M. L. Meistrich, manuscript in preparation). One group of these GnRH-ant+flutamide-treated irradiated rats received 0.8 μg rhFSH in PBS per day for 2 wk from sc implanted miniosmotic pumps, which were replaced at weekly intervals. Appropriate, sham injections, placebo pellets, or Teflon bars were used for rats not receiving all treatments. In experiment 1B, groups of irradiated rats were given single sc injections of the GnRH-ant cetrorelix acetate and cetrorelix pamoate at doses of 1.5 and 0.75 mg/kg, respectively, which is expected to suppress the gonadotropin and T levels for 2 wk (11). Flutamide was given by sc implanting an 84-mg pellet calculated to deliver a dose of 20 mg/kg body weight per day for 2 wk; the residual pellet was left in place after 2 wk (11). The rhFSH was given as in experiment 1A. In experiment 1C, designed to compare the spermatogenic inhibitory effect of T with that of FSH in experiment 1B, the treatment schedule, doses, and delivery of GnRH-ant and flutamide were the same, with the exception that the residual flutamide pellet was surgically removed at the end of 2 wk. T was given to some of the rats either in 2- or 6-cm SILASTIC brand capsules sc implanted (19).
The second experiment was designed to test the role of T in the absence of FSH and compare the effects of hypophysectomy with GnRH-ant. Groups of sham-hypophysectomized rats were given weekly sc injections of acyline at a dose of 1.5 mg/kg in sterile water starting 4 d after surgery. T was provided to two groups of rats in 6-cm SILASTIC brand capsules and left in place until they were killed.
Experiment 3 was designed to compare the effects of suppressing FSH by passive immunization with FSHAb with blocking T action with flutamide. Irradiated-only rats, irradiated rats treated with GnRH-ant+flutamide, and irradiated rats treated with GnRH-ant+T were used, with and without FSHAb treatment. The GnRH-ant acyline was given by a single sc injection of 1.5 mg/kg, which had been previously shown to suppress T for 10 d in unirradiated LBNF1 rats (18), some of the rats in the current experiment had begun to show recovery of T levels at d 10. Flutamide was given by sc implanting 60-mg pellets that release the drug at a calculated dose of 20-mg/kg body weight per day for 10 d. Testosterone was administered in 2-cm SILASTIC brand capsules. The rats in these three treatment groups were treated with either FSHAb for 10 d or control IgG. All treatments were started 27 d after irradiation, and the duration of treatment was limited to 10 d because longer treatments can result in the development of antibodies to the neutralizing antisera. The doses of FSHAb given in irradiated-only rats, irradiated rats treated with GnRH-ant+flutamide, and irradiated rats treated with GnRH-ant+T were 3.62, 0.9, and 1.74 mg/kg·d, respectively, based on the dose (2 mg/kg·d) required to neutralize more than 90% of FSH in rats with a mean FSH value of 5.9 ng/ml (14) and the expected FSH levels in these rats. To directly compare the relative spermatogenic inhibitory effects of FSH and T, one group of GnRH-ant+T-treated rats was given flutamide for 10 d in 60-mg pellets.
To confirm the activity of FSHAb in blocking FSH in LBNF1 rats, a separate experiment was performed. The spermatogenic inhibitory activity of the FSHAb was tested in GnRH-ant+T-treated unirradiated rats, using a protocol slightly modified from one previously employed in Sprague Dawley rats (14). Rats were given six weekly injections of acyline at 1.5 mg/kg to suppress gonadotropin and androgen levels. At the beginning of the seventh week they were implanted with 6-cm SILASTIC brand implants containing T to partially restore ITT and FSH levels and spermatogenesis, and given daily injections of either FSHAb or control IgG at 1.74 mg/kg for 7 d. The quantitative changes in the degree of spermatogenesis in these groups of rats was assessed by counting germ cell types in 25-μm-thick testicular sections embedded in methacrylate (JB4, Polysciences, Warrington, PA) by stereology.
Stereological analysis
The optical dissector method (20) was used to determine the total number of cells per testis. All estimates were performed using a x100 objective on a DML microscope (Leica Microsystems, Wetzlar, Germany) with a motorized stage controller and joystick. A software package, Stereo Investigator (MicroBrightField, Colchester, VT), was used to superimpose an unbiased counting frame on the video image. Fields were selected by a systematic uniform random sampling scheme as described previously (20).
A counting frame with an area of 686 μm2 was used to count Sertoli cells, type A spermatogonia/intermediate spermatogonia, B spermatogonia/preleptotene spermatocytes, leptotene/zygotene/pachytene spermatocytes, and round spermatids. We counted 40–200 of each cell type in each animal. Two sections at different regions of the each testis, selected by systematic random sampling, were analyzed.
Hormone measurements
We analyzed hormone levels in a minimum of five rats in each hormone- or hormone suppressor-treated group. In some cases blood was collected from the rats by sequential bleeding as described earlier (12). When the rats were killed, blood was collected by cardiac puncture under ketamine-acepromazine anesthesia. The serum was separated and stored at –20 C. In all rats the right testis was freed of the tunica, weighed, collected on ice, and homogenized. In some cases, an aliquot was removed and the sperm heads counted; the remainder of all samples was stored at –20 C for ITT analysis.
Serum levels of FSH and LH were measured using immunofluorometric assays (Delfia, Wallac Finland Oy, Turku, Finland) as previously described (21, 22). The standards used for rat LH and FSH were NIDDK-rLH-RP-3 and NIDDK-rFSH-RP-2 (AFP 4621B), respectively. For 25- and 30-μl samples, the minimum detection levels for LH and FSH by this method are 0.04 and 0.1 ng/ml, respectively. The standard used for human (h) FSH assay was calibrated against the second international reference preparation of human pituitary FSH/LH for bioassay. The minimum level of detection for human FSH was 1 U/liter. No detectable cross-reaction of rat FSH was observed in the human FSH assay for the range of FSH levels present in the experimental rats. However, there was cross-reactivity of the human FSH in the rat assay, which was quantified by running the same samples of rhFSH in a rat FSH assay (using rat FSH as standard for analysis) and a human FSH assay (using hFSH as standard). The cross-reactivity of human FSH in the rat assay run at a series of dilutions showed that 1 U/liter of rhFSH in the hFSH assay would give a reading of 0.54 ng/ml in the rat assay. In exogenous rhFSH-treated rats, the actual rat FSH values were calculated by subtracting the amount contributed by hFSH using this relationship. To compare the concentration of the human FSH in the rat serum with endogenous rat FSH levels, it was also necessary to determine the relationship between units per liter as measured by the immunofluorometric assay for human FSH and nanograms per milliliter of the rhFSH. A series of dilutions in buffer showed that 1 U/liter in the hFSH assay represents 4.94 ng/ml of rhFSH.
Serum T and ITT were assayed by using T-antiserum-coated tubes (Diagnostic Systems Laboratories, Webster, TX) as previously described in detail (11). The minimum T detection level was 0.04 ng/ml. To reflect the actual concentration of T to which the testicular cells were exposed, ITT was expressed as the amount per gram of testis.
Evaluation of spermatogenesis
To assess spermatogenic recovery, a minimum of five rats in each of the hormone-treated groups were killed at the times indicated in Fig. 1.
An aliquot from the right testicular homogenate was sonicated at 4 C for 4 min as described earlier (23). The sonication-resistant sperm heads, representing late spermatid nuclei, were counted in a hemocytometer. The detection limit of this assay is 3 x 103 sperm heads per testis.
The left testis was fixed in Bouin’s fluid and embedded in paraffin or methacrylate. Then 4-μm sections were cut and stained with hematoxylin. To evaluate recovery of spermatogenesis after irradiation and hormone treatment, the tubule differentiation index (TDI), which is the percentage of tubules showing differentiated germ cells, was computed in one section from each animal as described previously (13, 23).
Statistical analysis
The organ weights, TDI, and FSH were represented as arithmetic mean ± SEM. For sperm counts, LH, serum T, and ITT measurements, the averages and SEM were calculated on log-transformed data. The differences between the treatment groups were analyzed first by one-way ANOVA. If the difference was significant (P < 0.05), a Dunnett’s post hoc test was performed to determine the significance of the difference between the treated groups and a selected control group. When comparing multiple treatment groups simultaneously a Student’s t test with Bonferroni correction was applied; when only two groups were compared, the Student’s t test was performed without correction. In cases in which the data deviated from a normal distribution due to outliers, nonparametric statistics were applied. All analyses were performed with the SPSS (version 11.5, SPSS Inc., Chicago, IL) statistical package.
Results
Exogenous FSH treatment
To determine whether there is an inhibitory effect of FSH on spermatogonial differentiation, in experiment 1A, rhFSH was given to rats from wk 15 to 17 after the complete depletion of differentiating germ cells by radiation, whereas the levels and actions of androgens were suppressed with GnRH-ant and flutamide. The results showed that the GnRH-ant+flutamide stimulation of spermatogonial differentiation observed at wk 17 and 19 were reduced by rhFSH (Table 1). To confirm that FSH also inhibited the restoration of spermatogonial differentiation during the postirradiation depletion phase, rhFSH was given along with GnRH-ant+flutamide during wk 3–5 after irradiation. Treatment with GnRH-ant plus flutamide dramatically stimulated spermatogonial differentiation observed at wk 13 above that observed in irradiated-only rats (Fig. 2, A and B). Addition of rhFSH to this regimen significantly reduced the TDI and sperm head count, respectively, to 48 and 17% of the values observed with GnRH-ant plus flutamide alone.
We analyzed hormone levels in the rats in experiment 1B to confirm that the inhibitory effect was actually due to FSH. Treatment with a combination of GnRH-ant and flutamide, with and without additional rhFSH treatment, suppressed the serum levels of rat FSH to about 20% of irradiation-only levels (Fig. 2C), but the treatment with rhFSH produced hFSH levels of 10.9 U/liter (Fig. 2D). Based on the assay of known amounts of rhFSH in buffer, we estimated the concentration of rhFSH in treated rat serum to be 54 ng/ml, and the value might be higher if there were matrix effects of rat serum in the human FSH assay.
The LH levels were reduced by treatment with GnRH-ant+flutamide with or without rhFSH, but they were variable and the differences were not significant (data not shown). Nevertheless, the serum T levels were reduced to the limit of detection in these groups (Fig. 2F). GnRH-ant+flutamide treatment, without and with rhFSH, reduced the ITT concentration, respectively, to 3 and 4% (not significantly different) of the irradiated-only levels (Fig. 2E). Thus, the inhibition of spermatogenic recovery caused by exogenous rhFSH was unlikely to be due to a change in ITT levels.
To determine the effectiveness of T, compared with FSH, at inhibiting spermatogonial differentiation, rats were given GnRH-ant during wk 3–5 after irradiation with flutamide, no other treatment, or exogenous T (experiment 1C). GnRH-ant-only treatment for such a short period without blocking the action of the remaining low levels of T reduced the stimulation to only 10% of that observed in GnRH-ant+flutamide-treated irradiated rats (Fig. 3A). Moreover, addition of T in 2- and 6-cm capsules to GnRH-ant treatment further reduced the TDI to 5 and 1%, respectively, of that observed in GnRH-ant+flutamide-treated rats, with a concurrent reduction in sperm head counts (Fig. 3B). Such a severe inhibition by T, compared with only about 50% inhibition of TDI by rhFSH (Fig. 2A), suggests that these levels of T are more strongly inhibitory than the FSH treatment.
To confirm that the dose of rhFSH used was biologically active in LBNF1 rats, two groups of unirradiated LBNF1 rats were given GnRH-ant+flutamide for 2 wk, and one of these groups also received rhFSH at 0.8 μg/d. The GnRH-ant+flutamide treatment reduced the sperm head count and testis weights, but the addition of rhFSH produced significant increases in both end points (Fig. 4), consistent with its expected biological activity (24).
Hypophysectomy experiments
To confirm the independent spermatogenic inhibitory role of T after irradiation and to further examine whether pituitary hormones, especially FSH, are also involved in such inhibition, we analyzed the effects of T supplementation on spermatogenic recovery after irradiation in hypophysectomized and GnRH-ant-treated rats. Successful hypophysectomy was confirmed by the lack of body weight gain and assays of T levels 4 d after surgery (data not shown). No differences were observed between irradiated-only rats and sham-hypophysectomized irradiated rats in serum T at 4 d after surgery (not shown) or in ITT, serum T, LH, and FSH levels at the end of the experiment (Fig. 5, B–E).
Irradiated-only and sham-hypophysectomized irradiated rats showed negligible spermatogonial differentiation at 11 wk after irradiation (Fig. 5A). Hypophysectomy within a week after irradiation restored spermatogonial and spermatocyte differentiation in all the tubules. The histology of the testis was qualitatively similar to that observed in unirradiated rats after hypophysectomy (data not shown). Treatment of sham hypophysectomized, irradiated rats with GnRH-ant for 10 wk started at the time of hypophysectomy of the other group also stimulated differentiation in all tubules. When the hypophysectomized, irradiated rats were also treated with T in 6-cm SILASTIC brand capsules, starting immediately after hypophysectomy, the TDI was reduced to 56 ± 4%, whereas administration of 6 cm T to the GnRH-ant-treated rats inhibited the TDI at least as much to 47 ± 5%.
Hypophysectomy or GnRH-ant-treatment reduced the LH and serum T levels to below the limits of detection (Fig. 5, B and D). However, it was noted that the ITT levels in GnRH-suppressed, irradiated rats were significantly lower than those of hypophysectomized, irradiated rats (Fig. 5C). The more profound suppression of ITT by the GnRH-ant than hypophysectomy, despite undetectable levels of LH in both cases, suggests that GnRH-ant directly inhibits T production in Leydig cells of irradiated rats, possibly through GnRH receptors (25), as had been observed previously in other systems (26, 27).
Addition of 6 cm T increased the ITT concentrations to 19.5 ± 0.3 ng/g testis in hypophysectomized, irradiated rats but only to 11.0 ± 0.8 ng/g testis in GnRH-ant-treated irradiated rats (Fig. 5C). Although T treatment increased serum T levels in both hypophysectomized and GnRH-ant-treated rats, the levels were significantly higher in the hypophysectomized group (Fig. 5D). It should be noted that in the hypophysectomized rats the volume of blood was about half of the volume in nonhypophysectomized rats, which might have affected serum hormone concentrations.
Prolonged GnRH-ant treatment reduced serum FSH levels below the limit of detection, similar to that observed in hypophysectomized rats (Fig. 5E). The reduction of FSH to undetectable levels is attributed to the 10-wk treatment time because in a previous study, when the acyline was given for only 2 wk, FSH levels were still detectable (12). Although FSH levels were still below the limit of detection in T-treated, hypophysectomized irradiated rats, the addition of T partially restored the FSH levels in GnRH-ant-treated irradiated rats to 41% of those in irradiated-only rats.
To determine whether a relationship exists between the hormone levels and TDI, the TDIs were plotted against relevant hormone levels. The relationships between mean values of TDI and those of the ITT concentrations for intact irradiated and hypophysectomized irradiated rats, with and without additional treatments, is shown in Fig. 6A. The reduction of the TDI on addition of T to hypophysectomized rats, in the absence of FSH, demonstrates that T alone can inhibit spermatogonial differentiation in irradiated rats. However, an obvious shift in the curve to the left is observed for the intact irradiated rats, compared with the hypophysectomized irradiated rats. This suggests that the ITT concentration required to bring about a specific level of spermatogenic inhibition in irradiated rats with an intact pituitary is lower than that required in hypophysectomized irradiated rats. For example, to reduce the TDI to 56%, only about 10 ng T per gram testis were required in intact irradiated rats, compared with 20 ng T per gram testis in hypophysectomized-irradiated rats. Thus, a factor from the pituitary adds to the effect of ITT to facilitate inhibition of spermatogonial differentiation.
To model the possible additive inhibitory effect of FSH on spermatogonial differentiation in the irradiated rats, we plotted TDI against a linear combination of ITT concentration and 2 x FSH levels (Fig. 6B), which was the relationship we previously derived using irradiated rats treated with different combinations of GnRH-ant and other hormones (12). The curve relating TDI and [ITT + (2 x FSH)] for irradiated GnRH-ant-treated and irradiated hypophysectomized rats were very close, suggesting that this relationship approximates the additive effect of FSH to T in the inhibition of spermatogenic recovery in irradiated rats. In this study the best fit was obtained with the linear combination, ITT + (3.4 x FSH) (Fig. 6C).
FSH passive immunization
To further quantify the spermatogenic inhibitory activity of FSH in irradiated rats, circulating FSH was inactivated with FSHAb in experiment 3. We performed the treatment under three conditions: 1) in irradiated-only rats, which have high FSH and ITT; 2) in irradiated, GnRH-ant+flutamide-treated rats, which have low FSH and ITT and the action of T is blocked; and 3) in irradiated GnRH-ant+T-treated rats with intermediate levels of FSH and ITT. To compare the relative inhibitory effects of T and FSH, we also gave flutamide instead of FSHAb to an additional group of GnRH-ant+T-treated rats.
In irradiated-only rats the 10-d treatment with FSHAb did not increase the TDI or sperm head counts (Fig. 7, A and B), both of which were essentially zero. When gonadotropins and androgenic action were suppressed in the irradiated rats during this time period using GnRH-ant+flutamide, the TDI and sperm head counts significantly increased. However, the FSHAb treatment also did not alter the TDI or sperm head counts in these gonadotropin and androgen-suppressed rats. Third, the addition of T to the GnRH treatment, which increased ITT and FSH levels, significantly decreased the TDI and sperm head counts, but no significant increases in these measures were noted after FSHAb treatment. Serum levels of T were not altered by FSHAb treatment in any of these groups (Fig. 7C), indicating that changes in T were unlikely to have any effect on the outcome of spermatogenic recovery after FSHAb treatment.
Comparing the two groups treated with GnRH-ant and FSHAb, one treated with flutamide and one treated with T, shows T significantly reduces the TDI and sperm head counts. Because the FSH should be neutralized by the antibody, this result further demonstrates the inhibitory effects of T. Moreover, addition of flutamide, unlike FSHAb, to the GnRH-ant+T-treated rats reversed the spermatogenic inhibition, highlighting the greater inhibitory effect of endogenous levels of T than endogenous levels of FSH on spermatogonial differentiation.
To prove that the FSH antibody was indeed biologically active in LBNF1 rats, we examined its spermatogenic inhibitory activity in long-term GnRH-suppressed unirradiated rats that had been given T for 1 wk (14). Testosterone treatment increased the serum T levels to similar values of 7.6 ± 0.5 or 8.2 ± 0.4 ng/ml without or with daily injections of FSHAb, respectively, and raises FSH levels (14). The daily injections of FSHAb significantly (P < 0.001) suppressed the testis weight from 0.27 ± 0.01 to 0.20 ± 0.01 g and produced significant reductions in all the germ cell types analyzed in these rats (Fig. 8), demonstrating the biological activity of the FSHAb in blocking FSH action on the testis.
Discussion
The androgen and FSH inhibition of spermatogenic recovery after irradiation seems to contradict their well-studied roles in stimulating and maintaining spermatogenesis (14, 28). Although FSH and T have overlapping and synergistic effects on spermatogenesis (17), the role of T is more pronounced in supporting the differentiation of spermatids past step 7 (29), and the role of FSH is more important in supporting spermatogonial numbers and differentiation. Nevertheless, in normal rats spermatogonial differentiation is still qualitatively independent of both ITT and FSH (30). However, in irradiated rats we have now shown that the survival and differentiation of A spermatogonia are inhibited by moderate levels of ITT or high levels of FSH.
Through the use of hypophysectomy, this study unequivocally demonstrated for the first time that T alone can inhibit spermatogenic recovery in irradiated rats independently of the pituitary hormones. Although our previous studies showed that spermatogenic inhibition by exogenous androgens occurred through the androgen receptor, FSH mediation of such inhibition was not ruled out due to the parallel effects of exogenous androgens and antiandrogen on testicular androgen levels or activity and serum FSH levels (11, 13).
In the present study, we investigated the role of FSH in the inhibition of spermatogonial differentiation after irradiation using three different protocols. The first protocol, in which high levels of FSH were maintained by continuous administration of rhFSH for 2 wk, significantly inhibited spermatogenic recovery in androgen-suppressed irradiated rats. The second protocol, comparing hypophysectomized with GnRH-ant-suppressed rats, showed that a pituitary factor was involved in the inhibition of spermatogonial differentiation, and calculations showed that the results were consistent with this factor being FSH. In contrast, the third protocol, involving a reduction of endogenous FSH levels for 10 d under three different circumstances, failed to stimulate recovery of spermatogonial differentiation.
A quantitative comparison of the results of the three protocols is given in Table 2. Because we believe that the rhFSH is highly active in rats, the 54 ng/ml of rhFSH in the serum of rhFSH-treated rats in experiment 1B, which is 8 times that of rat FSH in irradiated-only rats, represents a supraphysiological level. The inhibitory effect of high FSH levels is observed with a 2-wk treatment under conditions in which the inhibition by T was minimized by the low ITT levels and the flutamide treatment.
In experiment 2 we were able to demonstrate an inhibitory effect of 10-wk exposure to endogenous FSH levels of 3 ng/ml in the intact rats treated with GnRH-ant and T, compared with the hypophysectomized rats treated with T in which there was no FSH present (Table 2). Despite the fact that the hypophysectomized, T-treated rats had higher ITT levels than the irradiated GnRH-ant+T-treated rats, they had slightly higher TDI values. Because we know that ITT is inhibitory, the slight increase in TDI (or at least the lack of any decrease) in the hypophysectomized, T-treated rats is most likely due to the absence of an inhibitory pituitary factor. The pituitary is responsible for the production of a variety of hormones, including the gonadotropins (LH and FSH), ACTH, TSH, GH, MSH, and prolactin. All the rats in this experiment received corticosteroids and T4, which should minimize the effects of loss of ACTH and TSH. Even so, corticosteroids act only on Leydig cells to inhibit T production (31), and this would be accounted for in our measurements of ITT. Thyroid hormones act only on immature, not adult, Sertoli cells, and their effect on Leydig cells is also primarily on steroidogenesis (32). Although thyroid hormone receptors are present on germ cells, the action of thyroid hormones does not appear to be critical for spermatogenesis in the adult. The effect of LH, which was suppressed to below the limit of detection in all hypophysectomized and GnRH-ant-treated groups, has been demonstrated to act only on spermatogenesis through T production (33). Whereas GH (34) and prolactin (35) have been reported to have stimulatory effects on spermatogenesis, their effects are mostly through steroidogenesis, and these effects are not as dramatic as the gonadotropins. Although we cannot rule out the possibility that a pituitary hormone besides FSH may have inhibitory effect on spermatogonial differentiation, we have shown that the outcomes can be explained by changes in FSH levels. The differences in the curves relating TDI to the ITT in intact and hypophysectomized rats (Fig. 6) could be nearly eliminated by replotting the data according to a previously developed linear relationship involving ITT and FSH (12).
In contrast, in experiment 3 no stimulation of spermatogenesis was observed by inhibiting FSH by passive immunization with an FSHAb preparation that we demonstrated to be active in unirradiated LBNF1 rats (Fig. 8). We believe that the failure of stimulation of spermatogonial differentiation in any of the three arms of this experiment could be attributed to stronger inhibition by high ITT levels, small changes in FSH levels, and the short time of the suppressive treatment (Table 2). In the irradiated-only rats, which had FSH levels of 6.8 ng/ml, the high levels of ITT exerted such a strong inhibitory effect that no recovery could be observed even with more than 90% suppression of FSH. In rats that received treatment with GnRH-ant+flutamide, the lack of enhancement of the percentage of tubules that differentiated by FSH immunoneutralization was attributed to the low levels of endogenous FSH in these animals. In the rats that received GnRH-ant+T, the inhibition of 3.3 ng/ml of FSH by FSHAb did not increase the TDI. The different result in this experiment from the enhancement of recovery in the hypophysectomized T-treated rats, compared with the GnRH-ant+T-treated rats, which also was a result of elimination of 3 ng/ml of FSH, is attributed to the short 10-d (experiment 3) vs. the long 10-wk (experiment 2) durations of FSH suppression. In addition, the greater inhibitory effects of the higher levels of ITT in this group of experiment 3 would further reduce detection of any effect of FSH modulation. Whereas the inhibitory effects of supraphysiological levels of FSH can be detected even after short exposure periods, exposure times must be long to detect a significant inhibitory effect of physiological or lower levels of FSH. However, the demonstration that flutamide but not FSHAb could stimulate the TDI in irradiated rats treated with GnRH-ant+T, even during this short treatment period (Fig 7) shows that even subphysiological levels of ITT can be more strongly inhibitory than physiological levels of FSH (Table 2).
The greater inhibitory effects of T caused by exogenous T (experiment 1C) compared with less inhibition caused by high levels of FSH (experiment 1B) further confirms the relative inhibitory action of the two hormones. The linear relationship derived from Fig. 6C can be used to quantify the relative effects of ITT and FSH on inhibition of spermatogonial differentiation in irradiated rats. The relationship shows that the inhibitory effect of 1 ng/ml FSH was equivalent to that of 3.4 ng T per gram testis. Because the level of ITT in the irradiated rats is 195 ng/g testis and FSH level was 6.8 ng/ml, we calculated that 90% of the inhibition of spermatogonial differentiation is a result of the ITT and only 10% is a result of the FSH.
Because androgen and FSH receptors are generally believed to be absent from germ cells including spermatogonia, T and FSH must produce their effects by acting on somatic cells. The actual physiological and molecular mechanisms of the T and FSH inhibition of spermatogonial differentiation after irradiation is under investigation.
T could be acting on multiple cell types that have androgen receptors (36). The action on the vasculature or other interstitial cells that affect fluid levels or composition deserves particular attention. This is supported by a strong correlation between the quantity of interstitial fluid per testis, which varies in response to ITT levels, and the inhibition of spermatogonial differentiation (18).
FSH can exert its direct inhibitory action only on Sertoli cells because they are the only ones with the receptor, and hence a reasonable approach is to examine FSH- and T-regulated Sertoli cell genes. Inhibin and activin are important Sertoli cell genes because activin stimulates spermatogonial proliferation in vitro (37) and while inhibin inhibits spermatogonial proliferation in vivo (38). Microarray analysis of total testicular RNA samples from experiment 1A (Bolden-Tiller, O. U., D. N. Stivers, C. C. Weng, and M. L. Meistrich, unpublished data) indicated that T/FSH suppression slightly (23%) but significantly (P < 0.001) up-regulated inhibin-, although inhibin-A was not expressed and inhibin-B was not identified as present on chips. In addition, activin inhibitors, follistatin, and bone morphogenetic protein and activin membrane-bound inhibitor, were, respectively, unchanged or slightly up-regulated (40%, P < 10–5) by T/FSH suppression. These data, which indicate that FSH would stimulate spermatogonial proliferation by reducing levels of inhibin and bone morphogenetic protein and activin membrane-bound inhibitor (39), suggest that these molecules are not involved in the inhibition of spermatogonial function by FSH and T.
Further research is required to resolve questions that remain about the application of these findings to other species. For example, although T also inhibits spermatogonial differentiation in jsd mice (40), FSH does not have any inhibitory role (41). Also, because it has recently been shown that T and FSH suppression was unsuccessful in stimulating spermatogenic recovery in irradiated nonhuman primates (42, 43), the focus of future research in this area should be on understanding the molecular mechanisms of T and FSH inhibition of spermatogonial differentiation through Sertoli cells in the rat. Although these hormonal treatments per se might not be clinically applicable, modulation of the downstream factors that they regulate could be useful in restoring fertility in patients who have undergone cancer therapy.
Acknowledgments
We thank Mr. Kuriakose Abraham for the histological preparations and Mr. Walter Pagel for editorial advice. We also thank Ms. Tarja Laiho for the skillful assistance in performing gonadotropin assays. We sincerely thank Drs. R. P. Blye and Hyun K. Kim (National Institute of Child Health and Human Development) for providing the acyline and Dr. A. F. Parlow (National Hormone and Peptide Program) for providing rhFSH. Hypophysectomy was performed by Ms. Kathy Rozek (Charles River Laboratories). We thank Dr. David Robertson for assistance in the generation of the sheep antisera raised against rat FSH.
Footnotes
This work was supported by Research Grant R01 ES-08075 from the National Institutes of Health (NIH)/National Institute of Environmental Health Sciences (to M.L.M.), Cancer Center Support Grant CA 16672 from the NIH, and a grant from the Lalor Foundation (to G.S.).
First Published Online October 6, 2005
Abbreviations: FSHAb, Antiserum raised against FSH; GnRH-ant, GnRH antagonist; h, human; ITT, intratesticular testosterone; LBNF1, F1 hybrids of Lewis and Brown-Norway; rh, recombinant human; T, testosterone; TDI, tubule differentiation index.
Accepted for publication September 28, 2005.
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