| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the * Department of Pathology and Laboratory
Medicine, Brown University, Providence, Rhode Island;
Novartis Pharmaceutical Corporation, East
Hanover, New Jersey; the Department of
Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer
Center, Houston, Texas; the Departments of
Radiation Oncology and ||
Surgery, Brown University, Providence, Rhode
Island; and the ¶ Oregon National Primate Center,
Beaverton, Oregon.
| Correspondence to: Dr Kim Boekelheide, Department of Pathology and Laboratory Medicine, Brown University, Box G-E504, Providence, RI 02912 (e-mail: Kim_Boekelheide{at}Brown.edu). |
| Received for publication July 6, 2004; accepted for publication September 29, 2004. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key words: Macaque, testis, spermatogenesis
We have shown in rats treated with radiation, cancer chemotherapeutic drugs, and environmental and occupational toxicants that many stem spermatogonia survive and that infertility is caused by the spermatogonia under-going apoptosis instead of differentiation (Allard and Boekelheide, 1996; Shuttlesworth et al, 2000; Meistrich and Shetty, 2003). However, in all of these instances and other instances of testicular injury produced by various chemical or physical agents, recovery of spermatogenesis can be induced by suppression of testosterone (T) and follicle-stimulating hormone (FSH) by treatment with gonadotropin-releasing hormone (GnRH) analogs (Kangasniemi et al, 1995a; Meistrich and Kangasniemi, 1997; Blanchard et al, 1998; Shoenfeld et al, 2001; Udagawa et al, 2002; Meistrich and Shetty, 2003). Furthermore, after cessation of GnRH treatment, differentiation continued and sperm were produced (Meistrich et al, 2001). In some cases, this resulted in essentially normal levels of fertility for at least 3 months. The mechanisms causing failure of spermatogonial differentiation after cytotoxic therapies and how the suppression of T restores this ability to differentiate are not yet known.
Several studies have tried to produce gonadal protection from radiation or chemotherapy via hormonal suppression in humans and nonhuman primates (Morris and Shalet, 1990). GnRH agonist (Brennemann et al, 1994), testosterone (Redman and Bajorunas, 1987), or medroxyprogesterone (Fossa et al, 1988) treatment failed to protect spermatogenesis in men treated with irradiation or cancer chemotherapy, although a clinical trial of hormonal suppression with testosterone to prevent cyclophosphamide-induced azoospermia in patients with nephrotic syndrome was encouraging (Masala et al, 1997). One study with a total of only 3 baboons (Lewis et al, 1985) suggested that GnRH-agonist treatment might protect against cyclophosphamide-induced oligospermia, and a larger study suggested that FSH treatment of macaques (rhesus) to stimulate spermatogonial stem cell proliferation enhanced spermatogonial numbers remaining after 1 Gy of irradiation (van Alphen et al, 1989b). However, a recent trial using either FSH or GnRH antagonist in macaques (rhesus) failed to show any protection of spermatogenesis from injury induced by 4 Gy of irradiation (Kamischke et al, 2003).
To explore this issue further, we sought to determine whether GnRH analog treatment of a nonhuman primate could enhance the maintenance or recovery of spermatogenesis with the use of a strategy that had been successful in the rat. The stump-tailed macaque, Macacca arctoides, was chosen for this study because spermatogenesis and the effects of radiation and GnRH antagonists have been well studied in macaques (Clermont and Antar, 1973; van Alphen et al, 1988a; Weinbauer et al, 1994). Radiation was chosen as the cytotoxic agent because it could be delivered locally, without systemic toxicity, in a precisely measured dose. Observations made by others in monkeys indicate that, like in the rat, most surviving A spermatogonia at 3 months after 2 Gy of radiation are found in colonies of A spermatogonia that did not undergo differentiation (van Alphen et al, 1988b). A GnRH antagonist was chosen to treat the monkeys because studies in the rat showed that it was more effective than a GnRH agonist at stimulating recovery of tubule differentiation and sperm counts in irradiated rats (Meistrich et al, 2001). In this study, the GnRH antagonist was given after cytotoxic insult for several reasons: 1) the level of maintenance of spermatogenesis was similar in rats whether the GnRH analog was given before or after cytotoxic insult (Kangasniemi et al, 1995a; Meistrich et al, 1999), 2) hormonal suppression prior to cytotoxic treatment appears to maintain spermatogenesis not by protecting the survival of stem cells but by the subsequent maintenance of their differentiation (Meistrich et al, 2000), and 3) hormonal treatment after toxic exposure is more logistically practical than treatment before exposure.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Radiation
The 10 monkeys in the radiation and radiation plus GnRH antagonist groups
were individually anesthetized with ketamine (10 mg/kg), temporarily
immobilized to a plastic frame, and administered a calculated midtestis dose
of 7 Gy x-radiation at a dose rate of 0.71 Gy/min with a Philips 250 kVp x-ray
machine. After half of the dose was delivered, the animals were rotated so
that the anterior and posterior aspects of the scrotum were equally exposed.
The dose rate was estimated on a phantom with a Victoreen chamber. In
addition, thermoluminescent dosimetry of each exposed animal was performed by
placing 2 to 4 detectors per animal on the anterior and posterior aspects of
the scrotum (Table). The
radiation-only and the radiation plus GnRH antagonist-treated monkeys were
given an average measured surface radiation dose of 7.3 Gy, which did not
differ significantly between the 2 groups
(Table). The dose to the center
of the testis was estimated to be 84% of the surface dose, and the average
tissue dose was 6.7 Gy. The 1 baseline and 2 GnRH antagonist-only monkeys were
anesthetized, immobilized, and sham irradiated.
Hormonal Suppression
Testosterone suppression was achieved by GnRH antagonist therapy
(Cetrorelix acetate, Asta Medica, Frankfurt, Germany). Beginning on the day of
irradiation, Cetrorelix acetate was delivered to the GnRH antagonist-only and
radiation plus GnRH antagonist groups as a combination of daily subcutaneous
injections at a dose rate of either 450 µg/kg/d (Monday through Thursday)
and a bolus of 1350 µg/kg/d (on Fridays) for a treatment duration of 84
days (12 weeks). We have shown that a variety of dosing regimens with GnRH
antagonists, including daily subcutaneous injection
(Kangasniemi et al, 1995b),
bolus weekly injections (Shetty et al,
2004), and infusion from osmotic minipumps
(Kangasniemi et al, 1996; Matsumiya et al, 1999;
Hild et al, 2004), are
effective in stimulating spermatogenic recovery in rodents. The baseline and
radiation-only monkeys were injected with vehicle.
Hormone Levels
Blood was drawn (
10 mL/sample) by venipuncture of the saphenous vein
under ketamine (10 mg/kg) sedation. Blood collection was performed twice
before treatment (7-17 days before start of treatment and at start of
treatment), at biweekly intervals during GnRH antagonist therapy, and at
monthly intervals through month 18. Tissue (40-92 mg) for intratesticular
testosterone measurements from the 18-month biopsy was also frozen immediately
in liquid nitrogen following removal from the monkeys.
Testosterone levels were measured by radioimmunoassay with a coated-tube kit (DSL-4000, Diagnostic Systems Laboratories, Webster, Tex) as described previously (Shetty et al, 2000). For serum T measurement, standards were prepared in dextran-coated charcoal (Sigma St Louis, Mo) stripped GnRH-suppressed rat serum. For intratesticular T measurements, standards were prepared in 0.1% gelatin in phosphate-buffered saline (GPBS). The lower limit of detection was 0.04 ng/mL and the intra- and interassay coefficients of variation were 8% and 15%, respectively.
The validity and the lower limits of the serum T assay were demonstrated by estimating radioimmunoassayable T added in known quantities to prepubertal and postmenopausal female monkey sera. When 5 ng of T was added to 1 mL of the above sera, an increase of 4.4 ng/mL of T was detected, which was within 12% of the actual amount added. However, we were surprised at the apparent levels of T in prepubertal and postmenopausal female monkey sera, which were 0.71 and 1.08 ng/mL, respectively. To determine whether the apparent T levels were a result of steroids or differential matrix effects between monkey and rat sera, the postmenopausal monkey serum and serum from 1 GnRH-suppressed male, which had an apparent T level of 0.53 ng/mL, were stripped of steroids with dextran-coated charcoal and assayed again, at which time the apparent T levels were 0.77 and 0.37 ng/mL, respectively. Thus, because of this matrix effect and the results from female monkeys, the serum T values for male monkeys less than 1 ng/mL are actually lower than they appear. The monkey serum contains some nonsteroidal substance in the matrix that causes nonspecific displacement in the RIA, accounting for about half of the apparent minimum T values in the GnRH antagonist-treated monkeys, and a steroidal component other than T also might give a slight reading in the assay.
The intratesticular testosterone levels were measured by RIA with homogenates of the testicular biopsy material. Samples from both testes were assayed and the results were averaged. A 1:10 dilution of all samples was made in GPBS. When additional T was added at 5 or 10 ng/mL to testicular homogenates, the assays indicated an additional 3.8 and 8.6 ng/mL, respectively, which were within 25% of the added amounts.
Bioactive luteinizing hormone (LH) was measured in duplicate serum samples with a mouse Leydig cell bioassay as described previously (Ellinwood and Resko, 1980) with either 10, 5, 2, 1, or 0.25 µL as required to fall within the linear range of the standard curve. The minimum detectable level at 10 µL was 1.0 ng/mL, and the intra- and interassay coefficients of variation for the 6 assays were 10.0% and 13.7%, respectively.
FSH was estimated in a single radioimmunoassay with the use of a protocol developed and reagents distributed by Dr A. F. Parlow (Harbor-UCLA Medical Center, Torrance, Calif) through the National Hormone and Peptide Program. Duplicate 100-µL serum samples or FSH standards (AFP 6940A) in triplicate were incubated with FSH antisera (AFP782594) and iodinated FSH (AFP 6940A) for 2 days. Bound FSH was precipitated with sheep anti-rabbit second antibody (lot 8TA 111, Antibodies Inc, Davis, Calif) for 3 days, separated by centrifugation at 3000 rpm, and counted for 2 min in a 10-channel gamma counter. FSH concentrations were calculated by interpolation after logit-log transformation of the data. The minimum detectable level was 0.4 ng/mL and the intra-assay coefficient of variation was 5.2%.
Inhibin B was estimated in 50-µL serum samples from selected time points (pretreatment, 9 months [40-43-week blood draws], and 18 months [75-80-week blood draws]) in a single enzyme-linked immunosorbent assay with the use of reagents obtained from Dr V. D. Castracane (Diagnostic Systems Laboratories). For each time point, 2 samples from each animal were measured and averaged. This assay, which has been validated for human and nonhuman primates by the manufacturer, had a minimum detectable level of 3 pg/mL in our hands and an intraassay coefficient of variation of 2.0%.
Testis Volume and Biopsies
Testis volume was determined by measuring the length and width of each
testis with calipers (±0.01 cm) and modeling the testis as an ellipse,
applying the following formula: testis volume = 
x width2
x length/6. Testis biopsies were performed under general anesthesia
before treatment in both testes at the 3- and 8-month time points, alternating
between left and right testis, and at 18 months from both testes; therefore,
the 2 uniorchid monkeys had 4 biopsies taken from their single testis, whereas
the other monkeys had 3 biopsies taken from each of their 2 testes. A small
incision was made in the scrotum and then in the tunica albuginea, and an
approximately 5-mm-diameter piece of tissue was excised with a sharp blade,
fixed in Bouins solution, embedded in glycol methacrylate, sectioned at 3
µm, and stained with hematoxylin and periodic acid Schiffs reagent for
histopathological analysis. Testis biopsy material obtained for determination
of intratesticular T concentration was immediately frozen in liquid nitrogen
and stored at -80°C until assayed (see "Hormone Levels").
Testis biopsy material at the 18-month time point was quantitatively assessed
by scoring each seminiferous tubule cross section for the presence or absence
of germ cells and the most advanced germ cell type present; seminiferous
tubule cross sections containing at least 1 germ cell of any type were scored
as positive. The mean number (and range) of seminiferous tubules counted per
testis for each group were as follows: radiation only, 944 (418-1822);
radiation plus GnRH antagonist, 1211 (580-1875); baseline, 360 (231-488); GnRH
antagonist only, 501 (328-700).
Sperm Collection and Semen Analysis
Semen was obtained from ketamine-sedated monkeys by rectal probe
electroejaculation with a Standard Precision Electronics, Inc, Ejaculator
(Littleton, Colo). Two ejaculates were collected pretreatment; once hormonal
therapy had begun, ejaculates were collected at biweekly or monthly intervals
through 18 months. Semen was diluted as needed with a Ringers solution
containing trypsin, fructose, or glucose and Hepes buffer. Sperm counts were
performed with a Petroff-Hauser counting chamber, and the percentage of motile
sperm was determined visually.
Data Analysis
For analysis and presentation of all the data, the group mean and standard
error of the mean (SEM) were determined. For serum testosterone, LH, and
inhibin B, these were calculated after log transformation of the data. For the
serum hormone data (T, FSH, LH, and inhibin B), the 2 pretreatment values were
averaged and are presented as a single value at the zero time point. For the
analysis and presentation of sperm counts, those semen collection attempts
that failed to yield an ejaculate were dropped from the determination of group
mean and SEM. Statistical comparisons between the radiation-only and radiation
plus GnRH antagonist groups used the Student's t test with
significance at P less than .05. The significance testing of the
relationship between seminiferous tubules with germ cells and irradiated
monkey age was performed by linear regression analysis.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Qualitative Testicular Histology
Testis biopsies collected at 4 times (before treatment, 3, 8, and 18
months) were evaluated morphologically for the extent of spermatogenesis
(Figure 2). When biopsied
before treatment, all monkeys had active spermatogenesis similar to that shown
in Figure 2A. The baseline
monkey maintained normal spermatogenesis at all time points
(Figure 2A).
|
The 2 GnRH antagonist-only monkeys had different responses. At the 3-month time point, 1 monkey had few residual germ cells, mostly spermatogonia with a few spermatocytes, in nearly all tubules, whereas the other had many germ cells with spermatocytes in nearly all the seminiferous tubules with occasional clusters of round and elongate spermatids (Figure 2B and C). At 18 months, in the first GnRH antagonist-only monkey (designated "failed to recover") most of the seminiferous tubules contained germ cells, but they were few in number, and the most advanced cells were generally spermatocytes, whereas the second GnRH antagonist-only monkey (designated "recovered") had normal spermatogenesis in most seminiferous tubules (Figure 2D).
In the radiation plus GnRH antagonist and radiation-only groups, there was a much more severe loss of germ cells than that seen in either of the GnRH antagonist-only monkeys. At the 3-month time point, the vast majority of the seminiferous tubules in these monkeys contained only Sertoli cells (not shown). No advanced germ cells were observed in the radiation plus GnRH antagonist group; only 1 tubule with 1 type A spermatogonium was noted. Only 1 monkey (in the radiation-only group) showed any evidence of ongoing differentiation, with spermatocytes in about 3% of the seminiferous tubules, some of which also had spermatogonia. Little recovery was observed at the 8-month time point; the vast majority of the tubules still contained only Sertoli cells. In both groups, only about 4% of the tubules had germ cells (not shown). At the 18-month time point, the radiation plus GnRH antagonist and radiation-only groups still showed marked testicular atrophy, with occasional seminiferous tubules showing repopulation (Figure 2E and F, respectively). A quantitative determination of germ cell content at the 18-month time point is described below.
Hormone Levels
Serum hormone levels (T, LH, and FSH shown in Figures
3,
4, and
5, respectively) were measured
throughout the experiment. Cetrorelix therapy was effective in lowering serum
T and serum LH during the time of its administration (Figures
3 and
4; GnRH antagonist-only and
radiation plus GnRH antagonist groups), and the effects were completely
reversible. There were no meaningful differences between groups in the initial
or final serum T levels. Most groups, including the baseline monkey, showed
higher T levels toward the end of the study than at the beginning.
|
|
|
Radiation induced a gradual and progressive increase in serum LH and FSH, which began within 2 weeks after exposure in the radiation-only group and about 8 weeks after cessation of Cetrorelix therapy in the radiation plus GnRH antagonist group (Figures 4 and 5). Such increases are expected in response to induced atrophy of the seminiferous epithelium. From the differences in FSH levels in the radiation-only and radiation plus GnRH antagonist groups during the time of Cetrorelix administration, we conclude that GnRH antagonist therapy was also effective at suppressing FSH. The GnRH antagonist-only monkey that failed to recover after GnRH antagonist therapy (see "Quantitative Measures of Lack of Spermatogenic Recovery"), also had progressive elevation in his serum FSH level after the suppressive effects of the Cetrorelix treatment had waned, whereas the recovered GnRH antagonist-only monkey still maintained a low serum FSH level similar to that observed in the baseline monkey.
In addition, one of the monkeys in the radiation-only group had an abnormal gonadotropin profile, characterized by LH and FSH levels that were elevated more than 15-fold and more than 2-fold, respectively, compared with other members of the group throughout the experiment (Figures 4 and 5). This monkey was considered separately in the subsequent analyses.
At the end of the experiment, testis biopsies were evaluated for the level of intratesticular T. The values were 739 ng/gm testis in the baseline monkey; 504 ng/gm testis in the GnRH antagonist-only recovered monkey; 1424 ng/gm testis in the GnRH antagonist-only monkey that failed to recover; 1560 ± 258 ng/gm testis in the radiation-only monkeys with normal hormonal profiles; 6525 ng/gm testis in the radiation-only monkey with the abnormal hormonal profile; and 1722 ± 476 ng/gm testis in the radiation plus GnRH antagonist monkeys. There were no statistically significant differences in intratesticular T between radiation-only vs radiation plus GnRH antagonist groups, although the groups with monkeys that had persistent atrophy tended to have higher intratesticular T levels than the groups with ongoing spermatogenesis, as observed previously with the rat (Meistrich and Kangasniemi, 1997).
Quantitative Measures of Lack of Spermatogenic Recovery
Testis volumes in the radiation-only monkeys declined rapidly such that by
12 weeks postexposure they were approximately 25% of pretreatment volumes
(Figure 6). Testis volumes were
further suppressed by GnRH antagonist therapy, such that by 12 weeks
postexposure in the radiation plus GnRH antagonist group, they were
approximately 20% of pretreatment volumes. These low testis volumes were
maintained by these 2 groups of monkeys through the end of the study. Testis
volumes were also suppressed in the GnRH antagonist-only animals, declining to
a similar level as in the radiation plus GnRH antagonist group. After
competing GnRH antagonist therapy, the GnRH antagonist-only monkey that failed
to recover maintained a testis volume of approximately 20% of the pretreatment
value, whereas the one that recovered rapidly increased his testis volume to
approximately 50% of the pretreatment value
(Figure 6).
|
Semen analysis was performed throughout the experiment (Figure 7). The effect of GnRH antagonist treatment on semen collection was demonstrated when 65% of semen collection attempts were unsuccessful in the GnRH antagonist-only and radiation plus GnRH antagonist groups (compared with only 17% of attempts in all monkeys at all time points) during weeks 8 through 27 (identified by the horizontal bars in Figure 7), which is during and shortly after the time of maximum GnRH antagonist suppression of T levels (Figure 3). The baseline monkey had an average count of 32 x 106 sperm/mL ejaculate with 38% motility. In the GnRH antagonist-only group, the recovered monkey had sperm counts that were approximately 50% of his pretreatment values (although this reduction was not statistically significant) and sperm motility that returned to pretreatment levels after cessation of therapy, whereas the monkey that failed to recover had persistent azoospermia. In the radiation-only group, which had an average pretreatment count of 64 x 106 sperm/mL ejaculate, a dramatic decrease in sperm counts to 0.2 x 106 sperm/mL ejaculate (about 0.4% of pretreatment value) was observed by week 20 postexposure, and counts remained at this plateau until the end of the study. In the radiation plus GnRH antagonist group, no sperm were present in the ejaculate after the first few weeks in 3 of 5 animals; in the remaining 2 monkeys in this group, less than 1 x 106 sperm/mL were sporadically present.
|
Serum inhibin B, which is a sensitive marker of damage to spermatogenesis (Foppiani et al, 1999), was determined at 3 time points during the experiment (Figure 8). All groups of animals had similar inhibin B levels before treatment, with the exception of the radiation-only monkey with an abnormal hormonal profile, who had a low pretreatment value. In the GnRH antagonist-only group, the recovered monkey maintained a baseline level of inhibin B at 9 and 18 months, whereas the monkey that failed to recover had about 20% of pretreatment levels at these time points. More dramatic decreases in inhibin B levels, to an average of 7% of pretreatment levels, were manifested by the radiation-only and radiation plus GnRH antagonist groups after exposure at 9 and 18 months; there were no significant differences between the 2 treatment groups.
|
A quantitative determination of germ cell content was conducted by microscopic analysis at the 18-month time point. In the baseline monkey, all the seminiferous tubules contained germ cells, including elongate spermatids. In the GnRH antagonist-only group, the recovered monkey showed complete repopulation of 1 testis (through elongate spermatids) and partial repopulation of the second testis (76.2% of seminiferous tubules positive for germ cells) for an average across both testes of 88.1% of the seminiferous tubules positive for germ cells. The GnRH antagonist-only animal that failed to recover had variable amounts of atrophy, with an average of 42.5% of the seminiferous tubules positive for germ cells, but only 5% of populated seminiferous tubule cross sections in each testis contained elongate spermatids. In the radiation-only group, only 3.0% ± 2.2% of seminiferous tubule cross sections contained germ cells. The radiation-only monkey with an abnormal hormonal profile had no germ cells present. In the radiation plus GnRH antagonist group, only 1.9% ± 0.9% of seminiferous tubule cross sections contained germ cells. Although few elongate spermatids were present in the recovering tubules, it should be noted that in the combined irradiated groups (10 monkeys), about 90% of the tubules containing germ cells showed at least some elongate spermatids. Of the approximately 500 recovering seminiferous tubules with germ cells of any type in these 10 irradiated monkeys, only 1 seminiferous tubule had spermatogonia alone without more differentiated germ cells. Hence, there does not appear to be any block in differentiation, as observed in the rat.
Because a previous report (Kamischke et al, 2003) indicated that the recovery of spermatogenesis after irradiation was lower in older monkeys, we examined whether there was any age dependence in the recovery of spermatogenesis after irradiation in this group. However, we found that the percentage of tubules with germ cells after irradiation increased with increasing age of the monkey in both groups of irradiated monkeys (with and without GnRH antagonist), and this correlation was significant (P < .01) when both groups were analyzed together.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
In the monkey study reported here, a slightly higher radiation dose (average tissue dose, 6.7 Gy) was chosen to minimize spontaneous recovery of spermatogenesis and more closely mimic the prolonged azoospermia observed in men. A slightly longer treatment period with a GnRH analog was chosen because of the slower response in primates to hormonal suppressive therapy, requiring 2 to 4 weeks to reach minimal values (Figure 3), compared with 3 to 7 days in the rat (Shuttlesworth et al, 2000). Despite the similarities in design, the primate seminiferous epithelium failed to respond to the hormonal intervention that successfully rescued spermatogenesis in rodents.
In the rodent model, successful reversal of injury-induced testicular atrophy depends on reductions in intratesticular T concentrations and possibly also serum FSH levels (Shetty et al, 2002). In this primate study, GnRH antagonist therapy produced a reduction in serum T levels to less than 10% of pretreatment levels and a resulting failure of ejaculation in the majority of treated animals that persisted for several months. No intratesticular T measurements were obtained during the time of hormonal suppression because of the limited testicular tissue available. The available rodent data shows that a marked suppression in serum T levels correlates with a reduction in intratesticular T (Shetty et al, 2000, 2002) and that GnRH antagonist can reduce intratesticular T concentrations to 1% of that observed in irradiated-only rats (Shuttlesworth et al, 2000), but other measurements made in monkeys indicate that GnRH antagonist treatment only lowers intratesticular T to about 30% of the pretreatment value (Weinbauer et al, 1988; Zhengwei et al, 1998). In our study, because Cetrorelix therapy rapidly lowered the serum LH level in unirradiated and irradiated monkeys to about one third of the pretreatment value, testicular T production and tissue levels should also be reduced. Similarly, although suppression of FSH could not be observed in unirradiated monkeys because values were at the limit of detection, in the radiation plus GnRH antagonist group, Cetrorelix therapy suppressed the atrophy-associated rise in serum FSH levels that was observed in the radiationonly monkeys. Thus, GnRH antagonist treatment produces suppression of gonadotropins and T in the monkey, but in the case of intratesticular T, the suppression might not be as great as in the rat.
Although the suppression of T, LH, and FSH achieved with Cetrorelix was completely reversible after treatment ended, spermatogenesis was only partially restored in the 2 GnRH antagonist-only monkeys. Previous studies have suggested that GnRH antagonist-induced inhibition of spermatogenesis is completely reversible in primates (Weinbauer et al, 1988, 1994). However, the Cetrorelix therapy in this study was of longer duration (12 weeks as opposed to 7 weeks in Weinbauer et al [1994]); it also differed in that it consisted of a work week (5 days out of 7) injection schedule with a 3-fold higher dose on Fridays. The other study that showed complete reversibility employed the GnRH antagonist Nal-Glu for 15 weeks at the same dose of 450 µg/kg/d (Weinbauer et al, 1988), but it has been shown that Cetrorelix is more potent (Weinbauer and Nieschlag, 1993). In addition, this study used stump-tailed macaques, whereas the others used cynomolgus macaques. Therefore, the lack of full recovery of spermatogenesis in our study after GnRH analog exposure might depend in part on species or age differences (Weinbauer and Nieschlag, 1989), the specific GnRH antagonist used, the duration or other unique aspects of the treatment protocol, or a combination of factors. In any case, our study indicated that high doses of a GnRH antagonist for 12 weeks could produce some prolonged and likely irreversible suppression of spermatogenesis.
Although 12 weeks of Cetrorelix therapy had some negative effect on spermatogenesis, it was minor compared with the marked inhibition of spermatogenesis produced by radiation only (3.0% of seminiferous tubules with germ cells in the radiation-only monkeys at 18 months). There was no evidence of a significant population of undifferentiated spermatogonia in the seminiferous tubules lacking differentiating germ cells at any of the postradiation biopsy times, as seen in the rat (Shuttlesworth et al, 2000). Our data of severe depletion of spermatogonia induced by a 6.7 Gy x-ray exposure, combined with previous studies in the rhesus (van Alphen et al, 1988a,b, 1989a) and cynomolgus (Kamischke et al, 2003) monkeys exposed to doses of up to 4 Gy, argue that primate stem spermatogonia are more sensitive to radiation injury and less capable of postinjury repopulation than those of the rodent.
In this study, 12 weeks of GnRH antagonist therapy beginning at the time of radiation exposure produced no improvement in testis volume, frequency of germ cell-positive seminiferous tubules, sperm counts, or the hormonal profile. Another recent study in primates that used GnRH analog treatment prior to and slightly after irradiation also failed to improve the postinjury outcome (Kamischke et al, 2003). Although there are many differences between these trials—including radiation dose; timing, duration, and dose schedule of GnRH analog treatment; length of posttreatment observation; and species of macaque—the conclusions were similar in that no amelioration by GnRH analog therapy of radiation-induced testicular injury was observed. In fact, the recovery from radiation damage appeared to be somewhat worse with GnRH analog treatment, with testis volume and sperm counts as endpoints, similar to the trend toward poorer recovery of sperm count in the group treated with GnRH analog observed previously (Kamischke et al, 2003). These results add to a literature reporting mixed results for gonadal protection from irradiation or chemotherapy by various hormonal manipulations in clinical trials (Fossa et al, 1988; Brennemann et al, 1994; Masala et al, 1997) and studies of nonhuman primates (Lewis et al, 1985; van Alphen et al, 1989b).
In rats, the mechanism by which GnRH analog therapy enhances postinjury recovery of spermatogenesis is unknown. One key factor seems to be that after testicular injury, and despite persistent atrophy, rats maintain a population of proliferating spermatogonia that fail to differentiate and instead undergo apoptosis (Boekelheide and Hall, 1991; Allard and Boekelheide, 1996; Shuttlesworth et al, 2000; Boekelheide and Schoenfeld, 2001). Various hormonal manipulations that lower intratesticular T levels, including GnRH analog therapy, enhance the survival potential of the differentiating spermatogonia in the atrophic rodent testis, leading to recovery of spermatogenesis (Shetty et al, 2000, 2002; Meistrich and Shetty, 2003).
Important differences in testicular physiology between primates and rodents might explain the variable recovery potential across species. For example, the spermatogonial populations themselves (Adark and Apale in primates vs As, Apr, and Aal spermatogonia in rodents) and the number of mitotic divisions during spermatogenesis are dissimilar between rodents and primates; such dissimilarities have been invoked to explain the altered response to radiation across species (van Alphen et al, 1988a). Although GnRH receptors are found on both rodent and primate Leydig cells (Bahk et al, 1995), the importance of this receptor to local paracrine control of T production and the effects of LH and exogenous GnRH antagonists on T production might differ across species (Huhtaniemi et al, 1987a,b; Weinbauer and Nieschlag, 1989). In addition, although testicular venous blood T levels were similar in rodents and primates (Maddocks et al, 1993), our measurements indicate higher intratesticular T levels in the stump-tailed macaques (740 ng/gm testis in the baseline monkey and 1600 ng/gm testis after irradiation) than in rats (50 ng/gm testis in control and 170 ng/gm testis after irradiation [Shuttlesworth et al, 2000]). Because lowering intratesticular T is important in restarting spermatogenesis in rodents (Shetty et al, 2000), the nadir in intratesticular T levels during GnRH analog therapy in primates (Weinbauer et al, 1988; Zhengwei et al, 1998) could be much higher than that of rodents and could still inhibit recovery of spermatogenesis. The use of more severe suppression of androgens could be tried but might result in an irreversible inhibitory effect on spermatogenesis.
The results of this study indicate that 6.7 Gy of radiation is killing many stem cells in monkeys, although appreciable numbers of stem spermatogonia survive similar doses in rats or mice (Kangasniemi et al, 1996). Nonetheless, there is evidence for spontaneous recovery of spermatogenesis in humans after long periods of azoospermia following cancer-related chemotherapy or radiotherapy, indicating that stem cells are present in some regions of the testis (Meistrich et al, 1992). In addition, there have been histopathological analyses that document a failure of completion of differentiation of surviving spermatogonia in the azoospermic human testes, similar to that observed in rodents (Kreuser et al, 1989; Meistrich and van Beek, 1990). These data support the potential for spermatogenesis to be restarted in the atrophic human testis, although the underlying testicular physiology differs from that of the rodent. A clearer understanding of the underlying mechanisms in rodents involved in the injury-associated block to spermatogenesis and the relief of this block by hormonal manipulations should be helpful in designing more rational intervention strategies to treat azoospermia in humans.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bahk JY, Hyun JS, Chung SH, Lee H, Kim MO, Lee BH, Choi WS. Stage specific identification of the expression of GnRH mRNA and localization of the GnRH receptor in mature rat and adult human testis. J Urol. 1995;154: 1958 -1961.[Medline]
Blanchard KT, Lee J, Boekelheide K. Leuprolide, a
gonadotropin-releasing hormone agonist, reestablishes spermatogenesis after
2,5-hexanedione-induced irreversible testicular injury in the rat, resulting
in normalized stem cell factor expression.
Endocrinology. 1998; 139: 236
-244.
Boekelheide K, Hall SJ. 2,5-Hexanedione exposure in the rat results
in long-term testicular atrophy despite the presence of residual
spermatogonia. J Androl. 1991; 12: 18
-26.
Boekelheide K, Schoenfeld HA. Spermatogenesis by Sisyphus: proliferating stem germ cells fail to repopulate the testis after `irreversible' injury. Adv Exp Med Biol. 2001; 500: 421 -428.[Medline]
Brennemann W, Brensing KA, Leipner N, Boldt I, Klingmuller D. Attempted protection of spermatogenesis from irradiation in patients with seminoma by D-tryptophan-6 luteinizing hormone releasing hormone. Clin Invest. 1994; 72: 838 -842.[Medline]
Clermont Y, Antar M. Duration of the cycle of the seminiferous epithelium and the spermatogonial renewal in the monkey Macaca arctoides. Am J Anat. 1973; 136: 153 -165.[Medline]
Clifton DK, Bremner WJ. The effect of testicular x-irradiation on
spermatogenesis in man. A comparison with the mouse. J
Androl. 1983;4: 387
-392.
Ellinwood WE, Resko JA. Sex differences in biologically active and immunoreactive gonadotropins in the fetal circulation of rhesus monkeys. Endocrinology. 1980; 107: 902 -907.[Abstract]
Foppiani L, Schlatt S, Simoni M, Weinbauer GF, Hacker-Klom U, Nieschlag E. Inhibin B is a more sensitive marker of spermatogenetic damage than FSH in the irradiated non-human primate model. J Endocrinol. 1999;162: 393 -400.[Abstract]
Fossa SD, Klepp O, Norman N. Lack of gonadal protection by medroxyprogesterone acetate-induced transient medical castration during chemotherapy for testicular cancer. Br J Urol. 1988; 62: 449 -453.[Medline]
Hild SA, Attardi BJ, Reel JR. The ability of a
gonadotropin-releasing hormone antagonist, acyline, to prevent irreversible
infertility induced by the indenopyridine, CDB-4022, in adult male rats: the
role of testosterone. Biol Reprod. 2004; 71: 348
-358.
Huhtaniemi I, Nikula H, Parvinen M, Rannikko S.
Pituitary-testicular function of prostatic cancer patients during treatment
with a gonadotropin-releasing hormone agonist analog. II. Endocrinology and
histology of the testis. J Androl. 1987a; 8: 363
-373.
Huhtaniemi IT, Nikula H, Detta A, Stewart JM, Clayton RN. Blockade of rat testicular gonadotropin releasing hormone (GnRH) receptors by infusion of a GnRH antagonist has no major effects of Leydig cell function in vivo. Mol Cell Endocrinol. 1987b; 49: 89 -97.[Medline]
Kamischke A, Kuhlmann M, Weinbauer GF, Luetjens M, Yeung CH, Kronholz HL, Nieschlag E. Gonadal protection from radiation by GnRH antagonist or recombinant human FSH: a controlled trial in a male nonhuman primate (Macaca fascicularis). J Endocrinol. 2003; 179: 183 -194.[Abstract]
Kangasniemi M, Dodge K, Pemberton AE, Huhtaniemi I, Meistrich ML. Suppression of mouse spermatogenesis by a gonadotropin-releasing hormone antagonist and antiandrogen: failure to protect against radiation-induced gonadal damage. Endocrinology. 1996; 137: 949 -955.[Abstract]
Kangasniemi M, Wilson G, Huhtaniemi I, Meistrich ML. Protection against procarbazine-induced testicular damage by GnRH-agonist and antiandrogen treatment in the rat. Endocrinology. 1995a; 136: 3677 -3680.[Abstract]
Kangasniemi M, Wilson G, Parchuri N, Huhtaniemi I, Meistrich ML. Rapid protection of rat spermatogenic stem cells against procarbazine by treatment with a gonadotropin-releasing hormone antagonist (Nal-Glu) and an antiandrogen (flutamide). Endocrinology. 1995b; 136: 2881 -2888.[Abstract]
Kreuser ED, Kurrle E, Hetzel WD, et al. Reversible germ cell toxicity following aggressive chemotherapy in patients with testicular tumors: results of a prospective study. Klin Wochenschr. 1989; 67: 367 -378.[Medline]
Lewis RW, Dowling KJ, Schally AV. D-Tryptophan-6 analog
of luteinizing hormone-releasing hormone as a protective agent against
testicular damage caused by cyclophosphamide in baboons. Proc Natl
Acad Sci U S A. 1985;82: 2975
-2979.
Maddocks S, Hargreave TB, Reddie K, Fraser HM, Kerr JB, Sharpe RM. Intratesticular hormone levels and the route of secretion of hormones from the testis of the rat, guinea pig, monkey and human. Int J Androl. 1993;16: 272 -278.[Medline]
Masala A, Faedda R, Alagna S, et al. Use of testosterone to prevent
cyclophosphamide-induced azoospermia. Ann Intern Med. 1997; 126: 292
-295.
Matsumiya K, Meistrich ML, Shetty G, Dohmae K, Tohda A, Okuyama A,
Nishimune Y. Stimulation of spermatogonial differentiation in juvenile
spermatogonial depletion (jsd) mutant mice by gonadotropin-releasing hormone
antagonist treatment. Endocrinology. 1999; 140: 4912
-4915.
Meistrich ML. Quantitative correlation between testicular stem cell survival, sperm production, and fertility in the mouse after treatment with different cytotoxic agents. J Androl. 1982; 3: 58 -68.[Abstract]
Meistrich ML, Kangasniemi M. Hormone treatment after irradiation
stimulates recovery of rat spermatogenesis from surviving spermatogonia.
J Androl. 1997;18: 80
-87.
Meistrich ML, Shetty G. Inhibition of spermatogonial
differentiation by testosterone. J Androl. 2003; 24: 135
-148.
Meistrich ML, Shuttlesworth G, Huhtaniemi I, Reissmann T. GnRH agonists and antagonists stimulate recovery of fertility in irradiated LBNF1 rats. J Androl. 2001; 22: 809 -817.[Abstract]
Meistrich ML, van Beek MEAB. Radiation sensitivity of the human testis. Adv Radiat Biol. 1990; 14: 227 -228.
Meistrich ML, Wilson G, Brown BW, da Cunha MF, Lipshultz LI. Impact of cyclophosphamide on long-term reduction in sperm count in men treated with combination chemotherapy for Ewing and soft tissue sarcomas. Cancer. 1992;70: 2703 -2712.[Medline]
Meistrich ML, Wilson G, Huhtaniemi I. Hormonal treatment after
cytotoxic therapy stimulates recovery of spermatogenesis. Cancer
Res. 1999;59: 3557
-3560.
Meistrich ML, Wilson G, Kangasniemi M, Huhtaniemi I. Mechanism of protection of rat spermatogenesis by hormonal pretreatment: stimulation of spermatogonial differentiation after irradiation. J Androl. 2000;21: 464 -469.[Abstract]
Morris ID, Shalet SM. Protection of gonadal function from cytotoxic chemotherapy and irradiation. Baillieres Clin Endocrinol Metab. 1990;4: 97 -118.[Medline]
Redman JR, Bajorunas DR. Suppression of germ cell proliferation to prevent gonadal toxicity associated with cancer treatment. In: Workshop on Psychosexual and Reproductive Issues Affecting Patients with Cancer. New York: American Cancer Society; 1987 : 90-94.
Schoenfeld HA, Hall SJ, Boekelheide K. Continuously proliferative
stem germ cells partially repopulate the aged, atrophic rat testis after
gonadotropin-releasing hormone agonist therapy. Biol
Reprod. 2001;64: 1273
-1282.
Schover LR, Brey K, Lichtin A, Lipshultz LI, Jeha S. Knowledge and
experience regarding cancer, infertility, and sperm banking in younger male
survivors. J Clin Oncol. 2002; 20: 1880
-1889.
Shetty G, Weng CC, Bolden-Tiller OU, Huhtaniemi I, Handelsman DJ,
Meistrich ML. Effects of medroxyprogesterone and estradiol on the recovery of
spermatogenesis in irradiated rats. Endocrinology. 2004; 145: 126
-133.
Shetty G, Wilson G, Hardy MP, Niu E, Huhtaniemi I, Meistrich ML.
Inhibition of recovery of spermatogenesis in irradiated rats by different
androgens. Endocrinology. 2002; 143: 3385
-3396.
Shetty G, Wilson G, Huhtaniemi I, Shuttlesworth GA, Reissmann T,
Meistrich ML. Gonadotropin-releasing hormone analogs stimulate and
testosterone inhibits the recovery of spermatogenesis in irradiated rats.
Endocrinology. 2000; 141: 1735
-1745.
Shuttlesworth GA, de Rooij DG, Huhtaniemi I, Reissmann T, Russell
LD, Shetty G, Wilson G, Meistrich ML. Enhancement of a spermatogonial
proliferation and differentiation in irradiated rats by gonadotropin-releasing
hormone antagonist administration. Endocrinology. 2000; 141: 37
-49.
Udagawa K, Takeda M, Hosaka M, Kubota Y, Ogawa T. Recovery of spermatogenesis by high dose gonadotropin-releasing hormone analogue treatment in rat cryptorchid testis after orchiopexy. J Urol. 2002; 168: 1279 -1283.[Medline]
van Alphen MM, van de Kant HJ, Davids JA, Warmer CJ, Bootsma AL, de Rooij DG. Dose-response studies on the spermatogonial stem cells of the rhesus monkey (Macaca mulatta) after X irradiation. Radiat Res. 1989a;119: 443 -451.[Medline]
van Alphen MM, van de Kant HJ, de Rooij DG. Depletion of the spermatogonia from the seminiferous epithelium of the rhesus monkey after X irradiation. Radiat Res. 1988a; 113: 473 -486.[Medline]
van Alphen MM, van de Kant HJ, de Rooij DG. Repopulation of the seminiferous epithelium of the rhesus monkey after X irradiation. Radiat Res. 1988b; 113: 487 -500.[Medline]
van Alphen MM, van de Kant HJ, de Rooij DG. Protection from
radiation-induced damage of spermatogenesis in the rhesus monkey (Macaca
mulatta) by follicle-stimulating hormone. Cancer
Res. 1989b;49: 533
-536.
Van Thiel DH, Sherins RJ, Myers GH Jr, De Vita VT Jr. Evidence for a specific seminiferous tubular factor affecting follicle-stimulating hormone secretion in man. J Clin Invest. 1972; 51: 1009 -1019.
Weinbauer GF, Gockeler E, Nieschlag E. Testosterone prevents complete suppression of spermatogenesis in the gonadotropin-releasing hormone antagonist-treated nonhuman primate (Macaca fasicularis). J Clin Endocrinol Metab. 1988; 284: 284 -290.
Weinbauer GF, Limberger A, Behre HM, Nieschlag E. Can testosterone alone maintain the gonadotrophin-releasing hormone antagonist-induced suppression of spermatogenesis in the non-human primate? J Endocrinol. 1994;142: 485 -495.[Abstract]
Weinbauer GF, Nieschlag E. Reversibility of GnRH agonist-induced inhibition of testicular function: differences between rats and primates. Prog Clin Biol Res. 1989; 303: 75 -87.[Medline]
Weinbauer GF, Nieschlag E. Comparison of the antigonadotropic activity of three GnRH antagonists (Nal-Glu, Antide and Cetrorelix) in a non-human primate model (Macaca fascicularis). Andrologia. 1993; 25: 141 -147.[Medline]
Zhengwei Y, Wreford NG, Schlatt S, Weinbauer GF, Nieschlag E, McLachlan RI. Acute and specific impairment of spermatogonial development by GnRH antagonist-induced gonadotrophin withdrawal in the adult macaque (Macaca fascicularis). J Reprod Fertil. 1998; 112: 139 -147.
This article has been cited by other articles:
|
|
 |
|
  Z. Zhang, S. Shao, and M. L. Meistrich Irradiated Mouse Testes Efficiently Support Spermatogenesis Derived From Donor Germ Cells of Mice and Rats J Androl, May 1, 2006; 27(3): 365 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Shetty, C. C. Y. Weng, S. J. Meachem, O. U. Bolden-Tiller, Z. Zhang, P. Pakarinen, I. Huhtaniemi, and M. L. Meistrich Both Testosterone and Follicle-Stimulating Hormone Independently Inhibit Spermatogonial Differentiation in Irradiated Rats Endocrinology, January 1, 2006; 147(1): 472 - 482. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
