* Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; and
Department of Physiology, University of Turku, 20520 Turku, Finland
Received July 2, 2003; accepted September 3, 2003
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ABSTRACT |
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Key Words: spermatogonia; gonadotropin-releasing hormone analogs; reproductive toxicants.
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INTRODUCTION |
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About half of the DBCP-exposed azoospermic men remained that way for many years (Eaton et al., 1986; Olsen et al., 1990
; Potashnik and Porath, 1995
), suggesting that all of the stem spermatogonia may have been killed. Others recovered sperm count, but in some cases the recovery did not occur until 3 to 6 years later (Eaton et al., 1986
; Olsen et al., 1990
; Potashnik, 1983
). This observation indicates that stem spermatogonia were present but unable to differentiate into spermatozoa for an extended period of time.
To develop methods for understanding and possibly reversing sterility in these men, we recently developed an animal model in which rats showed prolonged effects of DBCP on the germinal epithelium (Meistrich et al., 2003). Although spermatogenesis did not recover 20 weeks after DBCP exposure, the presence of type A spermatogonia in the tubules after DBCP treatment of these rats suggests that spermatogenesis could be restored.
The continued presence of type A spermatogonia that proliferate but die by apoptosis just prior to differentiation is a more general phenomenon (Meistrich and Shetty, 2003). For instance, it has been shown to be the case in rats that were treated with hexanedione (Boekelheide and Hall, 1991
), irradiation (Kangasniemi et al., 1996
), procarbazine (Meistrich et al., 1999
), boric acid (Ku et al., 1993
), or an indenopyridine (Hild et al., 2001
) and in aging rats (Schoenfeld et al., 2001
). We had also shown that GnRH-analog (agonist or antagonist) treatment given after irradiation stimulated the differentiation of these cells (Meistrich and Kangasniemi, 1997
; Shuttlesworth et al., 2000
). In several of the above cases (Blanchard et al., 1998
; Meistrich et al., 1999
; Schoenfeld et al., 2001
), as well as after testicular heating (Setchell et al., 2001
) or busulfan exposure (Udagawa et al., 2001
), spermatogenesis was also stimulated by the administration of a GnRH agonist after toxic exposure or the development of tubular atrophy, and in some cases fertility was restored.
Based on these observations, we tested whether a GnRH agonist administered to rats treated with DBCP could enhance spermatogenesis when given either immediately after DBCP exposure or after spermatogenesis had declined. We characterized the degree of recovery of differentiation within the seminiferous tubules during GnRH-agonist treatment. After the cessation of the hormone treatment, we also followed the maintenance of differentiation within the tubules as well as the degree of recovery of sperm production.
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MATERIALS AND METHODS |
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DBCP treatment.
Lots LA82202 and LA85851 of DBCP, purity 9798%, were obtained from Supelco Separation Technology (Bellefonte, PA). DBCP was dissolved in corn oil and given as four daily subcutaneous (sc) injections of 87.5 mg/kg body weight.
GnRH agonist treatment.
The GnRH agonist leuprolide acetate, ([D-Leu6, des-Gly-NH210, Pro-ethylamide9]-GnRH), was supplied as a nominal one-month depot formulation (Lupron) in microspheres of a lactic acidgylcolic acid copolymer by TAP Pharmaceuticals (Deerfield, IL). Leuprolide was suspended in a carboxymethyl cellulose vehicle provided by the manufacturer and given as 1.83-mg intramuscular (im) injections 2324 days apart. This timing was chosen based on data showing that the drug is released over a 4-week period with maintenance of serum leuprolide levels above 2 ng/ml and suppression of serum testosterone levels for that period of time (Ogawa et al., 1989). Serum testosterone was suppressed starting at 3 days after starting treatment (Ogawa et al., 1989
), and we have shown with a different GnRH agonist that follicle stimulating hormone (FSH) and intratesticular testosterone levels reached minimal values within 4 days (Kangasniemi et al., 1995
).
Two different treatment schedules were used. To study the maintenance of spermatogenesis after DBCP treatment (Experiments 1 and 2), the leuprolide treatment was started 1 day after the last DBCP injection, and a total of three injections, 2324 days apart, were given to provide exposure to the hormone for about 10 weeks (Fig. 1). Groups for comparisons and controls consisted of untreated rats, rats treated with DBCP alone, and rats treated with leuprolide only. Some rats were killed at week 10, near the end of the GnRH-agonist treatment, and others were killed at week 20 to assess whether spermatogenesis was still maintained after GnRH-agonist treatment was suspended or whether it regressed to the levels measured in DBCP-onlytreated rats. To study the restoration of spermatogenesis after it had declined (Experiments 3 and 4), leuprolide treatment was started 6 weeks after the last DBCP injection, and injections were given 2324 days apart until the rats were killed, 5.6 (Experiment 3) or 10 weeks later (Experiment 4). Because there was some variability in the effectiveness of DBCP between experiments (Meistrich et al., 2003
), results for some of the endpoints were plotted separately.
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The right testis was weighed after the removal of the tunica albuginea, and the tissue was then homogenized. Approximately 1 ml of the testis homogenate was removed from the rats killed at week 20 in Experiments 1 and 2 and sonicated (Meistrich and van Beek, 1993), and an aliquot was placed in a hemacytometer to count late spermatid nuclei.
To assess fertility, male rats in Experiments 1 and 2 were mated for 2 weeks before they were killed at week 20. Each male was caged with two females for 1 week, and then the females were replaced with two different females. The females were killed 14 days after the end of mating, and the embryos were counted. A male was considered fertile if he produced one or more offspring by any of the four females.
Characterization of type A spermatogonia.
Type A spermatogonial cells, mitoses, and cells with apoptotic morphology were counted in 200 atrophic seminiferous tubule cross sections, without additional morphological abnormalities, from the Bouins fixed tissue (Shuttlesworth et al., 2000). Type A spermatogonia were identified by their round or ovoid nuclei with fine granular chromatin and one to three nucleoli that are sometimes adjacent to the nuclear periphery (Chiarini-Garcia and Russell, 2001
). To normalize the count of type A spermatogonia to the numbers of Sertoli cells, the Sertoli cells with visible nucleoli were counted in 20 (every tenth) seminiferous tubular cross sections from each rat. The diameters of type A spermatogonia, mitoses, apoptotic cells, and Sertoli cell nucleoli previously measured were used to calculate the relative volume densities of the cells using the Abercrombie correction (Shuttlesworth et al., 2000
).
To determine the labeling index of type A spermatogonia, the rats were injected i.p. with bromodeoxyuridine (BrdU) at 30 mg/kg, 1 h prior to killing. The ethanol-fixed testicular tissue was processed for BrdU immunohistochemistry using protease digestion, clone B44 first antibody (Becton Dickinson Immunocytometric Systems, San Jose, CA), and the Vectastain kit (Vector Laboratories Inc., Burlingame, CA) as described in Shuttlesworth et al.(2000). Type A spermatogonia were counted in 200 seminiferous tubular cross sections to determine the labeling index.
Hormone measurements.
From some of the rats, blood was collected at the time they were killed by cardiac puncture under ketamineacepromazine anesthesia. The serum was separated and stored at -80°C.
The right testis was homogenized and stored at -80°C for analysis of intratesticular testosterone (ITT). Serum testosterone and ITT were assayed using coated tubes (DSL 4000, Diagnostic Systems Laboratories, Webster, TX) (Shetty et al., 2000). ITT was expressed as the amount per gram of testis rather than the amount per testis to reflect the concentration of testosterone to which the testicular cells were exposed. The minimum levels of detection for serum testosterone and ITT were 0.04 ng/ml and about 0.6 ng/g-testis, respectively.
The serum levels of FSH and luteinizing hormone (LH) were measured using immunofluorometric assays (Delfia, Wallac OY, Turku, Finland) as described in Haavisto et al.(1993) and van Casteren et al.(2000)
. The minimum levels of detection of LH and FSH by this method were 0.04 ng/ml and 0.1 ng/ml, respectively.
Statistics.
All data are presented as the mean ±1 SEM using either the untransformed data or log-transformed data for plots on a logarithmic scale. Pairwise comparisons between groups were done using the Students t-test. Comparisons involving multiple groups were done by first performing a one-way ANOVA and proceeding if there were significant differences between groups (P < 0.05). A single group or multiple groups were compared to a single control using the Students t-test or Dunnetts test, repsectively. The relationship between fertility and the testicular characteristics was tested by Spearmans rho, which is a measure rank order. All analyses were done using SPSS software (version 10.0, SPSS Inc., Chicago, IL).
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RESULTS |
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Restimulation of Spermatogenesis with Delayed GnRH Treatment
To determine whether spermatogonial differentiation could be stimulated after the TDI had declined, we initiated GnRH-agonist treatment 6 weeks after DBCP exposure. At this time the TDIs had already declined to below 30% (Fig. 6).
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In these two experiments, the incidence of abnormal tubules was between 5 and 19% in all DBCP-treated groups. The GnRH-agonist treatment did not induce any significant changes in the incidence of these tubular abnormalities.
Characterization of Type A Spermatogonia in GnRH-Agonist-Treated Rats
The type A spermatogonia and Sertoli cells were counted in tubules in which no differentiating cells were observed. Surprisingly, the GnRH treatment appeared to produce a small but significant decline in the numbers of Sertoli cells per tubule cross section (Table 1). However, stereological methods would be required to demonstrate an actual decrease in numbers of Sertoli cells per testis.
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DISCUSSION |
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Qualitatively, the ability of GnRH analogs to enhance spermatogenesis after DBCP-induced toxicity was similar to the case of other toxicants. Since the studies with radiation and procarbazine were done in this laboratory with the same rat strain, we can more quantitatively compare the degrees of stimulation of recovery (Table 2) for what we estimated to be equitoxic doses of the agents based on the TDIs without GnRH-agonist treatment. The degree of stimulation of spermatogonial differentiation at the end of the 10-week GnRH-agonist treatment observed when the block was induced by DBCP appeared to be slightly greater than with radiation but slightly less than with procarbazine treatment. However, spermatogonial differentiation progressively increased in the case of irradiated testes following the cessation of GnRH-agonist treatment, but it declined in the DBCP- or procarbazine-exposed testes. It should be noted, however, that in other experiments the restoration of spermatogonial differentiation after irradiation by GnRH analogs also eventually declined (Shuttlesworth and Meistrich, unpublished).
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One could expect that the testicular damage observed after DBCP would be similar to that observed after procarbazine or busulfan, since the proposed mechanisms of DBCP action also include alkylation and formation of adducts with DNA bases (Humphreys et al., 1991; Pearson et al., 1990
). However, the other agents and aging have different mechanisms of action, and some, like hexanedione, target the Sertoli cells whereas others, like radiation, primarily target the germ cells (Lee et al., 1999
). The common factor in the ability of GnRH analogs to stimulate the recovery of spermatogenesis after a wide variety of toxicants appears to be that in all cases the inhibition of spermatogenesis is at the level of the differentiation of type A spermatogonia.
The reversibility of the block in spermatogonial differentiation by GnRH-analog treatment after exposure to other toxicants led to the suggestion that it was largely the elevated intratesticular levels of testosterone, but possibly also FSH, acting on the testis that caused this inhibition (Meistrich et al., 1999; Meistrich and Shetty, 2003
; Shetty et al., 2000
, 2002
). The present results, showing the prevention and reversal of DBCP-induced testicular damage, support that hypothesis, since both the ITT and FSH levels were high in the DBCP-treated rats and were reduced by the GnRH-agonist treatment. But the reduction in ITT was greater when a 10-week GnRH-agonist treatment was given immediately after DBCP than when it was started 6 weeks later (Fig. 2d
). The differences in ITT reduction could be responsible for the greater stimulation of the TDI (to 94%) with immediate treatment (Fig. 3
) than with delayed treatment (52%) (Fig. 6
). A similar relationship was noted after irradiation: The suppression of ITT and the stimulation of recovery of spermatogenesis were both greater when the GnRH agonist was given immediately as opposed to 20 weeks after irradiation (Meistrich et al., 1999
). Preliminary data (G. Shetty, unpublished observations) also indicated that both the abilities to suppress ITT and to restore spermatogenesis in mutant jsd (juvenile spermatogonial depletion) mice with GnRH analogs were reduced with mouse age and further decline in spermatogenesis. Thus, the decreased effectiveness of delayed hormonal treatment may be due to its decreased ability to suppress ITT, rather than to a decrease in the intrinsic responsiveness of the cells. More effective suppression of testosterone levels or action might restore the effectiveness of stimulation of recovery.
In these instances of blocked spermatogonial differentiation, the spermatogonia actively proliferated but died by apoptosis instead of differentiating. We previously reported that GnRH-antagonist treatment initiated 15 weeks after irradiation altered spermatogonial kinetics (Shuttlesworth et al., 2000). The number of type A spermatogonia was increased within 1 week, and the increase was maintained. There was also an increase in the labeling index of the type A spermatogonia. Although there may have been a slight increase in the numbers of type A spermatogonia and a decrease in their apoptotic indices in the atrophic tubules with GnRH-agonist treatment after DBCP-induced testicular atrophy, these changes were not statistically significant, and there was no increase in labeling index of the spermatogonia (Table 1
). Whereas GnRH-antagonisttreated irradiated rats also showed a transient decline in mitotic and apoptotic indices for the first week or two after initiation of GnRH-analog treatment, these levels returned to those seen in the irradiated-only rats by about 5 weeks (Shuttlesworth et al., 2000
). Two reasons that changes in spermatogonial kinetics were not observed in the present study could be that we first counted type A spermatogonia after 5.6 weeks of GnRH-agonist treatment, perhaps missing evidence of transient changes, or that the changes occurred only in the subset of tubules in which repopulation was induced, but other explanations are possible too. Further studies are required to determine whether a decrease in apoptosis of type A spermatogonia at a particular stage of development is indeed responsible for the further development of spermatogenesis in a subset of tubules.
The mechanism by which testosterone and FSH inhibit spermatogonial differentiation in toxicant-treated rats and, consequently, how suppression of these hormones stimulates the recovery of spermatogenesis is still not known. Several important questions must be addressed in order to elucidate the mechanism as has been reviewed recently (Meistrich and Shetty, 2003). It must be determined whether the block to spermatogonial development is a result of a toxic effect on the spermatogonia themselves or on the Sertoli or other somatic cells. It also must be determined whether the failure of spermatogonial differentiation is a result of the loss of an essential growth or differentiation factor pathway or the induction of an apoptotic pathway that results in spermatogonial death before they are able to differentiate. In addition, the role of testicular edema, which occurs after both irradiation and DBCP treatment, needs to be addressed. Once the cellular and physiological targets for the testosterone-induced block in spermatogonial development is elucidated, it will be useful to screen for differentially expressed genes that are related to the block to spermatogonial development. We are currently addressing these questions using the irradiated rat model (Meistrich and Kangasniemi, 1997
) because of the greater reproducibility of the response and the absence of systemic toxicity.
The present study has emphasized that the same principles apply to a wider range of toxicants, including important occupational and environmental agents. Although DBCP has been banned, its environmental concentrations are decreasing, and many of the exposed individuals are aging beyond their reproductive years, similar compounds are still widely used. For example 1- and 2-bromopropanes are widely used solvents that cause reproductive toxicity in both rats and humans (Ichihara et al., 2000; Kim et al., 1996
). Like DBCP, 2-bromopropane is selectively toxic to spermatogonia and produces DNA damage (Omura et al., 1999
; Wu et al., 2002
).
These results indicate that there may be some possibility for the restoration of spermatogenesis in men rendered sterile by DBCP and other reproductive toxicants. The ability to reverse the decline in sperm counts depends on the existence of surviving stem spermatogonia that fail to complete differentiation. Although histological studies on testes of men exposed to high doses of DBCP often indicate the absence of any germ cells, some azoospermic individuals have occasional spermatogonia and even more advanced germ cells (Potashnik et al., 1978; Potashnik and Yanai-Inbar, 1987
). The occasional spontaneous reversal of DBCP-induced azoospermia or oligospermia further indicates that there are stem cells that are capable of additional activation and differentiation into spermatozoa. Further investigation is needed to determine whether the principles demonstrated here for the restoration of spermatogenesis in rats by transient suppression of gonadotropins and testosterone will apply to humans, since the potential applications are great and the toxicity of the treatment is low.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Synergos, Inc., 2202 Timberloch Place, Suite 230, The Woodlands, TX 77380.
3 Present address: Institute of Reproductive and Developmental Biology, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom.
4 Present address: Biogenex Laboratories, 4600 Norris Canyon Road, San Ramon, CA 94583.
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