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Regular Article |
a Pediatric Endocrinology Unit,
b Karolinska Institute; Karolinska Pharmacy, Karolinska Hospital, SE-171 76 Stockholm, Sweden
c Departments of Pediatrics and Anatomy, University of Turku, FIN-20520 Turku, Finland
ABSTRACT
A rapid in vitro method has been developed to detect early effects of cytostatic drugs on rat spermatogenesis. The induction of programmed cell death (apoptosis) and changes in DNA synthesis induced by doxorubicin and idarubicin were measured in specific stages of the cycle of seminiferous epithelium including mitotic (stage V) and meiotic (stage VIII-IX) S-phase cells. The model was used to investigate the protective effect of an organic thiophosphate, amifostine, against the toxicity of antracyclines. Premitotic DNA synthesis was found to be more sensitive than premeiotic DNA synthesis to antracyclines. Idarubicin was more toxic than doxorubicin to germ cells in inducing apoptosis and suppressing DNA synthesis. Amifostine had no protective effect against doxorubicin- or idarubicin-induced inhibition of DNA synthesis. In contrast, a significant stimulation of DNA synthesis in premitotic cells by amifostine was found, suggesting that this compound may have a stimulative effect on spermatogenic stem cells. These data show that stage-specific dissection of the seminiferous tubules and their in vitro exposure to predetermined doses of drugs may give us a unique possibility to detect drug action and protection against the cytotoxicity of antineoplastic agents at the cellular level of the spermatogenic cycle.
apoptosis, Sertoli cells, spermatid, spermatogenesis, testes
INTRODUCTION
The improved prognosis of children and young adults with cancer has led to a greater awareness of the late consequences of successful curative therapy [1, 2]. One of the major unresolved issues is the deleterious effects of chemotherapy on male reproductive function [3–6]. Despite the numerous clinical reports describing adverse effects of cytostatic agents on human fertility, an efficient method to study the toxic effects of these drugs in testicular tissue has not been available.
Spermatogenesis is a well-characterized process in which spermatogonia proliferate and differentiate into spermatozoa, starting with initial divisions of undifferentiated spermatogonia. The daughter cells enter spermatogenesis as differentiating spermatogonia and divide mitotically [7]. These differentiating spermatogonia can be morphologically identified as A1–A4, intermediate, and type B [8]. The final mitotic division of type B spermatogonia gives rise to preleptotene spermatocytes. This mitotic activity in rat spermatogenic epithelium is reflected by six peaks of DNA synthesis at stages VIII, XI, XII, I, II, and V [9] and meiotic phase by a peak at stage VIII-IX of the cycle [9]. The S-phase activity is reflected by 3H-thymidine incorporation by staged seminiferous tubule segments, showing prominent peaks at stages V (type B spermatogonia) and VIII (preleptotene spermatocytes) and background levels at stages VIIa–VIIc [10]. After two meiotic divisions, haploid round spermatids are formed. They undergo morphological changes and become spermatozoa. Programmed cell death (apoptosis) of germ cells is a physiological event during normal spermatogenesis [11]. Quantification of programmed cell death in different spermatogenic stages has revealed that spermatogonia of types A2–A4 in rodent testis are spontaneously deleted by apoptosis [11]. Either no apoptosis or a low extent of apoptosis seems to occur in intermediate and type B spermatogonia and some cell death is seen in spermatocytes and mature spermatids [8].
Knowledge of the effects of cytotoxic drugs in the complex process of spermatogenesis is poor. In vivo studies in rats have revealed that cytostatic drugs are able to induce apoptosis in the spermatogenic epithelium [12, 13]. The cell types most sensitive to this induction are spematogonia, zygotene and early pachytene spermatocytes, and meiotically dividing spermatocytes [12, 13]. In vivo treatment with cytotoxic drugs has also been shown to cause inhibition of stage-specific DNA synthesis in the rat testis [14, 15] and to induce DNA damage in spermatogonial cells, as measured by a micronucleus assay [16].
The organic thiophosphate, amifostine, is one of the promising pharmacological compounds showing selective protection in many human tissues against toxic side effects of radiation and cytotoxic drugs while preserving the antitumor efficacy of treatment [17]. The selectivity, in terms of cytoprotection of normal tissues, is believed to be related to perfusion-associated distribution and absorption of the amifostine and greater alkaline phosphatase activity in normal tissues than in malignant tissues [18]. The reports of the effect of amifostine on testicular tissue have been conflicting. Significant protection of testicular stem cells has been observed for high doses of radiation [19]. Repeated doses of amifostine given with or without fractionated radiation decreased stem cell survival, suggesting that amifostine by itself may also have cytotoxic effects in spermatogic cells [19, 20]. A detailed and stage-specific analysis of the protective capacity of amifostine in the spermatogenic cycle against cytostatic drug toxicity has not, however, been reported.
The aim of this study was to use the transillumination-assisted microdissection technique [21] to analyze the in vitro sensitivity of mitotic and meiotic germ cells in rat spermatogenesis to the toxicity of two antracyclines, doxorubicin and idarubicin. The antracyclines are known to bind and intercalate DNA, inhibit topoisomerase II activity, generate free radicals, and covalently bind to DNA following reductive metabolism [22]. A more lipophilic character, a more extensive formation, and slower elimination of the active metabolite and better intracellular penetration of idarubicin make it a more potent antitumor agent compared with doxorubicin [23]. The present in vitro model was also used to evaluate the protective effect of amifostine against the antracycline-induced toxicity on cultured germ cells. Squash preparations were used for quantification of stage-specific apoptosis [24, 25].
MATERIALS AND METHODS
Animals
Male Sprague Dawley rats from BK Universal, Stockholm, Sweden, aged 2–3 months were maintained in standard conditions at the Karolinska Institute, in Stockholm. The study was approved by the local animal ethics committee (PN 169/97).
Microdissection and Culture of Staged Seminiferous Tubule Segments
After removal and decapsulation of the testes, seminiferous tubule segments at stages V and VIII-IX of the cycle were microdissected under a transillumination stereomicroscope in a Petri dish containing PBS [21]. The isolated tubule segments (2 mm in length) were transferred from the Petri dish into wells of a 96-well culture plate in 10 µl of PBS. They were incubated at 34°C in 100 µl of Hams F12 Dulbeccos minimum essential Eagle's medium (MEM; Gibco BRL, Paisley, UK), supplemented with 0.1% BSA, penicillin G 60 µg/ml, and streptomycin 500 µg/ml (Gibco BRL) in a humidified atmosphere containing 5% CO2 for 1, 2, 4, 6, 18 and 24 h.
Treatment with Antracyclines and Amifostine
Methyl-3H-thymidine incorporation was measured in samples that were cultured with final concentrations of 0.1–750 ng/ml of doxorubicin (Adriamycin; Pharmacia-Upjohn, Stockholm, Sweden) and 0.008–2 ng/ml of idarubicin (Zavedos, Pharmacia-Upjohn) in order to find optimal dose-response concentrations (data not shown). In further studies, doxorubicin and idarubicin were added in 10 µl of 0.01 M H3PO4 at final concentrations of 0.1, 0.2, and 0.4 ng/ml and 0.008, 0.04, and 0.1 ng/ml of doxorubicin and idarubicin, respectively, for stage V; and, for stage VIII-IX, at concentrations of 1.2, 2, 5 ng/ml and 0.4, 1.2, 2 ng/ml. For in situ DNA 3'-end labeling, the final concentrations were selected to reflect the inhibitory effects of doxorubicin and idarubicin (i.e., 0.4 and 0.04 ng/ml for stage V and 5 and 2 ng/ml for stage VIII-IX, respectively). In control cultures, 10 µl of 0.01 M H3PO4 was added without drugs. Amifostine (WR-2721, S-2-[3-aminopropylamino] ethylphosphorothioic acid (Ethyol, Schering Plough, Stockholm, Sweden) was added to a final concentration of 2 µg/ml of medium (i.e., one tenth of the peak plasma concentration obtained after treatment with the standard amifostine dose of 740 mg/m2 in adult humans [26]). Two exposure sequences were made: 1) treatment for 15 min and then immediate removal by washing with PBS before incubation with the cytostatic drug for 18 h and 2) treatment for 18 h in combination with the cytostatic drug. In all experiments, at least two replicate samples from three animals were examined.
Concentration Measurements of Drugs
The concentrations of the antracyclines after 18 h of incubation in culture conditions were determined by reversed-phase liquid chromatography with fluorometric detection [27].
Measurement of DNA Synthesis
For measurement of DNA synthesis, 0.2 µCi of methyl-3H-thymidine was added into the medium for the last 4 h of incubation. Samples were harvested on filter discs and radioactivity was measured by liquid scintillation spectrometry.
Cell Preparations and In Situ DNA 3'-End Labeling
After culture, tubule segments were transferred in 10 µl of medium onto a microscope slide and carefully squashed under a coverslip. Slides were either examined under a phase contrast microscope to visualize apoptotic cells in living cell conditions or fixed for terminal deoxyribonucleotide-mediated dUTP nick-end labeling (TUNEL) staining [25]. Freshly isolated and squashed seminiferous tubule segments containing morphologically positive apoptotic cells in stage XII-I [24] were used as positive controls, stage VII tubule segments were used as negative controls. For fixation, slides were briefly frozen in liquid nitrogen. After the coverslip was removed, the slide was briefly dipped in ice cold ethanol, fixed for 10 min in 4% formalin, washed twice in PBS for 5 min each time, and incubated in ethanol:acetic acid (2:1) at -20°C for 5 min, washed in twice in PBS for 5 min each time, and finally dehydrated and stored at -70°C. DNA 3'-end labeling was performed with a standard method described by Billig [28]. Apoptotic cells were quantified from TUNEL-stained squash preparations and calculated per 1 mm of tubule length. Only cells with heavy staining or cells with a clear apoptotic nucleus were calculated.
Statistical Methods
Data in figures are presented as mean values ± SEM. The Mann-Whitney U-test was used for comparison of two independent sample populations. A Kruskall-Wallis analysis with a Dunns post-test was performed to obtain a multiple comparison of independent sample populations. A P value of less than 0.05 was considered statistically significant.
RESULTS
In Vitro Induction of Apoptosis in Seminiferous Tubules by Antracyclines
Only a few apoptotic cells stained with in situ end-labeling were observed in squash preparations prepared immediately after tissue collection. A rapid increase in germ cell apoptosis was seen in control samples after 2 h in culture, at stage V, and after 6 h at stage VIII-IX (Fig. 1). In stage V, apoptosis was significantly increased by doxorubicin at a concentration of 0.4 ng/ml and by idarubicin at a concentration 0.04 ng/ml after 1 h (Fig. 1). There was a significant increase in apoptosis in stage VIII-IX after 2 and 4 h of incubation with idarubicin at a concentration of 2 ng/ml and with doxorubicin at a concentration of 5 ng/ml (Fig. 1).
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Morphological Identification of Apoptotic Cells in Freshly Squashed Seminiferous Tubules
Degenerating germ cells were not always identifiable in stained and fixed squash preparations. Identification of the apoptotic cells with nuclear and cytoplasmic condensation was therefore made in unstained living squash preparations before fixation as described by Henriksén [24, 25]. In stage V segments of seminiferous tubules, type B spermatogonia showed intact morphology after 1 h of incubation under serum-free control conditions (Fig. 2A). Apoptosis was rapidly increased in the same cell type after incubation under serum-free conditions for more than 2 h. Incubation with doxorubicin (Fig. 2B) and idarubicin increased the apoptosis by time and magnitude. Sertoli cells, spermatogenic stem cells, spermatocytes, and round spermatids showed intact morphology (Fig. 2C). After 18 h of incubation with drugs, Sertoli cells, pachytene spermatocytes, and round spermatids were still showing intact morphology. There was no significant apoptosis in stage VIII-IX after culture under control conditions (Fig. 2D). Early changes of apoptosis were seen in some preleptotene and pachytene spermatocytes and in round spermatids after 2 h of culture. Similar changes were observed after doxorubicin and idarubicin treatments (Fig. 2, E and F). After 18 h of culture with drugs, most Sertoli cells, pachytene spermatocytes, and spermatids showed intact morphology.
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Effects of Antracyclines on DNA Synthesis in Cultured Seminiferous Tubules
3H-Thymidine incorporation in control conditions decreased only slightly during the fist 18 h but a significant drop was seen after 24 h of culture, especially in stage V (Fig. 3, A and B). The earliest sign of decreased 3H-thymidine incorporation was observed after 6 h of incubation of tubule segments in stage VIII-IX with the highest tested dose of idarubicin (Fig. 3D). After 18 h of incubation, both doxorubicin and idarubicin caused a dose-dependent inhibition of DNA synthesis in both tested stages (Fig. 3). A significant inhibition of 3H-thymidine incorporation by the drugs was seen also after 24 h of culture, but the decreasing DNA synthesis in control cultures made it difficult to further evaluate the kinetic response.
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Effects of Amifostine on DNA Synthesis in Cultured Spermatogenic Cells
No protective effect against the dose-dependent inhibition of 3H-thymidine incorporation by doxorubicin or idarubicin was seen by treatment with amifostine (Fig. 4). However, stage V seminiferous tubule segments that had been pretreated with amifostine showed significantly increased incorporation (P < 0.05) of 3H-thymidine compared with control cultures without amifostine (compare Fig. 3, A and B; and Fig. 4, A and B). The DNA synthesis in amifostine-pretreated control samples was also significantly higher (P < 0.05) compared with control samples after 6 h of culture. Exposure to amifostine during the entire incubation time did not affect the DNA synthesis in stages V and VIII-IX.
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Antracycline Concentration after Culture
Both doxorubicin and idarubicin degraded in the incubation medium. After incubation for 18 h, about 50% of the drugs remained intact.
DISCUSSION
These results show for the first time that it is possible to quantify dose-dependent toxicity of antracyclines at different stages of spermatogenesis. Concentrations of drugs needed to produce similar dose-dependent reduction of DNA synthesis were 10 times higher in stage VIII-IX compared with stage V. Premitotic DNA synthesis thus is more sensitive to antracycline toxicity than premeiotic DNA synthesis, supporting the earlier observations of Parvinen and Parvinen [14]. The cytostatic drugs induced apoptosis far more rapidly than inhibition of the 3H-thymidine incorporation. The first sign of toxic damage in stage V seminiferous tubule segments was induction of apoptosis in type B spermatogonia. This induction was seen earlier, and with a lower dose of drug compared with that seen in preleptotene spermatocytes and pachytene spermatocytes in stage VIII-IX. The anatomical location of the proliferating cells is one possible explanation for the observed stage-dependent toxicity of the antracyclines. The mitotically dividing spermatogonia in stage V are located at the basal membrane of the seminiferous tubules, which are easily reached by cytostatic drugs from the culture medium in vitro, or from testicular interstitial fluid in vivo [7]. In contrast, the meiotic spermatocytes in stage VIII-IX are in the process of being enveloped by Sertoli cells and are located in the intermediate compartment of seminiferous tubules [7, 29]. Although the Sertoli cell barrier in the seminiferous tubules does not restrict the penetration of cytostatic drugs into the tubular fluid [30], the close contact of the meiotic germ cells to the Sertoli cells may cause a cellular barrier to cytostatic drugs.
The lower sensitivity of stage VIII-IX to antracyclines may be a consequence of the production by Sertoli cells of survival factors for spermatocytes. Such a paracrine control of spermatocytes by Sertoli cells has previously been shown to be important because both FSH and testosterone are known to increase the survival of pachytene spermatocytes and spermatids in vitro [25, 31]. The protection has been proposed to be indirectly mediated by hormonal effects influencing Sertoli cell function [25]. In contrast, hormone treatment in vitro does not affect spermatogonial survival [31], suggesting that Sertoli cells do not have a similar protective effect to mitotic germ cells under culture conditions. Such absence of survival factors for spermatogonia may explain the poor survival of type B spermatogonia in vitro and the observed increased sensitivity to cytostatic drugs.
In agreement with earlier clinical observations [23], idarubicin was found to be a more potent cytotoxic agent than doxorubicin. Idarubicin decreased both mitotic and meiotic DNA synthesis and induced apoptosis at 2 to 10 times lower concentrations than doxorubicin. The major cell type affected by both of these drugs was type B spermatogonia in stage V. This is well in accordance with the earlier in vivo observations that the first cell types observed to be apoptotic after radiation are intermediate and type B spermatogonia [24]. In contrast, an earlier in vivo study failed to show sensitivity of type B spermatogonia to doxorubicin exposure. The main cell types affected were types A3–A4 spermatogonia and pachytene spermatocytes, when they were estimated 1 and 2 days after treatment [12]. The referred study used only 1 dose of doxorubicin and the results were calculated from histology sections of the testes, which may have affected the observations.
The present data support the earlier observations that testicular tissue rapidly degenerates when cultured in vitro [7, 31, 32]. Incubation of seminiferous tubule segments in serum-free medium without cytotoxic drugs induced apoptosis in germ cells as early as after 2 h in culture. This increase in apoptosis was 24 h later reflected by a decrease in DNA synthesis. Earlier studies have shown that human spermatocytes are prone to enter apoptosis in vitro [31]. The present data suggest that type B spermatogonia are even more sensitive to in vitro culture conditions. It must be pointed out that the culture-related apoptosis was observed in the same germ cell types in which toxic effects of cytostatic drugs were seen. The rapid kinetics of the apoptosis induced by cytostatic drugs gave a possibility of distinguishing these two phenomena. The concentration of the cytostatic drug used decreased rapidly during the culture time, which supports the relevance of our use of short-time studies for in vitro measurements of testicular toxicity.
In this investigation, we were unable to show any protective effects of amifostine in cultured spermatogenic cells when doxorubicin- or idarubicin-induced inhibition of DNA synthesis was studied. Previous in vitro studies with myocytes have shown that protection with amifostine was dependent on the timing of the treatment, and start of treatment either simultaneously or after doxorubicin exposure eliminated the cytoprotective effect [33]. In the present study there was no difference in DNA synthesis in germ cell cultures when amifostine was given before or simultaneously with cytostatic drug exposure, nor did amifostine change the stage-dependent sensitivity of the spermatogenic cells in vitro. The significant stimulation of DNA synthesis in stage V seen in seminiferous tubule segments by pretreatment with amifostine without cytostatic drug exposure suggests that amifostine is bioactive in the present in vitro model. It is interesting that the stimulated DNA synthesis was observed in the stage in which mitotically active spermatogonia were seen to apoptotically degenerate under serum-free culture. Amifostine is known to have a promotive effect on growth and survival of hematopoietic progenitor cells, which in clinical studies, has been detected as an improvement of hematological parameters in patients with myelodysplastic syndrome [34]. The present data suggest that this survival factor effect may also affect other types of differentiating cells. Whether amifostine could also be used as a promotive factor for malfunctioning spermatogonia in an infertile testis remains to be studied.
In conclusion, these data show, for the first time, that it is possible to measure and compare dose-dependent toxicity of different cytostatic drugs in defined spermatogenic stages in vitro. The technique is rapid and does not need in vivo animal experimentation, in contrast to traditional methods that employ histological morphology and fertility studies. Because of the discussed limitations of the technique, short-term cultures and detection of the earliest signs of toxic effects give the most reliable results. Amifostine lacked a protective effect against cytostatic toxicity in cultured spermatogenic cells. Amifostine may also have a direct effect on male germ cell survival in vitro. Whether this promotive effect can also be obtained in vivo remains to be studied.
ACKNOWLEDGMENTS
We thank Annika Lindberg for her skillful technical assistance.
FOOTNOTES
First decision: 15 December 1999.
1 This research was supported by the Swedish Child Cancer Fund, the Swedish Medical Research Council (PN 8282 and 11412), the Magnus Bergvall Foundation, Frimurare Barnhuset in Stockholm, the H.R.H. Crown Princess Lovisa Society of Pediatric Health Care, the Society for Child Care, the Swedish Medical Association, the Finnish Cancer Society, the Finnish Cultural Foundation, the Finnish Pediatric Research Foundation, and Sigrid Juselius Foundation. 
2 Correspondence: Kirsi Jahnukainen, Pediatric Endocrinology Unit, Astrid Lindgren Children's Hospital, Karolinska Institute, SE-171 76 Stockholm, Sweden. FAX: 46 8 51 77 51 28; kirsi.jahnukainen{at}tyks.fi
Accepted: April 5, 2000.
Received: November 8, 1999.
REFERENCES
. Endocrinology 1991; 129:1614–1620.This article has been cited by other articles:
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  M. Hou, D. Chrysis, M. Nurmio, M. Parvinen, S. Eksborg, O. Soder, and K. Jahnukainen Doxorubicin Induces Apoptosis in Germ Line Stem Cells in the Immature Rat Testis and Amifostine Cannot Protect against This Cytotoxicity Cancer Res., November 1, 2005; 65(21): 9999 - 10005. [Abstract] [Full Text] [PDF]
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 F.-p. Zhang, T. Pakarainen, F. Zhu, M. Poutanen, and I. Huhtaniemi Molecular Characterization of Postnatal Development of Testicular Steroidogenesis in Luteinizing Hormone Receptor Knockout Mice Endocrinology, March 1, 2004; 145(3): 1453 - 1463. [Abstract] [Full Text] [PDF]
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K. Jahnukainen, T. Jahnukainen, T. T. Salmi, K. Svechnikov, S. Eksborg, and O. Soder Amifostine Protects against Early but not Late Toxic Effects of Doxorubicin in Infant Rats Cancer Res., September 1, 2001; 61(17): 6423 - 6427. [Abstract] [Full Text] [PDF]
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