International Journal of Andrology, 1990, 13, pages 180- 189

Prolonged suppression of spermatogenesis by oestrogen does not preserve the seminiferous epithelium in procarbazine-treated rats I . D . MORRIS, C. W. BARDIN*, G . GUNSALUS* and

J. A . W A R D Reproduction Research Group, Department of Physiological Sciences, University of Manchester, Manchester, U.K. and *The Population Council, New York, U.S.A.

Summary We examined the hypothesis that induction of reversible testicular atrophy, subsequent to withdrawal of gonadotrophin support, would alleviate the testicular toxicity of the anti-cancer drug procarbazine. In rats, severe but reversible testicular atrophy and suppression of spermatogenesis were induced 56 days after the subcutaneous insertion of a silastic implant containing oestradiol-17P. The effect of this treatment upon the testicular toxicity of four weekly doses of procarbazine (200 mg kg-') was examined 56 days after the termination of procarbazine/oestrogen treatment. At this time the testicular endocrine and spermatogenic functions were close to normal in rats which has received only oestradiol-17b. Procarbazine produced severe testicular atrophy which was associated with azoospermia and destruction of the germinal epithelium. Serum L H and FSH concentrations were raised and were associated with low serum concentrations of both testosterone and androgen-binding protein. The combination of procarbazine with the oestrogen treatment did not change any of the testicular toxicity and in some cases it appeared to be exacerbated. In contrast to these experiments other studies have indicated that the testis can be protected if spermatogenesis is reversibly suppressed by other agents which are also active via the pituitary endocrine system. The data would therefore suggest that protection is achieved either by some testicular change other than withdraw1 of pituitary gonadotrophin support or that oestradiol-17P has additional activity which is permissive for the development of the testicular toxicity of procarbazine. Keywords: androgen-binding protein, cancer, cytotoxin, infertility, oestrogen, procarbazine, spermatogenesis, testis.

Introduction Infertility in men and women after treatment for cancer by cytotoxic drugs is a well documented adverse effect (Schilsky et al., 1980; Shalet, 1983; Gradishar & Schilsky, 1988). The impressive survival rate amongst men treated for malignancies such as Hodgkins disease and testicular cancer has focused attention upon treatment Correspondence: Dr I. D. Morris, Reproduction Research Group, Department of Physiological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.

180

Oestrogen and cytotoxic effects on testes 181 strategies designed to alleviate the adverse effects and improve the quality of life of the survivors. Destruction of the rapidly proliferating germinal epithelium by cancer treatment is related to the use of drugs and/or radiation designed to kill the equally rapidly proliferating tumour cells. Glode et al. (1981) proposed that if germ cell division in the seminiferous epithelium could be reduced selectively, by suppression of pituitary gonadotrophin secretion, the damaging testicular effects of cytotoxic treatment would be attenuated. The present study was designed to test this hypothesis in a rodent model. The animal experiment described by Glode et al. (1981) in the mouse, suggested that pretreatment with a G n R H agonist could prevent the testicular toxicity of cyclophosphamide. However, the suitability of this model has been questioned (Morris & Shalet, 1989). A recent study was unable to demonstrate a protective action of a G n R H agonist against the testicular toxicity of cyclophosphamide (Da Cunha et al., 1987). It is important to establish a mechanism on which to base a hypothesis for protective adjuvant therapy. In our study, oestrogen was selected as the adjuvant treatment because its ability to inhibit spermatogenesis is documented extensively (Steinberger & Duckett, 1967; Keeney et al., 1988; Morris et al., 1988). Oestrogen treatment was begun 56 days before the cytotoxic drug to ensure a stable degree of inhibition and to avoid dynamic interactions between adjuvant and the anti-tumour drug. As cyclophosphamide is inactive upon stem cells of the rat testis (Trasler et al., 1988) another anti-tumour drug, procarbazine, was chosen. Procarbazine can produce chronic infertility associated with destruction of the germinal epithelium in man and rodents (Hilscher & Reichelt, 1968; Schilsky et al., 1980; Shalet, 1983; Hodel et al., 1984; Gradishar & Schilsky, 1988; Ward et al., 1989). Materials and methods

Animals Outbred Wistar-derived albino rats (250-300 g) (ICI Pharmaceuticals plc, Macclesfield, U.K.) were used. They were assigned randomly to treatment groups and were housed four per cage under standard laboratory conditions and food and water were provided ad libitum. Drug treatment Oestradiol-l7(J (Sigma Chemical Co., Poole, U.K.) was administered in subcutaneous silastic implants (Dow Corning Corporation, Michigan, U.S.A.) 20 mm in length x 4.88 mm (external diameter) x 2.64 mm (internal diameter). Control implants were empty. Procarbazine HCI (Roche Products Ltd, Welwyn Garden City, U.K.) was diluted in distilled water to a concentration of 116 mg mi-' equivalent to 100 mg procarbazine ml-'. A weekly intraperitoneal injection was given of either procarbazine (200 mg kg-') or water (2 ml kg-' to controls) for 4 consecutive weeks. Experimental design Rats were divided into two groups which received either oestradiol or placebo subcutaneous implants by surgical implantation during ether anaesthesia, and were

182 I . D. Morris et al. then housed for 56 days to ensure that testicular atrophy was complete. Oestradiol z,?d placebo implanted rats (17 = 6 ) were killed at this time to indicate the level of testicular atrophy achieved. The remaining oestradiol-treated and control groups ( n = 11- 12) were then further subdivided into two groups ( n = 5-6) which received four weekly injections of either procarbazine or water. Seven days after the final injection the implants were removed under ether anaesthesia. Recovery from the effects of treatment was then allowed to take place for 56 days, a time which is slightly greater than the overall process of spermatogenesis. Therefore, any changes in the seminiferous epithelium observed at this time should reflect persistent spermatogonial damage. Rats were then killed by concussion and decapitation, blood was collected, allowed to clot, and serum stored at -20°C. Both testes, epididymides, seminal vesicles and the ventral prostate gland were removed and weighed. The left testis was fixed in Bouin's fluid and processed for histology. The right testis and both epididymides were stored at -20°C. When required, homogenates of testes or epididymides were prepared using 0.01 M Tris-buffer (pH 7.4) containing 10% (w/v) glycerine.

Radioimmunoassays Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in serum by double-antibody radioimmunoassays (RIAs) using reagents kindly provided by the NIADDK, Bethesda, U.S.A. Details of the protocol have been reported previously (Bowman et al., 1981). The antisera were LH-S-5, and FSH-S-11, the reference preparations were LH-RP-2, and FSH-1-6 and the hormones for iodination, LH-1-5 and FSH-1-6. All samples in this and the other assays were assayed together to avoid inter-assay variation. The intra-assay coefficient of variation was 11% for LH and 15% for FSH, whilst assay sensitivity was 2.5 ng ml-' for LH and 30 pg ml-' for FSH. Androgen-binding protein (ABP) was measured in serum and supernatants from testicular and epididymal homogenates, using a RIA described elsewhere (Gunsalus et al., 1986). ABP concentrations have been expressed as pl equivalents (pl eq) of an epididymal extract (GMB-E-1). Assay of the ABP level in GMB-E-1 using a steady-state polyacrylamide gel assay indicated that 1 p1 eq = 9.8 k 0.9 fmol of binding sites (Gunsalus et al., 1986). The intra-assay coefficient of variation was 10%. Testosterone was measured in serum and testicular homogenates following extraction with petroleum ether:diethyl ether (1 :1 viv), using a single-antibody RIA method (Furr ef a f . , 1987); the antiserum cross-reacted significantly with 5-ac-dihydrotestosterone (57.1%), 19-nortestosterone (11.4%) and 5-a-androstan3fl,17@-diol(10%) but showed neglible ( t o . 1%) cross-reaction with oestrogens, progestins and corticosteroids. The sensitivity of the assay was 16 pg ml-' and the intra-assay coefficient of variation was 7.9%. Light microscopy Testes were fixed in Bouin's fluid and embedded in paraffin wax. Cross sections (4 pm) across the longitudinal plane of the testis were cut from the centre of the testis, stained with haematoxylin and eosin and examined for qualitative changes in morphology.

Oestrogen and cytotoxic effects on testes

183

Stem cell survival index Quantitative assessment of the chronic testicular damage induced by procarbazine was performed using a modification of a stem cell survival method described by Withers et al. (1974). Counts were made of the total number of round tubules per section (approximately 400 tubules for each of 7-8 animalsigroup). The proportion of tubules with evidence of active spermatogenesis was calculated as a percentage of the total number of round tubules per section. Active spermatogenesis was considered to be present in a tubule if at least three spermatogonia were observed in close proximity to one another and to the basal lamina of the tubule. There is a good correlation between the stem cell survival index and the subsequent restoration of spermatogenesis (Meistrich, 1982). Sperm counts The method used for counts of testis and epididymal sperm content have been described previously (Meistrich et al., 1979). Briefly, the decapsulated right testis and both epididymides were homogenized on 0.01 M Tris buffer (pH 7.4) containing 10% (w/v) glycerine. The homogenates were diluted 1:25 hom0genate:buffer) in 1 % Triton X , incubated in an ice bath for 30 min and sperm counted in a Neubauer improved haemocytometer. Statistical analyses The effect of oestrogen was analysed by either Student’s f-test or the MannWhitney U-test. All other data except those from the stem cell survival method were analysed by a one-way analysis of variance utilizing a General Linear Models procedure. Post hoc Scheffe tests were performed to identify sources of significant differences between treatment groups. Hormone data were log-transformed before analysis because of non-homogeneity of variance and/or non-normal distribution. Data from the stem cell survival assessment were analysed by Kruskal-Wallis one-way analysis of variance followed by sequential Mann- Whitney U-tests to determine significant differences among the treatment groups. Results

Effect of oestradiol or procarbazine alone The testes were severely atrophied 56 days after the implantation of a silastic capsule containing oestradiol-17fi (Table 1). Rats receiving oestrogen did not gain weight as quickly as did control animals, which were significantly heavier at this time. Both testicular and epididymal weights were reduced and sperm were absent in homogenates of these tissues as well as in their histological sections (not shown). The androgen-dependent prostate gland and seminal vesicles were markedly smaller in oestrogen-treated rats. It was at this time that treatment with procarbazine or injection vehicle began. The effect of oestrogen pretreatment upon the testicular toxicity of procarbazine was examined 56 days after the end of treatment (Tables 2 and 3). Oestrogen treatment had some persistent effects. The testes, ventral prostate and seminal vesicles were smaller, changes which were similar but not significant if the weights

184 1. D. Morris et al. Table 1. The effect of a silastic subcutaneous implant of oestradiol-I7f!1upon body and tissue weights

(g) and sperm content determined 56 days after insertion Weight (mg per g body weight)

Weight (g) Parameter

Control

Oestradiol

Body weight Testis Epididymis Ventral prostate Seminal vesicle

508 f 21 1.76 f 0.10 0.57 f 0.02 0.54 i 0.07 1.50 f 0.14

377 0.31 0.09 0.05 0.11

Testis Epididymis

Tissue sperm content X lo6 290 f 20 O* 320 f 40 0’

Control

f 11** f 0.02** f 0.001** f 0.01** f 0.01**

-

3.47 f 0.08 1.12 f 0.03 1.06 f 0.24 2.93 f 0.16

Oestradiol -

0.82 f 0.06** 0.25 f 0.01** 0.13 f 0.05** 0.31 f 0.01**

Values represent means f SEM ( a = 5-6). *P

Prolonged suppression of spermatogenesis by oestrogen does not preserve the seminiferous epithelium in procarbazine-treated rats.

We examined the hypothesis that induction of reversible testicular atrophy, subsequent to withdrawal of gonadotrophin support, would alleviate the tes...
647KB Sizes 0 Downloads 0 Views