Effect of streptozotocin-induced hyperglycaemia on androgen-binding protein in rat testis and epididymis

Diabetologia, Apr 1984

Summary In adult male rats treated with streptozotocin 6 weeks before the experiments, androgen-binding protein concentration was increased in testicular tissue by 33% (p< 0.01) and reduced in epididymal tissue by 25% (p<0.005) in animals exhibiting severe hyperglycaemia as compared with animals remaining in normoglycaemia or moderate hyperglycaemia. Androgen-binding protein content was diminished in epididymal tissue by 40% (p<0.0005) but not changed in testicular tissue. If related to constant body weight, the sum of testicular and epididymal androgen-binding protein was identical in both normo- and hyperglycaemic animals. This disturbance in androgen-binding protein distribution may be the consequence of altered testicular secretion or impaired transport of androgen-binding protein from testes to epididymides.

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Effect of streptozotocin-induced hyperglycaemia on androgen-binding protein in rat testis and epididymis

Diabetologia Effect of streptozotocin-induced hyperglycaemia on androgen-binding protein in rat testis and epididymis N. KiJhn-Velten 0 1 R. Schermer 0 1 W. Staib 0 1 0 streptozotocin-diabetes , Sertoli cells 1 Institut fiir Physiologische Chemic II, University of Dtisseldorf, FRG Summary. In adult male rats treated with streptozotocin testicular and epididymal androgen-binding protein was iden6weeks before the experiments, androgen-binding protein concentration was increased in testicular tissue by 33% ( p < tical in both norrno- and hyperglycaemic animals. This disturbance in androgen-binding protein distribution may be the 0.01) and reduced in epididymal tissue by 25% (p < 0.005) in consequence of altered testicular secretion or impaired transanimals exhibiting severe hyperglycaemia as compared with port of androgen-binding protein from testes to epididymides. Androgen-binding protein; testis; epididymis - 9 Springer-Verlag 1984 Numerous studies have demonstrated impairment of fertility [ 1 ] and testicular endocrine function [ 1-4 ] to be late complications of streptozotocin-treated hypoinsulinaemic, hyperglycaemic male rats. In addition to reports on defective spermatogenesis [ 5-6 ] and indications o f disturbances of pituitary gonadotropin secretion [ 1-3, 5-7 ], dysfunction o f the Leydig cells resulting in diminished testosterone formation in these diabetic animals seems to be the most extensively investigated problem in this field [ 1-4 ]. In contrast, less attention has been drawn to Sertoli cell function in diabetic animals. Although a specific androgen-binding protein (ABP) was shown several years ago to be a marker of Sertoli cell function [ 8-13 ], ABP measurements in diabetic rats have been reported in only one study [2]. A paradoxical increase of ABP content (expressed as lxl-equivalent of epididymal cytosol standard) of the testes (the site o f production) but no change of ABP content of the epididymis (a possible site of action) has been found in rats 4 weeks after streptozotocin injection [ 2 ]. The aims of the present study on streptozotocintreated male rats were to re-evaluate those results, to distinguish between ABP concentrations and ABP content of testes and epididymides by consideration of changes in organ weights and to make correlations between testicular or epididymal ABP and blood glucose levels in order to prove possible diabetes-induced changes in testicular ABP secretion. Materials and methods Eighteen male Han: Wistar rats, weighing 214 + 5 g (mean + SEM) at the beginning of the study, received 65 mg streptozotocin (2-desoxy2-(3-methyl-3-nitrosurea)-la-glucopyranose; Calbiochem, Giel3en, FRG)/kg body weight, i.p. (experimental group). One rat died during week 3. Five animals (205 ___5 g initial body weight) were injected with citrate buffer only and served as a control group. All animals were allowed food (Altromin 1314; Altromin, Lage, FRG) and tap water ad libitum. Post-prandial blood glucose levels were examined routinely (seven times during the 6-week period of the experiment) using the Eyetone reflectometer system (Dextrostix; Ames-Miles, Frankfurt/Main, FRG). Urinary excretion of glucose and ketone bodies was measured with the Keto-Diabur 5000 test (Boehringer, Mannheim, FRG). The experimental group was divided into two subgroups. Group 1 consisted of nine animals exhibiting normo- or moderate hyperglycaemia (mean blood glucose levels <15 mmol/1; glycosuria> 3% in one, ketonuria in none of the animals). Group 2 consisted of eight animals exhibiting severe hyperglycaemia (mean blood glucose > 15 mmol/1; glycosuria> 3% in all, ketonuria + or + + in five of the animals). Animals were sacrificed 6 weeks after streptozotocin administration. Both epididymides and the left testis were removed for ABP measurement, decapsulated, trimmed of fat and weighed. Epididymides were minced with scissors and homogenized in six volumes, and testes in three volumes of Tris-HCl (50 mmol/1), Na2 EDTA (1 mmol/1), KC1 (80 mmol/1), 3-mercapto-l,2-propandiol (t2 mmol/ 1), 10% glycerol (v/v) pH 7.4 (TEMG-buffer). All reagents were of analytical grade and purchased either from Merck, Darmstadt, FRG or from Sigma Chemic, Mtinchen, FRG. A glass-glass Duall homogenizer was used (Kontes Scientific Glassware, Vineland, NJ, USA). Epididymis and testis homogenates were centrifuged at 150000 g for 45 min, the lipid layer discharged and the cytosol mixed with the 4000g pellet of an equal volume of charcoal (5 g/l), dextran T-70 N. Kfihn-Velten et al.: Androgen-binding protein in diabetic rats , 9 20 ~ 1,8 < 0 --.,' , , . . . . , , , , 0.6 0.8 tO 12 1.4 Weight of 2 epididymides [g] 40 6 ,~o +lbO +~ +26o Body weight increose [g/6 weeks] 16 2b Neon blood glucose [mrnol/I] 3b 1'0 2'0 Meon blood glucose [mmo[/I ] Jo < O"0 10 20 Neon blood glucose Immol/I] 30 o 1'o 2'0 Mean blood glucose [mmbl/I] 36 lb ~o Neon blood glucose [ m m o l / I ] 3b (0.5 g/l) (Pharmacia Fine Chemicals, Freiburg, FRG) in TEMG-buffer. After continuous shaking for 2 h at 2 ~ (in order to remove endogenous cytosolic steroids), the suspensions were centrifuged at 4 000 g for 10 min. Aliquots (400 gl) of the supernatants were incubated for 15 h at 2 ~ with a mixture of 50 ~tl glycerol containing 2 pmol [1,2-3H(N)]-dihydrotestosterone (DHT; 5a-androstan-17fl-ol-3-one; spec. act. 1.48 T B q / m m o l ; New England Nuclear, Dreieich, FRG) to achieve final 3H-DHT concentrations of 4 nmol/1. ABP concentrations in these cytosols equilibrated with 3H-DHT were determined by steady-state polyacrylamide gel electrophoresis as described elsewhere [ 9, 14, 15 ] with the following modifications: 100 gl of the 3H-DHT-labelled cytosol were layered on 7% acrylamide rod gels (85 m m x 5 mm) containing glycerol (2.5%) and 3H-DHT 4 nmol/1. Electrophoresis was run for 4 h at 3 mA/gel. Following electrophoresis, gels were frozen in n-hexane on dry ice and then thawing gels were sliced into 1-mm-thick pieces. Slices were counted for radioactivity 48 h after addition of scintillation fluid (Quickszint 212; Zinsser, Frankfurt/Main, FRG) with an efficiency of 32% in a TriCarb model 2650 spectrometer (Packard, Frankfurt/Main, FRG). The ABP content was normally calculated from five fractions (Rf=0.46) with elevated radioactivity (in comparison to the basal activity) and could be clearly distinguished [ 8 ] from a small peak representing se Results rum albumin (Rt = 0.67). A third peak of radioactivity reported to represent androgen receptor [ 15 ] could not be detected. The sensitivity of the method under these conditions is 15 fmol ABP/gel assuming one binding site/molecule. Correlation of ABP with control parameters, e.g. mean blood glucose levels, was proved by linear regression analysis with the least squares method. Differences between groups were analyzed using Student's t-test. Body weight increase (Fig. 1 A), weight ofepididymides (Fig. 1 B) as well as testicular weight (Fig. 1 C) showed negative correlations with mean blood glucose levels. Body weight increase and epididymal weight were reduced if group 2 was compared to group 1, but no difference existed between testicular weights in both groups (Table 1). The control group and group I did not differ significantly with regard to any of the variables tested. A distinct reduction of both ABP concentration (Fig. 1 D, Table 1) and ABP content (Fig. 1 E, Table 1) in epididymides occurred with increasing mean blood glucose levels irrespective of the method of testing significant differences (regression analysis or t-test). Due to the positive correlation of epididymal ABP content with the epididymidal weight (Fig. 1 F) the drop of ABP content in epididymides from hyperglycaemic rats resulted from both reduced ABP concentration and reduced epididymal weight. A positive correlation of epididymal ABP content with body weight increase (Fig. 1 G) was obtained. Nevertheless, epididymal ABP content was still significantly reduced in hyperglycaemic animals if related to constant body weight (Fig. 1 H, Table 1). In contrast to the epididymis, ABP concentrations in the testes were higher in severely hyperglycaemic than in normo- or moderate hyperglycaemic rats (Fig.l I, Table1). Due to slightly reduced testicular weight in hyperglycaemic animals (Fig. 1 C), the total ABP content of testicular tissue was not changed (Fig. 1 J, Table 1). However, testicular ABP content was increased in hyperglycaemic animals if related to constant body weight (Fig. 1 K, Table 1). The sum of ABP contents of both testes and epididymides was not influenced by hyperglycaemia (Fig. 1 L, Table 1). Therefore streptozotocin-induced hyperglycaemia resulted in a shift of ABP distribution from the epididymides back to the testes but not in a real increase or decrease of total ABE Discussion In most of the previous studies on reproductive and especially endocrine testicular function of streptozotocindiabetic male rats, animals not exhibiting glycosuria or other criteria of diabetes mellitus were excluded from the experiments and a control group was established N. Kiihn-Velten et al.: Androgen-binding protein in diabetic rats which received buffer injections only [ 1, 2, 4, 6 ]. The protocol chosen in the present study, however, excluded possible side-effects of the drug. With all streptozotocin-injected animals included in the study irrespective of the outcome of the treatment, it was possible to look into the dependence of reproductive function parameters on the degree of hyperglycaemia. On the other hand, the direct comparison of the untreated control group with group 1 one of the experimental group allows the conclusion that streptozotocin itself does not affect testicular or epididymal ABP (Table 1). With the protocol chosen for the present study, the effectiveness of diabetogenesis was comparable to that achieved previously by other authors [ 1 ]. A marked reduction of epididymal weight and only a slight reduction of testicular weight occurred in streptozotocin-diabetic animals (Fig.l). Under slightly different conditions, a 20% loss [ 1, 5, 6 ] or no change [ 2, 4, 7 ] of testicular weight has been reported, whereas epididyreal weight has been shown to be reduced by 50% [ 1 ] or by 15% [ 2 ]. Whether this loss in epididymal weight can be attributed to decreased serum testosterone levels in the diabetic rats remains questionable [ 1, 4, 7 ]. Alternatively, a slower increase of epididymal weight during development of diabetic rats may be the simple consequence of the slower increase in body weight. The reason why epididymal ABP concentration, as well as epididymal ABP content, were decreased in diabetic in comparison with normoglycaemic adult rats is at first unclear. Two hypotheses may be discussed: (1) is it possible that decreased epididymal ABP levels are due to decreased serum FSH levels? Serum FSH concentrations have been reported to be decreased in streptozotocin-diabetic animals [ 2 ], but this finding has not been confirmed by other authors [ 3, 7 ]. FSH has been proposed to enhance both testicular and epididymal ABP concentrations [ 8, 9, 11 ] as well as the ABP production by cultured Sertoli cells from immature rats [12]. Therefore possibly reduced circulating FSH concentrations in streptozotocin-diabetic male rats may result in a diminution of epididymal ABP but cannot explain increased testicular ABP concentrations. (2) Is it possible that decreased ABP levels are due to decreased serum testosterone levels? A reduction in circulating testosterone concentration has been observed in diabetic animals [ 1-4, 7 ]. Testosterone administration to normal rats results in elevated testicular and epididymal ABP levels either directly [ 10 ] or by stabilizing ABP activity [ 11 ]. But again, possibly reduced serum testosterone concentrations cannot explain the different development of testicular and epididymal ABP, so much the more as ABP production by Sertoli cells is unlikely to be affected in streptozotocin-hyperglycaemic rats (Fig. 1 L). Therefore, a reduction of Sertoli cell ABP secretion or impaired ABP transport from the testes to the epididymides seem to be more probable explanations for the results presented in this study. This assumption N. Kiihn-Velten et al.: Androgen-bindingprotein in diabetic rats is supported by the observation that hypophysectomy, as well as administration of anti-LH serum, affects fluid secretion by the Sertoli cells and causes loss of ABP in the caput epididymidis in the long term [161.A deficiency of LH might also be responsible for the impaired testicular ABP secretion in diabetes mellitus, although it cannot be excluded that testicular ABP secretion is affected via hormone-independent mechanisms. In conclusion, it is suggested that streptozotocin-induced hyperglycaemia does not cause impaired ABP production by the Sertoli cells, but affects ABP secretion by, or ABP transport from, the testis, the result being a relative elevation of testicular and a relative diminution of epididymal ABP in diabetic animals. Acknowledgement.Part of this study was supported financiallyby the Deutsche Forschungsgemeinschaft (SFB 113, C-3 and F-13). 1. Paz G , Homonnai ZT , Drasnin N , Sofer A , Kaplan R , Kraicer PF ( 1978 ) Fertility of the streptozotocin-diabetic male rat . Andrologia 10 : 127 - 136 2. Murray FT , Orth J , Gunsalus G , Weisz J , Li JB , Jefferson LS , Musto NA , Bardin CW ( 1981 ) The pituitary-testicular axis in the streptozotocin diabetic male rat: evidence for gonadotroph, Sertoli ceil and Leydig cell dysfunction . Int J Androl 4 : 265 - 280 3. Prrez Diaz J , Benitez A , Fern/mdez Galaz C ( 1982 ) Effect of streptozotocin diabetes on the pituitary-testicular axis in the rat . Horm Metab Res 14 : 479 - 482 4. Kfihn-Velten N , Waldenburger D , Staib W ( 1982 ) Evaluation of steroid biosynthetic lesions in isolated Leydig cells from the testes of streptozotocin-diabetic rats . Diabetologia 23 : 529 - 533 5. Oksanen A ( 1975 ) Testicular lesions of streptozotocin diabetic rats . Horm Res 6 : 138 - 144 6. Rossi GL , Aeschlimann M ( 1982 ) Morphometric studies of pituitary glands and testes in rats with streptozotocin-induced diabetes . Andrologia 14 : 532 - 542 7. Howland BE , Zebrowski EJ ( 1976 ) Some effects of experimentally-induced diabetes on pituitary-testicular relationships in rats . Horm Metab Res 8 : 465 - 469 8. Sanborn BM , Elkington JSH , Chowdhury M , Tcholakian RK , Steinberger E ( 1975 ) Hormonal influences on the level of testicular androgen binding activity: effect of FSH following hypophysectomy . Endocrinology96 : 304 - 312 9. Hagen~isL, Ritzrn EM , P16en L , Hansson V , French FS , Nayfeh SN ( 1975 ) Sertoli cell origin of testicular androgen-bindingprotein ABp . Mol Cell Endocrinol 2 : 339 - 350 10. Tindall DJ , Means AR ( 1976 ) Concerning the hormonal regulation of androgen binding protein in rat testis . Endocrinology 99 : 809 - 818 11. Tindall DJ , Mena CR , Means AR ( 1978 ) Hormonal regulation of androgen-binding protein in hypophysectomized rats . Endocrinology 103 : 589 - 594 12. Louis GB , Fritz IB ( 1979 ) Follicle-stimulating hormone and testosterone independently increase the production of androgenbinding protein by Sertoli cells in culture . Endocrinology 104 : 454 - 461 13. Gunsalus GL , Larrea F , Musto NA , Becker RR , Mather JP , Bardin CW ( 1981 ) Androgen binding protein as a marker for Sertoli cell function . J Steroid Biochem 15 : 99 - 106 14. Ritzrn EM , French FS , Weddington SC , Nayfeh SN ( 1974 ) Steroid binding in polyacrylamide gels: quantitation at steady state conditions . J Biol Chem 249 : 6597 - 6604 15. Kirchhoff J , Softie M , Rousseau GG ( 1979 ) Differences in the steroid-binding site specificities of rat prostate androgen receptor and epididymal androgen-binding protein ABp . J Steroid Biochem 10 : 487 - 497 16. Dym M , Madhwaraj HG ( 1977 ) Response of adult rat Sertoli cells and Leydig cellsto depletion of luteinizing hormone and testosterone . Biol Reprod 17 : 676 - 696 Received: 17June 1983 and in revised form: 6February 1984

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N. Kühn-Velten, R. Schermer, W. Staib. Effect of streptozotocin-induced hyperglycaemia on androgen-binding protein in rat testis and epididymis, Diabetologia, 1984, 300-303, DOI: 10.1007/BF00283654