0013-7227/91/1284-1857$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 4 Printed in U.S.A.
Growth Hormone and Insulin-Like Growth Factor I Treatment Increase Testicular Luteinizing Hormone Receptors and Steroidogenic Responsiveness of Growth Hormone Deficient Dwarf Mice* P. G. CHATELAIN, P. SANCHEZ, AND J. M. SAEZ INSERM U307, Hopital Debrousse, 69322 Lyon Cedex 05, France
ABSTRACT. To test the hypothesis that insulin-like growth factor (IGF-I) is required for the in vivo development of testicular Leydig cell function, either recombinant human GH [(hGH)(1.5 /ig/g BW) or recombinant IGF-I (1 ^g/g BW) was injected three times daily into immature Snell dwarf mice (dw/ dw) and into phenotypically normal control (Dw/—) for 7 days. In dw/dw mice hGH enhanced significantly body, liver, kidney, and testicular weight. In addition, hGH increased testicular LH receptors and the acute steroidogenic response to human CG, but there was no significant effect on basal plasma testosterone or plasma LH levels. The effects of IGF-I in body and kidney
T
he role of GH in testicular function has been suggested by the fact that in both humans (1-5) and experimental animals (6, 7), isolated GH deficiency and/ or a GH resistant state are associated with delayed puberty and a poor response to exogenous human CG (hCG). At least in humans, delayed puberty in isolated GH deficiency is very often improved following treatment with GH (1, 2, 5). These data suggest therefore that GH could be involved directly or indirectly in the development and maintenance of the gonadotropin responsiveness of Leydig cells. However, since many of the actions of GH in vivo are thought to be mediated by insulin-like growth factor I (IGF-I) (8), it appears likely that the action of GH on testicular function might be modulated by IGF-I. Immunoreactive IGF-I-like material (9) and IGF-I Received November 19, 1990. Address correspondence and requests for reprints to: Jose M. Saez, INSERM U307, Hopital Debrousse, 29 Rue Soeur Bouvier, 69322 Lyon Cedex 05, France. * This work was supported by research grants from Institut National de la Sante et de la Recherche Medicale, Nordisk Insulin Laboratorium, and Fondation pour la Recherche Medicale Francaise. The data of this paper were presented in part at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990 (Abstract 1343) and at the Vlth European Testis Workshop, Marieham, Finland, 1990.
weight were less pronounced than those produced by hGH, but its effects on testicular weight and LH receptors, as well as on the acute steroidogenic response to human CG, were similar to that observed after hGH treatment. In Dw/- mice hGH had no effect on either body or organ weight or on testicular function, despite the fact that it induced a significant increase in plasma IGF-I levels. These results indicated that IGF-I is able to induce the maturation of Leydig cell function and that the effects of hGH on the testis are probably mediated by IGF-I. They also suggest that the delayed puberty associated with GH deficiency or resistance is most likely related to an IGF-I deficiency. {Endocrinology 128: 1857-1862,1991)
mRNA (10) have been identified in the rat testis. The relevance of IGF-I to the physiology of testicular cells stems from observations indicating that both Sertoli (11, 12) and Leydig (13-15) cells contain specific IGF type I receptors and that this peptide greatly enhances the response of both cell types to gonadotropins. In addition, it has been shown that both Sertoli and Leydig cells from rat (16, 17) and pig (18, 19) secrete IGF-I and that this secretion is regulated by gonadotropins, but not by GH. The aim of the present study was to determine if IGFI plays a role in the in vivo development of the steroidogenic responsiveness of Leydig cells to gonadotropins. To address this question, we have studied the effects of recombinant human GH (hGH) and recombinant human IGF-I administration on several parameters, including testicular function, of immature Snell dwarf mice (dw/ dw). These mice are PRL and GH deficient and exhibit delayed testicular maturation (20-22).
Materials and Methods Snell male dwarfs (dw/dw) and phenotypically normal control mice (Dw/-), 6 to 7 weeks old, were purchased from CSEAL-CNRS (Orleans, France). Animals were maintained in standard condition for at least 3 days after arrival before the experiments were started. Recombinant hGH and recombinant IGF-I were a generous gift of Dr. L. Fryklund and Dr. R. 1857
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EFFECTS OF IGF-I IN TESTICULAR FUNCTION
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Gunnarson (Kabi, Stockholm, Sweden). Each hormone was dissolved in phosphate-saline buffer (pH 7.4) containing 0.1% bovine serum albumin. The hGH and IGF-I solutions were prepared at the beginning of the experiment and stored in small aliquots at —20 C in the dark. Animals were injected intramuscularly three times daily (0800 h, 1600 h, and 2400 h) for 7 days with either saline or hGH (1.5 ^g/g BW/injection) or IGF-I (1 Mg/g BW/injection; dw/dw mice only). The doses of both hormones were calculated for the body weight at the beginning of the experiment. Body weight was measured daily, just before the second injection. At the end of the experiment 0.1 ml saline or 10 IU (Dw/-) or 5 IU (dw/dw) hCG was given im and the animals were killed 2 h later (4-5 h after the last injection of hGH or IGF-I). Animals were bled under light ether anesthesia by cardiac puncture with a heparinized syringe. The liver, kidneys, and testes were removed, weighed, and stored at -70 C. Because the number of dw/dw mice of the same age available at the same time was limited, three independent experiments in January, April, and September were performed. Each time the animals were divided into two (Dw/-) (saline or hGH) or three (dw/dw) (saline, hGH, or IGF-I) groups of five to seven mice each. Because the body weights at the beginning of the experiments [Dw/- = 22.6 ± 0.2 g (n = 44) and dw/dw = 5.7 ± 0.08 g (n = 50)] were similar, and because the results from one experiment to another were also similar, the results from the three experiments performed at different times were pooled and analyzed as a single experiment. hCG binding assay hCG (CR 121; 13,450 IU/mg) was iodinated with iodogen (23) (SA 60 to 80 MCi/Mg). Testes from acute hCG-treated mice were homogenized in Tris-HCl buffer (50 mM, pH 7.4) containing 1 mM CaCl2, 1 mM MgCl2, and 250 mM sucrose and centrifuged at 600 X g for 10 min. The supernatant was decanted and centrifuged at 20,000 X g for 30 min. The supernatant was removed and the pellet was washed once with glycine buffer (50 mM glycine, 150 mM NaCl, pH 3) for 4 min at 4 C and twice with Tris-HCl buffer to remove the bound hCG (24). Using this procedure it was found in preliminary experiments that the number of hCG binding sites per testis, as well as the affinity of hCG for its receptors in Dw/- treated with 50 IU hCG 2 h before killing, was similar (97 ± 2%, n = 4) to that of untreated Dw/-. These results documented that even at this high dose of hCG there was no down-regulation of receptors during the 2 h the mice were exposed to hCG in vivo. All binding studies were performed in triplicate at 37 C for 4 h, using two concentrations of labeled hCG 9.10"10 and 9.10"9 M, which are close to the concentrations required to produce halfmaximal and maximal saturation of the binding sites. For each concentration, nonspecific binding was measured in the presence of a 500-fold excess of unlabeled hormone. Specific binding was computed by subtracting nonspecific from total binding. In preliminary studies the binding affinity of hCG for its receptor was determined in membranes prepared from Dw/-, dw/dw, and hGH-treated dw/dw. Scatchard analysis of the binding data indicated a single class of binding sites with a dissociation constant (KD) of 2.1 ± 0.2 X 10"9 M (n = 3). Moreover, the number of binding sites using this method was
Endo'1991 Vol 128 • No 4
similar to that found using a single saturating concentration of 125 I-hCG. Other methods Plasma IGF-I was measured by RIA (25) after acid-ethanol extraction of serum, using a nonequilibrium technique and a pool of sera from adult human males as standard, which was also extracted by acid-ethanol (26). Plasma testosterone was measured by the method described previously (27). LH in plasma was measured by RIA using the NIDDK-rat LH kit, which has been validated for measuring mouse LH (20). The results are expressed in terms of rat RP-1 standard. Because the amounts of plasma available in dwarf mice are small, a plasma pool from three animals was used for LH determinations.
Results Treatment with hGH had no effect on Dw/- body weight gain (Fig. 1). In contrast, this hormone produced a marked increase in the weight of dw/dw, which was significant after two days of treatment. IGF-I also enhanced body weight gain, but at a lower rate than hGH (Fig. 1). Table 1 summarizes the effect of hGH and IGFI on body weight gain and the weight of liver, kidney, 2,5 "*• Dw/- Control ••• Dw/hGII
2,0
1,5"
1,0"
0,5
0,0 0
1
2
3
5
6
7
8
Days of Treatment 2,5
2,0
1,5-
1,0"
0,5"
0,0 6
8
Days ol Treatment
FIG. 1. Body weight gain of normal (Dw/-) (top) and dwarf (dw/dw) (bottom) mice treated three times daily with saline (D), recombinant hGH (•) (1.5 Mg/g BW), or recombinant IGF-I (•) (1 Mg/g BW).
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TABLE 1. Effects of hGH and IGF-I on body and organ weight in normal (Dw/—) and dwarf (dw/dw) mice (mean ± SEM)
Dw/Saline (n = 22) BW(g) Liver (g) Kidneys (g) Testes (g)
24.85 ± 1.39 ± 0.405 ± 0.227 ±
0.18 0.03 0.011 0.006
dw/dw GH(n = 22) 24.95 1.39 0.411 0.229
± ± ± ±
0.41 0.03 0.010 0.006
Saline (n = 20) 6.12 ± 0.291 ± 0.081 ± 0.034 ±
0.08 0.012 0.002 0.003
GH (n = 15) 7.79 ± 0.441 ± 0.117 ± 0.057 ±
0.14° 0.012° 0.004" 0.004"
IGF-I (n = 15) 6.96 0.318 0.092 0.052
± ± ± ±
0.10"'6 0.0226 0.002"* 0.008"
Normal (Dw/—) and dwarf (dw/dw) mice were treated three times daily with saline, recombinant hGH (1.5 jtg/g of BW), or recombinant IGFl^g/gof BW) for 7 days. ° P < 0.001 compared to control. * P < 0.001 compared to hGH-treated group.
and testis after 7 days of treatment. In Dw/-, hGH had no significant effect on any of these parameters, despite the fact that the plasma levels of IGF-I were higher in hGH-treated than in control mice (2.80 ± 0.18 U/ml us. 1.64 ± 0.13, P < 0.001, n = 9). In dw/dw mice hGH increased significantly the body weight gain, and the weight of liver, kidney, and testis. IGF-I treatment had no significant effect on liver weight, but increased significantly the body and kidney weights when compared with those of saline-treated mice. However, its effects were lower than those induced by hGH. In contrast, its effects on testicular weight were similar to that produced by hGH. However, when the effects of hGH and IGF-I were analyzed by the organ weight/body weight ratios (see Table 3), the effects of IGF-I and hGH on testis only were similar. Due to the small volumes of plasma (0.1 to 0.19 ml for each mouse), we could not measure the plasma IGF-I levels in dw/dw mice. The effects of hGH and IGF-I on Leydig cell function are shown in Table 2. In Dw/-, hGH had no significant effects on plasma LH levels, on plasma testosterone levels under basal conditions or after hCG stimulation, or on testicular hCG binding sites. In contrast, in dw/dw mice both hGH and IGF-I pretreatments enhanced significantly the response to hCG evaluated by plasma testosterone levels, and the number of testicular hCG binding sites, expressed either by testis or per gram of testis, but had no effect on either plasma LH or basal plasma testosterone levels. These latter results indicate that both treatments not only increase testis weight but also the number of hCG receptors per unit weight. Indeed, the number of hCG receptors per unit weight in dw/dw mice treated with hGH or IGF-I was similar to that observed in Dw/- mice (Table 2). The enhanced ability of testis, in hGH- and IGF-I-treated mice, to produce testosterone in response to hCG, was also seen in the analysis of the ratio between plasma testosterone and testis weight (Table 3). The results indicated that the enhanced responsiveness to hCG was due not only to an increase in testis weight but also to an increase in
the capacity of Leydig cells to produce testosterone, and/ or to an increase in the number of Leydig cells. Discussion The present results clearly demonstrate that recombinant hGH and IGF-I stimulate body weight gain and enhance the weight of several organs of Snell dwarf mice. At the concentration used, hGH was more efficient than IGF-I on body and kidney weights, but had similar effects on testicular weight. These results are in agreement with those recently reported, showing that iv administration of hGH or IGF-I to hypophysectomized rats (28) or to mutant dwarf rats (29) promoted body weight gain and enhanced the weight of several organs. Similarly, GH or crude (30), semipurified (31), or recombinant human IGF-I (32) injected into Snell dwarf mice enhanced body and organ weights. Even though there are some differences resulting from the various models, different IGF-I preparations, or different routes of administration, both hGH and IGF-I promote body and bone growth, but they differ quantitatively and qualitatively in their pattern of actions. The lack of effects of hGH in normal mice is probably related to a high GH secretion during this period of life that is similar to that observed in the rat at the onset of puberty (33). The primary alteration in Snell dwarf mice is a defect of the anterior pituitary gland, which secretes little if any GH and PRL. This is associated with delayed puberty (22) and low levels of plasma testosterone at 4 to 6 weeks, a time where the normal mice reach adult levels (34). However, without any treatment, at the age of 3 months testosterone levels in dw/dw are similar to those of control animals (7, 34), and some of the mice became fertile (20) despite the fact that plasma levels of LH and FSH are lower than in control mice (7, 20). However, in adult animals the seminiferous tubules are often smaller, and contain fewer germ cells at different stages of maturation, than those of normal control mice (7, 20, 35). Several studies have been undertaken to investigate the effects of hormonal treatment on testicular function
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Endo«1991 Voll28«No4
TABLE 2. Effects of hGH and IGF-I treatments of normal (DW/-) and dwarf (dw/dw) mice on plasma testosterone and testicular hCG receptors (mean ± SEM) Dw/-
Plasma LH (ng/ml) Plasma testosterone (ng/ml) Plasma testosterone (ng/ml) hCG bound (fmol/testes) (fmol/g testis)
Basal Basal hCG
dw/dw
Saline
GH
32 ± 5 (n = 6) 1.86 ± 0.28 (n = 6) 79 ± 12 (n = 14)
34 ± 5 (n = 6) 2.61 ± 0.31 (n = 6) 92 ±16 (n = 13)
33.5 ± 2.5 147 ± 11 (n = 10)
29.6 ± 2.2 127 ± 10 (n = 10)
GH
Saline
IGF-I
12, 16"
13,17°
14,16°
0.21, 0.17°
0.19, 0.18°
0.18, 0.19°
7.9 ± 2.1 (n = 13)
32.1 ± 2.16 (n = 10)
25.2 ± 2.5* (n = 11)
2.5 ± 0.2 89 ± 7 (n = 10)
6.6 ± 0.4fc 120 ± 76 (n = 8)
5.6 ± 0.36 133 ± 7" (n = 8)
Normal (Dw/—) and dwarf (dw/dw) were treated for 7 days with as indicated in legent to Table 1. At the end of the experiment, some mice received im. 0.1 ml saline (basal) whereas others received 10 IU (Dw/-) or 5 IU (dw/dw) hCG and killed 2 h later. Plasma LH and testosterone were measured by RIA. hCG receptors were determined in testes from hCG-treated mice as described in Materials and Methods. " Values of two pools of three animals each. b P < 0.001 compared to saline treated animals. TABLE 3. Effects of hGH and IGF-I on the ratios between organ weight and BW (mg/g) and between plasma testosterone after hCG stimulation and testicular weight (ng/ml/g) (mean ± SEM) Dw/n Liver/BW Kidneys/BW Testes/BW Plasma testosterone/testes weight
22 22 22 13
Saline 55.4 ± 15.8 ± 8.9 ± 348 ±
1.6 0.4 0.2 43
dw/dw n
hGH 53.4 ± 15.6 ± 8.8 ± 401 ±
1.1 0.3 0.3 56
15 15 15
8
Saline 46.1 ± 12.6 ± 5.4 ± 232 ±
1.9 0.4 0.3 40
hGH 52.8 ± 1.5° 13.8 ± 0.6" 6.7 ± 0.4° 565 ± 41°
IGF-I 42.9 ± 12.5 ± 7.1 ± 486 ±
2.2 0.3 0.4° 58°
"P < 0.01 vs. saline of group.
in Snell dwarf mice (review in 22). Implants of normal pituitaries or administration of PRL or hGH cause an increase in testicular weight and in numbers of LH/hCG receptors (20, 36, 37), and improve spermatogenesis (35, 36), but there are some conflicting results concerning the effectiveness of hGH vs. PRL. One of the major problems associated with defining GH action on the reproductive system is the capacity of primate GHs to bind to both somatogenic (GH) and lactogenic (PRL) receptors in nonprimates (38), and the widespread distribution of PRL receptors in the reproductive system. However, the presence of somatogenic receptors in the rat testis has been demonstrated recently by immunohistochemistry (39) and by measuring GH receptor mRNA (40). The present results clearly demonstrate that hGH treatment was able to increase testicular weight and LH/ hCG receptors and improve the in vivo steroidogenic responsiveness to hCG. Whether these effects are direct (through somatogenic or lactogenic receptors in the testis) or indirect (through IGF-I) is unknown. However, since exogenous IGF-I was able to induce similar effects in the testis, we favor the latter hypothesis. In dwarf mice, testicular growth and plasma FSH, but not plasma LH, begin to increase slowly at about 4 to 5 weeks of age
(7). Thus the developmental process, which is presumably gonadotropin (mainly FSH) mediated, was under way in the dwarf mice that were used in the present study. The data in Table 2 support the notion of acceleration of testicular maturation (hCG responsiveness) by both GH and IGF-I, without significant changes in plasma LH or in basal plasma testosterone levels. It is likely that in vivo IGF-I could bring about these changes via direct action on Leydig cells, as has been shown in vitro (13-15). However, an indirect action of IGF-I (for example, on accelerated central activation of puberty) cannot be completely discarded. In vitro studies have shown that IGF-I is absolutely required for the maintenance and probably the expression of differentiated function of both pig (14, 15) and rat (13) Leydig cells. In particular, IGF-I, which has a weak mitogenic effect, dramatically enhances LH/hCG receptor number and the steroidogenic capacity of these cells (14, 15). Although the mechanisms by which IGF-I increases Leydig cell responsiveness to hCG in vivo are not completely understood, our results are compatible with those observed in vitro. However, the possibility remains that IGF-I might also increase Leydig cell number. In addition to this endocrine effect of IGF-I on
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EFFECTS OF IGF-I IN TESTICULAR FUNCTION
testicular maturation, IGF-I might play a paracrine/ autocrine role, because Leydig and Sertoli cells from both pig (18, 19) and rat (16, 17) secrete IGF-I-like material, and because this secretion is regulated by gonadotropins but not by GH. Thus, this local production of IGF-I (regulated by gonadotropin but not by GH) might be sufficient to induce an almost complete, although delayed, development of testicular growth and function observed in Laron dwarfism (3) due to an abnormal GH receptor (41) and in Snell dwarf mice (7, 20, 34) with GH deficiency. In contrast, under physiological conditions the GH-dependent IGF-I endocrine secretion adds its effects to accelerate the testicular maturation process and participates in the normal timing of puberty. These results raise the possibility that chronic exposure of tissues, such as testis, to high IGF-I levels could modify the timing of tissue maturation. This may be actually clinically relevant, due to the large utilization of GH therapy in short children without GH deficiency. Indeed, recent data indicate that hGH treatment in children accelerates the rate of pubertal maturation (42).
Acknowledgments The authors thank Drs. L. Fryklund and R. Gunnarson (Kabi, Stockholm, Sweden) for the generous gift of recombinant hGH and IGF-I, Dr. M. G. Forest for the anti-testosterone antibody, the Rat Pituitary Hormone Distribution Program, NIDDK, for the LH kit, Dr. S. Hall for reviewing the English manuscript, and J. Bois and M. A. di Carlo for their excellent assistance in the preparation of the manuscript.
References 1. Sheikholislan BM, Stempfel RS 1972 Hereditary isolated somatotropin deficiency: effects of human growth hormone administration. Pediatrics 49:362-374 2. Kulin HE, Samdjlike E, Santen R, Santner S 1981 The effects of growth hormone on the Leydig cell response to chorionic gonadotropin in boys with hypopituitarism. Clin Endocrinol 45:468-472 3. Laron Z 1984 Laron-type dwarfism (hereditary somatomedin deficiency). A review. Adv Int Med Pediatr 51:117-140 4. Tanner JM, Whitehouse RH 1975 A note on the bone age at which patients with true isolated hormone deficiency enter puberty. J Clin Endocrinol Metab 41:788-790 5. Rivarola MA, Heinrich JJ, Podesta EJ, Chondjnik MF, Bergada C 1972 Testicular function in hypopituitarism. Pediatr Res 6:634641 6. Odell WD, Swerdloff RS 1976 Etiologies of sexual maturation: a model system based on sexual maturating rat. Recent Prog Horm Res 32:246-288 7. Hochereau-de Reviers MT, de Reviers MM, Monet-Kuntz C, Perreau L, Fontaine I, Viguier-Martinez MC 1987 Testicular growth and hormonal parameters in the male Snell dwarf mouse. Acta Endocrinol 115:399-405 8. Froesch BA, Schmid CH, Schwauder J, Zapf J 1985 Action of insulin-like growth factors. Annu Rev Physiol 47:443-468 9. D'Ercole AJ, Stiles AL, Underwood LE 1984 Tissue concentration of somatomedin-C: Further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc Natl Acad Sci USA 81:935-939 10. Mathews LS, Norstedt G, Palmiter RD 1986 Regulation of insulinlike growth factor I gene expression by growth hormone. Proc Natl
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Acad Sci USA 83:9343-9347 11. Borland K, Mita M, Oppenheimer CL, Blinderman LA, Massague J, Hall PF, Czech MP 1984 The action of insulin-like growth factors I and II on cultured rat Sertoli cells. Endocrinology 114:240246 12. Jaillard C, Chatelain PG, Saez JM 1987 In vitro regulation of pig Sertoli cell growth and function. Effects of fibroblast growth factor and somatomedin-C. Biol Reprod 37:665-674 13. Lin T, Haskell J, Vinson N, Terracio L 1986 Characterization of insulin and insulin-like growth factor I receptors of purified Leydig cells and their role in steroidogenesis in primary culture. A comparative study. Endocrinology 119:1641-1647 14. Bernier M, Chatelain PG, Mather JP, Saez JM 1986 Regulation of gonadotropin receptors, gonadotropin responsiveness, and cell multiplication by somatomedin-C and insulin in cultured pig Leydig cells. J Cell Physiol 129:257-263 15. Perrard-Sapori MH, Chatelain PG, Jaillard C, Saez JM 1987 Characterization and regulation of somatomedin-C/insulin-like growth factor receptors on cultured pig Leydig cells. Effects of somatomedin-C on luteotropin receptor and steroidogenesis. Eur J Biochem 165:209-214 16. Smith EP, Svoboda ME, Van Wyk JJ, Kierszenbaum AL, Tres LL 1987 Partial characterization of a somatomedin-like peptide from medium of cultured rat Sertoli cells. Endocrinology 120:186-193 17. Cailleau J, Vermeire S, Verhoeven G 1990 Independent control of the production of insulin-like growth factor I and its binding protein by cultured testicular cell. Mol Cell Endocrinol 69:79-89 18. Chatelain PG, Naville D, Saez JM 1987 Somatomedin-C/insulinlike growth factor I-like material secreted by porcine Sertoli cells in vitro: characterization and regulation. Biochem Biophys Res Commun 146:1009-1017 19. Naville D, Chatelain PG, Avallet O, Saez JM 1990 Control of production of insulin-like growth factor I by pig Leydig and Sertoli cells cultured alone or together. Cell-cell interactions. Mol Cell Endocrinol 70:217-224 20. Bartke A, Goldman BD, Ber F, Dalterio S 1977 Effects of prolactin (PRL) on pituitary and testicular function in mice with hereditary PRL deficiency. Endocrinology 101:1760-1766 21. Barkley MS, Bartke A, Gross DS, Sinha YN 1982 Prolactin status of hereditary dwarf mice. Endocrinology 110:2088-2096 22. Van Buul-Offers S 1983 Hormonal and other inherited growth disturbances in mice with special reference to the Snell dwarf mouse. A review. Acta Endocrinol 103 [Suppl 258]: 1-47 23. Tuszynski GP, Knight LC, Kornecki E, Srivastava S 1983 Labeling of platelet surface proteins with 125I by the iodogen method. Anal Biochem 130:166-170 24. Segaloff DL, Ascoli M 1981 Removed of the surface-bound human choriogonadotropin. Result in cessation of hormonal responses in cultured Leydig tumor cells. J Biol Chem 256:11420-11423 25. Chatelain PG, Van Wyk JJ, Copeland KC, Blethen S, Underwood LE 1983 Effects of in vitro action of serum proteases or exposure to acid measurable immunoreactive somatomedin-C in serum. J Clin Endocrinol Metab 56:376-383 26. Daughaday WH, Mariz JK, Blethen SC 1980 Inhibition of access of bound somatomedin to membrane receptor and immunobinding sites: a comparison of radioreceptor and radioimmunoassay of somatomedin in native and acid-ethanol-extracted serum. J Clin Endocrinol Metab 51:781-788 27. Forest MG, Cathiard AM, Bertrand J 1973 Total and unbound testosterone levels in the newborn and in normal and hypogonadal children: use of a sensitive radioimmunoassay for testosterone. J Clin Endocrinol Metab 36:1132-1142 28. Guler HP, Zapf J, Scheiwiller E, Froesh ER 1988 Recombinant human insulin-like growth factor I stimulates growth and has distinct effects on organ size in hypophysectomized rats. Proc Natl Acad Sci USA 85:4889-4893 29. Skottner A, Clark RG, Fryklund L, Robinson ILAF 1989 Growth response in a mutant dwarf rat to human growth hormone and recombinant human insulin-like growth factor I. Endocrinology 124:2519-2520 30. Holder AT, Spencer EM, Preece MA 1981 Effects of bovine growth hormone and a partially pure preparation of somatomedin on
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EFFECTS OF IGF-I IN TESTICULAR FUNCTION
various growth parameters in hypopituitary dwarf mice. J Endocrinol 89:275-282 Van Buul-Offers S, Hoogerbrugge CM, Branger J, Feijlbrief M, Van der Brande JL 1988 Growth-stimulating effects of somatomedin/insulin-like peptides in Snell dwarf mice. Horm Res 29:229236 Van Buul-Offers S, Ueda I, Van der Brande JL 1966 Biosynthetic somatomedin-C (Sm-C/IGF-I) increases the length and weight of Snell dwarf mice. Pediatr Res 20:825-827 Jansson JO, Eden S, Isaksson 0 1985 Sexual dimorphism in the control of growth hormone secretion. Endocr Rev 6:128-150 Howe E, Howe C, Pollard I 1980 Plasma testosterone in the male, progesterone and estradiol-17/3 in the female, and 3/3-hydroxysteroid dehydrogenase (3/3-HSD) activity in the testis and ovary of the Snell dwarf mouse. Biol Reprod 23:887-892 Matsushima M, Kuroba K, Shirai M, Ando K, Sugisaki T, Noguchi T 1986 Spermatogenesis in Snell dwarf, little and congenitally hypothyroid mice. Int J Androl 9:132-140 Bohnet HG, Friesen HG 1976 Effects of prolactin and growth
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hormone on prolactin and LH receptors in the dwarf mice. J Reprod Fertil 48:307-311 Bartke A, Lloyd CW 1970 Influence of prolactin and pituitary isografts on spermatogenesis in dwarf mice and hypophysectomized rats. J Endocrinol 46:321-329 Posner BI, Kelly PA, Saiu RPL, Friesen HG 1974 Studies of insulin, growth hormone, and prolactin binding: tissue distribution, species variation and characterization. Endocrinology 95:521-531 Lobie PE, Breipohl W, Garcia JA, Waters MS 1990 Cellular localization of the growth hormone receptor/binding protein in the male and female reproductive systems. Endocrinology 126:22142221 Mathews LS, Enberg B, Norstedt G 1989 Regulation of rat growth hormone receptor gene expression. J Biol Chem 264:9905-9910 Amselem S, Duquesnoy P, Attree O, Novelli G, Bousniwa S, PostelVinay MC, Goossens M 1990 Laron dwarfism and mutations of the growth hormone-receptor gene. N Engl J Med 321:989-995 Darendeliler F, Hindmarsh PC, Brook CGD 1990 Dose-response curves for treatment with biosynthetic human growth hormone. J Endocrinol 125:311-318
Third International Symposium on Diabetic Angiopathy in Childhood From September 2-4, 1991 the Third International Workshop on Diabetic Angiopathy in Children will be held in Berlin, Germany. It is sponsored by the International Study Group on Diabetes in Children and Adolescents (ISGD). The symposium will feature presentations of selected abstracts as well as keynote lectures from internationally recognized researchers on the topics matrix metabolism, diagnostics, and therapy of diabetic angiopathy. It is designed as a workshop of basic scientists and clinicians and will provide ample time for discussion. The proceedings will be published. For more information and abstract forms contact Dr. Bruno Weber, Universitats-Kinderklinik, Heubnerweg 6, D-W 1000 Berlin 19, Germany. Tel.: 30-3035-1, Fax: 30-3035-4638. For further information or questions please do not hesitate to contact Dr. Weber (address above) or Dr. Danne at Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, Tel.: 617-732-2568, Fax: 617-7322593.
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Vol. 128, No. 4 Printed in U.S.A.
Growth Hormone and Insulin-Like Growth Factor I Treatment Increase Testicular Luteinizing Hormone Receptors and Steroidogenic Responsiveness of Growth Hormone Deficient Dwarf Mice* P. G. CHATELAIN, P. SANCHEZ, AND J. M. SAEZ INSERM U307, Hopital Debrousse, 69322 Lyon Cedex 05, France
ABSTRACT. To test the hypothesis that insulin-like growth factor (IGF-I) is required for the in vivo development of testicular Leydig cell function, either recombinant human GH [(hGH)(1.5 /ig/g BW) or recombinant IGF-I (1 ^g/g BW) was injected three times daily into immature Snell dwarf mice (dw/ dw) and into phenotypically normal control (Dw/—) for 7 days. In dw/dw mice hGH enhanced significantly body, liver, kidney, and testicular weight. In addition, hGH increased testicular LH receptors and the acute steroidogenic response to human CG, but there was no significant effect on basal plasma testosterone or plasma LH levels. The effects of IGF-I in body and kidney
T
he role of GH in testicular function has been suggested by the fact that in both humans (1-5) and experimental animals (6, 7), isolated GH deficiency and/ or a GH resistant state are associated with delayed puberty and a poor response to exogenous human CG (hCG). At least in humans, delayed puberty in isolated GH deficiency is very often improved following treatment with GH (1, 2, 5). These data suggest therefore that GH could be involved directly or indirectly in the development and maintenance of the gonadotropin responsiveness of Leydig cells. However, since many of the actions of GH in vivo are thought to be mediated by insulin-like growth factor I (IGF-I) (8), it appears likely that the action of GH on testicular function might be modulated by IGF-I. Immunoreactive IGF-I-like material (9) and IGF-I Received November 19, 1990. Address correspondence and requests for reprints to: Jose M. Saez, INSERM U307, Hopital Debrousse, 29 Rue Soeur Bouvier, 69322 Lyon Cedex 05, France. * This work was supported by research grants from Institut National de la Sante et de la Recherche Medicale, Nordisk Insulin Laboratorium, and Fondation pour la Recherche Medicale Francaise. The data of this paper were presented in part at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990 (Abstract 1343) and at the Vlth European Testis Workshop, Marieham, Finland, 1990.
weight were less pronounced than those produced by hGH, but its effects on testicular weight and LH receptors, as well as on the acute steroidogenic response to human CG, were similar to that observed after hGH treatment. In Dw/- mice hGH had no effect on either body or organ weight or on testicular function, despite the fact that it induced a significant increase in plasma IGF-I levels. These results indicated that IGF-I is able to induce the maturation of Leydig cell function and that the effects of hGH on the testis are probably mediated by IGF-I. They also suggest that the delayed puberty associated with GH deficiency or resistance is most likely related to an IGF-I deficiency. {Endocrinology 128: 1857-1862,1991)
mRNA (10) have been identified in the rat testis. The relevance of IGF-I to the physiology of testicular cells stems from observations indicating that both Sertoli (11, 12) and Leydig (13-15) cells contain specific IGF type I receptors and that this peptide greatly enhances the response of both cell types to gonadotropins. In addition, it has been shown that both Sertoli and Leydig cells from rat (16, 17) and pig (18, 19) secrete IGF-I and that this secretion is regulated by gonadotropins, but not by GH. The aim of the present study was to determine if IGFI plays a role in the in vivo development of the steroidogenic responsiveness of Leydig cells to gonadotropins. To address this question, we have studied the effects of recombinant human GH (hGH) and recombinant human IGF-I administration on several parameters, including testicular function, of immature Snell dwarf mice (dw/ dw). These mice are PRL and GH deficient and exhibit delayed testicular maturation (20-22).
Materials and Methods Snell male dwarfs (dw/dw) and phenotypically normal control mice (Dw/-), 6 to 7 weeks old, were purchased from CSEAL-CNRS (Orleans, France). Animals were maintained in standard condition for at least 3 days after arrival before the experiments were started. Recombinant hGH and recombinant IGF-I were a generous gift of Dr. L. Fryklund and Dr. R. 1857
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EFFECTS OF IGF-I IN TESTICULAR FUNCTION
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Gunnarson (Kabi, Stockholm, Sweden). Each hormone was dissolved in phosphate-saline buffer (pH 7.4) containing 0.1% bovine serum albumin. The hGH and IGF-I solutions were prepared at the beginning of the experiment and stored in small aliquots at —20 C in the dark. Animals were injected intramuscularly three times daily (0800 h, 1600 h, and 2400 h) for 7 days with either saline or hGH (1.5 ^g/g BW/injection) or IGF-I (1 Mg/g BW/injection; dw/dw mice only). The doses of both hormones were calculated for the body weight at the beginning of the experiment. Body weight was measured daily, just before the second injection. At the end of the experiment 0.1 ml saline or 10 IU (Dw/-) or 5 IU (dw/dw) hCG was given im and the animals were killed 2 h later (4-5 h after the last injection of hGH or IGF-I). Animals were bled under light ether anesthesia by cardiac puncture with a heparinized syringe. The liver, kidneys, and testes were removed, weighed, and stored at -70 C. Because the number of dw/dw mice of the same age available at the same time was limited, three independent experiments in January, April, and September were performed. Each time the animals were divided into two (Dw/-) (saline or hGH) or three (dw/dw) (saline, hGH, or IGF-I) groups of five to seven mice each. Because the body weights at the beginning of the experiments [Dw/- = 22.6 ± 0.2 g (n = 44) and dw/dw = 5.7 ± 0.08 g (n = 50)] were similar, and because the results from one experiment to another were also similar, the results from the three experiments performed at different times were pooled and analyzed as a single experiment. hCG binding assay hCG (CR 121; 13,450 IU/mg) was iodinated with iodogen (23) (SA 60 to 80 MCi/Mg). Testes from acute hCG-treated mice were homogenized in Tris-HCl buffer (50 mM, pH 7.4) containing 1 mM CaCl2, 1 mM MgCl2, and 250 mM sucrose and centrifuged at 600 X g for 10 min. The supernatant was decanted and centrifuged at 20,000 X g for 30 min. The supernatant was removed and the pellet was washed once with glycine buffer (50 mM glycine, 150 mM NaCl, pH 3) for 4 min at 4 C and twice with Tris-HCl buffer to remove the bound hCG (24). Using this procedure it was found in preliminary experiments that the number of hCG binding sites per testis, as well as the affinity of hCG for its receptors in Dw/- treated with 50 IU hCG 2 h before killing, was similar (97 ± 2%, n = 4) to that of untreated Dw/-. These results documented that even at this high dose of hCG there was no down-regulation of receptors during the 2 h the mice were exposed to hCG in vivo. All binding studies were performed in triplicate at 37 C for 4 h, using two concentrations of labeled hCG 9.10"10 and 9.10"9 M, which are close to the concentrations required to produce halfmaximal and maximal saturation of the binding sites. For each concentration, nonspecific binding was measured in the presence of a 500-fold excess of unlabeled hormone. Specific binding was computed by subtracting nonspecific from total binding. In preliminary studies the binding affinity of hCG for its receptor was determined in membranes prepared from Dw/-, dw/dw, and hGH-treated dw/dw. Scatchard analysis of the binding data indicated a single class of binding sites with a dissociation constant (KD) of 2.1 ± 0.2 X 10"9 M (n = 3). Moreover, the number of binding sites using this method was
Endo'1991 Vol 128 • No 4
similar to that found using a single saturating concentration of 125 I-hCG. Other methods Plasma IGF-I was measured by RIA (25) after acid-ethanol extraction of serum, using a nonequilibrium technique and a pool of sera from adult human males as standard, which was also extracted by acid-ethanol (26). Plasma testosterone was measured by the method described previously (27). LH in plasma was measured by RIA using the NIDDK-rat LH kit, which has been validated for measuring mouse LH (20). The results are expressed in terms of rat RP-1 standard. Because the amounts of plasma available in dwarf mice are small, a plasma pool from three animals was used for LH determinations.
Results Treatment with hGH had no effect on Dw/- body weight gain (Fig. 1). In contrast, this hormone produced a marked increase in the weight of dw/dw, which was significant after two days of treatment. IGF-I also enhanced body weight gain, but at a lower rate than hGH (Fig. 1). Table 1 summarizes the effect of hGH and IGFI on body weight gain and the weight of liver, kidney, 2,5 "*• Dw/- Control ••• Dw/hGII
2,0
1,5"
1,0"
0,5
0,0 0
1
2
3
5
6
7
8
Days of Treatment 2,5
2,0
1,5-
1,0"
0,5"
0,0 6
8
Days ol Treatment
FIG. 1. Body weight gain of normal (Dw/-) (top) and dwarf (dw/dw) (bottom) mice treated three times daily with saline (D), recombinant hGH (•) (1.5 Mg/g BW), or recombinant IGF-I (•) (1 Mg/g BW).
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EFFECTS OF IGF-I IN TESTICULAR FUNCTION
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TABLE 1. Effects of hGH and IGF-I on body and organ weight in normal (Dw/—) and dwarf (dw/dw) mice (mean ± SEM)
Dw/Saline (n = 22) BW(g) Liver (g) Kidneys (g) Testes (g)
24.85 ± 1.39 ± 0.405 ± 0.227 ±
0.18 0.03 0.011 0.006
dw/dw GH(n = 22) 24.95 1.39 0.411 0.229
± ± ± ±
0.41 0.03 0.010 0.006
Saline (n = 20) 6.12 ± 0.291 ± 0.081 ± 0.034 ±
0.08 0.012 0.002 0.003
GH (n = 15) 7.79 ± 0.441 ± 0.117 ± 0.057 ±
0.14° 0.012° 0.004" 0.004"
IGF-I (n = 15) 6.96 0.318 0.092 0.052
± ± ± ±
0.10"'6 0.0226 0.002"* 0.008"
Normal (Dw/—) and dwarf (dw/dw) mice were treated three times daily with saline, recombinant hGH (1.5 jtg/g of BW), or recombinant IGFl^g/gof BW) for 7 days. ° P < 0.001 compared to control. * P < 0.001 compared to hGH-treated group.
and testis after 7 days of treatment. In Dw/-, hGH had no significant effect on any of these parameters, despite the fact that the plasma levels of IGF-I were higher in hGH-treated than in control mice (2.80 ± 0.18 U/ml us. 1.64 ± 0.13, P < 0.001, n = 9). In dw/dw mice hGH increased significantly the body weight gain, and the weight of liver, kidney, and testis. IGF-I treatment had no significant effect on liver weight, but increased significantly the body and kidney weights when compared with those of saline-treated mice. However, its effects were lower than those induced by hGH. In contrast, its effects on testicular weight were similar to that produced by hGH. However, when the effects of hGH and IGF-I were analyzed by the organ weight/body weight ratios (see Table 3), the effects of IGF-I and hGH on testis only were similar. Due to the small volumes of plasma (0.1 to 0.19 ml for each mouse), we could not measure the plasma IGF-I levels in dw/dw mice. The effects of hGH and IGF-I on Leydig cell function are shown in Table 2. In Dw/-, hGH had no significant effects on plasma LH levels, on plasma testosterone levels under basal conditions or after hCG stimulation, or on testicular hCG binding sites. In contrast, in dw/dw mice both hGH and IGF-I pretreatments enhanced significantly the response to hCG evaluated by plasma testosterone levels, and the number of testicular hCG binding sites, expressed either by testis or per gram of testis, but had no effect on either plasma LH or basal plasma testosterone levels. These latter results indicate that both treatments not only increase testis weight but also the number of hCG receptors per unit weight. Indeed, the number of hCG receptors per unit weight in dw/dw mice treated with hGH or IGF-I was similar to that observed in Dw/- mice (Table 2). The enhanced ability of testis, in hGH- and IGF-I-treated mice, to produce testosterone in response to hCG, was also seen in the analysis of the ratio between plasma testosterone and testis weight (Table 3). The results indicated that the enhanced responsiveness to hCG was due not only to an increase in testis weight but also to an increase in
the capacity of Leydig cells to produce testosterone, and/ or to an increase in the number of Leydig cells. Discussion The present results clearly demonstrate that recombinant hGH and IGF-I stimulate body weight gain and enhance the weight of several organs of Snell dwarf mice. At the concentration used, hGH was more efficient than IGF-I on body and kidney weights, but had similar effects on testicular weight. These results are in agreement with those recently reported, showing that iv administration of hGH or IGF-I to hypophysectomized rats (28) or to mutant dwarf rats (29) promoted body weight gain and enhanced the weight of several organs. Similarly, GH or crude (30), semipurified (31), or recombinant human IGF-I (32) injected into Snell dwarf mice enhanced body and organ weights. Even though there are some differences resulting from the various models, different IGF-I preparations, or different routes of administration, both hGH and IGF-I promote body and bone growth, but they differ quantitatively and qualitatively in their pattern of actions. The lack of effects of hGH in normal mice is probably related to a high GH secretion during this period of life that is similar to that observed in the rat at the onset of puberty (33). The primary alteration in Snell dwarf mice is a defect of the anterior pituitary gland, which secretes little if any GH and PRL. This is associated with delayed puberty (22) and low levels of plasma testosterone at 4 to 6 weeks, a time where the normal mice reach adult levels (34). However, without any treatment, at the age of 3 months testosterone levels in dw/dw are similar to those of control animals (7, 34), and some of the mice became fertile (20) despite the fact that plasma levels of LH and FSH are lower than in control mice (7, 20). However, in adult animals the seminiferous tubules are often smaller, and contain fewer germ cells at different stages of maturation, than those of normal control mice (7, 20, 35). Several studies have been undertaken to investigate the effects of hormonal treatment on testicular function
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Endo«1991 Voll28«No4
TABLE 2. Effects of hGH and IGF-I treatments of normal (DW/-) and dwarf (dw/dw) mice on plasma testosterone and testicular hCG receptors (mean ± SEM) Dw/-
Plasma LH (ng/ml) Plasma testosterone (ng/ml) Plasma testosterone (ng/ml) hCG bound (fmol/testes) (fmol/g testis)
Basal Basal hCG
dw/dw
Saline
GH
32 ± 5 (n = 6) 1.86 ± 0.28 (n = 6) 79 ± 12 (n = 14)
34 ± 5 (n = 6) 2.61 ± 0.31 (n = 6) 92 ±16 (n = 13)
33.5 ± 2.5 147 ± 11 (n = 10)
29.6 ± 2.2 127 ± 10 (n = 10)
GH
Saline
IGF-I
12, 16"
13,17°
14,16°
0.21, 0.17°
0.19, 0.18°
0.18, 0.19°
7.9 ± 2.1 (n = 13)
32.1 ± 2.16 (n = 10)
25.2 ± 2.5* (n = 11)
2.5 ± 0.2 89 ± 7 (n = 10)
6.6 ± 0.4fc 120 ± 76 (n = 8)
5.6 ± 0.36 133 ± 7" (n = 8)
Normal (Dw/—) and dwarf (dw/dw) were treated for 7 days with as indicated in legent to Table 1. At the end of the experiment, some mice received im. 0.1 ml saline (basal) whereas others received 10 IU (Dw/-) or 5 IU (dw/dw) hCG and killed 2 h later. Plasma LH and testosterone were measured by RIA. hCG receptors were determined in testes from hCG-treated mice as described in Materials and Methods. " Values of two pools of three animals each. b P < 0.001 compared to saline treated animals. TABLE 3. Effects of hGH and IGF-I on the ratios between organ weight and BW (mg/g) and between plasma testosterone after hCG stimulation and testicular weight (ng/ml/g) (mean ± SEM) Dw/n Liver/BW Kidneys/BW Testes/BW Plasma testosterone/testes weight
22 22 22 13
Saline 55.4 ± 15.8 ± 8.9 ± 348 ±
1.6 0.4 0.2 43
dw/dw n
hGH 53.4 ± 15.6 ± 8.8 ± 401 ±
1.1 0.3 0.3 56
15 15 15
8
Saline 46.1 ± 12.6 ± 5.4 ± 232 ±
1.9 0.4 0.3 40
hGH 52.8 ± 1.5° 13.8 ± 0.6" 6.7 ± 0.4° 565 ± 41°
IGF-I 42.9 ± 12.5 ± 7.1 ± 486 ±
2.2 0.3 0.4° 58°
"P < 0.01 vs. saline of group.
in Snell dwarf mice (review in 22). Implants of normal pituitaries or administration of PRL or hGH cause an increase in testicular weight and in numbers of LH/hCG receptors (20, 36, 37), and improve spermatogenesis (35, 36), but there are some conflicting results concerning the effectiveness of hGH vs. PRL. One of the major problems associated with defining GH action on the reproductive system is the capacity of primate GHs to bind to both somatogenic (GH) and lactogenic (PRL) receptors in nonprimates (38), and the widespread distribution of PRL receptors in the reproductive system. However, the presence of somatogenic receptors in the rat testis has been demonstrated recently by immunohistochemistry (39) and by measuring GH receptor mRNA (40). The present results clearly demonstrate that hGH treatment was able to increase testicular weight and LH/ hCG receptors and improve the in vivo steroidogenic responsiveness to hCG. Whether these effects are direct (through somatogenic or lactogenic receptors in the testis) or indirect (through IGF-I) is unknown. However, since exogenous IGF-I was able to induce similar effects in the testis, we favor the latter hypothesis. In dwarf mice, testicular growth and plasma FSH, but not plasma LH, begin to increase slowly at about 4 to 5 weeks of age
(7). Thus the developmental process, which is presumably gonadotropin (mainly FSH) mediated, was under way in the dwarf mice that were used in the present study. The data in Table 2 support the notion of acceleration of testicular maturation (hCG responsiveness) by both GH and IGF-I, without significant changes in plasma LH or in basal plasma testosterone levels. It is likely that in vivo IGF-I could bring about these changes via direct action on Leydig cells, as has been shown in vitro (13-15). However, an indirect action of IGF-I (for example, on accelerated central activation of puberty) cannot be completely discarded. In vitro studies have shown that IGF-I is absolutely required for the maintenance and probably the expression of differentiated function of both pig (14, 15) and rat (13) Leydig cells. In particular, IGF-I, which has a weak mitogenic effect, dramatically enhances LH/hCG receptor number and the steroidogenic capacity of these cells (14, 15). Although the mechanisms by which IGF-I increases Leydig cell responsiveness to hCG in vivo are not completely understood, our results are compatible with those observed in vitro. However, the possibility remains that IGF-I might also increase Leydig cell number. In addition to this endocrine effect of IGF-I on
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EFFECTS OF IGF-I IN TESTICULAR FUNCTION
testicular maturation, IGF-I might play a paracrine/ autocrine role, because Leydig and Sertoli cells from both pig (18, 19) and rat (16, 17) secrete IGF-I-like material, and because this secretion is regulated by gonadotropins but not by GH. Thus, this local production of IGF-I (regulated by gonadotropin but not by GH) might be sufficient to induce an almost complete, although delayed, development of testicular growth and function observed in Laron dwarfism (3) due to an abnormal GH receptor (41) and in Snell dwarf mice (7, 20, 34) with GH deficiency. In contrast, under physiological conditions the GH-dependent IGF-I endocrine secretion adds its effects to accelerate the testicular maturation process and participates in the normal timing of puberty. These results raise the possibility that chronic exposure of tissues, such as testis, to high IGF-I levels could modify the timing of tissue maturation. This may be actually clinically relevant, due to the large utilization of GH therapy in short children without GH deficiency. Indeed, recent data indicate that hGH treatment in children accelerates the rate of pubertal maturation (42).
Acknowledgments The authors thank Drs. L. Fryklund and R. Gunnarson (Kabi, Stockholm, Sweden) for the generous gift of recombinant hGH and IGF-I, Dr. M. G. Forest for the anti-testosterone antibody, the Rat Pituitary Hormone Distribution Program, NIDDK, for the LH kit, Dr. S. Hall for reviewing the English manuscript, and J. Bois and M. A. di Carlo for their excellent assistance in the preparation of the manuscript.
References 1. Sheikholislan BM, Stempfel RS 1972 Hereditary isolated somatotropin deficiency: effects of human growth hormone administration. Pediatrics 49:362-374 2. Kulin HE, Samdjlike E, Santen R, Santner S 1981 The effects of growth hormone on the Leydig cell response to chorionic gonadotropin in boys with hypopituitarism. Clin Endocrinol 45:468-472 3. Laron Z 1984 Laron-type dwarfism (hereditary somatomedin deficiency). A review. Adv Int Med Pediatr 51:117-140 4. Tanner JM, Whitehouse RH 1975 A note on the bone age at which patients with true isolated hormone deficiency enter puberty. J Clin Endocrinol Metab 41:788-790 5. Rivarola MA, Heinrich JJ, Podesta EJ, Chondjnik MF, Bergada C 1972 Testicular function in hypopituitarism. Pediatr Res 6:634641 6. Odell WD, Swerdloff RS 1976 Etiologies of sexual maturation: a model system based on sexual maturating rat. Recent Prog Horm Res 32:246-288 7. Hochereau-de Reviers MT, de Reviers MM, Monet-Kuntz C, Perreau L, Fontaine I, Viguier-Martinez MC 1987 Testicular growth and hormonal parameters in the male Snell dwarf mouse. Acta Endocrinol 115:399-405 8. Froesch BA, Schmid CH, Schwauder J, Zapf J 1985 Action of insulin-like growth factors. Annu Rev Physiol 47:443-468 9. D'Ercole AJ, Stiles AL, Underwood LE 1984 Tissue concentration of somatomedin-C: Further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc Natl Acad Sci USA 81:935-939 10. Mathews LS, Norstedt G, Palmiter RD 1986 Regulation of insulinlike growth factor I gene expression by growth hormone. Proc Natl
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hormone on prolactin and LH receptors in the dwarf mice. J Reprod Fertil 48:307-311 Bartke A, Lloyd CW 1970 Influence of prolactin and pituitary isografts on spermatogenesis in dwarf mice and hypophysectomized rats. J Endocrinol 46:321-329 Posner BI, Kelly PA, Saiu RPL, Friesen HG 1974 Studies of insulin, growth hormone, and prolactin binding: tissue distribution, species variation and characterization. Endocrinology 95:521-531 Lobie PE, Breipohl W, Garcia JA, Waters MS 1990 Cellular localization of the growth hormone receptor/binding protein in the male and female reproductive systems. Endocrinology 126:22142221 Mathews LS, Enberg B, Norstedt G 1989 Regulation of rat growth hormone receptor gene expression. J Biol Chem 264:9905-9910 Amselem S, Duquesnoy P, Attree O, Novelli G, Bousniwa S, PostelVinay MC, Goossens M 1990 Laron dwarfism and mutations of the growth hormone-receptor gene. N Engl J Med 321:989-995 Darendeliler F, Hindmarsh PC, Brook CGD 1990 Dose-response curves for treatment with biosynthetic human growth hormone. J Endocrinol 125:311-318
Third International Symposium on Diabetic Angiopathy in Childhood From September 2-4, 1991 the Third International Workshop on Diabetic Angiopathy in Children will be held in Berlin, Germany. It is sponsored by the International Study Group on Diabetes in Children and Adolescents (ISGD). The symposium will feature presentations of selected abstracts as well as keynote lectures from internationally recognized researchers on the topics matrix metabolism, diagnostics, and therapy of diabetic angiopathy. It is designed as a workshop of basic scientists and clinicians and will provide ample time for discussion. The proceedings will be published. For more information and abstract forms contact Dr. Bruno Weber, Universitats-Kinderklinik, Heubnerweg 6, D-W 1000 Berlin 19, Germany. Tel.: 30-3035-1, Fax: 30-3035-4638. For further information or questions please do not hesitate to contact Dr. Weber (address above) or Dr. Danne at Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, Tel.: 617-732-2568, Fax: 617-7322593.
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