Anatomy of the insulin-like growth factor system in the human testis

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1 FERTILITY AND STERILITY Vol. 60, No.5, November 1993 Copyright 1993 The American Fertility Society Printed on acid-free paper in U. S. A. Anatomy of the insulin-like growth factor system in the human testis Jian Zhou, M.D., Ph.D.* Carolyn Bondy, M.D. Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland Objective: To study the cellular patterns of gene expression for insulin-like growth factors I and II (IGF-I and IGF-II), their receptors and specific high-affinity binding proteins (IGFBPs) in the human testis. Design: In situ hybridization histochemistry was used to localize messenger ribonucleic acids (mrnas) for IGF-I and IGF-II, the IGF-I and IGF-II receptors and IGFBP- 1 to 6 in fresh-frozen testis sections from healthy young men who died of trauma. Results: Insulin-like growth factor I mrna was not detected. Insulin-like growth factors II mrna was abundant in testicular blood vessels and peritubular connective tissue. Both IGF-I and IGF -II receptor mrn As were most abundant in the germinal epithelium. Insulin -like growth factor binding protein 1 mrna was not detected. Binding protein-2 mrna was expressed in both Leydig and Sertoli cells. Bindingprotein-3 mrna was detected only in the endothelium of testicular blood vessels. Binding protein-4 mrna was also localized in endothelium but, in addition, was present in Leydig cells and interstitial connective tissue. Binding protein-5 mrna was abundant in connective tissue and was detected at low levels in Leydig cells. Binding protein-6 mrna was present in some of the peritubular cells and in some cells of the interstitial compartment. Conclusion: Our results suggest that IGFs may play significant roles in testicular function and germ cell development. Fertit Steril1993;60: Key Words: IGF receptor, IGF binding protein, seminiferous tubules, spermatogenesis, Leydig cell, mrna, in situ hybridization Insulin-like growth factors I and II (IGF -I and IGF-II) are anabolic polypeptides that are thought to play autocrine and/or paracrine roles in the human ovary (1, 2), but there has been relatively little study of these factors in the human testis. An immunohistochemical study of biopsy specimens from infertile men showed that I G F -I immunoreactivity is preferentially localized in Sertoli cells and IGF-I receptor immunoreactivity in spermatocytes and in a minority of Leydig cells (3,4). Insulin-like growth Received April 27, 1993; revised and accepted July 13, * Reprint requests: Jian Zhou, M.D., Ph.D., Building 10, Room 10N262, National Institutes of Health, Bethesda, Maryland factors-i immunoreactivity is observed in spermatocytes ofthe rat (5) and IGF-I and IGF binding proteins (IGFBPs) are produced by Sertoli, Leydig, and peritubular cells derived from the immature rat testis and cultured in vitro (6, 7). Insulin-like growth factor binding protein-2 is synthesized by cultured peritubular cells and IGFBP-3 by Sertoli cells (8). Northern analysis has shown that IGFBP- 3 messenger RNA (mrna) is present in rat testis (9). To identify the cells synthesizing IGF-I and IGF-II, their receptors and specific high-affinity IGFBPs in the human testis, we used in situ hybridization histochemistry to map the patterns of gene expression for IGF-I and IGF-II, the IGF-I and IGF-II receptors, and IGFBPs- 1 to 6 in fresh frozen tissue obtained from healthy adult men who Vol. 60, No.5, November 1993 Zhou and Bondy Human testis IGF system 897

2 Figure 1 Insulin-like growth factor II gene expression in the human testis. The figure shows paired bright and dark field micrographs of sections hybridized to crna probe for IGF-II. The exposed silver grains representing the hybrid signal appear as clusters of white grains in the dark field view. Insulin-like growth factor I mrna is not detected, despite exposure of the sections for 40 days. Insulin -like growth factor II mrna is abundant in the endothelium and adventitia of testicular blood vessels (V) and the peritubular cells surrounding the seminiferous tubules and in some loose connective tissue cells (arrowheads). It is not detected in Leydig cells (L) or germinal epithelium (G). Bar, 50 I'm. died of trauma. To elucidate the function of the I G F system in the human gonad, we have compared IGF system localization in the testis with our previously published study of IGF system in the human ovary (10). MATERIALS AND METHODS Human testes were obtained through the National Disease Research Interchange (Philadelphia, PA) in accordance with a protocol for the use of human tissues approved by the Clinical Research Subcommittee of National Institute of Child Health and Human Development (National Institutes of Health, Bethesda, Maryland). The testes examined in the present study included five specimens obtained from healthy men ages 20, 24, 28, 35, and 44 years, who died oftrauma. Their bodies were refrigerated shortly after death, and tissue was snap-frozen 6 to 18 hours after death and stored at -70 C until sectioning. Serial10-J.lm-thick sections were cut at -15 C and thaw-mounted onto poly-llysine-coated slides. Clones Used for Riboprobe Synthesis The human IGF -I clone was a 442-base pair (bp) PstI-Rsal fragment subcloned into the plasmid vector pgem-3 (11). The human IGF-II clone consisted of 556-bp PstI-AccI fragment (12). The human IGF-I receptor clone contained 379-bp EcoRI Xhol fragment coding for part of the a-subunit of the receptor (13). The human type II IGF receptor riboprobe was generated by subcloning a 500-bp EcoRI -HindlII fragment of the corresponding complementary DNA (cdna) into pgem-2 (11). The human IGFBP-1 probe was synthesized from a PstI/BamHI coding region fragment from IGFBP- 1 cdna (14) subcloned into pgem3zf. The rat IGFBP-2 clone consisted of a 585-bp fragment corresponding to nucleotides 502 to 1,087 (15). The rat IGFBP-3 clone was a 440-bp coding region EcoR1-BamHl fragment (16). The human IGFBP- 4 probe was synthesized from a 462-bp cdna corresponding to the midportion of the proteincoding region (17). The mouse IGFBP-5 clone was a 463-bp fragment encoding the first 150 amino acids of the mature protein (Rotwein P, unpublished observations). These rodent clones all contain regions that are conserved between the species, and each one hybridizes to bands of the predicted size on Northern blots of human liver or placenta RNA (Zhou J, Bondy C, unpublished data). The human IGFBP-6 probe was derived from a 267-bp PstI fragment subcloned into Bluescript SK (18). In Situ Hybridization 35S-labeled RNA probes were synthesized to a specific activity of approximately equal to 2 X 10 8 disintegrations per mini J.lg in a protocol that has been previously described (19). Equal concentrations of labeled probe (l06 counts per min/100 J.lL) were applied to all sections, which were hybridized 898 Zhou and Bondy Human testis IGF system Fertility and Sterility

3 Figure 2 Insulin-like growth factor receptors gene expression in the seminiferous tubules of human testis. Both IGF-I (IR, A and B) and IGF H receptor mrnas (HR, C and D) are concentrated in the germinal epithelium, including Sertoli cells and primary spermatocytes, but little receptor mrna is detected in more mature spermatocytes or in the spermatids. Bar, 50 folm. and washed as previously described. Sections were first exposed to autoradiographic film screening to determine appropriate times for exposure to the photographic emulsion, which ranged between 10 and 30 days. RESULTS Insulin-like growth factor I mrna is not detected in the human testis; this negative finding is Vol. 60, No.5, November 1993 not due to an ineffective IGF-I probe because this same probe detects abundant IGF-I mrna in the proliferative endometrium (Zhou J, unpublished data). Insulin-like growth factor II mrna is localized in the endothelium and adventitia of testicular blood vessels and in the peritubular cells surrounding the seminiferous tubules but is not detected in the germinal epithelium or Leydig cells (Fig. 1A and B). Insulin-like growth factor I and IGF-II receptor mrnas are both most abundant in the germinal Zhou and Bondy Human testis IGF system 899

4 Figure 3 Distribution of IGFBP-2 to 5 mrnas in the human testis. Insulin-like growth factor binding protein- 2 (A and B) mrna is abundant in 8ertoli (8) and Leydig cells (L). Binding protein-3 mrn A (C and D) is detected only in the endothelium of testicular blood vessels (v). Binding protein-4 mrna (E and F) is also localized in endothelium of testicular blood vessels (v) but, in addition, is present in the interstitial compartment (IN). Binding protein-5 (G and H) mrna is also concentrated in the interstitial connective tissue (CT) and Leydig cells. Bar, 50 ~m. epithelium, especially in the large, pale staining, immature primary spermatocytes localized along the basal border of the seminiferous tubule, with little detected in more mature, smaller spermatocytes localized closer to the tubular lumen or in spermatids (Fig. 2A to D). Insulin-like growth factor I and IGF-II receptor mrnas are distinctly less abundant in the interstitial compartment and are inconsistantly detected in Leydig cells. 900 Zhou and Bondy Human testis IGF system Insulin -like growth factor binding protein-1 mrna is not detected, but IGFBPs-2 to 6 mrnas each demonstrate a distinctive testicular distribution. Binding protein-2 mrna is focally localized in Leydig and Sertoli cells (Fig. 3A and B), but little, if any, is detected in spermatocytes or interstitial connective tissue. Binding protein-3 mrna is detected only in the endothelium oftesticular blood vessels (Fig. 3C and D). Bindingprotein-4 mrnais Fertility and Sterility

5 Figure 3 (Continued) also localized in endothelium of testicular blood vessels but, in addition, is present in Leydig cells and connective tissue (Fig. 3E and F). Binding protein-5 mrna is highly abundant in the connective tissue between seminiferous tubules and in some Leydig cells (Fig. 3G and H). Binding protein-6 mrna is present in some peritubular cells and in some cells of the interstitial compartment (Fig. 4). The cellular distribution of IGF system mrnas in the human testis is summarized in Table 1. Vol. 60, No.5, November 1993 DISCUSSION To our knowledge, this is the first in situ hybridization study of IGF system expression in the human testis. Two studies have evaluated IGF-I and IGF-I receptor immunoreactivity in the human testis (3,4). Both reported that IGF-I immunoreactivity was concentrated in Sertoli cells, a few spermatocytes, and a few Leydig cells, and that IGF-I receptor immunoreactivity was localized primarily Zhou and Bondy Human testis IGF system 901

6 Figure 4 Binding protein-6 mrna is localized in peritubular cells (arrowhead) and in some cells of the interstitial compartment. Bar, 50 /Lm. in spermatocytes and a few isolated Leydig cells. The in situ hybridization and immunocytochemical findings agree with respect to the receptor localization, placing IGF-I receptor mrna most abundantly and consistently in developing spermatocytes and occasionally in Leydig cells. The co-localization of receptor mrna and protein is expected because the expression of the receptor is confined to the cell in which it is synthesized. Because, however, IGF-I is constitutively secreted in tandem with its synthesis and is not stored insidethe cells in which it is synthesized, the peptide's mrna and immunoreactivity are frequently dissociated. For example, IGF-I immunoreactivity is not detected in hepatocytes, the site of highest mrna levels and peptide production of any rat tissue, unless colchicine treatment is used to arrest secretion (20). Thus, the IGF-I immunoreactivity detected in seminiferous tubules is likely to be due to accumulation of IGF-I from the circulation, which may bind to IGFBP-2 or the IGF-I receptor localized in the germinal epithelial cells. Alternatively, because there is significant cross-reactivity in the immunological detection ofigf-i and IGF-II, the immunoreactive peptide detected within the human seminiferous tubule could be IGF-II derived from local testicular synthesis. Insulin-like growth factor II mrna is abundant in peritubular and vascular cells of the human testis. A study using prepubertal rat testicular cells found cultured peritubular cells producing mainly BP-3, whereas Sertoli cell-enriched populations yielded mainly BP-3 (8). The authors (Zhou J, Bondy C, unpublished data) confirm that IGFBP-2 mrna is concentrated in rat peritubular cells, but we have not yet evaluated IGFBP-310calization in the rat. Thus, it is apparent that there are significant differences between the rat and human in terms oftesticular IGF system gene expression,just as is the case with the ovary (10, 19). All the known physiological actions ofigf-i and IGF-II are mediated by the IGF-I receptor. This receptor is a transmembrane protein with ligandactivated tyrosine kinase activity, similar in structure and function to the insulin receptor (21, 22). Insulin -like growth factor I receptor mrn A is most abundant in the cytoplasm of large primary spermatocytes that are localized close to the base and away from the lumen of the seminiferous tubule. Little receptor mrna is detected in the smaller, more mature spermatocytes and spermatids closer to the luminal surface. Because primary spermatocytes are still on the abluminal side of the Sertoli cell barrier (23), it is likely that IGFs from the circulation or interstitium may reach them. Primary spermatocytes are engaged in active DNA and protein synthesis in preparation for meiosis; thus, IGF Table 1 Summary of IGF System Gene Expression in the Human Testis IGF-I IGF-II IGF-II receptor receptor IGFBP2 IGFBP3 IGFBP4 IGFBP5 IGFBP6 Spermatocytes Sertoli cells Leydig cells -/ -/ Connective tissue Blood vessel -/ 902 Zhou and Bondy Human testis IGF system Fertility and Sterility

7 stimulation may enhance spermatocyte biosynthetic activity. The IGF-II receptor, which has an entirely different structure from the IGF-I receptor, binds with high affinity to IGF-II and also binds mannose-6-phosphate groups, serving as a lysosomal enzyme transport protein (24). The role of this receptor in IGF-II-related signal transduction is still unclear. Insulin-like growth factor II receptor gene expression, similar to that of the IGF-I receptor, is localized primarily in primary spermatocytes. It is possible that this receptor functions in lysosomal enzyme trafficking during spermatocyte development, although its co-expression with the IGF-I receptor suggests a significant relationship with IGF action. We have also found co-localization of IGF-I and IGF-II receptor gene expression in placenta (25). Interestingly, IGF-I receptor mrna is also highly abundant in human and rat oocytes (19, 10), suggesting that IGFs playa role in both oocyte and spermatocyte development. Other elements of the IGF system demonstrate similar patterns of expression in analogous cell types in ovary and testis. Insulin-like growth factor bindingprotein-2 mrna is localized in granulosa and theca cells in the human ovary and in the Sertoli and Leydig cells of the human testis. Insulin -like growth factor binding protein-3 mrna is localized in vascular endothelium and IGFBP-4 and -5 in endothelium and interstitial tissue in both types of gonad. Although it is not known how these binding proteins function with respect to IGF action in local tissues, their similar pattern of expression suggests that their functions are similar in ovary and testis. Almost all of the IGF peptide present in the circulation is complexed with IGFBP-3, which binds both IGF-I and IGF-II with significantly higher affinity than does the IGF-I receptor. 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