Testis Epidermal Growth Factor and Spermatogenesis

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1 Archives of Andrology Journal of Reproductive Systems ISSN: (Print) (Online) Journal homepage: Testis Epidermal Growth Factor and Spermatogenesis Y.-C. Yan, Y.-P. Sun, M. L. Zhang & S. S. Koide To cite this article: Y.-C. Yan, Y.-P. Sun, M. L. Zhang & S. S. Koide (1998) Testis Epidermal Growth Factor and Spermatogenesis, Archives of Andrology, 40:2, , DOI: / To link to this article: Published online: 09 Jul Submit your article to this journal Article views: 159 Citing articles: 27 View citing articles Full Terms & Conditions of access and use can be found at

2 TESTIS EPIDERMAL GROWTH FACTOR AND SPERMATOGENESIS Y.-C. YAN Y.-P. SUN M. L. ZHANG Laboratory of Molecular Cell Biology, Shanghai Institute of Cell Biology, The Chinese Academy of Sciences, Shanghai, China S. S. KOIDE Center for Biomedical Research, Population Council, New York, New York, USA Epidermal growth factor (EGF) is a cytokine that promotes cell proliferation, regulates tissue differentiation, and modulates organogenesis. Although a rich source of EGF is the submaxillary gland, many tissues produce this cytokine, including the testis. Leydig cells are the principal source of EGF in the testis. On attainment of sexual maturation the germ cells, primarily spermatocytes and round spermatids, form EGF with the onset of spermatogenesis. EGF appears to be involved in the development of the testis and in spermatogenesis. The expression of the EGF gene in rat testis was determined by the application of the RT-PCR method and testis RNA as substrate. The results suggest that EGF produced by Leydig cells and germ cells may modulate spermatogenesis as an autocrine andor paracrine factor. Keywords cytokine, Leydig cells, spermatogenesis, submaxillary gland, testis The principal biological function of the testis is to produce sperm for the purpose of fertilizing the ova. Although gonadotrophins and testosterone are essential for the maintenance of normal mammalian spermatogenesis, cytokines, including epidermal growth factor (EGF), transforming growth factor-a (TGFa), fibroblast-like growth factors, insulin-like growth factors, interleukins, endorphins, inhibin, activin, mullerian inhibiting substance, and Sertoli cellsecreted growth factor, are produced by the testis and have been implicated as regulators of spermatogenesis [23]. They modulate organogenesis and complement the regulatory influence of the nervous system. One of the cytokines, EGF, is formed in the testis and is the topic of this review. Experimental data are presented, showing that the principal source of EGF in the testis is the Leydig cell and that the EGF gene is expressed early during the development of the fetal testis. Germ cells also produce EGF on attainment of sexual maturation and the onset of Address correspondence to S. S. Koide, Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021, USA. ARCHIVES OF ANDROLOGY 40: (1998) Copyright Taylor & Francis /98 $ OO 133

3 134 Y.-C. Yan et al. spermatogenesis. Testicular EGF may modulate spermatogenesis as an autocrine or a paracrine factor. PROPERTIES OF EGF EGF has been purified from the submaxillary gland (SMG) of male mouse and human urine and designated as urogastrone [6]. Kidney is probably the tissue source of urogastrone. EGF is a glycosylated polypeptide composed of 53 amino acid residues with an estimated M, of 6045 [28]. It promotes proliferation and differentiation of epithelial and somatic cells [I, 10, 22, 391 and is a potent inhibitor of HCl secretion by the stomach mucosa cells [ 101. The tissue sources of EGF are the tubular cells of mouse SMG; acinar cells of human SMG; epithelial cells of the renal proximal tubules, prostate gland, and seminal vesicle; glycopeptide-secreting cells of the anterior pituitary; and gastric and pyloric cells of the stomach, human duodenal glands, and the testis [4, 6, 16, 24, 25, 29, 401. The family of EGF-like factors includes the transforming growth factor-a (TGF,), pox virus growth factors, and amphiregulin. The members share three common characteristics: highaffinity binding to EGF receptor (EGFR), they trigger mitogenic response in sensitive cells, and they consist of 6 half cystines (three disulfide bonds) in their primary structure [6, 10, 281. Human EGF (53 amino acid polypeptide) is cleaved from a precursor molecule designated as prepro-egf, consisting of 1217 amino acid residues with an estimated M, of 113,000 and exists in cells as a glycosylated membrane protein. The EGF molecule is flanked by a 976 amino acid peptide segment at its amino terminus and a 188 amino acid peptide segment at the carboxyl terminus [28]. EGF-INDUCED BIOLOGICAL RESPONSES The primary biological effect of EGF in the intact animal and on cultured cells is to facilitate cell proliferation [l, 6, 101. EGF is a potent mitogen for cells and tissues of ectodermal and mesodermal origin, including chondrocytes, endothelial cells, conjunctival tissues, fibroblasts, granulosa cells, mammary glandular cells, liver cells, pharyngeal tissue, skin keratinocytes, thyroid follicular cells, and vascular smooth muscle cells. The biological responses to EGF are multiple. It invokes tooth opening in neonatal rodents, facilitates lung maturation in fetal mammals, promotes palatal development in organ culture, stimulates thyroid and adrenal growth, accelerates wound healing, supports mammary gland development, triggers pituitary secretion of adrenocorticotropic hormone (ACTH), prolactin, and growth hormone, and induces placental tissues to secrete choriogonado-tropin and lactogen. EGF AND MALE REPRODUCTIVE FUNCTION Tsutsumi et al. [38] showed that ablation of SG (sialoadenectomy) of adult male mice caused a marked decrease in the circulatory concentration of immunoreactive EGF, associated with a marked reduction in epididymal sperm count and in the number of spermatids in the testis. The administration of EGF to the sialoadenectomized mice restored the sperm count of the epididymis and spermatid number in the testis to normal values [2, 17, 341. Russell et al. [27] also demonstrated that sperm production parameters were lower in sialoadenectomized mice compared to sham-operated mice; however, the differences were not statistically significant and histological examination of the testis showed normal spermatogenesis.

4 TESTIS EGF Epidermal Growth Factor and Spermatogenesis 135 Tissue Distribution of Exogenous EGF in the Rat 12%EGF was administered IV to male rats and the radioactivity in various tissues was determined [15]. At 2.5 min after the injection, the liver had the highest radioactivity (52%), whereas the testis uptake was less than I%, indicating that circulating EGF has minimal influence on the testis. Rodent Testis EGF was identified in mouse testis by Radhakrishnan et al. [25] by metabolic labeling with [35S]methionine and analyzing the products by fluorography. Mouse testis contained the EGF precursor (EGFp), having an estimated Mrof 140,000 and 50,000, and the mature EGF (EGF,) with an estimated MI of The identity of EGFs was verified by immunoblot using specific antibodies against EGF. Both EGFp and EGF, were localized in Sertoli cells, pachytene spermatocytes, and round spermatids, while Sertoli cells contained only EGF,. By immunohistochemical staining, EGF was localized primarily to the Leydig cells and to a few spermatogonia, spermatocytes, and round spermatids of adult mouse and rat testis [36]. In immature rodent testis, EGF was localized only to the Leydig cells. EGF in Human Seminal Plasma and Testis A component with EGF-like activity was detected in human seminal plasma by isolating the factor by HPLC and assayed with a heterologous radioreceptor assay using murine radioiodinated EGF as ligand 191. Only the microsomal membranes of testis possessed the capacity to bind radiolabeled EGF. Although the membranes prepared from human prostate, seminal vesicle, epididymis, and spermatozoa were inactive, a component with EGF-like activity was detected in these tissues. EGF in Boar Testis EGF was immunolocalized in the germ cells and peritubular cells of postnatal and adult boar testis [7]. Various germ cells and somatic cell types of the testis contain EGF receptors, indicating that this cytokine maybe involved in spermatogenesis, particularly during the stages of meiosis and spermiogenesis. EGF in Seminoma Cells of seminoma stained intensely with anti-egf antibodies [35]. EGF produced by the seminoma may contribute to its invasive activity. Determination of EGF in testicular tumors may be useful in the staging of their malignancy. EGF Receptor The EGF receptor (EGFR) is a transmembrane glycoprotein with an estimated M, of 170,000 and is composed of a single polypeptide chain of 1186 amino acid residues, containing substantial amounts of N-linked oligosaccharides [6, 101. The extracellular ligand-binding domain and the cytoplasmic segment with the intrinsic tyrosine kinase activity are separated by a single hydrophobic membrane anchor sequence. The tyrosine kinase activity of the cytoplasmic segment is central to the cell proliferative activity of EGF. The extracellular domain contains 10-

5 136 Y.-C. Yan et al. 11 N-linked oligosaccharide chains and a high content of half-cystine residues. It interacts with EGF and other family members with high affinity. The distinguishing trait of the cytoplasmic segment of EGFR is the peptide sequence defining the tyrosine kinase domain. Substrates of the kinase include phospholipase, guanine nucleoside triphosphatase-activating protein, microtubule-associated protein kinase, rat kinase, lipocortin I, and c-erbb-2. These substrates have known functions and may mediate the EGF signal transduction pathway. EGF receptors are found in most cells types, including brain, thyroid, lung, skin, placenta, and fetal membrane, with the exception of hematopoietic cells. Rat Testis EGFR EGFR is expressed in a wide variety of cell types including testes of many species. Interstitial cells were prepared from intact and Leydig cell-depleted immature rat testis by treatment with ethane dimethane sulfonate (EDS) to assess their capacity to bind IZ5I-EGF, to incorporate [3H]thymidine into DNA and to produce testosterone [19]. Those Leydig cells capable of binding I2jI-hCG and possessing 3 P-hydroxysteroid dehydrogenase activity did not bind to 9-EGF. The fusiform mesenchymal cells isolated from EDS-treated testis bind IZ5I-EGF. The findings suggest that EGF action is on precursor Leydig cells rather than on the mature type. Mouse Testis EGFR EGFR was immunolocalized specifically in Leydig cells and Sertoli cells of mouse testis using a polyclonal anti-egfr antibody [33]. Immunoblot of membranes prepared from murine Sertoli and Leydig cell lines showed a prominent immunoreactive 170-kDa band which was also detected in a testis membrane preparation. The findings suggest that EGF may modulate spermatogenesis by acting on Leydig and Sertoli cells. EGFR in Monkey Testis EGFR was identified in the membrane preparations of testis, epididymis, and vas deferens of monkeys by immunoblot using a polyclonal antibody raised against a peptide-specific sequence of the intracellular domain of human EGFR [26]. The EGFR corresponded to a 170- kda band. Positive immunostaining of EGFR occurred in the Leydig cells, Sertoli cells, and peritubular cells of testis, and the basolateral and luminal borders of the epithelium of the epididymis and vas deferens. EGFR in Human Testis Leydig cells and peritubular cells of human testis showed positive immunoreactivity for EGF and EGF receptor [21, 321. An EGF receptor with a high-affinity, low-capacity, single binding site was located in the 105,OOOg particulate fraction of human testicular tissue [32]. Leydig cells showed strong TGF, immunostaining, while that of Sertoli cells was weak, which increased in association with a reduction in spermatogenesis; e.g., strong immunoreactivity was elicited with testis from Sertoli cell-only syndrome, suggesting a possible compensatory mechanism. On the other hand, EGF immunoreactivity was not associated with the degree of spermatogenesis. The findings suggest that TGF, is more directly involved with spermatogenesis than EGF.

6 EGFR and Human Infertility Epidermal Growth Factor and Spermatogenesis 137 Sertoli cells, peritubular basal cells, interstitial cells and germ cells in normal human testis showed weak immunostaining for EGFR [l 1,121. In spermatogenesis arrested testis, the immunostaining was intense and correlated with the reduced or arrested spermatogenesis as in Sertoli cell-only syndrome. Following treatment with FSH, the immunostaining reaction for EGFR on Sertoli and germ cells of the testis was intensified. The present findings suggest that EGF is involved in the growth and differentiation of germ cells and that FSH may modulate EGF expression. EGF ACTION ON THE TESTIS Spermatogenesis The effect of EGF on germ cell differentiation in the testis was studied using testis fragments obtained from surgically prepared cryptorchid testes in adult male mice and cultured for 9 days in a serum-free medium [14]. EGF added to the medium stimulated testis germ cell differentiation, whereas proliferation of type A spermatogonia induced by FSH was reduced. The results suggest that EGF modulates spermatogenesis by promoting differentiation of germ cells and reducing proliferation of spermatogonia. Stage synchronization of seminiferous epithelium of rat testis was regulated by the withdrawal and replenishment of vitamin A [3]. After initiation of stage synchrony of spermatogenesis, increased testicular EGF concentrations occurred after stage IX that correlated with the mitotic division of type A spermatogonia at stages IX, XII, and XIV of the cycle of the seminiferous epithelium. Retinol administered to control and vitamin A-deficient animals induced a marked increase in insulin-like growth factor I in the testis. Leydig Cells EGF may regulate testicular metabolism by acting as an autocrine factor in Leydig cells. EGF added to cultured porcine Leydig cells decreased gonadotropin receptor density, receptor mrna, and mrna levels encoding 17-a hydroxylase [S]. By these actions, EGF may modulate FSH action in stimulating steroidogenesis carried out by the Leydig cells of the testis. EGF added to a culture of purified porcine Leydig cells from immature intact animals inhibited hcg-induced dehydroepiandrosterone accumulation in the medium, whereas testosterone production was increased [3 13. The study shows that EGF enhances gonadotropin action on testosterone formation by increasing the availability of cholesterol for steroidogenesis in the mitochondria and by increasing the 3 P-hydroxysteroid dehydrogenase/isomerase activity. The interstitial fluid of rat testis contains detectable amounts of insulin-like growth factor I (IGF-I) and IGF binding proteins [20]. Insulin and EGF may act synergistically in stimulating [3H]thymidine incorporation into DNA of cultured interstial cells, suggesting that a biological response to EGF plus insulin is to facilitate proliferation of Leydig cells. Sertoli Cells and Protein Phosphorylation The signal transduction pathway of EGF action may evolve a cascade of protein phosphorylation steps. Sertoli cells isolated from immature rat testes express follistatin mrna [ 181. Both EGF and phorbol- 12-myristate-l3-acetate, an activator of protein kinase C, stimulates the

7 138 Y.-C. Yan et al. expression of follistatin gene, suggesting that action of EGF may involve protein kinase C pathway. A 56-kDa protein kinase (p56 KKIAMRE) related to the proline-directed protein kinase group of signal transducing enzymes is expressed in testis and activated by EGF [37]. The cytoplasmic tyrosine kinase activity of EGF, protein kinase C, and the 56 KKIAMRE may form a protein phosphorylating cascade in the signal transduction of EGF action. EGF and FSH Binding in the Mouse Testis Mouse EGFR and human FSH possess two homologous tetrapeptide sequences located in the hormone-specific P-subunit of FSH and within the 53-amino acid EGF purified from mouse SMG [30]. Mouse EGF and the two tetrapeptides blocked the binding of '"1-FSH to testis FSH receptors, suggesting that EGF acts in the testis by inhibiting FSH action. EGF IN DEVELOPING TESTIS Human Fetal, Adult, and Cryptorchid Testes Mouse anti-human EGF monoclonal antibody (Clone S 147) was used to immunolocalize EGF in testicular cells. Leydig cells in the testis from a 26-week old human fetus stained weakly (Figure la), whereas those in adult testis stained intensely (Figure 1B). In addition, some spermatogonia and a few peritubular myoid cells were stained. In cryptorchid testis, the seminiferous tubules were involuted and composed of arrested spermatogonia and clusters of Leydig cells in the interstitial regions (Figure IC). Staining of the interstitial cells was variable, ranging from weak to strong reactions (Figure 1D). EGF was immunolocalized in Leydig cells of fetal, adult, and cryptorchid testis. In the adult testis spermatocytes and spermatids were stained, showing that EGF is produced by germ cells with the occurrence of spermatogenesis. Developing Rat Testis Testis from days 15, 45, and 70 postnatal (pn) male rats were immunostained for EGF (Figure 2). Leydig cells in the testis of all three groups stained positively. In mature testis (day 70 pn group), spermatogonia and spermatocytes were also immunostained. Developing Mouse Testis Sections of testis from day 15 and 45 pn mice were immunostained for EGF (Figure 3). Leydig cells in testis from both groups showed positive reaction. Spermatogonia, spermatocytes, round spermatids, and peritubular myoid cells were stained in the testis from day 45 pn group (Figure 3C), showing that EGF is produced in germ cells with the onset of spermatogenesis. The above experimental studies support the thesis that EGF production in Leydig cells occur early in development, correlating with cell proliferation. EGF production in germ cells, however, occurs following the onset of spermatogenesis in association with meiosis and spermiogenesis. Development of Fetal Mouse Testis EGF may regulate testosterone-dependent fetal Wolffian duct differentiation [ 131. An immunoreactive 180-kDa component corresponding to EGFR was detected in the 18-day fetal male mouse reproductive tract by Western blot using a polyclonal anti-egfr antibody. Anti- EGFR antibody added to a culture of male reproductive tract blocked Wolffian duct differen-

8 Epidermal Growth Factor and Spermatogenesis 139 Figure 1. Immunolocalization of EGF in human fetal, adult and cryptorchid testes. (A) 26-week old fetal testis. Note intense (A) and weak (A) staining of Leydig cells, ~250. (B) Adult male testis. Note staining of Leydig cells (A), spermatogonia (A), and peritubular myoid cells (T), ~250. (C) Cryptorchid testis. Note staining of Leydig cells (A) in clusters, x 100. (D) Cryptorchid testis. Note varying staining intensities of Leydig cells: intense staining (A) and weak staining (A), ~250.

9 140 Y.-C. Yan et al. Figure 2. Immunolocalization of EGF in the testes of rats at different developmental stages. (A) Testis of 15-day postnatal rat. Note staining of Leydig cells (A), x50. (B) Testis of 45-day postnatal rat. Note staining of Leydig cells (A), x50. (C) Testis of 70-day postnatal rat. Note staining of Leydig cells (A), spermatogonia (A), and spermatocyte (?), ~100. (D) Testis of 70-day postnatal rat. Note staining of Leydig cell (A), spermatogonia (A), and spermatocyte (?), ~200.

10 141 Figure 3. Immunolocalization of EGF in the testes of mice at different development stages. (A) Testis of 15-day postnatal mouse. Note staining of Leydig cells (A), 60. (B) Testis of 45-day postnatal mouse. Note staining of Leydig cells (A), ~50. (C) Testis of 45-day postnatal mouse. Note staining of Leydig cells (A), spermatogonia (A), and spermatocyte (+), round spermatids (O), and peritubular myoid cells (?I, ~200.

11 142 Y.-C. Yan et al. tiation induced by fetal testis. Anti-EGFR antibody also prevented development of the Wolffian duct induced in female explants by exogenous EGF. The results suggest that EGFR mediates the action of EGF in promoting differentiation of the male reproductive tract. EGF and Cryptorchidism EGF administered to pregnant mice treated with flutamide, an antiandrogen, significantly decreased the incidence of undescended testes (74 to 24%) and epididymal anomalies (53 to 9%) [5]. EGF expression in the fetal testes or epididymis, however, was not altered by antiandrogen treatment. TESTIS EGF GENE Expression of the EGF gene in mouse testis was assessed by synthesizing the cdna using the RT-PCR method with specific synthetic oligonucleotides corresponding to segments found in EGF and utilizing mouse testis RNA as substrate [41]. Testis EGF cdna was separated by agarose gel electrophoresis, isolated, and sequenced. It was composed of 279 bp similar to that of SMG gene (Figure 4). To determine the developmental stage when the EGF gene is expressed, cdna was synthesized using RNA preparations from testes of male mice on days 15, 30, and 45 pn and the amounts were compared (Figure 5). The amount of EGF cdna synthe bp Figure 4. Electrophoresis analysis of EGF cdnas synthesized by the RT-PCR method using mouse testis and submaxillary gland (SMG) RNA. cdnas were synthesized by the RT-PCR method for 40 cycles and separated by electrophoresis on a 2% agarose gel. The cdna bands were stained with ethidium bromide. Lane 1, cdna synthesized from SMG RNA; lane 2, cdna synthesized from testis RNA; lane 3, polynucleotide molecular size markers: 657, 458, 434, 328, 289, 267, 174, 142, 102, 80 bp. The 279-bp band correspond to EGF.

12 Densitonietric Unit Epidermal Growth Factor and Spermatogenesis pn n " pg 2.5 P9 RNA Figure 5. Plot of the amounts of cdnas synthesized from RNAs prepared from mouse testis at different stages of development. RNAs were prepared from the testis of mice at days 15, 30, and 45 postnatal (pn). PCR amplification of serially diluted mrnas was performed for 40 cycles bp * 279 bp Figure 6. Electrophoresis analysis of EGF and (3-actin cdnas synthesized from RNAs prepared from mouse testis at different stages of sexual development. The RT-PCR method was performed in two steps. In the first step, reverse transcription of EGF and (3-actin RNAs was performed separately. In the second step, both cdnas were coamplified by PCR. Lane 1, cdna synthesized from mrna prepared from testis of day 45 pn mouse; lane 2, testis from day 30 pn mouse; lane 3, testis from days 15 pn mouse; lane 4, polynucleotide molecular size markers: 1543, 994, 695, 515, 377, 237 bp. The 279- and 550-bp bands correspond to EGF and (3-actin cdnas, respectively.

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