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Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)
Animal Reproduction Science 116 (2009) 155–161
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos) Junping He a, Changsheng Dong a,∗, Rongli You a, Zhiwei Zhu a, Lihua Lv a, George W. Smith a,b a
College of Animal Science and Technology, Shanxi Agricultural University, Taigu, Shanxi 030801, PR China Laboratory of Mammalian Reproductive Biology and Genomics, Departments of Animal Science and Physiology, Michigan State University, East Lansing, MI, USA
b
a r t i c l e
i n f o
Article history: Received 25 July 2008 Received in revised form 7 January 2009 Accepted 14 January 2009 Available online 20 January 2009 Keywords: Alpaca Epidermal growth factor Epidermal growth factor receptor Immunohistochemistry Testis
a b s t r a c t The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-monthold alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Postnatal development and function of the testes is controlled by a complex interaction of circulating gonadotropins with cytokines and growth factors (Skinner, 1991). Evidence supports a potential ∗ Corresponding author. Tel.: +86 354 628 8208. E-mail address: [email protected] (C. Dong). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.01.002
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regulatory role for epidermal growth factor (EGF) and the EGF receptor (EGFR) in control of testicular function in mice, rats and livestock species. EGF is present in the circulation of adult male mice at concentrations of approximately 5 ng/ml. The main tissue source of EGF is the submandibular glands (Suarez-Quian et al., 1994). Removal of the submandibular glands (sialoadenectomy) results in a decrease in circulating EGF to undetectable concentrations and is accompanied by an approximately 50% reduction in number of mature sperm in the epididymis and number of spermatids in the testes (Tsutsumi et al., 1986; Liu et al., 1994). Effects of submandibular gland removal on spermatogenesis are restored by EGF supplementation (Tsutsumi et al., 1986; Liu et al., 1994). Such results support a regulatory role for EGF in control of spermatogenesis, but also support regulation independent of circulating EGF. Based on results of transgenic and gene targeting studies, the role of EGF in regulation of male fertility is unclear or somewhat controversial. Male EGF null mutant mice do not have reduced fertility (Luetteke et al., 1999), but male transgenic mice overexpressing the EGF gene are infertile (Wong et al., 2000). The functional requirement of the EGFR is difficult to conclusively interpret because EGFR knockout mice display varying phenotypes, including prenatal or early postnatal mortality, depending on the background strain of mice utilized (Wong, 2003). Despite the lack of an overt reproductive phenotype in male EGF knockout mice, abundant evidence supports a potential local regulatory role for EGF in humans, rats and several agricultural species. Potent EGF regulation of multiple aspects of somatic and germ cell function (e.g. DNA synthesis, Leydig cell steroidogenesis) has been reported for human, rat and (or) porcine testicular cell types (Sordoillet et al., 1991; Syed et al., 1991; Nehar et al., 1993; Onoda and Suarez-Quian, 1994; Wahab-Wahlgren et al., 2003). EGF has been localized to Leydig cells of human testes (Nakazumi et al., 1996), in both germ and somatic cells of boar testes (Caussanel et al., 1996), and exclusively to the germ cells of bull testes (Kassab et al., 2007). Furthermore, the EGF receptor has been localized specifically to the Leydig and Sertoli cells of rat and mouse testes (Suarez-Quian et al., 1989; Suarez-Quian and Niklinski, 1990), in the Sertoli cells, peritubular cells and germ cells of human testes (Nakazumi et al., 1996), in both germ cells and somatic cells in boar testes (Caussanel et al., 1996) and specifically to germ cells in bull testes (Kassab et al., 2007). Knowledge of regulation of male reproductive (testicular) function in South American camelids, such as the alpaca, which exhibit a highly variable and late age at puberty is limited (Tibary and Vaughan, 2006). To our knowledge, the intratesticular localization of growth factors and growth factor receptors, such as EGF and EGFR, which may modulate testicular development/function in the alpaca, has not been examined previously. Hence, the objective of the present studies was to determine the temporal and cell specific localization of EGF and EGFR at two specific stages of postnatal development of the alpaca testes. 2. Materials and methods 2.1. Animals and tissue collection/processing Housing and care of animals and collection of testicular tissue for use in described experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals http://www.cioms.ch/frame 1985 texts of guidelines.htm. Testicular tissue was harvested from male alpacas at 12 and 24 months of age after castration under local anesthesia (n = 3 animals per group). Small pieces of testicular tissue (3–5 mm3 ) were fixed in Bouin’s solution for 24 h at 4 ◦ C and extensively washed in 70% ethanol. Thereafter, tissue samples were dehydrated in a graded series of ethanol (85%, 95% and 100%), cleared in xylene and embedded in paraffin wax. Sections (6 m) of testicular tissue were prepared and mounted onto 2% 3-aminopropyltriethoxysilanecoated slides for immunohistochemical localization of EGF and EGFR or haemotoxylin and eosin staining. 2.2. Immunohistochemical staining A polyclonal rabbit anti-EGF antibody and a polyclonal rabbit anti-EGFR antibody (Wuhan Boster Biological Technology Ltd., Wuhan, China) were utilized for described localization studies. All incuba-
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tions were performed in a humidified chamber. Sections of paraffin embedded testicular tissue were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 10 min. After three rinses (3 × 5 min each) in phosphate-buffered saline (PBS, pH 7.4), sections were incubated with 10% normal goat serum at room temperature for 20 min to block non-specific binding, and incubated overnight at 4 ◦ C in the presence of polyclonal rabbit anti-EGF or polyclonal rabbit anti-EGFR serum diluted 1:100 in PBS. For determination of non-specific staining, primary antibody was replaced by normal rabbit serum. Sections were rinsed in PBS (3 × 5 min) and incubated in the secondary antibody (polymerized HRP conjugated donkey-anti-rabbit IgG; Tianjin Haoyang Biological Manufacture Co., Ltd., Tianjin, China) at 37 ◦ C for 30 min, and washed 3× in PBS again (5 min each). Immunoreactivity was visualized by incubating sections in the presence of DAB substrate. The sections were counterstained with haemotoxylin and coverslips were sealed with neutral balsam. After completion of immunostaining, sections were examined using a Leica DMIRB (Leica, Wetzlar, Germany) computerized microscope with a Leica DFC 320 digital camera (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK) and images were captured and stored for analysis. The results of immunohistochemical staining are described as negative (no specific staining), weak (light brown staining), and strong staining (deep brown staining). 3. Results 3.1. Morphological characteristics of testes The testes of 12-month-old alpacas contained a pronounced interstitium and tubular component (Figs. 1 and 2). Leydig cells with a pronounced round morphology were prominent within the interstitium. Within the seminiferous tubules, large round spermatogonia enveloped by immature Sertoli cells were evident and pronounced lumen were not distinguishable. Such morphology is consistent with a prepubertal testis phenotype. In contrast, the testes of 24-month-old alpacas exhibited a postpubertal morphology (Figs. 1 and 2). A pronounced interstitial compartment and tubular component containing seminiferous tubules with pronounced lumen containing embedded spermatozoa were evident as were distinct basal and luminal compartments within the tubules. Spermatogonia and preleptotene spermatocytes with condensed chromatin were present in the basal compartment and in direct contact with the basal lamina and Sertoli cells. Postleptotene spermatocytes and spermatids supported by Sertoli cells were observed in the adluminal compartment. Peritubular myoid cells, surrounding the seminiferous tubules, were also easily identified. 3.2. Immunolocalization of EGF Specific staining for EGF was not evident within the testes of 12-month-old alpacas (Fig. 1A). However, specific staining was prominent within the testes of 24-month-old (postpubertal) alpacas (Fig. 1B–E). Dispersed, strong staining was noted in the interstitial compartment including the Leydig cells (Fig. 1B). Within the tubular component, pronounced cell specific localization to germ cells and somatic cells was evident. Within the basal compartment, localization to peritubular myoid cells and Sertoli cells was detected (Fig. 1C–E) and staining in spermatogonia was weak or isolated. In the adluminal compartment, strong staining for EGF in Sertoli cells and in germ cells at all stages was observed (Fig. 1B–E). EGF immunoreactivity was not detected when primary antibody was replaced by normal rabbit serum (Fig. 1F). 3.3. Immunolocalization of EGFR In the testes of 12-month-old alpacas, EGFR staining was specific to the interstitium (Fig. 2A). Strong staining was noted in the Leydig cells and no specific staining within cells within the seminiferous tubules was noted (Fig. 2A). In contrast, specific staining for EGFR was detected within both the interstitium and the tubular component of the testes of 24-month-old alpacas (Fig. 2B, C, and E). Within the seminiferous tubules, strong staining within spermatogonia of the basal compartment was observed, with less prominent staining within germ cells within the adluminal compartment. Strong
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Fig. 1. Localization of epidermal growth factor (EGF) within the alpaca testis. (A) Representative micrograph of a section through testis tissue collected from a 12-month-old alpaca and incubated with anti-EGF antibody. Note absence of staining in testis tissue collected from prepubertal animal. (B–E) Representative micrographs of sections of testis tissue collected from 24-month-old alpacas and incubated with anti-EGF antibody. Note dispersed strong staining within Leydig cells in the interstitial compartment and pronounced strong staining within germ cells and Sertoli cells in the adluminal compartment of the tubules. (F) Representative micrograph of section through testis tissue collected from a 24-month-old alpaca and incubated with normal rabbit serum (negative control). Note absence of immunoreactivity. Leydig cell (Le); Sertoli cell (Se); spermatogonia (Sg); germ cell (Gc); spermatocyte (Sc); round spermatid (RS); spermatozoa (Sp); peritubular myoid cell (PM). Bar = 25 m for panels A and C–F; 50 m for panel B.
staining within peritubular myoid cells and Leydig cells within the interstitial compartment was also noted within testes of 24-month-old alpacas. Specific staining for EGFR was not detected when primary antibody was replaced by normal rabbit serum (Fig. 2D and F). 4. Discussion Results of the present studies established a pronounced developmental and cell specific regulation of EGF and EGFR expression during postnatal development of the alpaca testis. Differences in morphological characteristics of alpaca testes collected at 12 and 24 months of age suggest that samples collected are representative of a pre- and postpubertal phenotype respectively and such transition was accompanied by an induction of localization of EGF in the testis. Most notably, pronounced lumen
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Fig. 2. Localization of epidermal growth factor (EGF) receptor (EGFR) within the alpaca testis. (A) Representative micrograph of a section through testis tissue collected from a 12-month-old alpaca and incubated with anti-EGFR antibody. Note strong staining for EGFR within Leydig cells in the interstitial compartment and absence of staining in the seminiferous tubules. (B, C, and E) Representative micrographs of sections through testis tissue collected from 24-month-old alpacas and incubated with antiEGFR antibody. Note specific staining for EGFR within both the interstitium and the tubular component, with strong staining within spermatogonia of the basal compartment of the tubules, less prominent staining to germ cells within the adluminal compartment and strong staining within peritubular myoid cells and Leydig cells within the interstitial compartment. (D and F) Representative micrographs of sections through testis tissue collected from 12- and 24-month-old alpacas respectively and incubated with normal rabbit serum (negative control). Note absence of immunoreactivity. Leydig cell (Le); spermatogonia (Sg); peritubular myoid cell (PM). Bar = 25 m for panels A–D and F, 50 m for panel E.
with embedded spermatozoa awaiting release were absent within the seminiferous tubules of 12 months, but not 24-month-old alpaca testis sections respectively. Furthermore, 24-month-old alpacas utilized in present study were free of preputial adhesions, a clear indicator of sexual maturity in this species (Tibary and Vaughan, 2006). While EGFR immunoreactivity was detected within the testis tissue collected from alpacas pre- and postpuberty, developmental regulation of EGF gene expression was noted. EGF immunoreactivity was undetectable in the interstitial and tubular components of the testes of alpacas at 12 months of age (before puberty), but readily detectable in the testes (interstitial and tubular components) of 24-month-old alpacas that had attained puberty. Such results suggest an association and potential local role for EGF in pubertal maturation in the testis. Expression of EGF gene coincident with the onset of spermatogenesis has been reported in other species (Yan et al., 1998). However, absence of EGF staining in the testes of prepubertal alpacas does not eliminate a potential role for EGF during testis development prior to puberty, given evidence supporting a role for circulating EGF in regulation of testicular function in mice (Tsutsumi et al., 1986; Wong et al., 2000) and the
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potential gene expression and action of other EGF family ligands (Xian, 2007) in testes prior to puberty cannot be discounted. The EGFR was localized to Leydig cells within the testes of both 12- and 24-month-old alpacas. Evidence supports a role for EGF in regulation of Leydig cell testosterone production. In other species, cultured Leydig cells from immature animals respond to EGF with an increase in testosterone (Verhoeven and Cailleau, 1986; Sordoillet et al., 1991), suggesting that EGFR signaling pathways are indeed functional prior to puberty and in the absence of local EGF production in the testis. EGF stimulated testosterone production by Leydig cells from mature animals has also been reported (Sordoillet et al., 1991; Syed et al., 1991). Localization of EGFR to rodent, pig and human Leydig cells (Nakazumi et al., 1996; Caussanel et al., 1996; Suarez-Quian and Niklinski, 1990; Suarez-Quian et al., 1989) suggests a potential common role for EGFR in regulation of testosterone production in diverse species. However, EGFR localization to Leydig cells was not observed in the bull testis (Kassab et al., 2007), suggesting that cell specific expression and function of the EGFR in the testis is species specific. Localization of EGF and EGFR to the peritubular myoid cells surrounding the seminiferous tubules of postpubertal (24-month-old) alpacas was noted in the present studies. Functions of peritubular myoid cells are not completely understood, but such cells contain abundant actin filaments and are thought to play a role in the transport of spermatozoa and testicular fluid within the tubule (Hoeben et al., 1995). These cells also secrete a number of substances including extracellular matrix components and growth factors (Maekawa et al., 1996). To our knowledge, potential paracrine and autocrine actions of EGF on peritubular myoid cells have not been described, but pronounced actions of TGF alpha on peritubular cells have been observed in vitro (Skinner et al., 1989). Within the seminiferous tubules of postpubertal alpaca testes, EGF staining was most prominent in all germ cells of the adluminal compartment and in Sertoli cells, whereas EGFR staining was prominent within the basal compartment. Localization of additional EGF related ligands and receptors will be necessary to gain a comprehensive understanding of the potential role for EGF/EGFR superfamily members (Xian, 2007) as a whole in regulation of alpaca germ cell development. However, results of the present studies establish the potential for direct actions of EGF within the seminiferous tubules of the alpaca. Evidence in other species supports a specific role for EGF in germ cell development (Niederberger et al., 1993; Wong et al., 2000; Bartlett et al., 1990; Liu et al., 1994). For instance, removal of circulating EGF by sialoadenectomy in mice results in reduced sperm counts attributed to a reduction in preleptotene and pachytene spermatocytes and round spermatids (Liu et al., 1994). It is unclear whether such effects are truly independent of changes in circulating testosterone, but EGF stimulation of spermatogonial proliferation in isolated rat seminiferous tubules in vitro has been reported (Wahab-Wahlgren et al., 2003). Furthermore, effects of EGF on Sertoli cell function in vitro have been documented (Onoda et al., 1994; Nehar et al., 1993) suggesting the potential for indirect effects of EGF on the seminiferous epithelium mediated by EGF regulation of Sertoli cell function. In conclusion, results of the present studies demonstrate pronounced developmental and cell specific regulation of EGF and EGFR expression within the developing and postpubertal testes of the alpaca. Results support the potential for a local role for EGF in regulation of testicular maturation and function in camelids. Acknowledgements The present research was supported by a grant from the Ministry of Agriculture of the People’s Republic of China (No. 2003-Z86). We also thank Ying Wang, the general manager of the Scientific Research Base of Shanxi Agricultural University (Shanxi, China), for provision of animals used in this study. References Bartlett, J.M., Spiteri-Grech, J., Nieschlag, E., 1990. Regulation of insulin-like growth factor I and stage-specific levels of epidermal growth factor in stage synchronized rat testes. Endocrinology 127, 747–758. Caussanel, V., Tabone, E., Mauduit, C., Dacheux, F., Benahmed, M., 1996. Cellular distribution of EGF, TGF-alpha and their receptor during postnatal development and spermatogenesis of the boar testis. Mol. Cell. Endocrinol. 123, 61–69.
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Hoeben, E., Briers, T., Vanderstichele, H., De Smet, W., Heyns, W., Deboel, L., Vanderhoydonck, F., Verhoeven, G., 1995. Characterization of newly established testicular peritubular and prostatic stromal cell lines: potential use in the study of mesenchymal-epithelial interactions. Endocrinology 136, 2862–2873. Kassab, M., Abd-Elmaksoud, A., Ali, M.A., 2007. Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor in the bovine testis. J. Mol. Histol. 38, 207–214. Liu, A., Flores, C., Kinkead, T., Carboni, A.A., Menon, M., Seethalakshmi, L., 1994. Effects of sialoadenectomy and epidermal growth factor on testicular function of sexually mature male mice. J. Urol. 152, 554–561. Luetteke, N.C., Qiu, T.H., Fenton, S.E., Troyer, K.L., Riedel, R.F., Chang, A., Lee, D.C., 1999. Targeted inactivation of EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development. Development 126, 2739–2750. Maekawa, M., Kamimura, K., Nagano, T., 1996. Peritubular myoid cells in the testis: their structure and function. Arch. Histol. Cytol. 59, 1–13. Nakazumi, H., Sasano, H., Maehara, I., Orikasa, S., 1996. Transforming growth factor-alpha, EGF, EGFR in human testis obtained from biopsy and castration: immunohistochemical study. Tohoku J. Exp. Med. 178, 381–388. Nehar, D., Mauduit, C., Revol, A., Morera, A.M., Behahmed, M., 1993. Effect of epidermal growth factor/transforming growth factor alpha on lactate production in porcine Sertoli cells: glucose transport and lactate dehydrogenase isozymes as potential sites of action. Mol. Cell. Endocrinol. 92, 45–53. Niederberger, C.S., Shubhada, S., Kim, S.J., Lamb, D.J., 1993. Paracrine factors and the regulation of spermatogenesis. World J. Urol. 11, 120–128. Onoda, M., Suarez-Quian, C.A., 1994. Modulation of transferrin secretion by epidermal growth factor in immature rat Sertoli cells in vitro. J. Reprod. Fertil. 100, 541–550. Skinner, M.K., Takacs, K., Coffey, R.J., 1989. Transforming growth factor-alpha gene expression and action in the seminiferous tubule: peritubular cell–Sertoli cell interactions. Endocrinology 124, 845–854. Skinner, M.K., 1991. Cell–cell interaction in the testis. Endocr. Rev. 12, 45–77. Sordoillet, C., Chauvin, M.A., de Peretti, E., Morera, A.M., Benahmed, M., 1991. Epidermal growth factor directly stimulates steroidogenesis in primary cultures of porcine Leydig cells: actions and sites of action. Endocrinology 128, 2160–2168. Suarez-Quian, C.A., Dai, M.Z., Onoda, M., Kriss, R.M., Dym, M., 1989. Epidermal growth factor receptor localization in the rat and monkey testes. Biol. Reprod. 41, 921–932. Suarez-Quian, C.A., Niklinski, W., 1990. Immunocytochemical localization of the epidermal growth factor receptor in mouse testis. Biol. Reprod. 43, 1087–1097. Suarez-Quian, C.A., Oke, B.O., Radhakrishnan, B., 1994. Relationship between submandibular gland epidermal growth factor and spermatogenesis in C3H mice. Tissue Cell 26, 285–298. Syed, V., Khan, S.A., Nieschlag, E., 1991. Epidermal growth factor stimulates testosterone production of human Leydig cells in vitro. J. Endocrinol. Invest. 14, 93–97. Tibary, A., Vaughan, J.L., 2006. Reproductive physiology and infertility in male South America camelids: a review and clinical observations. Small Ruminant Res. 61, 283–298. Tsutsumi, O., Kurachi, H., Oka, T., 1986. A physiological role of epidermal growth factor in male reproductive function. Science 233, 975–977. Verhoeven, G., Cailleau, J., 1986. Stimulatory effects of epidermal growth factor on steroidogenesis in Leydig cells. Mol. Cell. Endocrinol. 47, 99–106. Wahab-Wahlgren, A., Martinelle, N., Holst, M., Jahnukainen, K., Parvinen, M., Söder, O., 2003. EGF stimulates rat spermatogonial DNA synthesis in seminiferous tubule segments in vitro. Mol. Cell. Endocrinol. 201, 39–46. Wong, R.W., Kwan, R.W., Mak, P.H., Mak, K.K., Sham, M.H., Chan, S.Y., 2000. Overexpression of epidermal growth factor induced hypospermatogenesis in transgenic mice. J. Biol. Chem. 275, 18297–18301. Wong, R.W., 2003. Transgenic and knock-out mice for deciphering the roles of EGFR ligands. Cell. Mol. Life Sci. 60, 113–118. Xian, C.J., 2007. Roles of epidermal growth factor family in the regulation of postnatal somatic growth. Endocr. Rev. 28, 284–296. Yan, Y.C., Sun, Y.P., Zhang, M.L., 1998. Testis epidermal growth factor and spermatogenesis. Arch. Androl. 40, 133–146.
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos) Junping He a, Changsheng Dong a,∗, Rongli You a, Zhiwei Zhu a, Lihua Lv a, George W. Smith a,b a
College of Animal Science and Technology, Shanxi Agricultural University, Taigu, Shanxi 030801, PR China Laboratory of Mammalian Reproductive Biology and Genomics, Departments of Animal Science and Physiology, Michigan State University, East Lansing, MI, USA
b
a r t i c l e
i n f o
Article history: Received 25 July 2008 Received in revised form 7 January 2009 Accepted 14 January 2009 Available online 20 January 2009 Keywords: Alpaca Epidermal growth factor Epidermal growth factor receptor Immunohistochemistry Testis
a b s t r a c t The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-monthold alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Postnatal development and function of the testes is controlled by a complex interaction of circulating gonadotropins with cytokines and growth factors (Skinner, 1991). Evidence supports a potential ∗ Corresponding author. Tel.: +86 354 628 8208. E-mail address: [email protected] (C. Dong). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.01.002
156
J. He et al. / Animal Reproduction Science 116 (2009) 155–161
regulatory role for epidermal growth factor (EGF) and the EGF receptor (EGFR) in control of testicular function in mice, rats and livestock species. EGF is present in the circulation of adult male mice at concentrations of approximately 5 ng/ml. The main tissue source of EGF is the submandibular glands (Suarez-Quian et al., 1994). Removal of the submandibular glands (sialoadenectomy) results in a decrease in circulating EGF to undetectable concentrations and is accompanied by an approximately 50% reduction in number of mature sperm in the epididymis and number of spermatids in the testes (Tsutsumi et al., 1986; Liu et al., 1994). Effects of submandibular gland removal on spermatogenesis are restored by EGF supplementation (Tsutsumi et al., 1986; Liu et al., 1994). Such results support a regulatory role for EGF in control of spermatogenesis, but also support regulation independent of circulating EGF. Based on results of transgenic and gene targeting studies, the role of EGF in regulation of male fertility is unclear or somewhat controversial. Male EGF null mutant mice do not have reduced fertility (Luetteke et al., 1999), but male transgenic mice overexpressing the EGF gene are infertile (Wong et al., 2000). The functional requirement of the EGFR is difficult to conclusively interpret because EGFR knockout mice display varying phenotypes, including prenatal or early postnatal mortality, depending on the background strain of mice utilized (Wong, 2003). Despite the lack of an overt reproductive phenotype in male EGF knockout mice, abundant evidence supports a potential local regulatory role for EGF in humans, rats and several agricultural species. Potent EGF regulation of multiple aspects of somatic and germ cell function (e.g. DNA synthesis, Leydig cell steroidogenesis) has been reported for human, rat and (or) porcine testicular cell types (Sordoillet et al., 1991; Syed et al., 1991; Nehar et al., 1993; Onoda and Suarez-Quian, 1994; Wahab-Wahlgren et al., 2003). EGF has been localized to Leydig cells of human testes (Nakazumi et al., 1996), in both germ and somatic cells of boar testes (Caussanel et al., 1996), and exclusively to the germ cells of bull testes (Kassab et al., 2007). Furthermore, the EGF receptor has been localized specifically to the Leydig and Sertoli cells of rat and mouse testes (Suarez-Quian et al., 1989; Suarez-Quian and Niklinski, 1990), in the Sertoli cells, peritubular cells and germ cells of human testes (Nakazumi et al., 1996), in both germ cells and somatic cells in boar testes (Caussanel et al., 1996) and specifically to germ cells in bull testes (Kassab et al., 2007). Knowledge of regulation of male reproductive (testicular) function in South American camelids, such as the alpaca, which exhibit a highly variable and late age at puberty is limited (Tibary and Vaughan, 2006). To our knowledge, the intratesticular localization of growth factors and growth factor receptors, such as EGF and EGFR, which may modulate testicular development/function in the alpaca, has not been examined previously. Hence, the objective of the present studies was to determine the temporal and cell specific localization of EGF and EGFR at two specific stages of postnatal development of the alpaca testes. 2. Materials and methods 2.1. Animals and tissue collection/processing Housing and care of animals and collection of testicular tissue for use in described experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals http://www.cioms.ch/frame 1985 texts of guidelines.htm. Testicular tissue was harvested from male alpacas at 12 and 24 months of age after castration under local anesthesia (n = 3 animals per group). Small pieces of testicular tissue (3–5 mm3 ) were fixed in Bouin’s solution for 24 h at 4 ◦ C and extensively washed in 70% ethanol. Thereafter, tissue samples were dehydrated in a graded series of ethanol (85%, 95% and 100%), cleared in xylene and embedded in paraffin wax. Sections (6 m) of testicular tissue were prepared and mounted onto 2% 3-aminopropyltriethoxysilanecoated slides for immunohistochemical localization of EGF and EGFR or haemotoxylin and eosin staining. 2.2. Immunohistochemical staining A polyclonal rabbit anti-EGF antibody and a polyclonal rabbit anti-EGFR antibody (Wuhan Boster Biological Technology Ltd., Wuhan, China) were utilized for described localization studies. All incuba-
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tions were performed in a humidified chamber. Sections of paraffin embedded testicular tissue were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 10 min. After three rinses (3 × 5 min each) in phosphate-buffered saline (PBS, pH 7.4), sections were incubated with 10% normal goat serum at room temperature for 20 min to block non-specific binding, and incubated overnight at 4 ◦ C in the presence of polyclonal rabbit anti-EGF or polyclonal rabbit anti-EGFR serum diluted 1:100 in PBS. For determination of non-specific staining, primary antibody was replaced by normal rabbit serum. Sections were rinsed in PBS (3 × 5 min) and incubated in the secondary antibody (polymerized HRP conjugated donkey-anti-rabbit IgG; Tianjin Haoyang Biological Manufacture Co., Ltd., Tianjin, China) at 37 ◦ C for 30 min, and washed 3× in PBS again (5 min each). Immunoreactivity was visualized by incubating sections in the presence of DAB substrate. The sections were counterstained with haemotoxylin and coverslips were sealed with neutral balsam. After completion of immunostaining, sections were examined using a Leica DMIRB (Leica, Wetzlar, Germany) computerized microscope with a Leica DFC 320 digital camera (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK) and images were captured and stored for analysis. The results of immunohistochemical staining are described as negative (no specific staining), weak (light brown staining), and strong staining (deep brown staining). 3. Results 3.1. Morphological characteristics of testes The testes of 12-month-old alpacas contained a pronounced interstitium and tubular component (Figs. 1 and 2). Leydig cells with a pronounced round morphology were prominent within the interstitium. Within the seminiferous tubules, large round spermatogonia enveloped by immature Sertoli cells were evident and pronounced lumen were not distinguishable. Such morphology is consistent with a prepubertal testis phenotype. In contrast, the testes of 24-month-old alpacas exhibited a postpubertal morphology (Figs. 1 and 2). A pronounced interstitial compartment and tubular component containing seminiferous tubules with pronounced lumen containing embedded spermatozoa were evident as were distinct basal and luminal compartments within the tubules. Spermatogonia and preleptotene spermatocytes with condensed chromatin were present in the basal compartment and in direct contact with the basal lamina and Sertoli cells. Postleptotene spermatocytes and spermatids supported by Sertoli cells were observed in the adluminal compartment. Peritubular myoid cells, surrounding the seminiferous tubules, were also easily identified. 3.2. Immunolocalization of EGF Specific staining for EGF was not evident within the testes of 12-month-old alpacas (Fig. 1A). However, specific staining was prominent within the testes of 24-month-old (postpubertal) alpacas (Fig. 1B–E). Dispersed, strong staining was noted in the interstitial compartment including the Leydig cells (Fig. 1B). Within the tubular component, pronounced cell specific localization to germ cells and somatic cells was evident. Within the basal compartment, localization to peritubular myoid cells and Sertoli cells was detected (Fig. 1C–E) and staining in spermatogonia was weak or isolated. In the adluminal compartment, strong staining for EGF in Sertoli cells and in germ cells at all stages was observed (Fig. 1B–E). EGF immunoreactivity was not detected when primary antibody was replaced by normal rabbit serum (Fig. 1F). 3.3. Immunolocalization of EGFR In the testes of 12-month-old alpacas, EGFR staining was specific to the interstitium (Fig. 2A). Strong staining was noted in the Leydig cells and no specific staining within cells within the seminiferous tubules was noted (Fig. 2A). In contrast, specific staining for EGFR was detected within both the interstitium and the tubular component of the testes of 24-month-old alpacas (Fig. 2B, C, and E). Within the seminiferous tubules, strong staining within spermatogonia of the basal compartment was observed, with less prominent staining within germ cells within the adluminal compartment. Strong
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Fig. 1. Localization of epidermal growth factor (EGF) within the alpaca testis. (A) Representative micrograph of a section through testis tissue collected from a 12-month-old alpaca and incubated with anti-EGF antibody. Note absence of staining in testis tissue collected from prepubertal animal. (B–E) Representative micrographs of sections of testis tissue collected from 24-month-old alpacas and incubated with anti-EGF antibody. Note dispersed strong staining within Leydig cells in the interstitial compartment and pronounced strong staining within germ cells and Sertoli cells in the adluminal compartment of the tubules. (F) Representative micrograph of section through testis tissue collected from a 24-month-old alpaca and incubated with normal rabbit serum (negative control). Note absence of immunoreactivity. Leydig cell (Le); Sertoli cell (Se); spermatogonia (Sg); germ cell (Gc); spermatocyte (Sc); round spermatid (RS); spermatozoa (Sp); peritubular myoid cell (PM). Bar = 25 m for panels A and C–F; 50 m for panel B.
staining within peritubular myoid cells and Leydig cells within the interstitial compartment was also noted within testes of 24-month-old alpacas. Specific staining for EGFR was not detected when primary antibody was replaced by normal rabbit serum (Fig. 2D and F). 4. Discussion Results of the present studies established a pronounced developmental and cell specific regulation of EGF and EGFR expression during postnatal development of the alpaca testis. Differences in morphological characteristics of alpaca testes collected at 12 and 24 months of age suggest that samples collected are representative of a pre- and postpubertal phenotype respectively and such transition was accompanied by an induction of localization of EGF in the testis. Most notably, pronounced lumen
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Fig. 2. Localization of epidermal growth factor (EGF) receptor (EGFR) within the alpaca testis. (A) Representative micrograph of a section through testis tissue collected from a 12-month-old alpaca and incubated with anti-EGFR antibody. Note strong staining for EGFR within Leydig cells in the interstitial compartment and absence of staining in the seminiferous tubules. (B, C, and E) Representative micrographs of sections through testis tissue collected from 24-month-old alpacas and incubated with antiEGFR antibody. Note specific staining for EGFR within both the interstitium and the tubular component, with strong staining within spermatogonia of the basal compartment of the tubules, less prominent staining to germ cells within the adluminal compartment and strong staining within peritubular myoid cells and Leydig cells within the interstitial compartment. (D and F) Representative micrographs of sections through testis tissue collected from 12- and 24-month-old alpacas respectively and incubated with normal rabbit serum (negative control). Note absence of immunoreactivity. Leydig cell (Le); spermatogonia (Sg); peritubular myoid cell (PM). Bar = 25 m for panels A–D and F, 50 m for panel E.
with embedded spermatozoa awaiting release were absent within the seminiferous tubules of 12 months, but not 24-month-old alpaca testis sections respectively. Furthermore, 24-month-old alpacas utilized in present study were free of preputial adhesions, a clear indicator of sexual maturity in this species (Tibary and Vaughan, 2006). While EGFR immunoreactivity was detected within the testis tissue collected from alpacas pre- and postpuberty, developmental regulation of EGF gene expression was noted. EGF immunoreactivity was undetectable in the interstitial and tubular components of the testes of alpacas at 12 months of age (before puberty), but readily detectable in the testes (interstitial and tubular components) of 24-month-old alpacas that had attained puberty. Such results suggest an association and potential local role for EGF in pubertal maturation in the testis. Expression of EGF gene coincident with the onset of spermatogenesis has been reported in other species (Yan et al., 1998). However, absence of EGF staining in the testes of prepubertal alpacas does not eliminate a potential role for EGF during testis development prior to puberty, given evidence supporting a role for circulating EGF in regulation of testicular function in mice (Tsutsumi et al., 1986; Wong et al., 2000) and the
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potential gene expression and action of other EGF family ligands (Xian, 2007) in testes prior to puberty cannot be discounted. The EGFR was localized to Leydig cells within the testes of both 12- and 24-month-old alpacas. Evidence supports a role for EGF in regulation of Leydig cell testosterone production. In other species, cultured Leydig cells from immature animals respond to EGF with an increase in testosterone (Verhoeven and Cailleau, 1986; Sordoillet et al., 1991), suggesting that EGFR signaling pathways are indeed functional prior to puberty and in the absence of local EGF production in the testis. EGF stimulated testosterone production by Leydig cells from mature animals has also been reported (Sordoillet et al., 1991; Syed et al., 1991). Localization of EGFR to rodent, pig and human Leydig cells (Nakazumi et al., 1996; Caussanel et al., 1996; Suarez-Quian and Niklinski, 1990; Suarez-Quian et al., 1989) suggests a potential common role for EGFR in regulation of testosterone production in diverse species. However, EGFR localization to Leydig cells was not observed in the bull testis (Kassab et al., 2007), suggesting that cell specific expression and function of the EGFR in the testis is species specific. Localization of EGF and EGFR to the peritubular myoid cells surrounding the seminiferous tubules of postpubertal (24-month-old) alpacas was noted in the present studies. Functions of peritubular myoid cells are not completely understood, but such cells contain abundant actin filaments and are thought to play a role in the transport of spermatozoa and testicular fluid within the tubule (Hoeben et al., 1995). These cells also secrete a number of substances including extracellular matrix components and growth factors (Maekawa et al., 1996). To our knowledge, potential paracrine and autocrine actions of EGF on peritubular myoid cells have not been described, but pronounced actions of TGF alpha on peritubular cells have been observed in vitro (Skinner et al., 1989). Within the seminiferous tubules of postpubertal alpaca testes, EGF staining was most prominent in all germ cells of the adluminal compartment and in Sertoli cells, whereas EGFR staining was prominent within the basal compartment. Localization of additional EGF related ligands and receptors will be necessary to gain a comprehensive understanding of the potential role for EGF/EGFR superfamily members (Xian, 2007) as a whole in regulation of alpaca germ cell development. However, results of the present studies establish the potential for direct actions of EGF within the seminiferous tubules of the alpaca. Evidence in other species supports a specific role for EGF in germ cell development (Niederberger et al., 1993; Wong et al., 2000; Bartlett et al., 1990; Liu et al., 1994). For instance, removal of circulating EGF by sialoadenectomy in mice results in reduced sperm counts attributed to a reduction in preleptotene and pachytene spermatocytes and round spermatids (Liu et al., 1994). It is unclear whether such effects are truly independent of changes in circulating testosterone, but EGF stimulation of spermatogonial proliferation in isolated rat seminiferous tubules in vitro has been reported (Wahab-Wahlgren et al., 2003). Furthermore, effects of EGF on Sertoli cell function in vitro have been documented (Onoda et al., 1994; Nehar et al., 1993) suggesting the potential for indirect effects of EGF on the seminiferous epithelium mediated by EGF regulation of Sertoli cell function. In conclusion, results of the present studies demonstrate pronounced developmental and cell specific regulation of EGF and EGFR expression within the developing and postpubertal testes of the alpaca. Results support the potential for a local role for EGF in regulation of testicular maturation and function in camelids. Acknowledgements The present research was supported by a grant from the Ministry of Agriculture of the People’s Republic of China (No. 2003-Z86). We also thank Ying Wang, the general manager of the Scientific Research Base of Shanxi Agricultural University (Shanxi, China), for provision of animals used in this study. References Bartlett, J.M., Spiteri-Grech, J., Nieschlag, E., 1990. Regulation of insulin-like growth factor I and stage-specific levels of epidermal growth factor in stage synchronized rat testes. Endocrinology 127, 747–758. Caussanel, V., Tabone, E., Mauduit, C., Dacheux, F., Benahmed, M., 1996. Cellular distribution of EGF, TGF-alpha and their receptor during postnatal development and spermatogenesis of the boar testis. Mol. Cell. Endocrinol. 123, 61–69.
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