J Mol Hist (2007) 38:207–214 DOI 10.1007/s10735-007-9089-2
ORIGINAL PAPER
Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis M. Kassab Æ Ahmed Abd-Elmaksoud Æ Mona A. Ali
Received: 27 February 2007 / Accepted: 22 March 2007 / Published online: 11 May 2007 Springer Science+Business Media B.V. 2007
Abstract In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine. Keywords
EGF EGFR IHC Bovine Testis
M. Kassab M. A. Ali Department of Anatomy and Histology, Faculty of Veterinary Medicine, Kafr El-Sheikh University, Kafr El-Sheikh, Egypt A. Abd-Elmaksoud (&) Department of Histology and Cytology, Faculty of Veterinary Medicine, Mansoura University, Gihan street, Mansoura, Egypt e-mail:
[email protected]
Introduction In addition to hormonal control by gonadotrophins (LH and FSH) and testosterone, a number of locally produced growth factors and other paracrine mediators have been suggested to play an important role in the regulation of testicular function (for reviews see, Sharpe 1994; Gnessi et al. 1997; Huleihel and Lunenfeld 2004; Abd-Elmaksoud and Sinowatz 2005). Among such factors, EGF has been implicated as regulators of germ cell development (Niederberger et al. 1993). EGF is a polypeptide of 53 AA that was first isolated and purified from the submaxillary gland of male mice (Carpenter and Cohen 1990). Previously, some reports have demonstrated that testicular concentrations of EGF change during development of spermatogenesis (Bartlett et al. 1990) and overexpression of EGF results in disturbances of testicular function (Wong et al. 2000). EGF act through the epidermal growth factor receptors, the erbB family of tyrosine kinases. In mammals four members of these receptors (erbB1, erbB2, erbB3 and erbB4) are known and reported to be involved in the regulation of proliferation, differentiation, and migration in many tissues and cell types (Prigent and Lemoine 1992). EGF was detected in the testis of rat (Yan et al. 1998), mouse (Radhakrishnan et al. 1992; Yan et al. 1998) boar (Caussanel et al. 1996) and human (Nakazumi et al. 1996; Yan et al. 1998). Immunohistochemical approaches have also detected the EGFR in the testicular cells of different mammalian species including rat (Suarez-Quian et al. 1989; Kaloglu et al. 2000; Cupp and Skinner 2001), mouse (Suarez-Quian and Niklinski 1990; Suarez-Quian et al. 1994), monkey (Suarez-Quian et al. 1989; Radhakrishnan and Suarez-Quian 1992), boar (Caussanel et al. 1996), and human (Stubbs et al. 1990; Foresta and Varotto 1994; Nakazumi et al. 1996; Yang et al. 2002). EGF and its
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receptors have been reported to be involved in the regulation of male reproduction with effects on the developing testis as well as in adult spermatogenesis and steroidogenesis (Tsutsumi et al. 1986; Mullaney and Skinner 1992; Liu et al. 1994; Yan et al. 1998; Levine et al. 2000; Wong et al. 2000). Although several lines of evidence indicate that both EGF and EGFR are produced within the testis of several mammalian species, no data are to our knowledge available in bovine. Therefore, our study was aimed to localize the EGF and EGFR within the adult bovine testis.
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antibodies were visualized using a streptavidin-biotin peroxidase complex kit and diaminobenzidine (DAKO, Munich, Germany). All incubations were performed in a humidified chamber. Sections were left unstained or counterstained in Mayer’s haematoxylin, dehydrated, and mounted with DPX (Sigma, Munich, Germany). Negative controls were performed by omission of the primary antibody while sections of mouse prostate and seminal vesicles served as a positive control (Wu et al. 1993). Analysis of the adult seminiferous epithelium stages
Materials and methods Tissue preparation The testes of seven sexually mature bulls were obtained from Kafr El-sheikh abattoir within 20 min after slaughter. Small pieces of the testicular tissue (0.5–1 cm3) were fixed in Bouin’s solution for 24 h. The Bouin fixed pieces were extensively washed in 70% ethanol. Thereafter, the tissue samples were dehydrated in graded series of ethanol (80%, 95% and absolute), cleared in xylene and embedded in paraffin wax using standard techniques. Sections (5 lm) were cut on Leitz microtome and mounted on both coated and uncoated slides. For general histological structure, a selection of slides was stained routinely with haematoxylin-eosin. Immunohistochemical staining For the detection of EGF and its receptor, a mouse monoclonal antibody against EGF (E2520) and a mouse monoclonal antibody against EGFR (E3138) (Sigma, Munich, Germany) were used. Antigen localization was achieved using the avidin-biotin complex (ABC) technique (Hsu et al. 1981). Briefly, 5 lm sections of paraffinembedded testicular tissue were dewaxed, rehydrated, and rinsed in PBS pH 7.4 (3 · 5 min). Endogenous peroxidase was blocked by soaking the sections in 3 %v/v hydrogen peroxide/distilled water for 10 min at room temperature followed by washing them under running tap water for additional 10 min. Subsequently the slides were equilibrated in PBS pH 7.4 (2 · 5 min). Non-specific antibody binding was minimized by covering the slides with a serum-free protein blocking reagent (DAKO, Hamburg, Germany) for 10 min at room temperature. Sections were then incubated overnight at 4C with primary antibody against EGF and EGFR diluted 1:100 in antibody diluent (DAKO, Hamburg, Germany). The slides were subsequently rinsed in PBS pH 7.4 (2 · 5 min) followed by incubation with biotinylated rabbit anti-mouse IgG (diluted 1:300 in PBS) for 30 min at room temperature. Bound
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The seminiferous cycle is defined as a series of changes occurring in a cell line up to the re-emergence of the same phase at a given point in the tubule. Currently there are two main views on the criteria for staging the seminiferous cycle. The first is based on the development of the acrosomic system of spermatids beginning with the appearance of young spermatids and this has largely been applied in rodents (Leblond and Clermont 1952), while the second one depends on the morphological changes of all germ cell nuclei and begins after the release of spermatozoa into the tubular lumen and this has found wide application in domestic animals (Ortavant 1958; Russe and Sinowatz 1991). In our study we have used the second method where eight such stages are identified. Of the eight stages identified in this study, stages VI and VII were of relatively short duration and therefore considered as slight morphological variations of stage V. As a result, they are combined with stage V to form one main stage, stage V–VII. Consequently, bovine seminiferous cycle is condensed into six main divisions. The cycle begins with accomplished spermiation (stage I) and ends with the positioning of maturation phase spermatids at the Sertoli cell apex ready for release (stage VIII). This practice has previously gained wide application in bovine (Wrobel and Schimmel 1989), goat (Onyango et al. 2000), ram (Wrobel et al. 1995), and buffalo (Wrobel and Pawar 1992). The stage of a given seminiferous tubule is defined by a specific association of germ cell types. Germ cells were identified through the size and the shape of their nuclei, the distribution of the chromatin in the nucleus and their position in the tubule. Germ cell type staining was examined for each stage. According to Russe and Sinowatz (1991), bovine germ cells were classified into spermatogonia (type A, I, and B; stages I–VIII), young spermatocytes (primary spermatocytes in preleptotene, leptotene and zygotene stages, I–V), old spermatocytes (primary spermatocytes in pachytene, diplotene, and diakinesis; stages VI–III), meiotic cells (stage IV), secondary spermatocytes (not easy to observe because of their short life span in stage IV), spermatids (round from stage V–I, submitted to elongation at stage II, and totally elongated at stage VII), and maturing spermatozoa (stage
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VIII). Spermatogonia generally occupy the basal compartment while the spermatocytes and young spermatids are found in the mid and luminal portions of the tubule respectively. Round spermatids are exclusively located in the adluminal part of the seminiferous epithelium and undergo a complex series of cellular transformation (spermiogenesis). This process takes place via four well-defined phases, termed Golgi, cap, acrosomal, and maturation phase. Under light microscope, the first and second phases are characterized by spherical nuclei whereas the third and fourth phases have elongated nuclei (Wrobel and Schimmel 1989).
Results Immunolocalization of the ligand (EGF) Although specific EGF staining pattern was detected in bovine germ cells according to the stages of the seminiferous epithelium cycle, no staining was found in the somatic cells (i.e., Leydig, Sertoli, and myofibroblast cells). With the beginning of the cycle (stage I), a moderate EGF reaction was localized to the nuclei of type A spermatogonia in the basal compartment while intense staining was detected in the nuclei of round spermatids in the adluminal compartment. A weak reaction was additionally seen in some pachytene spermatocytes (Fig. 1). As the round spermatids start to elongate in stages II, III, and IV, they loss their specific staining and the EGF reaction was restricted only to spermatocytes (leptotene, zygotene, and pachytene) with varying degrees (Figs. 2, 3, 4). In the second half of the seminiferous epithelium cycle (stages V–VIII), where two generations of spermatids (round and elongating) are found, EGF staining was found in spermatogonia type A, pachytene spermatocytes, and round spermatids (Fig. 5). Importantly, elongating and elongated spermatids showed negative reaction throughout the cycle. In the excurrent duct system, EGF was expressed in the nuclei of modified Sertoli cells of the terminal segment (Fig. 6), and in the nuclei of the cuboidal cells lining the straight tubules (Fig. 7) and rete testis (Fig. 8). Immunolocalization of the receptor (EGFR) The EGFR was showed a specific reaction similar to distribution pattern of their ligand. The nuclei of type A spermatogonia was moderately labeled with EGFR during stages I, II, and V–VII (Figs. 9, 12). Interestingly, EGFR staining affinity of pachytene spermatocytes was found to increase in these cells from stage II until they accomplished the meiotic divisions in stage IV (Fig. 9, 10, 11). In addition, an intense EGFR staining was seen in the round
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spermatids in stages I, V–VII, and VIII of the seminiferous epithelium cycle (Figs. 12, 13). In contrast to the nuclear reaction of EGF in the rete testis, the EGFR staining was only cytoplasmic in a supranuclear position in these cells (Fig. 14). In a similar manner, the EGFR was not seen in any of the somatic cells.
Discussion Several lines of evidence have recently shown that growth factors are involved in the testicular spermatogenesis and steroidogenesis. Additionally, expression and localization data suggested that growth factors may alone or in association with hormones and other cytokines control the testicular activities (Gnessi et al. 1997; Huleihel and Lunenfeld 2004, Abd-Elmaksoud and Sinowatz 2005). EGF is a cytokine that promotes cell proliferation, regulates tissue differentiation, and modulates organogenesis (Yan et al. 1998). Moreover, EGF stimulates secretion of several hypothalamic and pituitary hormones, increases placental production of human chorionic gonadotropin and human chorionic somatomammotropin, increases adrenal cortisol production, and inhibits testicular, ovarian, and thyroid hormone secretion (Fisher and Lakshmanan 1990). In the present work, we have investigated the localization of the EGF and EGFR in the adult bovine testis by means of immunohistochemical technique. Our results showed that, EGF and EGFR staining pattern was detected in bovine germ cells, while no staining was found in the somatic cells (i.e., Leydig, Sertoli, and myofibroblast cells). Interestingly, the distribution of EGF and EGFR are dependant upon the cycle of seminiferous epithelium. In the first stage, EGF was localized in the spermatogonia type A, pachytene spermatocytes, and round spermatids. In stages II–IV, the reaction was only restricted to different stages of spermatocytes (leptotene, zygotene and pachytene). In the second half of the seminiferous epithelium cycle (stages V–VIII), where two generations of spermatids (round and elongating) are found, EGF staining was found in spermatogonia type A, pachytene spermatocytes, and round spermatids. These results are partially consistent with other findings that have previously been reported in rat, mouse, human (Yan et al. 1998) and pig (Caussanel et al. 1996) whereas; the spermatogonia, spermatocytes and round spermatids were the most clearly EGF reactive cells in these species (Table 1). However, a clear discrepancy was also observed between these approaches and our results in Sertoli and Leydig cells immunostaining, but this may be attributed to species differences. In a similar manner, several immunohistochemical approaches (Table 2) have also detected the EGFR in the testicular cells of different species including rat
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Figs. 1-8 (1) Stage I, Localization of EGF in the round spermatids (rspd), pachytene spermatocytes (p), and type A spermatogonia (spg). Sertoli cells (s) have no affinity. X400. (2) Stage II, EGF was mainly detected in the zygotene (z) and pachytene spermatocytes (p). Elongating spermatids (espd) and myofibroblast (open arrowhead) showed negative reaction. X400. ( 3) Stage III, the nuclei of zygotene (z) and pachytene spermatocytes (p) were moderately stained with EGF while myofibroblast (open arrowhead) showed negative reaction. X1000. ( 4) Stage IV at the very beginning, the EGF reaction was only restricted to the late pachytene spermatocyte (P).
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Notice, some cells are in the second maturation division (M). X400. (5) Stage V-VII, notice the presence of two generations of spermatids (round and elongating). EGF staining was confined to spermatogonia (spg), pachytene spermatocytes (p), and round spermatids (rspd). X1000. (6) Localization of EGF in the modified Sertoli cells (ms) of terminal segment. X400. (7) The lining epithelium of the straight tubules was moderately stained with EGF (black arrowheads). X400.(8) Intense EGF reaction was seen in the rete testis epithelium (black arrows). X400
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Figs. 9-14 (9) Stage II, EGFR was mainly observed in type A spermatogonia (spg) and pachytene spermatocytes (p). Sertoli cells (s), leptotene spermatocytes (L), zygotene spermatocytes (z) and elongating spermatids (espd) showed negative reaction. X1000. (10 and 11) Stage III, EGFR staining was only seen in the pachytene spermatocytes (p). Zygotene (z) and diplotene spermatocytes (d)
have no affinity. X1000. (12) Stage V-VII, spermatogonia (spg) and round spermatids (rspd) were the only EGFR reactive cells. X1000. (13) Stage VIII, the mature spermatozoa (black arrowhead) are ready to release. EGFR was solely localized to round spermatids (rspd). X1000. (14) EGFR was detected in the in a supranuclear position within the lining epithelium of rete testis. X1000
(Suarez-Quian et al. 1989; Kaloglu et al. 2000; Cupp and Skinner 2001), mouse (Suarez-Quian and Niklinski 1990; Suarez-Quian et al. 1994), monkey (Suarez-Quian et al. 1989; Radhakrishnan and Suarez-Quian 1992), boar (Caussanel et al. 1996), and human (Stubbs et al. 1990; Foresta and Varotto 1994; Nakazumi et al. 1996; Yang et al. 2002).The distribution of the EGFR in the bovine germ cells was also dependent upon the cycle of the seminiferous epithelium and these results are completely consistent with the findings of Caussanel et al. (1996) in boars. In both, the EGFR was detected in the spermatogonia type A. Additionally, a predominant EGFR staining was demonstrated during the meiotic and spermiogenic
process with a preponderant localization in pachytene spermatocytes and newly formed spermatids (Golgi and cap phase spermatids). Taken together, the localization of EGF and EGFR in the spermatogonia type A may point to an effect of such factor on the spermatogonia proliferation. This speculation is substantiated by the finding of a recent study, which showed that EGF stimulated the mitotic activity of spermatogonia in vitro (Wahab-Wahlgren et al. 2003). Our findings also suggest that, in germ cells, the EGF and EGFR might be implicated in the meiotic process. Such an observation, is supported by, at least, two other previous reports. First, Tsutsumi et al. (1986) reported the
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Table 1 Expression and localization of EGF in the mammalian testis Growth factor
Species
EGF
Rat Mouse Human Pig
Detected in
Sites of gene expression
Sites of protein localization
References Yan et al. 1998
Immature
–
Lc
Adult
–
Spg, Spc, Lc
Immature
Whole testis
Lc
Adult
Whole testis
Lc, Spg, Spc, rSpd, Pc
Foetus
–
Lc
Adult
–
Spc, Spd, Lc
Prepubertal, Adult
–
Perinatal, Spg, Sc, Lc
Yan et al. 1998 Yan et al. 1998 Caussanel et al. 1996
Lc: Leydig cells; Spg: spermatogonia; Spc: spermatocytes; rSpd: round spermatids; Pc: peritubular cells; Sc: Sertoli cells; Bv: blood vessels
accumulation of pachytene spermatocytes in EGF deprived-testis after sialoadenectomy in male mice. Second, Bartlett et al. (1990) showed an increase in the testicular concentration of EGF in rat testis synchronized between stages IX–II, which are closed to the meiotic division. Collectively, EGF may therefore play a role in the regulation of testicular function. This notion is supported by the findings that ablation of submaxillary gland (sialoadenectomy) of adult male mouse completely depleted the circulating levels of EGF and reduced body and reproductive organ weights. Quantitative analysis of spermatogenesis showed either an accumulation of pachytene spermatocytes (Tsutsumi et al. 1986) or a decrease in preleptotene and pachytene spermatocytes as well as in round spermatids (Liu et al. 1994), which resulted in a marked reduction in epididymal sperm count (Tsutsumi et al. 1986; Liu et al. 1994). Further on, sperm motility and fertility were also significantly decreased (Liu et al. 1994). Interestingly, the daily administration of EGF to sialoadenectomized mice restored the epididymal sperm count and testicular spermatids to the normal values. The association between EGF and EGFR in the same cells of the seminiferous epithelium may indicate either
autocrine and/or paracrine action of this growth factor in the bovine testis. This suggestion was augmented by Anklesaria et al. (1990) and Massague et al. (1990) who concluded that, one possible mechanism of EGF action within the seminiferous epithelium is juxtacrine form of cell-cell interaction. In this pathway a membrane anchored growth factor precursor binds to its receptor on adjacent cell leading to transduction of a signal. Importantly, the cells in the seminiferous epithelium observed to immunostain for EGF such as, spermatocytes and round spermatids are known to be antigenic (O’’Rand and Romerell 1977) thus; one possible role of germ cells EGF is that it may serve an immunosuppressive function in the event that the blood-testis barrier is breached (Radhakrishanan et al. 1992). Although EGF has been shown to influence steroidogenesis in rodent, pig, and human Leydig cells (Verhoeven and Cailleau 1986; Syed et al. 1991; Sordoillet et al. 1991), such an effect may be species-specific as the bovine Leydig cells were found to be EGF negative cells. Finally, a recent study showed that local and sustained EGF release after testicular torsion improves bilateral testicular injury (Uguralp et al. 2004). EGF administration
Table 2 Localization of EGFR receptors in the mammalian testis Receptors Species EGFR
Rat
Detected in
Expression and/or localization References
Embryos
Seminiferous cords
Cupp et al. 2001
Perinatal, Immature Gc, Sc, Lc, Pc
Suarez-Quian et al. 1989; Kaloglu et al. 2000; Cupp et al. 2001
Cell culture Intact testis
Spc, Spd, Sc, Lc, LcP, Pc
Suarez-Quian et al. 1989; Mullaney and Skinner 1992; Moore and Morris 1993
Mouse
Cell culture, Intact testis
Lc, Sc
Ascoli 1981; Suarez-Quian and Niklinski1990; Suarez-Quian et al. 1994
Human
Adult
Sc, Lc, Pc
Foresta et al. 1991
Pig
Cell culure,
Lc
Sordoillet et al. 1991
Perinatal
Sc, Lc, Spg
Caussanel et al. 1996
Prepubertal
Sc, Lc, Spg, Spc
Caussanel et al. 1996
Adult
Sc, Lc, Spg, Spc, Spd
Caussanel et al. 1996
Sc, Lc, Pc
Suarez-Quian et al. 1989; Radhakrishnan and Suarez-Quian 1992
Monkey Immature, adult
Gc: Germ cells (gonocytes in foetus); Sc: Sertoli cells; Lc: Leydig cells; Pc: peritubular cells; Spc: spermatocytes; Spd: spermatids; LcP: Leydig cells precursors
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may therefore be a new treatment choice for bilaterally injured testis after detorsion without removing the twisted testis. In conclusion the existence of normal hormonal level in several cases of male infertility greatly supports the notion that numerous mediators such as growth factors could play crucial roles in maintenance of normal fertility. Therefore, improper synthesis or release of these locally produced factors may, in part, influence the paracrine and/or autocrine pathways in the male gonads, which might result in testicular dysfunction. References Abd-Elmaksoud A, Sinowatz F (2005) Expression and localization of growth factors and their receptors in the mammalian testis. Review/Part I. Anat Histol Embryol 34(5):319–334 Anklesaria P, Teixido J, Laiho M et al (1990) Cell–cell adhesion mediated by binding of membrane-anchored transforming growth factor alpha to epidermal growth factor receptors promotes cell proliferation. Proc Natl Acad Sci U S A 87(9):3289–3293 Ascoli M (1981) Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor. J Biol Chem 256(1):179–183 Bartlett JM, 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(2):747–758 Carpenter G, Cohen S (1990) Epidermal growth factor. J Biol Chem 265(14):7709–7712 Caussanel V, Tabone E, Mauduit C et al (1996) Cellular distribution of EGF, TGFalpha and their receptor during postnatal development and spermatogenesis of the boar testis. Mol Cell Endocrinol 123(1):61–69 Cupp AS, Skinner MK (2001) Expression, action, and regulation of transforming growth factor alpha and epidermal growth factor receptor during embryonic and perinatal rat testis development. J Androl 22(6):1019–1029 Fisher DA, Lakshmanan J (1990) Metabolism and effects of epidermal growth factor and related growth factors in mammals. Endocr Rev 11(3):418–442 Foresta C, Caretto A, Varotto A et al (1991) Epidermal growth factor receptors (EGFR) localization in human testis. Arch Androl 27(1):17–24 Foresta C, Varotto A (1994) Immunocytochemical localization of epidermal growth factor receptors in human testis from infertile subjects. Fertil Steril 61(5):941–948 Gnessi L, Fabbri A, Spera G (1997) Gonadal peptides as mediators of development and functional control of the testis: an integrated system with hormones and local environment. Endocr Rev 18:541–609 Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577–580 Huleihel M, Lunenfeld E (2004) Regulation of spermatogenesis by paracrine/autocrine testicular factors. Asian J Androl 6:259–268 Kaloglu C, Bulut HE, Onarlioglu B (2000) Epidermal growth factor receptor (EGFR) immunolocalization in the male rat reproductive tract during pre- and postnatal periods. Turk J Vet Anim Sci 24:501–509
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