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EGFR ligands exert diverging effects on male reproductive organs
Experimental and Molecular Pathology 88 (2010) 216–218
Contents lists available at ScienceDirect
Experimental and Molecular Pathology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x m p
Case Report
EGFR ligands exert diverging effects on male reproductive organs Marlon R. Schneider a,⁎, Ana A. Gratao a, Maik Dahlhoff a, Auke Boersma b, Martin Hrabé de Angelis b, Cuong Hoang-Vu c, Eckhard Wolf a,d, Thomas Klonisch e a
Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany Clinics of Surgery, Faculty of Medicine, University of Halle-Wittenberg, Germany d Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Munich, Germany e Dept. of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Canada b c
a r t i c l e
i n f o
Article history: Received 28 October 2009 Available online 25 November 2009 Keywords: Epidermal growth factor EGFR Betacellulin Transgenic mice Male reproductive tract
a b s t r a c t While the EGFR and most of its ligands are expressed in the male reproductive tract, their functions in male reproduction are poorly understood. Interestingly, male transgenic mice overexpressing EGF are sterile, and transgenic mice overexpressing TGFA, another EGFR ligand, show an enlarged coagulation gland (anterior prostate) due to severe hyperplasia with focal dysplasia. We studied the male reproductive tract of transgenic mice overexpressing betacellulin (BTC-tg) under the control of a promoter conferring widespread transgene expression. Despite strong overexpression of BTC in different parts of the male reproductive tract, the gross appearance and histology of the reproductive organs of BTC-tg males were normal and the same were true for sperm parameters and the in vitro fertilization rate. Collectively, our findings demonstrate that excess of BTC exerts no deleterious effects on the structure or function of the male reproductive tract in mice and indicates unique, non-overlapping functions of specific EGFR ligands in male reproduction. © 2009 Elsevier Inc. All rights reserved.
Introduction The epidermal growth factor receptor (EGFR, ERBB1, HER1) is at the core of one of the most interesting and complex signaling networks in higher vertebrates, regulating an extraordinarily large number of processes in mammalian cells ranging from cell division to cell death, differentiation, adhesion, or malignant transformation (Citri and Yarden, 2006; Schneider et al., 2008; Sibilia et al., 2007; Yarden and Shilo, 2007). Canonical EGFR activation is accomplished by the interaction with seven related growth factors: amphiregulin (AREG), betacellulin (BTC), epidermal growth factor (EGF), epigen (EPGN), epiregulin (EREG), heparin-binding EGF-like growth factor (HBEGF), and transforming growth factor alpha (TGFA) (Harris et al., 2003; Schneider and Wolf, 2009). While the EGFR and most of its ligands are expressed in the male reproductive tract, their functions in male reproduction are poorly understood. Knockout mice lacking AREG, BTC, EGF, EREG, HBEGF, and TGFA have normal male reproductive tracts (reviewed in Schneider and Wolf, 2009). In contrast, male transgenic mice overexpressing human EGF are sterile. Seminiferous tubules have smaller diameter, enlarged lumen, germ layers of uneven thickness, reduced number of post-meiosis II gametes, and absence of sperm in the epididymis (Wong et al., 2000). Transgenic mice overexpressing TGFA, although having normal testis morphology
⁎ Corresponding author. E-mail address: [email protected] (M.R. Schneider). 0014-4800/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2009.11.005
and spermatogenesis, show an enlarged coagulation gland (anterior prostate) due to severe hyperplasia with focal dysplasia (Sandgren et al., 1990). Alterations of the male reproductive organs of mouse lines overexpressing additional EGFR ligands are unknown. Results and discussion Here, we studied the male reproductive tract of transgenic mice overexpressing betacellulin (BTC-tg) under the control of a promoter conferring widespread transgene expression (Schneider et al., 2005). BTC-tg males showed normal mating behavior and produced litters of usual size. Seminiferous tubules, epididymis, ductus deferens, seminal vesicles, coagulation gland, dorsal and ventral prostate, and urethra of BTC-tg males appeared normal (Fig. 1A). Absolute weights of testes, seminal vesicle, and preputial gland were significantly reduced in BTC-tg males, but the relative weights of all organs analyzed were not altered (Table 1). In contrast to TGFA-tg mice that display hyperplastic coagulation glands (Sandgren et al., 1990), no difference in weight or microscopic appearance of this organ was seen in up to 10-month-old BTC-tg mice and age-matched control males. Transgene-derived mRNA was readily detected in testis, epididymis, ductus deferens, and seminal vesicle of BTC-tg mice (Fig. 1B). Western blot analysis revealed increased BTC protein levels in testis, epididymis, seminal vesicle, and coagulation gland (Fig. 1C). In addition to the ∼36-kDa protein representing membrane-bound BTC, a ∼ 20-kDa protein, probably partially cleaved proBTC or the soluble form containing posttranslational modifications, was present in high
M.R. Schneider et al. / Experimental and Molecular Pathology 88 (2010) 216–218
217
Fig. 1. Transgene expression and reproductive phenotype of BTC-transgenic male mice. The generation and genotyping of transgenic mice overexpressing BTC have been described in detail elsewhere (Schneider et al., 2005). In brief, expression of BTC in a broad spectrum of tissues was achieved by placing a cDNA sequence coding for full-length BTC under the control of the cytomegalovirus immediate-early enhancer fused to the chicken β actin gene promoter. Transgenic and non-transgenic control mice from the strain FVB/N were maintained in a specific pathogen-free facility with controlled temperature (22–25 °C) and photoperiod (12 h of light, 12 h of dark), and received standard food and water ad libitum. The experimental procedures involving animals have been approved by the author's institutional committee on animal care and carried out with permission from the responsible veterinary authority. (A) Picture of the mouse male genital organs showing the location of the principal segments analyzed in this study. The five segments are the testis (1), epididymis (2), ductus deferens (3), seminal vesicle (4), and coagulation gland (5); Northern (B) and Western (C) blotting showed increased levels of BTC in the tissues of transgenic males. For Northern blots, tissues from control and transgenic males were homogenized in TriZol reagent (Invitrogen, Karlsruhe, Germany), and total RNA was isolated according to the manufacturer's instructions. Twelve micrograms of total RNA was separated by electrophoresis in a formaldehyde gel and blotted on positively charged nylon membranes. 32Plabeled probes were generated with the Rediprime labeling system (GE Healthcare, Buckinghamshire, UK) and hybridized in Rapid-hyb buffer (GE Healthcare). The Btc (Schneider et al., 2005) and the Gapdh (Schneider et al., 2001) probes have been described previously. For Western blots, tissues were homogenized in lysis buffer (Cell Signaling, Danvers, MA, USA), and protein concentration was determined using a modified BCA assay. Total protein (30 μg) was separated by SDS-PAGE and transferred to PVDF membranes by electroblotting. Loading of equal amounts of protein for each sample was verified by Ponceau S staining. The antibody to detect BTC (R&D Systems, Wiesbaden, Germany) and the secondary rabbit anti-goat HRP-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany) were employed as described previously (Schneider et al., 2005). Signals were detected using a chemiluminescence detection reagent (Luminol, Santa Cruz) and appropriate X-ray films (GE Healthcare). Note the signal at about 20 kDa in the seminal vesicle and coagulation gland protein extracts in C, probably representing partially cleaved BTC or the soluble form containing posttranslational modifications. (D) Sperm analyses were performed with BTC transgenic male mice and control males ranging from 3 to 5 months of age. The sperm quality was evaluated using a computerized semen analysis system (IVOS, V. 12.1c, Hamilton Thorne Research, Beverly, MA). (E) For in vitro fertilization, sperm were collected from the caudae epididymides and vasa deferentia of fertile non-transgenic or BTC transgenic mice in IVF medium (Vitrolife, Kungsbacka, Sweden). Collection of oocytes and in vitro fertilization was performed as described previously (Gratao et al., 2008). Six hours after fertilization, oocytes were denuded from the remaining cumulus cells by repeated gentle pipetting and evaluated for successful fertilization (appearance of two pronuclei) and cultured for detection of cleavage after further 24 h.
amounts in the protein extracts from the seminal vesicle and coagulation glands of BTC-tg males (Fig. 1C). Immunohistochemistry confirmed higher BTC levels in the testis of BTC-tg mice, particularly in interstitial cells (data not shown). BTC-tg mice showed normal spermatogenesis and mature sperm production. The concentration, motility, and progressivity of spermatozoa collected from the caudal part of the epididymidis were similar between BTC-tg and control mice (Fig. 1D), as was the in vitro fertilization capacity, performed to
assess the ability of spermatozoa from BTC-tg males to fertilize mature oocytes (Fig. 1E). Collectively, our findings demonstrate that excess of BTC exerts no deleterious effects on the structure or function of the male reproductive tract in mice and indicates unique, non-overlapping functions of specific EGFR ligands in male reproduction. Whether the differences in functional responsiveness towards specific EGFR ligands result from differential receptor expression profiles or the
218
M.R. Schneider et al. / Experimental and Molecular Pathology 88 (2010) 216–218
Table 1 Body weight and absolute and relative weights of the reproductive organs (paired) from betacellulin transgenic mice and control littermates (values shown are means ± SD). Weight
Body (g) Testis (mg) Epididymis (mg) Ductus deferens (mg) Seminal vesicle (mg) Coagulating gland (mg) Preputial gland (mg)
%Body weight
wt (n = 5)
tg (n = 6)
wt
tg
26.6 ± 0.7 179.4 ± 12.1 73.4 ± 14.4 25.9 ± 8.0 182.6 ± 25.1 22.3 ± 4.7 118.9 ± 11.2
24.5 ± 1.4⁎ 161.0 ± 7.3⁎ 59.3 ± 13.3 22.2 ± 6.8 148.6 ± 27.4⁎ 20.7 ± 3.8 101.0 ± 14.9⁎
0.68 ± 0.03 0.28 ± 0.05 0.10 ± 0.03 0.69 ± 0.06 0.08 ± 0.02 0.45 ± 0.04
0.66 ± 0.03 0.24 ± 0.04 0.09 ± 0.02 0.61 ± 0.09 0.08 ± 0.01 0.41 ± 0.05
⁎ P b 0.05; t-test.
activation of distinct signaling pathways in male reproductive tissues remains to be determined. Acknowledgments A.A.G. was a recipient of a fellowship from the CAPES, Brazil. M.R.S. and E.W. acknowledge the support from Deutsche Forschungsgemeinschaft (GRK 1023) and C.H.-V. and T.K. are grateful for the support by Deutsche Krebshilfe.
References Citri, A., Yarden, Y., 2006. EGF-ERBB signalling: towards the systems level. Nat. Rev. Mol. Cell Biol. 7, 505–516. Gratao, A.A., Dahlhoff, M., Sinowatz, F., Wolf, E., Schneider, M.R., 2008. Betacellulin overexpression in the mouse ovary leads to MAPK3/MAPK1 hyperactivation and reduces litter size by impairing fertilization. Biol. Reprod. 78, 43–52. Harris, R.C., Chung, E., Coffey, R.J., 2003. EGF receptor ligands. Exp. Cell Res. 284, 2–13. Sandgren, E.P., Luetteke, N.C., Palmiter, R.D., Brinster, R.L., Lee, D.C., 1990. Overexpression of TGF alpha in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61, 1121–1135. Schneider, M.R., Wolf, E., 2009. The epidermal growth factor receptor ligands at a glance. J. Cell. Physiol. 218, 460–466. Schneider, M.R., Zhou, R., Hoeflich, A., Krebs, O., Schmidt, J., Mohan, S., Wolf, E., Lahm, H., 2001. Insulin-like growth factor-binding protein-5 inhibits growth and induces differentiation of mouse osteosarcoma cells. Biochem. Biophys. Res. Commun. 288, 435–442. Schneider, M.R., Dahlhoff, M., Herbach, N., Renner-Mueller, I., Dalke, C., Puk, O., Graw, J., Wanke, R., Wolf, E., 2005. Betacellulin overexpression in transgenic mice causes disproportionate growth, pulmonary hemorrhage syndrome, and complex eye pathology. Endocrinology 146, 5237–5246. Schneider, M.R., Werner, S., Paus, R., Wolf, E., 2008. Beyond wavy hairs: the epidermal growth factor receptor and its ligands in skin biology and pathology. Am. J. Pathol. 173, 14–24. Sibilia, M., Kroismayr, R., Lichtenberger, B.M., Natarajan, A., Hecking, M., Holcmann, M., 2007. The epidermal growth factor receptor: from development to tumorigenesis. Differentiation 75, 770–787. 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. Yarden, Y., Shilo, B.Z., 2007. SnapShot: EGFR signaling pathway. Cell 131, 1018.
Contents lists available at ScienceDirect
Experimental and Molecular Pathology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x m p
Case Report
EGFR ligands exert diverging effects on male reproductive organs Marlon R. Schneider a,⁎, Ana A. Gratao a, Maik Dahlhoff a, Auke Boersma b, Martin Hrabé de Angelis b, Cuong Hoang-Vu c, Eckhard Wolf a,d, Thomas Klonisch e a
Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany Clinics of Surgery, Faculty of Medicine, University of Halle-Wittenberg, Germany d Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Munich, Germany e Dept. of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Canada b c
a r t i c l e
i n f o
Article history: Received 28 October 2009 Available online 25 November 2009 Keywords: Epidermal growth factor EGFR Betacellulin Transgenic mice Male reproductive tract
a b s t r a c t While the EGFR and most of its ligands are expressed in the male reproductive tract, their functions in male reproduction are poorly understood. Interestingly, male transgenic mice overexpressing EGF are sterile, and transgenic mice overexpressing TGFA, another EGFR ligand, show an enlarged coagulation gland (anterior prostate) due to severe hyperplasia with focal dysplasia. We studied the male reproductive tract of transgenic mice overexpressing betacellulin (BTC-tg) under the control of a promoter conferring widespread transgene expression. Despite strong overexpression of BTC in different parts of the male reproductive tract, the gross appearance and histology of the reproductive organs of BTC-tg males were normal and the same were true for sperm parameters and the in vitro fertilization rate. Collectively, our findings demonstrate that excess of BTC exerts no deleterious effects on the structure or function of the male reproductive tract in mice and indicates unique, non-overlapping functions of specific EGFR ligands in male reproduction. © 2009 Elsevier Inc. All rights reserved.
Introduction The epidermal growth factor receptor (EGFR, ERBB1, HER1) is at the core of one of the most interesting and complex signaling networks in higher vertebrates, regulating an extraordinarily large number of processes in mammalian cells ranging from cell division to cell death, differentiation, adhesion, or malignant transformation (Citri and Yarden, 2006; Schneider et al., 2008; Sibilia et al., 2007; Yarden and Shilo, 2007). Canonical EGFR activation is accomplished by the interaction with seven related growth factors: amphiregulin (AREG), betacellulin (BTC), epidermal growth factor (EGF), epigen (EPGN), epiregulin (EREG), heparin-binding EGF-like growth factor (HBEGF), and transforming growth factor alpha (TGFA) (Harris et al., 2003; Schneider and Wolf, 2009). While the EGFR and most of its ligands are expressed in the male reproductive tract, their functions in male reproduction are poorly understood. Knockout mice lacking AREG, BTC, EGF, EREG, HBEGF, and TGFA have normal male reproductive tracts (reviewed in Schneider and Wolf, 2009). In contrast, male transgenic mice overexpressing human EGF are sterile. Seminiferous tubules have smaller diameter, enlarged lumen, germ layers of uneven thickness, reduced number of post-meiosis II gametes, and absence of sperm in the epididymis (Wong et al., 2000). Transgenic mice overexpressing TGFA, although having normal testis morphology
⁎ Corresponding author. E-mail address: [email protected] (M.R. Schneider). 0014-4800/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2009.11.005
and spermatogenesis, show an enlarged coagulation gland (anterior prostate) due to severe hyperplasia with focal dysplasia (Sandgren et al., 1990). Alterations of the male reproductive organs of mouse lines overexpressing additional EGFR ligands are unknown. Results and discussion Here, we studied the male reproductive tract of transgenic mice overexpressing betacellulin (BTC-tg) under the control of a promoter conferring widespread transgene expression (Schneider et al., 2005). BTC-tg males showed normal mating behavior and produced litters of usual size. Seminiferous tubules, epididymis, ductus deferens, seminal vesicles, coagulation gland, dorsal and ventral prostate, and urethra of BTC-tg males appeared normal (Fig. 1A). Absolute weights of testes, seminal vesicle, and preputial gland were significantly reduced in BTC-tg males, but the relative weights of all organs analyzed were not altered (Table 1). In contrast to TGFA-tg mice that display hyperplastic coagulation glands (Sandgren et al., 1990), no difference in weight or microscopic appearance of this organ was seen in up to 10-month-old BTC-tg mice and age-matched control males. Transgene-derived mRNA was readily detected in testis, epididymis, ductus deferens, and seminal vesicle of BTC-tg mice (Fig. 1B). Western blot analysis revealed increased BTC protein levels in testis, epididymis, seminal vesicle, and coagulation gland (Fig. 1C). In addition to the ∼36-kDa protein representing membrane-bound BTC, a ∼ 20-kDa protein, probably partially cleaved proBTC or the soluble form containing posttranslational modifications, was present in high
M.R. Schneider et al. / Experimental and Molecular Pathology 88 (2010) 216–218
217
Fig. 1. Transgene expression and reproductive phenotype of BTC-transgenic male mice. The generation and genotyping of transgenic mice overexpressing BTC have been described in detail elsewhere (Schneider et al., 2005). In brief, expression of BTC in a broad spectrum of tissues was achieved by placing a cDNA sequence coding for full-length BTC under the control of the cytomegalovirus immediate-early enhancer fused to the chicken β actin gene promoter. Transgenic and non-transgenic control mice from the strain FVB/N were maintained in a specific pathogen-free facility with controlled temperature (22–25 °C) and photoperiod (12 h of light, 12 h of dark), and received standard food and water ad libitum. The experimental procedures involving animals have been approved by the author's institutional committee on animal care and carried out with permission from the responsible veterinary authority. (A) Picture of the mouse male genital organs showing the location of the principal segments analyzed in this study. The five segments are the testis (1), epididymis (2), ductus deferens (3), seminal vesicle (4), and coagulation gland (5); Northern (B) and Western (C) blotting showed increased levels of BTC in the tissues of transgenic males. For Northern blots, tissues from control and transgenic males were homogenized in TriZol reagent (Invitrogen, Karlsruhe, Germany), and total RNA was isolated according to the manufacturer's instructions. Twelve micrograms of total RNA was separated by electrophoresis in a formaldehyde gel and blotted on positively charged nylon membranes. 32Plabeled probes were generated with the Rediprime labeling system (GE Healthcare, Buckinghamshire, UK) and hybridized in Rapid-hyb buffer (GE Healthcare). The Btc (Schneider et al., 2005) and the Gapdh (Schneider et al., 2001) probes have been described previously. For Western blots, tissues were homogenized in lysis buffer (Cell Signaling, Danvers, MA, USA), and protein concentration was determined using a modified BCA assay. Total protein (30 μg) was separated by SDS-PAGE and transferred to PVDF membranes by electroblotting. Loading of equal amounts of protein for each sample was verified by Ponceau S staining. The antibody to detect BTC (R&D Systems, Wiesbaden, Germany) and the secondary rabbit anti-goat HRP-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany) were employed as described previously (Schneider et al., 2005). Signals were detected using a chemiluminescence detection reagent (Luminol, Santa Cruz) and appropriate X-ray films (GE Healthcare). Note the signal at about 20 kDa in the seminal vesicle and coagulation gland protein extracts in C, probably representing partially cleaved BTC or the soluble form containing posttranslational modifications. (D) Sperm analyses were performed with BTC transgenic male mice and control males ranging from 3 to 5 months of age. The sperm quality was evaluated using a computerized semen analysis system (IVOS, V. 12.1c, Hamilton Thorne Research, Beverly, MA). (E) For in vitro fertilization, sperm were collected from the caudae epididymides and vasa deferentia of fertile non-transgenic or BTC transgenic mice in IVF medium (Vitrolife, Kungsbacka, Sweden). Collection of oocytes and in vitro fertilization was performed as described previously (Gratao et al., 2008). Six hours after fertilization, oocytes were denuded from the remaining cumulus cells by repeated gentle pipetting and evaluated for successful fertilization (appearance of two pronuclei) and cultured for detection of cleavage after further 24 h.
amounts in the protein extracts from the seminal vesicle and coagulation glands of BTC-tg males (Fig. 1C). Immunohistochemistry confirmed higher BTC levels in the testis of BTC-tg mice, particularly in interstitial cells (data not shown). BTC-tg mice showed normal spermatogenesis and mature sperm production. The concentration, motility, and progressivity of spermatozoa collected from the caudal part of the epididymidis were similar between BTC-tg and control mice (Fig. 1D), as was the in vitro fertilization capacity, performed to
assess the ability of spermatozoa from BTC-tg males to fertilize mature oocytes (Fig. 1E). Collectively, our findings demonstrate that excess of BTC exerts no deleterious effects on the structure or function of the male reproductive tract in mice and indicates unique, non-overlapping functions of specific EGFR ligands in male reproduction. Whether the differences in functional responsiveness towards specific EGFR ligands result from differential receptor expression profiles or the
218
M.R. Schneider et al. / Experimental and Molecular Pathology 88 (2010) 216–218
Table 1 Body weight and absolute and relative weights of the reproductive organs (paired) from betacellulin transgenic mice and control littermates (values shown are means ± SD). Weight
Body (g) Testis (mg) Epididymis (mg) Ductus deferens (mg) Seminal vesicle (mg) Coagulating gland (mg) Preputial gland (mg)
%Body weight
wt (n = 5)
tg (n = 6)
wt
tg
26.6 ± 0.7 179.4 ± 12.1 73.4 ± 14.4 25.9 ± 8.0 182.6 ± 25.1 22.3 ± 4.7 118.9 ± 11.2
24.5 ± 1.4⁎ 161.0 ± 7.3⁎ 59.3 ± 13.3 22.2 ± 6.8 148.6 ± 27.4⁎ 20.7 ± 3.8 101.0 ± 14.9⁎
0.68 ± 0.03 0.28 ± 0.05 0.10 ± 0.03 0.69 ± 0.06 0.08 ± 0.02 0.45 ± 0.04
0.66 ± 0.03 0.24 ± 0.04 0.09 ± 0.02 0.61 ± 0.09 0.08 ± 0.01 0.41 ± 0.05
⁎ P b 0.05; t-test.
activation of distinct signaling pathways in male reproductive tissues remains to be determined. Acknowledgments A.A.G. was a recipient of a fellowship from the CAPES, Brazil. M.R.S. and E.W. acknowledge the support from Deutsche Forschungsgemeinschaft (GRK 1023) and C.H.-V. and T.K. are grateful for the support by Deutsche Krebshilfe.
References Citri, A., Yarden, Y., 2006. EGF-ERBB signalling: towards the systems level. Nat. Rev. Mol. Cell Biol. 7, 505–516. Gratao, A.A., Dahlhoff, M., Sinowatz, F., Wolf, E., Schneider, M.R., 2008. Betacellulin overexpression in the mouse ovary leads to MAPK3/MAPK1 hyperactivation and reduces litter size by impairing fertilization. Biol. Reprod. 78, 43–52. Harris, R.C., Chung, E., Coffey, R.J., 2003. EGF receptor ligands. Exp. Cell Res. 284, 2–13. Sandgren, E.P., Luetteke, N.C., Palmiter, R.D., Brinster, R.L., Lee, D.C., 1990. Overexpression of TGF alpha in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61, 1121–1135. Schneider, M.R., Wolf, E., 2009. The epidermal growth factor receptor ligands at a glance. J. Cell. Physiol. 218, 460–466. Schneider, M.R., Zhou, R., Hoeflich, A., Krebs, O., Schmidt, J., Mohan, S., Wolf, E., Lahm, H., 2001. Insulin-like growth factor-binding protein-5 inhibits growth and induces differentiation of mouse osteosarcoma cells. Biochem. Biophys. Res. Commun. 288, 435–442. Schneider, M.R., Dahlhoff, M., Herbach, N., Renner-Mueller, I., Dalke, C., Puk, O., Graw, J., Wanke, R., Wolf, E., 2005. Betacellulin overexpression in transgenic mice causes disproportionate growth, pulmonary hemorrhage syndrome, and complex eye pathology. Endocrinology 146, 5237–5246. Schneider, M.R., Werner, S., Paus, R., Wolf, E., 2008. Beyond wavy hairs: the epidermal growth factor receptor and its ligands in skin biology and pathology. Am. J. Pathol. 173, 14–24. Sibilia, M., Kroismayr, R., Lichtenberger, B.M., Natarajan, A., Hecking, M., Holcmann, M., 2007. The epidermal growth factor receptor: from development to tumorigenesis. Differentiation 75, 770–787. 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. Yarden, Y., Shilo, B.Z., 2007. SnapShot: EGFR signaling pathway. Cell 131, 1018.