- Email: [email protected]
Establishment of a Mouse Sertoli Cell Line Producing Rat Androgen-binding Protein (ABP)
Establishment of a mouse Sertoli cell line producing rat androgen-binding protein (ABP) Ange´lique Ducray,* Miche`le Bloquel,† Ketsia Hess,‡ Geoffrey L. Hammond,§ Hubert Ge´rard,* and Anne Ge´rard* *Unite´ de Ge´ne´tique et Interactions Cellulaires en Reproduction, De´partement de Cytologie Histologie–Embryologie, Faculte´ de Me´decine, Universite´ Henri Poincare´ Nancy I, Vandoeuvre cedex, France; †IUT BA Sante´; ‡Biochimie, Faculte´ de Me´decine, Universite´ Henri Poincare´ Nancy I; Vandoeuvre cedex, France; and §Department of Obstetrics and Gynecology and Pharmacology and Toxicology, The University of Western Ontario, LRCC Cancer Research Laboratories, London, Ontario, Canada The ultimate goal of this study was to compare the fate of rat testicular germ cells cocultured with mouse Sertoli cells that either do or do not produce rat androgen-binding protein (ABP). As a first step, we stably transfected a rat ABP expression construct into an immortalized mouse Sertoli cell line (TM4), which does not produce ABP when growing on plastic without hormones. The transfection of the pRc/CMV- rat ABP cDNA expression vector containing a neomycin resistance gene was made by either the liposome method (Dotap) or by polyethyleneimine transfection (PEI) into TM4 cell cultures. Neomycin-resistant clones were selected by adding Geneticin to the culture medium for 3 weeks. Analysis of over 25 clones revealed the presence of recombinant rat ABP when cell extracts and culture media were probed with a rabbit polyclonal antibody raised against rat testicular ABP, indicating the translation and secretion of a protein similar to rat testicular ABP. Transfected TM4 cells maintain the secretion of rat ABP for more than 40 days, with immunopositive rat ABP localized within cytoplasmic granules in the Golgi region and along cytoplasmic processes in TM4 transfected with either vector. Electron microscopic study revealed a higher development of cytoplasmic organelles involved in protein secretion. (Steroids 63:285–287, 1998) © 1998 by Elsevier Science Inc.
Keywords: androgen-binding protein; germ cells; Sertoli cells
Introduction Products of the ABP/SHBG gene interact with spermatogenic cells of different species by a receptor-mediated mechanism dependent on a maturation step,1– 4 and with mounting evidence for a role for ABP as paracrine factor during development and in adult spermatogenesis. Because germ cells do not survive without Sertoli cells and because Sertoli cells lose their ability to produce ABP in vitro even in the presence of follicle-stimulating hormone (FSH) and testosterone,5 we established an experimental germ cell/Sertoli cell coculture model with Sertoli cells that either produce or do not produce ABP. The TM4 mouse Sertoli cell line was used for this purpose because these cells do not produce ABP except in the presence of peritubular cells.6,7 As a first step, we
Address reprint requests to Anne Gerard, Faculte´ de Me´decine–Universite´ Henri Poincare´ Nancy I, De´partement de Cytologie, Histologie–Embryologie, 9, Avenue de la Fore´t BP 184, 54505 Vandoeuvre cedex, France. E-mail: [email protected] Steroids 63:285–287, 1998 © 1998 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
transfected this cell line with an expression vector that constitutively expresses rat ABP.
Culture conditions and transfections TM4 cells were obtained from ECACC (European Collection of Animal Cell Cultures, Valbonne-France) and maintained in a basal culture medium consisting in Ham F12/Dulbecco’s modified Eagle’s medium (Sigma), 10% fetal calf serum, insulin, transferrin, selenium, and glutamine—penicillin–streptomycin solution (Sigma) at 37°C in a 5% CO2/95% air atmosphere. The plasmid pRc/CMV- rat ABP contains the cloned 1.6 Kbp rat ABP cDNA inserted into a HindIII/XbaI-digested pRc/CMV for eukaryotic expression, the neomycin and the ampicillin resistance genes for selection in mammalian cells, and Escherichia Coli, respectively (Hammond et al., unpublished data). Adherent cells at 50% confluence (1 3 106 cells) were transfected by the liposome method8 using 6– 8 mg DNA in 36 mL of transfection reagent Dotap (Boehringer Mannheim) for 6–8 h in the absence of serum and antibiotics. The 0039-128X/98/$19.00 PII S0039-128X(98)00030-0
Steroids in maturity cells were then renewed with the basal culture medium without antibiotics to allow for recovery prior to the addition of 200 mg Geneticin/mL (G418, Life Technologies) for selection of neomycin-resistant cells. From this point, resistant clones were harvested for an additional 3 weeks in the presence of G418 and then again in basal medium. Another synthetic transfection vector,9 polyethyleneimine (PEI, Exgen, 500, Euromedex), was complexed to DNA (2 mg/1 mL PEI) and incubated with 1 3 106 TM4 cells in the presence of serum without antibiotics for 24 h, and then renewed in the selection medium (200 mg/mL G418). For each experiment, control cultures were performed with the vector only or without transfection. The media and transfected cells were collected and screened from 48 h post-transfection until 40 days after transfection.
Screening of transfected cells Detection of recombinant protein was either by immunoblot of cell extracts and the corresponding media or by immunocytochemistry on cells grown on coverslips.
Immnoblots Cell extracts (in 100 mM Tris; 2.5% Nonidet-P40; 2.5% Triton X-100; 4% deoxycholate; 0.2 mM phenylmethylsulfonyl fluoride; 0.01% NaN3; 0.14 M NaCl, pH 7.4) or media (concentrated about 20-fold) were blotted onto Hybond C-extra membranes and subjected to immunodetection using a polyclonal antiserum against rat testicular ABP (1:2000, kindly provided by J. Closset) and indirect peroxidase detection using diaminobenzidine as chromogen.
Figure 1 Immunoblot analysis with rat testicular ABP antibody of independent clones after Dotap or Exgen transfection after different selection periods. A, cell extracts; B, culture medium.
nant rat ABP in cell homogenates (Figure 1A) and in culture medium (Figure 1B) when probed with the antirat ABP antibody. Only a few cells were positive 48 h after transfection by immunocytochemistry (Figure 2A), while the cytoplasm of all cells was immunopositive after selection in the culture medium containing G418 (Figure 2B). The same pattern of ABP localisation was obtained after transfection with Exgen, with an accumulation of positive droplets in the perinuclear Golgi region and along the cytoplasmic pro-
Immunocytochemistry Neomycin resistant clones were plated onto 2% aminopropyltriethoxysilane-coated coverslips and grown for 2 days, fixed with 4% paraformaldehyde in PBS for 15 min before immunocytochemistry with the rat ABP antibody (1:1000) and developed with the avidin-biotin-peroxidase detection method with 3-amino, 9-ethylcarbazole (AEC) as the chromogen (Dako). Some fixed transfected cells were treated with an antimouse ABP antibody (1:800) instead of the rat antibody. Normal serum was substituted for the primary antibody in negative controls. Sections were counterstained with hematoxylin.
Ultrastructural analysis Cells were pelleted, fixed in 2.5% glutaraldehyde in cacodylate buffer for 1 h, and prepared for routine electron microscopy. Ultrathin sections were examined on a Phillips CM12 electron microscope.
Results Neomycin-resistant clones were selected with both transfection vectors and rat ABP cDNA. These clones were able to grow in the basal medium after 3 weeks of selection. Eighteen Dotap-clones and 15 Exgen-clones showed recombi-
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Figure 2 Immunohistochemical detection of recombinant rat ABP within cells from different clones. A, 48 h after transfection; B, 15 days after transfection.
Mouse Sertoli cell line producing rat ABP: Ducray et al. ABP antibody is used suggests that the protein obtained was not the result of reactivation of the endogenous ABP gene, but expression of the integrated rat ABP gene. The increase with time of the number of positive cells reflects the stable and continuous production of recombinant protein. Unmodified TM4 and transfected TM4 offer the opportunity to establish conditions of germ cell coculture which differ only by the presence or the absence of ABP, and should allow a fuller understanding of the role of this steroid binding protein in the testis.
Acknowledgments We thank M. Adam and B. Cunin for technical assistance.
References 1. 2. Figure 3 Ultrastructural features of positive Sertoli clone secreting rat ABPR. R, reticulum; M, mitochondrion; N , nucleus. Magnification 37000. Inset, high magnification showing cytoplasmic organelles involved in protein secretion. Magnification 320,000.
3.
4.
cesses. The use of mouse rather than rat ABP antibody consistently decreased the intensity of the immunopositive signal in transfected cells, and controls were completely negative. Electron microscopic examination showed enhancement of cytoplasmic organelles involved in protein secretion, with an increased number of granular reticulum profiles (Figure 3).
5.
6. 7.
Discussion TM4 mouse Sertoli cells have been obtained that constitutively express a rat ABP cDNA, and produce and secrete rat ABP. Morphologically, transfected TM4 acquire signs of enhanced protein synthesis. Immunocytochemistry indicates the translation and the secretion of a protein similar to rat testicular ABP. The faint reaction obtained when mouse
8.
9.
Gerard A, En Nya A, Egloff M, Domingo M, Degrelle H, Gerard H (1991). Endocytosis of human sex steroid-binding protein in monkey germ cells. Ann NY Acad Sci 637:258–277. Felden F, Gueant JL, Gerard A, En Nya A, Fremont S, Nicolas JP, Gerard H (1992). Photoaffinity labeled rat androgen-binding protein and human sex steroid-binding protein bind specifically to rat germ cells. Mol J Endocrinol 9:39–46. Gerard H, Gerard A, Felden F, En Nya A, Gueant JL (1994). Spermatogenic cells do internalize Sertoli androgen-binding protein: an electron microscope autoradiographic study in the rat. Endocrinology 134:1515–1527. Gerard A (1995). Endocytosis of steroid-binding proteins (ABP, SHBG) by spermatogenic cells. Steroid Biochem J Mol Biol 53:33– 542. Rommerts F, Kruger-Sewnarain B, Van Woerkom-Blick A, Grootegoed J, Van der Molen H (1978). Secretion of proteins by Sertoli cell enriched cultures: Effects of follicle stimulating hormone, dibutyryl cAMP and testosterone and correlation with secretion of oestradiol and androgen binding protein. Mol Cell Endocrinol 10:39. Mather JP (1980). Establishment and characterization of two distinct mouse testicular epithelial cell lines. Biol Reprod 23: 243–252. Mather JP, Perez-Infante V, Zhuang LZ, Phillips D (1982). Culture of testicular cells in hormone-supplemented scrum-free medium. Ann NY Acad Sci 383:44–68. Felgner PL, Gadek TR, Hoem M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987). Lipofection, a highly efficient, lipid-mediated DNA transfection procedure. Proc Natl Acad Sci USA 84:7413–7417. Boussif O, Lezouale’h F, Zanta M, Mergny MD, Demeneix B, Behr JP (1995). A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc Natl Acad Sci USA 92:7297–7301.
Steroids, 1998, vol. 63, May/June
287
Keywords: androgen-binding protein; germ cells; Sertoli cells
Introduction Products of the ABP/SHBG gene interact with spermatogenic cells of different species by a receptor-mediated mechanism dependent on a maturation step,1– 4 and with mounting evidence for a role for ABP as paracrine factor during development and in adult spermatogenesis. Because germ cells do not survive without Sertoli cells and because Sertoli cells lose their ability to produce ABP in vitro even in the presence of follicle-stimulating hormone (FSH) and testosterone,5 we established an experimental germ cell/Sertoli cell coculture model with Sertoli cells that either produce or do not produce ABP. The TM4 mouse Sertoli cell line was used for this purpose because these cells do not produce ABP except in the presence of peritubular cells.6,7 As a first step, we
Address reprint requests to Anne Gerard, Faculte´ de Me´decine–Universite´ Henri Poincare´ Nancy I, De´partement de Cytologie, Histologie–Embryologie, 9, Avenue de la Fore´t BP 184, 54505 Vandoeuvre cedex, France. E-mail: [email protected] Steroids 63:285–287, 1998 © 1998 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
transfected this cell line with an expression vector that constitutively expresses rat ABP.
Culture conditions and transfections TM4 cells were obtained from ECACC (European Collection of Animal Cell Cultures, Valbonne-France) and maintained in a basal culture medium consisting in Ham F12/Dulbecco’s modified Eagle’s medium (Sigma), 10% fetal calf serum, insulin, transferrin, selenium, and glutamine—penicillin–streptomycin solution (Sigma) at 37°C in a 5% CO2/95% air atmosphere. The plasmid pRc/CMV- rat ABP contains the cloned 1.6 Kbp rat ABP cDNA inserted into a HindIII/XbaI-digested pRc/CMV for eukaryotic expression, the neomycin and the ampicillin resistance genes for selection in mammalian cells, and Escherichia Coli, respectively (Hammond et al., unpublished data). Adherent cells at 50% confluence (1 3 106 cells) were transfected by the liposome method8 using 6– 8 mg DNA in 36 mL of transfection reagent Dotap (Boehringer Mannheim) for 6–8 h in the absence of serum and antibiotics. The 0039-128X/98/$19.00 PII S0039-128X(98)00030-0
Steroids in maturity cells were then renewed with the basal culture medium without antibiotics to allow for recovery prior to the addition of 200 mg Geneticin/mL (G418, Life Technologies) for selection of neomycin-resistant cells. From this point, resistant clones were harvested for an additional 3 weeks in the presence of G418 and then again in basal medium. Another synthetic transfection vector,9 polyethyleneimine (PEI, Exgen, 500, Euromedex), was complexed to DNA (2 mg/1 mL PEI) and incubated with 1 3 106 TM4 cells in the presence of serum without antibiotics for 24 h, and then renewed in the selection medium (200 mg/mL G418). For each experiment, control cultures were performed with the vector only or without transfection. The media and transfected cells were collected and screened from 48 h post-transfection until 40 days after transfection.
Screening of transfected cells Detection of recombinant protein was either by immunoblot of cell extracts and the corresponding media or by immunocytochemistry on cells grown on coverslips.
Immnoblots Cell extracts (in 100 mM Tris; 2.5% Nonidet-P40; 2.5% Triton X-100; 4% deoxycholate; 0.2 mM phenylmethylsulfonyl fluoride; 0.01% NaN3; 0.14 M NaCl, pH 7.4) or media (concentrated about 20-fold) were blotted onto Hybond C-extra membranes and subjected to immunodetection using a polyclonal antiserum against rat testicular ABP (1:2000, kindly provided by J. Closset) and indirect peroxidase detection using diaminobenzidine as chromogen.
Figure 1 Immunoblot analysis with rat testicular ABP antibody of independent clones after Dotap or Exgen transfection after different selection periods. A, cell extracts; B, culture medium.
nant rat ABP in cell homogenates (Figure 1A) and in culture medium (Figure 1B) when probed with the antirat ABP antibody. Only a few cells were positive 48 h after transfection by immunocytochemistry (Figure 2A), while the cytoplasm of all cells was immunopositive after selection in the culture medium containing G418 (Figure 2B). The same pattern of ABP localisation was obtained after transfection with Exgen, with an accumulation of positive droplets in the perinuclear Golgi region and along the cytoplasmic pro-
Immunocytochemistry Neomycin resistant clones were plated onto 2% aminopropyltriethoxysilane-coated coverslips and grown for 2 days, fixed with 4% paraformaldehyde in PBS for 15 min before immunocytochemistry with the rat ABP antibody (1:1000) and developed with the avidin-biotin-peroxidase detection method with 3-amino, 9-ethylcarbazole (AEC) as the chromogen (Dako). Some fixed transfected cells were treated with an antimouse ABP antibody (1:800) instead of the rat antibody. Normal serum was substituted for the primary antibody in negative controls. Sections were counterstained with hematoxylin.
Ultrastructural analysis Cells were pelleted, fixed in 2.5% glutaraldehyde in cacodylate buffer for 1 h, and prepared for routine electron microscopy. Ultrathin sections were examined on a Phillips CM12 electron microscope.
Results Neomycin-resistant clones were selected with both transfection vectors and rat ABP cDNA. These clones were able to grow in the basal medium after 3 weeks of selection. Eighteen Dotap-clones and 15 Exgen-clones showed recombi-
286
Steroids, 1998, vol. 63, May/June
Figure 2 Immunohistochemical detection of recombinant rat ABP within cells from different clones. A, 48 h after transfection; B, 15 days after transfection.
Mouse Sertoli cell line producing rat ABP: Ducray et al. ABP antibody is used suggests that the protein obtained was not the result of reactivation of the endogenous ABP gene, but expression of the integrated rat ABP gene. The increase with time of the number of positive cells reflects the stable and continuous production of recombinant protein. Unmodified TM4 and transfected TM4 offer the opportunity to establish conditions of germ cell coculture which differ only by the presence or the absence of ABP, and should allow a fuller understanding of the role of this steroid binding protein in the testis.
Acknowledgments We thank M. Adam and B. Cunin for technical assistance.
References 1. 2. Figure 3 Ultrastructural features of positive Sertoli clone secreting rat ABPR. R, reticulum; M, mitochondrion; N , nucleus. Magnification 37000. Inset, high magnification showing cytoplasmic organelles involved in protein secretion. Magnification 320,000.
3.
4.
cesses. The use of mouse rather than rat ABP antibody consistently decreased the intensity of the immunopositive signal in transfected cells, and controls were completely negative. Electron microscopic examination showed enhancement of cytoplasmic organelles involved in protein secretion, with an increased number of granular reticulum profiles (Figure 3).
5.
6. 7.
Discussion TM4 mouse Sertoli cells have been obtained that constitutively express a rat ABP cDNA, and produce and secrete rat ABP. Morphologically, transfected TM4 acquire signs of enhanced protein synthesis. Immunocytochemistry indicates the translation and the secretion of a protein similar to rat testicular ABP. The faint reaction obtained when mouse
8.
9.
Gerard A, En Nya A, Egloff M, Domingo M, Degrelle H, Gerard H (1991). Endocytosis of human sex steroid-binding protein in monkey germ cells. Ann NY Acad Sci 637:258–277. Felden F, Gueant JL, Gerard A, En Nya A, Fremont S, Nicolas JP, Gerard H (1992). Photoaffinity labeled rat androgen-binding protein and human sex steroid-binding protein bind specifically to rat germ cells. Mol J Endocrinol 9:39–46. Gerard H, Gerard A, Felden F, En Nya A, Gueant JL (1994). Spermatogenic cells do internalize Sertoli androgen-binding protein: an electron microscope autoradiographic study in the rat. Endocrinology 134:1515–1527. Gerard A (1995). Endocytosis of steroid-binding proteins (ABP, SHBG) by spermatogenic cells. Steroid Biochem J Mol Biol 53:33– 542. Rommerts F, Kruger-Sewnarain B, Van Woerkom-Blick A, Grootegoed J, Van der Molen H (1978). Secretion of proteins by Sertoli cell enriched cultures: Effects of follicle stimulating hormone, dibutyryl cAMP and testosterone and correlation with secretion of oestradiol and androgen binding protein. Mol Cell Endocrinol 10:39. Mather JP (1980). Establishment and characterization of two distinct mouse testicular epithelial cell lines. Biol Reprod 23: 243–252. Mather JP, Perez-Infante V, Zhuang LZ, Phillips D (1982). Culture of testicular cells in hormone-supplemented scrum-free medium. Ann NY Acad Sci 383:44–68. Felgner PL, Gadek TR, Hoem M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987). Lipofection, a highly efficient, lipid-mediated DNA transfection procedure. Proc Natl Acad Sci USA 84:7413–7417. Boussif O, Lezouale’h F, Zanta M, Mergny MD, Demeneix B, Behr JP (1995). A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc Natl Acad Sci USA 92:7297–7301.
Steroids, 1998, vol. 63, May/June
287