Dokument

Zusammenfassung:
Die Regulation der Spermatogenese beruht auf einem komplexen Netzwerk an endokrinen, parakrinen und zellulären Mediatoren. Der genaue Ablauf der Spermatogenese ist detailliert bekannt, doch trotz intensiver Forschung ist bis heute die genaue Regelung unbekannt. Viele Gruppen versuchten in vitro-Systeme zur Kultivierung von Keimzellen zu entwickeln und so zur Klärung der regulierenden Mechnanismen beizutragen. Es ist bis heute jedoch nicht möglich, Keimzellen in vitro bis zu elongierten Spermatiden oder Spermatozoen zu kultivieren. In früheren Studien unserer Arbeitsgruppe als auch anderer Gruppen wurde die Hypothese aufgestellt, dass TGF-β und die durch TGF-β induzierte Apoptose von Keimzellen bei Eintritt in die Pubertät eine essentielle Rolle in der Spermatogenese hat. In dieser Arbeit versuchten wir herauszufinden, ob die TGF-β Sekretion eventuell Androgen reguliert sein könnte. Über einen Zeitraum von 72h wurden reine Sertolizellen (SK11), eine Mischkultur aus Sertolizellen und Peritubulärzellen (WL3) und reine Keimzellen (GC2) mit Testosteron in verschiedenen Konzentrationen stimuliert. Anschließend wurde mittels ELISA die TGF-β Menge im Mediumüberstand dieser Zellen gemessen. Die Sertolizellen (SK11) sezernierten nur 20% so viel TGF-β1 wie die Mischkultur (WL3) und sogar nur 3% so viel TGF-β2 wie die Mischkultur (WL3). Die TGF-β1 und TGF-β2 Sekretion in reinen Sertolizellen (SK11) und der Mischkultur (WL3) konnte konzentrationsabhängig von Testosteron inhibiert werden. Dieser Effekt war durch eine antiandrogene Stimulierung mit Flutamid reversibel. Dies weist darauf hin, dass immortalisierte Sertolizellen einen funktionellen AR aufweisen. Interessanterweise maßen wir keine TGF-β3 Sekretion in präpubertären Keimzellen (GC2) und der Mischkultur (WL3). Außerdem konnten wir keine TGF-β2 Sekretion in Keimzellen (GC2) messen. Zur Bestätigung unserer Ergebnisse untersuchten wir zusätzlich die TGF-β2 Sekretion und die AR-Expression testikulärer Zellen an Hodenschnitten infertiler Männer mittels Immunhistochemie. Dabei beobachteten wir, dass es in den Hodenschnitten von Männern mit gestörter Spermatogenese zu einer signifikant stärkeren Anfärbung von TGF-β2 in den testikulären Zellen kam, als in den Schnitten von Männern mit normaler Spermatogenese. Ein Zusammenhang zwischen der AR-Expression und TGF-β2 Sekretion ließ sich mit dieser Methode nicht herstellen und wurde von uns mit anderweitiger Methodik noch nicht nachgegangen. Damit konnten wir aufzeigen, dass TGF-β1 und TGF-β2 androgen reguliert sind. Somit ist ein Regulationsmechanismus während der Spermatogenese verstanden und kann als Hilfe für weitere Studien zur Klärung des gesamten Mechanismus Spermatogenese beitragen und den Weg zur Realisierung verbesserter Therapieoptionen bei männlicher Infertilität ebnen.

Bibliographie / References

1. Roberts AB, Sporn MB (1985). Transforming growth factors. Cancer Surv 4:683-705
2. Kang HY, Huang KE, Chang SY, Ma WL, Lin WJ, Chang C (2002). Differential modulation of androgen receptor-mediated transactivation by Smad3 and tumor suppressor Smad4. J Biol Chem 277:43749-43756
3. Walther N, Jansen M, Ergün S, Kascheike B, Ivell R (1996). Sertoli cell lines established from H-2K b -tsA58 transgenic mice differentially regulate the expression of cell-specific genes. Exp Cell Res 225:411-421
4. Kerr JB, Savage GN, Millar M, Sharpe RM (1993). Response of the seminiferous epithelium of the rat testis to withdrawal of androgen: evidence for direct effect upon intercellular spaces associated with Sertoli cell junctional complexes. Cell Tissue Res 274:153-161
5. van der Poel HG (2005). Androgen receptor and TGFbeta1/Smad signaling are mutually inhibitory in prostate cancer. Eur Urol 48:1051-1058
6. Memon MA, Anway MD, Covert TR, Uzumcu M, Skinner MK (2008). Transforming Growth Factor Beta (TGFβ1, TGFβ2 and TGFβ3) Null-Mutant Phenotypes in Embryonic Gonadal Development. Mol Cell Endocrinol 294:70-80
7. Prepin J, Le Vigouroux P (1997). Inhibition by TGF-β1 of the in vitro thymulin- stimulated proliferation of gonocytes from fetal rat testes. Reprod Nutr Dev 37:203- 206
8. Wang H, Song K, Sponseller TL, Danielpour D (2004). Novel function of the androgen receptor-associated protein 55/Hic-5 as a negative regulator of smad3 signaling. J Biol Chem 280:5154-5162
9. Xia W, Mruk DD, Lee WM, Cheng CY (2006). Differential interactions between transforming growth factor-beta3/TbetaR1, TAB1, and CD2AP disrupt blood-testis barrier and Sertoli-germ cell adhesion. J Biol Chem 281:16799-16813
10. Hata A, Lagna G, Massague J, Hemmati-Brivanlou (1998). Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor.
11. Immunohistochemical quantitative study of peritubular lamina propria after induction of testicular atrophy induced by epinephrine. Int J Androl 18:295-306
12. Weinbauer GT, Wessels J (1999). 'Paracrine' control of spermatogenesis. Andrologia 31:249-262
13. Roelen BA, Cohen OS, Raychowdhury MK, Chadee DN, Zhang Y, Kyriakis JM, Alessandrini AA, Lin HY (2003). Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-beta-induced nuclear accumulation. Am J Physiol Cell Physiol 285:823-830
14. Vornberger W, Prins G, Musto NA, Suarez-Quian CA (1994). Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis.
15. Singh J, O'Neill C, Handelsman DJ (1995). Induction of spermatogenesis by androgens in gonadotropin-deficient (hpg) mice. J Endocrinol 136:5311-5321 LITERATURVERZEICHNIS
16. Konrad L, Albrecht M, Renneberg H, Aumüller G (2000). Transforming growth factor-β2 mediates mesenchymal-epithelial interactions of testicular somatic cells. Endocrinology 141:3679-3686
17. Zatelli MC, Rossi R, degli Uberti EC (2000). Androgen influences transforming growth-factor beta1 gene expression in human adrenocortical cells. J Clin Endocrinol Metab 85:847-852
18. Skinner MK, Moses HL (1989). Transforming growth factorβ gene expression and action in the seminiferous tubule: peritubular cell-Sertoli cell interactions. Mol Endocrinol 3:625-634
19. Lui WY, Lee WM, Cheng CY (2003b). TGF-βs: Their role in testicular function and Sertoli cell tight junction dynamics. Int J Androl 26:147-160
20. Konrad L, Keilani MM, Laible L, Nottelmann U, Hofmann R (2006). Effects of TGF- betas and a specific antagonist on apoptosis of immature rat male germ cells in vitro. Apoptosis 11:739-748
21. Wright WW, Frankel AI (1980). An androgen receptor in the nuclei of late spermatids in testes of male rats. J Endocrinol 107:314-318
22. Zhang S (1999). An Atlas of Histology. Springer-Verlag New York, Inc. ISBN 0-387- 94954-2
23. Quigley CA, De Bellis A, Marschke KB, El-Awady MK, Wison EM, French FS (1995). Androgen receptor defects: historical, clinical and molecular perspectives. Endocr Rev 16:271-321
24. Androgen receptor-Ets protein interaction is a novel mechanism for steroid hormone- mediated down-modulation of matrix metalloproteinase expression. J Biol Chem 271:23907-23913
25. Rodriguez I, Ody C, Araki K, Garcia I, Vassalli P (1997). An early and massive wave of germinal cell apoptosis is required fort he development of functional spermatogenesis. EMBO J 16:2262-2270
26. Monesi V (1962). Autoradiographic study of DNA synthesis and the cell cycle in spermatogonia and spermatocytes of mouse testis using tritiated thymidine. J Cell Biol 14:1-18
27. Wilson EM, French FS (1976). Binding properties of androgen receptors: evidence for identical receptors in rat testis, epididymis and prostate. J Biol Chem 251:5620-5629
28. Ozanne B, Stiles C (eds) Cancer Cells. Cold Spring Harbor Press, Cold Spring Harbor, NY, vol 3:65-71
29. Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ. Hruban RH, Kern SE (1996). DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271:350-353
30. Wikstrom P, Westin P, Stattin P, Damber JE, Bergh A (1999). Early castration- induced upregulation of transforming growth factor beta1 and its receptors is associated with tumor cell apoptosis and a major decline in serum prostate-specific antigen in prostate cancer patients. Prostate 38:268-277
31. gene expression and action during pubertal development of the seminiferous tubule: Potential role at the onset of spermatogenesis. Mol Endocrinol 7:67-76
32. Zhou X, Xing L, Boyce BF, Hung MC, Zhang S, Gan L, Chang C (2002). Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues. Proc Natl Acad Sci USA 99:13498-13503
33. ten Dijke P, Hansen P, Iwata KK, Pieler C, Foulkes JG (1988). Identification of a new member of the transforming groth factor type β gene family. Proc Natl Acad Sci USA 85:4715-4719
34. Mueller OM, Korach KS (2001). Immortalized testis cell lines from estrogen receptor (ER) α knock-out and wild-type mice expressing funcional ERα or ERβ. J Androl 22:652-664
35. Shan LX, Bardin CW, Hardy MP (1997). Immunohistochemical analysis of androgen effects on androgen receptor expression in developing Leydig and Sertoli cells. J Endocrinol 138:1259-1266
36. Teerds KJ, Dorrington JH (1993). Localization of transforming growth factor β1 and β2 during testicular development in the rat. Biol Reprod 48:40-45
37. Roberts AB (1998). Molecular and cell biology of TGF-β. Miner Electrolyte Metab 24:111-119
38. ten Dijke P, Hill CS (2004). New insights into TGF-β-Smad signalling. Trends Biochem Sci 29:265-273
39. Wang Y, Lui WY (2009). Opposite effects of interleukin-1alpha and transforming growth factor-beta2 induce stage-specific regulation of junctional adhesion molecule- B gene in Sertoli cells. Endocrinology 150:2404-2412
40. Tachibana I, Imoto M, Adjei PN, Gores GJ, Subramaniam M, Spelsberg TC, Urrutia R (1997). Overexpression of the TGFβ-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells. J Clin Invest 99:2365-2374
41. Guo Y, Kyprianou N (1999). Restoration of transforming growth factor beta signaling pathway in human prostate cancer cells suppresses tumorigenicity via induction of caspase-1-mediated apoptosis. Cancer Res 59:1366-1371
42. Mruk DD, Cheng CY (2000). Sertoli cell proteins in testicular paracriny. In: Jegou B, Pineau C (eds.), Testis, Epididymis and technologies in the year 2000. Heidelberg: Springer-Verlag; 197-228
43. Zhang Y, Feng XH, Derynck R (1998). Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta induced transcription. Nature 394:909-913
44. Smad3 represses androgen receptor-mediated transcription. Cancer Res 61:2112-2118
45. Postlethwaite AE, Keski-Oja J, Moses HL, Kang AH (1987). Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor-β. J Exp Med 165:251-256
46. Zhang M, Latham DE, Delaney MA, Chakravarti A (2005). Survivin mediates resistance to antiandrogen therapy in prostate cancer. Oncogene 24:2474-2482
47. Wilson JD (1992). Syndromes of androgen resistance. Biol Reprod 46:68-76
48. Massague J (1998). TGF-beta signal transduction. Annu Rev Biochem 67:753-791
49. Perey B, Clermont Y, Leblond CP (1961). The wave of the seminiferous epithelium in the rat. Am J Anat 108:47-77
50. Massague J, Wotton D (2000). Transcriptional control by the TGF-β/Smad signaling system. EMBO J 19:1745-1754
51. Mullaney BP, Skinner MK (1993). Transforming growth factor-β (β1, β2, and β3)
52. Keski-Oja J, Leof EB, Lyons RM, Coffey RJ Jr, Moses HL (1987). Transforming growth factors and control of neoplastic cell growth. J Cell Biochem 33:95-107
53. Monesi V (1965). Synthetic activities during spermatogenesis in the mouse RNA and protein. Exp Cell Res 39:197-224
54. Maire M, Florin A, Kaszas K, Regnier D, Contard P, Tabone E, Mauduit C, Bars R, Benahmed M (2005). Alteration of transforming growth factor-β signaling system expression in adult rat germ cells with a chronic apoptotic cell death process after fetal androgen disruption. Endocrinology 146:5135-5143
55. Sneddon SF, Walther N, Saunders PTK (2005). Expression of androgen and estrogen receptors in Sertoli cells: studies using the mouse SK11 cell line. Endocrinology 146:5304-5312
56. MacConell LA, Leal AM, Vale WW (2002). The distribution of betaglycan protein and mRNA in rat brain, pituitary, and gonads: Implications for a role for betaglycan in inhibin-mediated reproductive functions. Endocrinology 143:1066-1075
57. Effects of testosterone on spermatogenic cell populations in the adult rat. Biol Reprod 51:945-955
58. Konrad L, Scheiber JA, Völck-Badonin E, Keilani MM, Laible L, Brandt H, Schmidt A, Müller G, Hofmann R (2007). Alternative splicing of TGF-betas and their high- affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells. BMC Genomics 8:318
59. Valderrama-Carvajal H, Cocolakis E, Lacerte A, Lee EH, Krystal G, Ali S, Lebrun JJ (2002). Activin/TGF-β induce apoptosis through Smad-dependent expression of the lipid phosphatase SHIP. Nat Cell Biol 4:963-969
60. Kadrmas JL, Beckerle MC (2004). The LIM domain: from the cytoskeleton to the nucleus. Nat Rev Mol Cell Biol 5:920-931
61. Zhang C, Yeh S, Chen YT, Wu CC, Chuang KH, Lin HY, Wang RS, Chang YJ, Mendis-Handagama C, Hu L, Lardy H, Chang C (2006). Oligozoospermia with normal fertility in male mice lacking the androgen receptor in testis peritubular myoid cells. Proc Natl Acad Sci USA 103:17718-17723
62. Guyton AC, Hall JE, editors (2000). Textbook of Medical Physiology. WB Saunders Company, Philadelphia, pp 925-926
63. Kang HY, Lin HK, Hu YC, Yeh S, Huang KE, Chang C (2001). From transforming growth factor-β signaling to androgen action: Identification of Smad3 as an androgen receptor coregulator in prostate cancer cells. Proc Natl Acad Sci USA 98:3018-3023
64. Yeh S, Chang C (1996). Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells. Proc Natl Acad Sci USA 93:5517-5521
65. Sanderson N, Factor V, Nagy P, Kopp J, Kondaiah P, Wakefield L, Roberts AB, Sporn MB, Thorgeirsson SS (1995). Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions. PNAS 92:2572-2576
66. Shin I, Bakin AV, Rodeck U, Brunet A, Arteaga CL (2001). Transforming growth factor β enhances epithelial cell survival via Akt-dependent regulation of FKHRL1.
67. Absence of a nuclear androgen receptor in isolated germinal cells of rat testis. Mol Cell Endocrinol 9:159-167
68. Taipale J, Saharinen J, Keski-Oja J (1998). Extracellular matrix-associated transforming growth factor-β: role in cancer cell growth and invasion. Adv Cancer Res 75:87-134
69. Hayashi H, Abdollah S, Qui Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone Jr MA, Wrana JL, Falb D (1997). The MAD-related protein Smad7 associates with the TGFβ receptor and functions as an antagonist of TGFβ signalling. Cell 89:1165-1173
70. Murphy-Ullrich JE, Poczatek M (2000). Activation of latent TGF-β by thrombospondin-1: Mechanism and physiology. Cytokine Growth Factor Rev 11:59- LITERATURVERZEICHNIS
71. Haagmans BL, Hoogerbrugge JW, Themmen APN, Teerds KJ (2003). Rat testicular germ cells and sertoli cells release different types of bioactive transforming growth factor beta in vitro. Reprod Biol Endocrinol 1:3

Das Dokument ist im Internet frei zugänglich - Hinweise zu den Nutzungsrechten