Paracrine regulation of Sertoli cell proliferation

The Sertoli cell is a highly differentiated somatic cell in the seminiferous epithelium of the testis. This cell is of prime importance for both testicular development and spermatogenesis. Sertoli cells proliferate up to the start of puberty and, due to their nursing functions, the final Sertoli cell number is well correlated to the amount of germ cells produced by the adult testis. The physiological regulation of Sertoli cell growth is not fully understood, but follicle stimulating hormone (FSH) from the pituitary and testicular paracrine factors are known to be important. Testicular function may be harmed by local or general inflammatory diseases. Inflammatory stimuli induce testicular production of proinflammatory cytokines, which may be of pathogenic importance. Sertoli cells are possible targets of these proinflammatory cytokines. The aim of the present thesis was to set up a Sertoli cell proliferation assay, suitable for screening of putative mitogens, and use it to study the effects of growth factors and cytokines, thought to be of physiological or pathophysiological relevance. Sertoli cells from 8 to 9-day-old rats were isolated and cultured under serum-free conditions. Proliferation was assessed by three different methods; H-thymidine incorporation, BrdU immunocytochemistry and MTT supravital staining. Transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) were found to dose-dependently stimulate Sertoli cell proliferation, while insulin-like growth factor (IGF)-I and IGF-II, acidic and basic fibroblast growth factors (FGFa, FGFb) were negative in the system. In search for Sertoli cell growth factors in testicular tissue, proteins from testes of immature (5-day-old), pubertal (25-day-old) and adult (50-day-old) rats were purified by HPLC gel permeation chromatography. Pubertal and adult testes contained a 17 kDa protein with potent stimulatory effect on immature Sertoli cell multiplication in vitro. This protein was identified as interleukin-1α (IL-1α), which is constitutively produced by the adult testis. Immature rat testis produced a Sertoli cell growth stimulating factor(s) in the 45 kDa size range that still remains to be identified. The recombinant proinflammatory cytokines IL-1α, IL-1β and TNF-α showed potent Sertoli cell mitogenic effects. It was further demonstrated that the IL-1-induced Sertoli cell proliferation was mediated by the p38 MAPK intracellular signal transduction pathway. IL-1α and IL-1β, but not TNF-α, also produced a striking morphological change which may represent a change in Sertoli cell differentiation state. There was a synergistic increase in Sertoli cell proliferation by IL-1 or TNF-α together with the important endocrine Sertoli cell regulator FSH. Furthermore, the cytokines IL-6 and IFN-γ modulated the FSH effect in different ways. IL-6 increased, while IFN-γ inhibited FSHinduced Sertoli cell proliferation. Bacterial endotoxin directly stimulated Sertoli cell growth. Taken together, the results show that TGF-α is a Sertoli cell growth factor and that proinflammatory cytokines and bacterial endotoxin interfere with Sertoli cell proliferation in vitro. TGF-α may be a physiological paracrine Sertoli cell growth regulator while cytokines and endotoxin may potentially disturb testicular cytoarchitecture and development during inflammatory and infectious diseases in vivo. LIST OF PUBLICATIONS The present study is based on the following papers, which will be referred to by their Roman numerals I. Scarpino S., Morena A.R., Petersen C., Fröysa B., Söder O., Boitani C. A rapid method of Sertoli cell isolation by DSA lectin, allowing mitoitic analyses Molecular and Cellular Endocrinology 1998, 146: 121-127 II. Petersen C., Boitani C., Fröysa B., Söder O. Transforming growth factor-α stimulates proliferation of rat Sertoli cells Molecular and Cellular Endocrinology 2001, 181: 221-227 III. Petersen C., Boitani C., Fröysa B., Söder O. Interleukin-1 is a potent growth factor for immature rat Sertoli cells Molecular and Cellular Endocrinology 2002, 186: 37-47 IV. Petersen C., Svechnikov K., Fröysa B., Söder O. p38 MAPK pathway mediates interleukin-1-induced Sertoli cell proliferation Submitted manuscript V. Petersen C., Fröysa B., Söder O. Endotoxin and proinflammatory cytokines modulate Sertoli cell proliferation in vitro Manuscript