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  • Title: Regulation of follistatin production by rat granulosa cells in vitro.
    Author: Shintani Y, Dyson M, Drummond AE, Findlay JK.
    Journal: Endocrinology; 1997 Jun; 138(6):2544-51. PubMed ID: 9165047.
    Abstract:
    The aims of this study were to apply enzyme-linked immunosorbent assays (ELISA) for human follistatins (FS) to measure total immunoreactive (ir-) rat FS and free rat FS, and investigate the regulation of production of total ir-FS and free FS by rat granulosa cells (GC) in vitro. Production of ir-inhibin was monitored as an index of GC function. The ELISAs for total ir-FS, based on an immunoradiometric assay developed recently for human FS, and free FS, based on capture of FS by a monoclonal antibody and detection by activin A binding, had sensitivities of 0.4 and 0.8 ng recombinant human (rh-) FS 288/ml, respectively, and did not cross-react with inhibin A, rLH, or FSH. rh-Activin did not cross react in the total ir-FS ELISA, but interfered with the measurement of free FS. Dilutions of GC-conditioned medium were parallel to the standard curve of rh-FS 288 for each assay. The values obtained in the free FS assay were 10- to 20-fold higher than those in the total ir-FS ELISA, suggesting that rat FS may be recognized by the antibodies differently than the human standard. Both total ir-FS and free FS production by undifferentiated GC from diethylstilbestrol (DES)-treated, immature rats increased with cell number and time in culture and were stimulated dose dependently by FSH, rh-activin A (except free FS, which was not measured because of interference), forskolin, and phorbol 12-myristrate. The effects of FSH and activin on FS production by undifferentiated GC were additive. There were significant effects of degree of differentiation of GC on basal FS production and responsiveness to FSH, LH, and rh-activin A. Both total ir-FS and free basal FS production increased up to 4-fold with the degree of differentiation of GC, produced by treating rats in vivo with DES (undifferentiated), DES plus FSH (partially differentiated), or DES plus FSH plus hCG (fully differentiated). The addition of FSH in vitro increased FS production by undifferentiated and partially differentiated GC, but not by fully differentiated GC. The only detectable effect of LH on FS production was on partially differentiated GC. Activin A stimulated total ir-FS production by undifferentiated and partially differentiated GC, but inhibited total ir-FS production by fully differentiated GC. Ir-inhibin production in these experiments was similar to that of FS with the following exceptions; phorbol 12-myristrate inhibited ir-inhibin production by undifferentiated GC, basal ir-inhibin decreased in fully differentiated GC, FSH stimulated ir-inhibin only in undifferentiated GC, and rh-activin A stimulated ir-inhibin at all stages. It is concluded that 1) FS protein production by cultured undifferentiated rat GC is up-regulated by FSH and activin, possibly via both protein kinase A and C pathways; 2) increasing GC differentiation is associated with a significant increase in basal FS production by rat GC and a change in the hormonal regulation of FS production; and 3) FS and ir-inhibin production by cultured rat GC can be differentially regulated. The results are consistent with the hypothesis that activin tone decreases within follicles as they develop due to increased production of the activin-binding protein FS.
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