Insulin-like growth factor-I and -II binding and action on DNA synthesis in rainbow trout spermatogonia and...

Radio-labeled recombinant human insulin-like growth factor (125I-rhIGF-I) bound specifically to total testicular cells and to spermatogonia plus primary spermatocytes (Go+CI) that had been prepared from trout testes at various maturation stages by centrifugal elutriation and then cleared of somatic cells by preculture in the presence of 2% Ultroser G. Binding sites showed high affinity (Ka = 0.5 +/- 0.2 x 10(10) M-1) and low capacity (1.1 +/- 0.8 fmol/10(6) testicular cells) for 125I-IGF-I. (Gln3, Ala4, Tyr15, Leu16)IGF-I ([QAYL]IGF-I) was equipotent to IGF-I in competing with 125I-IGF-I for site occupancy on Go+CI. Insulin-like growth factor-II (IGF-II) was to 10-fold less potent than IGF-I or (QAYL)IGF-I, while bovine insulin competed only about 300-fold higher concentrations. Go+CI were cultured for 3 days in the presence or absence of mammalian IGF-I, IGF-II, (QAYL)IGF-I, and salmon or bovine insulin. All these molecules stimulated the incorporation of [3H]thymidine (added during the last 24 h in culture) by Go+CI in a dose-dependent manner. The mean ED50, independent of testicular maturation stage, was 5.9 +/- 4.9 ng/ml and 29.1 +/- 15.8 ng/ml for IGF-I and IGF-II, respectively. (QAYL)IGF-I was as potent as IGF-I. Concentrations of salmon or bovine insulin 100- to 300-fold higher were required to produce effects similar to those of IGF-I. While recombinant human IGF binding protein (IGFBP-3) had no effect by itself of basal [3H]thymidine incorporation, it inhibited the effect of IGF-I in a dose-dependent manner; however, it had no effect on the stimulation by (QAYL)IGF-F. Although combinations of low concentrations of IGF-I and IGF-II or salmon insulin had additive effects, combinations of maximum concentrations did not. We conclude that, in vitro, IGFs stimulate DNA synthesis of trout male germ cells by interacting directly with these cells through one IGF receptor.