Purification of ovine transferrin and study of the hormonal control of its secretion in enriched cultures of ovine Sertoli cells

Abstract

Ovine transferrin (o-transferrin) was purified from sheep serum by fractionated precipitation with ammonium sulphate, ion-exchange chromatography on DEAE trisacryl and finally by affinity chromatography on Affigel blue to remove albumin. Ovine transferrin was identified by its apparent molecular weight in sodium dodecyl sulphate polyacrylamide gel electrophoresis and by its N-terminal amino-acid sequence. The procedure presented in this report permits the preparation of highly purified o-transferrin with a good recovery (52% of initial total immunoactivity). An antiserum against o-transferrin was then raised in rabbits, using this highly purified preparation. A specific radioimmunoassay was set up using 125I-labelled o-transferrin. Its detection threshold (4 ng/ml) was low enough to measure o-transferrin in spent culture media of ovine Sertoli cells, which ranged between 15 and 600 ng/ml. Sheep seminiferous tubule cells, containing ~80% Sertoli cells, were cultured at a high density (1.5 x 106 cells/cm2) on a thin layer of reconstituted basement membrane. Kinetic studies showed that basal daily secretion of o-transferrin was reduced by half (-49%) between Day 1 and Day 2 of culture, and progressively decreased thereafter. Under FIRT (500 ng ovine follicle-stimulating hormone (FSH)/ml + 10 μg insulin/ml + 500 ng retinol/ml + 5 x 10-7 mol/l testosterone) stimulation, the ratio of stimulated to basal secretions increased 11-fold between Day 1 (1.1) and Day 6 (12). When 10% fetal calf serum was added, mean o-transferrin secretion was a third of that in serum-free medium, suggesting that fetal calf serum contains factors that inhibit secretion of ovine Sertoli cell transferrin. In the presence of serum, the ratio of FIRT-stimulated to basal secretions doubled between Day 1 (1.0) and Day 4-6 (2.0). Between Days 2 and 4 of culture, insulin had a slight stimulatory effect on o-transferrin secretion (128% of control at 10 μg insulin/ml), as well as epidermal growth factor (124% of control at 50 ng/ml). Testosterone at up to 5 x 10-7 mol/l had no effect: 500 ng retinol/ml doubled o-transferrin secretion (218% of control) as did 500 ng FSH/ml (220% of control). A combination of retinol and FSH increased the secretion 4-fold, indicating that maximal stimulation of o-transferrin secretion by ovine Sertoli cells requires the combined actions of mechanisms dependent and independent of cAMP.