Role of Insulin, Thyroxin and Cortisol in Luteinization of Porcine Granulosa Cells Grown in Chemically Defined Media1

Abstract

In order to determine the exact hormonal requirements for luteinization in vitro, porcine granulosa cells obtained from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles were grown in serum-free media in the presence of various combinations of luteinizing hormone (LH), follicle stimulating hormone (FSH), insulin, cortisol and thyroxin. Morphology as well as progestin secretion was monitored throughout an 8–10 day culture period. The cells were grown in either medium “199D,” which consisted of medium 199 plus 0.4 percent bovine serum albumin (BSA) and 0.2 percent lactalbumin hydrolysate, or the serumless medium of Neuman and Tytell (“SMD”), which already contains 0.2 percent lactalbumin hydrolysate, plus 0.4 percent BSA. Additional cells were grown in the presence of 15 percent pig serum in a balance of medium 199 (medium Y). Cell adherence to the glass and cell maintenance were both less in the serum free media compared to serum-containing medium Y. In contrast, progestin secretion expressed per cell was consistently greater when the cells were grown in serum-free compared to serum-containing medium Y. Cells harvested from large follicles were able to secrete more progestin compared to cells obtained from small or medium-sized follicles in both types of serum-free media. If cells were grown in serum-free media, addition of 1 mU/ml insulin had some stimulatory (50-200 percent) action upon cell maintenance as well as a small, slow developing stimulatory action upon progestin secretion. The insulin treatment led to an increase in cytoplasmic vacuoles and lipid droplets. Addition of either 10⁻⁷ M triiodothyronine or thyroxine or 0.01 µg/ml cortisol along with 1.0-10 mU/ml insulin led to a 3–5 fold stimulation of progestin secretion by cells harvested from small and large follicles. The thyroxin in combination with insulin brought about an increase in oil red O and hematoxylin and eosin stainable material present within the cytoplasmic. The cortisol brought about an increase in distinctness of nucleolar material. Addition of either cortisol or thyroxin in the absence of insulin consistently brought about cellular necrosis. In cultures of cells grown in serum-free medium obtained from small follicles, addition of a mixture of 0.1 µg/ml human LH plus 0.1 µg/ml human FSH, 1.0 mU/ml insulin plus either 10⁻⁷ M thyroxin or 0.01 µg/ml cortisol brought about morphological luteinization and a 4–14 fold increase in progestin secretion above when LH and FSH were added alone. The LH and FSH failed to bring about full morphological luteinization in the absence of these hormones. Addition of a complete mixture of all these hormones brought about the best morphological luteinization and stimulation in progestin secretion. It can be concluded that spontaneous and LH- and FSH-induced granulosa cell luteinization in serum-free culture media is enhanced by the inclusion of a mixture of insulin, cortisol and thyroxin.