Partial Purification and Characterization of Two Non-FSH Steroid-Modulating Factors in Rat Thymic Epithelial Cell-Conditioned Medium (TCM)

Abstract

Previously, we have reported that unknown factor(s) in rat thymic epithelial cell-conditioned medium (TCM) stimulates basal and follicle-stimulating hormone (FSH)-induced steroid hormone production and aromatase enzyme activity in cultured rat granulosa cells. Here we report the partial purification and characterization of two of these activities. Thymic epithelial cells were prepared from immature female rats and used for TCM production. Lyophilized aliquots of TCM were reconstituted with distilled water at 25% of the original volume, applied to a gel filtration column, and column fractions were tested for their stimulation of steroidogenesis in granulosa cells prepared from immature diethylstilbestrol-treated rats. Two distinct biologically active regions were identified that corresponded to apparent molecular weights of approximately 22,000 and less than 1,000. The <1 kDa activity (“TCM-1”) stimulated (P < 0.01) basal production of progestins [progesterone and 20α-hydroxypregn-4-en-4-one (20α-OH-progesterone)] and estrogen, and also induced dramatic morphological changes on the rat granulosa cells. In contrast, the ∼22 kDa activity (“TCM-22”) stimulated (P < 0.01) only basal progestins, and had no effect (P < 0.05) on basal estrogen production or morphology of the cultured rat granulosa cells. In the presence of 100 ng/ml FSH, TCM-1 stimulated (P < 0.01) estradiol and progesterone production, whereas TCM-22 stimulated (P < 0.01) progesterone, but inhibited (P < 0.01) estradiol production. When both activities were assayed together, they were synergistic in stimulating (P < 0.01) basal progesterone production, but TCM-22 antagonized (P < 0.01) TCM-1-induced estradiol production. The biologic and physico-chemical characteristics of TCM-1 and TCM-22 were distinct from one another, as well as from FSH. When subjected to C 8 reverse-phase HPLC, TCM-1 retained its characteristic biologic properties and was eluted (54% acetonitrile) as A 214-absorbing moiety with a peak retention time of 92–93 minutes. The elution of TCM-22 was not correlated with an identifiable protein peak. These results suggest that ovarian steroid production may be modified by non-FSH factors produced by thymic epithelial cells although amino acid sequencing of TCM-1 was unsuccessful. This highlights a potential role of the thymus gland in regulating ovarian function.