The configuration of the alpha and beta subunit domains in single-chain bovine LH analogs influences the secretion and steroidogenic response

The gonadotrophins LH, FSH and human (h) CG are non-covalent heterodimers composed of a common α and the hormone-unique β subunit. LH regulates the production of androgens and progestins in the follicle, and the levels of these steroids are critical for the ovarian function. Structural features of the gonadotrophins involved in the steroidogenic response of the ovary are not completely understood. As an approach to address how the topology of the ligand affects steroidogenesis we exploited the single-chain (SC) gonadotrophin methodology because manipulating the relative position of the tethered subunit domains in SC hCG analogs enabled to change in the conformation, secretion, receptor binding and adenylyl cyclase activity. We genetically engineered a SC bovine LH analog with a linker derived from the CTP domain of the hCGβ subunit, NH₂-alpha-CTP-LHbeta-COOH (denoted as αCTPLHβ; AB configuration) and evaluated the secretion form transfected CHO cells and steroidogenesis in follicular derived cells in comparison to the variant NH₂-LHbeta-CTP-alpha-COOH (LHβCTPα; BA configuration). The secretion of the analogs from CHO cells was quantitative, and that of αCTPLHβ was more efficient than that of LHβCTPαṡ The experiments suggested that both variants were N- and O- glycosylated, though the posttranslational modifications are likely to be non-identical in the AB and BA analogs. The analogs stimulated progesterone secretion by immortalized rat granulosa cells that express the rat LH receptor but the EC50 of αCTPLHβ (AB orientation) was higher by 20 fold, as compared to LHβCTPα (BA). In primary cultures of bovine theca cells, αCTPLHβ stimulated progesterone release with a reduced sensitivity (by at least 50 folds) and smaller magnitude over the basal levels (about 3 folds) relative to LHβCTPα. In contrast, the accumulation of androstenedione in the media of the same primary cultures appeared to be nearly identical. As a result, the androstenedione/progesterone ratio for the αCTPLHβ analog was significantly increased relative to LHβCTPα (2-3 folds). This unequal response suggests a distinct regulation of progesterone and androstenedione biosynthesis. Our data demonstrate major differences in steroid balance following stimulation of the receptor with structural LH analogs and provide further insight into gonadotrophin regulation of ovarian steroid production.