The steroidogenic effect of single-chain bovine LH analogs in cultured bovine follicular cells

Single-chain gonadotropin analogs had been constructed for the purpose of structure-function studies and analog design. Incorporation of a spacer derived from the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) β subunit between the tethered subunit domains of the human gonadotropins is beneficial for the secretion of the single-chain variants without compromising biocactivity. Although the CGβ subunit containing the CTP domain is expressed only in primates and equids, a CTP-like sequence exists in the untranslated region of the LHβ gene of several mammalian species, including the bovine species. The CTP encrypted in the bovine LHβ DNA (designated as 'boCTP') and the CTP derived from the human CGβ subunit (denoted as 'huCTP') served as a linker sequence in the design of bovine single-chain luteinizing hormone (LH) analogs. The purpose of the present study was to evaluate steroidogenesis in cultured bovine theca cells following stimulation with these single-chain analogs. The concentration of the LHβboCTPα and LHβhuCTPα analogs in the conditioned media of the expressing CHO cells was three- to six-fold higher than that of the “linkerless” LHβα and LHβ111α variants. The four analogs induced androstenedione and progesterone secretion from the primary theca cells in a dose-dependent manner, but differences in the steroidogenic response were observed. The LHβboCTPα analog (10 ng/ml) effectively induced androstenedione and progesterone secretion over unstimulated levels (4.0- and 4.4-fold increase for androstenedione and progesterone, respectively). The response to the pituitary bovine LH standard (10 ng/ml) was less pronounced for both steroids (two- to three-fold increase over basal levels). The activities of LHβhuCTPα, LHβα and LHβ111α were comparable and sightly reduced relative to the LHβboCTPα activity. The data suggested that LHβboCTPα was ranked as the most potent and this was even more prominent when analogs were used at a lower dose (1 ng/ml). These data suggest that the design, including the huCTP or boCTP linker, is favorable for the production of single-chain bovine LH analogs. Furthermore, spacing of the tethered subunit domains with the cryptic boCTP sequence that originated from the bovine LHβ gene appears advantageous for the purpose of stimulating steroid production in the species-specific bioassay. Thus, an effective strategy to produce bioactive single-chain LH analogs in non-primate, non-equid species would be the mutatation of the LHβ genes with the aim of expressing the cryptic CTP sequence as a spacer derived from the DNA of the same organism.