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

摘要

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) beta 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 CGbeta subunit containing the CTP domain is expressed only in primates and equids, a CTP-like sequence exists in the untranslated region of the LHbeta gene of several mammalian species, including the bovine species. The CTP encrypted in the bovine LHbeta DNA (designated as 'boCTP') and the CTP derived from the human CGbeta 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 LHbetaboCTPalpha and LHbetahuCTPalpha analogs in the conditioned media of the expressing CHO cells was three- to six-fold higher than that of the "linkerless" LHbetaalpha and LHbeta111alpha 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 LHbetaboCTPalpha 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 LHbetahuCTPalpha, LHbetaalpha and LHbeta111alpha were comparable and sightly reduced relative to the LHbetaboCTPalpha activity. The data suggested that LHbetaboCTPalpha 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 LHbeta 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 LHbeta genes with the aim of expressing the cryptic CTP sequence as a spacer derived from the DNA of the same organism.