Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked α- and β-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (Δ1, Δ2, Δ3, Δ4, and Δ5) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2～3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (Δ1, Δ4, and Δ5) were transcripted, but not translated into proteins. Rec-eCG Δ2 was secreted in much lower amounts than the wild type. Only the rec-eCG Δ3 (β-subunit: Gln94-Ile95-Lys96→Ala94-Ala95-Ala96) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered eCGβα. However, the FSH-like activity of rec-eCGβαΔ3 was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94～96 amino acid sequences in eCG β-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

The glycoprotein hormone family represents a class of heterodimers, that includes the placental hormone equine chorionic gonadotropin (eCG) and the anterior pituitary hormones‐ follitropin (FSH), lutropin (LH), and thyrotropin (TSH). The 4 hormones are heterodimers, with a common α‐subunit and unique β‐subunits. eCG is the most heavily glycosylated of the known pituitary and placental glycoprotein hormones. Recent observations using single chain glycoprotein hormone analogs in which, the β‐and α‐subunits are linked, implied that heterodimeric‐like quaternary configuration is not a prerequisite for receptor/signal transduction. To study the function and signal transduction of tethered rec‐eCG, a single chain eCG molecule was constructed and rec‐eCG protein was produced. Molecular mass of the single chain is about 45 kDa. All mice were ovulated by tethered rec‐eCG treatment. The dual activity of tethered rec‐eCG was determined in receptor cell lines of nonequid species; in fact, this dual activity was proven in species other than horse. Tethered rec‐eCG in equids does not bind to FSH receptors, suggesting that eCG is primarily an LH‐like hormone in the horse. Taken together, these data suggest that tethered rec‐eCG has dual activity in nonequid species in vitro. However, it has only LH‐like activity in equid species in vitro.