Signal Transduction of Eel Luteinizing Hormone Receptor (eelLHR) and Follicle Stimulating Hormone Receptor (eelFSHR) by Recombinant Equine Chorionic Gonadotropin (rec-eCG) and Native eCG

Abstract

Previous studies showed that recombinant equine chorionic gonadotropin (rec-eCGβ/α) exhibits both follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-like activities in rat LHR- and FSHR-expressing cells. In this study, we analyzed signal transduction by eelFSHR and eelLHR upon stimulation with rec-eCGβ/α and native eCG. The cyclic adenosine monophosphate (cAMP) stimulation in CHO-K1 cells expressing eelLHR was determined upon exposure to different doses (0–1,450 ng/mL) of rec-eCGβ/α and native eCG. The EC50 values of rec-eCGβ/α and native eCG were 172.4 and 786.6 ng/mL, respectively. The activity of rec-eCGβ/α was higher than that of native eCG. However, signal transduction in the CHO PathHunter Parental cells expressing eelFSHR was not enhanced by stimulation with both agonist rec-eCGβ/α and native eCG. We concluded that rec-eCGβ/α and native eCG were completely active in cells expressing eelLHR, similar to the activity in the mammalian cells expressing LHRs. However, rec-eCGβ/α and native eCG did not invoke any signaling response in the cells expressing eelFSHR. These results suggest that eCG has a potent activity in cells expressing eelLHR. Thus, we also suggest that rec-eCGβ/α can induce eel maturation by administering gonadotropic reagents (LH), such as salmon pituitary extract.

The C-terminal Phosphorylation Sites of eel Follicle-Stimulating Hormone Receptor are Important Role in the Signal Transduction

“…The rec-eelFSHβ/α proteins were expressed by transfecting the expression vector into CHO-S cells using the FreeSytle MAX reagent transfection method, according to the supplier's instructions and as described previously ( Lee et al, 2017 ; Byambaragchaa et al, 2018 ). Briefly, CHO-S cells were cultured with FreeStyle CHO expression medium at 1×10 7 cells/ 50 mL for 3 days.…”

“…eelFSHR cDNA was cloned using cDNA of eel testis and ovary, as previously reported ( Byambaragchaa et al, 2018 ). The PCR fragments were ligated into the pcDNA3 mammalian expressing vector by the Xho I and EcoR I enzyme sites (designated as pcDNA3-eelFSHR-WT).…”

“…The location of truncation site (t614) is also shown. The amino acid sequence was amplified with ovary and testis cDNA of eel from our laboratory and sequenced according to a previoius reported method ( Byambaragchaa et al, 2018 ). eelFSHR, eel follicle-stimulating hormone receptor.…”

“…Measurement of AMP accumulation in CHO cells and PathHunter CHO-K1 EA-Parental cells was performed using cAMP Dynamics 2 competitive immunoassay kits (Cisbio Bioassays) as described previously ( Byambaragchaa et al, 2018 ). The standard samples were prepared to cover an average range of 0.17-712 nM (final concentration of cAMP per well).…”

Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

“…Thus far, we have attempted to assess the different roles of R-eCG with respect to their attached oligosaccharides [2,24], glycosylation sites for LH-and FSH-like activity [2], tethered R-eCGs [6], internalization of rat FSH and LH receptors by R-eCG [25], and signal transduction through eel FSH receptor and LH receptor by R-eCG and Natural-eCG (N-eCG) [26]. Furthermore, we have analyzed the ovulation rates between N-eCG and deglycosylated R-eCGs in mice [27].…”