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, 22 (1), 55-64

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

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

Munkhzaya Byambaragchaa et al. Dev Reprod.

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.

Keywords: Native eCG; cAMP stimulation; eelFSHR; eelLHR; rec-eCGβ/α.

Figures

Fig. 1
Fig. 1. Schematic diagram of rec-eCGβ/α.
The tethered eCG cDNA was ligated into the pcDNA3 mammalian expressing vector by EcoRI and SalI sites. rec-eCG, recombinant equine chorionic gonadotropin; eCG, equine chorionic gonadotropin; cDNA, complementary DNA.
Fig. 2
Fig. 2. Cloning of eelLHR and eelFSHR into the mammalian expressing vector.
The cDNAs of eelLHR and eelFSHR were ligated into the pCMV-ARMS1-PK2 (NheI-SacI site) and pcDNA3 (EcoRI-XhoI site) mammalian expressing vectors. eelLHR, eel luteinizing hormone receptor; eelFSHR, eel follicle stimulating hormone receptor.
Fig. 3
Fig. 3. Dose-dependent inhibition and increase in cAMP accumulation induced by rec-eCGβ/α and native eCG in cells transiently expressing eelLH/CGR.
CHO-K1 EA Parental cells were transfected with eelLH/CGR. The transfection method is described in the Materials and Methods section. The transfected culture media were replaced with new CHO growth medium 24 h after transfection. Cells were subjected to cAMP analysis 48–72 h after transfection. The standard samples were prepared across a concentration range of 0.17–712 nM. The plate was incubated for 30 min after adding rec-eCGβ/α (0–1,500 ng/mL). cAMP d2 and anti cAMP-cryptate were added and incubated at RT for 1 h. Inhibition of cAMP accumulation was shown by Delta F%, and the cAMP concentration was calculated by GraphPad Prism. A) Standard curve, B) and C) Delta F% value was shown by inhibition of cAMP responsiveness (GraFit), cAMP nM (1 104 cells) calculated by GraphPad Prism. cAMP, cyclic adenosine monophosphate; rec-eCG, recombinant equine chorionic gonadotropin; eCG, equine chorionic gonadotropin; RT, reverse transcription.
Fig. 4
Fig. 4. Dose-dependent inhibition and increase in cAMP accumulation induced by a rec-eCGβ/α and native eCG in stably cells expressing eelFSHR.
PathHunter CHO-K1 EA Parental cells were transfected with eelFSHR. The stably expressing cells were seeded at 10,000 cells per well into 384-well plates. The standard samples were prepared across a concentration range of 0.17–712 nM. The plate was incubated for 30 min at RT after adding rec-eCGβ/α (0–1,500 ng/mL). A) Standard curve, B) and C) Delta F% value was shown by inhibition of cAMP responsiveness (GraFit). cAMP, cyclic adenosine monophosphate; rec-eCGβ/α, recombinant equine chorionic gonadotropin ; eCG, equine chorionic gonadotropin; eelFSHR, eel follicle stimulating hormone receptor; RT, reverse transcription.

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