Highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonadotropin allowing their measurement in plasma.

Danièle Klett,Yves Combarnous

doi:10.1530/raf-21-0045

Abstract

In previous studies, we had shown the synergistic effect of 10−5 M forskolin (FSK) on the detection threshold of the cyclic AMP response to luteinizing hormones (LH) and chorionic gonadotropins (CG) from various species in the mouse Leydig tumor cell (mLTC) cell line. Independently, we started to study the effect of 10−12–10−6 M oxytocin (OXT) also on the cyclic AMP response to LH and CG preparations on these same cells and found an amplifying effect on the luminescence response caused by gonadotropins. The aim was then to explore the effects of 10−12–10−6 M OXT on the gonadotropin-induced cAMP response, in the presence or absence of 10 µM FSK to optimize the assay down to a sensitivity compatible with the detection of the circulating concentrations of these hormones in various species. Finally, the optimization relies on three independent phenomena: (1) the inhibition of nucleotide phosphodiesterase by IBMX (3-isobutyl-1-methylxanthine) to avoid cAMP degradation; (2) the strong synergy of 10 µM forskolin with low concentrations of LH or CG during the 1-h luminescence measurement; (3) the stimulatory effect of 10−8M OXT on the amplitude of transfected cAMP-sensitive luciferase response. By doing this, the detectable concentrations are at the 1–10 pg/well (pM range) for the LHs and CGs from various species. The bioactivities of circulating LHs and CGs in blood or urine are therefore expected to be measurable in 10 µL-plasma samples from mammalian species and maybe others. Indeed, a preliminary study with equine and donkey plasma samples shows that the measured bioactivity was fully inhibited by a specific MAB against the receptor-binding region of equine LH (eLH) and equine CG (eCG), thus eliminating a possible response due to interfering substances other than eLH or eCG. From these data, it is expected that the bioactivity profiles of these hormones will be measurable in the blood of human, equine, and ovine species and very likely in rodents, ruminants, and hopefully in most other mammalian species.Lay summaryLuteinizing hormone (LH) plays a central role in controlling ovary and testicle functions in many animals, including humans. The highly sensitive method, known as an assay, described in this paper, measures the biological activity of LH in the blood of mammals. The assay is performed in culture of cells derived from mouse testicles in the presence of factors that diminish the detection threshold for LH. The knowledge of the bioactive LH concentration dynamics in the blood is very informative about the reproductive status of male and female mammals. This new in vitro bioassay provides a powerful tool to get this information.

Highlights

The mouse Leydig tumor cell line (Rebois 1982) has been used for years to study the signaling pathway downstream of the binding of luteinizing hormones (LH) and CGs to their LH receptor named LHR in almost all vertebrate species and LHCGR in the primates and equids, who are the only groups having chorionic gonadotropin (CG) (Rebois & Fishman 1984, Kellokumpu 1987, El-Hefnawy et al 2000, Wang et al 2007, Mendoza-Villarroel et al 2014)
The increasing effect of OXT preincubation on the cAMP response of mouse Leydig tumor cell line (mLTC) to human chorionic gonadotrophin (hCG) was observed with the other gonadotropins tested
In the third set of experiments, we explored whether the other human and animal gonadotropins in the presence of 10 μM FSK, exhibited enhanced cAMP responses after a 2-h preincubation of mLTC cells with 10−6M OXT

Summary

Introduction

The mouse Leydig tumor cell line (mLTC) (Rebois 1982) has been used for years to study the signaling pathway downstream of the binding of LHs and CGs to their LH receptor named LHR in almost all vertebrate species and LHCGR in the primates and equids, who are the only groups having chorionic gonadotropin (CG) (Rebois & Fishman 1984, Kellokumpu 1987, El-Hefnawy et al 2000, Wang et al 2007, Mendoza-Villarroel et al 2014). Since the mouse LHR can bind to LH and CG from numerous species, we and others have used this cell line to study the response to various LH and CG preparations at the levels of intracellular cyclic AMP accumulation (Dufau et al 1995, Nikula et al 1999, Evaul & Hammes 2008, Klett et al 2016, Nguyen et al 2018, 2019). FSK exhibits a strong synergy with very low concentrations of LH or CG that do not show any stimulating activity in its absence to reach a much lower threshold for LH and CG detection (Nguyen et al 2018) It has been known for a long time that Leydig cells possess oxytocin receptors (OXTR) (Frayne & Nicholson 1995, Einspanier & Ivell 1997). We have explored the effects of an extensive range of oxytocin (OXT) concentrations on the cAMP response of mLTCs and found that it strongly diminished the detection limit for gonadotropins down to a threshold compatible with their measurement in biological fluids

Methods

Results

Conclusion