Highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonadotropin allowing their measurement in plasma.
Abstract: In previous studies, we had shown the synergistic effect of 10 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 10-10 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 10-10 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 10M 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. Luteinizing 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 bioassay provides a powerful tool to get this information.
© The authors.
Publication Date: 2021-11-11 PubMed ID: 35118407PubMed Central: PMC8788580DOI: 10.1530/RAF-21-0045Google Scholar: Lookup
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Summary
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This research discusses a highly sensitive method to measure biological activity of luteinizing hormones (LH) and chorionic gonadotropins (CG) from various species in plasma. The method, described as an assay, involves the use of mouse Leydig tumor cell lines and factors that reduce detection threshold for the hormones. This approach offers valuable insights on the reproductive status of different mammals.
Background of the Research
- The study builds upon previous research demonstrating the synergistic effect of 10 M forskolin (FSK) on the detection threshold of the cyclic AMP response to luteinizing hormones (LH) and chorionic gonadotropins (CG) in a mouse Leydig tumor cell (mLTC) line.
- The researchers had also separately investigated the impact of 10-10 M oxytocin (OXT) on the cyclic AMP response to LH and CG preparations in the same cells. An amplifying effect on the luminescence response induced by the gonadotropins was observed.
Aim of the Study
- The objective was to measure the effects of 10-10 M OXT on the gonadotropin-induced cAMP response, in the presence or absence of 10 µM FSK.
- The goal was to optimize the assay and heighten its sensitivity to detect the circulating concentrations of these hormones in various species.
Findings and Conclusion
- The optimization leverages three independent phenomena: the inhibition of nucleotide phosphodiesterase by IBMX to prevent cAMP degradation, strong synergy of 10 µM forskolin with low concentrations of LH or CG during the 1-h luminescence measurement, and the stimulatory effect of 10M OXT on the amplitude of transfected cAMP-sensitive luciferase response.
- With this approach, detectable concentrations at the 1-10 pg/well (pM range) for the LHs and CGs from various species are achievable.
- The bioactivities of circulating LHs and CGs in plasma or urine samples can be measured using tiny amount of mammalian species plasma (10 µL).
- Preliminary studies with equine and donkey plasma samples demonstrate that the measured bioactivity was inhibited by a specific MAB against the receptor-binding region of equine LH (eLH) and equine CG (eCG), discounting any response due to interfering substances other than eLH or eCG.
- The researchers expect that the bioactivity profiles of these hormones will be measurable in the blood of humans, equine, ovines and very likely in rodents, ruminants, and hopefully in most other mammalian species.
Significance of the Research
- Luteinizing hormone (LH) plays a vital role in controlling ovary and testicle functions in various animals, including humans.
- The highly sensitive assay can be used to measure the biological activity of LH in the blood, providing information about the reproductive status of different mammals.
- The new bioassay could potentially serve as a powerful tool for researchers studying mammalian reproduction.
Cite This Article
APA
Klett D, Combarnous Y.
(2021).
Highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonadotropin allowing their measurement in plasma.
Reprod Fertil, 2(4), 300-307.
https://doi.org/10.1530/RAF-21-0045 Publication
Researcher Affiliations
- Institut National de la Recherche Agronomique et environnementale (INRAe), Centre National de la Recherche Scientifique (CNRS), Université de Tours, Joint Laboratory of Reproductive Physiology and Behaviors (PRC), Nouzilly, France.
- Institut National de la Recherche Agronomique et environnementale (INRAe), Centre National de la Recherche Scientifique (CNRS), Université de Tours, Joint Laboratory of Reproductive Physiology and Behaviors (PRC), Nouzilly, France.
MeSH Terms
- Animals
- Biological Assay
- Cell Line, Tumor
- Chorionic Gonadotropin
- Colforsin
- Cyclic AMP
- Female
- Horses
- Humans
- Luteinizing Hormone
- Male
- Mammals
- Mice
- Sheep
References
This article includes 15 references
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Citations
This article has been cited 1 times.- Klett D, Pellissier L, Lomet D, Derouin-Tochon F, Robert V, Nguyen TMD, Duittoz A, Reiter E, Locatelli Y, Dupont J, Dardente H, Jean-Alphonse F, Combarnous Y. Highly-Sensitive In Vitro Bioassays for FSH, TSH, PTH, Kp, and OT in Addition to LH in Mouse Leydig Tumor Cell.. Int J Mol Sci 2023 Jul 27;24(15).
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