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Correspondence

Lupeol and pristimerin do not inhibit activation of the human sperm CatSper Ca(2+)-channel

[version 1; peer review: 3 approved with reservations]
PUBLISHED 24 Feb 2022
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Abstract

Opposing findings have been published on the regulation of the sperm-specific Ca2+ channel CatSper (cation channel of sperm) in human sperm cells by the plant triterpenoids lupeol and pristimerin. While the original study on this topic found these triterpenoids to act as potent inhibitors of human CatSper, subsequent studies have failed to replicate such an inhibitory effect. It has been suggested that these issues could in part be due to purity issues and/or batch variation between the plant-derived extracts of lupeol and pristimerin obtained for the studies. The aim of this study was to elucidate this controversy by investigating the batches of lupeol and pristimerin used in our previous study with state-of-the-art 1H-, 13C- and 2D-nuclear magnetic resonance (NMR) methods to reveal potential purity and/or batch variation issues. When comparing the NMR-spectra obtained from 1H-NMR and 13C-NMR with previously published NMR-spectra for lupeol and pristimerin, we could confirm that both the lupeol and pristimerin batch were ≥95 % pure. These results confirm the validity of the findings in our previous study for lupeol and pristimerin, showing that lupeol and pristimerin do not inhibit activation of CatSper in human sperm. In conclusion, using 1H-, 13C- and 2D-NMR methods, we confirm that the lupeol and pristimerin batches used in our previous study were ≥95 % pure and thereby fail to identify any purity issues and/or batch variation that could explain the observed inability of lupeol and pristimerin to inhibit activation of CatSper in human sperm.

Keywords

Fertility, CatSper, Male reproduction, Lupeol, Pristimerin, Sperm function

The putative inhibitory action of the two plant triterpenoids lupeol and pristimerin on the activation of the human sperm CatSper Ca(2+)-channel has recently been debated in the scientific literature. The original study on this subject (Mannowetz et al., 2017) indicated that these triterpenoids act as very potent and efficacious inhibitors of progesterone-activated CatSper-currents in human sperm cells with IC50-values in the lower nM range, and a follow-up study by the same research group confirmed the inhibitory action of pristimerin on progesterone-induced Ca(2+)-influxes via CatSper through measurements in the principal piece of the flagellum in single human sperm cells (Mannowetz et al., 2018).

In contrast to these findings, two studies from independent research groups failed entirely to replicate any inhibitory action for neither lupeol nor pristimerin on progesterone-induced Ca(2+)-influxes through CatSper in populations of human sperm cells (Brenker et al., 2018; Rehfeld, 2020) and progesterone-activated CatSper-currents in single human sperm cells (Brenker et al., 2018), even when exposing the sperm cells to lupeol and pristimerin at much higher µM concentrations.

The complete failure of these studies to replicate the findings from (Mannowetz et al., 2017; Mannowetz et al., 2018) is highly concerning since a patent has been filed (Lishko & Mannowetz, 2018) and a company (YourChoice Therapeutics, CA, US) has been formed based on the original discovery by (Mannowetz et al., 2017; Mannowetz et al., 2018) that lupeol and pristimerin act as potent inhibitors of human CatSper and could thus potentially be used as novel male and female contraceptives.

Since the publication of the most recent study on this matter (Rehfeld, 2020), the corresponding author was contacted by researchers who questioned the validity of the results presented in the study for lupeol and pristimerin, i.e., the inability to reproduce the inhibitory action of these triterpenoids on human CatSper, and suggested that the failure to identify such an inhibitory effect on human CatSper could be due to purity issues and/or batch variation between the plant-derived extracts of lupeol and pristimerin obtained for the study from Cayman Chemicals (MI, USA).

Although Cayman Chemicals stated that the lupeol and pristimerin batches were delivered with a purity of ≥98 %, we fully agreed with these researchers that it would be good scientific conduct and of general interest of the field of human sperm physiology to examine the two stocks solutions used in (Rehfeld, 2020), i.e., a 5 mM pristimerin dimethylsulfoxid (DMSO) stock and a 1 mM lupeol ethanol stock, using state-of-the-art 1H-, 13C- and 2D-nuclear magnetic resonance (NMR) methods (Bruker 500 MHz Ultrashield Plus equipped with a CryoProbe, Bruker, Germany) to reveal potential purity and/or batch variation issues in these stocks.

To prepare the stocks for the NMR-measurements, we first evaporated the ethanol from the lupeol stock, removed the DMSO from the pristimerin stock using an evaporation system (V-10 evaporator, Biotage, Sweden), and exchanged the solvent for both triterpenoids to deuterated chloroform (CDCl3). The raw NMR data can be found as Underling data (Rehfeld, 2022a). When comparing the NMR-spectra obtained on the two stocks from 1H-NMR and especially 13C-NMR (see Extended data (Rehfeld, 2022b)) with previously published NMR-spectra for lupeol and pristimerin (Espindola et al., 2018; Shwe et al., 2019), we could confirm that Cayman Chemicals had indeed provided us with batches containing lupeol and pristimerin, respectively. Furthermore, the NMR-data showed that both lupeol and pristimerin were ≥95 % pure (Extended data (Rehfeld, 2022b)), despite the prolonged storage at -20 °C since conducting the experiments for (Rehfeld, 2020).

Taken together, the results provided here confirms the validity of the findings in our previous study for lupeol and pristimerin (Rehfeld, 2020), i.e., that the two plant triterpenoids lupeol and pristimerin do not inhibit activation of CatSper in human sperm. The findings in (Rehfeld, 2020) are therefore still in line with the observations by (Brenker et al., 2018) and still contradicting the putative inhibitory action of lupeol and pristimerin on human CatSper described in (Mannowetz et al., 2017; Mannowetz et al., 2018).

In conclusion, using state-of-the-art 1H-, 13C- and 2D-NMR methods, we confirm here that the lupeol and pristimerin stocks used in (Rehfeld, 2020) were ≥95 % pure and thereby fail to identify any purity issues and/or batch variation that could explain the observed inability of these triterpenoids to inhibit activation of CatSper in human sperm.

Data availability

Underlying data

Figshare. Raw 1H-, 13C- and 2D-NMR data for lupeol and pristimerin in MestReNova (Mnova) format. https://doi.org/10.6084/m9.figshare.19181087.v1 (Rehfeld, 2022a).

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Raw 1H-, 13C- and 2D-NMR data for lupeol and pristimerin in MestReNova (Mnova) format are also available at the BMRbig repository, part of the Biological Magnetic Resonance Data Bank (BMRB), with ID: BMRbig35, https://bmrbig.org/released/bmrbig35.

Extended data

Figshare: Supplementary file 1. https://doi.org/10.6084/m9.figshare.19134488.v1 (Rehfeld, 2022b).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

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Version 2
VERSION 2 PUBLISHED 24 Feb 2022
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CITE
how to cite this article
Rehfeld A and Marcus Pedersen C. Lupeol and pristimerin do not inhibit activation of the human sperm CatSper Ca(2+)-channel [version 1; peer review: 3 approved with reservations]. F1000Research 2022, 11:222 (https://doi.org/10.12688/f1000research.109279.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Open Peer Review

Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 24 Feb 2022
Views
16
Cite
Reviewer Report 04 May 2022
Huafeng Wang, Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA 
Approved with Reservations
VIEWS 16
The effects of lupeol and pristimerin on human CatSper have aroused a controversy since two PNAS papers were published from different groups (Mannowetz et al., 2017 and Brenker et al., 2018). This manuscript was aiming to uncover a potential possibility for a ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Wang H. Reviewer Report For: Lupeol and pristimerin do not inhibit activation of the human sperm CatSper Ca(2+)-channel [version 1; peer review: 3 approved with reservations]. F1000Research 2022, 11:222 (https://doi.org/10.5256/f1000research.120762.r129827)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 02 Aug 2022
    Anders Rehfeld, Department of Drug Design and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, 2200 Copenhagen N, Denmark
    02 Aug 2022
    Author Response
    We have now contacted these groups to offer them NMR-analyses of their lupeol and pristimerin batches.
    Competing Interests: No competing interests were disclosed.
COMMENTS ON THIS REPORT
  • Author Response 02 Aug 2022
    Anders Rehfeld, Department of Drug Design and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, 2200 Copenhagen N, Denmark
    02 Aug 2022
    Author Response
    We have now contacted these groups to offer them NMR-analyses of their lupeol and pristimerin batches.
    Competing Interests: No competing interests were disclosed.
Views
21
Cite
Reviewer Report 13 Apr 2022
Tao Luo, Institute of Life Science, School of Life Sciences, Nanchang University, Nanchang, China 
Approved with Reservations
VIEWS 21
Different research groups showed contradictory results on whether plant triterpenoids, lupeol and pristimerin, inhibit the activation of CatSper. It has been suggested that these issues could in part be due to purity issues and/or batch variation between the plant-derived extracts ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Luo T. Reviewer Report For: Lupeol and pristimerin do not inhibit activation of the human sperm CatSper Ca(2+)-channel [version 1; peer review: 3 approved with reservations]. F1000Research 2022, 11:222 (https://doi.org/10.5256/f1000research.120762.r129824)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 02 Aug 2022
    Anders Rehfeld, Department of Drug Design and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, 2200 Copenhagen N, Denmark
    02 Aug 2022
    Author Response
    We have now addressed these two issues in the discussion of the updated manuscript.
    Competing Interests: No competing interests were disclosed.
COMMENTS ON THIS REPORT
  • Author Response 02 Aug 2022
    Anders Rehfeld, Department of Drug Design and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, 2200 Copenhagen N, Denmark
    02 Aug 2022
    Author Response
    We have now addressed these two issues in the discussion of the updated manuscript.
    Competing Interests: No competing interests were disclosed.
Views
27
Cite
Reviewer Report 02 Mar 2022
João Ramalho-Santos, CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal;  DCV-Department of Life Sciences, University of Coimbra, Coimbra, Portugal;  CIBB-Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal 
Approved with Reservations
VIEWS 27
My comments have to do with the fact that, while the topic is very worthwhile, this is not exactly (in my view) a straightforward Correspondence. The steps are as follows:
  1. Originally a paper is published in
... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Ramalho-Santos J. Reviewer Report For: Lupeol and pristimerin do not inhibit activation of the human sperm CatSper Ca(2+)-channel [version 1; peer review: 3 approved with reservations]. F1000Research 2022, 11:222 (https://doi.org/10.5256/f1000research.120762.r125363)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 04 Mar 2022
    Anders Rehfeld, Department of Drug Design and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, 2200 Copenhagen N, Denmark
    04 Mar 2022
    Author Response
    I fully agree with the reviewer in his comment that this is not a straightforward Correspondence. I would have preferred to be able to publish all my data on this ... Continue reading
  • Author Response 02 Aug 2022
    Anders Rehfeld, Department of Drug Design and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, 2200 Copenhagen N, Denmark
    02 Aug 2022
    Author Response
    As the other reviewers also suggest that we discuss the issues raised in the 2018 PNAS exchange, we have now update our Correspondence manuscript to address these issues more thoroughly.
    Competing Interests: No competing interests were disclosed.
COMMENTS ON THIS REPORT
  • Author Response 04 Mar 2022
    Anders Rehfeld, Department of Drug Design and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, 2200 Copenhagen N, Denmark
    04 Mar 2022
    Author Response
    I fully agree with the reviewer in his comment that this is not a straightforward Correspondence. I would have preferred to be able to publish all my data on this ... Continue reading
  • Author Response 02 Aug 2022
    Anders Rehfeld, Department of Drug Design and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, 2200 Copenhagen N, Denmark
    02 Aug 2022
    Author Response
    As the other reviewers also suggest that we discuss the issues raised in the 2018 PNAS exchange, we have now update our Correspondence manuscript to address these issues more thoroughly.
    Competing Interests: No competing interests were disclosed.

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 24 Feb 2022
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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