Keywords
Fertility, CatSper, Male reproduction, Lupeol, Pristimerin, Sperm function
This article is included in the Cell & Molecular Biology gateway.
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.
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.
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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Is the rationale for commenting on the previous publication clearly described?
Yes
Are any opinions stated well-argued, clear and cogent?
Partly
Are arguments sufficiently supported by evidence from the published literature or by new data and results?
Partly
Is the conclusion balanced and justified on the basis of the presented arguments?
Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: sperm, CatSper, ion channel, electrophysiology
Is the rationale for commenting on the previous publication clearly described?
Yes
Are any opinions stated well-argued, clear and cogent?
Yes
Are arguments sufficiently supported by evidence from the published literature or by new data and results?
Yes
Is the conclusion balanced and justified on the basis of the presented arguments?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Male infertility
Is the rationale for commenting on the previous publication clearly described?
Partly
Are any opinions stated well-argued, clear and cogent?
Yes
Are arguments sufficiently supported by evidence from the published literature or by new data and results?
Partly
Is the conclusion balanced and justified on the basis of the presented arguments?
Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Sperm physiology, male and female infertility, reproductive and stem cell biology.
Alongside their report, reviewers assign a status to the article:
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