Elsevier

Reproductive Toxicology

Volume 94, June 2020, Pages 1-7
Reproductive Toxicology

Investigating the effects of fipronil on male fertility: Insight into the mechanism of capacitation

https://doi.org/10.1016/j.reprotox.2020.04.002Get rights and content

Highlights

  • GABA and GABAAR play various roles in the process of sperm capacitation.

  • FPN binds to GABAAR to block GABA in GABAergic pathway.

  • FPN inhibits phosphorylation of GABAAR in Intracellular interacting pathway.

  • FPN can directly and indirectly suppress various sperm functions in capacitation.

  • FPN can negatively affect male fertility leading to infertility.

Abstract

Fipronil (FPN) is a widely used phenylpyrazole pesticide for the control of insects and removal of veterinary pet fleas, ticks, etc. Although FPN presents moderate hazards to human health, people are readily exposed in daily life. FPN acts by impairing the central nervous systems of insects by blocking gamma-aminobutyric acid (GABA) and glutamate-activated chloride channels. A previous study demonstrated that GABA and GABAAR are present in spermatozoa and play various roles in the process of sperm capacitation, which is required for fertilization. However, the effects of FPN on mammalian fertility are not yet fully understood. Therefore, the present study was designed to investigate the effects of FPN on spermatozoa. Herein, we treated various concentrations of FPN (0.1, 1, 10, 100, and 300 μM) or a control treatment with mouse spermatozoa. FPN treatment significantly reduced sperm motility, motion kinematic parameters, and intracellular ATP level, whereas the acrosome reaction was enhanced. Levels of phospho-PKA and phospho-tyrosine substrate were significantly decreased in a dose-dependent manner. Meanwhile, there was no difference between control and treatment groups in the level of GABAAR β-3. Only the ratio of GABAAR β-3 pS408/pS409 was significantly decreased at higher concentrations of FPN. Moreover, cleavage and blastocyst formation rates were also significantly decreased by FPN treatments. Taken together, these data suggest that FPN can directly and indirectly suppress various sperm functions. Therefore, FPN can negatively affect male fertility leading to infertility. From these results, we suggest that the use of FPN as a pesticide requires the attention of reproductive toxicity.

Introduction

The phenylpyrazole known as fipronil (FPN) is a well-known pesticide that has been widely used since 1993 for the control of insects, dog fleas, ticks, etc. In 2017, FPN was detected in chicken eggs in Europe and Korea. According to the WHO, FPN has been classified as a ‘Moderately hazardous’ active pesticide ingredient, but humans are often readily exposed to it in daily life, and excessive doses are used in various places such as Taiwan. FPN disrupts the central nervous systems of insects by blocking gamma-aminobutyric acid (GABA) and glutamate-activated chloride (GluCl) channels, which do not exist in mammals [[1], [2], [3]]. GABA is an inhibitory neurotransmitter produced from l-glutamate by glutamate decarboxylase (GAD); binding of GABA to its receptor opens Cl or K+ channels. Recently, GABA was found to play an important role in regulating sperm function [[4], [5], [6], [7], [8]]. It is well known that the GABA type A receptor (GABAAR) is activated upon GABA binding. Binding of GABA to the receptor causes a shift in ion concentration via channel opening. FPN impairs this activity through inhibition of the channels by binding to GABAAR and GluCl receptors in insects.

Ejaculated sperm cells must undergo a maturation process within the female reproductive tract that has been defined as “Capacitation”, in which sperm acquire fertilization ability. This is important because only capacitated sperm can undergo the acrosome reaction and penetrate the zona pellucida. During capacitation, various proteins undergo phosphorylation [[9], [10], [11], [12]]. In particular, GABAAR, which regulates chloride channels for anion transport, requires phosphorylation to be activated. Furthermore, the beta-subunit of GABAAR is phosphorylated by cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), tyrosine kinase and others [13]. It has been reported that GABA and GABAAR are present in human spermatozoa and play a major role in sperm capacitation and hyperactivated motility in spermatozoa [4,5,8]. In addition, several studies have reported the adverse effects of FPN on fertility and related processes [[14], [15], [16], [17], [18], [19], [20]].

Despite numerous reports on the toxicity of FPN, there are few reports investigating its effects on spermatozoa and mammalian fertility. Therefore, the present study was designed to evaluate the effects of FPN on sperm functions and fertilization ability.

Section snippets

Chemicals and media

Modified Tyrode’s medium was used as the basic medium (BM) (97.84 mM NaCl, 1.42 mM KCl, 0.47 mM MgCl2·6H2O, 0.36 mM NaH2PO4·H2O, 5.56 mM d-glucose, 25 mM NaHCO3, 1.78 mM CaCl2.2H2O, 24.9 mM Na-lactate, 0.47 mM Na-pyruvate, 50 μg/ml gentamycin, and 0.005 mM phenol red). To induce sperm capacitation, 0.4 % bovine serum albumin was added to BM. All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA).

Preparation and treatment of spermatozoa

All animal procedures were performed in accordance with the guidelines for the ethical

Effects of FPN on sperm total motility and motion kinematics

The CASA program enables the evaluation of 10 sperm motility and motion kinematics parameters. Aside from Medium motility and Slow motility, 8 parameters were significantly decreased by FPN treatment in a concentration-dependent manner. Total motility, Rapid motility, Progressive motility, VCL, VAP, and ALH were reduced with high concentrations (1, 10, 100, and 300 μM), whereas, BCF significantly decreased starting at 10 μM FPN. Of note, VSL was significantly decreased only at 300 μM (P < 0.05;

Discussion

Since 1993, the phenylpyrazole insecticide FPN has been widely used for pest control in agriculture and stockbreeding, veterinary products and other applications [2]. In 2017, FPN was detected in chicken eggs in Europe and Korea, and in Taiwan, a notice of excessive FPN use was issued. Although FPN is classified as a ‘Moderately hazardous’ active insecticide ingredient, people are readily exposed in daily life. FPN acts by impairing the central nervous systems of insects by blocking

Conflict of interest

The authors have declared that no competing interests exist.

Acknowledgement

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT) (NRF-2019R1F1A1049216).

References (29)

  • C.C. Tingle et al.

    Fipronil: environmental fate, ecotoxicology, and human health concerns

    Rev. Environ. Contam. Toxicol.

    (2003)
  • X. Zhao et al.

    Fipronil is a potent open channel blocker of glutamate-activated chloride channels in cockroach neurons

    J. Pharmacol. Exp. Ther.

    (2004)
  • Q.X. Shi et al.

    γ-Aminobutyric acid (GABA) induces the acrosome reaction in human spermatozoa

    Mol. Hum. Reprod.

    (1997)
  • M.N. Ritta et al.

    Occurrence of GABA and GABA receptors in human spermatozoa

    Mol. Hum. Reprod.

    (1998)
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