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