Edited by: Ana Josefa Soler, University of Castilla–La Mancha, Spain
Reviewed by: David Martin Hidalgo, University of Porto, Portugal; Olga Garcia-Alvarez, University of Castilla–La Mancha, Spain
†These authors share senior authorship
This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Fifty percent of male subfertility diagnosis is idiopathic and is usually associated with genetic abnormalities or protein dysfunction, which are not detectable through the conventional spermiogram. Glutathione
In humans, about 30–50% of fertilizations fail because of male subfertility problems, usually related to abnormal sperm count, motility, and/or morphology (
Several studies reported the association between male idiopathic subfertility or infertility and some null genotypes of glutathione
Ezatiostat or Terrapin 199 (TER) is a specific inhibitor of the GSTP1–JNK heterocomplex, used as an anticancer drug (
Along these lines, understanding the molecular role of the GSTP1–JNK heterocomplex in mammalian sperm physiology is much warranted. Herein, cell biology and immunological approaches were performed through pharmacologically inhibiting the formation of the GSTP1–JNK heterocomplex, prior to analyzing sperm quality and functionality parameters, the presence and localization of GSTP1, and the activation of JNK. Therefore, the present study aimed to investigate the function of this heterocomplex in mammalian sperm physiology, using the pig as a model, which has recently been stablished as a suitable animal model for research in human reproduction (
Chemicals and reagents were purchased from Sigma-Aldrich (Saint Louis, MO, United States), unless otherwise indicated. TER was reconstituted in dimethyl sulfoxide (DMSO) to a stock solution of 64 mM. Fluorochromes [SYBR-14, propidium iodide (PI), merocyanine 540 (M540), Yo-Pro-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC1), Fluo3-AM (Fluo3), hydroethidine (HE), and fluorescein-conjugated peanut agglutinin/PI (PNA)] were purchased from Life Technologies (Thermo Fisher Scientific, Carlsbad, CA, United States). SYBR-14, M540, Yo-Pro-1, JC1, Fluo3, and HE were reconstituted in DMSO, whereas PI and PNA were diluted in phosphate-buffered saline (PBS) 1X. Antibody against GSTP1 (ref. MBS3209038) was purchased from MyBioSource (San Diego, CA, United States), whereas phospho-JNK (Thr183/Tyr185) antibody (pJNK, ref. 4668S) was purchased from Cell Signaling Technology (Danvers, MA, United States). Secondary anti-rabbit (ref. P0448) and anti-mouse (ref. P0260) antibodies conjugated with horseradish peroxidase for immunoblotting analysis were purchased from Dako (Derkman A/S, Denmark), whereas the secondary anti-rabbit antibody conjugated with Alexa Fluor 488 for immunofluorescence analysis was purchased from Thermo Fisher Scientific (ref. A32731).
Semen samples, commercially sold as pig artificial insemination (AI) seminal doses, were purchased from an authorized local AI center (Grup Gepork S.L., Masies de Roda, Spain) that followed ISO certification (ISO-9001:2008) and operates under commercial, standard conditions. Thirteen ejaculates (one ejaculate per boar,
All semen samples (
Sperm motility assessment was performed through a computer-assisted sperm analysis (CASA) system, using an Olympus BX41 microscope (Olympus, Tokyo, Japan) with a negative phase-contrast field (Olympus 10 × 0.30 PLAN objective, Olympus) connected to a personal computer containing the ISAS software (Integrated Sperm Analysis System V1.0, Proiser S.L., Valencia, Spain). Semen samples were incubated for 15 min at 38°C prior to motility assessment. Once incubated, 5 μl of each sample was examined in a prewarmed (38°C) Makler counting chamber (Sefi Medical Instruments, Haifa, Israel). Three technical replicates of at least 500 sperm per replicate were examined in each sample. Total motility (TMOT), progressive motility (PMOT), and average path velocity (VAP, μm/s) were used to evaluate sperm motility. A sperm cell was considered motile when VAP was ≥10 μm/s and progressively motile when the coefficient of straightness (STR) was ≥45%.
Sperm viability, plasma membrane stability, mitochondrial activity, intracellular calcium levels, intracellular superoxide levels, and acrosome membrane integrity assessments were conducted using a Cell Laboratory QuantaSC cytometer (Beckman Coulter, Fullerton, CA, United States) equipped with an argon-ion laser (488 nm) set at a power of 22 mW. Semen samples were diluted (2 × 106 sperm/ml) in prewarmed PBS to a final volume of 600 μl prior to staining with the corresponding protocol. Sperm viability (SYBR-14/PI) (
The electronic volume (EV) gain, PMT voltages of optical filters (FL-1, FL-2, and FL-3), and fluorescence overlapping were set using unstained and single-stained samples of each fluorochrome. Flow rate, laser voltage, and sperm concentration were constant throughout the experiment. Sperm cells from debris events were distinguished using EV. Three technical replicates of at least 10,000 sperm per replicate were examined for each sample. As recommended by the International Society for Advancement of Cytometry (ISAC), Flowing Software (Ver. 2.5.1, University of Turku, Finland) was used to analyze flow cytometry data.
Semen samples were diluted in PBS (3 × 106 sperm/ml) and fixed in 2% paraformaldehyde (Alfa Aesar, Haverhill, MA, United States) and washed twice. Two 150-μl aliquots of each sample were placed in an ethanol prerinsed slide and subsequently blocked and permeabilized for 40 min at room temperature (RT) with a blocking solution containing 0.25% (v:v) Triton X-100 and 3% (w:v) bovine serum albumin (BSA). Samples were incubated with anti-GSTP1 antibody (1:200, v:v) overnight, washed thrice, and subsequently incubated with an anti-rabbit antibody (1:400, v:v). In negative controls, the primary antibody was omitted. Then, 10 μl of Vectashield mounting medium containing 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was added prior to being covered and sealed with nail varnish. Finally, each sample was evaluated using a confocal laser scanning microscope (CLSM, Nikon A1R, Nikon Corp., Tokyo, Japan).
Semen samples were centrifuged twice (3,000 ×
Twenty micrograms of total protein was diluted (1:1, v:v) in Laemmli reducing buffer 4X (Bio-Rad) and heated at 95°C for 7 min prior to being loaded onto a 12% polyacrylamide gel (Mini-PROTEAN®, TGX Stain-FreeTM Precast Gels, Bio-Rad) and electrophoresed for 2 h at 120 V. Total protein was visualized using a G:BOX Chemi XL system (Syngene, Frederick, MD, United States). Mini-PROTEAN®, TGX Stain-FreeTM Precast Gels contain a trihalo compound that allows fluorescent detection of tryptophan residues. Thereafter, proteins from the gel were transferred onto polyvinylidene difluoride (PVDF) membranes using the Trans-Blot®, TurboTM (Bio-Rad). Transferred membranes were blocked using 5% BSA and incubated with the anti-GSTP1 (1:5,000, v:v) or anti-pJNK (1:2,000, v:v) antibodies for 1 h in agitation at RT. Next, membranes were rinsed thrice and incubated with the secondary anti-rabbit antibody 1:10,000 (v:v) for GSTP1 and 1:4,000 (v:v) for pJNK. Then, membranes were washed five times, and bands were visualized through incubation with a chemiluminescent substrate (ImmobilonTM Western Detection Reagents, Millipore, United States) prior to scanning with G:BOX Chemi XL 1.4 (Syngene, India). Finally, membranes were stripped, and the process was repeated by replacing the primary antibody for the anti-α-tubulin antibody (1:100,000, v:v) and the secondary antibody for the anti-mouse antibody (1:150,000, v:v), as loading control and for normalization. In the pJNK assessment, Quantity One software package (Version 4.6.2, Bio-Rad) was used to quantify the bands of two technical replicates per sample, normalized using α-tubulin.
Plotting and statistical analysis of the results were performed using GraphPad Prism v.8 (GraphPad Software, La Jolla, CA, United States) and IBM SPSS for Windows v. 25.0 (IBM Corp., Armonk, NY, United States). Each biological replicate was considered a statistical case, and data were checked for normal distribution (Shapiro–Wilk test) and homogeneity of variances (Levene test). Sperm quality and functionality parameters, as well as normalized pJNK relative levels, were compared between treatments (control-0h, control-72h, and TER-72h) using a one-way ANOVA followed by Tukey’s multiple-comparison test. Data are shown as mean ± standard error of the mean (SEM). The level of significance was set at
The presence and localization of GSTP1 in sperm samples are presented in
Immunoblotting analysis of GSTP1. Pig sperm lysates were incubated with
Immunolocalization of GSTP1 in pig sperm.
Immunoblotting analysis of Thr183 and Tyr185 phosphorylation of JNK revealed a double-band pattern showing both p46 and p54 splicing variants of JNK (
The percentage of viable sperm was higher (
Flow cytometry analysis of sperm viability.
As shown in
Computer-assisted sperm analysis (CASA) of pig semen samples. Mean, standard error of the mean (SEM), and sample distribution of the percentage of
The assessment of MMP is presented in
Flow cytometry analysis of pig sperm mitochondrial activity.
As shown in
Flow cytometry analysis of plasma sperm membrane stability.
Flow cytometry analysis of intracellular superoxide levels.
The relative Fluo3 fluorescence intensity (Fluo3+) of the viable sperm population (PI–) is presented in
Flow cytometry analysis of intracellular calcium levels.
Flow cytometry analysis of acrosome integrity.
Several studies have evidenced the essential role of GSTs as molecular regulators of mammalian sperm physiology and fertilizing capacity (
While the presence of GSTP1 in the sperm was established by proteomic studies in several mammalian species such as human (
As has been previously reported in the literature, the JNK activation is regulated by GSTP1 in somatic cells (
An interesting physiological effect of the activation of JNK was the significant decrease in sperm mitochondrial activity, viability, and motility. Activation of JNK has been reported in the literature to be related to mitochondrial dysfunction and cell death in somatic cells (
Related to sperm mitochondrial dysfunction, the results of the present study showed an increase in intracellular superoxide levels triggered by the GSTP1–JNK heterocomplex dissociation and subsequent activation of JNK. Similar results were reported in somatic cells, where JNK activation was related to increased superoxide formation (
Our results showed that pharmacological dissociation of the GSTP1–JNK heterocomplex in sperm cells significantly impaired the stability of lipidic membranes, although it did not affect the acrosome membrane. Previous studies utilizing general GST inhibitors in goat and pig sperm reported high levels of plasma membrane damage and destabilization, although they did not find any effect on the acrosomal membrane (
Interestingly, although GSTP1–JNK dissociation caused severe mitochondrial damage and membrane destabilization in sperm cells, it did not have any effects upon intracellular calcium reservoirs. In this sense, previous studies in pig sperm showed that general GST inhibitors caused a significant increase in calcium levels, predominantly in the sperm middle piece (
In conclusion, immunological and cell biology analyses confirmed that, as schematized in
Proposed diagram of the function of the GSTP1–JNK heterocomplex under physiological conditions and cellular stress in sperm cells. In physiological conditions, the GSTP1–JNK heterocomplex prevents JNK activation, maintaining sperm viability, motility, mitochondrial activity, and plasma membrane stability and increased superoxide levels. On the other hand, under cellular stress conditions, the GSTP1–JNK heterocomplex is dissociated, and thus, JNK is activated by tyrosine (pY) and threonine (pT) phosphorylation. The activation of JNK decreases sperm viability and motility, disrupts mitochondrial activity, causes plasma membrane destabilization, and increases intracellular superoxide levels.
The original contributions presented in the study are included in the article/
MY and ML: conceptualization and methodology. ML, SO, YM-O, AD-B, and SR: formal analysis and investigation. ML: writing-original draft preparation. MY and IB: writing-review and editing and supervision. MY: funding acquisition. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to thank the Technical Research Services (University of Girona) for their technical support. The authors would also like to thank the Servier Medical Art for their image bank used to create all figures.
The Supplementary Material for this article can be found online at:
Preliminary concentration test. Mean and standard error of the mean (SEM) of the percentage of
Raw data. Raw data of sperm quality and functionality parameters of all treatments and time points. TMOT, percentage of total motile sperm; PMOT, percentage of progressively motile sperm; VAP, sperm average path velocity (μm/s); VIABILITY, percentage of viable sperm (SYBR-14+/PI–); M540, percentage of membrane-destabilized cells (M540+) within the total viable sperm population (Yo-Pro-1–); HE, relative fluorescence intensity of viable sperm with high levels of intracellular O2–⋅ (E+/PI–); JC1, percentage of high mitochondrial membrane potential sperm (ΔΨm) resulted from the orange-stained (JC1
Supplementary information for Materials and Methods.