Regulation of Phosphorylation of Glycogen Synthase Kinase 3α and the Correlation with Sperm Motility in Human

Abstract

Purpose

To unravel the mechanism regulating the phosphorylation of glycogen synthase kinase 3 (GSK3) and the correlation between the inhibitory phosphorylation of GSK3α and sperm motility in human.

Materials and Methods

The phosphorylation and priming phosphorylated substrate-specific kinase activity of GSK3 were examined in human spermatozoa with various motility conditions.

Results

In human spermatozoa, GSK3α/β was localized in the head, midpiece, and principal piece of tail and p-GSK3α(Ser21) was enriched in the midpiece. The ratio of p-GSK3α(Ser21)/GSK3α was positively coupled with normal sperm motility criteria of World Health Organization. In high-motility spermatozoa, p-GSK3α(Ser21) phosphotyrosine (p-Tyr) proteins but p-GSK3α(Tyr279) markedly increased together with decreased kinase activity of GSK3 after incubation in Ca²⁺ containing medium. In high-motility spermatozoa, p-GSK3α(Ser21) levels were negatively coupled with kinase activity of GSK3, and which was deregulated in low-motility spermatozoa. In high-motility spermatozoa, 6-bromo-indirubin-3′-oxime, an inhibitor of kinase activity of GSK3 increased p-GSK3α(Ser21) and p-Tyr proteins. p-GSK3α(Ser21) and p-Tyr protein levels were decreased by inhibition of PKA and Akt. Calyculin A, a protein phosphatase-1/2A inhibitor, markedly increased the p-GSK3α(Ser21) and p-Tyr proteins, and significantly increased the motility of low-motility human spermatozoa.

Conclusions

Down regulation of kinase activity of GSK3α by inhibitory phosphorylation was positively coupled with human sperm motility, and which was regulated by Ca²⁺, PKA, Akt, and PP1. Small-molecule inhibitors of GSK3 and PP1 can be considered to potentiate human sperm motility.

INTRODUCTION

Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that is critically involved in multiple cellular processes, including cell fate determination and glucose metabolism [1,2,3]. Phosphorylation on serine residues of GSK3 decreases kinase activity but phosphorylation on tyrosine residues of GSK3 increases kinase activity [4]. GSK3 is conserved in all eukaryotes and exists in α and β isoforms in mammals. The testes highly express GSK3α and β at the mRNA level; however, at the protein level, GSK3β is minimally expressed due to tight regulation of its translation or extreme instability of the protein [5].

In human testis, GSK3β is predominant isoforms in spermatogonia, and which was switch to GSK3α during meiotic entry, and GSK3α is predominant in human spermatozoa [6]. In mice, targeted disruption of GSK3α affects sperm motility, resulting in infertility [7]. In epididymal spermatozoa of mice, post-transcriptional Wnt signaling reduces protein polyubiquitination through GSK3 and initiates sperm motility by inhibiting protein phosphatase-1 (PP1) [8]. GSK3 has been suggested as a key regulator of mammalian sperm motility. In mammalian spermatozoa, serine phosphorylation of GSK3α is associated with motility [9,10,11,12]. Nonetheless, regulation of phosphorylation of the GSK3α, GSK3 kinase activity, and correlation between inhibitory phosphorylation of GSK3α and sperm motility remain poorly understood in human spermatozoa. In this study, we evaluated the phosphorylation and kinase activity of GSK3 to priming the phosphorylation of specific substrates in human spermatozoa that were categorized according to the World Health Organization (WHO) criteria for human sperm motility, with a one-sided lower reference limit (LRL) of 40%. Moreover, to unravel the intracellular mechanisms that regulate GSK3 and sperm motility, we examined the roles of Ca²⁺, cAMP-PKA, Akt, and PP1 signaling on both the inhibitory phosphorylation and kinase activity of GSK3 in human spermatozoa.

MATERIALS AND METHODS

1. Ethics statement

Experiments with human samples were performed in accordance with the Institutional Review Board (IRB) of Cheil General Hospital (CGH) (CGH-IRB-2014-29). Animal study was approved by the Hanyang University Institutional Animal Care and Use Committee (IACUC) (IACUC No. 2015-0255A).

This study included male patients with infertility by oligospermia or asthenozoospermia as well as healthy male who visited Cheil General Hospital for semen analysis and received care from the same single physician. The semen samples were collected by masturbation after 3–5 days of abstinence and analyzed after liquefaction within 30 minutes. Semen analysis was performed according to the standard methodology proposed by the WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edition [13]. For the study, total 63 spermatozoa samples were subjected further experiments. For the biochemical analyses in this study, informed consent was obtained from every patient.

2. Animals

Eight-week-old male Institute of Cancer Research mice were obtained from DAEHAN Biolink. Brain, testes, and caudal epididymides were removed after asphyxiation in CO₂ and washed in phosphate-buffered saline (PBS) to eliminate blood. Brain and testes subjected to protein isolation, and caudal epididymides were subjected to sperm preparation.

3. Chemicals

LiCl, adenosine 5′-triphosphate (ATP) disodium salt hydrate, Akt inhibitor VIII, 8-bromoadenosine 3′,5′-cyclic monophosphate (8-br-cAMP), PKA inhibitor H89, and 6-bromo-indirubin-3′-oxime (BIO) were purchased from Sigma-Aldrich Korea. Tetramethyl-6-carboxyrhodamine (TAMRA)-labeled peptides (T-Pep, TAMRA-KEEPPSPPQSPR; T-Pep(p), TAMRA-KEEPPSPPQp-SPR) were synthesized by Peptron, Inc. ECL Prime Western Blotting Detection Reagent was obtained from Amersham Bioscience (RPN2232). Xpert protease and phosphatase inhibitor cocktail were purchased from GenDEPOT. Calyculin A was purchased from Abcam (ab141784).

4. Computer-assisted sperm analysis

Sperm motility analysis was conducted using video recording and computer-assisted sperm analysis. Briefly, 20-µL sperm samples in media or semen samples were dispensed onto a pre-warmed MAKLER^® counting chamber (Irvine Scientific) at 37 ℃. Movies were recorded on a Nikon Diaphot microscope equipped with a CoolSnap EZ CCD camera (Photometrics) controlled through iSPERM software (CNC Biotech). Sperm motility was classified according to the WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edition [13] and measured at least 200 spermatozoa.

5. Collection and drug treatment of spermatozoa

Human spermatozoa were isolated from freshly ejaculated semen samples (n=63) from male with various motility conditions (Supplement Table 1). Tyrode’s basal medium (TBM, 96 mM NaCl, 4.7 mM KCl, 0.4 mM MgSO₄, 0.3 mM NaH₂PO₄, 5.5 mM glucose, 1 mM sodium pyruvate, 21.6 mM sodium lactate, 20 mM HEPES, and pH 7.45) was overlaid on ejaculated semen, and spermatozoa were allowed to swim up for 20 minutes in a CO₂ incubator at 37 ℃. Motile spermatozoa were harvested, washed in TBM, and centrifuged at 500×g for 5 minutes. After a second wash and centrifugation, the sperm concentration was adjusted to 1×10⁶ cells/mL through addition of modified Tyrode’s complete medium (TCM, made by the addition of 1 mM CaCl₂, 10 mM NaHCO₃, and 3 mg/mL bovine serum albumin in TBM) or TBM. BIO, Akt inhibitor VIII, 8-br-cAMP, and Calyculin A were added to the sperm suspensions, and incubated for 90 minutes in a CO₂ incubator at 37 ℃. After incubation, spermatozoa were subjected to protein extraction. To confirm whether the GSK3α/β and p-GSK3α/β(Ser21/9) antibodies detected corresponding GSK3 isoforms, mouse tissues and spermatozoa were subjected to western blot. Mouse cauda epididymides were placed in TBM and squeezed to release spermatozoa. Motile spermatozoa were harvested after 20 minutes, centrifuged at 500×g for 5 minutes and subjected to protein extraction.

6. Western blot analysis

Human sperm samples and mouse tissues were sonicated 5 times for 5 seconds at 4 ℃ in PBS containing 1% Triton-X-100 and 1% (v/v) protease and phosphatase inhibitor cocktail. Lysates were centrifuged at 18,341×g for 20 minutes and the supernatants were collected. Protein concentration in the lysate was determined using BCA protein assay kit (23225; Thermo Scientific), and samples were subjected to SDS-PAGE. After transferred to nitrocellulose membranes, the blots were probed with GSK3α/β (#5676; Cell Signaling), p-GSK3α/β(Ser21/9) (#9327; Cell Signaling), phosphotyrosine (p-Tyr, 05-321; Millipore), β-tubulin (ab108342; Abcam), β-actin (ab8226; Abcam), GAPDH (sc-25778; Santa Cruz), and p-GSK3α/β(Tyr279/216) (sc-81496; Santa Cruz) antibodies were diluted in 5% skim milk in Tris-buffered saline (TBS) and incubated with the membranes overnight at 4 ℃. After three washes with TBS/0.1% Tween 20 (TBST) for 20 minutes, the membranes were incubated with peroxidase-labeled goat anti-rabbit IgG (ab6721; Abcam) or rabbit anti-mouse IgG (ab6728; Abcam) diluted in 5% skim milk in TBST for 1 hour. The membranes were washed three times with TBST for 20 minutes. The signals were detected with Fusion SL (Vilber Lourmat) and ECL Prime Western Blotting Detection Reagent (RPN2232; Amersham Bioscience) according to the manufacturers’ instructions. The band intensities were analyzed with ImageJ (Ver.1.51j8; National Institutes of Health) and are expressed in arbitrary units (AU). Antibodies specificity of GSK3α/β and p-GSK3α/β(Ser21/9) to corresponding GSK3 isoforms was confirmed (Supplement Fig. 1, 2).

7. Immunocytochemistry of GSK3 in human spermatozoa

Spermatozoa with normal motility were smeared on poly-L-lysine-coated slides, fixed in acetone: methanol (1:1) at 4 ℃ for 10 minutes, and air dried. The slides were blocked in 5% donkey serum (ab7475; Abcam) in PBS and incubated with GSK3α/β and p-GSK3α(Ser21) (sc-101690; Santa Cruz) antibody diluted in 1.5% donkey serum in PBS in a humidified chamber overnight at 4 ℃. Normal rabbit IgG (ab27478; Abcam) was used as a negative control to replace the primary antibodies. After washing three times in PBS, the signal was developed with Alexa Fluor 488–conjugated donkey anti-rabbit IgG (ab150061; Abcam) diluted in 1.5% donkey serum in PBS for 1 hour at room temperature. For acrosome visualization, the slides were incubated with rhodamine-labeled peanut agglutinin (RL-1072; Vector Laboratories) diluted in 1.5% donkey serum in PBS for 1 hour at room temperature. After washing three times in PBS, nuclear staining and mounting were conducted with ProLong™ Gold Antifade mounting medium containing DAPI (P36931; Invitrogen). Images were captured with a fluorescence microscope equipped with a cooled CCD (DP71; Olympus). The fluorescence intensities were analyzed with ImageJ Ver.1.51j8. No specific signal was detected in negative controls.

8. Gel-shift assay to measure GSK3 kinase activity using priming phosphorylated substrate

A fluorescent gel-shift assay was conducted according to the method of Choi et al [14] in 2015. Briefly, to ensure GSK3 activity, T-Pep(p) (1 µL at 100 µM), GSK3 (1 µl at 25 U), ATP (0.4 µL at 10 mM), and 10× kinase reaction buffer (2 µL) were mixed to a final volume of 20 µL in standard reaction buffer (20 mM Tris-HCl, pH 7.4). For the GSK3 kinase activity assay, washed sperm pellets were lysed in RIPA buffer (20 mM HEPES, 0.1% SDS, 0.5% deoxycholic acid, 150 mM NaCl, pH7.5) and quantified. For the kinase inhibition assay, LiCl, a GSK3 inhibitor (1–10 mM) was mixed with equal volume of reaction buffer containing the T-Pep(p), sperm extracts (8.8 µg), and ATP. A 5-µL aliquot of lysate was mixed with 1 µL of 100 µM T-Pep(p) and 0.4 µL of 10 mM ATP to a final volume of 20 µL in reaction buffer (20 mM Tris-HCl, pH 7.4). The reactants were further incubated for 120 minutes at 30 ℃, followed by gel electrophoresis. Fluorescent gel-shift assays were performed on a 1% agarose gel cast in 1× Tris-Borate Buffer without ethylenediaminetetraacetic acid at 50 V for 60 minutes. A fluorescent image of the agarose gel was produced using a UV transilluminator equipped with a digital camera (UVP Inc.).

9. Statistical analysis

Statistical analyses were performed by SPSS (version 17.0; SPSS Inc.) using an unpaired, two-tailed Mann–Whitney U-test. Statistical significance was defined as p<0.05 and p<0.01.

RESULTS

1. Phosphorylation of GSK3 isoforms in human spermatozoa with different motility

In acrosome-intact spermatozoa, GSK3α/β was found in the acrosomal and post-acrosomal regions of the head, midpiece, and principal piece of the tail. p-GSK3α(Ser21) was primarily found in the post-acrosomal region and midpiece. In acrosome-reacted spermatozoa, GSK3α/β was detected in the equatorial region of the head, the midpiece, and the principal piece of the tail whereas p-GSK3α(Ser21) was primarily found in the midpiece. Fluorescence intensity of GSK3α/β in the acrosome-intact spermatozoa was significantly higher than those in the acrosome-reacted spermatozoa whereas no significant change was observed in p-GSK3α(Ser21). No visible immunoreactivity was found in normal IgG (Fig. 1). In a western blot, GSK3α was predominant and was phosphorylated at the Ser21 and Tyr279 residues, whereas only a small amount of GSK3β was detected. Next, we used western blotting to analyze phosphorylation of the GSK3. p-GSK3β(Ser9) was not detected in human spermatozoa. In densitometric analysis, the ratio of p-GSK3α(Ser21)/GSK3α in the high-motility spermatozoa was significantly higher than those of low-motility spermatozoa whereas no significant difference was in the other parameters (Fig. 2, Supplement Fig. 3, Supplement Table 2).

Fig. 1. Expression and phosphorylation of glycogen synthase kinase 3 (GSK3) in human spermatozoa. (A) In acrosome-intact (left) spermatozoa, GSK3α/β was expressed in the head (asterisks) and in the midpiece (arrows) and principal piece of the tail (arrowheads). p-GSK3α(Ser21) was expressed in the post-acrosomal region of the head and midpiece and tail. In acrosome-reacted (right) spermatozoa, GSK3α/β disappeared from the acrosomal and post-acrosomal regions of the head and was found in the equatorial region of the head (asterisks) and in the midpiece (arrows) and principal piece (arrowheads) of the tail. p-GSK3α(Ser21) was found in the midpiece of the tail. (B) Fluorescence intensity of GSK3α/β in acrosome-intact spermatozoa was significantly higher than those in acrosome-reacted spermatozoa whereas no significant change was observed in p-GSK3α(Ser21). GSK3α/β and p-GSK3α(Ser21) are green. Nuclei were stained blue by DAPI. Scale bar=5 µm. AU: arbitrary units. ^*Significantly different by Mann–Whitney U-test at p<0.05 levels.

Fig. 2. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms in human spermatozoa with different motility. (A) Representative western blot of GSK3 in human spermatozoa with different motility conditions. (B) Sperm motility of 40% was used as the criterion for normal motility according to the World Health Organization lower reference limit of human sperm motility. The ratio of p-GSK3α(Ser21)/GSK3α was positively correlated with human sperm motility. No significant difference in the ratio of p-GSK3α(Tyr279)/GSK3α, p-GSK3β(Tyr216)/GSK3β, p-GSK3α/β(Ser21/9)/GSK3α/β, and p-GSK3α/β(Tyr279/216)/GSK3α/β was found between the high-motility spermatozoa and low-motility spermatozoa. AU: arbitrary units, ND: not detected. ^*Significantly different from low-motility spermatozoa by Mann–Whitney U-test at p<0.05 levels. X and circles indicate mean and individual values, respectively.

2. Change in serine phosphorylation and priming phosphorylated substrate-specific kinase activity of GSK3 after capacitation in human spermatozoa

In a fluorescent agarose gel-shift assay, the T-Pep(p) exhibited a distinct mobility shift when tested with recombinant rabbit GSK3 in the presence of ATP, but not in T-Pep alone. GSK3 in sperm lysates induced a shift in the fluorescent band of T-Pep(p), a GSK3 specific substrate, but it was visibly decreased by LiCl. The levels of p-GSK3α(Ser21) and p-Tyr proteins in spermatozoa incubated in TCM were higher than those of spermatozoa incubated in TBM. In the gel kinase assay, the kinase activity of GSK3 was visibly decreased after incubation in TCM, and which was lesser than that incubated in TBM (Fig. 3). The initial kinase activity of GSK3 of high-motility spermatozoa was higher than that of low-motility (<40%) spermatozoa. Following incubation in TCM, the kinase activity of GSK3 in high-motility spermatozoa decreased, but it did not decrease in the low-motility spermatozoa. In high-motility spermatozoa, p-GSK3α(Ser21)/GSK3α but not p-GSK3α(Tyr279)/GSK3α significantly increased following incubation in TCM. In low-motility spermatozoa, neither p-GSK3α(Ser21) nor p-GSK3α(Tyr279) changed after incubation in TCM. Treatment of BIO, an inhibitor of kinase activity of GSK3 increased the p-GSK3α(Ser21) and p-Tyr proteins in a dose-dependent manner (Fig. 4, Supplement Table 3).

Fig. 3. Change in Ser21 phosphorylation and priming phosphorylated substrate-specific kinase activity of glycogen synthase kinase 3 (GSK3) after capacitation in human spermatozoa. (A) Efficacy of the priming phosphorylated peptide substrate (T-Pep(p)) in determining GSK3 kinase activity in sperm extracts. T-Pep showed no remarkable change in phosphorylation by recombinant GSK3 regardless of ATP. In contrast, T-Pep(p) was phosphorylated by recombinant GSK3 and ATP. T-Pep and T-Pep(p) were loaded in control (Con) lane for comparison. (B) Fluorescent gel kinase assay for GSK3 substrate specific kinase activity to T-Pep(p) in fresh human spermatozoa lysates. LiCl (0–10 mM) decreased GSK3 kinase activity in dose dependent manner. T-Pep(p) was loaded in Con lane without sperm extracts and LiCl. (C) Western blots of p-GSK3α(Ser21) and phosphotyrosine (p-Tyr) proteins in human spermatozoa after incubation in Tyrode’s basal medium (TBM) or Tyrode’s complete medium (TCM). p-GSK3α(Ser21) and p-Tyr proteins levels in spermatozoa incubated in TBM were lower than those incubated in TCM. (D) Gel kinase assay of GSK3 in high-motility human spermatozoa after incubation in TBM or TCM. Reduction of GSK3 kinase activity of spermatozoa following incubation was apparent in TCM compared to TBM. ATP: adenosine 5′-triphosphate, TAMRA: tetramethyl-6-carboxyrhodamine.

Fig. 4. Change in Ser21 phosphorylation and priming phosphorylated substrate-specific kinase activity in human spermatozoa with different motility. (A) Fluorescent gel kinase assay of glycogen synthase kinase 3 (GSK3) from human spermatozoa with high and low-motility. After incubation in Tyrode’s complete medium (TCM), the kinase activity of GSK3 decreased in the high-motility spermatozoa but increased in the low-motility spermatozoa. T-Pep(pp) band intensities were measured by densitometric analysis and calculated as ratio compared to those of T-Pep(p). T-Pep, T-Pep(p), and T-Pep(pp) were loaded as marker. (B) Phosphorylation of GSK3 in low- and high-motility spermatozoa after incubation in TCM. p-GSK3α(Ser21), but not p-GSK3α(Tyr279), increased in the high-motility spermatozoa. In the low-motility spermatozoa, no visible change in p-GSK3 was observed. (C) The ratio of p-GSK3α(Ser21)/GSK3α was increased significantly after incubation in TCM, whereas the ratio of p-GSK3α(Tyr279)/GSK3α ratio was not. (D) Western blots of p-GSK3α(Ser21) and phosphotyrosine (p-Tyr) proteins after incubation with the 6-bromo-indirubin-3′-oxime (BIO), an inhibitor of GSK3 kinase activity in TCM. BIO (5–500 nM) increased p-GSK3α(Ser21) and p-Tyr proteins in a dose-dependent manner. AU: arbitrary units. ^**Significantly different from fresh spermatozoa by Mann–Whitney U-test (p<0.01).

3. Change in serine phosphorylation of GSK3 and p-Tyr proteins in human spermatozoa by 8-br-cAMP, H89, and Akt inhibitor VIII treatment

Following treatment with 1 mM 8-br-cAMP in either TBM or TCM, the levels of p-GSK3α(Ser21) and p-Tyr proteins were increased. In TCM, 100 µM H89 decreased the levels of p-GSK3α(Ser21) and p-Tyr proteins. Akt inhibitor VIII also decreased the levels of p-GSK3α(Ser21) and p-Tyr proteins in spermatozoa (Fig. 5).

Fig. 5. Change in Ser21 phosphorylation of glycogen synthase kinase 3 (GSK3) and phosphotyrosine (p-Tyr) proteins in human spermatozoa by 8-bromoadenosine 3′,5′-cyclic monophosphate (8-br-cAMP), H89, and Akt inhibitor VIII treatment. (A) Western blots of p-GSK3(Ser21) and p-Tyr proteins in human spermatozoa after incubation with 8-br-cAMP in Tyrode’s basal medium (TBM) or Tyrode’s complete medium (TCM). 8-brcAMP (1 mM) increased p-GSK3α(Ser21) and p-Tyr proteins levels, and which was apparent in the spermatozoa incubated in TCM compared to those incubated in TBM. (B) Western blots of p-GSK3α(Ser21) and p-Tyr proteins after incubation with the PKA inhibitor H89 in TCM. H89 (100 µM) markedly decreased the levels of p-GSK3α(Ser21) and p-Tyr proteins in human spermatozoa. (C) Western blots of p-GSK3α(Ser21) and p-Tyr proteins after incubation with Akt inhibitor VIII in TCM. Akt inhibitor VIII (50–100 µM) markedly decreased the levels of the p-GSK3α(Ser21) and p-Tyr proteins after incubation in TCM.

4. Change in serine phosphorylation of GSK3 and p-Tyr proteins in low-motility human spermatozoa by Calyculin A

In low-motility spermatozoa, PP1/2A inhibitor Calyculin A (10 nM) markedly increased the levels of p-GSK3α(Ser21) and p-Tyr proteins and significantly increased the progressive as well as total motility (Fig. 6, Supplement Fig. 4).

Fig. 6. Recovery of p-GSK3(Ser21), phosphotyrosine (p-Tyr) proteins and motility in low-motility human spermatozoa by Calyculin A. (A) Western blots of p-GSK3α(Ser21) and p-Tyr proteins in low-motility human spermatozoa following incubation with Calyculin A in Tyrode’s complete medium. Calyculin A (10 nM) markedly increased the levels of p-GSK3α(Ser21) and p-Tyr proteins in low-motility spermatozoa. (B) Effects of Calyculin A on low-motility human spermatozoa. Calyculin A (10 nM) significantly increased the progressive as well as total motility. GSK3: glycogen synthase kinase 3. ^*Significantly different from low-motility spermatozoa by Mann–Whitney U-test at p<0.05 levels.

DISCUSSION

1. Roles of GSK3α/β in human sperm motility

In acrosome-intact human spermatozoa, GSK3α/β were localized in the acrosomal and post-acrosomal regions, midpiece and principal piece of the tail. p-GSK3α(Ser21) was primarily localized in the post-acrosomal region and the midpiece of the tail. Similarly, in mouse spermatozoa, GSK3α/β is expressed in the peri-acrosomal region, where it could regulate acrosomal exocytosis by phosphorylating dynamin [15]. In acrosome-reacted spermatozoa, disappearance of GSK3α/β from the acrosomal region might be attributable to loss of peri-acrosomal cytoplasm. The equatorial region was positive for GSK3α/β but not for p-GSK3α(Ser21), suggesting that the kinase activity of GSK3α supports the interaction between sperm and with oolemma. In the midpiece of sperm, both p-GSK3α(Ser21) and GSK3α immunoreactivity were found. In neuron and cardiac muscle cells, GSK3β has been known to decrease mitochondrial membrane potential [16]. In boar spermatozoa, dephosphorylation of GSK3α impaired mitochondrial function, which was reversed by a GSK3 inhibitor [12]. Together, p-GSK3α(Ser21) in the midpiece might be coupled with mitochondrial activity to enable motility in human spermatozoa. In western blots, GSK3α and p-GSK3α(Ser21) were abundantly expressed but GSK3β was marginal. A positive correlation was found between sperm motility and levels of p-GSK3α(Ser21)/GSK3α. This suggests that the inhibitory phosphorylation of GSK3α might be important for sperm motility and could be useful as diagnostic markers for sperm motility in human.

2. Correlation between sperm motility and kinase activity of GSK3α/β and serine phosphorylation of GSK3α

Recently, the kinase activity of GSK3α/β of mouse spermatozoa was successfully measured by fluorescent gel-shift assays using the priming phosphorylated substrates [17]. In human spermatozoa, the kinase activity of GSK3α/β was reduced by GSK3α/β inhibitor LiCl. After incubation in TCM, p-GSK3α(Ser21) and p-Tyr proteins levels were increased, and which was higher than those of spermatozoa incubated in TBM. In mammalian spermatozoa, capacitive Ca²⁺ entry occurs during capacitation [18]. Based on these findings, increase in intracellular Ca²⁺ can stimulate inhibitory phosphorylation of GSK3α, thereby potentiating the sperm motility during capacitation. The kinase activity of GSK3α/β is governed by inhibitory phosphorylation on its serine residues, changes in substrate accessibility, and recognition through the phosphorylation of its tyrosine residues [4]. In bovine spermatozoa, the inhibitory phosphorylation of GSK3α was negatively coupled with GSK3α/β kinase activity [10]. In gel-shift assay, the GSK3α/β kinase activity of human spermatozoa incubated in TCM was lower than that of spermatozoa incubated in TBM, suggesting that Ca²⁺ can increase inhibitory phosphorylation of GSK3α, decreasing the kinase activity of GSK3α. In low-motility spermatozoa, the basal kinase activity of GSK3α/β and the ratio of p-GSK3α(Ser21)/GSK3α were lower than those of high-motility spermatozoa. Therefore, the low level of basal kinase activity of GSK3α in low-motility spermatozoa might be attributable to low GSK3α level. Following incubation in TCM, the kinase activity of GSK3α/β decreased together with an increase in p-GSK3α(Ser21) in high-motility spermatozoa, indicating that decreased kinase activity of GSK3α/β is coupled with inhibitory phosphorylation of GSK3α(Ser21). Of note, BIO, a specific inhibitor of kinase activity of GSK3 increased p-GSK3α(Ser21) and p-Tyr proteins levels, suggesting the tight coupling between kinase activity and inhibitory phosphorylation of GSK3α in spermatozoa. Similarly, in mouse proximal tubular cells, p-GSK3β(Ser9) was increased by BIO [19]. By contrast, in low-motility spermatozoa, the kinase activity of GSK3α/β increased without a visible change in p-GSK3α(Ser21) after incubation in TCM, indicative of decoupling of the kinase activity and inhibitory phosphorylation of GSK3α(Ser21). Importantly, in high-motility spermatozoa, p-GSK3α(Ser21) but p-GSK3α(Tyr279) was increased after incubation in TCM, suggesting that inhibitory phosphorylation of the Ser21 residue is the prime mechanism by which GSK3α activates sperm motility during capacitation.

3. Regulation of the inhibitory phosphorylation of GSK3α by upstream kinases

The PI3K–Akt and cAMP–PKA signaling pathways pivotal for capacitation have been known to be coupled with inhibitory phosphorylation of GSK3α/β in various cell types [20,21]. In human spermatozoa p-GSK3α(Ser21) and p-Tyr protein levels were increased by 8-br-cAMP but decreased by PKA inhibitor H89. This suggests that PKA promotes the inhibitory phosphorylation of GSK3α, leading to capacitation of in human spermatozoa. Akt inhibitor VIII decreased p-GSK3α(Ser21) and p-Tyr protein levels in human spermatozoa. This suggests that Akt may promote the inhibitory phosphorylation of GSK3α, leading to sperm capacitation. In support of this idea, GSK is a phosphorylation target of Akt in human smooth muscle and embryonic stem cells [22,23]. In human spermatozoa, phosphorylation of Ser21 residue of GSK3α and p-Tyr proteins were sensitive to Akt inhibitor VIII and 8-br-cAMP. This suggests that activation of PKA and Akt converges phosphorylation of Ser21 residue of GSK3α, stimulating the sperm motility. Together, low levels of p-GSK3α(Ser21) in low-motility human spermatozoa below the WHO LRL might be attributable to deregulation of upstream protein kinases such as PKA and Akt.

4. Regulation of the inhibitory phosphorylation of GSK3α by BIO and PP1

Calyculin A, a PP1/2A inhibitor, markedly increased the levels of both p-GSK3α(Ser21) and p-Tyr proteins, and significantly increased the motility of low-motility human spermatozoa. This also suggests that a decreased inhibitory phosphorylation of GSK3α might be attributable to activation of PP1 and Ca²⁺/CaM by decreased PP1 inhibitor I-1 activity [24,25]. Given that GSK3 inhibits PP1 inhibitor I-2 by phosphorylation [20], increased p-GSK3α(Ser21) may be attributable to increased I-2 activity and decreased PP1 activity. Together, activation of PP1 via Ca²⁺ entry can stimulate dephosphorylation of p-GSK3α(Ser21), compromising motility of human spermatozoa. Specific small-molecule inhibitors of PP1, preserving inhibitory phosphorylation of GSK3α, could be considered for therapeutic treatment of low motility sperm.

CONCLUSIONS

In human spermatozoa, regulatory circuits crucial for sperm capacitation tightly control the kinase activity of GSK3α via inhibitory phosphorylation, leading to motility activation. Specific small-molecule inhibitors of GSK3α and PP1, preserving inhibitory phosphorylation of GSK3α, could be considered for therapeutic treatment of low motility sperm (Fig. 7).

Fig. 7. Regulation of glycogen synthase kinase 3 (GSK3) in human spermatozoa. In human spermatozoa, inhibitory phosphorylation of GSK3α is tightly regulated by Ca²⁺, protein phosphatase-1 (PP1), PKA, and Akt, leading to capacitation and motility activation. Small-molecule inhibitors of GSK3 and PP1 can be considered as a potential therapeutic drug for treatment of low sperm motility in human.

Data Sharing Statement

The data analyzed for this study have been deposited in HARVARD Dataverse and are available at https://dataverse.harvard.edu/dataverse/wjmh.

Supplementary Materials

Supplementary materials can be found via https://doi.org/10.5534/wjmh.230004.

Supplement Fig. 1

Expression of GSK3α and GSK3β in mouse tissues and spermatozoa. To verify antibody specificities, mouse brain, testis, and spermatozoa lysates were subjected to western blot analysis. GSK3 isoforms in mouse brain, testis, and spermatozoa were detected by p-GSK3α/β(Ser21/9) and GSK3α/β antibodies. GSK3β levels in brain were higher than those in testis or spermatozoa whereas GSK3α levels in testis or spermatozoa were higher than those in brain. GSK3: glycogen synthase kinase 3.

Supplement Fig. 2

Full uncropped western blot images used detection of p-GSK3α(Ser21) and GSK3α/β in human spermatozoa. GSK3: glycogen synthase kinase 3.

Supplement Fig. 3

Expression of p-GSK3α(Ser21) and GSK3α/β in human spermatozoa with various motility. GSK3: glycogen synthase kinase 3.

Supplement Fig. 4

Effects of Calyculin A on p-GSK3(Ser21) and phosphotyrosine (p-Tyr) proteins in high-motility human spermatozoa by Calyculin A. Western blots of p-GSK3α(Ser21) and p-Tyr proteins in high-motility human spermatozoa following incubation with Calyculin A (10 nM) in Tyrode’s complete medium. Calyculin A markedly increased the levels of p-GSK3α(Ser21) and p-Tyr proteins in high-motility spermatozoa. GSK3: glycogen synthase kinase 3.

Supplement Table 1

The details for human spermatozoa samples

Supplement Table 2

The quantification data of the western blot in Fig. 2B

Supplement Table 3

The quantification data of the western blot in Fig. 4C