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. 2024 Apr;42(2):373-383.
doi: 10.5534/wjmh.230004. Epub 2023 Aug 9.

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

Seung Hyun Park #  1 , Young-Pil Kim #  1 , Jeong Min Lee  1 , Dong-Wook Park  2 , Ju Tae Seo  3 , Myung Chan Gye  4
Affiliations

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

Seung Hyun Park et al. World J Mens Health. 2024 Apr.

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.

Keywords: Glycogen synthase kinase 3; Humans; Phosphorylation; Sperm motility.

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Conflict of interest statement

The authors have nothing to disclose.

Figures

Fig. 1
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
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.
Fig. 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
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).
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.
Fig. 6
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.
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.
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