Roles of glycogen synthase kinase 3α in the regulation of mouse sperm motility

다국어 초록 (Multilingual Abstract)

Seung Hyun Park Department of Life Science The Graduate School Hanyang University Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase involved in various signaling pathways. Phosphorylation of serine and tyrosine residues of GSK3 inhibits and activates kinase activity, respectively. Among the two isoforms of GSK3α and β, GSK3α is the predominant isoform in spermatozoa and is known to play a crucial role in the regulation of sperm motility. However, the downstream pathway of GSK3α in spermatozoa is poorly understood. In somatic cells, GSK3β has been known to regulates mitochondrial activity via interaction with the cyclophilin D (CypD) which is a mitochondrial matrix protein and a key regulator of the mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential and ATP production. Dibutyl phthalate (DBP), endocrine-disrupting chemical found in plastics and various daily products is known to cause reproductive toxicity. However, the toxicity mechanisms of DBP in spermatozoa poorly understood. In this study, the role of CypD, as a downstream target of GSK3α was examined during sperm maturation and capacitation. In addition, involvement of GSK3α in the toxicity mechanism of DBP was investigated. In epididymal spermatozoa, the levels of CypD and GSK3α were increased during epididymal maturation. CypD and GSK3α were abundant in the caput epididymal exosomes but not in the cauda epididymal exosomes, suggesting the transfer of CypD and GSK3α to spermatozoa via exosome during sperm maturation in epididymis. During capacitation, both CypD and GSK3α levels decreased in spermatozoa. In the mitochondria of spermatozoa CypD and GSK3α directly interact. Cyclosporin A, CypD inhibitor increased sperm motility, mPTP closing, mitochondrial membrane potential, and ATP production. BIO, GSK3 inhibitor decreased CypD and increased mPTP closing, mitochondrial membrane potential, and ATP production. Of note, degradation of CypD was attenuated by proteasome inhibitor MG115. Therefore, GSK3α may mediate proteasomal degradation of CypD, and which is involved in sperm motility activation. In vitro exposure to DBP decreased sperm motility and increased bent tails. DBP altered intracellular Ca2+ levels and pH in spermatozoa. DBP increased ROS generation and lipid peroxidation, suggesting that DBP-induced oxidative stress in spermatozoa. DBP up-regulated phosphorylated PKA substrates and phosphotyrosine proteins in a dose-dependent manner. DBP decreased mitochondrial activity. DBP stimulated spontaneous acrosome reaction, potentially interrupting fertilization. DBP decreased serine phosphorylation of GSK3α and increased tyrosine phosphorylation and phosphatase activity, suggesting up-regulated kinase activity of GSK3α. Calyculin A, protein phosphatases 1 and 2A inhibitor increased inhibitory phosphorylation of GSK3α and sperm motility in DBP-treated spermatozoa. DBP increased the ubiquitination of sperm proteins including GSK3α and the degradation of GSK3α, which was attenuated by the proteasome inhibitor MG115. Together, DBP may decrease sperm motility by altering the intracellular signaling and decrease the inhibitory phosphorylation of GSK3α. In conclusion, GSK3α plays a critical role in the regulation of sperm motility via interaction with CypD. GSK3α is an important target associated with DBP toxicity in spermatozoa. BACKGROUND AND OBJECTIVE Metabolism for energy production is the most important and essential process for living cells. For this reason, various signal pathway in cells for metabolism has been studied. Glucose regulation, which is usually regulated by insulin, is based on biochemical action in cells (Ralston, 2002). Glycogen synthase kinase 3 (GSK3) is well-known serine/threonine kinase and which is participates in glucose regulation. In mammals, GSK3 encoded by two genes that translated by GSK3α and GSK3β (Frame and Cohen, 2001). The kinase activity of GSK3 is regulated via phosphorylation on its serine 21 and 9 residue of GSK3α and GSK3β, respectively (Beurel et al., 2015). In contrast, auto phosphorylation on tyrosine 279 and 216 residue of GSK3αand GSK3β increases its kinase activity (Medina and Wandosell, 2011; Wang et al., 1994). As shown in Fig. I, various factors participate in regulating the activity of GSK3 through serine and tyrosine phosphorylation. (Martelli et al., 2022).

목차 (Table of Contents)

• CONTENTS · 4
• LIST OF FIGURES · 10
• ABSTRACT · 13
• BACKGROUND AND OBJECTIVE · 16
• Reference · 25
• Chapter 1. Changes in glycogen synthase kinase 3α and cyclophilin D in mouse spermatozoa during maturation. · 31
• Abstract · 31
• 1.1. Introduction · 32
• 1.2. Materials and Methods · 33
• 1.2.1. Ethics statement · 33
• 1.2.2. Sperm preparation and incubation · 33
• 1.2.3. Immunoblotting · 34
• 1.2.4. Immunocytochemistry · 34
• 1.2.5. Immunofluorescence · 35
• 1.2.6. Epididymal exosome isolation · 36
• 1.2.7. Statistical anlysis · 36
• 1.3. Results · 37
• 1.3.1. Expression of GSK3 in epididymal mouse spermatozoa · 38
• 1.3.2. Expression of CypD in epididymal mouse spermatozoa · 40
• 1.3.3. Expression of CypD and GSK3 in caput and cauda epididymal exosome · 42
• 1.3.4. Expression of CypD in epididymis · 44
• 1.4. Disucussion · 46
• 1.4.1. Differential expression and phosphorylation of GSK3 isoforms in epididymal spermatozoa · 46
• 1.4.2. Change in CypD in epididymal spermatozoa during maturation · 47
• 1.4.3. Transfer of CypD to epididymal spermatozoa during sperm maturation · 48
• 1.5. Conclusion · 50
• 1.6. References · 52
• Chapter 2. Inhibition of mitochondrial cyclophilin D, a downstream target of glycogen synthase kinase 3α, improves sperm motility. · 58
• Abstract · 58
• 2.1. Introduction · 60
• 2.2. Materials and Methods · 61
• 2.2.1. Ethic statement · 61
• 2.2.2. Sperm preparation and incubation · 61
• 2.2.3. Immunoblotting · 62
• 2.2.4. Immunocytochemistry · 62
• 2.2.5. Live mitochondria isolation · 63
• 2.2.6. Co-immunoprecipitation assay · 63
• 2.2.7. Computer-assisted sperm analysis · 64
• 2.2.8. Mitochondrial membrane potential assay · 64
• 2.2.9. Measurement of mPTP opening · 65
• 2.2.10. ATP assay · 65
• 2.2.11. Statistical analysis · 66
• 2.3. Results · 67
• 2.3.1. Expression of CypD and GSK3 in spermatozoa · 67
• 2.3.2. Co-immunoprecipitation of CypD with GSK3α ·71
• 2.3.3. Change in sperm motility, phosphotyrosine proteins, mPTP opening, MMP, p-GSK3α(Ser21), and ATP production by cyclosporin A, a CypD inhibitor · 73
• 2.3.4. Change in p-GSK3α(Ser21), motility, CypD, mPTP opening, MMP, and ATP production by BIO, a GSK3 inhibitor · 76
• 2.3.5. Changes in CypD level and sperm motility after MG 115 treatment · 79
• 2.4. Disucussion · 81
• 2.4.1. Changes in CypD and GSK3α in spermatozoa during capacitation · 81
• 2.4.2. Inhibition of CypD stimulates sperm motility · 82
• 2.4.3. Role of GSK3α as a regulator of CypD in spermatozoa · 83
• 2.5. Conclusion · 85
• 2.6. References · 87
• Chapter 3. Dibutyl phthalate disrupts [Ca2+]i, reactive oxygen species, [pH]i, protein kinases and mitochondrial activity, impairing sperm function. · 91
• Abstract · 91
• 3.1. Introduction · 93
• 3.2. Materials and Methods · 95
• 3.2.1. Chemicals · 95
• 3.2.2. Animals · 95
• 3.2.3. Sperm collection and chemical treatment · 96
• 3.2.4. Measurement of intracellular Ca2+ · 97
• 3.2.5. Measurement of intracellular pH · 97
• 3.2.6. Sperm morphology and motility examination · 98
• 3.2.7. Measurement of ROS · 98
• 3.2.8. Measurement of lipid hydroperoxide in spermatozoa · 99
• 3.2.9. Measurement of mPTP opening · 99
• 3.2.10. Measurement of mitochondrial membrane potential · 100
• 3.2.11. Measurement of ATP · 101
• 3.2.12. Immunoblotting · 102
• 3.2.13. Acrosome labeling · 102
• 3.2.14. Statistical analysis · 103
• 3.3. Results · 104
• 3.3.1. Effects of DBP on [Ca2+]i, [pH]i, motility, and tail bending in spermatozoa · 104
• 3.3.2. Effects of DBP on ROS generation, lipid peroxidation, mPTP opening, MMP, and ATP production in spermatozoa · 108
• 3.3.3. Effects of DBP on phosphotyrosine proteins and phosphorylated PKA substrate proteins · 111
• 3.3.4. Effects of DBP on spontaneous acrosome reaction of spermatozoa · 113
• 3.4. Disucussion · 115
• 3.5. Conclusion · 119
• 3.6. References · 121
• Chapter 4. Dibutyl phthalate impaired protein stability and inhibitory phosphorylation of glycogen synthase kinase 3α crucial for sperm motility. · 129
• Abstract · 129
• 4.1. Introduction · 130
• 4.2. Materials and Methods · 132
• 4.2.1. Chemicals · 132
• 4.2.2. Sperm preparation and chemical treatment · 132
• 4.2.3. Immunoblotting · 133
• 4.2.4. Immunocytochemistry of p-GSK3α(Ser21) and GSK3α in spermatozoa · 134
• 4.2.5. Alkaline phosphatase activity assay · 135
• 4.2.6. Immunoprecipitation (IP) of Poly-Ubiquitinated GSK3α · 136
• 4.2.7. Statistical analysis · 137
• 4.3. Results · 138
• 4.3.1. DBP decreased cellular GSK3α, serine phosphorylation of GSK3α, and sperm motility · 138
• 4.3.2. Effects of DBP on phosphatase activity in spermatozoa · 140
• 4.3.3. Effects of calyculin A on inhibitory phosphorylation of GSK3α and sperm motility in DBP-treated spermatozoa · 142
• 4.3.4. DBP increased poly-ubiquitinated proteins and poly-ubiquitination of GSK3α · 144
• 4.3.5. Effects of the proteasome inhibitor MG115 on the stability and inhibitory phosphorylation of GSK3α and motility of DBP-treated spermatozoa · 147
• 4.4. Disucussion · 149
• 4.4.1. DBP increased ROS and phospho-tyrosine proteins but decreased motility in spermatozoa · 149
• 4.4.2. DBP decreased the serine phosphorylation of GSK3α in spermatozoa · 150
• 4.4.3. DBP increased the activity of protein phosphatases, decreasing the serine phosphorylation of GSK3α in spermatozoa · 151
• 4.4.4. DBP increased polyubiquitination of proteins including GSK3α in spermatozoa · 152
• 4.5. Conclusion · 153
• 4.6. References · 155
• ABSTRACT IN KOREAN · 167
• ACKNOWLEDGEMENT · 169