PP1γ2对树鼩精子成熟和运动性的调控作用研究
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摘要
为了阐明树鼩精子中是否存在丝氨酸/苏氨酸蛋白磷酸酶1γ2(PP1γ2)及其在附睾精子中的存在形式,进而探究PP1γ2对精子成熟和运动性的调控作用,本试验以树鼩为研究对象,采用Western blotting分析了不同条件下树鼩附睾头和附睾尾精子中PP1γ2的存在形式和磷酸化程度,探讨了双丁酰环腺苷酸(db-cAMP)、3-异丁基-1-甲基黄嘌呤(IBMX)或Ca~(2+)对树鼩精子中PP1γ2磷酸化表达水平的影响,并进一步研究了磷酸酶抑制剂冈田酸(okadaic acid,OA)和花萼海绵诱癌素A(calyculin A,CA)对树鼩精子中PP1γ2磷酸化程度的影响及其对树鼩附睾头和附睾尾精子运动度的影响。结果显示,在树鼩附睾头和附睾尾精子中均存在PP1γ2,且在等量的附睾头和附睾尾精子蛋白中,PP1γ2在附睾尾精子的磷酸化程度远高于附睾头精子;db-cAMP、IBMX或Ca~(2+)不改变PP1γ2的磷酸化水平;磷酸酶抑制剂OA和CA能明显提高附睾头和附睾尾中PP1γ2磷酸化的程度,且能显著提高精子(尤其是附睾头精子)的运动度(P<0.05),OA和CA的最佳作用浓度分别为1μmol/L和10 nmol/L,最佳作用时间分别为15、20 min。本研究结果表明,蛋白磷酸酶PP1γ2对树鼩精子成熟及运动性具有重要的调控作用,其主要通过磷酸化和去磷酸化的变化发挥作用。
In order to elucidate whether PP1γ2 is present or not in tree shrews' spermatozoa and its form in epididymal sperm,and further explore the regulation of PP1γ2 on sperm maturation and motility in tree shrews,the present form and phosphorylation levels of PP1γ2 in caput and cauda epididymal spermatozoa under various conditions have been analyzed by Western blotting.The effects of db-cAMP,IBMX or Ca~(2+) on the phosphorylation levels of PP1γ2 in tree shrew sperm were investigated.The effects of okadaic acid(OA) and calyculin A(CA) on the phosphorylation level of PP1γ2 and motility in caput and cauda epididymal spermatozoa were further studied with tree shrews as experimental animals in the present study.The results showed that PP1γ2 was present in caput and cauda epididymal spermatozoa of tree shrew,and the phosphorylation level of PP1γ2 was far higher in cauda epididymal spermatozoa than that of caput spermatozoa.db-cAMP,IBMX or Ca~(2+) did not change the phosphorylation level of PP1γ2,OA or CA could significantly increase not only the phosphorylation level of PP1γ2 in caput and cauda epididymal spermatozoa but also sperm motility(P<0.05),especially for caput epididymal spermatozoa.The optimal concentrations of OA and CA were 1 μmol/L and 10 nmol/L,respectively,and the optimal incubation time were 15 and 20 min,respectively.This study indicated that PP1γ2 played an important role in regulating sperm maturation and motility in tree shrews through phosphorylation and dephosphorylation.
In order to elucidate whether PP1γ2 is present or not in tree shrews' spermatozoa and its form in epididymal sperm,and further explore the regulation of PP1γ2 on sperm maturation and motility in tree shrews,the present form and phosphorylation levels of PP1γ2 in caput and cauda epididymal spermatozoa under various conditions have been analyzed by Western blotting.The effects of db-cAMP,IBMX or Ca~(2+) on the phosphorylation levels of PP1γ2 in tree shrew sperm were investigated.The effects of okadaic acid(OA) and calyculin A(CA) on the phosphorylation level of PP1γ2 and motility in caput and cauda epididymal spermatozoa were further studied with tree shrews as experimental animals in the present study.The results showed that PP1γ2 was present in caput and cauda epididymal spermatozoa of tree shrew,and the phosphorylation level of PP1γ2 was far higher in cauda epididymal spermatozoa than that of caput spermatozoa.db-cAMP,IBMX or Ca~(2+) did not change the phosphorylation level of PP1γ2,OA or CA could significantly increase not only the phosphorylation level of PP1γ2 in caput and cauda epididymal spermatozoa but also sperm motility(P<0.05),especially for caput epididymal spermatozoa.The optimal concentrations of OA and CA were 1 μmol/L and 10 nmol/L,respectively,and the optimal incubation time were 15 and 20 min,respectively.This study indicated that PP1γ2 played an important role in regulating sperm maturation and motility in tree shrews through phosphorylation and dephosphorylation.
引文
[1] 苏傲蕾,秦银鸽,郑禹,等.树鼩的生物学特性研究概述[J].动物医学进展,2014,5(10):115-118.SU A L,QIN Y G,ZHENG Y,et al.Introduction of biological characteristics of tree shrew[J].Progress in Veterinary Medicine,2014,5(10):115-118.(in Chinese)
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[5] 杨增明,孙青原,夏国良.生殖生物学[M].北京:科学出版社,2005.YANG Z M,SUN Q Y,XIA G L.Reproductive biology [M].Beijing:Science Press,2005.(in Chinese)
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[7] DEVI K U,JHA K,SHIVAJI S.Plasma membrane-associated protein tyrosine phosphatase activity in hamster spermatozoa[J].Molecular Reproduction & Development,1999,53(1):42-50.
[8] VISCONTI P E,WESTBROOK V A,CHERTIHIN O,et al.Novel signaling pathways involved in sperm acquisition of fertilizing capacity[J].Journal of Reproductive Immunology,2002,53(2):133-150.
[9] BOLLEN M,PETI W,RAGUSA M J,et al.The extended PP1 toolkit:Designed to create specificity[J].Trends in Biochemical Sciences,2010,35(8):450-458.
[10] COHEN P T.Protein phosphatase 1——Targeted in many directions[J].Journal of Cell Science,2002,115(2):241-256.
[11] FARDILHA M,ESTEVES S L,KORRODI-GREGORIO L,et al.The physiological relevance of protein phosphatase 1 and its interacting proteins to health and disease[J].Current Medicinal Chemistry,2010,17(33):3996-4017.
[12] HEROES E,LESAGE B,GORNEMANN J,et al.The PP1 binding code:A molecular-lego strategy that governs specificity[J].The FEBS Journal,2013,280(2):584-595.
[13] KORRODI-GREGORI L,ESTEVES S L,FARDILHA M.Protein phosphatase 1 catalytic isoforms:Specificity toward interacting proteins[J].Translational Cancer Research,2014,64(5):366-391.
[14] CHAKRABARTI R,CHENG L,PURI P,et al.Protein phosphatase PP1 gamma 2 in sperm morphogenesis and epididymal initiation of sperm motility[J].Asian Journal of Andrology,2007,9(4):445-452.
[15] KITAGAWA Y,SASAKI K,SHIMA H,et al.Protein phosphatases possibly involved in rat spermatogenesis[J].Biochemical and Biophysical Research Communications,1990,171(1):230-235.
[16] SHIMA H,HANEJI T,HATANO Y,et al.Protein phosphatase 1 gamma 2 is associated with nuclei of meiotic cells in rat testis[J].Biochemical and Biophysical Research Communications,1990,194(2):930-937.
[17] SINHA N,PURI P,NAIRN A C,et al.Selective ablation of Ppp1cc gene in testicular germ cells causes oligo-teratozoospermia and infertility in mice[J].Biology of Reproduction,2013,89(5):128.
[18] SMITH G D,WOLF D P,TRAUTMAN K C,et al.Primate sperm contain protein phosphatase 1,a biochemical mediator of motility[J].Biology of Reproduction,1996,54(3):719-727.
[19] VIJAYARAGHAVAN S,CHAKRABARTI R,MYERS K.Regulation of sperm function by protein phosphatase PP1gamma2[J].Society of Reproduction and Fertility Supplement,2007,63:111-121.
[20] VIJAYARAGHAVAN S,STEPHENS D T,TRAUTMAN K,et al.Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity[J].Biology of Reproduction,1996,54(3):709-718.
[21] BAVISTER B D,LEIBFRIED M L,LIEBERMAN G.Development of preimplantation embryos of the golden hamster in a defined culture medium[J].Biology of Reproduction,1983,28(1):235-247.
[22] VISCONTI P E,BAILEY J L,MOORE G D,et al.Capacitation of mouse spermatozoa.Ⅰ.Correlation between the capacitation state and protein tyrosine phosphorylation[J].Development,1995,121(4):1129-1137.
[23] GOSWAMI S,KORRODI-GREGORI L,SINHA N,et al.Regulators of the protein phosphatase PP1γ2,PPP1R2,PPP1R7,and PPP1R11 are involved in epididymal sperm maturation[J].Journal of Cellular Physiology,2019,234(3):3105-3118.
[24] 刘芳,杨明华,朱方方,等.PP1γ2对昆明小鼠精子成熟和运动性的调控作用[J].云南农业大学学报(自然科学版),2018,33(6):1054-1059.LIU F,YANG M H,ZHU F F,et al.Sperm maturation and motility in kunming mice is regulated by protein phosphatase PP1γ2[J].Journal of Yunnan Agricultural University (Natural Science),2018,33(6):1054-1059.(in Chinese)
[25] HUANG Z,VIJAYARAHAVAN S.Increased phosphorylation of a distinct subcellular pool of protein phosphatase,PP1γ2,during epididymal sperm maturation[J].Biology of Reproduction,2012,70(2):439-447.
[26] DUDIKI T,JOUDEH N,SINHA N,et al.The protein phosphatase isoform PP1γ1 substitutes for PP1γ2 to support spermatogenesis but not normal sperm function and fertility[J].Biology of Reproduction.2018.Epub Ahead of Print.
[27] SIGNORELLI J R,DIZA E S,FARA K,et al.Protein phosphatases decrease their activity during capacitation:A new requirement for this event[J].PLoS One,2013,8(12):e81286.
[2] 许凌,范宇,蒋学龙,等.树鼩进化分类地位的分子证据[J].动物学研究,2013,34(2):70-76.XU L,FAN Y,JIANG X L,et al.Molecular evidence on the phylogenetic position of tree shrews[J].Zoological Research,2013,34(2):70-76.(in Chinese)
[3] 徐林,张云,梁斌,等.实验动物树鼩和人类疾病的树鼩模型研究概述[J].动物学研究,2013,34(2):59-69.XU L,ZHANG Y,LIANG B,et al.Tree shrews under the spot light:Emerging model of human diseases[J].Zoological Research,2013,34(2):59-69.(in Chinese)
[4] FAN Y,HUANG Z Y,CAO C C,et al.Genome of the Chinese tree shrew[J].Nature Communications,2013,4:1426.
[5] 杨增明,孙青原,夏国良.生殖生物学[M].北京:科学出版社,2005.YANG Z M,SUN Q Y,XIA G L.Reproductive biology [M].Beijing:Science Press,2005.(in Chinese)
[6] SALICAONI A M,PLATT M D,WERTHEIMER E V,et al.Signalling pathways involved in sperm capacitation[J].Society of Reproduction and Fertility Supplement,2007,65:245-259.
[7] DEVI K U,JHA K,SHIVAJI S.Plasma membrane-associated protein tyrosine phosphatase activity in hamster spermatozoa[J].Molecular Reproduction & Development,1999,53(1):42-50.
[8] VISCONTI P E,WESTBROOK V A,CHERTIHIN O,et al.Novel signaling pathways involved in sperm acquisition of fertilizing capacity[J].Journal of Reproductive Immunology,2002,53(2):133-150.
[9] BOLLEN M,PETI W,RAGUSA M J,et al.The extended PP1 toolkit:Designed to create specificity[J].Trends in Biochemical Sciences,2010,35(8):450-458.
[10] COHEN P T.Protein phosphatase 1——Targeted in many directions[J].Journal of Cell Science,2002,115(2):241-256.
[11] FARDILHA M,ESTEVES S L,KORRODI-GREGORIO L,et al.The physiological relevance of protein phosphatase 1 and its interacting proteins to health and disease[J].Current Medicinal Chemistry,2010,17(33):3996-4017.
[12] HEROES E,LESAGE B,GORNEMANN J,et al.The PP1 binding code:A molecular-lego strategy that governs specificity[J].The FEBS Journal,2013,280(2):584-595.
[13] KORRODI-GREGORI L,ESTEVES S L,FARDILHA M.Protein phosphatase 1 catalytic isoforms:Specificity toward interacting proteins[J].Translational Cancer Research,2014,64(5):366-391.
[14] CHAKRABARTI R,CHENG L,PURI P,et al.Protein phosphatase PP1 gamma 2 in sperm morphogenesis and epididymal initiation of sperm motility[J].Asian Journal of Andrology,2007,9(4):445-452.
[15] KITAGAWA Y,SASAKI K,SHIMA H,et al.Protein phosphatases possibly involved in rat spermatogenesis[J].Biochemical and Biophysical Research Communications,1990,171(1):230-235.
[16] SHIMA H,HANEJI T,HATANO Y,et al.Protein phosphatase 1 gamma 2 is associated with nuclei of meiotic cells in rat testis[J].Biochemical and Biophysical Research Communications,1990,194(2):930-937.
[17] SINHA N,PURI P,NAIRN A C,et al.Selective ablation of Ppp1cc gene in testicular germ cells causes oligo-teratozoospermia and infertility in mice[J].Biology of Reproduction,2013,89(5):128.
[18] SMITH G D,WOLF D P,TRAUTMAN K C,et al.Primate sperm contain protein phosphatase 1,a biochemical mediator of motility[J].Biology of Reproduction,1996,54(3):719-727.
[19] VIJAYARAGHAVAN S,CHAKRABARTI R,MYERS K.Regulation of sperm function by protein phosphatase PP1gamma2[J].Society of Reproduction and Fertility Supplement,2007,63:111-121.
[20] VIJAYARAGHAVAN S,STEPHENS D T,TRAUTMAN K,et al.Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity[J].Biology of Reproduction,1996,54(3):709-718.
[21] BAVISTER B D,LEIBFRIED M L,LIEBERMAN G.Development of preimplantation embryos of the golden hamster in a defined culture medium[J].Biology of Reproduction,1983,28(1):235-247.
[22] VISCONTI P E,BAILEY J L,MOORE G D,et al.Capacitation of mouse spermatozoa.Ⅰ.Correlation between the capacitation state and protein tyrosine phosphorylation[J].Development,1995,121(4):1129-1137.
[23] GOSWAMI S,KORRODI-GREGORI L,SINHA N,et al.Regulators of the protein phosphatase PP1γ2,PPP1R2,PPP1R7,and PPP1R11 are involved in epididymal sperm maturation[J].Journal of Cellular Physiology,2019,234(3):3105-3118.
[24] 刘芳,杨明华,朱方方,等.PP1γ2对昆明小鼠精子成熟和运动性的调控作用[J].云南农业大学学报(自然科学版),2018,33(6):1054-1059.LIU F,YANG M H,ZHU F F,et al.Sperm maturation and motility in kunming mice is regulated by protein phosphatase PP1γ2[J].Journal of Yunnan Agricultural University (Natural Science),2018,33(6):1054-1059.(in Chinese)
[25] HUANG Z,VIJAYARAHAVAN S.Increased phosphorylation of a distinct subcellular pool of protein phosphatase,PP1γ2,during epididymal sperm maturation[J].Biology of Reproduction,2012,70(2):439-447.
[26] DUDIKI T,JOUDEH N,SINHA N,et al.The protein phosphatase isoform PP1γ1 substitutes for PP1γ2 to support spermatogenesis but not normal sperm function and fertility[J].Biology of Reproduction.2018.Epub Ahead of Print.
[27] SIGNORELLI J R,DIZA E S,FARA K,et al.Protein phosphatases decrease their activity during capacitation:A new requirement for this event[J].PLoS One,2013,8(12):e81286.