小鼠精子透明质酸酶SPAM1在受精过程中的功能研究

doi:10.11843/j.issn.0366-6964.2017.04.008

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (4): 652-659.doi: 10.11843/j.issn.0366-6964.2017.04.008

小鼠精子透明质酸酶SPAM1在受精过程中的功能研究

周崇^1,3#*, 黄莉^2,3#, 石德顺¹, 蒋建荣¹, 马场忠³

1. 广西大学, 亚热带生物资源保护利用国家重点实验室, 南宁 530004;
2. 广西壮族自治区兽医研究所, 广西兽医生物技术重点实验室, 南宁 530001;
3. 日本筑波大学, 生命环境系, 筑波 305-8572

收稿日期:2016-10-31 出版日期:2017-04-23 发布日期:2017-04-23
通讯作者: 周崇，E-mail：czhou720@126.com
作者简介:周崇（1976-），男，广西南宁人，博士，副教授，主要从事动物生殖生理学研究；黄莉（1978-），女，广西南宁人，博士，副研究员，主要从事动物病毒学研究，E-mail：lhuang405@126.com
基金资助:
广西自然科学基金（2014GXNSFCA118008）

Functional Analysis of Mouse Sperm Hyaluronidase SPAM1 in Fertilization

ZHOU Chong^1,3#*, HUANG Li^2,3#, SHI De-shun¹, JIANG Jian-rong¹, BABA Tadashi³

1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, China;
2. Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China;
3. Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City 305-8572, Japan

Received:2016-10-31 Online:2017-04-23 Published:2017-04-23

摘要/Abstract

摘要：

旨在研究小鼠精子透明质酸酶SPAM1（Sperm adhesion molecule 1）对受精过程中精子/卵丘互作的影响，并初步探讨其可能的作用机制。本研究抽提小鼠尾尖基因组，利用PCR法检测小鼠Spam基因型；筛选的野生型（WT）和Spam1敲除（KO）小鼠，提取附睾尾部精子蛋白进行Western blot和酶活性检测；经TYH培养液2 h获能后，分别对精子的运动性、穿透和分散卵丘细胞能力及体外受精（IVF）进行统计分析。结果表明，KO小鼠精子中未检测到SPAM1蛋白，透明质酸酶活性也极显著低于WT小鼠（P<0.01）；而获能后精子运动性，在KO和WT小鼠之间差异不显著（P>0.05）；与WT相比，KO小鼠精子缺失Spam1后，极显著地影响卵丘细胞层基质中精子顶体反应的发生比率（P<0.01），导致精子穿透卵丘细胞层的能力极显著降低（P<0.01），仅有少数精子能够到达卵子透明带表面，大量精子极易黏附于卵丘细胞层表面或外部边缘（P<0.01）；此外，KO小鼠精子IVF 2 h的卵丘细胞分散和受精率均呈现显著延迟（P<0.05）。综上表明，小鼠精子透明质酸酶SPAM1与顶体反应相关联并影响精子/卵丘互作。揭示SPAM1在穿卵过程中除了具有降解透明质酸的作用外，还存在其他的非酶活性功能。

Abstract:

This study was aimed to elucidate the possible functions of sperm adhesion molecule 1 (SPAM1) on the sperm/cumulus interaction during fertilization and explore its potential mechanism. Genomic DNA was extracted from the tails of mice and genotype for Spam1 null mutation was confirmed by PCR analysis. The protein of the screened wild type (WT) and Spam1-deficient (KO) sperm were extracted and confirmed by Western blot, and the hyaluronidase activity was measured by the colorimetric method. The epididymal sperm were capacitated by incubation for 2 h and subjected to the assays of sperm motility, sperm/cumulus interaction or in vitro fertilization (IVF). These results showed that the SPAM1 protein was readily absent in the KO sperm, and the hyaluronic acid-hydrolyzing enzymes activity was significantly reduced in the KO sperm, compared to that of the WT sperm (P<0.01). There were no significant differences in sperm motility between the KO and WT groups after capacitation (P>0.05). However, the loss of SPAM1 significantly influenced the percentage of acrosome-reacted sperm in the extracellular matrix of the cumulus (P<0.01), resulted in a markedly decrease in sperm entry into and/or penetration of the cumulus mass, only a few sperm succeeded in access to the surface of the oocyte zona pellucida (ZP) and contrastly a remarkably great deal of sperm accumulated on the surface or outer edge of the cumulus (P<0.01). In addition, sperm lacking SPAM1 caused the delayed cumulus disperse and fertilization at IVF 2 h (P<0.05). Therefore, mouse sperm hyaluronidase SPAM1 may be associated with the acrosome reaction and affect the sperm/cumulus interaction, which reveal that SPAM1 may play other possible roles in sperm access to the oocyte ZP except the hyaluronan-degrading activity.

中图分类号:

Q954.44

周崇, 黄莉, 石德顺, 蒋建荣, 马场忠. 小鼠精子透明质酸酶SPAM1在受精过程中的功能研究[J]. 畜牧兽医学报, 2017, 48(4): 652-659.

ZHOU Chong, HUANG Li, SHI De-shun, JIANG Jian-rong, BABA Tadashi. Functional Analysis of Mouse Sperm Hyaluronidase SPAM1 in Fertilization[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(4): 652-659.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2017.04.008

https://www.xmsyxb.com/CN/Y2017/V48/I4/652

参考文献

[1] KNOBIL E, NEILL J D. The physiology of reproduction[M]. 2nd ed. New York:Raven Press, 1994:189-317.
[2] WASSARMAN P M. Mammalian fertilization:molecular aspects of gamete adhesion, exocytosis, and fusion[J]. Cell, 1999, 96(2):175-183.
[3] WASSARMAN P M, JOVINE L, LITSCHER E S. A profile of fertilization in mammals[J]. Nat Cell Biol, 2001, 3(2):E59-E64.
[4] PRIMAKOFF P, MYLES D G. Penetration, adhesion, and fusion in mammalian sperm-egg interaction[J]. Science, 2002,296(5576):2183-2185.
[5] IKAWA M, INOUE N, BENHAM A M, et al. Fertilization:a sperm's journey to and interaction with the oocyte[J]. J Clin Invest, 2010, 120(4):984-994.
[6] VANSOFLA F N, ROSHANGAR L, MONTASERI A, et al. Impact of prunus cerasus on PGR and HAS2 in cumulus cells and fertility outcome[J].Adv Pharm Bull,2016, 6(1):65-69.
[7] NISHIMURA H, KIM E, NAKANISHI T, et al. Possible function of the ADAM1a/ADAM2 fertilin complex in the appearance of ADAM3 on the sperm surface[J]. J Biol Chem, 2004, 279(33):34957-34962.
[8] FATEHI A N, ZEINSTRA E C, KOOIJ R V, et al. Effect of cumulus cell removal of in vitro matured bovine oocytes prior to in vitro fertilization on subsequent cleavage rate[J]. Theriogenolngy, 2002, 57(4):1347-1355.
[9] LI R, NORMAN G R, ARMSTRONG D T,et al.Oocyte-secreted factor(s) determine functional differences between bovine mural granulosa cells and cumulus cells[J]. Biol Reprod, 2000, 63(3):839-845.
[10] ZHANG H,TIAN S,KLAUSEN C,et al.Differential activation of noncanonical SMAD2/SMAD3 signaling by bone morphogenetic proteins causes disproportionate induction of hyaluronan production in immortalized human granulosa cells[J]. Mol Cell Endocrinol, 2016, 428:17-27.
[11] CHEN X Y,BONFIGLIO R,BANERJI S,et al. Micromechanical analysis of the hyaluronan-rich matrix surrounding the oocyte reveals a uniquely soft and elastic composition[J]. Biophys J, 2016, 110(12):2779-2789.
[12] BRIGGS D C, BIRCHENOUGH H L, ALI T, et al.Metal Ion-dependent heavy chain transfer activity of TSG-6 mediates assembly of the cumulus-oocyte matrix[J].J Biol Chem, 2015, 290(48):28708-28723.
[13] SANTOS J D R,BATISTA R I T P,MAGALHÃES L C,et al.Overexpression of hyaluronan synthase 2 and gonadotropin receptors in cumulus cells of goats subjected to one-shot eCG/FSH hormonal treatment for ovarian stimulation[J]. Anim Reprod Sci, 2016, 170:15-24.
[14] RICHARDS J S. Ovulation:new factors that prepare the oocyte for fertilization[J]. Mol Cell Endocrinol, 2005, 234(1-2):75-79.
[15] PRIMAKOFF P, HYATT H, MYLES D G. A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida[J]. J Cell Biol, 1985, 101(6):2239-2244.
[16] MODELSKI M J,MENLAH G,WANG Y,et al.Hyaluronidase 2:a novel germ cell hyaluronidase with epididymal expression and functional roles in mammalian sperm[J].Biol Reprod, 2014,91(5):109.
[17] KIM E,BABA D,KIMURA M,et al. Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass[J]. Proc Natl Acad Sci USA, 2005, 102(50):18028-18033.
[18] BABA D,KASHIWABARA S I,HONDA A,et al.Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg[J]. J Biol Chem, 2002, 277(33):30310-30314.
[19] KIMURA M,KIM E,KANG W,et al.Functional roles of mouse sperm hyaluronidases, HYAL5 and SPAM1, in fertilization[J]. Biol Reprod, 2009, 81(5):939-947.
[20] LIN Y,MAHAN K,LATHROP W F, et al. A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg[J]. J Cell Biol, 1994, 125(5):1157-1163.
[21] MYLES D G, PRIMAKOFF P. Why did the sperm cross the cumulus? To get to the oocyte. Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg[J]. Biol Reprod, 1997, 56(2):320-327.
[22] YOON S,CHANG K T,CHO H,et al.Characterization of pig sperm hyaluronidase and improvement of the digestibility of cumulus cell mass by recombinant pSPAM1 hyaluronidase in an in vitro fertilization assay[J]. Anim Reprod Sci, 2014, 150(3-4):107-114.
[23] ZHOU C, KANG W, BABA T. Functional characterization of double-knockout mouse sperm lacking SPAM1 and ACR or SPAM1 and PRSS21 in fertilization[J]. J Reprod Dev, 2012, 58(3):330-337.
[24] YAMASHITA M, HONDA A, OGURA A, et al. Reduced fertility of mouse epididymal sperm lacking Prss21/Tesp5 is rescued by sperm exposure to uterine microenvironment[J].Genes Cells, 2008, 13(10):1001-1013.
[25] IKAWA M, TOKUHIRO K, YAMAGUCHI R, et al. Calsperin is a testis-specific chaperone required for sperm fertility[J]. J Biol Chem, 2011, 286(7):5639-5646.
[26] KANG W J, ZHOU C, KOGA Y, et al. Hyaluronan-degrading activity of mouse sperm hyaluronidase is not required for fertilization?[J].J Reprod Dev, 2010, 56(1):140-144.
[27] DURAN-REYNALS F. Tissue permeability and the spreading factors in infection:A contribution to the host:parasite problem[J]. Bacteriol Rev, 1942, 6(4):197-252.
[28] OREN-BENAROYA R, ORVIETO R, GAKAMSKY A, et al. The sperm chemoattractant secreted from human cumulus cells is progesterone[J].Hum Reprod, 2008, 23(10):2339-2345.
[29] LISHKO P V, BOTCHKINA I L, KIRICHOK Y. Progesterone activates the principal Ca²⁺ channel of human sperm[J]. Nature, 2011, 471(7338):387-391.
[30] SHIMADA M,YANAI Y,OKAZAKI T,et al.Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization[J]. Development, 2008, 135(11):2001-2011.
[31] JIN M, FUJIWARA E, KAKIUCHI Y, et al. Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization[J]. Proc Natl Acad Sci U S A, 2011, 108(12):4892-4896.
[32] INOUE N,SATOUH Y,IKAWA M,et al.Acrosome-reacted mouse spermatozoa recovered from the perivitelline space can fertilize other eggs[J]. Proc Natl Acad Sci U S A, 2011, 108(50):20008-20011.
[33] INOUE N,IKAWA M,NAKANISHI T,et al.Disruption of mouse CD46 causes an accelerated spontaneous acrosome reaction in sperm[J]. Mol Cell Biol, 2003, 23(7):2614-2622.

小鼠精子透明质酸酶SPAM1在受精过程中的功能研究

Functional Analysis of Mouse Sperm Hyaluronidase SPAM1 in Fertilization

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics

本文评价