Department of Medical Research, Mackay Memorial Hospital, Tamshui, New Taipei City, Taiwan.
J Cell Biochem. 2013 Apr;114(4):888-98. doi: 10.1002/jcb.24428.
SPINKL, a serine protease inhibitor kazal-type-like protein initially found in mouse seminal vesicle secretions, possesses structurally conserved six-cysteine residues of the kazal-type serine protease inhibitor family. However, it has no inhibitory activity against serine proteases. Previously, it was found to have the ability to suppress murine sperm capacitation in vitro. Herein, we investigated the mechanisms underlying the suppressive effect of SPINKL on sperm capacitation. Three in vitro capacitation-enhancing agents, including bovine serum albumin (BSA), methyl-beta-cyclodextrin (MBCD), and dibutyryl cyclic AMP (dbcAMP), coupled with 3-isobutyl-1-methylxanthine (IBMX), were used to evaluate the influence of SPINKL on capacitation signaling. Preincubation of sperm with SPINKL suppressed BSA- and MBCD-induced sperm capacitation by blocking three upstream signals of capacitation that is the cholesterol efflux from sperm plasma membranes, extracellular calcium ion influx into sperm, and increases in intracellular cAMP. Moreover, SPINKL also inhibited downstream signal transduction of capacitation since it suppressed dbcAMP/IBMX and N(6) -phenyl cAMP (6-Phe-cAMP)-activated cAMP-dependent protein kinase-associated protein tyrosine phosphorylation. Such inhibition is probably mediated by attenuation of SRC tyrosine kinase activity. Furthermore, SPINKL could not reverse capacitation once sperm had been capacitated by capacitation-enhancing agents or capacitated in vivo in the oviduct. SPINKL bound to sperm existed in the uterus but had disappeared from sperm in the oviduct during the sperm's transit through the female reproductive tract. Therefore, SPINKL may serve as an uncapacitation factor in the uterus to prevent sperm from precocious capacitation and the subsequent acrosome reaction and thus preserve the fertilization ability of sperm.
SPINKL 是一种丝氨酸蛋白酶抑制剂 Kazal 型类似蛋白，最初在小鼠精囊分泌物中发现，具有 Kazal 型丝氨酸蛋白酶抑制剂家族结构保守的六个半胱氨酸残基。然而，它对丝氨酸蛋白酶没有抑制活性。先前发现它具有抑制体外小鼠精子获能的能力。在此，我们研究了 SPINKL 抑制精子获能的机制。使用三种体外获能增强剂，包括牛血清白蛋白（BSA）、甲基-β-环糊精（MBCD）和二丁酰环 AMP（dbcAMP），结合 3-异丁基-1-甲基黄嘌呤（IBMX），评估 SPINKL 对获能信号的影响。SPINKL 与精子预孵育可通过阻断获能的三个上游信号来抑制 BSA 和 MBCD 诱导的精子获能，这三个上游信号是精子质膜中的胆固醇流出、胞外钙离子内流入精子和细胞内 cAMP 增加。此外，SPINKL 还抑制了获能的下游信号转导，因为它抑制了 dbcAMP/IBMX 和 N(6)-苯环 cAMP（6-Phe-cAMP）激活的 cAMP 依赖性蛋白激酶相关蛋白酪氨酸磷酸化。这种抑制可能是通过减弱 SRC 酪氨酸激酶活性介导的。此外，一旦精子被获能增强剂获能或在输卵管中体内获能，SPINKL 就不能逆转获能。SPINKL 结合的精子存在于子宫中，但在精子通过雌性生殖道的过程中从输卵管中的精子中消失。因此，SPINKL 可能作为子宫中的一种非获能因子，防止精子过早获能和随后的顶体反应，从而保持精子的受精能力。
