精子功能相關(guān)的蛋白質(zhì)調(diào)控受精過程的研究進(jìn)展

陳志林，馮美瑩，陳預(yù)明，衛(wèi)恒習(xí)，李莉，吳同山，張守全

1. 華南農(nóng)業(yè)大學(xué)動物科學(xué)學(xué)院，國家生豬種業(yè)工程技術(shù)研究中心，廣州 510642；

2. 東莞市畜牧科學(xué)研究所，東莞 523086

哺乳類動物受精是一個精細(xì)的程序化復(fù)雜過程。受精過程中任何的偏差往往會導(dǎo)致動物的低繁殖力或不育[1]。具有良好受精能力的精子是受精成功的先決條件。精子在睪丸曲精細(xì)管中不斷生成，隨后離開睪丸進(jìn)入到附睪中進(jìn)行成熟。在這一過程中，精子發(fā)生了若干的生理變化，其主要表現(xiàn)為附睪及副性腺分泌的蛋白因子在精子頭部的組裝和附著，精子質(zhì)膜表面蛋白構(gòu)象的改變，向前運(yùn)動能力的獲得等[2]。精子在雌性生殖道里需要依次經(jīng)歷超活化和獲能反應(yīng)，以獲得與卵子受精的能力。獲能的精子需要依次通過子宮與輸卵管接合部、輸卵管壺-峽連接部等屏障才可以與卵子相遇。只有運(yùn)動活力極強(qiáng)的精子才能夠通過雌性生殖道中的這一系列屏障。在精卵相遇后，精子仍需要經(jīng)歷復(fù)雜的過程才能完成受精過程。其中，精卵開始接觸的過程中，精子受卵丘細(xì)胞和透明帶的作用，逐步誘發(fā)了頂體反應(yīng)；反應(yīng)過程釋放多種頂體內(nèi)的水解酶以促進(jìn)精子溶解透明帶；同時，頂體膜與精子質(zhì)膜的融合暴露出精子頭部的卵識別和結(jié)合位點(diǎn)[3]，為隨后的精卵結(jié)合做好準(zhǔn)備；精子溶解透明帶后進(jìn)入到卵周間隙，精子頭部的識別和結(jié)合位點(diǎn)在若干蛋白因子的協(xié)助下，識別和結(jié)合卵膜受體蛋白[4]，從而導(dǎo)致精卵的膜融合(圖1)，并最終形成完整的受精卵。

圖1 受精過程

在整個受精過程中，精子功能相關(guān)的蛋白分子參與其中的信號通路調(diào)控以及一系列的生理生化反應(yīng)。隨著蛋白組學(xué)的發(fā)展和基因敲除技術(shù)的應(yīng)用，哺乳動物精子功能相關(guān)的蛋白質(zhì)不斷涌現(xiàn)。本文主要對精子相關(guān)的信號通路及其他受精過程相關(guān)的精子功能蛋白質(zhì)進(jìn)行總結(jié)，闡述這些蛋白質(zhì)分子與精子運(yùn)動活力、精子獲能、頂體反應(yīng)、透明帶穿入以及精卵融合方面的關(guān)系，并對其在改善哺乳動物繁殖力上的應(yīng)用前景提出展望。

1 精子運(yùn)動活力及獲能反應(yīng)的信號通路

在受精過程中，精子不僅需要在雌性生殖道中游動到與卵子結(jié)合的位點(diǎn)，而且還要獲得與卵子受精的能力，故精子運(yùn)動活力及獲能反應(yīng)是影響受精的兩個關(guān)鍵因素。精子運(yùn)動活力主要由精子尾部的長鞭毛擺動而形成[5]，除了受外界環(huán)境影響外，也受到內(nèi)部若干種信號通路的調(diào)節(jié)。精子獲能是精子獲得與卵子結(jié)合的能力的生理過程，是精子在受精前必須經(jīng)歷的一個重要反應(yīng)。環(huán)腺苷酸(Cyclic adenodine monophosphate, cAMP)-環(huán)腺苷酸依賴性蛋白激酶A(CAMP-dependent protein kinase, cAMPPKA)信號通路和鈣離子信號通路是調(diào)控哺乳動物精子運(yùn)動活力和獲能反應(yīng)的兩個最重要的信號通路，其信號通路調(diào)控模式相對比較清晰[6](圖2)。這兩種信號通路的活化過程分別是由精子細(xì)胞內(nèi)的 HCO3-和Ca2+水平來調(diào)節(jié)[7]。自精子從雄性生殖道射出后，精清富含的HCO3-可以通過Na+/ HCO3-協(xié)同轉(zhuǎn)運(yùn)蛋白(Cotransporter, nbc)進(jìn)入精子細(xì)胞內(nèi)，引起胞內(nèi)的pH改變[8]。與此同時，由精子膜產(chǎn)生的膽固醇外流作用促使膜通透性的增強(qiáng)，HCO3-也因此可以快速進(jìn)入精子細(xì)胞內(nèi)[9]，引起細(xì)胞內(nèi) HCO3-濃度的升高，導(dǎo)致精子胞質(zhì)的堿性化。胞質(zhì)堿性化在刺激精子加速新陳代謝的同時激活了胞內(nèi)的可溶性腺苷酸環(huán)化酶(Soluble adenyl cyclase, sAC)[10]。sAC的活化過程是增強(qiáng)精子運(yùn)動活力和誘發(fā)精子獲能的必要過程。sAC的主要作用是引起細(xì)胞內(nèi)cAMP水平的提高，并致使 PKA的激活[6]。活化的 PKA可以使精子細(xì)胞內(nèi)的蛋白磷酸酶(Protein phosphatases，PPs)活性受到抑制，同時也不斷地激活蛋白酪氨酸激酶(Protein tyrosine kinases, PTK)，使胞內(nèi)蛋白酪氨酸磷酸化作用得到增強(qiáng)。另外，精子內(nèi)PKA的活化還有利于鈣離子通道蛋白(CatSper)的激活，從而達(dá)到誘發(fā)精清中的Ca2+內(nèi)流的效果[11～13]。胞內(nèi)Ca2+濃度的升高不僅可以引起精子細(xì)胞膜的超極化[14]，增強(qiáng)精子鞭毛的運(yùn)動活力[15,16]，也能夠維持sAC的活化程度[6]，從而持續(xù)激活 PTK，增強(qiáng)胞內(nèi)蛋白酪氨酸磷酸化的水平。事實上，PTK的活化能夠促使凝溶膠蛋白(Gelsolin)發(fā)生磷酸化[17]，并使其處于抑制狀態(tài)，進(jìn)而引起肌動蛋白的聚合作用，最后引發(fā)精子發(fā)生超活化反應(yīng)[18,19]。另外，由于PPs去磷酸化活性被抑制以及PTK磷酸化作用的持續(xù)，細(xì)胞內(nèi)的蛋白酪氨酸磷酸化水平不斷提高，從而誘發(fā)精子超活化運(yùn)動和獲能反應(yīng)[20,21]。精子的超活化和獲能不僅直接活化了精子鞭毛軸絲[16]，增強(qiáng)鞭毛運(yùn)動性和尾部側(cè)擺幅度[15,22]，還解除了對精子頂體反應(yīng)的抑制，有利于精卵的結(jié)合。研究表明，胞內(nèi) HCO3-和 Ca2+高濃度水平的狀態(tài)不僅是維持精子超活化運(yùn)動和獲能反應(yīng)的關(guān)鍵[11,23]，也是精子發(fā)生頂體反應(yīng)的重要因素[6]。

圖2 cAMP/PKA信號通路和鈣離子信號通路

2 參與精子運(yùn)動活力調(diào)節(jié)及獲能反應(yīng)的精子功能蛋白

雖然cAMP/PKA信號通路和鈣離子信號通路的具體分子調(diào)控機(jī)理仍不明確，但近年來的研究結(jié)果表明，多種精子和精清蛋白質(zhì)參與這些過程(表 1)。其中鋅 a2糖蛋白(Zn-a2-glycoprotein, ZAG)是調(diào)節(jié)cAMP/PKA通路中的關(guān)鍵因子cAMP的蛋白分子。ZAG抗體能夠顯著地降低精子細(xì)胞內(nèi)的cAMP水平，致使PKA蛋白激酶不能維持活化狀態(tài)，從而抑制精子的超活化，影響精子的運(yùn)動活力[24]。附睪活力抑制因子II (Motility inhibiting factor, MIF-II)[25]和精漿活力抑制因子(Seminal plasma motility inhibitor,SPMI)[26]與 ZAG抗體作用相似，同樣具有降低精子細(xì)胞內(nèi)cAMP濃度的功能，但其具體作用靶點(diǎn)仍不清楚。此外，CatSper是鈣離子信號通路的關(guān)鍵蛋白?；罨?CatSper能夠引起 Ca2+內(nèi)流，誘發(fā)精子發(fā)生超活化反應(yīng)。研究表明，缺失catsper基因的小鼠精子失去超活化的能力以及表現(xiàn)出極弱的運(yùn)動力[27]。另外，小鼠附睪分泌的鈣離子-腺苷三磷酸膜蛋白(Plasma membrane Ca2+-ATPase 4a, PMCA4a)能夠作為一種 Ca2+流泵，影響鈣離子信號通路的激活并維持精子內(nèi)Ca2+的穩(wěn)態(tài)。缺失pmca4基因的精子不能夠發(fā)生超活化和獲能反應(yīng)[28]，因此PMCA4a對精子運(yùn)動活力和受精具有極其重要的作用。

表1 精子運(yùn)動活力及獲能相關(guān)的蛋白質(zhì)

公牛精清蛋白(Bovine seminal plasma, BSP)是與公牛繁殖力相關(guān)的蛋白質(zhì)。BSP可以改變精子膜表面的蛋白構(gòu)象，增強(qiáng)精子膜的流動性，調(diào)節(jié)膜表面的膽固醇外流，從而參與精子獲能的調(diào)節(jié)[29]。研究表明，BSP的主要成分PDC-109是引起膜膽固醇流的主要作用蛋白[30]。PDC-109能夠改變膜的通透性，提高胞內(nèi)的HCO3-和Ca2+-ATPase的濃度，影響精子的運(yùn)動活力以及獲能進(jìn)程[31]。

隨著對受精生物標(biāo)記的深入研究，多種精子活力及獲能相關(guān)的蛋白質(zhì)也隨之被發(fā)現(xiàn)。致密纖維蛋白(Outer dense fiber protein, ODF2)是精子尾部的組成蛋白，對精子尾部的擺動作用十分重要。研究顯示，odf2+/-嵌合體小鼠由于產(chǎn)生運(yùn)動活力極低的精子而重度不育[32]。附睪蛋白酶抑制蛋白(Epididymal protease inhibitor, EPPIN)是一種由睪丸支持細(xì)胞分泌的蛋白，能夠與精囊蛋白相互結(jié)合，抑制精子的活力[33]。Trophinin-binding Peptide是發(fā)現(xiàn)于精子尾部的一種內(nèi)源性蛋白質(zhì)，能夠促進(jìn)精子尾部的擺動，增強(qiáng)精子的直線游動能力[34]。絲氨酸蛋白酶抑制因子(Serine protease inhibitor kazal-type-like SPINKL)是一種由精囊分泌的蛋白因子[35]，能夠抑制精子膜的膽固醇外流，阻礙膜通透性的增強(qiáng)和胞質(zhì)的堿性化，從而抑制精子發(fā)生獲能反應(yīng)[36]。Sp32作為前頂體素的結(jié)合蛋白，發(fā)生磷酸化作用主要是與精子獲能相關(guān)[37]。表皮生長因子受體(Epidermal growth factor receptor, EGFR)獲能時的磷酸化則可能與獲能信號通路相關(guān)，因為EGFR與表皮生長因子(Epidermal growth factor, EGF)結(jié)合后，不僅能夠明顯地降低精子蛋白質(zhì)酪氨酸的磷酸化作用[38]，且抑制了PKA的活化狀態(tài)[9]。此外，酶檸檬酸蛋白酶、延胡索酸水化酶、蘋果酸酶和脫氫酶在公馬精液中存在，其含量與精子運(yùn)動活力和個體的受精能力呈正相關(guān)[39]；公牛精液的骨橋蛋白(Osteopontin, OPN)對于維持精子在母性生殖道中的活力及獲能狀態(tài)具有重要作用，其含量與雄性個體繁殖力呈正相關(guān)[40]。

上述蛋白對精子運(yùn)動活力或獲能都具有重要的作用，但其作用機(jī)理還有待考證。在今后的研究中，通過這些蛋白篩選高繁殖力的個體，檢測這些分子標(biāo)記以判斷是否留種，從而指導(dǎo)畜牧生產(chǎn)育種工作，提高選育工作效率。

3 精子功能蛋白質(zhì)與頂體反應(yīng)的關(guān)系

獲能后的精子在輸卵管中與卵子相遇后，接觸并溶解卵丘細(xì)胞層。在該過程中，精子運(yùn)動活力和透明質(zhì)酸酶的作用十分關(guān)鍵。在自然受精時精子穿越卵丘層的分子機(jī)理至今仍不清楚，一般認(rèn)為是精子頂體在穿透卵丘細(xì)胞時能夠誘發(fā)頂體膜的融合、頂體內(nèi)各種酶的釋放以及卵透明帶被水解等頂體反應(yīng)現(xiàn)象(圖3)。然而有研究顯示，在精子成功穿過卵丘細(xì)胞層后，頂體的配體與透明帶表面的受體相互識別，膜表面的半乳糖基轉(zhuǎn)移酶(GalT)等蛋白分子隨后與透明帶蛋白(Zona pellucida protein, ZP3)結(jié)合，引發(fā)頂體反應(yīng)[41]。盡管頂體反應(yīng)的發(fā)生仍存在爭議，但反應(yīng)形成的入卵通道以及過程中暴露出的卵膜識別位點(diǎn)對精卵融合過程必不可少。

圖3 頂體反應(yīng)的過程

頂體反應(yīng)是受精的先決條件，只有經(jīng)過獲能和完成頂體反應(yīng)的精子才能夠進(jìn)入卵周間隙，完成精卵膜融合過程。該反應(yīng)時間進(jìn)程長，多種蛋白分子參與其中(表2)。除了GalT能夠與ZP3結(jié)合外，乙酰膽堿受體(Acetylcholine receptor, α7nAChR)能夠致使 EGFR分子活化，介導(dǎo)EGFR和 ZP3結(jié)合[42]。EGFR和ZP3的結(jié)合有可能是刺激了鈣離子通路，影響 Ca2+調(diào)節(jié)的頂體反應(yīng)。另外，肌動蛋白結(jié)合蛋白(Secretory actin-binding protein, SABP)是一種位于精子尾部的蛋白分子，在頂體反應(yīng)時與肌動蛋白結(jié)合，從而阻斷肌動蛋白的聚合[43]。由于精子獲能后Gelsolin發(fā)生的去磷酸化能使肌動蛋白解聚，并引發(fā)頂體反應(yīng)，因此 SABP很可能是一種誘發(fā)頂體反應(yīng)的重要作用因子。此外，精子頭部還存在多種與頂體反應(yīng)相關(guān)但作用位點(diǎn)未知的蛋白。P14是一種附睪來源的頂體蛋白質(zhì)，在頂體膜與精子質(zhì)膜發(fā)生融合時發(fā)揮重要的作用，其抗體能夠顯著降低頂體反應(yīng)的發(fā)生率[44]。與P14作用相似的還有動力蛋白(Dynamin)，Dynamin能夠啟動頂體內(nèi)容物胞外分泌，調(diào)控頂體膜融合的發(fā)生[45]。此外，精子頭部的頂體相關(guān)蛋白分子(Sperm acrosome associated 7, SPACA7)的釋放也是誘發(fā)頂體反應(yīng)的關(guān)鍵因素[46,47]。

表2 精子頂體反應(yīng)相關(guān)的蛋白質(zhì)

4 精子功能蛋白質(zhì)與入卵通道的形成

哺乳動物卵透明帶一般由 3～4種糖蛋白(ZP1、ZP2和ZP3等)組成[49,50]，構(gòu)成了精卵受精過程的屏障。自發(fā)生頂體反應(yīng)后，頂體內(nèi)釋放的復(fù)合酶(頂體素(Acrosin)、卵結(jié)合蛋白、脂酶和唾液酸苷酶等)能溶解卵透明帶，協(xié)助精子突破此屏障，并形成一條入卵通道。事實上，在形成入卵通道之前，精子頭部需要結(jié)合在透明帶表面以形成一種牢固的狀態(tài)(圖4)。這種狀態(tài)對于精子隨后溶解堅硬的透明帶是十分必要的。精子頭部內(nèi)源性或附著的蛋白則是形成該狀態(tài)的關(guān)鍵分子(表3)。

圖4 精子穿入透明帶的過程

表3 精子穿入透明帶相關(guān)的蛋白質(zhì)

近年來相關(guān)的研究結(jié)果還發(fā)現(xiàn)了與穿入透明帶相關(guān)的 Acrosin[51]、RNase10[52]、Prss37[53]和 PMIS2[54]4種蛋白質(zhì)。這 4種蛋白質(zhì)在精子與透明帶形成穩(wěn)定的綁定或結(jié)合狀態(tài)時發(fā)揮著必不可少的作用。敲除編碼這 4種蛋白的任一基因后，小鼠精子均不能與卵子的透明帶產(chǎn)生相互作用，也無法完成兩者間的綁定與結(jié)合，從而導(dǎo)致受精失敗。哺乳動物附睪合成的精子粘附素(Heparin-binding spermadhesins,AQN-3)[55]和 Lactadherin[48]同樣能夠協(xié)助精子與透明帶發(fā)生結(jié)合，使精子可以牢固地綁定在卵子表面。精細(xì)胞內(nèi)源的Testase1蛋白因子則能夠發(fā)生磷酸化，并作為一種錨定蛋白來引導(dǎo)頂體復(fù)合酶溶解透明帶，實現(xiàn)入卵通道的形成[56]。雖然這些功能蛋白質(zhì)的具體作用方式不明確，但目前多數(shù)推斷是與透明帶表面受體相互結(jié)合，保證精子穩(wěn)定地結(jié)合在卵子上，為溶解透明帶提供一種穩(wěn)態(tài)，也為精子順利穿入卵子做好鋪墊。

5 精子功能蛋白質(zhì)與精卵融合過程的關(guān)系

精子穿過透明帶后進(jìn)入到卵周間隙。此時，精卵膜融合的成功是精子頭部內(nèi)的遺傳物質(zhì)輸送入卵細(xì)胞內(nèi)的保證，也是受精過程最重要的一步。雖然精卵融合的具體過程和分子機(jī)制目前尚不十分清楚，但是精子蛋白質(zhì)在這個過程中發(fā)揮著不可替代的作用(表4)。其中熱休克蛋白(Heat shock 70 kDa protein 2, HSPA2)與精子頭部粘附因子(Sperm adhesion molecule 1, SPAM1)形成識別復(fù)合物，介導(dǎo)精卵間的識別[57]；隨后 Phospholipase C-zeta[58]和 Prosaposin[59]能夠激活卵子，刺激精卵發(fā)生膜融合(圖5)，實現(xiàn)合子的形成。精卵膜融合的實質(zhì)是精子膜和卵膜表面互補(bǔ)配對的特異性蛋白分子介導(dǎo)的過程，如精子膜表面的受精素家族(ADAMs)和卵膜上的整合素(Integrin)配對[60,61]，精子頭部赤道區(qū)的IZUMO1蛋白和卵膜上JUNO分子的結(jié)合[62～64]，精子頂體后膜的 CRISP家族和卵膜上微絲的相互作用[65,66]，IAM38[67]和Zonadhedin[48]分別與不同卵膜受體蛋白相互結(jié)合等。另外，卵膜上CD9分子、微絲的長度及密度對精卵膜識別和融合至關(guān)重要[68]。當(dāng)CD9與特異性糖蛋白(Pregnancy-specific glycoprotein 17,PSG17)結(jié)合后，PSG17占據(jù)了配體融合結(jié)合位點(diǎn)，因而導(dǎo)致精卵不能發(fā)生融合[69]。

總之，只有精卵發(fā)生膜融合后，精子才得以穿入卵內(nèi)并逐漸形成雄原核，此時卵子受到激活并完成第二次減數(shù)分裂。隨著第二極體的排出，雌原核形成以及隨后兩性原核發(fā)生結(jié)合并形成受精卵。隨后受精卵不斷發(fā)育和成長，受精卵開始第一次卵裂并完成精卵受精過程。受精卵在此后也依次經(jīng)歷4-細(xì)胞期、8-細(xì)胞期、16-細(xì)胞期、桑葚胚期、囊胚期等過程，最終形成胚胎乃至動物個體。

表4 精子識別并融合卵膜的相關(guān)蛋白質(zhì)

圖5 精卵膜融合的過程

6 結(jié) 語

哺乳動物受精作用受到多種蛋白分子調(diào)控，目前大部分蛋白分子在精子運(yùn)動活力、精子獲能、頂體反應(yīng)以及精卵融合等方面的詳細(xì)作用機(jī)理仍不清楚。近年來，相關(guān)研究顯示，精子功能相關(guān)的蛋白質(zhì)能夠作為受精能力的標(biāo)記，評估或改善家畜的繁殖力。人工授精是應(yīng)用在畜牧業(yè)生產(chǎn)上最成功和廣泛的一項遺傳育種技術(shù)，但是如何客觀地評估和判斷精子受精能力是人工授精技術(shù)的關(guān)鍵之一。目前，畜牧生產(chǎn)上常用的評估方法是檢測精子的活力、密度以及形態(tài)等，然而這些傳統(tǒng)的評估方法并不能準(zhǔn)確判斷出公畜個體間受精能力的差異[70,71]。個體間受精能力的差異是由于精液中受精相關(guān)的蛋白分子組成不同所導(dǎo)致[72]，檢測出這些與受精能力相關(guān)的分子標(biāo)記或許是最為行之有效的評估方法。建立生物標(biāo)記檢測方法，人們可以有效地預(yù)測和評定公畜的繁殖力和受精性能，客觀地區(qū)分高低繁殖力的個體以獲得更好的生產(chǎn)效率。

雖然已證實一些蛋白分子(如 BSP、OPN、Acrosin、ADAMs和 CRISP1等)與精子功能以及公畜受精能力相關(guān)，但限于其實驗的重復(fù)性和實際生產(chǎn)的應(yīng)用性，這些受精生物標(biāo)記還不足以客觀地應(yīng)用于評估個體的繁殖力。對精子發(fā)生、精子成熟、精子獲能以及頂體反應(yīng)等一系列受精過程需要進(jìn)行更深入地研究和探討，以尋找出合適于畜牧生產(chǎn)的生物標(biāo)記分子，進(jìn)而建立起更加簡便、客觀和高效的受精能力評定方法。

[1]Ashrafzadeh A, Karsani SA, Nathan S. Mammalian sperm fertility related proteins. Int J Med Sci, 2013, 10(12):1649–1657.

[2]Escoffier J, Jemel I, Tanemoto A, Taketomi Y, Payre C, Coatrieux C, Sato H, Yamamoto K, Masuda S, Pernet- Gallay K,Pierre V, Hara S, Murakami M, De Waard M, Lambeau G,Arnoult C. Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice. J Clin Invest, 2010, 120(5): 1415–1428.

[3]Gupta SK, Bhandari B. Acrosome reaction: relevance of zona pellucida glycoproteins. Asian J Androl, 2011, 13(1): 97–105.

[4]Primakoff P, Myles DG. Cell-cell membrane fusion during mammalian fertilization. FEBS Lett, 2007, 581(11): 2174–2180.

[5]Vernon GG, Woolley DM. Basal sliding and the mechanics of oscillation in a mammalian sperm flagellum. Biophys J,2004, 87(6): 3934–3944.

[6]Signorelli J, Diaz ES, Morales P. Kinases, phosphatases and proteases during sperm capacitation. Cell Tissue Res, 2012,349(3): 765–782.

[7]Visconti PE. Understanding the molecular basis of sperm capacitation through kinase design. Proc Natl Acad Sci USA,2009, 106(3): 667–668.

[8]Demarco IA, Espinosa F, Edwards J, Sosnik J, De La Vega-Beltrán JL, Hockensmith JW, Kopf GS, Darszon A, Visconti PE. Involvement of a Na+/HCO-3cotransporter in mouse sperm capacitation. J Biol Chem, 2003, 278(9): 7001–7009.

[9]Ickowicz D, Finkelstein M, Breitbart H. Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases. Asian J Androl, 2012, 14(6): 816–821.

[10]Lishko PV, Botchkina IL, Fedorenko A, Kirichok Y. Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel. Cell, 2010, 140(3): 327–337.

[11]Marquez B, Suarez SS. Bovine sperm hyperactivation is promoted by alkaline-stimulated Ca2+influx. Biol Reprod,2007, 76(4): 660–665.

[12]Santi CM, Santos T, Hernández-Cruz A, Darszon A. Properties of a novel pH-dependent Ca2+permeation pathway present in male germ cells with possible roles in spermatogenesis and mature sperm function. J Gen Physiol, 1998,112(1): 33–53.

[13]Kirichok Y, Navarro B, Clapham DE. Whole-cell patchclamp measurements of spermatozoa reveal an alkaline- activated Ca2+channel. Nature, 2006, 439(7077): 737–740.

[14]Navarro B, Kirichok Y, Clapham DE. KSper, a pH-sensitive K+ current that controls sperm membrane potential. Proc Natl Acad Sci USA, 2007, 104(18): 7688–7692.

[15]Giroux-Widemann V, Jouannet P, Pignot-Paintrand I, Feneux D. Effects of pH on the reactivation of human spermatozoa demembranated with Triton X-100. Mol Reprod Dev, 1991,29(2): 157–162.

[16]Ho HC, Granish KA, Suarez SS. Hyperactivated motility of bull sperm is triggered at the axoneme by Ca2+and not cAMP.Dev Biol, 2002, 250(1): 208–217.

[17]Finkelstein M, Megnagi B, Ickowicz D, Breitbart H. Regulation of sperm motility by PIP2(4, 5) and actin polymerization.Dev Biol, 2013, 381(1): 62–72.

[18]Gremm D, Wegner A. Gelsolin as a calcium-regulated actin filament-capping protein. Eur J Biochem, 2000, 267(14):4339–4345.

[19]Janmey PA, Iida K, Yin HL, Stossel TP. Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin. J Biol Chem, 1987, 262(25):12228–12236.

[20]Flesch FM, Wijnand E, van de Lest CHA, Colenbrander B,van Golde LMG, Gadella BM. Capacitation dependent activation of tyrosine phosphorylation generates two sperm head plasma membrane proteins with high primary binding affinity for the zona pellucida. Mol Reprod Dev, 2001, 60(1): 107–115.

[21]Visconti PE, Moore GD, Bailey JL, Leclerc P, Connors SA,Pan D, Olds-Clarke P, Kopf GS. Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development, 1995, 121(4): 1139–1150.

[22]Goltz JS, Gardner TK, Kanous KS, Lindemann CB. The interaction of pH and cyclic adenosine 3', 5'-monophosphate on activation of motility in Triton X-100 extracted bull sperm.Biol Reprod, 1988, 39(5): 1129–1136.

[23]Suarez SS. Control of hyperactivation in sperm. Hum Reprod Update, 2008, 14(6): 647–657.

[24]Qu F, Ying X, Guo W, Guo Q, Chen G, Liu Y, Ding Z. The role of Zn-α2 glycoprotein in sperm motility is mediated by changes in cyclic AMP. Reproduction, 2007, 134(4):569–576.

[25]Das S, Saha S, Majumder GC, Dungdung SR. Purification and characterization of a sperm motility inhibiting factor from caprine epididymal plasma. PLoS ONE, 2010, 5(8):e12039.

[26]Iwamoto T, Hiroaki H, Furuichi Y, Wada K, Satoh M, Satoh M, Osada T, Gagnon C. Cloning of boar SPMI gene which is expressed specifically in seminal vesicle and codes for a sperm motility inhibitor protein. FEBS Lett, 1995, 368(3):420–424.

[27]Jin J, Jin N, Zheng H, Ro S, Tafolla D, Sanders KM, Yan W.Catsper3 and Catsper4 are essential for sperm hyperactivated motility and male fertility in the mouse. Biol Reprod, 2007,77(1): 37–44.

[28]Al-Dossary AA, Strehler EE, Martin-Deleon PA. Expression and secretion of plasma membrane Ca2+-ATPase 4a(PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm. PLoS ONE, 2013, 8(11):e80181.

[29]Hung PH, Suarez SS. Alterations to the bull sperm surface proteins that bind sperm to oviductal epithelium. Biol Reprod,2012, 87(4): 88.

[30]Srivastava N, Jerome A, Srivastava SK, Ghosh SK, Kumar A.Bovine seminal PDC-109 protein: An overview of biochemical and functional properties. Anim Reprod Sci, 2013,138(1-2): 1–13.

[31]Gwathmey TM, Ignotz GG, Suarez SS. PDC-109 (BSPA1/A2) promotes bull sperm binding to oviductal epithelium in vitro and may be involved in forming the oviductal sperm reservoir. Biol Reprod, 2003, 69(3): 809–815.

[32]Tarnasky H, Cheng M, Ou Y, Thundathil JC, Oko R, van der Hoorn FA. Gene trap mutation of murine outer dense fiber protein-2 gene can result in sperm tail abnormalities in mice with high percentage chimaerism. BMC Dev Biol, 2010, 10(1):67.

[33]O'Rand MG, Widgren EE, Hamil KG, Silva EJ, Richardson RT. Functional studies of eppin. Biochem Soc Trans, 2011,39(5): 1447–1449.

[34]Park SK, Yoon J, Wang L, Shibata TK, Motamedchaboki K,Shim KJ, Chang MS, Lee SH, Tamura N, Hatakeyama S,Nadano D, Sugihara K, Fukuda MN. Enhancement of mouse sperm motility by trophinin-binding peptide. Reprod Biol Endocrinol, 2012, 10(1): 101.

[35]Wang C, Wang L, Su B, Lu N, Song JJ, Yang XQ, Fu WW,Tan WW, Han B. Serine protease inhibitor Kazal type 1 promotes epithelial-mesenchymal transition through EGFR signaling pathway in prostate cancer. Prostate, 2014, 74(7):689–701.

[36]Lin MH, Lee RK, Hwu YM, Lu CH, Chu SL, Chen YJ,Chang WC, Li SH. SPINKL, a Kazal-type serine protease inhibitor-like protein purified from mouse seminal vesicle fluid,is able to inhibit sperm capacitation. Reproduction, 2008,136(5): 559–571.

[37]Dubé C, Leclerc P, Baba T, Reyes-Moreno C, Bailey JL. The proacrosin binding protein, sp32, is tyrosine phosphorylated during capacitation of pig sperm. J Androl, 2005, 26(4):519–528.

[38]Luna C, Colas C, Perez-Pe R, Cebrian-Perez JA, Muino-Blanco T. A novel epidermal growth factor-dependent extracellular signal-regulated MAP kinase cascade involved in sperm functionality in sheep. Biol Reprod, 2012, 87(4): 93.

[39]Novak S, Smith TA, Paradis F, Burwash L, Dyck MK, Foxcroft GR, Dixon WT. Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions. Theriogenology, 2010,74(6): 956–967.

[40]Boccia L, Di Francesco S, Neglia G, De Blasi M, Longobardi V, Campanile G, Gasparrini B. Osteopontin improves sperm capacitation and in vitro fertilization efficiency in buffalo(Bubalus bubalis). Theriogenology, 2013, 80(3): 212–217.

[41]Rodeheffer C, Shur BD. Sperm from β1, 4-galactosyltransferase I-null mice exhibit precocious capacitation. Development, 2004, 131(3): 491–501.

[42]Jaldety Y, Glick Y, Orr-Urtreger A, Ickowicz D, Gerber D,Breitbart H. Sperm epidermal growth factor receptor (EGFR)mediates α7 acetylcholine receptor (AChR) activation to pro-mote fertilization. J Biol Chem, 2012, 287(26): 22328– 22340.

[43]Capkova J, Elzeinova F, Novak P. Increased expression of secretory actin-binding protein on human spermatozoa is associated with poor semen quality. Hum Reprod, 2007, 22(5):1396–1404.

[44]Nandi P, Ghosh S, Jana K, Sen PC. Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa. PLoS ONE, 2012, 7(1): e30552.

[45]Reid AT, Lord T, Stanger SJ, Roman SD, Mccluskey A, Robinson PJ, Aitken RJ, Nixon B. Dynamin regulates specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa. J Biol Chem, 2012, 287(45):37659–37672.

[46]Korfanty J, Toma A, Wojtas A, Rusin A, Vydra N, Widlak W.Identification of a new mouse sperm acrosome-associated protein. Reproduction, 2012, 143(6): 749–757.

[47]Nguyen EB, Westmuckett AD, Moore KL. SPACA7 is a novel male germ cell-specific protein localized to the sperm acrosome that is involved in fertilization in mice. Biol Reprod,2014, 90(1): 16.

[48]Shur BD. Reassessing the role of protein-carbohydrate complementarity during sperm-egg interactions in the mouse. Int J Dev Biol, 2008, 52(5/6): 703–715.

[49]Conner SJ, Lefièvre L, Hughes DC, Barratt CLR. Cracking the egg: increased complexity in the zona pellucida. Hum Reprod, 2005, 20(5): 1148–1152.

[50]Wassarman P M. Zona pellucida glycoproteins. Annu Rev Biochem, 2008, 283: 24285–24289.

[51]Ferrer M, Rodriguez H, Zara L, Yu Y, Xu W, Oko R. MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization. Cell Tissue Res,2012, 349(3): 881–895.

[52]Krutskikh A, Poliandri A, Cabrera-Sharp V, Dacheux JL,Poutanen M, Huhtaniemi I. Epididymal protein Rnase10 is required for post-testicular sperm maturation and male ferti lity. FASEB J, 2012, 26(10): 4198–4209.

[53]Shen C, Kuang Y, Liu J, Feng J, Chen X, Wu W, Chi J, Tang L, Wang Y, Fei J, Wang Z. Prss37 is required for male ferti lity in the mouse. Biol Reprod, 2013, 88(5): 123.

[54]Yamaguchi R, Fujihara Y, Ikawa M, Okabe M. Mice expressing aberrant sperm-specific protein PMIS2 produce normal-looking but fertilization-incompetent spermatozoa.Mol Biol Cell, 2012, 23(14): 2671–2679.

[55]van Gestel RA, Brewis IA, Ashton PR, Brouwers JF, Gadella BM. Multiple proteins present in purified porcine sperm apical plasma membranes interact with the zona pellucida of the oocyte. Mol Hum Reprod, 2007, 13(7): 445–454.

[56]Zhu GZ, Myles DG, Primakoff P. Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization.J Cell Sci, 2001, 114(Pt 9): 1787–1794.

[57]Redgrove KA, Nixon B, Baker MA, Hetherington L, Baker G,Liu DY, Aitken RJ. The molecular chaperone HSPA2 plays a key role in regulating the expression of sperm surface receptors that mediate sperm-egg recognition. PLoS ONE, 2012,7(11): e50851.

[58]Theodoridou M, Nomikos M, Parthimos D, Gonzalez-Garcia JR, Elgmati K, Calver BL, Sideratou Z, Nounesis G, Swann K, Lai FA. Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+oscillations in mammalian eggs at fertilization. Mol Hum Reprod,2013, 19(12): 852–864.

[59]Morales CR, Hay N, El-Alfy M, Zhao Q. Distribution of mouse sulfated glycoprotein-1 (prosaposin) in the testis and other tissues. J Androl, 1998, 19(2): 156–164.

[60]Kim T, Oh J, Woo JM, Choi E, Im SH, Yoo YJ, Kim DH,Nishimura H, Cho C. Expression and relationship of male reproductive ADAMs in mouse. Biol Reprod, 2006, 74(4):744–750.

[61]Nishimura H, Kim E, Nakanishi T, Baba T. Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface. J Biol Chem, 2004, 279(33):34957–34962.

[62]Bianchi E, Doe B, Goulding D, Wright GJ. Juno is the egg Izumo receptor and is essential for mammalian fertilization.Nature, 2014, 508(7497): 483–487.

[63]Inoue N, Ikawa M, Okabe M. The mechanism of sperm- egg interaction and the involvement of IZUMO1 in fusion. Asian J Androl, 2011, 13(1): 81–87.

[64]Satouh Y, Inoue N, Ikawa M, Okabe M. Visualization of the moment of mouse sperm-egg fusion and dynamic localization of IZUMO1. J Cell Sci, 2012, 125(Pt 21): 4985–4990.

[65]Cohen DJ, Maldera JA, Weigel MM, Ernesto JI, Vasen G,Cuasnicu PS. Cysteine-rich secretory proteins (CRISP) and their role in mammalian fertilization. Biol Res, 2011, 44(2):135–138.

[66]Cohen DJ, Maldera JA, Vasen G, Ernesto JI, Munoz MW,Battistone MA, Cuasnicu PS. Epididymal protein CRISP1 plays different roles during the fertilization process. J Androl,2011, 32(6): 672–678.

[67]Sutovsky P. Sperm-egg adhesion and fusion in mammals.Expert Rev Mol Med, 2009, 11: e11.

[68]Kaji K, Kudo A. The mechanism of sperm-oocyte fusion in mammals. Reproduction, 2004, 127(4): 423–429.

[69]Ellerman DA, Ha C, Primakoff P, Myles DG, Dveksler GS.Direct binding of the ligand PSG17 to CD9 requires a CD9 site essential for sperm-egg fusion. Mol Biol Cell, 2003,14(12): 5098–5103.

[70]Januskauska A, Johannisson A, Rodriguez-Martinez H. Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen-thawed semen from Swedish AI bulls. Theriogenology,2001, 55(4): 947–961.

[71]Graham JK, Mocé E. Fertility evaluation of frozen/thawed semen. Theriogenology, 2005, 64(3): 492–504.

[72]Govindaraju A, Dogan S, Rodriguez-Osorio N, Grant K,Kaya A, Memili E. Delivering value from sperm proteomics for fertility. Cell Tissue Res, 2012, 349(3): 783–793.