Beagle犬卵母細(xì)胞體外成熟研究進(jìn)展

胡敏華，周治東，倪慶純，劉運(yùn)忠

(廣州醫(yī)藥研究總院有限公司實(shí)驗(yàn)動(dòng)物研究開發(fā)中心 國(guó)家犬類實(shí)驗(yàn)動(dòng)物種子中心)，廣州 510240

研究進(jìn)展

Beagle犬卵母細(xì)胞體外成熟研究進(jìn)展

胡敏華，周治東，倪慶純，劉運(yùn)忠

(廣州醫(yī)藥研究總院有限公司實(shí)驗(yàn)動(dòng)物研究開發(fā)中心 國(guó)家犬類實(shí)驗(yàn)動(dòng)物種子中心)，廣州 510240

國(guó)內(nèi)實(shí)驗(yàn)Beagle犬種質(zhì)資源保存利用及制備基因修飾人類疾病動(dòng)物模型，要求有充足的Beagle犬胚胎。目前誘導(dǎo)排卵技術(shù)在犬上效果不明顯，體內(nèi)獲取犬成熟卵母細(xì)胞有困難。同時(shí)，盡管科研人員對(duì)犬卵母細(xì)胞體外成熟培養(yǎng)進(jìn)行了多方面的嘗試，但尚未獲得突破，成熟率低，嚴(yán)重制約了其在種質(zhì)資源保存、基因修飾模型制備及生物醫(yī)學(xué)研究中的應(yīng)用。本文梳理了不同犬齡及生殖周期、卵母細(xì)胞形態(tài)與體積及脂滴在Beagle犬卵母細(xì)胞體外成熟中的影響，以為其體外成熟尋找新的突破口。

犬；疾病模型；卵母細(xì)胞；體外成熟

Beagle犬是國(guó)際公認(rèn)的新藥安評(píng)與研發(fā)首選用犬，作為國(guó)家犬類實(shí)驗(yàn)動(dòng)物種子中心，對(duì)國(guó)內(nèi)優(yōu)質(zhì)Beagle犬種質(zhì)資源或者瀕危犬種進(jìn)行保護(hù)利用，具有重要意義。目前國(guó)內(nèi)Beagle犬只有活體保種單一形式，同時(shí)經(jīng)多代的繁衍后，未免會(huì)對(duì)其遺傳穩(wěn)定性造成影響，而Beagle犬種子體外保存是解決上述問(wèn)題的最好方法之一。近年來(lái)基因修飾技術(shù)進(jìn)展很快，我國(guó)也已成功實(shí)現(xiàn)世界首例基因敲除犬，但該技術(shù)及體外保種要求有充足的實(shí)驗(yàn)材料(受精卵)，目前成熟卵母細(xì)胞一是從自然發(fā)情排卵的母犬輸卵管中手術(shù)獲取，二是從卵巢獲取卵丘-卵母細(xì)胞復(fù)合體(cumulus oocytes-complexs, COCs)再進(jìn)行體外成熟(in vitro maturation, IVM)。如方法一獲得成熟卵母細(xì)胞，其成本很高且非常困難，而方法二是比較適宜的選擇，因此需要急切解決犬COCs體外成熟、受精等技術(shù)壁壘，以盡快應(yīng)用于保種及疾病模型研究。

1 不同培養(yǎng)體系對(duì)犬卵丘-卵母細(xì)胞體外成熟的影響

犬是非季節(jié)性單次發(fā)情動(dòng)物，分為發(fā)情前期、發(fā)情期、發(fā)情后期及乏情期。母犬一年發(fā)情1～2次，犬發(fā)情間期平均7個(gè)月，母犬排卵雖然主要集中在發(fā)情期前1～3 d內(nèi)，但在發(fā)情期的7 d內(nèi)隨時(shí)都可發(fā)生排卵，排卵的確切時(shí)間難以掌握[1]。且犬排出的卵母細(xì)胞處于生發(fā)泡期(germinal vesicle, GV)，需在輸卵管內(nèi)完成成熟過(guò)程(48～72 h)，因此犬排出的卵母細(xì)胞在2～3 d后，才具有受精能力，再次啟動(dòng)減數(shù)分裂過(guò)程。多年來(lái)科研人員嘗試了各種各樣的卵母細(xì)胞體外成熟培養(yǎng)體系[2]，如往培養(yǎng)液添加促性腺激素(FSH，LG，PMSG，HCG)的[3-5]、甾類(雌二醇、孕酮)的[1, 6]、不同類型血清的[6, 7]、生長(zhǎng)因子的(IGF-1，EGF)[8]、各種蛋白源的[9]、透明質(zhì)酸酶的[10]、抗氧化劑的[7, 11]、細(xì)胞周期抑制劑的[12]、模擬輸卵管內(nèi)環(huán)境與犬輸卵管上皮細(xì)胞共培養(yǎng)的[13, 14]，甚至將卵母細(xì)胞注入體外培養(yǎng)的輸卵管內(nèi)等等[15]，但無(wú)論采用何種基礎(chǔ)培養(yǎng)液及添加各種生物活性物質(zhì)，仍只有20%左右的犬卵母細(xì)胞能成功發(fā)育至MII階段[16]。事實(shí)上，大概60%的COCs取出后就已停止發(fā)育，大概25%的卵母細(xì)胞退化，只有少量卵母細(xì)胞能成功發(fā)育至MII階段，但因質(zhì)量太差，其繼續(xù)體外受精及發(fā)育能力亦會(huì)大大降低[17, 18]。諸多研究表明，目前對(duì)犬卵母細(xì)胞體外成熟培養(yǎng)體系是不適宜的，到底COCs在體外缺少了何種因子的刺激，致其大部分停止發(fā)育？研究者們必須另辟途徑，尋找突破口。

2 不同犬齡及生殖周期對(duì)卵丘-卵母細(xì)胞體外成熟的影響

犬卵巢功能隨著年齡的增加而不斷下降，在國(guó)家犬類實(shí)驗(yàn)動(dòng)物種子中心，雖然也存在6歲以上的種母犬懷孕分娩情況，但其胎均產(chǎn)仔數(shù)明顯要比6歲以下的低，且出現(xiàn)病、弱仔機(jī)率上升。經(jīng)比較，從6歲以上母犬卵巢獲得的COCs數(shù)顯著低于6歲以下正常母犬(未發(fā)表數(shù)據(jù))。而Hewitt等[19]也發(fā)現(xiàn)供體母犬的年齡與COCs的獲取數(shù)呈現(xiàn)負(fù)相關(guān)，COCs的平均獲取數(shù)隨供體年齡的增長(zhǎng)每年下降4.7枚(Hewittetal., 1998)。Lopes等[ 20]對(duì)取自6歲以下和7歲以上母犬的卵母細(xì)胞體外培養(yǎng)比較，發(fā)現(xiàn)6歲以下母犬所取的卵母細(xì)胞在體外培養(yǎng)成熟的潛力更高。

有學(xué)者研究在不同生殖周期所取的COCs是否會(huì)對(duì)體外成熟率有影響，但結(jié)果不相一致。Rodrigues等[21]認(rèn)為COCs的體外成熟不受母犬生殖階段的影響，在不同生殖周期采集的COCs對(duì)其減數(shù)分裂的恢復(fù)沒(méi)有顯著差異。但更多的研究認(rèn)為生殖周期是COCs體外成熟的重要影響因素，供體母犬發(fā)情期卵巢的內(nèi)環(huán)境含有高濃度的雌激素、孕酮及一些未知因子，有利于其后的體外成熟[22]。卵泡期的犬COCs恢復(fù)減數(shù)分裂和達(dá)到MI、MII的能力比乏情期的高[23]，Oh等[24]認(rèn)為適當(dāng)生殖周期的卵母細(xì)胞對(duì)其減數(shù)分裂的恢復(fù)有重要影響。盡管如此，Yamada等[25]采集經(jīng)誘導(dǎo)超數(shù)排卵后的母犬排卵前COCs，其體外成熟率仍只有32%，對(duì)照組為12%，表明雖然生殖周期對(duì)COCs體外成熟存在一定的影響，但并不是關(guān)鍵因素之一。

3 卵丘-卵母細(xì)胞形態(tài)及體積與體外成熟的關(guān)系

卵丘細(xì)胞與卵母細(xì)胞緊密連接形成COCs，卵丘細(xì)胞通過(guò)縫隙連接(Gap junctions, GJs)與卵母細(xì)胞間發(fā)生營(yíng)養(yǎng)物質(zhì)、離子以及cAMP等調(diào)節(jié)小分子的交換，從排卵前到排卵后，卵丘細(xì)胞與卵母細(xì)胞間發(fā)生著復(fù)雜的信號(hào)傳遞，從而對(duì)卵母細(xì)胞的發(fā)育實(shí)現(xiàn)分子水平的調(diào)控。但對(duì)于犬類動(dòng)物GJs的研究?jī)H限于形態(tài)學(xué)上的研究。

為了提高COCs體外成熟率，學(xué)者們都遵循一個(gè)形態(tài)學(xué)標(biāo)準(zhǔn)，即胞質(zhì)顏色深暗而均一，直徑>100 μm，有兩層以上完整的顆粒細(xì)胞層[27]。對(duì)于為何以這個(gè)標(biāo)準(zhǔn)選取，也是經(jīng)過(guò)驗(yàn)證的。Lopes等[28]發(fā)現(xiàn)根據(jù)上述標(biāo)準(zhǔn)選取的犬卵母細(xì)胞核凋亡的比例低于15%。Otoi等[29]發(fā)現(xiàn)直徑小于100 μm的COCs成熟率僅為4%～10%，Songsasen等[30]發(fā)現(xiàn)僅有一層顆粒細(xì)胞的卵母細(xì)胞在培養(yǎng)48 h后退化。Hewitt等[19]也發(fā)現(xiàn)直徑大于100 μm的COCs發(fā)育至MI、MII階段的比例為20%，而直徑<100 μm的COCs其比例僅為4%～10%。這個(gè)標(biāo)準(zhǔn)在牛卵母細(xì)胞上也同樣適用，可根據(jù)其胞質(zhì)顏色判斷細(xì)胞的發(fā)育潛能[26]。上述研究表明卵母細(xì)胞體積大小、胞質(zhì)顏色等對(duì)其核體外成熟有一定的影響。

4 脂滴形態(tài)對(duì)卵丘-卵母細(xì)胞體外成熟的影響

脂滴(lipid droplets，LD)是富集在動(dòng)物脂肪組織中的動(dòng)態(tài)細(xì)胞器，是由磷脂單分子層、游離膽固醇和特殊蛋白覆蓋在核心的中性脂質(zhì)組成，控制著體脂的貯存。蛋白質(zhì)組學(xué)研究表明：LD參與脂類代謝和運(yùn)輸、細(xì)胞內(nèi)物質(zhì)交換、信號(hào)轉(zhuǎn)導(dǎo)及細(xì)胞骨架構(gòu)成。犬、豬、牛等卵母細(xì)胞和胚胎細(xì)胞內(nèi)以脂滴的形式儲(chǔ)存大量的內(nèi)源性脂質(zhì)，因此胞質(zhì)顏色深暗，這些脂質(zhì)為卵母細(xì)胞及早期胚胎發(fā)育供能[31]。LD在豬卵母細(xì)胞中的研究相對(duì)較多，豬、馬卵母細(xì)胞成熟過(guò)程中脂滴形態(tài)和含量是一個(gè)動(dòng)態(tài)變化過(guò)程[32-34]，且其脂質(zhì)代謝改變會(huì)干擾單精受精和胚胎發(fā)育[35]。

而關(guān)于犬卵母細(xì)胞LD的研究鮮有報(bào)道。2012年Apparicio等[36]通過(guò)基質(zhì)輔助激光解吸電離(matrix-assisted laser desorption mass spectrometry,MALDI-MS)法首次對(duì)犬和貓卵母細(xì)胞里L(fēng)D的化學(xué)構(gòu)成進(jìn)行研究，為改進(jìn)其體外培養(yǎng)及冷凍保存技術(shù)提供參考。2016年Ariu等[37]首次分析了犬COCs體積及生殖階段與LD分布的關(guān)系，結(jié)果表明在卵泡期，大部分卵母細(xì)胞LD呈現(xiàn)不規(guī)則分布，但在黃體期及乏情期，LD主要在核周邊分布，且不管犬處于哪個(gè)生殖階段，直徑大于120 μm的卵母細(xì)胞LD含量要顯著高于直徑在110～120 μm之間的卵母細(xì)胞。犬卵母細(xì)胞含有大量LD，推測(cè)其在卵母細(xì)胞體外成熟過(guò)程中有重要作用，需進(jìn)一步展開研究以探明其如何影響卵母細(xì)胞的發(fā)育過(guò)程。

5 展望

2005年，公布的犬的全基因組序列分析，發(fā)現(xiàn)犬基因組與人的相似性達(dá)95.7%，且有數(shù)百種遺傳疾病與人類相似[38]，是研究人類疾病機(jī)理機(jī)制的重要模型。但從2005年[39]首例克隆犬的誕生到2015年首例基因敲除犬的出生[40]，已過(guò)去10年，有關(guān)犬輔助生殖技術(shù)遠(yuǎn)遠(yuǎn)滯后于小鼠、豬等動(dòng)物的研究，尤其是卵母細(xì)胞體外成熟培養(yǎng)效率停滯不前，究其原因，一是其獨(dú)特的生殖生理，發(fā)情排卵時(shí)間難以把握，GV期及成熟卵母細(xì)胞獲取有困難；二是犬COCs現(xiàn)行體外成熟培養(yǎng)技術(shù)體系尚未滿足啟動(dòng)其恢復(fù)減數(shù)分裂的要求，提示需要從分子機(jī)制來(lái)展開研究。犬卵母細(xì)胞體內(nèi)成熟調(diào)節(jié)機(jī)制是什么？卵丘細(xì)胞、脂滴究竟通過(guò)什么通路影響著卵母細(xì)胞的成熟？輸卵管內(nèi)存在哪些關(guān)鍵因子促進(jìn)卵母細(xì)胞減數(shù)分裂的恢復(fù)？等等，這些問(wèn)題，都亟待解決。

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Research progress of in vitro maturation of Beagle dog oocytes

HU Min-hua*, ZHOU Zhi-dong, NI Qing-chun, LIU Yun-zhong

(Research and Development Center of Experimental Animal, Guangzhou General Pharmaceutical Research Institute Co., Ltd., (National Seed Center of Experimental Dogs) Guangzhou 510240, China)

Sufficient embryos are needed for the preservation of Beagle dogs germplasm resources and the preparation of gene-modified human disease animal models.Up to now, the induced ovulation technique has no effect on dogs,it is hard to obtain mature oocytes in vivo, although the scientists try a lot in many aspects, but still could not make a breakthrough. The in vitro maturation rate is too low to support the preservation of germplasm resources, application in gene-modified disease models and biomedical research. Aiming to provide useful information on breakthrough in dog oocytes maturation, this review will summarize the effect of different age and reproductive stage,different morphology and size of the oocytes and lipid droplet on the in vitro maturation of dog oocytes.

Beagle dogs; Disease model; Ooocytes; in vitro maturation

HU Min-hua, E-mail: myemail-cony@163.com

廣州市“珠江科技新星”項(xiàng)目(201610010144)；廣州市科技基礎(chǔ)條件平臺(tái)建設(shè)項(xiàng)目(201605040005)。

胡敏華(1983-)，男，畜牧師，博士，動(dòng)物疾病模型。Email: myemail-cony@163.com

Q95-33

A

1005-4847(2017) 01-0107-04

10.3969/j.issn.1005-4847.2017.01.020

2016-08-09