王永霞 伍園園馬娜 鐘興明 崔蓉 王小蘭 楊寧 苗竹林
1.廣東省計(jì)劃生育科學(xué)技術(shù)研究所,廣東廣州510600;2.同濟(jì)大學(xué)附屬第一婦嬰保健院生殖中心,上海200204;3.廣東藥學(xué)院基礎(chǔ)醫(yī)學(xué)院生理學(xué)系,廣東廣州510006
胎兒卵巢的形態(tài)學(xué)及細(xì)胞凋亡的觀察與研究
王永霞1伍園園2馬娜3▲鐘興明1崔蓉1王小蘭1楊寧1苗竹林1
1.廣東省計(jì)劃生育科學(xué)技術(shù)研究所,廣東廣州510600;2.同濟(jì)大學(xué)附屬第一婦嬰保健院生殖中心,上海200204;3.廣東藥學(xué)院基礎(chǔ)醫(yī)學(xué)院生理學(xué)系,廣東廣州510006
目的探討人胎兒卵泡的發(fā)育形成過程及卵細(xì)胞凋亡情況。方法收集2013年1~12月于廣東省計(jì)劃生育科學(xué)技術(shù)研究所行中期妊娠及晚期妊娠(24~32周)引產(chǎn)的女性胎兒卵巢組織18例。其中24~27周(孕中期)胎兒卵巢組織8例,28~32周(孕晚期)胎兒卵巢組織10例。采用免疫組化蘇木精-伊紅(HE)染色及原位末端標(biāo)記法(TUNEL),觀察胎兒卵巢卵泡發(fā)育及形成過程及該過程中卵細(xì)胞凋亡情況。結(jié)果HE染色顯微鏡下觀察,孕中期胎齡卵巢原始卵母細(xì)胞較多,卵巢間質(zhì)少,顆粒細(xì)胞較少。隨著胎齡的增加,顆粒細(xì)胞逐漸增多;圍繞卵母細(xì)胞,原始卵泡逐漸形成并開始增多,卵母細(xì)胞數(shù)量減少。孕晚期原始卵母細(xì)胞明顯減少,原始卵泡形成,數(shù)量進(jìn)一步增多,隨著孕齡增加,原始卵泡愈發(fā)完整。TUNEL染色觀察見陽性凋亡細(xì)胞多見于未形成卵泡的卵母細(xì)胞,卵細(xì)胞周圍的顆粒細(xì)胞在孕晚期可見少許凋亡,卵泡內(nèi)卵細(xì)胞未見陽性染色。孕中期卵巢卵母細(xì)胞凋亡明顯多于孕晚期,且凋亡主要發(fā)生于未形成原始卵泡的卵母細(xì)胞。結(jié)論胎兒卵巢發(fā)育過程中,原始卵母細(xì)胞數(shù)量逐步減少,原始卵泡形成,其發(fā)育過程中原始卵母細(xì)胞不斷發(fā)生凋亡,導(dǎo)致生殖細(xì)胞丟失。
胎兒卵巢;卵母細(xì)胞;細(xì)胞凋亡
在女性妊娠8周的女性胚胎卵原細(xì)胞開始增殖,妊娠20周左右達(dá)到峰值,之后開始進(jìn)入減數(shù)分裂期。在由有絲分裂向減數(shù)分裂過渡時(shí),細(xì)胞數(shù)目急劇下降,大量丟失[1]。初級(jí)卵母細(xì)胞繼續(xù)發(fā)育,此時(shí)它們被來自卵巢壁的前顆粒細(xì)胞所包圍,形成原始卵泡,這期間卵母細(xì)胞仍在不斷死亡,有三分之二的卵母細(xì)胞在出生前已被丟失[2]。大量實(shí)驗(yàn)證明,哺乳動(dòng)物生殖細(xì)胞在發(fā)生過程中的大量死亡或丟失屬于細(xì)胞凋亡[3-4]。但到目前為止人胎兒卵巢發(fā)育的相關(guān)研究較少,因此對(duì)于其發(fā)育過程中細(xì)胞凋亡的發(fā)生及機(jī)制的認(rèn)識(shí)還很少,凋亡因子在卵巢內(nèi)細(xì)胞定位、發(fā)生時(shí)相以及相互作用尚未完全闡明,調(diào)控卵母細(xì)胞凋亡的途徑尚不清楚,這一切均有待進(jìn)一步的深入研究。本研究采用蘇木精-伊紅染色(HE)觀察胎兒卵巢發(fā)育,使用原位末端標(biāo)記法(TUNEL)檢測(cè)胎兒卵細(xì)胞凋亡,旨在探討胎兒卵巢發(fā)育過程中卵細(xì)胞形成過程及卵細(xì)胞凋亡,為進(jìn)一步研究卵細(xì)胞凋亡機(jī)制及其調(diào)控提供線索。
1.1 對(duì)象
選擇2013年1~12月由于母親或社會(huì)因素于廣東省計(jì)劃生育科學(xué)技術(shù)研究所(以下簡稱“我院”)行妊娠終止的病例,所有患者均采用米非司酮聯(lián)合前列腺素進(jìn)行引產(chǎn)。收集孕24~32周引產(chǎn)的女性胎兒卵巢組織18例。其中24~27周(中期妊娠)胎兒卵巢8例,28~32周(晚期妊娠)胎兒卵巢10例。本研究經(jīng)我院醫(yī)學(xué)倫理委員會(huì)討論通過,收集的胎兒卵巢組織也均得到胎兒母親的同意并簽署知情同意書。
1.2 方法
卵巢組織投入10%福爾馬林溶液固定,經(jīng)脫水、浸蠟、包埋。最終切成3μm石蠟切片。石蠟切片經(jīng)脫蠟浸水后,常規(guī)HE染色后顯微鏡下觀察胎兒卵巢組織。細(xì)胞凋亡檢測(cè)試劑盒購自羅氏公司,具體操作步驟參照試劑盒說明書進(jìn)行,用熒光顯微鏡拍照后,二氨基聯(lián)苯胺-過氧化氫顯色,設(shè)立陽性對(duì)照(用DNA酶處理切片)和陰性對(duì)照(用PBS代替末端脫氧核糖核酸轉(zhuǎn)移酶)。結(jié)果判斷:熒光顯微鏡下陽性細(xì)胞呈黃綠色熒光,二氨基聯(lián)苯胺(DAB)顯色后陽性定位于細(xì)胞核,或細(xì)胞核、細(xì)胞漿同時(shí)著色,呈棕黃色。
2.1 HE染色結(jié)果觀察
顯微鏡下觀察,24~27周胎齡卵巢原始卵母細(xì)胞較多,顆粒細(xì)胞少,且大部分并未圍繞卵母細(xì)胞形成原始卵泡,但是隨著胎齡的增加,顆粒細(xì)胞圍繞卵母細(xì)胞數(shù)目增多,原始卵泡形成開始增多。27~28周胎齡,已有部分原始卵泡形成。28~32周胎齡原始卵母細(xì)胞明顯減少,顆粒細(xì)胞增多,原始卵泡形成。隨著孕齡增加,原始卵泡愈發(fā)完整。見圖1(封三)。
2.2 凋亡細(xì)胞觀察
陽性凋亡細(xì)胞的細(xì)胞標(biāo)記為黃綠色熒光小體,主要見于未形成卵泡的卵母細(xì)胞及早期原始卵泡。卵泡越成熟,凋亡發(fā)生越少見。孕中期卵巢卵母細(xì)胞明顯多于孕晚期,且凋亡主要發(fā)生于未形成原始卵泡的卵母細(xì)胞(圖2,封三)。部分孕晚期的胎兒卵巢中,凋亡主要發(fā)生于顆粒細(xì)胞(圖3,封三)。
卵泡發(fā)育是一個(gè)多階段的復(fù)雜過程,在妊娠11~12周時(shí)卵原細(xì)胞開始向初級(jí)卵母細(xì)胞轉(zhuǎn)化,在妊娠20周時(shí)卵母細(xì)胞總數(shù)達(dá)高峰,然而,有2/3的卵母細(xì)胞在出生前被丟失[2]。因此,細(xì)胞凋亡是卵巢功能和發(fā)育過程中不可或缺的組成部分。細(xì)胞凋亡,又稱程序性細(xì)胞死亡,凋亡在生物胚胎發(fā)生、器官形成發(fā)育、成熟細(xì)胞新舊交替、激素依賴性生理退化以及自身免疫性疾病和腫瘤等的發(fā)生發(fā)展中都發(fā)揮著不可替代的重要作用。卵泡的凋亡可能發(fā)生于卵母細(xì)胞或顆粒細(xì)胞,最終導(dǎo)致卵泡閉鎖。有學(xué)者認(rèn)為,原始卵泡、初級(jí)卵泡閉鎖主要是由卵母細(xì)胞凋亡引起,而在生長卵泡中,顆粒細(xì)胞的凋亡起主導(dǎo)作用[5],但也有學(xué)者認(rèn)為兩種凋亡同時(shí)發(fā)生于卵泡生長發(fā)育的任何一個(gè)階段[6]。
本研究發(fā)現(xiàn),孕中期(24~27周)胎兒卵巢發(fā)育與孕晚期(28~32周)胎兒卵巢有明顯區(qū)別。在孕中期胎兒卵巢組織,主要由多量的原始卵母細(xì)胞密集排列構(gòu)成,卵母細(xì)胞較小,核圓形深染,胞漿不多,顆粒細(xì)胞較少,卵巢間質(zhì)不明顯。隨著孕齡的增加,顆粒細(xì)胞增多,逐漸圍繞原始卵母細(xì)胞,形成原始卵泡。在孕27~28周的胎兒卵巢中,已可見部分原始卵泡形成,卵巢間質(zhì)開始增多。在孕晚期胎兒卵巢,原始卵母細(xì)胞明顯減少,多數(shù)已被顆粒細(xì)胞包繞,隨著孕齡增加,原始卵泡體積增大,卵母細(xì)胞核圓形,增大,胞漿豐富紅染,圍繞顆粒細(xì)胞增多,卵巢間質(zhì)增多,血管增生。隨著孕齡增加,卵母細(xì)胞進(jìn)一步減少,僅剩形態(tài)結(jié)構(gòu)清晰的原始卵泡。TUNEL染色發(fā)現(xiàn),細(xì)胞凋亡主要存在原始卵母細(xì)胞,顆粒細(xì)胞也存在凋亡現(xiàn)象。隨著孕齡增加,卵巢發(fā)育進(jìn)一步成熟,原始卵泡形成,數(shù)目逐漸減少,凋亡細(xì)胞相應(yīng)減少,且主要發(fā)生于顆粒細(xì)胞。說明在胎兒卵巢發(fā)育過程中,卵母細(xì)胞的數(shù)量持續(xù)減少,原始卵泡形成,在這個(gè)過程中,細(xì)胞凋亡起著至關(guān)重要作用。從而提示對(duì)于原始卵泡形成階段,卵細(xì)胞的減少多由于卵細(xì)胞本身的凋亡,而對(duì)于原始卵泡形成后,卵泡閉鎖可能基于顆粒細(xì)胞的凋亡。
近年來,對(duì)人胎兒卵巢細(xì)胞凋亡的研究已取得一些進(jìn)展,有研究者[7]利用TUNEL法觀察孕中、晚期胎兒卵細(xì)胞,發(fā)現(xiàn)部分卵細(xì)胞出現(xiàn)陽性反應(yīng),即發(fā)生凋亡,而且孕中期胎兒卵細(xì)胞凋亡發(fā)生率明顯高于孕晚期,且發(fā)現(xiàn)Caspase-3在胎兒卵細(xì)胞內(nèi)廣泛表達(dá),而且與卵細(xì)胞凋亡呈正相關(guān),提示Caspase-3可能參與卵細(xì)胞凋亡的調(diào)控。另外,胎兒早期卵泡的顆粒細(xì)胞內(nèi)未見Caspase-3表達(dá),TUNEL法檢測(cè)亦未見陽性顆粒細(xì)胞,提示早期卵泡閉鎖始于卵細(xì)胞凋亡。De Pol等[8]采用TUNEL法觀察到了孕18~20周的人類胚胎卵巢內(nèi)典型的凋亡細(xì)胞形態(tài),并且還證實(shí),這個(gè)時(shí)期的胚胎中只有生殖細(xì)胞發(fā)生了凋亡。這與本實(shí)驗(yàn)結(jié)果一致。說明在人胎兒卵巢的發(fā)育過程中,在卵細(xì)胞的選擇性發(fā)育成熟過程中,細(xì)胞凋亡起著關(guān)鍵作用。
女性卵母細(xì)胞儲(chǔ)備量一出生就已確定,在女性一生中只有少數(shù)卵泡能完成排卵。婦女在其成年期大約可以排400個(gè)卵母細(xì)胞[9-10],在早期卵泡閉鎖過程中,卵母細(xì)胞首先發(fā)生凋亡,卵泡顆粒細(xì)胞由內(nèi)層向外層逐漸凋亡;而生長晚期的卵泡,顆粒細(xì)胞首先凋亡,誘導(dǎo)卵母細(xì)胞凋亡,觸發(fā)了卵泡閉鎖[11]。胎兒時(shí)期卵母細(xì)胞的凋亡、每個(gè)卵泡周期非優(yōu)勢(shì)卵泡的清除以及植入前胚胎細(xì)胞的凋亡,均保證了卵巢、卵泡、卵母細(xì)胞和胚胎的正常發(fā)育,為獲得優(yōu)秀后代提供了保障。卵巢內(nèi)卵母細(xì)胞及顆粒細(xì)胞的凋亡不僅是引起卵泡閉鎖的原因,而且凋亡與卵巢正?;虍惓5墓δ芟嚓P(guān)。卵巢顆粒細(xì)胞的凋亡就是卵巢早衰發(fā)病的主要原因[12]。有學(xué)者從不同角度對(duì)其病因進(jìn)行了研究,無論從組織水平、分子水平,還是基因水平,都發(fā)現(xiàn)有顆粒細(xì)胞的凋亡[13-16]。
因此,研究卵巢卵母細(xì)胞與顆粒細(xì)胞凋亡,對(duì)改善和提高女性生育能力,保持卵巢功能有重要意義。尤其是對(duì)人胎兒卵巢發(fā)育過程、卵泡凋亡和閉鎖的機(jī)制研究,將為最終實(shí)現(xiàn)對(duì)人卵泡發(fā)育的調(diào)控提供線索,將有助于了解卵母細(xì)胞發(fā)育機(jī)制。通過人為誘導(dǎo),延緩或促進(jìn)卵母細(xì)胞凋亡,開發(fā)利用卵巢內(nèi)卵泡資源有重要意義,可為女性生殖系統(tǒng)疾病、輔助生殖技術(shù)提供基礎(chǔ)研究方向,有重大的理論意義和廣泛應(yīng)用價(jià)值。
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Observation and research of morphology and apoptosis in fetal ovary
WANG Yongxia1WU Yuanyuan2MA Na3▲ZHONG Xingming1CUI Rong1WANG Xiaolan1YANG Ning1MIAO Zhulin1
1.Guangdong Family Planning Institute of Science and Technology,Guangdong Province,Guangzhou 510600,China; 2.Reproductive Center,Shanghai First Maternity and Infant Hospital,Tongji University School of Medicine,Shanghai 200204,China;3.Department of Physiology,Basic School of Guangdong Pharmaceutical University,Guangdong Province,Guangzhou 510006,China
ObjectiveTo investigate the development and apoptosis of the oocyte during human fetal development.MethodsOvarian tissues from 18 fetuses(aged 24-32 weeks)from January 2013 to December 2013 in Guangdong Family Planning Institute of Science and Technology were obtained,including 8 fetuses aged 24-27 weeks(midpregnancy)and 10 fetusesaged 28-32 weeks(late-pregnancy).The morphology and developmentofoocyte and the occurrence ofoocyte apoptosis during human fetal follicular development were examined using HE-staining and TUNEL method.ResultsMicroscope observation after HE-staining,the ovarian tissues in the midpregnancy had more oocytes than the late-pregnancy,less ovarian stroma and granulosa cells.The granulosa cells increased gradually,and primordial follicles grown up and began to accumulate around the oocytes during human fetal development,while the number of oocytes decreased. The number of original oocytes decreased significantly in late-pregnancy,Primordial follicle grown up and began to accumulate with the increase of gestational age,primitive follicles more complete.TUNEL stained showed that,apoptotic cell was easily found in follicular oocytes that had not formed the follicle,granule there were a few apoptotic cells around egg cells in the late-pregnancy,no egg cell in follicle was positive stained.The number of apoptotic cell in midpregnancy was more than late-pregnancy,and apoptosis mainly happened on the oocytes that had not formed a primordialfollicle.ConclusionThe originaloocytes become less during the human fetalfollicular development,it is caused by the oocyte apoptotosis,the original follicular is formed in the fetal ovar of the late-pregnancy.
Fetal ovarian;Oocyte;Apoptosis
R735.7
A
1673-7210(2015)07(b)-0149-03
2015-03-20本文編輯:任念)
廣東省人口計(jì)生委科研課題項(xiàng)目(20110202)。
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