中緬樹(shù)鼩精子形態(tài)特征及冷凍損傷

張遠(yuǎn)旭, 平述煌, 楊世華,*

(1. 昆明理工大學(xué) 生命科學(xué)與技術(shù)學(xué)院衰老與腫瘤分子遺傳學(xué)實(shí)驗(yàn)室, 云南 昆明650500; 2. 中國(guó)科學(xué)院和云南省動(dòng)物模型與人類疾病機(jī)理重點(diǎn)實(shí)驗(yàn)室, 中國(guó)科學(xué)院昆明動(dòng)物研究所, 云南 昆明 650223)

中緬樹(shù)鼩精子形態(tài)特征及冷凍損傷

張遠(yuǎn)旭1,2, 平述煌1, 楊世華1,*

(1.昆明理工大學(xué) 生命科學(xué)與技術(shù)學(xué)院衰老與腫瘤分子遺傳學(xué)實(shí)驗(yàn)室,云南 昆明650500; 2.中國(guó)科學(xué)院和云南省動(dòng)物模型與人類疾病機(jī)理重點(diǎn)實(shí)驗(yàn)室,中國(guó)科學(xué)院昆明動(dòng)物研究所,云南 昆明650223)

樹(shù)鼩作為一種新型的、接近靈長(zhǎng)類的實(shí)驗(yàn)動(dòng)物, 在醫(yī)學(xué)生物學(xué)上的應(yīng)用受到越來(lái)越多的重視。精子的結(jié)構(gòu)特性研究及冷凍后結(jié)構(gòu)的完整性分析是精子生物學(xué)的主要內(nèi)容, 也有助于樹(shù)鼩的實(shí)驗(yàn)室快速繁殖。該研究采用人工飼養(yǎng)的中緬樹(shù)鼩(Tupaia belangeri chinensis), 結(jié)果顯示其睪丸占總體重的(1.05±0.07)%, 總體積為(1.12±0.10) mL。附睪尾及輸精管精子總量估計(jì)在2.2×107～8.8×107, 其運(yùn)動(dòng)度和頂體完整率分別為(68.8±3.9)%和(90.0±2.1)%。利用掃描電子顯微鏡和透射電子顯微鏡對(duì)樹(shù)鼩附睪精子的超微結(jié)構(gòu)進(jìn)行的觀察和分析顯示精子頭部呈圓形或卵圓形; 頭部長(zhǎng)度、寬度平均分別為6.65和5.82 μm; 精子尾部中段、主段、尾段和精子總長(zhǎng)度平均分別為13.39、52.35、65.74和73.05 μm; 尾部中段的線粒體螺旋數(shù)量為48個(gè), 其軸絲結(jié)構(gòu)為典型的“9+9+2”結(jié)構(gòu)。冷凍解凍后的精子主要表現(xiàn)在頂體與質(zhì)膜不完整、精子斷裂、尾部扭曲和膨大。上述結(jié)果提示樹(shù)鼩精子與其他哺乳動(dòng)物精子的結(jié)構(gòu)特征相似, 但是精子大小和線粒體螺旋數(shù)目有明顯的差別, 且超微結(jié)構(gòu)改變?nèi)允抢鋬鼍舆\(yùn)動(dòng)和受精能力下降的主要原因。

樹(shù)鼩; 睪丸; 精子; 形態(tài)結(jié)構(gòu); 冷凍損傷

樹(shù)鼩是一種晝行、樹(shù)棲的小型哺乳動(dòng)物, 曾被定義為攀鼩目、靈長(zhǎng)目(Liu et al, 2001; Wong & Kaas, 2009), 目前對(duì)樹(shù)鼩的分類地位仍然存在爭(zhēng)議。但無(wú)論如何, 樹(shù)鼩是最接近于靈長(zhǎng)類的小型哺乳動(dòng)物,具有許多靈長(zhǎng)類動(dòng)物共有的特征(Poonkhum et al, 2000)。樹(shù)鼩在人類醫(yī)學(xué)及生物學(xué)研究方面具有重要地位, 如神經(jīng)生物學(xué)(Remple et al, 2007)、人類肝炎病毒感染(Xu et al, 2007; Li et al, 2008)、視覺(jué)系統(tǒng)發(fā)育(Poveda & Kretz, 2009)及群體階層應(yīng)激(Kozicz et al, 2008; Zambello et al, 2010)等。目前, 有關(guān)雄性樹(shù)鼩生殖生物學(xué)研究的報(bào)道較少(Collins et al, 2007)。Collins et al于1982年報(bào)道了雄性樹(shù)鼩生殖道的解剖學(xué)特征和生理功能(Collins et al, 1982); 隨后, 有研究報(bào)道了雄性樹(shù)鼩從出生到性成熟過(guò)程的生殖器官生長(zhǎng)與發(fā)育(Collins & Tsang, 1987); 精子細(xì)胞在睪丸內(nèi)的成熟過(guò)程(Maeda et al, 1998); 雄性樹(shù)鼩精子的形成和染色質(zhì)凝聚(Suphamungmee et al, 2008)等結(jié)果。然而, 樹(shù)鼩睪丸、精子形態(tài)結(jié)構(gòu)特性的研究仍然很有限。我們?cè)鴪?bào)道樹(shù)鼩精子可以被冷凍保存(Ping et al, 2011), 但是精子冷凍后的超微結(jié)構(gòu)變化尚不清楚。為此, 本研究分析樹(shù)鼩的睪丸、附睪精子的形態(tài)學(xué)結(jié)構(gòu)特征, 以及樹(shù)鼩精子冷凍復(fù)蘇后的結(jié)構(gòu)變化。

1 材料和方法

1.1 實(shí)驗(yàn)材料

4只健康的成年雄性樹(shù)鼩, 均來(lái)自中科院昆明動(dòng)物研究所。所有實(shí)驗(yàn)均在當(dāng)年3—5月份完成。除特殊說(shuō)明外, 所用化學(xué)試劑均購(gòu)自Sigma公司(St. Louis, MO, USA)。精子稀釋液為TALP-HEPES (modified Tyrode’s solution with albumin, lactate and pyruvate) (Bavister et al, 1983), 冷凍液為TTE(Tris-TES-egg yolk)(Si et al, 2000), 均按照文獻(xiàn)中描述的方法配制。

1.2 實(shí)驗(yàn)方法

1.2.1 睪丸的稱重和測(cè)量 氯胺酮(60～70 mg/kg體重)經(jīng)肌肉注射, 待樹(shù)鼩麻醉后稱其體重。在麻醉狀態(tài)下, 外科手術(shù)暴露腹腔后取出睪丸、附睪及輸精管; 在附睪頭、尾相連處中間部分分離彼此, 并對(duì)上述各部分分別稱重; 游標(biāo)卡尺測(cè)定睪丸的縱橫長(zhǎng)度; 將分離得到的睪丸分別緩慢浸入含有3 mL生理鹽水的5 mL量筒中, 通過(guò)觀察量筒中液體體積的增加來(lái)確定睪丸的體積。

1.2.2 精子收集與分析 分離獲得的附睪尾和輸精管置于平皿中, 37 ℃TALP-HEPES洗滌; 隨后置于1 mL TALP-HEPES中剪至～1 cm, 用鑷子輕輕擠壓管腔以釋放儲(chǔ)存的精子; 微量移液器吸取所有精液, 混勻后取10 μL精液樣品檢測(cè)精子的數(shù)量、運(yùn)動(dòng)度和頂體完整性; 剩余的精子經(jīng)離心處理后將沉淀精子固定, 用于超微結(jié)構(gòu)分析。

精子計(jì)數(shù)和運(yùn)動(dòng)度測(cè)定 10 μL新鮮精子用TALP-HEPES稀釋100～500倍后取10 μL滴加到已預(yù)熱至37 ℃的血細(xì)胞計(jì)數(shù)板上, 在光學(xué)顯微鏡下計(jì)數(shù)前向運(yùn)動(dòng)精子和原地不動(dòng)精子, 用二者數(shù)量之和計(jì)算每只樹(shù)鼩獲得精子數(shù)量; 運(yùn)動(dòng)精子占所有計(jì)數(shù)精子的百分比即為精子運(yùn)動(dòng)度, 其操作步驟參考文獻(xiàn)報(bào)道(Saragusty et al, 2009)。每次至少計(jì)數(shù)200個(gè)精子, 重復(fù)兩次。

頂體完整率檢測(cè) 熒光染料FITC-PNA檢測(cè)精子的頂體完整率, 其操作步驟見(jiàn)文獻(xiàn)報(bào)道(Ping et al, 2011)。簡(jiǎn)要描述如下：精子樣品在TALP-HEPES中2 500 r/min、5 min離心和洗滌2次, 并稀釋至～1×105個(gè)/mL; 取20 μL滴在載玻片上, 自然風(fēng)干后,在通風(fēng)櫥中用甲醇溶液浸泡固定5 min; 載玻片取出風(fēng)干后, 加入40 μg/mL FITC-PNA 40 μL于精子樣品上并在37 ℃培養(yǎng)箱中避光孵育30 min; PBS(pH 7.2)洗去多余的染料, 在熒光顯微鏡下檢查,其激發(fā)光波長(zhǎng)和發(fā)射光波長(zhǎng)分別為490和515 nm。頂體完整的精子均勻地著上蘋果綠色, 而頂體不完整的精子部分著色或不著色。計(jì)數(shù)頂體完整和不完整的精子, 每次至少計(jì)數(shù)200個(gè), 頂體完整精子占所有計(jì)數(shù)精子的比率為精子頂體完整率。

1.2.3 精子冷凍程序 冷凍方法參照Ping et al (2011)。從雄性樹(shù)鼩附睪中獲取精子, 離心洗滌后TTE稀釋至濃度為4×106個(gè)/mL; 將其置于4 ℃冰箱中進(jìn)行預(yù)冷處理, 直至精子混合液降到4 ℃; 將精子混合液裝入0.25 mL冷凍麥管, 水平放置在15 cm×10 cm的鋁架上并將鋁架懸掛在離液氮面4 cm的位置上平衡10 min; 隨后將冷凍麥管投入液氮中冷凍保存。冷凍保存2 d后, 取出麥管, 直接投入37 ℃水浴中解凍; 解凍后將精子釋放到1.5 mL離心管中, 并用5×精液體積的TALP-HEPES稀釋;隨后以2 500 r/min、5 min離心洗滌1次, 將沉淀精子按電鏡樣品的制作方法處理。

1.2.4 電鏡樣品的處理與觀察 掃描電鏡樣品的處理方法參照文獻(xiàn)（Simeo et al, 2010)報(bào)道進(jìn)行。簡(jiǎn)述如下：冷凍?復(fù)蘇精子的沉淀中加入2.5%戊二醛PBS緩沖液(0.1 mol/L, pH 7.4, 4 ℃), 4 ℃前固定24 h; PBS緩沖液輕輕漂洗6次, 其中前3次每次10 min,后3次每次20 min; 質(zhì)量分?jǐn)?shù)為1%～2%的鋨酸4 ℃后固定1 h; 雙固定后, PBS緩沖液漂洗3次, 其中前2次每次5 min, 后1次30 min; 系列酒精逐級(jí)脫水; 冷凍干燥處理, 以及離子濺射儀噴金; 掃描電子顯微鏡觀察精子外部形態(tài)結(jié)構(gòu)并拍照和分析。

透射電鏡樣品的處理 樣品處理也參照文獻(xiàn)（Simeo et al，2010)報(bào)道進(jìn)行。簡(jiǎn)述如下：冷凍?復(fù)蘇精子的沉淀中加入2.5%戊二醛PBS緩沖液(0.1 mol/L, pH 7.4), 4 ℃前固定24 h; PBS緩沖液漂洗6次, 其中前3次每次10 min, 后3次每次20 min; 質(zhì)量分?jǐn)?shù)為1%的鋨酸4 ℃后固定1 h; 雙固定后, PBS緩沖液漂洗3次, 其中前2次每次5 min, 后1次30 min; 梯度酒精、丙酮逐級(jí)脫水; Epon618環(huán)氧樹(shù)脂滲透、包埋、半切片、光鏡定位、修塊; Lecia-R超薄切片機(jī)切片(切片厚約90 nm); 切片經(jīng)檸檬酸鉛和醋酸雙氧鈾雙重染色后用透射電子顯微鏡觀察精子超微結(jié)構(gòu)并拍照和分析。

2 結(jié) 果

2.1 樹(shù)鼩睪丸和附睪精子的形態(tài)與特征

測(cè)定結(jié)果見(jiàn)表1。

表1 樹(shù)鼩睪丸大小及附睪精子特征Tab. 1 Testis measurements and sperm characteristics of tree shrew

2.2 樹(shù)鼩精子的超微結(jié)構(gòu)

新鮮樹(shù)鼩精子超微結(jié)構(gòu)見(jiàn)表2。

樹(shù)鼩精子由頭、尾部組成, 尾部是精子最長(zhǎng)的部分, 可分為中段、主段和末段等三部分(表2和圖1A, B)。在掃描電鏡下, 精子頭部呈圓形或卵圓形;精子尾部明顯分為中段(mid-piece, MP)、主段(principal piece, PP)和末段(end piece)。在透射電鏡下, 整個(gè)精子由質(zhì)膜包裹, 其中頭部頂體區(qū)域的質(zhì)膜連接呈松弛狀態(tài); 頭部由核、頂體和后頂體鞘組成; 頂體為一位于頭頂部的薄層帽狀囊腔; 頂體和核之間存在頂體下間隙(subacrosomal space, SAS);核由核膜包被, 為頭部的主要組成部分, 占據(jù)頭部的絕大部分體積, 其電子密度大而均勻; 核膜外為電子密度小而均勻的頂體; 由頭后端向前端, 核膜逐漸變薄, 在赤道段區(qū)域最堅(jiān)固(圖1C, D)。

頭部核末端有一個(gè)凹面植入窩, 與頸部的小頭嵌合; 頸部, 又稱連接段, 由小頭、節(jié)柱和近端中心粒組成; 小頭的凸型致密纖維板結(jié)構(gòu), 與核末端的植入窩基板線相吻合; 稀疏、適度的電子致密物質(zhì)填充在基板線和小頭的間隙里(圖1C)。

表2 樹(shù)鼩精子細(xì)胞的形態(tài)測(cè)定參數(shù)Tab. 2 Morphometry of epididymal sperm of tree shrew

圖1 樹(shù)鼩精子的超微結(jié)構(gòu)Fig. 1 Tree shrew sperm morphology

尾主要由軸絲和外周致密纖維組成; 軸絲起于精子頸部, 延伸至尾部末段; 軸絲由9對(duì)雙聯(lián)體周圍微管和一對(duì)中央微管組成, 9對(duì)雙聯(lián)體周圍微管均勻環(huán)繞在中央微管周圍, 形成了“9+9+2”的微管構(gòu)造; 每一對(duì)雙聯(lián)體均由一個(gè)小的圓柱狀微管和一個(gè)不完整C形微管組成; 從中心微管到周圍微管呈現(xiàn)放射狀延伸, 一對(duì)中心微管由一個(gè)短橋連接。在尾部中段和主段, 軸絲均有9根外周致密纖維環(huán)繞,每一根外周致密纖維伴隨一對(duì)雙聯(lián)體微管; 纖維橫截面呈現(xiàn)花瓣?duì)?、黃豆?fàn)罨驁A形。以中心微管軸絲為基準(zhǔn)將外周致密纖維按順時(shí)針?lè)较蚓幪?hào)1～9, 結(jié)果發(fā)現(xiàn)外周致密纖維大小不同：尾部中段, 1、2、5、6外周致密纖維明顯較其他纖維粗; 尾部主段, 1、5、6外周致密纖維也明顯較其他纖維粗; 尾部中段靠近頸部的纖維較粗, 主段靠近尾部末段的纖維較細(xì)(圖1E?H)。

外周致密纖維在尾部中段由線粒體鞘環(huán)繞, 在主段由纖維鞘環(huán)繞。在尾部中段, 線粒體頭尾相接形成一個(gè)螺旋環(huán)繞致密纖維; 精子線粒體螺旋數(shù)為48; 在線粒體鞘的末端有一個(gè)三角狀環(huán), 連接質(zhì)膜基底和向內(nèi)突出的致密纖維頂端; 環(huán)位于螺旋狀線粒體末端和主段纖維鞘前端之間, 為中段和主段的分界線。纖維鞘從精子主段延伸到末段, 其電子密度高, 且越靠近末端越細(xì), 直至消失; 纖維鞘由背、腹側(cè)縱柱及其環(huán)形連接肋柱組成, 且這兩個(gè)縱柱似乎固定在外周致密纖維3和8上(圖1E?G)。

2.3 冷凍復(fù)蘇精子形態(tài)結(jié)構(gòu)的變化

鮮精精子運(yùn)動(dòng)度和頂體完整率分別為(68.82±3.95)%和(89.99±2.14)%, 解凍后精子復(fù)蘇運(yùn)動(dòng)度和頂體完整率分別為(44.73±3.67)%和(76.52±1.84)%。 FITC-PNA熒光染色后, 可見(jiàn)經(jīng)冷凍/解凍, 部分精子的頂體表現(xiàn)出部分或完全缺失(圖2A); 精子頭部核表面的頂體不勻稱, 表明經(jīng)冷凍/解凍后, 精子頂體結(jié)構(gòu)出現(xiàn)損傷(圖2 B,C); 精子頭部核表面無(wú)可見(jiàn)頂體, 表明頂體完全缺失(圖2D)。

圖2 冷凍/解凍精子頭部頂體的缺損Fig. 2 Sperm acrosome defects after freezing and thawing

經(jīng)過(guò)冷凍/解凍處理后, 精子膜結(jié)構(gòu)變化主要表現(xiàn)在以下幾個(gè)方面(圖2)：頭部頂體處質(zhì)膜破損;頭部和頸部連接處質(zhì)膜缺失; 尾部中段線粒體鞘外質(zhì)膜缺失; 尾部主段質(zhì)膜缺失; 線粒體膜不完整。

在冷凍/解凍過(guò)程中, 外來(lái)冷凍壓力會(huì)造成精子的機(jī)械性損傷, 導(dǎo)致其不完整結(jié)構(gòu)。頭、頸部斷裂致頭、尾部分離(圖4A, E); 尾部中段、主段斷裂;尾部主段、末段斷裂(圖4B?D)。

圖3 冷凍/解凍精子質(zhì)膜的缺損Fig. 3 Sperm plasma membrane defects after freezing and thawing

圖4 冷凍/解凍精子不同部位間的斷裂Fig. 4 Sperm breakage in different locus after freezing and thawing

冷凍/解凍對(duì)精子的另一個(gè)損傷體現(xiàn)于尾部的彎曲度。精子尾部彎曲通常發(fā)生于中段或主段(圖5), 致使主段和中段的線粒體被同一質(zhì)膜包裹, 造成軸絲結(jié)構(gòu)受損, 進(jìn)而導(dǎo)致精子運(yùn)動(dòng)功能喪失。

圖5 冷凍/解凍精子尾部的彎曲Fig. 5 Coiled sperm tail after freezing and thawing

解凍過(guò)程中, 滲透性防凍劑致使胞內(nèi)滲透壓高于胞外, 胞外水向胞內(nèi)流入, 有可能進(jìn)而導(dǎo)致細(xì)胞膨脹, 如精子尾部中段膨脹(圖6)。

4 討 論

與哺乳動(dòng)物及靈長(zhǎng)類精子相似, 樹(shù)鼩精子頭部呈卵圓形(Downing et al, 2005)。不同物種精子頭部的長(zhǎng)度和寬度各不相同, 如人類為4.5 μm和3 μm (Pedersen, 1969), 黑熊為6.57 μm和4.76 μm (Brito et al, 2010), 而樹(shù)鼩為6.65 μm和5.82 μm。樹(shù)鼩精子全長(zhǎng)為73.05 μm, 與眼鏡熊和河貍精子長(zhǎng)度相似(Gage, 1998; Bierla et al, 2007), 但與倉(cāng)鼠(119 μm) (Nagy, 1994)和人類(55～60 μm)差異顯著。另外,由于擠壓釋放附睪尾和輸精管中的精子, 所計(jì)算的精子總數(shù)可能存在差異, 但損失精子比例可能很小。

圖6 冷凍/解凍精子尾部中段的膨脹Fig. 6 Swollen sperm tail mid-piece after freezing and thawing

線粒體鞘在精子尾部中段呈螺旋形包繞外周致密纖維, 不同物種動(dòng)物精子尾部中段的線粒體螺旋數(shù)各異：人類為15個(gè); 倉(cāng)鼠為133個(gè)(Nagy, 1994);黑熊為59個(gè)(Gage, 1998), 而樹(shù)鼩為48個(gè)。

軸絲由與精子運(yùn)動(dòng)密切相關(guān)的微管組成。不同物種動(dòng)物精子尾部的軸絲結(jié)構(gòu)不同, 樹(shù)鼩精子的軸絲結(jié)構(gòu)類似于人類、蟹、長(zhǎng)鼻浣熊、野豬及蜜蜂, 即為“9+9+2”型(EI-Shoura et al, 1995; Báo et al, 2004; Fischman et al, 2009; Lima et al, 2009; Mancini et al, 2009)。

哺乳動(dòng)物冷凍精子的受精率和受孕率較鮮精為低(O'Meara et al, 2005), 因?yàn)樵诶鋬鲞^(guò)程中, 精子經(jīng)受著許多外來(lái)壓力的考驗(yàn), 如冷休克、膜相轉(zhuǎn)換、冰晶形成等(Aboagla & Terada, 2004; White, 1993)。冷休克會(huì)導(dǎo)致部分精子運(yùn)動(dòng)能力喪失、膜結(jié)構(gòu)損傷(Jones & Martin, 1973; Ortman & Rodriguez-Martinez, 1994)及線粒體膜電位改變(Watson, 1995)。冷凍時(shí), 由于細(xì)胞外冰晶形成將造成胞外液體滲透壓升高, 促使細(xì)胞內(nèi)水分外排, 導(dǎo)致細(xì)胞收縮, 如細(xì)胞膜不能承受如此劇烈的收縮變化, 將出現(xiàn)細(xì)胞損傷; 而解凍時(shí), 胞外水的流入又可致使細(xì)胞破裂(Mazur, 1984)。在對(duì)樹(shù)鼩冷凍/解凍精子超微結(jié)構(gòu)的觀察和分析中發(fā)現(xiàn)：樹(shù)鼩精子冷凍/解凍后, 質(zhì)膜和頂體最容易受到損傷, 其頂體會(huì)完全缺失或損傷;無(wú)論在頭部還是尾部中段和主段, 質(zhì)膜均有可能腫脹、變薄、破損、甚至缺失。同時(shí), 解凍時(shí), 胞內(nèi)的滲透性防凍劑將導(dǎo)致胞內(nèi)滲透壓高于胞外, 細(xì)胞吸水, 進(jìn)而使得細(xì)胞因滲透壓梯度作用而表現(xiàn)為不均一膨脹, 如部分精子尾部中段膨脹。再者, 由于機(jī)械性壓力影響，很容易導(dǎo)致樹(shù)鼩精子頭、頸部斷裂, 尾部中段、主段斷裂, 造成精子的致死性損傷。由此可見(jiàn), 質(zhì)膜和頂體完整性受損仍然是導(dǎo)致樹(shù)鼩冷凍/解凍精子受精率低下的主要原因。

因此, 通過(guò)研究樹(shù)鼩精子的超微結(jié)構(gòu)及冷凍對(duì)超微結(jié)構(gòu)的影響, 有助于改善現(xiàn)有的樹(shù)鼩精子冷凍方法和提高樹(shù)鼩的人工繁殖能力, 為低溫生物學(xué)發(fā)展提供理論依據(jù)。

Aboagla EME, Terada T. 2004. Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa[J]. Theriogenology, 62(6): 1160-1172.

Báo SN, Gon?alves Sim?es D, Lino-Neto J. 2004. Sperm ultrastructure of the bees Exomalopsis (Exomalopsis) auropilosa Spinola 1853 and Paratetrapedia (Lophopedia) sp. Michener & Moure 1957 (Hymenoptera, Apidae, Apinae)[J]. J Submicrosc Cytol Pathol, 36(1): 23-28.

Bavister BD, Leibfried ML, Lieberman G. 1983. Development of preimplantation embryos of the golden hamster in a defined culture medium[J]. Biol Reprod, 28(1): 235-247.

Bierla JB, Gizejewski Z, Leigh CM, Ekwall H, S?derquist L, Rodriguez-Martinez H, Zalewski K, Breed WG. 2007. Sperm morphology of the Eurasian beaver, Castor fiber: an example of a species of rodent with highly derived and pleiomorphic sperm populations[J]. J Morphol, 268(8): 683-689.

Brito LF, Sertich PL, Stull GB, Rives W, Knobbe M. 2010. Sperm ultrastructure, morphometry, and abnormal morphology in American black bears (Ursus americanus)[J]. Theriogenology, 74(8):1403-1413.

Collins PM, Tsang WN. 1987. Growth and reproductive development in the male tree shrew (Tupaia belangeri) from birth to sexual maturity[J]. Biol Reprod, 37(2): 261-267.

Collins PM, Tsang WN, Lofts B. 1982. Anatomy and function of the reproductive tract in the captive male tree shrew (Tupaia belangeri)[J]. Biol Reprod, 26(1): 169-182.

Collins PM, Tsang WN, Urbanski HF. 2007. Endocrine correlates of reproductive development in the male tree-shrew (Tupaia belangeri) and the effects of infantile exposure to exogenous androgens[J]. GenComp Endocrinol, 154(1-3): 22-30.

Downing Meisner A, Klaus AV, O'Leary MA. 2005. Sperm head morphology in 36 species of artiodactylans, perissodactylans, and cetaceans (Mammalia)[J]. J Morphol, 263(2): 179-202.

EI-Shoura SM, Abdel Aziz M, Ali ME, EI-Said MM, Ali KZM, Kemeir MA, Raoof AMS, Allam M, Elmalik EMA. 1995. Deleterious effects of khat addiction on semen parameters and sperm ultrastructure[J]. Hum Reprod, 10(9): 2295-2300.

Fischman ML, Bolondi A, Cisale H. 2009. Ultrastructure of sperm 'tail stump' defect in wild boar[J]. Andrologia, 41(1): 35-38.

Gage MJG. 1998. Mammalian sperm morphometry[J]. Proc Biol Sci, 265(1391): 97-103.

Jones RC, Martin ICA. 1973. The effects of dilution, egg yolk and cooling to 5°C on the ultrastructure of ram spermatozoa[J]. J Reprod Fertil, 35(2): 311-320.

Kozicz T, Bordewin LAP, Czéh B, Fuchs E, Roubos EW. 2008. Chronic psychosocial stress affects corticotropin-releasing factor in the paraventricular nucleus and central extended amygdala as well as urocortin 1 in the non-preganglionic Edinger-Westphal nucleus of the tree shrew[J]. Psychoneuroendocrinology, 33(6): 741-754.

Li Y, Wan DF, Wei W, Su JJ, Cao J, Qiu XK, Ou C, Ban KC, Yang C, Yue HF. 2008. Candidate genes responsible for human hepatocellular carcinoma identified from differentially expressed genes in hepatocarcinogenesis of the tree shrew (Tupaia belangeri chinesis)[J]. Hepatol Res, 38(1): 85-95.

Lima GL, Barros FFPC, Costa LLM, Castelo TS, Fontenele-Neto JD, Silva AR. 2009. Determination of semen characteristics and sperm cell ultrastructure of captive coatis (Nasua nasua) collected byelectroejaculation[J]. Anim Reprod Sci, 115(1-4): 225-230.

Liu FGR, Miyamoto MM, Freire NP, Ong PQ, Tennant MR, Young TS, Gugel KF. 2001. Molecular and morphological supertrees for eutherian (placental) mammals[J]. Science, 291(5509): 1786-1789.

Maeda S, Nakamuta N, Nam SY, Kimura J, Endo H, Rerkamnuaychoke W, Kurohmaru M, Yamada J, Hayashi Y, Nishida T. 1998. Monoclonal antibody TSd-1 is specific to elongating and matured spermatids in testis of common tree shrew (Tupaia glis)[J]. J Vet Med Sci, 60(3): 345-349.

Mancini K, Lino-Neto J, Dolder H, Dallai R. 2009. Sperm ultrastructure of the European hornet Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae)[J]. Arthropod Struct Dev, 38(1): 54-59.

Mazur P. 1984. Freezing of living cells: mechanisms and implications[J]. Am J Physiol, 247(3 Pt 1): C125-C142.

Nagy F. 1994. On the ultrastructure of the spermatozoa in the Siberian hamster (Phodopus sungorus campbelli)[J]. J Submicrosc Cytol Pathol, 26(4): 533-544.

O'Meara CM, Hanrahan JP, Donovan A, Fair S, Rizos D, Wade M, Boland MP, Evans ACO, Lonergan P. 2005. Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen[J]. Theriogenology, 64(8): 1797-1808.

Ortman K, Rodriguez-Martinez H. 1994. Membrane damage during dilution, cooling and freezing-thawing of boar spermatozoa packaged in plastic bags[J]. J Vet Med Ser, 41(1-10): 37-47.

Pedersen H. 1969. Ultrastructure of the ejaculated human sperm[J]. Z Zellforsch Mikrosk Anat, 94(4): 542-554.

Ping S, Wang F, Zhang Y, Wu C, Tang W, Luo Y, Yang S. 2011. Cryopreservation of epididymal sperm in tree shrews (Tupaia belangeri)[J]. Theriogenology, 76(1): 39-46.

Poonkhum R, Pongmayteegul S, Meeratana W, Pradidarcheep W, Thongpila S, Mingsakul T, Somana R. 2000. Cerebral microvascular architecture in the common tree shrew (Tupaia glis) revealed by plastic corrosion casts[J]. Microsc Res Tech, 50(5): 411-418.

Poveda A, Kretz R. 2009. c-Fos expression in the visual system of the tree shrew (Tupaia belangeri)[J]. J Chem Neuroanat, 37(4): 214-228.

Remple MS, Reed JL, Stepniewska I, Lyon DC, Kaas JH. 2007. The organization of frontoparietal cortex in the tree shrew (Tupaia belangeri): II. Connectional evidence for a frontal-posterior parietal network[J]. J Comp Neurol, 501(1): 121-149.

Saragusty J, Gacitua H, Rozenboim I, Arav A. 2009. Protective effects of iodixanol during bovine sperm cryopreservation[J]. Theriogenology, 71(9): 1425-1432.

Si W, Zheng P, Tang XH, He XC, Wang H, Bavister BD, Ji WZ. 2000. Cryopreservation of rhesus macaque (Macaca mulatta) spermatozoa and their functional assessment by in vitro fertilization[J]. Cryobiology, 41(3): 232-240.

Simeo CG, Kurtz K, Rotllant G, Chiva M, Ribes E. 2010. Sperm ultrastructure of the spider crab Maja brachydactyla (Decapoda: Brachyura)[J]. J Morphol, 271(4): 407-417.

Suphamungmee W, Wanichanon C, Vanichviriyakit R, Sobhon P. 2008. Spermiogenesis and chromatin condensation in the common tree shrew, Tupaia glis[J]. Cell Tissue Res, 331(3): 687-699.

Watson PF. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function[J]. Reprod Fertil Dev, 7(4): 871-891.

White IG. 1993. Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review[J]. Reprod Fertil Dev, 5(6): 639-658.

Wong PY, Kaas JH. 2009. Architectonic Subdivisions of Neocortex in the tree shrew (Tupaia belangeri)[J]. Anat Rec-Adv Int Anat Evol Biol, 292(7): 994-1027.

Xu XP, Chen HB, Cao XM, Ben KL. 2007. Efficient infection of tree shrew (Tupaia belangeri) with hepatitis C virus grown in cell culture or from patient plasma[J]. J Gen Virol, 88(9): 2504-2512.

Zambello E, Fuchs E, Abumaria N, Rygula R, Domenici E, Caberlotto L. 2010. Chronic psychosocial stress alters NPY system: different effects in rat and tree shrew[J]. Prog Neuropsychopharmacol Biol Psychiatry, 34(1): 122-130.

Morphological characteristics and cryodamage of Chinese tree shrew (Tupaia belangeri chinensis) sperm

ZHANG Yuan-Xu1,2, PING Shu-Huang1, YANG Shi-Hua1,*

(1. Laboratory of Molecular Genetics of Aging and Tumor, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China; 2. Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, China)

The tree shrew (Tupaia belangeri chinensis) is a small non-rodent mammal, which is a relatively new experimental animal in medicine due to its close evolutionary relationship to primates and its rapid propagation. Sperm characteristics and cryopreservation in the tree shrew were the main contents of our spermatological research. Epididymal sperm were surgically harvested from male tree shrews captured from the Kunming area. The rate of testis weight to body weight was (1.05±0.07)%, volume of both testis was (1.12±0.10) mL, total sperm from epididymis and vas deferens were 2.2?8.8×107, and sperm motility and acrosome integrity were (68.8±3.9)% and (90.0±2.1)%, respectively. Sperm ultrastructure of the tree shrew was examined by scanning electron microscopy and transmission electron microscopy. Tree shrew sperm had a round or oval shaped head of approximately 6.65×5.82 μm, and midpiece, principal piece, tail, and total sperm lengths were 13.39, 52.35, 65.74, and 73.05 μm, respectively. The mitochondria in the midpiece consisted of approximately 48 gyres and had a 9+9+2 axonemal pattern. After freezing and thawing, sperm showed partly intact acrosomes and plasma membrane defects, and sperm breakages, twists, and swellings were found. The tree shrew had similar ultrastructure with other mammalians except for the mitochondria number and the sperm size. Ultrastructural alteration is still the main cause resulting in poor sperm after cryopreservation.

Tree shrew; Testis; Sperm; Morphological structure; Cryo-damage

張遠(yuǎn)旭,男,碩士研究生,主要從事疾病動(dòng)物模型研究

Q418; Q954.43

A

0254-5853-(2012)01-0029-08

10.3724/SP.J.1141.2012.01029

2011-11-01；接受日期：2011-12-31

國(guó)家自然科學(xué)基金(31071279)

?通信作者(Corresponding author)，E-mail: huashiyang@hotmail.com