雌性三倍體太平洋牡蠣減數(shù)分裂中期染色體觀察

辛榮+李慷均+王昭萍

DOI:10.3969/j.issn.1004-6755.2014.04.015

摘 要：采用鐵礬蘇木精壓片的方法，光鏡下觀察了三倍體太平洋牡蠣卵細(xì)胞第一次減數(shù)分裂Ⅰ中期染色體的聚合形態(tài)。研究發(fā)現(xiàn)，聚合形態(tài)在不同細(xì)胞、不同個(gè)體間變化較大，但大多數(shù)聚合體以三價(jià)體為主，同時(shí)存在著單價(jià)體、二價(jià)體和超聚合體。由于聚合價(jià)態(tài)的不同導(dǎo)致細(xì)胞內(nèi)聚合體數(shù)目出現(xiàn)較大差異，10～13個(gè)的細(xì)胞占多數(shù)，其他數(shù)目的較少，細(xì)胞內(nèi)最高可達(dá)18個(gè)聚合體。

關(guān)鍵詞：太平洋牡蠣；三倍體；減數(shù)分裂；聚合體

多倍體一直是貝類細(xì)胞遺傳學(xué)研究的熱點(diǎn)領(lǐng)域之一。研究染色體行為、數(shù)目不僅對(duì)于了解其遺傳組成、發(fā)育機(jī)制有重要意義，而且對(duì)于預(yù)測多倍體育種結(jié)果具有重要的參考價(jià)值。因太平洋牡蠣四倍體通常是通過三倍體卵子與二倍體精子受精后抑制第一極體獲得。本研究對(duì)于三倍體卵子染色體行為的觀察，旨在為其提供初步的參考數(shù)據(jù)。

1 材料與方法

1.1 材料

實(shí)驗(yàn)所用三倍體太平洋牡蠣取自威海榮城尋山。所選個(gè)體均經(jīng)流式細(xì)胞以鑒定，證明為三倍體個(gè)體。

1.2 方法

1.2.1 卵細(xì)胞制片樣品的制備 將牡蠣解剖，經(jīng)性別鑒定后，挑選成熟度較好的個(gè)體，解剖取卵，卵子先后經(jīng)200目和500目篩絹網(wǎng)過濾去除較大的組織碎片、組織液和碎屑。洗好的卵置于24 ℃的海水中促熟40 min使其胚泡破裂，待卵子沉淀后，去掉海水，加0.075 mol/L的氯化鉀低滲40 min，再用卡諾固定液（甲醇：冰醋酸=3：1）固定[1]，此后每隔20 min，20 min，40 min，40 min更換固定液一次，最后置4 ℃冰箱中保存?zhèn)溆谩?/p>

1.2.2 壓片 用吸管吸取卵細(xì)胞懸液，在潔凈載玻片上滴2滴，稍后，在樣品的稠密處，滴2～3滴蘇木精-鐵礬染液（0.5 g蘇木精溶解于100 mL的體積分?jǐn)?shù)為45%的醋酸中，后加適量的鐵明礬使染液呈棕藍(lán)色），輕輕蓋上蓋玻片，避免有氣泡產(chǎn)生，酒精燈微熱，2～3 min后，用手指輕輕用力壓蓋玻片。鏡檢，將具有好的分裂相的片子冷凍揭片，中性樹膠封片。并利用Olympus顯微鏡拍照。

2 實(shí)驗(yàn)結(jié)果

圖1 分裂相中不同聚合體所占的百分比

試驗(yàn)對(duì)6個(gè)個(gè)體卵細(xì)胞內(nèi)聚合體數(shù)進(jìn)行統(tǒng)計(jì)顯示，三倍體太平洋牡蠣的卵子中期聚合體數(shù)目的變化較復(fù)雜，配對(duì)較為混亂。在所觀察的6個(gè)個(gè)體中，從8～18呈現(xiàn)不連續(xù)分布，最高可達(dá)18個(gè)，主要為10～13個(gè)聚合體，平均每細(xì)胞的聚合體數(shù)在10.59～12.08之間（表1）。在三倍體太平洋牡蠣的卵子中，只有少數(shù)細(xì)胞能判別聚合體的價(jià)態(tài)，大部分為三價(jià)體，同時(shí)并存有單價(jià)體和二價(jià)體甚至超聚合體（超過三價(jià)體的聚合形態(tài)）。細(xì)胞中聯(lián)會(huì)狀況細(xì)胞彼此間也非常復(fù)雜。大部分染色體能配對(duì)，染色較濃，收縮變粗，相互之間易區(qū)分，主要是三價(jià)體，少數(shù)可明顯看出是多價(jià)體；存在由兩條已聯(lián)會(huì)的二價(jià)體通過端部聯(lián)合形成四價(jià)體；也有的有多條同源染色體相互交叉折疊配對(duì)形成的多價(jià)復(fù)合體。已聯(lián)會(huì)的二價(jià)體，形態(tài)清晰，配對(duì)交叉清晰可見（見圖2）。據(jù)觀察在同一個(gè)體中，卵細(xì)胞發(fā)育并不完全同步。

在所觀察的所有細(xì)胞中，隨機(jī)挑選11個(gè)染色體形態(tài)較為清晰的分裂相進(jìn)行價(jià)態(tài)分析結(jié)果（圖1）。在123個(gè)聚合體的統(tǒng)計(jì)觀察表明三價(jià)體占總數(shù)的70.73%，其次是單價(jià)體和二價(jià)體，兩者所占比例基本相當(dāng)，最少的是超聚合體，僅占3.25%。

表1 三倍體卵子聚合體數(shù)目列表

個(gè)體

No. 觀察卵

細(xì)胞數(shù) 含不同聚合體數(shù)的卵細(xì)胞數(shù)

聚合體數(shù)

8 9 10 11 12 13 14 18

1 39 2 1 5 9 11 10 1

2 39 1 1 6 10 8 10

3 36 1 2 5 11 9 7 2

4 40 3 3 7 10 9 9

5 44 5 1 7 7 9 10 4 1

6 40 2 2 9 10 8 8 1

合計(jì) 238 14 10 39 57 54 54 8 1

百分比/% 5.88 4.2 16.32 23.95 22.69 22.69 3.36 0.42

a.示12個(gè)聚合體，示單價(jià)體,示“X”型二價(jià)體;b.示10個(gè)聚合體，示三價(jià)體，示一四價(jià)體形式;

c.示18個(gè)聚合體，示單價(jià)體，示三價(jià)體，示二價(jià)體d.示13個(gè)聚合體，示超聚合體

圖2 不同聚合體數(shù)目的三倍體太平洋牡蠣(Crassostrea gigas)卵細(xì)胞

3 討論

3.1 卵子發(fā)育狀況

在人工誘導(dǎo)的貝類三倍體中，生殖腺的不育性程度很高。國內(nèi)外學(xué)者曾用組織學(xué)的方法對(duì)三倍體長牡蠣生殖腺發(fā)育進(jìn)行過研究，發(fā)現(xiàn)雖然三倍體配子發(fā)育受阻，但雌性和雄性三倍體均發(fā)育分化出一些性腺組織，仍能形成一定量的配子[2-3]。據(jù)本實(shí)驗(yàn)觀察，三倍體卵子的發(fā)育同二倍體卵子發(fā)育相同，停滯在第一次減數(shù)分裂前期，發(fā)育較好，經(jīng)24 ℃海水促熟部分卵子可達(dá)到第一次減數(shù)分裂中期（見圖2）。中期同樣也有核仁的存在，而事實(shí)也證明三倍體卵子完全有能力受精[4-6]。

3.2 同源染色體的配對(duì)

三倍體卵子中有三套同源染色體，共30條染色體，原則上每個(gè)同源組的三條有可能出現(xiàn)以下三種情況：一是形成10個(gè)二價(jià)體，10個(gè)單價(jià)體；二是十個(gè)三價(jià)體；三是單價(jià)體，二價(jià)體、三價(jià)體甚至更高多價(jià)體同時(shí)并存。在三倍體減數(shù)分裂同源染色體配對(duì)中，單價(jià)體和二價(jià)體是聯(lián)會(huì)不完全的標(biāo)志，三價(jià)體數(shù)目的減少就意味著單價(jià)體和二價(jià)體的增多（見圖2-c）。此外，在三倍體中存在的超聚合現(xiàn)象，有可能存在非同源配對(duì)現(xiàn)象（見圖2-d）。

實(shí)驗(yàn)發(fā)現(xiàn)在三倍體同源染色體的聯(lián)會(huì)非常復(fù)雜，與前人的研究結(jié)果較為一致[7-8]。有些卵子能形成十個(gè)聚合體，絕大部分是十個(gè)三價(jià)體。有些多于或少于10個(gè)，似乎并無規(guī)律可循。聚合體則以單價(jià)體、二價(jià)體、三價(jià)體，甚至超聚合體的形式存在，甚至在同一細(xì)胞中也存在多種聚合現(xiàn)象（見圖2）。從其粗線期細(xì)胞中可觀察到正在配對(duì)的交叉以及不同聚合體的構(gòu)型來看，三倍體太平洋牡蠣可作為多倍體減數(shù)分裂染色體配對(duì)與聯(lián)會(huì)機(jī)制的理想材料。本研究發(fā)現(xiàn)該現(xiàn)象也同樣存在于三倍體太平洋牡蠣減數(shù)分裂同源聯(lián)會(huì)中。

3.3 同源染色體交叉與分離

在三倍體太平洋牡蠣中，這種復(fù)雜的聯(lián)會(huì)形式有可能直接影響以后減數(shù)分裂染色體分離的形式。據(jù)觀察，染色體分離似乎與聯(lián)會(huì)程度沒有關(guān)系，大多數(shù)卵子能完成兩次減數(shù)分裂，類似于二倍體卵子，只是三倍體中多出的一組染色體隨機(jī)分配到細(xì)胞兩端[9-10]。三倍體所產(chǎn)生的絕大多數(shù)配子的染色體數(shù)目在n和2n之間，在三倍體日本珍珠貝卵細(xì)胞的減數(shù)分裂中也觀察到了類似情況[11]。參考文獻(xiàn)：

[1] 辛榮，王昭萍，于瑞海，等．太平洋牡蠣卵母細(xì)胞減數(shù)分裂中期Ⅰ--染色體交叉配對(duì)觀察[J].中國海洋大學(xué)學(xué)報(bào)（自然科學(xué)版），2006，36（2）：265-268

[2] 李霞，張國范，王永平，等．三倍體牡蠣性腺組織學(xué)研究[J].大連水產(chǎn)學(xué)院學(xué)報(bào)，1999，14（2）：1-6

[3] 曾志南，林琪，吳建紹，等．長牡蠣二倍體和三倍體生殖腺發(fā)育的組織學(xué)觀察.水產(chǎn)學(xué)報(bào)，1999，18（3）：332-339

[4] Allen ,S.K., Jr., and S. l. Downing. Performance of triploid Pacific oysters.Crassostrea gigas Gametogenesis. Can. J. Fish[J]. Aquat. Sci. 1990,47: 1213-1222

[5] Allen, S.K., Jr.. Reproductive Sterility of Triploid Shellfish and Fish[D]. Ph.D. dissertation, University of Washington, Seattle, Washington 1987

[6] 鞏寧，張國范．長牡蠣非整倍體的制備胚胎發(fā)育及存活能力[J].中國水產(chǎn)科學(xué),2003,10(1):5-9

[7] Guo, X., and S.K.Allen,Jr. Reproductive potential and genetics of triploid pacific oyster[J]. Crassostrea gigas(Thunberg).boil.Bull. 1994,187:309-318

[8] Que，H., Guo,X., Zhang,F., Standish.K., and Allen,Jr.Chromosome Segregation in Fertilized Eggs From Triploid Pacific Oyster, Crassostrea gigas(Thunberg), Following Inhibition of Polar Body Ⅰ.Biol.bull.1997,193:14-19

[9] Guo,X. Studies on tetraploid induction in the Pacific oysters. Crassostrea gigas (Thunber). Ph.D. Dissertation. University of Washington. Seatle. WA 1991

[10] Guo, X., W. K. Hershberger, K.Cooper. and K. K. Chew. Genetic consequences of blocking polar body Ⅰwith cytochalasin B in fertilized eggs of the Pacific oyster. Crassostrea gigas:Ⅱ.Segregation of chromosomes. Biol.Bull. 1992,183:387-393

[11] Komaru,A., and K.T.Wada. Meiotic maturation and progeny of oocytes from triploid pearl oyster (Pinctada.fucata martensii) fertilized with spermatozoa from diploid. Aquaculture. 1994,120: 61-70

Observations of Chromosome Aggregates in Triploid Pacific

Oyster Eggs at Metaphase Ⅰduring Meiosis

XIN Rong1, LI Kang

________________________________________

jun1,WANG Zhao

________________________________________

ping2

( 1.The aquaculture department of Rizhao Polytechnic, 276823；

2. Dept. of life sciences and technology , Ocean University of China, Qingdao,266003)

Abstract：The chromosome synapsis configurations in triploid pacific oyster eggs at metaphase Ⅰduring meiosis were observed with acteo-haematoxylin stained under light microscope . Synapsis in eggs from triploid Pacific oyster were highly variable and chaotic, but most synapsis are trivalent, and univalent, bivalent and multivalent also occurred. Different synapsis formation contribute to the variations of synapsis number, most are 10-13, the greatest can reached 18.

Key words：Pacific oyster； triploid oyster；meiosis, synapsis

（收稿日期：2014-02-17）

[3] 曾志南，林琪，吳建紹，等．長牡蠣二倍體和三倍體生殖腺發(fā)育的組織學(xué)觀察.水產(chǎn)學(xué)報(bào)，1999，18（3）：332-339

[4] Allen ,S.K., Jr., and S. l. Downing. Performance of triploid Pacific oysters.Crassostrea gigas Gametogenesis. Can. J. Fish[J]. Aquat. Sci. 1990,47: 1213-1222

[5] Allen, S.K., Jr.. Reproductive Sterility of Triploid Shellfish and Fish[D]. Ph.D. dissertation, University of Washington, Seattle, Washington 1987

[6] 鞏寧，張國范．長牡蠣非整倍體的制備胚胎發(fā)育及存活能力[J].中國水產(chǎn)科學(xué),2003,10(1):5-9

[7] Guo, X., and S.K.Allen,Jr. Reproductive potential and genetics of triploid pacific oyster[J]. Crassostrea gigas(Thunberg).boil.Bull. 1994,187:309-318

[8] Que，H., Guo,X., Zhang,F., Standish.K., and Allen,Jr.Chromosome Segregation in Fertilized Eggs From Triploid Pacific Oyster, Crassostrea gigas(Thunberg), Following Inhibition of Polar Body Ⅰ.Biol.bull.1997,193:14-19

[9] Guo,X. Studies on tetraploid induction in the Pacific oysters. Crassostrea gigas (Thunber). Ph.D. Dissertation. University of Washington. Seatle. WA 1991

[10] Guo, X., W. K. Hershberger, K.Cooper. and K. K. Chew. Genetic consequences of blocking polar body Ⅰwith cytochalasin B in fertilized eggs of the Pacific oyster. Crassostrea gigas:Ⅱ.Segregation of chromosomes. Biol.Bull. 1992,183:387-393

[11] Komaru,A., and K.T.Wada. Meiotic maturation and progeny of oocytes from triploid pearl oyster (Pinctada.fucata martensii) fertilized with spermatozoa from diploid. Aquaculture. 1994,120: 61-70

Observations of Chromosome Aggregates in Triploid Pacific

Oyster Eggs at Metaphase Ⅰduring Meiosis

XIN Rong1, LI Kang

________________________________________

jun1,WANG Zhao

________________________________________

ping2

( 1.The aquaculture department of Rizhao Polytechnic, 276823；

2. Dept. of life sciences and technology , Ocean University of China, Qingdao,266003)

Abstract：The chromosome synapsis configurations in triploid pacific oyster eggs at metaphase Ⅰduring meiosis were observed with acteo-haematoxylin stained under light microscope . Synapsis in eggs from triploid Pacific oyster were highly variable and chaotic, but most synapsis are trivalent, and univalent, bivalent and multivalent also occurred. Different synapsis formation contribute to the variations of synapsis number, most are 10-13, the greatest can reached 18.

Key words：Pacific oyster； triploid oyster；meiosis, synapsis

（收稿日期：2014-02-17）

[3] 曾志南，林琪，吳建紹，等．長牡蠣二倍體和三倍體生殖腺發(fā)育的組織學(xué)觀察.水產(chǎn)學(xué)報(bào)，1999，18（3）：332-339

[4] Allen ,S.K., Jr., and S. l. Downing. Performance of triploid Pacific oysters.Crassostrea gigas Gametogenesis. Can. J. Fish[J]. Aquat. Sci. 1990,47: 1213-1222

[5] Allen, S.K., Jr.. Reproductive Sterility of Triploid Shellfish and Fish[D]. Ph.D. dissertation, University of Washington, Seattle, Washington 1987

[6] 鞏寧，張國范．長牡蠣非整倍體的制備胚胎發(fā)育及存活能力[J].中國水產(chǎn)科學(xué),2003,10(1):5-9

[7] Guo, X., and S.K.Allen,Jr. Reproductive potential and genetics of triploid pacific oyster[J]. Crassostrea gigas(Thunberg).boil.Bull. 1994,187:309-318

[8] Que，H., Guo,X., Zhang,F., Standish.K., and Allen,Jr.Chromosome Segregation in Fertilized Eggs From Triploid Pacific Oyster, Crassostrea gigas(Thunberg), Following Inhibition of Polar Body Ⅰ.Biol.bull.1997,193:14-19

[9] Guo,X. Studies on tetraploid induction in the Pacific oysters. Crassostrea gigas (Thunber). Ph.D. Dissertation. University of Washington. Seatle. WA 1991

[10] Guo, X., W. K. Hershberger, K.Cooper. and K. K. Chew. Genetic consequences of blocking polar body Ⅰwith cytochalasin B in fertilized eggs of the Pacific oyster. Crassostrea gigas:Ⅱ.Segregation of chromosomes. Biol.Bull. 1992,183:387-393

[11] Komaru,A., and K.T.Wada. Meiotic maturation and progeny of oocytes from triploid pearl oyster (Pinctada.fucata martensii) fertilized with spermatozoa from diploid. Aquaculture. 1994,120: 61-70

Observations of Chromosome Aggregates in Triploid Pacific

Oyster Eggs at Metaphase Ⅰduring Meiosis

XIN Rong1, LI Kang

________________________________________

jun1,WANG Zhao

________________________________________

ping2

( 1.The aquaculture department of Rizhao Polytechnic, 276823；

2. Dept. of life sciences and technology , Ocean University of China, Qingdao,266003)

Abstract：The chromosome synapsis configurations in triploid pacific oyster eggs at metaphase Ⅰduring meiosis were observed with acteo-haematoxylin stained under light microscope . Synapsis in eggs from triploid Pacific oyster were highly variable and chaotic, but most synapsis are trivalent, and univalent, bivalent and multivalent also occurred. Different synapsis formation contribute to the variations of synapsis number, most are 10-13, the greatest can reached 18.

Key words：Pacific oyster； triploid oyster；meiosis, synapsis

（收稿日期：2014-02-17）