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Evidence of DNA Replication Licensing and Paternal DNA Degradation by MCM7 and ORC2 in the Mouse One-cell Embryo
Biomed Sci Letters 2017;23:372-379
Published online December 31, 2017;  https://doi.org/10.15616/BSL.2017.23.4.372
© 2017 The Korean Society For Biomedical Laboratory Sciences.

Chang Jin Kim1, Tae Hoon Kim2, Eun-Woo Lee3, and Kyung-Bon Lee1,†

1Department of Biology Education, College of Education, Chonnam National University, Gwangju 61186, Korea,
2Department of Food Science and Biotechnology, College of Engineering, Daegu University, Gyeongsan 38453, Korea,
3Division of Applied Bioengineering, College of Engineering, Dong-eui University, Busan 47340, Korea
Correspondence to: Corresponding author: Kyung-Bon Lee. Department of Biology Education, College of Education, Chonnam National University, Gwangju 61186, Korea. Tel: +82-62-530-2507, Fax: +82-62-530-2509, e-mail: kblee@jnu.ac.kr
Received October 13, 2017; Revised November 6, 2017; Accepted November 7, 2017.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract

This study was investigated to test whether paternal DNA that was destined for degradation was properly licensed by testing for the presence of mini-chromosome maintenance protein (MCM) 7 and origin recognition complex (ORC) 2 in the paternal pronuclei. ORC2 is one of the first licensing protein to come on and MCM7 is one of the last licensing protein to come on. Zygotes were prepared by injection of control and treated sperm injection (ICSI). To control for DNA breakage, epididymal spermatozoa were treated with DNase I to fragment the DNA, then injected into oocytes. The presence of MCM7 and ORC2 in the pronuclei of mouse zygotes was tested by immunohistochemistry, just before the onset of DNA synthesis, at 5 h after fertilization, and after DNA synthesis began, at 9 h post fertilization. We found that in all cases, both MCM7 and ORC2 were present in both pronuclei at 5 h after sperm injection, just before DNA synthesis began. This indicates that no matter how extensive the DNA damage, recruitment of licensing proteins to the origins of replication was not inhibited. Sperm DNA fragmentation does not prevent licensing of DNA replication origins. Furthermore, the embryo recognizes DNA that is damaged by nucleases. Our data indicate that the one-cell embryo does harbor a mechanism to prevent the replication of severely damaged DNA from spermatozoa, even though the embryos do not undergo classical apoptosis.

Keywords : MCM7, ORC2, DNA breakage, DNA fragmentation, DNA replication
꽌濡

룷쑀룞臾쇱쓽 1꽭룷湲 닔젙쓽 寃쎌슦뿉뒗 룆듅븳 DNA 蹂듭젣 씪씠꽱떛 湲곗옉쑝濡 씤빐 DNA 蹂듭젣 愿젴맂 떎뼇븳 異붽쟻 뿰援щ 븯뒗뜲 留ㅼ슦 쟻빀븳 紐⑤뜽씠떎. 룷쑀룞臾쇱쓽 DNA 뿼湲곕뒗 왂 60뼲쓽 뿼湲곗뙇쑝濡 씠猷⑥뼱졇 엳떎(Pardoll et al., 1980; Vogelstein et al., 1980). 씠젃寃 留롮 뿼湲곗뙇 닔젙遺꽣 떆옉빐꽌 씠 媛쒖껜媛 깮議댄븯怨 엳뒗 룞븞 臾댁닔엳 留롮 蹂듭젣媛 씠猷⑥뼱吏硫, 쑀쟾泥 쟾泥댁뿉 嫄몄퀜 닔留롮 吏젏뿉꽌 꽌濡 떎瑜 떆湲곗뿉 媛쒕퀎쟻쑝濡 蹂듭젣媛 씠猷⑥뼱吏꾨떎(Stubblefield, 1975; Pardoll et al., 1980). 諛뺥뀒由ъ븘(Lemon and Grossman, 1998)굹 룷쑀룞臾(Pardoll et al., 1980) DNA 蹂듭젣媛 떆옉릺뒗 遺쐞씤 蹂듭젣湲곗젏씠씪뒗 怨좎젙쟻씤 옣냼뿉꽌 蹂듭젣蹂듯빀泥(replication complex)媛 삎꽦맂떎. 洹 썑, 씠 蹂듭젣蹂듯빀泥닿 二쇳삎 DNA瑜 걣뼱떦湲곕㈃꽌 蹂듭젣媛 씪뼱궃떎. 諛뺥뀒由ъ븘쓽 寃쎌슦뿉 蹂듭젣湲곗젏쓣 씤떇븯뒗 諛⑸쾿쑝濡 듅젙 DNA 꽌뿴쓣 씤떇븯뿬 蹂듭젣蹂듯빀泥닿 삎꽦릺吏留, 룷쑀룞臾쇱쓽 蹂듭젣湲곗젏 DNA 꽌뿴뿉 醫뚯슦릺吏 븡뒗떎(Gilbert, 2001; Robinson and Bell, 2005; Costa and Blow, 2007). 룷쑀룞臾쇱쓽 蹂듭젣湲곗젏 옞옱쟻씤 蹂듭젣湲곗젏뿉꽌 궗쟾蹂듭젣蹂듯빀泥(pre-replication complex, pre-RC)媛 삎꽦릺뼱빞 蹂듭젣湲곗젏쑝濡 씤떇맆 닔 엳떎. 씠泥섎읆 蹂듭젣湲곗젏뿉꽌쓽 궗쟾蹂듭젣蹂듯빀泥 삎꽦怨쇱젙쓣 DNA 蹂듭젣 씪씠꽱떛씠씪 븳떎.

蹂듭젣 씪씠꽱떛 怨쇱젙쓽 떆옉 옞옱쟻 蹂듭젣湲곗젏뿉 蹂듭젣씤떇蹂듯빀泥(origin recognition complex, ORC)쓽 寃고빀뿉 쓽빐 떆옉맂떎(Coster et al., 2014; Ticau et al., 2015). 蹂듭젣씤떇蹂듯빀泥댁쓽 寃고빀 蹂듭젣 쟾 G1湲 룞븞뿉 씪씠꽱떛 떒諛깆쭏(licensing protein)씠씪怨 紐낅챸릺뒗 씪젴쓽 떒諛깆쭏뱾씠 DNA긽뿉 젙솗븳 닚꽌濡 寃고빀릺뒗 湲곗옉뿉 쓽빐 씠猷⑥뼱吏꾨떎(Blow and Laskey, 1988; Leno et al., 1992; Blow, 1993). 씪씠꽱떛 怨쇱젙뿉 李몄뿬븯뒗 씪젴쓽 떒諛깆쭏 ORC1~ORC6源뚯 6媛쒖쓽 냼떒쐞泥 떒諛깆쭏씠 蹂듭젣씤떇蹂듯빀泥대 삎꽦븯怨 뿬湲곗뿉 異붽쟻쑝濡 CDT1, CDC6, MCM2~7씠 寃고빀븯뿬 궗쟾蹂듭젣蹂듯빀泥대 삎꽦븿쑝濡쒖뜥 씪씠꽱떛씠 씠猷⑥뼱吏꾨떎(Takeda and Dutta 2005; Krude, 2006).

援ъ껜쟻 DNA 蹂듭젣 씪씠꽱떛 怨쇱젙 ORC2, ORC3, ORC5濡 遺덈━뒗 꽭 媛쒖쓽 냼떒쐞泥닿 꽭룷吏덉뿉꽌 슦꽑 蹂듯빀泥대 삎꽦븳 썑, ORC4, ORC6怨 異붽쟻쑝濡 寃고빀븯뿬 빑쑝濡 닔넚맂떎(Ghosh et al., 2011). 빑쑝濡 닔넚맂 ORC2~6 ORC1씠 寃고빀븯湲 쟾(Takeda et al., 2005) 삉뒗 썑(Ghosh et al., 2011)뿉 蹂듭젣湲곗젏뿉 寃고빀븳 썑, 꽭룷 二쇨린 룞븞뿉뒗 DNA뿉 怨꾩냽 寃고빀븯뿬 궓븘 엳떎(Depamphils, 2005; Thomae et al., 2008). ORC1씠 ORC2~6뿉 寃고빀븳 썑 씠 蹂듯빀泥댁뿉 CDT1, CDC6씠 李⑤濡 寃고빀븯怨 洹 썑 mini-chromosome maintenance protein (MCM)2-7 蹂듯빀泥닿 寃고빀븿쑝濡쒖뜥 蹂듭젣湲곗젏뿉꽌 셿踰쏀븯寃 궗쟾蹂듭젣蹂듯빀泥닿 삎꽦맂떎(Rao and Stillman, 1995; Rowley et al., 1995; Speck et al., 2005). G1湲곕룞븞뿉 蹂듭젣湲곗젏뿉꽌쓽 궗쟾蹂듭젣蹂듯빀泥 삎꽦쓽 셿猷뚮뒗 DNA 蹂듭젣떆옉쓣 쐞븳 以鍮꾧낵젙씤 씪씠꽱떛씠 씠猷⑥뼱吏 寃껋쑝濡 媛꾩<릺怨 씠 썑 씪젴쓽 怨쇱젙쓣 嫄곗퀜 蹂듭젣媛 떆옉맂떎.

留덉슦뒪瑜 룷븿븳 씪遺 룷쑀룞臾쇱쓽 寃쎌슦 젙옄 삎꽦怨쇱젙뿉꽌 솚寃 삉뒗 솕븰쟻 뒪듃젅뒪뿉 끂異쒕릺硫 DNA 씠以묐굹꽑 젅떒(DNA double-stranded break)씠 씪뼱궃떎(Yamauchi et al., 2007). 留덉슦뒪 젙옄뿉 2媛 뼇씠삩씤 MnCl2 CaCl2쓣 泥섎━븯硫 씠윭븳 DNA 씠以묐굹꽑 젅떒씠 옒 씪뼱굹뒗 寃껋쑝濡 蹂닿퀬맂 諛 엳떎(Yamauchi et al., 2007). 留덉슦뒪 젙옄뿉꽌 諛쒖깮븯뒗 DNA 씠以묐굹꽑쓽 젅떒 겕寃 2떒怨꾩쓽 怨쇱젙쑝濡 굹닃 닔 엳떎. 泥 踰덉㎏ 怨쇱젙 Topoisomerase II B(TOP2B)뿉 쓽빐 왂 25~50 kb 겕湲곗쓽 젅렪쑝濡 굹돇뒗뜲 씠윭븳 DNA 젅렪쓣 젙옄 뿼깋吏 議곌컖(sperm chromatin fragmentation, SCF)씠씪 븯怨 씠윭븳 SCF뒗 EDTA뿉 쓽빐 떎떆 쉶蹂듭씠 媛뒫븯硫(Shaman et al., 2006) 씠윭븳 쉶蹂듬맂 DNA 젅렪쓣 SCF-religated씪 븳떎. 몢 踰덉㎏ 怨쇱젙쑝濡 젙옄 궡 빑궛遺꾪빐슚냼뿉 쓽빐 愿묐쾾쐞븯寃 DNA degradation씠 씪뼱굹硫 씠윭븳 degradation씠 씪뼱궃 젙옄 DNA瑜 젙옄 DNA degradation (sperm chromatin degradation, SDD)씠씪怨 遺瑜몃떎(Shaman et al., 2006). 듅엳 泥댁쇅뿉꽌 留덉슦뒪 젙옄뿉 MnCl2 CaCl2쓣 泥섎━븷 븣 젙옄媛 議댁옱븯뒗 쐞移섏뿉 뵲씪 SCF EDTA뿉 쓽븳 쉶蹂 젙룄媛 떖由 씪뼱궃떎. 洹 삁濡쒖뜥 vas deferens sperm씠 epididymal sperm蹂대떎 뜑슧 愿묐쾾쐞븯寃 SCF媛 씪뼱굹硫(Yamauchi et al., 2007), EDTA뿉 쓽븳 쉶蹂듬룄 vas deferens sperm蹂대떎 epididymal sperm뿉꽌 嫄곗쓽 셿踰쏀븯寃 쉶蹂듬릺뿀떎(Shaman et al., 2006). 씠뒗 vas deferens sperm뿉 빑궛遺꾪빐슚냼媛 넂 냽룄濡 議댁옱븯湲곗뿉 epididymal sperm蹂대떎 DNA degradation 젙룄媛 뜑 떖븯寃 씪뼱궓쓣 븣 닔 엳떎(Yamauchi et al., 2007). 뵲씪꽌 蹂 뿰援ъ뿉꽌뒗 닔젙쓽 degradation씠 삁젙맂 遺怨 쟾빑怨 紐④퀎 쟾빑뿉꽌 MCM7怨 ORC2쓽 議댁옱 뿬遺瑜 DNA 蹂듭젣 쟾怨 썑뿉 媛곴컖 솗씤븯뿬 DNA 蹂듭젣 씪씠꽱떛씠 릺뒗吏瑜 솗씤븯떎.

옱猷 諛 諛⑸쾿

怨듭떆옱猷

蹂 뿰援ъ뿉꽌 씠슜맂 留덉슦뒪뒗 B6D2F1 (C57BL/6N XDBA/2)쑝濡 삩룄 23±1°C, 뒿룄 50±1% 궡쇅, 洹몃━怨 12떆媛 紐낆븫 二쇨린(light 8:00~20:00, dark 20:00~8:00)濡 쑀吏릺뒗 룞臾 궗쑁떎뿉꽌 궗쑁븯떎. 1二 媛꾩쓽 쟻쓳 湲곌컙 룞븞 떎뿕룞臾쇱뿉寃 쓬떇怨 臾쇱쓣 옄쑀濡쒖씠 怨듦툒븯떎. 紐⑤뱺 떎뿕怨꾪쉷 쟾궓븰援 룞臾쇱쑄由ъ쐞썝쉶쓽 洹쒖젙뿉 쓽嫄고빐꽌 떎뿕븯떎. 蹂 옱猷 諛 諛⑸쾿뿉꽌 蹂꾨룄濡 쉶궗瑜 湲곗옱븯吏 븡 떆빟 紐⑤몢 Sigma-Aldrich 궗쓽 젣뭹쓣 궗슜븯떎.

젙옄 以鍮

8~12二쇰졊쓽 留덉슦뒪쓽 epididymis 諛 vas deferens쓽 궡媛 궡슜臾쇱쓣 遺꾨━ 異붿텧븯뿬 HEPES-CZB (HCZB) 諛곗뼇븸뿉 꽔뿀떎. 젙옄瑜 HCZB 諛곗뼇븸뿉 꽔 썑 뵾렖똿쑝濡 怨좊Ⅴ寃 꽎뼱以 썑 control, SCF 諛 SCF-religated 洹몃9쑝濡 媛곴컖 굹늻뿀떎. Control 洹몃9쓽 젙옄뒗 떎삩뿉꽌 90遺 룞븞 諛곗뼇븯떎. SCF 洹몃9怨 SCF-religated 洹몃9 10 mM MnCl2 CaCl2쓣 泥④븳 썑 떎삩뿉꽌 90遺 룞븞 諛곗뼇븯떎. 洹 썑 SCF-religated 洹몃9뿉뒗 100 mM EDTA瑜 泥④븯怨 떎떆 떎삩뿉꽌 30遺 룞븞 異붽 諛곗뼇븯떎. 諛곗뼇 썑 젙옄뱾 諛붾줈 ICSI뿉 씠슜븯떎.

怨쇰같 쑀룄 諛 궃옄 梨꾩랬

8~12二쇰졊쓽 꽦닕븳 븫而 留덉슦뒪뿉 5 IU eCG瑜 蹂듦컯 二쇱궗븯怨, 48떆媛 썑 5 IU hCG瑜 二쇱엯븯뿬 怨쇰같쓣 쑀룄븯떎. hCG 二쇱엯 썑 14~15떆媛꾩뿉 궃愿쓣 젅媛쒗븯뿬 諛곕맂 궃援ъ꽭룷-궃옄 蹂듯빀泥대 諛⑹텧떆耳 0.1% hyaluronidase媛 븿쑀맂 HCZB 諛곗뼇븸뿉 移⑥븯떎. 궃援ъ꽭룷媛 遺꾨━맂 궃옄뒗 HCZB 諛곗뼇븸쑝濡 3쉶 꽭泥 썑 利됱떆 ICSI뿉 씠슜븯떎.

꽭룷吏 궡 젙옄誘몄꽭二쇱엯

ICSI뒗 Szczygiel Yanagimachi (2003)쓽 諛⑸쾿쓣 蹂삎븯뿬 떎떆븯떎. 癒쇱 60 mm petri dish (Falcon, NJ, USA)뿉 12% polyvinyl pyrolidone (PVP)媛 룷븿맂 HCZB 諛곗뼇븸 냼쟻쓣 留뚮뱺 썑 씠 룞씪븳 遺뵾쓽 젙옄 遺쑀븸 냼쟻쓣 留뚮뱾뼱 몢 냼쟻쓣 꽎뼱二쇱뿀떎. 洹몃━怨 以鍮꾨맂 궃옄 1媛쒕떦 1媛쒖쓽 젙옄 癒몃━瑜 二쇱엯븯떎. 궃옄뿉 젙옄 癒몃━ 二쇱엯怨쇱젙 Micromanipulators (Micromanipulator Transferman, Eppendorf, Germany) Piezo-electric actuator (Prime Tech, Tsukuba, Japan)쓣 씠슜븯뿬 떎떆븯떎. 젣씪 癒쇱 븯굹쓽 젙옄瑜 二쇱엯슜 뵾렖쑝濡 瑗щ━瑜 슦꽑 씉씤븳 썑 븬쟾 쟾瑜섎 쓽젮 以묓렪怨 瑗щ━瑜 뼹뼱궡뿀떎. 洹 썑 癒몃━瑜 二쇱엯슜 뵾렖쑝濡 옱 씉씤븳 썑 궃옄 븞뿉 利됱떆 二쇱엯븯떎.

泥댁쇅 諛곗뼇

ICSI瑜 씠슜븯뿬 젙옄媛 二쇱엯맂 궃옄뒗 37°C, 5% CO2 議곌굔븯뿉 CZB 諛곗뼇븸쑝濡 諛곗뼇븯떎. 1꽭룷湲 닔젙뿉꽌 MCM7, ORC2 諛쒗쁽 솗씤 ICSI 썑 媛곴컖 5, 9떆媛꾩㎏뿉 씠瑜 솗씤븯떎.

MCM7 諛 ORC2쓽 硫댁뿭삎愿묒뿼깋

留덉슦뒪 1꽭룷湲 닔젙쓣 CZB 諛곗뼇븸뿉 諛곗뼇븳 썑 떎삩뿉꽌 30遺 룞븞 4% 뙆씪룷由꾩븣뜲엳뱶濡 怨좎젙븯떎. 뙆씪룷由꾩븣뜲엳뱶 옣슜븸 怨좎젙 쟾 16% 옣슜븸(Alfa Aesar stock no. 43368)쓣 씗꽍븯뿬 4%濡 留뚮뱺 썑 븯猷삳갇 룞븞 4°C뿉 蹂닿븳 썑 씠슜븯떎. 怨좎젙 썑, 꽭룷瑜 뵽뼱 궦 썑 10遺 룞븞 0.1% Tween씠 븿쑀맂 PBS (PBST)濡 2쉶 꽭泥숉븯떎. 洹 썑 1꽭룷湲 닔젙쓣 15遺 룞븞 0.5% Triton X-100쑝濡 permeabilization 떆궓 썑 0.5% BSA媛 븿쑀맂 PBST濡 10遺꾩뵫, 2쉶 꽭泥숉븯떎. 떎삩뿉꽌 1떆媛 룞븞 BSA濡 blocking 썑 4°C뿉꽌 븯猷삳갇 룞븞 1:300쑝濡 씗꽍떆궓 1李 빆泥댁뿉 諛곗뼇븯떎. 諛곗뼇 썑 1꽭룷湲 닔젙쓣 0.5% BSA媛 븿쑀맂 PBST濡 10遺꾩뵫 2쉶 꽭泥숉븯怨 1떆媛 룞븞 떎삩뿉꽌 1:1,000쑝濡 씗꽍맂 삎愿묐Ъ吏덉씠 遺숈뼱엳뒗 2李 빆泥닿 遺李⑸릺룄濡 諛곗뼇븯떎. 삎愿묓몴吏 썑, 1꽭룷湲 닔젙쓣 PBST濡 10遺꾩뵫, 3쉶 꽭泥숉븯떎. 뒳씪씠뱶湲씪뒪뿉 ProLong Gold antifade reagent with DAPI (P-36931, Invitrogen, USA)쓣 뼥뼱쑉젮 냼쟻쓣 留뚮뱾怨 닔젙쓣 닾뿬븳 썑, 而ㅻ쾭湲씪뒪濡 뜮 썑 삎愿묓쁽誘멸꼍쑝濡 愿李고븯떎.

1李 빆泥대줈 ORC2쓽 寃쎌슦 anti-ORC2 (sc-13238, Santa Cruz Biotechnology, USA)瑜 씠슜븯쑝硫, MCM7쓽 寃쎌슦뿉뒗 anti-MCM7 (sc-46687, Santa Cruz Biotechnology, USA)쓣 궗슜븯떎. 2李 빆泥대줈 Alexa Fluor 488 (A11078, Invitrogen, USA)怨 Alexa Fluor 546 (A21085, Invitrogen, USA)쓣 궗슜븯떎.

넻怨 遺꾩꽍

蹂 뿰援ъ뿉꽌뒗 媛 떎뿕援곗뿉 븯뿬 4쉶 씠긽 諛섎났떎뿕쓣 떎떆븯쑝硫, 뼸뼱吏 紐⑤뱺 떎뿕 寃곌낵쓽 넻怨꾩쿂由щ뒗 SPSS software (Version 13.0)瑜 씠슜븳 씪썝遺꾩궛遺꾩꽍踰뺤쑝濡 泥섎━援ш컙 쑀쓽꽦쓣 寃젙븯쑝硫, P<0.05쓽 쑀쓽꽦留뚯쓣 넻怨꾪븰쟻 李⑥씠媛 엳뒗 寃껋쑝濡 씤젙븯떎.

寃곌낵

Epididymal sperm쓣 씠슜븯뿬 留뚮뱺 1꽭룷湲 닔젙뿉꽌 DNA 빀꽦 쟾씤 젙옄 二쇱엯 썑 5떆媛꾩㎏ MCM7怨 ORC2쓽 諛쒗쁽 寃곌낵뒗 Fig. 1Table 1怨 媛숇떎. Epi-Con (epididymal sperm injection-control), Epi-SCF 諛 Epi-SCF-religated 洹몃9 紐⑤몢 遺怨꾩 紐④퀎 쟾빑 뼇履쎌뿉꽌 MCM7怨 ORC2媛 諛쒗쁽맂 寃쎌슦뒗 100%떎. 遺怨 쟾빑씠굹 紐④퀎 쟾빑뿉꽌留 MCM7怨 ORC2媛 媛곴컖 諛쒗쁽맂 寃쎌슦뒗 굹굹吏 븡븯떎. DNA 빀꽦씠 吏꾪뻾 以묒씤 젙옄 二쇱엯 썑 9떆媛꾩㎏뿉 MCM7怨 ORC2媛 諛쒗쁽맂 寃곌낵뒗 Fig. 3Table 3怨 媛숇떎. 씠 뿭떆 Epi-Con, Epi-SCF 諛 Epi-SCF-religated 洹몃9 紐⑤몢 遺怨꾩 紐④퀎 쟾빑 뼇履쎌뿉꽌 MCM7怨 ORC2媛 諛쒗쁽맂 寃쎌슦뒗 100%떎. 遺怨 쟾빑씠굹 紐④퀎 쟾빑뿉꽌留 MCM7怨 ORC2媛 媛곴컖 諛쒗쁽맂 寃쎌슦뒗 굹굹吏 븡븯떎.

Fig. 1.

Immunocytochemical localization of MCM7 and ORC2 in mouse one-cell embryos at 5 h after epididymal sperm injection into mouse oocyte (Before DNA synthesis). Embryos were fixed and double stained with antibodies to MCM7 (Green) and ORC2 (Red). Pronuclei were also stained with DAPI (Blue), and then visualized by confocal microscopy.


Fig. 2.

Immunocytochemical localization of MCM7 and ORC2 in mouse one-cell embryos at 9 h after epididymal sperm injection into mouse oocyte (During DNA synthesis). Embryos were fixed and double stained with antibodies to MCM7 (Green) and ORC2 (Red). Pronuclei were also stained with DAPI (Blue), and then visualized by confocal microscopy.


MCM7 and ORC2 expression at 5 h after epididymal sperm injection into mouse oocyte (Before DNA synthesis)

Group No. of injected oocytes No. of zygotes (%)

Stained with MPN only Stained with FPN only Stained with MPN and FPN
MCM 7 Control 113 0 (0.0) 0 (0.0) 113 (100)
SCF 110 0 (0.0) 0 (0.0) 110 (100)
SCF-religated 103 0 (0.0) 0 (0.0) 103 (100)

ORC2 Control 113 0 (0.0) 0 (0.0) 113 (100)
SCF 106 0 (0.0) 0 (0.0) 106 (100)
SCF-religated 104 0 (0.0) 0 (0.0) 104 (100)

MPN: male pronuclei, FPN: female pronuclei.


MCM7 and ORC2 expression at 9 h after epididymal sperm injection into mouse oocyte (During DNA synthesis)

Group No. of injected oocytes No. of zygotes (%)

Stained with MPN only Stained with FPN only Dtained with MPN and FPN
MCM7 Control 112 0 (0.0) 0 (0.0) 112 (100)
SCF 112 0 (0.0) 0 (0.0) 112 (100)
SCF-religated 110 0 (0.0) 0 (0.0) 110 (100)

ORC2 Control 113 0 (0.0) 0 (0.0) 113 (100)
SCF 105 0 (0.0) 0 (0.0) 105 (100)
SCF-religated 113 0 (0.0) 0 (0.0) 113 (100)

MPN: male pronuclei, FPN: female pronuclei.


Fig. 3.

Immunocytochemical localization of MCM7 and ORC2 in mouse one-cell embryos at 5 h after vas deferens sperm injection into mouse oocyte (Before DNA synthesis). Embryos were fixed and double stained with antibodies to MCM7 (Green) and ORC2 (Red). Pronuclei were also stained with DAPI (Blue), and then visualized by confocal microscopy.


MCM7 and ORC2 expression at 5 h after vas deferens sperm injection into mouse oocyte (Before DNA synthesis)

Group No. of injected oocytes No. of zygotes (%)

Stained with MPN only Stained with FPN only Stained with MPN and FPN
MCM7 Control 110 0 (0.0) 0 (0.0) 110 (100)
SCF 104 0 (0.0) 0 (0.0) 104 (100)
SCF-religated 94 0 (0.0) 0 (0.0) 94 (100)

ORC2 Control 108 0 (0.0) 0 (0.0) 108 (100)
SCF 107 0 (0.0) 0 (0.0) 107 (100)
SCF-religated 95 0 (0.0) 0 (0.0) 95 (100)

MPN: male pronuclei, FPN: female pronuclei.



Vas deferens sperm쓣 씠슜븯뿬 留뚮뱺 1꽭룷湲 닔젙뿉꽌 DNA 빀꽦 쟾씤 젙옄 二쇱엯 썑 5떆媛꾩㎏ MCM7怨 ORC2쓽 諛쒗쁽 寃곌낵뒗 Fig. 2Table 2 媛숇떎. 씠 뿭떆 epididymal sperm쓣 二쇱엯븳 寃쎌슦 留덉갔媛吏濡 Vas-Con (vas deferens sperm injection-Control), Vas-SCF 諛 Vas-SCF-religated 洹몃9 紐⑤몢 遺怨꾩 紐④퀎 쟾빑 뼇履쎌뿉꽌 MCM7怨 ORC2媛 諛쒗쁽맂 寃쎌슦뒗 100%떎. 遺怨 쟾빑씠굹 紐④퀎 쟾빑뿉꽌留 MCM7怨 ORC2媛 媛곴컖 諛쒗쁽맂 寃쎌슦뒗 굹굹吏 븡븯떎. 삉븳 젙옄 二쇱엯 썑 9떆媛꾩㎏(DNA 蹂듭젣 吏꾪뻾 以)뿉 MCM7怨 ORC2瑜 愿李고븳 寃곌낵뒗 Fig. 4Table 4 媛숇떎. Vas-Con, Vas-SCF 諛 Vas-SCF-religated 洹몃9 紐⑤몢 遺怨꾩 紐④퀎 쟾빑 뼇履쎌뿉꽌 MCM7怨 ORC2媛 愿李곕맂 寃쎌슦뒗 100%떎. 遺怨 쟾빑씠굹 紐④퀎 쟾빑뿉꽌留 MCM7怨 ORC2媛 媛곴컖 愿李곕맂 寃쎌슦뒗 굹굹吏 븡븯떎. 洹몃━怨 DNA 빀꽦씠 吏꾪뻾 以묒씤 젙옄 二쇱엯 썑 9떆媛꾩㎏ Vas-SCF Vas-SCF-religated 洹몃9쓽 DAPI 뿼깋쓣 蹂대㈃ DNA degradation씠 릺뼱 遺怨 쟾빑씠 愿李곕릺吏 븡븯떎.

Fig. 4.

Immunocytochemical localization of MCM7 and ORC2 in mouse one-cell embryos at 9 h after vas deferens sperm injection into mouse oocyte (During DNA synthesis). Embryos were fixed and double stained with antibodies to MCM7 (Green) and ORC2 (Red). Pronuclei were also stained with DAPI (Blue), and then visualized by confocal microscopy.


MCM7 and ORC2 expression at 9 h after vas deferens sperm injection into mouse oocyte (During DNA synthesis)

Group No. of injected oocytes No. of zygotes (%)

Stained with MPN only Stained with FPN only Stained with MPN and FPN
MCM7 Control 104 0 (0.0) 0 (0.0) 104 (100)
SCF 97 0 (0.0) 0 (0.0) 97 (100)
SCF-religated 115 0 (0.0) 0 (0.0) 115 (100)

ORC2 Control 105 0 (0.0) 0 (0.0) 105 (100)
SCF 105 0 (0.0) 0 (0.0) 105 (100)
SCF-religated 107 0 (0.0) 0 (0.0) 107 (100)

MPN: male pronuclei, FPN: female pronuclei.


怨좎같

룷쑀룞臾쇱쓽 닔젙씠 DNA 넀긽뿉 쓳븯뒗 諛⑹떇 븘吏 遺덈텇紐낇븯떎. Gawecka (2012)쓽 뿰援 寃곌낵뿉 뵲瑜대㈃ 닔젙씠 DNA 넀긽쓣 씤떇븯怨 DNA 蹂듭젣 吏뿰, 諛곗븘 諛쒕떖 吏뿰 諛 諛곗븘 諛쒕떖 以묎린媛 諛쒖깮븳떎怨 븳떎. 뵲씪꽌 諛곗븘 諛쒕떖怨쇱젙뿉 엳뼱꽌 떎瑜 닔以, 떎瑜 삎깭쓽 DNA 넀긽뿉 諛섏쓳븯뒗 諛⑹떇뿉 븳 씠빐뒗 뼢썑 뿰援ъ뿉 以묒슂븳 젙蹂대 젣怨듯븷 寃껋씠怨, 씠뒗 깉濡쒖슫 꽭룷 二쇨린 젙吏 삉뒗 꽭룷궗硫 湲곗쟾쓣 諛앺궡뒗뜲 룄씠 맆 寃껋씠떎. 蹂 뿰援щ뒗 DNA 넀긽뿉 뵲씪 DNA 蹂듭젣 媛쒖떆뿉 븵꽌 씪뼱굹뒗 DNA 씪씠꽱떛쓽 李⑥씠 닔젙쓽 DNA 넀긽 씤떇 李⑥씠, 諛곗븘諛쒕떖쓽 李⑥씠瑜 뿰援ы븯떎. 꽦닕븳 젙옄뿉 2媛 뼇씠삩씤 MnCl2 CaCl2쓣 泥섎━븯硫 젙옄 DNA뿉꽌 씠以묐굹꽑 젅떒씠 씪뼱굹 25~50 kb 겕湲곕줈 SCF媛 留뚮뱾뼱吏硫(Yamauchi et al., 2007), 씠윭븳 DNA 議곌컖 EDTA媛 泥섎━릺硫 떎떆 쉶蹂(SCF-religated)맂떎(Shaman et al., 2006). 삉븳 epididymal sperm怨 vas deferens sperm 빑궛遺꾪빐슚냼쓽 솢꽦 李⑥씠濡 SCF SCF-religated 젙룄媛 떎瑜대떎(Yamauchi et al., 2007). 뵲씪꽌 epididymal sperm怨 vas deferens sperm뿉 2媛 뼇씠삩쓣 泥섎━븯뿬 DNA 넀긽쓣 쑀諛쒗븯떎. 洹몃━怨 씠瑜 씠슜븯뿬 留뚮뱺 1꽭룷湲 닔젙뿉꽌 MCM7怨 ORC2쓽 諛쒗쁽 뿬遺瑜 넻빐 젙긽쟻씤 씪씠꽱떛씠 씪뼱굹뒗吏 븣븘蹂댁븯떎.

DNA 蹂듭젣 쟾 紐⑤뱺 洹몃9쓽 1꽭룷湲 닔젙뿉꽌 100% 鍮꾩쑉濡 遺怨꾩 紐④퀎 쟾빑뿉꽌 MCM7怨 ORC2媛 諛쒗쁽릺뿀떎(Table 1, 2). 삉븳 DNA 蹂듭젣 吏꾪뻾 以묒뿉룄 紐⑤뱺 洹몃9쓽 1꽭룷湲 닔젙뿉꽌 100% 鍮꾩쑉濡 遺怨꾩 紐④퀎 쟾빑뿉꽌 MCM7怨 ORC2媛 諛쒗쁽릺뿀떎(Table 3, 4). 씠뒗 젙옄쓽 DNA뿉 SCF 諛 SCF-religated 넀긽씠 씪뼱굹뜑씪룄 遺怨꾩 紐④퀎 쟾빑쓽 DNA 蹂듭젣湲곗젏뿉꽌 씪씠꽱떛 젙긽쟻쑝濡 씠猷⑥뼱議뚯쓬쓣 떆궗븳떎. 利 1꽭룷湲 닔젙쓽 遺怨꾩 紐④퀎 쟾빑 궡쓽 DNA 蹂듭젣湲곗젏뿉꽌 씠猷⑥뼱吏뒗 씪씠꽱떛 吏꾪뻾뿉 DNA 넀긽씠 쁺뼢쓣 겮移섏 븡뒗떎怨 寃곕줎쓣 궡由 닔 엳뿀떎.

삉븳 DNA 蹂듭젣 吏꾪뻾 以묒뿉 Vas-SCF Vas-SCF-religated 洹몃9쓽 遺怨 쟾빑씠 愿李곕릺吏 븡怨 degradation씠 愿李곕릺뿀뒗뜲, Yamauchi쓽 뿰援 寃곌낵씤 SCF媛 씪뼱궃 젙옄瑜 二쇱엯븯硫 遺怨 쟾빑뿉꽌 DNA 蹂듭젣 떆옉 떆 degradation맂떎뒗 뿰援 寃곌낵 씪移섑븯떎(Yamauchi et al., 2007).

寃곕줎쟻쑝濡 DNA 蹂듭젣 씪씠꽱떛 썑 蹂듭젣媛 吏꾪뻾릺硫 DNA 蹂듭젣 씪씠꽱떛 DNA 넀긽 뿬遺 愿怨꾧 뾾吏留 洹 씠썑 怨쇱젙뿉꽌뒗 넀긽맂 DNA쓽 蹂듭젣瑜 諛⑹븯湲 쐞빐 遺怨 쟾빑뿉꽌 쟾삎쟻씤 꽭룷옄궡寃쎈줈뒗 떎瑜 湲곗옉씠 議댁옱븯뒗 寃껋쑝濡 깮媛곷릺硫, 씠뿉 븳 援ъ껜쟻씤 寃쎈줈 뙆븙쓣 쐞븳 異붽 뿰援ш 븘슂븳 寃껋쑝濡 뙋떒맂떎.

ACKNOWLEDGEMENTS

This study was financially supported by Chonnam National University, 2014.

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