Havemeyer Foundati<strong>on</strong> M<strong>on</strong>ograph Series No. 3COMPARISON OF BOVINE AND <strong>EQUINE</strong> OOCYTESAS HOST CYTOPLASTS FOR <strong>EQUINE</strong> NUCLEARTRANSFERK. Hinrichs, T. Shin, C. C. Love, D. D. Varner and M. E. WesthusinCollege <str<strong>on</strong>g>of</str<strong>on</strong>g> Veterinary Medicine, Texas A&M University, College Stati<strong>on</strong>, Texas 77843-4466, USATransfer <str<strong>on</strong>g>of</str<strong>on</strong>g> adult somatic cell nuclei to enucleatedoocytes has resulted in producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g>fspring inmany species, including sheep, cattle and mice.The basic steps involved in nuclear transfer arematurati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytes to MII, enucleati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> MIIoocytes to form a host cytoplast, placement <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>somatic cell into <str<strong>on</strong>g>the</str<strong>on</strong>g> perivitelline space, fusi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g><str<strong>on</strong>g>the</str<strong>on</strong>g> cell membranes to introduce <str<strong>on</strong>g>the</str<strong>on</strong>g> somatic cellnucleus into <str<strong>on</strong>g>the</str<strong>on</strong>g> cytoplast, and activati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>rec<strong>on</strong>structed oocyte. No informati<strong>on</strong> is currentlyavailable <strong>on</strong> nuclear transfer in <str<strong>on</strong>g>the</str<strong>on</strong>g> horse, however,our preliminary studies suggested that many stepsinvolved in nuclear transfer may be more difficultin <str<strong>on</strong>g>the</str<strong>on</strong>g> horse than in o<str<strong>on</strong>g>the</str<strong>on</strong>g>r species. To define <str<strong>on</strong>g>the</str<strong>on</strong>g>areas which may present problems in work withhorse oocytes, we compared <str<strong>on</strong>g>the</str<strong>on</strong>g> efficiency <str<strong>on</strong>g>of</str<strong>on</strong>g>nuclear transfer procedures using ei<str<strong>on</strong>g>the</str<strong>on</strong>g>r enucleatedbovine or equine oocytes as host cytoplasts. Weevaluated <str<strong>on</strong>g>the</str<strong>on</strong>g>ir ability to dec<strong>on</strong>dense a transferredhorse somatic cell nucleus and to undergo cleavageand development after nuclear transfer. Bovineoocytes have been used successfully as hostcytoplasts for <str<strong>on</strong>g>the</str<strong>on</strong>g> transfer <str<strong>on</strong>g>of</str<strong>on</strong>g> nuclei from a variety<str<strong>on</strong>g>of</str<strong>on</strong>g> species, including sheep, pigs, m<strong>on</strong>keys and rats(Dominko et al. 1999). While development to <str<strong>on</strong>g>the</str<strong>on</strong>g>blastocyst stage was reported for interspeciesnuclear transfer embryos, in our experience andthat <str<strong>on</strong>g>of</str<strong>on</strong>g> o<str<strong>on</strong>g>the</str<strong>on</strong>g>rs, <str<strong>on</strong>g>the</str<strong>on</strong>g>se embryos rarely progress past<str<strong>on</strong>g>the</str<strong>on</strong>g> 16-cell stage.Mature bovine oocytes were obtained from acommercial source. One hundred fifty-nine invitro-matured Metaphase II bovine oocytesunderwent enucleati<strong>on</strong>, <str<strong>on</strong>g>of</str<strong>on</strong>g> which all (100%)survived intact. Micromanipulati<strong>on</strong> was performedas previously described (Hill et al. 2000). Theenucleated cells were recombined with equinecumulus or fibroblast cells and were subjected to 2x 25-µs 1.2 kv/cm DC pulses. The proporti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g>cells successfully fused was 109/159 (69%).Oocytes were activated using i<strong>on</strong>ophore A23187or i<strong>on</strong>omycin followed by culture in 6-DMAP.Ninety-three fused oocytes were cultured fur<str<strong>on</strong>g>the</str<strong>on</strong>g>rin vitro <strong>on</strong> Vero cell m<strong>on</strong>olayers; <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>se, 49(53%) cleaved and 36 (39%) developed to <str<strong>on</strong>g>the</str<strong>on</strong>g> 8 to16-cell stage. No morula or blastocystdevelopment was observed. There were nosignificant differences in fusi<strong>on</strong> or cleavage ratesbetween oocytes fused with cumulus cells (35/51,69% and 17/35, 49%, respectively) or fibroblasts(74/108, 69% and 32/58, 55%, respectively).Horse oocytes were recovered fromslaughterhouse-derived ovaries and were harvestedand matured in vitro as previously described(Hinrichs and Schmidt 2000) or were matured in100% equine follicular fluid derived in vivo from adominant preovulatory follicle 24 h after hCGadministrati<strong>on</strong>. Of 86 in vitro-matured MetaphaseII horse oocytes used for NT, 59 (69%) were intactafter enucleati<strong>on</strong>. The difficulty <str<strong>on</strong>g>of</str<strong>on</strong>g> enucleati<strong>on</strong> wasincreased because <str<strong>on</strong>g>of</str<strong>on</strong>g> various factors. Thec<strong>on</strong>sistency <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> z<strong>on</strong>a pellucida was more pliablethan that <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> bovine oocytes, and thus was moredifficult to penetrate with <str<strong>on</strong>g>the</str<strong>on</strong>g> large pipette neededfor enucleati<strong>on</strong> and for transfer <str<strong>on</strong>g>of</str<strong>on</strong>g> somatic cells.The metaphase plate and polar body were <str<strong>on</strong>g>of</str<strong>on</strong>g>tenwidely separated, necessitating 2 z<strong>on</strong>a puncturesto fully enucleate <str<strong>on</strong>g>the</str<strong>on</strong>g> oocyte. In additi<strong>on</strong>, <str<strong>on</strong>g>the</str<strong>on</strong>g> polarbody frequently appeared to be attached to <str<strong>on</strong>g>the</str<strong>on</strong>g>z<strong>on</strong>a pellucida, making it difficult to removewithout damage to <str<strong>on</strong>g>the</str<strong>on</strong>g> oocyte. No obviousdifferences in ease <str<strong>on</strong>g>of</str<strong>on</strong>g> oocyte manipulati<strong>on</strong> werenoted between maturati<strong>on</strong> treatments. Theenucleated oocytes were recombined with equinecumulus or fibroblast cells. Of recombined cells,28/59 (48%) successfully fused. In unfusedoocytes, ei<str<strong>on</strong>g>the</str<strong>on</strong>g>r <str<strong>on</strong>g>the</str<strong>on</strong>g> somatic cell lysed after <str<strong>on</strong>g>the</str<strong>on</strong>g>43
Equine Embryo Transferelectric pulse, or both oocyte and somatic cellmembranes were found to be still intact. Oocyteswere activated using ei<str<strong>on</strong>g>the</str<strong>on</strong>g>r i<strong>on</strong>omycin or calciumi<strong>on</strong>ophore A23187, followed by cycloheximidetreatment. Subsequent culture was performed <strong>on</strong> aVero cell m<strong>on</strong>olayer. Only 3/28 (11%) <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> fusedrecombined cells cleaved; no significantdifferences were noted between maturati<strong>on</strong>treatments or d<strong>on</strong>or cell source. These recombinedcells developed to <str<strong>on</strong>g>the</str<strong>on</strong>g> 2-, 3-, and 5-cell stage butdid not develop fur<str<strong>on</strong>g>the</str<strong>on</strong>g>r. Presence <str<strong>on</strong>g>of</str<strong>on</strong>g> nuclei wasc<strong>on</strong>firmed in <str<strong>on</strong>g>the</str<strong>on</strong>g>se embryos using Hoechst 33258.To our knowledge, this is <str<strong>on</strong>g>the</str<strong>on</strong>g> first reportdocumenting <str<strong>on</strong>g>the</str<strong>on</strong>g> producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> equine nucleartransfer embryos. The results <str<strong>on</strong>g>of</str<strong>on</strong>g> this study indicatethat rates <str<strong>on</strong>g>of</str<strong>on</strong>g> enucleati<strong>on</strong>, fusi<strong>on</strong> and subsequentcleavage are lower when equine IVM oocytes areused as cytoplasts than when bovine IVM oocytesare used. The difficulty in performing <str<strong>on</strong>g>the</str<strong>on</strong>g>procedures necessary for nuclear transfer in <str<strong>on</strong>g>the</str<strong>on</strong>g>horse is unfortunate in light <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> lowefficiency <str<strong>on</strong>g>of</str<strong>on</strong>g> methods for harvest <str<strong>on</strong>g>of</str<strong>on</strong>g> equineoocytes (by follicular slicing and scraping <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>granulosa cell layer or by follicle aspirati<strong>on</strong>),relatively low maturati<strong>on</strong> rates <str<strong>on</strong>g>of</str<strong>on</strong>g> equine oocytesin vitro, and labour-intensive methods needed fordenuding <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> equine cumulus (by individualpipetting, as mass vortexing is not effective).These factors combine to increase <str<strong>on</strong>g>the</str<strong>on</strong>g> difficulty <str<strong>on</strong>g>of</str<strong>on</strong>g>using horse oocytes as host cytoplasts, incomparis<strong>on</strong> with bovine oocytes. The successobtained with bovine host cytoplasts dem<strong>on</strong>stratesthat <str<strong>on</strong>g>the</str<strong>on</strong>g> use <str<strong>on</strong>g>of</str<strong>on</strong>g> equine somatic cells can result insuccessful fusi<strong>on</strong> and cleavage. Fur<str<strong>on</strong>g>the</str<strong>on</strong>g>r work isneeded to determine optimal parameters for invitro maturati<strong>on</strong> and nuclear transfer in horseoocytes.ACKNOWLEDGEMENTSThis work was supported by <str<strong>on</strong>g>the</str<strong>on</strong>g> Link EquineResearch Endowment Fund, Texas A&MUniversity.REFERENCESDominko, T., Mitalipova, M., Haley, B., Zeki, B.,Memili, E., McKusick, B. and First, N. (1999)Bovine oocyte cytoplasm supports development <str<strong>on</strong>g>of</str<strong>on</strong>g>embryos produced by nuclear transfer <str<strong>on</strong>g>of</str<strong>on</strong>g> somaticcell nuclei from various mammalian species. Biol.reprod. 60, 1496-1502.Hill, J.R., Winger, Q.A., L<strong>on</strong>g, C.R., Lo<strong>on</strong>ey, C.R.,Thomps<strong>on</strong>, J.A. and Westhusin, M.E. (2000)Development rates <str<strong>on</strong>g>of</str<strong>on</strong>g> male bovine nuclear transferembryos derived from adult and fetal cells. Biol.reprod. 62, 1135-1140.Hinrichs, K. and Schmidt, A.L. (2000) Meioticcompetence in horse oocytes: Interacti<strong>on</strong>s am<strong>on</strong>gchromatin c<strong>on</strong>figurati<strong>on</strong>, follicle size, cumulusmorphology and seas<strong>on</strong>. Biol. reprod. 62, 1402-1408.44
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