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Reliability of Hoechst 33342 Staining under Wide-Field Microscopy for Evaluation of the Nuclear Status of Living Dog Oocytes

  • Martine Chebrout (a1) (a2), Pierre-Gaël Adenot (a1), Karine Reynaud (a1) (a2) and Sylvie Chastant-Maillard (a1) (a2) (a3)

Due to the marked cytoplasmic opacity of canine oocytes, the diagnosis of their nuclear status is difficult. The objective of the present study was to evaluate the accuracy of Hoechst staining observed under epifluorescence wide-field microscopy [living oocyte observation (LivOO)] by comparison to a reference technique [DNA staining with ethidium homodimer-2 under confocal microscopy; fixed oocyte observation (FixOO)]. Four Hoechst 33342 concentrations (200 ng, 500 ng, 1 μg, 2 μg/mL) were tested and 1 μg/mL was the lowest one with the lowest proportion of oocytes in which DNA was missed. At this concentration, LivOO procedure did not affect the degeneration rate. On 379 oocytes observed individually with the two techniques successively, diagnosis of meiosis resumption by LivOO was exact in 87.3% of the cases, but the meiosis resumption rate was underestimated (23.5% versus 34.3% with FixOO; p < 0.001). Diagnosis for metaphase II was exact in 80% of the cases, but LivOO detected only 72.7% of the metaphase II oocytes present. Metaphase rates did not differ between LivOO and FixOO. This study contributes to a better interpretation of in vitro maturation results. The developmental potential of metaphase II canine oocytes sorted after Hoechst staining is to be evaluated.

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P.G. Adenot , E. Corteggiani , M. Geze , D. Besombes & P. Debey (1992). The DNA content of mouse two-cell embryos can be measured by microfluorimetric image analysis under conditions of cell viability. J Fluoresc 2, 181206.

P.G. Adenot , M.S. Szöllösi , M. Geze , J.P. Renard & P. Debey (1991). Dynamics of paternal chromatin changes in live one-cell mouse embryo after natural fertilization. Mol Reprod Dev 28, 2334.

A.K. Alhaider & P.F. Watson (2009). The effects of hCG and growth factors on in vitro nuclear maturation of dog oocytes obtained during anoestrus. Reprod Fert Dev 21, 538548.

D.J. Arndt-Jovin & T.M. Jovin (1977). Analysis and sorting of living cells according to deoxyribonucleic acid content. J Histochem Cytochem 25, 585589.

L. Bogliolo , M.T. Zedda , S. Ledda , G. Leoni , S. Naitana & S. Pau (2002). Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes. Reprod Nutr Dev 42, 265273.

S. Chastant-Maillard , M. Chebrout , S. Thoumire , M. Saint-Dizier , M. Chodkiewicz & K. Reynaud (2010). Embryo biotechnology in the dog: A review. Reprod Fert Dev 22, 10491056.

S. Chastant-Maillard , C. Viaris De Lesegno , M. Chebrout , S. Thoumire , T. Meylheuc , A. Fontbonne , M. Chodkiewicz , M. Saint-Dizier & K. Reynaud (2011). The canine oocyte: Uncommon features of in vivo and in vitro maturation. Reprod Fert Dev 23, 391402.

M. Chebrout , C.V. De Lesegno , K. Reynaud , S. Chat & S. Chastant-Maillard (2009). Nuclear and cytoplasmic maturation of canine oocytes related to in vitro denudation. Reprod Dom Anim 44(Suppl 2), 243246.

E.S. Critser & N.L. First (1986). Use of a fluorescent stain for visualization of nuclear material in living oocytes and early embryos. Stain Technol 61(1), 15.

X.S. Cui , Y.X. Jin , X.H. Shen , J.Y. Lee , H.S. Lee , X.J. Yin , I.K. Kong & N.H. Kim (2006). Epidermal growth factor enhances meiotic resumption of canine oocytes in the presence of BSA. Theriogenology 66, 267274.

P. Debey , J.P. Renard , M. Coppey-Moisan , I. Monnot & M. Geze (1989). Dynamics of chromatin changes in live one-cell mouse embryos: A continuous follow-up by fluorescence microscopy. Exp Cell Res 183(2), 413433.

C.V. De Lesegno , K. Reynaud , C. Pechoux , S. Thoumire & S. Chastant-Maillard (2008). Ultrastructure of canine oocytes during in vivo maturation. Mol Reprod Dev 75(1), 115125.

R.E. Durand & P.L. Olive (1982). Cytotoxicity, mutagenicity and DNA damage by Hoechst 33342. J Histochem Cytochem 30, 111116.

E. Hamori , D.J. Arndt-Jovin , B.G. Grimwade & T.M. Jovin (1980). Selection of viable cells with known DNA. Content Cytometry 1(2), 132135.

C. Hanna , S. Menges , D. Kraemer & C.R. Long (2008). Synchronisation of canine germinal vesicle stage oocytes prior to in vitro maturation alters the kinetics of nuclear progression during subsequent resumption of meiosis. Reprod Fertil Dev 20(5), 606614.

S. Hatoya , Y. Sugiyama , H. Nishida , T. Okuno , R. Torii , K. Sugiura , K. Kida , N. Kawate , H. Tamada & T. Inaba (2009). Canine oocyte maturation in culture: Significance of estrogen and EGF receptor gene expression in cumulus cells. Theriogenology 71(4), 560567.

D.A. Hewitt , P.F. Watson & G.C.W. England (1998). Nuclear staining and culture requirements for in vitro maturation of domestic bitch oocytes. Theriogenology 49, 10831101.

M.S. Hossein , M.K. Kim , G. Jang , H.J. Oh , O. Koo , J.J. Kim , S.K. Kang , B.C. Lee & W.S. Hwang (2007). Effects of thiol compounds on in vitro maturation of canine oocytes collected from different reproductive stages. Mol Reprod Dev 74(9), 12131220.

G. Jang , S. Hong , J. Kang , J. Park , H. Oh , C. Park , J. Ha , D. Kim , M. Kim & B. Lee (2009). Conservation of the Sapsaree (Canis familiaris), a Korean Natural Monument, using somatic cell nuclear transfer. J Vet Med Sci 71(9), 12171220.

M.K. Kim , Y.H. Fibrianto , H.J. Oh , G. Jang , H.J. Kim , K.S. Lee , S.K. Kang , B.C. Lee & W.S. Hwang (2005). Effects of estradiol-17b and progesterone supplementation on in vitro nuclear maturation of canine oocytes. Theriogenology 63, 13421353.

K. Kuetemeyer , A. Lucas-Hahn , B. Petersen , E. Lemme , P. Hassel , H. Niemann & A. Heisterkamp (2010). Combined multiphoton imaging and automated functional enucleation of porcine oocytes using femtosecond laser pulses. J Biomed Opt 15(4), 046006.

E. Lee , J.H. Kim , S.M. Park , Y.I. Jeong , J.Y. Lee , S.W. Park , J. Choi , H.S. Kim , Y.W. Jeong , S. Kim , S.H. Hyun & W.S. Hwang (2008). The analysis of chromatin remodeling and the staining for DNA methylation and histone acetylation do not provide definitive indicators of the developmental ability of inter-species cloned embryos. Anim Reprod Sci 105(3-4), 438450.

H.S. Lee , Y.I. Seo , X.J. Yin , S.G. Cho , S.S. Lee , N.H. Kim , S.K. Cho & I.K. Kong (2007a). Effect of follicle stimulation hormone and luteinizing hormone on cumulus cell expansion and in vitro nuclear maturation of canine oocytes. Reprod Domest Anim 42(6), 561565.

S.R. Lee , M.O. Kim , H.K. Sung , B.S. Kim , D.H. Yoo , Y.S. Park , Y.B. Park , J.H. Ha & Z.Y. Ryoo (2007c). Effect of conditioned medium of mouse embryonic fibroblasts produced from EC-SOD transgenic mice in nuclear maturation of canine oocytes in vitro. Anim Reprod Sci 99, 106116.

G.P. Li , K.L. White & T.D. Bunch (2004). Review of enucleation methods and procedures used in animal cloning: State of the art. Cloning Stem Cells 6(1), 513.

Y. Liu , H.S. Sui , H.L. Wang , J.H. Yuan , M.J. Luo , P. Xia & J.H. Tan (2006). Germinal vesicle chromatin configurations of bovine oocytes. Microsc Res Tech 69(10), 799807.

G.C. Luvoni , S. Chigioni , E. Allievi & D. Macis (2003). Meiosis resumption of canine oocytes cultured in the isolated oviduct. Reprod Domest Anim 38, 410414.

G.C. Luvoni , S. Chigioni , E. Allievi & D. Macis (2005). Factors involved in in vivo and in vitro maturation of canine oocyte. Theriogenology 63, 4159.

F. Miyara , C. Migne , M. Dumont-Hassan , A. Le Meur , P. Cohen-Bacrie , F.X. Aubriot , A. Glissant , C. Nathan , S. Douard , A. Stanovici & P. Debey (2003). Chromatin configuration and transcriptional control in human and mouse oocytes. Mol Reprod Dev 64(4), 458470.

T. Otoi , R. Shimizu , H. Naoi , P. Wongsrikeao , B. Agung & M. Taniguchi (2006). Meiotic competence of canine oocytes embedded in collagen gel. Reprod Dom Anim 41, 1721.

T. Otoi , T. Shin , D.C. Kraemer & M.E. Westhusin (2004). Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization. Reprod Nutr Dev 44(6), 631637.

T. Otoi , T. Shin , D.C. Kraemer & M.E. Westhusin (2007). Role of cumulus cells on in vitro maturation of canine oocytes. Reprod Domest 42(2), 184189.

T. Otoi , L. Willingham , T. Shin , D.C. Kraemer & M. Westhusin (2002). Effects of oocyte culture density on meiotic competence of canine oocytes. Reproduction 124(6), 775781.

K. Reynaud , A. Fontbonne , N. Marseloo , S. Thoumire , M. Chebrout , C.V. De Lesegno & S. Chastant-Maillard (2005). In vivo meiotic resumption, fertilization and early embryonic development in the bitch. Reproduction 130(2), 193201.

B. Rodrigues & J.L. Rodrigues (2003). Influence of reproductive status on in vitro oocyte maturation in dogs. Theriogenology 60(1), 5966.

B.A. Rodrigues , L.C. dos Santos & J.L. Rodrigues (2006). The effect of hyaluronan concentrations in hST-supplemented TCM 199 on in vitro nuclear maturation of bitch cumulus-oocyte complexes. Theriogenology 66(6-7), 16731676.

B. de A. Rodrigues , L.C. dos Santos & J.L. Rodrigues (2004). Embryonic development of in vitro matured and in vitro fertilized dog oocytes. Mol Reprod Dev 67(2), 215223.

J. Saikhun , S. Sriussadaporn , N. Thongtip , A. Pinyopummin & Y. Kitiyanant (2008). Nuclear maturation and development of IVM/IVF canine embryos in synthetic oviductal fluid or in co-culture with buffalo rat liver cells. Theriogenology 69, 11041110.

M. Saint-Dizier , J.P. Renard & S. Chastant-Maillard (2001). Induction of final maturation by sperm penetration in canine oocytes. Reproduction 121, 97105.

M. Saint-Dizier , K. Reynaud & S. Chastant-Maillard (2004). Chromatin, microtubules, and kinases activities during meiotic resumption in bitch oocytes. Mol Reprod Dev 68(2), 205212.

A.E. Silva , P. Rodriguez , L.F. Cavalcante , B.A. Rodrigues & J.L. Rodrigues (2009). The influence of oxygen tension on cumulus cell viability of canine COCs matured in high-glucose medium. Reprod Domest Anim 44(Suppl 2), 259262.

L.C. Smith (1993). Membrane and intracellular effects of ultraviolet irradiation with Hoechst 33342 on bovine secondary oocytes matured in vitro. J Reprod Fertil 99(1), 3944.

C. Suzukamo , M. Hoshina , H. Moryia , N. Hishiyama , S. Nakamura , F. Kawai , H. Sato , M. Ariga , J. Ito & N. Kashiwazaki (2009). Kinetics of nuclear status and kinase activities during in vitro maturation of canine oocytes. J Reprod Dev 55(2), 116120.

J.H. Tan , H.L. Wang , X.S. Sun , Y. Liu , H.S. Sui & J. Zhang (2009). Chromatin configurations in the germinal vesicle of mammalian oocytes. Mol Hum Reprod 15(1), 19.

J.V. Tesoriero (1982). A morphological, cytochemical, and chromatographic analysis of lipid yolk formation in the oocytes of the dog. Gamete Res 6, 267279.

Y. Tsunoda , M. Shioda , K. Onodera , K. Nakamura & T. Uchida (1988). Differential sensitivity of mouse pronuclei and zygote cytoplasm to Hoechst staining and ultraviolet irradiation. J Reprod Fert 82, 173178.

C.I. Vannucchi , C.M. de Oliveira , M.G. Marques , M.E. Assumpção & J.A. Visintin (2006). In vitro canine oocyte nuclear maturation in homologous oviductal cell co-culture with hormone-supplemented media. Theriogenology 66(6-7), 16771681.

C.I. Vannucchi , M. Faustino , M.G. Marques , M. Nichi , M.E. Assumpção & J.A. Visintin (2009). Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17beta on in vitro maturation of canine oocytes. In Vitro Cell Dev Biol Anim 45(7), 328333.

L.A. Willingham-Rocky , K. Hinrichs , M.E. Westhusin & D.C. Kraemer (2003). Effects of stage of oestrous cycle and progesterone supplementation during culture on maturation of canine oocytes in vitro. Reproduction 126, 501508.

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Microscopy and Microanalysis
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