Hostname: page-component-76fb5796d-vvkck Total loading time: 0 Render date: 2024-04-26T05:55:49.846Z Has data issue: false hasContentIssue false
  • English
  • Français

Non-invasive assessment of porcine oocyte quality by supravital staining of cumulus–oocyte complexes with lissamine green B

Published online by Cambridge University Press:  21 July 2015

Rahul Dutta ,
Shun Li ,
Konrad Fischer ,
Alexander Kind ,
Tatiana Flisikowska ,
Krzysztof Flisikowski ,
Oswald Rottmann  and
Angelika Schnieke
Rahul Dutta*
Affiliation:
Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.
Shun Li
Affiliation:
Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.
Konrad Fischer
Affiliation:
Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.
Alexander Kind
Affiliation:
Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.
Tatiana Flisikowska
Affiliation:
Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.
Krzysztof Flisikowski
Affiliation:
Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.
Oswald Rottmann
Affiliation:
Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.
Angelika Schnieke
Affiliation:
Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.
*
All correspondence to: Rahul Dutta. Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany. e-mail: doctordut@gmail.com
Get access Rights & Permissions [Opens in a new window]

Summary

We evaluated the usefulness of lissamine green B (LB) staining of cumulus–oocyte complexes (COC) as a non-invasive method of predicting maturational and developmental competence of slaughterhouse-derived porcine oocytes cultured in vitro. Cumulus cells of freshly aspirated COCs were evaluated either morphologically on the basis of thickness of cumulus cell layers, or stained with LB, which penetrates only non-viable cells. The extent of cumulus cell staining was taken as an inverse indicator of membrane integrity. The two methods of COC grading were then examined as predictors of nuclear maturation and development after parthenogenetic activation. In both cases LB staining proved a more reliable indicator than morphological assessment (P < 0.05). The relationship between LB staining and cumulus cell apoptosis was also examined. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for DNA fragmentation revealed that oocytes within COCs graded as low quality by either LB staining or visual morphology showed significantly greater DNA fragmentation (P < 0.05) than higher grades, and that LB and visual grading were of similar predictive value. Expression of the stress response gene TP53 showed significantly higher expression in COCs graded as low quality by LB staining. However expression of the apoptosis-associated genes BAK and CASP3 was not significantly different between high or low grade COCs, suggesting that mRNA expression of BAK and CASP3 is not a reliable method of detecting apoptosis in porcine COCs. Evaluation of cumulus cell membrane integrity by lissamine green B staining thus provides a useful new tool to gain information about the maturational and developmental competence of porcine oocytes.

Keywords

Cumulus cellsIVMLissamine green BMembrane integrityPigTP53TUNEL
Type
Research Article
Information
Zygote , Volume 24 , Issue 3 , June 2016 , pp. 418 - 427
Copyright
Copyright © Cambridge University Press 2015 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

3

Present address: Shanghai Public Health Clinical Center, Jin Shan District, Shanghai 201508, China.

References

Abeydeera, L.R. (2002). In vitro production of embryos in swine. Theriogenology 57, 256–73.Google Scholar
Abeydeera, L.R. & Day, B.N. (1997). Fertilization and subsequent development of in vitro pig oocytes inseminated in a modified tris-buffered medium with frozen–thawed ejaculated spermatozoa. Biol. Reprod. 57, 729–34.CrossRefGoogle Scholar
Akshey, Y.S., Malakar, D., De, A.K., Jena, M.K., Sahu, S. & Dutta, R. (2011). Study of the efficiency of chemically assisted enucleation method for handmade cloning in goat (Capra hircus). Reprod. Domest. Anim. 46, 699–704.Google Scholar
Assou, S., Haouzi, D., De Vos, J. & Hamamah, S. (2010). Human cumulus cells as biomarkers for embryo and pregnancy outcomes. Mol. Hum. Reprod. 16, 531–8.CrossRefGoogle ScholarPubMed
Brantom, P.G., Creasy, D.M. & Gaunt, I.F. (1987). Long-term toxicity study of green S in mice. Food Chem. Toxicol. 25, 977–83.CrossRefGoogle ScholarPubMed
Chandrakanthan, V., Li, A., Chami, O. & O’Neill, C. (2006). Effects of in vitro fertilization and embryo culture on TRP53 and Bax expression in B6 mouse embryos. Reprod. Biol. Endocrinol. 4, 61.CrossRefGoogle ScholarPubMed
Cheng, W.T.K., Polge, C. & Moor, R.M. (1986). In vitro fertilization of pig and sheep oocytes. Theriogenology 25, 146 (abstract).Google Scholar
Cheng, E., Chen, S., Lee, T.H., Pai, Y.P., Huang, L.S., Huang, C.C. & Lee, M.S. (2013). Evaluation of telomere length in cumulus cells as a potential biomarker of oocyte and embryo quality. Hum. Reprod. 28, 929–36.CrossRefGoogle ScholarPubMed
Clode, S.A. (1987). Teratogenicity and embryo toxicity study of green S in rats. Food Chem. Toxicol. 25, 995–7.Google Scholar
Corn, C.M., Hauser–Kronberger, C., Moser, M., Tews, G. & Ebner, T. (2005). Predictive value of cumulus cell apoptosis with regard to blastocyst development of corresponding gametes. Fertil. Steril. 84, 627–33.CrossRefGoogle ScholarPubMed
Coticchio, G., Sereni, E., Serrao, L., Mazzone, S., Iadarola, I. & Borini, A. (2004). What criteria for the definition of oocyte quality? New York Acad. Sci. 1034, 132–44.Google Scholar
Davachi Dadashpour, N., Kohram, H. & Zainoaldini, S. (2012). Cumulus cell layers as a critical factor in meiotic competence and cumulus expansion of ovine oocytes. Small Rum. Res. 10, 237–42.Google Scholar
Flisikowska, T., Kind, A. & Schnieke, A. (2013). The new pig on the block: modelling cancer in pigs. Transgenic Res. 22, 673–80.Google Scholar
Galeati, G., Modina, S., Lauria, A. & Mattioli, M. (1991). Follicle somatic cells influence pig oocyte penetrability and cortical granule distribution. Mol. Reprod. Dev. 29, 40–6.Google Scholar
Gandhi, A., Lane, M., Gardner, D. & Krisher, R. (2001). Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media. Mol. Reprod. Dev. 58, 269–75.Google Scholar
Garg, S., Dutta, R., Malakar, D., Jena, M.K., Kumar, D., Sahu, S. & Prakash, B. (2012). Cardiomyocytes rhythmically beating generated from goat embryonic stem cell. Theriogenology 77, 829–39.CrossRefGoogle ScholarPubMed
Grupen, C.G. (2014). The evolution of porcine embryo in vitro production. Theriogenology 81, 24–37.Google Scholar
Hamrah, P., Alipour, F., Jiang, S., Sohn, J.H. & Foulks, G.N. (2011). Optimizing evaluation of lissamine green parameters for ocular surface staining. Eye 25, 1429–34.Google Scholar
Hendriksen, P.J., Vos, P.L., Steenweg, W.N.M., Bevers, M.M. & Dielemans, S.J. (2000). Bovine follicular development and its effect on the in vitro competence of oocytes. Theriogenology 53, 11–20.Google Scholar
Hyttel, P., Fair, T., Callesen, H. & Greve, T. (1997). Oocyte growth, capacitation and final maturation in cattle. Theriogenology 47, 23–32.CrossRefGoogle Scholar
Jones, S.N., Roe, A.E., Donehower, L.A. & Bradley, A. (1995). Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206–8.Google Scholar
Kim, J. (2000). The use of vital dyes in corneal disease. Curr. Opin. Ophthalmol. 11, 241–7.CrossRefGoogle ScholarPubMed
Kim, J. & Foulks, G.N. (1999). Evaluation of the effect of lissamine green and rose Bengal on human corneal epithelial cells. Cornea 18, 328–32.Google Scholar
Lee, K.S., Joo, B.S., Na, Y.J., Yoon, M.S., Choi, O.H. & Kim, W.W. (2001). Cumulus cells apoptosis as an indicator to predict the quality of oocytes and the outcome of IVF-ET. J. Assist. Reprod. Genet. 18, 490–8.CrossRefGoogle ScholarPubMed
Livak, K.J. & Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔ C T method. Methods 25, 402–8.CrossRefGoogle Scholar
Luna, R.M.D., Wagner, D.S. & Lozano, G. (1995). Rescue of early embryonic lethality in Mdm2-deficient mice by deletion of P53. Nature 378, 203–6.Google Scholar
Manabe, N., Imai, Y., Ohno, H., Takahagi, Y., Sugimoto, M. & Miyamoto, H. (1996). Apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pig ovaries. Experientia 52, 647–51.Google Scholar
McElroy, S.L., Byrne, J.A., Chavez, S.L., Behr, B., Hsueh, A.J., Westphal, L.M. & Pera Reijo, R.A. (2010). Parthenogenetic blastocyst derived from cumulus-free in vitro matured human oocytes. PLoS One 5, e10979.Google Scholar
Meyer, M., Martin, A.H., Wurst, W. & Kühn, R.R. (2010). Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc. Natl. Acad. Sci. 107, 15022–6.Google Scholar
Mikkelsen, A.L., Host, E. & Lindenberg, S. (2001). Incidence of apoptosis in granulosa cells from immature human follicles. Reproduction 122, 481–6.Google Scholar
Miyano, T. & Manabe, N. (2007). Oocyte growth and acquisition of meiotic competence. Soc. Reprod. Fert. 63, 531–8.Google Scholar
Moorhouse, S.R., Creasy, D.M. & Gaunt, I.F. (1987). Three-generation toxicity study of rats ingesting green S in the diet. Food Chem. Toxicol. 25, 985–93.Google Scholar
Morita, Y. & Tilly, J.L. (1999). Oocyte apoptosis: like sand through an hourglass. Dev. Biol. 213, 1–17.Google Scholar
Nakai, M., Kashiwazaski, N., Takizawa, A., Hayashi, Y., Nakatsukasa, E., Fuchimoto, D., Noguchi, J., Kaneko, H., Shino, M. & Kikuchi, K. (2003). Viable piglets generated from porcine oocyte matured in vitro and fertilized by intracytoplasmic sperm head injection. Biol. Reprod. 68, 1003–8.Google Scholar
Prates, E.G., Nunes, J.T. & Pereira, R.M. (2014). A role of lipid metabolism during cumulus-oocyte complex maturation: impact of lipid modulators to improve embryo production. Med. Inflam. 11, 11.Google Scholar
Qian, Y., Shi, W., Ding, J., Sha, J. & Fan, B. (2003). Predictive value of the area of expanded cumulus mass on development of porcine oocytes matured and fertilized in vitro . J. Reprod. Dev. 49, 167–74.CrossRefGoogle ScholarPubMed
Rougier, N. & Werb, Z. (2001). Minireview, Parthenogenesis in mammals. Mol. Reprod. Dev. 59, 468–74.Google Scholar
Ruppert-Lingham, C.J., Paynter, S.J., Godfrey, J., Fuller, B.J. & Shaw, R.W. (2006). Membrane integrity and development of immature murine cumulus–oocyte complexes following slow cooling to –60°C: the effect of immediate rewarming, plunging into LN2 and two-controlled-rate-stage cooling. Cryobiology 52, 219–27.Google Scholar
Sirard, M.A., Richard, F., Blondin, P. & Robert, C. (2006). Contribution of the oocyte to embryo quality. Theriogenology 65, 126–36.Google Scholar
Sjunnesson, Y.C.B., Morrell, J.M. & González, R. (2013). Single layer centrifugation-selected boar spermatozoa are capable of fertilization in vitro . Acta Vet. Scand. 55, 20.CrossRefGoogle ScholarPubMed
Szoltys, M., Tabarowski, Z. & Pawlik, A. (2000). Apoptosis of postovulatory cumulus granulosa cells of the rat. Anat. Embryol. Berl. 202, 523–9.Google Scholar
Tanghe, S., Van Soom, A., Nauwynck, H., Coryn, M., de Kruif, A. (2002). Minireview: functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol. Reprod. Dev. 61, 414–24.Google Scholar
Tong, G.Q., Heng, B.C., Chen, N.Q., Yip, W.Y. & Ng, S.C. (2004). Effects of elevated temperature in vivo on the maturational and developmental competence of porcine germinal vesicle stage oocytes. J. Anim. Sci. 82, 3175–80.Google Scholar
Uchikura, K., Nagano, M. & Hishinuma, M. (2011). Prediction of maturational competence of feline oocytes using supravital staining of cumulus cells by propidium iodide. Zygote 20, 333–7.Google Scholar
Van Blerkom, J. (1996). The influence of intrinsic and extrinsic factors on the developmental potential and chromosomal normality of the human oocyte. J. Soc. Gynecol. Invest. 3, 3–10.Google Scholar
Vandaele, L., Goossens, K., Peelman, L. & Van Soom, A. (2008). mRNA expression of Bcl-2, Bax, caspase-3 and -7 cannot be used as a marker for apoptosis in bovine blastocysts. Anim. Reprod. Sci. 106 (1–2), 168–73.Google Scholar
Wang, W., Sun, Q., Hosoe, M., Shioya, Y. & Day, B.B. (1997). Quantified analysis of cortical granule distribution and exocytosis of porcine oocytes during meiotic maturation and activation. Biol. Reprod. 56, 1376–82.Google Scholar
Weil, M., Jacobson, M.D., Coles, H.S.R, Davies, T.J., Gardner, R.L., Raff, K.D. & Raff, M.C. (1996). Constitutive expression of the machinery for programmed cell death. J. Cell Biol. 133, 1053–9.Google Scholar
Yuana, Y.Q., Van Sooma, A., Leroya, J.L.M.R., Dewulfa, J., Van Zeverenb, A., de Kruifa, A. & Peelman, L.J. (2005). Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence. Theriogenology 63, 2147–63.Google Scholar
Yuge, M.M. (2003). Effects of cooling ovaries before oocyte aspiration on meiotic competence of porcine oocytes and of exposing in vitro matured oocytes to ambient temperature on in vitro fertilization and development of the oocytes. Cryobiology 47, 102–8.Google Scholar
Zeuner, A., Muller, K., Reguszynski, K. & Jewgenow, K. (2003). Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation. Theriogenology 59, 1421–33.Google Scholar