Application of saponin on differential staining examination in animal blastocysts

SUNG WOO KIM; NEELESH SHARMA; IN-SUL HWANG; CHANGYONG CHOE; DONGKYO KIM; HWAN-HOO SEONG; DONG KEE JEONG

doi:10.56093/ijans.v87i9.74292

Authors

SUNG WOO KIM National Institute of Animal Science, Unbongup Hwansanro 1214–13, Namwon 55717, Republic of Korea
NEELESH SHARMA National Institute of Animal Science, Unbongup Hwansanro 1214–13, Namwon 55717, Republic of Korea
IN-SUL HWANG National Institute of Animal Science, Unbongup Hwansanro 1214–13, Namwon 55717, Republic of Korea
CHANGYONG CHOE National Institute of Animal Science, Unbongup Hwansanro 1214–13, Namwon 55717, Republic of Korea
DONGKYO KIM National Institute of Animal Science, Unbongup Hwansanro 1214–13, Namwon 55717, Republic of Korea
HWAN-HOO SEONG National Institute of Animal Science, Unbongup Hwansanro 1214–13, Namwon 55717, Republic of Korea
DONG KEE JEONG National Institute of Animal Science, Unbongup Hwansanro 1214–13, Namwon 55717, Republic of Korea

https://doi.org/10.56093/ijans.v87i9.74292

Keywords:

Blastocysts staining, Bovine, Inner cell mass, Saponin, Trophectoderm cells

Abstract

Although there are several ways such as karyotyping to evaluate the quality and normality of embryos, the counting of total cell in blastocyst after the differential staining has been used as a simple indicator for quality of culture systems and normality of embryo itself. This differential staining method was regarded as a basic technique of early developmental biology of mammals, and it helps the scientific community to understand the signals regulating
morphological events of early developmental process. The present study was undertaken to develop a simple and fast differential staining method for inner cell mass (ICM) and trophectoderm (TE) cells of mammalian blastocysts using saponin as a permeabilizing agent without using species-specific antibodies and complements. The prestained blastocyst with SYTO-13 (green) was exposed to saponin solution for propidium iodide (PI) permeation into TE cells and examined for the differential staining patterns. Three dimensional confocal microscopy was used to demonstrate the process of successful staining and showed the high impact on saponin treatment. Although the fluorescent images of blastocysts showed that one or two cell of TE stained to yellowish green, ICM was protected from saponin/PI mixture with the short exposure time of SYTO-13 pre-stained blastocysts. The total staining
procedure did not exceed 30 min before examination under epi-fluorescence or confocal microscope. These results clearly demonstrate that saponin could be used as substituent molecule instead of species-specific antibodies and complements in differential staining examination for the first differentiation of mammalian embryos.

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References

Abdalla H, Shimoda M, Hirabayashi M and Hochi S. 2009. A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa. Theriogenology 72: 453–60. DOI: https://doi.org/10.1016/j.theriogenology.2009.03.011

Abeydeera L R, Wang W H, Cantley T C, Rieke A, Murphy C N, Prather R S and Day B N. 2000. Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor. Theriogenology 54: 787–97. DOI: https://doi.org/10.1016/S0093-691X(00)00390-3

Barlow P, Owen D A and Graham C. 1972. DNA synthesis in the preimplantation mouse embryo. Journal of Embryology and Experimental Morphology 27: 431–45. DOI: https://doi.org/10.1242/dev.27.2.431

Brackett B G and Oliphant G. 1975. Capacitation of rabbit spermatozoa in vitro. Biology of Reproduction 12: 260–74. DOI: https://doi.org/10.1095/biolreprod12.2.260

Dourmashkin R R, Dougherty R M and Harris R J C. 1962. Electron microscopic observations on Rous sarcoma virus and cell membranes. Nature 194: 1116–19. DOI: https://doi.org/10.1038/1941116a0

Ebert K M, Hammer R E and Papaioannou V E. 1985. A simple method for counting nuclei in the preimplantation mouse embryo. Experientia 41: 1207–09. DOI: https://doi.org/10.1007/BF01951732

Francis G, Levavi-Sivan B, Avitan A and Becker K. 2002. Effects of long-term feeding of Quillaja saponins on sex ratio, muscle and serum cholesterol and LH levels in Nile tilapia (Oreochromis niloticus (L). Comparative Biochemistry and Physiology - Part C: Toxicology and Pharmacology 133: 593– 603. DOI: https://doi.org/10.1016/S1532-0456(02)00167-9

Fuente de la R and King W A. 1997. Use of a chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos. Zygote 5(4): 309–20. DOI: https://doi.org/10.1017/S0967199400003890

Gardner R L, Papaioannou V E and Barton S C. 1973. Origin of the ectoplacental cone and secondary giant cells in mouse blastocysts reconstituted from the isolated trophoblast and inner cell mass. Journal of Embryology and Experimental Morphology 30: 561–72. DOI: https://doi.org/10.1242/dev.30.3.561

Giles J R and Foote R H. 1995. Rabbit blastocyst: allocation of cells to the inner cell mass and trophectoderm. Molecular Reproduction and Development 41: 204–11. DOI: https://doi.org/10.1002/mrd.1080410211

Giritharan G, Li M W, De Sebastiano F, Esteban F J, Horcajadas J A, Lloyd K C K, Donjacour A, Maltepe E and Rinaudo P F. 2010. Effect of ICSI on gene expression and development of mouse preimplantation embryos. Human Reproduction 25(12): 3012–24. DOI: https://doi.org/10.1093/humrep/deq266

Giritharan G, Talbi S, Donjacour A, Di Sebastiano F, Dobson A T and Rinaudo P F. 2007. Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos. Reproduction 134: 63–72. DOI: https://doi.org/10.1530/REP-06-0247

Handyside A H and Hunter S. 1984. A rapid procedure for visualizing the inner cell mass and trophectoderm nuclei of

mouse blastocysts in situ using polynucleotide-specific fluorochromes. Journal of Experimental Zoology 231: 429– 34.

Hardy K, Handyside A H and Winston R M. 1989. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 107: 597– 604. DOI: https://doi.org/10.1242/dev.107.3.597

Hyun S, Lee G, Kim D, Kim H, Lee S, Nam D, Jeong Y, Kim S, Yeom S, Kang S, Han J, Lee B and Hwang W S. 2003. Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein. Biology of Reproduction 69: 1060– 68. DOI: https://doi.org/10.1095/biolreprod.102.014886

Iwamura S, Sone M, Kawarasaki Ryu S T and Ogasa A. 1999. Precocious estrus and reproductive ability induced by PG 600 in prepuberal gilts. Journal of Veterinary Medical Science 61: 7–11. DOI: https://doi.org/10.1292/jvms.61.7

Iwasaki S, Yoshiba N, Ushijima H, Watanabe S and Nakahara T. 1990. Morphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo. Journal of Reproduction and Fertility 90: 279–84. DOI: https://doi.org/10.1530/jrf.0.0900279

Jalal F, Jumarie C, Bawab W, Corbeil D, Malo C, Berteloot A and Crine P. 1992. Polarized distribution of neutral endopeptidase 24.11 at the cell surface of cultured human intestinal epithelial Caco–2 cells. Biochemistry J 288 (Pt 3): 945–51. DOI: https://doi.org/10.1042/bj2880945

Kardel R, Ulfgren A K, Reinholt F P and Holmlund A. 2003. Inflammatory cell and cytokine patterns in patients with painful clicking and osteoarthritis in the temporomandibular joint. International Journal of Oral Maxillofacial Surgery 32: 390– 96. DOI: https://doi.org/10.1054/ijom.2002.0357

Koo D B, Kang Y K, Choi Y H, Park J S, Kim H N, Oh K B, Son D S, Park H, Lee K K and Han Y M. 2002. Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts. Biology of Reproduction 67: 487–92. DOI: https://doi.org/10.1095/biolreprod67.2.487

Leppens G, Gardner D K and Sakkas D. 1996. Co-culture of 1- cell outbred mouse embryos on bovine kidney epithelial cells: effect on development, glycolytic activity, inner cell mass: trophectoderm ratios and viability. Human Reproduction 11: 598–603. DOI: https://doi.org/10.1093/HUMREP/11.3.598

Machaty Z, Day B N and Prather R S. 1998. Development of early porcine embryos in vitro and in vivo. Biology of Reproduction 59: 451–55. DOI: https://doi.org/10.1095/biolreprod59.2.451

Papaioannou V E and Ebert K M. 1988. The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro. Development 102: 793–803. DOI: https://doi.org/10.1242/dev.102.4.793

Parrish J J, Susko-Parrish J L, Leibfried-Rutledge M L, Critser E S, Eyestone W H and First N L. 1986. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 25: 591–600. DOI: https://doi.org/10.1016/0093-691X(86)90143-3

Rosenkrans C F Jr, Zeng G Q, Mcnamara G T, Schoff P K and First N L. 1993. Development of bovine embryos in vitro as affected by energy substrates. Biology of Reproduction 49: 459–62. DOI: https://doi.org/10.1095/biolreprod49.3.459

Solter D and Knowles B B. 1975. Immunosurgery of mouse blastocyst. Proceeding of National Academy Science USA 72: 5099–102. DOI: https://doi.org/10.1073/pnas.72.12.5099

Summers M C, McGinnis L K, Lawitts J A, Raffin M and Biggers J D. 2000. IVF of mouse ova in a simplex optimized medium supplemented with amino acids. Human Reproduction 15: 1791–1801. DOI: https://doi.org/10.1093/humrep/15.8.1791

Tao T and Niemann H. 2000. Cellular characterization of blastocysts derived from rabbit 4-, 8- and 16-cell embryos and isolated blastomeres cultured in vitro. Human Reproduction 15: 881–89. DOI: https://doi.org/10.1093/humrep/15.4.881

Thouas G A, Korfiatis N A, French A J, Jones G M and Trounson A O. 2001. Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts. Reproductive BioMedicine Online 3: 25–29. DOI: https://doi.org/10.1016/S1472-6483(10)61960-8

Tremoleda J L, Stout T A, Lagutina I, Lazzari G, Bevers M M, Colenbrander B and Galli C. 2003. Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and blastocyst capsule formation. Biology of Reproduction 69: 1895–1906. DOI: https://doi.org/10.1095/biolreprod.103.018515

Van Soom A, Boerjan M, Ysebaert M T and De Kruif A. 1996. Cell allocation to the inner cell mass and the trophectoderm in bovine embryos cultured in two different media. Molecular Reproduction and Development 45: 171–82. DOI: https://doi.org/10.1002/(SICI)1098-2795(199610)45:2<171::AID-MRD10>3.0.CO;2-4

Webel S K and Day B N. 1982. The control of ovulation. Control of Pig Reproduction. pp 197–210. (Eds) Cole D J A and Foxcroft G R. Butterworths, London. DOI: https://doi.org/10.1016/B978-0-408-10768-6.50015-0