Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Simple Classification of Male Mouse Germ Cells using Hoechst 33258 Staining

Hoechst 33258 Staining을 이용한 웅성 생쥐 성세포의 간편 분류

  • Kim, Kyoung Guk (Dept. of Animal Science, Agricultural College of Yanbian University) ;
  • Park, Young Sik (Dept. of Animal Science and Biotechnology, College of Ecology and Environment Science, Kyungpook National University)
  • 김경국 (연변대학교 동물과학과) ;
  • 박영식 (경북대학교 축산생명공학과)
  • Received : 2015.05.07
  • Accepted : 2015.09.30
  • Published : 2015.09.30
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Abstract

In the study for a differentiation and development of spermatogonial cells, the researchers should commonly require a simple, fast and reasonable method that could evaluate the developmental stage of male germ cells without any damage and also relentlessly culture them so far as a cell stage aiming at experimental applications. For developing the efficient method to identify the stage of sperm cells, the morphological characteristics of sperm cells were investigated by staining the cells with blue fluorescent dye Hoechst 33258, and a criterion for male germ cell classification was elicited from results of the previous investigation, then the efficiency of the criterion was verified by applying it to assort the germ cells recovered from male mice in age from 6 to 35 days. As morphological characteristics, spermatogonia significantly differed from spermatocytes in size, appearance and fluorescent patches of nucleus, and spermatids could also be distinguished from spermatozoa by making a difference in the volume and shape of nucleus and the shape and fluorescence of tail. Aforesaid criterion was applicable for classifying in vitro cultured sperm cells by verifying its efficiency and propriety for assorting the stages of testicular germ cells. However, the fluorescent staining showed that germ cells in mouse testis should be dramatically differentiated and developed at 21 days and 35 days of age, which were known as times of sexual puberty and maturity in male mice, respectively. In conclusion, the results indicated that this simple criterion for sperm cell classification using fluorescence staining with Hoechst 33258 may be highly efficient and reasonable for spermatogenesis study.

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References

  1. de Franca LR, Ye SJ, Ying L, Sandberg M and Russell LD. 1995. Morphometry of rat germ cells during spermatogenesis. Anat. Rec. 241(2):181-204. https://doi.org/10.1002/ar.1092410206
  2. Drabent B, Bode C, Bramlage B and Doenecke D. 1996. Expression of the mouse testicular histone gene H1t during spermatogenesis. Histochem. Cell Biol. 106(2):247-51. https://doi.org/10.1007/BF02484408
  3. Drumond AL, Meistrich ML and Chiarini-Garcia H. 2011. Spermatogonial morphology and kinetics during testis development in mice: A high-resolution light microscopy approach. Reproduction. 142(1):145-55. https://doi.org/10.1530/REP-10-0431
  4. Feng LX, Chen Y, Dettin L, Reijo Pera RA, Herr JC and Goldberg E. 2002. Generation and in vitro differentiation of a spermatogonial cell line. J. Science. 297(5580):392-5. https://doi.org/10.1126/science.1073162
  5. Guan K, Wolf F, Becker A, Engel W, Nayernia K and Hasenfuss G. 2009. Isolation and cultivation of stem cells from adult mouse testes. Nat. Protoc. 4(2):143-54. https://doi.org/10.1038/nprot.2008.242
  6. Latt SA, Stetten G, Juergens LA, Willard HF and Scher CD. 1975. Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence. J. Histochem. Cytochem. 23 (7):493-505. https://doi.org/10.1177/23.7.1095650
  7. Lee DR, Kaproth MT and Parks JE. 2001. In vitro production of haploid germ cells from fresh or frozen-thawed testicular cells of neonatal bulls. J. Biol. Reprod. 65(3):873-8. https://doi.org/10.1095/biolreprod65.3.873
  8. Marh J, Tres LL, Yamazaki Y, Yanagimachi R and Kierszenbaum AL. 2003. Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes. J. Biol. Reprod. 69(1):169-76 https://doi.org/10.1095/biolreprod.102.015099
  9. McLachlan RI. 2000. The endocrine control of spermatogenesis. Baillieres Best Pract. Res. Clin. Endocrinol. Metab. 14(3): 345-362.
  10. Namekawa SH. 2014. Slide preparation method to preserve threedimensional chromatin architecture of testicular germ cells. J. Vis. Exp. 10;(83):e50819.
  11. Portugal J and Waring MJ. 1988. Assignment of DNA binding sites for 4',6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study. Biochimica. et Biophysica. Acta. 949 (2):158-168. https://doi.org/10.1016/0167-4781(88)90079-6
  12. Schmid M, Muller H, Stasch S and Engel W. 1983. Silver staining of nucleolus organizer regions during human spermatogenesis. Hum. Genet. 64(4):363-70. https://doi.org/10.1007/BF00292368
  13. Singer-Sam J, Robinson MO, Bellve AR, Simon MI and Riggs AD. 1990. Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis. Nucleic Acids Res. 18(5):1255-9. https://doi.org/10.1093/nar/18.5.1255
  14. Somfai T, Bodo S, Nagy S, Gocza E, Ivancsics J and Kovacs A. 2002. Simultaneous evaluation of viability and acrosome integrity of mouse spermatozoa using light microscopy. Biotech. Histochem. 77(3):117-20. https://doi.org/10.1080/bih.77.3.117.120
  15. Yang M, Zuo X, Li Y, Huang L and Xing X. 2014. A fast method for preparing the mouse germ cell slides. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 30(2):202-5.