Skip to main content Accessibility help

FACS selection of valuable mutant mouse round spermatids and strain rescue via round spermatid injection

  • Lian Zhu (a1), Wei Zhou (a2), Peng-Cheng Kong (a1), Mei-Shan Wang (a1) (a3), Yan Zhu (a4), Li-Xin Feng (a2), Xue-Jin Chen (a5) (a1) and Man-Xi Jiang (a5) (a1)...


Round spermatid injection (ROSI) into mammalian oocytes can result in the development of viable embryos and offspring. One current limitation to this technique is the identification of suitable round spermatids. In the current paper, round spermatids were selected from testicular cells with phase contrast microscopy (PCM) and fluorescence-activated cell sorting (FACS), and ROSI was performed in two strains of mice. The rates of fertilization, embryonic development and offspring achieved were the same in all strains. Significantly, round spermatids selected by PCM and FACS were effectively used to rescue the infertile Pten-null mouse. The current results indicate that FACS selection of round spermatids can not only provide high-purity and viable round spermatids for use in ROSI, but also has no harmful effects on the developmental capacity of subsequently fertilized embryos. It was concluded that round spermatids selected by FACS are useful for mouse strain rederivation and rescue of infertile males; ROSI should be considered as a powerful addition to the armamentarium of assisted reproduction techniques applicable in the mouse.


Corresponding author

All correspondence to: Man-Xi Jiang or Xue-Jin Chen. Department of Laboratory Animal Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. Tel: +86 21 63846590, ext. 776539. e-mail: or


Hide All
Barchi, M., Geremia, R., Magliozzi, R. & Bianchi, E. (2009). Isolation and analyses of enriched populations of male mouse germ cells by sedimentation velocity: the centrifugal elutriation. Methods Mol. Biol. 558, 299321.
Bos-Mikich, A., Swann, K. & Whittingham, D.G. (1995). Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes. Mol. Reprod. Dev. 41, 8490.
Chang, Y.F., Lee-Chang, J.S., Panneerdoss, S., MacLean, J.A. 2nd & Rao, M.K. (2011). Isolation of Sertoli, Leydig, and spermatogenic cells from the mouse testis. Biotechniques 51, 341–4.
Getun, I.V., Torres, B. & Bois, P.R. (2011). Flow cytometry purification of mouse meiotic cells. J. Vis. Exp. 50, e2602. doi:10.3791 /2602
Goodell, M.A. (2005). Stem cell identification and sorting using the Hoechst 33342 side population (SP.) In Current Protocols in Cytometry. New York: John Wiley & Sons, Inc. Chapter 9, Unit 9.18.
Hikichi, T., Kishigami, S., Thuan, N.V., Ohta, H., Mizutani, E., Wakayama, S. & Wakayama, T. (2005). Round spermatids stained with MitoTracker can be used to produce offspring more simply. Zygote 13, 5561.
Hirabayashi, M., Kato, M., Aoto, T., Ueda, M. & Hochi, S. (2002). Rescue of infertile transgenic rat lines by intracytoplasmic injection of cryopreserved round spermatids. Mol. Reprod. Dev. 62, 295–9.
Kimura, Y. & Yanagimachi, R. (1995a). Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring. Development 121, 2397–405.
Kimura, Y. & Yanagimachi, R. (1995b). Development of normal mice from oocytes injected with secondary spermatocyte nuclei. Biol. Reprod. 53, 855–62.
La Salle, S., Sun, F. & Handel, M.A. (2009). Isolation and short-term culture of mouse spermatocytes for analysis of meiosis. Methods Mol. Biol. 558, 279–97.
Lassalle, B., Ziyyat, A., Testart, J., Finaz, C. & Lefevre, A. (1999). Flow cytometric method to isolate round spermatids from mouse testis. Hum. Reprod. 14, 388–94.
Meng, X., Akutsu, H., Schoene, K., Reifsteck, C., Fox, E.P., Olson, S., Sariola, H., Yanagimachi, R. & Baetscher, M. (2002). Transgene insertion induced dominant male sterility and rescue of male fertility using round spermatid injection. Biol. Reprod. 66, 726–34.
Morena, A.R., Boitani, C., Pesce, M., De Felici, M. & Stefanini, M. (1996). Isolation of highly purified type A spermatogonia from prepubertal rat testis. J. Androl. 17, 708–17.
Ogura, A., Matsuda, J. & Yanagimachi, R. (1994). Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc. Natl. Acad. Sci. USA 91, 7460–2.
Ogura, A., Ogonuki, N., Takano, K. & Inoue, K. (2001). Microinsemination, nuclear transfer, and cytoplasmic transfer: the application of new reproductive engineering techniques to mouse genetics. Mamm. Genome 12, 803–12.
Palermo, G., Joris, H., Devroey, P. & Van Steirteghem, A.C. (1992). Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, 17–8.
Parrilla, I., Vazquez, J.M., Cuello, C., Gil, M.A., Roca, J., Di Berardino, D. & Martinez, E.A. (2004). Hoechst 33342 stain and u.v. laser exposure do not induce genotoxic effects in flow-sorted boar spermatozoa. Reproduction 128, 615–21.
Pelengaris, S.A. & Moore, H.D. (1995). Separation of round spermatids from the rat using an immunoselection panning technique. Mol. Reprod. Dev. 41, 348–54.
Romero, Y., Meikar, O., Papaioannou, M.D., Conne, B., Grey, C., Weier, M., Pralong, F., De Massy, B., Kaessmann, H., Vassalli, J.D., Kotaja, N. & Nef, S. (2011). Dicer1 depletion in male germ cells leads to infertility due to cumulative meiotic and spermiogenic defects. PLoS One 6, e25241.
Sasagawa, I. & Yanagimachi, R. (1997). Spermatids from mice after cryptorchid and reversal operations can initiate normal embryo development. J. Androl. 18, 203–9.
Sasagawa, I., Kuretake, S., Eppig, J.J. & Yanagimachi, R. (1998). Mouse primary spermatocytes can complete two meiotic divisions within the oocyte cytoplasm. Biol. Reprod. 58, 248–54.
Sharan, S.K., Pyle, A., Coppola, V., Babus, J., Swaminathan, S., Benedict, J., Swing, D., Martin, B.K., Tessarollo, L., Evans, J.P., Flaws, J.A. & Handel, M.A. (2004). BRCA2 deficiency in mice leads to meiotic impairment and infertility. Development 131, 131–42.
Sofikitis, N.V., Miyagawa, I., Agapitos, E., Pasyianos, P., Toda, T., Hellstrom, W.J. & Kawamura, H. (1994). Reproductive capacity of the nucleus of the male gamete after completion of meiosis. J. Assist. Reprod. Genet. 11, 335–41.
Sutovsky, P., Ramalho-Santos, J., Moreno, R.D., Oko, R., Hewitson, L. & Schatten, G. (1999). On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker™ and high resolution differential interference contrast microscopy. Hum. Reprod. 14, 2301–12.
Tesarik, J., Mendoza, C. & Testart, J. (1995). Viable embryos from injection of round spermatids into oocytes. N. Engl. J. Med. 333, 525.
Van Steirteghem, A.C., Nagy, Z., Joris, H., Liu, J., Staessen, C., Smitz, J., Wisanto, A. & Devroey, P. (1993). High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod. 8, 1061–6.
Wykes, S.M. & Krawetz, S.A. (2003). Separation of spermatogenic cells from adult transgenic mouse testes using unit-gravity sedimentation. Mol. Biotechnol. 25, 131–8.
Yanagimachi, R. (2001). Gamete manipulation for development: new methods for conception. Reprod. Fertil. Dev. 13, 314.



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed