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In vitro fertilization in inbred BALB/c mice II: effects of lactate, osmolarity and calcium on in vitro capacitation

Published online by Cambridge University Press:  01 August 2008

Seiji Kito*
Research Center for Radiation Protection, Department of Advanced Technologies for Radiation Protection Research, National Institute of Radiological Sciences, 4–9-1 Anagawa, Inage-ku, Chiba 263–8555, Japan. Department of Advanced Technologies for Radiation Protection Research, Research Center for Radiation Protection, National Institute of Radiological Sciences, Chiba, Japan.
Yuki Ohta
Science Services, Chiba, Japan.
All correspondence to: Seiji Kito, Research Center for Radiation Protection, Department of Advanced Technologies for Radiation Protection Research, National Institute of Radiological Sciences, 4–9-1 Anagawa, Inage-ku, Chiba 263–8555, Japan. Tel: +81 43 206 3059. Fax: +81 43 251 4138. e-mail:


To elucidate requirements for in vitro sperm capacitation in inbred BALB/c mice, osmolarity, calcium and lactate were optimized using modified simplex optimization medium (mKSOM). Modified human tubal fluid (mHTF), a capacitation-supporting medium, was used as a control. In the first series of experiments, the effects of calcium and osmolarity were studied in the presence of lactate. Although preincubation with ≥5 mM CaCl2 improved fertilization after insemination significantly, it was still significantly lower than incubation with mHTF. To obtain fertilization at the equivalent levels to that of mHTF, isotonic osmolarity (305 mOsmol) was required. Trehalose, an osmotic reagent, could substitute for NaCl partially. In the second series of experiments, the effects of lactate were examined using a concentration of 5 mM calcium and isotonic osmolarity. Preincubation with ≤2.5 mM lactate increased fertilization significantly (>75%), as well as the percentages of B (capacitated) pattern sperm (≥40%) in chlortetracycline (CTC) staining, as compared with incubation in mHTF (46% and 28%, respectively; p < 0.05). In the third series of experiments, the effects of osmolarity and calcium in the absence of lactate were examined. An increase in osmolarity during sperm preincubation increased both fertilization and B-pattern sperm significantly in a dose-dependent manner. Trehalose, sucrose and choline chloride could substitute for NaCl. An increase in CaCl2 concentration during preincubation had no effect on fertilization, but this increase reduced the percentages of B-pattern sperm. In vitro capacitation of inbred BALB/c mice is sensitive to lactate and osmolarity, but that sensitivity for calcium varies depending on the presence or absence of lactate.

Research Article
Copyright © Cambridge University Press 2008

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Bavister, B.D. & Yanagimachi, R. (1977). The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol. Reprod. 16, 228–37.CrossRefGoogle ScholarPubMed
Bielfeld, P., Jeyendran, R.S. & Zaneveld, L.J. (1993). Osmo-sensitivity of the human sperm acrosome reaction. Hum. Reprod. 8, 1235–9.CrossRefGoogle ScholarPubMed
Bleil, J.D. (1993). In vitro fertilization. Method Enzymol. 225, 253–63.CrossRefGoogle ScholarPubMed
Burruel, V.R., Yanagimachi, R. & Whitten, W.K. (1996). Normal mice develop from oocytes injected with spermatozoa with grossly misshapen heads. Biol. Reprod. 55, 709–14.CrossRefGoogle ScholarPubMed
Byers, S.L., Payson, S.J. & Taft, R.A. (2006). Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs). Theriogenology 65, 1716–26.CrossRefGoogle Scholar
Choi, Y-H., Seng, S. & Toyoda, Y. (2000). Comparison of capacitation and fertilizing ability of BALB/c and ICR mice epididymal spermatozoa: analysis by in vitro fertilization with cumulus-intact and zona-free mouse eggs. J. Mamm. Ova Res. 17, 914.CrossRefGoogle Scholar
Duan, C. & Goldberg, E. (2003). Inhibition of lactate dehydrogenase C4 (LDH-C4) blocks capacitation of mouse sperm in vitro. Cytogenet. Genome Res. 103, 352–9.CrossRefGoogle ScholarPubMed
Felix, R. (2005). Molecular physiology and disease of Ca2+-conducting channels in the plasma membrane of mammalian sperm. Reproduction 129, 251–62.CrossRefGoogle Scholar
Fraser, L.R. & Herod, J.E. (1990). Expression of capacitation-dependent changes in chlortetracycline fluorescence patterns in mouse spermatozoa requires a suitable glycol sable substrate. J. Reprod. Fertil. 88, 611–21.CrossRefGoogle Scholar
Fraser, L.R. (1993). In vitro capacitation and fertilization. Method Enzymol. 225, 239–53.CrossRefGoogle ScholarPubMed
Fraser, R.F. (1995). Ionic control of sperm function. Reprod. Fertil. Dev. 7, 905–25.CrossRefGoogle ScholarPubMed
Ho, Y., Wigglesworth, K., Eppig, J.J. & Schultz, R.M. (1995). Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Mol. Reprod. Dev. 41, 232–8.CrossRefGoogle ScholarPubMed
Hoppe, P.C. (1976). Glucose requirement for mouse sperm capacitation in vitro. Biol. Reprod. 15, 3945.CrossRefGoogle ScholarPubMed
Hoppe, P.C. & Pitts, S. (1973). Fertilization in vitro and development of mouse ova. Biol. Reprod. 8, 420–6.CrossRefGoogle ScholarPubMed
Jones, A.R. (1997). Metabolism of lactate by mature boar spermatozoa. Reprod. Fertil. Dev. 9, 227–32.CrossRefGoogle ScholarPubMed
Kito, S. & Ohta, Y. (2005). Medium effects on capacitation and sperm penetration through the zona pellucida in inbred BALB/c spermatozoa. Zygote 13, 145–53.CrossRefGoogle ScholarPubMed
Kito, S., Hayao, T., Noguchi-Kawasaki, Y., Ohta, Y., Hideki, U. & Tateno, S. (2004). Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice. Comp. Med. 54, 564–70.Google ScholarPubMed
Lawitts, J.A. & Biggers, J.D. (1993). Culture of preimplantation embryos. Methods Enzymol. 225, 153–64.CrossRefGoogle ScholarPubMed
Liu, D.Y., Clarke, G.N. & Baker, H.W. (2006). Tyrosine phosphorylation on capacitated human sperm tail detected by immunofluorescence correlates strongly with sperm–zona pellucida (ZP) binding but not with the ZP-induced acrosome reaction. Hum. Reprod. 21, 1002–8.CrossRefGoogle Scholar
Miller, D.J. & Hunter, A.G. (1986). Effect of osmolarity and glycosaminoglycans on motility, capacitation, acrosome reaction and in vitro fertilizability of bovine ejaculated sperm. J. Dairy Sci. 69, 2915–24.CrossRefGoogle ScholarPubMed
Miyamoto, H. & Chang, M.C. (1973a). Effect of osmolality on fertilization of mouse and golden hamster eggs in vitro.J. Reprod. Fertil. 33, 481–7.CrossRefGoogle ScholarPubMed
Miyamoto, H. & Chang, M.C. (1973b). The importance of serum albumin and metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J. Reprod. Fertil. 32, 193205.CrossRefGoogle ScholarPubMed
Mobraaten, L.E. (1986). Mouse embryo cryobanking. J. In vitro Fertil. Embryo Transf. 3, 2832.CrossRefGoogle ScholarPubMed
Nakanishi, T., Ikawa, M., Yamada, S., Toshimori, K. & Okabe, M. (2001). Alkalinization of acrosome measured by GFP as a pH indicator and its relation to sperm capacitation. Dev. Biol. 237, 222–31.CrossRefGoogle ScholarPubMed
Neill, J.M. & Olds-Clarke, P. (1988). Incubation of mouse sperm with lactate delays capacitation and hyperactivation and lowers fertilization levels in vitro. Gamete Res. 20, 459–73.CrossRefGoogle ScholarPubMed
Niwa, K. & Chang, M.C. (1975). Effect of pretreatment of rat spermatozoa with high concentrations of NaCl on sperm capacitation and fertilization of eggs in vitro. Biol. Reprod. 13, 187–9.CrossRefGoogle ScholarPubMed
Oh, S.H., Miyoshi, K. & Funahashi, H. (1998). Rat oocytes fertilized in modified rat 1-cell embryo culture medium containing a high sodium chloride concentration and bovine serum albumin maintain developmental ability to the blastocyst stage. Biol. Reprod. 59, 884–9.CrossRefGoogle ScholarPubMed
Oliphant, G. & Brackett, B.G. (1973). Capacitation of mouse spermatozoa in media with elevated ionic strength and reversible decapacitation with epididymal extracts. Fertil. Steril. 24, 948–55.CrossRefGoogle ScholarPubMed
Rossato, M., Di Virgilio, F. & Foresta, C. (1996). Involvement of osmo-sensitive calcium influx in human sperm activation. Mol. Hum. Reprod. 2, 903–9.CrossRefGoogle ScholarPubMed
Roudebush, W.E., & Duralia, D.R. (1996). Superovulation, fertilization and in vitro embryo development in BALB/cByJ, BALB/cJ, B6D2F1/J and CFW mouse strains. Lab. Anim. Sci. 46, 239–40.Google ScholarPubMed
Sakkas, D., Urner, F., Menezo, Y. & Leppens, G. (1993). Effects of glucose and fructose on fertilization, cleavage and viability of mouse embryos in vitro. Biol. Reprod. 49, 1288–92.CrossRefGoogle ScholarPubMed
Sekirina, G.G. & Neganova, I.E. (1995). The microenvironment created by nonblocking embryos in aggregates may rescue blocking embryos via cell–embryo adherent contacts. Zygote. 3, 313–24.CrossRefGoogle Scholar
Simpson, E.M., Linder, C.C., Sargent, E.E., Davisson, M.T., Mobraaten, L.E. & Sharp, J.J. (1997). Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat. Genet. 16, 1927.CrossRefGoogle ScholarPubMed
Summers, M.C., Bhatnagar, P.R., Lawitts, J.A. & Biggers, J.D. (1995). Fertilization in vitro of mouse ova from inbred and outbred strains: complete preimplantation embryo development in glucose-supplemented KSOM. Biol. Reprod. 53, 431–7.CrossRefGoogle ScholarPubMed
Szczygiel, M.A., Kusakabe, H., Yanagimachi, R. & Whittingham, D.G. (2002). Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains. Biol. Reprod. 67, 287–92.CrossRefGoogle ScholarPubMed
Sztein, J.M., Farley, J.S. & Mobraaten, L.E. (2000). In vitro fertilization with cryopreserved inbred mouse sperm. Biol. Reprod. 63, 1774–80.CrossRefGoogle ScholarPubMed
Thornton, C.E., Brown, S.D. & Glenister, P.H. (1999). Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens and mouse backcrosses. Mamm. Genome 10, 987–92.CrossRefGoogle ScholarPubMed
Toyoda, Y., Yokoyama, M. & Hoshi, T. (1971). Studies on fertilization of mouse eggs in vitro. I. In vitro fertilization of eggs by fresh epididymal sperm. Jpn. J. Anim. Reprod. 16, 147–51.CrossRefGoogle Scholar
Tsunoda, Y. & Chang, M.C. (1975). In vitro fertilization of rat and mouse eggs by ejaculated sperm and the effect of energy sources on in vitro fertilization of rat eggs. J. Exp. Zool. 193, 7986.CrossRefGoogle ScholarPubMed
Wu, H.T., Chou, C.K., Lin, C.S. & Huang, M.C. (2003). Effects of glucose concentration on in vitro fertilization in BALB/c mice. Reprod. Dom. Anim. 38, 470–4.CrossRefGoogle ScholarPubMed
Yanagimachi, R. (1982). Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in hamster. Gamete Res. 5, 324–44.CrossRefGoogle Scholar
Yanagimachi, R. (1994). Mammalian fertilization. In The Physiology of Reproduction, 2nd edn, (eds , E. Knobil & J.D. Neill), pp. 189317. New York: Raven Press.Google Scholar
Zar, J.H. (1996). Biostatistical Analysis, 3rd edn. pp. 277305. Englewood Cliffs, NJ: Prentice-Hall.Google Scholar
Zeng, Y., Oberdorf, J.A. & Florman, H.M. (1996). pH regulation in mouse sperm: identification of Na+-, Cl- and NaHCO3 -dependent and aryl aminobenzoate-dependent regulatory mechanisms and characterization of their roles in sperm capacitation. Dev. Biol. 173, 510–20.CrossRefGoogle Scholar