In vitro fertilization in inbred BALB/c mice II: effects of lactate, osmolarity and calcium on in vitro capacitation.
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 >or=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 <or=2.5 mM lactate increased fertilization significantly (>75%), as well as the percentages of B (capacitated) pattern sperm (>or=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.
Kito
Seiji
S
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. sk126@nirs.go.jp
Ohta
Yuki
Y
D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology*; D005260:Female; D005307:Fertilization in Vitro / Q000187:drug effects*; D007552:Isotonic Solutions; D019344:Lactic Acid / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009994:Osmolar Concentration; D013075:Sperm Capacitation / Q000187:drug effects* / Q000502:physiology; D013084:Sperm-Ovum Interactions / Q000187:drug effects / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*; D015044:Zona Pellucida / Q000502:physiology
D007552:Isotonic Solutions; D019344:Lactic Acid; D002122:Calcium Chloride