Kito Seiji, Ohta Yuki
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.
Zygote. 2008 Aug;16(3):259-70. doi: 10.1017/S0967199408004619. Epub 2008 Jun 26.
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.
为阐明近交系BALB/c小鼠体外精子获能的条件，使用改良单纯形优化培养基（mKSOM）对渗透压、钙和乳酸进行了优化。改良人输卵管液（mHTF）作为一种支持获能的培养基用作对照。在第一组实验中，研究了在乳酸存在的情况下钙和渗透压的影响。虽然预孵育氯化钙浓度≥5 mM可显著提高授精后的受精率，但仍显著低于在mHTF中孵育的受精率。为获得与mHTF相当的受精水平，需要等渗渗透压（305 mOsmol）。渗透剂海藻糖可部分替代氯化钠。在第二组实验中，使用5 mM钙浓度和等渗渗透压研究了乳酸的影响。与在mHTF中孵育相比（分别为46%和28%；p<0.05），预孵育乳酸浓度≤2.5 mM可显著提高受精率（>75%），以及金霉素（CTC）染色中B（获能）型精子的百分比（≥40%）。在第三组实验中，研究了在无乳酸情况下渗透压和钙的影响。精子预孵育期间渗透压的增加以剂量依赖的方式显著提高了受精率和B型精子数量。海藻糖、蔗糖和氯化胆碱可替代氯化钠。预孵育期间氯化钙浓度的增加对受精率没有影响，但这种增加降低了B型精子的百分比。近交系BALB/c小鼠的体外获能对乳酸和渗透压敏感，但对钙的敏感性因乳酸的存在与否而异。
