Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium
Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium
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DOI：
10.1095/biolreprod59.6.1328
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发表时间：
1998-12-01
影响因子：
3.6
通讯作者：
Toyoda, Y
中科院分区：
文献类型：
--
作者：
Choi, YH;Toyoda, Y
环糊精，刺激胆固醇流出细胞，检查其诱导小鼠精子获能的能力。一种化学成分确定的，无蛋白质的培养基用于体外受精的卵丘无小鼠卵。在添加1 mg/ml聚乙烯醇而不是BSA的改良Krebs-Ringer碳酸氢盐培养基（TYH）中未发生受精。然而，当精子与甲基-β-环糊精（MBCD）预孵育时，观察到受精;受精率从0.25到0.75 mM MBCD呈剂量依赖性增加，当0.75 mM MBCD加入预孵育和受精培养基时，受精率下降，而仅加入预孵育培养基（21%对53%）;与此形成鲜明对比的是，与仅预孵育培养基相比，当两种培养基中均存在4 mg/ml BSA时，受精增加（66%对25%）。在0.75 mM浓度下，2-羟基-β-环糊精在体外使精子获能的能力低于MBCD（14%对41%）。当在KSOM中培养时，用MBCD（0.75 mM）处理的精子受精的卵发育成囊胚（45%，80个中的36个）。当将160个受精卵转移到ICR受体时，出生了62个活的后代。小鼠精子在含0.75 mM MBCD的TYH培养基中孵育90 min后，精子的胆固醇含量显著升高，（p < 0.01）低于对照组（2.27 ± 0.09 vs. 4.13 ± 0.09 nmol未酯化胆固醇/10（7）个精子;平均值+/- SEM，n = 5），获能的比例（B模式）MBCD处理90 min后，金霉素荧光测定的精子数高于对照组（45%对15%; p < 0.01），顶体反应（AR模式）精子的比例在MBCD处理组和对照组之间没有差异。因此，MBCD增加获能，而不是顶体反应的精子。
Cyclodextrin, which stimulates cholesterol efflux from cells, was examined for its ability to induce capacitation of mouse spermatozoa. A chemically defined, protein-free medium was used for in vitro fertilization of cumulus-free mouse eggs. Fertilization did not occur in modified Krebs-Ringer bicarbonate medium (TYH) supplemented with 1 mg/ml polyvinylalcohol instead of BSA. However, fertilization was observed when spermatozoa were preincubated with methyl-beta-cyclodextrin (MBCD); fertilization rates increased dose-dependently from 0.25 to 0.75 mM MBCD, The fertilization rate decreased when 0.75 mM MBCD was added to both preincubation and fertilization media versus only the preincubation medium (21% vs. 53%); in sharp contrast, fertilization increased when 4 mg/ml BSA was present in both of the media versus the preincubation medium only (66% vs. 25%). At 0.75 mM, 2-hydroxy-beta-cyclodextrin had a lower ability to capacitate spermatozoa in vitro than MBCD (14% vs. 41%), Eggs fertilized by spermatozoa treated with MBCD (0.75 mM) developed to blastocysts (45%, 36 of 80) when cultured in KSOM, When 160 fertilized eggs were transferred to ICR recipients, 62 live offspring were born. After incubation of mouse spermatozoa for 90 min in 0.75 mM MBCD in TYH medium, the cholesterol content of the spermatozoa was significantly (p < 0.01) lower than that of the control (2.27 +/- 0.09 vs. 4.13 +/- 0.09 nmol unesterified cholesterol/10(7) sperm; mean +/- SEM, n = 5), The proportion of capacitated (B pattern) spermatozoa determined by chlortetracycline fluorescence was higher with MBCD treatment for 90 min than for the control (45% vs. 15%; p < 0.01), The proportion of acrosome-reacted (AR pattern) spermatozoa was not different between MBCD treatment and the control. Therefore, MBCD increased capacitation rather than the acrosome reaction of spermatozoa.