Vjugina Ulyana, Zhu Xiaoling, Oh Eugene, Bracero Nabal J, Evans Janice P
Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Biol Reprod. 2009 Apr;80(4):833-41. doi: 10.1095/biolreprod.108.075275. Epub 2009 Jan 7.
The involvement of egg integrins in mammalian sperm-egg interactions has been controversial, with data from integrin inhibitor studies contrasting with evidence from knockouts showing that specific integrin subunits are not essential for fertility. An alpha(4)/alpha(9) (ITGA4/ITGA9) integrin subfamily member has been implicated in fertilization but not extensively examined, so we tested the following three hypotheses: 1) an ITGA4/ITGA9 integrin participates in sperm-egg interactions, 2) short-term acute knockdown by RNA interference of integrin subunits would result in a fertilization phenotype differing from that of chronic depletion via knockout, and 3) detection of a fertilization phenotype is sensitive to in vitro fertilization (IVF) assay conditions. We show that mouse and human eggs express the alpha(9) integrin subunit (ITGA9). RNA interference-mediated knockdown resulted in reduced levels of Itga9 mRNA and surface protein in mouse eggs. RNA interference attempts to knockdown ITGA9's likely beta partner, beta(1) (ITGB1), resulted in reduced Itgb1 mRNA but no reduction in ITGB1 surface protein. Therefore, studies using a function-blocking anti-ITGB1 antibody tested the hypothesis that ITGB1 participates in gamete interactions. Analyses of sperm-egg interactions with Itga9-knockdown eggs and anti-ITGB1 antibody-treated eggs in IVF assays using specific sperm:egg ratios revealed the following: 1) a reduction, but not complete loss, of sperm-egg binding and fusion was observed and 2) the reduction of sperm-egg binding and fusion was not detected in inseminations with high sperm:egg ratios. These data demonstrate that ITGA9 and ITGB1 participate in sperm-egg interactions but clearly are not the only molecules involved. This also shows that careful design of IVF parameters allows detection of deficiencies in gamete interactions.
卵整合素在哺乳动物精卵相互作用中的作用一直存在争议，整合素抑制剂研究的数据与基因敲除的证据相互矛盾，基因敲除表明特定的整合素亚基对生育能力并非必不可少。一种α(4)/α(9)（ITGA4/ITGA9）整合素亚家族成员被认为与受精有关，但尚未得到广泛研究，因此我们测试了以下三个假设：1）ITGA4/ITGA9整合素参与精卵相互作用；2）通过RNA干扰对整合素亚基进行短期急性敲低会导致与基因敲除导致的慢性耗竭不同的受精表型；3）受精表型的检测对体外受精（IVF）试验条件敏感。我们发现小鼠和人类卵子表达α(9)整合素亚基（ITGA9）。RNA干扰介导的敲低导致小鼠卵子中Itga9 mRNA和表面蛋白水平降低。RNA干扰试图敲低ITGA9可能的β伴侣β(1)（ITGB1），导致Itgb1 mRNA减少，但ITGB1表面蛋白没有减少。因此，使用功能阻断性抗ITGB1抗体的研究检验了ITGB1参与配子相互作用的假设。在IVF试验中，使用特定的精子:卵子比例分析与Itga9敲低卵子和抗ITGB1抗体处理卵子的精卵相互作用，结果如下：1）观察到精卵结合和融合减少，但未完全丧失；2）在高精子:卵子比例的授精中未检测到精卵结合和融合的减少。这些数据表明ITGA9和ITGB1参与精卵相互作用，但显然不是唯一涉及的分子。这也表明精心设计IVF参数可以检测配子相互作用中的缺陷。
