Odorant Receptors and Desensitization Proteins Colocalize in Mammalian Sperm
Odorant Receptors and Desensitization Proteins Colocalize in Mammalian Sperm
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DOI：
10.1007/bf03401561
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发表时间：
1995
影响因子：
5.7
通讯作者：
L. Walensky;A. Roskams;R. J. Lefkowitz;Solomon H. Snyder;G. Ronnett
中科院分区：
文献类型：
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作者：
L. Walensky;A. Roskams;R. J. Lefkowitz;Solomon H. Snyder;G. Ronnett
背景哺乳动物生殖细胞中编码嗅觉受体的转录本的鉴定（1）产生了嗅觉受体可能在受精过程中精子趋化性中起化学感受作用的假说。我们已经试图确定和本地化这些受体和它们的调节机制，在大鼠精子，以获得进一步深入了解哺乳动物精子趋化性和气味受体physiology.Materials和MethodsWe进行逆转录-聚合酶链反应（RT-PCR），使用简并引物针对保守的已知气味受体家族的成员，以确定从睾丸和圆形精子细胞的转录本。免疫组化和Western分析显示，在成年大鼠睾丸和圆形精子细胞中均检测到编码气味受体的转录本。PCR产物的限制性内切酶分析表明存在多个基因产物。两个抗气味受体抗体特异性识别64 kD的带在大鼠精子制备的蛋白质印迹。蛋白质GRK 3和β-arrestin 2，涉及嗅觉脱敏，检测精子胞质提取物中使用Western分析。免疫组织化学共定位推定的气味受体，GRK 3和β-arrestin 2在睾丸中的精子细胞和成熟精子的中段。结论气味受体的特异性定位的成熟精子的呼吸中枢是一致的，这些蛋白质在转导趋化信号的作用。基于这种共定位，GRK 3和β-arrestin 2在精子中起着调节假定的化学感受器反应的作用是合理的
BackgroundThe identification of transcripts encoding putative olfactory receptors in mammalian germ cells (1) has generated the hypothesis that olfactory receptors may serve a chemosensory role in sperm Chemotaxis during fertilization. We have sought to identify and localize these receptors and their regulatory machinery in rat sperm in order to gain further insight into mammalian sperm Chemotaxis and odorant receptor physiology.Materials and MethodsWe conducted reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers directed against sequences conserved across members of the known odorant receptor family to identify transcripts from testis and round spermatids. Western analysis and immunohistochemistry were performed using antibodies raised against two peptide sequences conserved among odorant receptors and using fusion protein antibodies to G-protein receptor kinase 3 (GRK3/βARK2) andβ-arrestin2.ResultsWe detected transcripts encoding putative odorant receptors in both testis and round spermatids of the adult rat. Restriction digests of the PCR products demonstrated the existence of multiple gene products. Two anti-odorant receptor antibodies specifically recognized a 64 kD band in rat sperm preparations by Western blot. The proteins GRK3 andβ-arrestin2, implicated in olfactory desensitization, were detected in sperm cytosolic extracts using Western analysis. Immunohistochemistry colocalized putative odorant receptors, GRK3 andβ-arrestin2 to elongating spermatids in the testis and to the midpiece of mature sperm.Conclusions The specific localization of odorant receptors to the respiratory center of mature sperm is consistent with a role for these proteins in transducing chemotactic signals. Based on the colocalization, it is plausible that GRK3 andβ-arrestin2 function in sperm to regulate putative chemoreceptor responses