Walensky L D, Roskams A J, Lefkowitz R J, Snyder S H, Ronnett G V
Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Mol Med. 1995 Jan;1(2):130-41.
The 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.
We 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/beta ARK2) and beta-arrestin2.
We 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 and beta-arrestin2, implicated in olfactory desensitization, were detected in sperm cytosolic extracts using Western analysis. Immunohistochemistry colocalized putative odorant receptors, GRK3 and beta-arrestin2 to elongating spermatids in the testis and to the midpiece of mature sperm.
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 and beta-arrestin2 function in sperm to regulate putative chemoreceptor responses.
在哺乳动物生殖细胞中鉴定出编码假定嗅觉受体的转录本(1)，这引发了一种假说，即嗅觉受体可能在受精过程中精子趋化作用中发挥化学感应作用。我们试图在大鼠精子中鉴定并定位这些受体及其调节机制，以便更深入了解哺乳动物精子趋化作用和气味受体生理学。
我们使用针对已知气味受体家族成员间保守序列的简并引物进行逆转录聚合酶链反应（RT-PCR），以鉴定来自睾丸和圆形精子细胞的转录本。使用针对气味受体中两个保守肽序列产生的抗体以及针对G蛋白受体激酶3（GRK3/βARK2）和β-抑制蛋白2的融合蛋白抗体进行蛋白质印迹分析和免疫组织化学。
我们在成年大鼠的睾丸和圆形精子细胞中均检测到编码假定气味受体的转录本。PCR产物的限制性酶切证明存在多种基因产物。两种抗气味受体抗体通过蛋白质印迹在大鼠精子制剂中特异性识别一条64 kD的条带。使用蛋白质印迹分析在精子胞质提取物中检测到与嗅觉脱敏有关的GRK3和β-抑制蛋白2。免疫组织化学将假定气味受体、GRK3和β-抑制蛋白2共定位于睾丸中正在伸长的精子细胞以及成熟精子的中段。
气味受体在成熟精子呼吸中心的特异性定位与这些蛋白质在转导趋化信号中的作用一致。基于共定位，GRK3和β-抑制蛋白2在精子中发挥作用以调节假定化学感受器反应是合理的。
