Sutovsky P, Plummer W, Baska K, Peterman K, Diehl J R, Sutovsky M
Division of Animal Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA.
J Androl. 2007 Jan-Feb;28(1):92-108. doi: 10.2164/jandrol.106.000216. Epub 2006 Aug 23.
High content of the platelet activating factor (PAF) and its plasma membrane receptor (PAFr) in semen is thought to benefit fertility in farm animals and humans. We used flow cytometric, biochemical, and immunocytochemical analysis to examine PAFr levels alone (Trial 1, n = 156 bulls) or in a dual assay with sperm defect marker ubiquitin (UBI; Trial 2, n = 88 bulls), in semen samples from 160 yearling bulls undergoing Breeding Soundness Evaluations (BSE). In both trials, we observed increased PAFr levels in semen samples with high content of white blood cells (WBC). Consequently, PAFr levels within such semen samples correlated negatively with several subjective parameters of BSE, including palpation, satisfaction of evaluation, and scrotal circumference. Due to a high WBC content, increased semen sample dilution had to be applied for microscopic evaluation. There was a negative correlation between semen PAFr and conventional sperm morphology, while the increased levels of PAFr correlated positively with sperm UBI content. Immunofluorescence microcopy revealed high expression of PAFr on the surface of leukocytes and morphologically normal spermatozoa, while reduced immunoreactivity was observed in defective spermatozoa immunoreactive to anti-UBI antibodies. A single PAFr band of appropriate mass was observed in Western blots of ejaculated spermatozoa, while testicular and epididymal spermatozoa also displayed several larger bands indicative of posttranslational processing or modification. Collectively, these data suggest that high levels of semen PAFr in young bulls are indicative of semen contamination with WBC. In the future, objective protein marker-based semen analyses in young bulls will likely require additional parameters distinguishing between marker expression in the spermatozoa and in the contaminating WBC. While identification of high sperm PAFr levels may support fertility, this assay alone is not reliable, due to the expression of PAFr in WBC that contaminate semen samples.
精液中高含量的血小板活化因子（PAF）及其质膜受体（PAFr）被认为对家畜和人类的生育能力有益。我们使用流式细胞术、生化分析和免疫细胞化学分析，单独检测PAFr水平（试验1，n = 156头公牛），或与精子缺陷标志物泛素（UBI）进行双重检测（试验2，n = 88头公牛），对160头接受繁殖健全性评估（BSE）的一岁公牛的精液样本进行检测。在两项试验中，我们均观察到白细胞（WBC）含量高的精液样本中PAFr水平升高。因此，此类精液样本中的PAFr水平与BSE的几个主观参数呈负相关，包括触诊、评估满意度和阴囊周长。由于WBC含量高，必须增加精液样本稀释度以进行显微镜评估。精液PAFr与传统精子形态呈负相关，而PAFr水平升高与精子UBI含量呈正相关。免疫荧光显微镜检查显示，PAFr在白细胞和形态正常的精子表面高表达，而在对抗UBI抗体呈免疫反应的缺陷精子中观察到免疫反应性降低。在射出精子的蛋白质免疫印迹中观察到一条质量合适的单一PAFr条带，而睾丸和附睾精子也显示出几条较大的条带，表明存在翻译后加工或修饰。总体而言，这些数据表明，年轻公牛精液中高水平的PAFr表明精液受到WBC污染。未来，基于客观蛋白质标志物的年轻公牛精液分析可能需要额外的参数来区分精子和污染WBC中标志物的表达。虽然识别高精子PAFr水平可能有助于生育，但由于污染精液样本的WBC中也表达PAFr，仅靠该检测并不可靠。
