Glycans Antigen LewisX,LewisY Expression On The Testis Epididymis And Relation To Sperm Maturation

Glycoconjugate lie mainly in the cell membrane surface, play a mediating role in physiological process and pathology process such as sperm-egg recognition, lymphocyte homing, bacteria and host cell adhesion, inflammatory reaction and embryo implantation etc. Lewis glycans are fucosylated lactose series glycans, including Le~X Le~Y Le~a Le~b sLe~X etc. According to their structure of carbohydrate chain, the difference between Le~X and Le~Y only lie in extreme fucose Fuc α 1-2. Lewis glycans are relate to individual development, organ formation, hematopoietic cell differentiation and tumour cell diversion. Our laboratory have studied Le~Y glycan function at female reproductive system and found the antigen consecutively highly expression on the endometrium of implantation stage and blastocyst, and it might act as signal molecule participating in recognition, adhesion of uterus-embryo and regulating implantation by ovarian hormone. Gene of the key enzyme (fucosyltransferase, futs), which catalyze Lewis glycans synthesis showed the same tendency with Lewis glycans carrying proteins. The results suggest that Le~X and Le~Y synthesis were regulated by key enzyme gene futs.Having highly differentiated spermatozoa when left testis were not mature, interact with epididymal duct microenvironment during passing through epididymal lumen, and they acquired the capacity for forward motility and the ability to fertilize. Sperm maturation process mainly completed at epididymis, and sperm maturation mainly happened at epididymal caupt. Epididymis secreted proteins which participated in acrosome reaction, sperm-egg recognition, fusion at sperm plasma membrane and regulated sperm maturation and capacitation. It has reported that human seminal plasma contains 0.3-0.4mg/ml free glycans, which is rich in fucose carried Le~X and Le~Yepitopes and abundance fucosyltransferase , assumed that free glycans may be involved in strengthening the ability for sperm forward motion, promoting sperm binding to eggs in oviduct and increasing immunosuppressive activity of sperm. c57 mouses were as research object, utilized Le~X and Le~Y antibodies as a probe to analyze the source and distribution of the fucosylated glycans in the testis epididymis by immunohistochemistry, RT-PCR and Western-blot. To investigate whether the abundance fucosylated glycans in human seminal plasma are relate to sperm development and maturation. Results as follows:1. By immunohistochemistry detection of the expression of Lewis glycans in the mouse testis, different epididymal regions (caupt, corpus, cauda). The results revealed that Le~X and Le~Y were negative in the testis of mouse, but dramatically turned to strong expression on the caput epididymis, and on the sperm surface in the epididymal lumen. But the antigens showed light positive, different epididymal regions displayed region specificity. RT-PCR result revealed the fut9 gene, which was the key enzyme to catalyze the synthesis of Le~X, strongly expressed on epididymal caput, yet lightly expressed on epididymal corpus and epididymal cauda, fut1 and fut4 gene which is the key enzyme to catalyze the synthesis of Le~Y, gradually degraded from epididymal caupt, epididyml corpus to epididymal cauda. Gene expression presented region specificity too (these gene expressed in the testis), and this region specificity was identical with Lewis glycan protein expression. Le~X and Le~Y mainly expressed on epididymal caupt. Sperm maturation mainly happened on the same part of the epididymal, it indicates that Le~X and Le~Y have great role in promoting sperm maturation. Western-blot results revealed Le~X protein (30-40 kDa) expressed on the testis, and its expression gradually degraded from epididymal caupt, epididymal corpus to epididymal cauda, Le~X protein (40~60kDa) expression gradually heighten from epididymal caupt, epididyml corpus to epididymal cauda. Le~Y protein 50 kDa expressed on the testis, and its expressed highly at epididymal caupt and epididymal corpus ,lowly at epididymal cauda, Le~Y protein (60-70 kDa) expression gradually degraded from epididymal caupt, epididymal corpus to epididymal cauda. Le~X and Le~Y carried proteins have different molecular weight in the testis and epididymal different regions, but the expression tendency was also different. It presumed that high expression fucosylated glycans on epididymal caupt may be related to sperm maturate.2. RT-PCR technique analysis the gene expression of fucosyltransferase {futs) on mouse testis, epididymis at adolescence (5 week, 7 week, 11 week ), adult (6 month),aging (400 day), fut1 fut4 expression tendency were essentially identical to the expression on epididymal caupt, epididymal corpus, epididymal cauda, ie. adolescence low, adult the highest, ageing descent. It showed fluctuating tendency to company with age changing, and this tendency was identical with hormone changing at growth and development stage. It manifests that the gene expression may be controlled by hormone. Fut9 expression had no obviously tendency on epididymal caupt ,epididymal cauda, but its expression on epididymis corpus presents the same tendency as that of fut1 fut4. ie. it presents at adolescence low, the highest peak appears at aldult , descent at aging, it showed that it possibly made an effect on epididymal corpus, and the alteration tendency may be controlled by hormone.3. Le~X and Le~Y expression on the different development stage (3 week, 5 week, 7 week,adult)of mouse testis and epididymis were detected by Immunohistochemistry .Expression profile of Le~X and Le~Y on epididymal caupt as followed: low at 3 week, gradually increased from 5 week ,7 week to adult; Le~X expressed on epididymal corpus: weak at 3 week, strong at 7 week, weak at adult; but Le~Y strongly expressed on epididymal corpus at 5 week. Le~X expression was conformed to Le~Y on epididymal cauda.; strong at 3 week, gradually weaken from 5 week, 7 week, 11 week ,to adult, mouse epididymal epithelial cells of 3 week were in differential phase, cells were not different between epididymal caupt, epididymal corpus and epididymal cauda, their cells function mainly secreted Le~X and Le~Y, so Le~X and Le~Y expression level were not different on theirs, mouse epididymal epithelial cells of 5 week and 7 week were proliferous phase, these cells further differentiate, so epididymal caupt and epididymal corpus expressed strongly; their cell function showed some degree differently; epididymal epithelial cells of adult had fulfiled differentiation,so cells of epididymal caupt, corpus and cauda had obviously different. Le~X and Le~Y strongly expressed on epididymal caupt, and weakly expressed on epididymal corpus and cauda. Le~X and Le~Y expression showed tissue specificity. This research further proved Le~X and Le~Y expression mainly appears on epididymal caupt, and they may promote sperm maturation.Sperm maturation mechanism is very complicated physiological process. The study had found that fucosylated glycan antigen Le~X and Le~Y may be relate to sperm maturation, whereas specific mechanism on sperm maturation process still remained unknown. So further investigations need to be done to elucidate it.