| 摘要: | 精子游動力是目前被認為是受孕能力的重要指標。臨床上如何選擇高游動力或改善精子游動力，藉以提高受孕能力是極為重要的課題。骨橋蛋白(osteopontin , OPN)，在公牛被認為受孕的指標蛋白。OPN廣泛存在於壺腹部、副睾、儲精囊及輸卵管中, 此外OPN是一種磷酸蛋白質，主要調節許多細胞，包括巨噬細胞(macrophages)、蝕骨細胞(osteoclasts)及癌症細胞的發生和轉移有關。本研究主要為探討OPN是否具有調節精子細胞的游動能力及受孕力。在Western blotting的分析結果中，發現在25kD以及50kD處有OPN的表現，並且其表現量與精子的游動力有相關性，游動力佳的精子25kD OPN表現量較多(54.4%?b11.6% vs. 81.8%?b6.8%，p＜0.05)。並且依精子的游動力細分樣本為Group 1 為0~30%，Group 2為30%~60%，Group 3為60%~100%，並以電腦輔助精液分析儀(computer-assisted semen analysis)測定各項游動參數。當我們以添加0.1µM OPN與精子作用30分鐘前後游動力變化，發現在Group 1的精子中，游動力有增加的情形(15.6%?b2.1% vs. 39.6%?b4.6%，p＜0.01)，Group 2亦有相同的增加情形(51.7%?b1.5% vs. 69.5%?b6.0%，p＜0.05)。在CASA的分析結果中，共計8個精子的運動參數中，有4個參數，包括VCL、LIN、STR與BCF，在游動力較差的Group 1有明顯升高的情形，其中VCL提高為原始之175%?b18.0%倍、LIN為226.5%?b25.0%倍、STR為241.1%?b30.0%倍，以及BCF也成為原先的154.4%?b13.0% (p＜0.05)，不過在活動力較好的兩組(Group 2及Group 3)，OPN並沒有顯著的提升。進一步分析OPN對於精子受孕能力的影響，包括精子移動至卵子能力的測定(in vitro migration test)、精子穿透力試驗(sperm penetration assay)、頂體反應試驗(acrosomal reaction)，以及老鼠體外受精試驗等，發現OPN可以提高精子移動到卵子的能力(1.5%?b0.2% vs. 6.3%?b1.5%，p＜0.05)、穿透卵子的能力(1.4?b0.1個 vs. 2.3%?b0.1個，p＜0.05)以及增加體外受精率(62.0%?b3.6% vs. 78.0%?b2.1%，p＜0.05)。不過在精子頂體反應試驗中，結果並沒有發現有增加的情形。最後我們利用螢光染色分析方法，觀察到OPN表現於精子的頭部(head)與中節(mid-piece)，並且在加入OPN後會造成精子的細胞骨架細胞(cytoskeleton)的改變，在actin及tubulin有增加的情形，而spectrin螢光表現強度並無明顯增加。本研究證實OPN具有調控精子游動力及受孕力的能力。
The motility of the spermatozoa is reported as a critical indicator of fertilizing capacity。In clinical, to enhance the fertilization rate by selecting sperms with high motility or improving the sperm motility is an important issue. Osteopontin (OPN) was proposed as a fertility indexes protein of bulls. OPN is a secreted phosphoprotein, regulates biological functions of diverse cell types, such as macrophages and osteoclasts. It is also involved in onset and metastasis of cancer cells. OPN is expressed in the ampulla, seminal vesicles, epididymis, as well as oviduct. Aim of this study was to address the role of OPN in regulation of sperm motility and fertility potential. A significantly increase of the OPN expression by Western Blotting. A significant increased expression of 25 kD OPN was found in the sperm with good motility (54.4%?b11.6% vs. 81.8%?b6.8%, p<0.05). Then we divided all the sample into three groups, motility from 0% to 30% (Group 1), 30% to 60% (Group 2), and up to 60% (Group 3). Follow the supplementation of 0.1µM OPN for 30 minutes, the alteration of sperm motility and moving characteristics were examined by computer-assisted semen analysis (CASA).The sperm motility was significantly increased in Group 1(51.7?s?b???|???s?nvs. 69.5%?b6.0%, p<0.05). OPN caused a significant increase in the percentage of VCL (175.0%?b18.0%, p<0.05), LIN (226.5%?b?????s?z?np<0.05?w, STR (241.1%?b30.0%, p<0.05), and BCF (154.4%?b13.0%, p<0.05) in Group1 but not seen in Group 2 and 3. The effects of OPN on fertilizing capacity were evaluated by in vitro migration test, sperm penetration assay, acrosomal reaction, and mouse in vitro fertilization test. The result showed that the capability of sperm migration (1.5%?b0.2%vs. 6.3%?b1.5%, p<0.05), sperm penetration number (1.4?s?b?~?|???nspermatozoa vs. 2.3%?b0.1 spermatozoa, p<0.05), and in vitro fertilization rate (62.0%?b3.6% vs. 78.0%?b2.1%, p<0.05) was enhanced by OPN. However, there was no difference in terms of acrosomal reaction followed by OPN treatment. In addition, the distribution of actin, tubulin and spectrin was determined by immunofluorescent analysis. Sperm actin and tubulin increased display, but not in spectrin. In conclusion, OPN plays an important role in the regulation of sperm motility and fertilizing capacity. |
