皮質粒是位在於許多脊椎和無脊椎生物的未受精卵母細胞皮層部的膜結合分泌胞器。哺乳動物之皮質粒經由高基氏體在卵母細胞生長時衍生而成。在卵母細胞受精之後，皮質粒會經由胞吐作用，將其內含物釋出。此分泌過程中需鈣離子參與，稱之為皮質反應。胞吐作用之後，釋出的皮質粒蛋白是經由修飾卵母細胞的外基質阻斷多重受精，例如哺乳動物的透明帶和棘皮動物的卵黃周隙以及兩棲類的受精層。在先前的研究報告中甚至於顯示，皮質粒之內含物在受精之後的胚胎發生初期可能扮演重要角色。數種蛋白酶已經被推論或顯示存在於哺乳動物之皮質粒中。組織型胞漿素原活化劑和鈣網蛋白也已經被推論是皮質粒蛋白，因為它們被偵測存在於受精和/或活化之卵母細胞分泌物中。有一些蛋白質如N-乙醯葡萄糖胺酶、卵過氧化酶、及p32 被利用免疫細胞化學法定位存在卵母細胞皮質粒；但是，這些蛋白質還沒有被分離出來進行鑑定或充分的性質分析。亦有數個研究使用多糖體結合染劑或探針，如過碘酸希夫瓦(PAS)反應劑、釕紅（ruthenium red）、colloidal iron，顯示哺乳動物的皮質粒富含碳水化合物。但是，現今我們對於哺乳動物卵母細胞皮質粒的內含物所知還是很有限。許多報告顯示，適時的釋出皮質粒為成功受精與胚胎發育進行所必須的，有趣的是，經超低溫冷凍後老鼠卵母細胞的受精率低而其原因是超低溫冷凍誘導皮質粒提早釋放其內含物。先前研究也顯示，皮質粒提早釋放以及在受精之前建立多重受精的障礙，很可能是導致許多女性在接受人工受孕手術中失敗的原因。然而，鑑定皮質顆粒內含物有助於了解此胞吐作用的分子機轉並提供一個較理想可用於臨床檢定卵母細胞品質的準則。除此之外，老鼠皮質粒成分的鑑定可以協助我們確定人類皮質粒的生化成分，因為它們可能有相同的組成物質。最後，皮質粒成分適當的功能對於一個成功的受精作用是絕對需要的，因為它們是擔負建立多重受精的障礙。沒有此種障礙受精卵會成為多倍體最後導致死亡。如果我們能鑑定出皮質粒内負責此功能之物質，也將給予我們一個發展新非荷爾蒙避孕藥物之可能性。本計畫擬鑑定小鼠皮質粒之生化組成，而這一些資訊可以讓我們更進一步的了解這些物質在卵母細胞受精時以及在著床前胚胎發育所扮演之角色。最後，申請者希望藉執行本計畫，提供醫學界開發新非荷爾蒙之避孕藥物所需相關訊息。
Cortical granules (CG) are membrane bound secretory organelles located in the cortex of unfertilized oocytes in many invertebrates and vertebrates. Following fertilization, cortical granules undergo exocytosis to release their contents and the released cortical granule proteins are responsible for blocking polyspermy by modifying of the oocytes’ extracellular matrices, such as the zona pellucida in mammals, the vitelline envelope in echinoderms, and the fertilization layers in amphibians. Several proteinases have been inferred or demonstrated to be present in mammalian cortical granules. Some proteins such as N-acetylglucosaminidase, ovoperoxidase, and p32 have been immunocytochemically localized to oocyte cortical granules; however, these proteins have not yet been isolated for identification or fully characterized. Several studies using polysaccharides-binding dyes or probes, such as periodic acid Schiff’s reagents, ruthenium red, colloidal iron, reveal that mammalian cortical granules are rich in carbohydrates. However, relatively little is known about the contents of mammalian oocyte cortical granules. Timely release of these granules is essential for successful fertilization and embryo development to occur. Interestingly, low fertilization rate were observed following cryopreservation of mouse oocytes and this low rate was attributed to cryopreservant induced premature cortical granule discharge. Previous study also showed that premature release of cortical granules and early establishment of block to polyspermy prior to insemination may be the cause in some cases of unsuccessful fertilization in routine in vitro fertilization. Nevertheless, identification of granule contents will help us to better understand the molecular mechanism of this exocytotic event and provide as a better criterion to evaluate oocyte quality in the clinical setting. Furthermore, identification of mouse cortical granule components may help us in defining the biochemical composition of human cortical granules since homologous constituents may be present. Therefore, it is tempting to envision the possibility that newly identified cortical granule component(s) may be new targets for developing non-hormone based contraceptive medication. The goal of this proposed study is to identify mammalian cortical granule components and investigate their physiological role during as well as after fertilization.
