絲胺酸蛋白酶抑制蛋白E2在精子獲能作用與卵子成熟過程中所扮演的角色

The roles of the SERPINE2 protein in sperm capacitation and oocyte maturation

呂仲浩(Chung-Hao Lu)

指導教授 ： 林劭品

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.00534

摘要

絲胺酸蛋白酶抑制蛋白E2(SERPINE2)為一種可以抑制絲胺酸蛋白酶活性的蛋白質，其藉由抑制絲胺酸蛋白酶活性來參與許多生理功能。目前已知SERPINE2可以調控plasminogen activator及thrombin的酵素活性。在第一部份的研究中，首先我們從小鼠儲精囊分泌液中純化出具有可以抑制plasminogen activator活性的SERPINE2蛋白質。接著發現SERPINE2會結合在射出的小鼠精子以及已經游入輸卵管的精子，但是無法在已發生獲能反應的精子身上發現SERPINE2蛋白質的存在。此外，在體外試驗中發現SERPINE2也會抑制牛血清白蛋白(BSA)所引起的精子獲能反應，使得精子無法與卵子結合進而影響與卵子的受精作用。精子身上的膽固醇移除(cholesterol efflux)為發生獲能反應的重要步驟，而SERPINE2的存在會去抑制精子身上膽固醇的移除，因此SERPINE2可能扮演著精子去獲能因子(decapacitation factor)的角色。
Plasminogen activator已被證實存在於排卵過程中的濾泡壁上，但其在卵子成熟過程中的相關功能仍然未知。在第二部份的研究中我們證實了PLAU (urokinase plasminogen activator)及其抑制者SERPINE2參與小鼠卵丘細胞擴張(cumulus expansion)及卵子成熟。當高量的SERPINE2結合上卵丘細胞之細胞間質(ECM)時會降低PLAU的活性，導致卵丘細胞無法擴張，進而影響卵子成熟。因此在病人進行人工生殖技術過程中，額外加入PLAU蛋白質於體外培養系統中，或許可以幫助不成熟卵子進行最後階段之熟成而提高受精率。
綜合這些實驗結果，我們認為SERPINE2蛋白質可以在精子到達輸卵管之前保護精子並避免其過早發生獲能反應。此外，我們推測部分不孕症病人之卵丘細胞無法擴張及卵子無法成熟可能與SERPINE2蛋白質不正常的高量表現有關。

關鍵字

絲胺酸蛋白酶抑制蛋白E2 ； 絲胺酸蛋白酶 ； 精子 ； 獲能作用 ； 受精作用 ； 卵丘細胞擴張 ； 卵子成熟

並列摘要

SERPINE2, one of the potent serine protease inhibitors that modulate the activity of plasminogen activator and thrombin, is implicated in many biological processes. In the first study, we purified SERPINE2 from mouse seminal vesicle secretion based on its potent inhibitory activity against the urokinase-type plasminogen activator. A prominent amount of SERPINE2 was detected on ejaculated and oviductal spermatozoa, predominantly on uncapacitated sperm, suggesting the need to remove SERPINE2 before initiation of the capacitation process. Moreover, SERPINE2 could inhibit in vitro bovine serum albumin-induced sperm capacitation and prevent sperm binding to the egg, thus blocking fertilization. It acts through preventing cholesterol efflux, one of the initiation events of capacitation, from the sperm. These findings suggest that SERPINE2 protein may play a role as a sperm decapacitation factor.
Plasminogen activators play a crucial role in follicle wall rupture during ovulation; however, their function in oocyte maturation during pre-ovulation remained unclear. Our second study provides the first evidence that PLAU (urokinase plasminogen activator) and its inhibitor SERPINE2 are involved in murine cumulus expansion and oocyte maturation. High SERPINE2 levels bound to the extracellular matrix of cumulus cells could reduce PLAU activity, and ultimately suppressing cumulus expansion and oocyte maturation. PLAU supplementation to culture medium may assist the final maturation of the immature human oocytes collected during assisted reproductive technology procedures, thus providing a potential therapeutic strategy.
Based on these results, SERPINE2 protein possibly influencese on sperm to prevent precocious capacitation and the acrosome reaction before sperm reach the oviduct. In addition, we suppose that aberrantly high SERPINE2 protein levels in cumulus cells may be one of the etiologies for patients with defects in cumulus expansion and subsequent oocyte maturation.

並列關鍵字

SERPINE2 ； serine protease ； sperm ； capacitation ； fertilization ； plasminogen activator ； cumulus expansion ； oocyte maturation

參考文獻

1. Fülöp C, Szántó S, Mukhopadhyay D, Bárdos T, Kamath RV, et al. (2003) Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice. Development 130: 2253-2261.

2. Scarchilli L, Camaioni A, Bottazzi B, Negri V, Doni A, et al. (2007) PTX3 interacts with inter-alpha-trypsin inhibitor: implications for hyaluronan organization and cumulus oophorus expansion. J Biol Chem 282: 30161-30170.

3. Yokoo M, Sato E (2004) Cumulus-oocyte complex interactions during oocyte maturation. Int Rev Cytol 235: 251-291.

4. Chang MC (1951) Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168: 697-698.

5. Austin CR (1952) The capacitation of the mammalian sperm. Nature 170: 326.