以小白鼠為動物模式探討雄性附屬性腺分泌胰蛋白酶抑制蛋白 Spink3 調控哺乳類生殖的機制

Abstract

本研究室已從小白鼠儲精囊分泌液純化得到一個由 57 個胺基酸所組成的 Kazal 型的胰蛋白酶抑制因子，其對胰蛋白酶的抑制常數 (Ki) 為 0.15 nM。由於此胰蛋白酶抑制因子的 cDNA 及胺基酸序列與 Mills 等人已發表的 P12 cDNA 所推測的結構相同，因此本實驗室將此小分子量 (約 6000 Da) 的蛋白暫時稱作 P12；而根據目前的國際命名，P12 已正式定名為 Spink3。從本實驗室的研究顯示，Spink3 會專一地結合在精蟲頂體前端，以 Scatchard plot 的分析可知Spink3 對精蟲的結合強度以解離常數 (Kd) 表示約為 70 nM，且與精蟲的結合部位為單一形式，每隻精蟲的最大結合量為 1.49 x 106。但研究至今，尚無法得知 Spink3 在精蟲的結合部位為何。 本論文利用酵母菌雙雜合系統的方法 (yeast two-hybrid system)，鑑定出Spink3 在精蟲上的結合蛋白可能是TESPL (testis-specific protease-like protein)。由西方和北方墨點法顯示 TESPL 只專一地在睪丸表現且表現量會隨著發育成長而有增加的趨勢。具有 GPI 修飾部位的 TESPL 在一級結構的胺基酸序列與絲胺酸蛋白酶相似，都保留著活性區的 His 與 Asp，但 Ser 卻被 Pro 取代，因此被認為是失去胰蛋白酶活性的膜蛋白。觀察睪丸切片的免疫組織染色，TESPL 主要表現在只含有單套染色體的精細胞及成熟精蟲。以副睪尾部的精蟲作間接免疫螢光染色，TESPL 也會出現在精蟲的頂體前端。從這些數據提示 TESPL 是 Spink3 在精蟲的潛在結合部位。 Spink3 會經由射精時伴隨精液進入到雌鼠生殖道，子宮腔含結合 Spink3 的精蟲，但在輸卵管所發現的精蟲卻沒有 Spink3 的訊號。以體外實驗探討 Spink3 對精蟲的生理功能，顯示 Spink3 不會影響精蟲進行獲能效應而伴隨的反應如蛋白質酪胺酸磷酸化、精蟲的泳動力，但卻會降低頭部的鈣離子濃度及 A23187 所誘發的頂體反應。從體外受精的實驗，Spink3 卻會因為影響精蟲與卵子的結合能力，而進一步地降低受精率。失去胰蛋白酶抑制能力的 Spink3 突變種 R19L 也同樣地具有降低頂體反應及受精能力的功能，顯示 Spink3 對精蟲的影響並不是藉由抑制胰蛋白酶活性而產生的。 精蟲獲能效應不會移除精蟲上的 Spink3，但子宮分泌液的胰蛋白酶 (Spink3-Inhibiting trypsin-like activity, SITA) 卻有此能力；不過 SITA 會在交配後一開始會先受到進入子宮腔的 Spink3 抑制。因此 Spink3 與雌性生殖道的蛋白酶兩者相互作用的關係亦影響著正常的生殖功能。 本論文以小白鼠為動物模式，闡釋 Spink3扮演的生殖角色：1) 結合在精蟲頂體的 Spink3 會抑制獲能的精蟲發生自發性的頂體反應，避免精蟲遇到卵子之前，失去生殖能力；2) 由於 Spink3 會抑制受精過程，因此須藉由子宮液分泌 SITA將精蟲上的 Spink3移除，使精蟲到達輸卵管可以發生頂體反應而進一步與卵子結合達到授精；3) Spink3 會抑制 SITA，保護精蟲不受雌性生殖道蛋白酶的破壞。

Mice were used to study the involvement of reproductive-derived Spink from males in mammalian reproduction. A Kazal-type protease inhibitor purified from mouse seminal vesicle secretion by our group has an inhibitory constant (Ki) of 0.15 nM to trypsin and a primary structure consisting 57 amino acid residues. Since this rather small protein was derived from the P12 cDNA cloned from the mouse ventral prostate by Mills et al., it was tentatively named P12. According to the Mouse Genome Informatics nomenclature committee, P12 is now renamed mouse Spink3. Our previous study suggest that Spink3 has a single-type binding site (1.49 x 106 sites/cell) with a Kd value of 70 nM mainly on the plasma membrane overlaying the acrosomal region of mouse sperm cell. Yet, the membrane-anchored molecule of Spink3 on sperm head has not been established. We identified a testis-specific protease-like protein tentatively named TESPL from the clones of a yeast two-hybrid screen against a mouse testicular cDNA library using the trypsin inhibitor Spink3 from male accessory sexual glands as bait. We found that TESPL transcription was restricted to the testis and that the level of transcription was positively correlated with animal maturation. Alignment of the cDNA-deduced sequences of serine proteases showed the replacement of an essential serine residue in the catalytic triad of serine proteases by a proline residue in TESPL, which was demonstrated to be a membrane-bound protein devoid of proteolytic activity. The immunohistochemical staining patterns of seminiferous tubules in the testis revealed TESPL mainly on postmeiotic cells such as spermatids and spermatozoa. On the mouse sperm from caudal epididymis, TESPL was localized mainly on the plasma membrane overlaying the acrosomal region. Result of indirect immunofluorescence stain indicated that Spink3 was found in the secretion and seen on a considerable portion of sperm in the uterine cavity but disappeared in the oviduct lumen after coitus. The Spink3-sperm binding did not change the cell status and inhibit the capacitation-related protein tyrosine phosphorylation and cell motility enhancement, but reduced the head [Ca2+]i and the ionophore A23187-induced acrosome reaction. The sperm-egg interaction and fertility rate greatly suppressed after insemination of oocyte-cumulus complexes containing Spink3 in the capacitated sperm preparation. It is of interest to note that R19L, like its wild type, can bind sperm to suppress AR and reduce fertility. This substantiates that the reactive R19 on the Spink3 molecule for protease inhibition is not essential for its action on sperm. Not the membrane modification associated with the sperm capacitation but the Spink3-inhibiting trypsin-like activity (SITA) in the uterine fluid of estrous females was involved in releasing Spink3 from sperm to resume their fertilizing ability. Meanwhile, suppressing SITA by free Spink3 protected sperm from proteolytic damage in the uterine cavity, manifesting the important interplay of Spink and SITA during natural coitus. Using mice as experimental animals, this work was conducted to prove that: i) Spink3 binding on the apical hook of sperm head prevents them from becoming infertile before encountering an egg by diminishing the acrosome reaction of capacitated sperm; ii) Spink3 on sperm reduces in vitro fertility, and the Spink3-inhibiting trypsin-like activity (SITA) secreted from the uterus of estrous females during natural coitus releases Spink3 from sperm to restore their ability to fertilize; iii) the proteolytic damage to sperm from SITA is suppressed by free Spink3 in the uterine cavity.