紅螢光豬生殖母細胞之純化與特性分析

Abstract

生殖母細胞 (gonocyte) 係一介於始基生殖細胞 (primordial germ cell) 及精原母細胞 (spermatogonium) 之雄性生殖細胞種類。生殖母細胞與未分化之精原母細胞因可建立及維持生精作用，皆被稱為精原幹細胞 (spermatogonial stem cell)。小鼠精原幹細胞已被證實可於體外長期增殖培養，並保有其幹細胞特性，此外，亦有多方研究報告指出，部分精原幹細胞於培養過程中，會主動行再程序化 (reprogram) 轉為類似胚幹細胞 (embryonic stem cell) 特性之多能性生殖幹細胞 (pluripotent germline stem cell)。若於前述特性，精原幹細胞不僅成為後生遺傳學 (epigenetics) 及生殖生物學 (reproductive biology) 等相關研究之重要模型，更可做為治療性生殖產學之極佳技術新平台。 製備高純度之精原幹細胞為研究其分子及生理特性之重要步驟。生殖母細胞為二月齡前仔豬睪丸內唯一屬於生殖類型之細胞；職是之故，將雄性仔豬睪丸中之體細胞移除後，即可獲得高純度之雄性生殖母細胞。雖然目前尚未發現生殖母細胞之特異表面抗原，但可依據其低貼附特性和較大之細胞容積與體細胞區隔之。 源自本研究室成功產製攜帶有紅色螢光蛋白質DsRed之轉基因豬，乃藉由CAG啟動子有效驅動螢光蛋白質之表現，遂令其全身細胞均能穩定表此螢光蛋白；惟試驗結果證明在該等DsRed轉基因豬之睪丸中，各不同分化階段之生殖細胞中，其螢光表現遠不如彼等體細胞者然。進一步試驗依上述之生殖母細胞特性，配合細胞貼附性差異及細胞分選策略針對四至六週齡仔豬之生殖母細胞進行培養，再經流式細胞分選儀 (cell sorter) 完成篩選獲得具備強前散射光 (forward scatter, FSC) 及弱紅螢光之細胞，證明確能有效將生殖母細胞之純度由初始分離之1.0 ± 0.3% 顯著提升至90.4 ± 1.6% (p<0.01) 之譜；設若僅收集彼等具有強前散射光之細胞群，其細胞純度亦高達80.9 ± 2.6% (p<0.01)。 進一步試驗乃藉由精原幹細胞標誌PLZF抗體之使用，針對二至八週齡仔豬，完成睪丸切片之免疫染色鑑定；結果發現彼等表現有PLZF之生殖母細胞比例，係隨著週齡漸增，且與生殖母細胞之回位 (homing) 現象具正相關性；足見PLZF之表現確可提供做為豬生殖母細胞具幹細胞特性之標誌。職是之故，進一步試驗乃針對以PLZF之表現進行檢測，俾確認彼等業經純化之生殖母細胞的固有特性；試驗結果顯示，業經純化之生殖母細胞中具有表現PLZF之百分率高達67.2 ± 7.3%，分別係可能具幹細胞潛力者。 綜合上述，本研究完成兩階段之細胞純化技術，自單一睪丸成功製備高度純化之豬生殖母細胞，除可以提供未來最為早期生殖細胞分化特性研究之模式細胞，另外且依生殖母細胞之基因表現形象將之區分成為若干類別之生殖母細胞亞群 (subpopulation)，可供未來針對生殖母細胞之幹細胞特性及其細胞命運 (cell fate) 決定因子等分子機制，進行系列性詳實深入探討之使用。

Gonocytes have been known to be derived from primordial germ cells and to give rise to undifferentiated spermatogonia, or spermatogonial stem cells (SSCs), which establish and maintain spermatogenesis within the postnatal testes. Mouse SSCs were demonstrated as that they can long-term proliferate in vitro without losing the ability of differentiating into sperm after transplantation. Besides, a few SSCs can automatically reprogram to a pluripotent status which is similar to those of embryonic stem cells (ESCs). Due to these special features, SSC is a valuable cell model for epigenetic and reproductive studies, and will be a novel platform for transgenic animal production as well. Establishment of significantly enriched population of SSCs is a critical step that will facilitate the researches of their molecular and biological properties. Despite of the lack of specific surface marker, the weak adhesion ability and the large size of gonocytes suggested the possibility to purify gonocytes by differential plating and cell sorting. Most cells of our DsRed transgenic pigs, in which the transgene of DsRed fluorescent protein was driven by the ubiquitous CAG promoter, stably represent fluorescence. However, to our surprise, in all the male germ cells, the expression levels of DsRed were found to be much lower than those found in most of somatic cells. Hence, gonocytes, which were isolated from pigs between 4 to 6 weeks of age, were purified by differential plating following by cell sorting sequentially. The high forward scatter (FSC) and weak fluorescence cell population was collected, and the germ cells were labeled with an antibody targets a germ cell specific marker, VASA. The purity of gonocytes was significantly elevated from 1.0 ± 0.3% to 90.4 ± 1.6% (p<0.01), which is also significantly higher than the other ones (80.9 ± 2.6%) selected by FSC only (p<0.01). PLZF is a transcriptional regulator essential for self-renewal and maintenance of SSCs. Here in this study, immunostain was performed on testes sections from pigs at each 2, 4, 6 and 8 weeks of age, respectively. The ratio of PLZF positive germ cells appeared to be increased with ages and the relation between PLZF expression and germ cell homing was observed, suggesting PLZF is a marker for porcine SSCs. For the purpose to analyze the stem cell potential of purified gonocytes, the expression of PLZF was detected by immunocytochemistry. There were 67.2 ± 7.3% of VASA positive cells, found in those cells purified after cell sorting, and these cells were also shown positive to PLZF, indicating the stemness of them. Overall, a two-step purification method for preparing gonocyte-enriched testicular cells from single testis has been successfully established after this present study. The highly pure fluorescent gonocytes might include a specific germ cell population, which can derive to SSCs. As a consequence, this gonocyte-enriched cell population could be an excellent model for further studies related to both characterization of primitive porcine germ cells and the germ cell fate determination.