圈養雌性小貓熊卵巢之形態學與免疫組織化學分析

Abstract

小貓熊（red panda）學名Ailurus fulgens，屬肉食目浣熊科，2000年在國際自然保護聯盟瀕危物種紅皮書（IUCN 3.1）被列為易危物種（VU），至今仍不斷受到棲息地喪失和破碎化的威脅。野生族群不易掌握現況，圈養族群的調查則相較容易，2004年西方專家調查指出，全世界小貓熊圈養族群10年來未成長反而有萎縮跡象，其中小貓熊川西亞種球圈養族群僅295隻（北美與日本地區為主），而中國地區根據1997年一份調查圈養族群為256隻。 如何提升小貓熊之繁殖效率是改善圈養族群衰退的關鍵，然而現今我們對小貓熊的繁殖生理瞭解仍非常有限。本研究以中國福建省福州市中國海峽（福州）大貓熊研究中心2006∼2014年間死亡的10隻不同年齡與生殖生理狀態母小貓熊為樣本，摘取其卵巢利用蘇木紫－伊紅染色（H&E stain）進行濾泡與黃體之組織學觀察，並測量不同發育階段之濾泡以及其卵母細胞之直徑。結果發現未成年72日齡小貓熊初級濾泡之濾泡壁仍未發育超過一層細胞；而成年個體的濾泡直徑依不同發育階段分別為：始基濾泡至初級濾泡20－82 μm、次級濾泡68－248 μm、成熟濾泡380－2379 μm；而卵母細胞平均直徑：始基濾泡至初級濾泡卵母細胞11－59 μm、次級濾泡卵母細胞56－69 μm、成熟空腔濾泡卵母細胞77－128 μm。分析以上結果，濾泡尺寸在空腔期成長比例較空腔前期為大，濾泡卵母細胞尺寸則是在空腔前期成長比例較空腔期為大，此現象與多數物種相同。隨後進一步利用免疫組織染色法（Immunohistochemical stain）偵測組織切片中增生細胞核抗原（PCNA）、類固醇生成急性調控蛋白（StAR）、類固醇生成酵素（P450scc、3β-HSD）之表現，結果一隻8歲以上動情後期（metestrus）個體的卵巢觀察到濾泡組織呈現PCNA、StAR、P450scc與3β-HSD的陽性反應訊號，而黃體組織呈現StAR、P450scc與3β-HSD的陽性反應訊號，顯示濾泡組織正在增生成長，而濾泡與黃體組織皆在進行類固醇生成作用（steroidogenesis）。本結果證實免疫組織染色法可應用於小貓熊之後續生理研究。

The red panda (Ailurus fulgens), also called lesser panda, is a unique species that belongs to its own taxonomic family and is not closely related to any other living species. It has been classified as vulnerable (VU) species by the IUCN in 2000 as the global population in the wild is estimated less than 10,000 mature individuals distributed over two subspecies, A. f. fulgens and A. f. styani. The wild population continues to decline and is threatened by habitat loss and fragmentation, poaching, and inbreeding depression, although the red panda is protected by national laws in their range countries. The zoo population also declined over the past decade. Even though this small arboreal mammal is very significant biologically, is of high conservation value, and has the potential to serve as a flagship for conservation across its range, eastern Himalayas and southwestern China, research data on the species is so insufficient that the first workshop hasn’t been held until 2010. A viable zoo population of red pandas could play an important role in ensuring the red panda’s survival for future reintroduction, and also function as a research resource for learning more about red panda biology. Unlocking certain reproductive characteristics of the species would aid in providing an environment for the captive animals that mimics their natural environment. The red panda is a seasonal breeder with a mating season in the winter. It is important to estimate factors may affect breeding success for zoo management. To assess changes of various reproductive statuses in female red panda, ovaries were collected from ten captive female red pandas died of non-reproductive system diseases at various reproductive stages in Fuzhou Giant Panda Research Center. First, this research describes the histology of follicular and corpus luteal tissues either in immature or mature ovaries. The ovaries collected were fixed in 10% formalin, and tissue slices were made using standard histological techniques and stained with H&E. Cyclic changes can be found in individuals achieved sexual maturity, at 18 to 20 months of age. Fully developed corpus luteum is present in both postpartum and non-pregnant animals. We also measure the diameter of follicles and oocytes at each folliclular growing stages; diameter ranges of primordial follicle and primary follicle was 20－82 μm, secondary follicle was 68－248 μm, Graafian follicle was 380－2379 μm; diameter ranges of oocyte in primordial follicle and primary follicle was 11－59 μm, secondary follicle was 56－69 μm, Graafian follicle was 77－128 μm. Follicular size showed a higher increasing rate during antral phase, while oocyte size showed a higher increasing rate during preantral phase. Second, to identify the factors that regulate progesterone production, the three steroidogenic enzymes (P450scc, 3βHSD, StAR) and proliferating cell nuclear antigen (PCNA) were investigated using immunohistochemical (IHC) staining methods. Positive signals of P450scc, 3βHSD, StAR and PCNA were exhibited by granulosa cells and luteal cells in an individual at metestrus, representing these follicles and corpus luteum still proceeding steroidogenesis, which means the follicular and luteal tissue were secreting steroid hormone. In conclusion, the result showed IHC staining can apply to ovarian tissues of female red panda. This study is the first histology report of red panda using IHC staining method; our purpose is to provide reproduction physiological information of red panda for its further research, such as hormone tracking, or application to zoo management. More knowledge is demanded to improve the declining problem of zoo population, and further to protect this species in the wild.