山羊與牛在動情周期間黃體超微結構與生理功能之探討

The Study of the Ultrastructural and Physiological Functions of Corpora Lutea during the Estrous Cycle in Cattle and Goats

江逸凡(Yi-Fan Jiang)

指導教授 ： 邱智賢

共同指導教授 ： 吳兩新(Leang-Shin Wu)

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.00634

摘要

在雌性哺乳動物之生殖系統中，黃體（corpus luteum, CL）為一分泌孕酮（progesterone, P4）之暫時性組織，其功能在生殖系統運作過程中扮演極重要之角色。本研究即是以反芻家畜動物為研究對象，從形態學（morphology）、基因表現（gene expression）、以及內分泌功能等面向，探討動情周期中黃體組織與細胞之變化，以期更進一步了解黃體發育與退化之過程。本研究首先透過牛黃體組織切片，在早期黃體組織中發現大量紅血球之堆積；中期組織間黃體細胞密度增加，細胞間隙縮小；而至後期時，組織切片中黃體細胞所佔之比例則大幅下降。基因表現與孕酮濃度分析之結果亦顯示，中期黃體組織含大量類固醇生成細胞（steroidogenic cell），並具備最佳化之類固醇生成能力；暗示早期黃體組織發育可能透過細胞增生（proliferation）之過程，影響中期黃體之孕酮分泌能力。

由於生長中之組織可能處於組織缺氧（hypoxia）之狀態，本研究進一步分析缺氧誘導因子1（hypoxia inducible factor 1, HIF1）、增生細胞核抗原（proliferating cell nuclear antigen, PCNA）以及血管內皮生長因子A（vascular endothelial growth factor A, VEGFA）等基因在各時期牛黃體組織之表現，以釐清缺氧與黃體發育之關聯。所分析之基因在早期組織中有較高之表現量，顯示黃體自早期發育至中期時，可能經歷了缺氧、細胞增生、以及血管生成（angiogenesis）等過程。為模擬黃體組織於中期與早期之發育環境，本研究分別將中期牛黃體細胞與牛黃體化粒性細胞（luteinized granulosa cell）培養於缺氧環境，發現缺氧環境均可抑制此二種細胞之類固醇生成（steroidogenesis），但僅可刺激黃體化粒性細胞表現PCNA，顯示黃體細胞對於缺氧之反應可隨黃體發育之時期而有所改變，其中缺氧刺激早期黃體細胞之增生，則可能為黃體發育的重要因子之一。

為更進一步探討黃體細胞於動情周期間改變之過程，本研究以穿透式電子顯微鏡（transmission electron microscope, TEM），觀察山羊黃體組織中細胞形態之變化。動情周期中，中期山羊黃體組織中含適當分布之微血管組織；相對後期則可發現組織中出現形態萎縮之黃體細胞（luteal cell）、延伸的退化區，以及含多葉細胞核之免疫細胞分布。萎縮的黃體細胞內多呈現自噬作用（autophagy）與細胞凋亡（apoptosis）的典型特徵，顯示山羊黃體退化（CL regression）之過程中，可能包含了計畫性細胞死亡（programmed cell death）的現象。本研究亦發現山羊早期黃體組織中含活化之內皮細胞（endothelial cell），顯示早期山羊黃體處於血管生成旺盛之階段。另一方面，在後期黃體組織中，穩定的微血管結構以及在胞外基質（extracellular matrix, ECM）中大量堆積之纖維，則顯示在後期黃體組織中，血管生成已逐漸減少。

大黃體細胞（large luteal cell, LLC）是山羊黃體中具備孕酮分泌能力的細胞。以穿透式電子顯微鏡進行觀察，可於大黃體細胞表面觀察到彼此交疊排列之微絨毛通道（microvillar channel）。由分布於通道中之球狀結構，初步推測山羊大黃體細胞可透過選擇性膽固醇吸收作用（selective uptake pathway），利用細胞外膽固醇（cholesterol）之來源，進行類固醇生成。另一方面，雖然在各時期大黃體細胞之細胞質中，均可發現分泌顆粒（secretion granule）與粒線體相關膜系（mitochondria-associated membrane, MAM） 之分布，但僅在早期與中期之細胞中，可觀察到囊狀內質網（cisternal endoplasmic reticulum）呈現膨大之結構。而大黃體細胞之粒線體（mitochondria）在早期至中期之組織中多含管狀脊（tubular cristae）之結構；至後期時，粒線體則多呈腫脹之外觀，內部亦轉變為板狀脊（lamellar cristae）之構造。本試驗透過超微結構之觀察，發現並整理山羊黃體細胞於動情周期間之形態變化，顯示黃體細胞之胞器可塑性（plasticity）可能與細胞之類固醇生成能力相關。

為了探究胞器結構與類固醇生成之關聯，本研究進一步利用已建立之山羊黃體細胞株CLC-D，進行試驗處理與超微結構之觀察。初步結果顯示，CLC-D細胞在正常培養環境下，即具備微絨毛、內質網與粒線體等胞器。以22R-OHC（22R-hydroxycholesterol）處理黃體細胞後，可刺激CLC-D進行類固醇生成，並增加含管狀粒線體之細胞百分比。顯示在山羊黃體細胞中，超微結構之變化亦可能反應細胞類固醇生成能力之改變；然而更多的胞器變化，以及形態變化與類固醇生成之關聯性，仍待更多研究加以探討。

綜上而論，本研究結果顯示黃體早期經歷細胞增生與血管生成，至中期開始發揮最佳化之類固醇生成能力；隨後透過計畫性細胞死亡達成黃體生長與退化之周期。黃體細胞之結構以及其對於外界刺激的反應亦隨著周期而轉變，最終反應於內分泌功能上。然而細胞結構、功能與基因表現之連結，仍待進一步研究加以證實。

關鍵字

山羊 ； 黃體 ； 組織缺氧 ； 類固醇生合成 ； 血管生成 ； 超微結構 ； 粒線體

並列摘要

Corpora lutea (CLs) are transient progesterone(P4)-secreting glands necessary for reproduction. In present studies, we investigated the cellular events during the lifespan of CLs in ruminants from the views of morphology, gene expressions and P4 secretion. The histological observation of bovine CLs revealed abundant erythrocytes in the early stage, dense luteal cells without intercellular space in the middle, and the reduction in percentage of luteal cells in the late. The gene expression and P4 level in CLs confirmed the maximum cell number and steroidogenic abilities in the middle CLs, indicating that cell proliferation could be significant during luteal formation.
With growing tissue often under the stress of hypoxia, we tested the expressions of hypoxia inducible factor 1 (HIF1), proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor A (VEGFA) during estrous cycles in bovine CLs. The genes were highly expressed in the early stage, suggesting that proliferation, hypoxia and angiogenesis could contribute to the formation and function of bovine CLs. Further, we stimulated primary luteal cells from middle CLs and luteinized granulosa cells under hypoxia, to mimic the developing CL in middle and early stages, respectively. The hypoxia inhibited steroidogenesis in both cells, but stimulated cell proliferation and angiogenesis in luteinized granulosa cells. These results suggested that hypoxia could be a critical factor in luteal developments and that cellular response could vary from stage to stage.
For the details, the ultrastructure of goat CLs in various stages was observed. Our analyses revealed proper patterned capillaries in middle CLs, while shrunken luteal cells, expanding regression areas and immune cells with lobed nuclei were identified in the late. Those shrunken cells showed the figures of both autophagy and apoptosis, indicating that programmed cell death might involve CL regression. The active endothelial cells suggested activated angiogenesis in early CLs. On the other hand, the stabilized capillaries and the accumulation of extracellular matrix (ECM) fibers indicated the absence of angiogenesis in the late.
The large luteal cell (LLC) is one of the P4-secreting cells in goat CL. Under transmission electron microscope (TEM), LLCs assembled to form microvillar channels on their surface. With globular structures discerned in the channels, the selective uptake pathway for cholesterol utilization could be significant in goat LLCs. In the cytoplasm, secretion granules and the mitochondria-associated membrane (MAM) were recognized throughout the cycles, while some swollen cisternal ERs were identified only in the early and middle stages. The mitochondria with tubular cristae increased in early and middle LLCs. However, the mitochondria swelled and the cristae shifted to lamellar type in the late, suggesting that plasticity of organelles could contribute to steroidogenesis in goat CLs.
To make the connection between ultrastructure and steriodogenesis, we observed the ultrastructure of a caprine luteal cell line, CLC-D. Preliminary results indicated the morphological shift of mitochondria from lamellar to tubular cristae after steroidogenic treatment. Yet more connections remain to be identified.
In conclusion, our results suggested the cell proliferation and angiogenesis in early CLs, the steroidogenesis in the middle, and the programmed cell death in the late. Luteal cellular responses and ultrastructure vary depending on stages. However, their connections with function, structure and gene expressions call for further studies.

並列關鍵字

goat ； corpus luteum ； hypoxia ； steroidogenesis ； angiogenesis ； ultrastructure ； mitochondria

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