缺氧對牛黃體細胞增生與類固醇生成之影響

Abstract

雌性哺乳動物卵巢內濾泡排卵後，濾泡細胞隨即轉型成為黃體細胞而進入黃體期，隨著黃體的發育，黃體期可概分為前期（第0-6天）、中期（第8-12天）與後期（第15-17天）；黃體是雌性哺乳動物調控動情周期與維持懷孕之重要組織，主要功能為分泌孕酮，孕酮之生成需要類固醇生成急性調控蛋白（steroidogenic acute regulatory protein, StAR）、細胞色素P450膽固醇側鏈截切酶（P450 cholesterol side-chain cleavage enzyme, P450scc）及3b-羥化類固醇去氫酶（3b-hydroxysteroid dehydrogenase, 3b-HSD）等一連串類固醇生成相關蛋白及酵素的參與。現今已知黃體發育與實質性腫瘤（solidtumor）生長類似，而近年的研究發現實質性腫瘤生長過程中伴隨著缺氧狀況的發生，且已知細胞在缺氧的環境下將啟動相關轉錄因子；缺氧誘導因子-1（hypoxia inducible factor-1, HIF-1）是由缺氧誘導因子-1a（HIF-1a）與缺氧誘導因子-1a（HIF-1a）兩個次單元所組成，藉由調控其下游基因如血管內皮生長因子（vascular endothelial growth factor, VEGF）等，使細胞適應低氧環境。因此本試驗設計之目的在探討黃體發育過程中缺氧對黃體細胞生長與類固醇生成之影響。 本試驗利用取自屠宰場之牛黃體組織，應用免疫組織染色、西方墨染法及孕酮酵素免疫分析等技術，探討不同時期黃體之孕酮、類固醇生成相關蛋白及酵素、缺氧誘導因子、血管內皮生長因子及細胞核增生抗原蛋白（proliferating cell nuclear antigen, PCNA）等蛋白質表現變化。結果發現，HIF-1a、HIF-1b、VEGF與PCNA蛋白質表現量皆以前期較中期與後期高，顯示黃體發育初期細胞處於缺氧環境，且細胞增生快速，並促使血管新生成。同時，StAR與P450scc蛋白質表現於前期相對於中期低，顯示StAR與P450scc基因表現在缺氧條件下可能受到抑制，導致孕酮分泌量較低。 此外，以體外（in vitro）培養系統，將中期黃體細胞培養於正常氧（20 % 氧氣濃度）或缺氧（1 % 氧氣濃度）條件下，並添加PKA（protein kinase A）訊息傳遞路徑活化劑（oLH或8-bromo-cAMP）或孕酮生成前驅物（22R-羥化膽固醇或孕烯醇酮），測定其基礎與受激後孕酮分泌及類固醇生成相關蛋白及酵素之差異，結果顯示，黃體細胞於缺氧條件下StAR及3b-HSD蛋白質表現量較正常氧低，而P450scc蛋白質表現量雖無明顯變化，但其酵素活性卻受到抑制，致使黃體細胞孕酮分泌減少。除此之外，PCNA蛋白質於缺氧條件下表現量下降，顯示中期黃體細胞發育已臻成熟，缺氧將可能引導黃體走向解體的命運。 另一方面，由於中期黃體細胞發育已臻成熟，其細胞生理與黃體發育初期已然有所差異，因此本研究亦應用黃體化粒性細胞（luteinized granulosa cells）模擬發育初期之黃體細胞，並培養於正常氧（添加氯化鈷）或缺氧（未添加氯化鈷）條件下，添加PKA訊息傳遞路徑活化劑（oLH或8-bromo-cAMP）測定孕酮生成與相關蛋白質之表現差異。結果發現，黃體化粒性細胞於缺氧條件下其HIF-1a、HIF-1b與VEGF蛋白質表現量皆較正常氧高；同時，StAR及P450scc蛋白質表現量則降低，但3b-HSD不受缺氧影響；此外，藉由特異性抗體檢測PKA訊息傳遞路徑發現，PKA訊息傳遞路經於缺氧條件下磷酸化PKA（phospho-protein kinase A）蛋白質表現量下降，可能經此路徑的調控造成StAR及P450scc蛋白質表現量下降，導致黃體化粒性細胞孕酮分泌量減少。然而，意外地發現缺氧條件下黃體化粒性細胞之PCNA蛋白質表現量反而增加，此結果與中期黃體細胞之結果恰好相反，顯示此時之細胞分裂旺盛而快速增生，與黃體發育初期之細胞生理相似，且缺氧條件可能扮演著重要的調控角色。 本試驗初步發現缺氧條件下類固醇生成相關蛋白及酵素可能受到HIF-1直接或間接的影響，終使孕酮分泌量下降；而細胞增生指標的PCNA於不同時期黃體細胞對缺氧條件有截然不同的反應，是否亦受HIF-1所調控，尚待進一步實驗設計以證實之。

Different from other mammal reproduction researches, mostly which focus on the relation between a development of corpus luteum and the secretion of progesterone, this study specifically investigates the development of corpus luteum under the influence of hypoxia, which has not been paid much attention to in the field of reproduction. Results showed that hypoxia actively affected corpus luteum, particularly the growth of luteal cell and the secretion of steroid hormones. Hopefully, the result of my research will serve as a pathway to further exploration, like how hypoxia functionally affects corpus luteum during the process of luteal phase when the formation of corpus luteum is unavoidably accompanied by inadequate amounts of available oxygen. Corpus luteum is similar to solid tumorgenesis, which is a fast-growing tissue and its formation is always associated with hypoxic condition in which cells will automatically turn on the transcription factor -hypoxia inducible factor-1 (HIF-1) and leads to the enhancement of vascular endothelial growth factor (VEGF) expression to adapt cells to hypoxic condition. In this study, bovine corpus luteum was obtained in the slaughter house of Ya-Sheng frozen foods CO., LTD and immunohistochemistry, Western Blot and progesterone enzyme-linked immunoassay were used to exam the morphological and the functional changes of corpus luteum. Experiment was divided into three stages sequentially in coordination with three different developments of luteal phase: early (0-6 days), middle (8-12 days) and late (15-17 days). As to the objects, not only progesterone but also steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (P450scc), 3b-hydroxysteroid dehydrogenase (3b-HSD), HIF-1a, HIF-1b, VEGF and proliferating cell nuclear antigen (PCNA) were under investigation. Briefly speaking, initial samples from abattoir were analyzed, comparing the difference in cell proliferation and progesterone between in early stage and in middle stage. Furthermore, luteal cells, in vitro, were put under investigation, proving how hypoxia literally affected the performance of luteal cell and the secretion of progesterone as a result. There were more protein expression of HIF-1a, HIF-1b, VEGF and PCNA in early stage than in middle or late stage. Regardless of the condition of hypoxia, luteal cells instead performed a fast-growing proliferation in early stage and angiogenesis as a result. But less progestrone was secreted in early stage due to less expression of StAR and P450scc, both of which might be inhibited in the hypoxic condition. Some middle-aged luteal cells were cultured, developed in vitro, in hypoxic condition (1 % oxygen) while others in normoxic condition (20 % oxygen). The purpose of adding protein kinase A (PKA) signal transduction pathway activator (oLH or 8-bromo-cAMP) and progesterone precursor (22R-OHC or pregnenolone) were to have a clear picture of the changing secretion of progesterone in response to the different performance of related protein. The results in this case was literally meaningful, that luteal cells secreted relatively less progesterone in hypoxia than in normoxia, with decreasing protein expression of StAR and 3b-HSD. To some extent, the phenomenon that P450scc enzyme activity was inhibited also said something about less progesterone secretion. Besides, it is speculated that luteolysis could be inspired under the condition of hypoxia due to the observed decrease in PCNA caused by hypoxia. Likewise, there was a significant result in the case of investigating whether luteal cells in early stage was affected by hypoxia similarly to those in middle stage, given big difference in the development of cell physiology. In this experiment, luteinized granulosa cells were used to mimic the early stage luteal cells. And luteinized granulosa cells were also cultured under the normoxic conditon (without cobalt chloride) and the hypoxic condition (with cobalt chloride) individually. oLH or 8-bromo-cAMP were added to luteinized granulosa cells. Results indicated that, protein expression of HIF-1a, HIF-1b and VEGF had a better performance in hypoxia than in normoxic condition, but phospho-PKA, StAR and P450scc protein expression had weaker expression in the former condition than in the later one. As a result of decreasing secretion of progesterone the decreasing of phospho-PKA was suspected to have a role in the reduction of StAR and P450scc in the condition of hypoxia. Compared by middle-stage luteal cell, luteinized granulosa cells responded an increasion in PCNA even under hypoxic condition, which spoke for an important role of hypoxia in controlling the performance of cell proliferation. Similarity was proved between luteal cell and luteinized granulosa cells when it came to the performance of cell proliferation under a condition of hypoxia. In summary, we found HIF-1 indeed affected steroidogenic protein and enzyme in a direct or indirect way, leading to the changes of progesterone secretion. However, it will need more and further study to providing enough evidence to prove PCNA is also affected by HIF-1 when it comes to cell proliferation in normoxic conditon or in hypoxic condition.