The Effect of Hypoxia on Cell Proliferation and Steroidogenesis in Bovine Luteal Cell
Date Issued
2007
Date
2007
Author(s)
Lin, Cheng-Wei
DOI
zh-TW
Abstract
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.
Subjects
缺氧
缺氧誘導因子-1 alpha
黃體
牛
細胞增生
類固醇生成
hypoxia
HIF-1 alpha
corpus luteum
bovine
cell proliferation
steroidogenesis
Type
thesis
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