Paul Avishek, Bharati Jaya, Punetha Meeti, Kumar Sai, Mallesh Vidyalakshmi G, Chouhan Vikrant S, Sonwane Arvind, Bag Sadhan, Bhure Sanjeev Kumar, Maurya Vijai Prakash, Singh Gyanendra, Whitworth Kristin M, Sarkar Mihir
Indian Veterinary Research Institute, Physiology & Climatology Division, Bareilly, India.
Indian Veterinary Research Institute, Division of Animal Genetics, Bareilly, India.
Cell Physiol Biochem. 2019;52(3):532-552. doi: 10.33594/000000038.
BACKGROUND/AIMS: Thrombospondins (TSPs) are large multi-modular proteins, identified as natural angiogenesis inhibitors that exert their activity by binding to CD36 and CD47 receptors. The anti-angiogenic effect of TSPs in luteal regression of water buffalo has not been addressed. The present study characterized the expression pattern and localization of TSPs and their receptors in ovarian corpus luteum during different stages of development in buffalo. This study also elucidated the effect of exogenous Thrombospondin1 (TSP1) or the knocking out of the endogenous protein on luteal cell viability and function. Further, the in vitro transcriptional interaction of TSP1 with hormones, LH, PGF2α and angiogenic growth factors, VEGF and FGF2 were also evaluated.
First, the CLs were classified into four groups based on macroscopic observation and progesterone concentration. mRNA expression of examined factors was measured by qPCR, localization by immunoblotting and immunohistochemistry. TSP1 was knocked out (KO) in cultured luteal cells isolated from late luteal stage CLs (day 1116) by CRISPR/Cas9 mediated gene editing technology in order to functionally validate the TSP1 gene. Isolated cells from late stage CLs were also stimulated with different doses of TSP1, LH, PGF2α, VEGF and FGF2 for various time intervals to determine transcriptional regulation of thrombospondins.
mRNA expression of TSPs and their receptors were found to be significantly higher in late and regressed stage of CL as compared to other groups which was consistent with the findings of immunoblotting and immunolocalization experiments. It was observed that TSP1 induced apoptosis, down regulated angiogenic growth factors, VEGF and FGF2 and attenuated progesterone production in cultured luteal cells. However, knocking out of endogenous TSP1 with CRISPR/Cas9 system improved the viability of luteal cells, progesterone synthesis and upregulated the expression of VEGF and FGF2 in the KO luteal cells. PGF2α induced the upregulation of TSPs and Caspase 3 transcripts, whereas treatment with LH and angiogenic growth factors (VEGF and FGF2) down regulated the TSP system in luteal cells.
Collectively, these data provide evidence that thrombospondins along with their receptors are expressed at varying levels in different stages of CL progression with maximum expression during the late and regressing stages. These results are consistent with the hypothesis that thrombospondins stimulated by PGF2α plays an essential modulatory role in bringing about structural and functional luteolysis in buffalo.
背景/目的：血小板反应蛋白（TSPs）是大型多模块蛋白，被鉴定为天然血管生成抑制剂，通过与CD36和CD47受体结合发挥其活性。TSPs在水牛黄体退化中的抗血管生成作用尚未得到研究。本研究对水牛卵巢黄体在不同发育阶段TSPs及其受体的表达模式和定位进行了表征。本研究还阐明了外源性血小板反应蛋白1（TSP1）或内源性蛋白敲除对黄体细胞活力和功能的影响。此外，还评估了TSP1与激素、促黄体生成素（LH）、前列腺素F2α（PGF2α）以及血管生成生长因子血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（FGF2）的体外转录相互作用。
首先，根据宏观观察和孕酮浓度将黄体分为四组。通过定量聚合酶链反应（qPCR）检测所研究因子的mRNA表达，通过免疫印迹和免疫组织化学进行定位。为了在功能上验证TSP1基因，利用CRISPR/Cas9介导的基因编辑技术在从黄体晚期（第11 - 16天）黄体分离的培养黄体细胞中敲除TSP1。还对从晚期黄体分离的细胞用不同剂量的TSP1、LH、PGF2α、VEGF和FGF2进行不同时间间隔的刺激，以确定血小板反应蛋白的转录调控。
与其他组相比，发现TSPs及其受体的mRNA表达在黄体晚期和退化期显著更高，这与免疫印迹和免疫定位实验的结果一致。观察到TSP1诱导培养的黄体细胞凋亡，下调血管生成生长因子VEGF和FGF2，并减弱孕酮生成。然而，用CRISPR/Cas9系统敲除内源性TSP1可提高黄体细胞活力、孕酮合成，并上调敲除TSP1的黄体细胞中VEGF和FGF2的表达。PGF2α诱导TSPs和半胱天冬酶3转录本上调，而用LH和血管生成生长因子（VEGF和FGF2）处理可下调黄体细胞中的TSP系统。
总体而言，这些数据表明，血小板反应蛋白及其受体在黄体发育的不同阶段以不同水平表达，在晚期和退化期表达最高。这些结果与以下假设一致，即PGF2α刺激的血小板反应蛋白在水牛黄体的结构和功能溶解中起重要调节作用。
