黄体期不同营养水平对湖羊卵泡发育及相关葡萄糖代谢途径的影响

doi:10.11843/j.issn.0366-6964.2018.05.009

摘要/Abstract

摘要：

旨在研究黄体期短期营养水平对湖羊卵泡发育、卵巢细胞凋亡率、葡萄糖转运蛋白及葡萄糖代谢途径关键基因表达量的影响，以探讨黄体期营养水平调控绵羊卵泡发育的可能机制。本研究选择经产湖羊30只进行同期发情处理，撤栓后使用调教公羊试情，发情结束当日定义为下一个情期的第0天，随后6 d对所有试验羊给予1.0倍维持需要量水平饲喂，在情期的第7～14天，将试验羊随机分为1.0倍维持需要组（1.0M；n=10）；0.5倍维持需要量组（0.5M；n=10）和1.5倍维持需要量组（1.5M；n=10）；第15天分别从3组随机挑选屠宰6只试验羊并取卵巢组织待用，剩下的12只湖羊按1.0倍维持需要量词喂，并对原有各组试验羊进行发情鉴定观察，用以统计发情周期。结果表明，与1.0M组和1.5M组相比，限饲导致0.5M组试验羊出现显著的发情延迟现象（P<0.05），并伴随着<2.5 mm直径卵泡/总卵泡比例升高和≥2.5 mm卵泡/总卵泡比例的降低（P<0.05）；TUNEL结果显示，0.5M组试验羊卵巢胞凋亡率显著高于1.0M组和1.5M组（P<0.05）；葡萄糖转运蛋白-4（GLUT4）蛋白在卵巢组织上各级卵泡中均有表达，且其在1.5M组<2.5 mm卵泡中的表达量显著高于1.0M组和0.5M组（P<0.05）；通过对卵泡细胞葡萄糖代谢途径：多元醇途径（Polyol pathway）、糖酵解途径（Glycolysis pathway）、己糖胺生物合成途径（Hexosamine biosynthesis pathway；HBP）和磷酸戊糖途径（Pentose phosphate pathway；PPP）关键基因进行qRT-PCR检测后发现：HBP途径关键酶GFPT2基因表达量在<2.5 mm卵泡中随采食量水平升高而降低（P<0.05），但其在≥2.5 mm卵泡中则随营养水平的升高而显著升高（P<0.05）；0.5M组试验羊≥ 2.5 mm卵泡细胞PPP途径关键酶G6PDH基因表达显著低于1.0M组和1.5M组（P<0.05）。综上表明，黄体期限饲造成湖羊发情延迟，推测其可能与其卵巢细胞凋亡率的升高，<2.5mm卵泡细胞GLUT4蛋白表达量的降低和HBP途径的增强，≥2.5 mm卵泡细胞中HBP途径的减弱及PPP途径的增强有关。本研究通过分析湖羊发情周期、卵巢组织不同直径卵泡分布、卵巢组织细胞凋亡率；卵泡细胞葡萄糖转运蛋白及葡萄糖代谢通路关键基因表达量随黄体期营养水平的变化规律，丰富黄体期卵泡发育的营养调节机制，为空怀母羊繁殖力调控提供理论依据。

Abstract:

This research was designed to investigate the effect of nutrient treatments during luteal phase on follicle development, apoptotic rate of ovarian cells, protein expression of glucose transporter-4 (GLUT4), and expression levels of genes involved in glucose metabolic pathway, to explore the possible mechanism involved in follicle development response to nutrients. After estrous synchronization with intravaginal progestogen sponges, estrous behavior was detected by vasectomized rams from the second day of pessary removal. The end of estrous behavior was considered to be day 0 of the estrous cycle. All 30 multiparous Hu sheep received a total mixed ration diet based on the feeding standards for maintenance requirement (M) from day 0 to day 6 of estrous cycle, and were randomly divided to 0.5M, 1.5M and 1.0M groups from day 7 to day 14 of estrous cycle. Six ewes from each group were randomly selected to be slaughtered on day 15 of estrous cycle. The remaining ewes from all groups were fed the maintenance diet, and the estrous behavior was examined individually. The results indicated that the feed restriction of 0.5M significantly delayed the estrous cycle (P<0.05), which accompanied with significantly increased and decreased ratio of small follicle (<2.5 mm in diameter) and large follicle (≥ 2.5 mm in diameter) to total follicle (≥ 1.0 mm in diameter), respectively (P<0.05); likewise, the apoptotic rate of ovarian tissue in 0.5M group was greater than those in 1.5M and 1.0M groups (P<0.05; TUNEL method results). GLUT4 were immunolocalization detected in all stages of follicle development of ovarian tissue within each group, meanwhile, within <2.5 mm follicle, the protein expression level of GLUT4 of 1.5M group was significantly greater than that of 0.5M and 1.0M groups (P<0.05). Analysis of expression levels of key genes involved in glucose metabolism pathway(polyol pathway, glycolysis pathway, hexosamine biosynthesis pathway (HBP), and pentose phosphate pathway (PPP) by qRT-PCR showed that, the gene expression level of glutamine:fructose-6-phosphate amidotransferase-2 (GFPT2), the enzyme which catalyzes the rate-limiting enzyme of HBP, were significantly decreased by increasing of feed intake in <2.5 mm follicles (P<0.05), however, the expression levels of GFPT2 in ≥ 2.5 mm follicles were significantly increased by increasing of feed intake (P<0.05). Additionally, the gene expression level of Glucose-6-phosphate dehydrogenase (G6PDH), the enzyme which catalyzes the rate determining step of the PPP, was significantly lower in ≥ 2.5 mm follicles of the 0.5M group than that of 1.0M and 1.5M groups (P<0.05). Overall, the feed restriction during luteal phase prolonged estrous cycle of ewes, this response might be related to the increased of ovarian cell apoptosis rate and decrease of GLUT4 expression, but decreased HBP intensity within <2.5 mm follicles, and also be related to the (decreased HBP and enhanced PPP) intensity within ≥ 2.5 mm follicles, respectively. The current research will enrich the mechanism in follicle development regulated by different nutritional levels, by exploring the changing pattern of estrous cycle, ovarian cell apoptosis, intra-follicular glucose metabolic pathway, to provide a theoretical basis for reproductive improvement for non-pregnant ewes by feeding and management.

中图分类号:

S826.2

聂海涛, 黄欣爱, 刘浩, 郭艺璇, 王子玉, 张艳丽, 万永杰, 樊懿萱, 张国敏, 王锋, 王洁. 黄体期不同营养水平对湖羊卵泡发育及相关葡萄糖代谢途径的影响[J]. 畜牧兽医学报, 2018, 49(5): 942-953.

NIE Hai-tao, HUANG Xin-ai, LIU Hao, GUO Yi-xuan, WANG Zi-yu, ZHANG Yan-li, WAN Yong-jie, FAN Yi-xuan, ZHANG Guo-min, WANG Feng, WANG Jie. Effect of Different Nutrient Levels on Follicle Development and Glucose Metabolic Pathway Involved in Folliculogenesis of Hu Sheep during Luteal Phase[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(5): 942-953.

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