Pathogenesis of heat-induced infertility in mammals

哺乳动物热致不孕症的发病机制

• 批准号: RGPIN-2019-04823
• 负责人: Kastelic, John
• 金额: $ 2.4万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=737884
• 关键词: Pathogenesis; heat; induced; infertility; mammals

In male mammals, high ambient temperatures reduce normal and motile sperm and fertility. Bos taurus (European/British) bulls are more susceptible than Bos indicus (zebu). The paradigm is that mammalian testes operate near hypoxia, increased testicular temperature increases metabolism, but not blood flow and testes become hypoxic. However, our recent data refute that. My overall hypothesis is that deleterious effects of increased testicular temperature are primarily due to hyperthermia and not hypoxia. There are 3 objectives: Objective 1: Determine effects of increased testicular temperature in B. taurus and B. indicus bulls on testicular blood flow and oxygenation. Testicular temperatures will be serially increased (34 to 40ºC). We expect no anerobic metabolism and B. indicus bulls to have a significantly smaller increase in testicular blood flow. Objective 2: Determine effects of increased testicular temperature on mRNA and protein expression of specific gene targets in Sertoli and Leydig cells. Bulls will be castrated before and varying times after scrotal insulation. Cell-sorting (FACS) will be done to isolate Sertoli and Leydig and cells, using GATA4 and 3ßHSD antigens, respectively. We will isolate mRNA, create cDNA and do western blotting and immunohistochemistry to detect target genes and their proteins, as well as RNA sequencing (discovery approach). We expect Sertoli and Leydig cells to be differentially affected by heat stress (up- or down-regulation of various genes and their proteins). Objective 3: Determine: 1) associations between environmental and testicular temperatures; 2) effects of increased testicular temperature on semen quality, fertilization, development and epigenetic effects on sperm and embryos; and 3) differences among breeding bulls in ability to maintain semen quality and fertility under field conditions with warm ambient temperatures. We will monitor temperature with intratesticular sensors in testes of beef bulls; some will be used for natural breeding (~6 wk), with location (e.g. shade versus non-shade) measured with GPS and sire performance determined by calf birth dates and parentage testing. We will increase testicular temperature with scrotal insulation. Semen will be collected, assessed and used for in vitro fertilization. We expect testicular temperatures will be directly affected by ambient temperatures. Increases in testicular temperature beyond a threshold (which will vary among bulls) will reduce motility, morphology and fertilizing ability and cause epigenetic changes in sperm and embryos. New knowledge generated in my research program will be used to develop evidence-based biomarkers of susceptibility/resistance to testicular hyperthermia and ways to prevent or mitigate deleterious effects on spermatogenesis, sperm and fertility. This program will generate well-trained HQP specialized in reproductive biology, with a broad range of skills, preparing them for careers in academia, industry and elsewhere.

在雄性哺乳动物中，较高的环境温度会降低正常和能动的精子和生育力。牛(欧洲/英国)公牛比野牛(斑马)更容易受到影响。这种模式是，哺乳动物的睾丸在低氧附近运行，睾丸温度升高会增加新陈代谢，但不会导致血液流动，睾丸会变得缺氧。然而，我们最近的数据驳斥了这一点。我的总体假设是，睾丸温度升高的有害影响主要是由于体温过高，而不是缺氧。目标1：确定金牛和野牛睾丸温度升高对睾丸血流和氧合的影响。睾丸温度将连续升高(34至40摄氏度)。我们预计不会有厌氧代谢，而且公牛睾丸血流的增加幅度会小得多。目的：探讨睾丸温度升高对支持细胞和间质细胞特异性基因靶点基因表达的影响。公牛将在阴囊绝缘前后不同的时间被阉割。细胞分选(FACS)将分离支持细胞和间质细胞，分别使用GATA4和3？HSD抗原。我们将分离信使核糖核酸，构建cDNA，并进行免疫印迹和免疫组织化学检测目的基因及其蛋白，以及RNA测序(发现法)。我们预计支持细胞和间质细胞将受到不同的热应激(各种基因及其蛋白质的上调或下调)的影响。目的3：确定：1)环境温度与睾丸温度之间的关系；2)睾丸温度升高对精液质量、受精、发育的影响以及对精子和胚胎的表观遗传效应；3)在环境温度较高的野外条件下，种公牛保持精液质量和生育能力的差异。我们将使用睾丸内传感器监测公牛睾丸的温度；其中一些将用于自然繁殖(~6wk)，位置(例如阴凉处与非阴凉处)通过GPS测量，公牛性能由出生日期和亲子鉴定确定。我们会用阴囊绝缘体来提高睾丸温度。精液将被收集、评估并用于体外受精。我们预计睾丸温度将直接受到环境温度的影响。如果睾丸温度超过某一临界值(不同的公牛会有所不同)，将降低精子的活力、形态和受精能力，并导致精子和胚胎的表观遗传学变化。在我的研究计划中产生的新知识将被用于开发基于证据的生物标记物，用于检测睾丸过热的敏感性/抵抗力，以及预防或减轻对精子发生、精子和生育能力的有害影响的方法。该计划将培养出训练有素的生殖生物学专业HQP，拥有广泛的技能，为他们在学术界、工业界和其他地方的职业生涯做好准备。