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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=766026
• 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，高环境温度会降低正常和活动的精子和生育能力。 Bos taurus（欧洲/英国）公牛比 Bos indicus（瘤牛）更容易受到影响。该范例是，哺乳动物睾丸在缺氧状态下运行，睾丸温度升高会促进新陈代谢，但血流量不会增加，睾丸会缺氧。然而，我们最近的数据反驳了这一点。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。 有 3 个目标： 目标 1：确定金牛座牛和印度牛座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°C）。我们预计没有无氧代谢，并且公牛的睾丸血流量增加明显较小。 目标 2：确定睾丸温度升高对支持细胞和间质细胞中特定基因靶标的 mRNA 和蛋白质表达的影响。公牛将在阴囊保温之前和之后的不同时间被阉割。将分别使用 GATA4 和 3ßHSD 抗原进行细胞分选 (FACS) 以分离支持细胞和间质细胞。我们将分离 mRNA、创建 cDNA 并进行蛋白质印迹和免疫组织化学以检测目标基因及其蛋白质，以及 RNA 测序（发现方法）。我们预计支持细胞和间质细胞会受到热应激的不同影响（各种基因及其蛋白质的上调或下调）。 目标 3：确定：1) 环境温度和睾丸温度之间的关联； 2）睾丸温度升高对精液质量、受精、发育以及对精子和胚胎的表观遗传影响的影响； 3) 种公牛在温暖环境温度的田间条件下维持精液质量和生育能力的差异。我们将利用肉牛睾丸内的睾丸内传感器监测温度；有些将用于自然繁殖（约 6 周），通过 GPS 测量位置（例如遮荫与非遮荫），并通过小牛出生日期和亲子测试确定公牛性能。我们将通过阴囊隔热来提高睾丸温度。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。睾丸温度升高超过阈值（该阈值因公牛而异）将降低活力、形态和受精能力，并导致精子和胚胎的表观遗传变化。 我的研究项目中产生的新知识将用于开发对睾丸高温的易感性/抵抗力的循证生物标志物，以及预防或减轻对精子发生、精子和生育力的有害影响的方法。该计划将培养训练有素的生殖生物学专业人才，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。