Pathogenesis of heat-induced infertility in mammals

在雄性哺乳动物中，较高的环境温度会降低正常的、活动的精子和生育能力。牛（欧洲/英国）比牛（zebu）更易受感染。一般认为，哺乳动物的睾丸在缺氧状态下工作，睾丸温度升高会增加新陈代谢，但血液流量不会增加，睾丸就会缺氧。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。目的1：确定升高的金牛和印度公牛睾丸温度对睾丸血流量和氧合的影响。睾丸温度会逐渐升高（34 ~ 40℃）。我们预计没有无氧代谢和公牛公牛的睾丸血流量有明显较小的增加。目的2：确定睾丸温度升高对睾丸支持细胞和间质细胞特定基因靶点mRNA和蛋白表达的影响。公牛将在阴囊绝缘之前和之后的不同时间被阉割。细胞分选（FACS）将分别使用GATA4和3ßHSD抗原分离Sertoli和Leydig和细胞。我们将分离mRNA，创建cDNA，并进行western blotting和免疫组织化学检测靶基因及其蛋白，以及RNA测序（发现方法）。我们预计支持细胞和间质细胞受到热应激的不同影响（上调或下调各种基因及其蛋白质的调节）。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)不同种公牛在温暖环境条件下保持精液质量和生育能力的差异。我们将利用肉牛睾丸内的传感器监测温度；一些将用于自然育种（~6周），用GPS测量位置（例如荫蔽处与非荫蔽处），并通过小牛出生日期和亲子鉴定确定父系表现。我们会用阴囊保温来提高睾丸温度。精液将被收集、评估并用于体外受精。我们预计睾丸温度会直接受到环境温度的影响。睾丸温度升高超过某一阈值（不同公牛的睾丸温度有所不同），会降低活动性、形态和受精能力，并导致精子和胚胎的表观遗传变化。在我的研究项目中产生的新知识将用于开发睾丸高温易感性/抗性的循证生物标志物，以及预防或减轻对精子发生、精子和生育能力的有害影响的方法。该项目将培养训练有素的生殖生物学专业HQP，具备广泛的技能，为他们在学术界、工业界和其他地方的职业生涯做好准备。

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.