牛源犬新孢子虫对雄性小鼠睾丸的损伤机制研究

Neospora caninum mainly causes abortions in dams, fetal death, neonatal fetal movement disorders, and nervous system diseases. However, reproductive damage caused by N.caninum in the male is unknown. The objective of present study is to determine the effect of the parasites on the male testis damage on the basis of establishing animal models that male BABL/c mice infected with N.caninum. In the present study, TEM, gene chip, FCM, TUNEL, qRT-PCR and IHC were carried out to measure the pathological damage of mouse testis, apoptosis detection of testis sperm gene and oxidative gene. To certificated testis damage caused by N.caninum, sperm morphology, density and activity analysis, parasite in sperm, anti-sperm antibody and the level of NO were also detected. In the other hand, a total of 7 kinds of hormone levels including GnRH, FSH and T, were measured using RIA method to determine the effect of N.caninum on the function of hypothalamic-pituitary- testicular axis. Finally, to reveal the mechanism of testis damage caused by bovine N.caninum in male mice, the breeding test was performed to record the developmental index and infection of F1 offspring, pregnancy rate and survival rate. This project will settle the solid foundation for the pathogenic role of N.caninum.

犬新孢子虫能够引起孕畜流产、死胎、新生儿运动神经系统障碍，但犬新孢子虫对雄性生殖系统的损伤作用目前尚不清楚。为确定犬新孢子虫对雄性小鼠睾丸的损伤机制，本研究在建立牛源犬新孢子虫雄性BABL/c小鼠动物模型的基础上，拟应用透射电镜、基因芯片、FCM、TUNEL、qRT-PCR及IHC等技术对小鼠睾丸进行病理学损伤观察，睾丸生精功能相关基因、氧化凋亡相关基因及细胞凋亡检测，对睾丸产生精子进行形态学、密度及活性分析，精液中虫体、抗精子抗体及NO水平检测，阐明犬新孢子虫对小鼠睾丸及其精子的损伤作用；采用RIA等方法检测GnRH、FSH、T等七种激素水平，确定犬新孢子虫对小鼠下丘脑－垂体－睾丸轴的影响；通过繁殖试验，统计雌鼠受孕率、活胎率等，检测F1代仔鼠发育指标和染虫情况，明确犬新孢子虫对雄鼠生育能力的影响，进而揭示牛源犬新孢子虫对小鼠睾丸的损伤机制。以期为犬新孢子虫致病作用的研究奠定基础。

新孢子虫病(Neosporiasis)是由犬新孢子虫(Neospora caninum)引起多种家畜的一种原虫病。该病主要引起孕畜流产、死胎或新生儿运动障碍和神经系统疾病，尤其犬新孢子虫对雄性生殖系统的损伤作用尚不清楚。为确定犬新孢子虫对雄性小鼠睾丸的损伤机制，本研究在建立牛源犬新孢子虫雄性BALB/c小鼠动物模型基础上，开展了牛源犬新孢子虫对雄性小鼠睾丸组织细胞的损伤观察，牛源犬新孢子虫对雄性小鼠精子质量、下丘脑－垂体－睾丸轴以及生育能力的影响等研究。结果证实，牛源犬新孢子虫感染能够引起雄性小鼠各级生精细胞精子数量明显减少，多数生精细胞排列紊乱，损伤睾丸组织细胞，造成少精、无精现象；牛源犬新孢子虫感染造成精子密度、精子活力降低，精子畸形数增加，导致睾丸及其精子的损伤；牛源犬新孢子虫感染可造成雄性小鼠的激素水平分泌紊乱，影响下丘脑-垂体-睾丸轴的功能；牛源犬新孢子虫感染雄性小鼠造成合笼后雌鼠的受孕率和活胎率降低，死胎率升高，F1代仔鼠的发育指标降低，严重影响雄性小鼠的生育能力。表明牛源犬新孢子虫感染可造成雄性小鼠睾丸和精子的损伤，严重影响生殖功能。本研究为犬新孢子虫致病机制研究奠定了基础。