【作者】 李纯锦；

【导师】 周虚；

【作者基本信息】 吉林大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 本研究系统探索了BDNF、NGF及其受体在牛成熟精子中的表达及对精子功能的影响,并且研究了BDNF、NGF和TrkA在人成熟精子中的表达及它们与男性不育之间的关系。结果表明BDNF、TrkB、TrkA的mRNA均存在于牛的成熟精子中,而NGFmRNA却没有检测到;BDNF、NGF、TrkB、TrkA的蛋白均在牛的成熟精子中检测到,并且分布在精子不同的部位,同时发现精子自身能分泌BDNF。BDNF/TrkB、NGF/TrkA可以显著影响精子胰岛素和瘦素的分泌。BDNF/TrkB能影响精子活率、线粒体活性、凋亡和精子内钙离子浓度。NGF/TrkA能影响精子活率和凋亡。BDNF、TrkA的mRNA和蛋白均在人成熟精子中检测到。BDNF和TrkA的mRNA在弱精少精男性精子中的含量显著低于正常生育男性精子中的含量;弱精少精男性精浆中BDNF浓度显著低于弱精和正常生育男性中的浓度,虽然弱精少精男性精浆中NGF的浓度低于弱精和正常生育男性中的浓度,但差异不显著。不育男性和正常男性中BDNF基因的G196A和C270T基因型分布均符合Hardy–Weinberg平衡规律。不育男性与正常男性中BDNF基因G196A和C270T基因型频率和等位基因频率差异不显著。更多还原

【Abstract】 Brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), two members of neurotrophins, are not only required for the survival and differentiation of the nervous system but also important for the development of reproductive tissues. Recent studies showed that BDNF and NGF could regulate oocyte maturation and development of early embryo though acting on TrkB, TrkA, and also affect the reproductive actions by interacting with some steroid hormones. Until now, the expression of BDNF, NGF and their receptors had been detected in animals’testis, indicating the potential roles of neurotrophins in the testis maturation and spermatogenesis. Previous studies showed that there were no transcriptional actions in ejaculated sperm, because sperm was a terminally differentiated cells with highly condensed nucleus. With the presence of advanced technologies and specialized study, the theory had been doubted.The present study detected the expression of BDNF, NGF, and their receptor TrkB, TrkA, systematically studied the effects of BDNF and NGF on physiological functions of sperm, and investigated the relationship between BDNF, NGF and male infertility. The study would establish a foundation for demonstrating the roles BDNF and NGF in male reproduction and further learning the physiological actions of ejaculated sperm. The principal results were described as following:1. Expression and affects of BDNF, NGF and their receptors on physiological functions in bovine ejaculated spermatozoaAccording to the study that ejaculated sperm was a terminally differentiated cells with no transcriptional contents in nucleus, the study extracted the RNAs from bovine ejaculated spermatozoa by swim-up technology and micro-RNA extraction technology, and investigated the presence of mRNAs and proteins of BDNF, NGF and their receptors in bovine ejaculated spermatozoa with RT-PCR, Western Blot, and Immunofluorescence. The results showed that both mRNA and protein of BDNF, TrkB and TrkA were detected in bovine spermatozoa. However, only NGF protein was detected in bovine spermatozoa; immunolocalization of BDNF protein was mainly in the head, neck and tail of bovine spermatozoa, NGF protein was in the head and tail, TrkB protein was in the acrosome and tail, TrkA was in the acrosomal cap, nucleus, and tail. Secretion of BDNF from sperm increased in a sperm concentration dependent manner. Linear regression analysis showed that the correlation coefficient was 0.96. The time-course studies showed that BDNF concentration in incubation media was different, indicating that bovine ejaculated spermatozoa could secret BDNF by themselves.When bovine spermatozoa were treated with exogenous BDNF, the productions of insulin and leptin by sperm increased (P<0.01); Secretion of leptin increased when the exogenous NGF was added into the incubation media (P<0.01). However, the secretions of insulin and leptin were decreased when sperm were exposed to K252a which can inhibit tyrosine phosphorylation of TrkA and TrkB (P<0.05). Although the viability and mitochondrial activity became higher when adding exogenous BDNF, there were no statistical difference (P>0.05). However, NGF could increase the sperm viability (P<0.01). The viability and mitochondrial activity of sperm decreased when sperm were treated with K252a (P<0.05); BDNF and NGF could significant affect the apoptosis of sperm (P<0.05); Both intracellular calcium level and acrosome reaction of bovine sperm were not changed with treatment of exogenous NGF (P>0.05), however, BDNF could increase the intracellular calcium level in sperm (P<0.05).2. Expression profiles of BDNF and NGF in spermatozoa and seminal plasma from infertile and fertile manAccording to the important roles of BDNF and NGF in sperm function, the study investigated the relationship between BDNF and NGF and male infertility. Both mRNA and protein of BDNF were detected in human ejaculated spermatozoa, and BDNF protein was mainly in the head, neck, and tail of sperm. Due to the absence of NGF mRNA in spermatozoa, TrkA expression profile was studied. The mRNA and protein of TrkA were detected in human ejaculated spermatozoa, and TrkA protein mainly located in the head, neck, and middle piece of sperm. Real-time PCR showed that the level of BDNF and TrkA mRNA in spermatozoa from oligoasthenozoo- spermic sample significantly lower than those in spermatozoa from fertile sample (P<0.05); A significant decrease of BDNF concentration in seminal plasma was observed in male subjects with oligoasthenoteratozoospermia compared to those of fertile male subjects(P<0.05) and asthenozoospermic male subjects (P<0.05). Comparing to fertile semens, seminal plasmal NGF decreased in oligoasthenozoosp- ermic and asthenozoospermic semens, but the differences were not statistically significant (P>0.05). These data showed that the decrease in the transcript and protein of BDNF, NGF and TrkA in the oligoasthenozoospermic group may be associated with pathogenesis in some types of male infertility.3. BDNF polymorphisms and male infertilityThe study determined the genotype distribution of BDNF gene G196A and C270T in infertile group and fertile group with restriction fragment length polymorphism. The results showed that two polymorphisms of G196A and C270T were in Hardy-Weinberg equilibrium for both fertile and infertile group (P>0.05). For G196A and C270T polymorphism, there were no significant differences in the genotype distribution (P>0.05) and allele frequencies (P>0.05) between fertile and infertile group. When infertile group were divided into oligoasthenozoospermic group and asthenozoospermic group, there were no significant differences in the genotype distribution(P>0.05) and allele frequency (P>0.05) between oligoasthenozoospermic group and asthenozoospermic group. The BDNF gene G196A and C270T polymorphism had no significant difference between asthenozoospermic group and fertile group in either the genotype (P>0.05) or the allele frequency (P>0.05). There was also no significant difference in the genotype distribution (P>0.05) and allele frequencies (P>0.05) between oligoasthenozoospermic group and fertile group. These findings suggest that BDNF gene G196A and C270T polymorphisms did not play a major role in the male infertility in the studied groups.In a word, the study demonstrated that the mRNAs of BDNF, TrkB, and TrkA also existed in ejaculated spermatozoa, and bovine ejaculated spermatozoa could secret the BDNF by themselves; the study suggested that BDNF and NGF could be important to secretion of insulin and leptin, viability, mitochondrial activity, apoptosis, and intracellular calcium level of bovine sperm; There were different expression profiles of BDNF and NGF in spermatozoa and seminal plasma from fertile and infertile men; there were no significant association between BDNF gene G196A and C270T polymorphisms and male infertility.更多还原