Novel phenotype of mouse spermatozoa following deletion of nine β-defensin genes

β-defensin peptides are a large family of antimicrobial peptides. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. Despite their inducible presence at mucosal surfaces, their main site of expression is the epididymis. Recent evidence suggests that a major function of these peptides is in sperm maturation. In addition to previous work suggesting this, work at the MRC Human Genetics Unit, Edinburgh, has shown that homozygous deletion of a cluster of nine β-defensin genes in the mouse results in profound male sterility. The spermatozoa derived from the mutants had reduced motility and increased fragility. Epididymal spermatozoa isolated from the cauda region of the homozygous mutants demonstrated precocious capacitation and increased spontaneous acrosome reactions compared with those from wild-types. Despite this, these mutant spermatozoa had reduced ability to bind to the zona pellucida of oocytes. Ultrastructural examination revealed a disintegration of the microtubule structure of mutant-derived spermatozoa isolated from the epididymal cauda region, but not from the caput. Consistent with premature acrosome reaction and hyperactivation, spermatozoa from mutant animals had significantly increased intracellular calcium content. This work demonstrates that in vivo β-defensins are essential for successful sperm maturation, and that their disruption alters intracellular calcium levels, which most likely leads to premature activation and spontaneous acrosome reactions that result in hyperactivation and loss of microtubule structure of the axoneme. Determining which of the nine genes are responsible for the phenotype and the relevance to human sperm function is important for future work on male infertility.     

Hyperactivation of capacitated human sperm correlates with the zona pellucida-induced acrosome reaction of zona pellucida-bound sperm

BACKGROUND: The aim of this study was to determine the relationship between human sperm hyperactivation (HA), sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction (AR) of ZP-bound sperm in vitro. METHODS: Sperm samples from 129 infertile men were studied. Motile sperm (2 x 10(6)) selected by Pure sperm were incubated with four oocytes in 1 ml human tubal fluid supplemented with 10% human serum. After 2-h incubation, the number of sperm bound to the ZP and the AR of ZP-bound sperm were examined. Velocities and HA of sperm in insemination medium were assessed by Hamilton-Thorn Sperm Analyzer. RESULTS: The HA was highly correlated with the ZP-induced AR in all the subjects (rho = 0.626, P < 0.001). In the 69 men with < or = 100 sperm bound/ZP, allowing accurate counts, HA was not significantly correlated with sperm-ZP binding. Men with <7% HA sperm were more likely to have very low ZP-induced AR. Only normal sperm morphology was significantly correlated with sperm-ZP binding (rho = 0.346 and 0.446 in semen and insemination medium, respectively, both P < 0.001). Sperm motility and velocities were significantly correlated with sperm morphology but not with either sperm-ZP binding or the ZP-induced AR. CONCLUSIONS: The correlation of HA with the ZP-induced AR of ZP-bound sperm suggests a mechanistic link between HA and the physiological AR in humans. Assessment of HA of capacitated sperm in vitro may be a useful clinical test for male infertility associated with defective ZP-induced AR that does not require the use of human oocytes.     

Varicocele: effect on sperm functions

Despite the numerous studies published over the past decade, the role of varicocele in male infertility is still controversial. Although more frequent in infertile men, its influence on sperm production or function has not, as yet, been determined. Moreover, the exact mechanism of varicocele action is not clear. We have surveyed the literature, the correlation of varicocele to sperm parameters and to sperm function tests, such as binding capacity, hypo-osmotic swelling test, presence of reactive oxygen species, and in particular, the correlation to fertility potential. Almost every subject examined had contradictory results. Larger control studies may possibly elucidate and clarify the cases in which varicocele is associated to sperm function, and where treatment may improve fertility.     

中医药治疗男性免疫性不育症及顶体酶异常研究进展

精子膜表面存在的抗精子抗体及精子顶体酶异常是男性不育的重要原因,但其病因十分复杂,大部分患者原因不明,现代医学缺乏现成的、可选择的经验性药物,因此临床疗效不甚理想,且不良反应较多。中医药在该方面有一定的潜力,可在不同环节和机制上发挥不同的作用,近年来已有不少学者进行相关研究工作,并取得了较好的疗效。     

The complement regulatory proteins CD55 (decay accelerating factor) and CD59 are expressed on the inner acrosomal membrane of human spermatozoa as well as CD46 (membrane cofactor protein)

The complement regulatory proteins CD55 and CD59 are expressed on the plasma membrane of human spermatozoa, whereas CD46 is only on the inner acrosomal membrane (IAM) which becomes surfaced exposed after the acrosome reaction when sperm assume fertilisation-competence. CD55 & CD59, two glycosylphosphatidylinositol (GPI)-anchored proteins, have been detected previously in some studies also in the acrosomal region of chemically fixed spermatozoa but never demonstrated at this site on unfixed spermatozoa. Dual labelling immunofluorescence and confocal microscopy on fresh unfixed spermatozoa, with minimal subsequent time to fixation, has shown CD55 to be markedly expressed on the IAM, more than on the plasma membrane. However, unlike for CD46, CD55 displayed patchy staining over the acrosome, with some variation between individual spermatozoa. All IAM-associated CD55 was localised within GM1-containing lipid rafts. CD59 was expressed also on the IAM, but in a pronounced granular pattern with more variation observed from one spermatozoa to another. Both CD55 & CD59 were released from the IAM by PI-PLC, demonstrating them to be GPI-anchored. Analysis of acrosome-reacted spermatozoal CD55 by Western blotting revealed a novel single 55 kDa protein lacking significant oligosaccharides susceptible to glycosidases. Antibody-induced membrane rafting and release of CD55 & CD59 in vitro may have influenced previous results. Significant coexpression of CD55 & CD46 on the IAM suggests some functional cooperation at this site.     

PRESENCE OF N-ETHYL MALEIMIDE SENSITIVE FACTOR (NSF) ON THE ACROSOME OF MAMMALIAN SPERM

We have extended previous observations to show that the ATPase N-ethyl maleimide sensitive factor (NSF) an important regulator of membrane trafficking and fusion in somatic cells, is present on bovine, murine and rhesus macaque sperm. However, NSFs main effectors, alfa- and beta-SNAP, although present in the developing acrosome, could not be detected in the mature organelle. The fact that NSF localizes mainly to the acrosome suggests that this protein, together with other factors such as rabs and SNAREs, may be a common feature in the triggering/regulation of membrane merging during the mammalian acrosome reaction.     

精浆转铁蛋白含量与不育的关系

为探讨精浆转铁蛋白 (Tf)含量与男性生育力的关系 ,采用速率散射比浊法 ,对 2 0例正常生育男性和 96例不育男性精浆Tf含量进行了测定 ,同时做精子密度、精子活动率及精子顶体完整率 (PIA)分析。结果表明少精子组 (精子密度 <2 0× 10 6/ml)精浆Tf含量较生育组与正常密度组 (精子密度≥ 2 0× 10 6/ml)显著低 (P <0 .0 1) ;精浆Tf含量与精子活动率无关 ;正常精子密度的不育患者精子PIA <80 %组精浆Tf的含量显著低于PIA≥ 80 %组(P <0 .0 1)。提示精浆Tf含量的下降与男性生育力有关     

精子形态学分析中染色方法的比较

目的：评价改良巴氏染色法、瑞-吉染色法和考马斯亮蓝染色法对形态正常精子率和顶体完整率检测结果影响。方法：分别采用改良巴氏染色法、瑞-吉染色法和考马斯亮蓝染色法对55例男性不育症患者精液涂片染色,计算形态正常精子率和顶体完整率。结果：①3种染色方法检测的形态正常精子率两两比较,差异均无显著性(P>0.05),瑞-吉染色法评价颈部和中段缺陷精子率及尾部缺陷精子率显著低于改良巴氏染色法和考马斯亮蓝染色法（P<0.05）。②3种染色方法检测的Ⅰ型、Ⅳ型顶体完整率两两比较,差异均无显著性 (P>0.05),改良巴氏染色法检测的Ⅱ型和Ⅲ型顶体完整率明显高于考马斯亮蓝染色法和瑞-吉染色法（P<0.05）。③3种染色方法检测的形态正常精子率间均呈显著正相关关系(r=0.535、P<0.01, r=0.601、P<0.01,r=0.329、P<0.05)。3种染色方法检测的顶体完整率间均呈显著正相关关系（r=0.604,P<0.01, r=0.682、P<0.01, r=0.446、P<0.01）。结论：3种染色方法均可作为检测人精子形态和顶体完整率实用方法,其中改良巴氏染色法更适于精子形态学分析,瑞-吉染色法更适于顶体完整率判读。