棉酚对大白鼠精子发生影响的银染观察

大白鼠喂服棉酚后,对其附睾精子活动力、精子形态学及睾丸减数分裂制片进行银染观察。对大白鼠一次性喂服大剂量棉酚(400 mg/kg体重),喂后2～5周,精子活动力明显降低,不正常精子频率增加,附睾精子银染显示精子尾部有损伤,包括精子头尾分离、尾部成圈、精子尾部中段部分区域损伤,但精子头部形态正常。服药6周后,附睾精子全部恢复正常。短期体外孵育实验,显似类似结果,但较轻微。喂服棉酚2～4周期间,不同发育阶段精子细胞的嗜银结构消失。实验组睾丸组织切片及睾丸减数分裂制备片,并无异常发现。     

用双色荧光原位杂交检测人精子染色体非整倍体率

目的检测人精子染色体非整倍体率。方法采用双色荧光原位杂交（ＦＩＳＨ）方法,取少量精标本经洗后制片,用二硫苏糖醇（ＤＴＴ）和二碘水杨酸锂（ＬＩＳ）处理,使精子头部染色质去凝集。然后,与生物素标记的α卫星Ｘ染色体特异ＤＮＡ探针（ＤＸＺ１）和地高辛标记的α卫星Ｙ染色体特异ＤＮＡ探针（ＤＹＺ３）进行原位杂交。用ＣＹ３－链亲和素、山羊抗链亲和素检测Ｘ染色体探针杂交信号;用鼠抗地高辛抗体、与荧光素结合的兔抗鼠抗体检测Ｙ染色体探针杂交信号。结果在Ｎｉｋｏｎ荧光显微镜下可以清楚看到精子头部的杂交信号,头部有１个红色荧光杂交信号的精子为Ｘ染色体精子（Ｘ精子）,有１个绿色荧光杂交信号的精子为Ｙ染色体精子（Ｙ精子）。精子头部有２个荧光杂交信号的精子为染色体数目异常精子。若用１条常染色体探针和１条性染色体探针进行ＦＩＳＨ,可以区别头部有２个相同颜色荧光杂交信号的精子属非整倍体精子或二倍体精子。结论双色荧光原位杂交（ＦＩＳＨ）方法,可以用于测定接触致突变剂和非整倍体诱导剂后,人精子染色体非整倍体率的变化。     

环磷酰胺和雷公藤多苷对儿童性腺的远期影响

目的观察口服雷公藤多苷（GTW）和（或）静脉应用环磷酰胺（CTX）对儿童性腺的远期影响。方法回顾性收集1986年1月至2005年3月于首都儿科研究所附属儿童医院住院予GTW口服和（或）CTX静脉冲击治疗的原发性肾病综合征和过敏性紫癜肾炎患儿的资料,对停药后5年以上的患儿进行随访,内容包括开始用药时年龄,用药情况（累积剂量和疗程）,随访时年龄,随访时停药时间,婚育史,男性进行精液检测,女性随访乳房发育、初潮时间和月经史。结果符合纳入标准患儿136例,共随访到47例,其中男性21例,女性26例。①男性随访结果：单独应用GTW者12例,开始用药年龄7～14岁,累积剂量51～174mg·kg^-1,随访时停药9～17年,其中6例已婚育,子女均体健;2例女方有怀孕史;4例未婚者行精液检测示异常精子轻度升高1例,精子活动力略下降2例。单独应用CTX者6例,开始用药年龄3～14岁,累积剂量55～72mg·kg^-1,随访时停药6～17年,其中2例女方均有怀孕史;余4例行精液检测示异常精子轻度升高并精子活动力轻度下降1例。先后应用GTW和CTX者3例,应用间隔时间1d至7个月,精液检测示精子数量减少、精子活动率下降、异常精子升高或活动力下降2例。②女性随访结果：单独应用GTW者19例,开始用药年龄7～14岁,累积剂量36～190mg·kg^-1,随访时停药5～20年;单独应用CTX者6例,开始用药年龄12～15岁,累积剂量32～213mg·kg^-1,随访时停药5～9年;先后应用GTW和CTX者1例,应用间隔时间1年,累积剂量分别为87和160mg·kg^-1,随访时分别停药8和7年。已育者的子女均体健,未婚者月经和乳房发育均正常。结论单独口服GTW或CTX静脉冲击治疗累积量较低时可能对男性性腺的损害较轻,对女性生育、月经和乳房发育未见影响。GTW及CTX连续应用者,应用间隔时间较短可能对男性产生显著的性腺损害,应避免短期内连续应用GTW和CTX。     

Epididymis-specific pathologic disorders in rats exposed to gossypol from weaning through puberty

Previous work in our laboratory revealed that the pubertal period of reproductive development in the male rat was particularly vulnerable to gossypol exposure, with a higher frequency of round structures in the lumen of the cauda epididymidis in the treated rats. Herein, we utilized hemicastration and electron microscopy to confirm that the epididymis is a definitive target of gossypol. Although exposure to gossypol from weaning through puberty caused a significant decrease in daily sperm production, as well as in the concentration of sperm in the epididymis, serum testosterone levels and reproductive organ weights were not altered. In gossypol treated rats, sperm morphology was compromised severely, but the epithelium in testis and epididymis appeared morphologically normal. Ultrastructural examination revealed that round structures, present only in gossypol exposed males, represented: (1) principal cells exfoliated from the epididymal epithelium; (2) epididymal epithelial cell cytoplasm containing degenerating sperm; and (3) degenerating epithelial cells, consisting of vesicles and particles of different sizes, forms and densities. Taken together, the data confirm that gossypol targets the epididymis, disturbing both the structure and function of this organ, and presumably disrupts sperm maturation.     

低剂量棉酚与激素联合应用的抗生育作用位点的研究

目的 探讨低剂量棉酚与激素联合应用在8周内使大鼠达到不育效果的作用位点.方法 本实验采用大鼠喂服棉酚12.5mg/(kg·d) 去氧孕烯125μg/(kg·d) 炔雌醇25μg/(kg·d) 十一酸睾丸酮100mg/(kg·d)的联合用药方式,与单独喂服相同剂量的棉酚或激素的大鼠及喂服载体溶剂的大鼠相对照,通过一系列形态学观察和半定量检测,探讨联合用药的具体作用环节.结果 无论在睾丸组织形态还是睾丸精子数量,联合用药组的变化趋势都与单独应用激素组的相似,而不同于单独应用棉酚组和对照组.而在附睾中,3个用药组在精子活力和精子形态方面,都与对照组有显著差异.结论 联合用药组中激素主要使睾丸精子数量迅速下降;而在精子数量大大下降的基础上,棉酚和激素又对已成熟精子的结构和运动能力进一步破坏,从而使雄性大鼠受精能力迅速下降,联合用药组比单独应用其中一种成分更快、更有效地达到使大鼠不育的目的.     

Oligo‐astheno‐teratozoospermia in mice lacking ORP4, a sterol‐binding protein in the OSBP‐related protein family

Oligo‐astheno‐teratozoospermia (OAT), a condition that includes low sperm number, low sperm motility and abnormal sperm morphology, is the commonest cause of male infertility. Because genetic analysis is frequently impeded by the infertility phenotype, the genetic basis of many of OAT conditions has been hard to verify. Here, we show that deficiency of ORP4, a sterol‐binding protein in the oxysterol‐binding protein (OSBP)‐related protein family, causes male infertility due to severe OAT in mice. In ORP4‐deficient mice, spermatogonia proliferation and subsequent meiosis occurred normally, but the morphology of elongating and elongated spermatids was severely distorted, with round‐shaped head, curled back head or symplast. Spermatozoa derived from ORP4‐deficient mice had little or no motility and no fertilizing ability in vitro. In ORP4‐deficient testis, postmeiotic spermatids underwent extensive apoptosis, leading to a severely reduced number of spermatozoa. At the ultrastructural level, nascent acrosomes appeared to normally develop in round spermatids, but acrosomes were detached from the nucleus in elongating spermatids. These results suggest that ORP4 is essential for the postmeiotic differentiation of germ cells.     

The Hook1 gene is non-functional in the abnormal spermatozoon head shape (azh) mutant mouse

In mice carrying the autosomal recessive mutation 'abnormal spermatozoon head shape' (azh) all spermatozoa display a highly abnormal head morphology that differs drastically from the compact and hook-shaped head of the normal murine sperm. Moreover, the azh mutation causes tail abnormalities often resulting in coiled sperm tails or in the decapitation of the sperm head from the flagellum. We have isolated and characterized murine Hook1 cDNA and analyzed the corresponding genomic structure. Furthermore, the Hook1 gene was mapped to the same region on chromosome 4 to which the azh locus was previously linked. The Hook1 gene is predominantly expressed in haploid male germ cells, and immunohistochemical analysis revealed that Hook1 is responsible for the linkage of the microtubular manchette and the flagellum to cellular structures. Here, we report that the azh mutation is due to a deletion of exons 10 and 11 in the murine Hook1 gene leading to a non-functional protein. Our results indicate that loss of Hook1 function results in ectopic positioning of microtubular structures within the spermatid and causes the azh phenotype. Therefore, the human HOOK1 gene could serve as a candidate gene for male infertility due to teratozoospermia or decapitation defects.     

精子大头多尾畸形与性染色体异常

目的 探讨精子畸形与精子染色体异常的关系,了解畸形精子的病理学改变,方法 应用光学和电子显微镜,性染色体特异性探针荧光原位杂交（FISH）技术研究罕见的大头多尾畸形精子。结果 巴氏染色后观察的精子畸形率达98．75％（油镜下测量头部畸形达100％）,精子多尾率达60．25％（最多达8尾）,电镜观察证实,精子头部表面凹凸不平,核型极不规则,有大量细胞质结构,尾部除数量异常外,尚有中心粒,线粒体和鞭毛结构的异常,FISH结果证实,性染色体多体率为61．4％,与精子多尾的组成比有大致的平行关系。结论 尽管体细胞染色体正常,畸形精子人可伴有严重染色体异常。     

Ultrastructural nuclear defects and increased chromosome aneuploidies in spermatozoa with elongated heads

BACKGROUND: Cellular and molecular mechanisms leading to elongated sperm heads are not known. We have analysed the nuclear status of spermatozoa with elongated heads. METHODS: Fourteen men with at least 30% of spermatozoa with an elongated nucleus were studied and compared with five fertile men as controls. Sperm morphology was analysed by a quantitative ultrastructural analysis. Sperm chromosomal content was assessed by three-colour fluorescence in-situ hybridization (chromosomes X, Y, 18). Y chromosome microdeletion and karyotype were analysed. RESULTS: Elongated sperm head rates of the patients were 46.9% (30-75 versus 0-2% in the control group) by light microscopy and 34.4% by electron microscopy. In all patients, the chromatin was poorly condensed in elongated sperm heads (50% of elongated nuclei). No anomalies of sperm biochemical markers were found. All the men showed normal karyotype (46,XY) and absence of Y chromosome microdeletion. Aneuploidy rates of gonosomes and chromosome 18 were significantly increased in patients (1.64- and 3.6-fold, P = 0.006 and 0.026, respectively). CONCLUSIONS: This study demonstrates that impaired chromatin compaction and slightly increased chromosome aneuploidies are found in spermatozoa with an elongated head, suggesting possible mechanisms such as meiotic non-disjunctions or spermiogenesis anomalies.     

Gossypol induces DNA strand breaks in human fibroblasts and sister chromatid exchanges in human lymphocytes in vitro.

The male contraceptive agent gossypol was found to induce a dose related increase of DNA strand breaks in human fibroblasts in vitro at concentrations of 5 to 40 micrograms/ml. The effect was reduced in the presence of 2% fetal calf serum. A weak but reproducible increase in the SCE frequency was found in human lymphocytes treated for 1 hour in serum-free medium with 0.04 to 4 micrograms/ml of gossypol.