de Boer P, Giele M, Lock M T W T, de Rooij D G, Giltay J, Hochstenbach R, te Velde E R
Laboratory of Genetics, Wageningen Institute of Animal Sciences, ZODIAC, Wageningen, The Netherlands.
Cytogenet Genome Res. 2004;105(1):36-46. doi: 10.1159/000078007.
We have developed a protocol for the identification of aberrant chromosome behavior during human male meiosis up to metaphase of the secondary spermatocyte. Histological evaluation by the Johnsen score of a testicular biopsy was combined with immunofluorescence of first meiotic prophase spermatocytes, using antibodies against synaptonemal complex protein 3 (SYCP3) and the product of the ataxia telangiectasia and rad3-related gene (ATR). This combination enables accurate meiotic prophase substaging and the identification of pachytene spermatocytes with asynapsis. Furthermore, we also investigated the competence of late pachytene primary spermatocytes to complete the first meiotic division up to metaphase and of secondary spermatocytes to transform into metaphase by an in vitro challenge with okadaic acid (OA). We tested this protocol on five males with normal Johnsen scores that presented with obstructive azoospermia, five males with low Johnsen scores and non-obstructive azoospermia and six vasectomized control males of proven fertility and normal Johnsen scores. In all azoospermics, the profiling of meiotic prophase stages by immunofluorescence increases the resolving power of the Johnsen score. In both obstructive and non-obstructive azoospermic patients, relatively more leptotene meiotic prophase stages were counted compared to the controls. In non-obstructive azoospermics, a marked heterogeneity in spermatogenesis was found, after combining the results of all three approaches, pointing at functional mosaicism of the germinal epithelium. Asynaptic pachytene spermatocytes were rarely encountered. Also, when first meiotic metaphase could be induced by OA, chiasma counts were normal. In none of the non-obstructive azoospermic males did the pattern of spermatogenesis resemble that of knock-out mouse azoospermics. We conclude that this combined histological and cytological approach enables a detailed phenotypic classification of infertile males, at a level comparable to that applied for male-sterile knock-out mice with a meiotic defect. This may facilitate the identification of candidate genes for human male infertility.
我们已经开发出一种方案，用于鉴定人类男性减数分裂过程中直至次级精母细胞中期的异常染色体行为。通过睾丸活检的约翰森评分进行组织学评估，并结合对第一次减数分裂前期精母细胞的免疫荧光分析，使用针对联会复合体蛋白3（SYCP3）和共济失调毛细血管扩张症及Rad3相关基因（ATR）产物的抗体。这种结合能够实现减数分裂前期的准确分期，并识别出存在联会异常的粗线期精母细胞。此外，我们还通过用冈田酸（OA）进行体外刺激，研究了粗线期末期初级精母细胞完成第一次减数分裂直至中期的能力，以及次级精母细胞转化为中期的能力。我们在5名约翰森评分正常但患有梗阻性无精子症的男性、5名约翰森评分低且患有非梗阻性无精子症的男性以及6名经证实具有生育能力且约翰森评分正常的输精管结扎对照男性身上测试了该方案。在所有无精子症患者中，通过免疫荧光对减数分裂前期阶段进行分析提高了约翰森评分的分辨能力。与对照组相比，梗阻性和非梗阻性无精子症患者中计数到的细线期减数分裂前期阶段相对更多。在非梗阻性无精子症患者中，综合所有三种方法的结果后，发现精子发生存在明显的异质性，这表明生精上皮存在功能镶嵌现象。很少遇到联会异常的粗线期精母细胞。此外，当OA能够诱导第一次减数分裂中期时，交叉计数正常。在任何一名非梗阻性无精子症男性中，精子发生模式都与敲除小鼠无精子症的模式不同。我们得出结论，这种组织学和细胞学相结合的方法能够对不育男性进行详细的表型分类，其水平与应用于具有减数分裂缺陷的雄性不育敲除小鼠的水平相当。这可能有助于识别人类男性不育的候选基因。
