背景:非梗阻性无精子症(NOA)是男性不育的最严重形式,影响全球约1%的男性。范可尼贫血(FA)基因因其在DNA修复中的重要作用而闻名,越来越多的证据表明FA途径在NOA中的关键作用。然而,Fance缺乏导致男性生殖细胞严重缺陷和延迟成熟的潜在机制尚不清楚。
方法:我们使用Fance缺乏小鼠模型进行实验,并在8周(8W)时收集小鼠的睾丸或附睾,术后17.5天(dpc),产后11(P11)至P23。小鼠涉及三种基因型:野生型(Fance+/+),杂合(Fance+/-),和纯合(Fance-/-)。苏木精和伊红染色,免疫荧光染色,并进行精母细胞表面扩散,以探讨Fance-/-小鼠NOA的机制。每个实验最少进行三次生物学重复,并使用带有Dunn's校正的Kruskal-Wallis进行统计分析。
结果:在本研究中,我们发现,成年雄性Fance-/-小鼠在生精小管中表现出大量的生殖细胞丢失,在附睾中精子急剧减少。在胚胎期,Fance-/-繁殖质的数量明显减少,不影响增殖(Ki-67,PCNA)和凋亡(切割的PARP,裂解的胱天蛋白酶3)状态。DNA双链断裂(γH2AX)在Fance-/prospon-matogonia的细胞水平上增加,可能与非同源末端连接(53BP1)增加和同源重组(RAD51)活性降低有关。此外,Fance缺乏阻碍了精母细胞减数分裂前期I的进展。该机制需要减少瘦素上DNA末端切除蛋白RPA2的募集,并在合子上重组酶RAD51和DMC1。它还涉及受精素处的RPA2和粗线质处的FANCD2病灶的去除受损。在早期的粗线烯加速了交叉的初始形成,用MLH1表示。
结论:Fance缺乏导致大量雄性生殖细胞丢失,涉及繁殖体中DNA损伤修复的失衡和同源重组中蛋白质动力学的改变,DNA末端切除,和交叉,为NOA的病因和分子基础提供新的见解。
BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe form of male infertility and affects approximately 1% of men worldwide. Fanconi anemia (FA) genes were known for their essential role in DNA repair and growing evidence showed the crucial role of FA pathway in NOA. However, the underlying mechanisms for Fance deficiency lead to a serious deficit and delayed maturation of male germ cells remain unclear.
METHODS: We used Fance deficiency mouse model for experiments, and collected testes or epididymides from mice at 8 weeks (8W), 17.5 days post coitum (dpc), and postnatal 11 (P11) to P23. The mice referred to three genotypes: wildtype (Fance +/+), heterozygous (Fance +/-), and homozygous (Fance -/-). Hematoxylin and eosin staining, immunofluorescence staining, and surface spread of spermatocytes were performed to explore the mechanisms for NOA of Fance -/- mice. Each experiment was conducted with a minimum of three biological replicates and Kruskal-Wallis with Dunn\'s correction was used for statistical analysis.
RESULTS: In the present study, we found that the adult male Fance -/- mice exhibited massive germ cell loss in seminiferous tubules and dramatically decreased sperms in epididymides. During the embryonic period, the number of Fance -/- prospermatogonia decreased significantly, without impacts on the proliferation (Ki-67, PCNA) and apoptosis (cleaved PARP, cleaved Caspase 3) status. The DNA double-strand breaks (γH2AX) increased at the cellular level of Fance -/- prospermatogonia, potentially associated with the increased nonhomologous end joining (53BP1) and decreased homologous recombination (RAD51) activity. Besides, Fance deficiency impeded the progression of meiotic prophase I of spermatocytes. The mechanisms entailed the reduced recruitment of the DNA end resection protein RPA2 at leptotene and recombinases RAD51 and DMC1 at zygotene. It also involved impaired removal of RPA2 at zygotene and FANCD2 foci at pachytene. And the accelerated initial formation of crossover at early pachytene, which is indicated by MLH1.
CONCLUSIONS: Fance deficiency caused massive male germ cell loss involved in the imbalance of DNA damage repair in prospermatogonia and altered dynamics of proteins in homologous recombination, DNA end resection, and crossover, providing new insights into the etiology and molecular basis of NOA.