无精子症是至少两次精液检查均未发现精子，约占所有男性1%，其中近1/4由精母细胞发育停滞（SDA)导致。但对人SDA的分子基础和机制，了解甚少。我们假设：精母细胞发育关键基因突变导致了人SDA。为发现人SDA致病突变，中以在精子发生研究中取得了重要成绩且各具优势的研究者组成攻关团队，拟开展以下工作：1.病例鉴定-通过睾丸组织病理和精子发生进程分析，发现精子发生停滞于精母细胞期的无精子患者；2.遗传筛选-用全外显子测序和生物信息学分析发现候选SDA致病突变；3.功能鉴定-用培养生殖细胞和转基因小鼠精子发生分析，鉴定SDA致病突变；4.病理确认-用突变携带者睾丸组织，对SDA致病突变基因及关联分子进行表达定位分析。以期发现并证实人类SDA致病突变、揭示致病机制，为无精子症患者基因诊断、为人工辅助生殖所获胚胎遗传筛查，提供分子标志；发现调节精母细胞发育的新基因、新机制，为生育调节提供新思维。

Azoospermia is defined as the absence of sperm in at least two different ejaculate samples.Approximately, 1% of all men suffer from azoospermia induced infertility, of which about 22.6% are caused by spermatocyte developmental arrest (SDA). However, molecular basis and mechanisms underlying SDA are largely unknown. We hypothesize that the SDA is resulted from mutations of the genes that play critical roles during spermatocyte development. To identify the SDA-causing mutations, a joint research team is formed between Chinese and Israeli scientists with complementary advantages in spermatogenesis studies. We will firstly identify the patients whose spermatogenesis arrests at spermatocyte via the inspection of histological patterns, meiotic progression and chromosome behavior in testicular biopsy of idiopathic azoospermia cases. Then the DNAs from 20 SDA patients identified will be used for whole exome sequencing (WES) followed by extensive bioinformatics analysis to find the candidate SDA-causing genetic variants. The candidate variants will be re-sequenced in the affected SDA cases for accuracy of WES. If they are confirmed to be true, the frequency of mutations for the affected genes will be determined by sequencing all their exons in other 150 unrelated SDA cases, and by high-resolution melting.analysis in 300 normal men. The mutants with a significantly higher frequency in SDA cases than controls will subject to functional analysis by examining spermatocyte development in cultured germ cells conditionally expressing the mutant human CDS, using live cell imaging and spermatocyte micro-spreading techniques. For the mutants functionally confirmed in the above cultured germ cells, transgenic mice will be generated, and used for function study alone, if they are heterozygous and show dominant negative effects in patients, or in combination with the gene knockout mice if the latter are available. The functionally confirmed mutants will be analyzed for molecular mechanisms by which they cause defects using biochemistry and molecular biology approaches. The expression and localization of products of the mutated genes and/or their related molecules will be detected in testicular tissue sections of the SDA cases carrying the mutants. We believe that, using these unique research strategy and technology (genetic screening of well-characterized patients followed by functional analysis), novel mutations, even novel genes and molecular mechanisms underlying SDA will be revealed by this systemic study, which can be used as diagnostic and genetic screening biomarkers for NOA patients and early embryos derived from assistant reproductive technology using testicular sperm from NOA patients.

为发现人精母细胞发育停滞（SDA)的致病突变，我们开展了以下工作：通过睾丸组织病理和精子发生进程分析，发现精子发生停滞于精母细胞期的无精子患者；利用全外显子测序和生物信息学分析筛选了候选SDA致病突变；进而制备了携带患者突变的小鼠模型，并对其精子发生进行了分析，从功能上确定了SDA致病突变；最后对致病突变基因及关联分子进行了生化分子生物学分析等，揭示了致病突变导致精子发生障碍的分子机制。 在项目实施过程中，我们按统一的病例信息和标本收集要求，在国内多家生殖中心收集了200余例SDA样本，并对其中54个SDA患者进行了全外显子深度测序；利用我们建立的精子发生生物信息学分析平台对测序数据进行了分析、注释，发现了72个SDA候选致病突变；进而利用CRISPR/Cas9基因修饰技术，制备了20余种携带患者突变的基因修饰小鼠；对模型小鼠的精子发生减数分裂进行研究，发现了5种基因突变小鼠出现减数分裂异常。如，发现X染色体基因Rpl10的反转座基因Rpl10l（位于常染色体上）是精子发生减数分裂所必需的，但其在精子发生中的作用可以被异位表达的Rpl10替代，为减数分裂性染色体失活的基因功能补偿假说提供了直接证据。对其他SDA致病突变的分子作用机制还在进一步研究中。 在本项目资助下，发表SCI论文12篇；培养博士生8名、硕士生1名，博士后4人。 在项目实施过程中，中-以双方开展了广泛的交流合作，提高了两国在精子发生以及相关疾病发病原因和机制研究领域的国际影响力；项目所取得的相关研究成果也有望为无精子症患者的基因诊断、为人工辅助生殖所获胚胎遗传筛查，提供了潜在的分子标志。