目的:非梗阻性无精子症(NOA)的致病突变及其对精子发生的影响?
结论:ADAD2的双等位基因错义和移码突变破坏了人和小鼠中圆形精子向精子的分化。
背景:NOA是男性不育的最严重原因,其特征是由于精子发生受损而导致射精中缺乏精子。在老鼠身上,RNA结合蛋白ADAD2的缺乏导致精子在附睾中完全缺失,但ADAD2突变在人类NOA相关不育中的生精效应需要功能验证.
■来自三个无关家庭的六名不育男性患者在巴基斯坦当地医院根据不育史被诊断为NOA,性激素水平,两次精液分析和阴囊超声检查。六名患者中有两名进行了睾丸活检。使用CRISPR/Cas9基因组编辑工具产生携带与NOA患者中发现的突变相似的突变的Adad2突变小鼠(Adad2Mut/Mut)。在2月龄时验证Adad2Mut/Mut小鼠的生殖表型。随机选择野生型(WT)和Adad2Mut/Mut小鼠的同窝中的圆形精子细胞,并将其注射到刺激的WT卵母细胞中。该圆形精子细胞注射(ROSI)程序用三个生物学重复进行,并评估>400个ROSI来源的受精卵。在四只Adad2WT/Mut雄性小鼠和六只Adad2WT/Mut雌性小鼠中评估ROSI衍生后代的生育力达三个月。总共120个Adad2Mut/Mut,Adad2WT/Mut,和WT小鼠用于本研究。整个研究进行了3年。
方法:在6名受NOA影响的患者中进行全外显子组测序以检测潜在致病性突变。使用定量PCR在人睾丸组织和小鼠模型中评估并验证了鉴定的ADAD2突变的致病性,西方印迹,苏木精-伊红染色,周期性酸性希夫染色,和免疫荧光。通过荧光激活细胞分选收集WT和Adad2Mut/Mut小鼠的圆形精子细胞,并注射到刺激的WT卵母细胞中。在胚胎和出生后阶段评估了ROSI衍生后代的发育。
结果:在ADAD2中鉴定出三个隐性突变(MT1:c.G829T,p.G277C;MT2:c.G1192A,p.D398N;MT3:c.917_918del,p.Q306Rfs*43)来自三个无关巴基斯坦家庭的患者。MT1和MT2显著降低ADAD2的睾丸表达,可能导致NOA患者的精子发生失败。具有相应MT3突变的Adad2Mut/Mut雄性小鼠的免疫荧光分析显示ADAD2蛋白的不稳定性和过早降解,导致精子生成缺乏表型。通过ROSI,Adad2Mut/Mut小鼠可以产生具有类似胚胎发育的幼崽(Adad2Mut/Mut为46.7%,WT为50%)和出生率(Adad2Mut/Mut为21.45±10.43%,WT为27.5±3.536%,P=0.5044)对WT小鼠。来自ROSI的Adad2WT/Mut后代(通过三个ROSI重复总共17只幼崽)未显示明显的发育缺陷,并且具有正常的生育力。
方法:不适用。
结论:这是一份初步报告,表明ROSI可以有效治疗不育的Adad2Mut/Mut小鼠。在临床试验期间,需要在人体中仔细检查进一步的辅助生殖尝试。
结论:我们的工作提供了功能性证据,证明ADAD2基因的突变是有害的,并在人类和小鼠中引起一致的生精缺陷。此外,初步结果表明,ROSI可以帮助Adad2Mut/Mut产生生物后代。这些发现为人类男性ADAD2突变体相关不育的遗传咨询提供了有价值的线索。
背景:这项工作得到了国家自然科学基金(32000587,U21A20204和32061143006)的支持,国家重点研究发展计划(2019YFA0802600和2021YFC2700202)。这项工作也得到了健康与医学研究所的支持,合肥综合性国家科学中心,合肥,中国。作者宣布没有竞争利益。
OBJECTIVE: What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis?
CONCLUSIONS: Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice.
BACKGROUND: NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification.
UNASSIGNED: Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2Mut/Mut mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and Adad2Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2WT/Mut male mice and six Adad2WT/Mut female mice. A total of 120 Adad2Mut/Mut, Adad2WT/Mut, and WT mice were used in this study. The entire study was conducted over 3 years.
METHODS: Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages.
RESULTS: Three recessive mutations were identified in ADAD2 (MT1: c.G829T, p.G277C; MT2: c.G1192A, p.D398N; MT3: c.917_918del, p.Q306Rfs*43) in patients from three unrelated Pakistani families. MT1 and MT2 dramatically reduced the testicular expression of ADAD2, likely causing spermiogenesis failure in the NOA patients. Immunofluorescence analysis of the Adad2Mut/Mut male mice with the corresponding MT3 mutation showed instability and premature degradation of the ADAD2 protein, resulting in the spermiogenesis deficiency phenotype. Through ROSI, the Adad2Mut/Mut mice could produce pups with comparable embryonic development (46.7% in Adad2Mut/Mut versus 50% in WT) and birth rates (21.45 ± 10.43% in Adad2Mut/Mut versus 27.5 ± 3.536% in WT, P = 0.5044) to WT mice. The Adad2WT/Mut progeny from ROSI (17 pups in total via three ROSI replicates) did not show overt developmental defects and had normal fertility.
METHODS: N/A.
CONCLUSIONS: This is a preliminary report suggesting that ROSI can be an effective treatment for infertile Adad2Mut/Mut mice. Further assisted reproductive attempts need to be carefully examined in humans during clinical trials.
CONCLUSIONS: Our work provides functional evidence that mutations in the ADAD2 gene are deleterious and cause consistent spermiogenic defects in both humans and mice. In addition, preliminary results show that ROSI can help Adad2Mut/Mut to produce biological progeny. These findings provide valuable clues for genetic counselling on the ADAD2 mutants-associated infertility in human males.
BACKGROUND: This work was supported by the National Natural Science Foundation of
China (32000587, U21A20204, and 32061143006), and the National Key Research and Developmental Program of
China (2019YFA0802600 and 2021YFC2700202). This work was also supported by Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei,
China. The authors declare no competing interests.