Department of Obstetrics and Gynecology, Second Xiangya Hospital, Central South University, Changsha, China.
Clinical Research Center for Gynecological Disease in Hunan Province, Hunan Province, Changsha, China.
Reprod Biol Endocrinol. 2024 Aug 29;22(1):113. doi: 10.1186/s12958-024-01284-w.
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.
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.
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.
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.
非阻塞性无精子症（NOA）是男性不育症中最严重的一种形式，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 中起着至关重要的作用。然而，范可尼缺陷导致严重的男性生殖细胞缺陷和成熟延迟的潜在机制仍不清楚。
我们使用 Fance 缺陷小鼠模型进行实验，收集 8 周（8W）、妊娠后 17.5 天（dpc）、出生后 11 天（P11）至 P23 的小鼠睾丸或附睾。这些小鼠分为三种基因型：野生型（Fance ）、杂合型（Fance ）和纯合型（Fance ）。进行苏木精和伊红染色、免疫荧光染色和精母细胞表面铺展，以探讨 Fance 小鼠 NOA 的机制。每个实验均进行了至少三个生物学重复，使用 Kruskal-Wallis 检验加 Dunn 校正进行统计分析。
在本研究中，我们发现成年雄性 Fance 小鼠的生精小管中存在大量的生殖细胞丢失，附睾中的精子数量明显减少。在胚胎期，Fance 前体精原细胞的数量显著减少，但对增殖（Ki-67、PCNA）和凋亡（cleaved PARP、cleaved Caspase 3）状态没有影响。Fance 前体精原细胞的细胞水平上 DNA 双链断裂（γH2AX）增加，这可能与非同源末端连接（53BP1）的增加和同源重组（RAD51）活性的降低有关。此外，Fance 缺陷阻碍了精母细胞减数分裂前期 I 的进展。这些机制包括在细线期减少 DNA 末端切除蛋白 RPA2 的募集，以及在合线期减少重组酶 RAD51 和 DMC1 的募集。还包括在合线期无法去除 RPA2 和在粗线期无法去除 FANCD2 焦点。以及在早期粗线期加速形成交叉，这一点可以从 MLH1 中看出。
Fance 缺陷导致大量雄性生殖细胞丢失，涉及前体精原细胞中 DNA 损伤修复的失衡，以及同源重组、DNA 末端切除和交叉的蛋白质动力学改变，为 NOA 的病因和分子基础提供了新的见解。
