Primordial germ cells (PGCs) development is a subtle and complex regulatory process. Fance is an important substrate molecule necessary for the activation of the Fanconi anemia (FA) pathway, and its homozygous mutant causes massive oocyte loss as early as embryonic day 13.5 (E13.5). Here, we present histological and RNA-seq analysis of Fance deficient PGCs to explore the possible mechanisms responsible for its progressive depletion of germ cells. In Fance-/- embryos, the reduction of PGCs was already evident at E9.5 and the progressive loss of PGCs led to the PGCs being almost exhausted at E12.5. An increase of apoptotic cells was detected among Fance-/- PGCs, which may intuitively explain their reduced number in embryos. Moreover, abnormal cell proliferation and accumulating DNA damage were detected in E12.5 Fance-/- PGCs. We identified 3026 differentially expressed genes in E12.5 Fance-/- PGCs compared to Fance+/+ . KEGG pathway analysis revealed that the up-regulated genes were highly associated with ‘lysosome’, various metabolism pathways, whereas the down-regulated genes were mainly enriched in ‘cell cycle’, ‘oocyte meiosis’, ‘ribosome’, and various DNA repair pathways. In addition, multiple genes of various cell death pathways were found to be differentially expressed in E12.5 Fance-/- PGCs, indicating that PGCs death in Fance-/- embryos might diverge from canonical apoptosis. These findings indicate that Fance is essential for PGCs survival and the potential mechanisms involve cell cycle regulation, DNA damage repair, cell death prevention, and by regulating lysosome and ribosome function. Our results provide an important reference for further studies.
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Suye Suye, Huan Yin, Zhixian Zhou, Chunying Zheng, Zhen Ren, Liye Shi, and Chun Fu
Huan Yin, Suye Suye, Zhixian Zhou, Haiyi Cai, and Chun Fu
In brief
Fanconi anemia results in subfertility and primary ovarian deficiency in females. This study reveals that disrupted meiosis in oocytes is one of the mechanisms involved.
Abstract
Fance is an important factor participating in the repair of DNA interstrand cross-links and its defect causes severe follicle depletion in female mice. To explore the underlying mechanisms, we investigated the effects of Fance on ovarian development in embryonic and newborn mice. We found that the number of oocytes was significantly decreased in Fance −/− mice as early as 13.5 days post coitum (dpc). The continuous decrease of oocytes in Fance −/− mice compared with the Fance +/+ mice led to the primordial follicles being almost exhausted at 2 days postpartum (dpp). The mitotic–meiotic transition occurred normally, but the meiotic progression was arrested in pachytene in Fance −/− oocytes. We detected the expressions of RAD51 (homologous recombination repair factor), 53BP1 (non-homologous end-joining repair factor), and γH2AX by immunostaining analysis and chromosome spreads. The expressions of 53BP1 were increased and RAD51 decreased significantly in Fance −/− oocytes compared with Fance +/+ oocytes. Also, the meiotic crossover indicated by MLH1 foci was significantly increased in Fance −/− oocytes. Oocyte proliferation and apoptosis were comparable between Fance −/− and Fance +/+ mice (P > 0.05). The aberrant high expression at 17.5 dpc and low expressions at 1 and 2 dpp indicated that the expression pattern of pluripotent marker OCT4 (POU5F1) was disordered in Fance −/− oocytes. These findings elucidate that Fance mutation leads to a progressive reduction of oocytes and disrupts the progression of meiotic prophase I but not the initiation. And, our study reveals that the potential mechanisms involve DNA damage repair, meiotic crossover, and pluripotency of oocytes.
