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Suye Suye, Huan Yin, Zhixian Zhou, Chunying Zheng, Zhen Ren, Liye Shi, and Chun Fu

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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Li-Ying Yan, Jun-Cheng Huang, Zi-Yu Zhu, Zi-Li Lei, Li-Hong Shi, Chang-Long Nan, Zhen-Jun Zhao, Ying-Chun OuYang, Xiang-Fen Song, Qing-Yuan Sun, and Da-Yuan Chen

The assembly of microtubules and the distribution of NuMA were analyzed in rabbit oocytes and early cloned embryos. α-Tubulin was localized around the periphery of the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), multi-arrayed microtubules were found tightly associated with the condensed chromosomes and assembled into spindles. After the enucleated oocyte was fused with a fibroblast, microtubules were observed around the introduced nucleus in most reconstructed embryos and formed a transient spindle 2–4 h post-fusion (hpf). A mass of microtubules surrounded the swollen pseudo-pronucleus 5 hpf and a normal spindle was formed 13 hpf in cloned embryos. NuMAwas detected in the nucleus in germinal vesicle-stage oocytes, and it was concentrated at the spindle poles in both meiotic and mitotic metaphase. In both donor cell nucleus and enucleated oocyte cytoplasm, NuMA was not detected, while NuMA reappeared in pseudo-pronucleus as reconstructed embryo development proceeded. However, no evident NuMA staining was observed in the poles of transient spindle and first mitotic spindle in nuclear transfer eggs. These results indicate that NuMA localization and its spindle pole tethering function are different during rabbit oocyte meiosis and cloned embryo mitosis.