Loss of CENPF leads to developmental failure in mouse embryos

Aneuploidy caused by abnormal chromosome segregation during early embryo development leads to embryonic death or congenital malformation. Centromere protein F (CENPF) is a member of centromere protein family that regulates chromosome segregation during mitosis. However, its necessity in early embryo development has not been fully investigated. In this study, expression and function of CENPF was investigated in mouse early embryogenesis. Detection of CENPF expression and localization revealed a cytoplasm, spindle and nuclear membrane related dynamic pattern throughout mitotic progression. Farnesyltransferase inhibitor (FTI) was employed to inhibit CENPF farnesylation in zygotes. The results showed that CENPF degradation was inhibited and its specific localization on nuclear membranes in morula and blastocyst vanished after FTI treatment. Also, CAAX motif mutation leads to failure of CENPF-C630 localization in morula and blastocyst. These results indicate that farnesylation plays a key role during CENPF degradation and localization in early embryos. To further assess CENPF function in parthenogenetic or fertilized embryos development, morpholino (MO) and Trim-Away were used to disturb CENPF function. CENPF knockdown in Metaphase II (MII) oocytes, zygotes or embryos with MO approach resulted in failure to develop into morulae and blastocysts, revealing its indispensable role in both parthenogenetic and fertilized embryos. Disturbing of CENPF with Trim-Away approach in zygotes resulted in impaired development of 2-cell and 4-cell, but did not affect the morula and blastocyst formation because of the recovered expression of CENPF. Taken together, our data suggest CENPF plays an important role during early embryonic development in mice. Abbreviation: CENPF: centromere protein F; MO: morpholino; FTI: Farnesyltransferase inhibitor; CENPE: centromere protein E; IVF: in vitro fertilization; MII: metaphase II; SAC: spindle assembly checkpoint; Mad1: mitotic arrest deficient 1; BUB1: budding uninhibited by benzimidazole 1; BUBR1: BUB1 mitotic checkpoint serine/threonine kinase B; Cdc20: cell division cycle 20

早期胚胎发育过程中染色体分离异常引发的非整倍体（aneuploidy）会导致胚胎死亡或先天性畸形。着丝粒蛋白F（centromere protein F, CENPF）是着丝粒蛋白家族成员，在有丝分裂过程中调控染色体分离，但其在早期胚胎发育中的必要性尚未得到充分研究。

本研究针对小鼠早期胚胎发生过程中CENPF的表达与功能展开探究。对CENPF表达及定位的检测结果显示，其在整个有丝分裂进程中呈现与细胞质、纺锤体及核膜相关的动态分布模式。

本研究使用法尼基转移酶抑制剂（farnesyltransferase inhibitor, FTI）处理受精卵，以抑制CENPF的法尼基化修饰。结果显示，经FTI处理后，CENPF的降解受到抑制，且其在桑椹胚与囊胚阶段核膜上的特异性定位消失。此外，CAAX基序突变会导致CENPF-C630无法在桑椹胚和囊胚中正常定位。上述结果表明，法尼基化修饰在早期胚胎CENPF的降解与定位过程中发挥关键作用。

为进一步评估CENPF在孤雌生殖或受精胚胎发育中的功能，本研究使用吗啉代寡核苷酸（morpholino, MO）与Trim-Away技术干扰CENPF的功能。通过MO技术在减数分裂II期（metaphase II, MII）卵母细胞、受精卵或胚胎中敲低CENPF，会使其无法发育至桑椹胚与囊胚阶段，证实其在孤雌生殖与受精胚胎中均不可或缺。在受精卵中通过Trim-Away技术干扰CENPF，会导致2-细胞与4-细胞阶段发育受损，但由于CENPF的表达恢复，并未影响桑椹胚与囊胚的形成。

综上，本研究数据表明CENPF在小鼠早期胚胎发育过程中发挥重要作用。

缩写说明：CENPF：着丝粒蛋白F（centromere protein F）；MO：吗啉代寡核苷酸（morpholino）；FTI：法尼基转移酶抑制剂（farnesyltransferase inhibitor）；CENPE：着丝粒蛋白E（centromere protein E）；IVF：体外受精（in vitro fertilization）；MII：减数分裂II期（metaphase II）；SAC：纺锤体组装检验点（spindle assembly checkpoint）；Mad1：有丝分裂阻滞缺陷蛋白1（mitotic arrest deficient 1）；BUB1：苯并咪唑抗性蛋白1（budding uninhibited by benzimidazole 1）；BUBR1：BUB1有丝分裂检验点丝氨酸/苏氨酸激酶B（BUB1 mitotic checkpoint serine/threonine kinase B）；Cdc20：细胞分裂周期蛋白20（cell division cycle 20）