Farnesylation-Dependent Kinetochore Targeting of CENP-F Is Essential for Oocyte Meiotic Progression and Female Fertility.

Background: During mammalian oocyte meiosis, accurate chromosome segregation critically depends on precise regulation of kinetochore-microtubule (K-MT) attachments, a process monitored by the spindle assembly checkpoint (SAC). While CENP-F has been well characterized as a kinetochore-associated protein that stabilizes K-MT connections during mitosis, its functional mechanisms during meiosis remain poorly understood. In particular, there is still controversy over whether farnesylation modification governs localization and functionality of CENP-F. Concurrently, clinical investigations face a knowledge gap regarding the genetic basis of oocyte maturation arrest, a prevalent phenotype observed in female infertility patients.

Objective: This study aims to reveal the regulatory mechanism of CENP-F farnesylation modification on its meiotic function and explore the association between CENP-F gene mutations and female oocyte maturation disorders.

Study Design: Previous studies have shown that CENP-F is essential for chromosome segregation during mitosis, but its functional mechanism during meiosis remains poorly understood. Oocyte microinjection, western blotting, co-immunoprecipitation (Co-IP), and immunofluorescence were used to explore the localization and function of CENP-F in oocytes. The role of CENP-F farnesylation in mouse oocytes was investigated using pharmacological (farnesyltransferase inhibitor treatment) and genetic (C3111S point mutation) methods. Subsequently, four patients with CENP-F mutations were identified in the whole-exome sequencing (WES) dataset consisting of 179 infertile patients with oocyte maturation disorders. Mouse oocyte and 293T cell models were used to verify the mechanism of patient-derived CENP-F mutations causing oocyte maturation disorders.

Results: Microinjection of Cenp-f siRNA into mouse oocytes significantly reduced maturation rates (77.84±2.087% vs 34.26±4.748%, P<.01), with the majority arrested at metaphase I (MI) (17.69±2.207% vs 44.93±5.539%, P<.05). Time-course immunofluorescence analysis revealed dynamic CENP-F localization: initially dispersed across chromosome following nuclear envelope breakdown (NEBD), then progressively accumulating at kinetochores by MI. Co-IP assays confirmed a direct interaction between CENP-F and AURKB. Knockdown of AURKB would damage the kinetochore localization of CENP-F in oocytes. Farnesylation inhibition (via farnesyltransferase inhibitor or C3111S mutation) significantly decreased oocyte maturation rates (75.58±3.703% vs 46.18±1.282%, P<.01; 75.58±3.703% vs 44.04±2.541%, P<.01), concomitantly weakening interaction between CENP-F and AURKB (P<.01) and disrupting kinetochore localization. Genetic screening identified four CENP-F mutations in 179 infertile women with oocyte maturation arrest. Microinjection of patient-derived mutant CENP-F cRNAs into mouse oocytes significantly reduced maturation rates (77.00±2.411% vs 49.10±6.561%, P<.01; 77.00±2.411% vs35.43±1.035%, P<.01; 77.00±2.411% vs 55.43±1.288%, P<.05; 77.00±2.411% vs 40.00±4.187%, P<.01). Two of these mutations (K1708T/S1971fs) can reduce the farnesylation of CENP-F (P<.05/P<.01), damage its interaction with AURKB (P<0.05/P<0.01), and disrupt the kinetochore localization. Both CENP-F depletion and patient mutations induced constitutive SAC activation, and the treatment with SAC inhibitor partially rescued the meiotic arrest phenotype in oocytes (P<.05).

Conclusion: This study represents the first demonstration of a direct association between CENP-F genetic defects and human infertility, uncovering a novel farnesylation-dependent mechanism that governs meiotic progression, while simultaneously identifying CENP-F as a potential molecular marker for diagnosing oocyte maturation failure.