Farnesylation-dependent kinetochore targeting of centromere protein F is essential for oocyte meiotic progression and female fertility

Abstract

Background: During mammalian oocyte meiosis, accurate chromosome segregation critically depends on precise regulation of kinetochore-microtubule attachments, a process monitored by the spindle assembly checkpoint. While centromere protein F has been well characterized as a kinetochore-associated protein that stabilizes kinetochore-microtubule 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 centromere protein 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 centromere protein F farnesylation modification on its meiotic function and explore the association between centromere protein F gene mutations and female oocyte maturation disorders.

Study design: Previous studies have shown that centromere protein F is essential for chromosome segregation during mitosis, but its functional mechanism during meiosis remains poorly understood. Oocyte microinjection, western blotting, co-immunoprecipitation, and immunofluorescence were used to explore the localization and function of centromere protein F in oocytes. The role of centromere protein F farnesylation in mouse oocytes was investigated using pharmacological (farnesyltransferase inhibitor treatment) and genetic (C3111S point mutation) methods. Subsequently, 4 patients with centromere protein F mutations were identified in the whole-exome sequencing 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 centromere protein F mutations causing oocyte maturation disorders.

Results: Microinjection of centromere protein 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 (17.69±2.207% vs 44.93±5.539%, P <.05). Time-course immunofluorescence analysis revealed dynamic centromere protein F localization: initially dispersed across chromosome following nuclear envelope breakdown and then progressively accumulating at kinetochores by metaphase I. Co-immunoprecipitation assays confirmed a direct interaction between centromere protein F and Aurora kinase B. Knockdown of Aurora kinase B would damage the kinetochore localization of centromere protein 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 centromere protein F and Aurora kinase B (P <.01) and disrupting kinetochore localization. Genetic screening identified 4 centromere protein F mutations in 179 infertile women with oocyte maturation arrest. Microinjection of patient-derived mutant centromere protein F complementary RNAs into mouse oocytes significantly reduced maturation rates (77.00±2.411% vs 49.10±6.561%, P <.01; 77.00±2.411% vs 35.43±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 centromere protein F (P <.05/P <.01), damage its interaction with Aurora kinase B (P <.05/P <.01), and disrupt the kinetochore localization. Both centromere protein F depletion and patient mutations induced constitutive spindle assembly checkpoint activation, and the treatment with spindle assembly checkpoint inhibitor partially rescued the meiotic arrest phenotype in oocytes (P <.05).

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

Keywords: CENP-F; farnesylation; female infertility; kinetochore; oocyte metaphase I arrest; spindle assembly checkpoint.