P-558 Characterization of the morphology of the outer-kinetochore expandable module, aka fibrous corona, and its role in ensuring proper chromosome segregation in human and bovine oocytes
Authors: Baggio C , Larreategui-Aparicio A , Arends B , Pieters M , Kops G , De Ruijter-Villani M 
Published: July 2024

Abstract

Study question
How does the Fibrous Corona (FC) behave in human and bovine oocyte meiosis and what is its function in chromosome segregation during meiosis I?

Summary answer
In human and bovine oocytes, FC shows extreme plasticity expanding beyond the centromeric region of the chromosomes. Pharmacological inhibition of FC’s expansion induces chromosome mis-segregation.

What is known already
Embryonic aneuploidy of meiotic origin is one of the major causes of implantation failure and early pregnancy loss. Erroneous microtubule (MT)-kinetochore attachments in human meiosis are the main cause of meiotic chromosome segregation errors. In somatic cells, the FC, a multiprotein complex, assembles on kinetochores and expands around the centromere during prometaphase to facilitate MT interactions and stimulate correct MT-kinetochore attachment. We have shown that in bovine oocytes FC expand beyond the centrosomes and is a potent MTs nucleator (10.1016/j.cub.2023.01.010). However, it is still unknown how FC behaves and what are its functions in MT-kinetochore interactions in human oocytes.

Study design, size, duration
FC morphology was studied in immature human oocytes discarded during ICSI procedures and donated (informed consent) by the patients for research (Ethical permit TCBio.21-754) during the years 2021-2024. Because of ethical concerns and reduced availability of human oocytes, functional studies on FC were performed on bovine oocytes collected from slaughterhouse ovaries.

Participants/materials, setting, methods
180 immature human oocytes were matured in vitro and either fixed at different meiosis I stages or treated 2h with colcemid, a MT depolymerizing drug, to induce maximum FC expansion and fixed. The oocytes were then immuno-stained for chromatin(DAPI), FC(ZW10), MT(alpha tubulin) and kinetochores(CREST) and imaged with super-resolution confocal microscopy.
Bovine immature oocytes were matured in vitro with or without lonafarnib (inhibitor of FC expansion). After maturation chromosome spreads were performed to evaluate oocyte ploidy.

Main results and the role of chance
In human oocytes, after nuclear envelope breakdown, before microtubule appearance when the chromosomes appear compacted, FCs are visible on the top of the kinetochores. During MT nucleation, FCs form thick mesh in the center of the compacted chromosomes. During prometaphase I, FCs expand beyond the centromeric region of the chromosomes forming a cup around the sister kinetochores of the paired homologous chromosomes. When the chromosomes starts to align at the metaphase plate, FCs start to be stripped from the kinetochores and colocalize to the MTs. At metaphase I and II, only kinetochore localization is visible. When oocytes are treated with colcemid, FCs extend to partially cover chromosome arms. We observed a wide variability in FCs expansion between and within patients.
Inhibition of FC expansion in bovine oocytes increase the incidence of oocyte aneuploidy (90% in lonafarnib treated vs 37% in control oocytes).
Altogether, our data reveal that in human oocytes FCs expand more compared to human somatic cells. Considering that inhibition of FC expansion induces chromosome segregation errors, we propose that the large FC size in human oocytes may play a role in achieving correct chromosome-MT attachments within their extremely large cytoplasm thus ensuring proper chromosome segregation.

Limitations, reasons for caution
The limited availability of human oocytes requires the use of an animal model for in depth mechanistic studies. Although bovine oocytes have similar aneuploidy rate and spindle assembly mechanisms as human oocytes, they show morphological differences in FC expansion which could underlie intrinsic functional discrepancies between the two organisms.

Wider implications of the findings
A better understanding of the chromosome segregation mechanisms in human meiosis will help understanding what causes the high incidence of aneuploidies in human oocytes and possibly contribute to the development of interventions or of oocyte selection procedures to reduce the incidence of oocyte aneuploidy during in vitro maturation.

Trial registration number
Not applicable