The biological role of fetuin-B in inhibition of premature zona pellucida hardening during oocyte maturation

Sadr, Seyedeh Zeynab; Jahnen-Dechent, Wilhelm (Thesis advisor); Spehr, Marc (Thesis advisor)

Aachen : RWTH Aachen University (2022, 2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

Ovarian tissue contains different stages of follicles, which develop during folliculogenesis. At the last stage, the preovulatory follicle, which contains an immature oocyte, ovulates its oocyte, coinciding with oocyte maturation. The mature oocyte or metaphase II (MII) oocyte is ready to be fertilized by sperm. Before fertilization and during maturation, a small number of cortical granules which are located beneath the oocyte membrane release their contents. These granules comprise proteases including the metalloproteinase ovastacin. Ovastacin is inhibited by fetuin-B, which reaches the oocyte via blood circulation. In the absence of fetuin-B, ovastacin cleaves zona pellucida protein 2 (ZP2), and consequently, zona pellucida (ZP) hardening occurs. Therefore, no sperm can penetrate because of premature ZP hardening. ZP hardening is a known risk during in vitro maturation of oocytes before in vitro fertilization. To address this issue, fetuin-B deficient mouse oocytes, which showed premature ZP hardening and thus infertility, were matured in vitro by applying recombinant fetuin-B. Remarkably, fetuin-B deficient oocytes could be fertilized for the first time by conventional in vitro fertilization. However, fetuin-B deficient oocytes showed a lower two-cell embryo rate compared to fertile heterozygous oocytes (10 ± 1% vs. 49 ± 11%, p = 0.0063), suggesting that premature ZP hardening has already partially occurred before oocyte maturation. This assumption could be verified by ZP2 immunoblot as the ZP2 cleavage was visible in fetuin-B deficient antral follicle derived oocytes (AF oocytes). For individual analysis of oocytes regarding ZP hardening, we measured the elastic modulus of AF oocytes by nanoindentation. AF oocytes showed a low rate of soft ZP before maturation, which could explain the low fertilization rate of 10 ± 1% after oocyte in vitro maturation. Concerning the earlier stage of oocytes, secondary follicle derived oocytes (SF oocytes), did not exhibit premature ZP hardening. This result indicates that ovastacin is released between the transition of the secondary to antral follicle stage. Consequently, the use of recombinant fetuin-B is beneficial to prevent premature ZP hardening during in vitro maturation and could be used as an additive at the stage of secondary follicles.

Institutions

• Chemosensation Laboratory [163310]
• Department of Biology [160000]
• [529000-2]