Initial detachment of the mouse oocyte from the zona pellucida is mediated by metallopeptidase activity

Abstract

The fully grown mammalian oocyte is tightly attached to its extracellular matrix shell, the zona pellucida (ZP), but the oocyte detaches from the ZP shortly after ovulation is signaled. The mechanism by which the oocyte detaches from the ZP is unknown. Because ZP proteins are initially secreted as transmembrane proteins, we hypothesized that attachment of the oocyte to the ZP is mediated by transmembrane ZP proteins and that detachment occurs when these proteins are cleaved by peptidases. To identify potential candidates for the type of peptidase, we used mouse oocyte transcriptome data sets to identify candidate peptidases localized to the exterior of the oocyte. Screening with a set of small molecule inhibitors that broadly target the families of peptidases represented by the candidates, we found that only inhibitors of the M10 and M12 families of metallopeptidases prevented detachment. Using more selective inhibitors indicated that detachment was prevented by an inhibitor, GI254023X, developed to be selective for ADAM10 in the M12 family but not by those considered selective for the M10 family or for other M12 metallopeptidases expressed in oocytes. Using an antibody that binds to an epitope just distal to the likely cleavage site of murine ZP3 showed that this site was gradually lost from the oocyte surface during the period when detachment occurs, and that inhibiting metallopeptidase activity prevented the loss of this epitope. Taken together, these results indicate that detachment of the oocyte from the ZP is mediated by a metallopeptidase.

The cell volume‐regulatory glycine transporter GLYT1 is activated following metallopeptidase‐mediated detachment of the oocyte from the zona pellucida

“…Where specified, the inert trisaccharide D(+)-raffinose was added to increase osmolarity of the medium to 310 mOsM as previously described (Dawson & Baltz, 1997). COCs were released from excised ovaries by mincing with a razor blade and oocytes freed from the surrounding cumulus cells by repeated pipetting through a narrow bore flame-pulled Pasteur pipette in Hepes-mKSOM as previously described (Macaulay et al, 2023;Tartia et al, 2009). Oocytes were cultured in mKSOM and either allowed to spontaneously resume meiotic progression or were maintained in meiotic arrest at the GV stage with milrinone (10 µM), a selective phosphodiesterase 3 (PDE3) inhibitor.…”

“…Since oocyte-ZP detachment occurs before GLYT1 activation and individual oocytes exhibiting GLYT1 activity seem to be substantially detached, it was possible that detachment is required for GLYT1 to be activated. We recently found that detachment is mediated by metallopeptidase activity and identified small molecule inhibitors of M12 family metallopeptidases that prevent detachment (Macaulay et al, 2023). Therefore, we tested whether the same metallopeptidase inhibitors prevented GLYT1 activation.…”

“…It was previously found that the volume of the oocyte decreases substantially as it detaches from the ZP (Macaulay et al, 2023;Tartia et al, 2009). Therefore, it was possible that the activation of GLYT1, which is a volume-regulatory mechanism in eggs and early embryos (Steeves et al, 2003), might be activated by the decrease in volume that accompanies detachment.…”

“…We therefore tested whether inhibiting metallopeptidase activity in ZP-free oocytes affected GLYT1 activation. GV oocytes were cultured for 4 h in mKSOM after their ZPs were removed with acid Tyrode or with their ZP intact, in the presence or absence of the previously validated (Macaulay et al, 2023) metallopeptidase inhibitors GI254023X, batimastat, or marimastat (Figure 5d). There was no significant effect of inhibitor identity or the presence versus absence of the ZP (Figure 5d).…”

“…Recently, it was shown that oocyte-ZP detachment is mediated by a metallopeptidase that, when it becomes activated, is thought to cleave transmembrane ZP glycoproteins that form an attachment between the oocyte membrane and the inner ZP surface (Macaulay et al, 2023). The identity of the metallopeptidase that mediates detachment is not known, although it is likely to be a member of the M12 family of peptidases which includes the ADAM metallopeptidases (Macaulay et al, 2023). Coincident with detachment, the oocyte decreases substantially in volume (Tartia et al, 2009).…”