Crossing the barrier or how regulation of ovastacin controls fertilization and translates into clinical phenotypes

Abstract

The zona pellucida, a glycoprotein matrix enveloping the mammalian egg, exerts essential functions during fertilization and early embryonic development. Its safeguard property regulates sperm entry and thus indirectly controls fertility. Limited proteolysis by the metalloproteinase ovastacin, released from the egg during fertilization, induces hardening of the zona pellucida. This precludes sperm entry and protects the embryo until implantation. However, ovastacin leakage before fertilization causes premature hardening and infertility if activity is not inhibited. This highlights the importance of ovastacin regulation by its endogenous inhibitor, fetuin-B. Accordingly, both loss and excessive ovastacin activity are linked to infertility. Here, we review recent discoveries on how ovastacin is precisely controlled to preserve zona pellucida permeability prior to fertilization and prevent penetration afterward. Based on these molecular mechanisms, we propose explanations for clinical phenotypes of recently discovered genetic mutations in ovastacin and discuss how modulation of ovastacin activity might be employed to regulate fertilization.

Keywords: Biochemistry; Cell biology; Developmental biology.

Graphical abstract

Figure 1

Schematic overview of mammalian egg-sperm interaction and fertilization focusing on the function of ovastacin and fetuin-B (A) Schematic overview of cumulus-oocyte complex before fertilization including detailed illustration of the barriers surrounding the egg in temporal and spatial context. (B) Chronological events of egg-sperm interaction (marked by arrow) starting with (I) arrival of sperm at the cumulus-oocyte complex, followed by (II) acrosome reaction and adhesion of sperm to the zona pellucida (ZP; light blue), (III) penetration of the ZP, (IV) sperm accessing the perivitelline space, (V) binding of the sperm to the oolemma and gamete fusion leading to exocytosis of cortical granules including release of the metalloproteinase ovastacin, and (VI) subsequent cleavage of ZP2 (dark blue angles). Thereby, ovastacin abolishes sperm binding to the ZP and hardens the ZP (dark blue) (VII and VIII). Horizontal bars approximate the respective concentration of ovastacin and fetuin-B in the vicinity of the ZP and the perivitelline space. The vertical bar (ZP2 turnover) approximates the temporal ZP2 conversion (relative ovastacin activity). Estimation of ovastacin release and regulation by fetuin-B based on the mouse model.^,,,, Prematurely released ovastacin is blocked by fetuin-B to prevent premature ZPH; massive release of ovastacin as a consequence of the cortical reaction temporarily overcomes fetuin-B concentration, thus causing cleavage of ZP2 into ZP2_f and causing mechanical hardening of this extracellular matrix. Polar body (PB), pronuclei (PN).

Figure 2

Schematic overview of pathophysiologic zona pellucida (ZP) reaction during egg-sperm interaction in fetuin-B-deficient (Fetub^−/−, upper row) or ovastacin-deficient (Astl^−/−, lower row) mouse oocytes Chronological events during fertilization, starting with preovulation (germinal vesicle intact) oocytes (left). No inhibition of prematurely released ovastacin by fetuin-B causing premature conversion of ZP2 to ZP2_f (upper row right). This premature ZP hardening renders the ZP impassable for sperm and results in female infertility. Absence of ZP2 cleavage in ovastacin deficiency keeps the ZP passable for additional sperm even after fertilization without increasing the polyspermy rate in vivo. Horizontal bars approximate the respective concentration of ovastacin and fetuin-B in the vicinity of the ZP and the perivitelline space. The vertical bar (ZP2 turnover) approximates the temporal ZP2 conversion (relative ovastacin activity). Estimation of ovastacin release and regulation by fetuin-B based on the mouse model.^,,,,

Figure 3

Structures of ovastacin and fetuin-B and conservation of the region in fetuin-B relevant for inhibition as well as of the cleavage site in ZP2 (A) Upper section: schematic domain structure of murine proovastacin (propeptide [PRO], catalytic domain [CAT], and C-terminal region [CTR]) including positions of disulfide bonds. Lower section: structure of the catalytic domain of murine ovastacin (AlphaFold3 model¹²⁸) in standard orientation (left) and rotated 90° clockwise (right) depicting secondary structure elements (α-helices and β-strands), disulfide bonds labeled as yellow sticks, the histidine-coordinated catalytic zinc ion in magenta, and the termini. (B) Upper section: schematic domain structure of murine ZP2 including positions of disulfide bonds and indicating the ovastacin cleavage site. Lower section, left: sequence logo of the ovastacin cleavage sites of 30 mammalian ZP2 from different orders illustrates the total conservation of an aspartate residue in all mammals in position P1′. Positions in non-prime (P) and prime (P′) of the cleavage according to Schechter and Berger (1967). Lower section, right: sequence alignment of the cleavage site in ZP2 from selected mammalian species using ClustalX. (C) Upper section: schematic domain structure of murine fetuin-B (cystatin-like domain 1 [CY1], linker [LNK], cystatin-like domain 1 [CY1], and C-terminal region [CTR] including positions of disulfide bonds. Lower section: structure of murine fetuin-B (pdb 7AUW) depicting secondary structure elements (α helices and β-sheets), disulfide bonds as labeled yellow sticks and the termini. (D) Upper section: sequence logo of the linker of 38 vertebrate fetuin-B from different taxa illustrates the full conservation of CPDCP in all jawed vertebrates. Lower section: sequence alignment of the linker of fetuin-B from selected vertebrate with CPDCP in bold using ClustalX. (E) Structure of the complex of murine ovastacin (AlphaFold3 model) and fetuin-B (pdb 7UAW) in standard orientation rotated 90° counterclockwise. (F) Structure of the catalytic domain of human ovastacin (AlphaFold3 model) in standard orientation highlighting known clinical mutations and variants. Point mutations that cause a single amino acid exchange are marked in red; regions not translated as a result of a point mutation leading to a skipping of exon 6 are highlighted in orange.