The E-modulus of the oocyte is a non-destructive measure of zona pellucida hardening

Abstract

After fertilization, the oocyte-specific metalloproteinase ovastacin is released and cleaves the zona pellucida protein 2 (ZP2), making the zona pellucida impermeable to sperm. Before fertilization, the zona remains permeable because previously released ovastacin is inhibited by fetuin-B. Consequently, in the absence of fetuin-B, ZP2 cleavage occurs prematurely and leads to infertility of female fetuin-B deficient mice. In contrast, fetuin-B/ovastacin double-deficient oocytes show a permanently permeable zona with intact ZP2. In this study, we asked if the elastic modulus of the zona pellucida informs about ZP2 cleavage and thus could serve as a new reference of oocyte fertility. Therefore, we determined the elastic modulus of mouse oocytes by nanoindentation as a direct measure of mechanical zona hardening. The elastic modulus reflects ZP2 cleavage, but with more than double sensitivity compared to immunoblot analysis. The elastic modulus measurement allowed to define the range of zona hardening, confined by the extreme states of the zona pellucida in fetuin-B and ovastacin-deficient oocytes with cleaved and uncleaved ZP2, respectively. We present here nanoindentation as a method to quantify the effect of potential contributing factors on the zona hardening of individual oocytes. To demonstrate this, we showed that mechanical hardening of the zona pellucida is forced by recombinant ovastacin, inhibited by additional administration of fetuin-B, and unaffected by zinc. Since the change in elastic modulus is induced by ZP2 cleavage, an automated elastic modulus measurement of oocytes may serve as a novel sensitive, non-destructive, marker-free, and observer-unbiased method for assessing individual oocyte quality.

Substrate profiling of the metalloproteinase ovastacin uncovers specific enzyme–substrate interactions and discloses fertilization‐relevant substrates

“…Both the very high conservation level of ovastacin, including its surface properties relevant for substrate interaction, and the presence of the typical di‐acidic cleavage motif in all mammalian ZP2 suggest functional conservation of ZP2 cleavage and ZPH, respectively, in all mammalian species. This hypothesis is supported by strong conservation of the structural elements required for the inhibition of ovastacin in fetuin‐B [24,30]. Perhaps for this reason, the highly variable residues on the prime side (starting with P3′) in ZP2 (Fig.…”

“…We used the secretome proteins of mouse embryonic fibroblasts (MEFs) as a versatile resource for mass spectrometric N-TAILS analysis of the cleavage specificity of heterologously expressed murine ovastacin. We have favored this model, as ovastacin and its regulation by fetuin-B have been best studied for the mouse ortholog [1,17,22,24,[30][31][32][33]. Since the physiological function, ZP2 cleavage, per se, and its regulation by fetuin-B are highly conserved [24,25,30], we assume this model is valid for most theria and thus also for humans as well as domestic animals.…”

“…We have favored this model, as ovastacin and its regulation by fetuin-B have been best studied for the mouse ortholog [1,17,22,24,[30][31][32][33]. Since the physiological function, ZP2 cleavage, per se, and its regulation by fetuin-B are highly conserved [24,25,30], we assume this model is valid for most theria and thus also for humans as well as domestic animals. MEFs were chosen rather than germ cells to overcome the particularly low quantity of germ cell secretome and to avoid very high animal consumption.…”

Viscoelastic Properties of Zona Pellucida of Oocytes Characterized by Transient Electrical Impedance Spectroscopy

“…Mice were hormonally stimulated for the collection of the oocytes in the MII stage. The oocytes were separated, as previously described [ 11 , 28 ]. After isolation, the oocytes were transferred to the microfluidic chip for EIS measurements.…”

“…For both techniques, sophisticated electromechanical transducers had to be developed. Atomic force microscopy (AFM) as well as nanoindenter tools use stiff cantilever beams instead of needles [ 11 , 12 , 13 , 14 ]. The force that is induced by the cantilever is proportional to the beam’s deflection.…”