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

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.

Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes.

STUDY QUESTION: Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate? SUMMARY ANSWER: Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts. WHAT IS KNOWN ALREADY: Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate. STUDY DESIGN, SIZE, DURATION: We fertilized batches of 20-30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm. MAIN RESULTS AND THE ROLE OF CHANCE: In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM). LIMITATIONS, REASONS FOR CAUTION: Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS: The astacin-type protease ovastacin triggers definitive ZP hardening by cleaving the zona pellucida protein 2. Animal sera are known to inhibit premature ZP hardening. The addition of rFetuB to the culture medium of oocytes could increase IVF rates by the inhibition of premature ZP hardening. In this regard, the results could be useful for clinical activity. LARGE SCALE DATA: None. STUDY FUNDING/COMPETING INTERESTS: The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. The authors ED, JF and WJD are named inventors on a patent application of RWTH Aachen University covering the use of fetuin-B in ovary and oocyte culture.

The serum protein fetuin-B is involved in the development of acute myocardial infarction.

The rupture of an atherosclerotic plaque is one of the main causes of coronary artery thrombotic occlusion, leading to myocardial infarction. However, the exact mechanism and causal risk factors for plaque rupture remain unclear. To identify a potential molecule that can influence atherosclerotic plaque rupture, we investigated protein expression in serum from patients with acute myocardial infarction (AMI) and stable angina (SA), using proteomic analysis. The expression of six proteins, including fibrinogen, fetuin-B, keratin 9, proapolipoprotein and fibrinogen, were altered in serum from patients with AMI compared with serum from those with SA. Of these, fetuin-B, proapolipoprotein, fibrinogen γ-B-chain precursors and fibrinogen expression were greater in serum from patients with AMI than from patients with SA. Increased fetuin-B expression in serum from AMI patients was also confirmed by Western blot analysis. Treatment with recombinant human fetuin-B increased the migration in monocytes and macrophages in a concentration-dependent manner. Fetuin-B also affected vascular plaque-stabilizing factors, including lipid deposition and cytokine production in macrophages, the activation of matrix metalloproteinase (MMP)-2 in monocytes, and the activation of apoptosis and MMP-2 in vascular smooth muscle cells. Moreover, in vivo administration of fetuin-B decreased the collagen accumulation and smooth muscle cell content and showed an increased number of macrophages in the vascular plaque. From these results, we suggest that fetuin-B may act as a modulator in the development of AMI. This study may provide a therapeutic advantage for patients at high risk of AMI.