Role of the hepatic plasma protein Fetuin-B in fertility

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The zona pellucida (ZP) is a glycoprotein matrix surrounding mammalian oocytes. Upon fertilization, ZP hardening prevents sperm from binding to and penetrating the ZP. We reported that targeted gene deletion of the liver-derived plasma protein fetuin-B causes premature ZP hardening and, consequently, female infertility. Transplanting fetuin-B- deficient ovaries into wild-type recipients restored fertility, indicating that plasma fetuin-B is necessary and sufficient for fertilization. In vitro fertilization of oocytes from fetuin-B-deficient mice only worked after rendering the ZP penetrable by laser perforation. Mechanistically, fetuin-B sustains fertility by inhibiting ovastacin, a cortical granula protease known to trigger ZP hardening. Thus, plasma fetuin-B is necessary to restrain protease activity and thereby maintain ZP permeability until after gamete fusion. These results also show that premature ZP hardening can cause infertility in mice. To better understand the sequence of events that lead to infertility in the absence of fetuin-B, we will determine the exact timing of protease activation, minimum amount and duration of fetuin-B action required. To rescue fertility in fetuin-B deficient mice we will restore hepatic fetuin-B expression by hydroponic plasmid, and adenoviral gene transfer. To prove that ovastacin inhibition is necessary and sufficient to maintain fertility in the absence of fetuin-B, we will generate fetuin-B/ovastacin double deficient mice that should be fertile despite fetuin-B deficiency. To test if fetuin-B deficiency may be a viable alternative to hormonal contraception, we will reduce fetuin-B plasma levels by immune depletion, and by antisense oligonucleotide mediated down-regulation of expression. Test matings of female mice thus treated will show if, and how long infertility can be induced by therapeutic fetuin-B downregulation.We will collect blood, follicular fluid and DNA samples from patients undergoing infertility treatment in preparation of an association study of fetuin-B polymorphisms and infertility.We will determine the glycosylation pattern of fetuin-B, and produce up to 50 mg of homogeneously glycosylated fetuin-B to determine a high resolution 3-D structure of the protein in collaboration with renownded protein crystallographers.