Down-regulation of the liver-derived plasma protein fetuin-B mediates reversible female infertility.

STUDY QUESTION: Does antisense oligonucleotide (ASO)-mediated down-regulation of serum fetuin-B cause infertility like fetuin-B gene deficiency in female mice? SUMMARY ANSWER: Pharmacological fetuin-B down-regulation by ASO therapy results in reversible infertility in female mice. WHAT IS KNOWN ALREADY: Female fetuin-B deficient (Fetub-/-) mice are infertile owing to premature zona pellucida (ZP) hardening. Enzyme activity studies demonstrated that fetuin-B is a potent and highly specific inhibitor of the zona proteinase ovastacin, which cleaves ZP protein 2 (ZP2) and thus mediates definitive ZP hardening. STUDY DESIGN, SIZE, DURATION: Ten fetuin-B ASO boli (100 mg/kg) were injected s.c. over 20 days in 12 female mice, and 10 phosphate-buffered saline (PBS)-treated mice were used as control. At day 20 females were mated to evaluate fetuin-B as a potential molecular target for contraception. ASO and PBS treatment was continued for ten injections. After treatment cessation at day 50, mating was continued to investigate if infertility was reversible. PARTICIPANTS/MATERIALS, SETTING, METHODS: We generated fetuin-B/ovastacin double deficient (Fetub-/-, Astl-/-) mice by conventional breeding to test if fertility of Fetub-/- female mice was restored when the target proteinase would likewise be deleted. At least five matings with each female genotype (Fetub-/- single deficient, Astl-/- single deficient, Fetub-/-, Astl-/- double deficient) were performed. To test the contraceptive effect of fetuin-B down-regulation, 22 female mice (6-13 weeks old) were treated with repetitive boli of 100 mg/kg fetuin-B ASO (n = 12) or PBS (n = 10) and mated continuously. Serum fetuin-B was determined by immunoblot before, during and after the ASO treatment. After 3 weeks of ASO treatment, in 6 females Fetub mRNA in liver was analyzed by PCR, and six PBS-treated females were used as control. Aspartate (AST) and alanine aminotransferase (ALT) were also measured in serum of six mice in each group. To determine the minimum permissive serum fetuin-B concentration required for successful fertilization IVF was performed in five fetuin-B ASO-treated mice. As a control, six females were injected with control oligonucleotides and six females were left untreated. MAIN RESULTS AND THE ROLE OF CHANCE: Fertility of Fetub-/- female mice was restored by additional ovastacin deficiency (Astl-/-). Unlike Fetub-/- mice, female Fetub-/-, Astl-/- mice were fertile, confirming ovastacin as a primary molecular target of fetuin-B. At day 20, after receiving 10 fetuin-B ASO boli, serum fetuin-B was down-regulated to 8 ± 6% (mean ± SD) of baseline level. Fetuin-B down-regulation was confirmed at the mRNA level. Fetuin-B ASO-treated females had 12.1 ± 3.1% of the liver Fetub mRNA level seen in PBS-treated females. In the following mating study, 11 out of 12 mated females failed to become pregnant during 50 days of ASO treatment and continuous mating from day 20 onwards. IVF of oocytes derived from ASO-treated females suggested that a serum fetuin-B level of less than 10 µg/ml was required to prevent pregnancy. Withdrawal of ASO treatment normalized serum fetuin-B and restored fertility; all female mice became pregnant and had litters within 60.3 ± 35.9 days after cessation of ASO treatment. The first litter was significantly smaller than that of control mice (4.6 ± 2.3 versus 6.7 ± 1.8 pups, n = 20, P = 0.04) but the smaller litter size was only temporary. The size of the second litter was similar to the first litter of control mice (7.6 ± 1.3 versus 6.7 ± 1.8 pups, n = 18, P = 0.25). LIMITATIONS, REASONS FOR CAUTION: The repeated dose of 100 mg/kg fetuin-B ASO boli caused an increased serum ALT and AST activity, suggesting hepatotoxicity. Daily vaginal plug checks indicated successful mating, but mating plugs in ASO-treated mice were less stable (vaginal tract not closed) than in control mice. WIDER IMPLICATIONS OF THE FINDINGS: Pharmacological fetuin-B down-regulation in mice caused reversible infertility. Control of ovastacin proteinase activity by fetuin-B is a necessary determinant of female fertility that can serve as a target for female contraception. Although promising in terms of human contraception, further studies analyzing the balance between sufficient fetuin-B down-regulation and tolerable side effects are required to improve safety before transfer into human reproductive biology can be considered. LARGE SCALE DATA: None. STUDY FUNDING AND 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 E.D., J.F. and W.J.-D. are named inventors on a patent application of RWTH Aachen University covering the use of fetuin-B in ovary and oocyte culture. No conflict of interest is declared by C.S. and A.C.

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.