Down-regulation of the liver-derived plasma protein fetuin-B mediates reversible female infertility
Down-regulation of the liver-derived plasma protein fetuin-B mediates reversible female infertility
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DOI：
10.1093/molehr/gaw068
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发表时间：
2017-01-01
影响因子：
4
通讯作者：
Jahnen-Dechent, W.
中科院分区：
文献类型：
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作者：
Floehr, J.;Dietzel, E.;Jahnen-Dechent, W.
研究问题：反义寡核苷酸 (ASO) 介导的血清胎球蛋白 -B 下调是否会像雌性小鼠胎球蛋白 -B 基因缺陷一样导致不孕？总结答案：ASO 治疗药理学胎球蛋白 -B 下调会导致雌性小鼠出现可逆性不孕。已知信息：雌性胎球蛋白 -B 缺陷 (Fetub(-/-)) 小鼠由于透明带 (ZP) 过早硬化而导致不育。酶活性研究表明，胎球蛋白-B 是透明带蛋白酶 ovastacin 的有效且高度特异性的抑制剂，它会裂解 ZP 蛋白 2 (ZP2)，从而介导最终的 ZP 硬化。 研究设计、规模、持续时间：皮下注射 10 个胎球蛋白-B ASO boli (100 mg/kg)。在 12 只雌性小鼠中进行了 20 天以上的研究，并使用 10 只磷酸盐缓冲盐水 (PBS) 处理的小鼠作为对照。第 20 天时，对雌性进行交配，以评估胎球蛋白-B 作为避孕的潜在分子靶点。 ASO 和 PBS 处理继续进行十次注射。第50天停止治疗后，继续交配以研究不育是否可逆。 参与者/材料、设置、方法：我们通过常规育种产生了胎球蛋白-B/ovastacin双缺陷（Fetub（-/-）、Astl（-/-））小鼠，以测试当同样删除目标蛋白酶时，Fetub（-/-）雌性小鼠的生育能力是否恢复。每种雌性基因型（Fetub（-/-）单缺陷、Astl（-/-）单缺陷、Fetub（-/-）、Astl（-/-）双缺陷）至少进行五次交配。为了测试胎球蛋白 B 下调的避孕效果，对 22 只雌性小鼠（6-13 周龄）重复注射 100 mg/kg 胎球蛋白 B ASO（n = 12）或 PBS（n = 10）并连续交配。在 ASO 治疗之前、期间和之后通过免疫印迹测定血清胎球蛋白-B。 ASO处理3周后，通过PCR分析6名女性肝脏中Fetub mRNA，并使用6名PBS处理的女性作为对照。还测量了每组六只小鼠的血清中的天冬氨酸（AST）和丙氨酸转氨酶（ALT）。为了确定成功受精所需的最低容许血清胎球蛋白 B 浓度，对五只经胎球蛋白 B ASO 处理的小鼠进行了 IVF。作为对照，六只雌性小鼠注射了对照寡核苷酸，六只雌性小鼠不接受治疗。 主要结果和机会的作用： Fetub(-/-) 雌性小鼠的生育力通过额外的 ovastacin 缺乏 (Astl(-/-)) 得以恢复。与Fetub(-/-)小鼠不同，雌性Fetub(-/-)、Astl(-/-)小鼠具有生育能力，这证实了ovastacin是胎球蛋白-B的主要分子靶标。第20天，接受10个胎球蛋白-B ASO boli后，血清胎球蛋白-B下调至基线水平的8 +/- 6%（平均值+/- SD）。 Fetuin-B 下调在 mRNA 水平得到证实。 Fetuin-B ASO 处理的雌性肝脏 Fetub mRNA 水平是 PBS 处理的雌性的 12.1 +/- 3.1%。在接下来的交配研究中，12只交配的雌性中有11只在50天的ASO治疗和从第20天开始的连续交配期间未能怀孕。对来自经 ASO 处理的雌性的卵母细胞进行 IVF 表明，血清胎球蛋白 B 水平需要低于 10 +/- g/ml 才能预防妊娠。停止 ASO 治疗可使血清胎球蛋白 B 正常化并恢复生育能力；所有雌性小鼠在停止 ASO 治疗后 60.3 +/- 35.9 天内怀孕并产仔。第一窝小鼠的数量显着小于对照小鼠（4.6 +/- 2.3 只小鼠与 6.7 +/- 1.8 只幼崽，n = 20，P = 0.04），但较小的窝数只是暂时的。第二窝小鼠的大小与第一窝对照小鼠的大小相似（7.6 +/- 1.3 只与 6.7 +/- 1.8 只幼鼠，n = 18，P = 0.25）。 限制，注意原因：重复剂量 100 mg/kg 胎球蛋白-B ASO boli 导致血清 ALT 和 AST 活性增加，表明存在肝毒性。每日阴道塞检查表明交配成功，但 ASO 治疗小鼠的交配塞比对照小鼠稳定性差（阴道未闭合）。 研究结果的广泛影响：小鼠体内胎球蛋白 B 的药理学下调导致可逆性不孕。通过胎球蛋白-B 控制伐他星蛋白酶活性是女性生育能力的必要决定因素，可作为女性避孕的目标。尽管在人类避孕方面很有希望，但在考虑转移到人类生殖生物学之前，需要进一步研究分析足够的胎球蛋白 B 下调和可耐受的副作用之间的平衡，以提高安全性。大规模数据：无。
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 downregulation 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 +/- g/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.