Kyaw Hay Mar, Sato Hiroko, Tagami Takayoshi, Yanagawa Yojiro, Nagano Masashi, Katagiri Seiji
Laboratory of Theriogenology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, 060-0818, Japan.
Laboratory of Molecular Enzymology, Division of Fundamental AgriScience Research, Research Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan.
Theriogenology. 2022 May;184:26-33. doi: 10.1016/j.theriogenology.2022.02.008. Epub 2022 Feb 17.
Endometrial epidermal growth factor (EGF) shows a cyclic change with two peaks on Days 2-4 and 13-14 during the estrous cycle. An altered (i.e., loss of the two peaks) profile has been linked to reduced fertility in repeat breeder cows. We previously demonstrated that a form of osteopontin (OPN), with a molecular weight of 29 kDa and found in bull seminal plasma (SP), normalized the EGF profile and restored fertility in repeat breeder cows. OPN has many molecular forms due to post-translational modifications and is abundant in bovine milk. The purpose of the present study was to investigate whether mOPN normalizes the endometrial EGF profile and restores fertility in repeat breeder dairy cows with an altered EGF profile. OPN was separated by one-step anion-exchange column chromatography from the whey of bovine milk. Purified mOPN was verified by Western blotting and peptide mass fingerprinting analyses. The OPN fraction showed three major protein bands of 61, 37 and 31 kDa (peptides I, II, and III, respectively) on SDS-PAGE. All three major bands were identified as OPNs by Western blotting and their tryptic peptide masses were matched at approximately 50, 40, and 10%, respectively, to the bovine OPN amino acid sequence by a peptide mass finger printing analysis. The three bands accounted for approximately 85% of the total protein content and 6-23 mg of OPN was obtained from 1 L of bovine milk. A lyophilized eluate containing 1.3 mg of mOPN (171 cows), 0.5 mL of frozen SP (62 cows), and PBS (84 cows) was infused at estrus into the vagina of repeat breeder cows with an altered EGF profile. Some of the cows treated with mOPN, SP, and PBS (46, 50, and 45 cows, respectively) were inseminated immediately before the infusion and then examined for pregnancy between Days 60 and 65. The rate at which mOPN to normalize the EGF profile (56.1%) was similar to that of SP (58.1%) and higher than that of PBS (23.8%) (P < 0.05). The conception rate after the infusion of mOPN (43.5%) was similar to that of SP (40.0%) and higher than that of PBS (22.2%) (P < 0.05). The present results indicate that the infusion of mOPN into the vagina is a treatment option for repeat breeder cows with an altered EGF profile. Further studies are needed to compare the capacity of the three OPN molecules in milk to normalize the EGF profile, together with their molecular characteristics due to post-translational modifications.
子宫内膜表皮生长因子（EGF）在发情周期中呈现周期性变化，在第2 - 4天和第13 - 14天出现两个峰值。重复配种母牛的EGF变化模式改变（即两个峰值消失）与生育力降低有关。我们之前证明，公牛精液中存在的一种分子量为29 kDa的骨桥蛋白（OPN）形式可使EGF变化模式正常化，并恢复重复配种母牛的生育力。由于翻译后修饰，OPN有多种分子形式，且在牛乳中含量丰富。本研究的目的是调查单体OPN（mOPN）是否能使EGF变化模式改变的重复配种奶牛的子宫内膜EGF变化模式正常化并恢复生育力。通过一步阴离子交换柱色谱法从牛乳乳清中分离OPN。通过蛋白质免疫印迹法和肽质量指纹分析验证纯化的mOPN。OPN组分在SDS - PAGE上显示出61、37和31 kDa的三条主要蛋白带（分别为肽I、II和III）。通过蛋白质免疫印迹法确定所有这三条主要条带均为OPN，并且通过肽质量指纹分析，它们的胰蛋白酶肽质量分别约50%、40%和10%与牛OPN氨基酸序列匹配。这三条带约占总蛋白含量的85%，从1升牛乳中获得6 - 23毫克OPN。将含有1.3毫克mOPN的冻干洗脱液（171头奶牛）、0.5毫升冷冻精液（62头奶牛）和磷酸盐缓冲液（84头奶牛）在发情期注入EGF变化模式改变的重复配种奶牛的阴道。一些用mOPN、精液和磷酸盐缓冲液处理的奶牛（分别为46、50和45头奶牛）在注入前立即进行授精，然后在第60至65天之间检查是否怀孕。mOPN使EGF变化模式正常化的比例（56.1%）与精液（58.1%）相似，高于磷酸盐缓冲液组（23.8%）（P < 0.05）。注入mOPN后的受孕率（43.5%）与精液组（40.0%）相似，高于磷酸盐缓冲液组（22.2%）（P < 0.05）。目前的结果表明，向阴道内注入mOPN是EGF变化模式改变的重复配种奶牛的一种治疗选择。需要进一步研究比较牛乳中三种OPN分子使EGF变化模式正常化的能力，以及它们由于翻译后修饰而具有的分子特征。
