Molnárová M, Arendarcik J, Molnár P
Univerzita veterinárskeho lekárstva, Kosice.
Vet Med (Praha). 1992 Sep-Oct;37(9-10):517-30.
The function of proteins, peptides, proteases and inhibitors of proteases in modulations of regulation mechanisms of gonadotrophins during the development of ovarian folicles has not been fully explained up to now. We can see difference reactions of ewes to superovulation stimulations in oestrous and anoestrous periods as shown by the variation of the antiproteolytic activity of blood plasma and cervical mucus. Trypsin is used as a model for serine protease, and trypsin inhibitory activity (TIA) was measured from the reduced rate of trypsin hydrolysis of the chromogenic substrate N-alpha-tosyl-L-arginine-4-nitroanilide (TAPA, Bartík et al., 1974). Full hydrolytic activity was determined as a change in absorbency at 405 nm = 1.0 after ten-minute incubation at 25 degrees C and pH = 8.1, and inhibition was expressed in percentage of full activity. Statistical analyses were performed by Student's t-test. Twenty-three ewes in anoestrus and twenty-eight ewes in oestrus were included in this experiment. They were of the Slovak Merino breed, two to three years old, with the mean live weight of thirty to forty kg. The ewes were treated with Ageline vaginal sponges (20 mg chlorsuperlutin/sponge) to provide for synchronization of ovarian activity, in the interval of eleven to twelve days. After removal of sponges, the ewes were stimulated for superovulation as shown in Tab. I. Heparinized blood plasma samples were stored at -25 degrees C. Low molecular TIA activities were determined in HClO4--treated blood plasma. Samples of cervical mucus were taken on cotton-wool tampons which were evaluated in 0.2 M Tris-HCl buffer, pH = 8.1. Figs. 1-6 shows TIA changes in blood plasma and cervical mucus on particular days. Fig. 7 shows the mean values of controls (I-initial) after synchronization (A) and after stimulation (S). TIA changes were different in anoestrous and oestrous periods. Differences in TIA changes in blood plasma and cervical mucus in oestrous period suggested certain local regulation mechanisms of synthesis and/or secretion of this activity in the cervix. Some values of TIA were maximum at the time of expected heat or ovulation and may be some of the factor(s) which influenced fertility of females after superovulation stimulations.
蛋白质、肽、蛋白酶及蛋白酶抑制剂在卵泡发育过程中对促性腺激素调节机制的调控作用，至今尚未完全阐明。我们可以看到，母羊在发情期和乏情期对超数排卵刺激的反应不同，这表现为血浆和宫颈黏液抗蛋白水解活性的变化。以胰蛋白酶作为丝氨酸蛋白酶的模型，通过测定发色底物N-α-甲苯磺酰-L-精氨酸-4-硝基苯胺（TAPA，Bartík等人，1974）的胰蛋白酶水解速率降低来测量胰蛋白酶抑制活性（TIA）。在25℃和pH = 8.1下孵育10分钟后，将405nm处吸光度的变化确定为1.0时的完全水解活性，并以完全活性的百分比表示抑制率。采用学生t检验进行统计分析。本实验纳入了23只处于乏情期的母羊和28只处于发情期的母羊。它们为斯洛伐克美利奴品种，年龄在两到三岁，平均活重为30至40千克。使用Ageline阴道海绵栓（每栓含20mg氯地孕酮）对母羊进行处理，以同步卵巢活动，间隔为11至12天。取出海绵栓后，按照表I所示对母羊进行超数排卵刺激。肝素化血浆样本储存在-25℃。在经高氯酸处理的血浆中测定低分子TIA活性。在棉塞棉签上采集宫颈黏液样本，并在pH = 8.1的0.2M Tris-HCl缓冲液中进行评估。图1 - 6显示了特定日期血浆和宫颈黏液中TIA的变化。图7显示了同步化（A）和刺激（S）后对照组（I - 初始）的平均值。乏情期和发情期的TIA变化不同。发情期血浆和宫颈黏液中TIA变化的差异表明，宫颈中该活性的合成和/或分泌存在某些局部调节机制。某些TIA值在预期发情或排卵时达到最大值，可能是影响超数排卵刺激后母羊繁殖力的一些因素。
