猪卵泡液中抑制素α亚基的鉴定

利用SephadexG 2 0 0和SephadexG 1 0 0 2次凝胶过滤层析的方法 ,从猪卵泡液中初步分离得到抑制素及其α亚基的粗提物。通过免疫印迹的方法检测 ,表明粗提物中含有抑制素α亚基     

Effects of Immunization Against Inhibin on Egg-Laying Performance in Magang and Landaise Geese

This study aimed to improve egg-laying performance in incubating Magang geese of Guangdong origin and Landaise geese of French origin. In experiment 1, 50 adults, egg-laying Magang geese were inoculated intramuscularly （i.m.） on days 0, 22, and 45 with 1 mL of immunogen containing 1 mg of recombinant chicken inhibin fusion protein. Immunization significantly increased blood antibody titers against inhibin fusion protein, but did not affect the egg-laying performance within 10 days after the first inoculation. From day 15, the egg-laying rate in inhibin-immunized group increased and was significantly higher than the values of control geese from day 40 to 55. However, the reverse was true from day 55 to 75 when more immunized geese developed incubation. In the entire 120 days of the experiment, the immunized geese laid 17.3 eggs in contrast to 16.4 eggs laid by the control geese. From day 30 till the end of the experiment, weight of eggs in the control geese was significantly greater than that in inhibin-immunized birds. In experiment 2, 40 Landaise geese were immunized against inhibin, as described in experiment 1. These geese laid 9.0 eggs on average in contrast to 7.3 eggs laid by nonimmunized control geese over 90 days of egg laying. The above results demonstrated that immunization against recombinant chicken inhibin fusion protein improved egg-laying performance in geese, and the increment was higher in nonincubating geese.     

不同因素对抑制素基因免疫肉牛反应性的影响

为探讨佐剂、质粒纯度和免疫次数3种因素对抗抑制素抗体效价的影响，以分析抑制索基因免疫是否使内牛产生免疫应答，利用已构建的抑制索真核融合表达质粒pCISI对内牛进行了免疫试验．结果表明，抑制素pCISI基因免疫内牛后佐剂处理组比无佐剂组免疫效果明显．对抗体P/N（待测血样的吸光值P与阴性血样吸光值N的比值）来说，普鲁卡因佐剂显著优于脂质体，但就抗体阳性率来说，普鲁卡因和脂质体差异不显著．无论是抗体P/N值还是抗体阳性率，高纯度组的效果略高于低纯度组，但差异不明显．随着免疫次数的增加抗体P/N值和抗体阳性率均升高，两者差异显著，说明了抑制素基因免疫内牛可诱导产生抗抑制素抗体，纯度对抗体的产生和抗体阳性牛比例没有影响，但免疫佐剂与免疫次数有影响．     

黄牛对抑制素pCISI基因免疫的反应性

为分析抑制素基因免疫对黄牛的影响,优化免疫方法,将3.0 mg抑制素真核融合表达质粒pCISI以不同纯度并配以不同的免疫佐剂分2次导入72头母黄牛体内,采用ELISA法检测血样中抗抑制素抗体效价。结果表明:抗体阳性牛占59.72%(43/72);添加佐剂的重组质粒较空质粒组免疫效果明显(P<0.05),普鲁卡因佐剂显著优于脂质体(P<0.05);高纯度疫苗组阳性牛的比例略高于低纯度组,但差异不明显(P>0.05)。这说明应用抑制素质粒pCISI免疫黄牛后可产生抗抑制素抗体。     

免疫抑制素和卵泡抑素阻止夏季热应激导致的猪精液品质下降的研究

[目的]通过免疫抑制素和卵泡抑素促进精子发生,在夏季热应激状态下保持公猪精液品质.[方法]在夏季热应激状态下对精液品质出现下降的成年种公猪单独免疫抑制素、或联合免疫抑制素α亚基和卵泡抑素重组蛋白(n=6)3次,每次间隔21 d;然后在9周试验期间定期采血测定血液抗体效价、FSH和睾酮浓度,每周采精观察精子活力和精子密度作为精液品质指标.[结果]抑制素单独免疫、抑制素和卵泡抑素联合免疫都使猪血液中抗抑制素的抗体效价显著高于对照组值(P<0.01),联合免疫也使猪血液中抗卵泡抑素的抗体效价显著高于对照组值(P<0.01).单独免疫或联合免疫都显著升高了血液FSH浓度;联合免疫还升高了血液睾酮水平.在试验期间对照组中猪的精子活力持续下降,而两免疫组的精子活力都上升,其中联合免疫猪的精子活力显著高于对照组猪.试验开始时对照组猪精子密度显著高于两免疫组,但在试验期间持续下降;而免疫组中猪精子密度在试验期间未下降,特辑是在联合免疫组于试验后期还显著高于对照组猪.[结论]免疫抑制素和卵泡抑素可以促进公猪FSH和睾酮分泌,克服夏季热应激的不良影响,促进猪精子发生,并提高精液精子活力和密度,有望用于热应激状态下保持种用公猪的繁殖性能.     

The expression and localization of inhibin isotypes in mouse testis during postnatal development

Inhibin, which is important for normal gonadal function, acts on the pituitary gonadotropins to suppress follicle-stimulating hormone (FSH) secretion. The level and cellular localization of the inhibin isotypes, α, β_A and β_B, in the testis of mice were examined during postnatal development in order to determine if inhibin expression is related to testicular maturation. Mouse testes were sampled on postnatal days (PNDs) 1, 3, 6, 18, 48 and 120, and analyzed by Western blotting and immunofluorescence. Western blot analysis showed very low levels of inhibin α, β_A and β_B expression in the testes at days 1 to 6 after birth. The levels then increased gradually from PND 18 to 48-120, and there were significant peaks at PND 48. Inhibin α, β_A and β_B were detected in testicular cells during postnatal development using immunohistochemistry. The immunoreactivity of inhibin α was rarely observed in testicular cells during PND 1 to 6, or in the cytoplasmic process of Sertoli cells surrounding the germ cells and interstitial cells during PND 18 to 120. Inhibin β_A and β_B immunoreactivity was rarely observed in the testis from PND 1 to 6. On the other hand, it was observed in some spermatogonial cells, as well as in the interstitial space between PND 48 and PND 120. We conclude that the expression of inhibin isotypes increases progressively in the testis of mice with increasing postnatal age, suggesting that inhibin is associated with a negative feedback signal for FSH in testicular maturation.     

抑制素基因免疫的免疫反应性

通过应用抑制素真核表达质粒pcINH和抑制素融合表达质粒pCIS分别经肌肉注射免疫大鼠的2个系列试验，检测了免疫鼠抑制素抗体水平及抗体阳性鼠的比例。结果，抑制素质粒pcINH经脂质体介导免疫大鼠，50％（13／26）的个体产生了抑制素阳性抗体。40μg组产生的抗体水平最高；抑制素融合表达质粒pCIS免疫经盐酸普鲁卡因处理的大鼠，2次免疫获得了38.9％（23／54）的抗体阳性率。3次免疫获得55．6％（20／36）的抗体阳性率，100μg组产生的抗体水平最高；加强免疫可提高抗体的水平。但免疫剂量的增加并不一定能增加抗体阳性鼠的比例。     

Inhibin: Regulation of reproductive function and practical use in females

Inhibins are gonadal glycoprotein hormones selectively and potently inhibiting follicle‐stimulating hormone (FSH) secretion from the pituitary gland. Inhibins are produced mainly by the ovary and are purified from follicular fluid. Inhibins were shown to be produced in two forms through dimeric assembly of an α‐subunit and one of two closely related β‐subunits to form inhibin A (α‐βA) and inhibin B (α‐βB). Although inhibin subunits are expressed in various tissues, the gonads are the major source of circulating inhibins. While inhibins may act as a paracrine or autocrine factor in some tissues, their best understood roles are as endocrine regulators of pituitary FSH. In this review we focus our attention on more recent developments in inhibin research. We describe patterns of inhibin A and B secretion during the estrous cycle. We also review the immunization against inhibin α subunit as a practical method for superovulation. Superovulation has been induced successfully by passive or active immunization against the inhibin α‐subunit in several species such as mice, rats, hamsters, guinea pigs, cows, mares, ewes and goats. Furthermore, several studies have shown that oocytes superovulated with immunization against inhibin α‐subunit have the ability to develop normally, suggesting that inhibin immunization could be used as a practical method for superovulation in a wide range of animal species.     

抑制素(IB)主动免疫对牛超排效果的影响及机制

用猪精液抑制素 ( inhibin,IB)抗原主动免疫供体母牛 ,结合外源纯化 FSH超排处理 ,观察了抑制素主动免疫对牛超排效果的影响 ,并通过检测不同生理时期血液中相关生殖激素的水平 ,探讨了抑制素免疫影响牛超排效果的机制。选产后 50 d以上的 3～ 5岁荷斯坦母牛 1 2头和西杂青年母牛 1 2头 ,按其发情顺序随机分为试验组和对照组 ,每组 1 2头。试验组用完全福氏佐剂乳化的 IB抗原进行首免 ,间隔 2 0 d再用不完全福氏佐剂乳化的 IB抗原加免 1次 ,加免后 6 d开始采用纯化 FSH做超排处理 ,观察发情 ,适时进行配种 ,于配种后 7d进行非手术法采卵。对照组只是用纯化 FSH做超排处理。参试牛于不同生理时期颈静脉采血 ,分离血清 ,用放免法测定各生理时期血清中 FSH、LH、E2 、P4水平。结果表明 :( 1 )抑制素主动免疫可以提高牛超排效果 ,试验组头均胚胎数和头均可用胚胎数分别比对照组增加 2 .4枚和 1 .4枚 ( P<0 .0 5) ;( 2 )抑制素主动免疫可提高母牛血浆中 FSH水平 ,从而促使卵泡发育 ,增加胚胎数和可用胚胎数 ;( 3)在首免后到加免前 ,抑制素主动免疫对 LH、E2 、P4基础分泌基本无影响     

猪精液抑制素的提取与纯化及其生物活性的测定

新鲜猪精液经乙醇沉淀，丙酮清洗，得抑制素粗提物，再经ＳｅｐｈａｄｅｘＧ－１００凝胶过滤，收集蛋白质洗脱峰。采用小鼠ｈＣＧ卵巢增重抑制法（改良斯蒂尔曼－－波菜生物测定法）地其进行生物活性测定，粗品１０００μｇ剂量组对性成熟前雌性小鼠卵巢增重的抑制作用极显著（Ｐ〈０．０１），第二峰下降部分２００μｇ组的抑制效果显著（Ｐ〈０．０２）。表明从精液中可提取到具有生物活性的抑制素制品。