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Gene expression during pre- and peri-implantation embryonic development in pigs 猪着床前和着床期胚胎发育过程中的基因表达
Pub Date : 2020-10-12 DOI: 10.1530/biosciprocs.16.0013
P. Maddox‐Hyttel, A. Dinnyés, J. Laurinčík, D. Rath, H. Niemann, H. Rosenkranz, H. Wilmut
'Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, 1870 Frederiksberg C, Denmark; 2Department of Gene Expression and Development, Roslin Institute, Roslin, UK;3Constantin the Philosopher University and 4 Research Institute of Animal Production, Nitra, Slovak Republic; 5Department of Biotechnology, Institute for Animal Science and Behaviour (FAL), Mariensee, 31535 Neustadt, Germany; and 6 Veterinary University, Josef Baumann Cassel, A-1210 Vienna, Austria
1870年,丹麦弗雷德里克斯堡皇家兽医和农业大学解剖和生理学系;2英国罗斯林罗斯林研究所基因表达与发育系;3哲学大学和动物生产研究所,斯洛伐克共和国尼特拉;5动物科学与行为研究所生物技术系,马里恩塞,德国诺伊施塔特31535;和6兽医大学,Josef Baumann Cassel,A-1210,维也纳,奥地利
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引用次数: 11
The Control of Ovulation 排卵的控制
Pub Date : 2020-10-12 DOI: 10.1530/biosciprocs.11.0010
S. Webel, B. Day
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引用次数: 0
Changes in responsivenessto bicarbonate under capacitating conditions in liquid preserved boar spermatozoa in vitro 体外液体保存猪精子在能化条件下对碳酸氢盐反应性的变化
Pub Date : 2020-10-12 DOI: 10.1530/biosciprocs.18.0006
H. Henning, A. Petrunkina, R. Harrison, D. Waberski
Liquid-stored boar semen is commonly used for artificial insemination (Al) up to 72 h after dilution. Insemination with semen stored for longer periods generally results in reduced fertility. Standard semen parameters, i.e. motility and membrane integrity, usually give no indication of this reduction. Therefore, more sensitive methods are needed for detection of storage-induced changes in sperm quality. Capacitation has long been known to be an essential step in fertilization. In a number of studies bicarbonate hasbeen shown to be the key capacitating agent in boar sperm in vitro (reviewed in Harrison & Gadella 2005). The ability of sperm to respond to bicarbonate in vitro by undergoing capacitatory changes can be measured as a sperm property crucial to fertilization (Petrunkina et al. 2005a; Silva & Gadella 2006). In this study we used calcium influx as a parameter to investigate the responsiveness of stored semen samples to bicarbonate. This parameter has been shown to be sensitive with respect to evaluating detrimental effect of cooling during liquid storage of boar sperm (Petrunkina et al. 2005b). Three ejaculates from each of 14 boars of proven fertility were diluted in Beltsville Thawing Solution (BTS)extender to a concentration of 20 x 106sperm / ml and stored at 17°C. After 12, 24, 72, 120 and 168 h of storage, motility was assessedin the diluted semen with a CASA-system, and membrane integrity was checked with propidium iodide (PI)and FITC-conjugated peanut agglutinin (FITC-PNA) using a flow cytometer. Samples were then washed through Percoll, loaded with the calcium probe Fluo-3-AM and PI, and incubated at 38°C in parallel in two variants of a Tyrode's medium. Medium A contained 15 mM bicarbonate aswell as 2 mM Ca2+,whereas bicarbonate was omitted from medium B; incubation in medium A was performed under 5°/c.CO2. Changes in Ca2+ influx were assessedon a flow cytometer at 3, 20, 40, 60, 90, 120, 150 and 180 min. The resulting kinetics of cell sub-populations were compared between media and storage time points, based on analyses of the non-agglutinated population. During storage, motility declined only from 89.0 ± 3.2 to 74.4 ± 10.4°/c.(p —0.001) and membrane integrity from 83.1 ± 4.2 to 71.0 ± 20.9°/s (p —0.001). However, bicarbonate induced marked changes in membrane permeability, asmeasured by increases in the population of Ca2+-positiveand Pl-negative (live) cells as well as by increases in the population of PI-positive (dead) cells. After 12 and 24 h of storage, the population of Ca2+-positiveand Pknegative cells reached a maximum within 90 min of incubation in medium A, but as storage was prolonged the increase lessened although it reached its maximum more rapidly (after 40-60 min). A time point of 60 min was chosen for comparisons between storage periods and media. Values for the total % Ca2*-positive / PI-negative cells in medium A declined significantly from 21.6 ± 6.4% at 12 h to 15.7 ± 2.78% at 72 h of storage (p < 0.01) after
液体储存的猪精液通常用于人工授精(Al),稀释后可达72小时。将精液储存较长时间的人工授精通常会导致生育能力下降。标准精液参数,即活力和膜完整性,通常没有显示这种减少。因此,需要更灵敏的方法来检测储存引起的精子质量变化。能化一直被认为是受精的一个重要步骤。在许多研究中,碳酸氢盐已被证明是猪精子体外的关键激活剂(回顾在Harrison & Gadella 2005)。精子在体外对碳酸氢盐作出反应的能力可以通过经历容量变化来衡量,这是精子对受精至关重要的特性(Petrunkina等人,2005;Silva & Gadella 2006)。在这项研究中,我们使用钙内流作为一个参数来研究储存的精液样本对碳酸氢盐的反应性。在评估猪精子液体储存期间冷却的有害影响方面,该参数已被证明是敏感的(Petrunkina等人,2005b)。在贝尔茨维尔解冻液(BTS)扩展剂中稀释14头已证实生育能力的公猪的3次射精,浓度为20 x 106个精子/ ml,并在17°C保存。储存12、24、72、120和168 h后,用casa系统评估稀释后的精液的运动性,用流式细胞仪检测碘化丙啶(PI)和fitc -共轭花生凝集素(FITC-PNA)的膜完整性。然后将样品通过Percoll洗涤,装载钙探针Fluo-3-AM和PI,并在38°C的两种Tyrode培养基中平行孵育。培养基A中含有15mm碳酸氢盐和2mm Ca2+,而培养基B中没有碳酸氢盐;在5°/c.CO2条件下在培养基A中孵育。在3、20、40、60、90、120、150和180分钟时,用流式细胞仪评估Ca2+内流的变化。基于对非凝集细胞群的分析,比较了培养基和储存时间点之间细胞亚群的动力学结果。贮藏期间,运动度仅从89.0±3.2°/c下降到74.4±10.4°/c。(p -0.001),膜完整性从83.1±4.2°/s到71.0±20.9°/s (p -0.001)。然而,碳酸氢盐诱导了细胞膜通透性的显著变化,这可以通过Ca2+阳性和pl阴性(活)细胞数量的增加以及pi阳性(死)细胞数量的增加来测量。经过12和24 h的储存,Ca2+阳性和pk阴性细胞的数量在培养液a中培养90 min内达到最大值,但随着储存时间的延长,尽管更快(在40-60 min后)达到最大值,但增加幅度减小。选择一个60分钟的时间点来比较存储周期和介质。在A培养基中,Ca2*阳性/ pi -阴性细胞的总百分比从12 h时的21.6±6.4%显著下降到72 h时的15.7±2.78% (p < 0.01),之后保持不变。在培养3分钟时,培养基A中pl阳性(死亡)细胞的比例在12小时时为9.8±4.9%,在168小时时为14.7±3.3%,而在培养60分钟后,无论储存时间如何,这一比例都上升到46%左右。相反,在培养基B中,两种群体(即Ca2+阳性/ pl阴性和pl阳性细胞)的60 min值虽然最初远低于培养基A,但通过-显著增加
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引用次数: 2
Nutrition and Reproduction 营养与生殖
Pub Date : 2020-10-12 DOI: 10.1530/biosciprocs.11.0029
Dannis Cole
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引用次数: 0
The Endocrine Control of Parturition 分娩的内分泌控制
Pub Date : 2020-10-12 DOI: 10.1530/biosciprocs.11.0016
N. First, J. K. Lohse, B. Nara
The pig evolved as a litter bearing species with the maintenance of pregnancy dependent on the continued presence of the litter in utero and on continued production of ovarian progesterone. The length of gestation is reasonably precise, being approximately 112-116 days depending on the breed, size of litter and season (Cox, 1964; Bichard et al. , 1976; Aumaitre, Deglaire and LeBost, 1979). Parturition occurs slightly more frequently in the late afternoon and at night (Bichard et at , 1976; Boning, 1979) but no differences are seen in the frequency of day and night delivery when parturition is Artificially induced (Hammond and Matty, 1980). For an individual herd a knowledge of the average length of gestation and exact breeding dates are essential for optimal piglet survival and growth when parturition is to be induced. The entire parturition process requires 2-5 hours with piglets being delivered at approximately 12-16 minute intervals (Sprecher et aL, 1974; see Table 16.1). Piglets are delivered randomly from the two uterine horns (Dziuk and Harmon, 1969; Taverne et al., 1977). They sometimes pass each other in birth order (Taverne a aL , 1977) and the placentas are delivered either in part after the emptying of one uterine horn or within approximately four hours after the last piglet is delivered (Jones, 1966). This is at a time when plasma levels of oxytocin are elevated (Taverne, 1979). Parturition is normally preceded by udder oedema, attempted maternal nest building and a milk ejection response (First and Bosc, 1979). Parturition is not without complications. Body temperature increases 13±4.1 hours before delivery of the first piglet, reaches a peak of 0.6 °C-1.2 °C above the normal of 38.3 ±0.3 °C and in healthy sows returns to near normal within 24 hours (Elmore et al. , 1979). Parturition is often complicated by a disease called Mastitis—Metritis—Agalactia in which the sow's temperature remains greatly elevated for a prolonged period and she refuses to provide milk for the piglets. Not all piglets survive farrowing; at least 6% are born dead (Randall, 1972; Sprecher et , 1974; Leman, Hurtgen and Hilley, 1979) and the last piglet in each uterine horn has less than a 50% chance of survival (Sevier and Dziuk, 1976). The factors influencing piglet survival were reviewed recently by Leman, Hurtgen and Hilley (1979). It is apparent from their
猪是一种产仔品种,妊娠期的维持取决于产仔在子宫内的持续存在和卵巢孕酮的持续产生。妊娠期相当精确,根据品种、产仔数和季节的不同,约为112-116天(Cox,1964;Bichard等人,1976年;Aumaitre、Deglaire和LeBost,1979年)。分娩发生在下午晚些时候和晚上的频率略高(Bichard et at,1976;Boning,1979),但人工诱导分娩时,昼夜分娩频率没有差异(Hammond和Matty,1980)。对于一个个体群来说,了解平均妊娠期和确切的繁殖日期对于仔猪在分娩时的最佳生存和生长至关重要。整个分娩过程需要2-5小时,小猪每隔12-16分钟分娩一次(Sprecher等人,1974;见表16.1)。小猪从两个子宫角随机分娩(Dziuk和Harmon,1969;Taverne等人,1977)。它们有时会按出生顺序相互传递(Taverne a aL,1977),胎盘要么在一个子宫角排空后部分分娩,要么在最后一只小猪分娩后约四小时内分娩(Jones,1966)。这是在血浆催产素水平升高的时候(Taverne,1979)。分娩前通常会出现乳房水肿、试图建立母体巢穴和排奶反应(First和Bosc,1979)。分娩并非没有并发症。在第一头小猪分娩前13±4.1小时,体温升高,达到峰值0.6°C-1.2°C,高于正常值38.3±0.3°C,健康母猪在24小时内恢复到接近正常值(Elmore等人,1979)。分娩通常会因一种名为乳房炎的疾病而变得复杂。乳房炎是一种无乳症,母猪的体温在很长一段时间内都会大幅升高,并且拒绝为小猪挤奶。并不是所有的小猪都能在分娩后存活下来;至少6%的小猪出生时死亡(Randall,1972;Sprecher等人,1974;Leman、Hurtgen和Hilley,1979),每个子宫角的最后一只小猪存活的几率不到50%(Sevier和Dziuk,1976)。Leman、Hurtgen和Hilley(1979)最近对影响仔猪存活的因素进行了综述。从他们的
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引用次数: 0
Embryo Transplantation and Preservation 胚胎移植与保存
Pub Date : 2020-10-12 DOI: 10.1530/biosciprocs.11.0014
C. Polge
Effective techniques for embryo transplantation in some laboratory and farm animals are now well established. Methods for collection and transfer of embryos in the pig were first developed in the early 1960s (Hancock and Hovell, 1962; Dziuk, Polge and Rowson, 1964; Vincent, Robison and Ulberg, 1964) and since then they have been applied mainly in research. Embryo transplantation has proved to be a valuable experimental tool in a number of studies concerned with early embryonic development, the survival of embryos in vivo or in vitro, migration and spacing of embryos within the uterus and factors affecting the maintenance of pregnancy. Future research is also likely to be concerned increasingly with cellular and genetic manipulation of eggs and embryos in vitro and the application of these techniques depends to a large extent on having reliable methods for the culture of embryos and their subsequent transfer. Practical applications, particularly in farm animals, are also important and the best example is in cattle where methods developed for research have now been extended very successfully into the practice of animal breeding. Applications in pig husbandry have so far been on a relatively small scale. The high fecundity and reproductive rate in pigs compared with cattle will never provide the same economic incentive to apply such methods for the purpose of genetic improvement or to get more offspring from a few superior animals. On the other hand, strict control of disease, especially in large intensive units, is a most important aspect of modern pig husbandry and embryo transplantation should provide the safest method of introducting new genetic material into closed herds. It is mainly for this reason, and perhaps also for the possibility of transporting embryos between countries, that embryo transplantation in pigs is likely to be applied as a practical measure.
一些实验室和农场动物胚胎移植的有效技术现已建立。收集和移植猪胚胎的方法最早是在20世纪60年代早期发展起来的(Hancock和Hovell, 1962;Dziuk, Polge和Rowson, 1964;文森特,罗宾逊和乌尔伯格,1964年),从那时起,它们主要应用于研究。胚胎移植已被证明是一种有价值的实验工具,在许多有关早期胚胎发育、胚胎在体内或体外存活、胚胎在子宫内的迁移和间距以及影响妊娠维持的因素的研究中。未来的研究也可能越来越关注体外卵子和胚胎的细胞和遗传操作,这些技术的应用在很大程度上取决于胚胎培养及其随后转移的可靠方法。实际应用,特别是在农场动物上,也很重要,最好的例子是在牛身上,为研究而开发的方法现在已经非常成功地扩展到动物育种实践中。到目前为止,在养猪业中的应用规模相对较小。与牛相比,猪的高繁殖力和繁殖率永远不会提供同样的经济动机来应用这种方法来进行遗传改良或从少数优质动物中获得更多的后代。另一方面,严格控制疾病,特别是在大型集约化单位,是现代养猪业的一个最重要的方面,胚胎移植应该是向封闭猪群引入新遗传物质的最安全的方法。主要是由于这个原因,也可能是由于在国家之间运送胚胎的可能性,猪的胚胎移植很可能被作为一种实际措施加以应用。
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引用次数: 5
Introduction to Pregnancy in Waiting: Embryonic Diapause in Mammals: Proceedings of the Third International Symposium on Embryonic Diapause 等待怀孕的介绍:哺乳动物的胚胎滞育:第三届胚胎滞育国际研讨会论文集
Pub Date : 2020-06-03 DOI: 10.1530/biosciprocs.10.001
B. Murphy, K. Jewgenow, M. Renfree, S. Ulbrich
The capacity of the mammalian embryo to arrest development during early gestation is a topic that has fascinated biologists for over 150 years. The first known observation of this phenomenon was in a ruminant, the roe deer (Capreolus capreolus) in 1854, later confirmed in a number of studies in the last century [1]. The phenomenon, now known as embryonic diapause, was then found to be present in a wide range of species and across multiple taxa. Since that time, its biological mystery has attracted studies by scientists from around the globe.
哺乳动物胚胎在妊娠早期阻止发育的能力是一个150多年来一直吸引着生物学家的话题。1854年,人们首次在反刍动物狍身上观察到这种现象,后来在上个世纪的一系列研究中得到了证实。这种现象,现在被称为胚胎滞育,后来被发现存在于许多物种和多个分类群中。从那时起,它的生物学奥秘吸引了全球科学家的研究。
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引用次数: 1
Autophagy and multivesicular body formation in blastocysts during the experimental diapause in mice 小鼠实验滞育过程中囊胚自噬和多泡体的形成
Pub Date : 2020-06-03 DOI: 10.1530/biosciprocs.10.011
Hj Lim, H. Shin, J. J. Hyun, H. Song
In experimentally induced diapause model in mice, blastocysts remain dormant for an extended period but resume implantation competency upon estrogen injection. The underlying mechanism by which extended longevity of dormant blastocysts is maintained is unclear. We have previously shown that dormant blastocysts, during experimentally induced diapause, exhibit heightened autophagic activation. Activation of autophagy appears to be a crucial adaptive response for survival in the unfavorable uterine environment, as inhibiting autophagy reduces the survival rate of dormant blastocysts. As a unique cell biological change occurring following estrogen supplementation to activate dormant blastocysts, multivesicular bodies (MVBs) accumulate in the trophectoderm. In various cellular contexts, autophagy and MVB formation are linked cell biological phenomena. Herein, we discuss the implications of these cell biological changes in dormant and activated blastocysts.
在小鼠实验诱导滞育模型中,囊胚在较长时间内处于休眠状态,但在注射雌激素后恢复着床能力。维持休眠囊胚延长寿命的潜在机制尚不清楚。我们之前的研究表明,在实验诱导的滞育过程中,休眠囊胚表现出高度的自噬激活。自噬的激活似乎是在不利的子宫环境中生存的关键适应性反应,因为抑制自噬会降低休眠囊胚的存活率。作为补充雌激素激活休眠囊胚后发生的一种独特的细胞生物学变化,多泡体(multives水泡体,MVBs)在滋养外胚层积累。在各种细胞环境中,自噬和MVB的形成是相互关联的细胞生物学现象。在此,我们讨论了休眠和激活囊胚中这些细胞生物学变化的含义。
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引用次数: 0
Conserved mechanisms for putting pregnancy on hold in the mouse, mink and tammar wallaby 小鼠、水貂和灰袋鼠暂缓怀孕的保守机制
Pub Date : 2020-06-03 DOI: 10.1530/biosciprocs.10.003
J. Fenelon, G. Shaw, M. Renfree, B. Murphy
Embryonic diapause was first identified over 150 years ago, but many questions still remain about how the external and hormonal controls of embryonic diapause translate into how the uterus conveys information to the embryo. Current evidence suggests that the control of diapause is mediated by components of the uterine secretions. However, the identity of the essential signalling molecule(s) is unknown. The mouse (Mus musculus), the mink (Neovison vison) and the tammar wallaby (Macropus eugenii) are the three most extensively studied mammalian diapause species. Despite differences in the external and hormonal control of diapause between these three species, we have now found that there is conservation of numerous molecular factors around diapause and reactivation. This was first suggested via the conserved expression of various growth factors. The first evidence for a conserved mechanism resulted from a study on the muscle segment homeobox transcription factor (MSX) in the uterus during diapause, whose expression is conserved amongst the mouse, mink and wallaby. Following this was the evidence that inhibition of polyamines induces entry into diapause in both the mink and mouse. Thus, although the signalling mechanisms via which the uterus is induced into diapause vary amongst species, the molecular communication that occurs between the uterus and the embryo to control diapause is conserved. Given that these mechanisms are conserved across varying taxa, this implies a universal mechanism for maintaining embryo health amongst all mammals. New technologies are now allowing us to examine diapause from a global perspective and to increase our knowledge of this enigmatic stage of pregnancy.
胚胎滞育在150多年前首次被发现,但关于胚胎滞育的外部和激素控制如何转化为子宫如何向胚胎传递信息的许多问题仍然存在。目前的证据表明,滞育的控制是由子宫分泌物的成分介导的。然而,基本信号分子的身份是未知的。小鼠(Mus musculus)、水貂(Neovison vison)和袋鼠(Macropus eugenii)是研究最广泛的三种哺乳动物滞育物种。尽管这三个物种之间滞育的外部和激素控制存在差异,但我们现在已经发现,围绕滞育和再激活的许多分子因子是守恒的。这是通过各种生长因子的保守表达首次提出的。子宫肌段同源盒转录因子(MSX)在滞育过程中的表达在小鼠、水貂和小袋鼠中是保守的,这是子宫肌段同源盒转录因子(MSX)保守机制的第一个证据。随后的证据表明,抑制多胺诱导水貂和小鼠进入滞育。因此,尽管不同物种诱导子宫进入滞育的信号机制不同,但子宫和胚胎之间发生的控制滞育的分子通讯是保守的。鉴于这些机制在不同的分类群中是保守的,这意味着在所有哺乳动物中维持胚胎健康的普遍机制。现在,新技术使我们能够从全球的角度来研究滞育,并增加我们对怀孕这一神秘阶段的了解。
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引用次数: 0
Identification of mink (Neovison vison) fecal proteins during embryonic diapause and placental pregnancy for non-invasive pregnancy diagnosis in wildlife 水貂胚胎滞育和胎盘妊娠期间粪便蛋白的鉴定用于野生动物的非侵入性妊娠诊断
Pub Date : 2020-06-03 DOI: 10.1530/biosciprocs.10.007
E. Curry, J. Easley, J. Wojtusik, T. Roth
Currently, there is no method to diagnose pregnancy non-invasively in most wildlife species that experience delayed implantation and pseudopregnancy, either during embryonic diapause or placental pregnancy. The aim of this study was to utilize farmraised mink (Neovison vison) as a model species to evaluate changes in the fecal proteome associated with pregnancy. Specific objectives were to: 1) determine if fecal peptides were differentially abundant in parturient versus non-parturient mink and; 2) identify proteins of interest. Samples (n=12) were selected retrospectively from mink (n=6) that were parturient (n=3) or non-parturient (n=3) and were collected from parturient females during embryonic diapause and placental pregnancy or, on the same calendar dates from non-parturient females. Following protein extraction, twodimensional differential in-gel electrophoresis was utilized to assess differences in protein spot abundance among samples. The mean number of spots per gel was 2107±62.2 and spots meeting specific criteria (student’s t-test; P<0.10; >2.5 fold change between groups) were selected for identification via matrix-assisted laser desorption/ ionizationtime-of-flight mass spectrometry. During diapause, six spots (angiotensinconverting enzyme 2, interleukin-36 receptor antagonist, carboxypeptidase A1 (two spots), carboxypeptidase A2, and chymotrypsin-like protease CTRL-1) were higher in parturient and one spot (intestinal fatty acid-binding protein) was higher in nonparturient. During placental pregnancy, seven spots (cytosol aminopeptidase (three spots), calcium-activated chloride channel regulator 1, carboxypeptidase A1 (two spots), and chymotrypsin) were higher in parturient and two (ovalbumin and protein PRR14L) were higher in non-parturient. This is the first description of the mink fecal proteome related to pregnancy and of changes in specific fecal proteins during embryonic diapause in any species.
目前,在胚胎滞育或胎盘妊娠期间,大多数经历延迟着床和假妊娠的野生动物物种都没有非侵入性诊断妊娠的方法。本研究的目的是利用养殖水貂(Neovison-vison)作为模式物种来评估与妊娠相关的粪便蛋白质组的变化。具体目标是:1)确定分娩水貂和未分娩水貂的粪便肽含量是否存在差异;2) 鉴定感兴趣的蛋白质。样本(n=12)是从分娩(n=3)或未分娩(n=3)的水貂(n=6)中回顾性选择的,这些样本是从胚胎滞育和胎盘妊娠期间的分娩雌性或在同一日历日期从未分娩雌性采集的。蛋白质提取后,利用凝胶电泳中的二维差异来评估样品之间蛋白质斑点丰度的差异。每个凝胶的平均斑点数为2107±62.2,并通过基质辅助激光解吸/电离飞行时间质谱法选择符合特定标准的斑点(学生t检验;组间P2.5倍变化)进行鉴定。滞育期间,产妇体内有6个位点(血管紧张素转换酶2、白细胞介素-36受体拮抗剂、羧肽酶A1(两个位点)、羧肽蛋白酶A2和糜蛋白酶样蛋白酶CTRL-1)较高,非滞育者体内有1个位点(肠脂肪酸结合蛋白)较高。在胎盘妊娠期间,产妇中有7个斑点(胞浆氨肽酶(3个斑点)、钙激活的氯通道调节因子1、羧肽酶A1(2个斑点)和糜蛋白酶)较高,非产妇中有2个斑点(卵清蛋白和蛋白质PRR14L)较高。这是首次描述与妊娠有关的水貂粪便蛋白质组,以及任何物种胚胎滞育期间特定粪便蛋白质的变化。
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引用次数: 2
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