孕酮在犬子宫蓄脓发病过程中的作用

为探讨孕酮在犬子宫蓄脓发病中的作用,无菌采取健康犬与发病犬子宫角,进行常规石蜡切片、HE染色和孕酮受体的免疫组化检测(IHC);取健康犬子宫内膜上皮细胞进行原代培养。试验结果显示发病犬子宫内膜层不完整、脱落以及腺体数量增多、增大等;其子宫组织内孕酮受体阳性,并且集中分布在腺体周围;健康犬子宫组织内均未检测到孕酮受体。利用不同质量浓度的孕酮、雌激素对犬子宫内膜原代细胞进行刺激培养,发现孕酮刺激组的细胞体积明显增大。结果表明孕酮可导致内犬子宫内膜细胞病变,从而促进犬子宫蓄脓的发生。     

孕酮、干扰素τ对牛子宫内膜上细胞外基质及其相关配体表达的影响

为探索干扰素τ(IFN-τ)、孕酮对牛子宫内膜上细胞外基质(ECM)及其配基基因表达的影响,本研究在体外培养的牛子宫内膜细胞(bECs)培养液中,分别添加孕酮、bIFN-τ、孕酮和bIFN-τ混合物培养24h后,通过荧光定量PCR技术,检测bECs上Collagen、Laminin、Fibronectin、THBS和Tenascin等ECM基因和ECM配基基因Syndecan、CD44、CD47和RHAMM的表达。结果表明:在体外培养的牛子宫内膜细胞上,bIFN-τ显著地诱导了Tenascin、Collagen、RHAMM和Laminin基因的表达,孕酮显著地诱导了CD44、Fibronectin、RHAMM和Laminin基因的表达,但当bIFN-τ和孕酮联合作用时,CD44、THBS基因的转录水平受到抑制,Tenascin、Fibonectin、Colla-gen基因的转录水平恢复到对照组转录水平。研究结果显示,在胚胎植入过程中,IFN-τ和孕酮可能对牛子宫内膜上ECM及其配基基因表达具有调控作用。     

雌激素和孕酮对犬子宫内膜基质细胞增殖和孕酮受体表达的影响

本试验应用不同消化分离途径获取犬子宫内膜基质细胞,调节培养液中雌激素(E₂)和孕酮(P₄)的浓度,采用MTT法测定E₂和P₄浓度水平对犬子宫内膜基质细胞体外增殖的影响,利用细胞免疫组织化学法鉴定细胞并测定细胞孕酮受体(PR)表达与激素浓度水平的相关性。结果表明,E₂浓度变化(15、30、100 pg/mL)对犬子宫内膜基质细胞的增殖和PR的表达均没有显著的调节作用(P>0.05);P₄(15、30 ng/mL)对犬子宫内膜基质细胞的增殖有显著促进作用(P<0.05),P₄(3、15、30 ng/mL)对犬子宫内膜基质细胞PR的表达具有显著的抑制作用(P<0.05),其影响程度与浓度和作用时间关系密切。     

孕酮对奶牛子宫内膜上皮细胞Wnt/β-catenin信号通路关键蛋白表达的影响

为研究孕酮对奶牛子宫内膜上皮细胞Wnt/β-catenin信号通路关键蛋白表达的影响,本试验以原代奶牛子宫内膜上皮细胞为材料,添加不同浓度孕酮(0、1、3、5 ng/mL)对其培养,采用CCK-8法检测孕酮对细胞增殖的影响;免疫印迹法检测孕酮对Wnt/β-catenin信号通路关键蛋白表达的影响。结果表明:与对照组相比,添加1、3 ng/mL孕酮组奶牛子宫内膜上皮细胞增殖率提高(P>0.05),5 ng/mL孕酮组细胞增殖率降低(P>0.05);与对照组相比,1 ng/mL和3 ng/mL孕酮组Wnt/β-catenin信号通路关键蛋白β-catenin、cyclin D1和c-Myc的表达水平均升高(P<0.01),而5 ng/mL孕酮则抑制了β-catenin的表达(P<0.05),且cyclin D1和c-Myc蛋白表达较3ng/mL孕酮组有下降趋势(P>0.05)。综上,高浓度孕酮抑制了体外培养的原代奶牛子宫内膜上皮细胞Wnt/β-catenin信号通路的激活和表达,提示该信号通路参与了奶牛产后子宫复旧延迟进程,为进一步研究奶牛子宫内膜的发病机制提供思路。     

孕酮对奶牛子宫内膜上皮细胞MAPK信号通路的影响

本试验旨在探究孕酮对奶牛子宫内膜上皮细胞MAPK信号通路的影响。选取与奶牛产后体内相似浓度的孕酮作用于奶牛子宫内膜上皮细胞,模拟产后子宫内分泌环境,检测MAPK通路关键蛋白表达的变化。结果发现,不同浓度孕酮均未对细胞活性产生影响,且20 ng/m L浓度范围内的孕酮对奶牛子宫内膜上皮细胞无毒性作用。用0、1、3、5 ng/m L孕酮处理30 min后,并未对子宫内膜上皮细胞ERK、JNK和P38的磷酸化产生显著作用,且不同浓度孕酮之间也没有显著差异。由此得出结论,外源性孕酮不能通过直接抑制奶牛子宫内膜上皮细胞MAPK的活化起抗炎效应。     

奶牛子宫内膜组织体外培养方法的建立及前列腺素E_2和F_（2α）受体分布的研究

[目的]建立体外培养荷斯坦奶牛子宫内膜组织的方法及检测子宫内膜组织中前列腺素E_2和前列腺素F_（2α）受体的分布情况。[方法]挑选新鲜、健康的发情前期奶牛子宫内膜组织,采集双侧子宫角部位内膜组织,体外悬浮培养组织小块并通过组织学形态鉴定培养效果。通过RT-qPCR方法检测PGE_2受体EP1、EP2、EP3（EP3A、EP3B、EP3C、EP3D）、EP4和PGF_（2α）受体FP的表达情况。[结果]HE染色表明,体外培养的奶牛子宫内膜组织结构完整,细胞和腺体形态完好,细胞核完好;发情前期奶牛子宫角部位内膜组织中PGE_2和PGF_（2α）受体均有表达,且表达量存在差异,其中PGE_2受体EP4相对表达量最高,而EP1、EP3B及PGF_（2α）受体FP的相对表达量较低。[结论]成功地建立了奶牛子宫内膜组织的体外培养方法;PGE_2和PGF_（2α）的受体在奶牛子宫内膜组织中均有表达,但不同受体的表达量有所不同。     

孕酮对奶牛子宫内膜上皮细胞凋亡和容受性的影响

为研究孕酮对奶牛早期胚胎附植的影响,本试验使用不同质量浓度孕酮处理奶牛子宫内膜上皮细胞,CCK8检测细胞活力,流式细胞术分析细胞凋亡,实时荧光定量RT-PCR和Western blot检测凋亡相关基因(p53、Cyto-c、Bax、Bcl-2、Caspase-9、Caspase-8和Caspase-3)和容受性相关基因(TLR-4、NF-κB、IL-6、VEGF、LIF和EGF)的mRNA和蛋白表达变化。结果显示：10,25,50,100μg/L孕酮对细胞活力未见影响;10,100μg/L孕酮对细胞凋亡未见影响,对细胞p53、Cyto-c、Bax、Bcl-2、Caspase-9、Caspase-8和Caspase-3的mRNA及蛋白的表达未见影响,但显著下调了TLR-4 mRNA和蛋白表达水平,显著上调了LIF和VEGF mRNA和蛋白表达水平。结果表明孕酮不影响奶牛子宫内膜上皮细胞的凋亡,但能提高子宫容受性,降低免疫反应。     

孕酮对小鼠妊娠早期子宫内膜转化生长因子β1Ⅰ型受体表达的影响

采用免疫组织化学SABC法，研究了外源性孕酮对妊娠小鼠着床早期子宫内膜转化生长因子β1Ⅰ型受体(TGFβRⅠ)表达的影响。结果显示：(1)孕4d，注射2mg孕酮组，子宫表面上皮、腺上皮及基质中TGFpRI表达增加，与对照组和注射孕酮4mg组差异显著(P＜0．01)。注射孕酮4mg组，表面上皮TGFβRⅠ表达无显著增加；腺上皮细胞TGFβRⅠ表达稍有下降，但与对照组差异不显著；基质细胞TGFβRⅠ表达明显增加，与对照组差异显著(P＜0．01)。(2)孕5d，试验组表面上皮和腺上皮TGFβRⅠ随着孕酮剂量的加大，其表达下降；基质细胞TGFβRⅠ表达则随孕酮剂量的增加而增多，且3组间差异显著(P＜0．01)。(3)孕6d，注射2mg组，表面上皮及基质细胞TGFβRⅠ表达增加，与对照组差异显著(P＜0．01)，而腺上皮TGFβRⅠ表达呈下降趋势。注射4mg组，表面上皮、腺上皮及基质表达变化趋势同孕5d。结论：外源性孕酮对妊娠小鼠着床早期子宫内膜转化生长因子β1型受体表达有一定影响，即孕酮可显著提高基质TGFβRⅠ表达，但对腺上皮TGFβRⅠ的表达有下调作用；对表面上皮TGFβRⅠ的表达作用不定，2mg使表面上皮TGFβRⅠ表达增多，4mg使TGFβRⅠ表达降低。     

Aroclor 1254致孕鼠子宫内膜细胞损伤模型的建立

为建立Aroclor 1254致孕鼠子宫内膜细胞损伤模型,通过分离、培养怀孕大鼠的原代子宫内膜细胞,用不同浓度的Aroclor 1254处理子宫内膜细胞48 h,倒置显微镜下观察细胞形态,MTT法检测细胞的活力,Western blot法检测细胞中CYP450的表达,并通过ELISA法检测细胞培养基中TNF-α、IL-6、雌二醇(estradiol,E2)和孕酮(progesterone,P4)的含量,来确定Aroclor 1254是否对细胞造成损伤。结果表明,随着Aroclor 1254浓度的升高,子宫内膜细胞的损伤及抑制率也随之增高,CYP450的表达随Aroclor 1254浓度的升高而升高。TNF-α和IL-6的水平比对照组显著升高(P0.05),E2和P4的水平则比对照组显著降低(P0.05)。这表明Aroclor 1254对体外培养子宫内膜细胞损伤程度呈浓度依赖性,10 mg/L的质量浓度作用48 h可建立可靠的子宫内膜细胞损伤模型。     

绵羊妊娠识别、建立和维持的信号系统

妊娠的建立和维持归因于胚体的信号和黄体对孕酮的产生,在反刍动物,胚体滋养层分泌的激素抑制子宫内膜产生前列腺素PGF2α。在发情周期的绵羊,孕酮下调子宫内膜腔上皮和子宫腺上皮孕酮受体基因的表达,并伴随着上皮雌激素受体和催产素受体的增加。在催产素的作用下,子宫开始分泌溶黄体作用的前列腺素F2α。在妊娠绵羊,孕体滋养层产生的干扰素可作用于子宫内膜而直接抑制雌激素受体基因和催产素受体基因的转录。孕酮、干扰素、胎盘催乳素和生殖激素共同组成了一个"作用网络"来调节子宫的功能分化和子宫腺的形态发生,从而维持绵羊妊娠。本文综述了绵羊妊娠识别、建立和维持的信号系统。     

Endogenous Progesterone Concentrations Affect Progesterone Release from Intravaginal Devices Used for Oestrous Synchronization in Cattle

Intravaginal progesterone‐releasing devices are largely used both as contraceptives in humans and as a component of oestrous synchronization protocols in cattle. To reduce costs in large‐scale timed artificial insemination, the reuse of these releasing devices is common. Passive hormone diffusion, however, depends on the concentration gradient, which could affect the amount of residual progesterone present in these devices after a first use. To evaluate the effect of the presence of a corpus luteum in the release of progesterone from intravaginal devices, three synchronization protocols were designed to simulate the effects of inserting the device in the early dioestrus, late dioestrus or anoestrus. Holstein‐Zebu cross‐bred heifers were randomly allocated into one of these three treatments, and a series of blood samples was taken to evaluate the plasma progesterone concentrations. After 8 days, the intravaginal devices were removed and underwent a previously validated alcoholic extraction technique to measure the residual progesterone. Non‐used devices were used as controls. As expected, the simultaneous presence of the intravaginal device and a corpus luteum resulted in increased plasma progesterone concentrations. Conversely, the amount of residual progesterone in the devices after use was inversely proportional to the plasma progesterone concentration. These results demonstrate that the release rate of progesterone from intravaginal devices is affected by the endogenous concentration of this hormone; consequently, the strategy for reuse should account for the category and expected luteal cyclic activity of the animals undergoing synchronization protocols.     

Effects of Estrogen and Progesterone on Canine Endometrial Stromal Cells Proliferation and Progesterone Receptor Expression

In this study different digestion and isolation methods were applied to obtain canine endometrial stromal cells (ESCs), different concentration levels were set for estrogen (E₂) and progesterone (P₄), then MTT method was used to measure their effect on cell proliferation in vitro, also cell immunohistochemistry was used for cell identification and measurement of effect on progesterone receptors (PR) expression.The results indicated that E₂ (15, 30 and 100 pg/mL) showed no significant regulations on both cell proliferation and PR expression, P₄ (15 and 30 ng/mL) had significant promoting effect on proliferation of ESCs (P<0.05), P₄ (3, 15 and 30 ng/mL) showed significant inhibitory effect on PR expression (P<0.05), the regulation level was related to concentration and acting time.     

Progesterone receptors in feline mammary adenocarcinomas

Estradiol and progesterone receptors were measured in tumor cytosols from 3 intact and 4 neutered female cats with spontaneously occurring mammary adenocarcinomas. Serum from 2 of the intact cats which had been in estrus 4 and 4 to 6 weeks before tumor excision contained progesterone concentrations of 16.2 and 2.2 ng/ml, respectively; serum progesterone in the other cats was less than 2 ng/ml. Estradiol receptors were not detected in any cytosols. Progesterone receptors were detected in all of the cytosols, in concentrations ranging from 4.0 to 11.7 (mean = 7.2) fmol/mg of protein. Scatchard plot analysis of tumor cytosol from an 8th cat with mammary adenocarcinoma revealed presence of high affinity progesterone binding with a dissociation constant (Kd) of 3.47 nM. Tumor receptor content could not be correlated with stage of the estrous cycle nor with whether the cat was intact or neutered.     

孕酮对鸡蛋壳品质的影响

本试验旨在通过腿肌注射孕酮来研究孕酮对蛋壳品质的影响。选取72只40周龄的海兰褐蛋鸡,随机分为6组,每组12只蛋鸡,单笼饲养。对照组注射花生油;处理1在排卵后5 h注射1.00 mg/kg体重(BW)的孕酮;处理2和处理3分别在排卵后5 h和2 h注射0.25 mg/kg BW的孕酮;处理4和处理5分别在排卵后2 h和1 h注射0.15 mg/kg BW的孕酮。处理后第2天上午收集各组所产鸡蛋,测定蛋壳强度和厚度。结果表明:处理1所产蛋均为破壳蛋,处理2含部分破壳蛋和部分蛋壳完好的鸡蛋,处理3、处理4和处理5没有破壳蛋;与对照组相比,处理1和处理2的蛋壳厚度降低(P<0.05),处理4蛋壳强度和厚度增加(P<0.05),处理3和处理5的蛋壳强度和厚度无显著差异;与对照组比,处理1和处理4的蛋壳超微结构差异显著。结果显示,孕酮可以影响蛋壳强度和厚度,改变蛋壳超微结构,其影响程度与剂量和注射时间有关。     

Seasonal Changes in Luteal Progesterone Concentration and mRNA Expressions of Progesterone Synthesis-related Proteins in the Corpus Luteum of Mares

Although circulating progesterone (P(4)) levels tend to change with the season, little is known about the seasonal changes of P(4) synthesis-related proteins in the corpus luteum (CL) of mares. To examine these changes, seventy-four ovaries containing a CL were collected from Anglo-Norman mares at a local abattoir in Kumamoto, Japan (~N32°), five times during one year. The stages of the CLs were classified as early, mid and regressed by macroscopic observation of the CL and follicles. The mid CL, which had the highest P(4) concentration, was used to evaluate the seasonal changes in P(4) synthesis. The luteal P(4) concentration and mRNA expression of luteinizing hormone receptor (LHCGR) were lowest during early winter and highest during late winter. The mRNA expressions of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) were lowest during early winter and increased during late winter. These results suggest that P(4) synthesis in the CL is affected by the seasonal changes in the mRNA expressions of P(4) synthesis-related proteins in mares.     

绵羊孕酮受体基因的PCR-SSCP分析

作者设计3对引物,采用PCR-SSCP方法检测孕酮受体（progesterone receptor,PGR）基因DNA结合区和配体结合区序列在高繁殖力绵羊品种（小尾寒羊和湖羊）和低繁殖力绵羊品种（多赛特和特克赛尔）中的单核苷酸多态性,同时研究该基因对绵羊繁殖力的影响;对小尾寒羊PGR基因外显子5、6和9克隆测序,结合GenBank提供的绵羊PGR基因部分编码序列,拼接出绵羊PGR基因DNA结合区和配体结合区的完整序列,推导出编码的氨基酸序列;并将人、兔、狗、绵羊、小鼠和大鼠的PGR基因这两个区域的核苷酸与氨基酸序列进行比较。结果表明,PGR基因DNA结合区和配体结合区序列在所检测的4个绵羊品种中都不存在单核苷酸多态性;人、兔、狗、绵羊、小鼠、大鼠PGR基因DNA结合区和配体结合区的核苷酸序列同源性分别为88.6%~97.2%和84.4%~96.3%,氨基酸序列同源性分别为97.6%~100%和96.3%~99.6%。可见,哺乳动物PGR基因DNA结合区和配体结合区序列保守性很强,这两个区域可能不是影响绵羊高繁殖力的功能结构域。