基于IL-17/NF-κB信号通路探讨敦煌医方大补脾汤联合奥沙利铂对胃癌荷瘤小鼠炎症免疫的影响

目的：研究敦煌医方大补脾汤对胃癌荷瘤小鼠的抑瘤作用以及基于IL-17/NF-κB信号通路探讨大补脾汤联合奥沙利铂对胃癌荷瘤小鼠炎症免疫的影响。方法：建立MFC胃癌皮下荷瘤小鼠模型,随机分为模型组、奥沙利铂组、大补脾汤高、中、低剂量联合奥沙利铂组[21.58、10.79、5.40 g/（kg·d）],每组10只,雌雄分笼饲养,接种8 d后开始给药,连续给药14 d;末次给药后次日眼球取血,处死小鼠,摘取肿瘤组织称重,计算抑瘤率;ELISA检测小鼠血清IL-17和IL-6含量,免疫组化（IHC）、RT-qPCR和Western blot分别检测小鼠肿瘤组织IL-17、IL-6、NF-κB和pNF-κB mRNA和蛋白表达。结果：奥沙利铂组、大补脾汤高、中、低剂量联合奥沙利铂组抑瘤率分别为33.02%、52.92%、46.33%和39.52%,各给药组肿瘤质量均较模型组显著降低（P<0.01）,大补脾汤高、中、低剂量联合奥沙利铂组显著高于奥沙利铂组（P<0.01）;与模型组相比,各给药组血清IL-17和IL-6含量、肿瘤组织IL-17、IL-6、NF-κB p65和pNF-κB ...     

大补脾汤联合奥沙利铂对胃癌荷瘤小鼠瘤组织TLR4/MyD88/NF-κB信号通路及下游相关分子的影响北大核心CSCD

目的:研究大补脾汤联合奥沙利铂对胃癌荷瘤小鼠瘤组织TLR4/MyD88/NF-κB信号通路及下游相关分子的影响,探讨其可能的作用机制。方法:采用昆明小鼠建立胃癌荷瘤模型,造模成功后将小鼠随机分为5组:模型组、奥沙利铂组(10 mg/kg)、大补脾汤高、中、低剂量(21.58、10.79、5.40 g/kg)联合奥沙利铂组,每组10只。造模成功后开始给药,连续给药14 d;末次给药后次日眼球取血,处死小鼠,摘取肿瘤组织称重,计算抑瘤率;采用苏木素-伊红(HE)染色后观察瘤组织形态,实时荧光定量聚合酶链式反应(RT-qPCR)、免疫组化法(IHC)和Western blot分别检测小鼠肿瘤组织中Toll样受体4(TLR4)、髓样分化因子88(MyD88)、肿瘤坏死因子受体相关因子6(TRAF6)和核因子κB(NF-κB)mRNA和蛋白相对表达,采用RT-qPCR检测小鼠肿瘤组织中TNF-α和IL-6 mRNA相对表达。结果:奥沙利铂组、大补脾汤高、中、低剂量联合奥沙利铂组瘤质量均较模型组显著降低(P<0.01),抑瘤率分别为37.12%、49.48%、42.10%、40.65%,大补脾汤高剂量联合奥沙利铂组与奥沙利铂组相比差异具有统计学意义(P<0.05);HE结果显示:与模型组相比,各给药组能明显降低肿瘤细胞密度,引起肿瘤细胞坏死;与模型组相比,各给药组肿瘤组织中TLR4、MyD88、TRAF6和NF-κB p65 mRNA和蛋白相对表达均显著降低(P<0.01,P<0.05);与奥沙利铂组相比,大补脾汤高、中剂量联合奥沙利铂组肿瘤组织中TLR4、MyD88、TRAF6和NF-κB p65 mRNA和蛋白相对表达均显著降低(P<0.05);与模型组相比,各给药组肿瘤组织中TNF-α和IL-6 mRNA相对表达均显著降低(P<0.01);与奥沙利铂组相比,大补脾汤高剂量联合奥沙利铂组肿瘤组织中TNF-α和IL-6 mRNA相对表达均显著降低(P<0.05)。结论:大补脾汤可通过抑制TLR4/MyD88/NF-κB信号通路下调下游炎症因子TNF-α和IL-6的表达,调节免疫微环境,发挥抗肿瘤作用,抑制胃癌发生发展。     

温补脾肾法对脾肾阳虚型UC大鼠JAK2/STAT3-SOCS6信号通路相关蛋白及基因表达的影响北大核心CSCD

目的:观察以温补脾肾法为指导的理中汤合四神丸对脾肾阳虚型溃疡性结肠炎(UC)大鼠模型病变结肠组织中JAK2、STAT3、p-STAT3、SOCS6蛋白及基因表达的影响。方法:采用病证结合造模方法复制脾肾阳虚型UC模型,成模大鼠随机分为模型组、柳氮磺砒啶组(SASP组)、理中汤合四神丸高、中、低剂量组,16只/组,同时设16只大鼠作为空白组。空白组与模型组给予蒸馏水灌胃,各治疗组给予对应的药物及剂量灌胃治疗,连续21 d。HE染色观察大鼠结肠黏膜组织病理变化;免疫组化法检测大鼠结肠黏膜组织中JAK2、STAT3、p-STAT3、SOCS6蛋白表达情况;RT-qPCR检测大鼠JAK2、STAT3、SOCS6mRNA表达情况;ELISA检测大鼠组织和血清中TGF-β1、ICAM-1含量。结果:与空白组相比,模型组大鼠结肠黏膜组织JAK2、STAT3、p-STAT3蛋白表达显著升高(P<0.01),JAK2、STAT3、p-STAT3蛋白着色深,表达呈强阳性,SOCS6蛋白表达显著降低(P<0.01),SOCS6蛋白着色浅,表达呈弱阳性。JAK2、STAT3 mRNA表达显著升高,SOCS6 mRNA表达显著降低(P<0.01),组织和血清中TGF-β1含量显著降低(P<0.01),ICAM-1含量显著升高(P<0.01);与模型组相比,中药高、中剂量组及SASP组JAK2、STAT3、p-STAT3蛋白表达显著降低(P<0.01),JAK2、STAT3、p-STAT3蛋白着色浅,表达呈弱阳性,SOCS6蛋白表达显著升高(P<0.01),SOCS6蛋白着色深,表达呈强阳性。JAK2、STAT3 mRNA表达显著降低(P<0.01),SOCS6 mRNA表达显著升高(P<0.01),组织和血清中TGF-β1含量显著升高(P<0.01),ICAM-1含量显著降低(P<0.01)。结论:以温补脾肾法为指导的理中汤合四神丸可能通过抑制JAK2/STAT3-SOCS6信号通路的激活,调节免疫系统平衡,修复受损结肠黏膜,达到治疗UC的目的。     

苏铁总黄酮对Lewis 肺癌模型小鼠的抑制作用及免疫功能的影响

目的:探讨苏铁总黄酮对Lewis 肺癌模型小鼠的抑制作用及免疫功能的影响。方法:选取造模成功的荷瘤小鼠84 只,随机分为模型组、空白对照组、苏铁总黄酮高、中、低剂量组、顺铂组、联合组,每组12 只。分别给药干预14 d,于第15天摘眼球法取血,常规法剥取肿瘤组织、肺、脾、胸腺,并分别称重,计算抑瘤率、肺脏指数、脾脏指数和胸腺指数;采用ELISA法检测血清中IL-2、IL-10 的含量。结果:苏铁高、中、低各剂量组、顺铂组和联合组小鼠瘤质量与模型组比较均降低,差异具有显著意义(P<0.01),苏铁总黄酮高、中、低各剂量组的脾脏指数和胸腺指数明显增加,且远远高于顺铂组与联合组。ELISA 法检测结果显示,与模型组比较,苏铁总黄酮高中低各剂量组、顺铂组和联合组的IL-2 水平明显升高,而IL-10 水平除了苏铁总黄酮中剂量组之外的其他各组均明显升高。结论:苏铁总黄酮可以调节免疫细胞因子IL-2、IL-10 的表达水平,抑制Lewis 肺癌模型小鼠肿瘤细胞的生长与转移并提高其免疫功能。     

IGF-2通过调控Treg/Th17免疫平衡促进小鼠结肠癌细胞移植瘤生长

目的 探讨胰岛素样生长因子-2(IGF-2)对结肠癌荷瘤小鼠Treg免疫活性的影响。方法 将24只Balb/c小鼠左侧腋部皮下注射小鼠结肠癌细胞CT26制备结肠癌荷瘤小鼠模型,并随机分为对照组（生理盐水）和IGF-2低、中、高浓度组（25μg/kg、50μg/kg、100μg/kg IGF-2）。检测胸腺指数和脾脏指数,酶联免疫（ELISA）法检测血清IL-10、IL-18、TGF-β1、IFN-γ水平,流式细胞仪检测血液Treg、Th17细胞比例,瘤组织称质量、计算小鼠促瘤率,TUNEL染色检测瘤组织细胞凋亡率,Western blot法检测瘤组织中Ki67、Caspase-3蛋白表达。结果 与对照组相比,IGF-2低、中、高浓度组结肠癌荷瘤小鼠血清IL-10、TGF-β1水平、血液Treg、Th17、Treg/Th17细胞比例、促瘤率及瘤组织中Ki67蛋白水平显著升高（P<0.05）,血清IL-18、IFN-γ水平、胸腺指数和脾脏指数及瘤组织细胞凋亡率、Caspase-3蛋白水平显著降低（P<0.05）;随着IGF-2浓度的升高,结肠癌荷瘤小鼠血清IL-10、TGF-β...     

紫杉醇联合奥沙利铂对非小细胞肺癌模型小鼠的治疗作用

目的观察紫杉醇联合奥沙利铂对荷瘤裸鼠非小细胞肺癌治疗效果以及对PDCD5和XIAP表达的影响。方法制备裸鼠肺癌荷瘤模型,将荷瘤小鼠随机分为空白组、0.9%氯化钠溶液组、奥沙利铂组、紫杉醇组和紫杉醇联合奥沙利铂组;q-PCR检测各组PDCD5和XIAP基因表达水平;Western bolt分析各组PDCD5和XIAP蛋白表达情况;对比分析各组肿瘤组织重量。结果紫杉醇联合奥沙利铂组PDCD5 mRNA表达水平最高(P0.01),XIAP mRNA表达水平最低(P0.01);紫杉醇联合奥沙利铂组PDCD5蛋白表达最高(P0.01),XIAP蛋白表达最低(P0.01);对比各分组肿瘤组织重量,紫杉醇联合奥沙利铂组肿瘤质量最小(P0.01)。结论紫杉醇联合奥沙利铂化疗能显著增加PDCD5表达和降低XIAP表达,能使已发生的非小细胞肺癌组织质量显著减少。     

黄芪多糖通过JAK/STAT3通路抑制口腔鳞癌SCC-25裸鼠移植瘤

目的　阐明黄芪多糖（AP）对口腔鳞癌（oral squamous cell carcinoma）细胞系SCC-25移植瘤模型小鼠的影响和机制。 方法　皮下注射口腔鳞癌细胞系SCC-25建立异种移植瘤模型,将移植瘤BALB/c裸鼠模型随机分为4组：对照组（cTRL）、AP低剂量组（AP 10 mg）、AP中剂量组（AP 25 mg）和AP高剂量组（AP 50 mg）。AP治疗后,检测移植瘤体积和小鼠存活率;免疫组化检测Ki67和血管内皮生长因子（vascular endothelial growth factor,VEGF）表达水平;TUNEL实验检测移植瘤细胞凋亡情况;Western blot 检测JAK2/STAT3通路蛋白表达情况和磷酸化情况。 结果　与对照组相比较,AP低、中、高剂量组肿瘤体积明显减小（P<0.01）,小鼠存活率明显增加（P<0.01）,Ki67和VEGF表达水平明显下降（P<0.01）,JAK2/STAT3/c-myc磷酸化水平明显被抑制（P<0.01）。 结论　AP以剂量依赖抑制SCC-25移植瘤体积,提高移植瘤小鼠存活率,下调Ki67和VEGF表达水平和JAK2/STAT3磷酸化水平。AP可能通过抑制JAK2/STAT3/c-myc信号通路抑制口腔鳞癌细胞SCC-25移植瘤生长。     

刺五加多糖对Lewis 荷瘤小鼠抗肿瘤免疫调节作用及机制的研究

目的:探讨刺五加多糖(ASPS)对Lewis 荷瘤小鼠TLR4 信号转导通路的调控机制。方法:用C57BL/6 建立 Lewis 实体型荷瘤小鼠模型,随机分为生理盐水组(NS 组)、阿霉素组(ADM 组)和ASPS 低、中、高剂量组,灌胃给药25 d 后,称 重计算瘤重、抑瘤率、胸腺指数和脾脏指数。收集荷瘤小鼠眼球血,ELISA 检测外周血中细胞因子TNF-α、IL-1β、IL-6 和IL- 12p70 的分泌情况。采用实时荧光定量PCR(Q-PCR)和Western blot 法分别检测荷瘤小鼠脾细胞中TLR4 相关节点TLR4、 MyD88、TRAM、TRAF6、NF-κB p65、AP-1 基因和蛋白的表达水平。结果:与NS 组相比,ASPS 中剂量组荷瘤小鼠的瘤重明显降 低,抑瘤率和免疫器官指数显著升高(P<0.05)。与NS 组相比,ASPS 显著促进荷瘤小鼠外周血TNF-α、IL-1β和IL-6 的分泌 (P<0.05),对IL-12p70 的产生无显著影响(P>0.05)。ASPS 显著上调TLR4 信号通路中TLR4、MyD88、TRAF6、NF鄄资B p65、 AP-1 基因和蛋白在荷瘤小鼠脾脏中的表达(P<0.05),而对TRAM 无显著作用(P>0.05)。结论:TLR4 信号通路可能是ASPS 在Lewis 荷瘤小鼠体内发挥免疫调节及抑制肿瘤作用的通路之一。     

注射用免疫核糖核酸Ⅱ的免疫调节及对顺铂的增效减毒作用

目的探讨注射用免疫核糖核酸Ⅱ(BP素)对顺铂(DDP)的增效减毒作用及对S180荷瘤小鼠的免疫调节作用。方法建立小鼠S180皮下移植瘤模型,随机分组,采用腹腔注射给药,连续给药10 d,通过白细胞(WBC)计数、骨髓有核细胞计数,取肿瘤、胸腺、脾脏称重,计算抑瘤率、胸腺指数、脾脏指数以及MTT法检测刀豆蛋白A(Con A)诱导的小鼠脾脏T淋巴细胞增殖,观察BP素对顺铂的增效减毒作用。通过ELISA法检测血清中的IL-1β、IL-6、TNF-α水平,通过腹腔巨噬细胞对鸡红细胞吞噬率的测定评价BP素对S180荷瘤小鼠的免疫调节作用。结果 BP素与DDP联合应用可显著提高DDP对S180荷瘤小鼠的抗肿瘤作用(P0.01),明显改善DDP化疗所致的脾脏指数下降,外周血WBC数、骨髓有核细胞数的减少及脾脏T淋巴细胞增殖抑制等毒副作用(P0.01或P0.05);单用BP素可显著降低荷瘤小鼠血清中的IL-1β、IL-6、TNF-α水平(P0.01或P0.05),提高腹腔巨噬细胞吞噬鸡红细胞的吞噬率(P0.01)。结论 BP素对DDP具有增效减毒作用,并对S180荷瘤小鼠具有免疫调节作用。     

A reappraisal of the feedback effects of oestradiol uppm luteinizing hormone surge

The objective of this study was to assess the temporal relationshipsof serum oestradiol and luteinizing hormone (LH) with regardto the feedback mechanism leading to the LH surge. Daily measurementsof oestradiol, LH and follicle stimulating hormone were madein 65 women with ovarian failure undergoing an evaluation ofendometrial response to oral cycle stimulation. Patients receivedincremental oral oestradiol valerate for varying durations atthe in-vitro fertilization unit of the Sheba Medical Center,Tel Hashomer, Israel. The treatment protocol involved the administrationof daily oestradiol valerate, starting with 1 mg/day, followedby a daily increment of 1 mg for a total of 4, 6 and 8 days.After the last day, a daily maintenance dose of 2 mg was continued.Serum oestradiol concentrations correlated with the dose oforal oestradiol valerate. Peak oestradiol concentrations (mean± SEM) were 572 ± 60, 721 ± 42 and 797± 53 pg/ml for 4, 6 and 8 days of oestradiol valerateadministration respectively. Serum LH, after an initial suppression,peaked 2 days after maximal oestradiol valerate dose and 1 dayafter peak serum oestradiol. The magnitude of the LH peak wasproportional to the duration of incremental oestradiol valeratetreatment. In conclusion, the LH surge is temporally relatedto the cessation of serum oestradiol increase. Consequently,the occurrence of this surge can be explained by a simple negativefeedback inhibition of pituitary LH release rather than by adual negative/positive mechanism.     

Effect of estrogen on angiogenesis in co-cultures of human endometrial cells and microvascular endothelial cells

BACKGROUND: We recently showed that vascular endothelial growth factor (VEGF) expression by endometrial glandular epithelial and stromal cells, and endometrial microvascular endothelial cell permeability, an early step in angiogenesis, were rapidly increased by estradiol (E(2)) administration to ovariectomized baboons. We proposed that estrogen promotes endometrial angiogenesis by regulating VEGF expression by glandular epithelial and stromal cells. In the present study, we developed a co-culture of human endometrial cells and microvascular endothelial cells to determine whether the regulatory role shown for estrogen on endometrial angiogenesis in vivo in the non-human primate would be demonstrable in vitro in the human. METHODS AND RESULTS: Human endometrial glandular epithelial and stromal cells were co-cultured with human myometrial microvascular endothelial cells (HMMECs) and E(2). HMMEC tube formation (means +/- SEM, % endothelial tube area/total endothelial cell area), an index of angiogenesis, was 65% (P < 0.05) and 2-fold (P < 0.01) greater in cells co-cultured with human glandular epithelial cells (54 +/- 7%) and glandular epithelial cells plus E(2) (66 +/- 11%), respectively, compared with medium (33 +/- 4%). In contrast, endothelial tube formation was not altered in HMMECs incubated with endometrial stromal cells (32 +/- 4%), stromal cells plus E(2) (36 +/- 2%) or E(2) (39 +/- 3%). CONCLUSIONS: We propose that estrogen, by regulating expression and secretion of angiogenic factors such as VEGF by glandular epithelial cells of the endometrium, regulates endometrial angiogenesis.     

articles

Thromboembolic events are serious but rare complications followingovarian stimulation for in-vitro fertilization (IVF). We reporta case of severe ovarian hyperstimulation syndrome (OHSS), presentingin a second IVF cycle with a late complication of right internaljugular vein thrombosis despite mini-dose heparin prophylaxis.Thrombosis and thromboembolism as late complications of OHSShave been reported by others but not after prophylactic heparinization.The patient was successfully treated with heparin and the twinpregnancy is ongoing. In pregnant patients with severe OHSSconsideration should be given to treatment with low dose heparinthroughout the first trimester to prevent the serious complicationsof thrombosis and thromboembolism.     

Low-dose prednisolone does not improve the outcome of in-vitro fertilization in male immunological infertility

Early reports of male immunological infertility suggested adecline in antisperm antibody concentrations in some patientsafter even short-term (10 day) therapy with lowdose prednisolone.In the present study, 53 men with positive results in spermatozoalmixed antiglobulin reaction (MAR) and serum tray agglutinationtests (TAT), were randomized to receive either 20 mg of prednisoloneor placebo daily for 2 weeks prior to in-vitro fertilization(IVF) treatment. The antibody levels were also monitored byflow cytometry (FCM). There were no significant differencesbetween these groups as regards fertilization rates (35% withprednisolone; 39% with placebo) and pregnancy rates (29%; 32%).No significant changes occurred in either MAR or FCM resultsin relation to therapy. Patients with fertilization rates of<10% had significantly higher immunoglobulin G (IgG) MARvalues compared with those with better fertilization, whereasthere was no relationship between IgA levels and fertilizationresults. As regards FCM, the results were similar, but withoutstatistical significance. In conclusion, IVF is a good courseof action in severe male immune infertility, but low-dose prednisolonetherapy does not lower the sperm-bound antibody numbers anddoes not improve the IVF outcome.     

Information access and donated gametes: how much do we know about who wants to know?

This paper reviews the methodological adequacy of the psychosocialliterature on information access when donated gametes and embryosare used. In all, 10 major flaws were identified: (i) samplesizes were too small, (ii) sample selection procedures weread hoc, (iii) there were no comparisons between current andpast donors and recipients, (iv) there were no comparisons betweencurrent donors and recipients in the one study, (v) studiesrelied on just one partner from a recipient couple, (vi) donormotivation was assessed crudely, (vii) studies failed to clarifywhat was identifying and non-identifying information, (viii)links between researchers and clinics may have influenced respondents,(ix) response measurement was crude, and (x) data analysis waslimited and basic. It is argued that these flaws prohibit anyfirm conclusions to be drawn either way about whether donorsand recipients should disclose information, whether they shouldhave access to information, or even whether donors and recipientswant to have access to information about each other or to haveinformation about themselves disclosed to the other party.     

Cryoloop vitrification of rabbit oocytes

BACKGROUND: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimization. In this study, rabbit oocytes (fertilized by ICSI) were vitrified with cryoloops, and the effect of three different cryopreservation protocols on spindle configuration and embryo quality was assessed. METHODS: Metaphase II rabbit oocytes were randomly assigned to one of four groups: (i) control; (ii) E40 [40% ethylene glycol (EG)]; (iii) ED20 [20% EG + 20% dimethylsulphoxide (DMSO)]; and (iv) ED20 + M (20% EG + 20% DMSO + vitrification machine). After warming, one part of each group was fertilized by ICSI to examine the fertilization and embryo cleavage ability, and the others were immunostained for tubulin and chromatin before visualization using confocal microscopy. RESULTS: The survival rates after warming were 79.1, 83.1 and 82.3%, respectively. In protocols E40 and ED20, the spindles were severely injured and the embryo quality not good compared with those in the ED20 + M group. CONCLUSIONS: The fastest cooling rate in combination with EG and DMSO as cryoprotectants had the fewest adverse effects on the spindle configuration of rabbit oocytes and embryo development.     

Fertilization and early embryology: Intracytoplasmic sperm injection for Rhesus monkey fertilization results in unusual chromatin, cytoskeletal, and membrane events, but eventually leads to pronuclear development and sperm aster assembly

The disassembly and reorganization of sperm-derived structuresare landmarks for the onset of embryonic development. Sincecomplete information on these events is not yet available, weexamined the disassembly of the sperm axoneme, the formationof the sperm aster, and the decondensation and development ofthe male and female pronuclel in inseminated Rhesus monkey oocytesconceived by in-vitro fertilization (IVF) or by intracytoplassnicsperm injection. During IVF, the spermatozoa lose their acrosomesafter contacting the zona pellucida, and the plasma membraneand nuclear envelope disappear after fusion with the oolemma.Subsequently, a sperm aster of microtubules forms around theproximal centriole, which is bound to the sperm connecting piece.This process is then followed by the formation of both pronuclei.The single sperm centriole later duplicates and the bipolarmitotic apparatus is observed. Following sperm injection, thespermatozoa have both an intact plasma membrane and acrosome.Although the microtubules form the sperm aster in a fashionidentical to that seen during IVF, the presence of an intactacrosome appears to be associated with a heterogeneity in thedecondensation of sperm chromatin. While this may indicate anabnormal pattern of chromatin decondensation during the formationof the male pro-nucleus following sperm injection, the malepronucleus eventually fully decondenses, as during 1W. Spermmito chondria are displaced as the sperm centriole is exposed.Annulate lamellae and a previously undescribed organelle whichseems to contain annulate lamellae precursors, as well as maternalmitochondria, are found in association with the developing pronuclearenvelopes. This information increases understanding of fertilizationin primates, and may also be of significance for use in assistedhuman reproduction as well as in the preservation of endangeredmammalian species. In addition, these results demonstrate thesimilarities between fertilization in Rhesus monkeys and humans,providing additional evidence for the use of this non-humanprimate as a model system in which to investigate the cellularand molecular biological basis of human reproduction.     

Evolutionary conservation of MHC class I and class II molecules—different yet the same

Despite the underlying similarities, class I molecules seem to be more heterogeneous in structure and function than class II molecules. Many features which are shared between classical class I, certain nonclassical class I and classical class II molecules (including the patterns of dilsufide bonds and particular glycosylation sites) are also conserved in vertebrate evolution; some clearly reflect structural requirements but others may be due to similarities in function. In contrast, other features (for example, the residues involved in binding the mainchain atoms of the antigenic peptide) are different in classical class I and class II molecules, but nevertheless are highly conserved in vertebrate evolution. The residues implicated in interaction with the co-receptors CD4 and CD8 are not very well conserved in vertebrate evolution, and may reflect co-evolution of the MHC molecules with their particular co-receptors.     

Physiology: Growth rates and antrum formation of mouse ovarian follicles in vitro in response to follicle-stimulating hormone, relaxin, cyclic AMP and hypoxanthine

The culture of individual intact follicles in vitro, from smallpre-antral to pre-ovulatory stages, will improve our abilityto perform controlled experiments studying follicle growth andfemale gamete development. This study was undertaken to characterizefurther the conditions required for physiological follicle growthin vitro. We cultured a total of 398 pre-antral follicles ofimmature mice (aged 26–28 days) with an initial diameterof 140–340 µm for 4 days in vitro, using individualmicro-cultures under paraffin oil. Summarizing the results ofall groups, 50 follicles were damaged (12.6%) and, of thoseremaining intact (n = 348), 60 (17.2%) became atretic, 195 (56.0%)became antral and 26 (7.5%) ovulated. The most advanced folliclesgrew to 400–500 µm diameter. The presence of follicle-stimulatinghormone (FSH) in the medium significantly stimulated folliclegrowth in vitro (P < 0.03), in a manner proportional to theinitial diameter over the range of 140–250 µm initialdiameter, with larger follicles being refractory. FSH also significantlyincreased the proportion of follicles forming antra (P <0.001) and their likelihood of ovulating in vitro (P < 0.01),and reduced the frequency of atresia (P < 0.01). Dibutyryl-cyclicAMP mimicked FSH, significantly stimulating growth of largefollicles (P < 0.05) and antrum formation (P < 0.01).Hypoxanthine also stimulated antrum formation (P < 0.01)but did not significantly affect follicle growth. Porcine relaxinhad no significant effect on mouse follicle growth or antrumformation. The optimal conditions for mouse follicle growthin vitro have not yet been defined, but selection of folliclesof < 250 µm diameter and inclusion of FSH or dibutyryl-cyclicAMP in the culture medium are recommended.     

Gelatinases and their tissue inhibitors during human ovulation: increased expression of tissue inhibitor of matrix metalloproteinase-1

Remodelling of the extracellular matrix (ECM) of the follicular wall by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) has been suggested to be crucial in ovulation. To investigate the expression of the gelatinases, MMP-2 and MMP-9, together with their inhibitors, TIMP-2 and TIMP-1, in the perifollicular ovarian stroma from women just before and during ovulation, we obtained biopsies of the stroma adjacent to the leading follicle. Laparoscopic surgery was performed either before the LH peak or at any of three intervals after ovulation triggering by hCG. Immunoblotting, immunohistochemistry and quantitative RT-PCR were performed. All four proteins were expressed by immunoblots, with no detectable changes in the expression of MMP-2, MMP-9 and TIMP-2. Scattered immunostaining for MMP-9 and TIMP-2 was seen, and MMP-2 was demonstrated in a concentric layer. A significant increase in TIMP-1 protein and mRNA was seen during the three ovulatory phases, and a strong and patchy immunostaining for TIMP-1 was shown. This is the first study that has demonstrated an ovulation-associated expression of these ECM-remodelling enzymes around the human follicle at ovulation. The increased expression of TIMP-1 may reflect a specific temporal inhibition of collagenolysis and thereby a time-dependent regulation of ECM breakdown in areas surrounding the apex of the follicle.