鲍曼不动杆菌外膜蛋白A通过Akt/mTOR/p70S6K信号通路引起RAW264.7细胞自噬

目的:分析鲍曼不动杆菌标准株ATCC 19606(Acinetobacter baumannii ATCC 19606)外膜蛋白A(outer membrane protein A,Omp A)对RAW264. 7细胞的自噬诱导作用。方法:建立Omp A刺激RAW264. 7细胞的模型,通过细胞免疫荧光染色、Western blot和透射电子显微镜检测Omp A对RAW264. 7细胞自噬的影响。结果:Omp A可以引起自噬蛋白LC3B-II表达升高,并抑制Akt/mTOR/p70S6K的磷酸化水平;雷帕霉素可以进一步降低mTOR和p70S6K磷酸化,并提高Omp A引起的LC3B-II表达升高。结论:鲍曼不动杆菌Omp A通过Akt/mTOR/p70S6K信号通路引起RAW264. 7细胞自噬。这为将来进一步研究鲍曼不动杆菌引起自噬的分子机制及找到对抗其感染的新方法提供理论依据。     

蛇床子素对碘海醇所致大鼠急性肾损伤的保护作用及可能机制

目的 观察蛇床子素对碘海醇所致肾损伤的保护作用,探讨mTOR/p70S6K信号通路的调控机制.方法 40只SPF级SD大鼠随机分为4组,分别为对照组、模型组、蛇床子素低、高剂量组,每组10只.利用静脉注射碘海醇(3g/kg)建立急性肾损伤模型,肾损伤3d后生化检测大鼠血清肌酐(Scr)和尿素氮(BUN)的变化;ELISA方法检测各组大鼠尿液中肾损伤分子-1(KIM-1)、N-乙酰-D-氨基葡萄糖苷酶(NAG)含量;HE染色观察肾脏病理学变化;Western blot检测肾脏中自噬关键蛋白LC 3、Beclin 1表达及mTOR/p70S6K信号通路mTOR、p-mTOR、p70S6K及p-p70S6K蛋白的表达.结果 蛇床子素低、高剂量显著降低Scr、BUN水平,降低尿液中KIM-1、NAG的表达水平,改善肾功能,上调自噬相关蛋白LC3Ⅱ/Ⅰ和Beclin 1蛋白的表达水平,抑制p-mTOR及p-p70S6K蛋白的活性.结论 蛇床子素对碘海醇所致肾损伤大鼠模型具有保护作用,与其抑制mTOR/P70S6K信号通路活性,激活自噬有关.     

雷帕霉素激活自噬改善嘌呤霉素氨基核苷诱导的足细胞损伤

目的:通过观察雷帕霉素对PAN 诱导的足细胞损伤及自噬相关蛋白表达的影响,探讨自噬在雷帕霉素保护PAN 诱导的足细胞损伤中的作用及可能机制。方法:构建PAN 诱导的足细胞损伤模型,将足细胞分成对照组(Control 组),PAN 组(加入50 μg/ ml PAN),雷帕霉素组(RAP 组:分别加入100、200、300 ng/ ml 雷帕霉素),PAN+雷帕霉素组(PAN+RAP组:细胞在用含PAN 的培养液培养前1 h,分别用100、200、300 ng/ ml 雷帕霉素进行预处理1 h)。采用Annexin V/ PI 双染法检测细胞凋亡,透射电镜观察自噬小体,Western blot 检测LC3、p62、4EBP1、P70S6K、mTOR 蛋白表达。结果:与对照组比较,PAN组足细胞凋亡增加,自噬体减少,LC3域蛋白表达下调,p62 上调,mTOR、4EBP1、P70S6K 磷酸化水平上调;与PAN 组比较,PAN+RAP 组足细胞凋亡率下降,自噬体增加,LC3域蛋白表达上调,p62 下调,mTOR、4EBP1、P70S6K 磷酸化水平下调。结论:PAN 可以抑制足细胞自噬,促进足细胞凋亡;雷帕霉素可通过激活自噬改善PAN 诱导的足细胞损伤,这种作用可能与雷帕霉素抑制mTOR/4EBP1、P70S6K 信号通路有关。     

苦参碱通过 MAPK/mTOR 信号通路诱导 IMCD3 细胞自噬及机制研究

目的:通过观察苦参碱对小鼠肾脏内髓集合管上皮细胞(IMCD3)自噬的影响,探讨苦参碱诱导细胞自噬可能的分子机制。方法:在小鼠IMCD3中加入不同浓度的苦参碱(0.2、0.4、0.8、1.6 mg/ml),检测苦参碱对细胞活性、细胞的凋亡以及细胞自噬的影响;采用Western blot检测自噬相关的蛋白LC3、ERK、p-ERK、p53、Beclin-1、p70S6K、p-p70S6K、AKT、p-AKT表达水平的变化。结果:当苦参碱浓度为0、0.2、0.4、0.8 mg/ml时,IMCD3活性以及凋亡坏死没有明显变化,当苦参碱浓度为1.6 mg/ml时,细胞凋亡坏死明显增加;苦参碱处理后,细胞中自噬小体的数量明显增加;随着苦参碱处理浓度的增加,细胞中LC3蛋白表达明显升高,p-ERK、p-p70S6K表达明显减弱,ERK、p70S6K、p53、Beclin-1、AKT和p-AKT等蛋白表达无明显变化。结论:苦参碱可通过抑制MAPK/mTOR信号通路的活化促进小鼠IMCD3的自噬。     

TP53BP2/ASPP2通过p53非依赖性方式激活mTOR通路抑制HepG2人肝癌细胞自噬

目的研究肿瘤蛋白p53结合蛋白2/p53凋亡刺激蛋白2(TP53BP2/ASPP2)对HepG2人肝癌细胞自噬的调节作用和机制。方法通过腺病毒、慢病毒感染HepG2细胞上调或下调细胞中ASPP2的表达, HepG2细胞继续在不含胎牛血清的培养基中培养24 h,通过Western blot法检测自噬相关蛋白微管相关蛋白1轻链3(LC3)、 beclin1、 P62、自噬相关基因5(ATG5)、 ATG7和哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白mTOR、磷酸化的mTOR(p-mTOR)、真核转录起始因子4E结合蛋白1(4EBP1)、磷酸化的4EBP1(p-4EBP1)、核糖体蛋白S6、磷酸化的S6(p-S6)、核糖体S6激酶B1(RPS6KB1/p70S6K)、磷酸化的p70S6K(p-p70S6K)的蛋白表达,荧光显微镜检测细胞表达的绿色荧光蛋白-微管相关蛋白1轻链3(GFP-LC3)融合蛋白。同时,使用慢病毒感染不表达p53的Hep3B细胞,敲低细胞中的ASPP2水平, Western blot法检测Hep3B细胞ASPP2和mTOR通路相关蛋白mTOR、 p-mTOR、 4EBP1、 p-4EBP1、 S6、 p-S6、 RPS6KB1/p70S6K、 p-p70S6K的蛋白表达。结果当ASPP2的表达上调时, mTOR复合物1(mTORC1)通路被激活,并且自噬相关蛋白的表达和自噬体的数量减少。在下调ASPP2表达后, mTORC1通路受到抑制,自噬相关蛋白的表达水平和自噬体的数量增加。在p53表达沉默的Hep3B细胞中,当ASPP2下调后, mTORC1途径仍然受到抑制。结论 ASPP2以p53非依赖性方式激活mTOR通路抑制HepG2人肝癌细胞自噬。     

AMPK/mTOR信号通路介导的自噬在血根碱抑制鼻咽癌细胞增殖中的作用研究北大核心CSCD

目的:研究血根碱(SAN)对人鼻咽癌5-8F细胞自噬和增殖的影响,并探讨5-8F细胞自噬与增殖的关系及作用机制。方法:MTT和实时无标记细胞分析技术(RTCA)检测细胞增殖;单丹磺酰戊二胺(MDC)染色和透射电镜观察自噬情况;Western blot检测蛋白表达。结果:与Control组相比,SAN可显著抑制鼻咽癌细胞增殖,降低PCNA表达,诱导自噬小体形成,降低P62表达,提高Beclin-1表达、LC3Ⅱ/Ⅰ;而抑制自噬后(3-MA预处理),SAN诱导自噬和抑制细胞增殖作用显著减弱,同时,SAN对Beclin-1、LC3Ⅱ/Ⅰ、p62、PCNA的影响改变;SAN可激活AMPK/mTOR信号通路关键蛋白AMPK、p-AMPK并抑制mTOR表达;而抑制AMPK/mTOR信号通路后(抑制剂Compound C预处理),SAN诱导自噬和抑制细胞增殖作用显著减弱。结论:SAN能够抑制人鼻咽癌5-8F细胞增殖并诱导自噬,可能通过诱导细胞自噬抑制细胞增殖,其机制可能与激活AMPK/mTOR信号通路有关。     

四妙勇安汤含药血清对巨噬细胞TLR4/MyD88信号通路及其下游炎症因子的影响

目的研究四妙勇安汤含药血清对LPS诱导的小鼠腹腔巨噬细胞Toll样受体4(TLR4)、髓样分化因子88(MyD88)信号通路及TNF-α、IL-6等炎症因子表达的影响,初步探讨其抗动脉粥样硬化的作用机制。方法采用大、中、小剂量四妙勇安汤水煎剂灌胃干预清洁级SD大鼠,制备含药血清;经MTT法观察含药血清对细胞增殖的影响后,选择剂量浓度为20%的含药血清干预体外培养的经LPS活化的巨噬细胞,采用ELISA法检测细胞上清液中TNF-α及IL-6含量;荧光定量PCR及Westernblot法检测细胞TLR4、MyD88的mRNA及蛋白的表达变化。结果用LPS刺激细胞后,引起TLR4、MyD88、TNF-α及IL-6的高表达,与正常血清对照组相比,差异具有统计学意义(P0.01);而用四妙勇安汤含药血清干预后,能显著抑制各项指标的高表达,与模型组相比,差异具有统计学意义(P0.05或P0.01)。结论四妙勇安汤含药血清能够有效抑制LPS诱导的巨噬细胞炎症反应,其机制可能与调控TLR4/MyD88信号转导通路有关,这可能是该方防治动脉粥样硬化及免疫炎症性疾病作用的机制之一。     

黄连解毒汤含药血清对巨噬细胞自噬相关基因表达的影响

目的:考察黄连解毒汤含药血清对RAW264郾7 巨噬细胞自噬的影响,初步探讨其抗动脉粥样硬化的作用机制。方法:采用大、中、小剂量黄连解毒汤水煎剂灌胃干预SD 大鼠,制备含药血清;经MTT 法观察含药血清对细胞增殖的影响后,选择各剂量浓度为20% 的含药血清干预体外培养的巨噬细胞,采用荧光定量PCR 方法检测自噬相关基因Beclin1 和mTOR 的mRNA 表达水平;Western blot 检测Beclin1、p鄄mTOR 蛋白的表达变化。结果:与正常对照组血清相比,黄连解毒汤含药血清干预后诱导了Beclin1 mRNA 及蛋白的表达,抑制了mTOR mRNA 及p鄄mTOR 蛋白的表达。结论:黄连解毒汤含药血清可诱导RAW264郾7 巨噬细胞自噬相关基因Beclin1 的表达、抑制mTOR 的表达,这可能是该方抗动脉粥样硬化作用的重要机制之一。     

亚硒酸钠通过AMPK/mTOR通路调控白血病NB4细胞凋亡

 目的 研究亚硒酸钠对白血病NB4细胞中AMPK及其下游靶蛋白对细胞凋亡的调控作用。方法 分别用亚硒酸钠、AICAR和AMPK干扰序列处理NB4细胞。用Western blot检测细胞AMPK、mTOR及其下游蛋白P70S6K的磷酸化水平及激活和干扰AMPK后AMPK、mTOR及P70S6K的磷酸化水平、流式细胞术检测NB4细胞凋亡率;免疫共沉淀法检测AMPK和mTOR的相互作用。结果 亚硒酸钠可以上调NB4细胞中AMPK的磷酸化水平,下调mTOR及P70S6K的磷酸化水平;AICAR和亚硒酸钠单独处理具有类似的促进NB4细胞凋亡的效果;干扰AMPK后,mTOR及P70S6K的磷酸化水平上升,亚硒酸钠对NB4细胞的促凋亡作用被拮抗;AMPK和mTOR有直接相互作用。结论 亚硒酸钠可通过激活AMPK表达,抑制mTOR及P70S6K,促进NB4细胞凋亡。     

西格列汀对AGEs作用下系膜细胞自噬的影响

目的:探讨西格列汀对晚期糖基化终末产物(advanced glycation end products,AGEs)作用下系膜细胞细胞外基质和自噬相关蛋白表达的影响。方法:培养大鼠肾小球系膜细胞,共分5组,分别为正常对照(control)组、AGE组和不同浓度(5、10和20μmol/L)西格列汀组。培养48 h后采用MTT法测定细胞活力,采用ELISA法检测细胞培养上清胶原蛋白Ⅳ(collagen IV,Col IV)含量的变化。采用Western blot检测自噬相关蛋白beclin-1、腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)、p-AMPK、p70S6K和p-p70S6K的蛋白水平。结果:与control组比较,AGEs可明显引起系膜细胞活力和Col IV表达增加,不同浓度的西格列汀均可明显抑制AGEs诱导的系膜细胞活力和Col IV表达的增加;与control组比较,AGEs可引起系膜细胞自噬相关蛋白beclin-1表达和AMPK磷酸化水平下降,p70S6K磷酸化水平增加;不同浓度的西格列汀均可促进系膜细胞自噬相关蛋白beclin-1表达及AMPK磷酸化,抑制p70S6K磷酸化,并呈一定的浓度依赖性。结论:西格列汀可能通过引起系膜细胞的自噬发挥肾脏保护作用。     

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.     

Pharmacokinetics of different routes of administration of misoprostol

BACKGROUND: The pharmacokinetic parameters of four different routes of administration of a single dose of 400 microg of misoprostol were studied. METHODS: A total of 40 women undergoing termination of pregnancy by suction evacuation was randomized by computer model to receive 400 microg of misoprostol by one of four routes: (i) sublingual (ii) oral (iii) vaginal and (iv) vaginal with addition of water. Venous blood samples were taken at 0, 1, 2, 5, 10, 20, 30, 45, 60, 120, 240 and 360 min after the administration of misoprostol. Misoprostol acid (MPA) was determined in serum samples using gas chromatography/tandem mass spectrometry. RESULTS: Sublingual misoprostol achieved the highest serum peak concentration (Cmax) (574.8 +/- 250.7 pg/ml) of MPA and this was significantly higher than those in the other groups [Oral: 287.6 +/- 144.3 pg/ml (P < 0.01), vaginal: 125.2 +/- 53.8 pg/ml (P < 0.001) and vaginal with water: 162.8 +/- 57.1 pg/ml (P < 0.001)]. The time to peak concentration (Tmax) was similar in both the sublingual (26.0 +/- 11.5 min) and oral groups (27.5 +/- 14.8 min) and was significantly shorter than those in both vaginal groups. The area under the MPA concentration versus time curve up to 360 min in the sublingual group (743.7 +/- 291.2 pg.h/ml) was significantly greater than those in oral (402.8 +/- 151.6 pg.h/ml, P < 0.05) and vaginal (433.7 +/- 182.6 pg.h/ml, P < 0.05) groups, but no significant difference was found between sublingual and vaginal administration if water (649.3 +/- 333.8 pg.h/ml) was added. CONCLUSION: The new sublingual route of administration of misoprostol demonstrated a great potential to be developed into a method of medical abortion.     

Postzygotic diploidization of triploids as a source of unusual cases of mosaicism,chimerism and twinning

Triploidy is one of the most frequent chromosomal errors responsible for reproduction failure. This paper encompasses, in one conceptual frame, four recent findings in reproduction biology: predominant dispermic origin of triploids, paternal centrosome inheritance, eccentric cleavage divisions of dispermic triploid zygotes and certain intricate cases of mosaicism/chimerism. It is argued that dispermic zygotes, in contrast to digynic ones, are characterized by cytogenetic phenomenon described here as postzygotic diploidization of triploids (PDT). PDT embraces three main developmental scenarios: (i) the maintenance of the triploid state accompanied by regular segregation of 2n cells and the 2n/3n mixoploid populations; (ii) immediate diploidization with elimination of an odd haploid set of chromosomes and regular appearance of 1n/2n, 2n/3n and other mixoploids and (iii) tripolar spindle formation leading to gross aneuploidy, cell death with occasional survival of 2n+1 or 2n+1+1 trisomics and uniparental disomics. According to the PDT concept, a trisomy and disomy might occur due to generalized karyotype instability of dispermic triploids. PDT may provide a natural explanation for the regular appearance of 2n homozygous androgenic moles, various 2n/3n, 2n/2n molar/twin complexes without necessitating the concept of the 'empty' oocyte fertilization. Convincing evidence for a reservoir of anuclear oocytes does not exist. Peculiar implications are expected in the case of two rounds of diploidizations or involvement of triploid cell derivatives in the twinning process. Cryptic mosaic/chimeras and unusual twins intermediate between monozygotic (MZ) and dizygotic (DZ) are expected. Thus, PDT could have an explanation for the broad spectrum of odd reproductive cytogenetic events and might provide additional alternatives and definite predictions.     

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.     

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.     

Immunological aspects of endometriosis

The immune system probably plays a role in the onset and development of endometriosis. A general picture can be proposed. In some women refluxing endometrial cells are not destroyed, either because the patient is genetically programmed not to respond to endometrial antigens, or because the reflux is so abundant that the scavenging capacity of the peritoneal immune cells is overloaded. Refluxing cells could be protected due to an abnormal adherence to the mesothelium which exceptionally expresses certain adhesive molecules. Undestroyed, these endometrial cells would cause an inflammation with activation of macrophages. Not only does the peritoneum protect these endometrial cells, but it also produces abnormal quantities of chemotactic and angiogenic cytokines (interleukin-8). Macrophages facilitate development via growth factors such as transforming growth factor P. Immunosuppressive factors block the cytotoxic activity of natural killer (NK) cells. Activated macrophages present antigens of endometrial cells to T cells which will co-operate with B cells to synthesize autoantibodies. Synthesized antibodies protect the ectopic endometrium and could worsen the dysfunction of local NK cells. A vicious circle is set up involving all the partners of the immune system. It is as yet impossible to pinpoint the triggering mechanism. The primary defect could be localized on the endometrium, macrophages already activated by an extrinsic factor (infection, spermatozoa, chemical substances), the uterus or the tubo-uterine junction. The two pathophysiological theories put forward to explain endometriosis are linked by a defective immune system. Indeed, once the vicious circle is set up, growth and angiogenic factors could induce metaplasia of the already irritated mesothelium.     

Season of birth influences the timing of menopause

BACKGROUND: Seasons may influence prenatal growth and future fertility. This study investigated whether season and month of birth influenced the timing of menopause in a group of women attending three Italian menopause clinics. METHODS and RESULTS: Age at menopause of 2822 post-menopausal women (>12 months of amenorrhoea) was stratified by month and season of birth. Mean age at menopause was 49.42 years (SEM: 0.78 years). Menopause occurred earlier for women born in the spring (age 49.04+/-0.15 years) than in the autumn (49.97+/-0.14 years). The earliest menopause was found in women born in March (48.9+/-0.25 years) and the latest in women born in October (50.3+/-0.25 years). The effect of season of birth on age at menopause remained even when considering factors that in our analysis were capable of significantly interfering with the timing of menopause, such as age at menarche, body mass index, smoking habit, level of education and type of job. CONCLUSIONS: Taking into consideration the retrospective design of the study, and a possible recall bias, the present data seem to suggest that environmental factors linked to seasons are capable of interfering with the timing of a woman's ovarian exhaustion by an action exerted in the prenatal period.     

Physiology of meiosis-activating sterol: endogenous formation and mode of action

BACKGROUND: In the context of mammalian oocyte maturation, it has been suggested that intermediates of cholesterol biosynthesis may represent the physiological signal that instructs the oocyte to reinitiate meiosis. METHODS: Endogenous levels of follicular fluid meiosis-activating sterol (FF-MAS) were monitored in rabbit ovarian tissue, and the influence of exogenous gonadotrophins on sterol formation was assessed. The involvement of cAMP in FF-MAS-induced versus spontaneous oocyte maturation in vitro in mice was also investigated, as was the direct microinjection of FF-MAS into mouse oocytes. RESULTS: Levels of FF-MAS in rabbit ovaries were significantly elevated 1 h after hCG/LH induction and remained so for 4 and 12 h after induction. In naked oocytes undergoing spontaneous maturation, a significant decrease in cAMP was detected after 30 min of culture. However, FF-MAS-mediated induction of oocyte maturation in hypoxanthine-arrested naked oocytes was not associated with any detectable decrease in intracellular cAMP levels. Microinjected FF-MAS failed to induce any noticeable meiosis. CONCLUSIONS: A rapid increase in FF-MAS level occurred in vivo in the rabbit ovary in response to LH, and clear differences were seen in the cAMP pattern during spontaneous and induced oocyte maturation in mice.     

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.     

The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts

Blastocysts from 198 patients were frozen using glycerol ascryoprotectant. No difference in the post-thaw survival of blastocystsor implantation rates was found between 177 patients (122 transfers)with all surplus embryos cultured to blastocysts before freezingand 20 patients (12 transfers) whose embryos were consideredunsuitable for freezing during cleavage and were then frozenas blastocysts. Nineteen pregnancies were achieved, of whichsix aborted. Pre-freezing morphology was similar in blastocystsof patients in groups 1 and 2 and did not relate to their survivalafter cryopreservation. A significantly lower proportion withsuspected damage after thawing was present among patients becomingpregnant after transfers of single blastocysts (P < 0.01)and implanting embryos were in general more expanded at thetime of transfer. No differences were detected between blastocystsresulting in normal development and those leading to abortion.The developmental consequences of damage to human blastocystsare discussed.