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An unexpected role for anticoagulant heparan sulfate proteoglycans in reproduction. de Agostini A et al. Major tissue remodelling occurs in hormone responsive tissues of the female genital tract, at ovulation and during gestation, involving proteolysis and inflammation. Disorders of tissue remodelling events are associated with infertility in women with luteinized unruptured follicle syndrome and with gestational pathologies as preeclampsia. Ovulation impairment is an important factor of infertility and a major concern in reproductive medicine. The gonadotrophin discharge inducing ovulation triggers proteolytic activities involved in the breakdown of the follicular wall and elicits an acute inflammatory reaction in the ovary. Tight control of these reactions is required to allow successful ovulation while avoiding excessive tissue damage. Anticoagulant heparan sulfate proteoglycans (aHSPG), like heparin, possess a pentasaccharide sequence which binds and activates antithrombin III. These proteoglycans are produced by endothelial cells and are thought to endow the vascular wall with antithrombotic properties. aHSPG are also present in the reproductive tract; in the ovary they are strongly expressed in granulosa cells of preovulatory follicles and they are co-localised with serine protease inhibitors involved in the control of proteolytic activities at ovulation. The presence of aHSPG in the oviduct, in the uterus and in human follicular fluid, suggests that they could play additional distal roles in gestation. The females exhibited impaired ovarian function as well as intrauterine growth restriction linked to delayed placenta development. In these mice, the placenta is challenged by inflammation at mid-gestation, occasionally resulting in miscarriage and maternal death. Collectively, these observations suggest that aHSPG are involved in the control of inflammatory events occurring during tissue remodelling in hormone-responsive tissues. Further studies are needed to identify the inflammation mediators involved in this process. The use of R393C-Antithromib III mutant decreased the number of ovulated oocyte and ncreased fibrin deposition within follicles.
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Apolipoprotein A-IV as a novel gene associated with polycystic ovary syndrome. Kim YS et al. Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder, affecting 6-10% of women of reproductive age. The etiology remains poorly understood. To investigate the differentially expressed proteins from PCOS patients versus healthy women, the protein expression in follicular fluid was analyzed using two-dimensional electrophoresis (2-DE). Since follicular fluid contains a number of secretory proteins required for oocyte fertilization and follicle maturation, it is possible that follicular fluid can be used as a provisional source for identifying pivotal proteins associated with PCOS. In this study, six overexpressed proteins kininogen1, cytokeratin9, antithrombin, fibrinogen ?-chain, apolipoproteinA-IV (apoA-IV) precursor and a-1-B-glycoprotein (A1BG)] in follicular fluids from PCOS patients were identified with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and nano LC-MS/MS. Western blot analysis confirmed that the protein expression levels of apoA-IV precursor and A1BG were increased in follicular fluid from PCOS patients compared with those from normal controls. The analysis of protein expression for other proteins revealed individual variation. These results facilitate the understanding of the molecular mechanisms of PCOS and provide candidate biomarkers for the development of diagnostic and therapeutic tools.
Proteome mining of human follicular fluid reveals a crutial role of complement cascade and key biological pathways in women undergoing in vitro fertilisation. [Jarkovska K et al. In vitro fertilisation (IVF) is fraught with problems and currently proteomics approaches are being tried out to examine the microenvironment of the follicle in order to assess biological and immunological parameters that may affect its development. Additionaly, better understanding of reproductive process may help increase IVF birth rate per embryo transfer and at the same time avoid spontaneous miscarriages or life threatening conditions such as ovarian hyperstimulation syndrom. The primary aim of this study was to search for specific differences in protein composition of human follicular fluid (HFF) and plasma in order to identify proteins that accumulate or are absent in HFF. Depletion of abundant proteins combined with multi-dimensional protein fractionation allowed the study of middle and lower abundant proteins. Paired comparison study examining HFF with plasma/serum from women undergoing successful IVF revealed important differences in the protein composition which may improve our knowledge of the follicular microenvironment and its biological role. This study showed involvement of innate immune function of complement cascade in HFF. Complement inhibition and the presence of C-terminal fragment of perlecan suggested possible links to angiogenesis which is a vital process in folliculogenesis and placental development. Differences in proteins associated with blood coagulation were also found in the follicular milieu. Several specific proteins were observed, many of which have not yet been associated with follicle/oocyte maturation. These proteins together with their regulatory pathways may play a vital role in the reproductive process. THis protein is 2.5 fold higher in FF than plasma.
A proteomic analysis of IVF follicular fluid in women Estes SJ et al. OBJECTIVE: To address the lack of predictors of IVF success by using proteomic biometrics. DESIGN: Experimental study of follicular fluid specimens from a prospective cohort of IVF patients. SETTING: Academic research laboratory and IVF program. PATIENT(S): Women or=11 oocytes and live birth (10 pairs). Year of cycle start and IVF down-regulation protocol were also matched. INTERVENTION(S): Follicular fluid was separated by two-dimensional polyacrylamide gel electrophoresis followed by Sypro Ruby staining and comparison with PDQuest software. Logistic regression was incorporated to calculate the likelihood of live birth in relation to the protein spot of interest. MAIN OUTCOME MEASURE(S): Protein markers. RESULT(S): Liquid chromatography-tandem mass spectrometry and searching of sequence databases revealed 11 potential protein candidates. Haptoglobin alpha, predominantly fetal expressed T1 domain, mitochondrial integrity genome (ATPase), apolipoprotein H (beta-2 glycoprotein I), dihydrolipoyl dehydrogenase, lyzozyme C, fibrinogen alpha-chain, and immunoglobulin heavy chain V-III (region BRO) were found to have increased expression in the live birth group, whereas antithrombin, vitamin D-binding protein, and complement 3 were decreased. An ELISA confirmed a significantly lower level of antithrombin. CONCLUSION(S): Proteomic evaluation of follicular fluid is able to identify potential biomarkers of good versus poor responders in matched pairs of IVF patients.
Coordinate expression of anticoagulant heparan sulfate proteoglycans and serine protease inhibitors in the rat ovary: a potent system of proteolysis control. Hasan S et al. During the reproductive cycle, ovarian follicles undergo major tissue-remodeling involving vascular changes and proteolysis. Anticoagulant heparan sulfate proteoglycans (aHSPGs) are expressed by granulosa cells during the development of the ovarian follicle. The function of aHSPGs in the ovary is unknown, but they might be involved in proteolysis control through binding and activation of serine protease inhibitors. To identify functional interactions between aHSPGs and heparin-binding protease inhibitors in the follicle, we have coordinately localized aHSPGs, antithrombin III, protease nexin-1, and plasminogen activator inhibitor-1 in the rat ovary during natural and gonadotropin-stimulated cycles. Anticoagulant HSPGs were visualized by autoradiography of cryosections incubated with 125I-antithrombin III, and protease inhibitors were assessed by immunohistochemistry and Northern blot hybridization. Anticoagulant HSPGs were expressed in follicles before ovulation, were transiently decreased in postovulatory follicles, and were abundant in the corpus luteum, mainly on capillaries. Anticoagulant HSPGs were colocalized with protease nexin-1 in follicles from the early antral stage until ovulation, with antithrombin III in the preovulatory stage and after ovulation, and with plasminogen activator inhibitor-1 in the corpus luteum. These data demonstrate that aHSPGs are critically expressed in the ovary to interact sequentially with protease nexin-1, antithrombin III, and plasminogen activator inhibitor-1 during the cycle. The specificity of these inhibitors is shifted toward thrombin inhibition in the presence of heparin, suggesting that aHSPGs direct their action to control fibrin deposition in the follicle. The occupation of aHSPGs antithrombin-binding sites by mutant R393C antithrombin III, injected in the ovarian bursa, decreased ovulation efficiency, further supporting the involvement of aHSPGs in the ovulation process.
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