Effect of urokinase type plasminogen activator on in vitro bovine oocyte maturation

Reproduction ◽  
2017 ◽  
Vol 154 (3) ◽  
pp. 331-340 ◽  
Author(s):  
Roldán-Olarte Mariela ◽  
Maillo Verónica ◽  
Sánchez-Calabuig María Jesús ◽  
Beltrán-Breña Paula ◽  
Rizos Dimitrios ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasminogen Activator ◽  
Oocyte Maturation ◽  
Cell Junctions ◽  
Cortical Granules ◽  
Cleavage Rate ◽  
Nuclear Maturation ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

This study examines the impacts of the urokinase-type plasminogen activator (uPA) on thein vitromaturation (IVM) of bovine oocytes. Cumulus–oocyte complexes in IVM medium were treated with uPA, amiloride (an uPA inhibitor), dimethyl sulfoxide (DMSO) or left untreated (control group). After 24 h of IVM, oocytes were recovered for testing or werein vitrofertilized and cultured to the blastocyst stage. The factors examined in all groups were: (i) oocyte nuclear maturation (Hoëscht staining); (ii) oocyte cytoplasmic maturation (cortical granules, CGs, distribution assessed by LCA-FITC); (iii) oocyte and cumulus cell (CC) gene expression (RT-qPCR); and (iv) embryo development (cleavage rate and blastocyst yield). Oocytes subjected to uPA treatment showed rates of nuclear maturation and CG distribution patterns similar to controls (P > 0.05), whereas lower rates of oocyte maturation were recorded in the amiloride group (P < 0.05). Both in oocytes and CC, treatment with uPA did not affect the transcription of genes related to apoptosis, cell junctions, cell cycle or serpin protease inhibitors. In contrast, amiloride altered the expression of genes associated with cell junctions, cell cycle, oxidative stress and CC serpins. No differences were observed between the control and uPA group in cleavage rate or in blastocyst yield recorded on Days 7, 8 or 9 post-insemination. However, amiloride led to drastically reduced cleavage rate (28.5% vs 83.2%) and Day 9 embryo production (6.0% vs 21.0%) over the rates recorded for DMSO. These results indicate that the proteolytic activity of uPA is needed for successful oocyte maturation in bovine.

199 EFFECT OF UROKINASE TYPE PLASMINOGEN ACTIVATOR DURING IN VITRO MATURATION OF BOVINE OOCYTES AND EARLY EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 230
Author(s):  
M. Roldán-Olarte ◽  
V. Maillo ◽  
M. J. Sánchez-Calabuig ◽  
A. Tío-Castro ◽  
P. Beltrán ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Oocyte Maturation ◽  
Early Embryo ◽  
Developmental Competence ◽  
Cell Junctions ◽  
Cortical Granules ◽  
Cleavage Rate ◽  
Expression Of Genes ◽  
Type Plasminogen Activator

The study of molecules involved in the oocyte maturation and early embryo development is crucial to improve the conditions of in vitro embryo production. The plasminogen activation system is involved in the initial steps of reproduction and urokinase type plasminogen activator (uPA) is expressed in the granulosa cells (GC) of bovine cumulus-oocyte complexes (COC). The aim of this study was to analyse the effect of uPA in bovine oocyte in vitro maturation (IVM). We have analysed whether the addition of uPA or the inhibition of its proteolytic activity affects IVM. We have evaluated (1) nuclear and cytoplasmic maturation, (2) developmental competence, and (3) oocyte and GC gene expression. Immature COC were obtained by aspiration of ovarian follicles of slaughtered heifers. Selected COC were in vitro-matured in 4 groups: control, 10 nM uPA, dimethyl sulfoxide control, and 100 μg mL–1 amiloride, a specific inhibitor of uPA proteolytic activity (4 replicates). After 24 h of IVM, oocytes of each treatment were either fixed and stained with Hoëscht (to evaluate nuclear maturation) or LCA-FITC to analyse the cortical granules distribution as a marker of cytoplasmic maturation (n = 10/group per treatment per replicate). In addition, pools of 10 oocytes and their separated GC were snap-frozen to analyse by qRT-PCR their profile expression of genes related with apoptosis (BAX, BCL2, TP53, SHC1), cell junctions (GJA1, TJP1), cell cycle (CCNB1), oxidative stress (SOD2, GPX1), oocyte quality (BMP15, GDF9), and serpin proteases inhibitors (SRP1, SRP5), normalised respect 2 housekeeping genes (H2AFZ, ACTB). The remaining COC were fertilised (Day 0) and in vitro cultured to evaluate developmental competence in terms of cleavage rate (Day 2) and blastocyst yield (Days 7–9). All data were analysed by one-way ANOVA. In the presence of amiloride, a significant reduction in the oocyte maturation was observed at both levels; 83.33% of oocytes remained in vesicle stage, and 75.0% showed a cortical granules distribution of type I. The rest of the groups (62.67%, 62.65%, and 60.29%) reached metaphase II (MII), and 51.66%, 32.9%, and 25.44% showed granule distribution of type III. For embryo development, the amiloride group had a cleavage rate and blastocyst yield significantly lower compared with controls (23.23% v. 80.85% and 83.83%; 4.45% v. 25.21% and 25.21%, respectively), whereas uPA treatment had no effect (84.28% and 24.16%). In presence of amiloride, the transcript levels of TJP1, GJA1, and CCNB1 were up-regulated, whereas SOD2, SRP1, and SRP5 were down-regulated in GC compared with all other groups (P < 0.05). No differences were observed in oocytes gene expression between treatments. In conclusion, although the addition of exogenous uPA does not alter oocyte maturation, the specific inhibition of the proteolytic activity of uPA by amiloride reduced IVM of bovine oocytes and altered the expression of genes related to cell junctions, cell cycle, oxidative stress, and serpins of GC, indicating that proteolytic activity of uPA is critical for oocyte IVM in bovines.

ON THE MECHANISM OF THE IN VIVO SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR (t-PA) AND SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (scu-PA)

1987 ◽  
Author(s):  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Rabbit Model ◽  
Sequential Therapy ◽  
Tissue Type ◽  
Single Chain ◽  
Molar Ratios ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

t-PA and scu-PA, in molar ratios between 1:4 and 4:1 do not act synergically in vitro (Thromb. Haemost. 56,35,1986) but display marked synergism in a rabbit model (Circulation 74, 838, 1986) and in man (Am. Heart J. 112, 1083, 1986). To investigate the mechanism of in vivo synergism in the rabbit model (J. Clin. Invest. 71, 368, 1983), t-PA and scu-PA were infused 1) simultaneously over 4 hrs, 2) t-PA over 1 hr, then 15 min later scu-PA over 2 hrs and 3) scu-PA over 1 hr, then 15 min later t-PA over 2 hrs.Significant synergism on thrombolysis is observed when t-PA and scu-PA are infused simultaneously or when t-PA is followed by scu-PA but not when scu-PA is followed by t-PA. These results suggest that low dose t-PA induces some plasminogen activation, sufficient to partially degrade fibrin, exposing COOH-terminal lysines with high affinity for plasminogen (Eur. J. Biochem. 140, 513, 1984). scu-PA might then activate surface-bound Glu-pla-minogen more efficiently.Sequential therapy with t-PA (or any other agent which "predigests" the thrombus), followed by scu-PA might constitute an alternative to simultaneous infusion of synergistic thrombolytic agents.

Intracellular activation of the fibrinolytic cascade in the Quebec Platelet Disorder

2003 ◽  
Vol 90 (08) ◽  
pp. 293-298 ◽  
Author(s):  
Prameet Sheth ◽  
Walter Kahr ◽  
Anwar Haq ◽  
Dragoslava Veljkovic ◽  
Georges Rivard ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Small Proportion ◽  
Bleeding Disorder ◽  
Phenotypic Changes ◽  
Granule Protein ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Platelet Disorder ◽  
Granule Proteins

SummaryThe Quebec Platelet Disorder (QPD) is an unusual bleeding disorder associated with increased platelet stores of urokinase-type plasminogen activator (u-PA) and proteolysis of platelet α–granule proteins. The increased u-PA and proteolyzed plasmino-gen in QPD platelets led us to investigate possible contributions of intracellular plasmin generation to QPD α-granule proteolysis. ELISA indicated there were normal amounts of plasminogen and plasmin-α2-antiplasmin (PAP) complexes in QPD plasmas. Like normal platelets, QPD platelets contained only a small proportion of the blood plasminogen, however, they contained an increased amount of PAP complexes compared to normal platelets (P < 0.005). The quantities of plasminogen stored in platelets were important to induce QPD-like proteolysis of normal α-granule proteins by two chain u-PA (tcu-PA) in vitro. Moreover, adding supplemental plasminogen to QPD, but not to control, platelet lysates, triggered further α-granule protein proteolysis to forms that comigrated with plasmin degraded proteins. These data suggest the generation of increased but limiting amounts of plasmin within platelets is involved in producing the unique phenotypic changes to α-granule proteins in QPD platelets. The QPD is the only known bleeding disorder associated with chronic, intracellular activation of the fibrinolytic cascade.

scholarly journals Cross-talk of anosmin-1, the protein implicated in X-linked Kallmann's syndrome, with heparan sulphate and urokinase-type plasminogen activator

2004 ◽  
Vol 384 (3) ◽  
pp. 495-505 ◽  
Author(s):  
Youli HU ◽  
David GONZÁLEZ-MARTÍNEZ ◽  
Soo-Hyun KIM ◽  
Pierre Marc Gilles BOULOUX
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Molecular Mechanisms ◽  
Expression System ◽  
Heparan Sulphate ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Kallmann's Syndrome ◽  
Kallmann’S Syndrome

Defective function of anosmin-1, the protein encoded by KAL-1, underlies X-linked Kallmann's syndrome (X-KS), a human hereditary developmental disorder. Anosmin-1 appears to play a role in neurite outgrowth and axon branching, although molecular mechanisms of its action are still unknown. Anosmin-1 contains a WAP (whey acidic protein-like) domain and four contiguous FnIII (fibronectin-like type III) repeats; its WAP domain shows similarity to known serine protease inhibitors, whereas the FnIII domains contain HS (heparan sulphate)-binding sequences. To investigate the functional role of these domains, we have generated both wild-type and mutant recombinant anosmin-1 proteins using a Drosophila S2 cell expression system. Here we present the first biochemical evidence demonstrating the high-binding affinity between HS and anosmin-1, as measured by SPR (surface plasmon resonance) (Kd=2 nM). The FnIII domains, particularly the first, are essential for dose-dependent HS binding and HS-mediated cell surface association. Furthermore, we have identified uPA (urokinase-type plasminogen activator) as an anosmin-1 interactant. Anosmin-1 significantly enhances the amidolytic activity of uPA in vitro; and anosmin-1–HS–uPA co-operation induces cell proliferation in the PC-3 prostate carcinoma cell line. Both the HS interaction and an intact WAP domain are required for the mitogenic activity of anosmin-1. These effects appear to be mediated by a direct protein interaction between anosmin-1 and uPA, since anosmin-1–uPA could be co-immunoprecipitated from PC-3 cell lysates, and their direct binding with high affinity (Kd=6.91 nM) was demonstrated by SPR. We thus propose that anosmin-1 may modulate the catalytic activity of uPA and its signalling pathway, whereas HS determines cell surface localization of the anosmin-1–uPA complex.

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