Abstract
It is generally accepted that the in vitro embryo production (IVP) protocols are still far from ideal. The development of optimal conditions for IVP has been a step upon step procedure. The understanding of the mechanisms governing basic biological processes is necessary in this procedure. Plasminogen is an extracellular proenzyme, abundant in blood plasma and most extacellular fluids. Plasminogen activators (PAs) are proteolytic enzymes, capable of converting plasminogen into the broad-spectrum, trypsin-like proteinase, plasmin. The precise temporal and spatial regulation of plasminogen activator(s) or plasmin activity is controlled by plasminogen activator inhibitors and plasmin inhibitors, respectively. In many animal species and the bovine, members of the plasminogen activators/plasmin system or their activity have been shown in follicular fluid, cumulus-oocyte complexes (COCs), oocytes, and cumulus-cell cultures. Plasminogen activators and other members of the plasmin proteolytic ...
It is generally accepted that the in vitro embryo production (IVP) protocols are still far from ideal. The development of optimal conditions for IVP has been a step upon step procedure. The understanding of the mechanisms governing basic biological processes is necessary in this procedure. Plasminogen is an extracellular proenzyme, abundant in blood plasma and most extacellular fluids. Plasminogen activators (PAs) are proteolytic enzymes, capable of converting plasminogen into the broad-spectrum, trypsin-like proteinase, plasmin. The precise temporal and spatial regulation of plasminogen activator(s) or plasmin activity is controlled by plasminogen activator inhibitors and plasmin inhibitors, respectively. In many animal species and the bovine, members of the plasminogen activators/plasmin system or their activity have been shown in follicular fluid, cumulus-oocyte complexes (COCs), oocytes, and cumulus-cell cultures. Plasminogen activators and other members of the plasmin proteolytic system or their activity have been also shown in oviduct, in whole sperm extracts and in sperm plasma and outer acrosomal membrane extracts. Members of the plasmin system or their activity have been found in embryos from the zygote until the hatched blastocyst stage. Extracellullar proteolysis, linked with plasminogen activator/plasmin system activity, is probably implicated in physiological processes, such as cumulus-cell layer expansion, oocyte maturation, fertilization, zona reaction and embryo implantation. These findings provide evidence for a role of plasmin system in reproduction. Considering that in vitro embryo production (IVP) media emulate, to a degree, the in vivo conditions, the plasminogen activator/plasmin system presence and/or activity in the IVP procedure is possibly a factor that could affect IVP outcome. The aim of the present study was to examine the role of addition of urokinase plasminogen activator (u-PA), tissue type plasminogen activator (t-PA), plasmin or plasmin inhibitor (ε-ACA) into the different stages of bovine IVP procedure. In the first part of this study plasminogen activator activity, plasminogen activator inhibition and plasmin inhibition in preovulatory and mid-cycle dominant follicle follicular fluid, as well as in in vitro maturation, fertilization and embryo culture media were determined. In follicular fluid samples PAs activity was identified. In the second part of this study four major categories of experiments using 10.639 COCs were conducted. In the first one in vitro maturation medium was modified by addition of plasminogen (5 CU/0.1ml), u-PA (0.5 IU/0.1ml), t-PA (50 IU/0.1ml), plasmin (5 CU/0.1ml plasminogen + 1 IU/0.1ml u-PA and preincubation for 30 min) or ε-ACA (10 mM). After 18 or 24 hour incubation, oocytes were fixed and stained, in order to estimate the effect of IVM medium modification on nuclear maturation. In the second category after 18 or 24 hour incubation, as above, the oocytes underwent IVF and IVC, in order to estimate the effect of IVM medium modification on cytoplasmic maturation. In the third category, IVF medium was modified by addition of plasminogen (5 CU/0.1ml), u-PA (0.5 IU/0.1ml), t-PA (50 IU/0.1ml), plasmin (5 CU/0.1ml plasminogen + 1 IU/0.1ml u-PA and preincubation for 30 min) or ε-ACA (10 mM). After 24 hour incubation in the IVF medium, presumptive embryos were transferred in control IVC medium and 48 hour later incubation was terminated, in order to estimate the effect of IVF medium modification on fertilization and subsequent embryo development. In the fourth category IVC medium was modified by the addition of plasminogen (5 CU/0.1ml), u-PA (0.5 IU/0.1ml), t-PA (50 IU/0.1ml), plasmin (5 CU/0.1ml plasminogen + 1 IU/0.1ml u-PA and preincubation for 30 min) or ε-ACA (10 mM). After 24h incubation in controls IVM and IVF media presumptive embryos were transferred in the modified IVC medium. The estimation of embryo development was made at 48h of incubation. In each experiment, one standarized medium served as control, while a second plasminogen-supplemented medium was also included. The modification of the media was performed only to one phase of IVP each time. Our results are as follows: 1) a. PAA of preovulatory and mid-cycle dominant follicle follicular fluid was significantly higher compared to PAA detected in IVM, IVF and IVC media. PAI and PI of preovulatory and mid-cycle dominant follicle follicular fluid were significantly higher compared to PAI and PI detected in IVM, IVF and IVC media. b. PAA and PAI of the follicular fluid of preovulatory follicle were significantly higher compared to PAA and PAI of mid-cycle dominant follicle follicular fluid. c. In follicular fluid samples both plasminogen activators were identified
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