Production of Plasminogen Activators (PAs) in Bovine Cumulus-Oocyte Complexes during Maturation In Vitro: Effects of Epidermal Growth Factor on Production of PAs in Oocytes and Cumulus Cells1

Abstract

We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 +/- 3.5 kDa, 79.0 +/- 3.0 kDa, and 113.5 +/- 6.5 kDa) were observed. Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 +/- 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 +/- 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 +/- 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex. In COCs before culture, however, no activity of PAs was detected. At 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.

Urokinase-type plasminogen activator does not affect in vitro bovine embryo development and quality

“…PAs and u-PAR mRNA expression, as well as PAs activity, were found to be upregulated in a spatiotemporal manner after a GnRH-induced gonadotropin surge in the bovine periovulatory follicle, indicating their significant contribution to ovulation (Dow et al, 2002). In the same study, u-PAR mRNA was found to be primarily localised in the granulosa and theca cells, 6 h after the gonadotropin surge, while in vitro maturation of denuded bovine oocytes did not result in any increase in u-PA activity, suggesting that increased u-PA might not be of oocyte origin, but is physiologically provided by cumulus cells (Park et al, 1999).…”

“…Nowadays, the use of somatic cells in bovine IVP has been abandoned; here, cumulus cells were solely added to facilitate the study of possible effects of u-PA on embryo development. It has been proposed that u-PA activity is physiologically provided by cumulus cells instead of oocytes (Park et al, 1999). It is therefore possible that cumulus cells might moderate u-PA activity.…”

Fertilization outcome could be regulated by binding of oviductal plasminogen to oocytes and by releasing of plasminogen activators during interplay between gametes

“…Because integrin a 6 b 1 is present in the oocyte membrane and participates in human and mouse gamete fusion (36), the parallels between both models should be considered. The u-PA is synthesized by cumulus cells (37) and its activity has also been detected in in vitro matured bovine oocytes (38). Because u-PA receptors can bind integrins (39) and it has been suggested that oolemma-anchored integrins are receptors for sperm ADAM proteins, it would plausible that sperm binding to oolemma mediated by integrins triggers u-PA binding to its receptor, finally activating plasminogen.…”

“…In pigs, mRNA for PAI-1 has been also seen in the oviductal tissue (9) and plasminogen activator (PA) activity has been detected both in oviductal tissue and oviductal fluid (OF) of hamster (17). Bovine oocytes show u-PA activity (18). Mouse, rat, and Rhesus monkey oocytes show t-PA activity (12,16), and the presence of t-PA in cortical granules of rat and cow oocytes has been also proposed (19,20).…”

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

“…When the urokinase activity reached a particular threshold value, the cells were again induced to secrete urokinase into the medium leading to a temporary rise in the urokinase secretion profile. Park et al (1999) reported the production of plasminogen activators from oocytes and cumulus cells and according to their findings urokinase activity in the cultured broth increased at 12 h of culture without any further increase at 24 h. A minor decline in urokinase activity in the spent medium was observed after 144 h. Similar losses of urokinase activity on prolonged maintenance of the cultures have been reported previously by Lewis (1979). Further studies are needed to be performed on inhibitor secretion by this cell line in order to have a complete understanding of the urokinase secretion profile of the cell line.…”