EXPRESSION OF AVIAN UROKINASE AND TISSUE-TYPE PLASMINOGEN-ACTIVATOR MESSENGER-RIBONUCLEIC-ACID DURING FOLLICLE DEVELOPMENT AND ATRESIA

While several studies have documented the presence of plasminogen acti vator (PA) activity in hen ovarian follicle granulosa and theca tissue s, to date it has not been possible to conclusively distinguish betwee n the urokinase (u) and the tissue-type (t) form of the enzyme; this i nability is due, in part, to lack of the cloned or characterized chick en tPA gene or gene product. Thus, the present studies were conducted to identify a partial cDNA for chicken tPA and subsequently to evaluat e expression of uPA and tPA mRNA in granulosa and theca tissues in viv o and in vitro. Urokinase PA mRNA levels were highest in prehierarchic al-follicle granulosa (3- to 5- and 6- to 8-mm follicles) and theca (6 - to 8-mm follicles) tissue compared to hierarchical (9-12 mm through largest preovulatory) follicles. In vitro treatment with a phorbol est er (phorbol 12-myristate, 13-acetate), but not a cAMP analogue (8-brom o-cAMP), significantly increased uPA mRNA levels in both granulosa and theca tissue from the largest and second-largest preovulatory follicl es. Of special interest was the finding that levels of uPA mRNA were 1 0.9-fold higher in atretic compared to morphologically normal 3- to 5- mm follicles. Moreover, 4- to 8-mm-follicle granulosa cells, which spo ntaneously undergo apoptosis in vitro, demonstrated a rapid increase i n uPA mRNA levels after 1 h of incubation (prior to the detection of o ligonucleosome formation) while levels in preovulatory-follicle granul osa cells, which do not undergo spontaneous apoptosis, were not altere d after 18 h of incubation. By contrast, while tPA mRNA can be identif ied in granulosa and theca tissues from prehierarchical and preovulato ry follicles following polymerase chain reaction amplification, consti tutively expressed levels of the transcript were too low to reliably m easure by Northern blot analysis. These data indicate that while the c hicken expresses a tPA gene that is homologous to the mammalian tPA, u PA is the predominant PA expressed in the hen ovary. In addition, the higher levels of uPA mRNA found in granulosa cells actively undergoing apoptosis and in follicles most susceptible to atresia (4-8 mm) sugge st a role for this protease in mediating processes both during the ear ly stages of programmed cell death and in the later stages of follicle atresia and resorption.