Expression of a cytosolic phospholipase A2 by ovine endometrium on days 11-14 of a simulated oestrous cycle.

Abstract

Oxytocin stimulates the synthesis and secretion of PGF2 alpha from uterine tissues in vivo and in vitro late in the ovine oestrous cycle. The synthesis of eicosanoids is dependent upon the availability of free arachidonic acid which is released through the activity of arachidonate releasing phospholipases. In the present study, the following hypothesis was tested: the ovine endometrium expresses a cytosolic phospholipase A2 (cPLA2) and expression or activity of cPLA2 increases as uterine secretory responsiveness to oxytocin develops late in the oestrous cycle. Endometrial tissue was collected from cyclic ewes on day 15 of the oestrous cycle for the preparation of tissue homogenates and isolation of mRNA to determine whether ovine endometrium expressed a cPLA2. A 110 kDa band was detected by western blotting, indicating the presence of a putative ovine cPLA2. A 834 bp fragment of the ovine cPLA2 shared 87% homology with human and mouse cDNA, and northern blot hybridization analysis indicated a single 3.4 kb transcript. A total of 20 ewes were ovariectomized and treated with progesterone and oestrogen to simulate the oestrous cycle to determine whether the expression or activity of ovine cPLA2 changed during the onset of uterine secretory responsiveness to oxytocin in vivo. On days 11-14 (n = 5 per day) of a simulated oestrous cycle, caruncular endometrium was evaluated for expression of ovine cPLA2 mRNA and protein and the synthesis of PGF2 alpha in response to melittin (a potent stimulator of PLA2 activity). Immunoreactive cPLA2 and cPLA2 mRNA were observed on all days and did not increase during the development of uterine responsiveness to oxytocin in vivo. Similarly, melittin increased the synthesis of PGF2 alpha irrespective of day, indicating the presence of a functional cPLA2 on all days examined. These data indicate that the ovine endometrium expresses a functional cPLA2 and that ample concentrations of cPLA2 are present by day 11 of a simulated oestrous cycle.