The regulation of prostaglandin E2 and prostaglandin F2α in primary cultures of epithelial and stromal cells of bovine endometrium was investigated using two physiological agents, oxytocin and platelet-activating factor, and the protein kinase C activator, 4β-phorbol 12-myristate 13-acetate. At the basal level, epithelial cells produced more prostaglandin F2α than did stromal cells (P ≤ 0.0001) and stromal cells produced more prostaglandin E2 than did epithelial cells (P ≤ 0.0001). In the presence of oxytocin, production of prostaglandin E2 and F2α increased in a dose-dependent manner, only in epithelial cells. Stromal cells did not respond to oxytocin, suggesting that the oxytocin response is cell type specific, acting preferentially on the cell type in which prostaglandin F2α is the major prostaglandin produced. Platelet-activating factor increased prostaglandin E2 and prostaglandin F2α production in epithelial cells at 1 and 10 pmol l−1 (PGE2: 10 pmol l−1, P ≤ 0.01; PGF2α: 1 pmol l−1, P ≤ 0.02). Stromal cells also responded to platelet-activating factor, but only at high concentrations (PGE2: 0.1 μmol l−1, P ≤ 0.001; PGF2α: 0.1 μmol l−1, P ≤ 0.0001). These results further demonstrate the differences in epithelial and stromal cells; epithelial cells are more sensitive to platelet-activating factor than are stromal cells. Phorbol 12-myristate 13-acetate increased prostaglandin production in a dose-dependent manner in both epithelial and stromal cells, indicating that protein kinase C activation can increase prostaglandin production in these cells. Higher concentrations of phorbol 12-myristate 13-acetate down-modulated protein kinase C activity. The role of protein kinase C in oxytocin- and platelet-activating factor-induced increase in prostaglandin was investigated by down-modulating protein kinase C in epithelial cells and subsequently treating the cells with oxytocin and platelet activating factor. Oxytocin- and platelet-activating factorinduced increases in prostaglandin production were not found in protein kinase C-down-modulated cells, suggesting that protein kinase C activation is necessary for this response.
S. Bilodeau, M. A. Fortier and M. A. Sirard
The effect of adenylate cyclase stimulation via the components of the enzyme on nuclear maturation in bovine cumulus-enclosed and zona-free oocytes was examined. The stimulating agents were cholera toxin, pertussis toxin, forskolin, sodium fluoride and prostaglandin E2. Cyclic AMP contents were measured in cumulus-oocyte complexes, cumulus-enclosed oocytes and in zona-free oocytes after stimulation, to establish the relationship between cumulus cell and oocyte cAMP concentrations and the meiotic status of the oocyte. In cumulus-enclosed oocytes, forskolin alone and 3-isobutyl-1-methylxanthine (IBMX), at 0.5 mmol l−1, inhibited the resumption of meiosis after 8 h of culture; the other agents were without effect. After 24 h of culture, IBMX at 0.5 mmol l−1 was without effect, but at 2 mmol l−1 reduced the percentage of oocytes at the mature stage (51 versus 82% in control medium). Forskolin alone reduced the proportion of oocytes at the mature stage from 82 to 58%. Forskolin plus IBMX at 2 mmol l−1 and sodium fluoride plus IBMX at 2 mmol l−1 significantly diminished the maturation rate (6 and 17% mature oocytes, respectively). Cholera toxin (with IBMX) and forskolin (alone or with IBMX) stimulated the synthesis of high amounts of cAMP in complexes, but only forskolin had a significant effect on the cAMP contents of oocytes derived from complexes. Forskolin was more effective in zona-free oocytes than in cumulusenclosed oocytes in inhibiting nuclear maturation (24% mature oocytes versus 73% in control medium) even after 24 h of culture; its effect was potentiated by IBMX; forskolin also stimulated cAMP synthesis. IBMX was as effective as forskolin in delaying nuclear maturation, but did not cause an accumulation of cAMP above the control value. The other agents were without effect on meiosis and cAMP concentrations in zona-free oocytes. These results suggest that increases in cAMP concentration in denuded oocytes inhibit maturation; but, when cAMP concentrations are high in cumulus cells, a maturation signal can be generated that bypasses the inhibitory effect of cAMP in the oocyte. Bovine oocytes can synthesize high amounts of cAMP, but its adenylate cyclase may not be coupled to G proteins sensitive to cholera or pertussis toxin.
M. A. Fortier, A. P. Boulet and R. D. Lambert
Summary. Adenylate cyclase activity was measured in broken cell preparations of whole endometrial tissue from rabbits on Days 0, 1, 6·5, 9 and 15 of pseudopregnancy and in endometrial epithelial and stromal cells on Days 1 and 6·5 to assess the specific response of individual cell types. In dispersed cells, adenylate cyclase activity was higher (P < 0·01) in stromal than in epithelial cells and reduced on Day 6·5 compared to Day 1 in both cell types. The response of adenylate cyclase to isoproterenol appeared more important relative to the PGE-2 response in epithelial than in stromal cells and strongly reduced in the former on Day 6·5. In endometrium, the overall adenylate cyclase activity was increased significantly on Day 1 of pseudopregnancy compared to Day 0 (oestrus), only 18 h after injection of hCG. On the following days, the activity decreased progressively on Days 6·5 and 9 and exhibited a recovery on Day 15. Adenylate cyclase response to isoproterenol (% over GTP) was comparable on Days 0, 1 and 6·5, abolished on Day 9 and recovered on Day 15. Maximal response to PGE-2 (% over GTP) was observed on Day 6·5, at the time of implantation, maintained on Day 9 and reduced on Day 15 towards the low levels measured in oestrus and Day 1 of pseudopregnancy. Our results demonstrate a dramatic alteration of adenylate cyclase activity in rabbit endometrium during pseudopregnancy. It suggests a possible involvement of catecholamines and prostaglandin E-2 in the regulation of endometrial receptivity through a cAMP-mediated process.
Keywords: endometrium; prostaglandins; cyclic AMP; catecholamines; decidual reaction
M. A. Fortier, L. A. Guilbault and F. Grasso
Summary. Epithelial and stromal cells were isolated from endometrium of cyclic heifers by enzymic dispersion. These cells exhibited specific morphological and functional properties. Epithelial cells appeared cuboidal or columnal and showed contact inhibition as they reached confluence. Stromal cells were fibroblast-like and enlarged at the time of confluence after which they overgrew in multiple layers. The presence of specific receptors for PGE-2 and β-adrenergic catecholamines (isoproterenol) was estimated by activation of adenylate cyclase. Stromal cells had more adenylate cyclase activity (P < 0·01) than did epithelial cells before (basal) and after stimulation with guanosine triphosphate (GTP) and PGE-2. However, epithelial cells were much more responsive to isoproterenol (P < 0·01). Treatment of cultured cells with indomethacin to block PG synthesis increased the sensitivity and maximal response to PGE-2 in stromal (P < 0·01) but not in epithelial (P > 0·1) cells. The latter result suggested autologous desensitization of the PGE-2 response resulting from synthesis of PGs in cultured cells. Both cell types synthesized PGs in culture: PGF-2α was synthesized in greater quantity in epithelial than in stromal cells (P < 0·05) while stromal cells synthesized more PGE-2 than did epithelial cells (P < 0·001). Endometrial cells separated in this way should prove useful for study of their specific role in the processes of implantation and maternal recognition of pregnancy.
Keywords: cattle; endometrium; cell culture; adenylate cyclase; prostaglandins