Oxytocin stimulates secretion of prostaglandin F(2alpha) from endometrial cells of swine in the presence of progesterone.

Oxytocin (OT) stimulates endometrial secretion of prostaglandin (PG) F(2 alpha) during corpus luteum regression in swine but there is differential responsiveness to OT among endometrial cell types. To determine if progesterone influenced responsiveness of luminal epithelial, glandular epithelial, and stromal cells to 100 nM OT during luteolysis in swine, cells were isolated from endometrium of 15 gilts by differential enzymatic digestion and sieve filtration on day 16 postestrus and cultured continuously in the presence of 0, 10 or 100 nM progesterone. For phospholipase C (PLC) activity and PGF(2 alpha) secretion, stromal cells were most responsive to OT (P<0.01) in the absence of progesterone, whereas luminal epithelial cells were unresponsive and glandular epithelial cells displayed an intermediate response to OT (P<0.09). Progesterone enhanced PLC activity linearly in glandular epithelial cells (P<0.05) and influenced it quadratically in stromal cells (P=0.05). The effect of OT and progesterone on PLC activity in luminal epithelial cells was not significant, and progesterone did not increase PLC activity in response to OT in any cell type. Culture in the presence of progesterone, enhanced PGF(2 alpha) secretion in response to OT in luminal epithelial cells (P<0.05) but not in glandular epithelial or stromal cells. Progesterone also increased overall PGF(2 alpha) release from glandular epithelial (P<0.05) and stromal cells (P<0.06) across both levels of OT treatment. These results indicate that progesterone enhanced PGF(2 alpha) secretion from luminal epithelial cells in response to OT and increased basal PGF(2 alpha) release from glandular epithelial and stromal cells.

Oxytocin stimulates prostaglandin F2alpha secretion from porcine endometrial cells through activation of calcium-dependent protein kinase C.

The mechanism for oxytocin's (OT) stimulation of PGF2alpha secretion from porcine endometrium is not clear, but is thought to involve mobilization of intracellular Ca2+ and subsequent activation of protein kinase C (PKC). This study determined: (1) if mobilization of inositol trisphosphate-sensitive Ca2+ by thapsigargin or activation of PKC by phorbol 12-myristate 13-acetate (PMA) could stimulate PGF2alpha release from luminal epithelial, glandular epithelial and stromal cells of porcine endometrium and (2) if inhibitors of various PKC isotypes could attenuate the ability of OT, thapsigargin and PMA to stimulate PGF2alpha secretion from these cells. Thapsigargin and PMA each stimulated (P < 0.01) PGF2alpha secretion from all three endometrial cell types examined. However, the effects of thapsigargin and PMA were synergistic (P < 0.05) only in stromal cells. Three protein kinase C inhibitors (i.e. Gö6976, Gö6983 and Ro-31-8220) differentially attenuated (P < 0.05) the ability of OT, thapsigargin and PMA to stimulate PGF2alpha release. These results are consistent with the hypothesis that OT mobilizes Ca2+ to activate a Ca2+-dependent PKC pathway to promote PGF2alpha secretion from porcine endometrial cells. The differing pattern of response to isotype-specific inhibitors of PKC among cell types suggests that distinct PKC isoforms are differentially expressed in luminal epithelial, glandular epithelial and stromal cells.

Effects of arginine- and lysine-vasopressin on phospholipase C activity, intracellular calcium concentration and prostaglandin F2alpha secretion in pig endometrial cells.

Oxytocin and vasopressin are related peptides that have receptors in the uterus. Species from families other than Suidae produce only arginine-vasopressin; in contrast, pigs apparently express both arginine- and lysine-vasopressin. The aim of this study was to determine whether arginine- or lysine-vasopressin would activate phospholipase C, increase intracellular calcium concentration [Ca(2+)](i) and stimulate PGF(2alpha) production in enriched cultures of stromal, glandular epithelial and luminal epithelial cells from pig endometrium. Cells were obtained from gilts on day 16 after oestrus by differential enzymatic digestion and sieve separation. After 96 h in culture, the cells were treated with 0 or 100 nmol arginine- or lysine-vasopressin l(-1). The responses to 100 nmol oxytocin l(-1) and 100 nmol GnRH l(-1) were used as positive and negative controls, respectively. Consistent with previous results, oxytocin stimulated phospholipase C activity (P < 0.05), increased [Ca(2+)](i) (P < 0.05) and promoted PGF(2alpha) secretion (P < 0.05) from stromal and glandular epithelial cells. Activity of phospholipase C, [Ca(2+)](i) and PGF(2alpha) release were also increased (P < 0.05) by arginine-vasopressin in stromal cells, but the responses were less (P < 0.01) than those induced by oxytocin. An oxytocin antagonist attenuated the [Ca(2+)](i) response of stromal cells to both oxytocin and arginine-vasopressin. Sequential treatment of cells with oxytocin and arginine-vasopressin indicated that oxytocin desensitized the response to oxytocin, but arginine-vasopressin did not similarly desensitize the response to oxytocin. In glandular and luminal epithelial cells, arginine-vasopressin did not stimulate phospholipase C activity, [Ca(2+)](i) or PGF(2alpha) secretion. Neither GnRH nor lysine-vasopressin induced phospholipase C activity, increased [Ca(2+)](i) or stimulated PGF(2alpha) production in any endometrial cell type. These results indicate that oxytocin receptors can bind arginine-vasopressin more readily than they bind lysine-vasopressin. Type 1 vasopressin receptors may also exist in endometrium predominantly on cells other than stromal, glandular epithelial and luminal epithelial cells, as in previous studies both arginine-vasopressin and lysine-vasopressin stimulated phospholipase C activity in endometrial explants to a similar extent as oxytocin.

Directional secretion of prostaglandin F(2alpha) by polarized luminal epithelial cells from pig endometrium.

In swine, endometrial prostaglandin F(2alpha) (PGF(2alpha)) is the luteolysin. The capacity of luminal epithelial cells isolated from the endometrium of day 16 cyclic pigs, to secrete PGF(2alpha)500 Omega/cm(2)), they were treated on the apical, basal or both surfaces with 0 or 100 nM oxytocin (OT) in Experiment 1 or phorbol 12-myristate 13-acetate (PMA) in Experiment 2. In the absence of OT or PMA, PGF(2alpha) secretion occurred primarily from the basal surface and was approximately 12-fold greater (P < 0.001) than from the apical surface. Treatment with OT did not stimulate PGF(2alpha) secretion from either surface regardless of which surface was treated. In contrast, PMA increased PGF(2alpha) secretion from both surfaces. Treatment of the apical surface or both surfaces with PMA increased (P < 0.001) PGF(2alpha) secretion similarly from both surfaces. Treatment of only the basal surface with PMA increased (P < 0.01) PGF(2alpha) secretion from both surfaces, but tended (P = 0. 06) to increase its secretion from the basal surface more than from the apical surface. These results indicated that PGF(2alpha) secretion by luminal epithelial cells obtained from cyclic pigs occurs primarily toward a basal direction and is not stimulated by oxytocin. Activation of protein kinase C stimulates directional secretion of PGF(2alpha) from both surfaces of the epithelial cells.

Oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F2alpha secretion by luminal epithelial, glandular epithelial and stromal cells from pig endometrium. II. Responses of cyclic, pregnant and pseudopregnant pigs on days 12 and 16 post oestrus.

In pigs, the exact mechanism for the shift in endometrial PGF2alpha secretion from an endocrine to an exocrine mode during pregnancy recognition is not known. The objective of this study was to examine whether this shift involved a change in the responsiveness of luminal epithelial, glandular epithelial and stromal cells to 0 or 100 nM oxytocin. Luminal epithelial cells, glandular epithelial cells and stromal cells were isolated from cyclic, pregnant or oestrogen-induced pseudopregnant gilts on Day 12 (Experiment 1) or Day 16 (Experiment 2) post oestrus (oestrus = Day 0). For cells obtained on Day 12, oxytocin stimulated PGF2alpha secretion by stromal cells (P<0.01) similarly for each reproductive status, whereas oxytocin stimulated PGF2alpha secretion from luminal and glandular epithelial cells (P<0.05) from pregnant and pseudopregnant gilts but not from cyclic gilts. For both concentrations of oxytocin, mean PGF2alpha secretion was less (P<0.05) from stromal cells of pregnant than cyclic gilts. For cells obtained on Day 16, oxytocin stimulated PGF2alpha release from stromal cells of cyclic gilts but not from stromal cells of pregnant gilts. Mean PGF2alpha secretion also was less (P<0.05) from stromal cells of pregnant gilts than cyclic gilts. Oxytocin tended to stimulate PGF2alpha release (P<0.07) from glandular epithelial cells of cyclic but not pregnant or pseudopregnant gilts. Luminal epithelial cells from all reproductive statuses were similarly unresponsive to oxytocin. In conclusion, the increased PGF2alpha secretory response to oxytocin of luminal and glandular epithelial cells from pregnant gilts on Day 12, combined with the decreased response of stromal cells from pregnant gilts on Days 12 and 16, may contribute, in part, to the shift in endometrial PGF2alpha secretion from an endocrine to an exocrine direction during early pregnancy in pigs.

Intracellular free calcium in response to oxytocin in pig endometrial cells.

Intracellular free calcium concentration ([Ca2+]i) in response to oxytocin (OT) was studied in stromal, glandular epithelial and luminal epithelial cells obtained from the endometrium of gilts 16 days post-estrus. The amplitude of increased [Ca2+]i in response to 100 nM OT was greatest in stromal cells, intermediate in glandular epithelial cells and not evident in luminal epithelial cells. During continuous OT administration, stromal cells responded initially with a synchronous spike of [Ca2+]i that did not require extracellular Ca2+ and then displayed spontaneous asynchronous [Ca2+]i spikes that required extracellular Ca2+. Each cell possessed its own characteristic response. Increasing concentrations of OT induced an increasing percentage of stromal cells responding, with some cells having nearly equal [Ca2+]i responses at all concentrations and others having graded [Ca2+]i responses as the concentration of OT increased. These results are consistent with the proposed mechanism of OT action in pig endometrium involving activation of phosphoinositide-Ca2+ signaling pathway.

Oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F secretion by luminal epithelial, glandular epithelial, and stromal cells from pig endometrium. I. Response of cyclic pigs on day 16 postestrus.

Oxytocin (OT) is the physiological stimulus for pulsatile release of endometrial prostaglandin (PG) F2alpha during luteolysis in domestic ungulates, and the cellular mechanism for this appears to involve phosphoinositide (PI) hydrolysis. To determine which endometrial cell type(s) was responsive to OT during luteolysis in swine, luminal epithelial (LEC), glandular epithelial (GEC), and stromal cells (SC) were isolated from endometrium by differential enzymatic digestion and sieve filtration on Day 16 postestrus and cultured. For PI hydrolysis in experiment 1, SC were most responsive to 100 nM OT (p < 0.001), whereas LEC were least responsive and GEC had an intermediate response (p < 0.001). For PGF secretion in experiment 2, the response to OT was greatest for SC, least for LEC, and intermediate for GEC. In experiment 3, 100 nM OT increased PI hydrolysis in SC within 30 min (p < 0.05) and in GEC within 60 min (p < 0.05) but did not increase PI hydrolysis in LEC. In experiment 4, PI hydrolysis in SC was increased (p < 0.05) by 33-333 nM OT but was not increased by

FSH bioactivity in commercial preparations of gonadotropins.

During the past 2 decades, commercial preparations of FSH have been extensively used to superovulate cattle. The problems that have been encountered in superovulation of cattle include high variability in the ovulation rate and subsequent yield of viable embryos. The lack of predictability in superovulatory trials has been attributed to difficulties in standardizing the potency of commercial FSH preparations. Traditionally, FSH potency has been tested in bioassays that utilize specific responses in whole animals or primary cell cultures. Whole animal bioassays lack sensitivity, while primary cell culture bioassays, which use fresh cells, have inherent variability within each preparation. An FSH bioassay that employed a stable chimeric cell line expressing the human FSH-R was used to provide an accurate measurement of FSH bioactivity. The hormonal potency of 2 commercial preparations of FSH used to superovulate cattle was determined using FSH immuno- and bioassays. Commercial FSH preparations differed in potency. One commercial product, prepared in 4 different years, showed no difference in the immunoactive levels of FSH. In the same product stored under identical conditions, FSH bioactivity varied from year to year. There was variability in FSH bioactivity both between and within commercial products. The lack of correlation between bioactivity and immunoactivity of commercial FSH preparations may explain, in part, the variability observed in superovulation of cattle.

3ß-hydroxy-5-ene Steroid dehydrogenase gene expression regulation in porcine granulosa cells : Differential effect of FSH and LH on gene transcription.

The objective of this study was to investigate the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) on FSH- and LH-induced 3ß-HSD-gene expression in cultured porcine granulosa cells. FSH and LH induced a dose dependent increase in the accumulation of 3ß-HSD mRNA, measured by Northern blot. A 1.6- to 1.8-fold increase (p<0.01) was observed with 10 ng/mL of FSH or LH. Maximal levels of 2.5- to 2.9-fold increases, relative to control, were reached at 30 and 100 ng/mL of the gonadotropins. When granulosa cells were treated with PMA (100 nM) just before the addition of FSH, the 3ß-HSD rnRNA levels induced by 10 or 30 ng/mL of FSH were inhibited or partially inhibited, respectively. PMA did not inhibit elevated levels of 3ß-HSD mRNA induced by FSH at concentrations of 100, 300, and 1000 ng/mL. Alternatively, PMA added just before LH, inhibited LH-stimulated 3ß-HSD mRNA levels at all doses of LH tested (10, 30, 100, 300, and 1000 ng/mL). The protein kinase A-stimulators, dibutyryl-cAMP (cAMP) (0.5 mM) and forskolin (10 nM), also elevated the 3ß-HSD-gene transcription, 3.5- and 4.0-fold respectively. PMA prevented the stimulation of the 3ß-HSD-gene transcription when it was added just before cAMP or forskolin. We concluded that stimulation of PKC by PMA appears to have inhibited the gonadotropin-induced increase in 3ß-HSD mRNA levels by preventing cAMP-activated 3ß-HSD-gene transcription. The data also suggest that the effect of PMA appears to be more specific for regulation of LH-stimulated intracellular signals than those of FSH. This effect may indicate a site of differential regulation of FSH and LH on the stimulation of 3ß-HSD-gene transcription.

3ß-hydroxy-5-ene steroid dehydrogenase gene expression regulation in porcine granulosa cells. I: FSH- and LH-mediated transcriptional activation.

In this report we examined the effect of FSH and LH, on the steady state levels of 3ß-5-hydroxy-5-ene steroid dehydrogenase (3ß-HSD) mRNA and on the 3ß-HSD-gene transcriptional activation in porcine cultured granulosa cells. Exposure of granulosa cells to 100 ng/ml FSH or LH for 8 h, elevated to 3.0 and 2.5-fold respectively the levels of 3ß-HSD mRNA measured by Northern blot analyses. The withdrawal of FSH and LH induced a rapid decay of the 3ß-HSD levels, reaching the control values after 2 h. Re-addition of FSH and LH after 4 h withdrawal elevated the levels of 3ß-HSD mRNA to 4.8 and 5.3-fold respectively. Addition of actinomycin D, to granulosa cells previously treated with FSH or LH, induced a rapid decay in the levels of 3ß-HSD mRNA, reaching the control values after 2 h, with an estimated half life 1.3 and 1.2 h respectively. FSH and LH stimulated the 3ß-HSD-gene transcription, measured by nuclear run-on assays, by 1.7 and 1.9-fold respectively. Addition of cholera toxin (10 ng/ml) or forskolin (10NM: ) stimulated the 3ß-HSD-gene transcription by 2.15 and 2.4-fold respectively. We conclude that gonadotropins positively regulate 3ß-HSD transcriptional activation and appear to have no effect on the 3ß-HSD mRNA stability.