Interrelationships between metabolic hormones,leptin and ghrelin,and oil-related contaminants in control of oxytocin and prostaglandin F release by feline ovaries

We examined the effects of metabolic hormones leptin and ghrelin, and the oil-related environmental contaminants toluene and xylene on the release of ovarian hormones by gravid and non-gravid cats, as well as the functional interrelationships between metabolic hormones and contaminants. Ovarian fragments of non-gravid cats were cultured with and without leptin and toluene. Next, ovarian fragments of either non-gravid or gravid animals were cultured with and without ghrelin and xylene. Oxytocin (OT) and prostaglandin F (PGF) release was measured using ELISA.We confirm ovarian OT and PGF production by feline ovary, demonstrate the involvement of leptin and ghrelin in controlling OT and PGF release, show the direct influence of toluene and xylene on feline ovarian secretory activity, indicate the ability of leptin and ghrelin to mimic and promote the main contaminant effects, demonstrate that oil-related contaminants can prevent and even invert the effects of leptin and ghrelin on the ovary, and suggest the gravidity-associated changes in ability of ghrelin to promote xylene action on PGF (but not to OT), but not in basic ovarian OT and PGF release and their response to ghrelin or xylene.     

Prostaglandin F2 alpha, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effects of oxytocin, luteinizing hormone, prostaglandin F2 alpha and estradiol-17 beta

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 x 10(5) cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F2 alpha (PGF), oxytocin (OT), estradiol-17 beta (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 +/- 66.2, 111.1 +/- 37.8, 57.7 +/- 15.4 and 124.3 +/- 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P less than 0.01) than on Day 8, 14 and 18 (OT: 17.5 +/- 2.6 versus 5.6 +/- 0.7, 6.0 +/- 1.4 and 3.1 +/- 0.4 pg/ml; P: 138.9 +/- 19.5 versus 23.2 +/- 7.5, 35.4 +/- 6.5 and 43.6 +/- 8.1 ng/ml, respectively). Oxytocin increased (P less than 0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17 beta stimulated (P less than 0.05) PGF secretion on Days 8, 14 and 18, and LH increased (P less than 0.01) PGF production only on Day 14. Prostaglandin F2 alpha, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P less than 0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P less than 0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Prostaglandin F2α, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: Effects of oxytocin, luteinizing hormone, prostaglandin F2α and estradiol-17β

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 × 10⁵ cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F₂α (PGF), oxytocin (OT), estradiol-17β (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 ± 66.2, 111.1 ± 37.8, 57.7 ± 15.4 and 124.3 ± 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P<0.01) than on Day 8, 14 and 18 (rmOT: 17.5 ± 2.6 versus 5.6 ± 0.7, 6.0 ± 1.4 and 3.1 ± 0.4 pg/ml; P: 138.9 ± 19.5 versus 23.2 ± 7.5, 35.4 ± 6.5 and 43.6 ± 8.1 ng/ml, respectively). Oxytocin increased (P<0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17β stimulated (P<0.05) PGF secretion on Days 8, 14 and LH increased (P<0.01) PGF production only on Day 14. Prostaglandin F₂α, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P<0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P<0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Involvement of ovarian steroids in basal and oxytocin-stimulated prostaglandin (PG) F2 alpha secretion by the bovine endometrium in vitro

It is assumed that exposure of endometrium to spontaneously secreted luteal hormones stimulates PGF2 alpha secretion and modifies oxytocin (OT) influence on the bovine uterus. At first, the time-dependent effect of endogenous luteal products on endometrial PGF2 alpha secretion was examined. Endometrial strips (100 mg) from slaughtered heifers (Days 11 to 17 of the cycle) were incubated alone or with luteal cells (1 x 10(5) cells/mL). The highest PGF2 alpha secretion by the endometrium under influence of hormones secreted from luteal cells was observed after 12 h of incubation compared with the control (P < 0.001). Then, endometrium (Days 11 to 17) was incubated with luteal cells and concomitantly with antagonists of P4 and OT. The P4 antagonist prevented the stimulatory effect of endogenous luteal hormones on PGF2 alpha secretion (P < 0.05), but the OT antagonist did not. Further, direct effects of exogenous P4, OT and estradiol (E2) on endometrial PGF2 alpha secretion (Days 11 to 17) were examined. Both OT and P4 increased PGF2 alpha secretion (P < 0.05); E2 alone had no effect on PGF2 alpha secretion, but it amplified the P4 effect (P < 0.05). Finally, we studied the effect of endogenous luteal products on OT-stimulated PGF2 alpha secretion from endometrium. When endometrium (Days 11 to 17) was incubated without luteal cells, OT stimulated PGF2 alpha secretion (P < 0.001), whereas incubation of endometrium with luteal cells abolished the stimulatory effect of OT on PGF2 alpha secretion (P < 0.001). These treatments did not affect PGF2 alpha secretion from the endometrium collected on Days 1 to 4. In conclusion, P4 stimulates PGF2 alpha secretion by the endometrium and E2 amplifies this effect. As long as the endometrium is under the influence of P4, ovarian OT does not affect PGF2 alpha secretion.     

Leptin directly controls secretory activity of human ovarian granulosa cells: possible inter-relationship with the IGF/IGFBP system

AIMS: The aim of our in vitro studies was to understand the role of leptin and the insulin-like growth factor I/insulin-like growth factor protein (IGF/IGFBP) system in controlling human ovarian function. METHODS: We studied the action of leptin (0, 1, 10, or 100 ng/ml) and immunoneutralization of IGF-I using specific antiserum (0.1%) on the release of progesterone (P), estradiol (E), oxytocin (OT), IGF-I, IGFBP-3, and prostaglandins F (PGF) by these cells using radioimmunoassay/immunoradiometric assay. RESULTS: It was found that leptin stimulated the secretion of OT, IGFBP-3, and PGF. It suppressed the secretion of E and IGF-I, but not P, into the medium. The addition of antiserum against IGF-I decreased IGF-I output, increased P, OT, IGFBP-3, and PGF secretion, and had no effect on E release. Immunoneutralization of IGF-I also prevented or reversed the effects of leptin on P, E, IGF-I, IGFBP-3, PGF, but not on OT. CONCLUSIONS: These observations (1) demonstrate that leptin directly controls the secretory activity of human ovarian cells, (2) confirm the involvement of IGF-I in the regulation of ovarian cells, and (3) suggest an inter-relationship between leptin and the IGF/IGFBP system in the control of these functions and the involvement of IGF/IGFBP system in mediating leptin action on the ovary.     

Effect of ovarian steroids and oxytocin on the production of prostaglandin E2, prostaglandin F2alpha and endothelin-1 from cow oviductal epithelial cell monolayers in vitro.

Cyclic physio-anatomical variation in the oviducts is mediated by the local countercurrent transfer of ovarian products. Thus, in this study cow oviductal epithelial cells (COEC) culture were utilized to investigate the effects of ovarian products such as progesterone (P4), estradiol 17beta (E2) and oxytocin (OT) on local oviductal prostaglandin E2 (PGE2), F2alpha (PGF2alpha) and endothelin-1 (ET-1) production. COEC were collected from non-pregnant Holstein cows (n = 8) during the follicular phase and cultured in M199 under standard culture conditions until monolayer formation. Cells in first passage were incubated for 24 or 48 h with P4 (500 ng/ml), E2 (1 ng/ml), OT (10(-9) M) or combination of E2 + P4. Administration of E2 significantly increased the production of PGE2, PGF2alpha and ET-1. However, simultaneous administration of P4 blocked the effect of E2. OT did not show any effect on oviductal productions of either PGs or ET-1. The results of this study show that E2 stimulates PG and ET-1 production by COEC in vitro. Thus, it can be suggested that locally transferred E2 from the ovarian follicles may be important for oviductal contraction and gamete/zygote transport during the peri-ovulatory period.     

Growth hormone, but not luteinizing hormone, acts with luteal peptides on prostaglandin F2alpha and progesterone secretion by bovine corpora lutea in vitro*

Prostaglandin F2alpha (PGF2alpha) is a major physiological luteolysin in the cow. However, injection of PGF2alpha before day 5 (day 0 = estrus) of the estrous cycle dose not induce luteolysis. On the other hand, the early corpus luteum (CL) actively produces PGF2alpha. This indicates that luteal PGF2alpha may play a key role in the refractoriness to PGF2alpha injected during the early luteal phase when angiogenesis is active in the CL. Thus, this study aimed to investigate the possible interaction between pituitary hormones and local factors (luteal peptides) on secretion of PGF2alpha and progesterone (P) by the early bovine CL, and to evaluate the effect of growth hormone (GH) as well as its interactions on production of PGF2alpha in the developing CL. A RT-PCR analysis revealed that mRNA for GH receptor in CL was fully expressed from early in the luteal phase throughout the estrous cycle, while luteinizing hormone (LH) receptor mRNA was expressed less by the early and regressing CL than those at mid or late luteal phases (P < 0.05). For the stimulation test, an in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of GH, insulin-like growth factor-1 (IGF-1) and oxytocin (OT) increased (P < 0.05) PGF2alpha and P release. In contrast, LH had no effect (P > 0.05) on PGF2alpha secretion and little effect on P release. Unexpectedly, there was no distinct interaction between pituitary hormones and luteal peptides on secretion of PGF2alpha and P. These results indicate that GH is a more powerful stimulator of PGF2alpha and P production in the early bovine CL than LH and suggest that GH and luteal peptides, IGF-1 and OT, contribute to maintenance of elevated PGF2alpha production in the developing bovine CL.     

Growth hormone, but not luteinizing hormone, acts with luteal peptides on prostaglandin F2alpha and progesterone secretion by bovine corpora lutea in vitro

Prostaglandin F2alpha (PGF2alpha) is a major physiological luteolysin in the cow. However, injection of PGF2alpha before day 5 (day 0 = estrus) of the estrous cycle dose not induce luteolysis. On the other hand, the early corpus luteum (CL) actively produces PGF2alpha. This indicates that luteal PGF2alpha may play a key role in the refractoriness to PGF2alpha injected during the early luteal phase when angiogenesis is active in the CL. Thus, this study aimed to investigate the possible interaction between pituitary hormones and local factors (luteal peptides) on secretion of PGF2alpha and progesterone (P) by the early bovine CL, and to evaluate the effect of growth hormone (GH) as well as its interactions on production of PGF2alpha in the developing CL. A RT-PCR analysis revealed that mRNA for GH receptor in CL was fully expressed from early in the luteal phase throughout the estrous cycle, while luteinizing hormone (LH) receptor mRNA was expressed less by the early and regressing CL than those at mid or late luteal phases (P < 0.05). For the stimulation test, an in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of GH, insulin-like growth factor-I (IGF-I) and oxytocin (OT) increased (P < 0.05) PGF2alpha and P release. In contrast, LH had no effect (P > 0.05) on PGF2alpha secretion and little effect on P release. Unexpectedly, there was no distinct interaction between pituitary hormones and luteal peptides on secretion of PGF2alpha and P. These results indicate that GH is a more powerful stimulator of PGF2alpha and P production in the early bovine CL than LH and suggest that GH and luteal peptides, IGF-1 and OT, contribute to maintenance of elevated PGF2alpha production in the developing bovine CL.     

Role of intraluteal prostaglandin F(2alpha), progesterone and oxytocin in basal and pulsatile progesterone release from developing bovine corpus luteum

The present study examined the role of intra-luteal prostaglandin (PG) F(2alpha), progesterone (P4) and oxytocin (OT) on the corpus luteum function by using specific hormone antagonists. Luteal cells from the developing CL (days 5-7 of the estrous cycle) were exposed to P4 antagonist (onapristone, OP, 10(-4)M), OT antagonist (atosiban, AT; 10(-6)M) or indomethacin (INDO; 10(-4)M), for 12h and then stimulated with PGF(2alpha) (10(-8)M) for 4h. Pre-treatment of the cells with OP, AT or INDO resulted in an increase in P4 secretion in response to PGF(2alpha). To examine the temporal effects of P4, OT and PGs on P4 secretion, dispersed luteal cells were pre-exposed to OP, AT or INDO for 1, 2, 4, 6 or 12h. Prostaglandin F(2alpha) stimulated P4 secretion (P<0.05) after 2h of pre-exposition. In the microdyalisis study, the spontaneous release of P4 from developing CL tissue was of pulsatile nature with irregular peaks at 1-2h intervals. Treatment with OP increased the number of P4 peaks (P<0.05), whereas AT and INDO significantly reduced the number of P4 peaks detected (P<0.05). Interestingly, INDO completely blocked the pulsatile nature in the release of P4, but it secretion remained stable throughout the experimental period. These results demonstrate that luteal PGF(2alpha), OT, and P4 are components of an autocrine/paracrine intra-ovarian regulatory system responsible for the episodic (pulsatile) release of P4 from the bovine CL during the early luteal phase.     

Prostaglandin E2, prostaglandin F2 alpha and endothelin-1 production by cow oviductal epithelial cell monolayers: effect of progesterone, estradiol 17 beta, oxytocin and luteinizing hormone

The optimal oviductal environment, including contractile activity for gamete transport, fertilization and early embryonic development, is mediated by physiological and anatomical changes in the oviduct during the estrous cycle. Oviductal epithelial cell culture was utilized to investigate the effect of ovarian steroids (progesterone [P4] and estradiol 17 beta [E2]), oxytocin (OT) and luteinizing hormone (LH) on the local production of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha) and endothelin-1 (ET-1) in the cow oviduct. Epithelial cells isolated from oviducts collected during the follicular phase were cultured in M199 under standard culture conditions until monolayer formation. Then the cells were trypsinized and plated at a density of 3 x 10(4)/mL/well and cultured again until subconfluency, at which time the cells were incubated for 4 or 24 h with M199 only (control), high P4 (H-P4; 1 microgram/mL), low P4 (L-P4; 10 ng/mL), E2 (1 ng/mL), LH (10 ng/mL), OT (10(-9) M) ET-1 (10(-9) M), PGE2 (10(-8) M) PGF2 alpha (10(-9) M) or their combination (H-P4 + E2, L-P4 + E2, LH + E2, ET-1 + E2, L-P4 + E2 + LH and H-P4 + E2 + LH). The production of both PG and ET-1 was increased by E2 + low P4 and LH + E2 + low P4 (P < 0.05), while LH + E2 enhanced the production of PGF2 alpha and ET-1 (P < 0.05). Moreover, E2 + ET-1 stimulated PG production (P < 0.05). However, OT had no effect on the production of any of these substances. These results suggest that the preovulatory LH surge, together with locally re-circulated high levels of E2 from the Graafian follicle and basal P4 from regressing corpus luteum (CL), induces the maximum stimulatory effect on oviductal PGE2, PGF2 alpha and ET-1 production during the periovulatory period. Consequently, the elevated local ET-1 concentration during periovulatory period may induce the high contractile activity of the oviduct and, at the same time, the stimulation of PG production. Thus, ET-1 may act as a local amplifier for oviductal PG production stimulated by LH and ovarian steroids.     

Prostaglandin F2 alpha-induced release of oxytocin from bovine corpora lutea in vitro

Two experiments were conducted to study the in vitro effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2), and luteinizing hormone (LH) on oxytocin (OT) release from bovine luteal tissue. Luteal concentration of OT at different stages of the estrous cycle was also determined. In Experiment 1, sixteen beef heifers were assigned randomly in equal numbers (N = 4) to be killed on Days 4, 8, 12, and 16 of the estrous cycle (Day 0 = day of estrus). Corpora lutea were collected, an aliquot of each was removed for determination of initial OT concentration, and the remainder was sliced and incubated with vehicle (control) or with PGF2 alpha (10 ng/ml), PGE2 (10 ng/ml), or LH (5 ng/ml). Luteal tissue from heifers on Day 4 was sufficient only for determination of initial OT levels. Luteal OT concentrations (ng/g) increased from 414 +/- 84 on Day 4 to 2019 +/- 330 on Day 8 and then declined to 589 +/- 101 on Day 12 and 81 +/- 5 on Day 16. Prostaglandin F2 alpha induced a significant in vitro release of luteal OT (ng.g-1.2h-1) on Day 8 (2257 +/- 167 vs. control 1702 +/- 126) but not on Days 12 or 16 of the cycle. Prostaglandin E2 and LH did not affect OT release at any stage of the cycle studied. In Experiment 2, six heifers were used to investigate the in vitro dose-response relationship of 10, 20, and 40 ng PGF2 alpha/ml of medium on OT release from Day 8 luteal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxytocin-induced prostaglandin release from perifused bovine caruncular and intercaruncular endometrial tissue on days 20, 30 and at first estrus postpartum

Twenty-two multiparous Brahman x Hereford F1 cows were utilized to determine the effect of oxytocin (OT) on prostaglandin F2 alpha (PGF) release from caruncular and intercaruncular endometrial tissues and prostaglandin E2 (PGE) release from intercaruncular tissue. The previously gravid uterine horn was removed on d 20 postpartum (n = 7), on d 30 postpartum (n = 7) or the uterine horn ipsilateral to the dominant follicle was removed 12-18 h after onset of first behavioral estrus postpartum (ES; n = 8). Tissues (200 mg wet wt) were cultured in Nutrient Mixture F-10 medium in a perifusion system. The medium and tissues were aerated with 95% O2: 5% CO2 and temperatures were maintained at 39 degrees C. The flow rate was 100 microliters/min and fractions were collected at 20 min intervals for 400 min. After a 2 h settling phase, the tissues were challenged with 1, 2 or 4 micrograms [Asu1,6]-OT/ml of media for 1 h. Basal release of PGE and PGF on d 20 was greater than on d 30 and at ES (P less than .02) which were similar. All doses of OT increased PGE and PGF with both remaining elevated throughout the duration of the perifusion (P less than .008). However, there were no differences among doses. Release of PGE in response to OT on d 20 and 30, was higher than at ES (P less than .008). More PGF was released in response to OT from intercaruncular than caruncular tissue on d 20 (P less than .0001) and at ES (P less than .003). Release of PGF in response to OT on d 20 was higher (P less than .0001) than on d 30 and d 30 was higher than at ES (P less than .007). Basal and OT-induced release of PGE and PGF declined as day postpartum increased. We conclude that intercaruncular tissue released more PGF than caruncular tissue and both intercaruncular and caruncular tissue responded to OT with a sustained release of prostaglandins in a non-dose-dependent manner on d 20, 30 and at ES postpartum.     

Involvement of the cytoskeleton in oxytocin secretion by cultured bovine luteal cells

A number of substances have been implicated in the regulation of oxytocin (OT) secretion from bovine corpus luteum in vivo. However, isolated bovine luteal cells cultured in a monolayer lose the ability to secrete OT in response to stimulatory substances. The present study investigated how cell-to-cell contact and the cytoskeleton affect OT secretion by isolated bovine luteal cells. In experiment 1, bovine midluteal cells (Days 8-12 of the estrous cycle) were stimulated with prostaglandin F2alpha (PGF2alpha; 1 microM), noradrenaline (NA; 10 microM), or growth hormone (GH; 5 nM) in two culture systems: In one system, cell monolayers were incubated in 24-well culture plates, and in the other system, aggregates of cells were incubated in glass tubes in a shaking water bath. The cells cultured in a monolayer underwent considerable spreading and showed a variety of shapes, whereas the cells cultured in glass tubes remained fully rounded during the experimental period and soon formed aggregates of cells. Although PGF2alpha, NA, and GH did not stimulate OT secretion by the monolayer cells, all tested substances stimulated OT secretion by the aggregated cells (P < 0.01). In experiment 2, the monolayer cells were pre-exposed for 1 h to an antimicrofilament agent (cytochalasin B; 1 microM) or two antimicrotubule agents (colchicine or vinblastine; 1 microM) before stimulation with PGF2alpha, NA, or GH. Although PGF2alpha, NA, and GH did not stimulate OT secretion by the monolayer cells in the presence of colchicine or vinblastine, they all stimulated OT secretion in the presence of cytochalasin B (P < 0.001). The overall results show that OT secretion by bovine luteal cells depends on microfilament function and cell shape. Moreover, the aggregate culture system that allows three-dimensional, cell-to-cell contact seems to be a good model for studying OT secretion by isolated bovine luteal cells.     

Local distributions of oviductal estradiol, progesterone, prostaglandins, oxytocin and endothelin-1 in the cyclic cow

The cyclic patterns of hormones which regulate the activity of the oviduct in the cow have not been adequately reported. We studied progesterone (P4), estradiol 17 beta (E2), prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), oxytocin (OT) and endothelin-1 (ET-1) concentrations in the cow oviduct. Reproductive tracts from cyclic Holstein cows in the follicular phase (n = 5), post ovulation phase (n = 5) and luteal phase (n = 5) were collected at a slaughterhouse. Oviducts were separated from the uterus, the lumen vas washed with physiological saline, and the enveloping connective tissues were removed. The fimbria was then separated at first and then the rest was divided into 2 parts of equal length (proximal and distal). After extraction, levels of different hormones in the tissues were measured using double antibody enzyme immunoassays (EIAs). There were no differences in any hormone concentration between the 3 parts of the oviduct at any stage of the estrous cycle. The highest concentration of oviductal P4 was observed during the luteal phase and in the oviduct ipsilateral to the functioning CL. Oviductal OT was unchanged throughout the cycle. The highest E2 concentration was observed during the follicular phase in the oviduct ipsilateral to the dominant follicle. The oviduct ipsilateral to the dominant follicle during the follicular phase and ipsilateral to the ovulation site post ovulation showed higher levels of PGE2, PGF2 alpha and ET-1 than those on the contralateral side or during the luteal phase. The highest PGE2 was observed in the oviduct ipsilateral to the ovulation site during the post ovulation phase. The results suggest that the ovarian products (P4, OT and E2) and the local oviductal products (PGE2, PGF2 alpha, and ET-1) may synergistically control oviductal contraction for optimal embryo transport during the periovulatory period, and provide further evidence for the local delivery of ovarian steroids to the adjacent reproductive tract.     

Influence of nitric oxide and noradrenaline on prostaglandin F(2)(alpha)-induced oxytocin secretion and intracellular calcium mobilization in cultured bovine luteal cells

Although prostaglandin (PG) F(2alpha) released from the uterus has been shown to cause regression of the bovine corpus luteum (CL), the neuroendocrine, paracrine, and autocrine mechanisms regulating luteolysis and PGF(2alpha) action in the CL are not fully understood. A number of substances produced locally in the CL may be involved in maintaining the equilibrium between luteal development and its regression. The present study was carried out to determine whether noradrenaline (NA) and nitric oxide (NO) regulate the sensitivity of the bovine CL to PGF(2alpha) in vitro and modulate a positive feedback cascade between PGF(2alpha) and luteal oxytocin (OT) in cows. Bovine luteal cells (Days 8-12 of the estrous cycle) cultured in glass tubes were pre-exposed to NA (10(-5) M) or an NO donor (S-nitroso-N:-acetylpenicillamine [S-NAP]; 10(-4) M) before stimulation with PGF(2alpha) (10(-6) M). Noradrenaline significantly stimulated the release of progesterone (P(4)), OT, PGF(2alpha), and PGE(2) (P: < 0.01); however, S-NAP inhibited P(4) and OT secretion (P: < 0.05). Oxytocin secretion and the intracellular level of free Ca(2+) ([Ca(2+)](i)) were measured as indicators of CL sensitivity to PGF(2alpha). Prostaglandin F(2alpha) increased both the amount of OT secretion and [Ca(2+)](i) by approximately two times the amount before (both P: < 0.05). The S-NAP amplified the effect of PGF(2alpha) on [Ca(2+)](i) and OT secretion (both P: < 0.001), whereas NA diminished the stimulatory effects of PGF(2alpha) on [Ca(2+)](i) (P: < 0.05). Moreover, PGF(2alpha) did not exert any additionally effects on OT secretion in NA-pretreated cells. The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL function. While NA stimulates P(4) and OT secretion, NO may inhibit it in bovine CL. Both NA and NO are likely to stimulate the synthesis of luteal PGs and to modulate the action of PGF(2alpha). Noradrenaline may be the factor that is responsible for the limited action of PGF(2alpha) on CL and may be involved in the protection of the CL against premature luteolysis. In contrast, NO augments PGF(2alpha) action on CL and it may be involved in the course of luteolysis.     

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. G?6976, G?6983 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.     

The involvement of oxytocin in ovulation and in the outputs of cyclo-oxygenase and 5-lipoxygenase products from isolated rat ovaries

The effects on ovulation of a specific anti-oxytocin rabbit serum (anti-OT) (50.0 microliters) given by intrabursal injection into the right ovaries of etherized adult female rats at proestrus, were explored by counting the number of ovulated ova present within the right oviducts. Left ovaries were not treated and served as control ovaries. Control rats were treated with male normal rabbit serum (NRS) (50.0 microliters) given by intrabursal injections into the right ovaries of animals at proestrus. Ovulation was induced by injection of human chorionic gonadotrophin (hCG). Anti-OT administered into the right ovarian bursae of proestrous rat ovaries evoked a significant 51% inhibition of ovulation in comparison with that observed in control non-injected left ovaries (p less than 0.01). Also, when the ovulation of right ovaries injected with anti-OT was compared with that of left ovaries injected with NRS, the number of ovulated ova in the right side was significantly smaller (30%) than on the contralateral side (p less than 0.02). However, in rats pre-treated with hCG the intrabursal injection of oxytocin (OT) (50.0 mU/ml) into right and left ovaries failed to alter the number of ovulated ova compared with that of rats receiving intrabursal injections of saline. The basal control and the OT-evoked synthesis and release of endogenous prostaglandin E2 (PGE2) and PGF2 alpha were explored in ovaries isolated from prepuberal rats injected with pregnant mare's serum gonadotrophin (PMSG), two days prior to sacrifice. OT augmented the basal release of PGF2 alpha but did not influence that of PGE2. Moreover, the conversion of exogenous 14C-arachidonic acid (14C-AA) into different prostanoids and into 5-HETE, in the presence and in the absence of added OT (50.0 mU/ml), was studied in rat ovaries isolated in proestrus. The challenge with OT augmented the basal synthesis and release of PGF2 alpha and of 5-HETE from 14C-AA, but failed to influence the formation of products generated via the cyclo-oxygenase pathway, namely 6-keto-PGF1 alpha, PGE2 and thromboxane B2 (TXB2). Therefore, the present results suggest that ovarian OT may play a role in the ovulatory process, via generation of PGF2 alpha to enhance contractions of ovarian smooth muscle and of 5-HETE to promote follicular collagenolysis.