Real-time relationships in intraluteal release among Prostaglandin F2alpha, endothelin-1, and angiotensin II during spontaneous luteolysis in the cow

It is well known that prostaglandin F(2alpha) (PGF(2alpha)) is a physiological luteolysine, and that its pulsatile release from the endometrium is a luteolytic signal in many species. There is now clear evidence that the vasoactive peptides endothelin-1 (ET-1) and angiotensin II (Ang II) interact with PGF(2alpha) in the luteolytic cascade during PGF(2alpha)-induced luteolysis in the cow. Thus, we investigated the local secretion of PGF(2alpha), ET-1, and Ang II in the corpus luteum (CL) and their real-time relationships during spontaneous luteolysis in the cow. For this purpose, an in vivo microdialysis system (MDS) implanted in the CL was utilized to observe local secretion changes within the CL microenvironment. Each CL of cyclic Holstein cows (n = 6) was surgically implanted with MDS capillary membranes (18 lines/6 cows) on Day 15 (estrus = Day 0) of the estrous cycle. The concentrations of PGF(2alpha), ET-1, Ang II, and progesterone (P) in the MDS samples were determined by enzyme immunoassays. The intraluteal PGF(2alpha) secretion slightly increased from 12 h after the onset of luteolysis (0 h) and drastically increased (by about 300%) from 24 h. Intraluteal ET-1 secretion increased from 12 h. Intraluteal Ang II secretion was elevated from 0 h and was maintained at high levels (about 180%) toward estrus. In each MDS lines (in the same microenvironment) within the regressing CL, the local releasing profiles of PGF(2alpha), ET-1, and Ang II CL positively correlated with each other (P < 0.05) at high proportions in 18 MDS lines (PGF(2alpha) vs. ET-1, 44.4%; PGF(2alpha) vs. Ang II, 55.6%; ET-1 vs. Ang II, 38.9%). In contrast, there was no clear relationship among these substances released into different MDS lines implanted in the same CL (with different microenvironments). In conclusion, we propose that the increase of PGF(2alpha), ET-1, and Ang II within the CL during luteolysis is a common phenomenon for both PGF(2alpha)-induced and spontaneous luteolysis. Moreover, this study illustrated the in vivo relationships in intraluteal release among PGF(2alpha), ET-1, and Ang II during spontaneous luteolysis in the cow. The data suggest that these vasoactive substances may interact with each other in a local positive feedback manner to activate their secretion in the regressing CL, thus accelerating and completing luteolysis.     

Changes in prostaglandin secretion by the regressing bovine corpus luteum

Secretion of prostaglandins (PGs) by the regressing corpus luteum (CL) was investigated in the cow. Six cows were implanted with microcapillary dialysis membranes of a microdialysis system (MDS) into the CL during Days 8-9 (Day 0 = estrus), and a prostaglandin (PG) F2alpha analogue (Estrumate) was injected intramuscularly (i.m.) to induce luteolysis. Acute increases in intraluteal release of PGF2alpha and PGE2 were observed during the first 4 h, followed by decreases over the next 8 h. Intraluteal release of both PGs gradually increased again during the period 48-72 h. Concentrations of PGF2alpha in ovarian venous plasma (OVP) were 4-13 times higher than those of jugular venous plasma (JVP) (P < 0.001) during the period of the experiment, and increased from 24 h after treatment with Estrumate (P < 0.05). Cyclooxygenase (COX)-2 mRNA expression increased (P < 0.05) at 2 and 24 h after treatment with Estrumate. The results indicated that local release of PGF2alpha and PGE2, and COX-2 mRNA expression were increased by Estrumate in the regressing CL at the later stages of luteolysis. Thus, luteal secretion of PGs may be involved in the local mechanism for structural rather than functional luteolysis.     

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.     

Angiotensin II interacts with prostaglandin F2alpha and endothelin-1 as a local luteolytic factor in the bovine corpus luteum in vitro

Recent findings suggest that the ovarian renin-angiotensin system may regulate ovarian function through the paracrine/autocrine actions of angiotensin II (Ang II). In this study, we have examined and characterized the local effects of Ang II as a luteolytic factor and its interaction with prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1) in the bovine corpus luteum (CL) of the mid-luteal phase, by using an in vitro microdialysis system (MDS). Ang II was detected in the MDS perfusate (4 pg/ml), and infusion of PGF2alpha (10(-6) M) for 2 h increased the Ang II release by 50-100% during the following experimental period, in addition to its stimulation of ET-1 release. Two 2-h infusions of Ang II (10(-7)-10(-5) M) separated by a 2-h interval induced a dose- and time-dependent decrease of progesterone (P4) release by 41-66%. When the luteal explants were pre-perfused with PGF2alpha (10(-6) M) for 2 h, two consecutive perfusions of Ang II (10(-6) M) at a 2-h interval rapidly reduced the P4 release (by 50%). This reduction occurred 6 h earlier than those of infusions of PGF2alpha or Ang II alone. The simultaneous infusion of either 1) Ang II (10(-6) M) with PGF2alpha (10(-6) M), 2) ET-1 (10(-7) M) with PGF2alpha, or 3) Ang II + ET-1 with PGF2alpha (10(-6) M) for 2 h also induced a rapid and pronounced (60%) decrease in P4 release. Perfusion with the Ang II antagonist blocked the P4-suppressing activity of Ang II alone or PGF2alpha + Ang II infusion. Ang II stimulated the release of ET-1 and oxytocin during infusion but inhibited them after infusion. These results show that Ang II is released in the bovine midcycle CL in vitro, and this peptide, either alone or together with PGF2alpha, can suppress the release of P4. As PGF2alpha directly stimulated Ang II release, Ang II may influence the critical period for starting the cascade of functional luteolysis in vivo and might lead to structural luteolysis with ET-1 as a major vasoconstrictor. The overall results suggest that Ang II may have an important role at luteolysis in the 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.     

Intraluteal release of angiotensin II and progesterone in vivo during corpora lutea development in the cow: effect of vasoactive peptides.

The newly formed corpus luteum (CL) develops rapidly and has the features of active vascularization and mitosis of steroidogenic cells. Such local mechanisms must be strictly regulated by the complex relationship between angiogenic growth factors and vasoactive peptides such as angiotensin (Ang) II, atrial natriuretic peptide (ANP), and endothelin (ET)-1. Thus, the objective of the present study was to determine 1) the changes in vasoactive peptides and progesterone (P) concentrations within the developing CL, along with the changes in concentration in ovarian venous plasma (OVP) and jugular venous plasma (JVP) in the cow, 2) the effects of CL exposure to vasoactive peptides on Ang II and P secretion, and 3) the expression of mRNA for ANP type C receptor in the bovine CL and endothelial cells (ETC) from bovine developing CL. A microdialysis system (MDS) was surgically implanted into multiple CL of six cows on Day 3 after a GnRH injection that induced superovulation, and a catheter was simultaneously inserted into the ovarian vein. The Ang II concentration in OVP was higher than that in JVP throughout the experiment, while the intraluteal release of Ang II was stable. During the experimental period, the concentrations of other vasoactive peptides (ANP and ET-1) showed no clear changes in plasma and were below detectable levels in the MDS perfusate. Exposure of CL to Ang II using the MDS stimulated P release, while exposure to ANP enhanced Ang II release within the developing CL. However, ET-1 had no effect on either P or Ang II release. The expression of mRNA for ANP type C receptor was mainly observed in early CL and ETC. The results suggest that the ET-Ang-ANP system in the preovulatory follicle switches to an Ang-ANP system to enhance both the angiogenesis and steroidogenesis that are actively occurring in developing CL.     

Effect of local interaction of reactive oxygen species with prostaglandin F(2alpha) on the release of progesterone in ovine corpora lutea in vivo

Prostaglandin (PG) F(2alpha) is implicated in the process of luteal regression in many species, and has been shown to increase the generation of reactive oxygen species. In this study, the role of reactive oxygen species in the local regulatory mechanisms of functional luteolysis in the ewe was examined. In Experiment 1, we studied local effects of hydrogen peroxide (H(2)O(2)) and its interaction with PGF(2alpha) on P secretion in ovine corpus luteum (CL) in vivo. For this purpose, a microdialysis system (MDS) was used, where only the cells surrounding the capillary membrane in the microenvironment of the CL are exposed to these factors, and the P secretory ability of the CL is maintained as if intact. The study used a multiple CL model to implant the MDS, enabling us to examine in parallel several experimental infusions into the MDS implanted in different CLs (one MDS line per CL) developed after superovulation in one ewe. On Day 8 after GnRH treatment, the MDS were implanted into multiple CL in both ovaries of six ewes. A 4-h infusion with PGF(2alpha) (10(-6)M) at 8-12 h slightly increased P release during infusion, while a 4-h infusion with H(2)O(2) (10(-3)M) at 20-24 h decreased P release at 27-38 h. A pre-infusion with PGF(2alpha) for 4h at 8-12h, followed by infusion of H(2)O(2) at 20-24 h rapidly decreased the P release at 20-40 h (P<0.05); this decrease occurred 7h earlier than in the CL treated with H(2)O(2) alone. In Experiment 2, by utilizing the MDS we also applied free radical scavengers to examine their possible weakening effect on the inhibition of P secretion in the microenvironment within the regressing CL induced by PGF(2alpha) treatment. On Day 8 after estrus, the MDS were implanted into the CL (single CL model, two MDS lines per CL). Infusion of free radical scavengers, superoxide dismutase (SOD;50mg/ml)+catalase (CAT; 10mg/ml), at 0-28 h first increased P release until 12 h (P<0.05), and consequently delayed the decrease in P release until 30 h after administration of PGF(2alpha) i.m. (P<0.05). The present results support the concept that the leading pathway from PGF(2alpha) induces an increase of reactive oxygen species in luteolysis in the ewe.     

Regulation of intraluteal production of prostaglandins

There is clear evidence for intraluteal production of prostaglandins (PGs) in numerous species and under a variety of experimental conditions. In general, secretion of PGs appears to be elevated in the early corpus luteum (CL) and during the period of luteolysis. Regulation of intraluteal PG production is regulated by a variety of factors. An autoamplification pathway in which PGF-2alpha stimulates intraluteal production of PGF-2alpha has been identified in a number of species. The mechanisms underlying this autoamplification pathway appear to differ by species with expression of Cyclooxygenase-2 (Cox-2) and activity of phospholipase A2 acting as important physiological control points. In addition, a number of other responses that are induced by PGF-2alpha (decreased luteal progesterone, increased endothelin-1, increased cytokines) also have been found to increase intraluteal PGF-2alpha production. Thus, regulation of intraluteal PG production may serve to initiate or amplify physiological signals to the CL and may be important in specific aspects of luteal physiology particularly during luteal regression.     

Changes in steady-state concentrations of messenger ribonucleic acids in luteal tissue during prostaglandin F2alpha induced luteolysis in mares

Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2alpha on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares (n=8/group) were randomized into three groups: control (no treatment or biopsy), saline+biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2alpha+biopsy (5mg PGF2alpha at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline+biopsy group. At Hour 24 (12h after biopsy) there was a decrease in circulating progesterone in saline group to 56% of pre-biopsy values, indicating an effect of biopsy on luteal function. Mean plasma progesterone concentrations were lower (P<0.001) at Hour 12 in the PG group compared to the other two groups. The relative concentrations of mRNA for different genes in luteal tissue at Hour 12 was quantified by real time PCR. Compared to saline-treated mares, treatment with PGF2alpha increased mRNA for cyclooxygenase-2 (Cox-2, 310%, P<0.006), but decreased mRNA for LH receptor to 44% (P<0.05), steroidogenic acute regulatory protein to 22% (P<0.001), and aromatase to 43% (P<0.1) of controls. There was no difference in mRNA levels for PGF2alpha receptor between PG and saline-treated groups. Results indicated that luteal biopsy alters subsequent luteal function. However, the biopsy approach was effective for collecting CL tissue for demonstrating dynamic changes in steady-state levels of mRNAs during PGF2alpha-induced luteolysis. Increased Cox-2 mRNA concentrations suggested that exogenous PGF2alpha induced the synthesis of intraluteal PGF2alpha. Thus, the findings are consistent with the concept that an intraluteal autocrine loop augments the luteolytic effect of uterine PGF2alpha in mares.     

An intra-corpus luteum site for the luteolytic action of prostaglandin F2 alpha in the rhesus monkey

It has not been possible to demonstrate prostaglandin F2 alpha (PGF2 alpha) participation in primate luteolysis under conditions of systemic administration or of acute intraluteal injection. These study designs were hampered by the short biological half-life in the first instance and brevity of administration in the latter. In this study, luteolysis has resulted from chronic, intraluteal delivery of PGF2 alpha. Using the Alzet osmotic pump-cannula system, normally cycling rhesus monkeys were continuously infused, until menses occurred, with PGF2 alpha (10 ng/1/hr) directly into the corpus luteum (CL, n = 6), into the stroma of the ovary not bearing the corpus luteum (NCL, n = 3), or subcutaneously (SC, n = 5). An additional 5 monkeys received vehicle (V) into the corpus luteum. All experiments commenced 5-7 days after the preovulatory estradiol surge. Luteal function was assessed by the daily measurements of plasma progesterone, estradiol, and LH. Intraluteal PGF2 alpha caused premature functional luteolysis in all monkeys, as reflected by a highly significant decline in circulating progesterone and estradiol and the early onset of menstruation, when compared to the other groups. V, NCL, and SC infusions had no effect on either circulating steroid levels or luteal phase lengths. None of the experimental groups showed any change in plasma LH concentrations. These are the first data to indicate that PGF2 alpha can induce functional luteolysis in the primate, and the site of action appears to be the corpus luteum.     

Cooperative expression of monocyte chemoattractant protein 1 within the bovine corpus luteum: evidence of immune cell-endothelial cell interactions in a coculture system

Endothelial cells (EC) of the bovine corpus luteum (CL) are a known source of proinflammatory mediators, including monocyte chemoattractant protein 1 (CCL2) and endothelin 1 (EDN1). Here, a coculture system was devised to determine if immune cells and PGF 2alpha together affect CCL2 and EDN1 secretion by EC. Luteal EC were cultured either alone or together with peripheral blood mononuclear cells (PBMC), and treated without or with PGF 2alpha for 48 h (n = 6 experiments). Coculture of EC with PBMC increased CCL2 secretion an average of 5-fold higher compared with either cell type alone (P < 0.05). Basal secretion of EDN1 by EC was substantial (approximately 2 ng/ml), but was not affected by coculture with PBMC (P > 0.05). EC cocultured with concanavalin A-activated PBMC (ActPBMC) increased CCL2 secretion an average of 12-fold higher compared with controls (P < 0.05), but again, EDN1 secretion was unchanged (P > 0.05). Interestingly, PGF 2alpha did not alter either CCL2 or EDN1 secretion, regardless of culture conditions (P > 0.05). In a second series of experiments (n = 3 experiments), mixed luteal cells (MLC) were cultured alone or with PBMC as described above. Secretion of CCL2 and EDN1 was not affected by coculture or by PGF 2alpha (P > 0.05), but MLC produced less progesterone in the presence of ActPBMC (P < 0.05). Collectively, these results suggest that immune cells and EC can interact cooperatively to increase CCL2 secretion in the CL, but this interaction does not affect EDN1 secretion nor is it influenced by PGF 2alpha. Additionally, activated immune cells appear to produce a factor that impairs progesterone production by luteal steroidogenic cells.     

Sensitivity of bovine corpora lutea to prostaglandin F2alpha is dependent on progesterone, oxytocin, and prostaglandins.

Prostaglandin (PG) F2alpha that is released from the uterus is essential for spontaneous luteolysis in cattle. Although PGF2alpha and its analogues are extensively used to synchronize the estrous cycle by inducing luteolysis, corpora lutea (CL) at the early stage of the estrous cycle are resistant to the luteolytic effect of PGF2alpha. We examined the sensitivity of bovine CL to PGF2alpha treatment in vitro and determined whether the changes in the response of CL to PGF2alpha are dependent on progesterone (P4), oxytocin (OT), and PGs produced locally. Bovine luteal cells from early (Days 4-5 of the estrous cycle) and mid-cycle CL (Days 8-12 of the estrous cycle) were preexposed for 12 h to a P4 antagonist (onapristone: OP; 10(-4) M), an OT antagonist (atosiban: AT; 10(-6) M), or indomethacin (INDO; 10(-4) M) before stimulation with PGF2alpha. Although OP reduced P4 secretion (p < 0.001) only in early CL, it reduced OT secretion in the cells of both phases examined (p < 0.001). OP also reduced PGF2alpha and PGE2 secretion (p < 0.01) from early CL. However, it stimulated PGF2alpha secretion in mid-cycle luteal cells (p < 0.001). AT reduced P4 secretion in early and mid-cycle CL (p < 0.05). Moreover, PGF2alpha secretion was inhibited (p < 0.05) by AT in early CL. The OT secretion and the intracellular level of free Ca2+ ([Ca2+]i) were measured as indicators of CL sensitivity to PGF2alpha. PGF2alpha had no influence on OT secretion, although [Ca2+]i increased (p < 0.05) in the early CL. However, the effect of PGF2alpha was augmented (p < 0.01) in cells after pretreatment with OP, AT, and INDO in comparison with the controls. In mid-cycle luteal cells, PGF2alpha induced 2-fold increases in OT secretion and [Ca2+]i. However, in contrast to results in early CL, these increases were magnified only by preexposure of the cells to AT (p < 0.05). These results indicate that luteal P4, OT, and PGs are components of an autocrine/paracrine positive feedback cascade in bovine early to mid-cycle CL and may be responsible for the resistance of the early bovine CL to the exogenous PGF2alpha action.     

Effects of endothelin receptor type-A and type-B antagonists on prostaglandin F2alpha-induced luteolysis of the sheep corpus luteum

Three experiments were designed to examine the mechanisms that govern prostaglandin (PGF2alpha)-induced regression of the sheep corpus luteum. Evidence is presented supporting the involvement of endothelin 1 (EDN1) in PGF2alpha-induced luteolysis. Experiment 1 measured effects of PGF2alpha when actions of EDN1 were blocked by sustained administration of a type-A endothelin (EDNRA) or type-B endothelin (EDNRB) antagonist in vivo. Experiment 2 examined antisteroidogenic actions of PGF2alpha and EDN1 in the presence of an EDNRA or EDNRB antagonist in Day-8 luteal minces. In experiment 3, luteal cellular expression of EDNRA and EDNRB was determined immunohistochemically. Relative gene expression of EDNRA and EDNRB receptors was examined by real-time RT-PCR in Day-8 sheep corpora lutea. EDNRA, but not EDNRB, participated in antisteroidogenic actions of EDN1. During the first 12 h after PGF2alpha-induced luteolysis, EDNRA antagonist did not prevent a decline in serum progesterone concentrations. Early actions of PGF2alpha are either direct or mediated by something other than EDN1. However, beyond 12 h after PGF2alpha, serum progesterone concentrations increased in EDNRA antagonist-treated animals until they were the same as saline-treated controls, whereas an EDNRB antagonist had no effect in vivo or in vitro. The EDNRA antagonist negated the antisteroidogenic actions of EDN1 but only partially abolished the actions of PGF2alpha in vitro. In contrast, the EDNRB antagonist was ineffective in abolishing antisteroidogenic actions of EDN1 and PGF2alpha. Whereas real-time RT-PCR demonstrated high expression of EDNRA and low expression of EDNRB, immunohistochemically, only EDNRA was located in small steroidogenic, endothelial, and smooth muscle cells. In summary, studies in ovine corpora lutea provided strong evidence that: 1) EDNRA, but not EDNRB, mediates antisteroidogenic actions of EDN1, 2) actions of PGF2alpha are both independent of and dependent upon mediation by EDN1, and 3) small steroidogenic cells are targets for antisteroidogenic effects of EDN1. Furthermore, the results from experiment 1 suggest that the intermediary role of EDN1 may be more important in later stages of luteal regression.     

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.     

Involvement of endothelin-1 and its receptors in PGF2alpha-induced luteolysis in the rat

The possible mediatory role of endothelin-1 (ET-1) in prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis in the rat was examined. The effect of PGF(2alpha) was tested on day 9 of pregnancy either in vivo, by injecting cloprostenol, an analog of PGF(2alpha) or in vitro, in isolated intact corpora lutea incubated with PGF(2alpha). Luteolysis was confirmed by progesterone determination in the peripheral blood serum or in the culture medium, respectively. Administration of cloprostenol (.0025 mg/rat) induced within 1 hr, a significant fall (from 56.8 to 27.6 ng/ml, P < 0.0001) in serum progesterone concentrations that was associated with an increased expression of the mRNA to ET-1 and its protein product in rat luteal tissue. Elevated level of ET-1 were also determined at the spontaneous regression of the CL, upon parturition. Expression of the ET receptors, ETA and ETB was not affected by cloprostenol. On the other hand, this PGF(2alpha) analog induced expression of luteal VEGF mRNA. In vitro experiments demonstrate that the LH (100 ng/ml)-induced increase in luteal progesterone secretion was reduced by PGF(2alpha) (1 microg/ml). The inhibitory effect of PGF(2alpha) was reversed by BQ123 (10(- 7) M), that is a selective ETA receptor antagonist. We conclude that the PGF(2alpha)-induced elevation in luteal expression of ET-1 combined with the reversal of its luteolytic effect by an ETA receptor antagonist suggest that ET-1 may take part in the PGF(2alpha)-induced luteolysis in the rat.     

Local changes in blood flow within the early and midcycle corpus luteum after prostaglandin F(2 alpha) injection in the cow

One of the postulated main luteolytic actions of prostaglandin (PG) F(2 alpha) is to decrease ovarian blood flow. However, before Day 5 of the normal cycle, the corpus luteum (CL) is refractory to the luteolytic action of PGF(2 alpha). Therefore, we aimed to determine in detail the real-time changes in intraluteal blood flow after PGF(2 alpha) injection at the early and middle stages of the estrous cycle in the cow. Normally cycling cows at Day 4 (early CL, n = 5) or Days 10--12 (mid CL, n = 5) of the estrous cycle (estrus = Day 0) were examined by transrectal color and pulsed Doppler ultrasonography to determine the blood flow area, the time-averaged maximum velocity (TAMXV), and the volume of the CL after an i.m. injection of a PGF(2 alpha) analogue. Ultrasonographic examinations were carried out just before PG injection (0 h) and then at 0.5, 1, 2, 4, 8, 12, 24, and 48 h after the injection. Blood samples were collected at each of these times for progesterone (P) determination. The ratio of the colored area to a sectional plane at the maximum diameter of the CL was used as a quantitative index of the changes in blood flow within the luteal tissue. Blood flow within the midcycle CL initially increased (P < 0.05) at 0.5-2 h, decreased at 4 h to the same levels observed at 0 h, and then further decreased to a lower level from 8 h (P < 0.05) to 48 h (P < 0.001). Plasma P concentrations decreased (P < 0.05) from 4.7 +/- 0.5 ng/ml (0 h) to 0.6 +/- 0.2 ng/ml (24 h). The TAMXV and CL volume decreased at 8 h (P < 0.05) and further decreased (P < 0.001) from 12 to 24 h after PG injection, indicating structural luteolysis. These changes were not detected in the early CL, in which luteolysis did not occur. In the early CL, the blood flow gradually increased in parallel with the CL volume, plasma P concentration, and TAMXV from Day 4 to Day 6. The present results indicate that PGF(2 alpha) induces an acute blood flow increase followed by a decrease in the midcycle CL but not in the early CL. This transitory increase may trigger the luteolytic cascade. The lack of intraluteal vascular response to PG injection in the early CL appears to be directly correlated with the ability to be resistant to PG.     

Angiotensin II and atrial natriuretic peptide in the cow oviductal contraction in vitro: direct effect and local secretion of prostaglandins, endothelin-1, and angiotensin II

Angiotensin II (Ang II) and atrial natriuretic peptide (ANP) may be involved in local regulation of the oviductal contraction during the estrous cycle. Thus, the in vitro effects of Ang II and ANP on the secretion and contraction of bovine oviduct during the follicular, postovulatory, and luteal phases were investigated. An in vitro microdialysis system (MDS) was utilized to determine the intraluminal release of prostaglandins (PGs), Ang II, and endothelin-1 (ET-1) from the bovine oviducts as well as to observe the effect of Ang II and ANP on the local secretion of these substances. The basal release of PGs, ET-1, and Ang II was higher (P < 0.05) during the follicular and postovulatory phases than during the luteal phase. Stimulation by infusion of Ang II (10(-6) M) or ANP (10(-7) M) into the MDS was carried out for 4 h between 4 and 8 h of incubation. In the oviducts from the follicular and postovulatory phases, the infusion of ANP increased the release of Ang II, but not of ET-1. Infusion of Ang II stimulated the release of ET-1. Both Ang II and ANP increased PGE(2) and PGF(2alpha) release. In the contraction study, direct administration of Ang II (10(-7) M) or ANP (10(-8) M) into the medium during the follicular and postovulatory phases increased the amplitude of oviductal contraction. In contrast, these substances did not show any effect in the contraction and secretion of oviducts from cows during the midluteal phase. These results indicate that during the periovulatory period, Ang II and ANP stimulate the contractile amplitude of the oviduct in vitro. In addition to their direct action on oviductal contraction, Ang II may activate oviductal secretion of ET-1 and PGs. Likewise, ANP stimulates oviductal secretion of PGs and Ang II. Hence, the overall results suggest the existence of a functional endothelin-angiotensin-ANP system in the bovine oviduct during the periovulatory period, which may regulate the oviductal contraction to ensure maximum efficiency of gamete/embryo transport through the oviduct.