Dariusz J. Skarzynski

Administration of a Nitric Oxide Synthase Inhibitor Counteracts Prostaglandin F2-Induced Luteolysis in Cattle

Abstract The objective of this study was to determine whether nitric oxide (NO) is produced locally in the bovine corpus luteum (CL) and whether NO mediates prostaglandin F2α (PGF2α)-induced regression of the bovine CL in vivo. The local production of NO was determined in early I, early II, mid, late, and regressed stages of CL by determining NADPH-d activity and the presence of inducible and endothelial NO synthase immunolabeling. To determine whether inhibition of NO production counteracts the PGF2α-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (Nω-nitro-l-arginine methyl ester dihydrochloride [L-NAME]; 400 mg/h; n = 9) was infused for 2 h on Day 15 of the estrous cycle into the aorta abdominalis of Holstein/Polish Black and White heifers. After 30 min of infusion, saline or cloprostenol, an analogue of PGF2α (aPGF2α; 100 μg) was injected into the aorta abdominalis of animals infused with saline or L-NAME. NADPH-diaphorase activity was present in bovine CL, with the highest activity at mid and late luteal stages (P < 0.05). Inducible and endothelial NO synthases were observed with the strongest immunolabeling in the late CL (P < 0.05). Injection of aPGF2α increased nitrite/nitrate concentrations (P < 0.01) and inhibited P4 secretion (P < 0.05) in heifers that were infused with saline. Infusion of L-NAME stimulated P4 secretion (P < 0.05) and concomitantly inhibited plasma concentrations of nitrite/nitrate (P < 0.05). Concentrations of P4 in heifers infused with L-NAME and injected with aPGF2α were higher (P < 0.05) than in animals injected only with aPGF2α. The PGF2α analogue shortened the cycle length compared with that of saline (17.5 ± 0.22 days vs. 21.5 ± 0.65 days P < 0.05). L-NAME blocked the luteolytic action of the aPGF2α (22.6 ± 1.07 days vs. 17.5 ± 0.22 days, P < 0.05). These results suggest that NO is produced in the bovine CL. NO inhibits luteal steroidogenesis and it may be one of the components of an autocrine/paracrine luteolytic cascade induced by PGF2α.

Progesterone Is a Suppressor of Apoptosis in Bovine Luteal Cells

Abstract Progesterone is suggested to be a suppressor of apoptosis in bovine luteal cells. Fas antigen (Fas) is a cell surface receptor that triggers apoptosis in sensitive cells. Furthermore, apoptosis is known to be controlled by the bcl-2 gene/protein family and caspases. This study was undertaken to determine whether intraluteal progesterone (P4) is involved in Fas L–mediated luteal cell death in the bovine corpus luteum (CL) in vitro. Moreover, we studied whether an antagonist of P4 influences gene expression of the bcl-2 family and caspase-3 and the activity of caspase-3 in the bovine CL. Luteal cells obtained from the cows in the midluteal phase of the estrous cycle (Days 8–12 of the cycle) were exposed to a specific P4 antagonist (onapristone [OP], 10−4 M) with or without 100 ng/ml Fas L. Although Fas L alone did not show a cytotoxic effect, treatment of the cells with OP alone or in combination with Fas L resulted in killing of 30% and 45% of the cells, respectively (P < 0.05). DNA fragmentation was observed in the cells treated with Fas L in the presence of OP. The inhibition of P4 action by OP increased the expression of Fas mRNA (P < 0.01); however, it did not affect bax or bcl-2 mRNA expression (P > 0.05). Moreover, OP stimulated expression of caspase-3 mRNA (P < 0.01). The overall results indirectly show that intraluteal P4 suppresses apoptosis in bovine luteal cells through the inhibition of Fas and caspase-3 mRNA expression and inhibition of caspase-3 activation.

Different actions of noradrenaline and nitric oxide on the output of prostaglandins and progesterone in cultured bovine luteal cells.

The effects of noradrenaline (NA) and nitric oxide (NO) on prostaglandins (PGs) and progesterone (P4) secretion during the development of the bovine corpus luteum (CL) were investigated. Bovine luteal cells of early and mid-cycle CL were cultured for 20 to 24 h in medium containing 10% calf serum, washed, and treated with NA or nitrergic agents for an additional 16 h in a serum-free medium. NA (10(-5) M) stimulated P4 from early and mid-cycle CL by 238% and 154% (P < 0.01), respectively. Moreover, although NA induced a twofold increase in PGE2 secretion (P < 0.01) in both examined periods, the effect of NA on PGF2alpha secretion was approximately 1.5 times higher (P < 0.05) in early than in mid-cycle CL. Two NO synthase inhibitors, L-NAME and L-NOARG (both 10(-4) M), stimulated P4 secretion only in mid-luteal cells (P < 0.01), although they did not affect the cells from early CL. Although a NO donor, S-NAP (10(-4) M) inhibited P4 secretion from mid-cycle luteal cells (P < 0.05), it strongly stimulated PGE2 in both examined phases (P < 0.001). On the other hand, the output of PGF2alpha was stimulated by S-NAP only in the cells of the mid-cycle CL (P < 0.01). The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL functions. Whereas NA may play a supporting role in luteal development, NO may participate in the functional regression of the bovine CL by inhibiting steroidogenesis.

Regulation of Luteal Function and Corpus Luteum Regression in Cows: Hormonal Control, Immune Mechanisms and Intercellular Communication

The main function of the corpus luteum (CL) is production of progesterone (P4). Adequate luteal function to secrete P4 is crucial for determining the physiological duration of the oestrous cycle and for achieving a successful pregnancy. The bovine CL grows very fast and regresses within a few days at luteolysis. Mechanisms controlling development and secretory function of the bovine CL may involve many factors that are produced both within and outside the CL. Some of these regulators seem to be prostaglandins (PGs), oxytocin, growth and adrenergic factors. Moreover, there is evidence that P4 acts within the CL as an autocrine or paracrine regulator. Each of these factors may act on the CL independently or may modify the actions of others. Although uterine PGF(2 alpha) is known to be a principal luteolytic factor, its direct action on the CL is mediated by local factors: cytokines, endothelin-1, nitric oxide. The changes in ovarian blood flow have also been suggested to have some role in regulation of CL development, maintenance and regression.

Roles of Tumor Necrosis Factor-α of the Estrous Cycle in Cattle: An In Vivo Study

Abstract We have suggested in a previous in vitro study that tumor necrosis factor-α (TNFα) plays a role in the initiation of luteolysis in cattle. The aim of the present study was to examine the influence of different doses of TNFα on the estrous cycle in cattle by observing the standing behavior and measuring peripheral concentrations of progesterone (P4) during the estrous cycle. Moreover, we evaluated the secretion of P4, oxytocin (OT), nitric oxide (NO), and luteolytic (prostaglandin F2α [PGF2α] and leukotriene C4 [LTC4]) and luteotropic (PGE2) metabolites of arachidonic acid in peripheral blood plasma as parameters of TNFα actions. Mature Holstein/Polish black and white heifers (n = 36) were treated on Day 14 of the estrous cycle (Day 0 = estrus) by infusion into the aorta abdominalis of saline (n = 8), an analogue of PGF2α (cloprostenol, 100 μg; n = 3) or saline with TNFα at doses of 0.1 (n = 3), 1 (n = 8), 10 (n = 8), 25 (n = 3), or 50 μg (n = 3) per animal. Peripheral blood samples were collected frequently before, during, and up to 4 h after TNFα treatment. After Day 15 of the estrous cycle, blood was collected once daily until Day 22 following the first estrus. Lower doses of TNFα (0.1 and 1 μg) decreased the P4 level during the estrous cycle and consequently resulted in shortening of the estrous cycle (18.8 ± 0.9 and 18.0 ± 0.7 days, respectively) compared with the control (22.3 ± 0.3 days, P < 0.05). One microgram of TNFα increased the PGF2α (P < 0.001) and NO (P < 0.001) concentrations and decreased OT secretion (P < 0.01). Higher doses of TNFα (10, 25, 50 μg) stimulated synthesis of P4 (P < 0.001) and PGE2 (P < 0.001), inhibited LTC4 secreton (P < 0.05), and consequently resulted in prolongation of the estrous cycle (throughout 30 days, P < 0.05). Altogether, the results suggest that low concentrations of TNFα cause luteolysis, whereas high concentrations of TNFα activate corpus luteum function and prolong the estrous cycle in cattle.

Influence of Nitric Oxide and Noradrenaline on Prostaglandin F2α-Induced Oxytocin Secretion and Intracellular Calcium Mobilization in Cultured Bovine Luteal Cells

Abstract Although prostaglandin (PG) F2α 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 PGF2α 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 PGF2α in vitro and modulate a positive feedback cascade between PGF2α 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 PGF2α (10−6 M). Noradrenaline significantly stimulated the release of progesterone (P4), OT, PGF2α, and PGE2 (P < 0.01); however, S-NAP inhibited P4 and OT secretion (P < 0.05). Oxytocin secretion and the intracellular level of free Ca2+ ([Ca2+]i) were measured as indicators of CL sensitivity to PGF2α. Prostaglandin F2α increased both the amount of OT secretion and [Ca2+]i by approximately two times the amount before (both P < 0.05). The S-NAP amplified the effect of PGF2α on [Ca2+]i and OT secretion (both P < 0.001), whereas NA diminished the stimulatory effects of PGF2α on [Ca2+]i (P < 0.05). Moreover, PGF2α 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 P4 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 PGF2α. Noradrenaline may be the factor that is responsible for the limited action of PGF2α on CL and may be involved in the protection of the CL against premature luteolysis. In contrast, NO augments PGF2α action on CL and it may be involved in the course of luteolysis.

Soybean-derived phytoestrogens regulate prostaglandin secretion in endometrium during cattle estrous cycle and early pregnancy.

Phytoestrogens acting as endocrine disruptors may induce various pathologies in the female reproductive tract. The purpose of this study was to determine whether phytoestrogens present in the soybean and/or their metabolites are detectable in the plasma of cows fed a diet rich in soy and whether these phytoestrogens influence reproductive efficiency and prostaglandin (PG) synthesis during the estrous cycle and early pregnancy in the bovine endometrium. In in vivo Experiment 1, we found significant levels of daidzein and genistein in the fodder and their metabolites (equol and p-ethyl-phenol) in bovine serum and urine. The mean number of artificial inseminations (Als) and pregnancy rates in two kinds of herds, control and experimental (cows fed with soybean 2.5 kg/day), were almost double in the soy-diet herd in comparison with the control animals. In in vivo Experiment 2, three out of five heifers fed soybean (2.5 kg/day) became pregnant whereas four out of five heifers in the control group became pregnant. The concentrations of a metabolite of PGF2α (PGFM) were significantly higher in the blood plasma of heifers fed a diet rich in soybean than those in the control heifers throughout the first 21 days after ovulation and AI. The higher levels of PGFM were positively correlated with equol and p-ethyl phenol concentrations in the blood. In in vitro experiments, the influence of isoflavones on PG secretion in different stages of the estrous cycle was studied. Although all phytoestrogens augmented the output of both PGs throughout the estrous cycle, equol and p-ethyl-phenol preferentially stimulated PGF2α output. The results obtained lead to the conclusion that soy-derived phytoestrogens and their metabolites disrupt reproductive efficiency and uterus function by modulating the ratio of PGF2α to PGE2, which leads to high, nonphysiological production of luteolytic PGF2α in cattle during the estrous cycle and early pregnancy.

The use of an oxytocin antagonist to study the function of ovarian oxytocin during luteolysis in cattle

Abstract The importance of ovarian oxytocin (OT) in cattle during luteolysis and the mid-luteal phase using a highly specific OT antagonist (CAP-527) was studied. To establish the effective dose of CAP, heifers (n = 4) were infused with saline for 30 min, followed by 50 IU OT into the abdominal aorta on Days 17 and 18 of the estrous cycle. After 5.5 hours later, either 4, 6, 8 or 10 mg of CAP was infused for 30 min, followed by 50 IU OT. Plasma concentrations of 15-keto-13, 14-dihydro-prostagiandin F2α (PGFM) increased after 4 and 6 mg CAP. Therefore, in Experiment 2, 8 mg of CAP was infused and 50 IU OT was given after 3, 4, 6 and 9 h to define how long CAP saturates OT receptors. Concentrations of PGFM increased after 6 and 9 h of OT treatment only. We concluded that 8 mg CAP effectively blocked uterine OT receptors for 4 h in our model; hence in further experiments this dosage of CAP was used. In Experiment 3, CAP was given to 4 heifers every 4 h on Days 15 to 22 of the cycle, and 4 additional heifers received saline and served as the control. The CAP treatment changed neither the duration of the cycle, progesterone, PGFM nor OT plasma concentrations compared with that of the controls. Experiment 4 was designed to study the involvement of OT in noradrenaline (NA)-stimulated progesterone secretion. It was found earlier that ovarian OT stimulates progesterone secretion, and that NA was able to evoke concomitant release of both progesterone and OT. Therefore, in Experiment 4, NA was infused on Days 11 and 12 of the cycle in heifers (n = 4) but it was preceded with 8 mg CAP or with a saline (control) infusion. Concentrations of plasma progesterone concentrations increased after NA treatment in both the experimental and control groups. Thus, we conclude that if ovarian OT and uterine/ovarian OT receptors are involved in luteolysis and steroidogenesis in cattle, they play a more facilitating than mandatory role.

Involvement of ovarian steroids in basal and oxytocin-stimulated prostaglandin (PG) F2α 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.

Cytokines and Angiogenesis in the Corpus Luteum

In adults, physiological angiogenesis is a rare event, with few exceptions as the vasculogenesis needed for tissue growth and function in female reproductive organs. Particularly in the corpus luteum (CL), regulation of angiogenic process seems to be tightly controlled by opposite actions resultant from the balance between pro- and antiangiogenic factors. It is the extremely rapid sequence of events that determines the dramatic changes on vascular and nonvascular structures, qualifying the CL as a great model for angiogenesis studies. Using the mare CL as a model, reports on locally produced cytokines, such as tumor necrosis factor α (TNF), interferon gamma (IFNG), or Fas ligand (FASL), pointed out their role on angiogenic activity modulation throughout the luteal phase. Thus, the main purpose of this review is to highlight the interaction between immune, endothelial, and luteal steroidogenic cells, regarding vascular dynamics/changes during establishment and regression of the equine CL.