Mamadou M. Bah

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.

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.

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 influence of tumor necrosis factor α (TNF) on the secretory function of bovine corpus luteum: TNF and its receptors expression during the estrous cycle

Tumor necrosis factor alpha (TNF) inversely regulates the function of bovine corpus luteum (CL). Whereas the low doses of TNF induce luteolysis, the high doses prolong CL lifespan and prevent luteolysis in vivo. We suggest that the varying effects of TNF may be caused by its action exerted on CL via multiple signaling pathways involving two distinct receptors: TNFR-I (responsible for induction of the cell death) and TNFR-II (implicated in cell proliferation). In the study, we determined CL expressions of TNF, TNFR-I and TNFR-II mRNAs during the bovine estrous cycle using semi-quantitative RT-PCR. Specific transcripts for TNF, TNFR-I and TNFR-II were found in the CL with the highest (p<0.05) expression in the regressed CL. We also examined the TNF influence on the bovine CL function in vivo. On Day 15 of the estrous cycle, cows were infused (via aorta abdominalis) with saline, TNF (1 or 10 microg) or analogue of prostaglandin (PG)F(2alpha) (aPGF(2alpha) , 500 microg; a positive control). Four hours after infusions, CLs were collected by colpotomy and luteal contents of progesterone (P(4)), stable metabolites of nitric oxide (NO; nitrite/nitrate), leukotriene (LT)C(4), luteolytic PGF(2alpha),and luteotropic PGE(2) were determined. Moreover, caspase-3 activity was measured in the CLs as an indicator of apoptosis induction. The luteal content of P(4) decreased (p<0.05) after infusion of 1 microg of TNF. TNF inversely affected PGs content in CL: the low dose increased (p<0.01) the PGF(2alpha) level and the high dose increased (p<0.05) PGE(2) level. Contents of LTC(4) and nitrite/nitrate increased (p<0.01) after the low dose of TNF. Moreover, 1 microg of TNF induced apoptosis and increased (p<0.05) caspase-3 activity in the CLs collected during the late luteal phase. In conclusion, the high expressions of TNF and TNF receptors mRNAs were observed during or just after the luteolysis. A low concentration of TNF stimulated in vivo luteolytic factors such as PGF(2alpha), LTC(4) and NO as well as induced apoptosis; whereas the high concentration of TNF stimulated a survival pathway in the bovine CL increasing luteal content of P(4) and PGE(2).

Lysophosphatic acid modulates prostaglandin secretion in the bovine uterus.

Lysophosphatidic acid (LPA) modulates prostaglandin (PG) synthesis via LPA receptor 3 (LPAR3) in the murine endometrium. The lack of functional LPAR3 in mice may lead to embryo mortality. In the present study, we examined the role of LPA in the bovine uterus. We confirmed that LPA is locally produced and released from the bovine endometrium. Moreover, there are enzymes involved in LPA synthesis (phospholipase (PL) D(2) and PLA2G1B) in the bovine endometrium during estrous cycle and early pregnancy. Expression of the receptor for LPA (LPAR1) was positively correlated with the expression of PGE(2) synthase (PGES) and negatively correlated with the expression of PGF(2alpha) synthase (aldose reductase with 20 alpha-hydroxysteroid dehydrogenase activity - PGFS) during early pregnancy. In vivo LPA induced P4 and PGE(2) secretion was inhibited by LPAR1 antagonist (Ki16425). The overall results indicate that LPA is locally produced and released from the bovine endometrium. Moreover, LPAR1 gene expression in the endometrium during the estrous cycle and early pregnancy indicates that LPA may play autocrine and/or paracrine roles in the bovine uterus. LPAR1 gene expression is positively correlated with the expression of the enzyme responsible for luteotropic PGE(2) production (PGES) in endometrium. In cow, LPA stimulates P4 and PGE(2) secretion. Thus, LPA in the bovine reproductive tract may indirectly (via endometrium) or directly support corpus luteum action via the increase of P4 synthesis and the increase of PGE(2)/PGF(2)(alpha) ratio. It suggests that LPA may serve as an important factor in the maintenance of early pregnancy in cow.

Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms.

Prostaglandins (PGs) are known to modulate the proper cycllcity of bovine reproductive organs. The main luteolytlc agent in ruminants Is PGF2α, whereas PGE2 has luteotropic actions. Estradiol 17ß (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2α and PGE2synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2α synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2α than stromal cells, increasing the PGF2α, to PGE2 ratio. The epithelial and stromal cells were prelncubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a transiation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2- Although the action of E2 on PGF2α synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2action, phytoestrogens did not cause Intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2α and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2α synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2α to PGE2 ratio.

Infusion of Exogenous Tumor Necrosis Factor Dose Dependently Alters the Length of the Luteal Phase in Cattle: Differential Responses to Treatment with Indomethacin and L-NAME, a Nitric Oxide Synthase Inhibitor

Abstract We examined whether prostaglandins (PGs) and nitric oxide (NO) mediate tumor necrosis factor (TNF) actions in the estrus cycle. On Day 14 of the cycle, the following solutions were infused into the aorta abdominalis of a total of 51 heifers (Experiments 1 and 2): saline; 1 or 10 μg of TNF; 480 mg indomethacin (INDO), an inhibitor of prostaglandin H synthase; 800 mg L-NAME, an inhibitor of NO synthase; and TNF (1 or 10 μg) in combination with INDO or L-NAME. TNF at 1 μg infused directly into aorta abdominalis increased the level of PGF2alpha and decreased the level of progesterone (P4) in the peripheral blood and shortened the estrus cycle. The high TNF dose stimulated P4 and PGE2 and prolonged the corpus luteum (CL) lifespan. INDO blocked the effects of both TNF doses on the CL lifespan and hormone output. L-NAME completely blocked the effects of the luteolytic TNF dose, whereas the effects of the luteotropic TNF dose were not inhibited. In Experiment 3 (Day 14), saline or different TNF doses were infused into the jugular vein (n = 9) or into the uterine lumen (n = 18). The CL lifespans of the different groups were not different when TNF was infused into the jugular vein. Although high TNF doses (1 and 10 μg) infused into the uterine lumen prolonged the CL lifespan, low doses (0.01 and 0.1 μg) induced premature luteolysis. We suggest that the actions of exogenous TNF on the CL lifespan depend on PG synthesis stimulated by TNF in the uterus. TNF at low concentrations initiates a positive cascade between uterine PGF2alpha and various luteolytic factors, including NO, to complete premature luteolysis. PGE2 is a good candidate mediator of the luteotropic actions of exogenous TNF action.

Is interleukin-1α a luteotrophic or luteolytic agent in cattle?

Cytokines are thought to regulate prostaglandin (PG) secretion in the bovine endometrium. However, there is no consensus about the role of interleukin-1alpha (IL1A) on PG secretion. The objective of this study was to examine the influence of IL1A on basal and interferon-tau (IFNT)-regulated PG in vitro secretion, as well its effects on PG secretion, progesterone (P(4)) output, and corpus luteum (CL) in vivo lifespan. Explants of bovine endometrium (days 16-17 of the estrous cycle or early pregnancy) were stimulated with IL1A (10 ng/ml), IFNT (30 ng/ml), or IL1A combined with IFN. IL1A alone strongly stimulated luteotrophic PGE(2) secretion by endometrial tissues of both pregnant and nonpregnant cows. IL1A also stimulated luteolytic PGF(2alpha) output in the late luteal phase. IFNT augmented the stimulatory effect of IL1A on PGE(2) secretion. In an in vivo experiment, saline or IL1A at different doses (0.001-10 microg/per animal) was applied to the uterine lumen on day 16 of the cycle. Only the highest dose of IL1A caused a temporal increase in PGF(2alpha) secretion, while it had no effect on P(4) secretion or CL lifespan. Application of 0.1 and 1 microg IL1A stimulated P(4) and PGE(2) output and prolonged the CL lifespan. Although IL1A may stimulate in vitro luteolytic PGF(2alpha) secretion during the estrous cycle, it only acts as a luteotrophic factor in vivo. IL1A increased luteotrophic PGE(2) and P(4) output, inhibiting spontaneous luteolysis. These luteotrophic effects may result in appropriate luteal development and function in cows during the estrous cycle and early pregnancy.

Adenomyosis in the bovine uterus: correlation between frequency, age, and 17β-estradiol-progesterone equilibrium.

Adenomyosis of the uterus is characterized by the presence of islands of endometrial glands and stroma within the myometrium. Etiopathology of adenomyosis has not been clearly defined but it potentially interferes reproductive processes in cattle. The aim of this initial study was to evaluate the impact of age on the frequency of adenomyosis in cows. Endometrial tissues collected from cows slaughtered between Day 8 and 12 of the estrous cycle (N = 72) were divided into two age groups: (1) 2 to 4 years old (N = 36) and (2) 5 years old and older (N = 36). The tissues were stained with hematoxylin and eosin. The adenomyosis histopathomorphologic stage was classified on a four-point scale according to the penetration of endometrial structures inside the perimetrium. The protein expression of the 17-β estradiol (E2) and progesterone (P4) receptors were evaluated in the endometrial tissue samples by immunohistochemistry and Western blot analysis, and E2 and P4 concentrations were measured in the peripheral blood and uterine tissue. Adenomyosis was observed in 38 of the cows examined including 13 of the 2- to 4-year-old cows and 25 of the cows 5 years old or older. The frequency and intensity of adenomyosis increased with age. Higher E2 receptor protein expression was observed in adenomyotic cows and increased with disease development and increase of number of glands inside the uterus in the direction of perimetrium, and P4 receptor protein expression were unchanged in healthy and adenomyotic cows. An increase in the expression of E2 receptors and high, supraphysiological levels of E2 was detected in cows with III and IV degree of adenomyosis (P < 0.05). Overexpression of E2 receptor and alternations in E2 secretion might make the bovine uterus susceptible to a growth advantage of adenomyotic tissue over the surrounding myometrium. The pathogenesis and immunoendocrine mechanisms controlling adenomyosis in cattle warrant further study.