Administration of a nitric oxide synthase inhibitor counteracts prostaglandin F2-induced luteolysis in cattle

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 F2alpha (PGF2alpha)-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 PGF2alpha-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (Nomega-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 PGF2alpha (aPGF2alpha; 100 microg) 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 aPGF2alpha 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 aPGF2alpha were higher (P < 0.05) than in animals injected only with aPGF2alpha. The PGF2alpha 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 aPGF2alpha (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 PGF2alpha.     

Analysis of 5-methylcytosine in DNA of breast and colon cancer tissues

5-methylcytosine (m(5)C) can be used as a sensitive marker of progress of the tumor formation induced by the oxidative damage reactions. We have analyzed the amount of m(5)C in DNA of patients with breast and colon cancers. Two dimensional thin layer chromatography (TLC) has been used to monitor 5-methylcytosine level in DNA extracted from cancer tissues. The level of methylation of cytosine at C-5 position in DNA from breast cancer patients correlates well with the malignancy of tumors. Interestingly higher amount of m(5)C in DNA for the breast cancer patients treated with different chemotherapeutics was observed. It suggests an activation of DNA methyltransferase as well as a genomic suppression of the DNA repair genes expression. These differences clearly reflect the health condition of patients and support the global analysis of m(5)C in DNA as a good marker for diagnosis of neoplasia in clinical practice.     

Limitation of C3–CAM shift in the common ice plant under high irradiance

In the halophytic plant Mesembryanthemum crystallinum salinity or drought can change the mode of photosynthesis from C₃ to crassulacean acid metabolism (CAM). These two stress factors are linked to oxidative stress, however, the induction of CAM by oxidative stress per se is not straightforward. Treatment with high light (HL) did not lead to the induction of CAM, as documented by a low night/day difference in malate level and a low expression of the CAM-related form of phosphoenolcarboxylase (Ppc1), despite causing some oxidative damage (elevated MDA level, malondialdehyde). In contrast to the action of high salinity (0.4 M NaCl), HL treatment did not activate neither the cytosolic NADP-malic enzyme nor the chloroplastic form of NADP-dependent malate dehydrogenase (NADP-MDH). In plastids of HL-treated plants a huge amount of starch was accumulated. This was associated with a weak stimulation of hydrolytic and phosphorolytic starch-degrading enzymes, in contrast to their strong up-regulation under high salinity. It is concluded that HL alone is not able to activate starch degradation necessary for CAM performance. Moreover, in the absence of salinity in C₃M. crystallinum plants an age-dependent increase in energy dissipation from PSII was documented under high irradiance, as illustrated by non-photochemical quenching (NPQ). Obtained data suggest that in this halophytic species several photoprotective strategies are strictly salinity-dependent.     

Superoxide dismutase activity in C<Subscript>3</Subscript> and C<Subscript>3</Subscript>/CAM intermediate species of <Emphasis Type="Italic">Clusia</Emphasis>

The C₃-CAM intermediate Clusia minor L. and the C₃ obligate Clusia multiflora H.B.K. plants were exposed for 7 d to a combination of drought stress and high irradiance of about 1200 μmol m⁻² s⁻¹ for 12 h per day. In both species under these conditions a strong decrease in stomatal conductance was observed at dawn and dusk. Changes in stomatal behaviour of C. minor were accompanied by only a low nocturnal accumulation of malate and citrate. Thus, in C. minor drought stress applied in combination with high irradiance limited CAM expression, and possibly this is the main reason why C. minor prefers semi-shaded sites in the field. The mitochondrial MnSOD, in both well watered and stressed plants of two species showed strong diurnal oscillations with maximum activity at dusk. These oscillations can be explained by the engagement of mitochondria in dissipation of an excess of reducing equivalents. In plants which are able to carry out CAM metabolism tricarboxylic acid cycle is expected to be down regulated in the dark period to prevent breakdown of the entire malate and citrate.     

Daily rhythm of MnSOD in the C<Subscript>3</Subscript>-CAM intermediate <Emphasis Type="Italic">Clusia fluminensis</Emphasis> Planch. et Triana.

The C₃-CAM intermediate plant Clusia fluminensis under well-watered at low light conditions opens stomata during the light period. In leaf extracts of this plant we have found two copper-zinc superoxide dismutases (CuZnSODs) and two manganese SODs: MnSOD-like protein (MnSOD II) and MnSOD I. Daily rhythm of the MnSOD I shows maximum activity during the afternoon hours and it is accompanied by only a very small tendency to increase in catalase (CAT) activity and lowering of citrate level.     

Distribution of NADPH-diaphorase and nitric oxide synthase (NOS) in different regions of porcine oviduct during the estrous cycle.

Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs, including those of the reproductive tract. We investigated potential changes in NADPH-diaphorase (NADPH-d) activity (marker for NOS activity) and the presence and distribution of NOS in the porcine oviduct. Tissues were obtained from gilts (n=16) on different days of the estrous cycle. One fallopian tube was used for histo- and immunohistochemistry and the other for Western blotting analysis. NADPH-d activity was much higher in the epithelium of the mucosa than in the myosalpinx. The highest activity of NADPH-d was always found in the epithelium of the isthmus. The intensity of the reaction (arbitrary units +/- SEM) in isthmus epithelium increased from the postovulatory period until early proestrus (96.2 +/- 11.2) and then gradually decreased. The lowest intensity of NADPH-d reaction in the epithelium of the isthmus was seen at estrus (58.4 +/- 7.7). The most intense NADPH-d activity in myosalpinx of all parts of the oviduct was observed at the postovulatory stage of the estrous cycle (isthmus 38.3 +/- 2.5; ampulla 35.6 +/- 4.2; infundibulum 24.7 +/- 0.8) and then decreased during the remaining stages of the estrous cycle (p< 0.001). The presence of endothelial NOS (eNOS) was detected in epithelial cells of mucosa and in endothelium of vascular tissues and myosalpinx during all studied days of the estrous cycle. The positive reaction for inducible NOS (iNOS) was restricted only to the endothelium of lymph vessels and some blood vessels. Because our Western blotting analysis revealed that porcine oviduct contains eNOS but not iNOS, we suggest that eNOS is the main isoform of NOS expressed in the porcine oviduct. We concluded that the different activity of NADPH-d in the various regions of the oviduct, accompanied by changes in its activity during the course of the estrous cycle, could indicate an important role of NO in regulation of tubal function.     

Oestrogen-dependent expression of LH/hCG receptors in pig Fallopian tube and their role in relaxation of the oviduct.

The current studies investigated the concentration and distribution of LH receptors in the oviduct of ovariectomized gilts at various times after administration of oestradiol benzoate (10 micrograms kg-1 body weight) to determine whether LH participates in the regulation of oviductal contractions. Polyclonal antibodies to the LH receptor were used in immunocytochemical and western blot analyses of oviductal tissues. The mechanical activity of the isthmus and ampullar segments of oviduct, collected from 16 cyclic gilts, was recorded for 30 min after LH or hCG treatment. In the oviduct, there was little competition for receptor occupancy between hCG and pig FSH, bovine thyroid-stimulating hormone (TSH), pig growth hormone (GH) and pig prolactin (1.2, 0.1, 0.01 and < 0.001%, respectively) but pig LH could completely inhibit the binding of [125I]hCG. Oestradiol benzoate increased (P < 0.01) the number of LH binding sites in oviduct 24, 48 and 72 h (0.60 +/- 0.08, 1.62 +/- 0.15, 2.48 +/- 0.35 fmol mg-1 protein; n = 4 per treatment, respectively) after injection compared with the control gilts treated with corn oil (0.20 +/- 0.04 fmol mg-1 protein; n = 4). The affinity of oviductal LH/hCG binding sites (Ka) varied from 4.0 to 8.5 x 10(10) l mol-1 and was similar to that of luteal cell binding sites (6.1 x 10(10) l mol-1). Oestradiol benzoate also resulted in more intense LH receptor immunostaining of the tubal mucosal epithelium, smooth muscle cells and blood vessels as compared with controls. Western blotting has revealed that the pig oviduct, similar to the corpus luteum, contains 75, 48 and 45 kDa immunoreactive LH receptor proteins. Treatment with LH in vitro (100 ng ml-1) affected the contractility of oviduct. During the peri-ovulatory stage of the oestrous cycle, the amplitude, frequency and area under curve(s) of the isthmus decreased (P < 0.05), as did the frequency and area under curve (P < 0.05 and P < 0.01, respectively) of the ampulla (n = 4). The frequency and area under curve of the oviductal contractions were also significantly reduced during the early follicular phase of the oestrous cycle (P < 0.05). There was no effect of LH (or hCG) on the frequency and area under curve of the oviductal contractions during luteal stages of the oestrous cycle (n = 8). These data indicate that (1) the pig oviduct possesses immunoreactive and functional LH receptor, (2) oestradiol promotes the synthesis of LH receptor in the epithelium and smooth muscles, and (3) LH causes the relaxation of oviduct, especially during the peri-ovulatory stage of the oestrous cycle. In summary, the results of the present study indicate that LH can control oviductal contractions directly and may be partially responsible for the relaxation of isthmus during fertilization in pigs.     

Effect of estradiol and progesterone on oviductal LH-receptors and LH-dependent relaxation of the porcine oviduct

We have previously shown that the porcine oviduct possesses immunoreactive and functional LH receptors and that LH causes relaxation of the oviduct, especially during the periovulatory stage of estrous cycle. The current studies were undertaken to investigate the effects of estradiol and progesterone on LH receptor protein and LH-stimulated motility of the oviduct in steroid-primed ovariectomized gilts. Twenty-one cross-bred gilts were ovariectomized at 6 m.o. of age. Four weeks later gilts received daily intramuscular injection of either 2 mL corn oil (control n = 4), estradiol benzoate (EB) 1.5 mg (n = 6), progesterone 50 mg (n = 5), or 1.5 mg EB plus 50 mg progesterone (n = 6) for 4 consecutive days. The gilts were slaughtered on Day 5 after the first injection of steroids or vehicle. Rings of isthmus and ampulla were collected from each oviduct and placed in a tissue chamber perfused with Kreb's solution for 60 min. The mechanical activity was recorded for 30 min after LH treatment. Immunoreactivity of LHR in the Fallopian tube sections were detected in the epithelium of the tubal mucosa, smooth muscle cells and the blood vessel endothelium. Western blotting showed that porcine oviducts contain 75, 48 and 45 kDa immunoreactive LH receptor proteins, like the corpus luteum (CL). The lowest receptor expression was found in controls and in gilts treated with estradiol or progesterone. Combined treatment with estradiol and progesterone resulted in a significant increase of LH receptor protein concentrations when compared with control animals. In vitro LH treatment affected oviduct contractility of combined estradiol and progesterone treated gilts but not the oviduct of the remaining groups. It also caused a decrease in amplitude, frequency and areas under the curve (AUC) of ampulla (P < 0.05) and the amplitude and AUC of isthmus (P < 0.001) in combined estradiol and progesterone-primed gilts. These results indicate that estradiol and progesterone together, but not separately, increase LH receptor protein in the porcine oviduct and that combined estradiol and progesterone priming is necessary for LH-induced relaxation of the porcine oviduct.