Marek Bogacki

Scarless skin repair in immunodeficient mice

Scarring, the end result of the wound healing process in adult mammals, is a problem of significant clinical importance. We observed that athymic nude‐nu mice, similar to mammalian fetuses, are able to restore the structure and integrity of injured skin through a process resembling regeneration, where scar formation is absent. Among the postinjured skin tissues collected from athymic nude‐nu, wild‐type controls (C57BL/6J), severe‐combined immunodeficient, Rag (lack of B and T cells), athymic (thymectomized neonates and adult C57BL/6J), and mice treated with an immunosuppressant (cyclosporin A), only athymic nude‐nu mice showed: a lack of scar by histological examination (hematoxylin & eosin and Massons trichrome staining), low levels of collagen (as determined by hydroxyproline content), high levels of hyaluronic acid, a statistically significant increase in elastic modulus for injured samples over unwounded (biomechanical testing) and low levels of the pro‐scarring cytokines platelet‐derived growth factor‐B and transforming growth factor β1. Additionally, immunohistochemical and Western blot analyses of postinjured tissues as well as flow cytometry analysis of blood samples showed the presence of CD8‐positive cells in all studied animals except nude‐nu mice. We conclude that scarless skin healing in athymic nude‐nu mice provides a new model to study the influence of the immune system on tissue regeneration.

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.

Noradrenaline affects secretory function of corpus luteum independently on prostaglandins in conscious cattle

The aim of this study was to examine whether prostaglandins are involved in the effects of noradrenaline on corpus luteum (CL) function. To establish an effective dose of indomethacin (INDO) to prevent prostaglandin synthesis, different doses (120, 180, 240 and 300 mg) of drug were infused for 30 min on days 17-18 of the estrous cycle in four heifers and followed with 50 IU of OT. Plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) were measured, both to illustrate the concentrations of all prostaglandins and to establish the effective dose of INDO that can effectively block prostaglandins synthesis. In Experiment 2, on days 10-12 of the cycle, heifers (n = 6) were infused in a Latin square design with 4 mg of noradrenaline (NA) and pre-treated with 120 mg of INDO or with saline. In both cases, NA did stimulate progesterone and ovarian oxytocin secretion. We conclude that NA affects secretory function of the CL independently of prostaglandins.

Changes in ovarian oxytocin secretion as an indicator of corpus luteum response to prostaglandin F2α treatment in cattle

Exogenous prostaglandin F(2alpha) (PGF(2alpha)) rapidly increases ovarian oxytocin (OT) release and decreases progesterone (P4) secretion in cattle. Hence, the measurement of OT secretion (the area under the curve and the height of the peak) after different doses of Oestrophan - PGF(2alpha) analogue (aPGF(2alpha)) on Days 12 and 18 of the estrous cycle (estrus = day 0), could be a suitable indicator of corpus luteum (CL) sensitivity to PGF(2alpha) treatment. Mature heifers (n = 36) were used in this study. Blood samples were collected from the jugular vein for the estimation of OT, P4 and 13, 14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM). In Experiment 1, different doses of aPGF(2alpha) (400, 300, 200 and 100 microg) given on Day 12 of the estrous cycle (n = 8) shortened (P < 0.05) the cycle duration (15.2 +/- 0.6 d) compared with that of the control (21.7 +/- 0.4 d). Successive heifers were also treated on Day 12 with 200 (n = 2), 100 (n = 2), 75 (n = 2) or 50 microg aPGF(2alpha) (n = 2). Only the 50 microg aPGF(2alpha) dose did not cause CL regression, although it increased OT concentrations to levels comparable to those observed during spontaneous luteolysis (50 to 70 pg/ml). In Experiment 2, on Day 18 of the cycle heifers (n = 8) were treated with 50, 40, 30 and 20 microg aPGF(2alpha). There was a dose-dependent effect of aPGF(2alpha) on OT secretion on Day 18 of the estrous cycle (r = 0.77; P < 0.05). In Experiment 3, an injection of 500 microg aPGF(2alpha) on Day 12 (n = 4) and 50 microg aPGF(2alpha) on Day 18 (n = 4) caused a similar (P > 0.05) increase in the OT concentration (288.5 +/- 23.0 and 261.5 +/- 34.7 pg/ml, respectively). Thus the effect of the same dose of aPGF(2alpha) (50 microg) on OT secretion was different on Days 12 and 18 of the cycle. To evoke similar OT secretion on Days 12 and 18 the dose of aPGF(2alpha) on Day 18 could be reduced 10-fold, confirming that CL sensitivity to PGF(2alpha) appears to increase in the late luteal phase.

Conceptus Signals for Establishment and Maintenance of Pregnancy in Pigs : Lipid Signaling System

Establishment of pregnancy in pigs requires estrogen secretion by the conceptus. The developmental changes of embryo before implantation and embryo-uterine cross talk are dependent on various biological molecules secreted by the endometrium and conceptus. An integral part of maternal recognition of pregnancy seems to be also the lipid signaling system consisting of prostaglandin (PG) F2 alpha and E2 and/or lysophosphatic acid (LPA). The downstream enzymes in PG synthesis pathway are: microsomal PGE synthase-1 (mPGES-1), PGF synthase (PGFS) and prostaglandin 9-ketoreductase/carbonyl reductase (CBR1) which catalyzes conversion of PGE 2 into PGF2 alpha. In contrast to mPGES-1, endometrial PGFS is highly increased on days 13-15 similarly as CBR1 on days 16-17 of the estrous cycle. Potential mechanism by which a conceptus inhibits luteolysis is changing the PGE2/PGF2 alpha ratio in favor of PGE2. It may be result of high expression of mPGES-1 in trophoblast and endometrium on days 10-13 of pregnancy and simultaneously the down-regulation of PGFS and CBR1 in conceptuses during this period. The conceptus can alter expression of endometrial CBR1 to modulate the PGE2/PGF2 alpha ratio in the uterus during the maternal recognition of pregnancy. High expression of conceptus and endometrial terminal PG synthases and CBR1 after initiation of blastocyst attachment suggest their involvement in early placentation. The higher LPA3 receptor mRNA expression during the early pregnancy compared to corresponding period of estrous cycle could indicate an important role of LPA and its receptor during the peri-implantation stage of pregnancy in pigs. Above results suggest that the lipid signaling system is an integral part of establishment and maintenance of pregnancy in the pig.

Expression of prostaglandin synthesis pathway enzymes in the porcine corpus luteum during the oestrous cycle and early pregnancy

Prostaglandins (PGs) of luteal origin may have paracrine and/or autocrine actions on the functions of the corpus luteum (CL). Previously, we have shown that enzymes of PG synthesis pathway such as prostaglandin E synthase (mPGES-1), prostaglandin F synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1) are important in regulation of PG production in the conceptuses and endometrium of cyclic and pregnant pigs. Therefore, localization and expression patterns of these enzymes were determinated in porcine CL. The PGFS protein content was lower in metestrus and higher around luteolysis, and then decreased in late regressing CL. PGFS protein levels were lower on days 5-8 of pregnancy and did not differ between days 10 and 25. Elevated expression of mPGES-1 mRNA was found in early luteal phase. The mPGES-1 protein content, similarly to PGFS, was higher during luteolysis. mPGES-1 mRNA and protein levels were constant between days 5 and 25 of pregnancy. PGFS and mPGES-1 expression was down-regulated on days 16-17 of the oestrous cycle when compared to the corresponding days of pregnancy. Enhanced mPGES-1/PGFS ratio occurred during early luteal phase and days 5-8 of pregnancy. Expression of CBR1 mRNA and protein was constant during the cycle and pregnancy. Our studies revealed higher mPGES-1/PGFS ratios in the CL during early luteal phase and corresponding days of pregnancy that could favor PGE(2) synthesis and may be important in the control of luteal development. However, PG synthesis in the endometrium/conceptus rather than in the CL could be involved in luteolysis and maternal recognition of pregnancy in pigs.

The influence of embryo presence on prostaglandins synthesis and prostaglandin E2 and F2α content in corpora lutea during periimplantation period in the pig

We determined the expression of PGE2 synthase (mPGES‐1), PGF synthase (PGFS), carbonyl reductase/prostaglandin 9‐ketoreductase (CBR1) genes and the content of PGE2, PGF2α in porcine corpora lutea on Days 12–14 of pregnancy and Days 12–14 of the estrous cycle. For this study we used a surgically‐generated model in which one of the uterine horns was cut transversely and a part of this horn was detached from the uterine corpus. The expression of mPGES‐1, PGFS, and CBR1 genes and mPGES‐1/PGFS ratio were significantly higher in corpora lutea of the pregnant gilts compared to the corpora lutea from the parallel ovaries of the cyclic gilts. There was no difference in mPGES‐1, PGFS, CBR1 genes expression and mPGES‐1/PGFS ratio between corpora lutea ipsi‐(CL1) and contralateral (CL2) to the uterine horn with the developing embryos. The highest content of PGE2 was found in CL1 of the pregnant gilts. The PGE2/PGF2α ratio was significantly higher in CL1 of the pregnant gilts compared to corpora lutea from parallel ovary of the cyclic gilts. We suggest that the activity of the investigated genes is induced by compounds of embryonic origin which are not distributed only to the ipsilateral ovary but are transported within the mesometrium to both ovaries in a more systemic manner. Mol. Reprod. Dev. 75: 1208–1216, 2008.

Influence of dopamine as noradrenaline precursor on the secretory function of the bovine corpus luteum in vitro

1 . Dopamine is assumed to affect the ovary function after its conversion into noradrenaline (NA). 2 . To study this bovine luteal slices from 11–14 days of the oestrous cycle were preincubated for 24 h to recover β‐receptors and next they were incubated for 1, 2 or 4 h with (a) different doses of dopamine; (b) dopamine together with a β‐antagonist (propranolol) or with a dopamine receptor blocker (droperidol); (c) dopamine with a dopamine‐β‐hydroxylase inhibitor. 3 . Dopamine stimulated the luteal content of oxytocin (OT) and progesterone. This effect was inhibited by propanolol but not by droperidol. 4 . Dopamine added to the medium was followed by an increase of noradrenaline there. This rise was dose and time‐dependent. 5 . The dopamine‐β‐hydroxylase inhibitor, inhibited the stimulating effect of dopamine on luteal progesterone and OT content. 6 . Bovine corpus luteum can synthesize de novo NA from dopamine as a precursor.

Influence of noradrenaline on progesterone synthesis and posttranslational processing of oxytocin synthesis in the bovine corpus luteum

Noradrenaline (NA) influences secretory function of the bovine corpus luteum (CL), stimulating secretion of progesterone and ovarian oxytocin (OT). To study whether NA is able to stimulate progesterone synthesis and to affect post-translational OT processing, different doses of NA alone or in combination with different doses of OT were added to bovine CL slices from 8 to 13 d of the estrous cycle. To determine which receptors NA affects, and if dopamine (DA) also affects CL function, we used NA or DA combined with a beta-antagonist (propranolol). The results indicated that NA stimulates both luteal progesterone and OT content; furthermore, it increased the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA), terminal enzymes in synthesis of these 2 hormones. The stimulating effect of NA was inhibited by propranolol and by pre-treatment of CL slices with high OT doses. Post-translational processing of OT synthesis by PGA activation was also stimulated by DA, but this effect was inhibited by beta-receptor blocker. Thus DA acts in CL as a NA precursor. In conclusion, it can be assumed that the noradrenergic system affects CL secretory function on different levels of regulation. Furthermore, a high concentration of OT in CL prevents NA from activating PGA and thus decreases post-translational OT synthesis.