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J. Kotwica

Summary. On Day 14 of the oestrous cycle PGF-2α was infused into one anterior uterine vein (15 or 7·5 μg/kg body weight) or jugular vein (15 μg/kg) of pigs for 5 h. Progesterone levels were significantly decreased in the ipsilateral and contralateral utero-ovarian veins, the effect being greater on the side of infusion and with the higher dose. Infusion into the jugular vein did not cause luteolysis. It is suggested that the lymphatic circulation may be involved in the passage of PGF-2α from one uterine horn to the contralateral ovary.

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D. Skarzynski and J. Kotwica

Noradrenaline stimulates the concomitant release of ovarian oxytocin and progesterone in cattle within a few minutes, but the mechanism of its action is unknown. Changes in α- and β-receptors and blood pressure were considered as possible mechanisms of the noradrenaline effect. Heifers in group 1 (n = 4) were infused with noradrenaline (0.16 pg kg−1 min−1 for 30 min into the aorta abdominalis (cranial to the origin of the ovarian artery) on day 10. On days 11 and 12 before noradrenaline, phentolamine (α-blocker; 30 μg kg−1 min−1) or propranolol (β-blocker; 5 μg kg−1 min−1) were infused for 30 min. Four other heifers were infused with noradrenaline only as controls. Only propranolol inhibited the stimulatory effect of noradrenaline on the secretion of progesterone and oxytocin. In group 2, heifers (n = 4) were infused, making use of the latin square design, with vasoconstrictive (angiotensin; 0.042 μg kg−1 min−1) or vasodilatory (xanthinol–theophyline nicotinate; 250 μg kg−1 min−1) drugs that do not act through the adrenoceptors. Noradrenaline (0.3 μg kg−1 min−1) was given 1 h later as in group 1. Blood pressure changes were measured in the posterior aorta abdominalis and oxytocin and progesterone concentrations were determined in the blood samples collected from the jugular vein. Noradrenaline and angiotensin increased (P < 0·01), whereas xanthinol decreased (P < 0·01), blood pressure during their infusion. However, the rise of oxytocin and progesterone concentrations was observed only after noradrenaline infusion. We suggest that (i) noradrenaline enhances the secretion of progesterone and oxytocin from the corpus luteum acting through β-receptors; and (ii) increase of vascular blood pressure which does not occur concomitantly with β-receptor stimulation does not appear to be involved in the corpus luteum secretion.

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J. Kotwica and D. Skarzynski

Mature heifers (n = 31) were infused with either saline or noradrenaline (0.3 μg kg−1 min−1 via a cannula inserted into the aorta abdominalis through the coccygeal artery (cranial to the origin of the ovarian artery). Noradrenaline was infused for three periods of 30 min on days 11 and 12 and on days 15 and 16 of the oestrous cycle. After the noradrenaline treatment, saline was given for 1 h in the same way. During each infusion, peripheral blood samples were collected for progesterone and oxytocin determination every 5–10 min and then once a day until oestrus. As a control, four heifers were infused with saline in latin square design on days 11, 12 and on days 15, 16; they were bled once a day until oestrus. Other heifers were infused on the same days, but 1 h after the last infusion of noradrenaline, 500 μg of prostaglandin F (PGF) analogue was injected, to measure any remaining luteal oxytocin. For comparison four heifers were injected with PGF analogue alone on day 12 and four others on day 16. Blood samples were taken as described previously. Each infusion of noradrenaline stimulated (P < 0.01) progesterone secretion. There was a significant (P < 0.05) response of oxytocin to each noradrenaline stimulation on days 11 and 12, although on each day the response to the second infusion was reduced and further reduced after the third infusion. On days 15 and 16, only the first noradrenaline infusion caused a clear surge of oxytocin with much smaller increases in oxytocin secretion after subsequent infusions. Noradrenaline stimulation reduced the total content of oxytocin in the corpus luteum on day 12 by 68–82% (mean 73·3%) and by 74% on day 16. The duration of the oestrous cycle was 20.8 ± 0.6 and 21.0 ± 0.4 days, respectively, in heifers infused with noradrenaline compared with 21.0 ± 0.5 days in controls. Noradrenaline infusions markedly reduced the total amount of oxytocin in mid- and late-cycle bovine corpora lutea but this did not affect either spontaneous luteolysis or the duration of the oestrous cycle. It is therefore assumed that ovarian oxytocin may have a permissive role rather than a direct action on luteolysis in cattle.

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T. Krzymowski, J. Kotwica and S. Stefańczyk

Summary. [3H]Testosterone or 51Cr-labelled red blood cells were infused for 30 min through a cannula inserted into the ovarian vein below the hilus of the ovary of cyclic sows. The whole of the vascular pedicle was isolated by ligations from the systemic circulation except for the ovarian artery supplying the ovary. During and after the testosterone infusion, radioactivity was found in ovarian arterial blood and ovarian tissue, but not in peripheral blood. When 51Cr-labelled blood cells were infused in the same fashion there was no radioactivity in arterial blood or ovarian tissue. These results indicate the existence of a counter-current transfer for testosterone in the sow ovarian vascular pedicle.

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T. Krzymowski, J. Kotwica, S. Stefańczyk, J. Dȩbek and J. Czarnocki

Summary. The ovary and its vascular pedicle were isolated and transferred under a stereomicroscope to a heated surface (40°C). The ovary was supplied with the blood from the middle uterine artery through a cannula. On different days of the oestrous cycle [3H]testosterone, [3H]progesterone or [3H]oestradiol-17β were infused for 30 min into the ovarian vein 1–2 cm below the ovary. During and 30 min after the infusion radioactivity was found in ovarian arterial blood and ovarian tissue. Recovery of [3H]testosterone, [3H]oestradiol and [3H]progesterone infused into the ovarian vein from ovarian venous blood collected during 60 min of experiment was (mean ± s.e.m.) 26·8 ± 6·8%, 20·1 ± 6·1% and 23·7 ± 6·2% respectively. When 51Cr-labelled blood cells were infused in the same fashion as the steroids there was no radioactivity in arterial blood or ovarian tissue. These results indicate the existence of a countercurrent transfer mechanism and retention of the hormones in tissue(s) other than blood in the ovarian pedicle.

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T. Krzymowski, J. Kotwica, S. Stefańczyk, J. Czarnocki and J. Dȩbek

Summary. Sow ovaries with their ovarian pedicle were isolated and supplied with blood from the middle uterine artery. During the 30 min of infusion with [3H]testosterone into the ovarian vein 3 cm below the ovary and for the 30 min after the infusion, radioactivity was detected in tissue fluid 3 cm laterally from the ovarian vein and artery. When [3H]testosterone was infused into the muscles of the ovarian pedicle radioactivity was detected in the ovarian artery branches near the ovary. Of the total amont of blood entering the ovarian artery 63·7 ± 4·2% reached the ovary and 36·3 ± 3·1% travelled to the muscles and connective tissue of the ovarian pedicle. It was demonstrated that the ovarian arterial branches supplying the ovarian pedicle muscles after capillarization form the veins which descend within the ovarian artery network, redivide and create the venous mesh covering the spiralling ovarian artery branches.

It is suggested that a special subovarian exchanging mechanism exists in the ovarian pedicle for countercurrent transfer of ovarian steroid hormones and that ovarian function can thereby be regulated.

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T. Krzymowski, J. Kotwica, S. Okrasa, Teresa Doboszyńska and A. Zieçik

Summary. Prostaglandin F-2α (1·5 mg over 10 h) was infused into the anterior uterine vein of pigs on Days 6, 8, 10, 12, 14 and 15 of the oestrous cycle. At each stage of the cycle PGF-2α suppressed luteal function although the fall in progesterone secretion was much greater and statistically significant when the infusion was performed on Days 12,14 and 15 of the cycle than on Days 6, 8 and 10. The concentration of cAMP was depressed on Days 15 and 17 and fatty degeneration of luteal cells on Days 6–8 or 14 was more pronounced in the ovary ipsilateral to the PGF-2α infusion than in the contralateral ovary. The results are compatible with the local perfusion of PGF-2α from the anterior uterine vein to the ipsilateral ovary, but a systemic effect was also apparent.

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J. Kotwica, D. Schams, H. H. D. Meyer and Th. Mittermeier

Summary. In Exp. I oxytocin (60 μg/100 kg/day) was infused into the jugular vein of 3 heifers on Days 14–22, 15–18 and 16–19 of the oestrous cycle respectively. In Exp. II 5 heifers were infused with 12 μg oxytocin/100 kg/day from Day 15 of the oestrous cycle until clear signs of oestrus. Blood samples were taken from the contralateral jugular vein at 2-h intervals from the start of the infusion. The oestrous cycle before and after treatment served as the controls for each animal. Blood samples were taken less frequently during the control cycles. In Exp. III 3 heifers were infused with 12 pg oxytocin/100 kg/day for 50 h before expected oestrus and slaughtered 30–40 min after the end of infusion for determination of oxytocin receptor amounts in the endometrium. Three other heifers slaughtered at the same days of the cycle served as controls.

Peripheral concentrations of oxytocin during infusion ranged between 155 and 641 pg/ml in Exp. I and 18 and 25 pg/ml in Exp. II. In 4 out of 8 heifers of Exps I and II, one high pulse of 15-keto-13,14-dihydro-prostaglandin F-2α (PGFM) appeared soon after the start of oxytocin infusion followed by some irregular pulses. The first PGFM pulse was accompanied by a transient (10–14 h) decrease of blood progesterone concentration. High regular pulses of PGFM in all heifers examined were measured between Days 17 and 19 during spontaneous luteolysis. No change in length of the oestrous cycle or secretion patterns of progesterone, PGFM and LH was observed. The number of oxytocin receptors in endometrium was not affected by oxytocin infusion around the time of oestrus. These results suggest that luteolytic events were not significantly influenced by a constant infusion of oxytocin.

Keywords: oxytocin; infusion; luteolysis; oestrous cycle length

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A V Sirotkin, A Benčo, A Tandlmajerova, D Vašíček, J Kotwica, K Darlak and F Valenzuela

The aim of our in vitro experiments was to examine the role of transcription factor p53 in controlling the basic functions of ovarian cells and their response to hormonal treatments. Porcine ovarian granulosa cells, transfected and non-transfected with a gene construct encoding p53, were cultured with ghrelin and FSH (all at concentrations of 0, 1, 10, or 100 ng/ml). Accumulation of p53, of apoptosis-related (MAP3K5) and proliferation-related (cyclin B1) substances was evaluated by immunocytochemistry. The secretion of progesterone (P4), oxytocin (OT), prostaglandin F (PGF), and E (PGE) was measured by RIA. Transfection with the p53 gene construct promoted accumulation of this transcription factor within cells. It also stimulated the expression of a marker of apoptosis (MAP3K5). Over-expression of p53 resulted in reduced accumulation of a marker of proliferation (cyclin B1), P4, and PGF secretion and increased OT and PGE secretion. Ghrelin, when added alone, did not affect p53 or P4, but reduced MAP3K5 and increased PGF and PGE secretion. Over-expression of p53 reversed the effect of ghrelin on OT, caused it to be inhibitory to P4 secretion, but did not modify its action on MAP3K5, PGF, or PGE. FSH promoted the accumulation of p53, MAP3K5, and cyclin B1; these effects were unaffected by p53 transfection. These multiple effects of the p53 gene construct on luteinizing granulosa cells, cultured with and without hormones 1) demonstrate the effects of ghrelin and FSH on porcine ovarian cell apoptosis and secretory activity, 2) confirm the involvement of p53 in promoting apoptosis and inhibiting P4 secretion in these cells, 3) provide the first evidence that p53 suppress proliferation of ovarian cells, 4) provide the first evidence that p53 is involved in the control of ovarian peptide hormone (OT) and prostaglandin (PGF and PGE) secretion, and 5) suggest that p53 can modulate, but probably not mediate, the effects of ghrelin and FSH on the ovary.

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A Benčo, A V Sirotkin, D Vašíček, S Pavlová, J Zemanová, J Kotwica, K Darlak and F Valenzuela

The aim of our in vitro experiments was to study the role of the transcription factor STAT1 and the hormone ghrelin in controlling porcine ovarian function. The effects of treatment with ghrelin (0, 1, 10, 100 ng/ml), transfection-induced overexpression of transcription factor STAT1, and their combination on apoptosis (expression of apoptosis-related peptides caspase-3, BAX and anti-apoptotic peptide BCL2), proliferation (expression of proliferating cell nuclear antigene PCNA, proliferation-associated protein kinase MAPK/ERK1,2) and release of the hormones progesterone (P4), prostaglandin F (PGF) and oxytocin (OXT) in cultured porcine ovarian granulosa cells was evaluated using RIA, immunocytochemistry and SDS-PAGE–western immunoblotting. It was found that ghrelin, when given alone, increased the expression of proliferation-associated PCNA and MAPK/ERK1,2, decreased the accumulation of apoptosis-related substances caspase-3, BAX, BCL2, decreased P4, and increased PGF and OXT release. Ghrelin tended to promote accumulation of STAT1 in both control and transfected cells, although in transfected cells ghrelin at 1 ng/ml decreased STAT1 accumulation. Transfection of porcine granulosa cells by a gene construct encoding STAT1 promoted the expression of STAT1 and apoptosis-related-BAX but the expression of BCL2 did not, and decreased the accumulation of proliferation-associated MAPK/ERK1,2 but not that of PCNA. It also promoted PGF and OXT but not P4 release. Overexpression of STAT1 reversed the effect of ghrelin on STAT1, PCNA, PGF, OXT (from stimulatory to inhibitory), BCL2, P4 (from inhibitory to stimulatory), prevented ghrelin effect on caspase-3 and BAX, but did not affect ghrelin's effect on MAPK/ERK1,2 expression. These results suggest that ghrelin directly affects porcine ovarian cells function – stimulates proliferation, inhibits apoptosis and affects secretory activity. Furthermore, they demonstrated the involvement of the transcription factor STAT1 in controlling these functions, the promotion of some markers of apoptosis (BAX), inhibition of some markers of proliferation (MAPK/ERK1,2) and stimulation of PGF release. Finally, the obtained data failed to demonstrate that STAT1 is involved in mediating the action of ghrelin on ovarian cell functions.