Barbara Wąsowska

Localization and correlation between NADPH-diaphorase and nitric oxide synthase isoforms in the porcine uterus during the estrous cycle.

Nitric oxide (NO), a highly reactive free radical is involved in vasodilation, neurotransmission, hormone secretion, and reproduction. Since all known nitric oxide synthase (NOS) isoforms possess NADPH-diaphorase (NADPH-d) activity, NADPH-d histochemistry was used as a commonly accepted procedure for NOS identification. The aim of our study was to determine the cellular localization of NADPH-d, eNOS, and iNOS in the porcine uterus and the correlation between NADPH-d and NOS activity in the early, middle, late luteal, and follicular phase of the estrous cycle. Light-microscopic observations of the sections revealed the differential expression of the NADPH-d in the analyzed stages of the estrous cycle. The most intense staining was observed in the luminal epithelium in the late luteal phase and in some groups of the endometrial glands in all studied stages. Positive reaction was also found in the endothelial cells of blood vessels and in the myometrium itself. Immunostaining for eNOS was observed in the luminal and glandular epithelium in all studied stages, but no clear fluctuations were observed. The endothelium of both endometrial and myometrial blood vessels displayed pronounced eNOS immunostaining. Strong iNOS staining was observed in the luminal epithelium in the late luteal and follicular phase and in selected groups of endometrial glands. Thus, only NADPH-d and iNOS undergo cyclic changes in the studied stages of the estrous cycle. The differential expression of NADPH-d/NOS in the porcine uterine horn during the estrous cycle suggests a role for NO in modulating uterine function.

Effects of the phytoestrogen, genistein, and protein tyrosine kinase inhibitor-dependent mechanisms on steroidogenesis and estrogen receptor expression in porcine granulosa cells of medium follicles.

The use of soy-based products in pig diets had raised concerns regarding the reproductive toxicity of genistein, the predominant isoflavone in soybeans. Genistein was reported to exhibit weak estrogenic activity but its mechanism of action is not fully recognized. The aim of the study was to examine the in vitro effects of genistein on (1) progesterone (P(4)) and estradiol (E(2)) secretion by porcine granulosa cells harvested from medium follicles, (2) the viability of cultured granulosa cells, and (3) the mRNA and protein expression of estrogen receptors α and β (ERα and ERβ) in these cells. In addition, to verify the role of protein tyrosine kinase (PTK)-dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. We found that genistein inhibited (P < 0.05) basal P(4) secretion by granulosa cells harvested from medium follicles of pigs. In contrast, lavendustin C did not affect basal P(4) secretion by the cells. Moreover, genistein increased (P < 0.05) basal granulosal secretion of E(2). In contrast, lavendustin C did not alter basal E(2) secretion by porcine granulosa cells. In addition, we demonstrated that genistein increased mRNA and protein expression of ERβ (P < 0.05) in the examined cells. The expression of ERα mRNA was not affected by genistein and ERα protein was not detected in the cultured granulosa cells of pigs. In summary, the genistein action on follicular steroidogenesis in pigs involved changes in the granulosal expression of ERβ. However, the genistein action on P(4) and E(2) production by granulosa cells harvested from medium follicles did not seem to be associated with PTK.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters steroid secretion but does not affect cell viability and the incidence of apoptosis in porcine luteinised granulosa cells.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a by-product of human industrial activity, was found to affect ovarian steroidogenesis in animals, but the mechanism of its action is still unclear. The aims of the study were to examine the effect of TCDD on (1) progesterone (P4) and oestradiol (E2) production by granulosa cells isolated from medium (3-6 mm) and preovulatory (≥ 8 mm) porcine follicles, (2) the viability of the cells, and (3) the incidence of apoptosis. Porcine granulosa cells were cultured (48 h) with or without TCDD (100 pM, 100 nM). Steroid hormone concentrations in the medium were determined by radioimmunoassay. The viability of granulosa cells was tested spectrophotometrically (alamarBlue™ assay). Apoptosis was evaluated by flow cytometry using Annexin V and by TUNEL assay. The higher dose of TCDD (100 nM) significantly inhibited P4 and stimulated E2 production by luteinised granulosa cells isolated from medium follicles. The lower dose of TCDD (100 pM) significantly stimulated P4 and inhibited E2 secretion by the cells isolated from preovulatory follicles. None of the two TCDD doses affected cell viability or induced apoptosis in granulosa cells. In conclusion, TCDD directly affected steroid production by granulosa cells obtained from mature pigs, but the effect of TCDD was not due to its cytotoxicity.

A Possible Humoral Pathway for the Priming Action of the Male Pheromone Androstenol on Female Pigs

Intraspecific communication by chemical signals (pheromones) plays important behavioral and physiological roles in coordinating reproduction in mammals. Females of a variety of domestic species respond to pheromones from males by undergoing an earlier onset of puberty (Brooks and Cole, 1970; Kirkwood et al., 1983; Kalbrom, 1982; Pearce and Paterson, 1992), alterations in ovarian cycles (Oldham et al., 1979; Booth and Baldwin, 1983; Booth and Signoret, 1992) and estrous behavior (Signoret, 1970; Domes et al., 1997). The precocious attainment of puberty in gilts was studied after contact with the boar (Kirkwood et al., 1983; Kalbrom, 1982) or following the treatment with the male pheromone androstenone, commercially available as an aerosol spray called Suidor (Glei et al., 1989). According to Pearce and Paterson (1992), physical contact with the boar is essential for the maximal pubertal acceleration as it allows the direct transfer of the priming pheromone from the boar to the snout of the recipient gilt; among adult females, nosing and sniffing of the genitalia corresponded to 47% of interactions between the boar and anestrous gilts, while the remaining 43% of interactions involved head-to-head contacts (Signoret, 1970).

Pattern of expression of vascular endothelial growth factor and its receptors in the ovine choroid plexus during long and short photoperiods

Vascular endothelial growth factor (VEGF-A) plays an important role in maintaining cerebrospinal fluid (CSF) homeostasis and the function of the choroid plexuses (CPs). The objective of the study was to determine the expression of vascular endothelial growth factor (VEGF-A), tyrosine kinase receptors Flt-1 and KDR and KDR co-receptor neuropilin 1 (NRP-1) in ovine CPs during different photoperiods. CPs were collected from the lateral brain ventricles from ovariectomized, estradiol-treated ewes during long day (LD; 16L:8D, n = 5) and short day (SD; 8L:16D, n = 5) photoperiods. We analyzed mRNA expression levels of two VEGF-A isoforms, VEGF-A120 and VEGF-A164 and our results indicate that VEGF-A164 was the predominant isoform. Expression levels of VEGF-A and Flt-1 were similar during the SD and LD photoperiods. There were significant increases in KDR mRNA and protein expression (p < 0.05) and NRP-1 mRNA expression (p < 0.05) during SD. These data show that expression of KDR and its co-receptor NRP-1 are up-regulated by short photoperiod and that this effect is not dependent on ovarian steroids. Our results suggest that the VEGF-A-system may be involved in photoperiodic plasticity of CP capillaries and may therefore be responsible for photoperiodic changes in the CSF turnover rate in ewes.

Local effect of progesterone infusion into the porcine ovarian artery on androgen and estrogen secretion during the middle luteal phase

The present study was undertaken to elucidate whether an increased, but physiological, amount of progesterone (P(4)) supplied to the porcine corpus luteum (CL) affects luteal secretion of androgens and estrogens in freely moving gilts. On day 9 of the estrous cycle, the jugular veins as well as both ovarian arteries and both ovarian veins of gilts were cannulated. Progesterone was infused into the right ovarian arteries of experimental gilts (n=5) on days 10, 11 and 12 of the estrous cycle at a rate adequate to physiological retrograde transfer found during the middle luteal phase of the cycle. The left ovarian arteries of the experimental gilts were infused with saline. Both ovarian arteries of the control gilts (n=5) were infused with saline. The P(4) infusion rate was 0.62 microg/min (10 day), 2x0.62 microg/min (11 day) and 3x0.62 microg/min (12 day). Blood samples were collected from the jugular vein and both ovarian veins of the experimental and control gilts on days 10-12 of the estrous cycle before and after P(4) or saline infusion. The mean plasma androstenedione (A(4)) level in the ovarian vein ipsilateral to the P(4)-infused ovary was higher (p<0.01) on days 10-12 of the estrous cycle than those found in the contralateral ovarian vein of the experimental gilts as well as the control gilts. The ovarian venous level of testosterone (T) in the ovarian vein ipsilateral to the P(4)-infused ovary on days 10-12 of the estrous cycle was not significantly different (p>0.05) from those found in the contralateral ovarian vein of the experimental gilts and ovarian vein of the control gilts. However, on day 12, a decrease in T concentration was demonstrated in the ovarian vein ipsilateral to the P4-infused ovary in comparison to those of the contralateral and control ovarian veins. The mean plasma 17beta-estradiol (E(2)) level in the ovarian vein ipsilateral to the P(4)-infused ovary was lower on days 10-12 than those found in the contralateral ovarian vein of the experimental gilts and in the ovarian vein of the control gilts (p<0.001). The results of the present paper indicate that local elevation of P4 concentration in blood supplying the ovary during the middle luteal phase of the porcine estrous cycle affected local secretion of androgens and estrogens. The effect of P(4) on the secretion of androgens and estrogens suggested the existence of a short regulatory loop of a positive feedback for A(4) secretion and a negative feedback for E(2) secretion.

The effect of unilateral progesterone infusion into the ovarian artery during the middle luteal phase on progesterone secretion in gilts.

The aim of the study was to determine, in an experiment performed on conscious gilts, whether an increased amount of progesterone (P4) supplied to the porcine corpus luteum (CL), maintained within a physiological systemic concentration would influence its own secretion. On day 9 of the estrous cycle the jugular veins as well as both ovarian arteries and both ovarian veins were cannulated. In the experimental gilts (n=5), P4 was infused into the right ovarian arteries on days 10, 11 and 12 of the estrous cycle at a rate adequate for physiological retrograde transfer found during the middle luteal phase. The left ovarian arteries of these gilts were infused with saline. Both ovarian arteries of the control gilts (n=5) were infused with saline. The P4 infusion rate was 0.62 microg/min (10 day), 2 x 0.62 microg/min ( 11 day) and 3 x 0.62 microg/min (12 day) and physiological levels of the steroid were maintained. Blood samples were collected from the jugular vein and both ovarian veins in the experimental and control animals on days 10, 11 and 12 of the estrous cycle during two periods on each day: before and after P4 or saline infusion. The mean plasma P4 level in the ovarian vein ipsilateral to the P4-infused ovary was significantly (p<0.001) higher on days 10-12 of the estrous cycle than those found in contralateral ovarian vein of the experimental gilts and in the ovarian vein of the control gilts. This was also true for day 12 of the estrous cycle (p<0.001). However, on days 10 and 11 plasma P4 in the vein from the P4-infused ovary tended (p<0.061) to be higher than those in veins from the saline-treated ovaries. The mean P4 concentration in the ovarian vein ipsilateral to the P4-infused ovary did not vary significantly (p>0.05) among the particular days of the experiment. In contrast, mean P4 levels in the contralateral ovarian vein of the experimental gilts as well as in the ovarian vein of the control gilts tended to decrease (p<0.06) between day 10 and day 12. The results of the present paper indicate that during the middle luteal phase of the porcine estrous cycle (days 10-12), ovarian P4 secretion remained unaltered due to the elevation of P4 concentration in blood supplying the steroid-infused ovary, while a decrease in P4 concentration was observed in ovarian veins of the saline-infused ovaries. The influence of the progestagen on its own secretion suggests that on days 10-12 of the porcine estrous cycle there is a short regulatory loop of positive feedback between P4 being retrograde transferred into the ovary and P4 ovarian secretion.

Relationship Between Contractions of the Uterus and Concentration of PGF2α in Uterine Venous Blood after Luteolysis in Gilts

The origin and physiological significance of high pulses of prostaglandin F2α (PGF2α) in uterine venous blood that occur 2-3 days after luteolysis are not well understood. We studied the relationship between contractions of the uterus evoked by exogenous oxytocin (OT) and PGF2α concentration in uterine venous blood on day 17 of the porcine oestrous cycle. The infusion of OT into the uterine artery produced an immediate increase in the uterine intraluminal pressure (UIP) (p < 0.001) and a simultaneous elevation in PGF2α concentration in uterine venous blood (p < 0.0001). The infusion of indomethacin (IND) into the uterine artery slightly decreased PGF2α concentration in uterine venous blood, but it did not suppress uterine contraction or the rapid increase in PGF2α concentration in uterine venous blood just after OT infusion (p < 0.0001), which was lower that in gilts not treated with IND. We conclude that the spikes of PGF2α concentration in uterine venous blood occurring after OT infusion on day 17 of the porcine oestrous cycle are mainly caused by the excretion with venous blood from the remodelled uterus and that PGF2α synthesis may contribute to this. These results suggest that the high spikes in PGF2α concentration that occur 2-3 days after luteolysis in pigs, sheep, cows and mares all have a similar origin.

Expression of hypoxia inducible factor 1α and antioxidant enzymes: Superoxide dismutases-1 and -2 in ischemic porcine endometrium

The aim of this study was to determine how 60-min ischemia changes the expression of hypoxia inducible factor 1α (HIF-1α) and superoxide dismutases (SOD)-1 and -2 in selected regions of porcine uterine horns. The results showed that 60-min ischemia of the porcine uterus conducted at the mid-secretory estrous phase caused decreased HIF-1α and increased SOD-2 gene expression. Higher expression of SOD-2 suggests that this enzyme may play an important role in the suppression of HIF-1α accumulation in an ischemic endometrium.