Renata E. Ciereszko

Relationship between vitamin C and plasma concentrations of testosterone in female rainbow trout, Oncorhynchus mykiss

Two-year old rainbow trout females were fed diets containing 0, 30, 110, 220, 440 and 870 mg kg-1 ascorbyl-2-monophosphate Mg+ salt (groups 1, 2, 3, 4, 5, and 6, respectively) from August until March. At the time of spawning (February–March) blood was sampled and the ovulating females were hand stripped. Estradiol (E2) and testosterone (T) concentrations in plasma, and ascorbic acid (AA) concentrations in plasma and eggs were determined. The mean plasma concentrations of T were higher in group 4, 5, or 6 than in group 2 or 3 (p < 0.05). Moreover, the average plasma concentration of T in fish fed the diets with AA level below National Research Council (NRC) recommendations (groups 1, 2 and 3) was significantly lower (p<0.01) than the average plasma concentration in fish fed diets with AA level above NRC recommendations (groups 4, 5, and 6). These data are consistent with the hypothesis that AA can influence production of steroids in female rainbow trout.

Resveratrol inhibits reproductive toxicity induced by deoxynivalenol.

The aim of this in vitro study was to examine the release of progesterone by porcine ovarian granulosa cells (GCs) after exposure to toxic concentrations of deoxynivalenol (DON), resveratrol (RSV), and their combination (DON with RSV). Ovarian granulosa cells were incubated without (control) or with treatments of natural substances at various doses for 24 h: RSV (10, 30 and 50 μg/mL) / DON (2000, 3000 and 5000 ng/mL), and their combination (10 μg/mL of RSV with 2000 ng/mL of DON; 30 μg/mL of RSV with 3000 ng/mL of DON; 50 μg/mL of RSV with 5000 ng/mL of DON). Progesterone was determined by radioimmunoassay (RIA). Progesterone release was significantly (P < 0.05) stimulated by RSV at the doses 50 μg/mL but not at 30 and 10 μg/mL and by DON treatment at all used doses (2000, 3000 and 5000 ng/mL). RSV in combination with DON stimulated significantly (P < 0.05) the progesterone release by GCs at the highest doses (50 μg/mL of RSV with 5000 ng/mL of DON). On the other hand, the stimulatory effect of RSV in combination with DON was significantly (P < 0.05) lower in comparison with alone DON effect. In conclusion, our results indicate, (1) the dose-depended stimulatory effects of RSV, DON and combination of RSV with DON on release of steroid hormone progesterone and (2) reduction of the stimulatory effect of DON by RSV. Our in vitro results suggest that reproductive toxicity of animals induced by a mycotoxin - deoxynivalenol can be inhibited by a protective natural substance – resveratrol.

Prolactin signaling in porcine adrenocortical cells: involvement of protein kinases

Prolactin (PRL) was found to have a stimulatory effect on adrenal steroidogenesis in vivo and in vitro in several species including pigs. PRL signal transduction pathways, however, in adrenocortical cells are poorly recognized. Therefore, the goal of this paper is to ascertain the involvement of protein kinase C (PKC) and tyrosine kinases in PRL signaling in porcine adrenal cortex. Adrenals were harvested from locally slaughtered mature gilts. Cortical cells were dispersed by sequential treatment with collagenase. The cells were seeded into 24-well culture plates at a density of 3 x 10(5)/mL. Cells were incubated with or without PRL (500 ng/mL), ACTH (5 nM--a positive control), tyrosine kinase inhibitor--genistein (1; 2.5 or 5 microM), PKC inhibitor--sphingosine (20-1000 nM) and PKC activators--diacylglycerol (DiC8; 10-100 microM) and phorbol ester (PMA; 1-1000 nM). All incubations were performed for 8 h (95% air and 5% CO(2), 37 degrees C). PRL and ACTH (P < 0.05) increased cortisol and androstenedione (A(4)) secretion. DiC8 and PMA mimicked the stimulatory effect of PRL. Sphingosine (P < 0.05) suppressed basal and PRL-stimulated steroid secretion. Genistein inhibited (P < 0.05) PRL-stimulated cortisol secretion and enhanced (P < 0.05) basal and PRL-stimulated A(4) secretion. Moreover, PKC activation was assessed by measuring the specific association of [3H]phorbol dibutyrate ([3H]PDBu) with adrenocortical cells after treatment with PRL or ionomycin (a positive control). PRL (within 2-3 min) and ionomycin (within 2-5 min) increased (P < 0.05) specific binding of [3H]PDBu to the porcine adrenocortical cells. In addition, PRL did not augment the cortisol and A(4) secretion by PKC-deficient adrenocortical cells. In conclusion, presented results support the hypothesis that PKC and tyrosine kinases are involved in PRL signaling in adrenocortical cells in pigs. Moreover, activation of PKC is associated with the increased secretion of cortisol and A(4).

The involvement of prolactin in the regulation of adrenal cortex function in pigs.

We investigated the in vivo and in vitro effect of prolactin (PRL) on porcine adrenal cortex function. The in vivo study was performed on 10 multiparous sows. Blood was sampled every 4 h beginning on the 17th day of the estrous cycle and continuing for 6 subsequent days. Plasma was stored at -20 degrees C until steroid hormones analysis was completed. PRL or saline were administered iv for 48 h in 2 h intervals. Injections of PRL began 4-20 h after the preovulatory LH surge. At the end of the sampling period sows were slaughtered and adrenals were immediately dissected. Adrenals were frozen at -70 degrees C for determination of adrenal cortex steroid hormones content. At the end of PRL treatment period mean plasma level of cortisol in control sows was significantly lower than that of PRL-treated sows. Moreover, the area under the mean plasma cortisol concentration curve was significantly higher in PRL-treated sows in comparison to controls. The mean cortisol adrenal content was significantly higher in adrenal cortex of PRL-treated sows than that of controls. PRL did not affect adrenal cortex concentration of androstenedione (A(4)), testosterone (T), dehydroepiandrosterone (DHEA) and estradiol (E(2)). Dehydroepiandrosterone sulfate (DHEAS) was not found in porcine adrenal cortex. In the in vitro experiment adrenal glands were removed immediately after slaughter of 6 crossbred gilts. Dispersed adrenocortical cells were incubated for 8 h with or without porcine PRL. Prolactin stimulated cortisol secretion in a dose-dependent manner. These results suggest that PRL is one of the key factors involved in the regulation of adrenal cortex function in pigs.

Plasma concentrations of steroid hormones in male yellow perch, Perca flavescens : the effect of age and photothermal manipulation

Plasma steroid concentrations in two and three year-old male yellow perch maintained under two different photothermal regimes were investigated. Initially, all fish kept indoors were exposed to the same water temperature (22 °C) and photoperiod (15L:9D). By the end of August, following the first sampling, fish were exposed to different photothermal regimes. Groups A2 (2 year old) and A3 (3 year old) were maintained under photothermal conditions similar to those of southern Ohio. Groups B2 (2 year old) and B3 (3 year old) were exposed to a condensed light/temperature regime designed to accelerate maturation. Testosterone (T) was the major circulating androgen in all groups. In regime A fish, plasma concentrations of 11-ketotestosterone (11KT) and T were very low in August, increased in October and remained elevated until March. In regime B plasma androgens were high until February and then dropped abruptly in March. The elevated circulating levels of 11KT and T were associated with production of sperm. The highest sperm concentration in the groups A3 and B3 was observed in February and December, respectively. There were no major differences in profiles and levels of plasma steroids between two age categories within each photothermal regime. These data indicate that the compression of the photothermal cycle accelerated both the occurrence of the low postspawning levels of circulating steroids and the completion of milt production. Higher sperm concentration observed in B3 group earlier in the season compared to A3 group also support the notion that the condensed photothermal cycle accelerated gonadal maturation. It appears that modification of the environmental cues may be a useful tool for manipulation of reproductive processes in male yellow perch.

Prolactin involvement in the regulation of the hypothalamic–pituitary–ovarian axis during the early luteal phase of the porcine estrous cycle

Our previous in vivo and in vitro studies revealed that prolactin (PRL) affected luteal function during the first days of the porcine estrous cycle. Since the mechanism by which the luteotrophic action of PRL might be mediated was not elucidated, the goal of the present study is to investigate the effects of short term, in vivo administration of PRL on in vitro functions of hypothalamic explants, adenohypophyseal cells and luteal cells of sows. Injections of PRL or saline (performed every 2h) started shortly after the preovulatory LH surge and lasted for 2 or 3 days. Peripheral blood plasma for determination of LH, PRL and progesterone (P(4)) was sampled at 4h intervals. Ovaries, pituitaries and the stalk median eminence (SME) dissected after slaughter were used for in vitro studies. Luteal and adenohypophysial cells as well as hypothalamic tissue were incubated/cultured with different treatments. Medium and plasma levels of GnRH, LH and P(4) were quantified by radioimmunoassays (RIAs). Corpora lutea (CL) were used for LH/human chorionic gonadotrophin (hCG) receptor analysis. In vivo and in vitro treatment with PRL increased the in vitro GnRH release by hypothalamic explants (P<0.05). GnRH-stimulated LH production was enhanced in PRL-treated sows compared to that of control sows (P<0.05). PRL injections had no effect on plasma P(4) concentrations during the treatment period. However, luteal secretion of P(4) (P=0.06) and LH/hCG receptor concentration (P=0.079) tended to be higher in PRL-treated sows in comparison to those of controls. The results indicate that PRL may be involved in the regulation of the hypothalamic-pituitary-ovarian axis at the beginning of the luteal phase of the porcine estrous cycle.

Antiandrogen flutamide affects folliculogenesis during fetal development in pigs

Androgen deficiency during prenatal development may affect the expression of genes involved in the folliculogenesis regulation. In order to study the effect of antiandrogen on fetal ovarian development, pregnant gilts were injected with flutamide (for 7 days, 50 mg/kg bodyweight per day) or corn oil (control groups) starting on gestation days 43 (GD50), 83 (GD90), or 101 (GD108). The obtained fetal ovaries were fixed for histology and immunohistochemistry or frozen for real-time PCR. Morphological evaluation, TUNEL assay, and expression of selected factors (Ki-67, GATA binding transcription factor 4 (GATA4), E-Cadherin and tumor necrosis factor a (TNFa)) were performed. On GD90 and GD108, ovaries following flutamide administration showed a higher number of egg nests and lower number off ollicles than those in respective control groups. An increased mRNA and protein expression of Ki-67 was observed in flutamide-treated groups compared with controls on GD50 and GD108 but decreased expression was found on GD90. In comparison to control groups a higher percentage of TUNEL-positive cells was shown after flutamide exposure on GD50 and GD90 and a lower percentage of apoptotic cells was observed on GD108. These data were consistent with changes in TNF (TNFa) mRNA expression, which increased on GD90 and decreased on GD108. E-cadherin mRNA and protein expression was upregulated on GD50 and downregulated on GD90 and GD108. In conclusion diminished androgen action in porcine fetal ovaries during mid- and late gestation leads to changes in the expression of genes crucial for follicle formation. Consequently, delayed folliculogenesis was observed on GD90 and GD108. It seems however that androgens exhibit diverse biological effects depending on the gestational period.

TOTAL ASCORBATE AND DEHYDROASCORBATE CONCENTRATIONS IN PORCINE OVARIAN STROMA, FOLLICLES AND CORPORA LUTEA THROUGHOUT THE ESTROUS CYCLE AND PREGNANCY

Ovarian tissues are thought to require ascorbate as an antioxidant and enzymatic cofactor for the processes of steroid and collagen synthesis. We measured the concentrations of total ascorbate and oxidized ascorbate (dehydroascorbate, DHA) in ovarian stroma, follicles and corpora lutea (CL) throughout the estrous cycle and pregnancy of the sow. Both total ascorbate and DHA concentrations were greatest in luteal tissue and lowest in ovarian stroma across all stages examined. Within the CL, total ascorbate levels were lowest during the early, early-mid, and late luteal phase and were elevated during the mid-luteal phase. Luteal total ascorbate concentrations were further elevated during early pregnancy and were comparable to mid-luteal phase concentrations during the remainder of gestation. Luteal DHA concentrations decreased from mid to late luteal phase, and were elevated throughout pregnancy. As the CL aged during the cycle, the DHA/total ascorbate ratio decreased and remained low throughout pregnancy. Total ascorbate concentrations in follicular tissue increased during the follicular phase and were lowest during the early luteal phase. The DHA concentrations and DHA/total ascorbate ratios in follicular tissue did not differ with stage. Total ascorbate and DHA concentrations in ovarian stroma were low and did not vary with stage. We conclude that periods of maximal luteal and follicular function are associated with increased concentrations of total ascorbate within the tissue. Furthermore, luteolysis appears to be associated with depletion of luteal ascorbate species.

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.

In vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on ovarian, pituitary, and pineal function in pigs

The aims of the study were: (1) to examine 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and/or prolactin (PRL) effects on in vitro secretion of progesterone (P(4)) and estradiol (E(2)) by luteinized granulosa and theca cells from porcine preovulatory follicles; and (2) to determine the effects of TCDD on PRL, luteinizing hormone (LH), and melatonin luteal phase in pigs. We found that TCDD itself did not affect progesterone secretion, but it abolished the stimulatory effect of PRL in the follicular cells. TCDD stimulated PRL secretion during the luteal phase and inhibited during the follicular phase. Moreover, TCDD increased luteinizing hormone secretion by pituitary cells during the follicular phase. In contrast to protein and steroid hormones, melatonin secretion in vitro was not affected by TCDD. In conclusion, it was found that the pituitary-ovarian axis in pigs is sensitive to TCDD, and the dioxin exhibited a profound ability to disrupt the ovarian actions of prolactin.