Andrzej Sechman

Influence of triiodothyronine (T3) on secretion of steroids and thyroid hormone receptor expression in chicken ovarian follicles

The present study was designed to (1) assess the role of triiodothyronine (T(3)) with regard to in vitro steroid hormone secretion by chicken ovarian follicles; (2) determine whether T(3) influences the in vivo function of the pituitary-ovarian axis in the hen; and (3) detect expression of thyroid hormone receptor (TR) mRNA in chicken ovarian follicles. In the first experiment, laying hens were decapitated 22.5h before ovulation. White prehierarchical follicles (1-8mm) and fragments of theca and granulosa layers of the 3 largest yellow preovulatory follicles F3-F1 (22-35mm) were incubated in a medium supplemented with T(3) (0, 0.1, 1, 10, 100, or 1000ng/mL) or ovine luteinizing hormone (LH) (10ng/mL) in combination with doses of T(3) (1, 10, and 100ng/mL). Triiodothyronine decreased basal and LH-stimulated estradiol secretion by white follicles and the theca layer of all preovulatory follicles. On the other hand, it increased progesterone secretion by F2 and F1 follicles. In the second experiment, hens were injected 1h after ovulation with saline (control) or T(3) (10microg/100g body weight, intraperitoneally). Results indicated that exogenous T(3) decreased plasma concentrations of LH and estradiol and increased plasma concentrations of progesterone. In the third experiment, using reverse transcription polymerase chain reaction (RT-PCR) analysis, expression of thyroid hormone receptor (TRalpha and TRbeta0), mRNA was detected in all of the ovarian compartments. The expression of TRalpha mRNA was relatively greater in comparison with TRbeta0. There were no differences between white ovarian follicles in the expression of TRalpha and TRbeta0 mRNA. A considerably higher TRalpha and lower TRbeta0 expression was detected in the granulosa layer of preovulatory follicles in comparison with the theca layer. In conclusion, the data indicate that thyroid hormones acting via nuclear receptors are involved in regulation of the pituitary-ovarian axis and processes associated with follicle growth and maturation.

The role of thyroid hormones in regulation of chicken ovarian steroidogenesis.

In all vertebrates, including birds, the normal development of the ovary and ovarian follicles is under the regulatory influence of hormones produced by the reproductive axis. In recent years, it has become clear that in birds an adequate level of thyroid hormones (THs), i.e. thyroxine (T4) and triiodothyronine (T3), in blood circulation is of primary importance for normal female reproductive functions. In avian species, characterized by seasonal reproduction, THs are involved in the photoperiodic regulation of reproduction acting at the mediobasal hypothalamus. In domestic fowl, where the seasonality of reproduction has been eliminated, the role of THs in ovarian function is not fully elucidated. Recent studies have revealed that ovarian follicles of the laying hen express mRNAs of TH nuclear receptors (TRα and TRβ0) as well as integrin (αVβ3) plasma membrane receptors, indicating genomic and nongenomic action of THs in the chicken ovary. In vivo experiments carried out on laying hens have showed that the bolus injection of T3 decreases levels of luteinizing hormone (LH) and estradiol (E2) in blood, and a hyperthyroid state evoked by administration of T3 for few days diminishes LH, E2 and progesterone (P4) levels, reduces the weight of the ovary, induces atresia of preovulatory follicles and eventually causes stoppage of egg laying. In vitro studies have demonstrated that T3 decreases E2 secretion from white nonhierarchical follicles and the theca layer of yellow preovulatory follicles, while on the other hand, it elevates P4 production from the granulosa layer of these follicles. These effects have been associated with steroidogenic enzyme expression and cyclic AMP synthesis. This review summarizes the current knowledge concerning the role of THs in regulation of steroidogenesis in chicken ovarian follicles.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on secretion of steroids and STAR, HSD3B and CYP19A1 mRNA expression in chicken ovarian follicles

The aim of the study was to investigate the in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid hormone secretion by chicken ovarian follicles and mRNA expression of genes involved in steroids synthesis. In the first in vitro experiment, white (WF) and yellowish (YF) follicles and fragments of the theca (TL) and granulosa (GL) layers of the 3 largest yellow preovulatory follicles (F3-F1) were incubated in a medium supplemented with TCDD (0.01-100nM). In the second experiment, they were incubated in a medium with TCDD (10nM), ovine LH (10ng/mL; oLH) or a combination of oLH (10ng/mL) and TCDD (10nM). It was found that TCDD decreased estradiol (E2) secretion by WF and the TL of all preovulatory follicles, testosterone (T) secretion by WF, YF, and the TL of F2 and F1 follicles, and progesterone (P4) secretion by the GL of the preovulatory follicles. It also reduced oLH-stimulated E2 and P4 secretion by all examined follicles and T by WF. Real-time qPCR revealed that TCDD affected basal and oLH-stimulated expression of STAR, HSD3B and CYP19A1 mRNAs in all investigated ovarian follicles. In conclusion, the data obtained indicate that TCDD inhibits sex steroids secretion from chicken ovarian follicles. The effects of TCDD depend on its concentration and the stage of follicle maturation, and are associated with modulation of STAR, HSD3B and CYP19A1 mRNAs expression. These results indicate that the exposure of the laying hen to TCDD by influence of ovarian steroidogenesis may impair the selection of white follicles to preovulatory hierarchy and disturb their growth and preovulatory maturation.

The expression of pituitary FSHβ and LHβ mRNA and gonadal FSH and LH receptor mRNA in the chicken embryo

SUMMARY In avian species, synthesis of sex steroids by embryonic gonads is regulated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In order to elucidate the role of the two gonadotropins in gonadal axis development during the second half of chicken embryogenesis, pituitary expression of LH β subunit (LHβ) and FSH β subunit (FSHβ) mRNAs as well as gonadal expression of LH and FSH receptor (LHR and FSHR) mRNAs were determined on days 11 (E11) and 17 (E17) of embryonic development and after hatching (D1). In the pituitary of the female embryo, the gene expression of FSHβ was the lowest on E11 and increased on E17. In the male pituitary, the expression of FSHβ did not differ among the studied days. The FSHβ mRNA expression on E11 was higher in the male than in the female pituitary gland. The expression of LHβ mRNA in the female pituitary increased on D1 in comparison to E11. In the male pituitary gland, the expression of LHβ gene was relatively constant. The expression of mRNA encoding FSHR in the ovary increased on E17, while in testes it did not differ among the studied days. There were no significant alterations in LHR gene expression in the ovary or in the testes in the examined period however, the gene expression on E17 was higher in the ovary than in the testes. We observed positive correlations between the pituitary FSHβ mRNA expression and ovarian expression of FSHR mRNA (r=0.63; p

Effect of tamoxifen on sex steroid concentrations in chicken ovarian follicles

The aim of the present investigation was to study the effect of tamoxifen (TAM), an oestrogen receptor antagonist, on the concentrations of sex hormones in chicken ovarian follicles. The experiment was carried out on Hy-line hens which were randomly divided into two groups (control and experimental). TAM was given at a dose of 4 mg/hen (per os) at first once a day for 7 consecutive days, and subsequently four times a day for the next 6 days. Control hens received placebo. Birds were killed on the day after the last TAM treatment. From the dissected ovaries the following compartments were isolated: stroma with follicles < 1 mm, white non-hierarchical (1-4 mm and 4-8 mm) and yellow hierarchical follicles (F6-F1; 18-35 mm). The concentrations of the sex steroids progesterone (P4), testosterone (T) and oestradiol (E2) in the ovarian follicles were determined by radioimmunoassay. In the TAM-treated group, a gradual decrease in egg-laying rate was observed from the 4th day of the experiment. Eventually, egg laying stopped entirely on the 12th day of the experiment. TAM significantly decreased the weight of the ovary and affected the sex hormone concentrations in the ovarian follicles. Following TAM treatment (1) a significant increase in E2 and T concentrations in the stroma, white follicles and the F4 and F1 follicles, (2) a significant decrease in E2 and T concentrations in the F2 follicle, and (3) a significant decline of P4 in the F4 to F1 follicles were observed. The results indicate that the blockade of oestrogen receptors by TAM significantly modulates the process of chicken ovarian steroidogenesis.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid concentrations in blood and gonads of chicken embryo

The aim of the present study was to examine the effect of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) injected at the early stage of incubation on hormonal activity of the chicken ovary and testis during the second half of embryogenesis measured by steroid concentrations in blood plasma and gonads. The effect of TCDD on hatchability was also evaluated. In the first experiment TCDD was administrated to chicken eggs on day 7 of embryogenesis (E7) at doses of 0 (control), 2.5, 5 and 10ng/egg dissolved in 50μl of peanut oil. Blood plasma for estradiol (E2) and testosterone (T) determination was collected from embryos at E14, E18, E20 and at hatching (D1). In the second experiment TCDD at doses of 0 (control), 2.5, 5 and 10ng/egg dissolved in 50μl of DMSO was injected on E6. Blood plasma and gonads were collected on the day of hatching for progesterone (P4), T and E2 concentrations measured by means of the RIA method. It was found that TCDD injection: (1) increased the level of T and decreased the level of E2 in the plasma of female chicken embryos during the second half of embryogenesis, while on the other hand in male embryos the effect of TCDD was opposite, (2) increased T and E2 concentrations in plasma of newly hatched female chickens with a concomitant decrease of these hormones in the ovary; P4 in the ovary was elevated by TCDD, (3) decreased P4 and T in plasma and testes, whereas increased E2 concentration in the plasma of newly hatched male embryos (E2 concentration in the testes was below the sensitivity of the applied RIA method), and (4) decreased hatchability at all examined doses. The results obtained clearly demonstrate that exposure to TCDD at an early stage of embryogenesis affects steroid production and secretion by chicken gonads. The effect of TCDD depends on (1) the applied dose, (2) the day of embryogenesis, and (3) the sex. It is suggested that dioxins are potent modulators of the process of steroidogenesis in the chicken gonads. Moreover, the data obtained indicate that dioxins exert an effect on embryo development and the hatching process.

Expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1) and CYP1 family monooxygenase mRNAs and their activity in chicken ovarian follicles following in vitro exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The aim of this in vitro study was to determine the effect of TCDD and luteinizing hormone (LH) on mRNA expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1), and the CYP1 family monooxygenases (CYP1A4, CYP1A5, CYP1B1), and to assess the basal and TCDD-induced activity of these enzymes in chicken ovarian follicles. White (WF) and yellowish (YF) prehierarchical follicles and fragments of the theca (TL) and granulosa (GL) layers of the 3 largest preovulatory follicles (F3-F1) were exposed to TCDD (10nM), ovine LH (oLH; 10ng/mL) or a combination of TCDD (10nM) and oLH (10ng/mL), and increasing doses of TCDD (0.01-100nM). AHR1 and ARNT1 mRNA transcripts were found in all examined follicles. The effect of TCDD and oLH on AHR1 and ARNT1 mRNA expression depended on the maturational state of the follicle. CYP1A4 was predominantly expressed in the GL of the F3-F1 follicles; in comparison with the WF, a higher level of CYP1A5 mRNA was found both in the GL and TL of F3-F1 follicles. Alternatively, the highest level of CYP1B1 mRNA was noticed in the WF follicles. In different developmental stages of the follicle TCDD and oLH induced a different CYP1 isoform. TCDD increased EROD and MROD activities in all the investigated ovarian follicles. In conclusion, AHR1 and ARNT1 mRNA expression indicate that the chicken ovary is a target tissue for dioxin and dioxin-like compounds. The expression of CYP1-family genes and TCDD-inducible EROD and MROD activities in ovarian follicles suggest the possibility of xenobiotic detoxification in the chicken ovary.

Plasma thyroid hormones and corticosterone levels in blood of chicken embryos and post hatch chickens exposed during incubation to 1800 MHz electromagnetic field

IntroductionThis study attempted to determine the effect of a 1800 MHz electromagnetic field (EMF) (only carrier frequency) on thyroxine (T4), triiodothyronine (T3) and corticosterone (CORT) concentrations in the blood plasma of chick embryos, and to investigate the effect of electromagnetic field (EMF) exposure during embryogenesis on the level of these hormones in birds that are ready for slaughter.Material and MethodsThroughout the incubation period, embryos from the experimental group were exposed to a 1800 MHz EMF with power density of 0.1 W/m2, 10 times during 24 h for 4 min. Blood samples were collected to determine T4, T3 and CORT concentrations on the 12th (E12) and 18th (E18) day of incubation, from newly hatched chicks (D1) and from birds ready for slaughter (D42).ResultsThe experiment showed that T4 and T3 concentrations decreased markedly and CORT levels increased in the embryos and in the newly hatched chicks exposed to EMF during embryogenesis. However, no changes were found in the level of the analyzed hormones in the birds ready for slaughter. Differences in T4 and T3 plasma concentrations between the EMF-exposed group and the embryos incubated without additional EMF were the highest in the newly hatched chicks, which may be indicative of the cumulative effect of electromagnetic field on the hypothalamo-pituitary-thyroid axis (HPT).DiscussionThe obtained results suggest that additional 1800 MHz radio frequency electromagnetic field inhibits function of HPT axis, however, it stimulates hypothalamo-pituitary-adrenal axis by inducing adrenal steroidogenic cells to synthesize corticosterone. Further investigations are needed to elucidate the mechanisms by which radio EMFs affect HPT and HPA axis function in the chicken embryos.

Effects of PCB 126 and PCB 153 on secretion of steroid hormones and mRNA expression of steroidogenic genes (STAR, HSD3B, CYP19A1) and estrogen receptors (ERα, ERβ) in prehierarchical chicken ovarian follicles

The objective of this study was to assess the in vitro effects of dioxin-like PCB 126 and non-dioxin-like PCB 153 on basal and ovine LH (oLH)-stimulated testosterone (T) and estradiol (E2) secretion and expression of steroidogenic genes (STAR, HSD3B and CYP19A1) and estrogen receptors α (ERα) and β (ERβ) in white (WF) and yellowish (YF) prehierarchical follicles of the hen ovary. Steroid concentrations in a medium and gene expression in follicles following 6h of exposition were determined by RIA and real-time qPCR, respectively. Both PCBs increased basal and oLH-stimulated T secretion by the WF follicles. PCB 126 reduced basal E2 secretion by the WF follicles. PCB 153 elevated but PCB 126 reduced oLH-stimulated E2 secretion by the prehierarchical follicles. PCB 126 increased basal STAR and HSD3B and reduced CYP19A1 mRNA expression in these follicles. PCB 153 increased basal expression of STAR and HSD3B in YF follicles, but diminished HSD3B mRNA levels in the WF. The studied PCBs had an opposite effect on basal and oLH-stimulated CYP19A1 mRNA expression in prehierarchical follicles. Both PCBs modulated basal and inhibited oLH-stimulated ERα and ERβ gene expression in the prehierarchical follicles. In conclusion, data of the current study demonstrate the congener-specific effects of PCBs on sex steroid secretion by prehierarchical follicles of the chicken ovary, which are at least partly related to STAR, HSD3B and CYP19A1 gene expression. It is suggested that PCBs, by influencing follicular steroidogenesis and expression of estrogen receptors, may impair development and selection of yellowish follicles to the preovulatory hierarchy.

Effect of weak electromagnetic field on cardiac work, concentration of thyroid hormones and blood aminotransferase level in the chick embryo

The aim of the study was to determine the effect of alternating electromagnetic field (EMF; 50 Hz frequency, 50 and 100 μT induction) on cardiac work of the chick embryo. Eggs from the experimental groups were exposed to EMF throughout incubation. During the experiment, heart rate (ballistocardiographic method), thyroxine (T4) and triiodothyronine (T3) concentrations, heart weight, ventricle wall thickness, and levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. The results show, for the first time, that the exposure of chick embryos to EMF augments the heart rate, especially from 17 days of incubation. The increased heart rate in the embryos exposed to EMF was associated with considerable increases in plasma T4 and T3 concentrations, which were recorded during the final stage of embryogenesis. The significant effect of the 100-μT field on heart weight and blood AST levels in the embryos suggests that EMF has a direct effect on the physiological function of cardiac muscle.