Anna Hrabia

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.

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.

Growth hormone production and role in the reproductive system of female chicken

The expression and role of growth hormone (GH) in the reproductive system of mammals is rather well established. In birds the limited information thus far available suggests that GH is an endocrine or paracrine/autocrine regulator of ovarian and oviductal functions too. GH and its receptors are expressed in all compartments of the ovary and oviduct and change accordingly to physiological state. The intra-ovarian role of GH likely includes the regulation of steroidogenesis, cell proliferation and apoptosis, the modulation of LH action and the synthesis of IGFs (insulin-like growth factors). In the oviduct, GH is also involved in the regulation of oviduct-specific protein expression. The present study provides a review of current knowledge on the presence and action of GH in the female reproduction, in which it is likely that act in endocrine, autocrine or paracrine mechanisms.

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.

Preparation and characterization of recombinant chicken growth hormone (chGH) and its putative antagonist chGH G119R mutein

Abstract:  Synthetic cDNA of chicken GH (chGH) and its G119R mutein was synthesized after being optimized for expression in E. coli. The respective cDNAs were inserted into expression vector, expressed and found almost entirely in the insoluble inclusion bodies (IBs). The IBs were isolated, the proteins solubilized in 4.5 M urea, at pH 11.3 in presence of cysteine, refolded, and purified to homogeneity by anion‐exchange chromatography on Q‐Sepharose. The overall yields were 400 to 500 mg from 5 L of fermentation. Both proteins were > 98% pure, as evidenced by SDS‐PAGE, and contained at least 95% monomers, as documented by gel‐filtration chromatography under non‐denaturing conditions. Circular dichroism analysis revealed that both proteins have identical secondary structure characteristic of cytokines, namely > 50% of alpha helix content. Chicken GH was capable of forming a 1:2 complex with recombinant oGH receptor extracellular domain, but its affinity, as determined by RRA, was 11‐fold lower than that of ovine GH (oGH). Correspondingly, its bioactivity, assessed using FDC‐P1 3B9 cells stably transfected with rabbit GHR, was 30–40‐fold lower, whereas chGH G119R mutant did not bind to oGHR‐ECD and was devoid of any biological activity in FDC‐P1 3B9 cells. However, in binding experiments that were carried out using chicken liver membranes, both oGH and chGH showed similar IC50 values in competition with 125I‐oGH, while the IC50 of G119R mutein was 10‐fold higher. These results emphasize the importance of species specificity and indicate the possibility of antagonistic activity of chGH G119R.

Comparison of in vitro bioactivity of chicken prolactin and mammalian lactogenic hormones.

Recombinant chicken prolactin, expressed in Escherichia coli as an unfolded protein, was successfully refolded and purified to homogeneity as a monomeric protein. Its biological activity was evidenced by its ability to interact with rabbit prolactin receptor extracellular domain and stimulate prolactin receptor-mediated proliferation in three cell types possessing mammalian prolactin receptors. Chicken prolactin activity in those assays was 20-100-fold lower than that of mammalian lactogenic hormones, likely due to lower affinity for mammalian prolactin receptors and not to improper refolding, because in two homologous bioassays, chicken prolactin activity was equal to or higher than that of ovine prolactin and the CD spectra of chicken and human prolactin were almost identical. Our results using seven mammalian lactogenic hormones from five species in three bioassays revealed the major role of species specificity in testing biological activity in vitro. Heterologous bioassays may be misleading and homologous assays are strongly recommended for predicting the activity of species-specific lactogenic hormones in vivo.

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.

Localization of apoptotic and proliferating cells and mRNA expression of caspases and Bcl-2 in gonads of chicken embryos.

The aim of the present study was to analyze participation of apoptosis and proliferation in gonadal development in the chicken embryo by: (1) localization of apoptotic (TUNEL) and proliferating (PCNA immunoassay) cells in male and female gonads and (2) examination of mRNA expression (RT-PCR) of caspase-3, caspase-6 and Bcl-2 in the ovary and testis during the second half of embryogenesis and in newly hatched chickens. Apoptotic cells were found in gonads of both sexes. At E18 the percentage of apoptotic cells (the apoptotic index, AI) in the ovarian medulla and the testis was lower (p<0.05) than in the ovarian cortex. In the ovarian medulla, the AI at E18 was lower (p<0.05) than on E12. In the testis, the AI was significantly lower (p<0.05) at E18 than at E15 and 1D. The percentage of proliferating cells (the proliferation index: PI) within the ovary significantly increased from E15 to 1D in the cortex, while proliferating cells in the medulla were detected only at E15. In the testis, the PI gradually increased from E12 to 1D. The mRNA expression of caspase-3 and -6 as well as Bcl-2 was detected in male and female gonads at days 12 (E12), 15 (E15) and 18 (E18) of embryogenesis and the day after hatching (1D). The expression of all analyzed genes on E12 was significantly higher (p<0.05) in female than in male gonads. This difference was also observed at E15 and E18, but only for the caspase-6. The results obtained showed tissue- and sex-dependent differences in the number of apoptotic and proliferating cells as well as mRNA expression of caspase-3, -6 and Bcl-2 genes in the gonads of chicken embryos. Significant increase in the number of proliferating cells in the ovarian cortex and lack of these cells in the ovarian medulla (stages E12, E18, 1D) simultaneous with decrease in the intensity of apoptosis only in the medulla indicates that proliferation is the dominant process involved in the cortical development, which constitutes the majority of the functional structure of the fully developed ovary. No pronounced changes in the expression of apoptosis-related genes found during embryogenesis suggest that they cannot be considered as important indicators of gonad development. The molecular mechanisms of the regulation of balance between apoptosis and proliferation in developing avian gonads need to be further investigated.

Histamine affects blood flow through the reproductive organs of the domestic hen (Gallus domesticus).

The study was undertaken to determine the effect of histamine on blood flow to the ovary and oviduct in the domestic hen (Gallus domesticus). Cardiac output and blood flow were measured with 86RbCl through the ovarian stroma, white ovarian follicles, yellow preovulatory follicles, postovulatory follicles and four oviductal parts: infundibulum, magnum, isthmus and shell gland 1 min and 5 min after histamine treatment. In comparison with control hens which received 0.9% NaCl, histamine significantly increased (by 21.4%) cardiac output exclusively 5 min after its treatment. Blood flow (ml/min/g tissue) through the stroma, the infundibulum and the shell gland was significantly elevated both 1 min (54.3%, 84.3% and 64.2%, respectively) and 5 min (87.1%, 111.5% and 70.4%, respectively) after histamine administration and through the ovarian follicles (29.3%-61.9%) exclusively 5 min after histamine treatment. The increase in blood flow through the ovarian stroma, follicles and the oviductal parts following the administration of histamine was not the result of increased cardiac output but the consequence of local histamine action on blood flow through the ovary and oviduct. The results of the present study indicate that histamine, by influencing the hemodynamics of blood vessels and in consequence changing the blood flow through the reproductive organs, participates in the processes taking place in the ovary during growth, maturation and regression of the follicles, and in the oviduct during formation of the egg.