Helena E. Paczoska-Eliasiewicz

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.

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.

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 is involved in the regulation of ovarian steroidogenesis in the domestic hen ( Gallus domesticus )

Histamine is present in all compartments of the chicken ovary (Paczoska-Eliasiewicz and Rzasa, 1998; Paczoska-Eliasiewicz et al., 1999). The concentration of ovarian histamine changes significantly during sexual maturation (PaczoskaEliasiewicz and Rzasa, 1997), lay (PaczoskaEliasiewicz and Rzasa, 1998) and the pause in lay (Paczoska-Eliasiewicz et al., 1998). In the laying hen, within the hierarchy of yellow pre-ovulatory F7-F1 follicles, the initial decrease in histamine concentration from the F7 to the F4 follicle is followed by the gradual increase as follicle matures, reaching the highest level in F1 follicle. After ovulation there is a significant decrease in the histamine concentration of the post-ovulatory follicle. This suggests that ovarian histamine might be involved in follicular maturation and/or ovulation (Paczoska-Eliasiewicz and Rzasa, 1998). As in the mammalian pre-ovulatory follicle, histamine stimulates progesterone synthesis (Schmidt et al., 1987). It seems likely that in birds the action of this amine as an intra-ovarian regulator may occur via its association with steroidogenesis. Therefore, the aim of the present study was to examine the effect of histamine and histamine receptor blockers on in vitro and in vivo steroid production by pre-ovulatory follicles of the domestic hen. Studies were performed on ISA Brown laying hens at 28–36 weeks of age.