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.

The expression of pituitary FSHbeta and LHbeta mRNA and gonadal FSH and LH receptor mRNA in the chicken embryo.

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 beta subunit (LHbeta) and FSH beta subunit (FSHbeta) 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 FSHbeta was the lowest on E11 and increased on E17. In the male pituitary, the expression of FSHbeta did not differ among the studied days. The FSHbeta mRNA expression on E11 was higher in the male than in the female pituitary gland. The expression of LHbeta mRNA in the female pituitary increased on D1 in comparison to E11. In the male pituitary gland, the expression of LHbeta 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 FSHbeta mRNA expression and ovarian expression of FSHR mRNA (r = 0.63; p<0.01) as well as between LHbeta mRNA and LHR mRNA in the testes (r=0.65; p<0.01). The reported alterations in gene expression of FSHbeta, LHbeta and their receptors between sexes and among the stages of embryonic development indicate time- and sex-dependent action of gonadotropins in gonads of chicken embryos.

MRNA expression and immunocytochemical localization of leptin receptor in the oviduct of the laying hen (Gallus domesticus).

The role of leptin in female reproduction is fairly well established in mammals, whereas reports concerning leptin action in birds are scarce. The aim of the present study was to detect leptin receptor (LEP-R) mRNA and to localize the leptin receptor protein in the oviduct of laying hens 2h after ovulation by the RT-PCR method and immunocytochemical staining. The RT-PCR reaction demonstrated expression of the long form ofleptin receptor mRNA in all examined oviductal parts (infundibulum, magnum, isthmus and shell gland) and the weakest level was found in the isthmus. The expression of the short isoform was lower than the long form in all examined tissue samples and no differences between oviductal parts were observed. Immunostaining specific for leptin receptor was found in the walls of all examined oviductal parts. The intensity of the immunopositive reaction was the strongest in the epithelium of all examined parts of the oviduct and in the endothelium and muscles of blood vessels. The weakest immunopositive reaction was observed in tubular glands, the connective tissue layer and in circular and longitudinal muscles. The results obtained in this experiment suggest that the oviduct may be a target tissue for leptin, where this polypeptide hormon may participate in egg formation and/or its transport through the oviduct of the domestic hen.

Expression and localization of growth hormone and its receptors in the chicken ovary during sexual maturation.

Roles of pituitary growth hormone (GH) in female reproduction are well established. Autocrine and/or paracrine actions of GH in the mammalian ovary have additionally been proposed, although whether the ovary is an extra-pituitary site of GH expression in the laying hen is uncertain. This possibility has therefore been assessed in the ovaries of Hy-Line hens before (between 10-16 weeks of age) and after (week 17) the onset of egg laying. Reverse transcription/polymerase chain reaction (RT-PCR) analysis has consistently detected a full-length (690 bp) pituitary GH cDNA in ovarian stroma from 10 weeks of age, although GH expression is far lower than that in the pituitary gland or hypothalamus. GH mRNA is also present in small (>1-4 mm diameter) follicles after their ontogenetic appearance at 14 weeks of age and in all other developing follicles after 16 weeks of age (>4-30 mm diameter). Immunoreactivity for GH is similarly present in the ovarian stroma from 10 weeks of age and in small (<4 mm diameter) and large (>4-30 mm) follicles from 14 and 16 weeks of age, respectively. The relative intensity of GH staining in the ovarian follicles is consistently greater in the granulosa cells than in the thecal cells and is comparable with that in the follicular epithelium. A 321-bp fragment of GH receptor (GHR) cDNA, coding for the intracellular domain of the receptor, has also been detected by RT-PCR in the ovary and is present in stromal tissue by 10 weeks of age, in small follicles (<4 mm diameter) by 14 weeks of age, and in larger follicles (>4-30 mm diameter) from 16 weeks. GHR immunoreactivity has similarly been detected, like GH, in the developing ovary and in all follicles and is more intense in granulosa cells than in the theca interna or externa. The expression and location of the GH gene therefore parallels that of the GHR gene during ovarian development in the laying hen, as does the appearance of GH and GHR immunoreactivity. These results support the possibility that GH has autocrine and/or paracrine actions in ovarian function prior to and after the onset of lay in hens.

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

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 IC(50) values in competition with (125)I-oGH, while the IC(50) 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.