Function of TGF-beta2 in the growth of chicken primordial germ cells and germinal ridge stroma cells during embryonic development.

The development of chicken embryonic gonads is locally regulated by the systematic action of growth factors. Recently, we used suppressive subtraction cloning to identify transforming growth factor beta2 (TGF-beta2) as a growth factor gene preferentially expressed in chicken embryonic ovaries and testes during the early periods of development (Hattori et al. 2002a. Prominent expression of transforming growth factor beta2 gene in the chicken embryonic gonad as revealed by suppressive subtraction cloning. Gen Comp Endocrinol 125:311-316). In the present study, the function of TGF-beta2 in chicken embryonic gonads was investigated using a serum-free culture system in the presence of several growth factors, which may behave as mitogenic or survival factors of primordial germ cells (PGCs). Chicken germinal ridges containing PGCs and germinal ridge stroma cells (GRSCs) were collected from six-day embryos. Addition of TGF-beta2 caused a dose-dependent inhibition of the number of co-cultured PGCs and GRSCs in the presence of these growth factors. However, there was no obvious difference between embryonic ovaries and testes in the effects of TGF-beta2. Immunocytochemical analysis using anti-SSEA-1 antibody revealed that TGF-beta2 induced fragmentation of PGCs. Expression of the TGF-beta2 gene was estimated in the co-cultured PGCs and GRSCs by semi-quantitative RT-PCR. The mRNA level of TGF-beta2 was significantly suppressed in the presence of the growth factors. These results suggest that TGF-beta2 is a gonadal regulator preferentially expressed at the early stages of chicken embryonic development and reduces the growth of PGCs and GRSCs by suppressing proliferation. However, expression of TGF-beta2 may be controlled by mitogenic or survival factors of PGCs.

Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos.

This study was performed to investigate whether the embryonic somatic cells are capable of reconstituting and participating in the embryonic development of chickens to produce chimeras. In order to track the migration behavior of the donor cells, a cell line, originally isolated from an Indian peafowl embryo, was fluorescent-labeled by transfection of the cells with enhanced Green Fluorescent Protein (GFP) and Neomycin resistant (Neo) genes prior to injection into the stage X blastoderm of White Leghorn chickens. The injection was performed with a medium in the presence of 1-5% polyethylene glycol. The development of putative chimeric embryos between the stages three and 24 was examined for GFP expression under fluorescent light. To trace the peafowl cells in the developing chicken embryos, both a species-specific genetic marker originating from the mitochondrial DNA cytochrome b (cyt b) gene and a DNA fragment of GFP gene were used. Of the 185 fertile eggs manipulated, 173 developed into embryos. Fifty-five of them showed positive GFP patches in extra-embryonic tissues, and 15 expressed GFP in intra-embryonic tissues such as those of the head, heart, and gonad. PCR analysis revealed that PCR fragments for the peafowl mitochondrial DNA cyt b and GFP genes were detected in the samples of the GFP positive extra- and intra-embryonic tissues of the chimeras. The present results provide evidence that fluorescent-labeled peafowl embryonic cells carrying GFP and Neo genes are able to participate in the development of chicken embryos to generate chimeras.

Evaluation of deoxyribonuclease activity in seminal plasma of ejaculated chicken semen.

AIM: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents. METHODS: A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 degree C for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light. RESULTS: The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L approximately 5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents. CONCLUSION: The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.

Establishment of feather follicle stem cells as potential vehicles for delivering exogenous genes in birds.

The present study was performed to develop a culture system for feather keratinocyte stem cells to enable the genetic manipulation of endangered avian species. The feather follicle cells were isolated from growing feathers of adult White Leghorn chicken. Leukemia inhibitory factor (LIF) was used to maintain the characterization of the keratinocyte colony-forming cells (KCFCs). The EGFPN1 plasmid DNA retroviral vector was used to deliver Green Fluorescent Protein (GFP) gene, which was introduced to the KCFCs by lipofection. After removal of the fibroblast-like cells, the feather KCFCs attached to the substrate within 24 h of seeding. The cells continued to proliferate for at least 30 days in the presence of LIF. The cell-adhesion molecules such as integrin beta1 and CD49c were immunocytochemically positive in the cells. The KCFCs differentiated into barbular cells and pennaceous feather vane in the LIF-free medium. The GFP gene-transfected KCFCs stably expressed GFP. The present results indicate that the KCFCs derived from feather follicles are closely related to multipotent stem cells. In addition, gene manipulation of such stem cells may be useful for the production of chimera in avian species.

Formalin removal from archival tissue by critical point drying.

The extraction of high-quality nucleic acid may be problematic in formalin-fixed tissues because of cross-linking between proteins and DNA. Old fixed tissue specimens do produce fragmented DNA (<1.2 kb), which is only used for PCR amplification. Here we show that high molecular weight DNA (>194 kb) can be successfully extracted from fixed tissue samples (16-70 years old) by gradual dehydration and critical point drying. The reliability of extracted DNA was measured by its ability to serve as a template for the amplification of mtDNA fragments (403 and 1198 bp) and an nDNA fragment (1844 bp). In addition, fingerprinting analysis was performed using DNA from fixed human tissue to ensure the ability of extracted DNA to hybridize with the DNA probe. DNA derived by this method can be subject to amplification, complete digestion by restriction endonuclease, and hybridization.

Retinoic acid suppression of endothelial nitric oxide synthase in porcine oocyte.

The presence of endothelial nitric oxide synthase (eNOS) has been found in porcine oocytes, but its mRNA and protein levels remain relatively constant during hormonal stimulation. The present study was designed to determine the effect of retinoic acid on eNOS regulation in porcine oocytes during follicle-stimulating hormone (FSH) stimulation. Cumulus-oocyte complexes (COCs), prepared from small antral follicles of immature porcine ovaries, were cultured for 15 h and treated with FSH for an additional 48 h. eNOS mRNA and its protein were analyzed by reverse transcription - polymerase chain reaction and Western blotting, respectively. Retinoic acid had an inhibitory effect on the level of oocyte eNOS mRNA in a dose-dependent manner if COCs were exposed to retinoic acid before FSH stimulation. The inhibition of FSH action was reflected in a decrease in expression of c-fos mRNA. eNOS protein also decreased to approximately 50% of the control after exposure to 10 microM retinoic acid. However, the ability of NO synthesis was abolished in the oocytes prepared from retinoic acid pretreated COCs. These results suggest that retinoic acid has a strong inhibitory action on eNOS mRNA level and NO synthesis in the porcine oocyte.

Preferential expression of ADP-ribosylation factor gene in the chick embryonic gonads.

cDNA cloning from chick embryonic gonads subtracted from tissues of the brain, heart, liver, gizzard, mesonephros and skeletal muscle was performed to identify genes with expression unique to embryonic gonads. Several cDNA clones encoding characterized as well as many uncharacterized genes were obtained. ADP-ribosylation factor (ARF) of these identified genes was preferentially expressed in the chick embryonic ovary and testis as revealed by reverse transcription-polymerase chain reaction analysis. Expression of the ARF was evaluated through embryonic development, but no difference in the transcript (relative to glyceraldehyde-3-phosphate dehydrogenase transcript) was observed between the left and right ovaries, and between the ovary and testis. In addition, the ARF transcript was detected in the gonads on embryonic days 5 to 21. These findings indicate that the ARF is constantly, but preferentially expressed in the embryonic gonads during development.

Prominent expression of transforming growth factor beta2 gene in the chicken embryonic gonad as revealed by suppressive subtraction cloning.

cDNA cloning from chicken embryonic gonad subtracted from tissues of the brain, heart, liver, gizzard, mesonephros, and muscle was performed to identify growth factor genes with expression unique to embryonic ovary and testis. We obtained several cDNA clones encoding known and many unknown genes. We found for the first time that the transforming growth factor beta2 (TGF-beta2) is preferentially expressed in the chicken embryonic ovary and testis. cDNA subtraction cloning with respect to the selective expression of TGF-beta2 in the ovary and testis was further analyzed by reverse transcription-polymerase chain reaction analyses of other embryonic tissues. The ontogeny of TGF-beta2 was evaluated in chicken embryonic ovary and testis. In both testis and ovary, the levels of TGF-beta2 transcripts were high during the early period of embryonic development (E7), gradually decreased until the late embryonic days (E14--E17), and then slightly increased at the last embryonic day (E21). There was no difference in the TGF-beta2 transcripts per RNA between the left and the right ovaries. TGF-beta2 may have a critical role in the regulation of the development of chicken ovarian and testicular germ cells during the embryonic period.