Quantitative genotyping by amplifying the polymorphic sequences of Pre-Melanosomal Protein (PMEL17) gene using real-time polymerase chain reaction in chickens.

1. This study was conducted to develop a quantitative genotyping system of chimaeric chickens by real-time PCR. 2. The polymorphisms in exons 7 and 11 of PMEL17 gene, which is one of the major genes affecting plumage colour, were identified from White Leghorn, Barred Plymouth Rock and Rhode Island Red chickens. 3. Quantitative genotyping was successfully performed by real-time PCR using polymorphic sequence-specific TaqMan Probes. 4. This methodology can support future research of germline chimaeric chickens as well as the application of germ cell transfer technique.

Effect of soft X-ray irradiation on the migratory ability of primordial germ cells in chickens.

1. The present study was conducted to elucidate the effect of soft X-ray irradiation on the migratory ability of primordial germ cells (PGCs) to the germinal ridges of chicken embryos. 2. PGCs (Barred Plymouth Rock, BPR) were isolated from embryonic blood and irradiated with soft X-rays for 1-10 min. Then, the PGCs were transfected in vitro with GFP gene by lipofection. The manipulated PGCs were transferred to recipient embryos (White Leghorn, WL) and migration to the germinal ridges was analysed by examining GFP gene expression in the gonads of recipient embryos under UV light at x40 magnifications. The expression of GFP gene was detected in all the gonads of recipient embryos examined up to 10.5 d of culture. 3. Migration of PGCs irradiated with soft X-rays to the germinal ridges was also confirmed by detecting a single nucleotide polymorphism in the D-loop region of the mitochondrial DNA of BPR and WL chickens. Freshly collected PGCs (BPR) were transferred to the bloodstream of recipient embryos (WL). The fate of the transferred donor PGCs was traced by detecting the single nucleotide polymorphism in the D-loop region of the mitochondrial DNA in BPR and WL used in this study. Transferred donor PGC-derived cells were detected in all the gonads of 17-d cultured embryos by PCR. 4. The results suggest that PGCs irradiated with soft X-rays still retain the ability to migrate to the germinal ridges of recipient embryos.

Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR.

1. The present study was carried out to determine whether primordial germ cells isolated from embryonic blood can enter the bloodstream and successfully migrate to the germinal ridges of recipient embryos after transfer to stage X blastoderms, and also whether they can differentiate into blood cells, as is suggested in mice. 2. Primordial germ cells were transfected in vitro by lipofection and then transferred to stage X blastoderms. The introduced GFP gene was efficiently expressed in the gonads of 6-d incubated embryos. 3. Freshly collected primordial germ cells were transferred to stage X blastoderms. The fate of the transferred primordial germ cells was traced by detecting the single nucleotide polymorphism in the D-loop region of the mitochondrial DNA in White Leghorn and Barred Plymouth Rock chickens used in this study. The transferred donor primordial germ cell-derived cells were detected in the gonads, but not in the blood cells, of 17-d incubated embryos by PCR. 4. This procedure for primordial germ cell manipulation could provide a novel method of producing germline chimaeric chickens. 5. In conclusion, our findings indicate that primordial germ cells isolated from embryonic blood can migrate to the germinal ridges of recipient embryos after being transferred to stage X blastoderms. Although these transferred primordial germ cells differentiated into germ cells, no differentiation into blood cells was observed.

Introduction of exogenous DNA into gonads of chick embryos by lipofection and electroporation of stage X blastoderms in vivo.

1. In order to introduce exogenous DNA into gonads of chick embryos, stage X blastoderms of freshly laid and unincubated eggs were transfected by lipofection and electroporation in vivo. 2. The introduced DNA, green fluorescence protein (GFP) gene, was efficiently expressed in the blastoderms incubated for 24 h (78.8%, 78/99). 3. The GFP gene was present in most of the embryonic bodies and extra-embryonic membranes died by d 10 of incubation, when analysed by polymerase chain reaction. On d 16 to 20 of incubation, the GFP gene was detected in 7.0 to 20.9% of embryos in the heart, liver, stomach and brain, but not in the sartorius muscle. For the gonads, the GFP gene was detected in 22.2% (6/27) of the testes and 6.3% (1/18) of the ovaries examined. 4. These results suggest that it is possible to introduce exogenous DNA into gonads of chick embryos by lipofection and electroporation of stage X blastoderms in vivo.

Cloning of porcine IGF1 receptor cDNA and detection of sequence polymorphisms using RT-PCR.

A cDNA encoding the porcine type 1 insulin-like growth factor receptor (IGF1R) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The sequence of a 4.2-kb product was determined and had an open reading frame, encoding 1367 amino acids with 98.1 and 95.2% sequence similarity to the human and rat IGF1R, respectively. In the comparison of RT-PCR derived IGF1R sequences from 12 unrelated pigs, 12 silent sequence variants were found.

Localization of primordial germ cells or their precursors in stage X blastoderm of chickens and their ability to differentiate into functional gametes in opposite-sex recipient gonads.

This study was performed to determine the distribution of primordial germ cells and their precursors in stage X blastoderm of chickens. The blastoderm (Barred Plymouth Rock chickens) isolated from the yolk was separated into three portions: the central disc, the marginal zone and the area opaca. The dissociated blastodermal cells derived from the central disc, marginal zone and area opaca were transferred into a recipient blastoderm (White Leghorn chicken) from which a cell cluster was removed from the centre of the central disc. The manipulated embryos were cultured in host eggshells until hatching. The chicks were raised until sexual maturity and test mated with Barred Plymouth Rock chickens to assess the donor cell contribution to the recipient germline. Germline chimaeric chickens were produced efficiently (46.7%, 7/15) when the blastodermal cells derived from the central disc were transferred into recipient embryos of the same sex, whereas no germline chimaeric chickens were produced when the blastodermal cells derived from the marginal zone or area opaca were transferred into recipient embryos of the same sex (0/12). Germline chimaeric chickens were also produced by transfer of blastodermal cells derived from the central disc (6.7%, 1/15), marginal zone (10.0%, 1/10) or area opaca (11.1%, 1/9) into recipient embryos of the opposite sex. It is concluded that primordial germ cells are induced during or shortly after stage X and that the cells derived from the central disc have the highest potential to give rise to germ cells. Cells derived from the marginal zone and area opaca can also give rise to germ cells, although the frequency is low.

Separation and assay methods for melatonin and its precursors.

Melatonin is an indoleamine hormone that is synthesized from tryptophan via 5-hydroxytryptophan, serotonin and N-acetylserotonin in the vertebrate pineal gland. Many chromatographic and non-chromatographic techniques have been developed and improved for the determination and measurement of melatonin and its related indoleamines. At present, gas chromatography with mass spectrometry and reversed-phase high-performance liquid chromatography with fluorescence or electrochemical detection are widely used for indoleamine determinations in the pineal gland. This review will deal with methods for the separation and determination of the melatonin and its related indoleamines.

Differentiation of donor primordial germ cells into functional gametes in the gonads of mixed-sex germline chimaeric chickens produced by transfer of primordial germ cells isolated from embryonic blood.

This study was carried out to elucidate whether primordial germ cells, obtained from embryonic blood and transferred into partially sterilized male and female recipient embryos, could differentiate into functional gametes and give rise to viable offspring. Manipulated embryos were cultured until hatching and the chicks were raised until maturity, when they were mated. When the sex of the donor primordial germ cells and the recipient embryo was the same, 15 out of 22 male chimaeric chickens (68.2%) and 10 out of 16 female chimaeric chickens (62.5%) produced donor-derived offspring. When the sex of the donor primordial germ cells and the recipient embryo was different, 4 out of 18 male chimaeric chickens (22.2%) and 2 out of 18 female chimaeric chickens (11.1%) produced donor-derived offspring. The rates of donor-derived offspring from the chimaeric chickens were 0.6-40.0% in male donor and male recipient and 0.4-34.9% in female donor and female recipient. However, the rates of donor-derived offspring from the chimaeric chickens were 0.4-0.9% in male donor and female recipient and 0.1-0.3% in female donor and male recipient. The presence of W chromosome-specific repeating sequences was detected in the sperm samples of male chimaeric chickens produced by transfer of female primordial germ cells. These results indicate that primordial germ cells isolated from embryonic blood can differentiate into functional gametes giving rise to viable offspring in the gonads of opposite-sex recipient embryos and chickens, although the efficiency was very low.

Indoleamine metabolism in the pineal gland of the Chinese hamster, Cricetulus griseus.

Day- and nighttime contents of pineal melatonin and other indoleamines were measured in the Chinese hamster, Cricetulus griseus. The hamsters were inbred in our colony and kept in LD 14:10. Absolute levels of pineal melatonin and N-acetylserotonin contents were extremely low, but showed a significant circadian rhythm with a minor peak during the light period. Pineal hydroxytryptophan and serotonin (5HT) contents were also found to be higher in the light period. The activity of N-acetyltransferase in the pineal was higher in the light period than in the dark period, whereas there was no difference in hydroxyindole-O-methyltransferase activity between the light and dark periods. The uptake level of tryptophan into the pineal gland was higher in the light period than in the dark period. These results suggest that the 5HT synthesis pathway and/or tryptophan uptake may be involved in forming the circadian rhythm of 5HT content in the Chinese hamster pineal gland.

The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken.

A novel system has been developed to determine the origin and development of primordial germ cells (PGCs) in avian embryos directly. Approximately 700 cells were removed from the center of the area pellucida, the outer of the area pellucida, and the area opaca of the stage X blastoderm (Eyal-Giladi and Kochav, 1976; Dev Biol 49:321-337). When the cells were removed from the center of the area pellucida, the mean number of circulating PGCs per 1 microliter of blood was significantly decreased to 13 (P < 0.05) in the embryo at stage 15 (Hamburger and Hamilton, 1951: J Morphol 88:49-92) as compared to intact embryos of 51. When the removed recipient cells from the center of the area pellucida were replenished with 500 donor cells, no reduction in the PGC number was observed. The removal of cells from the outer of area pellucida or from the area opaca had no effect on the number of PGCs. When another set of the manipulated embryos were cultured ex vivo to hatching and reared to sexual maturity, the absence of germ cells and the degeneration of seminiferous tubules were observed in resulting chickens derived from the blastoderm from which the cells were removed from the center of the area pellucida. Chimeric embryos produced by the male donor cells and the female recipient contained the female-derived cells at 97.2% in the whole embryo and 94.3% in the erythrocytes at 5 days of incubation. At 5-7 days of incubation, masculinization was observed in about one half of the mixed-sex embryos. The proportions of the female-derived cells in the whole embryo and in the erythrocytes were 76.5% and 80.2% at 7 days to 55.7% and 62.5% at 10 days of incubation, respectively. When the chimeras reached their sexual maturity, they were test mated to assess donor contribution to their germline. Five of six male chimeras (83%) and three of five female chimeras (60%) from male donor cells and a female recipient embryo from which 700 cells at the center of area pellucida were removed were germline chimeras. Three of the five male germline chimeras (60%) and one of the three female germline chimeras (33%) transmitted exclusively (100%) donor-derived gametes into the offspring. When embryonic cells were removed from the outer of area pellucida or area opaca, regardless of the sex combination of the donor and the recipient, the transmission of the donor-derived gametes was essentially null. The findings in the present studies demonstrated, both in vivo and in vitro, that the PGCs originate in the central part of the area pellucida and that the developmental fate to germ cell (PGCs) had been destined at stage X blastoderm in chickens.

Chromosome localization of the mouse zonadhesin gene and the human zonadhesin gene (ZAN).

Zonadhesin is a recently discovered sperm membrane protein that binds to the extracellular matrix of the egg in a species-specific manner. The gene for zonadhesin maps to chromosome 5 in the mouse (Zan) and to chromosome 7 in the human (ZAN) in a position near the gene for the egg extracellular matrix protein ZP3.

Simultaneous determination of serotonin, N-acetylserotonin and melatonin in the pineal gland of the juvenile golden hamster by high-performance liquid chromatography with electrochemical detection.

A simple and simultaneous determination of melatonin and its precursors, serotonin (5-HT) and N-acetylserotonin, was achieved by reversed-phase high-performance liquid chromatography with electrochemical detection. The addition of an ion-pairing agent, sodium 1-octanesulfonate, to the chromatographic mobile phase caused an increase of the retention time of 5-HT, and resulted in the successful simultaneous resolution of these three indoleamines. This method was used to quantitate these indoleamines in the pineal gland of juvenile golden hamsters.

Survey on swine SINEs (PRE-1) as candidates for SSCP markers in genetic linkage analysis.

Of 310 random cosmid clones, 216 were positive for PRE-1 sequences by Southern hybridization. Thirty nine sub-fragments positive for the PRE-1 sequences were cloned from independent cosmid clones, and sequenced, with 17 complete PRE-1 elements found. Seven PRE-1 loci were amplified by polymerase chain reaction using genomic DNA of 12 unrelated pigs as template. The amplified fragments were then subjected to an analysis of single strand conformation polymorphism, with all the loci being polymorphic.

Localization and characterization of phosphatidylcholine in sea urchin spermatozoa.

Sea urchin spermatozoa obtain energy for movement through oxidation of endogenous phospholipids, particularly phosphatidylcholine (PC). This study was undertaken to determine the localization of PC available for utilization in energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Following incubation with sea water, the PC content in sperm heads decreased significantly, while that in sperm tails did not change. PC was abundant in sperm heads, particularly the midpieces. PC composed of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in the midpieces PC to be unsaturated. Phospholipase A2 activity was also distributed in sperm heads, particularly the midpieces. It thus appears that PC as a substrate for energy metabolism is located in the midpieces of sea urchin spermatozoa.

The crab-eating frog Rana cancrivora: serum mineral concentrations and histology of the ultimobranchial gland and the parathyroid gland.

In the crab-eating frog Rana cancrivora, taken from a full-strength seawater pond, the in situ levels of serum Na, Cl, K, Ca, Mg, Pi, urea, and osmolarity were examined. The levels were higher than those usually reported for freshwater anurans. However, values for the monovalent salts, urea, and osmolarity were lower than those reported by Gordon et al. (1961, J. Exp. Biol. 38, 659-678) following immersion of this species in 80% seawater for 7 days. The histological features of the ultimobranchial gland and parathyroid gland were coincident with those known in usual freshwater frogs, in spite of the peculiarity of the habitat of this species. The ultimobranchial gland was composed of a single follicle or multiple follicles. In most parenchymal cells, immunoreactive calcitonin was detected using the PAP method with anti-salmon calcitonin antiserum. In the parathyroid gland, cells in the central part of the gland were smaller than those in the peripheral part.

The influence of an egg-associated peptide on energy metabolism in sea-urchin spermatozoa: the peptide stimulates preferential hydrolysis of phosphatidylcholine and oxidation of fatty acid.

A study was made of the effects of a sperm-activating peptide (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) on energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. The swimming activity and respiratory rate in slightly acidic seawater (pH 6.6) have been shown to be somewhat less than in normal seawater (pH 8.2). Little change occurred in sperm lipid levels during incubation in seawater at pH 6.6. The addition of SAP-I to seawater at pH 6.6 enhanced the consumption of endogenous phosphatidylcholine (PC), with no change in the levels of other lipids. SAP-I also caused increase in 14CO2 production from exogenous [1-14C]oleic acid following incubation of spermatozoa at pH 6.6. However, the stimulated levels of PC consumption and fatty acid oxidation with SAP-I at pH 6.6 did not exceed those at pH 8.2. At pH 8.2, SAP-I had no effect on PC metabolism. Activities of phospholipase A2 and fatty acid oxidation were markedly influenced by pH and increased at pH exceeding 7. SAP-I is thus concluded to stimulate sea-urchin sperm energy metabolism which depends on the oxidation of endogenous PC. It follows from these results that PC metabolism is activated following increase in the intracellular pH of spermatozoa.