RESUMO
Although platelet-activating factor receptor (PAFr) gene was well characterized in the human, little was known about it in domestic animals. Porcine PAFr gene was mapped using fluorescence in situ hybridization (FISH). The structure of this gene was investigated using a 5' rapid amplification of cDNA ends (RACE) technique. Temporal expression of PAFr and estrogen receptor alpha genes (ER), and distribution of the PAFr transcripts in porcine endometrial and embryonic tissues on days 0, 10, 12, 14, 16, and 18 were analyzed using DNA competitors and reverse transcription and polymerase chain reaction (RT-PCR). The porcine PAFr gene was mapped to SSC6q26-27. Alternative splicing of primary transcripts of the PAFr gene produced two different transcripts. Transcript 1 was expressed in all tissues and cells, and transcript 2 was detected in all tissues but white blood cells. The temporal expression of the PAFr gene in endometrial (P > 0.05) and embryonic (P < 0.05) tissues of pregnant sows increased from day 10 to 16. The temporal expression of ER genes in endometrial tissues of pregnant sows decreased from day 10 to 18 (P < 0.05). In addition, ER expression was detectable in 20-60% of embryonic tissue samples, which generally decreased. In combination with previously obtained data on PAF and estradiol-17beta (E(2)) concentrations in pregnant uterine luminal fluids (pULF), endometrial and embryonic tissues, the present results indicated that the increasing PAFr transcripts were positively associated with increasing levels of PAF. Both ER transcripts and E(2) found in pULF decreased correspondingly from day 13 to 16. These results indicate that via PAFr, PAF could play a dominant role in peri-implantation development in pigs as compared to E(2).
Assuntos
Mapeamento Cromossômico , Embrião de Mamíferos/metabolismo , Endométrio/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Receptores de Superfície Celular/genética , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G , Animais , Receptor alfa de Estrogênio , Feminino , Hibridização in Situ Fluorescente , Glicoproteínas da Membrana de Plaquetas/biossíntese , Glicoproteínas da Membrana de Plaquetas/química , Gravidez , Isoformas de Proteínas , RNA Mensageiro , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/química , Receptores de Estrogênio/biossíntese , Análise de Sequência de DNA , SuínosRESUMO
Four exons of porcine platelet-activating factor receptor (PAFr) gene expressing transcript 1 and transcript 2 were determined previously. In this study, we cloned and sequenced a new exon, which also initiates transcript 2, and determined the order of 5 exons in the PAFr gene. In addition, two other variants of transcript 2 were found, but no additional variants of transcript 1 were found. Transcript 2 has three variants that were detected in porcine tissues other than in white blood cells.
Assuntos
Glicoproteínas da Membrana de Plaquetas/genética , Receptores Acoplados a Proteínas G/genética , Suínos/genética , Animais , Sequência de Bases , Éxons/genética , Dados de Sequência Molecular , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de DNA , Transcrição GênicaRESUMO
After the pig platelet-activating factor receptor (PAFr) gene was cloned and sequenced, the chromosomal location of this gene was studied using a pig/rodent somatic cell hybrid panel containing 27 cell lines. The results indicated that the pig PAFr gene is located on SSC6q22-23. Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is thought to be very important in the animal reproductive processes. Its function is mediated through a membrane-bound receptor. Pig PAFr mRNA distribution in different tissues was tested using reverse transcription and PCR (RT-PCR) reactions. All tissues examined expressed PAFr. Using a pig PAFr gene DNA competitor, PAFr expression was quantificated. The pig PAFr mRNA expression level was estimated to be from 1 x 10(2) to 1.2 x 10(4) copies of complementary DNA (cDNA) per 50 ng of total RNA. The highest level was found in lung, and the lowest in the skeletal muscle. These results demonstrated that PAFr was differentially expressed in pig tissues.
