Cloning of porcine signal transducer and activator of transcription 3 cDNA and its expression in reproductive tissues.
The signal transducer and activator of transcription 3 (Stat3) protein is a member of the Stat family that has a variety of biological functions including cell growth, anti-apoptosis, and cell motility, depending on the cell type and stimulus. Recent studies have suggested that Stat3 plays an important role in embryo development. Although the Stat3 gene has been cloned in humans, mice, cow, and rats, its sequence in pigs is unknown. In the present study, the 2476 bp Stat3 cDNA was cloned using real time reverse transcriptase (RT)-PCR. Comparison of sequences across species revealed that the porcine Stat3 cDNA is 93 and 90% homologous to human and mouse respectively. To study the expression pattern of Stat3, RNA and protein were isolated from heart, lung, kidney, ovary, oviduct, and uterus tissues. RT-PCR and western blot indicated that Stat3 is expressed in all the tissues tested, and the level of expression is relatively high in tissues from the reproductive system. In addition, immunohistochemistry studies suggested that the Stat3 protein was present in the oocyte, granulosa, theca, and interstitial cells of the ovary, the mucosal folds in the oviduct, and both the epithelium and stromal layers in the endometrium. To study whether Stat3 is functional in responding to growth factor stimulation in the ovary, granulosa cells were isolated from large follicles (>3 mm) and cultured in the presence of epidermal growth factor (EGF; 10 ng/ml) for 5, 10, 15, 30, and 60 min, following which western blots were performed using an antibody against the phosphorylated Stat3. Phosphorylated Stat3 was upregulated following 5 min of EGF challenge and was sustained during the 15-min stimulation, and decreased back to the control level following 60-min stimulation. The translocation of phosphorylated Stat3 from cytoplasm to nucleus following stimulation of EGF was also detected via immunocytochemistry. Our data suggests that Stat3 may play a role in porcine ovarian function.
Wen
Lihua
L
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada.
Craig
Jesse
J
Dyce
Paul W
PW
Li
Julang
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics*; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D005187:Fallopian Tubes / Q000737:chemistry; D005260:Female; D015870:Gene Expression; D006107:Granulosa Cells / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D010053:Ovary / Q000737:chemistry; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050796:STAT3 Transcription Factor / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013552:Swine / Q000378:metabolism*; D014599:Uterus / Q000737:chemistry
D018076:DNA, Complementary; D012333:RNA, Messenger; D050796:STAT3 Transcription Factor; D004815:Epidermal Growth Factor