Abundance Of Estrogen Receptor Alpha (ERα) MRNA In Porcine Throughout The Menstrual Cycle And Cloning Of E Domain CDNA

Xiao Meishan gilts with different ERa genotype (AA, AB, BB) identified by RT-PCR technique, were slaughtered to study reproductive parameters. No difference in ovary weight, uterus weight, ovary length, ovary width and uterine horn length were detected (P>0.05). Estrus cycle length was not affected by ERa genotype (P>0.05). Furthermore, nipple numbers were not different for three genotypes (P>0.05).In order to investigate localization and quantitative expression of ERamRNA in ovary and uterus of porcine on Day 2, 7, 12, 17 throughout menstrual cycle, nonradioactive in situ hybridization using DIG labelled oligonucleotide probes was applied. In the porcine ovary, positive signals for ERa were limited exclusively to the granulose cells of all types of follicles and were present in a few cells of early corpora lutea. Expression of ERamRNA in ovary remained low during the whole oestrus cycle but increased on Day 17. ERa positive signals were mainly detected in glandular epithelial cells, a few part of stromal cells and smooth muscle cells in the uterus. Changes of ERamRNA in porcine endometrium were highly menstrual cycle dependent. Expression of ERamRNA on Day 17 of the oestrus cycle was significant higher than that of Day 12 (P<0.01). E2 concentrations of serum were measured by radioimmunoassy (RIA) on Day 2, 7, 12, 17 of the menstrual cycle. The levels of ERamRNA synchronized with the changes of estrogen with positively relations (P~0.042).Partial E domain cDNA for porcine ERa gene was amplified from Xiao Meishan pig uterus. The cDNA, 546bp, encoded a peptide of 182 amino acids with a predicted molecular weight of 21.1 KDa and potential estrogen binding ability. The amino acid sequence of the porcine ERa E domain was 97.3%, 97.8%, 96.7%, and 95.1% identical to that of human, cattle, sheep and chicken. The porcine ERa gene E domain cDNA was amplified from recombinant plasmid pGEM-T- ERaE using PCR technique, and then transcloned into the prokaryotic expression vector pGEX-6p-l. ERaE fragment wasgenetically inserted at down-stream of the 3'-terminus of the gene encoding for enzyme glutathine S-transferase, which served as a carrier in this expression system. Recombinant expression plasmid pGEX-6p-l-ERaE was constructed and transferred into E.coli BL21. Induced by IPTG, ERaE gene was expressed as a fusion protein linked with the GST designated as GST- ERa E. The results of SDS-PAGE demonstrated that the expected fusion protein with a molecular weight (MW) about 49 KDa, was successfully obtained in E.coli. The fusion protein was detected up to 32% of the total bacterial protein expressed.The results all gained above explored the relationship between ERa genotype and porcine part traits of reproductive organs, obtained the ERamRNA expression pattern in different tissues throughout the menstrual cycle and discovered molecular structure of porcine ERa gene E domain in order to lay the groundwork for marker assisted selection (MAS), clarification of the molecular biological mechanisms of Meishan piglets' high reproductive traits and the research of protein immunity and application of ERa.