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Cloning and Characterization of Porcine Ovarian Estrogen Receptor ß Isoforms¹

^a Department of Cell Biology and Neuroscience, University of South Carolina School of Medicine, Columbia, South Carolina 29208

The cDNA for the full-length porcine estrogen receptor ß (ERß) and an alternatively spliced transcript with a deletion of exon 5 (ERß5) was cloned from pig ovary. RNase protection assays revealed that ERß mRNA was expressed in the preovulatory follicles and early, midluteal, and regressing corpora lutea (CL) of eCG ± hCG-primed gilts. ERß and ERß5 transcripts were shown by semiquantitative reverse transcription polymerase chain reaction to be expressed at a ratio of approximately 2:1 in granulosa cells, small, medium, and large antral follicles, and midluteal phase corpora lutea of unprimed animals. Immunoreactive ERß proteins corresponding to the size of in vitro translated ERß and ERß5 were detected by immunoblot. Full-length ERß was detected in granulosa, small, medium, and large antral follicles, and midluteal phase CL of unprimed animals. Putative ERß5 immunoreactive bands were abundant only in granulosa cell extracts. In COS-1 cells, transfected ERß5 had no effect on basal transcription of an estrogen-responsive reporter construct but did repress wild-type ERß transactivation when cotransfected at 10-fold excess plasmid. No repression of ER transactivation was observed. In primary granulosa cell cultures, transfected ERß5 plasmid did not inhibit basal reporter activation. ERß5 was shown by immunofluorescence to localize to the nucleus in transfected COS-1 cells. In vitro translated ERß5 proteins bound estrogen response elements in DNA in electrophoretic mobility shift assays, as indicated by supershift analysis. ERß is abundant in porcine ovary, and a naturally occurring splice variant missing exon 5 may have biological function.

First decision: 25 September 2001.

¹ This work was supported in part by NIH grant HD-38945 (to H.A.L.).

² Correspondence: Holly A. LaVoie, Department of Cell Biology and Neuroscience, Bldg. 1, School of Medicine, University of South Carolina, Columbia, SC 29209. FAX: 803 733 3212; hlavoie{at}med.sc.edu

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