Cloning and expression of bovine prodynorphin cDNA and investigation of its intrapituitary role in the regulation of LH secretion

Honglin Jiang, Purdue University

Abstract

Prodynorphin (proDYN) in the anterior pituitary (AP) gland appears to be processed differently from in the brain, and the proDYN-derived peptides may function differently in the AP than in the brain. The purpose of the present study was to investigate the potential role of proDYN-derived peptides in mediating the regulation of LH secretion by estradiol-17$\beta$ (E2) and luteinizing hormone releasing hormone (LHRH). In the present study, the complete cDNA sequence for bovine proDYN was determined and found to be identical in the AP and brain. However, expression of proDYN in the AP was much lower than in the brain. Exposure of bovine AP cells in primary suspension culture to E2 increased (p $<$ 0.05) the release of ir-DYN in a dosage- and time-dependent manner. However, there was no situation wherein mRNA for or cellular content of proDYN was altered by E2 exposure suggesting that non-transcriptional mechanisms were involved in the E2-induced release of AP ir-DYN from a large intracellular pool. Exposure to E2 for 10 h and 24 h, but not 2 h, also increased (p $<$ 0.05) LHRH-induced release of LH in a dosage-dependent manner. Exposure to relatively low dosages of E2 for 2 h and 10 h, but not 24 h, increased (p $<$ 0.05) the steady-state amount of mRNA for LH-$\beta$ subunit, suggesting that an increase in LH subunit gene expression may contribute to E2-augmented release of LH in response to LHRH but that different mechanisms probably mediate E2 augmentation of LH and ir-DYN release. The amount of mRNA for gonadotropin releasing hormone receptor (GnRH-R) was not altered by the present E2 treatments suggesting that transcriptional activation of GnRH-R was not involved in E2-augmented AP responsiveness to LHRH. Exposure of AP cells to proDYN antisense oligodeoxynucleotide decreased (p $<$ 0.01) LHRH-induced release of LH, but not expression of LH-$\beta$ subunit, synthesis or basal release of LH suggesting that a component(s) of LHRH signal transduction may be influenced by proDYN antisense treatment. The specificity of this proDYN antisense effect was also evaluated and discussed.

Degree

Ph.D.

Advisors

Malven, Purdue University.

Subject Area

Molecular biology|Anatomy & physiology|Animals|Neurology

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