Reproduction Abstracts

Introduction: Neuromedin B (NMB), a highly conserved bombesin-related decapeptide originally isolated from porcine spinal cord, has various physiological effects including the regulation of exocrine and endocrine secretions. The current aims were to investigate whether: (1) NMB is expressed in the bovine ovary (2) NMB or NMB antagonist can modulate ovarian steroidogenesis or proliferation by cultured bovine theca (TC) and granulosa (GC) cells.

Methods: GC (n=40) and TC (n=44) samples retrieved from bovine antral follicles (2–18 mm) were categorized into five size classes. Early, mid and regressing corpora lutea (CL) were also collected (n=17). Total RNA was harvested for qPCR analysis and data were analysed using the ΔΔC_T method using β-actin for normalization. Bovine TC and GC cultured under both non-luteinized (LH or FSH) and luteinized (forskolin) conditions were treated for 5 days with NMB (10⁻¹⁰–10⁻⁶M), NMB antagonist (BIM 23042 10⁻¹⁰–10⁻⁶M) or a combination of the two. Steroid secretion (androstenedione, oestradiol, progesterone) was measured by ELISA and viable cell number determined by neutral red uptake assay. Results are based on 3–8 independent cultures.

Results and Discussion: Two-way ANOVA showed a significant effect of follicle cell-type (P<0.01) and cell-type x follicle category interaction (P<0.05) with NMB expression declining in GC whilst increasing in TC during follicle development. NMB expression also varied according to CL stage (P<0.05). However, TC/GC culture experiments using NMB or its antagonist offered no evidence that NMB has a direct intra-ovarian role to modulate basal or LH-induced TC androgen production, basal or FSH-induced GC oestrogen production or basal or forskolin-induced progesterone production by luteinized TC/GC. However, NMB dose-dependently increased viable cell number by non-luteinized GC (~ 2.3-fold P<0.05) without affecting TC or luteinized TC/GC number. Further experiments are in progress to ascertain whether NMB enhances GC proliferation or reduces cell death.