Reproduction Abstracts

The pars tuberalis (PT) of the ovine pituitary contains high-density melatonin receptors (MT1) known to participate in the seasonal regulation of prolactin (PRL) output from lactotroph cells in the pars distalis (PD). However, the paracrine mechanism relaying the signal from the PT to the PD remains unresolved. We have recently shown that MT1-positive cells in the PT co-express vascular endothelial growth factor-A isoforms (VEGF₁₆₅/VEGF₁₆₅b), and that VEGF-receptors are expressed in the PD. In this study, individual primary PT and PD cell cultures were generated from ewes during the breeding season (BS) and non-breeding season (NBS). PT cells were treated with: i) media (24 h); ii) BS-like melatonin (16 h:8 h, melatonin:media); iii) NBS-like melatonin (8 h:16 h melatonin:media). Treatments were applied to match the nocturnal rise of endogenous melatonin. PD cells were treated with: i) media (24 h); ii) BS-like VEGF₁₆₅ (16 h:8 h, VEGF₁₆₅:media); iii) NBS-like VEGF₁₆₅ (8 h:16 h, VEGF₁₆₅:media); iv) NBS-like PT-conditioned media; v) BS-like PT-conditioned media; vi) melatonin; vii) TRH; viii) NBS-like conditioned media?anti-VEGF₁₆₅ antibody; and ix) BS-like conditioned media?anti-VEGF₁₆₅b antibody. Cell lysates were obtained after 6 days in culture for quantification of PRL and FSH mRNA by QPCR.

In PT cultures, BS-like melatonin treatment up-regulated VEGF₁₆₅b during both BS and NBS. Conversely, treatment with NBS-like melatonin caused an up-regulation of VEGF_165. Responses to BS and NBS-like melatonin regimens were greatest when melatonin matched the season of application.

In PD cultures, NBS-like VEGF₁₆₅ increased PRL expression, whilst BS-like VEGF₁₆₅ up-regulated FSH. NBS-like conditioned media (NBSCM) resulted in a greater increase in PRL than VEGF₁₆₅ alone. However, NBSCM?anti-VEGF₁₆₅ abolished the NBSCM-mediated PRL rise. Similarly, BS-like conditioned media (BSCM) caused a larger increase in FSH than VEGF₁₆₅ alone, which was blocked when BSCM+anti-VEGF₁₆₅b was applied.

These results reveal that VEGF-A isoforms are involved in relaying the photoperiodic signal from MT1-PT cells to endocrine cells of the ovine PD.