Cell type-specific expression and regulation of neuropilin-1 and -2 in the rhesus macaque endometrium suggests roles for VEGF-Neuropilin in endometrial remodeling during the menstrual cycle in primates

Abstract

Objective: Neuropilin-1 and -2 (NP-1/NP-2) are transmembrane receptors that play a role in axonal guidance by binding class III semaphorins, and in angiogenesis by binding vascular endothelial growth factor (VEGF). Although much is known about the biology of VEGF and its cognate receptors, VEGFR1 and VEGFR2, the roles of NP-1 and NP-2 in the primate endometrium are unknown. The goal of this study was to investigate the temporal and spatial expression of NP-1 and NP-2 in the rhesus macaque endometrium during the menstrual cycle. Design: We used in situ hybridization to investigate the cellular localization of NP-1 and NP-2 mRNA in the endometrium of ovarectomized, artificially cycled rhesus macaques. Materials and Methods: Twenty-four ovariectomized rhesus macaques were sequentially treated with estradiol (E) for 14 days and E plus progesterone (P) for 14 days. Then P was withdrawn from all animals to induce menstruation, and E was maintained. Uteri were obtained from one set of animals on days 5–6 (early-proliferative, n = 4), 8-10 (mid-proliferative, n = 3) and on day 14 (late-proliferative, n = 4) after P withdrawal. In the rest of the animals, P was added back after 14 days E treatment and tissue was sampled 3 (early-secretory, n = 4), 7/8 (mid-secretory, n = 4), and 14 (late-secretory, n = 3) days later. In situ hybridization was performed using self-designed probes by RT-PCR and cloning into GEM-T /Teasy vector systems to evaluate the expression and localization of NP-1 and -2 mRNA. Results: NP-1 mRNA was highly expressed in the endothelium of both arteries and veins, but there were no marked changes in its expression during the cycle. However, the luminal epithelium, which also expressed NP-1 mRNA, showed increased expression during the mid- to late-proliferative phase followed by a substantial decrease in expression of this receptor during the luteal phase. NP-1 mRNA was also abundantly expressed in the endometrial stromal cells (but not the glands) with a marked gradient of expression from the surface to the lower-functionalis. Stromal cell NP-1 mRNA changed little during the cycle except for a slight down-regulation during the mid- to late-secretory phase. NP-2 mRNA expression was strictly localized to the endothelium of the veins, was very low during the proliferative phase, and became substantially elevated during the secretory phase. Conclusion: This is the first report in the primate endometrium showing dynamic cell-specific expression and regulation of NP-1 and -2 mRNA during the menstrual cycle. The cyclic expression of NP-1 in the luminal epithelium suggests that VEGF-NP-1 interactions may influence the cyclic changes in the surface properties of this epithelium. In the vascular endothelium, VEGF-neuropilin interactions may result in proliferative effects when only NP-1 is present, and other effects (perhaps related to permeability) when both NP-1 and NP-2 are present during the secretory phase. Stromal NP-1 may mediate other, nonendothelial effects of VEGF through paracrine or autocrine routes. Supported by:German Research Foundation GE 1173/1-1 (AG) and Mellon Foundation (NRN).