Prokineticins Exert Anti-Apoptotic Effects in the Human Fetal Testis.

During fetal testis development, signaling cascades between various cell types regulate key events such as proliferation, differentiation, cell survival and vascularization, however the signaling pathways regulating these processes are poorly characterized. Prokineticins (PROK1 and PROK2) are recently described ligands that signal interchangeably via two G-coupled protein receptors (PROKR1 and PROKR2). PROK ligands have been demonstrated to regulate proliferation and vascularization in various organs (endometrium, kidney, heart) and are expressed during gonadal development. However, little is known about potential functional roles during fetal testis development. Therefore, the objective of this study was to investigate expression and function of the PROK signalling pathway during human fetal testis development. mRNA expression of PROK ligands and receptors was analysed across a range of gestations: first trimester (8-11 weeks), early second trimester (14-16 weeks), and late second trimester (17-21 weeks; n=4-6 samples per group). The expression of all PROK components increased significantly with gestation. PROK1 was highly expressed in the late second trimester, with relative mRNA levels 100-fold greater than those of PROK2. PROKR1 and 2 mRNA levels also increased significantly during late second trimester compared with first and early second trimester. Immunofluorescence analysis revealed PROK1 was expressed in most compartments of the fetal testis, with particularly intense staining in germ cells (identified by dual staining with VASA). PROK2 staining was confined to interstitial cells and endothelia of blood vessels. Both receptors were localized to the interstitium, but with distinct distributions. This was confirmed by dual staining with the Leydig cell marker 3β-HSD, which indicated that PROKR2 expression was restricted to Leydig cells, whilst PROKR1 was expressed in non-Leydig interstitial cells. To investigate PROK1 function, fragments of human fetal testes (14-19 weeks, n=5) were cultured for 24 hours in the presence/absence of 40 nM PROK1. Immunohistochemical analysis of apoptotic (cleaved-caspase 3-positive) and proliferating (BrdU-positive) cells was determined by image analysis. PROK1 treatment resulted in decreased apoptosis of intra-tubular cells (Sertoli and germ cells) relative to controls, but did not alter cell proliferation in either the tubular or interstitial compartments. These data indicate that prokineticins may be significant regulators of human testicular development. PROK1 signals both through paracrine and autocrine pathways via interstitial cells, which in turn signal to promote intra-tubular cell survival. Future work will investigate possible transcriptional targets of PROK1 in order to determine how it exerts its pro-survival effects. Supported by the University of Edinburgh and the UK Medical Research Council. Ethical approval for use of human tissue was given by Lothian Research Ethics Committee. (platform)