The role of the prokineticin 2 pathway in human reproduction: evidence from the study of human and murine gene mutations

Abstract

A widely dispersed network of hypothalamic GnRH neurons controls the reproductive axis in mammals. Genetic investigation of the human disease model of isolated GnRH deficiency has revealed several key genes crucial for GnRH neuronal ontogeny and GnRH secretion. Among these genes, prokineticin 2 (PROK2), and PROK2 receptor (PROKR2) have recently emerged as critical regulators of reproduction in both mice and humans. Both prok2- and prokr2-deficient mice recapitulate the human Kallmann syndrome phenotype. Additionally, PROK2 and PROKR2 mutations are seen in humans with Kallmann syndrome, thus implicating this pathway in GnRH neuronal migration. However, PROK2/PROKR2 mutations are also seen in normosmic GnRH deficiency, suggesting a role for the prokineticin signaling system in GnRH biology that is beyond neuronal migration. This observation is particularly surprising because mature GnRH neurons do not express PROKR2. Moreover, mutations in both PROK2 and PROKR2 are predominantly detected in the heterozygous state with incomplete penetrance or variable expressivity frequently seen within and across pedigrees. In some of these pedigrees, a "second hit" or oligogenicity has been documented. Besides reproduction, a pleiotropic physiological role for PROK2 is now recognized, including regulation of pain perception, circadian rhythms, hematopoiesis, and immune response. Therefore, further detailed clinical studies of patients with PROK2/PROKR2 mutations will help to map the broader biological role of the PROK2/PROKR2 pathway and identify other interacting genes/proteins that mediate its molecular effects in humans.

Fig. 1.

Genetic causes of isolated GnRH deficiency in a large series of patients at the Massachusetts General Hospital. A, Percentage of patients (n = 397) with isolated GnRH deficiency that harbor rare sequence variants of known genes. B, Histogram of percentage of patients with isolated GnRH deficiency harboring rare sequence variants in each known gene displayed according to their olfactory phenotype. Percentage of patients with normal sense of smell is shown on the left of y-axis, and percentage of patients with anosmia is shown on the right of y-axis. The total number of patients who have been screened for each gene is given on either side of the bar corresponding to their olfactory phenotype. Note that the total cohort of patients shown in panel B is larger than in panel A for some genes (i.e., FGFR1, FGF8, PROK2, and PROKR2). FGFR1, Fibroblast growth factor receptor; FGF8, fibroblast growth factor 8; GPR54, G-protein couple receptor 54; TAC3, tachykinin 3; TACR3, tachykinin receptor 3; KAL1 Kallmann syndrome 1; NELF, nasal embryonic LHRH factor.

Fig. 2.

Schematic representation of the gene and protein structures of the prokineticin ligands. A, PROK1 and PROK2 are encoded by three and four exons, respectively. Exons 1, 2, and 4 of PROK2 gene encode a mature protein of 81 amino acids. Exon 3 of PROK2 is represented in yellow and undergoes alternative splice processing. Both PROK1 and PROK2 encode a signal peptide that is shown in white. The gene sequence that encodes the AVITGA motif is shown in blue. The amino acid substitutions resulting in various mutations in the PROK2 gene identified to date in GnRH deficiency patients (12, 13, 16, 18) are shown below the schematic of the PROK2 gene. B, The PROK2 protein three-dimensional structure (Protein Data Bank code: 1IMT) was modeled using Cn3D 4.1 software (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). The mature prokineticin ligands exhibit a globular shape as a result of five disulfide bonds (shown in orange). Selected nonsynonymous mutations in PROK2 gene identified in GnRH-deficient patients are shown in green. The AVITGA sequence is shown in blue.

Fig. 3.

PROKR2 gene mutations identified in GnRH-deficient patients. Schematic of the PROKR2 protein generated using the SOSUI secondary structure software (http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html) showing the seven trans-membrane spans (blue cylinders) and the PROKR2 mutations identified to date in isolated GnRH-deficient patients. Mutations labeled in red have been identified in KS patients; mutations labeled in yellow have been identified in normosmic GnRH-deficient (nIHH) probands; and hatched red and yellow label shows mutations that have been identified in nIHH as well as KS patients (, –18, 135).

Fig. 4.

PROKR2 is not expressed in the GnRH neurons located in the median preoptic area. A, Double immunostaining showing the GnRH neurons (red) and PROKR2 immunoreactive cells (green) in the preoptic area of PROKR2-GFP transgenic mice. B, A higher magnification view of the boxed area in panel A is shown illustrating the absence of PROKR2 protein expression in GnRH expressing cells.

Fig. 5.

Variable expressivity and incomplete penetrance in subjects with PROKR2 mutations: representative pedigree and potential mechanisms of how heterozygous PROK2/PROKR2 mutations cause human GnRH deficiency. Schematic representation of potential mechanisms to explain how heterozygous mutations in the PROK2/PROKR2 system cause a broad spectrum of olfactory and reproductive phenotypes. The phenotype severity results from the combination of heterozygous PROK2/PROKR2 mutation along with several factors including haploinsufficiency, oligogenicity, and/or epigenetic modifiers.