Pig uterine luminal fluid contains the developmentally regulated neurotrophic factor, pleiotrophin

Abstract

The pig conceptus produces a non-invasive placenta which is probably dependent on uterine secretory products during both the pre- and post-attachment periods. Since some of these factors are also produced during the oestrous cycle, uterine luminal flushings (ULF) from adult nonpregnant pigs were analysed to identify potentially important maternally-secreted growth factors or cytokines. ULF contained several proteins that were detected on Western blots by an antibody raised against the N-terminal 14 amino acids of rat pleiotrophin (PTN). One of the proteins co-migrated on SDS-PAGE gels and co-eluted from heparin-affinity columns with 18 kDa recombinant human PTN, suggesting its identity as the intact native porcine PTN (pPTN) molecule. Additional immunoreactive forms of pPTN were identified that were of lower molecular mass (14-16 kDa), had lower heparin-affinities, were more hydrophobic, and were apparently C-terminally truncated. Native pPTN was isolated from ULF using cation-exchange chromatography, heparin-affinity fast protein liquid chromatography and C4 reverse-phase high performance liquid chromatography. Structural analysis of the purified protein resulted in definitive identification of 15 out of 17 N-terminal amino acids; these were 100% conserved with the corresponding residues of human, bovine and rat PTN. These results demonstrate various biochemical properties of pPTN and suggest that, in addition to the apparent involvement of PTN in differentiation during early neonatal life, it may be delivered in uterine secretory fluids to, as yet, undefined target cells in the reproductive tract of the adult female.

Purification and Characterization of Novel Heparin-binding Growth Factors in Uterine Secretory Fluids

“…Although we have previously shown that constituent growth factors in this cationic extract include PDGF, HB-EGF, bFGF, and PTN (2,8,12), heparin affinity chromatography revealed the presence of an additional unidentified HBGF that required 0.8 M NaCl for elution from an EconoPac heparin column and, in terms of the amount of bioactivity recovered from the column, appeared to be a principal cationic HBGF in ULF for 3T3 cells (Fig. 1A).…”

“…Although the presence of CTGF-like activity in uterine flushings was initially demonstrated several years ago (2,8,38), its identification and structural characterization was not possible until the development of the purification protocol described herein, which allowed for its separation from other unrelated HBGFs such as PDGF, bFGF, HB-EGF, and PTN (2,8,12). The data presented in this report show that a fibroblast and smooth muscle cell growth factor in uterine secretory fluids is a CTGF protein that is heparin-binding and substantially N-terminally truncated in comparison with the predicted CTGF primary translation product.…”

“…1A). The elution position of this factor (hereafter termed HBGF-0.8) from heparin affinity columns was clearly distinct from PDGF, HB-EGF, PTN, aFGF, bFGF, and amphiregulin (2,8,12,(22)(23)(24). Stability tests performed by exposing HBGF-0.8 to pH 1.5 for 2 min, 56°C for 30 min, or 100°C for 5 min demonstrated that the 3T3 cell mitogenic activity of HBGF-0.8 was highly susceptible to acid or heat inactivation (data not shown).…”

Mapping and microsatellite marker development for the porcine leukemia inhibitory factor receptor (LIFR) and epidermal growth factor receptor (EGFR) genes

“…All of these ligands are expressed by the pig endometrium during early pregnancy (Brigstock et al, 1990(Brigstock et al, , 1996a(Brigstock et al, , 1996bKennedy et al, 1994;Kim et al, 1995). A quantitative trait locus (QTL) for uterine capacity was identified on the long arm of chromosome 8 near 71 cM .…”

Upregulation of the angiogenic factor heparin affin regulatory peptide by progesterone in rat uterus

“…Recent studies suggest that, during development, HARP is involved in stroma-epithelium interactions (Vanderwinden et al 1992, Mitsiadis et al 1995b and in growth and cell differentiation steps (Peng et al 1995, Szabat & Rauvala 1996. HARP has been isolated from adult brain (Courty et al 1991), follicular fluid (Ohyama et al 1994), mature uterus (Milner et al 1989) and uterine luminal fluid (Brigstock et al 1996). In addition, HARP mRNA is upregulated by androgens in cultured prostatic epithelial cells .…”