Abstract
BackgroundEndogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.MethodsFITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis.ResultsPIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIPK site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.ConclusionCollectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo.
Highlights
After fertilization the embryo/allograft is surrounded by the zona-pellucida which physically separates the embryo from the maternal environment
We report that the specific PreImplantation Factor (PIF) targets identified protein disulfide-isomerases (PDI)/Heat-shock Proteins (HSPs) could have important supportive and protective roles in embryo development regulating oxidative stress and protein misfolding
The same or even four-fold higher concentration of the scrambled FITC-PIF which was tested in parallel as control did not show any detectable binding to the blastocyst. (Fig. 1b)
Summary
After fertilization the embryo/allograft is surrounded by the zona-pellucida which physically separates the embryo from the maternal environment. While maternal-derived compounds still can reach the embryo, access through the embryo cell membrane is more limited. Exogenous sPIF (synthetic identical structure) administered to singly cultured bovine embryos, enhances their development to the blastocyst stage [22]. PIF acts as a rescue factor, protecting cultured murine embryos against toxicity of serum (i.e. containing oxygen radicals, antibodies) derived from patients with a history of recurrent pregnancy loss (RPL) [22]. PIF regulates the maternal environment by promoting embryo receptivity acting on endometrium, implantation phase and first trimester decidua [23,24]. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were devised
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