Anti-HSP70 monoclonal antibody, clone 4B4

This work was designed to study whether HSP70-1A, HSP90 alpha, ezrin or PDI4, proteins previously identified in porcine oviductal secretions, have a role in zona pellucida (ZP) resistance to enzymatic digestion, in vitro fertilization (IVF) and sperm viability. In vitro matured porcine cumulus oocyte complexes were denuded and i) incubated for 1 h in TALP medium supplemented or not with each exogenous oviductal protein and in presence or absence of heparin to assess ZP digestion time by pronase; and ii) inseminated with fresh ejaculated boar spermatozoa in medium supplemented or not with each exogenous oviductal protein to assess their effect on fertilization results. Finally, spermatozoa were incubated in Tyrode's medium (0, 1 and 20 h) supplemented or not with HSP-701A, HSP-90 alpha or ezrin, to assess simultaneously sperm viability and acrosome status by means of flow cytometry. Although all proteins increased the ZP digestion time, this increase was lower than 1 min, being ezrin the protein with a stronger effect. Presence of heparin in the medium reinforced the ZP hardening effect of ezrin and HSP-701A up to one more min, but not HSP-90 alpha nor PDI4. Sperm penetration, but not IVF efficiency, increased when gametes were cocultured in medium containing PDIA4 whereas sperm penetration and polyspermy rates decreased in presence of ezrin and HSP proteins. This reduction was not the result of a detrimental effect of proteins on sperm viability or acrosome reaction. In conclusion, addition of exogenous proteins detected in oviductal secretions to artificial media does not reproduce the effect of adding such secretions nor improve the final efficiency of the porcine IVF system. (c) 2020 Elsevier Inc. All rights reserved.

Heat shock proteins (Hsp) are class of conserved and ubiquitous stress proteins present in all living organisms from primitive to higher level. Various studies have demonstrated multiple cellular functions of Hsp in living organisms as an important biomarker in response to abiotic and biotic stressors including temperature, salinity, pH, hypoxia, environmental pollutants, and pathogens. However, full understanding on the mechanism and pathway involved in the induction of Hsp still remains challenging, especially in aquatic invertebrates. In this study, the entire Hsp family and subfamily members in the marine rotifers Brachionus spp., one of the cosmopolitan ecotoxicological model organisms, have been genome-widely identified. In Brachionus spp. Hsp family was comprised of Hsp10, small hsp (sHsp), Hsp40, Hsp60, Hsp70/105, and Hsp90, with highest number of genes found within Hsp40 DnaJ homolog subfamily C members. Also, the differences in the orientation of the conserved motifs within Hsp family may have induced differences in transcriptional gene modulation in response to thermal stress in Brachionus koreanus. Overall, Hsp family-specific domains were highly conserved in all three Brachionus spp., relative to Homo sapiens and across other animal taxa and these findings will be helpful for future ecotoxicological studies focusing on Hsps.