167 Role of ADGRA2/TEM5/GPR124 protein during spermatogenesis and fertilisation events
M. Assumpção A B , L. Hamilton B , E. Díaz Miranda B C , M. Zigo B , A. Jones B , J. Rissman B , J. Taylor B D , R. Schnabel B E and P. Sutovsky B FA School of Veterinary Medicine and Animal Science, Sao Paulo, SP, Brazil
B Division of Animal Science, University of Missouri, Columbia, MO, USA
C Department of Animal Science, Federal University of Viçosa, Viçosa, MG, Brazil
D Genetics Area Program, University of Missouri, Columbia, MO, USA
E Institute of Data Science and Informatics, University of Missouri, Columbia, MO, USA
F Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, Columbia, MO, USA
Reproduction, Fertility and Development 35(2) 211-211 https://doi.org/10.1071/RDv35n2Ab167
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
The ADGRA2/TEM5/GPR124 protein is an adhesion G protein-coupled receptor and transmembrane protein with a large extracellular domain responsible for cell-cell and cell-matrix interactions. The role of ADGRA2 has been characterised in the neuronal endothelium, where it functions both in a cell-autonomous fashion regulating the development of the blood-brain barrier and as part of the Wnt/β-catenin pathway through the formation of the RECK/ADGRA2/FZD/LPR complex. Interestingly, ADGRA2 is also highly expressed within the testis; however, its role in sperm development and function has yet to be fully characterised. Here, we aim to investigate whether ADGRA2 acts through similar mechanisms within the testicular environment. The developmental and terminal localisation of ADGRA2 was investigated in isolated testicular cells, paraffin-embedded testicular sections, and mature spermatozoa (bovine, porcine, human) using a polyclonal anti-TEM5 antibody (Invitrogen, PA5-20442, dil.1:500) and a goat-anti-rabbit fluorescein isothiocyanate conjugated secondary antibody (GAR-FITC, dil.1:200). Cells were co-stained with DAPI and PNA-AF647, to visualise the nuclei and acrosomes, respectively. Results indicated that, developmentally, ADGRA2 has an increased expression in the basal compartment and surrounding pachytene spermatocytes. It also associates with both the heads and tails of fully differentiated testicular spermatozoa but is weakly expressed in round and elongating spermatids. In mature spermatozoa, immunoreactivity was observed in the equatorial segment region of the sperm head, and the sperm tail connecting and principal pieces, with a notable absence of labelling in the tail midpiece. Based on its localisation, we investigated whether this pattern changed during sperm capacitation as an indicator of the action of a noncanonical Wnt pathway, by using imaged-based flow cytometry. The ADGRA2 intensity and localisation were both investigated using 4 h in vitro capacitation studies in boars, using both PNA-AF647 and a whole sperm zinc signature assay to track capacitation progression. Samples were live imaged for zinc signatures and fixed for ADGRA2 and PNA staining. Results revealed decreased ADGRA2 labelling in the head of capacitated spermatozoa, commensurate with acrosome exocytosis and an increased intensity of the ADGRA2 tail labelling, which was further validated by indirect immunofluorescence. These findings suggest that ADGRA2 may facilitate signalling events during capacitation, but further experiments should be performed to elucidate whether its actions do, in fact, function through the Wnt/β-catenin pathway. Furthermore, both the developmental and functional patterning of ADGRA2 mirror that of integrin aVB3, a previously reported ADGRA2 interacting partner. This suggests that ADGRA2 may also have additional functions outside of the Wnt/β-catenin pathway and may facilitate processes such as the organisation of tetraspanin-enriched microdomains in the sperm head plasma membrane.
