Association of GLIPR1L1 and GLIPR1L2 with sperm, behaviour during capacitation and role in the fertilization process

ERNESTO JI; CURIA A; WEIGEL MUÑOZ M; COHEN DJ; CUASNICU PS

Congreso; 10th International Congress of Andrology; 2013

Based on reports suggesting the presence of GLIPR1L1 and GLIPRL2 proteins in sperm, we investigated their association with sperm, behaviour during capacitation and role in fertilization. Recombinant proteins produced by cloning of Glpr1l1 and Glprll2 in E. coli showed the predicted Mw of 30 kDa. and 37 kDa, respectively, when analyzed by SDS?PAGE. Western Blot (Wb) studies using the antibodies generated against these proteins revealed the presence of GLIPRL1 in testicular tissue and of GLIPRL2 in the testes and the epididymis. Wb and immunofluorescence (IIF) studies confirmed the presence of both proteins in sperm revealing their localization in the dorsal region of the head of live sperm and in both head and tail of permeabilized cells. Treatment of sperm with different agents showed that while neither NaCl nor DTT were able to extract the proteins from the cells, Triton X-100 completely removed both proteins. Exposure of sperm to PNGase-F and Phospholipase-C indicated that both proteins are glycosilated and that GLIPRL1 would be GPI-associated. Wb and IIF studies indicated that GLIPRL1 and GLIPRL2 remained in sperm after capacitation and acrosome reaction (AR) without undergoing post-traslational modifications. In vitro fertilization studies using zona pellucida (ZP)-intact and ZP-free eggs showed that while none of the antibodies affected gamete fusion, both significantly (p< 0,05) reduced sperm binding to the ZP without affecting sperm viability, motility or AR. Together, these results support the participation of GLIPRL1 and GLIPRL2 in sperm-ZP interaction contributing to a better understanding of the molecular mechanisms involved in mammalian fertilization.