Impact of the genetic background on the reproductive phenotype of CRISP1 and CRISP4 knockout mice

CUASNICÚ PS; BATTISTONE MA; WEIGEL MUÑOZ M; DA ROS VG; BRUKMAN NG; PIGNATARO O; CARVAJAL G

Conferencia; Gordon Research Conference, Fertilization & Activation of Development.; 2017

Epididymal CRISP1 and CRISP4 associate withsperm during maturation and participate in fertilization very likely throughtheir ability to regulate critical sperm Ca2+ channels such as CatSper andTRPM8.  In spite of this, CRISP1 andCRISP4 knockout (KO) mice are fertile. As it has been reported that a singlemutation can produce markedly different phenotypes depending on the geneticbackground of the animals, in the present work we studied the effect of thegenetic background on the reproductive phenotype of CRISP1 and CRISP4 KO mice.The new CRISP1 and CRISP4 KO colonies used in this study had a homogeneousC57BL6 background (generated by backcrossing the original 129SvEv/C57BL6Nmice), and a mixed C57BL6/DBA background, respectively. Fertility was evaluatedby mating, total motility by CASA, tyrosine phosphorylation (pTyR) by Westernblot, cAMP levels by RIA, intracellular Ca2+ levels by flow cytometry, andfertilizing ability by IVF. Results revealed that males from CRISP1 and CRISP4KO colonies remained fertile but their sperm exhibited changes in severalparameters compared to those observed for the original colonies. Differentlyfrom CRISP1 KO sperm from the mixed genetic background which had shown a clearreduced pTyR, CRISP1 KO sperm from the homogeneous colony had normal pTyR butsignificantly reduced sperm motility. Interestingly, both reduced pTyR andmotility could be restored by exposure of sperm to dbcAMP+IBMX, suggesting adeficiency in cAMP levels in cells from the two genetic backgrounds. Subsequentstudies confirmed the presence of lower cAMP levels in both groups, indicatingthat the same cAMP defect could be manifested in two different ways dependingon the genetic background of the animal. In the case of CRISP4, differentlyfrom what had been reported in C57BL6N, sperm from the new mixed C57BL6/DBA colonyshowed significantly lower pTyR levels, normal sperm-ZP binding, and lowerfusion ability than wild type sperm. These sperm also showed a lower increasein intracellular Ca2+ during capacitation and responded differentially to Ca2+ionophore than controls. Altogether, our results indicate that the phenotypesof CRISP1 and CRISP4 KO mice are not entirely controlled by the mutation at Crisp1or Crisp4 loci but are influenced by the genetic background of the animal.Considering the wide range of genetic backgrounds inherent to humans, theseresults highlight the relevance of analyzing the phenotype of a particularmutation in different inbred strains to improve the diagnosis and treatment ofhuman infertility