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

WEIGEL MUÑOZ M; BATTISTONE MA; CUASNICÚ P; CARVAJAL G; PIGNATARO O; BRUKMAN N; DA ROS VG

Miami

Conferencia; American Society of Andrology (ASA) 42nd Annual Conference; 2017

American Society of Andrology

INTRODUCTION. Epididymal CRISP1 and CRISP4 associate with sperm during maturation and participate in different stages of the fertilization process very likely through their ability to regulate critical sperm calcium channels such as CatSper and TRPM8. In spite of their important functional roles, the knockout (KO) mice for each of these proteins are fertile. One possible explanation for these observations may be related to the influence of the genetic background of the animals on their phenotype as it has been reported that a single mutation can produce markedly different phenotypes depending on the genetic background of the animals, including fertility. OBJECTIVE. Based on this, the aim of the present work was to study reproductive phenotype of CRISP1 and CRISP4 KO mice with a genetic background different from that of the original colonies. MATERIALS AND METHODS. CRISP1 and CRISP4 KO mice colonies used throughout the study had a homogeneous C57 background (generated by backcrossing of the original C57/129 CRISP1 KO mice) and a C57/DBA mixed background, respectively. Male fertility was evaluated by mating. The sperm parameters analyzed included number (by counting), viability (by eosin staining), total and hyperactivated motility, (by CASA), PTyR (by Western blot), cAMP levels (by RIA), sperm calcium levels (by flow cytometry). and fertilizing ability (by in vitro fertilization using cumulus surrounded eggs?). RESULTS. The new CRISP1 and CRISP4 KO mice remained fertile but their sperm exhibited changes in some functional parameters compared to those corresponding to the original colonies. Differently from KO sperm from mice of the mixed background showing severely reduced PTyR levels, sperm from the new homogeneous background had normal PTyR but reduced sperm motility. Interestingly, both parameters could be restored by exposure of sperm to dbcAMP+IBMX, suggesting a deficiency in cAMP levels in cells from the two genetic backgrounds. Subsequent studies confirmed the presence of lower cAMP levels in both groups, indicating that the same cAMP defect was manifested in two different ways depending on the genetic background of the animal. In the case of the CRISP4, we observed that, differently from the two reported Crisp4-/- colonies, sperm from the new CRISP4 colony showed significantly lower PTyR levels normal sperm-ZP binding, and lower fusion ability compared to wild type sperm. In addition, these CRISP4 KO sperm showed lower intracellular calcium levels during capacitation and responded differentially to their exposure to calcium ionophore than controls.CONCLUSIONS. Together, these observations revealed that CRISP1 and CRISP4 KOs constitute new examples of the influence of the genetic background on the reproductive phenotype. Considering the wide range of genetic backgrounds inherent to humans, these results highlight the relevance of analysing the phenotype of a particular mutation in different inbred strains to improve the diagnosis and treatment of human infertility.