Functional redundancy and compensation: Deletion of multiple murineCrispgenes reveals their essential role for male fertility

Abstract

Mammalian Cysteine-RIch Secretory Protein (CRISP) family includes four members present in sperm and reported to regulate Ca 2+ channels and fertilization. Based on our previous observations using single knockouts models and suggesting the existence of functional compensation among CRISP proteins, we investigated their relevance for male fertility by generating multiple Crisp gene mutants by CRISPR/ Cas9 technology. Whereas targeting of Crisp1 and Crisp3 yielded subfertile males with early embryo developmental defects, the same deletion in zygotes from fertile Crisp2 −/− .Crisp4 −/− mice led to the generation of both triple and quadruple knockout mice exhibiting a complete or severe disruption of male fertility due to a combination of sperm transport, fertilization, and embryo developmental defects linked to intracellular Ca 2+ dysregulation. These observations reveal that CRISP proteins are essential for male fertility and organize in functional modules that contribute distinctly to fertility success, bringing insights into the mechanisms underlying functional redundancy/compensation in protein families and emphasizing the importance of generating multiple and not just single knockout which might be masking the true functional relevance of family genes.

Cysteine-Rich Secretory Proteins (CRISP) are Key Players in Mammalian Fertilization and Fertility

“…In agreement with this, a lower number of Crisp2 −/− sperm was observed within the cumulus mass during cumulus penetration assays, and hyperactivated motility was significantly lower in mutant sperm ( Brukman et al, 2016 ). Subsequent studies supported that these motility defects could be due to a stiffness of the midpiece in CRISP2 mutant sperm that impairs hyperactivation development and thus, egg coat penetration ( Lim et al, 2019 ; Curci et al, 2020 ). Different from Crisp1 −/− animals, sperm lacking CRISP2 exhibit a clear dysregulation of Ca 2+ homeostasis ( Brukman et al, 2016 ) that could explain the molecular mechanisms underlying the capacitation-associated defects in the mutant cells.…”

“…The fact that Crisp2 and Crisp4 are located in different chromosomes together with the availability of CRISP2 and CRISP4 single knockout colonies, led our laboratory to generate double Crisp2 −/− /Crisp4 −/− mice by natural mating ( Curci et al, 2020 ). Analysis of animal fertility revealed that, in spite of the participation of both proteins in key stages of the fertilization process, males lacking both proteins were fertile under normal laboratory conditions ( Curci et al, 2020 ).…”

“…The fact that Crisp2 and Crisp4 are located in different chromosomes together with the availability of CRISP2 and CRISP4 single knockout colonies, led our laboratory to generate double Crisp2 −/− /Crisp4 −/− mice by natural mating ( Curci et al, 2020 ). Analysis of animal fertility revealed that, in spite of the participation of both proteins in key stages of the fertilization process, males lacking both proteins were fertile under normal laboratory conditions ( Curci et al, 2020 ). Nevertheless, when Crisp2 −/− /Crisp4 −/− males were mated under more demanding conditions such as using superovulated females, a clear decrease in in vivo fertilization rates was observed compared to controls and to either Crisp2 or Crisp4 single mutant males ( Curci et al, 2020 ).…”

“…Analysis of animal fertility revealed that, in spite of the participation of both proteins in key stages of the fertilization process, males lacking both proteins were fertile under normal laboratory conditions ( Curci et al, 2020 ). Nevertheless, when Crisp2 −/− /Crisp4 −/− males were mated under more demanding conditions such as using superovulated females, a clear decrease in in vivo fertilization rates was observed compared to controls and to either Crisp2 or Crisp4 single mutant males ( Curci et al, 2020 ). Consistent with this and with the reported roles of CRISP2 and CRISP4 in different stages of the fertilization process, Crisp2 −/− /Crisp4 −/− sperm showed severe in vitro fertilization defects likely due to the combination of the capacitation-associated defects observed in sperm from each single knockout model (i.e.…”

CRISPs Function to Boost Sperm Power Output and Motility

“…Exactly how this is achieved is unknown although based on biochemical data it is presumed that a significant proportion of activity will be mediated via ion channel, including at least some of which remain to be characterized ( Gibbs et al, 2006 , 2011 ; Ernesto et al, 2015 ). Deletion of all four CRISPs in mice leads to severe subfertility ( Curci et al, 2020 ). For more information on the CRISPs and their roles in fertility and their evolutionary origins, readers are referred to ( Gaikwad et al, 2020a ).…”