A low-calcium microenvironment is imperative for spermatozoa maturation within the epididymis. Our previous work has shown that γ-glutamyl carboxylase (GGCX), the carboxylation enzyme of the matrix Gla protein (MGP), plays an essential role in epididymal calcium homeostasis and sperm maturation in rats and that the GGCX SNP mutation rs699664 was associated with asthenozoospermia (AZS) in humans. Here, we investigated the expression patterns of GGCX and MGP in the mouse epididymis and generated knock-in (KI) mice. We also tested the effects of this mutation on epididymal calcium homeostasis, sperm function, and male fertility in mice. The results showed that both GGCX and MGP were enriched in all regions of the mouse epididymis, especially in the initial segment of the epididymis. Double immunofluorescence staining revealed that GGCX colocalized with MGP in the epithelial cells of the initial segment and caput regions as well as in the lumen of the corpus and cauda regions of the mouse epididymis. However, the mice were fertile with normal epididymal morphology, sperm functions, and epididymal calcium concentration. Overall, our findings revealed that the mutation does not exert any discernible effect on male fertility in mice.
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