Sperm IZUMO1-Dependent Gamete Fusion Influences Male Fertility in Mice.

Takako Saito,Ikuo Wada,Naokazu Inoue

doi:10.3390/ijms20194809

Takako Saito, Ikuo Wada + Show 1 more

Open Access

https://doi.org/10.3390/ijms20194809

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Abstract

Sperm–egg fusion is accomplished through the interaction of a specific set of membrane proteins in each gamete: sperm IZUMO1 and oocyte JUNO. Recently, we found that alternative splicing of the Izumo1 gene generates a novel IZUMO1 isoform (IZUMO1_v2). Here, we obtained four mouse lines, having graded different levels of IZUMO1 protein by combining an original IZUMO1 (IZUMO1_v1) knockout with IZUMO1-null (both IZUMO1_v1 and _v2 disrupted) genetic background, in order to determine how the quantity of IZUMO1 influences male fertility. Subsequently, we clarified that the signal intensity from two quantitative assays, western blot and immunostaining analyses with a monoclonal antibody against mouse IZUMO1, were strongly correlated with average litter size. These results suggest that evaluating IZUMO1 protein levels is useful for predicting fecundity, and is a suitable test for male fertility.

Highlights

Fertilization is a complex phenomenon containing multiple essential events
We reported that IZUMO1 knockout (KO/KO; both IZUMO1_v1 and _v2 are disrupted) male mice are sterile, whereas IZUMO1_v1 specific knockout (v1KO/v1KO) male mice still carrying functional IZUMO1_v2 are fertile and have a smaller litter size compared to wild-type mice [7,13]
We performed western blot analysis with an IZUMO1 monoclonal antibody (Mab18), using sperm BASIGIN as a normalizing protein to compare the amount of IZUMO1 protein in the spermatozoa of four mouse lines (Figure 1A)

Summary

Introduction

Fertilization is a complex phenomenon containing multiple essential events. Ejaculated spermatozoa undergo physiological changes, such as capacitation and hyperactivation, and morphological modification, acrosome reaction (AR), prior to reaching the oocyte. In bovine models, it has been suggested that a loss of tyrosine-phosphorylation of SPACA1-including proteins in spermatozoa is strongly linked to AI subfertility [6] Another acrosomal protein IZUMO1, an indispensable factor for gamete fusion, consists of an IZUMO domain, a N-glycosylated immunoglobulin-like domain, and a transmembrane region with a short cytoplasmic tail [7,8,9]. IZUMO1_v2 possesses identical mature protein and functional properties as the original IZUMO1 (IZUMO1_v1); an IZUMO1_v1-specific knockout mouse carried only 19% of IZUMO1 protein compared to wild-type mice, and still retained male fertility This suggested that a minimal amount of IZUMO1 is sufficient for completing the sperm–egg fusion [13]. Our data suggest that IZUMO1 is a useful biomarker for male fertility

Comparison of IZUMO1 Protein Quantity Among Each Mouse Line

Materials and Methods

Genotyping

Western Blot Analysis of Spermatozoa

Fluorescence Imaging for Spermatozoa

Image and Statistical Analyses