Monitoring dimeric status of IZUMO1 during the acrosome reaction in living spermatozoon

Abstract

The acrosome reaction (AR) is indispensable for successful spermatozoon-oocyte fusion. Recent studies have indicated that sperm IZUMO1 gradually gathers in the equatorial segment (EQ), which is the initiation site of sperm-egg fusion, after the AR. In addition, by examining the binding process of oocytes and Izumo1-expressing cultured cells to reconstitute the early steps of fertilization, we previously demonstrated that robust IZUMO1-dependent adhesion specifically occurs at the contact site along with the dimerization of IZUMO1. However, when IZUMO1 dimerizes after the AR in living spermatozoon is unknown. Here, we report dynamics of IZUMO1 dimerization during the AR in spermatozoa by combining transgenic mice and time-lapse imaging using a set of bimolecular fluorescence complementation (BiFC) probes. Surprisingly, dimeric IZUMO1 was already formed at the acrosomal cap region before the AR and redistributed into the EQ after the AR. We categorized the translocation of the dimer into two types: Type 1, the near-simultaneous appearance of BiFC signals with IZUMO1-mCherry; and Type 2, the delayed formation of dimer in the EQ. Those findings suggest that, before encountering oocytes, spermatozoa are prepared to boost their affinity with JUNO.

Keywords: Fertilization; IZUMO1; gamete fusion; oocyte; spermatozoon.

Figure 1.

Live sperm imaging of oligomerization of IZUMO1 tagged by BiFC. (a) Constructs of transgenes to express mouse Izumo1 under the control of Calmegin promoter. (b) Expression of IZUMO1-BiFC in spermatozoa. The intensity of 60–70 kDa doublet bands (asterisks) of IZUMO1-BiFC (IZUMO1-VN155 and -VC) was comparable to those of His-tagged IZUMO1 TG when a monoclonal antibody against IZUMO1 (Mab18) was used. In either case, treatment with PNGase F (New England Biolabs) caused slight mobility shift. BASIGIN antibody (sc-9757: Santa Cruz Biotechnology) was also used as a loading control. In each lane, 10 μg of sperm protein was used for SDS-PAGE. (c) Fluorescence images of a spermatozoon expressing IZUMO1-mCherry and -BiFC. The relative fluorescence intensity in the arrow of the merged images (acrosome intact and acrosome reacted spermatozoon) in c was analyzed using Fiji. AC and EQ stands for acrosomal cap region and equatorial segment, respectively. Magnified images (3×) of a boxed region after the AR are also shown in Fire LUT of Fiji. BiFC signals were almost absent in the acrosomal cap region. (d) Antibody staining of IZUMO1-BiFC expressing spermatozoon. Non-permeabilized spermatozoon expressing IZUMO1-BiFC was stained with Alexa-546 labeled anti-IZUMO1 monoclonal antibody (Mab18). Three times enlarged images as in d at the bottom. Scale bar, 2 μm.

Figure 2.

Time-lapse of IZUMO1 oligomerization upon the AR. (a) Translocation of IZUMO-mCherry. In Acro-GFP/IZUMO1-mCherry double transgenic mice, mCherry and GFP fluorescence images were captured before and after the AR. Immediately after the AR, no GFP signals were detected, indicating acrosomal exocytosis, and translocation of IZUMO1-mCherry to the EQ was observed. (b) Type 1 movement of IZUMO1-BiFC. Appearance of BiFC fluorescence (green) followed IZUMO1-mCherry (red) almost simultaneously. Arrow head indicates the region where IZUMO1 was clustered. (c) Type 2 movement of IZUMO1-BiFC. Formation of BiFC signals (green) was delayed compared to translocation of IZUMO1-mCherry (red) to the EQ after the AR. Scale bar, 2 μm. (d) Quantification of b and c. Fluorescence intensity was measured at an arrow in b and c. In Type 2 movement, BiFC was largely devoid in the EQ at 5 min, and caught up with mCherry signals over 25 min.