A model of the acrosome reaction progression via the acrosomal membrane-anchored protein equatorin

Abstract

It is important to establish a reliable and progressive model of the acrosome reaction. Here, we present a progression model of the acrosome reaction centering around the acrosomal membrane-anchored protein equatorin (MN9), comparing the staining pattern traced by MN9 antibody immunofluorescence with that traced by Arachis hypogaea agglutinin (PNA)-FITC. Prior to the acrosome reaction, equatorin was present in both the anterior acrosome and the equatorial segment. Since sperm on zona pellucida showed various staining patterns, MN9-immunostaining patterns were classified into four stages: initial, early, advanced, and final. As the acrosome reaction progressed from the initial to the early stage, equatorin spread from the peripheral region of the anterior acrosome toward the center of the equatorial segment, gradually over the entire region of the equatorial segment during the advanced stage, and finally uniformly at the equatorial segment at the final stage. In contrast, the PNA-FITC signals spread more quickly from the peripheral region of the acrosome toward the entire equatorial segment, while decreasing in staining intensity, and finally became weak at the final stage. MN9-immunogold electron microscopy showed equatorin on the hybrid vesicles surrounded by amorphous substances at advanced stage of acrosome reaction. Equatorin decreased in molecular mass from 40-60 to 35 kDa, and the signal intensity of 35 kDa equatorin increased as the acrosome reaction progressed. Thus, the established equatorin-based progression model will be useful for analyzing not only the behavior of equatorin but also of other molecules of interest involved in the acrosome reaction.

Equatorin-Related Subcellular and Molecular Events During Sperm Priming for Fertilization in Mice

“…IAM inner acrosomal membrane, N nucleus, OAM outer acrosomal membrane, PM plasma membrane. ( a-c and d , e reproduced with slight modifi cations from Yoshida et al 2010 ) and Yamatoya et al 2009 , respectively) by the MN9 antibody has a specifi c affi nity for the equatorial plasma membrane; however, a suffi cient amount of equatorin remains in the acrosome-reacted spermatozoa. Immunofl uorescence double-staining data using the anti-Izumo1 antibody and anti-MN9 antibody suggest that Izumo1 can spread rapidly and widely to the postacrosomal region in the acrosome-reacting spermatozoa, whereas the equatorin epitope region recognized by MN9 antibody remains confi ned to the anterior acrosomal region (unpublished data ).…”

“…The molecular size of mouse equatorin is approximately 65 kDa in testicular germ cells (Ito et al 2013 ), but the size decreases to 40-50 kDa in cauda epididymal spermatozoa when examined by Western blotting (Yoshida et al 2010 ).…”

“…Some amount of equatorin is relocated on the surface of the plasma membrane over the equatorial segment but not to the plasma membrane over the postacrosomal region (Yoshida et al 2010 ). This fact suggests that the epitope region recognized Immunogold particles (10-nm gold particles, arrowheads ) are abundant on the inner acrosomal membrane facing the acrosome lumen, as indicated with asterisks , but depleted (no gold particles in this photograph) on the outer acrosomal membrane in the anterior acrosomal region before or at the very initial stage of the acrosome reaction; gold particles are not present on the plasma membrane.…”

“…Immunofl uorescence double-staining data using the anti-Izumo1 antibody and anti-MN9 antibody suggest that Izumo1 can spread rapidly and widely to the postacrosomal region in the acrosome-reacting spermatozoa, whereas the equatorin epitope region recognized by MN9 antibody remains confi ned to the anterior acrosomal region (unpublished data ). The molecular size of equatorin appears to be reduced to 35 kDa at the end of the acrosome reaction (Yoshida et al 2010 ).…”

USP8/UBPy-regulated sorting and the development of sperm acrosome: the recruitment of MET

“…In parallel, we followed the progression of the AR at each time-point by Coomassie blue staining (Feng et al 1998). To classify the different patterns of USP8/UBPy and MET localization, we resorted to a nomenclature of AR stages in agreement with Yoshida et al (2010). At the initial stage (time 0 min), when w88% of spermatozoa were acrosome intact, USP8/ UBPy (Fig.…”