Identification and disruption of sperm-specific angiotensin converting enzyme-3 (ACE3) in mouse

Abstract

Background: IZUMO1 is the only sperm protein which is proven to be essential for sperm-egg fusion. However, the IZUMO1 is a structurally simple protein with single Ig domain and seems not to include either a "fusogenic peptide" or a fusion machinery domain. This led us to assume the existence of an IZUMO1-interacting protein(s) which makes a functional fusion machine interacting with IZUMO1.

Methodology/principal findings: We produced a transgenic mouse line which expresses His-tagged IZUMO1 in the Izumo1(-/-) genetic background. After solubilization of sperm membranes, we purified His-tagged IZUMO1 using anti-His affinity chromatography and found a protein that interacts with IZUMO1. After being separated on SDS-PAGE gel, the IZUMO1-interacting protein was subjected to LC-MS/MS analysis and from the partial fragments, we identified the protein as ACE3. We raised the antibody against ACE3 and found that ACE3 is localized on the acrosomal cap area as in the case of IZUMO1. However, ACE3 disappeared from sperm after acrosome reaction while IZUMO1 remained on sperm. In order to investigate the role of ACE3 in vivo, we generated Ace3-deficient mice by homologous recombination and examined the fertilizing ability of the males. Unexpectedly, the male mice showed no defect in fertilizing ability in in vivo or in an in vitro fertilization system.

Conclusions/significance: We identified an IZUMO1-interacting protein in sperm, which we identified as testis specific ACE homologue ACE3. We produced an Ace3 disrupted mouse line, and found the localization of IZUMO1 spread in a little wider area on sperm, but the elimination of ACE3 did not result in a loss of sperm fertilizing ability, differing from the case of ACE disruption.

Figure 1. Identification and characterization of the IZUMO1-interacting protein.

(A) The purified IZUMO1 protein complex was separated by SDS-PAGE and stained with silver. Two specific 80-kDa and 56-kDa bands appeared corresponding to tACE3 and IZUMO1, respectively. Proteins from wild-type sperm were used as controls. (B) Amino-acid sequences of mouse tACE3. The peptide sequences obtained by LC-MS/MS are shown in red. The putative signal peptide and transmembrane region are shown in green and orange, respectively. Antigen for producing tACE3-specific antibody is shown in blue. (C) Reverse transcription-PCR of Ace3 (upper panel) and Gapdh (control; lower panel) from 10 mouse cDNAs. (D) tACE3 was detected exclusively in testis and sperm by western blotting. All solubilized proteins were loaded at 30 µg on each lane and detected by 1 µg/ml anti-tACE3 antibody. (E) Interaction between IZUMO1 and tACE3. Total sperm proteins (∼100 µg) prepared from wild-type male mice were immunoprecipitated with anti-IZUMO1 or anti-tACE3 antibodies and blotted with anti-tACE3 or anti-IZUMO1 antibodies, respectively. (F) tACE3 protein exists in Izumo1-deficient sperm.

Figure 2. Subcellular localization of tACE3 protein in mature sperm.

Incubated sperm (left) and Izumo1 knockout sperm (right) were prepared from cauda epididymis. They were stained with 4 µg/ml anti-tACE3 (red) and anti-IZUMO1 (green) antibodies. The anti-tACE3 antibody stained acrosome-intact sperm head, but not reacted to acrosome-reacted sperm (asterisk).

Figure 3. Targeted disruption of Ace3 gene.

(A) Complete structures of the wild-type mouse Ace3 allele. Exons and introns are represented by boxes and horizontal lines, respectively. For the targeted disruption of mouse Ace3 allele, the first 9 exons (closed boxes) were replaced by the neomycin-resistant gene (Neo^r). A herpes simplex virus thymidine kinase gene (tk) was introduced into the targeting construct for negative control. (B) Genotyping of tail tip DNA by PCR amplification with primers indicated in the figure. (C) Western blotting analysis of sperm lysates from wild-type, Ace3 ^+/−, Ace3 ^−/−, Ace ^+/− and Ace ^−/− mice.

Figure 4. Fertility analysis of Ace3 ^−/− mice.

(A) Fecundity of Ace3 ^+/− and Ace3 ^−/− males and Ace3 ^−/− females. The numbers in parentheses indicate the numbers of mating pairs. Values are presented as mean ± standard error of mean (SEM). (B) in vitro fertilization of sperm from Ace3 ^+/− and Ace3 ^−/− mice using cumulus-free and –intact eggs (n = 5). (C) Comparison of the fusing ability of Ace3 ^+/− and Ace3 ^−/− sperm. Average numbers of fused sperm observed 30 minutes after insemination (n = 5). Values are presented as mean ± SEM.

Figure 5. Immnolocalization of IZUMO1 protein in Ace3 ^−/− sperm.

Wild-type and Ace3 ^−/− sperm were immunostained with 4 µg/ml anti-tACE3 (red) and anti-IZUMO1 (green) antibodies (A). The staining pattern of IZUMO1 in Ace3 ^−/− sperm showed a significantly broader staining area than that of wild-type sperm (B).