ACE2 Receptor and Its Isoform Short-ACE2 Are Expressed on Human Spermatozoa

Abstract

Angiotensin-converting enzyme 2 (ACE2) is a protein widely expressed in numerous cell types, with different biological roles mainly related to the renin-angiotensin system. Recently, ACE2 has been in the spotlight due to its involvement in the SARS-CoV-2 entry into cells. There are no data available regarding the expression of ACE2 and its short-ACE2 isoform at the protein level on human spermatozoa. Here, protein expression was demonstrated by western blot and the percentage of sperm displaying surface ACE2 was assessed by flow cytometry. Immunocytochemistry assays showed that full-length ACE2 was mainly expressed in sperm midpiece, while short ACE2 was preferentially distributed on the equatorial and post-acrosomal region of the sperm head. To our knowledge, this is the first study demonstrating the expression of protein ACE2 on spermatozoa. Further studies are warranted to determine the role of ACE2 isoforms in male reproduction.

Keywords: ACE2; SARS-CoV-2; fertility; male reproduction; short-ACE2; spermatozoa.

Figure 1

Western blot analysis of ACE2. The image illustrates (A) the glycosylated full-length ACE2 (120 KDa) and short-ACE2 isoforms (52 KDa) recognized with anti-ACE2-1 and (B) the glycosylated full-length ACE2 (120 KDa) detected by anti-ACE2-2. Antibodies were incubated on the same blot after membrane stripping and re-blotting. Blots were cut prior to hybridization. Each of the six lanes contains 20 µg of protein from semen of different donors. At least four independent experiments with different donors were performed. C+: protein from mouse testis.

Figure 2

Representative schema of the ACE2 and short-ACE2 sequences and anti-ACE2 antibodies. Full-length ACE2 is composed of 805 amino acids (aas), while short-ACE2 is 459 aas length, thus sharing the sequence between the aas 347–805 (including the C-terminal domain). Numbers in blue correspond to the aas present in each sequence, while numbers in grey correspond to the sequence of aas missing in the short-ACE2 isoform. Three different antibodies were used: anti-ACE2-1 (abcam, ab15348) that recognizes the sequence between the aas 750–805; anti-ACE2-2 (Novus, NBP2-67692), recognizing the aas 200–230 from the full-ACE2; and anti-ACE2-3 (AF933, R&D Systems) recognizing the aas 18–640, mainly present in the full-ACE2 isoform.

Figure 3

Immunocytochemistry analysis of ACE2 on ejaculated human sperm cells. Panel (A) shows the location of the C-terminal domain of ACE2 recognized by anti-ACE2-1 (Abcam, ab15348), shared between both isoforms; Panel (B) shows the fluorescence pattern after immunocytochemistry analysis with anti-ACE2-2 (Novus, NBP2-67692), recognizing the full-length ACE2 only. DAPI was used to stain the nuclei. Top: two-laser image (anti-ACE2 antibody + DAPI); middle: sperm cells stained only with DAPI; bottom: sperm cells illustrating only ACE2 fluorescence patterns (scale bar = 5 µm). Negative controls for immunocytochemistry assays are shown in Supplementary Figure S1.

Figure 4

Surface expression of ACE2 in viable motile human spermatozoa selected by the swim-up procedure as evaluated by flow cytometry. Typical histograms of fluorescence intensity (FL2-H) in spermatozoa incubated with (A) a non-specific serum from non-immunized goat (negative control) or (B,C) two representative sperm samples incubated with primary goat antibody against human ACE2 (anti-ACE2-3 polyclonal antibody, R&D Systems, AF933).