Angiotensin-converting enzyme and male fertility

Abstract

The angiotensin-converting enzyme (ACE; EC 3.4.15.1) gene (Ace) encodes both a somatic isozyme found in blood and several other tissues, including the epididymis, and a testis-specific isozyme (testis ACE) found only in developing spermatids and mature sperm. We recently used gene targeting to disrupt the gene coding for both ACE isozymes in mice and reported that male homozygous mutants mate normally but have reduced fertility; the mutant females are fertile. Here we explore the male fertility defect. We demonstrate that ACE is important for achieving in vivo fertilization and that sperm from mice lacking both ACE isozymes show defects in transport within the oviducts and in binding to zonae pellucidae. Males generated by gene targeting that lack somatic ACE but retain testis ACE are normally fertile, establishing that somatic ACE in males is not essential for their fertility. Furthermore, male and female mice lacking angiotensinogen have normal fertility, indicating that angiotensin I is not a necessary substrate for testis ACE. Males heterozygous for the mutation inactivating both ACE isozymes sire wild-type and heterozygous offspring at an indistinguishable frequency, indicating no selection against sperm carrying the mutation.

Figure 1

Targeting of the Ace gene to eliminate somatic ACE but leave testis ACE intact. (A) The endogenous Ace locus from exon 5 to 14 is shown; the horizontal bar indicates the probe used for cloning and Southern analysis; the numbered black boxes indicate exons, with 12T indicating the testis-specific exon; B, Sa, X, S, and H indicate BamHI, SacI, XbaI, SnaBI, and HindIII. (B) The targeting construct is shown. Sequences from the XbaI site to the SnaBI site have been replaced with the neomycin resistance gene (Neo) oriented oppositely from the Ace promoters. TK indicates the thymidine kinase gene oriented as indicated; wavy lines indicate plasmid sequences (not drawn to scale). (C) The targeted Ace locus with most of exon 10 replaced by an inserted copy of Neo is shown.

Figure 2

Percentage of harvested eggs from females inseminated by stst males (2 males, 6 females) or STST males (2 males, 5 females) that were fertilized and/or progressed to the 8-cell stage or beyond. Error bars show standard errors of the means. ∗, P < 0.02; ∗∗, P < 0.001 for stst vs. STST.

Figure 3

Location of sperm from stst (n = 5) and STST males (n = 4) in oviducts 1 hr after mating. Sperm in each oviduct region were counted (one section every 50 μm) in six experiments. The open symbols represent means for individual males; the open (stst) and solid (STST) bars indicate group means for each oviduct region. Very few sperm from stst males progress beyond the two oviduct regions nearest the uterus [colliculus tubaris (T) and intramural uterotubal junction (UTJ)]. Significantly more sperm from STST males reach the extramural uterotubal junction and the lower and upper isthmus regions of the oviducts (P < 0.001 by ANOVA; P < 0.02 by Wilcoxon rank sum tests).

Figure 4

ACE mRNA (A–E) and protein (F–J) expression in testis from wild-type and mutant mice (genotypes indicated at top) as shown by in situ hybridization to a rat cDNA riboprobe (dark field, upper panel) and immunohistochemistry (lower panel) by using a polyconal antibody against a C-terminal sequence common to human somatic and testis ACE with no counterstaining. Both mRNA and protein were detected only in spermatids, adjacent to the lumen of each seminiferous tubule. Magnification, ×80.