Local renin angiotensin system and sperm DNA fragmentation

Abstract

The renin angiotensin system (RAS) appears to influence male fertility at multiple levels. In this work, we analyzed the relationship between the RAS and DNA integrity. Fifty male volunteers were divided into two groups (25 each): control (DNA fragmentation ≤20%) and pathological (DNA fragmentation >20%) cases. Activities of five peptidases controlling RAS were measured fluorometrically: prolyl endopeptidase (which converts angiotensin [A] I and A II to A 1-7), neutral endopeptidase (NEP/CD10: A I to A 1-7), aminopeptidase N (APN/CD13: A III to A IV), aminopeptidase A (A II to A III) and aminopeptidase B (A III to A IV). Angiotensin-converting enzyme (A I to A II), APN/CD13 and NEP/CD10 were also assessed by semiquantitative cytometry and quantitative flow cytometry assays, as were the receptors of all RAS components: A II receptor type 1 (AT1R), A II receptor type 2 (AT2R), A IV receptor (AT4R or insulin-regulated aminopeptidase [IRAP]), (pro)renin receptor (PRR) and A 1-7 receptor or Mas receptor (MasR) None of the enzymes that regulate levels of RAS components, except for APN/CD13 (decrease in fragmented cells), showed significant differences between both groups. Micrographs of RAS receptors revealed no significant differences in immunolabeling patterns between normozoospermic and fragmented cells. Labeling of AT1R (94.3% normozoospermic vs 84.1% fragmented), AT4R (96.2% vs 95.3%) and MasR (97.4% vs 87.2%) was similar between the groups. AT2R (87.4% normozoospermic vs 63.1% fragmented) and PRR (96.4% vs 48.2%) were higher in non-fragmented spermatozoa. These findings suggest that fragmented DNA spermatozoa have a lower capacity to respond to bioactive RAS peptides.

Keywords: DNA fragmentation; local renin angiotensin system; sperm fertility.

Figure 1

Tested activities included (a) aminopeptidase N (APN/CD13; A III to A IV) and aminopeptidase B (APB; A III to A IV), (b) aminopeptidase A (APA; A II to A III), neutral endopeptidase (NEP; A I to A 1–7), and prolyl endopeptidase (PEP; which converts angiotensin [A] I and A II to A 1–7). NEP in seminal plasma and PEP activity in sperm were not found. A: angiotensin

Figure 2

Presence of aminopeptidase N (APN/CD13), angiotensin-converting enzyme (ACE/CD143) and neutral endopeptidase (NEP/CD10), in spermatozoa with DNA fragmentation ≤20% and DNA fragmentation >20% by flow cytometry, shown as (a) percentage of positive sperm and (b) marker intensity. ^*P<0.05.

Figure 3

Presence and intensity of angiotensin (A) II type 1 receptor (AT1R), A II type 2 receptor (AT2R), A IV receptor (AT4R/IRAP), (pro)renin receptor (PRR) and A 1–7 receptor (MasR) in human spermatozoa with DNA fragmentation ≤20% and DNA fragmentation >20% by flow cytometry, shown as (a) percentage of positive sperm and (b) marker intensity.

Figure 4

Results by immunocytochemistry of renin-angiotensin receptors in spermatozoa with DNA fragmentation ≤20% and with DNA fragmentation >20%: (a) angiotensin (A) II receptor type 1 (AT1R), (b) A II type 2 receptor (AT2R), (c) A IV receptor (AT4R/IRAP), (d) (pro)renin receptor (PRR), and (e) A 1–7 receptor or Mas receptor (MasR). H258: Hoechst 33258; Ac2: secondary antibody

Figure 5

Schematic representation of the sperm renin angiotensin system, with the alterations (in bold) that occur in its axes in spermatozoa with DNA fragmentation. Due to the principal location of the enzymes and receptors altered in this principal axis of renin angiotensin system (RAS) (the tail of spermatozoa), it could be suggested that the spermatic motility is modified in DNA fragmented cells. ACE: angiotensin-converting enzyme; NEP: neutral endopeptidase; PEP: prolyl endopeptidase; MasR: angiotensin (A) 1–7 or Mas receptor; APN: aminopeptidase N; APA: aminopeptidase A; APB: aminopeptidase B; AT1R: A II receptor type 1; AT2R: A II receptor type 2; AT4R: A IV receptor; PRR: (pro)renin receptor.