Luminal fluid of epididymis and vas deferens contributes to sperm chromatin fragmentation

Do the luminal fluids of the epididymis and the vas deferens contribute to sperm chromatin fragmentation (SCF) in mice? The luminal fluids of both organs are required for activating SCF in mice, but the vas deferens luminal fluid does this more efficiently than that of the epididymis. Mice sperm have the ability to degrade their DNA in an apoptotic-like fashion when treated with divalent cations in a process termed SCF. SCF has two steps: the induction of reversible double-strand DNA breaks at the nuclear matrix attachment sites, followed by the irreversible degradation of DNA by nuclease. Single stranded DNA breaks accompany SCF. Luminal fluids from two reproductive organs of the mouse (B6D2F1 strain), the epididymis and vas deferens, were extracted and tested for SCF activation with divalent cations using four different combinations of the sperm and the surrounding luminal fluids: (i) in situ—sperm were kept in their luminal fluid and activated directly; (ii) reconstituted—sperm were centrifuged and resuspended in their luminal fluid before SCF activation; (iii) mixed—sperm were centrifuged and resuspended in the luminal fluid of the other organ; (iv) no luminal fluid—sperm were centrifuged and reconstituted in buffer. All four experiments were performed without (controls) and with divalent cations (resulting in SCF). For each experimental condition, two different mice were used and the analyses averaged. DNA damage by SCF was analyzed by three different methods, the sperm chromatin structure assay (SCSA), terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) analysis and field inversion gel electrophoresis. In all three assays that we used, the vas deferens luminal fluid was much more efficient in stimulating SCF in the sperm from either source than that of the epididymis (P < 0.0001). Vas deferens sperm were capable of initiating lower levels of SCF in the absence of luminal fluid (P < 0.0001). Analyses were performed in only one species, the mouse, but we used three separate assays in our analysis. The data suggest that the luminal fluid of the male reproductive tract interacts with sperm during their transit providing a mechanism to degrade the DNA. We hypothesize that this is part of an apoptotic-like mechanism that allows the reproductive tract to eliminate defective sperm. The SCF model also allowed us to identify differences in the types of DNA lesions that the three tests can identify, providing important background information for the use of these tests clinically. Funding was obtained from the National Institutes of Health, USA Grant HD060722 to W.S.W. and SCSA Diagnostics, Brookings, SD, USA. Two of the authors work for SCSA Diagnostics, and one owns the company and the patents. Trial registration number is only required for clinical trials.