Sperm degradation after vasectomy follows a sperm chromatin fragmentation-dependent mechanism causing DNA breaks in the toroid linker regions

Abstract

Vasectomy is a widely used surgical technique creating an obstructive azoospermia. Although sperm cannot be ejaculated, the testis maintains sperm production in vasectomized males. The continuous accumulation of sperm deposited in the epididymis and the vas deferens fraction necessarily need to be degraded and eliminated. While the elimination process is carried out by granulomas that form after vasectomy, the detailed mechanisms of sperm degradation are still not known. The aim was to assess whether sperm chromatin fragmentation (SCF), a mechanism that degrades the entire sperm genome at the toroid linker regions (TLR), is activated after vasectomy in sperm cells. We vasectomized mice and evaluated the presence of TLR-specific double-strand breaks through pulsed-field gel electrophoresis and the Comet assay at 1, 2 and 3 weeks after surgery. Results for DNA damage (Olive tail moment) at single-cell level showed an increase of double-strand breaks after vasectomy for vas deferens sperm after 1, 2 and 3 weeks post-vasectomy (21.78 ± 2.29; 19.71 ± 1.79 and 32.59 ± 1.81, respectively), compared to mock surgery (7.04 ± 1.03; 10.10 ± 1.29 and 8.64 ± 0.85, respectively; P < 0.001). Similar findings were obtained for cauda epididymis sperm (P < 0.001), but not for caput epididymis (P > 0.05). Pulsed-field gel electrophoresis showed the presence of double-stranded breaks between 15 kb and 145 kb, indicating that DNA breaks were produced mainly in the sperm TLRs. Results presented here suggest that SCF is a mechanism activated in vas deferens after vasectomy to degrade sperm DNA when they cannot be ejaculated, preventing their function.

Low levels of mouse sperm chromatin fragmentation delay embryo development

“…While it is unclear whether H 2 O 2 can penetrate the DNA that is buried within the protamine toroids, it is reasonable to hypothesize that it could cause breaks in the DNA that is on the outer surface of the protamine toroids. The induction of DSBs by MnCl 2 through a mechanism we term SCF activates an endogenous nuclease in the lumen of the epididymis and vas deferens [16, 22] to cause DSBs in the toroid linker regions of the sperm chromatin. The SCF thus exploits the few in situ structures in compact sperm chromatin that remain accessible to nuclease digestion.…”

“…Imaging and comet images' analysis were performed as described [15, 16]. Briefly, an Olympus BX51 epifluorescence microscope (Tokyo, Japan) equipped with the FITC filter was used to visualize comets.…”

“…The MnCl 2 - or H 2 O 2 - treated samples and pulse-field gel electrophoresis (PFGE) were performed as described [16]. Briefly, epididymal sperm samples were collected in 25 mM Tris and 0.25% Tx-100.…”

“…We term this process as sperm chromatin fragmentation (SCF). The breaks that occur in SCF are largely double-stranded DNA breaks (DSBs), not SSBs [16]. In our previous experiments designed to understand the mechanisms and functions of SCF, in vitro [13, 14, 17] and in vivo [16], we digested all or most of the sperm chromatin to toroid-sized fragments.…”

“…The breaks that occur in SCF are largely double-stranded DNA breaks (DSBs), not SSBs [16]. In our previous experiments designed to understand the mechanisms and functions of SCF, in vitro [13, 14, 17] and in vivo [16], we digested all or most of the sperm chromatin to toroid-sized fragments. However, such a complete genomic degradation does not approach the much more minor fragmentation seen in clinical samples where some degradation is visible, but most of the DNA remains intact by this analysis [18].…”