Paternal DNA damage resulting from various sperm treatments persists after fertilization and is similar before and after DNA replication

Abstract

In spite of its highly condensed state, sperm DNA is vulnerable to damage that can originate from oxidative stress, the activity of sperm-specific nucleases, or both. After fertilization, in the oocyte, paternal chromatin undergoes dramatic changes, and during this extensive remodeling, it can be both repaired and degraded, and these processes can be linked to DNA synthesis. Here, we analyzed sperm response to damage-inducing treatments both before and after fertilization and before or after zygotic DNA replication. Epididymal mouse spermatozoa were either frozen without cryoprotection (FT) or treated with detergent Triton X-100 coupled with dithiothreitol (TX+DTT) to induce DNA damage. Fresh, untreated sperm served as control. Immediately after preparation, spermatozoa from 3 groups were taken for comet assay, or for intracytoplasmic sperm injection into prometaphase I oocytes to visualize prematurely condensed single-chromatid chromosomes, or into mature metaphase II oocytes to visualize chromosomes after DNA replication. Comet assay revealed increased DNA fragmentation in treated sperm when compared with control, with FT sperm more severely affected. Chromosome analysis demonstrated paternal DNA damage in oocytes injected with treated, but not with fresh, sperm, with FT and TX+DTT groups now yielding similar damage. There were no differences in the incidence of abnormal paternal karyoplates before and after DNA synthesis in all examined groups. This study provides evidence that subjecting sperm to DNA damage-inducing treatments results in degradation of highly condensed sperm chromatin when it is still packed within the sperm head, and that this DNA damage persists after fertilization. The difference in DNA damage in sperm subjected to 2 treatments was ameliorated in the fertilized oocytes, suggesting that some chromatin repair might have occurred. This process, however, was independent of DNA synthesis and took place during oocyte maturation.

Fig. 1. DNA fragmentation in sperm subjected to freeze thawing and treatment with TX+DTT assessed by comet assay

A: Distribution of comet tail lengths; B: Distribution of comet tail types; C: Examples of comet tail types (1, short tail; 2, long tail, with majority of DNA still in the head; 3, long tail with DNA evenly distributed through out; 4, long tail, with most of the DNA at the distal portion. The severity of DNA damage increases proportionately with tail length and with tail type, from 1 to 4. In A and B each graph bar represents an average percentage of sperm ± standard deviation of n=3 replicates, with 50 sperm scored per replicate. Bar in C = 50 μm.

Fig. 2. Morphological changes of the oocytes maturing in vitro

A: GV oocytes collected 48 h after eCG injection; B: GV oocytes denuded from cumulus cells after 2 h of culture; C: ProMI oocytes after 4 h culture; D: oocytes that were injected with sperm at proMI stage and subsequently cultured for 18-20 h, note that these are MII oocytes but in addition to PB1 they extruded additional ‘pseudo polar bodies’; E: as in D but note that some of the oocytes did not reached MII stage and arrested at MI; F: control oocytes cultured in vitro for the same time but without sperm injection either developed to MII and extrude PB1 or remained arrested at MI stage. Scale = 100 μm

Fig. 3. BrdU staining of sperm injected immature oocytes

MII (A&B) and ProMI (C&D) oocytes were injected with sperm and cultured for 7 and 20 h, respectively, in the presence of BrdU. The oocytes were then fixed and stained with anti-BrdU antibody (green, B&D) and with propidium iodide to visualize total DNA (red, A&C). No BrdU staining was noted in oocytes injected with sperm at proMI. Bar = 50 μm.

Fig. 4. Chromosome analysis of oocytes matured in vitro

A: a clump of paternal chromatin that failed to form separate chromosomes after ICSI into proMI oocyte; B: chromosomes of oocyte that matured in vitro reaching MII (n=20); C: chromosomes of oocyte that matured in vitro but arrested at MI (n=20); D: normal maternal (m, n=20) and paternal (p, n=20) zygote chromosome complements after fresh sperm ICSI into ovulated MII oocyte; the assignation of complement origin is arbitrary in this case, based previous experience that maternal chromosomes tend to be shorter than paternal, note characteristic morphology of zygotic chromosomes different than that of MI, MII maternal and prematurely condensed paternal chromosomes shown in other figure panels; E: normal maternal (m, n=20) and paternal (p, n=20) chromosome complement after fresh sperm ICSI into proMI oocytes; note that paternal chromosomes have distinctly different morphology that allows differentiating them from maternal chromosomes and that maternal chromosomes arrested at MI; F: normal maternal (n=20) and paternal (n=20) chromosome complements after ICSI into proMI oocytes, with maternal chromosomes reaching MII; G: normal maternal (n=20) and abnormal paternal chromosome complements after ICSI with TX+DTT treated sperm into proMI oocyte; note many abnormalities of the paternal complement such as associated chromosomes (long arrows), small chromosome fragments (short arrows) and ring chromosomes (arrowheads); H: normal maternal (n=20) and abnormal paternal (n=19 + 2 large chromosome fragments shown with arrows and 1 small fragment shown by arrowhead) chromosome complements after with frozen-thawed sperm ICSI of TX+DTT treated sperm into proMI oocytes. Scale = 20 μm.