Screening Ability of Female Reproductive Tract for Fragmented DNA Spermatozoa Depends on the Source of Damage.

In mammals, spermatozoa may undergo stringent selection processes within the female reproductive tract before they meet and fertilize the oocyte. The physiological bases of the sperm selection process are unknown. It has been reported that sperm DNA damage does not impair in vitro fertilization of the oocyte; however it has not been established if the in vivo selection processes for mammalian fertilization may screen the artificially induced DNA damage in spermatozoa. In this work, DNA damage of spermatozoa was artificially induced by two treatments, (a) a scrotal heat treatment (42°C for 30 min) and (b) irradiation with 137Cs γ-rays (4 Gy at 1.25 Gy/min). Twenty one days after the heat treatment and 35 days after the irradiation with 137Cs, spermatozoa were recovered from the epididymis caudae of CD1 mice and from the uterine horns near the cervix (Uc), from the uterine horns near the oviducts (Uo), and from the oviducts (Ov) of CD1 females 1-2 hours after mating with treated and control males. In each region we determined numbers of spermatozoa, motility, sperm DNA integrity by COMET assay (percentage of DNA in tail, tail lenght and the COMET moment was calculated; BOR Hourcade et al. 2007, Special issue, abstract 490, p201). In addition females naturally mated either with treated or control males were sacrificed at Day 14 of pregnancy, and number of fetuses and resorptions was recorded. One way ANOVA was used to compare the results form each group. Epididymal sperm count (12*106, 4.4*106, and 0.5*106; for control, heat treatment and 137Cs radiation treatment respectively), sperm motility (75%, 21%, and 18% respectively) and testis weight (133.90 mg, 68.76 mg and 67.16 mg respectively) were significantly reduced after heat or 137Cs radiation treatment (P<0.001). For the heat stress treatment, COMET values decreased significantly during the transit from Uc to Uo and from Uo to Ov (Tail DNA: 25.7, 23.5 and 14.4% respectively, P<0.01; Tail lenght: 38.4, 29.4 and 11.2 pixels, P<0.001; COMET Moment: 12.5, 8.5, and 2 respectively, p<0.001). For 137Cs rays treatment COMET values were similar between Uc and Uo, and only tail lenght decreased significant during the transit from Uc to Ov (Tail DNA: 32.2, 34.1 and 24.6%, P>0.05; Tail lenght: 47.1, 39.7, and 25.8 pixels respectively; P<0.001, COMET Moment: 17.2, 17.8, and 8.5 respectively, p>0.05). Heat treatment did not affect numbers of fetuses or resorptions obtained, whereas pregnancy rate and implantation rate were significantly reduced in those females mated to males exposed to 137Cs rays treatment. Our results indicate that the female reproductive tract could select spermatozoa with fragmented DNA when sperm DNA damage was produced by heat stress but could not discern sperm cells with fragmented DNA when damage was caused by irradiation with 137Cs rays. We hypothesize that female reproductive tract may not assess sperm DNA integrity directly, and thus selection should be based on sperm phenotype and/or function associated with the physical quality of DNA. The in vitro identification of these phenotypes and/or function would become ideal for the selection of spermatozoa in assisted reproductive technologies.