Behavior of transgenic mouse spermatozoa with galline protamine.

General morphology, physical and chemical stability of nuclei, and postfertilization behavior of spermatozoa from transgenic mice [TgN (Prml Gal) 223 Bri] containing nuclear avian protamine (galline) were compared to those in the spermatozoa of wild-type (Wild) mice. Galline to protamine I ratios in spermatozoal nuclei of transgenic mice, strains 3175 (T75) and 3177 (T77), were 1.94 and 5.62, respectively. Live T75 and T77 spermatozoa were indistinguishable in their gross morphology from Wild spermatozoa. However, unlike Wild and T75 spermatozoa, T77 spermatozoa were vulnerable to mechanical handling, as about 40% of heads and tails were separated after gentle pipetting in suspension. Motility of T77 spermatozoa was markedly inferior to that of T75 and Wild. Chromatin heterogeneity and instability of transgenic spermatozoal nuclei were evident by transmission electron microscopy, staining reaction to Giemsa, and, as apparent by both light microscopy and flow cytometry, reaction to SDS detergent. Wild and T75 spermatozoa fertilized 90% and 60% of zona-intact oocytes in vitro, respectively. T77 spermatozoa completely failed to fertilize and bound to zona surfaces very weakly, and none of them inserted their heads into the zona. Although inefficiently, T77 spermatozoa could fertilize zona-free oocytes in vitro, indicating some ability to undergo capacitation and spontaneous acrosome reaction in vitro. After microsurgical injection into oocytes, the rate of nuclear decondensation was the greatest in rooster spermatozoa, followed by T77, T75, and Wild spermatozoa.

Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis.

Susceptibility of mammalian sperm DNA to low pH- or heat-induced denaturation in situ has shown very strong dose-response relationships with animal and human exposure to chemical and physical toxicants and also fertility potential. In this study, 23 human semen samples representing a wide range in percentage (7-86%) of sperm exhibiting abnormally high susceptibility of DNA in situ to denaturation were studied for the integrity of their DNA using alkaline comet assay (single-cell microgel electrophoresis, pH 10.0). The percentage of comets observed for these samples ranged from 5 to 95%; these data correlated strongly with the percentage of sperm with increased DNA denaturability (r = 0.973; P < 0.001). Labeling of 3' ends of nicked DNA sites with 5-bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP) followed by tagging with FITC-BrdUTP monoclonal antibody and flow cytometry also indicated significantly strong correlations of BrdUTP incorporation with both abnormal susceptibility of DNA to denaturation (r = 0.859, P < 0.001) and comet assay (r = 0.812, P < 0.001). The relationship among susceptibility of sperm chromatin to acid denaturation in situ, BrdUTP incorporation, and formation of comets suggests that DNA fragmentation monitored by these assays may have important physiological relevance in terms of sperm quality and fertility potential.

Effects of heat stress on mouse testicular cells and sperm chromatin structure.

Scrotal regions of mice were exposed to a 38.0, 40.0, or 42.0 degrees C (+/-0.1) H2O bath for 60 minutes to determine the effects of elevated temperatures on testicular cells and sperm chromatin structure. Mice were killed on various days after exposure, and ratios of acridine orange-stained testicular cell populations were determined by flow cytometry. Testicular weights of mice exposed to 42.0 degrees C decreased significantly day 1 (P < 0.01) through 35 (P < 0.001). Also, a significant relative decrease in testicular haploid cells was seen on days 3-35 (P < 0.001) with a corresponding increase in the diploid population (P < 0.001). Testicular analyses of mice exposed to 38.0 degrees C were not significantly different from control values. Testis weights of mice exposed to 40.0 degrees C were not affected, but a relative decrease in percent haploid cells occurred on days 11 and 14 (P < 0.001). The sperm chromatin structure assay (SCSA) was used to measure the susceptibility of cauda epididymal sperm DNA to in situ denaturation at low pH. Caudal epididymides of mice exposed to 42.0 degrees C had no sperm. Caudal epididymal sperm from mice exposed to 40.0 degrees C were most susceptible to acid-induced DNA denaturation on days 3 (P < 0.05), 7, 11, and 14 (all P < 0.001). The 38.0 degrees C exposed mice showed some minor sperm chromatin abnormalities at later time points (days 11-35). When compared to sperm head morphology measurements, SCSA parameters were more sensitive indicators of heat-induced sperm abnormalities. These results show that mouse spermatogenesis is disrupted by scrotal exposure to environmental temperatures several degrees over normal physiological temperature and, of more biological interest, that some thermal ranges above normal allowed production of sperm with compromised nuclear chromatin structure.