179 Decondensation of bovine spermatozoa after dithiothreitol treatment

Abstract

Dithiothreitol (DTT) has been shown to reduce protamine disulfide bonds present in the outer acrosomal membrane of sperm. These disulfide bonds provide bull sperm with greater membrane stability and an increased resistance to decondensation compared with other species. (Hutchinson et al. 2017 Biophysical J. 113, 1925-1933). This can result in asynchronous fertilization due to the sperm nucleus failing to properly decondense and form male pronuclei after assisted reproductive techniques, such as IVF or intracytoplasmic sperm injection (ICSI) (Águila et al. 2017 Reproduction 154, 307-318). By reducing these bonds and disrupting the acrosomal membrane, DTT can minimize this complication, allowing the sperm nucleus to decondense more readily. This study aimed to assess bovine sperm, treated or not with DTT, for changes in morphology and to track the progress of decondensing sperm nuclei after treatment. Frozen-thawed bovine semen, utilising only egg yolk extender, was prepared by the swim-up method in the presence or absence of DTT at 5 mM. After a 1-h incubation time, permitting sperm to swim up, sperm in the top 0.5 mL in each culture tube were washed by centrifugation and observed hourly. Changes in morphology of DTT-treated and nontreated sperm were observed through a combination of phase-contrast and Hoffman modulation contrast microscopy. To assess DNA dispersal, DTT-treated sperm were stained with Hoechst and viewed under fluorescence microscopy. The DTT-treated sperm exhibited time-dependent changes in morphology, including altered movement, head folding at the equatorial segment, bending and swelling of the mid-piece, and expansion of the sperm head accompanied by a cratered appearance, signalling a partially decondensed nucleus, which progresses to full decondensation, and last, complete dispersal of DNA. These changes were seen over a 7-h incubation period in HEPES-TALP. No such changes were observed in nontreated sperm. The proportion of sperm in each stage of decondensation was estimated by a single observer at hourly time points in at least 8 repetitions. Mean estimated proportions of each stage of decondensation over time was analysed through factorial ANOVA and found significant (P < 0.001). An estimated 90% of treated sperm displayed early changes in morphology after the second hour of incubation. Sperm displaying partial decondensation of the nucleus was estimated as 42% by the third hour and increased to 62% by the fifth hour of incubation. Fully decondensed sperm or sperm with completely dispersed DNA increased from 20% at 5 h to 50% at 7 h. A remaining 10% of sperm remained unaffected and showed no changes to morphology. These results were found to be in agreement with other studies and indicate that 5 mM DTT treatment induces decondensation of bovine sperm nuclei, which may assist in male pronuclei formation for other assisted reproductive technologies.