Interspecies Differences in the Stability of Mammalian Sperm Nuclei Assessed in Vivo by Sperm Microinjection and in Vitro by Flow Cytometry1

Abstract

To assess the structural stability of mammalian sperm nuclei and make interspecies comparisons, we microinjected sperm nuclei from six different species into hamster oocytes and monitored the occurrence of sperm nuclear decondensation and male pronucleus formation. The time course of sperm decondensation varied considerably by species: human and mouse sperm nuclei decondensed within 15 to 30 min of injection, and chinchilla and hamster sperm nuclei did so within 45 to 60 min, but bull and rat sperm nuclei remained intact over this same period of time. Male pronuclei formed in oocytes injected with human, mouse, chinchilla, and hamster sperm nuclei, but rarely in oocytes injected with bull or rat sperm nuclei. However, when bull sperm nuclei were pretreated with dithiothreitol (DTT) in vitro to reduce protamine disulfide bonds prior to microinjection, they subsequently decondensed and formed pronuclei in the hamster ooplasm. Condensed rat spermatid nuclei, which lack disulfide bonds, behaved similarly. The same six species of sperm nuclei were induced to undergo decondensation in vitro by treatment with DTT and detergent, and the resulting changes in nuclear size were monitored by phase-contrast microscopy and flow cytometry. As occurred in the oocyte, human sperm nuclei decondensed the fastest in vitro, followed shortly by chinchilla, mouse, and hamster and, after a lag, by rat and bull sperm nuclei. Thus species differences in sperm nuclear stability exist and appear to be related to the extent and/or efficiency of disulfide bonding in the sperm nuclei, a feature that may, in turn, be determined by the type(s) of sperm nuclear protamine(s) present.

Condensation of DNA by Spermatid Basic Nuclear Proteins

“…These experiments also show that the off-rate determined for hamster protamine 2 is not significantly different from that observed for protamine 1. This indicates that the previously reported finding that sperm containing substantial amounts of protamine 2 decondenses more rapidly in the oocyte after fertilization (42) must not be caused by differences in the affinities of the two protamines for DNA.…”

Modification of equine sperm chromatin decondensation method to use fluorescence in situ hybridization (FISH).

“…For this reason TRIS, which is a buffer solution maintaining stable (slightly alkaline) pH was used, • the detergent disodium tetraborate, which dissolves lipids. Differences within series and between animals or species may result from the number of disulfide bonds in the sperm nucleus [9,13]. Bull spermatozoa are condensed more easily than stallion spermatozoa because bull sperm nuclei are characterized by a higher concentration of disulfide bonds compared to human or mouse sperm nuclei and therefore they seem more resistant to the action of chemical preparations [9].…”

“…Glutathione is the major intercellular free thiol in mammalian oocytes. It plays a major role in reducing disulfide bonds between protamine, which contributes to subsequent dispersion of sperm chromatin during fertilization [13]. The use of GSH combined with heparin produced the expected result of decondensation of bull sperm nucleus [12].…”

Changes in spermatozoal chromatin packaging and susceptibility to oxidative challenge during aging

“…Additionally, human spermatozoa express both protamine 1 and protamine 2 (whereas rats have only protamine 1). It has been shown that the chromatin of spermatozoa from species expressing both protamines is more susceptible to decondensation (71); this property might also make it more susceptible to damage associated with oxidant exposure.…”