Distribution of DNA, nuclear micro-heterogeneities and compaction of the chromatin in rabbit epididymal spermatozoa. Ultrastructural evaluation of the Feulgen-like technique using osmium ammine

Abstract

Summary ― An adaptation of the Feulgen procedure to visualise DNA at the ultrastructural level, using osmium ammine instead of Schiff reagent, was applied to ultrathin sections of rabbit epididymal spermatozoa, known to display increasing chromatin compaction as they progress through the epididymis. In contrast with the somatic cell nuclei of the epididymal epithelium, which display classical staining, the chromatin of spermatozoa is partly or fully destroyed during the hydrolysis step of the technique. The sperm chromatin resistance towards destruction is a function of the initial sperm nuclear compaction and of the duration of hydrolysis prior to Feulgen-like staining. For a given nucleus, the maximum staining intensity is only obtained after an optimal duration of hydrolysis. However, because of this duration and local differences in chromatin compaction, the total DNA of a section is never completely visualised.

CpG DNA methylation changes during epididymal sperm maturation in bulls

“…Corpus epididymis profile is likely to have an intermediate methylation level between caput and cauda, however we observed specific dynamic changes in DNA methylation in that region. Several direct and indirect observations can support our results: (i) our measurements show that epididymis corpus presents significant alterations in variables related to sperm with defect in acrosome integrity, and sperm morphology; (ii) chromatin changing was previously observed to specifically occur in the epididymis corpus [ 22 , 33 – 35 ]; (iii) non-coding RNA profiling of bull sperm isolated from testis to ejaculated sperm showed that small RNA sperm content is markedly remodeled as sperm matures along the epididymis, with specific enrichment in ribosomal RNA and also in 20 nt long piRNAs in the epididymis corpus [ 17 ], (iv) specific remodeling in histone post-translational modifications was observed in mouse and ram sperm isolated from caput and cauda, which is likely to shape the paternally inherited epigenome and may influence spatial organization of the genome in the early embryo [ 18 ]. Unfortunately, this study did not investigate histone modification in the corpus epididymis [ 18 ].…”

“…Analysis of sperm chromatin compaction by Feulgen-like technique showed variation in different epididymis tracts in rabbit [ 33 ]. Using the same technique, ram sperm isolated from the corpus of the epididymis showed the greatest decrease in Feulgen-DNA and surface area compared with spermatozoa from the other regions, which indicates a marked restructuration of the chromatin in this region of the epididymis [ 34 ].…”

Ultrastructural immunolocalisation of histones (H2B, H3, H4), transition protein (TP1) and protamine in rabbit spermatids and spermatozoa nuclei. Relation to condensation of the chromatin

“…The lack of one protamine species is also documented in human (De Yebra etal, 1993). In the rabbit, chromatin micro-heterogeneities, characterised by easily extractable protamine, have been described in spermatozoa as sites where the DNA is more accessible (Courtens et al, 1994a). However, in this species, the frequency of sperm nuclei which spontaneously decondense is the main factor related to low litter sizes (Courtens et al, 1994b), one of the main parameters of fertility.…”

“…Moreover, transition proteins (Akama et al, 1994a) and protamines (Chirat et al, 1991;Green et al, 1994) are phosphorylated/dephosphorylated to different degrees, which hypothetically leads to the final tightness of the chromatin. Rabbit sperm nuclei normally display microheterogeneities in protein/DNA concentrations (Courtens et al, 1991(Courtens et al, , 1994a and the condensation of sperm chromatin in the rabbit is also spontaneously reversible in 3-6% of the cells. A variable proportion of decondensing spermatozoa is found in all ejaculates and is inversely related to litter size (Courtens etal, 1994b).…”

A brief history of the Feulgen reaction

“…Interestingly, detection of DNA at the EM level showed some strange patterns in particular types of chromatin. In mature sperm nuclei in mouse (Biggiogera 1986 ) and other species (Courtens et al 1991 , 1994 ), the cell nucleus, homogeneously stained after exposure to uranyl and lead, becomes stained in spots, as if the DNA was unevenly distributed. The reason for this behaviour lies in microheterogeneities of the protamine–protamine interaction (Boutinard Rouelle-Rossier and Biggiogera 1992 ) rather than reflecting biological features.…”