Sue Hammoud

The aetiology of sperm protamine abnormalities and their potential impact on the sperm epigenome

During the elongating spermatid stage of spermatogenesis, there is a step-wise replacement of nuclear histones with protamines 1 and 2. In fertile men, the ratio of protamine 1/protamine 2 (P1/P2) is within the narrow range of 0.8-1.2. Ratios above or below that range are associated with infertility, exhibiting a wide range of defects including decreased sperm counts, morphology, fertilization ability, and embryo implantation capacity. In this review, we highlight studies evaluating potential causes of abnormal protamine expression, including the sequencing of genes relevant to protamine expression in both affected patients and controls. While the variants of the protamine genes themselves do not appear to be responsible for most observed defects, variants of the Contrin gene, a transcription factor and translation repressor, appear to be contributory to some cases of abnormal expression. Additionally, we explore the potential effects of abnormal protamine replacement on the epigenome of human sperm. Ongoing studies are evaluating the role of retained histones and DNA methylation in sperm, which may be affected in sperm with aberrant protamine replacement. This important area of epigenetic research has profound clinical implications.

Protamine ratio and the level of histone retention in sperm selected from a density gradient preparation

Fertile males express two forms of sperm nuclear proteins, protamine 1 (P1) and protamine 2 (P2), in roughly equal quantities, whereas some infertile men have been shown to have a reduction in protamine content and an increase in the level of histones retained in mature sperm. In this study, we assessed histone and protamine levels in spermatozoa isolated from different layers of a density gradient centrifugation column to evaluate the nuclear protein content of the sperm population selected. Protamine levels were measured using acid gel electrophoresis and immunofluorescence, and the percentage of cells retaining histones was evaluated using aniline staining and immunofluorescence. Our data suggests that there is an inverse correlation between P1/P2 ratio and the level of histone expression in the different layers of the density gradient. Paradoxically, the 90% layer had a lower P1/P2 ratio, which corresponded with an increase in histone expression. It is concluded that although the sperm population selected in the 90% layer of the density gradient columns had a lower P1/P2 ratio, it was yet similar to the P1/P2 ratio observed in previously screened fertile donors.

Methylation of AR locus does not always reflect X chromosome inactivation state

Clonality can be established by a lack of mosaicism in a female because of random inactivation of either the maternal or paternal X chromosome early in embryogenesis. The methylation status of CpG sites close to the trinucleotide repeats in exon 1 of the human androgen receptor (AR) X chromosome gene assay (HUMARA) has been used to determine clonality. This HUMARA at times indicated clonal hematopoiesis in healthy elderly women, thus precluding its applicability. We used a clonality assay based on quantitative expression of polymorphic X chromosome genes (qTCA) and found no evidence of clonal hematopoiesis in healthy nonanemic elderly persons. We found instances of discordance between HUMARA results and those obtained by pyrosequencing and qTCA methods, as well as by directly quantifying AR gene expression. To determine the basis of this discrepancy we examined the methylation pattern of the AR locus subject to HUMARA. Notably, we found the extent of DNA methylation to be highly variable at the AR gene in granulocytes of persons with discordant results and also in erythroid burst-forming unit colonies but not in those with clonal hematopoiesis. These data provide the molecular basis of incomplete correlation with the pattern of DNA methylation of this X chromosome AR gene locus.

Differential methylation of pluripotency gene promoters in in vitro matured and vitrified, in vivo-matured mouse oocytes

OBJECTIVE To assess the methylation patterns of four pluripotency gene promoters in mouse oocytes after in vivo maturation, in vitro maturation (IVM), and vitrification followed by IVM. DESIGN Experimental study. SETTING Research laboratory. ANIMAL(S) Three populations of metaphase II mouse oocytes were analyzed after in vivo maturation, IVM, and vitrification followed by IVM (V-IVM). Cumulus cells and blastocyst embryos were controls. INTERVENTION(S) The CpG methylation patterns (overall and CpG specific) in the promoters of four pluripotency genes (Oct4, Nanog, Foxd3, and Sox2) were analyzed for each cell type by traditional DNA bisulfite sequencing. MAIN OUTCOME MEASURE(S) Differences for overall methylation were evaluated using the Students t-test and for individual CpG sites by χ2 analysis. RESULT(S) Significantly lower levels of overall methylation in promoters of Oct4 (25%) and Sox2 (4.5%) were noted in V-IVM oocytes compared with in vivo-matured oocytes (62.5% and 8.5%, respectively). Cumulus cell promoters were generally hypomethylated at Nanog, Foxd3. and Sox2, but hypermethylated at Oct4. CONCLUSION(S) The methylation status of Oct4 and Sox2 promoters of V-IVM mouse oocytes are altered when compared with in vivo-matured oocytes. The biological risk and significance of these changes are unknown and this study indicates caution and that further analyses are warranted.

Identification of Genetic Variation in the 5′ and 3′ Non-coding Regions of the Protamine Genes in Patients with Protamine Deregulation

Deregulation of sperm nuclear protamine ratio (P1/P2) has been shown to correlate with male factor infertility in humans, but the cause of this abnormal protein expression has yet to be identified. Recent studies have shown that there is little genetic variability in the coding regions of either of the protamine gene sequences. However, these studies did not investigate the 5′ or 3′ non-coding regions of these genes for mutations that might account for changes in the transcriptional or translational regulation of the protamines. In an effort to determine if genetic variation in these non-coding regions may account for aberrant protamine expression, we have sequenced the 5′ and 3′ untranslated regions (UTRs) of both protamine 1 (P1) and protamine 2 (P2) genes in a population of infertile men with protamine deregulation, men presenting for infertility work-up with normal protamine ratios, and a population of unrelated, fertile men from the Utah Genetic Reference Project (UGRP). This analysis has identified 14 single nucleotide polymorphisms (SNPs), of which 13 were novel SNPs in the UTRs of P1 and P2, and verified the existence of a variable length repeat (VLR), GAn, in the P2 5′ region. The SNP frequencies and VLR allelic frequencies did not achieve statistical significance between the populations, however, one of the SNPs identified in the 3′ UTR of protamine 2 was found at a low frequency in the abnormal protamine patients, but was completely absent in men with verified normal protamine ratio and donors of known fertility. In conclusion, a number of SNPs have been reported in the protamine genes and the untranslated regions, however, these gene variants do not appear to be responsible for protamine deficiency. Hence, the underlying cause for aberrant protamine expression may possibly be due to abnormalities in candidate spermatogenic transcriptional/translational regulators, post-translational modifiers, or as-of-yet unidentified factors affecting the testicular environment.

The Emerging Role of the Sperm Epigenome and its Potential Role in Development

The sperm genome has traditionally been thought to lack chromatin structure significant to affect embryonic development, since during spermatogenesis nucleosomes are widely replaced by protamines, which are believed to silence the genome, and the sperm DNA was known to be hypermethylated in comparison to the egg. The notion of an irrelevant sperm epigenome has been widely challenged due to many recent reports that suggest that sperm chromatin is actually poised similar to an embryonic stem cell, a finding that has been reported in the germline of many organisms. The significance of the mature sperm cellular epigenome is unknown; however, one may foresee two potential roles: either a role in developing embryo or a reminiscent memory of the spermatogonial stem cell with no significance beyond ensuring proper sperm differentiation and maturation. If these marks do help guide the embryo, then perturbations to the epigenome may have implications on embryo quality, likelihood of maintaining a pregnancy, or disease onset later in life.