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Lewis, S.E.M. et al. (2013). The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment. Astract:Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes. It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage and childhood diseases. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the strengths, weaknesses and clinical applicability of current sperm DNA tests. Next, the biological significance of DNA damage in the male germ line is considered. Finally, as sperm DNA damage is often the result of oxidative stress in the male reproductive tract, the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. DNA damage in human spermatozoa is an important attribute of semen quality. It should be part of the clinical work up and properly controlled trials addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency. IntroductionMale factor infertility is implicated in more than 40% of couples presenting for assisted reproduction treatment. Conventional semen analysis continues to be the only routine test to diagnose this condition even though it is known that such descriptive assessments cannot discriminate between the spermatozoa of fertile and infertile men (Guzick et al., 2001). The shifting values for normality (all 'normal' values now lower) in the fifth edition of the WHO manual (World Health Organization, 2010) compared with the previous WHO editions may result in even less men being classified as infertile (Murray et al., 2012).

Nitric oxide (NO) enhances human sperm motility and capacitation associated with increased protein phosphorylation. NO activates soluble guanylyl cyclase, but can also modify protein function covalently via S-nitrosylation of cysteine. Remarkably, this mechanism remains unexplored in sperm although they depend on post-translational protein modification to achieve changes in function required for fertilisation. Our objective was to identify targets for Snitrosylation in human sperm. Spermatozoa were incubated with NO donors and S-nitrosylated proteins were identified using the biotin switch assay and a proteomic approach using tandem mass spectrometry. 240 S-nitrosylated proteins were detected in sperm incubated with Snitrosoglutathione. Minimal levels were observed in glutathione or untreated samples. Proteins identified consistently based on multiple peptides included established targets for S-nitrosylation in other cells e.g. tubulin,, glutathione-S-transferase and heat shock proteins but also novel targets including A-kinase anchoring protein (AKAP) types 3 and 4, voltage-dependent anion-selective channel protein 3 and semenogelin 1 and 2. In situ localisation revealed S-nitrosylated targets on the post-acrosomal region of the head and throughout the flagellum. Potential targets for Snitrosylation in human sperm include physiologically significant proteins not previously reported in other cells. Their identification will provide novel insight into the mechanism of action of NO in spermatozoa.

A functional zona pellucida is critical for both fertilization and the early stages of embryo development. Recent data from genomic and proteomic studies have questioned our simplistic view of the zona as being composed of three proteins whose functions are clearly defined. In the human, for example, the zona pellucida is composed of four proteins, not three. The increased complexity of the zona pellucida in humans and other species across the evolutionary tree now demands that we reconsider our reliance on the mouse model for understanding early fertilization events. Additionally, we are now well placed to examine, for the first time, potential defects in zona genes and their proteins associated with defined pathology.

Although sperm dysfunction is the single most common cause of infertility, we have poor methods of diagnosis and surprisingly no effective treatment (excluding assisted reproductive technology). In this review, we challenge the usefulness of a basic semen analysis and argue that a new paradigm is required immediately. We discuss the use of at-home screening to potentially improve the diagnosis of the male and to streamline the management of the sub-fertile couple. Additionally, we outline the recent progress in the field, for example, in proteomics, which will allow the development of new biomarkers of sperm function. This new knowledge will transform our understanding of the spermatozoon as a machine and is likely to lead to non-ART treatments for men with sperm dysfunction. Reproduction (2007) 133 675-684

The experiments presented in this study define the molecular basis of the bm 12 mutation. Initial characterization of an alloreactive T cell clone, 4.1.4, showed this clone to recognize an allodeterminant present on the E beta b and A beta bm12 chains, but not on the bm 12 parent A beta b chain. To define the extent of sequence shared between the I-E beta product and the mutant I-A beta product, we isolated a cDNA clone of the E beta b gene and determined its nucleotide sequence. Comparison of the nucleotide sequences of E beta b, A beta b, and A beta bm12 shows the the A beta bm12 gene to be identical to the E beta b gene in the region where it differs from its A beta b parent. We predict that the bm 12 mutation arose by gene conversion of this region, which spans 14 nucleotides between amino acid residues 67-71 of the mature A beta chain, from the E beta b locus to the corresponding position at the A beta b locus. Recognition of this region, which spans one of the previously defined E beta allelic "hypervariable" regions, by an alloreactive T cell clone provides the first direct evidence of the functional importance of these hypervariable regions in T cell stimulation. The identification of a gene conversion event involving one of these allelic variable regions implicates conversion as a mechanism that acts on class II beta genes to create sequence diversity in regions of Ia molecules that interact with foreign antigen or a T cell receptor, regions where protein sequence polymorphism would presumably be selected for by the expanded ability it affords the organism to mount effective immune responses against a wider variety of foreign antigens.

The vertebrate egg envelope is composed of a family of related proteins, the zona pellucida (ZP) proteins, which are characterized by the presence of a conserved zona pellucida domain. Analysis of teleost fish ZP gene sequences has demonstrated that there are no direct orthologues of the mammalian ZPB and ZP1 genes, but that teleost fish contain multiple copies of two classes of genes (ZPXa and ZPXb) that are equally related to ZPB and ZP1. The two classes of genes are further distinguished by expression in liver or ovary, respectively, indicating there was probably an initial duplication event, followed by a switch to hepatic expression of one of the paralogues. This switch was followed in some species by additional amplification of one of the paralogues with the subsequent loss of the other. It is proposed that the expansion of the number of ZPXa and ZPXb genes and the acquisition of dual sites of synthesis are the result of an ancient polyploidization event, followed by additional species-specific gene amplifications.

Introduction: Microarray gene-expression profiling is a powerful tool for global analysis of the transcriptional consequences of disease phenotypes. Understanding the genetic correlates of particular pathological states is important for more accurate diagnosis and screening of patients, and thus for suggesting appropriate avenues of treatment. As yet, there has been little research describing gene-expression profiling of infertile and subfertile men, and thus the underlying transcriptional events involved in loss of spermatogenesis remain unclear. Here we present the results of an initial screen of 33 patients with differing spermatogenic phenotypes. Methods: Oligonucleotide array expression profiling was performed on testis biopsies for 33 patients presenting for testicular sperm extraction. Significantly regulated genes were selected using a mixed model analysis of variance. Principle components analysis and hierarchical clustering were used to interpret the resulting dataset with reference to the patient history, clinical findings and histological composition of the biopsies. Results: Striking patterns of coordinated gene expression were found. The most significant contains multiple germ cell-specific genes and corresponds to the degree of successful spermatogenesis in each patient, whereas a second pattern corresponds to inflammatory activity within the testis. Smaller-scale patterns were also observed, relating to unique features of the individual biopsies.