Chronic testicular Chlamydia muridarum infection impairs mouse fertility and offspring development

With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality

Interaction between genetic and epigenetic variation defines gene expression patterns at the asthma-associated locus 17q12-q21 in lymphoblastoid cell lines

Phenotypic variation results from variation in gene expression, which is modulated by genetic and/or epigenetic factors. To understand the molecular basis of human disease, interaction between genetic and epigenetic factors needs to be taken into account. The asthma-associated region 17q12-q21 harbors three genes, the zona pellucida binding protein 2 (ZPBP2), gasdermin B (GSDMB) and ORM1-like 3 (ORMDL3), that show allele-specific differences in expression levels in lymphoblastoid cell lines (LCLs) and CD4+ T cells. Here, we report a molecular dissection of allele-specific transcriptional regulation of the genes within the chromosomal region 17q12-q21 combining in vitro transfection, formaldehyde-assisted isolation of regulatory elements, chromatin immunoprecipitation and DNA methylation assays in LCLs. We found that a single nucleotide polymorphism rs4795397 influences the activity of ZPBP2 promoter in vitro in an allele-dependent fashion, and also leads to nucleosome repositioning on the asthma-associated allele. However, variable methylation of exon 1 of ZPBP2 masks the strong genetic effect on ZPBP2 promoter activity in LCLs. In contrast, the ORMDL3 promoter is fully unmethylated, which allows detection of genetic effects on its transcription. We conclude that the cis-regulatory effects on 17q12-q21 gene expression result from interaction between several regulatory polymorphisms and epigenetic factors within the cis-regulatory haplotype region

New insights into the molecular basis of human sperm-egg interaction

Research Doctorate - Doctor of Philosophy (PhD)The recognition and binding of a free-swimming spermatozoon to an ovulated oocyte is one of the most important cellular interactions in biology. While traditionally viewed as a simple lock and key mechanism, emerging evidence suggests that this event may require the concerted action of several sperm proteins. In this thesis we examine the hypothesis that the activity of such proteins may be coordinated by their assembly into multimeric recognition complexes on the sperm surface. Through the novel application of blue native polyacrylamide gel electrophoresis, we tender the first direct evidence that human spermatozoa do indeed express a number of high molecular weight protein complexes on their surface. Furthermore, we demonstrate that a subset of these complexes display affinity for homologous zonae pellucidae. Proteomic analysis of two such complexes identified them as the 20S proteasome and chaperonin-containing TCP-1 (CCT) complexes. The later complex was also shown to harbour at least one putative zona pellucida binding protein, ZPBP2. Although localized in the right region of the sperm head, and appearing to play a role in primary sperm-ZP binding, neither of these complexes can account for the species-specific nature of this interaction, nor does their inhibition completely abolish the ability of mature spermatozoa to bind to homologous zonae pellucidae. While determining the precise mechanisms that underpin the binding of the two gametes is of vital importance in terms of aiding in the possible creation of new contraceptive methods, it is also significant in elucidating the reasons for the growing epidemic of male factor infertility. As one of the most common defects associated with this form of infertility is an idiopathic failure of the spermatozoon to bind to the oocyte, a study was performed to compare the proteomic profiles of spermatozoa exhibiting an impaired capacity for egg recognition with that of normal cells. This analysis indicated that impaired sperm-zona binding was associated with reduced expression of the molecular chaperone, heat shock 70kDa protein 2 (HSPA2), from the sperm proteome. Our characterisation of this chaperone revealed that it was present in the acrosomal domain of human spermatozoa and existed as a major component of 5 of the large molecular mass complexes previously isolated from human spermatozoa. The most dominant of these complexes was found to contain HSPA2 in close association with two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which have been previously implicated in sperm-egg interactions. Subsequent studies revealed that this complex undergoes a capacitation-associated translocation to facilitate the repositioning of ARSA to the apical region of the sperm head, a location compatible with a role in the mediation of sperm-ZP interactions. Conversely, SPAM1 appears to reorient away from the sperm surface, possibly reflecting its primary role in cumulus matrix dispersal preceding sperm-ZP recognition. Importantly, this dramatic relocation of the complex was abolished by incubation of capacitating spermatozoa in exogenous cholesterol or broad spectrum protein kinase A (PKA) and tyrosine kinase inhibitors, suggesting that it may be mediated by alterations in membrane fluidity and concurrently by the activation of capacitation-associated signal transduction pathways. Collectively, the data presented in this these afford novel insights into the sub-cellular localization and potential functions of a number of multimeric zona-pellucida receptor complexes in human spermatozoa

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