Rafael Oliva

VERTEBRATE PROTAMINE GENES AND THE HISTONE-TO-PROTAMINE REPLACEMENT REACTION

Publisher Summary This chapter discusses Blochs classes Type 1 (“true” protamines) and Type 2 (“stable” protamines), for which substantial molecular information is now available. Protamines are defined as having a cysteine-plus-arginine composition of 45–80 mol% and a serine plus- threonine content of 10–25 mol%. However, it should be mentioned that an additional class of sperm protamines found in the sperm nuclei of certain flat fish in the order Pleuronectiformes. These protamines are rich in arginine and serine, but are very large (80,000–200,000 daltons) compared to the typical true and stable protamines ranging from 5000 to 10,000 Da. In addition, the fact that distinctive amino acids, such as threonine or valine, present in chicken protamine polypeptides, are conserved in position in the amino acid sequence of quail only if the sequence redetermined by Oliva and Dixon is considered, makes the probability of different allelic variants in chicken much less likely.

Identification of proteomic differences in asthenozoospermic sperm samples

BACKGROUND Asthenozoospermia is one of the most common findings present in infertile males, but its aetiology remains unknown in most cases. Present proteomic tools now offer the opportunity to identify proteins which are differentially expressed in asthenozoospermic semen samples and potentially involved in infertility. METHODS We compared the expression of 101 sperm protein spots in 20 asthenozoospermic samples to that of 10 semen donor controls using two-dimensional proteomic analysis. RESULTS Seventeen protein spots have been identified at different amounts in the asthenozoospermic samples compared with controls. These are cytoskeletal actin-B, annexin-A5, cytochrome C oxidase-6B, histone H2A, prolactin-inducible protein and precursor, calcium binding protein-S100A9 (2 spots), clusterin precursor, dihydrolipoamide dehydrogenase precursor, fumarate hydratase precursor, heat shock protein-HSPA2, inositol-1 monophosphatase, 3-mercapto-pyruvate sulfurtransferase/dienoyl-CoA isomerase precursor, proteasome subunit-PSMB3, semenogelin 1 precursor and testis expressed sequence 12. The detected amount of these proteins enabled the grouping of asthenozoospermic sperm samples in an unsupervised clustering analysis. CONCLUSIONS We have identified several proteins present at different amount in asthenozoospermic sperm samples. These proteins could be candidates towards the development of diagnostic markers, and open up the opportunity to gain further insight into the pathogenic mechanisms involved in asthenozoospermia.

Familial atypical progressive supranuclear palsy associated with homozigosity for the delN296 mutation in the tau gene

Heterozygous missense and splice‐site mutations in the tau gene have been previously identified in familial frontotemporal dementia with autosomal dominant inheritance. Here we report a Spanish kindred in which two brothers born from a third‐degree consanguineous marriage were both affected with atypical progressive supranuclear palsy. A homozygous deletion at codon 296 (delN296) was identified in one of the affected siblings. Among the heterozygous carriers, two members with probable Parkinsons disease were identified, but none of heterozygotes developed atypical parkinsonism. The delN296 mutation lies in the sequence corresponding to the second tubulin‐binding repeat of tau protein and affects one asparagine residue absolutely conserved in other species. This finding indicates that homozygous mutations in the tau gene may also cause hereditary tauopathies. Ann Neurol 2001;49:263–267

SPERM CELL PROTEOMICS

The spermatozoon is an accessible cell which can be easily purified and therefore it is particularly well suited for proteomic analysis. It is also an extremely differentiated cell with very marked genetic, cellular, functional and chromatin changes as compared to other cells, and has profound implications for fertility, embryo development and heredity. The recent developments in MS have boosted the potential for identification and study of the sperm proteins. Catalogues of hundreds to thousands of spermatozoan proteins in human and in model species are becoming available setting up the basis for subsequent research, diagnostic applications and the development of specific treatments. The present article reviews the available scientific publications dealing with the composition and function of the sperm cell using an MS proteomic approach.

Significant association between the tau gene A0/A0 genotype and Parkinson's disease

A significant association between the tau gene A0/A0 genotype and progressive supranuclear palsy has been reported recently. To determine if the presence of a tau polymorphism could constitute a risk factor for the development of sporadic and familial Parkinson’s disease, a dinucleotide repeat marker at intron 11 was genotyped in 152 patients with PD, 52 patients with Alzheimers disease, and 150 healthy controls. We detected a significant difference in A0 allelic frequency in the Parkinsons disease group (79.27%) compared with the control group (71%) and the Alzheimers disease group (73.07%). Individuals homozygous for the A0 allele were also detected significantly more frequently in the Parkinsons disease group (63.8%) compared with the control group (52.66%) and the Alzheimers disease group (48.07%). These results suggest a possible involvement of the tau gene in the pathogenesis of some cases of Parkinsons disease. Ann Neurol 2000;47:242–245

The combined human sperm proteome: cellular pathways and implications for basic and clinical science

BACKGROUND The human sperm cell is very well suited for proteomic studies, as it is accessible, can be easily purified and is believed to be transcriptionally and translationally silent. The recent use of advanced proteomic approaches is clearly challenging the understanding of sperm biology. The aims of this review are to discuss the various human sperm proteomic studies, to create a compiled list of all the sperm proteins described to date and to re-assess the potential functional implications. METHODS A search of the scientific literature available in the PubMed/Medline database at 31 December 2012 was conducted for studies on human sperm proteomics. The complete list of proteins obtained was carefully analysed using different bioinformatics tools, including Reactome, a knowledgebase of biological pathways. RESULTS A total of 30 studies were identified. The proteomics studies have resulted in the identification of 6198 different proteins, an important proportion of which (around 30%) are known to be expressed in the testis. The proteins were assigned to various functional pathways, including metabolism, apoptosis, cell cycle, meiosis and membrane trafficking, among others. Unexpectedly, the sperm cell also contains a range of proteins involved in RNA metabolism and translational regulation, as well as proteins usually located in organelles believed to be absent in sperm, such as cytoplasmatic ribosomes and peroxisomes. Additionally, some proteins whose levels seem to be altered in low-quality sperm might have clinical relevance. CONCLUSIONS The analysis of the most complete sperm proteome available to date indicates the presence of several cellular protein pathways previously ignored in the male gamete. Confirming the activity of each of these pathways and understanding their biological significance will certainly boost the knowledge of human sperm and male fertility and infertility in the next years.

Prevalence of the Cys282Tyr and His63Asp HFE gene mutations in Spanish patients with hereditary hemochromatosis and in controls

Abstract Background/Aims: A mutation (Cys282Tyr) of the HFE gene has recently been reported to be present in most of the patients with hereditary hemochromatosis of Northern European ancestry, but in a lower frequency in Italy. No data are so far available on the prevalence of these mutations in Spain. Therefore, we initiated the present study to determine if the reported Cys282Tyr HFE mutations is also the main cause of hereditary hemochromatosis in Spain. In addition, we investigated the presence of the His63Asp HFE mutation in patients and in controls. Methods: Thirty-one hereditary hemochromatosis patients and 485 controls were screened for the Cys282Tyr and the His63Asp mutations, using polymerase chain reaction amplification of genomic DNA, followed by digestion with the restriction enzymes Rsa I or Dpn II, respectively, and the separation of the products by electrophoresis. Results: Twenty-seven out of 31 (87.1%) hereditary hemochromatosis patients were homozygous for the Cys282Tyr mutation. None of the patients was homozygous for the His63Asp mutation, and two patients (6.5%) were compound heterozygous (Cys282Tyr/His63Asp). Only one of 512 (0.2%) controls was homozygous for the Cys282Tyr mutation, and 29 (5.7%) were heterozygous. The Cys282Tyr mutation is present with an allelic frequency of 90.3±7.5% in patients with hereditary hemochromatosis and 3.0±1.1% in controls. Twenty out of 487 (4.1%) controls were His63Asp homozygous, while 171 (35.1%) were heterozygous. The His63Asp mutation is present with an allelic frequency of 21.7±2.7% in controls. Conclusions: The high frequency of the Cys282Tyr mutation in hereditary hemochromatosis patients indicates that this mutation is the most common defect associated with hereditary hemochromatosis in Spain. The finding of some patients with the wild genotype at position 282 suggests the existence of other changes in the HFE gene or in other loci involved in the disease. We have found one of the highest allelic frequencies reported for the His63Asp mutation in our controls (21.7±2.7%).

Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes

The exchange of histones with protamines in sperm DNA results in sperm chromatin compaction and protection. Variations in sperm protamine expression are associated with male infertility. The aim of this study was to investigate relationships between DNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradient centrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-urea polyacrylamide gels. Sperm DNA fragmentation and protamine content (P1-DNA, P2-DNA, P1+P2-DNA) decreased in spermatozoa after DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathic infertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or protamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was no correlation between protamine content (P1-DNA, P2-DNA, P1+P2-DNA) or P1/P2 ratios and IVF or ICSI pregnancies. Increased sperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo quality and reduced pregnancy rates.

Spanish Multicenter Normative Studies (NEURONORMA Project): Methods and Sample Characteristics

This paper describes the methods and sample characteristics of a series of Spanish normative studies (The NEURONORMA project). The primary objective of our research was to collect normative and psychometric information on a sample of people aged over 49 years. The normative information was based on a series of selected, but commonly used, neuropsychological tests covering attention, language, visuo-perceptual abilities, constructional tasks, memory, and executive functions. A sample of 356 community dwelling individuals was studied. Demographics, socio-cultural, and medical data were collected. Cognitive normality was validated via informants and a cognitive screening test. Norms were calculated for midpoint age groups. Effects of age, education, and sex were determined. The use of these norms should improve neuropsychological diagnostic accuracy in older Spanish subjects. These data may also be of considerable use for comparisons with other normative studies. Limitations of these normative data are also commented on.

Proteomic characterization of the human sperm nucleus.

Generating a catalogue of sperm nuclear proteins is an important first step towards the clarification of the function of the paternal chromatin transmitted to the oocyte upon fertilization. With this goal, sperm nuclei were obtained through CTAB treatment and isolated to over 99.9% purity without any tail fragments, acrosome or mitochondria as assessed by optical microscopy and transmission electron microscopy. The nuclear proteins were extracted and separated in 2‐D and 1‐D gels and the 2‐D spots and 1‐D bands were excised and analysed to identify the proteins through LC‐MS/MS. With this approach, 403 different proteins have been identified from the isolated sperm nuclei. The most abundant family of proteins identified are the histones, for which several novel members had not been reported previously as present in the spermatogenic cell line or in the human mature spermatozoa. More than half (52.6%) of the proteins had not been detected in the previous human whole sperm cell proteome reports. Of relevance, several chromatin‐related proteins, such as zinc fingers and transcription factors, so far not known to be associated with the sperm chromatin, have also been detected. This provides additional information about the nuclear proteins that are potentially relevant for epigenetic marking, proper fertilization and embryo development.