Sara de Mateo

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.

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.

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.

Protamine 2 precursors (Pre-P2), protamine 1 to protamine 2 ratio (P1/P2), and assisted reproduction outcome

OBJECTIVE To determine whether the presence of protamine 2 precursors (pre-P2/P2 ratio) and the protamine 1 to protamine 2 ratio (P1/P2) are related to the assisted reproduction outcome. DESIGN Prospective study. SETTING Assisted Reproduction Unit and University laboratory. PATIENT(S) One hundred two infertile patients undergoing treatment at the Assisted Reproduction Unit of the Hospital Clinic of Barcelona. INTERVENTION(S) Intracytoplasmic sperm injection (ICSI) and/or IVF treatment of the infertile patients, sperm protamine analysis through electrophoresis and densitometry, and pre-P2 analysis through Western blot. MAIN OUTCOME MEASURE(S) The presence of protamine 2 precursors (pre-P2/P2 ratio), sperm P1/P2 ratio, fertilization rates by IVF and/or ICSI, and pregnancy outcome. RESULT(S) Pre-P2/P2 and P1/P2 ratios are positively associated with the pregnancy rate. In addition, the P1/P2 ratio is positively associated with the proportion of embryos obtained by IVF, but not by ICSI. The pre-P2/P2 ratio was not related to the fertilization rate. CONCLUSION(S) Decreased pre-P2/P2 and P1/P2 ratios are related to a poor pregnancy outcome, but not with the proportion of embryos obtained after ISCI.

Protamine/DNA Ratios and DNA Damage in Native and Density Gradient Centrifuged Sperm From Infertile Patients

Protamines are the major nuclear proteins condensing DNA in the sperm nucleus. One of their proposed functions is the protection of the genetic message delivered by the sperm. To date, evidence of their involvement in DNA protection has been obtained by correlating the protamine P1/P2 ratio, protamine concentrations, or chromomycin A3 staining with DNA fragmentation. However, a correlation of the absolute protamine/DNA content with the DNA fragmentation in sperm from the same infertile patients as assessed with the comet assay has not been studied. Protamine/DNA ratios were calculated after protamine and DNA extraction, electrophoresis, and gel quantification of the protamines and DNA quantification in the sperm samples of 66 infertile patients before (native sample) and after a 2-step discontinuous PureSperm density gradient centrifuged (DGC) selection of the sperm. DNA fragmentation was assessed using the alkaline comet assay. In DGC sperm, the total protamine/DNA, P1/DNA, and P2/DNA ratios all correlated inversely with DNA damage in sperm from infertile patients. The detection of this inverse correlation between protamine/DNA ratios and DNA damage in DGC sperm adds support to the hypothesis that defective protamination is related to DNA damage in the clinically relevant subpopulation of sperm from infertile men.

A common protamine 1 promoter polymorphism (-190 C->A) correlates with abnormal sperm morphology and increased protamine P1/P2 ratio in infertile patients.

It is known that targeting the protamine 1 gene in mice leads to infertility, abnormal chromatin packaging, and abnormal sperm morphology. Because many infertile patients also have an abnormal sperm morphology and chromatin packaging, the human protamine 1 gene (PRM1) is an important candidate to screen for potential mutations. In this work, we have screened the PRM1 gene in search of potential mutations and determined the sperm morphology and the ratio between protamine 1 and protamine 2 (P1/P2 ratio). Direct sequencing of the PRM1 promoter led to the identification of a common single-nucleotide polymorphism (SNP; -190 C-->A). The -190 AA genotype was detected at a higher frequency (13.8%) in patients with markedly altered sperm morphology (<or=9% normal forms) compared with other patients (4.5%; P < .05) or compared with controls (2.97%; P < .005). The allelic frequency of the PRM1 -190 C-->A change was also consistently higher (.331) in infertile patients with a markedly altered morphology compared with population controls (.178; P < .01). Additionally, we have determined that the P1/P2 ratio is significantly increased in patients with the PRM1 -190 AA genotype compared with patients with the CA or CC genotypes (P = .006, Mann-Whitney). These findings indicate that the common PRM1 -190 C-->A polymorphism identified is associated with abnormal sperm head morphology and abnormal P1/P2 ratio in infertile patients.

Protamine 2 Precursors and Processing

Protamine 2 (P2) is synthesised as a precursor protein (pre-P2) which by proteolysis is processed to generate the mature components of the protamine 2 family of proteins (HP2, HP3 and HP4). In infertile patients, abnormal processing of the protamine 2 precursors has been suggested by the detection of an increased presence of precursor forms. However, the presence of small detectable amounts of precursor proteins has been demonstrated also in normal sperm samples, although the variation of pre-P2 in individual human sperm cells had not yet been explored. In the present manuscript we perform a mini-review describing what is known about protamine 2 precursors and P2 processing. In addition, we by immunofluorescence demonstrate the existence of a marked variation in the presence and abundance of pre-P2 in individual sperm cells.

Methods for the Analysis of the Sperm Proteome

Proteomics is the study of the proteins of cells or tissues. Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput for the identification and study of the sperm proteins. Catalogues of spermatozoal proteins in human and in model species are becoming available laying the groundwork for subsequent research, diagnostic applications, and the development of patient-specific treatments. A wide range of MS techniques is also rapidly becoming available for researchers. This chapter describes a methodological option to study the sperm cell using MS and provides a detailed protocol to identify the proteins extracted from a Percoll-purified human sperm population and separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using LC-MS/MS.

Methodological advances in sperm proteomics

Proteomics is the study of the proteins of cells or tissues. Sperm proteomics aims to identify the proteins that compose the sperm cell and the study of their function. Recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and study 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. A wide range of MS techniques are also rapidly becoming available for researchers. The present review summarises the different methodological options to study the sperm cell using MS and to provide a summary of some of the ongoing proteomic studies.

Medical Implications of Sperm Nuclear Quality

The sperm nucleus is essential in the appropriate transmission of the paternal genome. Therefore, it has long been recognized that one of the key components of the sperm nuclear quality is the quality of the genomic DNA delivered by the sperm cell. In addition, it has recently been uncovered that other important components of the quality of the sperm cell nucleus are represented by its proteome and its epigenome. Sperm genome quality implies a correct number of chromosomes and the integrity of the DNA sequence. Sperm nuclear proteome quality means an appropriate composition in nuclear proteins that organize and condense the male genome, including protamines and other chromatin-associated proteins. The specific organization of the male genome, together with appropriate DNA methylation, and other components of the epigenome such as modified histones and RNA, carried by the sperm cell, constitutes the sperm epigenome. This chapter reviews the current state of the art of the normal genomic, proteomic, and epigenetic sperm cell constitution and the proven and potential medical implications of its anomalies.