José Luis Ballescà

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.

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.

Human sperm tail proteome suggests new endogenous metabolic pathways

Proteomic studies are contributing greatly to our understanding of the sperm cell, and more detailed descriptions are expected to clarify additional cellular and molecular sperm attributes. The aim of this study was to characterize the subcellular proteome of the human sperm tail and, hopefully, identify less concentrated proteins (not found in whole cell proteome studies). Specifically, we were interested in characterizing the sperm metabolic proteome and gaining new insights into the sperm metabolism issue. Sperm were isolated from normozoospermic semen samples and depleted of any contaminating leukocytes. Tail fractions were obtained by means of sonication followed by sucrose-gradient ultracentrifugation, and their purity was confirmed via various techniques. Liquid chromatography and tandem mass spectrometry of isolated sperm tail peptides resulted in the identification of 1049 proteins, more than half of which had not been previously described in human sperm. The categorization of proteins according to their function revealed two main groups: proteins related to metabolism and energy production (26%), and proteins related to sperm tail structure and motility (11%). Interestingly, a great proportion of the metabolic proteome (24%) comprised enzymes involved in lipid metabolism, including enzymes for mitochondrial beta-oxidation. Unexpectedly, we also identified various peroxisomal proteins, some of which are known to be involved in the oxidation of very long chain fatty acids. Analysis of our data using Reactome suggests that both mitochondrial and peroxisomal pathways might indeed be active in sperm, and that the use of fatty acids as fuel might be more preponderant than previously thought. In addition, incubation of sperm with the fatty acid oxidation inhibitor etomoxir resulted in a significant decrease in sperm motility. Contradicting a common concept in the literature, we suggest that the male gamete might have the capacity to obtain energy from endogenous pools, and thus to adapt to putative exogenous fluctuations.

Human sperm DNA fragmentation: Correlation of TUNEL results as assessed by flow cytometry and optical microscopy

An association between DNA fragmentation in sperm determined by the terminal deoxynucleotidyl transferase [TdT]‐mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay and the incidence of reproductive failure has been reported, either using flow cytometry or optical microscopy. However, the results obtained using each of these two approaches are different. Since there is a relative lack of studies standardizing these two approaches, the direct comparison of the results described in the different articles is difficult at present. To allow the comparison of the TUNEL results obtained using flow cytometry and optical microscopy, we applied these two approaches in a total of 66 human sperm samples. A positive correlation is detected in the TUNEL results as measured by flow cytometry and optical microscopy (Spearman; r = 0.720, P < 0.001). The percentage of TUNEL‐positive spermatozoa assessed by flow cytometry is 2.6 times higher than that detected in optical microscopy (39.7% ± 23.1% versus 15.3% ± 10.3%). Although there is a good correlation of the TUNEL results obtained by flow cytometry and optical microscopy, the percentages obtained with either technique are different. Therefore, the TUNEL results described in the present work should be valuable to compare the results described in many independent articles, using either optical microscopy or flow cytometry.

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.

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.

THE ROLE OF LUTEINIZING HORMONE IN HUMAN FOLLICLE DEVELOPMENT AND OOCYTE FERTILITY : EVIDENCE FROM IN VITRO FERTILIZATION IN A WOMAN WITH LONG-STANDIN G HYPOGONADOTROPIC HYPOGONADISM AND USING RECOMBINANT HUMAN FOLLICLE-STIMUL ATING HORMONE

To evaluate the relative importance of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in follicular development and oocyte fertility in the human species, the use of recombinant human FSH, human menopausal gonadotrophin (HMG), and very highly purified urinary human FSH (FSH-HP) plus oestradiol valerate for ovarian stimulation and in-vitro fertilization (IVF) were compared in three cycles in a woman with isolated congenital gonadotrophin deficiency who had never been treated with ovarian stimulating agents. The total number of ampoules of gonadotrophins used was lower in the HMG treatment cycle. Ovarian response and IVF outcome in the three treatment cycles were as follows: (i) HMG cycle: normal follicular growth, normal pattern of oestradiol and inhibin through the menstrual cycle, high fertilization rate (93%); (ii) recombinant FSH cycle: normal follicular growth, low oestradiol and abnormal inhibin, finally poor rate of fertilization (28%); (iii) FSH-HP plus oestradiol valerate cycle: normal follicular growth, normal pattern of inhibin and poor fertilization rate (27%). Luteal plasma progesterone concentrations were much higher in the HMG treatment cycle. This case shows that FSH is the only factor required in order to induce follicular growth in the human, although LH or a product derived from its action may assist in order to achieve full follicular maturity and oocytes capable of fertilization. Though oestradiol might have a mediatory role in the process of follicular maturation, our results favour a direct primary role of LH in complete maturation of the follicle.

Identificación de diferencias proteómicas en muestras oligozoospérmicas

Resumen Objetivos La oligozoospermia es uno de los hallazgos mas comunes presentes en los varones con subfertilidad, pero su etiologia sigue siendo desconocida en la mayoria de los casos. En este trabajo se ha propuesto utilizar la metodologia de estudio proteomico para identificar proteinas diferenciales presentes en muestras de semen de pacientes oligozoospermicos y, por tanto, potencialmente implicados en la infertilidad. Metodos Se comparo la abundancia relativa de 75 proteinas de espermatozoides en 3 grupos de 5 muestras oligozoospermicas, con las presentes en los espermatozoides de 5 donantes de semen a traves de su identificacion en electroforesis bidimensionales. Resultados En los pacientes oligozoospermicos se han identificado 14 proteinas, con una abundancia relativa diferencial de al menos 2 veces en comparacion con los controles. Proteinas detectadas como aumentadas son la creatincinasa B (CKB), proteina HINT1 (HINT1), el precursor de la succinil CoA 3 cetoacido transferasa (OXCT1), transgelina-2 (TAGLN2), 2 puntos correspondientes a la actina citoesqueletica (ActB), la glutation S-transferasa Mu 3 (GSTM3), annexina A5 (ANXA5), y la cinasa dependiente de ciclina 5 (CDK5). Proteinas detectadas como disminuidas son el precursor mitocondrial de la cadena beta de la ATP sintasa (ATP5B), proteina ODF2 (ODF2), subunidad beta de la tubulina (TUBB2), proteina CAPZ beta (CAPZB), triosafosfato isomerasa 1 (TPI1). Discusion Se han identificado varias proteinas presentes en una abundancia distinta en muestras de pacientes oligozoospermicos en comparacion con las muestras de controles. Estas proteinas se podrian considerar como candidatas para el desarrollo de marcadores diagnosticos y abren la oportunidad de profundizar en los mecanismos patogenicos implicados en la oligozoospermia.

Estudio mutacional del gen HSPA2 en pacientes estériles y en controles

Mutational study of the HSPA2 gene in infertile patients and controls Objectives: We started the present work to perform a mutational study on the HSPA2 gene in different types of infertile patients. The rationale for this work is based on the results of previous studies which suggest that the HSPA2 gene may be a candidate gene to explain some of the cases of infertility.