Influence of Two Widely Used Solvents, Ethanol and Dimethyl Sulfoxide, on Human Sperm Parameters.

To study mechanisms involved in fertility, many experimental assays are conducted by incubating spermatozoa in the presence of molecules dissolved in solvents such as ethanol (EtOH) or dimethyl sulfoxide (DMSO). Although a vehicle control group is usually included in such studies, it does not allow to evaluate the intrinsic effect of the solvent on sperm parameters and its potential influence on the outcome of the experiment. In the present study, we incubated human spermatozoa for 4 h in a capacitation medium in the absence or the presence of different concentrations of EtOH and DMSO (0.1, 0.5, 1.0, and 2.0%) to assess the impact of these solvents on sperm motility, vitality, capacitation, and acrosome integrity. The presence of statistically significant relationships between increasing solvent concentrations and the investigated parameters was assessed using linear mixed models. A significant effect was observed with both solvents for total and progressive sperm motilities. We also evaluated the effect of time for these parameters and showed that the influence of the solvents was stable between 0 and 4 h, indicating an almost direct impact of the solvents. While EtOH did not influence sperm vitality and acrosome integrity, a significant effect of increasing DMSO concentrations was observed for these parameters. Finally, regarding capacitation, measured via phosphotyrosine content, although a dose-dependent effect was observed with both solvents, the statistical analysis did not allow to precisely evaluate the intensity of the effect. Based on the results obtained in the present study, and the corresponding linear mixed models, we calculated the concentration of both solvents which would result in a 5% decline in sperm parameters. For EtOH, these concentrations are 0.9, 0.7, and 0.3% for total motility, progressive motility, and capacitation, respectively, while for DMSO they are 1.5, 1.1, >2, 0.3 and >2% for total motility, progressive motility, vitality, capacitation, and acrosome integrity, respectively. We recommend using solvent concentrations below these values to dissolve molecules used to study sperm function in vitro, to limit side effects.

Targeted Analysis of HSP70 Isoforms in Human Spermatozoa in the Context of Capacitation and Motility.

HSP70s constitute a family of chaperones, some isoforms of which appear to play a role in sperm function. Notably, global proteomic studies analyzing proteins deregulated in asthenozoospermia, a main cause of male infertility characterized by low sperm motility, showed the dysregulation of some HSP70 isoforms. However, to date, no clear trend has been established since the variations in the abundance of HSP70 isoforms differed between studies. The HSPA2 isoform has been reported to play a key role in fertilization, but its dysregulation and possible relocation during capacitation, a maturation process making the spermatozoon capable of fertilizing an oocyte, is debated in the literature. The aim of the present study was to investigate the fate of all sperm HSP70 isoforms during capacitation and in relation to sperm motility. Using Multiple-Reaction Monitoring (MRM) mass spectrometry, we showed that the relative abundance of all detected isoforms was stable between non-capacitated and capacitated spermatozoa. Immunofluorescence using two different antibodies also demonstrated the stability of HSP70 isoform localization during capacitation. We also investigated spermatozoa purified from 20 sperm samples displaying various levels of total and progressive sperm motility. We showed that the abundance of HSP70 isoforms is not correlated to sperm total or progressive motility.

Influence of Risk Factors for Male Infertility on Sperm Protein Composition.

Male infertility is a common health problem that can be influenced by a host of lifestyle risk factors such as environment, nutrition, smoking, stress, and endocrine disruptors. These effects have been largely demonstrated on sperm parameters (e.g., motility, numeration, vitality, DNA integrity). In addition, several studies showed the deregulation of sperm proteins in relation to some of these factors. This review inventories the literature related to the identification of sperm proteins showing abundance variations in response to the four risk factors for male infertility that are the most investigated in this context: obesity, diabetes, tobacco smoking, and exposure to bisphenol-A (BPA). First, we provide an overview of the techniques used to identify deregulated proteins. Then, we summarise the main results obtained in the different studies and provide a compiled list of deregulated proteins in relation to each risk factor. Gene ontology analysis of these deregulated proteins shows that oxidative stress and immune and inflammatory responses are common mechanisms involved in sperm alterations encountered in relation to the risk factors.

Helicase-like transcription factor: a new marker of well-differentiated thyroid cancers.

BACKGROUND: The preoperative characterization of thyroid nodules is a challenge for the clinicians. Fine-needle aspiration (FNA) is the commonly used pre-operative technique for diagnosis of malignant thyroid tumor. However, many benign lesions, with indeterminate diagnosis following FNA, are referred to surgery. There is an urgent need to identify biomarkers that could be used with the FNA to distinguish benign thyroid nodules from malignant tumors. The purpose of the study is to examine the level of expression of the helicase-like transcription factor (HLTF) in relation to neoplastic progression of thyroid carcinomas. METHODS: The presence of HLTF was investigated using quantitative and semi-quantitative immunohistochemistry in a series of 149 thyroid lesion specimens. Our first clinical series was composed of 80 patients, including 20 patients presenting thyroid adenoma, 40 patients presenting thyroid papillary carcinoma, 12 patients presenting thyroid follicular carcinoma and 8 patients presenting anaplastic carcinoma. These specimens were assessed quantitatively using computer assisted microscopy. Our initial results were validated on a second clinical series composed of 40 benign thyroid lesions and 29 malignant thyroid lesions using a semi-quantitative approach. Finally, the HLTF protein expression was investigated by Western blotting in four thyroid cancer cell lines. RESULTS: The decrease of HLTF staining was statistically significant during thyroid tumor progression in terms of both the percentage of mean optical density (MOD), which corresponds to the mean staining intensity (Kruskall-Wallis: p < 0.0005), and the labelling index (LI), which corresponds to the percentage of immunopositive cells (Kruskall-Wallis: p < 10-6). Adenomas presented very pronounced nuclear HLTF immunostaining, whereas papillary carcinomas exhibited HLTF only in the cytoplasm. The number of HLTF positive nuclei was clearly higher in the adenomas group (30%) than in the papillary carcinomas group (5%).The 115-kDa full size HLTF protein was immunodetected in four studied thyroid cancer cell lines. Moreover, three truncated HLTF forms (95-kDa, 80-kDa and 70-kDa) were also found in these tumor cells. CONCLUSIONS: This study reveals an association between HLTF expression level and thyroid neoplastic progression. Nuclear HLTF immunostaining could be used with FNA in an attempt to better distinguish benign thyroid nodules from malignant tumors.

The ribosome inhibitor chloramphenicol induces motility deficits in human spermatozoa: A proteomic approach identifies potentially involved proteins.

Mature spermatozoa are almost completely devoid of cytoplasm; as such it has long been believed that they do not contain ribosomes and are therefore not capable of synthesising proteins. However, since the 1950s, various studies have shown translational activity within spermatozoa, particularly during their in vitro capacitation. But the type of ribosomes involved (cytoplasmic or mitochondrial) is still debated. Here, we investigate the presence and activity of the two types of ribosomes in mature human spermatozoa. By targeting ribosomal RNAs and proteins, we show that both types of ribosomes are localized in the midpiece as well as in the neck and the base of the head of the spermatozoa. We assessed the impact of cycloheximide (CHX) and chloramphenicol (CP), inhibitors of cytoplasmic and mitochondrial ribosomes, respectively, on different sperm parameters. Neither CHX, nor CP impacted sperm vitality, mitochondrial activity (measured through the ATP content), or capacitation (measured through the content in phosphotyrosines). However, increasing CP concentrations induced a decrease in total and progressive motilities as well as on some kinematic parameters while no effect was observed with CHX. A quantitative proteomic analysis was performed by mass spectrometry in SWATH mode to compare the proteomes of spermatozoa capacitated in the absence or presence of the two ribosome inhibitors. Among the ∼700 proteins identified in the different tested conditions, 3, 3 and 25 proteins presented a modified abundance in the presence of 1 and 2 mg/ml of CHX, and 1 mg/ml of CP, respectively. The observed abundance variations of some CP-down regulated proteins were validated using Multiple-Reaction Monitoring (MRM). Taken together, our results are in favor of an activity of mitochondrial ribosomes. Their inhibition by CP results in a decrease in the abundance of several proteins, at least FUNDC2 and QRICH2, and consequently induces sperm motility deficits.

Combination of galectin-3, CK19 and HBME-1 immunostaining improves the diagnosis of thyroid cancer.

Currently, fine-needle aspiration is the most frequently used pre-operative technique for diagnosis of malignant thyroid tumors, however, pathologists are unable to reach efficient and accurate differential diagnoses between benign and malignant thyroid nodules. To aid in resolving this issue, immunohistochemistry for galectins (gal)-1, -3, -7, -8, cytokeratin 19 (CK19), Hector Battifora Mesothelial Epitope-1 (HBME-1) and thyroid peroxidase (TPO) was performed on two tissue microarrays composed of 66 follicular adenomas (FA) and 66 papillary carcinomas (PC). The identification of optimal cut-off levels and the diagnostic value of single immunomarkers or combinations were evaluated using the receiver operating characteristic curve analysis. Signal intensities for gal-1, gal-3, CK19 and HBME-1 were significantly greater in PC compared with FA (P<0.001). Conversely, expression levels of TPO were significantly increased in FA compared with PC (P<0.001). Gal-3 and CK19 appeared to be the most sensitive markers (97 and 98%, respectively), whereas galectin-1 was the most specific (97%). The combination of gal-3, CK19 and HBME-1 acted as the most efficient and informative marker panel reaching the greatest specificity (97%) and sensitivity (95%) for the diagnosis of PCs. The findings suggest that this combination of markers may improve the reliability of diagnosis of thyroid cancer.

Galectin-1 is a diagnostic marker involved in thyroid cancer progression.

Fine-needle aspiration (FNA) is the most commonly used pre-operative technique for diagnosis of malignant thyroid tumor. However, many benign lesions, with indeterminate diagnosis following FNA, are referred to surgery. Based on multifunctionality of the endogenous galectin-1, we aimed to assess its status for early diagnosis of thyroid cancer. Immunohistochemistry for galectin-1 and -3 was performed on a clinical series of 69 cases of thyroid lesions. Galectin-1 expression was further examined in two additional tissue microarrays (TMA) composed of 66 follicular adenomas and 66 papillary carcinomas in comparison to galectin-3 and cytokeratin-19 (CK19). In addition, a knockdown of galectin-1 in papillary (TPC-1) and anaplastic (8505C) thyroid cancer cell lines was achieved by lentiviral transduction for in vitro experiments. A murine orthotopic thyroid cancer model was used to investigate tumor growth and metastatic ability. Immunohistochemical analyses of galectin-1 and -3 in the series of 69 cases of thyroid lesions revealed that galectin-1 was completely absent in the epithelial compartment of all benign thyroid lesions. Levels of both galectins significantly increased in the cytoplasmic compartment of malignant thyroid cells. Galectin-1 expression in the TMA yielded an excellent specificity (97%), while galectin-3 and CK19 presented a higher sensitivity (>97%) in discriminating benign from malignant thyroid lesions. In vitro experiments revealed that migration was negatively affected in TPC-1 galectin-1 knockdown (KD) cells, and that proliferation and invasion capacity of 8505C cells decreased after galectin-1 KD. Moreover, an orthotopic mouse model displayed a lower rate of tumor development with galectin-1 KD thyroid anaplastic cancer cells than in the control. Our findings support the introduction of galectin-1 as a reliable diagnostic marker for thyroid carcinomas. Its involvement in cell proliferation, migration, invasion and tumor growth also intimate functional involvement of galectin-1 in the progression of thyroid carcinoma, suggesting its potential as a therapeutic target.