RESUMO
In brief: Although common in many commercial extenders, supraphysiological concentrations of glucose in the media may be detrimental to stallion spermatozoa. In this study, we present evidence that these elevated glucose levels may predispose spermatozoa to ferroptosis. Abstract: Stallion spermatozoa depend on oxidative phosphorylation as their major source of ATP; however, the metabolism of these cells is complex and a great degree of metabolic plasticity exists. The composition of the media in which the spermatozoa are extended, or exposed to in the mare's reproductive tract, exerts a profound effect on sperm function and may even accelerate cell demise. Recent research indicates that high concentrations of glucose in the media, although common in commercial extenders, may be detrimental. To determine if supraphysiological glucose concentration may induce or predispose to ferroptosis (a caspase-independent form of programmed cell death, triggered by oxidative stress), stallion spermatozoa were incubated under different concentrations of glucose, 67 mM (HG) or 1 mM plus 10 mM pyruvate (LG-HP), in the presence or absence of known inductors of ferroptosis. Furthermore, we developed a single-cell flow metabolic assay to identify different metabolic phenotypes in spermatozoa. Storage and incubation of spermatozoa under high glucose concentrations led to an increase in the percentage of necrotic spermatozoa (P < 0.0001). Moreover, ferroptosis was induced more intensely in sperm in media with high glucose concentrations (P < 0.0001). Finally, we observed that induction of ferroptosis modified two proteins (oxoglutarate dehydrogenase and superoxide dismutase 2) in spermatozoa incubated in media containing 67 mM glucose but not in media containing 1 mM glucose and 10 mM pyruvate. The composition of the media, especially the concentration of glucose, exerts a major impact on the functionality and life span of the spermatozoa. The results reported here may pave the way for improvements in existing semen extenders.
Assuntos
Ferroptose , Preservação do Sêmen , Animais , Cavalos , Masculino , Feminino , Glucose/farmacologia , Glucose/metabolismo , Sêmen , Espermatozoides/metabolismo , Ácido Pirúvico/farmacologia , Ácido Pirúvico/metabolismo , Motilidade dos Espermatozoides , Preservação do Sêmen/métodosRESUMO
Although cryopreservation is widely used in animal breeding, the technique is still suboptimal. The population of spermatozoa surviving the procedure experiences changes attributed to alteration in their redox regulation. In order to expand our knowledge regarding this particular aspect, the proteome in fresh and frozen thawed aliquots of equine spermatozoa was studied to identify the proteins most severely affected by the procedure. If alteration of redox regulation is a major factor explaining cryodamage, proteins participating in redox regulation should be principally affected. Using a split sample design, 30 ejaculates from 10 different stallions were analyzed as fresh spermatozoa, and another aliquot from the same ejaculate was analyzed as a frozen thawed sample. The proteome was studied under both conditions using UHPLC-MS/MS and bioinformatic analysis conducted to identify discriminant variables between both conditions. Data are available through the ProteomeXchange Consortium with identifier PXD022236. The proteins most significantly reduced were Aldo-keto reductase family 1 member B (p = 2.2 × 10-17) and Superoxide dismutase (Cu-Zn) (p = 4.7 × 10-14). This is the first time that SOD1 has been identified as a discriminating variable using bioinformatic analysis, where it was one of the most highly significantly different proteins seen between fresh and frozen thawed semen. This finding strongly supports the theory that alteration in redox regulation and oxidative stress is a major factor involved in cryodamage and suggests that control of redox regulation should be a major target to improve current cryopreservation procedures.
Assuntos
Criopreservação , Espectrometria de Massas em Tandem , Aldeído Redutase , Animais , Cavalos , Masculino , Oxirredutases , Motilidade dos Espermatozoides , Espermatozoides , Superóxido Dismutase/genética , Superóxido Dismutase-1/genéticaRESUMO
The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design. Half of the ejaculate was analyzed as a fresh aliquot and the other half was frozen and then analyzed as a frozen-thawed aliquot. Computer-assisted sperm analysis and flow cytometry were used to analyze sperm quality. Detailed proteomic analysis was performed on fresh and frozen and thawed aliquots, and bioinformatic analysis was used to identify discriminant variables in fresh samples able to predict the outcome of cryopreservation. Those with a fold change > 3, a P = 8.2e-04, and a q = 0.074 (equivalent to False discovery rate (FDR)) were selected, and the following proteins were identified in fresh samples as discriminant variables of good motility post-thaw: F6YTG8, K9K273, A0A3Q2I7V9, F7CE45, F6YU15, and F6SKR3. Other discriminant variables were also identified as predictors of good mitochondrial membrane potential and viability post-thaw. We concluded that proteomic approaches are a powerful tool to improve current sperm biotechnologies.
Assuntos
Criopreservação/veterinária , Cavalos/fisiologia , Proteoma/química , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Masculino , Espermatozoides/químicaRESUMO
Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by centrifugation. In this study, the SP-proteome of 10 different stallions was defined through high-performance liquid chromatography with tandem mass spectrometry and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5°C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direct extension in a commercial extender (good), stallions requiring removal of seminal plasma (RSP) to maintain seminal quality (good-RSP), and stallions, unable to maintain good semen quality even after RSP (poor). Pathway enrichment analysis of the proteins identified in whole equine SP using human orthologs was performed using g: profiler showing enriched Reactome and the Kyoto Encyclopedia of Genes and Genomes pathways related to hexose metabolism, vesicle mediated transport, post translational modification of proteins and immune response. Specific proteins overrepresented in stallions tolerating conservation by refrigeration included a peroxiredoxin-6 like protein, and transcobalamin-2, a primary vitamin B12-binding, and transport protein. Also, the protein involved in protein glycosylation, ST3 beta-galactoside alpha-2,3-sialyltransferase 1 was present in good stallions. These proteins were nearly absent in poor stallions. Particularly, annexinA2 appeared as to be the most powerful discriminant variable for identification of stallions needing RSP prior to refrigeration, with a P = 0.002 and a q value = 0.005. Overall this is the first detailed study of the equine SP-proteome, showing the potential value of specific proteins as discriminant bio-markers for clinical classification of stallions for AI.
Assuntos
Anexinas/metabolismo , Cavalos/fisiologia , Refrigeração , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Biomarcadores/química , Sobrevivência Celular/fisiologia , Masculino , Proteínas de Plasma Seminal/química , Proteínas de Plasma Seminal/metabolismoRESUMO
The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not individually predictive of fertilizing potential. The aim of this study was to harness mass spectrometry to compare the proteomic profiles of high- and low-quality stallion spermatozoa, with the ultimate goal of identifying fertility biomarker candidates. Extended stallion semen (n = 12) was fractionated using Percoll density gradients to isolate low-quality and high-quality sperm populations. Motility and morphological assessments were carried out, and proteomic analyses was conducted using UHPLC-MS/MS. High-quality spermatozoa recorded higher total (95.2 ± 0.52% vs 70.6 ± 4.20%; P ≤ 0.001) and progressive motilities (43.4 ± 3.42% vs 27.3 ± 4.32%; P ≤ 0.05), and a higher proportion of morphologically normal cells (50.2 ± 4.34% vs 38.8 ± 2.72%; P ≤ 0.05). In total, 1069 proteins were quantified by UHPLC-MS/MS, of which 22 proteins were significantly more abundant in the high-quality sperm population (P ≤ 0.05). A-kinase anchor protein 4 (AKAP4) and Hexokinase 1 (HK1) were considered possible biomarker candidates and their differential expression was confirmed by immunoblot. Protein expression was significantly correlated with total (AKAP4 R2 = 0.38, P ≤ 0.01; HK1 R2 = 0.46, P ≤ 0.001) and progressive motilities (AKAP4 R 2 = 0.51, P ≤ 0.001; HK1 R2 = 0.55, P ≤ 0.01), percentage rapid (AKAP4 R2 = 0.29, P ≤ 0.05; HK1 R2 = 0.58, P ≤ 0.001), straight-line velocity (HK1 R2 = 0.50, P ≤ 0.01) and straightness (HK1 R2 = 0.40, P ≤ 0.01). Furthermore, AKAP4 was highly susceptible to adduction by 4-hydroxynonenal (4HNE), which resulted in a global reduction in the phosphorylation profiles following capacitation. In conclusion, the proteomic profiles of high- and low-quality stallion spermatozoa differ substantially, and proteins such as AKAP4 and HK1 could serve as biomarkers of ejaculate quality.
Assuntos
Proteoma/metabolismo , Análise do Sêmen/veterinária , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Cavalos , Masculino , Proteoma/análise , Espermatozoides/fisiologiaRESUMO
We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 µM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.
Assuntos
Aldeídos/metabolismo , Senescência Celular , Glutationa/fisiologia , Espermatozoides/fisiologia , Compostos de Sulfidrila/metabolismo , Aldeídos/farmacologia , Animais , Senescência Celular/efeitos dos fármacos , Glutationa/metabolismo , Cavalos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Análise do Sêmen , Preservação do Sêmen , Motilidade dos Espermatozoides , Espermatozoides/química , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismoRESUMO
If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are common in current commercial extenders could be counteracted. To test this hypothesis, spermatozoa were incubated in a 67 mM Glucose modified Tyrode's media in the presence of 1- or 10-mM pyruvate and in the Tyrode's basal media which contains 5 mM glucose. Spermatozoa incubated for 3 h at 37 °C in 67 mM Tyrode's media with 10 mM pyruvate showed increased motility in comparison with aliquots incubated in Tyrode's 5 mM glucose and Tyrode's 67 mM glucose (57.1 ± 3.5 and 58.1 ± 1.9 to 73.0 ± 1.1 %; P < 0.01). Spermatozoa incubated in Tyrode's with 67 mM glucose 10 mM pyruvate maintained the viability along the incubation (64.03 ± 15.4 vs 61.3 ± 10.2), while spermatozoa incubated in 67 mM Glucose-Tyrode's showed a decrease in viability (38.01 ± 11.2, P < 0.01). 40 mM oxamate, an inhibitor of the lactate dehydrogenase LDH, reduced sperm viability (P < 0.05, from 76 ± 5 in 67 mM Glucose/10 mM pyruvate to 68.0 ± 4.3 %, P < 0.05). Apoptotic markers increased in the presence of oxamate. (P < 0.01). UHPLC/MS/MS showed that 10 mM pyruvate increased pyruvate, lactate, ATP and NAD+ while phosphoenolpyruvate decreased. The mechanisms that explain the improvement of in presence of 10 mM pyruvate involve the conversion of lactate to pyruvate and increased NAD+ enhancing the efficiency of the glycolysis.
Assuntos
Ácido Pirúvico , Sêmen , Masculino , Animais , Cavalos , Ácido Pirúvico/farmacologia , Ácido Pirúvico/metabolismo , NAD/farmacologia , NAD/metabolismo , Espectrometria de Massas em Tandem/veterinária , Motilidade dos Espermatozoides , Espermatozoides , Lactatos/metabolismo , Lactatos/farmacologia , Glucose/farmacologia , Glucose/metabolismoRESUMO
It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6-19 rams at two very distant points in the mating season (July as Early Breeding Season -EBS- and November as Late Breeding Season -LBS-). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.
Assuntos
Proteômica , Sêmen , Masculino , Gravidez , Ovinos , Animais , Feminino , Estações do Ano , Carneiro Doméstico , Fertilidade , Espermatozoides , Proteínas do EspermatozoideRESUMO
BACKGROUND: Most commerce of equine seminal doses is carried out using commercial extenders under refrigeration at 5°C. OBJECTIVES: To determine if 10 mm pyruvate in a 67 mm glucose extender and storage at 22°C could be the basis of an alternative storage method to cooling to 5°C. MATERIAL AND METHODS: Stallion ejaculates were extendedin: INRA96 (67 mm glucose, non-pyruvate control), modified Tyrode's (67 mm glucose-10 mm pyruvate), supplemented with 0, 10, 50, and 100 µM itaconate. As itaconate was vehiculated in DMSO, a control vehicle was also included. Sperm motility, viability, mitochondrial membrane potential, and production of reactive oxygen species were measured after collection and again after 48 and 96 h of storage at 22°C. To disclose molecular metabolic changes, spermatozoa were incubated up to 3 h in modified Tyrode's 67 mm glucose-10 mm pyruvate and modified Tyrode's 67 mm glucose, and metabolic analysis conducted. RESULTS: After 96 h of storage aliquots stored in the control, INRA96 had a very poor total motility of 5.6% ± 2.3%, while in the 67 mm glucose-10 mm pyruvate/10 µm itaconate extender, total motility was 34.7% ± 3.8% (p = 0.0066). After 96 h, viability was better in most pyruvate-based media, and the mitochondrial membrane potential in spermatozoa extended in INRA96 was relatively lower (p < 0.0001). Metabolomics revealed that in the spermatozoa incubated in the high pyruvate media, there was an increase in the relative amounts of NAD+ , pyruvate, lactate, and ATP. DISCUSSION AND CONCLUSIONS: Aliquots stored in a 67 mm glucose-10 mm pyruvate-based medium supplemented with 10 µM itaconate, maintained a 35% total motility after 96 h of storage at 22°C, which is considered the minimum acceptable motility for commercialization. Improvements may be related to the conversion of pyruvate to lactate and regeneration of NAD+ .
RESUMO
This paper provides a detailed set of data on how the stallion sperm proteome differs among stallions with different sperm motilities, although within normal ranges. Findings distinguish proteins that may help to identify stallions of superior sperm motility. Sperm proteins were analyzed using a UHPLC/MS/MS system comprising of an Agilent 1290 infinity series UHPLC coupled to an Agilent 6550 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). These data can be used to disclose potential targets to identify good sperm samples and to study specific pathways involved in the regulation of sperm motility. This data article is linked to the paper "Proteins involved in mitochondrial metabolic functions and fertilization predominate in stallions with better motility Journal of Proteomics 247:104335 doi: 10.1016/j.jprot.2021.104335".
RESUMO
Seminal plasma plays an important role in sperm physiology. Seminal plasma proteins vehiculated in microvesicles, carry RNAs and proteins with a potential role in early embryo development. Additionally, proteins present in seminal plasma participate in redox regulation and energy metabolism. In view of these facts, we hypothesized that differences in protein composition of the seminal plasma among stallions may help to explain differences in freeze-ability seen among them. Three independent ejaculates from 10 different stallions of varying breeds were frozen using standard protocols in our laboratory. Aliquots of the ejaculate were separated and stored at -80 °C until further proteomic analysis. Semen analysis was performed using computer assisted sperm analysis and flow cytometry. Significant differences in proteome composition of seminal plasma were observed in the group of stallions showing better motility post thaw. 3116 proteins were identified, and of these, 34 were differentially expressed in stallions with better motility post thaw, 4 of them were also differentially expressed in stallions with different percentages of linearly motile sperm post thaw and 1 protein, Midasin, was expressed in stallions showing high circular velocity post thaw. Seminal plasma proteins may play a major role in sperm functionality; being vehiculated through extracellular vesicles and participating in sperm physiology. Bioinformatic analysis identifies discriminant proteins able to predict the outcome of cryopreservation, identifying potential new biomarkers to assess ejaculate quality.
Assuntos
Preservação do Sêmen , Adenina , Animais , Arginina , Criopreservação/veterinária , Cavalos , Masculino , Metiltransferases , Proteômica , Sêmen , Preservação do Sêmen/veterinária , Proteínas de Plasma Seminal , Motilidade dos Espermatozoides , EspermatozoidesRESUMO
Even in stallions with sperm quality within normal reference ranges at ejaculation, subtle differences in sperm quality exist that in many cases lead to reduced time frames for conservation of the ejaculate and/or reduced fertility. The spermatozoon is a cell highly suitable for proteomics studies, and the use of this technique is allowing rapid advances in the understanding of sperm biology. The aim of the present study was to investigate differences among stallions of variable sperm quality (based on motility and sperm velocities), although all horses had sperm characteristics within normal ranges. The proteome was studied using UHPLC/MS/MS and posterior bioinformatic and enrichment analysis; data are available via ProteomeXchange with identifier PXD025807. Sperm motility, linear motility and circular, straight line and average velocities (VCL, VSL, VAP) were measured using computer assisted sperm analysis (CASA). In stallions showing better percentages of motility, circular and average velocity predominated mitochondrial proteins with roles in the Citric acid cycle, pyruvate metabolism and oxidative phosphorylation. Interestingly, in stallions with better percentages of total motility, sperm proteins were also enriched in proteins within the gene ontology (G0) terms, single fertilization (G0: 0007338), fertilization (G0: 0009566), and zona pellucida receptor complex (GO:0002199). The enrichment of this proteins in samples with better percentages of total motility may offer a molecular explanation for the link between this parameter and fertility. SIGNIFICANCE: Proteomic analysis identified a high degree of specificity of stallion sperm proteins with discriminant power for motility, linear motility, and sperm velocities (VCL, VAP and VSL). These findings may represent an interesting outcome in relation to the molecular biology regulating the movement of the spermatozoa, and the biological meaning of the measurements that computer assisted sperm analysis (CASA) provide. Of a total of 903 proteins identified in stallion spermatozoa, 24 were related to the percentage of total motility in the sample; interestingly, gene ontology (G0) analysis revealed that these proteins were enriched in terms like single fertilization and fertilization, providing a molecular link between motility and fertility. Field studies indicate that the percentage of total motility is the CASA derived parameter with the best correlation with fertility in stallions.
Assuntos
Preservação do Sêmen , Motilidade dos Espermatozoides , Animais , Fertilização , Cavalos , Masculino , Proteômica , Espermatozoides , Espectrometria de Massas em TandemRESUMO
Proteomic technologies allow the detection of thousands of proteins at the same time, being a powerful technique to reveal molecular regulatory mechanisms in spermatozoa and also sperm damage linked to low fertility or specific biotechnologies. Modifications induced by the cryopreservation in the stallion sperm proteome were studied using UHPLC/MS/MS. Ejaculates from fertile stallions were collected and split in two subsamples, one was investigated as fresh (control) samples, and the other aliquot frozen and thawed using standard procedures and investigated as frozen thawed subsamples. UHPLC/MS/MS was used to study the sperm proteome under these two distinct conditions and bioinformatic enrichment analysis conducted. Gene Ontology (GO) and pathway enrichment analysis were performed revealing dramatic changes as consequence of cryopreservation. The terms oxidative phosphorylation, mitochondrial ATP synthesis coupled electron transport and electron transport chain were significantly enriched in fresh samples (Pâ¯=â¯5.50â¯×â¯10-12, 4.26â¯×â¯10-8 and 7.26â¯×â¯10-8, respectively), while were not significantly enriched in frozen thawed samples (Pâ¯=â¯1). The GO terms oxidation reduction process and oxidoreductase activity were enriched in fresh samples and the enrichment was reduced in frozen thawed samples (1.40â¯×â¯10-8, 1.69â¯×â¯10-6 versus 1.13â¯×â¯10-2 and 2-86â¯×â¯10-2 respectively). Reactome pathways (using human orthologs) significantly enriched in fresh sperm were TCA cycle and respiratory electron transport (Pâ¯=â¯1.867â¯×â¯10-8), Respiratory electron transport ATP synthesis by chemiosmosis coupling (Pâ¯=â¯2.124â¯×â¯10-5), Citric acid cycle (TCA cycle)(Pâ¯=â¯8.395â¯×â¯10-4) Pyruvate metabolism and TCA cycle (Pâ¯=â¯3.380â¯×â¯10-3), Respiratory electron transport (Pâ¯=â¯2.764â¯×â¯10-2) and Beta oxidation of laurolyl-CoA to decanoyl CoA-CoA (Pâ¯=â¯1.854â¯×â¯10-2) none of these pathways were enriched in thawed samples (Pâ¯=â¯1). We have provided the first detailed study on how the cryopreservation process impacts the stallion sperm proteome. Our findings identify the metabolic proteome and redoxome as the two key groups of proteins affected by the procedure. SIGNIFICANCE: In the present manuscript we investigated how the cryopreservation of stallion spermatozoa impacts the proteome of these cells. This procedure is routinely used in horse breeding and has a major impact in the industry, facilitating the trade of genetic material. This is still a suboptimal biotechnology, with numerous unresolved problems. The limited knowledge of the molecular insults occurring during cryopreservation is behind these problems. The application and development of proteomics to the spermatozoa, allow to obtain valuable information of the specific mechanisms affected by the procedure. In this paper, we report that cryopreservation impacts numerous proteins involved in metabolism regulation (mainly mitochondrial proteins involved in the TCA cycle, and oxidative phosphorylation) and also affects proteins with oxidoreductase activity. Moreover, specific proteins involved in the sperm-oocyte interaction are also affected by the procedure. The information gathered in this study, opens interesting questions and offer new lines of research for the improvement of the technology focusing the targets here identified, and the specific steps in the procedure (cooling, toxicity of antioxidants etc.) to be modified to reduce the damage.
Assuntos
Preservação do Sêmen , Animais , Criopreservação , Cavalos , Humanos , Masculino , Oxirredução , Proteômica , Motilidade dos Espermatozoides , Espermatozoides , Espectrometria de Massas em TandemRESUMO
This work presents the development of a method based on a voltammetric determination coupled to second-order data modeling with multivariate curve resolution-alternating least-square (MCR-ALS) and unfolded partial least squares regression followed by residual bilinearization (U-PLS/RBL) for the quantitation of the pesticide ethiofencarb in the presence of interferences in tap water. The determination of ethiofencarb by voltammetry is possible on the basis that this pesticide is hydrolysed in alkaline media giving rise to a compound which shows an oxidation peak at Ep=0.80 V. But some other pesticides belonging to the same family can be considered as interferences due to the high overlapping in their signals. The two such interferences are fenobucarb and bendiocarb, which have not been included in the calibration step. The possibility of second-order multivariate calibration was studied by using the hydrolysis time as the third variable, and MCR-ALS and U-PLS/RBL. Good results were obtained in this determination in spite of the high overlapping in voltammetric signals.
Assuntos
Algoritmos , Carbamatos/análise , Água Potável/química , Técnicas Eletroquímicas , Praguicidas/análise , Poluentes Químicos da Água/análise , Calibragem , Carbamatos/química , Humanos , Hidrólise , Análise dos Mínimos Quadrados , Praguicidas/química , Fenilcarbamatos/química , Poluentes Químicos da Água/químicaRESUMO
A method for the determination of several tricyclic antidepressants (imipramine, desipramine, amitriptyline, nortriptyline, clomipramine, norclomipramine, doxepine and nordoxepine) in breast milk has been developed. This assay consists of a common extraction process in an organic phase, which is evaporated until dried and finally reconstituted in the appropriate buffer for injection in a capillary electrophoresis system. The capillary electrophoresis method used is an "acetonitrile stacking" method previously reported for determining these drugs in serum samples. The method developed was applied to the analysis of these compounds in human breast milk at different concentration levels (50, 100 and 200 ppb of the TCAs hydrochlorides). An interference study of some ansiolitic drugs such as lorazepam and alprazolam was made.
Assuntos
Antidepressivos Tricíclicos/análise , Eletroforese Capilar/métodos , Leite Humano/química , Contaminação de Alimentos/análise , Humanos , Reprodutibilidade dos TestesRESUMO
A robustness study for a sensitive-stacking capillary electrophoresis method based on "acetonitrile-stacking" was carried out. Ten variables (pH, acetonitrile and triethanolamine in the buffer, injection time, injection pressure, acetonitrile and NaCl in the sample, capillary and tray temperature and separation voltage), whose levels were varied by 10% around the nominal level, were examined by a Plackett-Burman design (two-level design). The effects on corrected peak area and resolution (responses) were calculated and interpreted using three statistical approaches: dummy variables, distribution effects (Dong's algorithm) and calibration curve. Dong's method was found to be the most suitable to evaluate the robustness, since it considers qualitative (resolution) and quantitative (corrected peak area) responses and does not need a minimum number of dummy variables in the experimental design. From these studies, we can deduce that the first four variables were significant at 10% around the nominal level, and therefore a new design was made with those four variables at 5% nominal level. Then, only two variables proved to be significant for the resolution between some peaks, so the system suitability test limits were defined for these resolutions.
RESUMO
The work presented here explores the possibilities of the electrokinetic injection (EK) to achieve sensitive methods for the determination of tricyclic antidepressants in biological samples (serum). The addition of ACN to the sample, with high content in salts, causes stacking at the tip of the capillary, in a similar way as for hydrodynamic injection. An experimental design with the response surface methodology has been used to find the optimum composition of the matrix of the sample (sodium chloride and ACN percentages) and the conditions for the EK (water-plug length, time, and voltage of injection) in few experiments. The composition of the separation buffer was the same as utilized in a previous paper. The use of a bubble capillary to reach lower detection limits implies a loss of the resolution and requires a new optimization. Finally, a comparison between electrokinetic and hydrodynamic injections is made.
Assuntos
Antidepressivos Tricíclicos/análise , Eletroforese Capilar/métodos , Antidepressivos Tricíclicos/sangue , Análise Química do Sangue/métodos , Cromatografia Capilar Eletrocinética Micelar/métodos , Humanos , SaisRESUMO
A semiautomatic extraction-fluorimetric method for the determination of tricyclic antidepressant drugs (TCAs) based in the formation of ion pairs with 9,10-dimethoxyanthracene-2-sulphonate (DMAS) has been developed. The aqueous solutions of the TCAs (imipramine, desipramine, amitriptyline, nortriptyline, clomipramine or doxepine) are injected into a carrier composed by DMAS in an acid medium and the ion pair formed is extracted into dichloromethane where the fluorescence is measured. An experimental design (Central Composite Design) together with the Response Surface Methodology has been used to find the optimal instrumental FIA and chemical variables. We have considered as the response function the product of the peak height by the sampling frequency. The calibration curves were linear over the working range (0.25-3.00mgL(-1)). The limits of detection were lower than 0.30mgL(-1). The method has been satisfactorily applied to the determination of imipramine, amitriptyline, clomipramine and doxepin in pharmaceutical preparations.
RESUMO
Stacking methods are very important in overcoming the poor detection limits in capillary electrophoresis (CE). In this paper, the separation and determination of several tricyclic antidepressants by a stacking method is described. The inclusion of acetonitrile (ACN) in the sample causes stacking (transient pseudoisotachophoresis) especially in presence of sodium chloride. An experimental design (central composite design) together with the response surface methodology has been used to find the optimum composition of the separation buffer and the optimal stacking conditions in few experiments. The response functions used are the product of the total resolution by the number of peaks, for the optimization of the separation buffer, and the product of the total resolution by the mean of the peak heights, for the optimization of the stacking conditions. About 28% of the capillary volume is loaded with sample. The calibration curves are linear over the working range (50-300 ng/mL). With a bubble capillary, the limits of detection (LODs) are in the order of 5 ng/mL. For the analysis of serum samples, enrichment with sodium chloride and the protein precipitation with ACN are enough to avoid interferences and to get stacking. Recoveries between 91.6 and 104% and RSD between 0.6 and 12% are obtained in the analysis of samples of lyophilized human serum and non-lyophilized human serum, spiked with the drugs.