RESUMO
In vivo, the temperature inside preovulatory follicles of cows is approximately 1 °C lower than rectal temperature. However, standard bovine oocyte in vitro maturation (IVM) protocols use 38.5 °C based on rectal temperature. This study evaluated the effect of reducing IVM temperature to 37.5 °C on the proteomic profile of oocytes compared to the routine 38.5 °C. Nuclear maturation rate and cumulus cell (CC) expansion (30 COCs per group, 21 replicates) were assessed by observing the first polar body and using a subjective scoring method (0-4). Total nitrite concentrations in the culture medium were measured using the Griess method. Differential proteomics was performed using LC-MS/MS on pooled oocyte samples (500 matured oocytes per group, three replicates), followed by gene ontology enrichment, protein-protein interaction, and putative miRNA target analyses. No significant differences were observed between the groups in nuclear maturation, CC expansion, or nitrite concentration (P > 0.05). A total of 806 proteins were identified, with 7 up-regulated and 12 down-regulated in the treatment group compared to the control. Additionally, 12 proteins were unique to the control group, and 8 were unique to the treatment group. IVM at 37.5 °C resulted in the upregulation of proteins involved in protein folding and GTP binding, and the downregulation of enzymes with oxidoreductase activity and proteins involved in cytoskeletal fiber formation. Furthermore, 43 bovine miRNAs potentially regulating these genes (DES, HMOX2, KRT75, FARSA, IDH2, CARHSP1) were identified. We conclude that IVM of bovine oocytes at 37.5 °C induces significant proteomic changes without impacting nuclear maturation, cumulus cell expansion, or nitrite concentration in the IVM medium.
Assuntos
Técnicas de Maturação in Vitro de Oócitos , Oócitos , Proteômica , Animais , Bovinos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Feminino , Temperatura , ProteomaRESUMO
Canine mammary tumours (CMT) have histological, clinicopathological and molecular resemblances to human breast cancer (HBC), positioning them as viable models for studying the human disease. CMT initiation and progression occur spontaneously in immune-competent animals, which challenge the suggested limitations of genetically modified mice, also enabling the evaluation of immunotherapies in canine patients. Dogs have shorter life expectancy compared to humans, and cancer advances more rapidly in this species. This makes it possible to perform studies about the clinical efficacy of new therapeutic modalities in a much shorter time than in human patients. The identification of biomarkers for tumour subtypes, progression and treatment response paves the way for the development of novel therapeutic and diagnostic approaches. This review addresses the similarities between CMT and HBC and the molecular signatures identified in CMT samples that have been explored to date. We proposed a detailed molecular exploration of the CMT stroma using state-of-the-art methods in transcriptomics and proteomics. Using CMT as an analog for HBC not only helps to understand the complexities of the disease, but also to advance comparative oncology to the next level to prove the claim of dogs as a valid translational model.
Assuntos
Modelos Animais de Doenças , Doenças do Cão , Neoplasias Mamárias Animais , Cães , Animais , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/genética , Doenças do Cão/patologia , Doenças do Cão/genética , Feminino , Humanos , Neoplasias da Mama/veterinária , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Pesquisa Translacional BiomédicaRESUMO
The present study was conducted to investigate the global proteome of 8-day-old equine blastocysts. Follicular dynamics of eight adult mares were monitored by ultrasonography and inseminated 24 h after the detection of a preovulatory follicle. Four expanded blastocysts were recovered, pooled, and subjected to protein extraction and mass spectrometry. Protein identification was conducted based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, and PepExplorer). Enrichment analysis was performed using g:Profiler, Panther, and String platforms. After the elimination of identification redundancies among search tools (at three levels, based on identifiers, peptides, and cross-database mapping), 1977 proteins were reliably identified in the samples of equine embryos. Proteomic analysis unveiled robust metabolic activity in the 8-day equine embryo, highlighted by an abundance of proteins engaged in key metabolic pathways like the TCA cycle, ATP biosynthesis, and glycolysis. The prevalence of chaperones among highly abundant proteins suggests that regulation of protein folding, and degradation is a key process during embryo development. These findings pave the way for developing new strategies to improve equine embryo media and optimize in vitro fertilization techniques.
Assuntos
Blastocisto , Proteoma , Animais , Cavalos/embriologia , Feminino , Blastocisto/metabolismo , Desenvolvimento Embrionário , Estudos Prospectivos , Proteômica , Fertilização in vitro/veterináriaRESUMO
Boar seminal plasma (SP) proteins were associated with differences on sperm resistance to cooling at 17°C. However, information about seminal plasma proteins in boars classified by capacity of semen preservation and in vivo fertility remains lacking. Thus, the objective was to evaluate the SP proteome in boars classified by capacity of semen preservation and putative biomarkers for fertility. The ejaculates from high-preservation (HP) showed higher progressive motility during all 5 days than the low-preservation (LP) boars. There was no difference for farrowing rate between ejaculates from LP (89.7%) and HP boars (88.4%). The LP boars presented lower total piglets born (14.0 ± 0.2) than HP (14.8 ± 0.2; p < 0.01). A total of 257 proteins were identified, where 184 were present in both classes of boar, and 41 and 32 were identified only in LP and HP boars, respectively. Nine proteins were differently expressed: five were more abundant in HP (SPMI, ZPBP1, FN1, HPX, and C3) and four in LP boars (B2M, COL1A1, NKX3-2, and MPZL1). The HP boars had an increased abundance of SP proteins related to sperm resistance and fecundation process which explains the better TPB. LP boars had a higher abundance of SP proteins associated with impaired spermatogenesis.
Assuntos
Preservação do Sêmen , Sêmen , Suínos , Animais , Masculino , Sêmen/metabolismo , Preservação do Sêmen/veterinária , Proteômica , Inseminação Artificial , Espermatozoides , Fertilidade , Análise do Sêmen , Proteínas de Plasma Seminal/metabolismo , Motilidade dos EspermatozoidesRESUMO
Abstract Objective: Seminal cryopreservation causes significant damage to the sperm; therefore, different methods of cryopreservation have been studied. The aim of the study was to compare the effects of density gradient processing and washing/centrifugation with seminal plasma removal for cryopreservation in semen parameters. Methods: Seminal samples of 26 normozoospermic patients were divided into 3 parts: with seminal plasma; after washing/centrifugation; and after selection through density gradient. The samples were cryopreserved for at least two weeks. Motility, sperm count, morphology and viability were evaluated before cryopreservation and after thawing. Results: Density gradient processing selected motile and viable sperm with normal morphology in fresh samples (p<0.05). Cryopreservation negatively affected all sperm parameters regardless of the processing performed, and even if the sperm recovery was lower in the density gradient after the thawing, progressive motility, total motility, viability and morphology remained higher (p<0.05). Conclusion: Cryopreservation significantly compromises sperm parameters (motility, morphology, viability). In normozoospermic patients, the density gradients select better quality spermatozoa compared to other processing methods; this benefit was kept after thawing.
RESUMO
Over the years, reproductive efficiency in the swine industry has focused on reducing the sperm cell number required per sow. Recent advances have included the identification of subfertile boars, new studies in extended semen quality control, new catheters and cannulas for intrauterine artificial insemination (AI), and fixed-time AI under commercial use. Therefore, it is essential to link field demands with scientific studies. In this review, we intend to discuss the current status of porcine AI, pointing out challenges and opportunities to improve reproductive efficiency.
Assuntos
Preservação do Sêmen , Sêmen , Suínos , Animais , Masculino , Feminino , Análise do Sêmen , Fertilidade , Inseminação Artificial/veterinária , Inseminação Artificial/métodos , Contagem de Espermatozoides , Preservação do Sêmen/veterinária , EspermatozoidesRESUMO
O controle da qualidade seminal produzido por centrais e unidades de disseminação genética permite a maximização da produção de doses inseminantes por ejaculado, bem como organiza o acompanhamento da produtividade de cada macho. Este acompanhamento é crítico tanto quando o reprodutor jovem é introduzido na rotina de coleta de sêmen quanto na determinação do momento do descarte e reposição. Essencialmente, a avaliação do sêmen em suínos é a mesma há décadas, porém novas metodologias de análise individual da célula como a citometria de fluxo e de constituintes de tecidos e fluídos como a proteômica e metabolômica já estão trazendo avanços na andrologia animal. Na presente revisão, são abordados os aspectos técnicos, vantagens e limitações destas três análises avançadas aplicadas ao sêmen suíno, discutindo a sua implementação e impactos no manejo reprodutivo da espécie.(AU)
The quality control of the semen doses produced by artificial insemination centers and allows the maximization of the production of inseminating doses per ejaculate, as well as organizes the monitoring of the productivity of each male. This follow-up is critical when the young male is initiated in routine semen collection and in determining the moment of male replacement. Essentially, semen evaluation in pigs has been the same for decades, but new methodologies for individual cell analysis such as flow cytometry and tissue and fluid constituents such as proteomics and metabolomics are already bringing advances in animal andrology. In this review, the technical aspects, advantages, and limitations of these three advanced analyzes applied to swine semen are addressed, discussing their implementation and impacts on the reproductive management of the species.(AU)
Assuntos
Animais , Masculino , Suínos/fisiologia , Inseminação Artificial/veterinária , Análise do Sêmen/métodos , ProteômicaRESUMO
The present study evaluated the effects of in vitro maturation (IVM) on the proteome of cumulus-oocyte complexes (COCs) from ewes. Extracted COC proteins were analyzed by LC-MS/MS. Differences in protein abundances (p < 0.05) and functional enrichments in immature versus in vitro-matured COCs were evaluated using bioinformatics tools. There were 2550 proteins identified in the COCs, with 89 and 87 proteins exclusive to immature and mature COCs, respectively. IVM caused downregulation of 84 and upregulation of 34 proteins. Major upregulated proteins in mature COCs were dopey_N domain-containing protein, structural maintenance of chromosomes protein, ubiquitin-like modifier-activating enzyme 2. Main downregulated proteins in mature COCs were immunoglobulin heavy constant mu, inter-alpha-trypsin inhibitor heavy chain 2, alpha-2-macroglobulin. Proteins exclusive to mature COCs and upregulated after IVM related to immune response, complement cascade, vesicle-mediated transport, cell cycle, and extracellular matrix organization. Proteins of immature COCs and downregulated after IVM were linked to metabolic processes, immune response, and complement cascade. KEGG pathways and miRNA-regulated genes attributed to downregulated and mature COC proteins related to complement and coagulation cascades, metabolism, humoral response, and B cell-mediated immunity. Thus, IVM influenced the ovine COC proteome. This knowledge supports the future development of efficient IVM protocols for Ovis aries.
Assuntos
Células do Cúmulo , MicroRNAs , Ovinos , Animais , Feminino , Células do Cúmulo/metabolismo , Proteoma/metabolismo , Carneiro Doméstico , Cromatografia Líquida , Espectrometria de Massas em Tandem , Oócitos/metabolismo , Ubiquitinas/metabolismo , Ubiquitinas/farmacologia , Imunoglobulinas/metabolismo , Macroglobulinas/metabolismo , Macroglobulinas/farmacologia , MicroRNAs/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodosRESUMO
The present study was conducted to characterize the major proteome of preimplantation (D6) ovine embryos produced in vitro. COCs were aspirated from antral follicles (2-6 mm), matured and fertilized in vitro and cultured until day six. Proteins were extracted separately from three pools of 45 embryos and separately run in SDS-PAGE. Proteins from each pool were individually subjected to in-gel digestion followed by LC-MS/MS. Three 'raw files' and protein lists were produced by Pattern Lab software, but only proteins present in all three lists were used for the bioinformatics analyses. There were 2,262 proteins identified in the 6-day-old ovine embryos, including albumin, zona pellucida glycoprotein 2, 3 and 4, peptidyl arginine deiminase 6, actin cytoplasmic 1, gamma-actin 1, pyruvate kinase, heat shock protein 90 and protein disulfide isomerase, among others. Major biological processes linked to the sheep embryo proteome were translation, protein transport and protein stabilization, and molecular functions, defined as ATP binding, oxygen carrier activity and oxygen binding. There were 42 enriched functional clusters according to the 2,147 genes (UniProt database). Ten selected clusters with potential association with embryo development included translation, structural constituent of ribosomes, ribosomes, nucleosomes, structural constituent of the cytoskeleton, microtubule-based process, translation initiation factor activity, regulation of translational initiation, cell body and nucleotide biosynthetic process. The most representative KEEG pathways were ribosome, oxidative phosphorylation, glutathione metabolism, gap junction, mineral absorption, DNA replication and cGMP-PKG signalling pathway. Analyses of functional clusters clearly showed differences associated with the proteome of preimplantation (D6) sheep embryos generated after in vitro fertilization in comparison with in vivo counterparts (Sanchez et al., 2021; https://doi.org/10.1111/rda.13897), confirming that the quality of in vitro derived blastocysts are unlike those produced in vivo. The present study portrays the first comprehensive overview of the proteome of preimplantational ovine embryos grown in vitro.
Assuntos
Proteoma , Proteômica , Animais , Blastocisto/fisiologia , Cromatografia Líquida/veterinária , Fertilização in vitro/veterinária , Oxigênio , Ovinos , Espectrometria de Massas em Tandem/veterináriaRESUMO
Sperm cells rely on different substrates to fulfil thei energy demand for different functions and diverse moments of their life. Species specific mechanism involve both energy substrate transport and their utilization: hexose transporters, a protein family of facilitative passive transporters of glucose and other hexose, have been identified in spermatozoa of different species and, within the species, their localization has been identified and, in some cases, linked to specific glycilitic enzyme presence. The catabolism of hexose sources for energy purposes has been studied in various species, and recent advances has been made in the knowledge of metabolic strategies of sperm cells. In particular, the importance of aerobic metabolism has been defined and described in horse, boar and even mouse spermatozoa; bull sperm cells demonstrate to have a good adaptability and capacity to switch between glycolysis and oxidative phosphorylation; finally, dog sperm cells have been demonstrated to have a great plasticity in energy metabolism management, being also able to activate the anabolic pathway of glycogen syntesis. In conclusion, the study of energy management and mitochondrial function in spermatozoa of different specie furnishes important base knowledge to define new media for preservation as well as newbases for reproductive biotechnologies.
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Background: Superficial necrolytic dermatitis (SND), hepatocutaneous syndrome (HCS), metabolic epidermal necrosis (MEN), and necrolytic migratory erythema (NME) are useful terms to describe a disease that likely has a multifactorial etiopathogenesis. SND is a rare and fatal disease characterized by skin lesions and liver disease. Common skin lesions include hyperkeratosis, fissures, erosion, ulceration, crusting, exudation from the paws, face, perianal regions, and pressure points. This case report aimed to report the case of a bitch that developed the rare Superficial Necrolytic Dermatitis disease, emphasizing the clinical signs of the disease, and the importance of complementary exams such as abdominal ultrasound and skin biopsy for the definitive diagnosis. Case: A 9-year-old, mixed-breed, neutered female was referred for clinical examination with 5 months history of hyperkeratosis and ulceration of the paw pads, presenting pain, lameness and weight loss. Abdominal ultrasound revealed a liver with heterogeneous echotexture, mixed echogenicity, irregular and poorly delimited margins with hypoechoic nodules throughout like honeycombs. The gallbladder was visualized with a moderately thick layer. Histological analysis confirmed the diagnosis of SND. Skin biopsies showed an increase in thickness of the epidermis due to irregular hyperplasia and proliferation of keratinocytes in the basal layer of the epidermis, pallor of the spinous layer of the epidermis and important parakeratosis. Due to the progression of the disease, significant worsening of the patient's clinical condition and pain, associated with the impossibility of cure, the animal was submitted to euthanasia. A necropsy was performed to allow assessment of the liver and pancreas. The biopsies showed a severe proliferative chronic hepatitis, steatosis and cholestasis associated with pancreatitis and necrotic multifocal proliferative fibrinopurulent areas in the pancreas. Discussion: Clinical signs such as lethargy, inappetence, weight loss, as well as the dermatological signs presented by this bitch are nonspecific clinical signs and require a deeper clinical, pathological and histopathological diagnostic investigation to reach the diagnosis of this disease. The definitive diagnosis is made on the basis of a characteristic honeycomb pattern in the liver or associated with a neoplastic finding in the pancreas on ultrasound examination and confirmed by histopathological evaluation of skin biopsies. Palliative treatment with corticosteroid anti-inflammatories, improvement in feed quality, with higher nutritional and protein intake and intravenous amino acid supplementation are suggested by some authors as treatment alternatives. However, not all owners can afford a costly lifetime treatment. With the progressive worsening of the condition, many owners opt for euthanasia as a way to shorten the suffering of the animal. This decision is not an easy one to make. Despite the poor prognosis of the disease, treatment options should be tried by veterinarians and owners prior to the option of euthanasia. However, new affordable nutritional and pharmacological strategies to treat or control the disease are needed in order to improve quality of life of SND patients.
Assuntos
Animais , Feminino , Cães , Eritema Migratório Necrolítico/veterinária , Eritema Migratório Necrolítico/diagnóstico por imagem , Dermatopatias/veterinária , Ultrassonografia/veterináriaRESUMO
Sperm cells rely on different substrates to fulfil thei energy demand for different functions and diverse moments of their life. Species specific mechanism involve both energy substrate transport and their utilization: hexose transporters, a protein family of facilitative passive transporters of glucose and other hexose, have been identified in spermatozoa of different species and, within the species, their localization has been identified and, in some cases, linked to specific glycilitic enzyme presence. The catabolism of hexose sources for energy purposes has been studied in various species, and recent advances has been made in the knowledge of metabolic strategies of sperm cells. In particular, the importance of aerobic metabolism has been defined and described in horse, boar and even mouse spermatozoa; bull sperm cells demonstrate to have a good adaptability and capacity to switch between glycolysis and oxidative phosphorylation; finally, dog sperm cells have been demonstrated to have a great plasticity in energy metabolism management, being also able to activate the anabolic pathway of glycogen syntesis. In conclusion, the study of energy management and mitochondrial function in spermatozoa of different specie furnishes important base knowledge to define new media for preservation as well as newbases for reproductive biotechnologies.(AU)
Assuntos
Animais , Masculino , Sêmen/citologia , Mitocôndrias/fisiologia , Hexoses , MetabolismoRESUMO
The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.
Assuntos
Embrião de Mamíferos/química , Proteoma/análise , Carneiro Doméstico/embriologia , Animais , Feminino , Inseminação Artificial/veterinária , Masculino , MicroRNAs/genética , Proteoma/genética , Carneiro Doméstico/genética , Carneiro Doméstico/metabolismoRESUMO
Osteopontin (OPN) is a multifunctional phosphoprotein that has been linked to fertility in bulls. However, the exact mechanism by which OPN contributes to fertilisation is yet unknown. The biotechnological use of OPN in bovine reproduction is promising but some gaps remain unfilled. The present work aimed: (a) to verify whether the seminal plasma OPN is associated with seminal traits and a standard breeding soundness exam; (b) to predict OPN interactions with integrins, CD44 and glycosaminoglycans through molecular docking; and (c) to develop a protocol for recombinant expression of OPN from vesicular gland cDNA. Ejaculates from top ranked bulls had higher amounts of seminal plasma OPN in comparison with bulls classified as questionable (p < .01). The structural modelling and molecular docking predictions indicated that bovine OPN binds to heparin disaccharide, hyaluronic acid and hyaluronan. In addition, docking studies described the binding complexes of OPN with CD44 and the integrin heterodimers α5ß1 and αVß3. Finally, expression of rOPN-6His was successfully obtained after 3 hr of induction with 0.5 mM IPTG at 37°C and a denaturing purification protocol resulted in efficiently purified recombinant OPN. The present results contribute to the development of biotechnological uses of OPN as a biomarker in bovine reproduction.
Assuntos
Osteopontina , Análise do Sêmen/veterinária , Sêmen , Animais , Bovinos , Fertilidade , Masculino , Simulação de Acoplamento Molecular , Osteopontina/genéticaRESUMO
O estresse oxidativo é caracterizado pelo desequilíbrio entre a produção de espécies reativas de oxigênio e a atuação de sistemas antioxidantes da própria célula e tecido. O espermatozoide de mamíferos apresenta metabolismo oxidativo e por isso é um potencial produtor destas espécies reativas que podem causar danos a estrutura lipídicas, proteínas e mesmo ao DNA espermático. Na espécie suína, o plasma seminal confere importante proteção antioxidante ao espermatozoide ejaculado no útero da fêmea, onde os desafios à sua sobrevivência são inúmeros. Contudo, esta defesa natural do plasma seminal não é suficiente para garantir o máximo de resultado de fertilidade com o sêmen submetido a processos de refrigeração ou congelação e utilizado na inseminação artificial. Na presente revisão, serão abordados os temas estresse oxidativo e metabolismo espermático, bem como apresentados alguns dados sobre a aplicação de antioxidantes na andrologia suína.
Oxidative stress is characterized by an imbalance between the production of reactive oxygen species and the action of antioxidant systems present in the cell and tissue. Mammalian sperm have oxidative metabolism and are therefore a potential producers of these reactive species which can lead to damage to lipids, proteins and even sperm DNA. In the swine species, seminal plasma provides an important antioxidant protection to spermatozoa that are ejaculated and deposited in the uterus, where the challenges to their survival are numerous. However, this natural seminal plasma defense is not sufficient to guarantee maximum fertility when semen is processed for artificial insemination, and later used after preservation by refrigeration or freezing. In this review, the topics related to oxidative stress and sperm metabolism will be addressed, and results about the application of antioxidants in swine andrology will be presented as well as.
Assuntos
Animais , Antioxidantes/análise , Estresse Oxidativo , Inseminação Artificial/veterinária , Preservação do Sêmen , Suínos/embriologia , EspermatozoidesRESUMO
Os primeiros estudos de espermatozoides com citometria de fluxo com espermatozoides iniciaram no final da década de 1970. Com os avanços tecnológicos, hoje contamos com equipamentos com alta sensibilidade e eficiência que, em conjunto com amplo catálogo de sondas fluorescentes, podemos mensurar com alta precisão características celulares. Como exemplo, destacam-se dano em membrana plasmática, atividade mitocondrial, produção de espécies reativas de oxigênio, dano ao DNA espermático e muito mais. Na presente revisão, as potencialidades e limitação para a implementação da citometria de fluxo na análise seminal de espécies domésticas são exploradas e comentadas.
The first flow cytometry studies with spermatozoa were published in the late 1970s. With the technological advances in the following years, today we have equipments with high sensitivity and efficiency that, together with a large number of commercially available fluorescent probes, allow us to measure different cell characteristics with high accuracy. Some of the most evaluated characteristics are plasma membrane damage, mitochondrial activity, production of reactive oxygen species, sperm DNA damage, and much more. In this review, the potentials and limitations for the implementation of flow cytometry in the seminal analysis of domestic species are explored and commented.
Assuntos
Animais , Andrologia/educação , Citometria de Fluxo/métodos , Citometria de Fluxo/veterinária , Dano ao DNA , Membrana Celular , Mitocôndrias/genéticaRESUMO
Male infertility or subfertility is frequently associated with disruption of the hypothalamic-pituitary-testis axis events, like secondary hypogonadism. However, little is known how this condition affects the proteomic composition of the epididymal fluid. In the present study, we evaluated the proteomic changes in the cauda epididymal fluid (CEF) in a swine model of secondary hypogonadism induced by anti-GnRH immunization using multidimensional protein identification technology. Seven hundred and eighteen proteins were identified in both GnRH-immunized and control groups. GnRH immunization doubled the number of proteins in the CEF, with 417 proteins being found exclusively in samples from GnRH-immunized boars. CEF from GnRH-immunized boars presented an increase in the number of proteins related to cellular and metabolic processes, with affinity to organic cyclic compounds, small molecules, and heterocyclic compounds, as well changed the enzymatic profile of the CEF. Also, a significant increase in the number of proteins associated to the ubiquitin-proteasome system was identified in CEF from GnRH-immunized animals. These results bring strong evidence of the impact of secondary hypogonadism on the epididymal environment, which is responsible for sperm maturation and storage prior ejaculation. Finally, the differently expressed proteins in the CEF are putative seminal biomarkers for testicular and epididymal disorders caused by secondary hypogonadism.
Assuntos
Líquidos Corporais/metabolismo , Epididimo/metabolismo , Hipogonadismo/metabolismo , Infertilidade Masculina/metabolismo , Proteoma/metabolismo , Animais , Anticorpos/farmacologia , Líquidos Corporais/química , Líquidos Corporais/efeitos dos fármacos , Anticoncepção Imunológica/métodos , Anticoncepção Imunológica/veterinária , Epididimo/química , Epididimo/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/imunologia , Hormônio Liberador de Gonadotropina/metabolismo , Hipogonadismo/etiologia , Hipogonadismo/imunologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Infertilidade Masculina/etiologia , Infertilidade Masculina/imunologia , Infertilidade Masculina/veterinária , Masculino , Modelos Animais , Proteoma/análise , Proteoma/efeitos dos fármacos , Proteômica , Transdução de Sinais/efeitos dos fármacos , Suínos/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
The protein disulphide isomerase A1 (PDIA1) is an important chaperone involved in protein quality control and redox regulation. Also, the ability of PDIA1 to bind to oestrogens suggests that it may play a role in epididymal maturation and male fertility. The goals of this study were to (a) verify the possible interaction between 17ß-estradiol and equine PDIA1 using bioinformatics; (b) identify and quantify PDIA1 protein in equine cauda epididymis throughout peripuberty; and (c) determine whether the amounts of PDIA1 in equine seminal plasma and spermatozoa are associated with fertility. Using in silico analysis, we were able to predict the tertiary structure of equine PDIA1 and to demonstrate the interaction between 17ß-estradiol and the putative binding site in domains b and b'. Colts under 24 months of age had lower relative amounts of PDIA1 in cauda epididymal fluid in comparison with older males (p < .01). No difference was observed in seminal plasma PDIA1 between fertile and subfertile stallions. Our study demonstrates that PDIA1 expression in the epididymis increases during peripuberty. However, in the adult stallion, its quantity in seminal plasma is not associated with fertility.
Assuntos
Epididimo/metabolismo , Cavalos/fisiologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Sêmen/metabolismo , Maturidade Sexual/fisiologia , Animais , Biologia Computacional , Epididimo/química , Estradiol/química , Estradiol/metabolismo , Fertilidade , Masculino , Simulação de Acoplamento Molecular , Isomerases de Dissulfetos de Proteínas/análise , Isomerases de Dissulfetos de Proteínas/ultraestrutura , Estrutura Terciária de Proteína , Sêmen/químicaRESUMO
Seminal plasma (SP) contributes to sperm physiology and metabolism, prevents premature capacitation, and protects sperm against oxidative stress. In order to evaluate the impact of heat stress in the semen of tropically adapted Brangus breed and in their seminal plasma proteome, we studied the effects of scrotal insulation for 72 h. Semen samples from six bulls, between 7 and 8 years of age, were collected prior to scrotal insulation (pre-insulation), and at 4 and 11 wk after insulation. Seminal plasma samples were analyzed by 2D SDS-PAGE and liquid chromatography coupled with mass spectrometry (LC-MS/MS). Insulation caused decrease in vigour, gross and total motility after 4 wk of scrotal insult (P < 0.001). Total defects in sperm were higher after 4 wk compared to pre-insulation and 11 wk after scrotal insulation (P < 0.001). The analysis of the 2D protein profile of the SP resulted in the identification 183 unique protein spots in all gels evaluated. There was no difference in mean number of protein spots amongst time points. Eight protein spots were more abundant in SP after scrotal insulation, returning to the same expression level at 11 wk post-insulation. One spot had higher abundance at 11 wk post-insulation, and one spot had decreased abundance 4 wk after insulation. The ten protein spots with differential abundance amongst time points were identified as Seminal plasma protein PDC-109, Seminal plasma protein A3, Seminal plasma protein BSP-30 kDa, Spermadhesin-1 and Metalloproteinase inhibitor 2. The validation of these five proteins as biomarkers for thermal testicular stress in Brangus breed would allow the development of new biotechnologies that could improve bovine semen analysis in breeding systems in tropical and subtropical conditions. A close association between the identified BSP and Spermadhesin-1 was evidenced in protein-protein interaction analysis. Based on gene ontology analysis, variation in sperm function after insulation could be explained by variation in the expressed proteins in the SP. Further studies are required to verify if these proteins could be used as biomarkers for the identification of bulls with increased seminal resistance to heat stress in Brangus breed.
Assuntos
Bovinos/fisiologia , Proteoma/fisiologia , Escroto , Sêmen/fisiologia , Espermatozoides/fisiologia , Animais , Bovinos/genética , Masculino , Análise do Sêmen/veterinária , Motilidade dos EspermatozoidesRESUMO
In the present study, we describe the proteome of porcine cauda epididymis fluid and spermatozoa by means of Multidimensional Protein Identification Technology (MudPIT). Ten sexually mature healthy boars were surgically castrated and epididymides were dissected to obtain the cauda epididymal content. Polled protein extracts of cauda epididymal fluid (CEF) and spermatozoa (CESperm) were loaded in an Agilent 1100 quaternary HPLC and peptides eluted from the microcapillary column were electro-sprayed directly into a LTQ Orbitrap XL mass spectrometer. Using bioinformatics, identified proteins were classified by their molecular functions, involvement in biological processes and participation in relevant metabolic pathways associated with spermatozoa physiology, fertility potential and protection. A total of 645 proteins were identified in the CEF, with epididymal-specific lipocalin-5, beta-hexosaminidase subunit beta precursor and phosphatidylethanolamine-binding protein 4 being the most abundant proteins found. A total of 2886 proteins were identified in the CESperm proteome with 81 proteins being considered more abundant (spectral counts > 100). CEF and CESperm data were compared and 345 proteins were present in both proteomes. Phosphatidylethanolamine-binding protein 4 precursor was the only protein found most abundant in both CEF and CESperm proteomes. Based on Gene Ontology analysis, we identified CEF and CESperm proteins associated with sperm protection against ROS and immune mediated response, glycosaminoglycan degradation, ubiquitin-proteasome system, metabolic process and maturation, modulation of acrosome reaction and ZP binding and oocyte penetration. These results provide a better comprehension about the molecular process and biological pathways involved in sperm epididymis maturation and establishment of the cauda epididymis sperm reservoir.