RESUMO

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

RESUMO

The present study evaluated the effects of heat stress on reproductive parameters of hairy rams. Six animals were subjected to scrotal insulation during four consecutive nights (6 PM - 6 AM). Day (D) 0 was the first day of insulation. Scrotal circumference increased from 30.5 ± 0.3 cm (at pre-insulation) to 31.8 ± 0.4 cm on D4, decreased 3.9 cm on D28, returning to 30.6 ± 0.6 cm on D57. Sperm concentration decreased from 3.7 ± 0.12 ×109 sperm/mL before insulation to 2.6 ± 0.1 ×109 on D23, returning to normal on D57. Sperm motility averaged 75 ± 2.9% before insulation, was undetectable on D23, and became normal on D77. Sperm with normal morphology reached 5.9 ± 2.6% on D35 but recovered (86.8 ± 2.1%) on D91. Sperm DNA integrity decreased from 86.5 ± 4.7% before insulation to 11.1 ± 3.7% on D63, returning to pre-insulation values on D120. Sperm BSP immunostaining was reduced after scrotal insulation. Variations in seminal protein abundances coincided with changes in sperm parameters. Seminal plasma superoxide dismutase, carboxypeptidase Q-precursor and NPC intracellular cholesterol transporter 2 decreased on D18, returning to normal after D28. Albumin, inhibitor of carbonic anhydrase precursor, EGF-like repeat and discoid I-like domain-containing protein 3 and polymeric immunoglobulin receptor increased after insulation. In summary, intermittent scrotal insulation drastically altered ram sperm attributes and seminal proteins, especially those associated with oxidative stress. Knowledge of animal´s response to thermal stress is vital in the scenario of climate changes.

Assuntos

RESUMO

The present study evaluates the proteome of early antral follicles from Ovis aries. Fifty follicles were collected from ovaries of adult ewes and extracted proteins were trypsin-digested, desalted and analyzed by LC-MS/MS. Genes were screened for potential modulation by miRNAs and protein data, subjected to functional enrichment analysis. Label-free mass spectrometry allowed the identification of 2503 follicle proteins, confirming vimentin, actin, lamin, heat shock proteins and histones as the most abundant ones. In silico analyses indicated that miRNAs modulate the expression of genes coding proteins of the sheep follicles involved in cell cycle, cell differentiation, aging, apoptosis, cell death, adipocyte differentiation, cell division. The most important biological processes associated with the follicle proteins were innate immune response, translation, adaptive immune response and protein folding, while molecular functions linked to the proteome of sheep antral follicles related to metal ion binding, ATP binding, oxygen binding, RNA binding and GTP binding, among others. Upload of 2503 Uniport accession codes through DAVID platform matched 1274 genes, associated with translation, metabolic process, proteolysis involved in cellular protein catabolic process, zona pellucida receptor complex and others. KEEG pathways analysis indicated genes correlated with ovine follicular development, with major pathways listed as carbon metabolism, biosynthesis of amino acids, glutathione metabolism, oxidative phosphorylation, fatty acid degradation and oocyte meiosis. This represents a comprehensive atlas of proteins expressed in sheep early antral follicles and will contribute to future identification of biomarkers for follicular development and oocyte maturation.

Assuntos

RESUMO

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

RESUMO

Myelodysplastic syndrome (MDS) is a hematological disorder characterized by abnormal stem cell differentiation and a high risk of acute myeloid leukemia transformation. Treatment options for MDS are still limited, making the identification of molecular signatures for MDS progression a vital task. Thus, we evaluated the proteome of bone marrow plasma from patients (n = 28) diagnosed with MDS with ring sideroblasts (MDS-RS) and MDS with blasts in the bone marrow (MDS-EB) using label-free mass spectrometry. This strategy allowed the identification of 1,194 proteins in the bone marrow plasma samples. Polyubiquitin-C (UBC), moesin (MSN), and Talin-1 (TLN1) showed the highest abundances in MDS-EB, and centrosomal protein of 55 kDa (CEP55) showed the highest relative abundance in the bone marrow plasma of MDS-RS patients. In a follow-up, in the second phase of the study, expressions of UBC, MSN, TLN1, and CEP55 genes were evaluated in bone marrow mononuclear cells from 45 patients by using qPCR. This second cohort included only seven patients from the first study. CEP55, MSN, and UBC expressions were similar in mononuclear cells from MDS-RS and MDS-EB individuals. However, TLN1 gene expression was greater in mononuclear cells from MDS-RS (p = 0.049) as compared to MDS-EB patients. Irrespective of the MDS subtype, CEP55 expression was higher (p = 0.045) in MDS patients with abnormal karyotypes, while MSN, UBC, and TALIN1 transcripts were similar in MDS with normal vs. abnormal karyotypes. In conclusion, proteomic and gene expression approaches brought evidence of altered TLN1 and CEP55 expressions in cellular and non-cellular bone marrow compartments of patients with low-risk (MDS-RS) and high-risk (MDS-EB) MDSs and with normal vs. abnormal karyotypes. As MDS is characterized by disrupted apoptosis and chromosomal alterations, leading to mitotic slippage, TLN1 and CEP55 represent potential markers for MDS prognosis and/or targeted therapy.

RESUMO

Prediction of bull fertility is critical for the sustainability of both dairy and beef cattle production. Even though bulls produce ample amounts of sperm with normal parameters, some bulls may still suffer from subpar fertility. This causes major economic losses in the cattle industry because using artificial insemination, semen from one single bull can be used to inseminate hundreds of thousands of cows. Although there are several traditional methods to estimate bull fertility, such methods are not sufficient to explain and accurately predict the subfertility of individual bulls. Since fertility is a complex trait influenced by a number of factors including genetics, epigenetics, and environment, there is an urgent need for a comprehensive methodological approach to clarify uncertainty in male subfertility. The present review focuses on molecular and functional signatures of bull sperm associated with fertility. Potential roles of functional genomics (proteome, small noncoding RNAs, lipidome, metabolome) on determining male fertility and its potential as a fertility biomarker are discussed. This review provides a better understanding of the molecular signatures of viable and fertile sperm cells and their potential to be used as fertility biomarkers. This information will help uncover the underlying reasons for idiopathic subfertility.

RESUMO

Prediction of bull fertility is critical for the sustainability of both dairy and beef cattle production. Even though bulls produce ample amounts of sperm with normal parameters, some bulls may still suffer from subpar fertility. This causes major economic losses in the cattle industry because using artificial insemination, semen from one single bull can be used to inseminate hundreds of thousands of cows. Although there are several traditional methods to estimate bull fertility, such methods are not sufficient to explain and accurately predict the subfertility of individual bulls. Since fertility is a complex trait influenced by a number of factors including genetics, epigenetics, and environment, there is an urgent need for a comprehensive methodological approach to clarify uncertainty in male subfertility. The present review focuses on molecular and functional signatures of bull sperm associated with fertility. Potential roles of functional genomics (proteome, small noncoding RNAs, lipidome, metabolome) on determining male fertility and its potential as a fertility biomarker are discussed. This review provides a better understanding of the molecular signatures of viable and fertile sperm cells and their potential to be used as fertility biomarkers. This information will help uncover the underlying reasons for idiopathic subfertility.(AU)

Assuntos

RESUMO

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

RESUMO

The present study evaluated the major proteome of ram seminal plasma and the main secretions that contribute to its formation, such as the cauda epididymal and accessory sex gland fluids. The study also investigated sperm membrane protein profiles before and after ejaculation. First, semen was collected from six rams (using artificial vagina) to obtain seminal plasma and ejaculated sperm. Then, rams were vasectomized for collection of accessory sex gland fluid (using artificial vagina). Next, rams were slaughtered and cauda epididymal fluid (CEF), seminal vesicle fluid, bulbourethral gland fluid and cauda epididymal sperm were properly collected. Proteins from reproductive fluids and sperm membranes were analyzed by 2-D SDS-PAGE, tandem mass spectrometry and bioinformatics. There we 386 proteins and 256 isoforms identified in all samples. The most abundant seminal plasma proteins were BSP1, BSP5 and spermadhesins (bodhesin-2 and spermadhesin Z13-like). These proteins were present in similar patterns in maps of accessory sexgland fluid, with very low quantities in the CEF and absent in the bulbourethral gland secretion. Thus, practically all BSPs and spermadhesins come from seminal vesicles. Bulbourethral gland fluid brought bactericidal/permeability-increasing protein-containing Family A member 1 isoforms, superoxide dismutase [Cu-Zn] and betamicroseminoprotein to seminal plasma. CEF was the major provider of clusterin, epididymal-specific lipocalin-5-like isoform, epididymal secretory gluthathione peroxidase, epididymal secretory protein E1 and prostaglandin-H2 D-isomerase to seminal plasma. Albumin came from all reproductive fluids. BSPs and spermadhesins were present in 2-D maps of ejaculated sperm but absent in cauda epididymal sperm. These proteins come from the seminal vesicles and bind to sperm at the moment of ejaculation. Other proteins of ejaculated and epididymal sperm membranes were mostly associated to energy production, cell adhesion and proteolytic activity (ATP synthases, disintegrin, metalloproteinase domain-containing protein 32, carboxypeptidase Q and cytosol aminopeptidase). In conclusion, there is a well-orchestrated sequence of events to form the major seminal plasma proteome, with specific contributions from cauda epididymis, seminal vesicles and bulbourethral glands. The present data contribute to a better understanding of male reproductive biology and how sperm functions are affected by the noncellularmicro environment of semen.

Assuntos

RESUMO

The present study evaluated the effect of binder of sperm protein 1 (BSP1) and/or heparin on in vitro bovine capacitation and fertilization rates using epididymal and ejaculated bovine sperm. Frozen-thawed sperm were selected and used in the following treatments. Control group: Fert-TALP medium without heparin; heparin (HEP) group: Fert-TALP with heparin (10 UI/ml); BSP1 group: Fert-TALP medium with BSP1 (10 µg/ml for ejaculated sperm; 40 µg/ml for epididymal sperm); HEP + BSP1 group: Fert-TALP medium with heparin (5 UI/ml) and BSP1 (5 µg/ml for ejaculated sperm; 20 µg/ml for epididymal sperm) and determined in vitro capacitation rates in different interval times (0, 15, 30 and 60 min) using the chlortetracycline fluorescence (CTC) method. Also, we evaluated the development rates of oocytes fertilized with ejaculated or epididymal sperm into the same treatments. Capacitation was greater and faster when ejaculated sperm were treated for 60 min with heparin compared with other treatments. However, developmental rates were similar in all treatments. For epididymal sperm, the treatments with BSP1 presented higher capacitation and fertilization rates compared with heparin (P < 0.05). The effects of heparin + BSP1 on capacitation and developmental rates did not cause any increase in capacitation or blastocyst rates compared with other groups for ejaculated or epididymal sperm. In conclusion, this study confirmed that either BSP1 and heparin can be used as capacitator agents for bovine ejaculated sperm during IVF. However, BSP1 seems to be more efficient compared with heparin for epididymal sperm. Furthermore, BSP1 and heparin have no synergic effects on sperm capacitation.

Assuntos

RESUMO

The present study was conducted to characterize the metabolome of accessory gland fluid (AGF) of locally adapted Morada Nova rams, raised in the Brazilian Northeast. AGF was collected by an artificial vagina from five vasectomized rams. Metabolites were identified by gas chromatography-mass spectrometry (GC/MS) and high-performance liquid chromatography-mass spectrometry (LC/MS), with the support of Human Metabolome Database, PubChem, LIPID Metabolites, Pathways Strategy databases, and MetaboAnalyst platforms. There were 182 and 190 metabolites detected by GC/MS and LC/MS, respectively, with an overlap of one molecule. Lipids and lipid-like molecules were the most abundant class of metabolites in the ram AGF (127 compounds), followed by amino acids, peptides, and analogs(103 metabolites). Considering all GC/MS and LC/MS, fructose, glycerol, citric acid, d-mannitol, d-glucose, and l-(+)-lactic acid were the most abundant single metabolites present in the ram AGF. Meaningful pathways associated with AGF metabolites included glycine, serine and threonine metabolism; pantothenate and CoA biosynthesis; galactose metabolism; glutamate metabolism and phenylalanine metabolism, and so forth. In conclusion, the combined use of LC/MS and GC/MS was essential for getting a holistic view of the compounds embedded in the ram AGF. Chemical analysis of the accessory sex gland secretion is relevant for understanding sperm function and fertilization.

Assuntos

RESUMO

Saimiri collinsi is used as an animal model in biotechnology research for conservation of species from the genus Saimiri. However, the development of biotechnologies depends on a proper knowledge of the sperm morphology to understand the basic aspects of sperm physiology, as potential male fertility depends on different cellular sperm structures. With this purpose, this study characterized the micromorphological and ultrastructural characteristics of squirrel monkeys (Saimiri collinsi) sperm using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM electromyography revealed that a normal Saimiri collinsi sperm measures 71.7 ± 0.7 µm with lateral tail insertion, a paddle-shaped flattened head and an acrosome occupying most of the head. TEM also showed that the middle piece is characterized by a central 9 + 2 microtubule axoneme surrounded by nine dense fibres, and that the mitochondria were juxtaposed, forming the mitochondrial sheath. Here we provide the first micromorphological and ultrastructure description of S. collinsi sperm.

Assuntos

RESUMO

The aim of this study was to characterize the protein profile of ovarian follicular fluid (FF) of brown brocket deer (Mazama gouazoubira). Five adult females received an ovarian stimulation treatment and the FF was collected by laparoscopy from small/medium (≤3.5 mm) and large (>3.5 mm) follicles. Concentrations of soluble proteins in FF samples were measured and proteins were analyzed by 1-D SDS-PAGE followed by tryptic digestion and tandem mass spectrometry. Data from protein list defined after a Mascot database search were analyzed using the STRAP software tool. For the protein concentration, no significant difference (P > 0.05) was observed between small/medium and large follicles: 49.2 ± 22.8 and 56.7 ± 27.4 µg/µl, respectively. Mass spectrometry analysis identified 13 major proteins, but with no significant difference (P > 0.05) between follicle size class. This study provides insight into elucidating folliculogenesis in brown brocket deer.

Assuntos

RESUMO

The objective of the present study was to develop a methodology to obtain the enriched fraction of ram seminal vesicle protein 14 (RSVP14). The study was developed using Morada Nova rams, from which semen samples were collected weekly. Seminal plasma proteins were precipitated with cold ethanol, and then 6.15 mg/mL of total proteins were subjected to liquid gelatin affinity chromatography using a GelatinSepharose matrix coupled to an automated chromatographic system. Proteins were eluted into four fractions (A, B, C, and D), in which A and B contained non-gelatin-binding proteins, and C and D fractions contained gelatin-binding proteins. Gels were analyzed by Quantity One software, in which five protein bands were detected in fraction D, with molecular weights between 12 and 30 kDa. The gelatin-binding proteins (fraction D) were loaded into a HiTrap™ Heparin HP affinity column. Two chromatographic fractions were separated (D1 and D2), in which D1 contained non-heparin-binding proteins, and D2 contained heparin-binding proteins. Proteins from the last two peaks were subjected to 12.5% SDS-PAGE and Western Blot. Two bands with molecular weight of 14 and 24 kDa, contained in fraction D1, were excised from gel and subjected to tandem mass spectrometry, identifying the proteins RSVP14 and RSVP24. Thus, the chromatographic methods of the present study are efficient to capture the enriched fraction of RSVP14.(AU)

Assuntos

RESUMO

Brazilian Somalis is a locally-adapted breed of rams raised in tropical climate and native pastures. The present study was conducted to evaluate gene expression and proteome of the reproductive tract of such rams. Samples were collected from testes, epididymides, seminal vesicles and bulbourethral glands of four rams. Expression of clusterin (CLU), osteopontin (OPN) and prostaglandin D2 synthase (PGDS) genes were evaluated in all samples by real-time PCR. Shotgun proteomic analysis was performed using samples from the head, corpus and cauda epididymides and from all other structures as well. Gene ontology terms and protein interactions were obtained from UniProtKB databases and MetaCore v.6.8 platform. CLU trasncripts were detected in the testes, epididymides, seminal vesicles and bulbourethral glands of the Somalis rams. The initial region and body of the epididymis had the greatest CLU expression. OPN mRNA was localized in all tissues of the ram reproductive tract. PGDS mRNA was detected in the testes and epididymides. Lable-free mass spectrometry allowed the identification of 137 proteins in all samples. Proteins of the epididymis head mainly participate in cellular processes and response to stimulus, participating in catalityc activity and binding. Proteins of epididymis body acted as regulatory proteins and in cellular processes, with binding and catalytic activity. Cauda epididymis molecules were associated with cellular processes and regulation, with binding function and catalytic activity as well. Testis proteins were mainly linked to cell processes and response to stimuli, and had catalytic function. Seminal vesicle proteins were involved in regulation and mainly with binding functions. Most bulbourethral gland proteins participated in cellular processes. The present study is the first to evaluate the proteome and gene expressions in the reproductive tract of Brazilian Somalis rams. Such pieces of information bring significant cointribution for the understanding of the reproductive physiology of locally-adapted livestock.

Assuntos

RESUMO

The identification of biomarkers associated with seminal traits could aid in the selection of higher quality ejaculates and benefit the swine industry. The objective of this study was to identify boar seminal plasma proteins associated with sperm motility and morphology. Twenty ejaculates from fifteen adult boars from a commercial boar stud were used for this work. After routine semen collection and analysis, ejaculates were classified into two groups: high-quality semen (HQS) and low-quality semen (LQS), based on sperm motility and morphology. Semen samples were processed for seminal plasma separation and analysis by 2D SDS-PAGE. Total and progressive sperm motility differed between groups (p < 0.001), as well sperm morphology (p < 0.05). The intensity of spots identified as Major seminal plasma PSP-I (PSP-I) and cathepsin B (CTSB) was higher in LQS as compared to HQS samples (p < 0.05). Also, PSP-I was positively associated with major and sperm cauda defects. Sperm motility was negatively correlated with both PSP-I and cathepsin B. We conclude that high concentrations of Major seminal plasma PSP-I and cathepsin B in boar seminal plasma are associated with reduced total and progressive sperm motility and low sperm morphology and might be used as biomarkers for semen quality.

Assuntos

RESUMO

Ring-tailed coati is listed as a species of least concern in the International Union for Conservation of Nature (IUCN) Red List, however, there has been a sharp decline in their population. The present study was conducted to evaluate the major proteins of both seminal plasma and sperm in ring-tailed coatis. Semen sample was collected from three adult coatis and evaluated for their morphological characteristics. Further, the sample was centrifuged to separate spermatozoa from seminal plasma, and then stored in liquid nitrogen. The seminal plasma and sperm proteins were subjected to one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by mass spectrometry. Gene ontology and protein networks were analyzed using bioinformatics tools. Based on sperm concentration and average protein content of the semen, the concentration of protein/spermatozoon was found to be 104.69 ±â€¯44.43 µg. The analysis of SDS-PAGE gels showed 20.3 ±â€¯3.1 and 17 ±â€¯2 protein bands/lane for seminal plasma and sperm, respectively. In-gel protein digestion and peptide analysis by mass spectrometry revealed 238 and 246 proteins in the seminal plasma and sperm, respectively. The gene ontology analysis revealed that the proteins of seminal plasma mainly participated in cellular (35%) and regulatory (21%) processes. According to their cellular localization, seminal plasma proteins were categorized as structural (18%), extracellular (17%), and nuclear (14%) proteins with molecular functions, such as catalytic activity (43%) and binding (43%). The sperm proteins were also involved in cellular (38%) and regulatory (23%) processes, and mainly categorized as extracellular (17%), nuclear (13%), and cytoplasmic (10%) proteins. The major molecular functions of the sperm proteins were catalytic activity (44%) and binding (42%). These results indicated that the seminal plasma of ring-tailed coati has an array of proteins that can potentially modulate several sperm functions, from sperm protection to oocyte binding. However, further studies are necessary to interpret the roles of these major seminal plasma proteins in coatis.

Assuntos

RESUMO

The present study was conducted to characterize the major proteome of ovarian follicular fluid from locally-adapted, "Canindé" goats in the northeast of Brazil. Eight estrous cycling goats received a hormonal treatment consisting of medroxyprogesterone acetate, D-cloprostenol and FSH. Fluid was collected by laparoscopy from small (<3mm), medium (3-4mm) and large (>4mm) follicles and then, proteins were analyzed by 2-D SDS-PAGE and tandem mass spectrometry. Thirty-six proteins were identified in the goat follicular fluid, including albumin, immunoglobulins, ceruloplasmin, complement factor B, alpha-1B-glycoprotein precursor, serotransferrin, complement C3 and serpins, among others. Albumin and immunoglobulins were the most abundant proteins. Protein concentrations were similar in the fluid from small (45.3±3.1mg/mL), medium (44.2±3.3mg/mL) and large follicles (45.1±2.3mg/mL). The intensities of spots identified in 2-D gels as serotransferrin, zinc-alpha-2-glycoprotein-like, complement factor B and complement protein C3 differed (P<0.05) among follicle categories. The amount of serotransferrin was greater in the medium than small follicles (P<0.05). Content of zinc-alpha-2-glycoprotein-like, complement factor B and complement C3 was greater (P<0.05) in the fluid of large follicles than in medium follicles. Based on gene ontology, the major molecular functions associated with goat follicular fluid proteins were binding and catalytic activity, while the main biological processes were related to regulation, cellular processing, location and the immune system. In conclusion, the major proteome of the follicular fluid from goats subjected to hormonal stimulation was elucidated in the present study. Also, molecules associated with follicle development are potential biomarkers of oocyte competence were prevalent.

Assuntos

RESUMO

This study aimed to define sperm membrane protein markers of semen freezability of boars with the aid of a proteomic approach. Semen from fourteen adult boars were subjected to slow freezing and rapid thawing. After thawing, sperm vigor and motility were analyzed, and based on these results, animals were separated into two groups: good (GFEs) and poor freezability (PFEs). Sperm membrane proteins were extracted and subjected to two-dimensional electrophoresis. Stained gels were analyzed by computerized resources to indicate differentially expressed protein spots, that were identified by mass spectrometry. Six animals showed good freezability with average sperm vigor and motility of 2.2±0.8 and 41.8±22.9, respectively, whereas eight boars showed poor freezability, with 1.9±0.6 and 26.8±17.5 of sperm vigor sperm motility, respectively. An average of 263±62.2 spots per gel and 234.2±54.6 of spots consistently present in all gels were detected. The intensities of five spots were significantly different between groups. Fc fragment of IgG binding protein and lactadherin were more intense in the PFE group, while Arylsulfatase A and F-actin capping protein subunit alpha 1 were more expressed in the GEF group. Based on their functions and interactions with other proteins, we conclude that these four sperm membrane proteins may act as potential markers of boar semen freezability.

Assuntos

RESUMO

The aims of this study were to investigate the effects of medium replacement system (experiment I) and of FSH presentations (homeopathic - FSH 6cH and allopathic FSH - rFSH; experiment II) on the in vitro development, hormone production and gene expression of isolated ovine preantral follicles cultured for 6 days. In experiment I, secondary follicles were cultured in the α-MEM+ supplemented with FSH 6cH (0.05 fg/ml) or recombinant bovine FSH (100 ng/ml) without/with daily medium addition. The homeopathic FSH treatments with/without medium addition improved (p < .05) follicular development compared to rFSH100 treatment without addition. FSH 6cH with addition showed the highest (p < .05) estradiol production. To verify whether the effects of homeopathic FSH were not due to its vehicle, experiment II was performed. The α-MEM+ was supplemented or not with alcohol (0.2% grain ethanol, v/v), FSH 6cH or rFSH100 with daily medium addition. Surprisingly, we found that all treatments improved follicular development compared to the α-MEM+ (p < .05). Moreover, homeopathic FSH was similar to the other treatments including its vehicle. In conclusion, its vehicle (ethanol) causes the effect of homeopathic FSH on in vitro development of isolated ovine preantral follicles.

Assuntos