Identification of Novel Quinolone and Quinazoline Alkaloids as Phosphodiesterase 10A Inhibitors for Parkinson's Disease through a Computational Approach.

Phosphodiesterases (PDEs) are vital in signal transduction, specifically by hydrolyzing cAMP and cGMP. Within the PDE family, PDE10A is notable for its prominence in the striatum and its regulatory function over neurotransmitters in medium-spiny neurons. Given the dopamine deficiency in Parkinson's disease (PD) that affects striatal pathways, PDE10A inhibitors could offer therapeutic benefits by modulating D1 and D2 receptor signaling. This study was motivated by the successful history of quinazoline/quinazoline scaffolds in the inhibition of PDE10A. This study involved detailed in silico evaluations through docking followed by pharmacological, pharmacophoric, and pharmacokinetic analyses, prioritizing central nervous system (CNS)-active drug criteria. Seven cyclic peptides, those featuring the quinazoline/quinazoline moiety at both termini, exhibited notably enhanced docking scores compared to those of the remaining alkaloids within the screened library. We identified 7 quinolines and 1 quinazoline including Lepadin G, Aspernigerin, CJ-13536, Aurachin A, 2-Undecyl-4(1H)-quinolone, Huajiaosimuline 3-Prenyl-4-prenyloxyquinolin-2-one, and Isaindigotone that followed the standard CNS active drug criteria. The dominant quinoline ring in our study and its related quinazoline were central to our evaluations; therefore, the pharmacophoric features of these scaffolds were highlighted. The top alkaloids met all CNS-active drug properties; while nonmutagenic and without PAINS alerts, many indicated potential hepatotoxicity. Among the compounds, Huajiaosimuline was particularly significant due to its alignment with lead-likeness and CNS-active criteria. Aspernigerin demonstrated its affinity for numerous dopamine receptors, which signifies its potential to alter dopaminergic neurotransmission that is directly related to PD. Interestingly, the majority of these alkaloids had biological targets primarily associated with G protein-coupled receptors, critical in PD pathophysiology. They exhibit superior excretion parameters and toxicity end-points compared to the standard. Notably, selected alkaloids demonstrated stability in the binding pocket of PDE10A according to the molecular dynamic simulation results. Our findings emphasize the potential of these alkaloids as PDE10A inhibitors. Further experimental studies may be necessary to confirm their actual potency in inhibiting PDE10A before exploring their therapeutic potential in PD.

Decrypting the multi-genome data for chimeric vaccine designing against the antibiotic resistant Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a gram-negative bacterium that can be fatal if not treated properly. Three types of plague are currently known: bubonic, septicemic, and pneumonic plague, among which the fatality rate of septicemic and pneumonic plague is very high. Bubonic plague can be treated, but only if antibiotics are used at the initial stage of the infection. But unfortunately, Y. pestis has also shown resistance to certain antibiotics such as kanamycin, minocycline, tetracycline, streptomycin, sulfonamides, spectinomycin, and chloramphenicol. Despite tremendous progress in vaccine development against Y. pestis, there is no proper FDA-approved vaccine available to protect people from its infections. Therefore, effective broad-spectrum vaccine development against Y. pestis is indispensable. In this study, vaccinomics-assisted immunoinformatics techniques were used to find possible vaccine candidates by utilizing the core proteome prepared from 58 complete genomes of Y. pestis. Human non-homologous, pathogen-essential, virulent, and extracellular and membrane proteins are potential vaccine targets. Two antigenic proteins were prioritized for the prediction of lead epitopes by utilizing reverse vaccinology approaches. Four vaccine designs were formulated using the selected B- and T-cell epitopes coupled with appropriate linkers and adjuvant sequences capable of inducing potent immune responses. The HLA allele population coverage of the T-cell epitopes selected for vaccine construction was also analyzed. The V2 constructs were top-ranked and selected for further analysis on the basis of immunological, physicochemical, and immune-receptor docking interactions and scores. Docking and molecular dynamic simulations confirmed the stability of construct V2 interactions with the host immune receptors. Immune simulation analysis anticipated the strong immune profile of the prioritized construct. In silico restriction cloning ensured the feasible cloning ability of the V2 construct in the expression system of E. coli strain K12. It is anticipated that the designed vaccine construct may be safe, effective, and able to elicit strong immune responses against Y. pestis infections and may, therefore, merit investigation using in vitro and in vivo assays.

Sperm traits and seminal plasma proteome of locally adapted hairy rams subjected to intermittent scrotal insulation.

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.

Hypomethylation at H19DMR in penile squamous cell carcinoma is not related to HPV infection.

Penile squamous cell carcinoma (SCC) is a rare and aggressive tumour mainly related to lifestyle behaviour and human papillomavirus (HPV) infection. Environmentally induced loss of imprinting (LOI) at the H19 differentially methylated region (H19DMR) is associated with many cancers in the early events of tumorigenesis and may be involved in the pathogenesis of penile SCC. We sought to evaluate the DNA methylation pattern at H19DMR and its association with HPV infection in men with penile SCC by bisulfite sequencing (bis-seq). We observed an average methylation of 32.2% ± 11.6% at the H19DMR of penile SCC and did not observe an association between the p16INK4a+ (p = 0.59) and high-risk HPV+ (p = 0.338) markers with methylation level. The average methylation did not change according to HPV positive for p16INK4a+ or hrHPV+ (35.4% ± 10%) and negative for both markers (32.4% ± 10.1%) groups. As the region analysed has a binding site for the CTCF protein, the hypomethylation at the surrounding CpG sites might alter its insulator function. In addition, there was a positive correlation between intense polymorphonuclear cell infiltration and hypomethylation at H19DMR (p = 0.035). Here, we report that hypomethylation at H19DMR in penile SCC might contribute to tumour progression and aggressiveness regardless of HPV infection.

Metabolomic profile of seminal plasma from Guzerá bulls (Bos indicus) with contrasting sperm freezability phenotypes.

The present study evaluated the seminal plasma metabolome of Bos indicus Guzerá bulls with good (n = 4) and poor (n = 5) sperm freezability. Animals were raised in natural pasture of a 'Caatinga' ecosystem, in the semi-arid region of Brazil. Seminal plasma samples were subjected to gas chromatography coupled to mass spectrometry and data, analysed using bioinformatics tools (Cytoscape with the MetScape plug-in). Sixty-two metabolites were identified in the bovine seminal plasma. Fatty acids and conjugates and organic compounds were the predominant seminal fluid metabolites, followed by carboxylic acids and derivatives, amino acids, benzenes and steroids and derivatives, carbohydrates and carbohydrate conjugates and prenol lipids. Multivariate analysis indicated a distinct separation of seminal plasma metabolomes from bulls with contrasting sperm freezability. Abundances of propanoic acid, d-ribose and glycine were greater in the seminal plasma of bulls with good sperm freezability. Heptadecanoic acid and undecanoic acid were the predominant in bulls of poor sperm freezability. Propanoic acid is an energy source for spermatozoa and may act as an antimicrobial component in semen. Glycine acts against oxidizing and denaturing reactions. d-ribose is also an energy source and reduces apoptosis and oxidative stress. Undecanoic acid may protect sperm against fungal damage. This study provides fundamental information approximately the seminal plasma metabolome of tropically adapted bulls and its association with sperm freezability. However, further studies with larger groups of animals are needed to validate those metabolites as markers of sperm freezability. This strategy could support the selection of sires with superior sperm cryoresistance.

Comprehensive proteomic profiling of early antral follicles from sheep.

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.

Contributions of seminal plasma proteins to fertilizing ability of bull sperm: A meta-analytical review.

Seminal plasma is a dynamic, intricate combination of fluids from the testicles, epididymides, seminal vesicles, bulbourethral glands, and prostate, containing molecules that modulate sperm functions, post-fertilization events, and the female reproductive tract physiology. Significant variations in sperm parameters and fertility status of bulls relate to differences in the seminal plasma proteome. In this framework, a meta-analytical study was conducted examining 29 studies (published between 1990 and 2021) to ascertain the effects of seminal fluid proteins on parameters associated with bull fertility and the influence of distinct methodologies on such effects. Our results revealed that seminal proteins ameliorate sperm parameters, such as motility, integrity, capacitation, and fertilizing ability, and favours sperm protection. Seminal binder of sperm proteins and beta-defensin 126 highly favoured sperm protection when cells were collected from the epididymis by retrograde flux and analysed under room temperature conditions. Furthermore, seminal proteins improved the motility and quality of Bos taurus sperm collected by artificial vagina, mainly in the presence of heparin-binding proteins. The key limitations faced by this meta-analysis were the paucity of studies evaluating the effects of whole seminal fluid proteins and the limited number of studies conducted in vivo. In conclusion, the present meta-analytical study confirms that seminal proteins improve fertility-related parameters in the bovine species. However, methodological strategies used by authors are diverse, with distinct endpoints and methods. Thus, the translational aspects of seminal plasma research should be taken into consideration to precisely define how seminal proteins can be harnessed to advance reproductive biotechnology.

HPV infection and 5mC/5hmC epigenetic markers in penile squamous cell carcinoma: new insights into prognostics.

BACKGROUND: Penile cancer is one of the most aggressive male tumors. Although it is preventable, the main etiologic causes are lifestyle behaviors and viral infection, such as human papillomavirus (HPV). Long-term epigenetic changes due to environmental factors change cell fate and promote carcinogenesis, being an important marker of prognosis. We evaluated epidemiological aspects of penile squamous cell carcinoma (SCC) and the prevalence of HPV infection using high-risk HPV (hrHPV) and p16INK4A expression of 224 participants. Global DNA methylation was evaluated through 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). RESULTS: The incidence of HPV was 53.2% for hrHPV and 22.32% for p16INK4a. hrHPV was not related to systemic or lymph node metastasis and locoregional recurrence, nor influenced the survival rate. P16INK4a seems to be a protective factor for death, which does not affect metastasis or tumor recurrence. Lymph node and systemic metastases and locoregional recurrence increase the risk of death. An increased 5mC mark was observed in penile SCC regardless of HPV infection. However, there is a reduction of the 5hmC mark for p16INK4a + (P = 0.024). Increased 5mC/5hmC ratio (> 1) was observed in 94.2% of penile SCC, irrespective of HPV infection. Despite the increase in 5mC, it seems not to affect the survival rate (HR = 1.06; 95% CI 0.33-3.38). CONCLUSIONS: P16INK4a seems to be a good prognosis marker for penile SCC and the increase in 5mC, an epigenetic mark of genomic stability, may support tumor progression leading to poor prognosis.

Spermatozoa and seminal plasma proteomics: Too many molecules, too few markers. The case of bovine and porcine semen.

The study of the protein composition of semen (i.e., spermatozoa and seminal plasma) is not new. However, with development of proteomics technologies, our understanding of the roles of cellular and fluid proteins has expanded enormously. Today, several seminal proteins have already been suggested as biomarkers associated with semen traits (e.g., sperm motility and integrity) and fertility. Also, many others were associated with infertility, being identified in humans and domestic animals with poor semen quality (e.g., oligozoospermia) and fertility impairment. These proteins not only might explain the causes of fail in fertilization but also have potential as diagnostic tools, improving traditional semen analyses. However, despite characterization of thousands of seminal proteins, to date, few commercial kits based on protein biomarkers are available. In this article, not only the advances and advantages of semen proteomics will be discussed, but also limitations in its application in a commercial AI centre.

Global Proteomics Analysis of Bone Marrow: Establishing Talin-1 and Centrosomal Protein of 55 kDa as Potential Molecular Signatures for Myelodysplastic Syndromes.

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.

How in vitro maturation changes the proteome of ovine cumulus-oocyte complexes?

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.

Identification of biomarkers for bull fertility using functional genomics.

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.

Global proteomic analysis of preimplantational ovine embryos produced in vitro.

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.

Impact of Seminal Plasma Composition on Sperm Freezability in Wild Mammals: A Review.

This review was designed to summarize the most important information around seminal plasma composition and discuss its impact on the freezability of wild mammal semen samples. Seminal plasma is made up of various biochemical constituents, including ions, lipids, proteins, enzymes, and sugars, which vary between species in response to the presence and size of any relevant accessory glands. The biochemical constituents of seminal plasma may change as a result of age, individual variability, and seasonality. These constituents are responsible for supporting different functions in sperm cells, contributing to motility, acrosomal reaction, and fertilization events. A detailed understanding of seminal plasma biochemistry may help to optimize semen freezing protocols, enabling the dynamic alteration in diluents to allow for increased sperm viability rates after thawing.

Functional attributes of seminal proteins in bull fertility: a systematic review.

Proteomic approaches have been widely used in reproductive studies to uncover protein biomarkers of bull fertility. Seminal plasma is one of the most relevant sources of these proteins that may influence sperm physiology. Nonetheless, there are still gaps in existing knowledge in the functional attributes of seminal proteins. Thus, we reviewed the relationships between seminal plasma proteins and bull fertility by conducting a systematic review with data obtained from 71 studies. This review showed that the associations related to fertility improvement with the use of total seminal plasma proteins are still controversial. None of the studies explored the sperm fertilizing ability following these interactions. By contrast, the exposure to a single protein, such as osteopontin, binder of sperm proteins, and heparin binding proteins, can increment sperm motility, capacitation, and fertilizing ability by modulating intracellular calcium concentrations, removing lipids from sperm membranes, and regulating the acrosome reaction. Variations in protein analyses and the protein contents and their abundances between animals contributed to the difficulty of establishing protein biomarkers of fertilizing potential of the bull sperm. Indeed, the heterogenicity of methodologies was a limitation of this review. Standardized methods of seminal protein analyses, as well as sperm endpoints, may minimize such discrepancies. In conclusion, potential biomarkers of sperm parameters are still to be established. Future studies should evaluate protein isoforms and how they interact with sperm to ascertain their biological functions.

H2O2 priming induces proteomic responses to defense against salt stress in maize.

KEY MESSAGE: H2O2 priming reprograms essential proteins' expression to help plants survive, promoting responsive and unresponsive proteins adjustment to salt stress. ABSTACRT: Priming is a powerful strategy to enhance abiotic stress tolerance in plants. Despite this, there is scarce information about the mechanisms induced by H2O2 priming for salt stress tolerance, particularly on proteome modulation. Improving maize cultivation in areas subjected to salinity is imperative for the local economy and food security. Thereby, this study aimed to investigate physiological changes linked with post-translational protein events induced by foliar H2O2 priming of Zea mays plants under salt stress. As expected, salt treatment promoted a considerable accumulation of Na+ ions, a 12-fold increase. It drastically affected growth parameters and relative water content, as well as promoted adverse alteration in the proteome profile, when compared to the absence of salt conditions. Conversely, H2O2 priming was beneficial via specific proteome reprogramming, which promoted better response to salinity by 16% reduction in Na+ content and shoots growth improvement, increasing 61% in dry mass. The identified proteins were associated with photosynthesis and redox homeostasis, critical metabolic pathways for helping plants survive in saline stress by the protection of chloroplasts organization and carbon fixation, as well as state redox. This research provides new proteomic data to improve understanding and forward identifying biotechnological strategies to promote salt stress tolerance.

Morphological and ultrastructural changes in seminal coagulum of the squirrel monkey (Saimiri collinsi Osgood, 1916) before and after liquefaction.

Studies with squirrel monkey semen are of special interest due to the large amount of coagulation that is a component of the semen, which is a problem that has to be overcome when the objective is harvesting of gametes. In the present study, there was characterization of the seminal coagulum of captive S. collinsi. Four samples of ejaculates were collected using electroejaculation procedures from four animals. The aim in conducting this study was to evaluate seminal coagulum of S. collinsi using histological and scanning electron microscopy (SEM) procedures before and after semen liquefaction in an ACP-118® extender. Seminal coagulum of S. collinsi was composed of a superficial plate (external), which coats the spongy seminal plasma matrix of S. collinsi. Additionally, there were sperm in the external and internal components of the coagulum with these gametes being isolated or grouped and with there being a heterogeneous distribution of gametes. The supplementation of semen with ACP-118® resulted in a partial dissolution of the seminal plate and spongy matrix portions of the seminal coagulum within the first hour of incubation.

Proteomic profile of pre-implantational ovine embryos produced in vivo.

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.

In vitro long-term culture of isolated ovine preantral follicles: Influence of ethanol on steroid production, oocyte meiotic resumption, and metabolomic profile.

Ethanol is used routinely to dilute cell culture media supplements with little or no water solubility. This study evaluates the effect of low concentration of ethanol on the follicular development, oocyte maturation, hormone production, gene expression, and metabolomics profile of spent culture medium after long-term culture of isolated ovine preantral follicles. For this, follicles were cultured for 18 days in α-Minimum Essential Medium+ alone (control treatment) or supplemented with 100 ng/mL recombinant bovine FSH (rbFSH treatment) or with 0.2%-v/v ethanol (ethanol treatment). Ethanol treatment increased the percentage of degenerated follicles and oocytes significantly, however, it showed the highest estradiol secretion. Also, the rate of meiosis resumption was higher in ethanol treatment than Control treatment. Ethanol treatment decreased the mRNA levels of B-cell lymphoma 2 (BCL2), BCL2 associated X, Aquaporin 3, Connexin 43, Inhibin Subunit Beta A, kit ligand, Heat Shock Protein (HSP A1A) significantly when compared to the Control treatment. However, mRNA levels of cytochrome P450 family 19, and FSH receptors were significantly higher in ethanol treatment than in the Control treatment. The levels of some metabolites, which are likely amino acids, lipids, an analog of Cyclic guanosine monophosphate, and a derivative of phosphoinositol phosphate metabolism, had higher relative concentrations in ethanol and rbFSH treatments than the Control treatment. In conclusion, ethanol addition augmented the follicular and oocyte degeneration rates but increased the estradiol production and the meiotic resumption. Furthermore, the follicular metabolomic profile was similar between ethanol and rbFSH treatments being both treatments; however, different from the Control treatment.

Mapping the major proteome of reproductive fluids and sperm membranes of rams: From the cauda epididymis to ejaculation.

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.