Project description:Seminal plasma contributes to the safe environment for sperm maturation, sperm viability and fertilization in mammals (Kumar 2013). Moreover, seminal plasma is a promising source for the study of potential reproductive biomarkers, because it is a complex mixture of secretions from testis, epididymis and male accessory sex glands (González-Cadavid et al. 2014). The protein composition of mammalian seminal plasma varies among species, and has important effects on sperm function (Mortarino et al. 1998). In rabbits, seminal plasma has a positive effect in maintaining sperm motility and viability during in vitro storage (Castellini et al. 2000). To study proteins on a large scale, the application of a proteomic approach is mandatory (Dietrich 2014). Rabbit seminal plasma proteome has been proved different between rabbit genetic line A (maternal) and R (growth) (Viudes-de-Castro et al. (2004), Safaa et al. (2008), Casares-Crespo 2016) and between seasons (Casares-Crespo 2016). In these previous studies a traditional 1 dimension polyacrylamide gel was done, identifying only major proteins visible after Coomassie Colloidal Blue staining and obtaining the relative quantity of these protein bands. On the contrary, new approaches such as liquid chromatography in-gel or in-solution digestion coupled with mass spectrometric analysis are a powerful, yet relatively uncomplicated strategy to identify and characterize proteins. Mass spectrometry-based proteomics has become the tool of choice for identifying and quantifying the proteome of an organism (Karpievitch 2010). Therefore, the present study was conducted to describe the seminal plasma proteome of rabbits, compare the differentially expressed seminal plasma proteins between genetic lines A and R and study the effect of season in rabbit seminal plasma proteome.
Project description:A greater understanding of the proteins involved in reproduction can benefit animal production. New advances in proteomics are having a major impact on our understanding of how spermatozoa acquire their capacity for fertilization [1]. Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function [2]. The sperm cell is one of the most highly differentiated cells and is composed of a head with a highly compacted chromatin structure and a large flagellum with midpiece that contains the required machinery for movement and therefore to deliver the paternal genetic and epigenetic content to the oocyte [3]. By being so highly differentiated, spermatozoa are advantageous cells to study proteomics of specific compartments such as the membrane, which basically is the area of major importance for its role in interacting with the surroundings and the oocyte [4]. The fusion of a sperm and an oocyte is a sophisticated process that must be preceded by suitable changes in the sperm's membrane composition [5]. Recent studies of spermatozoa from the proteomic point of view have allowed the identification of different proteins in spermatozoa that are responsible for the regulation of normal/defective sperm functions [6]. While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics [7]. Using this method, detailed proteomic data are now available for human [8], macaque [9,10], mouse [11], rat [12], bull [13-15], stallion [16], fruit fly [17], Caenorhabditis elegans [18], carp [19], rainbow trout [20], mussel [21], ram [22], honeybee [23] and rooster [24] sperm membrane proteins. Rabbit (Oryctolagus cuniculus) is an important mammalian species worldwide, being at the same time of commercial interest and a research model animal. European rabbit meat production is approximately 500 thousand tons, corresponding to a 30% share of world production [25]. Besides, rabbits account for the seventh highest number of animals slaughtered per year in the European Union-27, with 347,603 × 1000 head in 2014 [26]. In a previous work, we identified and quantified rabbit seminal plasma proteins between two different genotypes [27], concluding the clear effect of genotype in the abundance of certain seminal plasma proteins. However, it is unknown at present whether these differences also exist at sperm proteome level. Therefore, the aim of the present study was to characterise rabbit sperm membrane proteins through NanoLC-MS/MS analysis focusing on the influence of the genetic origin.
Project description:Understanding how P4 signaling occurs in endometrial cells is important to guide the development of more effective therapeutic approaches to the numerous reproductive diseases caused by disrupted P4 signaling, resulting in subfertility or infertility (Bunch et al., 2013; Kim et al., 2013; Zhang and Murphy, 2014; Szekeres-Bartho, 2018; Hirota, 2019; Liao et al., 2019). Here, to fill these knowledge gaps, we characterized endometrial PGRMC2 expression throughout the human menstrual cycle and in hEnSCs subjected to an in vitro model of human endometrial decidualization. Additionally, we describe the functional implications of PGRMC2 with a silencing approach assay, integrating RNA-seq and proteomic techniques. We evaluated the functional implication of PGRMC2 in the embryo implantation process with an outgrowth in vitro model. Finally, we also compared endometrial PGRMC2 expression with PGRMC1 and PGR.
Project description:Solid State Fermentation (SSF) processes have been explored for yeast growth and protein and metabolites production. However, most of these processes lack standardization. In this work, we present a polylactic acid (PLA) 3D printed matrix that dramatically enhances yeast growth when embedded in liquid media compared to equivalent static cultures, and changes yeast expression patterns at the proteome level. Moreover, differences in sugar assimilation and ethanol production, as the main product of alcoholic fermentation, are observed. Our results suggest that these matrixes may be useful for a vast range of biotechnological applications based on yeast fermentation.
Project description:Since the first human conceived through in vitro fertilisation in 1978, over 8 million babies have been born by assisted reproductive technologies (ART). Although most ART babies and children seem healthy, in recent years, several human and animal model studies have evidenced a potential impact of ART on long-term development. However, the long-term follow-up data in this field is still limited. Till now, studies are mainly focused on techniques such as in vitro fertilisation or in vitro culture, being the information from gametes/embryos cryopreservation field practically missing. Herein, we have developed an animal model to determine whether vitrified-thawed embryo transfer procedure has long-term consequences over the offspring. The birth weight, growth performance and adult body weight of the rabbits derived from vitrified-thawed embryos was compared with that of the naturally-conceived animals. In adulthood, the liver, heart, kidneys, spleen, lungs, gonads and adrenal glands of the males were weighed and compared. Moreover, some haematological and biochemical parameters were assessed on peripheral blood. Besides, some liver samples were obtained to perform a comparative proteomic study. The embryo vitrification-transfer process modified the birth weight and the growth pattern of the offspring, reducing the growth performance in a sex-specific manner. In adulthood, animals derived from vitrified embryos showed a significantly lower body, liver and heart weight. Molecular analyses revealed that vitrified-thawed embryo transfer procedure triggers concordant reprogramming of the liver proteome. The most relevant metabolic alteration denoted by the protein profile was that related to the oxidative phosphorylation, suggesting an impaired oxidative metabolism in the mitochondria. Furthermore, hints of dysregulation in the zinc and lipid metabolism were identified. These results evidence long-term consequences in the offspring derived from cryopreserved-transferred embryos and represented an evident example of the phenotypic plasticity exhibited by the mammalian embryo.
Project description:In the ex situ conservation of chondrycthyan species, successful reproduction in aquaria is essential. However, aquatic species often exhibit reduced reproductive success under human care. Different factors, including water temperature, nutrition, and intrinsic genetic and epigenetic elements, influence their fertility. Conventional sperm analyses do not provide insights into the functional competence of semen. Therefore, proteomics is gaining relevance, increasing a better understanding of male physiology. In this study, we investigated the proteomic profiles of seminal plasma and spermatozoa from small-spotted catsharks in two different environments: natural environment and aquarium environment.
Project description:Prediction of male or semen fertility potential remains a persistent challenge that has yet to be fully resolved. This work analyzed several in vitro parameters and proteome of spermatozoa in bulls cataloged as high (HF; n=5) and low field (LF; n=5) fertility after more than a thousand artificial inseminations. Sperm motility was evaluated by Computer-Assisted Sperm Analysis. Sperm viability, mitochondrial membrane potential (MMP), and reactive oxygen species (mROS) of spermatozoa were assessed by flow cytometry. Proteome was evaluated by SWATH-MS procedure. Spermatozoa of HF bulls showed significantly higher total motility than the LF group (41.4% vs. 29.7%). Rates of healthy sperm (live, high MMP, and low mROS) for HF and LF bull groups were 49% and 43%, respectively (p > 0.05). Spermatozoa of HF bulls showed higher presence of differentially abundant proteins (DAPs) related to both energy production (COX7C), mainly OXPHOS pathway, and to the development of structures linked with the motility process (TPPP2, SSMEM1 and SPAG16). Furthermore, we observed that EQTN, together with other DAPs related to the interaction with the oocyte, were overrepresented in HF bull spermatozoa. The biological processes related to protein processing, catabolism, and protein folding were found to be overrepresented in LF bull sperm in which the HSP90AA1 chaperone was identified as the most DAP
Project description:Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How these proteome changes relate with the loss of sperm function during cryopreservation remains unsolved. The present study attempted to clarify this issue evaluating differential changes in the proteome of pig spermatozoa retrieved from the cauda epididymis and the ejaculate, with clear differences in cryotolerance, comparing fresh and frozen-thawed cells. Sperm samples were collected from 10 healthy, sexually mature and fertile boars, and cryopreserved using a standard 0.5 mL straw protocol. Total and progressive motility, viability and mitochondria membrane potential were higher and membrane fluidity and reactive oxygen species generation lower in frozen-thawed (FT) cauda epididymal than ejaculated spermatozoa. Quantitative proteomics of fresh and FT sperm samples were analyzed using a LC-ESI-MS/MS-based SWATH approach. Cryopreservation quantitatively altered more proteins in ejaculated than cauda epididymal spermatozoa. Differential protein-protein networks highlighted a set of proteins directly involved in mitochondrial functionality among those quantitatively altered in ejaculated spermatozoa, which would explain the worse post-thaw quality of ejaculated pig spermatozoa.
Project description:It has remained unknown how cells reduce cystine taken up from the extracellular space, which is a required step for further utilization of cysteine in key processes such as protein or glutathione synthesis. Here we show that the thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) is the rate limiting enzyme for intracellular cystine reduction. When TRP14 is genetically knocked out, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways becomes lethal in C. elegans subjected to proteostatic stress. TRP14 can also reduce cysteinyl moieties on proteins, rescuing their activities as here shown with cysteinylated peroxiredoxin 2. Txndc17 knock-out mice were, surprisingly, protected in an acute pancreatitis model, concomitant with activation of Nrf2-driven antioxidant pathways and upregulation of transsulfuration. We conclude that TRP14 is the evolutionary conserved enzyme principally responsible for intracellular cystine reduction in C. elegans, mice and humans.