Showing papers by "Thomas Fröhlich published in 2015"

Cryopreservation‐induced alterations in protein composition of rainbow trout semen

Abstract: The aim of this study was to detect cryopreservation-induced alterations in the protein composition of rainbow trout semen using two independent methods 1DE SDS-PAGE prefractionation combined with LC-MS/MS and 2D difference gel electrophoresis followed by MALDI-TOF/TOF identification. Here, we show the first comprehensive dataset of changes in rainbow trout semen proteome after cryopreservation, with a total of 73 identified proteins released from sperm to extracellular fluid, including mitochondrial, cytoskeletal, nuclear, and cytosolic proteins. Our study provides new information about proteins released from sperm, their relation to sperm structure and function, and changes of metabolism of sperm cells as a result of cryopreservation. The identified proteins represent potential markers of cryoinjures of sperm structures and markers of the disturbances of particular sperm metabolic pathways. Further studies will allow to decipher the precise function of the proteins altered during rainbow trout cryopreservation and are useful for the development of extensive diagnostic tests of sperm cryoinjures and for the successful improvement of sperm cryopreservation of this economically important species.

Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

Abstract: Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

Interclonal proteomic responses to predator exposure in Daphnia magna may depend on predator composition of habitats

Abstract: Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterizing the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the water flea, is a textbook example for predator-induced phenotypic plastic defences; however, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages. We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. To get a more comprehensive idea of this phenomenon, we studied four genotypes with five biological replicates each, originating from habitats characterized by different predator composition, ranging from predator-free habitats to habitats containing T. cancriformis. We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype-dependent manner. Proteins connected to genotype-dependent responses were related to the cuticle, protein synthesis and calcium binding, whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype-dependent responses at the proteome level were most distinct for the only genotype that shares its habitat with Triops. Altogether, our study provides new insights concerning genotype-dependent and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.

Shotgun proteomics of rainbow trout ovarian fluid.

Abstract: In the present study we used a shotgun proteomic approach to identify 54 proteins of rainbow trout ovarian fluid. The study has unravelled the identity of several proteins not previously reported in fish ovarian fluid. The proteome of trout ovarian fluid consists of diverse proteins participating in lipid binding and metabolism, carbohydrate and ion transport, innate immunity, maturation and ovulation processes. Most trout ovarian fluid proteins correspond to follicular fluid proteins of higher vertebrates, but 15% of the proteins were found to be different, such as those related to the immune system (precerebellin-like protein), proteolysis (myeloid cell lineage chitinase), carbohydrate and lipid binding and metabolism (vitellogenins), cell structure and shape (vitelline envelope protein gamma) and a protein with unknown functions (UPF0762 protein C6orf58 homologue). The present study could help in the decoding of the biological function of these proteins and in the discovery of potential biomarkers of oocyte quality.

Human testicular peritubular cells secrete pigment epithelium-derived factor (PEDF), which may be responsible for the avascularity of the seminiferous tubules

Abstract: Male fertility depends on spermatogenesis, which takes place in the seminiferous tubules of the testis. This compartment is devoid of blood vessels, which are however found in the wall of the seminiferous tubules. Our proteomic study using cultured human testicular peritubular cells (HTPCs) i.e. the cells, which form this wall, revealed that they constitutively secrete pigment epithelium-derived factor, PEDF, which is known to exert anti-angiogenic actions. Immunohistochemistry supports its presence in vivo, in the human tubular wall. Co-culture studies and analysis of cell migration patterns showed that human endothelial cells (HUVECs) are repulsed by HTPCs. The factor involved is likely PEDF, as a PEDF-antiserum blocked the repulsing action. Thus testicular peritubular cells, via PEDF, may prevent vascularization of human seminiferous tubules. Dihydrotestosterone (DHT) increased PEDF (qPCR) in HTPCs, however PEDF expression in the testis of a non-human primate occurs before puberty. Thus PEDF could be involved in the establishment of the avascular nature of seminiferous tubules and after puberty androgens may further reinforce this feature. Testicular microvessels and blood flow are known to contribute to the spermatogonial stem cell niche. Hence HTPCs via control of testicular microvessels may contribute to the regulation of spermatogonial stem cells, as well.

Proteome analysis of early lineage specification in bovine embryos

Abstract: During mammalian embryo development, the zygote undergoes embryonic cleavage in the oviduct and reaches the uterus at the morula stage, when compaction and early lineage specification take place. To increase knowledge about the associated changes of the embryonic protein repertoire, we performed a comprehensive proteomic analysis of in vitro produced bovine morulae and blastocysts (six biological replicates), using an iTRAQ-based approach. A total of 560 proteins were identified of which 502 were quantified. The abundance of 140 proteins was significantly different between morulae and blastocysts, among them nucleophosmin (NPM1), eukaryotic translation initiation factor 5A-1 (EIF5A), receptor of activated protein kinase C 1 (GNB2L1/RACK1), and annexin A6 (ANXA6) with increased, and glutathione S-transferase mu 3 (GSTM3), peroxiredoxin 2 (PRDX2), and aldo-keto reductase family 1 member B1 (AKR1B1) with decreased abundance in blastocysts. Seventy-three percent of abundance altered proteins increased, reflecting an increase of translation activity in this period. This is further supported by an increase in the abundance of proteins involved in the translation machinery and the synthesis of ATP. Additionally, a complementary 2D saturation DIGE analysis led to the detection of protein isoforms, e.g. of GSTM3 and PRDX2, relevant for this period of mammalian development, and exemplarily verified the results of the iTRAQ approach. In summary, our systematic differential proteome analysis of bovine morulae and blastocysts revealed new molecular correlates of early lineage specification and differentiation events during bovine embryogenesis.

The influence of simulated microgravity on the proteome of Daphnia magna.

Abstract: The waterflea Daphnia is an interesting candidate for bioregenerative life support systems (BLSS). These animals are particularly promising because of their central role in the limnic food web and its mode of reproduction. However, the response of Daphnia to altered gravity conditions has to be investigated, especially on the molecular level, to evaluate the suitability of Daphnia for BLSS in space. In this study, we applied a proteomic approach to identify key proteins and pathways involved in the response of Daphnia to simulated microgravity generated by a two-dimensional (2D) clinostat. We analyzed five biological replicates using 2D-difference gel electrophoresis proteomic analysis. We identified 109 protein spots differing in intensity (P<0.05). Substantial fractions of these proteins are involved in actin microfilament organization, indicating the disruption of cytoskeletal structures during clinorotation. Furthermore, proteins involved in protein folding were identified, suggesting altered gravity induced breakdown of protein structures in general. In addition, simulated microgravity increased the abundance of energy metabolism-related proteins, indicating an enhanced energy demand of Daphnia. The affected biological processes were also described in other studies using different organisms and systems either aiming to simulate microgravity conditions or providing real microgravity conditions. Moreover, most of the Daphnia protein sequences are well-conserved throughout taxa, indicating that the response to altered gravity conditions in Daphnia follows a general concept. Data are available via ProteomeXchange with identifier PXD002096. Low-gravity conditions could trigger physiological changes in tiny crustaceans, reports a German team. The planktonic crustacean Daphnia consumes algae and acts as food for fish, and could thus contribute to sustainable mini-ecosystems that provide oxygen and food for astronauts. In order to learn how microgravity might affect Daphnia, Christian Laforsch at Bayreuth University and colleagues subjected the crustaceans to simulated microgravity with a device known as a clinostat and examined the resulting changes in protein production. They identified striking differences in specific biological pathways—for example, simulated microgravity interfered with proteins that form the cellular infrastructure and facilitate movement, and appeared to increase overall energy consumption. These findings offer a peek into the stresses that cultured Daphnia are likely to experience in low-gravity environments, representing an important starting point for further research into space life-support systems.