The steady improvement of mammalian cell factories for the production of biopharmaceuticals is a key challenge for the biotechnology community. Recently, small regulatory microRNAs (miRNAs) were identified as novel targets for optimizing Chinese hamster ovary (CHO) production cells as they do not add any translational burden to the cell while being capable of regulating entire physiological pathways. The aim of the present study was to elucidate miRNA function in a recombinant CHO-SEAP cell line by means of a genome-wide high-content miRNA screen. This screen revealed that out of the 1, 139 miRNAs examined, 21% of the miRNAs enhanced cell-specific SEAP productivity mainly resulting in elevated volumetric yields, while cell proliferation was accelerated by 5% of the miRNAs. Conversely, cell death was diminished by 13% (apoptosis) or 4% (necrosis) of all transfected miRNAs. Besides these large number of identified target miRNAs, the outcome of our studies suggest that the entire miR-30 family substantially improves bioprocess performance of CHO cells. Stable miR-30 over expressing cells outperformed parental cells by increasing SEAP productivity or maximum cell density of approximately twofold. Our results highlight the application of miRNAs as powerful tools for CHO cell engineering, identified the miR-30 family as a critical component of cell proliferation, and support the notion that miRNAs are powerful determinants of cell viability.

H 2 O 2 is a reactive oxygen species (ROS), which can diffuse away from its site of generation and may act as a cell-to-cell signaling factor. The mechanisms responsible for the generation of H 2 O 2 in human ovarian follicles and possible signaling role(s) of H 2 O 2 are not well known. We identified a source of H 2 O 2 , the enzyme NADPH oxidase (NOX) 4, in isolated differentiated, in-vitro fertilisation-derived human granulosa-lutein cells (GCs), in proliferating human granulosa tumour cells (KGN), as well as in situ in cells of growing ovarian follicles. H 2 O 2 was readily detected in the supernatant of cultured GCs and KGN cells. H 2 O 2 levels were significantly lowered by the NOX4 blocker GKT137831, indicating a pronounced contribution of NOX4 to overall H 2 O 2 generation by these cells. We provide evidence that extracellular H 2 O 2 is taken up by GCs, which is facilitated by aquaporins (peroxiporins). We thus conclude that GC-derived H 2 O 2 might act as autocrine/paracrine factor. Addition of H 2 O 2 increased MAPK-phosphorylation in GCs. Moreover, reducing H 2 O 2 production with GKT137831 slowed proliferation of KGN cells. Our results pinpoint NOX4 and H 2 O 2 as physiological players in the regulation of GC functions.

No abstract

BackgroundA previous study showed that dopamine (DA), which is contained in follicular fluid (FF) from IVF patients, strongly increased the production of reactive oxygen species (ROS) by cultured human granulosa cells (GCs). ROS, including H2O2, are assumed to play roles in ovarian physiology and pathology. Ovarian DA could be derived from the circulation, ovarian innervation and/or unknown ovarian sources. L-DOPA is the direct precursor of DA in its synthetic pathway. It was not yet described in FF. We examined L-DOPA levels in FF from IVF patients. As it may exert anti-oxidative and ROS-scavenging functions, we studied whether it exerts such actions in human GCs and whether DOPA-decarboxylase (DDC), the enzyme converting L-DOPA to DA, is expressed in the human ovary.ResultsELISA measurements revealed that human IVF-derived FF contains L-DOPA. In cultured human GCs automated confluence analyses showed that L-DOPA enhanced their survival. This is in contrast to the actions of DA, which reduced cell survival. A dose-dependent mode of action of L-DOPA was identified using a fluorescent ROS indicator. The results showed that it antagonized intracellular ROS accumulation induced by exogenous H2O2. DDC was absent in follicular GCs, but immunohistochemistry identified it in theca cells (TCs) of large follicles in the human ovary. Laser micro-dissection followed by RT-PCR corroborated the expression. DDC was also identified in the steroidogenic cells of the corpus luteum.ConclusionsL-DOPA in FF is an antioxidant factor and exerts positive influences on GCs. Ovarian DA is derived from L-DOPA and has opposite actions. Exogenous L-DOPA is a standard therapy for Parkinson’s disease, and the results raise the possibility that it may be able to exert positive actions as an antioxidant in ovarian conditions, as well.Electronic supplementary materialThe online version of this article (doi:10.1186/s13048-016-0269-0) contains supplementary material, which is available to authorized users.

Recent studies showed that KGN cells, derived from a human granulosa cell tumor (GCT), express NADPH oxidase 4 (NOX4), an important source of H2O2. Transient receptor potential melastatin 2 (TRPM2) channel is a Ca2+ permeable cation channel that can be activated by H2O2 and plays an important role in cellular functions. It is also able to promote susceptibility to cell death. We studied expression and functionality of TRPM2 in KGN cells and examined GCT tissue microarrays (TMAs) to explore in vivo relevance. We employed live cell, calcium and mitochondrial imaging, viability assays, fluorescence activated cell sorting (FACS) analysis, Western blotting and immunohistochemistry. We confirmed that KGN cells produce H2O2 and found that they express functional TRPM2. H2O2 increased intracellular Ca2+ levels and N-(p-Amylcinnamoyl)anthranilic acid (ACA), a TRPM2 inhibitor, blocked this action. H2O2 caused mitochondrial fragmentation and apoptotic cell death, which could be attenuated by a scavenger (Trolox). Immunohistochemistry showed parallel expression of NOX4 and TRPM2 in all 73 tumor samples examined. The results suggest that GCTs can be endowed with a system that may convey susceptibility to cell death. If so, induction of oxidative stress may be beneficial in GCT therapy. Our results also imply a therapeutic potential for TRPM2 as a drug target in GCTs.

Chinese hamster ovary (CHO) suspension cells are the main production hosts for biopharmaceuticals. For the improvement of production processes, it is essential to understand the interaction between CHO cells and their microenvironment. While the cellular membrane is the crucial surface barrier between the inner and outer cell compartments, the subgroup of cell surface proteins (surfaceome) is of particular interest due to its potential to react to external factors and initiate cell communication and interaction pathways. Therefore, the CHO surfaceome was explored for the first time by enriching exposed N-glycosylated membrane proteins before tandem mass spectrometry (MS/MS) analyses, identifying a total of 449 surface proteins, including 34 proteins specific for production cells. Functional annotation and classification located most proteins to the cell surface belonging mainly to the protein classes of receptors, enzymes, and transporters. In addition, adhesion molecules as cadherins, integrins, Ig superfamily and extracellular matrix (ECM) proteins as collagens, laminins, thrombospondin, fibronectin, and tenascin were significantly enriched, which are involved in mechanisms for the formation of cell junctions, cell-cell and cell-ECM adhesion as focal adhesions. As cell adhesion and aggregation counteracts scalable production of biopharmaceuticals, experimental validation confirmed differential expression of integrin β1 (ITGB1) and β3, CD44, laminin, and fibronectin on the surface of aggregation-prone CHO production cells.The subsequent modulation of the central interaction protein ITGB1 by small interfering RNA knockdown substantially counteracted cell aggregation pointing toward novel engineering routes for aggregation reduction in biopharmaceuticalThis is an open access article under the terms of the Creative Commons Attribution -NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made.

Impairment of puerperal fertility may be a severe problem in some dairy herds. Within the last years, several diagnostic possibilities weredevelopedandvalidated.Theaimofthisstudy wastoevalu-atetheapplicabilityofbloodserumparameterssubstanceP(SP)and vasoactiveintestinalpolypeptide(VIP)fortheiruseinearlydiagnostics of uterine involution disturbances. Blood serum samples of 86 dairycowstakenwithinthefirst20dayspost-partumwereexamined using commercially available ELISA test kits. Animals were divided into two groups (healthy or diseased) depending on the results of clinicalandgynaecologicalexamination.Statisticalanalysisconsisted ofthetimelychangesinbloodserumlevelsaswellasthegroupcomparisonofhealthycowsandcowswithuterinedisease.Bloodserum concentrations for SP increased statistically significant within the first 20days after calving (p<0.04). There was no statistically significantdifferencebetweenthegroups.ConcerningVIP,neitherfor thetimelycoursenorforthegroupcomparisonasignificantdifferencecouldbeshown.SPrepresentsanaccreditedbiomarkerforpain in cattle. An increase of substance P within the first 20days postpartum suggests the presence of persisting pain presumably due to uterine involution processes. Nevertheless, both serum parametersdonotseemsuitableasindicatorsforthepresenceofuterine involutiondisorders. Theobjectivesofthisstudyweretoinvestigatethecasesofcamel dystocia in which fetotomy was indicated, the related complicationsandtheassociatedrisksformaternalmortalityandfertility. Fifty female dromedary camels were handled with fetotomy due to uncorrectable dystocia. The complications during and after fetotomyandpost-operativefertilityrateswererecorded.Logistic regressionwasperformedtoidentifyriskfactorsforthedependentvariablesofmaternalmortalityandfertilityafterfetotomy.The independentvariableswereparity,durationofdystocia,presence of foetal emphysema, number of cuts, duration of fetotomy and occurrence of complications during or after fetotomy. Common indications of fetotomy were head and neck deviations alone or with carpal or shoulder flexions (54%) and breech presentation (14%). Complications included tearing of the soft tissue of the birth canal (24%), uterine prolapse (6%) and retained placenta (4%). Maternal mortality occurred in 26% of the cases. A significantassociationwasdetectedbetweenmaternaldeathsandduration of dystocia (odds ratio=4.67, p=0.03), presence of foetal emphysema (odds ratio=3.93, p=0.04) and occurrence of complications during or after fetotomy (odds ratio=8.9, p=0.003). Post-operative fertility was 62.2%. None of the estimated factors showed a significant relationship with post-operative fertility. In conclusion, postural abnormalities constituted the most common indication of fetotomy in dromedary camels. The duration of dystocia, presenceoffoetal emphysema and occurrence of complications during or after fetotomy represented risks for maternal recovery. The fertilityrateafter fetotomy was generally encouraging. 3 | Effect of the ovarian superstimulation and ovum pick-up procedure on t...

Dinocampus coccinellae (Hymenoptera:Braconidae, Euphorinae) is a solitary, generalist Braconid parasitoid wasp of over fifty diverse species of coccinellid ladybeetles worldwide that reproduces through thelytokous parthenogenesis, an asexual process in which diploid daughters emerge from unfertilized eggs. Here we utilized a common garden and reciprocal transplant experiment using parthenogenetic lines of D. coccinellae presented with three different host ladybeetle species of varying sizes, across multiple generations to investigate heritability, plasticity, and environmental covariation of body size. Since unilineal (reared on same host species) lines restrict environmental variation on clones, we expected positively correlated parent-offspring parasitoid regressions, indicative of heritable size variation. Whereas multilineal (reared on different host species) lines would quantify phenotypic plasticity of clones reared in varying environments, we expected negatively correlated parent-offspring parasitoid regressions. Contrary to expectations, our results indicate (1) little heritable variation in body size, (2) strong dependence of offspring size on the host environment, (3) a consistent signal of size-host tradeoff wherein small mothers always produced larger offspring, and vice versa, independent of host environment. Our study offers support for a constrained fecundity advantage model of Cope's Law, wherein D. coccinellae maintains phenotypic plasticity in body size despite parthenogenetic reproduction.