Joanna Bukowska

Characterization of bovine immortalized luteal endothelial cells: action of cytokines on production and content of arachidonic acid metabolites

BackgroundThe interactions between luteal, vascular endothelial, immune cells and its products: steroids, peptide hormones, prostaglandins (PGs), growth factors and cytokines play a pivotal role in the regulation of corpus luteum (CL) function. Luteal endothelial cells undergo many dynamic morphological changes and their action is regulated by cytokines. The aims are: (1) to establish in vitro model for bovine luteal endothelial cells examination; (2) to study the effect of cytokines: tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) on cell viability, leukotrienes (LTs) and PG synthases, and endothelin-1 (EDN-1) mRNA, protein expression and their secretion in bovine immortalized luteal endothelial (EnCL-1) cells.MethodsThe primary cultures of bovine luteal endothelial cells were immortalized by transfection with vector carrying the Simian virus 40 T-antigen (SV40 T-ag) sequence. Expression of SV40 T-ag gene in EnCL-1 cells was confirmed by RT-PCR and immunofluorescence staining showed the presence of endothelial cell markers: VE-cadherin and von Willebrand factor. EnCL-1 cells were stimulated by TNFalpha with IFNgamma (50 ng/ml each) for 24 h. Cell viability, mRNA expression (real time RT-PCR), protein expression (western blotting) for LTC4 synthase (LTC4S), LTA4 hydrolase (LTA4H), PGE2 and PGF2alpha synthases and endothelin-1 (EDN-1), and levels of LTs (B4 and C4) and PGs (E2 and F2alpha) and EDN-1 in the medium (EIA) were evaluated.ResultsWe received immortalized luteal endothelial cell line (EnCL-1). Cytokines did not change EnCL-1 cell viability but increased mRNA expression of LTC4S, LTA4H, PGE2 and PGF2alpha synthases and EDN-1. EDN-1/2/3, LTC4 and PGF2alpha synthases protein expression were elevated in the presence of TNFalpha/IFNgamma, and accompanied by increased EDN-1, LTC4 and PGF2alpha secretion. Cytokines had no effect on PGES and LTA4H protein expression, and PGE2 and LTB4 release.ConclusionsTNFalpha and IFNgamma modulate EnCL-1 cell function. Moreover, established EnCL-1 cell line appears to be a good model for investigating the molecular mechanisms related to cytokines action and aa metabolites production in cattle.

Isolation and Characterization of an Ovoinhibitor, a Multidomain Kazal-Like Inhibitor from Turkey (Meleagris gallopavo) Seminal Plasma

ABSTRACT Turkey seminal plasma contains three serine proteinase inhibitors. Two of them, with low molecular masses (6 kDa), were identified as single-domain Kazal-type inhibitors responsible for regulating acrosin activity. Our experimental objective was to isolate and characterize the inhibitor with the high molecular weight from turkey seminal plasma. The inhibitor was purified using hydrophobic interaction and affinity chromatography. Pure preparations of the inhibitor were used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. Gene expression and immunohistochemical detection of the inhibitor were analyzed in the testis, epididymis, and ductus deferens. The inhibitor with a high molecular weight from turkey seminal plasma was identified as an ovoinhibitor, which was found in avian semen for the first time. The turkey seminal plasma ovoinhibitor was a six-tandem homologous Kazal-type domain serine proteinase inhibitor that targeted multiple proteases, including subtilisin, trypsin, and elastase, but not acrosin. Our results suggested that hepatocyte growth factor activator was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. The presence of the ovoinhibitor within the turkey reproductive tract suggested that its role was to maintain a microenvironment for sperm in the epididymis and ductus deferens. The turkey seminal plasma ovoinhibitor appeared to play a significant role in an antibacterial semen defense against Bacillus subtilis and Staphylococcus aureus.

Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma.

A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti-Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

Evidence for the presence of stem/progenitor cells in porcine endometrium

A population of adult stem cells responsible for cyclic reconstructing and remodeling has been proposed to reside in the highly regenerative mammalian endometrium. Recently, stem/progenitor cells have been identified in the human and mouse endometrium, but less is known about these cells in livestock animals. Using Hoechst 33342 fluorescent dye staining and flow cytometry, we identified an emerging cell side population that may be responsible for the regeneration process of the porcine endometrium. The percentage of side‐population cells on Day 19 of the estrous cycle was significantly higher than that on Days 2–4. Moreover, single cells were able to seed clones that could differentiate into three independent mesenchymal‐cell lineages. We also demonstrated the expression of specific markers of self‐renewal cells on these side‐population cells and the presence of a population of cells among the stromal cells that possess markers for mesenchymal stem cells. These results indicate that the porcine endometrium contains a population of cells with the capacity for self‐renewal and a high rate of proliferation, which depend on the phase of the estrous cycle. These cells could potentially be involved in the cyclic reconstruction of the porcine endometrium. Mol. Reprod. Dev. 82: 182–190, 2015.

Foxn1 and Mmp-9 expression in intact skin and during excisional wound repair in young, adult, and old C57Bl/6 mice

The transcription factor Foxn1 is essential for skin development. Our previous studies performed on young C57BL/6J mice model showed that Foxn1 acts as regulator of the skin wound healing process. The present study extended our initial research regarding the expression and potential role of Foxn1 in the intact and wounded skin as a function of animal age and stage of the wound healing process. We analyzed Foxn1 and Mmp‐9 expression in the intact and postinjured skin of young, adult, and old C57BL/6J and transgenic Foxn1::Egfp mice. The similar levels of epidermal Foxn1 mRNA expression were detected in young and adult C57BL/6J mice and higher levels in old animals. Postinjured skin tissues displayed a gradual decrease of Foxn1 mRNA expression at Days 1, 5, and 7 after injury. Foxn1‐eGFP positive cells were abundant at wound margin and in re‐epithelialized epidermis at postwounded Days 1, 5, and 7 and colocalized with E‐cadherin and Mmp‐9. Postwounded skin at Days 14–36 displayed Foxn1‐eGFP cells in the epidermis and in the dermal part of the skin (papillary dermis). A subset of Foxn1‐eGFP positive cells in the papillary dermis expressed the myofibroblast marker αSMA. Flow cytometric analysis of cells isolated from postwounded (Day 7) skin tissues showed a significant increase in the percentage of Foxn1‐eGFP positive cells with phenotype of double positivity for E‐cadherin/N‐cadherin (epithelial/mesenchymal markers). Collectively, these data identify the transcription factor Foxn1 as a potential key epidermal regulator modifying both epidermal and dermal healing processes after cutaneous wounding.

Foxn1 expression in keratinocytes is stimulated by hypoxia: further evidence of its role in skin wound healing

Recent studies have shown that the transcription factor Foxn1, which is expressed in keratinocytes, is involved in the skin wound healing process, yet how Foxn1 functions remains largely unknown. Our latest data indicate that Foxn1 drives skin healing via engagement in re-epithelization and the epithelial-mesenchymal transition (EMT) process. In the present study, 2D-DIGE proteomic profiling analysis of in vitro cultured keratinocytes transfected with adenoviral vector carrying Foxn1-GFP or GFP alone (control) revealed forty proteins with differential abundance between the compared groups. Among the proteins with Foxn1-dependent expression, several enable adaptation to hypoxia. Subsequent experiments revealed that hypoxic conditions (1% O2) stimulate endogenous and exogenous (transfected Ad-Foxn1) Foxn1 expression in cultured keratinocytes. A proteomics analysis also identified proteins that can act as a factors controlling the balance between cell proliferation, differentiation and apoptosis in response to Foxn1. We also showed that in C57BL/6 keratinocytes, the stimulation of Foxn1 by hypoxia is accompanied by increases in Mmp-9 expression. These data corroborate the detected co-localization of Foxn1 and Mmp-9 expression in vivo in post-wounding skin samples of Foxn1::Egfp transgenic mice. Together, our data indicate that Foxn1 orchestrates cellular changes in keratinocytes in both physiological (self-renewal) and pathological (skin wound healing) contexts.

Bone Marrow Adipocyte Developmental Origin and Biology

Purpose of ReviewThis review explores how the relationships between bone marrow adipose tissue (BMAT) adipogenesis with advancing age, obesity, and/or bone diseases (osteopenia or osteoporosis) contribute to mechanisms underlying musculoskeletal pathophysiology.Recent FindingsRecent studies have re-defined adipose tissue as a dynamic, vital organ with functions extending beyond its historic identity restricted solely to that of an energy reservoir or sink. “State of the art” methodologies provide novel insights into the developmental origin, physiology, and function of different adipose tissue depots. These include genetic tracking of adipose progenitors, viral vectors application, and sophisticated non-invasive imaging modalities.SummaryWhile constricted within the rigid bone cavity, BMAT vigorously contributes to local and systemic metabolic processes including hematopoiesis, osteogenesis, and energy metabolism and undergoes dynamic changes as a function of age, diet, bone topography, or sex. These insights will impact future research and therapies relating to osteoporosis.

Expression and secretion of albumin in male turkey (Meleagris gallopavo) reproductive tract in relation to yellow semen syndrome1

Yellow semen syndrome (YSS) is the most widely recognized problem among male turkeys. Yellow semen is of low quality and, when used for insemination, results in reduction of fertility and hatchability. Elevated level of serum albumin-like protein accession no. XP_003205725 is a characteristic feature of yellow seminal plasma suggesting albumin role in YSS pathology. However, knowledge regarding the expression of albumin in the reproductive tract in relation to YSS is very limited. The aim of this study was to identify albumin secretion and localization sites in the turkey reproductive tract in relation to YSS. Reproductive tract tissues and liver originating from turkeys producing white semen (WS) and YSS were used for analysis of albumin mRNA expression and its localization using immunohistochemistry. Moreover, albumin abundance in tissues, blood and seminal plasma was analyzed using two dimensional electrophoresis and western blot analysis. Albumin mRNA expression was found in all parts of the reproductive tract. Apart from the liver, the highest expression of albumin was found in the ductus deferens in YSS turkeys. The testicular spermatids, Leydig, and myoid cells and the epithelium of the epididymis and ductus deferens were the main secretion sites of albumin in the reproductive tract in turkeys. Higher albumin abundance was found in the reproductive tract and seminal plasma of YSS toms compared to WS toms. Our results demonstrated that germ cells from spermatocytes to spermatids, Leydig cells, and myoid cells synthesized and secreted albumin in turkey testis, and epithelial cells are the main secretion sites in epididymis and ductus deferens. Ductus deferens secretion of albumin seems to be mostly responsible for YSS. Over-secretion by the ductus deferens may be the main origin of albumin abundance in YSS semen. Knowledge regarding disturbances of albumin secretion in relation to YSS may be useful for future work on studies related to better understanding the molecular basis of YSS.ABSTRACT Yellow semen syndrome (YSS) is the most widely recognized problem among male turkeys. Yellow semen is of low quality and, when used for insemination, results in reduction of fertility and hatchability. Elevated level of serum albumin‐like protein accession no. XP_003205725 is a characteristic feature of yellow seminal plasma suggesting albumin role in YSS pathology. However, knowledge regarding the expression of albumin in the reproductive tract in relation to YSS is very limited. The aim of this study was to identify albumin secretion and localization sites in the turkey reproductive tract in relation to YSS. Reproductive tract tissues and liver originating from turkeys producing white semen (WS) and YSS were used for analysis of albumin mRNA expression and its localization using immunohistochemistry. Moreover, albumin abundance in tissues, blood and seminal plasma was analyzed using two dimensional electrophoresis and western blot analysis. Albumin mRNA expression was found in all parts of the reproductive tract. Apart from the liver, the highest expression of albumin was found in the ductus deferens in YSS turkeys. The testicular spermatids, Leydig, and myoid cells and the epithelium of the epididymis and ductus deferens were the main secretion sites of albumin in the reproductive tract in turkeys. Higher albumin abundance was found in the reproductive tract and seminal plasma of YSS toms compared to WS toms. Our results demonstrated that germ cells from spermatocytes to spermatids, Leydig cells, and myoid cells synthesized and secreted albumin in turkey testis, and epithelial cells are the main secretion sites in epididymis and ductus deferens. Ductus deferens secretion of albumin seems to be mostly responsible for YSS. Over‐secretion by the ductus deferens may be the main origin of albumin abundance in YSS semen. Knowledge regarding disturbances of albumin secretion in relation to YSS may be useful for future work on studies related to better understanding the molecular basis of YSS.

Foxn1 in Skin Development, Homeostasis and Wound Healing

Intensive research effort has focused on cellular and molecular mechanisms that regulate skin biology, including the phenomenon of scar-free skin healing during foetal life. Transcription factors are the key molecules that tune gene expression and either promote or suppress gene transcription. The epidermis is the source of transcription factors that regulate many functions of epidermal cells such as proliferation, differentiation, apoptosis, and migration. Furthermore, the activation of epidermal transcription factors also causes changes in the dermal compartment of the skin. This review focuses on the transcription factor Foxn1 and its role in skin biology. The regulatory function of Foxn1 in the skin relates to physiological (development and homeostasis) and pathological (skin wound healing) conditions. In particular, the pivotal role of Foxn1 in skin development and the acquisition of the adult skin phenotype, which coincides with losing the ability of scar-free healing, is discussed. Thus, genetic manipulations with Foxn1 expression, specifically those introducing conditional Foxn1 silencing in a Foxn1+/+ organism or its knock-in in a Foxn1−/− model, may provide future perspectives for regenerative medicine.

Effect of TGFβ1, TGFβ3 and keratinocyte conditioned media on functional characteristics of dermal fibroblasts derived from reparative (Balb/c) and regenerative (Foxn1 deficient; nude) mouse models

Skin injuries in mammals are healed through repair or regeneration. Our previous studies demonstrated that deficient expression of the transcription factor Foxn1 in epidermis of nude mice accounts for their skin’s pronounced regenerative properties. Since homeostasis within the skin depends on complex interactions between the epidermal and underlying dermal layers, the present study characterizes and compares isolated dermal fibroblasts (DFs) between regenerative nude (Foxn1 deficient) mice and their wild-type Balb/c counterparts. Nude DFs exhibited a higher cumulative number of population doublings (cumulative PD) at low seeding density and increased adipogenic differentiation capacity relative to their Balb/c DF counterparts. Nude DFs displayed reduced migration and gel contraction, functional features associated with wound healing. The comparison of transforming growth factor β family (TGFβ) expression showed significantly higher levels of Tgfβ3 transcript between nude and Balb/c mice but no differences were detected for Tgfβ1. Nude DFs were specifically sensitive to the presence of the pro-regenerative TGFβ3 isoform, showing increased collagen I deposition and alpha smooth muscle actin expression. Viability of Balb/c DFs was stimulated by keratinocyte conditioned media (KCM) from Balb/c (Foxn1 active) but inhibited by nude (Foxn1 deficient) KCM. In contrast, nude DFs did not respond to either KCMs with respect to their metabolic activity. Collectively, the enhanced plasticity and greater sensitivity of nude DFs to TGFβ3 stimulation are indicative of and consistent with their pro-regenerative characteristics. These data support the hypothesis that epidermal Foxn1 plays a critical role in determining the DFs regenerative phenotype.