Minimally Invasive Intradiscal Delivery of BM-MSCs via Fibrous Microscaffold Carriers.

Current treatments of degenerated intervertebral discs often provide only temporary relief or address specific causes, necessitating the exploration of alternative therapies. Cell-based regenerative approaches showed promise in many clinical trials, but limitations such as cell death during injection and a harsh disk environment hinder their effectiveness. Injectable microscaffolds offer a solution by providing a supportive microenvironment for cell delivery and enhancing bioactivity. This study evaluated the safety and feasibility of electrospun nanofibrous microscaffolds modified with chitosan (CH) and chondroitin sulfate (CS) for treating degenerated NP tissue in a large animal model. The microscaffolds facilitated cell attachment and acted as an effective delivery system, preventing cell leakage under a high disc pressure. Combining microscaffolds with bone marrow-derived mesenchymal stromal cells demonstrated no cytotoxic effects and proliferation over the entire microscaffolds. The administration of cells attached to microscaffolds into the NP positively influenced the regeneration process of the intervertebral disc. Injectable poly(l-lactide-co-glycolide) and poly(l-lactide) microscaffolds enriched with CH or CS, having a fibrous structure, showed the potential to promote intervertebral disc regeneration. These features collectively address critical challenges in the fields of tissue engineering and regenerative medicine, particularly in the context of intervertebral disc degeneration.

Laser-Assisted Fabrication of Injectable Nanofibrous Cell Carriers.

The use of injectable biomaterials for cell delivery is a rapidly expanding field which may revolutionize the medical treatments by making them less invasive. However, creating desirable cell carriers poses significant challenges to the clinical implementation of cell-based therapeutics. At the same time, no method has been developed to produce injectable microscaffolds (MSs) from electrospun materials. Here the fabrication of injectable electrospun nanofibers is reported on, which retain their fibrous structure to mimic the extracellular matrix. The laser-assisted micro-scaffold fabrication has produced tens of thousands of MSs in a short time. An efficient attachment of cells to the surface and their proliferation is observed, creating cell-populated MSs. The cytocompatibility assays proved their biocompatibility, safety, and potential as cell carriers. Ex vivo results with the use of bone and cartilage tissues proved that NaOH hydrolyzed and chitosan functionalized MSs are compatible with living tissues and readily populated with cells. Injectability studies of MSs showed a high injectability rate, while at the same time, the force needed to eject the load is no higher than 25 N. In the future, the produced MSs may be studied more in-depth as cell carriers in minimally invasive cell therapies and 3D bioprinting applications.

Death Processes in Bovine Theca and Granulosa Cells Modelled and Analysed Using a Systems Biology Approach.

In this paper, newly discovered mechanisms of atresia and cell death processes in bovine ovarian follicles are investigated. For this purpose the mRNA expression of receptor interacting protein kinases 1 and 3 (RIPK1 and RIPK3) of the granulosa and theca cells derived from healthy and atretic follicles are studied. The follicles were assigned as either healthy or atretic based on the estradiol to progesterone ratio. A statistically significant difference was recorded for the mRNA expression of a RIPK1 and RIPK3 between granulosa cells from healthy and atretic follicles. To further investigate this result a systems biology approach was used. The genes playing roles in necroptosis, apoptosis and atresia were chosen and a network was created based on human genes annotated by the IMEx database in Cytoscape to identify hubs and bottle-necks. Moreover, correlation networks were built in the Cluepedia plug-in. The networks were created separately for terms describing apoptosis and programmed cell death. We demonstrate that necroptosis (RIPK-dependent cell death pathway) is an alternative mechanism responsible for death of bovine granulosa and theca cells. We conclude that both apoptosis and necroptosis occur in the granulosa cells of dominant follicles undergoing luteinisation and in the theca cells from newly selected follicles.

Expression profile of developmental competence gene markers in comparison with prostaglandin F2α synthesis and action in the early- and late-cleaved pre-implantation bovine embryos.

The kinetics of early cleavage stages can affect embryo quality. The bovine model of early- and late-cleaved embryos has been described in the literature and is deemed a useful tool in the field of oocyte developmental competence studies. The expression of genes demonstrating developmental potential differs between early- and late-cleaved embryos. Previously, we demonstrated that prostaglandin F2α synthase (PGFS) and prostaglandin F2α receptor (PTGFR) expression depend on the developmental stage and embryo quality. In the present study, we used the same model to determine the mRNA expression profile of developmentally important genes (IGF1R, IGF2R, PLAC8, OCT4, SOX2) in early, expanded and hatched blastocysts obtained from the early- and late-cleaved group of embryos, as well as to correlate the transcription levels of these embryonic gene markers with the transcription levels of PGFS and PTGFR. The mRNA expression of PGFS, PTGFR and factors described as gene markers of embryonic implantation ability and developmental competence genes was determined by real-time PCR. The obtained results were analysed using statistical software GraphPad prism 6.05. During the course of our analyses, we observed that the transcript abundance of most analysed genes tends to be higher in the late-rather than in the early cleaved group of embryos, as well as in B and/or C grade embryos rather than in A grade embryos. On the other hand, for the early cleaved group of blastocysts with cavity, we detected higher PLAC8 mRNA expression for grade A embryos compared with grade C embryos. It suggests that the mRNA expression level of genes depends on the quality of embryos but differs according to various factors including the method of production or culture method. Moreover, numerous correlations between analysed gene markers and PGF2α synthase and PGF2α receptor suggest that PGF2α plays a role in the crucial steps of bovine embryo development.

mRNA Expression and Role of PPARγ and PPARδ in Bovine Preimplantation Embryos Depending on the Quality and Developmental Stage.

Peroxisome proliferator-activated receptors (PPARs), a nuclear receptors for prostacyclin (PGI2) have been recognized as being essential for early embryo development. The objectives of the present study were to determine if the bovine early- and late-cleaved embryos in different stages of early development express PPARγ and PPARδ. Since embryo developmental competence depends on numerous biological factors, we evaluated if the expression of PPARγ and PPARδ correlate with selected embryo quality markers (SOX2, OCT4, PLAC8, IGF1R) in the in vitro produced embryos at different stages of their development. Developmental rates and embryo quality for early- and late-cleaved embryos were provided according to International Embryo Transfer Society (IETS; developmental stages: 2-, 4-, 16-cell embryo, morula, blastocyst (1-early, 2-developing, 3-expanded, 4-hatched); quality stages: A-high quality, B-moderate quality, C-low quality). We found that bovine embryos expressed mRNA of PPARδ and PPARγ at all stages of early development, independently of their quality. In addition, the expression of PPARδ and PPARγ correlated with the expression of quality markers in bovine blastocysts. Positive correlations were stronger and more frequent in the group of early-cleaved embryos, whereas the negative correlations were typical for the group of late-cleaved embryos. Obtained results and available literature reports may indicate the participation of PGI2, via PPARδ and PPARγ, in the processes related to the early embryo development, through the participation of this factor in the modulation of blastocyst hatching, implantation, and post-implantation development.

Immune Response to COVID-19: Can We Benefit from the SARS-CoV and MERS-CoV Pandemic Experience?

The global range and high fatality rate of the newest human coronavirus (HCoV) pandemic has made SARS-CoV-2 the focus of the scientific world. Next-generation sequencing of the viral genome and a phylogenetic analysis have shown the high homology of SARS-CoV-2 to other HCoVs that have led to local epidemics in the past. The experience acquired in SARS and MERS epidemics may prove useful in understanding the SARS-CoV-2 pathomechanism and lead to effective treatment and potential vaccine development. This study summarizes the immune response to SARS-CoV, MERS-CoV, and SARS-CoV-2 and focuses on T cell response, humoral immunity, and complement system activation in different stages of HCoVs infections. The study also presents the quantity and frequency of T cell responses, particularly CD4+ and CD8+; the profile of cytokine production and secretion; and its relation to T cell type, disease severity, and utility in prognostics of the course of SARS, MERS, and COVID-19 outbreaks. The role of interferons in the therapy of these infections is also discussed. Moreover, the kinetics of specific antibody production, the correlation between humoral and cellular immune response and the immunogenicity of the structural HCoVs proteins and their utility in the development of a vaccine against SARS, MERS, and COVID-19 has been updated.

Influence of Bone Marrow-Derived Mesenchymal Stem Cell Therapy on Oxidative Stress Intensity in Minimally Conscious State Patients.

Neurological disorders, including minimally conscious state (MCS), may be associated with the presence of high concentrations of reactive oxygen species within the central nervous system. Regarding the documented role of mesenchymal stem cells (MSCs) in oxidative stress neutralization, the aim of this study is to evaluate the effect of bone marrow-derived MSC (BM-MSC) transplantation on selected markers of oxidative stress in MCS patients. Antioxidant capacity was measured in cerebrospinal fluid (CSF) and plasma collected from nine patients aged between 19 and 45 years, remaining in MCS for 3 to 14 months. Total antioxidant capacity, ascorbic acid and ascorbate concentrations, superoxide dismutase, catalase, and peroxidase activity were analyzed and the presence of tested antioxidants in the CSF and plasma was confirmed. Higher ascorbic acid (AA) content and catalase (CAT) activity were noted in CSF relative to plasma, whereas superoxide dismutase (SOD) activity and total antioxidant capacity were higher in plasma relative to CSF. Total antioxidant capacity measured in CSF was greater after BM-MSC transplantations. The content of ascorbates was lower and CAT activity was higher both in CSF and plasma after the administration of BM-MSC. The above results suggest that MSCs modulate oxidative stress intensity in MCS patients, mainly via ascorbates and CAT activity.

Prostaglandin F2α (PGF2α) production possibility and its receptors expression in the early- and late-cleaved preimplantation bovine embryos.

BACKGROUND: Prostaglandin F2α (PGF2α) is an important component for the physiology of female reproductive processes. In the literature, the data pertaining to the synthesis and action of PGF2α in early embryonic bovine development are limited. In our study, we used the bovine in vitro culture model based on the time of first cleavage to determine the mRNA expression and immunolocalization of PGF2α synthase and its receptor in bovine embryos from the 2-cell stage to the hatched blastocyst stage. We also evaluated PGF2α production at 2, 5 and 7 days of in vitro culture. RESULTS: We found a significantly higher proportion of blastocysts obtained from the early-cleaved embryos than from the late-cleaved embryos (37.7% vs. 26.1% respectively, P < 0.05). The PGFS mRNA expression was significantly higher in the late-cleaved group than in the early-cleaved group at the 2-, 4- and 16-cell stages (P < 0.05). For PTGFR, we observed that within the late-cleaved group, the mRNA abundance was significantly higher in embryos at the 2- and 16-cell stages than in embryos at the 4- and 8-cell stages (P < 0.05). We observed that PTGFR mRNA expression was significantly higher in the 2- and 16-cell embryos in the late-cleaved group than that in the early-cleaved group embryos (P < 0.05). Among the blastocysts, the PGFS and PTGFR expression levels showed a trend towards higher mRNA expression in the late-cleaved group than in the early-cleaved group. Analysis of PGF2α production showed that within the early-cleaved group, the content of PGF2α in the in vitro culture medium was significantly higher on day 7 than it was on day 2 (P < 0.05). CONCLUSIONS: The mRNA expression levels of PGF2α synthase and its receptor depend on the developmental stage and the embryo quality. Analyses of PGFS and PTGFR expression in bovine blastocysts and of PGF2α embryo production suggest that prostaglandin F2α can act in an autocrine and paracrine manner in bovine in vitro-produced preimplantation embryos. Moreover, the tendency of PTGFR and PGFS mRNA expression to be upregulated in embryos with low developmental potential can indicate a compensation mechanism related to high PGFS and PTGFR mRNA expression levels in low-quality embryos.

Expression of enzymes involved in the synthesis of prostaglandin E2 in early- and late-cleaved bovine embryos at different stages of preimplantation development.

Prostaglandin (PG) E2 plays a role in numerous aspects of mammalian reproduction, such as oviductal transport of gametes, hatching from the zona pellucida in blastocysts and early embryonic development. Despite the evident role of PGE2 in the regulation of female reproductive processes, in the literature, there is very little information concerning the expression of PGE2 synthesizing enzymes and the exact amount of PGE2 produced by bovine embryos in vitro. In the present study, we aimed to determine the mRNA levels and immunolocalization of the enzymes responsible for PGE2 synthesis (PTGS2, mPGES1, mPGES2 and cPGES) in embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, early blastocyst, blastocyst, expanded blastocyst and hatched blastocyst stages, using a well-defined bovine model of oocyte developmental competence based on the time of first cleavage. PTGS2, mPGES2 and cPGES transcripts and proteins were detected in all stages of embryos, whereas the mPGES1 transcript and protein were not detected in embryos from the 2- to 16-cell stage. The results showed different transcription profiles of the enzymes involved in PGE2 synthesis in early- and late-cleaved embryos during the early stages of their in vitro preimplantation development. We also found that all the analysed stages of bovine preimplantation embryos released PGE2, with the highest concentration on Day 7 of culture in both the early- and late-cleaved groups. The present study is the first to demonstrate PGE2 synthesis and production by bovine early- and late-cleaved embryos at different stages of preimplantation development. Bovine embryos can produce PGE2, which may exert paracrine regulation during development. The transcription levels of PGE2 synthases were affected by the embryonic stage of development and quality. Our results indicate that the different transcription profiles of PTGS2, mPGES1, mPGES2 and cPGES, as well as PGE2 concentration, in early-versus late-cleaved embryos are dependent on the quality of the oocytes from which the embryos were obtained, which could reveal the association of PGE2 production during bovine preimplantation development with more advanced stages of embryo development.

LPAR2 and LPAR4 are the Main Receptors Responsible for LPA Actions in Ovarian Endometriotic Cysts.

Endometriosis has been considered as an estrogen (E2)-dependent and progesterone (P4)-resistant disease. On the other hand, lysophosphatidic acid (LPA) has been suggested as a significant modulator of ovarian pathology, acting via both LPA levels and LPA receptor (LPAR) upregulation. Therefore, the objective of the present study was to evaluate LPA concentration as well as LPARs, autotaxin (ATX), and phospholipase A2 (PLA2) expression in ovarian endometriotic cysts and normal endometrium with correlation of the expression of E2 and P4 receptors in endometriotic cysts. The analyses were carried out using the tissues derived from 37 patients with ovarian endometriosis and 20 endometrial samples collected from women without endometriosis were used as a control. We found that ovarian endometriotic cysts are a site of LPA synthesis due to the presence of enzymes involved in LPA synthesis in the tissue. Additionally, when we compared endometriotic cysts versus normal endometrium, we were able to show overexpression of 3 from 6 examined LPARs and both enzymes responsible for LPA synthesis in endometriotic cysts. Finally, we found the correlations between LPARs, ATX, and PLA2 and the expression of E2 and P4 receptors in endometriotic cysts. Owing to the high LPAR2 and LPAR4 transcript and protein expression in endometriotic ovarian cysts and positive correlations of both these receptors with the PR-B and ERß, respectively, those receptors seem to be the most promising predictors of the endometriotic cysts as well as the main receptors responsible for LPA action in the ovarian endometriosis.

Expression of genes for enzymes synthesizing lysophosphatidic acid, its receptors and follicle developmental factors derived from the cumulus-oocyte complex is dependent on the ovarian follicle type in cows.

Cumulus-oocyte complexes (COCs) release factors potentially involved in follicular growth and development, such as growth and differentiation factor 9 (GDF9), bone-morphogenetic protein 15 (BMP15), follistatin (FST) and cathepsins (CTSs). Moreover, the quality of the oocytes and follicles may be related to both the lipid composition of the follicle cells and follicular fluid. One of the lipids, locally regulating the reproductive functions in ovaries of cattle, is lysophosphatidic acid (LPA). In this study, the expression was investigated of the genes for LPA and other factors in COCs of follicles at different stages of development and regression. The relative abundances of mRNA were determined by real-time PCR for receptors for LPA (LPARs), enzymes synthesizing LPA (autotaxin (AX) and phospholipase A2 (PLA2)), BMP15, GDF9, CTSZ, CTSB and FST in COCs isolated from healthy, transitional and atretic follicles. The expression of genes for the LPARs, AX, PLA2 and the factors involved in follicular development in cattle COCs is follicle-type dependent. Greater expression of LPAR1-3 and AX genes were detected in the healthy follicles compared to the atretic and transitional follicles (P < 0.05). The relative abundance of GDF9, BMP15, CTSZ and CTSB was also greater in COCs from healthy follicles than from transitional and atretic follicles (P < 0.05). It is postulated that the greater expression of LPARs and AX genes in healthy follicles compared with atretic follicles indicates an enhanced role of LPA in follicular development. Results of the present study also suggest the regulatory role of factors derived from the COCs in the growth and development of follicles.

Bovine ovarian follicular growth and development correlate with lysophosphatidic acid expression.

The basis of successful reproduction is proper ovarian follicular growth and development. In addition to prostaglandins and vascular endothelial growth factor, a number of novel factors are suggested as important regulators of follicular growth and development: PGES, TFG, CD36, RABGAP1, DBI and BTC. This study focuses on examining the expression of these factors in granulosa and thecal cells that originate from different ovarian follicle types and their link with the expression of lysophosphatidic acid (LPA), known local regulator of reproductive functions in the cow. Ovarian follicles were divided into healthy, transitional, and atretic categories. The mRNA expression levels for PGES, TFG, CD36, RABGAP1, DBI and BTC in granulosa and thecal cells in different follicle types were measured by real-time PCR. The correlations among expression of enzymes synthesizing LPA (autotaxin, phospholipase A2), receptors for LPA and examined factors were measured. Immunolocalization of PGES, TFG, CD36, RABGAP1, DBI and BTC was examined by immunohistochemistry. We investigated follicle-type dependent mRNA expression of factors potentially involved in ovarian follicular growth and development, both in granulosa and thecal cells of bovine ovarian follicles. Strong correlations among receptors for LPA, enzymes synthesizing LPA, and the examined factors in healthy and transitional follicles were observed, with its strongest interconnection with TFG, DBI and RABGAP1 in granulosa cells, and TFG in thecal cells; whereas no correlations in atretic follicles were detected. A greater number of correlations were found in thecal cells than in granulosa cells as well as in healthy follicles than in transitional follicles. These data indicate the role of LPA in the growth, development and physiology of the bovine ovarian follicle.

Expression of factors involved in apoptosis and cell survival is correlated with enzymes synthesizing lysophosphatidic acid and its receptors in granulosa cells originating from different types of bovine ovarian follicles.

BACKGROUND: Lysophosphatidic acid (LPA) regulates reproductive processes in the cow. Ovarian granulosa cells play a pivotal role in follicle growth and development. Nevertheless, the role of LPA in the local regulation of granulosa cell function in different follicle categories in the bovine ovary has not been investigated. METHODS: Ovarian follicles were divided into healthy, transitional and atretic categories. The expression levels of AX, PLA2, LPARs and factors involved in apoptosis and cell survival processes in granulosa cells in different types of follicles were measured by real-time PCR. The correlations between the expression levels of AX, PLA2, LPARs and the examined factors were measured. The immunolocalization of AX, PLA2 and LPARs in different ovarian follicles was examined by immunohistochemistry. Statistical analyses were conducted in GraphPad using a one-way ANOVA followed by the Kruskal-Wallis multiple comparison test or a correlation analysis followed by Pearson's test. RESULTS: The expression levels of AX, PLA2 and LPARs, with the major role of LPAR2 and PLA2, were found in the granulosa cells originating from different follicle types. The expression levels of the factors involved in cell apoptosis (TNFα and its receptors, FAS, FASL, CASP3, CASP8, ß-glycan, and DRAK2) were significantly higher in the granulosa cells of the atretic follicles compared to the healthy follicles. A number of correlations between LPARs, AX, PLA2 and factors associated with apoptosis were observed in the atretic but not in the healthy follicles. A greater expression of the factors involved in differentiation and proliferation in the granulosa cells (DICE1 and SOX2) was found in the healthy follicles in comparison with the atretic. A number of correlations between LPARs, AX, PLA2 and the factors associated with cell survival were observed in the healthy but not in the atretic follicles. CONCLUSIONS: Granulosa cells are the target of LPA action and the source of LPA synthesis in the bovine ovarian follicle. We suggest that the participation of LPA in apoptosis in the atretic follicles mainly occurs through the regulation of TNF-α-dependent and caspase-induced pathways. In the transitional follicles, LPA might influence the inhibins to shift the balance between the number of healthy and atretic follicles. In the healthy follicle type, LPA, acting via LPAR1, might regulate MCL1 and estradiol-stimulating ERß mRNA expression, leading to the stimulation of anti-apoptotic processes in the granulosa cells and their differentiation and proliferation.

The effect of lysophosphatidic acid together with interferon tau on the global transcriptomic profile in bovine endometrial cells.

In cows, lysophosphatidic acid (LPA), which acts in an auto/paracrine manner, serves as a luteotropic factor during early pregnancy by stimulating progesterone and prostaglandin E2 secretion, thus protecting the bovine corpus luteum and early embryo development. Our hypothesis was that LPA exerted some local effects on the bovine endometrium prior to early embryo-maternal interactions and that interferon tau (IFNτ), the pregnancy recognition signal, modulated this action. In the present study, we applied an in vitro model involving whole-transcriptomic profiling to examine the effects of LPA on gene expression in bovine endometrial cells. Microarray analyses revealed 36, 269 and 284 differentially expressed transcripts in bovine endometrial cells in the control vs. LPA, control vs. LPA + IFNτ and LPA vs. LPA + IFNτ groups, respectively. The expression of matrix metalloproteinase 13 (MMP13) and radical S-adenosyl methionine domain containing 2 (RSAD2) was increased in the LPA-treated endometrial cells. Among the transcripts differentially regulated by LPA together with IFNτ, many of the genes were classical- or novel-type I IFN-stimulated genes (ISGs). The results indicated that 10 of the 16 analyzed genes showed a positive correlation with their corresponding microarray data upon real-time PCR validation, indicating a considerable consistency between both techniques. In summary, these transcriptional profiling studies identified a number of genes that were regulated by LPA alone and LPA together with IFNτ in endometrial cells from the bovine uterus. Available studies support the idea that LPA, which acts in an auto/paracrine manner on the endometrium, alters the expression of genes that are probably important for uterine receptivity, maternal immune tolerance to the embryo and conceptus growth and development during early pregnancy. Moreover, the differentially expressed genes (DEGs) that increased in the LPA + IFNτ-treated endometrial cells are largely in response to IFNτ actions and are possibly associated with crucial biological processes during the peri-implantation period of pregnancy.

Local effect of lysophosphatidic acid on prostaglandin production in the bovine oviduct.

The mammalian oviduct plays an important role in the fertilisation and transport of gametes and embryo. Prostaglandins (PGs) are local mediators of oviductal functions and are involved in fertilisation and the transport of gametes and embryo. Lysophosphatidic acid (LPA), a kind of phospholipid, is involved in various physiological actions. We hypothesised that LPA regulates PG production in the bovine oviduct. To test this hypothesis, we examined the mRNA expression of LPA receptors (LPAR1-6) and LPA-producing enzymes (ATX, PLA1α, PLA1ß) in ampullary and isthmic tissues and in cultured epithelial and stromal cells isolated from the bovine oviduct. We also investigated the effects of LPA on PG synthase expression and PG production in cultured cells. The mRNA of LPAR1-4, 6, ATX and PLA1α were expressed in cultured epithelial and stromal cells. The expressions of LPAR1-3 were significantly lower and the expression of LPAR4 was significantly higher in the isthmic than in the ampullary tissues. Lysophosphatidic acid significantly stimulated PG production in the cultured isthmic stromal cells. The overall findings suggest that LPA stimulates PG production via LPAR4 in the bovine oviduct. Since PGs are important for fertilisation and the transport of gametes and embryo, these findings show that locally produced LPA regulates oviductal functions.

Lysophosphatidic acid modulates prostaglandin signalling in bovine steroidogenic luteal cells.

We examined whether lysophosphatidic acid affects prostaglandin biosynthesis, transport, and signalling in bovine steroidogenic luteal cells. The aim of the present study was to determine the influence of LPA on PGE2 and PGF2α synthesis and on the expression of enzymes involved in PG biosynthesis (PTGS2, mPGES-1, cPGES, mPGES-2, PGFS and 9-KPR), prostaglandin transporter (PGT), and prostaglandin receptors (EP1, EP2, EP3, EP4 and FP) in bovine steroidogenic luteal cells. We found that LPA inhibited PGF2α synthesis in steroidogenic luteal cells. Moreover, LPA increased mPGES1 and cPGES and decreased PGFS expression in cultured bovine steroidogenic luteal cells. Additionally, LPA stimulated EP2 and EP4 receptor and PGT expression. This study suggests that LPA activity in the bovine CL directs the physiological intraluteal balance between the two main prostanoids towards luteotropic PGE2.

The significance of the altered expression of lysophosphatidic acid receptors, autotaxin and phospholipase A2 as the potential biomarkers in type 1 endometrial cancer biology.

In order to study lysophosphatidic acid (LPA) signaling associated with type 1 endometrial carcinoma (EC), we evaluated the LPA receptors (LPARs), autotaxin (ATX) and phospholipase A2 (PLA2) expression in EC and normal endometrium with correlation to clinicopathological features. We investigated LPAR1, LPAR2, LPAR3, LPAR4, ATX and PLA2 expression at mRNA and protein levels using quantitative real-time PCR and western blot analyses in 37 ECs and 10 normal endometria. All the examined LPARs (except for LPAR3 protein), ATX and PLA2 were overexpressed in cancerous compared to healthy endometrium. The studied ECs showed the highest LPAR2 and LPAR1 expression. Statistically positive correlations were found between depth of myoinvasion and levels of LPAR1, LPAR2 and PLA2 transcripts and proteins. We also found positive correlations between LPAR1, LPAR2, LPAR4 and PLA2 expression with the International Federation of Gynecology and Obstetrics (FIGO) stage. The expression of LPAR1, LPAR2 and PLA2 was positively associated with the age of patients. Positive correlations were found between the expression of LPAR1 mRNA, LPAR2 mRNA and protein and LPAR3 mRNA and body mass index (BMI) of the examined patients. We found no association between the expression levels of the studied factors and diabetes or hypertension among the examined patients. Owing to the highest LPAR2 and LPAR1 expression in EC and positive correlations of these two receptors with the depth of myoinvasion and the FIGO stage, we believe that LPAR2 and LPAR1 show promise as predictors of the EC progression as well as the main receptors responsible for LPA action in the EC tissue.

The effect of lysophosphatidic acid during in vitro maturation of bovine cumulus-oocyte complexes: cumulus expansion, glucose metabolism and expression of genes involved in the ovulatory cascade, oocyte and blastocyst competence.

BACKGROUND: In the cow, lysophosphatidic acid (LPA) acts as an auto-/paracrine factor, through its receptors LPAR1-4, on oocytes and cumulus cells during in vitro maturation (IVM). The aim of the present work was to determine the effect of LPA during IVM of bovine oocytes on: 1) oocyte maturation; 2) apoptosis of COCs; 3) expression of genes involved in developmental competence and apoptosis in bovine oocytes and subsequent blastocysts; 4) cumulus expansion and expression of genes involved in the ovulatory cascade in cumulus cells; 5) glucose metabolism and expression of genes involved in glucose utilization in cumulus cells; 6) cleavage and blastocyst rates on Day 2 and Day 7 of in vitro culture, respectively. METHODS: Cumulus-oocyte complexes (COCs) were matured in vitro in the presence or absence of LPA (10(-5) M) for 24 h. Following maturation, we determined: oocyte maturation stage, cumulus expansion, COCs apoptosis and glucose and lactate levels in the maturation medium. Moreover, COCs were either used for gene expression analysis or fertilized in vitro. The embryos were cultured until Day 7 to assess cleavage and blastocyst rates. Oocytes, cumulus cells and blastocysts were used for gene expression analysis. RESULTS: Supplementation of the maturation medium with LPA enhanced oocyte maturation rates and stimulated the expression of developmental competence-related factors (OCT4, SOX2, IGF2R) in oocytes and subsequent blastocysts. Moreover, LPA reduced the occurrence of apoptosis in COCs and promoted an antiapoptotic balance in the transcription of genes involved in apoptosis (BAX and BCL2) either in oocytes or blastocysts. LPA increased glucose uptake by COCs via augmentation of GLUT1 expression in cumulus cells as well as stimulating lactate production via the enhancement of PFKP expression in cumulus cells. LPA did not affect cumulus expansion as visually assessed, however, it stimulated upstream genes of cumulus expansion cascade, AREG and EREG. CONCLUSIONS: Supplementation of the maturation medium with LPA improves oocyte maturation rates, decreases extent of apoptosis in COCs and sustains the expression of developmental competence related factors during oocyte maturation and subsequently affects gene expression profile at the blastocyst stage. We also demonstrate that LPA directs glucose metabolism toward the glycolytic pathway during IVM.

Lysophosphatidic acid signaling in late cleavage and blastocyst stage bovine embryos.

Lysophosphatidic acid (LPA) is a known cell signaling lipid mediator in reproductive tissues. In the cow, LPA is involved in luteal and early pregnancy maintenance. Here, we evaluated the presence and role of LPA in bovine early embryonic development. In relevant aspects, bovine embryos reflect more closely the scenario occurring in human embryos than the mouse model. Transcription of mRNA and protein expression of enzymes involved in LPA synthesis (ATX and cPLA2) and of LPA receptors (LPAR1-4) were detected in Days 5 and 8 in vitro produced embryos. Embryonic LPA production into culture medium was also detected at both stages of development. Supplementation of culture medium with LPA (10(-5) M) between Days 2 and 8 had no effect on embryo yield and quality and on blastocyst relative mRNA abundance of genes involved in prostaglandin synthesis (PTGS2, PGES, and PGFS) and steroidogenesis (3ßHSD). However, LPA treatment affected transcription levels of embryo quality markers, decreasing BAX (apoptotic) and increasing BCL2 (antiapoptotic) and IGF2R (growth marker) gene transcription levels. Blastocyst transcription of OCT4 (pluripotency marker) was not affected by LPA stimulation. In conclusion, LPA is an early bovine embryonic autocrine/paracrine signaling mediator, and LPA action may be relevant in early embryo-maternal interactions leading to embryonic survival.