Deregulation of oxidative phosphorylation pathways in embryos derived in vitro from prepubertal and pubertal heifers based on whole-transcriptome sequencing.

BACKGROUND: Although, oocytes from prepubertal donors are known to be less developmentally competent than those from adult donors it does not restrain their ability to produce full-term pregnancies. The transcriptomic profile of embryos could be used as a predictor for embryo's individual developmental competence. The aim of the study was to compare transcriptomic profile of blastocysts derived from prepubertal and pubertal heifers oocytes. Bovine cumulus-oocyte complexes (COCs) were obtained by ovum pick- up method from prepubertal and pubertal heifers. After in vitro maturation COCs were fertilized and cultured to the blastocyst stage. Total RNA was isolated from both groups of blastocysts and RNA-seq was performed. Gene ontology analysis was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery). RESULTS: A higher average blastocyst rate was obtained in the pubertal than in the pre-pubertal group. There were no differences in the quality of blastocysts between the examined groups. We identified 436 differentially expressed genes (DEGs) between blastocysts derived from researched groups, of which 247 DEGs were downregulated in blastocysts derived from pubertal compared to prepubertal heifers oocytes, and 189 DEGs were upregulated. The genes involved in mitochondrial function, including oxidative phosphorylation (OXPHOS) were found to be different in studied groups using Kyoto Encyclopedia of Genes (KEGG) pathway analysis and 8 of those DEGs were upregulated and 1 was downregulated in blastocysts derived from pubertal compared to prepubertal heifers oocytes. DEGs associated with mitochondrial function were found: ATP synthases (ATP5MF-ATP synthase membrane subunit f, ATP5PD- ATP synthase peripheral stalk subunit d, ATP12A- ATPase H+/K + transporting non-gastric alpha2 subunit), NADH dehydrogenases (NDUFS3- NADH: ubiquinone oxidoreductase subunit core subunit S3, NDUFA13- NADH: ubiquinone oxidoreductase subunit A13, NDUFA3- NADH: ubiquinone oxidoreductase subunit A3), cytochrome c oxidase (COX17), cytochrome c somatic (CYCS) and ubiquinol cytochrome c reductase core protein 1 (UQCRC1). We found lower number of apoptotic cells in blastocysts derived from oocytes collected from prepubertal than those obtained from pubertal donors. CONCLUSIONS: Despite decreased expression of genes associated with OXPHOS pathway in blastocysts from prepubertal heifers oocytes, the increased level of ATP12A together with the lower number of apoptotic cells in these blastocysts might support their survival after transfer.

Expression profile of proinflammatory mediators in the placenta of mares during physiological detachment and retention of fetal membranes.

Physiological parturition is characterized by sterile, inflammatory-like processes. During parturition, the placenta expresses various proinflammatory mediators, such as chemokines and IL-17. Nevertheless, inflammatory processes present in the parturient mare are poorly characterized. The aim of this study was to investigate the expression of selected chemokines and IL-17 in the allantochorion and the endometrium of mares that retained fetal membranes (RFM) and expelled them physiologically. We hypothesized that the expression of these mediators may be altered in the placenta of mares with RFM and result in RFM occurrence. Differences in mRNA expression in the placenta of investigated groups of mares were detected for CCL2, CCL3, CCL4, CCL8, CXCL1, CXCL8, CXCL10, CX3CL1 and IL-17. There were no differences in mRNA expression of CCL5 and CXCL6. Gene ontology network analysis showed enrichment in genes related to leukocyte migration, cell chemotaxis and response to chemokine in tissues of RFM mares. Analysis of association network suggested denotations between CXCL6, CXCL8, CXCL1, CCL5, CCL4, CX3CL1 and CXCL10. Moreover, possible inhibition of CXCL10 by IL-17A and prostaglandin peroxide synthase 2 (PTGS2) by CXCL1 was detected. Our results suggest that, based on differences in chemokines and IL-17 expression, recruited subsets of leukocytes might differ between the analyzed groups of mares, which in turn may impair the separation of fetal membranes in the group of RFM mares. In addition, the results of the expression analysis suggest that macrophages might be one of the most abundant cells infiltrating the equine placenta during the expulsion of fetal membranes. Furthermore, we suspect that the synthesis of PTGS2 might be inhibited in mares with RFM.

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.

Prostaglandin E2 affects in vitro maturation of bovine oocytes.

The role of prostaglandin E2 (PGE2) in the successful resumption of oocyte meiosis and cumulus expansion has been well-documented. However, there remains very little information available on the influence of PGE2 on other processes that occur during oocyte maturation. In this study, we supplemented a maturation medium with PGE2 and monitored oocyte quality markers, glucose metabolism, mitochondrial status, oxidative stress, and apoptosis in the cumulus-oocyte complexes (COCs), using a well-established in vitro model of embryo production in cattle. We found that this increased availability of PGE2 during maturation led to an increase in the expression of genes associated with oocyte competence and improved the quality of blastocysts produced. Prostaglandin E2 also appeared to stimulate glucose uptake and lactate production in the COCs, both influencing the expression of enzymes involved in glycolysis and the hexosamine biosynthetic pathway. We found that PGE2 reduced intracellular reactive oxygen species levels, and simultaneously increased glutathione concentration and stimulated antioxidant gene expression in the oocyte. These results indicate that PGE2 has an important role in the protection of oocytes against oxidative stress. Mitochondrial membrane potential was also improved in PGE2-treated oocytes, and there was a reduction in the occurrence of apoptosis in the COCs. Promotion of an anti-apoptotic balance in transcription of genes involved in apoptosis was present in both oocytes and the cumulus cells. In summary, PGE2 could represent a novel autocrine/paracrine player in the mechanisms that can facilitate successful oocyte maturation and oocyte survival in the cow.

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 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 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 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.

Influence of lysophosphatidic acid on nitric oxide-induced luteolysis in steroidogenic luteal cells in cows.

Lysophosphatidic acid (LPA) together with its active G protein-coupled receptors are present in the corpus luteum (CL) of the cow. Under in vivo conditions, LPA stimulated P4 and PGE2 secretion during the luteal phase of the estrous cycle in heifers. Furthermore, LPA maintained P4 synthesis and actions in the bovine CL in vitro. However, the effect of this phospholipid on nitric oxide (NO)-induced functional and structural luteolysis has not been investigated. The aim of the present work was to determine the effects of LPA on 1) NO-induced functional luteolysis, 2) NO-dependent PG synthesis, and 3) NO-induced structural luteolysis in cultured steroidogenic luteal cells. We documented that LPA reversed the inhibitory effect of NONOate, an NO donor, on P4 synthesis and PGE2/PGF2alpha ratio in cultured steroidogenic luteal cells. Additionally, LPA inhibited NO-induced apoptosis in cultured steroidogenic luteal cells via abrogation of the NO-dependent stimulatory influence on proapoptotic TNFalpha/TNFR1 and Fas/FasL expression, Caspase 3 activity, and the Bax/Bcl2 ratio during luteal regression in the bovine CL. In conclusion, this study proves that in the presence of LPA, NO cannot induce luteolytic capacity acquisition, leading to functional and structural luteolysis of bovine luteal cells.

Lysophosphatidic acid (LPA) signaling in human and ruminant reproductive tract.

Lysophosphatidic acid (LPA) through activating its G protein-coupled receptors (LPAR 1-6) exerts diverse cellular effects that in turn influence several physiological processes including reproductive function of the female. Studies in various species of animals and also in humans have identified important roles for the receptor-mediated LPA signaling in multiple aspects of human and animal reproductive tract function. These aspects range from ovarian and uterine function, estrous cycle regulation, early embryo development, embryo implantation, decidualization to pregnancy maintenance and parturition. LPA signaling can also have pathological consequences, influencing aspects of endometriosis and reproductive tissue associated tumors. The review describes recent progress in LPA signaling research relevant to human and ruminant reproduction, pointing at the cow as a relevant model to study LPA influence on the human reproductive performance.

The effect of lysophosphatidic acid during in vitro maturation of bovine oocytes: embryonic development and mRNA abundances of genes involved in apoptosis and oocyte competence.

In the present study we examined whether LPA can be synthesized and act during in vitro maturation of bovine cumulus oocyte complexes (COCs). We found transcription of genes coding for enzymes of LPA synthesis pathway (ATX and PLA2) and of LPA receptors (LPAR 1-4) in bovine oocytes and cumulus cells, following in vitro maturation. COCs were matured in vitro in presence or absence of LPA (10(-5) M) for 24 h. Supplementation of maturation medium with LPA increased mRNA abundance of FST and GDF9 in oocytes and decreased mRNA abundance of CTSs in cumulus cells. Additionally, oocytes stimulated with LPA had higher transcription levels of BCL2 and lower transcription levels of BAX resulting in the significantly lower BAX/BCL2 ratio. Blastocyst rates on day 7 were similar in the control and the LPA-stimulated COCs. Our study demonstrates for the first time that bovine COCs are a potential source and target of LPA action. We postulate that LPA exerts an autocrine and/or paracrine signaling, through several LPARs, between the oocyte and cumulus cells. LPA supplementation of maturation medium improves COC quality, and although this was not translated into an enhanced in vitro development until the blastocyst stage, improved oocyte competence may be relevant for subsequent in vivo survival.

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.

Lysophosphatidic acid action in the bovine corpus luteum -an in vitro study.

We examined whether the CL is a site for lysophosphatidic acid (LPA) synthesis and/or a target for LPA action in the bovine reproductive tract. LPA concentrations in the CL tissue increased towards the end of the cycle and were stable during early pregnancy. No changes in the expression of LPA receptors (LPARs) occurred during the estrous cycle. The expressions of LPAR2 and LPAR4 on days 17-19 of pregnancy were higher than those on the respective days of the estrous cycle and higher than those on days 8-10 of pregnancy. LPA stimulated P4 synthesis via 3ßHSD stimulation but did not modulate the interferon-tau (IFNτ) influence on P4 synthesis in steroidogenic cells. Moreover, we found LPA-dependent stimulation of IFNτ action on 2,5'-oligoadenylate synthase (OAS1) and ubiquitin-like IFN-stimulated gene 15-kDa protein (ISG15) expression. The present study demonstrated that the CL might be a site of LPA synthesis and target of LPA action in the bovine reproductive tract. We postulate that during the estrous cycle and early pregnancy, LPA exerts autocrine and paracrine effects on the CL mainly via LPAR2 and LPAR4. The stimulatory effect of LPA on P4 synthesis via 3ßHSD stimulation and LPA-dependent stimulation of IFNτ action on OAS1 and ISG15 expression suggest that LPA is an additional auxiliary luteosupportive factor in steroidogenic cells.

Mitochondrial DNA content and developmental competence of blastocysts derived from pre-pubertal heifer oocytes.

In the cattle-breeding industry, there is an increasing demand for in vitro embryo production from pre-pubertal heifers. In this study, we evaluated the differences in mitochondrial DNA content, oxidative stress, and developmental competence in blastocysts derived from pre-pubertal and pubertal heifers. We found higher mitochondrial DNA copy numbers in blastocysts produced from pre-pubertal heifers than from pubertal heifers. In the group of pre-pubertal animals, there was a significantly lower number of blastocysts produced in vitro from the same number of collected oocytes, and these blastocysts did not differ from those obtained from pubertal oocytes in terms of their morphological quality. The morphologically appropriate blastocysts derived from pre-pubertal heifers had higher concentrations of reactive oxygen species and glutathione. In blastocysts derived from pre-pubertal heifers, we found alterations in the expression of gene markers for developmental competence, which correlated with higher mitochondrial DNA content, suggesting a lower quality of blastocysts derived from pre-pubertal animals than from pubertal animals. The inadequate redox balance in blastocysts obtained from pre-pubertal females, along with higher mitochondrial DNA copy number, as well as differential gene expression of markers of developmental competence, elucidate the low quality of blastocysts derived from pre-pubertal animals, despite their unaltered morphology.

Hypothyroidism Affects Uterine Function via the Modulation of Prostaglandin Signaling.

Thyroid hormones control the functions of almost all body systems. Reproductive dysfunctions, such as abnormal sexual development, infertility, or irregularities in the reproductive cycle, might be associated with thyroid disorders. Uterine receptivity is the period when the uterus is receptive to the implantation of an embryo. During the receptivity period (implantation window), a newly formed blastocyst is incorporated into the uterine epithelium. Prostaglandins are well-known primary mediators of pathological conditions such as inflammation and cancer but are also essential for the physiology of female reproduction. The aim of this study was to evaluate the possible relationship between hypothyroidism and changes in the prostaglandin signaling pathways in the uterus and in the process of uterine receptivity in a rat model. The results show that hypothyroidism impaired uterine receptivity by decreasing the level of E2 as well as decreasing the expression of the uterine-receptivity factors homeobox A10 and osteopontin. Moreover, hypothyroidism caused changes in the expression of elements of the prostaglandin E2, F2α, and I2 signaling pathways and changed the levels of those prostaglandins in the uterine tissue. The results suggest that the mechanisms by which hypothyroidism affects female reproductive abnormalities might involve the prostaglandin signaling pathway, resulting in a subsequent reduction in uterine receptivity.

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.

Iloprost affects in vitro maturation and developmental competence of bovine oocytes.

Prostacyclin (PGI2) is synthesised in oviductal fluid and enhance the embryo development during the preimplantation period. The objective of the present study was to determine the effect of an analogue of prostacyclin (iloprost) on the in vitro maturation (IVM) and the developmental competence of bovine oocytes. Cumulus oocyte complexes (COCs) were cultured in maturation medium with iloprost (0.5 µM) for 24 h. We found that iloprost assisted maturation rates and cumulus cell expansion of bovine oocytes, and it increased the mRNA expression of genes related to cumulus expansion: ADAM17, AREG, and TNFAIP6 and cathepsin genes (CTSK and CTSS). Moreover, iloprost reduced the occurrence of apoptosis in COCs and promoted an antiapoptotic balance in the transcription of genes involved in apoptosis (BAX and BCL2). COCs treatment with iloprost during IVM also reduced intracellular reactive oxygen species (ROS) levels, while glutathione (GSH) levels and the mRNA expression of antioxidant genes CAT and GPx4 were markedly increased. We also showed that an analogue of PGI2 influenced the mitochondrial status via distribution rates of mitochondria and mitochondrial membrane potential in oocytes. Although, iloprost-enhanced maturation had no direct effect on number of embryos cleaved, it increased blastocyst rates of bovine embryos as well as proportion of expanded blastocysts. These results indicate that the supplementation of maturation medium with iloprost is beneficial for the maturation efficiency and developmental competence of bovine oocytes.

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 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.