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.

Follicular Fluid Proteomic Analysis to Identify Predictive Markers of Normal Embryonic Development.

Ageing populations, mass "baby-free" policies and children born to mothers at the age at which they are biologically expected to become grandmothers are growing problems in most developed societies. Therefore, any opportunity to improve the quality of infertility treatments seems important for the survival of societies. The possibility of indirectly studying the quality of developing oocytes by examining their follicular fluids (hFFs) offers new opportunities for progress in our understanding the processes of final oocyte maturation and, consequently, for predicting the quality of the resulting embryos and personalising their culture. Using mass spectrometry, we studied follicular fluids collected individually during in vitro fertilisation and compared their composition with the quality of the resulting embryos. We analysed 110 follicular fluids from 50 oocyte donors, from which we obtained 44 high-quality, 39 medium-quality, and 27 low-quality embryos. We identified 2182 proteins by Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) using a TripleTOF 5600+ hybrid mass spectrometer, of which 484 were suitable for quantification. We were able to identify several proteins whose concentrations varied between the follicular fluids of different oocytes from the same patient and between patients. Among them, the most important appear to be immunoglobulin heavy constant alpha 1 (IgA1hc) and dickkopf-related protein 3. The first one is found at higher concentrations in hFFs from which oocytes develop into poor-quality embryos, the other one exhibits the opposite pattern. None of these have, so far, had any specific links to fertility disorders. In light of these findings, these proteins should be considered a primary target for research aimed at developing a diagnostic tool for oocyte quality control and pre-fertilisation screening. This is particularly important in cases where the fertilisation of each egg is not an option for ethical or other reasons, or in countries where it is prohibited by law.

Comment on 'Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection' by Fumiaki Itoi et al.

We present a commentary on the article published in the Zygote FirstView: 'Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection' by Fumiaki Itoi and colleagues. We comment on the importance of providing the microscope setup details whenever sperm morphology visualization is discussed. The claim of ×6000-10,000 magnification is misleading as such levels of magnification are impossible to achieve.

Inflammation-Related Molecules at the Maternal-Fetal Interface during Pregnancy and in Pathologically Altered Endometrium.

The blastocyst expresses paternally derived alloantigens and induces inflammation during implantation. However, it is necessary for the onset of pregnancy. An abnormal response might result in a pathological course of pregnancy or pregnancy failure. On the other hand, a state of maternal immune tolerance is necessary to ensure the normal development of pregnancy by suppressing inflammatory processes. This article discusses recognized mechanisms and the significance of inflammatory processes for embryo implantation and pregnancy establishment. We would also like to present disorders involving excessive inflammatory response and their influence on events occurring during embryo implantation. The chain of correlation between the processes responsible for embryo implantation and the subsequent physiological course of pregnancy is complicated. Many of those interrelationships are still yet to be discovered. Undoubtedly, their recognition will give hope to infertile couples for the emergence of new treatments that will increase the chance of giving birth to a healthy child.

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.

Altered expression of lysophosphatidic acid receptors, in association with the synthesis of estrogens and androgens in type 1 endometrial cancer biology.

To establish association between two main lysophosphatidic acid (LPA) receptors (LPAR2 and LPAR1) with the synthesis of estrogens and androgens in type-1 endometrial carcinoma (EC), we evaluated correlation of LPARs expression with expression of steroid 5 alpha-reductase 2 - aromatase (SRD5A2), or cytochrome P450 family 19 subfamily A member 1-5α-reductase (CYP19A1) in EC. Moreover, we aimed to investigate SRD5A2 and CYP19A1 expression in type 1 endometrial cancer and normal endometrium with its correlation to selected clinicopathological features. The studied cancerous samples showed higher CYP19A1 and SRD5A2 expression comparing to normal endometria. We also documented positive correlations between LPAR1 and LPAR2 with responsible for proliferation SRD5A2 in EC tissue which suggests that intratumoral estrogen metabolism and synthesis are pivotal in endometrial carcinoma progression, with the involvement of LPA in this process. However, positive correlation between CYP19A1 and LPAR1 accounts for supporting role of LPA acting via LPAR1 in intratumoral DHT concentration and the ethiology of endometrial cancer progression. Also, owing to the highest expression of LPARs, CYP19A1 and SRD5A2 as well as their association with depth of myoinvasion and FIGO stage LPAR2 and LPAR1 seem to be the efficient candidate prognostic markers in the individual, targeted therapies for EC.

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.

Endothelin as a local regulating factor in the bovine oviduct.

Endothelin (EDN) is a possible regulating factor of oviductal motility, which is important for the transport of gametes and embryo. To clarify the factors that control the secretion of EDN in the bovine oviduct, the expression of EDNs, EDN-converting enzymes (ECEs) and EDN receptors (EDNRs) were investigated. All isoforms of EDN (EDN1-3), ECE (ECE1 and ECE2) and EDNR (EDNRA and EDNRB) were immunolocalised in the epithelial cells of the ampulla and the isthmus. EDNRs were also immunolocalised in smooth-muscle cells. The mRNA expression of EDN2 and ECE2 was higher in cultured ampullary oviductal epithelial cells than in isthmic cells. The expression of EDN1, EDN2 and ECE2 in the ampullary tissue was highest on the day of ovulation. Oestradiol-17ß increased EDN2 and ECE1 expression, while progesterone increased only ECE1 expression in cultured ampullary epithelial cells. These results indicate that EDNs are produced by epithelial cells and their target site is smooth-muscle and epithelial cells, and suggest that ovarian steroids are regulators of endothelin synthesis in ampullary oviductal epithelial cells.

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.

[First in the world application of next generation sequencing in preimplantation genetic diagnostics in clinical practice - a case report]. / Pierwsze w swiecie zastosowanie sekwencjonowania nastepnej generacji w diagnostyce przedimplantacyjnej w praktyce klinicznej - opis przypadku.

Preimplantation genetic diagnosis (PGD) is a well established method for detecting genetic abnormalities during the course of infertility treatment, resulting in thousands of healthy newborns delivered worldwide. PGD with next generation sequencing (NGS) provides new possibilities for diagnosis and new parameters for evaluation. The use of next-generation DNA sequencing technique has lead to great progress in the human genome analysis. The aim of this study was molecular analysis using next generation sequencing technique of embryos from a couple suffering from recurrent pregnancy losses. As a result of in vitro fertilization procedure, seven embryos were created. Seven blastomeres, one from each embryo, were analyzed. Transfer of two blastocysts in a fresh cycle resulted in the singleton pregnancy. Healthy baby girl was delivered via caesarean section after 28 weeks of gestation (weight: 1250g, Apgar score: 8/9). The reason for the premature labor was likely caused by mother's pneumonia. This is the first case of clinical use of the NGS in PGD in fresh cycle after blastomere biopsy.

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.

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.

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.