[Endometrial receptivity in assisted reproductive techniques: An aspect to investigate in embryo implantation failure]. / La réceptivité endométriale en aide médicale à la procréation : Une piste à ne pas oublier devant un échec d'implantation.

Infertility affects between 8 and 12% of reproductive-age couples worldwide. Despite improvements in assisted reproductive techniques (ART), live birth rates are still limited. In clinical practice, imaging and microscopy are currently widely used, but their diagnostic effectiveness remains limited. In research, the emergence of innovative techniques named OMICS would improve the identification of the implantation window, while progressing in the understanding of the pathophysiological mechanisms involved in embryo implantation failures. To date, transcriptomic analysis seems to be the most promising approach in clinical research. The objective of this review is to present the results obtained with the different approaches available in clinical practice and in research to assess endometrial receptivity in patients undergoing ART.

Global, Survival, and Apoptotic Transcriptome during Mouse and Human Early Embryonic Development.

Survival and cell death signals are crucial for mammalian embryo preimplantation development. However, the knowledge on the molecular mechanisms underlying their regulation is still limited. Mouse studies are widely used to understand preimplantation embryo development, but extrapolation of these results to humans is questionable. Therefore, we wanted to analyse the global expression profiles during early mouse and human development with a special focus on genes involved in the regulation of the apoptotic and survival pathways. We used DNA microarray technology to analyse the global gene expression profiles of preimplantation human and mouse embryos (metaphase II oocytes, embryos at the embryonic genome activation stage, and blastocysts). Components of the major apoptotic and survival signalling pathways were expressed during early human and mouse embryonic development; however, most expression profiles were species-specific. Particularly, the expression of genes encoding components and regulators of the apoptotic machinery were extremely stable in mouse embryos at all analysed stages, while it was more stage-specific in human embryos. CASP3, CASP9, and AIF were the only apoptosis-related genes expressed in both species and at all studied stages. Moreover, numerous transcripts related to the apoptotic and survival pathway were reported for the first time such as CASP6 and IL1RAPL1 that were specific to MII oocytes; CASP2, ENDOG, and GFER to blastocysts in human. These findings open new perspectives for the characterization and understanding of the survival and apoptotic signalling pathways that control early human and mouse embryonic development.

Cell-free nucleic acids as non-invasive biomarkers of gynecological cancers, ovarian, endometrial and obstetric disorders and fetal aneuploidy.

BACKGROUND: Proper folliculogenesis is fundamental to obtain a competent oocyte that, once fertilized, can support the acquisition of embryo developmental competence and pregnancy. MicroRNAs (miRNAs) are crucial regulators of folliculogenesis, which are expressed in the cumulus-oocyte complex and in granulosa cells and some can also be found in the bloodstream. These circulating miRNAs are intensively studied and used as diagnostic/prognostic markers of many diseases, including gynecological and pregnancy disorders. In addition, serum contains small amounts of cell-free DNA (cfDNA), presumably resulting from the release of genetic material from apoptotic/necrotic cells. The quantification of nucleic acids in serum samples could be used as a diagnostic tool for female infertility. METHODS: An overview of the published literature on miRNAs, and particularly on the use of circulating miRNAs and cfDNA as non-invasive biomarkers of gynecological diseases, was performed (up to January 2014). RESULTS: In the past decade, cell-free nucleic acids have been studied for potential use as biomarkers in many diseases, particularly in gynecological cancers, ovarian and endometrial disorders, as well as in pregnancy-related pathologies and fetal aneuploidy. The data strongly suggest that the concentration of cell-free nucleic acids in serum from IVF patients or in embryo culture medium could be related to the ovarian hormone status and embryo quality, respectively, and be used as a non-invasive biomarker of IVF outcome. CONCLUSIONS: The profiling of circulating nucleic acids, such as miRNAs and cfDNA, opens new perspectives for the diagnosis/prognosis of ovarian disorders and for the prediction of IVF outcomes, namely (embryo quality and pregnancy).

Developmental regulated expression of anti- and pro-apoptotic BCL-2 family genes during human early embryonic development.

Apoptotic cell death has been reported in human oocytes and preimplantation embryos under in vivo and in vitro conditions. BCL-2 family proteins comprise both anti- and pro-apoptotic members, which are likely to play a key role in controlling oocyte and early embryo survival. However, very limited data are available on their expression kinetics during human early embryonic development. Using our DNA microarray data, we analyzed the expression pattern of 21 BCL-2 family genes in human mature MII oocytes, day 3 embryos and day 5/6 blastocysts from patients who underwent in vitro fertilization (IVF). Selected genes were further validated by qRT-PCR and their subcellular localization analyzed by immunofluorescence confocal microscopy. Our results suggest a switch from oocyte-inherited BCL-2 family transcripts, such as BCL2L10, to embryo-produced transcripts after embryonic genome activation, including BIK, BCL2L11 and NOXA. Moreover, the pro-apoptotic gene BCL2L13 was constitutively expressed throughout human early embryonic development. Remarkably, day 3 embryos expressed more BCL-2 pro-apoptotic genes than mature MII oocytes and day 5/6 blastocysts, suggesting that embryos at this stage are more prone to apoptosis. This is further supported by an absence of cleaved Caspase-3 in the oocyte and its presence in the embryo. Using a drug that induces apoptosis (gambogic acid), we were able to show activated Caspase-3 in the oocyte in addition to an alteration of BCL2L13 protein localization. Similarly BCL2L13 localization was altered in degenerated oocytes. This study opens new perspectives for understanding the molecular regulation of human oocyte and pre-implantation embryo survival and death.

MicroRNAs: new candidates for the regulation of the human cumulus-oocyte complex.

STUDY QUESTION: What is the expression pattern of microRNAs (miRNAs) in human cumulus-oocyte complexes (COCs)? SUMMARY ANSWER: Several miRNAs are enriched in cumulus cells (CCs) or oocytes, and are predicted to target genes involved in biological functions of the COC. WHAT IS KNOWN ALREADY: The transcriptional profiles of human MII oocytes and the surrounding CCs are known. However, very limited data are available about post-transcriptional regulators, such as miRNAs. This is the first study focussing on the identification and quantification of small RNAs, including miRNAs, in human oocytes and CCs using a deep-sequencing approach. STUDY DESIGN, SIZE, DURATION: MII oocytes and CCs were collected from women who underwent IVF. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using the Illumina/deep-sequencing technology, we analyzed the small RNAome of pooled MII oocytes (n = 24) and CC samples (n = 20). The mRNA targets of CC and MII oocyte miRNAs were identified using in silico prediction algorithms. Using oligonucleotide microarrays, genome-wide gene expression was studied in oocytes (10 pools of 19 ± 3 oocytes/each) and 10 individual CC samples. TaqMan miRNA assays were used to confirm the sequencing results in independent pools of MII oocytes (3 pools of 8 ± 3 oocytes/each) and CC samples (3 pools of 7 ± 3 CCs/each). The functional role of one miRNA, MIR23a, was assessed in primary cultures of human CCs. MAIN RESULTS AND THE ROLE OF CHANCE: Deep sequencing of small RNAs yielded more than 1 million raw reads. By mapping reads with a single location to the human genome, known miRNAs that were abundant in MII oocytes (MIR184, MIR100 and MIR10A) or CCs (MIR29a, MIR30d, MIR21, MIR93, MIR320a, MIR125a and the LET7 family) were identified. Predicted target genes of the oocyte miRNAs were associated with the regulation of transcription and cell cycle, whereas genes targeted by CC miRNAs were involved in extracellular matrix and apoptosis. Comparison of the predicted miRNA target genes and mRNA microarray data resulted in a list of 224 target genes that were differentially expressed in MII oocytes and CCs, including PTGS2, CTGF and BMPR1B that are important for cumulus-oocyte communication. Functional analysis using primary CC cultures revealed that BCL2 and CYP19A1 mRNA levels were decreased upon MIR23a overexpression. LIMITATIONS, REASONS FOR CAUTION: Only known miRNAs were investigated in the present study on COCs. Moreover, the source of the material is MII oocytes that failed to fertilize. WIDER IMPLICATIONS OF THE FINDINGS: The present findings suggest that miRNA could play a role in the regulation of the oocyte and CC crosstalk. STUDY FUNDING/COMPETING INTEREST(S): This work was partially supported by a grant from Ferring Pharmaceuticals. The authors of the study have no conflict of interest to report. TRIAL REGISTRATION NUMBER: Not applicable.

Altered gene expression profile in cumulus cells of mature MII oocytes from patients with polycystic ovary syndrome.

STUDY QUESTION: Oocyte developmental competence is altered in patients with polycystic ovary syndrome (PCOS); is gene expression in cumulus cells (CCs) from mature metaphase II oocytes of patients with PCOS altered as well? SUMMARY ANSWER: Compared with CCs from non-PCOS patients, the gene expression profile of CCs isolated from mature oocytes of patients with PCOS present alterations that could explain the abnormal folliculogenesis and reduced oocyte competence in such patients. WHAT IS KNOWN ALREADY: Abnormal mRNA expression of several members of the insulin-like growth factor (IGF) family in CCs from PCOS patients was previously reported. Moreover, the whole transcriptome has been investigated in cultured CCs from PCOS patients. STUDY DESIGN, SIZE AND DURATION: This retrospective study included six PCOS patients diagnosed following the Rotterdam Criteria and six non-PCOS patients who all underwent ICSI for male infertility in the assisted reproduction technique (ART) Department of Montpellier University Hospital, between 2009 and 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS: CCs from PCOS and non-PCOS patients who underwent controlled ovarian stimulation (COS) were isolated mechanically before ICSI. Gene expression profiles were analysed using the microarray technology and the Significance Analysis of Microarray was applied to compare the expression profiles of CCs from PCOS and non-PCOS patients. MAIN RESULTS: The gene expression profile of CCs from patients with PCOS was significantly different from that of CCs from non-PCOS patients. Specifically, CCs from women with PCOS were characterized by abnormal expression of many growth factors, including members of the epidermal growth factor-like (EGFR, EREG and AREG) and IGF-like families (IGF1R, IGF2R, IGF2BP2 and IGFBP2), that are known to play a role in oocyte competence. In addition, mRNA transcripts of factors involved in steroid metabolism, such as CYP11A1, CYP1B1, CYP19A1 and CYP2B7P1, were deregulated in PCOS CCs, and this could explain the abnormal steroidogenesis observed in these women. Functional annotation of the differentially expressed genes suggests that defects in the transforming growth factor ß and estrogen receptors signalling cascades may contribute to the reduced oocyte developmental competence in patients with PCOS. LIMITATIONS AND REASONS FOR CAUTION: Owing to the strict selection criteria (similar age, weight and reasons for ART), this study included a small sample size (six cases and six controls), and thus, further investigations using a large cohort of patients are needed to confirm these results. WIDER IMPLICATIONS OF THE FINDINGS: This study opens a new perspective for understanding the pathogenesis of PCOS. STUDY FUNDING/COMPETING INTERESTS: This work was partially supported by a grant from the Ferring Pharmaceutical. The authors of the study have no competing interests to report. TRIAL REGISTRATION NUMBER: Not applicable.

Slow freezing and vitrification differentially modify the gene expression profile of human metaphase II oocytes.

BACKGROUND: Cryopreservation is now considered as an efficient way to store human oocytes to preserve fertility. However, little is known about the effects of this technology on oocyte gene expression. The aim of this study was to examine the effect of the two cryopreservation procedures, slow freezing and vitrification, on the gene expression profile of human metaphase II (MII) oocytes. METHODS: Unfertilized MII oocytes following ICSI failure were cryopreserved either by slow freezing or by the Cryotip method for vitrification. After thawing, total RNA was extracted and analyzed using Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays. The gene expression profiles and associated biological pathways in slowly frozen/thawed and vitrified MII oocytes were determined and compared with those of non-cryopreserved MII oocytes used as controls. RESULTS: Both cryopreservation procedures negatively affected the gene expression profile of human MII oocytes in comparison with controls. However, slowly frozen and vitrified MI oocytes displayed specific gene expression signatures. Slow freezing was associated with down-regulation of genes involved in chromosomal structure maintenance (KIF2C and KIF3A) and cell cycle regulation (CHEK2 and CDKN1B) that may lead to a reduction in the oocyte developmental competence. In vitrified oocytes, many genes of the ubiquitination pathway were down-regulated, including members of the ubiquitin-specific peptidase family and subunits of the 26S proteasome. Such inhibition of the degradation machinery might stabilize the maternal protein content that is necessary for oocyte developmental competence. CONCLUSIONS: The low pregnancy rates commonly observed when using human MII oocytes after slow freezing-thawing may be explained by the alterations of the oocyte gene expression profile.

Transcriptome analysis reveals dialogues between human trophectoderm and endometrial cells during the implantation period.

BACKGROUND: Crosstalk between human trophectoderm (TE) and endometrial cells during the implantation window is a complex and not well-understood process. The aims of this study were (i) to evaluate the global gene expression profile in TE cells from Day 5 human blastocysts issued from IVF, (ii) to compare these data with the transcriptomic profile of endometrial cells in stimulated cycles for IVF and (iii) to identify potential early dialogues between maternal and embryonic cells during the implantation window. METHODS: Endometrial biopsies (n = 18) from normal responder patients were performed on the day of embryo transfer (Day 5 after human chorionic gonadotrophin administration). TE biopsies from five blastocysts donated for research purposes were mechanically extracted. DNA microarray analysis was carried out to identify the specific gene expression profiles and the biological pathways activated during the implantation window in endometrial and TE cells. RESULTS: Several cytokines (such as PDGFA, placenta growth factor, IGF2BP1 and IGF2BP3) were up-regulated in human TE cells, whereas some of the corresponding receptors (PDGFRA and KDR) were over-expressed in the receptive endometrium, suggesting that these molecules are involved in the early dialogue between blastocyst and maternal endometrial cells. In addition, several adhesion molecules and extracellular matrix proteins (MCAM, ITGAE and LAMA1) were also over-expressed in the TE, while others (ALCAM, CEACAM1, PECAM1, ITGB8 and LAMA2) were restricted to the receptive endometrium. CONCLUSION: The present study shows that several growth factors, cytokines, integrins and adhesion molecules are expressed in the TE and endometrium at the time of implantation. These results could contribute to the understanding of the mechanisms involved in the early dialogue between blastocyst and endometrium during implantation. Such results should be confirmed by further studies.

LH/hCGR gene expression in human cumulus cells is linked to the expression of the extracellular matrix modifying gene TNFAIP6 and to serum estradiol levels on day of hCG administration.

BACKGROUND: Recent studies suggest a role for luteinizing hormone and human chorionic gonadotrophin receptor (LH/hCGR) signalling in the regulation of the oocyte-cumulus oophorus cell interplay. The present study aimed at assessing the LH/hCGR gene expression in cumulus cells (CCs) surrounding oocytes in patients undergoing controlled ovarian hyperstimulation (COS) before ICSI and to relate the LH/hCGR expression to other COS quality parameters. METHODS: CCs from single oocytes of normal responder patients were analysed by DNA microarrays. Concomitantly, estradiol levels on the day of hCG administration, CC morphology, total collected oocyte and metaphase II oocyte number were assessed in relation to LH/hCGR gene expression in CC. RESULTS: The transcriptome analysis of CC indicated a variable expression of LH/hCGR among the patients and intra-patients. LH/hCGR mRNA expression was negatively correlated with serum estradiol level on the day of hCG administration. Eighty-five genes were significantly modulated between CCs from patients with a high and a low LH/hCGR expression. These genes are involved principally in steroid metabolism and in the ovulation process and include TNFAIP6, a gene expressed during CC-oocyte complex (COC) expansion. There were no significant differences in LH/hCGR gene expression profile between COS protocols. CONCLUSIONS: LH/hCGR is expressed in CC under COS conditions. LH/hCGR expression level is associated with TNFAIP6 gene expression and negatively correlated with serum estradiol level on the day of hCG administration.

Pertinence of apoptosis markers for the improvement of in vitro fertilization (IVF).

In assisted reproductive technology (ART), the pregnancy and birth rates following in vitro fertilization (IVF) attempts are still low. Recently, apoptotic markers have been suggested as new criteria for oocyte and embryo quality selection. Many studies have provided evidence that poor oocyte and embryo quality can be associated with apoptosis. The aim of this review is to summarize our current knowledge on the apoptotic process in oocytes and embryos, and focus on the possibility for using apoptotic markers as a reliable and predictive marker to select competent oocytes and embryos during IVF. Moreover, it is currently accepted that IVF failures, linked to poor embryo quality, are, in part, associated with suboptimal in vitro culture conditions. Here, we also review the current state of knowledge concerning how the genetic control of apoptosis during folliculogenesis and pre-implantation embryonic development is affected by in vitro culture conditions during IVF. In the future, identification of apoptotic markers in ART for oocyte and embryo selection should result in the development of new agonistic or antagonistic molecules of apoptosis by medicinal chemistry.

Gene expression profile of human endometrial receptivity: comparison between natural and stimulated cycles for the same patients.

BACKGROUND: The adjunction of exogenous hormones for controlled ovarian stimulation (COS) may alter endometrial receptiveness. In order to identify the genes misregulated under COS, we compared the endometrium gene expression profiles, from the same patients, in a natural cycle and in a subsequent COS cycle. METHODS: For the same normal-responder patients (n = 21), endometrial biopsies (n = 84) were collected during the pre-receptive (LH + 2) and receptive stages (LH + 7) of a natural cycle and, subsequently, on oocyte retrieval day (hCG + 2) and on transfer day (hCG + 5) of a stimulated cycle. Samples were analyzed using DNA microarrays. Gene expression profiles and biological pathways involved in endometrial receptivity were analyzed. RESULTS: Although endometrium transition profiles from pre-receptive to receptive phases are similar between patients, COS regimens alter endometrial receptivity in comparison with natural cycle. Under COS conditions, two endometrial profiles were identified and were associated either with a moderately altered receptivity profile for the majority of the patients or a strongly altered profile for a sub-category of patients. The receptive endometrium transcription profile under COS was defective for biological functions such as TGFbeta signaling, leukocyte transendothelial migration and the cell cycle. CONCLUSIONS: Gonadotrophin treatments in COS cycles led to disruptions of the transcriptional activation of genes involved in normal endometrial receptivity. We propose that when the receptiveness of the endometrium is seriously compromised by the COS protocol, fresh embryo replacement should be cancelled, the embryo frozen and thawed embryo replacement should be performed under natural cycles.

Identification of new biomarkers of human endometrial receptivity in the natural cycle.

BACKGROUND: Identification of new markers assessing endometrial receptivity may help in improving the clinical outcome of IVF. This study aimed at identifying genes expressed in human endometrium during the implantation window that could be used as such markers. METHODS: A series of normoresponder patients (n = 31) underwent endometrial biopsies (n = 62, 2 per patient) during the early secretory phase, 2 days after the LH surge (LH + 2) and the mid-secretory phase (LH + 7) of the same natural cycle that preceded a new ICSI attempt for male infertility factor. Samples were analyzed using DNA microarrays and gene expression profiles at the time of the implantation window were computed. Systems biology analysis allowed the identification of biological pathways that were over-represented in this signature. A new approach for class prediction applied to microarray experiments was then used to identify biomarkers putatively involved in endometrial receptiveness. RESULTS: Five genes expressed during the implantation window were all up-regulated in the LH + 7 samples compared with LH + 2 [laminin beta3 (P = 0.002), microfibril-associated protein 5 (P = 0.009), angiopoietin-like 1 (P = 0.005), endocrine gland-derived vascular endothelial growth factor (P = 0.049) and nuclear localized factor 2 (P = 0.007)]. Increased expression was validated by quantitative RT-PCR. CONCLUSIONS: Five genes have been identified for the first time as being up-regulated during the implantation window and are proposed as new biomarkers for exploration of endometrial receptiveness. As the endometrial biopsy procedure can be performed during a natural cycle, it would be worth testing this approach as a novel strategy in patients with poor implantation after IVF or ICSI.

A non-invasive test for assessing embryo potential by gene expression profiles of human cumulus cells: a proof of concept study.

Identification of new criteria for embryo quality is required to improve the clinical outcome of in vitro fertilization. The aim of this study was to determine the gene expression profile of cumulus cells (CC) surrounding the oocyte as biomarkers for embryo potential and to identify genes to be used as prognostic indicators of successful pregnancy. CC from single oocytes were analysed using DNA microarrays. Gene expression profiles of CC surrounding the oocyte associated with good embryonic quality and pregnancy outcome were computed. We observed that CC issued from oocytes that developed into embryos with a good morphology had differing gene expression profile according to the pregnancy outcome of the embryo. We demonstrated that the expression of BCL2L11, PCK1 and NFIB in CC is significantly correlated with embryo potential and successful pregnancy. These results were confirmed by quantitative RT-PCR. The gene expression profiling of human CC correlates with embryo potential and pregnancy outcome. BCL2L11, PCK1 and NFIB genes are proposed as biomarkers for predicting pregnancy. Our findings suggest a non-invasive approach, offering a new potential strategy for competent embryo selection. This approach should be validated in single-embryo transfer programmes.

[Oocyte and embryo quality: do the apoptotic markers have a place in the preimplantation genetic diagnostic?]. / Qualité ovocytaire et embryonnaire : les marqueurs apoptotiques ont-ils leur place dans le potentiel préimplantatoire ?

In Assisted Reproductive Technology (ART), the pregnancy and birth rates following in vitro fertilization (IVF) attempts are still low. Recently, research in the field of ART has explored new oocyte and embryo quality selection criteria such as apoptotic markers. Many studies provided evidence that bad oocyte and embryo quality can be associated with apoptosis. The aim of this review is to summarize our current knowledge on the apoptosis process in oocytes and embryos, and focus on the possibility of using apoptotic markers as a reliable and predictive marker to select competent oocytes and embryos during IFV.

[Could apoptotic markers help the exploration of male infertility?]. / Les marqueurs apoptotiques ont-ils leur place comme marqueurs potentiels de l'exploration de l'infertilité masculine ?

Apoptosis is a cell death program involved in different steps of spermatogenesis, first at puberty, at the beginning of spermatogenesis, then in adult testicles by controlling normal spermatogenesis. As a result, apoptosis deregulation can affect spermatogenesis. Many studies have provided evidence that apoptosis deregulation in germinal cells resulted in male infertility. In addition, apoptosis detection in ejaculated spermatozoa arouses a growing interest in research as a reliable marker of spermatozoon quality. The aim of this review is to summarize our knowledge on physiological apoptosis during spermatogenesis, and then analyse the possibility of using apoptotic markers as selective markers of spermatozoon quality to optimize the rate of success of in vitro fertilization.

Mitochondrial permeability transition as a novel principle of hepatorenal toxicity in vivo.

Atractyloside (Atr) binds to the adenine nucleotide translocator (ANT) and inhibits ANT-mediated ATP/ADP exchange on the inner mitochondrial membrane. In addition, Atr can trigger opening of a non-specific ion channel, within the ANT-containing permeability transition pore complex (PTPC), which is subject to redox regulation and inhibited by cyclosporin A (CsA). Here we show that the cytotoxic effects of Atr, both in vivo and in vitro, are determined by its capacity to induce PTPC opening and consequent mitochondrial membrane permeabilization (MMP). Thus, the Atr-induced MMP and death of cultured liver cells are both inhibited by CsA as well as by glutathione (GSH) and enhanced by GSH depletion. Similarly, the hepatorenal toxicity of Atr, assessed in vivo, was reduced by treating mice with CsA or a diet rich in sulfur amino acids, a regime which enhances mitochondrial GSH levels. Atr injection induced MMP in hepatocytes and proximal renal tubular cells, and MMP was reduced by either CsA or GSH. Acetaminophen (paracetamol)-induced acute poisoning was also attenuated by CsA and GSH, both in vitro and in vivo. Altogether these data indicate that PTPC-mediated MMP may determine the hepatorenal toxicity of xenobiotics in vivo.

Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria.

The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, we show that P. acidipropionici and freudenreichii, two species which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted by the bacteria. Bacterial culture supernatants as well as pure SCFA induced typical signs of apoptosis including a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species, caspase-3 processing, and nuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.

Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437.

An increasing number of experimental chemotherapeutic agents induce apoptosis by directly triggering mitochondrial membrane permeabilization (MMP). Here we examined MMP induced by lonidamine, arsenite, and the retinoid derivative CD437. Cells overexpressing the cytomegalovirus-encoded protein vMIA, a protein which interacts with the adenine nucleotide translocator, were strongly protected against the MMP-inducing and apoptogenic effects of lonidamine, arsenite, and CD437. In a cell-free system, lonidamine, arsenite, and CD437 induced the permeabilization of ANT proteoliposomes, yet had no effect on protein-free liposomes. The ANT-dependent membrane permeabilization was inhibited by the two ANT ligands ATP and ADP, as well as by recombinant Bcl-2 protein. Lonidamine, arsenite, and CD437, added to synthetic planar lipid bilayers containing ANT, elicited ANT channel activities with clearly distinct conductance levels of 20+/-7, 100+/-30, and 47+/-7 pS, respectively. Altering the ATP/ADP gradient built up on the inner mitochondrial membrane by inhibition of glycolysis and/or oxidative phosphorylation differentially modulated the cytocidal potential of lonidamine, arsenite, and CD437. Inhibition of F(0)F(1)ATPase without glycolysis inhibition sensitized to lonidamine-induced cell death. In contrast, only the combined inhibition of glycolysis plus F(0)F(1)ATPase sensitized to arsenite-induced cell death. No sensitization to cell death induction by CD437 was achieved by glucose depletion and/or oligomycin addition. These results indicate that ANT is a target of lonidamine, arsenite, and CD437 and unravel an unexpected heterogeneity in the mode of action of these three compounds.

The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal.

Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.

Prolonged, but not acute, glutathione depletion promotes Fas-mediated mitochondrial permeability transition and apoptosis in mice.

Glutathione depletion either decreased or increased death-receptor-mediated apoptosis in previous studies. Comparison of the durations of glutathione depletion before death-receptor stimulation in these studies might suggest a different effect of prolonged versus acute thiol depletion. We compared the effects of the prolonged glutathione depletion caused by a sulfur amino acid-deficient (SAA(-)) diet and the acute depletion caused by a single dose of phorone on hepatic apoptosis triggered by the administration of an agonistic anti-Fas antibody. The chronic SAA(-) diet did not affect hepatic Fas or Bcl-XL, but increased p53 and Bax, and exacerbated Fas-mediated mitochondrial membrane depolarization, electron-microscopy-proven outer mitochondrial membrane rupture, cytochrome c translocation to the cytosol, and caspase 3 activation. These effects were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. The SAA(-) diet increased internucleosomal DNA fragmentation, the percentage of apoptotic hepatocytes, serum alanine transaminase (ALT) activity, and mortality after Fas stimulation. Despite a similar decrease in hepatic glutathione, administration of a single dose of phorone 1 hour before the anti-Fas antibody did not change p53 or Bax, and did not enhance Fas-induced mitochondrial permeability transition and toxicity. However, 4 repeated doses of phorone (causing more prolonged glutathione depletion) increased Bax and Fas-mediated toxicity. In conclusion, a chronic SAA(-) diet, but not acute phorone administration, increases p53 and Bax, and enhances Fas-induced mitochondrial permeability transition and apoptosis. Thiol depletion could cause oxidative stress that requires several hours to increase p53; the latter induces Bax, which translocates to mitochondria after Fas stimulation.