The human cumulus cell transcriptome provides poor predictive value for embryo transfer outcome.

RESEARCH QUESTION: Is the transcriptome of cumulus cells a good predictor of the embryo's developmental competence? DESIGN: Cumulus cells were collected from donor oocytes and their transcriptome was analysed by RNA sequencing analysis at >30 × 106 reads in samples grouped according to the developmental potential of their enclosed oocyte: not able to develop to the blastocyst stage (Bl-), able to develop to the blastocyst stage but failing to establish a pregnancy (P-), or able to develop to the blastocyst stage and to establish a clinical pregnancy (P+). RESULTS: The cumulus cell trancriptome was largely independent of the developmental potential as, using a false dscovery rate-adjusted P-value of <0.05, only 10, 11 and 5 genes were differentially expressed for the comparisons P+ versus P-, P+ versus Bl-, and P- versus Bl-, respectively, out of a total of 17,469 genes expressed. Between the differentially expressed genes, those showing little overlap between samples from different groups were CHAC1, up-regulated in the P- and P+ groups compared with the Bl- group, and CENPE, CD93, PECAM1 and HSPA1B, which showed the opposite expression pattern. Focusing on the pregnancy potential, only EPN3 was consistently downregulated in the P+ compared with the P- and Bl- groups. CONCLUSIONS: The cumulus cell transcriptome is largely unrelated to the establishment of clinical pregnancy following embryo transfer, although the expression level of a subset of genes in cumulus cells may indicate the ability to develop to the blastocyst stage.

mtDNA content in cumulus cells does not predict development to blastocyst or implantation.

STUDY QUESTION: Is relative mitochondrial DNA (mtDNA) content in cumulus cells (CCs) related to embryo developmental competence in humans and/or the bovine model? SUMMARY ANSWER: mtDNA content in CCs provides a poor predictive value of oocyte developmental potential, both in vitro and following embryo transfer. WHAT IS KNOWN ALREADY: CCs are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby providing interesting biological material on which to perform molecular analyses designed to identify markers that predict oocyte developmental competence. Previous studies have positively associated oocyte mtDNA content with developmental potential in animal models and women. However, it remains debatable whether mtDNA content in CCs could be used as a proxy to infer oocyte developmental potential. STUDY DESIGN SIZE DURATION: mtDNA content was analyzed in CCs obtained from 109 human oocytes unable to develop to blastocyst, able to develop to blastocyst but failing to establish pregnancy or able to develop to blastocyst and to establish pregnancy. mtDNA analysis was also performed on bovine cumulus samples collected from 120 oocytes unable to cleave, oocytes developing into cleaved embryos but arresting development prior to the blastocyst stage or oocytes developing to blastocysts. PARTICIPANTS/MATERIALS SETTING METHODS: Human CCs samples were obtained from women undergoing IVF. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Bovine samples were obtained from slaughtered cattle and individually matured, fertilized and cultured in vitro. Relative mtDNA was assessed by quantitative PCR analysis. MAIN RESULTS AND THE ROLE OF CHANCE: mtDNA content in human and bovine CCs did not differ according to the developmental potential of their enclosed oocyte. Moreover, mtDNA content in bovine oocytes did not correlate with that of their corresponding CCs. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The lack of correlation found between mtDNA content in human CCs and oocytes was also assessed in bovine samples. Although bovine folliculogenesis, mono-ovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, they may not be fully comparable. WIDER IMPLICATIONS OF THE FINDINGS: The use of molecular markers for oocyte developmental potential in CCs could be used to enhance success rates following single embryo transfer. However, our data indicate that mtDNA in CCs is not a good proxy for oocyte quality. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by the Madrid Region Government. The authors declare no competing interests.

RNA-sequencing reveals genes linked with oocyte developmental potential in bovine cumulus cells.

Cumulus cells provide an interesting biological material to perform analyses to understand the molecular clues determining oocyte competence. The objective of this study was to analyze the transcriptional differences between cumulus cells from oocytes exhibiting different developmental potentials following individual in vitro embryo production by RNA-seq. Cumulus cells were allocated into three groups according to the developmental potential of the oocyte following fertilization: (1) oocytes developing to blastocysts (Bl+), (2) oocytes cleaving but arresting development before the blastocyst stage (Bl-), and (3) oocytes not cleaving (Cl-). RNAseq was performed on 4 (Cl-) or 5 samples (Bl+ and Bl-) of cumulus cells pooled from 10 cumulus-oocyte complexes per group. A total of 49, 50, and 18 differentially expressed genes (DEGs) were detected in the comparisons Bl+ versus Bl-, Bl+ versus Cl- and Bl- versus Cl-, respectively, showing a fold change greater than 1.5 at an adjusted p value <0.05. Focussing on DEGs in cumulus cells from Bl+ group, 10 DEGs were common to both comparisons (10/49 from Bl+ vs. Bl-, 10/50 from Bl+ vs. Cl-). These DEGs correspond to 6 upregulated genes (HBE1, ITGA1, PAPPA, AKAP12, ITGA5, and SLC1A4), and 4 downregulated genes (GSTA1, PSMB8, FMOD, and SFRP4) in Bl+ compared to the other groups, from which 7 were validated by quantitative PCR (HBE1, ITGA1, PAPPA, AKAP12, ITGA5, PSMB8 and SFRP4). These genes are involved in critical biological functions such as integrin-mediated cell adhesion, oxygen availability, IGF and Wnt signaling or PKA pathway, highlighting specific biological processes altered in incompetent in vitro maturation oocytes.

Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species.

Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated 'The people behind the papers' interview.

Embryonic disc formation following post-hatching bovine embryo development in vitro.

Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

TMEM95 is a sperm membrane protein essential for mammalian fertilization.

The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male-specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization.

ZP4 confers structural properties to the zona pellucida essential for embryo development.

Zona pellucida (ZP), the extracellular matrix sheltering mammalian oocytes and embryos, is composed by 3 to 4 proteins. The roles of the three proteins present in mice have been elucidated by KO models, but the function of the fourth component (ZP4), present in all other eutherian mammals studied so far, has remained elusive. Herein, we report that ZP4 ablation impairs fertility in female rabbits. Ovulation, fertilization and in vitro development to blastocyst were not affected by ZP4 ablation. However, in vivo development is severely impaired in embryos covered by a ZP4-devoided zona, suggesting a defective ZP protective capacity in the absence of ZP4. ZP4-null ZP was significantly thinner, more permeable, and exhibited a more disorganized and fenestrated structure. The evolutionary conservation of ZP4 in other mammals, including humans, suggests that the structural properties conferred by this protein are required to ensure proper embryo sheltering during in vivo preimplantation development.

Intergenerational transmission of the positive effects of physical exercise on brain and cognition.

Physical exercise has positive effects on cognition, but very little is known about the inheritance of these effects to sedentary offspring and the mechanisms involved. Here, we use a patrilineal design in mice to test the transmission of effects from the same father (before or after training) and from different fathers to compare sedentary- and runner-father progenies. Behavioral, stereological, and whole-genome sequence analyses reveal that paternal cognition improvement is inherited by the offspring, along with increased adult neurogenesis, greater mitochondrial citrate synthase activity, and modulation of the adult hippocampal gene expression profile. These results demonstrate the inheritance of exercise-induced cognition enhancement through the germline, pointing to paternal physical activity as a direct factor driving offspring's brain physiology and cognitive behavior.

Mitochondrial and metabolic adjustments during the final phase of follicular development prior to IVM of bovine oocytes.

In vitro maturation (IVM) leads to reduced developmental rates compared to the use of in vivo matured oocytes. This reduction can be attributed to the suboptimal environment experienced during IVM, but the use of incompetent oocytes also plays a significant role. The objective of this study has been to characterize the mitochondrial and metabolic differences between competent and incompetent bovine oocytes selected prior to IVM based on Brilliant Cresyl Blue (BCB) staining. BCB selection allowed to sort two populations of cumulus-oocyte complexes (COCs) exhibiting diverse developmental competence despite showing a similar size and thereby being morphologically undistinguishable otherwise. Nuclear maturation rates were similar in both populations, but cleavage and blastocysts rates were significantly higher in BCB+ compared with BCB-. Mitochondrial distribution was similar between both groups, but mtDNA content experienced a 1.9-fold increase between BCB- and BCB+ oocytes, suggesting that a significant mtDNA synthesis must occur at the last stages of follicular development to achieve full competence prior to IVM. Consistently, transcriptional analysis in cumulus cells revealed an upregulation of the mitochondrial transcription factor TFAM in BCB-. Transcriptional analysis also suggested a decrease in both anaerobic glycolysis and pentose phosphate pathway (PPP) in BCB+ COCs, as the anaerobic glycolysis enzymes GAPDH and LDHA and the positive regulator of G6PD activity SIRT2 were upregulated in BCB- cumulus cells. These results suggest that during the final stages of follicular development a significant mtDNA replication must occur to achieve full oocyte developmental competence, and that this replication may be linked to anaerobic glycolysis and PPP activities.