From genes to environment in shaping of an embryo: understanding embryonic-extraembryonic interactions at the BSDB autumn meeting in Oxford

The British Society for Developmental Biology Autumn Meeting, held in Oxford in September 2018, was the third in a series of international workshops which have been focussed on development at the extraembryonic-embryonic interface. This workshop, entitled “Embryonic-Extraembryonic Interactions: from Genetics to Environment” built on the two previous workshops held in 2011 (Leuven, Belgium) and 2015 (Göttingen, Germany). This workshop brought together researchers utilising a diverse range of organisms (including both vertebrate and invertebrate species) and a range of experimental approaches to answer core questions in developmental biology. This meeting report highlights some of the major themes emerging from the workshop including an evolutionary perspective as well as recent advances that have been made through the adoption of emerging techniques and technologies

Delayed APC/C activation extends the first mitosis of mouse embryos

The correct temporal regulation of mitosis underpins genomic stability because it ensures the alignment of chromosomes on the mitotic spindle that is required for their proper segregation to the two daughter cells. Crucially, sister chromatid separation must be delayed until all the chromosomes have attached to the spindle; this is achieved by the Spindle Assembly Checkpoint (SAC) that inhibits the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase. In many species the first embryonic M-phase is significantly prolonged compared to the subsequent divisions, but the reason behind this has remained unclear. Here, we show that the first M-phase in the mouse embryo is significantly extended due to a delay in APC/C activation. Unlike in somatic cells, where the APC/C first targets cyclin A2 for degradation at nuclear envelope breakdown (NEBD), we find that in zygotes cyclin A2 remains stable for a significant period of time after NEBD. Our findings that the SAC prevents cyclin A2 degradation, whereas over-expressed Plk1 stimulates it, support our conclusion that the delay in cyclin A2 degradation is caused by low APC/C activity. As a consequence of delayed APC/C activation cyclin B1 stability in the first mitosis is also prolonged, leading to the unusual length of the first M-phase.We are grateful to the Wellcome Trust, which supported this work. AA was a beneficiary of the Kolumb programme and the subsequent Kolumb supporting grant of the Foundation for Polish Science. MZG is supported by the Wellcome Trust

An Epididymis-Specific Secretory Protein HongrES1 Critically Regulates Sperm Capacitation and Male Fertility

Mammalian sperm capacitation is an essential prerequisite to fertilizion. Although progress had been made in understanding the physiology and biochemistry of capacitation, little is known about the potential roles of epididymal proteins during this process. Here we report that HongrES1, a new member of the SERPIN (serine proteinase inhibitor) family exclusively expressed in the rat cauda epididymis and up-regulated by androgen, is secreted into the lumen and covers the sperm head. Co-culture of caudal sperms with HongrES1 antibody in vitro resulted in a significant increase in the percentage of capacitated spermatozoa. Furthermore, the percentage of capacitated spermatozoa clearly increased in rats when HongrES1 was down-regulated by RNAi in vivo. Remarkably, knockdown of HongrES1 in vivo led to reduced fertility accompanied with deformed appearance of fetuses and pups. These results identify HongrES1 as a novel and critical molecule in the regulation of sperm capacitation and male fertility

Gas6 Downregulation Impaired Cytoplasmic Maturation and Pronuclear Formation Independent to the MPF Activity

Previously, we found that the growth arrest-specific gene 6 (Gas6) is more highly expressed in germinal vesicle (GV) oocytes than in metaphase II (MII) oocytes using annealing control primer (ACP)-PCR technology. The current study was undertaken to investigate the role of Gas6 in oocyte maturation and fertilization using RNA interference (RNAi). Interestingly, despite the specific and marked decrease in Gas6 mRNA and protein expression in GVs after Gas6 RNAi, nuclear maturation including spindle structures and chromosome segregation was not affected. The only discernible effect induced by Gas6 RNAi was a change in maturation promoting factor (MPF) activity. After parthenogenetic activation, Gas6 RNAi-treated oocytes at the MII stage had not developed further and arrested at MII (90.0%). After stimulation with Sr2+, Gas6-silenced MII oocytes had markedly reduced Ca2+ oscillation and exhibited no exocytosis of cortical granules. In these oocytes, sperm penetration occurred during fertilization but not pronucleus (PN) formation. By roscovitine and colcemid treatment, we found that the Gas6 knockdown affected cytoplasmic maturation directly, independent to the changed MPF activity. These results strongly suggest that 1) the Gas6 signaling itself is important to the cytoplasmic maturation, but not nuclear maturation, and 2) the decreased Gas6 expression and decreased MPF activity separately or mutually influence sperm head decondensation and PN formation

P-221 Bisphenol A affects Ca2+ homeostasis in mouse oocytes through GPR30 receptor and MAPK/ERK signalling pathway

Abstract Study question Does bisphenol A (BPA), xenoestrogen present in plastics, affect Ca2+ homeostasis in mouse oocytes? If yes, what is the mechanism of BPA action? Summary answer BPA (2 ng/ml, concentration reported for human follicular fluid) alternates the Ca2+ pattern generated in fertilized oocytes acting through GPR30 receptor and MAPK/ERK signaling pathway. What is known already BPA is a monomeric organic compound widely used in the production of resins, polycarbonates, and plastics. It is a xenoestrogen, i.e. a synthetic compound exerting an estrogen-like effect on cells. It can therefore disrupt the functioning of animal (including human) organisms. Recent literature data indicate that BPA may affect fertility in mammals by disturbing several processes in oocytes and embryos, including epigenetic modifications, energy metabolism, and spindle assembly. However, its effect on Ca2+ homeostasis in oocytes, key for activation of embryonic development upon fertilization, has not been yet examined. Study design, size, duration The study was carried out on mouse (a C57Bl6/Tar x CBA/Tar cross) oocytes. Each experimental variant was repeated at least 3 times, the number of oocytes analyzed was 40-75 per group, depending on the experimental variant. Participants/materials, setting, methods BPA was added to M16 medium, in which mouse oocytes matured in vitro. Oocytes that reached metaphase II were labeled with Oregon Green 488 BAPTA-1 AM, a fluorescent Ca2+ indicator, fertilized in vitro, and subjected to time-lapse imaging. The fluorescence intensity was analyzed to assess the pattern of Ca2+ oscillations triggered in oocytes upon fertilization. In some experiments, specific inhibitors were used to analyze the molecular pathway mediating the BPA action in oocytes. Main results and the role of chance 2ng/ml BPA added to oocytes during their in vitro maturation decreased the maturation rate (from 77% in control to 52%). BPA shortened the total duration of Ca2+ oscillations induced in metaphase II oocytes by fertilization (183 ± 80.4 min in control vs. 118.2 ± 84.2 min in BPA group) and the mean interval between the subsequent Ca2+ transients (14.3 ± 4.9 min vs. 9.7 ± 4.5 min). As GPR30, G protein-coupled estrogen receptor, can bind BPA and is expressed in oocytes, we investigated whether it mediates BPA action. G1, a GPR30 activator, added to the oocyte maturation medium instead of BPA, mimicked the BPA effect only partially: it shortened the total duration of Ca2+ oscillations (175.6 ± 71.5 in control vs. 134.5 ± 90.8 min in G1 group), but increased the inter-transient interval (11.6 ± 5.2 min vs. 15.2 ± 4.7 min). On the other hand, G15, a GPR30 inhibitor, alleviated the BPA-induced alternations (total duration of Ca2+ oscillations: 192.7 ± 77.5 min, the interval between Ca2+ transients: 11.4 ± 4.6 min). Moreover, PD0325901, a MAPK/ERK pathway inhibitor, rescued BPA-induced changes in the duration and frequency of Ca2+ oscillations (195.9 ± 95 min and 18.2 ± 4.8 min, respectively). Limitations, reasons for caution The susceptibility of oocytes to BPA, and, in consequence, its effect on Ca2+ homeostasis, may be different for various mammalian species. Wider implications of the findings BPA disturbs the pattern of Ca2+ oscillations in fertilized mouse oocytes through GRP30 and MAP/ERK pathway, but it is possible that BPA acts also through other estrogen receptors. Ca2+ oscillations are crucial for activation of embryonic development, so our observations provide a novel mechanism of BPA action on mammalian fertility. Trial registration number n/a </jats:sec

P–204 Dynamics of cavitation as a potential non-invasive predictor of mammalian blastocyst quality

Abstract Study question We wished to investigate whether dynamics of cavity formation can be used in embryo quality assessment. Summary answer Dynamics of mouse embryo cavitation reflects to certain extent blastocysts’ developmental capabilities. It can be potentially used as a biomarker of mammalian embryo quality. What is known already During cavity expansion blastocyst pulsates, i.e. changes its volume in an oscillatory way. Recent studies performed on a mouse model have shown, that dynamics of cavitation, biomechanical properties of the trophectoderm (TE) and embryo size are intertwined. Presence or absence of blastocyst contractions has been linked to particular parameters related to positive outcome of the in vitro fertilization procedures, but the data on influence of contractions on human embryos’ developmental capabilities is often contradictory. Moreover, mostly in those studies only strong contractions (leading to a high volume loss) have been taken into consideration. Study design, size, duration We tested how postovulatory (in vitro or in vivo) or maternal aging of mouse oocytes affects dynamics of cavity formation and expansion in the resulting embryos (n = 27, n = 26 and n = 30, respectively). Furthermore, we also analyzed almost 100 mouse blastocysts in order to correlate dynamics of their cavitation with their ability to form correct outgrowths (in vitro model of implantation). Participants/materials, setting, methods Mouse oocytes subjected to postovulatory (either in vivo or in vitro) or maternal aging were fertilized in vitro. Dynamics of cavity formation and expansion was assessed by time-lapse imaging; equatorial images were taken every 10 minutes. Blastocyst area was measured over time and compared to the outcome from control embryos. In another set of experiments, after the filming mouse blastocysts were cultured for additional 4 days to test their ability to form outgrowths. Main results and the role of chance We noticed, that mouse embryos which represent limited developmental potential (obtained from either postovulatory or maternally aged oocytes) and blastocysts developed from freshly fertilized young females’ oocytes differ in terms of some parameters related to dynamics of cavitation, e.g. time of the initiation of cavity formation, frequency of contractions or mean loss of blastocyst’s area during contraction. We observed that embryos obtained from oocytes subjected to maternal or postovulatory aging have distinct dynamics of cavitation. Moreover, we noticed slightly different effect on particular parameters related to cavitation between in vivo and in vitro version of postovulatory ageing. We also showed that blastocysts, which are unable to create proper outgrowths (i.e. too small or without epiblast cells), differ from embryos that differentiate into correct outgrowths in terms of certain parameters of cavitation dynamics. Our data indicates, that dynamics of cavity formation and expansion might be related to developmental potential of mouse embryo. Limitations, reasons for caution Further studies with extended group size and testing embryos’ ability to implant in vivo are required to confirm our results. Moreover, we examined dynamics of cavitation only in a mouse model, so additional studies performed on other mammalian species are needed. Wider implications of the findings: Our data proves, that dynamics of embryo cavitation reflects, to certain extent, developmental capabilities of mouse blastocysts. Therefore, it is possible that it can be a biomarker of embryo quality (in combination with parameters provided by other methods or solely) of other mammalian species, including humans. Trial registration number Not applicable </jats:sec

Correction to: From genes to environment in shaping of an embryo: understanding embryonic-extraembryonic interactions at the BSDB autumn meeting in Oxford

The authors of the article Ajduk & Duncan 2019 sincerely apologize for specifying the incorrect institutional affiliation for Professor Ali Brivanlou and also the incorrect spelling of Professor Brivanlou's surname in the text of the article