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Dramatic nuclear reorganization occurs during early development to convert terminally differentiated gametes to a totipotent zygote, which then gives rise to an embryo. Aberrant epigenome resetting severely impairs embryo development and even leads to lethality. How the epigenomes are inherited, reprogrammed, and reestablished in this critical developmental period has gradually been unveiled through the rapid development of technologies including ultrasensitive chromatin analysis methods. In this review, we summarize the latest findings on epigenetic reprogramming in gametogenesis and embryogenesis, and how it contributes to gamete maturation and parental-to-zygotic transition. Finally, we highlight the key questions that remain to be answered to fully understand chromatin regulation and nuclear reprogramming in early development.

10 Pág., The most commonly used technique to vitrify pig embryos is the super open pulled straw (SOPS), where a maximum of 6 embryos can be vitrified simultaneously per device without compromising the minimum volume necessary for optimal preservation. Since optimal embryo transfer (ET) demands a transfer of 20-40 embryos per recipient, the customary use of SOPS complicates embryo warming and ET in field conditions. Such complications could be avoided when using the Cryotop® (OC) system, which has been proven to be an effective option for vitrifying at least 20 porcine embryos simultaneously. This study aimed to investigate the changes in the transcriptome of blastocysts caused by vitrification using both systems. In vivo-derived blastocysts were OC- (n = 60; 20 embryos/device) and SOPS- (n = 60; 4-6 embryos/device) vitrified and cultured for 24 h after warming. Nonvitrified blastocysts (n = 60) cultured for 24 h postcollection acted as controls. At the end of culture, 48 viable embryos from each group (6 pools of 8 embryos) were selected for microarray (GeneChip® Porcine Genome Array, P/N 900624, Affymetrix) analysis of differentially expressed genes (DEGs). The survival rate of embryos vitrified with the OC and SOPS systems (>97%) was similar to that of the control embryos (100%). Microarray analysis of each vitrification system compared to the control group showed 245 DEGs (89 downregulated and 156 upregulated) for the OC system and 210 (44 downregulated and 166 upregulated) for the SOPS system. Two pathways were enriched for the DEGs specifically altered in each vitrification system compared to the control (glycolysis/gluconeogenesis and carbon metabolism pathways for the OC system and amino sugar and nucleotide sugar metabolism and lysosome pathways in the SOPS group). The OC group showed 31 downregulated and 24 upregulated genes and two enriched pathways (mineral absorption and amino sugar and nucleotide sugar metabolism pathways) when compared to the SOPS group. In summary, vitrification with the OC system altered fewer genes related to apoptosis and activated genes related to cell proliferation. We conclude that vitrification with either the OC or SOPS system has a moderate to low effect on the transcriptome of in vivo-derived porcine blastocysts. Further investigation is needed to elucidate how the differences in the transcriptome of embryos vitrified with these systems affect their subsequent developmental ability after ET., The authors would like to acknowledge the financial support from the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033) and from ERDF “A way of making Europe” (RTI2018-093525-B-I00), Madrid, Spain; Fundacion Seneca (19892/GERM/15), Murcia, Spain and the Research Council FORMAS (project 2019-00288), Stockholm, Sweden. We are also grateful to the Spanish Ministry of Science and Innovation for the grant-based support to A Gonzalez-Plaza (PRE2019-090508). We would also like to thank the staff of the Agropor piggery (Murcia, Spain) for their excellent care of the animals.

Ananas ananassoides (Baker) L.B.Sm. is a wild pineapple, commonly found in the savannas. This study aimed to describe the morpho-anatomy and histochemistry of its seed. The observations were made in the longitudinal and transverse sections, using an optical microscope. The cell arrangement in the seed coat, ripples in the integument, the ratio of embryo size and endosperm amount, and the number of strata in the aleurone layer are anatomical characteristics that may contribute to distinguishing this species. The starch in the endosperm, lipids and proteins in the embryo, constitute the seed's main nutritional reserves. The homogeneous embryo and phenolic compounds present in the seed coat and in the aleurone layer possibly contribute to the dormancy in this species. This study presents information relevant to the taxonomy and physiology of A. ananassoides, which represents contributions to the global knowledge of this species with a high potential as ornamental. Resumo Ananas ananassoides (Baker) L.B.Sm. é um abacaxi silvestre, comumente encontrado nas savanas. Este estudo teve como objetivo descrever a morfoanatomia e histoquímica de sua semente. As observações foram feitas nos cortes longitudinais e transversais, em microscópio óptico. O arranjo celular no tegumento, as ondulações no tegumento, a relação entre o tamanho do embrião e a quantidade de endosperma, e o número de estratos na camada de aleurona são características anatômicas que podem contribuir para a distinção dessa espécie. O amido do endosperma e os lipídios e proteínas do embrião constituem as principais reservas nutricionais da semente. O embrião homogêneo e os compostos fenólicos presentes no tegumento e na camada de aleurona possivelmente contribuem para a dormência nessa espécie. Este estudo apresenta informações relevantes para a taxonomia e fisiologia de A. ananassoides, sendo contribuições valiosas para o conhecimento global dessa espécie com alto potencial ornamental.

The cryotolerance of equine blastocysts larger than 300 μm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 μm, n = 14 and > 550 μm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10% glycerol (n = 14), or vitrification (n = 13) in 16.5% ethylene glycol/16.5% DMSO/0.5 M sucrose. Immediately after thawing or warming, embryos were cultured for 24 h at 38 °C and then graded and measured to assess re-expansion. Control embryos (n = 6) were cultured for 24 h following aspiration of blastocoel fluid, without cryopreservation or exposure to cryoprotectants. Subsequently, embryos were stained to assess live/dead cell proportion (DAPI/TOPRO-3), cytoskeleton quality (Phalloidin) and capsule integrity (WGA). For 300-550 μm embryos, quality grade and re-expansion were impaired after slow-freezing but not affected by vitrification. Slow-freezing embryos >550 μm induced additional cell damage as indicated by a significant increase in dead cell proportion and disruption of the cytoskeleton; neither of these changes were observed in vitrified embryos. Capsule loss was not a significant consequence of either freezing method. In conclusion, slow-freezing of expanded equine blastocysts collapsed by blastocoel aspiration compromises post-thaw embryo quality more than vitrification.

9 Pág., The developmental failures occurring between blastocyst hatching and implantation in farm ungulates are a major cause of pregnancy losses. At the expanded blastocyst stage, three cell lineages emerge in the embryo: trophoblast, hypoblast and epiblast, the latter being the most vulnerable during post-hatching development. Transforming growth factor beta (TGFβ) signaling pathway is involved in hypoblast and epiblast development; however, previous in vitro functional studies are limited to the expanded blastocyst stage. In this study, we have analyzed the effect of TGFβ inhibition with 10, 20 or 40 μM SB431542 during ovine post-hatching developmental period using a recently developed culture system able to recapitulate major developmental landmarks. We have found a negative effect of TGFβ inhibition on hypoblast and epiblast development that could be partially reverted by Rho-associated protein kinase (ROCK) inhibitor Y-27632. Our findings provide new insights into the molecular networks regulating embryo development beyond the expanded blastocyst and could help to elucidate the causes of early pregnancy losses in farm ungulates., This work has been funded by the projects StG-757886-ELONGAN from the European Research Council to PBA and PID2021-122153NA-I00 from the Spanish Ministry of Science and Innovation to PRI.

In the past four decades, the bovine model has been highly informative and inspiring to assisted reproductive technologies (ART) in other species. Most of the recent advances in ART have come from studies in cattle, particularly those unveiling the importance of several processes that must be recapitulated in vitro to ensure the proper development of the oocyte. The maintenance of structural and functional communications between the cumulus cells and the oocyte and a well-orchestrated chromatin remodeling with the gradual silencing of transcriptional activity represent essential processes for the progressive acquisition of oocyte developmental competence. These markers are now considered the milestones of physiological approaches to increase the efficiency of reproductive technologies. Different in vitro approaches have been proposed. In particular, the so-called "pre-IVM" or "prematuration" is a culture step performed before in vitro maturation (IVM) to support the completion of the oocyte differentiation process. Although these attempts only partially improved the embryo quality and yield, they currently represent a proof of principle that oocytes retrieved from an ovary or an ovarian batch shouldn't be treated as a whole and that tailored approaches can be developed for culturing competent oocytes in several species, including humans. An advancement in ART's efficiency would be desirable in carnivores, where the success is still limited. Since the progress in reproductive medicine has often come from comparative studies, this review highlights aspects that have been critical in other species and how they may be extended to carnivores.

Hexafluoropropylene oxide-dimer acid (HFPO-DA) is one of the emerging replacements for the “forever” carcinogenic and toxic long-chain PFAS. HFPO-DA is a polymerization aid used for manufacturing fluoropolymers, whose global distribution and undetermined toxic properties are a concern regarding human and ecological health. To assess embryotoxic potential, zebrafish embryos were exposed to HFPO-DA at concentrations of 0.5–20,000 mg/L at 24-, 48-, and 72-h post-fertilization (hpf). Heart rate increased significantly in embryos exposed to 2 mg/L and 10 mg/L HFPO-DA across all time points. Spinal deformities and edema phenotypes were evident among embryos exposed to 1000–16,000 mg/L HFPO-DA at 72 hpf. A median lethal concentration (LC50) was derived as 7651 mg/L at 72 hpf. Shallow RNA sequencing analysis of 9465 transcripts identified 38 consistently differentially expressed genes at 0.5 mg/L, 1 mg/L, 2 mg/L, and 10 mg/L HFPO-DA exposures. Notably, seven downregulated genes were associated with visual response, and seven upregulated genes were expressed in or regulated the cardiovascular system. This study identifies biological targets and molecular pathways affected during animal development by an emerging, potentially problematic, and ubiquitous industrial chemical.

The biocide tetrakis(hydroxymethyl)phosphonium sulphate (THPS) and other members of the tetrakis(hydroxymethyl) phosphonium salts (THPX) family are associated with liver toxicity in several mammalian species and teratogenicity in rabbits. Malformations include skeletal changes and abnormalities in eye development and are very similar to those seen with vitamin A deficiency or excess. For this reason, it was hypothesized that teratogenicity of THPS(X) might be attributed to disturbances in retinol availability and/or metabolism as a result of maternal toxicity, for example, either due to insufficient dietary intake by the mothers or due to liver toxicity. Therefore, in the present study, liver toxicity and vitamin A homeostasis were studied in pregnant rabbits that were exposed to 13.8 or 46.0 mg/kg THPS during organogenesis and in precision-cut liver slices of rats and rabbits exposed to 0-70 mu M THPS. Results show that in vivo exposure to THPS leads to a marked reduction of food intake, increased plasma concentrations of gamma-glutamytransferase, degenerative changes in the liver and to changes in retinoid content in liver and plasma in the rabbits during organogenesis. In addition, THPS, both in vivo and ex vivo, caused a change in expression of proteins related to vitamin A metabolism and transport. Together, these observations could explain the birth defects observed in earlier teratogenicity studies.

Several decades have passed since the generation of the first embryonic stem cell (ESC) lines both in mice and in humans. Since then, stem cell biologists have tried to understand their potential biological and clinical uses for their implementation in regenerative medicine. The hematopoietic field was a pioneer in establishing the potential use for the development of blood cell products and clinical applications; however, early expectations have been truncated by the difficulty in generating bonafide hematopoietic stem cells (HSCs). Despite some progress in understanding the origin of HSCs during embryonic development, the reproduction of this process in vitro is still not possible, but the knowledge acquired in the embryo is slowly being implemented for mouse and human pluripotent stem cells (PSCs). In contrast, ESC-derived hematopoietic cells may recapitulate some leukemic transformation processes when exposed to oncogenic drivers. This would be especially useful to model prenatal leukemia development or other leukemia-predisposing syndromes, which are difficult to study. In this review, we will review the state of the art of the use of PSCs as a model for hematopoietic and leukemia development. This work was funded by grants from PID2019-104695RB-I00 and PDC2021-120817-I00from AgenciaEstatal de Investigación (AEI) to AB, ERA PerMed GATA2-HuMo Funding Mechanism (Spain: Acció instrumental de SLT011/18/00006 and SLT011/18/00007 of the Department of Health of the Government of Catalonia to AG and A, the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO PID2020-15591RB-100), La Marató de TV3 (202001-32) and CERCA Programme/Generalitat de Catalunya for institutional support to AG. LGP is a recipient of 2021 FI_B2 00188, AGAUR, Generalitat de Cataluña.

Plastic pollution in our environment is one of the most important global health concerns right now. Micro- and nanoplastics (MNPs) are taken up by both humans and animals, mainly via food and water, and can pass important epithelial barriers. Indications of plastics in the blood circulation have recently been shown in both humans and farm animals, but standardized methods to quantify the exact levels of MNPs to which we are exposed are currently lacking. Potential hazards of MNPs are being investigated very recently, including the impact that MNPs may have on reproduction. However, studies on mammalian reproduction are scarce, but a wealth of data from aquatic species indicates reproductive effects of MNPs. The first studies in rodent models demonstrate that MNPs reach the gonads after oral exposure and may impact offspring after maternal exposure during the gestational period. These effects may arise from the particles themselves or the presence of plastic contaminants that leach from plastics. Plastic contamination has been detected in human placentas, fetal fluid and the meconium of newborns, indicating the presence of plastics from the very first start of life. Currently there is a lack of studies that investigate the impact of MNP exposure during the periconception and embryonic period, whereas this is an extremely sensitive period that needs considerable attention with the growing amount of plastics in our environment.

Studies on the germination and establishment of plants are key pieces to understanding the reproductive success of plants. This work aimed to describe in vitro germination and reserve mobilization in the bromeliad Vriesea friburgensis through morphological, histochemical, and biochemical analysis. The conditions used in this study for the in vitro germination are adequate. From the third day of in vitro inoculation, a uniform germination of 98% was obtained, exhibiting a high physiological quality of the seeds and a high potential to produce seedlings (94%). There is early reserve mobilization, which began in the imbibition phase. The accumulated reserves in the endosperm cytoplasm are degraded by hydrolytic enzymes provided by the aleurone layer. It is possible that compounds in the cell walls of the endosperm contribute to a lesser extent in mobilization. Additionally, it was observed that starch accumulation in the cotyledon increases when the seedling has formed. Results from this study provide insights for future studies on ecology, seed technology, and conservation in this species. This study contributes to the limited knowledge of the dynamics of reserves during germination and seedling establishment in Bromeliaceae. To the best of our knowledge, this is the first study with this approach in the genus Vriesea. Resumo Estudos sobre germinação e estabelecimento de plantas são peças-chave para entender o sucesso reprodutivo das plantas. Este trabalho teve como objetivo descrever a germinação in vitro e a mobilização de reservas na bromélia Vriesea friburgensis por meio de análises morfológicas, histoquímicas e bioquímicas. As condições utilizadas neste estudo para a germinação in vitro são adequadas. A partir do terceiro dia de inoculação in vitro, obteve-se germinação uniforme de 98%, apresentando alta qualidade fisiológica das sementes e alto potencial de produção de plântulas (94%). Há uma mobilização precoce de reservas, iniciada na fase de embebição. As reservas acumuladas no citoplasma do endosperma são degradadas por enzimas hidrolíticas fornecidas pela camada de aleurona. É possível que compostos nas paredes celulares do endosperma contribuam em menor grau na mobilização. Além disso, observou-se que o acúmulo de amido no cotilédone aumenta com a formação da plântula. Os resultados deste estudo fornecem informações para estudos futuros sobre ecologia, tecnologia de sementes e conservação desta espécie. Este estudo contribui para o conhecimento limitado da dinâmica das reservas durante a germinação e estabelecimento de plântulas em Bromeliaceae. Até onde sabemos, este é o primeiro estudo com esta abordagem no gênero Vriesea.

Satureja pilosa Velen., S. coerulea Janka and S. kitaibelii Wierzb. ex Heuff. are Balkan endemic species, and they are distributed in restricted territories, primarily found in dry grasslands, rocky slopes, and stony habitats. This study presents the results of the first embryological and micromorphological analyses of three Satureja species (S. pilosa, S. kitaibelii, and S. coerulea) from the Bulgarian flora. The aim of this study was to establish the features of the male and female reproductive sphere, as well as surface characteristics of leaves, stem, and calyx in order to understand the mode of reproduction, character, size and state of species populations and delimitation. For the embryological study, flowers and flower buds in different developmental stages were collected from plants of natural populations and treated with the classic paraffin method. Reproductive capacity was assessed using the following approaches: (1) acetocarmine test for pollen viability, (2) tetrazolium test (TTZ) for seed viability, and (3) germination test. The surfaces characteristics of leaves, stem, and calyx were analyzed by scanning electron microscopy (SEM). As a result, the study revealed the structures of the male (M) and female (F) generative spheres as well as the processes of gamete development, pollination, and endosperm and embryo formation. It was found that the three Satureja species exhibited a high pollen viability but low seed viability and germination. The SEM analysis showed both common and distinct micromorphology features regarding epidermis, calyx and stem surfaces among the three Satureja species. Notably, the S. coerulea surfaces (leaves, calyx, stem) were clearly distinguishable from the other two Satureja species. Regarding the nutlet surfaces, it was observed that the exocarp surfaces of S. coerulea and S. kitaibelii have a reticulate convex type surface and comprise two types of cells: (1) highly bulging, bubble-like cells; and (2) flat cells with numerous striations. On the other hand, the exocarp of S. pilosa displayed rectangular or polygonal shapes without bubble-like cells, and it had a tabular to slightly convex type surface. Additionally, nutlets (seeds) of both S. coerulea and S. pilosa exhibited distinct papilla formations resembling non-glandular trichomes seen on the ends of the nutlets for S. coerulea and over the entire surface for S. pilosa. The characteristics of the embryological structures and processes, along with the absence of apomixis, characterized the three studied Satureja species as sexually reproducing. The established balanced processes and stable structures contribute to their high reproductive potential and population stability. However, these traits may also decrease their adaptability to environmental changes.

Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the vitelline membrane (VM). Here we report that the chicken VM characteristically downregulates tension and stiffness to facilitate stage-specific embryo morphogenesis. Experimental relaxation of the VM early in development impairs blastoderm expansion, while maintaining VM tension in later stages resists the convergence of the posterior body causing stalled elongation, failure of neural tube closure, and axis rupture. Biochemical and structural analysis shows that VM weakening is associated with the reduction of outer-layer glycoprotein fibers, which is caused by an increasing albumen pH due to CO2 release from the egg. Our results identify a previously unrecognized potential cause of body axis defects through mis-regulation of extraembryonic tissue tension.

Recent years have seen exciting progress across human embryo research, including new methods for culturing embryos, transcriptional profiling of embryogenesis and gastrulation, mapping lineage trajectories, and experimenting on stem cell-based embryo models. These advances are beginning to define the dynamical principles of development across stages, tissues and organs, enabling a better understanding of human development before birth in health and disease, and potentially leading to improved treatments for infertility and developmental disorders. However, there are still significant roadblocks en route to this goal. Here, we highlight technical challenges to studying early human development and propose ways and means to overcome some of these constraints.

The detrimental effects of endometriosis on IVF success are well known, but the underlying mechanisms are still uncertain. The objective of this study was to investigate the possible detrimental effects of the disease on ovarian response and embryo development.IVF cycles performed in the authors' unit between 2014 and 2020 were retrospectively reviewed. Women with a good ovarian reserve who underwent their first IVF cycle were included. A total of 248 women with endometriosis were identified and matched to 248 women without the disease, according to age, type of stimulation, anti-Müllerian hormone concentration and study period. The primary outcome was the number of women without good-quality embryos. Secondary outcomes included the rate of unexpected poor response (retrieval of ≤3 oocytes), the cumulative clinical pregnancy rate and the cumulative live birth rate.The number of women without good-quality embryos did not differ between women with endometriosis (exposed group) and those without it (unexposed group). The adjusted odds ratio was 0.85 (95% CI 0.51-1.44, P = 0.56). The clinical pregnancy rate and the live birth rate were also similar. Conversely, the rate of unexpected poor response was higher among women with endometriosis (23% versus 13%, P = 0.005).Overall, endometriosis does not markedly affect folliculogenesis and embryo development in the context of IVF. Only an increased risk of unexpected poor response emerged.

Retinoic acid (RA) is a lipid-soluble bioactive substance, which is not only an essential factor in organogenesis, but is also involved in the regulation of cell functions in adult tissues. RA is synthesized from retinoids through oxidation processes, in which the retinaldehyde dehydrogenase (RALDH) family converts from retinal to RA. We previously reported that RALDH2 and RALDH3 were expressed in the developing anterior pituitary gland of rats. However, these RALDH-expressing cells have not been characterized. The present study aimed to clarify the spatiotemporal relationship between mRNA expressions of RALDHs and pituitaryspecific transcription factors (Prop1 and Pit-1) in the pituitary primordium of rat embryos. By in situ hybridization using digoxigenin-labeled cRNA probes, we successfully detected mRNA expressions for RALDH2, RALDH3, Prop1, and Pit-1. The results showed that RALDH2 and RALDH3 were expressed specifically in Rathke’s pouch on embryonic day 11.5 (e11.5), and were subsequently distributed in the pituitary primordium. On the other hand, the expression of Prop1 was observed from e12.5, and then was decreased after e15.5. Pit-1 mRNA was first detected on e15.5, and became distributed throughout the entire pituitary primordium during development. Moreover, Pit-1-positive cells expressed RALDH2 and RALDH3 in the pituitary primordium on e17.5. These results suggest that stem/progenitor cells of the anterior pituitary gland express RALDH2 and RALDH3. RA may act in an autocrine and/or paracrine manner to induce hormone-producing cells in the pituitary., 原著 2021年度神奈川大学総合理学研究所共同研究助成論文

4 Pág., Ungulate embryos undergo critical cell differentiation and proliferation events around and after blastocyst hatching. Failures in these processes lead to early pregnancy losses, which generate an important economic impact on farming. Conventional embryo culture media, such as SOF, are unable to support embryo development beyond hatching. In contrast, N2B27 medium supports early post-hatching development, evidencing a swift in embryonic nutritional requirements during this developmental window. Here, we investigate if earlier exposure to N2B27 could improve embryo development after hatching. Embryo culture in N2B27 from day (D) 5, 6 or 7 significantly enhanced complete hypoblast migration (>45 vs. ∼24%) and epiblast development into an embryonic disc (ED)-like structure at D12 (>40 vs. 23%), compared to embryos cultured in SOF up to D9. Culture in N2B27 from D5 significantly increased epiblast and hypoblast cell number in D8 blastocysts, but post-hatching embryos cultured in N2B27 from D5 or 6 frequently showed a disorganized distribution of epiblast cells. In conclusion, bovine embryo culture in N2B27 from D7 onwards improves subsequent post-hatching development. This improved fully in vitro system will be very useful to functionally explore cell differentiation mechanisms and the bases of early pregnancy failures without requiring animal experimentation., This work has been funded by the projects StG-757886-ELONGAN from the European Research Council and PID2020-117501RB-I00 and ECQ2018-005184-P from the Spanish Ministry of Science and Innovation to PBA, and by the project PID2021-122153NA-I00 from the Spanish Ministry of Science and Innovation to PRI.

International audience; [No abstract available]

Background: The CREB1 gene encodes the cAMP response element binding protein 1 (CREB1), a leucine zipper transcription factor that regulates cellular gene expression in response to elevated levels of intracellular cAMP. When activated by phosphorylation, CREB1 binds to the cAMP response element (CRE) of the promoters of its target genes. CREB1 is an essential component in many physiological processes, and its function is correlated to neurodevelopment, plasticity and cell survival, and learning and memory. The NFATC2 gene codes for the nuclear factor of activated T-cells 2 protein. The NFATC2 protein is a DNA-binding protein that functions as an inducer of gene transcription during immune response. Methods and results: The aim of the present study was to examine the developmental expression of porcine CREB1 and NFACT2 transcripts. The expression of CREB1 and NFACT2 mRNA was examined by quantitative real-time RT-PCR. For the CREB1 transcript, we found significant reduction in transcript levels in the brain stem and basal ganglia during porcine embryo development, determined from day 60 to day 115 of gestation. In contrast, a significant increase in CREB1 mRNA was detected in the lungs during embryo development. No significant changes in the NFATC2 transcript were detected in porcine brain tissue during embryo development. Conclusions: Differential CREB1 mRNA expression was found in pig brain tissues during embryo development.

In vitro systems like those used for in vitro embryo production are invaluable for our understanding of embryogenesis and the processes that regulate it. However, extensive research has also highlighted that in vitro produced embryos negatively differ from their in vivo counterparts in various ways. Not surprisingly, there is ~20% decrease in pregnancy success from pregnancies established using in vitro produced embryos. Therefore, much research has relied on attempting to produce a better in vitro embryo that more closely resembles their in vivo counterparts. Our laboratory has investigated this by supplementing a cytokine, interleukin-6 (IL6), during in vitro embryo culture. My dissertation work expands upon those initial efforts by answering more detailed questions related to the biological role of IL6 during cattle embryogenesis. In the work presented herein, IL6 supplementation during in vitro culture was able to transform the transcriptome of resulting conceptuses post embryo transfer. The transcriptome of these conceptuses included an abundance of genes associated with survival. Indeed, we witnessed IL6-treated conceptuses resulted in a 20% increased survival rate and were longer than their non-treated counterparts. In the second research project, we employed CRISPR-Cas9 genome editing technology to understand the embryo phenotype after part of the IL6 receptor responsible for signal transduction, interleukin-6 signal transducer (IL6ST), is disrupted. We discovered that IL6ST is required for development before the blastocyst stage. In addition, IL6ST disrupted blastocysts, presumed to contain wildtype, presented with severe, abnormal morphology. Not only did this group of embryos have decreased ICM and TE cell numbers, but they also had an increased occurrence of cells within the TE region that were negative for its traditional marker, CDX2. This suggests IL6ST is likely involved in a pathway responsible for determining cell fate identity at the blastocyst stage. Collectively, IL6 in cooperation with IL6ST, is a key controller of embryogenesis in cattle. Doctor of Philosophy There are major events that an embryo must successfully advance from to continue development to form into an organism capable of survival after birth. Over 30% of pregnancies in cattle and humans will fail within the first 30 days of gestation. This time period coincides with several key developmental events that ultimately modify the morphology of the growing embryo. Our laboratory primarily focuses on embryo development around the blastocyst stage. If an embryo advances to this stage, it has a greater likelihood of maintaining viability. Therefore, my dissertation research has focused on early embryonic development from the time of first cleavage (~day 2 of gestation) through embryo elongation (~day 15 of gestation), which encompasses the blastocyst stage. Within this time frame, I have been investigating embryonic effects after supplementation of a protein, interleukin-6 (IL6). Previously, our laboratory has identified IL6 to cause favorable impacts on the developing embryo, but its mode of action was unknown. Therefore, my dissertation research has investigated the mechanistic actions of IL6, and its beta receptor subunit, interleukin-6 signal transducer (IL6ST). In my first research project, we discovered that supplementing IL6 during in vitro embryo culture resulted in increased embryo elongation and survival. In my second research project, we found IL6ST is an absolute requirement for embryo survival to the blastocyst stage. Together, these results indicate IL6 is a very important protein needed for sustained pregnancy viability.

Vanillic acid (VA) has shown antioxidant and anti-inflammatory activities in different cell types, but its biological effects in the context of early embryo development have not yet been clarified. In the current study, the impact of VA supplementation during in vitro maturation (IVM) and/or post-fertilization (in vitro culture; IVC) on redox homeostasis, mitochondrial function, AKT signaling, developmental competence, and the quality of bovine pre-implantation embryos was investigated. The results showed that dual exposure to VA during IVM and late embryo culture (IVC3) significantly improved the blastocyst development rate, reduced oxidative stress, and promoted fatty acid oxidation as well as mitochondrial activity. Additionally, the total numbers of cells and trophectoderm cells per blastocyst were higher in the VA-treated group compared to control (p < 0.05). The RT-qPCR results showed down-regulation of the mRNA of the apoptosis-specific markers and up-regulation of AKT2 and the redox homeostasis-related gene TXN in the treated group. Additionally, the immunofluorescence analysis showed high levels of pAKT-Ser473 and the fatty acid metabolism marker CPT1A in embryos developed following VA treatment. In conclusion, the study reports, for the first time, the embryotrophic effects of VA, and the potential linkage to AKT signaling pathway that could be used as an efficacious protocol in assisted reproductive technologies (ART) to improve human fertility.

The chaperone system (CS) canonical function is to maintain protein homeostasis. Proper folding of nascent peptides is crucial in developing tissue. The chief components of the CS are 95th National Congress of the Italian Society for Experimental Biology | Trieste, Italy, 12-15 April 2023Non-commercial use only the molecular chaperones, which play key roles in development as indicated by their presence in embryonic tissue as early as at two-cell stage. However, scarce information on the CS in developing tissues is available, especially at advanced stages of embryogenesis and its role is not fully understood. In our previous study, we reported the presence of molecular chaperones in the ducts and acini of human adult salivary glands. Here, we extend our work and report the distribution pattern of the molecular chaperones Hsp10, Hsp27, Hsp60, and Hsp90 in adult mice submandibular glands (SMG) and in human and mice embryos at late stages of development. We also report tissue levels and expression of these molecular chaperones determined by means of immunohistochemistry, Western blot, and RT-PCR. The four molecular chaperones were present in salivary gland tissue with Hsp90 levels being the highest at both stages, embryonic and adult. The data indicate that the CS is active throughout the ontogeny of salivary glands, with time-course changes in levels and distribution that reflect a dynamic functional involvement specific for each developmental step. The implications of these findings for understanding the pathology of salivary glands with roots in abnormal development are worth exploring, and the data presented here should serve as a basis for such studies and, consequently, boost the invention of novel diagnostic and treatment tools.

4 Pág., This work was financially supported by the National Key Research and Development Program of China (Grant No. 2021YFD1200403).

Normalizing RT-qPCR miRNA datasets that encompass numerous preimplantation embryo stages requires the identification of miRNAs that may be used as stable reference genes. A need has also arisen for the normalization of the accompanying conditioned culture media as extracellular miRNAs may serve as biomarkers of embryo developmental competence. Here, we evaluate the stability of six commonly used miRNA normalization candidates, as well as small nuclear U6, using five different means of evaluation (BestKeeper, NormFinder, geNorm, the comparative Delta Ct method and RefFinder comprehensive analysis) to assess their stability throughout murine preimplantation embryo development from the oocyte to the late blastocyst stages, both in whole embryos and the associated conditioned culture media. In descending order of effectiveness, miR-16, miR-191 and miR-106 were identified as the most stable individual reference miRNAs for developing whole CD1 murine preimplantation embryos, while miR-16, miR-106 and miR-103 were ideal for the conditioned culture media. Notably, the widely used U6 reference was among the least appropriate for normalizing both whole embryo and conditioned media miRNA datasets. Incorporating multiple reference miRNAs into the normalization basis via a geometric mean was deemed beneficial, and combinations of each set of stable miRNAs are further recommended, pending validation on a per experiment basis.

In ovo administration as a possible alternative method of 6/85 MG vaccination was assessed. After 18 days of incubation (doi), the eggs were administered a particular dosage of a live attenuated 6/85 MG vaccine in either the air cell (AC) or amnion (AM). The treatments included non-injected eggs and eggs injected into the AC or AM with diluent alone as controls. Treatments also included eggs injected with diluent, which contained 1.73 × 102, or 1.73 × 104 CFU of 6/85 MG. Hatchability of viable injected eggs (HI) and residual embryonic mortality were determined at 22 doi. At hatch and at three weeks posthatch, one hatched chick per treatment replicate was bled and swabbed for the detection of 6/85 MG in the choanal cleft using PCR, serum plate agglutination (SPA), and ELISA methods. The results show that AC in ovo injection of 6/85 MG had no negative impacts on HI or on the live performance of pullets, but that it failed to provide adequate protection (p ≤ 0.0001) in hatchlings or three-week-old pullets. The 1.73 × 104 6/85 MG CFU dosage injected into the AM decreased the hatchability of injected eggs containing viable embryos (HI; p = 0.009) and was associated with a significant increase in late dead mortality (p = 0.001). Hatchling and three-week-old chick mortalities (p = 0.008) were significantly greater in the 1.73 × 104 CFU-AM treatment group in comparison with the other treatment groups. In addition, the 1.73 and 1.73 × 102 6/85 MG-AM treatments had no negative effects on the hatching process or on posthatch growth, and the 1.73 × 102 6/85 MG-AM treatment was more effective in the protection of pullets against MG (p ≤ 0.0001) as compared with the low dosage and non-injected treatment groups. Further research is needed to examine the influence of the 6/85 MG in ovo vaccine on layer immune competence.

(1) Background: Semen analysis is a poor predictor of the fertilization potential of spermatozoa and a male factor may contribute to poor outcomes of the IVF procedure, despite a normal semen analysis. The microfluidic sperm selection (ZyMot-ICSI) is based on the selection of the spermatozoa with the lowest DNA fragmentation rate, but studies do not prove better clinical outcomes after this method. (2) Methods: We conducted a retrospective trial comparing 119 couples that were allocated to the classic gradient centrifugation sperm method (control group), and 120 couples that were allocated with the microfluidic technique being used (study group) at our university-level clinic, to go through IVF. (3) Results: The statistical analysis showed that there is no significant difference between the fertilization rate (study vs. control p = 0.87), but regarding blastocyst rate (study vs. control p = 0.046) and clinical pregnancy (p = 0.049), there is quite a significant statistical difference. Microfluidic preparation of spermatozoa seems to improve the results and it may be utilized more broadly for ICSI, and could also improve the workflow in standard IVF, decrease intervention by laboratory personnel and provide more consistent incubation conditions. (4) Conclusions: The patients that had the sperm preparation for ICSI with the microfluidic sperm selection had slightly better results compared with the gradient centrifugation selection.

Resumen El presente trabajo analiza los sentidos que las usuarias de TRHA construyen sobre los embriones y fetos de forma situada. Desde un método cualitativo y un enfoque biográfico, se exploran las maneras en que las usuarias dan sentido y subjetivan a los embriones en diferentes momentos: durante la gestación, ante la pérdida de un embarazo, como frente a las tecnologías de visualización y de criopreservación, dando cuenta de diferentes interpretaciones y resignificaciones. Situado en la Provincia de Córdoba, estas experiencias permiten dar cuenta de los sentidos locales de lo reproductivo y comprender las controversias que afrontan las usuarias un campo biomédico poco regulado en Argentina. Resumo Este documento analisa os significados que as usuárias de TRHA constroem sobre embriões e fetos de uma forma situada. Utilizando um método qualitativo e uma abordagem biográfica, exploramos as formas como as usuárias dão sentido e subjetividade aos embriões em diferentes momentos: durante a gestação, face à perda de gravidez, e face às tecnologias de visualização e criopreservação, dando conta de diferentes interpretações e ressignificações. Situadas na província de Córdoba, estas experiências nos permitem dar conta dos significados locais das questões reprodutivas e compreender as controvérsias enfrentadas pelos utilizadores de um campo biomédico mal regulado na Argentina. Abstract The present work analyzes the construction-meaning process in ART users about embryos and fetuses in a situated way. From a qualitative method and a biographical approach, it explores the ways that ART users construct and subject ART embryos at different times: during gestation, in the face of pregnancy loss, as well as in front of visualization and cryopreservation technologies, accounting for different interpretations and resignifications. Located in the Province of Córdoba, these experiences allow us to account for the local meanings of reproductive issues and to understand the controversies faced by users of a biomedical field that is not very regulated in Argentina.

The clinical use of mifepristone for medical abortions has been established in 1987 in France and since 2000 in the United States. Mifepristone has a limited medical period that lasts

Background: in 2020, SARS-CoV-2 and the COVID-19 pandemic had a huge impact on the access to and provision of ART treatments. Gradually, knowledge of the virus and its transmission has become available, allowing ART activities to resume. Still, questions on the impact of the virus on human gametes and fertility remain. Onjective and rationale: This article summarizes published data, aiming to clarify the impact of SARS-CoV-2 and the COVID-19 disease on human fertility and assisted reproduction, as well as the impact of vaccination, and from this, provide answers to questions that are relevant for people contemplating pregnancy and for health care professionals. Search/methods: PUBMED/MEDLINE and the WHO COVID-19 database were searched from inception to 5 October 2022 with search terms focusing on 'SARS-CoV-2' and gametes, embryos, reproductive function, fertility and ART. Non-English studies and papers published prior to 2020 were excluded, as well as reviews and non-peer reviewed publications. Full papers were assessed for relevance and quality, where feasible. Outcomes: from the 148 papers included, the following observations were made. The SARS-CoV-2-binding proteins, angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2), are expressed in the testis, but co-expression remains to be proven. There is some evidence of SARS-CoV-2 RNA in the ejaculate of COVID-19 patients with severe disease, but not in those with mild/moderate disease. SARS-CoV-2 infection can impair spermatogenesis, but this seems to resolve after one spermatogenic cycle. Testosterone levels seem to be lower during and after COVID-19, but long-term data are lacking; disease severity may be associated with testosterone levels. COVID-19 cannot be considered a sexually transmitted disease. There is no co-expression of ACE2 and TMPRSS2 in the myometrium, uterus, ovaries or fallopian tubes. Oocytes seem to have the receptors and protease machinery to be susceptible to SARS-CoV-2 infection; however, viral RNA in oocytes has not been detected so far. Women contemplating pregnancy following COVID-19 may benefit from screening for thyroid dysfunction. There is a possible (transient) impact of COVID-19 on menstrual patterns. Embryos, and particularly late blastocysts, seem to have the machinery to be susceptible to SARS-CoV-2 infection. Most studies have not reported a significant impact of COVID-19 on ovarian reserve, ovarian function or follicular fluid parameters. Previous asymptomatic or mild SARS-CoV-2 infection in females does not seem to negatively affect laboratory and clinical outcomes of ART. There are no data on the minimum required interval, if any, between COVID-19 recovery and ART. There is no evidence of a negative effect of SARS-CoV-2 vaccination on semen parameters or spermatogenesis, ovarian function, ovarian reserve or folliculogenesis. A transient effect on the menstrual cycle has been documented. Despite concerns, cross reactivity between anti-SARS-CoV-2 spike protein antibodies and Syncytin-1, an essential protein in human implantation, is absent. There is no influence of mRNA SARS-CoV-2 vaccine on patients' performance during their immediate subsequent ART cycle. Pregnancy rates post-vaccination are similar to those in unvaccinated patients. Wider implication: this review highlights existing knowledge on the impact of SARS-CoV-2 infection or COVID-19 on fertility and assisted reproduction, but also identifies gaps and offers suggestions for future research. The knowledge presented should help to provide evidence-based advice for practitioners and couples contemplating pregnancy alike, facilitating informed decision-making in an environment of significant emotional turmoil., There was no funding for the current paper, apart from technical support from ESHRE.

Histone variant H3.3 is encoded by two genes, H3f3a and H3f3b, which can be expressed differentially depending on tissue type. Previous work in our lab has shown that knockout of H3f3b causes some neonatal lethality and infertility in mice, and chromosomal defects in mouse embryonic fibroblasts (MEFs). Studies of H3f3a and H3f3b null mice by others have produced generally similar phenotypes to what we found in our H3f3b nulls, but the relative impacts of the loss of either H3f3a or H3f3b have varied depending on the approach and genetic background. Here we used a knockout-first approach to target the H3f3a gene for inactivation in C57BL6 mice. Homozygous H3f3a targeting produced a lethal phenotype at or before birth. E13.5 null embryos had some potential morphological differences from WT littermates including smaller size and reduced head size. An E18.5 null embryowas smaller than its control littermates with several potential defects including small head and brain size as well as small lungs, which would be consistent with a late gestation lethal phenotype. Despite a reduction in H3.3 and total H3 protein levels, the only histone H3 post-translational modification in the small panel assessed that was significantly altered was the unique H3.3 mark phospho-Serine31, which was consistently increased in null neurospheres. H3f3a null neurospheres also exhibited consistent gene expression changes including in protocadherins. Overall, our findings are consistent with the model that there are differential, cell-type-specific contributions of H3f3a and H3f3b to H3.3 functions in epigenetic and developmental processes.

Septate junctions (SJs) perform an occluding function in invertebrate epithelia and consist of parallel septa extending across the intercellular space between neighbouring cells. In addition, they are required for several morphogenetic processes in arthropods. The biogenesis of SJs during development is inadequately studied and it was characterised in detail only for various epithelia of Drosophila melanogaster. This paper provides a detailed analysis of the ultrastructural differentiation of SJs in the epidermis of the terrestrial isopod Porcellio scaber during embryonic and postembryonic development. In this study, mid-stage embryo S13 was the earliest stage in which single septa were observed basally to the adherens junction (AJ). Differentiation of SJs during further development includes gradual elongation of septa arrays and formation of continuous arrays of septa. The enlargement of SJs in the epidermis is most pronounced at the transition from embryonic to postembryonic development and after the release of mancae from the marsupium. SJs of postmarsupial mancae are similar to those of adults, but are not yet as extensive. Comparison of the differentiation of SJs in the epidermis and hindgut of P. scaber, reveals a similar sequence of events. In addition, remodelling of SJs was observed in the epidermis of late marsupial mancae, the stage of cuticle renewal. Common features of SJs’ biogenesis in P. scaber and D. melanogaster ectodermal epithelia are indicated.

The objective of this work was to identify and describe the morphological aspects of the pyrene and the different stages of developing embryos of Ilex paraguariensis. The pyrenes were collected in the year 2016 and 2017, of ripe fruits. The pyrenes, a total of 400 per site and year of collection, remained for 24 h immersed in water for the section and internal observation. The pyrenes, with the visualized embryo, were used for the anatomical description and fixed for the histological evaluation. They were fixed in paraformaldehyde solution and included in Leica™ historesin. The samples were sectioned in Leica microtome with a thickness of 7 μm, and the sections were stained with Toluidine Blue. The pyrenes are formed by endocarp, integument, endosperm, and embryo. The embryos were classified in the globular, heart (occupying approximately 10% of the length of the seed), post heart (15% of length), torpedo (20% of length), and mature (40% of length) stages; and were located below the micropyle, aligned to the external point. The region surrounding the embryo, called the cavity, consists of a lignified layer.

Failure of embryo implantation has been introduced as an important limiting parameter in early assisted reproduction and pregnancy. The embryo-maternal interactions, endometrial receptivity, and detections of implantation consist of the embryo viability. For regulating the implantation, multiple molecules may be consistent; however, their specific regulatory mechanisms still stand unclear. MicroRNAs (miRNAs) have attracted a lot of attention due to their important effect on human embryo implantation. MicroRNA (miRNA), which acts as the transcriptional regulator of gene expression, is consisted of embryo implantation. Recent studies indicated that miRNAs not only act inside the cells but also can be secreted by cells into the extracellular environment via multiple packaging forms, facilitating intercellular communication and providing indicative information related to various conditions. The detection of extracellular miRNAs provided new information in cases of implantation studies. For embryo-maternal communication, MiRNAs offered novel approaches. In addition, in assisted reproduction, for embryo choice and prediction of endometrial receptivity, they can act as non-invasive biomarkers and can enhance the accuracy in the process of reducing the mechanical damage for the tissue.

Objective: The objective of this study was to investigate the effects of different degrees of maternal dietary energy restriction on lipid deposition in embryonic tissues during the medium laying period (37 to 39 weeks) in Arbor Acres (AA) broiler breeders.Methods: A single factor design was adopted, and 400 AA broiler breeders (20 weeks of age) with a similar weight were randomly allocated into four groups. The birds in the control group were fed a corn-soybean meal based diet, and those in trial groups were fed diets with 80%, 70%, and 50% energy levels of the basal diet. Incubated eggs from the medium laying period were collected. Samples of developing embryos at various stages were prepared for composition analysis.Results: The embryo weight in the 80% energy group was higher than those of the other groups on embryonic day (E) 13, but at 21 E, they were significantly decreased with decreasing energy intake of the broiler breeders (p

FUSCA3 (FUS3) is a member of B3-domain transcription factor family and master regulator of seed development. It has potential roles in hormone biosynthesis and signaling pathways and therefore plays diverse roles in plant life cycle, especially in seed germination, dormancy, embryo formation, seed and fruit development, and maturation. However, there is limited information about its functions in seed and fruit development of grapevine. In this study, we expressed VvFUS3 in tomato for its functional characterization. Overexpression of VvFUS3 in tomato led to a reduction in seed number and seed weight without affecting the fruit size. Histological analysis found that both cell expansion and cell division in transgenic seed and fruit pericarp have been affected. However, there were no obvious differences in pollen size, shape, and viability, suggesting that VvFUS3 affects seed development but not the pollen grains. Moreover, the expression of several genes with presumed roles in seed development and hormone signaling pathways was also influenced by VvFUS3. These results suggest that VvFUS3 is involved in hormonal signaling pathways that regulate seed number and size. In conclusion, our study provides novel preliminary information about the pivotal roles of VvFUS3 in seed and fruit development and these findings can potentially serve as a reference for molecular breeding of seedless grapes.

Suitable reference genes are one of the necessary conditions for obtaining reliable results by real-time fluorescence quantitative PCR (qPCR). In this study, the expression of the 10 common candidate reference genes (18s rRNA, rps27, cnbp, rpl7, ube2, hsp-at, gapdh, β-actin, rpl13a, 1-ef1a) at different developmental stages of gonad and embryo of Takifugu bimaculatus were analyzed by qPCR. And the expression stability of these reference genes is analyzed by GeNorm, NormFinder and Bestkeeper softwares. The results showed that the expression stability of 1-ef1a was the highest (p

What is the reproductive potential of embryos that achieve blastulation on day 7 followed by preimplantation genetic testing for aneuploidies (PGT-A) for infertility patients with slow embryo development?This was a retrospective cohort study in a private IVF clinic of consecutive female infertility patients (n = 2966) aged 24-48 (36.3 ± 3.8) years who underwent frozen embryo transfer (FET) of a single euploid blastocyst.The women underwent single euploid FET of an embryo that achieved blastulation on day 5 (n = 1880), day 6 (n = 986) or day 7 (n = 100). Day 7 embryos resulted in lower implantation and live birth rates compared with both day 5 and day 6 embryos (P 0.001). The day 5, day 6 and day 7 groups had 68.5%, 55.2% and 36.0% live birth rates, respectively. The day 7 group was older than the day 5 group (P 0.001); comparing age-matched cohorts, the day 7 group still had lower implantation and live birth rates (P 0.0001 and P 0.001, respectively). Embryo grade was unrelated to live birth rates. Day 7 embryos of expansion grade 5 or 6 or trophectoderm grade A were more likely to be euploid compared with expansion grade 3 or trophectoderm grade B.Euploid day 7 embryos represented reduced implantation potential, even when controlling for maternal age. Of all day 7 embryos that underwent PGT-A, euploidy was associated with expansion grade 5 or 6 and trophectoderm grade A. These results can help providers manage patient expectations in cases where infertile women have slow embryo development.

In many but not all recalcitrant Quercus seeds, water loss during drying only occurs through the scar. Consequently, the embryo axis sitting on the opposite side of the scar is well protected from desiccation. However, whether such a mechanism is common throughout Fagaceae species is unexplored. Similarly, little is known about the desiccation response of other Fagaceae genera. Germination and desiccation tolerance of Castanopsis sclerophylla (Lindl. & Paxton) Schottky (Fagaceae) were studied by drying the seeds with silica gel. Fresh seeds had a moisture content (MC) of 36% and germinated to 92.5% when incubated at 15/20 °C. Drying seeds to 22.5% and 20.7% MC decreased germination to 27% and 5%, respectively, indicating that seeds are recalcitrant. X-ray computed tomography and photomicrographs showed that the embryo axis of C. sclerophylla is located just below the scar. Regardless of drying occurring either through only the scar or the whole pericarp, embryo drying was deemed unavoidable. Nevertheless, germination percentage during drying showed exceptional similarities with the other Fagaceae species.

By allowing for the creation of embryo banks, reproductive biotechnologies play an essential role in the preservation of endangered goat breeds' genetic diversity. This study focused on comparing both available embryo collection methods [laparotomy (LAP) vs. nonsurgical embryo recovery (NSER)] in Canindé goats to create an embryo bank for later use in a breed conservation program. Twelve females were superovulated and subjected to either the LAP or NSER technique for embryo recovery. The recovery rate was similar (

[Synopsis] Fibroblast growth factor 9 (FGF9) plays a key role in the regulation of cell differentiation, cell proliferation and embryonic development. It is also a survival factor for various cell types and interacts with two FGF receptors, FGFR2 and FGFR3. Furthermore, the absence of specific thecal-and oocyte-derived products during in vitro maturation (IVM) is suggested to be one reason for the reductions in the competency of oocytes used in assisted reproductive techniques. FGF9, as with some FGFs shown to promote oocyte competence and embryo development in several species is localize largely in theca cells, somewhat in the oocyte and its receptors in oocytes and cumulus cells. Therefore, to investigate the possible reproductiverelated implications of FGF9 supplementation, its effects on IVM oocytes after parthenogenetic activation (PA) using the pig model were examined. Five approaches were taken to explore the function of FGF9 during in vitro embryo production (IVP). First, the maturation medium was supplemented with exogenous FGF9 to investigate its effects on maturation of GV oocytes measured by the extrusion of the first polar body hence MII oocytes. The results showed that FGF9 significantly accelerated MII rates in 50 ng/ml treated group (p

A massive adaptive radiation on the Hawaiian archipelago has produced approximately one quarter of the fly species in the family Drosophilidae. The Hawaiian Drosophila clade has long been recognized as a model system for the study of both the ecology of island endemics and the evolution of developmental mechanisms, but relatively few genomic and transcriptomic datasets are available for this group. We present here a differential expression analysis of the transcriptional profiles of two highly conserved embryonic stages in the Hawaiian picture-wing fly Drosophila grimshawi. When we compared our results to previously published datasets across the family Drosophilidae, we identified cases of both gains and losses of gene representation in D. grimshawi, including an apparent delay in Hox gene activation. We also found high expression of unannotated genes. Most transcripts of unannotated genes with open reading frames do not have homologs in non-Hawaiian Drosophila species, although the vast majority have sequence matches in other genomes of the Hawaiian picture-wing flies. Some of these genes may have arisen from non-coding sequence in the ancestor of Hawaiian flies or during the evolution of the clade. Our results suggests that both the modified use of ancestral genes and the evolution of new ones may occur in rapid radiations.RESEARCH HIGHLIGHTSThe early embryonic transcriptome of the Hawaiian fly Drosophila grimshawi shows a loss of expression of conserved Stage 5 genes, including the Hox genesThe de novo evolution of embryonically expressed genes may be occurring in the Hawaiian Drosophila lineageAUTHORS’ STATEMENTThis paper is not being considered for publication elsewhere. This study formed part of Madeline Chenevert’s M.S. thesis.

Implantation timing is key for a successful pregnancy. Short delay in embryo implantation caused by targeted gene ablation produced a cascading problem in the later stages of the pregnancy. Although several delayed implantation models have been established in wild mice, almost none of them is suitable for investigating the delay on the late events of pregnancy. Here, we report a new delayed implantation model established by the intraperitoneally administration of letrozole at 5 mg/kg body weight on the day 3 of pregnancy. In these mice, initiation of implantation was induced at will by the injection of estradiol (E2). When the estradiol (3 ng) was injected on day 4 of pregnancy (i.e., without delay), the embryo implantation restarted, and the pregnancy continued normally. However, high dose of estrogen (25 ng) caused compromised implantation. We also found that only 67% of the female mice could be pregnant normally and finally gave birth when the injection of estradiol (3 ng) was on day 5 of pregnancy (i.e., one day delay). Most of the failed pregnancies had impaired decidualization, decreased plasma progesterone levels and compromised angiogenesis. Progesterone supplementation could rescue decidualization failure in the mice. Collectively, we established a new model of delayed implantation by letrozole, which can be easily used to study the effect and mechanisms of delay of embryo implantation on the progression of late pregnancy events.

The growth and development were Studied of the heart in common carp fishes by using several models of larvae and juvenile during artificial fertilization in the Marine Science Center hatchery of fishes. Temperature incubation at 26c°and the incubation period was 38hr.This study included the morphological and histological sides of the heart development, the primordium of the heart rudiment appeared after (22 hrs) of incubation, gradually the heart became more differentiated with the advent of ventricle and atrium in addition to the bulbus arteriosus, the histologically of the heart was appeared at age 24,28 hours from incubation as a small undifferentiated tube, while the ventricle was surrounded by two layers of cells. The atrium was surrounded with a single layer with the differentiate bulbus arteriosus, The heart at one day after hatching consist of the main parts , atrium, ventricle, sinus venous and bulbus arteriosus. Heart valves were appeared at the age of (5 – 8) days after hatching, the heart be completed once at the age of 30 days after hatching with the differentiation of the two layers of the mycardium , compact layer and trabacular layer.

What is the effect on mouse fetal gene expression of combined antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and alpha-lipoic acid; A3) when used in culture media and vitrification/warming solutions?A laboratory-based analysis of an animal model. Embryo transfers were conducted on in-vivo-flushed blastocysts, or blastocysts cultured or vitrified with and without A3. Transcriptional profiles of E14.5 fetal liver and placental tissue in all groups were quantified using RNA-Seq and functional analyses (gene ontology [GO] biological processes and Kyoto Encyclopedia of Genes and Genomes [KEGG] pathway analysis).Both in-vitro culture in the presence of 20% oxygen and vitrification of blastocysts significantly perturbed fetal liver and placental gene expression. Notably, supplementation of in-vitro culture media or vitrification/warming solutions with A3 reduced the number of differentially expressed genes (DEG) and biological processes altered, establishing a more in-vivo-like gene expression profile, particularly within the E14.5 placenta. Specifically, A3 supplementation significantly reduced the expression of genes associated with pre-eclampsia and intrauterine growth restriction, along with genes involved in metabolism, cell senescence and cancer associated pathways. However, despite these improvements, several biological processes remained over-represented following both in-vitro culture and vitrification, even in the presence of A3.Both in-vitro culture in the presence of 20% oxygen and vitrification of blastocysts significantly perturbed fetal liver and placental gene expression, with the number of DEG greater following vitrification. Supplementation with A3 reduced the number of DEG and biological processes altered, establishing a more in-vivo-like gene expression profile, particularly in the placenta. Notably, A3 supplementation of in-vitro culture media significantly reduced the expression of genes associated with pre-eclampsia and intrauterine growth restriction.

Wild species of the genus Cucumis, such as C. anguria, are resistant to various melon (C. melo) diseases such as Fusarium wilt race 1.2 and are anticipated as genetic resources of melon. However, in the interspecific crosses between C. anguria and melon, the abortion of the embryos was observed in the early growth stage and could not be regenerated even when cultured. Therefore, for embryo rescue, it is necessary to promote embryo growth during abortion occurrence. This study investigated the effects of fruit heating on embryo growth in interspecific hybrids of C. anguria and melon. In the heating device, the minimum night temperature around the fruit was maintained above 21.5°C. The ovule grain area of C. anguria × MR-1 with fruit heating was significantly enlarged compared to that without fruit-heating. It was also significantly enlarged compared with the ovule grain area of C. anguria self-pollination. Although, in C. anguria × MR-1 without fruit-heating, no embryo was observed until 10 days after pollination (DAP), and pre-embryos were observed at 13 DAP. Conversely, in C. anguria × MR-1 with fruit-heating, pre-embryos were even observed at 10 DAP. These results suggest that the possibility of embryo or ovule culture regeneration has increased due to embryo growth promotion.

In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.

Objective: The present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius.Methods: Camelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method.Results: The collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitromatured oocytes.Conclusion: In this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.

Intra-follicular oocyte transfer (IFOT) is a promising and innovative technique for in vivo embryo production previously described for equines and bovines. The aim of this study was to assess the feasibility of IFOT in the ovine species. Two preliminary in vivo and in vitro trials were performed to test the optimal procedures and timing for IFOT. In the in vivo trial, follicular growth was monitored with transrectal ultrasonography in ten adult ewes to preliminarily determine the ovulation and ideal timing for IFOT. The in vitro trial assessed i) the optimal inner diameter of the injection needle and ii) the recovery rate and integrity of injected cumulus-oocyte complexes (COCs) after follicle aspiration. For IFOT and embryo collection, five ewes were synchronized by CIDR insertion. Forty hours after CIDR removal, in ewes under sedation and general anesthesia, the ovaries were exposed by laparotomy, and the preovulatory follicle was injected with COCs previously collected from ovaries obtained from an abattoir. At 4 h after surgery, fully recovered ewes were housed in a paddock with a ram of proven fertility. Crayon marking on ram's chest was used to detect mating. Ovulation was assessed 40 h after the transfer of oocytes by transrectal ultrasonography. On day 6 after IFOT, embryo collection was performed by uterine flushing. In the in vitro testing, injection of >5 mm follicles with a 28 G needle loaded with 30 COCs in a 5 μL volume resulted in higher recovery rates and better preservation of COCs integrity. In the in vivo trial, ultrasound scanning revealed that ovulation occurred between 60 and 72 h after CIDR removal in all animals. In one ewe subjected to IFOT, 22/24 oocytes were effectively injected into the preovulatory follicle, but no embryos were collected after flushing. In the remaining four animals, 85/102 oocytes were injected, and six cleaved embryos, 12 morulae and 1 blastocyst were collected, including native embryos. This preliminary investigation indicated that IFOT in ovine species resulted in ovulation, fimbrial capture, tubal transport of heterologous oocytes and in vivo embryo production. Further studies are needed to optimize the embryo recovery rate and develop less invasive techniques for oocyte injection and uterine flushing, such as through a laparoscopic or transcervical approach.

Which of the two mainstream endometrial preparation regimens, assisted natural cycle (NC) or hormone replacement treatment cycle (HRT), help frozen-thawed embryo transfer (FET) cycles after preimplantation genetic testing (PGT) achieve better clinical outcomes?This retrospective analysis included 3400 vitrified-warmed single blastocyst transfer cycles after PGT from January 2011 to November 2020, and involved 2332 patients with regular menstrual cycles. The decision to proceed with an assisted NC (n = 827) or HRT (n = 2573) before FET was reached based on a combination of patient preference and physician guidance. Clinical pregnancy rate, live birth rate, early miscarriage rate and obstetric outcomes were compared.No significant difference was observed between the assisted NC and HRT groups in terms of clinical pregnancy rate (51.6% versus 50.7%, P = 0.634), live birth rate (44.0% versus 43.4%, P = 0.746) or early miscarriage rate (12.6% versus 12.0%, P = 0.707). Multivariate analysis indicated that the endometrial preparation protocol was not an independent factor for a clinical pregnancy or live birth. In the HRT group, the Caesarean section rate (64.7% versus 51.9%, P 0.001) and pregnancy complication rate (20.2% versus 13.8%, P = 0.003) were significantly higher. The two groups were not statistically different with respect to gestational age, early preterm birth rate, fetal weight or fetal birth defect rate.For patients undergoing a PGT-FET cycle involving a single blastocyst transfer, using assisted NC and HRT for the endometrial preparation could lead to comparable rates of clinical pregnancy and live birth. Additionally, NC is safer than HRT in terms of avoiding pregnancy complications and adverse obstetric outcomes.

The embryo in maize has a critical role in controlling kernel nutrition components and grain yield. We measured five embryo weight and size traits, six kernel weight and size traits, and five embryo-to-kernel ratio traits in a nested association mapping (NAM) population of 611 recombinant inbred lines (RILs) derived from four inbred lines including the high-oil, giant-embryo line BY815 as the common parent. Using three statistical methods, we identified 5–22 quantitative trait loci (QTL) for each trait, explaining 4.7%–46.7% of the phenotypic variation. The genetic architecture of maize embryo size and its related traits appeared to be dominated by multiple small-effect loci with little epistasis, and the genetic control underlying embryo size appeared to be distinct from that underlying kernel size. A trait–QTL association network included 205 nodes and 439 edges and revealed 28 key loci associated with at least three traits. Cloned maize genes including ZmUrb2, Emp12 and ZmBAM1d, maize orthologs of known rice genes that control seed size including BG1, XIAO and GS9, and 11 maize orthologs of Arabidopsis EMBRYO-DEFECTIVE (EMB) genes were identified as underlying these key loci. Further, the phenotypic and genetic relationships between embryo size and kernel size were evaluated, and genetic patterns for identified loci that control embryo size and its related traits were proposed. Our findings reveal distinct genetic architectures for embryo size, kernel size, and embryo-to-kernel ratio traits and establish a foundation for the improvement of embryo-size-mediated kernel nutrition and grain yield.