Early cleavage of preimplantation embryos is regulated by tRNAGln-TTG–derived small RNAs present in mature spermatozoa

tRNA-derived small RNAs (tsRNAs) from spermatozoa could act as acquired epigenetic factors and contribute to offspring phenotypes. However, the roles of specific tsRNAs in early embryo development remain to be elucidated. Here, using pigs as a research model, we probed the tsRNA dynamics during spermatogenesis and sperm maturation and demonstrated the delivery of tsRNAs from semen-derived exosomes to spermatozoa. By microinjection of antisense sequences into in vitro fertilized oocytes and subsequent single-cell RNA-seq of embryos, we identified a specific functional tsRNA group (termed here Gln-TTGs) that participate in the early cleavage of porcine preimplantation embryos, probably by regulating cell cycle–associated genes and retrotransposons. We conclude that specific tsRNAs present in mature spermatozoa play significant roles in preimplantation embryo development.

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Amino acid depletion and appearance during porcine preimplantation embryo development in vitro

Reproduction ◽

10.1530/rep.1.00727 ◽

2005 ◽

Vol 130 (5) ◽

pp. 655-668 ◽

Cited By ~ 37

Author(s):

Paul J Booth ◽

Peter G Humpherson ◽

Terry J Watson ◽

Henry J Leese

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Embryo Development ◽

Preimplantation Embryos ◽

Type I ◽

Preimplantation Embryo Development ◽

Type Iv ◽

Stage Of Development ◽

Amino Acid Depletion

Preimplantation embryos can consume and produce amino acids in a manner dependent upon the stage of development that may be predictive of subsequent viability. In order to examine these relationships in the pig, patterns of net depletion and appearance of amino acids byin vitroproduced porcine preimplantation embryos were examined. Cumulus oocyte complexes derived from slaughterhouse pre-pubertal pig ovaries were matured for 40 h in defined TCM-199 medium (containing PVA) before being fertilised (Day 0) with frozen-thawed semen in Tris–based medium. After 6 h, presumptive zygotes were denuded and cultured in groups of 20, in NCSU-23 medium modified to contain 0.1 mM glutamine plus a mixture of 19 amino acids (aa) at low concentrations (0.02–0.11 mM) (NCSU-23aa). Groups of 2–20 embryos were removed (dependent on stage) on Day 0 (1 cell), Day 1 (two- and four-cells), Day 4 (compact morulae) and Day 6 (blastocysts) and placed in 4 μl NCSU-23aafor 24 h. After incubation, the embryos were removed and the spent media was analysed by HPLC. The net rate of amino acid depletion or appearance varied according to amino acid (P< 0.001) and, apart from serine and histidine, stage of development (P< 0.014). Glycine, isoleucine, valine, phenylalanine, tryptophan, methionine, asparagine, lysine, glutamate and aspartate consistently appeared, whereas threonine, glutamine and arginine were consistently depleted. Five types of stage-dependent trends could be observed: Type I: amino acids having high rates of net appearance on Day 0 that reached a nadir on Day 1 or 4 but subsequently increased by Day 6 (glycine, glutamate); Type II: those that exhibited lower rates of net appearance on Days 0 and 6 compared with the intermediate Days 1 and 4 (isoleucine, valine, phenylalanine, methionine, arginine); Type III: amino acids which showed a continuous fall in net appearance (asparagine, aspartate); Type IV: those that exhibited a steady fall in net depletion from Day 0 to Day 6 (glutamine, threonine); Type V: those following no discernable trend. Analysis of further embryo types indicated that presumptive polyspermic embryos on Day 0 had increased (P< 0.05) net rates of leucine, isoleucine, valine and glutamate appearance, and reduced (P< 0.05) net rates of threonine and glutamine depletion compared with normally inseminated oocytes. These data suggest that the net rates of depletion and uptake of amino acids by pig embryos vary between a) amino acids, b) the day of embryo development and, c) the type of embryos present at a given stage of development. The results also suggested that the net depletion and appearance rates of amino acids by early pig embryos might be more similar to those of the human than those of the mouse and cow.

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Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development

Reproduction ◽

10.1530/rep.1.00241 ◽

2004 ◽

Vol 128 (3) ◽

pp. 281-291 ◽

Cited By ~ 78

Author(s):

Andrea Jurisicova ◽

Beth M Acton

Keyword(s):

Gene Expression ◽

Cell Death ◽

Cell Fate ◽

Embryo Development ◽

Preimplantation Embryo ◽

Culture Media ◽

Culture Conditions ◽

Early Embryo ◽

Preimplantation Embryo Development

Human preimplantation embryo development is prone to high rates of early embryo wastage, particularly under currentin vitroculture conditions. There are many possible underlying causes for embryo demise, including DNA damage, poor embryo metabolism and the effect of suboptimal culture media, all of which could result in an imbalance in gene expression and the failed execution of basic embryonic decisions. In view of the complex interactions involved in embryo development, a thorough understanding of these parameters is essential to improving embryo quality. An increasing body of evidence indicates that cell fate (i.e. survival/differentiation or death) is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins, many of which are expressed during oocyte and preimplantation embryo development. The recent availability of mutant mice lacking expression of various genes involved in the regulation of cell survival has enabled rapid progress towards identifying those molecules that are functionally important for normal oocyte and preimplantation embryo development. In this review we will discuss the current understanding of the regulation of cell death gene expression during preimplantation embryo development, with a focus on human embryology and a discussion of animal models where appropriate.

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Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development

Scientific Reports ◽

10.1038/s41598-021-99512-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Paul J. McKeegan ◽

Selina F. Boardman ◽

Amy A. Wanless ◽

Grace Boyd ◽

Laura J. Warwick ◽

...

Keyword(s):

Mitochondrial Dna ◽

Oxygen Consumption ◽

Oxygen Concentration ◽

Embryo Development ◽

Preimplantation Embryo ◽

Complex Iii ◽

Preimplantation Embryos ◽

Mitochondrial Oxygen Consumption ◽

Preimplantation Embryo Development

AbstractWe report a novel method to profile intrcellular oxygen concentration (icO2) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO2 and decreasing oxygen consumption to increase icO2. Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9–4 × 106 copies and did not correlate with icO2. These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.

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mRNA Expression and Role of PPARγ and PPARδ in Bovine Preimplantation Embryos Depending on the Quality and Developmental Stage

Animals ◽

10.3390/ani10122358 ◽

2020 ◽

Vol 10 (12) ◽

pp. 2358

Author(s):

Katarzyna Suwik ◽

Emilia Sinderewicz ◽

Dorota Boruszewska ◽

Ilona Kowalczyk-Zięba ◽

Joanna Staszkiewicz-Chodor ◽

...

Keyword(s):

Early Development ◽

Embryo Development ◽

Embryo Quality ◽

Early Embryo ◽

Developmental Competence ◽

Preimplantation Embryos ◽

Peroxisome Proliferator ◽

Early Embryo Development ◽

Quality Markers

Peroxisome proliferator-activated receptors (PPARs), a nuclear receptors for prostacyclin (PGI2) have been recognized as being essential for early embryo development. The objectives of the present study were to determine if the bovine early- and late-cleaved embryos in different stages of early development express PPARγ and PPARδ. Since embryo developmental competence depends on numerous biological factors, we evaluated if the expression of PPARγ and PPARδ correlate with selected embryo quality markers (SOX2, OCT4, PLAC8, IGF1R) in the in vitro produced embryos at different stages of their development. Developmental rates and embryo quality for early- and late-cleaved embryos were provided according to International Embryo Transfer Society (IETS; developmental stages: 2-, 4-, 16-cell embryo, morula, blastocyst (1—early, 2—developing, 3—expanded, 4—hatched); quality stages: A—high quality, B—moderate quality, C—low quality). We found that bovine embryos expressed mRNA of PPARδ and PPARγ at all stages of early development, independently of their quality. In addition, the expression of PPARδ and PPARγ correlated with the expression of quality markers in bovine blastocysts. Positive correlations were stronger and more frequent in the group of early-cleaved embryos, whereas the negative correlations were typical for the group of late-cleaved embryos. Obtained results and available literature reports may indicate the participation of PGI2, via PPARδ and PPARγ, in the processes related to the early embryo development, through the participation of this factor in the modulation of blastocyst hatching, implantation, and post-implantation development.

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277 EFFECTS OF ERYTHROCYTE AND ERYTHROCYTE HEMOLYSATE ON BOVINE PREIMPLANTATION EMBRYO DEVELOPMENT IN VITRO UNDER REACTIVE OXYGEN SPECIES CONDITION

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab277 ◽

2010 ◽

Vol 22 (1) ◽

pp. 295

Author(s):

A. Ideta ◽

K. Tsuchiya ◽

Y. Nakamura ◽

M. Urakawa ◽

M. Murakami ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Embryo Development ◽

Reactive Oxygen ◽

Hemoglobin Concentration ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Oxygen Species ◽

Preimplantation Embryo Development ◽

Presence And Absence

Reactive oxygen species (ROS) damage preimplantation embryos by increasing DNA fragmentation, leading to early embryonic death. Erythrocytes have been shown to protect other cells and tissues against ROS. In mice, erythrocytes were recently found to improve the early development of embryos by their antioxidant effect. The purpose of the present study was to examine the effect of erythrocytes on the in vitro development of bovine IVF embryos in medium supplemented with ROS. COCs were aspirated from ovaries collected from a local slaughterhouse and were cultured for 22 h in TCM-199 containing 5% fetal bovine serum. IVF was performed using an IVF100 (Research Institute for the Functional Peptides, Yamagata, Japan) according to the manufacturer’s instructions. In experiment 1, IVF embryos were cultured in CR1aa medium supplemented with an oxidizing agent, 0.5 mM hypoxanthine and 0.01 U mL-1 xanthine oxidase (HX/XOD), in the presence and absence of erythrocytes (5 × 104, 5× 105, 5×106, and 5 × 107 erythrocytes mL-1). In experiments 2 and 3, the development of embryos under the condition without ROS was assessed in the presence and absence of erythrocytes (5 × 106 erythrocytes mL-1) or erythrocyte hemolysate (hemoglobin concentration of 1.9 g L-1), respectively. At 7 days after in vitro culture, the development to the blastocyst stage of IVF embryos was examined using a stereomicroscope. Data were analyzed using Fisher’s PLSD test and Student’s t-test In experiment 1, the presence of HX/XOD significantly inhibited embryo development to the blastocyst stage in vitro (P < 0.05). The addition of erythrocytes to medium supplemented with HX/XOD markedly improved preimplantation development (Table 1). In experiments 2 and 3, supplementation of erythrocytes or erythrocyte hemolysate promoted the development of embryos to the blastocyst stage (experiment 2: erythrocyte 42.4 ± 3.1%, control 28.5 ± 5.7%, P < 0.1; experiment 3: erythrocyte hemolysate 39.1 ± 3.3%, control 30.2 ± 1.0%, P < 0.1). In conclusion, we suggest that the addition of erythrocytes to culture medium can counteract the negative effects of ROS on embryo development and blastocyst formation. Table 1.Effect of HX/XOD and erythrocyte supplementation on embryo development to blastocyst stage

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Maternal Underweight Does Not Adversely Affect The Outcomes of IVF/ICSI and Frozen Embryo Transfer Cycles or Early Embryo Development

10.21203/rs.3.rs-640651/v1 ◽

2021 ◽

Author(s):

Dana Hoffman ◽

Yael Kalma ◽

Nivin Samara ◽

Einat Haikin Herzberger ◽

Sagi Levi ◽

...

Keyword(s):

Embryo Development ◽

Preimplantation Embryo ◽

Morphological Characteristics ◽

Time Lapse ◽

Early Embryo ◽

Normal Weight ◽

Oocyte Maturity ◽

Vitro Fertilization ◽

Preimplantation Embryo Development

Abstract Purpose To compare assisted reproductive technology (ART) outcomes and preimplantation embryo development between underweight and normal weight women. Methods This retrospective cohort study included 26 underweight women (body mass index [BMI] < 18.50 kg/m2) and 104 normal weight women (BMI > 20 and < 24.9 kg/m2) who underwent a total of 204 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles and 358 fresh/frozen embryo transfers (ET) in our institution between January 2016 and December 2018. Statistical analyses compared selected ART outcomes (ovarian stimulation, fertilization, and pregnancy) between both weight groups. Morphokinetic and morphological parameters were also compared between 346 and 1467 embryos of underweight and normal weight women, respectively. Results The mean ± standard deviation age of the underweight and normal weight women was similar (31.6 ± 4.17 vs 32.4 ± 3.59 years; p = 0.323). There were no differences in the peak estradiol levels, the number of retrieved oocytes, the number of metaphase II oocytes, and the oocyte maturity rates between the two groups. The IVF/ICSI fertilization rates and the number of embryos suitable for transfer or cryopreservation were similar for both groups. All morphokinetic parameters that were evaluated by means of time-lapse imaging as well as the morphological characteristics were comparable between low and normal BMI categories. There were no significant differences in pregnancy achievement, clinical pregnancy, live births, and miscarriage rates between the suboptimal and optimal weight women. Conclusion Underweight status has no adverse impacts on the outcomes of IVF/ICSI with either fresh or frozen ET or on preimplantation embryo development and quality.

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BPA and BPS Affect the Expression of Anti-Mullerian Hormone (AMH) and Its Receptor During Bovine Oocyte Maturation and Early Embryo Development

10.21203/rs.3.rs-435109/v1 ◽

2021 ◽

Author(s):

Angela Christina Saleh ◽

Reem Sabry ◽

Gabriela Fabiana Mastromonaco ◽

Laura Alessandra Favetta

Keyword(s):

Embryonic Development ◽

Embryo Development ◽

Oocyte Maturation ◽

Quantitative Polymerase Chain Reaction ◽

Early Embryo ◽

Bisphenol S ◽

Cell Stage ◽

Vitro Fertilization ◽

Preimplantation Embryo Development

Abstract Background Exposure to endocrine-disrupting chemicals, such as Bisphenol A (BPA) and Bisphenol S (BPS), is widespread and has negative implications on embryonic development. Preliminary evidence revealed that in women undergoing IVF treatment, urinary BPA levels were associated with low serum anti-Mullerian hormone, however a definitive relationship between the two has not yet been characterized. Methods This study aimed to evaluate BPA and BPS effects on in vitro oocyte maturation and early preimplantation embryo development through i) analysis of anti-Mullerian hormone (AMH) and anti-Mullerian hormone receptor II (AMHRII), ii) investigation of developmental parameters, such as cleavage, blastocyst rates and developmental arrest, iii) detection of apoptosis and iv) assessment of possible sex ratio skew. An in vitro bovine model was used as a translational model for human early embryonic development. We first assessed AMH and AMHRII levels after bisphenol exposure during oocyte maturation. Zygotes were also analyzed during cleavage and blastocysts stages. Techniques used include in vitro fertilization, quantitative polymerase chain reaction (qPCR), western blotting, TUNEL and immunofluorescence. Results Our findings show that BPA significantly decreased cleavage (p < 0.001), blastocyst (p < 0.005) and overall developmental rates as well as significantly increased embryonic arrest at the 2–4 cell stage (p < 0.05). Additionally, both BPA and BPS significantly increased DNA fragmentation in 2–4 cells, 8–16 cells and blastocyst embryos (p < 0.05). Furthermore, BPA and BPS alter AMH and AMHRII at the mRNA and protein level in both oocytes and blastocysts. BPA, but not BPS, also significantly skews sex ratios towards female blastocysts (p < 0.05) Conclusion This study shows that BPA affects AMH and AMHRII expression during oocyte maturation and that BPS exerts its effects to a greater extent after fertilization and therefore may not be a safer alternative to BPA. Our data lay the foundation for future functional studies, such as receptor kinetics, downstream effectors, and promoter activation/inhibition to prove a functional relationship between bisphenols and the AMH signalling system.

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Hemoglobin: potential roles in the oocyte and early embryo†

Biology of Reproduction ◽

10.1093/biolre/ioz078 ◽

2019 ◽

Vol 101 (2) ◽

pp. 262-270 ◽

Cited By ~ 1

Author(s):

Megan Lim ◽

Hannah M Brown ◽

Karen L Kind ◽

Jeremy G Thompson ◽

Kylie R Dunning

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

Preimplantation Embryo ◽

Cell Types ◽

Cellular Function ◽

Preimplantation Embryos ◽

Low Oxygen ◽

Preimplantation Embryo Development ◽

Regulated Gene Expression

Abstract Hemoglobin (Hb) is commonly known for its capacity to bind and transport oxygen and carbon dioxide in erythroid cells. However, it plays additional roles in cellular function and health due to its capacity to bind other gases including nitric oxide. Further, Hb acts as a potent antioxidant, quenching reactive oxygen species. Despite its potential roles in cellular function, the preponderance of Hb research remains focused on its role in oxygen regulation. There is increasing evidence that Hb expression is more ubiquitous than previously thought, with Hb and its variants found in a myriad of cell types ranging from macrophages to spermatozoa. The majority of nonerythroid cell types that express Hb are situated within hypoxic environments, suggesting Hb may play a role in hypoxia-inducible factor-regulated gene expression by controlling the level of oxygen available or as an adaptation to low oxygen providing a mechanism to store oxygen. Oocyte maturation and preimplantation embryo development occur within the low oxygen environments of the antral follicle and oviduct/uterus, respectively. Interestingly, Hb was recently found in human cumulus and granulosa cells and murine cumulus–oocyte complexes and preimplantation embryos. Here, we consolidate and analyze the research generated todate on Hb expression in nonerythroid cells with a particular focus on reproductive cell types. We outline future directions of this research to elucidate the role of Hb during oocyte maturation and preimplantation embryo development and finally, we explore the potential clinical applications and benefits of Hb supplementation during the in vitro culture of gametes and embryos.

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Embryos generated from oocytes lacking complex N- and O-glycans have compromised development and implantation

Reproduction ◽

10.1530/rep-12-0084 ◽

2012 ◽

Vol 144 (4) ◽

pp. 455-465 ◽

Cited By ~ 11

Author(s):

Patricia Grasa ◽

Heidy Kaune ◽

Suzannah A Williams

Keyword(s):

Embryo Development ◽

Subsequent Development ◽

Early Embryo ◽

Preimplantation Development ◽

Ovulation Rate ◽

Preimplantation Embryos ◽

Small Litter ◽

Delayed Implantation

Female mice generating oocytes lacking complexN- andO-glycans (double mutants (DM)) produce only one small litter before undergoing premature ovarian failure (POF) by 3 months. Here we investigate the basis of the small litter by evaluating ovulation rate and embryo development in DM (Mgat1F/FC1galt1F/F:ZP3Cre) and Control (Mgat1F/FC1galt1F/F) females. Surprisingly, DM ovulation rate was normal at 6 weeks, but declined dramatically by 9 weeks.In vitrodevelopment of zygotes to blastocysts was equivalent to Controls although all embryos from DM females lacked a normal zona pellucida (ZP) and ∼30% lacked a ZP entirely. In contrast,in vivopreimplantation development resulted in less embryos recovered from DM females compared with Controls at 3.5 days post coitum (dpc) (3.2±1.3 vs 7.0±0.6). Furthermore, only 45% of mated DM females contained embryos at 3.5 dpc. Of the preimplantation embryos collected from DM females, approximately half were morulae unlike Controls where the majority were blastocysts, indicating delayed embryo development in DM females. Post-implantation development in DM females was analysed to determine whether delayed preimplantation development affected subsequent development. In DM females at 5.5 dpc, only ∼40% of embryos found at 3.5 dpc had implanted. However, at 6.5 dpc, implantation sites in DM females corresponded to embryo numbers at 3.5 dpc indicating delayed implantation. At 9.5 dpc, the number of decidua corresponded to embryo numbers 6 days earlier indicating that all implanted embryos progress to midgestation. Therefore, a lack of complexN- andO-glycans in oocytes during development impairs early embryo development and viabilityin vivoleading to delayed implantation and a small litter.

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BPA and BPS affect the expression of anti-Mullerian hormone (AMH) and its receptor during bovine oocyte maturation and early embryo development

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00773-6 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Angela Christina Saleh ◽

Reem Sabry ◽

Gabriela Fabiana Mastromonaco ◽

Laura Alessandra Favetta

Keyword(s):

Embryonic Development ◽

Embryo Development ◽

Oocyte Maturation ◽

Quantitative Polymerase Chain Reaction ◽

Early Embryo ◽

Bisphenol S ◽

Cell Stage ◽

Vitro Fertilization ◽

Preimplantation Embryo Development

Abstract Background Exposure to endocrine-disrupting chemicals, such as Bisphenol A (BPA) and Bisphenol S (BPS), is widespread and has negative implications on embryonic development. Preliminary evidence revealed that in women undergoing IVF treatment, urinary BPA levels were associated with low serum anti-Mullerian hormone, however a definitive relationship between the two has not yet been characterized. Methods This study aimed to evaluate BPA and BPS effects on in vitro oocyte maturation and early preimplantation embryo development through i) analysis of anti-Mullerian hormone (AMH) and anti-Mullerian hormone receptor II (AMHRII), ii) investigation of developmental parameters, such as cleavage, blastocyst rates and developmental arrest, iii) detection of apoptosis and iv) assessment of possible sex ratio skew. An in vitro bovine model was used as a translational model for human early embryonic development. We first assessed AMH and AMHRII levels after bisphenol exposure during oocyte maturation. Zygotes were also analyzed during cleavage and blastocysts stages. Techniques used include in vitro fertilization, quantitative polymerase chain reaction (qPCR), western blotting, TUNEL and immunofluorescence. Results Our findings show that BPA significantly decreased cleavage (p < 0.001), blastocyst (p < 0.005) and overall developmental rates as well as significantly increased embryonic arrest at the 2–4 cell stage (p < 0.05). Additionally, both BPA and BPS significantly increased DNA fragmentation in 2–4 cells, 8–16 cells and blastocyst embryos (p < 0.05). Furthermore, BPA and BPS alter AMH and AMHRII at the mRNA and protein level in both oocytes and blastocysts. BPA, but not BPS, also significantly skews sex ratios towards female blastocysts (p < 0.05). Conclusion This study shows that BPA affects AMH and AMHRII expression during oocyte maturation and that BPS exerts its effects to a greater extent after fertilization and therefore may not be a safer alternative to BPA. Our data lay the foundation for future functional studies, such as receptor kinetics, downstream effectors, and promoter activation/inhibition to prove a functional relationship between bisphenols and the AMH signalling system.

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