45 Expression patterns of PRDM family genes in porcine pre-implantation embryos

Establishing proper levels of pluripotency is essential for normal development. The genome of gametes is remodelled upon fertilisation and pluripotency-related genes are expressed in blastocysts. Multiple pluripotency-related genes are involved in the well-orchestrated process; however, detailed mechanistic actions remain elusive. The PRDM family genes are reported to be closely related to the pluripotency. A previous report noted that PRDM14 plays an important role in the maintenance of pluripotency in human embryonic stem cells (ESCs) and potentially murine ESCs; loss of PRDM14 was found to cause abnormalities in genome-wide epigenetic status. Similarly, PRDM15 was found to be a key regulator of pluripotency in mouse ESCs. Structural similarities among the PRDM family suggest that other PRDM family genes may help to establish and maintain pluripotency in embryos. Unfortunately, little is known about the expression profile of PRDM family in porcine embryos. To expand our understanding of the role of PRDM family in porcine embryos, expression patterns of PRDM gene family were investigated using reverse transcription quantitative (RTq)-PCR. Candidate PRDM family genes were selected based on previous RNA-Seq data in porcine oocytes/embryos. To conduct this study, germinal vesicle (GV), MII, zygote, 4-cell, and blastocyst samples were collected. Complementary DNA synthesised from the samples was used for RT-qPCR to analyse the expression pattern of selected PRDM family genes: PRDM2, PRDM4, PRDM6, PRDM14, and PRDM15. The expression of target genes was normalized to the YWHAG level, an internal control. Then, GV stage was used as a control for ΔΔCT analysis. Two technical replications and three biological replications were performed. Analysis of variance was used for statistical analysis and P-values<0.05 were considered significant. There was a significant decrease in PRDM2 expression in 4-cell and blastocyst, PRDM4 expression in 4-cell, and PRDM6 in all stages (MII, zygote, 4-cell, and blastocyst), compared with the GV stage. Because zygotic genome activation occurs at the 4-cell stage in the pig, the significant decrease in gene expression (PRDM2, PRDM4, and PRDM6) indicates they may be maternally originated and involved in the reprogramming process following fertilisation. On the other hand, there was a significant increase in PRDM15 expression in blastocysts and the PRDM14 transcript was only detected in blastocysts in all three biological replicates, suggesting that the genes are most likely involved in pluripotency maintenance, as was found in previous human studies. These results indicate that PRDM family genes are differentially expressed during early embryo development in pigs and may play a role in maintenance of pluripotency. For further study, we intend to evaluate the role of PRDM family genes during early embryo development in pigs.

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Comprehensive Transcriptome Analysis of mRNA Expression Patterns of Early Embryo Development in Goat under Hypoxic and Normoxic Conditions

Biology ◽

10.3390/biology10050381 ◽

2021 ◽

Vol 10 (5) ◽

pp. 381

Author(s):

Yongjie Wan ◽

Dongxu Li ◽

Mingtian Deng ◽

Zifei Liu ◽

Liang Liu ◽

...

Keyword(s):

Embryo Development ◽

Expression Patterns ◽

Interaction Network ◽

Blastocyst Stage ◽

Early Embryo ◽

Functional Enrichment ◽

Gene Interaction Network ◽

Cell Stage ◽

Early Embryo Development ◽

Pathway Gene

It has been reported that hypoxic environments were more suitable for the in vitro development of mammalian embryos, but the underlying mechanisms were still unclear. In the present study, RNA-seq was performed to compare 8-cell-stage and blastocyst-stage goat embryos under hypoxic and normoxic conditions; zygotes were checked at 72 and 168 h to 8-cell stage (L8C) and blastocyst stage (LM) in hypoxic conditions and 8-cell stage (H8C) and blastocyst stage (HM) in normoxic conditions. In the H8C and L8C groups, 399 DEGs were identified, including 348 up- and 51 down-regulated DEGs. In the HM and LM groups, 1710 DEGs were identified, including 1516 up- and 194 down-regulated DEGs. The expression levels of zygotic genes, transcription factors, and maternal genes, such as WEE2, GDF9, HSP70.1, BTG4, and UBE2S showed significant changes. Functional enrichment analysis indicated that these DEGs were mainly related to biological processes and function regulation. In addition, combined with the pathway–gene interaction network and protein–protein interaction network, twenty-two of the hub genes were identified and they are mainly involved in energy metabolism, immune stress response, cell cycle, receptor binding, and signal transduction pathways. The present study provides comprehensive insights into the effects of oxidative stress on early embryo development in goats.

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LIN28 coordinately promotes nucleolar/ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells

Protein & Cell ◽

10.1007/s13238-021-00864-5 ◽

2021 ◽

Author(s):

Zhen Sun ◽

Hua Yu ◽

Jing Zhao ◽

Tianyu Tan ◽

Hongru Pan ◽

...

Keyword(s):

Stem Cells ◽

Embryo Development ◽

Pluripotent Stem Cells ◽

Rna Binding ◽

Stem Cell Differentiation ◽

Early Embryo ◽

Transcriptional Program ◽

Cell Stage ◽

Early Embryo Development ◽

Nucleolar Stress

AbstractLIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28’s role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

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P–181 Morphine regulates BMP4 growth factor and is involved in in-vitro early embryo development and PGCs formation

Human Reproduction ◽

10.1093/humrep/deab130.180 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

I Muñoa ◽

M Araolaza-Lasa ◽

I Urizar-Arenaza ◽

M Gianzo Citores ◽

N Subiran Ciudad

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Environmental Factors ◽

Embryo Development ◽

Early Embryo ◽

Epigenetic Changes ◽

Morphine Treatment ◽

Early Embryo Development

Abstract Study question To elucidate if morphine can alter embryo development. Summary answer Chronic morphine treatment regulates BMP4 growth factor, in terms of gene expression and H3K27me3 enrichment and promotes in-vitro blastocysts development and PGC formation. What is known already BMP4 is a member of the bone morphogenetic protein family, which acts mainly through SMAD dependent pathway, to play an important role in early embryo development. Indeed, BMP4 enhances pluripotency in mouse embryonic stem cells (mESCs) and, specifically, is involved in blastocysts formation and primordial germ cells (PGCs) generation. Although, external morphine influence has been previously reported on the early embryo development, focus on implantation and uterus function, there is a big concern in understanding how environmental factors can cause stable epigenetic changes, which could be maintained during development and lead to health problems. Study design, size, duration First, OCT4-reported mESCs were chronically treated with morphine during 24h, 10–5mM. After morphine removal, mESCs were collected for RNA-seq and H3K27me3 ChIP-seq study. To elucidate the role of morphine in early embryo development, two cell- embryos stage were chronically treated with morphine for 24h and in-vitro cultured up to the blastocyst stage in the absence of morphine. Furthermore, after morphine treatment mESCs were differentiated to PGCs, to elucidate the role of morphine in PGC differentiation. Participants/materials, setting, methods Transcriptomic analyses and H3K27me3 genome wide distribution were carried out by RNA-Sequencing and Chip-Sequencing respectively. Validations were performed by RNA-RT-qPCR and Chip-RT-qPCR. Main results and the role of chance Dynamic transcriptional analyses identified a total of 932 differentially expressed genes (DEGs) after morphine treatment on mESCs, providing strong evidence of a transcriptional epigenetic effect induced by morphine. High-throughput screening approaches showed up Bmp4 as one of the main morphine targets on mESCs. Morphine caused an up-regulation of Bmp4 gene expression together with a decrease of H3K27me3 enrichment at promoter level. However, no significant differences were observed on gene expression and H3K27me3 enrichment on BMP4 signaling pathway components (such as Smad1, Smad4, Smad5, Smad7, Prdm1 and Prmd14) after morphine treatment. On the other hand, the Bmp4 gene expression was also up-regulated in in-vitro morphine treated blastocyst and in-vitro morphine treated PGCs. These results were consistent with the increase in blastocyst rate and PGC transformation rate observed after morphine chronic treatment. Limitations, reasons for caution To perform the in-vitro analysis. Further studies are needed to describe the whole signaling pathways underlying BMP4 epigenetic regulation after morphine treatment. Wider implications of the findings: Our findings confirmed that mESCs and two-cell embryos are able to memorize morphine exposure and promote both blastocyst development and PGCs formation through potentially BMP4 epigenetic regulation. These results provide insights understanding how environmental factors can cause epigenetic changes during the embryo development, leading to alterations and producing health problems/diseases Trial registration number Not applicable

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Role of ghrelin in fertilization, early embryo development, and implantation periods

Reproduction ◽

10.1530/rep-14-0129 ◽

2014 ◽

Vol 148 (2) ◽

pp. 159-167 ◽

Cited By ~ 12

Author(s):

Eugenia Mercedes Luque ◽

Pedro Javier Torres ◽

Nicolás de Loredo ◽

Laura María Vincenti ◽

Graciela Stutz ◽

...

Keyword(s):

Embryo Development ◽

Ovulation Induction ◽

Physiological Role ◽

Fertilization Rate ◽

Early Embryo ◽

Corpora Lutea ◽

Negative Effects ◽

Early Embryo Development ◽

And Control

In order to clarify the physiological role of ghrelin in gestation, we evaluated the effects of administration of exogenous ghrelin (2 or 4 nmol/animal per day) or its antagonist (6 nmol/animal per day of (d-Lys3)GHRP6) on fertilization, early embryo development, and implantation periods in mice. Three experiments were performed, treating female mice with ghrelin or its antagonist: i) starting from 1 week before copulation to 12 h after copulation, mice were killed at day 18 of gestation; ii) since ovulation induction until 80 h later, when we retrieved the embryos from oviducts/uterus, and iii) starting from days 3 to 7 of gestation (peri-implantation), mice were killed at day 18. In experiments 1 and 3, the antagonist and/or the highest dose of ghrelin significantly increased the percentage of atrophied fetuses and that of females exhibiting this finding or a higher amount of corpora lutea compared with fetuses (nCL/nF) (experiment 3: higher nCL/nF-atrophied fetuses: ghrelin 4, 71.4–71.4% and antagonist, 75.0–62.5% vs ghrelin 2, 46.2−15.4% and control, 10–0.0%;n=7–13 females/group;P<0.01). In experiment 2, the antagonist diminished the fertilization rate, and both, ghrelin and the antagonist, delayed embryo development (blastocysts: ghrelin 2, 62.5%; ghrelin 4, 50.6%; and antagonist, 61.0% vs control 78.4%;n=82–102 embryos/treatment;P<0.0001). In experiment 3, additionally, ghrelin (4 nmol/day) and the antagonist significantly diminished the weight gain of fetuses and dams during pregnancy. Our results indicate that not only hyperghrelinemia but also the inhibition of the endogenous ghrelin effects exerts negative effects on the fertilization, implantation, and embryo/fetal development periods, supporting the hypothesis that ghrelin (in ‘adequate’ concentrations) has a physiological role in early gestational events.

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Characterization of the Arabidopsis Nitrate Transporter NRT1.6 Reveals a Role of Nitrate in Early Embryo Development

The Plant Cell ◽

10.1105/tpc.107.056788 ◽

2008 ◽

Vol 20 (12) ◽

pp. 3289-3299 ◽

Cited By ~ 89

Author(s):

Anabel Almagro ◽

Shan Hua Lin ◽

Yi Fang Tsay

Keyword(s):

Embryo Development ◽

Early Embryo ◽

Nitrate Transporter ◽

Early Embryo Development

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24 EFFECTS OF HISTONE METHYLATION RELATED GENES ON EPIGENETIC REPROGRAMMING AND ZYGOTIC GENE ACTIVATION IN OVINE SOMATIC CELL NUCLEAR TRANSFER (SCNT) EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab24 ◽

2009 ◽

Vol 21 (1) ◽

pp. 112

Author(s):

I. Choi ◽

K. H. S. Campbell

Keyword(s):

Cell Cycle ◽

Somatic Cell ◽

Embryo Development ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Gene Activation ◽

Early Embryo ◽

Cell Stage ◽

Early Embryo Development ◽

Zygotic Gene

After fertilization, early embryo development is dependent upon maternally inherited proteins and protein synthesised from maternal mRNA until zygotic gene activation (ZGA) occurs. The transition of transcriptional activity from maternal to embryonic control occurs with the activation of rRNA genes and the formation of the nucleolus at the 8- to 16-cell stage that coincides with a prolonged fourth cell cycle in bovine and ovine embryos. However, previous studies have reported a shift in the longest cell cycle (fifth cell cycle) in bovine somatic cell nuclear transfer (SCNT) embryos, suggesting that the major genome activation is delayed, possibly due to incomplete changes in chromatin structure such as hypermethylation and hypoacetylation of histone (Memili and First 2000 Zygote 8, 87–96; Holm et al. 2003 Cloning Stem Cells 5, 133–142). Although global gene expression profile studies have been carried out in somatic cell nuclear transfer embryos, little is known about the expression of genes which can alter chromatin structure in early embryo development and possibly effect ZGA. To determine whether epigenetic reprogramming of donor nuclei affected ZGA and expression profiles in SCNT embryos, ZBTB33 (zinc finger and BTB domain containing 33, also known as kaiso, a methy-CpG specific repressor), BRG1(brahma-related gene 1, SWI/SNF family of the ATP-dependent chromatin remodeling complexes), JMJD1A (jumonji domain containing 1A, H3K9me2/1-specific demethylase), JMJD1C (putative H3K9-specific demethylase), and JMJD2C (H3K9me3-specific demethylase) were examined by RT-PCR at different developmental stages [germinal vesicle (GV), metaphase II (MII), 8- to 16-cell, 16- to 32-cell, and blastocyst in both parthenogenetic and SCNT embryos]. All genes were detected in parthenogenetic and SCNT blastocyts, and ZBTB33 was also expressed in all embryos at all stages tested. However, the onset of expression of JMJD1C, containing POU5F1 binding site at 5′-promoter region and BRG1 required for ZGA are delayed in SCNT embryos as compared to parthenotes (16- v. 8-cell, and blastoocyst v. 16-cell stage). Furthermore, JMJD2C containing NANOG binding sites at the 3′-flanking region was expressed in GV and MII oocytes and parthenogenetic blastocysts, whereas in SCNT embryos, JMJD2C was only observed from the 16-cell stage onwards. Interestingly, JMJD1A, which is positively regulated by POU5F1, was not detected in GV and MII oocytes but was present in blastocyst stage embryos of both groups. Taken together, these results suggest that incomplete epigenetic modifications of genomic DNA and histones lead to a delayed onset of ZGA which may affect further development and establishment of totipotency. Subsequently, aberrant expression patterns reported previously in SCNT embryos may be attributed to improper expression of histone H3K9 and H3K4 demethylase genes during early embryo development.

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45 THE EFFECT OF MEIOTIC STAGE OF BOVINE OOCYTES ON THE SURVIVAL OF VITRIFIED CUMULUS–OOCYTE COMPLEXES

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab45 ◽

2012 ◽

Vol 24 (1) ◽

pp. 135 ◽

Cited By ~ 1

Author(s):

J. R. Prentice ◽

J. Singh ◽

M. Anzar

Keyword(s):

Embryo Development ◽

Culture System ◽

In Vitro Maturation ◽

Germinal Vesicle ◽

Early Embryo ◽

Time Intervals ◽

Early Embryo Development ◽

In Vitro Culture System ◽

Bovine Oocytes

Vitrification is a rapid freezing method in which cells/tissues are frozen in a glass state without ice crystal formation. However, vitrification of bovine oocytes is challenging due to their complex structure and sensitivity to chilling. Oocytes at the germinal vesicle (GV) stage of maturation are thought to be less prone to chromosomal and microtubular damage during cryopreservation because no spindle is present and genetic material is contained within the nucleus. However, immature oocytes are thought to be more sensitive to osmotic stress and have lower cell membrane stability than mature, metaphase II (MII) stage oocytes. The present studies aimed to validate the in vitro culture system used in our laboratory and to evaluate the effect of vitrification of bovine cumulus-oocyte complexes (COC) at different meiotic stages on their in vitro maturation (IVM), cleavage and early embryo development. Analyses were conducted on each dataset with PROC GLIMMIX in SAS using binary distribution (for yes/no response variable) and considering replicate as a random factor. In Experiment 1, meiotic progression of oocytes was evaluated at different time intervals during IVM. The following COC stages were predominantly found at different IVM time intervals: GV (89%) at 0 h, GV (47%) and germinal vesicle breakdown (GVBD; 44%) at 6 h, metaphase I (MI; 90%) at 12 h and MII (84%) at 22 h (n > 62 oocytes at each time group). In Experiment 2, bovine COC at 0, 6, 12 and 22 h of IVM were exposed to vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5 M sucrose + 20% CS in TCM-199), loaded onto a cryotop device and vitrified by plunging in liquid nitrogen. Following warming (1 min in 0.5 M sucrose + 20% CS in TCM-199), COC completed 22 h of IVM and the nuclear stage was evaluated with lamin A/C-4′6-diamidino-2-phenylindole staining. Upon completion of 22 h of IVM, 23, 23, 35 and 89% of oocytes from 0-, 6-, 12- and 22-h groups, respectively were detected at MII (P < 0.0001). In Experiment 3, cleavage and embryo development of oocytes vitrified at 0, 12 and 22 h of IVM were evaluated. The cleavage rate did not differ among vitrification groups (i.e. 14% at 0 h, 17% at 12 h and 14% at 22 h; P = 0.825). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified (control) group than in vitrified groups (i.e. 73 vs 15% and 22 vs 0.3%, respectively). In conclusion, the maturation kinetics validated our in vitro culture system and vitrification adversely affected the ability of bovine oocytes to undergo in vitro maturation to the MII stage, in vitro fertilization and early embryo development. Vitrification of oocytes at GV, MI and MII stages of nuclear maturation did not differ in their subsequent survivability. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

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Lysine Deprivation during Maternal Consumption of Low-Protein Diets Could Adversely Affect Early Embryo Development and Health in Adulthood

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17155462 ◽

2020 ◽

Vol 17 (15) ◽

pp. 5462

Author(s):

Lon J. Van Winkle ◽

Vasiliy Galat ◽

Philip M. Iannaccone

Keyword(s):

Embryo Development ◽

Embryonic Stem ◽

Early Embryo ◽

Mammalian Embryo ◽

Early Embryo Development ◽

Low Protein ◽

Glutamate Production ◽

The Mean ◽

Hes Cells ◽

Protein Diets

The conversion of lysine to glutamate is needed for signaling in all plants and animals. In mouse embryonic stem (mES) cells, and probably their progenitors, endogenous glutamate production and signaling help maintain cellular pluripotency and proliferation, although the source of glutamate is yet to be determined. If the source of glutamate is lysine, then lysine deprivation caused by maternal low-protein diets could alter early embryo development and, consequently, the health of the offspring in adulthood. For these reasons, we measured three pertinent variables in human embryonic stem (hES) cells as a model for the inner cell masses of human blastocysts. We found that RNA encoding the alpha-aminoadipic semialdehyde synthase enzyme, which regulates glutamate production from lysine, was highly expressed in hES cells. Moreover, the mean amount of lysine consumed by hES cells was 50% greater than the mean amount of glutamate they produced, indicating that lysine is likely converted to glutamate in these cells. Finally, hES cells expressed RNA encoding at least two glutamate receptors. Since this may also be the case for hES progenitor cells in blastocysts, further studies are warranted to verify the presence of this signaling process in hES cells and to determine whether lysine deprivation alters early mammalian embryo development.

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