O-099 TEAD4 regulates trophectoderm differentiation upstream of CDX2 in human preimplantation embryos

Abstract Study question What is the main pathway regulating trophectoderm (TE) differentiation during pre-implantation development in mouse versus human embryos? Summary answer TEAD4 is acting upstream of CDX2 and is involved in TE differentiation, as TEAD4-null human embryos exhibit compromised TE lineage differentiation. What is known already TEAD4 is the earliest transcription factor during early embryo development, required for the expression of TE-associated genes leading to successful TE differentiation and subsequent blastocoel formation in mouse. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination have shown that Tead4-null embryos do not express TE specific genes, including Caudal-Type Homeobox Protein 2 (Cdx2) and GATA Binding Protein 3 (Gata3), but expression of inner cell mass (ICM)-specific genes, remains unaffected. Furthermore, ablation of Tead4 compromises embryonic development and subsequent blastocoel formation in mouse. The role of TEAD4, during human pre-implantation development has not been functionally characterized yet. Study design, size, duration CRISPR-Cas9 was introduced in mouse zygotes and editing efficiency was evaluated by next-generation sequencing (NGS) on 4.5dpc embryos (n = 55). Developmental kinetics were monitored in CRISPR-Cas9 targeted (n = 83), sham-injected (n = 26) and non-injected media-control (n = 51) mouse embryos. Immunofluorescence analysis was performed in Tead4 targeted (n = 57) and non-injected media-control embryos (n = 94). The same methodology was applied in human donated in vitro matured (IVM) metaphase-II (MII) oocytes, which were CRISPR-Cas9 targeted (n = 74) during ICSI or used as media-Control (n = 33). Participants/materials, setting, methods A gRNA-Cas9 mixture targeting exon 2 of Tead4/TEAD4 was microinjected in respectively mouse 2PN (pronuclear) stage zygotes, or human IVM MII oocytes along with the sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. Embryonic development and morphology were assessed daily, followed by a detailed scoring at the late blastocyst stage. Successful targeting following CRISPR-Cas9 introduction was assessed by immunostaining and NGS analysis of the targeted locus. Main results and the role of chance In mouse, we confirmed previous findings, as the developmental capacity of Tead4 targeted embryos was significantly reduced starting from the morula stage and blastocyst formation rates were 8.97% in the targeted group, compared to 87.23% in the control and 87.50% in the sham group, respectively. Immunofluorescence analysis of late morula and blastocyst stage embryos confirmed the absence of Tead4, Cdx2 and Gata3, resulting from the successful interruption of the Tead4 locus (n = 57). Exon 2 of TEAD4 was successfully targeted in human. In total, 21 embryos from various developmental stages were successfully NGS analyzed and 90,48% (19 out of 21) of the embryos carried genetic modifications as a result of CRISPR-Cas9 genome editing and seven blastocysts were identified carrying exclusively frameshift mutations. In contrast to mouse, the developmental capacity of human targeted embryos (25%) did not differ significantly from the control group (23%). However, the blastocyst morphology and quality were compromised in the targeted group showing mostly grade C TE scores, containing very few cells. Immunofluorescence analysis of targeted blastocysts (n = 6) confirmed successful editing by complete absence of TEAD4 and its downstream TE marker CDX2. Limitations, reasons for caution CRISPR-Cas9 germline genome editing results in multiple editing outcomes with variable phenotypic penetrance, the mosaic nature of which complicates the phenotypic analysis and developmental behaviour of the injected embryos. Wider implications of the findings Elucidation of the evolutionary conserved molecular mechanisms that regulate self-renewal of the trophoblast lineage can give us fundamental insights on early implantation failure. Trial registration number Not Applicable

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Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences

Human Reproduction ◽

10.1093/humrep/deab027 ◽

2021 ◽

Author(s):

P Stamatiadis ◽

A Boel ◽

G Cosemans ◽

M Popovic ◽

B Bekaert ◽

...

Keyword(s):

Blastocyst Stage ◽

S Phase ◽

Preimplantation Development ◽

Human Embryos ◽

Immunofluorescence Analysis ◽

Media Control ◽

Cas9 Protein ◽

M Phase ◽

Developmental Capacity

Abstract STUDY QUESTION What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model? SUMMARY ANSWER POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation. WHAT IS KNOWN ALREADY Clustered regularly interspaced short palindromic repeats—CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage. STUDY DESIGN, SIZE, DURATION The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain—B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts. LIMITATIONS, REASONS FOR CAUTION One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis. STUDY FUNDING/COMPETING INTEREST(S) The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

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374 THE EFFECTS OF POLYVINYLPYRROLIDONE ON BOVINE SPERM FUNCTION AND EMBRYO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab374 ◽

2007 ◽

Vol 19 (1) ◽

pp. 302 ◽

Cited By ~ 1

Author(s):

Y. Kato ◽

M. Fukushima ◽

A. Kenmotsu ◽

K. Chikazawa ◽

Y. Nagao

Keyword(s):

Embryonic Development ◽

Embryo Development ◽

Staining Method ◽

Blastocyst Stage ◽

Sperm Function ◽

Bovine Sperm ◽

Developmental Capacity ◽

Chromatin Integrity ◽

Triple Staining

In assisted reproduction by ICSI, PVP has been successfully used to replicate the viscosity of sperm solution, thus facilitating the handling and immobilization of spermatozoa. Sperm is suspended in medium containing polyvinylpyrrolidone (PVP), then injected into the oocytes together with a small amount of the medium in ICSI. However the effects of PVP on sperm function and embryo development have not been investigated in detail. In the present study, we investigated the effects of PVP solution on sperm function and embryonic development. Frozen–thawed spermatozoa from a Japanese Black bull and immature COCs from slaughterhouse bovine ovaries were used for all experiments. In experiment 1, bovine sperm was cultured in SOF or SOF containing 10% PVP. For detection of sperm acrosomal and chromatin integrity, sperm cultured in each medium were stained by the triple staining method and acridine orange after 0, 15, 30, and 60 min of culture. In experiment 2, zygotes were injected with PVP solution and cultured in vitro; subsequent cleavage and development to blastocysts were examined. In experiment 3, zygote injected with PVP solution was fixed by 4% paraformaldehyde after 1–3 h of PVP injection. The location of PVP solution in zygote was observed. In experiment 4, two-cell embryos were microinjected with a solution of dextran conjugated with fluorescein (FITC-dextran) and cultured in vitro. The location of FITC-dextran in the embryo was examined. In experiment 1, acrosome reactions of the sperm were enhanced after 15 min of incubation in PVP solution (P < 0.05), but chromatin integrity of the sperm was not influenced (P > 0.05). In experiment 2, PVP suppressed the development of the zygote to 2-cell, morula and blastocyst (75.0%, 35.1%, and 26.3% vs. 61.3%, 20.2%, and 12.9% for control and PVP group, respectively, P < 0.05). In experiment 3, the locations of PVP solution in the zygote were observed 1–3 h after injection. In experiment 4, FITC-dextran was observed in ICM at the blastocyst stage. These findings suggest that PVP affects the acrosome but not the chromatin of sperm in ICSI. PVP solution exists locally in embryos injected and affects the developmental capacity of the embryos.

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Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage

Zygote ◽

10.1017/s0967199400002100 ◽

1994 ◽

Vol 2 (4) ◽

pp. 281-287 ◽

Cited By ~ 76

Author(s):

Asangla Ao ◽

Robert P. Erickson ◽

Robert M.L. Winston ◽

Alan H Handysude

Keyword(s):

Mammalian Species ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Human Embryos ◽

Cell Stage ◽

Gonad Differentiation ◽

Embryonic Genome ◽

Human Preimplantation Embryos

SummaryGlobal activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

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55 The Role of Poly (ADP-Ribose) Polymerases in Porcine Cumulus - Oocyte Complex During In Vitro Maturation and Embryonic Development

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab55 ◽

2018 ◽

Vol 30 (1) ◽

pp. 166

Author(s):

D. H. Kim ◽

S. T. Shin ◽

H. T. Lee

Keyword(s):

Embryonic Development ◽

In Vitro Maturation ◽

Parp Inhibitor ◽

Development Program ◽

Cumulus Cells ◽

Treated Group ◽

Blastocyst Stage ◽

Mrna Levels

Poly(ADP-ribosyl)ation (PARylation) is related to DNA repair, chromatin modification, and apoptosis and is catalyzed by PARylation polymerases (PARP). Previous studies have shown that PARylation regulates pre-implantation development and participates autophagy mechanism in mouse and pig. However, the involvement of PARylation and pro-survival autophagy in pre-implantation development from cumulus–oocyte complexes (COC) to the blastocyst stage has not yet been documented. Thus, we investigated the role of PARylation during in vitro maturation (IVM) of porcine COC and their embryonic development. To study the effect of PARylation, COC were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during IVM. Nuclear maturation rates of oocytes were showed no significant differences between 2 groups in all stages (from GV to MII). However, the expansion rates of cumulus cells were significantly decreased in 3-ABA–treated COC compared with control (11.05 ± 1.09 v. 48.40 ± 0.67%). When we analysed mRNA levels of maturation- and expansion-related genes in cumulus cells, levels of PTX3, CX43, and COX-2 were increased but levels of HAS2 and TNFAIP6 were decreased in treatment group. In addition, expression levels of GDF9 and BMP15 in oocytes were up-regulated in treated group. Then, we examined the development of IVF embryos from IVM oocytes in the presence and absence of 3-ABA and their quality at the blastocyst stage. We found that the developmental rates of embryos were significantly decreased in 3-ABA-treated group. In particular, the proportion of expanded blastocysts was lower in the treated embryos (2.65 ± 1.53) compared with control embryos (12.27 ± 3.05). Furthermore, the transcript levels of autophagy-related genes (ATG5, BECLIN1, and LC3) in 3-ABA-treated embryos were lowered in all stages. In addition, we found a higher rate of apoptosis in treated blastocysts compared with the control (total apoptosis index; 15.65 ± 2.73 v. 4.89 ± 0.67). Finally, SQSTM1/p62 aggregate increased in 3-ABA-treated blastocysts, indicating that the inhibition of PARylation regulates selective autophagy pathways to utilise SQSTM1/p62. Therefore, these results indicate that PARylation by PARPs during IVM of COC is deeply involved in the pro-survival autophagy and influences the development and quality of porcine embryos. This research was supported by a Grant from the Bio & Medical Technology Development Program (2015M3A9C7030091) of the National Research Foundation (NRF) funded by the Korean government.

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The role of Trp53 in the mouse embryonic response to DNA damage

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz029 ◽

2019 ◽

Vol 25 (7) ◽

pp. 397-407

Author(s):

Yvonne Wilson ◽

Ian D Morris ◽

Susan J Kimber ◽

Daniel R Brison

Keyword(s):

Dna Damage ◽

Inner Cell Mass ◽

Cell Mass ◽

Blastocyst Stage ◽

Human Embryos ◽

Cell Stage ◽

X Irradiation

Abstract Apoptosis occurs primarily in the blastocyst inner cell mass, cells of which go on to form the foetus. Apoptosis is likely to play a role in ensuring the genetic integrity of the foetus, yet little is known about its regulation. In this study, the role of the mouse gene, transformation-related protein 53 (Trp53) in the response of embryos to in vitro culture and environmentally induced DNA damage was investigated using embryos from a Trp53 knockout mouse model. In vivo-derived blastocysts were compared to control embryos X-irradiated at the two-cell stage and cultured to Day 5. An analysis of DNA by comet assay demonstrated that 1.5 Gy X-irradiation directly induced damage in cultured two-cell mouse embryos; this was correlated with retarded development to blastocyst stage and increased apoptosis at the blastocyst stage but not prior to this. Trp53 null embryos developed to blastocysts at a higher frequency and with higher cell numbers than wild-type embryos. Trp53 also mediates apoptosis in conditions of low levels of DNA damage, in vivo or in vitro in the absence of irradiation. However, following DNA damage induced by X-irradiation, apoptosis is induced by Trp53 independent as well as dependent mechanisms. These data suggest that Trp53 and apoptosis play important roles in normal mouse embryonic development both in vitro and in vivo and in response to DNA damage. Therefore, clinical ART practices that alter apoptosis in human embryos and/or select embryos for transfer, which potentially lack a functional Trp53 gene, need to be carefully considered.

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Analyses of apoptosis and DNA damage in bovine cumulus cells afterin vitromaturation with different copper concentrations: consequences on early embryo development

Zygote ◽

10.1017/s0967199416000204 ◽

2016 ◽

Vol 24 (6) ◽

pp. 869-879 ◽

Cited By ~ 4

Author(s):

D.E. Rosa ◽

J.M. Anchordoquy ◽

J.P. Anchordoquy ◽

M.A. Sirini ◽

J.A. Testa ◽

...

Keyword(s):

Dna Damage ◽

Embryo Development ◽

Cumulus Cells ◽

Blastocyst Stage ◽

Early Embryo ◽

Dna Integrity ◽

Cumulus Expansion ◽

Developmental Capacity

SummaryThe aim of this study was to investigate the influence of copper (Cu) duringin vitromaturation (IVM) on apoptosis and DNA integrity of cumulus cells (CC); and oocyte viability. Also, the role of CC in the transport of Cu during IVM was evaluated on oocyte developmental capacity. Damage of DNA was higher in CC matured without Cu (0 µg/dl Cu,P< 0.01) with respect to cells treated with Cu for cumulus–oocyte complexes (COCs) exposed to 0, 20, 40, or 60 µg/dl Cu). The percentage of apoptotic cells was higher in CC matured without Cu than in CC matured with Cu. Cumulus expansion and viability of CC did not show differences in COC treated with 0, 20, 40, or 60 µg/dl Cu during IVM. Afterin vitrofertilization (IVF), cleavage rates were higher in COC and DO + CC (denuded oocytes + CC) with or without Cu than in DO. Independently of CC presence (COC, DO + CC or DO) the blastocyst rates were higher when 60 µg/dl Cu was added to IVM medium compared to medium alone. These results indicate that Cu supplementation to IVM medium: (i) decreased DNA damage and apoptosis in CC; (ii) did not modify oocyte viability and cumulus expansion; and (iii) improved subsequent embryo development up to blastocyst stage regardless of CC presence during IVM.

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Discovery of pluripotency-associated microRNAs in rabbit preimplantation embryos and embryonic stem-like cells

Reproduction ◽

10.1530/rep-12-0259 ◽

2013 ◽

Vol 145 (4) ◽

pp. 421-437 ◽

Cited By ~ 12

Author(s):

Pouneh Maraghechi ◽

László Hiripi ◽

Gábor Tóth ◽

Babett Bontovics ◽

Zsuzsanna Bősze ◽

...

Keyword(s):

Embryonic Development ◽

Cell Biology ◽

Embryonic Stem ◽

Blastocyst Stage ◽

Expression Level ◽

Preimplantation Embryos ◽

Regulatory Function ◽

Low Level ◽

Non Coding Rnas

MicroRNAs (miRNAs) are small non-coding RNAs that regulate multiple biological processes. Increasing experimental evidence implies an important regulatory role of miRNAs during embryonic development and in embryonic stem (ES) cell biology. In the current study, we have described and analyzed the expression profile of pluripotency-associated miRNAs in rabbit embryos and ES-like cells. The rabbit specific ocu-miR-302 and ocu-miR-290 clusters, and three homologs of the human C19MC cluster (ocu-miR-512, ocu-miR-520e, and ocu-miR-498) were identified in rabbit preimplantation embryos and ES-like cells. The ocu-miR-302 cluster was highly similar to its human homolog, while ocu-miR-290 revealed a low level of evolutionary conservation with its mouse homologous cluster. The expression of the ocu-miR-302 cluster began at the 3.5 days post-coitum early blastocyst stage and they stayed highly expressed in rabbit ES-like cells. In contrast, a high expression level of the ocu-miR-290 cluster was detected during preimplantation embryonic development, but a low level of expression was found in rabbit ES-like cells. Differential expression of the ocu-miR-302 cluster and ocu-miR-512 miRNA was detected in rabbit trophoblast and embryoblast. We also found that Lefty has two potential target sites in its 3′UTR for ocu-miR-302a and its expression level increased upon ocu-miR-302a inhibition. We suggest that the expression of the ocu-miR-302 cluster is characteristic of the rabbit ES-like cell, while the ocu-miR-290 cluster may play a crucial role during early embryonic development. This study presents the first identification, to our knowledge, of pluripotency-associated miRNAs in rabbit preimplantation embryos and ES-like cells, which can open up new avenues to investigate the regulatory function of ocu-miRNAs in embryonic development and stem cell biology.

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The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽

10.1242/dev.107.3.597 ◽

1989 ◽

Vol 107 (3) ◽

pp. 597-604 ◽

Cited By ~ 13

Author(s):

K. Hardy ◽

A.H. Handyside ◽

R.M. Winston

Keyword(s):

Cell Death ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Blastocyst Stage ◽

Human Embryos ◽

Cell Numbers ◽

Human Blastocyst ◽

Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

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131 MODIFICATION OF AMINO ACID CONCENTRATIONS IN MEDIUM BY PIG EMBRYOS FROM THE ZYGOTE TO THE BLASTOCYST STAGE

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab131 ◽

2005 ◽

Vol 17 (2) ◽

pp. 216

Author(s):

P. Booth ◽

T. Watson ◽

H. Leese

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Embryonic Development ◽

Amino Acid Profile ◽

Blastocyst Stage ◽

Morula Stage ◽

Amino Acid Profiles ◽

The Difference ◽

Amino Acid Concentrations

Pre-implantation embryos can produce and consume amino acids in a manner dependent upon stage of embryonic development (Partridge and Leese 1996 Reprod. Fert. Dev. 8, 945) that may also be predictive of subsequent viability (Houghton et al. 2002 Hum. Reprod. 17, 999). To examine these relationships in the pig, the appearance or depletion of 18 amino acids from a presumptive near-physiological mixture was determined by HPLC in porcine in vitro-produced embryos from the zygote to the blastocyst stage. Cumulus oocyte complexes derived from slaughterhouse prepubertal pig ovaries were matured for 40 h in modified TCM-199 before being fertilized (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 medium modified to contain a physiological mixture of 18 amino acids including 0.1 mM glutamine (NCSUaa). Groups of 2–10 embryos (dependent on stage) were removed on Day 0 (1 cell), Day 1 (2- and 4-cell), Day 4 (compact morula), and Day 6 (blastocyst) and placed in 4 μL NCSUaa for 24 h. After incubation, the embryos were removed and the medium analyzed by HPLC. Each stage was replicated 3–9 times. Since amino acid profiles of 2- and 4-cell embryos were not different, data were combined. Overall, arginine (1.19 ± 0.33), glutamine (0.78 ± 0.34) and threonine (0.05 ± 0.04) were significantly (P < 0.01) depleted from the medium whereas alanine (0.21 ± 0.1), glycine (0.20 ± 0.06), asparagine (0.13 ± 0.5), lysine (0.1 ± 0.03), isoleucine (0.08 ± 0.01), valine (0.05 ± 0.01), leucine (0.04 ± 0.02), phenylalanine (0.03 ± 0.01), and histidine (0.02 ± 0.04) significantly (P < 0.05) accumulated (mean of the 4 sampling timepoints; all values pmol/embryo/h ± SEM). The difference between amino acid accumulation and depletion (balance) was approximately equivalent between Day 0 and the morula stage although turnover (sum of depletion and accumulation) steadily decreased during this period from 3.1 on Day 0 to 1.35 pmol/embryo/h at the morula stage. However, at the blastocyst stage, turnover and balance increased to 6.32 and 2.42 pmol/embryo/h, respectively, i.e. net appearance occurred. Notable changes in amino acid profile during development included decreases in accumulation of asparagine, glutamate, and glycine in the medium and the depletion of glutamine over Days 0, 1, and 4, followed by reversal of these trends by Day 6. These data suggest that pig embryos can alter the accumulation and depletion rates of amino acids in a manner that is dependent on the specific amino acid and the stage of embryonic development. This work was supported by BBSRC.

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Baicalin improves the in vitro developmental capacity of pig embryos by inhibiting apoptosis, regulating mitochondrial activity and activating sonic hedgehog signaling

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz036 ◽

2019 ◽

Vol 25 (9) ◽

pp. 538-549 ◽

Cited By ~ 3

Author(s):

Qing Guo ◽

Mei-Fu Xuan ◽

Zhao-Bo Luo ◽

Jun-Xia Wang ◽

Sheng-Zhong Han ◽

...

Keyword(s):

Embryonic Development ◽

Signaling Pathway ◽

Sonic Hedgehog ◽

Hedgehog Signaling ◽

Control Group ◽

Mitochondrial Activity ◽

Cell Stage ◽

Shh Signaling ◽

Developmental Capacity

Abstract Baicalin, a traditional Chinese medicinal monomer whose chemical structure is known, can be used to treat female infertility. However, the effect of baicalin on embryonic development is unknown. This study investigated the effects of baicalin on in vitro development of parthenogenetically activated (PA) and in vitro fertilized (IVF) pig embryos and the underlying mechanisms involved. Treatment with 0.1 μg/ml baicalin significantly improved (P < 0.05) the in vitro developmental capacity of PA pig embryos by reducing the reactive oxygen species (ROS) levels and apoptosis and increasing the mitochondrial membrane potential (ΔΨm) and ATP level. mRNA and protein expression of sonic hedgehog (SHH) and GLI1, which are related to the SHH signaling pathway, in PA pig embryos at the 2-cell stage, were significantly higher in the baicalin-treated group than in the control group. To confirm that the SHH signaling pathway is involved in the mechanism by which baicalin improves embryonic development, we treated embryos with baicalin in the absence or presence of cyclopamine (Cy), an inhibitor of this pathway. Cy abolished the effects of baicalin on in vitro embryonic development. In conclusion, baicalin improves the in vitro developmental capacity of PA and IVF pig embryos by inhibiting ROS production and apoptosis, regulating mitochondrial activity and activating SHH signaling.

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