scholarly journals Glycine receptor α4 subunit facilitates the early embryonic development in mice

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 41 ◽  
Author(s):  
Hirofumi Nishizono ◽  
Mohamed Darwish ◽  
Takaho A Endo ◽  
Kyosuke Uno ◽  
Hiroyuki Abe ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Litter Size ◽  
Glycine Receptor ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Β Subunit ◽  
Neurotransmitter Receptor ◽  
Oviduct Fluid ◽  
Fertilized Egg ◽  
Number Of Cells

Oviduct fluid is essential for the fertilization and subsequent preimplantation development. Glycine is abundant in oviduct fluid and is reported to be critical for preimplantation development of fertilized eggs in mammals. However, the mechanism by which glycine exerts its action on fertilized eggs is yet to be understood. Here we show that glycine regulates the preimplantation development of mouse fertilized eggs via glycine receptors. Among them, the alpha-4 subunit (Glra4) and the β subunit are expressed in mouse fertilized eggs, and lacking Glra4 inhibits embryonic development to the blastocyst stage, decreases the number of cells in the blastocysts and the litter size. Thus, we identify a novel function of the glycine receptor, which is considered to act mainly as a neurotransmitter receptor, as a regulator of embryonic development and our data provide new insights into the interactions between oviduct milieu and mammalian fertilized egg.

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

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.

scholarly journals A Novel Glycine Receptor β Subunit Splice Variant Predicts an Unorthodox Transmembrane Topology

2006 ◽  
Vol 282 (5) ◽  
pp. 2798-2807 ◽  
Author(s):  
Jana Oertel ◽  
Carmen Villmann ◽  
Helmut Kettenmann ◽  
Frank Kirchhoff ◽  
Cord-Michael Becker
Keyword(s):  
Splice Variant ◽  
Glycine Receptor ◽  
Β Subunit ◽  
Transmembrane Topology

The effect of melatonin treatment during the seasonal anoestrus on the superovulatory response and embryo production of high-prolificacy Rasa Aragonesa ewes before culling

2003 ◽  
Vol 2003 ◽  
pp. 82-82
Author(s):  
F. Forcada ◽  
J.A. Abecia ◽  
J.A. Valares
Keyword(s):  
Embryonic Development ◽  
Breeding Season ◽  
Litter Size ◽  
Direct Effect ◽  
Ovulation Rate ◽  
Corpora Lutea ◽  
Melatonin Treatment ◽  
Embryo Production ◽  
Spring Equinox

The efficacy of melatonin implants inserted around the spring equinox to improve fertility and ovulation rate or litter size in Mediterranean ewes has been previously reported (Chemineau et al., 1996; Forcada et al., 2002a.), indicating the ability of the hormone to regulate the hypothalamic activity (Viguié et al., 1995). Moreover, a direct effect of melatonin on corpora lutea and embryonic development has been also reported (Wallace et al., 1988; Abecia et al., 2002). The use of prolific Rasa Aragonesa (RA) ewes (a Mediterranean breed) before culling as embryo donors has been previously tested in the breeding season (Forcada et al., 2002b.). The aim of this experiment was to improve embryo production during the seasonal anoestrus period in selected superovulated RA ewes at the end of their reproductive lives through the use of melatonin.

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

Reproduction ◽  
2013 ◽  
Vol 145 (4) ◽  
pp. 421-437 ◽  
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.

Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development

Development ◽  
1992 ◽  
Vol 115 (1) ◽  
pp. 305-312
Author(s):  
M. Aghayan ◽  
L.V. Rao ◽  
R.M. Smith ◽  
L. Jarett ◽  
M.J. Charron ◽  
...  
Keyword(s):  
Western Blot ◽  
Cellular Localization ◽  
Glucose Transporter ◽  
Developmental Stages ◽  
Molecular Level ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Developmental Expression

Two general mechanisms mediate glucose transport, one is a sodium-coupled glucose transporter found in the apical border of intestinal and kidney epithelia, while the other is a sodium-independent transport system. Of the latter, several facilitated transporters have been identified, including GLUT1 (erythrocyte/brain), GLUT2 (liver) and GLUT4 (adipose/muscle) isoforms. In this study, we used Western-blot analysis and high resolution immunoelectron microscopy (IEM) to investigate the stage-related expression and cellular localization of GLUT1, 2 and 4. The Western blot results demonstrate that GLUT1 is detectable in the oocyte and throughout preimplantation development. GLUT2 isoforms were not detectable until the blastocyst stage, while the GLUT4 isoform was undetectable in the oocyte through blastocyst stages. The present findings confirm previous studies at the molecular level which demonstrated that mRNAs encoding the same GLUT isoforms are detectable at corresponding developmental stages. GLUT1 and GLUT2 display different cellular distributions at the blastocyst stage as shown by IEM studies. GLUT1 has a widespread distribution in both trophectoderm and inner cell mass cells, while GLUT2 is located on trophectoderm membranes facing the blastocyst cavity. This observation suggests a different functional significance for these isoforms during mouse preimplantation development.

The roles of pyruvate, lactate and glucose during preimplantation development of embryos from F1 hybrid mice in vitro

Development ◽  
1991 ◽  
Vol 112 (1) ◽  
pp. 99-105 ◽  
Author(s):  
J.J. Brown ◽  
D.G. Whittingham
Keyword(s):  
Inbred Mouse ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
F1 Hybrids ◽  
Mouse Strains ◽  
F1 Hybrid ◽  
Morula Stage ◽  
Lactate And Pyruvate ◽  
Hybrid Mice

Embryos of certain inbred mouse strains, and their F1 hybrids, are able to develop from the 1-cell to blastocyst stage in simple chemically defined media containing lactate (L), pyruvate (P) and glucose (G). The individual roles of these substrates in supporting complete preimplantation development in vitro was examined with 1-cell F2 embryos from B6CBF1 hybrid mice. Embryos collected between 26 and 27 h post hCG were cultured in medium containing L, P, LP or LPG. After 50 h in culture, the proportions developing to the morula stage were 1%, 83%, 94% and 100%, respectively. In combination, lactate and pyruvate appeared to act synergistically and both the rate and level of development to the morula stage were unaffected by the absence of glucose. After a further 46 h in culture, only the embryos grown in the presence of glucose developed into blastocysts. In LP medium, embryos arrested at the compacted morula stage late on day 3 of development. As culture continued in the absence of glucose, embryos decompacted (approximately 82 h post hCG) and subsequently degenerated. Exposure to medium containing glucose for the first, second or third 24 h period in culture was sufficient to support the morula-to-blastocyst transition. Glucose still supported this transition when embryos were transferred to LPG medium 3 h after the completion of compaction (76 h post hCG), but was ineffective 6 h later (82 h post hCG) once decompaction had commenced. We conclude that lactate and pyruvate together are able to support normal development of 1-cell F2 embryos to the morula stage in vitro, but that glucose is an essential component of the culture medium for development to the blastocyst stage.

A method for isolating uncontaminated nuclei from all stages of developing Xenopus laevis embryos*

Development ◽  
1978 ◽  
Vol 48 (1) ◽  
pp. 101-108
Author(s):  
F. Farzaneh ◽  
C. K. Pearson
Keyword(s):  
Embryonic Development ◽  
Xenopus Laevis ◽  
Protein Content ◽  
Early Cleavage ◽  
Histone Protein ◽  
Pigment Granules ◽  
Xenopus Laevis Embryos ◽  
Number Of Cells

A method for isolating nuclei from Xenopus laevis embryos has been developed. This procedure enables the isolation of nuclei, free from contamination with yolk and pigment granules, at all stages of embryonic development. Using this method the nuclear yield is 60–70% of the estimated number of cells in the embryo. The DNA, RNA, histone and non-histone protein content of these nuclei during embryogenesis (from early cleavage to the swimming tadpole stage) has been measured.

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

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.

196 A NEW APPROACH TO PREDICT MOUSE EMBRYONIC DEVELOPMENTAL COMPETENCE USING A TIME-LAPSE SYSTEM AND THE 'SHORTEST HALF' analysis procedure

2007 ◽  
Vol 19 (1) ◽  
pp. 214 ◽  
Author(s):  
S. Yavin ◽  
A. Aroyo ◽  
Z. Roth ◽  
A. Arav
Keyword(s):  
Embryonic Development ◽  
Time Lapse ◽  
Time Frame ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Embryo Morphology ◽  
Cell Stage ◽  
Developmental Potential ◽  
Additional Tool ◽  
Embryonic Cleavage

Embryonic development is a dynamic process in which embryo morphology may change immensely within several hours. Therefore, identifying and selecting embryos with the highest probability of developing and achieving a pregnancy is a major challenge. The timing of embryonic cleavage may serve as an additional indicator for the identification of quality embryos. The aim of this study was to characterize the cleavage timing of mouse embryos and to identify the stage that is most indicative of blastocyst formation. Mated mice (CB6F1) were sacrificed 20 h after hCG administration; putative zygotes were recovered and cultured (50 embryos in each 20-µL drop of M16) in a time-lapse system (EmbryoGuard; IMT, Ltd., Ness-Ziona, Israel) inside the incubator. The time-lapse system was programmed to take photos at half-hour intervals such that culture dishes were not removed from the incubator. The ‘shortest half’ statistical procedure of JMPIN (SAS Institute, Inc., Cary, NC, USA) was utilized to evaluate the period during which at least 50% of the embryonic population cleaves within the shortest time frame. Captured images made it possible to search along the time axis for the densest 50% of cleavage observations. Developing embryos were categorized into 3 groups according to the time of cleavage after hCG administration: before, during, and after the ‘shortest half’ for each developmental stage. Two hundred thirty putative zygotes cleaved and created 2-cell-stage embryos, of which 55 arrested at various stages and 175 progressed to the blastocyst stage. During embryonic development, cleavage timing appeared to become less uniform and the ‘shortest half’ became longer for each successive cell division: Whereas the shortest period in which 50% of the 2-cell-stage embryos cleaved was a 2-h interval, cleavage into the 4-cell, 8-cell, and blastocyst stages took 2.5, 3.5, and 5 h, respectively. The ‘short half’ for the first cleavage appears to be a predictive time frame for subsequent embryonic development, because cleavage was closely synchronized with 80% of the embryos developing to the blastocyst stage. Note that only a small number of embryos were actually cleaving early, while the ‘shortest half’ consisted of 50% of the embryonic population. Moreover, late-cleaving embryos in the 2-cell stage expressed inferior developmental potential relative to those that cleaved within the ‘shortest half’ (see Table 1). In summary, 2-cell-stage embryos that cleaved within the ‘shortest half’ seemed to be better synchronized and consequently more competent than the rest of the embryonic population. Embryonic cleavage timing using the ‘shortest half’ parameter can be considered a biological indicator of embryo potential. It may be useful as an additional tool for selecting embryos for transfer and cryopreservation. Table 1. Cleavage timing distribution into the 2-cell stage according to the shortest half

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