167 EXPRESSION PATTERN OF GAP JUNCTIONAL CONNEXINS DURING IN VITRO AND IN VIVO PREIMPLANTATION EMBRYO DEVELOPMENT IN BOVINE

2008 ◽  
Vol 20 (1) ◽  
pp. 163
Author(s):  
S. Balasubramanian ◽  
W. J. Son ◽  
B. Mohana Kumar ◽  
Y. I. Yang ◽  
B. J. Jeon ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Expression Pattern ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
O2 Concentration ◽  
Gene Transcripts ◽  
High O2 ◽  
Gap Junctional

During preimplantation development, several connexin proteins are expressed and assembled into gap junctions in the plasma membrane at compaction but the functional significance of connexin diversity remains controversial. The present investigations were (i) to compare the expression pattern of a panel of gap junctional connexin (Cx) gene transcripts from in vitro-produced (IVP) under low (5%) and high (20%) oxygen (O2) concentrations and in vivo-derived (IVD) bovine embryos during various preimplantation stages, and (ii) to evaluate the expression of the same set of gene transcripts in blastocysts derived from IVP (low O2 concentration) and in vivo embryos following a conventional cryopreservation method using 1.5 m ethylene glycol (EG) (Hasler et al. 1997 Theriogenology 48, 563–579). Cumulus-oocyte complexes were matured in TCM199 supplemented with 10% FBS and hormones for 22 h at 39�C, 5% CO2 in air, and then inseminated and cultured in SOF medium for 7 days. IVD embryos were collected from 18 superovulated and artificially inseminated cows. In Experiment I, five pooled embryos from each developmental stage (2-, 4-, 8-, 16-cell, morula, and blastocyst) and embryo source (IVP under low and high O2 concentrations and IVD) and, in Experiment II, Day 7 IVP (low O2 concentration) and IVD blastocysts following cryopreservation and storage for at least 1 week were used for analyzing the expression pattern of gap junctional connexin (Cx30, Cx31, Cx32, Cx36, Cx43, and Cx45) gene transcripts using real-time RT-PCR (four replicates). Normalization of mRNAs at each developmental stage of bovine preimplantation embryos was performed by employing a similar amount of RNA at the reverse transcription (RT) step. This was followed by analyzing by qRT-PCR the target genes using GAPDH as a reference gene. Significant differences in gene expression were analyzed byANOVA and Student's t-test. Relative abundances (RA) of Cx30, Cx31, and Cx32 of IVD embryos were significantly (P < 0.05) higher at all stages compared to that of IVP embryos, except at the blastocyst stage for Cx32. Differences in Cx36 and Cx45 were observed at all stages, with the levels being higher (P < 0.05) in IVD than in IVP embryos. However, the differences at the 4-cell stage between the two embryo sources were not significant. The RA of Cx43 transcript in IVD embryos at 4- and 16-cell stages was higher (P < 0.05) than in IVP embryos, but did not differ at the 2-cell stage. Furthermore, there were differences at the 8-cell and blastocyst stages among IVD, and low and high O2 IVP embryos. Following cryopreservation, RA of all analyzed connexin transcripts of IVD blastocysts were significantly (P < 0.05) higher than that of the low O2 concentration. However, the expression levels in both embryo sources were lower compared to blastocysts before cryopreservation, except for Cx36. To the best of our knowledge, this is the first report on the differences in the expression pattern of a panel of gap junctional connexin gene transcripts during the key developmental stages of IVP embryos with direct comparisons of IVD counterparts.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

scholarly journals Effect of Increased Urea Levels on Mouse Preimplantation Embryos Developed in Vivo and in Vitro

2012 ◽  
Vol 56 (2) ◽  
pp. 211-216 ◽  
Author(s):  
Ján Bystriansky ◽  
Ján Burkuš ◽  
Štefan Juhás ◽  
Dušan Fabian ◽  
Juraj Koppel
Keyword(s):  
Apoptotic Cell ◽  
Nitrogen Concentration ◽  
High Plasma ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Plasma Urea ◽  
High Concentrations

Abstract High plasma urea nitrogen concentration has been proposed as an important factor contributing to the decline in reproductive parameters of domestic animals. The aim of this study was to evaluate the effect of urea on the development of preimplantation embryos in a mouse model. During in vivo tests, acute renal failure (ARF) accompanied by hyper-uraemia was induced by intramuscular administration of glycerol (50%) into hind limbs of fertilised dams. During in vitro tests, embryos collected from healthy dams were cultured in a medium with the addition of various concentrations of urea from the 4-cell stage to the blastocyst stage. Stereomicroscopic evaluation and fluorescence staining of embryos obtained from dams with ARF showed that high blood urea is connected with an increase in the number blastocysts containing at least one apoptotic cell and in the incidences of dead cells per blastocyst, but it did not affect their ability to reach the blastocyst stage. In vitro tests showed that culture of embryos with urea at concentration of 10 mM negatively affected the quality of obtained blastocysts. Blastocysts showed significantly lower numbers of cells and increased incidence of dead cells. An increase in apoptosis incidence was observed even in blastocysts obtained from cultures with 5 mM urea. Urea at concentrations 50 mM and higher negatively affected the ability of embryos to reach the blastocyst stage and the highest used concentrations (from 500 mM) caused overall developmental arrest of embryos at the 4- or 5- cell stage. These results show that elevated levels of urea may cause changes in the microenvironment of developing preimplantation embryos, which can negatively affect their quality. Embryo growth remains un-affected up to very high concentrations of urea.

scholarly journals Coordinated Formation of IMPDH2 Cytoophidium in Mouse Oocytes and Granulosa Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Shiwen Ni ◽  
Teng Zhang ◽  
Chenmin Zhou ◽  
Min Long ◽  
Xuan Hou ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
De Novo ◽  
Biological Significance ◽  
Preimplantation Embryos ◽  
Inosine Monophosphate Dehydrogenase ◽  
Cell Stage ◽  
Simultaneous Formation

Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme catalyzing de novo biosynthesis of guanine nucleotides, aggregates under certain circumstances into a type of non-membranous filamentous macrostructure termed “cytoophidium” or “rod and ring” in several types of cells. However, the biological significance and underlying mechanism of IMPDH assembling into cytoophidium remain elusive. In mouse ovaries, IMPDH is reported to be crucial for the maintenance of oocyte–follicle developmental synchrony by providing GTP substrate for granulosa cell natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system to produce cGMP for sustaining oocyte meiotic arrest. Oocytes and the associated somatic cells in the ovary hence render an exciting model system for exploring the functional significance of formation of IMPDH cytoophidium within the cell. We report here that IMPDH2 cytoophidium forms in vivo in the growing oocytes naturally and in vitro in the cumulus-enclosed oocytes treated with IMPDH inhibitor mycophenolic acid (MPA). Inhibition of IMPDH activity in oocytes and preimplantation embryos compromises oocyte meiotic and developmental competences and the development of embryos beyond the 4-cell stage, respectively. IMPDH cytoopidium also forms in vivo in the granulosa cells of the preovulatory follicles after the surge of luteinizing hormone (LH), which coincides with the resumption of oocyte meiosis and the reduction of IMPDH2 protein expression. In cultured COCs, MPA-treatment causes the simultaneous formation of IMPDH cytoopidium in cumulus cells and the resumption of meiosis in oocytes, which is mediated by the MTOR pathway and is prevented by guanosine supplementation. Therefore, our results indicate that cytoophidia do form in the oocytes and granulosa cells at particular stages of development, which may contribute to the oocyte acquisition of meiotic and developmental competences and the induction of meiosis re-initiation by the LH surge, respectively.

scholarly journals Involvement of Nlrp9a/b/c in mouse preimplantation development

Reproduction ◽  
2020 ◽  
Vol 160 (2) ◽  
pp. 181-191 ◽  
Author(s):  
Satoko Kanzaki ◽  
Shiori Tamura ◽  
Toshiaki Ito ◽  
Mizuki Wakabayashi ◽  
Koji Saito ◽  
...  
Keyword(s):  
Asymmetric Cell Division ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Dependent Manner ◽  
Blastocyst Development ◽  
Triple Mutant ◽  
Cell Stage

Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing proteins (NRLPs) are central components of the inflammasome. Accumulating evidence has shown that a reproductive clade of NRLPs is predominantly expressed in oocyte to cleavage stage embryos and participates in mammalian preimplantation development as a component of a multiprotein complex known as the subcortical maternal complex (SCMC). Nlrp9s belong to the reproductive class of NLRPs; Nlrp9b is unique in acting as an inflammasome against rotavirus in intestines. Here we generated mice carrying mutations in all three members of the Nlrp9a/b/c gene (Nlrp9 triple mutant (TMut) mice). When crossed with WT males, the Nlrp9 TMut females were fertile, but deliveries with fewer pups were increased in the mutants. Consistent with this, blastocyst development was retarded and lethality to the preimplantation embryos increased in the Nlrp9 TMut females in vivo. Under in vitro culture conditions, the fertilized eggs from the Nlrp9 TMut females exhibited developmental arrest at the two-cell stage, accompanied by asymmetric cell division. By contrast, double-mutant (DMut) oocytes (any genetic combination) did not exhibit the two-cell block in vitro, showing the functional redundancy of Nlrp9a/b/c. Finally, Nlrp9 could bind to components of the SCMC. These results show that Nlrp9 functions as an immune or reproductive NLRP in a cell-type-dependent manner.

170 EXPRESSION PATTERN OF THE Sox2 GENE IN BOVINE OOCYTES AND IN VITRO-DERIVED EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 165 ◽  
Author(s):  
T. A. L. Brevini ◽  
S. Antonini ◽  
F. Cillo ◽  
G. Pennarossa ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Inner Cell Mass ◽  
False Negative ◽  
Cell Mass ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Developmental Potential ◽  
Sox2 Expression ◽  
Bovine Oocytes

Sox2 is a member of the Sox (SRY-related HMGbox) family. It acts to maintain developmental potential and marks the pluripotent lineage of the early mouse embryo; in particular, as in the case of Oct-4 and Nanog, Sox2 is expressed specifically in the inner cell mass (ICM) and in the epiblast of this species. Moreover, it plays an important role in the transcription network that maintains stem cell pluripotency, interacting with other factors such as Oct-4 and Nanog. Little information is available on this gene in bovine; therefore aims of the present study were: a) to identify and characterize the Sox2 expression profile in bovine oocytes and preimplantation embryos; and b) to investigate its expression pattern in ICM and trophectoderm (TE). Bovine oocytes and embryos were obtained by in vitro maturation and fertilization; blastocysts at Day 7 post-insemination underwent microsurgery to separate TE from ICM. mRNA was isolated from 3 pools, each consisting of 5 MII oocytes, 2-, 4-, 8-, and 16-cell embryos, morulae, blastocysts, ICMs, and TEs. Semi-quantitative analysis of Sox2 expression was performed in the exponential phase of PCR amplification using rabbit globin as exogenous control. Data were analyzed with one-way ANOVA, followed by multiple pairwise comparisons with Tukey test (SigmaStat 2.03, SPSS, Inc., Chicago, IL, USA). Values are presented as mean � SEM and differences of P ≤ 0.05 are considered significant. In order to rule out false negative results, PCR amplifications of isolated ICMs and TEs were extended to the plateau phase. Fragment identity was confirmed by sequencing. Comparison of bovine Sox2 cDNA sequence (EMBL AM774325) with databases revealed a 98%, 93%, and 87% homology with sheep, human, and mouse, respectively. Sox2 mRNA was detectable in oocytes as well as in embryos at the different developmental stages analyzed. Semi-quantitative expression studies revealed that Sox2 was present as both maternal and embryonic transcript; in particular, a statistically significant increase from the 8-cell stage, concomitant with embryo genome activation, was observed. Differently from the mouse, Sox2 was expressed in both bovine ICM and TE, resembling the profile previously shown for Oct-4 (van Eijk et al. 1999 Biol. Reprod. 60, 1093–1103), and suggesting that Sox2 expression might be regulated by Oct-4 also in bovine, as described in mouse and human. These findings also suggest that its expression may become restricted to the ICM only at the expanded hatched stage, as previously described for Oct-4 in pig embryos (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718). This work was supported by PRIN 2006, FIRST 2005, TECLA-MIUR, and EUROSTELLS-ESF.

scholarly journals Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 546 ◽  
Author(s):  
Masahiro Sato ◽  
Rico Miyagasako ◽  
Shuji Takabayashi ◽  
Masato Ohtsuka ◽  
Izuho Hatada ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Preimplantation Embryos ◽  
Nucleic Acid Delivery ◽  
Cell Stage ◽  
Initial Development ◽  
Genetic Manipulations ◽  
Target Sites

Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4–1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4–1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called “sequential i-GONAD (si-GONAD).”

Expression pattern of GLUT 1, 5, 8 and citrate synthase transcripts in buffalo ( Bubalus bubalis ) preimplantation embryos produced in vitro and derived in vivo

2020 ◽  
Vol 55 (10) ◽  
pp. 1362-1370
Author(s):  
Gutulla Taru Sharma ◽  
Amar Nath ◽  
Shiv Prasad ◽  
Sumit Singhal ◽  
Vikash Chandra ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Citrate Synthase ◽  
Bubalus Bubalis ◽  
Preimplantation Embryos ◽  
Glut 1

scholarly journals Ribosomal RNA gene expression and chromosome aberrations in bovine oocytes and preimplantation embryos

Reproduction ◽  
2001 ◽  
pp. 21-30 ◽  
Author(s):  
P Hyttel ◽  
D Viuff ◽  
T Fair ◽  
J Laurincik ◽  
PD Thomsen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Aberrations ◽  
Rrna Genes ◽  
Preimplantation Embryos ◽  
Fertilization In Vitro ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oocyte Growth

This review focuses on the key features of development of the bovine oocyte and embryo, with comparisons of the developmental characteristics of embryos produced in vivo and in vitro. The oocyte is transcriptionally quiescent in the primordial and primary follicle. In the secondary follicle transcription is initiated in the oocyte and a ribosome-synthesizing nucleolus is established in this cell. Transcription and nucleolar activity are enhanced in the tertiary follicle during oocyte growth. When the oocyte reaches approximately 110 microm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription ceases and the nucleolus is inactivated, forming a dense spherical remnant. During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, it disperses. The rRNA genes are apparently re-activated during the four-cell stage, that is, the third cell cycle after fertilization, but a nucleolus is not formed. During the subsequent cell cycle, that is, during the eight-cell stage, ribosome-synthesizing nucleoli are again established. Bovine embryos produced in vitro apparently display the same pattern of nucleolus development as that in embryos developed in vivo. Examination of the ploidy of embryonic cells using fluorescence in situ hybridization has revealed that the production of bovine embryos in vitro is associated with increased chromosome aberrations in the embryos. Blastocysts produced in vitro display a significantly higher rate of mixoploidy, that is, when the embryo consists of both normal diploid and abnormal polyploid cells, than that in embryos developed in vivo. The rate of mixoploidy among embryos produced in vitro increases with increasing developmental stage. Moreover, after fertilization in vitro, initially there is a high rate of 'true' polyploidy, that is, when all cells of the embryos are polyploid. However, the polyploid embryos are eliminated before they cleave beyond the eight-cell stage, the stage at which major activation of the embryonic genome occurs in cattle.

In vivo and in vitro cultured mouse preimplantation embryos differ in their display of a teratocarcinoma cell surface antigen: possible binding of an oviduct factor

Development ◽  
1985 ◽  
Vol 88 (1) ◽  
pp. 55-69
Author(s):  
Stephen J. Gaunt
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Surface Antigen ◽  
Antigenic Site ◽  
Cell Surface Antigen ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Teratocarcinoma Cell

By use of a monoclonal antibody, 2B5, in indirect immunofluorescence experiments, it was found that both fertilized and unfertilized mouse eggs obtained directly from the oviduct commenced expression of a cell surface antigen at about 5h after ovulation. Surface labelling became intense by 16 h after ovulation and persisted over all blastomeres throughout preimplantation development. In contrast, embryos cultured in vitro did not show appearance of 2B5 antigen until about 48 h after ovulation, at which time they were at the 2- to 4-cell stage. Antigen expression in vitro commonly began on a single blastomere and did not appear consistently over all blastomeres until the 8-cell stage (72h after ovulation). Unfertilized eggs maintained for 72 h in culture did not acquire 2B5 antigen. It is postulated that the absence of 2B5 antigen on 1-cell eggs cultured in vitro may be due either to a failure of normal synthesis by eggs as a result of a deficiency in the culture medium, or alternatively, to absence of a soluble oviduct factor which carries the 2B5 antigen, and which normally becomes bound to the surface of eggs after ovulation. The second of these two possibilities was supported by egg transfer experiments which showed that unfertilized eggs within the oviduct became 2B5 antigenpositive even after their prior fixation in glutaraldehyde. By the 2- to 4-cell stage, however, embryos developed their own capacity for synthesis of 2B5 antigen-positive cell surface molecules. This synthesis was inhibited by tunicamycin, suggesting that the antigenic site involved the sugar component of glycoprotein. The range of tissues within the postimplantation embryo and adult reproductive tracts which labelled with 2B5 antibody was found to be very similar to that known for SSEA-1 monoclonal antibody (Solter & Knowles, 1978; Fox et al. 1981; Fox, Damjanov, Knowles & Solter, 1982), and as further evidence of a relationship between 2B5 and SSEA-1 antigens it was found that 125I SSEA-1 antibody could be blocked in its binding to teratocarcinoma cells by preincubation in 2B5 monoclonal antibody.

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