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

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).”

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Successful i-GONAD in Brown Norway Rats by Modification of in vivo Electroporation Conditions

10.21926/obm.genet.2004121 ◽

2020 ◽

Vol 4 (4) ◽

Author(s):

Shuji Takabayashi ◽

◽

Takuya Aoshima ◽

Yukari Kobayashi ◽

Hisayoshi Takagi ◽

...

Keyword(s):

Genome Editing ◽

Ex Vivo ◽

Preimplantation Embryos ◽

Brown Norway ◽

Sprague Dawley ◽

In Vivo Electroporation ◽

Nucleic Acids Delivery ◽

A Current

Improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) was developed for in situ genome editing of the preimplantation embryos present within the oviductal lumen of mice. This method is based on intra-oviductal instillation of genome editing components and subsequent in vivo electroporation (EP) in the entire oviduct. Therefore, i-GONAD differs from the previous methods (i.e., zygote microinjection and in vitro EP) in producing genome-edited mice, which relied on ex vivo handling of preimplantation embryos and egg transfer to the recipient females. We have previously demonstrated that i-GONAD can be successfully applied to produce genome-edited rats, including albino Sprague-Dawley and albino Lewis rats (however, not pigmented Brown Norway [BN] rats). We observed that the successful i-GONAD was dependent on the mouse strain used; for example, in random-bred mice, such as ICR and C3H/He × C57BL/6, it was successful under relatively stringent electrical conditions but not in the C57BL/6 strain. Under less stringent conditions, i-GONAD was successful in the C57BL/6 strain. We speculated that this would also be true for i-GONAD using BN rats. On applying a current of >500 mA, we failed to obtain rat offspring (fetuses/newborns); however, i-GONAD under a current of 100-300 mA using NEPA21 (NEPA GENE) led to the production of genome-edited BN rats with efficiencies of 75%-100%. Similarly, i-GONAD, under a current of 150-200 mA using CUY21EDIT II (BEX Co.) led to the production of genome-edited BN rats with efficiencies of 24%-55%. These experiments suggest the importance of selecting the appropriate current value, depending on the rat strain used, when performing i-GONAD.

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

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab180 ◽

2008 ◽

Vol 20 (1) ◽

pp. 169 ◽

Cited By ~ 1

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

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

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/v10213-012-0038-9 ◽

2012 ◽

Vol 56 (2) ◽

pp. 211-216 ◽

Cited By ~ 1

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.

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Coordinated Formation of IMPDH2 Cytoophidium in Mouse Oocytes and Granulosa Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.690536 ◽

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.

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Involvement of Nlrp9a/b/c in mouse preimplantation development

Reproduction ◽

10.1530/rep-19-0516 ◽

2020 ◽

Vol 160 (2) ◽

pp. 181-191 ◽

Cited By ~ 2

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.

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Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain

Cells ◽

10.3390/cells9040957 ◽

2020 ◽

Vol 9 (4) ◽

pp. 957 ◽

Cited By ~ 1

Author(s):

Yukari Kobayashi ◽

Takuya Aoshima ◽

Ryota Ito ◽

Ryota Shinmura ◽

Masato Ohtsuka ◽

...

Keyword(s):

Genome Editing ◽

Ex Vivo ◽

Inbred Mouse ◽

Inbred Mouse Strain ◽

Constant Current ◽

Mouse Strains ◽

Nucleic Acid Delivery ◽

Pregnant Females ◽

Novel Method

Improved genome editing via oviductal nucleic acid delivery (i-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. i-GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins via the oviductal wall of pregnant females at 0.7 days post-coitum, followed by in vivo electroporation (EP). Unlike outbred Institute of Cancer Research (ICR) and hybrid mouse strains, genome editing of the most widely used C57BL/6J (B6) strain with i-GONAD has been considered difficult but, recently, setting a constant current of 100 mA upon EP enabled successful i-GONAD in this strain. Unfortunately, the most widely used electroporators employ a constant voltage, and thus we explored conditions allowing the generation of a 100 mA current using two electroporators: NEPA21 (Nepa Gene Co., Ltd.) and GEB15 (BEX Co., Ltd.). When the current and resistance were set to 40 V and 350–400 Ω, respectively, the current was fixed to 100 mA. Another problem in using B6 mice for i-GONAD is the difficulty in obtaining pregnant B6 females consistently because estrous females often fail to be found. A single intraperitoneal injection of low-dose pregnant mare’s serum gonadotrophin (PMSG) led to synchronization of the estrous cycle of these mice. Consequently, approximately 51% of B6 females had plugs upon mating with males 2 days after PMSG administration, which contrasts with the case (≈26%) when B6 females were subjected to natural mating. i-GONAD performed on PMSG-treated pregnant B6 females under conditions of average resistance of 367 Ω and average voltage of 116 mA resulted in the production of pregnant females at a rate of 56% (5/9 mice), from which 23 fetuses were successfully delivered. Nine (39%) of these fetuses exhibited successful genome editing at the target locus.

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

Reproduction ◽

10.1530/rep.0.1220021 ◽

2001 ◽

pp. 21-30 ◽

Cited By ~ 54

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.

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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 ◽

10.1242/dev.88.1.55 ◽

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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Consumption of amino acids by bovine preimplantation embryos

Reproduction Fertility and Development ◽

10.1071/rd9960945 ◽

1996 ◽

Vol 8 (6) ◽

pp. 945 ◽

Cited By ~ 60

Author(s):

RJ Partridge ◽

HJ Leese

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Developmental Stages ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Cell Stage ◽

Amino Acid Requirement ◽

Zygote Stage

Bovine embryos produced in vitro from the putative zygote stage to the blastocyst stage, and blastocysts freshly flushed from the uterus, were cultured in a physiological mixture of amino acids. Depletion of amino acids from the medium and, in a few cases, their appearance, was measured by high performance liquid chromatography. Amino acids were depleted at widely differing rates. The depletion of amino acids was higher when embryos at later developmental stages were cultured, implying an increase in amino acid requirement with development. Threonine was the only amino acid to be depleted at all stages of development; depletion increased from 0.18 +/- 0.07 pmol embryo-1 h-1 at the putative zygote stage to 1.96 +/- 0.49 pmol embryo-1 h-1 at the blastocyst stage. Glutamine was depleted at the putative zygote stage and the 4-cell stage (0.76 +/- 0.05 and 0.94 +/- 0.10 pmol embryo-1 h-1 respectively), but was not significantly depleted at the later stages. Alanine was the only amino acid that appeared consistently in the medium and its production increased progressively throughout development. Aspartate, glutamate, threonine and lysine were depleted significantly by blastocysts derived both in vitro and in vivo; the embryos in vivo also depleted arginine, phenylalanine, isoleucine and tyrosine. These results indicate that individual amino acids are depleted at different rates by bovine preimplantation embryos and suggest that amino acid requirements change during development.

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

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab167 ◽

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.

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