scholarly journals Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain, embryo stage, sodium and the hexose transport inhibitor, phloridzin

Reproduction ◽  
2003 ◽  
pp. 111-118 ◽  
Author(s):  
BD Higgins ◽  
MT Kane
Keyword(s):  
Gene Activation ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Sodium Dependent ◽  
Morula Stage ◽  
Zygotic Gene ◽  
Zygotic Gene Activation ◽  
The One ◽  
Inositol Uptake

The uptake of myo-inositol by mouse oocytes and preimplantation embryos of a crossbred (DBA x C57BL/6) and a purebred outbred strain (MF1) was measured using [2-(3)H]myo-inositol. Uptake in crossbred embryos increased about 15-fold between the one- and two-cell stages and increased again by about sixfold at the blastocyst stage compared with the morula stage. Uptake in purebred embryos increased about 42-fold between the one- and two-cell stages and increased more than threefold at the blastocyst stage compared with the morula stage. In all stages examined, except two-cell crossbred embryos, inositol uptake was, depending on the stage, either largely or partly sodium dependent and could be inhibited by the sodium-dependent hexose transport inhibitor, phloridzin. This is consistent with the hypothesis that transport occurs via a sodium myo-inositol transporter (SMIT) protein. In addition, there was strong evidence that a sodium-independent mechanism of uptake, possibly a channel, was switched on at the two-cell stage coincident with zygotic gene activation which resulted in 141-fold and 71-fold increases in sodium-independent uptake from the one-cell to two-cell stages in crossbred and purebred embryos, respectively. This mechanism was either abolished or drastically downregulated at the blastocyst stage, whereas sodium-dependent uptake was markedly upregulated. In two-cell crossbred embryos, there was a complete abolition of sodium-dependent uptake, again possibly regulated by zygotic gene activation. The hypothesis that the changes in mechanism of inositol uptake at about the two-cell stage are due to zygotic gene activation was supported by the finding that these changes did not occur in parthenogenetic two-cell embryos.

scholarly journals Minor zygotic gene activation is essential for mouse preimplantation development

2018 ◽  
Vol 115 (29) ◽  
pp. E6780-E6788 ◽  
Author(s):  
Ken-ichiro Abe ◽  
Satoshi Funaya ◽  
Dai Tsukioka ◽  
Machika Kawamura ◽  
Yutaka Suzuki ◽  
...  
Keyword(s):  
Dna Replication ◽  
Gene Activation ◽  
Marked Change ◽  
Cell Stage ◽  
Maternal Mrna ◽  
Maternal To Zygotic Transition ◽  
Before And After ◽  
H3k4me3 Mark ◽  
Zygotic Gene ◽  
Zygotic Gene Activation

In mice, transcription initiates at the mid-one-cell stage and transcriptional activity dramatically increases during the two-cell stage, a process called zygotic gene activation (ZGA). Associated with ZGA is a marked change in the pattern of gene expression that occurs after the second round of DNA replication. To distinguish ZGA before and after the second-round DNA replication, the former and latter are called minor and major ZGA, respectively. Although major ZGA are required for development beyond the two-cell stage, the function of minor ZGA is not well understood. Transiently inhibiting minor ZGA with 5, 6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB) resulted in the majority of embryos arresting at the two-cell stage and retention of the H3K4me3 mark that normally decreases. After release from DRB, at which time major ZGA normally occurred, transcription initiated with characteristics of minor ZGA but not major ZGA, although degradation of maternal mRNA normally occurred. Thus, ZGA occurs sequentially starting with minor ZGA that is critical for the maternal-to-zygotic transition.

scholarly journals Embryo manipulation via assisted reproductive technology and epigenetic asymmetry in mammalian early development

2013 ◽  
Vol 368 (1609) ◽  
pp. 20120353 ◽  
Author(s):  
Takashi Kohda ◽  
Fumitoshi Ishino
Keyword(s):  
Assisted Reproductive Technology ◽  
Early Stage ◽  
Gene Activation ◽  
Reproductive Technology ◽  
Blastocyst Stage ◽  
Mammalian Development ◽  
Cell Stage ◽  
Genome Wide ◽  
Zygotic Gene ◽  
Developmental Risks

The early stage of mammalian development from fertilization to implantation is a period when global and differential changes in the epigenetic landscape occur in paternally and maternally derived genomes, respectively. The sperm and egg DNA methylation profiles are very different from each other, and just after fertilization, only the paternally derived genome is subjected to genome-wide hydroxylation of 5-methylcytosine, resulting in an epigenetic asymmetry in parentally derived genomes. Although most of these differences are not present by the blastocyst stage, presumably due to passive demethylation, the maintenance of genomic imprinting memory and X chromosome inactivation in this stage are of critical importance for post-implantation development. Zygotic gene activation from paternally or maternally derived genomes also starts around the two-cell stage, presumably in a different manner in each of them. It is during this period that embryo manipulation, including assisted reproductive technology, is normally performed; so it is critically important to determine whether embryo manipulation procedures increase developmental risks by disturbing subsequent gene expression during the embryonic and/or neonatal development stages. In this review, we discuss the effects of various embryo manipulation procedures applied at the fertilization stage in relation to the epigenetic asymmetry in pre-implantation development. In particular, we focus on the effects of intracytoplasmic sperm injection that can result in long-lasting transcriptome disturbances, at least in mice.

scholarly journals piRNA pathway is essential for generating functional oocytes in golden hamster

2021 ◽  
Author(s):  
Hongdao Zhang ◽  
Fengjuan Zhang ◽  
Jinghua Chen ◽  
Mingzhi Li ◽  
Xiaolong Lv ◽  
...  
Keyword(s):  
Golden Hamster ◽  
Small Rnas ◽  
Gene Activation ◽  
Endogenous Retroviruses ◽  
Cell Stage ◽  
Generating Functional ◽  
Early Embryos ◽  
Zygotic Gene ◽  
Zygotic Gene Activation ◽  
Pirna Pathway

AbstractPiwi-interacting RNAs (piRNAs) are small RNAs predominantly expressed in germ cells that are critical for gametogenesis in various species. However, PIWI-deficient female mice are fertile and mouse oocytes express a panel of small RNAs that do not appear widely representative of mammals, and piRNA function in the oogenesis of other mammals has therefore remained elusive. Recent studies revealed the small RNA andPIWItranscriptional profiles in golden hamster oocytes more closely resemble that of humans than mice. Herein, we generatedPIWIL1-,PLD6-andMOV10L1-deficient golden hamsters and found that all female mutants were sterile, with embryos arrested at the two-cell stage. InPIWIL1mutant oocytes, we observed transposon accumulation and broad transcriptomic dysregulation, while zygotic gene activation was impaired in early embryos. Intriguingly, PIWIL1-piRNAs exhibited a unique, preferential silencing of endogenous retroviruses (ERVs), whereas silencing LINE1s depended on both PIWIL1- and PIWIL3-piRNAs. Moreover, we showed that piRNAs participate in the degradation of maternal mRNAs in MII oocytes and embryos via partially complementary targets. Together, our findings demonstrate that piRNAs are indispensable for generating functional oocytes in golden hamster, and show the informative value of this model for functional and mechanistic investigations of piRNAs, especially those related to female infertility.

182 EXPRESSION PATTERN OF EMBRYONIC STEM CELL-SPECIFIC microRNAs IN MOUSE PREIMPLANTATION EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 190
Author(s):  
T.-Y. Fu ◽  
P.-C. Tang
Keyword(s):  
Expression Pattern ◽  
Es Cells ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Es Cell ◽  
Cell Stage ◽  
Morula Stage

The endogenous non-coding microRNAs (miRNAs) of 18–25 nucleotides (nt) have been shown to involve in a wide variety of cellular processes as the posttranscriptional regulators by repression of translation or cleavage of mRNAs. In mammals, there are approximately 250 miRNAs that have been identified, and the cluster of miRNA-290 s (miR-290 s) has been demonstrated to express dramatically from the 2-cell to the 4-cell stage in mouse embryos examined from oocytes to the 8-cell stage. The association of miR-290 to 295 with pluripotency has been reported according to their specific expression in embryonic stem (ES) cells. It is interesting to explore the roles of these ES cell-specific miRNAs during the preimplantation stages and early differentiation at the blastocyst stage. Therefore, the objective of this study was to profile the expression pattern of ES cell-specific miRNAs (miR-291-5p, miR-293-3p, and miR-294-3p) from the 4-cell, 8- to 16-cell, morula, and blastocyst stages of mouse embryos. CD-1 F1 embryos at various developmental stages were collected from superovulated and naturally mated CD-1 mice. Total miRNAs of each stage analyzed were collected from 3 embryos for every replicate. Real-time RT-PCR was performed by using the specific stem-loop primers and the embryo lysate as template, which was prepared by heating in 4 μL of PBS at 95°C. Additionally, the in situ expressions of miR-291-5p, miR-293-3p, and miR-294-3p in mouse preimplantation embryos were confirmed by LNA™ probes specific for individual miRNAs. The embryo was fixed with 4% paraformaldehyde for 2 h at room temperature, followed by 3 times wash in PBST (0.1% TritonX-100 in PBS). After hybridization with individual 5′-fluorescein-labeled LNA™ probe, the embryo was washed with 0.1 × SSC, 2 × SSC, and TN buffer (0.1 m Tris-HCl, pH 7.5, 0.15 m NaCl) subsequently. The in situ expressions of miRNAs were detected by immunocytochemical reaction. The results indicated that the expressions of miR-291-5p, miR-293-3p, and miR-294-3p were up-regulated from the 4-cell to the morula stage and then down-regulated afterwards. It was found that the signals of miR-293-5p in an expanded blastocyst were weaker than those at the early blastocyst stage. However, it showed that the intensity of expression at the morula stage was 2 to 4 folds higher compared to that at the 4-cell stage in each miRNA analyzed. Also, the result showed that the ES cell-specific miRNAs examined were expressed in all cells in a blastocyst, i.e. tropectoderm and inner cell mass. In conclusion, we have established the expression profile of ES cell-specific miRNAs during preimplantation stages in mouse embryos. The specific roles of these miRNAs would be further investigated in the short future.

scholarly journals 272OVUM RECOVERY AND BLASTOCYST DEVELOPMENT FOLLOWING INTRACYTOPLASMIC SPERM INJECTION IN CHIMPANZEES

2004 ◽  
Vol 16 (2) ◽  
pp. 256
Author(s):  
I. Hayasaka ◽  
N. Yoshimoto ◽  
Y. Mori ◽  
K. Suzuki ◽  
R. Honda ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Early Embryogenesis ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Morula Stage ◽  
The One ◽  
Estradiol Levels

In the present study, we report on oocyte collection, intracytoplasmic sperm injection and early embryogenesis in chimpanzees. Eight adult female chimpanzees, 11–27 years of age, received a single s.c. injection of 3.75mg GnRH (Leuplin, Takeda Co. Ltd., Osaka, Japan) 1 to 3 days after the beginning of menstruation. Daily i.m. injections of hMG (Humegon, Nippon Organon K.K., Tokyo, Japan) were initiated the following day. The dose of hMG was altered from 75 to 300IU according to serum estradiol levels. When at least one follicle of 17mm or more in diameter was observed, 10000IU of hCG (Pregnyl, Nippon Organon K.K.) were administered by i.m injection. Oocytes were recovered by ultrasound-guided transvaginal follicular aspiration 30.5 to 35.5h after hCG injection. Mature oocytes were denuded of cumulus cells by treatment with 0.1% hyaluronidase, and injected with a frozen-thawed or fresh spermatozoan using a Piezo-driven micromanipulator. Zygotes were cultured in Quinn’s Advantage Fertilization Medium (Cooper Surgical, Inc., Trumbull, CT, USA) with 10 serum protein substitute (SPS) at 37°C in a 5% CO2 atmosphere until the pronucleus stage. The medium was replaced by Quinn’s Advantage Cleavage Medium with 10 SPS from the pronuclear to 8-cell stage, and Quinn’s Advantage Blastcyst Medium with 10 SPS, thereafter. Mild ovarian hyperstimulation syndrome (OHSS) occurred in one female chimpanzee with estradiol levels of 7520pgmL−1. No oocytes were collected from 2 chimpanzees in which large follicles were observed. Thirty-five mature oocytes, one immature oocyte and 6 degenerate/fragmented oocytes were retrieved from 6 chimpanzees, including the one with OHSS. Among 35 mature oocytes injected with spermatozoa, 26 oocytes (74%) produced two pronuclei;; 23 zygotes (66%) cleaved to the 2-cell stage, 22 (63%) to the 4-cell stage, 14 (40%) to the 8-cell stage, and 9 (26%) to the morula stage. Seven zygotes (20%) developed to the blastocyst stage by 120h. There were no differences in fertilization rate or early embryogenesis between frozen and fresh spermatozoa. Results indicate that techniques used for human-assisted reproduction may be applicable to the chimpanzee to help preserve this endangered species.

scholarly journals Distinct roles of ROCK1 and ROCK2 during development of porcine preimplantation embryos

Reproduction ◽  
2014 ◽  
Vol 148 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Jin Yu Zhang ◽  
Huan Sheng Dong ◽  
Reza K Oqani ◽  
Tao Lin ◽  
Jung Won Kang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Coiled Coil ◽  
Specific Inhibitor ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Contact ◽  
Protein Distribution ◽  
Cell Stage ◽  
Oocyte Meiosis ◽  
Morula Stage

Cell-to-cell contact mediated by cell adhesion is fundamental to the compaction process that ensures blastocyst quality during embryonic development. In this study, we first showed that Rho-associated coiled-coil protein kinases (ROCK1 and ROCK2) were expressed both in porcine oocytes and IVF preimplantation embryos, playing different roles in oocytes maturation and embryo development. The amount of mRNA encoding ROCK1 and the protein concentration clearly increased between the eight-cell and morula stages, but decreased significantly when blastocysts were formed. Conversely, ROCK2 was more abundant in the blastocyst compared with other embryonic stages. Moreover, immunostaining showed that ROCK1 protein distribution changed as the embryo progressed through cleavage and compaction to the morula stage. Initially, the protein was predominantly associated with the plasma membrane but later became cytoplasmic. By contrast, ROCK2 protein was localized in both the cytoplasm and the spindle rotation region during oocyte meiosis, but in the cytoplasm and nucleus as the embryo developed. In addition, ROCK2 was present in the trophectoderm cells of the blastocyst. Treatment with 15 μM Y27632, a specific inhibitor of ROCKs, completely blocked further development of early four-cell stage embryos. Moreover, we did not detect the expression ofROCK1but did detectROCK2expression in blastocysts. Moreover, lysophosphatidic acid an activator of ROCKs significantly improved the rates of blastocyst formation. These data demonstrate that ROCKs are required for embryo development to the blastocyst stage. Together, our results indicate that ROCK1 and ROCK2 may exert different biological functions during the regulation of compaction and in ensuring development of porcine preimplantation embryos to the blastocyst stage.

scholarly journals Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos

Reproduction ◽  
2004 ◽  
Vol 128 (3) ◽  
pp. 301-311 ◽  
Author(s):  
Paolo Rinaudo ◽  
Richard M Schultz
Keyword(s):  
Gene Expression ◽  
Preimplantation Embryos ◽  
Expression Analysis Systematic Explorer ◽  
Cell Stage ◽  
Global Pattern ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Global Patterns ◽  
The One

Culture of preimplantation embryos affects gene expression. The magnitude of the effect on the global pattern of gene expression, however, is not known. We compared global patterns of gene expression in blastocysts cultured from the one-cell stage in either Whitten’s medium or KSOM + amino acids (KSOM/AA) with that of blastocysts that developed in vivo, using the Affymetrix MOE430A chip. The analysis revealed that expression of 114 genes was affected after culture in Whitten’s medium, whereas only 29 genes were mis-expressed after culture in KSOM/AA. Expression Analysis Systematic Explorer was used to identify biological and molecular processes that are perturbed after culture and indicated that genes involved in protein synthesis, cell proliferation and transporter function were down-regulated after culture in Whitten’s medium. A common set of genes involved in transporter function was also down-regulated after culture in KSOM/AA. These results provide insights as to why embryos develop better in KSOM/AA than in Whitten’s medium, and highlight the power of microarray analysis to assess global patterns of gene expression.

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