Nlrp4g is an oocyte-specific gene but is not required for oocyte maturation in the mouse

2014 ◽  
Vol 26 (5) ◽  
pp. 758 ◽  
Author(s):  
Hui Peng ◽  
Wenchang Zhang ◽  
Tianfang Xiao ◽  
Yong Zhang
Keyword(s):  
Electron Microscopy ◽  
Oocyte Maturation ◽  
In Situ Hybridisation ◽  
Gene Expression Pattern ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Immunogold Electron Microscopy ◽  
Specific Gene ◽  
Protein Distribution

The Nlrp gene family contains 20 members and plays a pivotal role in the innate immune and reproductive systems in the mouse. The aim of the present study was to analyse the Nlrp4g gene expression pattern, protein distribution and function in mouse oocyte maturation. Quantitative real-time polymerase chain reaction and in situ hybridisation were performed on Nlrp4g mRNA. Western blotting, immunohistochemistry and immunofluorescence were used to assess expression at the protein level. Confocal and immunogold electron microscopy analyses and RNA interference approach were used to determine the location of the NLRP4G protein and inhibit Nlrp4g function specifically in mouse germinal vesicle oocytes, respectively. Nlrp4g transcripts and proteins (~85 kDa) are specifically expressed in mouse ovaries, restricted to the oocytes at various follicular stages and decline with oocyte aging. There is a marked decline in transcript levels in preimplantation embryos before zygotic genome activation, but the protein remains present through to the blastocyst stage. Confocal microscopy demonstrated that this protein is localised in the cytoplasm. Immunogold electron microscopy further confirmed that NLRP4G protein was present in the cytosol rather than in oocyte cytoplasmic organelles. Furthermore, knockdown of Nlrp4g in germinal vesicle oocytes did not affect oocyte maturation. These results provide the first evidence that Nlrp4g is an oocyte-specific gene but dispensable for oocyte maturation, suggesting that this gene may play roles in mouse oogenesis and/or preimplantation development.

Identification of metaphase II-specific gene transcripts in porcine oocytes and their expression in early stage embryos

2005 ◽  
Vol 17 (6) ◽  
pp. 625 ◽  
Author(s):  
Xiang-Shun Cui ◽  
Hyuk Song ◽  
Nam-Hyung Kim
Keyword(s):  
Oocyte Maturation ◽  
Early Stage ◽  
Sequence Similarity ◽  
Germinal Vesicle ◽  
Specific Gene ◽  
Early Cleavage ◽  
Reverse Transcription Pcr ◽  
Gene Transcripts ◽  
Polymerase Chain ◽  
Inducible Protein

Annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) was used to identify the genes that are specifically or prominently expressed in porcine oocytes at the metaphase II (MII) and germinal vesicle (GV) stages. By using 60 ACPs, 13 differentially expressed genes (DEGs) were identified. The cloned genes or expressed sequence tags (ESTs) showed sequence similarity with known genes or ESTs of other species in GenBank. The mRNA expression during oocyte maturation and early embryonic development in both pigs and mice of four of these genes (namely transcription factor TZP, annexin A2, hypoxia-inducible protein 2, and ATPase 6) was further characterised by real-time quantitative reverse transcription–PCR. All four genes were markedly upregulated in pig and mouse MII oocytes compared with GV-stage oocytes. The expression levels of the four genes decreased gradually during early cleavage. Thus, these genes may play important roles during oocyte maturation and/or early cleavage in mammals. Although the detailed functions of these genes remain to be determined, their identification in the present study provides insights into meiotic maturation and fertilisation.

scholarly journals MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells

2015 ◽  
Vol 308 (6) ◽  
pp. E525-E534 ◽  
Author(s):  
Bo Pan ◽  
Derek Toms ◽  
Wei Shen ◽  
Julang Li
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Specific Effect ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Cumulus Expansion ◽  
Vitro Fertilization ◽  
And Function

We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion ( HAS2, PTGS2) and oocyte maturation ( CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 ( GDF9), bone morphogenetic protein 15 ( BMP15), zona pellucida 3 ( ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation.

scholarly journals Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential

2017 ◽  
Vol 29 (5) ◽  
pp. 876 ◽  
Author(s):  
Denise Laskowski ◽  
Ylva Sjunnesson ◽  
Patrice Humblot ◽  
Marc-André Sirard ◽  
Göran Andersson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Quantitative Polymerase Chain Reaction ◽  
Blastocyst Stage ◽  
Specific Gene ◽  
Bovine Oocyte ◽  
Developmental Potential ◽  
Metabolic Imbalance ◽  
Negative Effect

Metabolic imbalance impairs fertility, because changes in concentrations of metabolites and hormones in the blood and follicular fluid create an unfavourable environment for early embryonic development. Insulin is a key metabolic hormone known for its effects on fertility: insulin concentrations are increased during energy balance disturbances in diabetes or metabolic syndrome. Still, insulin is frequently used at supraphysiological concentrations for embryo in vitro culture with unknown consequences for the developmental potential of the offspring. In the present study we investigated the effects of insulin exposure during in vitro bovine oocyte maturation on developmental rates, embryo quality and gene expression. Supplementation of the maturation media with insulin at 10 or 0.1 µg mL–1 decreased blastocyst rates compared with an insulin-free control (19.8 ± 1.3% and 20.4 ± 1.3% vs 23.8 ± 1.3%, respectively; P < 0.05) and led to increased cell numbers (nearly 10% more cells on Day 8 compared with control; P < 0.05). Transcriptome analysis revealed significant upregulation of genes involved in lipid metabolism, nuclear factor (erythroid-derived 2)-like 2 (NRF2) stress response and cell differentiation, validated by quantitative polymerase chain reaction. To conclude, the results of the present study demonstrate that insulin exposure during in vitro oocyte maturation has a lasting effect on the embryo until the blastocyst stage, with a potential negative effect in the form of specific gene expression perturbations.

1 ABERRANT DNA METHYLATION IN PORCINE IN VITRO-, PARTHENOGENETIC-, AND NUCLEAR TRANSFER-PRODUCED BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 109 ◽  
Author(s):  
A. Bonk ◽  
M. Samuel ◽  
L. Lai ◽  
Y. Hao ◽  
R. Li ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
The Other ◽  
Specific Gene ◽  
Aberrant Dna Methylation

Aberrant DNA methylation of in vitro-, parthenogenetic-, and nuclear transfer-derived embryos has been implicated in the low developmental competence of early embryos. Demethylation of the genome occurs immediately after fertilization and continues through the blastocyst stage. Remethylation or reprogramming of the genome occurs around the time of implantation and is maintained in somatic tissues. The aim of this study was to analyze DNA methylation in porcine gametes and blastocysts. Differential DNA methylation hybridization was conducted to analyze the methylation status of the Bstu I site (CGCG) in the gamete and blastocyst epigenomes. Germinal vesicle oocytes were aspirated from ovaries collected at an abattoir, sperm was isolated from a fresh ejaculate, and blastocysts were derived and collected from in vivo, in vitro, nuclear transfer, and parthenogenetic sources. Genomic clones were selected from a porcine CpG Island library based on the presence of a Bstu I site. The inserts from these clones were PCR amplified and spotted on glass slides. DNA was digested with Mse I, ligated to linkers, and digested with Bstu I. Fragments with methylated Bstu I sites remained intact whereas fragments with unmethylated Bstu I sites were cut. Intact fragments were amplified by PCR and labeled with amino allyl-dUTP. Liver DNA served as the reference and was labeled with Cy5; the other samples were labeled with Cy3. An Axon Genepix 4000B scanner (Axon Instruments, Inc., Union City, CA, USA) was used to scan the slides. Initial analysis of the microarray image was performed with GenePix Pro 4.0 software. Additional analysis, performed by using Genespring 7.0 ANOVA (P < 0.05), identified 221 clones as being significantly different in at least one of the biological conditions of the gametes or the blastocysts. Forty-six clones were sequenced and BLAST analysis identified 18 clones that were unique, 16 clones that had no similarity, and 12 clones that had similarity to multiple genes. Ribosomal (RPS20, RPL18) and protoporphyrinogen oxidase (PPOX) genes were identified in several clones. Components of the immune system (CCRs, TLRs), a transcription factor (ATF2), and an embryo-specific gene (WNT8B) were also identified. A condition tree was created according to the standard correlation similarity measure for the spots identified as significantly different. The condition tree shows that the methylation profiles are most similar in the germinal vesicle oocyte, parthenogenetic blastocyst, nuclear transfer blastocyst, in vitro-produced blastocyst, and sperm. In vivo-produced blastocysts grouped separately from the other samples. These results are consistent with previous studies that have shown that gametes undergo demethylation after fertilization on through the blastocyst stage when the genome is remethylated. Additionally, these results suggest that the reprogramming events that occur during the development of the in vivo-produced blastocysts are less likely to occur in in vitro-, nuclear transfer-, and parthenogenetic-produced blastocysts. This work was funded by a grant from the NIH (RR13438) and Food for the 21st Century.

scholarly journals Bovine oocytes and early embryos express Staufen and ELAVL RNA-binding proteins

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 161-168 ◽  
Author(s):  
M.D. Calder ◽  
P. Madan ◽  
A.J. Watson
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Protein Distribution ◽  
Morula Stage ◽  
The Stability

SummaryRNA-binding proteins (RBP) influence RNA editing, localization, stability and translation and may contribute to oocyte developmental competence by regulating the stability and turnover of oogenetic mRNAs. The expression of Staufen 1 and 2 and ELAVL1, ELAVL2 RNA-binding proteins during cow early development was characterized. Cumulus–oocyte complexes were collected from slaughterhouse ovaries, matured, inseminated and subjected to embryo culture in vitro. Oocyte or preimplantation embryo pools were processed for RT-PCR and whole-mount immunofluorescence analysis of mRNA expression and protein distribution. STAU1 and STAU2 and ELAVL1 mRNAs and proteins were detected throughout cow preimplantation development from the germinal vesicle (GV) oocyte to the blastocyst stage. ELAVL2 mRNAs were detectable from the GV to the morula stage, whereas ELAVL2 protein was in all stages examined and localized to both cytoplasm and nuclei. The findings provide a foundation for investigating the role of RBPs during mammalian oocyte maturation and early embryogenesis.

scholarly journals Effects of WT1 down-regulation on oocyte maturation and preimplantation embryo development in pigs

Reproduction ◽  
2014 ◽  
Vol 148 (4) ◽  
pp. 377-387 ◽  
Author(s):  
Fei Gao ◽  
Jiyu Guan ◽  
Limei Liu ◽  
Sheng Zhang ◽  
Peipei An ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Preimplantation Embryo ◽  
Germinal Vesicle ◽  
Preimplantation Embryos ◽  
Down Regulation ◽  
Organ Growth ◽  
Preimplantation Embryo Development ◽  
Important Regulator ◽  
And Function

The Wilms' tumour 1 (WT1) gene originally identified as a tumour suppressor associated with WTs encodes a zinc finger-containing transcription factor that is expressed in multiple tissues and is an important regulator of cellular and organ growth, proliferation, development, migration and survival. However, there is a deficiency of data regarding the expression and function ofWT1during oocyte maturation and preimplantation embryonic development. Herein, we sought to define the expression characteristics and functions ofWT1during oocyte maturation and preimplantation embryonic development in pigs. We show thatWT1is expressed in porcine oocytes and at all preimplantation stages in embryos generated by ICSI. We then evaluated the effects of down-regulatingWT1expression at germinal vesicle and early ICSI stages using a recombinant plasmid (pGLV3-WT1-shRNA). Down-regulation ofWT1did not affect oocyte maturation but significantly decreased preimplantation embryonic development and increased apoptosis in blastocysts. These results indicate thatWT1plays important roles in the development of porcine preimplantation embryos.

scholarly journals Stimulation of Xenopus oocyte maturation by inhibition of the G-protein alpha S subunit, a component of the plasma membrane and yolk platelet membranes.

1995 ◽  
Vol 130 (2) ◽  
pp. 275-284 ◽  
Author(s):  
C J Gallo ◽  
A R Hand ◽  
T L Jones ◽  
L A Jaffe
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Oocyte Maturation ◽  
Time Course ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Immunogold Electron Microscopy ◽  
Germinal Vesicle Breakdown ◽  
Platelet Membranes ◽  
Yolk Platelet

Oocytes of Xenopus laevis undergo maturation when injected with an affinity-purified antibody against the COOH-terminal decapeptide of the alpha subunit of the G-protein Gs, an antibody that inhibits Gs activity. Germinal vesicle breakdown, chromosome condensation, and polar body formation occur, with a time course similar to that for oocytes treated with progesterone. The alpha S antibody-injected oocytes also acquire the ability to be activated by sperm. Coinjection of the catalytic subunit of cAMP-dependent protein kinase, or incubation with cycloheximide, inhibits maturation in response to injection of the alpha S antibody; these experiments show that the alpha S antibody acts at an early point in the pathway leading to oocyte maturation, before formation of maturation promoting factor, and like progesterone, its action requires protein synthesis. Immunogold electron microscopy shows that alpha S is present in the yolk platelet membranes as well as the plasma membrane. These results support the hypothesis that progesterone acts by inhibiting alpha S, and suggest that the target of progesterone could include yolk platelet membranes as well as the plasma membrane.

256 SWITCH IN THE EXPRESSION OF GENES INVOLVED IN LIPID METABOLISM FOR IN VIVO-MATURED BOVINE OOCYTES AND BLASTOCYSTS

2007 ◽  
Vol 19 (1) ◽  
pp. 244 ◽  
Author(s):  
O. Algriany ◽  
P. L. A. M. Vos ◽  
M. A. Sirard ◽  
S. J. Dieleman
Keyword(s):  
Lipid Metabolism ◽  
Oocyte Maturation ◽  
De Novo ◽  
Energy Requirement ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Germinal Vesicle Breakdown ◽  
Cpt I ◽  
Bovine Oocytes

The competence of oocytes to develop into healthy offspring is determined by many factors including oocyte quality, embryo culture conditions, and energy requirements. Although glucose metabolism has been studied extensively, little is known about lipid metabolism during oocyte maturation and in the blastocyst. In general, cells can obtain fatty acids (FA) by transport (1) from outside across the cell membrane or by synthesis (2) inside the cell, and loose FA by catabolic processes (3). Therefore, we analyzed mRNA abundance for (1) FA translocase (FAT/CD36) and FA transport protein (FATP1), (2) acetyl-CoA carboxylase (ACC) α and FA synthase (FAS), and (3) AMP-activated protein kinase gamma 1 (AMPK) and carnitine palmitoyltransferase I (CPT-I) in bovine oocytes during resumption of meiosis in vivo vs.in vivo blastocysts. Cyclic Holstein-Friesian cows (n = 16) were treated with oFSH and a controlled LH surge (Knijn et al. 2002 Reproduction 124, 365–375) to collect oocytes at onset of maturation, 2 h before LH, and after the start of germinal vesicle breakdown (6 h after LH). A second group of cows (n = 10) was treated with eCG/anti-eCG (Voset al. 1994 Theriogenology 41, 829–840) to flush blastocysts at Day 7 after ovulation. Following ovariectomy, oocytes from all follicles between 10 and 18 mm were denuded and stored separately at -80�C. Oocytes were selected on the basis of the relative steroid concentration in the follicular fluid, producing 3 replicate pools of 13 competent oocytes each, at both 2 h before and 6 h after LH. Four replicates of 5 expanded blastocysts grade 1 (IETS) each were stored at -80�C. Total RNA was isolated using a microspin column, and DNA was digested (Absolutely RNA Microprep Kit; Stratagene, San Diego, CA, USA) and reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). Amplification of cDNA was achieved by real-time PCR using specific primers and 2X iQ SYBR Green Supermix (Bio-Rad Laboratories) in duplicate. Relative levels of expression were analyzed using a modified delta-Ct method. Statistical analysis was done using ANOVA and the Tukey post-hoc test. Relative abundance of mRNA for FA transport and catabolism was &gt;10 times higher during oocyte maturation than in blastocysts; CPT-I was not detected in blastocysts. In contrast, transcripts of FA synthesis were sharply increased (&gt;5 times) during the blastocyst stage. Alterations in the expression of these mRNA indicate that they play a key role in mediating cell type-specific differences in energy requirement and that exogenous FA and FA catabolism could be a main source of energy during oocyte maturation. A high level of FA biosynthesis mRNA in blastocysts may reflect high demands of de novo long-chain FA synthesis and elongation that probably are essential for protection against stress.

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.

PLCz-induced Ca2+ oscillations are enhanced after germinal vesicle breakdown during mouse oocyte maturation

2016 ◽  
Author(s):  
Jessica Sanders ◽  
Ethan Bateson ◽  
Yuansong Yu ◽  
Michail Nomikos ◽  
Antony Lai ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Mouse Oocyte ◽  
Germinal Vesicle Breakdown
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