210 POST-TRANSLATIONAL MODIFICATION OF HISTONE H4 ACETYLATION DURING IN VITRO MATURATION OF CANINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 236 ◽  
Author(s):  
T. F. Motheo ◽  
D. R. Arnold ◽  
W. R. R. Vicente ◽  
B. I. Macente ◽  
F. G. F. Filgueira ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Least Square ◽  
Penicillin G ◽  
In Vitro Fertilisation ◽  
Histone H4 ◽  
Post Translational Modification ◽  
Germinal Vesicle Breakdown ◽  
Meiotic Process ◽  
Histone H4 Acetylation

Proper in vitro oocyte maturation, crucial for in vitro fertilisation, is poorly understood and inefficient in dogs. Post-translational modifications of histones are involved in the meiotic process and preparation of oocytes for fertilisation. The present study aimed to evaluate post-translational histone H4 acetylation at lysine 12 (H4K12ac) and 16 (H4K16ac) in canine oocytes during in vitro maturation (IVM). Ovaries were retrieved from 1–7 year-old bitches undergoing routine ovariohysterectomy (OH). Grade I cumulus-oocyte complexes (COC) were recovered and evaluated before and after IVM in TCM 199 + 0.025 mM sodium pyruvate + 100 000 IU mL–1 penicillin G sodium + 100 mg mL–1 streptomycin sulfate, 0.003 g mL–1 BSA + 50 μL mL–1 Pluset® (500 UI FSH + 500 UI LH) + 1 μL mL–1 insulin-like growth factor (IGF) at 38.5°C in 5% CO2 for 72 h. After IVM, the nuclear stage of oocytes was established using Hoechst 33342 staining, and the acetylation patterns were investigated by indirect immunofluorescence staining of fixed immature and post-IVM oocytes (4% paraformaldehyde) with specific antibodies against the acetylated lysine residues, H4K12ac and H4K16ac. All stages were collected and evaluated simultaneously, and the experiment was repeated 4 times, with a total of 5–14 of oocytes evaluated per stage. Immunofluorescence signal was quantified using the NIH ImageJ software 1.4, and data are presented as a percentage of the average fluorescence intensity of each specific antibody over the intensity of DNA, as determined by Hoescht staining. Immunofluorescence values were analysed using the least square ANOVA by the general linear model procedures of SAS (SAS Institute, Cary, NC, USA), and comparisons of means were further performed by Fisher’s Exact test. A probability level of P < 0.05 was considered significant. Our results indicate a variation in acetylation levels of H4K12 (P < 0.02) and H4K16 (P < 0.04) during meiosis. Fluorescence levels of H4K12ac decreased significantly when comparing immature (2.19 ± 0.62) with oocytes in metaphase I/II (MI/MII) (0.52 ± 0.14). Similarly, H4K16ac displayed a greater acetylation pattern in immature oocytes (1.20 ± 0.27) when compared to oocytes in germinal vesicle breakdown (GVBD; 0.58 ± 0.16) and MI/MII (0.62 ± 0.07) stages. These results support the hypothesis that, similar to other domestic species, post-translational modification of histone acetylation takes place during the meiotic process of oocyte IVM. Whether these histone modifications take place in a similar fashion during IVM remains to be investigated.

scholarly journals Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes

2011 ◽  
Vol 18 (5) ◽  
pp. 243-252 ◽  
Author(s):  
Federica Franciosi ◽  
Valentina Lodde ◽  
Ghylène Goudet ◽  
Guy Duchamp ◽  
Stefan Deleuze ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Histone H4 ◽  
Histone H4 Acetylation

scholarly journals CBIO-19. LOW GLOBAL HISTONE H4 ACETYLATION LEVELS REVEAL CANDIDATE QUIESCENT CELL POPULATIONS IN GLIOMA AND DISTINGUISH TUMORS BY GRADE

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii19-ii19
Author(s):  
Anca Mihalas ◽  
Heather Feldman ◽  
Anoop Patel ◽  
Patrick Paddison
Keyword(s):  
Cell Types ◽  
Strand Break ◽  
Cell Cycle Gene ◽  
Low Grade ◽  
Histone H4 ◽  
Current Standard ◽  
A Genome ◽  
Histone H4 Acetylation

Abstract Current standard of care therapy for glioblastoma (GBM) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, the presence of quiescent-like/G0 states, therefore, represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. To gain insight into how glioblastoma cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) harboring a G0-reporter to identify genes that when inhibited trap GSCs in G0-like states. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, which targets both histones (e.g., H4) and non-histone proteins for acetylation. NuA4 functions as a transcriptional activator, whose activities are coordinated with MYC in certain contexts, and also participates in DNA double-strand break repair by facilitating chromatin opening. However, currently little is known about the roles for NuA4 complex in GBM biology. Through modeling KAT5 function in GSC in vitro cultures and in vivo tumors, we find that KAT5 inhibition causes cells to arrest in a G0-like state with high p27 levels, G1-phase DNA content, low protein synthesis rates, low rRNA rates, lower metabolic rate, suppression of cell cycle gene expression, and low histone H4 acetylation. Interestingly, partial inhibition of KAT5 activity slows highly aggressive tumor growth, while increasing p27hi H4-aclow populations. Remarkably, we that low grade gliomas have significantly higher H4-aclow subpopulations and generally lower H4-ac levels than aggressive grade IV tumors. Taken together, our results suggest that NuA4/KAT5 activity may play a key role in quiescence ingress/egress in glioma and that targeting its activity in high grade tumors may effectively “down grade” them, thus, increase patient survival.

scholarly journals Activation Domain-Specific and General Transcription Stimulation by Native Histone Acetyltransferase Complexes

1999 ◽  
Vol 19 (1) ◽  
pp. 855-863 ◽  
Author(s):  
Keiko Ikeda ◽  
David J. Steger ◽  
Anton Eberharter ◽  
Jerry L. Workman
Keyword(s):  
Histone Acetyltransferase ◽  
Regulation Of Transcription ◽  
Dependent Manner ◽  
Saga Complex ◽  
Histone H4 ◽  
Activation Domain ◽  
Activation Domains ◽  
Nucleosomal Array ◽  
Histone H4 Acetylation

ABSTRACT Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription. Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro. Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains. On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner. In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation. However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription. SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain. These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation. In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions.

scholarly journals Changes in acetylation of lysine 5 on histone H4 in canine oocytes following in vitro maturation

2016 ◽  
Vol 52 ◽  
pp. 103-107 ◽  
Author(s):  
TF Motheo ◽  
DR Arnold ◽  
LC Padilha-Nakaghi ◽  
EA Pires-Buttler ◽  
AE Alves ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Histone H4

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

scholarly journals Protein kinase D1 phosphorylation of KAT7 enhances its protein stability and promotes replication licensing and cell proliferation

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Yao Liang ◽  
Yuanyuan Su ◽  
Chenzhong Xu ◽  
Na Zhang ◽  
Doudou Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Replication ◽  
Protein Kinase ◽  
Histone H4 ◽  
Replication Complex ◽  
Protein Kinase D1 ◽  
Defective Mutant ◽  
Histone H4 Acetylation

Abstract The histone acetyltransferase (HAT) KAT7/HBO1/MYST2 plays a crucial role in the pre-replication complex (pre-RC) formation, DNA replication and cell proliferation via acetylation of histone H4 and H3. In a search for protein kinase D1 (PKD1)-interacting proteins, we have identified KAT7 as a potential PKD1 substrate. We show that PKD1 directly interacts and phosphorylates KAT7 at Thr97 and Thr331 in vitro and in vivo. PKD1-mediated phosphorylation of KAT7 enhances its expression levels and stability by reducing its ubiquitination-mediated degradation. Significantly, the phospho-defective mutant KAT7-Thr97/331A attenuates histone H4 acetylation levels, MCM2/6 loading on the chromatin, DNA replication and cell proliferation. Similarly, PKD1 knockdown decreases, whereas the constitutive active mutant PKD1-CA increases histone H4 acetylation levels and MCM2/6 loading on the chromatin. Overall, these results suggest that PKD1-mediated phosphorylation of KAT7 may be required for pre-RC formation and DNA replication.

Putrescine supplementation during in vitro maturation of aged mouse oocytes improves the quality of blastocysts

2017 ◽  
Vol 29 (7) ◽  
pp. 1392 ◽  
Author(s):  
Dandan Liu ◽  
Guolong Mo ◽  
Yong Tao ◽  
Hongmei Wang ◽  
X. Johné Liu
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
In Vitro Fertilisation ◽  
Aged Mouse ◽  
Developmental Potential ◽  
The Impact

Mouse ovaries exhibit a peri-ovulatory rise of ornithine decarboxylase and its product putrescine concurrent with oocyte maturation. Older mice exhibit a deficiency of both the enzyme and putrescine. Peri-ovulatory putrescine supplementation in drinking water increases ovarian putrescine levels, reduces embryo resorption and increases live pups in older mice. However, it is unknown if putrescine acts in the ovaries to improve oocyte maturation. This study examined the impact of putrescine supplementation during oocyte in vitro maturation (IVM) on the developmental potential of aged oocytes. Cumulus–oocyte complexes from 9–12-month-old C57BL/6 mice were subjected to IVM with or without 0.5 mM putrescine, followed by in vitro fertilisation and culture to the blastocyst stage. Putrescine supplementation during IVM did not influence the proportion of oocyte maturation, fertilisation or blastocyst formation, but significantly increased blastocyst cell numbers (44.5 ± 1.9, compared with 36.5 ± 1.9 for control; P = 0.003). The putrescine group also had a significantly higher proportion of blastocysts with top-grade morphology (42.9%, compared with 26.1% for control; P = 0.041) and a greater proportion with octamer-binding transcription factor 4 (OCT4)-positive inner cell mass (38.3%, compared with 19.8% for control; P = 0.005). Therefore, putrescine supplementation during IVM improves egg quality of aged mice, providing proof of principle for possible application in human IVM procedures for older infertile women.

scholarly journals Delay of nuclear maturation and reduction in developmental competence of pig oocytes after mineral oil overlay of in vitro maturation media

Reproduction ◽  
2002 ◽  
pp. 557-564 ◽  
Author(s):  
M Shimada ◽  
N Kawano ◽  
T Terada
Keyword(s):  
Steroid Hormones ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Mineral Oil ◽  
Developmental Competence ◽  
Cdc2 Kinase ◽  
Germinal Vesicle Breakdown ◽  
High Concentrations ◽  
P34 Cdc2

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.

scholarly journals A Human Protein Complex Homologous to the Drosophila MSL Complex Is Responsible for the Majority of Histone H4 Acetylation at Lysine 16

2005 ◽  
Vol 25 (21) ◽  
pp. 9175-9188 ◽  
Author(s):  
Edwin R. Smith ◽  
Christelle Cayrou ◽  
Rong Huang ◽  
William S. Lane ◽  
Jacques Côté ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Interference ◽  
Histone Acetyltransferase ◽  
Histone H4 ◽  
M Cell ◽  
Cycle Arrest ◽  
Histone H4 Acetylation ◽  
Msl Complex ◽  
Host Cell Factor 1

ABSTRACT We describe a stable, multisubunit human histone acetyltransferase complex (hMSL) that contains homologs of the Drosophila dosage compensation proteins MOF, MSL1, MSL2, and MSL3. This complex shows strong specificity for histone H4 lysine 16 in chromatin in vitro, and RNA interference-mediated knockdown experiments reveal that it is responsible for the majority of H4 acetylation at lysine 16 in the cell. We also find that hMOF is a component of additional complexes, forming associations with host cell factor 1 and a protein distantly related to MSL1 (hMSL1v1). We find two versions of hMSL3 in the hMSL complex that differ by the presence of the chromodomain. Lastly, we find that reduction in the levels of hMSLs and acetylation of H4 at lysine 16 are correlated with reduced transcription of some genes and with a G2/M cell cycle arrest. This is of particular interest given the recent correlation of global loss of acetylation of lysine 16 in histone H4 with tumorigenesis.

Molecular Mechanisms of Prophase I Meiotic Arrest Maintenance and Meiotic Resumption in Mammalian Oocytes

2018 ◽  
Vol 26 (11) ◽  
pp. 1519-1537
Author(s):  
Maxim Filatov ◽  
Yulia Khramova ◽  
Maria Semenova
Keyword(s):  
In Vitro Maturation ◽  
Molecular Mechanisms ◽  
Germinal Vesicle ◽  
Follicular Cells ◽  
Meiotic Arrest ◽  
Germinal Vesicle Breakdown ◽  
Prophase I ◽  
The Stability ◽  
Multiple Molecules

Mechanisms of meiotic prophase I arrest maintenance (germinal vesicle [GV] stage) and meiotic resumption (germinal vesicle breakdown [GVBD] stage) in mammalian oocytes seem to be very complicated. These processes are regulated via multiple molecular cascades at transcriptional, translational, and posttranslational levels, and many of them are interrelated. There are many molecular cascades of meiosis maintaining and meiotic resumption in oocyte which are orchestrated by multiple molecules produced by pituitary gland and follicular cells. Furthermore, many of these molecular cascades are duplicated, thus ensuring the stability of the entire system. Understanding mechanisms of oocyte maturation is essential to assess the oocyte status, develop effective protocols of oocyte in vitro maturation, and design novel contraceptive drugs. Mechanisms of meiotic arrest maintenance at prophase I and meiotic resumption in mammalian oocytes are covered in the present article.

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