Peripheral administration of GH induces cell proliferation in the brain of adult hypophysectomized rats

IGF-I treatment has been shown to enhance cell genesis in the brains of adult GH- and IGF-I-deficient rodents; however, the influence of GH therapy remains poorly understood. The present study investigated the effects of peripheral recombinant bovine GH (bGH) on cellular proliferation and survival in the neurogenic regions (subventricular zone (SVZ), and dentate gyrus of the hippocampus), as well as the corpus callosum, striatum, parietal cortex, and piriform cortex. Hypopituitarism was induced in female rats by hypophysectomy, and the rats were supplemented with thyroxine and cortisone acetate. Subsequently, the rats received daily s.c. injections of bGH for either 6 or 28 days respectively. Following 5 days of peripheral bGH administration, the number of bromodeoxyuridine (BrdU)-positive cells was increased in the hippocampus, striatum, parietal cortex, and piriform cortex after 6 and 28 days. In the SVZ, however, BrdU-positive cells increased only after 28 days of bGH treatment. No significant change was observed in the corpus callosum. In the hippocampus, after 28 days of bGH treatment, the number of BrdU/NeuN-positive cells was increased proportionally to increase the number of BrdU-positive cells. 3H-thymidine incorporation in vitro revealed that 24 h of bGH exposure was sufficient to increase cell proliferation in adult hippocampal progenitor cells. This study shows for the first time that 1) peripheral bGH treatment increased the number of newborn cells in the adult brain and 2) bGH exerted a direct proliferative effect on neuronal progenitor cells in vitro.

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HNRNPA2B1 promotes multiple myeloma progression by increasing AKT3 expression via m6A-dependent stabilization of ILF3 mRNA

Journal of Hematology & Oncology ◽

10.1186/s13045-021-01066-6 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Fengjie Jiang ◽

Xiaozhu Tang ◽

Chao Tang ◽

Zhen Hua ◽

Mengying Ke ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Cellular Proliferation ◽

Aberrant Expression ◽

The Stability ◽

Induced Apoptosis ◽

Proliferation In Vitro

AbstractN6-methyladenosine (m6A) modification is the most prevalent modification in eukaryotic RNAs while accumulating studies suggest that m6A aberrant expression plays an important role in cancer. HNRNPA2B1 is a m6A reader which binds to nascent RNA and thus affects a perplexing array of RNA metabolism exquisitely. Despite unveiled facets that HNRNPA2B1 is deregulated in several tumors and facilitates tumor growth, a clear role of HNRNPA2B1 in multiple myeloma (MM) remains elusive. Herein, we analyzed the function and the regulatory mechanism of HNRNPA2B1 in MM. We found that HNRNPA2B1 was elevated in MM patients and negatively correlated with favorable prognosis. The depletion of HNRNPA2B1 in MM cells inhibited cell proliferation and induced apoptosis. On the contrary, the overexpression of HNRNPA2B1 promoted cell proliferation in vitro and in vivo. Mechanistic studies revealed that HNRNPA2B1 recognized the m6A sites of ILF3 and enhanced the stability of ILF3 mRNA transcripts, while AKT3 downregulation by siRNA abrogated the cellular proliferation induced by HNRNPA2B1 overexpression. Additionally, the expression of HNRNPA2B1, ILF3 and AKT3 was positively associated with each other in MM tissues tested by immunohistochemistry. In summary, our study highlights that HNRNPA2B1 potentially acts as a therapeutic target of MM through regulating AKT3 expression mediated by ILF3-dependent pattern.

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Cooperation between PDGF and FGF converts slowly dividing O-2Aadult progenitor cells to rapidly dividing cells with characteristics of O-2Aperinatal progenitor cells.

The Journal of Cell Biology ◽

10.1083/jcb.118.4.889 ◽

1992 ◽

Vol 118 (4) ◽

pp. 889-900 ◽

Cited By ~ 174

Author(s):

G Wolswijk ◽

M Noble

Keyword(s):

Progenitor Cells ◽

Adult Brain ◽

High Rate ◽

Adult Rat ◽

Large Numbers ◽

Dividing Cells ◽

Rapid Generation ◽

And Migration

We have shown previously that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells isolated from adult rat optic nerves can be distinguished in vitro from their perinatal counterparts on the basis of their much slower rates of division, differentiation, and migration when grown in the presence of cortical astrocytes or PDGF. This behavior is consistent with in vivo observations that there is only a modest production of oligodendrocytes in the adult CNS. As such a behavior is inconsistent with the likely need for a rapid generation of oligodendrocytes following demyelinating damage to the mature CNS, we have been concerned with identifying in vitro conditions that allow O-2Aadult progenitor cells to generate rapidly large numbers of progeny cells. We now provide evidence that many slowly dividing O-2Aadult progenitor cells can be converted to rapidly dividing cells by exposing adult optic nerve cultures to both PDGF and bFGF. In addition, these O-2Aadult progenitor cells appear to acquire other properties of O-2Aperinatal progenitor cells, such as bipolar morphology and high rate of migration. Although many O-2Aadult progenitor cells in cultures exposed to bFGF alone also divide rapidly, these cells are multipolar and migrate little in vitro. Oligodendrocytic differentiation of O-2Aadult progenitor cells, which express receptors for bFGF in vitro, is almost completely inhibited in cultures exposed to bFGF or bFGF plus PDGF. As bFGF and PDGF appear to be upregulated and/or released after injury to the adult brain, this particular in vitro response of O-2Aadult progenitor cells to PDGF and bFGF may be of importance in the generation of large numbers of new oligodendrocytes in vivo following demyelination.

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The Cold-Shock Domain Protein YB-1 Is Important for Cellular Proliferation and the Prevention of Premature Senescence.

Blood ◽

10.1182/blood.v104.11.2571.2571 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2571-2571

Author(s):

Zhi Hong Lu ◽

Jason T. Books ◽

Timothy James Ley

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Cycle Progression ◽

Binding Proteins ◽

Cellular Proliferation ◽

Cold Shock ◽

Premature Senescence ◽

Cycle Progression

Abstract Mammalian proteins containing “cold-shock” domains belong to the most evolutionarily conserved family of nucleic acid-binding proteins known in bacteria, plants, and animals. One of these proteins, YB-1, has been implicated in basic cellular functions such as cell proliferation and responses to environmental stresses. In mammalian cells, YB-1 has been shown to shuttle between the nuclear and cytoplasmic compartments. Within the nucleus, YB-1 interacts with several DNA-and pre-mRNA-binding proteins, and has been implicated in nuclear activities, including transcriptional regulation, chromatin remodeling, and pre-mRNA splicing. YB-1 is also abundant in the cytoplasm, where it binds nonspecifically to mRNA, and may act as a general regulator of mRNA stability, cytoplasmic localization, and translation. Thus, YB-1 has been proposed to function as a multifunctional regulator for the control of gene expression in both the nucleus and cytoplasm. YB-1 overexpression has been frequently detected in a variety of human cancers, often associated with unfavorable clinical outcomes. However, it remains unclear whether YB-1 overexpression contributes directly to the malignant phenotype, or whether it is simply a non-causal “marker” associated with rapid cell growth (and poor prognostic outcomes). To further assess the role of this protein in health and disease, we created mice deficient for YB-1. Complete loss of function of this gene results in fully-penetrant late embryonic and perinatal lethality. Morphological and histological analyses revealed that YB-1−/− embryos displayed major developmental and functional defects, including neurological abnormalities, hemorrhage, and respiratory failure, which probably contributed to lethality. Growth retardation occurred in all late-stage embryos, and was the result of hypoplasia in multiple organ systems. Consistent with these in vivo results, fibroblasts isolated from YB-1−/− embryos (MEFs) grew slowly and entered senescence prematurely in vitro; these defects were rescued by ectopic expression of a GFP-tagged human YB-1 cDNA. This data suggests that YB-1 plays an important cell-autonomous role in cell proliferation and prevention of premature senescence. We further showed that loss of YB-1 in early passage MEFs resulted a delay in G0/G1 to S-phase progression, and a defect in a transcriptional mechanism that normally represses the expression of the G1-specific CDK inhibitor gene p16Ink4a, and the p53 target genes p21Cip1 and Mdm2. However, YB-1 does not cause “global” changes in the transcriptome, the proteome, or protein synthesis efficiency. As predicted, p16Ink4a and p21Cip1 double knockdown by siRNA treatment led to an increase in the rate of cell proliferation, and an extension of proliferative capacity during late passages in YB-1−/− cells. Furthermore, YB-1 deficiency reduced the ability of MEFs to proliferate normally in response to c-Myc overexpression. In conclusion, our data has revealed that YB-1 is required for normal mouse development and survival, and that it plays an important role in supporting rapid cellular proliferation both in vivo and in vitro. Our data further suggests that YB-1 is a cell cycle progression regulator that is important for preventing the early onset of senescence in cultured MEF cells. This data raises the possibility that disregulated expression of YB-1 may contribute to malignant phenotypes by supporting rapid cell cycle progression, and by protecting cells from cytotoxic stresses.

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CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

Blood ◽

10.1182/blood.v110.11.1224.1224 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1224-1224

Author(s):

Jerry C. Cheng ◽

Dejah Judelson ◽

Kentaro Kinjo ◽

Jenny Chang ◽

Elliot Landaw ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemia Cells ◽

Mouse Bone Marrow ◽

T315i Mutation

Abstract The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

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UCP2 Modulates Cell Proliferation through the MAPK/ERK Pathway during Erythropoiesis and Has No Effect on Heme Biosynthesis

Blood ◽

10.1182/blood.v112.11.5372.5372 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5372-5372

Author(s):

Alvaro A Elorza ◽

Brigham B Hyde ◽

Hanna Mikkola ◽

Sheila Collins ◽

Orian S Shirihai

Keyword(s):

Cell Proliferation ◽

Progenitor Cells ◽

Fetal Liver ◽

Mitochondrial Protein ◽

Erythroid Progenitor ◽

Heme Synthesis ◽

Erythroid Progenitor Cells ◽

Deficient Mice

Abstract UCP2, an inner membrane mitochondrial protein, has been implicated in bioenergetics and Reactive Oxygen Species (ROS) modulation. UCP2 has been previously hypothesized to function as a facilitator of heme synthesis and iron metabolism by reducing ROS production. While UCP2 has been found to be induced by GATA1 during erythroid differentiation its role in erythropoiesis in vivo or in vitro has not been reported thus far. Here we report on the study of UCP2 role in erythropoiesis and the hematologic phenotype of UCP2 deficient mouse. In vivo we found that UCP2 protein peaks at early stages of erythroid maturation when cells are not fully committed in heme synthesis and then becomes undetectable at the reticulocyte stage. Iron incorporation into heme was unaltered in erythroid cells from UCP2 deficient mice. While heme synthesis was not influenced by UCP2 deficiency, mice lacking UCP2 had a delayed recovery from chemically induced hemolytic anemia. Analysis of the erythroid lineage from bone marrow and fetal liver revealed that in the UCP2 deficient mice the R3 (CD71high/Ter119high) population was reduced by 24%. The count of BFU-E and CFU-E colonies, scored in an erythroid colony assay, was unaffected, indicating an equivalent number of early erythroid progenitor cells in both UCP2 deficient and control cells. Ex-vivo differentiation assay revealed that UCP2 deficient c-kit+ progenitor cells expansion was overall reduced by 14% with population analysis determining that the main effect is at the R3 stage. No increased rate of apoptosis was found indicating that expansion rather than cell death is being compromised. Reduced expansion of c-kit+ cells was accompanied by 30% reduction in the phosphorylated form of ERK, a ROS dependent cytosolic regulator of cell proliferation. Analysis of ROS in UCP2 null erythroid progenitors revealed altered distribution of ROS resulting in 14% decrease in cytosolic and 32% increase in mitochondrial ROS. Restoration of the cytosolic oxidative state of erythroid progenitor cells by the pro-oxidant Paraquat reversed the effect of UCP2 deficiency on cell proliferation in in vitro differentiation assays. Together, these results indicate that UCP2 is a regulator of erythropoiesis and suggests that inhibition of UCP2 function may contribute to the development of anemia.

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Induction of endothelial proliferation and angiogenesis through activating the ERK1/2/EGF pathway mediate by CXC chemokine receptor 2 by chemokine (C-X-C motif) ligand 1.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.6_suppl.138 ◽

2013 ◽

Vol 31 (6_suppl) ◽

pp. 138-138 ◽

Cited By ~ 1

Author(s):

Makito Miyake ◽

Steve Goodison ◽

Evan Gomes ◽

Wasia Rizwani ◽

Shanti Ross ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Chemokine Receptor ◽

Cellular Proliferation ◽

Human Endothelial Cell ◽

Endothelial Cell Proliferation ◽

Cxc Chemokine ◽

Inhibition Of Angiogenesis

138 Background: Endothelial cell growth and proliferation are critical for tumoral angiogenesis. We report here that blockade of Chemokine (C-X-C motif) ligand 1 (CXCL1) results in reduction of human endothelial cell proliferation and its ability to induce angiogenesis. Methods: Two human endothelial cell lines, HUVEC and HDMEC, were used in the in vitro assays. Proliferation assay and matrigel tube formation assay were performed to test the inhibitory effect of anti-CXCL antibody on the activity of endothelial cells in vitro. Matrigel plug assay in nude mice was performed to test the in vivo angiogenic activity of CXCL1. Results: CXCL1 interacts with its receptor CXC chemokine Receptor 2 and induces endothelial cell proliferation, whereas blockade of CXCL1 is associated with reduction in cellular proliferation through a decrease in levels of cyclin D and cdk4 and inhibition of angiogenesis through EGF and ERK 1/2. Targeting CXCL1 inhibits neoangiogenesis but has no effect on disrupting established vasculature. Furthermore targeting CXCL1 is associated with reduction in migration of human endothelial cells in an in vitro model. Additionally, neutralizing antibody against CXCL1 in a xenograft angiogenesis model resulted in inhibition of angiogenesis. Conclusions: CXCL1-induced regulation of angiogenesis has not been studied extensively in human cancers, thus these findings illustrate a novel contribution of CXCL1 interactions in pathological angiogenesis. Therefore, the ability to selectively modulate CXCL1, specifically in tumoral angiogenesis, may promote the development of novel oncologic therapeutic strategies.

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Isocaloric maternal low-protein diet alters IGF-I, IGFBPs, and hepatocyte proliferation in the fetal rat

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00037.2003 ◽

2003 ◽

Vol 285 (5) ◽

pp. E991-E1000 ◽

Cited By ~ 44

Author(s):

Ilham El Khattabi ◽

Francine Grégoire ◽

Claude Remacle ◽

Brigitte Reusens

Keyword(s):

Cell Proliferation ◽

Fetal Liver ◽

Protein Diet ◽

Low Protein Diet ◽

Hepatocyte Proliferation ◽

Liver Growth ◽

Fetal Plasma ◽

Low Protein ◽

Igf I

We investigated the effect of an isocaloric maternal low-protein diet during pregnancy in rats on the proliferative capacity of cultured fetal hepatocytes. The potential roles of these changes on the IGF-IGF-binding protein (IGFBP) axis, and the role of insulin and glucocorticoids in liver growth retardation, were also evaluated. Pregnant Wistar rats were fed a control (C) diet (20% protein) or a low-protein (LP) diet (8%) throughout gestation. In primary culture, the DNA synthesis of hepatocytes derived from LP fetuses was decreased by ∼30% compared with control hepatocytes ( P < 0.05). In parallel, in vivo moderate protein restriction in the dam reduced the fetal liver weight and IGF-I level in fetal plasma ( P < 0.01) and augmented the abundance of 29- to 32-kDa IGFBPs in fetal plasma ( P < 0.01) and fetal liver ( P < 0.01). By contrast, the abundance of IGF-II mRNA in liver of LP fetuses was unaffected by the LP diet. In vitro, the LP-derived hepatocytes produced less IGF-I ( P < 0.01) and more 29- to 32-kDa IGFBPs ( P < 0.01) than hepatocytes derived from control fetuses. These alterations still appeared after 3–4 days of culture, indicating some persistence in programming. Dexamethasone treatment of control-derived hepatocytes decreased cell proliferation (54 ± 2.3%, P < 0.01) and stimulated 29- to 32-kDa IGFBPs, whereas insulin promoted fetal hepatocyte growth (127 ± 5.5%, P < 0.01) and inhibited 29- to 32-kDa IGFBPs. These results show that liver growth and cell proliferation in association with IGF-I and IGFBP levels are affected in utero by fetal undernutrition. It also suggests that glucocorticoids and insulin may modulate these effects.

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The novel BET inhibitor UM-002 reduces glioblastoma cell proliferation and invasion

Scientific Reports ◽

10.1038/s41598-021-02584-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Anna M. Jermakowicz ◽

Matthew J. Rybin ◽

Robert K. Suter ◽

Jann N. Sarkaria ◽

Zane Zeier ◽

...

Keyword(s):

Cell Proliferation ◽

Single Cell ◽

Rna Sequencing ◽

Ex Vivo ◽

Adult Brain ◽

Bet Inhibitor ◽

Bet Inhibitors ◽

Single Cell Rna Sequencing

AbstractBromodomain and extraterminal domain (BET) proteins have emerged as therapeutic targets in multiple cancers, including the most common primary adult brain tumor glioblastoma (GBM). Although several BET inhibitors have entered clinical trials, few are brain penetrant. We have generated UM-002, a novel brain penetrant BET inhibitor that reduces GBM cell proliferation in vitro and in a human cerebral brain organoid model. Since UM-002 is more potent than other BET inhibitors, it could potentially be developed for GBM treatment. Furthermore, UM-002 treatment reduces the expression of cell-cycle related genes in vivo and reduces the expression of invasion related genes within the non-proliferative cells present in tumors as measured by single cell RNA-sequencing. These studies suggest that BET inhibition alters the transcriptional landscape of GBM tumors, which has implications for designing combination therapies. Importantly, they also provide an integrated dataset that combines in vitro and ex vivo studies with in vivo single-cell RNA-sequencing to characterize a novel BET inhibitor in GBM.

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Characterization of the iPSC-derived conditioned medium that promotes the growth of bovine corneal endothelial cells

PeerJ ◽

10.7717/peerj.6734 ◽

2019 ◽

Vol 7 ◽

pp. e6734

Author(s):

Qing Liu ◽

Yonglong Guo ◽

Shiwei Liu ◽

Peiyuan Wang ◽

Yunxia Xue ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Conditioned Medium ◽

Cell Morphology ◽

Cell Cycle Progression ◽

Cellular Proliferation ◽

Activin A ◽

Corneal Endothelial Cells ◽

Induced Pluripotent

Corneal endothelial cells (CECs) maintain corneal transparency and visual acuity. However, the limited proliferative capability of these cells in vitro has prompted researchers to find efficient culturing techniques for them. The aim of our study was to evaluate the use of conditioned medium (CM) obtained from induced pluripotent stem cells (iPSCs) as a source for the effective proliferation of bovine CECs (B-CECs). In our study, the proliferative ability of B-CECs was moderately enhanced when the cells were grown in 25% iPSC conditioned medium (iPSC-CM). Additionally, hexagonal cell morphology was maintained until passage 4, as opposed to the irregular and enlarged shape observed in control corneal endothelial medium (CEM). B-CECs in both the 25% iPSC-CM and CEM groups expressed and Na+-K+-ATPase. The gene expression levels of NIFK, Na+-K+-ATPase, Col4A and Col8A and the percentage of cells entering S and G2 phases were higher in the iPSC-CM group. The number of apoptotic cells also decreased in the iPSC-CM group. In comparison to the control cultures, iPSC-CM facilitated cell migration, and these cells showed better barrier functions after several passages. The mechanism of cell proliferation mediated by iPSC-CM was also investigated, and phosphorylation of Akt was observed in B-CECs after exposure to iPSC-CM and showed sustained phosphorylation induced for up to 180 min in iPSC-CM. Our findings indicate that iPSC-CM may employ PI3-kinase signaling in regulating cell cycle progression, which can lead to enhanced cellular proliferation. Effective component analysis of the CM showed that in the iPSC-CM group, the expression of activin-A was significantly increased. If activin-A is added as a supplement, it could help to maintain the morphology of the cells, similar to that of CM. Hence, we conclude that activin-A is one of the effective components of CM in promoting cell proliferation and maintaining cell morphology.

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128 Evidence that Pregnancy-Associated Serum Protein A (PAPP-A) Plays Role on Bovine In Vitro Embryo Production

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab128 ◽

2018 ◽

Vol 30 (1) ◽

pp. 204

Author(s):

A. B. Giroto ◽

F. F. Franchi ◽

P. K. Fontes ◽

M. A. Maioli ◽

G. P. Nogueira ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Proliferation ◽

Dna Fragmentation ◽

Reference Genes ◽

Protein A ◽

Cumulus Cells ◽

Control Group ◽

Embryo Yield ◽

Igf I

The aim of present work was to assess the effects of pregnancy-associated serum protein A (PAPP-A) during oocyte in vitro maturation (IVM) on meiosis progression, DNA fragmentation, IGF-1 free bioavailability, as well as effects on embryo yield and transcriptional profile of matured cumulus–oocyte complexes (COC). First, the COC from a local abattoir were submitted to IVM for 24 h with TCM-199 serum-free medium supplemented with PAPP-A (100 ng mL−1: P100 group) or not (control group). The matured oocytes were submitted to evaluation of DNA fragmentation (TUNEL assay) and meiosis progression (Hoechst 33342; n = 5 replicates; 20 COC/replicate per group), and maturation medium was collected to measure levels of free IGF-1. Then, the oocytes were separated from their respective cumulus cells and followed for the transcriptional profile of 96 genes (3 reference genes; ACTH, GAPDH, PPIA) by RT-qPCR using Taqman® assays in the HD-Biomark System® (Fluidigm Corp., South San Francisco, CA, USA). Further, the matured oocytes were submitted to in vitro fertilization followed by in vitro culture for 7 days. On Days 3 and 7, the cleavage and blastocyst (BL) rates were verified. On Day 7, BL (3 BL/pool; control: n = 4 pools; P100: n = 5 pools) were collected to analyse the transcriptional pattern of 96 genes (4 reference genes; ACTH, GAPDH, PPIA, and SDHA) as described above for COC. The DNA fragmentation, meiosis progression, cleavage, and BL rates were calculated as percentages and transformed to arcsine. The mRNA abundance of target genes was normalized by geometric mean of reference genes and data were transformed to fold change. The free IGF-I concentration also was transformed to fold change. All data were tested by ANOVA and means were compared with t-test or Wilcoxon tests using JMP software (SAS Institute Inc., Cary, NC, USA). Differences were considered significant when P ≤ 0.05. The addition of PAPP-A increased free IGF-I concentration 1.27-fold in IVM medium. There were no alterations in the percentage of oocytes in metaphase II or oocyte DNA fragmentation. In cumulus cells, the genes BCL2, GPX1, RPLP0, and RPS25 (anti-apoptotic and anti-oxidative stress) was higher in the P100 group, whereas DICER, GREM1, GUCY1B3, and FOXO3 (cell proliferation, cumulus expansion, cGMP regulator, and apoptotic initiator, respectively) were higher in the control group. In oocytes, the mRNA relative abundance of ACACA, BCL2, H1FOO, TXNRD1, and VCAN (related with fatty acid synthesis, anti-apoptotic effect, chromatin regulation, oxidative stress processes, and cell proliferation, respectively) was higher in the P100 group. There was no difference in cleavage rate or embryo yield. The mRNA abundance of genes related to cellular stress (ATF4, GPX4, and HIF1A) and lipid metabolism (FASN and SREBF1) was lower in embryos of the P100 group. On the other hand, genes involved in cellular proliferation/differentiation (MAPK1) and pluripotency (POU5F1) were up-regulated in embryos of the P100 group. In conclusion, the addition of PAPP-A during the IVM increased free IGF-I and modulates the gene expression in COC and blastocysts, which could modify oocyte competence and embryo development.

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