185 Serum-dependent and serum-independent effects of prolactin and progesterone during the completion of invitro maturation on metaphase II chromosomes in bovine oocytes

2020 ◽  
Vol 32 (2) ◽  
pp. 220
Author(s):  
I. Lebedeva ◽  
G. Singina ◽  
E. Shedova ◽  
A. Lopukhov
Keyword(s):  
Calf Serum ◽  
Nuclear Material ◽  
Inhibitory Effect ◽  
Developmental Competence ◽  
Terminal Deoxynucleotidyl Transferase ◽  
H 26 ◽  
Independent Effects ◽  
Apoptotic Effects ◽  
Serum Dependent ◽  
Bovine Oocytes

The final stages of IVM are of great importance for the developmental competence of mammalian oocytes. The goal of the present work was to study effects of prolactin (PRL) and progesterone (PG) during the completion of IVM in serum-containing and serum-free media on destructive and apoptotic changes of MII chromosomes in bovine oocytes. Cumulus-oocyte complexes (COCs) were matured for 16h in TCM-199 containing 10% fetal calf serum (FCS), 10μgmL−1 porcine FSH, and 10μgmL−1 ovine LH at 38.5°C and 5% CO2. Thereafter, the COCs were transferred to and cultured for either 8 or 26h in the following systems: TCM-199 containing 10% FCS (Control-1) and TCM-199 containing 3mgmL−1 bovine serum albumin (BSA; Control-2). In both systems, the medium of experimental groups was supplemented with 50ngmL−1 bovine PRL or 50ngmL−1 PG. All treatments were repeated 4 to 5 times using 80 to 108 oocytes per group. The state of the oocyte nuclear material was evaluated by Tarkowski's method. Apoptosis in MII oocytes was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Arcsine-transformed data were analysed by ANOVA. At the end of culture, the rate of MII oocytes was similar in all the groups (83.4-93.9%). Following 8h of IVM, the frequency of MII chromosome abnormalities (decondensation, adherence, clumping) was reduced (P<0.01) in the PRL-1 and PG-1 groups compared with Control-1 (Table 1). This effect was still observed in the PG-1 group (P<0.001) but disappeared in the PRL-1 group after 26h of prolonged oocyte culture. In contrast, a decrease in the rate of MII oocytes with abnormal chromosomes occurred only in the PG-2 group by the end of 8h of culture (P<0.01), whereas the decrease was revealed in both the PG-2 and PRL-2 groups after 26h of culture (P<0.001). Furthermore, the rate of MII oocytes with apoptotic signs following 8h was lower in the PRL-1 and PG-1 groups than in Control-1 (P<0.05). During the 26-hculture, this rate increased in all groups, being higher in Control-1 than in the PG-1 group (P<0.05). The rate of apoptotic oocytes was 2.8 times (8h) and 1.8 times (26h) lower in Control-2 than in Control-1 (P<0.01). Neither PRL nor PG affected oocyte apoptosis in system 2. Thus, during the completion of IVM, PRL can exert a short-term and serum-dependent inhibitory effect on destructive changes of MII chromosomes in bovine oocytes, whereas the similar effect of PG is long-term and serum-independent. Anti-apoptotic effects of both PRL and P4 on the oocytes are determined by serum. Table 1.Effects of prolactin (PRL) and progesterone (PG) on MII chromosomes in bovine oocytes Group % of MII oocytes With abnormal chromosomes With apoptotic signs 8 h 26 h 8 h 26 h Control-1 36.5±3.1a 68.0±2.2a 19.4±1.1a 65.5±2.9a PRL-1 23.6±3.2bc 68.3±2.6a 9.8±3.3b 51.2±7.8abc PG-1 23.4±2.4bc 49.4±0.6b 8.0±3.1b 53.6±0.7b Control-2 31.7±2.7ac 62.7±1.0a 6.9±1.3b 37.3±3.3cd PRL-2 32.6±2.5ac 45.9±0.9b 7.4±2.9b 28.1±2.1d PG-2 20.0±2.5b 47.2±1.6b 6.8±0.9b 29.9±3.9cd a-dValues within a column differ (at least P<0.05). This study was supported by RFBR (No. 17-29-08035).

143 EFFECT OF FSH OR EPIDERMAL-GROWTH-FACTOR-LIKE PEPTIDE SUPPLEMENTATION TO MATURATION MEDIUM ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES DERIVED FROM FULLY DEVELOPED FOLLICLES INDUCED BY SUPER-STIMULATION

2017 ◽  
Vol 29 (1) ◽  
pp. 180
Author(s):  
T. Yamanouchi ◽  
S. Sugimura ◽  
H. Matsuda ◽  
M. Ohtake ◽  
Y. Goto ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Formation Rate ◽  
Calf Serum ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Maturation Medium ◽  
Epidermal Growth ◽  
Bovine Oocytes

Bovine oocytes obtained by ovum-pick-up (OPU) following follicle growth treatment (FGT) have improved quality and competence (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). However, the effect of the presence of FSH or epidermal growth factor (EGF) like peptide during in vitro maturation (IVM) on the developmental competence of FGT oocytes has not been well known. This study was undertaken to examine the developmental competence of FGT oocytes following IVM in the presence of FSH (recombinant human FSH) or EGF-like peptide (amphiregulin; Areg) and IVF. Japanese Black cows (n = 17) were used as donors. Five days after arbitrary OPU (opu group), follicles ≥8 mm in diameter were aspirated again, a controlled internal drug release (CIDR) was inserted into the vagina, and then pFSH was injected twice a day from the evening of Day 6 to the morning of Day 10 with decreasing doses (total of 20 AU; 4, 4, 3, 3, 2, 2, 1, 1 AU/day). On the evening of Day 8, PGF2α (0.5 mg of cloprostenol) was administered. On Day 11, oocytes were aspirated from follicles with ≥5 mm in diameter of the treated donors by OPU (fgt group). The cumulus-oocyte complexes (COC) were cultured in the absence (opu-cont and fgt-cont groups) or presence of 0.1 IU mL−1 FSH (opu-fsh and fgt-fsh groups) or 100 ng mL−1 Areg (opu-areg and fgt-areg groups) in IVM medium (mTCM199 containing 5 mg mL−1 BSA) for 20 to 22 h (1 COC/5 µL, total of 162–171 COC per group), and then co-cultured with 3 × 106 sperm/mL for 6 h. The presumptive zygotes were continued to culture in mCR1aa supplemented with 5% newborn calf serum for 216 h (1 zygote/5 µL) using micro-well culture dishes (Dai-Nippon-Print). When repeating this opu-fgt session in the same cow, an interval at least for 50 days was kept, and the session was performed 28 times. Statistical analysis was carried out by Mann-Whitney’s U-test (between opu and fgt groups) or Steel-Dwass test after Kruskal-Wallis test (among all groups). The number of follicles ≥5 mm increased in the fgt than opu group (17.8 v. 2.9; P < 0.01). The number of COC collected was not different between the opu and fgt groups (23.1 v. 19.6; P > 0.05). The blastocyst formation rate was higher in the fgt than opu group (36.9 v. 23.1%; P < 0.01). Within 6 groups, the blastocyst formation rate was higher in the fgt-fsh (43.3%; P < 0.01) and fgt-areg (39.5%; P < 0.05) groups than the opu-cont (16.3%) group. The rate in the fgt-fsh group was also higher than that in the opu-fsh group (43.3 v. 18.7%; P < 0.01). These results suggested that FGT improved the developmental competence of bovine oocytes, probably through improving the ability of the COC to react against FSH/Areg.

132 The Developmental Competence of Bovine Oocytes Exposed to Progesterone and Luteotrophic Hormones During the Second Step of Two-Step Maturation

2018 ◽  
Vol 30 (1) ◽  
pp. 206
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
T. Taradajnic ◽  
E. Shedova ◽  
A. Lopukhov ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Calf Serum ◽  
Developmental Competence ◽  
Second Step ◽  
Russian Science ◽  
Cleavage Rate ◽  
System 1 ◽  
And Control ◽  
Bovine Oocytes

Data on effects of progesterone (P4) during in vitro maturation of bovine oocytes on their capacity for embryonic development are contradictory. Our study was aimed at characterising effects of P4 and 2 luteotropic hormones, prolactin (PRL) and LH, on bovine oocyte developmental competence during the second step of two-step maturation (from metaphase (M)I to MII). Slaughterhouse-derived cumulus-enclosed oocytes (CEO) were matured for 12 or 24 h [one-step (OS) Control] in TCM-199 containing 10% fetal calf serum (FCS), 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH at 38.5°C and 5% CO2. The CEO cultured for 12 h were transferred to the following culture systems: (1) TCM-199 containing 10% FCS (Control 1) or (2) a monolayer of granulosa cells (GC) precultured for 12 h in TCM-199 containing 10% FCS (Control 2); then, the oocytes were matured for next 12 h. In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (25 and 50 ng mL−1) or ovine LH (5 μg mL−1). All treatments were repeated 5 to 6 times using 138 to 196 oocytes per group. Following IVM, all oocytes underwent IVF as described previously (Singina et al. 2014 Reprod. Fertil. Dev. 26, 154). Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured to Day 7. Embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst formation. Apoptosis was detected by the TUNEL method using 26 to 47 blastocysts per group (from 4 to 5 separate experiments). For each system, arcsine-transformed data were analysed by one-way ANOVA. In OS Control, the cleavage and blastocyst rates were 68.9 ± 4.4% and 22.0 ± 2.4%, respectively. Regardless of the system or medium of two-step culture, the cleavage rate did not differ from that for OS Control, varying between 57.6 and 68.4%. In the absence of GC (System 1), the blastocyst yield in the P4 group (30.4 ± 0.8%) was greater (P < 0.05) than in OS Control and Control 1 (20.2 ± 2.7%) as well as in the groups treated with LH (19.1 ± 3.0%) and 25 ng mL−1 PRL (20.1 ± 2.7%). In the presence of GC, P4 raised the yield from 16.7 ± 2.3% (Control 2) to 27.7 ± 2.4% (P < 0.05). Furthermore, in System 2, the blastocyst rate in groups treated with P4 and 50 ng mL−1 PRL (25.0 ± 2.8%) was higher (P < 0.05) than in the LH group (13.9 ± 2.6%). Meanwhile, the proportion of apoptotic nuclei (2.3-6.9%) was not associated with the system of oocyte maturation or effects of hormones studied. Our data indicate that P4 (50 ng mL−1) can enhance the developmental competence of bovine oocytes during the second step of two-step maturation regardless of the presence of granulosa cells, whereas the similar effect of PRL (50 ng mL−1) is less pronounced and depends on the granulosa-conditioned environment. This research was supported by the Russian Science Foundation (project 16-16-10069).

357 MOLECULAR AND SUBCELLULAR CHARACTERIZATION OF BOVINE OOCYTES AND THEIR SURROUNDING FOLLICULAR CELLS IN SUBJECT TO THEIR DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 335
Author(s):  
H. Torner ◽  
D. Janowski ◽  
N. Ghanem ◽  
D. Salilew-Wondim ◽  
H. Alm ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caspase 3 ◽  
Expression Profiles ◽  
Developmental Competence ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Altered Expression ◽  
Developmental Potential ◽  
Expression Of Genes ◽  
Bovine Oocytes ◽  
Oocyte Selection

Though many factors have been shown to affect the oocyte developmental potential, it remains difficult to draw clear and reliable criteria for oocyte selection. With the urgent need for establishing non-invasive means for oocyte selection, the brilliant cresyl blue (BCB) staining test based on glucose- 6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate competent and non-competent bovine oocytes (Alm et al. 2005 Theriogenology 63, 2194-2205). Also it has been hypothesized that there is a correlation between the appearance of light atretic granulosa cells (GC) in the follicle and an increased developmental competence of the oocyte. Here we aim to investigate whether different developmental competent oocytes show differences concerning the degree of apoptosis or in the gene expression pattern of their follicular environment [GC and cumulus cells (CC)]. After follicular aspiration, the immature COCs were separately stained with 26 μM BCB for 90 minutes. Based on their colouration, oocytes were grouped into BCB- (colourless cytoplasm, low developmental competence) and BCB+ (coloured cytoplasm, high developmental competence). The corresponding CC and GC were also grouped according to the colouration of the enclosed oocytes. BCB+ oocytes were found to result in a higher blastocyst rate at Day 8 of in vitro culture (34.1%) compared to BCB- ones (3.9%) (n = 601 COCs). Apoptosis in GC was determined either by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) or by Annexin-V-staining followed by flow cytometric measurement. The degree of apoptosis in GC of BCB+ oocytes was slightly increased in contrast to the BCB- group (17.0 v. 11.0%; P < 0.05). The abundance and activity of protein kinases Akt, MAP kinase, and Caspase-3 were estimated by western blot analysis. CC, GC, and oocytes from the BCB+ group showed a higher ratio of cleaved Caspase-3/Caspase-3 in contrast to all compartments of the BCB- group (P < 0.05). Moreover, a bovine Affymetrix microarray plate form (Affymetrix Inc., Santa Clara, CA, USA) was used to analyze the gene expression profiles in oocytes, CC, and GC. The BCB+ oocytes were found to be enriched with genes regulating the oocyte maturation (EIF3F, PRKCSH) and the transition from maternal to embryonic genome activation (HMG2L1). Also BCB+ derived follicular compartments showed elevated expression of genes related to steroidgenesis, cumulus expansion and gonadotropins. In conclusion, the results demonstrate that the developmental competence of oocytes is associated with the apoptotic level and altered expression of genes in cells of their follicular environment. This work was supported financially by Deutsche Forschungsgemeinschaft (To 138/5-1).

223 RELATIONSHIP BETWEEN THE LENGTH OF CELL CYCLES, CLEAVAGE PATTERN, AND DEVELOPMENTAL COMPETENCE DURING IN VITRO CULTURE OF IN VITRO-MATURED/IN VITRO-FERTILIZED BOVINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 209 ◽  
Author(s):  
T. Somfai ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
S. Kobayashi ◽  
...  
Keyword(s):  
Cell Division ◽  
In Vitro Culture ◽  
Cell Mass ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Pattern ◽  
Cell Cycles ◽  
Bovine Oocytes

In in vitro embryo production systems, there is a need to select embryos with good developmental competence at the early stages. This study was conducted to determine whether there was any relationship between the duration of the first 3 cell cycles, the cleavage pattern of the first cell division, and the developmental competence of embryos during in vitro culture. A total of 320 in vitro-matured and in vitro-fertilized bovine oocytes were cultured in microdrops of CR1aa medium supplemented with 5% calf serum covered by mineral oil in 5% CO2 and 20% O2 at 38.5°C. The kinetics of embryo development were measured by time-lapse cinematography. Embryos were classified according to their cleavage pattern at the first cell division. Of 285 cleaved embryos, 119 had 2 blastomeres of the same size (normal cleavage: NC), 49 had 2 blastomeres with multiple small fragments (multiple fragments: MF), 34 had 2 blastomeres and a protrusion (protrusion: PT), 45 showed direct cleavage from 1 cell to 3 or 4 blastomeres (3–4BL), and 60 oocytes cleaved to 2 blastomeres of different sizes (unequal blastomeres: UB). (Twenty-two embryos belonged to 2 classes.) After 175 h of culture, blastocysts were either subjected to differential inner cell mass/trophectoderm (ICM/TE) staining or karyotyped. The first and second cell cycles (mean ± SEM) of viable embryos (that could develop to the blastocyst stage) were significantly shorter than those of nonviable embryos (24.9 ± 0.3 h and 8.7 ± 0.1 h v. 26.6 ± 0.7 h and 10.0 ± 0.1 h, respectively); however, the length of the third cell cycle did not differ (P < 0.05, paired t-test). The duration of 1 cell stage in the NC group was significantly shorter than that of MF, PT, 3–4BL, and UB groups (24.7 ± 0.4 h, 26.6 ± 0.5 h, 26.3 ± 0.6 h, 26.0 ± 0.2 h, and 27.7 ± 0.9 h, respectively). The length of the second and third cell cycles did not differ among the groups. The percentage of NC embryos that developed to the blastocyst stage was similar to that of the 3–4BL group (66.9 and 56.7%, respectively) but was significantly higher than those of the MF, PT, and UB groups (40.5, 26.5, and 35.6%, respectively; P < 0.05, ANOVA). The mean cell numbers of NC blastocysts did not differ from those of the MF, 3–4BL, and UB groups but were higher than those of PT embryos (147.1, 155.6, 121.6, 146.4, and 115.1, respectively). There was no difference in ICM/TE rates between the groups. Unlike NC, MF, PT, and UB embryos, most (6 of 8 karyotyped) 3–4BL blastocysts had abnormal ploidy, such as haploid, triploid, mixoploid, or chaotic chromosome numbers, in blastomeres. Our results revealed that not only the length of the first cell cycles, but also the cleavage pattern during first cell division can be a marker of developmental competence and should be considered for the selection of good-quality embryos for embryo transfer. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

166 THE EFFECT OF TEMPERATURE DURING STORAGE OF IN VITRO-MATURED BOVINE OOCYTES IN A HEPES-BUFFERED MEDIUM ON DEVELOPMENTAL COMPETENCE

2016 ◽  
Vol 28 (2) ◽  
pp. 213
Author(s):  
T. Suttirojpattana ◽  
T. Somfai ◽  
S. Matoba ◽  
T. Nagai ◽  
R. Parnpai ◽  
...  
Keyword(s):  
Polar Body ◽  
Calf Serum ◽  
Cell Number ◽  
Developmental Competence ◽  
Effect Of Temperature ◽  
Control Group ◽  
Atp Content ◽  
Developmental Rates ◽  
Bovine Oocytes

The objective of this study was to clarify the effect of the temperature during liquid storage in in vitro matured (IVM) bovine oocytes. IVM bovine oocytes were stored in Eppendorf tube containing 1 mL HEPES TCM-199 supplemented with 10% (v/v) new born calf serum at different temperatures (4°C, 15°C, 25°C, and 38.5°C) for 20 h. The developmental rates of stored and not stored (control) oocytes to the blastocyst stage, cell numbers in resultant blastocysts, and fertilization normality were evaluated after in vitro fertilization and in vitro culture. The ATP content, reduced glutathione (GSH) content, and apoptosis rates in oocytes were also determined in stored and control groups. At least 3 replicates were conducted for each experiment. The data were analysed by 1-way ANOVA followed by post hoc Fisher’s protected least significantly difference test. Percentage data were transformed to arc-sine before analysis. All of the storage groups (4, 15, 25, and 38.5°C groups, respectively) showed significantly lower blastocyst developmental rates (8.5, 14.9, 19.3, and 24.5%, respectively) compared with the control group (39.8%; P < 0.05). Within the storage groups, the 25°C and the 38.5°C groups exhibited the greatest rate of blastocyst formation. In contrast, the total cell number of the 38.5°C group was significantly lower than that of control group (P < 0.05), whereas that of the 25°C group was similar with the control group. The frequency of normal emission of the second polar body (2PB) was significantly greater in the control group compared with the storage groups (P < 0.05). The 2PB emission rate was significantly lower in the 38.5°C group compared with the 4°C group (P < 0.05) but not different from those of the 15°C and 25°C storage groups. The percentage of male pronuclear formation in the control group was significantly higher than those in the stored groups (P < 0.05) except for the 25°C group. During storage at 4°C, the ATP content was significantly decreased compared with the control group (1.3 v. 1.7 pmol; P < 0.05); however, in the 25°C and 38.5°C groups, the ATP content (2.0 and 1.9 pmol, respectively) was significantly higher than that in the control group (1.7 pmol; P < 0.05); whereas the 15°C group showed the same ATP level compared with the control group. Storage of oocytes for 20 h reduced the GSH content compared with the control group without storage (P < 0.05); however, there were no significant differences among storage groups. Annexin-V staining revealed increased incidences of early apoptotic oocytes in the 4°C and 15°C groups (P < 0.05) compared with other groups. In conclusion, based on the embryo developmental competence of stored oocytes and quality of resultant blastocysts, 25°C was determined as the most suitable temperature for temporal storage of matured bovine oocytes. The study was supported by the NARO Institute of Livestock and Grassland Science, Japan (N32G4126), and the Royal Golden Jubilee-PhD scholarship (2.B.TS/53/F.2).

263 GENE EXPRESSION PROFILING OF IMMATURE AND IN VITRO-MATURED BOVINE OOCYTES USING AFFYMETRIX GENECHIP TECHNOLOGY

2007 ◽  
Vol 19 (1) ◽  
pp. 248 ◽  
Author(s):  
F. Carter ◽  
T. Fair ◽  
S. Park ◽  
M. Wade ◽  
A. C. O. Evans ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Bovine Genome ◽  
Calf Serum ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Total Rna ◽  
Rna Content ◽  
Bovine Oocytes

Previous studies by our group have demonstrated that oocyte maturation is a crucial event in the determination of subsequent developmental competence. The objective of the current study was to characterize changes in gene expression profiles of bovine oocytes during meiotic maturation. To this end, 5 replicate pools of 200 bovine cumulus–oocyte complexes (COCs)were collected from the ovaries of slaughtered heifers. Upon recovery, 100 COCs from each replicatewere immediately denuded, and the oocytes were snap frozen in liquid nitrogen. The remaining 100 COCs were matured in vitro in TCM-199 supplemented with 10% (v/v) fetal calf serum and 10 ngmL-1 epidermal growth factor for 24 h at 39�C under an atmosphere of 5% CO2 in air with maximum humidity. Following maturation, the remaining COCs were denuded and snap frozen. Total RNA was isolated (mean total RNA content 106.08�38.87 ng per 100 oocytes) and subjected to 2 rounds of amplification incorporating biotin-labeled nucleotides during the second in vitro transcription reaction (mean total RNA content 155.15�51.14 �g per 100 oocytes post-amplification). The resulting labeled antisense RNA was hybridized to a GeneChip Bovine Genome Arrays (Affymetrix, Inc., Santa Clara, CA, USA) (10 chips, 5 replicates each of immature and mature oocytes, n=100 oocytes/chip). Expression data were analysed using Genespring software (Agilent Technologies, Palo Alto, CA, USA), and data were normalized to the median. Overall, 54.9�1.3% and 53.3�3.3% of the 24 178 probe sets representing 23 000 transcripts spotted on the arrays were expressed in immature and in vitro-matured oocytes, respectively. Across the 5 array comparisons, 52 genes were consistently exclusively present in immature oocytes, whereas 16 genes were exclusively present in mature oocytes. A further 821 genes were found to be differentially expressed (≥2-fold) between the 2 groups (P &lt;0.05), of which 209 were up-regulated and 612 were down-regulated in the in vitro-matured oocytes compared with their immature counterparts. The differentially expressed transcripts were classified according to their gene ontology (http://benzer.ubic.ca/ermineJ). The existing Affymetrix annotation was updated by blasting the sequences against bovine, human, and murine databases (≥90% homology; increasing molecular function annotation from 14% to 42%). In terms of olecular function, the majority of these genes were associated with protein or nucleic acid binding (&gt;42%), catalytic activity (24%), signal transduction (7%), transporter activity (5%), and structural molecule activity (5%). In conclusion, we have stablished the molecular transcriptome blueprint of immature and in vitro-matured bovine oocytes. Through comparisons with in vivo-matured oocytes, this resource will be invaluable in determining genes that are involved in controlling the developmental competence of oocytes. This research was funded by the Science Foundation Ireland (02/IN1/B78).

292 INHIBITION OF HSP90 AGGRAVATES THE EFFECTS OF HEAT SHOCK ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 235
Author(s):  
E. D. Souza ◽  
N. C. Rabelo ◽  
T. D. Araujo ◽  
C. M. Assunção ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Oocyte Developmental Competence ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Bovine Oocytes

The heat shock protein 90kDa (HSP90) is a chaperone involved in protein homeostasis under normal and stress conditions. Its inhibition by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma, St. Louis, MO, USA) for 12 or 24 h during in vitro maturation reduces the oocyte's ability to develop after in vitro fertilization (Souza et al. 2014 Reprod. Fert. Dev. 26, 197). This study aimed to evaluate the effect of treatment with 17AAG during the heat shock on oocyte developmental competence. Immature bovine COC were randomly allocated in 4 treatments during IVM: control = no heat shock or 17AAG; HS = heat shock (41.5°C) for the first 12 h of IVM; 17AAG = 2 µM 17AAG for the first 12 h of IVM; and 17AAG + HS = 2 µM 17AAG plus heat shock for the first 12 h of IVM. In vitro maturation was performed in Nunc plate containing 400 µL of TCM199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2 in air, 95% humidity, and 38.5°C for 24 h. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) and oocytes were in vitro fertilized for 20 h with 2 × 106 spermatozoa mL–1 under the same IVM atmospheric conditions. Presumptive zygotes were completely denuded in a PBS solution with 0.1% hyaluronidase and then cultured in wells with 500 µL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h postfertilization and blastocyst rate was evaluated at Day 7 (D7) and 8 (D8). Data from 7 replicates were submitted to analysis of variance and means were compared by Student Newman Keul's test. There was no difference (P > 0.05) on cleavage rate among treatments. Heat shock or treatment with 17AAG, both for 12 h of IVM, decreased (P < 0.05) the blastocyst rate at D7 and D8 when compared to control but no significant difference between HS and 17AAG treatments was found (Table 1). However, the lowest (P < 0.05) blastocyst rate at D7 and D8 was achieved when oocytes were submitted simultaneously to 17AAG and heat shock for 12 h of IVM (17AAG + HS treatment, Table 1). In conclusion, the treatment with 17AAG during IVM worsens the deleterious effect of heat shock on oocyte developmental competence and suggests that HSP90 may also play role on cellular protection during heat shock in bovine oocytes. Table 1.Cleavage and blastocyst (Bl) rates at D7 and D8 for control, 17AAG, Heat Shock (HS), and 17AAG plus HS treatments Financial support comes from CNPq, FAPEMIG, and FAPES.

scholarly journals Apoptotic Effects of Xanthium strumarium via PI3K/AKT/mTOR Pathway in Hepatocellular Carcinoma

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Juyoung Kim ◽  
Kyung Hee Jung ◽  
Hyung Won Ryu ◽  
Doo-Young Kim ◽  
Sei-Ryang Oh ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Growth ◽  
Caspase 3 ◽  
Mtor Pathway ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Mechanistic Study ◽  
Migration And Invasion ◽  
Xanthium Strumarium ◽  
Apoptotic Effects ◽  
Hcc Cells

Xanthium strumarium (XS) has been traditionally used as a medicinal herb for treating inflammatory diseases, such as appendicitis, chronic bronchitis, rheumatism, and rhinitis. In this study, we yielded ethanol extracts from XS and investigated whether they could inhibit the progression of hepatocellular carcinoma (HCC) and its underlying mechanism. The XS-5 and XS-6 extracts dose-dependently inhibited the growth and proliferation in HCC cell lines. The apoptotic effects of them were observed via increased levels of cleaved caspase-3 and cleaved PARP, as well as elevated numbers of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling- (TUNEL-) positive apoptotic cells. They also decreased XIAP and Mcl-1 expression via loss of mitochondrial membrane potential. Additionally, they inhibited the invasion and migration of HCC cells. In an ex vivo model, the extracts significantly inhibited tumor cell growth and induced apoptosis by increasing the expression of the cleaved caspase-3. A mechanistic study revealed that they effectively suppressed PI3K/AKT/mTOR signaling pathways in HCC cells. Taken together, our findings demonstrate that they could efficiently not only induce apoptosis but also inhibit cell growth, migration, and invasion of human HCC cells by blocking the PI3K/AKT/mTOR pathway. We suggest XS-5 and XS-6 as novel natural anti-HCC agents.

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