Effect of Methylmercury Exposure on Bioaccumulation and Nonspecific Immune Respsonses in Hybrid Grouper Epinephelus fuscoguttatus × Epinephelus lanceolatus

Mercury (Hg) is a dangerous heavy metal that can accumulate in fish and is harmful when consumed by humans. This study investigated the bioaccumulation of mercury in the form of methylmercury (MeHg) and evaluated nonspecific immune responses such as phagocytic activity and superoxide anion (O2−) production in hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus). The hybrid grouper leukocytes were incubated with methylmercury chloride (CH3HgCl) at concentrations of 10–10,000 µg/L to determine cell viability, phagocytic activity, and O2− production in vitro. Subsequently, the grouper were exposed daily to CH3HgCl mixed in the experimental diets at concentrations of 0, 1, 5, and 10 mg/kg for 28 days. The bioaccumulation of MeHg in the liver, head kidney, and muscle tissue was measured, and the phagocytic activity and O2− production were evaluated. In vitro results indicated that cell viability was significantly lower than that of the control group at concentrations > 500 µg/L. The phagocytic rate and O2− production at concentrations ˃ 500 and ˃ 200 µg/L, respectively, were significantly lower than those of the control group. The dietary exposure demonstrated that MeHg accumulated more substantially in the liver and head kidney compared with the muscle tissue in the treatment groups. Moreover, the cumulative concentration significantly increased with higher concentrations and more days of exposure. The phagocytic rate and O2− production in the treatment groups were significantly lower than those in the control group from days 2 and 1, respectively. In conclusion, hybrid grouper accumulated significant MeHg in the liver and head kidney compared with the muscle tissue, and higher concentrations and more exposure days resulted in decreased cell viability, phagocytic activity, and O2− production.

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THE EFFECT OF VITAMIN C AND AEROMONAS VACCINE ON THE IMMUNE RESPONSE AND DISEASE RESISTANCE OF GROUPER (Epinephelus fuscoguttatus)

Marine Research in Indonesia ◽

10.14203/mri.v34i2.512 ◽

2009 ◽

Vol 34 (2) ◽

pp. 81-85

Author(s):

Ilmiah ◽

St. Hidayah Triana ◽

Asmi Citra Malina A.R. Tassakka ◽

Alex Rantetondok ◽

Hilal Anshary

Keyword(s):

Vitamin C ◽

Immune Responses ◽

Phagocytic Activity ◽

Head Kidney ◽

Treated Group ◽

Aeromonas Salmonicida ◽

Control Fish ◽

Control Group ◽

Epinephelus Fuscoguttatus ◽

Titre Antibody

We evaluated the effectiveness of vitamin C and Aeromonas salmonicida vaccine in grouper (Epinephelus fuscoguttatus) for increasing immune responses and protection against A .salmonicida. The vitamin C used was polyethoxylated ascorbic and tocopherol. The vaccine was prepared from formalin-killed cells and concentrated extracellular products of a single isolate A. salmonicida. Bath immersion vitamin C and vaccine trials were conducted for 60 min. Fish used had a mean weight 25 g. Control groupers were injected with tryptic soy broth. The results showed that vitamin C enhanced phagocytic activity in head kidney leucocytes of grouper 7, 14, 28 and 36 days after treatments. A significant different of the antibody titre was found between control fish and the treated fish at 42 days after treatments. In addition, at day 42, Relative Percent Survival (RPS) for control group was 53.3 %, vitamin C-treated group was 80.0 % and vaccinated group was 90.0 %. The results of this study suggest that bath immersion of vitamin C provided an increasing of phagocytic activity (non-specific immune responses), titre antibody (specific immune responses) and protection against A. salmonicida infection in grouper. A. salmonicida vaccine also en-hanced titre antibody and protection against A. salmonicida infection in grouper.

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Biological characterization of implant surfaces - in vitro study

Revista de Odontologia da UNESP ◽

10.1590/1807-2577.1087 ◽

2015 ◽

Vol 44 (4) ◽

pp. 195-199 ◽

Cited By ~ 1

Author(s):

Priscilla Barbosa Ferreira Soares ◽

Camilla Christian Gomes Moura ◽

Huberth Alexandre da Rocha Júnior ◽

Paula Dechichi ◽

Darceny Zanetta-Barbosa

Keyword(s):

Alkaline Phosphatase ◽

Cell Viability ◽

Total Protein ◽

Cell Attachment ◽

Control Group ◽

Mitochondrial Activity ◽

Serum Total Protein ◽

Trypan Blue Exclusion ◽

Experimental Surface

<title>Abstract</title><sec><title>Objective</title>Evaluate the biological performance of titanium alloys grade IV under different surface treatments: sandblasting and double etching (Experimental surface 1; Exp1, NEODENT); surface with wettability increase (Experimental surface 2; Exp2, NEODENT) on response of preliminary differentiation and cell maturation.</sec><sec><title>Material and method</title>Immortalized osteoblast cells were plated on Exp1 and Exp2 titanium discs. The polystyrene plate surface without disc was used as control group (C). Cell viability was assessed by measuring mitochondrial activity (MTT) at 4 and 24 h (n = 5), cell attachment was performed using trypan blue exclusion within 4 hours (n = 5), serum total protein and alkaline phosphatase normalization was performed at 4, 7 and 14 days (n = 5). Data were analyzed using one-way ANOVA and Tukey test.</sec><sec><title>Result</title>The values of cell viability were: 4h: C– 0.32±0.01A; Exp1– 0.34±0.08A; Exp2– 0.29±0.03A. 24h: C– 0.43±0.02A; Exp1– 0.39±0.01A; Exp2– 0.37±0.03A. The cell adhesion counting was: C– 85±10A; Exp1- 35±5B; Exp2– 20±2B. The amounts of serum total protein were 4d: C– 40±2B; Exp1– 120±10A; Exp2– 130±20A. 7d: C– 38±2B; Exp1– 75±4A; Exp2– 70±6A. 14 d: C– 100±3A; Exp1– 130±5A; Exp2– 137±9A. The values of alkaline phosphatase normalization were: 4d: C– 2.0±0.1C; Exp1– 5.1±0.8B; Exp2– 9.8±2.0A. 7d: C– 1.0±0.01C; Exp1– 5.3±0.5A; Exp2– 3.0±0.3B. 14 d: C– 4.1±0.3A; Exp1– 4.4±0.8A; Exp2– 2.2±0.2B. Different letters related to statistical differences.</sec><sec><title>Conclusion</title>The surfaces tested exhibit different behavior at dosage of alkaline phosphatase normalization showing that the Exp2 is more associated with induction of cell differentiation process and that Exp1 is more related to the mineralization process.</sec>

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Pharmacological Basis of CD101 Efficacy: Exposure Shape Matters

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00758-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 12

Author(s):

Elizabeth A. Lakota ◽

Justin C. Bader ◽

Voon Ong ◽

Ken Bartizal ◽

Lynn Miesel ◽

...

Keyword(s):

Time Course ◽

Fungicidal Activity ◽

Control Group ◽

Time Curve ◽

Content Type ◽

Treatment Groups ◽

Concentration Time ◽

Treatment Control Group ◽

Pharmacological Basis

ABSTRACT CD101 is a novel echinocandin with concentration-dependent fungicidal activity in vitro and a long half-life (∼133 h in humans, ∼70 to 80 h in mice). Given these characteristics, it is likely that the shape of the CD101 exposure (i.e., the time course of CD101 concentrations) influences efficacy. To test this hypothesis, doses which produce the same total area under the concentration-time curve (AUC) were administered to groups of neutropenic ICR mice infected with Candida albicans R303 using three different schedules. A total CD101 dose of 2 mg/kg was administered as a single intravenous (i.v.) dose or in equal divided doses of either 1 mg/kg twice weekly or 0.29 mg/kg/day over 7 days. The studies were performed using a murine disseminated candidiasis model. Animals were euthanized at 168 h following the start of treatment. Fungi grew well in the no-treatment control group and showed variable changes in fungal density in the treatment groups. When the CD101 AUC from 0 to 168 h (AUC0–168) was administered as a single dose, a >2 log10 CFU reduction from the baseline at 168 h was observed. When twice-weekly and daily regimens with similar AUC values were administered, net fungal stasis and a >1 log10 CFU increase from the baseline were observed, respectively. These data support the hypothesis that the shape of the CD101 AUC influences efficacy. Thus, CD101 administered once per week demonstrated a greater degree of fungal killing than the same dose divided into twice-weekly or daily regimens.

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136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab136 ◽

2005 ◽

Vol 17 (2) ◽

pp. 219 ◽

Cited By ~ 5

Author(s):

C.E. Ferguson ◽

T.R. Davidson ◽

M.R.B. Mello ◽

A.S. Lima ◽

D.J. Kesler ◽

...

Keyword(s):

Embryo Development ◽

Direct Effect ◽

Blastocyst Stage ◽

Control Group ◽

Bovine Embryo ◽

Bovine Embryos ◽

Direct Role ◽

Treatment Groups ◽

And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

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Silica/OCP Affects the Viability of Osteoblasts through ROS-Induced Autophagy

Journal of Nanomaterials ◽

10.1155/2021/3159848 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Jianghao Gong ◽

Shangjun Fu ◽

Zhenghao Zhou

Keyword(s):

Flow Cytometry ◽

Cell Viability ◽

Protein Level ◽

Reactive Oxygen ◽

Octacalcium Phosphate ◽

Control Group ◽

Substrate Protein ◽

Autophagy Inhibitor ◽

Osteoblast Cell Line

Objective. To explore the effects of silicone gel nanoparticles modified with octacalcium phosphate on the surface (silica/OCP) polymer drugs on the proliferation of osteoblasts and autophagy. Method. Silica/OCP was prepared in vitro, and the quality of the sample preparation was tested through characterization experiments. The osteoblast cell line (hFOB1.19) was treated with silica/OCP, autophagy inhibitor (3-methyladenine (3-MA)), and silica/OCP+3-MA, respectively. The proliferation of hFOB1.19 cells was detected through the methylthiazolyldiphenyl-tetrazolium bromide (MTT) kit. Flow cytometry was used to detect the cell apoptosis. The change in protein beclin1 and P62 expression in hFOB1.19 cells was observed in Western blot. An ROS detection kit was used to detect the content of reactive oxygen species in hFOB1.19 cells. Results. Silica/OCP was a sphere with a particle size of 50 nm to 130 nm and had an OCP phase in electron projection microscopy and X-ray diffraction techniques. The results indicated that OCP successfully modified silica and the material was successfully prepared. An MTT kit and flow cytometry test showed that the cell viability of the cells treated with silica/OCP increased significantly ( P < 0.05 ), and the intracellular apoptosis phenomenon was significantly decreased ( P < 0.05 ) compared to the control group. Moreover, the inhibition of cell viability and promotion of apoptosis caused by the autophagy inhibitor 3-MA can be rescued. Western blotting demonstrated that the protein level of beclin1 in osteoblasts reached the highest after six hours of treatment with silica/OCP, and the protein level of p62, the substrate protein of autophagy, reached the lowest. At the same time, treatment of cells with the autophagy inhibitor 3-MA and silica/OCP+3-MA found that the protein levels of beclin1 and p62 in the silica/OCP+3-MA group were adjusted back compared to the 3-MA group. After adding the autophagy inhibitor, the reactive oxygen content in the cell was significantly increased ( P < 0.05 ) in the silica/OCP group. In the presence of intracellular reactive oxygen inhibitors catalase and silica/OCP, the cell viability of osteoblasts was significantly lower than that of the silica/OCP group but significantly higher than that of the silica/OCP+3-MA group. The apoptosis level of the silica/OCP+catalase group was also significantly lower than that of the silica/OCP+3-MA group ( P < 0.05 ) but was significantly higher than that of the silica/OCP group ( P < 0.05 ). Conclusion. Silica/OCP nanoparticles can upregulate the level of autophagy in osteoblasts and promote the proliferation of osteoblasts.

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LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

Archives of Veterinary Science ◽

10.5380/avs.v9i1.4045 ◽

2004 ◽

Vol 9 (1) ◽

Author(s):

M.G.L. PINTO ◽

M.I.B. RUBIN ◽

C.A.M. SILVA ◽

T.F. HILGERT ◽

M.F. SÁ FILHO ◽

...

Keyword(s):

Follicular Fluid ◽

In Vitro Maturation ◽

Mineral Oil ◽

Control Group ◽

Sodium Pyruvate ◽

Vitro Fertilization ◽

Treatment Groups ◽

Maturation Medium ◽

Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

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357 CALCIUM RELEASE INDUCED BY THIMEROSAL AND INOSITOL 1,4,5-TRIPHOSPHATE IN HEAT-SHOCKED PORCINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab357 ◽

2007 ◽

Vol 19 (1) ◽

pp. 294

Author(s):

J.-K. Tseng ◽

P.-C. Tang ◽

J.-C. Ju

Keyword(s):

Heat Shock ◽

Calcium Release ◽

Prolonged Exposure ◽

Imaging System ◽

Developmental Competence ◽

Control Group ◽

Acetoxymethyl Ester ◽

Treatment Groups ◽

All Treatment

Elevated ambient temperature has been known to be deleterious to the developmental competence of mammalian oocytes and embryos, although the mechanism is still unclear. The objective of this study was to determine the effect of heat shock (HS) on the alteration of intracellular calcium concentrations ([Ca2+]i) of matured pig oocytes by two different calcium releasing agents. Porcine cumulus–oocyte complexes were aspirated from the follicles (3–6 mm) and subjected to standard in vitro maturation procedure for 42 h. Matured oocytes were then randomly allocated to different heat treatments at 41.5°C for 0 (Control, C0h), 1 (HS1h), 2 (HS2h), or 4 h (HS4h). An additional control group was cultured for 4 h without heat shock (C4h). Oocytes were incubated with 2 µM fura-2 acetoxymethyl ester (AM) and 0.02% pluronic F-127 in Ca2+-free PBS (40 min) following heat shock, and then washed with Ca2+-free PBS (30 min) for detection of [Ca2+]i. Fluorescent images were captured with alternative excitation wavelengths at 340/380 nm by a rotating chopper disk equipped with an Axon imaging system. Data from both experiments were analyzed by ANOVA using the General Linear Model (GLM) of the SAS (SAS Institute, Inc., Cary, NC, USA). In Experiment 1, matured oocytes were activated by 200 mM thimerosal (10 min) following heat treatment. The maximal [Ca2+]i in the HS2h group was the highest among all treatment groups. The lowest maximal peak of [Ca2+]i was observed in the HS4h group, but it was still higher than that in the C4h group (P < 0.05). The total amount of Ca2+ release represented by the total area of the peaks in C4h was lower than in any other groups except HS4h (P < 0.05). In Experiment 2, each matured oocyte was injected with approximately 10 pL of inositol 1,4,5-triphosphate (IP3, 0.5 mM); the Ca2+ transient was recorded as described in the previous experiment. The maximal value of [Ca2+]i in the C4h group was still the lowest among the heat-shocked and C0h groups (P < 0.05). The total Ca2+ release in the HS2h group was the highest among all treatment groups, but only significantly higher than the HS1h and C4h groups (P < 0.05). A similar pattern of Ca2+ release in HS-oocytes was induced by thimerosal and IP3 stimulations. These results indicate that Ca2+ releasing capacity of matured pig oocytes is enhanced by a shorter duration of heat shock, but declines after prolonged exposure of heat shock and/or in vitro culture. The differential Ca2+ releasing capacity of heat-shocked oocytes prior to fertilization revealed physiological changes of pig oocytes after heat shock. This finding provides further insight for the low fertilization and developmental competence that occurs in farm species during hot seasons.

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332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab332 ◽

2010 ◽

Vol 22 (1) ◽

pp. 322

Author(s):

D. D. Bücher ◽

M. A. Castro ◽

M. E. Silva ◽

M. A. Berland ◽

I. I. Concha ◽

...

Keyword(s):

Cell Viability ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Cumulus Cells ◽

Cell Number ◽

Control Group ◽

Cumulus Expansion ◽

Gm Csf ◽

Nuclear Stage

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles <8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P < 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P < 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P < 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

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215 DISTRIBUTION OF DIMETHYLATION OF HISTONE H3K9 IS NOT AFFECTED BY DNA, MESSENGER RNA, AND PROTEIN SYNTHESIS IN PRONUCLEAR-STAGE PORCINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab215 ◽

2009 ◽

Vol 21 (1) ◽

pp. 205

Author(s):

K. E. Park ◽

R. Cabot

Keyword(s):

Protein Synthesis ◽

Messenger Rna ◽

Control Group ◽

Mrna Synthesis ◽

Treatment Groups ◽

Asymmetrical Distribution ◽

Rna And Protein Synthesis ◽

H3k9 Dimethylation ◽

Differential Localization

Methylation of the lysine 9 residue of histone H3 (H3K9) is linked with repression of transcription. Dimethylated H3K9 adopts a strict asymmetrical distribution in murine zygotes, with dimethylated H3K9 detectable only on maternally derived chromatin. In contrast, both male and female pronuclei in porcine zygotes can possess dimethylated H3K9; however, some asymmetry in H3K9 dimethylation exists between individual pronuclei, particularly in polyspermic embryos. The objective of this study was to determine the extent that DNA, mRNA, and protein synthesis serve in maintaining the asymmetrical distribution of dimethylated H3K9 in porcine zygotes. We hypothesized that the distribution of dimethylated H3K9 between individual pronuclei would not depend on alternations in chromatin structure induced by DNA or mRNA synthesis but would be affected by protein synthesis. To test this hypothesis, in vitro-matured porcine oocytes were fertilized in vitro, cultured in porcine zygote medium-3 containing 3 mg mL–1 of BSA, and allocated to 1 of 4 treatment groups: (1) incubation with 25 μg mL–1 of α-amanitin (α-AM), (2) incubation with 3 μg mL–1 of aphidicolin (APH), (3) incubation with 50 μg mL–1 of cycloheximide (CYC), and (4) nontreated controls. Embryos were removed from each treatment group at 10, 15, 20, and 25 h post gamete mixing, fixed, and processed to detect dimethylated H3K9 immunocytochemically. For monospermic embryos in the control group, 24% (7/29), 31% (8/26), 30% (7/24), and 20% (4/20) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the control group, 82% (32/39), 78% (31/40), 74% (28/38), and 65% (24/37) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For monospermic embryos in the α-AM group, 29% (4/14), 14% (2/14), 8% (1/12), and 11% (1/9) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the α-AM group, 71% (15/21), 63% (12/19), 55% (10/18), and 47% (8/17) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For monospermic embryos in the APH group, 31% (4/13), 23% (3/13), 23% (3/13), and 18% (2/11) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the APH group, 75% (15/20), 67% (12/18), 63% (12/19), and 56% (10/18) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For monospermic embryos in the CYC group, 33% (5/15), 25% (4/16), 14% (2/14), and 9% (1/11) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the CYC group, 78% (18/23), 67% (16/24), 58% (14/24), and 59% (13/22) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. These results suggest that the distribution of dimethylated H3K9 between pronuclei is not affected by DNA, mRNA, or protein synthesis (P > 0.05), but is affected by the age of the pronuclei (P < 0.05).

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312 EFFECTS OF TRANSFORMING GROWTH FACTOR ALPHA AND INSULIN-LIKE GROWTH FACTOR 1 SUPPLEMENTATION ON IN VITRO MATURATION OF CANINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab312 ◽

2015 ◽

Vol 27 (1) ◽

pp. 245

Author(s):

A. Sato ◽

B. Sarentonglaga ◽

K. Ogata ◽

M. Yamaguchi ◽

A. Hara ◽

...

Keyword(s):

Growth Factor ◽

Synergistic Effect ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Transforming Growth Factor ◽

Germinal Vesicle ◽

Control Group ◽

Insulin Like Growth Factor ◽

Treatment Groups

Although in vitro maturation (IVM) of oocytes has been successfully established for many species, the efficiency of IVM in canine oocytes is still very low. As growth factors have been shown to promote oocyte maturation in some species, we investigated whether use of transforming growth factor α (TGF-a) and insulin-like growth factor 1 (IGF-1) might overcome the difficulties of achieving meiotic maturation in cultured canine cumulus-oocyte complexes (COC). Ovaries were obtained from bitches at 6 months to 7 years of age by ovariohysterectomy and were sliced repeatedly to release COC. In the first experiment, the COC were cultured at 38.8°C for 48 h in 5% CO2 in air in medium 199 supplemented with either TGF-a (0, 1, 10, or 100 ng mL–1) or IGF-1 (0, 0.5, 5, 10, or 50 µg mL–1). In the second experiment, the synergistic effect of TGF-a and IGF-1 was investigated by culturing COC in medium 199 supplemented with both TGF-a (0, 1, 10, or 100 ng mL–1) and IGF-1 (0, 0.5, 5, 10, or 50 µg mL–1). At the end of the culture period, the oocytes were denuded of cumulus cells by pipetting with a fine bore glass pipette; the denuded oocytes were then fixed in Carnoy's solution and stained with Hoechst 33342. The nuclear configuration and chromatin morphology of the oocytes were evaluated under confocal laser scanning microscopy. The cells were assigned to 1 of the following meiotic stages: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII). Data were analysed by ANOVA with Fisher's PLSD test. In experiment 1, no significant difference were observed in the rates of cells maturing to the MI and MII stages, but that in the 10 ng mL–1 of TGF-a group (56.3%) were larger than in the other treatment groups (38.8–51.0%). The frequencies of MII stage cells in the 5, 10, and 50 µg mL–1 of IGF-1 treatment groups (9.8, 13.3, and 12.2%, respectively) were significantly higher than in the 0.5 µg mL–1 of IGF-1 group and the control group (5.3 and 2.2%, respectively). In experiment 2, the frequency of MI and MII cells in the control, 1 ng mL–1 of TGF-a plus 0.5 µg mL–1 of IGF-1, 10 ng mL–1 of TGF-a plus 5 µg mL–1 of IGF-1, 10 ng mL–1 of TGF-a plus 10 µg mL–1 of IGF-1, and 100 ng mL–1 of TGF-a plus 50 µg mL–1 of IGF-1 group were 44.1, 36.1, 63.5, 70.8, and 50.8%, respectively. The frequency of MII cells in the control group and the same treatment groups were 2.8, 7.2, 10.4, 15.3, and 10.8%, respectively. Both frequencies in the 10 ng mL–1 of TGF-a plus 10 µg mL–1 of IGF-1 group were significantly higher than in the control group. The TGF-a may act in a paracrine fashion on the surrounding granulosa cells, and IGF-1 may play multiple roles in cellular metabolism, proliferation, growth, and differentiation in canine oocyte maturation, as has been reported for many other species. In conclusion, these results demonstrate that a synergistic effect between TGF-a and IGF-1 produces an increased rate of in vitro maturation to the MI and MII stages in canine oocytes.

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