Involvement of the sphingolipid ceramide in heat-shock-induced apoptosis of bovine oocytes

2011 ◽  
Vol 23 (7) ◽  
pp. 876 ◽  
Author(s):  
Dorit Kalo ◽  
Zvi Roth
Keyword(s):  
Heat Shock ◽  
De Novo ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Oocyte Developmental Competence ◽  
Induced Apoptosis

Programmed cell death via the sphingomyelin pathway has been suggested to underlie heat-shock disturbance of oocyte developmental competence. A series of experiments were performed to characterise the role of the sphingolipid ceramide in heat-shock-induced apoptosis, and to determine whether ceramide formation can be regulated. Bovine cumulus–oocyte complexes (COCs) were aspirated from ovaries collected in the cold season (November–April), in vitro-matured, fertilised and cultured for 8 days. Exposure of COCs to heat shock (41°C) during maturation reduced cleavage rate and blastocyst formation relative to the control group (38.5°C). Annexin-V binding (V-FITC assay), which is associated with the early apoptotic event of membrane phosphatidylserine turnover, was higher in oocytes exposed to short-term versus long-term heat shock, suggesting that heat-shock-induced apoptosis involves membrane alterations. Similar to heat exposure, oocyte maturation with C2-ceramide had a dose-dependent deleterious effect on the first cleavages and subsequent embryonic development in association with increased annexin-V binding. Blocking endogenous ceramide generation with fumonisin B1, a specific inhibitor of dihydroceramide synthase (i.e. de novo formation), moderated, to some extent, the effects of heat shock on oocyte developmental competence, suggesting that ceramide plays an important role in heat-shock-induced apoptosis.

248 INVOLVEMENT OF THE SPHINGOLIPID, CERAMIDE, IN HEAT SHOCK-INDUCED APOPTOSIS IN BOVINE OOCYTE

2009 ◽  
Vol 21 (1) ◽  
pp. 222 ◽  
Author(s):  
D. Kalo ◽  
Z. Roth
Keyword(s):  
Heat Shock ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Developmental Competence ◽  
Immunofluorescent Staining ◽  
Blastocyst Formation ◽  
Short Term ◽  
Oocyte Developmental Competence ◽  
Induced Apoptosis

Programmed cell death through the sphingomyelin pathway has been suggested to underlie the mechanism by which heat shock disturbs oocyte developmental competence. Serial experiments were performed to characterize the role of the sphingolipid ceramide in heat-shock-induced apoptosis, and to determine whether ceramide formation could be regulated. In all experiments, bovine cumulus–oocyte complexes (COC) were aspirated from ovaries collected at the abattoir. Cumulus–oocyte complexes were matured (22 h, 38.5°C, 5% CO2), in vitro fertilized (18 h, 38.5°C, 5% CO2), and cultured for 8 days (KSOM; 38.5°C, 5% CO2, 5% O2). In the first experiment, COC were matured at 38.5°C (n = 673) or 41°C (heat shock; n = 718). Exposure of COC to heat shock during maturation reduced cleavage (92.3 v. 84.2%; P < 0.05) and blastocyst formation rates (24.1 v. 14.6%; P < 0.05) relative to the control groups. In the second experiment, COC (n = 563) were exposed to either long-term (22 h) or short-term (0.5 to 3.5 h) heat shock during maturation. An Annexin V-FITC assay (Bender) was used to identify the turnover of phosphatidylserine, one of the features of early-stage apoptosis. In addition, a subgroup of matured oocytes (n = 384) were denuded and fixed in 2% paraformaldehyde, followed by immunofluorescent staining using anti-ceramide (Alexis) to detect ceramide levels and counterstaining with Hoechst (Sigma). Immunofluorescent staining showed no difference in endogenous ceramide levels between groups. Similarly, annexin expression did not differ between groups at the end of the maturation but was higher (P < 0.05) in oocytes exposed to short-term heat shock than in those subjected to long-term heat shock, suggesting that early features of apoptosis should be examined at a specific time of heat exposure. Another experiment was performed to examine the effect of ceramide on oocyte developmental competence. Cumulus–oocyte complexes (n = 1185) were matured with or without 10, 30, and 50 μm C2-ceramide (Sigma). An Annexin V-FITC assay was used with a subgroup of oocytes (n = 137) that were matured with or without 50 μm C2-ceramide for 2 h. To examine the major pathway of ceramide generation, heat-shocked COC were matured with or without 5 μm fumonisin B1 (FB1; Sigma), a specific inhibitor of dihydroceramide synthase (i.e. de novo formation) and ceramide synthase (i.e. salvage pathway), or with 5 μm desipramine hydrochloride (DH; Sigma), a specific inhibitor of the acid sphingomyelinase (i.e. hydrolysis). Results revealed that maturation in 50 μm C2-ceramide increased (P < 0.05) annexin expression in association with reducing cleavage rate and blastocyst formation (P < 0.05). Although not significant, maturation with FB1 moderated the heat-shock effect on oocyte developmental competence. Similarly, 5 μm DH increased the blastocyst formation rate for heat-shocked oocytes from a level of 17% to the control level (22.5%). In summary, ceramide appears to play an important role in heat-shock-induced apoptosis because blocking ceramide formation alleviated its deleterious effect. Research was supported in part by USDA Grant 2007-35203 and BARD Grant 3986-07

scholarly journals 121 HEAT SHOCK TO PIG OOCYTES DOES NOT INDUCE APOPTOSIS BUT REDUCES EMBRYO DEVELOPMENT

2005 ◽  
Vol 17 (2) ◽  
pp. 211
Author(s):  
J.-K. Tseng ◽  
P.-C. Tang ◽  
J.-C. Ju
Keyword(s):  
Heat Shock ◽  
Apoptotic Cell ◽  
Electric Pulse ◽  
Annexin V ◽  
Developmental Competence ◽  
Control Group ◽  
Hsp 70 ◽  
Cell Numbers ◽  
Significant Difference

Oocytes are susceptible to heat shock (HS) during the maturation process. It has been demonstrated that HS induces apoptosis and/or the expression of HS protein 70 (hsp 70) in in vitro-produced oocytes and embryos. The objectives of this study were to analyze the effects of HS on the development and apoptosis of pig oocytes and embryos. Porcine ovaries were collected from a local slaughterhouse and the cumulus-oocyte complexes (COCs) were aspirated from follicles 3–6 mm in diameter and subjected to standard in vitro maturation procedures at 39°C for 42 h. The in vitro matured oocytes were then randomly allocated to different HS treatments at 41.5°C for 0 (control, C0h), 1 (HS1h), 2 (HS2h), or 4 h (HS4h). An additional control group of oocytes was cultured for 4 h without HS (C4h). Data were analyzed by chi-square test. In Experiment 1, anti-hsp 70 (SPA-810AP, Stressgen, San Diego, CA, USA) and Western blotting were used to examine the expression of hsp 70. Results indicated that no significant difference of hsp 70 expression in metaphase II porcine oocytes occurred between controls and HS groups (P > 0.05, 7 replicates). In Experiment 2, apoptosis of metaphase II oocytes after HS was identified by annexin V-FITC (Sigma, St. Louis, MO, USA) staining and TUNEL (Roche, Indianapolis, IN, USA). No significant apoptotic signal was detected in the HS groups compared to the controls. The intensity of annexin V staining was not affected by HS, but it increased with the time of culture (P < 0.05, n = 24–37). In Experiment 3, the apoptotic rate and developmental competence of the HS-oocytes were evaluated by TUNEL assay (n = 123–137, 4 replicates). Parthenogenetic activation (n = 123–137) was performed by an electric pulse (2.2 kV cm−1) combined with 6-dimethyaminopurine treatment (6-DMAP, 2.5 μM, 4 h, Sigma). The cleavage rates in HS2h (43 ± 29%) and HS4h (35 ± 28%) decreased (P < 0.05) compared to those in C0h (62 ± 12%) and C4h (66 ± 8%). In addition, the blastocyst formation rates and total cell numbers reduced (P < 0.05) after 2 h (11 ± 10%, 20 ± 16) and 4 h (11 ± 8%, 19 ± 8) of HS treatments compared to those in C0h (23 ± 14%, 32 ± 22) and C4h (21 ± 11%, 27 ± 17), all respectively. The numbers of blastocysts with TUNEL-positive signals were not significantly different between the HS and control groups, but the signals increased (P < 0.05) before the 8-cell stage in HS groups (22–24%) compared to the C0h and C4h controls (16 and 11%), respectively. These results indicate that reduction in developmental competence of in vitro-matured pig oocytes after heat shock is not closely correlated to the expression of hsp 70 in the oocytes and to the apoptotic cell numbers in the blastocyst. Whether detection of apoptosis by TUNEL or annexin V-FITC in oocytes is a good indicator requires further investigation.

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 A Higher Frequency Administration of the Nontoxic Cycloartane-Type Triterpene Argentatin A Improved Its Anti-Tumor Activity

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1780 ◽  
Author(s):  
Zaira Tavarez-Santamaría ◽  
Nadia J. Jacobo-Herrera ◽  
Leticia Rocha-Zavaleta ◽  
Alejandro Zentella-Dehesa ◽  
Beatriz del Carmen Couder-García ◽  
...  
Keyword(s):  
Waste Product ◽  
Industrial Process ◽  
Annexin V ◽  
Control Group ◽  
Healthy Control ◽  
Hct 116 ◽  
Hct 116 Cells ◽  
Induced Apoptosis

Parthenium argentatum (Gray), commonly known as guayule, has been used to obtain natural rubber since the beginning of the 20th century. Additionally, the so called “resin” is a waste product derived from the industrial process. The cycloartane-type triterpene Argentatin A (AA) is one of the main constituents of the industrial waste resin. In this study we evaluated the AA anticancer activity both in vitro and in vivo in the HCT116 colon cancer cells. The apoptosis promotion of AA was assessed by the annexin V/propidium iodide (PI) assay. The senescence was evaluated for SA-β-galactosidase, and PCNA was used as a marker of proliferation. Its antitumor activity was evaluated using a xenograft mouse model. The results indicated that AA-induced apoptosis in HCT-116 cells and was positively stained for SA-β-galactosidase. In the xenografted mice test, the administration of AA at the dose of 250 mg/kg three times a week for 21 days reduced tumor growth by 78.1%. A comparable tumor reduction was achieved with cisplatin at the dose of 2 mg/kg administered three times a week for 21 days. However, nude mice treated with AA did not lose weight, as they did remarkably when treated with cisplatin. Furthermore, the animals treated with AA showed similar blood profiles as the healthy control group. These data indicate the low toxicity of AA compared to that shown by cisplatin.

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

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.

167 INHIBITION OF HEAT SHOCK PROTEIN 90 REDUCES DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 197
Author(s):  
E. D. Souza ◽  
F. B. E. Paula ◽  
C. C. R. Quintao ◽  
J. H. M. Viana ◽  
L. T. Iguma ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
In Vitro Maturation ◽  
Hsp90 Inhibitor ◽  
Stress Conditions ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Cleavage Rate ◽  
Bovine Oocytes

The 90-kDa heat shock protein (HSP90) is a chaperone that is important for maintaing protein homeostasis under stress conditions. HSP90 seems also to be required for maturation of Xenopus oocytes (Fisher et al. 2000 EMBO J. 19, 1516) and first cleavage of mouse zygotes (Audouard et al. 2011 PloS One 6, e17109). This study aimed to evaluate the effect of inhibition of HSP90 by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma St. Louis, MO, USA) during in vitro maturation (IVM) on bovine oocyte developmental competence. Immature cumulus–oocyte complexes (COC) were randomly allocated in 3 treatments during IVM: T0 (control; n = 240), no HSP90 inhibitor; T1: 2 μM HSP90 inhibitor (17AAG; n = 250) for the first 12 h of IVM; and T2: 2 μM HSP90 inhibitor (n = 188) for 24 h of IVM. In vitro maturation was performed in Nunc plates containing 400 μL of TCM-199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2, 95% humidity, and 38.5°C for 24 h. Oocytes were in vitro fertilized for 20 h and incubated under the same IVM conditions. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) an IVF performed with 2 × 106 spermatozoa mL–1. Presumptive zygotes were completely denuded in a PBS solution with 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 post-fertilization and blastocyst rates were evaluated at Day 7 and Day 8. Data from 6 repetitions were analysed by generalized linear model procedure of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA), and means were compared by Student-Newman-Keuls test. Values are shown as mean ± s.e.m. There was a tendency (P = 0.08) for a lower cleavage rate in T2 (52.6 ± 5.8%) than in T0 (control; 74.2 ± 4.1%). Inhibition of HSP90 by 17AAG for 12 h and 24 h of IVM (T1 and T2, respectively) decreased blastocyst rates at Day 7 (20.4 ± 3.0% and 14.3 ± 2.6%, respectively; P < 0.01) and Day 8 (22.6 ± 4.1% and 16.9 ± 2.7%, respectively; P < 0.05) when compared with control (T0 = 31.8 ± 2.5% and 34.1 ± 2.9% for Day 7 and Day 8, respectively). In addition, the inhibition of HSP90 for 24 h decreased (P < 0.05) the proportion of hatched blastocysts at Day 8 (9.5 ± 5.0% for T2, respectively) when compared with control (T0 = 35.8 ± 3.9%), indicating a reduction on embryo quality. In conclusion, inhibition of HSP90 by 17AAG during IVM results in lower developmental competence, suggesting that this protein is also important for bovine oocytes. Further studies are required to investigate if the role of HSP90 on developmental competence of bovine oocyte is affected when under stress conditions. The authors acknowledge CNPq 473484/2011-0, FAPEMIG and FAPES for financial support.

133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 184 ◽  
Author(s):  
T. Somfai ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Antioxidant Defense System ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Sperm Penetration ◽  
Second Polar Body ◽  
Polar Body Extrusion

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P &lt; 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P &lt; 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P &lt; 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P &lt; 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P &lt; 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).

351 POLARIZED LIGHT MICROSCOPY: DETECTION OF MICROTUBULES AND ITS EFFECTS ON THE VIABILITY OF IN VITRO-MATURED PORCINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 332 ◽  
Author(s):  
I. Molina ◽  
M. Muñoz ◽  
C. Díez ◽  
E. Gómez ◽  
E. A. Martínez ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
Developmental Competence ◽  
Meiotic Spindle ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Oocyte Developmental Competence ◽  
Blastocyst Rate

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) is used as a tool in human and, recently, in farm animals assisted reproductive technologies. PLM could be useful for a non-invasive evaluation of the meiotic spindle. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured porcine oocytes and to examine the effects of PLM on the oocyte developmental competence. Cumulus-oocyte complexes from slaughterhouse ovaries were matured in vitro for 42 h as described by Gil et al. (2004 Theriogenology 62, 544-552). In the first experiment, a total of 97 oocytes from 6 replicates were placed individually in 10-μL drops of TCM-199-Hepes-FCS in a glass Petri dish. PLM was used to detect the presence of polymerized protein which could be forming a meiotic spindle. The presence of polymerized protein and a meiotic spindle was confirmed in individual oocytes by inmunostaining and chromatin detection as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191-201). In the second experiment, a total of 160 oocytes from 4 replicates were exposed or not (controls) to PLM for 10 minutes. Thereafter, the oocytes were parthenogenetically activated and cultured in vitro. Cleavage rate, total blastocyst rate, expanded blastocyst rate on Day 7 and total cell numbers in expanded blastocysts were assessed. Data were analyzed by GLM procedure of SAS. There was a positive correlation (r = 1; P < 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by inmunostaining. A positive PLM signal was detected in 98.9% of the oocytes. A barrel-shape spindle was observed in 94.8% of the individual samples by inmunostaining and all of these oocytes were positive to PLM. Moreover, oocytes exposed to PLM did not differ significantly from controls on cleavage rate (83.7 ± 1.5 v. 84.4 ± 1.5), total blastocyst rate (36.9 ± 3.6 v. 41.2 ± 3.6) and expanded blastocyst rate on Day 7 (21.9 ± 1.7 v. 26.2 ± 1.7), respectively. There were also no differences in total cell numbers counted in expanded blastocysts (32.8 ± 2.6 v. 35.6 ± 2.5). These results indicate that polarized light microscopy did not exert detrimental effects on porcine oocyte developmental competence and it seems an efficient system to detect polymerized protein in in vitro-matured porcine oocytes. Grant support: INIA: RZ2007-00013-00-00. I. Molina, M. Muñoz, B. Trigal and D. Martín are sponsored by INIA, RYC08-03454, Cajastur and PTA2007-0268-I, respectively.

116 TAUROURSODEOXYCHOLIC ACID PREVENTS ENDOPLASMIC RETICULUM STRESS-INDUCED APOPTOSIS IN PREIMPLANTATION PIG EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 158
Author(s):  
J.-S. Kim ◽  
K.-S. Lee ◽  
B.-S. Song ◽  
J.-Y. Zhang ◽  
Y.-K. Choo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Control Group ◽  
Tauroursodeoxycholic Acid ◽  
Preimplantation Stage ◽  
Induced Apoptosis

Apoptosis is an important determinant for the normal development of preimplantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. The efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting classic pathways of apoptosis. Therefore, we explored the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis in preimplantation porcine embryos. Also, TM (tunicamycin; an ER stress inducing chemical reagent) was used to investigate the effect of ER-stress on pig embryo development. After in vitro maturation and fertilization, presumptive porcine embryos were cultured in NCSU23 medium supplemented with 200 μ g mL–1 TUDCA or 1 μg mL–1 (TM) for 6 days at 39°C, 5% CO2 in air. All data were analyzed by using Duncan test of ANOVA by Statistical Analysis System (SAS). When treated with TM during culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage compared with 27.4% (28/102) of the embryos in the control group (P < 0.05). We also confirmed that TM stimulates up-regulation of ER stress response genes, such as XBP-1 mRNA, and induces a high rate of apoptosis. Whereas the frequency of blastocyst formation in the TUDCA-treated group was increased compared with that in the control group (32.8%, 49/149 v. 22.2%, 32/144), P < 0.05). Furthermore, the blastocyst cell number was enhanced (30.6 v. 39.5) and apoptosis reduced (TUNEL positive nuclei number, 6.0 v. 3.2) by TUDCA treatment in pig embryos. As the result of real-time quantitative RT-PCR analysis, the expression of anti-apoptotic Bcl-xl gene was increased in the blastocyst stage by TUDCA treatment, whereas expression of pro-apoptotic Bax was decreased. In addition, we also confirmed that TUDCA decreases the rate of TM-induced apoptosis in preimplantation stage pig embryos. Our results indicate that TUDCA improves the developmental competence of porcine embryos by modulating the ER stress-induced apoptosis during preimplantation stage.

scholarly journals LIMK1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro

2020 ◽  
Vol 33 (10) ◽  
pp. 1579-1589
Author(s):  
Jeongwoo Kwon ◽  
Min-Jung Seong ◽  
Xuanjing Piao ◽  
Yu-Jin Jo ◽  
Nam-Hyung Kim
Keyword(s):  
Expression Patterns ◽  
Early Embryo ◽  
Developmental Competence ◽  
Cell Junction ◽  
Control Group ◽  
Cortical Region ◽  
Cortical Actin ◽  
Cleavage Rate ◽  
Morula Stage

Objective: This study was conducted to investigate the roles of LIM kinases (LIMK1 and LIMK2) during porcine early embryo development. We checked the mRNA expression patterns and localization of LIMK1/2 to evaluate their characterization. We further explored the function of LIMK1/2 in developmental competence and their relationship between actin assembly and cell junction integrity, specifically during the first cleavage and compaction.Methods: Pig ovaries were transferred from a local slaughterhouse within 1 h and cumulus oocyte complexes (COCs) were collected. COCs were matured in in vitro maturation medium in a CO2 incubator. Metaphase II oocytes were activated using an Electro Cell Manipulator 2001 and microinjected to insert LIMK1/2 dsRNA into the cytoplasm. To confirm the roles of LIMK1/2 during compaction and subsequent blastocyst formation, we employed a LIMK inhibitor (LIMKi3).Results: LIMK1/2 was localized in cytoplasm in embryos and co-localized with actin in cell-to-cell boundaries after the morula stage. LIMK1/2 knockdown using LIMK1/2 dsRNA significantly decreased the cleavage rate, compared to the control group. Protein levels of E-cadherin and β-catenin, present in adherens junctions, were reduced at the cell-to-cell boundaries in the LIMK1/2 knockdown embryos. Embryos treated with LIMKi3 at the morula stage failed to undergo compaction and could not develop into blastocysts. Actin intensity at the cortical region was considerably reduced in LIMKi3-treated embryos. LIMKi3-induced decrease in cortical actin levels was attributed to the disruption of adherens junction and tight junction assembly. Phosphorylation of cofilin was also reduced in LIMKi3-treated embryos.Conclusion: The above results suggest that LIMK1/2 is crucial for cleavage and compaction through regulation of actin organization and cell junction assembly.

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