scholarly journals In vitro maturation in the presence of Leukemia Inhibitory Factor modulates gene and miRNA expression in bovine oocytes and embryos

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Meritxell Vendrell-Flotats ◽  
Tania García-Martínez ◽  
Iris Martínez-Rodero ◽  
Manel López-Béjar ◽  
Jonathan LaMarre ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Leukemia Inhibitory Factor ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Inhibitory Factor ◽  
Hatching Rate ◽  
Cleavage Rate ◽  
Oocyte Competence ◽  
Bovine Oocytes

Abstract Members of the interleukin-6 (IL-6) family of cytokines are important for reproductive function that are mediated through changes in gene and miRNA expression. Herein, we characterized the expression of miR-21, miR-155, miR-34c and miR-146a in bovine oocytes and cumulus cells during in vitro maturation (IVM) with leukemia inhibitory factor (LIF), IL-6 and IL-11 or unsupplemented controls. LIF-exposed COCs showed higher expression of miR-21 and miR-155 in oocytes, whereas miR-146a expression was increased in oocytes matured with IL-6 and IL-11. In cumulus cells, miR-155 expression was elevated by all treatments while only LIF increased miR-21 expression. Based on these results, we next examined how LIF exposure during IVM affected oocyte competence, through IVF and the expression of specific genes in GV- and MII-oocytes, in 2- and 8-cell embryos, and in Day 8-blastocysts. LIF supplementation did not affect cleavage rate, blastocyst yield or several other developmental parameters, but did increase hatching rate. LIF suppressed DPPA3, ZAR1 and NPM2 expression in 2 cell- and/or 8-cell embryos. LIF increased the expression of KAT2A and HSPA1A in MII-oocytes, and that of HDAC1, KAT2A and HSP90AA1 and the BAX:BCL2L1 ratio in 2-cell embryos. In contrast, HDAC1, KAT2A and HSP90AA1 expression and BAX:BCL2L1 ratio was lower in 8-cell embryos derived from LIF oocytes. IVM with LIF also increased the expression of DNMT3A, HSPA1A and HSP90AA1 in blastocysts. In conclusion, supplementation with LIF during IVM was consistently associated with changes in the relative abundance of transcripts in mature bovine oocytes and in specific embryo developmental stages.

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

6 TIMING OF DEADENYLATION OF GDF-9 AND CYCLIN 3′ UTR CONSTRUCTS IN BOVINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 121
Author(s):  
D. J. Walker ◽  
C. J. Wilusz ◽  
G. E. Seidel Jr
Keyword(s):  
In Vitro Maturation ◽  
Control Point ◽  
Tail Length ◽  
Cumulus Cells ◽  
Cyclin B1 ◽  
Defined Medium ◽  
Untranslated Regions ◽  
Labeled Rna ◽  
Bovine Oocytes

The maternal pool of mRNA undergoes major changes during oocyte maturation and early embryonic development. Specific genes are activated or degraded in response to changes in poly-(A) tail length. However, little is known about how the oocyte targets specific transcripts for degradation or translation in a timely manner. The objective of this study was to determine how poly-(A) tail length of different transcripts is affected in bovine oocytes by time of in vitro maturation. Cyclin B1 and GDF-9 32 untranslated regions (UTRs) were cloned into modified p-GEM plasmids containing a poly-(A) tract of 60 or 0 adenosines (A60 or A0, respectively). Each 32 UTR was transcribed in vitro with (A60) or without (A0) a poly-(A) tail to generate UTP32-labeled RNA. Transcriptions producing at least 200 000 counts per min (cpm) per �L were used for subsequent injections into denuded bovine oocytes. Cumulus-oocyte complexes (COCs) recovered from slaughterhouse-derived ovaries (n = 216) were vortexed to remove cumulus cells immediately after aspiration, after 3 h of in vitro maturation, or after 19 h of maturation in a chemically defined medium supplemented with FSH, LH, EGF, and cysteamine. After vortexing, denuded oocytes were injected and snap frozen, or matured in vitro for 1 or 3 h. Eight oocytes were injected with ~0.5 nL (~100 cpm/oocyte) labeled RNA at each time point in 3 replicates. Total RNA was isolated from injected oocyte pools and loaded onto a 5% denaturing acrylamide gel for size separation. Radiolabeled A0 was used as a control point of reference for deadenylation. Gels were dried, and RNA was visualized on a phosphoimager after 24 h exposure to a phosphor screen. Changes in polyadenylation status (transcript size) were evaluated by comparing shifts in bands from gene-specific A60

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.

174 Effects of Melatonin on In Vitro Maturation of Bovine Oocytes and Gene Expression in Cumulus Cells: Preliminary Results

2018 ◽  
Vol 30 (1) ◽  
pp. 226
Author(s):  
F. C. Castro ◽  
L. Schefer ◽  
K. L. Schwarz ◽  
H. Fernandes ◽  
R. C. Botigelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Bovine Oocytes

Melatonin mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro. The aim of this study was to assess the effect of melatonin during the in vitro maturation (IVM) on nuclear maturation of bovine oocytes and gene expression in their corresponding cumulus cells (CC). Bovine cumulus–oocyte complexes (COC) were obtained by aspiration of follicles (2-6 mm) from slaughterhouse ovaries, selected (grades I and II) and transferred to 4 well plates (25-30 COC/well) containing IVM medium [TCM-199 supplemented with sodium bicarbonate (26 mM), sodium pyruvate (0.25 mM), FSH (0.5 µg mL−1), LH (5.0 µg mL−1), 0.3% BSA, and gentamicin (50 µg mL−1)] with 0, 10−5, 10−7, 10−9 or 10−11 M melatonin and cultured for 24 h at 38.5°C and 5% CO2. At the end of IVM, oocytes were stained with Hoechst 33342 (10 μg mL−1) and evaluated for nuclear maturation rate. The CC were evaluated for the expression of antioxidant (SOD1, SOD2, GPX4), pro-apoptotic (P53, BAX) and expansion-related genes (PTX3, HAS1, HAS2). For transcript detection in CC, RNA isolation was performed with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Relative quantification of transcripts was performed by RT-qPCR using 3 endogenous controls (β-actin, GAPDH, PPIA). Nuclear maturation rate and gene expression were tested by ANOVA and means were compared by Tukey’s test (6 replicates). In CC, the different concentrations of melatonin did not significantly alter expression of the investigated genes (P > 0.05), although all concentrations provided a numerical increase in the expression of the antioxidant SOD1 and of the expansion-related genes PTX3 and HAS2. Regarding the pro-apoptotic genes, concentrations of 10−11 and 10−9 M were able to reduce only numerically the expression of BAX and P53, respectively. In oocytes, the rate of nuclear maturation was not different among the tested treatments (P > 0.05), but it was numerically higher in the 10−7 M melatonin treated group compared with the control (69.71 ± 13.76% v. 88.1 ± 12.54%). In conclusion, under the studied conditions, melatonin was unable to improve maturation rate or to affect the expression of antioxidant, pro-apoptotic, and expansion-related genes in CC. Melatonin during IVM has shown variable results in different studies and appears to show different effects depending on culture conditions and parameters studied. In order to take advantage of the possible positive antioxidant effects of melatonin, other culture conditions and parameters should be investigated. In a next step, melatonin will be included during in vitro culture of embryos to evaluate its possible cytoprotective role, because such embryos are more exposed to oxidative stress during in vitro culture, and to investigate its benefits on developmental competence in vitro. This reaesrch was funded by FAPESP (2015/20379-0; 2014/17181-0).

Astaxanthin counteracts the effects of heat shock on the maturation of bovine oocytes

2018 ◽  
Vol 30 (9) ◽  
pp. 1169 ◽  
Author(s):  
J. Ispada ◽  
T. A. Rodrigues ◽  
P. H. B. Risolia ◽  
R. S. Lima ◽  
D. R. Gonçalves ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Negative Effects ◽  
Cellular Mechanisms ◽  
Sod Activity ◽  
Oocyte Competence ◽  
Maturation Medium ◽  
Bovine Oocytes

The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14 h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25 nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

148 Effect of concentration of methionine in maturation and culture medium on cleavage rate of oocytes from alpaca (Lama pacos)

2019 ◽  
Vol 31 (1) ◽  
pp. 199
Author(s):  
M. L. Uchuari ◽  
M. Artica ◽  
J. C. Villanueva ◽  
W. F. Huanca ◽  
W. Huanca
Keyword(s):  
In Vitro Maturation ◽  
Culture Medium ◽  
Egg Yolk ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Medium ◽  
Thermos Flask ◽  
Lama Pacos

Maturation time of oocytes from alpacas is around 38 to 40h (Huanca et al. 2009) that would induce an increase in reactive oxygen species during in vitro maturation and IVF and cause cytotoxic damage to gametes. The objective of this study was to determine the optimal concentration of methionine during in vitro maturation on cleavage rate of alpacas oocytes following IVF. Cumulus-oocyte complexes were collected from slaughterhouse ovaries and transported in a thermos flask containing a saline solution 0.9% and antibiotic, antimycotic at 35°C. Cumulus-oocyte complexes were aspirated from follicles &gt;2mm and evaluated with a stereomicroscope for selection. Only cumulus-oocyte complexes with a homogeneous cytoplasm and with 2 or more layers of cumulus cells were selected to be cultured in maturation medium TCM-199 supplemented with 10% FCS (v:v) plus 0.5μg mL−1 FSH, 10μg mL−1 hCG, 0.2mM sodium pyruvate, 50μg mL−1gentamycin and 1μg mL−1 oestradiol under mineral oil by 38h. Testes of mature males were collected from a slaughterhouse and transported to the laboratory. Caudal epididymide was isolated, and fluid, rich in spermatozoa, was aspirated in syringes containing 2mL of Tris-fructose-egg yolk extender. Motile spermatozoa were obtained by centrifugation at 700×g in a Percoll discontinuous gradient (22.5: 45.0%) for 10min. The supernatant was removed by aspiration, and the pellet was resuspended in TL stock and centrifuged again at 700×g for 5min. Spermatozoa and oocytes were co-incubated by 18h at 39°C with 5% CO2. Presumptive zygotes were culture in KSOMaa medium and evaluated at 72h. The treatments include 0, 14 and 21 μM of methionine in maturation and culture medium. Data were analysed by ANOVA, and results are presented in Table 1. The results suggest that addition of methionine in maturation and culture medium improve the cleavage rate in oocytes from alpacas. Table 1.Cleavage rate (%) following in vitro maturation at different concentrations of methionine Proyect 405-PNICP-PIAP-2014, INNOVATE-PERU, is acknowledged.

scholarly journals Development of a reliable in vitro maturation system for zebrafish oocytes

Reproduction ◽  
2008 ◽  
Vol 135 (3) ◽  
pp. 285-292 ◽  
Author(s):  
Shinsuke Seki ◽  
Toshimitsu Kouya ◽  
Ryoma Tsuchiya ◽  
Delgado M Valdez ◽  
Bo Jin ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Saline Solution ◽  
Stage Iii ◽  
Hatching Rate ◽  
Cleavage Rate ◽  
Immature Oocytes ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

In zebrafish oocytes, it has been reported that a 60 or 75% Leibovitz L-15 medium or simple balanced saline solution containing 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) is effective for nuclear maturation. However, most of the oocytes that matured under these conditions were not fertilized and did not hatch. Thus, thesein vitromaturation methods could not support the cytoplasmic maturation of zebrafish oocytes. Therefore, we tried to develop a reliablein vitromaturation method for zebrafish oocytes, which supports their ability to be fertilized and to develop till hatching. When zebrafish oocytes at stage III were cultured in 50–100% Leibovitz L-15 medium supplemented with DHP, the highest rates of cleavage (24%) and hatching (12%) were obtained from oocytes matured in 90% Leibovitz L-15 medium. When we examined the suitable pH (7.5–9.5) of the 90% medium, higher rates of cleavage (45%) and hatching (33%) were obtained in oocytes matured at pH 9.0 than at pH 7.5, 8.5, or 9.5 (cleavage rate, 16–29%; hatching rate, 8–21%). In oocytes matured in 90% Leibovitz L-15 medium at pH 9.0, high rates of cleavage (70%) and hatching (63%) were obtained when oocytes were cultured for 270 min with 0.5 mg/ml BSA. Thus, 90% Leibovitz L-15 medium at pH 9.0 containing 0.5 mg/ml BSA was effective for normal maturation of zebrafish oocytes. This method will become a powerful tool for understanding the mechanism ofin vitromaturation in zebrafish oocytes and for the practical use of immature oocytes.

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