Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 335-345 ◽  
Author(s):  
Masaki Iwamoto ◽  
Akira Onishi ◽  
Dai-ichiro Fuchimoto ◽  
Tamas Somfai ◽  
Shun-ichi Suzuki ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Blastocyst Stage ◽  
The Other ◽  
Control Group ◽  
Blastocyst Formation ◽  
Cell Nuclei ◽  
Somatic Nucleus ◽  
Membrane Breakdown ◽  
Caffeine Treatment ◽  
M Phase

The possibility of using aged porcine oocytes treated with caffeine, which inhibits the decrease in M-phase promoting factor activity, for pig cloning was evaluated. Cumulus–oocyte complexes (COCs) were cultured initially for 36h and subsequently with or without 5mM caffeine for 24h (in total for 60h: 60CA+ or 60CA– group, respectively). As a control group, COCs were cultured for 48h without caffeine (48CA–). The pronuclear formation rates at 10h after electrical stimulation in the 60CA+ and 60CA– groups decreased significantly (p<0.05) compared with the 48CA– group. However, the fragmentation rate was significantly higher (p<0.05) in the 60CA– group than in the 60CA+ and 48CA– groups. When the stimulated oocytes were cultured for 6 days, the 60CA+ group showed significantly lower blastocyst formation and higher fragmentation or degeneration rates (p<0.05) than the 48CA– group. However, the number of total cells in blastocysts was not affected by maturation period or caffeine treatment. When somatic cell nuclei were injected into the non-enucleated oocytes and exposed to cytoplasm for a certain duration (1–11h) before the completion of maturation (48 or 60h), the rate of nuclear membrane breakdown after exposure to cytoplasm for 1–2h in the 60CA– oocytes was significantly lower (p;<0.05) than in the other experimental groups. The rate of scattered chromosome formation in the same 60CA– group tended to be lower (p=0.08) than in the other groups. After the enucleation and transfer of nuclei, blastocyst formation rates in the 60CA+ and 60CA– groups were significantly lower (p<0.05) than in the 48CA– group. Blastocyst quality did not differ among all the groups. These results suggest that chromosome decondensation of the transplanted somatic nucleus is affected by both the duration of exposure to cytoplasm and the age of the recipient porcine oocytes, and that caffeine treatment promotes nuclear remodelling but does not prevent the decrease in the developmental ability of cloned embryos caused by oocyte aging.

43 PATTERN OF NUCLEOLI FORMATION IN RACCOON-PORCINE INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 169
Author(s):  
Y. H. Nam ◽  
Y. Jeon ◽  
S. A. Cheong ◽  
S. S. Kwak ◽  
S. H. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mammalian Species ◽  
Housekeeping Genes ◽  
Blastocyst Stage ◽  
Control Group ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Embryonic Genome

Recently, great focus has been on the rescue of endangered animals through somatic cell nuclear transfer (SCNT). Because it is difficult to obtain the oocytes of endangered species, interspecies SCNT (iSCNT) methods have been attempted. Numerous iSCNT embryos have shown unsuccessful development due to aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). In particular, aberrant EGA may cause the arrest of nucleoli formation and developmental block in embryos. According to this concept, we performed raccoon iSCNT using porcine oocytes and analyzed iSCNT embryo development pattern and formation of nucleoli. Enucleated porcine oocytes were fused with raccoon fibroblasts by electrofusion. Cleavage and blastocyst formation were evaluated under a stereomicroscope at 48 and 168 h post-activation (hpa), respectively. To confirm the formation of nucleoli, which can be detected by C23 antibody labeling in many mammalian species, C23 immunocytochemistry was performed at 48 and 72 hpa. A total of 158 iSCNT embryos were cultured; 68.5% of the raccoon iSCNT embryos were cleaved at 48 hpa (1-cell stage: 9.7%; 2-cell stage: 14.4%; 4-cell stage: 34.1%; 6-cell stage: 12.7%; 8-cell stage: 7.3%; fragmented: 21.8%). But, the embryos seen as 5- to 8-cell stage did not have the same number of nuclei as their blastomere number. When raccoon iSCNT embryos were stained by Hoechst 33342, 5- to 8-blastomere raccoon iSCNT embryos had only 4 nuclei. The raccoon iSCNT embryos did not develop past the 4-cell stage and failed to form blastocysts. In the control group, 65.2% of pig SCNT embryos were cleaved at 48 hpa (1-cell stage: 8.0%; 2-cell stage: 4.2%; 4-cell stage: 23.6%; 6-cell stage: 13.6%; 8-cell stage: 23.8%; fragmented: 26.8%), and 10.0% of pig SCNT embryos developed to blastocysts. In raccoon iSCNT embryos, raccoon nuclei failed to form nucleoli at 48 and 72 hpa. By contrast, pig SCNT embryos showed 18.8 and 87.9% nucleoli formation at 48 and 72 hpa. Our results demonstrate that 4-cell-stage embryos of raccoon-porcine hybrid embryos may be produced by SCNT methods. The pig oocytes partly supported the remodeling and reprogramming of the raccoon somatic cell nuclei, but they were unable to support nucleoli formation. Moreover, aberrant nucleoli formation caused the unsuccessful development of raccoon SCNT embryos to the blastocyst stage. This work was supported by a grant from the Next Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

29 TREATMENT OF DONOR CELLS WITH XENOPUS OOCYTE EXTRACT ENHANCED SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 126
Author(s):  
X. Yang ◽  
J. Mao ◽  
E. M. Walters ◽  
M. T. Zhao ◽  
K. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Natural Science ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Control Group ◽  
Blastocyst Formation ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Frog Oocyte

Somatic cell nuclear transfer (SCNT) efficiency in pigs and other species is still very low. This low efficiency and the occurrence of developmental abnormalities in offspring has been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei as well as gene expression to more resemble that of pluripotent cells. Rathbone et al. (2010) has showed that pretreating somatic donor cells with frog oocyte extract (FOE) increased live birth in ovine. Liu et al. (2011) also reported that treating donor cells with FOE enhanced handmade clone embryo development in pigs. The aim of this study was to evaluate the early development of cloned embryos produced with porcine GFP fibroblasts pre-treated with a permeabilizing agent, digitonin and matured frog oocyte extract. Frog egg cytoplasmic extract was prepared from one frog's oocytes after being matured in vitro to MII stage. The experiment included 2 groups. In the FOE-treated group, GFP-tagged fetal fibroblasts were permeabilized by digitonin (15 ng mL–1) and incubated in FOE containing an ATP-regenerating system (2.5 mM ATP, 125 μM GTP, 62.5 μg mL–1 of creatine kinase, 25 mM phosphocreatine and 1 mM NTP) at room temperature (24°C) for 2 h; cell membranes were re-sealed by culturing in 10% FBS in DMEM media for 2.5 h at 38.5°C before used as donor cells. In the control group, the same donor cells were treated with digitonin, but without frog oocyte extract incubation. The SCNT embryos were produced by using the 2 groups of donor cells as described above. In total, 305 control and 492 FOE oocytes were enucleated from 8 biological replicates. Two hundred fifty control and 370 FOE couplets were fused and cultured in porcine zygote medium 3. Percent cleavage was recorded on Day 2 and the percent blastocyst formation was determined on Day 7 (SCNT day = 0). In addition, the number of nuclei in the blastocysts was recorded on Day 7. Percent fusion, cleavage, blastocyst formation and number of nuclei in blastocysts were analysed by using SAS software (v9.2), with day and treatment class as main effects. There was no difference in percent fusion (FOE, 76.2 ± 2.5% vs control, 80.8 ± 2.8%) or in cleavage (FOE: 74.8 ± 2.5% vs control: 74.6 ± 2.9%). Only green blastocysts with 16 or more nuclei were considered to be a true SCNT blastocyst. The percent blastocyst was higher in the FOE group than that in the control (13.9 ± 0.8% vs 9.5 ± 0.9%, P < 0.05), whereas the number of nuclei in the blastocysts was not different between the 2 groups (39.7 ± 2.4, 35.9 ± 3.8 for FOE and control, respectively). In conclusion, our study demonstrated that pre-treatment of donor cells with digitonin and Xenopus MII oocyte extract increased porcine SCNT embryo development to blastocyst and cloning efficiency. Funded by the National Natural Science Foundation of China (NO. 31071311), Natural Science Foundation of Fujian Province of China (No. 2009J06017) and NIH U42 RR18877.

15 Combination of RepSox with histone deacetylation inhibitors on invitro development competence of porcine somatic cell nuclear transfer embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 133
Author(s):  
Z.-B. Luo ◽  
M.-F. Xuan ◽  
Z.-Y. Li ◽  
X.-J. Yin ◽  
J.-D. Kang
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone Deacetylase Inhibitors ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Histone Deacetylation ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). In this study, we compared histone deacetylase inhibitors combined with the pluripotency inducer RepSox on invitro development of porcine embryos produced via SCNT. Porcine embryos were treated with valproic acid (VPA), mocetinostat, M344 and panobinostat (LBH589) after SCNT, respectively. The porcine embryo invitro-development competence, histone modification level, and pluripotency-related genes expression were analysed. The results showed that LBH589 significantly increased the blastocyst formation rate compared with mocetinostat, M344, and control. In addition, VPA treatment increased the blastocyst formation rate of SCNT porcine embryos; both VPA-treated and the untreated clones developed to term, but offspring from VPA-treated embryos had a lower survival to adulthood than those from control embryos (18.2 vs. 67.0%; P&lt;0.05). Furthermore, cotreatment with 12.5mM RepSox and 50 nM LBH589 (RepSox+LBH589) for 24h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9 vs. 8.5%, respectively; P&lt;0.05). Moreover, RepSox + LBH589 improved epigenetic reprogramming by histone acetylation and methylation. The expression of pluripotency-related genes NANOG and SOX2 was found to be significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming, and improves the invitro development of porcine SCNT embryos.

27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
I. Hiriart ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Blastocyst Stage ◽  
Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

scholarly journals Diploid porcine parthenotes produced by inhibition of first polar body extrusion during in vitro maturation of follicular oocytes

Reproduction ◽  
2006 ◽  
Vol 132 (4) ◽  
pp. 559-570 ◽  
Author(s):  
Tamás Somfai ◽  
Manabu Ozawa ◽  
Junko Noguchi ◽  
Hiroyuki Kaneko ◽  
Katsuhiko Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Formation Rate ◽  
Metaphase Plate ◽  
Chromosome Analysis ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Formation ◽  
First Polar Body ◽  
Polar Body Extrusion

We investigated nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to cytochalasin B (CB) during in vitro maturation (IVM). Nuclear progression was similar in control oocytes and oocytes matured in the presence of 1 μg/ml CB (IVM-CB group) by 37 h IVM; at this time the proportion of oocytes that had reached or passed through the anaphase-I stage did not differ significantly between the IVM-CB and the control groups (61.3 and 69.9% respectively; P < 0.05). After IVM for 37 h, no polar body extrusion was observed in the IVM-CB group. In these oocytes, the two lumps of homologous chromosomes remained in the ooplasm after their segregation and turned into two irregular sets of condensed chromosomes. By 41 h IVM, the double sets of chromosomes had reunited in 89.5% IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached the metaphase-II stage by this time. When IVM-CB oocytes cultured for 46 h were stimulated with an electrical pulse and subsequently cultured for 8 h without CB, 39.0% of them extruded a polar body and 82.9% of them had a female pronucleus. Chromosome analysis revealed that the majority of oocytes that extruded a polar body were diploid in both the control and the IVM-CB groups. However, the incidence of polyploidy in the IVM-CB group was higher than that in the control group (P < 0.05). In vitro development of diploid parthenotes in the control and the IVM-CB groups was similar in terms of blastocyst formation rates (45.8 and 42.8% respectively), number of blastomeres (39.9 and 44.4 respectively), the percentage of dead cells (4.3 and 2.9% respectively), and the frequency of apoptotic cells (7.3 and 6.3% respectively). Tetraploid embryos had a lower blastocyst formation rate (25.5%) and number of cells (26.2); however, the proportion of apoptotic nuclei (7.0%) was similar to that in diploid parthenotes. These results suggest that the proportion of homozygous and heterozygous genes does not affect in vitro embryo development to the blastocyst stage.

Reprogramming cattle somatic cells by isolated nuclear injection

1998 ◽  
Vol 10 (8) ◽  
pp. 645 ◽  
Author(s):  
Alan Trounson ◽  
Orly Lacham-Kaplan ◽  
Maria Diamente ◽  
Tiki Gougoulidis
Keyword(s):  
Somatic Cell ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cell Nuclei ◽  
Surface Appearance ◽  
Intracytoplasmic Injection ◽  
Fetal Fibroblasts ◽  
Passage Number ◽  
Nuclear Injection ◽  
Apparent Influence

The development of a somatic cell nuclear transfer procedure for the production of blastocyst stage cattle embryos is described. Bovine fetal fibroblasts were used for fusion experiments with surgically enucleated oocytes (cytoplasts) following the establishment of optimal parameters for electrofusion from isofusion contours. Fusion rates were increased by decreasing size of the cytoplasts used but cleavage was decreased by decreasing size of the cytoplast used (quarter, half and whole cytoplasts). The use of double cytoplasts did not improve cleavage, and development to blastocysts could not be achieved. In a comparison of electrofusion of fibroblasts with cytoplasts in the subzonal perivitelline space with intracytoplasmic injection of nuclei and parthenogenetically activated oocytes, 2%, 14% and 24% developed to blastocysts respectively. In the group injected with isolated nuclei, the passage number (4 to 9) had no apparent influence on developmental competence to blastocysts. The embryos produced by nuclear injection of somatic cell nuclei showed the normal pattern of cell surface appearance of TEC-3 and TEC-4 stage-specific epitopes during development, as seen in fertilized oocytes. We conclude that the nuclear injection of somatic cell nuclei is a relatively efficient way to clone bovine embryos.

89 ZYGOTICALLY ACTIVATED GENES ARE SUPPRESSED IN MOUSE NUCLEAR-TRANSFERRED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 162
Author(s):  
T. Suzuki ◽  
N. Minami ◽  
H. Imai
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Early Stage ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Gene Activation ◽  
Blastocyst Stage ◽  
Success Rates ◽  
Cell Nuclei ◽  
Cell Stage

Mammalian oocytes have the ability to confer totipotency to terminally differentiated somatic cell nuclei. Viable cloned animals have been produced by somatic cell nuclear transfer (NT) into oocytes in many mammalian species including mouse. However, the success rates of the production were quite low in all species. Many studies have measured differences in gene expression between NT and fertilized embryos in relatively advanced stages of development such as pre- and post-natal stages or the blastocyst stage. In the mouse, major zygotic gene activation (ZGA) occurs at the 2-cell stage after fertilization and leads to the transition of gene regulation from maternal control to embryonic control. Suppression of the ZGA by a transcription inhibitor was shown to decrease the viability of embryos, and causes developmental arrest at the 2-cell stage. An abnormal ZGA may therefore affect the viability of NT embryos and cause further abnormalities in later embryonic development. In the present study, we compared gene expression patterns using differential display RT-PCR (DDRT-PCR) between the NT and IVF embryos at the 2-cell stage to detect some abnormalities affecting later development of NT embryos. The developmental rate of NT embryos to blastocysts (32.9%) was significantly lower than that of IVF (92.7%) or PA (92.8%). In addition, the cell numbers of NT embryos at the blastocyst stage (39.5 � 2.6; n = 19) were less than those of IVF (66.8 � 2.1; n = 30) or PA embryos (48.2 � 2.1; n = 30). Using these embryos, we first identified 4 genes that were differentially expressed between NT and IVF embryos at the 2-cell stage. Among the identified genes, Inpp5b and Chst12 were up-regulated, and MuERV-L and Dnaja2 were down-regulated in the NT embryos compared with IVF embryos. Further analysis showed that the expression of zygotically activated genes such as Interferon-γ, Dub-1, Spz1, DD2106, and DD2111 were not properly activated in NT embryos, suggesting that the cellular process involved in the control of the zygotic genome activation is not appropriately regulated. These results indicate that abnormal gene expression has already occurred at the early stage of pre-implantation development as a failure of nuclear reprogramming.

Effect of melatonin treatment on developmental potential of somatic cell nuclear-transferred mouse oocytes in vitro

Zygote ◽  
2013 ◽  
Vol 22 (2) ◽  
pp. 213-217 ◽  
Author(s):  
Mohammad Salehi ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Blastocyst Stage ◽  
Control Group ◽  
Developmental Potential ◽  
Significant Difference ◽  
Mouse Somatic Cell

SummaryThe beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10−6 to 10−12 M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10−12 M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.

scholarly journals 229 EFFECT OF CULTURE CONDITIONS ON AQUAPORIN mRNA ABUNDANCE IN MOUSE BLASTOCYSTS

2005 ◽  
Vol 17 (2) ◽  
pp. 265
Author(s):  
H. Offenberg ◽  
P.D. Thomsen
Keyword(s):  
Blastocyst Stage ◽  
Culture Conditions ◽  
Mrna Abundance ◽  
Control Group ◽  
Mrna Levels ◽  
Blastocyst Formation ◽  
Gapdh Mrna ◽  
The Media

It is known that culture conditions can alter gene expression of the pre-implantation embryo. We have previously shown that aquaporins (AQPs) are expressed in the mouse embryo and that they are involved in the passage of water across the trophoblast cells during blastocyst formation. This study was conducted to investigate whether AQP mRNA abundance is altered by culturing embryos in vitro compared to in vivo developed embryos. Furthermore we wanted to investigate if AQP mRNA abundance was influenced by the osmolality of the media. It is possible to compare the effect of hyperosmolality that the embryo may be able to compensate for by adding glycerol which can cross some AQPs, compared to the addition of sucrose which can not cross the membranes. Mouse embryos were obtained by superovulating B6D2F1 mice followed by culture of the flushed presumptive zygotes in KSOM to the blastocyst stage (in vitro) or by flushing blastocysts from the uterus (in vivo). For the study of the influence of osmolality on AQP mRNA abundance, zygotes were flushed and cultured to the compacted 8-cell stage and then placed in media of increasing osmolality, using either glycerol or sucrose. The osmolalities of the media were 243 (control), 300, 350, and 400 mOsm. Embryos were cultured to the blastocyst stage and frozen in liquid nitrogen. Embryonic RNA was extracted using a Dynabeads mRNA Capture kit (Dynal, Oslo, Norway). Real time PCR was performed on embryonic cDNA on a Lightcycler (Roche Diagnostics, 2650 Hvidovre, Denmark) using aquaporin-specific primers and primers for β-actin and GAPDH. The results of the quantitative RT-PCR analysis showed that in vitro-cultured embryos had a lower mRNA abundance for AQP 8, 9, and 11 compared to the in vivo-developed embryos but that the AQP 3 mRNA abundance was unaltered. Analysis of the housekeeping genes showed that GAPDH mRNA levels were unchanged in vitro, whereas β-actin was up-regulated in vitro. The osmotically challenged embryos showed the following blastocyst rates compared to the controls: glycerol 300: 100%; glycerol 350: 100%; glycerol 400: 100%; sucrose 300: 100%; sucrose 350: 78%; and sucrose 400: 0%. Thus, glycerol up to 400 mOsm had no effect on blastocyst rates, whereas addition of sucrose reduced blastocyst formation, with a total inhibition at 400 mOsm. Analysis of the mRNA abundance showed a reduction of AQP 8 in the glycerol solutions. The level was reduced to 30% of the control group at 300 mOsm, to 27% at 350 mOsm and to 8% at 400 mOsm. There was no corresponding reduction of AQP 8 mRNA abundance in sucrose solutions. Further, AQP 3, 7, 9, and 11 mRNA levels as well as β-actin and GAPDH mRNA levels were unaltered in the osmotically challenged embryos. In conclusion, this study shows that embryonic culture affects the abundance of several AQPs and that compensation of a glycerol-induced osmotical challenge induces down-regulation of AQP 8 expression. Embryos tolerate high glycerol concentrations better than high sucrose concentrations but the possible role of AQP 8 in this process is unclear at present.

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