Tricostatin A-treated round spermatid enhances preimplantation embryo developmental competency following round spermatid injection in mice

Significant Difference ◽

Round Spermatid Injection

Summary It has been documented that the inefficacy of round spermatid injection (ROSI) might be caused by abnormal epigenetic modifications. Therefore, this study aimed to evaluate the effect of trichostatin A (TSA) as an epigenetic modifier of preimplantation embryo development in activated ROSI oocytes. Matured oocytes were collected from superovulated female mice. Testes were placed in human tubal fluid medium and masses were then cut into small pieces to disperse spermatogenic cells. Round spermatids were treated with TSA and subsequently injected into oocytes. The expression level of the development-related genes including Oct4, Sox2, Nanog, Dnmt and Hdac transcripts were evaluated using qRT-PCR. Immunohistochemistry was performed to confirm the presence of Oct-4 protein at the blastocyst stage. There was no statistically significant difference in fertilization rate following ROSI/+TSA compared with the non-treated ROSI and intracytoplasmic sperm injection (ICSI) groups. Importantly, TSA treatment increased blastocyst formation from 38% in non-treated ROSI to 68%. The relative expression level of developmentally related genes increased and Dnmt transcripts decreased in ROSI/+TSA-derived embryos, similar to the expression levels observed in the ICSI-derived embryos. In conclusion, our results indicate that spermatid treatment with TSA prior to ROSI would increase the success rate of development to the blastocyst stage and proportion of pluripotent cells.

40 TRICHOSTATIN A TREATMENT EFFECTS ON IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER CAT EMBRYOS DEPEND ON RECIPIENT CYTOPLASM SPECIES

10.1071/rdv27n1ab40 ◽

2015 ◽

Vol 27 (1) ◽

pp. 113

Author(s):

L. T. K. Do ◽

Y. Sato ◽

M. Taniguchi ◽

T. Otoi

Keyword(s):

Nuclear Transfer ◽

Treatment Effects ◽

Trichostatin A ◽

Polar Body ◽

Blastocyst Stage ◽

Control Group ◽

Fluid Medium ◽

First Polar Body ◽

Bovine Oocytes

The developmental ability of interspecies somatic cell nuclear transfer (iSCNT) embryos decreases as the taxonomic distance between the donor and recipient species increases. Treatment of cat iSCNT embryos using bovine oocytes with 50 nM of trichostatin A (TSA) improves in vitro embryonic development (Wittayarat et al. 2013 Cell. Reprogram. 15, 301–308). This study investigated whether the TSA treatment effects differ between the development of cat iSCNT embryos reconstructed with porcine and bovine oocytes. Porcine and bovine cumulus-oocyte complexes were in vitro matured for 44 h and 24 h, respectively. After cumulus cell removal, enucleation was performed by aspiration of the metaphase II plate and the first polar body using a piezo-driven pipette. A cat fibroblast cell was then injected into cytoplasm of successfully enucleated oocyte. Reconstructed cybrids were electrically activated by a single 1.5 kV cm–1 pulse for 100 µs (pig-cat embryos), or a 2.3 kV cm–1 pulse for 30 µs (cow-cat embryos). Pig-cat and cow-cat embryos were cultured in porcine zygote medium (PZM)-5 and modified synthetic oviducal fluid medium (mSOF), respectively. After electrical activation, pig-cat and cow-cat embryos were cultured in medium supplemented with 5 µg mL–1 cytochalasin B + 50 nM TSA (TSA group) or without TSA (control group), and the cow-cat embryo medium was also supplemented with 10 µg mL–1 cycloheximide. After 2 h, TSA-treated pig-cat and cow-cat embryos were incubated in medium supplemented with TSA for 22 h, followed by 48 h incubation without TSA. Pig-cat and cow-cat control embryos were cultured in medium without TSA for 70 h after activation. Then, all pig-cat and cow-cat embryos were cultured in porcine blastocyst medium (PBM) or mSOF medium supplemented with 5% fetal bovine serum, respectively, for 5 additional days. Four to seven replicates were performed for each experiment. Data were analysed using Student's t-test. For pig-cat embryos, no difference was observed in cleavage rates between both groups, but development to the blastocyst stage was higher in the pig control group (n = 147, 8.0%) than that of pig TSA group (n = 131, 0.7%; P < 0.05). In contrast, development to the blastocyst stage in cow-cat embryos was not observed in the cow control group (n = 125, 0%), but it was observed in cow TSA group (n = 136, 3.7%). These results indicate that TSA treatment effects are species-specific, but those effects remain to be clarified.

Transfer at the blastocyst stage of embryos derived from testicular round spermatid injection

Human Reproduction ◽

10.1093/humrep/17.3.741 ◽

2002 ◽

Vol 17 (3) ◽

pp. 741-743 ◽

Cited By ~ 20

Author(s):

B. Urman ◽

C. Alatas ◽

S. Aksoy ◽

R. Mercan ◽

A. Nuhoglu ◽

...

Keyword(s):

Round Spermatid ◽

Blastocyst Stage ◽

Round Spermatid Injection

Harmful or Not: Trichostatin A treatment of embryos generated by ICSI or ROSI

Open Life Sciences ◽

10.2478/s11535-006-0023-5 ◽

2006 ◽

Vol 1 (3) ◽

pp. 376-385 ◽

Cited By ~ 10

Author(s):

Satoshi Kishigami ◽

Hiroshi Ohta ◽

Eiji Mizutani ◽

Sayaka Wakayama ◽

Hong-Thuy Bui ◽

...

Keyword(s):

Assisted Reproductive Technologies ◽

Trichostatin A ◽

Reproductive Technologies ◽

Round Spermatid ◽

Developmental Arrest ◽

Offspring Production ◽

Deacetylase Inhibitor ◽

Cloning Method ◽

Round Spermatid Injection ◽

Parthenogenetic Embryos

AbstractTrichostatin A (TSA), a histone deacetylase inhibitor, is a known teratogen causing malformations such as vertebral fusions when applied during the postimplantation period; TSA also causes developmental arrest when applied during the preimplantation period. Regardless of these hindrances, we have succeeded in the establishment of an efficient somatic cloning method for the mouse where reconstructed embryos are treated with TSA. To elucidate this apparent discrepancy, we treated fertilized mouse embryos generated either by intracytoplasmic sperm injection (ICSI) or round spermatid injection (ROSI) with 50 nM TSA for 20 h after fertilization as well as parthenogenetic embryos and found that TSA treatment inhibited the preimplantation development of ICSI embryos but not ROSI or parthenogenetic embryos. And, although we often observed hypomorphism following TSA treatment in embryos grown to full term produced by both ICSI (av. of body weight: 1.7 g vs. 1.5 g) and ROSI (1.6 g vs. 1.2 g), TSA treatment reduced the offspring production rate for ICSI from 57% to 34% but not for ROSI from 30% to 36%. Thus, these data indicate that the effects, harmful or not, of TSA treatment on embryonic development depend on their nuclear derivations. Also, the resulting hypomorphism after TSA treatment is a caveat for this procedure in current Assisted Reproductive Technologies.

Somatic cell nuclear transfer in buffalos: effect of the fusion and activation protocols and embryo culture system on preimplantation embryo development

10.1071/rd05079 ◽

2006 ◽

Vol 18 (4) ◽

pp. 439 ◽

Cited By ~ 14

Author(s):

Liz Simon ◽

C. Veerapandian ◽

S. Balasubramanian ◽

A. Subramanian

Keyword(s):

Culture System ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Preimplantation Embryo ◽

Blastocyst Stage ◽

Fetal Fibroblasts ◽

Oviduct Epithelial Cell ◽

Oviduct Fluid ◽

Better Than

The present study was conducted primarily to evaluate several factors that affect the nuclear transfer programme in water buffalos, in which relatively few studies have been performed. Embryos reconstructed with quiescent fetal fibroblasts and metaphase II cytoplasts were matured for 24 h, and activation was found to be comparatively better than in those matured for 30 h. A significantly higher proportion of embryos fused (52.0 ± 1.9) and cleaved (51.2 ± 1.7) when the couplets were fused 4–6 h before activation than when fused and activated simultaneously (46.5 ± 1.6 and 44.5 ± 2.0, respectively). Development of nuclear transfer embryos to the blastocyst stage (4.8 ± 2.2) was supported by a commercially available sequential medium, and cleavage (76.5 ± 2.8) was significantly higher in this medium compared with cleavage in TCM-199 with oviduct epithelial cell coculture (45.6 ± 1.5) and synthetic oviduct fluid (21.8 ± 6.6). Of the 16 cloned embryos transferred, none resulted in pregnancy. The present study demonstrates that optimal numbers of cloned buffalo blastocysts can be obtained from oocytes matured for 24 h, fused 3–4 h before activation and cultured in a commercially available sequential media (G1/G2), thus providing further information to enable successful nuclear transfer in buffalos.

Exposure to mono-n-butyl phthalate disrupts the development of preimplantation embryos

10.1071/rd12178 ◽

2013 ◽

Vol 25 (8) ◽

pp. 1174 ◽

Cited By ~ 15

Author(s):

Da-Peng Chu ◽

Shi Tian ◽

Da-Guang Sun ◽

Chan-Juan Hao ◽

Hong-Fei Xia ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Dna Methylation ◽

Dibutyl Phthalate ◽

Preimplantation Embryo ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Oxygen Species ◽

Immunofluorescent Analysis ◽

Butyl Phthalate

Dibutyl phthalate (DBP), a widely used phthalate, is known to cause many serious diseases, especially in the reproductive system. However, little is known about the effects of its metabolite, mono-n-butyl phthalate (MBP), on preimplantation embryo development. In the present study, we found that treatment of embryos with 10–3 M MBP impaired developmental competency, whereas exposure to 10–4 M MBP delayed the progression of preimplantation embryos to the blastocyst stage. Furthermore, reactive oxygen species (ROS) levels in embryos were significantly increased following treatment with 10–3 M MBP. In addition, 10–3 M MBP increased apoptosis via the release of cytochrome c, whereas immunofluorescent analysis revealed that exposure of preimplantation embryos to MBP concentration-dependently (10–5, 10–4 and 10–3 M) decreased DNA methylation. Together, the results indicate a possible relationship between MBP exposure and developmental failure in preimplantation embryos.

Corrigendum to: Exposure to mono-n-butyl phthalate disrupts the development of preimplantation embryos

10.1071/rd12178_co ◽

2014 ◽

Vol 26 (3) ◽

pp. 491 ◽

Cited By ~ 1

Author(s):

Da-Peng Chu ◽

Shi Tian ◽

Da-Guang Sun ◽

Chan-Juan Hao ◽

Hong-Fei Xia ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Dna Methylation ◽

Dibutyl Phthalate ◽

Preimplantation Embryo ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Oxygen Species ◽

Immunofluorescent Analysis ◽

Butyl Phthalate

Dibutyl phthalate (DBP), a widely used phthalate, is known to cause many serious diseases, especially in the reproductive system. However, little is known about the effects of its metabolite, mono-n-butyl phthalate (MBP), on preimplantation embryo development. In the present study, we found that treatment of embryos with 10–3M MBP impaired developmental competency, whereas exposure to 10–4M MBP delayed the progression of preimplantation embryos to the blastocyst stage. Furthermore, reactive oxygen species (ROS) levels in embryos were significantly increased following treatment with 10–3M MBP. In addition, 10–3M MBP increased apoptosis via the release of cytochrome c, whereas immunofluorescent analysis revealed that exposure of preimplantation embryos to MBP concentration-dependently (10–5, 10–4 and 10–3M) decreased DNA methylation. Together, the results indicate a possible relationship between MBP exposure and developmental failure in preimplantation embryos.

Fate of microinjected spermatid mitochondria in the mouse oocyte and embryo

Zygote ◽

10.1017/s0967199498000148 ◽

1998 ◽

Vol 6 (3) ◽

pp. 213-222 ◽

Cited By ~ 46

Author(s):

James M. Cummins ◽

Teruhiko Wakayama ◽

Ryuzo Yanagimachi

Keyword(s):

Significant Risk ◽

Primary Spermatocyte ◽

Round Spermatid ◽

Blastocyst Stage ◽

Infertile Men ◽

Oocyte Cytoplasm ◽

Almost All ◽

Cytoskeletal Elements ◽

Round Spermatid Injection ◽

Cell Transition

Mouse round spermatids labelled with MitoTracker were microinjected into Sr2+-activated mouse oocytes. The labelled mitochondria were tracked up to the morula/blastocyst stage using fluorescence microscopy. The overall incidence of embryos with labelled mitochondria fell from 80% in the 1-cell zygote to 25% in 2-cell, 9% in 4-cell and ~1% in 8-cell or later stages. Thus it appears that almost all round spermatid mitochondria finally disappear from embryos during the 4-cell to 8-cell transition, as happens for mature spermatozoa (Cummins et al.Zygote 1997, 5: 301–8). The spermatid mitochondria remained tightly bound together during this process. In contrast, labelled primary spermatocyte and cumulus mitochondria dispersed rapidly throughout the oocyte cytoplasm within 3 h. We hypothesise that spermatid mitochondria may be bound together by cytoskeletal elements produced in the early haploid spermatid. These elements, together with terminal differentiation of the sperm mitochondria, may be central to the processes by which the embryo ‘recognises’ the sperm mitochondria and inhibits inheritance of paternal mitochondrial DNA. These results suggest that round spermatid injection for infertile men will not pose a significant risk to offspring by transmitting abnormal mitochondrial genomes.

Expression and localization of Nlrp4g in mouse preimplantation embryo

Zygote ◽

10.1017/s0967199414000525 ◽

2014 ◽

Vol 23 (6) ◽

pp. 846-851 ◽

Cited By ~ 2

Author(s):

Hui Peng ◽

Xiujiao Lin ◽

Wenhao Li ◽

Wenchang Zhang

Keyword(s):

Preimplantation Embryo ◽

Expression Patterns ◽

Blastocyst Stage ◽

Gene Copies ◽

Mouse Preimplantation Embryo ◽

Cell Embryo ◽

Embryo Stage ◽

Genome Activation ◽

Zygotic Genome

SummaryThe Nlrp gene family contains 20 members and plays a pivotal role in the innate immune and reproductive systems in the mouse. During evolution, seven Nlrp4 gene copies (named from Nlrp4a to Nlrp4g). Nlrp4a–Nlrp4g have arisen that display specific or preferential ovarian expression patterns. However, the expression pattern and localization of Nlrp4g in mouse preimplantation embryo development are unknown. Here we report that Nlrp4g was highly expressed in mature oocytes and zygotes, then downregulated and not detected after the 2-cell embryo stage. NLRP4G protein remained present through the blastocyst stage and was mainly localized in the cytoplasm. Furthermore, overexpression of Nlrp4g in zygotes did not affect normal development in terms of the rate of blastocyst formation. These results provide the first evidence that NLRP4G is a maternal factor that may play essential role during zygotic genome activation in the mouse.

Postnatal weight of calves derived from in vitro matured and in vitro fertilized embryos developed under various oxygen concentrations

10.1071/rd00057 ◽

2000 ◽

Vol 12 (8) ◽

pp. 391 ◽

Cited By ~ 15

Author(s):

H. Iwata ◽

N. Minami ◽

H. Imai

Keyword(s):

Oxygen Tension ◽

Blastocyst Stage ◽

High Oxygen ◽

Gestation Length ◽

Low Oxygen ◽

Cell Stage ◽

Fluid Medium ◽

Significant Difference ◽

Oxygen Conditions

In the present study, weights of calves (14 days after birth) derived from embryos of a homogeneous line (Tajima line) of Japanese Black Cow, cultured in vitro under various oxygen conditions was examined. In vitro matured and fertilized oocytes were incubated for 48 h in modified synthetic oviduct fluid medium under 5% CO 2in air and embryos reaching at least the 5-cell stage were selected for further culture under various gas conditions (high oxygen tension: 5% CO 2 in air; low oxygen tension: 5% O 2 , 5% CO 2 , 90% N 2 ) for 5 days. Embryos that developed to the blastocyst stage were transferred to Holstein cows or cryopreserved until transfer. When embryos were cultured under high oxygen tension and cryopreserved, the weights of male calves (at 14 days) were significantly heavier than in the other groups. However, there was no significant difference in gestation lengths of male calves. In female calves, no difference was observed in either the weight or gestation length of calves irrespective of oxygen tension during the culture period or embryo conditions (fresh or frozen). From the results of the present study, it is suggested that the oxygen concentration during culture and cryopreservation synergistically induced the production of overweight male calves without influencing gestation length.

101. ISOLATION OF PUTATIVE EMBRYONIC STEM CELLS FROM CLONED PIG EMBRYOS

10.1071/srb09abs101 ◽

2009 ◽

Vol 21 (9) ◽

pp. 20 ◽

Cited By ~ 1

Author(s):

K. P. Truong ◽

I. Vassiliev ◽

L. F.S. Beebe ◽

S. M. McIlfatrick ◽

S. J. Harrison ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Trichostatin A ◽

Embryonic Stem ◽

Blastocyst Stage ◽

Electrical Pulse ◽

Blastocyst Development ◽

Significant Difference ◽

Cloned Pig

The isolation of embryonic stem cells from cloned embryos (NT-ESC) from domestic animals would have a number of biomedical and agricultural applications. Putative ESC lines from in vivo derived and in vitro produced pig embryos were recently established using a new isolation method1. The aim of the current study was to determine whether NT-ESC lines could be isolated from cloned pig embryos using this method. To do this we determined initially whether the treatment of embryos with Trichostatin A (TSA), a histone deacetylase inhibitor, could increase the number of cloned embryos that develop to the blastocyst stage because TSA has been shown to increase blastocyst development and NT-ESC isolation efficiencies in mice2. Cloned embryos were produced as described previously3. Briefly, in vitro matured sow oocytes were enucleated, fused with adult fibroblasts using an electrical pulse and activated about 1.5 hrs later with a second electrical pulse. Reconstructed embryos were then cultured in modified NCSU23 with or without 50nM TSA treatment for the initial 24 hours of culture. Embryo development was assessed on day 6. Treatment with TSA increased the number of cloned embryos that developed to the blastocyst stage (143/471; 30%) compared with control (54/353; 15%; P < 0.0001). Blastocysts were then plated by mechanical depression onto mitotically inactivated mouse embryonic fibroblast feeder layers in a serum-free culture system on day 7. There was no significant difference in the efficiencies of establishment of homogeneous primary outgrowths between TSA treated (17/96; 18%) and control blastocysts (8/43; 19%). Thirteen homogenous outgrowths from the TSA treated group were vitrified at passage 2 or 3. Sublines are currently being characterised to determine their pluripotency.