Developmental Outcome and Related Abnormalities in Goats: Comparison Between Somatic Cell Nuclear Transfer- and In Vivo-Derived Concepti During Pregnancy Through Term

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.

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44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab44 ◽

2006 ◽

Vol 18 (2) ◽

pp. 131

Author(s):

K. Kaneyama ◽

S. Kobayashi ◽

S. Matoba ◽

Y. Hashiyada ◽

K. Imai ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Survival Rates ◽

Calf Serum ◽

Cumulus Cells ◽

Nuclear Transplantation ◽

Embryo Cryopreservation

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved. Table 1. Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer

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24 BUFFALO (BUBALUS BUBALIS) SOMATIC CELL NUCLEAR TRANSFER EMBRYOS PRODUCED FROM FROZEN–THAWED SEMEN-DERIVED SOMATIC CELLS: EFFECT OF TRICHOSTATIN A ON THE IN VITRO AND IN VIVO DEVELOPMENTAL POTENTIAL, QUALITY, AND EPIGENETIC STATUS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab24 ◽

2015 ◽

Vol 27 (1) ◽

pp. 104

Author(s):

N. L. Selokar ◽

M. Saini ◽

H. Agrawal ◽

P. Palta ◽

M. S. Chauhan ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Trichostatin A ◽

Somatic Cells ◽

Developmental Potential ◽

Reconstructed Embryos ◽

Frozen Semen ◽

Significant Difference

Cryopreservation of semen allows preservation of somatic cells, which can be used for the production of progeny through somatic cell nuclear transfer (SCNT). This approach could enable restoration of valuable high-genetic-merit progeny-tested bulls, which may be dead but the cryopreserved semen is available. We have successfully produced a live buffalo calf by SCNT using somatic cells isolated from >10 year old frozen semen (Selokar et al. 2014 PLoS One 9, e90755). However, the calf survived only for 12 h, which indicates faulty reprogramming of these cells. The present study was, therefore, carried out to study the effect of treatment with trichostatin A (TSA), an epigenetic modifier, on reprogramming of these cells. Production of cloned embryos and determination of quality and level of epigenetic markers in blastocysts were performed according to the methods described previously (Selokar et al. 2014 PLoS One 9, e90755). To examine the effects of TSA (0, 50, and 75 nM), 10 separate experiments were performed on 125, 175, and 207 reconstructed embryos, respectively. The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher's least significant difference test for significance at P < 0.05. When the reconstructed buffalo embryos produced by hand-made clones were treated with 0, 50, or 75 nM TSA post-electrofusion for 10 h, the cleavage percentage (100.0 ± 0, 94.5 ± 2.3, and 96.1 ± 1.2, respectively) and blastocyst percentage (50.6 ± 2.3, 48.4 ± 2.7, and 48.1 ± 2.6, respectively), total cell number (274.9 ± 17.4, 289.1 ± 30.1, and 317.0 ± 24.2, respectively), and apoptotic index (3.4 ± 0.9, 4.5 ± 1.4, and 5.6 ± 0.7, respectively) in Day 8 blastocysts were not significantly different among different groups. The TSA treatment increased (P < 0.05) the global level of H4K5ac but not that of H3K18a in embryos treated with 50 or 75 nM TSA compared with that in controls. In contrast, the level of H3K27me3 was significantly lower (P < 0.05) in cloned embryos treated with 75 nM TSA than in embryos treated with 50 nM TSA or controls. The ultimate test of the reprogramming potential of any donor cell type is its ability to produce live offspring. To examine the in vivo developmental potential of the 0, 50, or 75 nM TSA treated embryos, we transferred Day 8 blastocysts, 2 each to 5, 6, and 5 recipients, respectively, which resulted in 2 pregnancies from 75 nM TSA treated embryos. However, one pregnancy was aborted in the first trimester and the other in the third trimester. In conclusion, TSA treatment of reconstructed embryos produced from semen-derived somatic cells alters their epigenetic status but does not improve the live birth rate. We are currently optimizing an effective strategy to improve the cloning efficiency of semen-derived somatic cells.

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59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab59 ◽

2007 ◽

Vol 19 (1) ◽

pp. 147

Author(s):

E. Lee ◽

K. Song ◽

Y. Jeong ◽

S. Hyun

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Cell Number ◽

Skin Fibroblasts ◽

Miniature Pig ◽

Donor Cells ◽

Fetal Fibroblasts

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P < 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P < 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

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Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

Reproduction ◽

10.1530/rep.1.00850 ◽

2005 ◽

Vol 130 (5) ◽

pp. 681-694 ◽

Cited By ~ 13

Author(s):

P Tveden-Nyborg ◽

T T Peura ◽

K M Hartwich ◽

S K Walker ◽

P Maddox-Hyttel

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Morphological Characterization ◽

Morphological Development ◽

Embryonic Disc ◽

Transmission Electron

The processes of cellular differentiation were studied in somatic cell nuclear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients on day 6. All embryos were processed for examination by light and transmission electron microscopy or immunohistochemical labelling for alpha-1-fetoprotein and vimentin. Overall, morphological development of in vivo embryos was superior to IVC and SCNT embryos. Day 7 and particularly day 9 IVC and SCNT embryos had impaired hypoblast development, some lacking identifiable inner cell masses. On day 11, only in vivo and IVC embryos had developed an embryonic disc, and gastrulation was evident in half of in vivo embryos and one IVC embryo. By day 13, all in vivo embryos had completed gastrulation whereas IVC and SCNT embryos remained retarded. On days 17 and 19, in vivo embryos had significantly more somites and a more developed allantois than IVC and SCNT embryos. We conclude that IVC and particularly SCNT procedures cause a retardation of embryo development and cell differentiation at days 7–19 of gestation.

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Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer

Zygote ◽

10.1017/s0967199408004620 ◽

2008 ◽

Vol 16 (3) ◽

pp. 211-222 ◽

Cited By ~ 26

Author(s):

Wakayama Sayaka ◽

Kishigami Satoshi ◽

Nguyen Van Thuan ◽

Ohta Hiroshi ◽

Hikichi Takafusa ◽

...

Keyword(s):

Somatic Cell ◽

Intracytoplasmic Sperm Injection ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Subsequent Development ◽

Developmental Potential ◽

Parthenogenetic Activation ◽

Somatic Cell Nucleus

SummaryAnimal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4–15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.

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33 PROPAGATION OF ELITE LIFESAVER DOGS BY SOMATIC CELL NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab33 ◽

2013 ◽

Vol 25 (1) ◽

pp. 164

Author(s):

B. C. Lee ◽

H. J. Oh ◽

M. J. Kim ◽

G. A. Kim ◽

E. J. Park ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Culture Media ◽

Calcium Ionophore ◽

Defined Medium ◽

German Shepherd ◽

Surrogate Mothers ◽

Working Dogs

Canine somatic cell nuclear transfer (cSCNT) has been used as a useful tool for propagation of elite working dogs. In 2009, 7 cloned dogs were successfully produced using somatic cells derived from the excellent drug-sniffing dog of Korea Customs Service. All cloned dogs perfectly performed drug detection in Incheon International Airport. The objective of the present study was to compare the efficiency of the 2 activation culture media to clone the retired Baekdu, a veteran rescue dog that performed lifesaving activities worldwide for 6 years in Korea National Emergency Management Agency (NEMA). Ear tissue was collected from a 10-year-old male German Shepherd and fibroblasts were cultured for cSCNT. The cells were injected into the perivitelline space of enucleated in vivo-matured dog oocytes, fused with electric stimulation using an electro cell fusion apparatus (Nepa Gene Co. Ltd.), and activated chemically. In the activation protocol, 2 different types of media were tested to investigate the effect of proteins with undefined functions. The first medium was a modified synthetic oviduct fluid (mSOF), which is a complex culture medium with BSA that includes undefined functions. The second medium was the porcine zygote medium (PZM-5), which is a chemically defined medium with polyvinyl alcohol (PVA). The fused couplets were activated by mSOF medium supplemented with 1.9 nM DMAP (SOF-DMAP), and PZM-5 supplemented with 1.9 nM DMAP (PZM-DMAP) for 4 h, followed by 4 min of calcium ionophore treatment. Then, reconstructed oocytes were transferred into the uterine tube of naturally estrus-synchronized surrogate dogs. In the PZM-DMAP group, a total of 56 activated cloned embryos were transferred into 3 female recipient dogs, and a total of 64 activated cloned embryos from the SOF-DMAP group were transferred into 4 female recipients. Pregnancy diagnosis was performed using a SONOACE 9900 (Medison, Seoul, Korea) ultrasound scanner with 7.0-MHz linear-array probe between 30 and 35 days after embryo transfer. As a result, pregnancy was detected in 1 out of 3 surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and 1 pregnancy (25%) was detected in 4 surrogate mothers receiving cloned embryos from the SOF-DMAP group. Two pregnant dogs each gave birth to 1 healthy cloned puppy by cesarean section. This study shows that existence of proteins with undefined functions in activation medium did not affect the dog cloning. In addition, the number of elite working dogs in diverse fields can be increased by the NT technique using donor cells derived from small tissue of elite working dogs. This study was supported by RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), IPET (no. 311062-04-1-SB010), Research Institute for Veterinary Science, and TS Corporation.

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27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab27 ◽

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

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Pseudophysiological transcomplementary activation of reconstructed oocytes as a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetal fibroblast cells

Polish Journal of Veterinary Sciences ◽

10.2478/v10181-012-0078-3 ◽

2012 ◽

Vol 15 (3) ◽

pp. 509-516 ◽

Cited By ~ 12

Author(s):

M. Samiec ◽

M. Skrzyszowska ◽

D. Lipiński

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Electrical Activation ◽

Developmental Outcome ◽

Fibroblast Cells ◽

Cleavage Activity ◽

New Strategy ◽

Cytoplasmic Hybrids ◽

Stimulation Of

Abstract The completely new strategy of pseudophysiological transcomplementary (transcytoplasmic) activation (PP-TCA) of nuclear-transferred oocytes, which had been derived from pWAPhGH-GFPBsd transfected foetal fibroblast cells, was recently applied to the somatic cell cloning of pigs. It resulted in the considerable enhancing not only the cleavage activity of cultured cloned embryos, but also their morula and blastocyst formation rates as compared to the use of standard simultaneous fusion and electrical activation of reconstituted oocytes (77% vs. 57%, 63% vs. 46% and 40% vs. 27%, respectively). Altogether, the use of cytosolic components descended from heterologous (rabbit) zygotes as the agents for stimulation of porcine clonal cytoplasmic hybrids (cybrids) turned out to be reliable and feasible strategy for the generation of transgenic blastocysts by somatic cell nuclear transfer (SCNT). Furthermore, to our knowledge, no previous study has reported the preimplantation developmental outcome of transgenic nuclear-transferred pig embryos following the PP-TCA that was developed and optimised in our laboratory.

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