375 UNEXPECTED SEVERE ABNORMALITIES IN MOUSE ROSI OFFSPRING

2007 ◽  
Vol 19 (1) ◽  
pp. 303
Author(s):  
P. N. Moreira ◽  
R. Fernández-González ◽  
M. Pérez-Crespo ◽  
P. Bermejo ◽  
J. D. Hourcade ◽  
...  
Keyword(s):  
Testicular Tumor ◽  
Round Spermatid ◽  
Sperm Cells ◽  
Healthy Children ◽  
Clinical Value ◽  
Cell Stage ◽  
Infertile Men ◽  
Vitro Development ◽  
Round Spermatid Injection

Live offspring resulting from round spermatid injection (ROSI) was first accomplished in the mouse, but similar success has been obtained in rat, hamster, rabbit, mastomys, pig, monkey, and human. ROSI has received clinical attention because some infertile men have no spermatozoa or just a very few in their testes, and these are difficult to harvest and are frequently dead and deformed. Although some clinicians were able to generate healthy children by ROSI, others could not (reviewed in Yanagimachi 2004 Reprod. Biomed. Online 9). The clinical value of ROSI has been widely debated. It remains unclear if post-meiotic and pre-fertilization modifications of sperm cells are necessary to ensure normal development. In order to answer this question, we decided to study and compare mouse offspring generated by ROSI and intracytoplasmic sperm injection (ICSI). ROSI and ICSI with fresh sperm cells were carried out in the B6D2 mouse strain as described (Marh et al. 2003 Biol. Reprod. 69, 169–176; Moreira et al. 2005 Hum. Reprod. 20, 3313–3317). In vitro-produced embryos were transferred at the 2-cell stage into Day 1 pseudopregnant females. As shown in Table 1 oocyte survival after injection was significantly higher (z-test, P < 0.05) with ICSI (91%) than with ROSI (68%). The proportion of live offspring obtained by ICSI was also significantly higher (26% vs. 6%; z-test, P < 0.05). Moreover, fertilization with spermatozoa produced healthy offspring more efficiently than with round spermatids. Out of 30 live offspring generated by ROSI, 6 (20%) presented severe abnormalities during their first 6–8 weeks of age. One ROSI animal presented an abnormally swollen skull (hydroencephaly) with a very thin and soft cranial wall. Another developed a subcutaneous engrossment of the forehead, producing a crest-like appearance. Three others presented deviations in their vertebral columns (hyperkyphosis and scoliosis), and recently a testicular tumor was detected in another animal. These types of malformations were not observed in the control offspring. In our experience, very rarely are they observed after ICSI or in naturally mated animals. To our knowledge, and although the risks of the ROSI procedure have been extensively highlighted in human and other species, the phenotypic abnormalities observed in this study have never been reported. Presently, we keep monitoring these animals as they age, as part of an ambitious plan that is also intended to characterize and understand the origin of the possible phenotypic consequences of the ROSI procedure. Table 1. In vitro development and development to term of B6D2 mouse embryos generated by ROSI or ICSI

scholarly journals Fourteen babies born after round spermatid injection into human oocytes

2015 ◽  
Vol 112 (47) ◽  
pp. 14629-14634 ◽  
Author(s):  
Atsushi Tanaka ◽  
Motoi Nagayoshi ◽  
Youichi Takemoto ◽  
Izumi Tanaka ◽  
Hiroshi Kusunoki ◽  
...  
Keyword(s):  
Germ Cells ◽  
Testicular Biopsy ◽  
Round Spermatid ◽  
Clinical Value ◽  
Vitro Fertilization ◽  
Infertile Men ◽  
Human In Vitro Fertilization ◽  
Haploid Male ◽  
Round Spermatid Injection

During the human in vitro fertilization procedure in the assisted reproductive technology, intracytoplasmic sperm injection is routinely used to inject a spermatozoon or a less mature elongating spermatid into the oocyte. In some infertile men, round spermatids (haploid male germ cells that have completed meiosis) are the most mature cells visible during testicular biopsy. The microsurgical injection of a round spermatid into an oocyte as a substitute is commonly referred to as round spermatid injection (ROSI). Currently, human ROSI is considered a very inefficient procedure and of no clinical value. Herein, we report the birth and development of 14 children born to 12 women following ROSI of 734 oocytes previously activated by an electric current. The round spermatids came from men who had been diagnosed as not having spermatozoa or elongated spermatids by andrologists at other hospitals after a first Micro-TESE. A key to our success was our ability to identify round spermatids accurately before oocyte injection. As of today, all children born after ROSI in our clinic are without any unusual physical, mental, or epigenetic problems. Thus, for men whose germ cells are unable to develop beyond the round spermatid stage, ROSI can, as a last resort, enable them to have their own genetic offspring.

In vitro development of haploid mouse embryos produced by bisection of one-cell fertilized eggs

Development ◽  
1977 ◽  
Vol 38 (1) ◽  
pp. 187-202
Author(s):  
Andrzej K. Tarkowski
Keyword(s):  
X Chromosome ◽  
Culture Conditions ◽  
Extrinsic Factors ◽  
Cell Stage ◽  
Glass Needle ◽  
Androgenetic Embryos ◽  
Haploid Embryos ◽  
Vitro Development ◽  
Further Development

F1(CBA × C57BL'10) mouse eggs originating from spontaneous or induced ovulation and fertilized by CBA-T6T6 or PO spermatozoa were bisected with a glass needle into halves each containing a pronucleus. This technique offers a unique opportunity of producing both androgenetic and gynogenetic haploid embryos from one egg. Out of 600 operated eggs, in 406 (67·7%) both halves survived. During 96 h of culture in vitro the fragments were inspected once daily and finally examined in air-dried preparations. Eighty-seven per cent of halves underwent first cleavage but their further development was to a large extent affected by extrinsic factors connected with experimental procedure (mainly by suboptimal and variable culture conditions) and by the origin of eggs (those from spontaneous ovulation being superior). For this reason developmental capabilities of egg halves were assessed in a selected group of pairs in which at least one partner reached the stage of four or more blastomeres. The observed ratio between pairs with both or only one sister embryo developing successfully suggests that androgenetic embryos carrying Y rather than X chromosome can cleave twice but do not survive beyond 4-cell stage. None of the metaphase plates from older embryos contained a Y chromosome. These observations imply that the X chromosome is genetically active during early cleavage and that a full haploid set is required for preimplantation development to be completed. Formation of blastocysts varied from batch to batch, with an average of 12·8% and maximal incidence of 29·5% . In 34 pairs both fragments developed beyond the 4-cell stage but in only one case did both form blastocysts. Haploid blastocysts were composed of 27 cells on average which was about a half of the number of cells in control diploid zona-free whole eggs. Ten out of 51 embryos with metaphase plates proved to be haploid/diploid mosaics.

297 PRODUCTION OF CHIMERIC PORCINE FETUSES BY AGGREGATION METHOD USING PARTHENOGENETIC EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 296
Author(s):  
K. Nakano ◽  
M. Watanabe ◽  
H. Matsunari ◽  
T. Matsuda ◽  
K. Honda ◽  
...  
Keyword(s):  
Cell Mass ◽  
Ips Cells ◽  
Large Animal ◽  
Aggregation Method ◽  
Cell Stage ◽  
In Vitro Development ◽  
Donor Cells ◽  
Vitro Development ◽  
Parthenogenetic Embryos

Porcine induced pluripotent stem (iPS) cells are considered to be an invaluable research tool in translational research with pigs as a large animal model. Pluripotency of the iPS cells needs to be verified by their competence to contribute to chimera formation. The aim of the present study is to establish feasible system to create chimeric pig fetuses using parthenogenetic embryos. In Experiment 1, inner cell mass (ICM) was isolated by immunosurgery from Day 6 blastocysts obtained by parthenogenetic activation of in vitro matured (IVM) oocytes. Isolated ICM were used as the donor cells after staining with fluorescent carbocyanine dye (DiI). Using parthenogenetic morulae or 4- to 8-cell embryos as the host embryos, chimeric embryos were prepared by injection or aggregation method. Injection of ICM was performed by micromanipulation: a single ICM was directly injected into the centre portion of the host morulae. In the aggregation method, a single ICM was aggregated with blastomeres isolated from 2 host embryos at the morula or 4- to 8-cell stage in a micro-well (400 µm diameter, 300 µm deep). The chimeric embryos were cultured in PZM-5 (Yoshioka et al. 2008) for 2 to 3 days to examine development to blastocysts and incorporation of donor ICM cells into the resultant blastocysts ICM (ICM chimerism). In Experiment 2, donor blastomeres isolated from a parthenogenetic morula or 4- to 8-cell embryo were stained by DiI and aggregated with a parthenogenetic host embryo at the morula or 4- to 8-cell stage, and the in vitro development to the blastocyst stage and the ICM chimerism were examined. In Experiment 3, ICM isolated from IVF blastocysts harboring humanized Kusabira-Orange (huKO) gene were used as donor cells. Donor ICM were aggregated with the host embryos at the morula or 4- to 8-cell stage, and the resultant blastocysts were transferred to 4 recipient gilts to collect fetuses on Day 18. Results of Experiments 1 and 2 are summarised in Table 1. Combination of the donor ICM and host morulae yielded high rates of blastocyst formation (~95%) and ICM chimerism (~85%), regardless of the method used (injection or aggregation). Transfer of 73 blastocysts developed from host morulae to 2 recipients (Experiment 3) gave rise to 25 (34.2%) fetuses, of which 6 (24.0%) were confirmed to be chimeric by their clear orange fluorescence and immunostaining by anti-huKO antibody. Of 22 (40.7%) fetuses obtained after transfer of 54 blastocysts derived from 4- to 8-cell host embryos to 2 recipients, 3 (13.6%) were chimeric. Contribution of the donor cells in the tissues of the chimeric fetuses measured by image analysis software (ImageJ, NIH, Bethesda, MD, USA) ranged between 16.1 and 65.2%. These results demonstrate that the aggregation method using parthenogenetic host embryos is an efficient means to produce chimeric pig fetuses, and thereby feasible for verification of pluripotent cells such as iPS cells. Table 1.In vitro development of injected or aggregated porcine embryos

scholarly journals 25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
S. Akagi ◽  
B. Tsuneishi ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Functional Peptide ◽  
Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

scholarly journals Effect of Ram Breed on the Efficiency of in vitroDevelopment of Sheep Embryos

2017 ◽  
Vol 14 (4) ◽  
pp. 1309-1313
Author(s):  
Y. Al-Anazi ◽  
M. G. Al-Mutary ◽  
M. M. Alfuraiji ◽  
M. Al-Ghadi ◽  
A. R. Al-himaidi ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Cell Stage ◽  
In Vitro Development ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Stage 4 ◽  
Before And After ◽  
Vitro Development ◽  
Fresh Semen

The aim of this work was to investigate the impacts of ram breed on in vitro embryo development from fresh or frozen semen. Semen was collected from Najdi and Naimi rams and frozen; the mass and progressive motility of the spermwere assessed in each trial before and after freezing. Then, 970 oocytes in six replicates were fertilized with fresh and frozen semen in vitro. Different stages of sheep embryos were recorded. There were no significant differences in mass and progressive sperm motility of fresh or frozen ram semen between Najdi and Naimi,but there were significant differences between frozen and fresh semen within each breed. Our results showed significant (P<0.05) differences in 2-cell stage, 4-cell stage, 8-cell stage, morula, fragmented embryos, cleavage and blastocyst rates in the frozen semen group compared to fresh semen group in both breeds. In addition, significant (P<0.05) differencesbetween the two breeds were shown in 8-cell and16-cell embryonic stages.In conclusion, there were slight breed effects on the efficiency of in vitro development of sheep embryos.

34 DEVELOPMENTAL ABILITY OF ZONA-FREE PORCINE CLONED EMBRYOS RECONSTRUCTED BY SOMATIC CELLS AND CENTRIFUGED CYTOPLASTS

2006 ◽  
Vol 18 (2) ◽  
pp. 125
Author(s):  
M. Fahrudin ◽  
K. Kikuchi ◽  
N. W. K. Karja ◽  
M. Ozawa ◽  
T. Somfai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Gradient Centrifugation ◽  
Somatic Cells ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

The combination of bulk enucleation and zona-free cloning will offer simplification of the conventional nuclear transfer technique. A bulk enucleation method such as enucleation by centrifugation could reduce the time of manipulation that is necessary for removing genetic materials from the oocytes. The present study was conducted to examine the ability of cytoplasts obtained by centrifugation of zona-free in vitro maturation (IVM) porcine oocytes to support remodeling of the somatic cell nucleus and the subsequent development in vitro of somatic cell nuclear transferred (SCNT) embryos. A primary culture of cumulus cells was used as the source of donor cells, and recipient cytoplasts were derived from IVM oocytes that were cultured for 48 h, denuded of zonae pellucidae, and subjected to gradient centrifugation in Percoll solution to separate the ooplasm into fragments. Fragments were stained with Hoechst-33342 and cytoplasts were selected under an epifluorescence microscope. Then two or three cytoplasts were aggregated with a single somatic cell in phytohemagglutinin solution (500 �g/mL). Fusion between somatic cell and cytoplasts was induced by two DC pulses of 1.5 kV/cm for 20 �s, and activation was accomplished by two DC pulses of 0.8 kV/cm for 30 �s at 1 h after fusion in 0.28 M mannitol solution supplemented with 0.05 mM CaCl2 and 0.1 mM MgSO4. The resultant embryos were transferred to a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) and cultured in glucose-free NCSU-37 containing 4 mg/mL BSA supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2; from Days 2 to 7 they were cultured in NCSU-37 supplemented with 5.55 mM {D}-glucose and 5% FCS. Some of the reconstructed embryos were fixed at 1, 10, and 24 h after activation and stained with 1% (w/v) orcein to display the morphology of the transferred somatic nuclei. The results showed that 53.6% (30/56) of the SCNT embryos underwent premature chromosome condensation at 1 h, 90.9% (50/55) formed pseudo-pronuclei at 10 h, and 21% (19/90) of them cleaved to the two-cell stage at 24 h after the activation. The development to the blastocyst stage of the embryos that were reconstructed by quartet cells (three cytoplasts and one somatic cell; 8.9%, 10/112) was significantly higher (P < 0.05) than that of the triplet ones (2.2%, 3/139). However, these blastocyst rates were significantly lower (P < 0.05) than the blastocyst development rate of parthenogenetic embryos with the intact zonae pellucidae (28.3%, 17/60). These results suggest that (1) cytoplasts obtained by gradient centrifugation could support reprogramming of somatic cells and in vitro development of SCNT embryos to the blastocyst stage, and (2) the volume of cytoplasts apparently affects their in vitro development in pigs.

Fate of microinjected spermatid mitochondria in the mouse oocyte and embryo

Zygote ◽  
1998 ◽  
Vol 6 (3) ◽  
pp. 213-222 ◽  
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.

Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos

2006 ◽  
Vol 73 (6) ◽  
pp. 700-708 ◽  
Author(s):  
Duangjai Boonkusol ◽  
Arpad Baji Gal ◽  
Szilard Bodo ◽  
Botond Gorhony ◽  
Yindee Kitiyanant ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Mouse Embryos ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

A novel method for isolating spermatid nuclei from cytoplasm prior to ROSI in the mouse

Zygote ◽  
2004 ◽  
Vol 12 (4) ◽  
pp. 321-327 ◽  
Author(s):  
Satoshi Kishigami ◽  
Nguyen Van Thuan ◽  
Sayaka Wakayama ◽  
Takafusa Hikichi ◽  
Teruhiko Wakayama
Keyword(s):  
Round Spermatid ◽  
Separation Method ◽  
New Method ◽  
Offspring Production ◽  
Spermatid Nucleus ◽  
Novel Method ◽  
Round Spermatid Injection ◽  
Normal Fertilization

In the current widely used round spermatid injection (ROSI) protocol for the mouse, the spermatid nucleus is separated from most of the cytoplasm before ROSI by drawing a spermatid in and out of a pipette. This results in the highest rate of normal fertilization. However, this separation method is not always consistent and can be time-consuming. An alternative separation method that cuts away the cytoplasm using the tip of an injection pipette was developed. After removing the cytoplasm, ROSI was performed following both post- and pre-activation protocols and development in vitro and in vivo were examined. The new method consistently removed the bulk of the cytoplasm, as shown by quantifying mitochondria. ROSI without the cytoplasm resulted in significantly higher rates of fertilization than ROSI with the cytoplasm into either post- or pre-activated oocytes. Furthermore, the offspring production rates of ROSI without the cytoplasm were also high (50% and 49% for the post- and pre-activation protocols, respectively). This new method for separating the cytoplasm is an alternative way of producing offspring using ROSI.

The effect of vitrification of immature bovine oocytes to the subsequent in vitro development and gene expression

Zygote ◽  
2014 ◽  
Vol 23 (6) ◽  
pp. 933-942 ◽  
Author(s):  
Marwa S. Faheem ◽  
E. Baron ◽  
I. Carvalhais ◽  
A. Chaveiro ◽  
K. Pavani ◽  
...  
Keyword(s):  
Molecular Level ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cell Stage ◽  
Maturation Rate ◽  
Developmental Rates ◽  
Polymerase Chain ◽  
Vitro Development ◽  
Bovine Oocytes

SummaryImmature bovine oocytes were vitrified using the cryotop method and their post-warming survivability and capability to undergo in vitro maturation, fertilization and subsequent embryonic development were evaluated. In addition throughout the embryonic 2-cell, 4-cell, morula and blastocyst stages, the expression of four developmentally important genes (Cx43, CDH1, DNMT1 and HSPA14) was analysed using the real-time polymerase chain reaction (PCR). Immature oocytes (n = 550) were randomly assigned to non-vitrified (fresh) or cryotop vitrification groups using ethylene glycol (EG) with 1,2 propanediol (PROH) or dimethylsulphoxide (DMSO). After warming, oocytes survivability, embryo cleavage and embryonic developmental rates were not statistically different between the two cryoprotectants groups. However, the DMSO group had a lower (P < 0.05) oocyte maturation rate compared with the fresh and PROH groups. For morula and blastocyst rates, the DMSO group achieved a lower (P < 0.05) morula rate compared with the fresh group, while at the blastocyst stage, there were no differences between fresh and both cryoprotectants groups. For molecular analysis, at the 4-cell stage, most studied genes showed an inconsistent pattern of expression either from the PROH or DMSO groups. Noteworthily, these differences were limited at the morula and blastocyst stages. In conclusion, the cryotop method is sufficient for vitrification of immature bovine oocytes, both for embryonic developmental competence and at the molecular level. Moreover, PROH showed some advantage over DMSO as a cryoprotectant.

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