Extra-uterine development of mouse blastocysts cultured in vitro from early cleavage stages

Development ◽  
1968 ◽  
Vol 20 (3) ◽  
pp. 391-400
Author(s):  
W. D. Billington ◽  
C. F. Graham ◽  
Anne McLaren
Keyword(s):  
Blastocyst Stage ◽  
Embryonic Tissue ◽  
Early Cleavage ◽  
Embryonic Tissues ◽  
Complete Development ◽  
Uterine Environment ◽  
Cleavage Stages ◽  
Mouse Eggs ◽  
Uterine Development

Many experiments indicate that oviducal mouse eggs are unable to form embryonic tissues following transplantation to extra-uterine sites; they give rise only to trophoblast and extra-embryonic membranes (Fawcett, Wislocki & Waldo, 1947; Fawcett, 1950; Chester-Jones, 1951; Whitten, 1958; Kirby, 1962; Billington, 1965). Possible rare exceptions exist in the reports by Runner (1947) and Stevens (1967). In contrast, about a quarter of uterine blastocysts form morphologically normal embryos when similarly transplanted (Kirby, 1963; Billington, 1965). The conditions under which the mouse eggs reach the blastocyst stage may also have developmental consequences. Eggs cultured in vitro to the blastocyst stage (Whitten, 1956) or tube-locked blastocysts (Kirby, 1962) did not develop embryonic tissue in extra-uterine sites, but were capable of complete development following transfer to a receptive uterus. These findings suggest that mouse eggs must be subjected to the uterine environment if they are to ‘realize totipotency’ in an extra-uterine site (Kirby, 1962, 1965).

scholarly journals IN-VITRO CULTURE OF RABBIT OVA FROM EARLY CLEAVAGE STAGES TO THE BLASTOCYST STAGE

Reproduction ◽  
1968 ◽  
Vol 16 (3) ◽  
pp. 491-493 ◽  
Author(s):  
H. ONUMA ◽  
R. R. MAURER ◽  
R. H. FOOTE
Keyword(s):  
In Vitro Culture ◽  
Blastocyst Stage ◽  
Early Cleavage ◽  
Cleavage Stages

Viability and growth of mouse embryos after in vitro culture and fusion

Development ◽  
1970 ◽  
Vol 23 (3) ◽  
pp. 693-704
Author(s):  
Patricia Bowman ◽  
Anne McLaren
Keyword(s):  
In Vitro Culture ◽  
Success Rate ◽  
Zona Pellucida ◽  
Growth Regulation ◽  
Excess Mortality ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Foster Mothers ◽  
Mouse Eggs

About 80 % of 8-cell mouse eggs developed to the blastocyst stage in culture, whether the zona pellucida was left intact, or removed with pronase (pre-incubated and dialysed) and the eggs then cultured singly or as fused pairs. When pronase was used without prior incubation and dialysis, the success rate was reduced to 50 %. After transfer to uterine foster-mothers, 20–30 % of apparently normal blastocysts cultured with or without the zona, singly or fused, developed into live foetuses, compared with over 50 % of control blastocysts taken directly from the uterus. Some of the excess mortality of cultured embryos took place before implantation and some soon after. The foetuses derived from cultured blastocysts averaged 0·1 g lighter than those derived from control uterine blastocysts similarly transferred. No differences in the weights of the placentae were observed. Foetal and placental weights were unaffected by whether the eggs had been cultured singly or fused, implying that growth regulation of fused embryos is complete by the 17th day of gestation. The longer the eggs were maintained in culture, the lower was their viability after transfer, and the lighter were the foetuses derived from them.

Growth and viability of human blastocysts in vitro

2000 ◽  
Vol 8 (3) ◽  
pp. 241-287 ◽  
Author(s):  
GM Jones
Keyword(s):  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cleavage Stage ◽  
Embryo Viability ◽  
Early Cleavage ◽  
Uterine Endometrium ◽  
Stage Of Development ◽  
Uterine Lavage

The transfer of a blastocyst established the first human clinical pregnancy following in vitro fertilization (IVF). Nine years later Cohen et al. reported pregnancies resulting from the transfer of cryopreserved human blastocysts. However, it was another six years before the first report of births resulting from the transfer of human blastocysts produced in vitro appeared in the medical literature. In the intervening period clinics have opted to transfer embryos at the early cleavage stage to the uterus, despite the fact that in vivo the embryo does not enter the uterus until two to three days later at the morula to blastocyst stage of development. The viability and potential for implantation of blastocysts is high, as indicated by the finding that more than 60% of in-vivo-derived blastocysts, recovered by uterine lavage following artificial insemination of fertile donors, implant and develop into viable fetuses when transferred to recipients. This is in stark contrast to the 10–20% of in-vitro-produced embryos transferred at the early cleavage stage of development that result in a live-birth. This reduction in viability following transfer of in-vitro-derived early cleavage stage embryos may have several possible explanations: (1) a failure of implantation due to poor synchronization between the embryo and the uterine endometrium; (2) a hostile environment in the uterus for early cleavage stage embryos; (3) sub-optimal in vitro culture conditions which result in a reduction in embryo viability; (4) the assumption that all oocytes retrieved in an IVF cycle have an equal ability to develop into viable embryos; and (5) the failure to identify the most viable embryo in a cohort. Certainly, improving culture conditions and laboratory techniques for developing high quality blastocysts routinely in vitro will not only address many of the above questions but will also improve the quality and viability of earlier stages of embryo development.

73 APPLICATION OF THE HOLLOW FIBER VITRIFICATION METHOD TO THE CRYOPRESERVATION OF HIGHLY CRYOSENSITIVE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 129 ◽  
Author(s):  
A. Uchikura ◽  
H. Matsunari ◽  
K. Nakano ◽  
S. Hatae ◽  
Y. Matsumura ◽  
...  
Keyword(s):  
Early Stage ◽  
Hollow Fibre ◽  
Membrane Thickness ◽  
Early Cleavage ◽  
Diverse Species ◽  
Organ Regeneration ◽  
Cleavage Stages ◽  
Vitro Development ◽  
Technological Options

We recently demonstrated that the hollow fibre vitrification (HFV) method (Matsunari et al. 2012) could effectively be applied to the cryopreservation of embryos from diverse species. In this study, we applied the HFV method to the cryopreservation of highly cryosensitive specimens, such as in vitro matured (IVM)/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, as well as IVM/IVF-derived cattle embryos at early cleavage stages. Porcine parthenogenetic morulae (d-4) derived from IVM oocytes were treated with 0.25% pronase to remove zona pellucidae. The resulting blastomeres were isolated from the zona-free morulae by a decompaction treatment followed by gentle pipetting. Bovine IVM-IVF embryos at the 2 to 4 cell (d-1), 8 to 16 cell (d-3), and morula stages (d-5) were then subjected to vitrification. The HFV procedure was performed as described previously using 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M trehalose as cryoprotectants. Four to twenty embryos, or all of the blastomeres isolated from a single morula, were individually loaded into a cellulose acetate hollow fibre (25 mm long, 185 μm φ, 15 μm membrane thickness) and vitrified. Survival of the vitrified embryos was assessed by in vitro development to blastocysts. Blastomeres recovered after vitrification were aggregated in micro-wells to examine their ability to form blastocysts. The HFV method was demonstrated to be effective for cryopreserving zona-free in vitro-produced porcine morulae and the blastomeres isolated from them (Table 1), as well as bovine IVM-IVF embryos at early cleavage stages. These data demonstrate that the HFV method is effective for highly cryosensitive specimens, such as IVM/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, and IVM/IVF-derived cattle embryos at early cleavage stages. These achievements may expand the technological options in the production of cloned and genetically modified pigs that are useful for biomedical research. Table 1.Survival of zona-free porcine morulae and isolated blastomeres after vitrification (top) and blastocyst formation rates in bovine early-stage in vitro matured-IVF embryos after vitrification (bottom) This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, and MUIIBR.

98 EXPERIMENTAL TRANSFER OF BOVINE IVF-DERIVED 32-CELL STAGE EMBRYOS INTO THE UTERUS: ENVIRONMENTAL EFFECTS ON DEVELOPMENTAL CHARACTERISTICS

2016 ◽  
Vol 28 (2) ◽  
pp. 179
Author(s):  
M. Hoelker ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
D. Tesfaye ◽  
K. Schellander
Keyword(s):  
Culture Media ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Considerable Proportion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Rates ◽  
Uterine Environment

Usually, in vitro-produced bovine embryos are cultured in vitro in static culture systems for 7 to 9 days in media composed according the oviducal fluid although it is well accepted that around Day 4.5–5 the bovine embryo enters the uterine cavity, providing environmental conditions different from the oviduct. Therefore, one has to raise the question whether changing culture media properties after Day 5 of culture could have beneficial effects on early development of bovine embryos. To answer that question, we transferred bovine IVF derived 32-cell stage embryos into the uterine cavity of synchronized recipients. All embryos had been matured and fertilized under routine standard conditions and were cultured in synthetic oviducal fluid supplemented with essential and nonessential amino acids (SOFaa) supplemented with either 0.3% fatty acid free bovine serum albumin (BSAfaf/Uterus) or 10% serum (serum/uterus) at 38.5°C, 5% O2, and 5% CO2 in humidified air prior transfer into the uterine environment, allowing further development to the blastocyst stage within the physiological environment prior recollection at Day 7 by routine uterine flushing followed by comparison with statically in vitro-developed embryos cultured in media supplemented with serum (serum/serum group) or BSAfaf (BSAfaf/BSAfaf group). All in all, a total of 1031 in vitro-derived 32-cell stage embryos were transferred to 21 synchronized Simmental recipient heifers. Of these, a total of 680 embryos (66%) could be recollected at Day 7. Embryos of the serum/serum group reached a higher blastocyst rate compared with embryos of the BSAfaf/BSAfaf group (68% v. 41%; P < 0.05, ANOVA, Tukey test), whereas the developmental rate to the blastocyst stage did not differ after 9 days of in vitro culture, indicating higher developmental kinetics of bovine 32-cell stage embryos when culture media is supplemented with serum. Moreover, embryos of the serum/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos of the serum/serum group (12.9% v. 68.4%). Likewise, embryos in the BSAfaf/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos in the BSAfaf/BSAfaf group (16.0% v. 40.1%). When allowed to develop for additional 48h in vitro, developmental rates to the blastocyst stage at Day 9 were still higher in BSAfaf/BSAfaf treatment compared with the BSAfaf/uterus treatment (91.4% v. 74.4%) and the serum/serum treatment compared with the serum/uterus treatment (92.5% v. 56.0%). Taken together, the results of our study demonstrate that uterine transfer of bovine 32-cell stage embryos results in reduction of developmental kinetics as well as lower developmental rates compared with embryos statically cultured in vitro. That might indicate, that a considerable proportion of bovine 32-cell stage embryos might not be able to adapt to the uterine environment.

scholarly journals Progesterone and conceptus elongation in cattle: a direct effect on the embryo or an indirect effect via the endometrium?

Reproduction ◽  
2009 ◽  
Vol 138 (3) ◽  
pp. 507-517 ◽  
Author(s):  
M Clemente ◽  
J de La Fuente ◽  
T Fair ◽  
A Al Naib ◽  
A Gutierrez-Adan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Fourfold Increase ◽  
Uterine Environment ◽  
High Concentrations

The steroid hormone progesterone (P4) plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating P4 in the immediate post-conception period have been associated with an advancement of conceptus elongation, an associated increase in interferon-τ production and higher pregnancy rates in cattle. Using in vitro and in vivo models and ∼8500 bovine oocytes across six experiments, the aim of this study was to establish the route through which P4 affects bovine embryo development in vitro and in vivo. mRNA for P4 receptors was present at all stages of embryo development raising the possibility of a direct effect of P4 on the embryo. Exposure to P4 in vitro in the absence or presence of oviduct epithelial cells did not affect the proportion of embryos developing to the blastocyst stage, blastocyst cell number or the relative abundance of selected transcripts in the blastocyst. Furthermore, exposure to P4 in vitro did not affect post-hatching elongation of the embryo following transfer to synchronized recipients and recovery on Day 14. By contrast, transfer of in vitro derived blastocysts to a uterine environment previously primed by elevated P4 resulted in a fourfold increase in conceptus length on Day 14. These data provide clear evidence to support the hypothesis that P4-induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle and that, interestingly, the embryo does not need to be present during the period of high P4 in order to exhibit advanced elongation.

The Influence of the Uterine Environment on the Development of Mouse Eggs

Development ◽  
1962 ◽  
Vol 10 (4) ◽  
pp. 496-506
Author(s):  
D. R. S. Kirby
Keyword(s):  
Anterior Chamber ◽  
Abdominal Cavity ◽  
Cell Stage ◽  
Kidney Capsule ◽  
Uterine Environment ◽  
Series Of Experiments ◽  
Mouse Eggs ◽  
Uterine Development

From the literature and the results of some preliminary experiments by the author (Kirby, 1960), it appeared that the ability of mouse eggs to develop into embryos in an extra-uterine site was at least partially dependent on the stage of the egg when transplanted. In most previous studies on the extra-uterine development of mouse eggs only tubal eggs have been transplanted. Fawcett, Wislocki, & Waldo (1947) transferred 8–10-cell-stage eggs to the anterior chamber of the eye, 3 or 4 eggs being injected in each case. In another series of experiments reported in the same paper the oviducts were transected, thereby causing the descending eggs to pass into the abdominal cavity. Fawcett (1950) transplanted 2–10-cell-stage eggs to a position beneath the kidney capsule, and, to compensate for possible loss, several eggs were usually introduced. Jones 1951) transplanted 2–10-cell-stage eggs to the eye and beneath the kidney capsule.

scholarly journals Gene Expression Profile of In Vitro Produced Bovine Blastocysts Exposed to Hyperglycemia During Early Cleavage Stages: Link with the Warburg Effect.

2011 ◽  
Vol 85 (Suppl_1) ◽  
pp. 81-81 ◽  
Author(s):  
Gael L.M. Cagnone ◽  
Isabelle Dufort ◽  
Christian Vigneault ◽  
Claude Robert ◽  
Marc-Andre Sirard
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Warburg Effect ◽  
Early Cleavage ◽  
Cleavage Stages ◽  
The Warburg Effect

The role of the zona pellucida in the development of mouse eggs in vivo

Development ◽  
1970 ◽  
Vol 23 (3) ◽  
pp. 539-547
Author(s):  
Jacek A. Modliński
Keyword(s):  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Development In Vitro ◽  
Single Blastomeres ◽  
Mammalian Eggs ◽  
Mouse Eggs

Up to the present time the function and significance of the zona pellucida in the development of mammalian eggs has not been fully explained. Zona-free mouse eggs will develop in vitro from the 2-cell stage, or later, up to the blastocyst stage (Tarkowski, 1961; Mintz, 1962; Gwatkin, 1963). Single blastomeres isolated at the 2-cell (Mulnard, 1965), 4- and 8-cell stage (Tarkowski & Wróblewska, 1967) will also develop in vitro up to the blastocyst stage. Similar experiments on development in vitro of 1- and 2-cell rabbit eggs (Edwards, 1964) showed that in this species also cleavage can occur when the zona pellucida is absent, although the blastomeres exhibit a tendency to fall away from each other. Tarkowski's observations (unpublished) would appear to show, however, that naked 1-, 2- and 4-cell mouse eggs do not develop when transferred to the oviduct. A few hours after transplanting the naked eggs none could be recovered by flushing the oviduct, whereas eggs surrounded by zonae which were transplanted simultaneously were recovered.

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