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PubMed1983

[Culture of mouse embryo for the toxicological evaluation of drugs in vitro. (1). Establishment of in vitro culture system and effect of 6-mercaptopurine on development of early stage embryo].

The basal conditions for culture of early mouse embryo in vitro were examined for drug evaluation. Among the media tested, BMOC-III was most suitable for mouse embryo culture. Changing the medium at 1 or 3 hr after culture was harmful to the survival and/or development of early staged embryos, whereas at 12 or 24 hr after culture, changing of the medium showed slight effect on embryo development. Eight-cell staged embryos were more resistant to changing medium that two-cell staged ones. Preliminary work using a suitable culture system established that exposure to 6-mercaptopurine for 12 or 24 hr resulted in a prolonged developmental rate at each stage. The effects showed a dose-dependent relationship with the 24 hr exposure. It was suggested that inhibition of the developmental rate during exposure resulted from a direct toxic effect on the cell and that the inhibition after exposure resulted from metabolic toxicity.

Animals↗
PubMed1980

Relation of protein synthesis in imbibing wheat embryos to the cell-free translational capacities of bulk mRNA from dry and imbibing embryos.

Dry wheat embryos were allowed to imbibe water in the presence or absence of an inhibitor of mRNA synthesis (alpha-amanitin). At each of a series of times after the onset of imbibition, newly synthesized polypeptides were labeled, isolated, and compared with those made by cell-free translation of RNA prepared from the same embryos. In the absence of alpha-amanitin, characteristic time-dependent changes in the relative proportions of many polypeptides in the electrophoretic distributions of proteins synthesized in imbibing wheat embryos could be correlated with parallel changes in the cell-free translational capacity of bulk mRNA from the same embryos. These changes were virtually eliminated when alpha-amanitin was present in the imbibing medium. The findings are consistent with the possibility that transcription of new mRNA, which begins with the onset of imbibition, is responsible for change in the electrophoretic distributions of nascent polypeptides between 40 min and 5 h postimbibition of dry wheat embryos (Cuming, A. C. & Lane, B. G (1979) Eur. J. Biochem. 99, 217-224). Allied with the principal investigation, a useful modification has been developed in order to obtain an improved field of resolution (encompassing a range of Mr values between less than 5 X 10(5) and greater than 200 X 10(3), without using a gradient in sodium dodecyl sulfate/polyacrylamide gel.

Animals↗
PubMed1991

Investigation of some antimicrobial procedures on the in vitro development of early murine embryos aimed toward developing methods for the disinfection of mammalian embryos prior to transfer.

Eight-cell, zona pellucida-intact mouse embryos were exposed to the following substances or procedures that have been reported to have germicidal effects to determine if the embryos would survive and develop under in vitro conditions: the photosensitive substances hematoporphyrin, hematoporphyrin derivative, 8-methoxypsoralen, 4,5',8-trimethylpsoralen, and thiopyronine; the enzymes lipase (0.5%), phospholipase C (2 U/ml), chymotrypsin (0.5%), and trypsin (0.5%); pH 5.0; and helium/neon laser light, visible light, ultraviolet A light, and ultraviolet C light. Under the conditions used, embryos were not adversely affected by hematoporphyrin and/or helium/neon laser light; methoxypsoralen and/or ultraviolet A light; lipase; trypsin; pH 5.0 for 20 min; and visible light. Variable results were obtained from hematoporphyrin derivative with laser light. Thiopyronine, trimethylpsoralen in combination with ultraviolet A light, and ultraviolet C light killed embryos, and chymotrypsin and phospholipase C were harmful at 10- and 15-min exposure times, respectively.

Animals↗
PubMed1984

The application of electron microscopy in the evaluation of two- to four-cell human embryos cultured in vitro for embryo transfer.

Eighteen two- to four-cell embryos, cultured in vitro for 32-65 hr after insemination, were examined by transmission electron microscopy to assess their normality and developmental potential. These stages are now being widely used for embryo transfer in in vitro fertilization clinics. They were obtained by inseminating preovulatory oocytes aspirated at laparoscopy, with or without ovarian stimulation, by methods which have yielded normal pregnancies. The organization of seven embryos was apparently normal and their blastomeres had cellular organelles usually present in fertilized ova. Details of their ultrastructure including subtle changes observed on prolonged culture are described. Other embryos showed some normal and obvious abnormal features, such as partial fragmentation and multinucleated blastomeres, or evidence of degeneration.

Blastomeres↗
PubMed2002

Eight-hydroxy-2'-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program.

OBJECTIVE: To determine the effects of oxidative stress on the quality of oocytes and embryos, 8-hydroxy-2'-deoxyguanosine (8-OHdG) in granulosa cells was quantitatively studied during an in vitro fertilization and embryo transfer (IVF-ET) program. DESIGN: Immunocytochemical staining of 8-OHdG in granulosa cells was quantitatively estimated using a charge-coupled device camera and analyzed using the National Institute of Health Image (NIH Image) freeware on a computer . SETTING: Obstetrics and gynecology department in a university hospital. PATIENT(S): Ninety-six infertile couples undergoing IVF-ET treatment and intracytoplasmic sperm injection (IVF, n = 72; intracytoplasmic sperm injection, n = 24). INTERVENTION(S): Oocytes, granulosa cells, and follicular fluids were collected 35-36 hours after the administration of hCG. MAIN OUTCOME MEASURE(S): 8-OHdG indices were obtained for mural [8-OHdG index (m)] and cumulus [8-OHdG index (c)] granulosa cells. RESULT(S): A negative correlation between the fertilization rate and both 8-OHdG indices (c and m) was found. The rate of production of good embryos also showed a negative correlation with the 8-OHdG index (m) and the 8-OHdG index (c). Negative correlations between the 8-OHdG index (c) and E2 levels in follicular fluid were observed. Endometriosis patients showed a higher 8-OHdG index (c) than did patients with other infertility causes, such as tubal, male factor, and unknown. CONCLUSION(S): Oxidative stress in granulosa cells lowered fertilization rates and subsequently led to a decrease in the quality of embryos. The quality of oocytes for endometriosis patients was impaired by the presence of 8-OHdG. This might be one causative factor in infertility in endometriosis patients.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

A comparison of in vitro fertilization results after embryo transfer after 2, 3, and 4 days of embryo culture.

Embryo transfer results after a 2 to 4 day period of embryo culture were compared. Two thousand two hundred ninety-seven ETs, performed in 1991 and 1992, were analyzed. Ongoing pregnancy rates after 2, 3, or 4 days of embryo culture were 23.3%, 21.9%, and 26.4%, respectively. Multiple pregnancy rates were 36.2%, 38.8%, and 32.6% per ongoing pregnancy for the three groups, respectively. The implantation rate of 73 cavitating morulae on day 4 was surprisingly high (41%) compared with that of other developmental stages. Transfer after 4 days of culture gives the ability to recognize embryos with a very high implantation potential.

Cells, Cultured↗
PubMed1996

Yeast infection of sperm, oocytes and embryos after intravaginal culture for embryo transfer.

OBJECTIVE: To report a case of an embryo culture infected with Candida albicans after intravaginal culture. DESIGN: Case report. SETTING: Private infertility practice and university medical center. PATIENT: A couple with tubal and male factor infertility. INTERVENTIONS: Superovulation, oocyte recovery, Percoll sperm preparation, and intravaginal culture of sperm and oocytes in a tissue culture tube. MAIN OUTCOME MEASURES: Yeast infection of sperm, oocyte, and embryo culture. RESULTS: Candida albicans infection occurred in the sperm, oocyte, and embryo culture when cultured in a sealed tube in the vagina. Candida albicans also was found in the prepared sperm suspension culture in a separate tube kept in a 37 degrees C incubator. CONCLUSIONS: Infection of the embryo culture with C. albicans probably occurred when contaminated sperm was added at the time of insemination. Sperm preparation by the Percoll gradient centrifugation failed to eliminate C. albicans in the semen.

Adult↗
PubMed2003

Plasma progesterone profiles, ovulation rate, donor embryo yield and recipient embryo survival in native Saloia sheep in the fall and spring breeding seasons.

The response to superovulatory (SOV) and estrus synchronization (ES) treatments and the fertility of donor (n=68) and recipient (n=118) Saloia ewes was evaluated in the fall and spring breeding seasons. The proportion of acyclic ewes at treatment time was significantly higher in the spring than in the fall (42.6% versus 4.0%, P<0.00001). Donors treated with eCG had a significantly higher mean number of follicles over 5mm in diameter in the ovaries at embryo recovery and a significantly lower mean efficiency of recovery than FSH-treated ewes. These negative effects were more pronounced in the fall than in the spring, which resulted in a significantly lower mean number of total and fertilized ova recovered from eCG-treated ewes, compared to FSH donors in the fall, but not in the spring. Season had no significant effect on the ovulation rate and plasma P4 concentrations of recipients treated with a progestagen plus eCG combination. Although the recipient lambing and embryo survival rates were higher in the fall than in the spring the differences were not significant. No significant differences were observed in the ovulation rate or P4 concentrations of recipients that lambed compared to those that did not lamb. These preliminary results show that, in Portugal, response of Saloia ewes to SOV or ES treatments and donor fertility following the SOV treatment were similar in the spring and the fall, which suggests that in the spring acyclic ewes are in moderate anestrus. The effect of season on fertility following embryo transfer should be confirmed in further studies involving a larger number of animals. The semilaparoscopic transfer method reported here allowed lambing and embryo survival rates higher (although not significantly) than a standard surgical approach.

Animals↗
PubMed2006

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

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.

Animals↗
PubMed2005

Single embryo transfer is an option in frozen embryo transfer.

BACKGROUND: A good strategy to decrease multiple pregnancy rate in assisted reproduction technology (ART) is the use of single embryo transfer (SET). METHODS: This retrospective study analysed 1647 frozen embryo transfers carried out during 1998-2003 in Helsinki University Central Hospital; of these, 872 were double embryo transfers (DETs) and 775 SETs. The SET group included 140 (18.1%) elective SETs (eSETs). RESULTS: The yearly rate of SETs in frozen cycles increased from 28 to 66%. Overall, the clinical pregnancy rate per frozen embryo transfer was 30.7% and the delivery rate 22.6%. The delivery rate was significantly higher in DET cryocycles than in SET cryocycles (25.7 versus 19.2%, respectively; P < 0.01). In DET cryocycles, the multiple delivery rate was 21.9%, 10 times higher than that observed in cryocycles with SET (2.0%) (P < 0.0001). When eSET was applied, no difference in delivery rate was observed when compared with cryocycles with DET (28.6 and 25.7%, respectively). CONCLUSIONS: SET can be used in frozen cycles to reduce multiple delivery rates.

Cryopreservation↗
PubMed1993

The applicability of the cumulative embryo score system for embryo selection and quality control in an in-vitro fertilization/embryo transfer programme.

The cumulative embryo score system involves three aspects of relevance in pregnancy achievement during in-vitro fertilization (IVF) and embryo transfer: cleavage rates, morphological qualities and the number of embryos transferred. The scores of 602 IVF/embryo transfer trials were calculated and analysed to determine the system's relationship to pregnancy rate, pregnancy outcome and the incidence of twin and triplet pregnancies. The system was also applied to cycles where endotoxins were either present in or absent from culture medium, in order to evaluate its validity in quality control analyses. Pregnancy rates were found to increase from 4%, with scores between 1 and 10, to 35% in the 41-50 group. The score of 20 was the criterion for separating patients into poor and good pregnancy prognosis groups (P = 0.00001). Biochemical abortions occurred more frequently with scores < 20 (P = 0.00978), but a similar relationship was not found in clinical abortion rates (P = 0.62206). Birth rates below and above a score of 20 (2.8 and 19.2%, respectively) differed significantly (P = 0.0005). The scores of twins overlapped extensively with those of singleton births, but those of all triplets were > 40. The system did not reflect a correlation between embryo quality and the presence of endotoxins in culture medium.

Culture Techniques↗
PubMed2004

Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture.

In mammals, embryonic development is more difficult to analyze than in non-mammalian species because this development occurs in utero. Interestingly, whole embryo culture allows the normal development of mouse post-implantation embryos for up to 2 days in vitro. One limitation of this technology has been the difficulty of performing loss-of-gene function studies in this system. RNA interference (RNAi), whereby double-stranded RNA molecules suppress the expression of complementary genes, has rapidly become a widely used tool for gene function analyses. We have combined the technologies of mouse whole embryo culture and RNAi to allow the molecular dissection of developmental processes. Here, we review the manipulation by topical injection followed by directional electroporation of endoribonuclease-prepared siRNA to demonstrate that this technology may be useful to knock down genes in a tissue- and region-specific manner in several organs of the developing mouse embryo.

Animals↗
PubMed1992

Factors promoting embryo implantation in in vitro fertilization and embryo transfer.

The low implantation rate after embryo transfer (ET) is the most important problem to be solved in in vitro fertilization and embryo transfer (IVF-ET). Various factors which may affect embryo implantation in the endometrium have been examined. In this study, the influence of the timing of luteinization on the establishment of pregnancy was investigated. Follicle stimulation was performed by luteinizing hormone-releasing hormone agonist, human menopausal gonadotropin and human chorionic gonadotropin (hCG). hCG was injected on the day when the mean diameter of two follicles exceeded 16 mm, and 36 h after the injection the oocyte was retrieved. Progesterone (P4) supplement was started on the day of ovum retrieval (50 mg), followed by 30 mg for 14 days. The patients were divided into three groups: (1) the pregnant patients (n = 20: group Pre); (2) the nonpregnant patients without bleeding during P4 injection (n = 35; group N), and (3) the nonpregnant patients with bleeding during P4 injection (n = 30; group B). The number of follicles that developed, endometrial thickness, the day of operation, the number of oocytes harvested, the number of good quality oocytes, the number of oocytes fertilized, the number of embryos transferred, estradiol (E2) and P4 levels, and the E2/P4 ratio were examined. The endometrial thickness of group Pre (10.9 +/- 0.6 mm; mean +/- SEM) on the day of hCG injection was significantly (p less than 0.05) greater than that of groups N (9.0 +/- 0.4 mm) and B (9.2 +/- 0.3 mm).(ABSTRACT TRUNCATED AT 250 WORDS)

Buserelin↗
PubMed1990

Antagonists of embryo-derived platelet-activating factor act by inhibiting the ability of the mouse embryo to implant.

This study utilized the transfer of preimplantation embryos to pseudo-pregnant mice to determine whether PAF-antagonists act primarily on the maternal or embryonic components of implantation. The first experiment used reciprocal embryo transfers, in which blastocysts from mice treated with PAF antagonist (SRI 63-441) or saline (controls), from Days 1 to 4 of pregnancy, were transferred to Day-3 pseudo-pregnant recipients which were also treated with SRI 63-441 or saline on Days 1-4 of pregnancy. The antagonist (40 micrograms) was administered at 16:00 h on Day 1 and at 09:00 h on Days 2-4 of pregnancy. The percentage of the transferred embryos which implanted was determined on Day 8 of pregnancy. Treatment of the recipient or the donor female with SRI 63-441 resulted in a reduction in implantation rate, from a control level of 45% to 33.8% or 34.7% (P less than 0.0002, P less than 0.007) respectively. These results suggest that the PAF antagonist affected implantation at the embryonic and maternal levels. However, when the blastocysts were transferred to Day-4 pseudopregnant recipients, treatment of the donor female had a dramatic effect on the implantation rate, resulting in a reduction of 64% (from 40% to 14.3%, P less than 0.04), while treatment of the recipient female had no significant effect. In this later experiment the transferred embryos were exposed to the recipient uterine environment for a shorter period before implantation. These results suggest that PAF antagonists affected implantation at the embryonic level and did not adversely affect maternal physiology.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Raphe of the posterior neural tube in the chick embryo: its closure and reopening as studied in living embryos with a high definition light microscope.

Chick embryos cultured on a curved substratum show a transient enlargement of the posterior neuropore (PN), mimicking the temporary delay of PN closure as seen in the curly tail (ct) mouse mutant (van Straaten et al. [1993] Development 117:1163-1172). In the present study the PN enlargement in the chick embryo was investigated further with a high definition light microscope (HDmic), allowing high resolution viewing of living embryos in vitro. The temporary PN enlargement appeared due to considerable reopening of the raphe of the posterior neural tube, which was followed by reclosure after several hours. The raphe was subsequently studied in detail. It appeared very irregular, with small zones of apposed, open and fused neural folds. During closure, these raphe features shifted posteriorly. A distinct fusion sequence between surface epithelium and neuroepithelium was not seen. During experimental reopening of the raphe in vitro, small bridges temporarily arose, broke and disappeared quickly; they likely represented the first adhesion sites between the neural folds. More prominent adhesion sites partly detached, resulting in bridging filopodia-like connections; they probably represented the first anteroposterior locations of neural fold fusion. Our observations in the living chick embryo in vitro thus show that the caudal neural tube has an irregular raphe with few adhesion sites, which can be readily reopened. As a result of the irregularity, the PN does not close zipper-like, but button-like by forming multiple closure sites.

Animals↗
PubMed2004

A pair of rosette glands in the embryo and zoeal larva of an estuarine crab Sesarma haematocheir, and classification of the tegumental glands in the embryos of other crabs.

A pair of rosette glands (one of the tegumental glands in crustaceans) is present at the root of the dorsal spine of the thorax in mature embryos of the estuarine crab Sesarma haematocheir. Each rosette gland is spherical, 45-50 microm in diameter. This gland consists of three types of cells: 18-20 secretory cells, one central cell, and one canal cell. The secretory cells are further classified into two types on the basis of the morphology of secretory granules. There are 17-19 a cells, and only one b cell per rosette gland. An a cell contains spherical secretory granules of 2-3 microm in diameter. The granules are filled with highly electron-dense materials near the nucleus but have lower electron-density near the central cell. The secretory granules contained in the b cell have an irregular shape and are 1-1.5 microm in diameter. The density of the materials in the granules is uniform throughout the cytoplasm. The secretory granules contained in both the a and b cells are produced by the rough endoplasmic reticulum. Materials in the granules are exocytotically discharged into the secretory apparatus inside the secretory cell, sent to the extracellular channels in the central cell, and secreted through the canal cell. The rosette gland can be distinguished from the epidermal cells 2 weeks after egg-laying and the gland matures just before hatching. Materials produced by this gland are secreted after hatching and secretion continues through five stages of zoeal larvae. These rosette glands were never found in the megalopal larva. Rosette glands are found in the embryos of Sesarma spp. and Uca spp. In other crabs, tegumental glands are also found at the same position as in the embryo of S. haematocheir, but the fine structure of their glands is largely different from that of the rosette gland. On the basis of the morphology of secretory cells (a-g cell types), the tegumental glands of a variety of crab embryos can be classified into four types, including rosette glands (type I-IV). The function of these tegumental glands is not yet known, but different types of the gland seem to reflect the phylogeny of the crabs rather than differences of habitat.

Animals↗
PubMed1984

Cell division in the ciliary ganglion of quail embryos in situ and after back-transplantation into the neural crest migration pathways of chick embryos.

Embryonic 4- to 15-day-old quail ciliary ganglia (CG) were grafted into the neural crest migration pathway of 2-day-old chick embryos at the adrenomedullary level of the neural axis. This back-transplantation results in dispersion of cells of the implanted ganglion, their migration in the host embryo, and subsequent promotion of their differentiation into a variety of neural-crest-derived cell types including adrenergic cells of the sympathetic ganglia and adrenal medulla. These cells can be recognized in the host through the nuclear marker that they carry. Here, we have analyzed quantitatively the expansion of CG-derived cell population after the graft, and compared cell division in CG after back-transplantation and during normal in situ development over the same period of time. Tritiated-thymidine [( 3H]TdR) incorporation showed that grafted CG cells proliferated during their migration and, to a greater extent, after they had homed to the host structures. Furthermore, proliferative activity of quail cells in the graft was found to be significantly higher than the growth rate of the CG cells in situ during the same period of development. In the quail donor embryo, the birthdate of the CG neurons occurred early in development; from 6 days onward, only nonneuronal cells were still dividing. When back-transplanted, the 4- to 5-day-old CG provided numerous quail cells located in autonomic structures of the host embryo. However, this increase of the total quail cell population and of cell division was reduced when CG were taken from quail donors at progressively later developmental stages. Postmitotic neurons from mature CG were found not to survive under the graft conditions. It is proposed that back-transplantation of the CG stimulates cell division and modifies the developmental programme of still undifferentiated precursor cells which then can give rise to a variety of cell types belonging either to the glial or the autonomic nerve and paraganglionic cell phenotypes, to the exclusion of sensory neurons which never derive from CG grafts.

Animals↗
PubMed1989

How do avian embryos breathe? Oxygen transport in the blood of early chick embryos.

1. Chick embryos with primary circulation, up to about 3 days of development, show no hemoglobin-mediated transport of oxygen. 2. In embryos with secondary circulation, between 3 and 6 days of incubation, the vascular area acts as the respiratory organ. Its efficiency in the oxygen uptake is less than that of the chorioallantois of later embryos. On the contrary, oxygen release to the tissues is highly efficient. 3. A full efficient hematic uptake of oxygen is reached at about the 6th incubation day, when chorioallantois acts as the embryonic respiratory organ. 4. The different respiratory mechanisms of developing chick embryo are closely related to the functional properties of the various hemoglobins which are produced during the embryonic life.

Animals↗