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PubMed2001

Postimplantation whole embryo culture assay for hamsters: an alternative to rat and mouse.

Postimplantation whole embryo culture (WEC) assay for rats and mice has been well established and introduced to many laboratories. Recently WEC technique for rabbits has been developed; however, information on culture of other species is very limited. Knowing the usefulness of hamsters in classical embryotoxicology, we reasoned that hamster WEC could be an alternative model for the most frequently used rat and mouse WEC. Previously we have optimized culture conditions for postimplantation hamster embryos. The aim of this study was to test the susceptibility of hamster embryos cultured in vitro to embryotoxic compounds and to compare our results with those reported by others on rat or mouse embryo culture. For that purpose we choose three known embryotoxic compounds--valproic acid, cadmium chloride, and diethylstilbestrol--and tested them using a postimplantation hamster whole embryo culture assay. Hamster embryos were cultured from 7.5 days gestation for 24 h in a medium consisting of 35% hamster serum and 65% synthetic culture medium (Iscove's or McCoy 5A). At the end of the culture period, the embryos were examined morphologically, measured with the aid of a computer image analysis system, and total protein content was assessed. All three compounds exhibited dose-related embryotoxic and teratogenic effects in hamster embryos. The malformations observed were similar to those reported on rat and mouse embryos. Comparison of the results with data reported by other authors indicates that hamster embryos cultured in vitro might be more susceptible to embryotoxic stimuli than rat and mouse embryos.

Abnormalities, Drug-Induced↗
PubMed1987

Prospects for the use of embryos in the control of disease and the transport of genotypes.

Transfer and low temperature storage of embryos are now proven techniques for a number of mammalian species. These techniques are useful in control of disease and in saving genotypes from infected animals. The place of embryos in the epidemiology of disease depends upon whether the causative organism can gain entry to the oocyte before or at fertilisation and on whether the young embryo can be invaded by organisms in the uterine environment. There is little evidence that important live-stock diseases are transmitted via gametes. The zona pellucida surrounding the embryo is an effective barrier against a number of important disease organisms; in some cases the embryo is susceptible once it has hatched from the zona pellucida. It is important therefore in considering the use of embryos in disease control, to ensure that virus is not attached to the surface of the zona pellucida from where it can infect the recipient and/or the embryo after hatching. Washing procedures have been devised together with the use of enzymes and antisera to remove virus from the surface of embryos. Some viruses enter pores and sperm tracks in the zona and removal of these may present a problem. African swine fever virus has been shown to resist removal by treatment with enzymes. There are no guidelines as to the likely interaction between a certain virus and embryos. Therefore each virus of interest must be tested to determine whether it can be transmitted via washed embryos. Nevertheless there are numerous instances of the use of embryo transfer to eradicate a specific disease or to save valuable genetic material from infected animals without transmitting disease.

Animal Diseases↗
PubMed1989

Adrenergic regulation of calcium-deficient hypertension in chick embryos.

Chick embryos rendered calcium deficient by long-term culture outside the eggshell develop hypertension and tachycardia (R. Tuan and H. Q. Nguyen, J. Exp. Med. 165: 1418-1423, 1987). To characterize the hypertension of cultured shell-less (SL) chick embryo, we have compared their cardiovascular response to adrenergic drugs with normal (NL) embryos at day 14 of incubation. Blood pressure and pulse rate were measured before and after administration of norepinephrine (NE), phentolamine (PA), isoproterenol (IP), and propranolol (PP). Base-line blood pressures (systolic and diastolic) were uniformly higher in SL than NL embryos. In both embryos, blood pressure was elevated by NE and PP but lowered by IP and PA; pulse rates were decreased by PP and PA and increased by NE and IP. Pharmacological sensitivity to NE and PA, based on their effective dosages and magnitude of response in blood pressure and pulse rate, was higher in SL embryos. On the other hand, the embryos did not differ significantly in their sensitivity to IP and PP. The plasma concentration of catecholamines was considerably higher in SL embryos. Although serum calcium was lower in SL embryos, myocardial calcium content was not significantly different. Thus the cardiovascular function of SL embryos is likely to be under more sensitive and potentiated alpha-adrenergic regulation, perhaps a result of different calcium handling by several tissues (e.g., myocardium). These properties of the SL chick embryo indicate that it is a novel and useful experimental system to study the relationship between calcium homeostasis and development of hypertension.

Animals↗
PubMed2004

Chromosomal constitution of embryos derived from tripronuclear zygotes studied by fluorescence in situ hybridization using probes for chromosomes 4, 13, 18, 21, X, and Y.

This study was designed to assess the chromosomal constitution and segregation patterns of cleaving embryos derived from tripronuclear zygotes. Thirty-two embryos obtained from 19 conventional IVF patients were analyzed by fluorescence in situ hybridization (FISH) using probes for chromosomes 4, 13, 18, 21, X, and Y. Sixteen embryos (50.0%) exhibited uniform, non-mosaic patterns. These embryos showed pure triploid (n = 7), pure diploid (n = 7), or pure haploid (n = 2). The remaining 16 embryos showed mosaic patterns; 1 was triploid-diploid mosaics, 9 were diploid-haploid, and 4 were haploid only. Autosomal aneuploidy occurred in 2 embryos showing a triploid complement. The sex chromosomal constituent XXX:XXY:XYY was 3:4:2 in embryos showing a pure triploid complement (including 2 embryos with aneuploidy). This ratio was not significantly different from the expected 1:2:1 (p = 0.96). Pure triploid was found in 41.7% of 2-cell embryos, but no triploid complement was found in 3-cell embryos. The present study also supports the diandric origin of tripronuclear zygotes in the conventional IVF, and, to our knowledge, is the first study to use simultaneous six-color FISH for chromosomes 4, 13, 18, 21, X, and Y in human embryos. However, no additive information was obtained about chromosome 4.

Chromosome Aberrations↗
PubMed1989

Analysis of the sex-chromosome constitution of digynic triploid mouse embryos.

LT/Sv strain mice regularly ovulate up to 50% of their eggs as primary oocytes, which are fertilisable and give rise to digynic triploid embryos. A similar number of eggs are ovulated as secondary oocytes and, following fertilisation, give rise to normal diploid embryos. Pregnant LT/Sv females were autopsied at about midday on day 10 of gestation, when normal diploid embryos would be expected to possess between 25 and 30 pairs of somites. While a few of the triploid embryos either consisted of disorganised embryonic masses or were resorbing, most were at readily recognisable embryonic stages. Just over half of the embryos recovered were "unturned," while the remainder had "turned" and possessed between 15 and 25 pairs of somites. The triploids were usually readily recognised, owing to their small size and because they often displayed neural tube and cardiac defects. All of the embryos recovered were analysed cytogenetically by G-banding to establish their ploidy and sex-chromosome constitution. The XY:XX sex ratio of the 105 diploid embryos recovered, all of which had "turned," was 1.06:1, while the overall XXY:XXX sex ratio of the 120 triploids was 1:1. Analysis of only the developmentally most advanced triploid embryos (i.e., the 49 that had "turned") revealed that the XXY:XXX sex ratio in this group was 1.13:1, which was not significantly different from the expected ratio of 1:1. The crown-rump lengths of the XY and XX "turned" embryos were almost identical, as were those of the XXY and XXX "turned" embryos, although the triploids were significantly smaller than the diploids. No obvious effect of sex-chromosome constitution on developmental potential was therefore observed in this study in relation to either the digynic triploid or the control diploid embryos.

Animals↗
PubMed1990

Sex-chromosome constitution of postimplantation tetraploid mouse embryos.

Tetraploid mouse embryos were produced at the two-cell stage by blastomere fusion induced by inactivated Sendai virus. The embryos were from chromosomally normal female mice that had been fertilised by homozygous Rb(1.3)1Bnr males carrying a pair of large metacentric marker chromosomes in their karyotype. These "reconstructed" one-cell tetraploid embryos were then transferred to the oviducts of pseudopregnant recipients, which were subsequently autopsied early on the 10th day of gestation. Two-cell stage embryos that did not undergo blastomere fusion after 4-5 h were transferred to a second group of recipients, which were also autopsied early on the 10th day of gestation. From a total of 153 tetraploid embryos transferred to females that subsequently became pregnant, 135 implanted. Sixty-eight implantation sites were found to contain resorptions, whereas 67 contained mostly headfold presomite-stage embryos. Four embryos possessed four to six pairs of somites. All 57 embryos that could be analysed cytogenetically were found to be tetraploid. G-banding analysis revealed that 30 of these embryos had an XXYY and 27 and XXXX sex-chromosome constitution. The presence of two marker chromosomes in all mitotic preparations from each of these tetraploid embryos confirmed that they had all been produced by duplication of their original XY or XX diploid chromosome constitution, respectively. The XXYY:XXXX sex ratio observed was not significantly different from unity. In the control series of transfers, all of the embryos recovered were at the forelimb bud stage and had a diploid chromosome constitution. The results reported here differ from human clinical findings, in which the XXYY:XXXX sex ratio of 120 human tetraploid spontaneous abortions recovered over the last 20 years is 45:75. Possible explanations for these differences are briefly discussed.

Animals↗
PubMed1986

Hemodynamic changes in chick embryos precede heart defects after cardiac neural crest ablation.

Neural crest cells are known to contribute to the normal architecture of the heart and aortic arch arteries. Ablation of neural crest cells over somites 1 to 3 in the chick embryo prevents conotruncal septation and results in persistent truncus arteriosus. To determine whether a deficiency of cardiac neural crest cells produces hemodynamic changes prior to the development of identifiable structural defects in the heart, we measured dorsal aortic blood velocity and vitelline artery blood pressure in lesioned and control embryos at a period of cardiac morphogenesis prior to septal formation. The internal diameter of the dorsal aorta at the level of the sinus venosus and the internal diameter of the aortic arch arteries at their midpoints were measured in embryos at Stage 18 of development using a filar micrometer eyepiece and a dissecting microscope. Embryos with neural crest lesions had significantly greater dorsal aortic blood flow velocity than control embryos. In addition, embryos lacking cardiac neural crest had significantly lower systolic and diastolic blood pressures than control embryos. There was no difference in heart rate, dorsal aortic diameter or internal diameter of the aortic arch arteries between lesioned and control embryos. Scanning electron micrographs revealed no gross morphological differences in cardiac looping or conotruncal wall development between lesioned and control embryos; however, embryos with cardiac neural crest ablations developed markedly hypoplastic 4th pharyngeal arches. This data suggests that hemodynamic changes precede the onset of structural heart defects in embryos with cardiac neural crest ablations.

Animals↗
PubMed2005

Expression of plasminogen activators in preimplantation rat embryos developed in vivo and in vitro.

BACKGROUND: Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs) have been implicated in mammalian fertilization, early stages of development and embryo implantation. The invasion of trophoblast cells into the endometrium during the implantation process can be blocked by inhibitors of serine proteases, illustrating the role of these enzymes in the invasion process. As in vitro developing embryos resulted in lower implantation rate than those developed in vivo we assume that a reduced PAs activity may lead to it. There is hardly any information regarding qualitative or quantitative differences in expression of PAs in preimplantation embryos, or comparisons between in vivo and in vitro developed embryos. The purpose of this study was to assess the expression of urokinase type (uPA) and tissue type (tPA) plasminogen activators in in vivo and in vitro preimplantation development in rat embryos using immunofluorescence confocal microscopy and computerized image analysis. METHODS: Zygotes, 2-cell, 4-cell, 8-cell, morula and blastocyst stages of development were flushed from the reproductive tract (control groups) of Wistar rats. Zygotes were flushed and grown in vitro to the above mentioned developmental stages and comprised the experimental groups. Immunofluorescence microscopy and computerized image analysis were used to evaluate both qualitative (localization) and quantitative expression of plasminogen activators. RESULTS: uPA and tPA were found to be expressed in rat embryos throughout their preimplantation development, both in vivo and in vitro. While uPA was localized mainly in the cell cytoplasm, the tPA was detected mainly on cell surface and in the perivitelline space. In blastocysts, both in vivo and in vitro, uPA and tPA were localized in the trophectoderm cells. Total uPA content per embryo was higher in the in vivo as compared with the in vitro developed embryos at all stages measured. Blastocyst uPA content was significantly low as compared with the four-cell, eight-cell, and morula stages. Total tPA content was higher in embryos developed in vivo than those developed in vitro except for the 4-cell and 8-cell stages. CONCLUSION: In vitro embryo development leads to lower PAs expression in a stage dependent manner as compared with in vivo developing controls. The enzymes studied vary probably in the ratio of their active and inactive forms as there is no correlation between their content and the activity observed in our previous study. The localization of both PAs in the blastocysts' trophectoderm supports the assumption that PAs plays a role in the implantation process in rats.

Animals↗
PubMed2005

Demilune cell and parotid protein from murine oviductal epithelium stimulates preimplantation embryo development.

In mammals, fertilization and early preimplantation embryo development occur in the oviduct. We hypothesized that interaction exists between the developing embryos and the maternal genital tract, such that the embryos modulate the physiology and gene expression of the oviduct so that it is conducive to their development. By comparing the gene expression patterns in mouse oviducts containing transferred preimplantation embryos with those of oviducts containing oocytes, we report here the characterization of demilune cell and parotid protein (Dcpp), which was up-regulated in the embryo-containing oviduct. Dcpp mRNA was highly expressed in the oviductal epithelium at the estrus stage. The Dcpp gene codes for a protein of 150 amino acids and contains a signal peptide suggestive of secretory function. The Dcpp mRNA level was maintained in the oviductal epithelium of pregnant females but decreased continuously in those of pseudopregnant mice. Exogenous estrogen stimulated the expression of Dcpp mRNA and protein in ovariectomized mice. The effect was abolished by an estrogen antagonist, ICI 182,780. Dcpp protein was present in mouse oviductal fluid but not in uterine fluid. More importantly, Dcpp immunoreactivity was found in embryos recovered from the oviduct but not in mature oocytes from the ovary. Supplementation of Dcpp to culture medium stimulated the development of mouse embryos to the blastocyst stage. Anti-Dcpp antibody decreased the beneficial effect of Dcpp on implantation of two-cell mouse embryos transferred to the oviducts of the foster mothers. In summary, our data demonstrated that Dcpp is highly expressed in the oviductal lumen in the presence of preimplantation embryos. It stimulates the growth of preimplantation embryos and may play an important role in embryo-maternal dialogue.

Animals↗
PubMed1980

Changes in hepatic iodothyronine metabolism during ontogeny of the chick embryo.

As a model of the changes in peripheral iodothyronine metabolism that occur during ontogeny, we have studied the metabolism of 125I-labeled T4 and several of its partially deiodinated derivatives by the liver of the chick embryo. Homogenates of livers obtained from chicks varying in embryonic age from 8 days to the time of hatching (20-21 days) were incubated with various iodothyronines, all labeled with 125I in their outer or phenolic ring. Rates and products of the reactions were analyzed by paper chromatography of whole homogenates. In livers from embryos of all ages studied, the addition of dithiothreitol (DTT; 2 mM) enhanced the rate of metabolism of the iodothyronines T4, T3, and rT3. Marked age-related changes in the metabolism of the iodothyronines were apparent; these were evident in the absence of DTT, but were most clearly seen in specimens to which DTT had been added. rT3 was degraded very rapidly, even in livers from 8-day-old embryos, but its rate of degradation increased progressively with increasing age of the embryo up to the time of hatching. Outer ring (5'-) monodeiodination, giving rise to 3,3'-diiodothyronine (3,3'-T2), was the predominant, and perhaps the sole, pathway of rT3 metabolism throughout this period of embryogenesis. Age-related changes in the metabolism of T4 and T3 were similar to one another, but differed greatly from those seen in the case of rT3. In specimens enriched with DTT, the rates of metabolism of T4 and T3 were moderately rapid in livers from 12-day-old embryos and then increased abruptly, remaining very rapid in livers from embryos through 18 days of age. Rapid degradation of T4 was not due to 5'-monodeiodination, since very little T3 was generated from T4. In addition, since results obtained were similar during incubations under air and N2, oxidative degradation of T4 was apparently not important. Rather, during this period, inner ring (5-) monodeiodination appeared to be by far the predominmant pathway of metabolism of both T4 and T3, leading to the formation of rT3 from T4 and 3,3'-T2 from T3. In livers from 19- and 20 day-old embryos, the latter obtained just before hatching, the overall metabolism of both T4 and T3 slowed abruptly and progressively owing to a decrease in the rate of 5-monodeiodination. Concomitantly, 5'-monodeiodination of T3 and T4 became more prominent, leading to increased generation of T3 from T4. These maturational changes in T4 and T3 metabolism coincided in time with penetration of the air sac by the embryo's beak and initiation of air breathing, a process termed internal pipping. Premature maturational changes in T4 and T3 metabolism, very similar to those that occurred spontaneously in 19- and especially 20-day-old embryos, were induced within 2 days by the single injection of 200 microgram hydrocortisone onto the allantoic membrane of immature embryos. It is concluded that hepatic iodothyronine metabolism in the immature embryo is directed so as to prevent the accumulation of T3 derived from T4...

Animals↗
PubMed1992

Commitment along the dorsoventral axis of the sea urchin embryo is altered in response to NiCl2.

Few treatments are known that perturb the dorsoventral axis of the sea urchin embryo. We report here that the dorsoventral polarity of the sea urchin embryo can be disrupted by treatment of embryos with NiCl2. Lytechinus variegatus embryos treated with 0.5 mM NiCl2 from fertilization until the early gastrula stage appear morphologically normal until the midgastrula stage, when they fail to acquire the overt dorsoventral polarity characteristic of untreated siblings. The ectoderm of normal embryos possesses two ventrolateral thickenings just above the vegetal plate region. In nickel-treated embryos, these become expanded as a circumferential belt around the vegetal plate. The ectoderm just ventral to the animal pole normally invaginates to form a stomodeum, which then fuses with the tip of the archenteron to produce the mouth. In nickel-treated embryos, the stomodeal invagination is expanded to become a circumferential constriction, and it eventually pinches off as the tip of the archenteron fuses with it to produce a mouth. Primary mesenchyme cells form a ring in the lateral ectoderm, but as many as a dozen spicule rudiments can form in a radial pattern. Dorsoventral differentiation of ectodermal tissues is profoundly perturbed: nickel-treated embryos underexpress transcripts of the dorsal (aboral) gene LvS1, they overexpress the ventral (oral) ectodermal gene product, EctoV, and the ciliated band is shifted to the vegetal margin of the embryo. Although some dorsoventral abnormalities are observed, animal-vegetal differentiation of the archenteron and associated structures seems largely normal, based on the localization of region-specific gene products. Gross differentiation of primary mesenchyme cells seems unaffected, since nickel-treated embryos possess the normal number of these cells. Furthermore, when all primary mesenchyme cells are removed from nickel-treated embryos, some secondary mesenchyme cells undergo the process of "conversion" (Ettensohn, C. A. and McClay, D. R. (1988) Dev. Biol. 125, 396-409), migrating to sites where the larval skeleton would ordinarily form and subsequently producing spicule rudiments. However, the skeletal pattern formed by the converted cells is completely radialized. Our data suggest that a major effect of NiCl2 is to alter commitment of ectodermal cells along the dorsoventral axis. Among the consequences appears to be a disruption of pattern formation by mesenchyme cells.

Animals↗
PubMed2000

Interruption of cardiac output does not affect short-term growth and metabolic rate in day 3 and 4 chick embryos.

The heart beat of vertebrate embryos has been assumed to begin when convective bulk transport by blood takes over from transport by simple diffusion. To test this hypothesis, we measured eye growth, cervical flexure and rates of oxygen consumption ( V(O2)) in day 3-4 chick embryos denied cardiac output by ligation of the outflow tract and compared them with those of embryos with an intact cardiovascular system. Eye diameter, used as the index for embryonic growth, increased at a rate of approximately 4.5-5 % h(-)(1) during the observation period. There was no significant difference (P>0.1) in the rate of increase in eye diameter between control (egg opened), sham-ligated (ligature present but not tied) and ligated embryos. Similarly, the normal progression of cervical flexure was not significantly altered by ligation (P>0.1). V(O2) (ml O(2 )g(-)(1 )h(-)(1)) at 38 degrees C, measured by closed respirometry, was not significantly different (P>0.1) on day 3 in sham-ligated (14.5+/-1.9 ml O(2 )g(-)(1 )h(-)(1)) and ligated 17.6+/-1.8 ml O(2 )g(-)(1 )h(-)(1)) embryos. Similarly, on day 4, V(O2) in sham-ligated and ligated embryos was statistically the same (sham-ligated 10. 5+/-2.9 ml O(2 )g(-)(1 )h(-)(1); ligated 9.7+/-2.9 ml O(2 )g(-)(1 )h(-)(1)). Expressed as a linear function of body mass (M), V(O2) in sham-ligated embryos was described by the equation V(O2)=-0.48M+24.06 (r(2)=0.36, N=18, P<0.01), while V(O2) in ligated embryos was described by the equation V(O2)=-0.53M+23.32 (r(2)=0.38, N=16, P<0.01). The regression line describing the relationship between body mass and V(O2) for pooled sham-ligated and ligated embryos (the two populations being statistically identical) was V(O2)=-0.47M+23.24. The slope of this regression line, which was significantly different from zero (r(2)=0.30, N=34, P<0.01), was similar to slopes calculated from previous studies over the same range of body mass.Collectively, these data indicate that growth and V(O2) are not dependent upon cardiac output and the convective blood flow it generates. Thus, early chick embryos join those of the zebrafish, clawed frog and axolotl in developing a heart beat and blood flow hours or days before required for convective oxygen and nutrient transport. We speculate that angiogenesis is the most likely role for the early development of a heart beat in vertebrate embryos.

Animals↗
PubMed2006

Rat embryo quality and production efficiency are dependent on gonadotrophin dose in superovulatory treatments.

The present study was performed to optimize a superovulation protocol in rats in order to produce a large number of good-quality embryos suitable to develop rat embryonic stem (rES) cells. We first evaluated the ovulation kinetics of three rat strains: Wistar, Fisher and ACI/N. Animals (n=30 per strain) were treated with 50 IU of pregnant mare serum gonadotrophin (PMSG), and ovulation was induced with 50 IU of human chorionic gonadotrophin (hCG) 50 h apart. Next, we evaluated the dose-response curves of PMSG and hCG in Wistar rats in order to obtain the highest number of embryos. The parameters evaluated for superovulation efficiency were: percentage of mated females, percentage of pregnant females and the average number of embryos collected per female. The results of these experiments suggested that the best dose combination was 50 IU for each hormone. Subsequent experiments, again with Wistar rats, were designed to test which of four hormonal combination treatments (30/30, 30/50, 50/30, and 50/50 IU of PMSG/hCG) will produce the largest numbers of good-quality embryos. Embryo quality was evaluated by embryo development uniformity, embryo morphology, embryo survival in an in vitro culture and embryo ability to generate rES-like cells. Results from these experiments showed that 30/50 IU of PMSG/hCG was the treatment that induced the best embryo quality. In conclusion, our results indicated that, in Wistar rats, the most appropriate hormonal combination dose for superovulation protocols with high number of good-quality embryos was 30 IU of PMSG and 50 IU of hCG given 50 h apart. We are performing further studies with rES-like cells produced with the present methodology to evaluate if they are able to participate in the production of germ-line chimeras.

Animal Husbandry↗
PubMed1983

Superovulation treatments and embryo transfer in Angora goats.

A high incidence of early luteal regression after PMSG superovulation was associated with low recovery of embryos from reproductive tracts of Angora goats flushed later than Day 5 after onset of oestrus. Embryos were successfully recovered (mean 7.9/female) by flushing on Days 2-5. Mean ovulation rate after an FSH regimen (16.1 +/- 0.8) was significantly higher than that after a single injection of PMSG (10.8 +/- 1.2). Fertilization rate and survival of embryos following transfer to naturally synchronized recipient feral goats did not differ between the two gonadotrophin regimens: the mean number of kids born to 47 donors treated with FSH (7.5 +/- 0.6) was significantly greater than that to 28 donors treated with PMSG (4.8 +/- 0.6). Irrespective of hormonal treatment, the numbers of embryos recovered and of kids born were correlated with ovulation rate (r = 0.82, P less than 0.001 for both). Embryo survival was influenced by ovulation rate in recipients, with 52%, 63% and 75% of transferred embryos being carried to term by recipients with 1,2 and 3 CL, respectively (P less than 0.01). More embryos survived (65%) when 2 embryos were transferred to each recipient than when 1 (51%) or 3 (48%) were transferred. In recipients receiving 2 embryos, survival was significantly improved by transfer of both embryos to the same oviduct (70%) than when one was transferred to each oviduct (62%). The percentage survival of embryos was optimal when oestrus of recipients was synchronized within +/- 1 day of oestrus in donors.

Animals↗
PubMed1983

Evidence for maternal control of blastocyst growth after asynchronous transfer of embryos to the uterus of the ewe.

The fate of embryos transferred asynchronously in the ewe was investigated when the functional life of the corpus luteum was prolonged by both hemi-hysterectomy and by the presence of a second synchronously transferred embryo. The development of asynchronously transferred embryos was assessed at progressively later stages after transfer. Prolongation of luteal function did not enable asynchronously transferred embryos to persist. Embryos from Day 4 donors were found to be retarded in their rate of development when placed in 'younger' Day 1 or 2 uteri and appeared unable to develop beyond the early blastocyst stage. Conversely, embryos from Day 4 donors placed in 'older' Day 6 or 7 uteri showed accelerated growth and development which was maintained until the uterus reached Day 12. Thereafter further growth of the asynchronously transferred embryos was retarded, although synchronously transferred embryos then entered the phase of rapid blastodermic vesicle elongation. Asynchronously transferred embryos disappeared from the uterus when the ewe entered pro-oestrus. The experiments demonstrate the existence of an active relationship between the embryo and the maternal environment during mid-cycle and an apparent lack of association between embryo size, growth rate and physiological maturation.

Animals↗
PubMed1990

Control of first cleavage in single-cell reconstituted mouse embryos.

Karyoplasts derived from mouse embryos at the initial and final stages of the first or second mitotic interphase were fused to early and late enucleated 1-cell embryos. The time of cleavage of reconstituted and control embryos was recorded at 1-h or 8-h intervals after manipulation. This enabled assessment of nuclear and cytoplasmic control over the mitotic apparatus of the 1-cell embryo. Early nuclei from 1- or 2-cell embryos fused to late enucleated embryos delayed cleavage but for only a few hours. However, late nuclei fused to early enucleated embryos were unable to advance the cytoplasmic timing of the next cleavage division. Furthermore, these reconstituted embryos stayed in interphase longer than did controls and many embryos with nuclei derived from late 2-cell embryos failed to cleave. These findings suggest that, allowing for a short period, early nuclei can synchronize with late cytoplasm with no major damage to the cleavage apparatus. It is proposed that this period is required for the completion of DNA synthesis by the early nuclei. However, late nuclei cannot induce mitosis before the expected cytoplasmic time, and, with 2-cell karyoplasts, this interaction causes many embryos to 'block' in interphase, without cleaving, suggesting incompatible nucleo-cytoplasmic interactions between late 2-cell karyoplast and early 1-cell stage cytoplasm.

Animals↗
PubMed1994

Effects of healthy pseudopregnant milieu on development of two-cell subdiabetic mouse embryos.

Female mice were injected with a single dose of streptozotocin (65 mg kg-1) 14-17 days before fertilization to investigate the significance of impaired insulin secretion induced by subdiabetic streptozotocin treatment on preimplantation embryo development. Subdiabetic mice (streptozotocin-treated) had significantly different glucose tolerance from that of control animals, despite similar basal glycaemia. Morphological analysis of preimplantation embryos collected on day 2 of pregnancy revealed no significant changes in the number of two-cell embryos recovered from streptozotocin-treated females compared with controls. Two-cell embryos were transferred into the oviducts of healthy, synchronous pseudopregnant females and recovered 24-28 h later. Morphological evaluation revealed a significantly greater percentage of degenerated embryos from streptozotocin-treated females than from control females. Morphological analysis of preimplantation embryos collected on day 2.5 of pregnancy revealed no significant changes in the number of two- to four-cell embryos recovered from streptozotocin-treated females compared with controls, but there was a significant increase in the number of degenerated embryos in streptozotocin-treated females that did not receive insulin therapy. Insulin (1-1.5 iu per 100 g) administered twice a day to streptozotocin-treated mice significantly improved the altered development of embryos in both experiments. It is possible that the impaired insulin secretion in female mice adversely affected the growth of preimplantation embryos. Almost half of the morphologically normal two-cell embryos isolated from subdiabetic females were incapable of development to the eight-cell stage even in a non-diabetic maternal environment. the morphologically distinct degenerative changes were first detected at the time of the second mitotic cleavage.

Animals↗
PubMed2005

Porcine embryo development and fragmentation and their relation to apoptotic markers: a cinematographic and confocal laser scanning microscopic study.

Porcine embryo selection prior to transfer is mainly influenced by morphological criteria. However, the relationship between embryonic morphology, developmental potential and cell death by apoptosis in porcine embryos is still unclear. The aim of this study was to establish embryo quality parameters for in vivo fertilised porcine embryos based on timing of development in vitro, embryo morphology and the presence of apoptosis. The kinetics of development and morphological parameters were investigated in a time-lapse cinematographic experiment. Possible links between embryo morphology and apoptosis were examined via a confocal laser scanning experiment, analysing nuclear changes, annexin V and terminal dUTP nick-end labelling. The timing of early cleavages was firmly linked to embryo developmental competence in vitro. Attainment of at least the 5-cell stage before 77 h post insemination and attainment of the morula stage before 102 h post insemination significantly increased the odds for reaching the early blastocyst stage. Overall, a negative effect of fragmentation percentage and fragmentation pattern on subsequent embryonic development was observed, but the developmental potential of embryos experiencing slight fragmentation (0-5%) was not different from embryos without fragmentation. Correlations detected between developmental arrest and fragmentation, and fragmentation and apoptosis were 0.60 and 0.87 (P < 0.05) respectively. Only a minority of the embryos arrested between the 1- and 4-cell stage displayed biochemical characteristics of apoptosis. Consequently, a significant correlation (0.57) between developmental arrest and apoptosis could only be established for embryos arrested after embryonic genome activation.

Animals↗