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A timetable of embryonic development, and ovarian and uterine changes during pregnancy, in the stripe-faced dunnart, Sminthopsis macroura (Marsupialia: Dasyuridae).

Aged stages (63) were available for establishment of a timetable of embryonic development of the stripe-faced dunnart. On Day 0 oocytes reaching maturity were found in the ovary. Within +/- 24 h of time 0 (time of minimum morning weight) polymorphonuclear leucocytes appeared and spermatozoa were last detected in the urine of 70% of females. Embryos were collected at intervals during pregnancy by hemihysterectomy and the embryos in the contralateral uterus either were examined at a later stage of pregnancy or allowed to develop to term. Cleavage to the unilaminar blastocyst stage with around 32 cells took 3 days with a cleavage arrest of 24 h at the 4-cell stage. Expansion of the unilaminar blastocyst occurred over the next 3 days. Primitive endoderm cells appeared on Day 6, fully bilaminar blastocysts by the end of Day 7 and trilaminar blastocysts on Day 8. Shell loss and implantation of 13-15-somite stage embryos occurred on Day 8 and organogenesis over the next 2-3 days. The gestation period was 9.5-12.0 days with most births occurring between 10.5 and 11.0 days. Major steps in embryonic development were correlated with stages in the development of the corpora lutea, which were maximal in size, and possibly in secretory activity, when the embryos were at the bilaminar blastocyst stage. Regression commenced when the embryos were at the primitive streak stage. At the time the corpora lutea were maximal the uterine epithelium reached its greatest height and the endometrium was thick and folded. Later in pregnancy villous-like projections of the epithelium formed, and the luminal epithelial cells became rounded. Two cell populations, a tier of 8 smaller cells above the yolk mass and a tier of 8 larger cells around the sides of the yolk mass appeared at the 16-cell stage. From the 16-cell stage to the blastocyst stage, with 150-200 cells, two cell populations distinguished by size, cell cycle time, cytoplasmic appearance and position relative to the yolk mass were present. The two populations were indistinguishable in blastocysts with greater than 200 and less than 2000 cells. They reappeared in blastocysts with greater than 2000 cells, as the darker cells of the embryoblast, and as the paler cells of the trophoblast. The darker cells lay in the yolky hemisphere and the paler cells in the non-yolky hemisphere.

Animals↗

Maspin plays an essential role in early embryonic development.

Maspin (Mp) is a member of the serpin family with inhibitory functions against cell migration, metastasis and angiogenesis. To identify its role in embryonic development in vivo, we generated maspin knockout mice by gene targeting. In this study, we showed that homozygous loss of maspin expression was lethal at the peri-implantation stage. Maspin was specifically expressed in the visceral endoderm after implantation; deletion of maspin interfered with the formation of the endodermal cell layer, thereby disrupting the morphogenesis of the epiblast. In vitro, the ICM of the Mp(-/-) blastocysts failed to grow out appropriately. Data from embryoid body formation studies indicated that the Mp(-/-) EBs had a disorganized, endodermal cell mass and lacked a basement membrane layer. We showed that the embryonic ectoderm lineage was lost in the Mp(-/-) EBs, compared with that of the Mp(+/+) EBs. Re-expression of maspin partially rescued the defects observed in the Mp(-/-) EBs, as evidenced by the appearance of ectoderm cells and a layer of endoderm cells surrounding the ectoderm. In addition, a maspin antibody specifically blocked normal EB formation, indicating that maspin controls the process through a cell surface event. Furthermore, we showed that maspin directly increased endodermal cell adhesion to laminin matrix but not to fibronectin. Mp(+/-) endodermal cells grew significantly slower than Mp(+/+) endodermal cells on laminin substrate. We conclude that deletion of maspin affects VE function by reducing cell proliferation and adhesion, thereby controlling early embryonic development.

Animals↗

Abnormal embryonic development diagnosed embryoscopically in early intrauterine deaths after in vitro fertilization: a preliminary report of 23 cases.

OBJECTIVE: To provide data about the phenotypic appearance of the embryo of early failed pregnancies after IVF. DESIGN: Clinical prospective descriptive study. SETTING: Tertiary care center. PATIENT(S): Twenty-three women who had conceived by IVF and had a missed abortion before 12 weeks of gestation. INTERVENTION(S): Embryoscopic examination of the embryo before curettage. Cytogenetic analysis of the chorionic villi by standard G-banding cytogenetic techniques or by comparative genomic hybridization in combination with flow cytometry analysis. MAIN OUTCOME MEASURE(S): Embryonic phenotype and karyotype were determined. RESULT(S): Twenty-one of 23 IVF embryos showed structural defects on embryoscopic examination. Seventeen of 23 specimens had a chromosomal abnormality. The majority were numerical aberrations such as monosomy X (2 cases). Trisomies for chromosomes 18 (one case), 16 (three cases), 15 (one case), 14 (two cases), 13 (one case), 12 (one case), 11 (one case), 10 (one case), 9 (one case), 8 (one case), and 3 (one case) were observed. A structural chromosome anomaly leading to a chromosomal trisomy was observed in one case. Aneuploidy explained the grossly abnormal embryonic development documented by embryoscopy in 15 of 21 cases. CONCLUSION(S): Aneuploidy is the major factor affecting normal embryonic development in missed abortions after IVF. Further investigation is needed to elucidate mechanisms that might prevent normal embryogenesis but evade detection by the cytogenetic techniques used in the present study.

Abortion, Missed↗

Progressive elimination of microinjected trehalose during mouse embryonic development.

Recently, sugars such as trehalose have been introduced into mammalian cells by overcoming the permeability barrier of cell membranes, and have provided improved tolerance against stresses associated with freezing and drying. However, the fate of the intracellular sugars has remained an open question. To address this issue, mouse oocytes were microinjected with 0.1 mol/l trehalose, and intracellular trehalose and glucose concentrations were determined during embryonic development using a high performance liquid chromatography and pulsed amperometric detection protocol. Trehalose was not detected in non-injected controls at any stage of development. In the microinjection group, the amount of intracellular trehalose progressively decreased as embryos developed. There was a corresponding increase in intracellular glucose concentration at the two-cell stage, suggesting cleavage of trehalose to two glucose molecules. In summary, this study presents a simple, highly sensitive protocol to determine intracellular sugars. The data reveal rapid elimination of microinjected trehalose during embryonic development. These findings have implications for designing osmolarity-optimized culture media for sugar-injected oocytes.

Animals↗

Decreased amount of ovarian tissue and maternal age affect embryonic development in old rats.

The effects of addition and/or reduction of ovarian tissue and maternal age on ovulation rates (number of corpora lutea) and embryonic development were evaluated in old, regularly cycling rats on Days 4 and 11 of gestation. Young and old control rats and old rats which were either unilaterally ovariectomized (ULO), intact with 2 additional ovaries transplanted under the kidney capsule or ULO with 2 additional ovaries transplanted under the kidney capsule were mated on proestrus of a 4- or 5-day cycle between the 3rd and 9th postoperative cycle. The percentages of normal embryos on Days 4 and 11 of gestation were decreased (P less than 0.05) in the ULO rats, while on a per ovary basis the ovulation rate and ovarian weight were significantly increased in all the ULO rats compared to the old intact rats. An increase in abnormal and retarded embryos each contributed to this decreased percentage of normal Day 4 and Day 11 embryos in the ULO rats (P less than 0.05). Transplantation of ovarian tissue into old intact and ULO rats did not affect either the ovulation rate or the percentage of normal embryos and did not reverse the detrimental effects of unilateral ovariectomy. This could be due to inadequate stimulation or function of the ovarian tissue remaining in the transplants and may arise from a smaller vascular bed and limited blood flow to the transplants. Although regularly cycling young and old control rats had similar ovulation rates, the old control animals had a decreased percentage of normal embryos on Day 11 of gestation, but not on Day 4 of gestation, compared to the young control rats. This decrease in percentage of normal Day 11 embryos in the old intact rats was due mainly to an increase in retarded rather than abnormal embryos. From this study, it is concluded that unilateral ovariectomy of old cycling rats was detrimental to embryonic development. A similar, but more gradual decrease in functional ovarian tissue with aging, could cause the increased incidence of anomalies in embryos of older females.

Aging↗

The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice.

The DNA-binding activity of AP-1 proteins is modulated, in vitro, by a posttranslational mechanism involving reduction oxidation. This mode of regulation has been proposed to control both the transcriptional activity and the oncogenic potential of Fos and Jun. Previous studies revealed that reduction of oxidized Fos and Jun by a cellular protein, Ref-1, stimulates sequence-specific AP-1 DNA-binding activity. Ref-1, a bifunctional protein, is also capable of initiating the repair of apurinic/apyrymidinic sites in damaged DNA. The relationship between the redox and DNA repair activities of Ref-1 is intriguing; both activities have been suggested to play an important role in the cellular response to oxidative stress. To investigate the physiological function of Ref-1, we used a gene targeting strategy to generate mice lacking a functional ref-1 gene. We report here that heterozygous mutant mice develop into adulthood without any apparent abnormalities. In contrast, homozygous mutant mice, lacking a functional ref-1 gene, die during embryonic development. Detailed analysis indicates that death occurs following blastocyst formation, shortly after the time of implantation. Degeneration of the mutant embryos is clearly evident at embryonic day 5.5. These findings demonstrate that Ref-1 is essential for early embryonic development.

Animals↗

Diabetes in pregnancy: uterine blood flow and embryonic development in the rat.

The uterine blood flow to individual implantation sites was evaluated in early normal and diabetic rat pregnancy, and related to maternal metabolic state, length of gestation, and embryonic outcome. The aim was to search for a possible coupling between the flow rate and embryonic development. We studied pregnant rats of a malformation-prone Sprague-Dawley strain on gestational d 9, 10, 11, and 12, a time period which roughly corresponds to postconception wk 3-6 in human gestation. The blood flow in the uterus was estimated with the aid of a microsphere technique, and the embryos were evaluated with respect to morphology and uterine position. We found increased blood flow in the uterine and decidual tissue of the pregnant diabetic animals compared with normal pregnant rats on all days studied. The blood perfusion peaked on gestational d 10, both in normal and diabetic pregnancy. The implantations tended to be fewer, whereas the resorption and malformation rates were higher, in the left horn than in the right horn. The blood flow in the uterine and decidual tissues was increased in the left horn in diabetic d 10 tissue, as well as d 12 tissues, thereby suggesting that compromised embryonic development is associated with increased rather than decreased supply of nutrients to the implantation site. These findings are in concert with previous in vitro results suggesting that enhanced oxidative stress due to increased substrate availability is an important factor in diabetic teratogenesis.

Animals↗

Effects of epidermal growth factor on early embryonic development after in vitro fertilization of oocytes collected from ewes treated with follicle stimulating hormone.

Epidermal growth factor (EGF) has been shown to enhance the in vitro rate of blastocyst formation in several species. Follicular development was induced in ewes (n=15) by twice daily administration of FSH-P on Days 13 and 14 of the estrous cycle. Cumulus oocyte complexes (COCs) were collected from all visible follicles (n=25+/-2.4/ewe) on Day 15. COCs from each ewe were cultured separately for 24h in maturation medium (containing 10% serum, LH, FSH and estradiol) with (8.2+/-0.9 per ewe) or without (7.8+/-0.8 per ewe) EGF (10 ng/ml). Oocytes were then denuded by hyaluronidase treatment, and healthy oocytes were cultured in the presence of frozen-thawed semen in synthetic oviductal fluid (SOF) medium containing 2% sheep serum. After 18-20 h, zygotes were transferred to SOF medium without glucose and cultured for about 36 h until they reached the 4-8 cell stage. Embryos were transferred to SOF medium with glucose for further development. Medium was changed every other day until blastocyst formation on Day 8 of culture (Day 1=day of fertilization). The rate of embryonic development was evaluated throughout the culture period. After maturation, cumulus cells were more expanded in the presence than in the absence of EGF. The rates of fertilization (overall 75.7+/-3.9%) and morula formation (overall 40.6+/-7.1%) were similar (P>0.05) for COCs cultured with or without EGF. However, EGF increased (P<0.01) the number of blastocysts (1.4+/-0.1 versus 0.6+/-0.2 per ewe) and tended to increase (P<0.1) the rate of blastocyst formation (21.0+/-6.6% versus 13.4+/-4.3% per ewe). These data demonstrate that EGF increases blastocyst formation in FSH-treated ewes. Therefore, EGF is recommended as a supplement to maturation medium to enhance embryonic development in vitro in FSH-treated sheep.

Animals↗

Nell-1 induces acrania-like cranioskeletal deformities during mouse embryonic development.

We previously reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with craniosynostosis (CS). Nell-1 overexpression also results in premature suture closure/craniosynostosis in newborn transgenic mice. On a cellular level, increased levels of Nell-1 induce osteoblast differentiation and apoptosis. In this report, mice over-expressing Nell-1 were examined during embryonic development as well as shortly after birth for further analysis of craniofacial defects including neural tube defects (NTDs). The results demonstrated that overexpression of Nell-1 could induce acrania at relatively late gestation stage (E15.5) in mouse embryos, through massive apoptosis in calvarial osteoblasts and neural cells. The induced apoptosis was associated with an increase in Fas and Fas-L production. In addition, transgenic E15.5 and newborn transgenic mice with the CS phenotype displayed distortion of the chondrocranium associated with premature hypertrophy and increased apoptosis of chondrocytes. These findings were also verified in vitro with primary chondrocytes transduced with AdNell-1. In conclusion, Nell-1 overexpression can induce craniofacial anomalies associated with neural tube defects during embryonic development and may involve mechanisms of massive apoptosis associated with the Fas/Fas-L signaling pathway. NELL-1: used when describing the human gene; NELL-1: used when describing the human protein; Nell-1: used when describing the rodent gene; Nell-1: used when describing the rodent protein.

Animals↗

Calcium metabolism in olive ridley turtle eggs during embryonic development.

Analyses of calcium, magnesium, sulphur, potassium and phosphorus content of the eggshell, yolk-albumen and embryos of olive ridley turtle, Lepidochelys olivacea, have been carried out at various stages of embryonic development. Calcium is the major inorganic constituent in the egg (shell and yolk-albumen) and embryos. Other elements are present either in trace or in minute trace amounts. The egg contents (yolk and albumen) provide only 40% of the embryonic calcium requirement of the hatchling. The remaining 60% is provided by the eggshell. The eggshell also undergoes a similar reduction in its calcium content from laying to hatching. Elements other than calcium present in the yolk-albumen are sufficient for normal embryonic development. The movement of calcium from the eggshell to the embryo starts at about the 40th day of development at 29.5 degrees C. Birds, turtles and crocodiles use their eggshell as the secondary source of embryonic calcium requirement. This dependence on the eggshell varies in different groups which is highest in birds and lowest in crocodiles.

Albumins↗

Comparison of embryonic development and metal contamination in several populations of the sea urchin Sphaerechinus granularis (Lamarck) exposed to anthropogenic pollution.

Over the past years the functional deficiencies observed in the sea urchin Sphaerechinus granularis from the Bay of Brest (France) have led us to select several populations within this species to assess water quality in the Bay of Brest. The present study reports on the comparison of the quality of embryonic development and of the level of metal contamination in sea urchin gonads and gut wall in four populations. Three of them lived in the Bay of Brest, and the last one was considered a reference, being settled in the Glénan Archipelago in southern Brittany (France). The sea urchin population living at the mouth of the River Aulne, at the Armorique Point, seemed to be the most affected, for it displayed the highest rate of blockage and the longest delay in embryonic development along with the highest concentrations in heavy metals, i.e., Fe, Cu, Pb, Cd, and Hg, in the gut wall and in the gonads. A comparison with previous data highlighted a degradation of health conditions in the population living at Marloux in the vicinity of Brest harbor. The comparison of the metal levels detected in individuals from these two populations with the data reported in the literature allowed us to conclude for a local contamination of the bay waters by Fe and Hg. In addition, this study also revealed that Zn and Cd concentrations were sometimes higher than the maximum values reported in the literature and evidenced an increase of contamination by Cu and Pb over the period of study; it should be noted, however, that these values were not excess levels. Our investigations also suggest that Zn, Cd, Pb, and Cu levels should be monitored in the future.

Animals↗

Embryonic development of folate binding protein-1 (Folbp1) knockout mice: Effects of the chemical form, dose, and timing of maternal folate supplementation.

Inactivation of folate binding protein-1 (Folbp1) adversely impacts murine embryonic development, as nullizygous embryos (Folbp1(-/-)) die in utero. Administration of folinic acid (N5-formyl-tetrahydrofolate) to Folbp1-deficient dams before and throughout gestation rescues the majority of embryos from premature death; however, a portion of surviving embryos develop structural malformations, including neural tube defects. We examined whether maternal supplementation with L-N5-methyl-tetrahydrofolate (L-5M-THF) has superior protective effects on embryonic development of Folbp1(-/-) fetuses compared with L-N5-formyl-tetrahydrofolate (L-5F-THF). We also examined the critical period during gestation when folate supplementation is most beneficial to the developing Folbp1(-/-) embryos. Folbp1(-/-) pups presented with a range of malformations involving the neural tube, craniofacies, eyes, and abdominal wall. The frequencies of these malformations decreased with increasing folate dose, regardless of the form used. There was no additional benefit provided by L-5M-THF compared with L-5F-THF. Despite rescuing the phenotype in Folbp1(-/-) embryos, no significant elevation of Folbp1(-/-) maternal folate levels was observed with supplementation.

Animals↗

Sequential acquisition of transcriptional control during early embryonic development in the rabbit.

Regulation of gene expression during early embryonic development in the rabbit was investigated by quantitative assay of firefly luciferase activity obtained by microinjection of three plasmid constructs using the regulatory region of polyomavirus promoter (PrPyV) with two different enhancer sequences (wild type or mutant "embryo-responsive," ER2) coupled to this reporter gene. Following injection at the 1-cell stage maximal level of expression of these genes was reached after three cell cycles. Two important regulatory steps that progressively limited gene expression were identified: the passage through the first mitosis and the transition from maternal to zygotic control of development (MZT) described at the 8- to 16-cell stage. The completion of the first mitosis was associated with the requirement of an enhancer sequence to stimulate expression of the weak PrPyV promoter while beyond the MZT, only particular enhancer sequences, such as ER2, allowed maintainance of the expression of PrPyV promoter. In addition, comparison of expression of constructs injected in pronuclei, 2-cell embryonic nuclei, and transplanted 32-cell blastomeres revealed that the nuclear environment could be a major effector in the regulation of embryonic gene expression. A schematic view is proposed describing the sequential establishment of the regulation exerted on early embryonic gene expression in progress from the onset of the zygotic genome activity to the MZT.

Animals↗

Reducing the amount of cytoplasm available for early embryonic development decreases the quality but not quantity of embryos produced by in vitro fertilization and nuclear transplantation.

The effect of reducing the amount of cytoplasm available for early embryonic development was investigated in embryos produced by in vitro fertilization (IVF) and nuclear transplantation. In Experiment 1, approximately 1/2 or 1/20 of the cytoplasm was removed from bovine embryos at the pronuclear-stage of development. The percentage of embryos developing to the compact morula or blastocyst stage was significantly higher in non-manipulated controls (26%) than in embryos with 1/20 of the cytoplasm removed (16%), and those with 1/2 of the cytoplasm removed (10%; P < 0.05). There was also a significant difference in the average number of cells between blastocysts in which 1/20 of their cytoplasm was removed (67), those with 1/2 of their cytoplasm removed (55), and nonmanipulated controls (77; P < 0.05). In Experiment 2, nuclear transfer embryos were produced in which approximately 1/2 or 1/20 of the cytoplasm was removed during oocyte enucleation. The percentage of embryos developing to the blastocyst stage was 17% for both groups of nuclear transfer embryos compared to 44% for control embryos (P < 0.05). The mean number of cells in blastocysts produced by nuclear transfer in which 1/20 of the cytoplasm was removed during oocyte enucleation (61) was no different than that in control embryos (66), but significantly higher than the mean number of cells in blastocysts produced by nuclear transfer in which 1/2 of the cytoplasm was removed (42; P < 0.05). There was no indication that altering the amount of cytoplasm available for early embryonic development of IVF embryos affected the timing of differentiation events, including those of embryo compaction and blastocyst formation.

Journal Article↗

Identification of possible mediators of embryonic mortality caused by mastitis: actions of lipopolysaccharide, prostaglandin F2alpha, and the nitric oxide generator, sodium nitroprusside dihydrate, on oocyte maturation and embryonic development in cattle.

PROBLEM: Mastitis and immunization against constituents of organisms causing mastitis can reduce fertility of cattle and sheep, respectively. For the current experiments, it was hypothesized that these effects are mediated via actions of lipopolysaccharide (LPS), prostaglandin F2alpha (PGF2), and nitric oxide on oocyte maturation and embryonic development. METHOD OF STUDY: To evaluate effects on oocyte maturation, oocytes were matured with various concentrations of LPS, PGF2alpha, or the nitric oxide (NO) generator, sodium nitroprusside (SNP). Following maturation, oocytes were fertilized and cultured until day 8 after fertilization. To test effects on embryo growth, oocytes were matured and fertilized and cultured after fertilization with LPS, PGF2alpha, or SNP. RESULTS: Addition of 100 and 1000 ng/mL LPS and 50 and 100 ng/mL PGF2alpha to oocyte maturation medium reduced the proportion of oocytes that became blastocysts at day 8 after fertilization. When added after fertilization, in contrast, neither LPS nor PGF2alpha reduced development to the blastocyst stage. Unlike for LPS and PGF2alpha, addition of SNP during oocyte maturation was without effect on the proportion of oocytes that became blastocysts at day 8 after fertilization. However, addition of 10 microM SNP to culture medium after fertilization completely prevented development to the blastocyst stage while 0.1 and 1 microM SNP did not affect development. CONCLUSIONS: Results indicate that increased local concentrations of LPS, PGF2alpha, and NO can have deleterious consequences on oocyte function (LPS, PGF2alpha) and embryonic development (NO). Thus, these molecules are putative mediators of effects of infectious disease or inflammation, including mastitis, on fertility of cattle.

Animals↗

Effect of gonadotrophin stimulation on mouse oocyte quality and subsequent embryonic development in vitro.

In-vivo-matured oocytes were collected from naturally ovulated and superovulated [pregnant mare's serum gonadotrophin (PMSG) + human chorionic gonadotrophin (HCG)] mice. Immature oocytes were retrieved from naturally cycling mice and from mice primed with PMSG. The percentages of cleavage and blastocyst formation were significantly different (P < 0.05) between in-vivo- and in-vitro-matured oocytes. Blastocyst formation rate was significantly higher (P < 0.05) in immature oocytes derived from PMSG-primed mice, and the percentages of oocytes with comet tails, and their length, were significantly higher and longer respectively in in-vitro-matured oocytes. Total cell numbers of blastocysts were also significantly different (P < 0.05) between in-vivo- and in-vitro-matured oocytes, but there were also no differences in ratio of trophectoderm (TE)/inner cell mass (ICM). In conclusion, in-vivo-matured mouse oocytes were more competent than those matured in-vitro, perhaps due to a lesser degree of DNA damage. Embryonic development capacity of in-vivo-matured oocytes is not promoted by ovarian stimulation. Gonadotrophin priming prior to immature mouse oocyte retrieval is beneficial to subsequent embryonic development.

Animals↗

Expression of reelin, the gene responsible for the reeler mutation, in embryonic development and adulthood in the mouse.

reelin has recently been isolated as a candidate gene, the mutation of which gives rise to the reeler phenotype in mice. In this study, we analyzed the expression of reelin during embryonic development in the mouse and in adult mouse tissues, by in situ hybridization. reelin transcripts were present on embryonic day (E) 8.5 in the somite, foregut, yolk sac, and unclosed neural plate. reelin was expressed in the brain, spinal cord, liver, and kidney throughout embryonic development, and transiently in many developing organs such as the optic cup, blood vessels, precartilage, stomach, pituitary, vibrissae, tooth germ, and in cells along growing nerve fibers. These observations indicate a role for reelin in development of organs in addition to that in neuronal migration. Furthermore, we demonstrated the existence of reelin mRNA and its cellular distribution in the adult brain, spinal cord, liver, kidney, testis, and ovary, suggesting additional roles for reelin in stabilizing the cyto-architecture and in remolding in adult organs. However, we detected no obvious phenotype of the reelin-expressing organs except for the brain in the reeler mouse, indicating the functional redundancy of this gene during the development of these organs.

Animals↗