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PubMed1996

Is direct cell-to-cell contact needed to improve embryonic development in co-culture?

In vitro co-culture of embryos and somatic cells is used to obtain well-developed embryos of humans and other species. However, it is not known whether direct cell-to-cell contact is essential to embryonic development. To elucidate this mechanism, we cultured mouse embryos using rabbit oviductal cell as a somatic cell. To avoid the direct contact we used a microporous membrane cell-culture insert. This cell-culture insert permits only the liquid portion of the culture medium to pass through it and was interposed between mouse embryos and cultured rabbit oviductal cells. When mouse embryos were placed on the cultured oviductal cells directly, 52.2% of two-cell-stage embryos developed to the blastocyst stage. In contrast, when the cell-culture insert was interposed between the embryos and the cultured oviductal cells, 50.0% of two-cell-stage embryos developed to that stage. There was no difference between the rate of blastocyst development in co-culture system with and without cell culture insert. Results indicate that oviductal factor (s), rather than a direct contact with oviductal cells, is essential to the enhancement of embryonic development in vitro.

Animals↗
PubMed1994

Protein phosphorylation pattern and role of products of c-erbB-1 and c-abl proto-oncogenes in murine preimplantation embryonic development.

PROBLEM: To investigate the protein phosphorylation pattern and role of products of c-erbB-1 and c-abl proto-oncogenes with known tyrosine kinase activity in preimplantation embryonic development in mice. METHOD: The protein phosphorylation pattern was studied by in vitro 32P metabolic labeling of murine ova/embryos as well as by in vitro kinase assay performed directly on various ova/embryos extracts. The role of products of c-erbB-1 (170 kDa, receptor for epidermal growth factor [EGF]) and c-abl proto-oncogenes (150 kDa) was examined by in vitro culturing murine embryos in the presence of monoclonal antibodies to respective protein products and by co-culturing with EGF, the ligand for EGF receptor (EGF-R). RESULTS: In vitro metabolic labeling of murine ova/embryos showed 32P incorporation into at least two protein bands of murine ova (M(r) 81 and 36 kDa), six protein bands of two-cell (M(r) 81, 36; and 97, 52, 22 and 19 kDa, respectively), six protein bands of morula (M(r) 81, 36; 97, 22, and 19; and 33 kDa, respectively), and eight protein bands of blastocyst (81, 36; 97, 22, 19; and 115, 58, and 15 kDa, respectively), stage embryos; there were some specific bands in each stage. Prolonged labeling from 2 to 4 h not only resulted in a relative increase in 32P incorporation into these proteins but also revealed additional bands in morula (M(r) 133 and 115 kD) and blastocyst (M(r) 49, 33, and 31 kD) stage embryos. In vitro kinase assays performed directly on various ova/embryos extracts revealed at least three phosphoproteins (M(r) 58, 36 and 33, respectively) that were common to ova, two-cell, morula, and early/late blastocyst stage embryos. Additionally, three protein bands each in murine ova and two-cell embryos (M(r) 108, 81, 73 kDa, respectively), and four protein bands of late blastocyst (M(r) 108, 73; 133 and 18 kDa, respectively) stage embryos were also revealed. Culture of two-cell embryos in the presence of EGF, the ligand for EGF-receptor, resulted in a concentration dependent increase (P < .001) in the number of cells per blastocyst. Monoclonal antibody to c-erbB-1 170 kDa protein (receptor for EGF) did not affect development of in vitro cultured murine embryos from two-cell to morula, but significantly (P < .001) inhibited the in vitro development of morula to late blastocyst stage. Monoclonal antibody to c-abl protein inhibited the development of murine embryos from two-cell to morula (P < .017), as well as, from morula to late blastocyst stage (P < .002 to .01). CONCLUSIONS: These results suggest that the stage-specific protein phosphorylation pattern and specific products of c-erB-1 and c-abl proto-oncogenes may have a role in preimplantation embryonic development in mice.

Animals↗
PubMed1977

Differentiation of primordial germ cells in the embryonic development of Thermobia domestica, Pack. (Thysanura): an ultrastructural study.

The primordial germ cells(PGCs) of Thermobia domestica undergo some morphological changes during the embryonic development. Most conspicuous are the changes in the ultrastructure of the nucleus, whose envelope shows a high degree of activity. Two types of vesicles bled off from the nucleus; the ones with the light interior are called the accessory nuclei, the others, with electron-opaque contents, have been termed the dense bodies. The nucleolus, initially clustered at the nucleus centre, undergoes dispersion and assembles again towards the end of embryonic development. At the same time, the sex differentiation of PGCs takes place. It is preceded by an increase in the activity of Golgi complexes and in the volume of lysosomes and lamellar bodies, the latter giving rise to lipid droplets. At the early stages of postembryonic development, preoogonia and prespermatogonia can readily be distinguished. Preoogonia have a wavy-surfaced nucleus and their cytoplasm contains dense bodies. In prespermatogonia, the nucleus is spherical with smooth envelope and there are no dense bodies in the cytoplasm. Throughout the period studied there occur nucleolus-like bodies and nuage material considered to be the germ-cell determinants in this species.

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PubMed1985

Embryonic development of glial cells and their junctions in the locust central nervous system.

The embryonic development of the specialized glial cells that form the perineurial blood-brain barrier in the locust CNS has been studied by freeze-fracture and tracer uptake. These cells migrate to form bracelet cell arrangements around the nervous tissues between day 4 to day 10 of embryonic differentiation which lasts 14 days in toto. A number of different kinds of intercellular junction form between the bracelet cells from day 8 to day 13 of development. These include gap junctions with features characteristic of arthropods, which seem to assemble by lateral migration of 13-nm E face intramembranous particles (IMPs), which ultimately cluster to form a large number of mature plaques of varying diameters. Less numerous are tight junctions which serve to restrict entry of exogenous molecules, including lanthanum and cationic ferritin, thereby forming the blood-brain barrier; these appear to assemble by migration of individual 8- to 10-nm P face IMPs into ridges which are found between the overlapping fingers of the perineurial bracelet cell processes. Septate junctions also mature at this stage in embryonic development by apparent assembly of IMPs into characteristic aligned rows; these may serve to slow down the entry of positively charged molecules as well as being adhesive, although anionic ferritin may leak into the CNS even after septate and tight junction formation. The observed changes in cellular associations and the formation of the blood-brain barrier coincide with the onset of mature neuronal electrical properties and spontaneous synaptic input.

Animals↗
PubMed1991

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↗
PubMed1982

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↗
PubMed1996

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↗
PubMed1995

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↗
PubMed1994

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↗
PubMed1997

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↗
PubMed1997

Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 microM in sea urchin eggs, 2 microM in Drosophila eggs, and only 0.11 microM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 microM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 microM apo-aequorin would take 50 days in zebrafish eggs, 2 days [corrected] in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 microm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.

Aequorin↗
PubMed1986

Does estradiol play a role in ovarian maturation or embryonic development of the silkworm?

Since estradiol has been detected in Bombyx ovaries effects of estradiol and other steroids on the growth and maturation of the silkworm ovary, rate of oviposition, and embryonic development were examined as a part of a study aimed at the clarification of physiological significance of estradiol in insects. These steroids were injected at various doses into the whole pupae and into the isolated pupal abdomens. No significant effect by the injections was observed on the ovarian development, as judged by increase in protein content or wet weight of ovaries and pattern of protein constituents including vitellin. However, rate of oviposition was considerably affected by the injection of estradiol at high doses. No clear effect was observed on embryonic development or determination of diapause by the injection of estradiol into the pupae. Effects of injection of anti-estrogen, nafoxidine, into the isolated abdomens and whole pupae were also examined. No effects were observed on ovarian development by injection. The relationship between physiological significance of the vertebrate steroids and metabolic activity of the ovary is discussed.

Animals↗
PubMed1991

Plasma patterns of prolactin, progesterone, and estradiol during early pregnancy in aging rats: relation to embryonic development.

Regularly cyclic, middle-aged female rats exhibit a decreased incidence of fertility, and those females that are fertile produce smaller litters. This decreased litter size is directly related to a reduced number of normal blastocysts available for implantation. Recent evidence indicates that embryonic abnormalities in middle-aged rats become apparent as early as Day 2 of pregnancy. Inasmuch as the semicircadian secretion of prolactin (PRL) is essential for the rescue of corpora lutea during early gestation and luteal production of progesterone (P) and estradiol (E2) in sufficient quantities is obligatory for embryonic development and implantation, the present study examined the profiles of plasma PRL, P, and E2 during the first 3 days of pregnancy in both young and middle-aged rats and assessed the embryonic development in these same animals. Regularly cyclic, middle-aged (9-11 mo) and young (4-5 mo) rats were cannulated via the right jugular vein on Diestrus Day 2 and mated with fertile males on proestrus. The next morning, sperm in the vaginal lavage confirmed mating, and that day was designated Day 1 of pregnancy. Beginning at 1400 h on Day 1 and continuing to 2400 h on Day 2, serial blood samples were taken at 2-h intervals for PRL assay. In the first experiment, samples were also collected at 8-h intervals during Days 1-3 for measurement of plasma P.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1976

Galactosyltransferase activities during embryonic development of chick neural tissue.

Galactosyltransferase specific activities in embryonic chick retina, optic tectum, and telencephalon were found to decline during embryonic development. Incorporation of galactose from nucleotide sugar into exogenously added glycoprotein acceptor was measured in the presence of excess of glycoprotein acceptor. This ensured that the specific activity measurements were reflections of tru enzyme specific activity rather than availability of acceptor. Moreover, we have shown that the decline in specific activity is not due to degradation of the nucleotide sugar, UDP-galactose, under our in vitro assay conditions. Enzymatic specific activity declined sharply with embryonic age for all tissues tested. This decline was not affected by the presence of 5-bromodeoxyuridine during in vitro culture of embryonic chick neural retina above that caused by the culturing alone. Galactosyltransferase activity was not found to be associated with the plasma membrane fraction from homogenized tissue but rather with the microsomal fraction. Thus, the changes in galactosyltransferase specific activity detected here do not reflect changes at the cell surface.

Animals↗
PubMed1996

The importance of cytoplasm in early embryonic development.

PURPOSE: A mouse model was used to evaluate the consequences of partial cytoplasm removal, which mimics the consequence of human early embryo fragmentation, at the one-cell stage on embryonic development to the morula and blastocyst stages. RESULTS: Opening the zona pellucida at the zygote stage has no detrimental effect on the number of differentiated or total cells at morula/blastocyst stage. However, reduced cytoplasm significantly affects the number of inner cell mass cells, 21.7 (10% cytoplasm removed) vs 27.6 (control) and 15.1 (20% cytoplasm removed) vs 24.7 (control). CONCLUSIONS: The contribution of cytoplasm of maternal origin is critical to early embryonic development.

Animals↗
PubMedDate not supplied

Effects of cyproheptadine on the rat yolk sac membrane and embryonic development in vitro.

Electron-microscopic examinations of rat embryonic yolk sacs treated in vitro with 1.5 X 10(-5) M cyproheptadine showed proliferation of the lysosomal structures; other organelles remained unaffected, and also overall yolk-sac growth and vascularization were comparable to non-treated samples. Radioactive measurements with 125I-labelled albumin showed that yolk sacs and embryos of the cyproheptadine-treated group incorporated less radioactivity than the controls. Embryos inside the yolk sacs, treated either for 24 or 48 h, were severely retarded in growth and differentiation (approximately 50% of the controls). It is suggested that the specific action of cyproheptadine on yolk-sac lysosomal structures, combined with reduced macromolecular transport, is the cause of inhibited embryonic development.

Animals↗
PubMed1983

Glycoprotein synthesis and embryonic development.

One of the most striking morphogenetic events during embryonic development is gastrulation, a process that leads to formation of the primitive gut. Using sea urchin embryos, we have studied the synthesis and function of glycoproteins during gastrulation. These studies have revealed that at least three processes are induced prior to gastrulation: de novo synthesis of dolichol; phosphorylation of dolichol by dolichol kinase, which may catalyze the final step in the de novo pathway; and initiation of the synthesis of N-linked glycoproteins. Whether or not activation of the glycosylation process results merely because of the production of dolichyl monophosphate or because, in addition, proteins containing glycosylatable-Asn-X-Ser/Thr-sequences are first translated just prior to gastrulation, is currently being investigated.

Animals↗