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Inhibition of in vitro fertilization and early embryonic development in hamsters by gossypol.

We have previously reported an inhibitory effect of gossypol and its metabolite on bovine and mouse early embryonic development. In the present study, eggs were collected from oviducts of superovulated hamsters. Epididymal sperm were used for in vitro fertilization (IVF). Gossypol at 5, 10, and 30 micrograms/ml significantly inhibited the formation of 2 pronuclei by 45, 65 and 95%, respectively. On the first day of pregnancy, hamsters were given an intrauterine treatment of 200 micrograms of gossypol in 100 microliters of corn oil per uterine horn. On day 3, embryos from controls were in morula (65%) and early morula (17%) stages, while less than 2% of embryos from the gossypol-treated hamsters were in the morula stage. The numbers of embryo implantation sites on day 8 and pups in controls (14 +/- 2.0 and 12 +/- 1.5, respectively) were significantly higher than those in the gossypol-treated hamsters (8.5 +/- 2.0 and 4.0 +/- 1.5, respectively). Our results suggest that gossypol is able to affect fertilization, embryonic development, embryo implantation, and the number of pups in hamsters through a not-yet-defined mechanism.

Animals

Differential responsiveness of somatotrophs to growth hormone-releasing hormone and thyrotropin-releasing hormone during chicken embryonic development.

This study was designed to evaluate responsiveness and sensitivity of pituitaries from chickens to growth hormone (GH) secretagogues during late embryonic development. Anterior pituitary cells from 16-, 18- and 20-day-old chicken embryos were subjected to reverse hemolytic plaque assays (RHPAs) for GH in the presence of GH-releasing hormone (GHRH) and thyrotropin-releasing hormone (TRH). The proportion of somatotrophs detected increased between embryonic days 16 and 20, from 16 to 19.5% of all pituitary cells. Sensitivity to GHRH and TRH was similar and increased between embryonic days 16 and 20. On embryonic day 16, about 50% of somatotrophs that were not detected under basal conditions released GH in the presence of GHRH at 2 and 6 h. In contrast, only 15 and 30% of day 16 somatotrophs released GH by 2 and 6 h, respectively, following exposure to TRH. Thus, at least one of five somatotrophs responded to GHRH but not to TRH. By embryonic day 20, the proportions of somatotrophs that responded to GHRH and TRH were approximately equal at about 40%. These results provide additional evidence that GHRH and TRH may be involved in hypothalamic regulation of GH secretion during chicken embryonic development. Furthermore, it appears that full differentiation of functional chicken somatotrophs does not occur abruptly but rather gradually between embryonic days 16 and 20, during which a subpopulation of GH cells undergoes changes in sensitivity and responsiveness to TRH.

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The post-embryonic development of cell properties and synaptic drive underlying locomotor rhythm generation in Xenopus larvae.

In the first 24 h of post-embryonic development, the motor rhythm underlying swimming in Xenopus laevis tadpoles changes from brief (ca. 7 ms) ventral root discharge in each cycle to bursts of activity lasting around 20 ms (Sillar et al. 1991). Because individual motoneurons in the spinal cord of newly hatched embryos normally fire only a single impulse per cycle, two possible changes underly the transition to motor bursts seen in larval ventral roots; desynchronization of neurons in a given ventral root which continue to fire once per cycle, or the developmental acquisition of a multiple spike capability in individual motoneurons. Here we have recorded intracellularly from ventrally positioned spinal neurons, presumed to be myotomal motoneurons, in stage 37/38 embryos and 24 h later in development in stage 42 larvae. We find that (i) larval neurons are able to fire more than one impulse per cycle of fictive swimming activity; (ii) unlike in the embryo, they generally will fire multiple impulses in response to injected depolarizing current; (iii) the synaptic drive to motoneurons during swimming increases dramatically in complexity, although it still consists of alternating phases of synaptic excitation and chloride-dependent inhibition, superimposed upon tonic synaptic depolarization. The results therefore suggest a developmental change in the membrane properties of rhythmically active neurons as a major factor in the post-embryonic development of swimming in Xenopus larvae. This change appears to occur in premotor rhythm generating interneurons as well as in the motoneurons themselves and may satisfy a demand for behavioural flexibility that allows larvae to survive in a complex and changing environment.

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Silver positivity of the NORs during embryonic development of Xenopus laevis.

Transcriptional activity of ribosomal RNA (rRNA) genes is detectable around blastula-gastrula transition during the embryonic development of amphibians and other non-mammalian systems. The silver staining reaction, known to selectively stain transcriptionally active nucleolus organizer regions (NORs) both in interphase and metaphase chromosomes allowed us to follow the activation of the NORs during the embryonic development of Xenopus laevis.

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The effect of a carbon dioxide pneumoperitoneum on rabbit follicular oocytes and early embryonic development.

The effect of a carbon dioxide (CO2) pneumoperitoneum and its duration on rabbit follicular oocytes was assessed by evaluating fertilization and subsequent embryonic development rates. CO2 may cross the plasma membrane and form carbonic acid, which liberates H+, thus lowering the intracellular pH. There were no significant differences in arterial pH and [HCO3-] between CO2 and air treatment groups, whereas arterial pCO2 and pO2 were significantly increased in the CO2 treatment group. We found that the duration of pneumoperitoneum, irrespective of type of gas used, was negatively correlated with success of embryonic development. These findings necessitate that more attention be given to the gas used for creation of a pneumoperitoneum during egg retrieval for in vitro fertilization and an attempt be made to minimize duration of the pneumoperitoneum.

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Apolipoprotein E (ApoE), a Bmp-2 (bone morphogenetic protein) upregulated gene in mesenchymal progenitors (C3H10T1/2), is highly expressed in murine embryonic development.

Apolipoprotein E (ApoE) was identified as upregulated by Bmp-2 (bone morphogenetic protein-2) in the murine mesenchymal progenitor cell line C3H10T1/2 by a subtractive cloning strategy. Expression of recombinant Bmps in mesenchymal C3H10T1/2 progenitors results in the differentiation into the osteogenic, the chondrogenic, and the adipogenic lineage. In addition, ApoE is also expressed in primary osteoblasts isolated from murine calvariae late in the in vitro osteoblast developmental sequence. To infer possible roles of ApoE in organogenesis and tissue differentiation, ApoE expression during mouse embryonic development was analyzed in murine midgestation and late embryonic development by in situ hybridization. ApoE is highly expressed at many sites of organ development (liver, brain, heart, eye, lung), probably in a subset of neural crest cells and ectodermal derivatives suggestive for important functions of ApoE during embryonic differentiation and organ development.

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Dynamics of the neurosecretory cells of the brain of Amsacta collaris Hampson (Lepidoptera: Artiidae) during post embryonic development.

A comprehensive account of the dynamics of the neurosecretory cells of the brain of Amsacta collaris Hampson, during post embryonic development has been given using largely PARF, as well as PF and PAVB techniques in whole mounts sections. On the basis of staining properties the neurosecretory cells have been distinguished into principal A, B and C types. These cell types have been further classified into A-1, A-2, A-3, and A-4; B-1, B-2, and C-1, C-2 subtypes. Occurrence of paired medial, lateral, optic, posterior, ventral and tritocerebral groups containing different types of cells in the brain of Amsacta collaris in specific development stages have been reported. It has been observed that in the 1st instar larvae, there is no distinction of subtypes of cells. The distinction of cells into respective subtypes starts from 2nd instar and onward. The number of cells are minimum in the 1st instar, which increases upto the sixth instar larva, and remain almost constant in the prepupa, and diapausing pupa. The maximum number of cells have been observed in the adult individuals. In addition to number and distribution, the secretory activity of different types of cells during post embryonic development have also been described.

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[Ultrastructural study of embryonic development in Grantia compressa F. (Porifera, Calcarea)].

The embryonic development of Grantia compressa is studied by means of the electron microscope from the blastula inside the mesenchyme to the mature amphi-blastula released in the excurrent canals. The study of the different cellular categories of the embryon shows the distribution of the vitellin inclusions and their evolution. The ultrastructure of the "cellules en croix" is not in favour of a photoreceptor part.

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Metamorphosis: an exquisite model for hormonal regulation of post-embryonic development.

Metamorphosis in invertebrates and vertebrates, a process highly conserved throughout evolution, is an ideal model for studying mechanisms of post-embryonic development regulated by external signals. Amphibian metamorphosis also shares many similarities with mammalian development in the perinatal period. The precocious induction in vivo and in culture of insect and amphibian metamorphosis by exogenous thyroid hormones, and its retardation or inhibition by prolactin (PRL), have allowed the analysis of such characteristic features of post-embryonic development as morphogenesis, tissue remodelling, gene reprogramming, and programmed cell death. Recent studies on metamorphosis have revealed the important role played by such processes as auto-and cross-regulation of thyroid hormone receptor (TR) genes and by cell death or apoptosis, as in the maturation of the central nervous system, tissue restructuring and organolysis.

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Proto-oncogenes and embryonic development.

The role of proto-oncogenes in embryonic development was investigated using one of the most characterized vertebrates, the amphibian Xenopus laevis. Genes which belong to the major proto-oncogene families have been detected in Xenopus genome. The developmental control of the myc gene was assayed using a characterized Xenopus myc probe and specific antibodies. The myc gene is highly expressed as a stable maternal mRNA in oocyte, and an unfertilized egg contains 5 X 10(5)-fold the myc RNA content of a proliferative somatic cell. The myc RNA store is evenly distributed in the oocyte and the egg. Fertilization triggers a post-transcriptional control of the gene and the RNA store is progressively degraded to a constitutive value of 10 to 30 myc RNA copies registered per gastrula embryonic cell. The 62K myc protein is accumulated late in oogenesis. This uncoupling of myc expression and cell proliferation appears as a specific developmental regulation of the myc gene, adapted to the series of rapid cell cleavages occurring after fertilization.

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Na+/K+ ATPase and cell growth: effect of epidermal growth factor on the enzymatic activity in chick embryo epidermis during the embryonal development.

1. The behaviour of ATPase activity during embryonic development of chick embryo epidermis has been studied in the absence or presence of a single inoculation of EGF at the fifth day from fertilization (0-day). 2. EGF strongly decreases ATPase activity by affecting Na+/K+ ATPase. This effect occurs only if begun at 0-day. 3. This effect is due to the EGF induced decrease of -SH groups that are active part of Na+/K+ ATPase.

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A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (DeltaCbfa1) in differentiated osteoblasts only postnatally. DeltaCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. DeltaCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that DeltaCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally.

Amino Acid Sequence

[Chronology of the embryonic development of the common frog].

The tables of embryonic development of the common frog (Dabagyan, Sleptsova, 1975) have been made more precise: more precise timing of successive developmental stages (in the number of tau 0) from fertilization till hatching was provided and new drawings of the embryos during gastrulation were given.

Animals

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

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

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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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

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.

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