Proliferation Of Embryonic Cells Research Articles

Effect of static magnetic field on development toxicity of rat embryonic midbrain neurons cells

To explore the effect of static magnetic field (SMF) on the differentiation and proliferation of rat embryonic midbrain neurons cells. The micromass culture of rat embryonic midbrain neurons cell was applied to study the effect of varieties of SMF (1.0, 10.0, 50.0, 100.0, 200.0 mT) and FACS. SMF inhibited the differentiation of the cell without affecting cell proliferation. The concentration of 50% inhibition of cell differentiation (ICD(50)) was 25 mT. The concentration of 50% inhibition of cell differentiation (IVD(50)) was 45 mT. The inhibition of SMF on embryonic midbrain neurons cells may be associated with impact of protein syntheses, and lipid peroxidation.

Effect of recombinant porcine IGFBP-3 on IGF-I and long-R3-IGF-I-stimulated proliferation and differentiation of L6 myogenic cells.

Insulin-like growth factor (IGF)-I stimulates both proliferation and differentiation of myogenic precursor cells. In vivo, IGFs are bound to one of the members of a family of six high-affinity IGF binding proteins (IGFBP 1-6) that regulate their biological activity. One of these binding proteins, IGFBP-3, affects cell proliferation via both IGF-dependent and IGF-independent mechanisms and it has generally been shown to suppress proliferation of cultured cells; however, it also may stimulate proliferation depending upon the cell type and the assay conditions. Cultured porcine embryonic myogenic cells (PEMCs) produce IGFBP-3 and its level drops significantly immediately prior to differentiation. Additionally, IGFBP-3 suppresses both IGF-I and Long-R3-IGF-I-stimulated proliferation of embryonic porcine myogenic cells. In this study, we have examined the effects of recombinant porcine IGFBP-3 (rpIGFBP-3) on IGF-I- and Long-R3-IGF-I-stimulated proliferation and differentiation of the L6 myogenic cell line. L6 cells potentially provide a good model for studying the actions of IGFBP-3 on muscle because they contain no non-muscle cells and they do not produce detectable levels of IGFBP-3. RpIGFBP-3 suppresses both IGF-I and Long-R3-IGF-I-stimulated proliferation of L6 cells, indicating that it suppresses proliferation via both IGF-dependent and IGF-independent mechanisms. Our data also show that rpIGFBP-3 causes IGF-independent suppression of proliferation without increasing the level of phosphosmad-2 in L6 cultures. Additionally, rpIGFBP-3 suppresses IGF-I-stimulated differentiation of L6 cells. In contrast, however, rpIGFBP-3 does not suppress Long-R3-IGF-I-stimulated differentiation. This suggests that rpIGFBP-3 does not have IGF-independent effects on L6 cell differentiation.

Hypoglycemia induced changes in cell death and cell proliferation in the organogenesis stage embryonic mouse heart.

Hypoglycemia is a side effect of diabetes therapy and causes abnormal heart development. Embryonic heart cells are largely resistant to teratogen-induced apoptosis. Hypoglycemia was tested for effects on cell death and cell proliferation in embryonic heart cells by exposing mouse embryos on embryonic day (E) 9.5 (plug = E0.5) to hypoglycemia (30-50 mg/dl glucose) in vivo or in vitro for 24 hr. Long-term effects of in vivo exposure on conceptus viability were evaluated at E18.5. Cell death was evaluated on E10.5 by: 1) two TUNEL assays in sectioned embryos to demonstrate DNA fragmentation; 2) confocal microscopy in whole embryos stained with Lysotracker; 3) flow cytometry in dispersed heart cells stained for TUNEL and myosin heavy chain (MHC) to quantify and characterize cell type susceptibility; and 4) immunohistochemistry (IHC) and Western analysis in sectioned embryos to evaluate potential involvement of caspase-3 active subunit and p53. Effects on cell proliferation were evaluated by IHC and Western analysis of proliferating cell nuclear antigen (PCNA). In vivo hypoglycemic exposure on E9.5 reduced viability in conceptuses examined on E18.5. Hearts examined on E10.5 demonstrated increased TUNEL and Lysotracker staining. In hearts of embryos exposed to hypoglycemia, flow cytometry demonstrated increased TUNEL-positive cells and cells dual-labeled for TUNEL and MHC. Protein expression of caspase-3 active subunit and p53 was increased and PCNA was markedly reduced in hearts of embryos exposed to hypoglycemia. Hypoglycemia reduces embryonic viability, induces significant cell death, and reduces cell proliferation in the E9.5 mouse heart, and these processes may involve active caspase-3 and p53.

Geminin Links Homeobox Domain Proteins to the Cell Cycle

Homeobox proteins play an essential role in pattern formation in development. Two groups report an antagonistic interaction between homeobox domain-containing proteins and the cell cycle regulator geminin. Geminin inhibits proliferation by binding to Cdt1, a protein required for the formation of prereplicative complexes needed for DNA replication. Del Bene et al. identified Xenopus geminin as an interacting partner of the homeobox domain-containing transcription factor Six3 in a yeast two-hybrid assay. Geminin binding to Six3 prevented Six3-dependent reporter gene activity but did not inhibit Six3 DNA binding. Six3 competed with Cdt1 for geminin binding, which may make Cdt1 available to participate in DNA replication and so allow cell proliferation. This activity appeared to be independent of the transcriptional regulatory activity of Six3. Various genetic assays confirmed the mutually antagonistic activities of Six3 for geminin's cell cycle suppression activity and geminin for Six3's proliferation activity. Luo et al. identified the Polycomb complex protein Scmh1 and various members of the Hox transcription factor family as partners of geminin in a yeast two-hybrid assay. Cultured cells exhibited colocalization of Polycomb complex proteins and geminin that was dependent on the phase of the cell cycle, with mitotic cells showing colocalization. Geminin also bound directly to DNA containing Hox regulatory elements and to the Hox proteins, which prevented their binding to DNA. Geminin inhibited Hox-stimulated reporter gene expression. Hox proteins also interact with Cdt1, and Hox and geminin competed for binding to Cdt1. These two papers link pathways controlling embryonic cell proliferation and pattern formation through geminin and suggest that not all transcription factor functions arise from regulation of gene expression (see Li and Rosenfeld for discussion). F. Del Bene, K. Tessmar-Raible, J. Wittbrodt, Direct interaction of geminin and Six3 in eye development. Nature 427 , 745-749 (2004). [Online Journal] L. Luo, X. Yang, Y. Takihara, H. Knoetgen, M. Kessel, The cell-cycle regulator geminin inhibits Hox function through direct and polycomb-mediated interactions. Nature 427 , 749-753 (2004). [Online Journal] X. Li, M. G. Rosenfeld, Origins of licensing control. Nature 427 , 687-688 (2004). [Online Journal]

Profiling gene expression in growth-arrested mouse embryos in diapause

A recent report has identified changes in embryonic gene expression that are associated with, and may halt, embryonic cell proliferation during diapause.

Effects of FGF9 on embryonic Sertoli cell proliferation and testicular cord formation in the mouse.

Proliferation and cord formation by embryonic Sertoli cells are pivotal events involved in testis morphogenesis. A number of growth factors have been implicated in mediating these events. However, the exact level of involvement and importance of each as yet remains elusive. We have adopted an in vitro approach to assess developing mouse Sertoli cells, whereby they are cultured in the presence or absence of fibroblast growth factor (FGF9) and/or extracellular matrix (ECM) gel, since previous studies have shown that ECM gel aids Sertoli cell differentiation. The present findings corroborate this effect, but in addition demonstrate that in the presence of FGF9 (10 ng/ml), cells undergo greater proliferation than those cultured on gel alone. They also display a differentiated epithelial phenotype, with appositional contact of cell membranes in cord-like aggregations. In addition we have shown that cultured Sertoli cells generally express a smaller truncated, nuclear form of the FGFr3, although in the presence of FGF9 and absence of gel, the larger, cytoplasmic form of the receptor is also expressed. Immunolocalisation of FGFr3 in Sertoli cells of whole testes revealed a temporal expression pattern profile, with high levels being abundant in the embryonic testicular cords and at puberty, but an absence in adult Sertoli cells. Our findings suggest that FGF9 plays an important role in proliferation and organisation of embryonic Sertoli cells during testis morphogenesis.

Un paradoxe et trois énigmes à propos du rôle de BRCA1 dans les cancers du sein et de l’ovaire

More than 50% of the hereditary forms are associated with germ line mutation in either BRCA1 or BRCA2 genes (BReast CAncer 1/2). The BRCA1 protein is expressed ubiquitously and is likely to play a role in several fundamental processes, including the maintenance of genomic integrity. Paradoxically, BRCA1 appears as a gene essential for proliferation of embryonic cells that simultaneously carries tumor suppressor activity. The nature of the role of BRCA1 in DNA repair and maintenance of genome integrity remains enigmatic. BRCA1 may indeed be a sensor of "abnormal" DNA structures that undergo heterochromatinisation. This model finds some support in the recent report that BRCA1 participates in the maintenance of X-chromosome inactivation, a paradigm for facultative heterochromatinisation. Why are epithelial cells from mammary glands and ovaries the privileged targets for tumorigenesis in women carrying germline mutations in BRCA1? The inheritance of a single defective copy of BRCA1 by women confers a status of susceptibility for developing breast and/or ovarian cancer. The loss of the wild-type allele inherited from the unaffected parent (LOH), commonly observed in the primary breast and ovarian tumors in these susceptible women, represents the event that initiates the tumorigenesis process. This classical two hit model, which assumes that heterozygote cells are "normal" until the LOH occurs stochastically, remains enigmatic.

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PTH2 receptor-mediated inhibitory effect of parathyroid hormone and TIP39 on cell proliferation

The parathyroid hormone (PTH) has dual mitogenic and inhibitory effects on cell proliferation, depending on the cell type and experimental conditions. PTH can signal via two different receptors, both positively coupled to the adenylyl cyclase/cyclic AMP pathway which can mimic some of the proliferative effects of PTH. We evaluated the role of the type-2 PTH (PTH2) receptor on cell proliferation in clonal human embryonic kidney HEK293 cells stably expressing the human PTH2 receptor. Using a cyclic AMP-responsive gene-reporter, we confirmed that the tuberoinfundibular peptide (TIP39) and various human (h) PTH fragments including hPTH-(1–34) were potent agonists (EC 50 in the range of 0.01–0.3 nM) whereas the bovine (b) PTH peptides b(Tyr 34)PTH-(7–34) and its tryptophan derivative b[ d-Trp 12,Tyr 34]PTH-(7–34) behaved as antagonists (IC 50=117 and 249 nM, respectively). hPTH-(1–34) produced a dose-dependent inhibition of cell proliferation (EC 50=8.5±0.4 nM) after 3 days and this effect was fully reversed by the tryptophan derivative antagonist. The same effect was observed with TIP39 which caused a 30% maximal inhibition. These findings reveal that PTH2 receptor activation can inhibit cell proliferation and might explain the dual functionality of PTH on cell proliferation.

The effects of various concentrations of FGF-2 on the proliferation and neuronal yield of murine embryonic neural precursor cells in vitro.

Embryonic neural precursors (ENPs), also termed neural stem cells or "neurospheres," are an attractive potential source of tissue for neural transplantation, because of their capacity to expand in number in vitro while retaining the ability to develop into the major phenotypes of the CNS. ENPs are isolated from the developing brain and proliferate in the presence of mitogens such as FGF-2 and EGF. Subsequent withdrawal of these mitogens and exposure to a suitable substrate results in differentiation into the major cell types of the CNS. As well as its role in precursor cell expansion, FGF-2 also plays a key role in the division of astrocytes, and in neuronal differentiation. Thus, it is important to establish the optimal concentrations of this factor for expansion and differentiation of neuronal phenotypes. Here we explore the effect of FGF-2 concentrations ranging from 1 to 20 ng/ml on the expansion and differentiation capacity of ENPs isolated from the cortex and striatum of E14 mice. ENP expansion was seen under all conditions, but was greatest at 10 and 20 ng/ml and least at 1 ng/ml. The numbers of neurons (as a proportion of total cell number) differentiating from these ENP populations appeared to be greatest at 1 ng/ml. However, once adjustments were made for the amount of expansion at each dose, final neuronal yield was maximum at the highest concentration of FGF-2 used (20 ng/ml).

Activation of RAF-1 through Ras and Protein Kinase Cα Mediates 1α,25(OH)2-Vitamin D3 Regulation of the Mitogen-activated Protein Kinase Pathway in Muscle Cells

We have previously shown that stimulation of proliferation of avian embryonic muscle cells (myoblasts) by 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)) is mediated by activation of the mitogen-activated protein kinase (MAPK; ERK1/2). To understand how 1alpha,25(OH)(2)D(3) up-regulates the MAPK cascade, we have investigated whether the hormone acts upstream through stimulation of Raf-1 and the signaling mechanism by which this effect might take place. Treatment of chick myoblasts with 1alpha,25(OH)(2)D(3) (1 nm) caused a fast increase of Raf-1 serine phosphorylation (1- and 3-fold over basal at 1 and 2 min, respectively), indicating activation of Raf-1 by the hormone. These effects were abolished by preincubation of cells with a specific Ras inhibitor peptide that involves Ras in 1alpha,25(OH)(2)D(3) stimulation of Raf-1. 1alpha,25(OH)(2)D(3) rapidly induced tyrosine de-phosphorylation of Ras-GTPase-activating protein, suggesting that inhibition of Ras-GTP hydrolysis is part of the mechanism by which 1alpha,25(OH)(2)D(3) activates Ras in myoblasts. The protein kinase C (PKC) inhibitors calphostin C, bisindolylmaleimide I, and Ro 318220 blocked 1alpha,25(OH)(2)D(3)-induced Raf-1 serine phosphorylation, revealing that hormone stimulation of Raf-1 also involves PKC. In addition, transfection of muscle cells with an antisense oligodeoxynucleotide against PKCalpha mRNA suppressed serine phosphorylation by 1alpha,25(OH)(2)D(3). The increase in MAPK activity and tyrosine phosphorylation caused by 1alpha,25(OH)(2)D(3) could be abolished by Ras inhibitor peptide, compound PD 98059, which prevents the activation of MEK by Raf-1, or incubation of cell lysates before 1alpha,25(OH)(2)D(3) exposure with an anti-Raf-1 antibody. In conclusion, these results demonstrate for the first time in a 1alpha,25(OH)(2)D(3) target cell that activation of Raf-1 via Ras and PKCalpha-dependent serine phosphorylation plays a central role in hormone stimulation of the MAPK-signaling pathway leading to muscle cell proliferation.

Developmental expression and functional significance of Kir channel subunits in ureteric bud and nephron epithelia.

Kir channel subunit expression during development of the rat collecting-duct epithelium was quantified by RT-PCR of primary monolayer cultures. mRNAs of the vascular-type K(ATP) (K(NDP)) channel-forming subunits Kir6.1/SUR2 were highly expressed in early ureteric bud generations (embryonic day E14) and downregulated thereafter, while Kir1.1b (ROMK2) mRNA increased fourfold during cortical collecting duct (CCD) maturation. As assessed by immunohistochemistry, Kir6.1 protein was abundant in the apical and basolateral plasma membranes of early ureteric buds and trunks (E15 to postnatal day P1), downregulated thereafter and not detectable in CCD and outer medullary collecting ducts (OMCD) (P7). During nephron development, Kir6.1 protein was expressed ubiquitously on plasma membranes of early nephron stages from mesenchymal condensations to S-shaped bodies. After fusion of nephron and CCD, Kir6.1 protein was restricted to the apical membrane of proximal tubule. The Kir6/SUR2 channel opener, pinacidil (100 microM/2 days), increased tubulogenesis in organ culture by a factor of 3. Cell proliferation of human embryonic kidney cells (HEK 293) which endogenously express Kir6.1/SUR2 mRNA was stimulated by pinacidil in a dose-dependent manner, an effect that was partially abolished by glibenclamide (3 microM). In summary, Kir6.1/SUR2 channel subunits are highly expressed during early development of ureteric bud and nephron epithelia where Kir6.1/SUR2 activity regulates cell proliferation.

(-)-Stepholidine promotes proliferation and neuronal differentiation of rat embryonic striatal precursor cells in vitro.

The present study investigated the influence of (-)-stepholidine, an effective dopamine D1 receptor agonist and D2 receptor antagonist, on the development of neural precursor cells. Incubation of striatal neural precursor cells with stepholidine resulted in significant increase in the number of proliferating precursor cell spheres when in the presence of fibroblast growth factor-2. This action can be blocked by application of haloperidol. Treatment with stepholidine also increased the number of microtubule-associated protein-2-immunoreactive cells in the cultures and promoted marked increases in tyrosine hydroxylase expression. These findings suggest that stepholidine is involved in the regulation of proliferation of precursor cells. The effect appears to be mediated by dopamine receptors. Stepholidine also promotes the differentiation of precursor cells, however, this action may be independent of its effect on dopaminergic receptors.

Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway.

The Shh signaling pathway is required in many mammalian tissues for embryonic patterning, cell proliferation and differentiation. In addition, inappropriate activation of the pathway has been implicated in many human tumors. Based on transfection assays and gain-of-function studies in frog and mouse, the transcription factor Gli1 has been proposed to be a major mediator of Shh signaling. To address whether this is the case in mouse, we generated a Gli1 null allele expressing lacZ. Strikingly, Gli1 is not required for mouse development or viability. Of relevance, we show that all transcription of Gli1 in the nervous system and limbs is dependent on Shh and, consequently, Gli1 protein is normally not present to transduce initial Shh signaling. To determine whether Gli1 contributes to the defects seen when the Shh pathway is inappropriately activated and Gli1 transcription is induced, Gli1;Ptc double mutants were generated. We show that Gli1 is not required for the ectopic activation of the Shh signaling pathway or to the early embryonic lethal phenotype in Ptc null mutants. Of significance, we found instead that Gli2 is required for mediating some of the inappropriate Shh signaling in Ptc mutants. Our studies demonstrate that, in mammals, Gli1 is not required for Shh signaling and that Gli2 mediates inappropriate activation of the pathway due to loss of the negative regulator Ptc.

Absolute numbers versus proportions in the assessment of differentiation of beta-cells in the embryonic avian pancreas.

Environmental factors may influence the proliferation and differentiation of embryonic pancreatic endocrine cells, creating a need for the quantification of such effects. The explanted dorsal pancreatic bud (DPB) of the 5-day chick embryo is a useful in vitro model. Since all explants cannot be assumed to have the same number of endocrine cells at the start of culture, the proportion of beta-cells with respect to alpha-cells may be a more meaningful measure than absolute numbers. This study aimed to establish baseline values for the proportion of beta-cells in both intact and mesoderm-depleted DPBs before culture. Buds were excised from 12 chick embryos and the surrounding mesoderm was removed from 6 buds following collagenase treatment. All the buds were freeze-dried, fixed in parabenzoquinone vapour, embedded in resin and sectioned at 1 micro m. alpha- and beta-cells were detected by an indirect immunoenzyme method. alpha-cells outnumbered beta-cells in 9 of the 12 buds. The proportion of beta-cells in the intact buds varied from 16% to 64% (mean 39.5%) and in the mesoderm-depleted buds from 17% to 66% (mean 39%). There was no significant difference between the absolute numbers or the proportions of cells in either case. The proportions of beta-cells in the 5-day DPBs were higher than those in buds cultured in previous studies for 7 days under various conditions. This result may reflect the role of apoptosis in response to the culture conditions.

Cyclin D-Cdk4 Is Not a Master Regulator of Cell Multiplication in Drosophila Embryos

Inactivation of Cyclin E-Cdk2 is essential for a timely arrest of the epidermal cell proliferation program during Drosophila embryogenesis. E-type cyclin-cdk complexes are thought to be activated by D-types titrating away inhibitors and inducing cyclin E transcription by activating E2F transcription factors via Rb phosphorylation. Therefore, we have analyzed whether the developmentally controlled inactivation of Cyclin E-Cdk2 required for the epidermal cell proliferation arrest occurs as a consequence of Cyclin D-Cdk4 inactivation. However, preventing Cyclin D-Cdk4 inactivation by overexpression has a minimal effect on Cyclin E expression and does not interfere with the initial G1 arrest, while it readily induces the E2F target RnrS in arresting epidermal cells. Prolonged Cyclin D-Cdk4 overexpression eventually interferes with maintenance of quiescence in some cells. Moreover, in Cdk4 mutant embryos, some RnrS expression is still induced by Cyclin E overexpression, and endogenous Cyclin E expression as well as cell cycle progression is not affected, except for late aspects of the endoreduplication program. These findings argue against the proposed necessity of complete Rb inactivation by sequential phosphorylation by D- and E-type cyclin-cdk complexes. They demonstrate that Cyclin D-Cdk4 does not function as the master regulator of the embryonic cell proliferation program.

Germline drift in chimeric male mice possessing an F2 component with a paternal F0 radiation history

In earlier work using mouse chimeric embryos we have shown that pre-implantation embryonic cells with a history of paternal gamma-irradiation (1 Gy) 6 weeks prior to conception demonstrated a competitive cell proliferation disadvantage when challenged by direct cell-cell contact with control pre-implantation embryos in chimeras even two generations after paternal irradiation. This effect on embryonic cell proliferation was associated with paternal irradiation of sensitive type A/B spermatogonia 6 weeks prior to conception but not from irradiation 5 weeks prior to conception. The purpose of this new study is to test the hypothesis that germ cell lines with a history of acute irradiation may also exhibit a selection disadvantage or germline chimeric drift over time in adult male mice two generations after paternal irradiation in chimeric mice. F(0) male CD1 mice received a 1.0 Gy whole body absorbed dose of attenuated (137)Cs gamma-rays and were mated at post-irradiation weeks 5 and 6. Chimeric XY<---->XY male CD1 mice were constructed with F(2) embryos from the F(1) offspring and with control embryos. To distinguish the control germline cells, the control embryos were heterozygous for the neo transgene, which served as a cell lineage marker. To test for germline chimeric drift, each XY<---->XY chimera was mated periodically from age 7 to 21 weeks with a different CD1 female. The fraction of offspring from each liter with the neo marker was used to quantify the relative contribution of the control germ cell lineage to the fertilizing sperm population. The results showed that there was a significant selection against germ cells with the paternal F(0) post-irradiation week 6 history. In contrast, there was a very small but significant selection in favor of the germ cells with the paternal F(0) post-irradiation week 5 history. These results indicate that the effects of a relatively non-toxic dose of radiation (1 Gy) on cell proliferation transmitted by ancestral type B spermatogonia to the embryo are manifested in the germline of the adult male mice even after two generations.

Endocan Is a Novel Chondroitin Sulfate/Dermatan Sulfate Proteoglycan That Promotes Hepatocyte Growth Factor/Scatter Factor Mitogenic Activity

Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458-20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression.

Exogenous platelet-activating factor stimulates cell proliferation in mouse pre-implantation embryos prior to the fourth cell cycle and shows isoform-specific stimulatory effects.

Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-derived PAF (EPAF), which act in an autocrine/paracrine fashion to stimulate embryonic development in vitro. Mouse EPAF is thought to consist predominantly of hexadecyl (C16) and octadecyl (C18) PAF-like components. Mouse pre-implantation embryos cultured with exogenous PAF from the early cleavage stages exhibit increased blastocyst cell numbers and rates of mitosis around the 8-cell stage. We investigated whether exogenous PAF could specifically stimulate embryonic cell proliferation prior to the blastocyst stage in the mouse and also compared the biological activities of the C16 and C18 PAF isoforms as follows. Embryos were cultured for either 24 h or 120 h from the 2-cell stage and their total cell numbers were determined or their development assessed in terms of their incidence of successful zona-hatching respectively. In each case, embryos were cultured in unsupplemented medium or in medium supplemented with either C16 or C18 PAF (0.5 microM). Compared with controls, culture with C16 PAF produced a significant stimulation of mean total per number per embryo and a significant increase in the incidence of successful zona-hatching, whilst culture with C18 PAF had no significant effect. We then cultured 1-cell zygotes for 48 h in unsupplemented medium or medium supplemented with either an equimolar mixture of C16 and C18 PF or with either C16 or C18 PF alone (each at 0.2 microM). Embryos were also scored for cell number at 4 h and 30 h of culture. Although no significant effect on mean cell number per embryo was seen following 4 h or 30 h of culture with a mixed C16/C18 PAF preparation, culture for 48 h with a mixed C16/C18 PAF preparation or with C16 PAF alone produced a significant increase in mean cell number per embryo compared with controls - an effect that is likely to be receptor-mediated, since culture with an equivalent concentration of C18 PAF had no significant effect compared with controls. We have demonstrated that mouse zygotes/embryos can respond in a specific manner to exogenous hexadecyl PAF in terms of increased rates of cell proliferation prior to cavitation, and must be capable of doing so at some time between the first and third, and also between the second and fourth, cell cycles. Such embryos presumably express one or more classes of functional PAF-receptor molecule during this period (i.e. as early as during the 1-, 2- or 4-cell stages). We have also demonstrated that embryonic response to exogenous PAF is significantly isoform-specific, which may reflect differences between the two isoforms either in affinity for binding to putative embryonic PAF-receptor molecules or in their ability to elicit a stimulatory response following binding. This observation calls into question the use of preparations containing a mixture of hexadecyl and octadecyl PAF isoforms, particularly in dose-response studies, in the mouse.

Regulation of the embryonic cell proliferation by Drosophila cyclin D and cyclin E complexes.

Cell proliferation during Drosophila development occurs in a well known spatial and temporal pattern which can readily be studied in vivo. The cells which form the larval epidermis exit from the cell division cycle during embryogenesis after the 16th round of mitosis when they enter for the first time into a G1/0 phase. We are interested in the mechanistic basis of this cell proliferation arrest. We have shown that the arrest requires the down-regulation of cyclin E/Cdk2 activity by inhibition of cyclin E expression and parallel activation of Dacapo/p27 expression. In addition, up-regulation of Fizzy-related is observed and is required for inhibition of Cdk1 activity. Do these processes result from the down-regulation of D-type cyclin/Cdk complexes? Extensive evidence from mammalian cells, and in particular from tumour cells has suggested that these complexes act as master regulators of cell proliferation upstream of cyclin E. Our genetic analyses indicate that Drosophila cyclin D/Cdk4, which interacts with the Drosophila Rb family member as expected, does not play an essential role in the regulation of cell proliferation.