Development of the fetal rat liver: ultrastructural and stereological study of hepatocytes

Qualitative and quantitative changes in the liver tissue composition have been studied during prenatal development of the Wistar rat by electron microscopy and stereologic methods. The absolute volume of the fetal liver is multiplied by 84 between days 13 and 20 of gestation. In the meantime, the average hepatocyte volume is multiplied by 1.5 between days 12 and 20. The volumetric fraction of hepatocytes increases from 35% of the volumetric fraction of the liver on day 12 to 66% on day 20 of gestation. The non-hepatocyte cells decrease from 49% on day 12 to 25% on day 20. By days 12 and 13, the rough endoplasmic reticulum and the Golgi apparatus are well differentiated, indicating that young fetal hepatocytes are able to synthesize and export plasma proteins. The volumetric fraction of free ribosomes decreases from 38% of the hepatocytic cytoplasm on days 12 and 13 to 6% on day 20. The mitochondrial compartment occupies about 10% of the hepatocyte cytoplasm. The mitochondria, small and round on days 12, 13 and 14, become oblong from day 18 of gestation. The shape of hepatocytes changes during the prenatal development, from potato-like on days 12, 13, 14 to cubic on day 20, with an intermediate, more spheric, stage on day 18.     

The Apoptotic and Nonapoptotic Nature of the Terminal Differentiation of Erythroid Cells

The morphology of erythroid cells changes dramatically during the course of their terminal differentiation. According to calculations made with cytospin preparations obtained from Syrian hamster yolk-sac-derived erythroid cells, the area of nuclei at day 10 of gestation ranges from 25 to 85 μm²and is reduced to 15–25 μm²on day 13 [K. Morioka and R. Minamikawa-Tachino,Dev. Growth Differ.35, 569–582, 1993]. The DNA and protein contents of each nucleus also decrease during this period. Nonspecific fragmentation of DNA was detected by agarose gel electrophoresis in all samples obtained from day 10 to day 13 of gestation, while distinct ladders of DNA fragments were not detected. DNA fragmentation was also detected by anin situDNA-end labeling (TUNEL) assay. As the terminal differentiation proceeded, gradual decreases in levels of both histone H1 and most nonhistone proteins were observed by SDS–polyacrylamide gel electrophoresis, while levels of core histones appeared to be constant. In particular, lamin B₂was almost completely lost from the nuclear matrix fraction on day 11. These results suggest that the terminal differentiation of erythroid cells and apoptosis might have common mechanisms. However, expansion of the cytoplasm during the terminal differentiation distinguishes these processes. In addition, in the erythroid terminal differentiation, nuclei never form lobules or become fragmented; no apoptotic bodies are formed, occurrence of the apoptosis-like cellular change is not sporadic but rather synchronous, and the process is slow, with at least several days being required for cell death. These characteristics are different from those of typical apoptosis. Thus, the terminal differentiation of nucleated embryonic erythroid cells exhibits both apoptotic and nonapoptotic features.     

Terminal differentiation of yolk-sac erythroid cells of the Syrian hamster in vitro

Summary The developmental fate of Syrian hamster yolk-sac (primitive) erythroid cells was examined in vitro. Highly purified yolk-sac erythroid cells at the polychromatophilic stage, obtained from the peripheral blood of embryos at day 10 of gestation, showed morphological and biochemical changes in our modified semi-solid culture system. Several morphological changes observed in the primitive erythroid cell cultures, such as nuclear condensation, approach of nuclei to the periphery of cells, development by cells of an extended pear-like shape, enucleation, and an increase in haemoglobin content, were quite similar to those of the terminal differentiation of fetal liver or adult bone marrow (definitive) erythroid cells. In addition, the transition of molecular species of haemoglobin from the embryonic to the fetal/adult pattern was also observed in our culture system. Thus we provide evidence, by the in vitro culture of yolk-sac erythroid cells, that primitive erythroid cells undergo terminal differentiation in a manner similar to that of definitive erythroid cells.     

Temporal Characteristics of the Differentiation of Embryonic Erythroid Cells in Fetal Peripheral Blood of the Syrian Hamster

The morphological changes in erythroid cells and their nuclei in the circulation of fetuses of the Syrian hamster were investigated by use of an image-processing system. The analysis included monitoring of nuclear condensation, nuclear periphralization (access of the nucleus to the cell membrane), enucleation, density of cells, and changes in cell size from day 9 of gestation to day 5 after birth. The yolk-sac-derived erythroid cells made rapid progress in nuclear condensation on day 11, while this process proceeded at a much lower rate after day 12 of gestation. The peripheralization of nuclei started on day 10 and reached a maximum on day 11. The frequency of enucleated cells was below 2% on day 11, while it increased to 30% on day 12. Extruded nuclei, most of which were accompanied by a small quantity of cytoplasm, appeared in the circulation on day 12. The most frequently observed diameter of enucleated erythrocytes, which was 10–10.5 μm on day 12, fell gradually to 8–9 μm on day 14. By contrast, the shift from fetal liver erythrocytes to adult erythrocytes occurred in a discontinuous manner. Adult-type erythrocytes were detected after birth with diameters of 5.5–6 μm. Our data allows us to present the schedule of morphological changes during embryonic erythropoiesis and show that the developmental behavior of "primitive" yolk-sac-derived erythroid cells is more closely correlated with that of the "definitive" fetal liver cells than has been considered to be the case to date.     

Heme deficiency in erythroid lineage causes differentiation arrest and cytoplasmic iron overload

Erythroid 5-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells. Mutation of human ALAS-E causes the disorder X-linked sideroblastic anemia. To examine the roles of heme during hematopoiesis, we disrupted the mouse ALAS-E gene. ALAS-E-null embryos showed no hemoglobinized cells and died by embryonic day 11.5, indicating that ALAS-E is the principal isozyme contributing to erythroid heme biosynthesis. In the ALAS-E-null mutant embryos, erythroid differentiation was arrested, and an abnormal hematopoietic cell fraction emerged that accumulated a large amount of iron diffusely in the cytoplasm. In contrast, we found typical ring sideroblasts that accumulated iron mostly in mitochondria in adult mice chimeric for ALAS-E-null mutant cells, indicating that the mode of iron accumulation caused by the lack of ALAS-E is different in primitive and definitive erythroid cells. These results demonstrate that ALAS-E, and hence heme supply, is necessary for differentiation and iron metabolism of erythroid cells.     

Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation

The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was colocalized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in nuclei. Pro-caspase-8 was found in the intermembrane space, inner membrane, and matrix of mitochondria after limited digestion of mitochondrial fractions, and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside, which opens the mitochondria permeability transition pore. Release was blocked by the mitochondria permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 h to an apoptotic inducer tumor necrosis factor-alpha (TNF-alpha), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8 cleavage products, in the cytoplasm of the TNF-alpha-treated striatal cells. CsA blocked the TNF-alpha-induced release of pro-caspase 8 but not cytochrome c. Internucleosomal DNA fragmentation started at 6 h and peaked 12 h after TNF-alpha treatment. These results suggest that pro-caspase-8 is predominantly localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.     

Observations on the ultrastructure of developing myocardium of rat embryos

Timed pregnancies were obtained in Sprague-Dawley rats and early ultrastructural differentiation of myocardium of embryos of 10, 11, 12, 13, and 14 days was investigated and compared with that of newborn. Ten-day myocardium is characterized by loosely packed cells; the cytoplasm is typified by a dearth of organelles. Both thick (myosin) and thin (actin) filaments become identifiable for the first time in the 10-day myocardium where the heart is pulsating but circulation is not established. These filaments are not visible in the embryos of 9-day-old myocardium. The formation of these filaments is observed to continue throughout the period covered in this investigation. Concomitant with the appearance of the myofilaments is the synthesis of Z band material. By the eleventh day of gestation and during the subsequent days there is a rapid proliferation and differentiation of most of the organelles. The myofilaments become organized into fully formed striated fibrils. Intercalated discs appear as. small wavy lines on the eleventh day and become plicated in later stages and serve as cell boundaries and points of attachment for myofilaments and fibrils. There is a perceptible change in the number and morphology of mitochondria from the tenth to eleventh day and later stages of development when the heart becomes functional. Similarly, there is a rapid proliferation and differentiation of granular endoplasmic reticulum and Golgi bodies. Large quantities of free ribosomes are dispersed in the cytoplasm of 10-day myocardium; however, in later stages there is a progressive reduction in the distribution of these particles. An intimate association of ribosomes and polysomes with the developing myofibrils is discernible. The T -system and sarcoplasmic reticulum begin to appear in II-day myocardium. The embryonic myocardium displays intense mitotic activity throughout its development and a unique feature of embryonic myocardial cells is the simultaneous occurrence of myofilament synthesis and mitotic activity within the same cells.     

Nucleoside phosphatase (ATPase) activity in myocardial cells of rat embryos. An electron-microscopic study.

Timed pregnancies were obtained in Sprague-Dawley rats, and cardiac tissues from embryos of days 10, 11, 12, 13, 14 and from newborn rats were used for the cytochemical localization of ATPase activity utilizing a lead phosphate precipitation procedure. Following incubation with ATP as the substrate, granular deposits of reaction product are discernible on the cell membranes of the embryonic myocardium. There is a noticeable decrease in the intensity of reaction product as visualized in the electron micrographs from the 10th day of gestation to the 14th day. No granular reaction product is recognizable in myofibrils, mitochondria or other organelles in the cytoplasm. It appears that there is a selective deposition of the reaction product on the cell membranes or structures derived from it. The intense ATPase activity seen on 10th and 11th days seems to be correlated with the initial appearance of myofilaments and fibrils in the myocardial cells.     

ULTRASTRUCTURAL CHANGES IN EMBRYONIC ECTODERMAL CELLS OF THE NEURULATING RAT EMBRYO

Embryonic ectodermal cells of rat embryos were examined by light and electron microscopy during the early stage of neurulation. Before the onset of neurulation (day 9–6 hr embryos), the cells underwent certain characteristic ultrastructural changes; that is, apical cytoplasmic protrusions and free spherules appeared, numerous vacuoles were formed in the cytoplasm, mitochondria showed ballooning, and the endoplasmic reticulum became dilated. The amniotic cells derived from the embryonic ectoderm exhibited the same ultrastructural changes, but those from the extraembryonic mesoderm did not. Embryonic mesodermal cells and neuroectodermal cells also did not show these changes. In the middle stage of neurulation (day 9–12 hr embryos), the embryonic ectodermal cells and the amniotic cells derived from the embryonic ectoderm assumed a flat squamous shape. None of the ultrastructural changes observed in day 9–6 hr embryos were noted in these cells. The functional significance of the production of apical cytoplasmic protrusions and free spherules in the embryonic ectodermal cells and amniotic cells is discussed in relation to similar phenomena reported to occur in other cell types.     

鹌鹑、红嘴鸥、黑翅长脚鹬胚胎期 小肠黏膜表面形态结构

应用扫描电镜观察了鹌鹑（Coturnix coturnix）、红嘴鸥（Larus ridibundus）、黑翅长脚鹬（Himantopus himantopus）胚胎期小肠黏膜的形态变化。鹌鹑与红嘴鸥胚胎发育过程中,小肠黏膜形态结构可分为3个阶段。第一阶段为鹌鹑卵孵育第10 ~ 11天、红嘴鸥卵孵育第13 ~ 14天（相当于胚胎发育60%的阶段）,小肠黏膜为山脊状纵行皱襞;第二阶段为鹌鹑卵孵育第12 ~ 13天、红嘴鸥卵孵育第15 ~ 16天（相当于胚胎发育70%的阶段）,小肠黏膜为“W”形板状皱襞;第三阶段为鹌鹑卵孵育第14 ~ 17天、红嘴鸥卵孵育第17 ~ 22天（相当于胚胎发育到80% ~ 100%阶段）,小肠黏膜为指状绒毛。黑翅长脚鹬卵孵育第10天（相当于胚胎发育到60%的阶段）,小肠黏膜为山脊状纵行皱襞;从卵孵育第12天（相当于胚胎发育到70%的阶段）一直到孵出小肠黏膜均为“W”形板状皱襞。初步判断3种鸟小肠黏膜形态发生这种有规律的变化可能是鸟类对其祖先系统发育的重演。     

Mitochondrial translocation of protein kinase C delta in phorbol ester-induced cytochrome c release and apoptosis

Apoptosis is induced by the release of cytochrome c from mitochondria to the cytoplasm. The present studies demonstrate that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces translocation of protein kinase C (PKC) delta from the cytoplasm to mitochondria. The results also show that translocation of PKCdelta results in release of cytochrome c. The functional significance of this event is further supported by the demonstration that PKCdelta translocation is required for TPA-induced apoptosis. These findings demonstrate that translocation of PKCdelta to mitochondria is responsible, at least in part, for inducing cytochrome c release and apoptosis.     

Haemopoietic modulation in tumour-bearing animals: enhanced progenitor-cell production in femoral marrow

Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFUc-GM compartment followed by an increase in the number of femoral CFUs. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFUe) compartment expanded to 168% of control, while the more differentiated (CFUe) compartment was reduced (45% of control at day 28). Reductions in both 59Fe-incorporation and erythroblasts/femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr X 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. The data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.     

The ontogeny of epidermal growth factor receptors during mouse development

In an attempt to understand the role(s) of epidermal growth factor (EGF) in vivo during murine development, we have examined the 125I-EGF binding characteristics of EGF-receptors in membrane preparations of tissues from the 12th day of gestation to parturition. Using autoradiography, the earliest time that we could detect EGF-receptors was on trophoblast cells cultured for 3 days as blastocyst outgrowths. Trophoblast eventually forms a large portion of the placenta, where EGF-receptors have long been recognized. We measured the number and affinity of EGF-receptors on tissues dissected from conceptuses from the 12th day of gestation in order to identify a stage when tissues may be most sensitive to EGF. Whereas the number of EGF receptors increases during gestation for all tissues examined, the affinity of the receptors declines for carcass and placenta and remains relatively unchanged for brain and liver. This suggests that EGF may function differently throughout development. Our hypothesis is that EGF (or its embryonic equivalent) initially stimulates proliferation in embryonic cells and then stimulates differentiation as the tissues mature. In the adult, its main role could be to stimulate tissue repair after damage.     

Ultrastructure of the sympathetic paravertebral ganglia in rat embryogenesis. I. Cytogenesis and cell differentiation

The development of sympathetic paravertebral ganglia was studied in rat embryos by electron microscopy. The main attention was paid to the initial stages of ganglion formation. The first aggregations of presumptive ganglionic cells were observed in 12 day-old embryos. Single preganglionic terminals appeared in contact with cell bodies sometime later. The appearance of large granular vesicles in the cytoplasm is the first ultrastructural feature of the beginning of neural differentiation of cells. Small granulated cells observed from the 12th day of gestation and neuroblasts differentiate earlier than glial cells. In the ganglia of late fetuses nerve cells varied in the electron density of the cytoplasm, in the degree of distention of rough endoplasmic reticulum and in vacuolization of mitochondria.     

Unreliability of the cytochrome c-enhanced green fluorescent fusion protein as a marker of cytochrome c release in cells that overexpress Bcl-2

A cytochrome c-enhanced green fluorescent protein chimera (cyt-c.EGFP) was used to monitor the release of cytochrome c from mitochondria in Bcl-2-negative and Bcl-2-positive MDA-MB-468 breast cancer cells. A comparison was made with the intracellular distribution of endogenous cytochrome c based on Western blotting of cell fractions and immunocytochemistry. The release of endogenous cytochrome c from mitochondria into the cytoplasm was detected in Bcl-2-negative cells treated with the kinase inhibitor staurosporine or the calcium-ATPase inhibitor thapsigargin. No release of endogenous cytochrome c was evident in Bcl-2-positive cells, consistent with earlier evidence that Bcl-2 overexpression inhibits cytochrome c release from mitochondria. Cyt-c.EGFP appeared to be localized to the mitochondria in Bcl-2-negative cells and to be released into the cytoplasm following treatment with either staurosporine or thapsigargin. However, in Bcl-2-positive cells the pattern of distribution of cytochrome c-EGFP was inconsistent with that of endogenous cytochrome c, due to accumulation of both cyt-c.EGFP and free EGFP in the cytoplasm of both treated and untreated cells. In summary, cyt-c.EGFP may be useful for monitoring cytochrome c release in living cells that do not express high levels of Bcl-2 but is an unreliable marker of cytochrome c release in cells that overexpress Bcl-2.     

Localization of Forssman glycolipid and GM1 ganglioside intracellularly and on the surface of germ cells during fetal testicular and ovarian development of mice

Changes in the expression pattern and intracellular localization of Forssman glycolipid (FA) and GM1 ganglioside (GM1) in fetal mouse gonads were examined during germ cell differentiation by immunofluorescence microscopy and immunoelectron microscopy. In male germ cells from the 12th to 14th day p.c., anti-FA binding was localized in granular structures aggregated on one side of the cytoplasm and/or in the plasma membrane. On day 16 p.c., some germ cells still showed patch-like positive reactions in the plasma membrane, but by day 18 p.c., positive reactions for FA had completely disappeared. The female germ cells showed granular bindings of anti-FA scattered throughout their cytoplasm during the 13th to 16th day p.c., although the positive reactions in female germ cells on day 12 p.c. tended to be found in one side of cytoplasm and/or plasma membrane similar to those in male germ cells from 12th to 14th day p.c. On day 18 p.c., positive reactions remained in the plasma membrane of some germ cells, but these positive reactions disappeared before birth. Immunoelectron microscopic observation showed that the sites of anti-FA bindings were equivalent to the "small dense bodies" (SDB) and the Golgi lamellae both in male and female germ cells. On the other hand, GM1 was not detected in male germ cells at any time during fetal testicular development, whereas an anti-GM1 reaction was detected in the plasma membrane of female germ cells from the 16th to 18th day p.c. (oocytes in the first meiotic prophase).(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical studies of enzymes of the energy metabolism in postimplantation rat embryos

Using histochemical procedures for the detection of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), and cytochrome c oxidase (cytox), we investigated the levels of these enzymes of the energy metabolism in postimplantation rat embryos (9.5-12.5 days of gestation). On day 10.5 of gestation, the neural tube, somites, myocardium, and mesenchyme displayed moderate levels of LDH activity; this activity gradually increased in strength, so that, on day 12.5 of gestation, intense LDH activity was uniformly distributed in these intraembryonic tissues. In contrast to LDH, distinct regional differences in the distribution of SDH and cytox were detected. On day 10.5 of gestation, the myocardium exhibited weak to moderate SDH and cytox activity, and on day 11.5, the myocardial activity of these enzymes had become moderate to intense. However, in all other embryonic tissues, e.g., the neural tube and somites, only weak SDH and cytox activity was present. On day 12.5 of gestation, the myocardium displayed very intense SDH and cytox activity, whereas the mantle layer of the neural tube, the spinal ganglia, and the myotomes exhibited only moderate levels of SDH and cytox activity. In the matrix of the neural tube and mesenchyme, these enzyme activities remained at low levels. At electron microscopy, cytox activity was detectable in the spaces between the inner and outer membranes as well as in the intracristal spaces of mitochondria. In general, cytox activity increased in parallel with the differentiation of mitochondria (i.e., increased mitochondrial numbers and size, and the development of mitochondrial cristae), but when the distribution of the cytox activity was considered in detail, it was found to differ among mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial targeting of JNK/SAPK in the phorbol ester response of myeloid leukemia cells

Treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with activation of the stress-activated protein kinase (SAPK) and induction of terminal monocytic differentiation. The present studies demonstrate that TPA targets SAPK to mitochondria by a mechanism dependent on activation of protein kinase C (PKC) beta. Translocation of SAPK to mitochondria in response to TPA is associated with release of cytochrome c, caspase-3 activation and induction of apoptosis. The results show that TPA induces the association of SAPK with the mitochondrial anti-apoptotic Bcl-x(L) protein. Overexpression of Bcl-x(L) attenuated the apoptotic response to TPA treatment. Moreover, expression of Bcl-x(L) mutated at sites of SAPK phosphorylation (Thr-47, -115) was more effective than wild-type Bcl-x(L) in abrogating TPA-induced cytochrome c release and apoptosis. By contrast, expression of Bcl-x(L) had little effect on induction of the monocytic phenotype. These findings indicate that myeloid leukemia cells respond to TPA with targeting of SAPK to mitochondria and that this response contributes to terminal differentiation through the release of cytochrome c and induction of apoptosis.     

Haemopoietic modulation in tumour-bearing animals: enhanced progenitor-cell production in femoral marrow

Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFU_c-GM compartment followed by an increase in the number of femoral CFU_s. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFU_e) compartment expanded to 168% of control, while the more differentiated (CFU_e) compartment was reduced (45% of control at day 28). Reductions in both ⁵⁹Fe-incorporation and erythroblasts/femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr × 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. the data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.