Incorporation of 3H-uridine into the large decidual cells of rats and their differentiation

The antimesometrial part of rat's decidua of the 9th day of gestation was divided into three zones. Cells of either zone display their own morphological and cytochemical properties. Different rates of 3H-uridine incorporation were observed in the cytoplasm and the nucleus in cells of either zone during 5, 30, 60 and 240 minutes after precursor injection. The largest member of silver grain accumulation was observed in the karyoplasm and nucleolus of cells of the transitional zone. The nucleus of basal zone cells had the smallest intensity of 3H-uridine incorporation. The nuclei of the epithelial zone cells are characterized by a lower intensity of 3H-uridine incorporation than those of the transition zone. The intensity to cytoplasmic accumulation of silver grains raised from cells of the basal zone up to cells of the epithelial zone. The largest quantity of cytoplasmic radioactivity was observed 240 minutes after 3H-uridine injection.     

Changes in the amount and synthesis of DNA in the nuclei of large decidual cells in rats during their differentiation

Using cytophotometry of the Feulgen-stained nuclei, the quantity of DNA was measured in the nuclei of rat's large decidua cells (LDC) on tissue sections of the antimesometrial region within days 7-13 of gestation. The quantity of nuclear DNA was expressed in units of ploidy, the haploid DNA standard being the quantity of DNA in rat's spermatid nucleus. On different days of gestation, the nuclear DNA was seen to vary in cells located in different zones of decidua. The maximum DNA content was found in the LDC located on days 9-12 of gestation somewhat in the middle of the decidua thickness. On day 11, the quantity of nuclear DNA in these cells reached in average, 22c. The quantity of DNA in the nuclei of the least differentiated LDC located on the periphery of decidua never exceeded 4.9c, whereas that in the nuclei of the most differentiated LDC, located close to the embryo, varied from 2.9c to 9.3c. On days 10 and 11 of rat's false gestation, the maximum DNA contents in the nuclei were registered in the LDC located in the middle of the decidua thickness. 3H-thymidine incorporation into the nuclei of the most differentiated LDC located nearest to the embryo stopped starting from day 10 of gestation. Phenomena of lesser quantities of nuclear DNA in most differentiated LDC, compared to that in LDC in the previous steps of differentiation, are discussed.     

Seasonal study of the adrenal gland of some Indian avian species.

Adrenal glands of eight Indian species of birds, namely Columba livia, Passer domesticus, Corvus splendens, Acridotheres tristis, Acridotheres ginginianus, Milvus migrans, Francolinus pondicerianus and Bubulcus ibis were examined during the sexually active and inactive phases of their annual reproductive cycles. Excepting A ginginianus and M. migrans, among members of either sex of the remaining six species the weight of the adrenal gland increases during the period of sexual activity. Histologically, the interrenal tissue of these birds could be divided into a peripheral subcapsular zone and a central zone. The cytochemical content of these two zones varies between sexual activity and inactivity. In sexually active birds of both sexes, interrenal cells of the central zone exhibit an increased concentration of alkaline phosphatase, glycogen, acid mucopolysaccharides and gross lipids, while in the subcapsular interrenal cells there is a prominent increase of ascorbic acid content. Cytochemical contents of chromaffin cells remain unchanged except acid phosphatase, which increases during the sexually active phase.     

The ultrastructure and histochemistry of the spermathecae of Tubifex tubifex (Annelida: Oligochaeta)

The wall of the spermathecal ampulla in Tubifex tubifex consists of epithelial, muscular and peritoneal layers. The epithelial surface contains closely microvilli while lateral and basal plasma membranes are extensively convoluted. Epithelial cytoplasm exhibits a vertical zonation of subcellular components. The distal zone contains filiform secretory particles which are orientated perpendicular to the apical surface; extrusion occurs by their fusion with the plasma membrane between the bases of neighbouring microvilli. Mitochondiral and Golgi zones, the latter containing the nucleus, subtend the distal zone. The basal zone, composed of vertical compartments formed by the folded plasma membrane, is rich in α-glycogen rosettes. The distal epithelium and lumen material contain neutral mucopolysaccharides and carboxylated acid mucopolysaccharides in conjunction with neutral protein. The ultrastructure of the spermathecal duct wall is comparable with that of the ampulla but is characterized by extremely long microvilli and a prominent musculature.     

Light and electron microscopic examination of the mature decidual cells of the rat with emphasis on the antimesometrial decidua and its degeneration

Rat gestation sites were obtained on days 10 through 16 of normal pregnancy. Light and electron microscopic examination of day-10 sites revealed a consistent complex pattern of stromal cell morphologies. Six distinct regions were identified: an antimesometrial region of epithelioid decidual cells that form the gestation chamber containing the embryo and extraembryonic membranes; an abembryonic antimesometrial decidual region, the decidual crypt, where the cells are separated by large extracellular spaces; a mesometrial region with granule-containing cells and mesometrial decidual cells; a region of spiny cells that are lateral to the antimesometrial decidual cells and continuous with the mesometrial decidual cells; and a region of undifferentiated stromal cells adjacent to the myometrium. Between days 12 and 16, the antimesometrial decidua becomes thinner and is eventually sloughed into the newly formed uterine lumen. The role of the antimesometrial decidual cells is discussed with reference to trophoblast invasiveness, protein synthesis, and especially remodeling of the gestation chamber. Differences between decidua and deciduoma are considered.     

Cytochemical studies of developmental stages during oogenesis in Culex pipiens molestus (Forsk?l). I. Lipids and carbohydrates

Lipids and carbohydrates were studied in the polytrophic ovaries of Culex pipiens molestus during oogenesis. The cytoplasm of both the oocyte and the nurse cells contains lipid structures at all stages of development--granules in the early stages and spheres in the later stages. Intranuclear lipid bodies can be demonstrated in the oocyte and in the nurse cells. After leaving the nucleus, lipids are deposited in the peripheral cytoplasm. From the third to the seventh adult phase, lipid granules are concentrated in the area of the nurse cell and oocyte junction, indicating that lipids originate in the nurse cells and are transported from these to the oocyte. The follicular epithelial cells provide the oocyte with lipid material for fatty yolk synthesis and formation of the egg envelopes. Lipids are distributed similarly to the Golgi apparatus, indicating that there is a relationship between this organelle and fat formation. In the early stages, the cytoplasm of the oocyte, the nurse cells and the follicular epithelium contains glycogen granules. In the later stages these cells also contain mucopolysaccharides. The mucopolysaccharide yolk spheres are enclosed in vacuoles, while the chorion is composed of acid mucopolysaccharides. The follicular epithelium and vitelline membrane are of a mucopolysaccharide nature. A topographical relationship exists between the Golgi apparatus and the glycogen granules, indicating that this organelle also plays a role in glycogen synthesis.     

Morphological and histochemical observations on the intestinal epithelium of Ascardia galli (Nematoda: Ascaridida).

The intestinal epithelium of Ascardia galli has been studied with various cytological and cytochemical techniques. It consists of large epithelial cells resting on a thick collagenous basal lamina. Their luminal surface is provided with microvilli. The intestinal cells store considerable amounts of glycogen and neutral lipids. Some intracellular granular inclusions, which stain for proteins, phospholipids and lipoproteins, are distributed throughout the cytoplasm. The brush border is composed of microvilli whereas the outer surface coat consists of saliva resistant PAS-positive material. The detailed histochemical analysis of surface material has revealed that it is composed of nonacetylated acid mucopolysaccharides rich in hyaluronic acid with carboxylate polyanions. The brush border shows intense activities of acid phosphatase and glucose-6-phosphatase, moderate of ATPase, and lipase, weak of 5'-nucleotidase. Acid phosphatase-positive intracellular structures are seen in the intestinal epithelium which form distinct aggregations.     

Ontogeny of adenosine deaminase in the mouse decidua and placenta: immunolocalization and embryo transfer studies

This study has determined the cellular site of adenosine deaminase (ADA) expression in the mouse during development from Days 5 through 13 (day vaginal plug was found = Day 0) of gestation. Developmental expression of ADA progressed in two overlapping phases defined genetically (maternal vs. embryonal) and according to region (decidual vs. placental). In the first phase, ADA enzyme activity increased almost 200-fold in the antimesometrial region (decidua capsularis + giant trophoblast cells) from Days 6 through 9 of gestation but remained low in the mesometrial region. Immunohistochemical staining revealed a major localization of ADA to the secondary decidua. In the second phase, ADA activity increased several-fold in the placenta (labyrinth + basal zones) from Days 9 through 13 of gestation but remained low in the embryo proper. Immunohistochemical staining revealed a major localization of ADA to secondary giant cells, spongiotrophoblast, and labyrinthine trophoblast. Regression of decidua capsularis and growth of the spongiotrophoblast population accounted for an antimesometrial to placental shift in both ADA enzyme activity and a 40-kDa immunoreactive protein band. To verify a shift from maternal to fetal expression, studies were performed with two strains of mice (ICR, Eday) homozygous for a different ADA isozyme (ADA-A, ADA-B). Blastocysts homozygous for Adab were transferred to the uterus of pseudopregnant female recipients homozygous for Adaa. The isozymic pattern in chimeric embryo-decidual units analyzed at Days 7, 9, 11, and 13 revealed a predominance of maternal-encoded enzyme at Days 7 through 11 of gestation and a shift to fetal-encoded enzyme by Day 13. Thus, maternal expression of ADA in the antimesometrial decidua may play a role during establishment of the embryo in the uterine environment, whereas fetal expression of ADA in the trophoblast might be important to placentation.     

Histochemical studies on the enzymes of glycogen metabolism in hamster epididymis

Summary Active and total (active + inactive) phosphorylase and glycogen synthetase (I- and D-form) were studied in hamster epididymis in relation to glycogen. Immature and adult, sexually active and regressed animals were examined.Epididymis in adult animals, based on their phosphorylase activity, may be divided into 5 zones. The zone 1 epithelium contains particulate glycogen, rich in phosphorylase and glycogen synthetase. The epithelial cytoplasm also contains moderate phosphorylase activity. The zone 2 epithelium is almost devoid of phosphorylase. The zone 3 epithelium shows considerable phosphorylase activity both in principal and holocrine cells. The epithelium of the zone 4 contains the highest total phosphorylase activity. In the zone 5 epithelium phosphorylase and glycogen are absent, but glycogen synthetase is often observed.Holocrine cells, particularly in zones 3 and 4, contain predominating active phosphorylase, some glycogen, but no synthetase activity. The lumen in the zone 4 often shows a faint staining for glycogen.In immature animals, low phosphorylase activity is always present in the epithelial cells. Holocrine cells are detectable, by their phosphorylase activity, in 4 week animals. The division of zones is usually established slightly before sexual maturation.During the period of sexual regression, phosphorylase diminishes considerably. Glycogen, phosphorylase and glycogen synthetase are, however, detectable in the zone 1 of these animals.     

Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate.

We have employed C2C12 myotubes to investigate lipid inhibition of insulin-stimulated signal transduction and glucose metabolism. Cells were preincubated for 18 h in the absence or presence of free fatty acids (FFAs) and stimulated with insulin, and the effects on glycogen synthesis and signaling intermediates were determined. While the unsaturated FFAs oleate and linoleate inhibited both basal and insulin-stimulated glycogen synthesis, the saturated FFA palmitate reduced only insulin-stimulated glycogen synthesis, and was found to inhibit insulin-stimulated phosphorylation of glycogen synthase kinase-3 and protein kinase B (PKB). However, no effect of palmitate was observed on tyrosine phosphorylation, p85 association, or phosphatidylinositol 3-kinase activity in IRS-1 immunoprecipitates. In contrast, palmitate promoted phosphorylation of mitogen-activated protein MAP) kinases. Ceramide, a derivative of palmitate, has recently been associated with similar inhibition of PKB, and here, ceramide levels were found to be elevated 2-fold in palmitate-treated C2C12 cells. Incubation of C2C12 cells with ceramide closely reproduced the effects of palmitate, leading to inhibition of glycogen synthesis and PKB and to stimulation of MAP kinase. We conclude that palmitate-induced insulin resistance occurs by a mechanism distinct from that of unsaturated FFAs, and involves elevation of ceramide by de novo synthesis, leading to PKB inhibition without affecting IRS-1 function.     

The differentiation of the decidua and the distribution of metrial gland cells in the pregnant mouse uterus

Summary A study was made with the light microscope of the cellular changes involved in the formation of the decidua in the pregnant mouse uterus up to day 11 of pregnancy. The differentiation sequence was similar to that found by previous workers but the morphology and development of the basal zone is described in more detail. In addition, the morphology of glycogen rich cells in an area termed the lateral decidual zone is described. The distribution of granulated metrial gland cells and their precursors is described. These cells are first found in the uterine stroma before the blastocyst has implanted. Later they occur in the decidua and in the circular smooth muscle zone beneath the mesometrial triangle prior to the formation of the metrial gland. Granulated metrial gland cells are also found in the maternal blood spaces of the implantation site.     

Ovarian steroid receptors and activated MAPK in the regional decidualization in rats

Though the decidua serves a critical function in implantation, the hormonal regulated pathway in decidualization is still elusive. Here we describe in detail the regional distribution and the effects of progesterone receptors (PGR), estrogen receptors (ESR), and MAPK activation on decidualization. We showed an increase in PGR A, PGR B, ESR1, and phosphorylated MAPK3-1 proteins (p-MAPK3-1), but not in ESR2, in the decidual tissue up to Day 8 of pregnancy. PGR was predominantly found in the nuclei of mesometrial decidual cells and of undifferentiated stromal cells where it colocalizes with ESR2 and ESR1. In the antimesometrial decidua, all the receptors showed cytoplasmic localization. MAPK was activated exclusively in undifferentiated stromal cells of the junctional zone between the antimesometrial and mesometrial decidua and at the border of the antimesometrial decidua. Treatment with the progesterone antagonist onapristone and/or the estrogen antagonist faslodex reduced the extent of decidual tissue and downregulated the levels of PGR and ESR1. The expression level of ESR2 was affected only by the progesterone receptor antagonist, while neither the antiprogestin nor the antiestrogen significantly modified the p-MAPK3-1 level. The inhibition of MAPK3-1 phosphorylation by PD98059 impaired the extent of decidualization and the closure reaction of the implantation chamber, and significantly downregulated ESR1. These results confirm a role of both steroid receptors in the growth and differentiation of the different decidual regions and suggest a new function for p-MAPK3-1 in regulating expression levels of ESR1, thereby maintaining the proliferation capacity of stromal cells and limiting the differentiation process in specified regions of decidual tissues.     

Histochemical studies of mucosubstances and lipids in normal human oesophageal epithelium

Synopsis The histochemistry of lipids and mucosubstances in normal human oesophageal epithelium were studies in biopsies obtained from 24 patients undergoing investigations of the upper gastro-intestinal tract. Neutral fat droplets, 1–2 gmm in diameter, were present in all layers, the greatest number being in the functional layer cells. No unsaturated lipids or fatty acids were demonstrable. Much glycogen was present in the cytoplasm of the prickle and functional cell layers as demonstrated by PAS (and diastase digestion) techniques. The intercellular space of the most superfical of the functional cell layer contained neutral and sialic acid-rich acid mucopolysaccharides. These may be important in protecting the epithelium against physical and chemical trauma.     

Effect of oxygen pressure on glycogen synthesis by rat-liver slices

1. Glycogen synthesized by rat-liver slices 0.5mm. thick incubated at 1atm. oxygen pressure in Hastings medium with glucose was localized in the cells of the periphery of the slice. Cells of the interior of this slice do not synthesize glycogen. 2. Inner cells of thin slices (about 0.3mm. thick) can synthesize glycogen when such slices are incubated under the same conditions, but oxygen pressures higher than 1atm. are required if inner cells of slices 0.5mm. or more thick are to be able to synthesize glycogen. 3. Localization of newly synthesized glycogen in rat-liver slices incubated in Hastings medium with glucose does not depend on glucose concentration. 4. Calculation of the minimum oxygen pressure required to synthesize glycogen gives values between 0.09 and 0.17atm. 5. The advantages of high oxygen pressures for the study of the synthesis of glycogen and other compounds that require ATP are discussed.     

Intrauterine growth retardation in experimental diabetes: possible role of the placenta

Fetal growth disorders are common in pregnancy complicated by diabetes. Whereas macrosomia often occurs in infants of diabetic women, growth retardation is almost a rule in spontaneous and experimental diabetes in animals. However, it is not clear when during development growth inhibition starts and how placental pathology might affect fetal growth in maternal diabetes. In this study pregnant Wistar rats were injected (ip) with a single dose of 50 mg/kg of streptozotocin (STZ) on gestation day (GD) 2 and a blood glucose level of 200 mg/dl or more determined 24 hrs later indicated diabetes. The controls were non-treated, buffer treated or, following confirmation of diabetes, injected with a single dose of 2--6 IU of insulin (Novo Ultralente) once daily. Fetuses and placentae were collected from GD 14--20. Intrauterine growth retardation (IUGR) in STZ group was significant as early as GD 15 and persisted to GD 20. Insulin produced a significant recovery in fetal weight gain. The placentas of STZ-treated group were significantly heavier than those of the control groups. The reduction in cord length of the STZ group became apparent on GD 16 and remained so to term. The placenta of GD 14 STZ group had a thicker decidua basalis and dilated maternal sinusoids. By GD 16, the decidua basalis contained glycogen-containing decidual cells and scattered glycogen cells confirmed by Best's carmine with or without diastase. The glycogen cells of the basal zone were more abundant, and had degenerated in some sites leaving behind cysts with eosinophilic mass. The giant cells had proliferated enormously. The labyrinthine zone appeared spongy with persistent fetal mesenchyme, peri-vascular fibrosis, and enhanced placental barrier. The trophoblasts of the labyrinths also contained traces of glycogen unlike the controls. By GD 18, the decidua basalis of the STZ group was thinner than that of the controls and contained necrotic giant cells and lymphocytic aggregations. In the basal zone, the giant cells had proliferated further; more glycogen cells had degenerated. Perivascular fibrosis was still extensive in the labyrinthine zone. Bloodless maternal sinusoids, extensive vacuolization, degeneration of glycogen islands and formation of cysts characterized the labyrinthine zone. These changes varied in intensity from one area to another in the same placenta and between placentas of the same and of different litters. The development of the upper and lower jaws, elevation and fusion of palatal shelves, reduction of physiological umbilical hernia, descent of the testes, fusion of the urethral folds and separation of digits of the paws were significantly delayed in the STZ group. The consistent association of placental pathology with fetal growth retardation is suggestive of an alteration in placental function possibly contributing to IUGR in STZ-induced diabetes in rats.     

Histochemistry of the glands associated with the reproductive tract ofLymnaea stagnalis

Summary The albumen gland, the muciparous gland and the oöthecal gland of female genital tract of Lymnaea stagnalis, collected in spring, autumn and winter have been studied.The reactions for polysaccharides, proteins and RNA have been performed in order to characterise the secretion of the glands.The albumen gland secretion consists almost exclusively of slightly acid polysaccharides whose histochemical reactions, according to Lison and Grainger and Shillitoe confirm the presence of galactogen. Proteins are also present in the secretion. The muciparous gland secretion consists of strongly acid mucopolysaccharides (non sulphated) produced by large cells among which small cells containing sulphated mucopolysaccharides are present.In the oöthecal gland two zones are present, one with a single type of cells containing acid mucopolysaccharides, and the other with two different types of cells: the first with mucopolysaccharides and the second with sulphated mucopolysaccharides, proteins and glycogen at the basis of the cell. Sialic acids are not present in the secretion of the glands studied.The polysaccharidic composition of the secretion of the glands is different from gland to gland. The secretion of the glands gradually changes and gets acid according to the composition of the various membranes and envelopes wrapping up the eggs.     

Chorioallantoic placenta formation in the rat: I. Luminal epithelial cell death and extracellular matrix modifications in the mesometrial region of implantation chambers.

On days 7 and 8 of pregnancy, mesometrial regions of rat gestation sites were examined by light microscopy and transmission electron microscopy to determine what changes occur before the chorioallantoic placenta forms in that region. By day 7, gestation sites contained a uterine lumen mesometrially and an antimesometrial extension of the uterine lumen, the implantation chamber. The implantation chamber consisted of a mesometrial chamber between the uterine lumen and the conceptus, an antimesometrial chamber that contained the conceptus, and a decidual crypt antimesometrial to the conceptus. Stromal cells that formed the walls of the implantation chamber were closely packed decidual cells, while those that surrounded the uterine lumen were loosely arranged. Late on day 7, a portion of the epithelium lining the mesometrial chamber was degenerating, but this area of initial degeneration was never adjacent to the antimesometrial chamber. By early day 8, most of the epithelial cells lining the mesometrial chamber were degenerating and were being sloughed into the chamber lumen. Although degeneration of these epithelial cells morphologically resembled necrosis, it was precisely controlled, since adjacent epithelial cells lining the uterine lumen remained healthy. The space that separated the denuded luminal surface of the mesometrial chamber from underlying decidual cells became wider and was occupied by an extracellular matrix rich in cross-banded collagen fibrils. Decidual cell processes, that earlier had penetrated the basal lamina beneath healthy epithelial cells, protruded into this matrix and penetrated the basal lamina at the luminal surface. By late day 8, large areas of denuded chamber wall were covered with decidual cell processes, little remained of the basal lamina, and cross-banded collagen fibrils were scarce in the area occupied by decidual cell processes. During the times studied, uterine tissues that formed the walls of the mesometrial chamber were not in direct contact with the conceptus. This study indicates that trophoblast does not play a direct role in epithelial degeneration, basal lamina penetration, or extracellular matrix modifications in the mesometrial region of implantation chambers where part of the chorioallantoic placenta forms, although trophoblast may be required to trigger or modulate some of the changes.     

Transcellular absorption of lipids in the midgut of the stablefly, Stomoxys calcitrans

The midgut of Stomoxys calcitrans is subdivided into several zones. The last of these, the lipoid zone is responsible for the absorption of digestion products. The ultrastructure of the lipoid zone cells is described. Analysis of E.M. autoradiographs of ³H-label derived from absorbed fatty acids shows a strong non-random distribution of label among the various cellular organelles. The majority of label in the lipoid zone cells is incorporated into triglycerides (ca. 60%) and phospholipids (ca. 20%). Triglyceride and phospholipid producing acyltransferases have been localized in the Golgi bodies. After a blood meal the Golgi bodies produce lipoid spheres for the temporary storage of fats and these spheres disappear after completion of meal digestion. Association of lipoid spheres and elements of endoplasmic reticulum is reported and it is suggested that this is the means by which the contents of the spheres are mobilized. The mitochondrial/basal infoldings junctional complex carries out the active concentration of ³H-label and may be important in the movement of fats from the cell to the haemolymph. Vesicles ca. 100 nm in diameter are reported in the extracellular space of the basal infoldings.A tentative proposal for a possible transcellular route (across the lipoid zone cell) for absorbed lipids is made based on the results reported in this communication.     

Localization of casein-rich, fat-rich and DNA-synthesizing cells in monolayer cultures of mid-pregnant mouse mammary epithelium.

Monolayers of 16-day pregnant BALB/cfC3H/Crl mouse mammary epithelial cells were examined for the occurrence and distribution of cells which contain large amounts of casein and fat and those which synthesize DNA. Cells within the central portions of epithelial cells appeared rich in casein and fat, whereas cells on the peripheral edges of the colonies synthesized DNA almost exclusively. Casein deposits and DNA synthesis were mutually exclusive phenomena, since only 2% of the cells synthesizing DNA also stained for casein. Of the casein-rich cells, 74% were also rich in fat, suggesting that cells wich contain large deposits of casein almost always contain large amounts of fat. These results indicate that a specialization of function exists between cells on the growing edge and those centrally located within a single colony of mammary epithelial cells.