Age-related changes in rat thymic epithelial cells

We investigated age-related changes in immunocytochemical localisation of cytokeratin 16 (CK16) in thymuses of female Wistar rats at various stages of adult life (months 1, 3, 6, 12). Within the 1 st month of life, distribution of CK typical for individual subsets of thymic epithelial cells (TEC) was observed. The most numerous CK16+ TEC were observed in the outer region of medulla, in the outer cells of Hassall's corpuscles and in the superficial epithelial layer neighbouring the connective tissue of the capsule, septa and vessels of the thymus. In the 3rd month of life, increased intensity of CK16 reaction in superficial TEC was accompanied by increased numbers of CK 16+ TEC in the outer region of the medulla. Age-related alterations in the distribution of the studied markers were evident beginning from the 6th month of life and involved increased expression of CK16 in the superficial layer of TEC, which at the interface with the septa formed stratified epithelium. In parallel, decreased numbers of CK16+ TEC were observed in the outer region of the medulla. Changes in CK16+ TEC distribution of a similar type developed in 12-month old rats and they probably reflected altered functions of some TEC populations and decreased or increased biological activity of other TEC populations.     

Histogenesis of the rat thymic medulla during first stages of development

We investigated first stages of thymic medulla organisation in foetuses of Wistar strain rats. between 13th and 17th days of foetal life (GD). Medullary cells were identified by immunocytochemical localisation of neuron-specific enolase (NSE) as well as by traits of ultrastructure. The first thymic medullary precursor cells which were reactive for NSE were at first spread all over the thymic primordium. In the period of thymus colonisation by lymphoid cells, the following stages were distinguished in medulla organisation: (1) migration of NSE+ cells to the central portion of the thymus (GD 14-15), (2) small medullary epithelial patches, distributed within the thymus (GD 16), and (3) expansion of medullary patches into medullary compartment (GD 17). At the second and third stages of the medulla organisation, an increase in the number of NSE+ cells, followed by differentiation of their ultrastructure and increase in their biological activity were observed. We conclude that formation of medullary architectural pattern is controlled by interactions between maturing epithelial cells and developing lymphoid cells and by angiogenesis in the region.     

Laminin ultrastructural immunolocalization in rat testis during ontogenesis

Summary The synthesis of one of the main glycoproteins of the basement membrane, the laminin, was demonstrated by ultrastructural immunolocalization during rat foetal (16th day to 20th day of gestation) and postnatal development of the testis. The lamina densa, part of seminiferous tubular basement membrane, is labeled uniformly at all studied stages. The lamina lucida is not well defined before the postnatal stages, at which times discrete immunostaining extends from the lamina densa to the adjacent seminiferous epithelial cells (spermatogonia and Sertoli cells). The extracellular matrix around the peritubular cells is not labeled before birth. Intracellular immunostaining was detected as early as the 16th day of gestation in both Sertoli cells and cells around the seminiferous tubules which will transform later into peritubular cells. It was located in rough endoplasmic reticulum (RER) cisternae and secretory vesicles. After 18–20 days of postnatal life, the immunostaining faints progressively. Some positive material is seen in the RER of the gonocytes at all studied stages.Sertoli cells and peritubular cells are the main producing cells of laminin after the 16th of gestation. The laminin secreted by gonocytes may play an important role in adhesion of gonocytes to the lamina densa and adjacent Sertoli cells before their transition from basal compartment to adluminal compartment.     

Growth hormone receptor gene expression in the skeletal muscle of normal and double-muscled bovines during foetal development

The expression of the growth hormone receptor (GHR) gene was investigated in semitendinosus muscle during bovine foetal development in both normal and double-muscled Charolais foetuses which differ with respect to muscle development. Northern-blot analysis of foetal muscle RNA preparations with a GHR cDNA probe identified the 4.5 kb GHR mRNA as early as 130 days post-conception. In double-muscled animals, the expression of GHR mRNA increased from 130 to 210 days of gestation while it stayed stable in normal ones. It was significantly higher (P < 0.05) in double-muscled foetuses compared to normal ones from the second third of gestation. Northern-blot analysis of foetal muscle RNA preparations from both genotypes with a beta-actin cDNA probe, revealed lower beta-actin gene expression in double-muscled foetuses than in normal ones, suggesting a delay in the differentiation of muscle cells. In situ hybridisation revealed the localisation of specific GHR mRNA in muscle cells at all gestation stages analysed (130, 170, 210 days post-conception) but not in connective tissue surrounding the muscle cells. At the adult stage, the hybridisation signal was also very high and observed in muscle cells only. These results show the ontogeny of GHR mRNA in bovine muscle and demonstrate a difference between normal and double-muscled animals.     

小鼠胚胎发育过程中表皮生长因子受体的免疫荧光定位研究

用间接免疫荧光法研究了第12—19天小鼠胚胎的表皮生长因子受体(EGFR)在各种组织中的分布。结果表明,特异性荧光出现在EGFR阳性细胞的细胞膜上。第14.5天鼠胚鼻粘膜上皮首先显示很强的EGFR特异性荧光,此后荧光稍为减弱,直至第19天后消失。消化系统中,舌味觉上皮在第16天、胃粘膜上皮在第17—18天间、十二指肠上皮在第17.5—18.5天、直肠粘膜上皮在第15.5—16天和肛管粘膜上皮在第15天均显示强特异性荧光。肝细胞从第14.5天起有弱阳性反应,随胎龄增大强度缓慢地增强。此外,在胚胎发育不同时期,还看到若干组织呈现阳性反应,包括膀胱粘膜变移上皮、眼睑原基、腺垂体上皮、舌下腺腺泡细胞、附属腺上皮细胞、胰岛细胞、颌下腺腺上皮和导管上皮细胞、降主动脉内皮以及卵巢髓质部富含血管的疏松结缔组织中的成纤维细胞等。     

Development of chequerboard pattern in human foetal skeletal muscle.

The study was undertaken to race the histochemical and electron microscopic development patterns of human myogenesis from the 9th to the 26th week of foetal life. Particular attention was paid to the possibility of appearance of metabolic or structural differences between individual skeletal muscle fibres in early periods of myogenesis. The 9th week of foetal life is the period when primitive myotubes are formed. Irregular distribution of the ATPase (pH 9-4) activity observed at this time is due to differences in the structure of fusing myoblasts. The early myotubes show a high activity of the oxidative enzymes and lack of phosphorylase. Conversion of immature muscle cells into structurally mature fibres begins between the 20th and the 24th week. The latter fibres exhibit a uniform activity of all the studied enzymes and thus resemble the intermediate type of fibres of mature muscles. From about the 26th week on the typical mosaic pattern of the enzymatic activity is observed. All the differences in enzymatic activity which appear in fibers prior to their full morphologicaal maturity result from differences in developmental stages of the fibres at the given moment. The present study also suggests that there are no morphological or metabolic differences between individual humanskeletal muscle fibres prior to their metabolic differentiation into types which occurs after their innervation.     

Cytokeratin expression in primary epithelial cell culture from bovine conjunctiva

Aim of the present study is to extend our previous observations on a model of primary epithelial cell culture obtained from bovine conjunctiva, and analyse the maintenance of the conjunctival phenotype, relative to cytokeratin (CK) expression, through extended periods of cultivation under different conditions. Conjunctival epithelial cells were grown in transwell filters, and cultured either under liquid covered (LC), or air-interface (AI) conditions. The physiological state of the cells was monitored daily by measurement of the trans-epithelial electrical resistance (TEER). Analysis of cytokeratin expression was then carried out at different time points (up until 14 days), and compared to the original profile of the conjunctival tissue in order to assess deviations from the primitive phenotype. Immunodetection studies, carried out by both western immunoblot and immunofluorescence analyses, revealed constant expression of the pan-epithelial marker AE3 (recognizing basic type cytokeratins), confirming the epithelial nature of the culture. Other cytokeratins characteristic of non-keratinized stratified epithelia (CK4 and CK13) were absent in corneal tissue, while in conjunctival epithelial cells were more expressed under AI than under LC culture conditions. Expression of CK12, a specific marker of corneal tissue, revealed by the antibody AE5, was never observed in conjunctival epithelial cells. These results indicate that the conjunctival phenotype is conserved during extended periods of culturing, making this system a reliable substitute of conjunctival tissue for pharmaceutical analyses.     

Critical and distinct roles of p16 and telomerase in regulating the proliferative life span of normal human prostate epithelial progenitor cells

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, alpha2beta1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.     

Laminin ultrastructural immunolocalization in rat testis during ontogenesis

The synthesis of one of the main glycoproteins of the basement membrane, the laminin, was demonstrated by ultrastructural immunolocalization during rat foetal (16th day to 20th day of gestation) and postnatal development of the testis. The lamina densa, part of seminiferous tubular basement membrane, is labeled uniformly at all studied stages. The lamina lucida is not well defined before the postnatal stages, at which times discrete immunostaining extends from the lamina densa to the adjacent seminiferous epithelial cells (spermatogonia and Sertoli cells). The extracellular matrix around the peritubular cells is not labeled before birth. Intracellular immunostaining was detected as early as the 16th day of gestation in both Sertoli cells and cells around the seminiferous tubules which will transform later into peritubular cells. It was located in rough endoplasmic reticulum (RER) cisternae and secretory vesicles. After 18-20 days of postnatal life, the immunostaining faints progressively. Some positive material is seen in the RER of the gonocytes at all studied stages. Sertoli cells and peritubular cells are the main producing cells of laminin after the 16th of gestation. The laminin secreted by gonocytes may play an important role in adhesion of gonocytes to the lamina densa and adjacent Sertoli cells before their transition from basal compartment to adluminal compartment.     

Generation of a Novel MMTV-tTA Transgenic Mouse Strain for the Targeted Expression of Genes in the Embryonic and Postnatal Mammary Gland

We have generated a new and improved transgenic mouse strain that permits a temporally controlled expression of transgenes throughout mammary gland development. High expression of the tetracycline-regulatible transactivator (tTA) under control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) is restricted to mammary epithelial cells and the salivary gland. The novel MMTV-tTA mouse strain induces a sustained transactivation of responder transgenes, which can be swiftly suppressed through administration of doxycycline (Dox). An important characteristic of this strain is its expression in early progenitor cells of mammary gland anlagen beginning at day 13.5 of embryonic development. We show here that the MMTV-tTA can be used in combination with GFP reporter strains to visualize CK8/CK14-dual positive progenitors in newborn females and their derived basal and luminal epithelial cell lineages in adult females. Our observations suggest that the novel MMTV-tTA can be utilized to express exogenous proteins in multipotent mammary progenitors during the earliest stages of mammary gland development to assess their biological significance throughout mammogenesis. Moreover, we demonstrate that the expression of the MMTV-tTA is sustained during mammary gland tumorigenesis in female mice expressing wildtype ErbB2. This makes this strain particular valuable to target the expression of exogenous proteins into developing mammary tumors to assess their significance in biological processes, such as tumor cell growth and survival, metabolism, and metastasis.     

Ontogeny of erythropoietin mRNA expression in liver, kidneys and testes of the foetal and the neonatal pig

Erythropoietin (EPO) mRNA expression in kidneys, liver and testes of foetal and neonatal pigs was analysed using a competitive RT-PCR assay. The results indicate that in the foetal pig, erythropoietin expression is greatest in the liver, at birth; hepatic and renal expression are nearly identical, and by 5 weeks of age there is mainly renal expression. The dynamics of the renal expression of EPO mRNA in the perinatal period provide a correlate for observations made earlier of plasma EPO concentrations. Early in foetal life (30 days after artificial insemination), the mesonephroi contained large amounts of EPO mRNA. As in the rat, the testes produced EPO mRNA in amounts comparable to the liver on a per gram tissue basis, though much less on a per organ basis.     

The development of the guinea pig gallbladder epithelial cells. II. Electron microscopical and enzymhistochemical investigations (author's transl)]

The development of the guinea pig gallbladder epithelium follows a distinct time schedule. During the first phase (up to 30th day of intrauterine life) the epithelial cells increase in number. They remain small and undifferentiated. In the second phase, from the 30th to the 44th day, cytodifferentiation is a dominating feature. The epithelial cells increase in height, the nuclei become more basal, the cells acquire their final zonal structure and the cell organelles exhibit their characteeristic appearance. Weak enzyme activities can be observed. In the third phase, from the 45th day until birth, there is functional differentiation and the adult pattern of enzyme distribution is established. Glycogen appears first on day 29, increases in amount and then disappears by day 57. There is a marked development of the Golgi apparatus associated with increased synthesis of secretory material. Between the 59th embryonic day and the 6th day of life mucoid cells with different functional states appear in the gallbladder epithelium. Later on these cells can be visualized only in the depths of the invaginations. After birth the epithelial cells become more columnar and by 10 days after birth the adult appearance of the epithelium is fully established.     

Modulation of the expression of the Cip/Kip family of cyclin-dependent kinase inhibitors in foetal developing lungs of hamsters

We examine the cell proliferation activity and expression of cyclin-dependent kinase inhibitors of the Cip/Kip family, p21Cip1, p27Kip1 and p57Kip2, in foetal hamster lungs to determine the expression patterns of the cyclin-dependent kinase inhibitors and to clarify the relationship between expression of the cyclin-dependent kinase inhibitors and lung development. Foetal hamster lungs on gestational days 12.5-16 (the day of birth) and adult lungs were fixed in 4% paraformaldehyde. Frozen sections were immunostained for the cyclin-dependent kinase inhibitors, and examined by immunostaining for Ki-67 and bromodeoxyuridine to determine the proliferation activity of the foetal lungs. During the foetal period, cell proliferation activity, as analysed by Ki-67 or bromodeoxyuridine labelling, decreased with development of the lung. In contrast to the gradual decrease of cell proliferation activity, cells with p27Kip1 immunoreactivity increased with development. On the other hand, p21Cip1-positive cells were most prominent around gestational day 14.5, while after birth positive cells decreased markedly. A few p57Kip2-positive cells were detected in the bronchiolar epithelium on gestational day 14.5. Western blotting analyses confirmed these immunostaining patterns. Thus, the levels of the cyclin-dependent kinase inhibitors of the Cip/Kip family are modulated in the lungs during the foetal period, and each shows a unique expression pattern. The cyclin-dependent kinase inhibitors may play roles not only in regulating cell proliferation activity but also in regulating other functions such as differentiation in the lung during the foetal period.     

Selective maintenance of cultured epithelial cells from DMBA-induced mammary tumours by bovine colostrum supplement

Growth kinetics in cultures of DMBA-induced mammary tumours supplemented with bovine colostrum were compared with the kinetics of cultures maintained with the conventional supplement of foetal calf serum. Although the latter permitted a greater degree of cell proliferation, a substantial amount of the cell growth was due to the fibroblastic proliferation. In the presence of bovine colostrum, epithelial islands surrounded by a few solitary cells became established. Unlike the foetal calf serum supplemented cultures, these cultures frequently did not become completely confluent within 7 days. The absence of fibroblasts in colostrum supplemented cultures was confirmed by electron microscopy. Results from this study suggest that colostrum may be useful in selective maintenance of primary cultures of epithelial origin.     

Optical and scanning electron microscopy observation of the mucosa of human fetal tongue

The structural features of the human foetal tongue have been studied in foetuses from 8th to 20th week of pregnancy. The characteristics of the developing papillae as well as of epithelial and mesenchymal layers have been pointed out. An early differentiation of the mesenchymal tissue has been observed, concerning phenomena of cellular condensation and reticular fibers organization both in superficial and deep layers. The hypothesis of the existence of straight interactions between epithelium and mesenchyme also in the developing human tongue mucosa has been suggested. Also the observations at SEM demonstrate that from the 8th to the 20th week the epithelial surface of the tongue reaches a stable structural pattern. From 11th week a characteristic cellular polymorphism occurs: cells with microvilli that diminish progressively, ciliated cells that disappear almost completely at the 20th week and cells whose free surface show microplicae, definitive stage of the tongue cell evolution.     

Ultrastructural features of the developing hypothalamo-hypophysial axis in the rat

Summary Nerve fibres containing granular vesicles first appear in the median eminence of the rat on the 16th foetal day while secretory granules in the cells of the adenohypophysis are not present till the 17th foetal day. These observations suggest that the differentiation and early activity of pars distalis cells may depend on substances elaborated at nerve terminals in the median eminence. Although the loops of the primary plexus of portal vessels do not develop until the 4th postnatal day, substances released by nerve fibres in the neurohypophysis could reach the pars distalis through vessels already present at the 15th foetal day in the mesenchyme between the diencephalon and the adenohypophysis. This view is supported by the fact that the earliest cells to exhibit ultrastructural evidence of secretory activity are in the rostral pole of the pars distalis, the first region of the gland to become vascularized. The earliest granules to appear in the cells of the pars distalis correspond to those which are considered to contain mucoprotein hormones; somatotrophin type granules were seen only in postnatal tissues.The finding that, in the median eminence, the development of granular vesicles precedes that of agranular vesicles is discussed with reference to the times at which neurosecretory materials and monoamines become detectable in the region.We should like to thank Miss Ann Pearson, Mr. D. Burns, and Mr. J. Nailon for their technical assistance, and Mr. J. Simmons, F.R.P.S., for his help in the preparation of illustrations. This work was supported by grants from the National Health and Medical Research Council of Australia.     

Bisphenol A induces permanent squamous change in mouse prostatic epithelium

Bisphenol A (BPA) is a monomer of plastic products widely used in daily life, and has weak estrogenic activity. In this study, male BALB/c mice were treated with BPA and diethylstilbestrol (DES) in adult and fetal periods to investigate whether BPA could affect prostatic epithelial differentiation. Eight-to 9-week-old mice treated for 3 weeks with subcutaneous implants of 0.2-200 mg BPA pellets induced the expression of cytokeratin 10 (CK10) in prostatic basal epithelial cells in a dose-dependent manner. Utilizing organ culture of adult prostate, 1 nM and 1 microM BPA also induced CK10 expression and squamous metaplasia with multilayering of basal epithelial cells, respectively. Fetal exposure to low-dose BPA (20 microg/kg/day) from gestation day (GD) 13 to GD18 induced permanent CK10 expression in basal cells of the adult prostate similar to DES (0.2 microg/kg/day). These results indicate that in mouse, BPA can directly elicit CK10 expression in prostatic epithelium, and that this change can be elicited by doses as low as 20 microg/kg/day. We speculate that low-dose BPA during fetal life may also induce permanent squamous change in human prostate.     

ERYTHROPOIETIC CELL POPULATION CHANGES DURING THE HEPATIC PHASE OF ERYTHROPOIESIS IN THE FOETAL MOUSE

Estimates have been made of the absolute numbers of hepatogenic erythropoietic cells from 12.5 days post fertilization onwards in the mouse. All stages of maturation up to reticulocytes are present in the earliest samples but the least mature cells (proerythroblasts and basophilic erythroblasts) predominate; more mature cells (orthochromatic erythroblasts, reticulocytes and erythrocytes) predominate later in development. The number of hepatogenic haemoglobinized cells increases exponentially with a population doubling time of about 8 hr until about 15.5 days post fertilization. There is then a sharp transition and the doubling time lengthens to about 2 days. The immature cells formed during the rapid phase of increase are poorly haemoglobinized; hence the increase in haemoglobin lags behind that of cells. Calculations of the rates of formation of hepatogenic haemoglobinized cells and haemoglobin per standard number of liver cells show maxima between 15 and 16 days; these findings are in accord with direct observations of rates of haemoglobin synthesis in cultured mouse foetal livers made previously.     

Expression of cytokeratin 8, vimentin,syndecan-1 and Ki-67 during human tooth development

Spatio-temporal immunolocalizations of cytokeratin 8 (CK8), vimentin, syndecan-1 and Ki-67 were analyzed in ten human incisors and canine tooth germs between the 7th and 20th developmental weeks. CK8 expression was mild to moderate in the epithelial tooth parts, while it shifted from absent or mild in its mesenchymal parts, but few cells, sparsely distributed throughout the tooth germ, strongly expressed CK8. As development progressed, CK8 expression increased to strong in preameloblasts, while expression of vimentin increased to moderate in the epithelial and mesenchymal tooth parts, particularly in the dental papilla and sac. Co-expression of CK8 and vimentin was observed in some parts of the tooth germ, and was increasing in the differentiating preameloblasts and preodontoblasts. Syndecan-1 showed characteristic shift of expression from epithelial to mesenchymal tooth parts, being particularly strong in dental papilla, sac and cervical loops, while co-expression of Ki-67/syndecan-1 was strong in the dental papilla. Our study demonstrated spatio-temporal expression and restricted co-expression of the investigated markers, indicating participation of CK8 and vimentin in cell proliferation and migration, and differentiation of preodontoblasts and preameloblasts. Our data also suggest involvement of syndecan-1 in morphogenesis of the developing tooth crown and cervical loops, and together with CK8 and vimentin in differentiation of preameloblasts and preodontoblasts.