Submandibular glands and the regulation of gastric proliferation and TGFα distribution during rat postnatal development

Rodent gastric mucosa grows and differentiates during suckling-weaning transition. Among the molecules in rat milk, EGF and TGFβ are important peptides in the control of cell proliferation, and together with TGFα, they are also produced by submandibular glands. We aimed to determine the effect of saliva and milk on epithelial cell proliferation in the stomach of rat pups. We also examined the distribution of TGFα in the gastric mucosa after sialoadenectomy (SIALO) and fasting in order to determine whether this growth factor is affected by the deprivation of molecules derived from saliva and milk. SIALO was performed at 14 days and fasting was induced 3 days later. Cell proliferation was evaluated through metaphasic index and TGFα was detected by immunohistochemistry. We observed that whereas SIALO did not alter cell division, since the metaphasic index (MI) was unchanged, fasting stimulated cell proliferation (P < 0.05). After SIALO and fasting, MI was reduced when compared to the fasted group (P < 0.05). We found that TGFα is distributed along gastric gland and SIALO did not interfere in the localization and number of immunolabeled cells, but fasting increased their density when compared to the control (P < 0.05). The association of SIALO and fasting reduced TGFα immunostaining (P < 0.05). Therefore, during fasting, high MI was parallel to increased TGFα in gastric epithelium, but interestingly, this effect was found only in the presence of submandibular glands. We suggest that during suckling, peptides derived from saliva and milk are important to regulate gastric growth.

EGFR is involved in control of gastric cell proliferation through activation of MAPK and Src signalling pathways in early-weaned rats.

OBJECTIVES: Early weaning (EW) increases proliferation of the gastric epithelium in parallel with higher expression of transforming growth factor alpha and its receptor epidermal growth factor receptor (EGFR). The primary objective of the present study was to examine involvement of EGFR signalling in regulating mucosal cell proliferation during the early weaning period. MATERIALS AND METHODS: Fifteen-day-old rats were split into two groups: suckling (control) and EW, in which pups were separated from the dam. Animals were killed daily until the 18th day, 3 days after onset of treatment. To investigate the role of EGFR in proliferation control, EW pups were injected with AG1478, an EGFR inhibitor; signalling molecules, proliferative indices and cell cycle-related proteins were evaluated. RESULTS: EW increased ERK1/2 and Src phosphorylation at 17 days, but p-Akt levels were unchanged. Moreover, at 17 days, AG1478 administration impaired ERK phosphorylation, whereas p-Src and p-Akt were not altered. AG1478 treatment reduced mitotic and DNA synthesis indices, which were determined on HE-stained and BrdU-labelled sections. Finally, AG1478 injection decreased p21 levels in the gastric mucosa at 17 days, while no changes were detected in p27, cyclin E, CDK2, cyclin D1 and CDK4 concentrations. CONCLUSIONS: EGFR is part of the mechanism that regulates cell proliferation in rat gastric mucosa during early weaning. We suggest that such responses might depend on activation of MAPK and/or Src signalling pathways and regulation of p21 levels.

Differential distribution of transforming growth factor beta and receptors in the hyper or hypoproliferative gastric mucosa of developing and adult rats.

Transforming growth factor beta (TGFbeta) isoforms are known for their antiproliferative effect on epithelial cells in vitro, but the role of each isoform in vivo is poorly understood, mainly when non-pathological conditions are considered. We correlated the presence and distribution of isoforms and receptors to physiological changes in gastric cell proliferation in developing and adult rats. We used fasting to induce either the hyper (14-day-old pups) or hypoproliferation (60-day-old rats) of the gastric epithelium. In 14-d-old pups fasting reduced only TGFbeta3 labelling in the gland. Conversely, in 60-d-old rats there was an increase of TGFbeta1 and TGFbeta3 immunolabelled cells. Receptors were detected at both ages. Therefore, the changes induced by fasting in the constitutive TGFbeta expression can be correlated to the differential epithelial proliferation in the stomach of developing and adult rats. These results suggest that one of the functional roles of TGFbeta in vivo is to locally regulate cell proliferation.

Ileal VIP submucous neurons: confocal study of the area enlargement induced by myenteric denervation in weanling rats.

Vasoactive Intestinal Peptide (VIP) neurons are maturing during suckling and weaning periods and the neuropeptide VIP is thought to be neurotrophic during ontogenesis. We have previously demonstrated that suckling rats with myenteric ablation have significantly higher mitotic index and an increase on villus height and crypt depth 15 days after treatment. In the current study, we measured the area of VIP neurons of submucous plexus in the ileum of weanling rats, in which myenteric neurons were ablated by serosal application of benzalkonium chloride (BAC). The area of VIP immunoreactive cell bodies, reconstructed under confocal microscope, was significantly increased in response to denervation. This result suggests that the myenteric plexus may have an inhibitory role over submucous plexus in the normal intestine. The enhanced production of VIP may be correlated with the increased epithelial proliferation induced by denervation in a critical period of life, from suckling to weaning time.

Goblet cell number in the ileum of rats denervated during suckling and weaning

The enteric nervous system plays a role on the stimulation of secretory cells of intestinal epithelia. We have demonstrated that ablation of ENS stimulates epithelial cell proliferation. As goblet cells are important constituents of the epithelial sheet, it is mandatory to investigate separately this cell type. The myenteric plexus of the ileum of rats in postnatal development was partially removed by the serosal application of benzalkonium chloride (BAC). Three groups of animals were used: those where BAC application was at 13 days and sacrifice was at 15 (13/28-day-old) or 23 days after treatment (13/36-day-old), and those where BAC was applied at 21 days and rats were killed 15 days after treatment (21/36-day-old) . The number of goblet cells in the ileum was estimated in sections stained by periodic acid-Schiff (PAS) histochemistry. In the 13/28 and 21/36 groups, the number of goblet cells was significantly higher after BAC treatment. These results suggest that the myenteric denervation may have an acute effect on the number of goblet cell in suckling and weanling rats, probably through submucous plexus.

Goblet cell number in the ileum of rats denervated during suckling and weaning

The enteric nervous system plays a role on the stimulation of secretory cells of intestinal epithelia. We have demonstrated that ablation of ENS stimulates epithelial cell proliferation. As goblet cells are important constituents of the epithelial sheet, it is mandatory to investigate separately this cell type. The myenteric plexus of the ileum of rats in postnatal development was partially removed by the serosal application of benzalkonium chloride (BAC). Three groups of animals were used: those where BAC application was at 13 days and sacrifice was at 15 (13/28-day-old) or 23 days after treatment (13/36-day-old), and those where BAC was applied at 21 days and rats were killed 15 days after treatment (21/36-day-old) . The number of goblet cells in the ileum was estimated in sections stained by periodic acid-Schiff (PAS) histochemistry. In the 13/28 and 21/36 groups, the number of goblet cells was significantly higher after BAC treatment. These results suggest that the myenteric denervation may have an acute effect on the number of goblet cell in suckling and weanling rats, probably through submucous plexus. (AU)

Serotonin in the nervous system of the head region of the land planarian Bipalium kewense.

The presence and distribution of serotonin (5-hydroxytryptamine, or 5-HT) in the head region of the land planarian Bipalium kewense has been investigated by an indirect immunofluorescence technique combined with confocal scanning laser microscopy (CSLM), and also by immunogold labeling at ultrastructural level. Serotonin immunoreactivity (IR) was restricted to elements of the nervous system, such as the cerebral ganglion, and the peripheral nerve net. Most of 5-HT-immunoreactive neurons are at the periphery of the brain; they were identified as unipolar, bipolar, and multipolar neurons. The ultrastructural results using immunogold labeling confirm the location of 5-HT within electron-dense vesicles (50-120 nm in diameter), clustered both in the cell bodies and in their processes. The intense 5-HT-IR herein demonstrated for B. kewense adds new data to the poorly studied nervous system of land planarians.

Localization of luteinizing-hormone releasing hormone binding sites in the gastric mucosa of suckling rats.

Luteinizing-hormone releasing hormone (LHRH) is a hypothalamic and milk-borne hormone that inhibits the cell proliferation of gastric epithelium in developing rats, although the mechanism of such action is unknown. We investigated the presence of binding sites for LHRH in the stomach of suckling rats after the injection of the hormone. Immunofluorescence at the confocal microscopy level revealed that LHRH binds to gastric cells, being particularly abundant over the gland. Different fluorescent lectins were used to identify gastric cell types and determine which were labeled by the hormone. Colocalization studies in these double-labeling experiments showed that LHRH staining colocalizes with parietal cells, suggesting the presence of binding sites in these cells. The three-dimensional (3-D) reconstruction of isolated parietal cells revealed the localization of the signal, which appears to be in the membrane of the canalicular region. These results suggest that there are binding sites for LHRH in the gastric epithelium, specifically in parietal cells, and they might play a role in the control of cell proliferation during suckling.

Cell proliferation and death in the gastric epithelium of developing rats after glucocorticoid treatments.

Glucocorticoids take part in the intense morphofunctional modifications that occur in the gastric mucosa during fetal and postnatal development. Two studies were designed to evaluate corticoids role in gastric cell proliferation and apoptosis in developing rats: in vivo, using suckling animals; in vitro, using gastric explants obtained from 20-day fetuses. These explants were cultured in DMEM/F12 medium treated or not with 50 ng/ml of corticosterone; after 22 hr, vincristine was added to the medium for 2 hr to block mitosis. The metaphasic index decreased significantly after the 24-hr treatment (controls: 1.52 +/- 0.53; treated: 0.40 +/- 0.21) and apoptotic cells were visualized under light and electron microscopy. Fifteen-day-old rats were treated with hydrocortisone (25 mg/Kg) for 3 days, and injected with BrDU (100 mg/Kg) 1 hr before sacrifice on the 18th day. BrDu-labeled and non-labeled cells were counted to determine the labeling index of epithelial cells. As apoptotic cells are rapidly eliminated, other animals were treated for only 2-3 hr. Sections were investigated for the presence of apoptotic cells, using morphological criteria and TUNEL labeling. Hydrocortisone significantly reduced the labeling index (controls: 15.6 +/- 1.6 vs. treated: 11.7 +/- 1.1), besides altering the body weight gain. Hydrocortisone treatment doubled the number of apoptotic cells after 2 hr, and quadruplicated it after 3 hr. The results demonstrated that glucocorticoids inhibit cell proliferation in the gastric epithelium of fetuses and suckling rats and increase apoptotic rates, suggesting the exit from cell cycle.

Early weaning and prolonged nursing induce changes in cell proliferation in the gastric epithelium of developing rats.

Food deprivation stimulates cell proliferation in the gastric epithelium of suckling, but not weanling rats. This study was designed to investigate the role of diet on proliferation in developing animals, using early weaning and prolonged nursing models. Rat pups were subjected to these dietary conditions at d 15 and were killed 3 or 7 d afterwards. One day before killing, half of pups were deprived of food. Body weights were recorded. After mitosis blockade, the histologic sections of the stomach were used for the evaluation of cell proliferation and methapasic cell distribution along the gland, and for the measurement of mucosa thickness. Body weight was impaired at 18 d by early weaning and at 22 d by prolonged nursing. Food restriction promoted a 10-15% weight loss regardless of dietary conditions. At 18 d, food deprivation inhibited cell division (P: < 0.01) and reduced the thickness of the mucosa (P: < 0.05) in rats that were weaned early. At 22 d, only the thickness of the mucosa was different between the groups that were subjected to early weaning and prolonged nursing (P: < 0.05), regardless of feeding state. The frequency of dividing cells along the gland was affected by early weaning in 18- and 22-d-old rats. These results suggest the following: 1) food deprivation effects are dependent on dietary condition at 18 d because different proliferative responses were achieved after early weaning and prolonged nursing; 2) the lack of changes after dietary manipulation in 22-d-old rats indicates a nonresponsive period during postnatal development. We conclude that milk is a modulatory factor for cell proliferation in the gastric mucosa of rats.

Effect of myenteric denervation on intestinal epithelium proliferation and migration of suckling and weanling rats.

The effects of myenteric denervation on the cell kinetics of the intestinal epithelium of suckling and weanling rats were investigated. The myenteric plexus of an ileal segment was partially ablated by serosal application of benzalkonium chloride (BAC) in three groups of rats: those that underwent surgery at 13 days and were killed 15 (13/28-day-old) or 23 (13/36-day-old) days after treatment, and those that were operated at 21 days (21/36-day-old) and were killed 15 days after treatment. The extent of denervation was assessed in whole-mount preparations. The cell bodies of myenteric neurones were stained by NADH-diaphorase histochemical technique. Cell proliferation was estimated by the mitotic index (MI) and morphometric analysis of villus and crypt lengths using an image analysis system. Thickness of the muscle layers was also assessed by morphometry. Cell migration on the villi was estimated by the position of the leading labelled cell 24 h after tritiated thymidine injection. The number of neurones was reduced by around 80% in rats operated at 13 days, and reduced by 98% in those operated at 21 days. The thickness of the muscle layers was increased in all groups of treated animals. MI was significantly higher 15 days after BAC-treatment in the 13/28 group. Morphological changes in the intestinal mucosa were observed 15 days after BAC-treatment, when there was an increase in villus height (13/28 group) and crypt depth (13/28 and 21/36 groups). Cell migration rate was accelerated in the 21/36 group. No differences where found in the 13/36 group. These results show the strong effect of myenteric ablation on cell proliferation and migration in the ileal epithelium in the first 15 days of treatment in suckling and in weanling rats, and the subsequent recovery of intestinal mucosa homeostasis later on.

LHRH antagonist inhibits gastric cell proliferation in suckling rats.

The cell proliferation of the gastric epithelium is stimulated by food deprivation in suckling rats, and LHRH inhibits this process as it does in other hyperproliferating tissues. However, as LHRH antagonist is also being used as a potent antiproliferative agent for tumors, this study aims to investigate whether it plays any role on the cell proliferation of the gastric epithelium. Seventeen-day-old rats were fasted for 20 h and treated with LHRH antagonist, LHRH, or both during this period. The following morning, animals were injected with BrDU to label proliferating cells, and were sacrificed 1 h later. Paraffin sections of the gastric mucosa were processed for immunohistochemistry and BrDU labeled and non-labeled epithelial cells were counted to determine the labeling index (LI). None of the hormone treatments changed body weight or the morphology of the stomach. Apoptotic cells were observed in the gastric gland in treated rats. The LI were inhibited by the antagonist, LHRH or both, showing that the LHRH antagonist plays an inhibitory role on the hyperproliferating gastric epithelium. These results suggest that both LHRH agonist and antagonist can influence the proliferative activity in suckling rats, though the mode of action remains to be determined.

A morphological study on posterior gills of the mangrove crab Ucides cordatus.

Morphological and histological studies on posterior gills of the mangrove crab Ucides cordatus showed that the 5th gill (of 7) has a larger surface area and a greater number of lamellae compared to the 6th gill. Regular separation of gill lamellae, important when the gill is in air, is maintained by enlargements of the marginal canals. Conical, spine-like structures along the efferent vessel of both 5th and 6th gills were also observed. In addition, pillar cells, a discontinuous lamellar septum and a hypobranchial artery were observed. The presence of valve-like structures near the efferent vessel was also indicated. These structures, together with the pillar cells, may have a role in directing the hemolymph flow towards certain gills during particular physiological states. Localization of osmoregulatory epithelia in the lamellae of both gills was inferred from dimethylaminostyrylethylpyridiniumiodine staining. Apparently gills 5 and 6 have osmoregulatory epithelial cell patches of similar area, corresponding to 43% and 38% of the total lamellae area, respectively. However, their localization is quite different. Gill number 5 osmoregulatory patches seem to be restricted to the afferent region of the lamella whereas in gill number 6, they are more dispersed over the entire lamella. These differences may be related to the particular functional characteristics of these gills.

Cell proliferation and migration in the jejunum of suckling rats submitted to progressive fasting.

Cell proliferation and migration in the intestinal crypts, and cell migration in the villus are controlled by different mechanisms in adult rats. In the present study, weanling rats and fasting rats were used to quantitatively study the correlation of cell cycle parameters and epithelial cell migration in crypts and intestinal villi. Eighteen-day-old rats received a single injection of tritiated thymidine [3H]TdR (23:00 h); half of the pups were submitted to fasting 5 h earlier. Cell proliferation was determined in radioautographs of jejunal crypts, on the basis of the labeling indices (LI) taken 1, 8, 13 and 19 h after [3H]TdR. The results showed that the labeling index did not differ 1 h or 19 h after [3H]TdR between the fed (38.7% or 48%) and fasting groups (34.6% or 50.4%). The modified method of grain count halving indicated that cell cycle time did not differ between fed (16.5 h) and fasting rats (17.8 h); the growth fraction, however, had lower values in fasting (59%) than in fed rats (77%). Cell migration in the crypt, estimated by the LI obtained for each cell position, did not change with treatment. As for the villi, the cell migration rate was significantly retarded by 3 cell positions (8%). These results suggest that the cell migration in the villi of weanling pups does not depend directly on the cell proliferation and migration in the intestinal crypt, but is directly affected by the absence of food in the lumen.

Feeding manipulation elicits different proliferative responses in the gastrointestinal tract of suckling and weanling rats.

Food deprivation has been found to stimulate cell proliferation in the gastric mucosa of suckling rats, whereas the weanling period has been reported to be unresponsive in terms of proliferative activity. In the present study we analyze regional differences in the effect of milk or food deprivation on cell proliferation of the epithelia of the esophagus and of five segments of small intestine in suckling, weanling and newly weaned Wistar rats of both sexes. DNA synthesis was determined using tritiated thymidine to obtain labeling indices (LI); crypt depth and villus height were also determined. Milk deprivation decreased LI by 50% in the esophagus (from 15 to 8.35%) and small intestine (from 40 to 20%) of 14-day-old rats. In 18-day-old rats, milk and food deprivation decreased LI in the esophagus (from 13 to 5%) and in the distal segments of the small intestine (from 36-40 to 24-32%). In contrast, the LI of the epithelia of the esophagus (5%) and of all small intestine segments (around 30%) of 22-day-old rats were not modified by food deprivation. Crypt depth did not change after treatment (80 to 120 microns in 14- and 22-day-old rats, respectively). Villus height decreased in some small intestine segments of unfed 14- (from 400 to 300 microns) and 18-day-old rats (from 480 to 360 microns). The results show that, contrary to the stomach response, milk deprivation inhibited cell proliferation in the esophagus and small intestine of suckling rats, demonstrating the regional variability of each segment of the gastrointestinal tract in suckling rats. In newly weaned rats, food deprivation did not alter the proliferation of these epithelia, similarly to the stomach, indicating that weanling is a period marked by the insensitivity of gastrointestinal epithelia to dietary alterations.

Feeding manipulation elicits different proliferative responses in the gastrointestinal tract of suckling and weanling rats

Food deprivation has been found to stimulate cell proliferation in the gastric mucosa of suckling rats, whereas the weanling period has been reported to be unresponsive in terms of proliferative activity. In the present study we analyze regional differences in the effect of milk or food deprivation on cell proliferation of the epithelia of the esophagus and of five segments of small intestine in suckling, weanling and newly weaned Wistar rats of both sexes. DNA synthesis was determined using tritiated thymidine to obtain labeling indices (LI); crypt depth and villus height were also determined. Milk deprivation decreased LI by 50 percent in the esophagus (from 15 to 8.35 percent) and small intestine (from 40 to 20 percent) of 14-days-old rats. In 18-days-old rats, milk and food deprivation decreased LI in the esophagus (from 13 to 5 percent) and in the distal segments of the small intestine (from 36-40 to 24-32 percent). In contrast, the LI of the epithelia of the esophagus (5 percent) and of all small intestine segments (around 30 percent) of 22-day-old rats were not modified by food deprivation. Crypt depth did not change after treatment (80 to 120 mum in 14- and 22-day-old rats, respectively). Villus height decreased in some small intestine segments of unfed 14- (from 400 to 300 mum) and 18-day-old rats (from 480 to 360 mum). The results show that, contrary to the stomach response, milk deprivation inhibited cell proliferation in the esophagus and small intestine of suckling rats, demonstrating the regional variability of each segment of the gastrointestinal tract in suckling rats. In newly weaned rats, food deprivation did not alter the proliferation of these epithelia, similarly to the stomach, indicating that weanling is a period marked by the insensitivity of gastrointestinal epithelia to dietary alterations.

Corticosterone treatment inhibits cell proliferation in the gastric epithelium of suckling rats.

During the 3rd postnatal week, the rat gastrointestinal tract undergoes major changes which depend on the different factors such as dietary transition and hormones. Glucocorticoids are closely involved in these processes, yet a clear proliferative response to these agents in the gastric epithelium has not yet been established. The aim of this study was to determine the effect of corticosterone on cell proliferation in the gastric mucosa of suckling rats. We also measured plasma corticosterone concentration in fed and fasted suckling (18-day-old) and weaning rats (22-day-old) observing that fasting increased the levels in animals of both ages (P < 0.05). Cell kinetic parameters, i.e., metaphase index, cell production rate, and potential doubling time, were obtained by vincristine blockade in control and corticosterone-treated 18-day-old pups. The metaphase index was strongly inhibited by corticosterone (P < 0.001), as was the cell production rate (P < 0.05), indicating a high potential doubling time in treated rats. Mucosal and glandular thickness were also measured, but no differences between treated and untreated animals were found. The results suggest that corticosterone treatment has an inhibitory effect on cell proliferation in the gastric mucosa in suckling rats, and that the high endogenous levels observed during fasting do not affect the proliferative activity.

Cell proliferation and migration in the jejunum of suckling rats submitted to progressive fasting

Cell proliferation and migration in the intestinal crypts, and cell migration in the villus are controlled by different mechanisms in adult rats. In the present study, weanling rats and fasting rats were used to quantitatively study the correlation of cell cycle parameters and epithelial cell migration in crypts and intestinal villi. Eighteen-day-old rats received a single injection of tritiated thymidine[3H]T dR (23:00 h); half of the pups were submitted to fasting 5 h earlier. Cell proliferation was determined in radioautographs of jejunal crypts, on the basis of the labeling indices (LI) taken 1,8,13 and 19 h after [3H]TdR. The results showed that the labeling index did not differ 1 h or 19 h after [3H]TdR between the fed (38.7 percent or 48 percent) and fasting groups (34.6 percent or 50.4 percent). The modified method of grain count halving indicated that cell cycle time did not differ between fed (16.5 h) and fasting rats (17.8 h); the growth fraction, however, had lower values in fasting (59 percent) than in fed rats (77 percent). Cell migration in the crypt, estimated by the LI obtained for each cell position, did not change with treatment. As for the villi, the cell migration rate was significantly retarded by 3 cell positions (8 percent). These results suggest that the cell migration in the villi of weanling pups does not depend directly on the cell proliferation and migration in the intestinal crypt, but is directly affected by the absence of food in the lumen.

LHRH and somatostatin effects on the cell proliferation of the gastric epithelium of suckling and weaning rats.

The aim of this study was to investigate the effects of LHRH and somatostatin on the cell proliferation of the gastric epithelium of suckling and weaning rats after fasting treatment. Previous studies on the cell proliferation of the gastric epithelium have shown that fasting stimulates this process in suckling, but not in weaning and adult rats. As milk is the most important source of nutrients and hormones during the suckling phase, and luteinizing hormone-releasing hormone (LHRH) and somatostatin are found in milk, their possible inhibitory roles on the gastric epithelium were investigated. Metaphasic index was achieved by vincristine blockade in 18- and 22-day-old treated and non-treated rats. The results showed that at 18 days, both hormones inhibited the enhanced proliferation activity due to fasting treatment, while at 22 days, no effect was detected. Therefore, LHRH and somatostatin were considered to have inhibitory roles on the cell proliferation of the gastric epithelium in suckling rats only.

Extensive networks of TMPase positive basal lysosomes are present in fetal rat gastric epithelium before overt differentiation.

The rat gastric epithelium of 17 to 19 days of intrauterine life changes from a pseudostratified epithelium to a simple columnar epithelium. From a flat epithelium at 17-18 days, groups of epithelial cells begin to invaginate towards a cellularly rich mesenchyma and foveolae appear at 19 fetal days. Ultrastructural differentiation of the first cells of gastric glands occurs at 19 days. The ultrastructure of the undifferentiated cells at 17-18 fetal days shows extensive distribution of granules in the basal cytoplasm, near the basal lamina. To investigate the nature of these structures, enzyme cytochemistry was carried out. Acid phosphatase and trimetaphosphatase activities were demonstrated cytochemically in the gastric epithelium of the fetal rat. Whereas acid phosphatase positive lysosomes were observed mainly in typical round lysosomes, near the Golgi, a strong positive reaction for trimetaphosphatase, at 17 and 18 fetal days, was present in granules and tubular structures located in the basal cytoplasm. This reaction was coincident with the observed basal granules. At 19 days, the number of basal TMPase positive lysosomes was diminished, but other lateral tubular and vesicular positive structures were seen. The presence of coated pits in the basal membrane and microtubules and coated vesicles between tubular lysosomes, at 17-18 days, just before overt differentiation, reinforces the possibility of the association of this kind of lysosome with endosomes and the internalization of receptors.