[Development of human fetal neuroretina grafted into nude mice]. / Développement de rétines foetales humaines après implantation de globes oculaires chez la souris nude.

PURPOSE: To determine the role of different ocular tissues in the development of the human fetal neuroretina heterotopically implanted in nude mice. MATERIAL AND METHODS: Fifty eight eyeballs obtained from legally aborted 6- to 7-week-old embryos or 8- to 10-week-old fetuses were heterotopically implanted in nude mice. Over a period of 1-245 days, all the grafts were removed for light and electron microscopy observations. RESULTS: All grafts were successful. Twenty six exhibited a normal histological organization of the choriocapillaris, the retinal pigment epithelium, and the neuroretina in the posterior part of the eye. Photoreceptor differentiation was observable approximately 80 days after transplantation and was complete at 166 days. The anterior part of the retina was always dysplasic. Twenty three eyes had a dysplasic neuroretina with folds, rosettes, and necrotized areas. In absence of retinal pigment epithelium, the neuroretina was always dysplasic or absent. Nine eyes were atrophic without any differentiation of the neuroretina. CONCLUSION: These results demonstrate that the development of a stratified neuroretina requires both rapid revascularization and normal development of retinal pigment epithelium. When these conditions are not met, the neuroretina becomes dysplasic or atrophic or disappears.

Paracellular transport of avidin saturated or not with biotinylated cobalamin through Caco-2 cell epithelium monolayer.

BACKGROUND: The cationic charge of molecules may promote their uptake across epithelia, which are rich in brush border anionic sites. The transport of unsaturated avidin and avidin saturated with a biotinylated compound was investigated across Caco-2 adenocarcinoma cell with fetal enterocyte phenotype. METHODS: The unsaturated avidin and avidin saturated with either biotin or a biotinyl-cobalamin conjugate (biotinyl-Cbl) were iodinated to follow their transport through the cell monolayer. Their apparent permeability coefficient (Papp) and transepithelial pathway were determined and compared to those for control radiolabeled markers [3H]-mannitol, [125I]-beta-lactoglobulin and [57Co]-cobalamin/intrinsic factor (Cbl/IF). RESULTS: The Papp of [125I]-avidin estimated at 2.8 x 10(-7) +/- 0.08 cm/s was close to that for mannitol that uses paracellular pathway. The binding of biotin or biotin conjugate to avidin enhanced its tetrameric conformation. The Papp for [125I]-avidin/biotin and [125I]- avidin/biotinyl-Cbl were respectively increased by 2-fold, compared to that for [125I]-avidin and 4-fold, compared to that for [125I]-beta-lactoglobulin and [54Co]-Cbl/IF. The protein was not accumulated in the cell and was found in intact form in the basolateral side, after its transport across the monolayer. Chloroquine (0.66 micromol/ml) did not significantly decrease the Papp for [125I]-avidin/biotinyl-Cbl. Conversely it decreased by 80% the Papp for Cbl/IF, that uses transepithelial pathway. CONCLUSIONS: Avidin (either saturated or not with biotin and biotinyl-Cbl) was able to cross the monolayer of Caco-2 cell line through a paracellular pathway. This study pointed out the interest for using this protein as a shuttle for increasing the transport rate of biotinylated compounds through fetal epithelial barriers.

Overexpression of folate binding protein alpha is one of the mechanism explaining the adaptation of HT29 cells to high concentration of methotrexate.

The human colon adenocarcinoma cell line HT29 can be adapted to 10(-7)- 10(-4) M concentrations of methotrexate (MTX). Cells adapted to 10(-4) M MTX have an enterocyte-like phenotype with DHFR gene amplification. Presently, we hypothetized that an increased expression of folate binding protein (FBP) may participate to the MTX resistance of 10(-4) MTX HT29 cells. The cDNA FBPalpha/beta-actin ratio of amplified transcripts was 4.8- and 1.5- fold higher in 10(-4) and in 10(-7) M MTX HT29 respectively, than in standard type HT29 cells. An increase of transcript level was observed when decreasing folic acid concentration. PI-PLC cleaved 7.7 times more membrane FBP in 10(-4) M than in 10(-7) M MTX and wild type HT29 cells. In contrast to 10(-7) M MTX cells, growth of 10(-4) M MTX cells was dependent on folic acid concentration and abolished at a concentration lower than 0.9 microM. In conclusion, the adaptive mechanism of HT29 cells resistant to 10(-4) M MTX is the result of the synergistic overexpression of both DHFR and FBPalpha. Overexpression of FBPalpha may be related to the enterocyte-like phenotype of the cells.

Morphologic and functional development of whole human fetal stomachs grafted into nude mice.

To study in vivo the cellular differentiation and secretion of human developing fetal stomach, ethically and technically impossible to perform in utero, 256 fetal stomachs were xenografted. Human stomachs from 6- to 10-week-old fetuses were grafted for 1-273 days into nude mice. Biopsies for immunohistochemistry, hybridization and electron microscopy were taken and a catheter introduced into the human stomach. Macroscopic growth was fast and cells in S phase were numerous during the first 9 weeks, then the stomach size was stable and the gastric mucosa, of adult type, remained normal. In situ hybridization detected only a minute mouse mesenchymal chimerism in the graft. Chromogranin A, intrinsic factor and H+/K+ adenosine triphosphatase were immunohistolocally detected in epithelial cells 20 days after grafting, gastrin was detected after 30 days and pepsinogen after 60 days. The pH in gastric juice, which was at 8.0 +/- 0.1 from days 10-25, dropped from 4.39 +/- 1.80 at 30 days to 1.58 +/- 0.29 at 90 days. Intrinsic factor was stable and pepsin ranged from 6.8 +/- 7.8 to 134 +/- 51 units at 90 days. The differentiation of the epithelial cells in xenografts was very accelerated in comparison to that in utero.

Bactericidal properties of the chloroform fraction from rhizomes of Aristolochia paucinervis Pomel.

The deffated chloroform fraction (APRC) obtained from the rhizomes of Aristolochia paucinervis Pomel (Aristolochiaceae) has a high bacteriostatic activity against bacterial strains like Clostridium perfringens ATCC 13124 and Enterococcus faecalis ATCC 29212. Here, we report the bactericidal activity of APRC against both strains which was evaluated by using time-to kill assays. The results showed that APRC produced an intense time-dependent bactericidal effect against C. perfringens, achieving over a 24 h-period a 5log10-unit decrease in CFU/ml at a concentration > or =1.25 x MIC. In contrast, when tested against E. faecalis, APRC exhibited a concentration-dependent killing activity at concentrations of 1.25 x MIC and 2.5 x MIC, yielding to a decrease of 1.5 and 2.5log10-unit in CFU/ml at 4 h, respectively. However, substantial regrowth of E. faecalis occurred within 24 h. Ultrastructural alterations were observed for both exposed microorganisms by scanning and transmission electron microscopy.

A 45 kDa protein related to PPARgamma2, induced by peroxisome proliferators, is located in the mitochondrial matrix.

Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARgamma2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance. Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARgamma2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance.

Transcytosis and coenzymatic conversion of [(57)Co]cobalamin bound to either endogenous transcobalamin II or exogenous intrinsic factor in caco-2 cells.

We have examined the intracellular route, coenzyme conversion and transcytosis rate of [(57) Co]-labeled cobalamin (Cbl) in function of its presentation to the apical side of Caco-2 cells, either free or bound to intrinsic factor (IF). The free-presented Cbl was progressively bound to endogenous transcobalamin II (TCII) which may stem, in part, from a basolateral to apical passage. Its transcytosis was TCII-mediated as it was abolished when antibodies to TCII were added to the apical medium. The apparent permeability coefficient (P(app)) was estimated at 20.8+/-3.6, 103.5+/-17.7, 0.9+/-0.3 x 10(-5) cm/h for TCII-Cbl, IF-Cbl and haptocorrin-Cbl, respectively. Chloroquine inhibited the transcytosis rate of both TCII and IF-bound Cbl in a dose-dependent manner. Approximately 80% of apical Cbl, bound to either exogenous IF or endogenous TCII, was transported to the basolateral side as intact cyano[(57)Co]Cbl whereas the remainder was converted into Ado-Cbl and CH(3)-Cbl within the cells, as shown by HPLC analyses of a 1,000-g pellet and a 12,000-g supernatant. Coenzymatic conversion was virtually abolished by chloroquine. In conclusion, we suggest that apically presented free Cbl is internalized via TCII-dependent transport. The apically internalized CN-Cbl, bound to either IF or TCII, is processed via an acidic vesicle and part of it is converted to coenzymes, whereas bulk of CN-Cbl is transcytosed intact.

Human embryonic gastric xenografts in nude mice: a new model of Helicobacter pylori infection.

In vitro or animal models have been used to investigate the pathogenesis of Helicobacter pylori infection. However, extrapolation to humans of results obtained with these heterologous models remains difficult. We have developed a new model for the study of H. pylori infection that uses human entire embryonic stomachs engrafted in nude mice. At 80 days after implantation, 22 of these xenografts, which exhibited a mature gastric epithelium, were inoculated with 10(7) to 10(8) CFU of either H. pylori LB1, a freshly isolated H. pylori strain (n = 12), or H. pylori ATCC 49503 (n = 10). After 12-week examination, H. pylori LB1 persistently colonized the antrum of all inoculated grafts, as assessed by culture (mucus and mucosa), immunohistochemistry (mucosa), and a rapid urease test (mucus). H. pylori ATCC 49503, either before or after in vivo passage, permitted only a transient 2-week colonization in one of the five inoculated grafts in both groups. Colonization was always associated with an increase of gastric juice pH. A mild neutrophil infiltration of the gastric mucosa was noted solely in infected grafts. Transmission electron microscopy showed adherence of H. pylori organisms to epithelial cell surface. In six animals, intracytoplasmic location of this bacterium was observed in the antrum or the fundus. These results allow us to propose this model as a new ex vivo model for the study of specific H. pylori-gastric cell interactions.

Effect of clofibrate on the peroxisomes of the intestine of the rat during foetal development.

The aim of the present work was to study the action of clofibrate, known as peroxisomal proliferator, on the intestinal peroxisomes in the foetus of treated pregnant females. The Novikoff technique (catalase activity detection) shows an increase in the number and size of intestinal peroxisomes in the treated females and in the foetus. Significant differences were observed between enterocyte peroxisomal enzymatic activities (catalase and PBE: peroxisomal bifunctional enzyme) in treated and control females on the one hand, and in the foetus of treated and control mothers on the other. The ultrastructural immunocytochemical study of the PPAR (peroxisome proliferator activated receptor) shows labelling of the enterocyte nucleus and mitochondria by the gold particles.

Gamma-GT activity in the rat epididymis: effect of LHRH agonist (D-Trp 6-LHRH).

The aim of the present study was to follow the gamma-GT activity changes in the rat epididymis after withdrawal of androgens. Treatment of adult rats with the LHRH agonist D-Trp 6-LHRH led to reduce serum testosterone concentrations below the limit of detection of the assay. In the treated rats, specific activity of gamma-GT (nmol/min mg protein-1) drastically decreased in caput epididymidis. Histochemical reactions for gamma-GT completely disappeared in the initial and proximal segments but weak activity remained on the luminal plasma membranes of middle segment of the caput. In the corpus and cauda epididymidis, residual reaction for gamma-GT could be seen on the basal part of epithelial cells and on peritubular cells: Thus gamma-GT is confirmed to be androgen-dependent, but there is a regional responsiveness to withdrawal of androgen in the epididymis.

[DIfferentiation of enthesis and the synovial membrane in the rat]. / Différenciation des enthèses et de la membrane synoviale chez le rat.

The development of the knee joint in Wistar rats was observed from the 14th fetal day to the 40th postnatal day by light microscopy, transmission electron microscopy and scanning electron microscopy. The differentiation of the capsular ligamentous and tendinous attachments, synovial cavity, and A and B cells were particularly compared. Capsular attachments appeared for the first time at the 15th day of fetal life. The formation of the cavity started at the 17th day of fetal life. The differentiation of A and B cells was observed by the 20th fetal day by T.E.M., and only by the 15th postnatal day by S.E.M.

Gamma-glutamyl transpeptidase activity in the epididymis during fetal and postnatal development in rats.

The histochemical and biochemical distributions of gamma-glutamyl transpeptidase (gamma-GT) were investigated in the epididymis of rats during fetal and postnatal development. In the epididymal homogenates, gamma-GT activity was detected on the fifth day after birth. A sharp increase was observed after 30 days of life in the caput homogenates. Moderate levels of the enzyme were found in the cauda epididymis. Gamma-GT is histochemically detected from the 15th day of gestation in Wolffian ducts and in 17- to 18-day-old fetuses in newly differentiated epididymal tubules. Enzyme activity, was associated with the plasma membranes (apical, lateral, and basal), was preponderant on the apical part of the epithelial cells. During the first 15 days of the postnatal life, the histochemical reaction intensities were identical from the caput to the cauda epididymidis. From the 18th day onwards, enzyme activity decreased in the corpus and in the cauda, while gamma-GT increased in the caput epididymidis, and a strong activity was found on the apical surface of epithelial cells. Weak or moderate gamma-GT activity of spermatozoa in the caput tubules, increasing steadily from caput to cauda epididymidis, suggests that gamma-GT may be related to the functional maturation of spermatozoa.

Autoradiographic localization of [3H]oestradiol in epididymis, seminal vesicles and prostate of the fetal guinea-pig.

The selective uptake and localization of radioactivity in the fetal male reproductive organs (epididymis, seminal vesicles and prostate) of the guinea-pig (50-60 days of gestation) after in-vivo and in-situ subcutaneous injection of [3H]oestradiol was investigated by autoradiography. In 50-day-old fetuses, the different areas of the epididymis showed selective retention of radioactivity in the nuclei of peritubular and stromal cells surrounding the epididymal duct; no retention was observed in the epididymal epithelium. A similar distribution of silver grains was observed in the 60-day-old fetus. Seminal vesicles and prostate sections from both 50- and 60-day-old fetuses showed concentration and retention of radioactivity only in stromal cells, whereas the epithelium did not exhibit silver grains. In all the tissues studied, the nuclear labelling was abolished after injection of [3H]oestradiol plus a 100-fold excess of non-labelled oestradiol. As the mesenchyme surrounding the epithelia of the epididymis, seminal vesicles and prostate were labelled selectively with [3H]oestradiol, it is suggested that during fetal life of the guinea-pig the mesenchymal stroma of these fetal male reproductive organs may be considered as a target tissue for oestrogen.

Effect of estradiol on the progesterone receptor and on morphological ultrastructures in the fetal and newborn uterus and ovary of the rat.

The effect provoked by estradiol after administration to pregnant rats (1 mg per day) was studied in fetal and newborn uteri and ovary. Estrogen receptors are found in the fetuses of non-treated animals. Their number (in fmol/mg DNA, +/- SD) in the fetal uterus (total sites, cytosol + nuclei) was at the age of 18 days: 63 +/- 15; at 20 days 101 +/- 13; and in the 24-h-old newborn; 415 +/- 120. The respective values in the ovary were: 105 +/- 25; 520 +/- 60 and 410 +/- 190. Estradiol stimulated significantly the progesterone receptor in the fetal uterus at 20 days old. The progesterone receptor (in fmol/mg DNA, +/- SD) which was 97 +/- 17 in the non-treated animals, increased to 790 +/- 90 in the E2-primed animals. Newborns, 24-h-old, had no detectable progesterone receptor, but in the E2-treated animals the value increased to 1210 +/- 120. In the fetal ovary of non-treated animals, progesterone receptor at the age of 18 days is: 90 +/- 19; at 20 days 132 +/- 47, and in the newborns 260 +/- 67; in the E2-treated animals, the values are respectively 330 +/- 49; 865 +/- 78 and 1280 +/- 307. In the fetal uteri of E2-treated animals, histological and ultrastructural studies showed an increase in the size of the uterine horn, the height of the epithelial cells, and stromal cell differentiation. It is suggested that, as was extensively demonstrated in the fetal compartment of the guinea-pig, the machinery for estrogen responses operates also during fetal development of the rat.

Ultrastructural study of the Sertoli cell and the limiting membrane in the seminiferous tubule of the adult cryptorchid rat.

Cryptorchidism was simulated in 13-15-day-old rats by severing the gubernaculum testis and fixing the testis to the abdominal wall. Ultrastructural examination of the testis was made 100 days after birth when a number of modifications to the seminiferous tubules were noted. Germ cells were scanty, with only occasional spermatogonia and primary spermatocytes persisting. The nuclei of Sertoli cells were regular and oval or indented in shape. Their cytoplasm was characterized by a rich smooth endoplasmic reticulum, lipid inclusions and mitochondria with tubulo-vesicular cristae indicative of steroïdogenic activity. The decrease in the number of the germ cells induced a membrane rearrangement with numerous tight junctions and interdigitations between the Sertoli cells. Sertoli cell-specific junctional complexes were very extensive. The lamina propria of the seminiferous tubule appeared thickened and folded and the multilayered basal lamina had complex folds. After fixation with glutaraldehyde containing lanthanum, the latter substance was identified in the basal intercellular spaces of the seminiferous tubules indicating that the blood-testis barrier remains functional in the intra-abdominal testis.

[Carbonic anhydrase activity in the epididymis of the developing rat]. / Activité de l'anhydrase carbonique dans l'épididyme de rat au cours du développement.

The enzyme, carbonic anhydrase (CA), has been studied by an histochemical procedure during the postnatal development of the rat epididymis. Its activity first appeared at Day 14 in the early differentiating cells, i.e. the narrow cells, and only in them. These cells, found in the initial segment and the head of the adult epididymis, showed considerable activity. At Day 35 they disappeared in the other segments (corpus and tail), while the light cells which became apparent immediately showed more discrete CA activity. No carbonic anhydrase activity was found in the seminiferous epithelium, the testicular interstitial tissue or the rete testis.

In vitro development of seminiferous tubules from immature rat testis: ultrastructural and morphometric studies.

Seminiferous tubules from 5 day-old rats maintained in a semi-solid culture system were examined for 4, 8 or 12 days. Morphometric and ultrastructural studies show a significant increase in the seminiferous tubule diameter and normal Sertoli cell differentiation. All the germinal cells degenerate except the spermatogonia.

[Postnatal development of oxidative metabolism of the epididymis in rats]. / Evolution postnatale du métabolisme oxydatif de l'épididyme de rat.

The oxidative metabolism of the epididymis decreases significantly from birth to 12 days and increases gradually after 20 days. Its evolution is contemporary with the development of the interstitial gland and follows the variations of plasmatic and tissular testosterone levels.

Ultrastructural study of seminiferous tubules in the rat after prenatal irradiation.

Is the presence of germinal cells necessary for the Sertoli cells to acquire normal features? To respond to this question we have studied the development of the Sertoli cells in rats irradiated at the end of the foetal life. In the prenatal irradiated rats, the lumen of the seminiferous tubules appears later than in the control rats. The Sertoli cells show numerous flexuose apical processes, with central microtubule bundles. These processes regress progressively after the 40th day of life when the tubular lumen appears; numerous junctional complexes differentiate with the same structure as those of control animals. There are important dilatations of the intercellular spaces. The cytoplasmic organelles show a normal development up to the 40th day of life. After this period, the rough endoplasmic reticulum and the Golgi apparatus clearly regress while important dilatations appear in the smooth endoplasmic reticulum and persist in the adult animal. From the 35th day on, the basal lamina of the seminiferous tubules is irregular and multilayered. The differentiation of the Sertoli cells seems to be independent of the presence of germinal cells until the 40th day of life and presents several particularities; thereafter the Sertoli cells show signs of regression.

Ultrastructural study of Sertoli cells in rat seminiferous tubules during intrauterine life and the postnatal period.

Sertoli cells have various functions: mechanical (creation of two compartments in the seminiferous tubules, migration of germinal cells), secretory (secretion of anti-Müllerian hormone, inhibin, androgen-binding-protein and estrogen) and phagocytic. We report an ultrastructural study of the rat Sertoli cell during maturation and consider possible correlations between the acquisition of certain morphological characteristics and certain functions. During fetal life, the Sertoli cell possesses differentiated zones of junction with the gonocytes and seems to have a role in the migration of the gonocytes towards the periphery of the seminiferous tubule. The Sertoli cell performs the phagocytosis of the gonocytes which degenerate during their migration, and seems to be the site of production of protein granules, whose presence can be related to the synthesis of anti-Müllerian hormone. After birth and before puberty, when the inclusions resembling secretory granules disappear, the Sertoli cell membranes in contact with spermatocytes II and spermatids differentiate, forming, through the differentiated junctional complexes, two compartments (adluminal and luminal) in the seminiferous tubules. Finally, they acquire the characteristics of active secretory cells, capable, in particular, of steroid synthesis.