Influence of the pituitary on Wolffian lens regeneration

Removal of the pituitary 3 days before lentectomy retards Wolffian lens regeneration in the adult newt, Notophthalmus viridescens, by two stages over a 21-day period. Hypophysectomy 5 or 10 days after lentectomy does not alter the progress of regeneration during the subsequent 10-day period. Hypophysectomy 3 days before lentectomy also significantly decreases the incorporation of [³H]thymidine by iris epithelial nuclei 5 days after lentectomy but has no statistically significant effect on the incorporation 7 days after lentectomy.Pituitary tissue from newts or frogs enhances the regenerative activity of newt iris epithelial cells in vitro and in many cases promotes lens fiber formation. To a lesser extent, other tissues, such as nerve ganglion, also enhance the production of lens fiber cells from iris epithelium in vitro, whereas muscle tissue does not; and under certain conditions iris epithelial cells were found to depigment and redifferentiate into lens cells in the absence of other tissues in vitro.     

DNA SYNTHESIS, MITOSIS, AND DIFFERENTIATION IN PANCREATIC ACINAR CELLS IN VITRO

Pieces of mouse embryonic pancreatic epithelium cultured in an inductive situation in vitro, or when examined at critical times in vivo, show a gradient of zymogen granule accumulation. Cells located internally in explants, or in central acini in vivo, show this overt differentiation first. As the epithelia age, the more peripheral cell population proceeds in a similar differentiation. Observations of autoradiograms of H³-thymidine-labeled tissues indicate that the first cells which cease incorporating the DNA-precursor are in the central regions that differentiate first. In older explants, thymidine incorporation is largely restricted to the periphery of the tissue as zymogen appears in the internal cells. Evidence suggests that cells or nuclei which have replicated DNA move inward before dividing. Some daughter cells apparently return peripherad to divide again, whereas others remain centrally where they undergo differentiation. During at least the first 24 hours of these maturational changes, mesenchyme has a stimulatory effect upon epithelial thymidine-incorporation frequencies. The presence of a post-DNA-synthetic population is seen in the form of a group of nonlabeling central cells that remains intact in the midst of a labeled epithelium for as long as 48 hours in vitro (from 72 to 120 hours). If explants are treated with 5-bromodeoxyuridine for any 24-hour segment of the 0 to 72-hour period, before the non-incorporating population arises, no subsequent overt zymogen formation occurs. If explants are treated continuously from 72 to 120 hours, on the other hand, zymogen still forms in some internal cells. Presumably, this differentiation is limited to the postmitotic population as revealed in the thymidine autoradiograms.     

Appearance and distribution of carbohydrate-rich macromolecules on the epithelial surface of the developing rat palatal shelf.

The synthesis and appearance of carbohydrate-rich macromolecules by epithelial cells of the developing secondary palate was examined with concanavalin A (CON A) binding and [³H]glucosamine labeling. The amount of [¹²⁵I]CON A bound to the epithelial surface of the rat palatal shelf in vitro increased from day 15 of gestation to day 16 when initial adhesion to the opposite shelf occurs in vivo. Visualization of CON A binding by electron microscopy using the peroxidase method revealed a dramatic increase in binding between days 15 and 16 of gestation, most apparent on the medial-edge epithelial surface. The incorporation in vivo of [³H]glucosamine during this period into the medial-edge epithelial cells was detected with autoradiography. These results show that a glycoprotein-rich surface material appears on the superficial cells of the medial-edge epithelium prior to adhesion of the apposing shelves.     

Histamine-induced changes in rat tracheal goblet cell mucin store and mucosal edema

The pathology of chronic asthma in human and mouse is characterized by inflammation and remodeling of airway tissues. As a result of repeated inflammatory insults to the lower airways, smooth muscle thickening, mucin secretion and airway hyperreactivity may develop. In ovalbumin (OVA)-sensitized mice with repeated challenges with OVA to the lower airways, the trachea and bronchi are characterized by goblet cell hyperplasia and mucus hypersecretion from goblet cells. Previous study reports that intravenous (i.v.) application of a high dose of capsaicin releases tachykinin from capsaicin-sensitive nerves, producing acute plasma leakage and mucosal edema formation and causing depletion of mucin granules in goblet cells that results in a reduction in the number and size of Alcian blue (AB)-positive goblet cells in the rat trachea within a few minute after capsaicin application. Histamine is an important non-neural mediator of asthma from mast cells. The present study investigated whether i.v. application of a high dose of histamine (18 μmol/ml/kg) could result in these acute changes and the similar time-course changes in rat trachea. The tracheal whole mounts stained with chloroacetate esterase reagent and AB and tracheal methacrylate sections stained with AB and periodic acid-Schiff reagent were used for evaluation of histological and cellular changes. At 5 min after histamine application, mucosal leaky venules were numerous and subepithelial edema ratio (% of length of edema along the mucosal epithelial circumference of tracheal cross section) was found to be 48.2 ± 4.9, which was greater (P < 0.01) than saline-treated rats. But, the number of AB-positive goblet cells, 2,030 ± 170/mm² of mucosal surface epithelium, was similar to saline-treated group (P > 0.05). One day later, edema ratio remained large and the number of AB-positive goblet cells was 1,140 ± 150/mm² epithelium, reduced to half the number of the group at 5 min after histamine (P < 0.01). It is suggested that mucus hypersecretion occurred at this time point. At 3 or 5 days after histamine, edema ratio gradually decreased. The number of AB-positive goblet cells continued to remain small on day 3. On day 5 after histamine, the number of AB-positive goblet cells restored to the level of rat group at 5 min after histamine application. At 7 days after histamine, edema ratio returned to the level of saline-treated group. It is concluded that degranulation and thinning of tracheal goblet cells and mucus hypersecretion lagged behind histamine-induced acute plasma leakage and edema, and restoration of mucin store in goblet cells was associated with remission of mucosal edema.     

Concentrations of putrescine and polyamines and their enzymic synthesis during androgen-induced prostatic growth

1. Castration of adult rats resulted in marked decreases in the amounts of putrescine, spermidine and spermine in the ventral prostate gland. Spermidine concentrations decline rapidly over the first 11 days after androgen withdrawal, reaching a value of only 12% of normal controls. Spermine concentrations diminish more slowly, reaching 24% of normal within 11 days. The spermidine/spermine molar ratio falls from 0.9 to 0.46 under these conditions. Putrescine concentrations decrease by 70% at 7 days after castration and then remain constant for some days. 2. After daily injections of testosterone propionate to rats castrated 7 days previously, prostatic spermidine and putrescine concentrations increase significantly within 24h; normal or even greater values are observed within 8 and 4 days respectively. In contrast, the spermine concentration does not increase until 5 days after commencement of androgen treatment. 3. The activities of two enzymes involved in polyamine biosynthesis (ornithine decarboxylase and a putrescine-activated S-adenosyl-l-methionine decarboxylase system) were greatly decreased soon after castration: after 7 days the respective values were 15% of normal for ornithine decarboxylase and 7% of normal for putrescine-dependent decarboxylation of S-adenosyl-l-methionine. Injection of testosterone propionate into animals castrated 7 days previously induced a rapid increase in both enzymic activities: ornithine decarboxylase was doubled in 6h, and increased three- to four-fold within 48h, whereas the putrescine-dependent decarboxylation of S-adenosyl-l-methionine doubled in 3h and increased tenfold within 48h of commencement of daily androgen treatments. 4. The activity of these enzyme systems was very low in the ventral prostates of hypophysectomized rats and was increased by administration of testosterone in a manner similar to that found in castrated rats. 5. Alterations in the activity of two ventral-prostate enzymes involved in ornithine production (arginase) and utilization (ornithine–2-oxoglutarate transaminase) that result from changes in the androgenic status of rats are described. 6. The findings presented suggest that the activities of ornithine decarboxylase and the putrescine-dependent S-adenosyl-l-methionine decarboxylase system, rather than ornithine concentrations, are rate-limiting for the formation of putrescine and polyamines in rat ventral prostate. 7. The relation of polyamines to androgen-induced prostatic growth is discussed with particular reference to the biosynthesis of proteins and nucleic acids.     

Transdifferentiation of outgrowth cells and cultured epithelial cells from swine trachea

Summary The morphologic and functional properties of explant out-growth cells and epithelial cells isolated from swine trachea epithelium by proteolysis were examined. A mixed population of ciliated, serous, and basal cells, obtained from out-growths, from proteolysis of trachea epithelium, and from unattached explants in organ culture, all yielded cell cultures that were composed almost entirely of mucus-secreting cells. When the cells were grown in primary or secondary culture on a modified collagen matrix in supplemented HAM:DMEM (1:1) medium they expressed a mucus-secreting phenotype with numerous mucus granules at various stages of maturation and incorporated [³H]GlcN and³⁵SO₄ into secreted mucin glycoproteins. Results obtained in these studies suggest that extensive transdifferentiation of ciliated and serous cells to mucus-secreting cells occurs after the release and during subsequent attachment and culture. Ciliated cells containing mucus granules were seen in various stages of cilia resorption. Basal cells containing mucus granules were also frequently observed. The number of mucus-secreting cells and the synthesis of mucin glycoproteins increased dramatically with time of attachment and culture, whereas cell proliferation, population doubling time of 72 h, and incorporation of [³H]thymidine into DNA increased much more slowly. The number of mucus-secreting cells correlated closely with the level of secretion of mucin glycoproteins. Taken collectively, these studies help to elucidate the transdifferentiation process, which dramatically increases the number of mucus-secreting cells after disruption and release of epithelial cells from swine tracheobronchial epithelium. A similar mechanism involving disruption of the extracellular matrix may be involved in the stimulation of hypersecretion of mucus and mucin glycoproteins by chemical and infections irritants.     

Further observations on uptake of S35-labelled sulfate by renal tissue in vitro

Additional studies have been made of the accumulation of S³⁵ by renal cortical tissue incubated in media containing radiosulfate. This process was found to occur in several mammalian species in addition to the rat, but was not observed as a significant occurrence in three species of lower vertebrates. In the case of rat renal tissue, S³⁵ uptake was found to be sensitive to the pH and osmolar concentration of the medium. The character of the anions present in conjunction with K⁺ affected it as well. Various factors known to be related to in vitro accumulative processes, as well as to renal sulfate reabsorption by the intact dog, were tested on rat kidney cortex to assess the effect on radiosulfate uptake. In general, all substances tested (amino acids, metabolic intermediates, ATP, metabolic inhibitors, competitive inhibitors for PAH accumulation in vitro) were found to lessen S³⁵ uptake, or to be without effect upon it. The one striking exception was phlorhizin, which enhanced markedly S³⁵ uptake in vitro, as it does sulfate reabsorption in vivo. Some implications of these findings have been discussed.     

The reduction of inorganic sulphate to inorganic sulphite in the small intestine of the rat

1. Whole scrapings of rat intestinal mucosa were incubated with carrier-free sodium [³⁵S]sulphate. Radioactivity was found in S-sulphocysteine and to a small extent in S-sulphoglutathione. 2. Whole scrapings of rat intestinal mucosa incubated with carrier-free sodium [³⁵S]sulphate and oxidized glutathione formed S[³⁵S]-sulphoglutathione as the main radioactive product. The amount of S[³⁵S]-sulphocysteine formed was considerably lower than in a control that contained no oxidized glutathione. 3. The supernatant fraction of homogenates of rat intestinal mucosa catalyses the NADPH-dependent reduction of adenosine 3′-phosphate 5′-sulphatophosphate to inorganic sulphite. NADH or GSH fail to replace NADPH as reducing agents. 4. The formation of inorganic [³⁵S]sulphite from inorganic [³⁵S]-sulphate may account for the incorporation of [³⁵S]sulphate into S-sulphoglutathione by the small intestine of the rat in vivo and in vitro.     

Regulation of the synthesis of mucin glycoproteins in swine trachea explants

Summary Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm² per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 μM glucosamine. A higher concentration of³⁵SO₄, 1000 μM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 μg/ml increased the rate of secretion twofold, whereas 0.1 to 100 μg/ml of hydrocortisone and 0.1 to 100 μg/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10⁻⁵ M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10⁻⁹ M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats. Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistingushable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio ofN-acetylgalactosamine,N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations. This investigation was supported by U.S. Public Health Service Grants HL 20868, HL 24688, and HL 24718 from the National Heart, Lung and Blood Institute, Bethesda, MD, and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, Bethesda, MD.     

Factors influencing the uptake and loss of radiosulfate by rat renal cortical tissue in vitro

Rat kidney cortical slices, during incubation in vitro, lose previously accumulated radiosulfur when exposed to conditions (e.g. addition to the medium of metabolic inhibitors) which normally depress the uptake of S³⁵. The extent of this loss is not affected significantly by the presence of phlorhizin, an agent which enhances markedly radiosulfate accumulation. On the other hand, when tissues are chilled to 1°C., loss is slight or negligible even in the presence of metabolic inhibitors. These data, and observations on the effect of pre-incubation of kidney slices in S³⁵-free media before the addition of radiosulfate, have been interpreted as evidence that S³⁵ accumulation in vitro may be resolved into at least two processes, namely (a) entrance of the isotope-labelled anion into the cells, by diffusion and/or active transport, and (b) complexing of S³⁵ (in ionic or other form) with an intracellular component. The postulated complex is stabilized, perhaps through inactivation of a specific enzyme, by chilling the tissue to 1°C. Possible relationships are discussed among the observations noted above, sulfur metabolism in general, and aspects of the known in vivo transport mechanism for sulfate ion; i.e., renal tubular reabsorption.     

Hamster tracheal organ culture in serum-free media: A quantitative comparison of in vitro epithelial morphology with that of in vivo controls

Summary The epithelial morphology of the hamster trachea in serum-free organ culture was compared with that of age-matched in vivo control tissues by collecting and statistically analyzing several quantifiable parameters. By this technique it was possible to detect both subtle and dramatic epithelial alterations. Midtracheal tissues from 6-wk-old male Syrian golden hamsters were used as the explants. Explants were placed on Gelfoam sponges and cultured for 1, 2, and 3 wk in CMRL 1066 alone and in CMRL 1066 to which seven factors were added: insulin and transferrin (5 μg/ml); hydrocortisone (5×10⁻⁷ M); epidermal growth facotr (5 ng/ml); bovine pituitary extract (0.5%); and phosphoethanolamine and ethanolamine (5×10⁻⁵ M). The following data were collected and statistically analyzed for each tracheal ring: number of epithelial cells; proportion and number of each cell type; basement membrane length; linear density of epithelial cells; epithelial height; and mitotic index. Compared to controls, ciliated cells decreased by 52% during washes in Leibovitz (L15) medium and tissue manipulation performed before culture and this loss persisted after cutlure for 1 wk. Explants culturedwithout the factors showed marked changes after 2 and 3 wk including epithelial thickening and folding, which was associated with increased linear density. Many cells in these specimens could not be categorized by type (22% were unidentifiable after 3 wk). Epithelial migration onto the outside of the explant was inhibited. In contrast, explants culturedwith the factors maintained a morphology similar to controls at 2 and 3 wk and epithelial migration onto the outside of the explant was supported. This study shows that explants in CMRL 1066 with the seven factors provide a useful biological model for the in vitro study of the mucociliary respiratory epithelium. This work was supported by grant HL24722 from the National Institutes of Health, Bethesda, Maryland.     

Explant organ culture of hamster alimentary tract epithelium

Adult Syrian Golden hamster alimentary tract maintained as explants in organ culture was studied using the model system for hamster pancreas described by Resau et al. (1983a). Explants of esophagus, stomach, duodenum and colon were maintained in organ culture on Gelfoam® sponge rafts in a high-oxygen atmosphere with serum-supplemented CMRL-1066 medium. All of the tissues were observed to show evidence of sublethal acute cell injury during the first several days of culture. Subsequently, the epithelial tissues recovered from this injury, repopulated the denuded areas of the explants and replicated within the sponge matrix. Explants were maintained in a differentiated state for 30+ days and sampled for morphology to examine the process of cell injury, repair, differentiation and replication which occurs in mucosal epithelia. The percentage of basement membrane covered by epithelia in the explants from various tissues was compared to the level of LDH in the media to reveal the relationship between viability determined by biochemical and by morphological methods. Restitution of the mucosal surface occurred in all of the explants. We conclude that adequate populations of replicating cells are maintained within the epithelium of the hamster alimentary tract tissues in vitro so that restitution can occur through migration and subsequent differentiation of the epithelial cells within the mucosa of the explants.Abbreviations 4F-1G 4% formaldehyde 1% glutaraldehyde fixative - LDH lactate dehydrogenase - OsO₄ osmium tetroxide - PAS/PLH periodic acid, periodic acid Shiff lead hematoxylin stain     

Anti-Tumor Action,Clinical Biochemistry Profile and Phytochemical Constituents of a Pharmacologically Active Fraction of S. crispus in NMU-Induced Rat Mammary Tumour Model

Cancer patients seek alternative remedies such as traditional medicinal plants for safe and effective treatment and help overcome the side effects of conventional therapy. Current knowledge indicates that extracts of Strobilanthes crispus of the Acanthaceae family exhibit potent anticancer properties in vitro and are non-toxic in vivo. S. crispus was also reported to be protective against chemical hepatocarcinogenesis. We previously showed that a bioactive fraction of S. crispus leaves also synergized with tamoxifen to cause apoptosis of human breast cancer cell lines without damaging non-malignant epithelial cells. The present study aimed to evaluate the antitumor effect of S. crispus dichloromethane fraction (F3) using N-methyl-N-Nitrosourea (NMU)-induced rat mammary tumor model. Tumor regression was observed in 75% of the rats following 8-week oral administration of F3 with no secondary tumour formation and no signs of anemia or infection. However, no improvement in the liver and renal function profiles was observed. Major constituents of F3 were identified as lutein, 13¹-hydroxy-13²-oxo-pheophytin a, campesterol, stigmasterol, β-sitosterol, pheophytin a and 13²-hydroxy-pheophytin a. These compounds however, may not significantly contribute to the antitumor effect of F3.     

Biotransformation of Externally Added Vanillin Related Compounds by Multiple Shoot Cultures of Vanilla planifolia L

The multiple shoots and callus cultures of Vanilla planifolia obtained from the nodal explant on MS medium supplemented with 6-benzylaminopurine (BAP) 2 mg l^?1 and α-naphthalene acetic acid (NAA) 2 mg l^?1 were maintained by regular subculturing every 30 days and also cultured liquid MS medium of the same hormonal combination. Shoots were transferred to the MS basal medium for rooting. Different explants along with vanilla pods and in vitro cultures were analyzed using HPLC for the presence of vanillin and related compounds. When the amount of these compounds was determined in explants and in in vitro cultures after precursor feeding and curing process, explants showed different profile after precursor feeding and after undergoing curing process. During further investigations we have applied a novel approach for curing in vitro tissues as done for vanilla beans. Curing of in vitro shoots resulted in a significant change in the aromatic compound profile.     

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant ^1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.     

Acid mucopolysaccharide synthesis in the secondary palate of the developing rat at the time of rotation and fusion

The amount of hexosamines and acid mucopolysaccharides present in the rat secondary palate increases during the critical stages of palatogenesis, namely, rotation and fusion. The synthesis of acid mucopolysaccharides in vivo and in vitro in the palate was determined by the incorporation of ³H-glucosamine and Na₂S³⁵O₄. The labeled mucopolysaccharides were isolated by DEAE-cellulose chromatography and were identified on the basis of several criteria as hyaluronic acid and sulfated acid mucopolysaccharides. Hyaluronic acid accounted for approximately 60% of the total acid mucopolysaccharides synthesized in the palate both in vivo and in vitro. DON (6-diazo-5-oxonorleucine), a known inhibitor of acid mucopolysaccharide synthesis, inhibited the incorporation of ³H-glucosamine and Na₂S³⁵O₄ by palatal shelves in vitro by 70%.     

The effect of cortisol on rabbit fetal lung maturation in vitro

Explants of lung tissue from 19-day gestational age fetal rabbits were maintained in organ culture in medium with or without fetal calf serum for 1 to 11 days. Based on the results of biochemical and morphological studies it was apparent that the type II pneumonocyte differentiated in vitro at a time similar to that which occurs with maturation in vivo. The epithelial cells of the presumptive alveoli were undifferentiated at the start of incubation, but within 9 days developed increased amounts of Golgi apparatus and rough endoplasmic reticulum, many microvilli on the luminal surface and numerous lamellar bodies. Secreted lamellar bodies and tubular myelin figures were observed in the lumina of cultured explants. The incorporation of [³H]choline into phosphatidylcholine by lung tissue explants maintained in medium containing 10% fetal calf serum remained relatively constant for 7 days of incubation but thereafter increased two-fold. When explants were maintained in fetal calf serum-containing medium and cortisol (10^?7M) or betamethasone (10^?7M), the incorporation of choline into phosphatidylcholine was two to three times greater than that of explants maintained in serum-containing medium without cortisol. When explants of fetal lung tissue were incubated in the presence of cortisol without fetal calf serum there was no stimulatory effect of cortisol on phosphatidylcholine biosynthesis. Therefore, serum cofactors are necessary for the stimulatory effects of cortisol on fetal lung development. The specific activity of phosphatidate phosphohydrolase (PAPase) increased to very high levels during the culture period. In the presence of serum, cortisol or betamethasone had no effect on the specific activity of phosphatidate phosphohydrolase.     

Reassembly of somatic cells and testicular organogenesis in vitro

Testicular organogenesis in vitro requires an environment allowing a reassembly of testicular cell types. Previous in vitro studies using male murine germ cells cultured in a defined three-dimensional environment demonstrated tubulogenesis and differentiation into spermatozoa. Combining scaffolds as artificial culture substrates with testicular cell culture, we analysed the colonization of collagen sponges by rat testicular cells focusing on cell survival and reassembly of tubule-like-structures in vitro. Isolated testicular cells obtained from juvenile Sprague Dawley and eGFP transgenic rats were cultured on collagen sponges (DMEM high glucose + Glutamax, 35 °C, 5% CO₂ with or without gonadotropins). Live cell imaging revealed the colonization of cells across the entire scaffold for up to 35 days. After two days, histology showed cell clusters attached to the collagen fibres and displaying signs of tubulogenesis. Clusters consisted mainly of Sertoli and peritubular cells which surrounded some undifferentiated spermatogonia. Flow cytometry confirmed lack of differentiation as no haploid cells were detected. Leydig cell activity was detected by a rise of testosterone after gonadotropin stimulation. Our approach provides a novel method which is in particular suitable to follow the somatic testicular cells in vitro an issue of growing importance for the analysis of germ line independent failure of spermatogenesis.     

In vitro stimulation of cartilage in embryonic chick neural crest cells by products of rentinal pigmented epithelium.

The retinal pigmented epithelium of the chick embryo influences head neural crest mesenchymal cells to form the scleral cartilage of the eye. The possible role of extracellular matrix in this interaction was studied. Extracellular matrix was deposited on Millipore filters in vitro by pigmented epithelial cells which were then killed by distilled water lysis. When grown on the Millipore filters which had carried pigmented epithelium, clonal neural crest and periocular mesenchyme “target” cells formed cartilage in 61 of 155 experiments. Cartilage was not formed when the cells were grown on naked filters nor did gels of purified Type I and Type II collagen promote chondrogenesis. It is concluded that extracellular matrix deposited by the pigmented epithelium in vitro is a potent stimulus for the induction of chondrogenesis in competent mesenchyme, and that living pigmented epithelial cells need not be present for such induction.