Explant Culture System Research Articles

Expression of human gonadotropin-releasing hormone receptor gene in the placenta and its functional relationship to human chorionic gonadotropin secretion.

Placental GnRH is one of the potential paracrine regulators of hCG secretion from the trophoblasts during pregnancy. Maternal serum hCG levels exhibit an exponential rise during the first 6 weeks of pregnancy, peak at 9-10 weeks, decline to a nadir at 20 weeks, and remain at low levels during the rest of pregnancy. However, the placental content of GnRH does not parallel the time course of hCG secretion, and GnRH messenger ribonucleic acid (mRNA) levels in the placenta remain unchanged during pregnancy. These data do not conform with a simple paracrine mechanism of GnRH as a regulator of hCG secretion. We, therefore, examined the potential variation in GnRH receptor gene expression in the placenta, which may account for the GnRH-mediated dynamic pattern of hCG secretion during gestation. First, we established a functional relationship of GnRH and hCG secretion. Using a placental explant culture system, a dose-response effect of hCG secretion was observed in the placental explant at 9 weeks when treated with GnRH ranging from 10(-9)-10(-7) mol/L. The effect of GnRH was completely blocked by a GnRH antagonist (Nal-Glu). The relative responsiveness of hCG secretion to GnRH stimulation at 10(-7) mol/L was further evaluated in placental explants at 6, 9, and 40 weeks gestation. Whereas the 9-week placenta showed a maximal response (> 300%) relative to the 6-week placenta, there was no response in term placenta. Again, the effects of GnRH on hCG secretion were blocked by Nal-Glu, supporting a receptor-mediated event. Localization of mRNA encoding human GnRH receptor in human placenta at 6, 9, 12, 20, and 40 weeks gestation was established by in situ hybridization. The mRNA signals were present in both cytotrophoblast and syncytiotrophoblast cell layers. Signal intensities varied with gestational ages and were abundant at 6 weeks, peaked at 9 weeks, declined at 12 and 20 weeks, and were undetectable at term. The present study demonstrates, for the first time, that GnRH receptor mRNA is expressed in both cytotrophoblasts and syncytiotrophoblasts and exhibits changes paralleling the time course of hCG secretion during pregnancy. These data provide a mechanistic understanding that the paracrine/autocrine regulation of hCG secretion by placental GnRH is mediated through an increase followed by a decline in GnRH receptor gene expression from the first trimester to term placenta.

The gingival immune response to periodontal pathogens in juvenile periodontitis

A gingival explant culture system was utilized to evaluate the reactivity of local immunoglobulins produced by juvenile periodontitis tissue. Gingival explant culture supernatant fluids were screened, via a standardized dot-immunobinding assay, for antibodies reactive to: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, Eikenella corrodens, Peptostreptococcus micros, Peptostreptococcus anaerobius, Capnocytophaga ochracea, Eubacterium nodatum and Fusobacterium nucleatum and one nonoral microorganism, Porphyromonas asaccharolytica. Of the 75 juvenile periodontitis supernatant fluids tested, the organisms that reacted with the highest numbers of supernatant fluids were E. nodatum (72%) and A. actinomycetemcomitans (49%). More juvenile periodontitis than healthy tissue samples showed supernatant fluid reactivity to P. intermedia, C. ochracea, E. nodatum and P. micros. No significant difference was observed between the juvenile periodontitis group supernatant fluids reactivity and the supernatant fluids of the other periodontal disease groups tested. Cluster analysis revealed the association, as determined by supernatant fluid reactivity, of P. micros and C. ochracea in the juvenile periodontitis group. The data from this investigation are consistent with a hypothesis of multiple possible etiologies of periodontal destruction in juvenile periodontitis and other forms of periodontal diseases.

Secretion of nerve growth factor from septum stimulates neurite outgrowth and release of the amyloid protein precursor of Alzheimer's disease from hippocampal explants.

Alzheimer's disease (AD) is characterized by the deposition of amyloid in the extracellular and intracellular compartments of the cerebral cortex. The extracellular amyloid consists of a protein (beta A4) which is derived from a larger precursor, the amyloid protein precursor (APP). Several studies have implicated APP in the regulation of neurite outgrowth during development, although the precise function of APP remains unknown. To examine the role of APP in the regulation of neurite outgrowth from hippocampal neurons, an explant culture system was developed. Explants of E18 mouse hippocampus were found to extend neurites when co-cultured with explants of E18 mouse septum. This finding demonstrated that the septum can release a neurite outgrowth-promoting factor (NOPF). As nerve growth factor (NGF) was also able to stimulate neurite outgrowth from the hippocampal explants, this suggested that the NOPF might be NGF. Immunoprecipitation of NGF from septal conditioned medium using a specific monoclonal antibody (27/21) completely blocked the neurite outgrowth-promoting effect, supporting this conclusion. Concomitant with its ability to stimulate neurite outgrowth, NGF stimulated the release of APP from the hippocampal explants. As previous studies have suggested that the binding of APP to heparan sulfate proteoglycans (HSPGs) in the extracellular matrix might be an important step in the regulation of neurite outgrowth by NGF, we examined the effect of APP on neurite outgrowth from dissociated hippocampal cells cultured on various protein substrates. When cells were cultured on a substrate of APP and HSPG, neurite outgrowth was markedly stimulated. No stimulation of neurite outgrowth was seen when neurons were cultured on substrates of either APP or HSPG alone. The results suggest that secreted forms of APP may be involved in stimulating neurite outgrowth from hippocampal neurons and that interactions between APP and HSPG may be important for a neurite outgrowth-promoting function.

Alternatively polyadenylated vasoactive intestinal peptide mRNAs are differentially regulated at the level of stability.

The role of cis-acting destabilizing RNA sequences in the determination of endocrine gene expression has been investigated using a novel paradigm, in which the differential regulation of two alternatively polyadenylated RNA transcripts may be observed both in vivo and in vitro. In the rat anterior pituitary gland in vivo, we have shown that, after the termination of an estrogen stimulus, a 1.7-kilobase (kb) vasoactive intestinal peptide (VIP) RNA containing an extensive 3'-untranslated region (UTR), is preferentially down-regulated with respect to a 1.0 kb VIP transcript that is uniquely abundant in this tissue. Differential regulation of the anterior pituitary VIP transcripts can be modeled in an explant culture system in which we defined both transcriptional and posttranscriptional phases of VIP gene regulation in vitro, and showed that selective down-regulation of the 1.7-kb transcript is posttranscriptional. Inhibitors of transcription and translation have also allowed us to show in vitro that differential regulation of VIP transcripts occurs through an active process that appears to involve the synthesis of a labile, destabilizing factor. In order to confirm the role of RNA destabilization as the primary mechanism of differential posttranscriptional regulation, we have also performed cell-free stability assays in which explant extracts were incubated with 32P-labeled run-off transcripts corresponding to the two alternatively polyadenylated VIP RNAs. The resultant estimates of RNA half-life showed significantly lower values for the synthetic VIP transcript containing the 3'-UTR. Our findings demonstrate the presence of functional destabilizing sequences in the 3'-UTR of the rat VIP RNA which appear to act in the physiological control of VIP gene expression.

Recombinant Bone Morphogenetic Protein-4, Transforming Growth Factor-β1, and Activin A Enhance the Cartilage Phenotype of Articular Chondrocytes in Vitro

Bone morphogenetic protein-4 (BMP-4) is a member of the transforming growth factor-β (TGF-β) supergene family and is characterized by its ability to induce singly de novo cartilage and bone in vivo. The influence of recombinant bone morphogenetic protein-4 and some related members, TGF-β1, activin A, and inhibin A, on articular chondrocyte metabolism in the presence and absence of extracellular matrix has been examined. BMP-4 and TGF-β1 stimulated [35S]-sulfate incorporation in a dose-dependent manner in short-term monolayer, micromass, and explant cultures. Activin A showed a slight but significant stimulation of proteoglycan synthesis while inhibin A decreased metabolic activity. The effects observed were most pronounced in the explant culture system. Although the relative influence of the growth factors was less apparent in chondrocytes isolated from adult cartilage, the qualitative responses were similar with cells obtained from young animals. The maintenance and enhancement of the cartilage phenotype was further investigated by Northern blot analysis. BMP-4 and TGF-β1 increased the levels of expression of type II collagen and proteoglycan aggrecan in short-term cultures, while activin A and inhibin A did not affect these parameters significantly when compared to serum-free control cultures. Binding experiments with 125I-BMP-4, revealed the presence of specific, high-affinity binding sites with an apparent dissociation constant of 110 pM and about 6000 receptors per cell. Chemical cross-linking showed the presence of three components (apparent size 200, 90, and 70 kDa), demonstrating the presence of functional receptors for BMP-4 on primary articular chondrocytes.

Effect of interleukin 1 on articular cartilage from young and aged horses and comparison with metabolism of osteoarthritic cartilage

Summary The effect of interleukin 1 (il-1) on equine articular cartilage was investigated, using a cartilage explant culture system. Measurement of [35S]O4 incorporation revealed synthesis of matrix proteoglycan by cartilage to be decreased 45, 59.7, and 37.5% after 1, 3, and 5 days, respectively, in culture in the presence of 5 U of il-1/ml. There was no change in proteoglycan degradation as determined by measurement of [35S]O4 release into the culture medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cartilage-conditioned medium indicated that exposure of cartilage to il-1 caused a decrease in total protein synthesis by 45, 68, and 87% after 1, 3, and 5 days, respectively, in culture while selectively inducing synthesis of the 57-kd neutral metalloproteinase stromelysin (matrix metallo-proteinase-3) in young and adult horses. Identification of stromelysin was confirmed by functional characterization and immunoprecipitation. Baseline total protein synthesis, as well as specific synthesis of stromelysin in cartilage from adult and aged horses, was markedly less than that of young horses. The il-1- induced reduction in total protein synthesis may not be a characteristic of equine articular cartilage from affected joints of horses with naturally acquired osteoarthritis as indicated by an overall increase in protein synthesis by osteoarthritic explants. Introduction of il-1 into an equine articular cartilage explant culture system resulted in decrease of matrix component synthesis and increase in specific degradative enzyme synthesis and activity. Articular cartilage from aged horses had markedly less overall metabolic activity, compared with cartilage from young horses. Articular cartilage from affected joints of horses with naturally acquired osteoarthritis did not have metabolic alterations identical to those of il-1-stimulated normal articular cartilage from the same individual, necessitating reevaluation of the validity of the il-1-induced model of osteoarthritis. Osteoar thritis is a common, naturally acquired disease of horses, and tissue from animals of all ages and stages of osteoarthritis is available. The equine model of osteoarthritis may afford an important means of studying the alterations in articular cartilage metabolism as a function of age and disease severity.

Decelerated migration of neocortical neurones in explant culture after exposure to radiation.

We describe a newly devised explant culture system employing rat neocortex in which neurones migrate in a fashion similar to that seen in vivo. Using neocortical explant culture we have demonstrated an effect of ionizing radiation on neuronal migration at doses as low as 10 cGy together with a changing pattern of expression of the neural cell adhesion molecule N-CAM. After a dose of more than 10 cGy there was a significant reduction in N-CAM immunoreactivity in the matrix cell zone. We do not know the reason why the reduction in N-CAM immunoreactivity is so localized, although it is not unlikely that the most active site in terms of N-CAM synthesis is significantly affected by radiation.

Skin Explant Culture: A Reliable Method for Detecting Pemphigoid Antibodies in Pemphigoid Sera that Are Negative by Standard Immunofluorescence and Immunoblotting

We investigated the presence of bullous pemphigoid antibodies in bullous pemphigoid sera that are negative by standard indirect immunofluorescence. We incubated each of four indirect immunofluorescence-positive bullous pemphigoid sera, seven indirect immunofluorescence-negative bullous pemphigoid sera, one indirect immunofluorescence-negative herpes gestationis serum, three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera, five indirect immunofluorescence-negative epidermolysis bullosa acquisita sera, and two normal human sera with fresh human skin explants in medium 199 at 4 degrees C for 48 h. All bullous pemphigoid sera, herpes gestations serum, and the three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera had IgG that bound the basement membrane zone of skin explants with moderate to marked intensity as demonstrated by immunofluorescence. Normal sera and indirect immunofluorescence-negative epidermolysis bullosa acquisita sera failed to bind the explant basement membrane zone. Immunoblotting of bullous pemphigoid sera showed five of seven indirect immunofluorescence-negative bullous pemphigoid sera to bind high-molecular weight and/or low-molecular weight bullous pemphigoid antigens from epidermal extracts. We conclude that the skin explant culture system is a very sensitive method for the detection of bullous pemphigoid antibodies in all bullous pemphigoid sera.

Regeneration from crayfish phasic and tonic motor axons in vitro.

An explant culture system is described that allows examination of axonal growth from the tonically and phasically active motoneurons of the abdominal nerve cord of the crayfish. In this preparation, growth occurs from the cut end of the axon while the remainder of the motoneuron is undisturbed. In vitro growth from the branches of the third roots, which contain the axons from the tonic and phasic motoneurons of abdominal ganglia one through four, was verified as axonal by retrograde labeling of axons and neuronal somata within the nerve cord. Growth from the axons of phasic and tonic cells was observed as early as 24 h after plating and continued for an additional 7-10 days. The morphology and growth rates of the motor terminals differed between the tonic and phasic axons. The phasic axons grew significantly faster and branched more often than did the tonic motor axons. These differences in growth may be related to differences in motoneuron size or, may result from differences in electrical activity. Tonic motoneurons show spontaneous impulse activity for up to 6 days in culture, whereas phasic motoneurons show no spontaneous impulse activity. In addition, the differences in growth may be related to the morphological differences in tonic and phasic motor terminals observed in situ.

Kinetics of Proteoglycan Turnover in Bovine Articular Cartilage Explants

The turnover of proteoglycans was studied in explant cultures of mature bovine articular cartilage. The aim of the study was to compare the in vitro turnover rates of newly synthesized proteoglycans and endogenous proteoglycans. Cartilage was maintained in the presence of various serum concentrations in order to determine the conditions of steady-state proteoglycan metabolism. The steady state was achieved in medium containing 20% fetal calf serum.The proteoglycan synthesis rate and the half-life of labeled proteoglycans in steady-state cultures were used to calculate the size of the metabolic pool of newly synthesized proteoglycans in steady state. This metabolic pool was shown to be equal to the total amount of proteoglycans in the matrix. It is concluded that all of the proteoglycans in the matrix have the same half-life in vitro.Taking another approach, aggrecan was isolated from the cartilage and the medium of steadystate cultures prelabeled with [35S] sulfate. The specific activity of the glycosaminoglycans from cartilage aggrecan were compared with that of glycosaminoglycans from medium aggrecan. These proved to be the same throughout the culture period. This shows that newly synthesized aggrecan and endogenous aggrecan have the same turnover rate in vitro.The significance of explant culture systems for the study of proteoglycan turnover is discussed.

Microglia-derived plasminogen enhances neurite outgrowth from explant cultures of rat brain

Recently, we reported the production and secretion of plasminogen (Pg) in cultured rat brain microglia [Nakajima et al., (1992) Fedn. Eur. Biochem. Socs Lett.308, 179–182]. To investigate the physiological significance of Pg, we determined the effect of Pg on neurite outgrowth of cultured neocortical explants of an embryonic rat brain in serum-free chemically defined medium. Pg markedly enhanced the neurite outgrowth. Although plasmin, which is derived from Pg by activation by urokinase (UK), had a similar effect in this explant culture system, UK itself did not show any effect. Furthermore, we studied the characteristics of Pg binding to cultured neocortical neurons dissociated from an embryonic 16-day-old rat brain by using 125I-Pg. Specific binding of Pg to neocortical neurons was detected and Scatchard plot analysis revealed high- and low-affinity binding sites on the neurons. The estimated dissociation constants of high- and low-affinity binding sites were approximately 16.1 and 124.2 nM, respectively. These results suggest that microglia-derived Pg plays certain roles in the regulation of neurite extension through binding to the surface of neocortical neurons.

Pancreatic Splenic Lobe Organ Culture System: Viability and Amylase Release

An organ explant culture system for the intact chick pancreatic splenic lobe (SL) was characterized for exocrine function. Organ cultures were prepared using the pancreatic splenic lobe from 9- and 15-day-old male broiler chicks (Arbor Acres × Arbor Acres) to characterize amylase release as well as tissue integrity during 2, 4, 8, and 12 h of incubation. Light microscopy studies indicated necrosis of the exocrine pancreatic acini after 4 h of incubation. Changes in islets of Langerhans were noted 4 h after incubation, but islet structural integrity remained intact for up to 12 h of incubation. Lactate dehydrogenase (LD) levels measured in the culture medium did not increase significantly from 2 to 4 h of incubation. After 4 h of incubation, total LD levels increased (P < .05) for the 9-day-old SL cultures, and LD levels increased (P < .01) per unit weight of SL for the 15-day-old cultures. Medium amylase activity did not increase after 2 h of incubation. Large increases occurred for total amylase activity and amylase activity per unit weight of SL between 4 and 12 h of incubation for both 9- and 15-day-old cultures. Histological examination as well as increases in LD and amylase activities for total and per unit weight of SL in the incubation medium suggest that the viability of the 9- and 15-day-old SL organ incubation system decreases after 4 h.

Inhibition of interleukin 1-mediated proteoglycan degradation in bovine articular cartilage explants by addition of sodium hyaluronate

Summary The effect of exogenous hyaluronate on normal cartilage metabolism and interleukin-1 (il-1)-induced cartilage matrix degradation was investigated in a bovine cartilage explant culture system. Addition of hyaluronate at a concentration of 1.5 mg/ml to cartilage culture explants consistently decreased normal proteoglycan release from the matrix to a value less than that found in control cultures. Addition of 1.5 mg of hyaluronate/ml to il-1 stimulated cartilage culture systems reduced proteoglycan release from the matrix by 83 to 113%. The reduction in control and il-1-stimulated proteoglycan degradation by hyaluronate had a concentration-dependent trend. Evaluation of alterations in protein (enzyme) release by il-1-stimulated chondrocytes after introduction of hyaluronate was evaluated by use of sodium dodecyl sulfate agar gel electrophoresis of cartilage-conditioned media. The quantity or the molecular weight profile of il-1-induced proteins did not differ after introduction of hyaluronate into the culture system. Results indicate that introduction of high molecular weight hyaluronate into cartilage culture systems results in a decrease in proteoglycan release from the matrix in control systems, as well as in cultures incubated with il-1. Because il-1-stimulated protein synthesis by chondrocytes remains unchanged after addition of exogenous hyaluronate, the mechanism of inhibition of matrix degradation does not appear to be interference with binding of il-1 to chondrocytes or to be inhibition of the production of neutral metalloproteases, including stromelysin.

Synthesis of uterine endometrial proteins during early diestrus in the cyclic and pregnant dog, and after estrogen and progesterone treatment.

The objectives of this study were to identify and characterize dog uterine endometrial proteins synthesized de novo in explant culture during early luteal phase, to examine distribution of these proteins prior to the embryo's entering the uterus and during its free-floating period prior to implantation, and to examine regulation of endometrial proteins by estrogen and progesterone (P4) treatments. Uterine endometrium was collected from cyclic and pregnant bitches on diestrus Days 3, 7, and 10 as determined by loss of cornification of vaginal epithelium, and from ovariectomized dogs after treatment with corn oil, estrogen, P4, or estrogen followed by 1 or 2 wk of P4. Tissue was incubated in an explant culture system in the presence of [3H]leucine or [35S]methionine. The rate of incorporation of [3H]leucine into nondialyzable macromolecules indicated no significant change in rates of incorporation by status (pregnant vs. nonpregnant), day, or steroid treatment. Uterine endometrial-conditioned culture medium, analyzed by two-dimensional SDS-PAGE and fluorography, revealed a complex array of at least ten proteins or protein complexes in cyclic and pregnant bitches. No difference in protein pattern was detected by status; however, differences in distribution were apparent by day of cycle or early pregnancy. Two major proteins, cP5 (M(r) 54,686) and cP6 (M(r) 23,010) appeared to be differentially expressed. Expression of cP5, maximal on diestrus Day 3, decreased as the cycle or pregnancy progressed to diestrus Day 10. In contrast, expression of cP6, a minor protein on diestrus Day 3, appeared to be up-regulated for each status to Day 10, with increased intensity and multiple isoelectric and molecular-weight variants. In ovariectomized steroid-treated dogs, two-dimensional SDS-PAGE showed that pattern and distribution of specific proteins were affected by treatment. Acidic protein cP1 (M(r) 87,600), synthesized after corn oil and P4 treatment, was suppressed with estradiol (E2). Proteins cP2 (M(r) 40,000 and M(r) 42,000), present with all treatments, were intensified with P4. A high-M(r) basic protein complex (cP3) and acidic protein cP4 were expressed with E2 and maintained with P4 treatment. Proteins cP5 and cP6, while not induced by E2 or P4 alone, required E2 priming for P4 induction. Protein cP5 was down-regulated while cP6 was up-regulated with P4 for 2 wk. Proteins induced by estrogen followed by 1 or 2 wk of P4 treatments were similar to those released by endometrial explants collected from pregnant and cyclic bitches on Days 3, 7, and 10 of spontaneous diestrus.(ABSTRACT TRUNCATED AT 400 WORDS)

GABA and glycine immunoreactivity in normal and kainic acid-treated retinas

We first demonstrated a temporal profile of retinal ganglion cell (RGC) death after axotomy in situ using a newly developed retinal explant culture system. 1,1′- dioctadecyl- 3,3,3′,3′-tetramethylindocarbocyanine perchlorate, a fluorescent tracer, was administered to the superior colliculi of 2 day old Wistar rats to label RGCs retrogradely. Small pieces of retinas were dissected and maintained at the interface between a 5% CO2 atmosphere and culture media, and temporally observed by fluorescent microscopy. The number of surviving RGCs, identified as fluorescent spots, gradually decreased during the course of experiments for up to 10 days in vitro. We identified apoptotic RGCs by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. Administration of cycloheximide, actinomycin D, or a caspase-3 inhibitor to media significantly decreased RGC death. This system provides a method of quantifying axotomized RGC death in relation to time-dependent changes in an identical retinal slip.

An in vitro model for studying growth and effect of trauma and external agents on the cricoid at the cellular level.

Over the past several years it has become evident that expansion laryngotracheal surgery is effective in the treatment of laryngotracheal stenosis. Several clinical and animal studies have been performed to study the process of laryngotracheal stenosis and its treatment. However, there are still many questions that cannot be addressed by currently used clinical and animal research. Further indepth study of the behavior of the subglottis at the cellular level is necessary. We present an in vitro model for studying chondrocyte metabolism of the bovine cricoid. Cartilage was successfully grown in an explant culture system, and it was shown that the chondrocytes were metabolically active and responded to external agents. This model will serve to study the mechanism of growth and effects of trauma and external agents on the cricoid at the cellular level.

Purification and structural analysis of hippocampal cholinergic neurostimulating peptide

Hippocampal soluble fraction stimulates acetylcholine (AcCho) synthesis of medial septal nuclei in explant culture system. This stimulating activity was purified from 10–12-day-old rat hippocampus. During purification, the activity was separated into two fractions and a previously unreported peptide was purified from one fraction. The structure of this novel peptide is acetyl-Ala-Ala-Asp-Ile-Ser-Gln-Trp-Ala-Gly-Pro-Leu and we designated it as hippocampal cholinergic neurostimulating peptide (HCNP). Synthesized HCNP and de-acetylated HCNP (free-HCNP) stimulated AcCho synthesis of medial septal nuclei culture, in a dose-dependent manner, but not cultures of corpus striatum or anterior spinal cord. Mean half-maximal concentrations of HCNP and free-HCNP in AcCho synthesis of medial septal nuclei culture were 1.0 ± 0.3 × 10 −10M and1.0 ± 0.6 × 10 −11M, respectively. Affinity purified polyclonal antibody to the free-HCNP neutralized the activity of crude hippocampal extract, as well as synthetic HCNP and free-HCNP. These observations suggested that HCNP was present in the hippocampal extract and was involved in development of specific cholinergic neuron in central nervous system.

The Gingival Immune Response to Actinobacillus actinomycetemcomitans in Juvenile Periodontitis

The established and advanced lesions of juvenile periodontitis-localized form (JP) are predominated by B-lymphocytes and plasma cells. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. Actinobacillus actinomycetemcomitans (A.a.) is implicated as a primary etiologic agent in JP. An in vitro gingival explant culture system was utilized to study the specificity of immunoglobulins produced by diseased JP tissues. A dot-immunobinding assay demonstrated that 46% of the supernatant fluids (SF) from explant cultures of diseased tissues (n = 39) were positive for the presence of antibody to A.a. Y4, while 61% of autologous JP sera (n = 39) tested positive. For rapidly progressive (RP) and adult periodontitis (AP) SF, 50% and 40% were positive for A.a. Y4, respectively. Seventeen percent of SF from healthy tissue were positive for A.a. Y4. There was no significant difference between JP SF reactivities to A.a. Y4 when compared to reactivities of SF from AP and RP patients. Only 10% of JP SF were positive for Porphyromonas asaccharolytica, a non-oral control microorganism. The de novo biosynthesis of antibody in JP tissue, reactive with A.a. Y4, was demonstrated with Staph Protein A isolated 14C-labeled IgG (SPAG) and the use of a dot-immunobinding assay and autoradiography. The in vitro gingival tissue explant culture system described provides a useful model for the study of the synthesis and specificity of localized immunoglobulins produced by diseased tissues of JP patients.

Response to fibronectin of mouse primordial germ cells before, during and after migration

The adhesive extracellular matrix glycoprotein fibronectin is thought to play a central role in cell migration during embryogenesis. In order to define this role, we have examined the response to fibronectin in cell culture of mouse primordial germ cells (PGCs) before, during and after their migration from the hindgut into their target tissue, the genital ridges. Using an explant culture system, we show that PGCs will emigrate from tissue fragments containing hindgut, and that fibronectin stimulates this migration. Adhesion assays show that the start of PGC migration is associated with a fall in adhesion to fibronectin. Double-labelling studies using in situ hybridization and histochemistry demonstrate that migrating PGCs do not contain detectable fibronectin mRNA, suggesting that they do not synthesize and secrete the fibronectin within their migratory substratum. Taken together, these findings are consistent with an important role for fibronectin in stimulating PGC migration. In addition, however, they suggest that the interaction between PGCs and fibronectin may be important in timing the start of migration, with the fall in adhesion allowing the PGCs to commence their migration towards the genital ridges.

Estrogen-independent growth of mouse vaginal epithelium in organ culture

A serum-free vaginal explant culture system was established to investigate the in vitro effect of estrogen on the growth of mouse vaginal epithelium. Vaginal explants were isolated from 40-day-old, ovariectomized BALB/cCrgl mice and cultured in a basal unsupplemented medium or in basal medium plus various doses of 17 beta-estradiol. Explants were processed for histology at the end of culture periods or were given 4-hour pulses of tritiated thymidine at various times and processed for autoradiography. Vaginal epithelium increased 3- to 5-fold in thickness and 2-fold in the number of epithelial cell layers during 72 hours of culture without estrogen; addition of estrogen did not significantly influence epithelial growth. Keratinization of vaginal epithelium occurred within 48 hours of culture in the absence of estrogen, and again addition of estrogen did not accelerate its appearance. Covering the explants with collagen decreased the estrogen-independent growth of vaginal epithelium. Autoradiography showed that ca. 70-90% of basal epithelial cells entered S phase during the initial 4 hours of culture and that this number declined rapidly after 48 hours to ca. 20%. Addition of 1.8 nM 17 beta-estradiol significantly decreased the labelling index of basal cells at 48 hours, but did not affect the labelling index at 24 and 72 hours. Stromal cells were not labelled at any time. Thus, DNA synthesis, cellular proliferation, and differentiation (keratinization) of vaginal epithelium in organ culture occurred without estrogen and were not stimulated by the addition of estrogen.