Characterization at the single-cell level of naive and primed CD8 T cell cytokine responses.

The aim of this study was to characterize differences between naive and primed CD8 T cells. Our results show that (i) naive and primed CD8 T cells display similar activation thresholds, with no direct evidence for a difference in their TCR signals, and (ii) primed cells differ mainly in their capacity to secrete IFN-gamma. A comparison of the two populations at the single-cell level demonstrated that the increased production of IFN-gamma by the primed cell subset is due to a larger proportion of single cells that are able to synthesize this cytokine early following activation. These results indicate that the intrinsic effector capabilities of individual CD8 T cells expressing the same TCR are heterogeneous and that cells with identical antigen specificity but increased effector capacities are generated or selected during the primary response.

Memory CD44(int) CD8 T cells show increased proliferative responses and IFN-gamma production following antigenic challenge in vitro.

F5 TCR transgenic mice challenged in vivo with peptide generate long-lived primed CD8 T cells that hyper-proliferate in response to peptide in vitro. These primed CD8 T cells can be subdivided into three distinct populations on the basis of CD44 cell surface expression. In this report, we show that among primed CD8 T cells, those expressing intermediate levels of CD44 appear to be true memory T cells by the measurement of a variety of characteristics. Indeed, these cells hyper-proliferate in response to peptide re-stimulation in vitro, and produce IFN-gamma with faster kinetics and at higher levels than naive populations in vitro. We also show that CD8 T cells expressing high levels of CD44 express several activation markers and cycle in vivo in the absence of antigen. However, this population is unable to respond to peptide stimulation in vitro as measured by both proliferation and IFN-gamma secretion. The origin and specificity of these cells is unknown. These results provide evidence that memory CD8 T cells are functionally different from naive CD8 T cells both in terms of proliferation and cytokine secretion. They identify the CD8/CD44(int) T cells as the population responsible for hyper-reactivity in vitro.

Tolerant CD8 T cells induced by multiple injections of peptide antigen show impaired TCR signaling and altered proliferative responses in vitro and in vivo.

The mechanisms responsible for peripheral CD8 T cell tolerance to foreign Ags remain poorly understood. In this study we have characterized the state of CD8 T cell tolerance induced in F5 TCR transgenic mice by multiple peptide injections in vivo. The tolerant state of CD8 T cells is characterized by impaired proliferative responses, increased sensitivity to cell death, and failure to acquire cytotoxic effector function after in vitro antigenic challenge. In vivo monitoring of CD8 T cell proliferation using 5-carboxyfluorescein diacetate succinimidyl ester showed that a large subset of the tolerant T cell population failed to divide in response to peptide. TCR down-regulation could not account for this loss of responsiveness to Ag since recombination-activating gene-1 (RAG-1)-/-F5 CD8 T cell responses were similar to those of RAG-1(-/-)F5 x RAG-1(-/-)F1 T lymphocytes, which express lower levels of the transgenic TCR. Analysis of early signal transduction in tolerant CD8 T cells revealed high basal levels of cytoplasmic calcium as well as impaired calcium mobilization and tyrosine phosphorylation after cross-linking of CD3epsilon and CD8alpha. Together these data indicate that repeated exposure to soluble antigenic peptide in vivo can induce a state of functional tolerance characterized by defective TCR signaling, impaired proliferation, and increased sensitivity to cell death.

Development and differentiation of pituitary cells.

Classically, it was thought that the adenohypophyseal gland originated from the oral ectoderm. Its development has been the object of numerous studies over many years. However, several questions are still raised about its origin, differentiation, and commitment. The adenohypophyseal gland could originate from the anterior ridge of the neural plate. Glandular adenohypophyseal cells are committed very early in embryonic life. Interactions between adenohypophyseal presumptive territory and neighboring tissues can exist very soon, as early as at the open neural stage. The expression of a given phenotype by the committed cells seems to be controlled by a number of differentiation and/or transcription factors. In view of all these studies, performed with the use of different in vivo and in vitro models, classical concepts of the embryology of the adenohypophyseal gland need to be reevaluated. Indeed, many questions remain unanswered concerning the molecular mechanisms of known and unknown factors controlling development of the adenohypophyseal gland.

Resting memory CD8+ T cells are hyperreactive to antigenic challenge in vitro.

The characteristics of CD8+ T cells responsible for memory responses are still largely unknown. Particularly, it has not been determined whether different activation thresholds distinguish naive from memory CD8+ T cell populations. In most experimental systems, heterogeneous populations of primed CD8+ T cells can be identified in vivo after immunization. These cells differ in terms of cell cycle status, surface phenotype, and/or effector function. This heterogeneity has made it difficult to assess the activation threshold and the relative role of these subpopulations in memory responses. In this study we have used F5 T cell receptor transgenic mice to generate a homogeneous population of primed CD8+ T cells. In the F5 transgenic mice, peptide injection in vivo leads to activation of most peripheral CD8+ T cells. In vivo BrdU labeling has been used to follow primed T cells over time periods spanning several weeks after peptide immunization. Our results show that the majority of primed CD8+ T cells generated in this system are not cycling and express increased levels of CD44 and Ly6C. These cells remain responsive to secondary peptide challenge in vivo as evidenced by short term upregulation of activation markers such as CD69 and CD44. The activation thresholds of naive and primed CD8+ T cells were compared in vitro. We found that CD8+ T cells from primed mice are activated by peptide concentrations 10-50-fold lower than naive mice. In addition, the kinetics of interleukin 2R alpha chain upregulation by primed CD8+ T cells differ from naive CD8+ T cells. These primed hyperresponsive CD8+ T cells might play an important role in the memory response.

Dexamethasone inhibits the early steps of antigen receptor signaling in activated T lymphocytes.

Glucocorticoids are very effective in inhibiting inflammatory and immune responses. In particular, the synthetic analogue dexamethasone has been shown to inhibit T cell proliferation and IL-2 production by interfering with the transcriptional activation of the IL-2 gene. The experiments described in this report were performed to determine whether dexamethasone treatment affects the early steps of TCR signal transduction in T cell hybrids. Incubation of murine T cell hybrids in the presence of dexamethasone prevents the intracellular calcium increase that normally follows TCR/CD3 aggregation. Accordingly, dexamethasone treatment decreases inositol phosphates production and phospholipase-Cgamma1 tyrosine phosphorylation induced after TCR/CD3 stimulation. Dexamethasone has no effect on cell surface expression of TCR-associated structures nor does it inhibit calcium responses induced by a heterologous G protein-coupled muscarinic receptor, suggesting that this hormone analogue specifically inhibits the TCR signaling pathway at a postreceptor stage. We also show that inhibition of membrane proximal events by dexamethasone requires binding to the intracellular glucocorticoid receptor and de novo protein synthesis. When splenic T cells were assayed, only activated but not resting T cells were found to be sensitive to this new immunomodulatory effect of dexamethasone. These findings indicate that in addition to their previously described inhibitory effects on cytokine gene transcription, glucocorticoids block IL-2 production in activated T cells by interfering with an early step of the signal transduction cascade initiated by TCR/CD3 cross-linking.

Repression of transforming growth factor beta 1 protein by antisense oligonucleotide-induced increase of adrenal cell differentiated functions.

Transforming growth factor beta 1 (TGF beta 1) is a potent inhibitor of several differentiated functions in bovine adrenal fasciculata cells (BAC). In addition, these cells express and secrete this factor. To determine whether this peptide plays an autocrine role in BAC, cells were transfected with 10 microM unmodified sense (SON) or antisense (AON) oligonucleotide complementary to the translation initiation region of the TGF beta 1 mRNA in an attempt to inhibit TGF beta 1 protein synthesis. We investigated first, the cellular uptake, the stability, and the intracellular distribution of 32P-labeled TGF beta 1 AON and SON; and second, the effects of both oligonucleotides on BAC specific functions. We have demonstrated that in BAC, the TGF beta 1 AON uptake reached a plateau after 8 h of transfection (16% of the radioactivity added) and remained fairly constant for at least 24 h. In contrast, the uptake of TGF beta 1 SON reached a plateau after 2 h of transfection (8% of the radioactivity added), remained stable for only 3 h, and then declined. After 8 h of transfection, followed by 44 h of culture without oligonucleotides, the intracellular level of TGF beta 1 AON was still high with about 8% of the radioactivity added, whereas that of TGF beta 1 SON represented only 1.2%. Moreover, AON was present in the cytoplasmic and nuclear fractions, and it was hybridized in both compartments. However, TGF beta 1 SON was present mainly in the cytoplasmic fraction where it was not hybridized. Neither TGF beta 1 AON nor SON modified TGF beta 1 mRNA levels; however, TGF beta 1 AON, but not SON, caused the disappearance of TGF beta 1 immunoreactivity inside the cells. Finally, the steroidogenic responsiveness of BAC transfected with TGF beta 1 AON increased about 2-fold, and this was associated with a 2-fold increase of the mRNA levels of both cytochrome P450 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase. Neither TGF beta 1 SON nor a scrambled oligonucleotide containing the same number of G nucleotides as TGF beta 1 AON had any effect on these parameters. Thus, these studies demonstrate that TGF beta 1 has an autocrine inhibitory effect on BAC differentiated functions, an effect that can be overcome by TGF beta 1 AON.

Early signal transduction by the receptor to the chemokine monocyte chemotactic protein-1 in a murine T cell hybrid.

Members of the C-C family of chemotactic cytokines promote chemotaxis and adhesion of leukocytes. In this study, we have identified a murine T cell hybrid that expresses receptors to the chemotactic cytokine monocyte chemotactic protein-1 (MCP-1). This cell line was used to examine MCP-1 receptor-mediated signal transduction events in a homologous system in the absence of interference with other receptors. Our results show that in the 3B4 M1.9 T cell hybrid, MCP-1 receptors mediate intracellular calcium mobilization and extracellular calcium import without detectable increases in total water-soluble inositol phosphates. In addition, MCP-1 regulates the tyrosine phosphorylation of specific substrates at 42 and 44 kDa and induces mobility shift of p42/44 mitogen-activated protein kinases. MCP-1-mediated calcium responses, tyrosine phosphorylation, and the electrophoretic mobility shift of p42/44 mitogen-activated protein kinases can be inhibited by pretreatment of cells with pertussis toxin, indicating a role for Gi-like G proteins in coupling the MCP-1R to signal transduction.

The impact of distribution of a patient-education pamphlet in a multidisciplinary breast clinic.

STUDY GOAL: To determine the impact of the distribution of patient-education pamphlets to women with benign breast conditions in a large urban multidisciplinary breast clinic. METHODS: It is clinic policy to administer such materials at the first patient visit for these conditions. A standardized telephone interview was conducted with a random sample of 50 such patients at a mean of five months after that visit (range 1-12 months). RESULTS: While only 29 of the 50 women interviewed reported receiving such materials, 27 of these 29 women reported reading them. The patient's level of education and whether the patient had friends/relatives with breast cancer were not different between the women who had read the pamphlets and the women who had not (p = NS). The women who had received and read the material scored significantly better than did women who had not on a brief breast cancer screening questionnaire (p = 0.027). Of the former, all but three found the information of use; three-fourths of such women rated the materials 8 or higher on an increasing-usefulness scale of 1-10. The most frequently reported benefit gained from the materials was the proper conduct of breast self-examination. Overall patient satisfaction with the pamphlets was 91%. Half of the women receiving such materials shared them with other women. CONCLUSIONS: Patient-education pamphlets distributed at a multidisciplinary breast clinic result in high patient satisfaction and better patient knowledge of breast health. However, these materials were received and read by patients less often than was expected by staff. Because 82% of all women interviewed reported that the clinic staff, the distributed pamphlets, and other reading materials were their best sources of information, it is further concluded that the distribution of such materials is an important function of a multidisciplinary breast clinic.

Embryology of the pituitary gland.

For some 40 years, the development of the pituitary gland has been the subject of numerous studies. Several aspects of the origin, differentiation, and commitment of the pituitary cells, however, are still in doubt. The pituitary gland of several species apparently originates from the anterior ridge of the neural plate. Glandular pituitary cells can be committed very soon in embryonic life, as early as at the open neural stages. Numbers of differentiation and transcription factors may then control the expression of a given phenotype from the committed cells. Many questions remain unanswered about factors underlying such cellular commitment. These issues call for further studies to elucidate the molecular mechanisms of known and unknown factors controlling the embryology of the pituitary gland.

Interactions between adenohypophyseal, hypothalamic and nasal presumptive territories during early neurulation process.

In chick embryo, the adenohypophysis shows close morphological relationships with hypothalamic and nasal presumptive territories. However, we do not know how long the adenohypophysis depends on its surrounding tissues for its development and differentiation nor do we know anything about factors and mechanisms invovled. This study was undertaken to investigate whether any interactions between these neighbouring tissues influence adenohypophyseal cell growth and differentiation. The ablation of the presumptive hypothalamus and neurohypophysis results in the failure of hypothalamic and infundibular process development. However, the adenohypophysis was present, although it was drastically modified. Moreover, gonadotrophs and corticotrophs can be detected in the developing adenohypophyseal tissue. After the ablation of nasal presumptive territory, from where GnRH neurons originate, the adenohypophyseal length and the number of gonadatrophs and corticotrophs are not significantly altered when compared to control embryos. These results suggest that the presumptive hypothalamus and neurohypophysis are committed during open neural stage. At the following stages, these territories may act to promote the future adenohypophysis development and morphogenesis. However, it seems that pituitary cells are committed from the very early embryonic stages, but interactions between the presumptive adenohypophysis and adjacent territories before the open neural stage cannot be ruled out.

CD45-mediated regulation of extracellular calcium influx in a CD4-transfected human T cell line.

Transfection of a CD4- Jurkat leukemic T cell line with the human wild-type CD4 gene resulted in the constitutive mobilization of calcium by these cells. The altered calcium phenotype was dependent on expression of a completely functional CD4 molecule on the cell surface. Transfectants receiving the vector alone or those in which a mutated CD4 gene lacking a functional Lck binding region failed to generate a constitutive calcium response. In addition, CD4 wild-type transfectants over time lost CD4 expression. These CD4- revertants failed to constitutively mobilize calcium. Treatment of CD4 wild-type transfected cells with either anti-CD45 mAb, EGTA, or PMA rapidly restored the cells to basal levels of intracellular calcium. Analysis of CD45 cross-linking on CD4+ and CD4- normal Jurkat lines demonstrated that CD4 expression was required for CD45-mediated inhibition of TCR induced calcium responses. CD45-mediated inhibition affected the duration of the response rather than its magnitude. These results, taken together with the observations obtained with CD4 transfected Jurkats suggested that influx of extracellular calcium was the predominant reason for the elevated levels of calcium within the cell. In support of this hypothesis, we could find no evidence of phosphorylated phospholipase C gamma 1 or constitutive inositol 1,4,5 trisphosphate generation in the CD4 wild-type transfected cells, yet both were detectable after anti-CD3 mAb-induced activation. Immunokinase assays of Lck and Fyn precipitated from untreated or anti-CD45-treated wild-type transfectants demonstrated that CD45 cross-linking dephosphorylated Lck and reduced its capacity to phosphorylate enolase. In contrast, neither Fyn autophosphorylation nor its activity was affected by CD45 cross-linking.

Induction of pituitary cell type differentiation by delta sleep-inducing peptide.

The effects of delta sleep-inducing peptide (DSIP) on pituitary cell differentiation was studied using an in vitro method and immunocytochemical techniques. Pituitary primordia were explanted from 11-day-old rat fetuses and cultured in a synthetic medium enriched with either DSIP at several concentrations, GnRH (10(-9) M) or TRH (10(-9) M). Expression of different pituitary phenotypes was quantified as the percentage of immunoreactive area per section of cultured primordia. Addition of DSIP during the first day of culture induced differentiation of LH and TSH cells only. The effect was dose-dependent. DSIP was less potent than GnRH and as potent as TRH in inducing LH and TSH differentiation. DSIP also induced lactotrope differentiation, but this effect may not be direct. DSIP had no effect on somatotrope and corticotrope differentiation. These results obtained in vitro suggest that DSIP exerts a direct action on the differentiation of several pituitary precursor cells.

Expression and regulation of transforming growth factor-beta 1 messenger ribonucleic acid and protein in cultured porcine Leydig and Sertoli cells.

In the present work, the expression and secretion of transforming growth factor-beta 1 (TGF beta 1) by immature pig Leydig and Sertoli cells were investigated. Both cell types express two TGF beta 1 mRNA transcripts of 2.5 and 3.5 kilobases, and the levels were 2.6-fold higher in Leydig than in Sertoli cells. In the latter cells, mRNA levels were enhanced when cultured cells were stimulated by epidermal growth factor and phorbol ester (4-beta-phorbol 12-myristate 13-acetate) and significantly decreased by FSH and testosterone. Using a polyclonal antibody raised against a synthetic peptide that corresponded to the carboxyl-terminal region of TGF beta 1 and recognized this peptide, but not TGF beta 2 or TGF beta 3, specific immunostaining of both Leydig and Sertoli cells was demonstrated in situ, after cell isolation, and during culture. The immunostaining was more marked in Leydig cells than in Sertoli cells. Western blot analysis of Leydig or Sertoli cell-conditioned medium demonstrated a band of 25 kilodaltons, which was shifted to 12.5 kilodaltons under reducing conditions. Using the mink lung epithelial cell bioassay for TGF beta 1, we could demonstrate the presence of TGF beta 1-like activity in Leydig and Sertoli cell-conditioned media after acid treatment, but not before activation. The inhibitory effects of both pure TGF beta 1 and acidified conditioned medium were almost completely blunted by the TGF beta 1 antibody. The amounts of TGF beta 1 secreted by Sertoli and Leydig cells were not significantly different and varied between 400-800 pg/48 h.10(6) cells. These studies demonstrate for the first time that both pig Leydig and Sertoli cells express TGF beta 1 mRNA, and the TGF beta 1-like activity secreted by these cells corresponds to TGF beta 1. As TGF beta 1 has been demonstrated to have strong effects on testicular cells, in particular on Leydig cell functions, it is suggested that local secreted TGF beta 1 may play a role in the autocrine/paracrine regulation of testicular functions.

Cross-linking of Fc gamma receptor to surface immunoglobulin on B cells provides an inhibitory signal that closes the plasma membrane calcium channel.

Stimulation of B lymphocytes by the cross-linking of surface Ig (sIg) with an F(ab')2 antibody fragment leads to the rapid activation of several tyrosine kinases. This gives rise to the activation of phospholipase C gamma (PLC gamma) and the generation of inositol phosphates. These, in turn, lead to a prolonged elevation of intracellular Ca2+ ([Ca2+]i) consisting of a rapid release of Ca2+ from intracellular stores and a sustained influx of extracellular Ca2+. In contrast, co-cross-linking sIg to Fc gamma receptor (Fc gamma RII) with intact anti-sIg induces a much more transient increase in [Ca2+]i. Stimulation of the murine B cell lymphoma, A20, with F(ab')2 anti-sIgG leads to the production of high levels of IL-2, while co-cross-linking of sIgG with Fc gamma RII blocks this response. In studies reported here, we show that co-cross-linking of Fc gamma RII with sIg prevents the influx of extracellular Ca2+ without significantly affecting the tyrosine phosphorylation of substrates including PLC gamma 1, PLC gamma 2, and Syk or the mobilization of Ca2+ from intracellular stores. In cells that had been previously activated with F(ab')2 anti-IgG, co-cross-linking of sIg to Fc gamma RII rapidly abrogated the influx of extracellular Ca2+ by closing the plasma membrane Ca2+ channel. Additionally, even 2-3 h after stimulation of the cells with F(ab')2 fragment, addition of intact anti-IgG to the cells, or removal of extracellular Ca2+, markedly inhibited (> 90%) IL-2 production. These results indicate that co-cross-linking sIg with Fc gamma RII both prevented the opening of and actively closed the Ca2+ channel, and, through this mechanism, Fc gamma RII was able to control production of IL-2. Overall, since influx of extracellular Ca2+ has been found to be necessary for the proliferation and differentiation of B cells, Fc gamma RII may play a critical role in controlling these responses by regulating the opening of the Ca2+ channel.

Description of a human papillary thyroid carcinoma cell line. Morphologic study and expression of tumoral markers.

BACKGROUND: The establishment of cell lines from thyroid carcinomas can provide an in vitro model of oncogenesis. B-CPAP is a new cell line that has been obtained from a differentiated papillary thyroid carcinoma. The data presented give a broader characterization and expression of tumoral markers of this cell line and identify the differentiated functions that are preserved. METHODS: An ultrastructural study was performed to confirm the thyroid nature of the new cell line. The cellular markers (thyroglobulin, S100, neuron-specific enolase [NSE]) and the oncogenes (mutated p53, H-ras, c-myc, PTC, trk) were studied by immunohistochemistry, Southern blot, or in situ hybridization. RESULTS: The cells were of a differentiated ultrastructural thyroid type. All of the cells proved immunoreactive with antibodies specific to thyroglobulin, S100 proteins, NSE, and mutant p53 protein. Mutations of H-ras, PTC, and trk were not observed. The c-myc gene was not amplified. CONCLUSIONS: The cell line described in these data provides a suitable model for the study of thyroid carcinogenesis, given that the cells present thyroid characteristics, and metabolic disorders not previously found in such cell lines. In addition, the coexpression of S100 proteins and mutant p53 proteins in the cells should permit the study of the interaction between these two proteins.

Re-evaluation of gonadotropin-releasing hormone (GnRH) action on pituitary cell differentiation with special regard to its effect on LH and TSH cell types.

We have previously demonstrated that TRH induces several hypophyseal cell type differentiation. In the present study, we re-evaluated the effect of GnRH on gonadotroph differentiation, and we determined whether gonadotropin is implicated in the appearance of other pituitary cell types. These effects were compared to the influence of TRH on LH and TSH cell differentiation. Pituitary primordia of 11 day-old rat embryos were cultured in a synthetic medium until the equivalent of term, and the LH and TSH cell differentiation was visualized using an immunohistochemical technique. The effect of GnRH on pituitary hormone cells was evaluated by measuring the percentage of immunoreactive area per section. GnRH, when added on the first day of culture, induced LH and TSH cell differentiation in a dose-dependent manner. GnRH was more effective than TRH in inducing gonadotropic phenotype expression. This suggests that GnRH, which evokes LH release, is the most effective factor inducing gonadotroph differentiation in early fetal life. However, GnRH was as effective as TRH in thyrotroph differentiation. Lactotrophs were detected in primordia cultured with GnRH. As lactotroph phenotype differentiation is controlled by the alpha-subunit of glycoproteic hormone, further studies are required to determine whether or not GnRH effect on lactotroph is direct. GnRH did not affect the corticotroph and somatotroph differentiation. These results confirm that hypothalamic factors such as TRH or GnRH are capable of inducing more than one pituitary cell phenotypes.

T cell long-term hyporesponsiveness follows antigen receptor engagement and results from defective signal transduction.

T cell receptor (TCR)-mediated stimulation of T hybridomas leads to cell activation and lymphokine production that is followed by a long-term hyporesponsiveness. To investigate the biochemical events involved in the induction and maintenance of this antigen receptor hyporesponsiveness or anergy, we have expressed a G protein/PLC beta 1-coupled muscarinic subtype 1 acetylcholine receptor in a murine T cell hybrid. Transfected cells were capable of responding to both muscarinic agonists and TCR ligands by inducing interleukin-2 secretion that was sensitive to cyclosporin A and dexamethasone. Both receptors induced tyrosine kinase (TK) activity, but muscarinic stimulation did not affect tyrosine phosphorylation of PLC gamma 1, nor did the TK inhibitor, herbimycin, block muscarinic receptor-mediated calcium mobilization. These data indicate that in T cells, the muscarinic receptor mediates T cell effector functions by regulating a TK-independent proximal pathway which later converges with the TCR pathway. Using these cells, we have explored the long-term consequences of T cell stimulation via antigen or muscarinic receptors. Our results show that hyporesponsiveness specifically follows TCR engagement and appears to result from a defect in the early signal transduction initiated by TCR cross-linking. A study of TCR-mediated signaling supports this model by showing that tyrosine phosphorylation and calcium mobilization are deficient in hyporesponsive T cells.

Intracellular gonadotropin-releasing hormone immunoreactivity in gonadotrophs of intact or castrated male rats: semiquantitative estimation of testosterone and GnRH antagonist treatment.

Changes in intracellular gonadotropin-releasing hormone (GnRH) immunoreactivity (GnRH-IR) in pituitary gonadotrophs were assessed by the use of a semiquantitative immunocytochemical method in male rats undergoing various manipulations known to greatly modify gonadotropin secretion. In basal conditions, immunoreactive GnRH was localized in the cytoplasmic matrix, the secretory granules and the nucleus of these cells. Following intravenous stimulation with GnRH (100 ng i.v.), the GnRH-IR increased rapidly in all these three subcellular compartments, peaking at 15 min. In untreated, long-term castrated rats, GnRH-IR increased both in the basal state and after administration of GnRH. Injection of the castrated rats with testosterone propionate reduced the observed GnRH-IR to the level observed in intact rats. Acute or chronic treatment of castrated rats with a potent GnRH antagonist rendered GnRH-IR completely undetectable in all the three previously positive subcellular compartments of gonadotrophs, and GnRH-IR did not reappear after stimulation with GnRH. In sum, the fact that modifications of GnRH immunoreactivity are observed in rat gonadotrophs: (1) confirms the GnRH internalization process; (2) suggests different sites of action for GnRH within the cell, and (3) demonstrates rapid clearance of intracellular GnRH after stimulation.