Immunohistochemical study on adenohypophysial primordia in organ culture

Summary Rathke's pouches isolated from rat fetuses on day 12 were maintained in organ culture for 9 days and investigated immunohistochemically to test whether or not the hypothalamus is involved in the cytodifferentiation of the adenohypophysis. The unlabeled antibody enzyme method demonstrated that the cultured tissue contains different types of glandular cells, i.e., adrenocorticotropin (ACTH)-, growth hormone (GH)-, luteinizing hormone (LH)-, thyrotropin (TSH)-, and prolactin-producing cells. Indirect evidence was also obtained to indicate the presence of melanocyte stimulating hormone (MSH)-cells. These findings suggest that adenohypophysial primordial cells of rats start to synthesize their respective hormones without stimuli from neurosecretory substances of the brain which are known to be essential for the maintenance of the secretory activity of the adult gland.We wish to express our thanks to Dr. A. Kawaoi for providing anti-porcine ^1–39ACTH, to Dr. S.S. Spicer for the supply of anti-porcine ^17–39ACTH and to Dr. P. Petrusz for the gift of antisera against bovine GH, bovine TSH, HCG and rat prolactin. We should also like to thank Mr. Y. Okamura for technical help and Mr. I. Shimada for preparation of the photographs.     

Intracellular pathways of receptor-bound GnRH agonist in pituitary gonadotropes

Summary Localization of GnRH receptors in rat pituitary gonadotropes was studied by use of ¹²⁵I-[azidobenzoyl-D-Lys⁶]GnRH which, upon photolysis, is covalently bound to the receptor molecule. Using high resolution autoradiography, it was found that, after a 90-min incubation of the analog with pituitary cells at 4° C, 93% of the silver grains were associated with the plasma membrane of the gonadotropes. After 45-min incubation of the cells at 37° C, clustering and internalization of the receptor-bound GnRH analog were evident. Silver grains were associated with coated pits, intracellular vesicles, Golgi complexes, lysosome-like structures and secretory granules. The data indicate that receptor-bound GnRH agonist is internalized, at least in part, via coated pits and is subsequently routed to lysosomes where degradation of the hormone-receptor complex may occur. The presence of a considerable amount of silver grains associated with secretory granules may suggest that some of the internalized receptor molecules can escape degradation and be recycled to the cell membrane.     

Proteolytic degradation of gonadotropin-releasing hormone (GnRH) by rat ovarian fractions in vitro

GnRH in physiological concentrations is highly degradable by both soluble and particulate fractions of rat ovarian homogenate in vitro. The two proteolytic enzyme activities differ strongly by the soluble activity showing a dithiothreitol optimum, high inhibition by diisopropyl fluorophospate (k_i=0.7 μM), and a relatively high affinity (K_m=1.1 μM) as opposed to the particulate fraction (K_i=3.5 mM and K_m=150 μM, respectively). The results of this study show that the rat ovary is differently endowed with GnRH-degrading activity at different sites. The involvement of these in terminating the biological activity of the hormone on the ovary may possibly depend on its exact pathway in this GnRH-target organ.     

Failure of luteinizing hormone-releasing hormone (LHRH) to affect the differentiation of LH cells in the rat hypophysial primordium in serum-free culture

Summary The aims of this study were to investigate the differentiating capacity of adenohypophysial LH cells in a serum-free culture medium and to test whether cytogenesis is affected by synthetic LHRH. The adenohypophysial primordia of fetal rats were isolated on days 11.5 and 12.5 of gestation and cultured without serum for 10 and 9 days, respectively, in synthetic Medium 199 or MEM. Immunohistochemical examination using the PAP method revealed that most culture expiants, apart from a few degenerate ones, contained LH cells. In comparison with Medium 199, which has been widely used as a culture medium for hypophysial explants, aMEM gave far better results and the primordia cultured in this medium showed better tissue growth and contained a greater number of LH cells.Administration of synthetic LHRH (10 ng/ml) on the first day of culturing had no effect on the number of LH cells, no matter whether or not the culture medium was supplemented with insulin, transferrin or thyroxine. These results suggest that at the early developmental stage LHRH is not essential for the differentiation and/or proliferation of LH cells.     

Mitotic activity of gonadotropes in the anterior pituitary of the castrated male rat

Summary The proliferation of gonadotropes in the anterior pituitary of the castrated male rat was examined immunohistochemically after colchicine treatment. The results show a more than 10-fold increase in mitotic frequency in gonadotropes 1 or 2 weeks after castration, as compared with controls. This result explains the increase in the population of immunoreactive LH cells in castrated male rats. The gonadotropes decreased significantly 1 month after castration. The mitotic activity of gonadotropes was almost completely suppressed in castrates implanted with a silastic tube filled with testosterone.     

Effects of brain and mesenchyme upon the cytogenesis of rat adenohypophysis in vitro

Summary The objective of this in-vitro study was to examine whether the diencephalic floor or the mesenchyme is involved in differentiation of LH cells in the developing rat adenohypophysis. Overall growth of the adenohypophysial tissue was retarded when the adenohypophysial primordium was cultivated after enzymatic removal of the diencephalic floor on days 11.5 and 12.5 of gestation. This malgrowth was more marked when the brain was separated on day 11.5; most expiants retained a simple cystiform structure that consisted of a few layers of undifferentiated cells. Removal of the brain also caused a highly significant decrease (P < 0.001) in the number of immunoreactive LH cells, if it was performed on day 11.5 but not day 12.5. Mesenchyme had little effect on the adenohypophysial growth or the number of immunopositive cells. Cultivation of the adenohypophysial primordium with the diencephalic floor resulted in the appearance of many immunoreactive LH cells. The number of LH cells significantly decreased, however, when the co-cultivated brain completely surrounded the adenohypophysial tissue.These results indicate that in 11.5-day-old fetal rats the diencephalic floor is indispensable for the initial proliferation of adenohypophysial primordial cells and for the early determinating process of LH cells. Once determined, the development of LH cells may proceed without the surrounding tissues. The cytodifferentiation seems to be rather inhibited when in contact with the brain. The significance of the intimate spatial relationship between developing LH cells and the surrounding mesenchyme is also discussed.     

Pituitary adenylate cyclase-activating peptide (PACAP) immunolocalization in lymphoid tissues of the rat

Pituitary adenylate cyclase activating peptide (PACAP) is a novel peptide isolated from the ovine hypothalamus. PACAP exists in 2 molecular forms with 27 (PACAP27) or 38 (PACAP38) amino acid residues. PACAP localization was studied by immunohistochemical methods in central (bone marrow and thymus) and peripheral (spleen, lymph nodes and duodenal mucosa) lymphoid tissues with antisera raised against PACAP27 or PACAP38. PACAP-positive cells were found in all lymphoid tissues examined. These cells were highly positive for PACAP38 but were negative for PACAP27. Morphologically, they were small mononuclear cells with relatively scarce cytoplasm and lymphocyte-like features. PACAP38-positive cells were abundant in peripheral lymphoid tissues (i.e., mesenteric lymph nodes). In the duodenal mucosa, PACAP38-positive cells were located either in the lamina propria or epithelium. These results suggest that PACAP38-positive cells are present within lymphoid tissues and may represent a lymphocyte-like cell subpopulation that has a potential role in cell-to-cell interactions in the immune system and in the integrated communication between neuroendocrine and immune systems.     

Immunohistochemical localization of gonadotropin-releasing hormone (GnRH) in the brain and infundibulum of the sheep

Summary To determine how neural influences control the function of the pineal gland, morphological and biochemical relationships after pharmacological treatment have been studied in rat pineal cells in monolayer cultures. Norepinephrine (NE) and dibutyryl cyclic 3,5-adenosine monophosphate (dBcAMP) treatment of cells that had been in culture for 5 and 21 days produced a stimulation in the enzyme activity of serotonin N-acetyl transferase, an enzyme important in indole synthesis. NE and dBcAMP also produced morphological changes which were dependent on the time of cells in culture. When 5 day-cultures were treated with NE and dBcAMP, light and dark cells were noted and endoplasmic reticulum increased and became more organized. Only dBcAMP treatment at 5 days produced an increase in dense granules and an elongation of cytoplasmic processes. Treatment of 21 day-cultures with dBcAMP also produced an increase in cytoplasmic processes while treatment with NE produced an increase in the synaptic ribbons and clear vesicles within the processes.     

Contractility and structure of adult rat seminiferous tubules in organ culture

Summary Isolated pieces of seminiferous tubules of adult rats were grown in organ culture for up to 8 weeks in Petri dishes on the surface of nutrient agar. The medium consisted of newborn calf serum, Eagle's minimum essential medium, glutamate and antibiotics. This method allowed observation of the contractions of the seminiferous tubules in the culture. Contractility, light and electron microscopic structure and histochemically demonstrable activities of alkaline phosphatase and ATPase of the tubule walls were studied at 1-week intervals. The contractility and alkaline phosphatase activity were maintained in the tubule wall for 3 weeks, and the activity of ATPase was maintained for 5 weeks. The thin filaments of the myoid cells, which are responsible for the contractility, were seen with the electron microscope in tubules cultured for 5 weeks. The organ culture method described in the present paper seems to be valuable for studies concerning the functioning of the myoid cells of the seminiferous tubules and the possibility that this is regulated by hormones.     

Neuropeptide glutamic-isoleucine (NEI) specifically stimulates the secretory activity of gonadotrophs in primary cultures of female rat pituitary cells

The neuropeptide EI (NEI) is derived from proMCH. It activates GnRH neurons, and has been shown to stimulate the LH release following intracerebroventricular administration in several experimental models. The aim of the present paper was to evaluate NEI actions on pituitary hormone secretion and cell morphology in vitro. Pituitary cells from female rats were treated with NEI for a wide range of concentrations (1–400 × 10⁻⁸ M) and time periods (1–5 h). The media were collected and LH, FSH, PRL, and GH measured by RIA. The interaction between NEI (1, 10 and 100 × 10⁻⁸ M) and GnRH (0.1 and 1 × 10⁻⁹ M) was also tested. Pituitary cells were harvested for electron microscopy, and the immunogold immunocytochemistry of LH was assayed after 2 and 4 h of NEI incubation. NEI (100 × 10⁻⁸ M) induced a significant LH secretion after 2 h of stimulus, reaching a maximum response 4 h later. A rapid and remarkable LH release was induced by NEI (400 × 10⁻⁸ M) 1 h after stimulus, attaining its highest level at 2 h. However, PRL, GH and FSH were not affected. NEI provoked ultrastructural changes in the gonadotrophs, which showed accumulations of LH-immunoreactive granules near the plasma membrane and exocytotic images, while the other populations exhibited no changes. Although NEI (10 × 10⁻⁸ M), caused no action when used alone, its co-incubation with GnRH (1 × 10⁻⁹ M), promoted a slight but significant increase in LH. These results demonstrate that NEI acts at the pituitary level through a direct action on gonadotrophs, as well as through interaction with GnRH.     

Influence on antiproliferative activity of structural modification and conjugation of gonadotropin-releasing hormone (GnRH) analogues

The effect of various GnRH analogues, and their conjugates on proliferation, clonogenicity and cell cycle phase distribution of MCF-7 and Ishikawa human cancer cell lines was studied. GnRH-III, a sea lamprey GnRH analogue reduced cell proliferation by 35% and clonogenicity by 55%. Structural modifications either decreased, or did not alter biological activity. Conjugation of GnRH analogues including MI-1544, MI-1892, and GnRH-III with poly(N-vinylpyrrolidone-co-maleic acid) (P) through a tetrapeptide spacer GFLG(X) substantially increased the inhibitory effect of the GnRH analogues. The conjugate P-X-GnRH-III induced significant accumulation of cells in the G2/M phase; from 8% to 15.6% at 24 h and 9.8% to 15% at 48 h. It was concluded that conjugation of various GnRH analogues substantially enhanced their antiproliferative activity, strongly reduced cell clonogenicity and retarded cell progression through the cell division cycle at the G2/M phase.     

Effects of prolactin on the morphology and function of rat Leydig cells: short-term versus long-term administration

Summary The bolus administration of prolactin (PRL) to adult rats did not cause any apparent change in the basal and luteinizing hormone (LH)-stimulated blood levels of testosterone (as estimated by radioimmune assay). Prolonged PRL infusion did not affect either basal testosterone plasma concentration or the morphology of Leydig cells (as evaluated by electron microscopy and stereology). Conversely, prolonged PRL treatment notably increased the gonadotrophic effects of chronic LH administration; this mainly consisted of a rise in the blood concentration of testosterone and a conspicuous hypertrophy of Leydig cells. The LH-induced increase in the volume of Leydig cells was the result of an increase in the volumes of all the organelles involved in steroid synthesis (i.e., smooth endoplasmic reticulum, peroxisomes and mitochondria). However, the trophic effects of PRL infusion exclusively concerned smooth endoplasmic reticulum and peroxisomes. In the light of these findings, the hypothesis is advanced that the mechanism underlying the gonadotrophic action of PRL involves an enhancement of the endogenous cholesterol synthesis, which could provide an abundance of precursors for testosterone synthesis, the post-cholesterol steps of which, in turn, would be exclusively controlled by LH.     

Stereological study of gonadotropes in the frog,Rana pipiens,after GnRH stimulation in vitro

Summary Previous physiological results have indicated the existence of two releasable pools of gonadotropins in amphibian pituitaries: an acute releasable pool that appears independent of protein synthesis, and a storage pool involved in chronic release that depends on protein synthesis. To elucidate the ultrastructural localization of these pools and the morphological changes induced in gonadotrope cells after treatment with gonadotropin-releasing hormone, we carried out a morphometric study of immuno-identified gonadotrope cells using an in vitro superfusion system. Treatment with gonadotropin-releasing hormone induced a degranulation of small (110–255 nm) and medium (236–360 nm) secretory granules as well as hypertrophy of the endoplasmic reticulum and Golgi complex. Simultaneous incubation with gonadotropin-releasing hormone and cycloheximide inhibited the release of secretory granules although the endoplasmic reticulum and Golgi complex were hypertrophied. These morphological results strongly suggest: (1) that gonadotropin-releasing hormone induces degranulation and hypertrophy of the biosynthetic machinery in gonadotrope cells; and (2) that the activation of the endoplasmic reticulum and Golgi complex by stimulation with gonadotropin-releasing hormone is independent of protein synthesis, while the release of secretory granules is protein synthesis-dependent. In addition, the second or storage pool of gonadotropin is associated mainly with the small and medium secretory granules.     

Effects of thymulin and GnRH on the release of gonadotropins by in vitro pituitary cells obtained from rats in each day of estrous cycle

The effects of thymulin and GnRH on FSH and LH release were studied in suspension cultures of anterior pituitary cells from female adult rats sacrificed on each day of the estrous cycle. The spontaneous release of gonadotropins by pituitaries, as well as their response to GnRH or thymulin addition, fluctuated during the estrous cycle. Adding thymulin to pituitary cells from rats in diestrus 1 increased the concentration of FSH; while in cells from rats in estrus, FSH level decreased. Thymulin had a stimulatory effect on the basal concentration of LH during most days of the estrous cycle. Adding GnRH increased FSH release in cells from rats in diestrus 1, diestrus 2, or proestrus, and resulted in higher LH levels in cells obtained from rats in all days of the estrous cycle. Compared to the GnRH treatment, the simultaneous addition of thymulin and GnRH to cells from rats in diestrus 1, diestrus 2, or proestrus resulted in lower FSH concentrations. Similar results were observed in the LH release by cells from rats in diestrus 1, while in cells from rats in proestrus or estrus, LH concentrations increased. A directly proportional relation between progesterone serum levels and the effects of thymulin on FSH release was observed. These data suggest that thymulin plays a dual role in the release of gonadotropins, and that its effects depend on the hormonal status of the donor's pituitary.     

Immunoreactivity to gonadotropin-releasing hormone and gonadotropic hormone in the brain and pituitary of the rainbow trout Salmo gairdneri

Summary Immunoreactivity to gonadotropin-releasing hormone (GnRH) and gonadotropic hormone (GTH) was studied at the light-microscopical level in the brain and pituitary of rainbow trout at different stages of the first reproductive cycle using antisera against synthetic mammalian GnRH and salmon GTH. GnRH perikarya were localized exclusively in the preoptic nucleus, both in the pars parvicellularis and the pars magnocellularis. A few somata contacted the cerebrospinal fluid. Not all neurosecretory cells were GnRH-positive, indicating at least a bifunctionality of the preoptic nucleus. We recorded no differences between sexes or stages of gonadal development in the location of GnRH perikarya, whereas gradual changes were found in staining intensity during the reproductive cycle. GnRH fibres ran from the partes parvicellularis and magnocellularis through the hypothalamus and merged into a common tract at the transverse commissure before entering the pituitary. In the pituitary, GnRH was localized in the neural tissue of the neurointermediate lobe and, to a lesser extent, in the neural protrusions penetrating the proximal pars distalis. The bulk of GTH-positive cells was situated in the proximal pars distalis. Some cells were found more rostrally amidst prolactin cells or in the neurointermediate lobe. Only a limited number of GTH cells appeared to be in close contact with GnRH-positive material.     

Ion channel properties and episodic activity in isolated immortalized gonadotropin-releasing hormone (GnRH) neurons

The mechanism of periodic gonadotropin-releasing hormone (GnRH) secretion from hypothalamic neurons is difficult to elucidate due to the diffuse distribution of GnRH neurons and the complex interaction of neuronal inputs onto them. Recent use of transgenic techniques allowed construction of an immortalized GnRH neuronal cell line (GT1), which has neuronal markers and secretes GnRH in a periodic fashion. Using the patch-clamp recording technique in the whole-cell and nystatin perforated-patch configuration, the present experiments show that this cell line expressed a tetrodotoxin-sensitive Na channel, two types of Ca channels, three types of outward K channels and a K inward rectifier. The latter current was suppressed in some cells by GnRH or somatostatin. In addition, a gamma-aminobutyric acid (GABA) response, presumably through GABA_A receptors, is recorded. In long-term current-clamp recordings, spontaneous depolarizing activity was found to increase, and then decrease, between 20–35 min after removal of the cells from serum- and steroid-containing medium. In some cases, more than one cycle of activity was seen. Under voltage clamp, an inward current was recorded at similar times, with reversal at about ?15 mV. Thus, two mechanisms of cell interaction, GABA_A responses and feedback through GnRH responses, and one mechanism of endogenous periodic electrical activity were observed in these cells, which could synchronize periodic GnRH release.     

Mitral cell differentiation and synaptogenesis of rat presumptive olfactory bulb in organ culture

Summary The ultrastructure of differentiating rat presumptive olfactory bulb in organ culture was investigated with particular reference to mitral cell differentiation and formation of synapses. The presumptive olfactory bulb and olfactory mucosa were dissected en bloc from rat embryos on the fifteenth day of gestation and cultured for 7 days, after which the expiants were examined by electron microscopy. The presumptive olfactory bulb had differentiated into a laminated structure with layers corresponding to the glomerular, external plexiform and mitral cell layers. Mitral-like cells were identified by their location and large cell size. Ultrastructural observations indicated that they were relatively well-differentiated. Their dendrites extended into the glomerular layer in which they were postsynaptic to incoming olfactory axons. The distal part of these dendrites frequently contained coated vesicles. Both asymmetrical and symmetrical synapses were found. The symmetrical synapses involved dendrodendritic contacts between periglomerular cells. Synapses in reciprocal arrangements were not observed in the organ cultures.     

Ontogenetic development of synovial A cells in fetal and neonatal rat knee joints

Summary Ontogenetic development of the synovial A cells in fetal rat knee joints was investigated by immunohistochemistry, immuno-electron microscopy, cultivation, and autoradiography. At day 17 of gestation, immature macrophages were first seen in the articular interzone, and thereafter they differentiated into macrophages (synovial A cells), which were found in the synovial intima. The degree of reactivity of macrophages with five monoclonal antibodies increased in the developing synovial membranes of fetal rats as shown by immunohistochemistry. Similar findings were obtained in organ cultures of fetal knee joints. A marked difference of proliferative potential was found between A and B cells during ontogeny. A cells after birth did not incorporate ³H-thymidine in contrast to B cells. Before birth, B cells had a labelling index which was at least five times larger than that of A cells. The results of this study indicate that the synovial A cells are derived from both monocytes and fetal macrophages circulating in peripheral blood and that they differ from the synovial B cells in morphology, differentiation, and proliferative potential.     

Synthesis and in vitro evaluation of small-molecule [18F] labeled gonadotropin-releasing hormone (GnRH) receptor antagonists as potential PET imaging agents for GnRH receptor expression

Two novel small molecule gonadotropin-releasing hormone (GnRH) receptor antagonists (12 and 13) of the furamide-class were synthesized and evaluated in vitro for their receptor binding affinities for the rat GnRH receptor. Radiolabeling with no carrier added fluorine-18 of the appropriate precursors was investigated in a one-step reaction. Log P (Octanol/PBS pH 7.4) and serum stability of the compounds were investigated. The antagonists showed low nM affinity for the rat GnRH receptor. ¹⁸F-radiolabled compounds were obtained in high radiochemical purity (>95%) and specific activity (>75 GBq/μmol). These findings suggest this class of compounds holds promise as potential probes for PET targeting of GnRH-receptor expression.