Autoradiographic localization of dihydrotestosterone and testosterone concentrating neurons in the brain of the oyster toadfish.

Vertebrate species with male mating calls or songs tend to have sexually dimorphic sonic neurons that concentrate gonadal steroids. The distribution of [3H]dihydrotestosterone- and testosterone-concentrating neurons was examined in oyster toadfish (Opsanus tau), males of which produce a courtship boatwhistle call. Labeled cells in the forebrain were found in the posterior nucleus of the dorsal telencephalon (Dp), a pallial structure, the supracommissural nucleus of the ventral telencephalon (Vs), nucleus propticus parvocellularis anterior (PPa) and other preoptic nuclei, the ventral, dorsal and caudal hypothalamus. Positive brainstem areas included the optic tectum, torus semicircularis, nucleus lateralis valvula, a periventricular nucleus of the rostral medulla and the inferior reticular formation. Compared to estrogen, androgens labeled fewer sites in the forebrain and more in the brainstem. Two of the positive sites, Vs and PPa, have been implicated in boatwhistle production. Many sites that connect to these areas in teleosts likewise concentrate steroids. Unlike the situation in frogs, birds, and one other teleost, the toadfish sonic motor nucleus did not concentrate androgens. Androgen labeling in the posterior nucleus of the dorsal telencephalon represents the first autoradiographic demonstration of steroid concentration in the pallium of a teleost forebrain.

Autoradiographic localization of estrogen-concentrating cells in the brain and pituitary of the oyster toadfish.

We examined the distribution of [3H] estradiol-concentrating neurons in gonadectomized male and female oyster toadfish (Opsanus tau) by thaw-mount autoradiography. Compared to other teleosts studied in this fashion, the toadfish is of interest because of its reliance on male sound production during courtship. Labeled cells were found in the pars distalis of the pituitary and in periventricular midline regions of the forebrain. These included the ventral, supracommissural and posterior nuclei of the ventral telencephalon, nucleus preopticus parvocellularis anterior, the ventral, lateral, dorsal and caudal hypothalamus, and the periventricular nucleus of the posterior tuberculum of the thalamus. For the first time in a teleost, estrogen-concentrating cells were recorded in the brainstem, in sites including the optic tectum and torus semicircularis. In contrast to a previous study with testosterone, two prominent nuclei in the medulla were negative. With some additions, the distribution of estrogen-concentrating neurons conformed to the general pattern in other teleosts.

Dihydrotestosterone induces a sexual dimorphism in estrogen uptake by specific anterior pituitary cell types in vivo.

Castrated male and female rats pretreated with dihydrotestosterone (DHT) were injected i.v. with 3H-estradiol (E2). Nuclear uptake and retention of 3H-E2 was measured in each of five cell types of the anterior pituitary gland using a combined quantitative autoradiographic and immunocytochemical procedure. In non-pretreated groups, each cell type bound a characteristic amount of ligand but no sex differences were apparent. DHT pretreatment, however, caused a significant decrease in 3H-E2 retention by gonadotrophs in both males and females. The treatment also caused a decrease in binding by lactotrophs and somatotrophs, but only in the females. No other cell types were altered. Thus, androgen appears to modulate E2 binding and retention by pituitary cells in both a cell-type and sex-dependent manner. Our results also indicate that the inhibitory effects of androgens on E2 binding by the pituitary gland is more complex than can be explained by simple competition for the estrogen receptor.

Prostate adenocarcinoma: effects of castration on in situ androgen uptake by individual cell types.

The cellular distribution of androgen uptake was investigated in normal prostate and in Dunning R3327H prostate tumors in Copenhagen rats in vivo at different time intervals after castration. Quantitative dry-autoradiography was used to demonstrate which cell types have androgen binding, and to quantify the amount of androgen binding per cell type during initial castration-induced tumor regression and subsequent tumor relapse. Regardless of time after castration, tumor stromal nuclei had significantly more 3H-dihydrotestosterone (DHT) labelling than did tumor epithelial nuclei (p less than .001). On the other hand, prostate gland epithelial nuclei showed more 3H-DHT binding than prostate gland stromal nuclei. Tumor stromal nuclei had greater DHT uptake than any other cell type measured in the tumor or in secondary sex organs at all times after castration. Different cell types responded differently to castration. The 3H-DHT uptake measured in tumor stromal nuclei after castration showed that one day after castration 14.6 +/- 2.1 silver grains were present, 14 days after castration 9.3 +/- 2.4 were seen, and 50 days after castration 18.9 +/- 1.8 were present. This significant increase from 14 days to 50 days is not seen in the other cell types. This study gives insight into the cellular androgen receptor distribution in normal and malignant rat prostate tissue.

Estrogen uptake capacity of individual pituitary cell types from male and female rats one, fourteen and fifty days after castration.

A quantitative autoradiographic technique was combined with immunocytochemical staining to compare 3H-estrogen uptake in individual pituitary cell types 1, 14 or 50 days after castration in both male and female rats. Silver grains were counted over nuclei of immunocytochemically stained cells and means were computed for each cell type. The order of labelling intensity for all groups was gonadotropes greater than or equal to lactotropes = somatotropes greater than thyrotropes = corticotropes. In male rats 3H-estrogen uptake capacity in all of these cell types remained unchanged over the post-castration interval. Only gonadotropes from female rats demonstrated a significant change in estrogen uptake capacity over the intervals examined. Uptake in these cells increased by 137% between 1 and 50 days after ovariectomy. At both 14 and 50 days post-ovariectomy, gonadotropes concentrated significantly more radioactive label than either lactotropes or somatotropes. One day after castration, gonadotropes from females concentrated less 3H-estrogen than males while at 50 days after castration they concentrated significantly more than gonadotropes from male rats.

Calcium, cAMP, and dopamine affect single mammotrophs assayed by hemolytic plaques.

We investigated the effects of dopamine, forskolin, and maitotoxin on prolactin release from individual rat and monkey mammotrophs. Forskolin increases cAMP levels, whereas maitotoxin amplifies the influx of extracellular calcium. Prolactin secretion from single mammotrophs was visualized by the reverse hemolytic plaque assay and then quantified by measuring the proportion of plaque-forming cells and the mean plaque area. In the presence of dopamine alone both the plaque proportion and mean area of the plaques formed by rat mammotrophs decreased by 50 and 40%, respectively. This inhibition of secretion was blocked by the dopaminergic antagonist spiperone as well as forskolin and maitotoxin. Forskolin and maitotoxin alone significantly elevated both the proportion (+30%) and area (+180% for forskolin and +250% for maitotoxin) of the plaques. These actions of maitotoxin were neutralized by D-600, a calcium channel blocker. All of these agents induced similar trends with monkey prolactin cells. We conclude that single mammotrophs in culture respond to perturbations in a differential manner and in a way predicted by earlier results based on macropopulation measurements.

Binding of [3H]aldosterone to a single population of cells within the rat epididymis.

Using the dry-mount autoradiographic technique, a single population of cells within the rat epididymis, the clear cells, have been shown to bind [3H]aldosterone at a nuclear site. Competitive binding experiments demonstrated that aldosterone was more potent than desoxycorticosterone than testosterone in reducing the nuclear uptake of radioactive aldosterone. Furthermore, the other epididymal cells (principal and basal cells) in all regions of the epididymis were not significantly labelled; occasional labelling was noted in some endothelial and stromal cells. It is suggested that aldosterone may play a role in controlling the intracellular and transcellular movement of ions and water necessary for concentrating absorbed macromolecules in the clear cell.

Biological activity of a growth hormone-releasing factor secreted by a human tumor.

A substance released by a pancreatic islet cell tumor induced signs and symptoms of acromegaly in a young woman. The culture medium in which the tumor was placed after resection was added to rat anterior pituitary cells and incubated in vitro. Both newly synthesized and total rat growth hormone (GH) release as well as cellular cyclic AMP accumulation were stimulated in a dose-dependent manner by the tumor medium. Coincubation with somatostatin blocked these effects. The increase of cyclic AMP preceded the enhanced GH release, indicating that cyclic AMP may be a second messenger for the tumor factor(s). Neither prolactin nor luteinizing hormone secretion was affected by the tumor medium. When measured by a perfused cell column apparatus, there was a rapid and dramatic release of GH by the dispersed rat pituitary cells during a 2.5-, 10-, and 40-min pulse of tumor medium; both the onset and termination of the GH response reached maximal or control values, respectively, within 5 min. Pretreatment of the tumor medium with pepsin markedly attenuated the tumor medium activity, indicating the peptide nature of the factor(s). Finally, ultrastructural analysis indicated that the somatotrophs were degranulated by the tumor medium, whereas there was no similar effect apparent on the mammotrophs. Whether this tumor polypeptide is identical to native hypothalamic GH-releasing hormone remains to be proved.

Evidence for protein absorption from the lumen of the seminiferous tubule and rete of the rat testis.

As luminal fluid moves from the seminiferous tubule and enters the rete testis, its protein concentration declines from approximately 6 mg/ml to 1 mg/ml. It was therefore suggested that protein is either 1) utilized by the spermatozoa, 2) transported across the epithelium of the terminal segment of the seminiferous tubule, the tubuli recti or rete testis, or 3) absorbed and degraded by the epithelium. Horseradish peroxidase (HRP), a protein marker, was microperfused into single seminiferous tubules or perfused directly into the rete. After fixation, the HRP was localized histochemically and the tissue observed under the light- and electron microscope. HRP was taken up via pinocytotic vesicles into the cytoplasm of the Sertoli cells and germ cells but did not permeate extracellularly beyond the tight junctions. Similar results were obtained in the cells lining the terminal segment and the tubuli recti. The rete epithelium showed uptake of HRP into coated and noncoated vesicles, while some cells additionally revealed diffuse cytoplasmic distribution of HRP. The terminal segment, tubuli recti, and rete testis may be important routes by which proteins may leave the testicular fluid either to be degraded or to enter the blood.

Acrolein/glutaraldehyde as a fixative for combined light and electron microscopic immunocytochemical detection of pituitary hormones in immersion-fixed tissue.

In an attempt to find a fixative that has a wide range of application in immunocytochemical studies of pituitary tissue, we have investigated the use of acrolein fixation followed by postfixation in osmium. Pituitary glands were excised and immersion-fixed in 2 or 5% acrolein/0.25% glutaraldehyde for 1, 2.5, 5, or 24 hr. Some tissues were subsequently postfixed in 1% OsO4 for 1.5 hr before embedding in Epon-Araldite. Sections were collected for both light and electron microscopic immunocytochemistry, employing several pituitary hormone antisera. Fixation for 2.5 hr with 2% acrolein/0.25% glutaraldehyde followed by osmication resulted in good staining for both light and electron microscopy with all antisera employed. Furthermore, the rapid penetration qualities of this fixative resulted in good ultrastructural preservation throughout the tissue.

Failure of dopamine and bromocriptine to affect prolactin release and cell growth in the dopamine receptor-deficient 235-1 clone.

The 235-1 clone was recently derived from the 7315a transplantable pituitary tumor and continues to secrete rat prolactin. The cells have a prominent Golgi apparatus which can be stained immunocytochemically for prolactin, but there were no 600-900 nm granules which are characteristic of normal mammotrophs. In a perfused cell-column apparatus, prolactin release from the clone was unchanged by dopaminergic agonists, thyrotropin-releasing hormone and estradiol but stimulated by dibutyryl cyclic AMP. Cellular cyclic AMP content was also not changed by dopamine but was dramatically enhanced by prostaglandin E1, indicating that at least one hormone-adenylate cyclase coupling mechanism was functional. In radioligand binding studies using the dopamine antagonist [3H]spiperone, no evidence of a dopamine receptor was obtained. The [3H]spiperone binding present was not stereoselective, and exceedingly high concentrations of other ligands were required to displace the binding. In addition, the induction of a prolactin-secreting hard tumor in rats by subcutaneous inoculation of the 235-1 cells failed to induce measurable dopamine receptors associated with the tumor cells. In order to address the possibility that there were functional dopamine receptors on these cells, but that they could not be resolved with either the cell column and cyclic AMP studies or the radioreceptor assay, the clone cells were incubated with 0.1-100 nM bromocriptine for up to 8 days. Bromocriptine had no effect on the growth rate or prolactin secretion of the 235-1 clone but inhibited prolactin release from anterior pituitary cells by over 73% in control studies. We conclude that the 235-1 clone does not express dopamine receptors and that the presence of dopamine receptors is obligatory for the typical inhibitory effects of bromocriptine on prolactin release and pituitary cell growth.

Immunocytochemical and ultrastructural identification of mitotic cells in the pituitary gland of ovariectomized rats.

Light microscopic immunocytochemistry was utilized on plastic-embedded, acrolein-fixed pituitary glands from intact rats and rats killed 14 days after ovariectomy to determine which cell types were undergoing cell division. A significant increase in the number of cell divisions in anterior pituitary cells was seen in ovariectomized rats compared to intact controls. Most of the dividing cells in ovariectomized rats were immunocytochemically identified as gonadotrophs, but dividing somatotrophs and non-immunoreactive cells were also found. All of the dividing gonadotrophs stained with both anti-FSH beta and anti-LH beta. These cells were large and ovoid with plentiful vesiculated rough endoplasmic reticulum and a single population of granules with a mean diameter of 200 nm. Many of the dividing cells which were not immunoreactive with any of the antisera employed contained small granules, 100 nm in diameter, but the content of these granules was not determined.

Dynamics of in situ estrogen uptake by nuclei of individual pituitary and uterine cell types.

This investigation was carried out to examine the temporal aspects of nuclear retention of 3H-estrogen by individual target cell types of the rat uterus and pituitary. A quantitative dry autoradiographic-immunocytochemical method was used to measure relative 3H-estrogen uptake by nuclei of seven different target cell types 15 m, 1 h, 3 h and 7 h after injection of 3H-estradiol-17 beta. All five target cell types in anterior pituitary showed maximal nuclear retention at 15 m. Possible cell-type differences were noted in the rate of release of the isotope from nuclei. In the uterus maximal uptake, occurred between 1 h and 3 h after injection with stromal cells consistently concentrating more 3H-estrogen than luminal epithelial cells. These findings indicate possible differences between the mechanisms controlling nuclear retention of estrogen by the uterus and the pituitary.

Testosterone uptake in the brainstem of a sound-producing fish.

Three nuclear areas in the medulla were implicated in the control of sound production in the oyster toadfish Opsanus tau. The sonic motor nucleus was labeled by retrograde transport of horseradish peroxidase injected into swimbladder sonic muscles, and an adjacent ventrolateral and a more anterior periventricular nucleus of the medulla were revealed by autoradiography with 3H-labeled testosterone. These androgen uptake sites occur in brainstem areas corresponding to areas predicted to contain the neural centers controlling the duration and fundamental frequency of the toadfish mating call.

Nuclear retention characteristics of [3H]estrogen by cells in four estrogen target regions of the rat brain.

Nuclear retention of radioactivity was studied in neural estrogen target cells 15 min-7 h after i.v. injection of [3H]estradiol. Maximal uptake occurred by 15 min. Cells of the medial preoptic nucleus retained the label longer than did those of the medial amygdaloid nucleus. Cells of the ventromedial nucleus and arcuate nucleus exhibited similar retention profiles which were intermediate between the medial preoptic and medial amygdaloid cells. These data are discussed in relation to observations that the duration of nuclear occupancy by estrogens is proportional to the magnitude of the cellular response.

Induction by progesterone of a sexual dimorphism of estrogen uptake by anterior pituitary cells in situ.

The effects of progesterone pretreatment on in vivo 3H-estrogen uptake by five anterior pituitary cell types was analyzed by means of a quantitative autoradiographic-immunocytochemical technique. Male and female rats castrated for 14 days show nuclear concentration of label in all five cell types one h after injection of 3H-estradiol, whether progesterone treated or not. The order of labeling intensity is gonadotropes greater than or equal to lactotropes = somatotropes greater than thyrotropes = corticotropes. Progesterone treatment induces a dramatic sexual dimorphism in estrogen uptake; it significantly increases 3H-estrogen uptake in all female cell types. In males, progesterone decreases uptake in gonadotropes while not altering uptake in other cell types.