The cells of origin of ACTH in man.

The evidence from ACTH-producing tumors, from the morphologic effects of excess glucorticoids on the hypophysis, and from immunocytochemistry all points to the predominant "basophils" of the human anterior lobe, the intensely PAS-possitive beta(R) cells, as the source of ACTH. Similar cells often also occur, sometimes in large numbers, in the pars nervosa. beta(R) cells can be immunostained with antibodies against alphaMSH and betaMSH, but it is likely that they actually contain the precursor molecule(s) of betaMSH, betaLPH (and may be gammaLPH). Once this is satisfactorily documented, the functional term corticolipotrops should replace the provisional name beta(R) cells. Electron microscopically, these cells contain randomly arranged granules with a maximum diameter of 300-500 nm, and occasionally small amounts of filamentous material, which increases dramatically in response to excess glucocorticoids. In the beta (R) cells of adults, a single set of granules stains for ACTH and with antibodies to betaMSH. In fetal life, ACTH cells appear early and material reactive with anti-betaMSH accumulates in them (and possibly also in cells not containing ACTH) only later. The posterior lobe beta(R) cells can be immunostained with both anti-betaMSH and antibodies against the COOH-terminal portion of ACTH, but the presence of bioactive ACTH in them remains to be shown.

Immunocytochemical localization of a folicle stimulating hormone-like molecule in the testis.

A follicle-stimulating hormone (FSH)-like molecule was localized in normal adult rat testes as well as testosterone-treated hypophysectomized rat tests with an unlabeled antibody (anti-FSH), peroxidase-antiperoxidase complex technique. Anti-FSH bound specifically to ultrathin sections of acrosomes of spermatids and intranuclear bodies of early spermatids. Quantitation of staining intensity demonstrated that FSH, used as an absorbing antigen, would significantly reduce this binding. There was less anti-FSH binding to the acrosomes of spermatozoa in the body and tail of the epididymis as compared to the less mature germ cells located in the testis and head of the epididymis. The acrosomal and nuclear staining of spermatids taken from hypophysectomized animals was similar to staining observed in sham injected animals. Taken together, these results suggest that there is a molecule within the acrosome that is immunologically similar to FSH. Most importantly, these results emphasize the importance of conducting physiologic experiments in conjunction with immunocytochemical studies.

Improvements of glycol methacrylate. I. Its use as an embedding medium for electron microscopic studies.

The technique for using the water-soluble embedding medium glycol methacrylate has been improved for ultrastructural studies by the simplification of the method of formation of prepolymers used in embedding the tissue, by the addition of a cross-linking agent so that sections are stable in the electron beam, and by improving the softness of the blocks by the addition of a plasticizing agent. The preservation of tissue morphology has been improved by complete dehydration in glycol methacrylate monomer prior to infiltration with the prepolymer. Preservations of tissue morphology is further enhanced by complete dehydration in ethanols and embedding in the improved glycol methacrylate medium.

Ultrastructural immunocytochemical characterization of the thyrotroph in rat and human pituitaries.

The beta chain of thyroid stimulating hormone (TSH) was localized in normal rat and human pituitaries with the unlabeled antibody--peroxidase-antiperoxidase complex technique and antisera to rat or bovine TSH beta. Araldite embedded tissues, immunoreactivity of TSH was best preserved after fixation with 1% glutaraldehyde or picric acid formaldehyde. In the TSH cells, the immunocytochemical stain was located on granules. However, there was a variation in staining of individual granules, and among the population of granules. Rat TSH cells were ovoid or angular to stellate, and contained granules ranging in size from 60-175 nm. The human TSH cell was polyhedral, and contained scattered granules of 150-300 nm diameter. In both species, granule distribution was either in 1-2 rows at the periphery, or scattered throughout the cytoplasm with some concentration in cytoplasmic processes. TSH cells in female rats in estrous contained more granules than those of other stages. TSH cells were distinguished from adrenocorticotropic hormone cells and luteinizing hormone cells on the basis of granule size and distribution, and cell shape.

An immunocytochemical study of TSH beta storage in rat thyroidectomy cells with and without D or L thyroxine treatment.

Binding sites to the beta chain of thyroid stimulating hormone (TSH) were localized in pituitaries of thyroidectomized rats. Immunocytochemical staining was observed in hypertrophied TSH cells ("thyroidectomy cells") and primarily located in dilated rough endoplasmic reticulum. Staining was also found on the few secretory granules and on some of the intracisternal granules. Some of the thyroidectomy cells stained intensely, while others exhibited very little staining. When thyroidectomized rats were treated with thyroxine 4 days before death, the TSH cells contained more secretory granules, and the intracisternal granules were larger and more numerous. L-thyroxine was 10 times as potent as D-thyroxine in promoting the build-up of granules. Both types of granules stained intensely.

Immunocytochemistry of the pituitary glycoprotein hormones.

The storage sites of the pituitary glycoprotein hormones were identified with the use of electron microscopic immunocytochemical techniques and antisera to the beta (beta) chains of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH). The TSH cells in normal rats is ovoid or angular and contains small granules 60-160 nm in diameter. In TSH cells hypertrophied 45 days after thyroidectomy, staining is in globular patches in granules or diffusely distributed in the expanded profiles of dilated rough endoplasmic reticulum. The gonadotrophs (FSH and LH cells) exhibited three different morphologies. Type I cells are ovoid with a population of large granules and a population of small granules. Staining for FSHbeta or LHbeta was intense and specific only in the large granules (diameter of 400 nm or greater). Type II cells are angular or stellate and contain numerous secretory granules averaging 200-220 nm in diameter. They predominate during stages in the estrous cycle when FSH or LH secretion is high. Type III cells look like adrenocorticotropin (ACTH) cells in that they are stellate with peripherally arranged granules. They generally stain only with anti-FSHbeta and their staining can not be abolished by the addition of 100 ng ACTH. In preliminary quantitative studies of cycling females, we found that on serial sections FSH cells and LH cells show similar shifts to a more angular population of cells during stages of active secretion. However, the shifts are not in phase with one another. Furthermore, there are at least 1.5 times more FSH cells than LH cells at all stages of the cycle. Our collection of serial cells shows that some cells (usually type I or II) stain for both gonadotropic hormones, whereas others (usually type II or III) contain only one.

Electron microscopic-immunocytochemical studies of rat pituitary gonadotrophs: a sex difference in morphology and cytochemistry of LH cells.

Morphological criteria have been used by Kurosumi and Oota to distinguish follicle-stimulating hormone (FSH) from luteinizing hormone (LH) cells in the rat pituitary. In this study the techniques of ultrastructural immunocytochemistry were used to determine if these cell types could be distinguished on the basis of their LH content. In female rats in diestrus a 1:125,000 dilution of anti-bLH-beta stained Kurosumi-Oota "LH cells," "FSH cells," and cells with some morphological characteristics of both types. Absorption with 10 ng of LH abolished the staining in all cell types. In pituitaries taken from rats in proestrus and estrus, the stained cells were mostly of the "LH" cell type. Such cells were generally poorly granulated in some of the estrous females. In males, predominantly one gonadotrophic cell type, the classical Kurosumi-Oota "FSH cell," reacted with anti-bLH-beta. Ten nanograms of LH was effective in abolishing the staining in the largest granules (larger than 400 nm). Both LH and TSH were equally effective in the neutralization of the staining of small granules. Our results suggest that female rats have two distinctive LH cell types and males have one. We also confirm other immunocytochemical reports which show that the classical morphological definition of a "FSH cell" (5,6) is not completely accurate since some of these cells contain LH. The small granules appear to contain an immunoreactive site common to both TSH and LH which is only weakly reactive with the anti-bLH-beta.

Bioactive and immunoactive ACTH in the rat pituitary: influence of stress and adrenalectomy.

Tissue levels of bioactive and immunoactive ACTH were measured in both the anterior and neuro-intermediate lobes of the rat pituitary. Similar concentrations of bioactive (65 ng/mg) and immunoactive (83 ng/mg) ACTH were found in the anterior lobes control rats. A 2-min ether stress had no effect on either bioactive or immunoactive ACTH levels in the anterior lobe. Twenty-four h after adrenalectomy the anterior lobe content of both bioactive and immunoactive ACTH decreased only to return to supranormal levels 21 days after the operation. A 30-min neurogenic stress had no effect on anterior lobe bioactive ACTH content but reduced the immunoactive ACTH level to 50 ng/mg. Synthetic alphah-17-39 ACTH was used in our radio-immunoassay in order to measure the C-terminal ACTH activity of the neuro-intermediate lobe. The concentration of such C-terminal activity in control rats (890 ng alphah-17-39 ACTH/mg) considerably exceeded the amount of bioactive ACTH (15 ng/mg). This is presumably due primarily to the presence of the so-called corticotropin-like intermediate lobe-peptide (CLIP). The amounts of bioactive or C-terminal immunoactive ACTH in the neuro-intermediate lobe were not affected by ether stress nor short term (24-h) or long term (21-day) adrenalectomy. Neuro-intermediate lobe bioactive ACTH decreased (to 8 ng/mg) only with the introduction of a 30-min neurogenic stress. Neurogenic stress had no effect on the concentration of CLIP, but when the stress was imposed 24 h after adrenalectomy, a significant reduction was observed. The data support the presence of bioactive ACTH in the intermediate lobe of the rat pituitary and suggest that such ACTH is preferentially released by neurogenic stress and not appreciably regulated by circulating levels of glucocorticoids. Until the biological function and/or target organ of CLIP is identified, the significance of the changes in tissue levels of C-terminal immunoactive ACTH will remain unknown.

The effect of stress on the cytology and immunocytochemistry of pars intermedia cells in the rat pituitary.

The purpose of this study was to determine if there were ultrastructural and immunocytochemical changes in intermediate lobe cells following stress. We found marked cytological changes in intermediate lobe cells after 30 min of stress with a buzzer and stroboscopic light (neurogenic). Following such treatment, the cytoplasm was filled with pale graules or empty vesicles. There was cytological evidence of increased secretory and synthetic activity. Innunocytochemical staining (for 17-39-ATCH) revelaed that there were fewer stained granules per cell in such rats although the individual granules stained as strongly as those in control rats. No cytological changes were observed following ether stress. These results correlated well with our bioassay and radioimmunoassay data which showed that only neurogenic stress resulted in depletion of ACTH from neuro-intermediate lobes (1). In agreement with the work of others, we found no obvious cytological changes in intermediate lobes from adrenalectomized or cortisol treated rats wheras anterior lobe ACTH cells hypertrophied following adrenalectomy and involuted following cortisol treatment. Anterior lobe ACTH cells from intact rats showed no degranulation following 30 min of buzzer stress or after 2 min of ether stress.