Douglas S. Gross

The mammalian hypophysial pars tuberalis: A comparative immunocytochemical study

Despite its occurrence in most vertebrate species, the function of the hypophysial pars tuberalis (PT) remains obscure. Recent immunocytochemical studies have demonstrated the presence of hormone-containing cells in the few species studied. In the present study the secretory cell composition of the PT was characterized in a variety of mammals using immunocytochemistry. Species studies were the mouse, rat, guinea pig, rabbit, sheep, rhesus monkey, baboon, and human. Antisera were chosen on the basis of their ability to identify a distinct cell population in the pars distalis. A total of 21 antisera were used to identify GH, PRL, ACTH, beta-endorphin, LH, FSH, and TSH. Gonadotropes were identified in the PT of all eight species and were the predominant immunoreactive cell type in the human, baboon, rhesus monkey, sheep, guinea pig, rabbit, and mouse. Thyrotropes were detected in all species except the sheep. They were the predominant cell type in the rat but were less common than gonadotropes in other species. No other secretory cell types were found, with the exception of occasional somatotropes and mammotropes in some human specimens, and small clusters of opiocorticotropes in the guinea pig. Thus the general pattern in the mammalian PT is the presence of gonadotropes and thyrotropes and the absence of other pituitary cell types. In the human, baboon, and rat, all PT parenchymal cells can be identified immunocytochemically. However, in the rhesus monkey, sheep, guinea pig, rabbit, and mouse, the majority of PT cells do not react with any antisera, and thus their function is unknown. Follicles are common in the PT of most mammalian species, however, the luminal contents do not react with antisera to adenohypophysial hormones.

Effects of somatostin on food intake in rats

Abstract We examined the possibility that somatostatin, a tetradecapeptide distributed in the gut and the central nervous system, may influence food intake and behavior in rats. Although intravenously infused somatostatin did not alter food intake in 8 hour fasted rats, intracerebroventricularly infused somatostatin resulted in a biphasic response, first increasing then decreasing food intake. We also observed that the effects of somatostatin vary depending upon whether animals are fed or fasted. In fed rats, food intake was decreased, while in fasted rats food intake was increased. These results suggests that somatostatin can act in the central nervous system to stimulate appetite; but that other factors, possibly related to gut motility or clearance, may inhibit further feeding once the stomach is full.

Quantitative Immunocytochemistry of Hypothalamic and Pituitary Hormones: Validation of an Automated, Computerized Image Analysis System'

A limiting factor in the use of immunocytochemistry in experimental endocrine studies has been the lack of a suitable procedure for quantification of immunoreactive hormones. The objective of the present study was the development of an automated, computerized image analysis system adapted to the quantitative analysis of light microscopic immunocytochemical reaction product. Reaction conditions that result in optimum, standardized, and quantitatively linear development of reaction deposit are described for H2O2 and diaminobenzidine concentrations, antiserum dilutions, and substrate incubation times. In addition, evaluation techniques, including the use of a standard control section to monitor variance and incorporate it into the statistical analysis of the results are documented. For each of the reaction variables, the immunostaining was linear over the range of specific staining. When the optimum conditions were exceeded, marked over-estimations of hormone levels occurred due to the detection of nonspecific background features reaching the detection threshold. Application of this quantitative immunocytochemical (QICC) method to the analysis of variations in hypothalamic and pituitary hormone levels was validated by comparing values obtained with QICC to those with radioimmunoassay (RIA). The relative changes in both hypothalamic gonadotropin-releasing hormone and pituitary luteinizing hormone induced by manipulation of gonadal steroid levels, as measured by RIA and QICC, were highly correlated. Two-way analysis of variance revealed that the two techniques were not significantly different in their detection of changes in either hormone. Thus, under optimally defined conditions, quantitative immunocytochemistry using computerized image analysis has been validated for the accurate measurement of pituitary and brain hormones in precise regions.

Neuronal responsiveness to gonadotropin-releasing hormone and its correlation with sexual receptivity in the rat

In the present study, an attempt was made to correlate the neuronal responsiveness of individual preoptic-septal (POA/S) units to iontophoretically applied GnRH with the onset of sexual receptivity. In both behavioral and electrophysiological studies, ovariectomized, estrogen-primed rats were used. In behaviorally tested rats, lordosis quotients (LQ) were determined at varying times following progesterone (P) injection. For electrophysiological studies, P was given 1 hr after the start of recording. GnRH was iontophoretically applied for 30 sec at 16 nA on spontaneously discharging cells. A unit was deemed excited or inhibited if a repeatable 30% change in discharge rate was observed. From 2-10 hours as the LQ increased from 17 to 90 the total number of GnRH sensitive cells did also. The majority of responsive cells were excited by the peptide. As receptivity displayed a sharp increase from 2 to 6 hours the mean responsiveness of cells excited by GnRH was significantly elevated over inhibitory responses. These findings confirm the E/P biasing effect on POA/S unit responses to GnRH. Moreover, they suggest that a dynamic relationship exists between GnRH responses at the cellular level and sexual behavior throughout the course of steroid-induced receptivity.

Gonadotropin-releasing hormone within the organum vasculosum lamina terminalis in the ovariectomized, estrogen/progesterone-treated rat: A quantitative immunocytochemical study using image analysis

No specific function has been ascribed to the high gonadotropin-releasing hormone (GnRH) content of the organum vasculosum lamina terminalis (OVLT). The objective of this study was to determine whether levels of GnRH within the OVLT are altered during cyclic gonadotropin secretion. GnRH levels were determined at various times during an estrogen/progesterone (E/P)-induced LH surge. Groups of E/P and sesame oil-treated animals were decapitated at 12.00 h, 14.00 h, 16.00 h, 18.00 h, and 22.00 h following the P or oil treatment. Morphological localization, as well as quantitation of immunoreactive GnRH within discrete regions of the brain was achieved by combining unlabeled antibody immunocytochemistry with computerized image analysis. Analysis of GnRH levels in the OVLT revealed that at any of the 5 times examined, there was: (1) no significant difference among controls, (2) no significant difference among E/P-treated animals, and (3) no significant difference between E/P-treated versus control animals. In contrast, ME GnRH levels in E/P-treated rats showed the expected decrease prior to the onset of the LH surge. These findings suggest that the level of GnRH detected in axon terminals within the OVLT cannot be related directly to the serum LH status of ovariectomized, E/P-treated rats. It is therefore possible that GnRH within the OVLT might function in a neuromodulatory role, rather than as a direct regulator of cyclic gonadotropin secretion.

Distribution of immunoreactive somatostatin in the primate hypothalamus

SummaryImmunocytochemical methods were used to compare the localization of somatostatin (SRIF) in the human and rhesus monkey hypothalamus. The distribution of SRIF-containing cell bodies and fibers is similar in the two species. Perikarya are located predominantly in the periventricular region and to a lesser extent in the ventromedial nucleus. Fibers occur in dense clusters within the periventricular region, ventromedial nucleus, arcuate nucleus, median eminence, and pericommissural area of both species. Analysis of serial sections suggests that fibers originate from cells in the periventricular region, extend ventrally through the ventromedial and arcuate nuclei to terminate around the portal vessels of the infundibular stalk, and thereby participate in the regulation of anterior pituitary function. Somatostatinergic fibers are also found surrounding non-immunoreactive perikarya in the ventromedial nucleus and periventricular region of both primates. This arrangement may support somatostatins postulated role as a neurotransmitter or neuromodulator. The strong similarity between the localization of hypothalamic SRIF in the human and rhesus monkey supports the use of the rhesus monkey as a model for the study of somatostatin as a neuroendocrine regulator in the human.

Effect of food-deprivation on rat pancreatic islet cells.

Somatostatin, insulin and glucagon concentrations in rat pancreas were measured following various intervals of food-deprivation. Tissue concentrations, as measured by radioimmunoassay, were correlated with A-, B-, and D-cell number and size using a scanning integrating image analyzer (Quantimet 720). Alterations in total islet hormone content were not correlated to changes in size or distribution of cells. This implies that changes in tissue content reflect changes in turnover of peptides rather than changes in cell size or number.