James A. Carr

Organization of tyrosine hydroxylase-immunoreactive neurons in the di- and mesencephalon of the American bullfrog (Rana catesbeiana) during metamorphosis

SummaryWe examined the immunocytochemical distribution of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, in the di-and mesencephalon of developing bullfrog tadpoles. Special attention was given to catecholaminergic innervation of the median eminence and pituitary. In premetamorphic tadpoles, tyrosine hydroxylase-immunoreactive neurons were visualized in the suprachiasmatic and infundibular hypothalamus, the ventral thalamus, and midbrain tegmentum by Taylor-Kollros stage V. The number of labeled neurons in all these areas increased as metamorphosis progressed. By mid-prometamorphosis, labeled neurons appeared in the preoptic recess organ as well as in the posterior thalamic nucleus. The majority of cells in the preoptic recess organ, as well as occasional neurons in the suprachiasmatic nucleus, exhibited labeled processes which projected through the ependymal lining of the preoptic recess to contact cerebrospinal fluid. The modified CSF-contacting neurons of the nucleus of the periventricular organ were devoid of specific staining. By late prometamorphosis, labeled fibers from the suprachiasmatic nucleus were observed projecting caudally to enter the hypothalamo-hypophysial-tract en route to innervating the median eminence and pituitary. Labeled fibers arising from the dorsal infundibular nucleus projected ventrolaterally to contribute to catecholaminergic innervation of the median eminence and pituitary. Immunoperoxidase staining of tyrosine hydroxylase-immunoreactive fibers and terminal arborizations in the median eminence were restricted to non-ependymal layers, while labeled fibers in the pituitary were observed in the pars intermedia and pars nervosa. Staining of tyrosine hydroxylase-immunoreactive fibers in the median eminence and pituitary was sparse or absent in premetamorphic tadpoles, but became increasingly more intense as metamorphosis progressed.

Benzodiazepine suppression of corticotropin-releasing factor (CRF)-induced beta-endorphin release from rat neurointermediate pituitary

Dopamine and gamma-aminobutyric acid (GABA) inhibit POMC peptide release from the pituitary intermediate lobe, via interaction with D2 or GABA-A/benzodiazepine receptors. Here, we examined the effects of an antianxiety triazolobenzodiazepine, adinazolam, on corticotropin-releasing factor (CRF)-stimulated POMC peptide secretion from the rat neurointermediate pituitary. Neurointermediate lobes (NILS) were incubated with CRF (10(-7) M), then adinazolam (10(-8) or (10(-9) M) was added, with CRF remaining in the medium. Aliquots were removed at 15-min intervals and frozen for radioimmunoassay of beta-endorphin. Adinazolam alone did not significantly affect secretion as compared to controls or CRF alone. Adinazolam incubated with CRF led to significant inhibition of beta-endorphin secretion, as compared to CRF alone. In addition, adinazolam was as effective as dopamine or the CRF antagonist, alpha-helical CRF, in preventing CRF-induced beta-endorphin release. Adinazolam appears to act directly on the pituitary to suppress hormone release induced by a stress-related hypothalamic peptide.

Interaction of corticotropin-releasing factor (CRF) and alpha-helical CRF on rat neurointermediate lobes: In vitro studies

Neurointermediate lobes (NILS) of the pituitary glands of adult male Sprague-Dawley rats were incubated in media in the presence of corticotropin-releasing factor (CRF), a stimulator of proopiomelanocortin (POMC) peptide release. Alpha-helical CRF, a peptide known to inhibit CRF induced POMC peptide release from the anterior pituitary, was incubated with NILS for a period of 90 min, to study its potential ability to modulate peptide release from the intermediate lobe. The alpha-helical peptide reduced beta-endorphin release from NILS, as measured by radioimmunoassay (RIA), when added for the entire incubation, or when added 30 min after start of the incubation period, with CRF present. Alpha-helical CRF alone reduced beta-endorphin release, as compared to control or CRF-treated lobes. Ultrastructural examination of intermediate lobes fixed at the end of incubations revealed a reduction in the numbers of Golgi-associated dense granules, an indicator of new peptide synthesis, in intermediate lobe tissue treated with alpha-helical CRF alone, both peptides together, or with CRF followed by alpha-helical peptide. The in vitro studies demonstrate the effectiveness of the antagonist peptide on intermediate lobe peptide secretion, thereby extending its effects to both POMC-secreting areas of the pituitary gland.

Stress-induced peptide release from rat intermediate pituitary : an ultrastructural analysis

SummaryWe tested the hypothesis that acute restraint stress results in ultrastructural evidence for enhanced release of alpha-melanocyte-stimulating hormone (α-MSH) and β-endorphin from the intermediate lobe (IL) of the rat pituitary. Measurements of plasma α-MSH-and β-endorphin-immunoreactivity (ir) were used to confirm ultrastructural findings. Plasma α-MSH-ir was elevated after 20 and 30 min of restraint while plasma β-endorphin-ir peaked 10 min after the onset of restraint. Ultrastructural analysis revealed a decrease in the content of secretory granules within IL cells of stressed rats. Analysis of Golgi-related immature secretory granules in IL cells indicated that new peptide synthesis was not enhanced after 30 min of restraint. These results confirm previous studies showing and elevation of plasma β-endorphin and α-MSH-ir during acute restraint. Furthermore, these results indicate that quantitative analysis at the ultrastructural level can be used to assess peptide release from IL secretory cells during stress.

Degeneration and regeneration of nerve terminals in the rat pituitary pars intermedia after 6-hydroxydopamine treatment.

6-hydroxydopamine (6-OHDA), a catecholamine neurotoxin, has been shown previously to induce degenerative changes in nerve terminals innervating proopiomelanocortin (POMC) cells of the pituitary intermediate lobe. The present study provides evidence for regeneration of nerve fibers to the pituitary within 4 weeks of drug treatment. Adult male Sprague-Dawley rats were treated with 6-hydroxydopamine (150 mg/kg) on Days 1 and 3 and then perfused for light or electron microscopy (EM) of the pituitary intermediate lobes at 1, 2, 3, or 4 weeks after the first injection. At 1, 2, and 3 weeks after drug treatment, the number of normal-appearing nerve terminals was significantly lower than that in controls, while at 4 weeks after 6-OHDA, the number of normal nerve terminals did not differ significantly from that of control rats. Tyrosine hydroxylase-immunoreactive fibers appeared more intensely stained at 1 and 2 weeks after 6-OHDA treatment, suggesting continued and perhaps enhanced synthesis of the enzyme by hypothalamic cell bodies to the remaining terminals. Serotonin immunoreactivity after 5-hydroxytryptophan pretreatment was not detectable at 1 week after drug treatment, but was clearly visible in fibers innervating the intermediate lobe at 3 weeks, indicating a return of uptake ability and conversion of the precursor to serotonin by the regenerating terminals. Chemical denervation of the intermediate lobe followed by regeneration of nerve fibers will be useful for examination of regulatory mechanisms for the POMC secretory cells.

The Hypothalamus–Pituitary–Thyroid (HPT) Axis of Non-Mammalian Vertebrates

The thyroid axis of non-mammalian vertebrates shows many parallels to mammals with respect to thyroid structure and to the synthesis and metabolism of thyroid hormones as well as to their actions. T 3 appears to be the more active form of thyroid hormones in all vertebrates. Thyroid hormones appear to interact with a variety of other endocrine-regulated systems where they play permissive or possibly synergistic roles, especially in development, growth, and reproduction. The system of deiodinases seems to be responsible for allowing surges in T 3 to occur at critical times. Direct actions of thyroid hormones on development occur in all vertebrates, but their participation in lipid metabolism and thermogenesis is correlated with homeothermy. Some notable phylogenetic differences stand out with respect to hypothalamic regulation in teleost fishes and amphibians. TRH appears to exert a negative control in many fishes over TSH release, and in amphibians evidence is mounting to identify the CRH peptide as the endogenous TSH-releasing hormone. Considerable work remains to be done on thyroid systems in non-mammalian vertebrates, especially in elasmobranchs, a greater variety of teleosts, and reptiles. Once this has been accomplished, a clearer pattern of vertebrate thyroid function should appear.

Comparative Aspects of Vertebrate Adrenals

Non-mammalian vertebrates do not exhibit the anatomical cortex-medullary relationship like mammals for the homologous adrenocortical cells and chromaffin cells. However, there is a general trend for a closer anatomical relationship between these tissues from being entirely separate in ancient fishes with direct associations with the kidneys to the evolution of discrete, separate glands in reptiles and birds. The synthesis of corticosteroids is the same as in mammals with the exception of elasmobranchs that produce a unique corticosteroid, 1α-hydroxycorticosterone. Teleosts rely on cortisol as the principal mineralocorticoid and glucocorticoid. Amphibians, reptiles, and birds secrete corticosterone and aldosterone as their glucocorticoid and mineralocorticoid, respectively. Some larval and aquatic amphibians, however, rely on cortisol. The role in non-mammals for cortisol and corticosterone in gluconeogenesis, the stress response, and aging are similar to those of mammals. Aldosterone secretion is regulated by a renin-angiotensin system in non-mammalian tetrapods. Blood pressure regulation involves the renin-angiotensin system in non-mammals similar to that of mammals and natriuretic peptides antagonize Ang-II actions. Chromaffin cells participate in emergency reactions and stress responses as described for mammals. In non-mammals, chromaffin cells generally secrete more norepinephrine than epinephrine. Clearly, cells of non-mammals have the capacity to convert norepinephrine to epinephrine, indicating that this biosynthetic step is phylogenetically ancient.

The Endocrinology of Mammalian Reproduction

Abstract The reproductive system includes the HPG axis and sex accessory structures. Primary control resides in pulsatile production of GnRH, which controls pituitary production of FSH and LH, which in turn cause gamete formation, gonadal steroid secretion, and regulation of sexual characters and reproductive behaviors. FSH is involved primarily with gamete production whereas LH is responsible for initiating steroid secretion and release of mature gametes (ovulation and spermiation). FSH and testosterone work cooperatively to produce mature sperm. Sex steroids stimulate other primary reproductive structures and secondary sex characters as well as mating behavior In mammals, the male sex is determined by the sry gene, which is responsible for production of AMH and androgens by the testis. AMH causes regression of some potential female structures and androgen production allows development of male sex accessory structures and reprograms hypothalamic reproductive centers in the brain to the male secretory pattern. The default sex in mammals is female although a number of transcription factors and nuclear receptor proteins are required for ovary development and estrogens are essential for completely normal female development. Mammalian reproductive cycles show great variations. Monotremes are egg-laying mammals that nourish their hatchlings with milk from their mammary glands. Marsupials allow their young to develop in a pouch after a short gestation period involving a nonendocrine placenta, relying on the mammary glands to support continued development of an exteriorized fetus. Eutherian mammals have a prolonged period of intrauterine fetal development supported by an endocrine placenta. Mammary glands are used for nutritional support after birth in all mammals. Most female mammals exhibit an ovarian-based estrous cycle characterized by a period of enhanced receptivity of the female to the male called estrus. In some species a special phase of the uterine cycle occurs, the menses, which involves sloughing and discharge of a portion of the endometrium and trapped blood. Because of this special uterine cycle, their reproductive cycles are typically called menstrual cycles and most may (e.g., rhesus monkey) or may not (human) exhibit a distinct period of estrus. The proliferative phase of the uterine cycle corresponds closely to the follicular phase of the ovarian cycle, and the secretory phase of the uterine cycle that follows ovulation coincides exactly with the luteral phase of the ovarian cycle. The uterine secretory phase is followed by a quiescent phase called diestrus in most mammals or by the menses in species having a uterine menstrual cycle. The menses corresponds to the first few days of the next follicular phase in the ovary. In eutherians, the cycle can be separated into a follicular phase during which one or more ova develop in follicles and a luteal phase that prepares the uterus for implantation of the blastocyst. The luteal phase is named for one or more corpora lutea that develop from ruptured follicles and possibly some atretic follicles that continue to secrete estrogens and progestogens. During pregnancy, the eutherian placenta in cooperation with the fetal adrenal functions as an endocrine gland to maintain pregnancy, initiate birth, and prepare the mammary glands for postnatal functions. The corpus luteum performs various roles in pregnancy depending on the species. Postnatal functions of the mammary gland are controlled by PRL and OXY from the pituitary gland. Gonadal secretory activities involve two special cell types responsive to FSH and LH, respectively. Ovarian granulosa cells and testicular Leydig cells are responsive primarily to LH and synthesize androgens. Ovarian thecal cells and testicular Sertoli cells as well as Leydig cells respond to FSH with conversion of androgens into estrogens . FSH also stimulates Sertoli cells . Gonadal cells make many other local regulators in response to GTHs or independently, and these regulators may have autocrine and/or paracrine actions that control local events in the gonads.

Chemical Regulation of Feeding, Digestion and Metabolism

Feeding is regulated by bioregulators secreted from adipose tissue and the GI tract as well as by local neuropeptides and endocannabinoids secreted by neurons in appetite-regulating areas of the NTS and hypothalamus. Peripheral factors affecting feeding are largely inhibitory (anorexic). Only one peptide produced in the periphery, ghrelin, stimulates feeding (an orexic factor). CNS factors stimulating appetite include orexin A and B, NPY, AgRP, and endocannabinoids. Anorexic factors produced in the CNS include CCK8, a-MSH,CART, CRH, Ucn-2, PYY, and PP. In addition, numerous adipokine proteins produced by adipose tissue have been implicated in appetite regulation. The existence of three major GI hormones (gastrin, secretin, and CCK) postulated to be present in mammals has been established. Gastrin is produced by the G cell of the gastric mucosa in response to food. Parietal cells in the stomach secrete HCl in response to gastrin or direct neural (vagal) stimulation. Histamine plays a role as an intermediate in the action of gastrin on the parietal cell. Parasympathetic stimulation stimulates acid secretion and also evokes secretion of pepsinogen from the chief cell in the gastric mucosa. SST produced in gastric D cells blocks gastric secretion. Secretin is produced by the S cell of the duodenal mucosa in response to the presence of acid. The action of secretin is to cause release of basic juices from the exocrine pancreas. The presence of peptides, amino acids, or fats in the chyme causes release of CCK from the I cell of the intestinal mucosa, which in turn stimulates secretion of pancreatic enzymes and release of bile from the gallbladder. Four additional peptides have been isolated from the mucosa of the small intestine and established as GI hormones.

The Hypothalamus–Pituitary System in Non-Mammalian Vertebrates

The progressive evolution of hypothalamic centers in vertebrates is evident from examination of different vertebrates. The pattern of regulation has branched within the fishes, with the most recent fishes (teleosts) exhibiting predominantly neuroglandular control of tropic hormone release. Primitive fishes, including the chondrichthyeans, and tetrapods have specialized in neurovascular control with the development of a distinct median eminence and a hypophysial portal blood system. The distinct regionalization of tropic cell types in the fish adenohypophysis is less marked in tetrapods. The adenohypophysis of fishes is usually separable into a rostral pars distalis, a proximal pars distalis, and a pars intermedia. The pars tuberalis of tetrapods is absent in fishes but a unique ependymal structure is evident, the saccus vasculosus. Lungfishes lack both a pars tuberalis and the saccus vasculosus.