James N. Fryer

Hormonal regulation of the ligand for c-kit in the rat ovary and its effects on spontaneous oocyte meiotic maturation.

Kit ligand (KL, c‐kit ligand) mRNA was detected in the ovaries of 26‐day‐old prepubertal rats using in situ hybridization. In antral follicles there was a gradient in the intensity of the hybridization signal across the layers of granulosa cells, with greatest intensity observed in the cumulus granulosa cells enclosing the oocyte, and less signal occurring in the granulosa cells furthest from the oocyte. In age‐matched rats 40 hr after injection of pregnant mare serum gonadotropin (PMSG), the pattern of distribution of KL resembled that in the untreated ovaries, although the intensity of the hybridization signal was greater in the PMSG‐primed ovaries. This morphological observation was confirmed using Northern blot analysis, which indicated that granulosa cells of PMSG‐treated rats had 3.5‐fold greater abundance of KL mRNA compared to untreated rats. The abundance of KL mRNA further increased to 7‐fold over control levels at 6 hr after PMSG‐primed rats were treated with human chorionic gonadotropin (hCG). By contrast, treatment of rats with diethylstilbestrol to stimulate follicular growth did not cause any change in the abundance of KL transcripts. To investigate a potential role for KL in oocyte meiotic maturation, fully grown oocytes were cultured for 24 hr with or without KL (50 or 500 ng/ml). The presence of KL resulted in a significant, albeit transient, delay in the progression of spontaneous meiotic maturation, using the indices of germinal vesicle breakdown and polar body formation. The inhibitory effects of KL were specifically blocked by ACK2, an antibody to the extracellular domain of the c‐kit receptor. These results indicate that KL is produced in rat granulosa cells at particularly high levels in the cells closest to the oocyte and that this production may be regulated directly by gonadotropic hormones. Furthermore, KL inhibits the progression of meiosis in cultured oocytes, which suggests a possible role in the maintenance of meiotic arrest that occurs throughout oocyte growth.

Gill Morphology and Acid-Base Regulation in Freshwater Fishes

This review examines the recent advances in our understanding of the mechanisms of ion transport and acid-base regulation in the freshwater fish gill. The application of a combination of morphological, immunocytochemical and biochemical techniques has yielded considerable insight into the field. An important mechanism for regulation of Cl- uptake/base excretion is by morphological modification of the gill epithelium. During acidosis, the chloride cell associated Cl-/HCO3- exchanger is effectively removed from the apical epithelium because of a covering by adjacent pavement cells; this mechanism reduces base excretion and contributes to the compensation of the acidosis. In addition, acidosis induces changes in both the surface structure and ultrastructure of pavement cells. Evidence is accumulating to support the hypothesis that Na+ uptake/H+ excretion is accomplished by the pavement cell. Further, specific localization of a V-type H+-ATPase on the pavement cell epithelium and an increased expression during acidosis provides support for the model originally proposed, that this exchange is accomplished by an electrochemically coupled H+-ATPase/Na+ channel mechanism.

ACTH-releasing activity of urotensin I and ovine CRF: Interactions with arginine vasotocin, isotocin and arginine vasopressin

The release of ACTH from superfused dispersed goldfish anterior pituitary cells was examined to determine if the neurohypophyseal peptides arginine vasotocin (AVT), isotocin (IST) or arginine vasopressin (AVP) potentiate the ACTH-releasing activities of the structurally homologous peptides urotensin I (UI) or ovine corticotropin-releasing factor (CRF). The ACTH-releasing activities of the neurohypophyseal peptides and UI or CRF were additive. AVT, IST or AVP failed to potentiate the ACTH-releasing activity of UI or CRF. These results suggest that in teleost fishes neurohypophyseal peptides have intrinsic ACTH-releasing activity but, unlike mammals, do not potentiate the release of ACTH evoked by CRF, or by the piscian CRF-like peptide, UI.

Cortisol inhibits the ACTH-releasing activity of urotensin I, CRF and sauvagine observed with superfused goldfish pituitary cells

The structurally homologous peptides urotensin I, ovine CRF and sauvagine stimulate the release of immunoreactive ACTH from a superfused dispersed goldfish anterior pituitary cell column. The addition of cortisol to the superfusion buffer resulted, following a latent period, in a decrease in basal release of ACTH from the pituitary cell column and a diminution in the ACTH-releasing activities of urotensin I, CRF and sauvagine. The removal of cortisol from the superfusion buffer resulted in a slow recovery of basal ACTH release and a recovery of the ACTH-releasing activities of urotensin I, CRF and sauvagine. These results are supportive of the view that urotensin I, or a urotensin I-like peptide, serves as a physiological regulator of ACTH release in teleost fishes.

Distributions and colocalization of neuropeptide Y and somatostatin in the goldfish brain

The distributions of single- and double-labelled neuropeptide Y- (NPY-) and somatostatin-immunoreactive (SOM-IR) perikarya and processes were determined in the goldfish brain using immunoperoxidase and immunofluorescence techniques, respectively. In double-labelled material, it was evident that although these two peptides showed markedly similar distributions, they were colocalized in very few instances. A high degree of colocalization of NPY and SOM was noted in the neurons of the ventrolateral telencephalon (VI), the entopenduncular nucleus (NE) and, to a lesser extent, in the dorsocentral nucleus of the telencephalon (Dc). In Vl and NE, neurons showing NPY-IR displayed SOM-IR and vice versa. The only other instance of colocalization was that noted in the brainstem, where SOM and NPY were colocalized in the large cell bodies of the medial column of the vagal motor complex. Single-labelled SOM- and NPY-IR neurons shared a very similar distribution in various nuclei in the diencephalon and in the optic tectum. Colocalization was also noted within fibers throughout many nuclei of the telencephalon and within fibers innervating the swim bladder, one of the peripheral organs to which neurons of the medial column of the vagal motor complex project. Processes in the torus semicircularis and vagal lobe showed single-labelled immunoreactivity for both SOM and NPY in distinct laminar patterns. Large single-labelled SOM-IR terminals appeared to form pericellular baskets in the eminentia granularis of the cerebellum. Single-labelled NPY- or SOM-IR fibers were also found in the secondary gustatory nucleus and tract, the facial lobe, descending trigeminal tract, reticular formation and spinal cord. As in mammalian species, select groups of neurons in teleosts colocalize the neuropeptides SOM and NPY.

CRF, Urotensin I, and sauvagine stimulate the release of POMC-derived peptides from goldfish neurointermediate lobe cells

In teleost fishes, the melanotropes of the neurointermediate lobe of the pituitary gland release numerous peptides--adrenocorticotropin (ACTH), melanotropin (MSH), lipotropin (LPH), corticotropin-like intermediate lobe peptide (CLIP), and endorphin--which are derived from the precursor molecule proopiomelanocortin. Superfused, isolated, dispersed goldfish neurointermediate lobe cell columns were used to investigate the release of immunoreactive (ir) alpha-MSH and ir ACTH from goldfish melanotropes. Stimulation of neurointermediate lobe cell columns with pulses of the structurally homologous peptides, Catostomus urotensin I (UI), ovine corticotropin-releasing factor (oCRF), or sauvagine, produced a significant increase in the concomitant release of ir alpha-MSH and ir ACTH. UI was two to three times as potent as ovine CRF or sauvagine. These studies suggest that CRF- and UI-like peptides stimulate the secretory activity of teleost melanotropes.

The fish neuropeptide urotensin I: Its physiology and pharmacology

Significant structural and biological homologies between urotensin I (UI), ovine hypothalamic corticotropin releasing factor (oCRF) and the frog skin peptide sauvagine (SVG) have been investigated and compared in fishes and mammals. In mammals, urotensin and the related peptides exert uniquely selective mesenteric vasodilatation, oCRF having approximately equal to 4% the activity of the other two. All three peptides are equipotent in stimulation of ACTH secretion in the rat in vivo and in vitro. UI is significantly more potent than the other two related peptides in stimulation of ACTH secretion in the goldfish pituitary. Immunocytochemical demonstration of UI not only in the caudal spinal cord but also in the brain, mainly in the lateral tuberal region and of an oCRF-like substance in the preoptic nucleus and pituitary, suggests that ACTH secretion in fishes may be controlled by two similar but distinct UI- or oCRF-like peptides.

Neurohypophysial hormonal control of cortisol secretion in the teleost Carassius auratus

Abstract Intravenous injections of arginine vasopressin (AVP), arginine vasotocin (AVT), and isotocin (IST) produced dose-dependent increments in circulating levels of plasma cortisol in goldfish in which endogenous ACTH secretion was suppressed by prior administration of dexamethasone. Injections of met 5 -enkephalin (ENK) or saline had no significant effect on plasma cortisol in dexamethasone-blocked fish. These results demonstrate that AVT and IST stimulate cortisol secretion in the goldfish and suggest that in teleosts AVT and IST possess corticotrophin-releasing factor (CRF) activity.

Proton pumps in the fish gill and kidney

The proton pump or vacuolar type H+-ATPase is an oligomeric protein responsible for electrogenic H+ secretion in a variety of acid-secreting epithelia. Recently, the proton pump was identified in both the gill and kidney of freshwater-adapted rainbow trout (Oncorhynchus mykiss). Using immunocytochemistry, H+-ATPase has been localized in the pavement cells and chloride cells of the lamellar epithelium. During periods of internal acidosis, there is a marked increase in the expression of the branchial proton pump as identified by Western analysis, immunocytochemistry and in situ hybridization. This augmented expression of proton pumps occurs concomitantly with a marked increase in branchial acid excretion and Na+ uptake. Immunocytochemical studies suggest that the pavement cell, rather than the chloride cell, is the predominant site of acid excretion during periods of acidosis. These findings are consistent with the notion that in freshwater teleosts, Na+ uptake and H+ excretion are linked via the coupling of the electrogenic proton pump to apical membrane Na+ channels. This mechanism may be controlled by hormones including cortisol and/or growth hormone. The fish kidney plays an important role in regulating acidosis via the re-absorption of filtered HCO3-. Recently, we have demonstrated using Western analysis and immunocytochemistry, the presence of proton pump in rainbow trout kidney and observed increased H+-ATPase expression during respiratory acidosis. These new findings suggest a role for the renal proton pump in acid-base regulation.

Hypophysiotropic neurons in the goldfish hypothalamus demonstrated by retrograde transport of horseradish peroxidase

SummaryThe horseradish-peroxidase (HRP) technique was used to visualize the cell bodies of axons projecting to the goldfish pituitary. Following intravenous injections of HRP, HRP reaction products were observed in axons of the rostral pars distalis, proximal pars distalis, neurointermediate lobe, pituitary stalk and in axons coursing from the pituitary into the hypothalamus. HRP-labelled cells in the brain were localized in two regions only — the nucleus preopticus (NPO) pars magnocellularis and pars parvocellularis, and the nucleus lateralis tuberis (NLT) of the hypothalamus. These observations suggest that the NPO and NLT are the source of the neurosecretory innervation of the goldfish pituitary.