Nervous control of reproduction in Octopus vulgaris: a new model

The classic study of Wells and Wells on the control of reproduction in Octopus demonstrated that the activity of the subpedunculate lobe of the brain and environmental illumination both inhibit the release of an unknown gonadotropin from the optic gland. This inhibitory control may be exerted by the neuropeptide Phe-Met-Arg-Phe-NH₂ (FMRFamide). It was later demonstrated that the olfactory lobe is also likely to be involved in the control of optic gland activity. The presence of gonadotropin-releasing hormone in the olfactory lobe suggested that it might exert an excitatory action on optic gland activity. Other neuropeptides have now been localised in the olfactory lobe: neuropeptide Y, galanin, corticotropin-releasing factor, Ala-Pro-Gly-Trp-NH₂ (APGWamide), as well as steroidogenic enzymes and an oestrogen receptor orthologue. This supports the hypothesis that this lobe may also play a part in the control of reproduction in Octopus. The olfactory lobe receives distant chemical stimuli and also appears to be an integrative centre containing a variety of neuropeptides involved in controlling the onset of sexual maturation of Octopus, via the optic gland hormone. This review attempts to summarise current knowledge about the role of the olfactory lobe and optic gland in the control of sexual maturation in Octopus, in the light of new findings and in the context of molluscan comparative physiology.     

Steroid transport through the surface of the prothoracic gland cells in Galleria mellonella L.

Steroid transport through the cell surface of the giant polyploid prothoracic gland cells of Galleria mellonella L. was studied by an ultracytochemical method. The alkaloid digitonin, known to form a complex with all sterols having a free-OH radical in position 3, proved to be suitable for studying the interiorisation of moulting hormone precursors and the release of synthesized hormones. The results suggest that cholesterol uptake in the last larval instar occurs by macropinocytosis during the feeding period, while the release of the steroids produced by the gland occurs by reverse micropinocytosis mostly on days 5-7 of the instar. The two processes are not simultaneous. The intracytoplasmic localisation of the reaction product confirms the steroidogenic role of the prothoracic gland.     

An in vitro bioassay for a mulluscan gonadotropin.

1. Protein synthesis occurs at a high rate in the ovaries of maturing Octopus vulgaris and can be measured from the incorporation of [14C]leucine in vivo and in isolated groups of eggs in vitro. 2. Removal of the optic glands in vivo 1--3 days prior to testing markedly reduces amino acid incorporation in vivo or in vitro. After 5 days in vivo incorporation stops. 3. The rate of incorporation in vitro is increased by the addition of optic gland extract. 4. Analysis of the kinetics of leucine uptake and incorporation in vitro indicates that the hormone has an effect on the inward transport of leucine which is independent of its action on protein synthesis. 5. Electron-microscope studies of the follicle cells and ova show that the former are the site of protein synthesis. 6. Changes in either uptake or incorporation into protein by the follicle cells can be used as a qualitative biolobical assay for the optic gland hormone. Uptake is very easy to measure but incorporation is the more sensitive parameter. Either is potentially suitable as a quantitative assay for this and perhaps also for other molluscan gonadotropins.     

Mechanistic considerations for the relevance of animal data on thyroid neoplasia to human risk assessment

There are two basic mechanisms whereby chemicals produce thyroid gland neoplasia in rodents. The first involves chemicals that exert a direct carcinogenic effect in the thyroid gland and the other involves chemicals which, through a variety of mechanisms, disrupt thyroid function and produce thyroid gland neoplasia secondary to hormone imbalance. These secondary mechanisms predominantly involve effects on thyroid hormone synthesis or peripheral hormone disposition. There are important species differences in thyroid gland physiology between rodents and humans that may account for a marked species difference in the inherent susceptibility for neoplasia to hormone imbalance. Thyroid gland neoplasia, secondary to chemically induced hormone imbalance, is mediated by thyroid-stimulating hormone (TSH) in response to altered thyroid gland function. The effect of TSH on cell proliferation and other aspects of thyroid gland function is a receptor mediated process and the plasma membrane surface of the follicular cell has receptors for TSH and other growth factors. Small organic molecules are not known to be direct TSH receptor agonists or antagonists; however, various antibodies found in autoimmune disease such as Graves' disease can directly stimulate or inhibit the TSH receptor. Certain chemicals can modulate the TSH response for autoregulation of follicular cell function and thereby increase or decrease the response of the follicular cell to TSH. It is thus important to consider mechanisms for the evaluation of potential cancer risks. There would be little if any risk for non-genotoxic chemicals that act secondary to hormone imbalance at exposure levels that do not disrupt thyroid function. Furthermore, the degree of thyroid dysfunction produced by a chemical would present a significant toxicological problem before such exposure would increase the risk for neoplasia in humans.     

The neurosecretory system of the eyestalk of Carcinus maenas (Crustacea: Decapoda)

The optic ganglia neurosecretory cells of male and female Carcinus maenas during intermoult are distinguishable into six types based on size, location, appearance and method of secretory material release from the perikaryon. Release occurs via the sinus gland and also, in one case, directly into blood capillaries among the neurosecretory cells themselves. The sinus gland consists of axonal extensions of the neurosecretory cells; no secretory granules are produced there and nuclei observed between the axonal endings are those of ill-defined glial cells.     

Interactions of the ring gland, overies, and juvenile hormone with brain neurosecretory cells in Musca domestica.

The staining intensity (median neurosecretory cell index) of the median neurosecretory cells (MNC) in Musca domestica increased as oögenesis progressed from stages 2 to 10. The amount of neurosecretory material within the MNC was dependent upon the presence of ovaries with developing or mature follicles. Ovariectomized flies had a median neurosecretory index that was 50 per cent less than that of control flies with mature eggs. In addition, we found that ring gland removal decreased the staining frequency of three different neurosecretory cell groups; increased staining frequency in another; increased the amount of neurosecretory material within the MNC fibre tract; increased the cytoplasmic area of types A and A′ MNC. Furthermore, neither the juvenile hormone analogue nor the ring gland had a direct effect on the median neurosecretory cell index but did influence neurosecretory activity indirectly by activating the ovaries. We hypothesize that an ovarian hormone—the oöstatic hormone—regulates either the release from or synthesis of neurosecretory material within the MNC.     

Cholinergic and VIPergic effects on thyroid hormone secretion in the mouse

The thyroid gland is known to harbor cholinergic and VIPergic nerves. In the present study, the influences of cholinergic stimulation by carbachol, cholinergic blockade by methylatropine and stimulation with various VIP sequences on basal, TSH-induced and VIP-induced thyroid hormone section were investigated in vivo in mice. The mice were pretreated with 125I and thyroxine; the subsequent release of 125I is an estimation of thyroid hormone secretion. It was found that basal radioiodine secretion was inhibited by both carbachol and methylatropine. Furthermore, TSH-induced radioiodine secretion was inhibited already by a low dose of carbachol. Moreover, a high dose of carbachol could inhibit VIP-induced radioiodine secretion. Methylatropine did not influence TSH- or VIP-stimulated radioiodine secretion, but counteracted the inhibitory action of carbachol on TSH- and VIP-induced radioiodine release. In addition, contrary to VIP, six various synthesized VIP fragments had no effect on basal or stimulated radioiodine release. It is concluded that basal thyroid hormone secretion is inhibited by both cholinergic activation and blockade. Furthermore, TSH-induced thyroid hormone secretion is more sensitive to inhibition with cholinergic stimulation than is VIP-induced thyroid hormone secretion. In addition, the VIP stimulation of thyroid hormone secretion seems to require the full VIP sequence.     

A novel avian hypothalamic peptide inhibiting gonadotropin release

The neuropeptide control of gonadotropin secretion at the level of the anterior pituitary gland is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), which was originally isolated from mammals and subsequently from non-mammals. To date, however, an inhibitory peptide of gonadotropin release is unknown in vertebrates. Here we show, in a bird, that the hypothalamus also contains a novel peptide which inhibits gonadotropin release. Acetic acid extracts of quail brains were passed through C-18 reversed-phase cartridges, and then the retained material was subjected to the reversed-phase and cation-exchange high-performance liquid chromatography (HPLC). The peptide was isolated from avian brain and shown to have the sequence Ser-Ile-Lys-Pro-Ser-Ala-Tyr-Leu-Pro-Leu-Arg-Phe-NH(2). Cell bodies and terminals containing this peptide were localized immunohistochemically in the paraventricular nucleus and median eminence, respectively. This peptide inhibited, in a dose-related way, gonadotropin release from cultured quail anterior pituitaries. This is the first hypothalamic peptide inhibiting gonadotropin release reported in a vertebrate. We therefore term it gonadotropin-inhibitory hormone (GnIH).     

Levels of epidermal growth factor in mice: tissues measured by a specific radioreceptor assay.

A radioreceptor assay of Epidermal Growth Factor (EGF), which uses as binder plasma membranes prepared from target tissues, instead of specific antibodies, is described. The amount of the polypeptide hormone present in the homogenate has been measured in various tissues. Submaxillary gland and parotid are confirmed to possess the highest levels of the factor. Results obtained incubating sliced tissues with or without pilocarpine, a drug which stimulates the hormone release, suggest that the tissues under investigation can be classified in two groups: a - “target tissues” (i.e. epidermis and corneal epithelium) b- “synthetizing” tissues (i.e. submaxillary gland, parotid, liver), which release the factor under pilocarpine stimulation.     

Neuroendocrine mechanisms in reproductive physiology

The estrous cycle of the female rat is understood fairly well. Determinations of pituitary and target gland hormones, and neurochemical and neurophysiologic studies provided some information allowing the following conclusion. Estradiol and progesterone in proper quantity and timing are signals for the central nervous system-pituitary axis to evoke preovulatory gonadotropin release. The steroids most likely interact with neurotransmitter regulating mechanisms at extra- and intrahypothalamic levels. Neural activity in the medial preoptic area — which plays a key role in regulating the estrous cycle—is increased during the time of preovulatory gonadotropin release. This forebrain structure has been shown to receive neural inputs from limbic and midbrain areas that are known to have modulatory functions on gonadotropin release. The neurochemical basis for increased release of hypothalamic hormone (s), which control gonadotropin release in female, male, and immature animals, are well integrated changes in turnovers of different neurotransmitters. Direct actions of hormones at the pituitary level to modulate the action of hypothalamic hormones are also possible. The molecular basis of these interactions is not completely understood.     

Genes expressed in the ring gland,the major endocrine organ of Drosophila melanogaster.

We have used an enhancer-trap approach to begin characterizing the function of the Drosophila endocrine system during larval development. Five hundred and ten different lethal PZ element insertions were screened to identify those in which a reporter gene within the P element showed strong expression in part or all of the ring gland, the major site of production and release of developmental hormones, and which had a mutant phenotype consistent with an endocrine defect. Nine strong candidate genes were identified in this screen, and eight of these are expressed in the lateral cells of the ring gland that produce ecdysteroid molting hormone (EC). We have confirmed that the genes detected by these enhancer traps are expressed in patterns similar to those detected by the reporter gene. Two of the genes encode proteins, protein kinase A and calmodulin, that have previously been implicated in the signaling pathway leading to EC synthesis and release in other insects. A third gene product, the translational elongation factor EF-1alpha F1, could play a role in the translational regulation of EC production. The screen also identified the genes couch potato and tramtrack, previously known from their roles in peripheral nervous system development, as being expressed in the ring gland. One enhancer trap revealed expression of the gene encoding the C subunit of vacuolar ATPase (V-ATPase) in the medial cells of the ring gland, which produce the juvenile hormone that controls progression through developmental stages. This could reveal a function of V-ATPase in the response of this part of the ring gland to adenotropic neuropeptides. However, the gene identified by this enhancer trap is ubiquitously expressed, suggesting that the enhancer trap is detecting only a subset of its control elements. The results show that the enhancer trap approach can be a productive way of exploring tissue-specific genetic functions in Drosophila.     

A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat

Experiments were undertaken to investigate if changes occur at the level of the anterior pituitary gland to result in selective follicle-stimulating hormone (FSH) release during late proestrus in the cyclic rat. At 1200 h proestrus, prior to the preovulatory luteinizing hormone (LH) surge in serum and the accompanying first phase of FSH release, serum LH and FSH concentrations were low. At 2400 h proestrus, after the LH surge and shortly after the onset of the second or selective phase of FSH release, serum LH was low, serum FSH was elevated about 4-fold, pituitary LH concentration was decreased about one-half and pituitary FSH concentration was not significantly decreased. During a two hour $\text{in}$$\text{vitro}$ incubation, pituitaries collected at 2400 h released nearly two-thirds less LH and 2.5 times more FSH than did pituitaries collected at 1200 h. Addition of luteinizing hormone releasing hormone (LHRH) to the incubations caused increased pituitary LH and FSH release. However, the LH and FSH increments due to LHRH in the 2400 h pituitaries were not different from those in the 1200 h pituitaries. The results indicate that a change occurs in the rat anterior pituitary gland during the period of the LH surge and first phase of FSH release which results in a selective increase in the basal FSH secretory rate. It is suggested that this change is primarily responsible for the selective increase in serum FSH which occurs during the second phase of FSH release.     

Studies of the neurohaemal organ (sinus gland) of the portunid crab Portunus sanguinolentus (Herbst) (Brachyura). 1. Morphology and histology in relation to moulting

The morphological and histological characters of the neurohaemal organ (sinus gland) of Portunus sanguinolentus are described in detail. The sinus gland lies on the dorsal surface of the optic ganglia, opposite the medulla interna. Histological techniques showed the presence of three tinctorially different secretory granules in the sinus gland. The predominant type of secretory material is basophilic and occurs as large granules, while two types of acidophilic material occur near the basement membrane. Cyclic changes in the relative amounts of acidophilic and basophilic material in correlation to moulting are also discussed. Allochthonous cells present in the sinus gland are identified.     

Increased synthesis of prolactin and growth hormone during incubation in the pituitary of broody Nagoya hens

Synthesis and release rates of prolactin and growth hormone (GH) in the anterior pituitary of laying and incubating broody chickens (Nagoya breed) were determined by a disc electrophoretic technique after in vitro incubation of anterior pituitaries with a labeled amino acid. Prolactin synthesis and release were two-fold higher in incubating than in laying hens, resulting in twofold increase in the concentration of prolactin in the gland. GH synthesis was three-fold higher in incubating than in laying hens but GH release was not affected by the incubation. GH concentration in the pituitary gland also increased in incubating hens. It is suggested that these changes in hormone synthesis, release, and concentrations are related to nesting behaviour and nutritional condition of incubating hens.     

Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release.

Using two specific and sensitive fluorometric/HPLC methods and a GC-MS method, alone and in combination with D-aspartate oxidase, we have demonstrated for the first time that N-methyl-D-aspartate (NMDA), in addition to D-aspartate (D-Asp), is endogenously present as a natural molecule in rat nervous system and endocrine glands. Both of these amino acids are mostly concentrated at nmol/g levels in the adenohypophysis, hypothalamus, brain, and testis. The adenohypophysis maximally showed the ability to accumulate D-Asp when the latter is exogenously administered. In vivo experiments, consisting of the i.p. injection of D-Asp, showed that D-Asp induced both growth hormone and luteinizing hormone (LH) release. However, in vitro experiments showed that D-Asp was able to induce LH release from adenohypophysis only when this gland was co-incubated with the hypothalamus. This is because D-Asp also induces the release of GnRH from the hypothalamus, which in turn is directly responsible for the D-Asp-induced LH secretion from the pituitary gland. Compared to D-Asp, NMDA elicits its hormone release action at concentrations approximately 100-fold lower than D-Asp. D-AP5, a specific NMDA receptor antagonist, inhibited D-Asp and NMDA hormonal activity, demonstrating that these actions are mediated by NMDA receptors. NMDA is biosynthesized from D-Asp by an S-adenosylmethionine-dependent enzyme, which we tentatively denominated as NMDA synthase.     

A new member of the PBAN family in Spodoptera littoralis: molecular cloning and immunovisualisation in scotophase hemolymph

In this article, we report evidence suggesting that the immunoreactive factor previously detected in Spodoptera littoralis scotophase hemolymph is PBAN, which supports a humoral route of the hormone to the pheromone gland. Western blot after native-PAGE of prepurified scotophase hemolymph extracts yielded an immunoreactive band with the same mobility as S. littoralis Br-SOG factor and the expected mobility for a noctuid PBAN. This band was not detected in photophase hemolymph extract. The identity of S. littoralis Br-SOG factor as PBAN was obtained from cDNA cloning using RT-PCR strategy. This allowed us to deduce the amino acid sequence of Spl-PBAN, which is highly homologous to other known PBANs. Moreover, we found that the PBAN encoding cDNA also encoded four other putative amidated peptides (Spl-DH homologue, Spl-alpha-NP, Spl-beta-NP and Spl-gamma-NP) that are identical or highly conserved among noctuids, and two non amidated peptides of unknown function. This cDNA organization is common to all known cDNAs encoding PBANs, leading to the release of different peptides after putative enzymatic cleavage of the preprohormone.     

Is octopamine a transmitter mediating hormone release in insects?

The release of hyperlipemic hormone from the glandular cells of the corpus cardiacum (CC) of Locusta migratoria is under the synaptic control of axons in nervus corpus cardiacum II (NCC II). The effects of aminergic agonists and antagonists on the release of the hyperlipemic hormone induced by electrical stimulation of NCC II have been examined. CC isolated from reserpine-injected locusts did not release hormone when subjected to electrical stimulation of NCC II but continued to release hormone in response to high-potassium saline. The electrically stimulated release of hormone from isolated CC was abolished by the alpha-adrenergic blocking agent, phenoxybenzamine, but potentiated by the beta-adrenergic blocking agent, propranolol. Phenoxybenzamine did not interfere with release induced by high-potassium saline. It is suggested that the postsynaptic receptors on the glandular cells are similar to the alpha-adrenergic receptors of vertebrates. Octopamine was found to be present in the glandular lobe of the CC at concentrations of 0.62 pmole per gland pair. Reserpine depleted the content to 0.3 pmole per pair. Bathing the CC in 10(-7) M octopamine resulted in the release of hyperlipemic hormone, and this release was blocked by phenoxybenzamine. It is concluded that the neurotransmitter involved in the synapse between axons of NCC II and the cells releasing hyperlipemic hormone is aminergic, possibly octopaminergic. Octopamine may well be a transmitter mediating hormone release in insects.     

Effects of acute ovariectomy on anterior pituitary gland follicle-stimulating hormone and luteinizing hormone secretion in the metestrous rat

Changes at the anterior pituitary gland level which result in follicle-stimulating hormone (FSH) release after ovariectomy in metestrous rats were investigated. Experimental rats were ovariectomized at 0900 h of metestrus and decapitated at 1000, 1100, 1300, 1500, 1700 or 1900 h of metestrus. Controls consisted of untreated rats killed at 0900 or 1700 h and rats sham ovariectomized at 0900 h and killed at 1700 h. Trunk blood was collected and the serum assayed for FSH and luteinizing hormone (LH) concentrations. The anterior pituitary gland was bisected. One-half was used to assay for FSH concentration. The other half was placed in culture medium for a 30-min preincubation and then placed in fresh medium for a 2-h incubation (basal FSH and LH release rates). The basal FSH release rate and the serum FSH concentration rose significantly by 4 h postovariectomy and remained high for an additional 6 h. The basal FSH release rate and the serum FSH concentration correlated positively (r=0.71 with 72 degrees of freedom) and did not change between 0900 and 1700 h in untreated or sham-ovariectomized rats. In contrast, the serum LH concentration and the basal LH release rate did not increase after ovariectomy. Ovariectomy had no significant effect on anterior pituitary gland FSH concentration. The results suggest that the postovariectomy rise in serum FSH concentration is the result, at least in part, of changes which cause an increase in the basal FSH secretion rate (secretion independent of the immediate presence of any hormones of nonanterior pituitary gland origin). The similarities between the selective rises in the basal FSH release rate and the serum FSH concentration in the ovariectomized metestrous rat and in the cyclic rat during late proestrus and estrus raise the possibility that an increase in the basal FSH release rate may be involved in many or all situations in which serum FSH concentration rises independently of LH.     

Distribution and functional significance of Leu-callatostatins in the blowfly Calliphora vomitoria

The Leu-callatostatins are a series of four neuropeptides isolated from nervous tissues of the blowfly Calliphora vomitoria that show C-terminal sequence homology to the allatostatins of cockroaches. The allatostatins have an important role in the reproductive processes of insects as inhibitors of the synthesis and release of juvenile hormone from the corpus allatum. In this study, the distribution of the Leu-callatostatin-immunoreactive neurones and endocrine cells has been mapped in C. vomitoria and, in contrast to the cockroach allatostatins, it has been shown that there is no cytological basis to suggest that the dipteran peptides act as regulators of juvenile hormone. Although occurring in various neurones in the brain and thoracico-abdominal ganglion, there is no evidence of Leu-callatostatin-immunoreactive pathways linking the brain to the corpus allatum, or of immunoreactive terminals in this gland. Three different types of functions for the Leu-callatostatins are suggested by the occurrence of immunoreactive material in cells and by the pathways that have been identified. (1) A role in neurotransmission or neuromodulation appears evident from immunoreactive neurones in the medulla of the optic lobes, and from immunoreactive material in the central body and in descending interneurones in the suboesophageal ganglion that project to the neuropile of the thoracico-abdominal ganglion. (2) Leu-callatostatin neurones directly innervate muscles of the hindgut and the heart. Immunoreactive fibres from neurones of the abdominal ganglion pass by way of the median abdominal nerve to ramify extensively over several areas of the hindgut. Physiological experiments with synthetic peptides show that the Leu-callatostatins are potent inhibitors of peristaltic movements of the ileum. Leu-callatostatin 3 is active at 10^-16 to 10^-13 M. This form or regulatory control over gut motility appears to be highly specific since the patterns of contraction in other regions are unaffected by these peptides. (3) Evidence that the Leu-callatostatins act as neurohormones comes from the presence of varicosities in axons passing through the corpus cardiacum (but not the corpus allatum) and also from material in extraganglionic neurosecretory cells in the thorax. Fibres from these peripheral neurones are especially prominent over the large nerve bundles supplying the legs. There are also a considerable number of Leu-callatostatin-immunoreactive endocrine cells in a specific region of the midgut. The conclusion from this study is that although conservation of the structure of the allatostatin-type of peptides is evident through a long period of evolution it cannot be assumed that all of their functions have also been conserved. Several different types of functions for the Leu-callatostatins of the blowfly are proposed in this study, but there is no evidence to suggest a role in the regulation of juvenile hormone synthesis and release.