Triiodothyronine induces lipid oxidation and mitochondrial biogenesis in rat Harderian gland.

The rat Harderian gland (HG) is an orbital gland producing a copious lipid secretion. Recent studies indicate that its secretory activity is regulated by thyroid hormones. In this study, we found that both isoforms of the thyroid hormone receptor (Trα (Thra) and Trß (Thrb)) are expressed in rat HGs. Although Thra is expressed at a higher level, only Thrb is regulated by triiodothyronine (T3). Because T3 induces an increase in lipid metabolism in rat HGs, we investigated the effects of an animal's thyroid state on the expression levels of carnitine palmitoyltransferase-1A (Cpt1a) and carnitine palmitoyltransferase-1B (Cpt1b) and acyl-CoA oxidase (Acox1) (rate-limiting enzymes in mitochondrial and peroxisomal fatty acid oxidation respectively), as well as on the mitochondrial compartment, thereby correlating mitochondrial activity and biogenesis with morphological analysis. We found that hypothyroidism decreased the expression of Cpt1b and Acox1 mRNA, whereas the administration of T3 to hypothyroid rats increased transcript levels. Respiratory parameters and catalase protein levels provided further evidence that T3 modulates mitochondrial and peroxisomal activities. Furthermore, in hypothyroid rat HGs, the mitochondrial number and their total area decreased with respect to the controls, whereas the average area of the individual mitochondrion did not change. However, the average area of the individual mitochondrion was reduced by ∼50% in hypothyroid T3-treated HGs, and the mitochondrial number and the total area of the mitochondrial compartment increased. The mitochondrial morphometric data correlated well with the molecular results. Indeed, hypothyroid status did not modify the expression of mitochondrial biogenesis genes such as Ppargc1a, Nrf1 and Tfam, whereas T3 treatment increased the expression level of these genes.

Morphological changes in frog mast cells induced by nerve stimulation in vivo.

We investigated at both histochemical and ultrastructural levels the effects of unilateral electrical stimulation in vivo of the frog hypoglossal nerve on the mast cells (MCs) within the nerve fascicles and among the axon terminals. The right ventral root of the hypoglossal nerve in different experiments was stimulated respectively for 1, 3, 5, 10 min with over-threshold stimuli (10 Hz; 2 ms duration). The stimulations at 3, 5 and 10 min caused a progressive degranulation and histochemical and ultrastructural changes of the MCs at the stimulated side. The morphological changes consisted of the loss of Alcian Blue secretory content and of a progressive release of safranin+ secretory granules, depending upon duration of stimulation. The ultrastructural study showed that granules are discharged whole into the microenvironment or may release their content through exocytosis. A functional relationship between nerve and MCs is also suggested by the close anatomical association between MCs and pre-terminal axons observed following 10 min of hypoglossal stimulation. No changes in MC morphology occurred after 1 min of electrical stimulation. The results suggest that active cholinergic fibres can modulate MC secretion.

In situ characterization of mast cells in the frog Rana esculenta.

The number, distribution, and ultrastructural characteristics of mast cells were assessed in the tongue, heart, and kidney of the frog Rana esculenta. The density of tongue mast cells (253+/-45 mast cells/mm2) was significantly higher than that of the heart (5.3+/-0. 4/mm2) and kidney (15.3+/-1.4 /mm2). A striking feature of this study was the remarkable association of frog mast cells to nerves. The ultrastructural study of the mast cell/nerve association demonstrated that mast cells were closely apposed to or even embedded in nerves. Mast cells were also physically associated with melanocytes even in the heart. Mast cells were Alcian blue+/safranin+ in the tongue and in the peritoneum, whereas in the heart and in the kidney they were Alcian blue-/safranin+. The mast cells in the lamina propria of the gastrointestinal tract were Alcian blue+/safranin-. The cytoplasm of frog mast cells was packed with numerous heterogeneous, membrane-bound granules. The ultrastructure of these cytoplasmic granules was unique, being totally unlike any other previously described granules in other animal species as well as in man. The histamine content/frog mast cell ( approximately 0.1 pg/cell) was approximately 30 times lower than that of human mast cells isolated from different tissues ( approximately 3 pg/cell). A monoclonal anti-histamine antibody was used to confirm the ultrastructural localization of histamine in secretory granules in frog mast cells.

Atrial natriuretic peptide, bradykinin, and angiotensin II-like immunoreactivity in the harderian gland of the terrapin Pseudemys scripta: response to osmotic stress.

The Harderian gland of the terrapin Pseudemys scripta has four types of acinar cells. Type IV cells are very similar to the salt secreting cells of the salt secretory glands of various marine vertebrates. The presence and localization of the Ile5-Angiotensin II, Atrial Natriuretic Peptide, and Bradykinin has been investigated by immunohistochemical methods. Immunoreactivity is confined to the type IV cells. Changes in the environmental salinity resulted in different patterns in the immunoreactivity especially after incubation with Ab-Angiotensin II and Ab-Atrial Natriuretic Peptide. Immunoreactive Angiotensin II cells are more numerous in animals maintained in distilled water, when reabsorption of sodium is needed. In contrast, immunoreactive Angiotensin II cells are very few in animals maintained in seawater. On the contrary, the number of immunoreactive cells for Atrial Natriuretic Peptide is high in seawater maintained animals, and weaker in animals in distilled water. The type IV cell may be considered a candidate for ion regulation in the terrapin Harderian gland.

The effects of testosterone and estradiol on mast cell number in the harderian gland of the frog, Rana esculenta.

The Harderian gland (HG) of the frog Rana esculenta contains mast cells in the interstitial tissue. The mast cell number (MCN) is influenced by sex hormones. Gonadectomy in both sexes provoked a decrease in MCN in January, while no effect was observed in September. Sex hormone-replacement therapy gave different results; estradiol treatment in castrated males and females always increased MCN, while testosterone did not. Acute estradiol treatment provoked an increase in MCN on days 2 and 4 of treatment and the morphology of the glandular compartment appeared normal. On days 8, 10 and 12 of treatment the MCN drastically decreased. The majority of glandular acini appeared strongly disorganized and the interstitial tissue became hypertrophic in concomitance with an increased vascularization. Our results suggest that estradiol acts by stimulating mast cells and acute estradiol treatment provokes proliferation of interstitial connective tissue together with glandular cells damage.

Organogenesis of the orbital glands in the lizard Podarcis s. sicula: a histological, histochemical and ultrastructural study.

The orbital glands of the lizard Podarcis s. sicula are represented by the anterior and posterior lacrimal glands and the Harderian gland. The anlage of the Harderian gland appears on about the 22nd day of development in the form of a short tubule projecting from the conjunctival epithelium. This event is coincident with the appearance of the nictitating membrane. At this stage the mesenchymal cells surrounding the glandular blastema proliferate at a high rate and form a definite sac, later occupied by both the Harderian gland and the anterior lacrimal glands. At the 26th day of development, the glandular blastema forms acini at its distal end. The prospective glandular cells are not yet differentiated histologically. At the 36th day of development, differentiated serous glandular cells become visible. At the 41st day of development, the acini fill up the preformed mesenchymal sac. Only at this stage does the most medial part of the gland differentiate into mucous-secreting anterior lacrimal gland. At the same time, a small primordium of the posterior lacrimal gland can be seen in the posterior commissure of the eye. The appearance of junctional complexes between epithelial cells and mesenchymal cells in the early developmental stages supports the role of the mesenchyme in the differentiation of the glandular cells. Since the glandular anlage differentiates laterally into Harderian gland and medially into anterior lacrimal gland, spatial and temporal differences seem to exist in the inductive process. Furthermore, a concentration gradient of the inductive substance(s) may be envisaged, since an intermediate zone is present between the Harderian gland and the anterior lacrimal gland, consisting of mixed glandular cells containing both mucous and serous secretory granules.

The effect of sex hormones on lipid content and mast cell number in the harderian gland of the female toad, Bufo viridis.

The Harderian gland of the toad Bufo viridis is a dimorphic gland owing to the presence of lipid droplets in the female glandular cells present only during summer months. Ovariectomy causes the disappearance of sudanophilia and estrogen-treatment completely prevents this change, while testosterone-injection has little effect. Estradiol-treatment also provokes a proliferation of the interstitial connective tissue concomitantly with the mast cell number increase. Our results suggest that estradiol acts, stimulating both mast cell and connective tissue proliferation, and plays a role in determining the expression of the female type of the toad Harderian gland.

The immunosuppressant FK506 inhibits the damage to mouse pancreatic islets induced by low dose streptozocin.

Diabetes mellitus was induced in 40 male C57BL6 mice by injection of a low dose of streptozocin (45 mg/kg body weight) on 5 consecutive days. Twenty four of the mice were immunosuppressed by administration of 1.5 mg FK506/kg body weight daily for 10, 15, 18 and 24 days. Administration of FK506 almost completely inhibited the streptozocin-induced islet damage, and consequently glycaemia remained normal. In FK506-treated animals any inflammatory infiltrate was very sparse and was limited to the vascular pole of the islets. Immunocytochemical results demonstrated that infiltrating cells were Ia-immunoreactive, but were not activated. Ultrastructural observations confirmed the absence of B cell necrosis and degranulation in FK506-treated mice; the few infiltrating elements encountered did not contain phagocytic vesicles or show other signs of activation.

Regeneration of the testicular interstitial compartment after ethane dimethane sulfonate treatment in the hypophysectomized frog Rana esculenta: independence of pituitary control.

The effects of ethane dimethane sulfonate (EDS) on the testes of hypophysectomized frogs (Rana esculenta) were investigated by light and electron microscopy. Initial signs of interstitial cell damage were observed in EDS- and EDS plus pituitary homogenate (PH)-treated animals 5 days after a single injection of EDS (100 mg/Kg body weight). The germinal compartment in these two groups appeared disorganized adjacent to the damaged interstitial tissue only in the EDS-treated animals, and by Day 8, spermatogenesis seemed to be affected in the EDS + PH-treated frogs in which Leydig cells had disappeared in some areas. On Day 28, regeneration of the interstitial tissue was complete and spermatogenesis was restored to normal. These data suggest that, in hypophysectomized frogs, the regeneration of the interstitial compartment is independent of pituitary activity and that the lack of interstitial cells activates the production of local factors responsible for the differentiation and proliferation of new Leydig cells. It is concluded that in addition to gonadotropins, the intratesticular environment is fundamental in the maintenance and regulation of testicular structure and function.

Regional and seasonal variations of RNA synthesis in the brain of the green frog, Rana esculenta.

Changes of RNA synthesis were demonstrated in neurons and ependymal cells of the green frog Rana esculenta during the annual cycle using the Mallory's trichrome stain as histochemical marker and autoradiography. Since the higher affinity of the nuclei for aniline blue is consistent with the increase of RNA content, the increase of RNA synthesis was expressed as percentage of the blue stained nuclei (% BSN). Neuronal transcription starts slowly in March or April, reaches a maximum in July and declines from September to November or December, depending on the brain region. In the ependymal cells, RNA synthesis starts in March and lasts until October. Neuronal transcriptional activity is found mostly in the glomerular layer of the olfactory bulb, in the striatum, nucleus accumbens septi, lateral and medial septal nuclei of the telencephalon, in the habenulae and various nuclei of the diencephalon, in the tectum opticum (particularly in the stratum griseum centrale), in the molecular layer of the cerebellum and in various nuclei of the rhombencephalon. The transcriptional activity of the ependymal cells is quite uniform in the lateral ventricles and the fourth ventricle, while it shows regional symmetric distribution in the third ventricle. Seasonal differences in transcriptional activity appear to be independent of seasonal thermic and photoperiodic fluctuations. In fact, temperature and photoperiod manipulations do not modify significantly the number of active nuclei. It is likely that the increase of RNA synthesis in nerve and ependymal cells corresponds to the resumption of neurotransmitter biosynthesis after hibernation. The simple Mallory's trichrome stain provides a reliable method for revealing increased transcriptional activity in histological sections.

The effects of gonadectomy and testosterone treatment on the Harderian gland of the green frog, Rana esculenta.

The effects of gonadectomy and testosterone treatment on the fine structure of the Harderian gland in male and female green frogs were investigated in different periods of the year. Gonadectomy, carried out when the glands are in the lowest secretory phase (September), causes degenerative changes consisting of a reduction of the rough endoplasmic reticulum, the appearance of autolysosomes, and an increase of nuclear heterochromatin. These effects can be prevented by testosterone treatment. No castration effects are found during the recovery (November) and enhancement (April-May) phases of secretory activity. The results suggest that the frog Harderian gland's sensitivity to testosterone changes during the annual cycle. The androgen dependence of the Harderian gland is correlated with the presence of androgen receptors in both male and female frogs.

Extraislet infiltration in NOD mouse pancreas: observations after immunomodulation.

This study describes the morphology, time appearance, persistence, and immunomodulation of extraislet infiltration in the nonobese diabetic (NOD) mouse pancreas. Female NOD mice were subdivided into two groups: untreated, killed at week 5 (n = 8), 10 (n = 8), or 21 (n = 8) of life; ganglioside treated (200 mg/kg body wt/day given from week 4 until week 21 of life, plus complete Freund's adjuvant at week 4), killed at week 10 (n = 6), 21 (n = 6), or 25 (4 weeks after the end of the ganglioside treatment; n = 6). Mononuclear elements were seen around small ductules located both in the vicinity of and far from the islets. Larger ducts were completely devoid of inflammation, thus evidencing that periductulitis is specific only to small ductules. Moreover, an exocrine focal pancreatitis was seen along septa and may demonstrate that macrophage activation takes place throughout the pancreatic connective tissue. Immunomodulation determines some suppression of insulitis; nevertheless, after treatment withdrawal, a reinfiltration occurs both within and outside the islets.

Morphological observations on pancreatic islet blood vessels in low-dose streptozocin-treated mice.

The aim of this study was to observe modifications occurring within the blood vessels of pancreatic islets in low-dose streptozocin (STZ)-treated mice. Forty-two male C57BL/6J mice were given 40 mg STZ on 5 consecutive days and killed 6 or 10 d after the first STZ injection. Immunocytochemistry revealed a dense accumulation of cells carrying the 7D10 epitope, related to macrophage inhibitor factor, arranged around the 'vascular pole' of the islets on d 6, and a small number of positive cells within the islets on d 10. Many Ia positive cells (equivalent to MHC class II antigen positive elements) were also found at the periphery of the islet. Ultrastructural observations showed the presence of monocytes within almost all the capillary vessels encircling the islets and entering them at the 'vascular pole' (d 6). On d 10 capillary lumina no longer contained marginating monocytes but activated macrophages were seen in the islet parenchyma.

In vivo effect of gangliosides on non-obese diabetic mice.

Non-obese diabetic mice were administered 150 mg/kg body weight of Cronassial daily from the 5th to the 11th week of life. Animals were killed either at the end of the treatment with Cronassial (week 11) or at week 21. The results obtained demonstrated that the appearance of the insulitis in control and treated animals at 11 and 21 weeks did not show a significant difference. The only effect of ganglioside treatment was to slow the rate of rise in glycaemia and the progression of the islet cyto-architectural derangement. In our opinion, this is not sufficient to prove the effectiveness of this drug in reducing the final diabetes incidence.

Immunomodulation of low dose streptozocin diabetes in mice reveals that insulitis is not obligatory for B cell destruction.

Twenty male C57Bl/J mice were injected with 50 microliters complete Freund's adjuvant (CFA) 1 wk before and 1 wk after induction of diabetes with 45 mg streptozocin (STZ)/kg body weight i.p. over 5 d. CFA administration prevented islet infiltration. Inflammatory cells were not seen within any of the islets observed. However, islet B cell destruction still occurred. These cells showed evidence of considerable damage, containing swollen mitochondria, contracted nuclei and areas of vacuolation and degranulation. Inflammation is therefore not obligatory for the development of low dose STZ induced diabetes.

Immunocytochemical identification of some regulatory peptides (gastrin, gastrin-releasing peptide, neurotensin and vasoactive intestinal polypeptide) in the Harderian gland of the green frog, Rana esculenta.

The presence and distribution of gastrin-, gastrin-releasing peptide-, neurotensin- and vasoactive intestinal polypeptide-like immunoreactivity in the Harderian gland of Rana esculenta were studied at different times of the annual cycle. Gastrin-releasing peptide, neurotensin and vasoactive intestinal polypeptide-like substances were found either in the glandular cells, or in the nerve fibers surrounding the glandular acini. Gastrin-like immunoreactivity was confined to the glandular cells. The immunoreactivity varied during the annual cycle, with the greatest concentration being noted during the recovery phase of glandular secretory activity.

Resumption of testicular activity in Gobius paganellus after administration of ethane 1,2-dimethane sulfonate (EDS).

1. The effect of a single injection of ethane-1,2-dimethane sulfonate (EDS) was studied in the teleost fish, Gobius paganellus in two different periods of the year. 2. During June EDS did not induce any change, while during December the drug was highly effective in promoting testicular activity. 3. Nucleus/cytoplasm ratio of interstitial cells strongly decreased concomitantly with the detection of high testicular androgen levels. 4. The germinal compartment was well developed showing the appearance of all spermatogenic stages and the cavity of lobular compartments filled of spermatozoa. 5. Our data are the first evidence of a stimulatory activity of EDS on testes of a vertebrate species.

Alterations of islet microvasculature in mice treated with low-dose streptozocin.

Islet capillary area was followed daily in mice after treatment with low-dose streptozocin (LDS), in order to elucidate the exact period during which the insular vascular bed undergoes a significant reduction. Forty C57BL6/J mice were diabetized with 5 x 40 mg streptozocin (STZ)/kg body wt and killed 6, 8, 9, 10, 11, 12, 15 or 18 days after the first STZ injection. Pancreases were sectioned and processed by staining for alkaline phosphatases using a method devised by Gomori. The percentage of the islet parenchymal area occupied by intra-islet capillaries was measured using a Videoplan videoanalyzer. LDS treatment did not significantly alter the islet capillary area up to day 8; the first signs of reduction were seen on days 9 and 10 (islet capillary area at days 9 and 10 respectively was 2.68% and 2.60% of controls). At day 11 a dramatic decrease in islet capillary area was seen (1.38%), which was not accompanied by a similar reduction of the islet parenchymal area. The reduction in islet capillary area continued to progress up to day 15 by which time it had achieved the lowest level (0.72%). On day 18, values remained practically unchanged.

Ultrastructural investigation of the corpora atretica of the electric ray, Torpedo marmorata.

Follicular atresia was studied in the ovary of the electric ray, Torpedo marmorata, by light and electron microscopy. The course of atresia may be divided into four stages. The first two comprise the dissolution of the oocyte and its phagocytosis by the small cells of the granulosa epithelium. The third stage consists of the transformation of the granulosa epithelium into an active glandular structure and is accompanied by the development of a smooth endoplasmic reticulum. The fourth stage is marked by sclerosis and pigmentary degeneration of the atretic follicle. Together these observations suggest an endocrine steroidogenic role for the corpora atretica (preovulatory corpora lutea) in T. marmorata.

Testosterone induction of poly(A)(+)-RNA synthesis and [35S]methionine incorporation into proteins of Rana esculenta Harderian gland.

The role of androgens in the cyclic secretory activity of the Rana esculenta Harderian gland (HG) was studied. Total RNA showed a dramatic increase in October and May when the nuclear androgen receptors peak. During the resumption of the secretory activity a gradual increase of poly(A)(+)-RNA was detected; during the enhancement phase (May) a peak of the poly(A)(+)-RNA fraction was found. In in vitro experiments testosterone increased the incorporation of [3H]uridine into the poly(A)(+)-RNA fraction and also that of [35S]methionine into a newly synthesized protein fraction (100 kDa). The latter effect is prevented by the exposure of the cells to the antiandrogen, cyproterone acetate (CPA). These findings reveal that, besides hamsters, the HG is a target for androgens in the frog.