Cell biology of the harderian gland.

The harderian gland is an orbital gland of the majority of land vertebrates. It is the only orbital gland in anuran amphibians since the lacrimal gland develops later during phylogenesis in some reptilian species. Perhaps because it is not found in man, little interest was paid to this gland until about four decades ago. In recent years, however, the scientific community has shown new interest in analyzing the ontogenetic and morphofunctional aspects of the harderian gland, particularly in rodents, which are the preferred experimental model for physiologists and pathologists. One of the main characteristics of the gland is the extreme variety not only in its morphology, but also in its biochemical properties. This most likely reflects the versatility of functions related to different adaptations of the species considered. The complexity of the harderian gland is further shown in its control by many exogenous and endogenous factors, which vary from species to species. The information gained so far points to the following functions for the gland: (1) lubrication of the eye and nictitating membrane, (2) a site of immune response, particularly in birds, (3) a source of pheromones, (4) a source of saliva in some chelonians, (5) osmoregulation in some reptiles, (6) photoreception in rodents, (7) thermoregulation in some rodents, and (8) a source of growth factors.

Androgen receptor in the thumb pad of Rana esculenta: dynamic aspects.

The androgen receptor (dissociation constant, 6 x 10(-9) mol/l) present in the 105 000 g cytosol fraction of the thumb pad of Rana esculenta is hormone dependent. The number of testosterone binding sites decreased in the absence of the testes and in long-term castrated animals (75 days) became undetectable. Administration of testosterone (50 ng) induced the replenishment of the cytophasmic androgen receptor; its concentration also varied during the annual reproductive cycle.

Androgen receptor in the Harderian gland of Rana esculenta.

An androgen receptor has been identified in the cytosolic and nuclear extracts of the Harderian gland of the frog, Rana esculenta. A single class of high-affinity binding sites was found: Kd = 1.9 +/- 1.3 (S.D.) nmol/l (n = 26) for the cytosolic extract and Kd = 0.9 +/- 0.8 nmol/l (n = 15) for the nuclear extract. The presence of binding activity in both nuclear and cytosolic extracts and the low rate of ligand-receptor dissociation are characteristics that distinguish this receptor from a steroid-binding protein. The Kd did not show any sex difference and did not exhibit any secretory activity-related change. Binding in both cytosolic and nuclear extracts was specific for androgens (testosterone = 5 alpha-dihydrotestosterone); oestradiol-17 beta showed a 30% cross-reaction; moreover, specific binding of [3H]oestradiol-17 beta was not detectable. The binding capacity of the Harderian gland increased progressively in both fractions from October to December, reaching a peak in May, and decreased suddenly during July to August. The lack of any morphological sex-related difference in the Harderian gland of the green frog might be accounted for by the high amount of circulating androgens as well as a similar concentration of androgen receptor in both sexes.

Seasonal testosterone profile and testicular responsiveness to pituitary factors and gonadotrophin releasing hormone during two different phases of the sexual cycle of the frog (Rana esculenta).

Plasma and testicular testosterone concentrations in the frog, Rana esculenta, were studied by radioimmunoassay and showed similar seasonal fluctuations. The increase in testicular androgen during November preceded that occurring in the plasma by 2 months. Pituitary products and gonadotrophin releasing hormone, and the responsiveness of the testis to these substances play an important role in determining the hormone profile.

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.

Pineal response to isoproterenol in rats chronically treated with electroconvulsive shock.

Chronic electroconvulsive shock (ECS) has been shown to induce a downregulation of beta 1-adrenergic receptors in the rat cerebral cortex. Because the secretion of melatonin in the pineal gland is regulated primarily by beta 1-adrenoceptors, in the present study we investigated the effect of chronic administration of ECS on pineal beta-adrenergic responsiveness to isoproterenol. To this purpose, young adult male rats received once daily for 8 days ECS (80 mA, 0.5 s) or sham ECS. On the day after the last ECS or sham treatment, they were injected with isoproterenol hydrochloride (1 mg/kg SC) or volume-matched saline at 1600 h. Two hours later they were killed by decapitation. Results showed that the isoproterenol-induced increase in the pineal melatonin content was blunted in rats treated with ECS as compared to sham-treated animals (shock x drug interaction = p < 0.01). These data indicate that chronic ECS treatment affects beta 1 receptor-mediated melatonin production in the pineal gland. Further studies need to elucidate whether the blunted melatonin response to isoproterenol in ECS-treated rats is due to a downregulation of pinealocyte beta-adrenergic receptors.

Role of seizure activity in the decreased pineal response to isoproterenol in rats chronically treated with electroconvulsive shock.

Chronic electroconvulsive shock (ECS) has been previously reported to blunt the melatonin response to acute isoproterenol administration in rats. To assess whether electrically induced seizures are indeed required for the appearance of the blunted pineal response to isoproterenol, pineal and serum melatonin levels were measured after isoproterenol stimulation in rats treated with ECS (80 mA, 0.5 sec), subconvulsive shock (15 mA, 0.5 sec), or sham-ECS once per day at 11:30-12:00 h for 8 days. In ECS-treated rats, both pineal and serum melatonin levels after isoproterenol administration were significantly lower than those in sham-treated animals and in rats receiving subconvulsive shock. Moreover, as compared with sham treatment, chronic subconvulsive shock did not affect the melatonin response to isoproterenol. These data show that seizure activity is indeed required for the ECS-induced decrease in the pineal response to acute beta-adrenergic stimulation.

Effects of single and repeated electroconvulsive shock on isoproterenol-stimulated pineal N-acetyltransferase activity and melatonin production in rats.

The response of the pineal gland to acute isoproterenol administration represents a useful tool to investigate beta 1-adrenoceptor function, because the production of melatonin and the activity of its main synthesizing enzyme, N-acetyltransferase (NAT), are regulated by beta 1-adrenergic receptors. In the present study, rats underwent single electroconvulsive shock (ECS) administration (0.80 mA, 0.5 s, at midday), chronic ECS treatment (0.80 mA, 0.5 s, once daily for 8 days), or sham treatments. On the day after the last ECS or sham ECS, animals were injected with isoproterenol hydrochloride (1 mg.kg-1 SC) or volume-matched saline at 1600 h. After single ECS, isoproterenol injection induced a clear-cut increase in both pineal NAT activity and melatonin levels with no significant differences between ECS-treated rats and the sham-treated ones. In rats chronically treated with ECS, the isoproterenol-induced increases in both pineal NAT activity and melatonin content were significantly lower than in sham-treated animals (p < 0.001 for NAT activity; p < 0.005 for melatonin levels; Turkey's test). These data show that the pinealocyte beta-adrenoceptor function is reduced by chronic, but not acute ECS administration, and that this change is not due to the nonspecific stress effect of animal handling or to the acute effects of the last of a series of ECS.

Effects of gonadectomy and temperature on the N-acetyltransferase activity in the harderian gland of the green frog Rana esculenta.

Since, in the Harderian gland (HG) of the hamster, the N-acetyltransferase (NAT), the specific enzyme involved in the biosynthesis of melatonin, exhibits a sexual dimorphism, in the present study, we investigated whether such a dimorphism is present also in the HG of the green frog Rana esculenta. In intact frogs, no significant differences emerged between males and females in the HG NAT activity under both cold (10 degrees C) and warm (22 degrees C) temperature conditions. In female frogs, the HG NAT activity was significantly decreased by both gonadectomy (P < 0.001) and warm temperature (P < 0.001), the two effects being not additive. In male animals, neither gonadectomy nor temperature alone significantly affected the activity of the NAT enzyme in the HG. However, gonadectomized male frogs exposed to warm temperature exhibited a significant drop in the HG NAT activity (P < 0.005). These data show that, in Rana esculenta, although no sexual dimorphism exists in the HG NAT activity, a sex difference is evident in the modulation of the enzyme activity by gonads and temperature, the female frogs being more sensitive to the impairing effects of both gonadectomy and higher temperature.

Mallory stain may indicate differential rates of RNA synthesis: II. Comparative observations in vertebrate nuclei.

The differential staining of nuclei by the use of the Mallory trichrome method was investigated in a variety of tissues of representative vertebrates. By this method nuclei stained orange or blue; erythrocyte nuclei stained red. Since the higher affinity for aniline blue is due to an increased RNA synthesis, it was possible to reveal not only the changing metabolic status of a cell type, as shown for instance in the liver parenchyma and other glandular tissues, and nervous tissue, but also in different cell populations in the same tissue, such as the spleen.

Mallory stain may indicate differential rates of RNA synthesis: I. A seasonal cycle in the harderian gland of the green frog (Rana esculenta).

When Mallory's trichrome stain is used, acinar nuclei of the Harderian gland of Rana esculenta display different affinities for the dye. Some of the orangiophilic nuclei show affinity for aniline blue (blue nuclei). In the Harderian gland of Rana esculenta their number and the intensity of staining with aniline blue may vary during the year. The affinity for aniline blue disappears following digestion of paraffin sections with RNAase, but not with DNAase or trypsin. Furthermore, in vitro incubation with [5, 6-3H]-Uridine shows a selective incorporation by the majority of blue nuclei. Therefore, the affinity for aniline blue is likely due to increased RNA synthesis. The increment of nuclear RNA shown by these methods is supported by the quantitative determination of total RNAs during the resumption (October) and enhancement (May) of secretory activity, when the percentage of blue nuclei of the acinar cells is at its highest levels of the year. The affinity of RNA-rich nuclei for aniline blue, while others are strictly orangiophil, is discussed on the basis of molecular structure of the dyes used in the staining mixture. Mallory's trichrome stain appears to be an useful tool for detecting changes in cell nuclear status.

Hypothalamic-pituitary-ovarian axis in women with operable breast cancer treated with adjuvant CMF and tamoxifen.

The effect of adjuvant CMF (cyclophosphamide, methotrexate, and 5-fluorouracil) and tamoxifen (TM) on hypothalamic-pituitary-ovarian function was studied in 120 women with stage I-II operable breast cancer. Sixty patients were premenopausal, of whom 25 were treated with CMF for 9 cycles, 25 with CMF for 9 cycles + TM for 2 years, started concurrently, and 10 with TM alone for 2 years. Sixty patients were postmenopausal and they were all treated with TM alone for 2 years. In all groups treatment was started within 4 weeks of mastectomy. Plasma levels of estrone (E1), estradiol-17 beta (E2), follicle-stimulating hormone, luteinizing hormone (LH), prolactin (Prl), testosterone (T) and thyroid-stimulating hormone (TSH) were determined in all patients before surgery and again at 3-month intervals from initiation of the adjuvant therapy. In ten patients of each treatment group FSH-LH and Prl-TSH release was determined following stimulation with releasing hormones. CMF and CMF+TM therapy resulted in amenorrhea in 42/50 premenopausal patients with decrease of E1+E2 (p less than 0.001) and elevation of FSH (p less than 0.001) and LH (p less than 0.01) plasma concentration to postmenopausal levels. In premenopausal women treated with TM a marked increase of E1+E2 (p less than 0.001) was observed with unaltered FSH-LH plasma concentration. A significant fall of Prl also occurred in these patients. In postmenopausal women and premenopausal patients with CMF-induced amenorrhea TM produced a marked fall of FSH-LH and a decrease of Prl plasma level. Plasma TSH and T were not affected in any patient by any of the treatment regimens. The results of the stimulatory tests are in agreement with the hormonal changes observed under basal conditions and indicate that, whereas CMF suppresses the ovary and does not alter hypothalamic-pituitary function, TM induces profound changes of the hypothalamic-pituitary-ovarian axis.

Connexin43 expression in the testis of frog Rana esculenta: sex hormonal regulation.

Spermatogenesis is the process of proliferating and differentiating germ cells that require highly coordinated cellular interactions. Intercellular junctions are important in maintaining communication between testicular cells. In particular, gap junctions play an important role in this event. Connexin43 (Cx43) is the most abundant protein forming a gap junction in a vertebrate testis. It is expressed in several cells types of Rana esculenta testes. The use of reverse transcriptase-PCR analysis demonstrates that expression levels of Cx43 are regulated by estradiol and testosterone in both in vivo and in vitro experiments.

Connexin 43 expression in the testis of the frog Rana esculenta.

Testicular cell-to-cell interactions play a key role in the regulation of spermatogenesis. In the testis, cell contacts are mediated through several mechanisms, including paracrine and direct contacts depending on gap junctional pathways. Gap junctions require connexin (Cx) channels and connexin-43 (Cx43) represent the most abundant Cx found in mammalian testis. Little is known about Cx expression in non-mammalian testis. Here we report the partial cloning of a Cx43 transcript of 381 bp from Rana esculenta testis. We also demonstrate that, in the frog testis, Cx43 transcript and protein show a parallel temporal and spatial pattern of expression throughout the reproductive annual cycle, with higher levels from September to January (when spermatogenesis is at a maximum level). In situ hybridization, carried out on testis collected in October, indicated that Leydig cells (LC) and Sertoli cells express Cx43 transcript, while the hybridization signal was less intense in germ cells. To obtain more information on Cx43 expression in the frog testis, we have used ethane-dimethane sulphonate (EDS), a toxin that specifically destroys LC. RT-PCR analysis shows a progressive decrease in Cx43 expression in EDS-treated testis from day 1 to day 4 after the injection, associated with LC destruction. Moreover, Cx43 expression returns to normal on day 28, when a new population of LC reappear in the interstitium, indicating that Cx43 is mainly expressed by LC. Taken together our data provide evidence that Cx43 is present in the frog testis with an important role in spermatogenesis.

Seasonal variations in the daily rhythm of melatonin and NAT activity in the Harderian gland, retina, pineal gland, and serum of the green frog, Rana esculenta.

Day-night variations of melatonin content and N-acetyltransferase (NAT) activity were studied in the Harderian gland (HG), retina, pineal gland, and serum of the green frog Rana esculenta. Throughout the year the retinal melatonin content was correlated with retinal NAT activity and was always higher than those in the pineal gland and HG. On the other hand, in these structures diurnal fluctuations in NAT activity were observed. There were clear seasonal differences in the magnitude of the nocturnal increase of retinal melatonin levels as well as in the nocturnal pattern of retinal NAT activity. In summer day-night variations of melatonin and NAT are absent. The prevailing photoperiod seems to affect melatonin and NAT circadian rhythms in R. esculenta.

Purification of a human progesterone receptor.

The cytoplasmic progesterone receptor form human uterus has been purified to apparent homogeneity by a combination of ammonium sulfate fractionation and affinity chromatography. Affinity resins prepared by conventional means were compared to those prepared by a modified method. The latter give more reproducible results. A consistent finding was that low capacity resins gave the highest fold purification of the receptor. The pure receptor sedimented at 3.6 S on sucrose density gradient centrifugation, was eluted as a single band by 0.2 M KCl from DEAE-cellulose, and migrated as a single band of molecular weight 42 000 on NaDodSO4-polyacrylamide gel electrophoresis. Molecular weight determinations, obtained from Strokes' radii and sucrose gradient centrifugation, the receptors' behavior on ion exchange resins, and hormone binding specificity were all similar to those of the receptor found in crude cytosol. When the crude cytosol receptor was photoaffinity labeled by using 3H-labeled 17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione followed by NaDodSO4-polyacrylamide gel electrophoresis, only protein of Mr 42 000 was labeled. This is consistent with our previous findings that alkylation of the pure receptor using 11-deoxycorticosterone bromo[3H]acetate showed labeling of a single protein of Mr 42 000. These properties confirm that the identity and integrity of the receptor have been maintained throughout its purification.

Prolactin receptors in the male Rana esculenta.

The binding of 125I-labeled ovine prolactin (oPRL) to membrane preparations of several tissues from the male green frog, Rana esculenta, collected during the year is reported. PRL binding to kidney fractions was generally high (range 5-45%). A maximum was observed in the month of October, whereas the lowest value was found during the summer season. The binding to skin fractions was equally high (range 5-25%) and the annual profile parallels that of renal fractions. In the liver, a lower specific binding (range 3-4%) occurred consistently during the year, whereas no detectable binding was found in the muscle. The 125I-oPRL binding was inhibited by oPRL and oGH but not by oFSH or oLH. Scatchard analysis gave dissociation constants of 0.4-1 x 10(-10) M and binding capacity of about 20 fmol/mg of membrane proteins was observed in both the skin and kidney fractions. No receptor sites were detectable in 30-day hypophysectomized animals. The administration of oPRL or a crude homogenate of the frog hypophysis induced the appearance of specific PRL binding. Testosterone is able to restore prolactin binding in hypophysectomized animals, as PRL treatment does.

A comparative study of melatonin production in the retina, pineal gland and harderian gland of Bufo viridis and Rana esculenta.

1. The circadian patterns of melatonin and of its synthesizing enzyme N-acetyltransferase (NAT) were investigated in the serum, retina, pineal gland and Harderian gland (HG) of two amphibian species, Bufo viridis and Rana esculenta. 2. Serum melatonin levels showed no diurnal fluctuations in Bufo viridis, whereas, in Rana esculenta, they exhibited a circadian rhythm, with the highest values occurring during the night. Retina melatonin exhibited characteristic circadian patterns in both species, with the highest values occurring during the day, in Bufo, and the highest concentrations occurring at night in Rana. 3. In the retina, NAT activity peaked at night in both amphibians, but in Bufo the levels were up to 30 times higher than in Rana. In the HG and in the pineal gland, NAT activity showed different patterns in the two species with no diurnal variations in Bufo, and characteristic circadian rhythms in Rana. 4. In the HG and pineal gland of both species, melatonin was only occasionally detectable over the 24-hr period. 5. This is the first report exploring melatonin production in Bufo viridis and Rana esculenta. In our experimental conditions, marked differences emerged between the two species.

Appearance of sex hormone receptors in frog (Rana esculenta) tadpole skin during metamorphosis.

In tadpole skin of Rana esculenta, a specific testosterone receptor was detected during the climax in both males and females. The Kass ranged between 1 and 2.79 x 10(9)M-1.

(+)-N-allylnormetazocine enhances N-acetyltransferase activity and melatonin synthesis: preliminary evidence for a functional role of sigma receptors in the rat pineal gland.

In the present study, to evaluate the role that sigma receptors play in the physiology of the pineal gland, we assessed the effects of the sigma receptor ligand (+)-N-allylnormetazocine on the gland activity during either the day or the night. As compared to saline, (+)-N-allylnormetazocine enhanced the physiological increases in both pineal N-acetyltransferase (NAT) activity and melatonin content at night, but it did not affect the biosynthetic activity of the gland during the day. Moreover, (+)-N-allylnormetazocine potentiated the enhancement of NAT activity and pineal melatonin content induced by isoproterenol administration during the day. The nocturnal stimulation of pineal NAT activity and melatonin levels by (+)-N-allylnormetazocine was prevented by pretreatment with rimcazole, a specific sigma receptor antagonist. These results demonstrate that sigma receptor activation by (+)-N-allylnormetazocine is not able, by itself, to stimulate pineal melatonin production, whereas it potentiates the biosynthetic activity of the pineal gland when this is stimulated noradrenergically.