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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.

The Harderian gland in Rana esculenta has been studied during the annual cycle at the histological, histochemical and ultrastructural levels. The Harderian gland has an acinar structure and is the only orbital gland in anuran amphibia. It develops at the medial corner of the orbit from the conjunctival epithelium at the premetamorphic stage. In the adult the glandular secretion reaches a maximum during the months of July and August, drops in September and resumes slowly from October onwards. The secretion is seromucoid and the secretory granules are released into the acinar lumen, mainly by exocytosis. Porphyrins were not detected. No sexual dimorphism was observed in the glandular cells. The resumption of secretory activity in October and the enhancement of secretion in May are marked by the appearance of "blue nuclei" (Mallory stain) in a relatively high percentage of glandular cells. This unusual blue colour, using the Mallory stain (by which nuclei stain red), disappears after digestion of paraffin sections with RNAase, but not with DNAase and trypsin. The blue staining may, therefore, indicate an increased amount of nuclear RNA. The Harderian gland in the frog most probably serves to lubricate and moisten the eye in the absence of the lacrimal gland. However, the gland may also represent an immunoactive organ owing to the presence of numerous mast cells and plasma cells in the interacinar spaces.

Prothymosin alpha (PTMA) is a highly acidic small polypeptide, that is, widely distributed and conserved among mammals. Its possible involvement in male gametogenesis has been mentioned but not clarified yet; in particular, it has been suggested that, in non-mammalian vertebrates, it could play a role during GC meiosis and differentiation. In the present work we investigated the possible association between PTMA and meiotic and post-meiotic phases of mammalian spermatogenesis. Three different time points during postnatal development of rat testis were analyzed, that is, 27 dpp (completed meiosis), 35 dpp (occurring spermiogenesis), and 60 dpp (first wave of spermatogenesis definitely ended). RT-PCR and Western blot analyses showed that the expression levels of both Ptma mRNA and corresponding protein decrease in total extracts from 27 to 60 dpp. The in situ hybridization localized the transcript in interstitial Leydig cells, peritubular myoid cells and, inside the tubules, in germ cells from pachytene spermatocytes to newly formed haploid spermatids. The immunohistochemistry analysis localized the protein in the same cell types at 27 dpp, while at 35 and 60 dpp the haploid cells remain the only germ cells that still express it. In particular, PTMA specific localization in the heads of spermatids and epididymal spermatozoa, associated with the acrosome system, supports for the first time the hypothesis of a direct function in male germ cells.

Prothymosin alpha (PTMA) is a small acidic protein abundantly and ubiquitously expressed in mammals and involved in different biological activities. Until now, its specific function in spermatogenesis has never been properly investigated. Recently, the isolation of a cDNA encoding for PTMA from the testis of the frog Rana esculenta has been reported: ptma transcript is highly expressed throughout the frog reproductive cycle, peaking in September/October, in concomitance with the germ cell maturation; it is specifically localized in the cytoplasm of primary and secondary spermatocytes and, at a lower level, in the interstitial compartment, in Leydig cells.In this article we support the involvement of PTMA in the meiotic phases of frog spermatogenesis. The expression of ptma mRNA increases in the testis of frogs treated with the antiandrogen cyproterone acetate, which blocks the II meiotic division and induces an increase in SPC cysts; on the contrary, it highly decreases in the testis of animals kept at 4 degrees C and treated with human corionic gonadotropin, in concomitance with the induced block of spermatogenesis and the disappearance of meiotic cells in the tubules. Furthermore, for the first time we have also evidenced by immunohistochemistry the expression of PTMA in the nuclei of secondary spermatocytes, spermatids, and spermatozoa, as well as in the cytoplasm of interstitial Leydig cells. Taken together our data suggest for an important role of PTMA in germ cell maturation and/or differentiation during R. esculenta spermatogenesis.

This study was conducted to verify the effect(s) of melatonin treatment on frog Leydig cells. Morphological observation after melatonin treatment indicates that many frog Leydig cells show degenerative changes (i.e. heterochromatic nuclei, loss of cellular adhesion) while in adjacent germinal tubules several Sertoli cells show heterochromatic nuclei, confirming the presence of a paracrine effect between interstitial and germinal compartments. The effect of melatonin on frog Leydig cell steroidogenesis was investigated in in vitro experiments; after 6 h of incubation melatonin severely inhibits both control and GnRH-induced testosterone secretion. In addition, in order to verify the effect of indolamine on frog Leydig cell activity, we investigated, by in situ hybridization, the presence of frog relaxin (fRLX, a transcript specifically expressed by these cells) in the testes of melatonin-injected animals after 48 h. fRLX signal completely disappeared from the testis of melatonin-injected frogs. The results of the present study indicate that melatonin treatment provokes Leydig cell morphological changes, blocks GnRH-antagonist-induced testosterone secretion and decreases fRLX expression. Taken together these results strongly indicate that melatonin acts on Leydig cells in the testis of the frog Rana esculenta.

SUMMARY In the present study, we have utilized 17β-estradiol to induce the increase of mast cell number in order to verify the melatonin effect on mast cell accumulation in the frog testicular interstitium. Data obtained from in vivo experiments confirm that 17β-estradiol increases the mast cell number and indicate a melatonin-inhibitory role in their accumulation in the frog testis. In addition, melatonin interferes with the effects of estradiol on the increase of mast cell number in short-term cultured testes, and this result has also been obtained in a dose-response experiment at physiological concentration. The data suggest that melatonin acts on mast cell number directly via its local action in the frog gonads. In conclusion, our study shows, for the first time, that melatonin may interfere, probably via estrogen receptors, with the differentiation and/or proliferation of mast cells induced by estradiol treatment either in vivo or in vitro in the testis of the frog Rana esculenta.

Ethane 1,2-dimethane sulphonate (EDS), a toxin which specifically destroys Leydig cells (LC), has been used to study cellular interactions in the testis of the frog Rana esculenta. Animals received three consecutive EDS injections and were sacrificed on day 4, 8, and 28 from the first injection. No significant morphological differences were observed between present observation and that obtained, in a previous experiment, after four consecutive EDS injections. In fact, on day 4, in the germinal tubules adjacent to apparently normal LC, Sertoli cells surrounding primary spermatogonia (I SPG) show heterochromatic nuclei and loss of cellular adhesion. Interestingly, I SPG surrounded by the heterochromatic Sertoli cells present grossly swollen mitochondria with ballooned cristae. On day 8, sometimes in the interstitium many LC appear strongly damaged and the germinal tubules appear disorganized; the only cell type still distinguishable is the I SPG. On day 28 from the first EDS injection a new population of LC reappear in the interstitium and spermatogenesis normalizes. These data confirm the close relationship between the interstitial and the geminal compartments. Immunocytochemical data obtained using a polyclonal antibody anticonnexin-43 (Cx-43, the most abundant Cx found in mammalian testis) demonstrate the presence of Cx-43 in the frog testis. In particular, Cx-43 is present between LC in the interstitium, between Sertoli and germ cells in the cysts and between Sertoli cells and I SPG. Cx-43 immunopositivity sharply decreases on day 4 from the first EDS injection simultaneously with the loss of cellular adhesion between Sertoli and germ cells. On day 8 and 28 from the first EDS injection Cx-43, immunopositivity is restored and, this data is also supported by Western blot analysis. Our data provide, for the first time, evidence that Cx-43 protein is present in the frog testis and confirm that EDS is a useful tool for studying cellular communication at the paracrine pathway or through direct contact depending on the gap junctional pathway in R. esculenta testis

Melatonin, nocturnally secreted by the pineal gland, regulates a variety of physiological functions, including reproduction. Here, we investigated the evidence of melatonin binding sites in frog tissue (brain, retina, and testis) through saturation and competition binding experiments. In the frog, Pelophylax esculentus, our results confirm the presence of a single class of melatonin-specific binding sites in the brain and retina, but not in the testis. Further experiments have been done using biomolecular approaches (PCR analysis). Here, we report the isolation of a cDNA encoding for a melatonin receptor type (mel1b) from brain, retina, and testis of the P. esculentus. PCR analysis revealed that melatonin expression is higher in the brain and retina, whereas it is lower in the testis. The presence of a melatonin receptor transcript in the frog testis corroborates our previous results obtained in in vitro experiments that suggest that melatonin might act directly in male vertebrate gonads, and indicates that the frog testis may be a suitable model to verify the role of indolamine in testicular activity. J. Exp. Zool. 315:520–526, 2011. © 2011 Wiley Periodicals, Inc.

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 forgs. © 1993 Springer-Verlag.

SummaryIt is well known that melatonin provokes reproductive alterations in response to changes in hours of daylight in seasonally breeding mammals, exerting a regulatory role at different levels of the hypothalamic–pituitary–gonadal axis. Although it has also been demonstrated that melatonin may affect testicular activity in vertebrates, until now, very few data support the hypothesis of a local action of melatonin in the male gonads. The aim of this study was to investigate whether MT1, MT2 melatonin receptors and the H9 melatonin-related receptor, are expressed in the adult rat testes and during development. A semi-quantitative RT-PCR method was used to analyse the expression of MT1, MT2 and H9 receptors mRNAs in several rat tissues, mainly focusing on testes during development and adult life. Our results provide molecular evidences of the presence of both MT1 and, for the first time, MT2 melatonin receptors as well as of the H9 melatonin-related receptor in the examined tissues, including adult testes. During development MT1 and MT2 transcripts are expressed at lower levels in testes of rats from 1 day to 1 week of age, lightly increased at 2 weeks of age and remained permanently expressed throughout development until 6 months. These data strongly support the hypothesis that melatonin acts directly in male vertebrate gonads suggesting that rat testes may be a suitable model to verify the role of indolamine in vertebrate testicular activity.

1. 1. The secretory activity of the Harderian gland in Rana esculenta varies during the year, reaching its highest activity during the hottest period (July-August). Therefore, secretion may be modulated by temperature and/or photoperiod. 2. 2. Adult males and females were placed under several combinations of light and temperature in two different periods of the year (February and July) in order to elucidate their respective roles, if any, on the stimulation of secretion. 3. 3. Under experimental conditions, high temperature (24°C), irrespective of the photoperiod selected, stimulates secretion shown both at histological and histochemical levels. 4. 4. Low temperature (8°C) impairs secretory activity, again independently of the photoperiod selected. 5. 5. This data suggests that the secretion of the Harderian gland in Rana esculenta is modulated mainly by temperature. © 1990.

Melatonin has a direct inhibitory effect on the basal and oestradiol-stimulated mitotic activity of primary spermatogonia in the testis of the frog, Rana esculenta. In this study oestradiol was used to induce spermatogonial proliferation to verify the anti-proliferative effect of melatonin. The colchicine metaphase arrest technique was used. The results obtained from in vivo experiments confirm that oestradiol increases the mitotic index of primary spermatogonia and, for the first time, indicate that melatonin has an inhibitory role on the proliferation of primary spermatogonia in the frog testis. Similar results were obtained from testes of melatonin-injected frogs that were exposed to oestradiol in vitro; in fact spermatogonia were unresponsive to hormonal stimulation. In addition, in short-term cultured testes, melatonin (at physiological concentration) interferes with the effects of oestradiol on spermatogonial proliferation, supporting the hypothesis that melatonin exerts the inhibitory effect directly via its local action on the frog gonads. Morphological observation after in vivo or in vitro melatonin treatments indicates that Leydig cells display degenerative features, whereas in adjacent germinal tubules, Sertoli cells show heterochromatic nuclei. These results indicate that melatonin may act on Leydig cells and confirm that there is a paracrine interaction between interstitial and germinal compartments. The results of the present study indicate, for the first time, that melatonin may be directly involved in the inhibitory control of spermatogonial proliferation in the testis of the frog, R. esculenta.

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 (p0.001 for NAT activity; p0.005 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.

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 (P0.001) and warm temperature (P0.001), 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 (P0.005). 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.

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.

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 β-adrenergic stimulation.

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.

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.

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.

Androgen, progesterone and estrogen receptors were analyzed in 12 primary colonic cancers and 16 primary rectal cancers. Androgen and progesterone receptors were positive in some colonic cancers and rectal carcinomas; however, none of the specimens analyzed showed estradiol receptor. Sourcerecord Id Scopus 21088-------http://apps.webofknowledge.com/full_record.do?product=UA&search_mode=GeneralSearch&qid=1&SID=Q1i@gpCm9b3mdEmO@n6&page=6&doc=60------- 25 citazioni Web of Knowledge

The interactions between thyroid hormones and receptors for steroid hormones and prolactin in dorsal skin and caudal fin of Triturus cristatus carnifex were studied during the annual cycle. Thyroidectomy induces an increase of prolactin binding in the dorsal skin and caudal fin in the animals captured in March. In these thyrodectomized animals the androgen receptors became undetectable. Results indicate that in Triturus cristatus carnifex the thyroid induces an increase of androgen receptors and a decrease, that is removed by thyroidectomy, of prolactin receptors.

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. J. Endocr. (1984) 102, 387–392

Because sigma receptors are richly concentrated in the rat pineal gland, the present study was performed to investigate their possible role in the modulation of melatonin production. To this purpose, we assessed in vivo the effects of the sigma-receptor ligands 1,3-di(2-tolyl)guanidine and (+)-N-allylnormetazocine on the rat pineal gland activity during either the daytime or the nighttime. Compared with vehicle, 1,3-di(2-tolyl)guanidine and (+)-N-allylnormetazocine potentiated the enhancement of N-acetyltransferase activity and pineal melatonin content induced by isoproterenol administration during the daytime, whereas they did not affect the diurnal basal biosynthetic activity of the gland. Conversely, at night, 1,3-di(2-tolyl)guanidine and (+)-N-allylnormetazocine enhanced significantly the physiological increases in both pineal N-acetyltransferase activity and melatonin levels. This enhancement was prevented by pretreatment with rimcazole, a specific sigma-receptor antagonist. These findings suggest that, in rats, the activation of pineal sigma-receptor sites does not affect the biosynthetic activity of the pineal gland during daytime, whereas it potentiates the production of melatonin when the gland is noradrenergically stimulated either by isoproterenol administration or by the endogenously released norepinephrine at nighttime.

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 = 5α-dihydrotestosterone); oestradiol-17β showed a 30% cross-reaction; moreover, specific binding of [3H]oestradiol-17β 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. Journal of Endocrinology (1991) 129, 227–232