Could melatonin unbalance the equilibrium between autophagy and invasive processes?

The Syrian hamster Harderian gland (HG) is a juxtaorbital organ exhibiting marked gender-associated morphological differences. Regarding contents of porphyrins, this gland is a good model for studying physiological oxidative stress effects, since both sexes present strong (in females) and moderate (in males) levels of this stress in normal conditions. We have recently showed that autophagic processes are in the Syrian hamster HG as the first result of an elevated porphyrin metabolism observed in both sexes. In this case, autophagy is not a cell death mechanism per se but a constant renovation system which allows to continuing with the normal gland activity. Moreover, we have also reported that this gland presents invasive processes, resembling to tumoral progression, and are, additionally, a consequence of a strong oxidative stress environment that is mainly observed in female Syrian hamster HG and in minor proportion in male HG. Here, we present additional data and discuss a model of melatonin action on these cited processes by which melatonin would be able to destroy the equilibrium between both detoxifying actions. We postulate that melatonin reduces oxidative stress level into HG as direct antioxidant. This decrease of free radicals produces the autophagy inhibition due to outbreak signal disappearance in HG. Under these events and regarding the huge contents of porphyrins that this gland supports, the invasive process triggers.     

Autophagic Cell Death and its Importance for Fungal Developmental Biology and Pathogenesis

The Syrian hamster Harderian gland (HG) is a juxtaorbital organ exhibiting marked gender-associatedmorphological differences. Regarding contents of porphyrins, this gland is a good model for studyingphysiological oxidative stress effects, since both sexes present strong (in females) and moderate (inmales) levels of this stress in normal conditions. We have recently showed that autophagic processes arein the Syrian hamster HG as the first result of an elevated porphyrin metabolism observed in both sexes.In this case, autophagy is not a cell death mechanism per se but a constant renovation system whichallows to continuing with the normal gland activity. Moreover, we have also reported that this glandpresents invasive processes, resembling to tumoral progression, and are, additionally, a consequence of astrong oxidative stress environment that is mainly observed in female Syrian hamster HG and in minorproportion in male HG. Here, we present additional data and discuss a model of melatonin action on thesecited processes by which melatonin would be able to destroy the equilibrium between both detoxifyingactions. We postulate that melatonin reduces oxidative stress level into HG as direct antioxidant. Thisdecrease of free radicals produces the autophagy inhibition due to outbreak signal disappearance in HG.Under these events and regarding the huge contents of porphyrins that this gland supports, the invasiveprocess triggers.     

Autophagic and proteolytic processes in the Harderian gland are modulated during the estrous cycle

The Syrian hamster Harderian gland (HG) is an organ that undergoes physiological autophagy in response to oxidative stress induced by porphyrin production. Porphyrin production in the HG has marked sex differences and is closely linked to reproductive function. In the present study, we observed that the estrous cycle and associated estrogen variations may affect oxidative-stress-induced proteolytic processes. In particular, significant changes in autophagic activity were detected during the estrous cycle. Notably, increased activation of macroautophagy as well as chaperone-mediated autophagy in the estrus phase coincided with a minimal antioxidant capability and the highest protein damage levels. By contrast, autophagic machinery was found to be blocked in the diestrus phase, likely due to mammalian target of rapamycin activation, which could be corroborated by the subsequent pS6K activation. Analogous results were observed regarding proteasome activity, which also showed maximal activity in the estrus phase. Interestingly, all these mechanisms were associated with important morphological changes in the HG during the estrous cycle. We observed statistically significant increases in Type II cells, which may be related to extensive autophagy in the estrus phase. Physiologically, this would result in a significant release of porphyrins specifically when females are more receptive. These data support the role of porphyrins as pheromones, as other authors have previously suggested, thus making the HG a scent organ. In addition, these results suggest a porphyrin-based approach to the treatment of porphyria during pregnancy, a condition for which no treatment is currently known.     

Studies on the local activity of antiandrogens at the molecular and histological level

Androgen dependent skin disorders are important in clinical practice. Effective topical antiandrogens would lead to a breakthrough in their treatment. Although many attempts have been performed to develop such compounds, major successes have not been forthcoming. In the present study three existing antiandrogenic molecules have been compared with regard to their effect on androgen metabolism, receptor competition and on histological parameters in the hamster flank organ test. It appears that the effect on the hamster pigmented spot can be predicted on the basis of molecular mechanism. However, the effects on histological parameters are apparently dependent on additional factors such as metabolism of the active substance before reaching the sebaceous structure or limited penetration through the skin surface. The results indicate that in the development of new antiandrogens pre-screening can be performed with the aid of metabolic and receptor studies, while the histological parameters in the hamster flank organ test provide an animal model with a good predictive value.     

Steroid hormone receptor studies in melanoma model systems

The Transplantable B-16 melanotic melanoma carried in syngeneic C57B1/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrosedensity gradient centrifugation of cytosol after incubation with [³H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5–35 fmoles per mg cytosol protein. Scatchard analysis of [³H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 × 10^?10 M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [³H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [³H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high affinity sites with a dissociation constant of 2 × 10^?9 M. Binding levels from 70–610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.     

Synthesis and biological evaluation of amino-pyridines as androgen receptor antagonists for stimulating hair growth and reducing sebum production

A series of amino-pyridines were synthesized and evaluated for androgen antagonist activities. Among these compounds, (R)-(+)-6-[methyl-(1-phenyl-ethyl)-amino]-4-trifluoromethyl-nicotinonitrile was the most active example of this class. This compound displayed potent androgen receptor antagonist activity as well as favorable pharmacokinetic characteristics for a potential topical agent. It also demonstrated remarkable potency for stimulating hair growth in a male C3H mouse model as well as reducing sebum production in the male Syrian hamster ear model.     

Ovarian hormones alter the behavioral response of the medial preoptic anterior hypothalamus to arginine-vasopressin

In Syrian hamsters (Mesocricetus autatus) arginine-vasopressin (AVP) within the medial preoptic-anterior hypothalamus (MPOA-AH) plays a critical role in the control of a hormone-dependent behavior called flank marking. The present study investigated whether ovarian hormones influence flank marking by altering the response of the MPOA-AH to AVP. The amount of flank marking stimulated by microinjection of AVP (9 μM in 200 nl saline) into the MPOA-AH varied significantly over the 4 days of the estrous cycle with the lowest levels of flank marking observed on estrus. A second experiment demonstrated that administration of progesterone significantly reduced AVP-stimulated flank marking in estradiol-treated ovariectomized hamsters. These data support the hypothesis that the changing levels of estradiol and progesterone during the estrous cycle influence flank marking by altering the sensitivity or response of the MPOA-AH to AVP.     

5 alpha-dihydrotestosterone administration converts indolamine metabolism and porphyrin content of the female Syrian hamster Harderian gland to the male type

The effects of ovariectomy and exogenous androgen administration on the indole and porphyrin metabolism of Syrian hamster Harderian glands were studied. Ovariectomy alone had no effect on any of the parameters analyzed. The administration of either testosterone or 5 alpha-dihydrotestosterone increased the activity of N-acetyltransferase in the Harderian glands. However, androgen treatment failed to change the activity of hydroxyindole-O-methyltransferase. Melatonin content of the glands dropped 20 days after treatment with testosterone and 10 days after the administration of 5 alpha-dihydrotestosterone. The porphyrin content of the Harderian glands was dramatically depressed after the administration of either androgen. It is concluded that the Harderian glands of Syrian hamsters are under an androgenic control involving 5 alpha-dihydrotestosterone.     

Partial maintenance of testes and accessory organs in blinded hamsters by homoplastic anterior pituitary grafts or exogenous prolactin

When blinded, golden Syrian hamsters undergo marked gonadal atrophy. This phenomenon is prevented by pinealectomy. The mechanisms involved in this pineal-mediated response were investigated through either the transplantation of pituitary homografts or treatment of blinded, male hamsters with exogenous prolactin. It was found that anterior pituitary homografts placed beneath the kidney capsule on the day of bilateral optic enucleation partially maintained testicular and accessory organ weights. Serum prolactin levels were reduced in blinded animals below that of intact controls. On the other hand, blinded hamsters bearing anterior pituitary homografts showed serum prolactin levels comparable to those of intact controls. In other experiments, the injection of either 3.2 or 6.4 I.U. of ovine prolactin/hamster/ day for a period up to seven weeks partially inhibited the atrophy of testes and accessory organ weights in blinded hamsters. These data suggest a possible role for prolactin in the pineal-mediated atrophic response to light deprivation.     

Myometrial characteristics of the syrian hamster uterine smooth muscle cell line,SHM

Summary We describe several characteristics of a novel smooth muscle cell line, SHM (Syrian hamster myometrium) derived from a primary uterine leiomyosarcoma which was induced by chronic estrogen plus androgen treatment of a female Syrian (golden) hamster. To determine the usefulness of the SHM cell line as a model for understanding myometrial function and its regulation, we have examined the morphologic and immunocytochemical properties of these cells, and the ability of uterotonic agonists to activate transmembrane signaling via phosphoinositide hydrolysis. The SHM cells exhibited a spindle-shape, smooth musclelike morphology when subconfluent, and a more compact, stellate shape at confluence. Like primary myocytes, SHM cells expressed the intermediate filament desmin and the contractile protein alpha smooth muscle actin, but not the epithelial antigen cytokeratin. Norepinephrine and bradykinin, which stimulate contraction and inositol polyphosphate production in the uterus, also stimulated inositol polyphosphate production in SHM cells. The maximal phosphoinositide signaling responses were lower in SHM cells compared with primary hamster uterine myocytes. We conclude that the SHM cell line exhibits primary uterine myocyte characteristics, and may therefore be a useful system for examining the mechanisms through which myometrial functions are regulated.     

Intracellular partitioning of androgen receptor immunoreactivity in the brain of the male syrian hamster: Effects of castration and steroid replacement

The effect of castration and steroid replacement on the intracellular partitioning of the androgen receptor in the brain of the male Syrian hamster was determined using immunocytochemistry. Androgen receptors were visualized using the PG-21 antibody (G. S. Prins) on 40-μm coronal brain sections from hamsters perfused with 4% paraformaldehyde with or without 0.4% glutaraldehyde. Control studies confirmed antibody specificity in gonad-intact and castrate males. In the normal adult male, androgen receptor immunocytochemistry reveals intense staining confined to the cell nucleus. Castration caused a gradual increase in cytoplasmic labelling within 2 weeks, accompanied by a reduction in nuclear staining intensity in androgen receptor-containing neurons throughout the brain. Cytoplasmic androgen receptor staining was eliminated after treatment of orchidectomized males for only 8 h with exogenous testosterone. Likewise, long-term exposure to testosterone and dihydrotestosterone, a nonaromatizable androgen, maintained nuclear androgen receptor immunoreactivity. However, exposure to low physiologic concentrations of estrogen was not effective in this regard. In addition, we determined that nuclear androgen receptor immunoreactivity decreases in response to inhibitory short-day photoperiod, but without an increase in cytoplasmic immunostaining. This appears to be due to the decrease in androgen production by the testis, rather than a direct photoperiodic effect, because testosterone supplementation to short-day males restored the intensity of nuclear androgen receptor immuno-reactivity to levels comparable to those in the intact male. These findings are compatible with a new model for the intracellular localization of androgen receptors, in which a subset of unoccupied receptors is located in the cell cytoplasm in the absence of ligand. They further demonstrate the repartitioning of such cytoplasmic receptors, thereby confirming and extending previous observations using biochemical techniques on the regulation of neuronal androgen receptors. © 1993 John Wiley & Sons, Inc.     

Effect of hamster pregnancy on female protein, a homolog of serum amyloid P component.

Pentraxins such as human serum amyloid P component (SAP) and C reactive protein (CRP) represent an ancient family of proteins that are ubiquitous in nature and have evolved with little change in structure or regulation. The pentraxin in the Syrian hamster (Mesocricetus auratus) is unique because it is preferentially expressed in the female at high constitutive levels and accordingly called female protein (FP) or FP(SAP) due to its close homology with human SAP. The high levels of FP in female serum (100-fold greater than male serum) suggested its role in hamster pregnancy, one of the shortest of any eutherian mammal. We determined the serum FP concentration in pregnant Syrian hamsters and found a marked decrease (>80%) at term with the nadir at parturition with subsequent increase. A similar downregulation of FP was found in the normal female Syrian hamster after injury (acute phase response), so in both cases the assumed beneficial effects were achieved with less, rather than more pentraxin, a paradoxical pentraxin response. The fall in serum FP concentration could represent a response to protect the fetus from the high and potentially toxic level of FP normally found in the female, that is harmful because of its association with amyloidosis. An FP that is 97.5% identical to Syrian hamster FP is found in the Turkish hamster (Mesocricetus brandti), although serum levels in females are much lower, and amyloid is very rare. During pregnancy/parturition of Turkish hamsters, the serum level of FP remained remarkably constant. In a more distantly related hamster, the Armenian hamster (Cricetulus migratorius), serum FP actually increased during pregnancy and at parturition in a manner similar to that found in the Armenian hamster during an acute phase response. The heterogeneity of FP kinetics during pregnancy in these three species of hamster indicates pleomorphic gene structure for regulation of their similar FPs, and suggests that this protein may have a different function in the pregnancy of each species.     

Comparison of the sequence organization of related retrovirus-like multigene families in three evolutionarily distant rodent genomes.

Sequences related to mouse intracisternal A-particle (IAP) genes have been isolated from rat and Syrian hamster gene libraries as recombinants in lambda phage. The sequences are moderately reiterated in both these genomes but their sequence organization in the hamster genome is different from that in the rat genome. Restriction analysis and electron microscopy indicate that the Syrian hamster IAP sequences represent a family of relatively homogeneous well-conserved units; in this, they resemble the mouse IAP genes. The rat sequences, in contrast, are heterogeneous. Both the hamster and rat IAP sequences contain regions homologous to mouse IAP genes interspersed with regions of apparent non-homology. The interspersed regions range in size from 0.5-1.0 kilobases (Kb). The regions of homology among the mouse, rat and Syrian hamster IAP sequences have been mapped to a 5-6 Kb internal region on the mouse IAP genes. Mouse IAP long terminal repeat (LTR) sequences were not detected in the rat and Syrian hamster genomes. We used the thermal stability of hybrids between cloned and genomic IAP sequences to measure family homogeneity. Mouse and Syrian hamster IAP sequences are homogeneous by this criterion, but the rat IAP sequences are heterogeneous with a Tm 6 degrees C below the self-hybrid. The contrasting organization of IAP-related elements in the genomes of these rodents indicates that amplification or homogenization of this sequence family has occurred independently and at different periods of time during their evolution.     

Facilitation of Male Sexual Behavior in Syrian Hamsters by the Combined Action of Dihydrotestosterone and Testosterone

Background

Testosterone (T) controls male Syrian hamster sexual behavior, however, neither of T''s primary metabolites, dihydrotestosterone (DHT) and estradiol (E₂), even in highly supraphysiological doses, fully restores sexual behavior in castrated hamsters. DHT and T apparently interact with androgen receptors differentially to control male sexual behavior (MSB), but whether these two hormones act synergistically or antagonistically to control MSB has received scant experimental attention and is addressed in the present study.

Methodology/Principal Findings

Sexually experienced male Syrian hamsters were gonadectomized and monitored 5 weeks later to confirm elimination of the ejaculatory reflex (week 0), at which time they received subcutaneous DHT-filled or empty capsules that remained in situ for the duration of the experiment. Daily injections of a physiological dose of 25 µg T or vehicle commenced two weeks after capsule implantation. MSB was tested 2, 4 and 5 weeks after T treatment began. DHT capsules were no more effective than control treatment for long-term restoration of ejaculation. Combined DHT + T treatment, however, restored the ejaculatory reflex more effectively than T alone, as evidenced by more rapid recovery of ejaculatory behavior, shorter ejaculation latencies, and a greater number of ejaculations in 30 minute tests.

Conclusions/Significance

DHT and T administered together restored sexual behavior to pre-castration levels more rapidly than did T alone, whereas DHT and vehicle were largely ineffective. The additive actions of DHT and T on MSB are discussed in relation to different effects of these androgens on androgen receptors in the male hamster brain mating circuit.     

Nitric oxide synthase in mating behavior circuitry of male Syrian hamster brain

Chemosensory and hormonal stimuli are essential for mating in the male Syrian hamster. These signals are processed in a neural circuit that includes the medial amygdaloid nucleus (Me), bed nucleus of the stria terminalis (BNST), and medial preoptic area (MPOA). Nitric oxide is implicated in the regulation of male sexual behavior, and nitric oxide synthase (NOS), the enzyme that catalyzes the production of nitric oxide, is present in the limbic system. In this study, the distribution of NOS-containing neurons in mating behavior circuitry of the male Syrian hamster brain was determined using labeling for brain NOS (bNOS) and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d). bNOS and NADPH-d labeled equivalent populations of neurons. NOS-containing neurons were clustered in specific subnuclei within the Me, BNST, and MPOA. NOS-positive fibers and neurons were seen in the stria terminalis and ventral amygdalofugal pathway, which link the Me with BNST and MPOA. Many NOS-positive neurons in the posterior subdivision of the Me, the medial preoptic nucleus (MPN), and the ventral premammillary nucleus contain androgen receptors. Castration reduced NOS-positive neurons in the MPN, implying a selective regulation of NOS by gonadal steroids. Together, these results suggest that NOS may contribute to the regulation of male sexual behavior by influencing the central neural processing of hormonal and chemosensory signals in the hamster limbic system. © 1996 John Wiley & Sons, Inc.     

Regulation of melanin synthesis in mammalian cells, as studied by somatic hybridization. I. Evidence for negative control

Somatic hybrids between pigmented Syrian hamster cells and unpigmented mouse cells were isolated and propagated in vitro. These hybrids are unpigmented and lack dopa oxidase (and tyrosinase) activity, which is correlated with the pigmentation of the Syrian hamster cells. In contrast, the presence of three other enzymes (LDH, MDH, and thymidine kinase) also specified by the hamster genome but unrelated to pigment synthesis is observed in the hybrid cells. This suggests that the repression of dopa oxidase in these cells is a specific effect on the enzyme associated with the differentiated state of pigment cells. It is concluded that the genetic control of differentiation in this case involves a diffusible regulator substance which functions negatively.