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The Harderian gland (HG) of Rattus norvegicus is an orbital gland secreting lipids that accumulate in excess under condition of increased lipid metabolism. To study the response elicitated by lipid overload in rat HG, we housed the animals in thermoneutral conditions (28-30°C) in association to high fat diet (HFD). In HFD rats alterated blood lipid levels result in lipid accumulation in HG as demonstrated by the increased gland weight and histochemical/ultrastructural analyses. The HFD-caused oxidative stress forces the gland to trigger antioxidant defense mechanisms and autophagic process, such as lipophagy and mitophagy. Induction of mitochondrial DNA (mtDNA) damage and repair was stronger in HFD-rat HGs. An increase in marker expression levels of mitochondrial biogenesis, fission, and fusion occurred to counteract mtDNA copy number reduction and mitophagy. Therefore, the results demonstrate that rat HG activates autophagy as survival strategy under conditions of increased lipid metabolism and suggest a key role for mitophagy and membrane dynamics in the mitochondrial adaptive response to HFD.

High-fat diet (HFD) affects the physiology of reproduction in males, and many studies have investigated its detrimental effects. In this study, we investigated the cellular response induced by an HFD in the rat testis, focusing on the mitochondrial compartment. After five weeks of HFD, an increase in the levels of malondialdehyde and of reduced form of glutathione in the rat testis indicated an increase in lipid peroxidation. The results showed an increase in autophagy, apoptosis, and mitochondrial damage in the testis of HFD rats. We found a decrease in the protein expression of mitochondrial antioxidant enzymes, such as catalase and SOD2. Immunohistochemical analysis revealed a decrease in the immunofluorescent signal of SOD2, mainly in the spermatogonia and spermatocytes of HFD rats. HFD-induced mitochondrial damage caused a reduction in mitochondria, as evidenced by a decrease in the protein expression of TOM20, a mitochondrial outer membrane receptor. Consistently, HFD enhanced the levels of the PINK1 protein, a mitophagy marker, suggesting the removal of damaged mitochondria under these conditions. Induction of mtDNA damage and repair was stronger in the HFD rat testis. Finally, we found a decrease in the mtDNA copy number and expression of the POLG enzyme, which is involved in mtDNA replication. In conclusion, our results showed that autophagy and apoptosis are activated in the testis of HFD rats as a survival strategy to cope with oxidative stress. Furthermore, HFD-induced oxidative stress affects the mitochondria, inducing mtDNA damage and mtDNA copy number reduction. Mitophagy and mtDNA repair mechanisms might represent a mitochondrial adaptive response.

Mitogen activated protein kinase (MAPK) activation and neurogenesis are known to play a role in neuronal survival during hibernation. Herein, we investigate the activity of c-Jun N-terminal kinases (JNK) and Ets like-1 protein (Elk1) kinase involved in cell survival, as well as the expression of proliferating cell nuclear antigen (PCNA), a cell proliferation marker, in the brain of the frog Pelophylax esculentus. The study was conducted on female and male frogs collected during the annual cycle. Our results demonstrated that JNK activity increased during the hibernating phase in relation to the active phase. Interestingly, P-Elk1 levels were positively correlated with P-JNK levels, suggesting that the JNK/Elk1 pathway is pivotal in mediating neuroprotective adaptations that are essential to successful hibernation. On the contrary, we detected higher PCNA expression levels during the active period compared with the hibernating period. A sex dimorphism was observed in the expression levels of P-JNK/P-Elk1 that were specifically higher in males, and in the expression of PCNA reporting higher levels in female brains. Much remains to be learned regarding the regulation of hibernation, however, our findings provide new insights into the role of MAPK and proliferative pathways in hibernation, adding new knowledge concerning the mechanisms activated in the brain of ectothermic species to counteract the damage resulting from extreme temperatures.

D-Aspartate (D-Asp) and its methylated form N-methyl-d-aspartate (NMDA) promote spermatogenesis by stimulating the biosynthesis of sex steroid hormones. d-Asp also induces spermatogonia proliferation directly by activating the ERK/Aurora B pathway. In the present study, a mouse spermatocyte-derived cell line (GC-2) which represents a stage between preleptotene spermatocyte and round spermatids was exposed to 200 μM d-Asp or 50 μM NMDA for 30 min, 2 h, and 4 h to explore the influence of these amino acids on cell proliferation and mitochondrial activities occurring during this process. By Western blotting analyses, the expressions of AMPAR (GluA1-GluA2/3 subunits), cell proliferation as well as mitochondria functionality markers were determined at different incubation times. The results revealed that d-Asp or NMDA stimulate proliferation and meiosis in the GC-2 cells via the AMPAR/ERK/Akt pathway, which led to increased levels of the PCNA, p-H3, and SYCP3 proteins. The effects of d-Asp and NMDA on the mitochondrial functionality of the GC-2 cells strongly suggested an active role of these amino acids in germ cell maturation. In both d-Asp- and NMDA-treated GC-2 cells mitochondrial biogenesis as well as mitochondrial fusion are increased while mitochondria fission is inhibited. Finally, the findings showed that NMDA significantly increased the expressions of the CII, CIII, CIV, and CV complexes of oxidative phosphorylation system (OXPHOS), whereas d-Asp induced a significant increase in the expressions only of the CIV and CV complexes. The present study provides novel insights into the mechanisms underlying the role of d-Asp and NMDA in promoting spermatogenesis.

It is a widely held belief that environmental contaminants contribute to the decline of amphibian populations. By spending most of their early life in water and later stages on the land, amphibians face a constant risk of exposure to pesticides and other chemical pollutants in both aquatic and terrestrial environments. This review presents an overview of the studies carried out in Italian amphibians to highlight hazardous effects of bioaccumulation of chemical pollutants in juveniles and adults in various contaminated environments. Further, the studies in the laboratory setting assessing the effects of chemical pollutants on reproductive and developmental processes are reported. These studies and their relative references have been summarized in a tabular form. Three prominent contaminant groups were identified: herbicides, insecticides, and fungicides; and only a few works reported the effects of other chemical pollutants. Each pollutant group has been delegated to a section. All through the literature survey, it is seen that interest in this topic in Italy is very recent and sparse, where only a few anuran and caudata species and only some chemical pollutants have been studied.

The Harderian gland (HG) is an exocrine gland located within the eye socket in a variety of tetrapods. During the 1980s and 1990s the HG elicited great interest in the scientific community due to its morphological and functional complexity, and from a phylogenetic point of view. A comparative approach has contributed to a better understanding of its physiology. Whereas the chemical nature of its secretions (mucous, serous or lipids) varies between different groups of tetrapods, the lipids represent the more common component among different species. Indeed, besides being an accessory to lubricate the nictitating membrane, the lipids may have a pheromonal function. Porphyrins and melatonin secretion is a feature of the rodent HG. The porphyrins, being phototransducers, could modulate HG melatonin production. The melatonin synthesis suggests an involvement of the HG in the retinal-pineal axis. Finally, StAR protein and steroidogenic enzyme activities in the rat HG suggests that the gland contributes to steroid hormone synthesis. Over the past twenty years, much has become known on the hamster (Mesocricetus auratus) HG, unique among rodents in displaying a remarkable sexual dimorphism concerning the contents of porphyrins and melatonin. Mainly for this reason, the hamster HG has been used as a model to compare, under normal conditions, the physiological oxidative stress between females (strong) and males (moderate). Androgens are responsible for the sexual dimorphism in hamster and they are known to control the HG secretory activity in different species. Furthermore, HG is a target of pituitary, pineal and thyroid hormones. This review offers a comparative panorama of the endocrine activity of the HG as well as the hormonal control of its secretory activity, with a particular emphasis on the sex dimorphic aspects of the hamster HG.

Cell differentiation during spermatogenesis requires a proper actin dynamic, regulated by several proteins, including formins. Disheveled-Associated-Activator of Morphogenesis1 (DAAM1) belongs to the formins and promotes actin polymerization. Our results showed that oral D-Aspartate (D-Asp) administration, an excitatory amino acid, increased DAAM1 protein levels in germ cells cytoplasm of rat testis. Interestingly, after the treatment, DAAM1 also localized in rat spermatogonia (SPG) and mouse GC-1 cells nuclei. We provided bioinformatic evidence that DAAM1 sequence has two predicted NLS, supporting its nuclear localization. The data also suggested a role of D-Asp in promoting DAAM1 shuttling to the nuclear compartment of those proliferative cells. In addition, the proliferative action induced by D-Asp is confirmed by the increased levels of PCNA, a protein expressed in the nucleus of cells in the S phase and p-H3, a histone crucial for chromatin condensation during mitosis and meiosis. In conclusion, we demonstrated, for the first time, an increased DAAM1 protein levels following D-Asp treatment in rat testis and also its localization in the nucleus of rat SPG and in mouse GC-1 cells. Our results suggest an assumed role for this formin as a regulator of actin dynamics in both cytoplasm and nuclei of the germ cells.

Exercise under fasting conditions induces a switch to lipid metabolism, eliciting beneficial metabolic effects. Knowledge of signaling responses underlying metabolic adjustments in such conditions may help to identify therapeutic strategies. Therefore, we studied the effect of mild exercise on rats submitted to food withdrawal at thermoneutrality (28°C) for 3 days. Animals were housed at thermoneutrality rather than the standard housing temperature (22°C) to avoid beta‐adrenergic signaling responses that themselves affect metabolism and well‐being. Quantitative analysis of multi‐organ mRNA levels, myofibers, and serum metabolites shows that this protocol (a) boosts fat oxidation in muscle and liver, (b) reduces lipogenesis and increases gluconeogenesis in liver, (c) increases serum acylcarnitines (especially C4OH) and ketone bodies and the use of the latter as fuel in muscle, (d) increases Type I myofibers, and (e) is associated with an increased thyroid hormone uptake and metabolism in muscle. In addition, stool microbiome DNA analysis revealed that food withdrawal dramatically alters the presence of bacterial genera associated with ketone metabolism. Taken together, this protocol induces a drastic switch toward increased lipid and ketone metabolism compared to exercise or food withdrawal alone, which may prove beneficial and may involve local thyroid hormones, which may be regarded as exercise mimetics., Environmental temperature influences metabolic responses. The results of the present study show that the relatively mild metabolic effects of exercise at thermoneutrality are boosted when combined with food withdrawal. To our knowledge, this is the first study to reveal this, studying the whole animal's response ranging from microbiota to muscle, liver, and serum, suggesting a contributing role for thyroid hormone.

The endogenous amino acids serine and aspartate occur at high concentrations in free D-form in mammalian organs, including the central nervous system and endocrine glands. D-serine (D-Ser) is largely localized in the forebrain structures throughout pre and postnatal life. Pharmacologically, D-Ser plays a functional role by acting as an endogenous coagonist at N-methyl-D-aspartate receptors (NMDARs). Less is known about the role of free D-aspartate (D-Asp) in mammals. Notably, D-Asp has a specific temporal pattern of occurrence. In fact, free D-Asp is abundant during prenatal life and decreases greatly after birth in concomitance with the postnatal onset of D-Asp oxidase expression, which is the only enzyme known to control endogenous levels of this molecule. Conversely, in the endocrine system, D-Asp concentrations enhance after birth during its functional development, thereby suggesting an involvement of the amino acid in the regulation of hormone biosynthesis. The substantial binding affinity for the NMDAR glutamate site has led us to investigate the in vivo implications of D-Asp on NMDAR-mediated responses. Herein we review the physiological function of free D-Asp and of its metabolizing enzyme in regulating the functions of the brain and of the neuroendocrine system based on recent genetic and pharmacological human and animal studies.

D-Aspartate, D-serine and D-alanine are a regular occurrence in mammalian endocrine tissues, though in amounts varying with the type of gland. The pituitary gland, pineal gland, thyroid, adrenal glands and testis contain relatively large amounts of D-aspartate in all species examined. D-alanine is relatively abundant in the pituitary gland and pancreas. High levels of D-serine characterize the hypothalamus. D-leucine, D-proline and D-glutamate are generally low. The current knowledge of physiological roles of D-amino acids in endocrine tissues is far from exhaustive, yet the topic is attracting increasing interest because of its potential in pharmacological application. D-aspartate is known to act at all levels of the hypothalamus-pituitary-testis axis, playing a key role in reproductive biology in several vertebrate classes. An involvement of D-amino acids in the endocrine function of the pancreas is emerging. D-Aspartate has been immunolocalized in insulin-containing secretory granules in INS-1 E clonal β cells and is co-secreted with insulin by exocytosis. Specific immunolocalization of D-alanine in pituitary ACTH-secreting cells and pancreatic β-cells suggests that this amino acid participates in blood glucose regulation in mammals. By modulating insulin secretion, D-serine probably participates in the control of systemic glucose metabolism by modulating insulin secretion. We anticipate that future investigation will significantly increase the functional repertoire of D-amino acids in homeostatic control.

This issue is dedicated to the late Professor Giovanni Chieffi, and this article is an overview of the research on Comparative Endocrinology of reproduction using Rana esculenta (alias Pelophylax esculentus) as a model system. Starting from the early 1970s till today, a large quantity of work have been conducted both in the fields of experimental endocrinology and in the definition of the diffuse neuroendocrine system, with a major focus on the increasing role of regulatory peptides. The various aspects investigated concerned the histological descriptions of principal endocrine glands of the hypothalamic–pituitary–gonadal (HPG) axis, the localization and distribution in the HPG of several different substances (i.e. neurosteroids, hypothalamic peptide hormones, pituitary gonadotropins, gonadal sex steroids, and other molecules), the determination of sex hormone concentrations in both serum and tissues, the hormone manipulations, as well as the gene and protein expression of steroidogenic enzymes and their respective receptors. All together these researches, often conducted considering different periods of the annual reproductive cycle of the green frog, allowed to understand the mechanism of cascade control/regulation of the HPG axis of R. esculenta, characterizing the role of different hormones in the two sexes, and testing the hypotheses about the function of single hormones in different target organs. It becomes evident from the review that, in their simplest form, several features of this species are specular as compared to those of other vertebrate species and that reproduction in this frog species is either under endogenous multi-hormonal control or by a wide array of different factors. Our excursus of this research, spanning almost five decades, shows that R. esculenta has been intensively and successfully used as an animal model in reproductive endocrinology as well as several field studies such as those involving environmental concerns that focus on the effects of endocrine disruptors and other environmental contaminants.

The present investigation was undertaken to increase our insight into the molecular basis of the physiological changes in rat testis induced by food withdrawal, and to clarify whether reduced testicular function can be ameliorated by mild exercise. Male rats were selected for four separate experiments. The first of each group was chow-fed, the second was chow-fed and submitted to exercise (5 bouts in total for 30 min at 15 m/min, and 0° inclination), the third was submitted to food withdrawal (66 h) and the fourth was submitted to food withdrawal and to exercise. At the end of experiments, we investigated (i) serum and testicular sex hormone levels; (ii) protein levels of StAR, 3β-Hydroxysteroid dehydrogenase (3β-HSD) and P450 aromatase, which play a key role in steroid hormone biosynthesis; and (iii) protein levels of mitotic and meiotic markers of spermatogenesis in rats, in relation to testis morphology and morphometry. We found that mild exercise or food withdrawal alone induced a significant increase or decrease in both serum and testis testosterone levels, respectively. Interestingly, we found that these levels were brought back to basal levels when food withdrawal was combined with mild exercise. The changes in testosterone levels observed in our experimental groups correlated well with the expression of steroidogenic enzymes as well as with spermatogenic activity. With mild exercise the increased testosterone/17β-estradiol (T/E2) ratio in the testis correlated with an increased spermatogenic activity. The T/E2 ratio dropped in fasted rats and was significantly reversed when food withdrawal was combined with exercise. Histological and morphometric analyses confirmed that spermatogenic activity varied in concomitance with each experimental condition. Importantly, the testis and serum T/E2 ratios correlated, confirming that exercise rescues the decline in food withdrawal-induced spermatogenesis. In conclusion, this study highlights that mild exercise normalizes the reduced spermatogenic activity caused by food withdrawal through the modulation of the steroidogenic pathway and restoring the T/E2 ratio, underlining the beneficial effects of mild exercise on the prevention and/or amelioration of reduced testis function caused by restricted caloric intake.

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a protein belonging to the formin family, which regulates, together with the small GTPase RhoA, the nucleation and the assembly of actin fibres through Wnt-Dishevelled PCP pathway. Its role has been investigated in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. In this work, we studied the expression of DAAM1 mRNA and protein by PCR and Western blot analyses and its co-localization with actin in adult mouse seminal vesicles by immunofluorescence. We show that both proteins are cytoplasmic: actin is evident at cell–cell junctions and at cell cortex; DAAM1 had a more diffused localization, but is also prominent at the apical plasmatic membrane of epithelial cells. These findings support our hypothesis of a role of DAAM1 in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular concerning actin filaments. We were also able to detect DAAM1 and actin association in the smooth muscle cells that surround the epithelium too. In this case, we could only speculate the possible involvement of this formin in muscular cells in the maintenance and the regulation of the contractile structures. The present results strongly suggest that DAAM1 could have a pivotal role in vesicle exocytosis and in the physiology of mouse seminal vesicles.

D-Aspartate (D-Asp) is an endogenous amino acid present in the central nervous system and endocrine glands of various animal taxa. D-Asp is implicated in neurotransmission, physiology of learning, and memory processes. In gonads, it plays a crucial role in sex hormone synthesis. We have investigated the effects of chronic (30 days D-Asp drinking solution) and acute (i.p. injection of 2 mmol/g bw D-Asp) treatments on sex steroid synthesis in rat brain. Furthermore, to verify the direct effect of D-Asp on neurosteroidogenic enzyme activities, brain homogenates were incubated with different substrates (cholesterol, progesterone, or testosterone) with or without the addition of D-Asp. Enzyme activities were measured by evaluating the in vitro conversion rate of (i) cholesterol to progesterone, testosterone, and 17b-estradiol, (ii) progesterone to testosterone and 17bestradiol, (iii) testosterone to 17b-estradiol. We found that D-Asp oral administration produced an increase of approximately 40% in progesterone, 110% in testosterone, and 35% in 17b-estradiol. Similarly, the results of the acute experiment showed that at 30 min after D-Asp treatment, the progesterone, testosterone, and 17b-estradiol levels increased by 29–35%, and at 8 h they further increased by a 100% increment. In vitro experiments demonstrate that the addition of D-Asp to brain homogenate + substrate induces a significant increase in progesterone, testosterone and 17b-estradiol suggesting that the amino acid upregulates the local activity of steroidogenic enzymes. D-Aspartate (D-Asp) is an endogenous amino acid present in the central nervous system and endocrine glands of various animal taxa. D-Asp is implicated in neurotransmission, physiology of learning, and memory processes. In gonads, it plays a crucial role in sex hormone synthesis. We have investigated the effects of chronic (30 days D-Asp drinking solution) and acute (i.p. injection of 2 μmol/g bw D-Asp) treatments on sex steroid synthesis in rat brain. Furthermore, to verify the direct effect of D-Asp on neurosteroidogenic enzyme activities, brain homogenates were incubated with different substrates (cholesterol, progesterone, or testosterone) with or without the addition of D-Asp. Enzyme activities were measured by evaluating the in vitro conversion rate of (i) cholesterol to progesterone, testosterone, and 17β-estradiol, (ii) progesterone to testosterone and 17β-estradiol, (iii) testosterone to 17β-estradiol. We found that D-Asp oral administration produced an increase of approximately 40% in progesterone, 110% in testosterone, and 35% in 17β-estradiol. Similarly, the results of the acute experiment showed that at 30 min after D-Asp treatment, the progesterone, testosterone, and 17β-estradiol levels increased by 29–35%, and at 8 h they further increased by a 100% increment. In vitro experiments demonstrate that the addition of D-Asp to brain homogenate + substrate induces a significant increase in progesterone, testosterone and 17β-estradiol suggesting that the amino acid upregulates the local activity of steroidogenic enzymes.

Steroidogenic Acute Regulatory protein (StAR), 3b-hydroxysteroid dehydrogenase (3b- HSD), 17b-hydroxysteroid dehydrogenase (17b-HSD), 5a-Reductase (5a-Red), P450 aromatase are key enzymes involved in steroidogenesis. Recently, we showed the expression and the localization of P450 aromatase in Podarcis sicula testis during the different phases of the reproductive cycle, showing its involvement in the control of steroidogenesis, particularly in 17b-estradiol synthesis. Now, we have investigated the presence and distribution of the other enzymes involved in steroidogenesis, i.e. StAR, 3b- HSD, 17b-HSD and 5a-Red, during three significant periods of the reproductive cycle: summer stasis (July–August), autumnal resumption (November) and reproductive period (May–June). We demonstrated for the first time that all these enzymes are always present in somatic cells (Leydig and Sertoli) and germ cells (spermatogonia, spermatocytes I and II, spermatids and spermatozoa) of Podarcis testis, mainly in spermatids and spermatozoa. The present results strongly suggest that in Podarcis testis both somatic and germ cells could be involved in local sex hormone synthesis and that 5a-Red and P450 could carry out a pivot role. Steroidogenic Acute Regulatory protein (StAR), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), 5 alpha-Reductase (5 alpha-Red), P450 aromatase are key enzymes involved in steroidogenesis. Recently, we showed the expression and the localization of P450 aromatase in Podarcis sicula testis during the different phases of the reproductive cycle, showing its involvement in the control of steroidogenesis, particularly in 17 beta-estradiol synthesis. Now, we have investigated the presence and distribution of the other enzymes involved in steroidogenesis, i.e. StAR, 3 beta-HSD, 17 beta-HSD and alpha-Red, during three significant periods of the reproductive cycle: summer stasis (July-August), autumnal resumption (November) and reproductive period (May- June). We demonstrated for the first time that all these enzymes are always present in somatic cells (Leydig and Sertoli) and germ cells (spermatogonia, spermatocytes I and II, spermatids and spermatozoa) of Podarcis testis, mainly in spermatids and spermatozoa. The present results strongly suggest that in Podarcis testis both somatic and germ cells could be involved in local sex hormone synthesis and that 5a-Red and P450 could carry out a pivot role. (C) 2017 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Prolyl endopeptidase (PREP) is a post-proline cleaving enzyme. It is involved in the regulation of multiple inositol polyphosphate phosphatase activity implicated in the pathway of inositol 1,4,5 trisphosphate, resulting in the modulation of cytosolic Ca2+ levels. Besides its peptidase activity, PREP was identified as binding partner of tubulin, suggesting that it may participate in microtubule-associate processes. In this paper, we evaluated the expression of PREP mRNA and protein by PCR and Western blot analyses and its colocalization with tubulin by immunofluorescence in adult mouse seminal vesicles. We show that both proteins are cytoplasmic: tubulin is localized at the apical half part of the cell; while PREP has a more diffuse localization, showing a prominent distribution at the apical cytoplasm. These findings support our hypothesis of a specific role of PREP in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular its association with tubulin filaments. Moreover, it may regulate the Ca2+ levels, which promotes the final step of the vesicular exocytosis, namely the fusion of the vesicles with the plasma membrane. These results strongly suggest for a pivotal role of PREP in vesicle exocytosis as well as in the physiology of mouse seminal vesicles. Summary Prolyl endopeptidase (PREP) is a post-proline cleaving enzyme. It is involved in the regulation of multiple inositol polyphosphate phosphatase activity implicated in the pathway of inositol 1,4,5-trisphosphate, resulting in the modulation of cytosolic Ca2+ levels. Besides its peptidase activity, PREP was identified as a binding partner of tubulin, suggesting that it may participate in microtubule-associate processes. In this paper, we evaluated the expression of PREP mRNA and protein by polymerase chain reaction and western blot analyses and its co-localization with tubulin by immunofluorescence in adult mouse seminal vesicles. We showed that both proteins are cytoplasmic: tubulin is localized at the apical half part of the cell, while PREP has a more diffuse localization, showing a prominent distribution at the apical cytoplasm. These findings support our hypothesis of a specific role for PREP in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular its association with tubulin filaments. Moreover, it may regulate Ca2+ levels, and promote the final step of vesicular exocytosis, namely the fusion of the vesicles with the plasma membrane. These results strongly suggest that there is a pivotal role for PREP in vesicle exocytosis, as well as in the physiology of mouse seminal vesicles.

D-Aspartate (D-Asp) is an endogenous amino acid that plays a central role in the development of the central nervous system (CNS) and functioning of the neuroendocrine system. In line with its functions, it is abundantly present in the CNS and reproductive systems of vertebrates and invertebrates. It has been implicated in the biosynthesis and/or secretion of hormones and factors that are involved in various reproductive functions, such as GnRH from the hypothalamus and testosterone from the testis. We conducted an in vivo study consisting of acute (i.p. injection of 2 μmol/g body weight) and chronic (15 days drinking solution) administration of D-Asp to adult rats to understand the signaling pathways elicited by D-Asp in the rat testis. We found that D-Asp upregulated the expression of prolyl endopeptidase (PREP), a serine protease having a pivotal role in the regulation of mammalian spermatogenesis and spermiogenesis. Immunofluorescence analysis revealed its overexpression in Leydig cells, Sertoli cells and spermatogonia. Moreover, PREP was found to co-localize with GluA2/3, an AMPA receptor subunit, whose protein expression also increased after D-Asp treatments. Finally, we found a significant increase in ERK and Akt activities in the testis of rats treated with D-Asp. Since PREP is known to be involved in regulating GnRH levels and in germ cell differentiation, we hypothesize D-Asp to play a pivotal role in regulating hormone homeostasis and spermatogenesis through activation of PREP, AMPAR, ERK and Akt. D-Aspartate (D-Asp) is an endogenous amino acid that plays a central role in the development of the central nervous system (CNS) and functioning of the neuroendocrine system. In line with its functions, it is abundantly present in the CNS and reproductive systems of vertebrates and invertebrates. It has been implicated in the biosynthesis and/or secretion of hormones and factors that are involved in various reproductive functions, such as GnRH from the hypothalamus and testosterone from the testis. We conducted an in vivo study consisting of acute (i.p. injection of 2 µmol/g body weight) and chronic (15 days drinking solution) administration of D-Asp to adult rats to understand the signaling pathways elicited by D-Asp in the rat testis. We found that D-Asp upregulated the expression of prolyl endopeptidase (PREP), a serine protease having a pivotal role in the regulation of mammalian spermatogenesis and spermiogenesis. Immunofluorescence analysis revealed its overexpression in Leydig cells, Sertoli cells and spermatogonia. Moreover, PREP was found to co-localize with GluA2/3, an AMPA receptor subunit, whose protein expression also increased after D-Asp treatments. Finally, we found a significant increase in ERK and Akt activities in the testis of rats treated with D-Asp. Since PREP is known to be involved in regulating GnRH levels and in germ cell differentiation, we hypothesize D-Asp to play a pivotal role in regulating hormone homeostasis and spermatogenesis through activation of PREP, AMPAR, ERK and Akt.

The purpose of the present study is to highlight the role of aromatase in the neuroendocrine control of the reproductive cycle of the male lizard Podarcis sicula during the three significant phases, i.e. the pre-reproductive, reproductive, and post-reproductive stages. Using immunohistochemical, biochemical, and hormonal tools, we have determined the localization and the activity of P450 aromatase (P450 aro) in the lizard's brain together with the determination of hormonal profile of sex steroids, i.e. testosterone and 1713-estradiol. The present data demonstrated that the localization of P450 is shown in brain regions involved in the regulation of the reproductive behavior (medial septum, preoptic area, and hypothalamus). Its activity, as well as the intensity of the signal, is modified according to the period of reproduction, resulting in functional dynamic changes. P450 aro activity and signal intensity decrease in the pre-reproductive period and progressively increase during the reproductive stage until it reaches the maximum peak level at the post-reproductive phase. P450 aro determines a local estrogen synthesis, balancing the testosterone and estradiol levels, and therefore its role is crucial, since it plays an important role in the neuroendocrine/behavioral regulation of the reproductive processes in the male lizard P. sicula. (C) 2019 Published by Elsevier Masson SAS on behalf of Academie des sciences.

The Harderian gland (HG) of the rat (Rattus norvegicus) secretes copious amounts of lipids, such as cholesterol. Here we report a study of the expressions of the StAR protein and key steroidogenic enzymes in the HG of male and female rats. The objective of the present investigation was to ascertain (a) whether the rat HG is involved in steroid production starting with cholesterol, and (b) whether the pattern of gene and protein expressions together with the enzymatic activities display sexual dimorphism. The results demonstrate, for the first time, the expression of StAR gene and protein, and Cyp11a1, Hsd3b1, Hsd17b3, Srd5a1, Srd5a2 and Cyp19a1 genes in the rat HG. StAR mRNA and protein expressions were much greater in males than in females. Immunohistochemical analysis demonstrated a non-homogeneous StAR distribution among glandular cells. Hsd17b3 and Cyp19a1 mRNA levels were higher in males than in females, whereas Srd5a1 mRNA levels were higher in females than in males. No significant differences were observed in mRNA levels of Cyp11a1, Hsd3b1 and Srd5a2 between sexes. Furthermore, the in vitro experiments demonstrated a higher 5 alpha-reductase activity in the female as compared to the male HG vice versa a higher P450 aro activity in males as compared to females. These results suggest that the Harderian gland can be classified as a steroidogenic tissue because it synthesizes cholesterol, expresses StAR and steroidogenic enzymes involved in both androgen and estrogen synthesis. The dimorphic expression and activity of the steroidogenic enzymes may suggest sex-specific hormonal effects into the HG physiology. (C) 2018 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Relaxin (RLN) and insulin (INSL)-like peptides are member of the INSL/RLN superfamily, which are encoded by seven genes in humans and can activate the G-protein coupled receptor RXFP 1–4. These peptides evolved from a common ancestor, RLN3-like gene. Two rounds of whole genome duplication (WGD) in early vertebrate evolution, together with an additional WGD in the teleost lineage, caused an expansion of RLN genes set in the genome of Danio rerio. In particular, six RLN genes are present: a single copy of rln and insl3 genes, and two paralogs for the rln3 gene (rln3a and rln3b), and the insl5 gene (insl5a and insl5b). We have already reported the presence of rln3a and rln3b genes in the developing zebrafish brain, as well as the expression of rln gene in the developing zebrafish brain and extraneural territories, such as thyroid gland and pancreas. Here, we report for the first time the expression of the two parologs genes for insl5, insl5a, and insl5b in D. rerio embryonic development. The corresponding transcripts of both the paralogs are present in all embryonic stages analyzed by RT-qPCR. In situ hybridization analyses showed a restricted signal in intestinal cells and the pancreatic region at 72 hpf for insl5a, while at 96 hpf both genes are expressed in specific intestinal cells. Furthermore, in adult zebrafish intestine tissue, in situ hybridation experiments showed that insl5a transcript is specifically localized in the goblet cells, while insl5b transcript is in enteroendocrine cells. These data revealed a high degree of gene expression pattern conservation for such genes in vertebrate evolution.

The brain of amphibians contains all the key enzymes of steroidogenesis and has a high steroidogenic activity. In seasonally-breeding amphibian species brain steroid levels fluctuate synchronously with the reproductive cycle. Here we report a study of gene expression of StAR protein, key steroidogenic enzymes and sex hormone receptors in the telencephalon (T) and diencephalon-mesencephalon (D-M) of male and female reproductive and post-reproductive Pelophylax esculentus, a seasonally breeding anuran amphibian. Significant differences in gene expression were observed between (a) the reproductive and post-reproductive phase, (b) the two brain regions and (c) male and female frogs. During the reproductive phase, star gene expression increased in the male (both T and D-M) but not in the female brain. Seasonal fluctuations in expression levels of hsd3b1, hsd17b1, srd5a1 and cypl9a1 genes for neurosteroidogenic enzymes occurred in D-M region of both sexes, with the higher levels in reproductive period. Moreover, the D-M region generally showed higher levels of gene expression than the T region in both sexes. Gene expression was higher in females than males for most genes, suggesting higher neurosteroid production in female brain. Seasonal and sex-linked changes were also observed in gene expression for androgen (ar) and estrogen (esr1, esr2) receptors, with the males showing the highest ar levels in reproductive phase and the highest esri and esr2 levels in post-reproductive phase; in contrast, females showed the maximum expression for all three genes in reproductive phase. The results are the first evidence for seasonal changes and sexual dimorphism of gene expression of the neurosteroidogenic pathway in amphibians. (C) 2016 Elsevier Inc. All rights reserved.

This article reviews the animal models and experimental designs that have been used during the past twenty years to demonstrate the prominent role played by d-aspartate (d-Asp) in the reproduction of vertebrates, from amphibians to humans. We have tabulated the findings of in vivo and in vitro experiments that demonstrate the effects of d-Asp uptake on hormone production and gametogenesis in vertebrate animal models. The contribution of each animal model to the existing knowledge on the role of d-Asp in reproductive processes has been discussed. A critical analysis of experimental designs has also been carried out. Experiments performed on wild animal species suggest a role of d-Asp in the mechanisms that regulate the reproductive cycle. Several in vivo and in vitro studies carried out on mouse and rat models have facilitated an understanding of the molecular pathways activated by D-Asp in both steroidogenesis and spermatogenesis, with particular emphasis on testosterone biosynthesis. Some attempts using d-Asp for the improvement of reproductive activity in animals of commercial interest have yielded mixed results. The increased transcriptome activity of enzymes and receptors involved in the reproductive activity in d-Asp-treated broiler roosters revealed further details on the mechanism of action of d-Asp on the reproductive processes. The close relationship between d-Asp and reproductive activity has emerged, particularly in relation to its effects exerted on semen quality, proposing therapeutic applications of this amino acid in andrology and in medically-assisted procreation techniques. This article reviews the animal models and experimental designs that have been used during the past twenty years to demonstrate the prominent role played by D-aspartate (D-Asp) in the reproduction of vertebrates, from amphibians to humans. We have tabulated the findings of in vivo and in vitro experiments that demonstrate the effects of D-Asp uptake on hormone production and gametogenesis in vertebrate animal models. The contribution of each animal model to the existing knowledge on the role of D-Asp in reproductive processes has been discussed. A critical analysis of experimental designs has also been carried out. Experiments performed on wild animal species suggest a role of D-Asp in the mechanisms that regulate the reproductive cycle. Several in vivo and in vitro studies carried out on mouse and rat models have facilitated an understanding of the molecular pathways activated by D-Asp in both steroidogenesis and spermatogenesis, with particular emphasis on testosterone biosynthesis. Some attempts using D-Asp for the improvement of reproductive activity in animals of commercial interest have yielded mixed results. The increased transcriptome activity of enzymes and receptors involved in the reproductive activity in D-Asptreated broiler roosters revealed further details on the mechanism of action of D-Asp on the reproductive processes. The close relationship between D-Asp and reproductive activity has emerged, particularly in relation to its effects exerted on semen quality, proposing therapeutic applications of this amino acid in andrology and in medically-assisted procreation techniques.

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 Trβ (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.

A bulk of evidence suggests that D-aspartate (D-Asp) regulates steroidogenesis and spermatogenesis in vertebrate testes. This review article focuses on intracellular signaling mechanisms elicited by D-Asp possibly via binding to the N-methyl-D-aspartate receptor (NMDAR) in both Leydig cells, and spermatogonia. In Leydig cells, the amino acid upregulates androgen production by eliciting the adenylate cyclase-cAMP and/or mitogen-activated protein kinase (MAPK) pathways. D-Asp treatment enhances gene and protein expression of enzymes involved in the steroidogenic cascade. D-Asp also directly affects spermatogonial mitotic activity. In spermatogonial GC-1 cells, D-Asp induces phosphorylation of MAPK and AKT serine-threonine kinase proteins, and stimulates expression of proliferating cell nuclear antigen (PCNA) and aurora kinase B (AURKB). Further stimulation of spermatogonial GC-1 cell proliferation might come from estradiol/estrogen receptor beta (ESR2) interaction. D-Asp modulates androgen and estrogen levels as well as the expression of their receptors in the rat epididymis by acting on mRNA levels of Srd5a1 and Cyp19a1 enzymes, hence suggesting involvement in spermatozoa maturation. A bulk of evidence suggests that d-aspartate (d-Asp) regulates steroidogenesis and spermatogenesis in vertebrate testes. This review article focuses on intracellular signaling mechanisms elicited by d-Asp possibly via binding to the N-methyl-d-aspartate receptor (NMDAR) in both Leydig cells, and spermatogonia. In Leydig cells, the amino acid upregulates androgen production by eliciting the adenylate cyclase-cAMP and/or mitogen-activated protein kinase (MAPK) pathways. d-Asp treatment enhances gene and protein expression of enzymes involved in the steroidogenic cascade. d-Asp also directly affects spermatogonial mitotic activity. In spermatogonial GC-1 cells, d-Asp induces phosphorylation of MAPK and AKT serine-threonine kinase proteins, and stimulates expression of proliferating cell nuclear antigen (PCNA) and aurora kinase B (AURKB). Further stimulation of spermatogonial GC-1 cell proliferation might come from estradiol/estrogen receptor β (ESR2) interaction. d-Asp modulates androgen and estrogen levels as well as the expression of their receptors in the rat epididymis by acting on mRNA levels of Srd5a1 and Cyp19a1 enzymes, hence suggesting involvement in spermatozoa maturation.

D-aspartate (D-Asp) is an endogenous amino acid present in vertebrate tissues, with particularly high levels in the testis. In vivo studies indicate that D-Asp indirectly stimulates spermatogenesis through the hypothalamic-pituitary-gonadal axis. Moreover, in vitro studies have demonstrated that D-Asp up-regulates testosterone production in Leydig cells by enhancing expression of the steroidogenic acute regulatory protein. In this study, a cell line derived from immortalized type-B mouse spermatogonia retaining markers of mitotic germ cells (GC-1) was employed to explore more direct involvement of D-Asp in spermatogenesis. Activity and protein expression of markers of cell proliferation were determined at intervals during incubation in D-Asp-containing medium. D-Asp induced phosphorylation of ERK and Akt proteins, stimulated expression of PCNA and Aurora B, and enhanced mRNA synthesis and protein expression of P450 aromatase and protein expression of Estrogen Receptor β (ERβ). These results are the first demonstration of a direct effect of D-Asp on spermatogonial mitotic activity. Considering that spermatogonia express the NR1 subunit of the N-Methyl-D-Aspartic Acid receptor (NMDAR), we suggest that their response to D-Asp depends on NMDAR-mediated activation of the ERK and Akt pathways and is further enhanced by activation of the P450 aromatase/ERβ pathway.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide that in mammalian testis is involved in the control of testosterone and 17β-estradiol synthesis. A similar involvement was recently postulated in the testis of a nonmammalian vertebrate, the wall lizard Podarcis sicula. Indeed, we reported the presence of PACAP and its receptors throughout the reproductive cycle within both germ and somatic cells. Now, we investigated the effects of PACAP on steroidogenesis in significant periods of Podarcis reproductive cycle:winter stasis, reproductive period and summer stasis. Using different in vitro treatments, in the absence or presence of receptor antagonists, we demonstrated that in P. sicula testis PACAP is involved in the control of testosterone and 17β- estradiol production. In particular we demonstrated that treatment with PACAP induced a testosterone increase only in stasis periods (winter and summer stasis); differently they induced a 17β-estradiol production in all periods analyzed (summer stasis, winter stasis and reproductive period). Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide that in mammalian testis is involved in the control of testosterone and 17 beta-estradiol synthesis. A similar involvement was recently postulated in the testis of a nonmammalian vertebrate, the wall lizard Podarcis sicula. Indeed, we reported the presence of PACAP and its receptors throughout the reproductive cycle within both germ and somatic cells. Now, we investigated the effects of PACAP on steroidogenesis in significant periods of Podarcis reproductive cycle: winter stasis, reproductive period and summer stasis. Using different in vitro treatments, in the absence or presence of receptor antagonists, we demonstrated that in P. sicula testis PACAP is involved in the control of testosterone and 17 beta-estradiol production. In particular we demonstrated that treatment with PACAP induced a testosterone increase only in stasis periods (winter and summer stasis); differently they induced a 17 beta-estradiol production in all periods analyzed (summer stasis, winter stasis and reproductive period). (c) 2015 Elsevier Inc All rights reserved.

Although D-aspartate (D-Asp) has been recognized to have a physiological role within different organs, high concentrations could elicit detrimental effects on those same organs. In this study, we examined the D-aspartate oxidase (D-AspO) activity and the expression of superoxide dismutase 1 (SOD1) and caspase 3 in different tissues of the frog Rana esculenta after chronic D-Asp treatment. Our in vivo experiments, consisting of intraperitoneal (ip) injections of D-Asp (2.0 μmol/g b.w.) in frogs for ten consecutive days, revealed that all examined tissues can take up and accumulate D-Asp. Further, in D-Asp treated frogs, i) the D-AspO activity significantly increased in all tissues (kidney, heart, testis, liver, and brain), ii) the SOD1 expression (antioxidant enzyme) significantly increased in the kidney, and iii) the caspase 3 level (indicative of apoptosis) increased in both brain and heart. Particularly, after the D-Asp treatment we found in both brain and heart (which showed the lowest SOD1 levels) a significant increase of the caspase 3 expression and, vice versa, in the kidney (which showed the highest SOD1 expression) a significant decrease of the caspase 3 expression. Therefore, we speculate that, in frog tissue, D-AspO plays an essential role in modulating the D-Asp concentration. In addition, exaggerated D-Asp concentrations activated SOD1 as cytoprotective mechanism in the kidney, whereas, in the brain and in the heart, where the antioxidant action of SOD1 is limited, caspase 3 was activated. © 2010 Verlag Helvetica Chimica Acta AG.

Radioligand binding of D-[(3)H]aspartic and L-[(3)H]glutamic acids to plasma membranes from rat Harderian gland was evaluated. Binding was optimal under physiological conditions of pH and temperature, and equilibrium was reached within 50 min. Specific binding for D-Asp and L-Glu was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (for D-Asp K(d) = 860 +/- 28 nM, B(max) = 27.2 +/- 0.5 pmol/mg protein; for L-Glu, K(d) = 580 +/- 15 nM and B(max) = 51.3 +/- 0.8 pmol/mg protein). L-[(3)H]glutamate had higher affinity and a greater percentage of specific binding than did D-[(3)H]aspartate. The pharmacological binding specificity of L-[(3)H]glutamate indicated an interaction with NMDA-type receptors. Specifically, the order of potency of the displacing compound tested was L-Glu > D-Asp > NMDA > MK801 > D-AP5 > glycine. For D-[(3)H]aspartate, the data revealed an interaction of D: -Asp with either NMDA-type receptors or putative specific binding sites.

Free D-aspartate (D-Asp) occurs in substantial amounts in glandular tissues. This paper reviews the existing work on D-Asp in vertebrate exocrine and endocrine glands, with emphasis on functional roles. Endogenous D-Asp was detected in salivary glands. High D-Asp levels in the parotid gland during development suggest an involvement of the amino acid in the regulation of early developmental phases and/or differentiation processes. D-Asp has a prominent role in the Harderian gland, where it elicits exocrine secretion through activation of the ERK1/2 pathway. Interestingly, the increase in NOS activity associated with D-Asp administration in the Harderian gland suggests a potential capability of D-Asp to induce vasodilatation. In mammals, an increase in local concentrations of D-Asp facilitates the secretion of anterior pituitary hormones, i.e., PRL, LH and GH, whereas it inhibits the secretion of POMC/α-MSH from the intermediate pituitary and of oxytocin from the posterior pituitary. D-Asp also acts as a negative regulator for melatonin synthesis in the pineal gland. Further, D-Asp can stereo-specifically modulate the production of sex steroids, thus taking part in the endocrine control of reproductive activity. Although D-Asp receptors remain to be characterized, gene expression of NR1 and NR2 subunits of NMDAr responds to D-Asp in the testis. © 2014 Springer-Verlag.

Previous studies have demonstrated that d-aspartic acid (d-Asp) has a role in regulating the release and synthesis of testosterone in rats. In this study, we investigated the molecular pathway by which this amino acid triggers its action in the rat testis. We found expression of N-Methyl-D-Aspartic Acid (NMDA) receptor messenger RNAs for NR1, NR2A, and NR2D receptor subunits. After d-Asp administration, NR1 and NR2A messenger RNA levels were significantly higher than those of controls, whereas NR2D levels remained unchanged. Expression of extracellular signal-regulated kinase (ERK) 1 protein was higher than that of ERK2 protein in the testis of both d-Asp-treated rats and controls. d-Asp administration increased testis levels of both phosphorylated ERK (P-ERK) 1 and 2. Using immunohistochemical technique, NR1 and P-ERK 1 or 2 proteins were preferentially localized within the spermatogonia. Moreover, d-Asp administration increased both serum and testis testosterone levels but not estradiol levels. Finally, in d-Asp-treated rats, testicular androgen receptor protein levels were significantly increased, whereas both estrogen receptor α and P-450 aromatase levels were significantly decreased. Conclusively, our results, besides strengthening the evidence that d-Asp administration in rats induces testosterone synthesis, demonstrate for the first time that d-Asp (1) induces testicular NMDA receptor-ERK pathway, (2) upregulates androgen receptor expression, and (3) downregulates estrogen receptor expression. © 2014 Elsevier Inc.

Testosterone (T) synthesised in Leydig cells enters the epididymis and may there be converted into dihydrotestosterone (DHT) by 5α-reductase (5α-red) or into 17β-oestradiol (E2) by P450 aromatase (P450-aro). D-aspartate (D-Asp) is known to induce T synthesis in the testis. In this study, we investigated the effects of in vivo D-Asp administration in two major regions of the rat epididymis (Region I: initial segment, caput, corpus; Region II: cauda). The results suggest that exogenous D-Asp was taken up by both regions of rat epididymis. D-Asp administration induced a rapid increase in T, followed by a more gradual decrease in the T : DHT ratio in Region I. In Region II, T levels rapidly decreased and the T : DHT ratio was consistently lower relative to the control. Expression of 5α-red and androgen receptor genes showed a good correlation with DHT levels in both regions. D-Asp treatment also induced an increase of both E2 levels and oestradiol receptor-α (ERα) expression in Region I, whereas neither E2 levels nor ERα expression were affected in Region II. The early increase of P450-aro expression in Region I and late increase in Region II suggests a direct involvement of D-Asp modulation in P450-aro gene expression. Our results suggest that D-Asp modulates androgen and oestrogen levels and expression of androgen and oestrogen receptors in the rat epididymis by acting on the expression of 5α-red and P450-aro genes.

D-Aspartic acid is an endogenous amino acid occurring in the endocrine glands as well as in the nervous system of various animal phyla. Our previous studies have provided evidence that D-aspartate plays a role in the induction of estradiol synthesis in gonads. Recently, we have also demonstrated that D-aspartic acid induces P450 aromatase mRNA expression in the frog (Pelophylax esculentus) testis. P450 aromatase is the key enzyme in the estrogen synthetic pathway and irreversibly converts testosterone into 17β-estradiol. In this study, we firstly investigated the immunolocalisation of P450 aromatase in the brain of P. esculentus, which has never previously been described in amphibians. Therefore, to test the hypothesis that D-aspartate mediates a local synthesis of P450 aromatase in the frog brain, we administered D-aspartate in vivo to male frogs and then assessed brain aromatase expression, sex hormone levels and sex hormone receptor expression. We found that D-aspartate enhances brain aromatase expression (mRNA and protein) through the CREB pathway. Then, P450 aromatase induces 17β-estradiol production from testosterone, with a consequent increase of its receptor. Therefore, the regulation of D-aspartate-mediated P450 aromatase expression could be an important step in the control of neuroendocrine regulation of the reproductive axis. Accordingly, we found that the sites of P450 aromatase immunoreactivity in the frog brain correspond to the areas known to be involved in neurosteroid synthesis. © 2012. Published by The Company of Biologists Ltd.

There is evidence that d -aspartate ( d -Asp) modulates sex hormone levels in frog testis by regulating the activity of P450 aromatase (P450 aro), the key enzyme which converts Testosterone (T) in 17s-Estradiol (E2). Here we report, for the first time, that there is a direct correlation among brain levels of d -Asp, P450 aro, E2 and Estradiol Receptor (ERα) in the male frogs during the reproductive as well as the post-reproductive phases of the breeding cycle, with highest levels being observed in the post-reproductive period. d -Asp i.p. administration to frogs ready for reproduction, induced an increase of brain P450 aro protein expression with concomitant enhancement of both E2 levels and ERα expression; at the same time, brain T levels and Androgen receptor expression decreased. In contrast, in the post-reproductive frogs, d -Asp treatment did not modify any of these parameters. Taken together, these results imply that the regulation of P450 aro expression by d -Asp could be an important step in the control of E2 levels in the frog brain.

Thyroid hormone receptor-β (trβ) cDNA was identified in the adult of Pelophylax esculentus (previously: Rana esculenta), a seasonally breeding species, in order to detect spatial brain trβ expression, its levels through the seasons and in response to 6-n-propyl-2-thiouracil, T(4) and T(3) administrations as well as to thermal manipulations. The deduced amino acid sequence of P. esculentus trβ showed a high similarity to the homologous of other vertebrates. By in situ hybridization we found trβ mRNA signal in the anterior preoptic nucleus, the habenulae, the hypothalamic-pituitary region and the ependyma. Brain trβ transcript levels varied through the seasons, and they were well correlated with brain T(4) levels but only partially with T(3) levels. Experimentally-induced hypothyroidism decreased brain trβ expression. The administration of exogenous thyroid hormones increased brain trβ expression, with T(4) appearing more potent than T(3). The experiments of thermal manipulations further strengthen the hypothesis that T(4) is more effective than T(3) in brain trβ regulation. This study also shows that, as in other vertebrates, deiodinase enzymes could modulate trβ expression via thyroid hormone regulation.

Mast cells are best known as multifunctional entities that may confer a benefit on immune system. This review presents the known facts on mast-cell system and breakthroughs in mast-cell biology in fish, amphibians, reptiles, and birds. As compared to mammals, there are relatively few data available on mast cells in many nonmammalian vertebrates. Nevertheless, like in mammals, mast cells in nonmammalian vertebrates contain a wide range of bioactive compounds including histamine, heparin, neuropeptides, and neutral proteases. In bony fishes, these cells secrete antimicrobial peptides as well. Mast cells have a widespread distribution in the brain, endocrine glands, intestine, liver, kidney, skin, tongue, and lungs, the highest concentration occurring in different tissues in the different taxa. Currently, researchers are grappling with the nature of scientific support to substantiate the functional importance of mast cells in nonmammalian vertebrates. Ultimately, the origin and evolution of vertebrate mast cell is of great interest to comparative immunologists seeking an underlying trend in the phylogenetic development of immunity.

Although d -aspartate ( d -Asp) has been recognized as having an important physiological role within different organs, high concentrations could elicit detrimental effects on those same organs. In this study, we evaluated the oxidative stress response to d -Asp treatment in rat Harderian gland (HG) by measuring total cellular hydroperoxide levels. Further, we examined the effect of d -Asp uptake on the expression of the mitochondrial uncoupling protein-3 (UCP3), β-actin, and α-tubulin. In rat HG, elevated levels of d -Asp significantly increased hydroperoxide production. This phenomenon was probably due to d -Asp uptake as well as lipid and porphyrin increased levels. Higher UCP3 levels and lower α-tubulin expression were also observed after d -Asp treatment. On the contrary, β-actin expression was unchanged. Given the possible role of UCP3 in lipid handling, the higher expression of mitochondria UCP3 protein in d -Asp-treated HG may reflect a major need to export excessive amounts of hydroperoxides deriving from a greater fatty acid flux across these organelles and higher mitochondrial porphyrin levels. Moreover, abundance of hydroperoxides in d -Asp treated rat HG could determine the decrease of α-tubulin expression. Thus, our findings indicate that a high concentration of d -Asp is critical in initiating a cascade of events determined by oxidative stress.

SUMMARY In the developing frog brain, the majority of mast cells (MC) are distributed in the pia mater, and some immature MC are located adjacent to the blood capillaries in and around the neuropil. In the adult brain, MC are more numerous than in pre- and pro-metamorphic tadpoles; they are mainly located within the pia mater and are particularly numerous in the choroid plexuses. Many MC are found within the brain ventricles juxtaposed to the ependymal lining. MC are rarely observed in the brain parenchyma. In the adult brain, MC number is much higher than in the brain of post-metamorphic froglets. In the latter, MC number is nearly 2-fold over that found in the pre-metamorphic brain. Treatment of pre- and pro-metamorphic tadpoles with 3,5,3′-triiodothyronine (T3) and thyroxine (T4) stimulates overall larval development but does not induce a significant change in MC population within the brain. By contrast, treatment with 6-n-propyl-2-thiouracil (PTU) delays larval development and leads to a significant numerical increase of brain MC. In the adult, PTU treatment also has a similar effect whereas hypophysectomy causes a drastic decrease of MC population. The negative effects of hypophysectomy are successfully counteracted by a two-week replacement therapy with homologous pars distalis homogenate. In the adult frog, MC population seems to be refractory to thyroid hormone treatment. The present study on frog brain suggests that pituitary–thyroid axis may be involved in the regulation of MC frequency.

In this paper, we examined the distribution pattern of D-aspartic acid (D-Asp), as well as (D-Asp), as well as In this paper, we examined the distribution pattern of D-aspartic acid D-aspartate oxidase (D-AspO), D-amino acid oxidase (D-AAO), and L-amino acid oxidase (L-AAO) activities in different tissues of frog, Rana esculenta. High concentrations of free D-Asp were found in the testes (0.21 ± 0.02 μmol/g b.w), in the liver (0.20 ± 0.03 μmol/g b.w), and in the Harderian gland (HG) (0.19 ± 0.03 μmol/g b.w). A higher activity of both D-AspO and D-AAO with respect to L-AAO was endogenously present in all examined frog tissues, particularly within the kidney, liver, and brain. Our in vivo experiments, consisting of i.p. injections of 2.0 μmol/g b.w. D-Asp in frogs, revealed that all examined tissues can take up and accumulate D-Asp and that this amino acid specifically triggers D-AspO activity. Indeed, no increase in both D-AAO and L-AAO was found in all frog tissues after D-Asp treatment. The optimum pH for D-AspO activity was around 8.2 and the optimum temperature was about 37°C. Furthermore, its activity linearly increased with increasing D-Asp incubation times. In vitro experiments assaying the substrate specificity of D-AspO indicated that the enzyme had greater affinity for N-methyl-D-aspartate than for D-Asp and D-glutamate. This study provides evidence of the presence of free D-Asp in frog R. esculenta tissues, along with its role in triggering D-AspO activity. These findings suggest that D-AspO could play an essential role in decreasing excessive amounts of D-Asp in frog tissues, a phenomenon that, if left unchecked, could have detrimental physiological effects on the animal. © 2009 Wiley-Liss, Inc.

This is the first descriptive study of ontogenesis and anatomical distribution of mast cells in the developing brain of three different amphibian species. In the toad and the green frog, mast cells are preferentially located in: (i) the meningeal lining (pia mater), (ii) the choroid plexuses, both anterior and posterior, and (iii) the neuropil, in close association with the epithelial cell lining of blood vessels. It is only in the perennially aquatic African clawed frog that mast cells never appear inside brain ventricles and within the neuropil. Mast cells first become identifiable in brain of different species in different stages of development. While there are differences in the number of mast cells in different species at different stages of development, the number nearly doubles in all three species during the transition from pro-metamorphic stage of larval development to the peak of metamorphic climax. Furthermore, the number of mast cells is comparatively higher in the toad and remarkably lower in the fully aquatic Xenopus laevis, in which species the first appearance of identifiable mast cells during larval development occurs much later than in equivalent stages of development of the toad and the green frog. The secretory nature of mast cells can be assumed by the presence of cytoplasmic granules, which may show species-specific texture. Further experimental analyses are required to unveil the usefulness of mast cells in the amphibian brain. © 2010 The Authors. Journal compilation © 2010 Anatomical Society of Great Britain and Ireland.

Neuroanatomical mapping as well as the influence of thyroid status on brain mast cell distribution and detectable mast cell number in adult Rana esculenta is studied. Treatment with tizoxin (T4) does not modify number or activational state of brain mast cells, whereas administration of the antithyroid agent 6-n-propyl-2-thiouracil induces a significant increase (up to 40%) in the mast cell number within the telencephalon and diencephalon. Hypophysectomy induces a significant decrease (up to 65%) of mast cells in all brain regions, whereas the pituitary homogenate augments their number. The results suggest that the pituitary‐thyroid axis may be involved in the regulation of brain mast cell population.

In this paper, the role of d-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous d-Asp were detected, along with aspartate racemases that convert d-Asp to l-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered d-Asp. d-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since d-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that d-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity. © 2008 Springer-Verlag.

Hamster (Mesocricetus auratus) harderian gland (HG) is a dimorphic orbital gland producing a copious lipid secretion. Two cell-types are present in hamster HG, type I in both sexes, type II only in males. In hamster HGs, we found a marked sexual dichotomy in the expression of uncoupling protein-3 (UCP3), a mitochondrial protein carrier, that probably exports fatty acid anions and fatty acid peroxides from the mitochondrial matrix. Following castration and/or testosterone treatment: (1) UCP3 levels correlated with the type II-cell percentage, not with testosterone levels, (2) in male HGs, UCP3 was comparable to female levels at 30 days post-castration (when the type II-cell percentage had fallen from 50% to 5%), although testosterone was already near zero at 15 days (when neither the type II-cell percentage nor the UCP3 level had fallen), and testosterone-replacement therapy prevented these changes. Testosterone-treated females possessed type II cells and a UCP3 level about twofold higher than in control females. Males displayed more intense UCP3 immunohistochemical positivity in type I HG cells than females. Hence, testosterone may indirectly control UCP3 expression by regulating the gland’s morphological and lipid dimorphism. Straight-chain fatty acids [found in alkyl diacylglycerols (ADGs) in males] are oxidized predominantly in mitochondria, branched-chain fatty acids (abundant in ADGs in females) predominantly in peroxisomes, so we speculate that the higher UCP3 expression in males reflects greater fatty acid flux in HG mitochondria. This is supported by our finding that in female (not male) HGs, the peroxisome-rich fraction contained a-methylacyl-CoA racemase (AMACR), an enzyme important in the beta-oxidation of branched-chain fatty acids. Hamster (Mesocricetus auratus) harderian gland (HG) is a dimorphic orbital gland producing a copious lipid secretion. Two cell-types are present in hamster HG, type I in both sexes, type II only in males. In hamster HGs, we found a marked sexual dichotomy in the expression of uncoupling protein-3 (UCP3), a mitochondrial protein carrier, that probably exports fatty acid anions and fatty acid peroxides from the mitochondrial matrix. Following castration and/or testosterone treatment: (1) UCP3 levels correlated with the type II-cell percentage, not with testosterone levels, (2) in male HGs, UCP3 was comparable to female levels at 30 days post-castration (when the type II-cell percentage had fallen from 50% to 5%), although testosterone was already near zero at 15 days (when neither the type II-cell percentage nor the UCP3 level had fallen), and testosterone-replacement therapy prevented these changes. Testosterone-treated females possessed type II cells and a UCP3 level about twofold higher than in control females. Males displayed more intense UCP3 immunohistochemical positivity in type I HG cells than females. Hence, testosterone may indirectly control UCP3 expression by regulating the gland's morphological and lipid dimorphism. Straight-chain fatty acids [found in alkyl diacylglycerols (ADGs) in males] are oxidized predominantly in mitochondria, branched-chain fatty acids (abundant in ADGs in females) predominantly in peroxisomes, so we speculate that the higher UCP3 expression in males reflects greater fatty acid flux in HG mitochondria. This is supported by our finding that in female (not male) HGs, the peroxisome-rich fraction contained α-methylacyl-CoA racemase (AMACR), an enzyme important in the β-oxidation of branched-chain fatty acids. © 2008 Wiley-Liss, Inc.

SUMMARY The secretory activity of the Harderian gland (HG) is influenced by both exogenous (such as light and temperature) and endogenous (such as prolactin,thyroid hormones and steroid hormones) factors, which vary among species. In the present study, the effects of hypothyroidism on the rat HG were examined at morphological and biochemical levels. The decrease in cytoplasmic lipoproteic vacuoles and the increase in mucosubstance secretion in the acinar lumina were the most notable histological effects elicited by hypothyroidism. The release of all granules with nuclei and cellular debris suggested the occurrence of holocrine secretion. Electron microscopy revealed in the glandular cells of hypothyroid rat an increased condensation of chromatin in the nuclei, mitochondria with decreased cristae and vacuolisation, decreased glycogen granules, autophagic vacuoles, and lipofuscins in the cytoplasm. TUNEL reaction indicated DNA fragmentation in hypothyroid HG, indicative of an underlying apoptotic process. Translocation of cytochrome c from mitochondria to cytosol strongly supported this hypothesis. In conclusion, these findings indicate that thyroid hormones play a pivotal role in preserving the structural integrity of the rat HG and, hence, its secretory activity.

High concentrations of free D-aspartate (D-Asp), an amino acid well known for its neuroexcitatory activity, are endogeneously present in the Harderian gland (HG) of the lizard Podarcis s. sicula. This orbital gland consists of two different parts: the medial part, which is prevalently a mucous acinar gland, and the lateral part, which is a serous tubulo-acinar gland. To determine the physiological effect of D-Asp on exocrine secretion in HG, D-Asp (2.0 µmol/g b.w.) was injected intraperitoneally into lizards. We found that highest accumulations of exogenous D-Asp in HGs occurred 15 hr after the injection. Specifically, exogenous D-Asp prevalently stimulated serous secretion from the lateral portion of the gland, where immunohistochemical analysis revealed a major accumulation. Similarly, in the medial part of the gland, highly sulfated mucosubstances were observed after D-Asp injection. Further, in both parts of the HG, the electron microscope revealed euchromatic nuclei, a prominent rough endoplasmic reticulum, as well as numerous secretory granules within the acinar cells. Thus, following D-Asp injection, a 60% increase in HG total protein was detected. In addition, exogenous D-Asp induced changes in the electrophoretic pattern of HG. In conclusion, although further investigations are still needed to clarify the molecular pathway induced by D-Asp in exocrine secretion, this study does indicate that free D-Asp plays a significant role in the secretory activity of this gland. J. Exp. Zool. 305A, 2006. © 2006 Wiley-Liss, Inc.

In the green frog, Rana esculenta, a substantial amount Of D-aspartate (D-Asp) is found enclogenously within the Harderian gland (HG) following its synthesis from (L)-aspartate ((L)-Asp) by an aspartate racemase. The frog HG is an orbital seromucoid gland that displays seasonal changes in secretory activity. Our in vivo experiments, consisting of i.p. injection of 2.0 mu mol/g.b.w. (D)-Asp in frogs collected during two periods of differing glandular activity (high or medium-low secretory activity), revealed that HG can to take up and accumulate D-Asp and that this amino acid may modulate the exocrine secretion through a kinase pathway. Atatirnewhenthe gland shows relatively low secretory activity, i.p. administration of (D)-Asp rapidly induced activation of ERK1 and an increase in cells active in RNA synthesis. This increase in transcriptional activity was followed by a significant increase in Mucous secretion. By contrast, administration of exogenous (D)-Asp when HG was showing high activity rapidly induced inhibition of both ERK1 and transcriptional activity. Since D-Asp is known to be recognized by receptors for N-methyl-(D)-aspartic acid (NMDA), it is possible that in the HG, D-Asp mediated NMDA activation may enhance the kinase pathway. The above activation of opposing stimulatory and inhibitory processes could reflect different levels of NMDA-receptor activity, which could vary as a function of the level of gland activity. This study provides the first evidence of a role for this excitatory amino acid in exocrine secretion. The effects of (D)-Asp in HG appear to be specific since they were not seen in frogs treated with other (D)- or (L)-amino acids with known excitatory effects on neurosecretion. (c) 2005 Wiley-Liss, Inc.