Search

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.

Simple Summary The aromatase–estrogen system plays a key role in gonadal sex differentiation in amphibians, reptiles, and birds during development. In adults of seasonal breeding species, aromatase activity and estrogen levels can act as an “on/off” switch for spermatogenesis and can also promote spermiogenesis. Abstract Estrogens are important physiological regulators of testicular activity in vertebrates. Estrogen levels depend on the activity of P450 aromatase, the enzyme responsible for the irreversible conversion of testosterone into 17β-estradiol. Therefore, P450 aromatase is the key player in the aromatase–estrogen system. The present review offers a comparative overview of P450 aromatase activity in male gonads of amphibians, reptiles, and birds, with a particular emphasis on the functions of the aromatase–estrogen system in these organisms during their developmental and adult stages. The aromatase–estrogen system appears to be crucial for the sex differentiation of gonads in vertebrates. Administration of aromatase inhibitors prior to sexual differentiation of gonads results in the development of males rather than females. In adults, both aromatase and estrogen receptors are expressed in somatic cells, Leydig and Sertoli cells, as well as germ cells, with certain differences among different species. In seasonal breeding species, the aromatase–estrogen system serves as an “on/off” switch for spermatogenesis. In some amphibian and reptilian species, increased estrogen levels in post-reproductive testes are responsible for blocking spermatogenesis, whereas, in some species of birds, estrogens function synergistically with testosterone to promote spermatogenesis. Recent evidence indicates that the production of the aromatase enzyme in excessive amounts reduces the reproductive performance in avian species of commercial interest. The use of aromatase inhibitors to improve fertility has yielded suitable positive results. Therefore, it appears that the role of the aromatase–estrogen system in regulating the testicular activity differs not only among the different classes of vertebrates but also among different species within the same class.

Simple Summary The quail Coturnix coturnix exhibits an annual cycle of testis size, sexual steroid production, and spermatogenesis. The testicular levels of both 17β-estradiol (E2) and androgens are higher during the reproductive period compared to the non-reproductive period, suggesting that estrogens act in synergy with the androgens for the initiation of spermatogenesis. Therefore, the present study aimed to investigate the estrogen responsive system in quail testis in relation to the reproduction seasons, with a focus on the molecular pathways activated in both active and regressive quail testes. The results indicated that estrogens participated in the activation of mitotic and meiotic events during the reproductive period by activating the ERK1/2 and Akt-1 pathways. In the non-reproductive period, when the E2/ERα levels are low, ERK1/2 and Akt-1 pathways remain inactive and apoptotic events occur. Our results suggest that the activation or inhibition of these molecular pathways plays a crucial role in the physiological switch “on/off” of the testicular activity in male quail during the seasonal reproductive cycle. Abstract The quail Coturnix coturnix is a seasonal breeding species, with the annual reproductive cycle of its testes comprising an activation phase and a regression phase. Our previous results have proven that the testicular levels of both 17β-estradiol (E2) and androgens are higher during the reproductive period compared to the non-reproductive period, which led us to hypothesize that estrogens and androgens may act synergistically to initiate spermatogenesis. The present study was, therefore, aimed to investigate the estrogen responsive system in quail testis in relation to the reproduction seasonality, with a focus on the molecular pathways elicited in both active and regressive quail testes. Western blotting and immunohistochemistry analysis revealed that the expression of ERα, which is the predominant form of estrogen receptors in quail testis, was correlated with E2 concentration, suggesting that increased levels of E2-induced ERα could play a key role in the resumption of spermatogenesis during the reproductive period, when both PCNA and SYCP3, the mitotic and meiotic markers, respectively, were also increased. In the reproductive period we also found the activation of the ERK1/2 and Akt-1 kinase pathways and an increase in second messengers cAMP and cGMP levels. In the non-reproductive phase, when the E2/ERα levels were low, the inactivation of ERK1/2 and Akt-1 pathways favored apoptotic events due to an increase in the levels of Bax and cytochrome C, with a consequent regression of the gonad.

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.

Food-waste is produced throughout all the food supply chain, with a large part already achieved at farm level. In fact, fruits and vegetables, which do not satisfy aesthetic demands, cannot be marketed, but their recovery could favour their valorisation for the obtainment of highly qualified goods. In this context, faulty zucchini fruits (cultivar &lsquo, Lungo Fiorentino&rsquo, ), intended for disposal, were rescued as effective, inexpensive and bio-sustainable source for cosmeceutical purposes. Zucchini fruits underwent extraction and fractionation to obtain ZLF-O and ZLF-A extracts, which were chemically characterized by UHPLC-HRMS. ZLF-A extract, rich in flavonols and flavones, scavenged massively DPPH&bull, and ABTS&bull, +, and was not cytotoxic at doses up to 200 &mu, g/mL. Thus, ZLF-A was incorporated into a base cream formula. Zucchini-based emulsion was deeply screened for its antiradical properties and cytotoxicity towards human keratinocytes and fibroblasts. ZLF-A-enriched cream, whose chemical stability was assessed over time and mimicking different storage conditions, was further tested on reconstructed epidermis disks (EpiskinTM). The recovery of valuable chemical substances from zucchini agro-food waste, complying with the principles of valorisation and sustainable development, can represent a new market force for local farmers. Data acquired were eager to convey a suitable reuse of nutraceuticals rich zucchini waste.

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.

Excitatory amino acids (EAAs) are found present in the nervous and reproductive systems of animals. Numerous studies have demonstrated a regulatory role for Glutamate (Glu), d-aspartate ( d-Asp) and N-methyl- d-aspartate (NMDA) in the control of spermatogenesis. EAAs are able to stimulate the Glutamate receptors, including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Here in, we assess expression of the main AMPAR subunits, GluA1 and GluA2/3, in the mouse testis and in spermatogonial GC-1 cells. The results showed that both GluA1 and GluA2/3 were localized in mouse testis prevalently in spermatogonia. The subunit GluA2/3 was more highly expressed compared with GluA1 in both the testis and the GC-1 cells. Subsequently, GC-1 cells were incubated with medium containing l-Glu, d-Glu, d-Asp or NMDA to determine GluA1 and GluA2/3 expressions. At 30 minutes and 2 hours of incubation, EAA-treated GC-1 cells showed significantly higher expression levels of both GluA1 and GluA2/3. Furthermore, p-extracellular signal-regulated kinase (ERK), p-Akt, proliferating cell nuclear antigen (PCNA), and Aurora B expressions were assayed in l-Glu-, d-Glu-, and NMDA-treated GC-1 cells. At 30 minutes and 2 hours of incubation, treated GC-1 cells showed significantly higher expression levels of p-ERK and p-Akt. A consequent increase of PCNA and Aurora B expressions was induced by l-Glu and NMDA, but not by d-Glu. Our study demonstrates a direct effect of the EAAs on spermatogonial activity. In addition, the increased protein expression levels of GluA1 and GluA2/3 in EAA-treated GC-1 cells suggest that EAAs could activate ERK and Akt pathways through the AMPAR. Finally, the increased PCNA and Aurora B levels may imply an enhanced proliferative activity.

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.

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.

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide involved in different functions, including testosterone synthesis. Recently, we reported the presence of VIP in the testis of Podarcis sicula, throughout the reproductive cycle. Now, we investigated the effects of the VIP on steroidogenesis in significant periods of the Podarcis reproductive cycle: winter stasis, reproductive period, and summer stasis. Using VIP treatments in testis culture in absence or presence of receptors antagonists, we demonstrated for the first time that in P. sicula, VIP is involved not only in testosterone synthesis, as in mammals, but in 17β-estradiol synthesis too. J. Exp. Zool. 323A: 714-721, 2015. © 2015 Wiley Periodicals, Inc.

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.