Polystyrene microplastics impair the functions of cultured mouse Leydig (TM3) and Sertoli (TM4) cells by inducing mitochondrial-endoplasmic reticulum damage.

Many laboratory studies demonstrated that the exposure to microplastics causes testosterone deficiency and spermatogenic impairment in mammals; however, the mechanism underlying this process remains still unclear. In this study, we investigated the effects of polystyrene microplastics (PS-MP) on the proliferation and functionality of cultured Leydig (TM3) and Sertoli (TM4) cells, focusing on the mitochondrial compartment and its association with the endoplasmic reticulum (ER). The in vitro exposure to PS-MP caused a substantial reduction in cellular viability in TM3 and TM4 cells. In TM3 cells PS-MP inhibited the protein levels of StAR and of steroidogenic enzymes 3ß-HSD and 17ß-HSD, and in TM4 cells PS-MP inhibited the protein levels of the androgen receptors other than the activity of lactate dehydrogenase (LDH). PS-MP inhibited the functions of TM3 and TM4, as evidenced by the decrease of the phosphorylation of ERK1/2 and Akt in both cell lines. The oxidative stress caused by PS-MP decreased antioxidant defense in TM3 and TM4 cells, promoting autophagic and apoptotic processes. Furthermore, we found mitochondrial dysfunction and activation of ER stress. It is known that mitochondria are closely associated with ER to form the Mitochondrial-Associated Endoplasmic Reticulum Membranes (MAM), the site of calcium ions transfer as well as of lipid biosynthesis-involved enzymes and cholesterol transport from ER to the mitochondria. For the first time, we studied this aspect in PS-MP-treated TM3 and TM4 cells and MAMs dysregulation was observed. This study is the first to elucidate the intracellular mechanism underlying the effects of PS-MPs in somatic testicular cells, corroborating that PS-MP might be one of the causes of an increase in male infertility through the impairment of steroidogenesis in Leydig cells and of the nurse function of Sertoli cells. Thus, our findings contributed with new information to the mechanism underlying the effects of PS-MP on the male reproductive system.

Steroidogenesis Upregulation through Mitochondria-Associated Endoplasmic Reticulum Membranes and Mitochondrial Dynamics in Rat Testes: The Role of D-Aspartate.

Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs) mediate the communication between the Endoplasmic Reticulum (ER) and the mitochondria, playing a fundamental role in steroidogenesis. This study aimed to understand how D-aspartate (D-Asp), a well-known stimulator of testosterone biosynthesis and spermatogenesis, affects the mechanism of steroidogenesis in rat testes. Our results suggested that D-Asp exerts this function through MAMs, affecting lipid trafficking, calcium signaling, ER stress, and mitochondrial dynamics. After 15 days of oral administration of D-Asp to rats, there was an increase in both antioxidant enzymes (SOD and Catalase) and in the protein expression levels of ATAD3A, FACL4, and SOAT1, which are markers of lipid transfer, as well as VDAC and GRP75, which are markers of calcium signaling. Additionally, there was a decrease in protein expression levels of GRP78, a marker of aging that counteracts ER stress. The effects of D-Asp on mitochondrial dynamics strongly suggested its active role as well. It induced the expression levels of proteins involved in fusion (MFN1, MFN2, and OPA1) and in biogenesis (NRF1 and TFAM), as well as in mitochondrial mass (TOMM20), and decreased the expression level of DRP1, a crucial mitochondrial fission marker. These findings suggested D-Asp involvement in the functional improvement of mitochondria during steroidogenesis. Immunofluorescent signals of ATAD3A, MFN1/2, TFAM, and TOMM20 confirmed their localization in Leydig cells showing an intensity upgrade in D-Asp-treated rat testes. Taken together, our results demonstrate the involvement of D-Asp in the steroidogenesis of rat testes, acting at multiple stages of both MAMs and mitochondrial dynamics, opening new opportunities for future investigation in other steroidogenic tissues.

A short-term high-fat diet alters rat testicular activity and blood-testis barrier integrity through the SIRT1/NRF2/MAPKs signaling pathways.

Background: Overweight/obesity are metabolic disorder resulting from behavioral, environmental, and heritable causes. WHO estimates that 50% of adults and 30% of children and adolescents are overweight or obese, and, in parallel, an ongoing decline in sperm quality and male fertility has been described. Numerous studies demonstrated the intimate association between overweight/obesity and reproductive dysfunction due to a highly intricate network of causes not yet completely understood. This study expands the knowledge on the impact of a short-term high-fat diet (st-HFD) on rat testicular activity, specifically on steroidogenesis and spermatogenesis, focusing on the involved molecular mechanisms related to mitochondrial dynamics, blood-testis barrier (BTB) integrity, and SIRT1/NRF2/MAPKs pathways. Methods: Ten adult Male Wistar rats were divided into two groups of five and treated with a standard diet or an HFD for five weeks. At the end of the treatment, rats were anesthetized and sacrificed by decapitation. Blood was collected for serum sex hormone assay; one testis was stored at -80ÅãC for western blot analysis, and the other, was fixed for histological and immunofluorescence analysis. Results: Five weeks of HFD results in reduced steroidogenesis, increased apoptosis of spermatogenic cells, and altered spermatogenesis, as highlighted by reduced protein levels ofmeiotic and post-meiotic markers. Further, we evidenced the compromission of the BTB integrity, as revealed by the downregulation of structural proteins (N-Cadherin, ZO-1, occludin, connexin 43, and VANGL2) other than the phosphorylation of regulative kinases (Src and FAK). At the molecular level, the impairment of mitochondrial dynamics (fission, fusion, andbiogenesis), and the dysregulation of the SIRT1/NRF2/MAPKs signaling pathways, were evidenced. Interestingly, no change was observed in the levels of pro-inflammatory markers (TNFα, NF-kB, and IL-6). Conclusions: The combined data led us to confirm that overweight is a less severe state than obesity. Furthermore, understanding the molecular mechanisms behind the association between metabolic disorders and male fertility could improve the possibility of identifying novel targets to prevent and treat fertility disorders related to overweight/obesity.

D-Aspartate Depletion Perturbs Steroidogenesis and Spermatogenesis in Mice.

High levels of free D-aspartate (D-Asp) are present in vertebrate testis during post-natal development, coinciding with the onset of testosterone production, which suggests that this atypical amino acid might participate in the regulation of hormone biosynthesis. To elucidate the unknown role of D-Asp on testicular function, we investigated steroidogenesis and spermatogenesis in a one-month-old knockin mouse model with the constitutive depletion of D-Asp levels due to the targeted overexpression of D-aspartate oxidase (DDO), which catalyzes the deaminative oxidation of D-Asp to generate the corresponding α-keto acid, oxaloacetate, hydrogen peroxide, and ammonium ions. In the Ddo knockin mice, we found a dramatic reduction in testicular D-Asp levels, accompanied by a significant decrease in the serum testosterone levels and testicular 17ß-HSD, the enzyme involved in testosterone biosynthesis. Additionally, in the testes of these Ddo knockin mice, the expression of PCNA and SYCP3 proteins decreased, suggesting alterations in spermatogenesis-related processes, as well as an increase in the cytosolic cytochrome c protein levels and TUNEL-positive cell number, which indicate an increase in apoptosis. To further investigate the histological and morphometric testicular alterations in Ddo knockin mice, we analyzed the expression and localization of prolyl endopeptidase (PREP) and disheveled-associated activator of morphogenesis 1 (DAAM1), two proteins involved in cytoskeletal organization. Our results showed that the testicular levels of DAAM1 and PREP in Ddo knockin mice were different from those in wild-type animals, suggesting that the deficiency of D-Asp is associated with overall cytoskeletal disorganization. Our findings confirmed that physiological D-Asp influences testosterone biosynthesis and plays a crucial role in germ cell proliferation and differentiation, which are required for successful reproduction.

Effects of a high-fat diet on rat epididymis.

The epididymis plays an essential role in reproduction, promoting sperm cell maturation. In this study, we investigated the effects of a high-fat diet (HFD) in the three regions of the epididymis of rats, including caput, corpus, and cauda. Our results showed an increase in malondialdehyde and a decrease in superoxide dismutase, which indicated an increase in oxidative stress in all segments of the epididymis. The cellular response mechanisms were mostly detected in the corpus/cauda regions, which showed an increase in apoptosis, probably for eliminating dysfunctional cells arising from HFD-induced oxidative stress, and a decrease in mitophagy. Additionally, an increase in lipophagy to prevent lipid accumulation and a decrease in cell proliferation were recorded in the corpus.

High-fat diet affects autophagy and mitochondrial compartment in rat Harderian gland.

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.

Mast Cell Population and Histamine Content in Hypothyroid Rat Tissues.

The morphological features and relative number of mast cells (MCs) were studied in the skin and exorbital lacrimal glands of hypothyroid Wistar rats, Rattus norvegicus. Hypothyroidism significantly increased the number of MCs (up to 4.5-fold) and histamine content (up to 50%) in the examined tissues. The magnitude of the increase in the number of MCs was greater in the cheek skin and exorbital lacrimal glands than in the back skin. In the skin, the MCs were mainly located within the hypodermis and closely associated with the blood vessels, nerve fascicles, and adipocytes. In the exorbital lacrimal gland, which is a seromucous gland located lateral to the cheek below the ear, the MCs were distributed in the connective tissue surrounding the acini. The secretory granules of MCs showed histochemical characteristics of connective tissue MCs. They were metachromatic with Toluidine blue and safranin positive with the Alcian blue/safranin reactions. Finally, a significant increase in degranulating MCs was observed in hypothyroid tissues, relative to euthyroid tissues. At the ultrastructural level, the MCs of euthyroid rats were predominantly non-degranulating (Stage I). In hypothyroid animals, numerous MCs showed partial degranulation (Stage II-III) or were in a stage of complete degranulation. Our results concerning the skin and exorbital lacrimal gland suggested that the thyroid status might be involved in regulating the frequency and activation state of MCs.

Comparative insights of the neuroanatomical distribution of the gonadotropin-inhibitory hormone (GnIH) in fish and amphibians.

This paper intends to apprise the reader regarding the existing knowledge on the neuroanatomical distribution of GnIH-like peptides in in fish and amphibians in both the adult stage and during ontogenesis. The neuroanatomical distribution of GnIH-like neuropeptides appears quite different in the studied species, irrespective of the evolutionary closeness. The topology of the olfactory bulbs can affect the distribution of neurons producing the GnIH-like peptides, with a tendency to show a more extended distribution into the brains with pedunculate olfactory bulbs. Therefore, the variability of the GnIH-like system could also reflect specific adaptations rather than evolutionary patterns. The onset of GnIH expression was detected very early during development suggesting its precocious roles, and the neuroanatomical distribution of GnIH-like elements showed a generally increasing trend. This review highlights some critical technical aspects and the need to increase the number of species to be studied to obtain a complete neuroanatomical picture of the GnIH-like system.

d-aspartate and N-methyl-d-aspartate promote proliferative activity in mouse spermatocyte GC-2 cells.

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.

Proliferative and Apoptotic Pathways in the Testis of Quail Coturnix coturnix during the Seasonal Reproductive Cycle.

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 17ß-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.

The Aromatase-Estrogen System in the Testes of Non-Mammalian Vertebrates.

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 17ß-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.

JNK/Elk1 signaling and PCNA protein expression in the brain of hibernating frog Pelophylax esculentus.

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.

New Evidence on the Role of D-Aspartate Metabolism in Regulating Brain and Endocrine System Physiology: From Preclinical Observations to Clinical Applications.

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-Amino acids in mammalian endocrine tissues.

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 ß cells and is co-secreted with insulin by exocytosis. Specific immunolocalization of D-alanine in pituitary ACTH-secreting cells and pancreatic ß-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.

Celebrating 50+ years of research on the reproductive biology and endocrinology of the green frog: An overview.

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 Harderian gland: Endocrine function and hormonal control.

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.

Mild Exercise Rescues Steroidogenesis and Spermatogenesis in Rats Submitted to Food Withdrawal.

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, 3ß-Hydroxysteroid dehydrogenase (3ß-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/17ß-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.

D-Aspartate Upregulates DAAM1 Protein Levels in the Rat Testis and Induces Its Localization in Spermatogonia Nucleus.

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.

D-Aspartic Acid in Vertebrate Reproduction: Animal Models and Experimental Designs‡.

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.

D-Asp upregulates PREP and GluA2/3 expressions and induces p-ERK1/2 and p-Akt in rat testis.

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.