Glyphosate Interference in Follicular Organization in the Wall Lizard Podarcis siculus.

Glyphosate (Gly) is a broad-spectrum herbicide widely used thanks to its high efficiency and low toxicity. However, evidence exists of its toxic effects on non-target organisms. Among these, the animals inhabiting agricultural fields are particularly threatened. Recent studies demonstrated that exposure to Gly markedly affected the morphophysiology of the liver and testis of the Italian field lizard Podarcis siculus. The present study aimed to investigate the effects of the herbicide on the female reproductive system of this lizard in order to have a full picture of Gly-induced reproductive impairment. The animals were exposed to 0.05 and 0.5 µg/kg of pure Gly by gavage for 3 weeks. The results demonstrated that Gly, at both doses tested, profoundly interfered with ovarian function. It induced germ cells' recruitment and altered follicular anatomy by anticipating apoptotic regression of the pyriform cells. It also induced thecal fibrosis and affected oocyte cytoplasm and zona pellucida organizations. At the functional levels, Gly stimulated the synthesis of estrogen receptors, suggesting a serious endocrine-disrupting effect. Overall, the follicular alterations, combined with those found at the level of the seminiferous tubules in males, suggest serious damage to the reproductive fitness of these non-target organisms, which over time could lead to a decline in survival.

Molecular and Histological Effects of Glyphosate on Testicular Tissue of the Lizard Podarcis siculus.

The expansion of agriculture produces a steady increase in habitat fragmentation and degradation due to the increased use of pesticides and herbicides. Habitat loss and alteration associated with crop production play an important role in reptile decline, among which lizards are particularly endangered. In this study, we evaluated testicular structure, steroidogenesis, and estrogen receptor expression/localization after three weeks of oral exposure to glyphosate at 0.05 and 0.5 µg/kg body weight every other day in the field lizard Podarcis siculus. Our results show that glyphosate affected testicular morphology, reduced spermatogenesis, altered gap junctions and changed the localization of estrogen receptors in germ cells, increasing their expression; the effects were mostly dose-dependent. The result also demonstrates that glyphosate, at least at these concentrations, did not influence steroidogenesis. Overall, the data indicate that this herbicide can disturb the morphophysiology of the male lizard's reproductive system, with obviously detrimental effects on their reproductive fitness. The effects of glyphosate must be considered biologically relevant and could endanger the reproductive capacity not only of lizards but also of other vertebrates, including humans; a more controlled and less intensive use of glyphosate in areas devoted to crop production would therefore be advisable.

How Glyphosate Impairs Liver Condition in the Field Lizard Podarcis siculus (Rafinesque-Schmaltz, 1810): Histological and Molecular Evidence.

The potential toxicity of glyphosate, a widely used broad-spectrum herbicide, is currently a great matter of debate. As vertebrate insectivores, lizards protect plants from herbivorous insects increasing plant biomass via the trophic cascade and represent an important link between invertebrates and higher predators. A negative effect of glyphosate on lizards' survival could have major impacts at the ecological levels. In this study, we investigated the effects of the exposure to low doses of glyphosate on the liver of the wall lizard Podarcis siculus, a suitable bioindicator of soil pollution. Two different doses of pure glyphosate (0.05 and 0.5 µg/kg body weight) were orally administered every other day for 3 weeks to sexually mature males and females. The results demonstrated that both doses, despite being very low, are toxic for the liver that showed clear signs of suffering, regardless of sex. The histological analysis provided a scenario of severe hepatic condition, which degenerated until the appearance of fibrotic formations. The morphological observations were consistent with a loss of liver physiological functions. Immunocytochemical investigations allowed us to detect an involvement of antioxidant/cytoprotective proteins, such as superoxide dismutase 1 (Cu/Zn SOD, known as SOD1), glutathione peroxidase 1 (GPx1), metallothionein (MT), and tumor suppressor protein 53, (p53) suggesting that the liver was trying to react against stress signals and damage induced by glyphosate. Finally, in situ hybridization and Real-Time PCR analysis showed the upregulation of estrogen receptor α and vitellogenin gene expression, thus demonstrating the xenoestrogenic action of glyphosate. The imbalance of the hormonal homeostasis could threaten the lizards' reproductive fitness and survival, altering the trophic cascade.

Health status of the lizard Podarcis siculus (Rafinesque-Schmaltz, 1810) subject to different anthropogenic pressures.

The terrestrial lizard Podarcis siculus is the most abundant reptile in Italy, where is considered a ubiquitous species. This lizard is widely distributed from the islands to the Apennines, from cultivated fields to anthropized areas such as gardens and city parks. For this reason, these animals are exposed to extensive physical and chemical stresses, as well as to the possibility of coming into contact with industrial pollutants and substances used in agricultural practices. Here, we review the health status of lizard specimens inhabiting natural non-anthropized areas and fields devoted to organic farming, considering the condition of (1) liver, representing the main detoxifying organ, directly influenced by feeding, and (2) gonads, essential for reproduction and, therefore, for the survival of the species. The morphological and biomolecular condition of these organs was then compared with those obtained from lizard specimens experimentally treated with nonylphenol, a co-formulant of many insecticides and plant protection products widely used in conventional farming, known to have harmful estrogenic effects. Taken together, data demonstrate that lizards inhabiting manured soil are in good health status and show a regular morphology of liver, testis, and ovary. Animals are found to be less exposed to the toxic heavy metals cadmium and lead if compared with specimens collected in areas not devoted to agriculture, but probably more exposed to vehicular traffic. However, manure, as well as nonylphenol, exerts a xeno-estrogenic effect, particularly evident in male specimens, more sensitive to estrogenic contamination.

Role of estrogen receptors, P450 aromatase, PCNA and p53 in high-fat-induced impairment of spermatogenesis in rats.

Obesity and overweight are frequently associated with male subfertility. To address new findings on the players involved in the obesity-induced impairment of spermatogenesis, we used a high-fat diet-induced overweight-rat model. Following four weeks of high-fat diet, the organization of seminiferous epithelium was affected, and tubules lumen showed immature/degenerated cells, typical signs of hormonal imbalance and testicular damage. Real-time PCR analysis allowed us to detect increased levels of ERα and decreased levels of aromatase CYP19 transcripts in testis, suggesting an increase in circulating estrogens derived from the accumulating adipose tissue rather than the induction of testicular estrogen synthesis. Moreover, in situ hybridization analysis showed an increased susceptibility towards estrogens in testis from high-fat fed rats, being ERs expressed not only in spermatogonia, as in control testis, but also in spermatids. Western blot and immunohistochemical analyses revealed an increase in the amount of p53 and PCNA, together with a change in their immunodetection, being the proteins localised on germ cells at different stages of maturation. Differences in p53 and PCNA expression may give evidence and be part of a cellular response to stress conditions and damage caused by the excessive intake of saturated fatty acids.

A comparative review on estrogen receptors in the reproductive male tract of non mammalian vertebrates.

Estrogen receptors alpha (ERα) and beta (ERß) are transcription factors known to be involved in the regulation of many complex physiological processes in mammals. They are expressed primarily in the reproductive tract of all vertebrates females, thus indicating important and conserved functions in female reproductive success. ERs are also present in physiological different tissues as bone, brain, liver, skin and adipose tissues, in both females and males. In the latter, ERs have been found also in the genital tract, supporting the findings of a complex role for estrogen in spermatogenesis and, more generally, in male reproduction. This review provides an overview and update on ERα and ERß expression and synthesis in male reproductive tract of non-mammalian vertebrates, with focus on their role in germ cells proliferation, maturation and survival. Data from studies on fish, amphibians, reptiles and birds were collated and common or species-specific distribution highlighted. The widespread distribution of estrogen receptors in testicular cells and ducts of all vertebrates so far investigated suggests that whatever are the roles that estrogens may exert on these structures, they are phylogenetically conserved and are possibly related to the physiological support given to achieve male reproductive success.

Gene expression profile of estrogen receptors alpha and beta in rat brain during aging and following high fat diet.

The "sex-hormone" estrogen-17ß promotes several cognitive functions and is a master regulator of brain bioenergetics via the estrogen receptors α and ß (ERα and ERß). In this work, by using Real-Time PCR analysis, we evaluated the effect of aging and high fat diet (HFD) on ERα and ERß expression in rat hippocampus and cortex. In young rats, ERß is abundant in cortex and ERα in hippocampus. During the aging, in the cortex, we observe a general decrease in ERα and ERß expression; in hippocampus ERα increases and ERß decreases. ER expression patterns in rat brain are also affected by the administration of an HFD. In cortex, after 4weeks of HFD, ERß transcripts are down-regulated, whereas ERα levels remain unchanged; after 12weeks, both ERα and ERß expression is up-regulated. In the hippocampus, the level of ERß transcripts does not change following HDF, whereas ERα expression is affected by HDF, in a time-dependent manner: it increases after the 4-week treatment and decreases after 12weeks. Possible involvements of these receptors in the control of cortex and hippocampus functions during aging and in the modulation of energetic metabolism and feeding behaviour are discussed.

Metallothionein expression and synthesis in the testis of the lizard Podarcis sicula under natural conditions and following estrogenic exposure.

Metallothionein (MT) is the main protein involved in the homeostasis of metallic micronutrients and in cellular defence against heavy metals and reactive oxygen species. Found in almost all vertebrate tissues, MT presence and localization in the testis has been controversial. In the present study, by using in situ hybridization and immunohistochemical analysis we assessed the localization of both MT transcript and protein in Podarcis sicula testes during two different phases of the reproductive cycle: the autumnal resumption and the spring-early summer mating period. In addition, with the same methodological approach, we verified the effect of estradiol-17ß and nonylphenol, a potent xenoestrogen, on MT expression and synthesis.  These results, the first collected in a non-mammalian oviparous vertebrate, demonstrated that the expression profile of MT mRNA and protein changes during the reproductive cycle. In the fall resumption, MT transcripts are absent in spermatogonia and present in all the other cells of tubules, including spermatozoa; vice versa, the MT protein is evident only in spermatozoa and somatic cells. In the mating period, both MT transcripts and proteins are present in spermatogonia, spermatocytes and spermatids, whereas in the spermatozoa only the proteins are detected, thus suggesting that the MTs translated in the earlier germinal stages are stored up to spermatozoa. Results also demonstrated that in lizard testis the expression of MT gene undergoes a cell-specific regulation after estrogenic exposure; the possible role and the mechanism by which this regulation occurs have been discussed.

Estrogen-dependent, extrahepatic synthesis of vitellogenin in male vertebrates: A mini-review.

In the last years, the hormonal balance is threatened by the interferences of substances with hormone-like action (endocrine disruptor chemicals, EDCs) that may harm animal reproduction. Most EDCs are resistant to environmental degradation and are considered ubiquitous contaminants. EDCs may have synthetic or natural origins. Pesticides used in intensive agriculture contain large amounts of chemicals with estrogenic properties, such as the alkylphenol nonylphenol (NP). Besides, animal feeding operations are important sources of natural estrogen metabolites introduced into the environment through manure application in organic farming. In both cases, EDCs can reach animals, including humans particularly at risk due to their position in the food chain. This is the reason for which it is important to use terrestrial vertebrates as sentinels in soil biomonitoring programmes. Today, the most validated biomarker of estrogenic exposure is the expression in male liver of the vitellogenin (VTG), an estrogen-dependent glycolipophosphoprotein naturally expressed only in the liver of oviparous females during the reproductive season. This report summarizes the data available on the EDC-dependent expression and the synthesis of VTG in male vertebrates, highlighting our latest studies that demonstrate the ability of testis and epididymis of the lacertid Podarcis sicula to synthesize VTG following estrogenic exposure. These findings provide, for the first time, evidence on an extrahepatic expression and synthesis of VTG in a terrestrial vertebrate and lay the groundwork for a new value of the VTG as a biomarker of environmental contamination. In addition, the results open a new scenario on the role of VTG in cells other than oocytes.

Age-related changes of metallothionein 1/2 and metallothionein 3 expression in rat brain.

Neurodegeneration is one of the main physiological consequences of aging on brain. Metallothioneins (MTs), low molecular weight, cysteine-rich proteins that bind heavy-metal ions and oxygen-free radicals, are commonly expressed in various tissues of mammals. MTs are involved in the regulation of cell proliferation and protection, and may be engaged in aging. Expression of the ubiquitous MTs (1 and 2) and the brain specific MT3 have been studied in many neurodegenerative disorders. The research results indicate that MTs may play important, although not yet fully known, roles in brain diseases; in addition, data lack the ability to identify the MT isoforms functionally involved. The aim of this study was to analyse the level of gene expression of selected MT isoforms during brain aging. By using real-time PCR analysis, we determined the MT1/2 and MT3 expression profiles in cerebral cortex and hippocampus of adolescent (2months), adult (4 and 8months), and middle-aged (16months) rats. We show that the relative abundance of all types of MT transcripts changes during aging in both hippocampus and cortex; the first effect is a generalized decrease in the content of MTs transcripts from 2- to 8-months-old rats. After passing middle age, at 16months, we observe a huge increase in MT3 transcripts in both cortical and hippocampal areas, while the MT1/2 mRNA content increases slightly, returning to the levels measured in adolescent rats. These findings demonstrate an age-related expression of the MT3 gene. A possible link between the increasing amount of MT3 in brain aging and its different metal-binding behaviour is discussed.

Ectopic synthesis of vitellogenin in testis and epididymis of estrogen-treated lizard Podarcis sicula.

In oviparous vertebrates, vitellogenin (VTG) is the major yolk precursor synthesized in the liver of sexually mature females during the reproductive period. In males, the VTG gene is silent, but it may be activated by estradiol-17ß (E2) or estrogen-like substances. Until now, extra-hepatic expression and synthesis of VTG after estrogen exposure has been reported only for aquatic vertebrates. This study demonstrates the ability of testis and epididymis of the terrestrial oviparous lacertid Podarcis sicula to synthesize VTG following E2 exposure. The results of in situ hybridization and immunohistochemistry analysis show the presence of both VTG mRNA and protein in these districts besides the known induction in the liver. The possible contemporaneous uptake of the E2-induced hepatic VTG by means of the specific vitellogenin receptor has been also evaluated. Finally, histological analysis shows that the E2-treatment during the mating season impairs spermatogenesis.

Estrogenic contamination by manure fertilizer in organic farming: a case study with the lizard Podarcis sicula.

In the last years, worldwide organic farming has grown exponentially; as a consequence, the use of animal manure as a soil fertility source has become the principal agricultural choice. However, the use of manure as fertilizer can increase the amount of steroid hormone metabolites in the soil. In southern Italy, lacertidae lizards are the most abundant vertebrate group in agroecosystems and have been identified as potential model species for ecotoxicological studies. The aim of this study was to understand if the manure applied in organic farming has estrogen-like effects in the lizard Podarcis sicula. Adult male lizards were captured in two organic agricultural fields (manure-treated sites) and in an uncultivated field (control site). Lizards from the two organic farms displayed hepatic biosynthetic alterations typical of an estrogenic contamination; hepatocytes contained both vitellogenin and estrogen receptor alpha transcripts and proteins, detected by in situ hybridization and immunocytochemistry. The same cells did not show cadmium, lead and metallothionein accumulation, indicative of the lack of inorganic contamination. These findings suggest that exogenous estrogens, arising from the use of manure, could affect the welfare of wild animals and animal breeding, leading to bioaccumulation of estrogens in food chain, with possible risk for human consumers. For this reason, organic farming should implement the use of sustainable practices such as crop rotation to preserve the soil biological activity, rather than organic manure as fertilizer.

Interferences of an environmental pollutant with estrogen-like action in the male reproductive system of the terrestrial vertebrate Podarcis sicula.

Nonylphenol (NP) is classified among the endocrine disruptor chemicals with estrogen-like properties. It is widely used in many industries and to dilute pesticides in agriculture, and is known to affect the reproductive system of many aquatic and semi-aquatic organisms. This study aimed to verify how NP, administered via food and water, may interfere with the reproductive cycle of a terrestrial vertebrate. Our model was the male Italian wall lizard Podarcis sicula, a seasonal breeding species that may be naturally exposed to environmental pollution. From our findings it emerges that an NP-polluted diet administered during the mating period causes in this lizard a slowdown of spermatogenesis and affects the testicular and epididymal structure, making it similar to that of the non-reproductive period. The distribution in the testis and epididymis of mRNA for steroid hormone receptors, i.e., estrogen α and ß and androgen receptors, was also investigated. NP treatment inhibits the expression of AR, ERα, and ERß-mRNA in spermatogonia and primary spermatocytes and causes a switch-off of the secretory activity of the epididymal corpus by inducing the expression of ERα.

Expression of estrogen receptor alpha switches off secretory activity in the epididymal channel of the lizard Podarcis sicula.

The epididymis in the male reproductive tract allows the survival, viability, and storage of spermatozoa from the testis. In the lizard Podarcis sicula, the epididymis can be regionalized to an initial segment called the caput that comprises the efferent ductules, followed by the middle and terminal segments, respectively termed the corpus and cauda. By means of in situ hybridization and immunocytochemistry, we analyzed the expression of the estrogen receptors of the alpha and beta type (ERα and ERß) in Podarcis to test the responsiveness of the epididymal regions to estrogen in the annual reproductive cycle of this seasonal breeder. The results show that the efferent ductules and the cauda always express both ERα and ERß throughout the year. In the corpus, the expression of ERα takes place only at the end of the mating period and continues in the non-reproductive season whereas ERß is expressed in all phases of the cycle. During the mating season, the cells of the corpus are engaged in massive secretory activity and do not express ERα. Experimental administration of E(2) during this season does not change the expression of ERß, nor does it affect the efferent ductules and cauda; instead, it inhibits the secretory activity in the corpus and induces the expression of ERα. Taken together, our findings suggest that in the epididymis of Podarcis, the expression of ERα may act as a switch for the secretory activity of the epididymal corpus.

Expression of vitellogenin in the testis and kidney of the spotted ray Torpedo marmorata exposed to 17ß-estradiol.

In vertebrates, the liver was long thought to be the only site of vitellogenin (Vtg) production, but recent studies demonstrated that Vtg is also expressed in extrahepatic districts. The aim of this paper is to assess, by in situ hybridization and immunohistochemistry, the expression of Vtg in the testis and kidney of Torpedo marmorata exposed to 17ß-estradiol (E(2)). In treated samples vtg mRNA and Vtg were detected contemporaneously only in the testis; differently the kidney cells were positive to Vtg antibody, but negative to vtg mRNA. This is the first study to assess that male germ cells, after an exposure to E(2), synthesize Vtg in a stage-dependent manner. The presence of Vtg and the modifications observed in the kidney after E(2) treatment are discussed.

Experimentally nonylphenol-polluted diet induces the expression of silent genes VTG and ERα in the liver of male lizard Podarcis sicula.

Endocrine Disruptor Chemicals (EDCs) with estrogen-like properties i.e nonylphenol (NP) induce vitellogenin (VTG) synthesis in males of aquatic and semi-aquatic species. In the oviparous species VTG is a female-specific oestrogen dependent protein. Males are unable to synthesize VTG except after E2 treatment. This study aimed to verify if NP, administered via food and water, is able to induce the expression of VTG even in males of vertebrates with a terrestrial habitat such as the lizard Podarcis. By means of ICC, ISH, W/B and ELISA we demonstrated that NP induces the presence of VTG in the plasma and its expression in the liver. VTG, undetectable in untreated males, reaches the value of 4.34 µg/µl in the experimental ones. Expression analysis and ISH in the liver showed that an NP-polluted diet also elicits the expression of ERα in the liver which is known to be related to VTG synthesis in Podarcis.

Molecular identification of estrogen receptors (ERalpha and ERbeta) and their differential expression during VTG synthesis in the liver of lizard Podarcis sicula.

In non-mammalian vertebrates yolk deposition in the oocytes is a hormone-dependent, gender-specific process. Produced by the ovary under gonadotropin stimulation, Estradiol 17-beta (E(2)) plays a key role in the liver synthesis of vitellogenin (VTG) which in turn is taken up by vitellogenic oocytes in the ovary. In many species a negative role in liver synthesis of VTG in females is also played by progesterone. Experimental administration of E(2) induces the expression of the VTG silent gene also in the liver of males of all the species studied. However, the role of the two isoforms of estrogen receptors, ERalpha and beta, in this process is still unclear. In order to elucidate what kind of ER is involved in the liver synthesis of VTG in the lizard Podarcis sicula, we obtained by means of RT-PCR two fragments of 430bp and 130bp from total ovarian mRNA, encoding respectively for ERalpha and ERbeta. Expression analysis of these two specific isoforms of ERs in the liver showed that in non-breeding females, and in wildlife untreated males only ERbeta is expressed. In breeding vitellogenic females and in E(2)-treated males both alpha and beta receptors are expressed. Furthermore, in females experimentally treated with progesterone during the breeding period, expression of ERalpha disappears. Conversely, treatment of females with E(2) in the non-breeding period induces expression of ERalpha. Immunohistochemical analysis and Western blotting showed that the presence of irVTG in liver and plasma is always parallel to hepatic expression of ERalpha in all the different experimental conditions. Our data strongly suggest that expression of ERalpha may be necessary for VTG synthesis in Podarcis. The possible modulatory role of ERbeta is also discussed.