No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Seasonal and pharmaceutical‐induced changes in selenoprotein glutathione peroxidase 4 activity in the reproductive dynamics of the soil biosentinel Podarcis sicula (Chordata: Reptilia )
Abstract
There is rising concern for the interaction of environmental contaminants with brain transcriptome and the potential effect on reproductive processes. The present study sought to determine selenoprotein glutathione peroxidase 4 (gpx4) transcriptional activity in the brain and testis of the soil biosentinel, Podarcis sicula, through the main phases of the reproductive cycle and whether pharmaceuticals exert an endocrine disruption. Based on gpx4 cloned amminoacids sequence (GenBank AEX09236.1.), we used a bioinformatic approach to assess the structural role. Specifically, we detected seasonally the reactive oxygen species (ROS) level using electron spin resonance spectroscopy and gpx4 transcriptional activity using quantitative real‐time polymerase chain reaction. In addition, the impact of pharmaceuticals was assessed after 21‐days of treatment with ICI 182,780 and human chorionic gonadotropin administration in mating and winter stasis, respectively. Bioinformatic data shows the gpx4 proteic activity and a phylogenetic profile. ROS contents in lizard brain are significantly less than in testis and display higher levels after treatments. Brain gpx4 expression gives statistically significant seasonal differences, opposite trends in testis and altered expression in both tissues, with evidence of testis morphological and DNA disruption. Taken together, these results provide direct evidence that gpx4 in P. sicula plays a seasonal regulatory role and may be a reliable biomarker for reproductive health toxicity screening.
... Among the major health concerns worldwide is the massive release of toxic compounds into the natural environment including soil and water [1,2]. Many of these compounds are defined as metals and are dangerous even at minimal concentrations and which may be cytotoxic, carcinogenic and mutagenic in nature [3,4]. ...
... They occur in the environment from natural and anthropogenic sources [5,6]. Dietary contamination by these chemical elements gives rise to numerous adverse effects on human and animal physiology [1,2,7,8]. These compounds may seriously affect cellular processes [7]. ...
... Heavy metal accumulation in fish has the potential to induce toxicological effects [1,2] and cause oxidative damage to tissues determining cell function loss [11,12]. As ionic Cu and Zn inhibit a number of enzymes, it follows that the basis for their toxicity may be due to their diminished activity. ...
A twofold integrated research study was conducted; firstly, to understand the effects of copper (Cu) and zinc (Zn) on the growth and oxidative stress in Nile tilapia, Oreochromis niloticus; secondly, to study the beneficial effects of the duckweed Lemna minor L. as a heavy metal remover in wastewater. Experiments were conducted in mesocosms with and without duckweed. Tilapia fingerlings were exposed to Cu (0.004 and 0.02 mg L −1) and Zn (0.5 and 1.5 mg L −1) and fish fed for four weeks. We evaluated the fish growth performance, the hepatic DNA structure using comet assay, the expression of antioxidative genes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPx and glutathione-S-transferase, GST) and GPx and GST enzymatic activity. The results showed that Zn exhibited more pronounced toxic effects than Cu. A low dose of Cu did not influence the growth whereas higher doses of Cu and Zn significantly reduced the growth rate of tilapia compared to the control, but the addition of duckweed prevented weight loss. Furthermore, in the presence of a high dose of Cu and Zn, DNA damage decreased, antioxidant gene expressions and enzymatic activities increased. In conclusion, the results suggest that duckweed and Nile tilapia can be suitable candidates in metal remediation wastewater assessment programs.
... It is now well known that, in the coming decades, the loss of biodiversity will have a dramatic impact on the life cycles of organisms [1][2][3]. Anthropogenic human activities and xenobiotics released into the environment such as metals, pesticides, herbicides, drugs, antifouling compounds, nanoparticles, and plastics directly influence migration, seasonal breeding, and reproduction [4][5][6]. These factors also have secondary deleterious effects contributing to climate change, as evidenced in global warming and ocean acidification, raising an alarm in the scientific community and government environmental agencies about reprotoxicity [7,8]. ...
... These factors also have secondary deleterious effects contributing to climate change, as evidenced in global warming and ocean acidification, raising an alarm in the scientific community and government environmental agencies about reprotoxicity [7,8]. In particular, there is growing evidence on how higher temperatures can act together with even low chemical concentrations of pollutants to elicit significant effects, undermining the traditional risk assessment paradigm for establishing threshold conditions below which a compound is not considered a threat [5,9]. Furthermore, "environmental endocrinology", focusing on active hormonal compounds, is bringing to light the hormonal mechanisms acting in response to changing environments [5,10,11]. ...
... In particular, there is growing evidence on how higher temperatures can act together with even low chemical concentrations of pollutants to elicit significant effects, undermining the traditional risk assessment paradigm for establishing threshold conditions below which a compound is not considered a threat [5,9]. Furthermore, "environmental endocrinology", focusing on active hormonal compounds, is bringing to light the hormonal mechanisms acting in response to changing environments [5,10,11]. ...
The objective of this review is to briefly summarize the recent progress in studies done on the assessment of reprotoxicity risk posed by global warming for the foundation of strategic tool in ecosystem-based adaptation. The selected animal data analysis that was used in this paper focuses on antioxidative markers and fertility rate estimated over the period 2000–2019. We followed a phylogenetic methodology in order to report data on a panel of selected organisms that show dangerous effects. The oxidative damage studies related to temperature fluctuation occurring in biosentinels of different invertebrate and vertebrate classes show a consistently maintained physiological defense. Furthermore, the results from homeothermic and poikilothermic species in our study highlight the influence of temperature rise on reprotoxicity.
... The quality of groundwater and surface water in rural and urban environments is influenced by natural and anthropogenic processes (Tang et al. 2014;Fasulo et al. 2015;Lecomte et al. 2017;Vardhan et al. 2019). With anthropogenic human activities, there has been a significant increase in the discharge of industrial waste into the environment, especially in urban areas, which creates an alarming situation for human life and aquatic biota (Jan et al. 2015;Lecomte et al. 2017;Guerriero et al. 2019). The anthropogenic factors include discharge of organic pollutants such as drugs, personal care products, steroids, hormones, endocrine disruptors, surfactants, phosphoric esters, flame retardants, industrial additives and siloxanes into natural freshwater bodies (Jaouadi et al. 2012;Khatri and Tyagi 2015;Lecomte et al. 2017;Parisi and Guerriero 2019). ...
... The documented effects of pollutants on biosentinels, such as amphibians, range from lethal to sublethal effects and include decreased growth and fertility, increased frequency of developmental abnormalities, increased susceptibility to disease, behavioral changes, and damage to germ lines (Ortiz et al. 2004;Karraker et al. 2008;Shinn et al. 2008;Snodgrass et al. 2008;Guerriero et al. 2009;Guerriero et al. 2011;Guerriero et al. 2019;Parisi and Guerriero 2019). Furthermore, due to the feeding habits of larval microphages of most species, toads frequently ingest sediments through which metals can be accumulated (Hopkins and Rowe 2010;Burlibaşa and Gavrilȃ 2011;James and Semlitsch 2011). ...
Our study attempted to monitor the quality of water in Medjarda basin (Northeastern Algeria) and to provide baseline information of heavy metals in the water as well as in a potential amphibian biosentinel, the spiny toad, Bufo spinosus. We measured pH, temperature, dissolved oxygen and biological oxygen demand of water and levels of heavy metals in toad skin using an atomic absorption flame spectrophotometer. Lead (Pb) concentration in water and in toad skin at all sites exceeded respectively 60 and 96 times the standard reference values. The heavy metal concentrations, in descending order, in water and in male toad skin were as follows: Pb > Fe > Cu > Zn and Fe > Pb > Zn > Cu respectively. This study highlights the ecological status of the surrounding areas upstream of the Medjarda basin as being a point source of heavy metal pollution. It is further stated that a non-inva-sive skin removal is an ethically sound technique to evaluate heavy metal accumulation in aquatic animals like toad, without euthanizing the specimens and making any loss to biodiversity of the species.
... The quality of groundwater and surface water in rural and urban environments is influenced by natural and anthropogenic processes (Tang et al. 2014;Fasulo et al. 2015;Lecomte et al. 2017;Vardhan et al. 2019). With anthropogenic human activities, there has been a significant increase in the discharge of industrial waste into the environment, especially in urban areas, which creates an alarming situation for human life and aquatic biota (Jan et al. 2015;Lecomte et al. 2017;Guerriero et al. 2019). The anthropogenic factors include discharge of organic pollutants such as drugs, personal care products, steroids, hormones, endocrine disruptors, surfactants, phosphoric esters, flame retardants, industrial additives and siloxanes into natural freshwater bodies (Jaouadi et al. 2012;Khatri and Tyagi 2015;Lecomte et al. 2017;Parisi and Guerriero 2019). ...
... The documented effects of pollutants on biosentinels, such as amphibians, range from lethal to sublethal effects and include decreased growth and fertility, increased frequency of developmental abnormalities, increased susceptibility to disease, behavioral changes, and damage to germ lines (Ortiz et al. 2004;Karraker et al. 2008;Shinn et al. 2008;Snodgrass et al. 2008;Guerriero et al. 2009;Guerriero et al. 2011;Guerriero et al. 2019;Parisi and Guerriero 2019). Furthermore, due to the feeding habits of larval microphages of most species, toads frequently ingest sediments through which metals can be accumulated (Hopkins and Rowe 2010;Burlibaşa and Gavrilȃ 2011;James and Semlitsch 2011). ...
Our study attempted to monitor the quality of water in Medjarda basin (Northeastern Algeria) and to provide baseline information of heavy metals in the water as well as in a potential amphibian biosentinel, the spiny toad, Bufo spinosus. We measured pH, temperature, dissolved
oxygen and biological oxygen demand of water and levels of heavy metals in toad skin using an atomic absorption flame pectrophotometer. Lead (Pb) concentration in water and in toad skin at all sites exceeded respectively 60 and 96 times the standard reference values.
The heavy metal concentrations, in descending order, in water and in male toad skin were as follows: Pb[Fe[ Cu[Zn and Fe[Pb[Zn[Cu respectively. This study highlights the ecological status of the surrounding areas upstream of the Medjarda basin as being a point source of heavy metal pollution. It is further stated that a non-invasive
skin removal is an ethically sound technique to evaluate heavy metal accumulation in aquatic animals like toad, without euthanizing the specimens and making any loss to biodiversity of the species.
... Mussels are also known to exhibit signs of oxidative stress by overexpression of free radicals when exposed to toxic compounds, which are harmful to cells because they cause disruption of cell membrane fluidity by peroxidation, as well as DNA damage in the form of degradation, base deletions and mutations [46][47][48]. Indeed, antioxidant enzymes which counteract oxidative stress are used on this biofouling model as contaminant/stress-related biomarkers [49][50][51][52]. Thus, the primary objective of the present review is to summarise and collect information available in the literature on the effect of eco-friendly and biocide AFs and their role in the antioxidative defence and settlement in M. galloprovincialis as a biofouling model organism. ...
Antifoulant paints were developed to prevent and reduce biofouling on surfaces immersed in seawater. The widespread use of these substances over the years has led to a significant increase of their presence in the marine environment. These compounds were identified as environmental and human threats. As a result of an international ban, research in the last decade has focused on developing a new generation of benign antifoulant paints. This review outlines the detrimental effects associated with biocide versus eco-friendly antifoulants, highlighting what are effective antifoulants and why there is a need to monitor them. We examine the effects of biocide and eco-friendly antifoulants on the antioxidative defence mechanism and settlement in a higher sessile organism, specifically the Mediterranean mussel, Mytilus galloprovincialis. These antifoulants can indirectly assess the potential of these two parameters in order to outline implementation of sustainable antifoulants.
... Indeed, the expression of gpx4 transcript increases following treatment with 17b-estradiol (Nam et al. 2003) and in conditions of oxidative stress. It suggests that like gpx, gpx4 plays an antioxidant role in male fertility (Guthrie and Welch 2012;Guerriero et al. 2014Guerriero et al. , 2018cGuerriero et al. , 2019. ...
The most representative organisms of the Harbor of Gaeta Gulf in South Italy were analyzed for biofouling by visual census and confirmed later by molecular approach on an artificial Conatex panel dipped 3 m into a eutrophic area during the Covid-19 pandemic. Mitochondrial Cytochrome C oxidase subunit 1(COI) gene was sequenced from 20 different marine species (flora: 2 families, 2 orders; fauna: 16 families, 11 orders) to test whether the morphology-based assignment of the most common biofouling member was supported by DNA-based species identification. Twelve months of submersion resulted in generation of sufficient data to obtain a facies climax represented mainly by the bivalve mollusk, Mytilus galloprovincialis. Specific diversity and variations of the biofouling biomasses were analyzed using two different anti-biofouling paints: an endocrine disrupting chemical (EDC)-containing metal biocide, and a biocide-free paint. Also, their effects on detoxification and reproductive health of M. galloprovincialis were evaluated using glutathione S-transferase enzymatic activity and RTqPCR expression of the fertility antioxidant gene glutathione peroxidase 4 (gpx4). The obtained data provide useful indications on which future investigations may be focused and may become a potential management tool for a harbor biofouling database to assist local administrations in EDCs protection of autochthonous benthic communities and their fertility using innovative antifouling paints.
... Our prior study also showed that obesity was related to male infertility [11]. Some studies and our prior study considered that oxidative stress may play the most important role [11,12] and that oxidative stress induced by obesity damages male reproduction maybe through superoxide generation, oxidative phosphorylation, and so on [13][14][15]. Therefore, an antioxidant substance may provide a protective effect on the damage induced by obesity. ...
(1) Background: In recent decades, the prevalence of obesity has grown rapidly worldwide, thus causing many diseases, including male hypogonadism. Sulforaphane (SFN), an isothiocyanate compound, has been reported to protect the reproductive system. This research investigated the protective effect of SFN against obesity-induced impairment in the male reproductive system and explored the potential mechanism involved in mice. (2) Methods: One hundred thirty mice were divided into 5 groups (Control, DIO (diet-induced obesity), DIO + SFN 5 mg/kg, DIO + SFN 10 mg/kg, and DIO + SFN 20 mg/kg). The effects of SFN on the male reproductive system were determined based on the sperm count and motility, relative testes and epididymis weights, hormone levels, and pathological analyses. Oxidative stress was determined by measuring malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione (GSH), H2O2, catalase (CAT), and glutathione peroxidase (GSH-PX) levels. Protein expression of nuclear factor erythroid-2 related factor 2 (Nrf2), Kelch-like ECH-associated protein-1 (Keap1), Microtubule-associated protein light chain 3 (LC3), Beclin1, and P62 were determined by western blotting. (3) Results: High-fat diet (HFD)-induced obesity significantly decreased relative testes and epididymis weights, sperm count and motility, and testosterone levels but increased leptin and estradiol levels. SFN supplementation ameliorated these effects. Additionally, SFN administration inhibited the obesity-induced MDA accumulation and increased the SOD level. Western blot indicated that SFN had an important role in the downregulation of Keap1. Moreover, SFN treatment attenuated obesity-induced autophagy, as detected by LC3 and Beclin1. (4) Conclusions: SFN ameliorated the reproductive toxicity associated with obesity by inhibiting oxidative stress mediated by the nuclear factor erythroid-2 related factor 2/ antioxidant response element (Nrf2/ARE) signaling pathway and recovery of normal autophagy.
The removal of heavy metals from industrial waste has become crucial in order to maintain water quality levels that are suitable for environmental and species reproductive health. The biosorption of Zn+2 and Fe+3 ions from aqueous solution was investigated using Ulva lactuca green algal biomass and Corallina officinalis red algal biomass, as well as their activated carbons. The effects of biosorbent dosage, pH, contact time, initial metal concentration, and temperature on biosorption were evaluated. The maximum monolayer capacity of Ulva lactuca and Corallina officinalis dry algal powder and algal activated carbon was reached at pH 5 and 3 for Zn+2 and Fe+3, respectively, while the other factors were similar for both algae, which were: contact time 120 min, adsorbent dose 1 g, temperature 40 °C and initial concentrations of metal ion 50 mg·L−1. The batch experimental data can be modelled using the Langmuir and Freundlich isotherm models. Thermodynamic characteristics revealed that the adsorption process occurs naturally and is endothermic and spontaneous. For the adsorption of Zn+2 and Fe+3 ions, the value of G° was found to be negative, confirming the practicality of the spontaneous adsorption process, which could be helpful for remediation in the era of temperature increases.
Purpose
In this study, a novel glutathione (GSH) surface molecular imprinting polymer (SMIP) was successfully prepared by using macroporous adsorption resins (MAR) as substrate, which could separate and purify GSH efficiently.
Design/methodology/approach
SMIP was synthesized by chloromethylated modified MAR (LX1180-Cl) as the substrate, N, N’-methylenebisacrylamide (NMBA) as a crosslinker, GSH as a template, acrylamide (AM) and N-vinylpyrrolidone (NVP) as functional monomers. The morphology and structure of the polymer were characterized by scanning electron microscope and Fourier transforms infrared spectroscopy.
Findings
The maximum adsorption capacity toward GSH was 39.0 mg/g and the separation decree had relation to L-cysteine (L-cys) was 4.2. The optimal operation conditions were studied in detail and the got as follows: the molar ratios of NMBA, AM, GSH and NVP, were 7.0, 0.8 and 0.5. The optimal time and temperature were 14 h and 40°C, respectively. The Langmuir and pseudo-first-order model were fitting these adsorption characteristics well.
Practical implications
GSH has a diversity of medicinal and bioactive functions, so the purpose of this study representing a method in separate and purify technology of GSH, which provided a way for the development of medicine.
Originality/value
This contribution provided a novel way to separate GSH from L-cys. Under the optimal conditions, the maximum adsorption capacity toward GSH was 39.0 mg/g and the separation decree had relation to L-cys was 4.2.
In this study, temperature variations and invasion on marine fauna are investigated to ascertain their relationship in the Mediterranean at the Gaeta Gulf (Tyrrhenian Sea). Sea temperatures were collected from 1986 to 2009, the only period in which the AVHRR sensor installed on NOAA satellite was available. Changes in the sea surface temperature from satellite using GIS software were estimated. Research on marine species was carried out using the barcoding technique. Nine organisms of which one Anthozoa and one Malacostraca and eight belonging to Actinopterygii Class were barcoded from 2005 to 2019 and four of these were detected as non-indigenous species. The first sightings are related to Fistularia commersonii, and Sphoeroides pachygaster identified since 2009 and well reported in our previous publication. They have penetrated the Mediterranean from the Indian Ocean (through the Suez Canal) and from the Atlantic Ocean (Strait of Gibraltar) respectively and are still sporadic. These bioinvasions were linked for the first time to water heating, which increased by about 0.4 °C between 1989–1999 and 1999–2009. In addition, there are other identifications, such as Arothron diadematus from the Suez Canal, Percnon gibbesi from the Strait of Gibraltar; thermophilus indigenous species such as Pomadasys incisus and Astroides calycularis and sporadic autochthonous species such as Sudis hyalina, Tetragonurus cuvieri, Trachipterus trachipterus, and Remora osteochir. Non-native species and their invasions could lead to radical and sudden upheavals in the Mediterranean marine ecosystem and therefore require constant alert of sea surface temperature change.
Soil contaminants can cause direct harm to lizards due to their regular swallowing of soil particles. As the world's fastest growing insecticide with long half-life in soil, the endocrine disrupting effect of neonicotinoids on lizards deserves more attention. In this report, we assessed the endocrine disrupting effect of imidacloprid on Eremias argus during 28 days of continuous exposure. Among the imidacloprid and its metabolites, only the metabolite 6-chloropyridic acid had a significant accumulation in the gonads and was positively correlated with its blood concentration. Imidacloprid might cause endocrine disrupting effects on lizards in two ways. First, the desnitro metabolites of imidacloprid could accumulate in the brain, inhibited the secretion of gonadotropin-releasing hormone, and ultimately affected the feedback regulation of hypothalamic-pituitary-gonadal related hormones. Secondly, imidacloprid severely inhibited the gene expression of the corresponding enzymes in the gonadal anti-oxidative stress system, causing histological damage to the gonads and ultimately affecting gonadal function. Specifically, exposure to imidacloprid resulted in abnormal arrangement of spermatogenic epithelial epithelium, hyperplasia of epididymal wall, and oligospermia of male lizard. Meanwhile, gene expressions of cyp17, cyp19, and hsd17β were severely inhibited in the imidacloprid exposure group, consistent with decreased levels of testosterone and estradiol in plasma. Imidacloprid exposure could cause insufficient androgen secretion and less spermatogenesis in male lizards. The risk of imidacloprid exposure to female lizards was not as severe as that of male lizards, but it still inhibited the expression of cyp19 in the ovaries and led to a decrease in the synthesis of estradiol. This study firstly reported the endocrine disruption of imidacloprid to lizards, providing new data for limiting the use of neonicotinoids.
Selenium (Se) is an essential micronutrient that has several important functions in animal and human health. The biological functions of Se are carried out by selenoproteins (encoded by twenty-five genes in human and twenty-four in mice), which are reportedly present in all three domains of life. As a component of selenoproteins, Se has structural and enzymatic functions; in the latter context it is best recognized for its catalytic and antioxidant activities. In this review, we highlight the biological functions of Se and selenoproteins followed by an elaborated review of the relationship between Se and female reproductive function. Data pertaining to Se status and female fertility and reproduction are sparse, with most such studies focusing on the role of Se in pregnancy. Only recently has some light been shed on its potential role in ovarian physiology. The exact underlying molecular and biochemical mechanisms through which Se or selenoproteins modulate female reproduction are largely unknown; their role in human pregnancy and related complications is not yet sufficiently understood. Properly powered, randomized, controlled trials (intervention vs. control) in populations of relatively low Se status will be essential to clarify their role. In the meantime, studies elucidating the potential effect of Se supplementation and selenoproteins (i.e., GPX1, SELENOP, and SELENOS) in ovarian function and overall female reproductive efficiency would be of great value.
Aims
Ferroptosis, a new form of iron‐dependent programmed cell death, has been shown to be involved in a range of diseases. However, the role of ferroptosis in traumatic brain injury (TBI) has yet to be elucidated. We aimed to investigate whether ferroptosis is induced after TBI and whether the inhibition of ferroptosis would protect against traumatic brain injury in a controlled cortical impact injury (CCI) mouse model.
Methods
After establishing the TBI model in mice, we determined the biochemical and morphological changes associated with ferroptosis, including iron accumulation with Perl's staining, neuronal cell death with Fluoro‐Jade B (FJB) staining, iron metabolism dysfunction with Western blotting, reactive oxygen species (ROS) accumulation with malondialdehyde (MDA) assays, and shrunken mitochondria with transmission electron microscopy. Furthermore, a specific inhibitor of ferroptosis, ferrostatin‐1(fer‐1), was administrated by cerebral ventricular injection after CCI. We used cresyl violet (CV) staining to assess lesion volume, along with the Morris water maze and beam walk test to evaluate long‐term outcomes.
Results
TBI was followed by iron accumulation, dysfunctional iron metabolism, the upregulation of ferroptosis‐related genes, reduced glutathione peroxidase (GPx) activity, and the accumulation of lipid‐reactive oxygen species (ROS). Three days (d) after TBI, transmission electron microscopy (TEM) confirmed that the mitochondria had shrunk a typical characteristic of ferroptosis. Importantly, the administration of Fer‐1 by cerebral ventricular injection significantly reduced iron deposition and neuronal degeneration while attenuating injury lesions and improving long‐term motor and cognitive function.
Conclusion
This study demonstrated an effective method with which to treat TBI by targeting ferroptosis.
Estradiol plays a critical role in stimulating the fetal hypothalamus-pituitary-adrenal axis at the end of gestation. Estradiol action is mediated through nuclear and membrane receptors that can be modulated by ICI 182,780, a pure antiestrogen compound. The objective of this study was to evaluate the transcriptomic profile of estradiol and ICI 182,780, testing the hypothesis that ICI 182,780 antagonizes the action of estradiol in the fetal hypothalamus. Chronically catheterized ovine fetuses were infused for 48 h with: vehicle (Control, n = 6), 17β-estradiol 500 μg/kg/day (Estradiol, n = 4), ICI 182,780 5 μg/kg/day (ICI 5 μg, n = 4) and ICI 182,780 5 mg/kg/day (ICI 5 mg, n = 5). Fetal hypothalami were collected afterward, and gene expression was measured through microarray. Statistical analysis of transcriptomic data was performed with Bioconductor-R and Cytoscape software. Unexpectedly, 35% and 15.5% of the upregulated differentially expressed genes (DEG) by Estradiol significantly overlapped (P < 0.05) with upregulated DEG by ICI 5 mg and ICI 5 μg, respectively. For the downregulated DEG, these percentages were 29.9% and 15.5%, respectively. There was almost no overlap for DEG following opposite directions between Estradiol and ICI ICI 5 mg or ICI 5 μg. Furthermore, most of the genes in the estrogen signaling pathway - after activation of the epidermal growth factor receptor - followed the same direction in Estradiol, ICI 5 μg or ICI 5 mg compared to Control. In conclusion, estradiol and ICI 182,780 have estrogenic genomic effects in the developing brain, suggesting the possibility that the major action of estradiol on the fetal hypothalamus involves another receptor system rather than estrogen receptors.
Over the last few decades, due to its relevant function in male reproduction assessment, important molecular achievements have been made in the molecular characterization of estrogen receptor genes in various species. Our work focuses on a male seasonal breeder, the bioindicator Podarcis sicula, because of its peculiar gonadal anatomy, similar to that of humans. Based on the cloned lizard’s gene sequence fragment of estrogen receptor beta, esr2 (GenBank JN705543.1), we found DNA binding domain identity of 99% as well as a homologous sequence with humans. Furthermore, in order to better illustrate how this gene is regulated in the lizard’s reproductive system organs, we investigated the transcriptional activity of esr2 in brain and testis tissues during mating and winter stasis phases of the reproductive cycle. Quantitative real time-polymerase chain reaction (qRT-PCR) analyses performed on male gonadal tissues demonstrate a significant increase in esr2 expression during mating compared to the winter stasis period, while in the brain, esr2 shows the opposite trend. Next, we provide morphological evidence of the detrimental effect on spermatogenesis of a pure anti-estrogen treatment (ICI 182,780) and the corresponding effect on esr2 expression in lizard specimens during the mating period which, upon treatment, was found to be no different from the expression levels in winter stasis both in the brain and in the testis. In this study, we explore the potential use of Podarcis sicula as a model for human testis development and maturation, as well as esr2 expression for toxicological screening in one-testis gonadectomy.
Background:
Reactive oxygen species (ROS) in biological tissues of elected biosentinels represent an optimal biomarker for eco-monitoring of polluted areas. Electron spin resonance (ESR) is the most definitive method for detecting, quantifying and possibly identifying radicals in complex systems.
Objective:
A non-invasive method for monitoring polluted areas by the quantitative determination of ROS in frog skin biopsy is presented.
Methods:
We assessed by ESR spectroscopy the ROS level in adult male of Pelophylax bergeri, specie not a risk of extinction, collected from the polluted Sarno River (SA, Italy) basin. The spin-trap ESR method was validated by immunohistochemical analysis of the well-assessed pollution biomarkers cytochrome P450 aromatase 1A (CYP1A) and glutathione S-transferase (GST), and by determining the poly(ADPribose) polymerase (PARP) and GST enzymatic activity.
Results:
ROS concentration in skin samples from frogs collected in the polluted area is significantly higher than that determined for the unpolluted reference area. Immunohistochemical analysis of CYP1A and GST supported the reliability of our approach, even in the absence of evident morphological and ultrastructural differences. PARP activity assay, connected to possible oxidative DNA damage, and the detoxification index by GST enzymatic assay give statistically significant evidence that higher levels of ROS are associated to alterations of the different biomarkers.
Conclusions:
ROS concentration, measured by ESR on isolated frog skin, through the presented non-lethal method, is a reliable biomarker for toxicity screening and represents a useful basic datum for future modelling studies on environmental monitoring and biodiversity loss prevention.
Introduction
Though the human fetus is exposed to placentally derived human chorionic gonadotropin (hCG) throughout gestation, the role of hCG on the fetal brain is unknown. Review of the available literature appears to indicate that groups of women with higher mean levels of hCG during pregnancy tend to have offspring with lower cerebral palsy (CP) risk. Given that newborn cerebral injury often precedes the development of CP, we aimed to determine whether hCG may protect against the neurodegenerative effects of neonatal brain injury.
Methods
We utilized the Rice–Vannucci model of neonatal cerebral hypoxia-ischemia (HI) in postnatal day 7 mice to examine whether intraperitoneal administration of hCG 15–18 h prior, 1 h after or immediately following HI decrease brain tissue loss 7 days after injury. We next studied whether hCG has pro-survival and trophic properties in neurons by exposing immature cortical and hippocampal neurons to hCG in vitro and examining neurite sprouting and neuronal survival prior and after glutamate receptor-mediated excitotoxic injury.
Results
We found that intraperitoneal injection of hCG 15 h prior to HI, but not at or 1 h after HI induction, resulted in a significant decrease in hippocampal and striatal tissue loss 7 days following brain injury. Furthermore, hCG reduced N-methyl-d-aspartate (NMDA)-mediated neuronal excitotoxicity in vitro when neurons were continuously exposed to this hormone for 10 days or when given at the time and following neuronal injury. In addition, continuous in vitro administration of hCG for 6–9 days increased neurite sprouting and basal neuronal survival as assessed by at least a 1-fold increase in MAP2 immunoreactivity and a 2.5-fold increase in NeuN + immunoreactivity.
Conclusion
Our findings suggest that hCG can decrease HI-associated immature neural degeneration. The mechanism of action for this neuroprotective effect may partly involve inhibition of NMDA-dependent excitotoxic injury. This study supports the hypothesis that hCG during pregnancy has the potential for protecting the developing brain against HI, an important CP risk factor.
Central nervous system (CNS) chemical protection depends upon discrete control of small-molecule access by the blood-brain barrier (BBB). Curiously, some drugs cause CNS side-effects despite negligible transit past the BBB. To investigate this phenomenon, we asked whether the highly BBB-enriched drug efflux transporter MDR1 has dual functions in controlling drug and endogenous molecule CNS homeostasis. If this is true, then brain-impermeable drugs could induce behavioral changes by affecting brain levels of endogenous molecules. Using computational, genetic, and pharmacologic approaches across diverse organisms, we demonstrate that BBB-localized efflux transporters are critical for regulating brain levels of endogenous steroids and steroid-regulated behaviors (sleep in Drosophila and anxiety in mice). Furthermore, we show that MDR1-interacting drugs are associated with anxiety-related behaviors in humans. We propose a general mechanism for common behavioral side effects of prescription drugs: pharmacologically challenging BBB efflux transporters disrupts brain levels of endogenous substrates and implicates the BBB in behavioral regulation.
Important toxicological achievements have been made during the last decades using reptiles. We focus our investigation on gonadal reproductive health of the soil biosentinel Podarcis sicula which is very sensitive to endocrine-disrupting chemicals. The aim of this study is to quantitatively detect, by sensitive microassays, reactive oxygen species and the glutathione antioxidants in the testis and investigate if they are differentially expressed before and after remediation of a site of the "Land of Fires" (Campania, Italy) subject to illicit dumping of unknown material. The oxidative stress level was evaluated by electron spin resonance spectroscopy applying a spin-trapping procedure able to detect products of lipid peroxidation, DNA damage and repair by relative mobility shift, and poly(ADP-ribose) polymerase enzymatic activity, respectively, the expression of glutathione peroxidase 4 transcript by real-time quantitative PCR analysis, the antioxidant glutathione S-transferase, a well-assessed pollution index, by enzymatic assay and the total soluble antioxidant capacity. Experimental evidences from the different techniques qualitatively agree, thus confirming the robustness of the combined experimental approach. Collected data, compared to those from a reference unpolluted site constitute evidence that the reproductive health of this lizard is impacted by pollution exposure. Remediation caused significant reduction of reactive oxygen species and downregulation of glutathione peroxidase 4 mRNAs in correspondence of reduced levels of glutathione S-transferase, increase of antioxidant capacity, and repair of DNA integrity. Taken together, our results indicate directions to define new screening approaches in remediation assessment.
The number and amount of man-made chemicals present in the aquatic environment has increased considerably over the past 50 years. Among these contaminants, endocrine-disrupting chemicals (EDCs) represent a significant proportion. This family of compounds interferes with normal hormonal processes through multiple molecular pathways. They represent a potential risk for human and wildlife as they are suspected to be involved in the development of diseases including, but not limited to, reprotoxicity, metabolic disorders, and cancers. More precisely, several studies have suggested that the increase of breast cancers in industrialized countries is linked to exposure to EDCs, particularly estrogen-like compounds. Estrogen receptors alpha (ERα) and beta (ERβ) are the two main transducers of estrogen action and therefore important targets for these estrogen-like endocrine disrupters. More than 70% of human breast cancers are ERα-positive and estrogen-dependent, and their development and growth are not only influenced by endogenous estrogens but also likely by environmental estrogen-like endocrine disrupters. It is, therefore, of major importance to characterize the potential estrogenic activity from contaminated surface water and identify the molecules responsible for the hormonal effects. This information will help us understand how environmental contaminants can potentially impact the development of breast cancer and allow us to fix a maximal limit to the concentration of estrogen-like compounds that should be found in the environment. The aim of this review is to provide an overview of emerging estrogen-like compounds in the environment, sum up studies demonstrating their direct or indirect interactions with ERs, and link their presence to the development of breast cancer. Finally, we emphasize the use of in vitro and in vivo methods based on the zebrafish model to identify and characterize environmental estrogens.
Oxidative stress is an important factor for development of male infertility because of very high rate of cell division and mitochondrial
oxygen consumption in testicular tissue as well as comparably higher levels of unsaturated fatty acids in this tissue than in other tissues.
Moreover, the level of oxygen pressure is low due to the weakness of testicular artery; therefore, there is a severe cell competition for
oxygen. Therefore, the testicular tissue and male reproductive system are particularly susceptible to oxidative stress. On the other
hand, exposure to X-ray, toxins and chemicals found in the environment as well as specific physical conditions such as varicocele
can exacerbate the oxidative stress and induce apoptosis of germ cells and subsequently spermatogenesis. However, under normal
conditions, the body's capacity to produce antioxidants for inhibiting adverse effects of oxidative stress is affected by metabolic process
and genetic structure. Besides that, environmental factors such as diet, pollutants, and chemicals can affect this capacity. Thus, the
body's antioxidant system alone is not able to neutralize all free radicals and prevent harmful complications of oxidative stress. Therefore,
use of antioxidants and development of antioxidant therapy can break down the oxidative chain reaction and play a very significant role
in increasing the body's capacity to fight free radical-induced oxidative stress, and therefore improve the process of spermatogenesis.
Synaptic loss and neuron death are the underlying cause of neurodegenerative diseases such as Alzheimer's disease (AD); however, the modalities of cell death in those diseases remain unclear. Ferroptosis, a newly identified oxidative cell death mechanism triggered by massive lipid peroxidation, is implicated in the degeneration of neurons populations such as spinal motor neurons and midbrain neurons. Here, we investigated whether neurons in forebrain regions (cerebral cortex and hippocampus) that are severely afflicted in AD patients might be vulnerable to ferroptosis. To this end, we generated Gpx4BIKO mouse, a mouse model with conditional deletion in forebrain neurons of glutathione peroxidase 4 (Gpx4), a key regulator of ferroptosis, and showed that treatment with tamoxifen led to deletion of Gpx4 primarily in forebrain neurons of adult Gpx4BIKO mice. Starting at 12 weeks after tamoxifen treatment, Gpx4BIKO mice exhibited significant deficits in spatial learning and memory function versus Control mice as determined by the Morris water maze task. Further examinations revealed that the cognitively impaired Gpx4BIKO mice exhibited hippocampal neurodegeneration. Notably, markers associated with ferroptosis, such as elevated lipid peroxidation, ERK activation and augmented neuroinflammation, were observed in Gpx4BIKO mice. We also showed that Gpx4BIKO mice fed a diet deficient in vitamin E, a lipid soluble antioxidant with anti-ferroptosis activity, had an expedited rate of hippocampal neurodegeneration and behavior dysfunction, and that treatment with a small-molecule ferroptosis inhibitor ameliorated neurodegeneration in those mice. Taken together, our results indicate that forebrain neurons are susceptible to ferroptosis, suggesting that ferroptosis may be an important neurodegenerative mechanism in diseases such as AD.
The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4 and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine-R-sulfoxide reductase 1) and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15 kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV) and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.
Genes of selenoproteome have been increasingly implicated in various aspects of neurobiology and neurological disorders, but remain largely elusive in Parkinson's disease (PD). In this study, we investigated the selenotranscriptome (24 selenoproteins in total) in five brain regions (cerebellum, substantia nigra, cortex, pons and hippocampus) by real time qPCR in a two-phase manner using a mouse model of chronic PD. A wide range of changes in selenotranscriptome was observed in a manner depending on selenoproteins and brain regions. While Selv mRNA was not detectable and Dio1& 3 mRNA levels were not affected, 1, 11 and 9 selenoproteins displayed patterns of increase only, decrease only, and mixed response, respectively, in these brain regions of PD mice. In particular, the mRNA expression of Gpx1-4 showed only a decreased trend in the PD mouse brains. In substantia nigra, levels of 17 selenoprotein mRNAs were significantly decreased whereas no selenoprotein was up-regulated in the PD mice. In contrast, the majority of selenotranscriptome did not change and a few selenoprotein mRNAs that respond displayed a mixed pattern of up- and down-regulation in cerebellum, cortex, hippocampus, and/or pons of the PD mice. Gpx4, Sep15, Selm, Sepw1, and Sepp1 mRNAs were most abundant across all these five brain regions. Our results showed differential responses of selenoproteins in various brain regions of the PD mouse model, providing critical selenotranscriptomic profiling for future functional investigation of individual selenoprotein in PD etiology.
A key computational principle for encoding time-varying signals in auditory and somatosensory cortices of monkeys is the opponent model of rate coding by two distinct populations of neurons. However, the subthreshold mechanisms that give rise to this computation have not been revealed. Because the rate-coding neurons are only observed in awake conditions, it is especially challenging to probe their underlying cellular mechanisms. Using a novel intracellular recording technique that we developed in awake marmosets, we found that the two types of rate-coding neurons in auditory cortex exhibited distinct subthreshold responses. While the positive-monotonic neurons (monotonically increasing firing rate with increasing stimulus repetition frequency) displayed sustained depolarization at high repetition frequency, the negative-monotonic neurons (opposite trend) instead exhibited hyperpolarization at high repetition frequency but sustained depolarization at low repetition frequency. The combination of excitatory and inhibitory subthreshold events allows the cortex to represent time-varying signals through these two opponent neuronal populations.
The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 (mGpx4) was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with targeted mutation of the active site selenocysteine (Sec) of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4-/- embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breedings and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoan midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mGpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared to Sec at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Since the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and being a structural protein, tightly controlled expression of functional Gpx4 emerges being key for full male fertility.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
In a growing deterioration of the ecosystem both in terms of biodiversity and ecological features, even more problematic is the conservation of species. The aim of this work was to study seasonal variations of biochemical and molecular DNA damage markers in Pelophylax bergeri exposed to potential endocrine disrupting chemicals. Frogs were collected in the main phase of the reproductive cycle: in April and May, during active spermatogenesis and the breeding season, and in October and November, in spermatogenetic regression, from a sampling site in the polluted Sarno River and from a clean reference site (Matese Lake). DNA profile and poly(ADPribose) polymerase (PARP) activity were evaluated in the testis. In the main phases of the reproductive cycle, all specimens from Sarno River showed a PARP activity higher than that measured in the same frog’s testis from Matese Lake. In addition, the PARP activity in active spermatogenesis was always higher compared with the activity in the spermatogenetic regression in Sarno River frogs. PARP2, usually activated in response to exposure to heavy metals, was expressed in all testes. In the species examined from Sarno River, no evident correlation between testis DNA damage and PARP activation was found. The working hypothesis is that PARP, implicated in genome surveillance and protection, might represent in frog spermatogenesis an appealing tool for genotoxic risk assessment useful to define a warning alarm for its survival.
Spermatogenesis is a complex process of male germ cells proliferation and maturation from diploid spermatogonia, through meiosis, to mature haploid spermatozoa. The process involves dynamic interactions between the developing germ cells and their supporting Sertoli cells. The gonadal tissue, with abundance of highly unsaturated fatty acids, high rates of cell division, and variety of testis enzymes results very vulnerable to the overexpression of reactive oxygen species (ROS). In order to address this risk, testis has developed a sophisticated array of antioxidant systems comprising both enzymes and free radical scavengers. This chapter sets out the major pathways of testis generation, the metabolism of ROS, and highlights the transcriptional regulation by steroid receptors of antioxidant stress enzymes and their functional implications. It also deals with of the advantages of the system biology for an antioxidant under steroid control, the major selenoprotein expressed by germ cells in the testis, the phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4) having multiple functions and representing the pivotal link between selenium, sperm quality, and species preservation.
Human chorionic gonadotropin (hCG) is an important marker for pregnancy, pregnancy-related disorders, and various cancers. Different molecular forms of hCG occur in different clinical conditions, and these can be distinguished with immunoassays using well-characterized monoclonal antibodies. Exact knowledge of the epitopes of the antibodies used is crucial for the design of assays with desired specificity. The epitopes of many hCG antibodies have been determined by comparing their reactivity with six 1st International Reference Reagents (IRRs) for hCG, but the specificity of some antibodies remains to be exactly defined. We have therefore studied the reactivity of 30 monoclonal antibodies (mAbs) with the six 1st IRRs for hCG, and variants were investigated using immunoaffinity extraction combined with liquid chromatography-mass spectrometry (LC-MS/MS) for the detection of hCG variants by specific tryptic signature peptides. Each of the mAbs had previously been characterized with regard to epitope specificity in the 2nd Tissue Differentiation Workshop on hCG of the International Society of Oncology and BioMarkers (ISOBM). Simultaneous identification of different hCG variants by LC-MS/MS confirmed that two standards used for mAb characterization, nicked hCG (hCGn, 1st IRR 99/642) and nicked β subunit of hCG (hCGβn, 1st IRR 99/692), are heterogeneous, being composed of two major variants each: hCGn44/45 and hCGn47/48 as well as hCGβn44/45 and hCGβ47/48. Furthermore, MS revealed cross-contamination by non-nicked hCG of the 1st IRR hCGn (99/642) standard. This information enabled fine-tuning of the previous epitope classifications of mAbs specific for heterodimeric hCG (c-mAbs). LC-MS/MS confirmed that c2-mAbs and most c1-mAbs did not recognize hCGn as the observed response in radioimmunoassays obviously resulted from the contamination of hCGn with hCG. Thus, c1 and c2 epitopes are partially dependent on hCGβ peptide loop 2. c3-mAbs recognized both hCG and hCGn. It appeared that c-mAbs cannot discriminate between hCGn44/45 and hCGn47/48 as they either recognize both or neither variant. For most mAbs directed against hCGβ, epitope specificity determined by LC-MS/MS was highly concordant with that obtained using standard immunological methods. In analogy to c-mAbs, hCGβ-mAbs cannot discern between hCGβn44/45, hCGβn47/48, or intact hCGβ as all 15 mAbs recognizing hCGβ also recognized both nicked variants irrespective of which of the three major hCGβ antigenic domains their epitopes were located within: on the caps of peptide loops 1 and 3, around the cystine knot, or along the hCGβCTP. LC-MS/MS confirmed that their epitopes were not located on hCGβ peptide loop 2. Thus, LC-MS/MS provided in-depth information on hCG variant composition of hCGn (99/642) and hCGβn (99/692) and hCG variant specificity profiles and facilitated precise classification of the epitopes of anti-hCG mAbs. This has impact on the design of selective immunoassays.
“Phospholipid Hydroperoxide Glutathione Peroxidase” (PHGPX) is a new selenoenzyme recently identified and purified from mammalian tissues that reduces membrane hydroperoxides. This monomeric enzyme (M.W. 21,000), different from all previously known glutathione peroxidases and glutathione transferases, has been purified from different mammalian tissues and accounts for the cytosolic inhibition of microsomal lipid peroxidation induced by NADPH or ascorbate and iron. A selenocysteine is present at the active site, as suggested by the kinetics of the inhibition in the presence of iodoacetate. The kinetics, as in the case of GPX, is compatible with a ter-uni ping pong mechanism. The kinetic comparison between PHGPX and GPX in the presence of different substrates and detergents, indicates that, while GPX is more active on soluble hydrophilic substrates, PHGPX is more active on the hydroperoxides in the membranes.
We applied PCR–RFLP (Polymerase Chain Reaction – Restriction Fragment Length Polymorphism) analysis to identify seven gadoid
species of different biogeographical origin and commercial relevance, namely Gadus morhua (Atlantic ocean); Trisopterus minutus capelanus, Trisopterus minutus minutus, Molva elongata, Phycis blennoides, Micromesistius poutassou (Atlantic ocean and Mediterranean sea); Theragra chalcogramma (Pacific ocean). Two DNA fragments belonging to mitochondrial 12S and 16S rRNA genes of about 430 and 630bp, respectively,
were isolated by PCR amplification. Their direct sequencing showed a significant genotypic diversity among gadoid species,
useful for species identification. Digestion of 16S rRNA gene PCR fragment with MvaI or Bsh1285I restriction enzymes, followed by agarose gel electrophoresis of the cleaved products, yielded specific restriction profiles
that enabled direct, visual identification of the species analyzed. This PCR–RFLP method allowed a clear and rapid discrimination
of the gadoid species studied.
Until recently, the histology and ultrastructural events of spermatogenesis in reptiles were relatively unknown. Most of the available morphological information focuses on specific stages of spermatogenesis, spermiogenesis, and/or of the mature spermatozoa. No study to date has provided complete ultrastructural information on the early events of spermatogenesis, proliferation and meiosis in class Reptilia. Furthermore, no comprehensive data set exists that describes the ultrastructure of the entire ontogenic progression of germ cells through the phases of reptilian spermatogenesis (mitosis, meiosis and spermiogenesis). The purpose of this review is to provide an ultrastructural and histological atlas of spermatogenesis in reptiles. The morphological details provided here are the first of their kind and can hopefully provide histological information on spermatogenesis that can be compared to that already known for anamniotes (fish and amphibians), birds and mammals. The data supplied in this review will provide a basic model that can be utilized for the study of sperm development in other reptiles. The use of such an atlas will hopefully stimulate more interest in collecting histological and ultrastructural data sets on spermatogenesis that may play important roles in future nontraditional phylogenetic analyses and histopathological studies in reptiles.
The PSIPRED protein structure prediction server allows users to submit a protein sequence, perform a prediction of their choice and receive the results of the prediction both textually via e-mail and graphically via the web. The user may select one of three prediction methods to apply to their sequence: PSIPRED, a highly accurate secondary structure prediction method; MEMSAT 2, a new version of a widely used transmembrane topology prediction method; or GenTHREADER, a sequence profile based fold recognition method.
Availability:
Freely available to non-commercial users at http://globin.bio.warwick.ac.uk/psipred/
NCBI’s Conserved Domain Database (CDD) is a resource for the annotation of protein sequences with the location of conserved
domain footprints, and functional sites inferred from these footprints. CDD includes manually curated domain models that make
use of protein 3D structure to refine domain models and provide insights into sequence/structure/function relationships. Manually
curated models are organized hierarchically if they describe domain families that are clearly related by common descent. As
CDD also imports domain family models from a variety of external sources, it is a partially redundant collection. To simplify
protein annotation, redundant models and models describing homologous families are clustered into superfamilies. By default,
domain footprints are annotated with the corresponding superfamily designation, on top of which specific annotation may indicate
high-confidence assignment of family membership. Pre-computed domain annotation is available for proteins in the Entrez/Protein
dataset, and a novel interface, Batch CD-Search, allows the computation and download of annotation for large sets of protein
queries. CDD can be accessed via http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.
N-myristoylation, one of the co- or post-translational modifications of proteins, has so far been regarded as necessary for anchoring of proteins to membranes. Recently, we have revealed that N(alpha)-myristoylation of several brain proteins unambiguously regulates certain protein-protein interactions that may affect signaling pathways in brain. Comparison of the amino acid sequences of myristoylated proteins including those in other organs suggests that this regulation is involved in signaling pathways not only in brain but also in other organs. Thus, it has been shown that myristoylated proteins in cells regulate the signal transduction between membranes and cytoplasmic fractions. An algorithm we have developed to identify myristoylated proteins in cells predicts the presence of hundreds of myristoylated proteins. Interestingly, a large portion of the myristoylated proteins thought to take part in signal transduction between membranes and cytoplasmic fractions are included in the predicted myristoylated proteins. If the proteins functionally regulated by myristoylation, a posttranslational protein modification, were understood as cross-talk points within the intracellular signal transduction system, known signaling pathways could thus be linked to each other, and a novel map of this intracellular network could be constructed. On the basis of our recent results, this review will highlight the multifunctional aspects of protein N-myristoylation in brain.
This study assessed metals in water and different tissues of Labeo rohita and the impact of these metals on DNA and proteins as biomarkers of gills and muscles of these fish from three different polluted sites (reference or low = KW, medium = CH and high = SK) of the Indus River, Pakistan. The Mn, Pb, Cu, Zn, Hg, and Cr levels in water, gills, liver, muscles, and skin of these fish were compared with the international permissible levels. All metals except Pb and Hg in water were within the acceptable limits of drinking water. In contrast, the Mn, Hg, and Cr levels in the fish tissues were higher than their permissible limits for fish as a human food. Here, the gills contained higher metals than the other tissues. Different patterns of biomarkers were found in fish from these sites. While the gills did not show four protein bands (55, 30, 18.4, and 16.4 kDa), the muscles showed four new protein bands (100, 85, 45, and 20 kDa) for fish from the medium and high polluted sites as compared to the reference or low polluted site. The fish from the CH and SK sites of the Indus River contained low molecular weight DNA in their gills but high molecular weight DNA in their muscles when compared with the KW site. This study suggests that the proteins and DNA profiles of L. rohita could be used as biomarkers to assess the impact of potential environmental stressors such as metals on the freshwater systems.
During the periimplantation period, human chorionic gonadotropin (hCG) plays a key role by increasing the uterine blood flow through uterine vessel vasodilatation but also through angiogenesis. Indeed, we previously demonstrated that hCG contributes to endothelial cell recruitment and vessel formation.
In this study, hCG was proposed as an arteriogenic factor that could promote perivascular cell recruitment and vessel stabilization.
The aortic ring assay, a three-dimensional ex vivo angiogenesis system mimicking all the steps of the angiogenesis process was used to study the impact of hCG on pericyte recruitment and vessel maturation.
The study was conducted at a university hospital laboratory.
Perivascular cell proliferation, migration, and apposition were quantified by computerized image analysis.
Physiological concentrations of hCG (10-400 IU/ml) significantly enhanced pericyte sprouting and migration and gave rise to the maturation and coverage of endothelial capillaries. In a three-dimensional coculture model of endothelial and perivascular cells, hCG enhanced vessel tube formation and endothelial/mural cell adhesion. In addition, hCG stimulated the proliferation of human umbilical vein endothelial cells and smooth muscle cells. The specificity of these effects was determined by using an anti-hCG blocking antibody. Signaling pathways implicated on this hCG effect is protein kinase A and phospholipase C/protein kinase C dependent for the proliferative effect but only phospholipase C/protein kinase C for the migrative process.
Our findings highlight a novel paracrine role of this early embryonic signal in vessel maturation by stimulating perivascular cell recruitment, migration, and proliferation.
Glutathione peroxidase 4 (Gpx4) is an essential antioxidant enzyme having multiple functions. A long form Gpx4 protein and
a short form Gpx4 protein, which are distinguishable by the presence or lack of a mitochondrial signal peptide at the N terminus,
are generated from the Gpx4 gene. In this study, we generated transgenic mice using mutated GPX4 genes encoding either the
long form Gpx4 (lGPX4 gene) or the short form Gpx4 (sGPX4 gene). Our results showed that transgenic mice with the sGPX4 gene
had increased Gpx4 protein in all tissues and were protected against diquat-induced apoptosis in liver. Moreover, the sGPX4
gene was able to rescue the lethal phenotype of the mouse Gpx4-null mutation. In contrast, transgenic mice with the lGPX4 gene had increased Gpx4 protein only in the testes, and the lGPX4
gene failed to rescue the lethal phenotype of the mouse Gpx4-null mutation. In Gpx4-null mice rescued by the sGPX4 gene, the Gpx4 protein was present in mitochondria isolated from somatic tissues, and the
submitochondrial distribution pattern of the Gpx4 protein in these mice was identical to that in wild-type mice. Interestingly,
the male Gpx4-null mice rescued by the sGPX4 gene were infertile and exhibited sperm malformation. Together, our results demonstrated for
the first time that the short form Gpx4 protein is present in somatic tissue mitochondria and is essential for survival and
protection against apoptosis in mice, whereas the long form Gpx4 protein is important for male fertility.
This review focuses on our current understanding of vertebrate sex steroid receptors, with an emphasis on their evolutionary relationships. These relationships are discussed based on nucleotide and amino acid sequence data, which provide clues to the process by which structure-function relations have originated, evolved, and been maintained over time. The importance of the distribution of sex steroid receptors in the vertebrate brain is discussed using the example of androgen receptor sites and their relatively conserved localizations in the vertebrate brain.
Selenium is linked to male fertility. Glutathione peroxidase 4 (GPx4), first described as an antioxidant enzyme, is the predominant selenoenzyme in testis and has been suspected of being vital for spermatogenesis. Cytosolic, mitochondrial, and nuclear isoforms are all encoded by the same gene. While disruption of entire GPx4 causes early embryonic lethality in mice, inactivation of nuclear GPx4 does not impair embryonic development or fertility. Here, we show that deletion of mitochondrial GPx4 (mGPx4) allows both normal embryogenesis and postnatal development, but causes male infertility. Infertility was associated with impaired sperm quality and severe structural abnormalities in the midpiece of spermatozoa. Knockout sperm display higher protein thiol content and recapitulate features typical of severe selenodeficiency. Interestingly, male infertility induced by mGPx4 depletion could be bypassed by intracytoplasmic sperm injection. We also show for the first time that mGPx4 is the prevailing GPx4 product in male germ cells and that mGPx4 disruption has no effect on proliferation or apoptosis of germinal or somatic tissue. Our study finally establishes that mitochondrial GPx4 confers the vital role of selenium in mammalian male fertility and identifies cytosolic GPx4 as the only GPx4 isoform being essential for embryonic development and apoptosis regulation.
The aim of this study was to develop and to validate a methodology based on biomarker responses and residue analysis on the terrestrial lizard Podarcis sicula to assess the ecotoxicological effects associated with on-shore oil extraction. The oil treatment plant investigated is located in Val d'Agri (southern Italy). Italian wall lizards were sampled on four stations along a transect determined on the basis of prevailing winds downwind of the oil plant. Cytochrome P450 1A1 activities (EROD and BPMO), AChE activity, PAH bile metabolites and contaminant levels (PAHs and trace elements) were measured. Major results in the evaluation of toxicological impact of oil field activity in the Italian wall lizards were obtained for Cd, Hg, total and carcinogenic PAH levels, and PAH metabolites in bile. Results obtained validate, for the first time, P. sicula as a terrestrial bioindicator for the assessment of the toxicological impact of on-shore extraction activity.
The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.
In mammals endorphinergic systems have been shown to modulate reproductive processes and β-endorphin (β-EP) has been found to influence sexual functions, acting at the hypothalamus-pituitary-gonadal axis level. Using immunocytochemical and in vitro studies, evidence for a diffuse pro-opiomelanocortin-related opioid system in the lizard Podarcis s. sicula was produced. In the testis, β-EP immunoreactivity showed seasonal variation, being most pronounced in the interstitial cells of sexually quiescent lizards (December). Reverse-phase high-performance liquid chromatography, coupled with radioimmunoassay and immunocytochemistry, showed that β-EP and acetyl β-EP increased during December, while their concentrations were low during April, when the highest testicular activity occurred.
Using in vivo studies, it was found that naltrexone treatment, blocking pituitary opioid receptor, increased androgen levels in the plasma and in the testis. It was also found with in vitro studies that the endogenous opioid system inhibits gonadotrophin release and therefore androgen production by the testis.
The data reported here provide evidence for the physiological role played by opioid peptides at the pituitary level to regulate the seasonal reproductive activity of the lizard Podarcis s. sicula .
Journal of Endocrinology (1994) 143, 565–571
Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx were subjected to functional analysis. The rate constants k+1 and k'+2, for the oxidation and the regeneration of the ground state enzyme were estimated, respectively. Moreover, the alkylation rate of the reactive centre by iodoacetate (kinact.) was also analysed. The substitution of the catalytically competent selenocysteine 46 by cysteine (PHGPxcys46) decreased k+1 and k'+2 by about three orders of magnitude, although leaving unaffected kinact.. Furthermore, mutations of PHGPxcys46 involving the other residues of the triad decreased both kinact. and k+1, thus highlighting the involvement of Gln 81 and Trp 136 in the dissociation/activation of the nucleophilic cysteine thiol. In general, substitutions of Gln 81 or Trp 136 by acidic residues in PHGPxcys46 most dramatically depressed the k+1 values, because they practically prevented the dissociation of the thiol group, while neutral or positively charged residues in these positions allowed an intermediate dissociation and induced a corresponding reactivity of the thiol. Our data, for the first time, reveal that the presumed triad of selenocysteine, glutamine and tryptophan residues represents a novel type of catalytic centre, whose integrity is essential for the full catalytic function of glutathione peroxidases.
Selenium deficiency is known to be associated with male infertility, and the selenoprotein PHGPx has been shown to increase in rat testis after puberty and to depend on gonadotropin stimulation in hypophysectomized rats [Roveri et al. (1992) J. Biol. Chem. 267, 6142 6146]. Exposure of decapsulated whole testis, however, failed to reveal any transcriptional activation or inhibition of the PHGPx gene by testosterone, human chorionic gonadotropin, or forskolin. Nevertheless, it was verified that the specific activity of PHGPx in testis, but not of cGPx, correlated with sexual maturation. Leydig cell destruction in vivo by ethane dimethane sulfonate (EDS) resulted in a delayed decrease in PHGPx activity and mRNA that could be completely prevented by testosterone substitution. cGPx transiently increased upon EDS treatment, probably as a result of reactive macrophage augmentation. In situ mRNA hybridization studies demonstrated an uncharacteristic low level of cGPx transcription in testis, whereas PHGPx mRNA was abundantly and preferentially expressed in round spermatids. The data show that the age or gonadotropin-dependent expression of PHGPx in testis does not result from direct transcriptional gene activation by testosterone, but is due to differentiation stage-specific expression in late spermatids, which are under the control of Leydig cell-derived testosterone. The striking burst of PHGPx expression at the transition of round to elongated spermatids suggests an involvement of this selenoprotein in sperm maturation.
A third of the mammalian selenoprotein repertoire accounts for the two main cellular redox systems in mammals, i.e., the glutathione- and thioredoxin-dependent systems. All three thioredoxin reductases contain selenocysteine as their penultimate amino acid and keep thioredoxins in their reduced, active state. Cytosolic thioredoxin reductase (TXNRD1) and mitochondrial thioredoxin reductase (TXNRD2) are directly involved in cell proliferation and cell protection of somatic cells, respectively, whereas thioredoxin-glutathione reductase (TXNRD3) contributes to sperm development. Five out of eight glutathione peroxidases (GPX) are selenoproteins in humans and are part of antioxidant network by keeping the levels of cellular peroxides in check. Among these, glutathione peroxidase 4 (GPX4) is unusual as it confers a moonlighting and essential function in sperm development because of its promiscuity towards reducing substrates. Moreover, due to its unique activity to efficiently reduce phospholipid hydroperoxides, GPX4 has emerged as one of the most important selenoproteins in mammals. In fact, we and others found that GPX4 is the key regulator of a recently described form of regulated necrotic cell death, called ferroptosis. Hence, this chapter aims at illuminating the importance of the main mammalian antioxidant systems in health and disease.
Cadmium (Cd) is an environmental contaminant that poses serious risks to aquatic organisms and their associated ecosystem. The mechanisms underlying Cd-induced oxidative stress and immunotoxicity in fish remain largely unknown. In this study, adult female zebrafish were exposed to 0 (control), 1 mg L⁻¹ Cd for 24 h and 96 h, and the oxidative stress and inflammatory responses induced by Cd were evaluated in the brain, liver and ovary. Reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) increased in a time-dependent manner after treatment with Cd in the brain and liver. The increase may result from the disturbance of genes including copper and zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), inducible nitric oxide synthase (iNOS), and ciclooxigenase-2 (COX-2) at mRNA, protein and activity levels. Although ROS, NO and MDA were not significantly affected by Cd in the ovary, the up-regulation of Cu/Zn-SOD, CAT, iNOS, and COX-2 was observed. Exposure to Cd induced a sharp increase in the protein levels of tumor necrosis factor alpha (TNF-α) in the brain, liver and ovary, possibly contributing to activate inflammatory responses. Furthermore, we also found a dramatic increase in mRNA levels of NF-E2-related factor 2 (Nrf2) and nuclear transcription factor κB (NF-κB) at 24 h in the liver and ovary. The corresponding changes in the mRNA levels of Kelch-like-ECH-associated protein 1 (Keap1a and Keap1b) and the inhibitor of κBα (IκBαa and IκBαb) may contribute to regulate the transcriptional activity of Nrf2 and NF-κB, respectively. Contrarily, mRNA levels of Nrf2, NF-κB, Keap1, Keap1b, IκBαa and IκBαb remained stable at 24 and 96 h in the brain. Taken together, we demonstrated Cd-induced oxidative stress and immunotoxicity in fish, possibly through transcriptional regulation of Nrf2 and NF-κB and gene modifications at transcriptional, translational, post-translational levels, which would greatly extend our understanding on the Cd toxicity.
The authors were asked by the Editors of ACS Chemical Biology to write an article titled "Why Nature Chose Selenium" for the occasion of the upcoming bicentennial of the discovery of selenium by the Swedish chemist Jöns Jacob Berzelius in 1817, and styled after the famous work of Frank Westheimer on the biological chemistry of phosphate [Westheimer, F. H. (1987) Why Nature chose phosphates, Science 235, 1173-1178.]. This work gives a history of the important discoveries of the biological processes that selenium participates in, and a point-by-point comparison of the chemistry of selenium with the atom it replaces in biology, sulfur. This analysis shows that redox chemistry is the largest chemical difference between the two chalcogens. This difference is very large for both one-electron and two-electron redox reactions. Much of this difference is due the inability of selenium to form pi-bonds of all types. The outer valence electrons of selenium are also more loosely held than those of sulfur. As a result, selenium is a better nucleophile and will react with reactive oxygen species faster than sulfur, but the resulting lack of pi-bond character in the Se-O bond means that the Se-oxide can be much more readily reduced in comparison to S-oxides. The combination of these properties means that replacement of sulfur with selenium in Nature results in a selenium-containing biomolecule that resists permanent oxidation. Multiple examples of this gain of function behavior from the literature are discussed.
The hybrid sol-gel zirconia-acetylacetonate amorphous material (HSGZ) shows high catalytic activity in oxidative degradation reactions without light or thermal pre-treatment. This peculiar HSGZ ability derives from the generation of highly reactive oxide radical species (ROS) upon exposure to air at room conditions. We disclose the origin of such unique feature by combining EPR and DRUV measurements with first-principles calculations. The organic ligand (acac) plays a pivotal role of in generating and stabilizing the superoxide radical species at the HSGZ-air interfaces. Our results lead the path toward further development of HSGZ and related hybrid materials for ROS-based energy and environmental applications.
The estrogen receptor-α (ERα) knockout mouse (αERKO) lacks ERα throughout development; therefore, an adult model for the study of estrogen effects in male mice was recently developed using the antiestrogen ICI 182,780. However, differences between species have been noted during immunostaining for ERα in the male tract as well as in response to treatments with antiestrogens. Therefore, we developed the antiestrogen model in the adult male rat to test, in another species, the hypothesis that estrogen regulates fluid reabsorption in efferent ductules. Estrogen receptor in the rat was blocked using ICI 182,780 for 100–150 days. Male Sprague-Dawley rats were treated weekly with s.c. injections of ICI 182,780 (10 mg) or castor oil (as control). The effects of ICI included testicular atrophy and infertility, similar to terminal effects in the αERKO male. Additionally, ICI induced dilations of the rete testis and efferent ductules and a reduction in the height of the ductule epithelium, which are changes similar to those in both αERKO and ICI-treated mice. One difference between species was a large variation in effects on the rat efferent ductule epithelium, including a transient increase in the number of periodic acid-Schiff-positive, lysosomal-like granules. These data confirm that estrogen is required for normal function of the efferent ductules and is essential for long-term fertility in the male rodent.
Biometric evaluation, ovary histomorphometry, plasma l7/i-estradiol (E2) and androgens (A) concentrations were investigated through the gonadal maturation and spawning of the wild chwb, Leuciscus cephalus. A lower concentration of plasma E2 was recorded from January (oogonia) to March (previtellogenic oocytes), while estradiol concentrations increased in April, when the ovary mostly contained vitellogenic oocytes. In July, when it was prevalently composed of postovulatory follicles, there was a decline in E2 plasma concentrations. Low concentrations of this steroid remained in the plasma until January, while high concentrations of androgens reached a peak in February and April preceding and following E2. The observed seasonal changes in plasma steroids, reflecting the pattern of follicles size development, were to a large extent consistent with observations made in other cyprinids. Since they define the reproductive cycle of the European female chub, they will be useful in the construction of a maturity schedule for wild populations.
We evaluated the activity and expression of antioxidant enzymes in the cerebellum and cortex of Swiss adult male mice exposed to methylmercury (MeHg) in drinking water (40mg/L) during 21 days. The activity of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and thioredoxin reductase (TrxR) were determined spectrophotometrically. The expression (protein levels) of GPx1 and GPx4 isoforms, TrxR1 as well as heat shock protein 70 (HSP70) were evaluated using specific antibodies and normalized by actin levels. The exposure of mice to MeHg caused a significant impairment in locomotors performance in the open field test (crossings and rearing). This result was followed by a significant reduction of GPx and TrxR activities in the cerebellum and cortex when compared to untreated animals. We also observed a substantial decrease in GPx1, GPx4 and TrxR1 protein levels in the cerebellum, while in the cerebral cortex, only GPx4 and TrxR1 were decreased after MeHg treatment. The activities of the antioxidant enzymes GR, GST, CAT and SOD were increased in the cerebellum after MeHg administration to mice. In contrast, only CAT was increased in the cerebral cortex of MeHg-treated animals. The expression of HSP70 was up-regulated only in the cerebellum where MeHg-exposed mice showed a significant increase in the immunocontent of HSP70 when compared to controls. This is the first report showing a role for GPx4 in the neurotoxicity induced by MeHg in vivo. In addition, our data indicates that the selenoproteins GPx and TrxR as main targets during MeHg exposure, which may be considered in biomarker studies.
The rise in plasma testosterone levels following stimulation with human chorionic gonadotrophin (3000 i.u./day for 4 days) has been used as a test of interstitial cell function in normal men and men with testicular disorders. The large majority of patients with testicular disorders resulting in infertility, showed a subnormal testosterone response to stimulation. The most marked impairment was noted in those patients with the biopsy appearance of germinal cell arrest and Sertoli cell only syndrome.
Given the highly dynamic nature of mRNA transcription and the potential variables introduced in sample handling and in the downstream processing steps (Garson et al. (2009)), a standardized approach to each step of the RT-qPCR workflow is critical for reliable and reproducible results. The MIQE provides this approach with a checklist that contains 85 parameters to assure quality results that will meet the acceptance criteria of any journal (Bustin et al. (2009)). In this paper we demonstrate how to apply the MIQE guidelines (www.rdml.org/miqe) to establish a solid experimental approach.
We report the presence of a progesterone receptor (PR) in the brain of the female amphibian Rana esculenta and changes in the levels of this PR during the gonadal recovery phase in relation to different circulating steroid levels and steroid treatment. The highest level of the PR in the nuclear brain extract corresponded to a low level of plasma progesterone and occurred when vitellogenin synthesis was at a minimum. The lowest level was found during follicular growth, concomitant with increased plasma 17beta-estradiol and progesterone levels. The PR levels were significantly higher in ovariectomized female R. esculenta than in intact and sham-operated female frogs. Treatment with 17beta-estradiol and progesterone downregulated the frog brain PR, indicating that a possible progesterone-receptor interaction is involved in the modulation of vitellogenin.
The rise in plasma testosterone levels following stimulation with human chorionic gonadotrophin (3000 i.u./day for 4 days) has been used as a test of interstitial cell function in normal men and men with testicular disorders. The large majority of patients with testicular disorders resulting in infertility, showed a subnormal testosterone response to stimulation. The most marked impairment was noted in those patients with the biopsy appearance of germinal cell arrest and Sertoli cell only syndrome.
Hydrophobic insertion of the acyl chain into the bilayer is necessary but not sufficient for the membrane binding of a myristoylated protein. The myristoylated alanine-rich C kinase substrate (MARCKS), Src, ADP-ribosylation factor and human immunodeficiency virus-1 matrix proteins also contain a cluster of basic residues that bind to acidic phospholipids; the hydrophobic and electrostatic interactions act together to anchor the protein to a membrane. For MARCKS, and perhaps other proteins, phosphorylation of serines within its basic cluster reduces the electrostatic attraction, producing translocation of the protein from the membrane to the cytosol by a simple 'electrostatic switch' mechanism.
Treatment of exogenous gonadotrophins(PMSG, hCG and PMSG+hCG) to male C. versicolor during nonbreeding phase resulted in increased weight of testis and its diameter. The seminiferous tubular diameter was also increased and tubules were filled with spermatogenic elements like spermatogonia, primary and secondary spermatocytes and spermatids. The results suggest that mammalian nonpituitary gonadotrophins(PMSG and hCG) also posses the potency to stimulate spermatogenesis in reptilian testis. Lowered cholesterol levels of testis, increased weight and protein content of epididymis after gonadotrophins treatment indicate that the exogenous gonadotrophins could initiate the steroidogenesis in testis. Interstitial space was enlarged and filled with active Leydig cells. Spermatogenesis was not complete as no spermatozoa were observed in the lumen of seminiferous tubule. The factors essential for the completion of spermatogenesis are discussed.