Imidacloprid induces morphological and molecular damages on testis of lizard (Podarcis sicula).

The insecticide imidacloprid was evaluated under laboratory conditions in the adult male Italian wall lizards (Podarcis sicula) to assess its potential toxicity. By an acute oral test, LD50 was 503.76 mg/kg. Changes in spermatogenesis, plasma sex hormone levels and androgen and oestrogen receptor mRNAs were analysed by subchronic test and simulated environmental exposure. 15-days subchronic test, in which lizards were orally dosed on alternate days at 0, 10, 50, 100 mg/kg bw, showed a dose-dependent changes of testicular architecture and an increase of apoptotic processes. In a 30-days simulated environmental exposure spermatogenesis was arrested at secondary spermatocyte level and only few primary spermatocytes were TUNEL-positive. In all experimental groups imidacloprid was able to decrease both the level of sex hormones and the steroid receptor mRNAs. The results demonstrate that imidacloprid affects reproduction function of male lizards therefore precautions must be taken to minimize the harmful effects of this compound.

Testicular toxicity of methyl thiophanate in the Italian wall lizard (Podarcis sicula): morphological and molecular evaluation.

The effects of the fungicide methyl thiophanate (MT) on testis were determined in the Italian wall lizard (Podarcis sicula) using morphological and molecular analyzes. Three experimental trials were performed: an acute test using six doses, a two-week chronic test, and "ecotoxicological" exposure (3 weeks). The minimal lethal dose (LD(50)) of pure MT, reached by the acute test, was 100 mg/kg body weight. Testicular histopathology of surviving animals showed a reduced lumen and several multinucleated giant cells 24 h after injection followed by large decreases in spermatogonia (72%) and secondary spermatocytes (58%) and a loss of spermatids and sperms 7 days after. In the chronic test, a dose equivalent to 1/100 of LD(50) was injected on alternate days. Complete shutting of the lumen and a great decrease in spermatogonia (82%) were observed. In "ecotoxicological" exposure, achieved with a commercial MT compound, testis showed a decrease in primary spermatocytes (20%) and several vacuoles. An increase in germ cell apoptosis was observed in all experimental groups using TUNEL assay. A decrease in expression of androgen and estrogen receptor (AR and ER) mRNAs was seen in all experimental groups. The reduction in AR and ER mRNAs was correlated to exposure time. Indeed, in the "ecotoxicological" treatment (30 days), the decrease reached 82 and 90% for AR and ER mRNAs, respectively. These data strongly indicate that treatment with MT, damaging the seminiferous epithelium and decreasing steroid receptor expression, might render exposed lizards infertile.

Poly(ADP-ribosyl)ation of proteins and germ cell development in hyperthyroid rat testes.

The effect of increased serum levels of thyroid hormone (triiodothyronine, T3) on young rat testis spermatogenesis was studied by analysing molecular and morphological parameters. Hyperthyroidism was induced by either T3-treatment or 2- and 10-day cold exposure. The poly(ADP-ribosyl)ation of proteins catalysed by poly(ADP-ribose) polymerase, which is particularly active at specific stages of rat spermatogenesis, was analysed as molecular index of DNA damage and cell stress. Poly(ADP-ribose) polymerase activity rose after both T3-treatment and 2- and 10-day cold exposure, with a trend of 10-day cold-exposed rats towards control values. In all hyperthyroid rats poly(ADP-ribose) turnover, as a contribution of both poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase), was enhanced with respect to euthyroid animals. Poly(ADP-ribosyl)ation of proteins occurred with long and branched polymers suggesting an increased involvement of the modification system in DNA repair. Morphological changes of germ tissue were observed in hyperthyroid rats, mainly a high reduction of mature cells in the seminiferous tubule, and evidence of germ cell apoptosis was obtained by TUNEL method. In control animals germ cell apoptosis was within physiological levels. Conversely, in hyperthyroid rats a dramatic increase in the number of TUNEL-positive cells (some spermatogonia and numerous primary spermatocytes) was found, even though the increase was lower in 10-day than in 2-day cold-exposed animals.

Spermatogenesis, epididymis morphology and plasma sex steroid secretion in the male lizard Podarcis sicula exposed to diuron.

The present study investigates the effects of diuron, a substituted urea-based herbicide, in the male lizard Podarcis sicula utilizing quantitative and qualitative morphological features of the reproductive system and endocrinological analysis. Besides the control group, lizards were divided into three groups ([a-c]) (n=6/group) and placed for 3 weeks in terraria on polluted soil substrate sprayed with 3.75 L/ha of herbicide Toterbane 50F (50% diuron). Each terrarium was supplemented either with drinking water contaminated by herbicide (i.e. 1.08 microg/mL of diuron; group [a]), or with food contaminated by herbicide (i.e. 5.4 mg of diuron; group [b]), or with drinking water and food contaminated as described above (group [c]). None of the animals exposed to the contaminant showed any signs of general toxicity or death during the course of the experiments. Severe testicular effects are evidenced in all herbicide-treated groups, although, such effects are of a greater magnitude in lizards exposed to contaminated water (groups [a] and [c]). The main degenerative changes observed include: (1) a significant decrease in the mean gonadosomatic index of 55% in group [a] (P<0.001), 21% in group [b] (P<0.01) and 34% in group [c] (P<0.001) compared with control group; (2) a significant shrinking (P<0.001) of seminiferous tubule diameter (more than 60% of the control) in groups [a] and [c], and about 18% in group [b] (P<0.01); (3) a significant decrease in the crude numbers of spermatogonia of 92% in group [a] (P<0.001), 27% in group [b] (P<0.01) and 62% in group [c] (P<0.001) compared with control group. A complete loss of meiotic and mature germ cells in groups [a] and [c], and a reduction of primary spermatocytes, secondary spermatocytes and spermatids (more than 27% of the control) and a decrease of spermatozoa (more than 90% of the control) in group [b]; and (4) an hypertrophy of interstitial connective tissue which contains numerous lymphocytes, neutrophils and monocytes. The decrease and/or loss of germ cells seems to be related to an induction of inflammation (necrosis) rather than to apoptotic processes. Indeed, this hypothesis is supported by a TUNEL-assay, which failed to reveal any apoptotic cells either in the seminiferous epithelium or in the interstitial space in the testis of all exposed groups. Also the epididymis appears affected by diuron exposure. In particular, in experimental groups [a] and [c] it is regressed with abundant connective tissue and low epithelial cells without secretory granules, whereas in group [b] it appears partially regressed, with some secretory granules still present. At the same time, an impairment of the plasma sex-hormone levels is observed in treated lizards, as evidenced by RIA analysis. Testosterone values significantly decreased by 43% in group [a] (P<0.001), 34% in group [b] (P<0.01) and 52% in group [c] compared with control group. Instead, 17beta-estradiol plasma content is undetectable in all diuron-exposed lizards. Taken together, the results presented here indicate that diuron exposure resulted in direct male reproductive toxicity and reveal that this lizard is suitable as a laboratory reptile species for toxicological investigations.

Sequence analysis of retinoic acid receptor alpha, beta and gamma isoforms in the lizard, Podarcis sicula.

Vitamin A and its principal biologically active derivative, retinoic acid (RA), play a fundamental role in diverse processes, such as proliferation, differentiation, morphogenesis, metabolism and apoptosis of many types of cells. In addition, RA has been shown to be involved in the regulation of testicular function. These effects are mediated by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), each with three subtypes alpha, beta and gamma. The physiological involvement of retinoids in testicular function has been conducted mainly in mammals. Recently, we found that exogenous all-trans-retinoic acid impairs spermatogenesis and enhance testicular germ cell apoptosis in the lizard, Podarcis sicula, a seasonal breeder. To further investigate the role of retinoic acid in lizard, we focus this work principally on the characterization of lizard retinoic acid receptors (alpha, beta and gamma isoforms). RARalpha is 2720 bp long with a putative ORF between 699 and 2133. A Kozac sequence is present at 696 and a putative poly-adenilation site is present in position 2612. The RARalpha sequence shares 87% homology with mouse RARalpha mRNA while it has 76 and 80% homology with lizard RARbeta and gamma mRNAs. RARbeta is 2478 bp long showing a putative ORF between 196 and 1543. A canonical Kozac sequence is present at 193 and a putative poly-adenilation site is present at 2294. RARbeta shares 91% homology with mouse RARbeta mRNA and has 76% homology with both RARalpha and gamma. RARgamma is 2416bp long. With a putative ORF between 444 and 1818. A Kozac sequence is present at 441 and a putative poly-adenilation site is present at 2288. RARgamma shares 86% homology with mouse RARbeta mRNA and has 80 and 76% homology with both RARalpha and beta respectively. It is worth to note that, as in mouse, the 5'UTR of all isoforms is TATA and CAAT less. Both Northern blot and PCR analyses indicate that lizard testis expresses only RARalpha and RARbeta mRNAs, while RARgamma mRNA transcript was not found. In the period analysed, RARbeta was expressed during the gonadal full activity (May) and RARalpha was present in the post-reproductive period (August). During the autumnal recrudescence (October) RARalpha and RARbeta are co-expressed and, as indicated by quantitative PCR analysis, RARbeta mRNA levels are lower than RARalpha ones. Thus, the appearance and abundance of each receptor correspond to a specific phase of lizard reproductive cycle, allow us to hypothesize that each RAR subtype could play a specific role in the regulation of spermatogenetic activity. The results of the present study show, for the first time, the characterization of RAR mRNAs in the testis of lizard P. sicula, whose expression is related to the different phase of reproductive cycle. Moreover, the gamma form, is principally expressed in the skin during the March-July period, having probably a role in regulating skin homeostasis and colour livery, which are important factor in mating during the reproductive cycle.

Impairment of spermatogenesis and enhancement of testicular germ cell apoptosis induced by exogenous all-trans-retinoic acid in adult lizard Podarcis sicula.

In mammals, retinoic acid is involved in the regulation of testicular function by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR). Among RAR isoforms, the testicular cells of the lizard were found to express only RARalpha (3.7 kb) and RARbeta (3.4 kb) mRNAs, as reported here. In this study, the effects of exogenous all-trans-retinoic acid (atRA) on spermatogenesis of a non-mammalian seasonal reproducer were investigated. Daily intraperitoneal injections of atRA or atRA plus testosterone (atRA+T) were given for 2 weeks to adult males of the lizard Podarcis sicula. In animals treated with atRA, the seminiferous tubules were markedly reduced in cross-area. The seminiferous epithelium collapse was responsible for a sensible reduction in the number of germ cells and disruption in normal epithelial organization. In comparison, in atRA+T-treated lizards the loss of germinal cells was significantly less. The loss of germ cells observed in both experimental groups results from an induction of apoptotic process, as revealed by TUNEL analysis. Although low in number, apoptotic germ cells were also observed in the control groups (saline- and T-treated lizard), where the main germ cells undergoing apoptosis are primary spermatocytes (most frequently) and some spermatogonia. In conclusion, it is shown here that retinoic acid has deleterious effects on lizard spermatogenesis, causing a severe depletion of seminiferous epithelium, probably via induction of apoptotic processes. These effects are not completely inhibited by simultaneous administration of testosterone, although this hormone, once injected, is able to stimulate spermatogenesis and protect germinal cells from apoptotic cell death.

Molecular cloning and characterization of the clock gene period2 in the testis of lizard Podarcis sicula and its expression during seasonal reproductive cycle.

Clock genes are known to oscillate with circadian rhythmicity in the central clock structure, the suprachiasmatic nucleus of the hypothalamus, and also in peripheral tissues. Reproduction is a peripheral activity that is strongly influenced by a circadian clock in many organisms. Most mammals that exhibit a seasonal cycle are able to decode the daily changes in light across the year and to translate these in hormonal signals that regulate reproductive cycles. Expression of many clock genes has been revealed in mouse testis, although transcription of these genes seems to be constitutive in 24 h, suggesting that these genes may play in the testis a different role with regard to the central clockwork function. The seasonal breeding lizard Podarcis sicula represents an attractive model for studying some developmental and differentiation phenomena, such as gonadal maturation, since in the adult male the testis shows a spring full activity and a complete summer regression. Experimental data seem to suggest that in lizard the environmental factors, as photoperiod and temperature, affect the endogenous elements, although the interaction mechanisms are unknown. It is known that temperature signals have a direct influence on clock processes such as transcription, translation, protein phosphorylation and degradation. In addition, most data show that the expression of circadian clock genes, such as period2, is affected by length of photoperiod. In this way, the core clockwork may also decode seasonal information. Here we report the cloning, sequencing and bioinformatic analysis of period2 gene, isolated from the testis of lizard P. sicula, and its expression both in the testis and in other tissues during the different phases of the seasonal cycle. RT-PCR assays enlighten the presence of transcript in testis, brain, heart, liver and kidney in all the phases analysed. Moreover, real time quantitative PCR assays detect a peak of per2 testicular expression during gonadal regression. Our preliminary results cannot clearly demonstrate the involvement of per2 gene in seasonal reproductive cycle of male lizard P. sicula, but its presence in the testis may suggest a role of this gene during spermatogenesis. Besides, our work can provide numerous starting points to clarify the role of per2 during seasonal reproductive cycle.

Temporal expression of thyroid hormone receptor alpha1 in the liver of the lizard Podarcis sicula.

The effects of thyroid hormones on metabolism and development are mediated by thyroid hormone receptors (TRs). To gain a better understanding of the potential role of thyroid hormone receptors in the liver of the lizard Podarcis sicula, we have evaluated the expression of TRs during the more critical periods of the annual variations of thyroid activity. The results obtained have indicated that in the liver of the lizard P. sicula there are three transcripts: mRNA of 5.0 kb for TRalpha1, mRNA of 2.6 kb for TRalpha2, and 6.0 kb band, which represent unprocessed heteronuclear RNA, encoding unspliced primary transcripts of RNA prior to their processing into the mature TRalpha1 and TRalpha2. By means of slot-blot, we are able to determine that there is a change in the expression of TRs that occurs in the liver during the annual cycle of thyroid activity. A major expression registers in May, when the lizard thyroid gland shows the maximal activity. The combination of molecular biology with immunohistochemistry revealed that hepatic cells were also TRalpha IR positive. Particularly intense immunostaining was present in the cell nuclei of animals sacrified in May. These observations suggest that in lizard P. sicula the thyroid hormone (T3) might regulate hepatic activity, modulating TR mRNA levels.

Apoptosis during spermatogenesis in the spotted ray Torpedo marmorata.

This article is a cytological and molecular investigation on the occurrence of apoptosis during spermatogenesis in Torpedo, a cartilaginous fish characterised by a typical cystic testis. Using DNA fragmentation and Bak gene expression, it demonstrated that germ cells undergo apoptosis only at the stages of spermatocyte and spermatid, and degeneration also involves Sertoli but not Leydig cells. In immature cysts, this cellular process probably occurs when the ratio of germ cells to the only Sertoli cell (SC) forming the spermatoblast changes. Apoptosis also takes place in mature cysts after sperm release to eliminate most of the SCs. Few of them, however, become cytoplasts and probably continue secreting androgens so as to control the final events of spermatogenesis, i.e., passage of spermatozoa through the ductus deferentes. Finally, the present investigation demonstrated that, in Torpedo testis, Bak mRNA is expressed during spermatogenesis, thus suggesting that the mitochondrial pathway might be active. This observation in one of the oldest vertebrate classes indicates that, in all vertebrates, the apoptotic process during spermatogenesis is conserved, contributing to testicular homeostasis.

Effects of the aromatase inhibitor fadrozole on plasma sex steroid secretion, spermatogenesis and epididymis morphology in the lizard, Podarcis sicula.

Recently, increasing importance has been attached to the role of estrogens and their receptors in male reproduction, since they have been found to be abundant in the male reproductive tract. In the lizard, Podarcis sicula, a seasonal breeder, estrogens seem to be involved in the regulation of testicular activity. Particularly, it has been hypothesized that the block of spermatogenesis and the complete regression of the epididymis and other secondary sexual characters (SSCs) in autumn might be due to high estrogen levels. To investigate the role of estrogens in the reproductive process of male lizards, we utilized Fadrozole ((AI) [4-(5,6,7,8-tetrahydroimidazole [1,5-a] pyridin-5-yl)-benzonitrile monohydrochloride] (CGS 16949A)), a nonsteroidal inhibitor of aromatase, the enzyme involved in the aromatization of androgens to estrogens, evaluating its effects on plasma sex-hormone release, spermatogenesis and epididymis morphology. For this purpose, adult male lizards, captured during the autumnal recrudescence, were intraperitoneally injected with 0.5 microg and 5 microg/g/body weight of AI for 15 and 30 days. In the animals treated with the higher AI dose, estrogen levels decreased if compared to the control groups, whereas androgen levels increased. Furthermore, histologic sections of testes and epididymes showed that the 30-day treatment with AI-induced spermatogenesis resumption with release of sperms into the large lumen of the seminiferous tubules, and the epididymes appeared more developed with moderately secreting columnar canal cells. Therefore, it is proposed that failure of spermatogenesis in autumn might be due to high estrogen levels.