Anna Cardone

The expression of androgen receptor messenger RNA is regulated by tri-iodothyronine in lizard testis.

The network of hormonal and non-hormonal signals required for testicular activity during the reproductive cycle of the seasonal breeding lizard, Podarcis sicula, are not yet well understood. Androgens are significantly involved in meiosis and spermiogenesis, and such an effect is mediated through their receptor (AR). Estrogens also affect the testicular activity down-regulating the expression of AR mRNA. Since over the last few years, extensive works have reported, in mammals, a clear influence of tri-iodothyronine (T(3)), the biologically active thyroid hormone, on Sertoli cell activities, we carried out a study to shead light on the effect/s exerted by T(3) in lizard testis. A thyroid hormone receptor mRNA (TR mRNA) has been found in the testis indicating that T(3) might be involved in the regulation of gonadal activity. In in vivo experiments, injection of T(3) to male lizards, captured during the recrudescence period (March) and maintained under experimental photothermal conditions (24 degrees C and 15 h daylight), increased the expression of AR mRNA. The in vitro results confirmed the stimulatory effect of T(3) on AR mRNA levels. Thus, in testosterone (T) exposed cells, the highest values of AR mRNA were observed in T(3)-primed animals, indicating that T and T(3) increase AR gene transcription independently. The present data suggest that, in lizards, the combined action of androgens, estrogen and T(3) might regulate testicular activity, modulating AR mRNA levels.

Regulation of androgen receptor mRNA expression in primary culture of Harderian gland cells: cross-talk between steroid hormones

The androgen receptor (AR) must be considered a transcription factor belonging to the steroid-thyroid hormones receptor superfamily. Previous results gained from the Harderian gland, a tubulo-alveolar gland located in the orbital cavity of the golden hamster, indicate that Harderian gland cells express mRNAs encoding for androgen, glucocorticoid, thyroid hormone (T(3)), and estrogen receptors, respectively. Since in other systems, these receptors have been related to the expression of the androgen receptor, we have studied the regulation of AR expression in primary cultures of the male hamster Harderian gland. Our in vitro experiments show that androgen, and thyroid hormones increase the expression of AR. Retinoic acids also show a positive effect on AR expression, while exposure to glucocorticoid or estrogen blocks AR expression. Since these steroids differently modulate AR expression, our results must be considered in the context of multi-hormonal control of gene expression that could act through cross-talk between members of the steroid-thyroid hormones.

Involvement of PGC-1, NRF-1, and NRF-2 in metabolic response by rat liver to hormonal and environmental signals

We studied liver oxidative capacity and O2 consumption in hypothyroid rats treated for 10 days with T4, or T3, or treated for 10 days with T3 and exposed to cold for the last 2 days. The metabolic response of homogenates and mitochondria indicated that all treatments increased the synthesis of respiratory chain components, whereas only the cold-induced mitochondrial proliferation. Determination of mRNA and protein expression of transcription factor activators, such as NRF-1 and NRF-2, and coactivators, such as PGC-1, showed that mRNA levels, except PGC-1 ones, were not related to aerobic capacities. Conversely, a strong correlation was found between cytochrome oxidase activity and PGC-1 or NRF-2 protein levels. Such a correlation was not found for NRF-1. Our results strongly support the view that in rat liver PGC-1 and NRFs are responsible for the iodothyronine-induced increases in respiratory chain components, whereas their role in cold-induced mitochondrial proliferation needs to be further on clarified.

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.

Further data on the occurrence and evolution of satellite DNA families in the lacertid genome

This paper reports the isolation and characterization of twoHindIII repetitive DNA families from the genome of two lacertid lizards,Podarcis sicula andLacerta saxicola. These satellites did not appear to be related to each other. The consensus sequences of their monomeric units did not show any similarity, though both DNAs were A-T rich. Moreover, each of them was found only in closely related species. The monomeric unit of theHindIII DNA family isolated fromP. sicula (pLHS) showed a close resemblance to pLCS, a centromeric satellite DNA previously isolated from the same species; it was, however, mainly localized at pericentromeric, interstitial and telomeric levels. The results also provide interesting information on the systematics of the lacertids studied.

EVOLUTION OF A CENTROMERIC SATELLITE DNA AND PHYLOGENY OF LACERTID LIZARDS

1. The composition and phyletic distribution of a highly repetitive satellite DNA, isolated from Podarcis sicula, was studied. 2. This DNA was rich in adenine and thymine and displayed frequent adenine stretches. It was always located on the centromeric heterochromatin even in quite taxonomically distant species. 3. Southern blot hybridization of the Taq I satellite on various species of lacertid families showed a close affinity among Podarcis, Algyroides and Lacerta dugesii. 4. All the other taxa investigated did not seem to possess this repeated sequence.

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.

In vitro effects of beta-endorphin on testicular release of androgens in the lizard podarcis sicula raf

The effects of the proopiomelanocortin‐derived opioid peptide, beta‐endorphin (β‐EP), and of the opioid antagonist, naloxone (NAL), on both basal and pituitary‐stimulated androgen secretion from superfused quiescent and active testes were assessed in the adult lizard, Podarcis sicula. In the absence of the homologous pituitary, in vitro treatment with β‐EP and/or NAL did not affect basal secretion of androgens from quiescent and active testes. Conversely, in the presence of the homologous pituitary, treatment with β‐EP brought about a decrease in androgen secretion in active testes, but no effect on quiescent ones Naloxone counteracted the inhibitory effect of β‐EP in active testes, and enhanced maximal pituitary‐stimulated secretion of androgens in quiescent but not in active testes. The effects produces by β‐endorphin and naloxone were reversible. These results suggest that, in this lizard, opioids might be involved in the control of androgen release. The lack of effect of β‐EP and naloxone when added directly to the testes seems to suggest that the opioid agonist and antagonist act on androgen release by modulating pituitary gonadotrophin output.

In vitro poly(ADPribosylation) of estrogen-induced proteins from the oviduct of the lizard Podarcis s. sicula Raf.

Poly(ADPribosylation) of nuclear proteins has been investigated in the nuclei from growing oviducts of intact and estrogen-treated spayed females of the lizard Podarcis s. sicula Raf. Isolated nuclei were incubated with [14C] NAD and nuclear proteins extracted in 0.2 m H2SO4. Labeled acid-soluble proteins were analysed by reverse-phase high-performance liquid chromatography (HPLC) and acetic acid-urea gel electrophoresis. The results reported here indicate that the ADPribosylation reaction is involved in modifying besides histone H2b, tissue specific proteins (SNPs and LMG-O). Moreover, comparable results have been obtained from nuclei prepared from the fully active oviduct of intact animals and spayed lizards stimulated with 17 beta-estradiol. It is concluded that the poly-(ADPribosylation) of hormone-induced proteins might play a role in the differentiation of the lizard oviduct.