| Name | Bruno |
| Surname | Varriale |
| Institution | Università degli Studi della Campania Luigi Vanvitelli |
| bruno.varriale@unicampania.it | |
| Address | Department of Experimental Medicine, Molecular Genetics Laboratory, University of Campania "Luigi Vanvitelli", Via Costantinopoli 16, 80138, Naples, Italy |
The development of brain and behaviour is controlled by the interaction of genetic determinants and environmental factors. To study genetic determinants, model systems such as the Naples rat lines, i.e. Naples high (NHE) and low excitability (NLE), are useful. They have been selectively bred for divergent behaviour arousal to novelty. Aim of this study was to assess the extent of the genetic control of the selection trait. Thus adult albino rats of NHE and NLE lines have been used throughout. According to a classical Mendelian cross-breeding design, a first experiment was carried out with hybrids obtained from parental lines P1 (NHE) and P2 (NLE) as F1, F2 and related backcrosses B1 (F1xP1) and B2 (F1xP2). Young adults (60-80 days) offspring of both gender were exposed separately for two 10min tests to a spatial novelty (Lát-maze). To verify a possible sex link of the trait, a second experiment was carried out adding to the Mendelian cross design parental gender. Behavioural variables were horizontal (corner-crossings: HA), vertical (rearings on hindlimbs: VA) or total activity (HVA: HA+VA) scores. The heritability of HVA trait was estimated across the 20 generations of selection and Mendelian cross hybrids. Quantitative-genetic analysis on this trait and its HA and VA components, was applied by the Lynch and Walsh joint-scaling test procedure to evaluate underlying genetic mechanism. The correlation between experimental data of hybrids and estimated values from different heritability models were also computed. Results indicated that (i) the activity scores by Mendelian hybrids were intermediate between the two parental lines and were also graded; (ii) there was no sex effect on the heritability of trait but only a general tendency of females to higher activity levels; (iii) the heritability of HVA trait was very high (h2 index=0.824); (iv) heritability model of HVA and HA trait was polygenic with a marked epistatic control where as VA trait was fitted by simpler model with less genes and lower epistatic effect. In conclusion the Naples lines reveal strong genetic determinants for behavioural traits associated with polygenic pattern. Moreover, HA and VA activity components with prevailing cognitive and non-cognitive meaning, respectively, show differential genetic control.
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.
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.
The steroid/thyroid hormone receptors are members of a very large family of nuclear-activated transcription factors. These receptors play a crucial role in most biological function, including regulation of development, metabolism, behaviour and reproduction. Among androgen receptor (AR), we have recently demonstrated that its expression in the Harderian gland (HG) of the male hamster is under a well-co-ordinated cross-talk between various steroid hormone receptors. Here, are presented data on the sequence of hamster AR promoter region (5'UTR) and the molecular tools of its regulation. The 5'UTR is 1585 bp. The promoter region shows various responsive elements. Two putative CREM elements are present at -71 and -1576 bp. A putative retinoic acid responsive element is present at -1476 bp. An androgen/glucocorticoid responsive element is present at -473 bp. A putative thyroid hormone-responsive element at -381 bp and an estrogen responsive element at -230 bp. Also, a homopurinic stretch is evident between -1199 and -1118. Furthermore, Sp1 sites are also spread along the sequence. As well as for human, mouse, rat and pig, the hamster lacks the canonical promoter TATA and CCAAT boxes. Gel retardation experiments confirm the presence of active responsive elements for AR, estrogen receptor, glucocorticoid receptor and thyroid hormone receptor. Previous data on the regulation of expression of AR by other members of steroid/thyroid hormone receptors well correlate with sequence analysis and gel retardation experiments. Thus, androgens, thyroid hormone, stimulate the AR transcription, while synthetic glucocorticoid (Dex) and estrogen are potent inhibitors of AR expression. The comparison of hamster AR promoter sequence with other AR promoter shows an 89, 82, 84 and 84% identity with human, rat, mouse and pig AR promoter, respectively. These results, in the light of the extreme plasticity of hamster HG, suggest that the comparative study of expression and regulation of AR gene in the HG of the hamster offers a useful tool to approach the normal and pathological phenotype in human.
There is increasing evidence that 17beta-estradiol is necessary for normal male fertility. The aim of the present study was to characterize estrogen receptor beta (ERbeta) expression in a non-mammalian vertebrate model, the lizard (Podarcis s. sicula) testis. Immunocytochemical analysis shows that ERbeta proteins are present among germ cells in the nucleus of the spermatogonia, in primary spermatocytes and spermatids. Western blot analysis with antibodies against the ERbeta gene product revealed an isoform with a specific weight of 55 kDa. In conclusion, the widespread expression of ERbeta in the Podarcis s. sicula testis is consistent with a role for estrogens in modulating spermatogenesis in the male.
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.
The hamster Harderian gland (HG), a compound tubuloalveolar gland located in the orbital cavity, displays sex dimorphism. The present study focuses on the sequence analysis of a cDNA clone named MHG07 and on the regulation of its expression by steroid hormones. MHG07 mRNA (5.0 kb) is expressed in male HG only. The MHG07 cDNA (1.74 kb) shows an ORF of 94 amino acids and has no significant homologies with other polypeptides/genes. Castration leads to the disappearance of MHG07 mRNA after 4 days, whereas treatment with testosterone impairs the effect of castration. No MHG07 mRNA has been found in either rat or murine HGs. Androgen (A) administration to female hamsters induces the appearance of MHG07 mRNA. In primary culture of male hamster HG, androgens increase the MHG07 expression and this effect is blocked by both flutamide and cycloheximide. Dose-response experiments show that, at low A concentration (10(-12) M), the MHG07 was higher than that of the control (2-fold). This effect reaches its zenith at 10(-8) M (10-fold). This picture is paralleled by androgen receptor mRNA expression. It is argued that the expression of MHG07 is under androgenic control.
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.
Steroid hormones regulate many developmental and physiological processes via specific receptors whose number can be up- or downregulated. The regulation of estrogen (ER) and androgen (AR) receptor mRNAs in primary cultures of lizard testis is described. The high degree of homology between the probes used and the receptor mRNAs in lizard testis was consistent with the high-stringency hybridisation conditions and the molecular size of ER mRNAs (7.4 and 4.5 kb) and AR mRNA (9.5 kb). Primary cultures of testis cells revealed a time- and drug-dependent relationship between ER and AR mRNAs. 17beta-oestradiol (E) autoregulated ER mRNA and downregulated AR mRNA. The antiestrogen ICI 164,384 reversed the latter effect. Cycloheximide (Cy), to inhibit protein synthesis, in combination with E, impaired the AR mRNA expression. Testosterone (T) autoregulated the expression of its own receptor mRNA whereas this effect was reversed by both flutamide (F) and Cy. Dose-response experiments showed that low concentrations of steroids (E or T 10(-12) M) increased ER or AR mRNA levels, respectively. These results suggest that both estrogen and androgen may autoregulate the expression of their own receptor mRNAs. Since in lizard testis androgens are significantly involved in meiosis and spermiogenesis and E dramatically impairs the AR mRNA expression, the latter effect may be key in regulating certain phases of reproduction.
Xenopus laevis shows a sexual dimorphism of the electrophoretic pattern of Harderian gland (HG) proteins. The male pattern displays three protein fractions whose molecular sizes are approx. 205, 180 and 78 kDa, respectively, and which are absent in the female pattern. Conversely, the female pattern displays two protein fractions of approx. 190 and 76 kDa, respectively. This sexual dimorphism led us to hypothesize a sex steroid control of the HG. Administration of 17beta-oestradiol to male Xenopus converts the male protein pattern into the female one, while the administration of testosterone to the female has no effect. In this respect neither Northern analysis nor the RNase-protection assay performed using a 213 bp encoding for the androgen-binding domain reveals the presence of an androgen receptor mRNA in Xenopus HG. Conversely, Northern analysis has shown an oestrogen receptor mRNA whose size is approx. 6.5 kb and the RNase-protection assay performed by using a 197 bp encoding for the oestrogen-binding domain has also displayed the presence of an oestrogen receptor mRNA in the female HG but not in the male one. In addition, the oestrogen administration to male Xenopus induces the appearance of an oestrogen receptor mRNA. Androgen administration to female toad is ineffective. Taken together, all these findings suggest that in Xenopus laevis oestrogens are involved into the HG physiology. The appearance of an oestrogen receptor mRNA in the oestradiol treated males supports the hypothesis of the occurrence of autoinduction of oestrogen receptor mRNA expression in the HG.