Francesco Angelini

Localization of FMRFamide-Like Immunoreactivity in the Brain of the Viviparous Skink (Chalcides chalcides)

Neuroanatomical distribution of FMRFamide-like immunoreactivity was investigated in the brain and olfactory system of the viviparous skink, Chalcides chalcides. In the adult brain FMRFamide immunoreactive (ir) perikarya were observed in the diagonal band of Broca, medial septal nucleus, accumbens nucleus, bed nucleus of the anterior commissure, periventricular hypothalamic nucleus, lateral forebrain bundle, and lateral preoptic, subcommissural, suprachiasmatic and lateral hypothalamic areas. This pattern was seen in both male and female brains. Though all major brain areas showed FMRFamide-ir innervation, the densest ir fiber network was observed in the hypothalamus. During development, ir elements were observed for the first time in embryos at mid-pregnancy. FMRFamide perikarya were located along the ventral surface of the vomeronasal nerve, in the olfactory peduncle mediobasally, as well as in the anterior olfactory nucleus and olfactory tubercle. Furthermore, some ir neurons were observed in the rhombencephalic reticular substance; however, the ir fiber network was poorly developed. Later in development FMRFamide-ir neurons appeared also in the bed nucleus of the anterior commissure as well as the rhombencephalic nucleus of solitary tract and the dorsal motor nucleus of vagus nerve. In juveniles, the distribution profile of FMRFamide immunoreactivity was substantially similar to that of the adults, with a less widespread neuronal distribution and a more developed fiber network. Ontogenetic presence of FMRFamide immunoreactivity in the nasal area has been linked to the presence of a nervus terminalis in this reptile.

Reproductive modes and strategies in vertebrate evolution

Abstract Vertebrate reproductive modes, which are characterized by egg and development type, have been compared and, in some cases, re-defined. Reproductive strategies regulate reproductive effort. Reproductive modes and strategies have been selected in species history: more anciently and with more conservative characters the former; more recently and with some flexibility in response to environmental changes the latter. Semelparity is the expression of entire reproductive effort in a single event in the life cycle; iteroparity, in several events. In iteroparous species living in uniform, aseasonal environments, continuous reproduction can be also found. However, in fluctuating environments, seasonality concentrates reproduction only in the most favourable period of the year (seasonal reproduction). Several mechanisms (i.e. refractoriness) can block reproductive processes in unfavourable periods, avoiding energy waste. Different strategies such as male sperm storage, delayed fertilization, embryonic diapa...

Plasma vitellogenin and 17β-estradiol levels during the annual reproductive cycle of Podarcis s. sicula Raf

Plasma vitellogenin and 17 beta-estradiol concentration were determined during the annual reproductive cycle of the female lizard Podarcis s. sicula Raf. living around Naples. Plasma vitellogenin was purified from estrogenized males for characterization and to raise specific immune serum. Using ELISA, plasma vitellogenin titers were determined in relation to ovary weight; plasma 17 beta-estradiol was measured by RIA method. Native vitellogenin was present as two polypeptide bands: alpha and beta. The electrophoretic patterns, studied in normal male and estrogenized male and female, showed vitellogenin to be a protein present in female and in estrogenized male plasma but not in normal males. Lizard monomeric VTG, determined by SDS-PAGE, was about 200 kDa. Correlations between seasonal ovarian weight variations and plasma vitellogenin and 17 beta-estradiol suggest that ovarian development in Podarcis depends on plasma vitellogenin synthesis, which in turn relies on plasma estradiol levels. The two ovulatory waves observed in this study coincided with the two peak values of plasma vitellogenin and 17 beta-estradiol.

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.

Detection of DNA in Ancient Bones Using Histochemical Methods

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian “Casti Amanti” house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4′ -′ 6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.

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.

Pituitary adenylate cyclase-activating polypeptide and its receptor PAC1 in the testis of Triturus carnifex and Podarcis sicula.

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon-related family that occurs in two amidated forms with 38 (PACAP38) and 27 (PACAP27) amino acids. First discovered in the brain, it was then localized in several peripheral tissues of mammals, including the testis. However, current knowledge of the expression and function of PACAP and its receptor PAC(1) in the reproductive system of non-mammalian vertebrates, and particularly in the testis, is still limited. The aim of this work was to study the presence of PACAP and its receptor PAC(1) in the testis of two non-mammalian vertebrates during the breeding season: the crested newt Triturus carnifex and the wall lizard Podarcis sicula. The expression and distribution of this neuropeptide and its receptor PAC(1) were investigated by using in situ hybridization and immunohistochemistry techniques. Our results demonstrated that PACAP and its receptor PAC(1) were highly represented in the testis of these two species. In particular, we showed that they are present within some germ cells and that PACAP, unlike in mammals, is expressed also in the somatic cells (Sertoli and Leydig cells) of the testis of these two non-mammalian vertebrates, suggesting that this neuropeptide is involved in the hormonal control of spermatogenesis and steroidogenesis.

Expression of Prothymosin alpha during the spermatogenesis of the spotted ray Torpedo marmorata

In this study, we show that Prothymosin alpha (Ptma), a small, unfolded, negatively charged protein, is present in the cartilaginous fish Torpedo marmorata. The ptma gene is functional and peculiarly controlled during the male spermatogenesis of T. marmorata, as revealed by in situ hybridization and by immunocytochemistry studies. The data show that the ptma transcript is present in stage-specific germ cells, i.e. spermatocytes II and round spermatids. The Ptma protein is detectable in spermatocytes II, in round and elongated spermatids as well as in spermatozoa before their release from cysts, while it is not evident in spermatozoa located in male genital tracts. The ptma transcript and protein are also evident in some Leydig cells, located among maturing cysts containing meiotic and differentiating male cells. No expression for ptma is observed within Sertoli cells. Furthermore, immunolocalization procedures demonstrate that the protein is preferentially localized in the cytoplasm, whereas a nuclear localization is observed in round and elongated spermatids. The possibility that Ptma is involved in testis activity is discussed.

Reproductive activity of bombina pachypus from southern Italy

Abstract Seasonal changes in gonadal morphology, secondary sexual characters, and plasma sex steroids were studied in male and female Bombina pachypus from southern Italy. In this species, frogs are active and breed from May to September. During the long breeding season, sperm were always present in the testes. However, the testes did not always exhibit all stages of spermatogene‐sis. In July, spermatogenesis did not exceed the stage of sperma‐tocyte I, whereas in August it included all stages. It is possible that males use sperm originating from the preceding fall for the earliest spring matings. Throughout the breeding season, females showed many follicles in advanced or complete vitellogenesis and many atretic vitellogenic follicles. Postovulatory follicles, if any, were rare. Females laid few eggs at each oviposition. Concerning male sex steroids, testosterone was at comparable levels from May to September, and it markedly decreased in October. In females, wide variations in plasma estradiol and proge...

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

The epididymis in the male reproductive tract allows the survival, viability, and storage of spermatozoa from the testis. In the lizard Podarcis sicula, the epididymis can be regionalized to an initial segment called the caput that comprises the efferent ductules, followed by the middle and terminal segments, respectively termed the corpus and cauda. By means of in situ hybridization and immunocytochemistry, we analyzed the expression of the estrogen receptors of the alpha and beta type (ERα and ERβ) in Podarcis to test the responsiveness of the epididymal regions to estrogen in the annual reproductive cycle of this seasonal breeder. The results show that the efferent ductules and the cauda always express both ERα and ERβ throughout the year. In the corpus, the expression of ERα takes place only at the end of the mating period and continues in the non‐reproductive season whereas ERβ is expressed in all phases of the cycle. During the mating season, the cells of the corpus are engaged in massive secretory activity and do not express ERα. Experimental administration of E2 during this season does not change the expression of ERβ, nor does it affect the efferent ductules and cauda; instead, it inhibits the secretory activity in the corpus and induces the expression of ERα. Taken together, our findings suggest that in the epididymis of Podarcis, the expression of ERα may act as a switch for the secretory activity of the epididymal corpus. Mol. Reprod. Dev. 79:107–117, 2012.