Mariailaria Verderame

Molecular identification of estrogen receptors (ERα and ERβ) and their differential expression during VTG synthesis in the liver of lizard Podarcis sicula

In non-mammalian vertebrates yolk deposition in the oocytes is a hormone-dependent, gender-specific process. Produced by the ovary under gonadotropin stimulation, Estradiol 17-beta (E(2)) plays a key role in the liver synthesis of vitellogenin (VTG) which in turn is taken up by vitellogenic oocytes in the ovary. In many species a negative role in liver synthesis of VTG in females is also played by progesterone. Experimental administration of E(2) induces the expression of the VTG silent gene also in the liver of males of all the species studied. However, the role of the two isoforms of estrogen receptors, ERalpha and beta, in this process is still unclear. In order to elucidate what kind of ER is involved in the liver synthesis of VTG in the lizard Podarcis sicula, we obtained by means of RT-PCR two fragments of 430bp and 130bp from total ovarian mRNA, encoding respectively for ERalpha and ERbeta. Expression analysis of these two specific isoforms of ERs in the liver showed that in non-breeding females, and in wildlife untreated males only ERbeta is expressed. In breeding vitellogenic females and in E(2)-treated males both alpha and beta receptors are expressed. Furthermore, in females experimentally treated with progesterone during the breeding period, expression of ERalpha disappears. Conversely, treatment of females with E(2) in the non-breeding period induces expression of ERalpha. Immunohistochemical analysis and Western blotting showed that the presence of irVTG in liver and plasma is always parallel to hepatic expression of ERalpha in all the different experimental conditions. Our data strongly suggest that expression of ERalpha may be necessary for VTG synthesis in Podarcis. The possible modulatory role of ERbeta is also discussed.

A network system for vitellogenin synthesis in the mussel Mytilus galloprovincialis (L.)

The aim of this study is to assess, by RT‐PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis). J. Cell. Physiol. 228: 547–555, 2013.

Experimentally nonylphenol-polluted diet induces the expression of silent genes VTG and ERα in the liver of male lizard Podarcis sicula

Endocrine Disruptor Chemicals (EDCs) with estrogen-like properties i.e nonylphenol (NP) induce vitellogenin (VTG) synthesis in males of aquatic and semi-aquatic species. In the oviparous species VTG is a female-specific oestrogen dependent protein. Males are unable to synthesize VTG except after E2 treatment. This study aimed to verify if NP, administered via food and water, is able to induce the expression of VTG even in males of vertebrates with a terrestrial habitat such as the lizard Podarcis. By means of ICC, ISH, W/B and ELISA we demonstrated that NP induces the presence of VTG in the plasma and its expression in the liver. VTG, undetectable in untreated males, reaches the value of 4.34 μg/μl in the experimental ones. Expression analysis and ISH in the liver showed that an NP-polluted diet also elicits the expression of ERα in the liver which is known to be related to VTG synthesis in Podarcis.

Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo marmorata

The aim of this investigation was to assess the effects of nonylphenol (NP), an oestrogen-like environmental pollutant, on the vitellogenin (VTG) synthesis in adult males of the aplacental viviparous cartilaginous fish Torpedo marmorata. The VTG recovery in males is considered a biomarker of xeno-oestrogenic pollution as this lipophosphoglycoprotein is physiologically induced by oestrogens only in females of oviparous and ovoviparous vertebrates. Using in situ hybridization and immunohistochemistry, T. marmorata males injected with nonylphenol showed the presence of VTG in the liver and the kidney. In particular, vtg messenger (m)RNA and VTG protein were expressed in the liver, whereas in the kidney cells only the presence of VTG was recorded. By contrast, no expression for VTG was detected in the testis. These results demonstrate that in T. marmorata NP induces the expression of vtg only in the liver; the presence of VTG in the kidney and its absence in the testis are discussed.

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.

Interferences of an environmental pollutant with estrogen-like action in the male reproductive system of the terrestrial vertebrate Podarcis sicula

Nonylphenol (NP) is classified among the endocrine disruptor chemicals with estrogen-like properties. It is widely used in many industries and to dilute pesticides in agriculture, and is known to affect the reproductive system of many aquatic and semi-aquatic organisms. This study aimed to verify how NP, administered via food and water, may interfere with the reproductive cycle of a terrestrial vertebrate. Our model was the male Italian wall lizard Podarcis sicula, a seasonal breeding species that may be naturally exposed to environmental pollution. From our findings it emerges that an NP-polluted diet administered during the mating period causes in this lizard a slowdown of spermatogenesis and affects the testicular and epididymal structure, making it similar to that of the non-reproductive period. The distribution in the testis and epididymis of mRNA for steroid hormone receptors, i.e., estrogen α and β and androgen receptors, was also investigated. NP treatment inhibits the expression of AR, ERα, and ERβ-mRNA in spermatogonia and primary spermatocytes and causes a switch-off of the secretory activity of the epididymal corpus by inducing the expression of ERα.

Spermatogenic Waves and Expression of AR and ERs in Germ Cells of Podarcis sicula

Androgens are considered the main hormones that regulate spermatogenesis, but a lot of evidence confers to estrogen a key role in this process. In the testis of the seasonal breeder lizard Podarcis sicula we analyzed by means of in situ hybridization the expression of the androgen receptor (AR) and of the two types, α and β, of the estrogen receptors (ERs) throughout the three periods of the annual cycle: mating (spring-early summer), postmating-refractory (late summer), and autumnal recrudescence. The results show that during the mating period AR and ERs are expressed in all germ cells present in the tubules from spermatogonia to spermatozoa. During the postmating-refractory period, when only spermatogonia are present in the tubules, almost all express ERs and very few AR mRNA. During the autumnal recrudescence the localization of AR and ERs is the same of the mating period except for the population of the primary spermatocytes. The expression of the investigated receptors is peculiar in these cells that are positive in the middle-late pachytene stage of the meiotic prophase and negative in preleptotene. A possible functional role of the observed differences during spermatogenesis and in the spermatozoa is also discussed.

Expression of vitellogenin in the testis and kidney of the spotted ray Torpedo marmorata exposed to 17β-estradiol

In vertebrates, the liver was long thought to be the only site of vitellogenin (Vtg) production, but recent studies demonstrated that Vtg is also expressed in extrahepatic districts. The aim of this paper is to assess, by in situ hybridization and immunohistochemistry, the expression of Vtg in the testis and kidney of Torpedo marmorata exposed to 17β-estradiol (E(2)). In treated samples vtg mRNA and Vtg were detected contemporaneously only in the testis; differently the kidney cells were positive to Vtg antibody, but negative to vtg mRNA. This is the first study to assess that male germ cells, after an exposure to E(2), synthesize Vtg in a stage-dependent manner. The presence of Vtg and the modifications observed in the kidney after E(2) treatment are discussed.

Estrogenic contamination by manure fertilizer in organic farming: a case study with the lizard Podarcis sicula.

In the last years, worldwide organic farming has grown exponentially; as a consequence, the use of animal manure as a soil fertility source has become the principal agricultural choice. However, the use of manure as fertilizer can increase the amount of steroid hormone metabolites in the soil. In southern Italy, lacertidae lizards are the most abundant vertebrate group in agroecosystems and have been identified as potential model species for ecotoxicological studies. The aim of this study was to understand if the manure applied in organic farming has estrogen-like effects in the lizard Podarcis sicula. Adult male lizards were captured in two organic agricultural fields (manure-treated sites) and in an uncultivated field (control site). Lizards from the two organic farms displayed hepatic biosynthetic alterations typical of an estrogenic contamination; hepatocytes contained both vitellogenin and estrogen receptor alpha transcripts and proteins, detected by in situ hybridization and immunocytochemistry. The same cells did not show cadmium, lead and metallothionein accumulation, indicative of the lack of inorganic contamination. These findings suggest that exogenous estrogens, arising from the use of manure, could affect the welfare of wild animals and animal breeding, leading to bioaccumulation of estrogens in food chain, with possible risk for human consumers. For this reason, organic farming should implement the use of sustainable practices such as crop rotation to preserve the soil biological activity, rather than organic manure as fertilizer.

The Involvement of the Androgen Receptor in the Secretion of the Epididymal corpus in the Lizard Podarcis sicula

A crucial role in the maintenance of male reproductive functions is carried out by the androgen through its receptor in balance with the estrogen receptors (ERs). The distribution of the androgen receptor (AR) is well documented in the testis and in the reproductive tissues of mammals but the findings about the AR in nonmammalian vertebrates and in particular in reptiles are very scarce. Here by means of in situ hybridization (ISH) we investigated the AR expression along the epididymal channel (efferent ductules, corpus, and cauda) of Podarcis sicula during the mating and nonmating period. The results show that in this seasonal breeding species the AR expression pattern is always constant throughout the epididymis. The administration of estradiol-17β in the mating period does not affect the AR expression but inhibits the secretory activity of the epididymal corpus. To verify the expression pattern of ERs, we also conducted ISH investigations on adjacent sections with ERs probes. The findings suggest that AR induces the secretory activity in the epithelial cells of the epididymal corpus and confirm our previous results that showed the role of ERalpha (ERα) as switch off for the secretion of this compartment.