Metallothionein expression and synthesis in the testis of the lizard Podarcis sicula under natural conditions and following estrogenic exposure.

Metallothionein (MT) is the main protein involved in the homeostasis of metallic micronutrients and in cellular defence against heavy metals and reactive oxygen species. Found in almost all vertebrate tissues, MT presence and localization in the testis has been controversial. In the present study, by using in situ hybridization and immunohistochemical analysis we assessed the localization of both MT transcript and protein in Podarcis sicula testes during two different phases of the reproductive cycle: the autumnal resumption and the spring-early summer mating period. In addition, with the same methodological approach, we verified the effect of estradiol-17ß and nonylphenol, a potent xenoestrogen, on MT expression and synthesis.  These results, the first collected in a non-mammalian oviparous vertebrate, demonstrated that the expression profile of MT mRNA and protein changes during the reproductive cycle. In the fall resumption, MT transcripts are absent in spermatogonia and present in all the other cells of tubules, including spermatozoa; vice versa, the MT protein is evident only in spermatozoa and somatic cells. In the mating period, both MT transcripts and proteins are present in spermatogonia, spermatocytes and spermatids, whereas in the spermatozoa only the proteins are detected, thus suggesting that the MTs translated in the earlier germinal stages are stored up to spermatozoa. Results also demonstrated that in lizard testis the expression of MT gene undergoes a cell-specific regulation after estrogenic exposure; the possible role and the mechanism by which this regulation occurs have been discussed.

Exploring the Role of Estrogens in Lizard Spermatogenesis through the Study of Clomiphene and FSH Effects.

Spermatogenesis is a fascinating biological process aiming to generate haploid spermatozoa from diploid spermatogonia through a specific hormonal network between gonadotropins and steroids. Increasing evidence suggests that the primary female sex hormone estrogen plays an active role in this process. This research points out on the role of estrogen during lizard spermatogenesis by using three experimental approaches: (1) exposure to an analogue of nonsteroidal estrogen as Clomiphene citrate that acts both as estrogen agonist and antagonist; (2) exposure to the gonadotropin FSH; and (3) exposures to FSH followed by Clomiphene. Histological and immunohistochemical results demonstrate that in the lizard Podarcis sicula during the mating period, Clomiphene as well as FSH determines the breakdown of spermatogenesis and the epididymal regression, presumably through estrogens input as indirectly demonstrated by the appearance of ERα and vitellogenin in the liver. The ability of Clomiphene to restore the gonadal natural condition after FSH treatment is also demonstrated. Finally, data indicate that lizard testis and epididymis control their morphophysiology regulating the intracellular presence of ERα.

Ectopic synthesis of vitellogenin in testis and epididymis of estrogen-treated lizard Podarcis sicula.

In oviparous vertebrates, vitellogenin (VTG) is the major yolk precursor synthesized in the liver of sexually mature females during the reproductive period. In males, the VTG gene is silent, but it may be activated by estradiol-17ß (E2) or estrogen-like substances. Until now, extra-hepatic expression and synthesis of VTG after estrogen exposure has been reported only for aquatic vertebrates. This study demonstrates the ability of testis and epididymis of the terrestrial oviparous lacertid Podarcis sicula to synthesize VTG following E2 exposure. The results of in situ hybridization and immunohistochemistry analysis show the presence of both VTG mRNA and protein in these districts besides the known induction in the liver. The possible contemporaneous uptake of the E2-induced hepatic VTG by means of the specific vitellogenin receptor has been also evaluated. Finally, histological analysis shows that the E2-treatment during the mating season impairs spermatogenesis.

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.

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 ß and androgen receptors, was also investigated. NP treatment inhibits the expression of AR, ERα, and ERß-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.

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).

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 ERß) 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 ERß 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 ERß 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 E(2) during this season does not change the expression of ERß, 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.

Expression of vitellogenin receptor in the ovarian follicles during the reproductive cycle of the spotted ray Torpedo marmorata Risso 1880.

The aim of this investigation was to identify the encoding sequence of vitellogenin receptor gene (vtgr), and its expression during the oogenesis in the spotted ray, Torpedo marmorata, in different phases of reproductive cycle. From an ovarian cDNA of vitellogenic female, we obtained a fragment of 581 bp, which corresponds to a partial sequence encoding the vitellogenin receptor (VTGR) in Torpedo (accession number: gi/193244760). This sequence shows a high identity with the VTGR of other vertebrates, particularly Leucoraja erinacea (89% identity) and Squalus acanthias (84% identity). We also showed that vtgr mRNA expression in the ovary modifies during the oogenesis and throughout the reproductive cycle. Indeed, in immature females, whose ovary contains only previtellogenic follicles, vtgr mRNA occurred in the oocyte cortex as well as within intermediate and pyriform cells. In mature females, whose ovary contains pre- and vitellogenic follicles, vtgr mRNA was detectable not only in the oocyte cortex and in intermediate and pyriform cells but also in small follicle cells present in the follicular epithelium of vitellogenic follicles. In ovulating females, that, as pregnant ones, show pre-and vitellogenic follicles, vtgr mRNA was evident in the oocyte cortex only, whereas in pregnant females, no vtgr mRNA was evident. The role of VTGR in the control of Torpedo vitellogenesis is discussed.

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

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.

Molecular identification of estrogen receptors (ERalpha and ERbeta) 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.

Effect of 17beta-estradiol and progesterone on vitellogenesis in the spotted ray Torpedo marmorata Risso 1810 (Elasmobranchii: Torpediniformes): studies on females and on estrogen-treated males.

The influence of 17beta-estradiol (E(2)) on vertebrate vitellogenesis is well ascertained. The aim of the present paper is to study the involvement of E(2) and progesterone (P) in the induction and regulation of vitellogenesis in females and experimental E(2)-treated males of Torpedo marmorata. We analyzed females in various stages of the reproductive cycle and E(2) experimentally treated males. The presence of vitellogenin was investigated in the plasma and in the liver by western blot and immunohistochemistry; its site of synthesis was investigated by in situ hybridization. The steroid levels in the plasma were measured by Enzyme Immunoassay. In treated males, E(2) induces in the liver the synthesis of VTG which is then secreted into the bloodstream as a 205-kDa polypeptide, the same that is found in the plasma of non-pregnant vitellogenic females. In females, E(2) is naturally present in the plasma and its level is correlated with VTG synthesis in the liver and with the female reproductive cycle. Indeed, large amounts of E(2) are only found in mature vitellogenic females, whose liver is involved in VTG synthesis and secretion. By contrast, small amounts of E(2) are evident in juveniles whose ovaries are lacking in vitellogenic follicles and in females preparing for ovulation. Low titers are also found in gravid females, whose liver is not engaged in VTG synthesis. We show that P, which is absent in untreated males and juvenile females, is evident in the blood serum of E(2)-treated males and sexually mature females. Interestingly, in treated males P appears in the plasma just 24h after the first injection of E(2) and its titer increases; a week after the last injections, the P level is similar to that recorded in non-gravid vitellogenic females. Finally, it is noteworthy that the highest titer of P was recorded in pregnant females. We demonstrate that in Torpedo vitellogenin synthesis, as in other vertebrates, is under the control of E(2) but also that this synthesis is probably under the control of progesterone.

An ultrastructural study on the vitellogenesis in the spotted ray Torpedo marmorata.

The present investigation strongly suggests that in Torpedo the oocyte growth is not only due to the uptake of exogenous molecules, but also by the oocyte itself and the granulosa cells. The oocyte, starting from the early previtellogenic follicles (see also Mol. Reprod. Dev. 61 (2002) 78), synthesizes large amounts of glycogen. Later, as the oocyte growth goes on, the cytoplasm of granulosa cells progressively bears numerous islets of glycogen, which are also evident inside the intercellular bridges and in the oocyte cortex, suggesting that they may flow from granulosa cells to the oocyte. The contribution of granulosa cells seems to become most relevant during the vitellogenesis. In vitellogenic follicles, both small, intermediate, and pyriform-like cells bear numerous vacuoles containing vitellogenin-like material, suggesting strongly that in Torpedo, differently from other vertebrate species, granulosa cells could be engaged in vitellogenesis. The present investigation does not allow us to know if such a material is due to a transcytosis process and/or is synthesized inside them. The organization of granulosa seems to exclude the possibility that it is transferred to granulosa via transcytosis. On the contrary, granulosa cells, especially in vitellogenic follicles, display the morphological organization of metabolically active cells, so they could be engaged in vitellogenin synthesis. This interpretation is consistent with the observation that granulosa cells are positively stained by OZI (osmium tetroxide-zinc iodide) and that the same positivity is evident on intercellular spaces, containing vitellogenin-like material, and on nascent yolk globules.

Lipovitellins and phosvitins of the fertilized eggs during embryo growth in the oviparous lizard Podarcis sicula.

In the lizard Podarcis sicula, the major vitellogenin (VTG)-derived yolk proteins, lipovitellins and phosvitins, were extracted from the yolk globules of laid and fertilized eggs at different periods of incubation up to 44 days close to hatching. Embryonic development was almost over at this time. Yolk proteins were isolated by precipitation in saturated (NH(4))(2)SO(4), separated on SDS-PAGE and detected by Western blotting with homologous polyclonal anti/VTG antibody. Two lipovitellins of 110 and 116 kDa were always present in the yolk of laid eggs after 1, 10, 18, and 44 days from oviposition. Both these proteins were glycosylated and were recognized by the anti/VTG antibody; their N-terminal sequences were analyzed. Four phosvitins were detected in freshly laid eggs, but their number decreased during incubation, and after 44 days only a single protein of approximately 6.5 kDa was present. The results indicated that, in this lizard, during embryonic development, lipovitellins remain unchanged, whereas the phosphorylated components of yolk undergo continuous degradation.

Vitellogenin precursors in the liver of the oviparous lizard, Podarcis sicula.

In reptiles, as in the other oviparous vertebrates, vitellogenin (VTG) synthesis is stimulated in the liver by ovarian estrogens. In this article, the presence of VTG precursors was detected in liver subcellular fractions of the oviparous lizard, Podarcis sicula, in the reproductive period. The rough endoplasmic reticulum (RER) and the smooth microsomal fraction (SMF), which includes smooth endoplasmic reticulum and Golgi complex, were separated by means of two different sucrose gradients. The successful separation was controlled at the electron microscope. The contents of the different compartments were extracted by means of n-octyl-beta-D-glucopiranoside detergent and subjected to SDS-PAGE. Western Blotting with homologous anti/VTG antibody revealed two immunoreactive proteins of about 84 and 70 kDa in the RER, and four proteins of about 180, 150, 60, 50 kDa in the SMF; all these proteins appeared phosphorylated and glycosylated. The differences in the molecular weight of these VTG precursors are discussed.

Oocyte growth and follicular hierarchy may be locally controlled by an inhibin-like protein in the lizard Podarcis sicula.

The lizard Podarcis shows an ovarian annual cycle with three to four ovulatory waves between April and July (reproductive period). In August to September, a refractory stage occurs, followed by a nonreproductive period (October to March), during which the oocytes undergo slow growth and prepare themselves for vitellogenesis and ovulation. In the reproductive period, only a certain number of oocytes start growing, giving rise to a follicular hierarchy, which is controlled by still unknown mechanisms. In the present paper, immunoreactive inhibin was detected in previtellogenetic follicles of the reproductive period, and in particular, in the pyriform cells of the follicular epithelium. As the follicle grew and the pyriform cells disappeared, immunostaining shifted to the oocyte cytoplasm. The smaller follicles did not show any immunoreactivity. In the nonreproductive period, no follicles were labeled. We conclude that in the reproductive period, inhibin characterizes the follicles destined to ovulation and might be one of the main factors controlling follicular hierarchy.