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Many of the current procedures for territory management (see Horizon 2020) and marine organism reproductive health assessment are based upon receptor-mediated responses of target genes and are founded on the assumption that these responses accurately reflect exposure of endocrine disrupter that can be linked to different pathological effects. The role of estrogens and their functional mechanism of action in bivalves are therefore far from clear. Building on these observations, the aim of this study, in the frame work of an Italian National Research Project (PRIN 2010 – 2011), was to identify estrogen receptor genes (ER1 and ER2) and to quantify any changes in expression levels using the real-time quantitative PCR technique in response of marine bivalve, Mytilus galloprovincialis at different pollution degree areas. Using gametes obtained by mantle (gonad) tissue of mature bivalves we demonstrated, after sequencing and alignments, the presence of differentially expressed transcripts for ER1 and ER2, quantitative differences in male and female gamete gene expression and an increase in both gametes of ER levels in polluted area. The significance of these findings in terms of the utility of estrogen receptors as biomarkers of reproductive health assessment in bivalve species is discussed.

Sexual behavior in vertebrates depends on the cyclic release of steroids and their binding to the brain receptors. Previously, we demonstrated the presence of specific binding of (3)H-testosterone and staining with PG-21 in the brain of the adult male frog, Rana esculenta. Here, we report our further receptor characterization using an anti-androgen receptor antiserum, PG-21, and the androgen site of action in frog brain. Nuclei, which contained cells labeled for the androgen receptor (AR), were mainly identified in the olfactory bulbs, preoptic-septal region, infundibulum, amygdala, thalamus, tectum, torus semicircularis, and medulla. The neuroanatomical AR staining appears similar to that in other lower vertebrates.

Steroids secreted by the ovary, specifically estrogen and progesterone, influence the expression of behaviors associated with reproduction by interacting with a specific binding protein, or receptor, located in target cells in certain hypothalamic nuclei. The present paper reviews the progesterone receptor studies in the vertebrates brain, the progesterone receptor fluctuations throughout the reproductive cycle and suggests a role for progesterone receptors in the regulation of hypothalamic functions in amphibians. Furthermore, we report here a combined biochemical and immunohistochemical analysis of the hypothalamic progesterone receptor during the reproductive cycle of a lower vertebrate, the female amphibian anura Rana esculenta. 3H-Progesterone binding activity was found in both cytosol and nuclear extract samples. The progesterone binding moiety showed typical characteristics of a true receptor, such as high affinity, low capacity and specificity for progesterone. Further characterization was performed by using monoclonal antiserum raised against both the subunits A and B of the chicken progesterone receptor. Immunostained neurons were located mainly in two specific regions of the hypothalamus: the preoptic area and the infundibular hypothalamus. An immunoreactive band of about 67 kDa was observed using Western blotting, both in the cytosol and in the nuclear extract. Progesterone receptor levels fluctuated throughout the cycle along with plasma steroids and vitellogenin synthesis.

In this paper we report the effect of gonadectomy and/or long-term sex steroid (testosterone and estradiol-17β) treatment and prolonged captivity (two months) on testosterone and estradiol-17β binding proteins (TBP and EBP, respectively) in the plasma of the male of the green frog Rana esculenta. Experiments were carried out during different periods of the reproductive cycle. Gonadectomy and prolonged captivity were carried out in winter, when the spermatogenic activity slowed down and the concentration of circulating androgens was high. Both gonadectomy and prolonged captivity resulted in a significant decrease in TBP binding activity, which could not be restored by the hormonal treatment. On the contrary, when the hormonal treatment was carried out in the early summer, when the spermatogenesis was active but the concentration of circulating androgens was low, a significant increase in TBP binding activity was observed. Neither gonadectomy, nor the prolonged captivity, nor the hormonal treatment affected EBP levels. Our data indicate that TBP apparent changes in response to testosterone and estradiol-17β treatment varied according to the period of the reproductive cycle, an indication that studies on sex steroid binding proteins regulation should take into consideration the internal endocrine condition before drawing any final conclusion especially in species with a seasonal mode of reproduction.

In this paper we report the effect of gonadectomy and/or long-term sex steroid (17β-estradiol and testosterone) treatment on binding activity of 17β-estradiol and testosterone binding proteins (EBP and TBP, respectively) in the plasma of the female of the green frog Rana esculenta. Experiments were carried out during different periods of the reproductive cycle when circulating levels of 17β-estradiol and androgens were : 1) low, 2) medium; 3) medium-high; 4) high. This study shows that EBP, but not TBP activity were affected by 17β-estradiol and testosterone treatment. The effect of the hormonal treatment changed according to the period of the reproductive cycle when it was carried out. Both 17β-estradiol and testosterone were ineffective when the circulating levels of 17β-estradiol and androgens were medium-high and high. On the contrary, the maximum effect was registered when circulating 17β-estradiol and androgens were at their minimum levels. Thus, our data indicate that binding activity of E...

Progesterone is a versatile hormone showing an ample variety of effects. One of the numerous functions attributed to progesterone is the modulation of vitellogenesis in oviparous vertebrates. As a prerequisite for the possible involvement of progesterone in vitellogenesis modulation, we investigated the presence of a progesterone receptor (PR) in the liver of the female green frog Rana esculenta. 3H-Progesterone (3H-P) binding activity was found in both cytosol and nuclear extract of the liver of Rana esculenta. The progesterone-binding moiety showed the typical characteristics of a true receptor, such as high affinity, low capacity, and specificity for progesterone. It also bound to DNA-cellulose and was eluted with a linear salt gradient at a concentration of 0.05 M of NaCl. The progesterone-binding moiety was down regulated by steroid hormones, in that ovariectomy resulted in a significant increase, in both cytosol and nuclear extract, of 3H-P binding activity with respect to intact females. On the contrary, 3H-P binding activity was almost undetectable after estradiol and/or progesterone treatment. The progesterone binding moiety of Rana esculenta was analyzed by Western blotting with the aid of a monoclonal antibody raised against the subunits A and B of the chicken PR. An immunoreactive band of about 67 kDa was observed in the liver of both intact and treated females. The 67 kDa band showed an increased intensity in ovariectomized animals, while it was faint following treatment with estradiol and/or progesterone. This is the first report on the presence of a progesterone receptor (PR) in the liver of an amphibian. PR of Rana esculenta is down regulated by estradiol and/or progesterone and shows peculiar immunological and biochemical characteristics, which make it rather different from the PR of other vertebrates.

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. © 1996 Wiley-Liss, Inc.

Phospholipid hydroperoxide glutathione peroxidase (GPx4/PHGPx) is a most abundant selenoprotein in the brain and GPx4 null mice die in utero at midgestation or knockdown of GPx4 during mammalian embryogenesis disturbs brain development. The current experiments were designed to investigate, by semiquantitative RT-PCR, the cerebral function of GPx4 of the lizards collected in both polluted (Sarno River, Italy) and unpolluted (Lago Matese, Italy) areas in order to provide useful molecular information on this widely distributed seleno-enzyme in adult as well through the development of the lizard, Podarcis sicula. Our research demonstrates the lizard GPx4 hormonal control. Treatments with HCG increases immunoreactivity while the anti-estrogen ICI 182-780 induces its reduction. The partial region of the cloned cDNA is of 340 bp; conserved protein domains reports its membership to selenoprotein and analysis amino acid residues the value as an antioxidant. The semiquantitative expression of GPx 4 of adult lizard brain in polluted station resulted generally very high and let sustain its importance as lizard neuroendocrine disruptor alarm.

In mammals, proopiomelanocortin (POMC)-related peptides are involved in reproductive processes at both the hypothalamopituitary and ovarian levels. Through immunocytochemical and physiological in vitro studies, evidence for a diffuse POMC-related opioid system in the lizard Podarcis s sicula is provided. In the lizard ovary, ,-endorphin (-EP)-like immunoreactive cells were observed within the granulosa layer; the immunoresponse showed seasonal variation, being most pronounced in the winter ovary. HPLC followed by immunoassay showed that acetyl P-EP is the main form of POMC-related peptide in both pituitary and ovary. In vitro studies showed that picomolar amounts of P-EP stimulate follicular estrogen production during both the reproductive and winter phases; induction was found to be higher in the reproductive phase. The data reported here provide evidence for the physiological role played by -EP in the reproductive function of Podarcis s sicula via induction of ovarian production of estradiol-17, which is the main factor responsible for the vitellogenic process.

An in vitro superfusion method was used to test sex hormone release from different kinds of ovarian follicle (growing follicles, postovulatory follicles, and atretic follicles) in the lizard Podarcis sicula sicula. Sex hormone output changes with the stage of follicle evolution and sexual cycle. Previtellogenetic follicles prevail in early‐spring quiescent ovaries and secrete mainly progesterone, which is probably utilized at that phase to delay ovarian resumption. In the active ovary, progesterone output from previtellogenetic follicles decreases, whereas vitellogenetic follicles produce a significant amount of 17β‐estradiol, which is necessary for sustaining vitellogenin synthesis by the liver and oviduct growth. As follicles become ripe, progesterone production is resumed, and it increases in young postovulatory follicles. This is in line with the functions assigned to the hormone at that phase of the sexual cycle, i.e., the induction of oocyte maturation and the regulation of egg retention in the oviduct. Postovulatory follicles can also synthetize 17β‐estradiol. After oviposition, this hormone, which is secreted by the old postovulatory follicles, can reinitiate vitellogenin synthesis, allowing the development of a new oocyte set. Our data confirm that active, although ephemeral, corpora lutea are also formed in oviparous species. A limited contribution to ovarian sex steroid production derives also from atretic follicles, at least at the early stages of the breeding cycle. © 1993 Wiley‐Liss, Inc. Copyright © 1993 Wiley‐Liss, Inc.

Progesterone (P), 17-OH-progesterone (17-OH-P), androstenedione (A), dehydroepiandrosterone (DHEA), testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), and 17 beta-estradiol (E2) were measured by RIA in plasma and testes of 114 males of the oviparous lizard Podarcis s. sicula raf, a species that displays annual hibernating cycles. Hormones were determined each month from January until December, except for August. Testosterone peaked at 174.8 ng/ml of plasma after emergence (March), while 5 alpha-DHT and A peaked in April. Plasma DHEA increased during hibernation. During the refractory period there were progressive increases in P and E2 plasma levels. The testicular peak of T, in March, coincided with that observed in plasma. The striking increases in testicular T and A in early July occurred at a time when plasma androgen concentrations were low. 5 alpha-DHT increased in April when spermatogenesis with spermiation occurred and then decreased alongside a second peak of T. There is an apparent separation of plasma and testicular androgen concentrations during the reproductive cycle.

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.

Progesterone, 17-hydroxyprogesterone, androstenedione, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, testosterone and oestradiol concentrations in the plasma were measured by simultaneous radioimmunoassay in males of the lizard Podarcis s. sicula. Hormonal determinations were performed at monthly intervals from January to December (except for August). Testosterone and androstenedione reached peak values of 174.8 ng/ml and 21.4 ng/ml in the mating season (spring) and then testosterone fell abruptly to 5.9 ng/ml in June remaining at this level during hibernation when dehydroepiandrosterone (DHA) reached a maximal level of 28.5 +/- 9.3 ng/ml. Castration resulted in a marked decrease of testosterone, androstenedione, dihydrotestosterone and DHA values, with DHA being significantly lowered only during the winter season. In castrated animals, however, testosterone and androstenedione persisted conspicuously in the plasma during the breeding period, suggesting that adrenal sex steroid output may change during the annual reproductive cycle. In intact animals, progesterone and oestradiol exhibited peak values during the refractory period after the mating season. We suggest a probable role of oestradiol in the induction of the refractory period in this lizard.

It is well known that certain actions of androgen are mediated through in situ aromatization to estrogen in neural target tissues. This study was undertaken to investigate androgen utilization in the hypothalamus of the female frog, Rana esculenta, through a quantification of estrogen receptors and aromatase activity during the reproductive cycle. 3H-estradiol-binding molecules were present in both the cytosol and the nuclear extract of the hypothalamus. These molecules bound specifically 3H-estradiol with high affinity (Kd 10(-10) M) and low capacity (cytosol: 1.2+/-0.4 fmol/mg protein; nuclear extract: 7.9+/-0.6 fmol/mg protein). Aromatase activity was detected in the microsomal fraction of the hypothalamus using a sensitive in vitro radiometric assay. Both aromatase activity and nuclear estrogen receptor binding fluctuated in synchrony throughout the reproductive cycle. Western blot analysis of aromatase protein revealed one immunoreactive band with a molecular weight of approximately 56 kDa. In contrast to aromatase enzyme activity, the relative levels of aromatase protein changed little during the reproductive cycle suggesting that post-translational mechanisms may be involved in regulating estrogen synthesis in the frog brain. A possible role for estrogens in the modulation of the reproductive behavior in this species is suggested.

In mammals endorphinergic systems have been shown to modulate reproductive processes and β-endorphin (β-EP) has been found to influence sexual functions, acting at the hypothalamus-pituitary-gonadal axis level. Using immunocytochemical and in vitro studies, evidence for a diffuse pro-opiomelanocortin-related opioid system in the lizard Podarcis s. sicula was produced. In the testis, β-EP immunoreactivity showed seasonal variation, being most pronounced in the interstitial cells of sexually quiescent lizards (December). Reverse-phase high-performance liquid chromatography, coupled with radioimmunoassay and immunocytochemistry, showed that β-EP and acetyl β-EP increased during December, while their concentrations were low during April, when the highest testicular activity occurred. Using in vivo studies, it was found that naltrexone treatment, blocking pituitary opioid receptor, increased androgen levels in the plasma and in the testis. It was also found with in vitro studies that the endogenous opioid system inhibits gonadotrophin release and therefore androgen production by the testis. The data reported here provide evidence for the physiological role played by opioid peptides at the pituitary level to regulate the seasonal reproductive activity of the lizard Podarcis s. sicula. Journal of Endocrinology (1994) 143, 565–571

The presence and activity of brain, pituitary and testicular β-endorphin (β-EP)-like material have been studied in the frog, Rana esculenta, using reverse-phase high-pressure liquid chromatography, coupled with radioimmunoassay and immunocytochemistry. In-vivo and in-vitro treatments with naltrexone were carried out to assess the putative physiological activity of opioid peptides. β(1–31) and (1–27), together with their acetylated forms, have been identified in brain, pituitary and testis. In particular, β-EP(1–31) concentrations peaked during July in the brain and pituitary, whilst in testes maximum concentrations were found in April and November. β-EP immunoreactivity was present in the brain within the nucleus preopticus and nucleus infundibularis ventralis while positive fibres in the retrochiasmatic regions projected to the median eminence. In the testis, interstitial cells, canaliculi of the efferent system, spermatogonia and spermatocytes showed positive immunostaining for β-EP. In intact animals, naltrexone treatment increased plasma and testicular androgen levels and this effect was confirmed in in-vitro incubations of minced testes. Naltrexone also induced a significant increase in germ cell degeneration. Our results indicated that an opioid system modulates the hypothalamus-pituitary-gonadal axis in the frog, Rana esculenta and, for the first time, we have shown that the testicular activity of a non-mammalian species may be regulated by opiates locally. Journal of Endocrinology (1993) 137, 49–57

In male Podarcis s. sicula plasma, a sex steroid binding protein [SSBP(s)] binds testosterone (T) and estradiol-17β (E 2 ) with moderate affinity ( K d = 0.23 ± 0.08 × 10 −8 for 3 H-E 2 , and 0.24 ± 0.07 × 10 −8 for 3 H-T) and high capacity. The SSBP binding affinity is unchanged throughout the sexual cycle, although its capacity is higher in nonreproductive males (winter and postreproductive period). This change may be related to changes in plasma T and E 2 levels, and is likely to be involved in mechanisms whereby free steroid is delivered to target organs. SSBP, under isoelectrofocusing, is distributed between pH 5.5–6.5 and pH 7.1–7.5. The concentration of these two forms varies during the annual cycle.

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 17β-estradiol. It is concluded that the poly-(ADPribosylation) of hormone-induced proteins might play a role in the differentiation of the lizard oviduct.

The evolution of sex steroids in the plasma and gonads of the lizard Podarcis s. sicula during the postreproductive period shows that these hormones could be involved in determining refractoriness. In the male, during this phase, the oestradiol could lower the hypothala‐mo‐hypophyseal system through a negative feed‐back. In the female a similar role could be assigned to progesterone.

The AA. have studied the effects of testosterone administration and of temperature increase on the subcommissural organ (SCO) of Lacerta s. sicula Raf. castrated during the winter. Testosterone administered to specimens at environment temperature did not cause stimulation of the SCO. It was sufficient to raise the temperature to obtain stimulation. At 28° C the SCO of castrated animals was also stimulated, but after more time and less intensely than in normal animals.

Adult males of the lizard, Podarcis s. sicula Raf., were treated with arginine-vasotocin (AVT) during the reproductive period to investigate its effects on the spermatogenic and endocrine activities of the testis. Results showed that AVT can affect gonadal activity by blocking spermiohistogenesis and inhibiting interstitial tissue.

Gonadotropin-releasing hormone (GnRH) molecular forms in the brains of three reptiles, Alligator mississippiensis (alligator), Calcides ocellatus tiligugu (skink) and Podarcis s. sicula (lizard) were characterized by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera, and by assessment of luteinizing (LH)-releasing activity in chicken dispersed pituitary cells. In alligator brain two GnRHs had identical properties to the two known forms of chicken hypothalamic GnRH (Gln8-GnRH and His5,Trp7,Tyr8-GnRH) in their elution on two reverse phase HPLC systems, cross-reaction with region-specific GnRH antisera, and ability to release LH. In skink brain, one immunoreactive and bioactive GnRH form, which eluted in the same position as His5,Trp7,Tyr8-GnRH on reverse phase HPLC, was identified. Three bioactive and immunoreactive GnRHs were detected in lizard brain. One form had similar properties to salmon brain GnRH (Trp7,Leu8-GnRH). The other two GnRH-like peptides are novel forms. One of these forms eluted in the same position as Gln8-GnRH on HPLC but had different immunological properties, while the third form was a rather hydrophobic species which appeared to be modified in the middle region of the molecule.