Neurodistribution of androgen receptor immunoreactivity in the male frog, Rana esculenta.

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.

Vitamin E in early stages of sea bass (Dicentrarchus labrax) development.

This study reports titration of vitamin E levels in the sea bass (Dicentrarchus labrax) using high-pressure liquid chromatography. The first part of the work is devoted to vitamin E detection in: (1) plasma of maturing females and males characterized by different body sizes; (2) seminal fluid and eggs; and (3) developing embryos of sea bass fed with vitamin E. In the second part of the study, variations of vitamin E levels during larval development are analyzed. The results show a direct correlation between plasma vitamin E content and body size for both adult male and female sea bass. High vitamin E levels were found in seminal fluid, in eggs before and after fertilization, and in embryos during development and at hatching, whereas vitamin E level was low in dead embryos and in embryos with limited survival. During larval development, the vitamin E content decreased slowly but steadily during the first four days of larval growth; subsequently, it progressively increased from day 9 to day 40. In teratogenic larvae, vitamin E content was significantly higher than in normal larvae. This study provides evidence on how vitamin E exerts an antioxidant defense in sea bass reproduction.

Progesterone receptor: some viewpoints on hypothalamic seasonal fluctuations in a lower vertebrate.

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.

Estrogen receptors and aromatase activity in the hypothalamus of the female frog, Rana esculenta. Fluctuations throughout the reproductive cycle.

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.

Characterization of SNP, a novel tissue- and phase-specific nuclear protein expressed during the proliferative phase in the oviduct of the lizard Podarcis sicula Raf.

The amino acid sequence of a novel tissue-and phase-specific nuclear protein (SNP) has been determined, after purification from the nuclei of the oviduct of the lizard Podarcis sicula Raf. during the reproductive period of the seasonal growth. SNP has a pI of 9.0 and contains 81 amino acid residues with a molecular weight of 9211.88 +/- 0.09. It shows a bipartite organization as the first 40 amino acids contain all 8 cysteinyl residues, while the last 41 amino acids contain 16 prolyl residues. Two more components have also been identified and characterized, with the first 79 amino acids matching SNP and missing one or two residues at the C-terminus. They have thus been named [des-(Ala81) SNP1] and [des-(Lys80-Ala81) SNP2], respectively. The molecular weights are 9140.21 +/- 0.83 for [des-(Ala81) SNP1] and 9011 +/- 0.09 for [des-(Lys80-Ala81) SNP2].

Effect of testosterone and 17beta-estradiol treatment on sex steroid binding proteins in the male of the green frog Rana esculenta.

In this paper we report the effect of gonadectomy and/or long-term sex steroid (testosterone and estradiol-17beta) treatment and prolonged captivity (two months) on testosterone and estradiol-17beta 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-17beta 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.

Evidence of a progesterone receptor in the liver of the green frog Rana esculenta and its down-regulation by 17 beta estradiol and progesterone.

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.

Apolipoproteins and their electrophoretic pattern throughout the reproductive cycle in the green frog Rana esculenta.

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.

Progesterone receptor in the reproductive system of the female of Octopus vulgaris: characterization and immunolocalization.

In this study for the first time we have characterized a progesterone receptor in the reproductive system of the female of Octopus vulgaris. Scatchard analysis revealed that one binding component with high affinity and low capacity for the ligand was present only in the nuclear extract. Competition experiments showed that the progesterone receptor was strictly specific for progesterone. DNA-cellulose binding and DEAE-Sephacel both confirmed the presence of one 3H-progesterone binding component which eluted at a salt concentration of 0.14 +/- 0.05 M NaCl and 0.15 +/- 0.05 M NaCl respectively. By using monoclonal antibodies against chicken progesterone receptor (subunits A and B), we have localized on Western Blot one band of about 70 kDa. Immunoreactivity for progesterone binding molecules has been localized in the nuclei of the follicle cells of the ovary, of the proximal portion of the oviduct and of the outer region of the nidimental gland. These data, taken together, provide evidence that in Octopus vulgaris the progesterone receptor has biochemical and immunohistochemical characteristics resembling those of progesterone receptor in vertebrates.

In vitro effects of beta-endorphin on testicular release of androgens in the lizard Podarcis sicula Raf.

The effects of the proopiomelanocortin-derived opioid peptide, beta-endorphin (beta-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 beta-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 beta-EP brought about a decrease in androgen secretion in active testes, but no effect on quiescent ones. Naloxone counteracted the inhibitor effect of beta-EP in active testes, and enhanced maximal pituitary-stimulated secretion of androgens in quiescent but not in active testes. The effects produces by beta-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 beta-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.

Poly(ADPribosyl)ation system in rat germinal cells at different stages of differentiation.

In order to study the possible functional relationship between poly(ADP-ribosyl)ation and spermatogenesis, the three main germinal cell types have been isolated and characterized as haploid spermatids and diploid and tetraploid spermatocytes. Purified germinal cell populations and rats of different age were used for activity-, immuno-, and Northern blot experiments, to determine at which level poly(ADPR)polymerase (PARP) is regulated at various stages of spermatogenesis. Poly(ADPR)glycohydrolase (PARG) activity was also determined, as was the subcellular distribution of both PARP and PARG enzymes. The results show that the maximum of both PARP amount and PARP activity can be detected on tetraploid spermatocytes which undergo meiotic division, whereas PARG activity does not differ in germinal cells; the cytoplasmic form of this enzyme is prevalent in testis. Moreover, a difference in timing was observed in maximal level between PARP expression, determined on testis from 60-day-old rats, and PARP activity, detected on testis from 30-day-old animals. It seems that different mechanisms modulate the poly(ADPribosyl)ation system during spermatogenesis. Regulation of the poly(ADPribose) turnover, variations of PARP amount, as well as changes of PARP transcription level, seem to accompany germinal cell differentiation, possibly being implicated in DNA replication, repair, and transcription.

Opioid peptides and testicular activity in the lizard Podarcis s. sicula Raf.

In mammals endorphinergic systems have been shown to modulate reproductive processes and beta-endorphin (beta-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, beta-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 beta-EP and acetyl beta-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.

Presence and steroidogenetic activity of beta-endorphin in the ovary of the lizard, Podarcis s. sicula raf.

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, beta-endorphin (beta-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 beta-EP is the main form of POMC-related peptide in both pituitary and ovary. In vitro studies showed that picomolar amounts of beta-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 beta-EP in the reproductive function of Podarcis s. sicula via induction of ovarian production of estradiol-17 beta, which is the main factor responsible for the vitellogenic process.

Changes in ovarian follicles and in vitro sex hormone release in the lizard Podarcis sicula sicula.

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 acute ovary, progesterone output from previtellogenetic follicles decreases, whereas vitellogenetic follicles produce a significant amount of 17 beta-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 17 beta-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.

Opioids and testicular activity in the frog, Rana esculenta.

The presence and activity of brain, pituitary and testicular beta-endorphin (beta-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. beta-EP(1-31) and (1-27), together with their acetylated forms, have been identified in brain, pituitary and testis. In particular, beta-EP(1-31) concentrations peaked during July in the brain and pituitary, whilst in testes maximum concentrations were found in April and November. beta-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 beta-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.

Plasma sex steroid binding proteins (SSBP) in the male lizard, Podarcis s. sicula, during the reproductive cycle.

In male Podarcis s. sicula plasma, a sex steroid binding protein [SSBP(s)] binds testosterone (T) and estradiol-17 beta (E2) with moderate affinity (Kd = 0.23 +/- 0.08 x 10(-8) for 3H-E2, and 0.24 +/- 0.07 x 10(-8) for 3H-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 E2 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.

Immunohistochemical demonstration of the presence and localization of diverse molecular forms of gonadotropin-releasing hormone in the lizard (Podarcis s. sicula) brain.

The immunohistochemical presence and the distribution pattern of four different molecular forms of gonadotropin-releasing hormone (GnRH) were investigated in the brain of both sexes of the lizard, Podarcis s. sicula. Animals used in this study were collected in November and April, representing two different periods of the reproductive cycle. The antisera used were those raised against synthetic mammalian GnRH, chicken GnRH-I and II, and salmon GnRH. Strong immunoreaction was obtained for salmon, chicken-I, and chicken-II GnRHs, whereas a very weak reaction was seen for the mammalian form of GnRH. The distribution of immunoreactive-GnRH perikarya and fibers did not vary with the sex, the reproductive condition of the animals, or the antiserum used. Also, the intensity of immunoreaction with any one antiserum was quite similar in both periods of the year and in all brains examined. The immunoreactive perikarya was seen as two distinct groups, one in the mesencephalon and the other in the infundibulum. Immunoreactive fiber endings were seen in the telencephalon, the optic tectum, the anterior preoptic area, the median eminence, the central grey matter, the rhombencephalon, and the cerebellum. No immunoreactive perikarya were seen in the telencephalon or the anterior preoptic area.

Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s. sicula Raf.

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 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.