Evaluation of Rana snk esculenta blood cell response to chemical stressors in the environment during the larval and adult phases.

The assessment of the biological effects on aquatic vertebrate species is frequently employed to monitor water pollution, as it provides significant information on bioavailability and actual concentration levels. In anamniote vertebrates (fish and amphibians), significant correlations have been observed between exposure to contaminants - both natural and experimental - and blood modification. We investigated the changes in some circulating blood cell parameters of green frog (Rana snk esculenta) tadpoles and adults collected at two sample rice fields, one heavily polluted and the other relatively unpolluted. The frequency of eosinophilic leucocytes, mitotic, anucleated and micronucleated erythrocytes was evaluated also regarding the haemopoietic/haemocatheretic and NOS expression of the liver. Haematological indicators in polluted samples were found to be significantly different from controls as regards both larval and adult exposure, and provided information on long-term background pollution of the habitats under investigation. The population of the polluted area showed evident effects of chronic exposure to contaminants, to a degree which could lead to sub-lethal alterations of their health status. The general nature of responses to this kind of stress emphasizes the role of amphibian peripheral blood as a sensitive indicator regarding contamination in aquatic environments.

Plasma sex steroid and thyroid hormones profile in male water frogs of the Rana esculenta complex from agricultural and pristine areas.

Some chemical compounds used in intensive agriculture have been found to induce estrogenic effects; therefore a histological analysis of the testes and an evaluation of plasma levels of sex steroid, thyroid hormones, and vitellogenin were carried out in adult male water frogs of two coexisting taxa (Rana lessonae and the hemiclonal hybrid Rana esculenta) sampled in agricultural and pristine areas. Differences in seasonal profiles of hormones were found in water frogs living in the agricultural area where the presence of endocrine disrupting compounds was suspected on the basis of a previous study. In R. esculenta, sampled in the pristine area, high androgen levels were found in May; the opposite trend was found for R. esculenta sampled in agricultural areas in which the highest androgen levels were found in September, significantly lower compared with those found in R. esculenta sampled in the pristine area. Low androgen levels were also recorded in R. lessonae males sampled both in pristine and agricultural areas, while the highest levels were found in September. Regarding the trend of estradiol-17beta, an increase of this hormone was found in July both in esculenta and lessonae sampled in the agricultural area, and in the same month an estradiol-17beta peak, even though lower, was also found both in esculenta and lessonae males captured in the pristine area; detectable vitellogenin was found neither in males captured in the agricultural area, nor in those sampled in the pristine one. Moreover, while no significant changes of thyroid hormones were found either in the esculenta or lessonae males sampled in the pristine area, increased T3 and T4 titers were found in July in both esculenta and lessonae captured in the agricultural area. Morphological differences of the testes in males of parental species captured in the agricultural area were also observed. These findings indicate alterations in endocrine and reproductive function in frogs in the agricultural area, that could suggest the presence of endocrine disrupting compounds.

On the evolution of erythrocyte programmed cell death: apoptosis of Rana esculenta nucleated red blood cells involves cysteine proteinase activation and mitochondrion permeabilization.

Batracian Rana esculenta erythrocytes cell death induced by either calcium influx, or staurosporine, involves typical apoptotic phenotype. Our data reveal: (i) a drastic modification of the cell morphology with loss of the ellipsoidal form as assessed by phase contrast microscopy and scanning electron microscopy; (ii) an exposure of the phosphatidylserine residues in the outer leaflet of the cell membrane; (iii) a caspase-3-like activity; (iv) a mitochondrial membrane potential (Delta Psi m) loss; and (v) a chromatin condensation and fragmentation. Erythrocyte chromatin condensation and fragmentation are prevented by caspase and calpain peptide inhibitors. These inhibitors also prevent Delta Psi m loss supporting the idea that mitochondria is a central sensor for Rana erythrocytes cell death. Our observations highlight the conservation of the programmed cell death machinery in erythrocytes across kingdom.

Ontogenetic profile of FSH and LH in Rana esculenta.

Circulating levels and pituitary content of FSH and LH were determined by specific radioimmunoassays in Rana esculenta starting a few days after hatching until the completion of metamorphosis. Both gonadotropins were found in the pituitary as well as in the blood plasma at all stages of development examined here. The plasma concentrations of FSH and LH were more or less uniform during pre- and prometamorphosis, but increased significantly at the onset of metamorphic climax. The plasma levels of FSH and LH remained high at the completion of metamorphosis. The pituitary content of FSH and LH was low in early premetamorphosis. It increased slightly through prometamorphosis and metamorphic climax, following which a highly significant increase occurred. Whereas plasma concentrations of FSH and LH were essentially similar within a single stage of development, the pituitary FSH content was severalfold higher than pituitary LH. The significance of these results is discussed in relation to the functional maturation of the brain-pituitary-gonadal axis in the frog.

L-alanine uptake by frog (Rana esculenta) red blood cells.

L-Alanine uptake has been studied in frog red blood cells. The present study shows the presence of different carriers for this amino acid in these cells. In the physiological concentration range, most L-alanine is taken up through the Na(+)-dependent system ASC, although the sodium-independent systems asc and L are also active. The competitive inhibition data obtained makes difficult to differentiate the two Na(+)-independent activities in a clear contrast with data from fish or mammalian erythrocytes, indicating that despite its widespread occurrence in vertebrates, these carriers show characteristics that are species specific.

Presence of casein immunoreactive epitopes in molluscs, fish and frog.

The presence of a group of peptides derived from milk proteins (caseins) was examined by immunocytochemistry in various tissues from invertebrates and lower vertebrates. Phagocytic hemocytes from different species of molluscs, and cells located in the intestine wall or in related glands of invertebrates and lower vertebrates showed immunoreactivity to antibodies to whole casein and related fragments. Several functional tests (cell migration, inhibition test, phagocytosis) using these peptides were performed on the mollusc hemocytes. Only ovine caseinoglycopeptide was able to increase the phagocytic activity of the hemocytes towards bacteria.

Sex steroid binding proteins in the plasma of the green frog, Rana esculenta: changes during the reproductive cycle and dependence on pituitary gland and gonads.

Sex steroid binding proteins (SSBP) have been reported in the plasma of female and male Rana esculenta. In both sexes SSBP bind [3H]estradiol and [3H]testosterone with medium-high affinity and high specificity. Using ion-exchange chromatography SSBP resolve into two peaks eluting at 0.27 M (peak I) and 0.36 M (peak II) NaCl. Both peaks bind [3H]estradiol and [3H]testosterone equally well. Isoelectrofocusing showed that peak I focused at pH 6.0 and 7.5, whereas peak II focused at pH 6.0. SSBP capacity for [3H]estradiol and [3H]testosterone changes throughout the reproductive cycle, showing low levels during the nonbreeding period and increasing levels during the breeding period. Hypophysectomy and/or gonadectomy result in small changes in SSBP capacity. Short-term steroid hormone treatment of gonadectomized animals does not modify SSBP capacity.

Opioid peptide modulation of stress-induced plasma steroid changes in the frog Rana esculenta.

The present paper aims to evaluate whether a neuroendocrine system including opioids accounts for the effects of stress in the frog, Rana esculenta. Using an acute-stress paradigm, the involvement of hypothalamic opioid peptides was investigated biochemically as well as by in vivo studies. HPLC and RIA investigations confirm the presence of beta-endorphin-like peptides in the brain of this frog. Several immunoreactive peaks are present, two of them coeluting with beta-endorphin and acetyl beta-endorphin reference peptides. Quantitative evaluation revealed that the beta-EP content of 24-hr captured animals was higher than that in fresh captured ones. The stress paradigm applied here was consistent with the measurement of plasma androgens and corticosterone levels in females after short-captivity confinement. In comparison with fresh-captured animals, a sharp decrease of these levels was found within 10-24 hr after capture. These effects were reversed by naltrexone, a long-acting opioid antagonist, after 24 hr of treatment. In fact, in the captive animals injected with 100 ng of naltrexone, the plasma androgen titers remained low at the 10th hour postcapture, but rose to the control levels within 24 hr. A similar effect was found for the plasma corticosterone levels. These data demonstrate that, in this anuran, endogenous opioids could mediate the stress-induced inhibition of gonadal function, and perhaps the inhibition of the hypothalamus-pituitary-adrenal axis.

Presence of X-AG carrier in frog (Rana esculenta) red blood cells.

Evidence is presented that the high levels of internal L-glutamic and L-aspartic acid in frog (Rana esculenta) red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity (Km = 3 microM) and low capacity (Vmax 0.4 mumol L-Glu.Kg-1 dry cell mass.10 min-1). It is Na+ dependent and the incorporation of L-glutamic acid can be inhibited by L- and D-aspartate and L-cysteic acid, while D-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X-AG family. Besides the incorporation through this system, L-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity (Km 39 microM) but high capacity (Vmax 1.8 mumol L-Glu.Kg-1 dry cell mass.10 min-1).

Seasonal changes in plasma growth hormone and prolactin concentrations of the frog Rana esculenta.

Plasma growth hormone (GH), prolactin (PRL), and vitellogenin (VTG) concentrations were determined during the annual reproductive cycle of the frog Rana esculenta. Plasma GH and PRL were measured using a RIA that employed purified bullfrog (Rana catesbeiana) GH and PRL as standards and radioligand, and their respective antibodies. Using ELISA, plasma VTG titers were related to ovarian weight. GH, PRL, and VTG displayed different trends related to season and sex. In male frogs the GH and PRL trends have been found parallel, showing the highest concentrations (35 and 85 ng/ml, respectively) during the winter months. In the female frogs, the GH trend behaved differently from that in males; in fact, plasma GH changed during the annual reproductive cycle, showing two main peaks occurring during the reproductive period and the autumn ovarian recrudescence that are well correlated with the vitellogenesis as well as with the main changes in ovarian weight.

Lipoprotein metabolism in the frog Rana esculenta.

1. The isoprenoid metabolism of the green frog has been studied, taking into consideration the transport and uptake mechanisms of plasma lipoproteins. 2. The lipoprotein complexes separated on KBr gradient showed six discrete peaks in both sexes. 3. The mechanisms of cellular uptake have been studied by immunological procedures. A molecule homologous to rat LDL receptor, and sharing its ability to bind only specific lipoproteins, has been shown. 4. Homology at mRNA level has also been demonstrated by Northern blot analysis and two different messengers have been shown in both male and female frog.

Seasonal changes in plasma prostaglandin F2 alpha and sex hormones in the male water frog, Rana esculenta.

Prostaglandin F2 alpha (PGF2 alpha), progesterone, androgens (testosterone + dihydrotestosterone), and 17 beta-estradiol were measured in the plasma of male frogs, Rana esculenta, by radioimmunoassays. Plasma concentrations of PGF2 alpha were higher from October to December and peaked in March (prereproduction) and in June (postreproduction). Plasma progesterone levels were relatively low but showed an increase from October to December and in June. Plasma androgen titres rapidly increased in early spring, started to fall during the reproductive period (May), and were lowest in July. 17 beta-Estradiol levels peaked in March and in June. The annual profile of the plasma PGF2 alpha levels was positively correlated with those of progesterone and androgens, while it was not correlated to the estradiol plasma pattern, except in March and June. The increase in plasma PGF2 alpha in the autumn may be related to gonadal recovery. The simultaneous increases in PGF2 alpha and 17 beta-estradiol, both in March and June, suggest a PGF2 alpha-dependent estradiol synthesis, a possibility also supported by the increased plasma 17 beta-estradiol previously observed in PGF2 alpha-treated postreproductive females. The effects of captivity and castration on plasma PGF2 alpha concentrations were also studied during the annual cycle. Captivity was associated with a reduced PGF2 alpha titre, while castration did not modify prostaglandin synthesis, which may point to an extragonadal source of plasma PGF2 alpha.

Prostaglandin F2 alpha in female water frog, Rana esculenta: plasma levels during the annual cycle and effects of exogenous PGF2 alpha on circulating sex hormones.

Prostaglandin F2 alpha (PGF2 alpha) and sex hormones (progesterone, androgens, and estradiol-17 beta) have been determined in the plasma of female water frog, Rana esculenta, utilizing radioimmunoassay methods (RIA). Plasma PGF2 alpha level increases in autumn-winter when recovery processes interest both gonad and oviduct. The PGF2 alpha plasma highest value has been recorded in October (4.52 +/- 0.58 ng/ml) and the lowest in May (1.43 +/- 0.12). The PGF2 alpha annual pattern is positively correlated to that of estradiol (but not to those of progesterone and androgens). Exogenous PGF2 alpha, injected into intact females during the various phases of the annual cycle, induces an increase of ovarian weight in frogs treated in December and February and an increase of plasma estradiol in postreproductive animals (June). Although preliminary, such results propose a putative implication of PGF2 alpha in ovarian recovery processes and in the interruption of reproductive processes in late summer.

Biochemical investigation of the Rana esculenta complex in the Kis-Balaton Nature Reserve, Hungary.

In this study it has been proved by PAGE of serum proteins that all the three members of the Rana esculenta complex occur in the Kis-Balaton Nature Reserve (Hungary). On the basis of the LDH isoenzyme pattern which is characteristic in green frogs we could distinguish all three variations of R. ridibunda and R. lessonae and one type of R. esculenta. The mobility of serum albumins on SDS-PAGE implies that the R. esculenta comes from hybridization of the two other species. The PAGE methods provide a reliable basis for the rapid taxonomic identification of both adults and immature speciments of the three forms of frogs.

Changes in plasma thyroxine levels during and after spontaneous metamorphosis in a natural population of the green frog, Rana clamitans.

Plasma thyroxine (T4) levels were measured by radioimmunoassay in a spontaneously metamorphosing population of green frog, Rana clamitans, tadpoles. In accordance with species previously studied, a sharp rise in plasma T4 was seen at metamorphic climax, followed by a decline at final transformation. Unlike other species studied thus far, T4 levels were also moderately high in growing tadpoles (stages X-XIII). These early elevated levels may be due to daily rhythmicity in thyroid secretion coupled with the restricted hours of blood collection used in this study.

Plasma sex hormones and post-reproductive period in the green frog, Rana esculenta complex.

The plasmatic profiles of androgens, estradiol, and progesterone, together with gonads and SSC modifications, have been followed, throughout the post-reproductive period in two Rana esculenta populations, inhabiting, respectively, a mountain pond (Colfiorito) and a sea level lagoon (Lesina). Testosterone and progesterone progressively decrease in the blood until July, while estradiol shows, in both sexes, an increase in the same month. Testosterone depletion accounts for thumb pad atrophy in the male and probably, in both sexes, for the summer interruption of sexual behavior. The estradiol increase could be linked to the induction of vitellogenin synthesis by the liver or, alternatively, could act through negative feed-back on the brain centers involved in GnRF synthesis and therefore it could be responsible for an inhibition of LH release and, in turn, of androgen synthesis/secretion by the gonads. The last function can be framed in the endocrine regulation of the so-called "refractory period" which interrupts the breeding during the summer. The hematic level of progesterone is higher during the ovulation period and this is consistent with the role assigned to the hormone, i.e., the induction of jelly release from oviductal glands. The hormonal trends in the blood of the two frog populations are very similar, although some differences exist in the levels of testosterone and progesterone.

Seasonal plasma sex steroid levels in the female Rana esculenta.

Seasonal plasma progesterone, androstenedione, estrone, and 17 beta-estradiol concentrations in the female Rana esculenta were determined by radioimmunoassay during the 1979 and 1981 seasons. Plasma levels of these steroids were highest just before the first ovulatory wave in spring and lowest after the breeding season. In the 1979 season (during the 1981 season hormones were not assayed in January, November, and December) progesterone, androstenedione, and estradiol levels showed another peak in November-December. During the breeding months, i.e., late March to late June, progesterone, androstenedione, and estradiol levels showed intermittent ups and downs corresponding roughly to the ovulatory waves. In addition, during the breeding season progesterone and androstenedione levels had a higher average in frogs with "ripe" ovaries than in those with "spent" ovaries. Relationships between seasonal steroid levels and ovarian activity are briefly discussed.

Variations in the vitellogenin titre during the reproductive cycle of Rana esculenta L.

1. Vitellogenin titre has been measured by rocket immunoelectrophoresis in specimens of Rana esculenta L. caught at three different locations in southern Italy. 2. Variations in the serum level of vitellogenin are temporally related to ovarian growth during the reproductive cycle. 3. With the exception of the FL population, vitellogenin titre is low at spawning time but increases prior to ovarian recovery. 4. It eventually levels off, or undergoes a slight decline during winter, when the ovary is in a static phase. 5. The evidence presented in this study is consonant with the experimental model on hormonal control of vitellogenesis. 6. The vitellogenin patterns observed in the populations examined in this study are interpreted as due to different environmental conditions even though the hybridogenetic nature of Rana esculenta has also to be taken into account.