The effects of gonadectomy and testosterone treatment on the Harderian gland of the green frog,Rana esculenta

The effects of gonadectomy and testosterone treatment on the fine structure of the Harderian gland in male and female green frogs were investigated in different periods of the year. Gonadectomy, carried out when the glands are in the lowest secretory phase (September), causes degenerative changes consisting of a reduction of the rough endoplasmic reticulum, the appearance of autolysosomes, and an increase of nuclear heterochromatin. These effects can be prevented by testosterone treatment. No castration effects are found during the recovery (November) and enhancement (April-May) phases of secretory activity. The results suggest that the frog Harderian gland's sensitivity to testosterone changes during the annual cycle. The androgen dependence of the Harderian gland is correlated with the presence of androgen receptors in both male and female forgs.     

Contraceptive steroid treatment affects steroid binding proteins and the percentage of free 17 beta-estradiol and testosterone in the cynomolgus monkey (Macaca fascicularis)

The levels of steroid binding globulins were characterized in cynomolgus monkeys that were treated with contraceptive steroid preparations delivered either by intravaginal rings (CVR) or orally (OC) in the diet. Levonorgestrel (dNG) was the bioactive progestin and the estrogen was either 17 beta-estradiol (E2) in the CVR treatment or ethinyl estradiol (EE) in the OC treatment. Both contraceptive treatments lowered sex hormone binding globulin (SHBG) levels below those observed in males (P less than 0.05) and normal females (P less than 0.01). Corticosteroid binding globulin (CBG) was elevated (P less than 0.01) in the OC treatment, demonstrating the potency of EE. The distribution of E2 and testosterone (T) between binding to SHBG or albumin and the unbound fraction was calculated after the determination of the percentage of free steroid by centrifugal ultrafiltration. Both contraceptive treatments increased the percentage of free T and E2 (P less than 0.01) in the subset of monkeys that were evaluated, but the percentage bound to SHBG and albumin were different only for the CVR group (P less than 0.05). Decreased total T concentrations in the treatment groups offset any increase in free T concentrations associated with an increase in the percentage of free T. The differences in the distribution of binding to SHBG associated with these contraceptive steroid treatments was influenced more by the reduction in the binding capacity of SHBG than by the displacement of E2 and T from SHBG by dNG.     

Relationships between liver testosterone receptor isoforms and aromatase activity in female green frog, Rana esculenta

Testosterone receptors (AR) are present in the liver of the female green frog, Rana esculenta, which resolve into two fractions (A and B) by ion-exchange chromatography. Fraction A is primarily located in the nuclei, fraction B predominates in the cytosols, and both fractions show a high affinity and specificity for testosterone. Liver AR fraction levels vary dramatically during the frog sexual cycle. Fraction A levels are high only when the liver is engaged in vitellogenin production and the plasma testosterone levels are high: they are maximal when aromatase activity is most intense. Fraction B levels are high when the liver is not producing vitellogenin and the plasma testosterone levels are minimal. In addition, in vivo experiments carried out on ovariectomized females treated with testosterone show that testosterone induces both fraction A and liver aromatase activity. This induction may be a step in the process that allows the liver to obtain estrogen from plasma testosterone which induces vitellogenin synthesis.     

An androgen receptor in the brain of the green frog Rana esculenta

An androgen receptor (AR) has been detected in both the hypothalamic and extra-hypothalamic area of the brain of the male of the green frog Rana esculenta. 3H-T binding activity was detected separately in the hypothalamic and extra-hypothalamic areas. Experiments of binding assay showed 3H-T binding activity in the nuclear extract but not in the cytosol of both the hypothalamic and the extra-hypothalamic areas. The androgen binding moiety was not strictly specific for androgens, binding also 17 beta-estradiol, although to a lesser extent. 3H-testosterone binding activity fluctuated in both the hypothalamic and the extra-hypothalamic areas throughout the reproductive cycle, and paralleled androgen plasma levels.     

Effects of sex steroid hormones and their antagonists on mast cell number in the testis of the frog, Rana esculenta

This study confirms our previous data on the effects of sex hormones on mast cell number (MCN) in the testis of frog Rana esculenta. After 15 days of treatment with oestradiol (E2) MCN strongly increases, while testosterone has no effect. After 30 days only a small increase in MCN is observed. These differences could be due to the non-physiological effect of E2 over a prolonged period. We also confirmed a massive increase in MCN after 15 or 30 days of treatment with cyproterone acetate (CPA). This increase in MCN is also observed after administration of CPA with tamoxifen. Ultrastructural analysis of testis shows empty spaces with degenerating Leydig cells in the interstitial compartment and numerous germinal cells completely degenerated, probably apoptotic, in the adjacent germinal compartment. The same effects were observed in testes after treatment with only CPA. Chronic E2 treatment provokes an increase in MCN on day 2. From day 4 to 12 of the treatment, MCN decreases dramatically and many germinal tubules appear strongly disorganised. In conclusion, the present results confirm that E2 treatment induces changes in MCN and chronic E2 treatment modifies the morphology of the frog testes. In addition, blocking androgen receptors with CPA, alone or in combination with tamoxifen, causes a significant increase in MCN, confirming the involvement of androgens in mast cell proliferation and/or differentiation.     

Fat body involvement in vitellogenin fate in the green frog, Rana esculenta

1. Since, in Rana esculenta, fat bodies contain vitellogenin, the present study was performed in order to determine whether or not fat bodies are involved in the fate of vitellogenin. 2. The experiment of November shows that fat body excision provokes plasma vitellogenin increase even in animals treated with estradion-17 beta + pituitary crude homogenate (as compared with relative control). The same picture has been shown in the April experiment. 3. The result on protein-bound phosphate in ovaries from the April experiment has shown that fat body extirpation causes a decrease of protein-bound phosphate in the ovary. 4. This results indicates that fat bodies play an important role in sequestrating circulating vitellogenin by the ovary.     

The annual cycle of activity of the subfornical organ in the green frog Rana esculenta

Abstract

The nuclear volume in cells of the subfornical organ of Rana esculenta shows a cyclic annual size change. It is pronounced both in the so‐called Gomori‐positive cells and the ependymal covering of the organ. There appears a maximum in April in the epen‐dyma and in May in the Gomori‐positive cells. The amount of neurosecretory material present in the subependymal layer also shows a maximum in April.     

Nociceptin binding sites in frog (Rana esculenta) brain membranes.

The recently discovered natural heptadecapeptide nociceptin (orphanin FQ) shares some homology with the opioid peptides but it binds to a distinct receptor type, termed nociceptin receptor. This study demonstrates the presence of specific nociceptin recognition sites in brain membrane fractions of an amphibian, Rana esculenta. Para-iodo-Phe(1)-nociceptin-amide was radiolabelled by catalytic dehalotritiation, resulting in p[(3)H]Phe(1)-nociceptin-amide of 25 Ci/mmol specific radioactivity. Specific binding of [(3)H]nociceptin-amide to frog brain membranes was found to be saturable and of high affinity with equilibrium K(d) values in the low nanomolar range. A single set of binding sites with about 180 fmol/mg protein maximal binding capacity was obtained in saturation and competition experiments. [(3)H]Nociceptin-amide binding could easily be inhibited by synthetic nociceptin compounds but not by opioid ligands. Both sodium ions and 5'-guanylylimidodiphosphate decreased the binding of the radioligand by transferring the receptor to a lower affinity state. Nociceptin dose-dependently stimulated the binding of the nonhydrolysable, radiolabeled GTP-analogue guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS) to G-proteins in frog brain membranes. Addition of 1 microM naloxone caused no significant change in the curves, indicating that nociceptin-mediated activation of G-proteins occurred through nonopioid mechanism.     

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.     

Sex steroid hormone binding globulin levels and free 17 beta-estradiol and testosterone in cynomolgus monkeys during different reproductive states

The effect of differences in sex hormone binding globulin (SHBG) levels associated with reproductive status on free 17 beta-estradiol (E2) and testosterone (T) was characterized in cynomolgus monkeys. Cycling female cynomolgus monkeys had higher SHBG levels than males, pregnant and lactating females (P less than 0.05). Ovariectomized females were not different than control females, suggesting no hypoestrogenic effect. The percentage of free T was elevated in pregnant animals (P less than 0.05) compared to normal males and females, but the percentage of free E2 was similar between these groups. Although a gender difference in the percentages of free E2 and T was not detected, there was a gender difference in the free T and E2 concentrations due to endogenous secretion. Increased free E2 concentrations during pregnancy were the result of endogenous secretion rather than the decreased binding capacity of SHBG; the increased percentage of free T during pregnancy significantly increased free T concentrations. These data suggest that the gender difference in SHBG levels in cynomolgus monkeys is due to androgenic influences and that estrogens have minimal influence. Furthermore, the decrease in SHBG levels during pregnancy and lactation may not be entirely dependent on these androgenic influences.     

Hormonal steroidogenesis in liver and small intestine of the green frog, Rana esculenta L.

We have investigated the potential of autonomous hormonal steroidogenesis in liver and small intestine of male and female frogs, Rana esculenta, during the recovery phase. After incubation of mitochondrial fractions with [4-14C]cholesterol, female liver and intestine formed pregnenolone at a rate of 0.63 and 2.3 pmol/mg protein/h, respectively, whereas conversion by male organs was only c. 0.03 pmol/mg protein/h. Minced tissues preparations transformed [4-14C]pregnenolone into progesterone and 17alpha-hydroxypregnenolone, the former prevailing in the liver, the latter in the intestine. Moreover, both tissues produced 20alpha-dihydropregnenolone, 20alpha-dihydroprogesterone and dehydroepiandrosterone. From incubates with [4-14C]dehydroepiandrosterone, androstenedione and androst-5-ene-3beta, 17beta-diol were identified, the former being more abundant in the liver, the latter in the intestine. These results indicate that both liver and intestine in frog can be independent sources of hormonally active steroids in both sexes.     

Cardiac role of frog ANF: negative inotropism and binding sites in Rana esculenta

To elucidate the role of atrial natriuretic peptides (NPs) in the amphibian heart, the myotropic effects and the cardiac distribution of frog atrial natriuretic factor (fANF) have been studied in Rana esculenta. Spontaneously, beating in vitro isolated working heart preparations were treated with increased concentrations (10(-11)-10(-8) M) of fANF-(1-24). The peptide at 10(-9) and 10(-8) M significantly reduced heart rate (HR) and, on the electrically paced preparations, decreased cardiac output (CO), stroke volume (SV) and work. Such negative inotropism was abolished by pretreatment with the pertussis toxin or by blocking the particulate guanylate cyclase (GC) with anantin while it was independent both from the functional impairment of the endocardium-endothelium by Triton X-100 and the inhibition of the soluble guanylate cyclase by 1 H-(1,2,4,) oxadiazolo-(4,3-a) quinoxalin-1-one (ODQ). By autoradiography, two classes of high and low affinity NPs binding sites were detected in the ventricular endocardium and myocardium and in the bulbus arteriosus. The analysis of displacement binding data using the radioligand [125I]-rat atrial natriuretic peptide [125I-rANP-(1-28)], its cold counterpart and the fANF-(1-24) showed that in the ventricular myocardium, the low affinity NPs sites bound both the heterologous and the homologous ligands at a concentration close to that responsible for the negative inotropism and chronotropism.     

Ovarian activity and reproduction in the frog, Rana esculenta

Rana esculenta specimens were collected, during the last 13 years, in well-defined areas around Naples. The annual ovarian cycle shows distinct phases of recrudescence (starting September; vitellogenesis), breeding (late March-early July; egg deposition and active oogenesis) and quiescence (July-August; no follicular growth). Previtellogenic follicles are recruited for vitellogenesis in early September and in between two successive ovulatory waves. Breeding congregations are generally formed after a heavy rain fall and eggs are laid in standing waters, temporary or permanent. A maximum of three clutches of eggs is produced during the breeding season, at roughly monthly intervals. All mature females reproduce to some extent. Ovarian weight and clutch size are positively correlated to body weight. Depending upon the body size, the potential clutch size ranges from 1000 to 3500 eggs during the first wave of ovulation and it is notably smaller in the successive wave(s) of ovulation. Egg masses and tadpoles are left unprotected and mortality is high. The life cycle from the fertilized egg to completion of metamorphosis is 2 months and oogenesis in the ovary starts in the larva before the onset of metamorphic climax. Young females hatching from the first clutch of eggs may reach sexual maturity and breed in May the following year; those hatching from the last clutch require nearly 20 months to reach sexual maturity. The importance of some endocrine and exocrine factors for the regulation of ovarian activity and reproduction is discussed.