Effect of photoperiod and temperature on ovarian cycle of the frogRana tigrina (Daud.)

The effect of varying photoperiod regimes (LD: 20,4; 4,20; 6,18; 18,6 and 12,12) on ovarian follicular development was analysed in the frogRana tigrina maintained at ambient and constant 30° ± l°C for 3 months. The experiments were conducted in early recrudescent and quiescent phases. The frogs were fed guppiesad libitum on alternate day. None of the photoperiod regimes had any effect on the ovaries or the fat bodies, whereas exposure to constant high temperature (regardless of photoperiod) during recrudescent phase induced production of greater number of eggs (∼ 18000 vs 13000 in controls) of ovulatory sizes (> 1400 μm) compared to the corresponding controls maintained at ambient temperature. Hence, ovarian mass also increased in these frogs. In the quiescent phase, high temperature merely enhanced growth of previtellogenic oocytes. In both the phases high temperature caused a reduction in the fat bodies over the respective controls, possibly due to increased metabolic activity. The above findings indicate that temperature plays a key role in the regulation of ovarian cycle ofRana tigrina and that the photoperiodic mechanisms may not govern the annual recrudescence of ovaries in the frog. The study also shows that the frog exhibits the phenomenon of “phenotypic plasticity” in its reproductive behaviour by producing significantly greater number of eggs in response to elevated temperature.     

Dynamics of oogenesis in the tropical anuranRana tigrina (Amphibia: Ranidae) with special reference to vitellogenic cycles in wild-caught and captive flogs

The ovarian cycle ofRana tigrina was analysed by quantifying the developing oocytes (classified into stages on the basis of diameter) and atretic ones at monthly intervals. Stages I to IV represent oocytes in the first growth phase and the remaining ones the vitellogenic or second growth phase. Stages I–III occurred year round but exhibited significant variation in their number. The number of stage II oocytes always dominated the other stages. Recruitment of oocytes to stages IV and V in April marked the initiation of vitellogenic growth in all specimens. Of the 30 to 35% second growth phase oocytes, 25 to 28% reached ovulatory sizes by June. After spawning the ovarian mass declined drastically from 15 to 0.2% of body mass in July. Atresia was maximal (5%) in August. In other months, it was less than 1.5% of the total oocytes. Oogenic episodes occurred in March and July yielding new oocytes. The number of first growth phase oocytes fluctuated from 65 to 95%. The fluctuation was inversely correlated with the second growth phase oocytes indicating a 30 to 35% annual turnover rate of oocytes in the frog. The final egg number/ovarian mass is positively correlated with the snout-vent length as well as body mass of the frogs.R. tigrina produces about 4000 eggs/100g body mass. Further, the mean number of yolky eggs/100 g body mass and the total volume (V) of eggs/frog were highly correlated. Frogs living in captivity produced fewer eggs compared to the wild ones (3594 ± 227 in captivevs 4704 ± 317 in wild frogs). Also, these frogs failed to breed though they showed amplexus with breeding males. Injection of desoxycorticosterone acetate however induced spawning in 4 out of 5 frogs. They released about 3000 eggs each. Captivity seems to mainly impair breeding and to a little extent the vitellogenic growth of oocytes inR. tigrina.     

Effect of androgens on oviductal growth in skipper frog Rana cyanophlyctis

Effects of exogenous androgens (testosterone, testosterone propionate and dihydrotestosterone) and estradiol-17beta on the oviductal growth/hypertrophy were studied in young and bilaterally ovariectomized (BLO) adult frogs (Rana cyanophlyctis) during postbreeding phase of the reproductive cycle. Estradiol-17beta injections induced oviductal hypertrophy to the maximal extent among hormone treated groups. In androgen treated frogs also there was an increase in the oviductal dry weight and protein content both in young and BLO adult frogs, suggesting the role of endogenous androgens in controlling the growth of oviduct in R. cyanophlyctis.     

Effect of progesterone administration on the distribution of oviductal carbohydrates in <Emphasis Type="Italic">Rana tigrina</Emphasis>

Our aim has been to determine whether carbohydrate distribution in the oviducts of progesterone-treated animals is comparable with that of seasonal breeders in Rana tigrina. Like many other anurans, R. tigrina oviduct exhibits a short straight portion (pars recta, pr) at the beginning followed by a long, highly coiled portion (pars convoluta, pc). Histologically, the oviduct of this species revealed some unique features, one of which was intense toluidine blue staining, specifically in the upper mucosal glands of pc4. Based on lectin reactivities in the epithelial cells and mucosal glands, patterns of lectin staining in the seasonal breeders were classified into seven types: R1-R3 (for pr) and C1-C4 (for pc). Typically, some lectins reacted selectively either with ciliated cells (concanavalin A) or non-cialiated cells (Ricinus communis agglutinin I and wheatgerm agglutinin); however, Bandeiraea simplicifolia agglutinin I reacted with both cell types. These staining patterns were different in the progesterone-treated animals. Differences in glycan distribution in the oviductal secretions were revealed by lectin blotting. Compared with the seasonal breeders, an enhanced staining of some lectins was noted in the hormone-treated animals: either an increased staining intensity of existing protein bands or additional staining of new protein bands. Inversely, the staining of wheatgerm agglutinin was markedly diminished in the hormone-treated animals, suggesting the inhibitory effect of progesterone on oviductal glycan distribution. Whether alteration in glycan distribution upon progesterone treatment affects the physiological properties of the released jelly substances remains to be addressed. This research was supported by Thailand Research Funds (to W.W.), a Research Initiate Grant from Kasetsart University (to A.T.), and Mahidol University.     

The participation of corticosterone in luteinizing hormone releasing hormone (LH-RH) action on luteinizing hormone (LH) release from anterior pituitary cells in vitro

Corticosterone alone was not able to stimulate release of luteinizing hormone (LH) from anterior pituitary cells $\text{in}$$\text{vitro}$, but corticosterone in combination with luteinizing hormone releasing hormone (LHRH) augmented the release of LH into the culture media. These results may indicate that corticosterone may have the capacity to activate membrane receptors for LHRH in the gonadotrophs.     

Hypothalamic-pituitary-adrenal and -gonadal axis function after exercise in sedentary and endurance trained elderly males

The aim of this study was to investigate hypothalamic-pituitary-adrenal (HPAA) and -gonadal (HPGA) axis responses to post-exercise (30 min at 65% V˙O_2max) combined corticotrophin, luteinizing hormone and thyrotrophin releasing hormone challenge (0.7 μg/kg body mass) in elderly distance runners (DR; age: 68.9 ± 4.2 year) and sedentary individuals (SI; age: 69.1 ± 2.6 year). Plasma cortisol, growth hormone, prolactin, luteinizing hormone, follicle stimulating hormone and total testosterone (T) concentrations pre- and post-exercise as well as in response to stimulation did not differ between DR and SI. Plasma adrenocorticotropic hormone returned to pre-exercise level in DR 60 min and in SI 90 min post-stimulation. Free T was lower in DR at all time points. Our results do not support the notion of altered releasing hormone-stimulable HPAA and HPGA synthesis-secretion capacity in elderly males after endurance training. Accepted: 18 November 1997     

Age related changes in ovarian follicular kinetics in the Indian skipper frogRana cyanophlyctis (Schn.)

Changes in ovarian follicular kinetics were studied in relation to aging in the Indian skipper frog Rana cyanophlyctis.Age was determined by skeletochronology, by counting the number of growth rings and lines of arrest of growth from the cross sections of 4th phalange of 4th toe. For follicular kinetics study oocytes were counted under binocular using 10% of Bouin’s fixed ovary and they were classified into first growth phase, medium-sized second growth phase, large-sized second growth phase and atretic follicles. Analysis of phalangeal cross sections indicated that frogs ranging 14–54 g in body weight and 4.9–8.9 cm in body size showed 1–7 year rings. Frogs that weighed 14–16 g showed 1 year ring, and contained immature ovaries; those with 18 g body weight had one to two year rings, in which second growth phase oocytes appeared for the first time in the primiparous ovary. Frogs with 20–54 g body weight showed 2–5 year rings in which ovary contained 5–24% of second growth phase oocytes. Further, body weight, body size, ovarian weight, number and size of second growth phase oocytes and total number of oocytes showed a significant (P < 0.05) positive correlation, while, the number of first growth phase and atretic follicles showed a poor correlation with age. The results suggest that in nature, the age of Rana cyanophlyctis ranges between 1–7 years. Phalangeal growth rings are formed annually. Females attain sexual maturity in 2nd year. Frogs with 2–5 years of age may constitute breeding females. Body weight, body size, ovarian mass, number of second growth phase and total oocytes, and egg size increase with age up to 5 years.     

Theoretical prediction of antigenic sites in the Β-subunits of human choriogonadotropin and luteinizing hormone

Using hydrophilicity and recognition values of amino acids, the antigenic sites of theΒ-subunits of human choriogonadotropin and luteinizing hormone were computed from their amino acid sequences. Six antigenic sites were calculated for human choriogonadotropinΒ-subunits: residues 3–8, 17–22, 59–65,100–106,110–116 and 134–139. For luteinizing hormoneΒ-chain three antigenic sites were calculated: residues 17–22,59–65, and 100–106; all these three sites of luteinizing hormoneΒ being identical to the corresponding sites in human choriogonadotropinΒ. There was no antigenic site in luteinizing hormone that was also not found in human choriogonadotropin. On the other hand, there were unique determinants in human choriogonadotropin that were not found in luteinizing hormone; these determinants were residues 3–8, 110–116 and 134–139     

Degeneration of germ line cells in amphibian ovary

Ogielska, M., Rozenblut, B., Augustyńska, R., Kotusz, A. 2010. Degeneration of germ line cells in amphibian ovary. —Acta Zoologica (Stockholm) 91 : 319–327 We studied the morphology of degenerating ovarian follicles in juvenile and adult frogs Rana temporaria, Rana lessonae and Rana ridibunda. Degeneration of primordial germ cells was never observed and was extremely rare in oogonia and early oocytes in a cyst phase in juveniles. Previtellogenic oocytes were rarely affected. Three main types of atresia were identified. In type I (subdivided into stages A–D), vitellogenic oocytes are digested by proliferating follicle cells that hypertrophy and become phagocytic. A – germinal vesicle shrinks, nucleoli fuse, oocyte envelope interrupts, and follicular cells hypertrophy; B – follicular cells multiply and invade the oocyte; C – entire vesicle is filled by phagocytic cells; D – degenerating phagocytes accumulate black pigment. Type II is rare and resembles breakdown of follicles and release of ooplasm. In type III, observed in previtellogenic and early vitellogenic oocytes, ooplasm and germinal vesicle shrink, follicle cells do not invade the vesicle, and condensed ooplasm becomes fragmented. The residual oogonia in adult ovaries (germ patches) multiply, but soon degenerate.     

Expression of a mammalian aquaporin 3 homolog in the anterior pituitary gonadotrophs of the tree frog, <Emphasis Type="Italic">Hyla japonica</Emphasis>

Aquaporins (AQPs) are a family of water channel proteins that play a major role in maintaining water homeostasis in various organisms. Several AQPs have been identified in the tree frog, Hyla japonica. Of these, AQP-h3BL, which is expressed in the basolateral membrane of the epithelial cells, is a homolog of mammalian AQP3. Using immunohistochemistry and in situ RT-PCR, we have demonstrated that AQP-h3BL is expressed in the anterior pituitary gonadotrophs of the tree frog but not in the other hormone-producing cells of the anterior pituitary. In gonadotrophs labeled for luteinizing hormone subunit-β (LHβ), AQP-h3BL protein was found to reside in the plasma membrane, the nuclear membrane and the cytoplasm. Double-labeling of AQP-h3BL mRNA and LHβ protein revealed that AQP-h3BL mRNA is expressed in the gonadotrophs. Following stimulation by gonadotropin-releasing hormone (GnRH), the label for AQP-h3BL localized in the plasma membrane became more intense, concomitant with the transport of LHβ-positive materials to the plasma membrane. These developments coincided with a decrease in the labeling density in the cytoplasm and near the nuclear membrane, suggesting that the latter localizations may function as “storage area“ for AQP-h3BL. Immunoelectron microscopy also confirmed these localizations of AQP-h3BL protein. Based on these results, we suggest that AQP-h3BL protein in the frog gonadotrophs is involved in the formation of secretory granules, the swelling and increase in the volume of the granules and exocytosis.     

Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

Summary The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human--thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thryrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.This work has been supported by grants no. 2184-83 and PB 86-0095 from the Comisión Interministerial para la Ciencia y Tecnología, Spain     

Ontention of antisera against a hypothalamic decapeptide (luteinizing hormone/follicle stimulating hormone releasing hormone) which stimulates the release of pituitary gonadotropins and development of its radioimmunoassay

Using the classical approach, a decapeptide was synthesized with the structure of porcine luteinizing hormone/follicle stimulating hormone releasing hormone reported by Matsuo, H., Baba, Y., Nair, R. M. G., Arimura, A. and Schally, A. V. (1971) Biochem. Biophys. Res. Commun. 43, 1393–1399. As already reported, this peptide was capable of inducing in vitro the release of luteinizing hormone and follicle stimulating hormone from rat pituitary glands. A specific antiserum against luteinizing hormone/follicle stimulating hormone releasing hormone has been generated in the guinea pig and this allowed the development of a radioimmunoassay for this peptide. The antisera, at a final dilution of to depending on the antiserum used, were able to bind 35% of the ¹³¹I-labelled antigen. The sensitivity of this assay method was 50 pg of luteinizing hormone/follicle stimulating hormone releasing hormone. The following substances did not cross-react: oxytocin, lysine-vasopressin, synthetic thyroid stimulating hormone releasing hormone, ovine luteinizing hormone, follicle stimulating hormone and prolactin. Des-Trp³ luteinizing hormone/follicle stimulating hormone releasing hormone, pyroglutamyl-histidyl-tryptophan and seryl-tyrosyl-glycyl-leucyl-arginyl-prolyl-glycinamide, exhibited flatter curves than luteinizing hormone/follicle stimulating hormone releasing hormone with a cross-reactivity of about . Using this method, luteinizing hormone/follicle stimulating hormone releasing hormone was assayed in extracts of the sheep stalk-median eminence and of the hypothalamus and in jugular vein blood from a normal ram and from normal male rats, from cyclic ewe and from hypophysectomized ram and rats. It was concluded that luteinizing hormone/follicle stimulating hormone releasing hormone is present in hypothalamic extracts and in plasma of sheep and rat.     

Immunocytochemistry of somatotrophs,gonadotrophs, prolactin and adrenocorticotropin cells in larval sea bream (Sparus auratus) pituitaries

Summary The chronological appearance of endocrine cells in the pituitary of sea-bream (Sparus auratus) larvae was studied using antisera against salmon prolactin, trout growth hormone, salmon gonadotropin and N-terminal human adrenocorticotropin. The larval pituitary (1–12 days after hatching) was oval in shape and was composed of a dense mass of cells with few neurohypophysial fibres. By 60 days after hatching it began to resemble the adult and was divisible into a distinct rostral pars distalis containing prolactin and adrenocorticotropin cells; a proximal pars distalis containing somatotrophs and gonadotrophs and a pars intermedia. Cells immunoreactive with antisera against growth hormone were observed immediately after hatching (2 days post-fertilization). Weakly staining prolactin cells were observed 2 days later in the region corresponding to the rostral pars distalis. Cells immunoreactive with anti-gonadotropin and anti-adrenocorticotropin sera were observed in the pituitary 6 and 8 days after hatching, respectively. All the cell-types studied were immunoreactive from the time they were first identified until the final samples 90 days after hatching.     

Subcellular localization of gonadotrophic hormones LH and FSH in frog adenohypophysis using double-staining immunocytochemistry

We applied double post-embedding immunocytochemical methods using specific antibodies against bullfrog (Rana catesbeiana) luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with immunogold staining (5- and 20-nm particles) to determine the subcellular localization of both gonadotropins and to observe their immunostaining patterns in anterior pituitary of the frog Rana pipiens. Results showed that individual gonadotrophs may store either one or both gonadotropins in a given secretory granule and in large globules (lysosomes?). Most gonadotrophs (50-88%) contain both hormones; 12-50% contain only FSH, and only a few (0-7%) contain LH alone. Individual secretory granules, even in cells that contain both hormones, may contain only one or both gonadotropin molecules. Evaluation of the percentage of monohormonal and multihormonal secretory granules revealed that multihormonal secretory granules were the most numerous and that LH monohormonal secretory granules were the least numerous. These results indicate that cellular storage of gonadotropin in amphibian pituitary is similar to that described for mammals, where a single cell type containing both gonadotropins predominates. Variability in hormone content both of cells and of granules in all individuals is consistent with the hypothesis that frog pituitary possesses a single multipotential gonadotroph.     

Improved solid phase synthesis of luteinizing hormone releasing hormone analogues using 9-fluorenylmethyloxycarbonyl amino acid active esters and catalytic transfer hydrogenation with minimal side-chain protection and their biological activities

Using mainly 9-fluorenylmethyloxycarbonyl amino acid 2, 4, 5-trichlorophenyl esters in the presence of 1-hydroxybenzotriazole and the solid supportp-alkoxybenzyl alcohol resin, synthesis of luteinizing hormone releasing hormone analogues was carried out with minimal side-chain protection. Catalytic transfer hydrogenation was employed for removal of NO₂ and Z-groups from Arg and < Glu respectively avoiding the use of HF and this led to good yields. An aromatic, hydrophilic amino acid, D-(p-hydroxyphenyl) glycine was incorporated into luteinizing hormone releasing hormone molecule along with other modifications. The agonistic as well as antagonistic activities of all the peptides have been studied     

Phosphorylation of the Protein Encoded by the First Open Reading Frame of the MDG4 Transposable Element (gypsy) by Homologous and Heterologous Casein Kinases Type 2

Homogeneous casein kinase type 2 (CK2) was obtained from oocytes of Rana temporaria and cells of Drosophila melanogaster by chromatography on heparin-Sepharose, phosphocellulose, and Mono Q columns using a Pharmacia FPLC system. The procedure was first successfully used for the purification of CK2 from the Drosophila melanogaster cell culture. It has been shown that the protein encoded by the first open reading frame (ORF) of the gypsy transposable element (MDG4) is an effective protein substrate both for homologous and heterologous CK2 from the oocytes of Rana temporaria in vitro. Both enzymes catalyze the incorporation of two moles of phosphate per mole of protein. The K_m and V_max values for the reaction catalyzed by CK2 from the Drosophila cell culture were 32.5 ± 2.1 nM and 70.97 ± 1.89 nmol/min per µg, respectively, and for CK2 from oocytes, these values were 37.6 ± 2.8 nM and 66.02 ± 2.15 nmol/min per µg, respectively.     

Advertisement calls of two anuran amphibians,Rana tigrina andTomopterna breviceps

Rana tigrina andTomopterna breviceps occur as sympatric species at Dharwad, India. Sexually mature males produce advertisement calls. The advertisement call of both the species consist of a number of calls produced in series forming a call group. Each call group ofRana tigrina comprises 10–40 calls, whereas that ofTomopterna breviceps consists 13–141 calls. Each call consists of a pulse group with variable number of pulses which lack pulse interval. Calls of both the species exhibit similarities in (i) call consisting of series of calls with a pulse group in each call, (ii) absence of pulse interval within the pulse group, (iii) the amplitude of the first pulse being always small, and (iv) the frequency spectrum beginning from 200 Hz. Based on the similarities in the spectral features of the calls, it is suggested that the two species may be closely related to each other.     

Influence of hormones and growth factors on viability,DNA, and protein content of adult hepatocytes in primary culture

Summary The survival of adult rat hepatocytes in monolayer culture was studied in the presence of different hormones (neurotensin, oxytocin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, cholecalciferol, bradykinin, substance P, aldosterone, melanocyte stimulating hormone, 3,3′,5-triiodo-1-thyronine, corticosterone, human growth hormone, glucagon, insulin, progesterone, testosterone, estradiol, and dexamethasone phosphate) or growth factors (fetal bovine serum). For this purpose trypan blue exclusion, lactate dehydrogenase, and DNA and protein content were measured at 24 and 72 h of culture. 10⁻⁷ M Dexamethasone, a mixture of eight hormones, 10% fetal bovine serum, and a combination of the latter two supplements caused a more than 64% higher DNA content at 72 h when compared to control cultures. A striking agreement of these results with changes of lactate dehydrogenase leakage was observed, whereas trypan blue exclusion gave erratic results. Considerable changes of cell arrangement apparently specific for each supplement were ovserved by low magnification microscopy. It is concluded that glucocorticoids and fetal bovine serum have an outstanding effect on cell viability and that DNA or protein content or both are reliable indicators of cell viability in amitotic cultures.     

Different patterns of expression of five neuropeptides in the adrenal gland and kidney of two species of frog

The aim of this study was to demonstrate in the adrenocortical and renal tissues of two species of frog, Rana italica and Rana esculenta, the presence and distribution of five neuropeptides: atrial natriuretic peptide (ANP), Leu-enkephalin (Leu-ENK), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal peptide (VIP).In anurans, the adrenal medulla is the site for the synthesis, storage and secretion of not only catecholamines but also various peptides. These peptides should not be regarded only as neurotransmitters or modulators for the secretion of catecholamines, but also as hormonal substances that induce systemic effects.All the peptides studied (ANP, Leu-ENK, NPY, SP and VIP) are present in both organs. However, different patterns of expression were observed for some of the peptides in two frogs.Immunopositivity to ANP was found in small clusters of chromaffin cells in both frogs whereas a clear strong positivity was present only in Rana esculenta kidney. Large clusters of chromaffin cells were immunoreactive to Leu-ENK in Rana italica but there were approximately 25% fewer compared to the positive cells present in Rana esculenta. Epithelial cells of renal tubules showed strong immunopositivity to Leu-ENK in Rana esculenta but not in Rana italica. A large number of adrenal cells (70–80%) were immunoreactive to NPY in Rana italica, while in Rana esculenta this peptide was localized in small clusters of chromaffin cells. Both frogs showed many NPY-positive cells in kidney. Many chromaffin cells were found positive to SP and VIP. A strong positivity was also observed in kidney in both frogs. These observations suggest a possible role of these peptides in the control of the physiological functions of adrenal glands and kidney of the two species of frogs studied.     

Evidence of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Infection in Water Frogs of the <Emphasis Type="Italic">Rana esculenta</Emphasis> Complex in Central Italy

Batrachochytrium dendrobatidis (phylum Chytridiomycota, order Chytridiales) is the causative organism of chytridiomycosis in amphibians, a disease associated with their population decline worldwide. In this work, we report a cutaneous infection in water frogs of the Rana esculenta complex in agricultural areas of Umbria, central Italy. Histological, immunohistochemical, ultrastructural, and molecular analyses demonstrated for the first time the presence of the Batrachochytrium dendrobatidis in this complex; to date, no association between the presence of chytrid fungal infection and mortality has been found, to our knowledge. However, the presence of Batrachochytrium dendrobatidis infection in the water frogs of the Rana esculenta complex is of concern because the frogs could act as a reservoir species and contribute to the decline of less resistant species.