Calcium, Ca2+-ATPase, Calmodulin, and Calbindin D-28KD Localization in Testis of Leptodactylus chaquensis (Anura: Leptodactylidae).

The intracellular localization of Ca2+, Ca2+-ATPase, Calmodulin, and Calbindin D-28KD have been studied in testes of the toad Leptodactylus chaquensis, using ultracytochemical and immunohistochemical techniques. The Ca2+ presences in the nucleus and into the mitochondria of the germ cells, together with the activity of Ca2+-ATPase detected in the nuclear envelope and mitochondrial crests, suggest the participation of this transporter in the storage of Ca2+. In Sertoli cells, Ca2+ deposits were also found in vesicles and lamellar bodies. Calmodulin and Calbindin D-28KD were revealed in the cytoplasm of both cell types. At the spermatozoon level, the cation deposits were located in the subacrosomal space and in the acrosomal vesicle. Ca2+-ATPase activity was observed in the acrosomal and plasma membranes of the gamete that suggests the existence of a transport system responsible for maintaining low cytoplasmic Ca2+ levels. The activity of Ca2+-ATPase and the location of Ca2+ deposits in gamete tail would be related to flagellar movement. The colocalization of Ca2+ deposits and their binding proteins in efferent duct cells would probably be associated with secretory activity. Considering that intracellular Ca2+ is present in different gonadal cells, this work would provide a better understanding of the cation importance in the testicular functions of this species.

Effect of nervous stimulation on ovarian steroid secretion in amphibians.

In the present study, the effect of nerve stimulation on the secretory activity of the ovary of adult females was analyzed for the first time in amphibians. Results revealed that in Rhinella arenarum the stimulation of nerves that supply the gonad induced an increase in estradiol and progesterone secretion, this response showing differences during the reproductive cycle of the species. During the postreproductive period, an increase in estradiol secretion was observed while, in the reproductive period, progesterone secretion increased. Our results suggest that the sympathetic division of the autonomic nervous system would be responsible for this increase, taking into account that, under our experimental conditions, acetylcholine did not affect the endocrine activity of the gonad, while adrenaline (epinephrine) was effective in inducing steroid secretion an effect that could be due to interaction with ß receptors. On the other hand, our data show that the association of adrenaline with follicle-stimulating hormone increased estradiol secretion during the postreproductive period, while the association of catecholamine with LH or hCG increased progesterone secretion during the reproductive period. Our results would suggest that nerve stimulation, mediated by the release of adrenaline, would act synergistically with gonadotrophins to stimulate steroid secretion.

Effects of nerve stimulation on amphibian oviductal activity.

The present study describes, for the first time in an anuran amphibian, the nerve stimulation effects on the secretory and motor activity of the oviduct of adult females. The results reveal that in Rhinella arenarum oviducts, the epithelial and glandular secretory cells of the mucosa of the pars convoluta respond to nerve stimulation secreting the products synthetized and stored in their cytoplasm. The ultrastructural analysis showed that the cell content released is made up of granular, fibrillar and floccular material, exocytosis being the main secretory mechanism found in epithelial secretory cells, although apocrine and holocrine processes could also be observed. In contrast, in glandular cells only exocytosis processes were found. With respect to the participation of the nervous system in the motility of the duct, observations under our experimental conditions indicated that oviductal nerve stimulation promotes motor activity as manifested by a succession of coordinated contractions and relaxations that generate movements similar to peristaltic waves. These results were observed in oviducts from animals captured during the reproductive and post reproductive periods. However, it is important to note that both the secretory response and duct motility are markedly decreased during the post reproductive period of the species.

Electron microscopy observations on testis and spermatozoa of Leptodactylus chaquensis (Anura, Leptodactylidae).

Scanning and transmission electron microscopy were used to investigate the fine structure of the testis and spermatozoa of toad Leptodactylus chaquensis. Our observations indicate that germinal compartment contains Sertoli (SC) and germ cells. The tight junctions and desmosomes among SC indicate the existence of an hematotesticular barrier in charge of maintaining the differences in the composition of the germinal and interstitial compartments. During spermatogenesis, SC acts as a structural support for germ cells. Secondary spermatogonias, spermatocytes and spermatids are joined by cytoplasmic bridges that allow communication between cells in the same cyst. Spermatids at the subcellular level two well-defined morphological stages can be observed: primary spermatids are rounded cells with an acrosomal vesicle attached to the nucleus which has a diameter of about 4.39±0.36µm. Secondary spermatids are elongated with a nucleus of about 19.50±0.92µm in diameter and the acrosome and the axoneme are located in opposite poles of the cells. At the apical end of the spermatozoon we can observe a large arrowhead-shaped acrosome (6.26±0.28µm in length) that takes up about one third of the gamete head with 17.33±0.29µm in length. The proximal centriole is located in the nuclear fossa while the distal centriole gives rise to the flagellar axoneme. The flagellum exhibits a typical pattern of "9+2" and adjacent to it is the axial fiber, and an undulating membrane stretches between both structures. Transmission electron microscopy observations allowed us to produce a diagram of the structure of the spermatozoon of L. chaquensis. Leydig cells, located in the interstitial compartment, show scarce cytoplasm, mitochondria and large-sized lipid droplets which would provide the raw matter for the synthesis of steroid hormones.

Ultrastructure of the oviductal mucosa of Leptodactylus chaquensis. Analysis of the preovulatory and postovulatory periods.

In the present study we analysed the ultrastructural characteristics of the oviductal mucosa of Leptodactylus chaquensis during the preovulatory period and immediately after ovulation. Epithelial secretory cells, ciliated cells, basal cells and glandular secretory cells are described. During the preovulatory period, the oviduct exhibits its maximum degree of development at both the epithelial and the glandular levels, with numerous secretory cells that contain a large number of secretory granules whose contents are released into the oviductal lumen by apocrine and exocytotic secretory processes. The secretory cells present throughout the oviduct display considerable variability in the characteristics of their secretory granules, which show different shapes, sizes, organization of the material contained and electron density. The different cell types are distributed following a characteristic pattern for each oviductal zone, thus creating an ultrastructural mosaic along the oviduct. During the postovulatory period, the number of secretory cells decreases and the remaining ones exhibit a marked reduction in secretory granules. Ciliated cells show a typical ultrastructural organization that is not modified throughout the reproductive cycle. Basal cells, located at the basal region of the epithelium, are characterized by their heterochromatic nuclei and electron-lucent cytoplasm, while glandular secretory cells exhibit oval, round or polyhedric granules, most of them with a prominent core. Our results, which indicate a high heterogeneity of secretory cell contents, allow us to suggest differential synthesis and secretion of specific products in each oviductal zone.

Homeostasis and secretion of calcium in the oviductal mucosa of toad Rhinella arenarum.

The presence of a calcium pump, calbindin D-28KD, and calmodulin in the secretory cells (SC) of the oviductal pars convoluta (PC) of Rhinella arenarum was established for the first time in amphibians using immunohistochemical techniques. Marked variations were observed in the localization and degree of expression of these proteins according to the duct segment and the period of the sexual cycle analyzed. During the preovulatory and ovulatory periods the calcium pump colocalized with calbindin D-28KD can be seen mainly in the apical border of the SC, which are located in the first zones of PC and synthesize and secrete the components of the inner jelly coat layers. These envelopes, which surround the oocytes, contain the molecules indispensable for fertilization, probably inducing the sperm acrosome reaction (AR). Our results suggest that calmodulin, colocalized with the calcium pump at the SC cytoplasmic level, would be involved in the active transport of the cation inside the secretory granules, maintaining adequate levels of intracellular Ca(2+) . During the postreproductive period, a calcium pump colocalized with calbindin D-28KD appears for the first time in the cycle in the basal zones of the SC. This system may be related to the replenishing of intracellular Ca(2+) stores. In contrast, in R. arenarum the Ca(2+) present in the jelly coats that surround the oocytes participates in the AR during fertilization, suggesting that this secretion system of the cation provided by the oviductal mucosa is functionally more active during the reproductive period of this species.

Innervation of amphibian reproductive system. Histological and ultrastructural studies.

In the present study we describe for the first time in anuran amphibians the histological and ultrastructural characteristics of innervation in the female reproductive organs. The observations in Rhinella arenarum revealed the presence of nerve fibers located predominantly in the ovarian hilium and in the oviduct wall. In both organs the nerves fibers are placed near blood vessels and smooth muscles fibers. In the present study the histological observations were confirmed using antibodies against peripherin and neurofilament 200 proteins. Ultrastructural analyses demonstrated that the innervation of the reproductive organs is constituted by unmyelinated nerve fibers surrounded by Schwann cells. Axon terminals contain a population of small, clear, translucent vesicles that coexist with a few dense cored vesicles. The ultrastructural characteristics together with the immunopositive reaction to tyrosine hydroxylase of the nerve fibers and the type of synaptic vesicles present in the axon terminal would indicate that the reproductive organs of R. arenarum females are innervated by the sympathetic division of the autonomic nervous system.

Histopathological and biological studies of the effect of cadmium on Rhinella arenarum gonads.

This study was to determine the lethal dose 50 (LD(50)) of CdCl(2) in adult Rhinella arenarum and analyzed the effect of two sublethal doses (0.5 and 5 mg/kg) of the xenobiotic in gonads. The 48 h LD(50) were 50.0 and 49.8 mg/kg for males and females respectively. Alterations in the ovary were evidenced by nuclear pleomorphism and cytoplasmic vacuolization of the oocytes at the early stages of development with the highest dose and an increase in the population of atretic oocytes. In the interstitial tissue we noticed congestion, edema and fibroblast proliferation. The nuclear maturation of the oocytes was affected by the xenobiotic in a dose- and time-dependent manner. In males, treatment with 5 mg/kg of cadmium (Cd) caused a decrease in the concentration, viability and straight progressive motility of sperm while there was an increase in immotile sperm. Testis histopathology revealed dilated seminiferous tubules, disappearance of cysts, tissue disorganization and leukocyte infiltration. Numerous germ cells showed hydropic tumefaction or signs of focal necrosis. The Cd content in animals intoxicated gonads with the highest sublethal dose was significantly higher than in the control. Results indicate that R. arenarum gonads are target for the xenobiotic, compromising the formation of gametes competent for fertilization, the effective CdCl(2) dose being 5 mg/kg.

Analysis of Bufo arenarum oviductal secretion during the sexual cycle.

SummaryBufo arenarum oocytes are oviposited surrounded by jelly coats, one component of the extracellular matrix required for fertilization. The secretion, released to the oviductal lumen, was analysed by SDS-PAGE. The coomassie blue staining evidenced an electrophoretic pattern with molecules ranging between 300 and 19 kDa that showed variations in their secretion profiles during the sexual cycle. In the preovulatory period the densitometric analysis showed the presence of nine peaks with marked predominance of the 74 kDa molecule. Once ovulation has occurred, the jelly coats become arranged around the oocytes during their transit throughout the oviductal pars convoluta (PC), revealing the addition of three proteins only observed during this period, which suggests a differential secretion. Some of these proteins could not diffuse under any extraction treatment, indicating for them a structural or in situ function. Proteins of low molecular mass diffused totally while others showed a partial diffusing capacity. After ovulation a marked decrease in the relative amount of all the proteins released to the lumen, especially the 74 kDa protein, could be detected. During this period, unlike the other stages of the sexual cycle, a differential secretion pattern was observed along the PC. The histochemical analysis performed during the ovulatory period showed the presence of glycoconjugates including both acidic and neutral groups. The present results are in agreement with previous ultrastructural and histochemical studies that describe the role of Bufo arenarum jelly coats in fertilization.

Subcellular localization of calcium and Ca-ATPase activity during nuclear maturation in Bufo arenarum oocytes.

The localization of calcium and Ca-ATPase activity in Bufo arenarum oocytes was investigated by ultracytochemical techniques during progesterone-induced nuclear maturation, under in vitro conditions. No Ca2+ deposits were detected in either control oocytes or progesterone-treated ones for 1-2 h. At the time when nuclear migration started, electron dense deposits of Ca2+ were visible in vesicles, endoplasmic reticulum cisternae and in the space between the annulate lamellae membranes. Furthermore, Ca-ATPase activity was also detected in these membrane structures. As maturation progressed, the cation deposits were observed in the cytomembrane structures, which underwent an important reorganization and redistribution. Thus, they moved from the subcortex and became located predominantly in the oocyte cortex area when nuclear maturation ended. Ca2+ stores were observed in vesicles surrounding or between the cortical granules, which are aligned close to the plasma membrane. The positive Ca-ATPase reaction in these membrane structures could indicate that the calcium deposit is an ATP-dependent process. Our results suggest that during oocyte maturation calcium would be stored in membrane structures where it remains available for release at the time of fertilization. Data obtained under our experimental conditions indicate that calcium from the extracellular medium would be important for the oocyte maturation process.

Modulators of Bufo arenarum ovulation.

In this study we investigated ovulation in vitro using ovary samples from Bufo arenarum with respect to their response to stimulation with homologous pituitary homogenate (HPH) or with progesterone and prostaglandins (PGF2alpha and PGE1) as intermediates of pituitary action. Ovary samples were obtained from animals captured during the breeding period. Our results demonstrate that the ovulatory response to all different inducers was dose dependent, the highest percentage of ovulated oocytes being obtained with HPH treatment. An important increase in the ovulatory response was obtained by the association of PGF2alpha with either HPH or progesterone at suboptimal doses, indicating that this prostaglandin induced a synergistic potentiating effect. Incubation with cyclooxygenase inhibitors (indomethacin or diclofenac sodium) produced a significant decrease in the ovulation induced by HPH, demonstrating that prostaglandins are involved in the action of the pituitary gland in this process. According to our results, PGE1 not only had no participation in the ovulatory process, but also produced an inhibitory effect on ovulation induced by HPH treatment.

Role of prolactin in nuclear maturation and ovulation in amphibian oocytes.

The present study was undertaken to determine the effect of prolactin (Prl) on Bufo arenarum oocyte maturation and ovulation, two characteristic events of the breeding period, the stage of the sexual cycle in which gamete growth is complete. We observed that Prl, at the doses assayed, did not affect nuclear maturation per se. In addition, when follicles were pretreated with Prl and progesterone was later added to the medium as a physiological nuclear maturation inducer, the percentage of germinal vesicle breakdown obtained with the steroid was unaffected by Prl. The analysis of the in vitro ovulation process demonstrated that pituitary homologous homogenate (PHH) produced a dose- and month-dependent stimulating effect. The maximum percentage of ovulated oocytes was obtained from the end of July to October, the period in which oviposition naturally occurs. Prl by itself did not affect the ovulation process, but when both the hormone and PHH were present in the incubation medium, a significant increase in ovulated oocytes was observed. The results suggest that Prl does not participate in oocyte maturation; however, its presence in the incubation medium would increase oocyte sensitivity to the action of the physiological ovulation inducers.

Changes in serum sex steroid levels throughout the reproductive cycle of Bufo arenarum females.

The changes in the serum levels of the sexual steroids estradiol-17beta (E(2)), testosterone (T), dihydrotestosterone (DHT), and progesterone (P) in Bufo arenarum females were determined by radioimmunoassay (RIA) during 3 consecutive cycles (1999-2001). The serum concentrations of T and DHT, which showed a close parallelism during the annual reproductive cycle, exhibited the highest levels during the preovulatory period, when oogenesis is advanced, while lowest serum levels of these hormones were found during the ovulatory period. The data obtained for E(2) showed a pattern contrary to that determined for androgens. The maximum E(2) concentrations detected in the early postovulatory period might be associated with vitellogenesis and follicular growth. Lowest E(2) concentrations were reached during the period in which B. arenarum undergoes its final hibernation stage. Serum P showed a peak during the preovulatoy period, related to the induction of nuclear maturation in full grown oocytes. A strong decrease in the levels of the circulating hormones was observed after ovariectomy. Our results showed that, out of the four hormones examined, T and DHT were the best indicators of ovarian and oviductal stage, as shown by the strong positive correlation found between androgen levels and organ weight, while E(2) showed a weak negative correlation with ovarian and oviductal weight.

Involvement of catecholamines in the regulation of oocyte maturation in frogs.

The present study investigates the role of catecholamines in the regulation of Bufo arenarum oocyte maturation. The metabolic changes in the oxidation of carbohydrates and the meiotic resumption evinced by the germinal vesicle breakdown were used as indicators of cytoplasmic and nuclear maturation, respectively. The results obtained suggest that noradrenaline (norepinephrine) could be one of the factors responsible for the metabolic behaviour that characterises cytoplasmically immature oocytes. The use of adrenaline (epinephrine), on the other hand, induced a metabolic change which made oocytes cytoplasmically mature. The effect of both catecholamines, which was dose-dependent, was observed in ovarian oocytes (surrounded by follicle cells) as well as in coelomic oocytes (free from follicle cells), suggesting the presence of adrenergic receptors in the gamete. The results obtained using adrenergic agonists and antagonists suggest that the effect of adrenaline would be due to an interaction with beta2-receptors. Although catecholamines have an influence on the determination of the stage of cytoplasmic maturation of the oocytes, they do not affect nuclear maturation by themselves. Nevertheless, pretreatment of follicles with adrenaline caused a significant inhibition in progesterone-induced nuclear maturation even though this effect was markedly weaker when using noradrenaline.