Cadmium-calcium interference in the Rhinella arenarum oviduct.

Given that the cadmium (Cd) toxicity could be due to its interference with the calcium (Ca) homeostasis, the aim of this work was to study the effect of Cd over the presence, distribution and volume density (Vv) of Ca and Ca-ATPase in the secretory cells of the pars preconvoluta (PPC) and the pars convoluta (pc) in Rhinella arenarum. The severe effect of the xenobiotic (CdCl2 2.5 mg/kg) in sexually matured females was evaluated. Co-localization, as well as a marked reduction of Ca and Ca-ATPase, was observed in treated animals, in the areas analyzed, compared to control. Low calcium deposits were found in the secreting granules (SG) of the epithelial (ESC) and glandular secretory cells (GSC), while an increase in their cytoplasm and intracellular space was observed. The Ca-ATPase in treated and control animals was detected at the SG and the plasmatic membrane of the ESC and GSC. In relation to the Vv estimates, a substantial reduction of Ca deposits and Ca-ATPase activity was observed in the treated group, with respect to the control. Both amounts of Vv of Ca and Ca-ATPase activity were higher in PPC than in pc, and, higher in ESC than in GSC. These results were associated with the Cd concentration in the oviductal PC, determining that it is a bioaccumulator organ. Thus, this work demonstrated that the Cd interacted with Ca-ATPase, leading to an increase of cytosolic Ca, which is responsible for the possible disruptions in cellular metabolism.

Early signs of toxicity in testes and sperm of rats exposed to low cadmium doses.

The cadmium (Cd) concentration in the environment has increased as a consequence of anthropogenic activity. The objective of this study was to determine early signs of Cd toxicity in testes and sperm as possible biomarkers. The dose orally administered to Wistar rats was within the range where chronic toxicity can appear. At the light microscopic level, gonads presented preserved cytoarchitecture throughout treatment; however, after the second month, transmission electron microscopy (TEM) revealed disruption of the blood-testis barrier. The study of sperm with light microscopy showed defects in gamete morphology after 2 months of treatment. Another parameter that revealed alteration was sperm motility after 3 months of treatment. TEM was used to analyze the flagellum, which in the midpiece showed aberrant mitochondria and displacement of outer dense fibers in relation to the central axoneme after 2 months. The data obtained were associated with Cd concentration in the testes, an increase in its levels being observed in a time-dependent manner. The results provided in this study demonstrated that early signs of Cd toxicity were observed in gonads and gametes during the second month of the treatment, generating morphological and functional alterations in the sperm that could lead to infertility.

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.

Spermatogenesis in Leptodactylus chaquensis. Histological study.

The organization and the histological characteristics of Leptodactylus chaquensis testis throughout the reproductive cycle were analyzed in the presented study. Gonads of adult males, processed with routine techniques for optical microscopy, revealed that during the reproductive period the seminiferous tubules were characterized by presentation of a large number of cysts, germ cells at the same maturation stage supported by Sertoli cells. All the germ line cells were also present in the postreproductive period and maintained their morphological characteristics. Primary spermatogonia were large-sized cells found isolated or in small groups. The rest of the cells of the germ line formed cysts. Secondary spermatogonia showed morphological characteristics similar to their predecessors, although they were smaller. Primary and secondary spermatocytes showed images of the different stages of the first and second meiotic division respectively. One finding was the presence of intercytoplasmic bridges between the secondary spermatocytes. Primary spermatids were rounded cells with an acrosomal vesicle associated with the nucleus and had cysts that were characterized by large intercellular spaces. Secondary spermatids were elongated cells with a well defined acrosome, which in the spermatozoa had the shape of an arrowhead. Another peculiar characteristic of this species was the fusion of the walls of the seminiferous tubule with the efferent duct that formed a path for spermatozoa during spermiation. The presence in the seminiferous tubules of all stages of the spermatogenic line during the two periods of the cycle studied indicated that Leptodactylus chaquensis had a potentially continuous reproductive cycle.

Structural analysis of toad oviductal mucosa in relation to jelly components secretion throughout the reproductive cycle.

In amphibians, the components of the jelly coats that surround the oocytes at the time of fertilization and coordinate gamete interaction are secreted by the oviduct. We analysed the histological variations in the mucosa of the oviductal pars convoluta (PC) of Rhinella arenarum during the reproductive cycle and its relationship with secretion. During the preovulatory period, the mucosa reaches a high degree of morphological and functional development, with a large number of epithelial (ESC) and glandular secretory cells (GSC) loaded with contents that are secreted into the oviductal lumen. During the ovulatory period, the secretory cells (SC) of both layers present maximum secretory activity through apocrinia and merocrinia. While the ESC located at the tips of the folds release their content directly in contact with the oocytes, the GSC secrete material from the bottom of the epithelial folds that, by interaction with the secretion of the ESC in the lateral faces, form a product with a certain degree of organization. Secretion is a continuous process with formation of coats of increasing complexity from the intermediate proximal zone (IPZ) to the pars convoluta (pc) itself, and the passage of the oocyte is a requisite for the organization of the jelly coats around the gamete. During the early postovulatory period, although there is a marked decrease in the number and volume of the SC, the ESC still release material into the oviductal lumen. In the late postovulatory period the morphological characteristics of the PC begin to recovery although there is no evidence of secretion.

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.

Effect of oviductal secretion components on the fertilizing capacity of amphibian sperm: biological and ultrastructural studies.

The present study was carried out to analyze the fertilization-supporting activity of Rhinella arenarum egg-jelly components. Spermatozoa were treated with diffusible factor (DF) constituted by the components released from the jelly coat into deionized water or with full jelly (FJ) containing all the components secreted by the oviductal pars convoluta (PC) during the transit of the oocytes through the duct, or with washed jelly (WJ) constituted only by structural components. Both jellies were solubilized by ultraviolet irradiation. These gametes were used for ultrastructural and biological studies in order to determine the acrosome state and the fertilizing capacity. Additional experiments were performed by using Ca(2+), a diffusible cation present in R. arenarum jelly envelopes. Results demonstrated a marked increase in the acrosome reaction (AR) of sperm treated with FJ or DF compared to the controls (Ringer's solution), no significant differences being observed between both treatments, while WJ showed low AR percentages similar to the ones obtained with the controls. The addition of Ca(2+) induced an increase in this parameter in a dose-dependent manner, although the values reached with FJ or DF were not attained. The results of the "in vitro" fertilization show a strong inverse association to the acrosome reaction (AR) rate. Treatment with Ca(2+) at the concentration present in the jelly (6.3 ± 0.9 mM) inhibited the fertilizing capacity as a function of incubation time, showing that at 2 min there was a decrease in the fertilization percentages compared to 10% Ringer's. Data indicated that Ca(2+) present in jelly is involved in the AR induction but suggests the possible participation of other diffusible and/or structural components of the oviductal secretion in this process.

Role of cations as components of jelly coats in Bufo arenarum fertilization.

The role of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations in Bufo arenarum fertilization was analysed. Our results showed that the highest fertilization percentages were obtained when strings of uterine oocytes (UO) were inseminated. Under these conditions, full jelly (FJ), which represents the jelly coats surrounding the oocytes at the time of deposition, contained 68.5 +/- 7.0 mM Na+, 27.4 +/- 2.4 mM K+, 6.3 +/- 0.9 mM Ca2+ and 6.9 +/- 0.9 mM Mg2+. When the strings of oocytes were washed in deionized water, these cations diffused into the liquid medium surrounding them. There was a marked similarity between the loss of Ca2+ in the jelly and the decrease in the fertilizability of the UO. Furthermore, the use of chelating agents of divalent cations showed the importance of the Ca2+ contained in the jelly. When Ca2+ was sequestered from the jelly coats by the addition of the chelating agents to the insemination medium as well as by pretreatment of the UO strings, a decrease in fertilization percentages occurred, this effect being dose dependent and more marked with EGTA. These results demonstrate that the Ca2+ in the jelly plays a role in fertilization. Nevertheless, taking into account that during the washing of the jelly other jelly coat components were diffused and considering that the addition of Ca2+ to the insemination medium reverted significantly, but only partially, the loss of fertilizability of jellied UO (washed), the participation of other components in the fertilization mechanism is suggested.

Effect of steroid hormones on Bufo arenarum oviduct. Ultrastructural study.

The endocrine regulation of the mucosa of the oviductal pars convoluta was analyzed by ultrastructural studies demonstrating that ovariectomy, together with a decrease in ovarian steroids circulating levels, caused a marked regression in this portion of Bufo arenarum oviduct. Twenty-five days after ovariectomy, a decrease in the depth of the epithelial and glandular layers was observed due to the notable loss of secretory cells, whose number was clearly smaller than in nonovariectomized females. The remaining secretory cells showed involution signs, with few secretory granules in their cytoplasm, little endoplasmic reticulum near poorly developed Golgi complexes and a large amount of lipid droplets. Cells in an advanced autolysis state were found in the lumen. These characteristics evidence a nonfunctional state of the pars convoluta. Treatment with 5alpha-dihydrotestosterone (DHT) completely reversed the ovariectomy effect, inducing pars convoluta growths and restoring the characteristics of epithelial and glandular secretory cells in the whole pars convoluta, with micrographs similar to the control. These same effects were observed after treatment with estradiol-17beta (E2), progesterone (P) o E(2)+P in the glandular layer of the whole pars convoluta, but only in the epithelial layer of the most anterior region of this duct. In the secretory cells of other segments these treatments induced the formation of granules of high electron density and homogeneous aspect. Each steroid had a particular effect on the pars convoluta. Although E2 and DHT induced the development of the organoids involved in the proteins biosynthesis, P and DHT acted as secretagogues.