Membrane rafts regulate sperm acrosome reaction via cAMP-dependent pathway in chickens (Gallus gallus domesticus).

Both transcriptionally and translationally inactive sperm need preassembled pathways into specific cellular compartments to function. Although initiation of the acrosome reaction (AR) involves several signaling pathways including protein kinase A (PKA) activation, how these are regulated remains poorly understood in avian sperm. Membrane rafts are specific membrane regions enriched in sterols and functional proteins and play important roles in diverse cellular processes, including signal transduction. Our recent studies on chicken sperm demonstrated that membrane rafts exist and play a role in multistage fertilization. These, combined with the functional importance of membrane rafts in mammalian sperm AR, prompted us to investigate the roles of membrane rafts in signaling pathways leading to AR in chicken sperm. Using 2-hydroxypropyl-ß-cyclodextrin (2-OHCD), we found that the disruption of membrane rafts inhibits PKA activity and AR without affecting protein tyrosine phosphorylation; however, these inhibitions were abolished in the presence of a cyclic 3,5-adenosine monophosphate (cAMP) analog. In addition, biochemical experiments showed a decrease in cAMP content in 2-OHCD-treated sperm, suggesting the involvement of soluble adenylyl cyclase (sAC) and transmembrane adenylyl cyclase (tmAC). Pharmacological experiments, combined with transcriptome analysis, showed that sAC and tmAC are present and involved in AR induction in chicken sperm. Furthermore, stimulation of both isoforms reversed the inhibition of PKA activity and AR in 2-OHCD-treated sperm. In conclusion, our results demonstrated that membrane rafts play an important role in AR induction by regulating the cAMP-dependent pathway and that they provide a mechanistic insight into membrane regulation of AR and sperm function in birds.

Crescimento vascular em membrana do saco vitelínico e desenvolvimento embrionário de codornas japonesas (Coturnix japonica) expostas a campo magnético de baixa frequência / Vascular growth in yolk sac membrane and embryonic development of Japanese quail (Coturnix japonica) exposed to low frequency magnetic field

O objetivo deste trabalho foi observar a influência do campo magnético (CM) de baixa frequência na membrana do saco vitelínico (MSV) e no desenvolvimento do embrião de codornas japonesas (Coturnix japonica) em 72 horas de incubação. Ovos fertilizados foram expostos a nove horas consecutivas de CM, sendo um grupo a partir das 48 horas e o outro a partir das 63 horas de incubação. A quantificação da vascularização da MSV foi determinada pela obtenção da dimensão fractal por meio dos métodos de box-counting e de dimensão de informação, enquanto o peso corporal e o percentual de comprimento cefálico dos embriões foram utilizados como parâmetros de desenvolvimento embrionário. O CM não causou diferenças significativas na densidade vascular da MSV nem no desenvolvimento embrionário, quando comparados ao grupo controle...

The aim of this study was to observe the influence of the low frequency magnetic field (MF) on the yolk sac membrane (YSM) and embryonic development of Japanese quail (Coturnix japonica) in 72 hours of incubation. Fertilized eggs were exposed to 9 consecutive hours of MF, with a group from 48 hours and the second group from 63 hours of incubation. The evaluation of YSM vascularization was determined by the fractal dimensions obtained through box-counting method and information dimension, while body weight of the embryo and percentage of cephalic length were used as parameters for embryo development. The MF caused no significant differences in vessel density in the YSM, nor in the embryonic development considering the body weight and percentage cephalic length, when were compared to the control group...

Early ontogenesis of the angelfish, Pterophyllum scalare Schultze, 1823 (Cichlidae)

This study describes the egg membrane structures of angelfish (Pterophyllum scalare), morpho-physiological changes during angelfish embryogenesis from activation to hatching under optimal conditions (28°C; pH 6.8), the developing larvae and fry, the effect of alkaline pH on the early developmental stages of the species, the relationship between food item size and fry survival. Egg membranes (thin, transparent, 1.67-2.18 µm thick) are covered by a sticky substance. The amber-coloured angelfish eggs were oval in shape, with average diameters of 1.436 and 1.171 mm, i.e., a mean volume of 1.033 ± 0.095 mm³. The survival rate of embryos and larvae kept in water with an elevated, slightly alkaline pH was very low: as few as 2% of the embryos survived, while in the batch kept in optimal water conditions very few eggs died. The first larvae hatched after 1288 h of embryonic development. The newly hatched larvae measured on average 2.60 ± 0.093 mm and had large (0.64 ± 0.077 mm³) yolk sacs. They attached themselves to the substrate with a secretion of thin, viscous threads, which was released from glands situated on the top of the head. The glands vanished on day 5. The 1-day-old larvae showed the first pigment cells on the body and the eyes of the 2-day-olds were already fully pigmented. Between day 4 and 5 of larval life, the larvae began feeding on live food. The 23-day-old fry looked like a miniature versions of the adults. Mortality of the angelfish larvae during their first days after hatching was higher in those fed brine shrimp (Artemia salina) nauplii than those fed protozoans and rotifers.

En este trabajo se ha descrito la estructura de las túnicas ovulares del escalar o pez ángel (Pterophyllum scalare), las modificaciones morfo-fisiológicas que transcurren durante la embriogénesis del escalar desde el momento de activización para el desove en condiciones ambientales óptimas (28ºC y pH 6,8) y, se han caracterizado las larvas y los alevines desarrollándose. Adicionalmente, se ha estudiado el efecto del pH básico del agua sobre los primeros estadios de desarrollo y la dependencia entre la cantidad de alimento y la supervivencia de los alevines. Las túnicas ovulares del escalar son finas (1,67-2,18 mm), transparentes, cubiertas de una sustancia viscosa. Los huevos de color ámbar tienen forma ovalada de diámetros medios 1,436 y 1,171 mm y de un volumen medio de 1,033 ± 0,095mm³. El porcentaje de supervivencia de embriones y de larvas en agua con pH aumentado, ligeramente básico, fue muy bajo, ya que sólo el 2% de los embriones sobrevivió, mientras que en agua de parámetros óptimos sólo algunos huevos palidecieron. Las larvas recién salidas del huevo medían 2,60 ± 0,093 mm por término medio y poseían grandes (0,64 ± 0,077 mm³) sacos vitelinos. Se han adherido al substrato mediante una secreción en forma de filamentos finos y viscosos. Esta secreción se ha desprendido de las glándulas ubicadas en la cumbre de la cabeza. Las glándulas desaparecieron al 5º día de vida de las larvas. Las larvas de un día poseían ya las primeras células pigmentarias en el cuerpo, los ojos de las larvas de dos días estaban pigmentadas plenamente. Entre el 4º y 5º día de vida las larvas empezaron a tomar alimento. Las larvas de 23 días se parecían a una versión miniaturizada de los adultos. La mortalidad de las larvas del escalar alimentadas durante los primeros días a partir del desove con larvas de artemia salina (Artemia salina) fue mayor que la de las alimentadas con protozoarios y rotíferos.

Drosophila vitelline membrane assembly: a critical role for an evolutionarily conserved cysteine in the "VM domain" of sV23.

The vitelline membrane (VM), the oocyte proximal layer of the Drosophila eggshell, contains four major proteins (VMPs) that possess a highly conserved "VM domain" which includes three precisely spaced, evolutionarily conserved, cysteines (CX7CX8C). Focusing on sV23, this study showed that the three cysteines are not functionally equivalent. While substitution mutations at the first (C123S) or third (C140S) cysteines were tolerated, females with a substitution at the second position (C131S) were sterile. Fractionation studies showed that sV23 incorporates into a large disulfide linked network well after its secretion ceases, suggesting that post-depositional mechanisms are in place to restrict disulfide bond formation until late oogenesis, when the oocyte no longer experiences large volume increases. Affinity chromatography utilizing histidine tagged sV23 alleles revealed small sV23 disulfide linked complexes during the early stages of eggshell formation that included other VMPs, namely sV17 and Vml. The early presence but late loss of these associations in an sV23 double cysteine mutant suggests that reorganization of disulfide bonds may underlie the regulated growth of disulfide linked networks in the vitelline membrane. Found within the context of a putative thioredoxin active site (CXXS) C131, the critical cysteine in sV23, may play an important enzymatic role in isomerizing intermolecular disulfide bonds during eggshell assembly.

Egg envelope synthesis and chorion modification after oviposition in the dragonfly Libellula depressa (Odonata, Libellulidae).

Libellula depressa (Odonata, Libellulidae) is an exophytic dragonfly ovidepositing eggs in clutches on the surface of floating plants and algae. The present work investigates, at ultrastructural level, the gradual differentiation of the egg envelopes and the chorionic changes after egg deposition in water. The ovary of the mature female of L. depressa is composed of numerous strings of panoistic ovarioles, where the eggshell formation takes place gradually throughout the activity of the follicle cells. The present data show that the egg envelopes are constituted of a very thick electrondense vitelline envelope, a thin endochorion and an extremely thick exochorion composed of a fibrillar matrix resting on a thin electrondense layer. After deposition in water, L. depressa eggs, initially white and almost transparent, gradually become brown spots in a semitransparent jelly coat, rich of incorporated debris. The jelly coat enveloping the eggs of L. depressa derives exclusively from the exochorion, constituted of a fibrillar matrix, which swell at contact with water. The jelly-like coat performs an adhesive function and presumably a protective role during egg segmentation and ensuing larval hatching.

Mechanical stimulation by osmotic and hydrostatic pressure activates Drosophila oocytes in vitro in a calcium-dependent manner.

Embryogenesis in vertebrates and marine invertebrates begins when a mature oocyte is fertilized, resulting in a rise in intracellular calcium (Ca(2+)) that activates development. Insect eggs activate without fertilization via an unknown signal imparted to the egg during ovulation or egg laying. One hypothesis for the activating signal is that deformation of eggs as they pass through a tight orifice provides a mechanical stimulus to trigger activation. Ovulation could produce two forms of mechanical stimulus: external pressure resulting from the passage of oocytes from the ovary into the narrow oviducts, and osmotic pressure caused by hydration-induced swelling of the oocyte within the oviducts. Ovulation could also trigger activation by placing the oocyte in a new environment that contains an activating substance, such as a particular ion. Here, we provide the first evidence that Drosophila oocytes require Ca(2+) for activation, and that activation can be triggered in vitro by mechanical stimuli, specifically osmotic and hydrostatic pressure. Our results suggest that activation in Drosophila is triggered by a mechanosensitive process that allows external Ca(2+) to enter the oocyte and drive the events of activation. This will allow exploitation of Drosophila genetics to dissect molecular pathways involving Ca(2+) and the activation of development.

Analysis of vitelline envelope synthesis and composition during early oocyte development in gilthead seabream (Sparus aurata).

The oocyte vitelline envelope (VE) of gilthead seabream is composed of four known zona pellucida (ZP) proteins, ZPBa, ZPBb, ZPC, and ZPX. We have previously shown that the gilthead seabream ZP proteins are differentially transcribed in liver and ovary, with the expression in liver being under estrogenic control. However, although mRNA was found in both liver and ovary, only low ZPBa protein levels were detected in liver and plasma. Using isoform-specific ZP antibodies we show that ZPBa and ZPX translation products are present in the cytosol of stage I and II oocytes. In addition, the zpBa and zpX mRNAs were detected in early developing oocytes. During oocyte growth (vitellogenesis), the VE increased in thickness (>10 microm), and we show that the four ZP isoforms are present in different regions of the VE. ZPX was detected closest to the oocyte plasma membrane while the intermediate region was composed of ZPBa, ZPBb, and ZPC. At the outer layer, only ZPC was detected. When oocytes reach the fully grown stage they resume meiosis and hydration. As the oocyte expands, thinning to 4 microm, the VE acquire a striped and compact appearance at the electron microscopy level. This study provides further evidence for the oocyte origin of some ZP proteins in the gilthead seabream and suggests that the ZP proteins are differentially distributed within the VE.

[Eggs fertilizability in domestic hens Gallus gallus domesticus as a function of the yolk surface area]. / Zavisimost' oplodotvoriaemosti iaitz domashnikh kur Gallus gallus domesticus ot ploshchadi poverkhnosti zheltka.

The increase of egg mass and reliable decrease of egg fertilizability were observed when the mass surface area of yolk (egg) increased. The results obtained suggest that, in addition to the number of viable spermatozoa penetrating across the perivitelline membrane within 15-20 min after ovulation, the probability of fertilization depends on the area of egg surface, which approximately corresponds to the area of perivitelline membrane. Apparently, the ratio of receptors' numbers and spermatozoa, which contact with them on the germ disc surface, to their number on the rest part of perivitelline membrane decreases with the increase of yolk size. The decreased egg fertilizability concomitant with the increased area of perivitelline membrane suggests that the egg size is one of the factors of fertility of the female gametes as concerns both variability of the egg composition and age.

Developing follicles of the spotted ray Torpedo marmorata express different glycoside residues in relation to granulosa differentiation and vitelline envelope formation.

Lectins constitute a class of proteins/glycoproteins that specifically bind to terminal glycoside residues. The present investigation aimed to identify lectin-binding sites in developing follicles of Torpedo marmorata. Using eleven lectins (WGA, GSI-A4, GSI-B4, PSA, UEA-I, PNA, MPA, Con-A, DBA, LCA, BPA, SBA), we demonstrated that the biochemical nature and the distribution of carbohydrate residues significantly change during oogenesis in the granulosa cells and the vitelline envelope. In fact, a progressive appearance of surface glycoproteins bearing terminated ss-GlcNAc O-linked side chains was observed in the granulosa during the differentiation of pyriform-like cells from the small ones via intermediate cells simultaneously with a significant reduction of the D-Gal chains present in their nucleus. Glycoproteins bearing ss-GlcNAc O-linked side chains were first evident on the surface of small cells in contact with the oocyte, then on the intermediate ones, and finally on pyriform-like cells. The distribution pattern of such glycoproteins over the differentiated granulosa cells remained unchanged during the subsequent stages of the oocyte growth so granulosa cells preserved the same sugar distribution pattern. Furthermore, a progressive loss of D-Gal residues was evident in the nucleus of granulosa cells. In fact, staining for D-Gal was intense in the nucleus of small follicle cells and progressively reduced till disappearing in differentiated pyriform-like cells. Conversely, the small follicle cells located under the basal lamina were devoid of ss-GlcNAc residues, and the nuclear content in D-Gal remained unchanged. This finding strongly suggests that surface glycoproteins containing ss-GlcNAc residues, and the nuclear content in D-Gal might be related to the differentiation of pyriform-like cells. The present investigation also demonstrates that the content of the sugar residues of the vitelline envelope (VE) changes during oocyte growth, suggesting that pyriform-like cells may contribute to its formation.

Ovarian follicle ultrastructure in the teleost Synbranchus marmoratus (Bloch, 1795), with special reference to the vitelline envelope development.

Synbranchus marmoratus is a protogynous diandric teleost fish widely distributed throughout South America. The aim of this work was to study the ultrastructure of the vitelline envelope and the relationship among oocyte and their follicular cells during oogenesis. During perinucleolar stage, the oocyte and the follicular cells form microvillar processes that project into the perivitelline space. The oocyte secretes a dense and amorphous material, which appears as the first evidence of the vitelline envelope (VE) development. The VE passes from a double to a multilayered structure during oocyte growth. In mature oocytes, the VE reach a mean thickness of 11 microm, having up to 30 layers. Oocyte microvilli are thinner than the follicular ones and were seen in contact with the follicular plasmalema, however we could not find any contact between the follicular microvilli and the oolemma. Before ovulation, microvillar processes retract and the pore canals seem to collapse. An outer electron dense layer occludes the superficial pore and forms a continuous layer. No jelly or adhesive coatings were seen at least in ovulated eggs sampled from ovarian lumen. Follicular cell and oocyte cytological characteristics do not differ from those described in other teleosts species.

Structure of ovaries and formation of egg envelopes in the stonefly, Leuctra autumnalis Aubert, 1948 (Plecoptera: Leuctridae). Ultrastructural studies.

The investigation of ovaries and the formation of egg envelopes of the stonefly Leuctra autumnalis was carried out with light and transmission electron microscopes. The ovary of the studied species is paired and consists of several dozen panoistic ovarioles opening individually to the oviduct. The process of egg capsule formation already begins in previtellogenesis. At this time the follicular cells secrete precursors of the vitelline envelope. Analysis of the presented data suggests that the oocyte itself also takes part in the formation of the vitelline envelope during late vitellogenesis. Simultaneously, the follicular cells produce precursors of further layers of the egg capsule, i.e. two-layered chorion and extrachorion, consisting of two gelatinous layers and a flocculent one. The completely developed capsule contains channels, probably micropylar ones.

Observations on the mechanism of eggshell formation in the liver fluke, Fasciola hepatica.

A mechanism for eggshell production in Schistosoma mansoni has been proposed (Wells & Cordingley, 1991), and suggests that the release of eggshell protein globules from the vitelline cells occurs under alkaline conditions within the ootype followed by their subsequent fusion to form the eggshell. Fusion and tanning of these components produces eggshell which autofluoresces. The present study was carried out to determine whether a similar process operates in Fasciola hepatica. A number of drug treatments were used to disrupt key steps in the maturation of vitelline cells. Treatment with the calcium ionophore lasalocid (1 x 10(-5) M) led to the premature release of eggshell globules from the vitelline cells but not their fusion. Incubation in monensin (1 x 10(-6)M), a sodium ionophore and ammonium chloride (NH4Cl) (5 x 10(-2) M), a weak base, resulted in the premature fusion of eggshell protein globules within the vitelline cells and premature tanning of the eggshell protein material. The copper-containing enzyme, phenol oxidase, is thought to be involved in the tanning process during the production of eggs. Diethyldithiocarbamate, (DDC, 1 x 10(-3) M) is a phenol oxidase inhibitor and treatment with this compound, in combination treatments with monensin and NH4Cl, prevented fusion of the vitelline cell globules and tanning of the shell protein material. The results of the study suggest that the mechanism for eggshell formation in F. hepatica is similar to that proposed for S. mansoni and may be common to other trematodes as well.

Xenopus laevis sperm-egg adhesion is regulated by modifications in the sperm receptor and the egg vitelline envelope.

The biochemical and ultrastructural changes in the envelope of the Xenopus laevis egg that occur during oviposition and fertilization have been thoroughly studied (Hedrick, J. L., and Nishihara, D. M., Methods Cell Biol. 36, 231-247, 1991; Larabell, C. A., and Chandler, D. E., J. Electron Microsc. Tech. 17, 294-318, 1991). However, the biological significance of these changes with respect to gamete interaction has been unclear. In the current study, it was found that changes in the envelope are directly responsible for regulating sperm-egg adhesion, an initial step of fertilization. As a result of these transformations, sperm bind only to unfertilized oviposited eggs, not to oocytes or coelomic eggs. In addition, they do not bind to fertilized eggs. The molecular and cellular basis of the regulation of the sperm binding process was investigated in the context of our recent findings that two structurally related envelope glycoproteins, gp69/64, serve as sperm receptors during fertilization (Tian, J.-D., Gong, H., Thomsen, G. H., and Lennarz, W. J., J. Cell Biol. 136, 1099-1108, 1997). Although the purified gp69/64 glycoproteins isolated from the oocyte or coelomic egg envelopes exhibited sperm binding activity, when these proteins are part of the intact oocyte or coelomic egg envelopes, they are not accessible to either anti-gp69/64 antibodies or to sperm. During the conversion from the coelomic to the vitelline envelope, the gp69/64 sperm receptors become exposed on the surface, an event that correlates with proteolytic cleavage of gp43 and accompanying ultrastructural alterations in the envelope. Conversely, after fertilization, when the vitelline envelope of the egg is converted to the fertilization envelope of the zygote, limited proteolytic cleavage of the sperm receptor results in loss of sperm binding activity. In addition, formation of a fertilization layer on top of the structurally altered VE adds another physical block to sperm binding. These results provide new insights into structure-function relationships between envelope components of the anuran egg, and provide further evidence supporting the key role of gp69/64 as sperm receptors during X. laevis fertilization.

Early embryonic angiogenesis in the chick area vasculosa.

The rate and pattern of growth as well as vessel ultrastructure of the area vasculosa were examined in the chick. The embryos were grown in shell-less culture after 3 d in ovo and staged according to Hamburger & Hamilton (1951) and the rate of increase in the diameter of the area vasculosa was measured. This revealed an increase in the area vasculosa diameter of 0.4 +/- 0.02 mm h-1 (n = 62) for embryos between stages 15 and 20. To determine the growth pattern of the sinus terminalis (the advancing edge of the area vasculosa), a marked length of the sinus was photographed at hourly intervals over a period of 9 h. It was found that this vessel grows by new vessels forming external to the sinus in the form of parallel plexuses, one of which then replaces the original sinus as the major route of bloodflow. Ultrastructurally the capillaries of the area vasculosa were simple tubes of endothelial cells, lacking a basement membrane. The endothelial cell cytoplasm contained only a few organelles, mainly mitochondria and rough endoplasmic reticulum. These findings indicate that the chick area vasculosa capillaries bear similar structural and growth characteristics to those associated with tumour angiogenesis and suggest that they may prove to be a useful model system for studying the factors involved in pathological angiogenesis.