Localization of plasminogen in the extracellular matrix of hamster eggs: exogenous activation by streptokinase

The plasminogen activator (PA)/plasminogen/plasmin proteolytic system has begun to be taken into account in the fertilization process. In this study, we demonstrated the presence of plasminogen in the extracellular matrix (ECM) of hamster oocytes by indirect immunofluorescence and immunoperoxidase assays using human anti-plasminogen. Plasminogen appeared first on the zona pellucida (ZP) of ovarian oocytes and later on the plasma membrane (PM) of oviducal eggs. This would suggest that oviducal oocytes modulate the expression of plasminogen binding sites on the PM. Human plasminogen as well as that of other species, known to be activated by streptokinase (SK), is rapidly converted to a plasmin-SK complex. We demonstrated the rapid formation of a SK-plasminogen complex that yields plasmin in the blood plasma of hamsters. Both the in vivo and in vitro SK treatment of eggs from superovulated female hamsters caused a decreased in the ZP dissolution time (ZPdt), probably either due to the proteolytic effect of plasmin or due to the SK-Plasminogen. Extracellular proteolysis assays carried out on agar-casein plates confirmed the proteolytic activity of SK-incubated eggs; the controls, on the contrary, failed to display a halo. These studies show that (1) superovulated hamster eggs contain plasminogen in their ECM, (2) oviducal eggs exhibit plasminogen on their PMs, indicating the presence of their corresponding binding sites, (3) in hamsters, SK, a non-enzymatic exogenous protein would be capable of activating ECM plasminogen to plasmin, and (4) the complex SK-plasminogen and/or the plasmin are capable of changing the ZPdt with alpha-chymotrypsin.     

A direct effect of some rifamycin derivatives on the morphology of mammalian mitochondria

Protein synthesis has been investigated in cell-free preparations from mature ovarian oocytes, unfertilized and fertilized eggs, and early embryos of Drosophila melanogaster. Preparations from unfertilized eggs have a specific activity that is 5- to 6-fold higher than the activity of fractions from ovarian oocytes. There is an additional small increase in activity of preparations from fertilized eggs. The specific activity that is rapidly attained in the fertilized egg remains essentially constant for 2 to 2.5 h after fertilization, decreases sharply during blastoderm formation, and again increases during gastrulation. The activities of unfertilized eggs decline slightly during the first 2 h after oviposition, and then decrease more sharply. About 35 % of the ribosomes in preparations from both unfertilized and fertilized eggs sediment in the polyribosome region of sucrose density gradients, whereas no polyribosomes could be detected in preparations from ovarian oocytes. In both ovarian oocytes and fertilized eggs, less than 1 % of the ribosome populations were present as subunits. Additional ribonucleoprotein material of buoyant densities different from those of ribosomal subunits or ribosomes was found throughout the sucrose gradients. About 3.5 % of the ribosomes were found to be membrane-bound in preparations from both unfertilized and fertilized eggs.     

Inhibition of the onset of vitellogenesis by abnormal retention of mature eggs in the stick insect,Carausius morosus

Mature eggs stored in the lateral oviducts are unusual in Carausius morosus. When egg laying is experimentally prevented, ovarian production is inhibited by the accumulation of mature eggs within the lateral oviducts. Such storage affects only the very small terminal oocytes and not the vitellogenic follicles, which attain to term and are ovulated. The fact that unilateral retention of eggs affects only the corresponding ovary strongly suggests that the mature eggs themselves inhibit ovarian production in the stick insect without the involvement of an endocrine organ. It is likely that this mode of action, different from that suggested for the housefly or Rhodnius prolixus, is correlated with the independence of ovarian development from the corpus allatum hormone in the stick insect.     

Characterization of urea transport in Bufo arenarum oocytes

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water.     

Sperm binding activity of the zona pellucida of immature mouse oocytes

Immature oocytes taken from ovarian follicles are sometimes used in studies of sperm-zona interaction in species for which it is difficult to obtain ovulated eggs. As yet, however, there has been no quantitative comparison of the sperm binding capacities of immature and ovulated oocytes. We report here that in mice there is no significant difference in the numbers of sperm which bind to the zonae pellucidae of immature and ovulated oocytes in vitro. These results support the use of immature oocytes in studies of sperm-zona interaction. We have also analyzed the sources of variability in sperm binding assays, and we make suggestions for the most efficient design of experiments.     

Leucine aminopeptidase,HlLAP, from the ixodid tick Haemaphysalis longicornis,plays vital roles in the development of oocytes

Female ixodid ticks are amazing invertebrate animals which efficiently convert a large amount of nutrients derived from their ingested blood meals into eggs. Although oocyte development (vitellogenesis) in ticks is triggered by a blood meal and is assumed to be supported by nutrition derived from ovarian cells connecting oocytes, little is known about the ovarian molecules processing nutrient materials for the vitellogenesis. In this study, we have suggested a putative function of leucine aminopeptidase (HlLAP) in the ovary of parthenogenetic adult ixodid tick Haemaphysalis longicornis regarding a negative output of reproduction following disruption of HlLAP gene by RNA interference. Endogenous HlLAP was shown to be localized in the ovarian cells, including ovarian epithelial and pedicel cells which were assumed to provide nutrients for the developing oocytes. Histological studies demonstrated that a majority of immature oocytes in HlLAP gene knockdown ticks were transformed into abnormal morpho-histological oocytes with vacuolated cytoplasm and/or condensed nucleus. Taken together, a reduction of the numbers of laid eggs in the HlLAP gene knockdown ticks may be due to the degeneration of immature oocytes following deprivation of nutrients such as amino acids supplied not only by midgut HlLAP but also by the ovarian HlLAP. Regulation of the tick molecules involved in nutrient metabolism for the reproduction, including blood digestion and vitellogenesis, would help in controlling the tick population and tick-borne pathogens.     

Relationship between oocyte morphology and reproductive strategy in loricariid catfishes of the Paraná River, Brazil

Relationships between ovarian structure, oocyte structure/development, and parental care/life history strategies of six loricariid catfishes common in the upper Paraná River, Brazil were examined with analysis of catch data, relative gonad weight, histology, and microscopy. Three life history strategies were observed. Loricariichthys platymetopon , Loricariichthys sp. And Loricaria sp. produce several small clutches of large eggs over a protracted spawning period. Males of these species guard their eggs and larvae, which are transported as a mass on the ventral surface of the male's body. Hypostomus ternetzi and Megalancistrus aculeatus produce the largest mature eggs and the smallest clutches relative to adult mass. The spawning periods of these species are short, and males guard their broods in excavations. Rhinelepis aspera has high fecundity, high relative mass of mature gonads (both sexes), small mature eggs, and broadcast spawning with no parental care. This species migrates to spawn over firm substrates in channel areas during a contracted period. Mature oocytes of external bearers had the thickest zona radiata, followed by the egg scatterer, and cavity nesters. The thickness of the zona radiata probably is an adaptation to protect the developing egg from injury from abrasion. The zona granulosa appeared to be associated with production of secretions responsible for egg adhesion, and this layer was thickest in mature oocytes of the cavity nesting species, followed by the external bearers. All six species have wide distributions in the Paraná River, tributaries, floodplain lagoons, and the Itaipu Reservoir, but brood guarders tended to be most common in lentic habitats.     

Females of a blood-sucking bug may adjust their mating decisions according to the risk of ovipositing infertile eggs

Females should adjust their mating preferences when the costs of being selective vary. Here, we focus on the cost of laying unfertilized eggs. Oviparous species lose their fertilizable oocytes if remain virgin for too long. Thus, females who are at a high risk of laying infertile eggs should be selected against rejecting a mating opportunity under such circumstances. We test this prediction using the blood-sucking bug Rhodnius prolixus females, which exhibit stereotyped discrimination behaviour to undesired copula. Under laboratory conditions, we compared the copulatory rejection behaviour between females at their first ovarian cycle (12 days old adults, at low risk of ovipositing infertile eggs) and females at their second ovarian cycle (26 days old adults, at high risk of ovipositing infertile eggs). Since R. prolixus female rejection behaviour is mainly exhibited in the presence of potential mates, we also manipulated the presence and absence of additional males. We found that rejection behaviour significantly decreases both when females enter their second ovarian cycle (and are at high risk of losing unfertilized eggs) and under the absence of additional males. We discuss the possible effects of the risk of losing fertilizable eggs on female mating preferences.     

Follicular dysfunction induced by autoimmunity to zona pellucida

The mammalian zona pellucida is an extracellular matrix that occurs in growing oocytes, ovulated eggs and pre-implantation embryos, and is known to be involved in several important events during ovarian folliculogenesis and fertilization. Since the zona pellucida is formed at an early stage of oocyte growth, circulating antibodies against zona pellucida may impair ovarian function. In this article we discuss whether anti-zona antibodies cause ovarian dysfunction and infertility. The discussion is based on clinical examination and animal experiments including the following approaches: 1/ immunological method using solubilized human zona pellucida detected anti-zona antibody with a high frequency in infertile patients, especially premature ovarian failure syndrome; 2/ in vivo experiment using hamsters showed that some, but not all, animals experienced ovarian failure after immunization with hamster recombinant zona proteins; 3/ in vitro experiment using mouse isolated ovarian follicles showed significant inhibitory effects on follicular growth and oocyte development. We concluded that anti-zona antibody may be involved in causing ovarian failure.     

A symbiont of the tick Ixodes ricinus invades and consumes mitochondria in a mode similar to that of the parasitic bacterium Bdellovibrio bacteriovorus

We have recently performed molecular characterisation of an intracellular alpha-proteobacterium, named IricES1, which resides in the ovarian tissue of female Ixodes ricinus ticks from Italy. A unique characteristic of this bacterium is its ability to invade the mitochondria of the cells in which it resides. Although some ultrastructural studies have been performed on close relatives of this bacterium from I. ricinus in England and Switzerland, a number of questions remain about its movement within ovarian tissues and mitochondria. We have performed the first detailed ultrastructural examination of IricES1 in engorged female adult I. ricinus. Among our findings was that the bacterium enters mitochondria in a similar way to that employed by the 'predatory' bacterium Bdellovibro bacteriovorus, that is, between the inner and outer membranes. It then appears to multiply, with the new 'colony' consuming the mitochondrial matrix. Despite having many of their mitochondria consumed, oocytes appear to develop normally, and the bacteria are likely to be vertically transferred to all eggs.     

pH in the Jelly Layer of Starfish Oocytes

Using liquid ion exchanger micro-electrodes we have studied the pH in the vicinity of starfish oocytes and eggs and also in their jelly layers. Although highly variable, the pH in these zones was consistently lower (6.3–7.6) than the pH of sea water (7.8–8.2). Aged oocytes/eggs presented more acid environments than fresh oocytes. The pH indicator Phenol Red shows that the ovarian fluid is more acid than seawater, ranging from 6.0 to 7.5. Our data suggests that starfish oocytes, from their time in the ovary until several hr after their release, are surrounded by an acid environment. This micro-environment may play a role in metabolic repression in the ovary and in the regulation of sperm-egg interaction following spawning.     

Quantitative aspects of the regulation of ovarian development in selected anautogenous Diptera: integration of endocrinology and nutrition

Aspects of the influence of nutrition on the degree of ovarian development in selected anautogenous Diptera are reviewed. The Diptera considered are several mosquito species, with emphasis on Aedes aegypti, the house fly, Musca domestica, the Australian bush fly, M. vetustissima and the blowflies, Lucilia cuprina and Phormia regina. All the selected species display discrete ovarian cycles in which all oocytes destined to reach maturity in a particular ovarian cycle develop synchronously. In these species, the proportion of females maturing oocytes and, where such data exist, the number of oocytes they mature are positively correlated with the amount of any particular nitrogen-containing material ingested or given by enema or by infusion. In addition, the degree of ovarian development may be affected by the chemical composition of nitrogen-containing food. Possible physiological bases for the observed relationships are discussed. Available evidence suggests that whether or not a female matures any oocytes is hormonally regulated and that the number of oocytes matured is probably regulated by the availability of nutrients. Some approaches that might further elucidate the physiological regulatory mechanisms involved in ovarian development are outlined.     

Morphology and ultrastructure of the somatic cells in Astacus leptodactylus ovary

We defined the somatic environment in which female germinal cells develop, and performed ultrastructural analyses of various somatic cell types, with particular reference to muscle cells and follicle cells, that reside within the ovary at different stages of oogenesis. Our findings show that ovarian wall of the crayfish is composed of long muscle cells, blood cells, blood vessels and hemal sinuses. The follicle and germinal cells lie within a common compartment of ovarian follicles that is defined by a continuous basal matrix. The follicle cells form branching cords and migrate to surround the developing oocytes. A thick basal matrix separates the ovarian interstitium from ovarian follicles compartment. Transmission electron microscopy shows that inner layer of basal matrix invaginates deeply into the ovarian compartment. Our results suggest that before being surrounded by follicle cells to form follicles, oogonia and early previtellogenic oocytes reside within a niche surrounded by a basal matrix that separates them from ovarian interstitium. We found coated pits and coated vesicles in the cortical cytoplasm of previtellogenic and vitellogenic oocytes, suggesting the receptor mediated endocytosis for transfer of material from the outside of the oocytes, via follicle cells. The interstitial compartment between the inner muscular layer of the ovarian wall and the basal matrix of the ovarian follicle compartment contains muscle cells, hemal sinuses, blood vessels and blood cells. Granular hemocytes, within and outside the vessels, were the most abundant cell population in the ovarian interstitium of crayfish after spawning and in the immature ovary. J. Morphol. 277:118–127, 2016. © 2015 Wiley Periodicals, Inc.     

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.     

Talin and vinculin in the oocytes, eggs, and early embryos of Xenopus laevis: a developmentally regulated change in distribution

We have investigated the expression and distribution of talin and vinculin in the oocytes, eggs, and embryos of Xenopus laevis. Antibodies to the previously characterized avian proteins stain several different Xenopus cell types identically by immunofluorescence: adhesion plaques of cultured kidney (A6) cells, the cell peripheries of oviduct cells, and the postsynaptic neuromuscular junctions of tadpole tail muscle fibers. These antibodies also identify cognate proteins of the appropriate sizes on immunoblots of A6 cell and oviduct lysates. Using these antibodies on ovarian tissue, we find talin to be highly localized at the cortices of oocytes and vinculin to be in the oocyte cytoplasm and absent from the oocyte cortex. In the cells of the ovarian layers that surround the oocytes, talin and vinculin can be detected as soluble and cytoskeletal components. Vinculin is first detectable as a cytoskeletal component in eggs, appearing some time during or between oocyte maturation and oviposition. During early embryo development, talin and vinculin are colocalized in the cortex of cleavage furrows and blastomeres. Thus, Xenopus oocytes and eggs display different distributions of talin and vinculin. The change from unlinked localization to colocalization appears to be developmentally regulated, occurring during the transition from oocyte to egg.     

The chorion defect of the ocelliless mutation caused by abnormal follicle cell function

Homozygous Drosophila females bearing the ocelliless mutation are sterile and produce oocytes with abnormal chorions. It has been possible to determine in which tissues these defects reside by generating ovarian chimeras. Pole cells from ocelliless female embryos can give rise to functional oocytes surrounded by normal chorions when placed in a wild-type environment. Conversely, when wild-type pole cells are placed in homozygous ocelliless females, the oocytes that form from them have abnormal chorions and never give rise to progeny. Thus the chorion defect and sterility of the ocelliless mutation are not germ-line autonomous. Homozygous ocelliless ovaries will attach to the uterus when placed in a wild-type third instar larva, but few eggs are ever laid, and the chorions of stage 14 oocytes remain ocelliless in morphology. Wild-type ovaries continue to produce oocytes with normal chorion morphology when placed into ocelliless hosts, indicating that the ocelliless chorion defect is ovary autonomous. Thus the chorion defect of the ocelliless mutation resides in the ovarian somatic tissue, presumably the follicle cells.     

Morphological differences in nuclear materials released from hamster sperm heads at an early stage of incorporation into immature oocytes,mature oocytes,or fertilized eggs

To elucidate the effects of ooplasmic factors on the early morphological changes in hamster sperm heads within the ooplasm, immature ovarian oocytes at the germinal vesicle stage (GV oocytes), ovulated fully mature oocytes, and fertilized eggs at anaphase II or the pronuclear stage (PN eggs) were examined in detail 15–30 min after insemination or reinsemination. Thin-sectioning studies demonstrated distinct materials released from the sperm nucleus over the entire postacrosomal nuclear surface immediately after disappearance of the sperm nuclear envelope. The release occurred in all of the oocytes and eggs prior to or even in the absence of subsequent chromatin decondensation. Depending upon the stage of the penetrated oocyte or egg, however, the materials varied in morphology: several hemispherical projections of amorphous material within mature oocytes; a number of electron-dense globules within GV oocytes and PN eggs; and both forms within eggs at anaphase II-telophase II. These observations and the fact that only the release of the amorphous material was accompanied by sperm chromatin decondensation indicate that this release was the initial process of chromatin decondensation, whereas the release of the globules resulted from a deficiency or lack of ooplasmic factors affecting the sperm nucleus. Restriction of the release in both forms of material to the late meiotic phase suggests changes in the factors associated with progression of meiosis. To approach an understanding of the mechanism of successful decondensation of sperm chromatin, the ooplasmic factors considered responsible for the stage-dependent release of nuclear materials are discussed. © 1996 Wiley-Liss, Inc.     

Amino acid cues emanating from Pacific salmon eggs and ovarian fluid

The eggs of salmonid fishes are an important food source for many aquatic predators that detect eggs using olfaction. Moreover, chemicals from eggs and ovarian fluid aid sperm cells in detecting and locating eggs for fertilization, and ovarian fluid is attractive to conspecific males. Thus chemicals from eggs and ovarian fluid may facilitate reproduction but may also attract egg predators. The authors sampled mature females of three Pacific salmon species – Chinook (Oncorhynchus tshawytscha), coho (Oncorhynchus kisutch) and sockeye (Oncorhynchus nerka) – and determined the proportional representation of amino acids, potent fish odorants, from their eggs and ovarian fluid (Chinook and coho salmon only). They then tested juvenile coho salmon, an egg predator, for responses to ovarian fluid and egg odours using the electro-olfactogram (EOG) recording technique. The amino acid compositions of the salmon species were significantly and positively correlated with each other, and the interspecific differences were comparable to those between individuals of the same species. The egg water samples were, on average, dominated by lysine, alanine and glutamine (12.6%, 12.4% and 10.9%, respectively). The ovarian fluid samples were dominated by lysine (20.5%), followed by threonine (9.7%), glycine (9.2%) and arginine (8.8%). EOG recordings demonstrated the ability of juvenile coho salmon to detect the chemical traces of eggs and ovarian fluid. It is concluded that salmon eggs are a potent source of odours for potential predators but likely not highly differentiated among salmon species.     

Oogenesis and ovarian morphology in pollinating and non-pollinating fig wasps: evidence from adult and immature stages

Hymenopteran insects have meroistic polytrophic ovaries characterised by trophocytes associated with oocytes inside the follicles. In pro-ovigenic species, all oocytes mature before emergence and no trace of oogenesis is visible in adult females. Pro-ovigeny is a rare condition among Hymenoptera, but common in pollinating fig wasps. In the present investigation, we studied adult and pupa females of three fig wasp species with different trophic strategies. We demonstrated that females of Pegoscapus aerumnosus and Idarnes spp. have an unusual ovarian organisation (i.e. each ovariole has only one mature egg and no oocyte) that has led to misleading interpretation of fig wasp reproductive anatomy. The ovaries of these studied species have several ovarioles, recognisable by the presence of nuclei of tunica propria cells surrounding them. Each adult wasp ovariole had one mature egg. None of the pupae had mature eggs, but all of them had follicles with oocytes in different developmental stages. The studied fig wasps are pro-ovigenic, irrespective of their trophic strategy, since there were no signs of ovigeny in adult females. We discuss ecological and phylogenetic factors that could play a role in fig wasps reproductive strategies.     

In vitro activation of Drosophila eggs

Mature ovarian eggs of Drosophila can be activated by treatment with hypotonic buffers. Well-fed, 4-day-old virgin flies contain large numbers of partially dehydrated mature eggs. When these eggs are transferred to a hypotonic culture medium, the ovarian eggs swell immediately and within minutes up to 70% become impermeable. The following cellular events ensue: meiosis, which had been arrested at metaphase I, is completed and the "polar body" nuclei fuse; cortical multivesicular bodies with acid phosphatase activity appear within minutes; polar granules fragment, dissociate from mitochondria, and become associated with polysomes; finally, monosomal ribosomes move to the polysomal region of a sucrose gradient. Each of these events corresponds to the normal in vivo effects of ovulation of oocytes, whether or not they are fertilized. When ovarian eggs of a parthenogenetic strain of D. mercatorum were activated by hypotonic treatment, some eggs developed into normal embryos. The presence of high potassium, low pH, and polyethylene glycol enhanced the frequency of normal development. Thus, we suggest that the rehydration of mature oocytes, as they move from the ovary to the uterus, activates the maternal program of the oocyte.