Induction of mictic females in the rotifer Brachionus: oocytes of amictic females respond individually to population-density signal only during oogenesis shortly before oviposition

1. One at a time during the reproductive period of amictic females, oocytes fill with yolk and undergo a mitotic maturation division (oogenesis), are oviposited as single cells, and then develop parthenogenetically into females. Sexual reproduction in Brachionus and several other genera is initiated when amictic females are crowded and oviposit some eggs induced to differentiate into mictic females. Mictic females produce haploid eggs that can develop parthenogentically into males or be fertilised and develop into diapausing embryos called resting eggs. 2. This study examines the time when oocytes in amictic females respond to maternal population density. Is the fate of all oocytes in the germarium irreversibly determined during the early postnatal life of the mother, or is each oocyte labile until just before oviposition? In the former case, the probability of an amictic female producing a mictic daughter at any time throughout her reproductive period would reflect the population density she experienced while young and not that at the time she oviposited an egg. 3. Amictic females of two clones of a Florida strain of B. calyciflorus were cultured singly from birth at a low or high density (in a large or small volume) until about halfway through their reproductive period and then switched (experimental treatment), or not (control treatment), to the other density condition. The results indicate that the female fate of an oocyte is determined by maternal population density during oogenesis. Eggs oviposited soon after transfer from low to high density had the same, or a higher, probability of becoming mictic females compared with those produced by control females kept at the high density; eggs oviposited after transfer from the high to the low density had the same low probability of becoming mictic females as those produced by control females kept at the low density. 4. Control females kept at the high density were less likely to produce mictic daughters as they aged. This decline is not because of a decreased propensity of older females to respond to crowding, as older females responded maximally when transferred from a low to a high population density. 5. As oocytes in amictic females respond to maternal population density only during oogenesis, there is a negligible lag between the population‐density signal in the environment and the commitment to sexual reproduction. This minimises the obligatory two‐generation lag between this signal and production of resting eggs, and thus reduces the possibility that crowding will lead to food limitation before production of these eggs.     

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.     

Free intracellular cations in echinoderm oocytes and eggs

The concentrations of Ca²⁺, Na⁺ and H⁺ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca²⁺ was about 10^-7 M. We did not detect any change in Ca²⁺ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na⁺ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na⁺ and pH.     

Age‐related changes in the development of oocytes and testicular follicles after emergence in Stavsolus japonicus (Plecoptera)

Abstract Female stoneflies oviposit several times during the adult stage of their life cycle. The number of eggs within the deposited egg masses decreases at successive ovipositions. To clarify the reason for this decrease and to determine the conditions of testicular follicles, the patterns of development of oocytes and testicular follicles on different days after emergence are investigated in the systellognathan species Stavsolus japonicus (Okamoto) (Perlodidae). The size of the mature oocytes in the ovariole peaks a few weeks after emergence but decreases to the lowest level by 35 days after emergence. Several maturing oocytes can be observed in the ovarioles of individuals a few weeks after emergence but only one mature oocyte is observed at 35 days after emergence. The decreased number of eggs laid per mass by older individuals may therefore be due to the lower maturation of all the ovarioles.     

Egg dumping by predatory insects

Synovigenic insects resorb oocytes when food is scarce and mature oocytes when food is plentiful. These two antagonistic processes allow an optimal allocation of resources to reproduction and somatic functions. Unlike hymenopteran parasitoids, ladybirds cannot resorb mature oocytes present in the oviducts. Is the energy contained in these oocytes lost or is there a mechanism for recovering it when needed? Females of two species of ladybird beetles Adalia bipunctata (L.) and Adalia decempunctata (L.) that are starved for >24 h lay single infertile eggs, which they immediately eat, and these eggs comprise the mature oocytes in the oviducts at the onset of starvation. This behaviour has some similarities to egg dumping reported in herbivorous insects and is part, in ladybird beetles, of a process to retrieve energy invested in reproduction. Such behaviour may exist in other predatory synovigenic insects species that do not invest in maternal care.     

Energy Metabolism and Pyridine Nucleotide Levels during Xenopus Oocyte Maturation*, 1

The rate of oxygen consumption increased in maturing Xenopus oocytes within 2 hr after progesterone addition, well before GVBD. This suggested an early requirement for energy metabolism during maturation, similar to the situation in sea urchin eggs during fertilization. Yet, the absence of similar increases in glucose-6-phosphate levels, glucose-6-phosphate dehydrogenase activity, glucose conversion to CO₂, and the conversion of NAD(H) to NADP(H), indicated that carbohydrate metabolism was not being stimulated in Xenopus oocytes during maturation. The oxidation of other energy yielding substrates is discussed which might account for the finding that, within 5 min of progesterone addition, both reduced forms of the pyridine nucleotides increased 20% over control levels. This was later followed by a drop in NADH levels and a rise in NAD relative to controls. The significance of these changes in pyridine nucleotide levels and their relationship to a number of maturation events are discussed.     

Fertilization of the starfish Astropecten aurantiacus

In the starfish Astropecten aurantiacus the acrosome reaction occurs when the spermatozoon contacts the outer surface of the jelly layer. A long thin acrosomal filament is extruded from the anterior region of the spermatozoon and establishes contact with the oocyte surface. This latter interaction initiates the movement of the spermatozoon to the oocyte surface, formation of the fertilization cone and the cortical reaction. The first detectable electrical change across the oocyte plasma membrane during interaction with the spermatozoon is the fertilization potential (FP) which occurs simultaneously with the cortical reaction. The FP is probably the electrical result of the modification of the oocyte plasma membrane during cortical exocytosis. There are no primary step-like depolarizations during fertilization of starfish oocytes, which contrasts with the situation in sea urchin eggs [see 13]. We suggest that the difference in electrical response to fertilization of starfish oocytes and sea urchin eggs may be attributed to the location of the acrosome reaction in these animals and not to their different meiotic states.     

A Highlights from MBoC Selection: Drosha protein levels are translationally regulated during Xenopus oocyte maturation

MicroRNAs (miRNAs) are ∼21-nucleotide-long, single-stranded noncoding RNAs that regulate gene expression. Biogenesis of miRNAs is mediated by the two RNase III-like enzymes, Drosha and Dicer. Here we study miRNA biogenesis during maturation of Xenopus oocytes to eggs using microinjection of pri-miRNAs. We show that processing of exogenous and endogenous primary miRNAs (pri-miRNAs) is strongly enhanced upon maturation of oocytes to eggs. Overexpression of cloned Xenopus Drosha in oocytes, however, boosts pri-miRNA processing dramatically, indicating that Drosha is a rate-limiting factor in Xenopus oocytes. This developmental regulation of Drosha is controlled by poly(A) length addition to the Drosha mRNA, which boosts translation upon transition from oocytes to eggs. Processing of pri-miRNAs by Drosha and Dicer has been shown to be affected by adenosine-to-inosine deamination–type RNA editing. Using activated Xenopus eggs for microinjection experiments, we demonstrate that RNA editing can reduce pri-miRNA processing in vivo. This processing block is determined by the structural but not sequence changes introduced by RNA editing.     

Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 μM in sea urchin eggs, 2 μM in Drosophila eggs, and only 0.11 μM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 μM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 μM apo-aequorin would take 50 days in zebrafish eggs, 19 days in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 gm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.     

Comparative life histories and microhabitat use in three sympatric sculpins (Cottidae:Cottus) in northeastern California

Synopsis Life histories of three sculpins endemic to the Pit River system, northeastern California, are detailed.Cottus pitensis is widely distributed throughout the drainage. This species is typically found in rapidly flowing, shallow water on coarse substrate. It grows to a large size, >100 mm, and lives to 5 years. Spawning occurs annually, in early spring. Fecundity is relatively low, <320 oocytes.Cottus asperrimus is restricted to a large population in Fall River and smaller concentrations in Hat Creek and Pit River. It is a relatively small fish, <80 mm, and lives to 5 years. It occurs in deep, slow-moving, often spring-fed streams, typically on fine substrates. Spawning lasts from autumn to early spring. Males establish nests on hard substrates and guard several clutches of eggs. Fecundity ranges from 140–580 oocytes.Cottus klamathensis macrops is relatively rare in the drainage. The largest concentration is in the Hat Creek system, smaller populations exist in Fall River and Pit River. This sculpin is found on coarse substrate, often where aquatic vegetation is abundant. It is typically found in slow, relatively shallow water. It grows to >100mm and lives to 5 years. Males guard eggs during the short spawning season beginning in late winter. Fecundity in this species reaches a maximum of 650 oocytes. Life history strategies vary within the genus, yet the life histories of these sculpins are more similar to each other than they are to many of their congeners. This within-group similarity may have resulted from exposure of all three species to similar environmental pressures. This is predicted by life-history theory.     

Ovarian Structure in Milnesium tardigradum (Tardigrada, Milnesiidae) during Early Vitellogenesis

The ultrastructure of the ovary of Milnesium tardigradum during early vitellogenesis is described. Within the ovary, there were large multinuclear cells surrounded by many mononuclear oocytes. Observation of serial sections revealed four multinuclear cells that were connected to each other by cytoplasmic bridges. Each peripheral oocyte was connected to the multinuclear cell. An enormous ER-like structure was conspicuous in the centre of the multinuclear cell. The presence of large numbers of lipid droplets and yolk granules in both multinuclear cells and many mononuclear oocytes suggested a role as nurse cells. A small number of these oocytes grow to be eggs. The structural features of the multinuclear nurse cell were compared with other known examples.     

Comparison of fecundity and longevity of Anoplophora malasiaca (Coleoptera: Cerambycidae) adults fed on three different host‐plants

Oocyte development, age at first reproduction, ovipositing activity and longevity were compared among three groups of Anoplophora malasiaca (Coleoptera: Cerambycidae) female adults that were grown on an artificial diet and provided with mandarin orange (MO), blueberry (BB) and willow (WI) branches after adult emergence. The female adults that were fed with MO began to lay eggs earlier, produced more eggs and lived longer than those that were fed with BB or WI. None of the female adults that were fed with BB laid eggs. The BB‐fed female adults developed the lowest number of oocytes after one and three weeks of feeding. The female adults that were fed with WI and MO for one week developed similar numbers of oocytes. However, after three weeks the MO‐fed female adults developed significantly more oocytes than WI‐fed females. When female adults were first fed with MO for one week and then supplied with BB, subsequent ovarian development was strongly suppressed and the number of immature oocytes decreased. The results suggested that some immature oocytes degenerated. From these results it was concluded that MO was the most suitable adult host‐plant, followed by WI and BB. It is possible that adults that emerge from BB trees may shift their host‐plant during the adult stage in the field.     

DNA synthesis in ocytes and eggs of Xenopus laevis injected with DNA.

Single-stranded calf thymus DNA injected into preovulation oocytes, postovulation oocytes or eggs of Xenopus laevis induces synthesis of double-stranded DNA of similar base composition. In contrast, native (double-stranded) calf thymus DNA injected into oocytes does not stimulate DNA synthesis, though it does do so in eggs. The buoyant density of normal or IUdR-substituted newly-synthesized DNA on neutral or alkaline CsCl gradients suggests that the injected DNA is replicated.The amount of synthesis induced by injecting single-stranded DNA is five times greater in eggs than in oocytes. The maximum synthesis observed in eggs injected with native DNA is 50 pg/hr; this is sufficient for nuclear DNA replication in uninjected fertilised eggs, but not in midcleavage. However in vitro studies (reported elsewhere) indicate the presence of a large store of DNA polymerase activity in eggs. We conclude that only a small proportion of the total DNA polymerase activity in an egg is available for DNA synthesis during the first 2 hr of development.     

Disappearance of a Novel Protein Component of the 26S Proteasome duringXenopusOocyte Maturation

We have prepared polyclonal antibodies againstXenopus20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development ofXenopus laevis.A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.     

The role of cAMP in oocyte maturation and the role of the germinal vesicle contents in mediating maturation and subsequent developmental events in hydrozoans

Summary Externally applied membrane permeable cAMP derivatives and the injection of cAMP induce oocyte maturation in several species of hydrozoans. This technique for inducing oocyte maturation has been used to study ion permeability changes, maturation promoting factor activity and surface tension changes during maturation. Oocyte membrane potential remains constant during maturation. Cyclic AMP induced maturation proceeds in the absence of external Ca²⁺, K⁻, Mg²⁺ or Na⁺. Cytoplasm from maturing oocytes that induces oocyte maturation when it is injected into untreated oocytes is produced during cAMP induced maturation. Surface tension, as measured by the application of a standardized force that mechanically deforms individual oocytes, declines during the first part of maturation. This is followed by a sharp rise and fall of surface tension at first and second polar body formation that accompanies a slow rise in the resistance of oocytes to deformation during the last part of maturation. The production of maturation promoting factor activity and some of the changes in surface tension during maturation can occur in the absence of germinal vesicle material. Two early developmental events that follow oocyte maturation are the production of sperm chemoattractant and calcium channel function. Neither of these events occurs in eggs that have undergone maturation in the absence of germinal vesicle material. The addition of germinal vesicle contents from oocytes to eggs that have undergone maturation in the absence of germinal vesicle material initiates calcium channel function. This experiment indicates that the germinal vesicle contains factors that are necessary for post-maturation developmental events.     

Lipid accumulation and utilization by oocytes and eggs of Rhodnius prolixus

Insect eggs must contain the necessary nutrients for embryonic growth. In this article, we investigated the accumulation of triacylglycerol (TAG) in growing oocytes and its utilization during embryonic development. TAG makes up about 60% of the neutral lipids in oocytes and accumulates as oocytes grow, from 2.2 ± 0.1 µg in follicles containing 1.0 mm length oocytes to 10.2 ± 0.8 µg in 2.0 mm length oocytes. Lipophorin (Lp), the hemolymphatic lipoprotein, radioactively labeled in free fatty acid (FFA) or diacylglycerol (DAG), was used to follow the transport of these lipids to the ovary. Radioactivity from both lipid classes accumulated in the oocytes, which was abolished at 4°C. The capacity of the ovary to receive FFA or DAG from Lp varied according to time after a blood meal and reached a maximum around the second day. ³H‐DAG supplied by Lp to the ovaries was used in the synthesis of TAG as, 48 hr after injection, most of the radioactivity was found in TAG (85.7% of labeling in neutral lipids). During embryogenesis, lipid stores were mobilized, and the TAG content decreased from 16.4 ± 2.1 µg/egg on the first day to 10.0 ± 1.3 µg on day 15, just before hatching. Of these, 7.4 ± 0.9 µg were found in the newly emerged nymphs. In unfertilized eggs, the TAG content did not change. Although the TAG content decreased during embryogenesis, the relative lipid composition of the egg did not change. The amount of TAG in the nymph slowly decreased during the days after hatching. © 2011 Wiley Periodicals, Inc.     

Redistribution and Increase in Cortical Inositol 1,4,5-Trisphosphate Receptors after Meiotic Maturation of the Mouse Oocyte

Mouse oocytes develop sensitivity to inositol 1,4,5-trisphosphate (IP₃) during oocyte maturation. We recently reported that a change in the organization of the endoplasmic reticulum (ER) during oocyte maturation may contribute to this enhanced sensitivity (Mehlmannet al.,1995,Dev. Biol.170, 607–615). Here, we investigated whether there is an increase in the number of available IP₃receptors after maturation and whether there is a redistribution of IP₃receptors similar to the redistribution of the ER that occurs during maturation. Western blot analysis of the IP₃receptor in oocytes and eggs demonstrated a 1.8-fold increase in immunoreactive mass of the IP₃receptor following oocyte maturation. Microinjection of the function-blocking monoclonal antibody 18A10 inhibited IP₃-induced Ca²⁺release in a concentration-dependent manner in both eggs and oocytes. More antibody was required to inhibit Ca²⁺release to the same extent in eggs compared to oocytes when both were injected with the same concentration of IP₃, suggesting that eggs contain a greater number of functional IP₃receptors. Immunolocalization of the IP₃receptor revealed that receptors were present in large clusters, 1–2 μm in diameter, in the cortex of the mature egg except in a ring-shaped band of cortex adjacent to the meiotic spindle. In contrast, receptor clusters were located around the entire cortex of the immature oocyte and were much smaller (<1 μm); larger patches were sometimes seen, but they did not display the same spherical organization as those in eggs. These results suggest that the number of cortical IP₃receptors increases during mouse oocyte maturation and that this increase may contribute to enhanced Ca²⁺release at fertilization.     

Stages of oocyte development in the zebrafish,Brachydanio rerio

Oocyte development has been divided into five stages in the zebrafish Brachydanio rerio, based on morphological criteria and on physiological and biochemical events. In stage I (primary growth stage), oocytes reside in nests with other oocytes (Stage IA) and then within a definitive follicle (Stage IB), where they greatly increase in size. In stage II (cortical alveolus stage), oocytes are distinguished by the appearance of variably sized cortical alveoli and the vitelline envelope becomes prominent. In stage III (vitellogenesis), yolk proteins appear in oocytes and yolk bodies with crystalline yolk accrue during this major growth stage. Ooctes develop the capacity to respond in vitro to the steroid 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) by undergoing oocyte maturation. In stage IV (oocyte maturation), oocytes increase slightly in size, become translucent, and their yolk becomes non-crystalline as they undergo final meiotic maturation in vivo (and in response to DHP in vitro). In stage V (mature egg), eggs (approx. 0.75 mm) are ovulated into the ovarian lumen and are capable of fertilization. This staging series lays the foundation for future studies on the cellular processes occurring during oocyte development in zebrafish and should be useful for experimentation that requires an understanding of stage-specific events. © 1993 Wiley-Liss, Inc.     

SIRT7 promotes chromosome synapsis during prophase I of female meiosis

Sirtuins are NAD⁺-dependent protein deacylases and ADP-ribosyltransferases that are involved in a wide range of cellular processes including genome homeostasis and metabolism. Sirtuins are expressed in human and mouse oocytes yet their role during female gamete development are not fully understood. Here, we investigated the role of a mammalian sirtuin member, SIRT7, in oocytes using a mouse knockout (KO) model. Sirt7 KO females have compromised fecundity characterized by a rapid fertility decline with age, suggesting the existence of a diminished oocyte pool. Accordingly, Sirt7 KO females produced fewer oocytes and ovulated fewer eggs. Because of the documented role of SIRT7 in DNA repair, we investigated whether SIRT7 regulates prophase I when meiotic recombination occurs. Sirt7 KO pachynema-like staged oocytes had approximately twofold increased γH2AX signals associated with regions with unsynapsed chromosomes. Consistent with the presence of asynaptic chromosome regions, Sirt7 KO oocytes had fewer MLH1 foci (~one less), a mark of crossover-mediated repair, than WT oocytes. Moreover, this reduced level of crossing over is consistent with an observed twofold increased incidence of aneuploidy in Metaphase II eggs. In addition, we found that acetylated lysine 18 of histone H3 (H3K18ac), an established SIRT7 substrate, was increased at asynaptic chromosome regions suggesting a functional relationship between this epigenetic mark and chromosome synapsis. Taken together, our findings demonstrate a pivotal role for SIRT7 in oocyte meiosis by promoting chromosome synapsis and have unveiled the importance of SIRT7 as novel regulator of the reproductive lifespan.

     

Effects of several inhibitors of macromolecule synthesis upon maturation of marine invertebrate oocytes

Protein, RNA and DNA syntheses, during oocyte maturation in Asterias glacialis and Chaetopterus, have been studied with cytochemical and biochemical methods. The effects of several inhibitors of the biosynthesis of these macromolecules have been investigated. The results show that protein synthesis is required for maturation: fusidic acid and puromycin, which strongly inhibit protein synthesis, prevent maturation. However, a paradoxical effect of cycloheximide was observed in Chaetopterus oocytes: this drug, like KCl, induces activation of the eggs. DNA and RNA are synthesized during maturation, but studies with inhibitors show that these syntheses are not necessary for the completion of maturation. Protein synthesis was followed during maturation and activation of Chaetopterus oocytes. It was observed that protein synthesis, which stops at the end of maturation, is not readily restored by activation. The significance of these results is discussed.