Maturation promoting factor destabilization facilitates postovulatory aging‐mediated abortive spontaneous egg activation in rat

The present study was designed to investigate whether destabilization of maturation promoting factor (MPF) leads to postovulatory aging‐mediated abortive spontaneous egg activation (SEA). If so, we wished to determine whether changes in Wee‐1 as well as Emi2 levels are associated with MPF destabilization during postovulatory aging‐mediated abortive SEA in rats eggs aged in vivo. For this purpose, sexually immature female rats were given a single injection (20 IU IM) of pregnant mare serum gonadotropin for 48 h followed by single injection of human chorionic gonadotropin (20 IU). Ovulated eggs were collected after 14, 17, 19 and 21 h post‐hCG surge to induce postovulatory aging in vivo. The morphological changes, Wee1, phosphorylation status of cyclin dependent kinase 1(Cdk1), early mitotic inhibitor 2 (Emi2), anaphase promoting complex/cyclosome (APC/C), cyclin B1, mitotic arrest deficient protein (MAD2) levels and Cdk1 activity were analyzed. The increased Wee 1 level triggered phosphorylation of Thr‐14/Tyr‐15 and dephosphorylation of Thr‐161 residues of Cdk1. The decrease of Emi2 level was associated with increased APC/C level and decreased cyclin B1 level. Changes in phosphorylation status of Cdk1 and reduced cyclin B1 level resulted in destabilization of MPF. The destabilized MPF finally led to postovulatory aging‐mediated abortive SEA in rat eggs. It was concluded that the increase of Wee 1 but decrease of Emi2 level triggers MPF destabilization and thereby postovulatory aging‐mediated abortive SEA in rat eggs.     

RyR channel-mediated increase of cytosolic free calcium level signals cyclin B1 degradation during abortive spontaneous egg activation in rat

In few mammalian species including rat, post-ovulatory aging induces abortive spontaneous egg activation (SEA), which is morphologically characterized by exit from metaphase-II (M-II) arrest. A possibility exists that the RyR channel-mediated insufficient increase of cytosolic free Ca²⁺ level could be one of the causes for post-ovulatory aging-induced abortive SEA. To test this possibility, eggs collected after 17 h post-hCG surge were cultured with or without various concentrations of nifedipine (NF), ruthenium red (RR), and KN-93 for 3 h in vitro. Morphological changes characteristic of abortive SEA, cytosolic free Ca²⁺ level, cyclin B1 level, and meiotic status were analyzed. Data of the present study indicate that NF and RR inhibited post-ovulatory aging-induced abortive SEA in a concentration-dependent manner. Further, RR protected against RyR channel as well as caffeine-mediated increase of cytosolic free Ca²⁺ level. In addition, KN-93 inhibited post-ovulatory aging-induced abortive SEA in a concentration-dependent manner. An increase of cytosolic free Ca²⁺ level was associated with a reduction of cyclin B1 level during post-ovulatory aging-induced abortive SEA. These data indirectly suggest the involvement of RyR channels in the increase of cytosolic free Ca²⁺ level. The increased cytosolic free Ca²⁺ level triggers cyclin B1 degradation possibly through CaMK-II activity during post-ovulatory aging-induced abortive SEA in rat eggs cultured in vitro.     

Nitric oxide signals postovulatory aging-induced abortive spontaneous egg activation in rats

Abstract

Objective

The aim of this study was to determine whether an increase of intracellular nitric oxide (NO) level signals postovulatory aging-induced abortive spontaneous egg activation (SEA) in rats.

Methods

Freshly ovulated eggs (arrested at metaphase-II stage; M-II) were cultured in vitro for 3 hours to induce postovulatory egg aging. The morphological changes, inducible nitric oxide synthase (iNOS) expression, NO, cytosolic free Ca²⁺, 3′,5′ cyclic guanosine monophosphate (cGMP), cell division cycle 25B (Cdc25B) and Wee1 levels, specific phosphorylation (pThr-14/Tyr-15) as well as total cyclin-dependent kinases-1 (Cdk1) (PSTAIRE) levels were analyzed.

Results

Postovulatory aging induced generation of NO possibly through an iNOS-mediated pathway. The increase in NO level was associated with augmented cytosolic free Ca²⁺ as well as cGMP levels in aged eggs. A significant increase in Wee1 level and decrease of Cdc25B level were observed in aged eggs. An accumulation of phosphorylated Cdk1 (pThr-14/Tyr-15) level was observed in aged eggs, while total Cdk1 (PSTAIR) level remained unchanged.

Conclusion

Our study demonstrates that generation of NO through an iNOS-mediated pathway increases cytosolic free Ca2+and cGMP levels. High levels of these signal molecules trigger the accumulation of phosphorylated Cdk1 in aged eggs. Thus, NO signals the accumulation of phosphorylated Cdk1 and induces postovulatory aging-induced abortive SEA in the rat.     

Biochemical and cellular insights into the temporal window of normal fertilization

Normal development depends on both the timing of fertilization and gamete quality, especially in assisted reproductive procedures. Recent studies of the proteins involved in the polyspermy block and cell cycle progression provide a cellular and biochemical basis for the short fertilizable lifespan of the mammalian egg in several species. Specifically, the status of cortical granules, zona proteins, cell cycle kinases, and intracellular calcium stores form a powerful panel of assays to monitor egg activation competence in eggs undergoing maturation and spontaneous activation events in mature eggs. An understanding of how these indicators are influenced by in vitro conditions and exogenous follicular stimulation should provide useful information for optimizing assisted reproductive procedures.     

Histology of abortive egg sites in the uterus of a viviparous,placentotrophic lizard,the skink Chalcides chalcides

Although abortive uterine eggs are often assumed to be resorbed by females of the viviparous skink Chalcides chalcides, little microscopic evidence of resorption of such eggs is available. Oviducts from pregnant female C. chalcides in which egg resorption was inferred were examined histologically to seek a morphological basis for resorption. Uterine histology at the site of abortive eggs was very similar to that of lizards in early pregnancy. The uterine epithelium consisted of a monolayer of pseudostratifed columnar cells that showed no evidence of yolk phagocytosis. The uterine lamina propria exhibited shell glands and modest vascularity, typical of early gestation, and contained neither yolk droplets nor accumulating leukocytes. Unattenuated regions of the lamina propria contained occasional macrophages and mast cells, some of the latter of which were undergoing degranulation. The abortive eggs often were collapsed with ruptured shell membranes, and some were undergoing extrusion from the incubation chambers down the oviduct. In eggs that had begun developing, extraembryonic ectoderm and endoderm were atypical in location, and had failed to enclose yolk leaking from the eggs. Oviducts sampled from later in the reproductive season were reproductively inactive, and showed no trace of abortive eggs or egg components. We postulate that abortive eggs are extruded from the oviduct by pregnant females under conditions of physiological stress, as a means of enhancing future reproductive effort. J. Morphol. 235:97–108, 1998. © 1998 Wiley-Liss, Inc.     

Effects of egg aging on in vitro fertilization and first cleavage division in the hamster

The postovulatory fertile life of mammalian eggs is remarkably short (approximately 6-36h). Anomalies of embryogenesis may result from fertilization of aged, defective eggs. Attempts to study this problem using whole-animal models are complicated by chances in the natural milieu of the gametes. In the present study, postovulatory hamster eggs were allowed to agein vivo then fertilized in vitro. Cumulus-intact eggs recovered from superovulated hamsters either 2 or 9 h after the estiated time of ovulation (12 h postHCG) were incubated for 4 h with preincubated sperm suspentions in a modified Tyrode's solution devised for in vitro fertilization. Eggs were either fixed or cultured for another 20h in fresh medium to allow cleavage to occur, then examined by light microscopy (phase and interference-contrast). No significant difference was found in the ablities of fresh and aged eggs to be penetrated by spermatozoa (94% vs 90%, respectively; 8 replicated experiments), but only 59% of penetrated aged eggs were found to undergo morphologically normally fertilization (2 polar bodies, 2 prounclei) compared with 75% of fresh eggs (difference significant, P< 0.01). About 13% of eggs were polyspermic in both categories. The most common anomaly in aged fertilied egges was failure to extrude the second polor body (23% off eggs vs 8% of fresh eggs, P < 0.01). Only 21% of aged eggs underwent first cleavaage, and only 74% of these appeared morphologically normal, compared with value of 68% and 98%, respectively, for fresh eggs. These data show that in the hamster, abnormal fertilization and cleavage failure can, in part, be directly attributed to postovulatory deterioration of eggs. We also infer that the apparently very short penetrable life of hamster eggs in vivo shown by previous investigators is an indirect effect of postovulatory changes in the female reproductive tract that are unfavorable for sperm-egg interactions.     

Caffeine alleviates the deterioration of Ca(2+) release mechanisms and fragmentation of in vitro-aged mouse eggs

The developmental competence of mammalian eggs is compromised by postovulatory aging. We and others have found that in these eggs, the intracellular calcium ([Ca(2+)](i)) responses required for egg activation and initiation of development are altered. Nevertheless, the mechanism(s) underlying this defective Ca(2+) release is not well known. Here, we investigated if the function of IP(3)R1, the major Ca(2+) release channel at fertilization, was undermined in in vitro-aged mouse eggs. We found that in aged eggs, IP(3)R1 displayed reduced function as many of the changes acquired during maturation that enhance IP(3)R1 Ca(2+) conductivity, such as phosphorylation, receptor reorganization and increased Ca(2+) store content ([Ca(2+)](ER)), were lost with increasing postovulatory time. IP(3)R1 fragmentation, possibly associated with the activation of caspase-3, was also observed in these eggs. Many of these changes were prevented when the postovulatory aging of eggs was carried out in the presence of caffeine, which minimized the decline in IP(3)R(1) function and maintained [Ca(2+)](ER) content. Caffeine also maintained mitochondrial membrane potential, as measured by JC-1 fluorescence. We therefore conclude that [Ca(2+)](i) responses in aged eggs are undermined by reduced IP(3)R1 sensitivity, decreased [Ca(2+)](ER) , and compromised mitochondrial function, and that addition of caffeine ameliorates most of these aging-associated changes. Understanding the molecular basis of the protective effects of caffeine will be useful in elucidating, and possibly reversing, the signaling pathway(s) compromised by in vitro culture of eggs.     

Mammalian fertilization as seen with the scanning electron microscope

For several years we have been looking at mammalian gametes and their interactions with the scanning electron microscope (SEM). Examining the images produced by the SEM has given us a three-dimensional view of sperm, eggs, and egg investments. We are particularly impressed with the structural variation among gametes of different mammalian species. In this short report we examine the structure of mammalian spermatozoa, eggs, zonae pellucidae, and cumuli. Our observations and those of others have led us to believe that variation in gamete structure and function may have evolved as a mechanism for reproductive isolation of mammalian species.     

A phylogenetic analysis of egg size,clutch size,spawning mode,adult body size,and latitude in reef fishes

Theoretical treatments of egg size in fishes suggest that constraints on reproductive output should create trade-offs between the size and number of eggs produced per spawn. For marine reef fishes, the observation of distinct reproductive care strategies (demersal guarding, egg scattering, and pelagic spawning) has additionally prompted speculation that these strategies reflect alternative fitness optima with selection on egg size differing by reproductive mode and perhaps latitude. Here, we aggregate data from 278 reef fish species and test whether clutch size, reproductive care, adult body size, and latitudinal bands (i.e., tropical, subtropical, and temperate) predict egg size, using a statistically unified framework that accounts for phylogenetic correlations among traits. We find no inverse relationship between species egg size and clutch size, but rather that egg size differs by reproductive mode (mean volume for demersal eggs = 1.22 mm³, scattered eggs = 0.18 mm³, pelagic eggs = 0.52 mm³) and that clutch size is strongly correlated with adult body size. Larger eggs were found in temperate species compared with tropical species in both demersal guarders and pelagic spawners, but this difference was not strong when accounting for phylogenetic correlations, suggesting that differences in species composition underlies regional differences in egg size. In summary, demersal guarders are generally small fishes with small clutch sizes that produce large eggs. Pelagic spawners and egg scatterers are variable in adult and clutch size. Although pelagic spawned eggs are variable in size, those of scatterers are consistently small.     

Identification of Mitochondrial Genome-Encoded Small RNAs Related to Egg Deterioration Caused by Postovulatory Aging in Rainbow Trout

Many factors have been reported to affect rainbow trout egg quality, among which, postovulatory aging is one of the most significant causes as reared rainbow trout do not usually volitionally oviposit the ovulated eggs. In order to uncover the genetic regulation underling egg deterioration caused by postovulatory aging in rainbow trout, mitochondrial genome-encoded small RNA (mitosRNAs) were analyzed from unfertilized eggs on Days 1, 7, and 14 postovulation with fertilization rates of 91.8, 73.4, and less than 50 %, respectively. A total of 248 mitosRNAs were identified from Illumina high-throughput sequencing of the small RNA libraries derived from the eggs of ten females. Ninety-eight of the small RNAs exhibited more than a threefold difference in expression between eggs from females exhibiting high fertilization rates at Day 1 and low fertilization rates at Day 14. The differentially expressed mitosRNAs were predominantly derived from mitochondrial D-loop, tRNA, rRNA, COII, and Cytb gene regions. Real-time quantitative PCR analysis was carried out for 14 differentially expressed mitosRNAs, of which, 12 were confirmed to be consistent with the sequencing reads. Further characterization of the differentially expressed mitosRNAs may lead to the development of new biomarkers for egg quality in rainbow trout.     

Extracellular calcium protects against verapamil-induced metaphase-II arrest and initiation of apoptosis in aged rat eggs

Non-specific L-type calcium channel blockers, such as verapamil (≥50 μM), induce metaphase-II (M-II) arrest and apoptosis in aged rat eggs cultured in Ca²⁺-deficient medium. However, the effects of extracellular Ca²⁺ on verapamil-induced M-II arrest and apoptosis have not yet been reported. We have demonstrated that postovulatory aging induced exit from M-II arrest by extruding a second polar body, a morphological sign of spontaneous egg activation (SEA). Verapamil inhibited SEA and induced egg apoptosis in a dose-dependent manner in Ca²⁺-deficient medium. The initiation of apoptotic features was observed at 50 μM of verapamil. Extracellular Ca²⁺ (1.80 mM) reduced intracellular H₂O₂ level, bax protein expression, caspase-3 activity, DNA fragmentation and protected against 50 μM, but not higher concentrations of ≥100 μM in verapamil-induced egg apoptosis. These results suggest that extracellular Ca²⁺ ions have a role during SEA and protect against verapamil-induced apoptosis in aged rat eggs.     

Effects of maternal age on reproductive traits and fitness components of the offspring in the bruchid beetle, Callosobruchus chinensis (Coleoptera: Bruchidae)

In many insect species, the size and number of eggs decrease with maternal age. Thus, both the size and number of eggs must be considered to know the exact cost of reproduction with maternal age. The resource depletion hypothesis was examined in the bruchid beetle Callosobruchus chinensis. The hypothesis explains why the egg size decreases with maternal age based on the decline of the female's reproductive capacity. A decrease was found in reproductive effort (= egg size × the number of eggs) and the fitness component of offspring with maternal age. The effects of the female's nutritional status on the relationship between maternal age and the reproductive effort of females with and without food and water were also examined. The results indicate that the decrease in size and number of eggs with maternal age can be explained by the resource depletion hypothesis in C. chinensis.     

The Drosophila calcipressin sarah is required for several aspects of egg activation

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.     

Reactive oxygen and nitrogen species during meiotic resumption from diplotene arrest in mammalian oocytes

Mammalian ovary is metabolically active organ and generates by‐products such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) on an extraordinary scale. Both follicular somatic cells as well as oocyte generate ROS and RNS synchronously and their effects are neutralized by intricate array of antioxidants. ROS such as hydrogen peroxide (H₂O₂) and RNS such as nitric oxide (NO) act as signaling molecules and modulate various aspects of oocyte physiology including meiotic cell cycle arrest and resumption. Generation of intraoocyte H₂O₂ can induce meiotic resumption from diplotene arrest probably by the activation of adenosine monophosphate (AMP)‐activated protein kinase A (PRKA)—or Ca²⁺‐mediated pathway. However, reduced intraoocyte NO level may inactivate guanylyl cyclase‐mediated pathway that results in the reduced production of cyclic 3′,5′‐guanosine monophosphate (cGMP). The reduced level of cGMP results in the activation of cyclic 3′,5′‐adenosine monophosphate (cAMP)‐phosphodiesterase 3A (PDE3A), which hydrolyses cAMP. The reduced intraoocyte cAMP results in the activation of maturation promoting factor (MPF) that finally induces meiotic resumption. Thus, a transient increase of intraoocyte H₂O₂ level and decrease of NO level may signal meiotic resumption from diplotene arrest in mammalian oocytes. J. Cell. Biochem. 111: 521–528, 2010. © 2010 Wiley‐Liss, Inc.     

Changes in intracellular pH following egg activation and during the early cell cycle of the amphibian Pleurodeles waltlii, coincide with changes in MPF activity.

Previous work on Xenopus laevis suggests a temporal coincidence between inactivation of the M-phase promoting factor (MPF) and intracellular pH (pHi) increase during egg activation. In addition, we recently showed that during the early cell cycle of Xenopus eggs, MPF activity cycling and pHi oscillations were temporally and functionally related. In the present work, using eggs of another amphibian, Pleurodeles waltlii, which has a natural cell cycle considerably longer than that of Xenopus laevis, we show a temporal coincidence between MPF activity and pHi changes, both at the time of egg activation and at each of the following cell cycles. Egg activation-induced pHi changes in Pleurodeles did not involve classical plasma membrane ion exchangers, and were not due to the activation of a H+ conductance. On the other hand, the pHi oscillations intervening at each cell cycle were suppressed by inhibitors of protein synthesis or phosphorylation, as were their counterparts in Xenopus eggs. We propose that physiological pHi changes in Pleurodeles and Xenopus eggs might have a metabolic origin, in direct relation with the cascade of phosphorylations-dephosphorylations of proteins implicated in the control of the cell cycle.     

Ovarian follicle dynamics of female Greater Scaup during egg production

ABSTRACT.   Studies of female waterfowl nutrient reserve use during egg production require a precise understanding of ovarian follicle dynamics to correctly interpret breeding status, and, therefore, derive proper inference. Concerns over numerical declines of North American scaup have increased the need to better understand the role of female condition in reproductive performance. We quantified ovarian follicle dynamics of female Greater Scaup ( Aythya marila ) breeding on the Yukon–Kuskokwim Delta, Alaska, using a method that accounts for within day variation in follicle size. We considered several models for describing changes in follicle growth with the best supported model estimating the duration of rapid follicle growth (RFG) to be 5.20 ± 0.52 days (±95% confidence intervals) for each developing follicle. Average diameter and dry mass of preovulatory follicles were estimated to be 9.36 mm and 0.26 g, respectively, at the onset of RFG, and these follicle characteristics were 41.47 mm and 15.57 g, respectively, at ovulation. The average diameter of postovulatory follicles immediately following ovulation was estimated to be 17.35 mm, regressing quickly over several days. In addition, we derived predictive equations using diameter and dry mass to estimate the number of days before, and after, ovulation for pre- and postovulatory follicles, as well as an equation to estimate dry mass of damaged follicles. Our results allow precise definition of RFG and nest initiation dates, clutch size, and the daily energetic and nutritional demands of egg production at the individual level. This study provides the necessary foundation for additional work on Greater Scaup reproductive energetics and physiology, and offers an approach for quantifying ovarian follicle dynamics in other species.     

A brood parasite selects for its own egg traits

Many brood parasitic birds lay eggs that mimic their hosts'' eggs in appearance. This typically arises from selection from discriminating hosts that reject eggs which differ from their own. However, selection on parasitic eggs may also arise from parasites themselves, because it should pay a laying parasitic female to detect and destroy another parasitic egg previously laid in the same host nest by a different female. In this study, I experimentally test the source of selection on greater honeyguide (Indicator indicator) egg size and shape, which is correlated with that of its several host species, all of which breed in dark holes. Its commonest host species did not discriminate against experimental eggs that differed from their own in size and shape, but laying female honeyguides preferentially punctured experimental eggs more than host or control eggs. This should improve offspring survival given that multiple parasitism by this species is common, and that honeyguide chicks kill all other nest occupants. Hence, selection on egg size in greater honeyguides parasitizing bee-eaters appears to be imposed not by host defences but by interference competition among parasites themselves.     

DNA barcoding reveals the temporal community composition of drifting fish eggs in the lower Hongshui River,China

Determining the temporal community composition of fish eggs in particular regions and understanding the reproductive times of regional fish taxa are key aspects of the management and regulation of regional fish stocks. However, it is extremely difficult to accurately identify fish eggs due to the absence of diagnostic morphological characters. We sampled fish eggs in the lower Hongshuihe River (an upper mainstem of the Pearl River) between May and September 2020. We then used DNA barcoding to determine the species composition of the egg pool and to predict the spawning periods of the identified species. A total of 641 eggs and 17 larvae were chosen for molecular identification; 397 eggs and 17 larvae yielded high‐quality barcoding sequences. The high failure rate (~38%) was most likely due to long‐term storage in low concentrations of ethanol prior to molecular analysis. We successfully classified 392 eggs into 10 species and 13 larvae into four species using public databases. Most of the species identified in the egg pool were small and/or benthic, and migratory species were rare. This may partially reflect the adverse effects of hydropower cascade development in this river section. We also found that spawning periods tended to be species‐specific. Our study provides a reference for the conservation and management of regional fishery stocks.     

Vertebrate extracellular preovulatory and postovulatory egg coats

Extracellular egg coats deposited by maternal or embryonic tissues surround all vertebrate conceptuses during early development. In oviparous species, the time of hatching from extracellular coats can be considered equivalent to the time of birth in viviparous species. Extracellular coats must be lost during gestation for implantation and placentation to occur in some viviparous species. In the most recent classification of vertebrate extracellular coats, Boyd and Hamilton (Cleavage, early development and implantation of the egg. In: Parkes AS (ed.), Marshall's Physiology of Reproduction, vol. 2, 3rd ed. London: Longmans, Green & Co; 1961:1-126) defined the coat synthesized by the oocyte during oogenesis as primary and the coat deposited by follicle cells surrounding the oocyte as secondary. Tertiary egg coats are those synthesized and deposited around the primary or secondary coat by the maternal reproductive tract. This classification is difficult to reconcile with recent data collected using modern molecular biological techniques that can accurately establish the site of coat precursor synthesis and secretion. We propose that a modification to the classification by Boyd and Hamilton is required. Vertebrate egg coats should be classed as belonging to the following two broad groups: the preovulatory coat, which is deposited during oogenesis by the oocyte or follicle cells, and the postovulatory coats, which are deposited after fertilization by the reproductive tract or conceptus. This review discusses the origin and classification of vertebrate extracellular preovulatory and postovulatory coats and illustrates what is known about coat homology between the vertebrate groups.