Vertebrate Eggs Research Articles

158 LEADING BLASTOMERE OF A 2-CELL-STAGE PORCINE PARTHENOGENETIC EMBRYO CONTRIBUTES TO TROPHECTODERM FIRST

Embryonic axis formation is important in normal animal development. Some vertebrate and invertebrate eggs display undeniable polarity along a plane known as the animal–vegetal axis. However, axis formation in mammals, which is studied only in mice, is still a point of issue (Hiiragi and Solter 2004 Nature 430, 360–364; Zernicka-Goetz et al. 2005 Nature 434, 391–395). In the present study, we investigated the embryonic–abembryonic axis formation in porcine species. We used porcine parthenogenetic embryos to prevent a topological change of the 2 apposing pronuclei in the egg center caused by polyspermy and to avoid the influence of the fertilization cone, which indicates the sperm entry position. For lineage tracing, DiI, a lipophilic fluorescence dye, was injected into one blastomere per embryo at the 2-cell stage. After this process, as embryos developed into the 3-cell stage, all the embryos were divided into 2 groups named Leading and Lagging, following the location of the DiI oil drop (Leading: the oil drop was positioned in the first-dividing blastomere; Lagging: the drop was positioned in the late-dividing blastomere). The embryos developed into blastocysts after 6 to 7 days of culture in vitro. Only the inner cell mass (ICM) was labeled in 50% (8/16) of the Lagging group. In some Lagging embryos (2/16), ICMs with a small portion of adjacent trophectodermal cells (TE) were labeled and 62.5% (10/16) of Lagging embryos formed the ICM part in total. On the other hand, 60% (6/10) of the Leading embryos formed only distal TE (opposite side of the ICM). Only one of the Leading embryos formed the ICM. The rest of Lagging embryos (37.5%, 6/16) or the Leading group (40.0%, 4/10) showed an even distribution of blastocysts, regardless of the ICM or TE. Oocytes, zygotes, and 2-cell-stage embryos in porcine species tended to show unequally distributed lipid contents and could be distinguished by their different colors and brightness, although not all of them showed significant differences. We injected DiI into a relatively brighter blastomere of the 2-cell-stage embryos and the results were very similar to those obtained from the Leading group (distal TE: 70.6%, 24/34). High lipid contents may be the cause of delayed development of the blastomere of an embryo. Our findings indicated that the Leading blastomere of 2-cell porcine parthenotes formed distal TE first, and that afterward, the Lagging blastomere not only filled the rest of the TE but also contributed to the ICM. This study was supported by the Research Project on the Production of Bio-organs (No. 200506030601) and Technology Developmental Program (High-Technology Development Project No. 204117-3) for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea

Cdc42 Activation Couples Spindle Positioning to First Polar Body Formation in Oocyte Maturation

During vertebrate egg maturation, cytokinesis initiates after one pole of the bipolar metaphase I spindle attaches to the oocyte cortex, resulting in the formation of a polar body and the mature egg. It is not known what signal couples the spindle pole positioning to polar body formation. We approached this question by drawing an analogy to mitotic exit in budding yeast, as asymmetric spindle attachment to the appropriate cortical region is the common regulatory cue. In budding yeast, the small G protein Cdc42 plays an important role in mitotic exit following the spindle pole attachment . We show here that inhibition of Cdc42 activation blocks polar body formation. The oocytes initiate anaphase but fail to properly form and direct a contractile ring. Endogenous Cdc42 is activated at the spindle pole-cortical contact site immediately prior to polar body formation. The cortical Cdc42 activity zone, which directly overlays the spindle pole, is circumscribed by a cortical RhoA activity zone; the latter defines the cytokinetic contractile furrow . As the RhoA ring contracts during cytokinesis, the Cdc42 zone expands, maintaining its complementary relationship with the RhoA ring. Cdc42 signaling may thus be an evolutionarily conserved mechanism that couples spindle positioning to asymmetric cytokinesis.

Maternal allocation of androgens and antagonistic effects of yolk androgens on sons and daughters

Mothers can influence the phenotype of their offspring by adjusting the quality of their eggs in relation to sex and reproductive value of the progeny. Maternal androgens in the eggs of vertebrates may mediate such adaptive early maternal effects. However, the evolution of early maternal effects mediated by egg androgens may be constrained by the inability of mothers to differentially allocate androgens to eggs with a male or a female if androgens have different effects on sons and daughters. In this study, we increased the concentration of androgens in the eggs of barn swallows (Hirundo rustica) within the physiological range of variation and analyzed the effect on nestling growth and begging behavior. Egg androgens increased body size and mass of sons but reduced these characters in daughters when compared to two control groups in a repeated-measures analysis of variance of data collected at different ages. However, the differential effect of androgen on the two sexes was no longer significant when the analysis was restricted to the age of 12 days, when final body size is attained. In a second experiment, we tested whether mothers differentially allocated androgens to eggs with sons rather than daughters while manipulating a paternal secondary sexual character. Androgen concentration did not vary in relation to paternal ornamentation or embryo sex. Hence, antagonistic effects of egg androgens on sons and daughters may exist in the very early posthatching life and may constrain the evolution of adaptive maternal effects because mothers do not differentially allocate androgens in relation to embryo sex. Key words: begging display, early maternal effects, growth, Hirundo rustica, laying order, recruitment, viability. [Behav Ecol]

Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation

Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.

Effects of elevated egg corticosterone levels on behavior, growth, and immunity of yellow-legged gull ( Larus michahellis) chicks

Eggs of vertebrates contain steroid hormones of maternal origin that may influence offspring performance. Recently, it has been shown that glucocorticoids, which are the main hormones mediating the stress response in vertebrates, are transmitted from the mother to the egg in birds. In addition, mothers with experimentally elevated corticosterone levels lay eggs with larger concentrations of the hormone, which produce slow growing offspring with high activity of the hypothalamo–adrenal axis under acute stress. However, the effects and function of transfer of maternal corticosterone to the eggs are largely unknown. In the present study, we injected corticosterone in freshly laid eggs of yellow-legged gulls ( Larus michahellis), thus increasing the concentration of the hormone within its natural range of variation, and analyzed the effect of manipulation on behavioral, morphological, and immune traits of the offspring in the wild. Eggs injected with corticosterone had similar hatching success to controls, but hatched later. Mass loss during incubation was greater for corticosterone-treated eggs, except for the last laid ones. Corticosterone injection reduced rate and loudness of late embryonic vocalizations and the intensity of chick begging display. Tonic immobility response, reflecting innate fearfulness, was unaffected by hormone treatment. Elevated egg corticosterone concentrations depressed T-cell-mediated immunity but had no detectable effects on humoral immune response to a novel antigen, viability at day 10, or growth. Present results suggest that egg corticosterone can affect the behavior and immunity of offspring in birds and disclose a mechanism mediating early maternal effects whereby stress experienced by females may negatively translate to offspring phenotypic quality.

A role for the anaphase-promoting complex inhibitor Emi2/XErp1, a homolog of early mitotic inhibitor 1, in cytostatic factor arrest of Xenopus eggs

Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCF(betaTrCP) ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.

Emi1 and Erp1: Who Can Stop These Eggs?

Vertebrate eggs prevent parthenogenetic development by producing cytostatic factor (CSF), which blocks exit from metaphase of meiosis II until fertilization. CSF was never purified but recently suspected to inhibit the anaphase-promoting complex (APC), an ubiquitin ligase required for entry into anaphase. In a recent paper in Genes & Development, Schmidt et al. describe the Xenopus APC inhibitor Erp1, which seems to be the best candidate yet for the downstream effector of CSF activity.

Functional interaction between p90Rsk2 and Emi1 contributes to the metaphase arrest of mouse oocytes

Vertebrate eggs arrest at metaphase of the second meiotic division before fertilization under the effect of a cytostatic factor (CSF). This arrest is established during oocyte maturation by the MAPK kinase module, comprised of Mos, MEK, MAPKs and p90Rsk. Maintenance of CSF arrest at metaphase requires inhibitors of the anaphase-promoting complex (APC) like Emi1, which sequesters the APC activator Cdc20. Although it was proposed that the Mos pathway and Emi1 act independently, neither one alone is sufficient to entirely reproduce CSF arrest. Herein we demonstrate that p90Rsk2 associates with and phosphorylates Emi1 upstream of the binding region for Cdc20, thus stabilizing their interaction. Experiments in transfected cells and two-cell embryos indicate that Emi1 and p90Rsk2 cooperate to induce the metaphase arrest. Moreover, oocyte maturation was impaired by interfering with the interaction between p90Rsk2 and Emi1 or by RNA interference of Emi1. Our results indicate that p90Rsk2 and Emi1 functionally interact during oocyte maturation and that the Mos pathway establishes CSF activity through stabilization of an APC-inhibitory complex composed by Emi1 and Cdc20 before fertilization.

Yolk testosterone organizes behavior and male plumage coloration in house sparrows (Passer domesticus)

Vertebrate eggs contain maternal steroid hormones in their yolks; and in avian species the concentration of these steroids vary within and among clutches. The organizational actions of these variable doses of maternal steroids in the avian egg, analogous to those regulating the development of sexually dimorphic traits, are little explored. In this study, we examined the organizational effects of yolk testosterone in the house sparrow, Passer domesticus, a sexually dichromatic passerine bird in which males are characterized by the presence of a throat patch or “badge” of black feathers that varies in size among males. We injected a physiological dose of testosterone or vehicle into eggs in the field, removed nestlings, and hand-raised them in the laboratory. At 5 months of age, we investigated treatment effects on plumage and behavior. Males that hatched from testosterone-injected eggs developed a larger badge than males that hatched from vehicle-injected (control) eggs. However, testosterone did not induce the expression of a badge in females. In staged dyadic encounters, both males and females hatched from testosterone-injected eggs were more successful at obtaining and defending a food source than individuals of the same sex hatched from control eggs. The results suggest that variable concentrations of maternal testosterone in the eggs of the house sparrow organize the expression of a plumage trait in males and behavior in both sexes.

A PKC wave follows the calcium wave after activation of Xenopus eggs

Calcium waves are well-known hallmarks of egg activation that trigger resumption of the cell cycle and development of the embryo. These waves rapidly and efficiently assure that activation signals are transmitted to all regions of the egg. Although the mechanism by which the calcium wave propagates across an egg as large as that of Xenopus is not known, two models prevail. One model is a wave of calcium-induced calcium release (CICR) and the other is propagation by inositol-induced calcium release (IICR). IICR requires a wave of phosphatidylinositol 4,5-bisphosphate (PIP 2) hydrolysis, generating two second messengers, IP 3, which then releases calcium and DAG, which activates protein kinase C (PKC). We show here that a wave of PKC-green fluorescent protein travels across the egg immediately following, and at the same velocity as, the calcium wave. This is the first example of a PKC wave in a vertebrate egg and supports the IICR model of wave propagation.

Hatching Events in the California Grunion, Leuresthes tenuis

This study provides the first detailed description of the events at hatching for an anamniotic vertebrate egg. California Grunion are ideally suited for this study, because these fish eggs incubate fully terrestrially and delay hatching until presented with an environmental trigger. Grunion eggs can be induced to hatch on demand within a few minutes, by mechanical agitation in seawater. We suggest that the process of hatching in grunion involves an enzymatic weakening of the chorion followed by the embryo's active efforts to escape. Using a dissecting microscope, microscope camera, and digital video camera, we compared embryonic movements within the chorion prior to hatching. Then, we recorded the exact moment of hatching with digital video to identify events that occur consistently. Prior to exposure to the hatching trigger, Grunion embryos move minimally within the egg. After the hatching trigger, embryos significantly increase the amount and types of movements within the egg. The chorion distorts in a location superior to the caudal region, and fluid escapes from the egg. Embryonic activity usually culminates in a vigorous tail lash that splits the chorion open. Ultimately, usually within two minutes, the Grunion larva emerges from the chorion into its new aquatic environment.

Metabolism of selenomethionine by rainbow trout ( Oncorhynchus mykiss) embryos can generate oxidative stress

Although selenium is required by vertebrates, toxicity can arise at concentrations only slightly greater than those they require. The toxicity of Se is thought to arise from its ability to substitute for sulfur during the assembly of proteins. However, recent studies also indicate that some forms of selenium are capable of generating oxidative stress in an in vitro test system that includes glutathione. l-Selenomethionine, the predominant form of selenium in the eggs of oviparous vertebrates, does not generate oxidative radicals in this system, but lesions consistent with oxidative stress have been identified in fish and birds with high concentrations of Se. Here we report on the ability of rainbow trout embryos to transform l-Selenomethionine to a form capable of producing a superoxide radical. Oxidative stress appears to be generated by methioninase enzyme activity in the embryos that liberates methylselenol from l-Selenomethionine. Methylselenol redox cycles in the presence of glutathione producing superoxide and likely accounts for oxidative lesions present in fish and birds environmentally exposed to excessive loads of selenomethionine.

Analysis of fish ZP1/ZPB homologous genes--evidence for both genome duplication and species-specific amplification models of evolution

The vertebrate egg envelope is composed of a family of related proteins, the zona pellucida (ZP) proteins, which are characterized by the presence of a conserved zona pellucida domain. Analysis of teleost fish ZP gene sequences has demonstrated that there are no direct orthologues of the mammalian ZPB and ZP1 genes, but that teleost fish contain multiple copies of two classes of genes (ZPXa and ZPXb) that are equally related to ZPB and ZP1. The two classes of genes are further distinguished by expression in liver or ovary, respectively, indicating there was probably an initial duplication event, followed by a switch to hepatic expression of one of the paralogues. This switch was followed in some species by additional amplification of one of the paralogues with the subsequent loss of the other. It is proposed that the expansion of the number of ZPXa and ZPXb genes and the acquisition of dual sites of synthesis are the result of an ancient polyploidization event, followed by additional species-specific gene amplifications.

Analysis of fish ZP1/ZPB homologous genes--evidence for both genome duplication and species-specific amplification models of evolution.

The vertebrate egg envelope is composed of a family of related proteins, the zona pellucida (ZP) proteins, which are characterized by the presence of a conserved zona pellucida domain. Analysis of teleost fish ZP gene sequences has demonstrated that there are no direct orthologues of the mammalian ZPB and ZP1 genes, but that teleost fish contain multiple copies of two classes of genes (ZPXa and ZPXb) that are equally related to ZPB and ZP1. The two classes of genes are further distinguished by expression in liver or ovary, respectively, indicating there was probably an initial duplication event, followed by a switch to hepatic expression of one of the paralogues. This switch was followed in some species by additional amplification of one of the paralogues with the subsequent loss of the other. It is proposed that the expansion of the number of ZPXa and ZPXb genes and the acquisition of dual sites of synthesis are the result of an ancient polyploidization event, followed by additional species-specific gene amplifications.

Under arrest: cytostatic factor (CSF)-mediated metaphase arrest in vertebrate eggs.

Under arrest: cytostatic factor (CSF)-mediated metaphase arrest in vertebrate eggs.

CpZPC, a newt ZPC molecule, localizes to the inner surface of the egg envelope.

Zona pellucida-3 is an essential molecule for the binding of sperm to the egg envelope and for induction of the acrosome reaction in mice. Its homologous molecules, ZPCs, have been widely identified in the eggs of many vertebrates, except for urodeles. In this study, to investigate the participation of ZPC in newt fertilization, we cloned the cDNA of newt ZPC from the ovary of Cynops pyrrhogaster by reverse transcription polymerase chain reaction (RT-PCR). The cDNA was constructed from 1,397 nucleotides and included one open reading frame corresponding to a sequence of 439 amino acids. The deduced amino acid sequence had identities at 52, 47 and 45% with Xenopus gp41, mouse ZP3 and medaka L-SF, respectively. It included four potential N-linked glycosylation sites and 12 highly conserved cysteine residues of mammalian ZP3/ZPC molecules. This result suggests that CynopsZPC (cpZPC) has molecular features similar to those of mammalian ZP3/ZPCs. Messenger RNA for cpZPC was detected in the ovary and faintly in the testis. Two bands corresponding to 84 kDa and 70 kDa in the egg envelopes were detected by immunoblotting with an antiserum raised against a 9 amino acid peptide in the C-terminus domain of cpZPC. The molecular size of 84 kDa fits with the size of a putative sperm-binding protein reported by Nakai et al. (1999), suggesting that cpZPC may contribute to sperm binding to the egg envelope in C. pyrrhogaster. The results of immunohistochemistry suggest that cpZPC was localized in the inner surface of the egg envelope. Similar localization is seen only in fish, suggesting that cpZPC is a unique molecule which may allow us to investigate the functional evolution of the egg envelope in vertebrates.

Storage of retinal in the eggs of the ascidian, Halocynthia roretzi

Retinoids in the eggs of the solitary ascidian, Halocynthia roretzi, were analyzed by high performance liquid chromatography. Retinal was the almost exclusive retinoid (≫99%), and the concentration of retinal was 25.9–40.1 (30.6 on average) ng/mg of protein. The egg retinal consisted of four isomers: all- trans (50.9%), 9- cis (6.8%), 11- cis (20.4%) and 13- cis (21.9%). The presence of retinal in the eggs of this ascidian is a characteristic shared with the wide range of oviparous vertebrates, although the isomer composition differs between ascidian eggs and vertebrate eggs; in vertebrate eggs, almost all the retinal is in the all- trans form. The egg retinal was bound to a protein complex via a Schiff base linkage. The electrophoretic characteristics of the protein complex were similar to that of egg yolk proteins of oviparous vertebrates. The results presented in this study strongly suggest that, as is found with oviparous vertebrates, retinal in the ascidian eggs is the essential mode of retinoid storage, and is the precursor of photoreceptive pigment chromophores and retinoic acid during development.

Emi1 is required for cytostatic factor arrest in vertebrate eggs

Vertebrate eggs are arrested at metaphase of meiosis II with stable cyclin B and high cyclin B/Cdc2 kinase activity. The ability of the anaphase-promoting complex/cyclosome (APC), an E3 ubiquitin ligase, to trigger cyclin B destruction and metaphase exit is blocked in eggs by the activity of cytostatic factor (CSF) (reviewed in ref. 1). CSF was defined as an activity in mature oocytes that caused mitotic arrest when injected into dividing embryos. Fertilization causes a transient increase in cytoplasmic calcium concentration leading to CSF inactivation, APC activation, cyclin B destruction and mitotic exit. The APC activator Cdc20 is required for APC activation after fertilization. We show here that the APC(cdc20) inhibitor Emi1 (ref. 6) is necessary and sufficient to inhibit the APC and to prevent mitotic exit in CSF-arrested eggs. CSF extracts immunodepleted of Emi1 degrade cyclin B, and exit from mitosis prematurely in the absence of calcium. Addition of Emi1 to these Emi1-depleted extracts blocks premature inactivation of the CSF-arrested state. Emi1 is required to arrest unfertilized eggs at metaphase of meiosis II and seems to be the long-sought mediator of CSF activity.

The Allure of Egg for Sperm

Although sperm binding proteins on the inner layers of the extracellular matrix of vertebrate eggs have received a good deal of attention, sperm attractants in the outer jelly area have been detected in bioassays, but have not been identified. Now Olson et al. have purified a sperm chemoattractant from the egg jelly of Xenopus eggs. The protein, which they name allurin, turns out to have sequence similarity to members of the CRISP (cysteine-rich secretory protein) family. The new results indicate that CRISP family members escort or guide sperm on their voyage from the testes to the egg. The CRISP protein TPX-1 functions to promote adhesion of sperm to Sertoli cells in the testes. During maturation in the epididymis, sperm bind another set of CRISP proteins, the acidic epididymal secretory proteins, or AEGs. And finally, they encounter the new CRISP member, allurin, the first to be identified as a product of the female. The authors suggest that receptors on the surface of the sperm likely sense a gradient in the concentration of allurin that guides the sperm to their target. J. H. Olson, X. Xiang, T. Ziegert, A. Kittelson, A. Rawls, A. L. Bieber, D. E. Chandler. Allurin, a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins. Proc. Natl. Acad. Sci. U.S.A. 98 , 11205-11210 (2001). [Abstract] [Full Text]

Cyclin B/cdc2 induces c-Mos stability by direct phosphorylation in Xenopus oocytes.

The c-Mos proto-oncogene product plays an essential role during meiotic divisions in vertebrate eggs. In Xenopus, it is required for progression of oocyte maturation and meiotic arrest of unfertilized eggs. Its degradation after fertilization is essential to early embryogenesis. In this study we investigated the mechanisms involved in c-Mos degradation. We present in vivo evidence for ubiquitin-dependent degradation of c-Mos in activated eggs. We found that c-Mos degradation is not directly dependent on the anaphase-promoting factor activator Fizzy/cdc20 but requires cyclin degradation. We demonstrate that cyclin B/cdc2 controls in vivo c-Mos phosphorylation and stabilization. Moreover, we show that cyclin B/cdc2 is capable of directly phosphorylating c-Mos in vitro, inducing a similar mobility shift to the one observed in vivo. Tryptic phosphopeptide analysis revealed a practically identical in vivo and in vitro phosphopeptide map and allowed identification of serine-3 as the largely preferential phosphorylation site as previously described (Freeman et al., 1992). Altogether, these results demonstrate that, in vivo, stability of c-Mos is directly regulated by cyclin B/cdc2 kinase activity.