Yolk androgens and embryo sex: maternal effects or confounding factors?

Maternal effects occur when offspring phenotype is affected by environmental factors experienced by the mother and, in egg-laying species, are often mediated via egg resources. There is currently great interest among behavioural ecologists in maternally allocated yolk androgens, especially their relationship with offspring sex and development. Such studies need embryonic tissue for sexing, however, requiring eggs to be incubated (usually for 3 days). Therefore, there are concerns about whether the androgen concentrations assayed reflect those allocated by the mother. In addition, studies showing sex biases in maternal allocation of androgens could be confounded if male and female embryos uptake or metabolise androgens at different rates. We ran a series of experiments using zebra finch (Taeniopygia guttata) eggs to address these potential confounding factors. First we showed, using eggs naturally incubated for up to 5 days, that eggs containing embryos had lower yolk androgen concentrations than eggs that had failed to form embryos. We then tested various hypotheses for this difference using controlled incubation treatments. Our results suggested that (a) embryo development causes the yolk to become progressively more diluted with albumin; and (b) between 3 and 5 days of incubation embryos start uptaking or metabolising androgens. Crucially, we found no decline in yolk androgen concentration at 3 days incubation, and no evidence for sex-specific rates of uptake or metabolism of androgens. This strongly suggests that yolk androgen levels up to 3 days incubation do reflect those allocated by the mother, and that studies of sex biased maternal allocation of yolk androgens are not confounded by sex differences in embryo development.     

Periodic cooling of bird eggs reduces embryonic growth efficiency

For many bird embryos, periodic cooling occurs when the incubating adult leaves the nest to forage, but the effects of periodic cooling on embryo growth, yolk use, and metabolism are poorly known. To address this question, we conducted incubation experiments on eggs of zebra finches (Taeniopygia guttata) that were frequently cooled and then rewarmed or were allowed to develop at a constant temperature. After 12 d of incubation, embryo mass and yolk reserves were less in eggs that experienced periodic cooling than in controls incubated constantly at 37.5 degrees Celsius. Embryos that regularly cooled to 20 degrees Celsius had higher mass-specific metabolic rates than embryos incubated constantly at 37.5 degrees Celsius. Periodic cooling delayed development and increased metabolic costs, reducing the efficiency with which egg nutrients were converted into embryo tissue. Avian embryos can tolerate periodic cooling, possibly by adjusting their physiology to variable thermal conditions, but at a cost to growth efficiency as well as rate of development. This reduction in embryo growth efficiency adds a new dimension to the fitness consequences of variation in adult nest attentiveness.     

Incubation temperature and hemoglobin dielectric of chicken embryos incubated under the influence of electric field

Eggs from a layer-type breeder flock (Baladi, King Saud University) between 61 and 63 weeks of age were used in 3 trials to study the effects of electric field (EF) during incubation on the internal temperature of incubation, and eggs and hemoglobin (Hb) dielectric of chicken embryos at 18 days of age. Dielectric relative permittivity (epsilon') and conductivity (sigma) of Hb were examined in the range of frequency from 20 to 100 kHz. The values of dielectric increment (Deltaepsilon') and the relaxation times (tau) of Hb molecules were calculated. The internal temperature of eggs was measured in empty (following the removal of egg contents) and fertilized eggs in trials 1 and 2, respectively. The level of the EF was 30 kV/m, 60 Hz. EF incubation of embryos influenced the temperature of incubation and electrical properties of Hb molecules and did not influence the temperature of incubation and internal environment of eggs when empty eggs were incubated. EF incubation of fertilized eggs significantly raised the temperature of incubation, egg air cell, and at the surface of the egg yolk by approximately 0.09, 0.60, and 0.61 degrees F, respectively and Hb epsilon', sigma, Deltaepsilon', and tau as a function of the range of frequency of 20 to 100 kHz when compared with their counterparts of the control group. It was concluded that the exposure of fertilized chicken eggs to EF of 30 kV/m, 60 Hz, during incubation altered dielectric properties of Hb and that probably affected cell to cell communication and created the right environment for enhancing the growing process and heat production of embryos consequently increasing the temperature of the internal environment of the egg, and incubation.     

乌骨鸡胚胎发育过程中卵内蛋白质脂肪的变化研究

对乌骨鸡胚胎发育过程中卵内各组成成分(蛋清、蛋黄、胚胎)在不同胚龄(即入孵的第0、3、6、9、12、15、18d和出壳)时蛋白质、脂肪含量变化进行测定与分析．结果表明：在不同胚龄时,蛋清中蛋白质含量显著高于蛋黄,蛋黄中脂肪含量始终高于蛋清;而胚胎中的蛋白质、脂肪逐渐增加。另外,胚胎发育过程中胚胎蛋白质的主要来源是蛋黄中的蛋白质,说明蛋黄是禽类胚胎发育过程中营养需要的主要来源。     

Metabolism of cholesterol in the tissues and blood of the chick embryo

Three artificially inseminated laying White Leghorn hens were given 35-50 micro c of cholesterol-4-(14)C intravenously. Their subsequently produced eggs contained cholesterol-(14)C-labeled yolks. Some of the fertilized eggs were analyzed for cholesterol content and radioactivity. Other eggs were incubated until hatching. The specific activity of the cholesterol contained in the serum and tissues of newly hatched chicks was determined and compared with that of yolk sac, which was taken as representative of egg yolk cholesterol before its metabolic transfer into the chick embryo. The specific activities of cholesterol in intestine, liver, serum, heart, and skeletal muscle and the whole chick were 95-98% of that in yolk sac, but that of brain cholesterol was only 11% of this value. These results indicate that whereas most of the cholesterol in the chick originated from the egg yolk, cholesterol biosynthesis was active in the brain and provided about 90% of its cholestero content. Newly hatched chicks were found to be hyperlipemic compared with older chicks and had fatty livers with a high cholesterol content. Desmosterol was found in 9- and 15-day old chick embryos but not in the newly hatched chicks, in which the only sterol was cholesterol.     

Egg Turning During Incubation has no Effect Upon the Growth of Embryos of Alligator mississippiensis

Alligator eggs are not turned during incubation, instead the embryo adheres to the top inside of the shell. Turning is alleged to shear off the embryo and kill it. Avian egg turning allegedly facilitates embryonic development by stimulating growth of the area vasculosa and minimizing the effects of unstirred yolk and albumen layers. From day 10 to day 45 of incubation, alligator eggs were experimentally turned, gently, through ± 60° in an hourly cycle. This turning regime killed only 6 out of 25 embryos. Compared with unturned controls, no significant effects were observed on the growth, production of extraembryonic fluids or utilization of albumen and yolk for those embryos that survived turning. The protein concentration of amniotic fluid at various stages of alligator development was examined in eggs incubated at 30 and 33°C. The fluid contained very little protein (max <8 mg) at any time: the protein concentration did not change consistently as development progressed. Differences in response to egg turning in birds and reptiles may be associated with the length of the incubation period, the protein content of the albumen and the mechanism of albumen utilization.     

Envelope surface ultrastructure and specific gravity of artificially fertilized Pacific cod Gadus macrocephalus eggs

The envelope surface ultrastructure and specific gravity of artificially fertilized eggs of the Pacific cod Gadus macrocephalus were examined. The unfertilized, demersal and slightly adhesive eggs of G. macrocephalus were almost spherical and had no oil globules. Wrinkled envelope surface with elaborated hexagonal reticulated patterns and type I micropyle were observed under a scanning electron microscope. The adhesiveness of the eggs was lost at the blastodermal‐cap stage after fertilization. The micropylar canal was sealed by secretion of the perivitelline fluid, and the entire surface became rough. Numerous bacilli were deposited at the micropyle and the outer envelope surface at the late germ‐ring stage and at the embryo five‐eighths around the yolk stage. The micropyle was completely deformed at the embryo seven‐eighths around the yolk stage. The specific gravity of the fertilized G. macrocephalus eggs ranged from c. 1·0316 to 1·0454. These values, however, sharply decreased towards the end stages of egg development to produce pelagic larvae. The ultrastructural changes in the micropyle and envelope surface of the G. macrocephalus eggs protected the embryo from microorganism infections and mechanical stress during the long incubation period. The adhesiveness and specific gravity of the eggs influenced their dispersion potential.     

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12–15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.     

Egg size manipulation: a technique for investigating maternal effects on the hatching characteristics of herring

A technique was developed for manipulating egg size of recently fertilized Atlantic herring Clupea harengus embryos. Larvae hatching from eggs with reduced yolk volume were shorter than predicted for the volume of yolk removed. The reduction in both body and yolk mass of hatchlings was less than predicted, partly because larval yolk-sac mass was unaffected by yolk removal.     

Fate of egg white trypsin inhibitor and start of proteolysis in developing chick embryo and newly hatched chick

Trypsin inhibitor and proteolytic activities were studied in incubated eggs, embryos, and newly hatched chicks. After rupture of the secondary seroamniotic suture at 11 days, the trypsin inhibitor content of the albumen gradually passes into the amniotic cavity; from there it is taken up orally by the chick embryo. It is supposed that between 11 and 18 days of embryonic development the trypsin inhibitor passes from the gut to the yolk sac through the vitellointestinal duct. The thin yolk contained only traces of trypsin inhibitor, and the allantoic fluid was entirely free from it. The amylase activity demonstrable in the liquid intestinal contents of the chick embryo indicates the presence of pancreatic secretion. The trypsin inhibitor probably suppresses the proteases not only directly, but also through prevention of the activation of zymogens. Enterocytes of chick embryos showed no morphological indication of the absorption of undigested proteins on histological examination. The cloacal membrane of the newly hatched chick ruptures shortly after the bird has dried up, and the trypsin inhibitor is subsequently eliminated along with the intestinal contents. The intestinal proteolytic enzymes appear immediately afterward. The proteolytic activity appeared regardless of whether the birds were or were not fed. Maximum proteolytic activity was measured in the small intestine of chicks that were fasted for 2 days after hatching. The pattern of proteolytic enzymes as well as their sensitivity to protease inhibitors did not notably differ from that of mammals.     

Is water uptake by reptilian eggs regulated by physiological processes of embryos or a passive hydraulic response to developmental environments?

Moisture availability is critical for successful embryonic development in many organisms. In most oviparous reptiles, for example, water exchange between eggs and the surrounding environment can have substantial fitness consequences, but regulation of this process is unclear. Here, we evaluate whether water uptake by eggs of the lizard Anolis sagrei is regulated by the presence of a live embryo or is a passive hydraulic response to substrate moisture conditions. Many eggs laid in our captive colony were infertile or contained embryos that died during early stages of development, yet these 'dead' eggs continued to gain mass similar to that of 'live' eggs at least during the first half of incubation. Our results suggest that water uptake by eggs is largely a passive hydraulic process during the first half of incubation, but active regulation by embryos may be necessary during latter stages. Maternal effects (e.g., deposition of salts into yolk) could influence this passive process during early incubation.     

PCR扩增同源重组片段筛选转基因胚胎

使用小鼠乳清酸蛋白基因（ＷＡＰ）启动子控制下的人集落刺激因子（Ｇ－ＣＳＦ）基因为显微注射片段,采用ＰＣＲ方法检测了转基因胚,为消除ＰＣＲ扩增中的假阳性结果,构建了两个具有部分同源性的亚克隆片段进行共注射．ＰＣＲ扩增片段跨越这一同源区域,仅当注射的片段能够整合并发生正常重组,转基因整合胚才能以相对高的比例扩增出特异性片段．结果表明,１、２和８细胞期的阳性率分别为１１．１％、５５．５％和４４．４％,较常规ＰＣＲ检测获得更为明确的结论,为在大动物转基因胚胎检测提供了依据     

Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells

We have examined the role of germline-specific chromosomal determinants of development in the mouse. Studies were carried out using aggregation chimaeras between androgenetic----fertilized embryos and compared with similar parthenogenetic----fertilized chimaeras. Several adult chimaeras were found with parthenogenetic cells but none were found with androgenetic cells. Analysis of chimaeras at mid-gestation showed that parthenogenetic cells were detected in the embryo and yolk sac but that androgenetic cells were found only in the trophoblast and yolk sac and not in the embryo. The contribution of parthenogenetic cells to the embryo and yolk sac was increased by aggregating 2-cell parthenogenetic and 4-cell fertilized embryos but the contribution of parthenogenetic cells in extraembryonic tissues remained negligible even after aggregation of 4-cell parthenogenetic and 2-cell fertilized embryos. Furthermore, parthenogenetic cells were primarily found in the yolk sac mesoderm and not in the yolk sac endoderm. These results suggest that maternal chromosomes in parthenogenetic cells permit their participation in the primitive ectoderm lineage but these cells are presumably eliminated by selective pressure or autonomous cell lethality from the primitive endoderm and trophectoderm lineages. Conversely paternal chromosomes in androgenetic cells confer opposite properties since the embryonic cells can be detected in the trophoblast and the yolk sac but not in the embryos, presumably because they are eliminated from the primitive ectoderm lineage. The spatial distribution of cells with different parental chromosomes may occur partly because of differential expression of some genes, such as proto-oncogenes, and partly due to their ability to respond to a variety of diffusible growth factors.     

Entamoeba histolytica: genetic control of susceptibility in chicken eggs.

Five strains of axenized Entamoeba histolytica were established in eggs from chickens of different genetic backgrounds. The eggs of outbred and inbred chickens varied in the proportion of embryos from which the strains of E. histolytica were recovered and there were variations in the numbers of trophozoites recovered from individual positive eggs. It was possible, however, to find individual, single-mated hens which laid only uniformly susceptible or resistant eggs. Susceptibility and resistance to E. histolytica could be demonstrated in embryonated eggs derived both from outbred heterogenous and in highly inbred chickens but eggs from a genetically stable source were relatively constant in the distribution of susceptible and resistant embryos. Except for gross hydrocephalus, tissue reactions were not observed in the chick embryos or in the chorioallantoic, amniotic, or yolk sac membranes. The yolk sac inoculation route and 37 C incubation temperature provided the optimal survival conditions for the amoebae. Infection with avian leukosis virus and interferon production did not seem to play a role in susceptibility and resistance. Neither the presence of E. histolytica cross-reacting, maternal antibody from the hen transmitted transovarially via the yolk sac, or other antiamoebic substances appeared to play a role in the resistance observed in nonsusceptible eggs. Multiple alternating passages of trophozoites in hen's eggs/TPS-1 broth led to an increase in the number of positive embryos and a decrease in embryo deaths.     

Effects of incubation temperature on growth and development of embryos ofAlligator mississippiensis

Summary Eggs ofAlligator mississippiensis were incubated at 30 °C and 33 °C throughout incubation up to hatching. Every four days several eggs were opened and the albumen, yolk and extra-embryonic fluids removed and weighed. The embryo was removed and fixed prior to being staged, weighted and measured for various morphometric criteria. Development at 33 °C was accelerated compared with 30 °C in terms of yolk and albumen utilization and embryo growth. Significant losses in yolk mass did not occur until stage 22 at 33 °C but occurred at stage 18 at 30 °C. Different patterns in growth were observed in embryos at the two temperatures at similar morphological stages: between stages 18 and 22 embryos at 33 °C were smaller (in mass and length) compared with embryos at 30 °C despite being morphologically similar. The differences in growth and physiology between embryos at 30 °C (females) and 33 °C (males) were dependent on incubation temperature but not sex. Incubation at 33 °C accelerated both growth and development inAlligator; initially morphogenesis was accelerated by the higher temperature but later, growth rate was accelerated.     

Accumulation of calcium and phosphorus in pigeon (Columba livia) embryos

Summary Calcium and phosphorus were measured in the yolk and albumen of fertile pigeon (Columba livia) eggs incubated for 0–17 days, and in embryos and hatchlings. Shell provided most of the calcium for skeletal mineralization of the embryos, whereas phosphorus was derived from the yolk and albumen. Mobilization of calcium from the shell to the embryo commenced at approximately day 11 of incubation, accumulating both in the embryo and the yolk sac. There was 1.4 times more calcium in squab yolk sacs than that contained in newly laid egg yolks. The results suggest that whereas general patterns of calcium and phosphorus accumulation during embryogenesis in altricial birds closely resemble those of precocial birds, calcium mobilization from the shell begins later, proceeds at a slower rate and results in a less mineralized hatchling.CIDA/NSERC Visiting Research Associate Permanent address: Department of Animal Science, University of Peradeniya, Peradeniya, Sri Lanka     

Isolation of cDNA Clones for Epidermis-Specific Genes of the Ascidian Embryo

The present investigation was conducted to isolate cDNA clones that correspond to epidermis-specific genes of the ascidian embryo. When cleavage of fertilized eggs of Halocynthia roretzi is blocked by treatment with cytochalasin B and the arrested eggs are reared as one-celled embryos for about 30 hr, they develop features of differentiation of the epidermis only. Translation in vitro of poly(A)⁺ RNA from cleavage-arrested embryos and analysis of the products by two-dimensional gel electrophoresis revealed several predominant polypeptides that were not detected in a similar analysis of fertilized eggs, suggesting the appearance of epidermis-specific mRNAs in cleavage-arrested embryos. A cDNA library was constructed from arrested one-celled embryos. Differential screening of the library with a total cDNA probe from cleavage-arrested embryos and with a similar probe from fertilized eggs yielded eight different cDNA clones specific for the cleavage-arrested embryos. Northern blot analysis revealed that the mRNAs that corresponded to these cDNAs were present in normal tailbud embryos. In addition, in situ hybridization of whole-mount specimens showed that the mRNAs were restricted to the epidermal cells of tailbud embryos.     

Characterization of yolk platelets isolated from developing embryos of Arbacia punctulata

Yolk platelets, a major organelle of sea urchin eggs and embryos, were isolated from Arbacia punctulata and biochemically characterized over the course of development to the pluteus stage. Fractionation by sucrose gradient centrifugation revealed yolk platelets in two major density classes. The low-density yolk platelet fraction could be obtained as a very homogeneous preparation and was highly enriched in acid phosphatase activity, while depleted of mitochondrial (cytochrome c oxidase) and plasma membrane (phosphodiesterase) marker enzymes. The chemical composition of low-density yolk platelets prepared from eggs and embryos at various stages of development remained unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and protein. However, analysis of the major yolk platelet glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a number of stage-specific changes. These glycoproteins were found to be major glycoproteins of crude embryo lysates and were predominantly of the polymannose, N-linked type. The predominance of polymannose-type glycoproteins in yolk platelets was further demonstrated by their staining with concanavalin A-colloidal gold in Lowicryl-embedded sections of embryos. These studies represent the first systematic biochemical characterization of intact yolk platelets and the changes in them during early embryonic development.     

Removal of vegetal yolk causes dorsal deficencies and impairs dorsal-inducing ability of the yolk cell in zebrafish.

To examine the nature of cytoplasm determinants for dorsal specification in zebrafish, we have developed a method in which we remove the vegetal yolk hemisphere of early fertilized eggs (vegetal removed embryos). When the vegetal yolk mass was removed at the 1-cell stage, the embryos frequently exhibited typical ventralized phenotypes: no axial structures developed. The frequency of dorsal defects decreased when the operation was performed at later stages. Furthermore, the yolk cell obtained from the vegetal-removed embryos lost the ability to induce goosecoid in normal blastomeres while the normal yolk cell frequently did so in normal and vegetal-removed embryos. These results suggested that the vegetal yolk cell mass contains the dorsal determinants, and that the dorsal-inducing ability of the yolk cell is dependent on the determinants.