Differential regulation of G protein subunit expression in mouse oocytes, eggs, and early embryos

Pertussis toxin ADP-ribosylation and Western blot analysis using G protein-specific antibodies were used to study G protein expression in mouse oocytes, eggs, and early embryos. A pertussis toxin (PT) substrate of about 40 kDa was observed in all stages, but its level was stage dependent. It decreased dramatically between germinal vesicle stage oocytes and unfertilized eggs, remained relatively constant through the early 2-cell stage, and then declined again with each cell division, reaching the lowest level at the 8- to 16-cell stage. Its level, or perhaps that of a different substrate, then increased at the blastocyst stage. Western blot analysis with antisera to the G protein alpha subunit indicated that the decrease between germinal vesicle stage oocytes and unfertilized eggs was less pronounced for the alpha subunit itself than for the PT substrate. Antisera to G protein beta subunit revealed that the difference in the amount of this subunit in germinal vesicle-stage oocytes versus unfertilized eggs was even greater than that of the PT ADP-ribosylation substrate. These results suggest that during oocyte maturation G protein beta gamma levels decline to a greater extent than alpha levels. Additional evidence supporting this hypothesis was obtained by showing that addition of exogenous beta gamma to unfertilized egg preparations increased the amount of PT substrate. These results indicate that G protein subunit expression is differentially regulated during oocyte maturation.     

Outer egg coats of the marsupial conceptus: secretion and protein composition

Little is known of the composition of the outer egg coats. We aimed to quantify secretion during embryonic development, identify precursor secreting cells and investigate protein composition. The study was based on 259 egg coats and 14 reproductive tracts of 104 T. vulpecula undergoing natural and induced cycles and 341 coats from 35 Sminthopsis macroura undergoing natural cycles. Following PAGE, Western blotting, and amino acid sequencing of egg coats, the short peptide sequences obtained from T. vulpecula and S. macroura coats were found to be dissimilar to each other and to any known protein. However, in T. vulpecula, S. macroura coat polyclonal antibody cross-reacted with coat precursors, suggesting similar epitopes, and showed mucoid precursors in secretory cells in oviduct epithelia and shell precursors in glands in the utero-tubal junction and uterus. Immuno-electron microscopy located shell coat precursors in various previously unidentified cell types, including pre-ovulatory apoptotic cells, early post-ovulatory holocrine cells, and milk-producing cells, found at blastocyst stages. Ultrastructural and quantitative volumetric analysis of the intact shell coat suggested a second wave of secretion at the blastocyst stages in T. vulpecula. Despite differences in protein composition, it was concluded that marsupial egg coats are homologous to each other because of similarities in ultrastructure and time and location of secretion.     

Uptake of microperoxidase by segmenting rat ova in vitro

Summary Uptake and incidence of microperoxidase (Sigma) in the rat ova during cleavage at the 1-, 2-, 8-cell and blastocyst stages were studied after 30 min incubation with the enzyme (molecular weight of 1,900 and size of molecule of 2 nm).Evidence was furnished of the presence of a small amount of reaction product of microperoxidase in the zona pellucida and its local concentration in some parts of the perivitelline space. The larger part of surface of the ovum, however, was free of microperoxidase. In the cytoplasm microperoxidase was found in pinocytotic vesicles, less frequently in large vacuoles and starting with the eight-cell stage in secondary lysosomes. The largest amount of microperoxidase was ascertained at the stage of blastocyst, chiefly in the cells of the trophoblast. In the cells of the embryoblast, on the contrary, microperoxidase was found but occasionally. The reaction product of microperoxidase was also present in the intercellular space of ova in negligible amount, and likewise on the side of blastocysts' cavity. In comparison with the ingestion of horseradish peroxidase the incidence of microperoxidase in the segmenting rat ova was less frequent.     

Differential protein expression in Colletotrichum acutatum: changes associated with reactive oxygen species and nitrogen starvation implicated in pathogenicity on strawberry

The cellular outcome of changes in nitrogen availability in the context of development and early stages of pathogenicity was studied by quantitative analysis of two-dimensional gel electrophoresis of Colletotrichum acutatum infecting strawberry. Significant alterations occurred in the abundance of proteins synthesized during appressorium formation under nitrogen-limiting conditions compared with a complete nutrient supply. Proteins that were up- or down-regulated were involved in energy metabolism, nitrogen and amino acid metabolism, protein synthesis and degradation, response to stress and reactive oxygen scavenging. Members belonging to the reactive oxygen species (ROS) scavenger machinery, superoxide dismutase and glutathione peroxidase, were up-regulated at the appressorium formation stage, as well as under nitrogen-limiting conditions relative to growth with a complete nutrient supply, whereas abundance of bifunctional catalase was up-regulated predominantly at the appressorium formation stage. Fungal ROS were detected within germinating conidia during host pre-penetration, penetration and colonization stages, accompanied by plant ROS, which were abundant in the apoplastic space. Application of exogenous antioxidants quenched ROS production and reduced the frequency of appressorium formation. Up-regulation in metabolic activity was detected during appressorium formation and nutrient deficiency compared with growth under complete nutrient supply. Enhanced levels of proteins related to the glyoxylate cycle and lipid metabolism (malate dehydrogenase, formate dehydrogenase and acetyl-CoA acetyltransferase) were observed at the appressorium formation stage, in contrast to down-regulation of isocitrate dehydrogenase. The present study demonstrates that appressoria formation processes, occurring under nutritional deprivation, are accompanied by metabolic shifts, and that ROS production is an early fungal response that may modulate initial stages of pathogen development.     

Uptake of peroxidose by the cockroach central nervous system

The central nervous system of the cockroach has been incubated with solutions of an exogenous tracer substance, horseradish peroxidase, and the sites of its penetration and uptake have been studied by electron microscopy. When the nervous tissue is intact, or intact but stretched, the peroxidase is taken up throughout the neural lamella and also penetrates short distances into the extracellular space between adjacent perineurial cells. When the ganglia have been desheathed, reaction product for peroxidase is found in the neural lamella, perineurial cells, and within the cytoplasmic substance of the glial cells adjacent to the desheathed area. This uptake of peroxidase by the injured glial cells in desheathed preparations may reflect an alteration in the normal diffusion pathway from the external medium to the axonal surfaces.     

尖唇散白蚁胚胎发育激光共聚焦扫描显微镜观察

【目的】本研究旨在探究尖唇散白蚁Reticulitermes aculabialis胚胎在不同发育阶段的变化特征。【方法】每日收集尖唇散白蚁的卵,并固定其胚胎发育状态,采用DAPI染剂对白蚁胚胎进行染色,通过激光共聚焦扫描显微镜观察记录尖唇散白蚁胚胎在不同发育阶段的形态特征。【结果】在25℃下尖唇散白蚁胚胎发育过程历经25~30 d,按照发育特征将其划分为12个阶段。胚胎发育早期,卵黄细胞均匀分布在卵内部,卵内细胞核向卵的中间浓缩,在细胞到达卵的后表面时形成浓缩的囊胚细胞作为胚盘;胚胎发育中期,胚胎开始进行“反转型”的囊胚运动,头部和前后轴从后极到前极反转,胚带出现明显的“双弯”结构。胚胎发育中后期,胚胎变宽,内部器官逐渐开始发育,出现明显的伸长与分节;胚胎发育后期,附肢发育明显,内部器官发育成熟。【结论】尖唇散白蚁胚胎发育过程历经12个阶段,属于短胚带型,胚带出现“双弯”结构,发育中期经历两次囊胚反转。本研究为真社会性昆虫白蚁的胚胎发育过程提供了形态学和生物学依据。     

Determination of protein concentration in cleaving mammalian ova by the capillary spectrophotometry method]

A micromodification of the Lowry's method is described which allows to measure reliably the protein content in 5-20 embryos of white rats and CBA mice. Differences in the protein content in rat embryos at the stages of 2 blastomeres and blastocyst were shown to be statistically unreliable (20.2 +/- 1.4 and 18.9 +/- 1.3 ng, resp.). The protein content in the mouse embryos at the same two stages differs reliably (19.1 +/- 1.1 and 22.0 +/- +/- 1.7 ng, resp.). The protein content in zona pellucida does not differ reliably from those in rat embryos both at the stages of 2 blastomeres (4.5 ng) and blastocyst (2.3 ng). The protein content in embryos devoid of zona pellucida decreased after 3 hours incubation in the medium 199 at 37 degrees at the stages of morula and blastocyst by 17-20% in rats and by 50% in mice. Addition of 1% serum albumin to the incubation medium did not prevent the partial "loss" of protein by the embryos. The protein content in the rat and mouse embryos at the stage of 2 blastomeres suffered no changes under long-term incubation in the medium 199.     

Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos

In vitro techniques for production of bovine embryos including in vitro oocyte maturation (IVM), fertilization (IVF) and culture (IVC) are becoming increasingly employed for a variety of research purposes. However, decreased viability following cryopreservation by conventional methods has limited commercial applications of these technologies. A practical alternative to facilitate transport would be to arrest development by chilling without freezing. The present research was undertaken to evaluate chilling sensitivity of IVM-IVF embryos at different stages of development, and to determine possible beneficial effects of cysteamine treatment during IVM, previously shown to enhance embryo development in culture, on survival following chilling at different stages. Embryos produced by standard IVM-IVF-IVC methods were chilled to 0 degrees C for 30 min at 2-cell (30-34 h post-insemination, hpi), 8-cell (48-52 hpi) or blastocyst (166-170 hpi) stages. Viability after chilling was assessed by IVC with development to expanded blastocyst stage determined on days 7 and 8 post-insemination (pi) and hatching blastocyst stage determined on days 9 and 10 pi. Control embryos at the same stages were handled similarly, but without chilling, and development during culture similarly assessed. The effect of cysteamine supplementation (100 microM) of the IVM medium was determined for both chilled and non-chilled (control) embryos. Cysteamine supplementation during IVM had no significant effect on oocyte maturation or fertilization, but increased the proportions of oocytes developing to blastocyst stage by day 7 (13.7+/-0.9% versus 7.2+/-0.9%; P<0.05), total blastocysts (20.5+/-0.9% versus 15.3+/-1.3%; P<0.05), and hatching blastocysts (16.8+/-1.6% versus 12.0+/-1.5%; P<0.05). The greater survival in terms of hatching (78.6+/-7.0) following chilling of blastocysts produced by IVM-IVF of oocytes matured in media supplemented with cysteamine offers promise for applications requiring short-term storage to facilitate transport of in vitro produced bovine embryos.     

Interleukin-1 beta induces autophagy of mouse preimplantation embryos and improves blastocyst quality

Autophagy is one of the basic cellular mechanism during preimplantation development of mammalian embryos, and it plays crucial role in several physiological processes. It is induced by interleukin (IL)-1β in mammalian cells. Our present study shows that IL-1β is important for autophagy activation in embryo development. Our in vitro culture system analysis shows effect of IL-1β in medium on the development of mouse embryos and it was found to be concentration dependent. A preimplantation embryo culture using medium containing IL-1β did not improve cleavage and blastocyst development rates of mouse embryos; however, blastocyst quality was significantly improved by increasing total cell number, especially in supplementary 20 ng/mL IL-1β. Furthermore, autophagy activation mainly occurs in 2 to 4 cell embryo and blastocyst, 20 ng/mL IL-1β into culture medium can effectively enhance levels of messenger RNA and protein of autophagy-related-factors in 2 to 4 cell embryos and blastocyst, while these factors reduce in VGX-1027 (IL-1β inhibitor) groups that also reduce the quality of blastocyst. Effects of IL-1β on the development of embryo reduced in 20 ng/mL IL-1β supplemented group when 5 mM 3-methyladenine (3-MA) was also added, which used to inhibit autophagy activation in endogenous PtdIns3Ks signal pathway. Our current results show that exogenous IL-1β can effectively induce autophagy in mouse embryos at stages of 2 to 8 cell and blastocyst, that also help to improve the quality of blastocyst.     

Cyclooxygenases and prostaglandin E synthases in preimplantation mouse embryos

Prostaglandin E2 (PGE2) is shown to be essential for female reproduction. Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandin synthesis from arachidonic acid and exists in two isoforms: COX-1 and COX-2. Prostaglandin E synthase (PGES) is a terminal prostanoid synthase and can catalyse the isomerization of the COX product PGH2 to PGE2, including microsomal PGES-1 (mPGES-1), cytosolic PGES (cPGES) and mPGES-2. This study examined the protein expression of COX-1, COX-2, mPGES-1, cPGES and mPGES-2 in preimplantation mouse embryos by immunohistochemistry. Embryos at different stages collected from oviducts or uteri were transferred into a flushed oviduct of non-pregnant mice. The oviducts containing embryos were paraffin-embedded and processed for immunostaining. COX-1 immunostaining was at a basal level in zygotes and a low level at the 2-cell stage, reaching a high level from the 4-cell to blastocyst stage. COX-2 immunostaining was at a low level at the zygote stage and was maintained at a high level from the 2-cell to blastocyst stages. A low level of mPGES-1 immunostaining was observed from the zygote to 8-cell stages. The signal for mPGES-1 immunostaining became stronger at the morula stage and was strongly seen at the blastocyst stage. cPGES immunostaining was strongly observed in zygotes, 2-cell and 8-cell embryos. There was a slight decrease in cPGES immunostaining at the 4-cell, morula and blastocyst stages. mPGES-2 immunostaining was at a low level from the zygote to morula stages and at a high level at the blastocyst stage. We found that the COX-1, COX-2, mPGES-1, cPGES and mPGES-2 protein signals were all at a high level at the blastocyst stage. PGE2 produced during the preimplantation development may play roles during embryo transport and implantation.     

Postimplantation development of mitomycin C-treated mouse blastocysts

Treatment of morula-stage mouse embryos with mitomycin C (0.004-0.5 microgram/ml) in vitro resulted in a decrease in the number of inner cell mass (ICM) cells at the blastocyst stage. The trophectoderm population was reduced only at the highest dosage (0.5 microgram/ml) tested. Postblastocyst development in vitro was retarded: Fewer embryos formed trophoblastic outgrowth, and the ICM was poorly developed. The embryo transfer experiments demonstrated that a reduction in ICM cell numbers diminished the potential of embryogenesis. The presence of a sufficient number of trophoblasts and ICM cells in the blastocyst is therefore a prerequisite for successful implantation and embryogenesis. The mitomycin-treated blastocysts with only 70% of normal ICM cells developed to egg cylinders that were about half normal size, but by days 12-14 the body size of the surviving embryo was similar to that of the control embryo. Morphogenesis was retarded during the early organogenesis stages, but only a slight delay was seen in the treated embryo on day 12. Such observation strongly suggests that a restorative phase of growth and morphogenesis has occurred during the immediate postimplantation period.     

Oct4 protein remains in trophectoderm until late stages of mouse blastocyst development

Oct-4, the marker of pluripotent cells, is crucial for murine preimplantation development. During the formation of the blastocyst Oct-4 is downregulated in the trophectoderm (TE) and its expression becomes restricted to the inner cell mass (ICM). In order to determine the exact timing of the disappearance of Oct-4 protein from TE we analyzed the localization and level of Oct-4 at different stages of blastocyst development. The presence of Oct-4 protein was determined by immunohistochemistry using confocal microscopy. We found that the downregulation of Oct-4 protein in TE of mouse blastocysts progresses gradually during development, and Oct-4 protein persists in some of the TE cells at least until the expanded blastocyst (120-140 cells) stage. Our findings indicate that the switching-off of the Oct-4 expression is not necessary for the trophectoderm formation. The complete elimination of Oct-4 protein from TE occurs at the period of blastocyst implantation, when lack of Oct-4 is required for the proper functioning of the trophectoderm.     

Effect of egg composition on the developmental capacity of androgenetic mouse embryos.

Analysis of the developmental capacities of androgenetic and gynogenetic mouse embryos (bearing two paternal or two maternal pronuclei, respectively) revealed a defect in blastocyst formation of androgenetic, but not gynogenetic, embryos that was a function of the maternal genotype. Androgenetic embryos constructed using fertilized eggs from C57BL/6 or (B6D2)F1 mice developed to the blastocyst stage at frequencies similar to those previously reported, whereas androgenetic embryos constructed with fertilized eggs from DBA/2 mice developed poorly, the majority failing to progress beyond the 16-cell stage and unable to form a blastocoel-like cavity, regardless of whether the male pronuclei were of C57BL6 or DBA/2 origin. This impaired development was observed even in androgenetic embryos constructed by transplanting two male pronuclei from fertilized DBA/2 eggs to enucleated C57BL/6 eggs, indicating that the defect cannot be explained as the lack of some essential component in the DBA/2 cytoplasm that might otherwise compensate for androgeny. Rather, the DBA/2 egg cytoplasm apparently modifies the incoming male pronuclei differently than does C57BL/6 egg cytoplasm. Several specific alterations in the protein synthesis pattern of DBA/2 androgenones were observed that reflect a defect in the regulatory mechanisms that normally modulate the synthesis of these proteins between the 8-cell and blastocyst stages. These results are consistent with a model in which cytoplasmic factors present in the egg direct a strain-dependent modification of paternal genome function in response to epigenetic modifications (genomic imprinting) established during gametogenesis and indicate that preimplantation development can be affected by these modifications at both the morphological and biochemical levels.     

Recovery of penetration ability in protease-treated zona-free mouse eggs occurs coincident with recovery of a cell surface 94 kD protein.

Previous studies have demonstrated that protease treatment of zona-free mouse eggs impairs sperm-egg interaction (Boldt et al.: Biol Reprod 39:19-27, 1988) and causes modification of a 94 kD egg plasma membrane protein (Boldt et al., Gamete Res 23:91-101, 1989). In this report, the ability of eggs to recover penetration ability following protease treatment was examined. Zona-free mouse eggs were isolated and treated with either trypsin or chymotrypsin (1 mg/ml, 20 min), then cultured for 0, 3, or 6 hr before insemination. Eggs cultured for 3 or 6 hr displayed significantly higher penetration levels than eggs inseminated immediately after protease treatment, indicating a recovery of penetration ability during the 3 or 6 hr incubation period. The recovery of penetration ability was not blocked by inclusion of cyclohexamide (50 micrograms/ml) during the 3 or 6 hr culture period, indicating that protein synthesis was not required for recovery of fusion ability. Cell surface radiolabeling studies with 125I revealed that a 94 kD cell surface protein was lost immediately following trypsin or chymotrypsin treatment but was found on the egg surface after the 3 or 6 hr recovery period. Recovery of the 94 kD egg surface protein occurred in the presence of cyclohexamide, and metabolic radiolabeling studies with 35S-methionine confirmed that synthesis of a 94 kD protein was blocked by cyclohexamide. These results suggest that the recovery of penetration ability after protease treatment of zona-free eggs is due to recovery of the 94 kD cell surface protein, providing further evidence for the involvement of the 94 kD protein in sperm-egg interaction.     

Biological Relationship of Rotyleinchulus borealis on Several Plant Cultivars

The embryogenic development of Rolylenchulus borealis, at 24-26 C, was completed on corn, in 12-15 days, and the life-cycle of the nematode from egg to egg required 35-40 days at 20-25 C. Juveniles remained in the soil as preinfective stages for 17-19 days before becoming adults. Only immature vermiform and swollen egg-laying females were found attached to corn roots. Eggs were laid in a gelatinous matrix on the root surface; the number of eggs per egg mass was 45 ± 28 on corn roots. Bean, green pea, potato, sorghum, and sweet potato were also found to be hosts of R. borealis. The nematode established a permanent feeding site on corn root in an endodermal cell that became hypertrophied. Pericyclic cells close to the feeding site showed granular cytoplasm and nuclei with hypertrophied nucleoli. A cell wall ingrowth was also noted around the area of stylet penetration into the endodermal cell.     

Study of yolk precursor transport in the avian ovary with the use of horseradish peroxidase.

Ovaries of adult Japanese quails were exposed in vivo to the exogenous protein horseradish peroxidase (HRP) for varying lengths of time to investigate ultrastructurally the permeability of the wall of these follicles, the protein uptake capacity of granulosa and oocyte and the kinetics of protein uptake in different stages. There is a sudden increase in permeability of the follicle wall from previtellogenesis to vitellogenesis. This is not due to a loss of sealing (tight) junctions in the granulosa cell layer, but is probably related to a permeability change in the basement membrane. The transition from the slow growth phase to the rapid growth during vitellogenesis is accompanied by a limited widening of the intercellular channels and the concomitant development of a complex endocytotic apparatus in the ooplasm. The slowing down of yolk deposition during the last day before ovulation is accompanied by a narrowing of the intercellular channel width. The granulosa cells show a high intracellular HRP uptake during intermediary yolk formation. Transcytosis through the granulosa cannot be excluded but is probably a minor pathway at certain stages. The light microscopically detectable uptake of HRP by the oocyte coincides with the start of exogenous vitellogenesis. After 90 sec of exposure to HRP (intravenous injection) the tracer can be found in the intercellular channels of the granulosa and in superficially located yolk spheres. On the other hand it takes 10 min for the tracer to traverse the cortex of the oocyte.     

Comparison of hybrid and purebred in vitro-derived cattle embryos during in vitro culture

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P<0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P<0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.     

鳙鱼ARISTICHTHYS NOBILIS早期发育的一些生物化学上的变化

本实验第一部分对鳙鱼卵及胚胎在不同的发育时期蛋白质合成的速度进行了研究,实验采用微量克氏定氮法,对鳙卵从未受精卵到受精卵、尾芽期和出膜期,共测定了十三个不同的发育时期。实验结果表明了在鱼的不同胚胎发育时期蛋白质合成速度有着明显的差异。 实验的第二部分对鳙鱼胚胎发育过程氨基酸组成和氨基酸含量进行了测定,其结果显示出鳙鱼卵和胚胎的各发育时期氨基酸的组分十分相似,但氨基酸的含量在各个时期是有差异的。