The encysted dormant embryo proteome of Artemia sinica

The possibility of the brine shrimp Artemia to produce dormant embryo (cysts) in diapause is a key feature in its life history. In the present study, we obtained a proteomic reference map for the diapause embryo of Artemia sinica using two-dimensional gel electrophoresis with a pH range of 4-7 and a molecular weight range of 10-100 kDa. Approximately 233 proteins were detected, and 60 of them were analyzed by capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Of these, 39 spots representing 33 unique proteins were identified, which are categorized into functional groups, including cell defense, cell structure, metabolism, protein synthesis, proteolysis, and other processes. This reference map will contribute toward understanding the state of the diapause embryo and lay the basis and serve as a useful tool for further profound studies in the proteomics of Artemia at different developmental stages and physiological conditions.

中华卤虫滞育卵发育和重金属刺激相关蛋白质组学研究

卤虫(Artemia)是一种广温、耐高盐的小型甲壳动物，广泛分布于内陆盐湖和沿海盐田中。卤虫的无节幼体作为重要的蛋白优质饵料，被广泛的应用于水产养殖生产。卤虫具有特殊的生物学特性，是研究甲壳动物胚胎发育的良好的实验材料，同时也是一种研究动物抗逆机制的模式动物。卤虫有卵生和卵胎生两种繁殖后代的方式，当环境条件适宜时，卤虫倾向于采取卵胎生方式，即直接产生无节幼体；而在恶劣的环境条件下，卵生方式占主要地位，产生处于滞育状态的、具有复杂外壳的休眠卵。卤虫的滞育卵具有独特的生物学特性和特殊的生理生化特点。其发育停滞，细胞分裂停止，酶活力下降，代谢活动受到抑制并可耐受各种极端恶劣环境，如缺氧、低温、紫外线、干燥等。即使在最适的环境中滞育卵的孵化率也很低，只有受到某些特定的非生物信号的刺激才自能终止这种滞育状态，恢复生理代谢；当环境条件适宜时，能够继续发育孵化成无节幼体。因此，卤虫的滞育卵在卤虫的整个生活史中占有重要的地位。另一方面，卤虫是极端环境生物，能够抵抗各种恶劣环境胁迫刺激，因此是研究抗逆机理的良好的实验动物。 本论文利用蛋白质组学技术，研究了卤虫滞育卵及滞育卵发育过程中的蛋白质组表达情况，并研究了卤虫幼体在重金属刺激后蛋白表达的变化情况。得到如下结果： 建立了中华卤虫滞育卵可溶性总蛋白的双向凝胶电泳对照图谱。在pH 4–7、分子量10-100 kDa范围内，检测到约 233个蛋白点，并利用高效液相色谱-质谱联用（LC-ESI-MS/MS）技术鉴定了其中的48个丰度较大及感兴趣的蛋白点，根据这些蛋白的生物学功能进行分类，功能类别包括细胞防御蛋白、抗氧化蛋白、细胞骨架蛋白、代谢相关蛋白等。在卤虫滞育卵中共分离鉴定到6个分子量和等电点存在差异的小热休克蛋白p26的异构体，生物信息学分析表明该蛋白有三种不同的功能位点，分别是蛋白激酶C磷酸化位点，Casein 激酶II磷酸化位点及 N-myristoylation 位点。 采用低温脱水的方法对滞育卵进行激活刺激，并对活化卵和滞育卵蛋白表达图谱进行了对比分析。结果表明对卤虫滞育卵的激活刺激引起了其蛋白表达的明显变化。活化卵图谱中蛋白点总数比滞育卵中明显增多，特别是在pI<5.5范围内。约70个蛋白点在激活刺激后上调表达，包括部分只在激活卵中表达的蛋白；25个下调表达，包括部分只在滞育卵中表达的蛋白；其余约60％（占滞育卵蛋白点数目百分比）的蛋白点表达量基本恒定。热休克蛋白家族、抗氧化蛋白家族成员等蛋白变化明显，小热休克蛋白p26、小热休克蛋白ArHsp21蛋白以及过氧化物还原酶异构体在激活卵中特异表达。 活化卵孵化过程中不同发育时期的蛋白表达又呈现出不同的特点，分别在孵化后6h、12h、18h和24h的蛋白质组学图谱上检测到267、285、195和210个蛋白点。孵化后6h和12h休眠卵蛋白表达个数相对较多，与胚胎发育过程中的器官发生和剧烈的形态变化相适应；孵化后18h和24h休眠卵蛋白表达明显下降，部分蛋白的表达关闭，部分蛋白开始富集表达。 利用双向凝胶电泳技术分析了中华卤虫幼体受到急性硫酸铜刺激后的蛋白表达变化情况。通过图谱对比分析，检测到了5mM硫酸铜刺激24h后，卤虫幼体中14个差异表达的蛋白点。利用LC-ESI-MS/MS技术鉴定了其中的7个蛋白，其中3个蛋白上调表达，分别是热休克蛋白70（7.5倍）, 肌动蛋白（2.3倍）和伴侣分子亚基1（3.0倍）。3个蛋白下调表达，分别是：精氨酸激酶（2.8倍）, 延伸因子2 (2.0倍） 和富含甘氨酸蛋白(2.0倍）。硫酸铜刺激后特异表达的一个蛋白被鉴定为过氧化物还原酶(Peroxiredoxin，Prx)。根据质谱检测提供的蛋白肽段信息和其他生物过氧化物还原酶保守氨基酸序列设计简并引物，结合RACE技术，从中华卤虫幼体中克隆到了过氧化物还原酶基因，该基因的cDNA全长为756个碱基，其中开放阅读框为594个碱基，编码198个氨基酸，其蛋白理论分子量为22.0 kDa，理论等电点为6.98。多序列比对结果显示中华卤虫Prx基因的推导氨基酸序列与美国卤虫和中国对虾的同源性高达98%和94%。实时荧光定量PCR结果显示，硫酸铜刺激后，该基因在卤虫无节幼体中的转录水平明显升高，在24h达到正常水平的3.0倍。

Les voies de signalisation utérines à l'émergence de la diapause embryonnaire chez le vison américain

La diapause embryonnaire se manifeste par un arrêt réversible du développement embryonnaire durant la période de préimplantation et induit un retard de l’implantation. Chez le vison américain, une diapause embryonnaire obligatoire caractérise chaque gestation. Si les mécanismes de contrôle de la diapause embryonnaire obligatoire chez cette espèce sont bien connus, le rôle utérin impliqué dans la réactivation de l’embryon demeure, quant à lui, encore inconnu. Le sujet de ce doctorat a consisté dans un premier temps à explorer l’environnement utérin à la sortie de la diapause embryonnaire afin de caractériser, dans un deuxième temps, les principaux acteurs utérins qui provoquent la réactivation de l’embryon. Nous avons effectué une analyse du transcriptome utérin à l’émergence de la diapause embryonnaire ce qui a permis de construire une librairie de 123 séquences d’ADNc utérines différentiellement exprimées à la réactivation de l’embryon et homologues à des séquences de gènes connues chez d’autres espèces. Ces gènes sont impliqués dans la régulation du métabolisme (25 %), de l’expression génique (21 %), de la transduction de signal (15 %), du cycle cellulaire (15 %), du transport (10 %) et de la structure cellulaire (9 %), reflétant ainsi d’importantes modifications utérines à la réactivation embryonnaire. Nous avons validé l’expression différentielle de dix gènes ainsi identifiés : GDF3 (growth and differentiation 3), ALCAM (activated leukocyte cell adhesion molecule), ADIPOR1 (adiponectin receptor 1), HMGN1 (high mobility group N1), TXNL1 (thioredoxin like 1), TGM2 (tissue transglutaminase 2), SPARC (secreted protein acidic rich in cystein), et trois gènes codant pour AZIN1 (antizyme inhibitor 1), ODC1 (ornithine decarboxylase 1) et SAT1 (spermidine/spermine N1-acetyltransferase), des enzymes impliquées dans la biosynthèse des polyamines. Le patron de l’expression spatio-temporel de SPARC et d’HMGN1 illustrent spécifiquement un remodelage tissulaire et de la chromatine au niveau utérin à la sortie de la diapause embryonnaire. Ayant mesuré une augmentation des concentrations utérines en polyamines à la reprise du développement embryonnaire, nous avons émis l’hypothèse que les polyamines seraient impliquées dans les événements menant à la sortie de la diapause. L’inhibition de la biosynthèse des polyamines par un traitement à l’ α-difluoromethylornithine (DFMO) a provoqué une diminution significative de la proliferation cellulaire dans les embryons à la réactivation, un retard du moment de l’implantation, mais n’a pas affecté le succès de la reproduction. De manière similaire, nous avons induit un état de dormance dans les cellules de trophoblaste de vison en présence DFMO dans le milieu de culture, et constaté que cet état était réversible. En conclusion, cette étude a non seulement ouvert de nouveaux horizons quant à la compréhension du rôle utérin dans les événements menant à la sortie de la diapause embryonnaire, mais a démontré pour la première fois, l’existence de facteurs utérins indispensables à la réactivation de l’embryon: les polyamines.

Reproductive cycle of Urolophus cruciatus in south-eastern Australia: does the species exhibit obligate or facultative diapause?

Observations of synchronous rapid growth of embryos and ovarian follicles in pregnant females during the half-year December–May leading to parturition, ovulation, mating, and fertilization suggest Urolophus cruciatus has the capacity for an annual reproductive cycle. Conversely, the higher proportion of the pregnant females in the population carrying eggs than carrying embryos in utero during December–May and all pregnant females in the population only carrying eggs in utero during June–November indicate a longer reproductive cycle. Analysis based on the usual assumptions implies that the species most likely exhibits a biennial cycle with ~18-month period of diapause following ovulation prior to ~6-month period of rapid embryogenesis. However, it is feasible that the period of the cycle is triennial with ~30-month period of diapause or alternatively diapause varies among individuals and varies from year to year. Rather than exhibiting a fixed-term reproductive cycle where obligatory diapause leads to parturition timed every year to provide favourable conditions for neonates, as suggested for several other chondrichthyan species, U. cruciatus may exhibit facultative diapause where the period of diapause and hence the reproductive cycle varies depending on the prevailing environmental conditions or density-dependent factors as described for many terrestrial species. U. cruciatus is highly matrotrophic (>4000 % wet mass gain from ovum to full-term embryo), litter size (1–4) increases with maternal length, sex ratio among embryos is 1:1, and male breeding condition varies seasonally with peak sperm production coinciding with female ovulation.

Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo

Background Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdhex4/5 huntingtin deficient embryos. Results In the absence of huntingtin, expression of nutritive genes appears normal but E7.0–7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. Conclusion Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease.

Freedom to choose? Embryo selection, reproductive decision-making and the role of the state

Involving the biopsy of an eight-cell embryo, PGD has been hailed as a means of making reproductive decisions without having to face the heart-wrenching decision to abort an affected foetus. However, controversy around the kinds of traits for which testing can be done, and who has access to the technology, has led to questions about the way in which the technology is developing. Women who are allowed to access in vitro fertilisation (IVF) services can currently also access PGD in limited circumstances.

The human embryo as property? Cryopreservation and the challenges for law

The development of the new reproductive technologies has presented significant challenges for policy makers and law reformers. This article focuses on the particular challenges posed by cryopreservation of embryos. These issues are analysed through discussion of relevant Australian statutory provisions and United States case law. The article concludes with a consideration of whether the property model provides an appropriate framework for reproductive material.

The potentiality of the embryo and the somatic cell

Recent arguments on the ethics of stem cell research have taken a novel approach to the question of the moral status of the embryo. One influential argument focuses on a property that the embryo is said to posses—namely, the property of being an entity with a rational nature or, less controversially, an entity that has the potential to acquire a rational nature—and claims that this property is also possessed by a somatic cell. Since nobody seriously thinks that we have a duty to preserve the countless such cells we wash off our body every day in the shower, the argument is intended as a reductio ad absurdum of the claim that the embryo should be afforded the same moral status as a fully developed human being. This article argues that this argument is not successful and that it consequently plays into the hands of those who oppose embryonic stem cell research. It is therefore better to abandon this argument and focus instead on the different argument that potentiality, as such, is not a sufficient ground for the creation of moral obligations towards the embryo.

Effect of soil burial depth and wetting on mortality of diapausing larvae and patterns of post-diapause adult emergence of sorghum midge, Stenodiplosis sorghicola (Coquillett) (Diptera: Cecidomyiidae)

In south-eastern Queensland, Australia, sorghum planted in early spring usually escapes sorghum midge, Stenodiplosis sorghicola, attack. Experiments were conducted to better understand the role of winter diapause in the population dynamics of this pest. Emergence patterns of adult midge from diapausing larvae on the soil surface and at various depths were investigated during spring to autumn of 1987/88–1989/90. From 1987/88 to 1989/90, 89%, 65% and 98% of adult emergence, respectively, occurred during November and December. Adult emergence from larvae diapausing on the soil surface was severely reduced due to high mortality attributed to surface soil temperatures in excess of 40°C, with much of this mortality occurring between mid-September and mid-October. Emergence of adults from the soil surface was considerably delayed in the 1988/89 season compared with larvae buried at 5 or 10 cm which had similar emergence patterns for all three seasons. In 1989/90, when a 1-cm-deep treatment was included, there was a 392% increase in adult emergence from this treatment compared with deeper treatments. Some diapausing larvae on the surface did not emerge at the end of summer in only 1 year (1989/90), when 28.0% of the larvae on the surface remained in diapause, whereas only 0.8% of the buried larvae remained in diapause. We conclude that the pattern of emergence explains why spring plantings of sorghum in south-eastern Queensland usually escape sorghum midge attack.

Vitamin A economy of the developing chick embryo and of the freshly hatched chick

. The changes in the net amounts of retinol, retinyl esters and retinal in both the developing chick embryo and the newly hatched chick were investigated. The embryo requires about 68nmol of the vitamin for its growth, whereas the baby chick requires about 108nmol during the first 7 days after hatching. 2. Retinal was present in the egg in fairly high concentrations at the beginning of the incubation but it virtually disappeared from the extra-embryonic tissue after day 17 of incubation. It was not found in the liver of the embryo or of the newly hatched chick up until day 7.

Studies on the fractionation of total transfer ribonucleic acid and purification of an alanine transfer ribonucleic acid from chick embryo

Chick embryo tRNA, prepared by a simple large-scale method, was fractionated on three different ion-exchange columns. In all cases simple chromatographic patterns for various tRNA species were observed, indicating the presence of only a few major species of tRNA for each amino acid. By repeated chromatography one species of alanine tRNA was purified to approx. 80% purity. T1 ribonuclease digest of this purified tRNA gave a simple chromatographic pattern. Because of the simplicity of the method of preparation of tRNA from this readily available source and the presence of only a few species of tRNA for each amino acid, chick embryo is suited for the study of tRNA and its various functions in higher systems.

A simple method for large-scale preparation of chick embryo tRNA

A simple method for preparing bulk quantities of tRNA from chick embryo has been developed. In this method chick embryos were homogenized in a buffer of pH 4.5, followed by deproteinization with phenol. The aqueous layer was allowed to separate under gravity. The resulting aqueous layer, after two more phenol treatments, was directly passed through a DEAE-cellulose column and the tRNA eluted therefrom with 1 Image NaCl. The tRNA prepared by this method was as active as the one prepared at neutral pH.

Succinate and malate improve development of hamster eight-cell embryos in vitro: Confirmation of viability by embryo transfer

The in vitro development of hamster preimplantation embryos is supported by non-glucose energy substrates. To investigate the importance of embryonic metabolism, influence of succinate and malate on the development of hamster 8-cell embryos to blastocysts was examined using a chemically defined protein-free modified hamster embryo culture medium-2 (HECM-2m). There was a dose-dependent influence of succinate on blastocyst development; 0.5 mM succinate was optimal (85.1% ± 3.9 vs. 54.5% ± 3.5). In succinate-supplemented HECM-2m, blastocyst development was reduced by omission of lactate (68.5% ± 7.2), but not pyruvate (85.8% ± 6.2) or glutamine (84.1% ± 2.1). Succinate along with either glutamine or lactate or pyruvate poorly supported blastocyst development (28%-58%). Malate also stimulated blastocyst development; 0.01 mM malate was optimal (86.3% ± 2.8). Supplementation of both succinate and malate to HECM-2m supported maximal (100%) blastocyst development, which was inhibited 4-fold by the addition of glucose/phosphate. The mean cell numbers (MCN) of blastocysts cultured in succinate-supplemented HECM-2m was higher (28.3 ± 1.1) than it was for those cultured in the absence of glutamine or pyruvate (range 20-24). The MCN was the highest (33.4 ± 1.6) for blastocysts cultured in succinate-malate-supplemented HECM-2m followed by those in succinate (28.3 ± 1.1) or malate (24.7 ± 0.5) supplemented HECM-2m. Embryo transfer experiments showed that 29.8% (±4.5) of transferred blastocysts cultured in succinate-malate-supplemented HECM-2m produced live births, similar (P > 0.1) to the control transfers of freshly recovered 8-cells (33.5% ± 2.0) or blastocysts (28.9% ± 3.0). These data show that supplementation of succinate and malate to HECM-2m supports 100% development of hamster 8-cell embryos to high quality viable blastocysts and that non-glucose oxidizable energy substrates are the most preferred components in hamster embryo culture medium. Mol. Reprod. Dev. 47:440-447, 1997. © 1997 Wiley-Liss, Inc.