Pattern of Brachyury gene expression in starfish embryos resembles that of hemichordate embryos but not of sea urchin embryos.

Echinoderms, hemichordates and chordates are deuterostomes and share a number of developmental features. The Brachyury gene is responsible for formation of the notochord, the most defining feature of chordates, and thus may be a key to understanding the origin and evolution of the chordates. Previous studies have shown that the ascidian Brachyury (As-T and Ci-Bra) is expressed in the notochord and that a sea urchin Brachyury (HpTa) is expressed in the secondary mesenchyme founder cells. A recent study by [Tagawa et al. (1998)], however, revealed that a hemichordate Brachyury (PfBra) is expressed in a novel pattern in an archenteron invagination region and a stomodaeum invagination region in the gastrula. The present study demonstrated that the expression pattern of Brachyury (ApBra) of starfish embryos resembles that of PfBra in hemichordate embryos but not of HpTa in sea urchin embryos. Namely, ApBra is expressed in an archenteron invagination region and a stomodaeum invagination region.     

Cryopreservation of mammalian embryos

As an innovative method for embryo cryopreservation, vitrification not only reduced the cooling stage duration to a minimum, but also eliminated any injuries cased by extracellular ice, which is a major cause of cell injury. Therefore, if embryos are treated adequately, high survival can be obtained. As a component of a vitrification solution, a permeating cryoprotective agent is essential, and additional inclusion of a macromolecule and a small saccharide makes the solution more effective. The author’s group composed a solution, designated EFS40, with ethylene glycol. Ficoll, and sucrose. This solution proved effective for the cryopreservation of various stages of embryos in many species. In this article, the author describes the detailed procedure for the vitrification of mouse morulae; related information is also described.     

Quick-splitting of bovine embryos

Described is a simplified method of bovine embryo bisection amenable to on-farm embryo transfer. Using a microblade operated by a hand-held micromanipulator, Day 7 bovine embryos were bisected while in the zona pellucida. With a vertical motion, the embryo was pinned between the blade and the bottom of a plastic petri dish and bisected. Demi-embryos were transferred nonsurgically (without zonae pellucidae) into synchronized recipients. Pregnancy rates were normal with 5 13 (38%) and 9 20 (45%) of recipients confirmed pregnant 70 to 80 d after receiving either twin or single half embryos, respectively. This compared to 12 28 (43%) of recipients becoming pregnant from transfer of whole embryos. These data confirm that bovine demi-embryos do not need zonae pellucidae on Day 7 and that simplified field methods of bisection give normal pregnancy results.     

Cryopreservation of canine embryos

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.     

Electrorotation of axolotl embryos

The frequency dependent dielectric properties of individual axolotl embryos (Ambystoma mexicanum) were investigated experimentally utilizing the technique of electrorotation. Individual axolotl embryos, immersed in low conductivity media, were subjected to a known frequency and fixed amplitude rotating AC electric field and the ensuing rotational motion of the embryo was monitored using a conventional optical microscope. None of the embryos in the pregastrulation or neurulation stages of development exhibited any rotational motion over the field frequency range (10 Hz-5 MHz). Over the same frequency range, the embryos in the gastrulation stage of development exhibited both co-field and counterfield rotation over different ranges of the applied field frequency. Typically, the counterfield rotation exhibited a peak in the rotation spectrum at similar 1 KHz while the co-field peak was located at similar 1-2 MHz. The rotational spectral data was analyzed using a multishelled spherical embryo model to determine the electrical character of embryos during the early development stages (Stages 5-16; i.e., 16 cell through open neural plate stages).     

Development of reconstituted mouse embryos produced from bisected and electrofused pronuclear-stage embryos

The present study was designed to investigate the in vitro and in vivo development potential of reconstituted mouse embryos produced by bisection and electrofusion of pronuclear stage embryos (PN-E). Pronuclear-stage ICR and F1 (C57BL x CBA) strain mouse embryos were bisected manually with a fine glass needle under the dissecting microscope to produce karyoplasts (KP) and cytoplasts (CP). The KP of ICR PN-E and CP of F1 PN-E (KP: ICR + CP:F1) or the KP of F1 PN-E and CP of F1 PN-E (KP:F1 + CP:ICR) were attached using phytohemagglutinin-P (PHA-P) and then electrofused. High fusion rates of the KP and CP of PN-E were obtained (93.5%). The fused embryos were encapsulated in alginate gel and cultured for 72 or 96 hours. The cleavage rates of reconstituted embryos were also high (98.8%). Developmental rates to the blastocyst stage in vitro for the 96-hour culture of reconstituted embryos were 68.9% (KP:ICR + CP:F1) and 78.4% (KP:F1 + CP:ICR). Furthermore, the developmental ability of reconstituted embryos in vivo was investigated, and some live young were obtained (KP:ICR + CP:F1, 7.5% and KP:F1 + CP:ICR, 10.8%). In this study, it was confirmed that reconstituted embryos produced by bisection and electrofusion of pronuclear stage embryos were able to develop into blastocysts in vitro and into live young in vivo.     

利用IVF废弃胚胎为核移植受体构建人体细胞克隆胚胎

目的：探讨利用IVF废弃胚胎构建人体细胞克隆胚胎的发育潜能及其在人治疗性克隆应用的可能性。方法：收集2008年7-12月在广州医学院第三附属医院进行体外受精-胚胎移植周期中的多精受精胚胎和MII期体外受精失败卵母细胞,运用显微操作技术构建人体细胞克隆胚胎,观察胚胎发育情况。结果：多精受精胚胎为核移植受体的克隆胚胎能够发育到8-细胞期,受精失败MII期卵母细胞为核移植受体的克隆胚胎能够激活,但不能够卵裂。两种IVF废弃的胚胎构建的人体细胞克隆胚胎在去核成功率,注核成功率上无显著差异（P＆gt;0.05）,但卵裂率和8细胞率上具有显著差异（P＆lt;0.05）。结论：多精受精胚胎比MII期体外受精失败卵母细胞更适合作为人核移植受体细胞。     

In vitro culture of embryos of Cucumis spp.: heart-stage embryos have a higher ability of direct plant formation than advanced-stage embryos

Summary The ability of embryos at different developmental stages to form plants in vitro has been studied in cultivated Cucumis sativus L. and in the wild species C. zeyheri 2 x Sond. and C. metuliferus Naud. On MS medium containing 3.5% sucrose, 0.1 mg 1^–1 kinetin (Kn) and 0.01 mg 1^–1 indoleacetic acid (IAA), proembryos (0.03–0.05 mm) and early globular embryos (0.05–0.08 mm) from the wild species developed into plants in low frequencies of 8% and 21%, respectively. These embryos should be surrounded by the embryo sac tissue. On the same medium late globular (0.08–0.1 mm) and early heart-stage embryos (0.1–0.3 mm) developed into plants in moderately high and high frequencies of 48% and 83%, respectively. The presence of the embryo sac at these stages was still beneficial, but no longer a prerequisite. Late heart-stage embryos (0.3–0.8 mm) also showed high frequencies of plant formation, 63%, if Kn was applied at a concentration of 1 mg 1^–1. From the early cotyledon stage onwards, the frequency of plant formation gradually decreased, reaching a minimum at the late cotyledon stage. Subsequently it began to increase again up to the late maturation stage. The poor plant formation shown by the intermediate-aged embryos could be improved slightly by lowering the sucrose concentration to 0.5% and by increasing the Kn concentration to 10 mg 1^–1. Relative to the wild species, embryos of C. sativus showed lower percentages of plant formation. The optimum sucrose concentration was 2% for the heart-stage C. sativus embryos. In all three species the ability to form plants strongly decreased with increasing embryo age, from early to late cotyledon. This is thought to be caused by the increasing tendency of the embryos at these stages to continue in vitro the normal embryo development.     

Induction of somatic embryos by macrosalt stress from mature zygotic embryos of Panax ginseng

Mature zygotic embryos of ginseng (Panax ginseng C. A. Meyer) were germinated on a Murashige and Skoog medium lacking growth regulators. However, when the zygotic embryos were cultured on MS medium containing increased levels of macrosalts (NH₄NO₃, KNO₃, KH₂PO₄, MgSO₄, or CaCl₂) to result is a mild salt stress, growth of zygotic embryos was strongly suppressed and eventually browning occurred. Somatic embryos or embryogenic calli were formed directly from these abnormal stressed zygotic embryos. Cotyledons were the most competent tissue for somatic embryo production. The highest frequency of somatic embryo formation (56.3%) was observed on medium containing 61.8 mM of NH₄NO₃. The highest frequency of somatic embryo formation by five different macrosalt treatments occurred in the following order: NH₄NO₃> KNO₃> KH₂PO₄> MgSO₄> CaCl₂. Somatic embryos were regenerated into plants with a shoot and root, and the plants survived on soil in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transfer of cultured bovine embryos

A total of 126 bovine embryos were surgically collected from 16 superovulated donor heifers 5 days after estrus and randomly selected for either immediate transfer to synchronized recipients or $\text{in}$$\text{vitro}$ culture at 37°C for 24 hours and subsequent transfer. Twenty-four of 56 (42.8%) embryos maintained for 24 hours in Ham's F10 medium supplemented with 10% heat treated fetal calf serum (HTFCS) and transferred to 32 recipients produced live calves. Survival of 70 noncultured embryos transferred to 35 recipients was 55.7% (39 calves). The percentages of recipients that were diagnosed pregnant at 42 days with cultured and control embryos were 59.4% ($\text{19}\text{32}$) and 74.3% ($\text{26}\text{35}$), respectively. No statistical difference was observed between the $\text{in}$$\text{vitro}$ cultured and control embryos for viability following transfer to recipient females.In a second study, Day 7 embryos maintained in Ham's F10 medium supplemented with 10% HTFC serum for various culture periods were tested for viability following nonsurgical transfer to recipient females. A total of $\text{1}\text{5}$, $\text{1}\text{3}$ and $\text{0}\text{4}$ embryos cultured for 24, 48 and 72 hours, respectively, resulted in pregnant recipients following transfer.     

Transfer of cultured rabbit embryos

Embryo transfer experiments were carried out to study the developmental capacity of cultured rabbit embryos when transferred to recipients of variable postovulatory maturity. Rabbit embryos were flushed from the oviduct at 26 hours postcoitum (pc) and cultured in a modified Ham's F-10 medium supplemented with bovine serum albumin (BSA) for a period of 70 hours. At 96 hours pc the cultured embryos, which ranged from the early morula to the expanding blastocyst stage, were transferred to pseudopregnant recipients mated to vasectomized males 36 to 96 hours prior to the transfer procedure. Greatest embryo survival occurred when transfers were made to either the oviducts or uterine horns of recipients at 48 hours pc. Intermediate results for both implantation rates and number of young born were obtained with recipients at 36, 60, 72, and 84 hours pc. Transferred embryos consistently failed to survive the uterine environment of recipients 96 hours pc at transfer although this group was synchronous with embryonic chronological age. Oviductal transfers were generally more successful than uterine transfers. Markedly higher rates of embryo survival resulted from embryos that were collected 60 and 72 hours pc and transferred directly to synchronous recipients without an interim period of culture. Dissimilarity of development for in vivo grown rabbit embryos and those cultured in synthetic medium is demonstrated.     

Development of reconstituted mouse embryos produced from the cytoplast of bisected oocytes or pronuclear-stage embryos and single blastomeres of 2-cell stage embryos

The present study investigated the in vitro developmental potential of reconstituted mouse embryos produced from the cytoplast of pronuclear-stage embryos or oocytes and single blastomeres of 2-cell stage embryos by electrofusion. The cytoplast of pronuclear-stage embryos and oocytes were obtained by manual bisection with a fine glass needle under a dissecting microscope. The fusion rates of the reconstituted embryos produced from the cytoplast of oocytes and single blastomeres of 2-cell stage embryos (O-SB2: 38.1 and 41.5%) were significantly lower than those produced from the cytoplast of pronuclear-stage embryos and single blastomeres of 2-cell stage embryos (P-SB2: 91.2 and 97.6%; P<0.001). Reconstituted embryos were encapsulated in alginate gel and were cultured for 96 hours. Similarly, the cleavage and development rates to the blastocyst stage of O-SB2 (56.3, 61.2 and 2.0, 3.1%, respectively) were significantly lower than those of the P-SB2 (91.0, 91.2 and 18.6, 20.7%; respectively, P<0.05). The cleavage and development rates to the blastocyst stage (61.2 and 2.0%) of reconstituted embryos produced from single blastomeres of late 2-cell stage embryos and oocyte cytoplast improved after activation by ethanol treatment (76.1 and 21.7%). However, the use of single blastomeres of early 2-cell stage embryos as nuclear donors did not enhance the cleavage and development rates of the reconstituted embryos to the blastocyst stage.