Fertilization and Embryo Development in Hybridization Between Wheat and Leymus

Observations were made of the fertilization and embryo development in intergeneric cross between Triticum aestivum L. cv. Chinese Spring and Leyrnus secalinus Tzrel. The pollen germination of Leymus secalinus appeared normal on the stigma of Triticum aestivum and the pollen tubes grew into the style and entered the embryo sacs. Double and simple fertilization were observed in the pollinated florets. Of the 319 ovaries examed 62 (19.44%) had double fertilization and had embryo and endosperm, but endosperm development was slower than that of the embryos, 49 (15.36%) had only embryo and 7 (2.19%) had only endosperm. The total percentage of fertilization was as high as 36.99%. However, only I seed was obtained from 150 wheat ftorets pollinated with Leymus secalinus. This was obviously due to the absence or poor development of the endosperm. It may be suggested that the potential of increasing the frequency of hybrid plant obtainment was great in the cross between wheat and leymus, if embryo culture technique is employed at the early stage of hybrid embryo development.     

Observation on Rice Embryo Sac Development with Confocal Laser Scanning Microscopy

An easy and rapid procedure has been launched for observafion of embryo sac development of Oryza sativa L. Following fixation and dehydration, whole mounted ovaries were cleared in methyl sulieylate, finally sealed under coverslips with clove oil. With the confocal laser scanmng microscopy procedure, the embryo sac exhibited autofluorescence and could be clearly observed. FAA and 4% glutaraldehyde as fixative produced different results but the latter seemed to be better.     

Development of Ovules and Embryo sacs in Ostrya virginiana (Betulaceae) and Its Systematic Significance

The development of ovules and embryo sacs in Ostrya virginiana was studied for the first time. Most ovaries had two ovules which were anatropous, unitegmic and crassinucellate. The ovule usually possessed several archesporial ceils which divided periclinally into the upper parietal cell and the lower sporogenous cell. The sporogenous cell functioned directly as megaspore mother cell. The tetrad of megaspores was linear in arrangement, and every megaspore might be functional. One ovule often contained 2- 6 embryo sacs and the embryo sac belongs to Polygonum type. It can be concluded from the present data that all ovules among the genera of the Betulaceae are unitegrnic. There are more groups with the phenomenon of multiple embryo sacs in anemophic plants such as Betulaceae, Casuarinaceae, Graminae, Jnglandaceae, Myricaceae, Simaroubaceae, Ulmaceae, than in entomophilous plants. Multiple embryo sacs also occur among some parasitic plants and saprophytes, e.g. Orobanchaceae, Cassytha in Lauraceae, Cuscuta in Convolvulaceae and Utricularia in Lentibulariaceae. It may be inferred that the characteristic of multiple embryo sacs be an evolutionary adaptation of those plants with lower pollination rate to increase the rate of fertilization. Finally, a comparison of embryological characters among the genera of the Betulaceae shows that the family is of a number of common embryological characters, such as multicellular archesporium, multiple embryo sacs in one ovule, and a long interval between pollination and fertilization. The diversity and systematic significance of several embryological characters among the “higher” hamamelid families are also discussed. It is still hard to explain the phy-logenetic relationships among those families clearly only with.     

Embryo technology in conservation efforts for endangered felids

Most of the 36 species of wild cats are classified as threatened, vulnerable or endangered due to poaching and habitat loss. The important role of assisted reproduction techniques (ART) as part of a multifaceted captive breeding program for selected wild cat species is gradually gaining acceptance. This recognition is a result of the progress made during the last decade in which the feasibility of oocyte recovery from gonadotropin-treated females, in vitro fertilization, embryo cryopreservation and embryo transfer (ET) was demonstrated in the domestic cat (Felis catus). Additionally, embryos have been produced in vitro from oocytes matured in vitro after recovery from ex situ ovaries of both domestic and non-domestic cat species and domestic kittens have been born following transfer of these embryos. In vitro fertilization has been successful in at least one-third of wild cat species and kittens were born after transfer of Indian desert cat (Felis sylvestris ornata) embryos into a domestic cat and con-specific transfer of tiger (Panthera tigris) embryos. The domestic cat is not only a valuable model for development of in vitro techniques but may serve as a recipient of embryos from several species of small wild cats.     

In Vitro Embryogenesis in Unfertilized Embryo Sacs of Oryza sativa L.

Haploid rice plantlets were induced from cultured ovaries in our previously reported experiment. The present paper is an embryological study on this subject. Young flowers of two japonica cultivars were excised and cultured just in the same manner as before. Liquid medium used for float culture was N6+3% sucrose+0.125 ppm MCPA. The inoculated materials were checked to be at late uninuclcate pollen stage which corresponded mainly to the uuinuelcate embryo sac stage, but as well as some 2- or 4-nucleate embryo sacs. Samples were fixed at 23 days intervals in acetomethanol (1:3), stained in toto with diluted Ehrlich's hematoxylin and sectioned by paraffin method for microscopical observation. 4 days after inoculation most of the embryo sacs developed up to 8-nucleate stage with polarized differentiation of the egg apparatus, central cell and antipodals. From 7th day on, proembryos of various sizes and shapes appeared in the micropylar region of some embryo sacs; some consisted of meristcmatic ceils, others were highly vacuo]ated. One-celled as well as linear multicellular suspensors atypical of in vivo zygote proembryos were observed, ttowever, it was uncertain whether the proembryos originated from the egg cell, the synergids, or the differentiating egg apparatus as a whole. Another peculiar event occured during culture was the formation of endosperm-like free nuclei from the unfertilized polar nuclei in some embryo sacs. Sometimes the free nuclei were numerous and showed a tendency of cell formation in localized areas. 12–15 days after inoculation, the proembryos developed into mieroseopieal ealli with globular or pearlike shape, wbieh continued enlarging to visible size with naked eyes at about 18–24 th day. Further growth eventually led the ealli protruding out the ovary wall beyond 32–35 the day. These observations indicate that the embryo sue, similarly as the pollen, can be induced to embryogenesis in vitro. This may open a new way to study the mechanism controlling gametophytie and sporophytie developmental pathways of embryo sac and provide means for large-scale production of "embryo sac plants" in future.     

Embryo production after in vitro fertilization with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm

The objective of this study was to determine the in vitro fertilizing capacity of bull sperm derived from fresh or frozen samples and subjected to sex sorting and re-cryopreservation. Four sperm types were assessed for their ability to fertilize and sustain early embryo development in vitro. Semen from three Bos taurus bulls of different breeds (Jersey, Holstein and Simmental) was collected and either sorted immediately and then frozen (SF) or frozen for later sorting. Frozen sperm destined for sorting were thawed, sex-sorted, and re-frozen (FSF) or thawed, sex-sorted (FS), and used immediately for in vitro fertilization (IVF). Frozen-thawed nonsorted semen from the same ejaculate was used as a control. Oocytes from donor cows were aspirated via ovum pick-up and matured in vitro prior to IVF and culture. On average, 19.0 ± 1.7 (mean ± SEM) oocytes were aspirated per donor cow, of which 74.4 ± 2.2% were selected for maturation. The proportion of cleaved embryos (Day 3) did not differ between sperm groups (P = 0.91). Likewise, IVF with FSF sperm resulted in similar Day 7 blastocyst rates (as a percentage of total oocytes) as those of control, SF, and FS sperm (FSF, 34.5 ± 4.7; control, 32.2 ± 4.6; SF, 35.9 ± 4.8; and FS, 26.9 ± 4.1%; P = 0.23). These encouraging results show that frozen-thawed sex-sorted sperm may be re-frozen and used for in vitro embryo production with similar blastocyst production as that of nonsorted frozen-thawed and sex-sorted frozen-thawed sperm.     

体外生产胚胎技术

体外生产胚胎技术是胚胎工程技术的基础,其过程涉及到卵母细胞的体外成熟、精子获能和体外受精、胚胎的体外培养这三个方面。主要综述了卵母细胞体外成熟的影响因素、精子分离和获能的方法、早期胚胎体外培养的发育“阻滞”现象和体外培养体系,同时展望了该技术的发展前景 。     

莴苣胚囊细胞分离

用酶解和解剖方法分离了莴苣的卵细胞,助细胞,中央细胞和合子。莴苣子房先在酶液中酶解40～50min,然后在不含酶的分离液中用解剖针解剖子房。在解剖出的胚囊中,可看到卵细胞,两个助细胞和中央细胞的轮廓。将胚囊的合点端切破,轻轻挤压胚囊的珠孔端,四个细胞即可逸出。在最佳条件下,90min可从40个子房中分离出29个胚囊,进一步从中分离出11个卵细胞。分离出的胚囊细胞用显微操作仪收集备用。莴苣卵细胞的成功分离为进行离体受精探索创造了条件。     

家猫的胚胎工程

家猫是惟一一种没有被列为珍稀或濒危的猫科动物。通过家猫的胚胎工程研究,对保护其它濒危猫科物种有重要的借鉴意义。本文描述了家猫的一般生殖特点,着床前的胚胎在体内的发育概况;综述了近年来对家猫的超数排卵,卵母细胞的体外成熟,体外受精,胚胎的体外培养,胚胎移植,冷冻保存和胚胎克隆等方面的研究进展。     

Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos.

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

小麦与赖草远缘杂交的受精和胚胎发育

对小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）和赖草（Ｌｅｙｍ ｕｓｓｅｃａｌｉｎｕｓＴｚｒｅｌ．）杂交的受精和胚胎发育进行了观察．赖草花粉在小麦柱头上萌发良好,花粉管可顺利长入花柱和胚囊,在检查过的３１９个小麦子房中,６２个（１９．４４％ ）发生了双受精,产生了胚和胚乳,但胚乳的发育往往落后于胚的发育;４９个（１５．３６％ ）发生了单卵受精,只产生胚而无胚乳;７个（２．１９％ ）发生了单极核受精,只产生胚乳而无胚．小麦×赖草虽然总受精率可高达３６．９９％ ,然而由于胚乳的缺乏或发育不完全,致使最后结实率很低．从１５０朵授过赖草花粉的小麦颖花中,只得到１粒种子．表明利用胚培养技术对杂种胚进行早期离体培养,可望提高杂种植株的获得率     

水曲柳雌花发育、大孢子发生及胚胎发育的解剖学观察

用石蜡切片法对水曲柳雌花发育、大孢子发生及胚胎发育的过程进行了解剖学观察。水曲柳在东北地区花期一般在5月,雌雄异株,雄蕊先熟,从散粉盛期到雌花开放间隔约一周,雌花一旦开放即可接受花粉。雌花雌蕊两侧具有退化雄蕊,子房2心皮2室,每室具2枚倒生胚珠,单珠被。薄珠心,单孢原,蓼型胚囊。大孢子四分体线形排列,通常合点端的1个大孢子为功能大孢子,但有极少数功能大孢子发生于合点端第2个或4分体两端的2个。核型胚乳。胚胎发育紫菀型,经历了合子、原胚、球形胚、心形胚和鱼雷形胚等不同阶段,6月末种胚形态分化完全,每子房中只有1个胚珠发育为成熟种子,种子单胚。对水曲柳典型的胚胎学特征和胚胎发生发育过程中的一些异常现象作了讨论。     

Comparison of cytoskeletal integrity,fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.     

兰属种间杂交胚拯救研究初报

取兰属大花蕙兰为母本、墨兰为父本进行种间杂交并进行杂种胚拯救研究。结果显示,子房离体培养明显优于胚珠离体培养,是胚抢救培养的有效手段。不同取材时间及在不同培养基中子房培养情况差异明显。在授粉后60 d子房离体培养即可获得成功,但以授粉后90 d子房离体培养最佳,萌发率高,达80%。子房培养的最适培养基为VW+BA 0.2 mg/L+NAA 0.2 mg/L+蛋白胨3 g/L+水解酪蛋白500 mg/L+椰子汁10%。     

Mayer's hemalum-methyl salicylate: a stain-clearing technique for observations within whole ovules

Nondissected ovaries of tuber-bearing Solanum sp. were stained with Mayer's hemalum, a positive stain for chromatin and nucleoli, and then optically cleared with methyl salicylate, a clearing agent. Clarity, resolution and contrast within the ovules dissected from ovaries were comparable to those of sectioned, paraffin embedded ovaries. Contrast within ovules greatly exceeded that of unstained and nonspecifically stained clearings, and eliminated the need of special optics, i.e., Nomarski interference-contrast optics, for optimal viewing and photography. Much less time and labor were required than for embedded specimens. Usefulness of the technique for cytogenetic and cytological research was verified by analyzing meiosis and other features of megasporogenesis and megagametogenesis in normal, and in two meiotic mutants, of Solanum. The results illustrate the usefulness of combined Mayer's hemalum staining and methyl salicylate clearing, and suggest additional stain-clearing agent combinations have potential for cytological and cytogenetic research. Preliminary results with other species suggest the technique may also be useful for classroom instruction.     

Fertilization and Embryo Development of Populus lasiocarpa Oliv

The double fertilization and embryo development of P. lasiocarpa were studied using cytochemical method for DNA, RNA, polysaccharides and proteins. Stigmas were covered by secretion stained positively with PAS and mercuric bromphenol blue at receptive stage. The pollen grains germinated on the stigma in large quantities 6 hours after pollination. Fertilization started at the 6th days after pollination. The sperm nucleus fused with the secondary nucleus faster than the sperm nucleus with the egg nucleus. The syngamy belonged to peremitotic type. A great deal of starch grains in the embryo sac disappeared during fertilization. The endosperm was nuclear type and becomed a cel 31 days after pollination. The endosperm was characterized by a dense cytoplasm rich in protein. No discrete starch grains were observed in endosperm. Afterwards, the endosperm was consumed by the developing embryo, thus the mature seeds were non-endospermous. The zygote was dormant for 6–8 days. During the dormency, many striking changes took place, and then, the zygotes showed more pronounced polarity. These changes included the shinkage of the large vacuole, the reduced size, the reappearance of large vacuole, the enlarging of the size. The embryogenesis conformed to the Solanad type. The ovules matured into seeds successively 44 days after pollination. The mature embryo was straight. Two cotyledons folded each other.     

In vitro maturation,fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.     

多花地宝兰胚囊和胚发育的细胞学研究

为探讨多花地宝兰(Geodorum recurvum)胚胎发育的系统分类学意义,采用石蜡制片法对多花地宝兰胚囊和胚的发育进行解剖学观察。结果表明,在开花前,多花地宝兰胚珠原基发育缓慢,开花授粉后胚珠原基快速发育成"树状二杈分枝结构",随后在"分枝结构"末端形成孢原细胞,开始胚囊发育。多花地宝兰的胚囊发育属于单孢蓼型胚囊,胚珠具有双层珠被。孢原细胞形成后,经过细胞膨大延长发育形成胚囊母细胞,胚囊母细胞经过减数分裂形成线性四分体,在珠孔端形成1个功能大孢子,功能大孢子经过3次有丝分裂形成8核胚囊。多花地宝兰的胚发育具有藜型和紫苑型两种方式。双受精完成后,多花地宝兰合子进行一次橫裂后形成基细胞和顶细胞;基细胞经过多次分裂形成细胞团,细胞团中的细胞向不同方向膨大延长形成多个胚柄细胞;顶细胞有两种分裂方式,一种是横裂形成藜型胚,一种是纵裂形成紫苑型胚。因此,推测多花地宝兰在兰科植物系统分类学上属于较为原始种。     

Embryo development rates after transfer of oocytes matured in vivo, in vitro, or within oviducts of mares

Objectives of the present study were to use oocyte transfer: 1) to compare the developmental ability of oocytes collected from ovaries of live mares with those collected from slaughterhouse ovaries; and 2) to compare the viability of oocytes matured in vivo, in vitro, or within the oviduct. Oocytes were collected by transvaginal, ultrasound-guided follicular aspiration (TVA) from live mares or from slicing slaughterhouse ovaries. Four groups of oocytes were transferred into the oviducts of recipients that were inseminated: 1) oocytes matured in vivo and collected by TVA from preovulatory follicles of estrous mares 32 to 36 h after administration of hCG; 2) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and matured in vitro for 36 to 38 h; 3) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and transferred into a recipient's oviduct <1 h after collection; and 4) im mature oocytes collected from slaughterhouse ovaries containing a corpus luteum and matured in vitro for 36 to 38 hours. Embryo development rates were higher (P < 0.001) for oocytes matured in vivo (82%) than for oocytes matured in vitro (9%) or within the oviduct (0%). However, neither the method of maturation nor the source of oocytes affected (P > 0.1) embryo development rates after the transfer of immature oocytes.     

卵母细胞透明带异常及其对助孕结局影响的研究进展

人类透明带(zona pellucida,ZP)是在卵泡发生过程中由卵母细胞和颗粒细胞共同分泌的由ZP1-ZP4四种糖蛋白分子组成的高度有序结构,它与卵母细胞的成熟、受精、胚胎发育及妊娠结局等预后紧密关联。许多生殖中心实验室发现有些患者的卵出现全部或部分的透明带异常,而且不同实验室发现的透明带异常类型各异。研究发现这些透明带异常与卵母细胞受精、胚胎发育及临床结局有一定的相关性。本文综述了关于透明带异常及其对卵母细胞受精、胚胎发育潜能和临床结局的影响的研究进展。