Cloning of bovine embryos by multiple nuclear transfer

The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2 +/- 4.3, twice: 19.8 +/- 9.2 and three times: 30.0 +/- 14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts,respectively.     

Reconstruction of enucleated mouse germinal vesicle oocytes with blastomere nuclei

We have investigated the possibility that mitotic nuclei originating from preimplantation stage embryos and placed in the oocyte cytoplasm can undergo remodelling that allows them to undergo meiosis in the mouse. To address this question, we have used enucleated germinal vesicle (GV) ooplasts as recipients and blastomeres from the 2-, 4- or 8-cell stage as nuclear donors. We employed two methods to obtain ooplasts from GV oocytes: cutting and enucleation. Although efficiency of the reconstruction process was higher after enucleation than after cutting (90% and 70% respectively), the developmental potential of the oocytes was independent of how they had been produced. Nuclei from the 2-, 4-, or 8-cell stage embryos supported maturation in about 35%, 55% and 60% of cases, respectively. The time between nuclear envelope breakdown and the first meiotic division was shortened by up to 5 h in reconstructed oocytes, a period equivalent to the mitotic division of control blastomeres. About one-third of oocytes reconstituted with blastomere nuclei divided symmetrically instead of extruding a polar body; however, in the majority of them metaphase plates were found, suggesting that reconstructed oocytes (cybrids) underwent a meiotic rather than mitotic division. The highest percentage of asymmetric divisions accompanied by metaphase plates was found in cybrids with 8-cell-stage blastomere nuclei, suggesting that the nuclei from this stage appear to conform best to the cytoplasmic environment of GV ooplasts. Our results indicate that the oocyte cytoplasm is capable of remodelling blastomere nuclei, allowing them to follow the path of the meiotic cell cycle.     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

室温下保存的小鼠屠体卵巢GV卵的发育能力

将ICR系雌性小鼠处死并在10℃、15℃、20℃和25℃下依次保存8、14、24和48 h后,采集其体内的卵巢GV期卵,采用常规方法进行体外成熟和体外受精,获得的2细胞期胚经体外培养或胚胎移植观察其发育能力.其结果,在10℃下保存24 h、15℃下保存14 h、20℃下保存8h和25℃下保存4 h后,其体内附有卵丘细胞的GV卵的体外成熟-体外受精后的2细胞率分别为14%、9%、10%和10%,随着保存温度的提高和保存时间的延长,带有颗粒细胞GV期卵的比率明显降低,同时其GV期卵经体外成熟及体外受精后的2细胞率明显降低.在20℃下保存24 h和25℃下保存14 h时,难以获得形态正常的GV期卵;体外受精获得的2细胞期胚经体外培养,总体上有64%的胚胎发育至扩张囊胚,未见有保存温度和保存时间的显著影响,且利用在15℃保存8 h后的GV卵获得2细胞期胚的移植获得正常新生小鼠.上述结果表明,雌性动物室温条件下死亡后,若能短时间及时采集其体内GV期卵并体外成熟、体外受精,体外培养及胚胎移植技术,就有可能获得新生后代.     

Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.     

Effect of ooplast activation on the development of oocytes following nucleus transfer in cattle

We assessed the effect of ooplast (enucleated oocytes) activation prior to receiving a donor nucleus on the development of nucleus transferred oocytes in cattle. The ooplasts were activated by electric stimulus at 30, 33, 36 and 39 h after being placed in culture medium for meiotic maturation. The activated ooplasts were further cultured in vitro, for a total 42 h from the beginning of maturation, 16- to 32-cell stage embryos produced by in vitro fertilization were used as donor embryos. The nucleus transferred oocytes were co-cultured with bovine oviductal epithelial cells in vitro. The fusion rate was not different between the activated (90%) and aged (94%) ooplasts 42 h after culture. Activated ooplasts receiving a donor nucleus showed a higher developmental rate than the aged ooplasts. Maximal development of the oocytes was obtained if the ooplast was activated at 9 h prior to receiving a donor nucleus. Thirty-nine percent developed to morulae and 24% to blastocysts. This compares (P<0.01) with 13% of the aged ooplasts developing to morulae and 8% to blastocysts. Of the activated ooplasts at 3, 6 and 12 h prior to fusion with a donor blastomere, 12, 16 and 13% developed to blastocysts, respectively. Of the 17 recipient cows receiving nucleus transferred embryos, 9 (53%) were diagnosed pregnant by palpation per rectum examination, and 3 normal offspring were obtained.     

Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

Maturity at collection and the developmental potential of rhesus monkey oocytes

The purpose of this study was to evaluate the in vitro fertilizability of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 h after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin [hCG] or 3 x 100 micrograms gonadotropin-releasing hormone [GnRH]). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstimulation, 45 germinal vesicle-intact (GV), 106 metaphase I (MI), and 24 metaphase II (MII) oocytes were collected and cultured in vitro. Upon reaching MII, oocytes were inseminated with 5 x 10(4) motile sperm/ml. Twenty-four percent of GV oocytes cultured in vitro matured to MII with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured to MII in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MII stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early luteal phase (1-3 days post LH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.     

Nuclear maturation and development of IVM/IVF canine embryos in synthetic oviductal fluid or in co-culture with buffalo rat liver cells

In vitro embryo production in the domestic bitch can provide valuable insights for conservation of endangered canids. In the present study, canine oocytes underwent in vitro maturation (IVM) in simple or complex media, with production of in vitro matured and fertilized (IVM/IVF) canine embryos. Cumulus–oocyte complexes (COCs) were harvested from ovaries by slicing and subjected to IVM in four media (SOF, TCM 199, Ham-F10, and DMEM/F12). After culture for 48 h, oocytes were stained and examined for nuclear maturation. There were no significant differences in the mean (±S.D.) percentage of nuclear maturation (metaphase II) of oocytes cultured in SOF (18.6 ± 7.6%), TCM 199 (18.3 ± 4.5%), Ham-F10 (13.9 ± 8.2%), or DMEM/F12 (11.9 ± 4.2%). For assessment of embryo development, oocytes were matured for 48 h in synthetic oviductal fluid (SOF), fertilized with frozen-thawed sperm, and presumptive zygotes were cultured for 7 d, either in SOF or as co-cultures with BRL cells in TCM 199. Percentages of IVM/IVF oocytes that developed to the 2-cell, 3–4-cell, and 5–7-cell stages were higher (P < 0.05) following culture in SOF versus BRL cell co-cultures (33.6 ± 1.2% vs 13.7 ± 1.2%, 24.7 ± 0.5% vs 8.7 ± 1.1%, and 15.1 ± 2.2% vs 4.3 ± 1.3%, respectively). However, none of the embryos developed beyond the 8–16-cell stage. In conclusion, simple or complex media successfully induced resumption of meiosis and nuclear maturation of canine oocytes. Furthermore, SOF supported in vitro development of IVM/IVF canine embryos to the 8–16-cell stage.     

Cytoplasmic modification of the nuclear lamina during pronuclear-like transformation of mouse blastomere nuclei.

During the successive interphases of cleaving mouse embryos the nuclear periphery diminishes its reactivity to anti-lamin A and C antibodies. This developmentally regulated characteristic can be modified by exposure of the blastomere nuclei to metaphase II (M II) oocyte cytoplasm followed by activation. In the current study we define the cytoplasmic conditions necessary for this modification of 8-cell and 16-cell stage nuclei in hybrids obtained by fusion with metaphase II arrested oocytes, oocytes at various time points after parthenogenetic activation, naturally fertilized eggs (zygotes) and interphase 2-cell embryo blastomeres. The intensity of fluorescence obtained with anti-lamins A/C in the blastomere nuclei increases as a result of fusion with freshly activated oocytes or early zygotes (first 3.0-5.5 h in the case of parthenogenetic activation), and not when eggs or 2-cell blastomeres advanced in interphase are used as partners for fusion. This transformation of the A/C lamin pattern is correlated with the ability to promote pronucleus-like growth of blastomere nuclei in hybrids. Blastomere nuclei introduced into M II-arrested oocytes undergo premature chromatin condensation and dissolution of the nuclear lamina. The results are discussed with regard to certain particularities of the first embryonic interphase of the mouse and the potential involvement of nuclear lamins in pronuclear growth.     

Injection of frozen-thawed porcine first polar bodies into enucleated oocytes results in fertilization and embryonic development

The objective was to determine whether enucleated oocytes injected with frozen porcine first polar bodies (pPB1s) could be fertilized and developed into viable embryos in vitro. Metaphase II (MII) oocytes with pPB1s were frozen (vitrified) and stored for 2 mo. The pPB1s were isolated from thawed MII oocytes and injected into enucleated recipient oocytes by micromanipulation. All recipients injected with thawed pPB1s were fertilized by intracytoplasmic sperm injection (ICSI), and the resulting recombinant zygotes were incubated to assess their developmental competence in vitro. Furthermore, double-antibody immunohistochemistry was used to verify that the nucleus of the pPB1 participated in fertilization and supported embryonic development. Porcine embryos (2- to 8-cell stage) were obtained from the recombinants. The average in vitro cleavage rate of 2-, 4-, and 8-cell stage recombinant embryos was 25.3, 17.7, and 9.3% (P < 0.05), respectively. Chromosomes in the labeled pPB1 participated in the formation of the two blastomere nuclei of 2-cell stage embryos derived from recombinant oocytes. In conclusion, nuclear materials of frozen-thawed pPB1 supported oocyte fertilization and subsequent embryonic development, thereby providing a new way to use frozen PB1s for preservation and reproduction of mammals.     

Bovine oocyte maturation, fertilization and further development in vitro and after transfer into recipients

Bovine oocytes were aspirated from ovaries within 1.6 to 2 hours after slaughter. They were then matured in TCM-199 medium drops under oil in CO(2)/air incubator at 39 degrees C. Spermatozoa were capacitated in SP-TALP medium with heparin. The percentage of embryos that developed in vitro to the 4- and 6- cell stages 48 hours post insemination and then reached the morula or blastocyst stage was 64.3% and 59.2%, respectively, while only 3.6% of the embryos that reached the 2-cell stage became morula or blastocysts. An average of 6.3+/-3.2 total in vitro fertilized embryos per cow were obtained (range 2 to 11). Maturation of bovine oocytes in vitro for 18 or 24 hours did not influence the percentage of cleaved embryos (71.0 and 75.9%, respectively) or that developed to the blastocyst stage (25.6 and 24.2%, respectively). The use of reindeer blood serum for in vitro culture of immature bovine oocytes and of dividing of embryos gave the following results: 57.4% of the oocytes cleaved after fertilization and 16.2% developed further to the blastocyst stage. In contrast in the control group, where cow serum was used, the values were 73.4% and 24.8%, respectively. Rabbit oviduct epithelium cell monolayers were able to support the development of 16.3% of the cleaved bovine embryos to the blastocyst stage as compared with 24.0% of the embryos on cow oviduct epithelium cell monolayers. After nonsurgical transplantation, 12 calves were produced from 91 in vitro fertilized embryos.     

小鼠卵母细胞体外成熟、体外受精的效果观察

目的　研究不同培养条件对小鼠卵母细胞体外成熟及体外受精率的影响。方法　小鼠卵母细胞分别在含有FSH、BSA和胰岛素的培养液中体外成熟,在Whitten 氏液中体外受精,比较体外成熟率、体外受精率。结果　1- 裸卵(DO) 的体外成熟率、体外受精率(81-4% ,31-0 % ) 均高于卵丘卵母细胞复合体(COC)(48-6 % ,27-1% ) 。2- 在培养液中添加FSH、胰岛素和BSA,卵母细胞的体外成熟率为77-9 % ,82-3% 、60-7% ;体外受精率为77-2 % 、72-6 % 、26-7% ;2 - 细胞率为49-2 % 、34-2 % 、10-0% 。胰岛素组的卵母细胞IVM 率最高,但IVF率、2 - 细胞率低于FSH 组。3- 添加BSA的两组的体外受精率只有26-7 % 、25-8 % ,显著低于其他组,其体外成熟率也较添加FSH 和胰岛素的组成。4- 排出第一极体(PbI) 的卵母细胞的体外受精率和2 - 细胞率(85-9 % ,22-4% ) 均高于GV期卵母细胞(71-1 % ,12-9 % ) 。结论　1- 卵丘卵母细胞(COC) 较裸卵(DO) 的体外成熟率、体外受精率都低,差异显著(P成熟< 0-01;P受精< 0-05) 。2-FSH 和胰岛素均能提高小鼠卵母细胞的体外成熟率、体外受精率。3-BSA可以降低小鼠卵母细胞体外受精率,差异极显著。4-GV 期卵母细胞的体外受精率显著低于体外培养的排出第一极体的卵母细胞(P2 - cell < 0-05,P受精<0-05)     

Bovine parthenogenetic blastocysts following in vitro maturation and oocyte activation with ethanol

The appropriate in vitro bovine oocyte maturation and ethanol activation conditions for preimplantation bovine embryo parthenogenetic development to the blastocyst stage were investigated. A 7% ethanol concentration significantly enhanced (P<0.05) the proportion of activated, in vitro-matured bovine oocytes (7% ethanol, 83.4 +/- 3.2% versus 0% ethanol, 63.9 +/- 2.0%). The proportion of activated oocytes was significantly higher (P<0.05) by treatment with 7% ethanol for a minimum of 2 minutes (2 minutes, 89.8 +/- 4.0% versus 0.5 minutes 63.4 +/- 4.9%). Oocyte maturation for periods ranging from 30, 34, 38 and 44 hours resulted in a significant increase (P<0.05) in the proportion of activated oocytes, and in oocytes displaying 2 or 3 pronuclei versus oocytes matured for 26 hours. The proportion of cleaved, activated oocytes (2-cell stage), 4 -cell stage and parthenogenetic morula/blastocysts was significantly higher (P<0.05) within the 34-hour oocyte maturation treatment group. Although the 44-hour oocyte maturation treatment group displayed the highest proportion of activated oocytes with 2 pronuclei, it did not display the highest cleavage frequency, possibly due to the effects of postovulatory aging. Several morphologically normal parthenogenetic bovine blastocysts developed from oocytes that were in vitro matured for 34 hours. The ability to produce such parthenogenetic embryos will eventually facilitate investigation into the role(s) of the maternal and paternal genomes during bovine early development.     

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (相似文献   

Embryonic development of fertilized rat oocytes induced to mature by an analogue of gonadotrophin-releasing hormone

A gonadotrophin-releasing hormone analogue (GnRHa) was administered to hypophysectomized immature rats. Postovulatory mature oocytes obtained under these conditions were exposed in vitro to a sperm suspension for fertilization. Developmental ability of the fertilized ova was studied by transfer of the 2-cell stage embryos to oviducts of foster mothers. The potential of oocytes, undergoing maturation in response to GnRHa, to develop into 2-cell embryos was similar to that of oocytes stimulated by hCG (76.4% and 83.1% respectively). The 2-cell stage embryos obtained from such oocytes were equally able to implant in the uteri of foster mothers (25.7% and 21.2% respectively) and subsequently develop into live embryos (15.3% and 15.2%, respectively, at Day 20 of pregnancy).     

Growth of mouse oocytes to maturity from premeiotic germ cells in vitro

In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12-14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6-7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16-18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.     

A separate procedure of fusion and activation in an ear fibroblast nuclear transfer program improves preimplantation development of bovine reconstituted oocytes

This study was conducted to examine whether preimplantation development of bovine ("HanWoo," Bos Taurus corcanae) oocytes reconstituted with ear fibroblasts could be improved by a modified procedure of fusion and activation. In Experiment 1, enucleated oocytes were reconstituted with ear fibroblast by a combined procedure of electric fusion and activation at either 24 or 28 hours after IVM. The 28 hours reconstitution yielded more blastocysts (4% vs. 21%, P = 0.0025) and higher ratio of blastocysts per 2-cell embryos (0.05 vs. 0.25, P = 0.003) than the 24 hours. In Experiment 2, enucleated oocytes were reconstituted by one of the three fusion and activation protocols; 1) a combined procedure of electric fusion and activation at 28 hours after IVM, 2) a combined procedure of electric fusion and chemical activation at 28 hours, and 3) a separate procedure of electric fusion at 24 hours and chemical activation at 28 hours. When compared two combined procedures, chemical activation with 5 microM ionomycin for 4 minutes did not promote embryo development and significantly reduced the fusion rate (42% vs. 53%, P = 0.0395). However, significant (P < 0.0113) increases in the development to 2-cell (90% vs. 70 to 74%) and blastocyst (47% vs. 7 to 13%) stages and in the ratio of blastocysts per 2-cell embryos (0.52 vs. 0.11 to 0.18) were obtained by a separate procedure of electric fusion and chemical activation than by the combined procedures. This separate protocol did not reduce the fusion rate compared with the combined procedures (58%). In conclusion, improved development of oocytes reconstituted with ear fibroblasts was achieved by undergoing a separate procedure of electric fusion and chemical activation 4 hours apart.