Early cleavage to formation of the unilaminar blastocyst in the marsupial Antechinus stuartii: ultrastructure

1997 ◽  
Vol 9 (2) ◽  
pp. 201 ◽  
Author(s):  
Henry Sathananthan ◽  
Lynne Selwood ◽  
Isabel Douglas ◽  
Kamani Nanayakkara
Keyword(s):  
Smooth Endoplasmic Reticulum ◽  
Spindle Pole ◽  
Blastocyst Stage ◽  
Cell Junctions ◽  
Cell Stage ◽  
Transmission Electron ◽  
Embryonic Genome ◽  
Antechinus Stuartii ◽  
First Time

The development of Antechinus stuartiifrom the 2-cell stage to the blastocyst stage in vivo was examined by routine transmission electron microscopy. The 2–8-cell stages had a similar organization of organelles, whereas the 16- to 32-cell stages had pluriblast cells and trophoblast cells forming an epithelium closely apposed to the zona pellucida. Specialized cell–zona plugs were formed at the 8-cell stage, and primitive cell junctions appeared in later conceptuses. The cytoplasmic organelles included mitochondria, lysosomes, aggregates of smooth endoplasmic reticulum, lipid and protein yolk bodies and fibrillar arrays, possibly contractile in function. Nuclei had uniformly-dispersed dense chromatin. Nucleoli of 2–4-cell conceptuses were dense, compact and fibrillar, and those of 8-cell conceptuses and later conceptuses were finely granular and became progressively reticulated. The embryonic genome is probably not switched on before the 8-cell stage. Sperm tails were detected in cells in several early conceptuses. The yolk mass had the same organelles as cells. Centrioles were discovered for the first time in marsupial conceptuses. These were prominently situated at a spindle pole in a 32-cell blastomere and were associated with a nucleus and sperm tail at the 4-cell stage. It is very likely that the paternal centrosome is inherited at fertilization and perpetuated in Antechinus embryos during cleavage.

Synthesis and phosphorylation of uvomorulin during mouse early development

Development ◽  
1992 ◽  
Vol 115 (1) ◽  
pp. 313-318 ◽  
Author(s):  
M. Sefton ◽  
M.H. Johnson ◽  
L. Clayton
Keyword(s):  
Cell Adhesion ◽  
Early Development ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Blastocyst Stage ◽  
Embryonic Stage ◽  
Cell Stage ◽  
Mouse Oocytes ◽  
Maternal Mrna ◽  
Embryonic Genome

The cell adhesion molecule, uvomorulin, is synthesised in both the 135 × 10(3) M(r) precursor and 120 × 10(3) M(r) mature forms on maternal mRNA templates in unfertilized and newly fertilized mouse oocytes. Synthesis on maternal message ceases during the 2-cell stage to resume later on mRNA encoded presumptively by the embryonic genome. Uvomorulin is detectable by immunoblotting at all stages upto the blastocyst stage, but shows variations in its total amount and processing with embryonic stage. Whilst only trace levels of phosphorylated uvomorulin are detectable in early and late 4-cell embryos, uvomorulin in 8-cell embryos is phosphorylated.

scholarly journals The production of mature oocytes from adult ovaries following primary follicle culture in a marsupial

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 247-255 ◽  
Author(s):  
A Nation ◽  
L Selwood
Keyword(s):  
Normal Growth ◽  
Primary Follicle ◽  
Follicle Culture ◽  
Nuclear Maturation ◽  
Transmission Electron ◽  
Progressive Growth ◽  
Sminthopsis Macroura ◽  
First Time

A model marsupial culture system has been developed whereby individual primary follicles, obtained from adult ovaries, can be grown in vitro to the antral stage and oocytes retrieved from these follicles can achieve nuclear maturation (metaphase II) in the presence of LH. Primary follicles isolated from adult Sminthopsis macroura ovaries were cultured individually in one of four systems: microdrops under oil, upright, inverted, or roller culture. After 6 days of culture, cumulus–oocyte complexes (COCs) were excised from early antral follicles and incubated for an additional 24 h to assess meiotic competence and the effects of LH and lithium on oocyte maturation. Histology and transmission electron microscopy established normal in vivo standards and verified oocyte and follicular integrity following culture. On day 6 of culture, follicle viability was significantly greater in the inverted system (73%) than in the other three systems (10–46%). The inverted system was the most effective in supporting development with follicles demonstrating progressive growth during culture and showing antral signs by day 4. Meiotic resumption during COC culture was facilitated by LH, but hindered by lithium. The ability to resume meiosis and progress to metaphase II was equivalent in oocytes retrieved following follicle culture and those matured in vivo. This study highlights the importance of oxygen and nutrient availability during marsupial follicle culture, and demonstrates for the first time that primary follicles isolated from adult mammalian ovaries can undergo normal growth and development in vitro, to produce mature, meiotically competent oocytes.

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

42 DEVELOPMENT OF PIG EMBRYOS CLONED FROM DONOR CELLS TREATED WITH TRICHOSTATIN A

2008 ◽  
Vol 20 (1) ◽  
pp. 101 ◽  
Author(s):  
J. Li ◽  
Y. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
K. Villemoes ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Embryonic Genome ◽  
Donor Cells ◽  
Deacetylase Inhibitor ◽  
Vitro Development

Development to the blastocyst stage following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to a totipotent state. Reprogramming of the transferred somatic nuclei must be completed by the time normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, Enright et al. (2003 Biol. Reprod. 69, 896–901) reported that in vitro development of cloned cow embryos was improved by treatment of donor cells with a histone deacetylase inhibitor, TrichostatinA (TSA). So far, there are no reports available for adult pig fibroblast cells treated with TSA. The objective of this study was to investigate whether the development of handmade cloned embryos in pig could be improved by using TSA-treated donor cells. Adult pig fibroblast cells were treated with 100, 150, or 200 nm TSA for 24 h, compared to untreated controls, and were then used as donor cells. The cells were electrofused with handmade enucleated pig oocytes separately and were activated with calcium ionophore and cycloheximide. They were subsequently cultured in porcine zygote medium 3 (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) using the well of the well system (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Experiments were repeated 4 times and the data were analyzed with AVEDEV and t-test in Excel (Microsoft Excel 2007). The cleavage rates and the total cell numbers per blastocyst were similar between groups (P > 0.05), as shown in Table 1. However, the cloned blastocyst rate using donor cells treated with 100 nm TSA was higher than in the other groups (69.9 ± 4.7% v. 43.6 ± 4.3%, 43.1 ± 5.8%, or 46.6 ± 3.6%; P < 0.05), as shown in Table 1. These data suggest that proper TSA treatment for donor cells before somatic cloning improves the rate of development of porcine handmade cloned embryos to the blastocyst stage. Further research is needed to examine the in vivo development of embryos reconstructed with TSA-treated donor cells. Table 1. Developmental ability of cloned pig embryos derived fromTSA-treated donor cells

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 283-293 ◽  
Author(s):  
M. Popelková ◽  
P. Chrenek ◽  
J. Pivko ◽  
A.V. Makarevich ◽  
E. Kubovičová ◽  
...  
Keyword(s):  
Ultrastructural Level ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Hatching Rate ◽  
Transmission Electron ◽  
Cellular Debris ◽  
Or Gene ◽  
Rabbit Gene

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p<0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not siginificantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.

scholarly journals Production of viable piglets for the first time using sperm derived from ectopic testicular xenografts

Reproduction ◽  
2010 ◽  
Vol 139 (2) ◽  
pp. 331-335 ◽  
Author(s):  
Michiko Nakai ◽  
Hiroyuki Kaneko ◽  
Tamas Somfai ◽  
Naoki Maedomari ◽  
Manabu Ozawa ◽  
...  
Keyword(s):  
Testicular Tissue ◽  
Blastocyst Stage ◽  
Large Mammals ◽  
Immunodeficient Mice ◽  
Neonatal Piglets ◽  
Porcine Oocyte ◽  
Large Animals ◽  
First Time

Xenografting of testicular tissue into immunodeficient mice is known to be a valuable tool for facilitating the development of immature germ cells present in mammalian gonads. Spermatogenesis in xenografts and/or in vitro embryonic development to the blastocyst stage after ICSI of xenogeneic sperm has already been reported in large animals, including pigs; however, development of the embryos to term has not yet been confirmed. Therefore, in pigs, we evaluated the in vivo developmental ability of oocytes injected after ICSI of xenogeneic sperm. Testicular tissues prepared from neonatal piglets, which contain seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 133 and 280 days after xenografting, morphologically normal sperm were recovered, and a single spermatozoon was then injected into an in vitro matured porcine oocyte. After ICSI, the oocytes were electrostimulated and transferred into estrus-synchronized recipients. Two out of 23 recipient gilts gave birth to six piglets. Here, we describe for the first time that oocytes fertilized with a sperm from ectopic xenografts have the ability to develop to viable offspring in large mammals.

scholarly journals Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage

Zygote ◽  
1994 ◽  
Vol 2 (4) ◽  
pp. 281-287 ◽  
Author(s):  
Asangla Ao ◽  
Robert P. Erickson ◽  
Robert M.L. Winston ◽  
Alan H Handysude
Keyword(s):  
Mammalian Species ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Cell Stage ◽  
Gonad Differentiation ◽  
Embryonic Genome ◽  
Human Preimplantation Embryos

SummaryGlobal activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 193
Author(s):  
J. Caudle ◽  
C. K. Hamilton ◽  
F. A. Ashkar ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Male Embryo ◽  
Male Development ◽  
Embryonic Genome ◽  
Genome Activation

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

159 USE OF PHYTOHEMAGGLUTININ-L, AS AN AGGLUTINATOR AGENT, TO PRODUCE CHIMERAS OF IVF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 238
Author(s):  
I. P. Emanuelli ◽  
B. F. Agostinho ◽  
M. P. M. Mancini ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Stage Of Development ◽  
Fisher’S Exact Test

Embryonic chimeras have been used as a tool to understand embryogenesis and organogenesis, as well as to prove, in vivo, the pluripotency of the embryonic stem cells. One of the techniques used to obtain embryonic chimeras is aggregation, which can be performed with intact or half-embryos and in different stages of the development, produced by in vivo or in vitro systems and in different wells. However, its efficiency tends to reduce when advanced stages, such as morulae and blastocysts, are used. The aim of this work was to evaluate the effect of the treatment with an agglutinating agent (phytohemagglutinin-L; PHA) in the percentage of chimeras produced with IVF bovine embryos. Bovine ovaries (from abattoir) were used to obtain 270 COC that were matured in drops (90 μL) of TCM-199 bicarbonate medium, supplemented with 10% of FCS, and incubated in vitro for 22 to 24 h. The fertilization occurred in TALP-IVF medium, and the COC were maintained in the incubator for 18 h. After fertilization, the presumptive zygotes were transferred to SOF culture medium to in vitro culture. In vitro maturation, fertilization, and culture were performed under 38.5°C, 5% CO2 in air and saturated humidity. The chimerism by aggregation was tested between 2 intact (zona-free) 8- to 16-cell stage embryos in the presence (G1, n = 16) or absence of PHA (G2, n = 14) and between one half-morula and one half-blastocyst with (G3, n = 15) or without PHA (G4, n = 12). The embryos in groups G1 and G3 were treated with PHA in a concentration of 500 μLg mL-1 for 3 min. After PHA treatment, the pairs of embryos were allocated in wells, under previously described culture conditions, until expanded blastocyst stage could be observed (Day 7 of culture). At 24 h of culture, embryonic aggregation pairs were first evaluated to detect only cohesive masses of cells. The results (chimerism rate) were 62.5%, 42.9%, 40.0%, and 25.0%, respectively, for groups G1, G2, G3, and G4. There were no significant differences neither among groups (chi-square, P = 0.252) nor between G1 and G2 (P = 0.464), G3, and G4 (P = 0.683; Fisher’s exact test). Main effects as use of PHA (G1 + G3 v. G2 + G4, P = 0.284) and stage of embryos (G1 + G2 v. G3 + G4, P = 0.183; Fisher’s exact test) were not statistically significant. However, when all groups were compared, the power of the performed test (0.354) was below the desired power of 0.800 (i.e. one must be cautious in over-interpreting the lack of difference among them). In the conditions of this study, it was concluded that the treatment with PHA did not increase the rate of aggregation in the embryonic chimera production, even for half-embryos in advanced stage of development (morulae and blastocysts). Granted by FAPESP, Brazil: 06/06491-2 and 07/07705-9 (MFGN) and 07/04291-9 (MPMM).

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