Pools Of Embryos Research Articles

Early Cleavage of Embryos to Two-Cell Stage Is Associated With Improved Progression to Blastocyst Stage, Implantation and Pregnancy Rates

Objectives: Recently embryos that cleave early were found to result in higher pregnancy rates. In the present study we compared the progression of the cleavage stage embryos to the blastocyst stage, implantation, pregnancy and abortion rates between cycles with and without early cleaved embryos.Design: Prospective study.Materials and Methods: Embryo cleavage to the two-cell stage approximately 27 hours after insemination was defined as early cleavage. Early cleavage was observed in 48 of 78 cycles. Excess embryos were left in sequential media to observe the rate of blastocyst formation.Results: Implantation and pregnancy rates were significantly higher in the early cleavage group. Excess embryos derived from the pool showing at least one embryo with early cleavage progressed to the blastocyst stage at a significantly higher rate. Tabled 1No early cleavageEarly cleavagePNo of cycles3048Mean age of patients34.233.1NSMean no of oocytes11.912.8NSFertilization rate74.877.3NSEarly two cells025.6<0.05Clevage rate95.996.8NSNo of embryos transferred3.94.0NSImplantation rate5.117.9<0.01Clin pregnancy rate16.7%47.9%<0.05Abortion rate20%13%NSBlastocyst progression25%56.7%<0.05 Open table in a new tab Conclusion: These findings suggest that early clevage of embryos can be used as good marker of implantation potential of cleavage stage embryos. Furthermore, sibling embryos in the pool of embryos showing early cleavage progress to the blastocyst stage at a higher rate suggesting that the potential of the embryo is evident from the early stages of development. Objectives: Recently embryos that cleave early were found to result in higher pregnancy rates. In the present study we compared the progression of the cleavage stage embryos to the blastocyst stage, implantation, pregnancy and abortion rates between cycles with and without early cleaved embryos. Design: Prospective study. Materials and Methods: Embryo cleavage to the two-cell stage approximately 27 hours after insemination was defined as early cleavage. Early cleavage was observed in 48 of 78 cycles. Excess embryos were left in sequential media to observe the rate of blastocyst formation. Results: Implantation and pregnancy rates were significantly higher in the early cleavage group. Excess embryos derived from the pool showing at least one embryo with early cleavage progressed to the blastocyst stage at a significantly higher rate. Tabled 1No early cleavageEarly cleavagePNo of cycles3048Mean age of patients34.233.1NSMean no of oocytes11.912.8NSFertilization rate74.877.3NSEarly two cells025.6<0.05Clevage rate95.996.8NSNo of embryos transferred3.94.0NSImplantation rate5.117.9<0.01Clin pregnancy rate16.7%47.9%<0.05Abortion rate20%13%NSBlastocyst progression25%56.7%<0.05 Open table in a new tab Conclusion: These findings suggest that early clevage of embryos can be used as good marker of implantation potential of cleavage stage embryos. Furthermore, sibling embryos in the pool of embryos showing early cleavage progress to the blastocyst stage at a higher rate suggesting that the potential of the embryo is evident from the early stages of development.

MRNAs encoding aquaporins are present during murine preimplantation development.

The present study was conducted to investigate the mechanisms underlying fluid movement across the trophectoderm during blastocyst formation by determining whether aquaporins (AQPs) are expressed during early mammalian development. AQPs belong to a family of major intrinsic membrane proteins and function as molecular water channels that allow water to flow rapidly across plasma membranes in the direction of osmotic gradients. Ten different AQPs have been identified to date. Murine preimplantation stage embryos were flushed from the oviducts and uteri of superovulated CD1 mice. Reverse transcription-polymerase chain reaction (RT-PCR) methods employing primer sets designed to amplify conserved sequences of AQPs (1-9) were applied to murine embryo cDNA samples. PCR reactions were conducted for up to 40 cycles involving denaturation of DNA hybrids at 95 degrees C, primer annealing at 52-60 degrees C and extension at 72 degrees C. PCR products were separated on 2% agarose gels and were stained with ethidium bromide. AQP PCR product identity was confirmed by sequence analysis. mRNAs encoding AQPs 1, 3, 5, 6, 7, and 9 were detected in murine embryos from the one-cell stage up to the blastocyst stage. AQP 8 mRNAs were not detected in early cleavage stages but were present in morula and blastocyst stage embryos. The results were confirmed in experimental replicates applied to separate embryo pools of each embryo stage. These results demonstrate that transcripts encoding seven AQP gene products are detectable during murine preimplantation development. These findings predict that AQPs may function as conduits for trophectoderm fluid transport during blastocyst formation.

P-156. The application of CE-LIF in quantifying the endogenous amino acid pool of mouse embryos

P-156. The application of CE-LIF in quantifying the endogenous amino acid pool of mouse embryos

Analysis of variation in relative mRNA abundance for specific gene transcripts in single bovine oocytes and early embryos

Variation in the abundance of a specific gene transcript was assessed in single bovine oocytes and in vitro–derived blastocysts. Transcripts encoding the Na+,K+-ATPase α1 subunit were detected by reverse-transcription polymerase chain reaction (RT-PCR) and quantified relative to an exogenously supplied rabbit α-globin mRNA using laser-induced fluorescence capillary electrophoresis (LIF-CE). The precision of this relative abundance (RA) calculation was predicted and shown to resolve 2-fold differences in transcript abundance between individual blastocysts and predicted in oocytes to resolve 3-fold differences. The RA of the α1 subunit transcript differed by 2- to 3-fold among blastocysts, and 3- to 6-fold among oocytes. Comparison of a general population of oocytes with blastocysts revealed little overlap in RA values between the two groups, with a 8- to 14-fold increase in the mean RA for each group with development observed in two successive experiments (P ≤ 0.05). In contrast, oocytes selected for their developmental competence on the basis of morphologic criteria exhibited only a 1.6- to 1.7-fold developmental increase when the assay was performed on cDNA generated from either embryo pools (n = 6 versus 6) or individuals (n = 7 versus 7), respectively. These results provide the first characterization of the degree of heterogeneity in the abundance of a specific mRNA transcript among individual mammalian oocytes and preimplantation embryos and demonstrate that transcript relative abundance can be correlated with bovine oocyte morphology. Mol. Reprod. Dev. 49:119–130, 1998. © 1998 Wiley-Liss, Inc.

Ontogeny of somatolactin mRNA in the gilthead sea bream Sparus aurata

Expression of somatolactin (SL) gene during embryonal and early stages of larval development of the teleost Sparus aurata was determined by Northern blot analysis. Poly(A+)RNA was prepared from a pool of embryos collected at early and late stages or from larvae collected on different days after hatching. When hybridized to Sparus aurata SL cDNA, SL specific mRNA was seen both in embryos and in larvae. Levels of SL increased from day 1 onwards and reached the highest levels on day 21. Re-hybridization with Sparus aurata growth hormone (GH) cDNA revealed GH specific mRNA first on day 6 post-hatching. Levels of GH increased to maximal levels on day 10 and then decreased on days 15 and 21, thereby confirming the pattern of GH expression (Funkenstein and Cohen 1996). The patterns of SL and GH gene expression are different in gilthead sea bream during embryonal and larval development. Furthermore, the presence of SL transcript in embryos, prior to pituitary gland development, suggests the possibility that SL mRNA of maternal origin may be present in the oocyte.

Coordinate expression of splice variants of the murine pregnancy-specific glycoprotein (PSG) gene family during placental development.

The human and murine pregnancy-specific glycoprotein (PSG) gene families encode a large number of closely related proteins which are abundantly expressed in the fetal trophoblast and secreted into the maternal circulation. Although the presence of a well conserved tripeptide sequence His or Arg-Gly-Asp or Glu or Lys (H/RGD/E/K) similar to the RGD motif found in extracellular matrix proteins hints towards a possible interaction with integrin-type receptors, the function of this group of proteins related to the carcinoembryonic antigen family is still unknown. It is also not clear whether the various members of the PSG family exert the same function. Here we describe the cloning of two splice variants of Cea4 (Cea4a, Cea4b), a murine PSG family member, which lacks the RGD-related consensus motif. Cea4a, like most of the other rodent PSG members, is composed of three immunoglobulin (Ig) variable-like domains (N1-N3) and and one Ig constant-like domain (A). In contrast, Cea4b lacks the N2 domain (N1N3A), demonstrating for the first time that PSG isoforms produced by alternative splicing also exist in mice. The mRNAs coding for Cea4a and Cea4b exhibit the same expression kinetics during placental development as found for two other murine PSGs, Cea2 and Cea3, which contain the RGD-like motif. Expression starts after day 12.5 of embryonic development (E12.5) and maximum steady-state levels are reached around E15.5-E17.5 as determined by RNase protection analyses. At E17.5, PSG transcripts can be detected exclusively in the spongiotrophoblast of the placenta. In addition, PCR analyses revealed that Cea2, Cea3, and Cea4 transcripts are also found in RNA from a pool of embryos (E12-E15) but are absent from a number of adult tissues tested (kidney, lung, testis, ovary, liver, brain, thymus, heart, spleen). These results indicate that the various PSG isoforms exert their function(s) at the same time during placental and embryonic development.

Transcription of Y‐ and X‐linked genes in preimplantation ovine embryos

Because male ovine embryos develop faster than female embryos, the transcription of SRY and ZFY, two genes located on the Y chromosome, was examined in preimplantation stages using the reverse transcriptase polymerase chain reaction (RT-PCR). RNA was extracted from pools of ovine embryos matured and fertilized in vitro then cultured in synthetic oviduct fluid medium and recovered from 24 to 207 hr post-insemination (two-cell up to hatched blastocyst stage). Since primers used to amplify ZFY also amplify the homologue ZFX, located on the X chromosome, transcripts were differentiated by digestion with restriction enzymes. ZFY and ZFX transcripts were present in all stages examined following RT-PCR, whereas transcripts for SRY were undetectable in all investigated stages following either RT nested PCR or Southern analysis. The presence of ZFY transcripts suggests that Y chromosome is transcriptionally active during early ovine preimplantation development. The possible relationship between a faster growth of male embryos and the transcription of Y-linked genes at early stages of development is discussed. © 1996 Wiley-Liss, Inc.

Transcription of Y- and X-linked genes in preimplantation ovine embryos.

Because male ovine embryos develop faster than female embryos, the transcription of SRY and ZFY, two genes located on the Y chromosome, was examined in preimplantation stages using the reverse transcriptase polymerase chain reaction (RT-PCR). RNA was extracted from pools of ovine embryos matured and fertilized in vitro then cultured in synthetic oviduct fluid medium and recovered from 24 to 207 hr post-insemination (two-cell up to hatched blastocyst stage). Since primers used to amplify ZFY also amplify the homologue ZFX, located on the X chromosome, transcripts were differentiated by digestion with restriction enzymes. ZFY and ZFX transcripts were present in all stages examined following RT-PCR, whereas transcripts for SRY were undetectable in all investigated stages following either RT nested PCR or Southern analysis. The presence of ZFY transcripts suggests that Y chromosome is transcriptionally active during early ovine preimplantation development. The possible relationship between a faster growth of male embryos and the transcription of Y-linked genes at early stages of development is discussed.

Measurements of the specific activity of the nucleoside triphosphate pool of sea-urchin embryos following 8- 3H-guanosine administration

Abstract The nucleoside triphosphate (NTP) pool during the early development of sea-urchin, Paracentrotus lividus, was measured using high-performance liquid chromatography (HPLC). Each NTP pool was measured at four stages between the eight-blastomere stage and the early blastula stage, as was the specific activity of each NTP following an initial administration of 8- 3H-guanosine. Unexpectedly high UTP (uridine-5′-triphosphate) concentrations and radioactivity levels in UTP were observed. Studies of the sea-urchin, Strongilocentrotus purpuratus, also revealed elevated levels of 8- 3H-guanosine incorporation into UTP. The present procedure was found to be suitable for the unequivocal identification of UTP.

Cytoplasmic distributions of translatable messenger RNA species and the regulation of patterns of protein synthesis during sea urchin embryogenesis

The cytoplasmic messenger RNA population of eggs and embryos of Strongylocentrotus purpuratus was characterized by cell-free translation and two-dimensional electrophoresis. The timing and extent of changes in patterns of translation products correspond closely to changes during development in patterns of protein synthesis in vivo. The increased synthesis of most up-regulated proteins, including those identified as tissue specific, is due to concommitant increases in translatable mRNA in the cytoplasm. In a few exceptional cases, notably the mRNAs encoding actin and the heat-shock protein hsp90, stored maternal mRNAs are not rapidly recruited into polysomes upon fertilization; instead, these mRNAs remain enriched in the pool of free RNPs until they appear on polysomes and become actively translated in vivo during early cleavage. It is thus likely that selective translational activation of maternal mRNA is required for these rare examples of increased protein synthesis during early development. The free RNP pool of early embryos apparently serves as a reservoir for mRNAs later recruited into polysomes. Analysis by translation and hybridization of cloned cDNA probes to RNA blots indicates that a few mRNA species are always relatively enriched in the free RNP pool of posthatching embryos and do not serve as a store of mRNA for later translation.

Metabolism of Glucose by Preimplantation Mouse Embryos in the Presence of Glucagon, Insulin, Epinephrine, cAMP, Theophylline and Caffeine

Neither insulin nor epinephrine influenced the incorporation of glucose into the acid-soluble or acid-insoluble glycogen pool of mouse embryos at the morula-early blastocyst stage during 5 h culture in the presence of radiolabelled glucose. During a 5 h chase culture of pulse-labelled embryos at this stage of development, acid-soluble glycogen labelled during the pulse was not utilized by the embryo but acid-insoluble glycogen was reduced. Addition of glucagon, insulin, epinephrine, cAMP, theophylline or caffeine during chase culture had no effect on the turnover of label in the glycogen pools of the embryo. These results indicate that the turnover of embryonic glycogen observed in vivo is not due to the direct effect of the hormones that regulate glycogen metabolism in the mother. Insulin was found to stimulate incorporation of glucose into non-glycogen macromolecules during both pulse and chase culture. Thus, whilst an effect of insulin on glycogen metabolism was absent, the anabolic effects of this hormone appear to have been expressed in the embryo at this stage of development.

Changes in the quantity of histone and actin messenger RNA during the development of preimplantation mouse embryos

Actin and histone H3 mRNA levels in mouse eggs and early embryos have been measured by use of recombinant DNA probes having sequence homology to these mRNA species. Total nucleic acid was extracted from pools of unfertilized eggs, two-cell embryos, eight-cell embryos, and blastocysts. The nucleic acids were resolved electrophoretically, bound to diazotized paper following Northern transfer, and hybridized with 32P-labeled histone or actin DNA probes. Our findings demonstrate that there is a maternal store of histone and actin mRNA in the unfertilized egg but that this mRNA pool is reduced roughly 10-fold on an embryo basis by the mid-two-cell stage. Following this reduction of maternal mRNA, histone and actin mRNA accumulation from the eight-cell cleavage stage to the blastocyst increases proportionally to cell number and appears to be controlled by zygote genome transcription.

NAD turnover during early development of Xenopus laevis

The NAD pools of Xenopus laevis oocytes and early embryos can be radioactively labelled by microinjection of [ adenine- 3H]NAD. This technique is used to study the metabolism of NAD in oocytes and during early development. The rate at which NAD is degraded in vivo has been monitored by determining the rate of transfer of adenine residues from the NAD pool into other nucleotides and polynucleotides. In oocytes, NAD turnover is extremely slow, with a half-life of about 400 h. NAD turnover increases dramatically after fertilisation, and the half-life of the compound decreases to 37 h in 5-h-old embryos and to 10 h in 40-h-old embryos. 2 mM 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase, reduces the NAD turnover rate by about 20%, whereas 5 mM isonicotinic acid hydrazide, a specific inhibitor of NAD glycohydrolase, produces no significant inhibition. This indicates that a significant fraction of the considerable NAD turnover observed involves poly(ADP-ribose) polymerase. Our results indicate that poly(ADP-ribose) polymerase is active during early development and suggest that this activity may be involved in one or more aspects of the nuclear metabolism of the embryo.

The uptake of valine and cytidine by sea-urchin embryos and its relation to the cell surface.

ABSTRACT The rate of uptake of [14C] valine and [3H]cytidine into the metabolic pool of early embryos of the sea urchin Paracentrotus lividus was measured by giving 5-min pulses of these precursors. The uptake rate rose sharply after fertilization, reached a maximum before first cleavage and then stayed constant until at least the fifth interphase. There were no changes in uptake rate at cell division. A longer-term experiment with valine showed that the uptake rate remained approximately constant until the pluteus stage, and was not altered for the first 10 h by a actinomycin D. Continuous labelling experiments indicated that the uptake rate may be controlled by the level of precursor in the pool. The following experiments indicated that the uptake was by specific transport mechanisms. Both precursors showed an uptake inhibited by competitors, an apparent concentration within the cells, and no back-exchange. In addition, the uptake of valine was inhibited by dinitrophenol and azide, and showed a concentration response with Michaelis-Menten kinetics. Valine uptake was not inhibited by puromycin added at fertilization, though it was somewhat affected if the eggs were pretreated. It is suggested that the transport mechanisms are mediated by specific carrier molecules at the cell surface. These mechanisms are activated at fertilization through links with metabolism. There is no change in the number of carriers per embryo throughout early development. Since the number of cells and the total cell surface are increasing, this implies that the number of carriers per cell or per unit of surface is decreasing. There is a possible reason for this differentiation of the cell surface.

THE YIELD OF RABIES VIRUS IN THE CHICK EMBRYO

Rabies virus was inoculated intracerebrally in 8 day old chick embryos and the virus activity of pools of embryos titered after incubation at 35-36 degrees C. for different lengths of time. The virus reached a titer of 10(-5.5) to 10(-6.5) in 5 to to 6 days and remained at a rather constant level until the 9th day of the infection. The report by Kligler and Bernkopf that rabies virus will invade the very young embryo after inoculation on the chorioallantoic membrane was confirmed.