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G Vajta

Publications and source records attributed to G Vajta.

At least 19 recordsLinked to original sources
PubMed2003

Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos.

The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphasized the need for basic knowledge about epiblast formation and gastrulation in bovine embryos. The aims of this study were to define stages of bovine post-hatching embryonic development and to analyse functional mechanisms of germ-layer formation. Embryos developed in vivo were collected after slaughter from superovulated cows on days 9, 11, 14 and 21 after insemination and processed for transmission electron microscopy (n = 26) or immunohistochemistry (n = 27) for potential germ-layer characterization (cytokeratin 8 for potential ectoderm; alpha-1-fetoprotein for potential endoderm; and vimentin for potential mesoderm). On day 9, the embryos were devoid of zona pellucida and presented a well-defined inner cell mass (ICM), which was covered by a thin layer of trophoblast cells (the Rauber's layer). Formation of the hypoblast from the inside of the ICM was ongoing. On day 11, the Rauber's layer was focally interrupted and adjacent underlying ICM cells formed tight junctions. The hypoblast, which formed a thin confluent cell layer, was separated from the ICM and the tropho-blast by intercellular matrix. The embryos were ovoid to tubular and displayed a confluent hypoblast on day 14. The epiblast was inserted into the trophoblast epithelium and tight junctions and desmosomes were present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells. In some embryos, the epiblast was more or less covered by foldings of trophoblast in the process of forming the amniotic cavity. Cytokeratin 8 was localized to the trophoblast and the hypoblast underlying the epiblast; alpha-1-fetoprotein was localized to most hypoblast cells underlying the trophoblast; and vimentin was localized to most epiblast cells. On day 21, the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites and allantois development. These embryos depicted the gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate and lateral plate mesoderm. Cytokeratin 8 was localized to the trophoblast, the hypoblast and the surface and neural ectoderm; and alpha-1-fetoprotein was localized to the hypoblast, but not the definitive endoderm, the intensity increasing with development. Vimentin was initially localized to some, but not all, cells positioned particularly in the ventral region of the primitive streak, to presumptive definitive endoderm cells inserted into the hypoblast, and to mesoderm. In conclusion, within 2 weeks of hatching, bovine embryos complete formation of the hypoblast and the epiblast, establishment of the amniotic cavity, ingression of epiblast cells for primitive streak formation, involution of cells through the node and the streak for endoderm and mesoderm fomation, neurulation and differentiation of the mesoderm. The recruitment of cells from the epiblast to form the primitive streak as well as the endoderm and mesoderm is associated with expression of the intermediate filament vimentin.

Animals↗
PubMed2002

Calves born after open pulled straw vitrification of immature bovine oocytes.

The aim of this study was to evaluate the developmental capacity of immature bovine oocytes after vitrification with 20% ethylene glycol (EG)+20% dimethyl sulfoxide (Me(2)SO) and 0.5M sucrose (SUC), by open pulled straw (OPS) technology. The effect of treatment with cytochalasin D before vitrification was also examined. No differences were observed in cleavage and blastocyst rates among the group vitrified without cytochalasin D treatment (Vitri) (49.0% and 6.1%) and that with cytochalasin D treatment before vitrification (CDVitri) (46.4% and 3.6%), but both were lower (P<0.05) than the unvitrified control group (85.1 and 45.9%). Calves were obtained after transfer of fresh and vitrified blastocysts from the Vitri group and after transfer of vitrified blastocysts from the CDVitri group. Cytochalasin D treatment does not improve the development of immature bovine vitrified oocytes. The results show that a small proportion of immature oocytes vitrified with this technology are fully competent to produce blastocysts, which may be transferred immediately or vitrified before transfer, and go on to develop healthy offspring.

Animals↗
PubMed2001

Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos.

The yield and quality of (a) parthenogenetic blastocysts produced by two activation treatments (cycloheximide [CHX] or 6-dimethylaminopurine [DMAP]) and (b) nuclear transfer blastocysts generated using these two activation treatments and three different ages of karyoplast derived from day 3, 4, or 5 in vitro produced donor embryos, were examined in order to define an optimal nuclear transfer protocol. The two activation protocols comprised calcium ionophore followed by either CHX or DMAP. Parthenogenetic blastocyst yields were greater (P < 0.001) following activation with DMAP than CHX (59.7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P < 0.0001) using cytoplasts activated with DMAP. The individual rates using day 3, 4, and 5 donors and using CHX and DMAP activation treatments were 31.9 +/- 5.0, 31.7 +/- 6.2, 20.4 +/- 7.3 and 27.8 +/- 4.7, 20.1 +/- 7.5, 12.7 +/- 8.3, respectively. Blastocyst rate per fused embryo was negatively correlated (P = 0.0091) with the total number of blastomeres per donor embryo. Despite this inverse relationship, the calculated potential blastocyst yield per donor embryo was positively correlated (P < 0.0048) to karyoplast age. The individual potential yields on days 3, 4, and 5 and for the two activation protocols (CHX and DMAP) were 4.7 +/- 0.8, 7.2 +/- 1.2, 10.1 +/- 2.1 and 3.8 +/- 0.8, 5.5 +/- 2.1, 7.3 +/- 4.1, respectively. One possible explanation for the observed inverse relationship is that differentiation events during early cleavage are able to reduce the ability of the cytoplast to reprogram the transferred karyoplast and hence reduce blastocyst yields. The mechanism that mediates the differential effect of the CHX and DMAP on blastocysts yields between parthenogenetic and nuclear transfer embryos remains to be elucidated. In conclusion, the results indicate that although activation of oocytes with DMAP can produce a higher percentage of blastocysts, CHX activation is superior for use in nuclear transfer.

Animals↗
PubMed2001

Somatic cell cloning without micromanipulators.

Until now, micromanipulators have been regarded as indispensable for somatic cell nuclear transfer. This paper describes an improved zona-free nuclear transfer procedure with manual bisection of oocytes, selection of cytoplasts by Hoechst staining, and two-step fusion of somatic cells from primary granulosa cell cultures with two cytoplasts. Blastocyst rates in the three systems tested for zona-free embryo culture were 0%, 18%, and 36% for microdrops, well of the wells (WOW system), and microcapillaries (GO system), respectively. This simple, rapid, and inexpensive procedure may become a useful alternative to the existing techniques for somatic cell nuclear transfer for large-scale application of the technology.

Animals↗
PubMed2001

Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method.

BACKGROUND: Human embryonic stem (ES) cells originate from the inner cell mass of the blastocyst, and retain in culture the properties of pluripotent cells of the early embryo. The study aim was to determine whether the open pulled straw (OPS) vitrification method, which is highly effective for the cryopreservation of embryos, might be also efficient for human ES cells. METHODS AND RESULTS: All human ES cell clumps that were vitrified by the OPS method could be recovered upon thawing, and gave rise to ES cell colonies after plating. Vitrified colonies were significantly smaller and showed an increased level of background differentiation compared with control colonies. However, these unwanted effects could be overcome by additional cultivation of the colonies for 1 and 2 days respectively. The vitrified human ES cells were cultivated for prolonged periods and retained the properties of pluripotent cells, including a normal karyotype, expression of the transcription factor Oct-4 and surface markers that are characteristic to human ES cells. When grafted into SCID mice, the vitrified cells gave rise to teratomas containing derivatives of all three embryonic germ layers. CONCLUSIONS: Vitrification by the OPS method is reliable and effective for the cryopreservation of human pluripotent embryonic stem cells.

Animals↗
PubMed2000

Vitrification of the oocytes and embryos of domestic animals.

After the first successful application of vitrification for embryo cryopreservation 15 years ago, a rapid application of the method in domestic animal embryology was presumed. However, although the advantages of vitrification (simplicity, cost efficiency, speed of the procedure) were widely acknowledged, its use has been mainly restricted to experimental studies. For commercial embryo transfer purposes, the traditional slow-rate or equilibrium freezing has been used. This review attempts to explain the reasons for this phenomenon and discusses the theoretical and practical differences between the two technologies as well as their commercial prospects. Recent developments that improve the efficiency of vitrification and applications to other reproductive technologies are also summarized. These advances may result in considerable advantage and could lead to widespread application of vitrification in certain areas of domestic animal embryology.

Animals↗
PubMed2000

New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.

Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 x 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 microl medium, 200 microl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 microl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P < 0.05 for all). The developmental rate was independent of the number of WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production.

Animals↗
PubMed2000

Vitrification of bovine oocytes with the open pulled straw method: ultrastructural consequences.

The objective of the present study was to investigate the ultrastructural consequences of vitrification of bovine oocytes at the metaphase II (MII) stage by the so-called "Open Pulled Straw" method. Oocytes were matured in vitro for 22 hr and cryopreserved by vitrification. After warming and additional 2 hr of culture, the oocytes were inseminated in vitro. Oocytes were fixed for transmission electron microscopy immediately after warming, at 4 hr after warming (i.e., 2 hr post insemination [hpi]), at 26 hr after warming (i.e., 24 hpi), and at 74 hr after warming (i.e., 72 hpi). Control oocytes (i.e., nonvitrified oocytes) were processed at 22 hr after in vitro maturation and at 2, 22, and 72 hpi. Compared to the controls, the vitrified oocytes fixed immediately after warming presented an additional category of small membrane-bound vesicles and lacked the typical compartment of solitary cortical granules aligned along the oolemma. Instead, they presented clusters of cortical granules that displayed varying degrees of degeneration. In vitrified oocytes fixed at 2 hpi, the small vesicles were less abundant, and more advanced degeneration of the cortical granule clusters was noted. In vitrified oocytes fixed at 24 hpi, the small vesicles were practically absent, and polyspermic penetration was observed as were vacuoles containing degraded cortical granule content. In vitrified oocytes fixed at 72 hpi, lack of cleavage as well as vacuolization and degeneration of blastomeres were noted. Moreover, the nucleolar ultrastructure signaled aberrant activation of the ribosomal RNA genes. In conclusion, vitrification of bovine oocytes at the MII stage resulted in cell biological alterations in the oocyte after warming that apparently were reflected in the subsequent fertilization and embryonic development.

Animals↗
PubMed2000

Body dimensions and birth and organ weights of calves derived from in vitro produced embryos cultured with or without serum and oviduct epithelium cells.

Body dimensions, birth and organ weights of calves derived from embryos produced in 2 in vitro culture systems (modified SOFaa with 20% cattle serum and co-cultured with oviduct-epithelium cells [IVPserum, n=8], and modified SOFaa with 3 mg/mL PVA [IVPdefined, n=6]) were compared with calves originating from artificial insemination (AI, n=85). Three additional IVP calves were included which had been vitrified as mature oocytes by the open pulled straw (OPS) method, warmed, fertilized and cultured to the blastocyst stage in modified SOFaa with 5% cattle serum, then again OPS-vitrified and warmed prior to transfer (IVPops, n=3). At birth, gestation length and birth weights were registered for all calves. At 1 wk of age all 17 IVP and 7 of the AI calves were killed, and their body dimensions and organ weights recorded. Birth weight was higher for the IVPserum and IVPops calves than for AI control calves (kg +/- SEM: IVPserum 46.9+/-1.8, IVPops 50.6+/-2.4, AI 41.8+/-0.8; P < 0.002). There was no difference between IVP and AI calves regarding gestation length and no effect of culture conditions on body dimensions or organ weights, except for longer hind legs in IVPdefined calves compared with AI calves (cm +/- SEM: IVPdefined 93+/-2, AI 87+/-2; P < 0.04). The IVPops calves had an increased liver weight compared with AI and the other IVP calves (g +/- SEM: IVPops 1.457+/-59; AI 1,117+/-37; IVPserum 1,159+/-34, IVPdefined 1,073+/-39; P < 0.0003). It is concluded that in vitro culture of bovine embryos in the presence of serum and oviduct epithelium cells increased birth weight but not organ weight and body dimension in 1-wk-old calves. However, vitrification of the ova as oocyte and again as blastocysts increased birth weight and liver size. This possible effect of cryopreservation of oocytes on subsequent fetal development awaits further investigation.

Animals↗
PubMed1999

The effect of media, serum and temperature on in vitro survival of bovine blastocysts after Open Pulled Straw (OPS) vitrification.

The recently introduced Open Pulled Straw (OPS) vitrification technique has successfully been used for cryopreserving porcine embryos as well as for bovine embryos and oocytes. The aim of this work is to investigate several factors on the in vitro survival of bovine blastocysts. In 5 experiments, a total of 862 in vitro produced blastocysts and expanded blastocysts was vitrified and warmed using the OPS technology, then cultured in vitro for an additional 3 days. The culture medium in Experiments 1 to 4 was SOFaa with supplements and 5% calf serum (CS). In Experiment 1, the replacement of TCM-199 + 20% CS with PBS + 20% CS in the holding medium during vitrification and warming did not result in significant differences in the re-expansion (92 vs 95%) and hatching rates (79 vs 72%). In Experiment 2, the PBS holding medium was supplemented with either 20% CS, 5 mg/mL bovine serum albumin (BSA) or 3 mg/mL polyvinylalcohol (PVA). Although the re-expansion rates did not differ (98, 95 and 93%, respectively), there was a decrease in the hatching rate after vitrification with PVA (77 and 78 vs 51%, respectively). In Experiment 3, the influence of temperature of equilibration media prior to and rehydration media after the vitrification was investigated. When the temperature of these media was adjusted to 20 degrees C instead of the standard 35 degrees C, both the re-expansion and the hatching rates decreased markedly. However, increasing the time of equilibration with the diluted cryoprotectant solution at 20 degrees C eliminated these differences. In Experiment 4, the ethylene-glycol and dimethyl sulfoxide cryoprotectant mixture was replaced with ethylene glycol-ficoll-trehalose solution. No difference in the re-expansion (89 vs 96%, respectively) or hatching rate (79 vs 84%, respectively) was detected. In Experiment 5, the vitrified-warmed blastocysts were cultured in SOFaa medium supplemented with 5% CS or 5 mg/mL BSA. Although the re-expansion rates were identical in the 2 groups (95%), the hatching rates were lower when embryos were cultured in BSA (71 and 47%, respectively). These findings indicated the possible broader application for OPS, as they demonstrated that the physical advantages of rapid cooling and warming may be accompanied by different chemical composition (holding media, cryoprotective additives) according to the requirements of the biological structure. Our study also shows the need for serum supplementation of the medium for hatching to occur after OPS vitrification.

Animals↗
PubMed1999

Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts: work in progress.

Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 microM, 5 min) and cycloheximide (10 microg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified,in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% +/- SEM) were not affected (83.7+/-9.2 vs. 79.8+/-4.6; P>0.05), but cleavage (55.7+/-2.9 vs. 92.8+/-3.9; P = 0.0002) and blastocyst rates (7.2+/-5.0 vs. 32.6+/-7.8; P = 0.0025, vitrified vs. nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos.

Animals↗
PubMed1999

Cloning of bovine embryos from vitrified donor blastomeres.

The use of cryopreserved in vitro produced bovine embryos as nuclear transfer donors was assessed. Day 4 or 5 morulae were vitrified and warmed using the open pulled straw method and used as donors for nuclear transfer. Although the proportion of morulae and blastocysts that developed from nuclear transfer embryos derived from day 5 vitrified embryos did not differ from that derived from fresh embryos (16.7 and 24.3%, respectively), development to blastocysts was reduced when vitrified donor cells were used (8.3 and 19.1%, respectively). Likewise, development to morulae and blastocysts was not different between nuclear transfer embryos derived from day 4 vitrified embryos allowed to recover for 24 h, and day 5 vitrified embryos allowed to recover for 1-2 h (27.7 and 15.6%, respectively), but the development to blastocysts was reduced when day 5 vitrified donor cells were used (23.2 and 10.0%, respectively). However, in nuclear transfer embryos derived from either day 4 vitrified or day 5 fresh donors, no differences were observed in development rates to morulae and blastocysts (34.3 and 36.3%, respectively) or to blastocysts alone (20.2 and 18.1%, respectively). Nor were there differences in the development rates of fresh or day 4 or day 5 vitrified in vitro produced (non-nuclear transfer) embryos (47.9, 51.0 and 35.5% developing to blastocysts at day 7, respectively). In vitro produced embryos and nuclear transfer embryos derived from day 4 vitrified or day 5 fresh donors were transferred to recipients at morula or blastocyst stage at day 6 or 7. The pregnancy rates were similar in both groups of nuclear transfer embryos, but higher in the control group consisting of in vitro produced embryos (47, 42 and 67%, respectively). In conclusion, if vitrified donor embryos are allowed to recover for 24 h after warming, their use in nuclear transfer results in similar efficiencies to those achieved with fresh embryos.

Animals↗
PubMed1998

Long-term stability of the bovine block to polyspermy.

Under most circumstances in eutherian mammals, mating occurs before ovulation and a defense against multiple sperm penetration of the zygote is established rapidly after fertilisation. It is generally assumed that this block to polyspermy is both stable and long-lasting, but these points have not been specifically tested. They assume particular significance in domestic farm animals in which procedures of artificial insemination could mistakenly introduce sperm suspensions into a genital tract already containing a fertilised egg. Bovine blastocysts generated in vitro were further exposed in vitro to new suspensions of capacitated bull spermatozoa containing 1.5 x 10(6) cells per ml on days 7 or 8 of development, that is, shortly before or after hatching from the zona pellucida. Inseminated and control blastocysts were examined by light or electron microscopy. Whereas accessory spermatozoa were frequently bound to the surface of the zona pellucida, non had penetrated into the substance of the zona. Similarly, whilst a few spermatozoa had attached to the trophoblast of hatched blastocysts on day 8, none had penetrated through to the blastocoele. No difference was found in the incidence of hatching or rate of expansion between experimental (re-inseminated) and control groups. Accordingly, it is concluded that the bovine block to polyspermy is both stable and long-lasting when tested in vitro in the presence of freshly capacitated bull spermatozoa. Early embryonic loss, which may reach a level of 30% or more in this species, is thus unlikely to be a consequence of instability in the block to polyspermy and accessory sperm penetration of the zygote.

Animals↗
PubMed1998

Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos.

Although cryopreservation of certain mammalian embryos is now a routine procedure, considerable differences of efficiency exist depending on stage, species and origin (in vivo or in vitro produced). Factors that are suspected to cause most of these differences are the amount of the intracellular lipid droplets and the different microtubular structure leading to chilling injury as well as the volume/surface ratio influencing the penetration of cryoprotectants. A new approach, the Open Pulled Straw (OPS) method, which renders very high cooling and warming rates (over 20,000 degrees C/min) and short contact with concentrated cryoprotective additives (less than 30 sec over -180 degrees C) offers a possibility to circumvent chilling injury and to decrease toxic and osmotic damage. In this paper we report the vitrification by the OPS method of in vitro produced bovine embryos at various stages of development. Embryos cryopreserved from Day 3 to Day 7 (Day 0 = day of fertilization) exhibited development into blastocysts at rates equivalent to those of control embryos; even those cryopreserved on Day 1 or 2 exhibited only somewhat reduced survival. Eighty-one percent of Day 8 hatched blastocysts also survived the procedure. The method was also successfully used for bovine oocytes; of 184 vitrified oocytes, 25% developed into blastocysts after fertilization and culture for 7 days. Pregnancies were achieved following transfer after vitrification at both the oocyte and blastocyst stage. The OPS vitrification offers a new way to solve basic problems of reproductive cryobiology and may have practical impact on animal biotechnology and human assisted reproduction.

Animals↗
PubMed1998

Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex.

The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period were included (n = 392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38%). The respective lengths of the first 4 cell cycles of viable embryos were 32.0 +/- 3.9, 8.8 +/- 1.6, 10.8 +/- 4.7 and 47.7 +/- 11.8 h. The subsequent intervals between the 9- to 16-cell, early morula, CM and BL stages lasted 16.2 to 18.2 h. Blastomeres of 2-, 4- and 8-cell embryos cleaved asynchronously with < 1, 2.6 +/- 2.5 and 9.2 +/- 4.5 h intervals, respectively, between the first and last blastomere to cleave. The interval from insemination to tight compaction and formation of a blastocoel was 128.4 +/- 10.7 and 145.8 +/- 12.5 h, respectively. The first 3 cell cycles were approximately 3 h shorter (P < 0.1) while the fourth cycle was 5 h shorter (P = 0.06) for the viable vs nonviable embryos. On this basis it was possible to define time windows in which the proportion of viable 2-, 3- to 4-, 5- to 8- and 9- to 16- cell embryos were at their highest. No differences were found between the cleavage intervals of male and female embryos. We conclude 1) that the time-lapse culture system allows for detailed observation of the developmental kinetics of several embryo groups at the same time, and 2) that these embryos can be manipulated at the end of culture, thus allowing a linkage between early cleavage events and other developmental parameters such as embryo sex or viability after transfer.

Animals↗
PubMed1998

Ribosomal ribonucleic acid is transcribed at the 4-cell stage in in vitro-produced bovine embryos.

Ribosomal RNA, rRNA genes, and silver-staining nucleolar proteins were visualized in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst using a sequential fluorescent in situ hybridization (FISH) and a silver-staining procedure. At FISH, the rRNA was differentiated from the signal of the rRNA genes through comparison of RNase- and non-RNase-treated embryos. Both RNase- and non-RNase-treated 2-cell embryos revealed up to 10 small clusters of fluorescein isothiocynate (FITC) labeling in interphase nuclei. The RNase-treated 4-cell embryos displayed the same FITC pattern as the 2-cell embryos. In the non-RNase-treated 4-cell embryos, in contrast, the clusters were larger and included numerous small spots. In 2-cell as well as 4-cell embryos, almost all FITC-labeled clusters colocalized with silver-stained spots. In the RNase-treated 8- to 16-cell embryos, up to 10 clusters of FITC labeling were organized as one or more large spots surrounding a central faint but homogeneously labeled area. The non-RNase-treated 8- to 16-cell embryos displayed similar complexes, but the central areas consisted of small labeled spots. In 8- to 16-cell embryos, all FITC-labeled clusters were again colocalized with silver-stained areas. In the blastocysts, 1-6 big clusters of FITC labeling colocalized with silver staining. In the RNase-treated blastocysts, the FITC labeling was typically located at the edges of the silver-stained areas, whereas in the non-RNase-treated blastocysts, the FITC labeling totally covered the silver-stained areas. In conclusion, there is a close association between the rRNA genes and silver-staining nucleolar proteins in in vitro-produced bovine embryos from the second cell cycle, i.e., the 2-cell stage; the first rRNA is apparently transcribed during the third cell cycle, and during the fourth cell cycle the molecular composition of functional nucleoli is established.

Animals↗