Biomedical subjects

T Fair

Publications and source records attributed to T Fair.

At least 19 recordsLinked to original sources

PubMed2006

Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos.

In vivo-derived bovine embryos are of higher quality than those derived in vitro. Many of the differences in quality can be related to culture environment-induced changes in mRNA abundance. The aim of this study was to identify a range of mRNA transcripts that are differentially expressed between bovine blastocysts derived from in vitro versus in vivo culture. Microarray (BOTL5) comparison between in vivo- and in vitro-cultured bovine blastocysts identified 384 genes and expressed sequence tags (ESTs) that were differentially expressed; 85% of these were down-regulated in in vitro cultured blastocysts, showing a much reduced overall level of mRNA expression in in vitro- compared with in vivo-cultured blastocysts. Relative expression of 16 out of 23 (70%) differentially expressed genes (according to P value) were verified in new pools of in vivo- and in vitro-cultured blastocysts, using quantitative real-time PCR. Most (10 out of 16) are involved in transcription and translation events, suggesting that the reason why in vitro-derived embryos are of inferior quality compared with in vivo-derived embryos is due to a deficiency of the machinery associated with transcription and translation.

Animals↗

PubMed2005

Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos.

It is generally accepted that mammalian preimplantation embryos are sensitive to their environment and that conditions of culture can affect future growth and developmental potential both pre- and postnatally. Evidence suggests that while culture conditions during bovine in vitro embryo production can impact somewhat on the developmental potential of the early embryo, the intrinsic quality of the oocyte is the key factor determining the proportion of oocytes developing to the blastocyst stage. In addition, evidence suggests that the period of post fertilization embryo culture is the most critical period affecting blastocyst quality assessed in terms of cryotolerance, gene expression pattern and ability to establish a pregnancy. This paper reviews the current literature, with emphasis on the bovine model, demonstrating evidence for an effect of post fertilization culture environment on embryo gene expression and quality.

Animals↗

PubMed2004

Effect of speed of development on mRNA expression pattern in early bovine embryos cultured in vivo or in vitro.

Recent data have demonstrated that fast-cleaving embryos produced in vitro are more likely to develop to blastocyst stage, and that the postfertilization culture system used impacts considerably on the mRNA expression and quality of blastocysts produced. The present study is the first to investigate the relationship between the developmental speed of embryos produced in vivo or in vitro and the temporal transcription pattern. Genes related to important preimplantation events are monitored during the first 4 days of embryo development in embryos with fast or slow development. The set of genes analyzed in the present study characterizes several important physiological processes including: transport and metabolism of fructose (Glut-5), stress (SOX), mitochondrial activity and detoxification of reactive oxygen species (MnSOD), cell communication (Cx43), maternal recognition of pregnancy (IFN-tau), imprinting (IGF-II), apoptosis (Bax), growth factor binding and metabolism (IGF-IR), and oxidative stress (G6PD). Using real time PCR, we have found that for all the genes analyzed there are differences in mRNA expression between embryos with fast and slow developmental speed produced both in vitro and in vivo. Frequently, genes that may be stress induced such as SOX, MnSOD, BAX, IFtau, and G6PD were highly transcribed in in vitro produced embryos and in embryos with slow developmental speed. On the other side, transcripts from genes related with metabolism, growth, and differentiation (Glut-5, Cx 43, IGF-II, and IGF-IR) were detected in higher amounts in in vivo produced embryos and in embryos with fast developmental speed. Moreover, it is interesting to stand out that for some genetic markers (such as SOX and G6PD) there are in vivo and in vitro differences that can be observed even before materno-zygotic transition, which probably reflects a differential mRNA degradation. These transcription patterns reflects the embryonic response to the adverse in vitro culture conditions, and connect the low quality of embryos which slow developmental speed produced in vivo and in vitro, with the mRNA expression pattern of some embryonic genes.

Animals↗

PubMed2003

Effect of culture environment on embryo quality and gene expression - experience from animal studies.

Recent studies comparing bovine oocyte maturation, fertilization and embryo culture in vivo and in vitro have demonstrated that the origin of the oocyte is the main factor affecting blastocyst yield, while the post-fertilization culture environment is critical in determining blastocyst quality, measured in terms of cryotolerance and relative transcript abundance, irrespective of the origin of the oocyte. Production of embryos in vitro, particularly when using an extended period of in-vitro culture, may predispose the embryo to phenomena such as the large offspring syndrome, which is likely to alter gene expression, particularly of imprinted genes. It is clear now that the post-fertilization culture environment has a profound effect on the relative abundance of gene transcripts within the embryo, and culture under suboptimal conditions for as little as 1 day can lead to perturbations in the pattern of expression.

Animals↗

PubMed2003

Oocyte and embryo quality: effect of origin, culture conditions and gene expression patterns.

In general, the majority of immature bovine oocytes fail to develop to the blastocyst stage following maturation, fertilization and culture in vitro. The evidence suggests that while culture conditions during in vitro embryo production can impact on the developmental potential of the early embryo, the intrinsic quality of the oocyte is the key factor determining the proportion of oocytes developing to the blastocyst stage. In addition, evidence suggests that the period of post-fertilization embryo culture is the most critical in determining blastocyst quality. This paper reviews the current literature, with emphasis on the bovine model, demonstrating evidence for an effect of oocyte origin and/or in vitro maturation conditions on the developmental capacity and gene expression patterns in the oocyte. Furthermore, the well-documented effects of post-fertilization culture environment on embryo gene expression and quality are highlighted.

Animals↗

PubMed2002

Evolution of mRNA polyadenylation between oocyte maturation and first embryonic cleavage in cattle and its relation with developmental competence.

In this study we analyzed the pattern of polyadenylation changes that takes place between the resumption of meiosis and the first cleavage of bovine oocytes. Moreover, we investigated whether the delayed occurrence of the first cleavage division, which characterizes embryos of low developmental competence, is accompanied by an altered polyadenylation pattern of individual transcripts. We determined the polyadenylation status of a group of genes that characterize physiological processes, involved in early differentiation (Oct-4), compaction, and cavitation (beta-actin, plakophilin, connexin-32, connexin-43), energy metabolism (glucose transporter type 1, pyruvate dehydrogenase phosphatase), RNA processing (RNA poly(A) polymerase), and stress (heat shock protein 70). RNA was isolated from pools of 20 oocytes or embryos at the germinal vesicle (GV) stage, at the end of in vitro maturation, at the end of in vitro fertilization, and at the time of the first cleavage. Cleavage was assessed 27, 30, 36, 42 hr post insemination (hpi), and at the latter time the remaining uncleaved oocytes were retained as a group. Between oocyte isolation and first cleavage at 27 hpi (best quality embryos), the poly(A) tail of individual transcripts followed four patterns: no changes (beta-actin, PDP); gradual reduction (Cx-43, Oct-4, Plako); gradual elongation (Cx-32, TPA); reduction followed by elongation (PAP, HSP-70, Glut-1). If the interval between insemination and first cleavage was longer than 27 hpi (progressively lower quality embryos) further changes of polyadenylation were observed, which differed for each gene considered. These data indicated that specific changes in polyadenylation contribute to the modulation of gene expression in bovine embryos at this stage of development. Defective developmental competence is accompanied by abnormal polyadenylation levels of specific maternal mRNAs with synchrony between polyadenylation and cleavage emerging as an apparently important factor.

Animals↗

PubMed2001

Ultrastructure of bovine blastocysts following cryopreservation: effect of method of blastocyst production.

The objective of this study was to describe the ultrastructure of blastocysts derived by in vivo and in vitro methods and to investigate how the morphology is affected by exposure to cryoprotectant (10% glycerol) or cryopreservation by conventional slow freezing. In vivo derived blastocysts were characterized by a narrow perivitelline space (PvS), a continuous cover of numerous stacked microvilli (MV) on the plasma membrane, a well-defined system of cell-to-cell coupling and a large population of round or elongated mitochondria with numerous transverse cristae. Exposure of these blastocysts to cryoprotectant was manifested by shrinkage of the blastocysts and swelling of the mitochondria. Cryopreservation resulted in further shrinkage, damage to the MV, and accumulation of cellular debris. In comparison, the in vitro matured (IVM)/in vitro fertilized (IVF) in vivo cultured blastocysts displayed a wider PvS; they appeared to possess less MV and all blastocysts displayed some cellular debris in their PvS. There was also a decrease in the number of junctional contacts between the trophoblastic cells. The reaction of these blastocysts to exposure to cryoprotectant was similar to that of the in vivo derived blastocysts. However, they appeared to be more susceptible to cryopreservation. The totally in vitro produced (IVP) blastocysts displayed a wider PvS, no stacking of the MV, increased numbers of lipid droplets and a further reduction in the junctional contacts between trophoblastic cells. The IVP blastocysts sustained breakage of the zona pellucida on exposure to cryoprotectant and were extremely sensitive to cryopreservation, losing all cell structure and organization. The findings of the present study indicate that in vivo derived blastocysts possess certain structural characteristics that confer a greater tolerance on them to exposure to cryoprotectant and cryopreservation.

Animals↗

PubMed2001

Immunolocalization of nucleolar proteins during bovine oocyte growth, meiotic maturation, and fertilization.

During the growth phase of the bovine oocyte transcripts, polypeptides and ribosomes are accumulated in the oocyte to drive and sustain future meiotic maturation, fertilization, and early embryonic development. The oocyte also furnishes the early embryo with the components required to establish a functional transcriptionally active nucleolus at the time of maternal embryonic transition. The aim of the present study was to describe the behavior of key components of the nucleolus. The temporal localization of nucleolar proteins fibrillarin, nucleophosmin, nucleolin, RNA polymerase I (RNA pol I), topoisomerase I, upstream binding factor (UBF), and coilin 5P10 was investigated in growing and fully grown immature bovine oocytes during in vitro maturation and during the first postfertilization cell cycle using whole-mount immunocytochemistry and confocal microscopy. During the oocyte growth phase, fibrillarin, nucleophosmin, nucleolin, RNA pol I, and UBF were localized to the oocyte nucleolus. On completion of the growth phase, nucleolin and nucleophosmin appeared to migrate to the periphery of the nucleolus and into the nucleoplasm, and the proportion of oocytes displaying RNA pol I localization had decreased. Topoisomerase I was not detected at any stage. Fibrillarin appeared to be localized to large foci within the nucleolus and/or nucleoplasm. Nucleophosmin and nucleolin labeling was characterized by a homogeneous signal over the nucleolus. RNA pol I and UBF were characterized by the localization of the antibodies to individual or clustered foci in the nucleolus and/or nucleoplasm. Following oocyte nucleus breakdown (ONBD), the proteins appeared to disperse into the cytoplasm. All proteins were undetectable during meiotic maturation and were not relocalized until 5-10 h postinsemination (hpi). UBF was localized to the fertilizing sperm head of most zygotes at 5 hpi. By 10 hpi, all proteins were detected in most oocytes displaying two pronuclei. Nucleolar protein localization was exclusive to or more abundant in one pronucleus up to 20 hpi; thereafter, the pattern was more evenly distributed. Fibrillarin, nucleophosmin, nucleolin, UBF, and Pol I are present in the nuclei of growing and fully grown bovine oocytes until ONBD. They reappear at the late telophase stage of meiosis II and continue to be present up to the first mitotic division of embryo development.

Animals↗

PubMed2001

Ribosomal RNA gene expression and chromosome aberrations in bovine oocytes and preimplantation embryos.

This review focuses on the key features of development of the bovine oocyte and embryo, with comparisons of the developmental characteristics of embryos produced in vivo and in vitro. The oocyte is transcriptionally quiescent in the primordial and primary follicle. In the secondary follicle transcription is initiated in the oocyte and a ribosome-synthesizing nucleolus is established in this cell. Transcription and nucleolar activity are enhanced in the tertiary follicle during oocyte growth. When the oocyte reaches approximately 110 microm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription ceases and the nucleolus is inactivated, forming a dense spherical remnant. During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, it disperses. The rRNA genes are apparently re-activated during the four-cell stage, that is, the third cell cycle after fertilization, but a nucleolus is not formed. During the subsequent cell cycle, that is, during the eight-cell stage, ribosome-synthesizing nucleoli are again established. Bovine embryos produced in vitro apparently display the same pattern of nucleolus development as that in embryos developed in vivo. Examination of the ploidy of embryonic cells using fluorescence in situ hybridization has revealed that the production of bovine embryos in vitro is associated with increased chromosome aberrations in the embryos. Blastocysts produced in vitro display a significantly higher rate of mixoploidy, that is, when the embryo consists of both normal diploid and abnormal polyploid cells, than that in embryos developed in vivo. The rate of mixoploidy among embryos produced in vitro increases with increasing developmental stage. Moreover, after fertilization in vitro, initially there is a high rate of 'true' polyploidy, that is, when all cells of the embryos are polyploid. However, the polyploid embryos are eliminated before they cleave beyond the eight-cell stage, the stage at which major activation of the embryonic genome occurs in cattle.

Animals↗

PubMed2000

Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality.

The objectives of this study were to examine the effect of culture system on bovine blastocyst formation rates and quality. Presumptive IVM/IVF bovine zygotes were cultured either in vitro in synthetic oviduct fluid (SOF, 25 embryos/25 microL in 5% CO2, 5% O2, 90% N2 at 39 degrees C) or in vivo in the ewe oviduct (approximately 100 embryos per oviduct). The recovery rate after in vivo culture was 53% (813/1,530). The blastocyst rate on Day 7 was significantly higher for the in vitro system (28%, 362/1,278 vs 17%, 37/813; P< 0.0001). However, after culture in vitro for a further 24 h, there was no difference in Day 8 yields (36%, 457/1,278 vs 32%, 258/813, for in vitro and in vivo culture, respectively). There was no difference in blastocyst cell number between treatments (Day 7: 96 vs 103; Day 8: 78 vs 85 for in vitro and in vivo culture, respectively). Irrespective of culture system, Day 7 blastocysts had a significantly higher cell number than those appearing on Day 8. There was no difference in pregnancy rate at Day 35 after fresh transfer of a single Day 7 blastocyst (37.5%, 21/56 vs 45.3%/, 24/53 for in vitro and in vivo culture, respectively). After cryopreservation by freezing in 10% glycerol, VS3a vitrification or solid surface vitrification, the survival of in vitro cultured embryos was significantly lower than survival of embryos cultured in the ewe oviduct or those produced by superovulation of donors. In conclusion, these findings demonstrate that while bovine zygotes cultured in vitro are capable of rates of development similar to those of their in vivo cultured counterparts (in terms of Day 8 blastocyst yield, cell number and early pregnancy rate), there are significant differences in embryo cryosurvival. This suggests that current in vitro culture systems need to be improved to optimize embryo quality and pregnancy rates.

Animals↗

PubMed2000

Activation of ribosomal RNA genes in preimplantation cattle and swine embryos.

Transcription of ribosomal RNA (rRNA) genes occurs in the nucleolus resulting in ribosome synthesis. In cattle and swine embryos, functional ribosome-synthesizing nucleoli become structurally recognizable towards the end of the fourth and third post-fertilization cell cycle, respectively. In cattle, a range of important nucleolar proteins become localized to the nucleolar anlage over several cell cycles and this localization is apparently completed towards the end of the fourth cell cycle. In swine, the localization of these proteins to the anlage is more synchronous and occurs towards the end of the third cell cycle and is apparently completed at the onset of the fourth. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus, serve to evaluate the developmental potential of embryos originating from different embryo technological procedures. By this approach, we have demonstrated that in vitro produced porcine embryos display a lack of localization of nucleolar proteins to the nucleolar anlage as compared with in vivo developed counterparts. Similarly, bovine embryos produced by nuclear transfer from morulae display such deviations as compared with in vitro produced counterparts. Collectively, this information may help to explain the appearance of abnormalities seen in a certain proportion of offspring derived from in vitro produced embryos and after cloning.

Animals↗

PubMed2000

Relationship between time of first cleavage and the expression of IGF-I growth factor, its receptor, and two housekeeping genes in bovine two-cell embryos and blastocysts produced in vitro.

We have previously demonstrated that there is a clear relationship between the time interval between insemination and first cleavage in vitro and the development to the blastocyst stage of bovine embryos. In addition we have shown that this developmental ability can be linked to the stability of the mRNA for several gene transcripts measured in 2-cell bovine embryos cleaving at different times. The aim of this study was to examine the relationship between bovine embryo developmental competence, assessed in terms of time of first cleavage, and the expression of insulin-like growth factor-I (IGF-I) ligand and receptor, hypoxanthine phosphoribosyl transferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD). The expression of beta-actin was used as a reference value. No differences were observed in the mRNA expression of G6PD and HPRT genes between male and female 2-cell embryos. However, the expression of these two genes was significantly higher in female blastocysts than in male blastocysts. Moreover, when the relative amount of G6PD and HPRT mRNA detected in these groups of male and female embryos was compared, there was a significant relationship between the time of first cleavage and the relative amount of mRNA: 2-cell embryos and blastocysts derived from oocytes that cleaved at 27 and 30 hr post insemination had higher levels of mRNA for G6PD and HPRT than those that cleaved after 33 hr. IGF-I ligand and receptor was detected in all blastocysts analyzed, irrespective of stage of development or time of first cleavage. In addition, the receptor was detected in all 2-cell embryos examined. In contrast, while IGF-I ligand was found in all 2-cell embryos that cleaved at 27 and 30 hpi, it was only found in some of those cleaving between 33 and 36 hpi and in none of those cleaving after 36 hr. In conclusion, we have demonstrated differences in gene expression in the early embryo that are reflective of differences in developmental competence between early- and late-cleaving zygotes.

Animals↗

PubMed2000

Bovine oocyte and embryo development following meiotic inhibition with butyrolactone I.

In this study we have shown that butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, inhibits meiotic resumption in bovine oocytes by blocking germinal vesicle breakdown in a dose-dependent manner. A concentration 100 microM blocked over 60% of oocytes, while 150 microM inhibited almost all oocytes compared to the control in which over 80% resumed meiosis. Following a second 24 hr culture under conditions permissive to normal maturation, almost all (95%) of blocked oocytes resumed meiosis and progressed to metaphase II. In terms of developmental competence, oocytes maintained in meiotic arrest for 24 hr with 100 microM exhibited a similar capacity to develop to the blastocyst stage as nonblocked control oocytes following maturation, fertilization, and culture in vitro. Cryopreservation was employed as a tool to detect differences in the oocyte viability between blocked and control oocytes. Cleavage of oocytes was significantly reduced following vitrification and activation both in BL-I treated (40.2% vs. 71.9%, P < 0.05) and the control groups (45.6% vs. 81.7%, P < 0.05). However, BL-I treated oocytes were less likely to develop into blastocysts following vitrification (20.0% from vitrified vs 42.5% from nonvitrified cleaved oocytes, P < 0.05, based on cleaved oocytes) compared to nontreated oocytes (34.0% from vitrified vs. 42. 9% from nonvitrified oocytes, P < 0.05). These results demonstrate the feasibility of maintaining bovine oocytes in artificial meiotic arrest without compromising their subsequent developmental competence and may represent a tool for improving the development of less competent oocytes.

4-Butyrolactone↗

PubMed1999

Timing of the first cleavage post-insemination affects cryosurvival of in vitro-produced bovine blastocysts.

The time of the first cleavage of bovine zygotes during in vitro culture can affect the rate of development and cell number of the blastocysts. The aim of this work was to study the effect of the timing of first cleavage on the cryosurvival of the resulting blastocysts. Following standard IVM and IVF, zygotes were cultured in modified synthetic oviduct fluid (SOF), with 10% fetal calf serum (FCS) added 48 hr post insemination, in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2. Embryos which cleaved by 24, 27, 30, 33, or 36 hr after insemination (IVF) were harvested and further cultured to the blastocyst stage (day 7 or day 8 post IVF). All developing blastocysts on days 7 and 8 were classified into three groups and were cryopreserved by vitrification. Group A consisted of blastocysts (<150 microm, small blastocysts); group B consisted of expanded or hatching blastocysts (>150 microm, large blastocysts); and group C consisted of morphologically poor quality blastocysts. The vitrification solution consisted of 6.5 M glycerol and 6% bovine serum albumin in PBS (VS3a). Thawed embryos were cultured further and survival was defined as the re-expansion and maintenance of the blastocoel over 24, 48, and 72 hr, respectively. Overall survival and hatching at 72 hr post-thawing was higher in blastocysts formed by day 7 than those formed by day 8 (60% vs. 40% survival; 63% vs. 45% hatching). Large blastocysts from day-7 and day-8 groups survived significantly better than small or poor quality blastocysts (76% vs. 63% and 31%; 72% vs. 30% and 26%, respectively; P < 0.05). Day-7 blastocysts from the 27- and 30-hr cleavage groups survived significantly better than those from the 36-hr group (63% and 66% vs. 25%, P < 0.05). Day-8 blastocysts from later cleaved (30 hr) zygotes had a higher survival than the 27-hr cleavage groups (52% vs. 26%, P < 0.05). These results indicate that the day of blastocyst appearance, developmental stage, and timing of the first cleavage post-insemination can influence the cryosurvival of bovine blastocysts following vitrification.

Animals↗

PubMed1998

Effect of protein synthesis inhibition before or during in vitro maturation on subsequent development of bovine oocytes.

The overall objective of this study was to assess the effect of maintaining meiotic arrest in bovine oocytes in vitro on developmental competence. In Experiment 1 the effect of inhibition of meiotic resumption using cycloheximide (CX), on subsequent was examined. Immature cumulus oocyte complexes (COCs, n = 804) were cultured in the absence (24 h) or presence of CX for 6, 12, 18 or 24 h. The control was inseminated 24 h later, while CX-treated oocytes were cultured for a further 24 h before insemination. In Experiment 2 the effect of exposing the oocyte (n = 1239) during meiotic arrest to putative stimulatory substances (pFSH and FCS) was examined. In Experiment 3, to study the importance of protein synthesis during maturation, synthesis was blocked for a 6-h period at various times (6, 12, 18 h) after start of culture (n = 1117). In Experiment 1, there was no difference in cleavage rate between treatments. However, the percentage of 5 to 8 cell embryos at 72 h post insemination was significantly lower after CX treatment (64 vs 42 to 51%; P < 0.05). This was reflected in a lower rate of blastocysts at Day 6 (9 to 15 vs 31%, P < 0.002). While the blastocyst rate at Day 8 was lower in CX-treated oocytes, the effect was only significant when CX was present for longer than 12 h. A marked decrease in development was noted following inhibition for 18 h or more compared with the control (17 to 19 vs 40%; P < 0.0002). In Experiment 2, addition of either FSH or FCS to oocytes in the presence of CX had no effect on any of the parameters studied, even though there was a positive effect in control oocytes. In Experiment 3, treatment with CX after the oocytes had matured for varying periods resulted in decreased blastocyst rates at Days 6 and 8 of culture. The most significant drop in development occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 40%; P < 0.0001). In conclusion, CX-blocked oocytes retained their developmental competence, although final blastocyst yields were reduced.

Animals↗

PubMed1997

Nucleus ultrastructure and transcriptional activity of bovine oocytes in preantral and early antral follicles.

An understanding of the recruitment and growth of follicles within the bovine ovary is crucial to their successful exploitation in vitro. The aim of the present study was to describe the nuclear ultrastructure and transcriptional activity of primordial to early tertiary follicular oocytes from bovine adult ovaries. Small blocks of ovarian cortex were incubated in medium enriched with 3H-uridine for 30 min. Subsequently, the tissue blocks were fixed in Karnowsky's fixative, dehydrated, epon embedded, sectioned (2 microns), processed for autoradiography, and examined under light microscopy. Sections showing preantral follicles with presumptive oocyte nucleoli were reembedded for transmission electron microscopy. The follicles were divided into five categories: 1) resting primordial, with a single layer of flattened granulosa cells, 2) activated primordial, with a single layer of flattened and some cuboidal granulosa cells, 3) primary, with a single layer of cuboidal granulosa cells, 4) secondary, with a complete or incomplete bilayer of cuboidal cells, and 5) tertiary, with more than two layers of granulosa cells delineating one or more intercellular cavities. The granulosa cells of all follicle classes were transcriptionally active. However, the oocytes did not display transcriptional activity, as measured by the present means, until the secondary and tertiary follicular stages. The oocyte nucleolus was granular in the primordial follicles. Following follicular activation, fibrillar centres invaded the nucleolus and, in the early tertiary follicle, numerous fibrillar centres were distributed throughout the nucleolus. In conclusion, the oocyte nucleolar function is gradually activated at follicle activation, and oocyte transcription is initiated at approximately the time of the secondary follicle stage.

Animals↗

PubMed1997

Oocyte ultrastructure in bovine primordial to early tertiary follicles.

The aim of the present study was to describe in detail the changes occurring in the cytoplasmic ultrastructure of the bovine oocyte from the onset of growth in the primordial follicle until the completion of growth in the tertiary follicle. Bovine oocytes from primordial, primary, secondary and early to mid-antral follicles were processed and analysed by light and transmission electron microscopy. The primordial follicular oocyte was characterized by numerous coated pits on the oolemma and the accumulation of free and organelle-related smooth (SER) and rough (RER) endoplasmic reticulum, round mitochondria and Golgi complexes around the nucleus, which was located slightly off centre. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, elongated mitochondria and Golgi complexes. During the secondary follicular stage, formation of the zona pellucida, development of gap junctions between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and reduction in the number of coated pits on the oolemma were seen. In the tertiary follicular oocyte up to 100 microm in diameter, the number of Golgi complexes and lipid droplets increased and the organelles were dislocated to the deep cortical region. During the final growth of the oocyte up to >120 microm, the organelles were dislocated further to the peripheral region, the extent of the free SER and RER compartments were reduced, the number of individual cortical granules increased, hooded mitochondria became abundant and the perivitelline space developed. In conclusion, the growth of the bovine oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte specific structures such as the zona pellucida and cortical granules.

Animals↗

PubMed1996

Nucleus structure and transcriptional activity in relation to oocyte diameter in cattle.

Bovine abattoir ovaries were sliced, and recovered oocytes were washed and incubated in medium enriched with 3H-uridine for 30 min. Uridine incorporation was stopped by washing at 4 degrees C in PBS supplemented with cold uridine. The oocytes were grouped according to their inside diameter- < 100, 100- < 110, 110- < 120, and > or = 120 microns-and processed for autoradiography and transmission electron microscopy. Oocytes < 110 microns in diameter typically presented fibrillogranular nucleoli and were actively transcribing; in contrast, most oocytes > 110 microns displayed electron-dense fibrillar nucleoli and lacked transcriptional activity, as measured by the present means. Based on morphological and transcriptional information, a dynamic model of nucleolus inactivation is proposed. The degree of chromatin condensation varied among oocytes. Fibrillogranular nucleoli were most frequently accompanied by lightly condensed chromatin. The dense fibrillar nucleoli were usually encapsulated by heavily condensed chromatin. The oocyte nuclei underwent a peripheral translocation as the oocyte diameter increased from < 100 to 110 microns. In conclusion, RNA synthesis appeared to cease as the oocyte diameter exceeded 110 microns, and concomitantly the nucleoli restructured from fibrillogranular to dense fibrillar.

Animals↗