Glucose and pyruvate metabolism of preimplantation goat blastocysts following in vitro fertilization and parthenogenetic activation

The energy metabolism of preimplantation embryos can be used to predict viability and postimplantation development. Although preimplantation development and mean blastocyst cell numbers of goat in vitro-fertilized (IVF) embryos and chemically activated parthenogenotes are comparable, mammalian parthenogenotes are not viable, with most dying shortly after implantation. The objective of this study was to compare glucose and pyruvate metabolism of IVF goat blastocysts with that of parthenogenetic blastocysts developing from chemically activated oocytes. Embryos derived from IVF and parthenogenotes produced by exposing oocytes to either ionomycin or ethanol followed by 6-dimethylaminopurine (6-DMAP) were cultured in G1.2/G2.2 sequential culture media. Metabolism was determined for individual blastocysts using [5-3H]glucose and [2-14C]pyruvate to determine glycolytic and Kreb's cycle activity, respectively. Data were analyzed by ANOVA. A significantly higher percentage of activated oocytes underwent cleavage and developed to the blastocyst stage compared to IVF oocytes (p < 0.05). There was no significant difference in glucose or pyruvate metabolism between IVF and parthenogenetically activated blastocysts. Mean glucose metabolism through glycolysis was 154.9 +/- 29.1, 130.3 +/- 17.1, and 129 +/- 16.5 pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively. Mean pyruvate metabolism through the Kreb's cycle was 28.1 +/- 8.0, 15.8 +/- 4.2, and 24.4 +/- 4.4 in pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively. Our results suggest that known differences in postimplantation development observed in IVF versus parthenogenetic embryos cannot be attributed to differences in pyruvate or glucose metabolism in the preimplantation blastocysts. Thus, these activation protocols result in embryos capable of appropriate regulation of key metabolic enzymes.     

In vitro development of polyspermic porcine oocytes: Relationship between early fragmentation and excessive number of penetrating spermatozoa

Embryo development during in vitro culture of polyspermic porcine oocytes was investigated in the present study. After in vitro fertilization (IVF) of in vitro matured oocytes, putative zygotes were centrifuged to visualize pronuclei. Two pronuclear (2PN) and poly-pronuclear (PPN) zygotes were selected and cultured in vitro. Their development to the blastocyst stage and total cell numbers, dead cell rates and ploidy at the blastocyst stage and morphology of resultant embryos after first cleavage were compared. A cleavage rate of PPN embryos was lower than that of 2PN (61.3% and 82.2%, respectively), however, the ability of cleaved embryos to develop to the blastocyst stage did not differ between the PPN and the 2PN groups (22.4% and 32.9%, respectively). Also there was no difference in total cell numbers and rates of dead cells between PPN and 2PN blastocysts. The majority of blastocysts in 2PN group were found to be diploid. In contrast, blastocysts in PPN group showed heterogeneous status in their ploidy including polyploidy and mixoploidy, whereas a remarkable proportion (31.3%) of them was found to be diploid. After the first cleavage (at 36 h after IVF), there was no difference in the number of nuclei/embryo between the two groups, nevertheless embryos in PPN group had significantly higher numbers of blastomeres than that of embryos in 2PN group, mainly due to an increased frequency of anuclear blastomeres. The present results indicate that correction of embryo ploidy in polyspermic embryos can occur during IVC. Nevertheless the frequency of partial fragmentation in polyspermic embryos is increased.     

Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

Glucose transporter 8 expression and translocation are critical for murine blastocyst survival

Glucose transporter (GLUT) 8 is an insulin-responsive facilitative glucose transporter expressed predominantly in the murine blastocyst. To determine the physiologic role of GLUT8, two-cell embryos were cultured to a blastocyst stage in antisense or sense oligonucleotides to GLUT8. Apoptosis was assessed using the TUNEL techniques and recorded as the percentage of TUNEL-positive nuclei/total nuclei. Embryos cultured in GLUT8 antisense experienced increased TUNEL-positive nuclei, whereas sense embryos did not. Embryos cultured in a control AS oligonucleotide, specific for heat shock protein 70-2, showed a rate of apoptosis similar to sense. To determine the outcome of these apoptotic embryos, blastocysts exposed to sense vs. antisense were transferred back into foster mice and the pregnancy continued until Day 14.5, at which time the uteri were examined for normal gestational sacs and resorptions. Embryos exposed to GLUT8 antisense experienced higher rates of resorptions and lower normal pregnancy rates compared to embryos cultured in GLUT8 sense. To examine the insulin growth factor (IGF)-1/insulin intracellular signaling pathways involved in GLUT8 translocation, IGF-1 receptor (IGF-1R) expression was decreased in the blastocysts with antisense oligonucleotides. Using confocal immunofluorescent microscopy, GLUT8 translocation in response to insulin was observed. Exposure to insulin in the embryos exposed to IGF-1R sense induced translocation of GLUT8 from intracellular compartments to the plasma membrane. Blastocysts exposed to IGF-1R antisense, however, failed to demonstrate any change in the intracellular location of GLUT8 with insulin treatment. The IGF-1R antisense embryos also displayed significantly greater TUNEL staining compared to sense embryos. These data suggest that GLUT8 expression and translocation in response to insulin are critical for blastocyst survival.     

Energy substrate requirements for in vitro development of early cleavage-stage bovine embryos

Energy substrate preferences of bovine cleavage-stage embryos produced by in vitro maturation and in vitro fertilization were examined in a chemically-defined (protein-free) culture medium modified hamster embryo culture medium-3, (mHECM3). Few inseminated ova cleaved without energy substrates. Glucose and/or glutamine could not support embryo development, but lactate alone was effective (37% 5–8-cells), equivalent to complex medium TCM-199 (44%). Addition of 11 selected amino acids to lactate increased embryo cleavages, although this treatment was not significantly different from pyruvate alone. Addition of glucose to lactate or to pyruvate depressed development. Lactate + amino acids was significantly better than TCM-199 (54% and 26% ≤8-cells, respectively). Blastocyst development was evaluated after transferring ≤8-cell embryos into a complex medium (TCM-199) containing serum. Cleavage-stage embryos produced with pyruvate alone or with lactate + amino acids yielded the highest proportions of blastocysts (36% and 41%, respectively, of inseminated ova). Between 33–63% of blastocysts derived from embryos that were initially developed in mHECM-3 supplemented with various substrates escaped from their zonae (hatched) depending on the treatment, but none of the embryos from the pyruvate + glucose combination hatched. This study shows that optimal energy substrates for bovine cleavage-stage embryo development can be determined using a chemically-defined culture medium, that a simple medium with selected substrates can support early development as well as or better than a complex medium, that a two-step culture system can be used to evaluate blastocyst development from these cleavage-stage embryos, and that timing and hatching of embryos may provide additional information about discriminating between the suitabilities of different substrates for early embryo development. © 1996 Wiley-Liss, Inc.     

Characterization and regulation of monocarboxylate cotransporters Slc16a7 and Slc16a3 in preimplantation mouse embryos

Concurrent with compaction, preimplantation mouse embryos switch from the high pyruvate consumption that prevailed during cleavage stages to glucose consumption against a constant background of pyruvate uptake. However, zygotes exposed to and subsequently deprived of glucose can form blastocysts by increasing pyruvate uptake. This metabolic switch requires cleavage-stage exposure to glucose and is one aspect of metabolic differentiation that normally occurs in vivo. Monocarboxylates, such as pyruvate and lactate, are transported across membranes via the SLC16 family of H(+)-monocarboxylate cotransporter (MCT) proteins. Thus, the increase in pyruvate uptake in embryos developing without glucose must involve changes in activity and localization of MCT. In mouse embryos, continued expression of Slc16a1 (MCT1) requires glucose supply. Messenger RNA for Slc17a7 (MCT2) and Slc16a3 (MCT4) has been detected in mouse preimplantation embryos; however, protein function, localization, and regulation of expression at the basis of these net pyruvate uptake changes remain unclear. The expression and localization of SLC16A7 and SLC16A3 have therefore been examined to clarify their respective roles in embryos derived from the reproductive tract and cultured under varied conditions. SLC16A3 appears localized to the plasma membrane until the morula stage and also maintains a nuclear distribution throughout preimplantation development. However, continued Slc16a3 mRNA expression is dependent on prior exposure to glucose. SLC16A7 localizes to apical cortical regions with punctate, vesicular expression throughout blastomeres, partially colocalizing in peroxisomes with peroxisomal catalase (CAT). In contrast to SLC16A3 and SLC16A1, SLC16A7 and CAT demonstrate upregulation in the absence of glucose. These striking differences between the two isoforms in expression localization and regulation suggest unique roles for each in monocarboxylate transport and pH regulation during preimplantation development, and implicate peroxisomal SLC16A7 as an important redox regulator in the absence of glucose.     

Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro

This study compares the effects of conventional controlled-rate freezing and vitrification on the morphology and metabolism of in vitro-produced bovine blastocysts. Day 7 expanded blastocysts cultured in synthetic oviduct fluid with 5% fetal calf serum were frozen in 1.36 M glycerol, 0.25 M sucrose or vitrified in 25% glycerol, 25% ethylene glycol. Cell alterations and in vitro development were evaluated immediately after thawing or after 72 h. The effect of cryopreservation on inner cell mass and trophectoderm (TE) cell number as well as glucose, pyruvate, and oxygen uptakes, and lactate release by blastocysts were evaluated. Immediately after thawing, blastocysts showed equivalent cell membrane permeabilization after both cryopreservation procedures, while alterations in nuclear staining were more frequent in vitrified embryos. After culture, similar survival and hatching rates were observed. Both procedures decreased cell number immediately after thawing and after 72 h. However, the number of TE cells was lower in frozen embryos than in vitrified ones. In relation to this, frozen blastocysts showed a decrease in glucose, pyruvate, and oxygen uptake, although those parameters were not altered in vitrified embryos. An increased glycolytic activity was also observed in frozen embryos, indicating a stress response to this procedure.     

Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos

Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.     

Differential acetylation of histone H4 lysine during development of in vitro fertilized,cloned and parthenogenetically activated bovine embryos

The oocyte is remarkable in its ability to remodel parental genomes following fertilization and to reprogram somatic nuclei after nuclear transfer (NT). To characterise the patterns of histone H4 acetylation and DNA methylation during development of bovine gametogenesis and embryogenesis, specific antibodies for histone H4 acetylated at lysine 5 (K5), K8, K12 and K16 residues and for methylated cytosine of CpG dinucleotides were used. Oocytes and sperm lacked the staining for histone acetylation, when DNA methylation staining was intense. In IVF zygotes, both pronuclei were transiently hyper-acetylated. However, the male pronucleus was faster in acquiring acetylated histones, and concurrently it was rapidly demethylated. Both pronuclei were equally acetylated during the S to G2-phase transition, while methylation staining was only still observed in the female pronucleus. In parthenogenetically activated oocytes, acetylation of the female pronucleus was enriched faster, while DNA remained methylated. A transient de-acetylation was observed in NT embryos reconstructed using a non-activated ooplast of a metaphase second arrested oocyte. Remarkably, the intensity of acetylation staining of most H4 lysine residues peaked at the 8-cell stage in IVF embryos, which coincided with zygotic genome activation and with lowest DNA methylation staining. At the blastocyst stage, trophectodermal cells of IVF and parthenogenetic embryos generally demonstrated more intense staining for most acetylated H4 lysine, whilst ICM cells stained very weakly. In contrast methylation of the DNA stained more intensely in ICM. NT blastocysts showed differential acetylation of blastomeres but not methylation. The inverse association of histone lysine acetylation and DNA methylation at different vital embryo stages suggests a mechanistically significant relationship. The complexities of these epigenetic interactions are discussed.     

Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos.

The present study characterized the profile of nuclear remodeling in nuclear transplant rabbit embryos and investigated the relationship between chromatin behavior after transfer and embryo development. The developmental potential and pattern of remodeling of donor nuclei from cleavage-, morula-, and blastocyst- (inner cell mass ICM, and trophectoderm, TE) stage donors were evaluated. In addition, we determined whether a modification in the synchrony between blastomere fusion and oocyte activation altered the profile of nuclear remodeling and affected development of reconstituted embryos. Development to blastocysts was similar with 8- and 32-cell-stage donor nuclei (42% and 33%, respectively, p greater than 0.1). However, it was reduced with ICM transplants (17%, p less than 0.05), and development of TE transplants did not progress beyond the 8-cell stage. Upon blastomere fusion into nonactivated oocyte cytoplasm, nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by pronuclear (PN) formation and swelling. PCC occurred synchronously within 1.2-1.5 h post-fusion with all stages of donor nuclei (p greater than 0.1). PN formation in 8- and 32-cell transplants occurred approximately 4 h after fusion, and was synchronous to that of female pronuclei in activated oocytes; however, it was delayed in ICM and TE transplants (p less than 0.01). With all stages of donor nuclei, final nuclear diameter was similar to, or larger than, that of female pronuclei. Fusion to activated oocyte cytoplasm, as opposed to nonactivated cytoplasm, prevented PCC and extensive nuclear swelling (16.0 +/- 0.7 vs. 30 +/- 0.7 microns, respectively, p less than 0.01). Nuclear diameter in early embryos was smaller (p less than 0.01), and development to blastocysts was reduced (p less than 0.05). The results indicate that remodeling of the donor nucleus is not essential for development to blastocysts; however, it is beneficial. Furthermore, complete reprogramming seems possible only after remodeling of the donor nucleus, i.e., PCC in nonactivated cytoplasm, followed by nuclear swelling upon activation of the oocyte.     

Meiotic and developmental competence of prepubertal and adult swine oocytes

The present study was conducted to compare meiotic and cytoplasmic competence of prepubertal and adult porcine oocytes, and the effects of EGF (0 to 100 ng/mL), FSH (0 to 400 ng/mL) and prepubertal pFF (0 to 10%) on nuclear maturation. Prepubertal oocytes were less responsive to FSH and pFF than were adult oocytes in terms of stimulation of nuclear maturation. The best nuclear maturation rates for prepubertal oocytes were obtained with 10 ng/mL EGF and 400 ng/mL FSH, whereas for adult oocytes no additional effect of EGF was seen in the presence of 400 ng/mL FSH. Supplementation with pFF had no additional effect on MII yield over that obtained with EGF plus FSH. After maturation in the presence of EGF, FSH and cysteamine, fertilization rates were not different between adult and prepubertal oocytes, but polyspermy was more frequent in prepubertal oocytes (31 +/- 17% vs. 17 +/- 7% in prepubertal and adult oocytes, respectively, P < 0.05). The addition of pFF to maturation medium decreased oocyte fertilization of adult oocytes and polyspermic fertilization in prepubertal oocytes. Blastocyst yield and developmental competence were significantly reduced in prepubertal oocytes compared to adult oocytes. The mean cell numbers in blastocysts cultured for 7 days ranged from 61 to 74, and did not differ among groups. Finally, the viability of the 2- to 4-cell embryos and blastocysts produced was assessed by embryo transfer experiments. One offspring was obtained after transfer of 2- to 4-cell embryos, and one after transfer of in vitro-produced blastocysts. In conclusion, although prepubertal gilt oocytes appeared less meiotically and developmentally competent than their adult counterparts, they can be used to produce blastocysts able to develop to term.     

Oocytes selected using BCB staining enhance nuclear reprogramming and the in vivo development of SCNT embryos in cattle

The selection of good quality oocytes is crucial for in vitro fertilization and somatic cloning. Brilliant cresyl blue (BCB) staining has been used for selection of oocytes from several mammalian species. However, the effects of differential oocyte selection by BCB staining on nuclear reprogramming and in vivo development of SCNT embryos are not well understood. Immature compact cumulus-oocyte complexes (COCs) were divided into control (not exposed to BCB), BCB+ (blue cytoplasm) and BCB- (colorless cytoplasm) groups. We found that BCB+ oocytes yielded a significantly higher somatic cell nuclear transfer (SCNT) blastocyst rate and full term development rate of bovine SCNT embryos than the BCB- and control oocytes. BCB+ embryos (embryos developed from BCB+ oocytes) showed increased acetylation levels of histone H3 at K9 and K18 (AcH3K9, AcH3K18), and methylation levels of histone H3 at K4 (H3K4me2) than BCB- embryos (embryos developed from BCB- oocytes) at the two-cell stage. Furthermore, BCB+ embryos generated more total cells, trophectoderm (TE) cells, and inner cell mass (ICM) cells, and fewer apoptotic cells than BCB- embryos. The expression of SOX2, CDX2, and anti-apoptotic microRNA-21 were up-regulated in the BCB+ blastocysts compared with BCB- blastocysts, whereas the expression of pro-apoptotic gene Bax was down-regulated in BCB+ blastocysts. These results strongly suggest that BCB+ oocytes have a higher nuclear reprogramming capacity, and that BCB staining can be used to select developmentally competent oocytes for nuclear transfer.     

Effects of beta-mercaptoethanol on formation of pronuclei and developmental competence of swamp buffalo oocytes

This study was conducted to determine the effect of supplementing maturation medium with beta-mercaptoethanol (betaME) on pronuclei formation and developmental competence of swamp buffalo oocytes. Buffalo oocytes were matured in TCM199 medium either with 10mM betaME or without betaME supplementation for 24h. In Experiment 1, oocytes were fixed and stained for cytological evaluation after in vitro fertilization (IVF). In Experiment 2, presumptive zygotes were cultured and their developmental competency was assessed. It was found that betaME significantly improved the proportion of oocytes that exhibited synchronous pronuclei formation (31.8+/-5.1% versus 17.9+/-3.3%, P<0.05). There were no significant differences between oocytes matured with or without betaME in their capability of developing into blastocyst-stage embryos (3.0+/-1.3% versus 1.8+/-0.9%). However, blastocysts produced from oocytes matured in the presence of betaME appeared to develop faster than those from oocytes matured in the absence of betaME (P<0.05). Cavitation of embryos from oocytes matured in the presence of betaME occurred at 156 hpi, whereas those matured in the absence of betaME occurred at 180 hpi. Although in vitro production of blastocysts did not increase by addition of betaME to maturation medium, quality of blastocysts produced from oocytes matured in the presence of betaME was improved. This study provides information for further investigations on optimizing a system for in vitro production of swamp buffalo embryos.     

The localization of LAP2beta in bovine oocytes after in vitro activation and fertilization

The present study was designed to clarify the localization of LAP2beta and to compare it with those of lamins A/C and B in bovine oocytes after activation and in vitro fertilization (IVF). After fertilization, LAP2beta was not found until telophase II, and was observed around condensed chromatin after the extrusion of the second polar body, but not in activated oocytes. Although the reaction of LAP2beta was temporally negative or weak on the membrane of the growing small pronuclei, it became strong on the fully grown pronuclei of both activated and fertilized oocytes. Examination of the timing of DNA synthesis using bromodeoxyuridine revealed that the expression of LAP2beta on the pronuclear membrane became strong around the end of the DNA synthesis in both activated and fertilized oocytes. Both male and female pronuclei exhibited the same reactivity to all nuclear proteins examined. It was also shown that LAP2beta first assembled around condensed chromatin, followed by the integration of lamins B and A/C as in somatic cells. LAP2beta staining was maintained on the nuclear membrane of the embryonic cells at interphase until the later stage of preimplantational development. There were no differences between parthenogenetic and fertilized embryos in the expression and localization of LAP2beta from the PN-stage oocyte to the blastocyst. The assembly of LAP2beta was observed around the telophase chromatin of both blastocyst and cumulus cells. Thus, it was shown that the timing of the aggregation of LAP2beta at the second meiosis was different from that in the mitosis of blastocyst and somatic cells. LAP2beta was constantly expressed in the nuclear membrane in in vitro fertilized and parthenogenetic embryos as was lamin B, and lamin A/C was expressed stage-dependently in both types of embryos. Lamin A/C was positive in some inner cell mass cells of parthenogenetic blastocysts, but not those of in vitro fertilized embryos.     

Developmental potential of selectively enucleated immature mouse oocytes upon nuclear transfer

Selective enucleation (SE) was applied to germinal vesicle (GV) oocytes by removing the chromatin attached to nuclear envelope, and leaving the liquid contents of GV in the cytoplast. However, after reconstruction with 1/8 blastomeres or fetal fibroblasts (FFs) neither the maturation efficiency nor the frequency of normal (asymmetric) division was improved as compared with completely enucleated (CE) oocytes. Chromosomal aberrations introduced with somatic nuclei were not rescued in SE oocytes either. On the other hand, timing of maturation division in SE GV oocytes, but not in CE GV oocytes, reconstructed with GV-karyoplasts was like in the control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomeres developed nucleolated donor pronuclei, contrary to CE oocytes. The latter could be rescued with nucleoli-containing nucleus, but not anucleolate nucleus, from a 1/2 blastomere. SE oocytes reconstructed with FFs contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes reconstructed with 1/8 blastomeres or FFs failed to cleave after activation or in vitro fertilization. Control GV oocytes enucleolated before fertilization seized cleavage at the 6-cell stage, as oppose to intact GV oocytes, which in 50.9% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the cleavage divisions. Activation of cumulus-enclosed SE GV oocytes matured in hormone-supplemented medium and fused to 1/2 blastomere-karyoplasts, yielded morulae, and blastocysts in 45.5% and 23.4% of reconstructed oocytes, respectively.     

Effect of follicle-stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro

A series of experiments were conducted to evaluate the effects of FSH supplementation during IVM on porcine oocyte nuclear maturation, and subsequent fertilization, cleavage and embryo development. Cumulus-oocyte complexes (COCs) were cultured 40 h without FSH (control), 40 h with FSH (FSH 0-40 h), or 20 h with FSH followed by a 20-h culture period without FSH (FSH 0-20 h). Nuclear stage of oocytes was assessed at intervals from 12 to 40 h of IVM. Furthermore, oocytes were in vitro fertilized, fixed and stained to determine normally fertilized and polyspermic oocytes. Additionally, COCs were matured with FSH, fertilized and zygotes cultured in NCSU-23. The percentage of cleaved embryos and blastocysts were determined and the number of nuclei was counted. The presence of FSH during the first 20 h of IVM retarded germinal vesicle breakdown. After 40 h of culture 84, 67 and 58% MII oocytes were observed in the FSH 0-20 h, FSH 0-40 h and control groups, respectively. After IVF, penetration rates were similar at 27, 26 and 29%, while the proportion of polyspermic oocytes was 7, 19 and 11% of penetrated oocytes for control, FSH 0-40 and FSH 0-20 h groups, respectively. Cleavage and blastocyst rates differed among treatments (21, 29 and 38%, and 7, 15 and 20% for control, FSH 0-40 and FSH 0-20 h groups, respectively). No differences in blastocyst cell number were found among groups. Blastocyst rates, based on number of cleaved embryos, were 51 and 52% for the FSH 0-40 and FSH 0-20 h groups, which differed significantly from the control group (31%). The results indicate that FSH has a stimulatory effect on nuclear and cytoplasmic maturation of sow oocytes. Addition of FSH for the first 20 h of culture was most beneficial, based on cleavage and blastocyst development rates.     

Glucose and glutamine metabolism in pre-attachment cattle embryos in relation to sex and stage of development

Individual Day-7 embryos (morulae to expanded blastocysts) were incubated with radiolabelled substrates and karyotyped to determine the sex. In Exp. 1, embryos were incubated for 3 h with D-[1-14C]glucose, as a measure of the activity of the pentose-phosphate pathway (PPP) and D-[5-3H]glucose, as a measure of total glucose metabolism. The labelled products 14CO2 and 3H2O were collected throughout the measurement period. Total glucose metabolism in male embryos was twice that in female embryos and increased between the morula and expanded-blastocyst stages. Relative to total glucose metabolism, PPP activity was four times greater in female than in male embryos. In Exp. 2, embryos were cultured with D-[1-14C]glucose, and L-[3,4-3H(N)]glutamine (a measure of Krebs cycle activity) in the presence of brilliant cresyl blue, a stimulator of the PPP. Glutamine metabolism increased from the morula to expanded-blastocyst stages. Relative to the metabolism of glutamine, the activity of the PPP was one-third greater in female than in male embryos.     

Measurement of the metabolism of energy substrates in individual bovine blastocysts

Individual blastocysts from cows were cultured for 3 h under 5% CO2 in air, in 4 microliters droplets of Ham's F-10 medium containing D-[5-3H]glucose, D-[1-14C]-glucose, D-[6-14C]glucose, [2-14C]pyruvate, or L-[U-14C]glutamine, and with or without 2,4-dinitrophenol (DNP) or phenazine ethosulphate (PES). The 14CO2 or 3H2O produced were collected by exchange with an outer bath of 400 microliter 25 mM-NaHCO3. All combinations of substrate and treatment (control, DNP or PES) produced measurable quantities of labelled product except for D-[6-14C]glucose in the presence of PES. Untreated and DNP-treated embryos developed normally during a subsequent 48-h culture period in fresh medium, but PES-treated embryos degenerated. Pyruvate and glutamine metabolism both increased markedly in the presence of DNP, indicating that the Krebs' cycle is active, and that glutamine can be used as an energy substrate. Conversely, DNP has no significant effect on glucose metabolism, indicating that glycolysis is blocked in the bovine blastocyst due to a lack or inhibition of pyruvate kinase. The production of 14CO2 from D-[1-14C]glucose increased significantly in the presence of PES, indicating that the activity of the pentose shunt is less than maximal.     

Expression of cytokeratin polypeptides in mouse oocytes and preimplantation embryos

The presence and distribution of intermediate filament proteins in mouse oocytes and preimplantation embryos was studied. In immunoblotting analysis of electrophoretically separated polypeptides, a distinct doublet of polypeptides with Mr of 54K and 57K, reactive with cytokeratin antibodies, was detected in oocytes and in cleavage-stage embryos. A similar doublet of polypeptides, reactive with cytokeratin antibodies, was also detected in late morula-and blastocyst-stage embryos, and in a mouse embryo epithelial cell line (MMC-E). A third polypeptide with Mr of 50K, present in oocytes only as a minor component, was additionally detected in the blastocyst-stage embryos. No cytokeratin polypeptides could be detected in granulosa cells. Immunoblotting with vimentin antibodies gave negative results in both cleavage-stage and blastocyst-stage embryos. In electron microscopy, scattered filaments, 10-11 nm in diameter, were seen in detergent-extracted cleavage-stage embryos. Abundant 10-nm filaments were present in the blastocyst outgrowth cells. In indirect immunofluorescence microscopy (IIF) of oocytes and cleavage-stage embryos, diffuse cytoplasmic staining was seen with antibodies to cytokeratin polypeptides but not with antibodies to vimentin, glial fibrillary acidic protein, or neurofilament protein. Similarly, the inner cell mass (ICM) cells in blastocyst outgrowths showed diffuse cytokeratin-specific fluorescence. We could not detect any significant fibrillar staining in cleavage-stage cells or ICM cells by the IIF method. The first outgrowing trophectoderm cells already had a strong fibrillar cytokeratin organization. These immunoblotting and -fluorescence results suggest that cytokeratin-like polypeptides are present in mouse oocytes and preimplantation-stage embryos, and the electron microscopy observations show that these early stages also contain detergent-resistant 10- to 11-nm filaments. The relative scarcity of these filaments, as compared to the high intensity in the immunoblotting and immunofluorescence stainings, speaks in favor of a nonfilamentous pool of cytokeratin in oocytes and cleavage-stage embryos.