Identification of a male-specific histocompatibility protein on preimplantation porcine embryos

An indirect immunofluorescence assay was used to detect the presence of male-specific protein(s) on various stages of preimplantation porcine embryos. Embryos were collected at slaughter from the reproductive tracts of day?2.5, ?4, ?5, ?6, and ?8 (day 0 = first day of estrus) sows and gilts. Embryos were placed in medium containing an anti-male primary antibody, washed, and transferred to culture drops containing a fluorescein isothiocyanate (FITC)-labeled secondary antibody. Embryos were classified as either fluorescent (H-Y positive) or nonfluorescent (H-Y negative), transferred to coded drops, and karyotyped to examine sex chromosomes. A total of 91 eight-cell to blastocyst stage embryos were evaluated; of these, 46% were classified as fluorescent and 54% as nonfluorescent. Of readable metaphase spreads (65%) from these embryos, 81% (48 of 59, P < 0.005) were correctly sexed by immunological detection of the male-specific antigen. Although 13 % (2/15)of four-cell embryos evaluated were classified as fluorescent, the accuracy with which embryos at this stage were sexed by detection of H-Y antigen was not different from 50%. Fifty percent of eight-cell embryos were classified as H-Y positive with 78% of embryos correctly sexed. It was concluded that the eight-cell embryo is the earliest stage of development for which there is evidence for expression of H-Y antigen. Detection of the male-specific protein was difficult at the expanded blastocyst stage.     

Uptake and incorporation of myo-inositol by bovine preimplantation embryos from two-cell to early blastocyst stages

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo preimplantation cattle embryos was investigated using [(3)H] myo-inositol. Uptake of inositol was examined in two-cell and four-cell embryos (day 2 after insemination), morulae (day 6) and early blastocysts (day 7). Uptake in all stages examined was largely sodium-dependent indicating the presence of a sodium-dependent inositol transporter. Uptake of inositol did not vary significantly from two-cell to early blastocyst stages when expressed either on a per embryo or a per microg of protein basis. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP, and PtdInsP(2), was detectable at all stages examined. In contrast, incorporation of inositol into inositol phosphates was not detected until blastocyst formation at day 7. The second messenger, Ins(1,4,5)P(3), was first detected in day 7 blastocysts.     

Leukemia inhibitory factor antisense oligonucleotide inhibits the development of murine embryos at preimplantation stages

Leukemia inhibitory factor (LIF) is an essential factor for implantation and establishment of pregnancy. However, its role in the development of preimplantation embryos remains controversial. In this study, changes in preimplantation embryos were determined after microinjection of LIF antisense oligonucleotide at the two-pronucleus stage. Although no significant differences were found in the percentages between the untreated group and the 0.25-fmol-treated group, the 0.5- or 1.0-fmol-treated groups had significantly lower percentages of embryos developed to the morula or blastocyst stage and the 2.0-fmol-treated group had significantly lower percentages of embryos developed to the four-cell, morula, or blastocyst stage. No embryos developed to the four-cell stage in the 4.0-fmol-treated group. Moreover, there was a decreasing trend in the levels of LIF immunoactivity with the increasing amount of LIF antisense oligonucleotide injected. The diameter of blastocysts in the 2.0-fmol-treated group was significantly smaller than that in the untreated group. The blastocysts in this group had significantly lower numbers of blastomeres and cells in the inner cell mass (ICM) or trophectoderm (TE) and ICM:TE ratio. The 1.0- or 2.0-fmol-treated groups had significantly lower implantation rates than their corresponding control groups. In the 2.0-fmol groups with supplementing exogenous LIF, significantly lower percentages were also observed in the four-cell, morula, and blastocyst stages. However, blastocysts treated with 50 ng/ml LIF had a significantly higher percentage than those in the LIF gene-impaired group without LIF supplement. These results indicate that LIF is a critical factor for the normal development of embryos at the preimplantation stages.     

Effects of thioredoxin on the preimplantation development of bovine embryos

Thioredoxin (TRX) is an ubiquitous protein disulfide reductase, which is known to be involved in the implantation development of mouse embryos. In the present study, recombinant human TRX was used to evaluate its effect on the promotion of preimplantation development of bovine embryos derived from in vitro maturation and fertilization. Supplementation of the medium 24h post insemination with TRX significantly (P<0.05) enhanced the frequency of development to the blastocyst stage in 5% O(2) concentration. The optimal concentration was 0.5 microg/ml (P<0.05, compared with 0, 0.1 and 1.0 microg/ml). This effect of TRX was evident only when added around the time of the first cleavage stage (24 h post insemination); no promotion was found with treatment at 6h (one-cell) or 44 h (six- to eight-cell) after insemination. Moreover, it is of interest that even with the best combination of the dose and timing of TRX treatment (0.5 microg/ml, at 24 h post insemination), no promotion of development was observed when embryos were cultured under 20% O(2). However, a preincubation of TRX in the culture medium under 20% oxygen for 24h did not diminish the promoting effect in the subsequent TRX treatment under optimal conditions, thus suggesting that the possible oxidation of TRX alone may not be the reason for the disappearance of the effect under a high oxygen concentration. These results indicate that TRX does improve the development of bovine embryos in vitro, though unlike the general reducing reagents such as beta-mercaptoethanol or cysteamine, TRX may have to exert its effect at specific times and in more physiologic oxygen environments.     

Effects of epidermal growth factor on preimplantation mouse embryos

When epidermal growth factor (EGF) was added to the medium for culture of preimplantation embryos, morphological development as determined by microscopic observation was unaffected, but 333 nM-EGF stimulated total uptake of [3H]leucine by late morulae/blastocysts which had been cultured for 24 h from morulae. Incorporation of [3H]leucine into protein by these embryos was increased by 0.33, 3.3 and 33 nM-EGF, following a quadratic relationship producing less stimulation at 333 nM, which may indicate down regulation of receptors. The estimated EC50 was approximately 0.25 nM. Manipulation of the culture period indicated that the embryos responded to EGF at the morula/blastocyst transition period and immunosurgery was used to show that the increased protein synthesis was restricted to the trophectoderm cells. No mitogenic effect was observed. The effective concentration of EGF is close to that of serum and to values which stimulate other tissues. It is suggested that EGF receptors appear at compaction and that EGF may have a role in differentiation of the trophectoderm cells.     

Platelet-activating factor: a paracrine factor in preimplantation stages of reproduction?

Platelet-activating factor (PAF) exerts its actions through activation of specific membrane binding sites found in a variety of tissues, including hypothalamus and endometrium. PAF has been identified in several reproductive tissues (i.e. embryo, ovary, uterus, and spermatozoon). In the uterus, PAF levels are hormonally controlled, being elevated by progesterone and prostaglandin E2 (PGE2). Antagonists of PAF interfere with sperm function, ovulation, and implantation. The available evidence suggests that PAF may be an important physiological regulator in reproduction.     

Effect of sericin on preimplantation development of bovine embryos cultured individually

The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H₂O₂), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H₂O₂. However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H₂O₂. When embryos were exposed to 100 μm H₂O₂ during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress.     

Effects of growth hormone on the ultrastructure of bovine preimplantation embryos

Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.This work was supported by the Deutsche Forschungsgemeinschaft (FOR 478/1)     

Detrimental effects of prostaglandin F2alpha on preimplantation bovine embryos

Two studies were performed to determine effects of prostaglandin F2alpha (PGF2alpha) on continued development of pre-compacted (in vitro-produced) and compacted (in vivo-derived) bovine embryos. In Experiment 1, pre-compacted embryos were placed in KSOM media supplemented with polyvinyl alcohol (0.3%) and assigned to the following treatments: (1) control; (2) PGF-1 (1 ng/mL PGF2alpha); (3) PGF-10 (10 ng/mL PGF2alpha); (4) PGF-100 (l00 ng/mL PGF2alpha); or (5) PGE-5 (5 ng/mL PGE2). Following 4 days of incubation in assigned treatments, continued development of pre-compacted embryos to blastocysts was reduced by addition of PGF2alpha in culture medium (P = 0.002). Development did not differ between control and PGE2 treatments (P > 0.10). In Experiment 2, compacted morula' s were placed in KSOM-PVA supplemented media and assigned to one of four treatments: (1) control; (2) PGF-0.1 (0.1 ng/mL PGF2alpha); (3) PGF-1 (1 ng/mL PGF2alpha); and (4) PGF-10 (10 ng/mL PGF2alpha). After 24h in culture, embryos were washed and placed in KSOM-BSA (0.5%) without PGF2alpha for an additional 48 h until assessment for development. Continued development of compacted morula to blastocyst was not affected by addition of PGF2alpha to the culture medium (P > 0.10). However, hatching rates of embryos cultured with PGF2alpha were lower (P = 0.05). In conclusion, it is suggested that PGF2alpha has a direct negative effect on continued embryonic development of pre-compacted and compacted bovine embryos.     

Heat shock-induced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon

Apoptosis is a form of cell death that can function to eliminate cells damaged by environmental stress. One stress that can compromise embryonic development is elevated temperature (i.e., heat shock). For the current studies, we hypothesized that heat shock induces apoptosis in bovine embryos in a developmentally regulated manner. Studies were performed to 1) determine whether heat shock can induce apoptosis in preimplantation embryos, 2) test whether heat-induced apoptosis is developmentally regulated, 3) evaluate whether heat shock-induced changes in caspase activity parallel patterns of apoptosis, and 4) ascertain whether exposure to a mild heat shock can protect embryos from heat-induced apoptosis. As determined by TUNEL reaction, exposure of bovine embryos > or =16 cells on Day 5 after insemination to 41 or 42 degrees C for 9 h increased the percentage of cells undergoing apoptosis. In addition, there was a duration-dependent increase in the proportion of blastomeres that were apoptotic when embryos were exposed to temperatures of 40 or 41 degrees C, which are more characteristic of temperatures experienced by heat-stressed cows. Heat shock also increased caspase activity in Day 5 embryos. However, heat shock did not induce apoptosis in 2- or 4-cell embryos, nor did it increase caspase activity in 2-cell embryos. The apoptotic response of 8- to 16-cell-stage bovine embryos to heat shock depended upon the day after insemination that heat shock occurred. When 8- to 16-cell embryos were collected on Day 3 after insemination, heat shock of 41 degrees C for 9 h did not induce apoptosis. In contrast, when 8- to 16-cell embryos were collected on Day 4 after insemination and exposed to heat shock, there was an increase in the percentage of cells undergoing apoptosis. Exposure of 8- to 16-cell embryos at Day 4 to a mild heat shock of 40 degrees C for 80 min blocked the apoptotic response to a subsequent, more-severe heat shock of 41 degrees C for 9 h. In conclusion, apoptosis is a developmentally acquired phenomenon that occurs in embryos exposed to elevated temperature, and it can be prevented by induced thermotolerance.     

Expression of paternal glucose phosphate isomerase-1 (Gpi-1) in preimplantation stages of mouse embryos

Electrophoretic variants of glucose phosphate isomerase have been used to study the time of paternal gene activation during early embryogenesis of the mouse. Hybrid embryos obtained from matings of GPI-1A ♀ X GPI-1B ♂ were examined electrophoretically, and assayed for GPI activity during preimplantation stages. The heteropolymeric GPI-1AB band was detected in late blastocysts and all three bands of the hybrid pattern were discernible in samples of expanded blastocysts, day 6. These findings indicate that the Gpi-1 paternal locus is expressed by day 5. Activity levels of GPI were comparable to values reported for G6PD. The activity of GPI was constant for days 1, 2, and 3; however, a marked decrease in activity occurred by day 4. A slight decrease in activity was observed in embryos from days 5 and 6. Our results demonstrate the value of using electrophoretic variants to pinpoint synthesis of new enzyme which may not be reflected in changes in levels of activity.