Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

Nuclear transfer in cattle using in vivo-derived vs. in vitro-produced donor embryos: Effect of developmental stage

To determine the best developmental stage of donor embryos for yielding the highest number of clones per embryo, we compared the efficiencies of nuclear transfer when using blastomeres from morulae or morulae at cavitation, or when using inner-cell-mass cells of blastocysts as nuclear donors. This comparison was done both on in vivo-derived and in vitro-produced donor embryos. In experiment 1, with in vivo-derived donor embryos, nuclei from morulae at cavitation supported the development of nuclear transfer embryos to the blastocyst stage (36%) at a rate similar to that of nuclei from morulae (27%), blastomeres from morulae at cavitation being superior (P < 0.05) to inner-cell-mass cells from blastocysts (21%). The number of blastocysts per donor embryo was significantly (P < 0.05) higher when using nuclei from morulae at cavitation (15.7 ± 4.1) rather than nuclei from morulae (9.8 ± 5.5) or blastocysts (6.3 ± 3.3). With in vitro-produced donor embryos (experiment 2), nuclei from morulae yielded slightly more blastocysts (32%) than nuclei from morulae at cavitation (29%), both stages being superior to nuclei from blastocysts (15% development to the blastocyst stage). Morulae at cavitation yielded a higher number of cloned blastocysts per donor embryo (11.5 ± 5.9) than did morulae (9.3 ± 3.2) and blastocysts (3.3 ± 1.4). Transfer of cloned embryos originating from in vivo-derived morulae, morulae at cavitation, and blastocysts resulted in four pregnancies (10%), three pregnancies (7%), and one (17%) pregnancy on day 45. The corresponding numbers of calves born were 3 (4%), 3 (7%), and 0, respectively. After transfer of blastocysts derived from in vitro nuclear donor morulae (n = 16) and morulae at cavitation (n = 7), two (20%) and two (50%) recipients, respectively, were pregnant on day 45. However, transfer of seven cloned embryos from in vitro donor blastocysts to three recipients did not result in a pregnancy. Using in vitro-produced donor embryos, calves were only obtained from morula-stage donors (13%). Our results indicate that the developmental stage of donor embryos affects the efficiency of nuclear transfer, with morulae at cavitation yielding a high number of cloned blastocysts. © 1996 Wiley-Liss, Inc.     

Effect of stage of development on survival of mouse embryos frozen-thawed rapidly

Embryos were recovered on Day 4 of pregnancy from superovulated random-bred OF₁ Swiss albino mice. They were classified into four categories based on their stage of development: expanding blastocyst, blastocyst, early blastocyst, and compacted morula. They were then cooled at 2 °C/min from ?7 to ?25 °C in a freezing medium containing 1.36 M glycerol and 0.25 M sucrose in phosphate-buffered saline (PBS). At ?25 °C, they were plunged into LN₂ and thawed a few hours later in water at 20 °C. After washing in PBS, recovered embryos were cultured for 20 to 24 hr and the number of embryos that had developed normally was recorded. The results showed a clear effect of the stage of development on survival. Survival of expanding blastocysts and blastocysts was very low (1.4 and 21.8%, respectively) compared to that of early blastocysts and compacted morulae (69.4 and 73.5%). The more differentiated stage of the blastocyst (two kinds of cells) and the presence of a blastocoelic cavity may explain the differences observed under our cooling conditions. As a further test of viability, 93 blastocysts that had developed in culture for 20 hr from 153 frozen-thawed early blastocysts and compacted morulae (60.8%) were transferred to 8 recipient mice. Seven became pregnant, yielding $\text{38}\text{82}$ normal live young (46.3%).     

In vitro culture of goat morulae to blastocysts before freezing

The results of transfer of frozen-thawed caprine embryos that were collected either as blastocysts or morulae and cultured to the blastocyst stage prior to freezing were compared. After thawing, the embryos collected as blastocysts appeared to be of marginally better quality than those that had been cultured from morulae (89 vs 72% rated as good; P > 0.05). The transfer of 24 frozen-thawed embryos collected as blastocysts to 12 recipients resulted in a pregnancy rate of 83% (10/12) and an embryo survival rate of 67%. Corresponding results for frozen-thawed blastocysts that had been cultured from morulae and were transferred to 11 recipients were 54% (6/11) and 41%, respectively. Since an earlier investigation had shown that the transfer of frozen caprine morulae yields very poor results, in our laboratory all morulae are now cultured to the blastocyst stage before being cryopreserved.     

Carbohydrate changes in pre- and peri-implantation mouse embryos as detected by a monoclonal antibody

We have examined the tissue and embryonic distribution of an antigen on a large polysaccharide that is recognized by a monoclonal antibody, IIC3, prepared against F9 teratocarcinoma cells. By immunofluorescence the antigen is first detected on compacted morulae and early blastocysts. It is strongly expressed on the primary endoderm and trophoblast of expanded blastocysts, but then disappears from the trophoblast of attached blastocysts in vitro. The binding of the antibody is completely inhibited by D-galactose and N-acetylgalactosamine. Fluoresceinated lectins were used to study further the changes in cell surface carbohydrates on trophoblast during implantation. Ricinus I, specific for terminal galactose, binds to preimplantation stages but does not bind to the trophoblast of the attached blastocyst. On the other hand, wheat germ agglutinin, specific for N-acetylglucosamine and sialic acid, binds to all preimplantation embryos and also to attached blastocysts (embryo proper and trophoblast). Neuraminidase treatment of blastocyst outgrowths enhances binding of both IIC3 and Ricinus I to the trophoblast; conversely, the binding of wheat germ agglutinin is decreased by this treatment. The results obtained in this study show changes of cell surface carbohydrates during early mouse development and suggest that sialic acid may be masking molecules on the surface of the trophoblast at the time of implantation.     

The effects of water-soluble vitamins on the expansion of rabbit blastocysts in vitro

The vitamin requirements for culture of rabbit morulae to expanded blastocysts were examined. Early morulae were cultured for 5 days either in a control complete medium containing all the 11 water-soluble vitamins of F10 culture medium (biotin, pantothenate, choline, inositol, niacinamide, pyridoxine, riboflavin, thiamine, folic acid, B12, and lipoic acid) or in media with each vitamin omitted individually. Blastocyst diameters were measured at the end of culture. The omission of inositol, pyridoxine, riboflavin, and niacinamide resulted in large statistically significant decreases in blastocyst expansion. The omission of B12 resulted in a significant increase in blastocyst expansion indicating that the level present in F10 is toxic to rabbit blastocysts.     

Blastocyst formation by pig embryos resulting from in-vitro fertilization of oocytes matured in vitro

Follicular oocytes collected from prepubertal gilts at a local slaughter house were matured (36 h), fertilized and developed in vitro. Of 785 embryos, 190 (24%) embryos cleaved to the 2-4 cell stages with blastomeres of regular size by 33 h after insemination. These cleaved embryos were surgically transferred into the oviducts of 4 synchronized recipient gilts and recovered from the uterine horns 4 or 7 days later: 13 morulae, 2 blastocysts and 1 expanded blastocyst were recovered after 4 days and 3 hatched blastocysts were recovered 7 days after transfer. Re-culture in vitro sustained further development of morulae recovered 4 days after transfer: 11 of 13 morulae had developed to the blastocyst/hatched blastocyst stages. Overall, 17 of 190 (9%) embryos developed to the blastocyst stage. The results indicate that pig oocytes can be matured and fertilized in vitro, and subsequently develop to the blastocyst stage.     

Oxygen tension affects histone remodeling of in vitro–produced embryos in a bovine model

In vitro production of bovine embryos is a biotechnology of great economic impact. Epigenetic processes, such as histone remodeling, control gene expression and are essential for proper embryo development. Given the importance of IVP as a reproductive biotechnology, the role of epigenetic processes during embryo development, and the important correlation between culture conditions and epigenetic patterns, the present study was designed as a 2 × 2 factorial to investigate the influence of varying oxygen tensions (O₂; 5% and 20%) and concentrations of fetal bovine serum (0% and 2.5%), during IVC, in the epigenetic remodeling of H3K9me2 (repressive) and H3K4me2 (permissive) in bovine embryos. Bovine oocytes were used for IVP of embryos, cleavage and blastocyst rates were evaluated, and expanded blastocysts were used for evaluation of the histone marks H3K9me2 and H3K4me2. Morulae and expanded blastocysts were also used to evaluate the expression of remodeling enzymes, specific to the aforementioned marks, by real-time polymerase chain reaction. Embryos produced in the presence of fetal bovine serum (2.5%) had a 10% higher rate of blastocyst formation. Global staining for the residues H3K9me2 and H3K4me2 was not affected significantly by the presence of serum. Notwithstanding, the main effect of oxygen tension was significant for both histone marks, with both repressive and permissive marks being higher in embryos cultured at the higher oxygen tension; however, expression of the remodeling enzymes did not differ in morulae or blastocysts in response to the varying oxygen tension. These results suggest that the use of serum during IVC of embryos increases blastocyst rate without affecting the evaluated histone marks and that oxygen tension has an important effect on the histone marks H3K9me2 and H3K4me2 in bovine blastocysts.     

The Chinese hamster gene map. Assignment of four genes (DTS, PGM2, 6PGD, EnoI) to chromosome 2

Mouse morulae and blastocysts express cell surface antigens that fortuitously cross-react with antisera to human chorionic gonadotropin (hCG). In the present study, the cell surface and cytoplasmic expression of these antigens was followed in mouse unfertilized oocytes, different stages of preimplantation embryos and in early post-implantation embryos cultured from blastocysts. In addition to their known stage-dependent cell surface expression on morulae and blastocysts, these antigens (1) were already present in the cytoplasm of mature unfertilized oocytes and pre-morula stages of embryos; (2) remained expressed as cell surface antigens on cells of the inner cell mass (ICM), but not on the surface of trophectodermal cells with further blastocyst development although (3) they persisted as cytoplasmic antigens in trophectodermal cells. In addition, these antigens were also detectable by antiserum to the alpha subunit of hCG.     

Developmental ability of bovine embryos after nuclear transfer based on the nuclear source: in vivo versus in vitro

Bovine nuclear transfer embryos reconsitituted from in vitro-matured recipient oocyte cytoplasm and different sources of donor nuclei (in vivo, in vitro-produced or frozen-thawed) were evaluated for their ability to develop in vitro. Their cleavage rate and blastocyst formation are compared with those of control IVF embryos derived from the same batches of in vitro-matured oocytes that were used for nuclear transfer and were co-cultured under the same conditions on bovine oviducal epithelial cell monolayers for 7 d. Using fresh donor morulae as the source of nuclei resulted in 30.2% blastocyst formation (150 497 ), which was similar to that of control IVM-IVF embryos (33.8% blastocysts, 222 657 ). When IVF embryos were used as the source of nuclei for cloning, a slightly lower blastocyst formation rate (22.6%, 41 181 ) was obtained but not significantly different from that using fresh donor morulae. Nuclear transfer embryos derived from vitrified donor embryos showed poor development in vitro (7.1%, 11 154 ). No difference in morphology or cell number was observed after 7 d of co-culture between blastocysts derived from nuclear transfer or control IVF embryos. The viability of 34 in vitro-developed nuclear transfer blastocysts was tested in vivo and resulted in the birth of 11 live calves (32.3%).     

Inhibitors of mitochondrial ATP production at the time of compaction improve development of in vitro produced porcine embryos

The relationship between partial inhibition of mitochondrial ATP production during the peri-compaction stage and porcine embryonic development was studied. In vitro produced porcine compact morulae were cultured for two days under conditions that should inhibit ATP production via oxidative phosphorylation. The culture conditions included supplementation of the culture medium with sodium azide (NaN3), an oxidative phosphorylation inhibitor; incubation in the presence of 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation; or incubation under 5% O2 concentration. NaN3 (10-20 microM) increased the average nuclear number found in the resulting blastocysts (P<0.05). The embryos developed in the presence of 100 microM DNP formed blastocysts at a significantly higher incidence than the control embryos (P<0.001); the average nuclear number found in these blastocysts was also higher (P<0.005). When these treatments were applied from the 1-cell stage they proved to be detrimental. Elevations in the frequency of blastocyst formation (P<0.05), and in the average nuclear number per blastocyst (P<0.001) were also measured when compact morulae were incubated in an atmosphere containing 5% vs. 20% O2. NaN3 or DNP did not have negative effects on long term development: the treated embryos were able to form viable conceptuses by day 30 after being transferred into recipients. The data indicate that transient inhibition of mitochondrial ATP production is advantageous for porcine embryonic development in vitro.     

Isolation and characterization of embryonic stem-like cells from canine blastocysts

Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. Here we describe the isolation of ES-like cells from canine blastocysts. Canine embryos were collected from beagle bitches at day 11-16 of first estrus. A total of 80 normal embryos were obtained from 15 dogs. Of the embryos, 13 were at the morulae stage, 39 at the blastocyst stage, and 28 at the hatched blastocyst stage. The inside of morulae or inner cell masses (ICMs) of blastocysts were isolated mechanically, and cultured onto mouse embryonic fibroblasts (MEF) as feeder layers. Primary cell colonies were formed in 0% (0/13) of morulae, 25.6% (10/39) of blastocysts, and 67.9% (19/28) of hatched blastocysts. These colonies were separated either by enzymatic dissociation or by mechanical disaggregation. Dissociation with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remained undifferentiated, and two ES-like cell lines (cES1, cES2) continued to grow in culture after eight passages. These cells had typical stem cell-like morphology and expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4. These cells formed embryoid bodies (EBs) in a suspension culture; extended culture of EBs resulted in the formation of cystic EBs. When the simple EBs were cultured on tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells. These observations indicate that we successfully isolated and characterized canine ES-like cells.     

Vitrification of Mouse Embryos at Various Stages by Open-Pulled Straw (OPS) Method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure—that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method—that is, embryos were first pretreated in 10%E+10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E+10%D for 0.5 min, exposed to EDFS30 for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

Vitrification of mouse embryos at various stages by open-pulled straw (OPS) method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure-that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method-that is, embryos were first pretreated in 10%E + 10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E + 10%D for 0.5 min, exposed to EDFS30for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation

Culture of bovine zygotes in medium SOFaa supplemented with 100 microM EDTA significantly increased cleavage rates during the first 72 hr of development compared to development in SOFaa. However, continued culture in the presence of EDTA for a further 72 hr (total of 6 days of culture) resulted in significantly reduced development to the morulae/blastocyst and blastocyst stages compared to culture without EDTA. Highest rates of development to the morulae/blastocyst stage (56.5%) and to the blastocyst stage (43.2%) were achieved when zygotes were cultured for 72 hr with EDTA before transfer to medium SOFaa without EDTA. Resultant blastocysts also had significantly increased blastocyst cell number and ICM cell number compared to those cultured without EDTA in the first 72 hr. EDTA was shown to inhibit glycolytic activity of the cleavage stage embryo, thereby preventing the premature stimulation of glycolysis and enhancing development. However, EDTA should not be used for the later stage embryo as the inhibition of glycolysis reduces energy production at the blastocyst stage and significantly inhibits inner cell mass development.     

Effects of membrane piercing and the type of pronuclear injection fluid on development of in vitro-produced bovine embryos

We studied the effects of mechanical damage of plasma and pronuclear membranes on the development of in vitro-produced bovine zygotes. The effects of the type of injection fluid and the presence of DNA in the zygotes were also studied. In the first experiment, either the plasma membrane or both the plasma and pronuclear membranes of zygotes were pierced with a capillary filled with DNA-buffer. Additionally, pronuclear microinjections with either MilliQ-water or buffer were performed. In the second experiment, pronuclear microinjections with buffer containing either none or 2 microg/ml of DNA were performed. Development of cleaved embryos to compact morulae and blastocysts at Day 7 was monitored. Results of Experiment 1 indicate that membrane piercing does not decrease development of cleaved embryos as compared with that of the controls (30.8, 28.8 and 29.9% compact morulae and blastocysts for controls, plasma membrane and pronuclear membrane pierced groups, respectively). Pronuclear microinjections decreased development significantly as compared with that of the controls, but no differences were observed between the effects of water and buffer (29.9, 18.4 and 15.5% compact morulae and blastocyst, respectively). Results of Experiment 2 showed that inclusion of DNA into the injection buffer decreased development even more drastically (36.7, 27.5 and 14.5% compact morulae and blastocyst in the control, buffer-injected and DNA-injected groups, respectively).     

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

Characterization of UDP-galactosyl:asialo-mucin transferase activity in the Golgi system of rat liver.

UDPgalactosyltransferase activity (UDPgalactose:mucopolysaccharide galactosyltransferase, EC 2.4.1.74) was measured in a well-characterized fraction of Golgi membranes in the presence of UDPgalactose and exogenous acceptor sites. Substrate saturation for 0.05 mg Golgi protein was achieved at a concentration of 4.6 mM UDPgalactose. Desialylated mucin proved to be the most suitable acceptor protein. Access to galactose acceptor sites was not rate limiting for the reaction when 20 mg of asialo-mucin/ml of incubation mixture was used. With these concentrations of substrates the use of nucleotides to inhibit pyrophosphatases and of detergents to perturb the membrane structure was not necessary and proved, in fact, to be inhibitory to galactose transfer. UDPgalactosyl:asialo-mucin transferase activity in Golgi membranes was 230 nmol galactose transferred/mg Golgi protein per 30 min.     

Role of embryonic oestrogen in rabbit blastocyst development and metabolism

Rabbit morulae were grown for 24 h in Ham's F12 medium supplemented with BSA. CI-628 citrate (1.5 micrograms/ml), a specific oestrogen antagonist, significantly inhibited the transformation of morulae to blastocysts. This inhibition was reversed with oestradiol-17 beta (1 micrograms/ml) but not oestradiol-17 alpha (1 micrograms/ml) added to the culture medium. The specific activities of phosphofructokinase, lactic dehydrogenase, malate dehydrogenase and alkaline phosphatase in blastocysts grown in vitro for 24 h in medium TC 199 + BSA showed significant elevation with blastocyst growth and expansion, while that of acid phosphatase revealed no change, and leucine aminopeptidase activity declined significantly. These changes were markedly inhibited by CI-628 citrate (2 micrograms/ml) and were reversed by oestradiol-17 beta (0.4 micrograms/ml) but not by oestradiol-17 alpha (0.4 micrograms/ml). Our findings suggest a role of oestrogen present in the rabbit morula and blastocyst in the triggering of embryonic differentiation and metabolic functions.     

Effects of the putative phospholipid precursors, inositol, choline, serine and ethanolamine, on formation and expansion of rabbit blastocysts in vitro

Rabbit morulae were cultured to blastocysts in various concentrations of the potential phospholipid precursors, myo-inositol, choline, serine and ethanolamine. Serine (20-2500 microM) had a significant stimulatory effect on blastocyst formation and blastocyst expansion and inositol (3-375 microM) had a significant stimulatory effect on blastocyst expansion. There was no significant stimulatory effect of choline or ethanolamine.