Factors influencing the in vitro hatching of mouse blastocysts

The influence of bovine serum albumin (BSA) concentration on embryo hatching and the number of embryos cultured per drop of culture medium was examined in F1 (C57BL/6J × DBA/2J), C3HeB/FeJ strain and Line E mice. Embryos collected from F1 and Line E mice exhibited uniform hatching rates at BSA concentrations between 1 and 10 mg/ml, and embryo numbers ranging from 1 to 10 per 3 μ1 of culture medium. The hatching of C3HeB/FeJ blastocysts was greater at the higher concentrations of BSA and higher embryo densities. When the C3HeB/FeJ embryos were grown at high densities until morula and then cultured singly in fresh media they hatched at a low rate. However, when allowed to develop until the blastocyst stage before replotting, the embryos hatched at a high rate. C3HeB/FeJ embryos cultured singly until morula and then placed in groups of 10 hatched at a high rate. Single C3HeB/FeJ embryos, cultured in medium conditioned by the prior presence of embryos at high densities, hatched at a slightly higher frequency than those cultured in fresh medium. There was no tendency of embryos developing from the two-cell to the eight-cell stages to hatch when cultured in the presence of high densities of hatching blastocysts.     

Lectin-induced abnormalities of mouse blastocyst hatching and outgrowth in vitro

We have examined the role of cell surface glycoconjugates during mouse blastocyst maturation, hatching, attachment, and outgrowth by monitoring the influence of six lectins on blastocyst development in vitro. Two lectins, concanavalin A and wheat germ agglutinin were toxic to blastocysts at the concentrations used. Bandierea simplicifolia lectin 1 (BSL-1) induced abnormal growth, developmental arrest at the hatching stage, and some disruption of cell contacts. Culture with Lotus tetragonolobus lectin-1 (LTA-1) also disrupted cell contacts and caused developmental arrest. The remaining lectins, Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin (UEA), retarded blastocyst hatching and outgrowth but did not induce any major defects, although differentiation of the inner cell mass was limited by both. This study demonstrates that very low concentrations of lectins can disrupt blastocyst development, suggesting that exposed surface saccharide moieties may be involved in interactions between blastomeres and their environment.     

Fibronectin and laminin promote in vitro attachment and outgrowth of mouse blastocysts

The process of mammalian implantation has been investigated using an in vitro model system wherein the trophoblast cells of mouse blastocysts attach to and outgrowth on tissue culture plates containing a complex medium. We now report that two extracellular matrix glycoproteins, fibronectin and laminin, when individually precoated on tissue culture plates promoted in vitro attachment and outgrowth of mouse blastocysts in serum-free medium. The kinetics of attachment and outgrowth processes in the presence of either of these two proteins were identical to that observed in complex, serum-containing medium. In contrast, plates containing a collagen matrix or pretreated with a variety of other serum proteins or various lectins failed to support in vitro attachment and outgrowth of blastocysts. Because all components of the culture medium are defined and both fibronectin and laminin are known components of the basement membrane of the endometrium, this in vitro system offers considerable advantages over the serum supplemented system to study in vitro implantation.     

Short-term urea feeding decreases in vitro hatching of bovine blastocysts

Cows fed high-protein diets may have impaired reproductive performance. Although the pathogenesis has not been completely elucidated, it appears that not only the uterus, but also the follicle and oocyte, are affected by excessive plasma urea nitrogen (PUN) concentrations. Thus, the objective was to determine the effects of short-term urea feeding on the competence of bovine oocytes. Forty crossbred heifers (Bos indicus vs Bos taurus) were allocated to two groups, namely CONTROL (maintenance diet) and UREA (maintenance diet supplemented with 75 g of urea/day), following a cross-over design. Heifers received their respective diets for 6 d (without adaptation). On the sixth day, blood samples were harvested both before and 3 h after feeding, and cumulus oocyte complexes (COCs) were collected by ovum pick-up. Although PUN concentrations were higher in UREA than CONTROL heifers (31.31 mg/dL ± 1.13 vs 22.12 mg/dL ± 0.86; mean ± SEM), neither the number of COCs recovered (8.8 ± 1.0 vs 9.2 ± 0.8, UREA vs CONTROL, respectively) nor their quality (based on morphology) differed significantly between groups. Next, oocytes were fertilized and cultured in vitro to assess developmental rates. There was an absence of significant differences between groups for rates of cleavage (Day 3) or blastocyst formation (Days 6, 7 and 9), but the hatched blastocyst rate on Day 11 after fertilization was lower (P < 0.05) in the UREA than the CONTROL groups (64.3 vs 83.5%). Therefore, we inferred that the effects of urea were only manifest later in development. In conclusion, high PUN concentrations decreased oocyte competence in heifers, reinforcing the hypothesis that poor reproductive performance in cows with high PUN was due, at least in part, to a deleterious effect on oocytes.     

Effect of removal of the zona pellucida on subsequent development of mouse blastocysts in vitro.

Removal of the zona pellucida allowed mouse blastocysts incapable of hatching in vitro to develop in culture. Blastocysts denuded by pronase always developed further than those of identical age that had hatched spontaneously. More blastocysts mechanically denuded and treated with pronase developed egg cylinder-like structures than did blastocysts mechanically denuded and not treated with pronase. Plastic support gave better development of blastocysts than did glass.     

Influence of reduced glucose levels on the in vitro hatching,attachment, and trophoblast outgrowth of the mouse blastocyst

The influence of glucose on the in vitro hatching, attachment, and trophoblast outgrowth of 856 mouse blastocysts has been examined. Results indicate that glucose is a necessary factor for the in vitro hatching of the mouse blastocyst and that a delay in hatching can be accomplished by culturing blastocysts in culture media devoid of glucose.     

Pulsatile activity and hatching of in vitro produced cow blastocysts: effects of serum supplementation.

The effect of serum added to a modified SOF medium on pulsatile activity and hatching of in vitro produced cow blastocysts was investigated by time-lapse cinematography. Embryos were generated from abattoir material and cultured in mSOF without serum or with 10% FCS added at 42h pi. Addition of serum significantly increases pulsatile activity before zona rupture and reduces the time of hatching. Pulsatile activity does not seem to be involved in the hatching process.     

Effects of culture milieus on the development of mouse blastocysts in vitro.

Various culture milieus were examined for their support of mouse blastocyst development. Two important variables were the time at which human cord serum was added to the medium and the concentration of amino acids. In the best medium. Eagle's Minimum Essential Medium (fortified with six times the usual amino-acid concentration plus 20 percent fetal bovine serum, replaced after 48 hr with human cord serum), 83 percent of the blastocysts shed the zona pellucida, 58 percent developed to the early egg cylinder stage, 42 percent to the advanced egg cylinder stage and 22 percent attained the primitive streak stage after 6 to 8 days of culture.     

Vitamin requirements for development of eight-cell hamster embryos to hatching blastocysts in vitro

We have shown in previous studies that development of 8-cell hamster embryos to hatching and hatched blastocysts in vitro is stimulated by the addition to the culture medium of a group of 11 water-soluble vitamins and growth factors from Ham's F10 medium. In the present study, the requirement for each of these vitamins for blastocyst hatching was examined by using a chemically defined protein-free medium. Eight-cell hamster embryos were cultured for 3 days either in medium with all 11 vitamins or in media with a single vitamin omitted at a time or in medium without any vitamins. The only vitamins whose omission caused a significant decrease in blastocyst hatching at any stage were inositol, pantothenate, and choline, with the omission of inositol having the most severe effect. This finding was confirmed in a subsequent experiment in which the addition of these 3 vitamins stimulated the same degree of hatching as all 11 vitamins.     

Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo.

The present studies were performed to establish the effects of the size and number of artificial holes produced in the zona pellucida (ZP) on hatching and trophoblast outgrowth in vitro. Limited partial zona dissection (PZD) produced small, narrow incisions, and zona drilling with acidic Tyrode's (AT) across a larger area in the ZP was used to produce bigger round holes. Some embryos were micromanipulated once; others were micromanipulated several times. Blastocysts hatched preferentially through the artificial gaps, but completion of hatching was dependent on the size of the hole. Only 16% (26/167) of PZD embryos migrating through narrow holes hatched completely; the remainder were trapped in a typical figure-eight shape. Seventy-two percent (43/60) of those migrating through larger PZD holes hatched, but trophoblast outgrowth was not observed. Significantly (P less than 0.001) more AT-blastocysts hatched (248/270; 92%) and showed trophoblast outgrowth (176/248; 70%). Simultaneous hatching through several openings was rarely observed in AT-embryos (14/167; 8%), but this did occur in 36% (73/201) of the PZD embryos. Trapping of PZD-embryos could be almost entirely avoided by drilling with AT elsewhere on the ZP. Embryos with multiple holes in their zonae preferentially hatched through the largest opening. The results suggest that the ability of microsurgically treated human embryos to fully hatch in vitro, should be carefully (re)assessed prior to application of clinical micromanipulation systems. Micromanipulated embryos with small holes in their zonae may be rescued by performing an additional more aggressive opening procedure elsewhere on the ZP.     

An ultrastructural study of in-vitro interaction of guinea-pig and mouse blastocysts with extracellular matrices.

Guinea-pig (intrusive) and mouse (displacement) blastocysts display different cellular mechanisms of implantation. Blastocysts were placed in CMRL-1066 supplemented with either 10 or 20% fetal calf serum, 0.1M L-glutamine and antibiotics and then transferred to dishes previously coated with either Matrigel or type I collagen. After culture for 48 or 72 h, the dishes were processed for transmission electron microscopy. Blastocysts had attached to both extracellular matrices by 48 h. Matrigel elicited minimal trophoblast cell activity. Trophoblast cell projections were oriented parallel to the Matrigel and displayed little invasive activity, but trophoblast cells displayed active interaction with type I collagen. By 72 h, trophoblast cells exhibited slender, anastomosing projections which extended into the collagen matrix. Bundles of microfilaments running parallel with the long axis of the projections were observed. The morphology of type I collagen was altered in the immediate vicinity of the trophoblast projections. The projections interdigitated and desmosomes developed between processes. Projections appeared to meet, fuse and entrap matrix. These results suggest that trophoblast cells do not significantly interact with Matrigel, but penetrate into type I collagen.     

Significance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts in vitro versus in vivo

We investigated the course of mouse blastocyst hatching in vitro after experimental modulation of the hatching process by growth hormone or by laser treatment and compared it to embryos grown in vivo. When embryos were grown in vitro, successful hatching was dependent on blastocyst expansion and was based on a minimum number of embryonic cells. Embryos grown in the presence of growth hormone were more advanced in their development and hatched earlier. When an artificial opening was laser-drilled into the zona pellucida, hatching occurred at lower numbers of embryonic cells. In vivo, escape from the zona pellucida occurred earlier and independent of blastocyst expansion. However, when we isolated in vivo-grown blastocysts with intact zonae that had developed in vivo and then cultured them in vitro, blastocysts started to expand and hatched the following day when a sufficiently high number of embryonic cells was present. Our data show that successful hatching in vitro is dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding. In vivo, the lower number of embryonic cells detected in zona-free blastocysts indicates that the underlying mechanism of zona escape is different, does not depend on blastocyst expansion, and presumably involves lytic factors from the uterus.     

Effects of culture milieus on the development of mouse blastocysts in vitro

Summary Various culture milieus were examined for their support of mouse blastocyst development. Two important variables were the time at which human cord serum was added to the medium and the concentration of amino acids. In the best medium, Eagle's Minimum Essential Medium (fortified with six times the usual amino-acid concentration plus 20% fetal bovine serum, replaced after 48 hr with human cord serum), 83% of the blastocysts shed the zona pellucida, 58% developed to the early egg cylinder stage, 42% to the advanced egg cylinder stage and 22% attained the primitive streak stage after 6 to 8 days of culture. A preliminary account was given at the Tissue Culture Association Meeting in 1976 and the abstract published in its proceedings (1). This work was supported by MRC Grant No. MA4235.     

Enhancement of hatching and trophoblastic outgrowth by mouse embryos cultured in Whitten's medium containing plasmin and plasminogen

Mice were induced to superovulate and 2-cell embryos were cultured in Whitten's medium with 10 mg bovine serum albumin/ml (WM) as control, Medium WM with 2.3, 4.6, 23.1 or 46.2 micrograms plasmin/ml, Medium WM with 14.6, 29.1 or 145.7 micrograms plasminogen/ml, Medium WM with 0.1, 0.2, 1.1 or 2.2 micrograms trypsin/ml; Medium WM with 0.2, 0.3, 1.6 or 3.3 micrograms pronase/ml and Medium WM with 10% heat-treated bovine serum (HTBS). Proteolytic activities in the culture media were evaluated at the start of the culture period and 10 days later. Blastocyst formation was significantly reduced in cultures supplemented with pronase and in the two higher levels of trypsin when compared to that in Medium WM. More embryos developed to the blastocyst stage in Medium WM + 2.3 or 23.1 micrograms plasmin/ml and Medium WM + 14.6 micrograms plasminogen/ml than in Medium WM (P less than 0.05). The incidence of hatching was significantly greater in Medium WM than in all plasminogen- and plasmin-supplemented media except for Medium WM + 29.1 micrograms plasminogen/ml. Although not significantly different, hatching was lower in Medium WM and Medium WM + 0.1 microgram trypsin/ml when compared to Medium WM + HTBS. Similar numbers of embryos completed the hatching process in Media WM, WM + 0.1 or 0.2 micrograms trypsin/ml and WM + 0.3 micrograms pronase/ml. Since dissolution of the zona pellucida occurred within 96 h for embryos cultured in Media WM + 1.6 or 3.3 micrograms pronase/ml and WM + 1.1 or 2.2 micrograms trypsin/ml, hatching could not be evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of human uterine flushings collected at various stages of the menstrual cycle on mouse blastocysts in vitro

Mouse blastocysts were cultured in vitro in a defined medium supplemented with uterine flushings (containing 500 microgram protein/ml) obtained from normal women at various stages of the menstrual cycle. With one exception (uterine flushing collected on the last day of a menstrual period) blastocyst hatching and attachment were not impaired by flushings collected before or after ovulation.