In utero and in vitro proteinase activity during the Mesocricetus auratus embryo zona escape time window.

The goal of the present study was to investigate proteinase activity in uterine flushates collected during the zona loss time window (68-80 h post-egg activation) in both pregnant and pseudopregnant hamsters and in culture medium conditioned by hatching blastocysts. Several prominent enzyme activities appeared in all pregnant and pseudopregnant uterine flushates. However, only a 45, 43 x 10(-3) M:(r) doublet coincided with the zona loss time window; these bands were absent outside of this time window and were not found in conditioned medium. In medium conditioned by hatching blastocysts, enzyme activity was represented by a 70, 65 x 10(-3) M:(r) doublet identical to a doublet seen in all uterine flushates collected and in serum. There were 12 pregnant and 8 pseudopregnant uterine flushates that were capable of zona lytic activity in vitro (positive bioassays). Of these positive bioassays, five pregnant and four pseudopregnant uterine flushates exhibited the 45, 43 x 10(-3) M:(r) doublet (correlative positive bioassays). These data suggest that there is an important uterine contribution to blastocyst escape from the zona pellucida, consisting of proteinases secreted during a finite time window prior to blastocyst attachment that are different from the proteinases responsible for the zona lytic activity in vitro.

Trophectoderm projections: a potential means for locomotion, attachment and implantation of bovine, equine and human blastocysts.

The behaviour of bovine, equine and human blastocysts was studied in vitro by time-lapse videomicrography and computer imaging. This study revealed that cytoplasmic extensions of the trophectoderm ['trophectoderm projections' (TEP)] were expressed by embryos of all three species, prior to or during zona escape. Bovine and human blastocysts escaped their zonae with a combination of blastocoele expansion, collapse and re-expansion coupled with the penetration of the zona pellucida by TEP. In equine embryos, after several cycles of blastocoele expansion and collapse, trophectoderm ruptured the zona with the concomitant appearance of TEP. This study provides documentation that TEP are expressed by a diverse range of mammalian species, bringing the total number of species in which this phenomenon is found to six, since TEP are also known to be expressed by guinea-pig, hamster and rhesus monkey blastocysts, representing rodents, ungulates and primates. In all species studied, the dynamic nature (extension, retraction, and angular movement) of the TEP was similar, moving in an undulating manner with rapid cycles of extension and retraction. Because TEP appear to be a general feature of mammalian blastocysts, they are implicated in one or more key events in early development, namely zona escape, attachment and/or implantation.

Kinematics of trophectoderm projections and locomotion in the peri-implantation hamster blastocyst.

The behavior of golden hamster blastocysts was studied in vitro by continuous time-lapse videomicrography and computer imaging, during and immediately following escape from the zona pellucida. This study revealed numerous small cytoplasmic trophectoderm projections (TEPs) approximately 18 microns long that penetrated the zona pellucida both radically and tangentially and appeared to be actively involved in zona escape in vitro. After escape from their zonae, some blastocysts moved across the culture dish by an endogenous means of locomotion, most likely involving activity of the small TEPs. Several hours after zona escape, embryos expressed large TEPs up to 46 microns long that moved in an undulating manner and showed rapid cycles of extension and retraction; the timing of their appearance suggested that these TEPs are normally involved in attachment to the uterine epithelium. Embryos fixed in utero, during the developmental interval between zona loss and embryo attachment, exhibited large TEPs similar in morphology to those expressed by cultured blastocysts. These observations document for the first time that mammalian blastocysts are capable of endogenous locomotion, confirm TEPs as components of normal blastocyst activity, reveal that there are two kinds of TEPs that differ temporally and morphologically, and extend earlier reports of TEP activity in guinea-pig embryos to the hamster.

Prediction of the developmental potential of hamster embryos in vitro by precise timing of the third cell cycle.

Time-lapse videomicrography was used to determine the timing of early developmental events in hamster embryos in vitro. The time intervals from pronuclear envelope breakdown to the completion of the first cleavage (Dt2), second cleavage (Dt4 = 2-4 cells), third cleavage (Dt8 = 4-8 cells), blastocyst formation, and zona escape were precisely measured to determine whether the variable 'time' (t) can be used to predict the developmental potential of preimplantation embryos. The range of the developmental time interval (Dt) from the second to the third cleavage divisions (Dt8) provided the best indicator for predicting the probabilities of blastocyst formation and zona escape (P = 0.015 and 0.041, respectively). Dt8 was subdivided into consecutive time cutoff points of < or = 750, < or = 800, < or = 850 and < or = 900 min. Of the embryos that took < or = 750 min to complete the third cleavage division, 92% developed into blastocysts and 69% escaped from their zonae pellucidae. When the completion of Dt8 extended to < or = 900 min, the percentages decreased to 75% and 49% for blastocyst formation and zona escape, respectively. This study identifies a specific developmental time interval and a model whereby time can be used as a noninvasive parameter to predict embryo developmental potential in vitro.

Zona pellucida escape by hamster blastocysts in vitro is delayed and morphologically different compared with zona escape in vivo.

The timing and mode of hamster blastocyst escape from the zona pellucida in vitro ("hatching") and in vivo were compared on the basis of dynamic and static images captured on videotape and computer, respectively. In vivo, most embryos (> or = 80%) escaped between 0030 h and 0600 h on Day 4 of pregnancy. The zona gradually thinned globally and greatly increased in diameter, eventually disappearing. Attachment of blastocysts to the uterine epithelium followed within 4 h. Retarded embryos also showed global zona lysis. In contrast, zona escape in vitro was delayed > or = 29 h both in blastocysts cultured from 1-cell embryos and in blastocysts flushed from uteri of mated, naturally cycling hamsters at 2330 h on Day 3. Zonae were focally lysed; then blastocysts egressed through the hole, leaving most of the zona intact. Global zona lysis never occurred, and retarded embryos did not escape. In conclusion, the striking morphological and temporal differences in zona escape indicate that the hatching behavior routinely observed in cultured hamster blastocysts is not representative of normal events, and implicate a uterine contribution to normal zona escape that is lacking in culture.

Manipulation of avian primordial germ cells and gonadal differentiation.

The authors hypothesized that donor primordial germ cells (PGC) are useful as vehicles of gene transfer in birds. The PGC have been identified in the blastula prior to incubation. They have been isolated from the Stage 7 germinal crescents, Stage 17 blood, and Stage 30 gonads. The percentages of PGC in these three embryonic sources were 2, .003 and 1.5%, respectively. The isolated PGC have been used as antigens to produce specific antibodies that have served as major tools in identification, isolation, and forming enriched in vitro cultures of PGC. Sterile hosts for normal donor PGC have been induced by use of ultraviolet irradiation, treatment with the chemical busulfan, and crossing rooster with female quail to form a sterile host termed "quickens." Micromanipulator technology, along with refinement of techniques to avoid trauma, will allow 80% hatchability. Integration of all the above biotechnology has led to successful use of PGC as vehicles of gene transfer and production of autogenic quail.