Previous reports have demonstrated that culture of mouse preimplantation embryos at high density stimulates their rate of development. The molecular basis of this phenomenon was investigated. Culture of embryos from the four-cell stage at high density in normal medium, or at low density either in embryo-conditioned medium or medium containing platelet-activating factor (PAF), significantly advanced the timing of compaction, initiation of cavitation and/or completion of zona hatching, and also increased the number of cells in blastocysts. In contrast, Lyso-PAF, an inactive metabolite of PAF, and Enantio-PAF, an enantiomer of PAF, did not have a stimulatory effect at low embryo density, but did not inhibit the stimulation of development at high embryo density. The stimulatory effect of culture at high density was inhibited in the presence of either CV-3988 or SDZ 64-412, two structurally distinct competitive PAF-receptor antagonists, while the development rate at low density was not affected. We conclude that an embryo-derived factor related to PAF is secreted by blastomeres during in vitro culture and acts in a receptor-mediated manner to stimulate the rate of development.

During cleavage, the mouse embryo expresses a variety of cell adhesion systems on its cell surfaces. We have reviewed biogenetic and assembly criteria for the formation of the uvomorulin/catenin, tight junction and desmosome adhesion systems as the trophectoderm differentiates. Each system reveals different mechanisms regulating molecular maturation. Adhesion processes contribute to the generation of distinct tissues in the blastocyst by modifying the expression pattern of blastomeres entering the non-epithelial inner cell mass lineage. Cell adhesion also influences the spatial organisation, but rarely the timing of expression, of proteins involved in trophectoderm differentiation.

Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-derived PAF (EPAF), which act in an autocrine/paracrine fashion to stimulate embryonic development in vitro. Mouse EPAF is thought to consist predominantly of hexadecyl (C16) and octadecyl (C18) PAF-like components. Mouse pre-implantation embryos cultured with exogenous PAF from the early cleavage stages exhibit increased blastocyst cell numbers and rates of mitosis around the 8-cell stage. We investigated whether exogenous PAF could specifically stimulate embryonic cell proliferation prior to the blastocyst stage in the mouse and also compared the biological activities of the C16 and C18 PAF isoforms as follows. Embryos were cultured for either 24 h or 120 h from the 2-cell stage and their total cell numbers were determined or their development assessed in terms of their incidence of successful zona-hatching respectively. In each case, embryos were cultured in unsupplemented medium or in medium supplemented with either C16 or C18 PAF (0.5 μM). Compared with controls, culture with C16 PAF produced a significant stimulation of mean total per number per embryo and a significant increase in the incidence of successful zona-hatching, whilst culture with C18 PAF had no significant effect. We then cultured 1-cell zygotes for 48 h in unsupplemented medium or medium supplemented with either an equimolar mixture of C16 and C18 PF or with either C16 or C18 PF alone (each at 0.2 μM). Embryos were also scored for cell number at 4 h and 30 h of culture. Although no significant effect on mean cell number per embryo was seen following 4 h or 30 h of culture with a mixed C16/C18 PAF preparation, culture for 48 h with a mixed C16/C18 PAF preparation or with C16 PAF alone produced a significant increase in mean cell number per embryo compared with controls - an effect that is likely to be receptor-mediated, since culture with an equivalent concentration of C18 PAF had no significant effect compared with controls. We have demonstrated that mouse zygotes/embryos can respond in a specific manner to exogenous hexadecyl PAF in terms of increased rates of cell proliferation prior to cavitation, and must be capable of doing so at some time between the first and third, and also between the second and fourth, cell cycles. Such embryos presumably express one or more classes of functional PAF-receptor molecule during this period (i.e. as early as during the 1-, 2- or 4-cell stages). We have also demonstrated that embryonic response to exogenous PAF is significantly isoform-specific, which may reflect differences between the two isoforms either in affinity for binding to putative embyronic PAF-receptor molecules or in their ability to elicit a stimulatory response following binding. This observation calls into question the use of preparations containing a mixture of hexadecyl and octadecyl PAF isoforms, particularly in dose-response studies, in the mouse.

No abstract

This study was designed to clarify whether orientation of the first polar body (PB) of the oocyte at 6 o'clock rather than 12 o'clock, during intracytoplasmic sperm injection (ICSI), significantly affects a number of clinically important outcome measures including fertilization, zygote cleavage, embryonic morphology, and clinical pregnancy and implantation rates. In all, 114 patients were allocated to one of two groups on the basis of strict alternation, both groups being treated by the same ICSI practitioner. In one group, all oocytes were injected with their first PB at 6 o'clock, whereas in the other, the orientation of the PB was reversed (12 o'clock). In all cases, a normally bevelled injection pipette was inserted into the oocyte in the 3 towards 9 o'clock direction. The orientation of the PB did not significantly affect the proportion of oocytes that failed to survive injection or the proportion scored as having either zero, one, two or three pronuclei. The proportion of normally fertilized zygotes that cleaved was not significantly different between the two groups, nor was the proportion of embryos classified as either grade 1, 2 or 3. However, the proportion of grade 4 embryos (the poorest grade) was significantly lower in the 12 o'clock, compared to the 6 o'clock group. Most importantly, there was no significant difference between the two groups in the proportion of patients having a positive clinical pregnancy test, nor in either the implantation rate or the mean number of fetal hearts detected per patient presenting with a clinical pregnancy.