The present study evaluates the role of apoptotic cell death and DNA methylation reprogramming in early developmental failures occurring in embryos at the 2-cell stage. Mouse 2-cell embryos were cultured in vitro and treated with chemicals that cause developmental arrest and apoptosis (α-amanitin, actinomycin D, TNF-α). After 24 h, 48 h and 72 h culture, embryos were analysed using cell-death assays (annexin V staining, TUNEL labelling and immunodetection of active caspase-3) and genome methylation assay (immunodetection of 5-methylcytosine). The ability of embryos at the 2-cell stage to undergo apoptotic processes was very low. In arrested embryos, the presence of all evaluated features of apoptosis was recorded only after 72 h culture and their incidence was sporadical. Interestingly, the most frequently observed apoptotic sign was nuclear condensation and the timing of its appearance preceded even the phosphatidylserine flip. Both normally developing and arrested embryos displayed reduction in DNA cytosine methylation. In arrested embryos, this process was independent of cellular cleavage, was more pronounced and finished in almost complete demethylation of the embryonic genome. The timing of the demethylation overlapped with the onset of major apoptotic events. Although observed apoptotic cells showed either demethylated or methylated DNA cytosine in their nuclei, at blastocyst stage the demethylated status appeared more frequently in them.

The phenomenon of developmental arrest at the 2-cell stage of zygotes obtained from certain mouse strains during in vitro culture is known as the 2-cell block. The effect of conditioned medium (CM) with rat hepatoma BRL cells on the 2-cell block of CD-1 mouse zygotes was investigated in comparison with that of CM with rat hepatoma Reuber H-35 cells. In control medium with EDTA, 75.4% of 2-cell embryos developed to the 4- to 8-cell stages. In the same conditions, the BRL Mr <10000 fraction inhibited the development of 2-cell embryos to the 4- to 8-cell stages (57.7%), although the inhibition by this fraction was weaker than by the Reuber Mr <10000 fraction (19.8%). As a result of reversed-phase column chromatography, a 2-cell stage specific inhibitor of the cleavage of mouse embryos (Fr.B-25), which separated into the Mr <10000 fraction of the Reuber CM, was detected at a low level in the BRL Mr <10000 fraction. On the other hand, the Mr >10000 fraction of BRL CM accelerated the development of the embryos (90.3%). This beneficial effect was also evident even in the absence of EDTA. RT-PCR analysis revealed that mRNAs encoding the β-A or β-B subunit of activins (Mr ~29000), which are well characterized cytokines that act as releasers of the 2-cell block, were expressed in BRL cells. These results indicate that BRL cells synthesize Fr.B-25 at low levels, and that activins contained in the BRL CM probably contributed to overcoming the 2-cell block of CD-1 zygotes cultured in vitro.

In ICSI procedures, it is well known that the selection of viable (live) spermatozoa and certain types of immobilization prior to injection is very important for obtaining successful results, but unfortunately there are rare situations when only immotile spermatozoa are available (such as in severe asthenozoospermia or necrozoospermia). In such cases, failure of oocyte activation after ICSI often occurs and may be due to the lack of SOAF (sperm-borne oocyte activating factor) activity. In order to investigate the SOAF activities of dead spermatozoa, mouse and human spermatozoa were immobilized (killed by sonication), maintained in THF medium for varying time intervals (up to 72 h) and then injected into mature unfertilized mouse oocytes. Injected mouse oocytes were examined for their activation, development into blastocysts and Ca2+ responses by imaging and confocal laser scanning microscope. The rates of oocyte activation, blastocyst development and normal patterns of Ca2+ oscillation from the killed-sperm-injected oocytes decreased gradually in accordance with the maintenance interval between sonication and injection. For injection with mouse sonicated spermatozoa, the rate of normal Ca2+ oscillations declined first (after a 3 h maintenance interval) and then blastocyst development was gradually obstructed (after approx. 10 h). The oocyte activation-inducing ability of dead spermatozoa was maintained for a relatively long period, but began to decline after 20 h. The activation rates and Ca2+ response of the oocytes that were injected with human sonicated spermatozoa decreased earlier than those injected with mouse spermatozoa. Although the oocyte activation-inducing ability was maintained for a relatively long time after the death of the spermatozoa, embryo development into blastocysts and the rate of normal Ca2+ oscillations declined after a short maintenance interval between sonication and injection. The Ca2+ response seemed to be the most sensitive indicator for the evaluating the SOAF activity of dead (killed) spermatozoa.

The authors apologise for the wrong affiliations being assigned to some of the authors, given on the title page of their paper (Liu, et al., 2009) published in Zygote.