We have previously demonstrated that initiation of intracellular calcium ([Ca2+]i) oscillations in mouse eggs signals activation or apoptotic death depending on the age of the eggs in which the oscillations are induced. To extend these studies, mouse eggs were aged in vitro to 24, 32, and 40 h post-hCG and injected with sperm cytosolic factor (SF), adenophostin A, or sperm (intracytoplasmic sperm injection), and the times at which signs of apoptosis first appeared were examined. These treatments, which induced [Ca2+]i oscillations, caused fragmentation and other signs of programmed cell death in eggs as early as 32 h post-hCG. The susceptibility of aged eggs to apoptosis appeared to be due to cytoplasmic deficiencies, because fusion of recently ovulated eggs with aged, SF-injected eggs prevented fragmentation. Evaluation of mRNA and protein levels of the apoptotic regulatory proteins Bcl-2 and Bax showed a prominent decrease in the amounts of Bcl-2 mRNA and protein in aged eggs, whereas Bax mRNA levels did not appear to be changed. Lastly, the Ca2+ responses induced by the aforementioned Ca2+ agonists ceased in advance in aged eggs. Together, these results suggest that one or several critical cytosolic molecules involved in the regulation of Ca2+ homeostasis, and in maintaining the equilibrium between anti- and proapoptotic proteins, is either lost or inactivated during postovulatory egg aging, rendering the fertilizing Ca2+ signal into an apoptosis-inducing signal.

Apoptosis, as determined by blastomere and DNA fragmentation, occurs in many preimplantation mouse embryos. To investigate which genes contribute to apoptosis in preimplantation embryos, we used the reverse transcription-polymerase chain reaction to assess mRNA levels for seven genes in the caspase family and seven genes in the BCL-2 family. All caspase mRNAs were detectable in oocytes, while expression in preimplantation embryos varied in a stage-specific manner. An assay for group II caspase enzymatic activity showed that although transcripts for these caspases could not be detected in zygotes, proteolytic activity could be detected in polar bodies, fragmented zygotes, and zygotes treated with staurosporine. This suggests that maternal caspases are inherited during oogenesis. Transcripts for some members of the BCL-2 family could be detected at every stage of preimplantation development. Transcripts for other members were rarely detected. When BCL-2 and BAX protein levels were assessed using immunofluorescence, both proteins were detected in zygotes and in blastocysts. When fragmented blastocysts were compared to normal blastocysts, levels of BCL-2 immunofluorescence tended to be lower in fragmented blastocysts. This result supports a model in which the ratio of BCL-2 to BAX is altered in apoptotic embryos.

In both mice and humans, 15-50% of embryos die during the preimplantation period from mechanisms that are largely unknown. Two major criteria predict preimplantation embryo quality, the rate of development and the degree of fragmentation. We review evidence that both of these criteria have a genetic basis. Rate of development and subsequent embryo survival are controlled by a gene, Ped, we discovered in the mouse. Although progress is being made in the search for the human homologue of the mouse Ped gene, it has not yet been identified. Fragmentation, observed in both mouse and human embryos, is probably the result of apoptosis. We analysed transcription of two genes that regulate apoptosis, bcl-2 and bax, and found that both are transcribed in mouse and human preimplantation embryos. Overall, the literature reviewed and new data presented in this paper support the concept that there is a genetic basis for preimplantation egg and embryo survival.

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.

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