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Developmental delay, as the result of ovariectomy, causes mouse blastocyst embryos obtained from +/t6 inter se matings to separate into two distinct populations when placed into outgrowth medium. One population remains as free floating embryos for a significantly longer period of time than the other population. Based upon their phenotypic expression following attachment and outgrowth, the former population was considered to be composed entirely of t6/t6 embryos and the latter, to be composed of +/+ and +/t6 embryos (Nadijcka et al., '81). In the present study, two-dimensional gel electrophoresis identified the embryos which were delayed in attachment as t6/t6 embryos since they synthesize only p63/6.9a, a product of the Tcp-1a locus which is unique to t-haplotypes. The early attaching embryos, assumed to be +/+ and +/t6, synthesize both p63/6.9a and b. The p63/6.9b protein is coded for by Tcp-1b on the wild-type homologous chromosome. Control +/+ blastocyst embryos synthesize only p63/6.9b. The data show that t6/t6 embryos can be identified prior to their lethal period and, thus, subjected to comparative studies to determine the cause of their lethality. Developmental delay is the first method established to enable one to unambiguously identify t6/t6 embryos prior to developmental arrest.

The effects of different types of insemination (normal and delayed matings and in vitro fertilization) on the transmission ratio distortion (TRD) of three t haplotypes were determined. The tw73 haplotype which contains all of the loci known to affect TRD is transmitted at equivalent frequencies in normal matings and in in vitro fertilizations (0.84 and 0.85, respectively) but at a significantly lower frequency (0.62) in delayed matings. The distal partial th18 haplotype is transmitted at equivalent frequencies in all types of insemination (0.66 to 0.70) while the proximal partial tw18 haplotype is transmitted in Mendelian frequencies in normal matings and in in vitro inseminations but at a significantly lower frequency in delayed matings. The results are discussed with reference to the current genetic model for transmission ratio distortion.

The tw5 haplotype is a recessive mutation which is lethal when homozygous in mouse embryos following implantation. This series of studies was undertaken to determine the effect of the tw5/tw5 genotype on embryos developing in vitro. Blastocyst embryos from +/tw5 inter se matings were compared with control blastocysts obtained from matings between T/+ and +/+ females and +/tw5 males for their abilities to continue development in vitro in two culture media. The data show that there are no significant differences between the percentages of experimental and control blastocyst embryos which attach and outgrow or which contain inner cell masses on any day of culture up to equivalent gestation day 21 in either media. These findings show that the life span of cells from tw5/tw5 embryos can be extended significantly by in vitro culture.

The recessive tw5-haplotype, a complete haplotype, is transmitted by heterozygous male mice at very high frequencies (greater than 0.90) in normal matings. The present studies were undertaken to determine the effects of delayed matings and in vitro fertilizations on this phenotypic expression. Males carrying the tw5-haplotype (+/tw5) were first tested for their frequencies of transmission of the mutant 17th chromosome in both normal and delayed matings. Spermatozoa obtained from these same males were then used to fertilize eggs in vitro. The in vivo and in vitro transmission frequencies were found to be statistically equivalent in all types of inseminations. An in vitro fertilization time course study showed that the same percentages of eggs are fertilized by tw5-bearing spermatozoa when the gametes are coincubated for either 2 or 6 h. The data lead to the conclusion that the transmission frequency of the tw5-haplotype is not affected either by the length of time elapsing between insemination and fertilization or by the environment in which fertilization occurs.

The th2-haplotype is transmitted at low frequencies (less than 0.30) by +/th2 males in normal matings. In the studies described here, the transmission frequency of the th2-haplotype from Rb7/th2 males was determined for normal and delayed matings and in vitro inseminations. The data show the transmission frequency from the two in vivo inseminations to be less than 0.30 and to be statistically equivalent. However, the in vitro transmission frequency (0.80) is significantly greater than either of the in vivo frequencies. The results show that the environment in which fertilization occurs affects the transmission frequency of this specific t-haplotype significantly.