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A comparative light and electron microscopy study was done on cauda epididymal spermatozoa obtained from correspondingly-aged sterile t6/tw32, and fertile T/tw32, T/t6 and BALB/c, mice. The results show that all of the males, regardless of age, contained defective gametes and that all contained the same types of aberrant gametes. The oldest males of each genotype contained more abnormal gametes than the younger males of the same genotype. No unique spermatozoan defect and no increased frequency of a specific spermatozoan defect was noted which could be correlated with the sterility of the t6/tw32 animals.

In vivo, t6/t6 embryos are developmentally arrested between gestation days 5.5 (short-egg-cylinder stage) and 6.75 (long-egg-cylinder stage). In the present series of studies we used both in vivo and in vitro blastocyst delay followed by in vitro outgrowth to determine whether the t6/t6 lethality is time- or stage-specific. The results show that the t6/t6 genome is expressed differently in vivo and in vitro and that the in vitro expression of the homozygous t6 genome differs with different methods of effecting developmental delay. Although delay increases the life span of t6/t6 embryos it does not alter the stage of lethality. One method used to effect delay (ovariectomy) causes the t6/t6 embryos to remain as blastocysts for a significantly longer period of time than their wild-type littermates when placed into outgrowth medium. This distinction provides a unique method for obtaining a sample composed entirely of t6/t6 embryos at a stage prior to the lethal period.

Total ATP, ATP/ADP ratios, and the rates of synthesis and turnover of ATP were determined for t12-, tw32- and T+/T+-litters at the 2-cell, 4-cell, 8-cell, early morula, late morula and late blastocyst stages. The results show that tn-litters have excessive rates of ATP metabolism up to the cleavage stage immediately before the stage of lethality for most tw32/tw32 embryos (early morula) and most t12/t12 embryos (late morula); mutant ATP metabolism then falls below control levels. After the death of the tn homozygotes, ATP metabolism in the tn-litters (composed of only T+/T+ and T+/tn embryos) returns to control levels. These data are discussed in relation to the phenotypes of the homozygous tn embryos.

High-resolution autoradiographic studies were used to determine whether t12/t12 and tw32/tw32 mouse embryos synthesize the excessive lipid which distinguishes these embryos prior to their death. The studies show that the tn homozygotes synthesize neutral lipid which is stored in intracellular lipid droplets. Cholesterol and phospholipid precursors are not incorporated into these droplets.

Homozygous tw32/tw32 mouse embryos, obtained from both spontaneously ovulated and superovulated T+/tw32 females mated to T+/tw32 males, have a lethal period which extends from the 8-12 cell stage to the late morula stage. Most of the homozygous mutant embryos die at the early morula stage and are characterized by excessive amounts of cytoplasmic lipid, mitochondrial abnormalities, binucleated cells and nuclear lipid droplets. The excessive cytoplasmic lipid and nuclear lipid droplets distinguish 35-50% of the embryos (presumably tw32 homozygotes) from their litter-mates prior to the lethal period. The remainder of the distinguishing characteristics appear in the later (8-cell to late morula) tw32/tw32 embryos in frequencies high enough to be considered phenotypic expressions of the mutant genome. The present study indicates that the non-complementary tw32 and t12 alleles are in fact separate T locus recessive alleles.

The results of a comparative ultrastructural study of spermiogenesis in T/tx, +/tx, +/T, C57BL/LJ, BALB/c and randomly breeding Swiss Albino mice are reported. The observations show that aberrant spermiogenesis occurs in males of all strains and genotypes and that the same specific types of abnormal spermatids are found in all of the males examined. No unique morphological defect which could be correlated with the increased transmission frequency of tx-bearing gametes can be found in males heterozygous for the tx allele.

Male mice which are heterozygous for a recessive lethal mutation (+/tL) transmit the tL mutation in non-Mendelian ratios. In the present studies, spermatozoa obtained from +/t12 males were used to fertilize ova from +/t12 females in vitro. The frequency of transmission of the t12 mutation determined from these in vitro studies was compared with the frequency of transmission of this mutation in normal and delayed matings. The data show that the transmission frequency of the t12 mutation, in vitro, is Mendelian and is the same as the transmission frequency of the t12 mutation in vivo when matings are delayed until the time of ovulation.

The results from a comparative ultrastructural study of spermiogenesis in 6-, 10-, 14- and 17-month-old sterile t6/tw32, and fertile T/t6, T/tw32 and BALB/c, mice are reported. The studies show that all of the males contained the same types of defective spermatids and that defects were not limited to specific spermatid stages. Younger males had fewer abnormal spermatids than older males of the same genotype and at each age the BALB/c and t6/tw32 males appeared to contain more abnormal spermatids than the other males. No unique spermatid defect or increased frequency of a specific defect was found which can be correlated with infertility of the t6/tw32 males.

A comparative light and electron microscopic study was done on cauda epididymal spermatozoa from +/tx, T/+, T/tx, C57BL/6J, BALB/c and randomly breeding Swiss Albino mice. The results show that all of the males contain abnormal spermatozoa and that all contain the same types of defective gametes. No unique defect was found which can be correlated with the increased transmission frequency of the tx-bearing allele.

In vivo, embryos which are homozygous for the t6 mutation die during egg cylinder development (gestation days 5.5--6.5). In vitro, these mutant embryos can be distinguished from their wild-type littermates by their developmental arrest and by the failure of their trophoblast cells to transform to giant cells. We have investigated the nature of this lethality by constructing triploid embryos with varying combinations of the t6 mutant chromosome. The phenotypes of outgrowths from these triploid embryos were examined and compared with the phenotypes of outgrowths from +/+, +/t6, and t6/t6 embryos. The data show 1) that +/t6/t6 embryos are phenotypically wild-type, while t6/t6/t6 embryos are phenotypically mutant and 2) that t6/t6/t6 and t6/t6 embryos are developmentally arrested at the same stage of outgrowth.

The phenocritical period of the t6/t6 genome extends from the late blastocyst substages through the elongated egg-cylinder stage. Most of the homozygous mouse embryos die during the short egg-cylinder stage. The viable egg-cylinder staged t6/t6 embryos can be distinguished from their phenotypically wild-type litter-mates at both the light and electron microscopic levels. The distinguishing characteristics of these embryos are aberrantly arranged entodermal cells, excessive cytoplasmic lipid and crystal-containing mitochondria. These same features are also characteristic of those mutant embryos which are developmentally arrested at both the short and elongated egg-cylinder stages. Over 50% of the t6/t6 embryos can be identified as early as the late blastocyst substages by the presence of large, electron-dense cytoplasmic lipid droplets.

Mouse embryos which are homozygous for the c25H deletion at the albino locus are developmentally arrested at the 2- to 6-cell cleavage stages. This study reveals that the mutant embryos cease development before they can be distinguished ultrastructurally from their normal litter-mates. After prolonged developmental delay (24--48 h), the nuclei of the mutant embryos become extremely aberrant in shape, whereas other subcellular organelles remain normal and there are no signs of pyknosis. Eventually, the mutant embryos do become pyknotic and begin to degenerate. The striking effects of this deletion on nuclear ultrastructure of cleavage-stage embryos are discussed in relation to biochemical and ultrastructural defects caused by other lethal deletions at the albino locus.

The utilization of fatty acids from the culture medium has been examined in preimplantation mouse embryos developing in vitro. Incorporation of exogenous fatty acid into embryo lipids was examined by culturing 8-cell mouse embryos for 2h in a medium containing 0.1 mM [9, 10-3H]palmitic acid (900 mCi/mmol). Lipids were extracted from the embryos, and the total lipid extract was fractionated into various neutral lipid and polar lipid classes by thin-layer chromatography. Most of the radioactivity, over 93%, was recovered in neutral glycerides (mono- di-, and triacylglycerols). About 2% of the total radioactivity was recovered in other neutral lipid species including fatty acids, fatty alcohols, and sterol esters. The remainder of the radioactivity was recovered in polar lipids. Seventy-four per cent of the polar lipid radioactivity was present in the amine phosphatides, inositol and/or serine phosphatides, sphingomyelin, choline lysophosphatides, sulfatides, cerebrosides, and monoglycosylglycerides. Chemical degradation studies of labelled embryo lipids indicated that the tritium lable was entering into embryo lipids as the fatty acid and not via metabolic recycling. The oxidation of exogenous fatty acids by mouse embryos was assessed by incubating variously staged embryos for 4h in medium containing 0.1 mM [U-14C]palmitic acid (50 mCi/mmol) and quantitating the production of 14CO2. The rate of fatty acid oxidation was found to be relatively constant from the unfertilized egg up to the 8-cell stage and then increase significantly between the 8-cell and late blastocyst stages. The results suggest that preimplantation mouse embryos developing in vitro can utilize fatty acids from the medium both for incorporation into embryo lipids and for energy production via oxidation.

Mice which are heterozygous for two complementary lethal t mutations (t6/tw32) exhibit complete male sterility. Experiments have been conducted to determine if spermatozoa from these heterozygous males can fertilize oviducal ova in vitro. The experiments have been designed so that specific barriers which are present during in vivo fertilization can be sequentially removed and the fertilizing ability of the spermatozoa tested after each barrier is eliminated. Our results show that spermatozoa from t6/tw32 males are unable to effect fertilization even after all of the barriers normally imposed by the female reproductive tract have been removed.