Nina Hillman

Separation of dead and live mouse spermatozoa

Abstract A modified glass bead column technique is described for separating live from dead mouse spermatozoa. The technique allows one to separate either epididymal or vasa deferentia cells into three fractions: broken spermatozoa and cellular debris; predominantly live spermatozoa; predominantly dead spermatozoa and contaminant cells. This separation method can facilitate those studies which require the initial use of live populations of spermatozoa.

The in vivo and in vitro transmission frequencies of the tw5-haplotype in mice

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.

Competent Chick Ectoderm: Nonspecific Response to RNA

Presumptive chick neuroectoderm responds to RNA from brain and heart by forming neural tubes, but it does not respond to liver RNA. This dif ferential response can be correlated with the presence of Folin-positive material in those RNA preparations which elicit the formation of neural structures.

The transmission ratio distortion of the th2-haplotype in vivo and in vitro

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.

The in vivo and in vitro transmission ratio distortion of one complete and two partial t haplotypes in mice.

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.

Glass-bead column separation of motile and nonmotile human spermatozoa

Glass-bead columns were tested for their efficiency in concentrating motile human spermatozoa from frozen semen samples. The data show that glass-bead filtration concentrates the motile gametes in each sample and is significantly more efficient than the swim-up method for obtaining populations of motile spermatozoa. The data suggest that this method can be applied clinically to obtain motile spermatozoa from poor-quality semen for use in in vitro fertilization.

The extended survival of tw5/tw5 mouse embryo cells in vitro.

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 ability of glass-bead column-filtered mouse spermatozoa to fertilize homologous eggs in vitro.

Glass-bead columns have been used to separate mouse spermatozoa into motile and nonmotile fractions. The spermatozoa in the motile fraction are able to fertilize eggs in vitro. Significantly more of the two-cell embryos which develop from these zygotes reach the blastocyst stage than do two-cell embryos developing from eggs fertilized in vitro with unfiltered control spermatozoa.

Acrosin activity in spermatozoa from sterile t6/tw32 and fertile control mice

Spermatozoa from sterile t6/tw32 and control fertile +/+, T/tw32, T/t6 mice were compared for their abilities to hydrolyse protein matrices and for their levels of acrosin activity. The data show that the immature and mature gametes from both the experimental and control males hydrolyse protein matrices. The quantitative acrosin assays show, however, that the mature gametes from the intercomplement males have significantly less total acrosin activity than any of the control groups of gametes. These findings suggest that this reduced acrosin activity is an additional phenotypic expression of the intercomplement genotype which results in male sterility.

The effect of experimentally induced triploidy on the lethal expression of the t12 mutation in mouse embryos

Diploid embryos which are homozygous for the t12 mutation die at the morula stage. In the current studies, ova from heterozygous (+t12) females were fertilized in vitro with spermatozoa from +t12 males. The fertilized ova were immediately placed into media containing cytochalasin B to prevent second polar body formation, producing +/+/+, +/+/t12, +/t12/t12, and t12/t12/t12 embryos. The subsequent development of these triploid embryos was compared with that of diploid +/+, +t12, and t12t12 embryos developing from ova which were also fertilized in vitro with spermatozoa from +t12 males but which were not treated with cytochalasin B immediately following gamete coincubation. The data show that those triploid embryos which possess a wild-type allele and two mutant alleles are phenotypically wild type while those possessing three mutant alleles are not phenotypically distinguishable from their diploid (t12t12) counterparts. Like t12t12 embryos, t12/t12/t12 embryos die at the morula stage, prior to blastocoelic cavity formation.