Erma Z. Drobnis

Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy.

Lipid phase transitions in membranes are thought to be a major damaging event during cooling of cells prior to cryopreservation or during warming after freeze-thaw has been completed. Although there is abundant evidence that such transitions occur in isolated phospholipids, the evidence that they are found in membranes in intact cells is less clear, due largely to technical difficulties in detecting such transitions in the complex mixtures of lipids and proteins found in natural membranes. We show here that Fourier transform infrared spectroscopy provides a rapid, convenient method for detecting these transitions in intact cells. We have used intact pollen grains of cattail (Typha latifolia) as a primary experimental subject. Spectra taken of the intact pollen grains show most of the features commonly seen in natural membrane vesicles or pure phospholipids. Shifts in the vibrational frequency and width of the CH2 bands with temperature can be used to detect lipid phase transitions. Biochemical analysis, coupled with the spectroscopy, was used to assign transitions to nonpolar and polar lipids. Finally, although assignment of the melting lipid unambiguously in other cells has not yet been made, we show that the transitions can nevertheless be detected in other intact cells, including those of four plant species and sperm of three animals.

Hamster sperm penetration of the zona pellucida: Kinematic analysis and mechanical implications

There have been few direct observations of penetration of the zona pellucida by spermatozoa, and no detailed description of the kinematics of this process. Such information is important in evaluating the contribution of mechanical thrust by the sperm flagellum to the mechanism of zona penetration by the sperm head. To make such observations, small numbers of hamster spermatozoa were inseminated to cumulus masses slightly compressed (150 micron) between a slide and coverglass. Observations were made with interference contrast optics and videorecorded at 60 fields/sec. A total of 63 penetrating spermatozoa were recorded, of which 21 were penetrating completely cumulus-intact zonae. Direct comparison of penetration angles for cumulus-intact and cumulus-dispersed zonae suggested that the cumulus may be important in reorientation of penetrating spermatozoa, which initially lie flat on the zona surface. The beat shape during zona penetration was more complex than the simple sinusoidal waves used previously in modeling the mechanics of sperm-zona interaction. Motility during zona penetration was bimodal, having high-amplitude, low-frequency lever strokes, alternating with low-amplitude, high-frequency propagated sinusoidal waves. The completely asymmetric lever mode and the oscillatory motions of the curved leading edge of the sperm head within the zona may afford significant mechanical advantages to spermatozoa in forcing their way through that matrix. Initial calculations of the maximum force exerted by the sperm head against the zona material during lever strokes predicted values as high as 2700 mu dyn. This result is two orders of magnitude higher than that previously estimated assuming more simple flagellar motility. Although not conclusive, our observations and analysis support the concept that zona penetration is more efficient when the cumulus is present, and that this may be due, in part, to a mechanical advantage conferred upon the sperm by the cumulus material.

Zona pellucida binding and zona-induced acrosome reactions in horse spermatozoa: Comparisons between fertile and subfertile stallions

Diagnostic tests that probe sperm function are needed to determine the potential etiologies of subfertility and to explore treatments of subfertility in stallions. Using epifluorescence and phase contrast microscopy, a comparison was made between ejaculates from 3 fertile and 3 subfertile stallions in which sperm-zona pellucida binding and acrosomal status were measured. Motile spermatozoa were selected by Percoll gradient centrifugation and were capacitated in vitro using TEST:TALP capacitation medium at 39 degrees C under humidified air containing 5% CO2. Concentration of motile spermatozoa was held constant during co-incubation with oocytes for fertile and subfertile ejaculates. The total number of zona pellucida-bound spermatozoa was higher for fertile stallions than for subfertile stallions (P < 0.05). Similarly, the percentage of acrosome reactions in zona pellucida-bound spermatozoa was higher for the 3 fertile stallions than for the 3 subfertile stallions (P < 0.05). These results indicate that spermatozoa from fertile stallions may interact with female gametes differently from that of subfertile stallions and suggest that sperm functions are measurable and may vary with fertility.

Evaluation of relative fertility of cryopreserved goat sperm

This study was designed to compare differences in the in vivo fertility of cryopreserved goat semen assessed by heterospermic insemination with differences in in vitro analyses. Five groups of does were inseminated with mixed frozen-thawed semen from different pairs of bucks. The percentage of offspring sired by each buck in the pair was compared with the relative ability of spermatozoa from that frozen-thawed ejaculate to penetrate zona-free hamster ova, relative post-thaw acrosomal integrity, ability to undergo an acrosome reaction during in vitro capacitation, and assessments of sperm motility. In 4 of the 5 different insemination pairs, the ratio of offspring born was other than 1:1. Acrosomal integrity, ability of spermatozoa to undergo an acrosome reaction, and parameters of sperm motility were not correlated with differences in relative fertility in this experiment using ejaculates from fertile bucks. The ability of spermatozoa to fuse with the oocyte plasma membrane was highly correlated with relative in vivo fertility (R2=0.78, P=0.04). This suggests that fusion with the oocyte plasma membrane is an event in the fertilization process in which significant variation exists among fertile bucks. Assessment of ability of spermatozoa to fuse with zona-free hamster ova may contribute to analysis of post-thaw fertility of frozen-thawed buck semen.