Wylie I. Lee

Selection of human spermatozoa according to their relative motility and their interaction with zona-free hamster eggs

Human spermatozoa were separated according to their motility by centrifuging semen on discontinuous Percoll gradients. Fractions of the gradients were examined for sperm motility, velocity, viability, morphology, bacteria, and sperm function using the hamster ova sperm penetration assay. The percentage of motile sperm increased from 40% to 60% motile sperm in 60% Percoll to 90% to 100% Percoll. Sperm velocity increased proportionately. Staining showed that greater than 90% of sperm in the 100% Percoll were alive and had normal morphology, and that only sperm cells were found in Percoll concentrations greater than 80%. Sperm isolated in the 80% to 100% Percoll fractions penetrated hamster ova much more frequently than sperm found in the 60% to 70% fractions or than sperm that had not been separated on a Percoll gradient.

Ciliary Activity by Laser Light-Scattering Spectroscopy ~

The frequency of ciliary beat was measured by laser light-scattering spectroscopy in cultures of ciliated cells of the rabbit oviduct. Measurements performed by this new method agree with those obtained by high speed cinematography. When beating cilia are illuminated by a laser beam, the scattered light shows a frequency modulation due to the oscillatory motion of cilia. The spectral structure of the scattered light depends on the frequency and time-space coherence of ciliary beat. This paper reports the experimental validation of this technique and the theoretical basis for obtaining the frequency and coherence of ciliary beat from the autocorrelation function of the spectrum of light scattered from moving cilia. Fiber optic light transmission could permit the extension of this method to assess ciliary activityin situ for applications in animal experimentation and clinical studies.

Intensity autocorrelation function for a flexible polymer

Abstract An exact expression for the autocorrelation function of the intensity of light scattered from a solution of large, flexible polymers is obtained using a simple “mean-force” model. A Q 2 dependence of the reciprocal relaxation time is found for both small values of QR G and also very large values of QR G . At small QR G the relaxation time is proportional to the translational diffusion coefficient D of the entire polymer, while at large QR G the relaxation time is proportional to the sum of D and an effective segment diffusion coefficient kt / f .

Effect of long-range hydrodynamic and direct intermacromolecular forces on translational diffusion

Abstract Within the framework of recently formulated microscopic theories of macromolecular diffusion it is shown that hydrodynamic forces act always to diminish the influence of direct forces, but never to reverse the sign of the correction term due to direct forces alone. Although the correction term D ( k ) to the intrinsic diffusion coefficient may vary with scattering vector | k |, it is shown that a reversal in sigh of the correction term with increasing | k |, if it occurs, must be associated with an amplitude of less than 10% of the correction term at | k | = 0. At | k | = 0 direct repulsive forces are predicted to always increase the apparent diffusion constant, even after accounting for hydrodynamic interactions. Although experiments on polylysine (1 mg/ml) at salt concentrations above 0.01 M are in qualitative accord with the theory, below 10 −3 M salt the apparent diffusion coefficient is reduced by a factor of about 20, concomitant with a much reduced intensity of scattered light. The strong contradiction of the theory implied by this observation is attributed to a dramatic rise in Stokes friction arising from long-range interionic forces in the low-salt solutions.

Dynamic Laser Scattering Study of Sperm Migration Through Cervical Mucus

Migration of spermatozoa through the cervix is of great interest in human reproductive biology, and is important in fertility studies. The penetration of spermatozoa into cervical mucus depends on sperm motility and also on the molecular structure of cervical mucus. We have used the technique of dynamic laser scattering to measure sperm motility and to study the structure of cervical mucus by analyzing the molecular dynamics of the mucus. The correlation of the two is evaluated by a modified Kremer test. The clinical significance of the test is quantitatively expressed by the penetration factor and the sperm-mucus interaction coefficient, which are derived from theory of transport phenomena.

Studies on Sperm Motility by Laser-Light Scattering

Measurement of sperm motility has received considerable attention of many investigators since the earliest observations of Leeuwenhoek. However, sperm motility has often been judged subjectively by an individual. Therefore, a suitable definition for the measurement of sperm motility continues to confuse the observer. Many methods of measurement have been developed in recent years (1,2). Essentially, these techniques can be divided into two categories depending upon the objectives of the study, which may be associated with the mechanism of flagellation of single cells or may be concerned with the evaluation of relevant statistical parameters which describe the quality of sperm motility. In the studies of effects of exogenous factors on sperm motility and clinical evaluations of semen quality, the microphoto-graphic technique for studying single cells becomes cumbersome due to its inherent disadvantage of being time-consuming. This paper summarizes some of our recent studies of sperm motility by laser-light scattering. Results of these measurements show that this simple and objective method has considerable potential for both basic research and clinical use in biomedical studies.

Laser light-scattering study of the effect of washing on sperm motility**Supported in part by grant HD-12629 to Dr. Wylie I. Lee and grant HD-13075 to Dr. Penelope Gaddum-Rosse, from the National Institutes of Health. Presented in part at the Thirty-Eighth Annual Meeting of The American Fertility Society, March 20 to 24, 1982, Las Vegas, Nevada.

The effect of washing on human sperm motility was measured by means of dynamic laser light-scattering spectroscopy. Semen samples from 24 fertile donors were diluted with Biggers, Whitten and Whittingham (BWW) medium and subsequently centrifuged at one of the following forces: 235 x g, 325 x g, 400 x g, 470 x g, 500 x g, 600 x g, and 800 x g. The duration of centrifugation was 8 minutes for the first wash, 6 minutes for the second wash, and 3 minutes for the third wash. Sperm motility was evaluated in terms of the root mean square swimming speed of the spermatozoa and the mean migration rate of washed spermatozoa in estrous bovine cervical mucus (BCM). It was found that sperm motility and viability were improved when semen samples were washed at 235 x g, even after three washes. However, washing at forces of 600 x g or more reduced sperm motility and also their ability to penetrate cervical mucus in vitro. Repeated washing at forces between 300 x g and 500 x g had little deleterious effect on sperm motility.

Sperm penetration into cervical mucus in vitro. III. effect of freezing on estrous bovine cervical mucus**Supported in part by grants HD-03752 and HD-12629 from the National Institutes of Health.††Presented at the Thirty-seventh Annual Meeting of The American Fertility Society, Atlanta Hilton, Atlanta, Georgia, March 14 to 18, 1981.

The influence of the storages period on estrous bovine cervical mucus after it was stored in the freezing compartment of the laboratory refrigerator was evaluated by an in vitro sperm penetration test with human spermatozoa, laser light-scattering, and a spinnbarkeit test. Data obtained from the sperm penetration test were analyzed by a mathematical model that correlates the sperm motility with the sperm transport rate and the penetrability of the mucus. The tests showed that estrous bovine cervical mucus can be stored for up to 4 weeks at -12 degrees C without a change in its physical properties. The results of this study strengthen the suggestion that bovine mucus could be employed as a substitute for human cervical mucus.

Sperm Penetration into Cervical Mucus In Vitro. II. Human Spermatozoa in Bovine Mucus**Supported by United States Public Health Service Grant HD-03752 and by Contract HD4-2826 from the National Institutes of Health.

Human spermatozoa pentrate estrous bovine cervical mucus readily in vitro and maintain good motility and viability for a number of hours. They show pronounced unidirectional motion in mucus that has been aligned linearly. Data from tube preparations indicate that human spermatozoa from a given ejaculate travel more rapidly in estrous bovine mucus than in human midcyle mucus. They are prevented from penetrating luteal phase bovine mucus. The results are discussed in relation to a model of the molecular structure of cervical mucus, derived from laser light-scattering spectroscopy. In addition, it is suggested that bovine cervical mucus could be developed as a possible substitute for human cervical mucus in cases of infertility due to deficient endogenous mucus.

Sperm Penetration into Cervical Mucus In Vitro. I. Comparative Studies**Supported by United States Public Health Service Grant HD-03752 and by Contract HD4-2826 from the National Institutes of Health.

Comparative studies have been carried out on the behavior of human and bovine spermatozoa toward homologous cervical mucus in vitro. In both cases the degree of sperm penetration and the pattern of sperm motility were influenced in a characteristic fashion by prior manipulation of the mucus: the most rapid and extensive penetration, and pronounced unidirectional motion, were seen in mucus that had been aligned linearly. By contrast, spermatozoa from rabbits, guinea pigs, rats, and mice were largely prevented from entering either midcycle human or estrous bovine cervical mucus, regardless of its physical arrangement. The observations on sperm motility patterns and the degree of penetration are discussed in relation to a model of the molecular arrangement of cervical mucus, derived in our laboratory from laser light-scattering spectroscopy.