Kathryn A. Dougherty

A Comparison of Subjective Measurements of Human Sperm Motility and Viability with Two Live-Dead Staining Techniques*

Two commonly used live-dead stains (eosin-nigrosin (EN) and eosin-opal blue (EOB)) were compared with the estimated active spermatozoa in semen samples from patients attending an infertility service. Twenty-eight semen samples were analyzed throughout the day of their collection by estimating the number of active spermatozoa and by staining a portion of the incubated sample (37 degrees C) with each stain. The samples were analyzed 30, 60, 120, 240, and 360 minutes after the initial collection. At 30 minutes there were no significant differences between the estimated values and those of either stain. The slope of the EN stain closely paralleled that of the estimated measurements throughout the remainder of the time periods, while the EOB slope was somewhat steeper. Repeatability of values for semen samples obtained on different days was generally good for each stain. A comparison of semen samples from 85 patients, 30 minutes after collection, showed no significant differences between the numbers of estimated active sperm and the percentage live using the EN stain. Studies of 132 semen samples using the EN stain showed a positive correlation with the over-all quality of sperm motility. The results indicate that there is a role for live-dead staining in assessing semen quality.

Semen Analysis: A Review of Samples from 225 Men Seen at an Infertility Clinic

The results from 225 men attending an infertility clinic are presented. The percentage of oval, viable and active sperm cells, and the motility scores were lower in samples with counts less than 10 times 10(6) per ml., increased in counts or 10 to 40 times 10(6) per ml. and again increased in counts more than 40 times 10(6) per ml. The percentage of semen samples with abnormalities in measured parameters dramatically increased as the sperm count decreased. The percentage of samples with significant numbers of white blood cells was higher in samples with sperm counts less than 10 times 10(6) per ml. and in the azoospermic patients, while viscosity problems seemed to be associated with counts less than 10 times 10(6) per ml. Agglutination was more of a problem in samples with counts more than 40 times 10(6) per ml.

Inhibition of rat spermatogenesis and seminiferous tubule growth after short-term and long-term administration of a monoamine oxidase inhibitor.

Seminiferous tubules from rats killed 24 hours after injection of pargyline, a monoamine oxidase inhibitor, did not grow well in tissue culture when compared to control tubules. Treated tubules showed severe tubular degeneration and loss of cellular detail after nine days in culture. Animals injected with pargyline for ten days had varying degrees of semi-niferous tubule degeneration with depletion of the spermatogenic elements. It is suggested that pargyline has a detrimental effect on spermatogenesis. Pargyline possibly acts by decreasing MAO levels which, in turn, may increase potentially damaging amines which may be responsible for the testicular damage.

Correlation between follicle stimulating hormone luteinizing hormone testosterone and 5-hydroxyindole acetic acid with sperm cell concentration.

Plasma follicle stimulating hormone, luteininzing hormone, testosterone, urinary 5-hydroxyindole acetic acid and 17-ketosteroids were measured in patients seen at an infertility clinic. Plasma folicle stimulating hormone and luteinizing hormone levels, and urinary 5-hydroxyindole acetic acid levels were increased in patients with sperm concentrations less than 10 times 10(6) per ml. Plasma testosterone levels were lower in patients with sperm concentrations less than 10 times 10(6) per ml. The results suggest that in patients with sperm counts less than 10 times 10(6) per ml. there is not only impaired spermatogenesis but also decreased Leydig cell function. Urinary 17-ketosteroid levels were not related to sperm cell concentration.

Simultaneous determination of human sperm morphology and viability: Simple office technique

Determinations of sperm morphology and viability are useful techniques for assessment of semen quality. In this report eosin-nigrosin is compared with hematoxylin-eosin for effectiveness in characterizing spermatozoa. The results indicate that eosin-nigrosin and hematoxylineosin give comparable results in differentiating spermatozoa. In addition, eosin-nigrosin allows the determination of the per cent of viable cells in each morphologic category. The eosin-nigrosin staining technique is a simple, rapid method which may give additional information in the assessment of male infertility.

Alterations with age in rat seminiferous tubule monoamine oxidase activity when compared with whole testicular tissue.

Monoamine oxidase, a deaminating enzyme, (MAO, monoamine: O2 oxidore-ductase [deaminating] EC. 1.4.3.4.) is normally present in the rat testes (1-3). Testicular MAO activity has been studied in rats during the late embryonic and the early postnatal periods (4). Seminiferous tubules had more MAO activity during the early postnatal period than did tubules from slightly older animals. The most intense MAO activity was observed in the testes of the neonate and foetuses during the late embryonic period (4). Recently MAO activity was found to be high in testicular preparations from neonatal animals, was reduced in preparations from slightly older animals, was elevated during sexual development, and decreased again with advanced age (after 365 days) (5). Testicular MAO activity was also found to be positively correlated with changes in andro-gen synthesis and testicular development with respect to aging (5). There have been no attempts to separate the seminiferous tubules from the interstitial elements of the testes and to follow MAO activity simultaneously in these two tissues with age. We therefore desired to determine the relative amounts of MAO activity in these miniferous tubules and whole testicular tissue in animals of various ages. We also wanted to determine the localization of testicular MAO activity. Materials and Methods. Male rats (Sprague-Dawley strain) were maintained under controlled conditions in an animal laboratory. The temperature was maintained at 72°F and the approximate relative humidity was 32 %. Artificial lights were used 12 hr each day followed by 12 hr of darkness. Rats used for the following studies were sacrificed by decapitation and their testes quickly removed, decapsulated, and prepared for later assay as described below.

Effect of amylase on sperm motility and viability.

The effect of increasing concentrations of amylase on the percentage of active spermatozoa, the quality of their motility and the percentage of viable cells was studied in semen samples in vitro. The amount of amylase needed to liquefy viscous semen samples in vitro also was determined. The percentage of active spermatozoa and viable cells, and the quality of sperm motility were altered in relationship to the amylase levels. Significant decreases in these parameters compared to control values were seen at the higher concentrations of amylase. The lowest level of amylase did not alter these parameters significantly and was sufficient to liquefy 80 per cent of the viscous semen samples. Amylase appears to be effective at low concentrations for use in liquefying viscous semen samples, thus making them easier to analyze during routine semen examination. The level of amylase used and the interval between addition and analysis must be controlled carefully.

Supravital staining of spermatozoa: relationship of eosin concentration to the percentage of cells staining live.

Supravital staining of human spermatozoa is a useful technique to assess semen quality. We compared 3 concentrations of eosin (1, 2.5 and 5 per cent) for their effectiveness to differentiate viable and non-viable spermatozoa. The percentage of viable cells determined by each concentration was compared as well as the percentage of cells estimated to be active. The results indicate that the percentage of spermatozoa determined to be viable with the supravital stains can be altered by changing the percentage of eosin in the stains. Use of 1 per cent eosin gave values that were significantly higher than the percentage of cells determined to be viable with 5 per cent eosin and the percentage of cells estimated to be active. Better quality slides were produced with 5 per cent eosin, which provided values that correlated favorably with motility estimations.

Vasectomy and Vasovasostomy. I. Timing of Histologic Changes in Immature and Mature Dog Testis After Vasectomy**Supported by the Henry C. Buswell Fund for Urology Research.

The effects of vasectomy on the development and maintenance of spermatogenesis was studied using immature and mature dog testes. Bilateral vasectomy in immature dogs delayed the development of advanced spermatocytes, spermatids, and spermatozoa for about 3 months postsurgery. Spermatogenesis appeared to be recovered to control levels by 4 months postvasectomy. Spermatogenesis in mature dogs was also altered after bilateral vasectomy. Decreased numbers of advanced spermatocytes as well as maturation arrest was observed by 3 weeks postvasectomy. Seminiferous tubule cell layers quickly decreased to one to three layers as the lumina became occluded with sloughed cells by 3 to 6 weeks postvasectomy. Recovery in terms of the numbers of spermatocytes, spermatids, and spermatozoa was evident by 13 weeks postvasectomy, although occasional dog testes did not recover and appeared to be more sensitive to vasectomy-induced damage. It thus appears that vasectomy temporarily inhibits both the development and maintenance of spermatogenesis in immature as well as mature dog testes. Spermatogenesis does recover but may be maintained at somewhat lower levels after vasectomy. Changes are rapid in onset and take several weeks to be reversed. Some dog testes appear to be more sensitive to the damage and it may be irreversible in these testes.

Age-Related changes in Male Rat Reproductive Organ Weights and Plasma Testosterone Concentrations After Administration of a Monoamine Oxidase Inhibitors*†*Supported by the Henry C. Buswell Fund for Urology Research.†Presented at the Thirty-Second Annual Meeting of The American Fertility Society, April 5 to 9, 1976, Las Vegas, Nev.

Experiments were conducted to determine the effects of pargyline, a monoamine oxidase inhibitor, onmale rat reproductive organ weights, testicular histology, and plasma testosterone levels. Pargyline was administered to various groups of rats by implanting it, either dissolved or in a powder form, in Silastic tubing capsules. Additional rats were injected with pargyline solutions. The effects of pargyline.appeared to be age-related with younger animals, weighing under 400 gm (less than 100 days of age), showing a decrease in testicular and accessory organ weights and plasma testosterone. Older animals did not show a decrease in organ weights. Testicular histology in the pargyline-treated groups showed sloughage of cellular material into the tubular lumina, decreases in the number of cell layers, and a reduction of the number of mature spermatocytes. Other areas in the same testis were normal, however, and resembled control histology. The results suggest that pargyline can have detrimental effects on testicular function and that the effects may be age-related.