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The amount of nuclear sulphydryl and disulphide groups was determined by cytofluorometry on single mouse spermatozoa from the caput, corpus and cauda epididymidis, and the vas deferens. N-(7-Dimethylamino-4-methylcoumarinyl) maleimide was used as a specific and quantitative fluorescent reagent for thiols. The sperm nuclear content of free sulphydryl groups decreased sharply from the caput epididymidis to the vas deferens. The amounts of cysteine residues present as the thiol form in the spermatozoa from the caput, corpus and cauda epididymidis and vas deferens were 50, 15, 5 and 3% respectively.

In order to identify high-order structures and pitch length of deoxyribonucleic acid (DNA), poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) were adsorbed on Cu(111) substrates by the pulse injection method and were observed by ultra high vacuum (UHV) scanning tunneling microscopy (STM). In large-scale STM images, the high-order structure of poly(dG-dC)·poly(dG-dC) has a higher divergence and shorter dimension compared with those of poly(dA-dT)·poly(dA-dT). High-resolution STM images revealed that the pitch lengths of poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) adsorbed onto the surface were longer than those possessed in the aqueous solution.

Toxicity of Ultrafine Nickel Particles in Lungs after Intratracheal Instillation: Qunwei ZHANG, et al. Department of Environment Health, Fukui Medical University-To study the lung acute and subacute toxicity of ultrafine nickel particles, rats were intratracheally instilled with 0, 0.1, 0.5, 1 and 5 mg ultrafine nickel (Uf-Ni), respectively. At 3 days after injection, the body weight and wet lung weight were determined. At the same time, bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH), total protein (TP), and total cell and differential cell counts. The results showed that indicators of lung injury and inflammation in BALF were markedly raised with increased Uf-Ni from 0 to 1 mg, and there were no differences in the indices between injection of Uf-Ni at 1 mg and at 5 mg. Rats were intratracheally instilled with 1 mg Uf-Ni, and wet lung weight, and bronchoalveolar lavage fluid (BALF) profiles were analyzed 1, 3, 7, 15 and 30 days later. The effects of Uf-Ni on indices that can be presumed to reflect epithelial injury and permeability (LDH or TP) were dramatically increased from day 1 up to 30 days after injection. Lung histology findings generally confirmed the BALF data, showing severe lung inflammation at 1 day after injection of UfNi, and epithelial hyperplasia and inflammation still present at 30 days after injection. Our findings suggest that Uf-Ni causes persistent inflammation following instillation of a small dose. (J Occup Health 1998; 40: 171-176)

In previous studies we have shown differences in the function of morphologically normal and abnormal sperm by evaluating their flagellar movements and swimming trajectories. In this study we have compared the capability of morphologically normal and abnormal human sperm to undergo an acrosome reaction after incubation with human follicular fluid. Semen samples were studied from 6 research donors and 21 semen evaluation patients. All men had normal semen by clinical criteria. Semen was prepared either by a two-step Percoll gradient centrifugation or the sperm were diluted, washed, and centrifuged three times. Sperm suspensions were incubated for 24 hours in a modified Tyrode's medium, containing 2.6% bovine serum albumin, prior to dilution with human follicular fluid. The percentage of acrosome reactions among viable sperm was assessed after 15 minutes using the supravital Hoescht stain and fluoresceinated pea lectin. Sperm head size was measured with an ocular micrometer and normal values were defined as length 3-5 microns and width 2-3 microns. At least 25 viable normal sperm, and 25 viable abnormal sperm were analyzed for acrosome reactions on each slide. With Percoll separation the percentage of acrosome reactions (mean +/- sem) for normal sperm was 38 +/- 3% vs. 22 +/- 2% for abnormal sperm (P less than 0.005). After washing, the comparable values were 12 +/- 1% vs. 5 +/- 1% (P less than 0.005). The incidence of spontaneous acrosome reactions (24 hours of incubation, no follicular fluid) was also higher for normal sperm than abnormal sperm (9 +/- 1% vs. 4 +/- 1%, P less than 0.01). These data demonstrate an association between normal sperm morphology and acrosomal function.

Flow cytometry was used in the scoring of acrosome-reacted human sperm. Propidium iodide was used for detection of the nonviability of the sperm. Fluoresceinated pea lectin was used to detect acrosome-reacted sperm. The results obtained by flow cytometry and those obtained by fluorescence microscopy were compared to determine if flow cytometry can serve as a more accurate, faster, and simpler method. It was possible to detect human sperm by flow cytometry. The percentage of propidium iodide labeled sperm determined by flow cytometry was close to that obtained by fluorescence microscopy. Comparison of the percentage of acrosome-reacted sperm determined by flow cytometry and fluorescence microscopy showed that these methods gave very similar results (r = 0.98, p less than 0.001). Objective scoring of more sperm was possible by flow cytometry than by fluorescence microscopy, and flow cytometry was useful as a simple method for evaluation of acrosome-reacted human sperm.

The acrosome of Macaca fascicularis sperm cannot be distinguished by conventional light microscopy, so determining whether sperm are acrosome‐intact or‐reacted is difficult. We describe methods for labeling the acrosomal region of sperm with two different probes: fluoresceinated Pisum sativum agglutinin and anti‐sperm antiserum. Acrosome‐intact sperm are much more heavily labeled in the acrosomal region than are acrosome‐reacted sperm, providing a simple means of differentiating the two types of sperm. The two probes detect similar numbers of acrosome‐reacted sperm following treatment with the divalent cation ionophore, A23187.