Gelatin-substrate film technique for detection of acrosin in single mammalian sperm

Ficsor, G.; Ginsberg, Leonard C.; Oldford, Gregory M.; Snoke, Roy E.; Becker, Richard W.

doi:10.1016/s0015-0282(16)46949-9

Cited by 20 publications

(1 citation statement)

References 7 publications

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“…This test was performed to determine the ability of bull spermatozoa to undergo acrosomal exocytosis (Fiscor et al, 1983). Slides were cooled by keeping in a moist chamber at four o C, for two h. Hundred microlitre of 2.5% gelatin suspension (dissolved in boiling distilled water) was placed on one end of a pre-cooled microscopic slide and smeared with another slide towards the other end.…”

Section: Gelatin Digestion Testmentioning

confidence: 99%

The Role of Mercury in the Etiology of Sperm Dysfunction in Holstein Bulls

Arabi¹

2006

Asian Australas. J. Anim. Sci

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A large number of toxicological substances and pharmacological and physical agents can cause reproductive intervention at the cellular and molecular level. The present study was designed to assess the effect of mercury (HgCl 2 ) at 50 to 550 µM concentration ranges, in vitro, on the sperm membrane and DNA integrity, viability, and acrosomal status of normal bull spermatozoa. The samples were processed for sperm analyses using semen-diluting fluid (PBS, pH 7.2). We recorded a sharp increase in the lipid peroxidation/LPO rate; the highest was at 550 µM mercury concentration, indicating a deleterious effect of mercury on the sperm membrane intactness. There was also a strong negative correlation between LPO rate and % viable spermatozoa (R = 0.987, p<0.001). Data obtained from a comet assay technique revealed that mercury is capable of inducing DNA breaks in the sperm nuclei. Interestingly, 92% of DNA breaks were double-stranded. The correlation between LPO rate and % DNA breaks was 0.984. Performing the gelatin test indicates that mercury is able to alter the integrity of acrosomal membranes showing an abnormal acrosome reaction. In this regard, a strong link was found between LPO rate and % halos (R = 0.990, p<0.001). Collectively, mercury proved to be a potent oxidant in the category of environmental factors affecting bull spermatozoa. Hence, considering the wide spread use of mercury and its compounds, these metals should be regarded with more concern.

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Section: Gelatin Digestion Testmentioning

confidence: 99%

The Role of Mercury in the Etiology of Sperm Dysfunction in Holstein Bulls

Arabi¹

2006

Asian Australas. J. Anim. Sci

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Comparison of methods for detecting mitomycin C‐ and ethyl nitrosourea‐induced germ cell damage in mice: Sperm enzyme activities, sperm motility, testis weight

et al. 1984

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Testes weights, sperm motility and enzyme activities in single sperm were compared with respect to their ability to detect either developmental or mutational damage to germ cells. Male mice were injected i.p. with 2.5 mg/kg mitomycin C (MC) or 50 or 100 mg/kg ethylnitrosourea (ENU) or saline and were then killed at times such that sperm derived from treated vas sperm (SZ), spermatids (ST), preleptotene-late-spermatogonial cells (PLSG), spermatogonial cells (SG), or spermatogonial stem cells (SGS) could be evaluated. Testis weights decreased significantly as early as 1 wk after treatment, with the greatest decrease reached 3-4 wk after treatment, followed by recovery to normal levels 10-15 wk after treatment. We conclude that testis weight, which is easily obtained, is a sensitive indicator of germ cell damage by these agents. Sperm from each animal were evaluated for sperm motility, acrosin activity, succinic dehydrogenase (SDH) activity with or without the competitive inhibitor malonate or after exposure to 60 degrees C for 10 min. The latter two assays were to detect sperm enzymes resistant to the inhibitor or heat. The presence of the acrosin protein was also detected immunologically. Sperm motility decreased most from treatment of PLSG and SG. After MC or ENU treatment, the greatest loss of acrosin activity and of the acrosin protein was also noted in sperm derived from treated PLSG and SG. MC and ENU failed to induce SDH activity in single sperm resistant to 60 degrees C heat inhibition or to inhibition by malonate. Of the sperm assays, acrosin activity proved to be the most sensitive indicator of germ cell damage and was the simplest to measure.

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A Simple, Clinical Assay to Evaluate The Acrosin Activity of Human Spermatozoa

Kennedy

Kaminski

Ven

et al. 1989

Journal of Andrology

142

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Acrosin, a sperm-specific acrosomal proteinase, has an essential role in the fertilization process. Low levels of acrosin appear to be associated with subfertility and infertility, and the acrosin activity of spermatozoa may potentially be a useful indicator of semen quality. The standard acrosin tests employed by research laboratories are too complicated and/or time consuming for clinical use; therefore, a simple assay has been developed to assess total acrosin activity (acrosin and activatable proacrosin). To perform the test, liquefied semen is centrifuged over Ficoll, the washed sperm pellet is suspended in a detergent (Triton X-100)-substrate (N-alpha-benzoyl-DL-arginine p-nitroanilide) buffer, pH. 8.0, and the amidase activity is determined spectrophotometrically after a 3-hour incubation period. Amidase activity can be inhibited with benzamidine, indicating that the activity is primarily or entirely due to acrosin. The absence of detergent in the incubation medium results in greatly reduced activity. The assay is repeatable, linear with increasing sperm concentration, sensitive to a lower limit of 2 x 10(6) spermatozoa, and the results correspond to those obtained with a standard acrosin extraction and assay technique. Storage of ejaculates at 3 to 6 C or at 22 to 24 C for 24 hours does not affect the acrosin activity significantly but much higher temperatures can cause a loss of activity. Freezing ejaculates results in a large decrease in sperm acrosin activity. Leukocytes show minimal activity in the assay. Sperm populations prepared by a swim-up procedure average approximately a 2-fold higher acrosin activity than the original ejaculates. Preliminary experiments indicate that the average sperm acrosin activity of ejaculates whose spermatozoa successfully fertilize human eggs in vitro is significantly higher than those that do not fertilize eggs.

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Gelatin-substrate film technique for detection of acrosin in single mammalian sperm

Cited by 20 publications

References 7 publications

The Role of Mercury in the Etiology of Sperm Dysfunction in Holstein Bulls

The Role of Mercury in the Etiology of Sperm Dysfunction in Holstein Bulls

Comparison of methods for detecting mitomycin C‐ and ethyl nitrosourea‐induced germ cell damage in mice: Sperm enzyme activities, sperm motility, testis weight

A Simple, Clinical Assay to Evaluate The Acrosin Activity of Human Spermatozoa

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