Semen Characteristics After Overnight Shipping: Preservation of Sperm Concentrations, HspA2 Ratios, CK Activity, Cytoplasmic Retention, Chromatin Maturity, DNA Integrity, and Sperm Shape

Abstract

ABSTRACT:We tested several approaches that can be used to preserve sperm attributes and the objective biochemical markers of sperm maturity and function for assessment in a remote centralized laboratory after overnight shipping of semen samples. Addition of phenyl-methyl-sulfonyl-fluoride (PMSF) to a final concentration of 20 g/mL semen at 4ЊC has preserved sperm concentrations and HspA2 isoform ratios, even at room temperature, simulating a shipping delay in moderate ambient temperatures. Regarding the attributes of individual spermatozoa, the patterns of CK-immunocytochemistry (demonstrates cytoplasmic retention in diminished-maturity spermatozoa); aniline blue staining pattern (tests chromatin maturity); sperm shape assessed by both Kruger strict morphology and computer assisted morphometry; and sperm DNA integrity, as tested by DNA nick translation, all remained unchanged. Thus, the PMSF-4ЊC conditions preserved sperm concentrations and the cytoplasmic and nuclear biomarkers of sperm cellular maturity and function for next-day analysis. This shipping method will facilitate the early detection of subtle changes in semen quality that can affect sperm function, even when there has been no decline in sperm concentrations to signal possible toxic effects. Furthermore, sample preservation will enable investigators to evaluate semen for toxicology studies and for diagnosis of male infertility from remote locations. Home collection of semen should enhance study participation, and semen assessment in centralized laboratories will address concerns regarding interlaboratory variations and quality control.Key words: Reproductive toxicity, male infertility, cytoplasmic and nuclear biomarkers.J Androl 2004;25:593-604 E xposures to environmental toxicants can have detrimental effects on human male reproduction (Schrader et al, 1992;Wyrobek et al, 1997). In field studies, two levels of semen analysis are usually carried out: primary studies, which can include the conventional semen parameters of sperm concentration, motility, and morphology, as well as measurement of specific biomarkers selected by the expected health effects of the environmental toxicants causing the exposure, and secondary studies based on archived samples (ie, frozen samples, videotaped microscopic fields, or sperm smears) that are used later to further evaluate specific findings. We are developing a potential third approach, which recognizes that each semen sample is composed of sperm populations of various levels of cellular maturity. The proportions of mature and diminished-maturity sperm, which define the functional efficacy of a semen sample, can be measured by objective biochemical methods, independently from sperm concentrations and motility. This approach provides two potential advantages: 1) reliable sperm biochemical measurements, which could reflect changes in fertility and in the paternal contribution of sperm DNA to zygote development, even when a man has not reached a level of exposure that would affect his sperm concentration, and 2) detec...

Cumulus cells and their extracellular matrix affect the quality of the spermatozoa penetrating the cumulus mass

“…The cells were resuspended in an appropriate volume of phosphate-buffered salin supplemented with 0.25 mmol/L phenylmethylsulfonyl fluoride to give a viable cell concentration of 8 Â 10 6 cells/mL. Our unpublished data show that phenylmethylsulfonyl fluoride at the concentration used did not affect sperm viability and motility, as determined by computer-assisted sperm analysis, consistent with earlier observations (31). The cell suspensions underwent freeze-thaw cycle described above three times.…”

High risk of temporary alteration of semen parameters after recent acute febrile illness

“…In addition, because standard cryoprotectants (freezing medium) do not contain a proteolytic enzyme inhibitor such as phenyl-methyl-sulfonyl-fluoride (PMSF), it might be possible that thawed straws were associated with deterioration of Hsp (36). In fact, it would be valuable to further investigate the functions of the Hsp gene family in the spermatogenic cell cycle and cell death, particularly in human sperm.…”

Standardization and Quality Control for the Determination of Alpha-Glucosidase in Seminal Plasma

“…There was no change of alpha-glucosidase activity in seminal plasma regardless of the presence of PMSF (p > 0.05). PMSF is a protease inhibitor, which can inhibit proteases degrading other enzyme proteins in seminal plasma [19,35]. We found that alpha-glucosidase in seminal plasma was very stable, and remained its activity for a long time even without PMSF.…”