Rheologic Behavior of Spermatozoa Exposed to Hypoosmotic Conditions at Varying Oncotic Pressure Environments: Possible Protective Mechanisms Against Sperm Swelling by the Addition of Protein Supplements

Abstract

Objective: Exposure of spermatozoa to hypoosmotic conditions that cause optimal swelling for assessment of sperm membrane functional integrity causes sudden and irreversible changes in membrane structure. In almost all cases, spermatozoa cannot support viability as observed by lack of motility during and at the end of the incubation period (30 to 60 min). This behavior of human spermatozoa exposed to hypoosmotic conditions has been previously assessed, which resulted in the development of a sperm functional test (hypoosmotic swelling (HOS) test. J Reprod Fertil 1984;70:219). The aim of this study was to assess possible osmoregulatory mechanisms of substances contributing to oncotic pressure (exerted by colloids in solution) such as protein supplements against exposure to hypoosmotic conditions (exerted by dissolved substances in solution). Design: Exposure of processed spermatozoa to controlled hypoosmotic conditions (150 mOsm/L) with varying serum supplement (Synthetic serum substitute; SSS, Irvine Scientific) concentrations contributing to variability in the oncotic pressure environment. Stock solutions consisted of HOS solution and hypoosmotic SSS (H-SSS) at 150 mOsm/L. The various stock and mixtures of solutions tested consisted of: 1) HOS solution 100%, 2) H-SSS 100%, 3) 75% H-SSS/25% HOS, 4) 50% H-SSS/50% HOS, and 5) 25% H-SSS/75% HOS. Materials and Methods: Semen specimens from 18 men were obtained for routine semen analysis and pooled (2 to 3 specimens each time). Pooled specimens were processed via density gradient centrifugation and reconstituted in human tubal fluid (HTF; Irvine Sci) media containing 0% SSS. Aliquots of the reconstituted specimens were diluted 1:10 (v/v) with each of the testing solutions and incubated for 60 min (37°C/5% CO2). Specimens were assessed on wet mounts for total swelling and swelling patterns, which included maximal (>50% tail swelling) and minimal swelling (<50% tail swelling), and motile sperm undergoing swelling of any type. Results: The results obtained are shown in the table below: legendType of swellinglegend∗, †, ‡, §, ∥. Denotes significant differences (P<0.05) between rows.H-SSS 100%75% H-SSS/ 25% HOS50% H-SSS/ 50% HOS25% H-SSS/ 75% HOSHOS 100%Maximal swelling13.4% ± 2.0∗20.6% ± 1.7†35.9% ± 1.7‡43.1% ± 2.7§50.8% ± 2.0∥Minimal swelling23.9% ± 1.6∗34.2% ± 1.8†33.3% ± 1.5†33% ± 1.8†27.9% ± 1.6‡Motile/swelling51.4% ± 2.5∗31.8 ± 2.3†15.4% ± 1.2‡6.3% ± 1.0§1.1% ± 0.4∥Total swelling88.8%86.6%84.7%82.3%79.8%legend ∗, †, ‡, §, ∥. Denotes significant differences (P<0.05) between rows. Open table in a new tab Conclusions: The addition of increasing concentrations of protein supplements resulted in a decrease in the distribution of maximal swelling patterns and an increase in the proportion of minimal swelling and motile spermatozoa undergoing minimal swelling. Furthermore, the overall sperm swelling patterns increased with an increase in the protein supplement concentration. Thus, addition of serum supplements may be beneficial to provide protection against changes in osmotic pressure in culture media, but caution is advised when processing spermatozoa for freezing or thawing due to the possible interaction between protein molecules and free-water, which freezes or thaws first, causing the opposite effect of further increasing the osmotic pressure of the medium.