7UTILIZATION OF CELL PROFILING TO EVALUATE BOVINE SPERMATOZOA IN NORMAL AND SIMULATED MICROGRAVITY

Abstract

We developed a method to evaluate bovine sperm membranes in normal (1G) and simulated microgravity (Sim-μG). Bovine spermatozoa are used as a model system because they have cellular membranes analogous to those of other cell types, and yet are much simpler because they have no cytoplasm and do not participate in DNA transcription or mRNA translation. They can be cultured as single cells and are easily evaluated for membrane characteristics using flow cytometry. These features make the mammalian spermatozoon a useful model for exploring the principles of membrane structure/function in the presence of a variety of environmental challenges such as simulated microgravity. Cryopreserved, washed beef bull sperm (4–8×106mL−1)were incubated under non-capacitating conditions (modified glucose-free Tyrode’s medium containing low bicarbonate, HEPES buffer, pyruvate and 3mgmL−1 BSA V; 23°C in air), and these spermatozoa remained alive for 24–48h at 1G. To simulate μG, spermatozoa were incubated under the same conditions, in a HARV 10 rotating wall vessel (RWV, Synthecon, Inc, Houston, TX, USA) at 9rpms. Spermatozoa were incubated in 1G and Sim-μG environments for 2.5–4.5h and subsequently exposed to 0, 60 or 80μgmL−1 LC for 0, 4, 8, 12, 16 and 20min. Three fluorochrome combinations were used as probes at each [LC]/time point: (1) propidium Iodide (dead status)+SYBR 14 (live status); (2) PI+FITC-PSA (acrosome reactions [ARs]); (3) PI+MitoTracker Deep Red (mitochondrial activity). Approximately 1million spermatozoa from 3 bulls were evaluated over 4 days. Data were acquired on a FACSVantage SE flow cytometer, and initially analyzed (quality control) using the bundled FACSVantage SE software package (Cell Quest, BD BioSciences, San Jose, CA, USA). This provided graphics of simple cell relations (fluorescence v. LC exposure time). For further statistical analysis, and incorporation of non-parametric statistical tools (including pattern recognition using Support Vector Machines), the data were processed using a collection of Perl scripts and C programs. Results: Live/dead status: When Sim-μG+60μgmL−1 LC sperm were compared to 1G+60μgmL−1LC, and 80μgmL−1 LC sperm, their profiles were more similar to the 1G 80μgmL−1 LC profiles. AR status: the Sim-μG+60μgmL−1 LC profiles were similar to the 1G+60μgmL−1 LC profiles. Mitochondrial Status: the Sim-μG+60μgmL−1LC profiles were more similar to 1G+80μgmL−1 LC profiles. Summary: although Sim-μG sperm lost their motility within 3h, they were alive. Cell profiles indicate that Sim-μG sperm nuclear membranes are less stable and their mitochondria are less functional than the 1G controls, but their acrosomes are intact indicating that fertilizing potential may remain. Additional experiments are needed to determine the time course for Sim-μG, induced changes, and whether Sim-μG sperm can penetrate eggs. Funding: NASA (2002)-Stennis-24 and The University of New Orleans.