223 CHANGE IN THE DISTRIBUTION OF RNA IN BOAR SPERM DURING CULTURE IN A CAPACITATION MEDIUM CONTAINING CAFFEINE

Abstract

Mammalian sperm are known to contain various types of RNA. Recently, translation of RNA to proteins occurring during capacitation was reported to be important for sperm function and fertilization. The objective of this study was to examine the change in the distribution of RNA in boar sperm during culture in a capacitation medium containing caffeine by using fluorescence specifically binding to RNA. The sperm-rich fraction from Berkshire boars (n = 4) was diluted (cells mL^–1) with modified Modena solution containing 20% seminal fluid, cooled to 15°C for 4 h, and kept at the same temperature until use. Stored, diluted semen was washed by centrifugation (1500 rpm for 35 min at room temperature) in a Percoll gradient (45/90%). A sperm suspension (concentration 1 × 10⁷ cells mL^–1) in modified TCM-199 containing 0.4% BSA and 5 mM caffeine was then prepared and cultured in an atmosphere of 5% CO₂ in air at 39°C for 1 or 4 h. Before and after culture, sperm were stained by using SYTO RNA Select Green Fluorescence Cell Stain (Molecular Probes, Eugene, OR) according to the manufacturer’s protocol, and then the mounted specimens were observed and the intensity of fluorescence images was measured under a fluorescence microscope (BIOREVO, Keyence, Osaka, Japan). Viability of sperm was also determined following SYBR-Green–propidium iodide staining under a fluorescence microscope. Statistical analyses were carried out by ANOVA and with a Bonferroni-Dunn post-hoc test (P < 0.05). Although the viability of sperm decreased before (96.7%) and 1 h after the start of culture (79.8%), it did not decline until 4 h after the start of culture (80.9%). Before culture, fluorescence indicating the presence of RNA was observed at the head, especially the postacrosomal region and the midpiece region of the sperm. The intensity of fluorescence changed during culture. The fluorescence intensity of RNA at the sperm head region was higher (P < 0.01) at 1 h of culture (36.11 × 10⁴) than before culture (30.50 × 10⁴) and at 4 h of culture (28.60 × 10⁴). The intensity of RNA at the midpiece region was higher (P < 0.01) at 1 h (11.42 × 10⁴) and was lower (P < 0.01) at 4 h of culture (4.93 × 10⁴) than before culture (8.45 × 10⁴). From these results, we concluded that the distribution and content of RNA changes drastically during culture in a capacitation medium containing caffeine. Additional study of the kinetics of sperm RNA during capacitation is ongoing to further understand the post-transcriptional regulation.