The University of Georgia College of Veterinary Medicine

Georgia Veterinary Scholar Program

Georgia Veterinary Scholar	Faculty Mentor

Kathryn Shelton University of Georgia Class of 2004	Dr. Benjamin Brackett

Progesterone and b-Cyclodextrin Induced Bull Sperm Capacitation assessed by Chlortetracycline Staining

Among many agents, heparin, progesterone, and b-cyclodextrins have been shown to induce capacitation, i.e. the acquisition of sperm fertilizing ability. Fluorescent staining of one particular bull’s heparin-treated spermatozoa with chlortetracycline (CTC) unexpectedly revealed no changes in the proportion of cells exhibiting the B (capacitated) pattern when compared with untreated samples; also, the CTC assessment of suboptimal capacitation was corroborated by inferior in vitro fertilization (IVF) results (Dinkins, MB & Brackett, BG Zygote 8: 245-256, 2000). Our objective was to devise a novel capacitating treatment for this bull’s “heparin resistant” spermatozoa by combining agents in completely chemically defined conditions. Based on earlier independent responses, 100mM of progesterone and 0.005% of 2-hydroxypropyl-b-cyclodextrin were selected for a combined treatment (P+HPCD). Frozen-thawed spermatozoa were diluted to 20 x 106 sperm/ml after swim-up in mDM (Dinkins, MB, ibid.) with 5 mM HEPES. The sperm suspension was divided into untreated (mDM), heparin (200mg/ml), and P+HPCD treated groups. Motility and CTC staining were assessed (100X) under UV light at 15 minute intervals. Five replications, each involving assessment of 100 to 250 sperm cells, led to confirmation of findings reported earlier, and the additional data reported here as mean percentage ± SEM. An increase in B pattern beginning at 2 minutes became significant by 15 minutes with P+HPCD compared to the heparin and untreated sperm, i.e. 57.8 ± 6.6, 22.2 ± 3, and 23.7 ± 1.9 respectively. Motility in the P+HPCD group decreased sharply after 15 minutes and remained consistently low concurrent with decreased B pattern, increase in F (uncapacitated) pattern, and a fairly constant AR (acrosome reacted) pattern. By contrast, along with gradually decreasing motility, the B and AR patterns increased and the F pattern decreased over time in the untreated and heparin groups. Further investigation utilizing IVF will determine whether the observed increase in B pattern reflects a functional change in capacitation status for this bull’s cryopreserved sperm. Future extension of this research promises a chemically defined “universal” sperm capacitation method. (Authors gratefully acknowledge support of Merck-Merial, Ltd. and Genex/CRI, Ithaca , NY ).