Biology of Reproduction, Vol 11, 644-653, Copyright © 1974 by Society for the Study of Reproduction

The In Vivo Incorporation of [¹⁴C] and [³H] Choline into Phospholipids of Maturing Rabbit Spermatozoa and Reproductive Tract Tissues

¹ Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, 1960 East-West Road, Honolulu, Hawaii 96822

The in vivo incorporation of [¹⁴C] and [³H] choline into spermatozoa and reproductive tract tissues of the rabbit was studied using two methods of isotope extraction. Tissues and spermatozoa were first fixed with glutaraldehyde which extracts malachite green stainable lipids, and then treated with chloroform—methanol to remove the remaining lipids. Peaks of activity were observed in ejaculated spermatozoa on Days 8 and 16 after injection, and autoradiographs of thin-layer chromatograms demonstrated that the labeled compounds were choline phosphatides. Glutaraldehyde soluble activity from caput and corpus epididymis and from spermatozoa consistently represented 50% or more of the total extractable radioactivity in the tissues, while as little as 10% of the total activity was present in the glutaraldehyde fractions from lung, seminal vesicle, and adrenal gland. The presence of considerable quantities of labeled phospholipid in glutaraldehyde extracts of Day 8 spermatozoa gives further evidence that malachite green stainable phospholipid, which accumulates in rabbit spermatozoa as they transit the epididymis, is a choline phosphatide. Also, the time required for labeled choline to be detected in ejaculated spermatozoa indicates that incorporation occurs in the epididymis.