Induction of thumbtack sperm during coculture with oviduct epithelial cell monolayers in a marsupial, the brushtail possum (Trichosurus vulpecula).

Abstract

A reorientation of the sperm head so that it is perpendicular to the sperm tail (i.e., T-shape or thumbtack) is considered an indicator of sperm capacitation in the Australian marsupial the brushtail possum (Trichosurus vulpecula). This study describes a method of oviduct epithelial cell monolayer and sperm coculture in the brushtail possum to obtain a high percentage of thumbtack sperm. The oviduct epithelial cell (OEC) monolayers were prepared in vitro from the isthmal and ampullary segments of eCG- and LH-primed brushtail possum oviducts. Coculture experiments demonstrated that cauda epididymidal sperm from the brushtail possum attached equally to the OEC monolayers derived from the isthmal and ampullary segments of the oviduct. After 2 h of coculture, a large number of sperm attached to OEC monolayers (ampulla, 60.1+/-4.7% and isthmus, 63.1+/-5.7%) as well as to controls (tracheal epithelial cell monolayer, 46.2+/-3.7%; Matrigel, 57.4+/-7.7%; plastic, 29.2+/-3.2%). After 6 h, fewer sperm were attached to tracheal epithelial cell monolayers (1.2+/-0.2%; P<0.01) and Matrigel (10.2+/-2.5%; P<0.01), compared to those attached to ampullary and isthmal OEC monolayers (37.9+/-7.2% and 44.6+/-2.2%, respectively), and none were attached to the plastic surface. Fewer sperm were released from the ampullary and isthmal OEC monolayers compared to those from controls (P<0.05). At 6 h of coculture with ampullary and isthmal OEC, the percentage motility of both attached and unattached spermatozoa was maintained at 40-50%, which was higher (P<0.05) than in controls. Progressive motility of unattached sperm was maintained at about 2 (on an arbitrary scale of 1-5) and was not different among treatments until 6 h. More than 60-70% sperm were viable at 6 h of coculture in all the treatments. Coculture of brushtail possum epididymal sperm with OEC monolayers transformed 60% of motile streamlined spermatozoa to thumbtack orientation at 2 h compared to approximately 25% in controls. No acrosomal modifications were induced in spermatozoa in any of the treatments. This study has demonstrated a role of the oviduct in transforming a large number of sperm from a streamlined to thumbtack orientation, which may have relevance in sperm capacitation and fertilization in this species.