Cholesterol and phospholipid concentrations and phospholipase activity were measured in fluid from cannulae collected from the bovine oviductal isthmus and ampulla at different stages of the oestrous cycle. The cholesterol concentration and cholesterol normalized by protein were significantly (P = 0.03) greater in isthmic oviductal fluid (224.3 ± 42.7 μg ml−1 over all stages) than in ampullary oviductal fluid (164.5 ± 11.3 μg ml−1), and maximal concentrations (284.5 ± 25.5 μg ml−1) were found during the luteal stage (serum progesterone concentration ≥ 1.5 ng ml−1). The concentrations of the phospholipids sphingomyelin and lysophosphatidylcholine increased at different stages of the cycle and in different regions. In the ampulla, the concentration of sphingomyelin was significantly (P < 0.05) greater in oviductal fluid collected during the luteal phase (12.1 ± 2.7% of total phospholipids) than in fluid collected near oestrus and ovulation (7.5 ± 1.5% and 6.9 ± 1%, respectively). The concentration of lysophosphatidylcholine was greater (P < 0.01) in ampullary (19.2 ± 1.6% of total phospholipids) than in isthmic oviductal fluid (9.9 ± 1.1%) collected near ovulation. The ratio of cholesterol to total phospholipid was highest in oviductal fluid collected from the isthmus during all stages (2.3 μg ml−1:% total phospholipid), while the minimal ratio was found in ampullary fluid collected near ovulation (1.5). Phospholipase activity was higher (P = 0.03) in isthmic oviductal fluid (20.4 ± 3.2% product formed) than in ampullary oviductal fluid (14.6 ± 1.4%); the lowest activity (12.6 ± 1.7% product formed) was in fluid collected during the phase of the oestrous cycle immediately before ovulation. We conclude that the regional and temporal differences in the concentrations of lipids in oviductal fluid provide general support for the concept that the isthmus serves as a sperm reservoir, while the ampulla is the site of the bovine sperm acrosome reaction.
A. A. Grippo, S. H. Anderson, D. A. Chapman, M. A. Henault and G. J. Killian
S. M. Skinner, G. J. Killian, L. A. Miller and B. S. Dunbar
The effectiveness of zona pellucida antigens in immunizing white-tailed deer to reduce fertility was evaluated by analysing the constituent deer zona pellucida proteins and their immunogenicity. Does were immunized with porcine zona pellucida antigens. The antibodies were characterized using immunohistochemical and immunoblot analysis, in which zona pellucida proteins were separated by one-dimensional and two-dimensional PAGE. Deer anti-porcine zona pellucida antibodies were found to recognize all the major proteins of the porcine zona pellucida. These antibodies also recognized several proteins of deer zona pellucida, indicating that it is possible to break immune tolerance in the deer using such a protocol. The antibodies were also found to recognize peptides of 55 and 75 kDa that were produced by expressing cDNA clones containing antigens of major glycoproteins of rabbit zona pellucida. Furthermore, antibodies against rabbit zonae pellucidae recognized antigens in zonae of paraffin-embedded deer ovaries. Taken together, these experiments demonstrate the crossreactive nature of a number of zona pellucida epitopes found in deer and in several other species. They also illustrate the immunogenicity possible in such an immunization protocol, and provide valuable probes for the investigation of follicular development in this and other species.
E. K. Topper, G. J. Killian, A. Way, B. Engel and H. Woelders
Before fertilization, inseminated spermatozoa acquire the ability to fertilize an egg, a phenomenon called capacitation. Bovine sperm capacitation is influenced by factors originating from both the male and female genital tract, and results in intracellular and membrane changes of the spermatozoa that facilitate the induction of the acrosome reaction. However, the effects of reproductive tract secretions and capacitation on the binding of spermatozoa to the zona pellucida have not been investigated. In this study, a sperm–egg binding assay was used to determine whether the ability of bull spermatozoa to bind to the zona pellucida was altered during in vitro capacitation by heparin or oviductal fluid, or by treatment of spermatozoa from the cauda epididymidis with accessory sex gland fluid. In addition, biotinylated solubilized zona pellucida proteins were used to visualize zona binding on spermatozoa. The ability of bull spermatozoa to bind to the zona pellucida was increased after both heparin and oviductal fluid induced in vitro capacitation. Exposure of spermatozoa from the cauda epididymidis to accessory sex gland fluid resulted in a direct increase in zona binding ability, followed by a further increase during capacitation in vitro. Binding of solubilized zona proteins was restricted to the acrosomal cap of bull spermatozoa. It is suggested that the observed increased ability of bull spermatozoa to bind to the zona pellucida enables optimal sperm–egg attachment, which also relates to the induction of the acrosome reaction by the zona pellucida. Thus, increased zona binding ability is likely to be an essential part of the process of capacitation.