Techniques for detecting vitellogenesis in the tuataraSphenodon punctatus

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Abstract
Techniques for detecting plasma vitellogenin (the precursor to egg-yolk proteins) were investigated in a rare reptile, the tuatara (Sphenodon punctatus). A single injection of oestradiol-17β (1 μg/g bodyweight) was used to induce or increase vitellogenesis in three captive animals. In the single treated male, a unique protein (molecular weight approximately 218 kDa), with a mobility during sodium dodecyl sulphate/ polyacrylamide-gel electrophoresis (SDS/PAGE), almost identical to that of partially purified vitellogenin from the turtle Chrysemys picta, appeared in the plasma 7 days after treatment. This protein was present in the plasma of both female tuatarabefore treatment, but increased as a percentage of total plasma protein (as assessed by laser densitometry) within 2 days of oestradiol treatment. Further increases in the level of this protein occurred over at least 12 days in the three treated animals. The protein was not detected in plasma from either of two control animals (one male and one female) during the 57 days of the experiment. The protein has been named vitellogenin-like protein (Vg-lp) and is likely to represent the tuatara form of vitellogenin, a calcium-binding glycolipophos- phoprotein ubiquitous in egg-laying vertebrates. No pronounced changes in plasma concentrations of total protein, calcium, inorganic phosphate, or cholesterol were observed over 29-34 days following oestradiol treatment. Giemsastaining of blood smears also did not detect oestradiol-induced vitellogenesis. These results indicate that SDS/PAGE is the most sensitive technique of those tested for detecting plasma vitellogenin in tuatara. Analysis by SDS/ PAGE of tuatara plasma samples collected monthly between October 1986 and May 1987 (excluding April) from wild tuatara on Stephens Island showed that with one exception, Vg-lp was never present in plasma of male tuatara. Between 40% and 60% of female tuatara had detectable Vg-lp in each month, suggesting a prolonged period of natural vitellogenesis. Another protein (190 kDa) was shown by SDS/PAGE to be a major protein component in the plasma of 11% of wild tuatara sampled and may represent an unusual genetic polymorphism.