Vitellogenesis in Tuatara (Sphenodon)

Abstract

1—A series of studies was made on vitellogenesis in tuatara(Sphenodon), a rare oviparous reptile endemic to New Zealand with a relict distribution on only about 30 small islands. 2—An assay for vitellogenin (Vg; the major precursor protein to yolk), based on densitometric analysis of plasma proteins resolved in SDS/PA gels, was used in collaborative studies to measure Vg levels in blood samples collected from tuatara on Stephens Island, Cook Strait. These studies revealed that: (i) tuatara do not exhibit daily cycles in plasma levels of sex steroids, (ii) intuatara, determinations of plasma levels of Vg or several other biochemical indicators of vitellogenesis are not useful for estimating the percentage of female tuatara nesting each year, (iii) in female tuatara on Stephens Island, vitellogenesis occurs gradually throughout the first 3 years of a 4-year ovarian cycle. Vg is present perenially in plasma at comparatively low to moderate levels during vitellogenesis (<meant ± SE of 10.9 ± 4.5% of total plasma protein), (iv) no marked peaks were observed in Vg levels even though oestradiol peaks at around mating time, (v) in contrast to other oviparous species, no large year-to-year variations were observed in plasma concentrations of Vg or related biochemical indicators of vitellogenesis, (vi) Vg levels were not correlated with any of the other plasma constituents measured, i.e. oestradiol, calcium, inorganic phosphate, total protein and cholesterol. The extraordinarily prolonged vitellogenic cycle revealed by these studies is unique among reptiles. 3—The induction of vitellogenesis was observed in male and female tuatara following administration of oestradiol implants. Measurements of plasma levels of Vg, calcium, inorganic phosphate and total protein showed that the observed response is comparatively very slow. Some indicators of vitellogenesis do not become elevated until weeks or months after receiving implants. 4—Vg was purified from plasma by selective precipitation and DEAE-cellulose chromatography and an e.l.i.s.a. developed. This was used to measure Vg levels in plasma samples from 11 populations of tuatara (S. p. punctatus) at the northern extremity of the tuatara's distribution. Levels ranged from non-detectable to 2.9 mg/ml in samples collected just prior to ovulation. Surprisingly, Vg concentrations were not significantly correlated with plasma levels of oestradiol, progesterone or testosterone levels in the same females. The results suggest that, as in S. punctatus on Stephens Island, female S. p. punctatus on northern islands do not ovulate each year. 5—The amino acid sequences of the NH2-terminus of mature Vg from tuatara (33 residues), and of several tryptic and CNBr-generated peptides were determined. Considerable similarities were observed to sequences of Vgs from other species. The amino acid composition of tuatara Vg is similar to that of other vertebrate Vgs, is almost identical to that of tuatara egg-yolk, and contains a large proportion of serine (13.7 mol/100 mol of amino acid) and alanine (7.9 mol/100 mol). 6—The quaternary structure of tuatara Vg was partially characterised. Inter-chain disulfide bonds do not appear to be utilised but subunits may form oligomers with apparent Ms >670 kDa. lmmunoblot analyses indicated that tertiary structure is conserved among Vgs of turtle, alligator and tuatara. The heterogeneity apparent in Vg subunits during protein chemical analyses suggests that tuatara Vg may be synthesised from multiple genes. 7—A precursor-product relationship between tuatara Vg and egg-yolk polypeptides was demonstrated. Vg is synthesised and secreted by the liver (as shown by incubations in vitro of liver explants from an oestradiol-treated female tuatara). It is transported in the bloodstream to the ovary but is not found intact in egg-yolk. Four polypeptides in tuatara egg-yolk (approx. 116, 105, 50 and 35 kDa in SDS/PA gels) cross-reacted strongly with anti-tuatara Vg antiserum in immunoblot experiments. These may correspond to the lipovitellins and phosvitin(s) obserued in other species. 8—An unusual putative polymorphism in a major plasma protein of tuatara was characterised. Immunochemical, electrophoretic and NH2-terminal sequence analysis (20 residues), revealed it to be a novel alloalbuminaemia. Both forms of tuatara albumin are immunochemically related to those of chicken and other reptiles. The 'normal' form of albumin in tuatara (approx. 130 kDa in reducing SDS/PA gels) is about twice the M of albumin from any other terrestrial vertebrate. The M of the 'variant' form (170 kDa in reducing SDS/PA gels) suggests mutation to a larger size has occurred. On Stephens Island, tuatara possessing the three alternative albumin genotypes are present at Hardy-Weinberg frequencies. Surveys of 101 tuatara in 11 populations outside Stephens Island identified only 3 tuatara (all heterozygotes) apparently possessing the same variant albumin. The NH2-terminal sequence of tuatara albumin is conserved with respect to other veftebrate albumins, but a Glu at position +3 of tuatara, chicken and lamprey albumins appears to have been lost from mammalian albumins. Determination of the first 15 residues of turtle (Trachemys scripta) albumins revealed that Glu is also present at this position in T. scripta alb-1 but that T. scripta alb-2 contains Asp instead. These appear to be the first sequences available for albumins of reptiles. 9—Tuatara plasma α2-macroglobulin (α2M) was identified by NH2-terminal sequence analysis and by specific assay. NH2-terminal sequence analyses revealed two α2M subunits of different lengths that are identical over at least 12 residues. This type of heterogeneity has not been previously reported for α2M. In tuatara, the intact protein appears to be composed of four or more covalently bonded subunits of approx. 160 kDa each. 10 An immunochemical analysis of tuatara lens crystallins revealed that ε-crystallin, previously found in lenses of only crocodilians and some birds, is also present in tuatara lenses. The taxonomic position of tuatara relative to other reptiles and birds was examined further by using αA-, β1-, and β11-haemoglobin protein sequences to construct a reptilian phylogeny by the maximum parsimony method. The interesting implications of these studies for reptilian taxonomy are discussed.