Endocrinology of oviposition in the tuatara (Sphenodon punctatus)—II. Plasma arginine vasotocin concentrations during natural nesting

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Fhysid. Vol. lOOA, No. 4, pp. 819-822, 1991

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ENDOCRINOLOGY OF OVIPOSITION IN THE TUATARA (~~H~~~~~N ~U~C~~~U~ )--II. PLASMA ARGININE VASOTOCIN CONCENTRATIONS DURING NATURAL NESTING J. GUILLETIE,JR,* CATHERINE R.PR~PPER,‘Q ALISONCREEC~II and ROBERTM. DOREST *Laboratory of Vertebrate Reproduction, Department of Zoology, University of Florida, Gainesville, FL 32611 U.S.A.; Telephone: (904)-392-1098;Fax: (904)-392-3704;tLaboratory of Comparative Reproduction, Department of EPO Biology, University of Colorado, Boulder, CO 80309, U.S.A.; #School of LOUIS

Biological Sciences, P.O. Box 600, Victoria University of Wellington, Wellington, New Zealand; and IDepartment of Biological Sciences, University of Denver, Denver, CO 80208, U.S.A. (Received 27 March i 99 1) Abstract-l. Plasma concentrations of the neurohypophysial arginine vasotocin (AVT) were measured during three stages of natural nesting [nest digging (N = 16), oviposition (N = 6), nest guarding (N = 6)) in the rare New Zealand reptile, the tuatara (Sphenodon puncfafus). 2. Nest digging females (X & 1 SE: 134.9 + 15.0 pg/ml) exhibited elevated but significantly lower concentrations of plasma AVT compared to those observed during oviposition (216.0 + 28.1 pg/ml). Nest guarding females (54.6 f 29.8 pg/rl) had plasma AVT concentrations significantly lower than other stages. 3. These data are similar to those reporting elevated plasma AVT con~ntrations during oviposition in sea turtles but tuatara exhibit significantly lower plasma AVT values (IO-fold difference). Plasma AVT

concentrations reported for tuatara and birds are comparable.

The endocrine control of reptilian oviposition involves the complex interaction of steroids, neurohypophysial peptides and prostaglandins. Until recently, almost nothing was known concerning plasma levels of these hormones during natural nesting in reptiles. Previous studies examining plasma levels of hormones during natural nesting in the tuatara (@hen&on punctatus; Guillette et al., 1990a) and sea turtles (Caretta curetta, Lepidochelys olivacea; Figler et al., 1989; Guillette et al., 1991a) have supported experimental studies (see Jones and Guillette, 1982; Guillette, 1990) indicating that arginine vasotocin (AVT), prostaglandms (PGF,, , PGEz) and steroids influence the uterine contractions associated with oviposition. In the loggerhead turtle (C. caretta), AVT rises at the onset of nest digging and peaks at oviposition, whereas plasma concentrations of PGF rise as the next chamber is dug, just prior to oviposition (Figler et al., 1989; Guillette et al., 1991a). These data suggest that AVT induces PGF release in uivo as it does from the lizard oviduct in vitro (Guillette et al., 1990b). However, beside the data reported above for sea turtles (Figler et al., 1989), we have no data on plasma concentrations of AVT during oviposition in any other reptile, SPresent address: Department of Biology, University of Northern Arizona, Flagstaff, Arizona, U.S.A. l/Present address: Department of Zoology, University of Otago, Dunedin, New Zealand.

This study examined plasma concentrations of AVT in female tuatara exhibiting three behavioral stages of nesting: nest digging, oviposition and nest guarding. Tuatara represent the last surviving representative of the ancient reptilian order Sphenodontida (Fraser, 1988). This species has a restricted ~stribution, occurring on approximately 30 islands surrounding New Zealand (Daugherty et al., in press). Nesting is concentrated in rookeries and occurs primarily during the month of November (Cree and Thompson, 1988; Cree et al., in press). Tuatara are unique in having the longest egg retention period described for oviparous reptiles; females ovulate a clutch of eggs during March-April and retain these eggs for approximately 6-8 months, until the following October-December (Cree et al,, in press). Eggs are laid with late gastrula-staged embryos (Moffat, 1985). Nest construction may take up to two weeks to complete depending on prevailing weather conditions (Cree and Thompson, 1988) during which time females return most nights to dig. Oviposition occurs on a single night (Cree and Guillette, unpublished data), and is followed by about 4-8 days of nest guarding (Cree, unpublished data). MATERIALS

AND METHODS

Animals and sample collection Adult femaletuatara (Sp~enudonp~c~utus) from Stephens Island, a wildlife sanctuary in Cook Strait, New Zealand, were studied. This site has previously been described in detail (Cree ef al., 1990a,b; Guillette er al., 1990a). Females exhibiting nest digging, oviposition or nest guarding were obtained from rookery areas during November 1987. A 819

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blood sample (approximately I ml) was collected from a caudal blood vessel using a heparinized syringe. Blood was then taken immediately to the New Zealand Department of Conservation field station on Stephens Island where it was centrifuged. The plasma was initially snap frozen in liquid nitrogen within 15 min of collection, and later transported to the U.S.A. on dry ice and stored at -80°C. Following collection of the blood sample, all females were weighed, measured (snout-vent length) and marked on each flank with a permanent ink marker for later identification. On subsequent nights, several of these females were collected exhibiting similar or different behaviors, and additional blood samples were obtained. These samples were used to determine temporal changes in plasma hormone concentrations.

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Arginine vasotocin was iodinated using the Chloramin-T method (Rees et al., 1971). Four micrograms of AVT were dissolved in 20~1 of potassium phosphate buffer (KPB, 250 mM, pH 7.5). To -this AVT-solution, 1 mCi of ‘*‘I (Amersham Corn.) in 50~1 KPB was added followed immediately by 2Oij of Chioramin-T (3.5 mg/ml KPB). The solutions were mixed by tapping the microcentrifuge tube gently, and then were allowed to react for 30 set before 20 ~1 of sodium meta-bisulfate solution (4.0 mg/ml KPB) was added to stop the reaction. After 2min, the solution was applied to a G-10 column and the radiolabelled peptide was eluted in 2 N acetic acid (0.1% BSA). The direct radioimmunoassay procedure was conducted using techniques similar to those described by Dores (1982). All standards, samples, antibody and ‘251-AVT solutions were prepared in 0.01 M dipotassium phosphate buffer (pH 7.5). A serial standard dilution was prepared ranging from 1.5pg to 10,OOOpg AVT (Sigma Chemical Co., St. Louis, MO, U.S.A.) in lOOn buffer. Plasma samples (200 ~1) from female tuatara (Sphenodon punctatus) were dried in a Savant speed-vat centrifuge, and 100~1 of buffer was added to each. All standards and samples received an additional 100 nl of buffer containing 10 000 cpm ‘**I-AVT, 1: 4000 anti-AVT antiserum 593 (kindly provided by Dr K. Lederis, Calgary, Canada to Dr R. E. Jones, University of Colorado), and I :50 normal rabbit serum (Lot 10705P; Arnel). Tubes were incubated for 24 hr at 4”C, after which 12 ~1 of 1: 1.5 goat anti-rabbit serum (Lot 41965 Biotek) was added to all standards and samples. Tubes were incubated for an additional 3 hr at 4°C followed by the addition of 1.5 ml of 0.05% Triton X. Standards and samples were immediately spun in a precooled centrifuge at 5,000 rpm for 10 min. The supernatant was aspirated and the pellet counted. All samples were run in a single radioimmunoassay. A three-point serial dilution of tuatara plasma was determined to be parallel to the standard curve. The antibody does not cross react with mesotocin and has previously been shown to be specific for AVT (Sawyer ef al., 1984). Total binding was 24.5% whereas nonspecific binding was 1.2%; sensitivity of this assay was 1.5 pg AVT/tube with an intra-assay coefficient of variation of 19%. Plasma concentrations were averaged for all samples from each behavioural group (only initial samples from each female were used in this analysis). Data were analysed statistically using a one-way ANOVA followed by Scheffe F-tests (Stat-view II, Abacus Concepts, Inc., Berkeley, CA, 1988). We obtained repeat blood samples from six females exhibiting nest digging and the initial concentration of plasma AVT was compared to the later samples using a f-test for repeated samples.

Guarding

STAGE

Radioimmunossay for A VT

Fig. 1. Mean (+ 1 SEM) plasma AVT concentrations in nest digging, ovipositing and nest guarding tuatara (Sphenodon punctatus). Bars having different superscripts are significantly different.

were more common than those ovipositing (N = 6) or nest guarding (N = 6). During nest digging, females had elevated plasma AVT concentrations (Fig. 1) when compared to nest guarding females. A further significant increase (F2,25= 6.39; P = 0.006) in mean plasma AVT concentrations occurred during oviposition, followed be a significant decline during nest guarding (Fig. 1). Repeat blood samples obtained at least a day apart from nest digging females showed no significant change in plasma AVT levels (Fig. 2). DISCUSSION

Nest digging and ovipositing tuatara had plasma concentrations of AVT that were elevated above those seen during nest guarding. These data resemble those reported for sea turtles, in which plasma AVT concentrations were elevated at oviposition compared with those seen during post-ovipositional nestfilling and return to the sea (Figler et al., 1989). However, female tuatara appear to differ from loggerhead turtles (C. caretta) in the temporal relationships between changes in plasma AVT and plasma PGF concentrations during nesting (Guillette et al., 1990a).

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125 = E a

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75

50

25 0 Second

First

Blood

Sample

RESULTS

Female tuatara were obtained in all three stages of nesting, but females exhibiting nest digging (N = 16)

Fig. 2. Mean (+ 1 SEM) AVT concentrations in initial and second (>24 hr later) plasma samples taken from nest digging female tuatara (Sphenodon punctam).

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AVT in Sphenodon

Female loggerhead turtles emerge from the sea with non-detectable plasma concentrations of AVT, PGF and PGE, . With the onset of digging, elevations in plasma AVT and neurophysin are observed (Figler et al., 1989). However, it is not until the female has completed digging the body pit and has begun to excavate the nest chamber that significant elevations in plasma PGF and PGE, concentrations are observed (Guillette et al., 199la). The concentrations of all four hormones reach their maxima at oviposition, and then decline during the covering of the nest and body pit (Figler er al., 1989; Guillette et al., 1991a). When females return to the ocean, plasma concentrations of AVT, PGF and PGE, are similar to values recorded during nest digging. No significant change in plasma progesterone (P) or estradioLl7jl (E,) occurs during nesting in sea turtles (Guillette et al., 199la). Tuatara exhibit a different pattern for mean plasma PGF and PGE, concentrations during nesting than observed in sea turtles (Guillette et al., 1990a). As in sea turtles, tuatara exhibiting nesting behavior (which may last for several weeks) have plasma PGF and PGE, concentrations elevated above levels seen during nest guarding. At oviposition, however, plasma PGF and AVT rise significantly above levels seen during digging, whereas plasma PGE, does not (Guillette et al., 1990a, present study). Following oviposition, plasma concentrations of both PGs and AVT decrease significantly below levels seen during nest digging and oviposition. Female tuatara (S. punctatus) resemble loggerhead turtles in exhibiting no variation in plasma concentrations of E, or P during nesting events (nest digging, oviposition or nest guarding; Guillette et al., 1990a), although plasma concentrations of E, rise during the month preceding nesting (Cree, Cockrem and Guillette, unpublished data). The relative timing of the appearance of AVT and PGF in the blood of loggerhead turtles during oviposition suggests that AVT may stimulate uterine contractions and induce the release of oviducal PGF (Guillette et al., 199la) as reported during parturition in mammals (Challis and Olson, 1988) and oviposition in birds (Hertelendy et al., 1984). Synthesis of PGF from the reptilian reproductive tract can be stimulated in vitro by AVT (Guillette er al., 1990b) as observed in birds (Rzasa, 1984) and the occurrence of natural birth or the effectiveness of AVT-induced parturition and oviposition in reptiles is decreased by pretreatment with indomethacin, a prostaglandin synthesis inhibitor (Guillette et al., 1990~; 1991b). These data suggest that AVT stimulates the release of PGF from the reproductive tract, which culminates in oviposition. Data from the tuatara suggest that this relationship may be ubiquitous among reptiles. In tuatara, plasma PGF concentrations rose significantly between nest digging and oviposition as did AVT. Unfortunately, we do not have extensive sampling immediately prior, during or immediately after oviposition in the tuatara which would help clarify the relationship between these hormones. Additional data from naturally ovipositing reptiles and further experimental work is needed to understand not only the interactions among these hormones at oviposition but also to determine the role

of environmental signals controlling the timing of oviposition in reptiles. Finally, the plasma concentration of AVT we observed in tuatara during oviposition is significantly less than that recorded in sea turtles (Figler et al., 1989). Sea turtles exhibit a 250-fold increase in AVT titer whereas we observed a 4-fold difference between maximal and minimal concentrations. Our changes, however, are consistent with that noted in the hen which shows a 47-fold increase during oviposition (Arad and Skadhauge, 1984; Tanaka et al., 1984). Figler et al. (1989) hypothesized that the large increase in AVT was due to the large clutch size of sea turtles and suggested that reptiles with smaller clutches would exhibit a less dramatic change in circulating AVT levels. Data are now available for plasma concentrations of prostaglandin F (PGF) and prostaglandin Ez (PGE,) during oviposition in loggerhead turtles (Guillette et al., 199la) and tuatara (Guillette et al., 1990a). Interestingly, these hormones exhibit a similar discrepancy in plasma concentrations with sea turtles, for example, having a maximal mean PGF concentration of 5236.5 pg/ml whereas ovipositing tuatara have mean plasma PGF concentrations of 529.7pg/ml. These data support the hypothesis that oviposition of larger clutches is associated with greater circulating AVT, PGF and PGE, concentrations. It is intriguing to note that oviposition of the complete clutch in both species, loggerhead and tuatara, takes approximately 30-60 min. Higher concentrations of AVT and prostaglandins could influence oviposition rate, thus maximizing the number of eggs laid in a specific period of time. Additional work needs to examine the basis for the difference observed among species in circulating concentrations of AVT. Acknowledgements-We

thank Mike Thompson and Jennie Hay for excellent field assistance and companionship. Thanks are also due to the NZ Department of Conservation, especially Don Newman, Noel Hellyer and Ian Govey, for assistance with permits. This work was supported in part by grants from: National Science Foundation-USA (DCB 84-16707), NZ Department of Conservation, NZ University Grants Committee, NZ Lottery Board, University of Florida (Division of Sponsored Research), Victoria University of Wellington (Internal Research Committee and School of Biological Sciences) and WWF-NZ.

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