Plasma arginine vasotocin, progesterone, and luteal development during pregnancy in the viviparous lizard Tiliqua rugosa

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

82, 140-151 (191)

Plasma Arginine Vasotocin, Progesterone, and Luteal Development during Pregnancy in the Viviparous Lizard Tiliqua rugosa’ B. FERGUSSONAND S.D. BRADSHAW Department

of Zoology,

University

of Western

Australia,

Perth,

Western

Australia

6009,

Australia

Accepted June 16, 1990 The relationship between plasma levels of arginine vasotocin (AVT), progesterone, and corpus luteum formation and degeneration was studied in the viviparous lizard Tiliqua rugosa. Hormone levels were monitored in free-ranging, pregnant females which were located for sampling by means of attached radio transmitters. There was an increase in plasma AVT levels in the 30 days immediately prior to parturition. Concurrent with this event was a decline in plasma progesterone levels from relatively high levels in mid-term to basal levels prior to parturition. This is associated with degenerative changes in the corpus luteum which include pyknosis of the nuclei of the cells of the cell mass and increasing prevalence of intercellular spaces, while the thecal layer became increasingly compacted. Ovariectomy experiments indicate that the major source of progesterone during pregnancy in T. rugosa is OVarian.

0 1991 Academic Press,

Inc

The neurohypophysial hormone arginine vasotocin (AVT) is hypothesized to play an endocrinological role in reptilian oviposition and parturition. AVT has been shown to induce oviducal or uterine contractions in a number of species (Munsick et al., 1960; La Pointe, 1969, 1977; Heller, 1972; Callard and Hirsch, 1976; Guillette and Jones, 1980). The mammalian neurohypophysial peptide oxytocin and mammalian neurohypophysial extracts have been found to induce oviposition in a number of oviparous reptiles (La Pointe, 1964; Ewert and Legler, 1978) and parturition in a number of viviparous species (Clausen, 1940; Panigel, 1956). However, AVT is more potent in this regard (La Pointe, 1969) and has since been shown to induce oviposition or parturition in a number of lizards (Guillette, 1979; Guillette and Jones, 1982). It is pre’ Preliminary reports of some of this material were presented at the 16th Annual Conference of the Australian Society for Reproductive Biology, Melbourne, 1984, and at the Annual Meeting of the Federation of American Societies for Experimental Biology, St. Louis, 1986.

mature, however, to attribute critical ovipositional or parturient functions to AVT in reptiles in the absence of corroborative evidence concerning the circulating plasma concentrations of AVT before, during, and after oviducal or uterine contractions. In birds, AVT is also believed to play a role in oviposition since it induces oviducal contractions, an increase in intraoviducal pressure, and egg laying (Munsick et al., 1960; Heller and Pickering, 1961; Rzasa and Ewy, 1971) and stimulates prostaglandin production in oviducal tissue (Rzasa, 1984). There is also evidence that the AVT content of the neurohypophysis of the hen decreases at oviposition (Tanaka and Nakajo, 1962) and that there is a concomitant increase in plasma concentrations of AVT (Sturkie and Lin, 1966; Niezgoda et al., 1973; Shimada et al., 1986). Similar data are required for reptilian species in order to enhance our understanding of the hormonal control of oviposition and parturition. The morphological similarities between reptilian and mammalian corpora lutea (Browning, 1973; Fox, 1977) and the obser140

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AVT

AND

PROGESTERONE

vation that luteal bodies are maintained throughout pregnancy in many viviparous reptiles have led to the assumption that the corpus luteum is an endocrine gland secreting progesterone (Matthews, 1955; Chan et al., 1973). The enzyme 3p-hydroxysteroid dehydrogenase, necessary for the synthesis of progesterone, has been localized histochemically in corpora lutea of Lacerta vivipara, Natrix sipedon, Sceloporus cyanogenys, Xantusia vigilis, and Mabuya capensis (Callard, 1966; Callard et al., 1972; Yaron,

1972, 1985). The presence of the steroid enzyme 21-hydroxylase has been demonstrated in the corpora lutea of the viviparous snake Storeria dekayi (Colombo and Yaron, 1976) and this activity was interpreted as a sign of progestin secretion. In viviparous reptiles, the timing of luteal regression varies to a large extent (Fox, 1977) and ranges from the end of the lirst trimester until soon after parturition (Panigel, 1956; Miller, 1959; Callard et al., 1972; Highlill and Mead, 1975; Guillette et al., 1981; Jones and Guillette, 1982). Plasma progesterone levels in most viviparous snakes (Chan et al., 1973; Highfill and Mead, 1975) and lizards (Callard et al., 1972; Veith, 1974) are high during gestation and then fall near the time of luteal regression and parturition. However, in the viviparous lizard Sceloporus jarrovi, there is a small rise in plasma progesterone levels in the first trimester when the corpora lutea are active and then a larger peak in later pregnancy when the corpora lutea are regressed (Guillette et al., 1981). It was suggested that the chorioallantoic placenta, corpora atretica, or the adrenal glands were possible nonluteal sources of progesterone during late pregnancy in S. jarrovi. In the viviparous skink Tiliqua rugosa, Bourne et al. (1986) have detected a peak in plasma progesterone concentration during the second trimester of pregnancy. However, previous studies (Bourne and Seamark, 1972; Bourne, 1980) indicated that circulating levels measured by competitive protein-

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binding radioassay were unaffected by ovariectomy in either pregnant or nonpregnant T. rugosa. Thus, the adrenals are proposed as possible significant contributors to progesterone secretion during pregnancy in this species (Boume et al., 1986). In contrast, luteectomy of the viviparous snake Thamnophis elegans (Highlill and Mead, 1975) and the oviparous lizard Anolis carolinensis (Guillette and Fox, 1985) resulted in reduction of plasma progesterone concentrations to levels approximately equal to those of nonpregnant or nongravid animals. This investigation examines changes in plasma concentrations of AVT and progesterone in free-ranging T. rugosa during late pregnancy in order to elucidate some of the hormonal events that lead up to parturition. The formation and degeneration of the corpus luteum were studied and evidence is presented that the major source of progesterone during pregnancy in this species is ovarian. MATERIALS

AND METHODS

Animals and Sampling The capture of T. rugosa, determination of pregnant status, radio-tracking procedures, and the study site on the Harry Waring Marsupial Reserve have been described in detail previously (Fergusson and Algar, 1986). After pregnancy had been determined, radiotransmitters were attached and the animals were released at the site of capture the next day. In all, six females were monitored in the field until the advent of parturition. The animals were caught and released in January or early February. An initial blood sample of approximately 2 ml was obtained by cardiac puncture using an heparinized syringe with a 22-gauge needle. The blood was centrifuged immediately and 1 ml plasma, for measurement of AVT concentration, was transferred to a S-ml plastic vial containing 0.1 ml 1% acetic acid (HAc) and stored at - 20”. The remaining plasma, about 0.5 ml, was stored at - 20” until progesterone concentrations were measured. Subsequent blood samples were obtained fortnightly in February and weekly in March until parturition when an immediate postpartum sample was obtained (deemed as Day 0). As the time of parturition approached, the animals were checked more frequently and the day 0 sample could have been no more than 2-days postpartum in any animal.

142

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HAc. The eluate was evaporated to dryness under a stream of air at 37”, reconstituted in 0.5 ml 0.01 M The measurement of circulating levels of plasma phosphate buffer (pH 7.5), and stored overnight at 4” AVT in T. rugosa was achieved using the assay origfor assay the next day. Recovery of 1251-AVT from T. inally developed by Rice (1982) for application to rugosa plasma was consistently around 85%. Varanus gouldii. In applying the assay to T. rugosa Separation of free and bound fractions. The double and the consequent optimization procedures, several antibody technique (Morgan and Lazarow, 1963) was modifications were employed. These were primarily in used to separate free and bound fractions in the incuthe areas of iodination of AVT, extraction of AVT bation mixture. In early assays, the secondary comfrom plasma, and the separation of free and bound plex often failed to precipitate, perhaps due to the fractions. The method, including modifications, is de- “prozone” phenomenon (Chard, 1982). Therefore, in scribed below. order to determine optimal conditions for precipitaIodination. Synthetic 8-arginine vasotocin (AVT) tion, varying concentrations of the second antibody (Ferring: 260 IUimg) was iodinated with carrier-free (donkey anti-rabbit, Wellcome) were mixed with vary‘251-labeled Na (Amersham) using a modification of ing concentrations of the carrier protein (rabbit the chloramine-T oxidation method of Hunter and y-globulin) and incubated with ‘*‘I-AVT following priGreenwood (1962). The reaction mixture consisted of mary incubation with antibody R: 19 (l/5000 dilution). 18.5 MBq ‘25I (5 JLI),2 p,g AVT in 20 )LI 0.2 M HAc, 2.5 The precipitation profiles revealed that maximal prePg chloramine-T in 5 pl distilled water, and 20 ~10.5 M cipitation could be achieved with as little as l/97,222 phosphate buffer, pH 7.5. The concentrations of lz51, dilution of rabbit y-globulin and l/336 dilution of donAVT, and chloramine-T used were suggested by Rice key anti-rabbit serum. These concentrations save con(personal communication). The reaction vial was vorsiderable expense in the use of the Wellcome antibody texed for 3 and 15 set after the addition of chloramineand nonspecific binding was consistently reduced to T, and the reaction was terminated by the addition of less than 2%, thereby enhancing statistical analysis of 100 ~125% bovine serum albumin (BSA). The reaction the results. mixture was transferred to a glass test tube containing Assay standards. Synthetic AVT (Ferring: 260 IUI 150 mg Dowex 2X8-50 and 1 ml distilled water. These mg) dissolved in 0.2 M HAc and 0.1% BSA was used were mixed for 10 min to bind unreacted free iodine. for the standards in the assay. This was stored as OSThe supernatant fluid was layered onto a 30 x 0.9-cm ml ahquots at -20” at a concentration of 640 pg/ml Sephadex G-25(Fine) column equilibrated with 0.2 M which facilitated easy dilution to the eight standards HAc and 0.1% BSAand prewashed with 0.5 ml normal used in the standard curve: 32, 16, 8, 4, 2, 1, 0.5, and rabbit serum. The column was eluted with 0.2 M HAc, 0.25 pg/tube. A separate batch of AVT purchased from and 0.1% BSA and 0.5 ml fractions were collected. Ferring and prepared in the same manner produced Aliquots (10 ~1) of each fraction were counted in a identical standard curves. Fresh standards were prePackard Prias autogamma counter and rechromatopared for each assay. Assay procedure. Triplicate 50-pl ahquots of the apgraphed on a second 30 x 0.9~cm Sephadex GZS(Fine) column and eluted with 0.2 M HAc and 0.1% BSA. propriate standard and duplicate 200~pl aliquots of The peak and two postpeak fractions were pooled and plasma extracts were incubated at 4” in 10 X 75-mm plastic test tubes (3DT, Disposable Products) with 50 stored at -20” as loo-p1 aliquots. Undamaged t.~l of R:19 antibody (at a final dilution of l/600,000, monoiodinated AVT was identified by its ability to which bound approximately 40% of the label in the 0 bind to an excess of a rabbit antibody raised against standard), 10 ~1 of 0.07% rabbit y-globulin, and 0.01 M 8-arginine vasopressin (Ferring AB, R: 19, l/5000 diluphosphate buffer (pH 7.5) to a final volume of 450 ~1. tion) and retained immunoreactivity for 6-10 weeks. Extracfion. Plasma extracts were prepared by highFifty microliters (ca. 2000 cpm) of iZ51-AVT was added 24 hr later, mixed for 3 set on a vortex mixer, and pressure liquid chromatography on octadecasilylincubated at 4 ’ for a further 72 hr. Then 200~pl donkey silica microcartridges (Sep Pak C18; Waters Associanti-rabbit serum (l/96 dilution) was added, and the ates Inc., Milford, MA.) (La Rochelle et a/., 1980). All solutions were pushed through the cartridges with dis- tubes were vortexed, incubated at 4” for a further 18 posable syringes. The cartridge was prewet with 5 ml hr, and then centrifuged at 2000g for 45 min at 4”. The ethanol followed by a wash with 10 ml distilled water. supematant was aspirated and the precipitate counted for 30 min or to 1% error in a Packard Prias autogaIf a cartridge was being reused it was flushed between mma counter. The standard curve was logit-log transextractions with 5 ml 8 M urea followed by 10 ml disformed (Rodbard and Lewald, 1970) and the sample tilled water prior to the methanol wash. Thawed values were calculated from the linear regression plasma (1 ml) was acidified with 100 p1 1 M HCl, transferred to a l-ml syringe, and pushed slowly, over a equation. Assay characteristics. A standard curve for AVT is period of 1 min, through the cartridge. Following a shown in Fig. 1. The sensitivity of the assay, assessed rinse with 20 ml 4% HAc, the peptides were eluted with two 3-ml vol of 75% aqueous acetonitrile and 4% as the smallest amount of AVT that could be signifi-

Radioimmunoassay

for

Plasma

AVT

AVT

AND

PROGESTERONE

IN

A VIVIPAROUS

LIZARD

143

the limited supply of plasma from pregnant animals it was not possible to validate the assay for this species. The assay was validated using plasma from female quokkas (Setonx bruchyurus) which have plasma progesterone concentrations roughly equivalent to those found in pregnant T. rugosa (Cake et al., 1980). The sensitivity of the assay, again assessed as the smallest amount of progesterone that could be significantly distinguished from zero with 95% confidence limits (Frankel et al., 1967), was calculated from five replicates of pooled plasma in five different assays and averaged 11.4 pg . ml-‘. Using these replicates. the intraassay coefficient of variation averaged 9.6%, the interassay coefficient of variation was 15.7%.

Corpus Luteum

FIG. 1. AVT standard curve, mesotocin crossreaction curve, and plasma dilution curve for the AVT assay using the R: 19 antibody.

cantly distinguished from zero with 95% confidence limits (Frankel et al., 1967), was calculated from four replicates for plasma in four assays and was 2.3 pg . ml-‘. Intra- and interassay coefficients of variation calculated from these replicates were 9.6 and 13.2%, respectively. Dilutions of 7’. rugosa plasma incubated in one assay showed apparent parallelism with the standard curve (Fig. 1). Standard concentrations of synthetic mesotocin (Bachem Fine Chemicals) incubated in the assay system showed no significant crossreaction with antibody R:19 in the presence of ‘*‘IAVT (Fig. 1).

Morphology

Throughout the study, the sets of ovaries containing a corpus luteum were surgically excised from 17 pregnant females which were not used to monitor hormone levels. Ovaries were also obtained from three postpartum females (April-May). The longest and shortest diameters of each corpus luteum were measured using vernier calipers and an average diameter was calculated. The ovaries were fixed in standard Bouin’s fluid (Humason, 1972), embedded in wax, serially sectioned at IO pm, and stained using either Harris’ haemotoxylin and eosin or Mallory’s triple connective tissue stain (Humason, 1972). For each corpus luteum, a section through the center was obtained and the width of the thecal layer was calculated by averaging five measurements which were placed equidistantly around the corpus luteum. Measurements were obtained with an eyepiece grid that was calibrated with a stage-piece micrometer. The diameter of the luteal cell mass was calculated by subtracting twice the width of the thecal layer from the corpus luteum diameter. Estimates of the relative density of the luteal cells within the luteal cell mass were gained using the eyepiece grid.

Radioimmunoassay for Plasma Progesterone

Ovariectomy

Plasma samples (50 or 100 ~1) were assayed following the method of Cake et al. (1980). Briefly, the assay utilizes antiprogesterone-l I-succinyl-BSA serum (antiserum No. Pl l-192, Endocrine Sciences Laboratories, CA). This antiserum has cross-reactivity with 17a-hydroxyprogesterone, 1 I-deoxycorticosterone, corticosterone, 20a-dihydroprogesterone, pregnenolone, and estradiol of 6.3, 5.3, 2.3, 0.6, 0.5, and O.l%, respectively. Progesterone was extracted with 5 ml redistilled n-hexane and separated from other steroids by chromatography on Lipidex-5000 minicolumns (Fairclough et al., 1977). For 7’. rugosa plasma with added [3H]progesterone there was an average of 90% recovery of the Iabeled steroid in the fraction collected. Nonpregnant T. rugosa generally have low levels of circulating plasma progesterone and because of

To test for the effect of ovariectomy on plasma progesterone levels, three groups of animals were established in the laboratory; pregnant-ovariectomized, pregnant sham-operated, and nonpregnant shamoperated females. The animals, four in each group, were collected in December. In January, blood samples were obtained by cardiac puncture prior to ovariectomy or sham-operation. In each operation, each animal was anesthetized with an intracardiac injection of methahexitone sodium (Brietal sodium, Eli Lilly, 1 ml/kg body wt) and a lower paramedian incision, approximately 3 cm long, was cut in the ventral body wall. Both uteri were inspected for the presence or absence of developing embryos. In pregnantovariectomized females, the ovarian blood vessels were tied off with 6-O silk suture and cauterized distal

Experiment

144

FERGUSSON

AND BRADSHAW

to the tie. The ovaries were then excised with fine scissors. The peritoneum was dusted with antibacterial powder (Neotracin) and the incision in the body wall was closed with 4-O silk suture. The wound was sealed with a thin layer of Supa-glue (Selleys). Shamoperated animals underwent the same procedure but the ovarian blood vessels were not tied off or cauterized and the ovaries were not excised. Three to 4 weeks later a further blood sample was obtained from each animal.

Sequential data from the same animals for circulating levels of plasma AVT are also shown in Fig. 2. There is an increase in plasma AVT concentrations 30 days immediately prior to parturition as indicated by a significant fit of the logarithmically converted AVT values to a quadratic regression model (1nAVT = 3.26 - 0.03 day + 0.0003 day’, r = 0.39, P < 0.05). T. rugosa corpora lutea are yellow, plateRESULTS let-shaped structures which form discrete bodies that are easily distinguished from The sequential data from the radiotracked pregnant females are presented, for the smooth, white ovarian lobes. Luteal formation occurs during November, followclarity, as means of IO-day intervals prior ing ovulation. They immediately begin to to parturition and 20 days following partudecrease in diameter and a significant rerition in Fig. 2. There is a highly significant reduction in plasma progesterone concen- duction occurs in December and January (Table 1). Following this, the reduction in tration from 60-50 days prior to parturition diameter is small and not statistically signifto 39-30 days prior to parturition (P < 0.01; icant. The decrease in diameter is due to a Newman-Keuls test; Zar, 1974). None of decrease in both the width of the thecal the mean values following this are signifilayer and the diameter of the luteal cell cantly different from each other at P < 0.05 mass. Significant reduction in the width of per comparison, although a steady decline the thecal layer occurs during the period of in levels occurs. For individual data points November to January (Table 1). Following there is a highly significant correlation beovulation, hypertrophy of the follicular tween plasma progesterone concentration granulosa forms the luteal cell mass. The and days prior to parturition (r = 0.947, P cell boundaries were poorly defined (Fig. < 0.001). 3a), but on average, the cells were 15 km in diameter and this appeared to change little during pregnancy; significant compaction of the luteal cell mass does not occur until after parturition (Table 1). However, the Y cell nuclei, which are initially large and ovoid in shape (Fig. 3a), begin to degenerate in February (Fig. 3b), and continue degenerating (Fig. 3c), until and after parturition when nuclei are exclusively pyknotic (Fig. 3d). Intercellular spaces start forming , / 39-M 29-20 19-10 9-O -l-(-201 60 -50 in February and increase until postparturition (Fig. 3d). Plasma progesterone concentrations in I+& 2. Circulating levels of plasma AVT (broken pregnant-ovariectomized, pregnant shamline) and progesterone (full line) in free-ranging, radiomonitored, pregnant Tiliqua rugosa. Means -C SE are operated, and nonpregnant sham-operated reported for IO-day intervals prior to parturition and females before and after surgery are shown for a 20-day period postpartum (progesterone only). in Fig. 4. Levels were significantly higher in For the progesterone data, values having different supregnant females relative to nonpregnant perscripts are significantly different (Newman-Keuls females prior to surgery. Ovariectomy sigtest, P < 0.01).

AVT AND PROGESTERONE

IN A VIVIPAROUS

TABLE 1 DATA FOR THE CORPORA LUTEA OF Tiliqua

MONTHLY

Month

n

Corpus luteum diameter (mm)

November December January February March April-May (postpartum)

3 4 3 1 6

19.5 ix 1.4” 10.5 2 l.Sb 7.3 2 0.4’ 5.9 6.1 2 0.2’

3

5.8 2 0.03’

LIZARD

145

rugosa

Luteal cell mass diameter (mm)

Luteal cell mass density (per mm*)

Thecal width (t.4

17.1 ‘- 1.5” 9.1 2 1.36 7.1 t O.lb 5.5 5.4 c 0.4b

4.9 f 1.3” 3.6 k 0.2” 4.9 2 0.4” 8.9 6.0 -c 0.7”

1211 -c 64” 695 -t- Ill’ 309 2 19’ 212 321 2 87”

9.7 +- o.4b

193 27 15”

5.4 -+ O.Olb

Note. Means +- SE are reported. Within a column of data, values having different superscripts are significantly different (based on Newman-Keuls multiple range test at P < 0.05 per comparison).

nificantly reduced progesterone levels in pregnant females (paired t test, P < 0.051, whereas sham operations did not affect levels in either nonpregnant or pregnant animals. DISCUSSION

The data presented in this paper show that there is an increase in circulating levels of plasma AVT in pregnant T. rugosa as the time of parturition approaches. This increase is over and above the chronic elevation due to the shortage of water during the summer period (Fergusson, 1986). Concurrent with increasing AVT levels in pregnant females is a reduction in plasma progesterone concentration and degeneration of the corpus luteum. An important aspect of this study is that hormone levels have been monitored in free-ranging lizards in which gestation and parturition have run their course while normal environmental conditions were experienced and handling was minimal. The timing of oviposition is probably largely a genotypic adaptation to climatic and environmental conditions (Shine and Bull, 1979). It is possible, however, that the timing of these events can be unnaturally advanced or delayed in direct response to adverse environmental conditions, such as captivity and excessive handling, leading to stress. An example may be

the oviparous lizard A. carolinensis in which epinephrine and P-adrenoreceptor agonist (isoproteronol) inhibit oviducal contractions, the P-adrenoreceptor antagonist (dichloroisoproteronol) facilitates AVT-induced oviposition which does not always occur (Jones et al., 1983). These results suggest that stress-induced sympathetic nervous system action can act as a short-term inhibitor of oviposition in A. carolinensis. The observation of elevated progesterone levels in pregnant females and a progressive decline in levels in the final 60 days of gestation is in agreement with the tindings of Bourne (1980), although there is a considerable discrepancy in the absolute values of levels measured in the two studies. The reasons for this are not clear but they may be due to differences in the two radioimmunoassays employed. There is also considerable variation in plasma progesterone concentrations reported for a number of different species. The levels reported for T. rugosa in this study are much lower than those observed in the viviparous species L. vivipara (Xavier, 1982) and, to a lesser extent, lower than those reported in S. cyanogenys (Callard et al., 1972), Chamaeleo pumilis (Veith, 1974), and the snakes Natrix sipedon pictiventris (Chan et al., 1973) and T. elegans (Highfill and Mead, 1975). This apparent species differ-

146

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FIG. 3. Photomicrographs of corpora lutea of pregnant Tiliqua rugosa at various stages of pregnancy. (a) Corpus luteum in December, soon after ovulation. The nuclei of the luteal cells are large and round and the cell boundaries are poorly defined. (b) In February the nuclei of the luteal cells are still large and, in most cases, ovoid in shape. However, pyknosis has commenced in some nuclei and intercellular spaces can be observed. (c) Prepartum corpus luteum in March. The nuclei of the luteal cells are predominantly pyknotic and more intercellular spaces can be observed. There is significant compaction of the cells of the thecal layer. (d) Postpartum corpus luteum in April. The nuclei of the luteal cells are exclusively pyknotic and intercellular spaces are prevalent. The thecal cells are highly compacted. For all photomicrographs magnification is 400X and the sections were stained with Mallory’s trichrome. LCM, luteal cell mass; T, theta.

AVT

AND

PROGESTERONE

ence is similar to the situation found in mammals and may be related to levels of progesterone-binding plasma proteins (Xavier, 1982). The decline in plasma progesterone levels in the last 2 months of pregnancy in T. rugosa is similar to the pattern observed in the viviparous snake T. elegans (Highfill and Mead, 1975) in which progesterone

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concentrations reach their highest levels in midterm and decrease in late pregnancy. These changes correlate with degenerative changes of the corpus luteum in T. elegans. In a number of other species, maximal levels of plasma progesterone are observed in late pregnancy. In C. pumilis (Veith, 1974) and S. cyanogenys (Callard et al., 1972), plasma progesterone concentrations fall

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just prior to parturition when the corpora lutea regress. In L. vivipara the late peak corresponds with luteal capacity to produce progesterone in vitro (Xavier, 1982). In S. jurrovi the late peak occurs after regression of the corpus luteum, suggesting that, during late pregnancy at least, the major source of progesterone in this species is nonluteal (Guillette et al., 1981). In T. rugosa the decline in progesterone concentrations in pregnant animals is correlated with degenerative changes in the corpus luteum, suggesting that the major source of progesterone during gestation is luteal. This contention is supported by the data showing that ovariectomy of pregnant females in midterm reduces plasma progesterone concentrations to levels approaching those found in nonpregnant animals (Fig. 4). This study reports the first measurements of plasma AVT concentrations for a viviparous reptile in the period leading up to parturition. AVT levels have been shown to increase at the time of oviposition in sea turtles (Figler et al., 1989). The elevation of plasma AVT levels in pregnant T. rugosa occurs over a much longer time period, during the 30 days leading up to parturition (Fig. 2). This is a similar pattern to that observed in some eutherian species such as the rat in which plasma oxytocin levels rise progressively to high concentrations near term and do not rise further with the onset of parturition (Liggins, 1981). It was widely accepted that oxytocin was unlikely to be involved in the initiation of labor because, in most species, plasma oxytocin concentrations show no acute elevation until late in parturition (Liggins, 1980). However, the discovery of the sudden appearance of oxytocin binding at term in rat uterine myometrium indicated that oxytocic activity without acute elevation of plasma levels of oxytocin could occur (Soloff et al., 1979). In the rat, this increased sensitivity correlates directly with changes in plasma levels of progesterone and estrogen. There is considerable evidence from reptilian species

that myometrial sensitivity to AVT is modulated by steroids or reproductive status. In pregnant females of the lizard Lioluemus gravenhorti the sensitivity of the uterus to oxytocin is greater in late pregnancy than in early pregnancy (Lemus et al., 1970; cited in Jones and Guillette, 1982). Progesterone administration can delay parturition in some viviparous reptiles (Panigel, 1956; Veith, 1974), and pretreatment with ovarian steroids can affect the sensitivity of the oviduct or uterus to AVT-induced contractions in vitro (Callard and Hirsch, 1976; Guillette and Jones, 1980). Recent investigations have shown that progesterone and estrogen administration to T. rugosa affects uterine sensitivity to AVT (Fergusson and Bradshaw, unpublished data). If the with-

I-

P
200

Pre

Post NP

Pre

Post

P-SO

Pre

Post

P-ovx

FIG. 4. Plasma progesterone concentrations in pregnant Tiliqua rugosa before and after ovariectomy (POVX), pregnant females before and after sham operations (P-SO), and nonpregnant females before and after sham operations (NP). Points connected by lines represent a single animal. Open bars indicate the mean value for the plasma progesterone concentration at that condition and are shown for illustrative purposes only since paired t-statistics were utilized to analyze the data. Probability level is indicated above pairs of pre- and postoperative groups where P < 0.05

AVT

AND

IN A VIVIPAROUS

PROGESTERONE

drawal of progesterone observed in T. rugosa is associated with an increase in numbers of uterine AVT receptors then a tonic elevation of plasma AVT levels near the time of parturition may occur in order to ensure sufficient occupation of these receptors at term. ACKNOWLEDGMENTS This work was supported by a research grant from the University of Western Australia. Thanks are due to Dr. G. E. Rice for providing helpful suggestions on the AVT assay methodology and to Felicity Bradshaw for similar assistance with the progesterone assay. We are most grateful to Tom Stewart for his valuable contribution with the histological procedures. Dr. David Algar assisted with the field work. The manuscript benefited from the comments of two anonymous referees. B. Fergusson received financial assistance from the Commonwealth Postgraduate Research Awards Scheme.

REFERENCES Boume, A. R. (1980). Progesterone-like activity in the plasma of the viviparous &ink, Trachydosaurus rugosus (Stump-tailed lizard). In “Proceedings of the Melbourne Herpetological Symposium” (C. B. Banks and A. A. Martin, Eds.), pp. 15-16. Dominion Press, Blackbum, Victoria. Boume, A. R., and Seamark, R. F. (1972). Progestins in the plasma of a viviparous lizard, Tiliqua rugosa. J. Reprod. Fertil. 28, 156-157. Bourne, A. R., Stewart, B. J., and Watson, T. G. (1986). Changes in blood progesterone concentration during pregnancy in the lizard Tiliqua (Truchydosaurus)

rugosa.

Comp.

Biochem.

Physiol.

A

84, 581-583. Browning, H. C. (1973). The evolutionary history of the corpus luteum. Biol. Reprod. 8, 128-157. Cake, M. H., Owen, F. J., and Bradshaw, S. D. (1980). Difference in concentration of progesterone in plasma between pregnant and non-pregnant quokkas (Setonix bruchyurus). .I. Endocrinol. 84, 153-158. Callard, I. P. (1966). Reptilian gonadal steroid synthesis. Excerpta Med. Int. Congr. Ser. 111, 216. Callard, I. P., Doolittle, J., Banks, W. I., and Chan, W. C. (1972). Recent studies on the control of the reptilian ovarian cycle. Gen. Comp. Endocrinol. Suppl. 3, 65-75. Callard, I. P., and Hirsch, M. (1976). The influence of oestradiol-17P and progesterone on the contractility of the oviduct of the turtle, Chrysemys picta, in vitro. J. Endocrinol. 68. 147-152.

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