GENERAL

AND

COMPARATIVE

In Vitro

ENDOCRINOLOGY

rine Contractions ects of Gestation

86, 203-210 (1992)

in the Viviparous Lizar and Steroid Pretreat

B. FERGUSSON~ AND S.D. Department

of Zoology,

University

of Western

Australia,

Perth,

?Vesiern

Australia

6009, Austvalia

Accepted July 19. 1991 Uterine contractility was investigated in the viviparous lizard Tiliqua rugosa. Arginine vasotocin (AVT) induces rhythmic contractions in vitro in strips of uterine tissue from ovariectomized female T. rugosa. The strength of these contractions was related to the dosage of AVT and reduced by pretreatment in viva with both progesterone and estradiol17p. The frequency of spontaneous and AVT-induced contractions was enhanced by estradiol-17P pretreatment. The strength of AVT-induced contractions in pregnant females was not significantly different from that recorded in nonpregnant females. Spontaneous rhythmic contractions were present only in pregnant females. Ovariectomy did not affect either spontaneous or AVT-induced contractions in pregnant females. The data indicate that ovarian steroids modulate uterine contractility in 7’. rugosa. It is suggested that, following a decline in plasma progesterone levels, estrogen may be involved in the onset of parturition. D 1992 Academic

Press, Inc.

The reptilian neurohypophysis secretes two pharmacologically active octapeptides: arginine vasotocin (AVT) and mesotocin (Sawyer and Pang, 1979). Studies with chickens (Saito et al., 1990) suggest that the ovaries may also be a source of these octapeptides. Mammalian neurohypophysial extracts and the mammalian octapeptide oxytocin 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; Guillette, 1979). AVT has since been shown to induce oviducal uterine contractions in vitro in a number of reptilian species (Munsick et ak., 1960; Heller, 1972; Callard and Hirsch, 1976; Guillette and Jones, 1980). AVT is far more potent than mesotocin, oxytocin, or vasopressin in stimulating contractions of the isolated uterus of the viviparous lizard Xantusia riversiana (La ’ Present address: Department of Applied Science, Kalgoorlie College, P.M.B. 22, Kalgoorlie, Western Australia 6430, Australia.

Pointe, 1969, 1977). Plasma levels of AVT increase at the time of oviposition in sea turtles (Figler et al., 1989) and are elevate in the period leading up to parturition in t viviparous lizard Tiliqua rugosa ~~~~~~§~~~ and Bradshaw, 1991). hese data that AVT is the imary ~euro~~pop~~~ia principle involv with oviposition or parturition in reptiles. The ovarian steroids progesterone and estradiol are known to influence the uteri myometrial response to oxytocin in eutherians (Heap et al., 1973). The available for reptiles indicate steroids can influence AVT-induced in vitro uterine contractions in oviparous species. Pretreatment of turtles (Chvysemys picka) with estradiol-17B increases the contractile amplitude and decreases the rest period between contr progesterone reduces the nu tion of contractions (Callard an 1976). In contrast, in An01 progesterone augments AV tractions while estradiol-l7@ inhibits contractions (Guillette and Jones, 1980). Lu203 0016~648Ol92 $1.50 Copyright 0 1992 by Academic Press, Inc. AIi rights of reproducrion in any form reserved.

FERGUSSON

204

AND BRADSHAW

teectomy in the oviparous species Sceloporus undulatus and Cnemidophorus uniparens causes early oviposition (Roth et al., 1973; Cuellar, 1979), suggesting that changing steroid levels during gestation may influence oviductal contractility. However, in the viviparous lizard X. riversiana, pretreatment with progesterone and estrone had no effect on AVT-induced uterine contractions (La Pointe, 1969). Ovariectomy or luteectomy of a number of viviparous snakes and lizards has no effect on gestation and parturition, impairs embryonic development and parturition, or delays parturition (Yaron, 1985). Removal of ovarian tissue therefore does not elicit early parturition in viviparous reptiles. T. rugosa is a large viviparous skink which has one short breeding season during the austral spring (Weekes, 1935) with parturition usually occurring sometime during late March or early April (Fergusson and Algar, 1986), gestation lasting for approximately 150 days. Plasma progesterone concentrations are elevated in pregnant females midterm and decline to basal levels prior to parturition (Bourne et al., 1986; Fergusson and Bradshaw, 1991). Ovariectomy of pregnant females reduces plasma progesterone concentrations to levels found in nonpregnant females (Fergusson and Bradshaw, 1991). This study was designed to determine whether AVT induces in vitro uterine contractions in T. rugosa and if pretreatment in vivo with progesterone and estradiol- 17p influences uterine contractility in a viviparous lizard. The in vitro uterine contractility of pregnant females and the effect of ovariectomy were also examined. MATERIALS

AND METHODS

T. rugosa (average body wt, 475 g) were collected by hand in the vicinity of Perth and maintained in the laboratory in tanks with a 250-W heat lamp activated on a lOL:14D regimen. Water dishes were present in the tanks and the lizards were fed a combination of tinned dog food and fruit.

Experiment

1

This experiment tested the responsiveness of uteri from nonpregnant ovariectomized females following treatment with different ovarian steroid regimes. Fifteen nonpregnant females were collected in October, prior to ovulation, and ovariectomized using methods previously described (Fergusson and Bradshaw, 1991). The animals were then held in a large communal tank for at least 2 weeks, after which a 2-ml blood sample was obtained without anaesthesia by cardiac puncture using an heparinized syringe with a 22-gauge needle. They were then randomly assigned to four pretreatment groups and treated daily for 14 days with one of the following regimens: (1) progesterone (100 kg . kg body wt-I), (2) estradiol-17s (10 ug . kg body wt-I), (3) both progesterone and estradiol-17B at the same dosages, (4) saline. The above pretreatment groups contained four animals, except for the saline group of three animals. The steroid solutions were prepared as fine crystalline suspensions in sterile saline (0.9% NaCl) and the solutions were injected subcutaneously at 1 ml kg body wt- ‘. The relative amounts of circulating progesterone and estradiol-17B and the interaction between them are important influences on uterine contractility (Soloff et al., 1979). Thus, a combined progesteroneiestradiol-17B treatment group was included in the experiment. On the day after the final injection animals were killed by decapitation and further blood was obtained by draining blood from the severed vessels in the neck. Plasma was separated, stored, and assayed for progesterone using the procedures detailed in Fergusson and Bradshaw (1991). A 5-mm-wide and 30-mm-long strip was then immediately dissected from the uterus (in vivo length, transverse orientation) and suspended in a IO-ml saline bath. One end was attached by silk thread to an isometric myographic transducer (Narco BioSystems F-2000). Two commonly-earthed 9-V dry cell batteries (Eveready No. 266) were wired to provide a voltage input to the transducer and output (1 mV . g-i) was recorded on a Mace FBQ 100 chart recorder. Internal calibration on the transducer indicated linear displacement, and this was further confirmed for the range of tensions recorded with a set of measured weights. The saline solution used was a modification of Munsick’s solution, containing 0.5 mM Mg*+ (La Pointe, 1969). Following addition of 60 ml distilled water the osmolality of this solution, measured on a Wescor vapor pressure osmometer (Model SlOOB), was 274 mOsm . kg-‘, which is approximately isosmotic with T. rugosa plasma. The saline was oxygenated by slow-bubbling oxygen, which also assisted in mixing the solution. The organ chamber was immersed in a water bath at 32”. After the uterine strip was placed in the saline bath the tension was set at 0.5g (g is the mean in vitro

UTERINE

CONTRACTIONS

contraction strength) and this was set as the baseline on the chart recorder. The preparation was then allowed to stabilize for up to 30 min. The suspended uterine strip was then exposed to a sequence of increasing doses of AVT (0.5, 1, 2, 4, 8, 16, and 32 ng ml-’ final concentrations). After each response the chamber was rinsed twice with saline and allowed to stabilize again before introducing the subsequent dose. AVT usually induced a tonic increase in tension over which rhythmic contractions were superimposed. A sample trace from Experiment 2 is shown in Fig. 1. Following the final dose of AVT, acetylcholine was introduced into the saline bath at a final concentration of 100 ug . ml-’ to verify the integrity of the preparation. All of the uterine strip preparations responded to the addition of acetylcholine with a strong tonic contraction. Two parameters of contraction were obtained; contraction strength (g) was measured as the maximal peak of the successive rhythmic contractions in response to a given dose of AVT, and contraction frequency was measured as the average number of contractions during a 5-min period in response to a given dose of AVT. A 2 x 2 x 8 (progesterone or no progesterone x estradiol-17B or no estradiol-17B x dose of AVT), mixed, repeated measure design was utilized to analyze the data (Bruning and Kintz, 1977).

Experiment 2 This experiment compared the uterine sensitivity to AVT of pregnant-ovariectomized, pregnant-shamoperated, and nonpregnant-sham-operated females. The animals, four in each group, were collected in December. In January, blood samples were obtained prior to ovariectomy or sham operations. Three to four weeks later uterine strips were prepared for measurement of in vitro contractions as in Experiment 1. After

IN A VIVIPAROUS

205

LIZA

stabilization preparations were exposed to doses of AVT (Ferring, 32 ng . ml-‘, 8.3 mU . ml-‘), oxytocin (Sigma, 10 mU . ml-‘), arginine vasopressin (Sigma, 9.8 mU . ml-‘), and mesotocin (Bachem Fine Cbemicals, 100 ng . ml-‘) in randomized sequences. After each response the preparation was rinsed three times before introducing the next peptide.

Experiment 1

The effectiveness of the steroid tration procedure is shown by plas gesterone concentrations before a treatment. Progesterone levels were elevated by progesterone only admi~~st~at~~~ from 129 k 59 pg . ml - 1 (mean + SEM) to 876 t 131 pg . ml-’ (B = 0.0025, paired f test). Combined e ioiiprogesterone administration elevat plasma pr~gesterQ~e from117t3~4t0843i293pg~mlW’(P= 0.0472). Progesterone levels were not significantly affected by saline treatment or treatment with estradiol-17P al mean in vitro contraction strengt ine strips from the four pretreatm is shown in Fig. 2. A dose-related response to AVT is evident in the control group while each of the steroid pretreatment showed little response . The analysis of varian cant effect of dose 0 ,004) and this response was . .._ -.

‘.

Frc. 1. Isometric in vitro contractions of a uterine strip from a pregnant female T. rncgosa in response to 32 ng arginine vasotocin/mI (8.3 mU . ml-‘).

206

FERGUSSON

L-

l

AND

Control

0

Progesterone

m

Estradiol

q

Prog.

+ Estradiol

3-

+

0 Dose

2

L

AVT

Ing/mll

8

16

32

FIG. 2. Contraction strength (g) of uterine strips from ovariectomized, nonpregnant T. rugosa in response to increasing dosages of AVT. The four groups were treated daily for 14 days with one of the following regimens: (1) progesterone (100 p,g . kg body weight-‘), (2) estradiol-17P (10 p,g. kg body wt-I), (3) both progesterone and estradiol-17P at the same dosages, (4) saline. The steroid solutions were prepared as fine crystalline suspensions in sterile saline (0.9% NaCl) and the solutions were delivered subcutaneously at a volume of 1 ml . kg body wt-r. For all steroid pretreatment groups the symbols represent the mean of a sample of four animals. For the saline pretreatment animals n = 3 (mean 2 SEM).

dependent on pretreatment with both progesterone (F(7,77) = 8.79, P = 0.015) and estradiol-176 (F(7,77) = 14.27, P = 0.005). There was also a significant main effect of pretreatment with both progesterone (F(l,ll) = 9.36, P = 0.034) and estradiol17p (F(l,ll) = 11.52, P = 0.021). An interaction between progesterone and estradioL17P was computed (F(1 ,ll) = 14.22, P = 0.003). The apparent cause of this interaction effect was the preset baseline level of uterine tension which constrained the measurable inhibitory effect of pretreatment with one or both steroids. A threeway interaction was also calculated (F(l,ll) = 10.24, P = 0.01). This was a reflection of the design of the experiment whereby, for example, the dose-response curves of animals receiving progesterone or

BRADSHAW

no progesterone were very different. The different response following progesterone pretreatment was effectively eliminated if animals received estradiol-17P. The mean in vitro contraction frequencies of the uterine preparations are shown in Fig. 3. The analysis of variance showed no significant effect of dosage of AVT on contraction frequency. There was a significant effect of estradiol-17P pretreatment (F(l,ll) = 16.15, P = 0.002). In the groups pretreated with estradiol-17P alone or both progesterone and estradiol-17l3, the freS quency of contraction was enhanced. Experiment

2

Figure 4 shows the initial in vitro contraction strength and the response to AVT in each of the three groups of animals. In this experiment, spontaneous rhythmic contractions, before the addition of neurohypophysial peptide to the bath, were absent in the nonpregnant females but occurred in both the sham-operated and the ovariectomized pregnant females. The mean value for AVT-induced contraction strength was similar in nonpregnant, pregnant shaml

C

10

2

FIG. 3. Contraction frequency (contractions per 5 min) of uterine strips from ovariectomized, nonpregnant T. rugosa in response to increasing dosages of AVT. Steroid pretreatments, figure symbols, and sample sizes are as for Fig. 2.

UTERINE

CONTRACTIONS

31 e-e 0

NP -0

P-SO

Sal Saline

bath

regimen

4. Strength (g) of spontaneous and AVTinduced contractions of uterine strips of T. rugosa. NP, nonpregnant females; P-SO, sham-operated pregnant females: P-OVX, ovariectomized pregnant females. FIG.

operated, and pregnant-ovariectomized animals (P > 0.05; SNK multiple comparison, or t test, nonpregnant vs pregnant). In all animals the rhythmic frequency of both spontaneous and AVT-induced contractions was approximately one per minute. Oxytocin, arginine vasopressin, and mesotocin, at the dosages used, were ineffective in initiating or enhancing contractions in all but two animals. In one shamoperated pregnant female, oxytocin and mesotocin induced stronger contractions, 0.75 and 1.4g, respectively, compared with an AVT-induced response of 1Sg. In another sham-operated pregnant animal arginine vasopressin induced stronger contractions: 1.3g compared with AVT-induced contractions of 1.4g. ISCUSSION

The results of these experiments demonstrate that AVT induces in vitro contractions of the uterus of T. rugosa. Pretreatment with progesterone, estradiol-17p, or both steroids combined reduces the strength of these contractions (Fig. 2). The progesterone treatments resulted in circulating progesterone levels that were within

IN A VIVIPAROUS

LIZARD

207

the range found in midterm pregnant females (Fergusson and contrast, progesterone tradiol- 178 reduces, AV tions in A. carolinensis Jones, 1980). In anoline lizards ovdati a single egg occurs ovary (Jones et al., 19 the ipsilateral corpus luteum which develops following ovulation secretes estradiol17p, which is directly delivered by uteroovarian blood vessels to the oviduct containing the nonmatured egg (Jones et a&,, 1982). This would unilaterally inhibit mature oviposition of the ~ns~~l~e~ Therefore the effect of ovarian steroid AVT-induced contractions can be reconciled with the characteristic repr iVtZ cycle of A. carolinensis. Compara ata for a viviparous species, X. riversiam, indicate that both es&one and progesterone did not influence the response of the uteri to AVT (La Pointe, 1969). The doses given to X. riversiana were, however, effective in increasing the weight of both the oviducts ies. Significant species ferences thus appear to occur Arnold viviparous lizards. In T. rugosa plasma progesterone co centrations are elevated in rnid~re~~a~~~ and decline, at the same time as the corpus luteum degienerates, prior to parturition (Bourne et e7I., 19%; Fergusson and Bradshaw, 19911). In the viviparous snake (Nero& spp.) uterine progesterone receptors reach a maximum concentration earIy in gestation and decrease well ahead of parturition (Kleis-San Francisco an 1986). It is therefore not s~~~si~ gesterone inhibits uterine contrac this could be perceived as a way of preventing premature parturition, similar to t progesteron’e “block” of mysmetrial contractions in rabbits (Heap et al., EW). It would be expected then that T-induce uterine contractions would inhibite during midpregnancy when ~ro~ester~~e levels are highest. The results of Ex

208

FERGUSSON

AND

ment 2 indeed suggest this (Fig. 4). However, the reduction in contraction strength in the pregnant females is not statistically significant at (Y = 0.05. Furthermore, ovariectomy of pregnant females, which reduces plasma progesterone concentrations to levels found in nonpregnant animals (Fergusson and Bradshaw, 1991), has no effect on AVT-induced contractions (Fig. 4). Although it is possible that the effects of progesterone on uterine responsiveness to AVT may persist for several weeks after ovariectomy, it seems that high plasma concentrations of progesterone in gestation in vivo do not simply block uterine contractions. It is perhaps significant that while estradiol-17P reduces the strength of uterine contractions in T. rugosa it also enhances the frequency at which contractions occur (Fig. 3). Estradiol-17B treatment has a similar effect in Chrysemys picta, for which it decreases the duration of the rest period between AVT-induced in vitro oviductal contractions (Callard and Hirsch, 1976). Alterations in resting membrane potential and threshold potential are observed in mammalian uterine muscle cells following estrogen stimulation (Heap et al., 1973). These changes could make it easier to initiate myometrial contractions, which would nonetheless be relatively weak, and might explain the observations in C. picta and T. rugosa. It is also of interest to note that in experiment 2, spontaneous in vitro uterine contractions are found only in pregnant T. rugosa (Fig. 4). In the viviparous lizards Liolaemus gravenhorti and Liolaemus tenuis the proportion of oviducts which spontaneously contract and the amplitude of the contractions increase progressively during gestation and decline only after parturition (Lemus et al., 1970). Specific temporal changes in both relative and absolute plasma levels of progesterone and estrogen regulate the sensitivity of mammalian myometrium to oxytocin (Heap et al., 1973;

BRADSHAW

Soloff et al., 1979; Fuchs and Fuchs, 1980). It is, therefore, not surprising that removal of the corpus luteum in viviparous reptiles delays, rather than advances, the timing of parturition (Yaron, 1985). Removal of the corpus luteum not only withdraws the major source of progesterone, but precludes other luteal secretions, including possibly estradiol-17B, which may initiate parturition. Plasma levels of estradiol-17P have not been reported for viviparous reptiles. Regulation of oviposition and parturition in reptiles is a complex process, involving interaction between ovarian steroids, AVT, and prostaglandins (Guillette, 1987). Recent experiments suggest that adrenal corticosterone may delay parturition in Lacerta vivipara (Dauphin-Villemant et al., 1990). It has been postulated that adrenal steroids block the synthesis of prostaglandins in lizards (Guillette, 1985). Prostaglandins induce luteolysis in the gravid oviparous lizard A. carolinensis (Guillette et al., 1984) and in recently ovulated turtles (Mahmoud et al., 1988). They also influence smooth muscle contractions in the reproductive tract of A. carolinensis (Guillette, 1987), and indomethacin, an inhibitor of prostaglandin synthesis and release, delays parturition in the viviparous lizard Sceloporusjarrovi (Guillette et al., 1991b). AVT induces prostaglandin release by the uterus of S. jarrovi (Guillette et al., 1990), although this may be due to mechanical contractions of the tissue. AVT and prostaglandin F,, have separate mechanisms of action in cultured avian uterine smooth muscle cells (Hertelendy and Molnar, 1991), and the two hormones may act in concert to initiate oviposition in birds and oviparous reptiles. In the loggerhead turtle (Caretta caretta) estradiol-17P probably promotes a uterine environment responsive to stimulation by AVT and prostaglandins (Guillette et al., 1991a). In a number of eutherian species, it is the synthesis of prostaglandin F,, which is stimulated by estrogens (First,

UTERINE

CONTRACTIONS

1980). It is possible that, following a decline in plasma progesterone levels, this also occurs in viviparous reptiles. ACKNOWLEDGMENTS This work was supported by a research grant from the University of Western Australia. Dr. Kate Creed allowed B. Fergusson to use her laboratory at the Veterinary Sciences Department, Murdoch University, for the initial experiments. B. Fergusson received financial assistance from the Commonwealth Postgraduate Research Awards Scheme. 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.

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A VIVIPAROUS

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Sawyer, W. H., and Pang, P. K. T. (1979). Responses of vertebrates to neurohypophysial principles. In “Hormones and Evolution” (E. J. W. Barrington, Ed.) pp. 493-523. Academic Press, New York. Soloff, M. S., Alexandrova, M., and Fernstrom, M. J. (1979). Oxytocin receptors: Triggers for parturition and lactation? Science 204, 1313-1315. Weekes, H. C. (1935). A review of placentation among reptiles, with particular regard to the function and evolution of the placenta. Proc. Zool. Sot. London. 1935, 625-645. Yaron, Z. (1985). Reptilian placentation and gestation: Structure, function and endocrine control. In “Biology of the Reptilia” (C. Gans and F. Billett, Eds.), Vol. 15, pp. 527-603. Wiley, New York.