GENERAL
AND
COMPARATIVE
Exogenous
ENDOCRINOLOGY
Progesterone or lndomethacin Delays Parturition Viviparous Lizard Sceloporus jarrovi
LOUIS J. GUILLETTE, Laboratory
81, 105-l 12 (1991)
of Vertebrate
JR., VINCENT DEMARCO, Reproduction, Department Gainesville, Florida
Accepted January
in the
AND BRENT D. PALMER
of Zoology, 32611
University
of Florida,
26, 1990
Female Sceloporusjarrovi in late pregnancy given an ip injection of progesterone (50 rig/g body wt) or indomethacin (4 pg/g body wt) exhibited no change in the length of pregnancy when compared to saline-treated controls. In contrast, pregnant females given subcutaneus implants (constant release pellets) containing progesterone or indomethacin exhibited significantly delayed parturition when compared with females given control implants. Indomethacin treatment also disrupted the normal birth process when it occurred, as all females recieving this compound exhibited parturition of only a portion (24-32%) of the total clutch. A similar phenomenon occurred in one experiment following implantation of progesterone pellets. Histological examination of the corpora lutea and oviduct indicated no obvious difference in structure among any of the treatment groups. 0 1991 Academic FXYSS. IX.
Gestation length in reptiles is thought to be controlled by the activity of the corpus luteum (for review, see Xavier, 1987). A correlation between luteal activity and gestation length is well established in oviparous squamates whereas it is less obvious in viviparous species (Jones and Guillette, 1982; Guillette, 1987). Surgical deluteinization induces premature oviposition in oviparous lizards (Sceloporus undulatus, Roth et al., 1973; Cnemidophorus uniparens, Cuellar, 1979; Anolis carolinensis, Guillette
and Fox, 1985). Prostaglandin F,,(PGF,,)induced luteolysis does not stimulate premature oviposition within 24 hr of treatment in an oviparous lizard (A. carolinensis, Guillette et al., 1984) or turtle (Chelydra serpentina, Mahmoud et al., 1988) but does in a viviparous lizard (Sceloporus jurrovi, Guillette and Matter, unpublished). In these species, a significant decline in plasma progesterone concentration and distinct morphological changes in luteal tissue occur following prostaglandin-induced luteolysis. Guillette and Fox (1985) observed that oviposition occurred in three of eight females following surgical deluteinization
in A. carolinensis even though plasma progesterone levels remained elevated (presumably due to the release of adrenal progesterone). In the unique monoallochronic reproductive cycle of A. caroiinesis, progesterone appears to remain elevated up to oviposition (Jones et al., 1983). Progesterone pretreatment in A. carolinensis induces a significant increase in the strength of uterine contractions in vitro in response to stimulation by arginine vasotocin (AVT) (Guillette and Jones, 1980). This study was undertaken to examine whether parturition could be delayed in the viviparous lizard S. jurrovi, a species in which progesterone falls markedly at parturition (Guillette et al., 1981), by maintaining an elevated plasma progesterone concentration. Additionally, since available data suggest that exogenous PGF,, causes luteolysis in several reptiles (see above), stimulates uterine contractions and premature parturition in S. jarrovi (Guillette, DeMarco and Palmer, unpublished) and is synthesized by the oviduct of lizards (Guillette et al., 1988, 1990a), we examined the influence of indomethacin on the mainte105
106
GUILLETTE,
DEMARCO.
nance of pregnancy. Indomethacin, a potent blocker of cyclooxygenase activity, inhibits the synthesis and release of prostaglandins (PGs) in the reproductive tract of the lizard S. jarrovi (Guillette et al., 1988, 1990a). Two methods of treatment were tested (chronic versus acute) to determine whether differences in administration influenced the response. MATERIALS
AND METHODS
Animals. Pregnant female 5. jarrovi were collected from the Dragoon and Chiricahua Mountains of southeastern Arizona during April, May, and June of 1986 and 1987. Females were returned to the University of Florida where they were housed on a 14L:lOD photoperiod in large tanks (2000 liter) containing cement blocks for cover and refuge. Heat lamps and plant lights were on during the photophase, and a gradient in temperature existed during the photophase of 20-50”. Prior to treatment, each female was weighed, measured to the nearest millimeter (snout-vent length), and given a unique toe-clip pattern. Females then were ranked on the basis of weight and divided sequentially into treatment groups. Experimenf I. Fifteen females, in the last week of pregnancy, were partitioned into three equal groups and treated with (1) progesterone (50 nglg body wt), (2) indomethacin (4 pg/g body wt), or (3) lizard Ringer’s solution (0.05 ml) (Guillette 1982). Treatments were given as ip injections twice daily: the first injection was given 2 hr after the onset of the photophase and the second 2 hr prior to the onset of the scotophase. Animals were housed individually in plastic boxes (15 x 30 x 10 cm) in an incubator set for a photoperiod and temperature regime of 14L/32”: lOD/24”. Animals were fed every other day with crickets and misted with water daily. Treatment continued for 16 days unless parturition occurred. If parturition occurred, the date of birth, quantity, and condition of the neonates were recorded. All females remaining pregnant on Day 16 were killed: the number and condition of in urero young were recorded. Experiments 2 and 3. Females were divided into three treatment groups as above. The differences between Experiments 2 and 3 consisted of (1) the date of capture, (2) location of capture, (3) time in captivity before the onset of treatment, and (4) date of initiation of treatment. Experiment 2 began on 6 June 1987. after animals obtained from the Dragoon Mountains had been housed in the laboratory for 4 weeks, whereas experiment 3 began on 26 June 1987, 4 days after females were captured in the Chiricahua Mountains. Treatments were as stated in Experiment 1, except that the administration of chemicals was by time-
AND
PALMER
release pellets (Innovative Research of America, Toledo, OH). Pellets were designed to provide a constant release over a 24-hr period for 21 days for a 10-g mammal. Progesterone pellets were designed to release enough hormone to elevate plasma progesterone to at least 5 rig/ml (similar to maximal levels measured during midpregnancy in this species by radioimmunoassay (Guillette ef al., 1981)). Indomethacin pellets were designed to release 40 p,g over a 24-hr period. Control pellets contained only the carrier, cholesterol, and binder material (methylcellulose; o-lactose). The pellet was implanted under the skin above the left scapula with a trochar. The wound caused by implantation of the pellet was swabbed with ethyl alcohol and covered with New-skin Liquid Bandage (Medtech Lab, Inc., Cody, WY). Implanted pellets were recovered after treatment was terminated to ensure that they had remained intact, thus releasing the contents as designed. Following implantation, females were housed in groups of three in aquaria (75 liters) provided with heat and broad spectrum lights (14L:lOD). A temperature gradient existed in the tank during the photophase of 28-45”. Females were observed twice daily to determine if parturition had occurred. If young were found in the tank, each female in that tank was examined visually. In most cases, the postpartum female was obvious owing to its reduction in girth and its depressed sides. If it was not visually apparent which female had given birth, the females in the tank were weighed to determine the postpartum female. The number and condition of young delivered by each female were recorded. Females were killed by cervical dislocation as soon as parturition was observed (even if it was still underway) and blood was collected. Plasma samples were obtained and frozen until assayed by radioimmunoassay for progesterone. Females that had not exhibited birth were killed on Day 21 and their ovaries and oviducts were removed. Embryos remaining in ulero were staged using the embryo staging sequence of Dufaure and Hubert (1961). Oviducts and ovaries were fixed in Bouin’s fixative. Following 24-hr fixation, tissues were rinsed in ethyl alcohol, dehydrated, and embedded in paraffin. Tissues were serially sectioned at 8 km, stained with alcian blue, and counterstained with hematoxylin and eosin (see Humason. 1979. for standard histological procedures). Sectioned tissues were examined using light and differential interference contrast (DIG) microscopy. Specifically, luteal structure and diameter were compared among the treatment groups as was the structure of the oviduct. Radioimmunoassays. Progesterone concentration in the plasma of females in Experiments 2 and 3 was analyzed in duplicate using an RIA kit (Farmos Diagnostica). Methods provided with the kit were used except the plasma volume was reduced from 100 to 25 ~1 and, thus, volumes of other reagents were reduced accordingly. Mean recovery of “‘I-progesterone from
PARTURITION
CONTROL
spiked lizard plasma was 98.4% (n = 5). Parallelism to the standard curve was exhibited by lizard samples diluted up to one-ftith with stripped, progesterone-free female plasma. Using triplicate samples at binding levels of 25.50, and 80% of maximum binding, inter- and intraassay coefficients of variation (CV) were determined. The mean intrassay CV for this assay was 6.5%, whereas interassay CV was 11.3% (5 assays). The minimum detectable progesterone concentration was 200 pg/ml. Statistics. Mean (*SE) number of days following the onset of treatment until parturition was calculated for each treatment group in each experiment. If a female had not exhibited parturition by the end of the experiment, she was given a score equal to the maximum number of days (Experiment I, 16 days; Experiments 2 and 3, 21 days) of observation. Differences among groups were statistically analyzed using a oneway ANOVA followed by Duncan’s new multiple range tests (Bruning and Kintz, 1977). Plasma progesterone values were log-transformed to obtain homogeneity of variance, and significance was tested using ANOVA and Duncan’s new multiple range tests (P < 0.05 per comparison).
RESULTS Experiment 1. Females given injections of progesterone or indomethacin exhibited
IN
107
LIZARD
no difference (F(2,ll) = 2.22, P > 0.05) in mean latency to birth when compared to control females. However, although no statistical difference was noted in latency to birth, three of four females remaining alive in the indomethacin group at the end of treatment had not given birth and, thus, a significant difference (F(2,ll) = 16.8, P < 0.05) in number of young born was noted (Table 1). The fifth indomethacin-treated female died during the experiment but was not representative of the health of other females. Females treated with either saline or progesterone gave birth to healthy, active neonates, whereas indomethacin-treated females which retained their young had fully formed (Stage 40), dehydrated, dead young in utero. Experiments 2 and 3. Females treated with progesterone or indomethacin implants exhibited a response different than that of those given injections of these chemicals. Females given implants containing progesterone or indomethacin retained
TABLE 1 CLUTCH SIZE AND BIRTH DATAFORSALINE,PROGESTERONE,ANDINDOMETHACIN-TREATEDFEMALE Sceloporus
Treatment Experiment I Saline Indomethacin Progesterone Experiment 2 Saline Indomethacin Progesterone Experiment 3 Saline Indomethacin Progesterone
jarrovi
iv
Female weight” (g)
Total clutch“,’
Number born”
Number of in utero embryos (alive/dead)
5 4 5
15.3 2 2.3d 15.1 2 2.Sd 16.8 k 2.2d
6.6 f 0.7d 6.3 ‘- 0.5d 7.0 2 0.4d
6.6 i 0.7‘+ 1.5 f 1.2 5.8 k 0.4d
11/8 5/l
5 5 6
12.2 * 1.5d 12.6 k l.5d 10.8 2 l.Od
3.6 f 0.4d 4.4 + 0.9 3.3 * 0.2d
3.4 2 0.5d 1.4 f 0.5’ 0.3 t 02
l/O 3/10 10/8
5 5 5
19.0 * 1.4d 17.4 f 1.6d 20.0 k 2.0d
9.4 2 1.5d 8.2 + 0.7d 8.0 k 0.6d
9.0 k 1.2d 2.6 2 0.9’ 5.4 + 1.6d
2/o 2126 2110
010
a Mean f 1 SE. b Total clutch represents total of young born and remaining in utero young. Differences in total clutch size between Experiments 2 and 3 are due to differences in size of the females. c This represents the total number of embryos remaining in utero in all females after 21 days of treatment and their condition (alive or dead). d.ef Values within a column of data for each experiment and having different superscripts are significantly different (Duncan’s multiple range test; P < 0.05 per comparison).
108
GUILLETTE,
DEMARCO,
their young for a significantly longer time (Experiment 2, F(2,15) = 7.11, P < .Ol; Experiment 3, F(2,13) = 8.87, P < 0.005) than control females (Figs. IA, 1B). Plasma progesterone concentration was measured in all females at the time of birth (Fig. 2). We observed that the pellets released progesterone as designed (proposed = 5 rig/ml; observed = 4.8 + 0.7 rig/ml), providing plasma concentrations similar to peak levels reported for this species during midpregnancy (Guillette et al., 1981). Indomethacin-treated females did not have plasma progesterone concentrations significantly different from those in control females (Fig. 2). All females which retained young in the progesteroneand indomethacin-treated groups had some dead, fully formed (Stage 40) in utero young (Table 1). Although many of the experimentally treated females exhibited parturition, they did not give birth to complete litters, whereas most control females did (Table 1). If parturition occurred in progesterone- or indomethacintreated animals, they gave birth to both normal and stillborn young. Histological examination of the corpora lutea revealed no obvious differences
25
among the treatment groups at the light microscopy level. That is, corpora lutea exhibited structural characteristics similar to those reported for this species undergoing natural luteolysis (Guillette et al., 1981). That is, many luteal cells had pycnotic nuclei and early fibroblast invasion of the luteal cell mass was noted. No significant difference in luteal diameter was observed (control = 775 + 25 pm; indomethacin = 795 * 23 pm; progesterone = 815 + 30 km). Likewise, no differences in oviductal structure were noted when the progesterone- or indomethacin-treated females that gave birth were compared to the controls that gave birth. However, one indomethatin-treated female which retained young exhibited tissue necrosis, with ruptured oviducts and abdominally located dead embryos. DISCUSSION
Previous studies suggest that luteal function is associated with the maintenance of gestation in oviparous and viviparous lizards (for review, see Xavier, 1987). The corpus luteum (CL) presumably maintains pregnancy in many reptiles by synthesizing 20
lmdant
20
I5
co 15 2 IO
F 2 IO 5
5 0 09
AND PALMER
0
control itldmmhchl plug&mm CATEQORIES
Go
control -
pogeetaone
CATEQORKZS
FIG. 1. Mean (*SE) number of days pregnancy continued following surgical implanting of hormone pellets in Sceloporus jarrovi. (A) Females housed in laboratory for 4 weeks, (B) females housed in lab for 4 days. Treatments having different superscripts are significantly different (Duncan’s new multiple range test, P < 0.05 per comparison).
PARTURITION
CONTROL
51 4-
3-
2-
a r$$>;gg x:::*:.:::::.::: #$g$. .E’
I-
control
lif-l
hdahch
m
TREATMENT
FIG. 2. Mean (-t-SE) plasma progesterone concentration from female Sceloporus jarrovi implanted with control, indomethacin, and progesterone pellets. As no differences were noted, values from females in both experiments (2 and 3) were consolidated. Treatments having different superscripts are significantly different (Duncan’s new multiple range test, P < 0.05 per comparison).
and releasing progesterone. Progesterone supports oviductal and uterine secretory activity and inhibits uterine contractions in some mammals either directly or indirectly by inhibiting the synthesis and/or release of uterotonic factors such as prostaglandins (Poyser, 1981; Challis and Olson, 1988). In reptiles, exogenous progesterone can maintain pregnancy following deluteinization or ovariectomy in two viviparous species (Bradypodion pumilus, Veith, 1974; Sceioporus cyanogenys, Callard et al., 1972). Our data demonstrate that elevated plasma progesterone can inhibit normal parturition and cause the retention of the in utero young. Prostaglandins exhibit several major functions during parturition or oviposition in vertebrates, including (1) luteolysis, (2) cervical dilatation, and (3) myometrial activity (for discussion see Poyser, 1981;
IN
LIZARD
109
Challis and Olson, 1988). The functions of PGs in reptilian reproduction are poorly understood, but PGs are synthesized by the reproductive tract of reptiles (Guillette et al., 1988; 199Oa) and elevated plasma levels of PGF and PGE, are observed during natural oviposition in several reptiles (Sphenodon puncratus, Guillette et al., 199Ob; Caretta caretta, Guillette et al., 1989). Prostaglandin F,, is luteolytic in reptiles, causing a significant decline in plasma progesterone (Guillette et al., 1984; Mahmoud et al., 1988). As with progesterone, chronic treatment with indomethacin maintains and prolongs normal pregnancy. Our results are similar to those reported for birds and mammals, in which treatment with indomethacin blocks or delays oviposition (Hertelendy, 1973; Day and Nalbandov, 1977; Hertelendy and Biellier, 1978; Shimada and Asai, 1979) or parturition (Aiken, 1972; Chester et al., 1972; Challis et al., 1975). Of interest is the observation of Aiken (1972) that aspirin or indomethacin administration during late pregnancy in rats not only extends parturition but also is correlated with an increase in the number of stillbirths. These stillbirths were associated with death of the pups during birth (premature separation of the placenta) not prior to birth. He also noted that the drug-treated groups gave birth to significantly fewer pups (Aiken, 1972). These data are similar to our observations showing that indomethacin-treated females did not exhibit complete birth. However, indomethacin- and progesterone-treated female S. jarrovi had both live and dead fully formed in utero young suggesting that our observations of stillborn young may be due to causes different than those reported by Aiken (1972). One major difference may account for the contrast in the data sets for stillbirths. Aiken (1972) and others (see above) have observed that in mammals, parturition can only be delayed for a few hours to a few days past the normal period. In contrast, our data suggest that the in-
110
GUILLETTE,
DEMARCO,
domethacin-treated reptilian females can retain young an average of 14 or 9 days longer than the control females. This extensive period of retention may account for the large number of in utero deaths, due primarily to asphyxia. That is, this species has a well-developed chorioallantoic placenta (Guillette et al., 1981) which presumably provides for the gas exchange needed by the in utero young. Separation of the placenta from the uterine wall or gas exchange needs greater than the capacity of the placenta (due to large, fully developed young) would explain these deaths. We observed that neither exogenous indomethacin nor progesterone maintained the histological appearance of the corpora lutea. This was unexpected, as inhibition of PG synthesis by indomethacin or progesterone was hypothesized to delay parturition by maintaining the CL (blocking luteolysis). The CL of this species exhibits a significant decline in diameter during midpregnancy, although plasma progesterone concentration does not decrease (Guillette et al., 1981). The CL of all control and experimental females exhibited a luteal diameter similar to that reported earlier for females of this species that had undergone normal luteolysis (Guillette et al., 1981). The mechanism by which elevated plasma progesterone or indomethacin impairs normal parturition is unknown in reptiles. The role of prostaglandins as uterotonic agents in birds and mammals has been well documented (see Hertelendy et al., 1984, and Challis and Olson, 1988, for reviews). The release of PGs from the mammalian uterus or ovary at the time of birth is thought to be controlled by another uterotonic agent, oxytocin (Chan, 1983). In reptiles, the related neuropeptide, arginine vasotocin (AVT), is capable of inducing strong uterine muscle contractions and premature parturition or oviposition (see Guillette, 1987) as it does in birds (Tanaka and Nakajo, 1962; Rzasa and Ewy, 1970). The stimulatory features of AVT on the avian
AND
PALMER
oviduct are mediated by PGs (Hertelendy, 1973; Rzasa, 1984). Recent data show that a similar phenomenon of AVT-induced PGF,, synthesis exists in the reptilian reproductive tract (Guillette et al., 1990a). The mechanism controlling the release of AVT at the time of oviposition or parturition in reptiles is still unknown. In mammals, progesterone from the CL maintains uterine hypertrophy and inhibits the release of uterine and ovarian PGs (Poyser, 1981; Challis and Olson, 1988). As plasma progesterone levels decline at the end of pregnancy, the oviduct and ovary produce PGs stimulating luteolysis in some species (Rothchild, 1981). Luteolysis creates an environment whereby progesterone synthesis is further reduced and uterotonic PG synthesis is stimulated. Raising uterine levels of PG induces oviductal contractions which helps promote parturition (Poyser, 1981). Our data do not address this mechanism or other possible scenarios but they do suggest that progesterone concentrations in the plasma must decline or at least exhibit episodic decreases for normal parturition to occur. Furthermore, PGs appear to have an active role during parturition, as a reduction in their synthesis impairs or inhibits birth in S. jarrovi. Future studies must examine the interactions of steroids, AVT, and PGs during natural and induced oviposition or parturition if we are to understand the mechanism controlling gestation length in reptiles. ACKNOWLEDGMENTS We thank J. Matter, A. Cree, and A. Hensley for helpful comments concerning this manuscript, and S. Shatie of Innovative Research of America for help in pellet formulation. Animals were collected under permit from the Arizona Fish and Game Commission. This research was funded by NSF Grant DCB-8416707 awarded to L.J.G.
REFERENCES Aiken, J. W. (1972). Aspirin and indomethacin prolong parturition in rats: Evidence that prostaglan-
PARTURITION
CONTROL
dins contribute to expulsion of foetus. Nature (London) 240, 21-25. Bruning, J. L., and Kintz, B. L. (1977). “Computational Handbook of Statistics.” Scott, Foresman, Glenview, IL. Callard, I. P., Chan, S. W. C., and Potts, M. A. (1972). The control of the reptilian gonad. Amer. Zool.
12, 273-287.
64, 363-370.
Challis, J. R. G., and Olson, D. M. (1988). Parturition. In The Physiology of Reproduction” (E. Knobil and J. Neill. Eds.), pp. 2177-2216. Raven Press, New York. Chan, W. Y. (1983). Uterine and placental prostaglandins and their modulation of oxytocin sensitivity and contractility in the parturient uterus. Biol. Reprod.
29, 680-688.
Chester, R., Dukes, M., Slater, S. R., and Walpole, A. I. (1972). Delay of parturition in the rat by antiinflammatory agents which inhibit the biosynthesis of prostaglandins. Nature (London) 240, 3738. Cuellar, H. S. (1979). Disruption of gestation and egg shelling in deluteinized oviparous whiptail lizards Cnemidophorus uniparens (Reptilia: Teiidae). Gen. Comp. Endocrinol. 39, 150-157. Day, S. L., and Nalbandov, A. V. (1977). Presence of prostaglandin F (PGF) in hen follicles and its physiological role in ovulation and oviposition. Biol. Reprod. 16, 48w94. Dufaure, J. P., and Hubert, J. (1961). Table de developpement du lezard vivipare, Lacerta viviparu Jacquin. Arch. Anat. Microsc. Morphol. Exp. 50, 309-327. Guillette, L. J., Jr. (1982). A physiological (Ringer’s) solution for anoline lizards. Herpetol. Rev. 13, 37-38. Guillette, L. J., Jr. (1987). The evolution of viviparity in fishes, amphibians and reptiles: An endocrine perspective. In “Hormones and Reproduction in Fishes, Amphibians and Reptiles” (D. 0. Norris and R. E. Jones, Eds.), pp. 523-562. Plenum, New York. Guillette, L. J., Jr., Bjomdal, K., Bolten, A., and Gross, T. S. (1989). Plasma prostaglandin and steroid concentrations during natural nesting of the loggerhead sea turtle (Caretta caretta). Biol. Reprod. 4O(Suppl. l), 157. Guillette, L. J., Jr., and Fox, S. L. (1985). Effect of deluteinization on plasma progsterone concentration and gestation in the lizard, Anolis carolinensis. Comp.
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303-306.
Guillette, L. J., Jr., Gross, T. S., Matter, J. M., and Palmer, B. D. (1990a). Arginine vasotocin-
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induced prostaglandin synthesis in vitro by the reproductive tract of the viviparous lizard Sceloporus jarrovi. Prostaglandins 31, 39-5 1. Guillette, L. J., Jr., Cree, A., and Gross, T. S. (1990b). Endocrinology of oviposition in the tuatara (Sphenodon punctatus). I. Plasma steroids and prostaglandins during natural nesting. Biol. Reprod.
Challis, J. R. G., Davies, I. J., and Ryan, K. J. (1975). The effects of dexamethasone and indomethacin on the outcome of pregnancy in the rabbit. J. Endocrinol.
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Guillette, L. J., Jr., and Jones, R. E. (1980). Arginine vasotocin-induced in vitro oviductal contractions in Anolis carolinensis: Effects of steroid hormone pretreatment in vivo. J. Exp. Zoo/. 212, 147-152. Guillette, L. J., Jr., Herman, C., and Dickey, D. (1988). Synthesis of prostaglandins by tissues of the viviparous lizard, Sceloporus jarrovi. J. Herpetal. 22, 180-185. Guillette, L. J., Jr., Lavia, L. A., Walker, N., and Roberts, D. (1984). Luteolysis induced by prostaglandin F,, in the lizard Anolis carolinensis. Cert. Camp.
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56, 271-277.
Guillette, L. J., Jr., Spielvogel, S., and Moore, F. L. (1981). Luteal development, placentation, and plasma progesterone concentration in the viviparous lizard Sceloporus jarrovi. Cert. Camp. Endocrinol.
43, 20-29.
Hertelendy, F. (1973). Block of oxytocin-induced parturition and oviposition by prostaglandin inhibitors. Life Sci. 13, 1581-1589. Hertelendy, F., and Biellier, H. V. (1978). Evidence for a physiological role of prostaglandins in oviposition by the hen. .l. Reprod. Fertil. 58, 71-74. Hertelendy, F., Olson, D. M., Todd, H., Hammond, R. W., Toth, M., and Asboth, G. (1984). Role of prostaglandins in oviposition and ovulation. In “Reproductive Biology of Poultry” (F. J. Cunningham, P. E. Lake, and D. Hewitt, Eds.), pp. 89-102. Longmans Green, New York. Humason, G. L. (1979). “Animal Tissue Techniques,” 4th ed. Freeman, San Francisco. Jones, R. E., and Guillette, L. J., Jr. (1982). Hormonal control of oviposition and parturition in lizards. Herpetologica 38, 80-93. Jones, R. E., Guillette, L. J., Jr., Summers, C. H., Tokarz, R. R., and Crews, D. (1983). The relationship among ovarian condition, steroid hormones, and estrous behavior in Anolis carolinensis. J. Exp. Zool. 221, 145-154. Mahmoud, I. Y., Cyrus, R. V., McAsey, M. E., Cady, D., and Woller, M. J. (1988). The role of arginine vasotocin and prostaglandin F,, on oviposition and luteolysis in the common snapping turtle Chelydra serpentina. Gen. Camp. Endocrinol.
69, 56-64.
Poyser, N. L. (1981). “Prostaglandins in Reproduction.” Research Studies Press, Chichester.
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Roth, J. J., Jones, R. E., and Gerrard, A. M. (1973). Corpora lutea and oviposition in the lizard Scelopot-us
undulatus.
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21,
569-572. Rothchild, I. (1981). The regulation of the mammalian corpus luteum. Rec. Prog. Horm. Res. 37, 183-298. Rzasa, J. (1984). The effect of arginine vasotocin on prostaglandin production of the hen uterus. Gen. Comp.
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Rzasa, J., and Ewy, Z. (1970). Effect of vasotocin and oxytocin on oviposition in the hen. J. Reprod. Fertil.
21, 549-550.
Shimada, K., and Asai, I. (1979). Effects of prosta-
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glandin F,, and indomethacin on uterine contraction in hens. Biol. Reprod. 21, 523-527. Tanaka, K., and Nakajo, S. (1962). Participation of neurohypophyseal hormones in oviposition in the hen. Endocrinology 70, 453-458. Veith, W. J. (1974). Reproductive biology of Chameleo pumilis pumilis with special reference to the role of the corpus luteum and progesterone. Zool. A$-. 9, 161-183. Xavier, F. (1987). Functional morphology and regulation of the corpus luteum. In “Hormones and Reproduction in Fishes, Amphibians, and Reptiles” (D. 0. Norris, and R. E. Jones, Eds.), pp. 241282. Plenum, New York.
AND
COMPARATIVE
Exogenous
ENDOCRINOLOGY
Progesterone or lndomethacin Delays Parturition Viviparous Lizard Sceloporus jarrovi
LOUIS J. GUILLETTE, Laboratory
81, 105-l 12 (1991)
of Vertebrate
JR., VINCENT DEMARCO, Reproduction, Department Gainesville, Florida
Accepted January
in the
AND BRENT D. PALMER
of Zoology, 32611
University
of Florida,
26, 1990
Female Sceloporusjarrovi in late pregnancy given an ip injection of progesterone (50 rig/g body wt) or indomethacin (4 pg/g body wt) exhibited no change in the length of pregnancy when compared to saline-treated controls. In contrast, pregnant females given subcutaneus implants (constant release pellets) containing progesterone or indomethacin exhibited significantly delayed parturition when compared with females given control implants. Indomethacin treatment also disrupted the normal birth process when it occurred, as all females recieving this compound exhibited parturition of only a portion (24-32%) of the total clutch. A similar phenomenon occurred in one experiment following implantation of progesterone pellets. Histological examination of the corpora lutea and oviduct indicated no obvious difference in structure among any of the treatment groups. 0 1991 Academic FXYSS. IX.
Gestation length in reptiles is thought to be controlled by the activity of the corpus luteum (for review, see Xavier, 1987). A correlation between luteal activity and gestation length is well established in oviparous squamates whereas it is less obvious in viviparous species (Jones and Guillette, 1982; Guillette, 1987). Surgical deluteinization induces premature oviposition in oviparous lizards (Sceloporus undulatus, Roth et al., 1973; Cnemidophorus uniparens, Cuellar, 1979; Anolis carolinensis, Guillette
and Fox, 1985). Prostaglandin F,,(PGF,,)induced luteolysis does not stimulate premature oviposition within 24 hr of treatment in an oviparous lizard (A. carolinensis, Guillette et al., 1984) or turtle (Chelydra serpentina, Mahmoud et al., 1988) but does in a viviparous lizard (Sceloporus jurrovi, Guillette and Matter, unpublished). In these species, a significant decline in plasma progesterone concentration and distinct morphological changes in luteal tissue occur following prostaglandin-induced luteolysis. Guillette and Fox (1985) observed that oviposition occurred in three of eight females following surgical deluteinization
in A. carolinensis even though plasma progesterone levels remained elevated (presumably due to the release of adrenal progesterone). In the unique monoallochronic reproductive cycle of A. caroiinesis, progesterone appears to remain elevated up to oviposition (Jones et al., 1983). Progesterone pretreatment in A. carolinensis induces a significant increase in the strength of uterine contractions in vitro in response to stimulation by arginine vasotocin (AVT) (Guillette and Jones, 1980). This study was undertaken to examine whether parturition could be delayed in the viviparous lizard S. jurrovi, a species in which progesterone falls markedly at parturition (Guillette et al., 1981), by maintaining an elevated plasma progesterone concentration. Additionally, since available data suggest that exogenous PGF,, causes luteolysis in several reptiles (see above), stimulates uterine contractions and premature parturition in S. jarrovi (Guillette, DeMarco and Palmer, unpublished) and is synthesized by the oviduct of lizards (Guillette et al., 1988, 1990a), we examined the influence of indomethacin on the mainte105
106
GUILLETTE,
DEMARCO.
nance of pregnancy. Indomethacin, a potent blocker of cyclooxygenase activity, inhibits the synthesis and release of prostaglandins (PGs) in the reproductive tract of the lizard S. jarrovi (Guillette et al., 1988, 1990a). Two methods of treatment were tested (chronic versus acute) to determine whether differences in administration influenced the response. MATERIALS
AND METHODS
Animals. Pregnant female 5. jarrovi were collected from the Dragoon and Chiricahua Mountains of southeastern Arizona during April, May, and June of 1986 and 1987. Females were returned to the University of Florida where they were housed on a 14L:lOD photoperiod in large tanks (2000 liter) containing cement blocks for cover and refuge. Heat lamps and plant lights were on during the photophase, and a gradient in temperature existed during the photophase of 20-50”. Prior to treatment, each female was weighed, measured to the nearest millimeter (snout-vent length), and given a unique toe-clip pattern. Females then were ranked on the basis of weight and divided sequentially into treatment groups. Experimenf I. Fifteen females, in the last week of pregnancy, were partitioned into three equal groups and treated with (1) progesterone (50 nglg body wt), (2) indomethacin (4 pg/g body wt), or (3) lizard Ringer’s solution (0.05 ml) (Guillette 1982). Treatments were given as ip injections twice daily: the first injection was given 2 hr after the onset of the photophase and the second 2 hr prior to the onset of the scotophase. Animals were housed individually in plastic boxes (15 x 30 x 10 cm) in an incubator set for a photoperiod and temperature regime of 14L/32”: lOD/24”. Animals were fed every other day with crickets and misted with water daily. Treatment continued for 16 days unless parturition occurred. If parturition occurred, the date of birth, quantity, and condition of the neonates were recorded. All females remaining pregnant on Day 16 were killed: the number and condition of in urero young were recorded. Experiments 2 and 3. Females were divided into three treatment groups as above. The differences between Experiments 2 and 3 consisted of (1) the date of capture, (2) location of capture, (3) time in captivity before the onset of treatment, and (4) date of initiation of treatment. Experiment 2 began on 6 June 1987. after animals obtained from the Dragoon Mountains had been housed in the laboratory for 4 weeks, whereas experiment 3 began on 26 June 1987, 4 days after females were captured in the Chiricahua Mountains. Treatments were as stated in Experiment 1, except that the administration of chemicals was by time-
AND
PALMER
release pellets (Innovative Research of America, Toledo, OH). Pellets were designed to provide a constant release over a 24-hr period for 21 days for a 10-g mammal. Progesterone pellets were designed to release enough hormone to elevate plasma progesterone to at least 5 rig/ml (similar to maximal levels measured during midpregnancy in this species by radioimmunoassay (Guillette ef al., 1981)). Indomethacin pellets were designed to release 40 p,g over a 24-hr period. Control pellets contained only the carrier, cholesterol, and binder material (methylcellulose; o-lactose). The pellet was implanted under the skin above the left scapula with a trochar. The wound caused by implantation of the pellet was swabbed with ethyl alcohol and covered with New-skin Liquid Bandage (Medtech Lab, Inc., Cody, WY). Implanted pellets were recovered after treatment was terminated to ensure that they had remained intact, thus releasing the contents as designed. Following implantation, females were housed in groups of three in aquaria (75 liters) provided with heat and broad spectrum lights (14L:lOD). A temperature gradient existed in the tank during the photophase of 28-45”. Females were observed twice daily to determine if parturition had occurred. If young were found in the tank, each female in that tank was examined visually. In most cases, the postpartum female was obvious owing to its reduction in girth and its depressed sides. If it was not visually apparent which female had given birth, the females in the tank were weighed to determine the postpartum female. The number and condition of young delivered by each female were recorded. Females were killed by cervical dislocation as soon as parturition was observed (even if it was still underway) and blood was collected. Plasma samples were obtained and frozen until assayed by radioimmunoassay for progesterone. Females that had not exhibited birth were killed on Day 21 and their ovaries and oviducts were removed. Embryos remaining in ulero were staged using the embryo staging sequence of Dufaure and Hubert (1961). Oviducts and ovaries were fixed in Bouin’s fixative. Following 24-hr fixation, tissues were rinsed in ethyl alcohol, dehydrated, and embedded in paraffin. Tissues were serially sectioned at 8 km, stained with alcian blue, and counterstained with hematoxylin and eosin (see Humason. 1979. for standard histological procedures). Sectioned tissues were examined using light and differential interference contrast (DIG) microscopy. Specifically, luteal structure and diameter were compared among the treatment groups as was the structure of the oviduct. Radioimmunoassays. Progesterone concentration in the plasma of females in Experiments 2 and 3 was analyzed in duplicate using an RIA kit (Farmos Diagnostica). Methods provided with the kit were used except the plasma volume was reduced from 100 to 25 ~1 and, thus, volumes of other reagents were reduced accordingly. Mean recovery of “‘I-progesterone from
PARTURITION
CONTROL
spiked lizard plasma was 98.4% (n = 5). Parallelism to the standard curve was exhibited by lizard samples diluted up to one-ftith with stripped, progesterone-free female plasma. Using triplicate samples at binding levels of 25.50, and 80% of maximum binding, inter- and intraassay coefficients of variation (CV) were determined. The mean intrassay CV for this assay was 6.5%, whereas interassay CV was 11.3% (5 assays). The minimum detectable progesterone concentration was 200 pg/ml. Statistics. Mean (*SE) number of days following the onset of treatment until parturition was calculated for each treatment group in each experiment. If a female had not exhibited parturition by the end of the experiment, she was given a score equal to the maximum number of days (Experiment I, 16 days; Experiments 2 and 3, 21 days) of observation. Differences among groups were statistically analyzed using a oneway ANOVA followed by Duncan’s new multiple range tests (Bruning and Kintz, 1977). Plasma progesterone values were log-transformed to obtain homogeneity of variance, and significance was tested using ANOVA and Duncan’s new multiple range tests (P < 0.05 per comparison).
RESULTS Experiment 1. Females given injections of progesterone or indomethacin exhibited
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no difference (F(2,ll) = 2.22, P > 0.05) in mean latency to birth when compared to control females. However, although no statistical difference was noted in latency to birth, three of four females remaining alive in the indomethacin group at the end of treatment had not given birth and, thus, a significant difference (F(2,ll) = 16.8, P < 0.05) in number of young born was noted (Table 1). The fifth indomethacin-treated female died during the experiment but was not representative of the health of other females. Females treated with either saline or progesterone gave birth to healthy, active neonates, whereas indomethacin-treated females which retained their young had fully formed (Stage 40), dehydrated, dead young in utero. Experiments 2 and 3. Females treated with progesterone or indomethacin implants exhibited a response different than that of those given injections of these chemicals. Females given implants containing progesterone or indomethacin retained
TABLE 1 CLUTCH SIZE AND BIRTH DATAFORSALINE,PROGESTERONE,ANDINDOMETHACIN-TREATEDFEMALE Sceloporus
Treatment Experiment I Saline Indomethacin Progesterone Experiment 2 Saline Indomethacin Progesterone Experiment 3 Saline Indomethacin Progesterone
jarrovi
iv
Female weight” (g)
Total clutch“,’
Number born”
Number of in utero embryos (alive/dead)
5 4 5
15.3 2 2.3d 15.1 2 2.Sd 16.8 k 2.2d
6.6 f 0.7d 6.3 ‘- 0.5d 7.0 2 0.4d
6.6 i 0.7‘+ 1.5 f 1.2 5.8 k 0.4d
11/8 5/l
5 5 6
12.2 * 1.5d 12.6 k l.5d 10.8 2 l.Od
3.6 f 0.4d 4.4 + 0.9 3.3 * 0.2d
3.4 2 0.5d 1.4 f 0.5’ 0.3 t 02
l/O 3/10 10/8
5 5 5
19.0 * 1.4d 17.4 f 1.6d 20.0 k 2.0d
9.4 2 1.5d 8.2 + 0.7d 8.0 k 0.6d
9.0 k 1.2d 2.6 2 0.9’ 5.4 + 1.6d
2/o 2126 2110
010
a Mean f 1 SE. b Total clutch represents total of young born and remaining in utero young. Differences in total clutch size between Experiments 2 and 3 are due to differences in size of the females. c This represents the total number of embryos remaining in utero in all females after 21 days of treatment and their condition (alive or dead). d.ef Values within a column of data for each experiment and having different superscripts are significantly different (Duncan’s multiple range test; P < 0.05 per comparison).
108
GUILLETTE,
DEMARCO,
their young for a significantly longer time (Experiment 2, F(2,15) = 7.11, P < .Ol; Experiment 3, F(2,13) = 8.87, P < 0.005) than control females (Figs. IA, 1B). Plasma progesterone concentration was measured in all females at the time of birth (Fig. 2). We observed that the pellets released progesterone as designed (proposed = 5 rig/ml; observed = 4.8 + 0.7 rig/ml), providing plasma concentrations similar to peak levels reported for this species during midpregnancy (Guillette et al., 1981). Indomethacin-treated females did not have plasma progesterone concentrations significantly different from those in control females (Fig. 2). All females which retained young in the progesteroneand indomethacin-treated groups had some dead, fully formed (Stage 40) in utero young (Table 1). Although many of the experimentally treated females exhibited parturition, they did not give birth to complete litters, whereas most control females did (Table 1). If parturition occurred in progesterone- or indomethacintreated animals, they gave birth to both normal and stillborn young. Histological examination of the corpora lutea revealed no obvious differences
25
among the treatment groups at the light microscopy level. That is, corpora lutea exhibited structural characteristics similar to those reported for this species undergoing natural luteolysis (Guillette et al., 1981). That is, many luteal cells had pycnotic nuclei and early fibroblast invasion of the luteal cell mass was noted. No significant difference in luteal diameter was observed (control = 775 + 25 pm; indomethacin = 795 * 23 pm; progesterone = 815 + 30 km). Likewise, no differences in oviductal structure were noted when the progesterone- or indomethacin-treated females that gave birth were compared to the controls that gave birth. However, one indomethatin-treated female which retained young exhibited tissue necrosis, with ruptured oviducts and abdominally located dead embryos. DISCUSSION
Previous studies suggest that luteal function is associated with the maintenance of gestation in oviparous and viviparous lizards (for review, see Xavier, 1987). The corpus luteum (CL) presumably maintains pregnancy in many reptiles by synthesizing 20
lmdant
20
I5
co 15 2 IO
F 2 IO 5
5 0 09
AND PALMER
0
control itldmmhchl plug&mm CATEQORIES
Go
control -
pogeetaone
CATEQORKZS
FIG. 1. Mean (*SE) number of days pregnancy continued following surgical implanting of hormone pellets in Sceloporus jarrovi. (A) Females housed in laboratory for 4 weeks, (B) females housed in lab for 4 days. Treatments having different superscripts are significantly different (Duncan’s new multiple range test, P < 0.05 per comparison).
PARTURITION
CONTROL
51 4-
3-
2-
a r$$>;gg x:::*:.:::::.::: #$g$. .E’
I-
control
lif-l
hdahch
m
TREATMENT
FIG. 2. Mean (-t-SE) plasma progesterone concentration from female Sceloporus jarrovi implanted with control, indomethacin, and progesterone pellets. As no differences were noted, values from females in both experiments (2 and 3) were consolidated. Treatments having different superscripts are significantly different (Duncan’s new multiple range test, P < 0.05 per comparison).
and releasing progesterone. Progesterone supports oviductal and uterine secretory activity and inhibits uterine contractions in some mammals either directly or indirectly by inhibiting the synthesis and/or release of uterotonic factors such as prostaglandins (Poyser, 1981; Challis and Olson, 1988). In reptiles, exogenous progesterone can maintain pregnancy following deluteinization or ovariectomy in two viviparous species (Bradypodion pumilus, Veith, 1974; Sceioporus cyanogenys, Callard et al., 1972). Our data demonstrate that elevated plasma progesterone can inhibit normal parturition and cause the retention of the in utero young. Prostaglandins exhibit several major functions during parturition or oviposition in vertebrates, including (1) luteolysis, (2) cervical dilatation, and (3) myometrial activity (for discussion see Poyser, 1981;
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109
Challis and Olson, 1988). The functions of PGs in reptilian reproduction are poorly understood, but PGs are synthesized by the reproductive tract of reptiles (Guillette et al., 1988; 199Oa) and elevated plasma levels of PGF and PGE, are observed during natural oviposition in several reptiles (Sphenodon puncratus, Guillette et al., 199Ob; Caretta caretta, Guillette et al., 1989). Prostaglandin F,, is luteolytic in reptiles, causing a significant decline in plasma progesterone (Guillette et al., 1984; Mahmoud et al., 1988). As with progesterone, chronic treatment with indomethacin maintains and prolongs normal pregnancy. Our results are similar to those reported for birds and mammals, in which treatment with indomethacin blocks or delays oviposition (Hertelendy, 1973; Day and Nalbandov, 1977; Hertelendy and Biellier, 1978; Shimada and Asai, 1979) or parturition (Aiken, 1972; Chester et al., 1972; Challis et al., 1975). Of interest is the observation of Aiken (1972) that aspirin or indomethacin administration during late pregnancy in rats not only extends parturition but also is correlated with an increase in the number of stillbirths. These stillbirths were associated with death of the pups during birth (premature separation of the placenta) not prior to birth. He also noted that the drug-treated groups gave birth to significantly fewer pups (Aiken, 1972). These data are similar to our observations showing that indomethacin-treated females did not exhibit complete birth. However, indomethacin- and progesterone-treated female S. jarrovi had both live and dead fully formed in utero young suggesting that our observations of stillborn young may be due to causes different than those reported by Aiken (1972). One major difference may account for the contrast in the data sets for stillbirths. Aiken (1972) and others (see above) have observed that in mammals, parturition can only be delayed for a few hours to a few days past the normal period. In contrast, our data suggest that the in-
110
GUILLETTE,
DEMARCO,
domethacin-treated reptilian females can retain young an average of 14 or 9 days longer than the control females. This extensive period of retention may account for the large number of in utero deaths, due primarily to asphyxia. That is, this species has a well-developed chorioallantoic placenta (Guillette et al., 1981) which presumably provides for the gas exchange needed by the in utero young. Separation of the placenta from the uterine wall or gas exchange needs greater than the capacity of the placenta (due to large, fully developed young) would explain these deaths. We observed that neither exogenous indomethacin nor progesterone maintained the histological appearance of the corpora lutea. This was unexpected, as inhibition of PG synthesis by indomethacin or progesterone was hypothesized to delay parturition by maintaining the CL (blocking luteolysis). The CL of this species exhibits a significant decline in diameter during midpregnancy, although plasma progesterone concentration does not decrease (Guillette et al., 1981). The CL of all control and experimental females exhibited a luteal diameter similar to that reported earlier for females of this species that had undergone normal luteolysis (Guillette et al., 1981). The mechanism by which elevated plasma progesterone or indomethacin impairs normal parturition is unknown in reptiles. The role of prostaglandins as uterotonic agents in birds and mammals has been well documented (see Hertelendy et al., 1984, and Challis and Olson, 1988, for reviews). The release of PGs from the mammalian uterus or ovary at the time of birth is thought to be controlled by another uterotonic agent, oxytocin (Chan, 1983). In reptiles, the related neuropeptide, arginine vasotocin (AVT), is capable of inducing strong uterine muscle contractions and premature parturition or oviposition (see Guillette, 1987) as it does in birds (Tanaka and Nakajo, 1962; Rzasa and Ewy, 1970). The stimulatory features of AVT on the avian
AND
PALMER
oviduct are mediated by PGs (Hertelendy, 1973; Rzasa, 1984). Recent data show that a similar phenomenon of AVT-induced PGF,, synthesis exists in the reptilian reproductive tract (Guillette et al., 1990a). The mechanism controlling the release of AVT at the time of oviposition or parturition in reptiles is still unknown. In mammals, progesterone from the CL maintains uterine hypertrophy and inhibits the release of uterine and ovarian PGs (Poyser, 1981; Challis and Olson, 1988). As plasma progesterone levels decline at the end of pregnancy, the oviduct and ovary produce PGs stimulating luteolysis in some species (Rothchild, 1981). Luteolysis creates an environment whereby progesterone synthesis is further reduced and uterotonic PG synthesis is stimulated. Raising uterine levels of PG induces oviductal contractions which helps promote parturition (Poyser, 1981). Our data do not address this mechanism or other possible scenarios but they do suggest that progesterone concentrations in the plasma must decline or at least exhibit episodic decreases for normal parturition to occur. Furthermore, PGs appear to have an active role during parturition, as a reduction in their synthesis impairs or inhibits birth in S. jarrovi. Future studies must examine the interactions of steroids, AVT, and PGs during natural and induced oviposition or parturition if we are to understand the mechanism controlling gestation length in reptiles. ACKNOWLEDGMENTS We thank J. Matter, A. Cree, and A. Hensley for helpful comments concerning this manuscript, and S. Shatie of Innovative Research of America for help in pellet formulation. Animals were collected under permit from the Arizona Fish and Game Commission. This research was funded by NSF Grant DCB-8416707 awarded to L.J.G.
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PARTURITION
CONTROL
dins contribute to expulsion of foetus. Nature (London) 240, 21-25. Bruning, J. L., and Kintz, B. L. (1977). “Computational Handbook of Statistics.” Scott, Foresman, Glenview, IL. Callard, I. P., Chan, S. W. C., and Potts, M. A. (1972). The control of the reptilian gonad. Amer. Zool.
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Chester, R., Dukes, M., Slater, S. R., and Walpole, A. I. (1972). Delay of parturition in the rat by antiinflammatory agents which inhibit the biosynthesis of prostaglandins. Nature (London) 240, 3738. Cuellar, H. S. (1979). Disruption of gestation and egg shelling in deluteinized oviparous whiptail lizards Cnemidophorus uniparens (Reptilia: Teiidae). Gen. Comp. Endocrinol. 39, 150-157. Day, S. L., and Nalbandov, A. V. (1977). Presence of prostaglandin F (PGF) in hen follicles and its physiological role in ovulation and oviposition. Biol. Reprod. 16, 48w94. Dufaure, J. P., and Hubert, J. (1961). Table de developpement du lezard vivipare, Lacerta viviparu Jacquin. Arch. Anat. Microsc. Morphol. Exp. 50, 309-327. Guillette, L. J., Jr. (1982). A physiological (Ringer’s) solution for anoline lizards. Herpetol. Rev. 13, 37-38. Guillette, L. J., Jr. (1987). The evolution of viviparity in fishes, amphibians and reptiles: An endocrine perspective. In “Hormones and Reproduction in Fishes, Amphibians and Reptiles” (D. 0. Norris and R. E. Jones, Eds.), pp. 523-562. Plenum, New York. Guillette, L. J., Jr., Bjomdal, K., Bolten, A., and Gross, T. S. (1989). Plasma prostaglandin and steroid concentrations during natural nesting of the loggerhead sea turtle (Caretta caretta). Biol. Reprod. 4O(Suppl. l), 157. Guillette, L. J., Jr., and Fox, S. L. (1985). Effect of deluteinization on plasma progsterone concentration and gestation in the lizard, Anolis carolinensis. Comp.
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