- Email: [email protected]
Effect of deluteinization on plasma progesterone concentration and gestation in the lizard, Anolis carolinensis
Camp. Biochem. Physiol. Vol. 80A, No. 3, pp. 303-306, 1985 Printed in Great Britain
0
0300-9629/85 $3.00 + 0.00 1985 Pergamon Press Ltd
EFFECT OF DELUTEINIZATION ON PLASMA PROGESTERONE CONCENTRATION AND GESTATION IN THE LIZARD, ANOLIS CAROLINENSIS LOUIS J.
GUILLETTE,
JR.*
and
SANDRA
L. Fox
Department of Biological Sciences, Wichita State University, Wichita, KS 67208, USA. Telephone: (316) 689-3 111 (Received 9 May 1984) Abstract-l.
Plasma progesterone concentration in deluteinized female lizards, as determined by radioimmunoassay, did not decrease significantly, as compared to non-surgical controls until 48hr after surgery. 2. Sham surgery induced a significant increase in plasma progesterone concentration 24 hr after surgery. Progesterone concentration then declined to an intermediate level not significantly different from non-surgical control or deluteinized females. 3. Plasma progesterone concentration in non-surgical control specimens exhibited no significant changes during the 96 hr in which animals were examined. 4. Oviposition did not occur in the control or sham groups whereas three of eight of the deluteinized females did oviposit by 48 hr.
INTRODUCTION
Corpora lutea are transitory in gravid female vertebrates
endocrine organs found (Browning, 1973). These
structures develop from the postovulatory follicular tissues, the granulosa and theta.. Reptilian luteal tissue possesses the enzymes necessary to synthesize progesterone and possibly androgens and estrogens (Lance and Callard, 1978). These hormones have a variety of functions during pregnancy (Browning, 1973; Jones and Guillette, 1982). One role of progesterone is to prepare and maintain the uterus for pregnancy (Yaron, 1972; Browning, 1973). In addition, it influences uterine sensitivity to the neurohypophyseal hormone arginine vasotocin which is involved with uterine contractions and birth (Callard and Hirsch, 1976; Guillette and Jones, 1980). Among oviparous lizards, a strong positive correlation exists between luteal function and maintenance of gestation (Jones and Guillette, 1982). That is, luteolysis occurs just prior to or just after oviposition. Deluteinization in the oviparous lizards Sceloporus undulatus (Roth et al., 1973) and Cnemidophorus uniparens (Cuellar, 1979) induces early oviposition. However, this response is not observed in oviparous females of the species, d. carolinensis (Jones et al., 1982). A weaker correlation exists between parturition and Iuteal function in viviparous lizards. Luteolysis may occur at any time during pregnancy, although the corpus luteum typically remains active until late in the 2nd or 3rd trimester (Jones and Guillette, 1982). Removal of luteal tissue from gravid, viviparous lizards appears to have no effect on embryonic development in some species (Panigel, 1956; Badir, 1968; Callard et al., 1972; Sekharappa and Deveraj Sarkar, 1978). However, the
*To whom correspondence
should be addressed.
operation induces spontaneous abortion if performed during early pregnancy; in contrast, if performed during late pregnancy it delays parturition. Besides species differences, the varying results in viviparous lizards may be due to several factors, including: (1) stage of pregnancy at which surgery was performed, (2) possible presence of an endocrine placenta and (3) differences in surgical technique. Deluteinization-induced oviposition or parturition in lizards is thought to occur due to the withdrawal of or a decline in plasma progesterone (see Jones and Guillette, 1982, for review). Data are not available, however, to test this hypothesis. No systematic studies examining plasma progesterone concentration following deluteinization are available. This study was designed to examine the effect of deluteinization on plasma progesterone concentration in the oviparous lizard A. carolinensis. MATERIALS AND METHODS
Gravid female Anolis carolinensis were obtained from The Snake Farm, LaPlace, Louisiana in May 1982. Females were palpated to determine reproductive state. Only those females exhibiting the one-egg stage were utilized. That is, Anolis alternates ovulation so that one oviduct possesses an egg undergoing shelling, whereas the contralateral oviduct has no egg. An active corpus luteum is found in the ovary ipsilateral to the gravid oviduct (for a detailed description of the reproductive cycle of A. carolinensis,see Jones et al., 1983b). Following determination of the reproductive state, females were partitioned into three experimental groups, (1) non-surgical control (IV = 15), (2) sham-operation (N = 15), and (3) deluteinization (N = 20). Operations were performed using low-temperature anesthesia. Deluteinization involved making a 5-mm incision on the ventral surface, exposing the ovary containing the corpus luteum, and removing the corpus luteum by cauterization. Sham operations were performed as above but the corpus luteum was left intact. Incisions were then sutured and covered with
303
Lours J.
304
GUILLETTE
JR. and
antibiotic. Following surgery, groups were further subdivided, as animals were sacrificed at 24, 48 and 96 hr after treatment. This design was possible for the non-surgical and sham control groups, as no animals were lost due to surgery or housing. However, four of the 20 deluteinized females were removed from the experiment due to death or poor health following surgery. The remaining 16 healthy animals were divided into two groups (24 and 48 hr), with no 96-hr group. We felt at the time that these two groups were the most critical, based on the results of previous deluteinization experiments. Thus, in the results, no plasma progesterone concentration data are available at 96 hr. Animals were housed individually in plastic boxes (6 x 12 x 4 in.) in a controlled temperature incubator. Ambient temperature was 32°C during the photophase (12 hr) and 20°C during the scotophase (12 hr). Animals were fed mealworms and watered ad bbitum. After the alloted time, animals were sacrificed by decapitation. Blood was collected in heparinized capillary tubes, centrifuged for 5min in a hematocrit centrifuge, and hematocrit recorded. Plasma was then collected for each animal and stored, frozen, at - 80°C until assayed for plasma progesterone concentration by radioimmunoassay (RIA). The radioimmunoassay procedure is that used by Guillette et al. (1981, 1984). Following blood collection, ovarian tissue was removed and fixed in 10% neutral buffered formalin. Ovaries were examined using a dissecting microscope to ascertain that all did (sham and non-surgical controls) or did not (deluteinization) possess luteal tissue. Finally, oviducts were examined to determine the presence or absence of an egg. Data collected were analyzed using a two-way ANOVA followed by Duncan’s multiple-range tests (Sokal and Rohlf, 1981).
RESULTS Examination of ovarian and oviductal tissue indicated that all females had one egg, and that all luteal tissue was removed during deluteinization. Hematocrit values for all groups were not significantly indicating that plasma concentration different, differences were not due to plasma volume changes. Data concerning plasma progesterone are shown in Fig. 1. Non-surgical control females exhibited a mean ( f SE) plasma progesterone concentration of 1.71 (2 0.25) ng/ml (N = 15). This value was not significantly different from the plasma progesterone concentrations obtained 24 hr after deluteinization (1.44 &-0.50 ng/ml; N = 8). However, by 48 hr postsurgery, deluteinized females exhibited a significant decline [F(2,39) = 6.12; P < 0.051 in plasma progesterone (0.41 f 0.27 ng/ml; N = 8). This value was not significantly different from sham values. Data for the 96-hr post-deluteinization group are not available (as reported in Materials and Methods). Plasma progesterone concentrations were also obtained for the females that underwent sham surgery. At 24 hr after sham surgery, plasma progesterone concentration was significantly higher than all other groups. In fact, this concentration (6.89 + 1.77 ng/ml; N = 5) was four times the level exhibited by the non-surgical control females. A decline was observed at 48 hr (0.94 k 0.21 ng/ml; N = 4), which produced concentrations lower than the non-surgical controls. However, at 96 hr, plasma progesterone concentration (2.51 + 0.81 ng/ml; N = 4) in the sham-operated
females was similar to control females.
SANDRA
L. Fox
Oviposition was not exhibited by any females in the control or sham groups. Three of eight deluteinized females, however, did oviposit within 48 hr of surgery (one within 24 hr, and two by 48 hr). None of these females, however, possessed an egg and an active corpus luteum on the contralateral side as would occur during the normal reproductive cycle. DISCUSSION
Deluteinization of gravid A. carolinensis females caused a significant decline in plasma progesterone concentration within 48 hr of surgery, but not by 24 hr. Feder et al. (1968) demonstrated that plasma progesterone declined to non-detectable levels within 8 hr of ovariectomy and adrenalectomy in rats. These data indicate an alternative source (extraluteal) of progesterone in Anolis. Several possible extraluteal sources of progesterone exist, including the adrenal glands and/or ovarian follicles. In mammals, surgery induces an acute stress the release of gluresponse, and consequently cocorticoids, adrenalcorticotropic hormone (ACTH), prolactin and possibly androgens (Ramaley, 1981). Progesterone is secreted by the mammalian adrenal gland, and plasma progesterone concentration increases during stress (Holzbauer and Newport, 1967, 1969; Feder et al., 1968, 1971; Feder and Ruf, 1969; Holzbauer et al., 1969; Resko, 1969). In fact, as a result of stress or exogenous ACTH administration, progesterone of adrenal origin attained plasma concentrations 2-10 times greater than that produced by the ovary (Feder et al., 1968). Bourne and Seamark (1972) observed that ovariectomy had little or no effect on plasma progesterone concentration in the viviparous lizard Tiliqua rugosa. They concluded that in this species, the adrenal glands are capable of producing significant amounts of progesterone and may be the main source of this hormone. The synthesis of corticosterone, the major adrenal corticosteroid in reptiles, requires the synthesis of progesterone (Licht and Bradshaw, 1969; Gist and Kaplan, 1976). It is not unlikely then, that as adrenal
24
48
96
HOURS
Fig. 1. Plasma progesterone concentrations in non-surgical, deluteinized and sham-operated female Anolis carolinensis. Values having different superscripts are significantly different (Duncan’s multiple-range test; P -c0.05per comparison).
Anolis
305
deluteinization
activity increases secretion of progesterone by the adrenal also would increase. In addition to an adrenal source of progesterone, this hormone is synthesized by vitellogenic follicles in lizards (Arslan et al., 1973). Anolis alternates ovulation, having a developing vitellogenic follicle at all times during the reproductive cycle (Jones et al., 1983b). Thus, it is not safe to presume that deluteinization will produce an immediate decline in plasma progesterone concentration, because extraluteal sources of this hormone probably exist. The observation that progesterone does not decline until 48 hr after deluteinization is of interest. Previous studies examining the effects of deluteinization on oviparous and viviparous reptilian females hypothesized that a decrease in plasma progesterone stimulated birth within 24 hr (Roth et al., 1973; Veith, 1974; Cuellar, 1979). This hypothesis was based upon the assumption that plasma progesterone concentration declined rapidly after deluteinization. Although the present study was performed on a different species (A. carolinensis) than those upon which the above hypothesis was based, the response observed to deluteinization may be common. Anolis is unique, however, in that deluteinization does not induce premature oviposition as readily as in other species (Jones et al., 1982; present study). Only three of eight females oviposited in response to the removal of luteal tissue and minimal plasma progesterone levels in this experiment. This observation is consistent with the hypothesis that gestation and oviposition in Anolis is controlled by a complex series of events (Guillette and Jones, 1980, 1982; Jones et al., 1982, 1983a,b). In A. carolinensis, exogenously administered arginine vasotocin (AVT), a potent stimulator of uterine contractions, does not induce oviposition in vivo (Guillette and Jones, 1982), but will stimulate uterine contractions in vitro (Guillette and Jones, 1980; Jones et al., 1982). Jones et al. (1983a) observed that AVT induces oviposition only if females were pretreated with dichloroisoproterenol, a beta adrenergic blocker. Thus, adrenal secretions also appear important in controlling the timing of oviposition in A. carolinensis. Jones et al. (1982) demonstrated that an active corpus luteum would block AVT-induced uterine contractions of the ipsilateral oviduct in vitro. However, deluteinization removed this block within 24 hr. Data obtained in the present study indicate that progesterone is not the blocking agent as it is still elevated 24 hr after deluteinization. A previous study using A. carolinensis observed that treatment with progesterone strengthened AVT-induced uterine contractions (Guillette and Jones, 1980). Thus, progesterone alone is not the main controller of the onset of parturition or oviposition as previously hypothesized. Interesting data also were obtained during this study concerning sham surgery. We observed a fourfold increase in plasma progesterone concentration 24 hr after sham surgery. Part of this value is due to the continued output of progesterone from the corpus luteum (approx. 2ng/ml). In addition, approx. 1.5 ng/ml, or the amount of progesterone measured in deluteinized animals, may be derived from an alternative source as discussed above. However, 3 ng/ml are
still unaccounted for. Several hypotheses can be proposed to account for this additional hormone. First, sham surgery may be more stressful than deluteinization and thus elicits a greater luteal or adrenal response. Sod-Moriah and Bedrak (1973) observed that mammalian corpora lutea may become hyperactive, secreting large amounts of progesterone under certain stressful conditions. Second, the corpus luteum may be responsive to ACTH or corticosteroid stimulation. That is, surgery stimulates the onset of a stress response and the release of ACTH as observed in mammals (Ramaley, 1981). Under ACTH stimulation, luteal tissue could secrete increased amounts of progesterone. Callard et al. (1976), however, observed no ,effect of mammalian ACTH on reptilian luteal tissue in vitro. This does not preclude the possibility that reptilian ACTH is effective in vivo, but does make it unlikely. A third hypothesis is that ovarian follicles release progesterone in response to stress. It is known that follicles are capable of synthesizing and secreting progesterone (Lance and Callard, 1978) and that under stressful conditions, follicles from rats secrete progesterone (Feder et al., 1971). No conclusive data, however, are available to support or reject any of the above hypotheses. These data and other recent studies indicate that the control of gestation in reptiles is complex and involves several systems and numerous hormones. Which, if any, of the above hypotheses are correct in describing the role of stress on gestation can not be determined at this time. Studies examining the stress response in reptiles and its interaction with the reproductive system are needed. Acknowledgements-The
authors would like to thank F. Moore and S. Speilvogel for assistance with the RIAs, and R. E. Jones, D. 0. Norris. M. Rand. G. Masson and D. Dickey for comments on this manusdript. This work was supported in part by a grant from the Wichita State University Faculty Grant program.
REFERENCES
Badir N. (1968) Structure and function of corpus luteum during gestation in the viviparous lizard Chalcides ocellatas. Anat. Anz. 122, l-10. Bourne A. R. and Seamark R. F. (1972) Progestins in the plasma of the viviparous lizard, Tiliqua rugosa. J. Reprod. Fert. 28, 156-157.
Browning H. C. (1973) The evolutionary history of the corpus luteum. Biol. Reprod. 8, 128-157. Callard I. P., Chan S. W. C. and Potts M. A. (1972) The control of the reptilian gonad. Am. Zoo/. 12, 273-287. Callard I. P., MO Chesney I., Scanes C. and Callard G. V. (1976) The influence of mammalian and avian gonadotropins on in vitro ovarian steroid synthesis in the turtle (Chrysemys picta). Gen. camp. Endocr. 28, 2-9.
Cuellar H. (1979) Disruption of gestation and egg shelling in deluteinized oviparous whiptail lizards Cnemidophorus uniparens. Gen. camp. Endocr. 39, 150-157. Feder H. H. and Ruf K. B. (1969) Stimulation of progesterone release and estrous behaviour by ACTH in ovariectomized rodents. Endocrinology 84, 171-174. Feder H. H., Brown-Grant K. and Corker C. S. (1971) Pre-ovulatory progesterone, the adrenal cortex and the “critical period” for luteinizing hormone release in rats. J. Endocr. 50, 29-39.
306
LOUISJ. GUILLETTE JR. and SANDRA L. Fox
Feder H. H., Resko J. A. and Goy R. W. (1968) Progesterone levels in the adrenal ulasma of me-ovulatorv and ovariectomized rats. J. Endocr. 41, 563-569. Gist D. H. and Kaplan M. L. (1976) Effects of stress and ACTH on plasma corticosterone levels in the Caiman Caiman crocodilus.
Gen. camp. Endocr. 28, 413419.
Guillette L. J. Jr and Jones R. E. (1980) Arainine vasotocininduced in vitro oviductal contractions in Anolis carolinensis: Effects of steroid hormone pretreatment in viuo. J. exp. Zool. 212, 147-152.
Guillette L. J. Jr and Jones R. E. (1982) Further observations on arginine vasotocin-induced oviposition and parturition in lizards. J. Herpetol. 16, 14&144. Guillette L. J. Jr, Spielvogel S. and Moore F. L. (1981) Luteal development, placentation and plasma progesterone concentration in the viviparous lizard Sceloporus jarrovi.
Gen. camp. Endocr. 43, 21329.
Guillette L. J. Jr, Lavia L. A., Walker N. J. and Roberts D. K. (1984) Luteolysis induced by prostaglandin F2 alpha in the lizard, Anolis Caroline&s. Gen. camp. Endocr., in press. Holzbauer M. and Newport H. M. (1967) The effect of stress on the concentration of 3/?-hydroxypregn-5en-20-one (pregnenolone) and pregn-4-ene-3,20-dione (progesterone) in the adrenal gland of the rat. J. Physiol. 193, 131-140. Holzbauer M. and Newport H. M. (1969) Adrenal secretion rates and adrenal tissue concentrations of pregnenolone, progesterone, 118 OH-androstenedione and some other steroids in young pigs and dogs. J. Physiol. 200, 821-848. Holzbauer M., Newport H. M., Birmingham M. K. and Traikov H. (1969) Secretion of pregn-4-ene-3,20-dione (progesterone) in vivo by the adrenal gland of the rat. Nature,
Lond. 221, 572-573.
Jones R. E. and Guillette L. J. Jr (1982) Hormonal control of oviposition and parturition in lizards. Herpetologica 38, 8(t93. Jones R. E., Summers C. H. and Lopez K. H. (1983a) Adrenergic inhibition of uterine contractions and oviposition in the lizard, Anolis carolinensis. Gen. camp. Endocr. 51, 77-83.
Jones R. E., Guillette L. J., Norman M. and Roth J. J. (1982) Corpus luteum-uterine relationships in the control of uterine contraction in the lizard Anohs carolinensis. Gen. camp. Endocr. 48, 104-112.
Jones R. E., Guillette L. J. Jr, Summers C. H., Tokarz R. R. and Crews D. (1983b) The relationship among ovarian condition, steroid hormones, and estrous behavior in Anolis carolinensis.
J. exp. Zool. 227, 145-154.
Lance V. and Callard I. P. (1978) Hormonal control of ovarian steroidogenesis in nonmammalian vertebrates. In The Vertebrate
Ovary: Comparative
Biology and Evolution
(Edited by Jones R. E.). Plenum Press, New York. Licht P. and Bradshaw S. D. (1969) A demonstration of corticotropic activity and its distribution in the pars distalis of the reptile. Gen. camp. Endocr. 13, 226235. Panigel M. (1956) Contribution a l’etude de l’ovoviviparite chez les reptiles: gestation at parturition chez le lezard vivipare Zootoca vivipara. Annls Sci. nat. (Zoo/.) 11, 569-668.
Ramaley J. A. (1981) Stress and fertility. In Environmental Factors in Mammal Reproduction (Edited by Gilmore D. and Cook B.). University Park Press, Baltimore. Resko J. A. (1969) Endocrine control of adrenal progesterone secretion in the ovariectomized rat. Science, Wash. 164, 70-71. Roth J. J., Jones R. E. and Gerrard A. (1973) Corpora lutea and oviposition in the lizard Sceloporus undulatus. Gen. camp. Endocr.
21, 569-572.
Sekharappa B. M. and Devarai Sarkar H. B. (1978) Role of corpora lutea m gestation in the skink Mabuya carinata (Schn.). Indian J. exp. Biol. 16. 1097-1098. Sod-Moriah U. A. and Bedrak ‘E. (1973) Corpora lutea activity in heat-acclimatized (HA) female rats. Acta endoer., Copemh. (Suppl.)
177, 337.
Sokal R. R. and Rohlf F. J. (1981) Biometry, 2nd edn.. W. H. Freeman, San Francisco. Veith W. J. (1974) Reproductive biology of Chumeleo pumilus pumilus with specific reference to the role of the corpus luteum and progesterone. Zool. Afr. 9, 161-183. Yaron Z. (1972) Endocrine aspects of gestation in viviparous reptiles. Gen. camp. Endocr. (Suppl.) 3, 668-674.
0
0300-9629/85 $3.00 + 0.00 1985 Pergamon Press Ltd
EFFECT OF DELUTEINIZATION ON PLASMA PROGESTERONE CONCENTRATION AND GESTATION IN THE LIZARD, ANOLIS CAROLINENSIS LOUIS J.
GUILLETTE,
JR.*
and
SANDRA
L. Fox
Department of Biological Sciences, Wichita State University, Wichita, KS 67208, USA. Telephone: (316) 689-3 111 (Received 9 May 1984) Abstract-l.
Plasma progesterone concentration in deluteinized female lizards, as determined by radioimmunoassay, did not decrease significantly, as compared to non-surgical controls until 48hr after surgery. 2. Sham surgery induced a significant increase in plasma progesterone concentration 24 hr after surgery. Progesterone concentration then declined to an intermediate level not significantly different from non-surgical control or deluteinized females. 3. Plasma progesterone concentration in non-surgical control specimens exhibited no significant changes during the 96 hr in which animals were examined. 4. Oviposition did not occur in the control or sham groups whereas three of eight of the deluteinized females did oviposit by 48 hr.
INTRODUCTION
Corpora lutea are transitory in gravid female vertebrates
endocrine organs found (Browning, 1973). These
structures develop from the postovulatory follicular tissues, the granulosa and theta.. Reptilian luteal tissue possesses the enzymes necessary to synthesize progesterone and possibly androgens and estrogens (Lance and Callard, 1978). These hormones have a variety of functions during pregnancy (Browning, 1973; Jones and Guillette, 1982). One role of progesterone is to prepare and maintain the uterus for pregnancy (Yaron, 1972; Browning, 1973). In addition, it influences uterine sensitivity to the neurohypophyseal hormone arginine vasotocin which is involved with uterine contractions and birth (Callard and Hirsch, 1976; Guillette and Jones, 1980). Among oviparous lizards, a strong positive correlation exists between luteal function and maintenance of gestation (Jones and Guillette, 1982). That is, luteolysis occurs just prior to or just after oviposition. Deluteinization in the oviparous lizards Sceloporus undulatus (Roth et al., 1973) and Cnemidophorus uniparens (Cuellar, 1979) induces early oviposition. However, this response is not observed in oviparous females of the species, d. carolinensis (Jones et al., 1982). A weaker correlation exists between parturition and Iuteal function in viviparous lizards. Luteolysis may occur at any time during pregnancy, although the corpus luteum typically remains active until late in the 2nd or 3rd trimester (Jones and Guillette, 1982). Removal of luteal tissue from gravid, viviparous lizards appears to have no effect on embryonic development in some species (Panigel, 1956; Badir, 1968; Callard et al., 1972; Sekharappa and Deveraj Sarkar, 1978). However, the
*To whom correspondence
should be addressed.
operation induces spontaneous abortion if performed during early pregnancy; in contrast, if performed during late pregnancy it delays parturition. Besides species differences, the varying results in viviparous lizards may be due to several factors, including: (1) stage of pregnancy at which surgery was performed, (2) possible presence of an endocrine placenta and (3) differences in surgical technique. Deluteinization-induced oviposition or parturition in lizards is thought to occur due to the withdrawal of or a decline in plasma progesterone (see Jones and Guillette, 1982, for review). Data are not available, however, to test this hypothesis. No systematic studies examining plasma progesterone concentration following deluteinization are available. This study was designed to examine the effect of deluteinization on plasma progesterone concentration in the oviparous lizard A. carolinensis. MATERIALS AND METHODS
Gravid female Anolis carolinensis were obtained from The Snake Farm, LaPlace, Louisiana in May 1982. Females were palpated to determine reproductive state. Only those females exhibiting the one-egg stage were utilized. That is, Anolis alternates ovulation so that one oviduct possesses an egg undergoing shelling, whereas the contralateral oviduct has no egg. An active corpus luteum is found in the ovary ipsilateral to the gravid oviduct (for a detailed description of the reproductive cycle of A. carolinensis,see Jones et al., 1983b). Following determination of the reproductive state, females were partitioned into three experimental groups, (1) non-surgical control (IV = 15), (2) sham-operation (N = 15), and (3) deluteinization (N = 20). Operations were performed using low-temperature anesthesia. Deluteinization involved making a 5-mm incision on the ventral surface, exposing the ovary containing the corpus luteum, and removing the corpus luteum by cauterization. Sham operations were performed as above but the corpus luteum was left intact. Incisions were then sutured and covered with
303
Lours J.
304
GUILLETTE
JR. and
antibiotic. Following surgery, groups were further subdivided, as animals were sacrificed at 24, 48 and 96 hr after treatment. This design was possible for the non-surgical and sham control groups, as no animals were lost due to surgery or housing. However, four of the 20 deluteinized females were removed from the experiment due to death or poor health following surgery. The remaining 16 healthy animals were divided into two groups (24 and 48 hr), with no 96-hr group. We felt at the time that these two groups were the most critical, based on the results of previous deluteinization experiments. Thus, in the results, no plasma progesterone concentration data are available at 96 hr. Animals were housed individually in plastic boxes (6 x 12 x 4 in.) in a controlled temperature incubator. Ambient temperature was 32°C during the photophase (12 hr) and 20°C during the scotophase (12 hr). Animals were fed mealworms and watered ad bbitum. After the alloted time, animals were sacrificed by decapitation. Blood was collected in heparinized capillary tubes, centrifuged for 5min in a hematocrit centrifuge, and hematocrit recorded. Plasma was then collected for each animal and stored, frozen, at - 80°C until assayed for plasma progesterone concentration by radioimmunoassay (RIA). The radioimmunoassay procedure is that used by Guillette et al. (1981, 1984). Following blood collection, ovarian tissue was removed and fixed in 10% neutral buffered formalin. Ovaries were examined using a dissecting microscope to ascertain that all did (sham and non-surgical controls) or did not (deluteinization) possess luteal tissue. Finally, oviducts were examined to determine the presence or absence of an egg. Data collected were analyzed using a two-way ANOVA followed by Duncan’s multiple-range tests (Sokal and Rohlf, 1981).
RESULTS Examination of ovarian and oviductal tissue indicated that all females had one egg, and that all luteal tissue was removed during deluteinization. Hematocrit values for all groups were not significantly indicating that plasma concentration different, differences were not due to plasma volume changes. Data concerning plasma progesterone are shown in Fig. 1. Non-surgical control females exhibited a mean ( f SE) plasma progesterone concentration of 1.71 (2 0.25) ng/ml (N = 15). This value was not significantly different from the plasma progesterone concentrations obtained 24 hr after deluteinization (1.44 &-0.50 ng/ml; N = 8). However, by 48 hr postsurgery, deluteinized females exhibited a significant decline [F(2,39) = 6.12; P < 0.051 in plasma progesterone (0.41 f 0.27 ng/ml; N = 8). This value was not significantly different from sham values. Data for the 96-hr post-deluteinization group are not available (as reported in Materials and Methods). Plasma progesterone concentrations were also obtained for the females that underwent sham surgery. At 24 hr after sham surgery, plasma progesterone concentration was significantly higher than all other groups. In fact, this concentration (6.89 + 1.77 ng/ml; N = 5) was four times the level exhibited by the non-surgical control females. A decline was observed at 48 hr (0.94 k 0.21 ng/ml; N = 4), which produced concentrations lower than the non-surgical controls. However, at 96 hr, plasma progesterone concentration (2.51 + 0.81 ng/ml; N = 4) in the sham-operated
females was similar to control females.
SANDRA
L. Fox
Oviposition was not exhibited by any females in the control or sham groups. Three of eight deluteinized females, however, did oviposit within 48 hr of surgery (one within 24 hr, and two by 48 hr). None of these females, however, possessed an egg and an active corpus luteum on the contralateral side as would occur during the normal reproductive cycle. DISCUSSION
Deluteinization of gravid A. carolinensis females caused a significant decline in plasma progesterone concentration within 48 hr of surgery, but not by 24 hr. Feder et al. (1968) demonstrated that plasma progesterone declined to non-detectable levels within 8 hr of ovariectomy and adrenalectomy in rats. These data indicate an alternative source (extraluteal) of progesterone in Anolis. Several possible extraluteal sources of progesterone exist, including the adrenal glands and/or ovarian follicles. In mammals, surgery induces an acute stress the release of gluresponse, and consequently cocorticoids, adrenalcorticotropic hormone (ACTH), prolactin and possibly androgens (Ramaley, 1981). Progesterone is secreted by the mammalian adrenal gland, and plasma progesterone concentration increases during stress (Holzbauer and Newport, 1967, 1969; Feder et al., 1968, 1971; Feder and Ruf, 1969; Holzbauer et al., 1969; Resko, 1969). In fact, as a result of stress or exogenous ACTH administration, progesterone of adrenal origin attained plasma concentrations 2-10 times greater than that produced by the ovary (Feder et al., 1968). Bourne and Seamark (1972) observed that ovariectomy had little or no effect on plasma progesterone concentration in the viviparous lizard Tiliqua rugosa. They concluded that in this species, the adrenal glands are capable of producing significant amounts of progesterone and may be the main source of this hormone. The synthesis of corticosterone, the major adrenal corticosteroid in reptiles, requires the synthesis of progesterone (Licht and Bradshaw, 1969; Gist and Kaplan, 1976). It is not unlikely then, that as adrenal
24
48
96
HOURS
Fig. 1. Plasma progesterone concentrations in non-surgical, deluteinized and sham-operated female Anolis carolinensis. Values having different superscripts are significantly different (Duncan’s multiple-range test; P -c0.05per comparison).
Anolis
305
deluteinization
activity increases secretion of progesterone by the adrenal also would increase. In addition to an adrenal source of progesterone, this hormone is synthesized by vitellogenic follicles in lizards (Arslan et al., 1973). Anolis alternates ovulation, having a developing vitellogenic follicle at all times during the reproductive cycle (Jones et al., 1983b). Thus, it is not safe to presume that deluteinization will produce an immediate decline in plasma progesterone concentration, because extraluteal sources of this hormone probably exist. The observation that progesterone does not decline until 48 hr after deluteinization is of interest. Previous studies examining the effects of deluteinization on oviparous and viviparous reptilian females hypothesized that a decrease in plasma progesterone stimulated birth within 24 hr (Roth et al., 1973; Veith, 1974; Cuellar, 1979). This hypothesis was based upon the assumption that plasma progesterone concentration declined rapidly after deluteinization. Although the present study was performed on a different species (A. carolinensis) than those upon which the above hypothesis was based, the response observed to deluteinization may be common. Anolis is unique, however, in that deluteinization does not induce premature oviposition as readily as in other species (Jones et al., 1982; present study). Only three of eight females oviposited in response to the removal of luteal tissue and minimal plasma progesterone levels in this experiment. This observation is consistent with the hypothesis that gestation and oviposition in Anolis is controlled by a complex series of events (Guillette and Jones, 1980, 1982; Jones et al., 1982, 1983a,b). In A. carolinensis, exogenously administered arginine vasotocin (AVT), a potent stimulator of uterine contractions, does not induce oviposition in vivo (Guillette and Jones, 1982), but will stimulate uterine contractions in vitro (Guillette and Jones, 1980; Jones et al., 1982). Jones et al. (1983a) observed that AVT induces oviposition only if females were pretreated with dichloroisoproterenol, a beta adrenergic blocker. Thus, adrenal secretions also appear important in controlling the timing of oviposition in A. carolinensis. Jones et al. (1982) demonstrated that an active corpus luteum would block AVT-induced uterine contractions of the ipsilateral oviduct in vitro. However, deluteinization removed this block within 24 hr. Data obtained in the present study indicate that progesterone is not the blocking agent as it is still elevated 24 hr after deluteinization. A previous study using A. carolinensis observed that treatment with progesterone strengthened AVT-induced uterine contractions (Guillette and Jones, 1980). Thus, progesterone alone is not the main controller of the onset of parturition or oviposition as previously hypothesized. Interesting data also were obtained during this study concerning sham surgery. We observed a fourfold increase in plasma progesterone concentration 24 hr after sham surgery. Part of this value is due to the continued output of progesterone from the corpus luteum (approx. 2ng/ml). In addition, approx. 1.5 ng/ml, or the amount of progesterone measured in deluteinized animals, may be derived from an alternative source as discussed above. However, 3 ng/ml are
still unaccounted for. Several hypotheses can be proposed to account for this additional hormone. First, sham surgery may be more stressful than deluteinization and thus elicits a greater luteal or adrenal response. Sod-Moriah and Bedrak (1973) observed that mammalian corpora lutea may become hyperactive, secreting large amounts of progesterone under certain stressful conditions. Second, the corpus luteum may be responsive to ACTH or corticosteroid stimulation. That is, surgery stimulates the onset of a stress response and the release of ACTH as observed in mammals (Ramaley, 1981). Under ACTH stimulation, luteal tissue could secrete increased amounts of progesterone. Callard et al. (1976), however, observed no ,effect of mammalian ACTH on reptilian luteal tissue in vitro. This does not preclude the possibility that reptilian ACTH is effective in vivo, but does make it unlikely. A third hypothesis is that ovarian follicles release progesterone in response to stress. It is known that follicles are capable of synthesizing and secreting progesterone (Lance and Callard, 1978) and that under stressful conditions, follicles from rats secrete progesterone (Feder et al., 1971). No conclusive data, however, are available to support or reject any of the above hypotheses. These data and other recent studies indicate that the control of gestation in reptiles is complex and involves several systems and numerous hormones. Which, if any, of the above hypotheses are correct in describing the role of stress on gestation can not be determined at this time. Studies examining the stress response in reptiles and its interaction with the reproductive system are needed. Acknowledgements-The
authors would like to thank F. Moore and S. Speilvogel for assistance with the RIAs, and R. E. Jones, D. 0. Norris. M. Rand. G. Masson and D. Dickey for comments on this manusdript. This work was supported in part by a grant from the Wichita State University Faculty Grant program.
REFERENCES
Badir N. (1968) Structure and function of corpus luteum during gestation in the viviparous lizard Chalcides ocellatas. Anat. Anz. 122, l-10. Bourne A. R. and Seamark R. F. (1972) Progestins in the plasma of the viviparous lizard, Tiliqua rugosa. J. Reprod. Fert. 28, 156-157.
Browning H. C. (1973) The evolutionary history of the corpus luteum. Biol. Reprod. 8, 128-157. Callard I. P., Chan S. W. C. and Potts M. A. (1972) The control of the reptilian gonad. Am. Zoo/. 12, 273-287. Callard I. P., MO Chesney I., Scanes C. and Callard G. V. (1976) The influence of mammalian and avian gonadotropins on in vitro ovarian steroid synthesis in the turtle (Chrysemys picta). Gen. camp. Endocr. 28, 2-9.
Cuellar H. (1979) Disruption of gestation and egg shelling in deluteinized oviparous whiptail lizards Cnemidophorus uniparens. Gen. camp. Endocr. 39, 150-157. Feder H. H. and Ruf K. B. (1969) Stimulation of progesterone release and estrous behaviour by ACTH in ovariectomized rodents. Endocrinology 84, 171-174. Feder H. H., Brown-Grant K. and Corker C. S. (1971) Pre-ovulatory progesterone, the adrenal cortex and the “critical period” for luteinizing hormone release in rats. J. Endocr. 50, 29-39.
306
LOUISJ. GUILLETTE JR. and SANDRA L. Fox
Feder H. H., Resko J. A. and Goy R. W. (1968) Progesterone levels in the adrenal ulasma of me-ovulatorv and ovariectomized rats. J. Endocr. 41, 563-569. Gist D. H. and Kaplan M. L. (1976) Effects of stress and ACTH on plasma corticosterone levels in the Caiman Caiman crocodilus.
Gen. camp. Endocr. 28, 413419.
Guillette L. J. Jr and Jones R. E. (1980) Arainine vasotocininduced in vitro oviductal contractions in Anolis carolinensis: Effects of steroid hormone pretreatment in viuo. J. exp. Zool. 212, 147-152.
Guillette L. J. Jr and Jones R. E. (1982) Further observations on arginine vasotocin-induced oviposition and parturition in lizards. J. Herpetol. 16, 14&144. Guillette L. J. Jr, Spielvogel S. and Moore F. L. (1981) Luteal development, placentation and plasma progesterone concentration in the viviparous lizard Sceloporus jarrovi.
Gen. camp. Endocr. 43, 21329.
Guillette L. J. Jr, Lavia L. A., Walker N. J. and Roberts D. K. (1984) Luteolysis induced by prostaglandin F2 alpha in the lizard, Anolis Caroline&s. Gen. camp. Endocr., in press. Holzbauer M. and Newport H. M. (1967) The effect of stress on the concentration of 3/?-hydroxypregn-5en-20-one (pregnenolone) and pregn-4-ene-3,20-dione (progesterone) in the adrenal gland of the rat. J. Physiol. 193, 131-140. Holzbauer M. and Newport H. M. (1969) Adrenal secretion rates and adrenal tissue concentrations of pregnenolone, progesterone, 118 OH-androstenedione and some other steroids in young pigs and dogs. J. Physiol. 200, 821-848. Holzbauer M., Newport H. M., Birmingham M. K. and Traikov H. (1969) Secretion of pregn-4-ene-3,20-dione (progesterone) in vivo by the adrenal gland of the rat. Nature,
Lond. 221, 572-573.
Jones R. E. and Guillette L. J. Jr (1982) Hormonal control of oviposition and parturition in lizards. Herpetologica 38, 8(t93. Jones R. E., Summers C. H. and Lopez K. H. (1983a) Adrenergic inhibition of uterine contractions and oviposition in the lizard, Anolis carolinensis. Gen. camp. Endocr. 51, 77-83.
Jones R. E., Guillette L. J., Norman M. and Roth J. J. (1982) Corpus luteum-uterine relationships in the control of uterine contraction in the lizard Anohs carolinensis. Gen. camp. Endocr. 48, 104-112.
Jones R. E., Guillette L. J. Jr, Summers C. H., Tokarz R. R. and Crews D. (1983b) The relationship among ovarian condition, steroid hormones, and estrous behavior in Anolis carolinensis.
J. exp. Zool. 227, 145-154.
Lance V. and Callard I. P. (1978) Hormonal control of ovarian steroidogenesis in nonmammalian vertebrates. In The Vertebrate
Ovary: Comparative
Biology and Evolution
(Edited by Jones R. E.). Plenum Press, New York. Licht P. and Bradshaw S. D. (1969) A demonstration of corticotropic activity and its distribution in the pars distalis of the reptile. Gen. camp. Endocr. 13, 226235. Panigel M. (1956) Contribution a l’etude de l’ovoviviparite chez les reptiles: gestation at parturition chez le lezard vivipare Zootoca vivipara. Annls Sci. nat. (Zoo/.) 11, 569-668.
Ramaley J. A. (1981) Stress and fertility. In Environmental Factors in Mammal Reproduction (Edited by Gilmore D. and Cook B.). University Park Press, Baltimore. Resko J. A. (1969) Endocrine control of adrenal progesterone secretion in the ovariectomized rat. Science, Wash. 164, 70-71. Roth J. J., Jones R. E. and Gerrard A. (1973) Corpora lutea and oviposition in the lizard Sceloporus undulatus. Gen. camp. Endocr.
21, 569-572.
Sekharappa B. M. and Devarai Sarkar H. B. (1978) Role of corpora lutea m gestation in the skink Mabuya carinata (Schn.). Indian J. exp. Biol. 16. 1097-1098. Sod-Moriah U. A. and Bedrak ‘E. (1973) Corpora lutea activity in heat-acclimatized (HA) female rats. Acta endoer., Copemh. (Suppl.)
177, 337.
Sokal R. R. and Rohlf F. J. (1981) Biometry, 2nd edn.. W. H. Freeman, San Francisco. Veith W. J. (1974) Reproductive biology of Chumeleo pumilus pumilus with specific reference to the role of the corpus luteum and progesterone. Zool. Afr. 9, 161-183. Yaron Z. (1972) Endocrine aspects of gestation in viviparous reptiles. Gen. camp. Endocr. (Suppl.) 3, 668-674.