THE JOURNAL OF EXPERIMENTAL ZOOLOGY 264461-467 (1992)
Arginine Vasotocin Concentrations in the Supraoptic Nucleus of the Lizard Anolis carolinensis Are Associated With Reproductive State but Not Oviposition CATHERINE R. PROPPER, RICHARD E. JONES, ROBERT M. DORES, AND KRISTIN H. LOPEZ Laboratory of Comparative Reproduction, Department of EPU Biology, University of Colorado, Boulder, Colorado 80309 (C.R.P., R.E.J., K.H.L.) and Department of Biology, University of Denver, Denver, Colorado 80210 (R.M.D.) Arginine vasotocin (AVT)is a neuropeptide involved in reproductive function in many ABSTRACT nonmammalian vertebrates. We determinedbrain and plasma AVT concentrationsduring the estrous cycle and oviposition in the lizard Anolis carolinensis. There were no differences in AVT concentrations in the plasma or any brain region during the ovipositional sequence. However, we found that females with an egg in each oviduct and a large pre-ovulatory follicle (diameter > 4.5 mm) in oneovary had significantly higher AVT concentrations in the supraoptic nucleus (SON) of the hypothalamus than did females with small pre-ovulatory follicles in both ovaries. In a second study, females with an egg in each oviduct and a large pre-ovulatory follicle had significantly greater AVT concentrations in the SON than females with only one oviductal egg and a large pre-ovulatory follicle or females with an egg in each oviduct and a small pre-ovulatoryfollicle in each ovary. Concentrations of AVT in other brain regions and in the plasma did not differ among these groups. Changes in steroid profiles during estrous and/or direct neural communication between the uterus, ovary, and brain may account for the changes in AVT concentrations seen in the supraoptic nucleus during the estrous cycle of Anolis carolinensis. o 1992 Wiley-Liss, Inc. Arginine vasotocin (AVT), a small neuropeptide synthesized in the hypothalamus of many nonmammalian vertebrates, is evolutionarily and functionally related to the peptides oxytocin (OXY) and arginine vasopressin (AVP) which are produced in the hypothalamus of mammals (Sawyer, '77; Acher, '80; Acher et al., '85). In many species ofbirds, reptiles, and amphibians, AVT induces oviductal contractions and oviposition (LaPointe, '77; Guillette and Jones, '82; Jones and Guillette, '82), and OXY plays a similar role i n mammalian parturition (Cross, '58; Fuchs, '64; Fuchs et al., '83). Plasma levels of AVT are elevated during oviposition in chickens (Shimada et al., '86; Koike et al., '88), sea turtles (Figler et al., '89), and the tuatara Sphenodon punctutus (Guillette et al., '91), and in the viviparous lizard Tiliqua rugosa prior to parturition (Fergusson and Bradshaw, '91). Brain concentrations of OXY also change over time. In rats, OXY levels are elevated in specific hypothalamic areas during late pregnancy (Caldwell et al., '87) and change during the estrous cycle (Crowley e t al., '78; Greer et al., '86). These studies demonstrate that there are dramatic changes in brain and plasma neuropeptide concentrations 0 1992 WILEY-LISS, INC.
around the time of parturition or oviposition. However, nothing is known about how concentrations of AVT change i n the brain during oviposition of any nonmammalian vertebrate. Arginine vasotocin induces oviductal contractions (Guillette and Jones, '80) and oviposition (Summers et al., '85) in the lizard Anolis carolinensis, suggesting that changes in plasma AVT levels may be responsible for oviposition in this species. The ovarian cycle of A. carolinensis is unusual for lizards because females ovulate a single egg from each ovary alternately (Noble and Greenberg, '41;Smith et al., '73). Because of this ovulatory pattern, only one egg is shelled and ready for oviposition at any one time. Thus, if endogenous AVT is responsible for oviposition, the timing of release of this neuropeptide from the pars nervosa may correspond to oviductal sensitivity to AVT. Because we know that immunoreactive AVT cells and fibers are present in several brain areas in A. carolinensis (Propper et al.,
Received January 7,1992;revision accepted July 26,1992. Address reprint requests to Catherine R. Propper, Department of Biological Sciences, Box 5640, Northern Arizona University, Flagstaff, A2 86011.
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'921, the purpose of this study was to determine whether there are changes not only in AVT in the plasma, but also in the brain during the estrous cycle and oviposition in A. carolinensis.
METHODS Animals All animals were purchased from Buck's Live Animals, La Place, Louisiana.
Experiment 1 To determine whether there were differences in brain and plasma AVT concentrations associated with oviposition behavior, we conducted this experiment in June and July 1989. Immediately upon arrival in the laboratory, we palpated females to determine whether they contained a n egg in each oviduct or an egg in only one oviduct. For this experiment we used only females with a n egg in both oviducts (2-egg state). These females were released into a large (61 x 121 x 42 cm) terrarium containing planting soil 5 cm in depth, artificial plants for perches, and water and meal worms ad libitum. The 1ight:dark cycle was maintained at 14:10, with lights on at 0600. Females were sacrificed within 3 days of arrival in the laboratory at a time determined by the display of ovipositionbehavior. Females were sacrificed by decapitation during one of the following 3 stages: 1)controls; females in 2-egg state that were not showing any oviposition related behaviors, 2) oviposition; females that had dug a nest and had deposited an egg in the nest within the previous 30 sec, and 3) post-oviposition; females that had completed nesting 25 min prior to sacrifice. After sacrifice, the diameter of the largest follicle in each ovary was determined. Females were categorized according to the diameter of the largest pre-ovulatory follicle: 1)follicle diameter greater than 4.5 mm, and 2) follicle diameter less t h a n 4.5 mm. Experiment 2 To determine if there are differences in brain and plasma AVT concentration among females at different reproductive stages, immediately upon arrival in July 1990 we palpated females in order to determine the number of eggs they contained and then placed them individually in plastic boxes (16 x 31 x 8 cm) containing water and meal worms ad libitum. All females were placed in a Percival incubator with a 14:lO 1ight:dark cycle and a temperature cycle of 32°C during the light phase and 20°C during the dark phase. Females were sacrificed within 2 days of arrival in the laboratory. After sacrifice,
females in this study were placed in 1of 3 categories: 1)1-egg state; females had one egg in one oviduct and no egg in the other; the follicle ipsilateral to the empty oviduct was large (> 4.5 mm), 2) 2-egg state, follicle > 4.5 mm; females had an egg in each oviduct and the largest follicle was greater than 4.5 mm, and 3) 2-egg state, follicle < 4.5 mm; females had a n egg in each oviduct and the largest follicle was smaller than 4.5 mm.
Tissue preparation Trunk blood was collected in heparinized capillary tubes and immediately centrifuged for 3 min in a microhematocrit centrifuge. Plasma was removed and stored frozen at - 80°C until assayed for AVT by radioimmunoassay (see below). Brains were immediately removed and frozen in OTC compound (Tissue Tek). Brains were sectioned coronally into 100 pm slices, which were thawmounted onto microscope slides and refrozen until specific brain regions were removed using the Palkovits and Brownstein ('82) punch technique as modified by Zoeller and Moore ('85).Procedures for extraction of AVT from the punch samples were as in Zoeller and Moore ('85). Brain areas analyzed were the supraoptic nucleus (SON), the paraventricular nucleus (PVN), the arcuate nucleus (ARC), and the pars nervosa (PN). These areas contain AVT immunoreactive cells and/or fibers in A. carolinensis (Propper et al., '92). For radioimmunoassay, AVT (Bachem, Torrence, CA) was iodinated using the Chloramin-T method of Rees et al. (71).The direct radioimmunoassay procedure of Dores ('82) was used to measure plasma and brain AVT concentrations. Sample, standard, antibody, and 1251-AVTsolutions were prepared in 0.01 M dipotassium phosphate buffer (pH = 7.5). A standard dilution was prepared ranging from 1.5 pg to 10,000 pg AVT (Sigma Chem. Co.) in 100 pl buffer. Plasma samples (20-30 pl) were brought to 100 pl with assay buffer. Lyophilized brain region samples were reconstituted in 100 p1 of assay buffer. All samples and standards received a n additional 100 pl of buffer containing 1:4,000 anti-AVT antiserum 593 (kindly provided by Dr. K. Lederis, Calgary, Canada to R.E. Jones), 10,000 cpm lZ5I-AVT, and 1:50 normal rabbit serum (Arnel). Tubes were incubated at 4°C for 24 hr, after which 12 pl of 1:1.5 goat anti-rabbit serum (Biotek) was added to all standards and samples. Tubes were incubated at 4°C for an additional 3 hr, followed by the addition of 1.4 ml of 0.05% Triton X. All standards and samples were spun immediately in a pre-cooled centri-
463
CHANGES IN BRAIN AVT CONCENTRATION DURING ESTROUS
fuge at 5,000 rpm for 10 min. The supernatant was aspirated and the pellet counted. All samples for a given brain region were run in a single radioimmunoassay. A 5-point, serial dilution of brain and plasma was parallel t o the standard curve. In experiment 1, total binding was 31.3%, non-specific binding was 1.8%,sensitivity was 5.5 pg, and the intraassay coefficient of variation was 2.2%. In experiment 2, total binding was 30.4%,non-specificbinding was 1.3%, sensitivity was 6.8 pg, and the intraassay coefficient of variation was 3.6%.
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RESULTS
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Experiment 1 Among the three groups in the different ovipositional stages, there were no significant differences in plasma or brain AVT concentrations (Fig. 1). There was a difference between groups of females of different largest follicle diameter sizes. Females that had largest follicles greater than 4.5 mm in diameter had significantly higher concentrations of AVT in the SON than did females with largest follicle diameters less than 4.5 mm (Fig. 2a; t = 2.42, P = 0.02). In all other brain regions and in the plasma, there were no differences in AVT concentrations between the two groups (Fig. 2b,c).
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There were differences in AVT concentrations in the SON among groups of females at different stages of estrous. Concentrations of AVT in the SON DISCUSSION were significantly higher in females in the 2-egg state with largest follicle diameters greater than 4.5 Unlike chickens (Shimada et al., '86; Koike et al., mm when compared t o females in the 2-egg state '881, sea turtles (Figler et al., '891, and the tuatara with largest follicle diameters less than 4.5 mm and Sphenodonpunctatus (Guillette et al., ,911,plasma females in the 1-egg state (Fig. 3a; F = 4.69, P = concentrations of immunoreactive AVT did not 0.02). There were no significant differences among change during oviposition in female A. carolinensis. the groups in AVT concentrations in any other brain In A. carolinensis, control of oviposition may be regulated by another mechanism rather than by a region or in the plasma (Fig. 3b,c).
C.R. PROPPER ET AL.
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Fig. 2. Arginine vasotocin concentrations in the brain and plasma of females with an egg in each oviduct and a small preovulatory follicle or a large pre-ovulatory follicle (Mean t SE). Females with a large pre-ovulatory follicle have higher concentrations of AVT in the supraoptic nucleus (SON) than do females with a small pre-ovulatory follicle (a).There are no significant differences between the two groups in any other brain area or in the plasma.
surge of AVT into the plasma to induce oviductal contractions. In this species, females have a single egg in each oviduct. One egg is ready to be oviposited while the other is only recently ovulated and is therefore
PLASMA
Fig. 3. Arginine vasotocin concentrations in the brain and plasma of females with an egg in each oviduct and a large or small pre-ovulatory follicle and females with an egg in one oviduct and a large pre-ovulatory follicle in one ovary (Mean k SE).Females with 2 eggs and a large pre-ovulatory follicle have higher concentrations of AVT in the SON than do females in either other group (a). There are no significant differences in AVT concentration in any other brain region or in the plasma.
unshelled. If a surge in AVT is necessary to induce oviductal contractions and consequently oviposition of the shelled egg, such a surge may induce contractions in the contralateral oviduct and therefore induce premature oviposition of the unshelled egg.
CHANGES IN BRAIN AVT CONCENTRATION DURING ESTROUS
Another controlling mechanism may include a difference in sensitivity of each oviduct t o endogeous AVT levels. Indeed, in female A. carolinensis the oviduct that no longer contains a shelled egg is the most sensitive t o AVT (Jones et al., '82). Perhaps changes in oviductal sensitivity to AVT regulate the timing of oviductal contractions and oviposition in this species. In a similar way, changes in OXY receptor concentration in the myometrium of mammals may regulate the timing of labor onset (Soloffet al., '79; Alexandrova and Soloff, '80). Concentrations of AVT in the SON are higher in females in the 2-egg state that have large (greater than 4.5 mm) pre-ovulatory follicles. The results suggest that AVT concentrations in the SON increase sometime after the large follicle reaches a diameter of 4.5 mm and then decrease soon after oviposition (female now in the 1-egg state with a large pre-ovulatory follicle). In rats, AVP and OXY concentrations change during the estrous cycle in the PVN and SON (Greer et al., '86) and in the pituitary (Crowley et al., '78). The results found here demonstrate that AVT concentrations in the SON of A . carolinensis also change throughout the estrous cycle. Other physiological changes are associated with growth of the large pre-ovulatory follicle in female A. carolinensis. Females with follicles less than 5 mm in diameter will not ovulate in response to exogenous follicle stimulating hormone (FSH) treatment, whereas females with larger follicle diameters will ovulate in response to FSH treatment (Jones et al., '88). Also, the amount of 3P-hydroxysteroid dehydrogenase (3p-HSD)activity is higher in large versus small follicles (Jones et al., '74). In the brain, there is a shift in hypothalamic catecholamine metabolism from the side ipsilateral to the ovary with the smaller follicle to the side ipsilateral t o the ovary with the larger follicle (Desan et al., '92). Behavioral changes are also associated with follicular development: as follicles grow from 3.5 mm t o 6.0 mm, femalesbecome sexuallyreceptive (Crews,'73). Changes in plasma estradiol concentrations may be responsible for changes in brain AVT concentrations. Estradiol concentrations are higher in A. carolinensis females in the 2-egg state than in females in the 1-egg state (Jones et al., '83). Estradiol is known to influence AVP content in the brain of rats. Estradiol-treated rats have more immunoreactive staining for AVP in the SON than do controls, but estradiol has no influence on AVP staining in the PVN (Jirikowski et al., '88). Pituitary OXY content is greater in rats pre-treated with estradiol (Crowley et al., '78; Jirikowski et al., '881, and
465
treatment of rats with estradiol increases oxytocin mRNA levels in the preoptic area (Caldwell et al., ,891, suggesting that changes in OXY content may be related t o an increase in OXY synthesis rather than a decline in OXY release. Estradiol may play a similar regulatory role in AVT synthesis and processing in A. carolinensis. The observed differences in AVT concentrations in the SON may also be regulated by direct neural input from the ovaries or the oviduct. In A. carolinensis, there are changes in monoamine metabolism in the hypothalamus that are associated with follicular development, and these changes may be under direct sensory input from the ovary (Jones et al., '90; Desan et al., '92). In rats, distension of the uterine horn causes excitation of cells in the PVN (Akaishi et al., '88). Increasing intraovarian pressure in the fish Clarias batrachus (Subhedar et al., '87) and the frog Rana tigrina (Subhedar and Rama Krishna, '90) causes cell hypertrophy and an increase in the presence of neurosecretory material in the preoptic region. In A. carolinensis, there may be an interaction between the oviductal pressure caused by the presence of a shelled egg and the growth of the large pre-ovulatory follicle that could influence the observed changes in AVT content in the SON. The fact that AVT content in the SON, but not the pars nervosa or plasma, varies with reproductive state suggests that AVT in the SON may play other than a neuroendocrine role in A. carolinensis. In mammals, hypothalamic OXY and AVP cells project t o several extrahypothalamic sites (see Swanson and Sawchenko, '80; Buijs, '85).Furthermore, AVP may have neurotransmitter-like or neuromodulatory-like actions in the hippocampus of rats (Albeck and Smock, '88; Brinton and McEwen, '89). In A. carolinensis, it is not unlikely that AVT may have similar types of action in the brain.
ACKNOWLEDGMENTS We thank Patricia Van Buskirk for helping to observe the lizards. We also appreciate William Johnson and Kiisa Nishikawa for their helpful comments on this manuscript. This study was supported by NIMH NRSA 5 F 31 MH09656-02 t o C.R.P. LITERATURE CITED Acher, R. (1980) Evolution of neuropeptides. Trends Neurosci., 4:225-229. Acher, R., J. Chauvet, M.T. Chauvet, and D. Hurpet (1985)Evolution of neurohypophsial hormones and their precursors. In: Current Trends in Comparative Endocrinology. B. Lofts and W.N. Holmes, eds. Hong Kong University Press, Hong Kong, pp. 1147-1152.
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Swanson, L.W., and P.E. Sawchenko (1980) Paraventricular nucleus: A site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology, 31 :410-417. Zoeller, R.T, and F.L. Moore (1985) Seasonal changes in luteinizing hormone-releasing hormone concentrations in microdissected brain regions of male rough-skinned newts (Taricha granulosa). Gen. Comp. Endocrinol., 58: 222-230.
Arginine Vasotocin Concentrations in the Supraoptic Nucleus of the Lizard Anolis carolinensis Are Associated With Reproductive State but Not Oviposition CATHERINE R. PROPPER, RICHARD E. JONES, ROBERT M. DORES, AND KRISTIN H. LOPEZ Laboratory of Comparative Reproduction, Department of EPU Biology, University of Colorado, Boulder, Colorado 80309 (C.R.P., R.E.J., K.H.L.) and Department of Biology, University of Denver, Denver, Colorado 80210 (R.M.D.) Arginine vasotocin (AVT)is a neuropeptide involved in reproductive function in many ABSTRACT nonmammalian vertebrates. We determinedbrain and plasma AVT concentrationsduring the estrous cycle and oviposition in the lizard Anolis carolinensis. There were no differences in AVT concentrations in the plasma or any brain region during the ovipositional sequence. However, we found that females with an egg in each oviduct and a large pre-ovulatory follicle (diameter > 4.5 mm) in oneovary had significantly higher AVT concentrations in the supraoptic nucleus (SON) of the hypothalamus than did females with small pre-ovulatory follicles in both ovaries. In a second study, females with an egg in each oviduct and a large pre-ovulatory follicle had significantly greater AVT concentrations in the SON than females with only one oviductal egg and a large pre-ovulatory follicle or females with an egg in each oviduct and a small pre-ovulatoryfollicle in each ovary. Concentrations of AVT in other brain regions and in the plasma did not differ among these groups. Changes in steroid profiles during estrous and/or direct neural communication between the uterus, ovary, and brain may account for the changes in AVT concentrations seen in the supraoptic nucleus during the estrous cycle of Anolis carolinensis. o 1992 Wiley-Liss, Inc. Arginine vasotocin (AVT), a small neuropeptide synthesized in the hypothalamus of many nonmammalian vertebrates, is evolutionarily and functionally related to the peptides oxytocin (OXY) and arginine vasopressin (AVP) which are produced in the hypothalamus of mammals (Sawyer, '77; Acher, '80; Acher et al., '85). In many species ofbirds, reptiles, and amphibians, AVT induces oviductal contractions and oviposition (LaPointe, '77; Guillette and Jones, '82; Jones and Guillette, '82), and OXY plays a similar role i n mammalian parturition (Cross, '58; Fuchs, '64; Fuchs et al., '83). Plasma levels of AVT are elevated during oviposition in chickens (Shimada et al., '86; Koike et al., '88), sea turtles (Figler et al., '89), and the tuatara Sphenodon punctutus (Guillette et al., '91), and in the viviparous lizard Tiliqua rugosa prior to parturition (Fergusson and Bradshaw, '91). Brain concentrations of OXY also change over time. In rats, OXY levels are elevated in specific hypothalamic areas during late pregnancy (Caldwell et al., '87) and change during the estrous cycle (Crowley e t al., '78; Greer et al., '86). These studies demonstrate that there are dramatic changes in brain and plasma neuropeptide concentrations 0 1992 WILEY-LISS, INC.
around the time of parturition or oviposition. However, nothing is known about how concentrations of AVT change i n the brain during oviposition of any nonmammalian vertebrate. Arginine vasotocin induces oviductal contractions (Guillette and Jones, '80) and oviposition (Summers et al., '85) in the lizard Anolis carolinensis, suggesting that changes in plasma AVT levels may be responsible for oviposition in this species. The ovarian cycle of A. carolinensis is unusual for lizards because females ovulate a single egg from each ovary alternately (Noble and Greenberg, '41;Smith et al., '73). Because of this ovulatory pattern, only one egg is shelled and ready for oviposition at any one time. Thus, if endogenous AVT is responsible for oviposition, the timing of release of this neuropeptide from the pars nervosa may correspond to oviductal sensitivity to AVT. Because we know that immunoreactive AVT cells and fibers are present in several brain areas in A. carolinensis (Propper et al.,
Received January 7,1992;revision accepted July 26,1992. Address reprint requests to Catherine R. Propper, Department of Biological Sciences, Box 5640, Northern Arizona University, Flagstaff, A2 86011.
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'921, the purpose of this study was to determine whether there are changes not only in AVT in the plasma, but also in the brain during the estrous cycle and oviposition in A. carolinensis.
METHODS Animals All animals were purchased from Buck's Live Animals, La Place, Louisiana.
Experiment 1 To determine whether there were differences in brain and plasma AVT concentrations associated with oviposition behavior, we conducted this experiment in June and July 1989. Immediately upon arrival in the laboratory, we palpated females to determine whether they contained a n egg in each oviduct or an egg in only one oviduct. For this experiment we used only females with a n egg in both oviducts (2-egg state). These females were released into a large (61 x 121 x 42 cm) terrarium containing planting soil 5 cm in depth, artificial plants for perches, and water and meal worms ad libitum. The 1ight:dark cycle was maintained at 14:10, with lights on at 0600. Females were sacrificed within 3 days of arrival in the laboratory at a time determined by the display of ovipositionbehavior. Females were sacrificed by decapitation during one of the following 3 stages: 1)controls; females in 2-egg state that were not showing any oviposition related behaviors, 2) oviposition; females that had dug a nest and had deposited an egg in the nest within the previous 30 sec, and 3) post-oviposition; females that had completed nesting 25 min prior to sacrifice. After sacrifice, the diameter of the largest follicle in each ovary was determined. Females were categorized according to the diameter of the largest pre-ovulatory follicle: 1)follicle diameter greater than 4.5 mm, and 2) follicle diameter less t h a n 4.5 mm. Experiment 2 To determine if there are differences in brain and plasma AVT concentration among females at different reproductive stages, immediately upon arrival in July 1990 we palpated females in order to determine the number of eggs they contained and then placed them individually in plastic boxes (16 x 31 x 8 cm) containing water and meal worms ad libitum. All females were placed in a Percival incubator with a 14:lO 1ight:dark cycle and a temperature cycle of 32°C during the light phase and 20°C during the dark phase. Females were sacrificed within 2 days of arrival in the laboratory. After sacrifice,
females in this study were placed in 1of 3 categories: 1)1-egg state; females had one egg in one oviduct and no egg in the other; the follicle ipsilateral to the empty oviduct was large (> 4.5 mm), 2) 2-egg state, follicle > 4.5 mm; females had an egg in each oviduct and the largest follicle was greater than 4.5 mm, and 3) 2-egg state, follicle < 4.5 mm; females had a n egg in each oviduct and the largest follicle was smaller than 4.5 mm.
Tissue preparation Trunk blood was collected in heparinized capillary tubes and immediately centrifuged for 3 min in a microhematocrit centrifuge. Plasma was removed and stored frozen at - 80°C until assayed for AVT by radioimmunoassay (see below). Brains were immediately removed and frozen in OTC compound (Tissue Tek). Brains were sectioned coronally into 100 pm slices, which were thawmounted onto microscope slides and refrozen until specific brain regions were removed using the Palkovits and Brownstein ('82) punch technique as modified by Zoeller and Moore ('85).Procedures for extraction of AVT from the punch samples were as in Zoeller and Moore ('85). Brain areas analyzed were the supraoptic nucleus (SON), the paraventricular nucleus (PVN), the arcuate nucleus (ARC), and the pars nervosa (PN). These areas contain AVT immunoreactive cells and/or fibers in A. carolinensis (Propper et al., '92). For radioimmunoassay, AVT (Bachem, Torrence, CA) was iodinated using the Chloramin-T method of Rees et al. (71).The direct radioimmunoassay procedure of Dores ('82) was used to measure plasma and brain AVT concentrations. Sample, standard, antibody, and 1251-AVTsolutions were prepared in 0.01 M dipotassium phosphate buffer (pH = 7.5). A standard dilution was prepared ranging from 1.5 pg to 10,000 pg AVT (Sigma Chem. Co.) in 100 pl buffer. Plasma samples (20-30 pl) were brought to 100 pl with assay buffer. Lyophilized brain region samples were reconstituted in 100 p1 of assay buffer. All samples and standards received a n additional 100 pl of buffer containing 1:4,000 anti-AVT antiserum 593 (kindly provided by Dr. K. Lederis, Calgary, Canada to R.E. Jones), 10,000 cpm lZ5I-AVT, and 1:50 normal rabbit serum (Arnel). Tubes were incubated at 4°C for 24 hr, after which 12 pl of 1:1.5 goat anti-rabbit serum (Biotek) was added to all standards and samples. Tubes were incubated at 4°C for an additional 3 hr, followed by the addition of 1.4 ml of 0.05% Triton X. All standards and samples were spun immediately in a pre-cooled centri-
463
CHANGES IN BRAIN AVT CONCENTRATION DURING ESTROUS
fuge at 5,000 rpm for 10 min. The supernatant was aspirated and the pellet counted. All samples for a given brain region were run in a single radioimmunoassay. A 5-point, serial dilution of brain and plasma was parallel t o the standard curve. In experiment 1, total binding was 31.3%, non-specific binding was 1.8%,sensitivity was 5.5 pg, and the intraassay coefficient of variation was 2.2%. In experiment 2, total binding was 30.4%,non-specificbinding was 1.3%, sensitivity was 6.8 pg, and the intraassay coefficient of variation was 3.6%.
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RESULTS
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Experiment 1 Among the three groups in the different ovipositional stages, there were no significant differences in plasma or brain AVT concentrations (Fig. 1). There was a difference between groups of females of different largest follicle diameter sizes. Females that had largest follicles greater than 4.5 mm in diameter had significantly higher concentrations of AVT in the SON than did females with largest follicle diameters less than 4.5 mm (Fig. 2a; t = 2.42, P = 0.02). In all other brain regions and in the plasma, there were no differences in AVT concentrations between the two groups (Fig. 2b,c).
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There were differences in AVT concentrations in the SON among groups of females at different stages of estrous. Concentrations of AVT in the SON DISCUSSION were significantly higher in females in the 2-egg state with largest follicle diameters greater than 4.5 Unlike chickens (Shimada et al., '86; Koike et al., mm when compared t o females in the 2-egg state '881, sea turtles (Figler et al., '891, and the tuatara with largest follicle diameters less than 4.5 mm and Sphenodonpunctatus (Guillette et al., ,911,plasma females in the 1-egg state (Fig. 3a; F = 4.69, P = concentrations of immunoreactive AVT did not 0.02). There were no significant differences among change during oviposition in female A. carolinensis. the groups in AVT concentrations in any other brain In A. carolinensis, control of oviposition may be regulated by another mechanism rather than by a region or in the plasma (Fig. 3b,c).
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Fig. 2. Arginine vasotocin concentrations in the brain and plasma of females with an egg in each oviduct and a small preovulatory follicle or a large pre-ovulatory follicle (Mean t SE). Females with a large pre-ovulatory follicle have higher concentrations of AVT in the supraoptic nucleus (SON) than do females with a small pre-ovulatory follicle (a).There are no significant differences between the two groups in any other brain area or in the plasma.
surge of AVT into the plasma to induce oviductal contractions. In this species, females have a single egg in each oviduct. One egg is ready to be oviposited while the other is only recently ovulated and is therefore
PLASMA
Fig. 3. Arginine vasotocin concentrations in the brain and plasma of females with an egg in each oviduct and a large or small pre-ovulatory follicle and females with an egg in one oviduct and a large pre-ovulatory follicle in one ovary (Mean k SE).Females with 2 eggs and a large pre-ovulatory follicle have higher concentrations of AVT in the SON than do females in either other group (a). There are no significant differences in AVT concentration in any other brain region or in the plasma.
unshelled. If a surge in AVT is necessary to induce oviductal contractions and consequently oviposition of the shelled egg, such a surge may induce contractions in the contralateral oviduct and therefore induce premature oviposition of the unshelled egg.
CHANGES IN BRAIN AVT CONCENTRATION DURING ESTROUS
Another controlling mechanism may include a difference in sensitivity of each oviduct t o endogeous AVT levels. Indeed, in female A. carolinensis the oviduct that no longer contains a shelled egg is the most sensitive t o AVT (Jones et al., '82). Perhaps changes in oviductal sensitivity to AVT regulate the timing of oviductal contractions and oviposition in this species. In a similar way, changes in OXY receptor concentration in the myometrium of mammals may regulate the timing of labor onset (Soloffet al., '79; Alexandrova and Soloff, '80). Concentrations of AVT in the SON are higher in females in the 2-egg state that have large (greater than 4.5 mm) pre-ovulatory follicles. The results suggest that AVT concentrations in the SON increase sometime after the large follicle reaches a diameter of 4.5 mm and then decrease soon after oviposition (female now in the 1-egg state with a large pre-ovulatory follicle). In rats, AVP and OXY concentrations change during the estrous cycle in the PVN and SON (Greer et al., '86) and in the pituitary (Crowley et al., '78). The results found here demonstrate that AVT concentrations in the SON of A . carolinensis also change throughout the estrous cycle. Other physiological changes are associated with growth of the large pre-ovulatory follicle in female A. carolinensis. Females with follicles less than 5 mm in diameter will not ovulate in response to exogenous follicle stimulating hormone (FSH) treatment, whereas females with larger follicle diameters will ovulate in response to FSH treatment (Jones et al., '88). Also, the amount of 3P-hydroxysteroid dehydrogenase (3p-HSD)activity is higher in large versus small follicles (Jones et al., '74). In the brain, there is a shift in hypothalamic catecholamine metabolism from the side ipsilateral to the ovary with the smaller follicle to the side ipsilateral t o the ovary with the larger follicle (Desan et al., '92). Behavioral changes are also associated with follicular development: as follicles grow from 3.5 mm t o 6.0 mm, femalesbecome sexuallyreceptive (Crews,'73). Changes in plasma estradiol concentrations may be responsible for changes in brain AVT concentrations. Estradiol concentrations are higher in A. carolinensis females in the 2-egg state than in females in the 1-egg state (Jones et al., '83). Estradiol is known to influence AVP content in the brain of rats. Estradiol-treated rats have more immunoreactive staining for AVP in the SON than do controls, but estradiol has no influence on AVP staining in the PVN (Jirikowski et al., '88). Pituitary OXY content is greater in rats pre-treated with estradiol (Crowley et al., '78; Jirikowski et al., '881, and
465
treatment of rats with estradiol increases oxytocin mRNA levels in the preoptic area (Caldwell et al., ,891, suggesting that changes in OXY content may be related t o an increase in OXY synthesis rather than a decline in OXY release. Estradiol may play a similar regulatory role in AVT synthesis and processing in A. carolinensis. The observed differences in AVT concentrations in the SON may also be regulated by direct neural input from the ovaries or the oviduct. In A. carolinensis, there are changes in monoamine metabolism in the hypothalamus that are associated with follicular development, and these changes may be under direct sensory input from the ovary (Jones et al., '90; Desan et al., '92). In rats, distension of the uterine horn causes excitation of cells in the PVN (Akaishi et al., '88). Increasing intraovarian pressure in the fish Clarias batrachus (Subhedar et al., '87) and the frog Rana tigrina (Subhedar and Rama Krishna, '90) causes cell hypertrophy and an increase in the presence of neurosecretory material in the preoptic region. In A. carolinensis, there may be an interaction between the oviductal pressure caused by the presence of a shelled egg and the growth of the large pre-ovulatory follicle that could influence the observed changes in AVT content in the SON. The fact that AVT content in the SON, but not the pars nervosa or plasma, varies with reproductive state suggests that AVT in the SON may play other than a neuroendocrine role in A. carolinensis. In mammals, hypothalamic OXY and AVP cells project t o several extrahypothalamic sites (see Swanson and Sawchenko, '80; Buijs, '85).Furthermore, AVP may have neurotransmitter-like or neuromodulatory-like actions in the hippocampus of rats (Albeck and Smock, '88; Brinton and McEwen, '89). In A. carolinensis, it is not unlikely that AVT may have similar types of action in the brain.
ACKNOWLEDGMENTS We thank Patricia Van Buskirk for helping to observe the lizards. We also appreciate William Johnson and Kiisa Nishikawa for their helpful comments on this manuscript. This study was supported by NIMH NRSA 5 F 31 MH09656-02 t o C.R.P. LITERATURE CITED Acher, R. (1980) Evolution of neuropeptides. Trends Neurosci., 4:225-229. Acher, R., J. Chauvet, M.T. Chauvet, and D. Hurpet (1985)Evolution of neurohypophsial hormones and their precursors. In: Current Trends in Comparative Endocrinology. B. Lofts and W.N. Holmes, eds. Hong Kong University Press, Hong Kong, pp. 1147-1152.
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Akaishi, T., A. Robbins, Y. Sakuma, and Y. Sat0 (1988) Neural inputs from the uterus to the paraventricular magnocellular neurons in the rat. Neurosci. Lett., 84:57-62. Albeck, D., and T. Smock (1988) A mechanism for vasopressin action in the hippocampus. Brain Res., 463:394-397. Alexandrova, M., and M.S. Soloff (1980) Oxytocin receptors and parturition in the guinea pig. Biol. Reprod., 22:1106-1111. Brinton, R.E., and B.S. McEwen (1989) Vasopressin neuromodulation in the hippocampus. J. Neurosci., 9:752-759. Buijs, R.M. (1985) Extrahypothalamic pathways of a neurosecretory system:Their role in neurotransmission. In: Neurosecretion and the Biology of Neuropeptides. H. Kobayashi, H.A. Bern, and A. Urano, eds. Springer-Verlag, Berlin, pp. 279-286. Caldwell, J.D., P.J. Brooks, G.F. Jirikowski, A.S. Barakat, P.K. Lund, and C.A. Pedersen (1989) Estrogen alters oxytocin mRNA levels in the preoptic area. J . Neuroendocrinol., 1:273-278. Caldwell, J.D., E.R. Greer, M.F. Johnson, A.J. Prange Jr., and C.A. Pedersen (1987) Oxytocin and vasopressin immunoreactivity in hypothalamic and extrahypothalamic sites in late pregnant and postpartum rats. Neuroendocrinology,46:39-47. Crews, D. (1973) Behavioral correlates to gonadal state in the lizard, Anolis carolinensis. Horm. Behav., 4:307-313. Cross, B.A. (1958) On the mechanism of labour in the rabbit. J . Endocrinol., 16:261-276. Crowley,W.R., T.L. ODonohue, J.M. George, and D.M. Jacobowitz (1978) Changes in pituitary oxytocin and vasopressin during the estrous cycle and after ovarian hormones: Evidence for mediation by norepinephrine. Life Sci., 23:2579-2586. Desan, P.H., K.H. Lopez, H.B. Austin, and R.E. Jones (1992) Asymmetric metabolism of hypothalamic catecholamines alternates with side of ovulation in a lizard (Anolis carolinensis). J . Exp. Zool.,262:105-112. Dores, R.M. (1982) Localization of multiple forms of ACTH- and beta-endorphin-related substances in the pituitary of the reptile, Anolis carolinensis. Peptides, 3:913-924. Fergusson, B., and S.D. Bradshaw (1991)Plasma arginine vasotocin, progesterone, and luteal development during pregnancy in the viviparous lizard Tiliqua rugosa. Gen. Comp. Endocrinol., 82:140-151. Figler, R.A., D.S. MacKenzie, D.W. Owens, P. Licht, and M.S. Amoss (1989) Increased levels of arginine vasotocin and neurophysin during nesting in sea turtles. Gen. Comp. Endocrinol., 73:223-232. Fuchs, A-R. (1964) Oxytocin and the onset of labour in rabbits. J. Endocrinol., 30:217-224. Fuchs, A-R., K. Goeschen, P. Husslein, A.B. Rasmussen, and F. Fuchs (1983) Oxytocin and the initiation of human parturition: 111. Plasma concentrations of oxytocin and 13, 14-dihydro-15-keto-prostaglandin Fz, in spontaneous and oxytocin-induced labor a t term. Am J. Obstet. Gynecol., 147:497-502. Greer, E.R., J.D. Caldwell, M.F. Johnson, A.J. Prange Jr., and C.A. Pedersen (1986) Variations in concentration of oxytocin and vasopressin in the paraventricular nucleus of the hypothalamus during the estrous cycle in rats. Life Sci., 38: 2311-2318. Guillette, Jr., L.J., and R.E. Jones (1980) Arginine vasotocininduced in vitro oviductal contractions in Anolis carolinensis: Effects of steroid hormone pretreatment in vivo. J . Exp. Zool., 212:147-152. Guillette, Jr., L.J., and R.E. Jones (1982) Further observations on arginine vasotocin-induced oviposition and parturition in lizards. J. Herpetol., 16:140-144.
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CHANGES IN BRAIN AVT CONCENTRATIONDURING ESTROUS Subhedar, N., N.S.R. Krishna, andM.K. Deshmukh (1987) The response of nucleus preopticus neurosecretory cells to ovarian pressure in the teleost Clarias batrachus. Gen. Comp. Endocrinol., 68:357-368. Summers, C.H., H.B. Austin, and R.E. Jones (1985) Induction of oviposition in cycling Anolis carolinensis requires a padrenoreceptor blocker and a high dosage of arginine vasotocin. Gen. Comp. Endocrinol., 57:389-392.
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Swanson, L.W., and P.E. Sawchenko (1980) Paraventricular nucleus: A site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology, 31 :410-417. Zoeller, R.T, and F.L. Moore (1985) Seasonal changes in luteinizing hormone-releasing hormone concentrations in microdissected brain regions of male rough-skinned newts (Taricha granulosa). Gen. Comp. Endocrinol., 58: 222-230.
