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Function of corpora lutea of pregnancy in the viviparous garter snake, Thamnophis elegans
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
27, 401-407
(1975)
Function of Corpora Lutea of Pregnancy in the Viviparous Garter Snake, Thamnophis elegans DICK R. HIGHFILL AND RODNEY A. MEAD Department of Biological Sciences, University of Idaho, Moscow, Idaho 83843
Accepted July 12, 1975 Although corpora lutea of the viviparous garter snake, Thamnophis elegant, are the major source of plasma progesterone during pregnancy, a specific function for this steroid or any other luteal factor has yet to be shown. Thirty pregnant snakes weighing 80-200 g were divided into three equal groups so that all groups contained snakes of comparable size. Equal numbers of snakes were luteectomized, sham luteectomized, or received no treatment during early pregnancy and were then allowed to complete gestation. Duration of pregnancy was significantly (P < 0.01) longer in luteectomized snakes than in either control group. Luteectomized snakes gave birth to a lower percentage of living young and fewer birth products, which also weighed significantly less. However, luteectomy did not prevent normal embryonic development and did not appear to affect the rate of embryonic development. There were no significant differences between intact and sham luteectomized snakes in any of the above parameters. We conclude that CL in the viviparous garter snake, Thamnophis elegans, function through the mediation of a hormone which ensures that parturition occurs at the normal time and with the delivery of a normal number of young. The mechanism or the compound responsible for this action remains unknown,
The function of corpora lutea (CL) in but hypophysectomy of Storeria and Naovoviviparous and viviparous snakes re- trix at this time resulted in abortion. Rahn mains controversial. It seems likely that (1939) reported that ovariectomy failed to CL in live-bearing snakes are endocrine produce resorption or abortion in 14 glands that function during pregnancy snakes (Thamnophis and Natrix) 25 days through the mediation of progesterone se- after treatment. However, castration procretion. The reasons for this latter supposiduced abortion in two out of six snakes tion have been presented elsewhere (Highduring the previous summer (Rahn, 1938). fall and Mead, 1975; Browning, 1973). Bragdon (1951) found that T. sirtalis or N. Data accessing luteal function in snakes is sipedon failed to abort or resorb their emcontradictory. Clausen (1935, 1940) found bryos after ovariectomy or hypophysecthat hypophysectomy of Natrix, Storeria, tomy in early, mid-, or late pregnancy. and Thamnophis sirtalis or ovariectomy of However, he did note an increase in the Natrix in early pregnancy resulted in re- length of pregnancy which was more comsorption of embryos. Hypophysectomy of plete subsequent to hypophysectomy than Natrix or ovariectomy of Natrix and ovariectomy . Unfortunately, these studies Storeria in midpregnancy resulted in abor- fail to clarify the function of CL in snakes tion or resorption of embryos. Ovariecnot only because the data are contradictomy of Natrix in late pregnancy failed to tory but also because they fail to deal prevent delivery of normal young at term, directly with CL. Ovariectomy or hypo401 Cwtiht All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
402
HIGHFILL
physectomy may produce effects which depend upon nonluteal structures. In addition, the inability to precisely determine the stage of pregnancy at which a treatment was performed makes it difficult to compare the results from different experiments. We have chosen to work with T. elegans because the CL of this species are known to be the primary source of progesterone during pregnancy (Highfill and Mead, 1975); hence removal of these glands should clarify the hormonal role of this steroid. Moreover, the stage of pregnancy can be determined accurately by classifying the developing embryos using the method of Zehr (1962). Our purpose was to determine the effect of luteectomy upon embryo survival, embryo development, the number birth products delivered, and the duration of pregnancy in Thamnophis elegans. MATERIALS
AND
MEAD
experiment. Snakes were maintained on a natural photoperiod (sunlight only) from June through midSeptember and temperature was not controlled. However, due to a prolonged cold spell in September the snakes were maintained at approximately 70 degrees on a photoperiod of 14 hr light and 10 hr dark for the remainder of the experiment. The date, number of birth products (live embryos, dead embryos, or eggs), and weight of fully developed young (alive or dead) were recorded within 24 hr after parturition. The snakes were killed and preserved in AFA (three parts 95% ethanol, one part acetic acid, one part formalin, and five parts water) at the end of the experiment and the number of birth products remaining in utero and their stage of development were recorded. During the previous year nine pregnant T. .&guns weighing 60-120 g were luteectomized (five snakes) or sham luteectomized (four snakes) in an attempt to determine the effect of luteectomy on the rate of embryonic development. At the time of treatment, an embryo was taken and classified according to Zehr stage. After approximately equal times had elapsed, one snake from each treatment was laparotomized and another embryo was taken and classified according to Zehr stage. All surgical treatments were carried out under ether anesthesia and the animals were housed and fed as described above.
AND METHODS
Thirty female Thamnophis elegans (weighing N-200 g) were captured near Moscow, Idaho, from April through June and brought into the laboratory. Prior to ovulation, snakes were kept in a large screen covered enclosure and provided with Styrofoam containers in which to hide. Snakes were palpated to determine growth of follicles and time of ovulation. After all snakes had ovulated, 10 snakes of approximately equal weight were randomly assigned to each of three treatment groups: luteectomy, sham luteectomy, and intact (no treatment). Snakes were anesthetized with ether and an incision made over each ovary. In the luteectomy group, the ovary was exposed and the CL were surgically removed. Snakes receiving sham luteectomies were treated in an indentical manner, except that CL were not removed. The intact snakes received no surgical treatment. The embryo nearest the right ovary was removed from the right oviduct in snakes which received surgical treatments and the stage of embryo development determined by the method of Zehr (1%2). Snakes received an injection (im) of 5000 IU of penicillin G and 6.25 mg of dihydrostreptomycin on the day following surgery. Snakes were then housed individually in separate cages with screen covers. Snakes were given free access to water and were force-fed canned dog food at 3- to 4-wk intervals for the duration of the
RESULTS
A one way analysis of variance indicated that snakes receiving no treatment or sham luteectomy did not differ in any of the parameters tested, thus verifying that surgical treatment did not adversely affect the measurements made in this study (Table 1). However, luteectomy significantly reduced the percentage of young born alive (P < 0.005) as well as the number of birth products delivered (P < 0.025) (Table 1). Luteectomy also significantly lengthened gestation (P < 0.01) and reduced the mean weight oi fully developed progeny (P < 0.005) (Table 1). All progeny delivered in this experiment were fully developed but about one-half were attached to placentae in which the yolk sac contained considerable amounts of yolk, whereas the others were devoid of yolk. The particular Zehr stage at which luteectomy was performed during
8.8 (41.2) 4.7 (+1.3)*
10 9
7-22 (11.4) 7-23 (12.6)
10.4 (-c1.8Y
7
-
Mean total number of birth productsb*e 75 (15.5) [55i73] 64 (k9.9) [56/87] 14 (*5.9)*** [5/37]
Mean % young born viabled 96 (22.7) [7Oi73] 97 (k2.8) [84/87] 89 (k6.7) [33/373
Mean % born fully developede
106.4 (r2.4)**
97.4 (22.6)
94.3 (~2.3)
Mean duration of pregnancy in days
a This refers to the range of Zehr stages at the time snakes were treated and the mean Zehr stage for the treatment group. b Birth products (dead young + live young + eggs). c One embryo removed after luteectomy of sham luteectomy, thus making these values somewhat lower than intact animals. d The numbers in brackets in this column represent the total number of viable young/total number of birth products. e The numbers in brackets in this column represent the total number of fully developed young/total number of birth products. f AU numbers in parentheses represent the standard errors of the means. * (P < 0.025). ** (P < .OlO). *** (P < .ooS).
Luteectomy
Sham luteectomy
None
Treatment
Number of snakes
ZehP stage (mean and raw39
1.70 (-eo.z)***
2.31 (20.10)
2.65 (-0.18)
Mean weight of fully developed young 62)
TABLE 1 THE EFFECT OF LUTEECTOMY, SHAMLUTEECTOMY, ORNOTREATMENT ONVARIOUSPARAMETERS OF PREGNANCY
2
? s K
$
2 3 :! 8 z
2 3 F
HIGHFILL
404 TABLE
2
COMPARISON OF ZEHR STAGES ELAPSED AND DAYS ELAPSED IN LUTEECTOMIZED AND SHAM LUTEECTOMIZED GARTER SNAKES
Treatment Sham luteectomy Luteectomy Lureectomy Sham luteectomy Luteectomy Sham luteectomy Luteectomy Sham luteectomy Luteectomy
Zehr stage at first laparotomy
Zehr stage at second laparotomy
Number of days elapsed from first to second laparotomy
7 7 7
28 27 28
23 22 20
7 7
29 29
35 35
7 7
30 30
26 35
7 7
35 35
67 67
trimester 1 did not appear to affect the reproductive parameters observed (Table 1). Rate of embryonic development between Zehr stages 7 and 35 was not significantly affected by luteectomy (Table 2). Of the 10 snakes that were initially included in the intact group, two were subsequently found to be not pregnant and the third escaped. They therefore were omitted from the statistical analysis. One of the luteectomized snakes died shortly after treatment and this animal was also eliminated from the study. DISCUSSION
Earlier experiments concerning luteal function in live-bearing snakes, as determined by ovariectomy or hypophysectomy (Clausen, 1935, 1940; Rahn, 1938, 1939; Bragdon, 1951) must be interpreted with caution. These earlier experiments related the stage of pregnancy to time of year rather than embryological development. Since the time of ovulation is highly variable in garter snakes, errors up to one-
AND MEAD
third the duration of pregnancy may have occurred (Bragdon, Lazo-Wasem, Zarrow, and Hisaw, 1954). In addition, ovariectomy removed interstitial tissue and ovarian follicles as well as the CL, while hypophysectomy presumably altered luteal function as well as that of other endocrine glands. Consequently, these earlier experiments with live-bearing snakes may have obscured the true function of CL by also altering the function of other nonluteal tissues. Luteectomy early in trimester I lengthened pregnancy in T. elegans, thus supporting results of ovariectomy studies by Bragdon (1951). Unfortunately he failed to present data regarding duration of pregnancy for the three Natrix and three T. sirtalk which were ovariectomized. Bragdon assumed CL were dependent upon pituitary hormones, thus hypophysectomy would remove luteal function. Interpreted in this way, his hypophysectomy data also support our findings. However, hypophysectomy undoubtedly removed posterior pituitary function too. Since pituitary extracts (Clausen, 1940) or oxytocin (unpublished data) will initiate parturition in livebearing snakes, Bragdon’s observations could also be attributed to the lack of a potent inducer of parturition. Contrary to our findings and those of Bragdon (1951), Clausen (1935, 1940) reported that hypophysectomy or bilateral ovariectomy of live-bearing snakes in early or midpregnancy produced abortions. Rahn (1938, 1939) found castration produced mixed results in Natrix and Storeria. In one experiment two of six snakes aborted, but in the second none of 11 snakes aborted. It is possible that the surgery performed by Clausen (1935, 1940) and Rahn (1939) induced release of oxytocin, which in turn produced abortions. We observed no abortions in the experiments we describe here. However, in previous years, we occassionally observed abortions following surgery even in snakes with intact CL, which is
LUTEAL
FUNCTION
IN
consistent with the idea of trauma induced oxytocin release. However, abortions following hypophysectomy are difficult to explain in terms of oxytocin release. The fact that Clausen failed to histologically confirm the success of his hypophysectomies may indicate that sufficient posterior pituitary tissue remained to effect oxytocininduced abortions. Duration of pregnancy in our garter snakes did not differ significantly (Table 1) in untreated and sham luteectomized snakes, thus indicating that surgical treatment alone did not prolong gestation. However, we noted that captive intact snakes had a longer gestation than that reported for this species in the wild (Hebard, 1950; Zehr, 1%2). It is possible that captive snakes lacked the ability to behaviorally thermoregulate adequately, thus prolonging pregnancy. The fact that 120 pregnant garter snakes out of a total of 160 males and females collected during midMay to early July were found sunning themselves near protected areas during the morning when temperatures were rising rapidly after a relatively cold night (less that 50 degrees F) indicates behavior is an important factor in temperature regulation. These data support the contentions of others who have found temperature to be an important factor in the reproduction of garter snakes (Clark, Florio, and Hurowitz, 1955; Fox, 1954) and other reptiles (Licht, 1972). It should also be noted that the mean number of birth products delivered (10.4) did not appear to differ greatly from other observations (9.7) in this species (Hebard, 1950). Snakes luteectomized during early pregnancy gave birth to a lower percentage of living young than intact or sham luteectomized garter snakes (Table 1). Thus normal luteal function appears to enhance the percentage of viable offspring produced. We concur with investigators who found that luteal function was not essential for completion of embryo development in live-
THE
GARTER
SNAKE
405
bearing snakes (Bragdon, 1951; Rahn, 1939). However, Clausen (1940) reported ovariectomy of T. sirtalis, Natrix, and Storeria retarded embryo growth but did not explain how he came to this conclusion. Previous investigations have been designed to determine if embryos can develop in absence of ovarian function, but not to detect fine differences in the rate of embryonic development. It is difficult to obtain snakes which contain embryos in the same stage with which to make a comparison. In addition, some embryological stages last so long that embryos might easily differ by more than a week (Zehr, 1962). However data summarized in Table 2, which were obtained from pairs of luteectomized and sham luteectomized snakes containing embryos in the same embryonic stage of development, suggest that luteectomy had no effect on the rate of embryonic development between Zehr stages 7 and 35. It has been suggested that CL of livebearing reptiles function through the mediation of progesterone to inhibit follicular growth during pregnancy (Callard, Doolittle, Banks, and Chan, 1972; Browning, 1973). Our macroscopic observations of ovarian follicles in garter snakes with intact CL and those devoid of CL failed to reveal any evidence of subsequent ovulations or increases in follicular size. Thus luteal hormones do not by themselves appear to significantly influence follicular growth during gestation; however we cannot eliminate the possibility that they, in conjunction with other factors, might play some role in this effect. Luteectomy experiments in livebearing lizards may or may not be relevant to studies in live-bearing snakes. Investigators of the former have been consistent in reporting normal embryonic development following luteectomy thus supporting our finding in snakes; however their data relating to gestation differ greatly. Luteectomy produced abortion in Xanthusia
406
HIGHFILL
(Yaron, 1972), increased gestation in Zoococa (Panigel, 1956), and had no effect on gestation length in Chalcides (Badir, 1968). Such data indicate considerable species differences in luteal function in livebearing lizards, and consequently caution should be exercised in comparing studies between species of live-bearing reptiles. All reproductive malfunctions observed in this study following luteectomy of pregnant garter snakes can perhaps be explained as the result of prolonged pregnancy. For example, increased embryo death as well as decreased weight of young delivered would be expected when gestation was prolonged and the developing young were forced to exhaust their yolk supply (Callard et al., 1972) or catabolize their own tissues. But the fact that some young were born dead but still possessed considerable quantities of yolk at birth may indicate problems in elimination of nitrogenous wastes or respiratory exchange when embryos of an advanced age were subjected to a prolonged stay in utero. The fact that fewer birth products were delivered (Table 1) clearly indicates a basic upset in the process of parturition. It is, however, more difficult to adequately explain how luteectomy delayed parturition. It has been postulated that progesterone (Panigel, 1956) or progesterone and estrogen (Yaron, 1972) may stimulate development of uterine smooth muscle as reported in live-bearing lizards. Therefore inadequate uterine muscle development resulting from reduced hormone levels may have delayed parturition. Luteal hormones may stimulate uterine vascularity (Browning, 1973) as in some mammals. Thus luteectomy may have decreased uterine circulation which in turn affected gas and nutrient exchange or nitrogenous waste elimination. Luteal hormones could conceivably have a unique effect on parturition in reptiles that is unprecedented in other vertebrate classes. Arguments for and against each of these
AND
MEAD
possibilities can be presented, but unfortunately the data now available make it impossible to choose between them. We conclude that the function of garter snake CL is to secrete progesterone and perhaps other factors that either directly or indirectly influence the length of parturition and the number of birth products delivered. The mechanism or hormones responsible for this remain unknown. REFERENCES Badir, N. (1968). Structures and function of corpus luteum during gestation in the viviparous lizard Chalcides
ocellatus.
Anat.
Anzeiger
122,
l-10.
Bragdon, D. E. (1951). The non-essentiality of the corpora lutea for the maintenance of gestation in certain live-bearing snakes. J. Exp. 2001. 118, 41w35. Bragdon, D. E., Lazo-Wasem, E. A., Zarrow, M. X., and Hisaw, F. L. (1954). Progesterone-like activity in the plasma of ovoviviparous snakes. Proc. Sot.
Exp.
Eiol.
Med.
86, 477-480.
Browning, H. C. (1973). The evolutionary history of the corpus luteum. Biol. Reprod. 8, 128-157. Callard, I. P., Doolittle, J. P., Banks, W. L., Jr., and Chan, S. W. (1972). Recent studies on the control of the reptilian gonad cycle. Gen. Comp. Endocrinol.
Suppl.
3, 65-74.
Clark H., Florio, B., and Hurowitz, R. (1955). Embryonic growth of Thamnophis s. sirtalis in relation to fertilization date and placental function. Copeia, 9-13. Clausen, H. J. (1935). The effects of ovariotomy and hypophysectomy on parturition in snakes. Anat. Rec. 64, Suppl., 88. Clausen, H. J. (1940). Studies on the effect of ovariotomy and hypophysectomy on gestation in snakes. Endocrinology 27,700-704. Fox, W. (1954). Genetic and environmental variation in the timing of the reproductive cycles of male garter snakes. J. Morphol. 95,415-450. Hebard, W. B. (1950). A dimorphic color pattern of the garter snake Thamnophis elegans vagrans in the Puget Sound region. Copeia, 217-219. Highfill, D. R. and Mead, R. A. (197.5). Source and levels of progesterone during pregnancy in the garter snake, Thamnophis elegans. Gen. Corn. Endocrinol.
27,389-400.
Licht, P. (1972). Environmental physiology of reptilian breeding cycles: Effect of temperature. Gen. Corn.
Endocrinol.
Suppl.
3,477+88.
Pamgel, M. (1956). Contribution a I’ttude de 1’ ovoviviparite chez les reptiles: gestation et parturi-
LUTEAL
FUNCTION
tion chez le lezard vivpare Zooroca Ann.
Sci. Nat.
Zool.
vivipara.
18, 569-668.
Rahn, H. (1938). The corpus luteum of reptiles. Anat. Rec. 72, Suppl., 55. Rahn, H. (1939). Structure and function of placenta and corpus luteum in viviparous snakes. Proc. SW. Exp. Biol. Med. 40, 381-382.
IN THE
GARTER
407
SNAKE
Yaron, Z. (1972). Effects of ovariectomy and steroid replacement on the genital tract of the viviparous lizard, Xantusia vigilis. J. Morphol. 136, 313-326.
Zehr, D. R. (1%2). “Stages-in the normal development of the common garter snake, Thamnophis sirtalis
sirtalis.”
Copeia,
322-329.
AND
COMPARATIVE
ENDOCRINOLOGY
27, 401-407
(1975)
Function of Corpora Lutea of Pregnancy in the Viviparous Garter Snake, Thamnophis elegans DICK R. HIGHFILL AND RODNEY A. MEAD Department of Biological Sciences, University of Idaho, Moscow, Idaho 83843
Accepted July 12, 1975 Although corpora lutea of the viviparous garter snake, Thamnophis elegant, are the major source of plasma progesterone during pregnancy, a specific function for this steroid or any other luteal factor has yet to be shown. Thirty pregnant snakes weighing 80-200 g were divided into three equal groups so that all groups contained snakes of comparable size. Equal numbers of snakes were luteectomized, sham luteectomized, or received no treatment during early pregnancy and were then allowed to complete gestation. Duration of pregnancy was significantly (P < 0.01) longer in luteectomized snakes than in either control group. Luteectomized snakes gave birth to a lower percentage of living young and fewer birth products, which also weighed significantly less. However, luteectomy did not prevent normal embryonic development and did not appear to affect the rate of embryonic development. There were no significant differences between intact and sham luteectomized snakes in any of the above parameters. We conclude that CL in the viviparous garter snake, Thamnophis elegans, function through the mediation of a hormone which ensures that parturition occurs at the normal time and with the delivery of a normal number of young. The mechanism or the compound responsible for this action remains unknown,
The function of corpora lutea (CL) in but hypophysectomy of Storeria and Naovoviviparous and viviparous snakes re- trix at this time resulted in abortion. Rahn mains controversial. It seems likely that (1939) reported that ovariectomy failed to CL in live-bearing snakes are endocrine produce resorption or abortion in 14 glands that function during pregnancy snakes (Thamnophis and Natrix) 25 days through the mediation of progesterone se- after treatment. However, castration procretion. The reasons for this latter supposiduced abortion in two out of six snakes tion have been presented elsewhere (Highduring the previous summer (Rahn, 1938). fall and Mead, 1975; Browning, 1973). Bragdon (1951) found that T. sirtalis or N. Data accessing luteal function in snakes is sipedon failed to abort or resorb their emcontradictory. Clausen (1935, 1940) found bryos after ovariectomy or hypophysecthat hypophysectomy of Natrix, Storeria, tomy in early, mid-, or late pregnancy. and Thamnophis sirtalis or ovariectomy of However, he did note an increase in the Natrix in early pregnancy resulted in re- length of pregnancy which was more comsorption of embryos. Hypophysectomy of plete subsequent to hypophysectomy than Natrix or ovariectomy of Natrix and ovariectomy . Unfortunately, these studies Storeria in midpregnancy resulted in abor- fail to clarify the function of CL in snakes tion or resorption of embryos. Ovariecnot only because the data are contradictomy of Natrix in late pregnancy failed to tory but also because they fail to deal prevent delivery of normal young at term, directly with CL. Ovariectomy or hypo401 Cwtiht All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
402
HIGHFILL
physectomy may produce effects which depend upon nonluteal structures. In addition, the inability to precisely determine the stage of pregnancy at which a treatment was performed makes it difficult to compare the results from different experiments. We have chosen to work with T. elegans because the CL of this species are known to be the primary source of progesterone during pregnancy (Highfill and Mead, 1975); hence removal of these glands should clarify the hormonal role of this steroid. Moreover, the stage of pregnancy can be determined accurately by classifying the developing embryos using the method of Zehr (1962). Our purpose was to determine the effect of luteectomy upon embryo survival, embryo development, the number birth products delivered, and the duration of pregnancy in Thamnophis elegans. MATERIALS
AND
MEAD
experiment. Snakes were maintained on a natural photoperiod (sunlight only) from June through midSeptember and temperature was not controlled. However, due to a prolonged cold spell in September the snakes were maintained at approximately 70 degrees on a photoperiod of 14 hr light and 10 hr dark for the remainder of the experiment. The date, number of birth products (live embryos, dead embryos, or eggs), and weight of fully developed young (alive or dead) were recorded within 24 hr after parturition. The snakes were killed and preserved in AFA (three parts 95% ethanol, one part acetic acid, one part formalin, and five parts water) at the end of the experiment and the number of birth products remaining in utero and their stage of development were recorded. During the previous year nine pregnant T. .&guns weighing 60-120 g were luteectomized (five snakes) or sham luteectomized (four snakes) in an attempt to determine the effect of luteectomy on the rate of embryonic development. At the time of treatment, an embryo was taken and classified according to Zehr stage. After approximately equal times had elapsed, one snake from each treatment was laparotomized and another embryo was taken and classified according to Zehr stage. All surgical treatments were carried out under ether anesthesia and the animals were housed and fed as described above.
AND METHODS
Thirty female Thamnophis elegans (weighing N-200 g) were captured near Moscow, Idaho, from April through June and brought into the laboratory. Prior to ovulation, snakes were kept in a large screen covered enclosure and provided with Styrofoam containers in which to hide. Snakes were palpated to determine growth of follicles and time of ovulation. After all snakes had ovulated, 10 snakes of approximately equal weight were randomly assigned to each of three treatment groups: luteectomy, sham luteectomy, and intact (no treatment). Snakes were anesthetized with ether and an incision made over each ovary. In the luteectomy group, the ovary was exposed and the CL were surgically removed. Snakes receiving sham luteectomies were treated in an indentical manner, except that CL were not removed. The intact snakes received no surgical treatment. The embryo nearest the right ovary was removed from the right oviduct in snakes which received surgical treatments and the stage of embryo development determined by the method of Zehr (1%2). Snakes received an injection (im) of 5000 IU of penicillin G and 6.25 mg of dihydrostreptomycin on the day following surgery. Snakes were then housed individually in separate cages with screen covers. Snakes were given free access to water and were force-fed canned dog food at 3- to 4-wk intervals for the duration of the
RESULTS
A one way analysis of variance indicated that snakes receiving no treatment or sham luteectomy did not differ in any of the parameters tested, thus verifying that surgical treatment did not adversely affect the measurements made in this study (Table 1). However, luteectomy significantly reduced the percentage of young born alive (P < 0.005) as well as the number of birth products delivered (P < 0.025) (Table 1). Luteectomy also significantly lengthened gestation (P < 0.01) and reduced the mean weight oi fully developed progeny (P < 0.005) (Table 1). All progeny delivered in this experiment were fully developed but about one-half were attached to placentae in which the yolk sac contained considerable amounts of yolk, whereas the others were devoid of yolk. The particular Zehr stage at which luteectomy was performed during
8.8 (41.2) 4.7 (+1.3)*
10 9
7-22 (11.4) 7-23 (12.6)
10.4 (-c1.8Y
7
-
Mean total number of birth productsb*e 75 (15.5) [55i73] 64 (k9.9) [56/87] 14 (*5.9)*** [5/37]
Mean % young born viabled 96 (22.7) [7Oi73] 97 (k2.8) [84/87] 89 (k6.7) [33/373
Mean % born fully developede
106.4 (r2.4)**
97.4 (22.6)
94.3 (~2.3)
Mean duration of pregnancy in days
a This refers to the range of Zehr stages at the time snakes were treated and the mean Zehr stage for the treatment group. b Birth products (dead young + live young + eggs). c One embryo removed after luteectomy of sham luteectomy, thus making these values somewhat lower than intact animals. d The numbers in brackets in this column represent the total number of viable young/total number of birth products. e The numbers in brackets in this column represent the total number of fully developed young/total number of birth products. f AU numbers in parentheses represent the standard errors of the means. * (P < 0.025). ** (P < .OlO). *** (P < .ooS).
Luteectomy
Sham luteectomy
None
Treatment
Number of snakes
ZehP stage (mean and raw39
1.70 (-eo.z)***
2.31 (20.10)
2.65 (-0.18)
Mean weight of fully developed young 62)
TABLE 1 THE EFFECT OF LUTEECTOMY, SHAMLUTEECTOMY, ORNOTREATMENT ONVARIOUSPARAMETERS OF PREGNANCY
2
? s K
$
2 3 :! 8 z
2 3 F
HIGHFILL
404 TABLE
2
COMPARISON OF ZEHR STAGES ELAPSED AND DAYS ELAPSED IN LUTEECTOMIZED AND SHAM LUTEECTOMIZED GARTER SNAKES
Treatment Sham luteectomy Luteectomy Lureectomy Sham luteectomy Luteectomy Sham luteectomy Luteectomy Sham luteectomy Luteectomy
Zehr stage at first laparotomy
Zehr stage at second laparotomy
Number of days elapsed from first to second laparotomy
7 7 7
28 27 28
23 22 20
7 7
29 29
35 35
7 7
30 30
26 35
7 7
35 35
67 67
trimester 1 did not appear to affect the reproductive parameters observed (Table 1). Rate of embryonic development between Zehr stages 7 and 35 was not significantly affected by luteectomy (Table 2). Of the 10 snakes that were initially included in the intact group, two were subsequently found to be not pregnant and the third escaped. They therefore were omitted from the statistical analysis. One of the luteectomized snakes died shortly after treatment and this animal was also eliminated from the study. DISCUSSION
Earlier experiments concerning luteal function in live-bearing snakes, as determined by ovariectomy or hypophysectomy (Clausen, 1935, 1940; Rahn, 1938, 1939; Bragdon, 1951) must be interpreted with caution. These earlier experiments related the stage of pregnancy to time of year rather than embryological development. Since the time of ovulation is highly variable in garter snakes, errors up to one-
AND MEAD
third the duration of pregnancy may have occurred (Bragdon, Lazo-Wasem, Zarrow, and Hisaw, 1954). In addition, ovariectomy removed interstitial tissue and ovarian follicles as well as the CL, while hypophysectomy presumably altered luteal function as well as that of other endocrine glands. Consequently, these earlier experiments with live-bearing snakes may have obscured the true function of CL by also altering the function of other nonluteal tissues. Luteectomy early in trimester I lengthened pregnancy in T. elegans, thus supporting results of ovariectomy studies by Bragdon (1951). Unfortunately he failed to present data regarding duration of pregnancy for the three Natrix and three T. sirtalk which were ovariectomized. Bragdon assumed CL were dependent upon pituitary hormones, thus hypophysectomy would remove luteal function. Interpreted in this way, his hypophysectomy data also support our findings. However, hypophysectomy undoubtedly removed posterior pituitary function too. Since pituitary extracts (Clausen, 1940) or oxytocin (unpublished data) will initiate parturition in livebearing snakes, Bragdon’s observations could also be attributed to the lack of a potent inducer of parturition. Contrary to our findings and those of Bragdon (1951), Clausen (1935, 1940) reported that hypophysectomy or bilateral ovariectomy of live-bearing snakes in early or midpregnancy produced abortions. Rahn (1938, 1939) found castration produced mixed results in Natrix and Storeria. In one experiment two of six snakes aborted, but in the second none of 11 snakes aborted. It is possible that the surgery performed by Clausen (1935, 1940) and Rahn (1939) induced release of oxytocin, which in turn produced abortions. We observed no abortions in the experiments we describe here. However, in previous years, we occassionally observed abortions following surgery even in snakes with intact CL, which is
LUTEAL
FUNCTION
IN
consistent with the idea of trauma induced oxytocin release. However, abortions following hypophysectomy are difficult to explain in terms of oxytocin release. The fact that Clausen failed to histologically confirm the success of his hypophysectomies may indicate that sufficient posterior pituitary tissue remained to effect oxytocininduced abortions. Duration of pregnancy in our garter snakes did not differ significantly (Table 1) in untreated and sham luteectomized snakes, thus indicating that surgical treatment alone did not prolong gestation. However, we noted that captive intact snakes had a longer gestation than that reported for this species in the wild (Hebard, 1950; Zehr, 1%2). It is possible that captive snakes lacked the ability to behaviorally thermoregulate adequately, thus prolonging pregnancy. The fact that 120 pregnant garter snakes out of a total of 160 males and females collected during midMay to early July were found sunning themselves near protected areas during the morning when temperatures were rising rapidly after a relatively cold night (less that 50 degrees F) indicates behavior is an important factor in temperature regulation. These data support the contentions of others who have found temperature to be an important factor in the reproduction of garter snakes (Clark, Florio, and Hurowitz, 1955; Fox, 1954) and other reptiles (Licht, 1972). It should also be noted that the mean number of birth products delivered (10.4) did not appear to differ greatly from other observations (9.7) in this species (Hebard, 1950). Snakes luteectomized during early pregnancy gave birth to a lower percentage of living young than intact or sham luteectomized garter snakes (Table 1). Thus normal luteal function appears to enhance the percentage of viable offspring produced. We concur with investigators who found that luteal function was not essential for completion of embryo development in live-
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bearing snakes (Bragdon, 1951; Rahn, 1939). However, Clausen (1940) reported ovariectomy of T. sirtalis, Natrix, and Storeria retarded embryo growth but did not explain how he came to this conclusion. Previous investigations have been designed to determine if embryos can develop in absence of ovarian function, but not to detect fine differences in the rate of embryonic development. It is difficult to obtain snakes which contain embryos in the same stage with which to make a comparison. In addition, some embryological stages last so long that embryos might easily differ by more than a week (Zehr, 1962). However data summarized in Table 2, which were obtained from pairs of luteectomized and sham luteectomized snakes containing embryos in the same embryonic stage of development, suggest that luteectomy had no effect on the rate of embryonic development between Zehr stages 7 and 35. It has been suggested that CL of livebearing reptiles function through the mediation of progesterone to inhibit follicular growth during pregnancy (Callard, Doolittle, Banks, and Chan, 1972; Browning, 1973). Our macroscopic observations of ovarian follicles in garter snakes with intact CL and those devoid of CL failed to reveal any evidence of subsequent ovulations or increases in follicular size. Thus luteal hormones do not by themselves appear to significantly influence follicular growth during gestation; however we cannot eliminate the possibility that they, in conjunction with other factors, might play some role in this effect. Luteectomy experiments in livebearing lizards may or may not be relevant to studies in live-bearing snakes. Investigators of the former have been consistent in reporting normal embryonic development following luteectomy thus supporting our finding in snakes; however their data relating to gestation differ greatly. Luteectomy produced abortion in Xanthusia
406
HIGHFILL
(Yaron, 1972), increased gestation in Zoococa (Panigel, 1956), and had no effect on gestation length in Chalcides (Badir, 1968). Such data indicate considerable species differences in luteal function in livebearing lizards, and consequently caution should be exercised in comparing studies between species of live-bearing reptiles. All reproductive malfunctions observed in this study following luteectomy of pregnant garter snakes can perhaps be explained as the result of prolonged pregnancy. For example, increased embryo death as well as decreased weight of young delivered would be expected when gestation was prolonged and the developing young were forced to exhaust their yolk supply (Callard et al., 1972) or catabolize their own tissues. But the fact that some young were born dead but still possessed considerable quantities of yolk at birth may indicate problems in elimination of nitrogenous wastes or respiratory exchange when embryos of an advanced age were subjected to a prolonged stay in utero. The fact that fewer birth products were delivered (Table 1) clearly indicates a basic upset in the process of parturition. It is, however, more difficult to adequately explain how luteectomy delayed parturition. It has been postulated that progesterone (Panigel, 1956) or progesterone and estrogen (Yaron, 1972) may stimulate development of uterine smooth muscle as reported in live-bearing lizards. Therefore inadequate uterine muscle development resulting from reduced hormone levels may have delayed parturition. Luteal hormones may stimulate uterine vascularity (Browning, 1973) as in some mammals. Thus luteectomy may have decreased uterine circulation which in turn affected gas and nutrient exchange or nitrogenous waste elimination. Luteal hormones could conceivably have a unique effect on parturition in reptiles that is unprecedented in other vertebrate classes. Arguments for and against each of these
AND
MEAD
possibilities can be presented, but unfortunately the data now available make it impossible to choose between them. We conclude that the function of garter snake CL is to secrete progesterone and perhaps other factors that either directly or indirectly influence the length of parturition and the number of birth products delivered. The mechanism or hormones responsible for this remain unknown. REFERENCES Badir, N. (1968). Structures and function of corpus luteum during gestation in the viviparous lizard Chalcides
ocellatus.
Anat.
Anzeiger
122,
l-10.
Bragdon, D. E. (1951). The non-essentiality of the corpora lutea for the maintenance of gestation in certain live-bearing snakes. J. Exp. 2001. 118, 41w35. Bragdon, D. E., Lazo-Wasem, E. A., Zarrow, M. X., and Hisaw, F. L. (1954). Progesterone-like activity in the plasma of ovoviviparous snakes. Proc. Sot.
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Browning, H. C. (1973). The evolutionary history of the corpus luteum. Biol. Reprod. 8, 128-157. Callard, I. P., Doolittle, J. P., Banks, W. L., Jr., and Chan, S. W. (1972). Recent studies on the control of the reptilian gonad cycle. Gen. Comp. Endocrinol.
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Clark H., Florio, B., and Hurowitz, R. (1955). Embryonic growth of Thamnophis s. sirtalis in relation to fertilization date and placental function. Copeia, 9-13. Clausen, H. J. (1935). The effects of ovariotomy and hypophysectomy on parturition in snakes. Anat. Rec. 64, Suppl., 88. Clausen, H. J. (1940). Studies on the effect of ovariotomy and hypophysectomy on gestation in snakes. Endocrinology 27,700-704. Fox, W. (1954). Genetic and environmental variation in the timing of the reproductive cycles of male garter snakes. J. Morphol. 95,415-450. Hebard, W. B. (1950). A dimorphic color pattern of the garter snake Thamnophis elegans vagrans in the Puget Sound region. Copeia, 217-219. Highfill, D. R. and Mead, R. A. (197.5). Source and levels of progesterone during pregnancy in the garter snake, Thamnophis elegans. Gen. Corn. Endocrinol.
27,389-400.
Licht, P. (1972). Environmental physiology of reptilian breeding cycles: Effect of temperature. Gen. Corn.
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Pamgel, M. (1956). Contribution a I’ttude de 1’ ovoviviparite chez les reptiles: gestation et parturi-
LUTEAL
FUNCTION
tion chez le lezard vivpare Zooroca Ann.
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18, 569-668.
Rahn, H. (1938). The corpus luteum of reptiles. Anat. Rec. 72, Suppl., 55. Rahn, H. (1939). Structure and function of placenta and corpus luteum in viviparous snakes. Proc. SW. Exp. Biol. Med. 40, 381-382.
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Yaron, Z. (1972). Effects of ovariectomy and steroid replacement on the genital tract of the viviparous lizard, Xantusia vigilis. J. Morphol. 136, 313-326.
Zehr, D. R. (1%2). “Stages-in the normal development of the common garter snake, Thamnophis sirtalis
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