Mating-induced ovarian recrudescence in the red ... - Springer Link

J Comp Physiol A (1990) 166:629-632

~'nal

of Neu~l,

and

9 Springer-Verlag 1990

Mating-induced ovarian recrudescence in the red-sided garter snake M a r y T. M e n d o n ~ a and David Crews Institute of Reproductive Biology, University of Texas at Austin, Austin, Texas 78712, USA Accepted October 26, 1989

Summary. In the female red-sided garter snake (Thamnophis sirtalis parietalis) mating initiates a neuroendocrine reflex that has both a short-term (within hours) effect on circulating estrogen concentrations and a long-term (6-7 weeks) effect on ovarian development. The perception o f mating appears at least facultative, if not obligatory, for the initiation and maintenance o f vitellogenesis and hence successful reproduction. Key words: Neuroendocrine reflex - Ovarian recrudescence - Proximates cues

Introduction Communication between the sexes is necessary to ensure successful reproduction. Vertebrate males often utilize a variety of sensory modalities (visual, auditory, chemical and/or tactile) to activate specific neuroendocrine responses in the female to modulate reproduction (Wingfield and Marler 1988). Some signals affect the timing of gamete release, such as in the case of spawning in some fish (visual and pheromonal cues), amplexus in most frogs (tactile cue), and reflex ovulation in some mammals (tactile cue). Other signals only affect gamete maturation. For example, courtship displays (visual or auditory) can affect ovarian development in a variety of vertebrates (Wingfield and Marler 1988), and primer pheromones (chemical) can accelerate the onset of puberty (Vandenbergh 1988). The perception of these signals is thought to activate certain neuroendocrine pathways which modulate the secretion of gonadotropin releasing hormone ( G n R H ) which in turn influences both gonadotropin secretion and gonadal state (Bronson 1988).

Abbreviations: E estradiol-17fl; GnRH gonadotropin releasing hormone; LH luteinizing hormone

Female red-sided garter snakes in Manitoba, Canada exhibit a dissociated pattern of reproduction (Crews 1984). Mating occurs when ovaries are regressed and circulating estradiol-17/3 (E) concentrations minimal (Garstka et al. 1985; Whittier et al. 1987). Mating is primarily observed in the spring, when receptive females emerge from the hibernacula. Within 4 h of mating, females display an elevation in E which peaks 24 h after mating (Garstka et al. 1985; Whittier et al. 1987). This E surge is presumably mediated through an increase in gonadotropin (Licht 1984; Crews and Silver 1985). In some years, ovaries of mated females become vitellogenic at a higher frequency than those of unmated females (Whittier and Crews 1986). In other years, unmated females almost never become vitellogenic (Bona Gallo and Licht 1983; Garstka et al. 1985). It appears that mating may play an important role in ovarian activation. We document here that the red-sided garter snake (Thamnophis sirtalis parietalis) exhibits an unique signalling system in which the perception of a tactile stimulus (intromission) initiates a neuroendocrine reflex which has both immediate (within hours) and long-term (within 6-7 weeks) effects on female reproduction. These responses indicate that mating is facultative to or, in some years, obligatory for the initiation and maintenance of vitellogenesis and for ovulation.

Materials and methods Freshly emerged, unmated females were collected from dens surrounding Narcisse, in the Interlake region of Manitoba, Canada in the spring of 1987 and 1988. Females were placed in one of 3 experimental treatments to directly test what role the perception of copulation plays in initiating both the E surge and future ovarian development. In order to disrupt the neural transmission of the signals from the cloaca during mating, females either underwent spinal transection, received a topical administration to the cloaca of lidocaine, or a short-acting local anesthetic that affects neuronal sodium channels (Cohen et al. 1986), or received an injection of tetracaine, a more potent, longer-acting local anesthetic which affects calcium channels (Feldman et al. 1986).

630

M.T. Mendonga and D. Crews: Mating-induced ovarian recrudescence in garter snake

In the first experimental set, transections were done 24 h before behavioral testing. We anesthetized females with a sub-cutaneous injection of Brevitol (15 mg/kg body mass) in the neck region. A dorsal incision was then made midway between the level of the ovary and the cloaca and the spinal cord cut in the transected animals or pinched in the shams. The wound was then cauterized and sutured. Animals had completely recovered from the anesthesia by the time of behavioral testing. In the spinal transection experiment, results from a pilot study in spring, 1987 were combined with a more extensive study done in spring 1988 after circulating estrogen level data were found not to differ significantly between years. Data from the two other experiments were collected in spring, 1988. In the lidocaine experiment, a 2% solution of lidocaine was swabbed onto and into (approximately 2 cm) the cloaca of the female just prior to placing her with courting males. Sham females were swabbed with distilled water. In the final experiment, 0.2 ml of a 0.5% tetracaine solution was injected intramuscularly at the cloaca approximately 10 min before courtship began. Behavior tests were conducted between 1000 and 1500 on sunny days. Treated or sham females were placed into 1 • 1 x 1 m arenas with 20 courting males to each female. Females were either allowed to mate naturally or, after 20 min of courtship, assisted by lifting their tail, thus gaping their cloaca, which permitted intromission by the male. There was no significant difference in E levels 24 h after mating between sham females that had been naturally mated or those that had been assisted (16.8___8.45 vs. 9.03+8.68 ng/ml, df=14, t=1.35, P=0.10). Treated and sham-operated or shaminjected females were courted and allowed to copulate. Blood samples were taken immediately following the end of intromission, and again 24 h after mating. Plasma was analyzed for estrogen by cellitte column chromatography and radioimmunoassay (Whittier et al. 1987). Variances of estrogen levels were found to be heterogeneous. Therefore E plasma level data were either analyzed by non-parametric statistical methods (Wilcoxon signed rank test) or log transformed for analysis by parametric statistics (t-test, ANOVA). Initial (+ 0 h) estrogen levels did differ significantly among the groups (F=2.60; dr=7,71; P=0.02). A Duncan's multiple comparison test found that the initial E levels of the Lidocaine Treated and Control Mated groups were significantly lower from those of the other treatment groups but did not differ significantly from one another. Since snakes were randomly collected upon emergence and randomly assigned to groups, we do not believe that these lower estrogen levels reflect a difference in these groups' initial physiological condition. Rather, the values for all groups were at the edge of the detectable level range of the assay, and the Lidocaine Treated and Control Mated groups had higher number of values in the non-detectable range. Females were laparotomized 7 weeks after mating to assess ovarian development. Fisher's Exact test was used to determine significant differences in ovarian development between number of sham and treated females in the 3 experimental (control, spinal, lidocaine, tetracaine) groups.

Results and discussion In all 3 manipulations, s h a m females experienced significant increases in levels o f E 24 h after m a t i n g (Table 1). These elevated concentrations did n o t differ significantly f r o m those seen in this time period for control u n m a n i pulated m a t e d females ( F = 0 . 4 7 , d f = 3 9 , P = 0 . 7 0 ) . Females with interrupted (either anatomically or chemically) sensory input, did n o t exhibit an estrogen surge (Table 1). Their circulating E concentrations 24 h after mating did n o t differ significantly f r o m females that were courted but n o t m a t e d ( t = 1 . 9 8 , P = 0 . 0 7 ) . Lidocainetreated females displayed a slight but significant increase

in E within 24 h o f mating, but this elevation was significantly lower (P = 0.0004) than those observed for control or sham m a t e d females and was not considered a surge (Table 1). This blockage o f an estrogen surge by interr u p t i o n o f afferent p a t h w a y s is the first direct evidence for a neural reflex mediating a short-term h o r m o n a l response via the spinal c o r d to the h y p o t h a l a m o - h y p o p h y seal-gonadal axis in reptiles. Sensory perception o f intromission also significantly affected ovarian vitellogenesis as m u c h as 7 weeks after mating. In all 3 experiments, significantly fewer treated females had vitellogenic follicles (transition f r o m ' w h i t e ' to ' y e l l o w ' follicles which occurs at approximately 5 m m in length) 6 weeks after copulation (Fig. 1). The n u m b e r o f s h a m females with ovarian development was statistically equivalent to that o f control m a t e d females (Z 2 = 0.38, d f = 3 , P = 0 . 9 4 ) . The average m a x i m u m follicle length o f all 3 treated g r o u p s was u n d e r 5.0 m m or essentially previtellogenic (x___l SE; Lidocaine 4.18_+0.49; Tetracaine 3.25 ___0.83 ; Spinal 4.65 ___0.45). Some lidocaine-treated and spinally transected females exhibited ovarian development (and, in the case o f the lidocaine treatment, one female ovulated). This result m a y be due to the incomplete blockage o f the transmission o f c o p u l a t o r y stimuli. In the rabbit, blockage o f vaginal stimuli alone (as w o u l d occur with the topical administration o f lidocaine) is n o t sufficient to prevent a L H surge (Fee a n d Parkes 1930; B r o o k s 1985). Other c o p u l a t o r y cues such as stimulation o f skin o f the female's tail and cloaca by the male m a y also contribute to stimulating ovarian growth. It should be noted that n o n e o f the females treated with tetracaine, the longer-lasting and m o r e pervasive anesthetic, exhibited any ovarian development in response to mating. Also, the administration o f anesthetic alone does n o t affect ovarian development (a general anesthetic is routinely used for laparotomies, after which females still p r o d u c e viable young). Tactile stimuli associated with m a t i n g have been k n o w n to activate a transient n e u r o e n d o c r i n e reflex that results in a relatively immediate surge in L H (luteinizing h o r m o n e ) and ovulation o f m a t u r e follicles in a variety o f species (Ramirez and Beyer 1988). M a t i n g has n o t been d o c u m e n t e d to have long lasting effects u p o n ovarian maturation. Indeed, in species with an associated pattern o f r e p r o d u c t i o n (mating occurs while g o n a d s are enlarged) ovarian recrudescence is primarily influenced by environmental rather than behavioral cues. Furthermore, species in which courtship displays m o d u l a t e ovarian development often require these displays to occur over an extended time period in order to be effective (Barfield 1971). The n e u r o e n d o c r i n e response seen in female garter snakes appears unique in that a single set o f tactile stimuli (intromission) can cause n o t only a relatively immediate and acute change in circulating h o r m o n e levels but also influence ovarian recrudescence for weeks after the stimulus. This system is unusual because a cue associated with mating, rather than abiotic cues, appears to induce ovarian development. However, in some years, u n m a t e d


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Table 1. Mean circulating estrogen levels at the end of mating (+ 0 h) and at + 24 h for different experimental treatments. Numbers are mean (1 standard error) and sample size. Significance levels and Z and t statistics were calculated from Wilcoxon signed ranked test and Student's t-test, respectively Experimental treatment

Mated control Unmated control

Sham Spinal transection

Sham

Mean estrogen level (ng/ml) +0 h

+24 h

0.26 (0.23) 7 0.56 (0.26) 7

13.32 (5.01) 6 0.50 (0.27) 7

0.65 (0.38) 12 0.50 (0.23) 10 0.22 (0.12) 13 0.05 (0.01) 15

Lidocaine

7.98 (2.91) 13 0.32 (0.09) 15

0.42 (0.18) 8 0.17 (0.09) 7

Sham Tetracaine

El00

10.89 (3.98) 8 0.40 (0.16) 7

I

Test statistic

P

0 h vs 24 h

Z=

2.2

0.01

0 h vs 24 h

Z = -0.51

0.31

24hvs24h

t =

3.13

0.005

0h vs 24h

Z=

3.06

0.001

0hvs24h

Z=

1.27

0.10

24 hvs 24h

t =

7.32

>0.0001

0 h vs 24h

Z=

3.06

0.001

0hvs24h

Z=

2.07

0.02

24hvs24h

t =

3.98

0.0002

0vs24h

Z=

1.68

0.05

0hvs24h

Z=

1.32

0.09

24hvs24h

t =

1.71

0.05

Control mafed Sham I Treafmen,

p=O.05

r

~

12.08 (2.75) 12 0.20 (0.10) 10

Statistical comparison

90

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p=O.01

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Spinal

Lidocaine

Tefracaine

Fig. 1. Percent of female red-sided garter snakes exhibiting ovarian development (either secondary vitellogenesis or ovulation) 6 weeks after mating. Four experimental groups are represented: Unmanipulated mated, Spinal (transected and sham-operated), Lidocaine (swabbed vs. sham-swabbed), and Tetracaine (injected vs. shaminjected). Fisher's Exact Test was used to calculate significance levels

females can also b e c o m e vitellogenic (Whittier and Crews 1986). Apparently, (an)other cue(s) m u s t be able to initiate n e u r o e n d o c r i n e changes similiar to those induced by the mating-induced n e u r o e n d o c r i n e reflex. It is u n k n o w n w h a t cue(s) is (are) stimulating vitellogenesis in u n m a t e d females but the females' energetic state has been postulated as a factor (Whittier and Crews 1989). It seems clear, however, that m a t i n g is a facultative and, in certain circumstances obligatory, stimulus for vitellogenesis in the female red-sided garter snake. This mating-induced n e u r o e n d o c r i n e reflex appears to be a function o f the dissociated pattern o f reproduction o f this species. In typical reflex ovulators, m a t i n g initiates the ovulation o f already m a t u r e follicles by m o dulating G n R H release. In contrast, m a t i n g in the garter snake, by apparently co-opting the same G n R H m o d u lating mechanism, influences the onset o f vitellogenesis itself. Given the harsh e n v i r o n m e n t o f n o r t h e r n C a n a d a where ovarian growth, in utero development o f y o u n g and a c c u m u l a t i o n o f fat reserves for an 8 m o n t h hibernation m u s t all be accomplished in a very short time span, early and correct allocation o f energy resources is criti-

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cal. M a t i n g u p o n emergence f r o m the h i b e r n a c u l u m provides a n i m m e d i a t e a n d reliable cue for o v a r i a n developm e n t . This f u n c t i o n a l m o d i f i c a t i o n of the m a t i n g - i n duced n e u r o e n d o c r i n e reflex m a y reflect the e v o l u t i o n of a n e u r a l response to the challenge o f the limiting e n v i r o n m e n t o f these animals.

Acknowledgments. We thank M. Shoesmith and W. Koonz of the Manitoba Department of Natural Resources for their assistance in this research. We also thank J. Bull, J.M. Manning, M.J. Ryan, J. Wade, and W. Wilczynski for comments on various manuscript drafts. MTM was supported by NICHD Training Grant HDO7264. This research was funded by a grant from the Texas Higher Education Coordinating Board Advanced Research Program and a NIMH Research Scientist Development Award 00135 to DC.

References Barfield RJ (1971) Gonadotrophic hormone secretion in the female ring dove in response to visual and auditory stimulation by the male. J Endocrinol 49:305-310 Bona Gallo A, Licht P (1983) Effects of temperature on sexual receptivity and ovarian recrudescence in the garter snake Thamnophis sirtalis parietalis. Herpetologica 39:173-182 Bronson FH (1988) Seasonal breeding. In: Knobil E, Neill Jet al. (eds) Physiology of Reproduction. Raven Press, New York, pp 1831-1871 Brooks CM (1985) Studies in the neural basis of ovulation in the rabbit. Am J Physiol 113 : 18-19 Cohen JB, Correll LA, Dryer EB, Kuisk IR, Medynski DC, Strnad NP (1986) Interactions of local anesthetics with torpedo nicotine acetylcholine receptors. In: Roth SH, Miller KW (eds) Molecular and cellular mechanisms of anesthetics, Plenum, New York, pp 111-124 Crews D (1984) Gamete production, sex hormone secretion and mating behavior uncoupled. Horm Behav 18:22-28 Crews D, Silver R (1985) Reproductive physiology and behavior

interactions in nonmammalian vertebrates. In: Adler N, Pfaff D, Goy RW (eds) Handbook of Behavioral Neurobiology, vol 7. Plenum Press, New York, pp 101-182 Fee AR, Parkes AS (1930) Studies on ovulation III. Effect of vaginal anesthesia on ovulation in the rabbit. J Physiol 70:385-388 Feldman HS, Arthur GR, Covino BG (1986) Toxicity of intravenously administered local anesthetic agents in the dog: cardiovascular and central nervous system effects. In: Roth SH, Miller KW (eds) Molecular and cellular mechanisms of anesthetics. Plenum, New York, pp 395-414 Garstka WR, Tokarz R, Diamond M, Halpert A, Crews D (1985) Behavioral and physiological control of yolk synthesis and deposit in the red-sided garter snake (Thamnophis sirtalis parietalis). Horm Behav 19:137-153 Licht P (1984) Reptiles. In: Lamming GE (ed) Marshall's physiology of reproduction. Churchill Livingston, Edinburgh, pp 206282 Ramirez VD, Beyer C (1988) The ovarian cycle of the rabbit: its neuroendocrine control. In: Knobil E, Neill J e t al. (eds) Physiology of reproduction. Raven Press, New York, pp 18731892 Vandenbergh JG (1988) Pheromones and mammalian reproduction. In: Knobil E, Neill Jet al. (eds) Physiology of reproduction. Raven Press, New York, pp 1679-1696 Whittier JM, Crews D (1986) Ovarian development in red-sided garter snakes, Thamnophis sirtalis parietalis: relationship to mating. Gen Comp Endocrinol 61:5-12 Whittier JM, Crews D (in press) Body mass and reproduction in female red-sided garter snakes (Thamnophis sirtalis parietalis). Herpetologica Whittier JM, Mason RT, Crews D (1987) Plasma steroid hormone levels of female red-sided garter snakes, Thamnophis sirtalis parietalis: relationship to mating and gestation. Gen Comp Endocrinol 67:33-43 Whittier JM, Mason RT, Licht P, Crews D (1987) Role of light and temperature in the regulation of reproduction of the redsided garter snake, Thamnophis sirtalis parietalis. Can J Zool 65 : 2090-2096 Wingfield JC, Marler P (1988) Endocrine basis of communication. In: Knobil E, Neill J e t al. (eds) Physiology of reproduction. Raven Press, New York, pp 1647-1677