Reproduction in Female Copperhead Snakes (Agkistrodon contortrix): Plasma Steroid Profiles during Gestation and Post-Birth Periods

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1 Reproduction in Female Copperhead Snakes (Agkistrodon contortrix): Plasma Steroid Profiles during Gestation and Post-Birth Periods Author(s) :Charles F. Smith, Gordon W. Schuett and Shannon K. Hoss Source: Zoological Science, 29(4): Published By: Zoological Society of Japan DOI: URL: BioOne ( is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

2 ZOOLOGICAL SCIENCE 29: (2012) 2012 Zoological Society of Japan Reproduction in Female Copperhead Snakes (Agkistrodon contortrix): Plasma Steroid Profiles during Gestation and Post-birth Periods Charles F. Smith 1,4 *, Gordon W. Schuett 2, and Shannon K. Hoss 3 1 Department of Ecology and Evolutionary Biology, 75 N Eagle Road Unit 3043, The University of Connecticut, Storrs, Connecticut , USA 2 Department of Biology and Center for Behavioral Neuroscience, Georgia State University, 33 Gilmer Street, SE, Unit 8, Atlanta, Georgia , USA 3 Department of Biology, San Diego State University, 5500 Campanile Drive, San Diego, California , USA 4 Department of Biology, Wofford College, 429 North Church Street, Spartanburg, South Carolina 29303, USA We investigated levels of plasma progesterone (P4), 17β-estradiol (E2), testosterone (T), and corticosterone (CORT) during gestation and post-birth periods in wild-collected female copperhead snakes (Viperidae; Agkistrodon contortrix). We also sought to determine whether CORT levels at (or near) birth dramatically increase and were correlated with duration of labor and litter size. Specifically, pregnant subjects (N = 14) were collected during early- to mid-gestation, held in the laboratory, and repeatedly bled to obtain plasma for steroid analyses. Progesterone showed significant changes during gestation, with the highest levels at the onset of sampling (circa 50 days prior to birth); P4 progressively declined up to parturition, and basal levels were observed thereafter. At the onset of sampling, E2 was at peak levels and fell sharply at circa 30 days prior to birth, a trend observed throughout the post-birth sampling period. Throughout the entire sampling period, T was undetectable. Although CORT showed no significant changes during gestation and several days following parturition, there was a highly significant peak at the time of birth. Our findings mirror the results of previous studies on pregnancy and steroid hormones of other live-bearing snakes, lizards, and mammals. As expected, there was a significant relationship between duration of labor and litter size; however, although levels of CORT did not achieve significance, there was a positive trend with litter size. We suggest that elevation of CORT at birth is involved in the mobilization and regulation of energy stores necessary for the physiological process of parturition and as a possible mechanism to trigger birth. Key words: pregnancy, reproduction, reptiles, snakes, steroid hormones, viviparity * Corresponding author. Tel. : ; Fax : ; smithcf@wofford.edu doi: /zsj INTRODUCTION Live-birth (viviparity) in reptiles is an ancient reproductive strategy (Qiang et al., 2010; Wang and Evans, 2011), and among extant taxa persists only in lizards, amphisbaenians, and snakes (Blackburn, 1982, 1985; Shine, 1985; Guillette, 1987; Murphy and Thompson, 2011). The endocrinology of gestation and birth has been studied rather extensively in lizards (Guillette, 1979; Jones and Guillette, 1982; Cree and Guillette, 1991; Guillette et al., 1991; Rooney et al., 1992; Cree et al., 2003; Martínez-Torres et al., 2003; Atkins et al., 2006) but less so in snakes (Mead et al., 1981; Kleis-San Francisco and Callard, 1986; Whittier and Tokarz, 1992; Robert et al., 2009; Taylor and DeNardo, 2011). Concentrations and temporal occurrences of steroid hormones (e.g., androgens, estrogens, gluccocorticoids, progestins) during gestation in lizards and snakes are similar and resemble the patterns described for some fishes (elasmobranchs) and mammals (Norris and Jones, 1987). Accordingly, these convergent similarities are hallmarks for common function and adaptive evolution of pregnancy. Here, we report the profiles of several circulating (plasma) steroid hormones (progesterone [P4], 17β-estradiol [E2], testosterone [T], and corticosterone [CORT]) during gestation and post-birth periods in wild-collected copperheads (Agkistrodon contortrix) from the northeastern extreme of their range (Gloyd and Conant, 1990; Campbell and Lamar, 2004; Douglas et al., 2009). In this population, there is a single mating season restricted to late summer; females show obligate long-term sperm storage until ovulation and fertilization in spring, and births occur from mid August to mid September (Smith et al., 2009, 2010). In addition to documenting profiles of P4, E2, T and CORT during gestation,

3 274 C. F. Smith et al. we attempted to determine whether CORT is elevated during the period at or near birth, which in reptiles has been reported in the rattlesnake Crotalus atrox (Schuett et al., 2004a; Taylor and DeNardo, 2005, 2011). Elevation of CORT (and cortisol) at birth has been reported in mammals, including humans (Bell et al., 1997; Smith, 1999, 2007; Weiss, 2000; Beshay et al., 2007; McLean et al., 2007; Wada, 2008). Furthermore, to determine whether duration of parturition is influenced by litter size, and whether this relationship is related to levels of maternal CORT, duration of birth and litter size were obtained from most mothers. MATERIALS AND METHODS Study site The study site was located in a 485 ha parcel of basalt trap rock ridge ecosystem situated 4.75 km NW of Meriden, Connecticut. Topography is consistent with trap rock systems found throughout the Central Connecticut River Valley. North and south oriented ridges, 200 m in elevation, are bordered to the west by steep cliff faces and extensive talus slides, and to the east by gently sloping woodlands. Two prominent basalt ridges are located at this site. Details of the topography and climate of this area are presented elsewhere (Smith, 2007; Smith et al., 2009). Research subjects Females were located in the field by periodically visiting known gestation (rookery) sites (Smith, 2007). Females suspected of being pregnant (N = 14) were gently captured, brought to the UCONN laboratory, processed, and provided private enclosures, which consisted of plastic cages (61 cm L 40 cm W 12 cm H), supplied with newsprint as a floor covering and substrate heating by heat tape (8 cm wide) situated beneath and across the front end of the cage (35 C). Collection dates for females brought into the laboratory ranged from 5 June to 28 July. Measurements for each snake included body mass (BM: ± 0.5 g using a triple beam balance), and snout-vent length and tail length (SVL: ± 0.2 cm; TL: ± 0.2 cm, using a non-stretchable cloth measuring tape while the snake was restrained in a clear acrylic tube). Artificial lighting (eight 40 W fluorescent tubes) positioned 3 m above the cage was timer-controlled to simulate natural (Connecticut time) photoperiod. Laboratory rodents (mice) were offered as food every 10 days until parturition. However, none were consumed until after parturition. Water was available in glass bowls ad libitum. Collection of blood and plasma Sampling was conducted from early- to mid gestation (mean start of sampling pre-parturition ± 1.25 SE; range days) to 20 days post-parturition to document profiles of the steroid hormones described above. To obtain a blood sample, each subject was gently removed from their private enclosure using grabber tongs and secured in a clear plastic tube of appropriate size. This procedure was done quickly (1 3 min) to minimize handling stress and possible effects on steroid levels (Schuett et al., 2004b). Once secured, 1.0 ml of blood was harvested via cardiocentesis using 1.0 ml sterile tuberculin syringes (26-G5/8 ) treated with porcinederived heparin sodium (1,000 units/ml). Subjects were returned immediately to their enclosures following this procedure. Blood samples were placed in disposable microcentrifuge tubes (1 ml) and centrifuged at 2.3 g. at room temperature (~21 23 C) for 5 min. Plasma was collected using a micropipette fitted with a sterile disposable tip and transferred to another microcentrifuge tube that was permanently labeled with the specimen identification code and date. Plasma samples were placed in an ultra-low freezer ( 80 C) until radioimmunoassays (RIAs) could be performed. Radioimmunoassays (RIAs) of plasma for steroids The general procedures we followed for conducting radioimmunoassay (RIAs) for measurements of plasma P4, E2, T, and CORT are published (Schuett et al., 1996, 1997, 2002, 2004a, 2004b, 2005, 2006; Schuett and Grober, 2000; Taylor and Schuett, 2004). Briefly, all RIAs of total P4, E2, T and CORT included validation (quantitative recovery and parallelism). Total hormone levels were measured to permit comparison to our earlier studies (i.e. Schuett and Grober, 2000; Schuett et al., 1996, 2004b) and to those of others. A single RIA was performed for P4 and assay samples were run in duplicate (n = 394). The intra-assay coefficient of variation (CV) was 8.7%. Two RIAs were performed for E2; the intra-assay CVs were 7.9% and 12.5%, and the inter-assay CV was 11.9%. Two RIAs were performed for T; the intra-assay coefficients of variation (CV) were 9.1% and 11.1%. One RIA was performed for CORT, and the intra-assay CV was 2.4%. Values for all steroids are presented as arithmetic means ± 1 SE (ng/ml). Duration of parturition Duration of parturition was investigated to determine whether litter size influences maternal CORT levels. Females were observed about every 3 4 hr from 0700 to 0200 h through the study period. Imminent parturition was recognized by the onset of body contractions, and duration of parturition was recorded as the beginning of body contractions (Time 0) until the last neonate was completely expelled (Time 1). Parturition was observed for nine of 14 (64.3%) females, and all births occurred between 1300 and 1900 h. The 14 mothers produced a total of 67 neonates. After birth, blood was obtained from the mothers and subjected to abovementioned procedures for RIA analysis of steroids. Statistical analysis All statistical analyses were conducted using SAS 9.1 (SAS Institute Inc., 2004). Prior to analysis, we ensured that the assumptions of normality and homogeneity of variances were met, and whether outliers could be detected (Zar, 1999). To determine whether plasma P4 and CORT levels varied among the sampling dates (i.e., from 40 days pre- to 12 days post-parturition), we conducted separate repeated measures analyses of variance on logtransformed hormone values using PROC MIXED, with snake ID as the repeated unit and sampling date as the main effect. We used the variance components covariance structure in our models, as this provided the best fit to our data out of 11 possible covariance structures, as assessed by Akaike and Bayesian information criterion (Wolfinger and Chang, 1995). Post hoc tests were conducted to determine least-squares mean differences in hormone levels between all possible pairs of sampling dates; p-values were adjusted for multiple comparisons using the Tukey-Kramer method. Owing to the fact that levels of E2 were undetectable on the majority of sampling dates, repeated measures analysis was not performed; nonetheless, we do include these data in Fig. 1. Similarly, levels of T were not detectable in any of the sampling dates, but were not depicted in Fig. 1. We used Pearson correlation to examine the relationship between duration of parturition and litter size (i.e., number of neonates per litter) for nine of 14 females (Table 1). We were unable to witness births for five females. We conducted an additional correlation analysis to determine whether length of parturition was related to CORT levels measured at Day 1. Day 1 was selected over Day 0 owing to larger sample size. RESULTS Data on mothers (N = 14) and offspring (N = 67) are provided in Table 1. Offspring were removed from their mothers immediately after birth. Steroid profiles are provided in Fig. 1. In the repeated measures analysis of P4, the main effect

4 of sampling date was significant (F 14, 158 = 99.34; P < 0.001), indicating that mean levels of P4 varied throughout pregnancy and following parturition. Post hoc comparisons revealed that all pre-parturient levels of P4 were significantly higher than all post-parturient levels (P < 0.05). In the repeated measures analysis of CORT, we found a significant main effect of sampling date (F 14,158 = 18.28; P < 0.001), and mean levels of CORT on the day of parturition (Day 0) and the day after parturition (Day 1) were significantly greater than on all other sampling dates (P < 0.05) but were not significantly different. Between 30 and 40 days pre-parturition, mean E2 levels were elevated but quickly dropped to mostly undetectable levels throughout the remainder of pregnancy to 12 days post-parturition. T levels were not detected throughout the entire sampling period, and thus not depicted in Fig. 1. Fig. 1. Mean (± 1 SEM) plasma sex steroid profiles during and following pregnancy of female copperheads (Agkistrodon contortrix) based on 14 wild-collected individuals held in the laboratory. CORT = corticosterone; E2 = 17β-estradiol; P4 = progesterone. Testosterone (T) levels were undetectable and thus not depicted (see text). Significance is designated by different letters. Agkistrodon Pregnancy Hormones 275 We found a highly significant correlation between duration of parturition (Time 0 to Time 1) and litter size (r = 0.88, P = 0.009, N = 9), which indicated that duration of labor was longer in females with larger litters. Although a significant correlation was not found between duration of parturition and CORT levels at Day 0 (P > 0.05, N = 7), a positive trend was detected at Day 1 (r = 0.67, P = 0.09, N = 9). DISCUSSION Over the past several decades, sources and functional roles of circulating steroid hormones (e.g., androgens, estrogens, glucocorticoids, progestins) in females, particularly in viviparous taxa, are better understood (Murphy and Thompson, 2011). For example, various reproductive tissues of females, such as gonads, follicles, yolk, adrenals, and corpus luteum even the placenta and extra-embryonic membranes can synthesize and/or store steroids (e.g., Schwabl, 1993; Albergotti et al., 2009). Moreover, androgens, estrogens, and progestins can have relatively straightforward effects on behaviors associated with sexual receptivity (McNicol and Crews, 1979; Wu et al., 1985; Whittier et al., 1987; Whittier and Tokarz, 1992), vitellogenesis and yolk synthesis (Ho et al., 1982; Garstka et al., 1985; Ho, 1987; Wilson and Wingfield, 1992), and maintenance of pregnancy (Chan et al., 1973; Highfill and Mead, 1975a, b; Xavier, 1987; Bonnet et al., 1994, 2001; Edwards and Jones, 2001; Tsai and Tu, 2001; Custodia-Lora and Callard, 2002). Also, a growing number of studies provide information on patterns of circulating sex steroids related to gestation in livebearing snakes (Whittier and Tokarz, 1992; Schuett et al., 2004a; Taylor et al., 2004; Lind et al., 2010; reviewed by Taylor and DeNardo, Table 1. Data on reproduction of female copperheads (Agkistrodon contortrix) in this study. ID Date of collection SVL (cm) Mass (g) pre-parturition Mass (g) post-parturition Range of sampling dates Number of times sampled Date of parturition Duration of parturition Number of neonates Mass (g) (range, mean) Total mass (g) 622B 2001/7/ Jul 14 sep Aug 105 mins 3 live (7.94) Unk /6/ Jul 26 Aug Aug 135 mins 4 live (8.60) Unk /6/ Jul 6 Sep Aug * 3 live (7.49) /7/ Jul 3 Sep Aug 175 mins 6 live (9.08) C 2001/7/ Aug 16 Sep 13 5-Sep * 3 live (7.93) D 2001/6/ Jul 20 Aug Aug 140 mins 5 live, 1 stillborn* (8.24) Unk /6/ Jul 4 Sep Aug 120 mins 4 live (7.31) Unk /6/ Jul 17 Sep 15 3-Sep 145 mins 5 live (8.23) Unk /6/ Jul 28 Aug Aug 160 mins 6 live (9.54) 57.5 Unk /7/ Aug 22 Sep 15 9-Sep * 4 live (8.74) Unk /6/ Jul 14 Sep 14 1-Sep 115 mins 4 live (8.86) Unk /7/ Aug 26 Sep Sep * 6 live (8.73) Unk /6/ Jul 31 Aug Aug 185 mins 8 live (6.88) Unk /6/ Jul 7 Sep Aug * 5 live (8.58) * The still born individual was fully developed.

5 276 C. F. Smith et al. 2011). However, these studies represent less than threetenths of one percent of the approximately 3,000 extant species of snakes. Accordingly, studies of reproduction in female snakes are in their infancy (Taylor and DeNardo, 2011). Thus, conclusions of endocrine control are limited and preliminary, due to the fact that experimental research is largely limited to a single genus (Thamnophis), and predominately the taxon Thamnophis sirtalis parietalis. Expanding research to a range of taxa from various major lineages is needed for a robust synthesis (Shine, 2003; Taylor and DeNardo, 2011). Here, we documented profiles of several plasma sex steroids (P4, E2, T, and CORT) during pregnancy, at or near birth, and post-birth periods in a population of copperheads (Agkistrodon contortrix) from the extreme northeastern area of their distribution (Smith et al., 2009, 2010). Our study is among the few that provides individual-based steroid profiles in a live-bearing snake (see Murphy and Thompson, 2011). Based on extensive field observations of this population (Smith et al., 2009, 2010), the females described in the present study likely were mated in late summer, showed long-term sperm storage during winter, and ovulated in mid spring (late May or early June). Thus, based on the dates of collection (range: 5 June 28 July), our study documented steroid profiles of pregnancy from early- to mid gestation to birth. The timing of ovulation and parturition in captive-held females were indistinguishable from females we observed in the wild (Smith et al., 2009). The steroid profiles of female A. contortrix in this study were similar to those reported in other live-bearing vipers (Bonnet et al., 2001, 2002; Tsai and Tu, 2001; Schuett et al., 2004a; Taylor et al., 2004), as well as live-bearing lizards (Xavier, 1987; Edwards and Jones, 2001; Martínez-Torres et al., 2003). Furthermore, patterns of P4 and CORT were similar to those reported in mammals, including humans (Smith, 1999, 2007; Weiss, 2000; Zhang et al., 2008). Therefore, we believe that the hormone profiles documented here are a result of pregnancy, and not an artifact of captivity or capture stress. In snakes, most reports show elevated E2 levels during vitellogenesis, yolk synthesis/deposition, and follicular maturation, with subsequent declines at ovulation and throughout gestation (Bonnet et al., 2001; Edwards and Jones, 2001; Taylor et al., 2004; Moore and Johnston, 2008). Recently, studies by Van Dyke and Beaupre (2011) on female A. contortrix and other live-bearing snakes have documented that metabolic costs associated with vitellogenesis and follicular development exceed the cost of pregnancy. Consequently, the dynamics of steroid hormones (e.g. E2, T) and metabolism of female reproduction is a rich topic for future exploration. We detected that levels of E2 fell sharply at about 30 Day, and levels remained basal throughout sampling. Several authors have suggested that high post-ovulatory levels of P4 suppress levels of E2 during pregnancy (Bonnet et al., 1994; Edwards and Jones, 2001), and this association was detected in the present study; however, this relationship remains to be tested experimentally. The role of P4 in the maintenance of pregnancy in squamates (snakes and lizards) has been reviewed (Edwards and Jones, 2001; Custodia-Lora and Callard, 2002; Martínez-Torres et al., 2003), but its involvement in labor is less well understood. Nonetheless, we detected the characteristic rapid decline in P4 levels just preceding birth, and assume that this change was due to luteolysis, regression of corpora lutea (Highfill and Mead, 1975a, b; Jones and Guillette, 1982; Xavier, 1987; Martínez-Torres et al., 2003). Relatively few studies report on T and other androgen levels in female snakes (Saint Girons et al., 1993; Taylor and DeNardo, 2011), but these levels tend to be high only during vitellogenesis and follicular growth, and similar to E2, and are generally low (basal) following ovulation and during gestation. Because we initiated sampling at early- to mid gestation, the fact that T levels were not detected in our samples was not unexpected. The function of CORT and corticotropin-releasing hormone (CRH) during labor has been studied rather extensively in animal (e.g., sheep) models and humans (Norwitz et al., 1999; Beshay et al., 2007). The characteristic sharp spike in plasma CORT at parturition is possibly derived from multiple sources (Bell et al., 1997; Petershack et al., 1999; Wada, 2008). The source of plasma CORT in pregnant snakes has not been experimentally investigated, but the involvement of the adrenals and other sources (e.g., fetuses and placenta) are implicated (Smith, 1999; Weiss, 2000; Girling and Jones, 2006; Wada, 2008). The significance of plasma CORT to reproductive processes in live-bearing reptiles is not well understood, especially in snakes, and results vary (Wilson and Wingfield, 1992; Girling and Cree, 1995; Schuett et al., 2004a; Taylor et al., 2004; Taylor and DeNardo, 2011). Several studies show that elevated levels are associated with the occurrence of sexual behavior (Schuett et al., 2004a; Taylor et al., 2004), as well as vitellogenesis and yolk deposition (Wilson and Wingfield, 1992; Schuett et al., 2004a; Taylor et al., 2004). Levels of CORT in this study were largely unchanged before birth (see Girling and Cree, 1995), a possible maternal response that might protect developing embryos from high levels of CORT and stress (Cree et al., 2003; Cartledge and Jones, 2007; Robert et al., 2009; Itonaga et al., 2011), and in the post-parturient period of sampling. There was, however, a highly significant surge at the time of birth. Because we had no samples at Day-1, we cannot be certain whether the surge of CORT preceded birth; however, CORT levels were not elevated in samples between Day-5 and -2. Comparison of CORT levels at the time of birth was essentially identical to levels reported in C. atrox (Schuett et al., 2004a; Taylor et al., 2004), but comparison to other livebearing viperid species is not possible owing to lack of information. In many temperate pitvipers, neonates remain with their mothers until they undergo their first ecdysis (Greene et al., 2002; Reiserer et al., 2008), and the presence of neonates could potentially affect post-birth CORT patterns in mothers. However, because we immediately removed neonates from their mothers after birth, mostly to make bleeding mothers easier, we potentially confounded our ability to interpret post-birth hormone levels (e.g., CORT) of mothers. Nonetheless, this remains to be ascertained in vipers and other snakes. As expected, we showed that the duration of parturition is positively correlated with litter size (number of progeny), and that circulating levels of CORT derived from female plasma showed a positive trend with litter size; however, sig-

6 Agkistrodon Pregnancy Hormones 277 nificance was not achieved. The high levels of maternal CORT we detected at birth are unlikely a flight or fight response (Greenberg and Wingfield, 1987; Romero, 2002; McEwen and Wingfield, 2003). Rather, we suggest that elevated levels of CORT and duration of parturition are linked to the mobilization and regulation of energy stores (Munck et al., 1984; Guillette et al., 1995; Wada, 2008). Also, elevated levels of CORT might act as a mechanism to trigger the birth process (Smith, 1999). Despite the fact that proximate control of labor (parturition) has not been thoroughly studied in live-bearing snakes, there is a growing body of information on live-bearing lizards, especially in phrynosomatids (Guillette, 1979) and the scincid genera Niveoscincus and Tiliqua (Furgusson and Bradshaw, 1992; Girling and Jones, 2003; Atkins et al., 2006; Girling and Jones, 2006). In such species, a cascade of endocrine events lead to labor and birth, which involves neurohypophysial involvement (e.g., arginine vasotocin, AVT) on local prostaglandin production (i.e., PGF 2α), which, in turn, appears to be modulated by the innervation of uterine tissues by the β-adrenergic system (Cree and Guillette, 1991; Atkins et al., 2006). Possibly, the action of AVT is glucocorticoid-mediated (Goodson and Bass, 2001), indicating a dynamic role for CORT in the birth process. Furthermore, in lizards, these mechanisms appear to be influenced by environmental factors, such as temperature (Girling and Jones, 2003; Atkins et al., 2006; Girling and Jones, 2006; Rock, 2006). Clearly, further research will be required to understand the role of CORT and other plasma sex steroids, as well as environmental factors, in regulating female reproduction in copperheads and other live-bearing snakes. ACKNOWLEDGMENTS We thank J. Victoria and L. 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