REALMS study: real-world effectiveness and safety of fingolimod in patients with relapsing-remitting multiple sclerosis in Portugal.

BACKGROUND: Fingolimod, an oral sphingosine 1-phosphate receptor modulator, is approved by EMA for relapsing-remitting multiple sclerosis (RRMS). OBJECTIVES: To assess the effectiveness and safety of fingolimod in patients with RRMS in real-world clinical practice in Portugal. METHODS: Retrospective, multicentre, non-interventional study, reporting 3 years follow-up of data collected from October 2015 to July 2016. Sociodemographic data and previous treatments at baseline and data regarding disease evolution, including number of relapses, annualised relapse rates (ARR) and Expanded Disability Status Scale (EDSS), were collected. RESULTS: Two-hundred and seventy-five participants were enrolled in the REALMS study. Results showed that the main reason to switch to fingolimod was failure of previous treatment (56.7%) and only 3.6% were naïve patients. In the total population, there was a significant decrease in ARR of 64.6% in the first year of treatment, 79.7% in the second year and 82.3% in the third year, compared with baseline. More than 67.0% of patients had no relapses during the 3 years after switching to fingolimod. EDSS remained stable throughout the study. CONCLUSIONS: Therapy with fingolimod showed a sustained effectiveness and safety over the 3 years, particularly on patients switched from first-line drugs (BRACE). No new safety issues were reported.

Evolution of sex-biased maternal effects in birds: III. Adjustment of ovulation order can enable sex-specific allocation of hormones, carotenoids, and vitamins.

Overlap in growth of offspring should constrain the opportunity for sex-biased maternal effects, yet sex-specific allocation of maternal resources among simultaneously growing ova is often observed in vertebrates. In birds, such allocation can be accomplished either by temporal clustering of ova that become the same sex, resulting in sex-biased egg-laying order, or by follicle-specific delivery of maternal resources. Two house finch populations at the northern and southern boundaries of the species range have opposite ovulation sequences of male and female eggs, and thus, in the absence of sex differences in ova growth or sex-specific maternal strategies, would be expected to have opposite sex-specific accumulation of maternal products. We found that the populations had strong and similar gradients of steroid distribution in relation to ovulation order, whereas distribution of carotenoids and vitamins correlated with each follicle's accumulation of steroids. In both populations, temporal bias in production of sons and daughters within a clutch enabled strongly sex-specific acquisition of maternal products, and oocytes of the same sex were highly interdependent in their accumulation of steroids. Moreover, in nests where the sex-bias in relation to ovulation order deviated from population-specific patterns, eggs had highly distinct concentrations of steroids, carotenoids and vitamins. These results and previous findings of sex-specific yolk partitioning among oocytes suggest that oocytes that become males and females are temporally or spatially clustered during their ovarian growth. We discuss the implication of these findings for the evolution of sex-specific maternal resource allocation.

Chronic exposure to coal fly ash causes minimal changes in corticosterone and testosterone concentrations in male southern toads Bufo terrestris.

More than 50% of the electricity in the United States is produced by coal-burning power plants. The byproduct of coal-burning plants is coal fly ash, which contains increased concentrations of trace metals and is disposed of in collection basins. Southern toads (Bufo terrestris) frequently use these basins for reproduction. Male toads were collected in spring 2001 and 2002 from an ash basin and a reference site and divided into four groups: toads collected at the control site and maintained on (1) control substrate and food or (2) ash and contaminated food and toads collected at the ash site and maintained in (3) control or (4) ash conditions. Blood was collected periodically during 5 months to determine testosterone and corticosterone concentrations. Reference to ash toads exhibited a significant, transient increase in corticosterone at 4 weeks, but neither corticosterone nor testosterone continued to increase beyond this time. In contrast, toads caught and maintained on ash did not exhibit increased corticosterone. Testosterone in these toads appeared to be unrelated to ash exposure. This unexpected lack of a corticosterone response and no effect on testosterone suggests that toads chronically exposed to trace metals can acclimate to a polluted environment, but they may still experience subtle long-term consequences.

A condition dependent link between testosterone and disease resistance in the house finch.

Testosterone has recently been proposed as a link between male quality and health and the expression of sexual traits. We investigated the relationship between testosterone and measures of the individual condition and health of males in a natural population of house finches (Carpodacus mexicanus). We also conducted a captive experiment in order to test for the effects of testosterone on resistance to coccidia, which is a common parasite of house finches. Free-living males in better condition had higher testosterone levels and lower corticosterone levels than free-living males in poor condition. In our captive experiment, increased testosterone accelerated the rate of coccidial infection as compared with sham-implanted or gonadectomized males. Although the differences were not significant, free-living males infected with coccidia had lower levels of testosterone and higher levels of corticosterone than males that were not infected. Thus, experimentally elevating testosterone levels in captive males resulted in a higher percentage of infected males, while free-living males with coccidial infection had low testosterone levels. This apparent discrepancy between captive and free-living males in the association of testosterone and disease may be explained by the condition dependence of testosterone. These results suggest that the testosterone-dependent sexual traits reliably indicate male overall condition and health and, thus, females could benefit from assessing potential mates based on these traits.

Comparison of reproductive parameters in male yellow-blotched map turtles (Graptemys flavimaculata) from a historically contaminated site and a reference site.

From May to September of 1998, we collected monthly plasma samples from male yellow-blotched map turtles captured at two sites in the Pascagoula River drainage, Mississippi. One site (Vancleave) has a documented history of pollution from industrial sources (principally 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD). Fish consumption advisories at the Vancleave site were lifted in 1996 and current impacts appear minimal. However, the yellow-blotched map turtle, a federally protected species, continues to decline in numbers. To determine if endocrine disruption could be a factor in the low reproductive rates observed in Vancleave turtles, we examined levels of plasma testosterone (T) and estradiol-17beta (E(2)) from males at this site and a second site (Leaksville), which has no known source of industrial pollution. Plasma was also tested for vitellogenin (VTG), which, in males, can be a biomarker of exposure to estrogenic contaminants. No males had detectable plasma VTG nor did mean monthly E(2) levels differ between sites. However, 10% of males from the historically polluted site were found to have high levels of E(2) (equivalent to levels found in females) and T was significantly lower for males captured at this site for 3 of 5 months. Our data suggest that the current impact of contaminants on reproduction in this population is limited. However, a portion of the population may have been affected developmentally, as represented by differences in reproductive parameters detected between sites.

Control of attractivity and receptivity in female red-sided garter snakes.

Female red-sided garter snakes emerge from their hibernacula in the spring attractive and receptive to males. Attractivity is communicated by a pheromone released through the female's skin and is a consequence of ovarian recrudescence the previous summer. Receptivity, on the other hand, is stimulated by ovarian estrogen secretion during emergence itself. Mating renders females both unattractive and unreceptive. Another "mating" pheromone of male origin is important in making females unattractive after mating. To investigate the role of cloacal stimulation in the loss of attractivity and receptivity we injected a local anesthetic (lidocaine or tetracaine) in the cloacal region of females before mating. This does not prevent mating, although it blocks neural transmission of copulatory sensory stimuli. The time course of transition from attractive and receptive states was then observed. Females treated with local anesthetic as well as control females were unattractive within 15 min of mating. However, when retested 2-3 and 24 h after mating, a significantly higher proportion of treated females regained their attractivity, while mated control females remained unattractive. This restorative effect was transient, though, as treated females retested 48 h after mating were as unattractive as the controls. Both anesthetized and control females were unreceptive when tested following mating and did not regain receptivity with time. Last, the mating-induced surge in circulating concentrations of prostaglandin was diminished in females that received a local anesthetic prior to mating. Taken together these results indicate that the loss of attractivity and receptivity following mating in the red-sided garter snake is due to combined effects of a mating pheromone and a physiological, neurally mediated response to the sensation of stimuli associated with the act of mating.

Seasonal variation in reproductive steroids of male and female yellow-blotched map turtles, Graptemys flavimaculata.

From April to October of 1996 and 1997, we measured seasonal changes in plasma testosterone (T) in male and plasma T and estradiol-17beta (E(2)) in female yellow-blotched map turtles, Graptemys flavimaculata, from the Pascagoula River, Mississippi. In 1996, plasma from females was analyzed for progesterone. In 1997, ovaries of adult females were ultrasound imaged to determine stages of follicular development. Males exhibited peak T levels in September and October of both years, indicating fall gonadal activity. Males also exhibited a low level spring peak in T during April. Female E(2) levels increased significantly in May and June, the primary period of ovarian development. Females with preovulatory follicles did not have significantly higher E(2) levels than females with medium- or small-sized follicles. When compared to other freshwater turtle species, peak levels of E(2) were low. Testosterone levels did not follow a distinct yearly pattern in females. Progesterone levels were elevated in June when peak nesting was observed. Ultrasound and hormone data indicate that females lack ovarian development in the fall and produce on average only one clutch per year.

Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and organochlorine pesticides in yellow-blotched map turtle from the Pascagoula River basin, Mississippi, USA.

Concentrations of polychlorinated-dibenzo-p-dioxins (PCDDs), -dibenzofurans (PCDFs), -biphenyls (PCBs), and organochlorine pesticides were measured in tissues of map turtles collected from two locations in the Pascagoula River drainage of Mississippi, USA. PCBs were most predominant among the organochlorines with a concentration of up to 99 ng/g, wet weight (580 ng/g, lipid weight) in livers. The greatest concentration of PCDDs/DFs of 1.1 pg/g, wet weight (15.76 pg/g, lipid weight) was found in the liver of a male turtle. The measured concentrations of organochlorines were less than those reported for turtles from the Great Lakes Basin and upper St. Lawrence River. PCBs contributed 90-99% of the total estimated 2, 3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs). Particularly, PCB congeners 105, 118, and 156 accounted for 68-80% of the estimated toxic potency of PCBs in turtles.

Seasonal changes in sex and adrenal steroid hormones of gopher tortoises (Gopherus polyphemus).

We sampled a population of gopher tortoises (Gopherus polyphemus) from May to October 1997 to determine seasonal cycles of steroid hormones (testosterone, T; 17beta-estradiol, E; and progesterone, P) and related them to observations of mating behavior. In males, plasma T levels peaked in July and August and remained elevated through October. This coincides with the reported time of peak mating and spermatogenesis, indicating that males display an associated pattern of reproduction. In females, E levels were high in September and October. Plasma T levels in females were elevated in May, decreased to basal levels in June and July, and rose again in August and September. Elevated E and T levels correspond to the reported time of peak vitellogenic activity, indicating that females also display an associated cycle. Plasma P in females remained basal throughout the active season, suggesting that ovulation occurs in late winter. We also determined levels of corticosterone (B) to assess the influence of capture stress on tortoises and correlated B levels with tortoise activity patterns and sex steroid levels. We found no seasonal variation in levels of B in males or females. Plasma B levels were not correlated with levels of T or E, but were positively correlated with female P levels. Further, we found no relationship between plasma B levels in males and mean distance moved, mean number of burrows used, or mean home range size. However, there was a significant negative correlation between plasma B levels and male body size. In females, there was no relationship between B levels and mean distance moved, but B levels were significantly negatively correlated with the number of burrows females occupied. Lastly, there was no relationship between levels of B and the number of minutes required to obtain blood from an animal. However, B levels increased with the length of time that a tortoise spent in a trap, suggesting that trapped tortoises do exhibit capture stress.

Responsiveness of the hypothalamo-pituitary-interrenal axis in an amphibian (Bufo terrestris) exposed to coal combustion wastes.

To assess the responsiveness of the interrenal axis to stress, we injected toads exposed to coal combustion wastes and toads from an unpolluted reference site with adrenocorticotropic hormone (ACTH), as well as the vehicle alone (saline). Initial circulating levels of corticosterone in toads captured at the polluted area were significantly higher than levels in toads from the reference site. Corticosterone levels in toads from the polluted site remained high even after 2 weeks of laboratory acclimation and injection with saline. The results may suggest disruption of hepatic enzymes responsible for the metabolic clearance of steroid hormones. Injection of toads from the polluted site with ACTH had no effect on plasma corticosterone levels, whereas a similar treatment of toads from the reference site stimulated a marked increase in corticosterone. Our study provides evidence that toads exposed to coal combustion wastes may be less efficient at responding to additional environmental stressors.

Elevated trace element concentrations in southern toads, Bufo terrestris, exposed to coal combustion waste.

A number of recent studies have linked developmental, physiological, and behavioral abnormalities in amphibians to coal combustion wastes (coal ash). Few studies, however, have determined trace element concentrations in amphibians exposed to coal ash. In the current study we compare total body concentrations of 20 trace elements in adult southern toads, Bufo terrestris, inhabiting coal ash settling basins with toads that were not exposed to the combustion wastes (reference). In addition, we document the accumulation of trace elements in toads transplanted from reference sites to field enclosures in an ash settling basin for 7 and 12 weeks. Arsenic, selenium, and vanadium levels were significantly elevated in toads captured at the ash-contaminated site in comparison to toads from the reference site. All three of these elements were also significantly elevated in toads exposed to the contaminated habitat for only 7 weeks. Our study suggests that adult anurans can bioaccumulate particularly high levels of selenium and may be useful bioindicators in agricultural and coal ash-impacted habitats.

Increased circulating levels of testosterone and corticosterone in southern toads, Bufo terrestris, exposed to coal combustion waste.

This study describes an interrenal stress response in adult toads, Bufo terrestris, after exposure to coal combustion waste (characterized by a variety of trace elements). In the first portion of this study, free-ranging male toads captured at the coal ash polluted site exhibited significantly higher circulating levels of corticosterone (B) in both June/July and August than conspecifics captured at uncontaminated sites. In addition, both calling and noncalling males from the polluted site had higher B levels than conspecifics engaged in the same behaviors at reference sites. Testosterone levels were elevated in toads from the polluted site, regardless of capture month or behavioral state, suggesting altered androgen production, utilization, and/or clearance. In the second portion of this study, male toads from reference sites were transplanted to enclosures at the polluted site or an uncontaminated site. Toads held at the polluted site exhibited significant increases in B after 10 days of exposure compared to toads held at the reference site. B levels remained significantly elevated in toads transplanted to the polluted site after 12 weeks. We hypothesize that high concentrations of various trace elements in the polluted site are responsible for these hormonal responses.

Effects of arousal from hibernation and plasma androgen levels on mating behavior in the male big brown bat, Eptesicus fuscus.

The effects of arousal from hibernation and presence of plasma androgen on the expression of mating behavior in male big brown bats (Eptesicus fuscus) were tested in a captive population exposed to seminatural conditions in central Alabama. In the mild winter of 1994-1995, flight cage temperatures never fell below 10 degrees C. Bats were never observed to enter sustained (over 2 d) torpor. They were also never observed to mate. Unmanipulated, sham-operated, and gonadectomized males and unmanipulated females were exposed to 6 d of 4 degrees C. All individuals appeared torpid, and body temperatures of monitored bats fell at least 15 degrees-20 degrees C. Plasma androgen levels of torpid unmanipulated, sham-operated, and gonadectomized males averaged 25.4 +/- 9.2, 19.7 +/- 9.1, and 1.5 +/- 0.25 ng/mL, respectively, and did not differ significantly from levels for the same groups 1 mo previous to induced torpor. When animals were returned to 23 degrees C, 57% of unmanipulated, 40% of the sham-operated, and 33% of gonadectomized males displayed mating behavior upon arousal. Almost all matings occurred within 48 h of arousal, the majority in the first 3 h. Males not exposed to low temperatures were not observed to mate. Although individuals from all three treatments mated, gonadectomized males averaged fewer mounts and copulations per individual. Androgen levels declined significantly from torpor levels in all groups 48 h after arousal. Thus, an extended period of low body temperature and arousal appeared to be a short-term activator of sexual behavior in the big brown bat; unmanipulated males were more strongly affected by this stimulus than gonadectomized males.

Courtship behavior and plasma levels of androgens and corticosterone in male marbled salamanders, Ambystoma opacum (ambystomatidae).

We measured plasma levels of testosterone, dihydrotestosterone (DHT), and corticosterone for male marbled salamanders (Ambystoma opacum) collected during the breeding season. Our goal was to ascertain whether steroid levels changed in response to particular reproductive behaviors or laboratory confinement. Six groups of salamanders were examined: (a) MIGRATING, males migrating toward the pond basin during the breeding season; (b) LABORATORY, males kept under confined conditions in the laboratory for 10 days; (c) LAB-FIELD, laboratory males that were later released into seminatural enclosures in the field; (d) COURTING, males from male-female pairs in which the male actively courted the female (and deposited at least one spermatophore); (e) SOLO, males that were individually isolated from conspecifics; and (f) MALE-MALE, males that were placed together in pairs, and in which one male actively courted the other male. In three groups (COURTING, SOLO, and MALE-MALE), salamanders were placed in containers for observation and each male was observed for at least 2 hr prior to a plasma sample being taken. Circulating levels of testosterone, DHT, and corticosterone did not differ significantly for males in these groups. The similarity of androgen levels among the three groups indicated a lack of behaviorally evoked change under experimental conditions designed to reveal a behavior-androgen response. Male A. opacum differ taxonomically from other amphibians showing a behavior-androgen response (three species of toads in the genus Bufo) and also lack amplexus and male-male combat during competition for mates. The effects of confinement were indicated by levels of testosterone and DHT in LABORATORY males that were significantly lower than average levels of males in the following groups: MIGRATING, LAB-FIELD, and MALE-MALE. We inferred that LAB-FIELD males, following their release to seminatural enclosures, were able to regain plasma androgen levels typical of migrating males. This increase is one of very few demonstrations for amphibians of an increase in androgen levels upon release from laboratory confinement. Levels of corticosterone did not differ significantly between males that were active in the field and males that were kept in the laboratory. The similarity of corticosterone levels among these groups differs from the typical pattern of elevated corticosterone and depressed androgen levels in captive amphibians. Maximal corticosterone levels in breeding male A. opacum may act differently than in other species in which chronic elevations inhibit the pituitary-gonadal axis.

Effects of gonadal sex steroids on sexual behavior in the big brown bat, Eptesicus fuscus, upon arousal from hibernation.

Vespertilionid bats are thought to have a dissociated pattern of reproduction; mating occurs in autumn as well as during periodic arousals from hibernation when testes are regressed and ovaries are "in stasis." Sex steroid levels in both sexes are basal at this time. This pattern would indicate that sex steroids per se may not be activating sexual behavior. Gonadectomy of male and female big brown bats, Eptesicus fuscus, months before the mating season, did not significantly affect the probability of expressing sexual behavior in either sex when staged behavior tests were conducted. Ovariectomized females, in fact, mated significantly more and exhibited proceptive behavior. Males mated at the same frequency whether they were intact, orchidectomized, or given testosterone implants. These results argue that gonadal sex steroids are not the direct activators of sexual behavior in this species. However, when males were allowed to mate freely within a large flight cage, orchidectomized males were not seen to mate, while intact males mated at the same frequency as in the staged trials. Females are apparently either differentiating between the two groups of males or there is male-male competition for mates within a system that has multiple matings with different individuals in both sexes.

Courting and noncourting male red-sided garter snakes, Thamnophis sirtalis parietalis: plasma melatonin levels and the effects of pinealectomy.

Previous studies found that pinealectomy of male Canadian red-sided garter snakes (Thamnophis sirtalis parietalis) in the autumn, before prolonged exposure to low temperatures (hibernation), significantly impaired the expression of courtship behavior upon emergence in the spring. Additionally, pinealectomized animals with a disrupted diel cycle of plasma melatonin did not court while those exhibiting a more typical diel pattern did. These results suggested that the pineal gland functions in the transduction of a temperature cue which stimulates courtship. To test this hypothesis, we pinealectomized males in the spring after they had undergone a normal hibernation but were still courting. Pinealectomy of courting males in the spring, in each of the 3 years of study, had no effect on courtship. This result suggests that once the cue is transduced, the pineal gland no longer has a modulatory effect on courtship behavior. Finally, we took advantage of the fact that, in the laboratory, there is always a small percentage of males that do not court upon emergence. Pinealectomy of these noncourters greatly increased the percent of males expressing courtship behavior in each of the study years. Plasma melatonin levels of unmanipulated courting and noncourting males was measured after emergence in successive years. In both years, courters had a typical pattern of melatonin secretion (low in the photophase, high in the scotophase) while persistent noncourters displayed the opposite pattern.

Pinealectomy, melatonin, and courtship behavior in male red-sided garter snakes (Thamnophis sirtalis parietalis).

Activation of courtship behavior in male red-sided garter snakes is independent of androgens. Only exposure to extended periods of low temperature with subsequent warming stimulates courtship in males. The pineal gland is thought to transduce temperature as well as photoperiodic information in reptiles. Therefore, we explored the relationship of the pineal and melatonin to sexual behavior in this species. Pinealectomy of male garter snakes disrupted sexual behavior upon emergence from a 17-week period of low temperature in approximately 60% of treated individuals in each of the 3 years of study. However, 40% of the males were unaffected by the pinealectomy, engaging in vigorous courtship. Administration of exogenous, chronic melatonin did not significantly modulate the effect of pinealectomy. Upon pinealectomy in the autumn (before hibernation), plasma levels of melatonin fell. However, upon emergence from hibernation, melatonin levels in pinealectomized (PINX) and sham-treated (SHAM) animals were equivalent, indicating extrapineal source(s) of melatonin. However, PINX males did not exhibit a diel cycle in melatonin levels upon emergence. Instead, melatonin remained elevated through the subsequent 24-hr period. SHAMs did exhibit a diel cycle. Ten days after emergence, PINX animals either had a disrupted/abnormal melatonin cycle and were non-courters or had a cycle similar to SHAM males and courted. Therefore, a normal diel cycle of melatonin appeared necessary for the proper expression of courtship behavior. These results suggest that the pineal in snakes 1) is part of a complex, multi-oscillator system as it is in birds and lizards and 2) may play a role in maintaining polymorphism in timing of reproductive behavior.

Effects of ovariectomy and estrogen replacement on attractivity and receptivity in the red-sided garter snake (Thamnophis sirtalis parietalis).

Activation of courtship behavior in male red-sided garter snakes, Thamnophis sirtalis parietalis, is independent of the presence of sex steroids. The only consistent treatment that stimulates courtship behavior in males is prolonged exposure to low temperature followed by subsequent warming, mimicking the emergence from hibernation. We investigated whether attractivity and receptivity in female red-sided garter snakes is similarly steriod independent. Female red-sided garter snakes are attractive when they emerge from hibernation and are courted by males; most mate within an hour of emergence. In a series of experiments, groups of females were either ovariectomized (OVEX) in the late spring, fall or while in hibernation. They were tested for attractivity and receptivity upon emergence from hibernation. Females OVEX in the spring were unattractive whereas those OVEX in fall or while in hibernation were attractive. Thus, attractivity appears determined the year before emergence and is dependent on the presence of the ovaries. All OVEX females were unreceptive upon emergence. OVEX females were also given replacement estradiol (E) treatment (either in Silastic capsules or single injections) at various points of their annual cycle. The only treatment that resulted in reinstating receptivity in OVEX females was the injection of E (20 micrograms) one hour prior to emergence. The effectiveness of E in reinstating receptivity was time dependent: the longer the period between emergence and injection, the less effective the same dosage was in stimulating receptive behavior. These experiments suggest that sexual behavior in female red-sided garter snakes is, unlike males, dependent on the presence sex steroid hormones. Although E is naturally at its lowest seasonal level upon emergence, the concentration is sufficient to stimulate receptivity. However, it appears that temperature regulates a time-limited window of sensitivity to E.

Seasonal changes and annual variability in daily plasma melatonin in the red-sided garter snake (Thamnophis sirtalis parietalis).

We report seasonal and annual variation in the daily cycle of plasma melatonin levels in male red-sided garter snakes, Thamnophis sirtalis parietalis. In autumn of 1989 and 1990, levels averaged a maximum of 210 pg/ml during scotophase and a minimum of 45 pg/ml during photophase and had a similar diel pattern. Under hibernation conditions (4 degrees, 0:24 L:D), melatonin was undetectable and a diel cycle could not be determined. In Spring 1990, melatonin levels rose rapidly and precipitously within an hour of emergence (while in photophase), peaked at levels significantly higher than those seen in the autumn (approximately 900 pg/ml) and remained significantly high for 24 hr after emergence (though the majority of animals did have decreased levels at the 0400 sample). By the 10th day after emergence, a diel cycle was reestablished and absolute melatonin levels had decreased. The next spring (1991), melatonin again rose within an hour after emergence, while in photophase, but not as high as the previous year. Also unlike the previous year, a diel cycle was observed within 24 hr of emergence. Melatonin levels at emergence were significantly higher than those observed 10 days later. Disruption of a diel rhythm of plasma melatonin (by pinealectomy the previous autumn) inhibits courtship behavior by males the next spring, implying a role for melatonin in the stimulation of sexual behavior. Males in 1991 (with quickly established melatonin cycles) courted much sooner after emergence than did males in 1990. Therefore, the initial day/night difference in melatonin levels at emergence (i.e., establishment of a normal diel cycle) may function in synchronizing and modulating reproductive behavior in male red-sided garter snakes.