Circulating prolactin in free-living California ground squirrels (Spermophilus beecheyi)

GENERAL

AND

COMPARATIVE

Circulating

ENDOCRINOLOGY

Prolactin

71, 48@92

in Free-Living

(1988)

California

Ground

Squirrels

(Spermophilus beecheyi) KAY E. HOLEKAMP,'SCOTTNUNES,ANDFRANKTALAMANTES' Thimann Laboratories,

University of California,

Santa Cruz, California

95064

Accepted April 13, 1988 During a long-term field study of a free-living population of California ground squirrels (Sperrnophilus beecheyi), blood samples were drawn at regular intervals from marked animals via femoral venipuncture, and plasma prolactin (PRL) was measured by homologous radioimmunoassay. Marked fluctuations with season and reproductive condition occurred in circulating PRL levels in both males and females, with peak levels occurring during the spring breeding season. Peak PRL levels in females were reached 4 weeks after peak male levels, but mean peak values did not differ significantly between the sexes. Among females, juveniles had lower mean PRL levels than adults, and yearlings had lower mean levels throughout their initial reproductive episodes than did older females. Q 1988 Academic Press. Inc.

Colosi et al. (1987) recently reported the isolation and purification of secreted prolactin (PRL) from the California ground squirrel (Spermophilus beecheyi). Our laboratory subsequently developed a sensitive, homologous radioimmunoassay (RIA) for S. beecheyi PRL (sbPRL) and used this assay to measure plasma PRL concentrations in small samples of male and female S. beecheyi sacrificed at various stages of their annual reproductive cycles (Thordarson et al., 1987). Those data indicated that sbPRL levels were higher during pregnancy and lactation than during nonreproductive phases of the annual cycle in either males or females. Interestingly, however, mean sbPRL levels in reproductively active males were also very high, and in fact did not differ significantly from peak levels in females. We offer here a more detailed analysis of changing sbPRL levels in male and female S. beecheyi during the annual activity cy-

cle, with particular attention to the breeding season. We present data describing PRL levels in plasma samples obtained repeatedly from members of a closely monitored, free-living S. beecheyi population in coastal California. MATERIALS

Subject nnimals. S. beecheyi is a group-living diurnal rodent inhabiting open grasslands throughout California and northern Mexico. Between November 1983 and January 1986, we studied the reproductive biology of a large S. beecheyi population located on the campus of the University of California at Santa Cruz (37”N, 122”W; Santa Cruz County, CA). Members of this free-living population were captured at weekly or biweekly intervals in Tomahawk live traps baited with grain. Trapped animals were marked with numbered metal eartags and with individually distinct patterns of black hair dye. At each capture all animals were weighed (22 g) with a Pesola spring balance and examined for wounds, and their reproductive conditions were recorded. Indices of reproductive condition noted for females included nipple size, shape, color, exposure, and vulva condition. For males these included testis length (2 1 mm), descent, and coloration. Detailed descriptions of reproductive indices are presented elsewhere (Holekamp, 1983). Throughout the study period behavior of marked animals was observed from elevated posts through 8- to

’ Current address: Department of Ornithology and Mammalogy, California Academy of Sciences, Golden Gate Park, San Francisco, CA 94118. ’ To whom reprint requests should be addressed. 484 0016-6480/88 $1.50 Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

AND METHODS

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IO-power binoculars. From January through June we observed their behavior for approximately 30 hr per week and for 5-10 hr per week in August through December. Thirty-four complete litters were captured at initial emergence from their natal burrow using methods described by Sherman (1976). Marked animals were assigned to age classes at the outset of each breeding season on the basis of whether they had been born within the past 12 months (yearlings) or earlier (adults). Plasma samples and assay procedure. Whole blood samples (1 cc) were collected weekly or biweekly in the field via femoral venipuncture in heparinized syringes from trapped 5. beecheyi. All blood samples were drawn between 1330 and 1730 hr, after animals had been held in traps l-3 hr. All trapped animals were handled in identical fashion, without anesthetic. Whole blood was stored on ice until transported to the lab (l-2 hr), where it was centrifuged for 8 min at 7000g. Plasma was then collected and stored frozen until assayed for sbPRL content. All samples were assayed in duplicate. The assay used to measure sbPRL content of plasma samples was the double-antibody RIA for sbPRL developed and previously described by Thordarson et al. (1987). Mean sensitivity of this assay, estimated as the lowest standard hormone concentration differing from replicate determinations of zero by two standard deviations, was 0.21 rig/ml (N = 9). The intraassay co-

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efficient of variation, estimated from replicate determinations of two quality control plasmas, ranged from 2.3 to 5.9%. Mean concentrations of sbPRL in the two quality control plasmas were 1.8 2 0.39 and 9.4 -C 1.7 rig/ml (mean + SD), with coefftcients of variation between assays of 13.6 and 14.9% (N = 9), respectively. Statistical analyses. Statistical data were expressed as means -+ standard error (SE), and comparisons of data grouped by sex and age class were evaluated using ANOVA, as described by Keppel (1973). Differences between group means were considered signilicant when P < 0.05. Except during the annual period of initial juvenile emergence from their natal burrows (May-June), 94 2 5% of animals sampled each month had been previously trapped. To determine whether plasma sbPRL concentrations of animals experiencing their first blood samples differed from those of resampled animals trapped during the same time period, we compared mean sbPRL values for initial and subsequent samples of adult 5. beecheyi during each month when both types were available. We found no significant differences in mean sbPRL values (P < 0.05; Student’s t test) between initial and subsequent samples. so data were pooled for each time period.

RESULTS Figure 1 presents annual cycles of surface activity, reproduction, and sbPRL

FIG. 1. Annual cycles of surface activity, reproduction, and sbPRL concentrations in the 5. beecheyi study population. (A) Plasma sbPRL concentrations (&ml) in male (dashed lines) and female (solid lines) 5. beecheyi. Data points represent monthly means for the number of animals (N) indicated; vertical lines indicate SE. (B) Percentage of trapped males (N) with large (>15 mm), descended testes in heavily pigmented scrotal sacs. (C) Surface activity (solid line) and estivation (dashed line) of adult males. (D) Surface activity and estivation of adult females. (E) Mating. (F) Pregnancy. (G) Lactation. (H) Surface activity of juveniles, starting at weaning. (I) Molt.

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concentrations in our S. beecheyi population. In accordance with reports from previous field studies of this species (Evans and Holdenreid, 1943; Fitch, 1948; Dobson, 1979), we found that most animals of both sexes first mated as yearlings, and that all males dispersed from their natal sites before they first bred, but females were philopatric. The females in our study population were all monestrous, producing one litter each year. Copulations took place above ground and could therefore be easily observed. Mating occurred only during late February and early March. The gestation period was 4 weeks and the lactation interval was 5 to 6 weeks. Juveniles first emerged from their natal burrows in May. All juveniles remained continually active on the surface from the time of initial emergence, at weaning, until the end of their first breeding season the following spring. All adult males estivated from early May until late October, and all but one of the adult females estivated from late July until early January. All surface-active animals molted in April, June, and July.

AND TALAMANTES

On the basis of residual enlargement and darkening of nipples, and/or on reproductive behavior observed the previous season, 96% (N = 45) of adult females appeared to have borne previous litters. All yearlings (N = 31) were primiparous. The mean size of 20 litters born to multiparous females was 8.3 pups. The mean size of 14 litters produced by primiparous females was 6.9 pups. The curves in Fig. 1 describing sbPRL concentrations represent all plasma samples for each month for all males and females in the population, without regard for age class or parity. Plasma sbPRL concentrations in both sexes exhibited striking annual cycles, with peak levels occurring in March for males and in April for females. This sex difference in the timing of peak sbPRL values was significant (F = 7.541; P < O.Ol), but sex differences in peak values themselves were not (see below). Weekly sbPRL values for males and females during the breeding season (January through June) are presented in Fig. 2. Peak sbPRL levels in males occurred near the 22 T

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LACTATION

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FIG. 2. Mean (&SE) weekly sbPRL values for male (dashed lines) and female (solid lines) S. heecheyi during the breeding season. * Indicates means are significantly different (P < 0.05; Student’s t test).

PRL

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end of the mating period, whereas peak female levels occurred 4 weeks later, during lactation. Temporal trends again differed significantly between the sexes (F = 13.042; P < O.Ol), but peak levels did not (Student’s I test, 0.362; P > 0.50). Repeated sbPRL measures for five individual males and females are plotted relative to the time of mating in Figs. 3 and 4, respectively. Mating occurred on only one day for each female, but males mated repeatedly over a period of 3 to 4 weeks, so the horizontal axes differ in these figures. Peak sbPRL levels in males occurred during the last week of, or immediately after, mating. Peak levels occurred during the third week of lactation in four of five females. Figure 5 compares plasma sbPRL levels in adult and yearling females from May 1984 through June 1985. Mean yearling sbPRL levels rose 1-2 weeks later in the spring, and their peaks remained significantly lower than those observed in older

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females (F = 8.624; P < 0.05). The two curves become statistically indistinguishable exactly 12 months after the yearlings were weaned, late in the lactation interval. DISCUSSION These data confirm and extend the earlier finding reported by Thordarson et al. (1987) that mean sbPRL levels fluctuate according to reproductive state in both male and female S. bercheyi. The current, more detailed analysis reveals that peak sbPRL levels in both sexes are approximately 10 times higher than trough values. To our knowledge, these data represent the only existing annual profiles of circulating PRL levels in any free-living species. These data are also unique in that no previous studies have used homologous RIA to monitor seasonal and physiological changes in protein hormone levels in any sciurid rodent. Considering that ground squirrels. prairie dogs, and marmots are now frequently used as subjects in studies of reproductive physiol-

FIG. 3. Repeated sbPRL measures for five adult male S. hrrc~heyi (ID numbers 3510, 3663, 3684. 3686, and 3698) during the 1985 breeding season. All of these males were observed to copulate during the indicated mating period. Numbers on the horizonatal axis represent weeks before and after the middle weeks of the mating period.

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WEEKS FIG. 4. Repeated sbPRL measures for five adult female S. beecheyi (ID numbers 3584, 3592, 3603, 3697, and 3779) during the 1985 breeding season, plotted relative to the week during which each of these females was observed to copulate.

culating PRL levels in members of both sexes. Furthermore, interpretation of earlier results presented by different investigators for male and female conspecifics is often obfuscated by discrepancies in assay technique between studies. Nevertheless, the available data suggest that, although PRL concentrations in males may exhibit marked annual cycles, male plasma levels never reach the peak values found in preg-

ogy and behavior (Young and Sims, 1979; Concannon et al., 1984; Foreman and Garris, 1984; Murie and Michener, 1984; Barnes, 1986; Barnes et al., 1986), our data should be useful to a large group of investigators. Previous studies of other mammalian species have rarely reported data comparable to those presented here, documenting seasonal and physiological changes in cir-

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1985 FIG. 5. Mean plasma sbPRL levels in adult (dashed lines) and yearling (solid lines) females from May 1984, when this cohort of yearlings was weaned, through June 1985, when both groups weaned their own 1985 litters. * Indicates means are significantly different (P < 0.05; Student’s t test).

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nant or lactating females in sheep (Rhind et al., 1972; Lincoln et al., 1982; Webster and Haresign, 1983; Almeida and Lincoln, 1984), goats (Buttle et al., 1972; Buttle, 1974), minks (Martinet and Allain, 1985), rats (Amenomori et al., 1970; Mock and Frankel, 1978), mice (Yanai and Nagasawa, 1974; Michael, 1976; Barkley, 1979), or humans (Ehara et al., 1973; Bunner et al., 1979). However, our finding that plasma PRL levels were similar in reproductively active members of both sexes is not unique. Using an heterologous RIA (for rat PRL), Bast and Greenwald (1974) found that PRL levels in pregnant and lactating hamsters ranged from 20 to 70 rig/ml and 30 to 40 &ml, respectively. Using the same RIA, Bex et al. (1978) found that plasma of male hamsters maintained under long-day photoperiod conditions contained approximately 50 rig/ml of PRL. Among avian species, circulating PRL levels have been found to be virtually identical in reproductively active males and females in ring doves (Goldsmith et al., 1981; Silver, 1984), black swans (Goldsmith and Follett, 1980), and snow geese (Campbell et al., 1981). Our data describe unambiguous annual cycles of reproduction, surface activity, and PRL secretion in S. beecheyi. However, it is not known whether these cycles are endogenous in this species. Whereas most seasonally breeding mammals require changes in photoperiod for expression of circannual rhythms of reproductive function and plasma hormone concentration (Sadlier, 1969; Zucker et al., 1980), these rhythms persist in various sciurid rodents even when animals are maintained under constant environmental conditions (Heller and Poulson, 1970; Kenagy, 1980; Barnes, 1986). Data presented in our previous report (Thordarson et al., 1987) revealed no differences in mean plasma sbPRL concentrations between pregnant and lactating S. beecheyi, whereas the current data indicate that sbPRL levels are substantially higher in lactating than in pregnant females, with

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489

peak concentraions occurring approximately midway through the lactation interval. Furthermore, mean peak levels of sbPRL in lactating females and breeding males are higher in this study than in our earlier report. We suggest these differences may be attributable to methodological discrepancies between our two studies. The earlier data were generated by RIA of plasma obtained in the laboratory from anesthetized, sacrificed S. beecheyi that had been captured in areas peripheral to our study site. Dates of mating and parturition were not known for squirrels used in that study, so samples from lactating animals may have come from females at any point within the 5- to 6-week lactation interval. Similarly, samples obtained previously from reproductively active males were collected during late March and early April, 2 to 3 weeks after mating was last observed in our population. PRL functions importantly in the regulation of pelage growth and molting cycles in several mammalian species (e.g., Webster and Barrell, 1985). Although additional molts might have occurred underground while adults were estivating, three molt periods were observed each year in surfaceactive animals. None of the three molt periods were coincident with peak sbPRL levels in males, but peak plasma levels in females occurred concurrently with the April molt (Fig. 1). The roles of PRL in regulating mammary gland function (Cowie et al., 1980), and as an essential part of the luteotrophic complex (Smith, 1980), have been extensively documented in various mammalian species. PRL apparently also functions to mediate production and secretion of both gonadotrophins (McNeilly, 1980) and steroid hormones (Fortune and Vincent, 1986; Fortune et al., 1986) in other species. Finally, PRL has repeatedly been implicated in control of maternal care and other reproductive behaviors in some species (Bridges et al., 1985; Siegel, 1986; Silver, 1974). Nothing is currently known about the role of PRL in

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either physiological or behavioral processes in any sciurid rodent. However, the patterns of PRL secretion observed in female S. beecheyi in the present study suggest that sbPRL might function importantly in any or all of the contexts mentioned above. Studies are currently underway in this laboratory documenting effects of sbPRL on behavioral and physiological events in free-living S. beecheyi. Our data confirm the earlier report by Thordarson et al. (1987) that peak sbPRL levels in breeding males did not differ significantly from peak levels in females. This result suggests that sbPRL may function importantly in male reproduction in this species. The role of PRL in reproductive processes of male mammals is very poorly understood. However, normal PRL levels in males of some species appear to support testis function, whereas high PRL levels suppress copulatory behavior and inhibit testis function (e.g., Almeida and Lincoln, 1984). The marked rise in plasma sbPRL levels in male S. beecheyi at the conclusion of the mating period suggests perhaps sbPRL might function similarly in this species to shut down behavioral and physiological processes of reproduction. For example, if annual patterns of gonadotropin secretion in S. beecheyi resemble those Barnes (1986) observed in S. lateralis, it is possible that elevated PRL mediates reduction in circulating levels of FSH or LH. PRL clearly does not function to facilitate paternal behavior in S. beecheyi, as it appears to do in various avian species (e.g., Silver, 1974; Goldsmith et al., 1981), because male S. beecheyi never participate in parental care. Barnes (1986) observed that timing of increases in plasma gonadotropin levels in S. lateralis varied according to whether or not animals had hibernated. It is possible that the temporal differences we observed between yearling and older female S. beecheyi (Fig. 5) might similarly have been influenced by whether or not estivation had occurred. Free-living juvenile ground squir-

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rels often enter and emerge from hibernation later than do adults, and juveniles may exhibit a generally delayed schedule of reproductive maturation (Murie and Michener, 1984), but S. beecheyi juveniles may be unique in that their surface activity persists throughout the entire first year of life. Previous reports have suggested a role of PRL in the initiation of puberty in other rodents. For example, reduced PRL levels delay the onset of puberty in female rats (Advis et al., 1981), whereas increased levels accelerate its onset (Admis and Ojeda, 1978). The slight rise in plasma PRL concentrations we observed in immature female S. beecheyi in November and December (Fig. 5) might indicate the occurrence of puberty in these animals. More detailed analyses of this question, and of temporal relationships between annual cycles of plasma testosterone, gonadal mass, body mass, behavior, and indices of reproductive maturity in males, are currently in preparation. ACKNOWLEDGMENTS We thank June Cheng, Margaret Conway, Katherine Green, David Polzine, Tamara Sloan, and Lisa Soon for their excellent assistance in the field. We also thank Linda Ogren for her many valuable suggestions during all phases of this research. Finally, we thank Laura Smale and Irving Zucker for helpful comments on an earlier version of this manuscript. This work was supported by National Research Service Award Postdoctoral Fellowship HD06553-02 to K.E.H.

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misurak, H. I. Siegel, M. Cheng, and H. F. Feder, Eds.), Annals. N.Y. Acad. Sci. 414, 202215. Silver, R. (1984). Prolactin and parenting in the pigeon family. J. Exp. Zool. 232, 617-625. Smith, M. S. (1980). Role of prolactin in regulating gonadotrophin secretion and gonad function in female rats. Fed. Proc. 39, 2571-2576. Thordarson, G., Holekamp, K. E., and Talamantes, F. (1987). Development of an homologous radioimmunoassay for secreted prolactin from the California ground squirrel (Spermophilus beecheyi). Biol. Reprod. 36, 1186-1190. Webster, J. R., and Barrell, G. K. (1985). Advancement of reproductive activity, seasonal reduction in prolactin secretion and seasonal pelage changes in pubertal red deer hinds (Cervus elaphus) subjected to artificially shortened photoperiod or daily melatonin treatments. J. Reprod. Fertil. 73, 255-260. Webster, J. R., and Haresign, W. (1983). Seasonal changes in LH and prolactin concentrations in ewes of two breeds. J. Reprod. Fertil. 67, 465471. Yanai, R., and Nagasawa, H. (1974). Radioimmunoassay of pituitary and plasma prolactin during the oestrous cycle in mice. J. Endocrinol. 62, 685686. Young, R. A., and Sims, E. A. H. (1979). The woodchuck, Marmota monax, as a laboratory animal. Lab. Anim. Sci. 29, 770-780. Zucker, I., Johnston, P. G., and Frost, D. (1980). Comparative, physiological, and biochronometric analyses of rodent seasonal reproductive cycles. Prog. Reprod. Biol. 5, 102-133.