Endocrine correlates of pregnancy in the ring-tailed lemur (Lemur catta): Implications for the masculinization of daughters

Hormones and Behavior 59 (2011) 417–427

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Endocrine correlates of pregnancy in the ring-tailed lemur (Lemur catta): Implications for the masculinization of daughters Christine M. Drea ⁎ Departments of Evolutionary Anthropology and Biology, Duke University, Durham, NC 27708-0383, USA

a r t i c l e

i n f o

Article history: Received 8 June 2010 Revised 27 September 2010 Accepted 30 September 2010 Available online 13 October 2010 Keywords: Sexual differentiation Masculinization Pregnancy Development Androgen Estrogen Strepsirrhine primate Spotted hyena Female dominance

a b s t r a c t Female ring-tailed lemurs (Lemur catta) are Malagasy primates that are size monomorphic with males, socially dominate males, and exhibit a long, pendulous clitoris, channeled by the urethra. These masculine traits evoke certain attributes of female spotted hyenas (Crocuta crocuta) and draw attention to the potential role of androgens in lemur sexual differentiation. Here, hormonal correlates of prenatal development were assessed to explore the possibility that maternal androgens may shape the masculine morphological and behavioral features of developing female lemurs. Maternal serum 17α-hydroxyprogesterone, dehydroepiandrosterone sulphate (DHEA-S), Δ4 androstenedione (androst-4-ene-3,17,dione), testosterone, and 17βestradiol were charted throughout the 19 pregnancies of 11 ring-tailed lemurs. As in spotted hyenas, lemur pregnancies were associated with an immediate increase in androgen concentrations (implicating early maternal derivation), followed by continued increases across stages of gestation. Pregnancies that produced singleton males, twin males, or mixed-sex twins were marked by greater androgen and estrogen concentrations than were pregnancies that produced singleton or twin females, especially in the third trimester, implicating the fetal testes in late-term steroid profiles. Concentrations of DHEA-S were mostly below detectable limits, suggesting a minor role for the adrenals in androgen biosynthesis. Androgen concentrations of pregnant lemurs bearing female fetuses, although less than those of pregnant hyenas, exceeded preconception and postpartum values and peaked in the third trimester. Although a maternal (and, on occasion, fraternal) source of androgen may exist for fetal lemurs, further research is required to confirm that these steroids would reach the developing female and contribute to her masculinization. © 2010 Elsevier Inc. All rights reserved.

Introduction The female ring-tailed lemur (Lemur catta) is unusual among mammals in that she possesses masculinized external genitalia (Drea and Weil, 2008; Hill, 1953), is size monomorphic with males (Drea and Weil, 2008; Kappeler, 1991), and is socially dominant over males (Jolly, 1966). In this regard, she displays a suite of masculine morphological and behavioral traits that are similar to, albeit less extreme than, those displayed by the female spotted hyena (Crocuta crocuta). Female spotted hyenas have the most highly masculinized external genitalia of any female mammal (Frank et al., 1990; Matthews, 1939; Neaves et al., 1980), are larger than males (Glickman et al., 1992; Kruuk, 1972; Matthews, 1939), and are more aggressive than and socially dominant over adult males (Frank, 1986; Kruuk, 1972; Smale et al., 1993). Detailed study of the spotted hyena has revealed a largely unexplored set of steroid metabolic pathways (Glickman et al., 1987; Licht et al., 1992, 1998; Yalcinkaya et al., 1993), potentially functional in other mammals (Drea, 2009), including

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humans (Conte et al., 1994; Shozu et al., 1991), through which maternal androgens might influence normal female sexual differentiation. Despite the intriguing genital morphology and unusual social relations of lemurs, their reproductive development and behavioral endocrinology have received relatively little attention. Here, I examine the endocrine profiles of gestating ring-tailed lemurs to search for commonalities with gestating spotted hyenas, specifically to explore whether maternal androgens might provide a mechanism for masculinizing lemur daughters. The study of sexual differentiation has focused on the active role of testicular androgens, specifically testosterone (T) and its metabolites, in the developing male. Likewise, T has played a central role in the study of male aggression (Demas et al., 2007). In exceptional cases, female exposure to exogenous androgens during appropriate sensitive periods of development promotes masculinization of female morphology, physiology, and, ultimately, behavior (including aggression). Supporting evidence of female masculinization derives primarily from experimental manipulations in various mammalian species (reviewed in Goy and Robinson, 1982; Wallen, 2005) and from clinical case studies of humans (including women with congenital adrenal hyperplasia and those exposed to male co-twins: reviewed in Collaer and Hines, 1995; White et al., 1987). Otherwise, the comparative

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dearth of information on normal feminine development stems from the view that, unlike male development, female sexual differentiation occurs in the absence of testicular androgens. Female mammals are naturally exposed to meaningful concentrations of androgens, however, both prenatally (Herman et al., 2000) and postnatally (Sherwin, 1988). These can metabolize into a variety of other steroids, including estrogens, any of which may play an active role in female differentiation (Collaer and Hines, 1995; Fitch and Denenberg, 1998). As all hormones have multiple effects and modes of action (Falkenstein et al., 2000), it is likely that development is influenced by the synergistic action of multiple hormones, such that both classes of steroids (i.e., androgens and estrogens) may be functionally significant in both sexes (Staub and De Beer, 1997; Whalen, 1984). For instance, novel and complex neuroendocrine mechanisms have been recently implicated in the regulation of aggressive behavior (Demas et al., 2007). Nevertheless, in contrast to studies on the role of estrogens in males, the role of naturally circulating androgens in vertebrate females (beyond their biochemical role in the synthesis of estrogens) generally has been overlooked (Staub and De Beer, 1997). Studies of the female spotted hyena have been exceptional in this regard and have shown that maternal androgens provide a contributory mechanism for the female's natural expression of male-typical morphology and behavior. Delivery of T to the developing female fetus involves the maternal ovary and its extreme production of Δ4 androstenedione (androst-4-ene-3,17,dione; A4). A4 is traditionally considered to be a prohormone—one that is only one metabolic step from becoming either an active androgen (i.e., T) or an active estrogen, depending on local enzymes—but it is also a weak androgen that binds to the androgen receptor (Jasuja et al., 2005). In rodents, exogenous A4 can be effective as a masculinizing agent (Popolow and Ward, 1978) and substrate for the maintenance of aggression (Erpino and Chappelle, 1971) and, under the appropriate administration regimen, has the unusual property of masculinizing without defeminizing (Goldfoot et al., 1969). A4 is the principal androgen in the female hyena (Racey and Skinner, 1979), circulating at higher concentrations than in males throughout most of postnatal life (Glickman et al., 1987, 1992; Licht et al., 1992). Pregnancy markedly alters the hormonal profiles of adult female spotted hyenas (Licht et al., 1992; Lindeque et al., 1986): In addition to facilitating the aromatization of A4 to estrogen, the hyena placenta is characterized by high activity levels of 17β-hydroxy-dehydrogenase (17β-HSD) that converts A4 to T. In earlier work, Yalcinkaya et al. (1993) proposed that increasing plasma T concentrations in pregnant hyenas were transferred to developing fetuses of both sexes, thereby producing organizing and activating effects in fetal females. Nevertheless, manipulating androgen concentrations pre- and postnatally has relatively subtle effects on phallic development in both sexes (Drea et al., 1998; Glickman et al., 1998). Moreover, significant placental aromatase cytochrome P450 activity occurs specifically during the sensitive period of genital differentiation, thereby largely protecting the developing female's genitalia from early androgen excesses (Conley et al., 2007). Thus, more recent findings implicate nonsteroidal mechanisms in the genital development of spotted hyenas (Conley et al., 2007; Cunha et al., 2005; Drea et al., 1998; Glickman et al., 1998, 2006). Nevertheless, an important role still exists for endogenous prenatal androgens, and possibly estrogens (Glickman et al., 1998; Place and Glickman, 2004), in the morphological (Drea et al., 1998, 2002) and behavioral (Browne et al., 2006; Dloniak et al., 2006) traits of female hyenas. The role of naturally circulating androgens in the normal development of other female mammals remains an open question, however (Drea, 2009). Whereas numerous researchers have measured progestins and estrogens in pregnant primates, for example, fewer have evaluated concomitant androgen concentrations (e.g.,

lemurs or strepsirrhines: Ostner et al., 2003; New World monkeys or platyrrhines: Chambers and Hearn, 1979; Old World monkeys or catarrhines: Altmann et al., 2004; Castracane and Goldzieher, 1983; humans: Castracane et al., 1998; Dawood and Saxena, 1977; Glass and Klein, 1981; Klinga et al., 1978). Despite the near ubiquity of masculinized genitalia and female dominance in strepsirrhine primates (reviewed in Drea, 2007), information about gestational hormones in this clade remains scant. Existing studies are typically focused on progestins and estrogens, often rely on urinary or fecal assay techniques, or represent only brief portions of gestation in only a few individuals (e.g., Brockman et al., 1995; Curtis et al., 2000; Gerber et al., 2004; Jurke et al., 1998; Ostner and Heistermann, 2003; Perry et al., 1992; Shideler et al., 1983). To begin to address the potential role and source of prenatal hormones, particularly androgens, in the unusual traits of female strepsirrhines, I measured serum 17α-hydroxyprogesterone (OHP), dehydroepiandrosterone sulphate (DHEA-S), A4, T, and 17β-estradiol (E2) in multiple ring-tailed lemurs sampled before conception, throughout gestation, and during the postpartum period. In a prior study of circulating steroid concentrations in nonpregnant, adult female L. catta, I identified a potential role for hormones in female dominance, showing that seasonal increases in female aggression were associated with concomitant increases in A4 and E2 (Drea, 2007). The main objective here is to examine whether maternal androgens are also elevated during gestation. More specifically, to influence the external genitalia and the neural substrates underlying sexually dimorphic behavior, naturally circulating androgens in female L. catta should be available during critical stages of fetal development. The stages associated with genital and behavioral differentiation typically include the late first and third trimesters, respectively. In the galago, for instance, the gonads remain indifferent through day 33 of a 133-day gestation (Yoshinaga et al., 1988). Thus, in L. catta, T concentrations in maternal blood are expected to be greater during early–mid and late pregnancy than during preconception or early postpartum periods, regardless of fetal sex. Moreover, if endogenous androgens derive from a maternal source (as opposed to a fetal source), increased androgen concentrations could be evident early in the first trimester of pregnancy, before differentiation of the fetal gonads or adrenals. Lastly, if the putative source of androgens were ovarian, rather than adrenal, it would implicate the progesterone or Δ4 pathway more so than the dehydroepiandrosterone or Δ5 pathway (Castracane et al., 1998). Accordingly, elevated A4 concentrations, if accompanied by an increase in OHP, would be consistent with an ovarian source, whereas if accompanied by an increase in DHEA-S, would be consistent with an adrenal source. Beyond these main predictions, a related aim was to evaluate whether the hormone profiles of pregnant L. catta differed by maternal parity, dominance status, litter size or sex ratio, and fetal sex. Maternal parity and dominance status can influence hormone concentrations during pregnancy, with, for example, primiparous female baboons showing greater mean estrogen concentrations compared to multiparous females (Altmann et al., 2004) and dominant female carnivores showing greater mean testosterone concentrations compared to subordinate females (spotted hyenas: Dloniak et al., 2006; meerkats: Clutton-Brock et al., 2006). In hyenas, at least, the T concentrations of pregnant females correlate positively with the aggressive behavior of their offspring, providing evidence to suggest a maternally driven mechanism for the behavioral masculinization of daughters that could contribute to rank inheritance (Dloniak et al., 2006). Moreover, twinning, which is fairly common in captive strepsirrhines (Pasztor and Van Horn, 1976; Parga and Lessnau, 2005) and produces a greater percentage of mixed-sex than same-sex pairs (Pasztor and Van Horn, 1976), can influence maternal estrogen profiles (Jurke et al., 1998). As male fetuses are known to affect the hormonal milieu of developing female littermates or co-twins in various species (Vandenbergh, 2003; vom Saal, 1989), including humans

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(McFadden, 1993), a male co-twin might additionally influence the masculinization of female lemurs. Therefore, as sample sizes permitted, I considered effects of twinning and twin sex ratio on maternal endocrine profiles. After the sexually indifferent stage of fetal development, sexual dimorphism of the external genitalia typically owes to the additional fetal T experienced by developing males. In some cases, fetal sex cannot be predicted by maternal androgen profiles, particularly if sample sizes are small (e.g., strepsirrhines: Ostner et al., 2003) or if one exams early pregnancy only (humans: Glass and Klein, 1981). In other cases, increases in androgen concentrations can be reliably detected in mothers bearing late-term sons (baboons: Altmann et al., 2004; humans: Meulenberg and Hofman, 1991). Likewise, male fetuses can sometimes be predicted by an increase in gestational E2 during late-term pregnancy, presumably reflecting aromatase activity in the fetoplacental unit (Jurke et al., 1998). This phenomenon is particularly evident in strepsirrhines (including Varecia, Hapalemur, and various Eulemur: Gerber et al., 2004; Ostner and Heistermann, 2003; Shideler et al., 1983). Thus, a final aim here was to examine if androgens and estrogens reliably predict fetal sex in L. catta.

Table 1 Maternal variables and reproductive outcome of 19 pregnancies and 4 time-matched nonconceptive cycles in 11 female Lemur catta. Maternal variables

Parity Statusa Sexb Weight Day Age range (g) weighed (years)

5984f (Clei)

19–20

12

D

F

78

0

16–17

12

D

F

89

0

10.5– 11 4–5

8

D

F

79

1

3

S

F

53

7–8

2

S

F

83

2

7–8

1

S

F

69

1

3.5–4

2

D

F

82

4

2.5– 3.5 14–15

1

D

F

62

0

7

I

FF

54, 56

1

18–19

11

D

M

72

0

3–4

2

I

M

1.5–2

1

S- N I

M

75

1

6–7

1

S

MM

64, —

1

15.5– 16 14.5– 15 12.5– 13 15–16

11

I

MF

85, 76

1

10

D

MF

83, 92

3

9

S

MF

—, 84

3

8

I

MF

67, 59

0

5–5.5

4

S

MF

52, —

0

16–17

10

D

U

—

17–18

—

D

N/A

—

6140f (Kati) 6280f (Ninn) 6761f (Dori) 6711f (Sosi) 6709f (Appo) 6796f (Arte) 6796f (Arte) 6276f (Dory) 5984f (Clei)

The focal subjects were 11 reproductively intact, adult female ringtailed lemurs (age range: 1.5–20 years), studied over the course of 7 years. Given the varying availability of subjects, this period of study represented 23 individual breeding cycles (range 1–4 per adult female) and included 19 pregnancies and 4 nonconceptive cycles (Table 1). The pregnancies included 12 singleton and 7 twin litters, producing 26 fetuses (15 females, 10 males, and 1 unsexed). Sexual maturity in lemurs occurs somewhat earlier in captivity than in the wild (Drea, 2007; Parga and Lessnau, 2005). In this study, a 1.5-year-old female (#6832) conceived, carried to term, and successfully weaned her infant. Two other females (#6761f and #6796f) showed no reproductive activity at that age. One pregnancy (initially detected by abdominal palpation and later confirmed by ultrasound) presumably resulted in a stillborn singleton, but the fetus was not retrieved, so its sex and date of birth were unknown. The remaining 18 pregnancies produced live births (n = 25 infants), including eight singleton females (F), one set of twin females (FF), three singleton males (M), one set of twin males (MM), and five sets of mixed-sex twins (MF: Table 1). Based on the outcome of aggressive interactions observed year round between group members (e.g., Drea and Scordato, 2008; Scordato and Drea, 2007; Starling et al., 2010), intrasexual dominance status was assigned to each dam at the time of her pregnancy (Table 1). Social status was relatively stable, although a few females experienced dominance reversals during the course of the study (Table 1). As the social groups studied were relatively small, the classification of adult status was expressed simply as dominant, intermediary, or subordinate for groups containing three breeding females, or as dominant and subordinate for groups containing two breeding females. An additional 12 reproductively intact, adult males (age range: 2.5–20 years), which were subjects in a prior endocrine study (Drea, 2007), provided a basis for intersexual comparisons. All of these animals were captive-born members of three semi-free ranging social groups, housed in large forested enclosures (1.5, 3.3, or 5.8 ha), with attached indoor areas, at the Duke Lemur Center (DLC), in Durham, N. C. The animals foraged freely in their outdoor habitat and were fed daily rations of monkey chow, supplemented with fresh fruit and vegetables. Additional details on housing and animal management have been provided elsewhere (Drea, 2007). The animals were maintained in accordance with United States Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Fetal variables

Duke animal ID Dates (four-letter code) studied

Methods Subjects

419

6761f (Dori)

c

6832 (Nebb) 6711f (Sosi) 5847f (Cori) 6140f (Kati) 6140f (Kati) 6276f (Dory) 6761f (Dori) 6159f (Cleo) 6159f (Cleo) 6709f (Appo) 6796f (Arte)

d

6761f (Dori)

d

8/04–6/ 05 8/03–7/ 04 12/99–7/ 00 8/04–6/ 05 8/04–6/ 05 8/04–7/ 05 10/05–6/ 06 8/04–7/ 05 9/03–8/ 04 9/03–8/ 04 8/03–8/ 04 10/05–6/ 06 9/03–8/ 04 12/99–7/ 00 10/01–3/ 02 12/99–7/ 00 8/04–6/ 05 10/05–6/ 06 8/03–8/ 04 8/04–6/ 05 8/03–8/ 04 9/03–8/ 04 10/01–3/ 02

6–7

0

S

N/A

—

1.5– 2.5 1.5–2

0

S

N/A

—

0

I

N/A

—

Dates of study are provided in continuous bins and subject ages during these periods are given to the nearest half-year. a Social status is given at the time of pregnancy: D = dominant, I = intermediate, S = subordinate, —N = reversal. b M = male, F = female, U = unknown (pregnancy presumably resulted in a stillborn singleton of unknown sex, as the body was not retrieved), N/A = no fetus owing to nonconceptive cycle. ‘Nonconceptive’ refers to females that may have lacked mating opportunities or mated but failed to conceive (as in Drea, 2007). None of these subjects had an unusual reproductive history and, to date, each has conceived at least once. c Early in the breeding season, the reproductive cycle of 6761f was disrupted for health concerns by administration of a single injection of medroxyprogesterone acetate (Depo-Provera®, Pfizer Inc, New York, NY 10017, USA: 5 mg/kg). Conception occurred later in the season (data collection on this female was suspended during the period of contraception). d Hormone concentrations during the first ‘nonconceptive’ cycles of two young females (6796f and 6761f) did not differ appreciably from those displayed during the nonconceptive cycles of two older females (6159f and 6709f).

Study periods I studied the dams and adult males during five time periods: an 8-mo period from Dec 1999 to Jul 2000, a 6-mo period from Oct 2001 to Mar 2002, and three contiguous 12-mo periods from Aug 2003 to Jul 2006. As

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C.M. Drea / Hormones and Behavior 59 (2011) 417–427

reproduction in L. catta is strictly seasonal, these periods targeted critical portions of the breeding cycle. Females are polyestrus, cycling somewhat synchronously (Pereira, 1991) up to three times per year, at about 40-day intervals (Evans and Goy, 1968; Jolly, 1966; Sauther et al., 1999). Here, the ‘breeding season’ refers to the continuous span of time that encompasses potentially three breeding peaks and the ‘nonbreeding season’ refers to the remaining portion of the year. In captive populations housed in the Northern Hemisphere, seasons are shifted by about 6 months from those in Madagascar (Drea, 2007; Parga and Lessnau, 2005), such that the peak of breeding occurs in early November and the peak of birthing occurs in mid March. For the purposes of this study, a yearly ‘reproductive cycle’ began before the onset of the breeding season (Aug) and ended after the weaning of infants (Jul), at 4–5 mo of age (Sauther et al., 1999). Blood sampling I obtained morning blood samples approximately once (sometimes twice) monthly per adult subject. The blood-draw techniques and schedule (that controlled for circadian rhythms) have been detailed elsewhere (Drea, 2007). This regimen provided n = 77 samples from pregnant females, n = 123 samples from nonpregnant females, and n = 141 samples from adult males. Although all months are represented in the data set, the richest sampling regimen occurred during the period encompassing gestation, spanning the onset of breeding through the early birthing season (Oct–Mar). The blood sampling protocol incorporated the following considerations: (1) samples (typically 3 ml) were reduced to 2 ml for pregnant females in their third trimester and (2) sampling was suspended for females nearing parturition and during early lactation, resuming after their infants were at least 1 mo of age. All research protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Duke University (protocol nos. A457-99-09 and A245-03-07). Hormone assays The hormone assays were performed in the Biomarkers Core Laboratory at the Yerkes National Primate Research Center, using previously validated procedures. To control for interassay variation, (1) all samples from a given animal's breeding cycle were run within the same assay, (2) maternal samples involving different fetal sexes and litter sizes were distributed equally between assays, and (3) multiple pregnancies involving the same female were run within the same assay if they produced offspring of different sexes but were run in different assays if they produced offspring of the same sex. Given the limited serum volume from late-term sampling, priority was given to analyzing A4, T, and E2 over OHP or DHEA-S. The sensitivities of the A4, T, and E2 assays and intra- and interassay coefficients of variation for these assays were reported previously (Drea, 2007). For analyses, I assigned the minimum detectable limit of the assay to the few samples that had undetectable E2 concentrations. As with E2, serum OHP was assayed in females only using commercial radioimmunoassay (RIA) kits (Diagnostic Systems Laboratories, DSL, Webster, TX 77598, USA). The OHP assay has a sensitivity of 0.1 ng ml− 1 using a 50-μl dose, with intra- and interassay coefficients of variation of 7.7% at 0.95 ng ml− 1 and 2.5% at 5.56 ng ml− 1, respectively. DHEA-S was first assayed using three different kits. The Coat-aCount DHEA-S RIA kit (Diagnostic Products Corp., DPC, Los Angeles, CA 90045, USA) has a sensitivity range of 25–10,000 ng ml− 1 and was used on 90 samples (from pregnant and nonpregnant females, as well as males), but revealed detectable DHEA-S concentrations for two samples only; all remaining samples were below 25 ng ml− 1. Two additional kits, including the Active DHEA-S RIA kit and the DHEA-S-7 RIA kit (DSL), which have sensitivity ranges of 5–800 ng ml− 1 and 50–8000 ng ml− 1, respectively, were tested on a subset of 10 samples

each, none of which revealed detectable concentrations of DHEA-S. The random sample pools from rhesus monkey (Macaca mulatta) that were also tested in these assays revealed both measurable DHEA-S concentrations and good dose–response curves; however, DHEA-S concentrations were greater in the DSL kits as compared to the DPC kit. Lastly, DHEA-S was assayed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/ MS). This approach was applied to samples from three male lemurs, each run in triplicate, using 50 μl of serum and slight modifications to previously published procedures (Higashi et al., 2007; Hsing et al., 2007; Luu-The et al., 2007; Nakajima et al., 1998): We used the solvent treatment (ethanol) and centrifugation, followed by decanting the supernatant. The samples were then processed (under evaluation) and dried down before resolubilizing them for introduction to the HPLC. No solid-phase cleanup occurred before LC-MS/MS. The method uses a sulfated steroid from horse urine as an internal standard. The DHEA-S was separated from the internal standard on a reverse phase column. Ions and their subsequent fragments were subsequently detected in negative ion mode. Although these procedures applied to chimpanzee (Pan troglodytes) serum revealed detectable concentrations of DHEA-S (e.g., 54 μg dl− 1) and spiking of those samples with extra DHEA-S (low and high amounts) showed appropriate signal responses in the MS, no DHEA-S was detected in lemur samples. No significant matrix effect was observed. Lemur DHEAS was at or below the limit of detection and limit of quantitation in the assay. Statistical analyses I examined variation in hormone profiles across the annual reproductive cycle but specifically targeted the period encompassing gestation (additional information on the annual sex steroid profiles of adult males and nonpregnant females are presented elsewhere: Drea, 2007). Using Student's t-tests, I first evaluated the effects of pregnancy on serum hormone concentrations by comparing pregnant females to nonpregnant females and adult males, independently. For these analyses, I collapsed all stages of gestation and all pregnancies, regardless of fetal sex or litter size. I also controlled for the annual variation in adult endocrine function (Drea, 2007) by considering only those time points for all subjects (i.e., including nonpregnant females and males) that corresponded to gestation months (i.e., Nov–Jun). The small sample sizes and empty cells for certain maternal and fetal variables (Table 1) precluded performing a multivariate analysis; however, as parity was evenly distributed between female- and malebearing litters (Table 1), I tested for effects of the dam's reproductive experience (n = 4 primiparae, n = 15 multiparae) on maternal hormone profiles, using Student's t-tests. Likewise, by using only the pregnancies that produced singleton females, I could test for effects of the dam's dominance status (n = 5 dominant, n = 3 subordinate; Table 1) on maternal hormone profiles, using Student's t-tests. Hereafter, I excluded from analyses of maternal hormone profiles the single pregnancy that resulted in a stillbirth. For the remaining 18 pregnancies, I looked for an effect of litter size, first by comparing the mean steroid concentrations in each same-sex twin against the range of means expressed by pregnancies that bore singletons of the same sex, respectively. Next, I excluded same-sex twins but compared singleton male and singleton female litters to mixed-sex twins using Student's t-tests. In subsequent analyses, I evaluated hormone profiles across stages of pregnancy. Stages approximating the first, second, and third trimesters ideally would have been represented by gestation days 1–45, 46–90, and 91–135, respectively; however, to minimize the number of empty cells, the actual days used were 1–48 (‘early’), 49–85 (‘mid’), and 86–135 (‘late’), respectively. I obtained a preconception (‘pre’) value for 15 of the 18 pregnancies during the 1-mo interval preceding the estimated

C.M. Drea / Hormones and Behavior 59 (2011) 417–427

conception date (at mean± SEM = 14.2± 3.3 days preconception) and a postpartum (‘post’) sample for 17 of the 18 pregnancies a minimum of 1 mo after delivery (at mean± SEM= 5.8 ± 0.3 weeks postpartum). Complete data sets therefore included five reproductive stages (pre, early, mid, late, and post gestation) and were obtained for seven ‘female’ litters and three ‘male’ litters. For these litters, I tested for relationships between fetal sex and maternal hormone profiles across reproductive stages by two-factor rANOVA, resolving main effects using NewmanKeuls' multiple-range tests (Bruning and Kintz, 1977) and significant interactions using F-tests for simple effects (Sokal and Rohlf, 1981). I predicted gestational increases in androgens, over preconception and postpartum ‘baseline’ concentrations, as well as over trimesters. In all of these analyses, I considered differences at α b 0.05 to be significant.

421

singleton females, there were no effects of dominance status on any maternal hormone concentrations (all ts6 b 2.14, all Ps N 0.19). Endocrine correlates of litter size: Comparison of singleton and twin litters

Pregnancy in ring-tailed lemurs was accompanied by an increase in sex steroids (Fig. 1). Using time-matched samples (i.e., Nov–Jun), serum A4 concentrations in pregnant females (range= 0.41–9.11 ng ml− 1) were elevated over those of nonpregnant females (range = 0.11– 5.23 ng ml− 1; t10 = 4.26, P b 0.005), falling within the adult male range (range = 0.41–9.49 ng ml− 1; t21 = 0.32, ns; Fig. 1a). Serum T concentrations in pregnant females (range= 0.05–2.13 ng ml− 1) were also elevated over those of nonpregnant females (range = 0.05– 0.69 ng ml− 1; t10 = 3.94, P b 0.005) but remained much lower than those of adult males (range= 0.46–48.79 ng ml− 1; t21 = 7.54, P b 0.001; Fig. 1a). Serum E2 concentrations in pregnant females (range = 5– 615.36 pg ml− 1) also surpassed those of nonpregnant females (range = 5–69.18 pg ml− 1; t10 = 3.42, P b 0.01; Fig. 1b). Likewise, serum OHP concentrations in pregnant females (range = 2.37– 15.55 ng ml − 1 ) exceeded those of nonpregnant females (range = 0.59–8.68 ng ml− 1; t9 = 5.71, P b 0.001; Fig. 1c). Lastly, the only two samples with detectable concentrations of DHEA-S derived from pregnant females carrying singleton females, one at midterm (33.4 ng ml− 1) and one in late gestation (39.9 ng ml− 1). Thus, while A4 and OHP concentrations were significantly elevated during pregnancy, DHEA-S was virtually undetectable.

To examine the possibility that steroid concentrations might be increased in twin pregnancies, I first examined same-sex litters. Mean hormone concentrations in the sole twin female litter (FF) were generally elevated by comparison to the eight singleton female litters (F), excepting for A4, which showed no difference (Fig. 2a). The FF litter fell three standard deviations (SD) above the F mean for T (Fig. 2b), over two SD above the F mean for E2 (Fig. 2c), and nearly four SD above the F mean for OHP (data not shown). In the FF litter, T concentrations were in the range of singleton males (M), whereas E2 concentrations were intermediate between the F and M ranges. The differences between the sole twin male (MM) and the three M litters were less conspicuous: T and E2 in the MM litter were about one SD above the M mean (Fig. 2). Next, I excluded the FF and MM litters and compared the singleton F (n = 8) and M (n = 3) litters to mixed-sex twins (MF, n = 5). In all cases, MF litters showed steroid concentrations that significantly exceeded those of F litters (A4: t11 = 4.51, P b 0.001; T: t11 = 3.10, P b 0.01; E2: t11 = 4.33, P b 0.001; OHP: t11 = 4.87, P b 0.001; Fig. 2). With the exception of having greater OHP concentrations (t6 = 2.91, P b 0.05), MF litters showed steroid concentrations similar to those of M litters (A4: t6 = 0.53, P = 0.61; T: t6 = 0.51, P = 0.63; E2: t6 = 1.36, P = 0.22; Fig. 2). Both the size and sex composition of the litter appeared to influence gestational endocrine profiles, but with variable effects for the different hormones. Whereas OHP concentrations may partially increase with the number of offspring (singleton b twin), steroid concentrations of A4, T, and E2 may reflect both the number and sex of offspring, in approximately the following incremental order: F ≤ FF ≤ M ≤ MF = MM (Fig. 2). Therefore, in subsequent analyses by ‘fetal sex,’ I defined ‘female’ litters as singleton or twin females and ‘male’ litters as singleton or twin males, plus mixed-sex twins. It should be noted, however, that the patterns observed in the following analyses would not have differed had twin females and twin males been excluded from the ‘female’ and ‘male’ categories, respectively.

Endocrine correlates of maternal variables: Parity and dominance status

Maternal changes by reproductive stage and ‘fetal sex’

Across all 19 pregnancies, there were no effects of parity on maternal A4, T, or E2 concentrations (all ts17 b 0.63, all Ps N 0.50) and only a nonsignificant trend for greater OHP concentrations in multiparae than primiparae (t15 = 1.74, P = 0.103). For dams bearing

All dams showed elevations in steroid concentrations during pregnancy (Fig. 1), but the temporal pattern and magnitude of the increases in A4, T, E2, and OHP differed by ‘fetal sex.’ The endocrine profiles across consecutive reproductive cycles of representative dams

Results Endocrine correlates of pregnancy: Comparison of time-matched adult profiles

a

b

c

Fig. 1. Effect of pregnancy on mean ± SEM serum concentrations of (a) androstenedione and testosterone, (b) estradiol, and (c) hydroxyprogesterone in female L. catta (n = 11). (a) Androgen concentrations in age-matched, adult male L. catta (n = 12) are provided for comparison (⁎P b 0.01, ⁎⁎P b 0.005, ⁎⁎⁎P b 0.001; t tests).

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a

b

c

Fig. 2. Maternal mean ± SEM serum concentrations of (a) androstenedione, (b) testosterone, and (c) estradiol in pregnant L. catta bearing various litter types. Singleton and twin female fetuses (F + FF) are considered ‘female’ litters; singleton male, twin male, and mixed-sex twins (M + MM + MF) are considered ‘male’ litters. Numbers above the bars in (a) represent sample sizes for each litter type in (a–c).

are shown in Fig. 3. Noteworthy is the rapid rise in T concentrations with the onset of pregnancy, regardless of fetal sex (e.g., Fig. 3b). The mean steroid concentrations in dams that bore either ‘female’ or ‘male’ litters are illustrated for each reproductive stage (using all available data from the 18 pregnancies in which fetal sex was known; Fig. 4). For statistical analyses of these data, I conservatively used twofactor rANOVAs (reproductive stage X fetal sex) on the subset of pregnancies sampled at all five time points (which generally included eight ‘female’ litters and five ‘male’ litters). Nonetheless, partial and complete data sets showed the same patterns, revealing that A4 and T concentrations could be used to predict fetal sex as early as the second trimester, whereas E2 concentrations could be used to predict fetal sex in the third trimester (Fig. 4). The analyses revealed significant main effects of reproductive stage for serum A4 (F4,44 = 15.47, P b 0.001; Fig. 4a), T (F4,44 = 11.35, P b 0.001; Fig. 4b), E2 (F4,44 = 10.68, P b 0.001; Fig. 4c), and OHP (F4,28 = 14.53, P b 0.001; Fig. 4d), with the greatest A4 and T concentrations occurring in the second and third trimesters, and the greatest E2 and OHP concentrations occurring in the third trimester. There were fewer significant main effects of fetal sex: Relative to dams bearing female fetuses, dams bearing at least one male fetus showed increased maternal concentrations of A4 (F1,11 = 7.39, P = 0.020; Fig. 4a) and E2 (F1,11 = 9.25, P = 0.011; Fig. 4c) across all reproductive stages combined, but relatively equal concentrations of T (F1,11 = 1.96, P = 0.190, ns; Fig. 4b) and OHP (F1,7 = 0.23, ns; Fig. 4d). Lastly, these analyses revealed significant interaction effects of reproductive stage by fetal sex for A4 (F4,44 = 10.99, P b 0.001; Fig. 4a), T (F4,44 = 5.91, P = 0.001; Fig. 4b), and E2 (F4,44 = 14.50, P b 0.001; Fig. 4c), but not for OHP (F4,28 = 1.99, ns; Fig. 4d). Thus, whereas dams bearing fetuses of either sex showed consistent gestational increases in OHP over preconception and postpartum concentrations, the developmental patterns for A4, T, and E2 differed by fetal sex in the following ways. For pregnancies involving female fetuses only, the gestational rise in A4 did not exceed preconception concentrations, but was greater throughout early (P b 0.005), mid (P b 0.001), and late (P b 0.01) gestation than during the postpartum period (Fig. 4a). Serum T concentrations rose slightly, but steadily, across gestation, reaching statistical significance over preconception concentrations during the latest stage of gestation (P b 0.05; Fig. 4b). As with A4, T concentrations were significantly elevated throughout early (P b 0.025), mid (P b 0.01), and late (P b 0.001) gestation, compared to the postpartum period. Lastly, dams bearing female fetuses showed no statistical differences in E2 across reproductive stages (Fig. 4c). For pregnancies involving at least one male fetus, A4 and T concentrations showed similar developmental patterns, with the greatest increases over preconception and postpartum concentrations occurring at mid and late gestation (all P values b 0.001; Figs. 4a and b). Both androgens were also elevated in mid and late gestation

relative to early gestation (P values b 0.001). Interestingly, concentrations of both androgens dipped in late gestation relative to mid gestation (A4: P b 0.025; T: P b 0.01). This dip was coincident with a sharp and dramatic elevation in maternal E2 concentrations during late gestation, relative to all other phases (all P values b 0.001; Fig. 4c). Endocrine correlates of pregnancy: Gestational effects versus seasonal fluctuation The pregnancies represented above derived from three conception cycles separated by roughly 40 days (as in Evans and Goy, 1968). Of the 18 firm estimates of conception, the first conception cycle (n = 11) occurred within the first 3-week period in November, the second conception cycle (n = 5) occurred within a 3-week period in December–January, and the final conception cycle (n = 2) occurred within a 1-week period in early February. Because of the span of time represented by these pregnancies and because of the seasonal fluctuation in androgen production by female L. catta (Figs. 3a–f; see also Fig. 4 in Drea, 2007), I examined maternal endocrine patterns for potential seasonal influence. Fig. 5 presents female androgen concentrations, matched for time of year (rather than gestation length), for three groups of females: females that were not pregnant, dams bearing female fetuses, and dams bearing male fetuses. It is apparent from this figure that androgen concentrations during gestation involving fetuses of either sex exceeded any baseline seasonal fluctuation, particularly during late gestation. Discussion Increasing a female mammal's exposure to androgens prenatally or neonatally, during sensitive periods of development, masculinizes her genitalia, enhances body size and growth, alters neuroendocrine function, augments aggression, and invigorates play (reviewed in Goy and Robinson, 1982; Wallen, 2005). The timing of the ‘inappropriate’ action of androgens independently influences morphological and behavioral differentiation, such that androgen exposure early in gestation masculinizes both female external genitalia and some components of behavior (e.g., Herman et al., 2000), whereas late prenatal androgens masculinize behavior only (e.g., Goy et al., 1988). To explore the possibility that, through prolonged prenatal exposure to endogenous androgens, a syndrome of masculine morphological and behavioral traits might occur naturally in a female mammal other than the spotted hyena, this study provides the first serum profiles of maternal sex steroids throughout gestation in the ring-tailed lemur. Concentrations of A4, T, E2, and OHP were elevated in pregnant lemurs, relative to nonpregnant females and relative to seasonal fluctuations, with gestational A4 achieving concentrations comparable to those of adult male conspecifics in peak breeding season. The

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a

e

b

f

c

g

d

h

Fig. 3. Serum concentrations of (a and e) androstenedione, (b and f) testosterone, (c and g) estradiol, and (d and h) hydroxyprogesterone during consecutive reproductive cycles of two representative female L. catta: (a–d) # 6711 and (e–h) # 6140. The reproductive cycles include periods when the adult female was not pregnant, when she was pregnant with a singleton female fetus, and when she was pregnant with either a set of male–male (a–d) or male–female (e–h) twins. Values represent a single morning sample, usually taken during the first week of each month. When applicable, the second month's sample was obtained after a 2-week interval. Numbers above the bars in (a) and (e) indicate the estimated day of gestation for each female and are consistent across the different steroids, respectively. Given variation in gestation length, hatched bars represent uncertainty, i.e., days that could have been ‘preconception’ days or days when the female may have already conceived.

gestational rise in A4 and T expressed in pregnant lemurs was respectively nearly 50% and over 20% that experienced by pregnant spotted hyenas. It should be noted that increased androgen concentrations characterize pregnancy in various species, including humans (e.g., Dawood and Saxena, 1977), but the timing and persistence of androgen secretion may well differ by species (as occurs with other steroids: e.g., Gerber et al., 2004). Whereas the increasing T concentrations across all stages of gestation observed in lemurs modestly replicate the pattern observed in pregnant spotted hyenas, similar profiles are absent in other gestating females (e.g.,

marmosets: Chambers and Hearn, 1979; dogs: Concannon and Castracane, 1985). Also, as in spotted hyenas, the rise in androgen concentrations at the onset of lemur pregnancy precedes the development of the fetal adrenals or gonads, consistent, at least initially, with maternal derivation. Gestational endocrine profiles were unrelated to maternal traits, including parity or dominance status; however, as the sample sizes for these analyses were small, the negative findings should be interpreted with caution. By contrast, gestational endocrine profiles were influenced by litter size, but most strongly related to fetal sex. In

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a

b

Fig. 5. Mean ± SEM monthly serum concentrations of testosterone in dams bearing ‘female’ or ‘male’ litters (as defined in Fig. 2), regardless of gestational stage. Provided for comparison is the annual variation in testosterone experienced by nonpregnant females. Relative to the data points, sample sizes for ‘female’ litters are provided to the right and italicized, sample sizes for ‘male’ litters are to the left, and sample sizes for nonpregnant females are beneath the curve.

c

d

Fig. 4. Mean ± SEM maternal serum concentrations of (a) androstenedione, (b) testosterone, (c) estradiol, and (d) hydroxyprogesterone across reproductive phases and by fetal sex (as defined in Fig. 2). Reproductive phases include preconception (‘pre) and postpartum (‘post’), as well as approximations of the first, second, and third trimester of gestation, termed ‘early,’ ‘mid,’ and ‘late,’ respectively. Numbers above the bars in (a) represent sample sizes at each reproductive phase in (a–c). Samples available for hydroxyprogesterone assay were slightly reduced, as indicated by the numbers above the bars in (d).

particular, the observed increments in maternal A4, T, and E2 were most pronounced in lemurs if pregnancies involved at least one male fetus (i.e., M, MF, or MM litters). Importantly, relative to preconception and postpartum periods, gestational increments in A4, T, and E2 were also evident in lemur pregnancies involving a sole female fetus, particularly during organizational periods typically characterized by differentiation of the external genitalia and the neural substrates

underlying behavior. Nonetheless, fetal sex differences at different stages of lemur gestation were nonoverlapping, such that concentrations of A4, T, and E2 in lemur dams reliably predicted fetal sex either by the second or third trimester. During human pregnancy, maternal T sometimes shows a similar pattern of differentiation by fetal sex, but with significant overlap in the variances between women bearing male versus female fetuses (Bolton et al., 1989; Meulenberg and Hofman, 1991). Consequently, reliable antenatal sex determination is precluded in a large percentage of human pregnancies. Moreover, a human male fetus' exposure to androgens at mid term might not be detectable via maternal serum endocrine profiles, requiring instead assay of either fetal plasma (Rodeck et al., 1985) or amniotic fluid (van de Beek et al., 2004). Thus, in humans, maternal endocrine profiles may not accurately reflect the fetal endocrine environment. By contrast, the present data on strong fetal sex differences in lemurs suggest that maternal serum endocrine profiles may well provide reliable information about fetal androgen (and estrogen) exposure, although various factors preclude drawing such a conclusion at this stage. As studies of gestational hormones in most species typically exclude measurement of androgens, the degree to which the endocrine profiles reported herein are unique to female-dominant species remains unknown. Minimally, therefore, a broader comparative examination of prenatal endocrine environments would be required. Another hurdle to understanding the ‘uniqueness’ of the pattern reported in hyenas and lemurs is that many other mammalian species studied produce larger litters, thereby increasing the fetal female's risk of exposure to male hormones. In such cases, the influence of fraternal androgens may confound any potential influence of maternal androgens. As seen here, the presence of a single male co-twin was sufficient to dramatically alter maternal endocrine profiles. Future studies could be aimed at assessing whether female lemurs derived from mixed-sex twins show greater genital or behavioral masculinization than do females that were either singletons or derived from same-sex twins. Most importantly, the hypothesis that prenatal exposure to maternal androgens may play a role in the morphological (Drea and Weil, 2008) and behavioral (Drea, 2007) masculinization of female ring-tailed lemurs and other strepsirrhines awaits more detailed information about the maternal–placental–fetal unit. Specifically, determining whether the maternal ovary produces androgens that actually reach the developing fetal lemur requires identification of the

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androgen substrates available to the strepsirrhine placenta, clarification of the role of placental enzyme activity (as in Conley et al., 2007; Yalcinkaya et al., 1993) and experimental manipulation of prenatal androgen concentrations (as in Drea et al., 1998), accompanied by postnatal developmental studies. Given the conservation status of lemurs and the potential risks associated with collecting these data, such studies present logistical challenges. Despite the limitations associated with the study of fetal development, it is clear that the reproductive biology of strepsirrhines is distinctive from that of haplorrhines in several ways (Martin, 1990). In particular, strepsirrhine placentation is least invasive or epitheliochorial, whereas haplorrhine placentation is most invasive or hemochorial (Martin, 1990, 2008; as is the case for spotted hyenas: Wynn and Amoroso, 1995). Although it is commonly assumed that epitheliochorial placentation is associated with lower rates of exchange between the mother and fetus, the relationship between type of placentation and efficiency of nutrient (and presumably hormonal) transfer may not be so simple (Martin, 2008). For instance, when the effects of maternal body weight and gestation length are taken into account, strepsirrhine fetuses show higher rates of development than do haplorrhine fetuses, potentially owing to lower maternal basal metabolic rate (BMR: Martin, 1990, 2008). Interestingly, the link between maternal BMR and fetal development appears to be most pronounced in strepsirrhine species characterized by female dominance (Young et al., 1990), but the mechanism to explain this association remains a mystery. The endocrine environment of pregnant females in these two primate clades may also differ substantially. For instance, haplorrhine pregnancy is characterized by a progressive rise in estrogens, beginning early and continuing throughout gestation (Albrecht and Pepe, 1999). Although maternal ovaries secrete estrogens that contribute to the maternal endocrine profile, particularly early in gestation, placental estrogen formation largely depends on C19 steroid precursors (e.g., DHEA-S) that later derive from the fetal adrenals (Albrecht and Pepe, 1999). By contrast, strepsirrhine pregnancies involve substantial elevations in estrogens only late in gestation, implicating the fetoplacental unit more so than the maternal ovaries (Jurke et al., 1998). As noted earlier, the late-term elevations in serum E2 observed in ring-tailed lemur dams were strongly biased by fetal sex, replicating the pattern previously reported for other strepsirrhines, using fecal or urinary assays (Gerber et al., 2004; Ostner and Heistermann, 2003; Shideler et al., 1983). Although the relationship between maternal estrogens and fetal sex cannot be viewed as a unique characteristic of lemurs, as it recently has been described in a catarrhine (Altmann et al., 2004), the magnitude of the fetal sex difference in E2 appears to be unusually pronounced in strepsirrhines. The increased E2 concentrations in male-bearing lemur pregnancies, relative to female-bearing pregnancies, probably reflect a combined effect of increased androgen precursor from the fetal testes, plus the progressive increase in the capacity for placental aromatization (Albrecht and Pepe, 1999; Shideler et al., 1983). Accordingly, strepsirrhine pregnancies may also differ from haplorrhine pregnancies in their respective roles for adrenal androgens, notably DHEA-S. In various catarrhine primates studied to date (e.g., macaques, chimpanzees, humans), DHEA and DHEA-S are the principal androgens secreted from the adrenals (Nguyen and Conley, 2008) and become the major precursors of estrogens during pregnancy (Albrecht and Pepe, 1999). In the present study, DHEA-S concentrations in pregnant female lemurs, as in adult male conspecifics, were at or below the limit of detectability (using RIA and LC-MS/MS procedures). Although it is possible that these analytical approaches may not have been well tuned to detecting lemur DHEA-S, prior researchers measuring DHEA-S via immunoenzymoassay also reported low concentrations in the mouse lemur (Microcebus murinus)—lower than in some haplorrhine species (Perret and Aujard, 2005). Together, these data suggest a potentially diminished role for adrenal androgens in strepsirrhine primates. Also in contrast with

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happlorrhine pregnancies, these data reveal a clear mid–late term contribution of androgens from the fetal testes. The combined results reported herein on OHP, DHEA-S, and A4 are most consistent with the Δ4 pathway of androgen derivation, suggesting a maternal ovarian source of androgen during lemur pregnancy. To the extent that hormone profiles in pregnant lemurs reflect maternal ovarian secretion and duplicate the temporal pattern seen in gestating hyenas, a potential role for prenatal, endogenous androgens may exist in the masculinization of lemur daughters. Testing this hypothesis nevertheless requires further study of the fetal endocrine environment in female-dominant lemurs. Acknowledgments I am grateful to the staff of the Duke Lemur Center, particularly C. Williams, DVM, J. Hurley, DVM, B. Schopler, DVM, D. Brewer, S. Combes, J. Ives, and J. Taylor. I am also indebted to S. Cork, C. Fitzpatrick, E. Scordato, and A. Starling for assistance with data collection. S.E. Glickman and P. Brown provided valuable discussion. This is DLC publication #1189. References Albrecht, E.D., Pepe, G.J., 1999. Placental steroidogenesis in primate pregnancy. In: Knobil, E., Neill, J.D. (Eds.), Encyclopedia of Reproduction. Vol. III. Academic Press, London, pp. 889–898. Altmann, J., Lynch, J.W., Nguyen, N., Alberts, S.C., Gesquiere, L.R., 2004. Life-history correlates of steroid concentrations in wild peripartum baboons. Am. J. Primatol. 64, 95–106. Bolton, N.J., Tapanainin, J., Koivisto, M., Vihko, R., 1989. Circulating sex hormonebinding globulin and testosterone in newborns and infants. Clin. Endocrinol. 31, 201–207. Brockman, D.K., Whitten, P.L., Russell, E., Richard, A.F., Izard, M.K., 1995. Application of fecal steroid techniques to the reproductive endocrinology of female Verreaux's sifaka (Propithecus verreauxi). Am. J. Primatol. 36, 313–325. Browne, P., Place, N.J., Vidal, J.D., Moore, I.T., Cunha, G.R., Glickman, S.E., Conley, A.J., 2006. Endocrined differentiation of fetal ovaries and testes of the spotted hyena (Crocuta crocuta): timing of androgen-independents versus androgen-driven genital development. Reproduction 132, 649–659. Bruning, J.L., Kintz, B.L., 1977. Computational Handbook of Statistics, 2nd Ed. Scott, Foresman, Glenview. Castracane, V.D., Goldzieher, J.W., 1983. Plasma androgens during early-pregnancy in the baboon (Papio cynocephalus). Fertil. Steril. 39, 553–559. Castracane, V.D., Stewart, D.R., Gimpel, T., Overstreet, J.W., 1998. Maternal serum androgens in human pregnancy: early increases within the cycle of conception. Hum. Reprod. 13, 460–464. Chambers, L.C., Hearn, J.P., 1979. Peripheral plasma levels of progesterone, oestradiol17b, oestrone, testosterone, androstenedione, and chorionic gonadotrophin during pregnancy in the marmoset monkey, Callithrix jacchus. J. Reprod. Fertil. 56, 23–32. Clutton-Brock, T.H., Hodge, S.J., Spong, G., Russell, A.F., Jordan, N.R., Bennett, N.C., Sharpe, L.L., Manser, M.B., 2006. Intrasexual competition and sexual selection in cooperative mammals. Nature 444, 1065–1068. Collaer, M.L., Hines, M., 1995. Human behavioral sex differences: a role for gonadal hormones during early development? Psychol. Bull. 118, 55–107. Concannon, P.W., Castracane, V.D., 1985. Serum androstenedione and testosterone concentrations during pregnancy and nonpregnant cycles in dogs. Biol. Reprod. 33, 1078–1083. Conley, A.J., Corbin, C.J., Browne, P., Mapes, S.M., Place, N.J., Hughes, A.L., Glickman, S.E., 2007. Placental expression and molecular characterization of aromatase cytochrome P450 in the spotted hyena (Crocuta crocuta). Placenta 28, 668–675. Conte, F.A., Grumbach, M.M., Ito, Y., Fisher, C.R., Simpson, E.R., 1994. A syndrome of female pseudohermaphrodism, hypergonadotropic hypogonadism, and multicystic ovaries associated with missense mutations in the gene encoding aromatase (P450arom). J. Clin. Endocrinol. Metab. 78, 1287–1292. Cunha, J.R., Place, N.J., Baskin, L., Conley, A., Weldele, M., Cunha, T.J., Wang, Y.Z., Cao, M., Glickman, S.E., 2005. The ontogeny of the urogenital system of the spotted hyena (Crocuta crocuta Erxleben). Biol. Reprod. 73, 554–564. Curtis, D.J., Zaramody, A., Green, D.I., Pickard, A.R., 2000. Non-invasive monitoring of reproductive status in wild mongoose lemurs (Eulemur mongoz). Reprod. Fert. Dev. 12, 21–29. Dawood, M.Y., Saxena, B.B., 1977. Testosterone and dihydrotestosterone in maternal and cord blood and in amniotic fluid. Am. J. Obstet. Gynecol. 129, 37–42. Demas, G.E., Cooper, M.A., Albers, H.E., Soma, K.K., 2007. Novel mechanisms underlying neuroendocrine regulation of aggression: a synthesis of rodent, avian and primate studies. In: Blaustein, J.D. (Ed.), Behavioral Neurochemistry, Neuroendocrinology and Molecular Neurobiology. Springer-Verlag, Berlin, pp. 337–372. Dloniak, S.M., French, J.A., Holekamp, K.E., 2006. Rank-related effects of androgens on behaviour in wild spotted hyenas. Nature 440, 1190–1193.

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