Exposure to developing females induces polyuria, polydipsia, and altered urinary levels of creatinine, 17β-estradiol, and testosterone in adult male mice (Mus musculus)

Hormones and Behavior 55 (2009) 240–247

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Exposure to developing females induces polyuria, polydipsia, and altered urinary levels of creatinine, 17β-estradiol, and testosterone in adult male mice (Mus musculus) Denys deCatanzaro ⁎, Ayesha Khan, Robert G. Berger, Elaine Lewis Department of Psychology, Neuroscience and Behaviour, McMaster University, Hamilton, Ontario L8S 4K1, Canada

a r t i c l e

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Article history: Received 11 June 2008 Revised 23 October 2008 Accepted 24 October 2008 Available online 3 November 2008 Keywords: Mice Polyuria Polydipsia Vandenbergh effect Creatinine Estradiol Testosterone

a b s t r a c t Novel male mice can accelerate reproductive maturation in proximal developing females, an effect mediated by the chemistry of the males' urine. Exogenous estrogens can similarly accelerate female sexual development. In Experiment 1, adult male mice were housed across wire grid from either empty compartments or those containing post-weanling females. Proximity of females caused males to urinate more, progressively over days of exposure, with most urination directed towards females' compartments. Male urine collected after 5 days in these conditions was analyzed by enzyme immunoassay for 17β-estradiol, testosterone, and creatinine. Urinary creatinine of isolated males significantly exceeded that of female-exposed males. Unadjusted urinary steroids also trended toward higher levels in isolates, but creatinine-adjusted estradiol and testosterone of female-exposed males significantly exceeded that of isolated males. In ,Experiment 2, measurement of water consumption indicated significantly greater drinking by female-exposed as opposed to isolated males. In , Experiment 3, males were housed in isolation or beside post-weanling intact (sham-operated) females, ovariectomized females, or intact (sham-operated) males. Male water consumption was elevated in all conditions involving social contact. Urinary creatinine was significantly lower in female-exposed males compared to isolated controls, while unadjusted testosterone was significantly lower in males in all social conditions. Again, creatinine-adjusted estradiol in female-exposed males significantly exceeded that of isolates. These data indicate that adult males drink and urinate more, have more dilute urine, and have a higher ratio of estradiol to creatinine when they are near developing females. These dynamics increase females' exposure to urinary steroids and other urinary constituents that can hasten sexual maturity. © 2008 Elsevier Inc. All rights reserved.

Introduction Proximity to novel adult males can accelerate sexual maturation in juvenile females of several mammalian species, including mice (Vandenbergh, 1967), rats (Vandenbergh, 1976), deer mice (Teague and Bradley, 1978), voles (Spears and Clarke, 1986), lemmings (Hasler and Banks, 1975), opossums (Harder and Jackson, 2003), and cattle (Roberson et al., 1991). In post-weanling female mice, exposure to adult males can advance sexual maturity by as much as twenty days (Vandenbergh, 1967). This is reflected in increased vaginal opening (Vandenbergh, 1967), cyclical changes in vaginal cytology (Bingel 1972; Vandenbergh 1967, 1976), increased reproductive tissue mass (Beaton et al., 2006; Khan et al., 2008b), and earlier display of sexual receptivity and more rapid insemination during direct exposure to adult males (Khan et al., 2008a). Sexual maturation in female mice and other mammals is dependent to a large degree upon ovarian steroid secretion. Growth of the uterus during development and within estrous cycles is highly dependent upon estrogens, with some species-specific variations in ⁎ Corresponding author. E-mail address: [email protected] (D. deCatanzaro). 0018-506X/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2008.10.013

timing (Gray et al., 2001). In mice, exogenous estradiol causes uterine cells to proliferate (Ogasawara et al., 1983). Estrogens regulate growth hormone and IGF-1 activity (Kahlert et al., 2000; Leung et al., 2004), and local IGF-1 activity mediates uterine growth in response to estradiol (Sato et al., 2002). Estrogens also play critical roles in the preparation of female sexual receptivity (e.g. deCatanzaro, 1987; Pfaff, 1980). Given that male mouse urine and other excretions contain abundant quantities of unconjugated estradiol (Beaton et al., 2006; deCatanzaro et al., 2006; Vella and deCatanzaro 2001), and male mice actively direct urine droplets at proximate females (deCatanzaro et al., 2006; Hurst, 1990b; Reynolds 1971), estrogens in males' excretions could contribute to male-induced pubertal acceleration in proximal females. Previous investigations have indicated that males' excretions may contain higher quantities of creatinine-adjusted estradiol and testosterone after males have been exposed for 3 or more days to postweanling, pre-pubertal females (Beaton et al., 2006) or females inseminated by other males around the time of intrauterine implantation (deCatanzaro et al., 2006). It is common practice in conducting urinary steroid analyses to compensate for variations in hydration by adjusting sample values for creatinine, an index of metabolic activity, due to variation within and among animals in fluid

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intake and excretion (e.g. deCatanzaro et al., 2003; Erb et al., 1970; Muir et al., 2001; Munro et al., 1991). However, creatinine itself can vary, at least in laboratory mice, depending on environmental, background, and social variables (Beaton et al., 2006; Khan et al., 2008b). It has also been observed that adult males that are proximal to females can be extraordinarily aroused, active, and aggressive (deCatanzaro et al., 1996, 2000), which could impact their delivery of urine and its bioactive constituents, such as estradiol, to nearby females. On this basis, we hypothesized that there might be differences between isolated males and males housed in proximity to developing females in fluid intake, urination, and urinary content of creatinine and sex steroids. The current experiments were designed to shed further light on the causation of male-induced pubertal acceleration in mice, specifically the possibility that this is mediated by delivery of exogenous estradiol and other steroids by males to females through urine. As it is already established that exogenous estrogens can accelerate female sexual maturation (cf. Bronson, 1975; Khan et al., 2008b; Ogasawara et al., 1983), that male urine contains unconjugated estradiol and other steroids (Beaton et al., 2006; deCatanzaro et al., 2006; Muir et al., 2001), and that males deliver their urine to proximal females (deCatanzaro et al., 2006; Reynolds, 1971), the validity of this hypothesis depends upon quantitative issues. Thus it is important to establish the dynamics of male urination during exposure to developing females and the concentrations of unconjugated estrogens in male urine. Fluid intake is also quite relevant given its likely impact upon urinary quantity, while creatinine levels in male urine should also reflect fluid intake and excretion. Accordingly, we compared adult males that were housed alone to those housed by developing females in frequency of urination, location of urination, water intake, and urinary concentrations of 17β-estradiol, testosterone, and creatinine. We also compared water intake, creatinine, and steroid dynamics in males' urine in the presence of developing intact females, ovariectomized females, and males. Methods Subjects Heterogeneous strain (HS) mice (Mus musculus) had previously been produced by interbreeding C57-B6, Swiss Webster, CF1, and DBA-2 strains obtained from Charles River Breeding Farms (Québec, Canada). HS male subjects, aged 7–9 months with mean (±SE) weight of 46.7 ± 1.5 g at the commencement of procedures, had been housed individually since weaning in standard polypropylene cages measuring 28 × 16 × 11 (height) cm and had no previous sexual experience. CF-1 strain stimulus female mice were of stock from Charles River Breeding Farms. Stimulus females were weaned from full litters at 28 days, with no more than two weanling females chosen per litter, then randomly placed in compartments of an experimental apparatus as described below. Prior to and throughout experimental procedures, animals had continuous access to water and 8640 Teklad Certified Rodent Chow. The animal colony was maintained under a reversed 10:14 hour dark:light cycle at 21 °C. This research was approved by the McMaster University Animal Research Ethics Board, conforming to standards of the Canadian Council on Animal Care. Experimental apparatus A urinary collection apparatus, constructed of Plexiglas and stainless-steel wire-mesh grid, was divided into three compartments. The male's rectangular compartment measured 30 × 9 × 15(height) cm. Two adjacent square compartments for developing stimulus animals measured 15 × 15 × 15 cm such that each compartment had a 225 cm2 interface through vertical grid with each other compartment. Vertical wire mesh between the two square compartments had grid of

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0.25 cm2, while that between each square (stimulus animal) compartment and the rectangular (male) compartment had a grid of 1 cm2, allowing limited interactions between the male and each developing stimulus animal. Each compartment had an outset closet away from the grid walls that provided continuous access to food and water. All compartments had wire-grid floor with squares of 0.5 cm2 raised 2 cm above a Teflon-coated collection tray. Experiment 1 This experiment was designed to compare urination patterns and urinary creatinine, estradiol, and testosterone in isolated males and those exposed to developing females. On the first day of experimental procedures (day 1), at 1 h after commencement of the dark phase of the lighting cycle, an HS male subject was placed in the rectangular compartment of the apparatus. For 12 randomly-selected isolated control males, the two adjacent square compartments remained empty. For 12 other female-exposed males, a 28-day old female was placed in each of the two square compartments of the apparatus. Two developing females were used for each male in order to ensure robust effects and to reduce the potential influence of inter-female variance. Behavioral observations were conducted for each animal under dim illumination on each of days 1–4 of experimental procedures. Each session lasted 30 min and commenced approximately 1 to 2.5 h after the start of the dark phase of the lighting cycle, with time of sessions counterbalanced across conditions and within conditions over days, such that each animal was tested at each of four possible half-hour intervals. A trained observer recorded each instance of male urination, and the location of urination in terms of three categories: toward the floor of the male's compartment, toward a Plexiglas wall, or toward an adjacent compartment through grid. Instances in which the male made nasal contact with the grid interface on one or the other adjacent compartments were also recorded for each male. Each session was videotaped via a Sony model TVR33 digital video camera equipped with an infrared camera mounted spotlight. Videotapes were reviewed as necessary to confirm counts. On day 5 of the experiment, at the commencement of the dark phase of the lighting cycle, each cage was gently lifted from the existing collection tray and placed on a clean tray which was covered with wax paper to facilitate urine droplet identification and collection. Males were monitored in dim illumination until urination was observed. Urine samples that were uncontaminated by feces, water, or food residue were aspirated using a 1 ml syringe with a 26-gauge needle and stored in labeled 1 ml microtubes. If necessary, males were observed and samples collected up until 5 h after commencement of the dark phase of the lighting cycle. On day 6 of the experiment, this whole procedure was repeated in order to gain a second urine sample for each male. All samples remained frozen at −20 °C until they were assayed for 17β-estradiol, testosterone, and creatinine as described below. Assays were conducted on all samples, simultaneously for each substance. Experiment 2 This experiment was designed to assess fluid intake in isolated males and those exposed to developing females. Sixteen HS males were exposed to 28-day-old females, while 16 others remained isolated in the experimental apparatus. Each male was given a graduated water bottle with a measured quantity of 200 ml water on the initial day of the experiment at the point of introduction to the apparatus, approximately 1 h after commencement of the dark phase of the light cycle. After 5 days (120 h) of undisturbed habitation in these conditions, the volume remaining in the water bottle was measured for each male, and subtracted from the initial quantity to calculate water consumption for each animal.

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given a graduated water bottle with a measured quantity of 200 ml water at the point of introduction to the apparatus. After 3 days in these conditions, at 2–5 h after commencement of the dark phase of the animals' light cycle, each male subject was observed in the experimental apparatus by a trained observer during a 30-min session, with time of observation counterbalanced over conditions. During this session, the observer recorded the frequency of male snout contacts made with the grid of an adjacent compartment, with the snout of a stimulus animal, with the genitals of a stimulus animal, and with any other part of the body of a stimulus animal (excluding snout and genitals). After 4 days (96 h) in the apparatus, the volume of water consumed was calculated as in Experiment 2. At this time urine samples were collected as in Experiment 1, and repeated the following day if necessary to gain a sufficient sample for chemical analysis. Samples remained frozen until analyzed for creatinine, 17β-estradiol, and testosterone via enzyme immunoassay as described below.

Fig. 1. Mean (±S.E.) observations of urination per 30-min interval sample directed toward adjacent compartments among males housed either with empty adjacent compartments (isolated) or with two females in the adjacent compartments (female-exposed) in Experiment 1. ⁎ differs from isolated controls, p b 0.05.

Experiment 3 This experiment was designed as a replication of a selection of measures of Experiment 1, with inclusion of additional conditions where adult males were exposed to developing males or ovariectomized developing females. To prepare stimulus animals, 16 adult CF-1 females were inseminated by same-strain males, with the sire removed following detection of a sperm plug. At 18 days of age, pups were individually anesthetized via isofluorane gas. Within each litter, one or two female pups were ovariectomized via bilateral flank incisions, one or two other females were given sham surgery involving flank incisions and sutures without removal of the ovary, and one or two male pups were subjected to such sham surgery. Each pup was labeled according to treatment by a colored marking on its fur and tail. After recovery from anesthesia on a heating pad, these surgicallytreated pups were each returned to their own dam and litter, and the remaining pups were culled such that eight pups remained in each litter. The litters were then left undisturbed but visually inspected on a daily basis until weaning at 28 days of age. Commencing at 32 days of age (2 weeks following surgery), surgically-treated pups served as stimulus animals for the adult male experimental subjects. Male HS subjects were placed in the experimental apparatus 2–3 h after commencement of the dark phase of the lighting cycle. These males were randomly assigned to four conditions: 1) isolation in the apparatus, with no stimulus animals in the adjacent compartments (control condition), 2) exposure to two intact (sham-surgery) females, 3) exposure to two ovariectomized females, and 4) exposure to two intact (sham-surgery) males. There were 18 males in the isolated control condition and 12 males in each other condition. Each male was

Assay procedures Enzyme immunoassay methods were previously validated (Muir et al., 2001; Munro et al.,1991). Creatinine,17β-estradiol, and testosterone were obtained from Sigma Chemical Co. Antibodies to 17β-estradiol, testosterone, and corresponding horseradish peroxidase conjugates were obtained from the Department of Population Health and Reproduction at the University of California, Davis. Cross reactivities for anti-17β-estradiol are: 17β-estradiol 100%, estrone 3.3%, progesterone 0.8%, testosterone 1.0%, androstenedione 1.0% and all other measured steroids b0.1%. Cross reactivities for anti-testosterone are: testosterone 100%, 5a-dihydrotestosterone 57.4%, androstenedione 0.27%, androsterone, and DHEA, cholesterol, 17β-estradiol, progesterone, and pregnenolone b0.05%. Steroid assays were conducted with duplicate readings per sample; the average for each sample was used in statistical analysis. NUNC Maxisorb plates were first coated with 50 ml antibody stock diluted 1:10,000 in a coating buffer (50 mmol/l bicarbonate buffer, pH 9.6), then stored for 12–14 h at 4 °C. Wash solution (0.15 mol/l NaCl solution containing 0.5 ml Tween 20 per l) was added to each well to rinse away any unbound antibody, then 50 ml phosphate buffer/well was added. The plates were incubated at room temperature for 2 h for 17βestradiol and 30 min for testosterone before adding standards, samples, or controls. Urine samples were diluted 1:9 in phosphate buffer (0.1 mol/l sodium phosphate buffer, pH 7.0 containing 8.7 g NaCl and 1 g BSA/l) before being added to the plate. Standard curves were derived by serial dilution from a known stock solution. For all assays, 50 ml estradiol or testosterone horseradish peroxidase was added to each well, with 20 ml standard, sample, or control for estradiol or 50 ml standard, sample, or control for testosterone. The plates were incubated for 2 h at room temperature, then washed, and then 100 ml substrate solution of citrate buffer, H2O2 and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid), was added to each well. The plates were then covered and incubated, while shaking at room temperature for 30–60 min. The plates were then read with a single

Table 1 Mean (±S.E.) total number of urinations, urinations toward the floor, urinations toward a wall (excluding grid to adjacent compartments), and instances in which males made snout (nasal) contact with the wire-grid interface with adjacent compartments, during 30-min interval samples during the first four days in which males either had two females in these compartments (female-exposed) or had empty adjacent compartments (isolated) in Experiment 1 Measure

Condition

Day 1

Day 2

Day 3

Day 4

Total urinations

Isolated Female-exposed Isolated Female-exposed Isolated Female-exposed Isolated Female-exposed

1.00 ± 0.28 1.50 ± 0.51 0.92 ± 0.23 0.75 ± 0.35 0.08 ± 0.08 0.08 ± 0.08 41.3 ± 10.2 124.9 ± 19.4⁎

0.17 ± 0.11 2.58 ± 0.76⁎ 0.17 ± 0.11 0.67 ± 0.28 0.00 ± 0.00 0.17 ± 0.11 17.6 ± 4.0 110.4 ± 25.6⁎

0.50 ± 0.15 2.83 ± 0.60 ⁎ 0.41 ± 0.15 0.67 ± 0.26 0.08 ± 0.08 0.17 ± 0.11 21.8 ± 5.3 127.4 ± 35.5⁎

0.50 ± 0.19 3.83 ± 0.90⁎ 0.50 ± 0.00 0.92 ± 0.36 0.00 ± 0.00 0.33 ± 0.19 30.2 ± 14.6 69.1 ± 19.9⁎

Floor urinations Wall urinations Snout to grid

⁎ differs from isolated controls, p b 0.05.

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filter at 405 nm on a microplate reader (Bio-Tek Instruments Inc, ELx 808). In all assays, optical densities were obtained, standard curves were generated, a regression line fit, and samples interpolated into the equation to get a value in pg per well. Creatinine measures were also taken, with the average of duplicates taken for each sample. All urine samples were diluted 1:40.77 urine:phosphate buffer. Using Dynatech Immulon flat bottom plates, 50 ml/well of standard was added with 50 ml distilled water,

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50 ml 0.75 M NaOH and 50 ml 0.4 M picric acid. The plate was then shaken and incubated at room temperature for 30 min. The plate was measured for optical density on the plate reader with a single filter at 490 nm. Standard curves were generated, regression lines were fit, and the regression equation was applied to the optical density for each sample. Where steroid measures were adjusted for creatinine, this was achieved by dividing the obtained value by the measure of creatinine per ml of urine for the particular sample.

Fig. 2. Mean (±S.E.) urinary levels of creatinine, 17β-estradiol, testosterone, creatinine-adjusted 17β-estradiol, and creatinine-adjusted testosterone among males housed either with empty adjacent compartments (isolated) or with two females in the adjacent compartments (female-exposed) in Experiment 1. ⁎ differs from isolated controls, p b 0.05.

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Results Experiment 1 Fig. 1 shows the number of male urinations per 30-min session toward either of the two adjacent compartments. There were no such urinations observed in the case of the isolated animals, while in the female-exposed animals the number increased progressively. Analysis of variance, treating condition as between-subjects and day of observation as within subjects, indicated significant main effects of condition, F(1,22) = 19.84, p = 0.0004, and day, F(3,66) = 2.88, p = 0.0414, and a significant interaction, F(3,66) = 2.88, p = 0.0414. Multiple comparisons (Newman–Keuls, p b 0.05) indicated that female-exposed males on days 2, 3, and 4 differed from isolated animals on all days and also from female-exposed males on day 1. Table 1 gives remaining behavioral measures from this experiment. For total urinations, analysis of variance showed a significant main effect of condition, F(1,22) = 15.39, p = 0.0010, and a significant interaction, F(3,66) = 4.33, p = 0.0078, but no main effect of day. Multiple comparisons indicated that urination levels of femaleexposed males on days 2, 3, and 4 exceeded those of isolated males on each day. Urination frequencies toward the floor and walls were similar among conditions and did not show any significant differences. The number of nasal contacts made by males to the wire grid interface to either adjacent compartment was greater for female-exposed males than for isolated males. Analysis of variance showed a significant main effect of condition, F(1,22) = 26.45, p = 0.0002, but the main effect of day and the interaction were not significant. Fig. 2 shows the results for enzyme immunoassay measures, including creatinine, 17β-estradiol, testosterone, creatinine-adjusted 17β-estradiol, and creatinine-adjusted testosterone. Creatinine levels were substantially higher in isolated males than in those exposed to females, t(22) = 2.83, p = 0.0095. Each of the unadjusted steroid measures showed a trend toward higher levels in isolated males than in female-exposed males; for estradiol, t(22) = 2.01, p = 0.0545, and for testosterone, t(22) = 1.44, p = 0.1612. Creatinine-adjusted estradiol and testosterone in urine of female-exposed males exceeded that of isolated males; achieving significance for both creatinine-adjusted estradiol, t(22) = 3.45, p = 0.0026, and creatinine-adjusted testosterone, t(22) = 2.44, p = 0.0219. Experiment 2 Female-exposed males consumed more water than did isolated males. The difference between the initial volume in the water bottle and the volume remaining after 5 days was 42.89 ± 1.80 ml among isolated (control) males, whereas that for female-exposed males was 55.78 ± 2.76 ml. This difference reached statistical significance, t(34) = 3.91, p = 0.0007.

Table 2 Mean (±S.E.) number of instances in which males made snout (nasal) contact with the wire-grid interface with adjacent compartments, or with the snout, body (excluding snout and genitals) or genitals of an adjacent animal during a 30-min interval sample on the third day in which males either had empty adjacent compartments (control), two intact (sham-operated) females, two ovariectomized females, or two intact (sham-operated) males in these compartments in Experiment 3 Stimulus animal

Grid

Snout

Body

Genitals

Control (Isolated) Female (Sham) Ovariectomized female Male (Sham)

25.33 ± 6.55 29.92 ± 7.80 27.83 ± 13.50 63.50 ± 14.12⁎

15.67 ± 3.67 2.75 ± 1.72⁎⁎ 13.75 ± 4.69

10.50 ± 3.67 8.75 ± 5.07 16.08 ± 10.48

1.08 ± 0.47 0.25 ± 0.25 0.58 ± 0.43

⁎ differs from isolated controls, p b 0.05. ⁎⁎ differs from remaining conditions, p b 0.05.

Fig. 3. Mean (±S.E.) difference between initial volume in the water bottle and the volume remaining after 4 days (96 h) among males with empty adjacent compartments (isolated), or two sham-operated females, two ovariectomized (OVX) females, or two sham-operated males in adjacent compartments in Experiment 3. ⁎ differs from isolated controls, p b 0.05; ⁎⁎ differs all other conditions, p b 0.05.

Experiment 3 Table 2 shows the frequency of male snout (nasal) contacts with the wire-grid partition of adjacent compartments. It also gives the frequency of such snout contacts with the snout, body area (excluding snout or genitals), or genitals of either of the animals in the adjacent compartments that contained intact females, ovariectomized females, and intact males. One way analysis of variance on snout to grid contacts showed a significant effect of condition, F(3,50) = 2.88, p = 0.0443; multiple comparisons showed that male-exposed males showed more such contacts than did animals in any other condition. The effect of condition was also significant for snout to snout contacts, F(2,35) = 3.80, p = 0.0319; multiple comparisons showed that the males exposed to ovariectomized females showed significantly fewer contacts with genitals of stimulus animals than did those exposed to intact females or males. The effect of condition was not significant for snout to body contacts, F(2,33) = 0.30, p = 0.7497. Although there were trends toward more contact with genitals of intact females than those of ovariectomized females, analysis of variance did not reach significance, F(2,33) = 1.12, p = 0.3384. Fig. 3 shows the difference between the initial volume in the water bottle and the volume remaining after 5 days. The reduction of water in the bottle was clearly greater for males housed with stimulus animals, and greatest for adult males housed adjacent to stimulus developing males. Analysis of variance showed a significant effect of condition, F(3,49) = 4.86, p = 0.0052. Multiple comparisons indicated that the isolated control males consumed less water than did males from any other group, and that males exposed to developing males consumed more water than did males in other groups. Fig. 4 gives the outcome for creatinine, estradiol, testosterone, creatinine-adjusted estradiol, and creatinine-adjusted testosterone for all conditions. Usable samples were obtained for 46 males (18 control, 11 female exposed, 11 ovariectomized female exposed, and 6 male exposed). Creatinine was substantially affected by the presence of developing conspecifics, significantly so for females. One-way analysis of variance indicated a significant effect, F(3,42) = 4.16, p = 0.0114; multiple comparisons showed differences between the control condition and both female-exposed conditions. Unadjusted estradiol and testosterone showed trends toward reduced levels in

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males exposed to conspecifics. Analysis of variance was significant for estradiol, F(3,42) = 3.05, p = 0.0383, and testosterone, F(3,42) = 4.54, p = 0.0074; for estradiol, multiple comparisons showed that only the male-exposed males differed from controls, while for testosterone all conspecific-exposed groups differed from controls. Creatinineadjusted estradiol was higher among female-exposed males; analysis of variance indicated an effect of condition, F(3,42) = 2.85, p = 0.0481, while multiple comparisons showed a difference between males exposed to intact females and control males. There was a trend toward lower creatinine-adjusted testosterone levels among males exposed to developing males, but the main effect of condition was not significant, F(3,42) = 2.28, p = 0.0918.

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Discussion These data indicate that adult male mice are stimulated by nearby developing females to increase urination. They progressively direct more urine toward females over the first four days of exposure. Water consumption is also enhanced among female-exposed males. Creatinine levels are reduced in urine of female-exposed males, indicating dilution of urine as males drink and urinate more. Male urine reliably contains 17β-estradiol and testosterone. Estradiol levels rise relative to creatinine levels as urine becomes progressively more dilute during female exposure. Creatinine-adjusted estradiol levels in urine of femaleexposed males significantly exceed those of isolated males.

Fig. 4. Mean (±S.E.) urinary levels of creatinine, 17β-estradiol, testosterone, creatinine-adjusted 17β-estradiol, and creatinine-adjusted testosterone among males with empty adjacent compartments (isolated), or with two sham-operated females, two ovariectomized (OVX) females, or two sham-operated males in adjacent compartments in Experiment 3. ⁎ differs from isolated controls, p b 0.05.

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Urination in male mice is clearly a social response (cf. Arakawa et al., 2007; Drickamer, 2001; Hurst, 1987, 1990a, 1990b; Reynolds, 1971). In the presence of both stimulus males and stimulus females, adult males disperse their urine in multiple droplets (cf. Arakawa et al., 2007; Desjardins et al., 1973; Maruniak et al., 1974). This complicates urine collection in mice in social contexts (cf. deCatanzaro et al., 2006). It would be technically challenging to measure total urinary volume among female-exposed males. One estimate for isolated adult males comes from Drickamer (1995), who found that dominant adult males averaged 5.4 ml of urine/day, while evidence suggests that isolated males resemble dominant males on many physiological measures (Brain, 1975). Female-exposed males in the present study probably excreted substantially higher levels of urine, given evidence of polyuria, polydipsia, and reduced creatinine. We observed that female exposure increased water consumption by 30% in Experiment 2 and 20% in Experiment 3, and to some extent the increment in urination should reflect that in water consumption. However, water loss can also reflect spillage/evaporation, excretion through defecation and palmer and nasal excretion (cf. deCatanzaro et al., 2006). Arousal-induced polydipsia has also been observed under various other circumstances in laboratory animals, with some relationship to pituitary–adrenal variables (Levine and Levine, 1989). The results of Experiment 3 confirm the drop in creatinine and rise in creatinine-adjusted estradiol induced by exposure to intact females in Experiment 1. Males in Experiment 3 exposed to ovariectomized females showed creatinine and steroid dynamics resembling those observed in males exposed to intact females, but males made fewer snout contacts with ovariectomized females. Immaturity of the females might help to account for this; perinatal sexual differentiation would not be impacted by ovariectomy at 18 days of age, while full ovarian activity would occur later than the age (32–37 days) when females were in contact with males. Maruniak et al. (1974) observed that ovariectomized as well as intact mature females stimulated urinary marking in adult male mice. Stimulus males in Experiment 3 clearly induced greater water consumption, but curiously this was not reflected in urinary creatinine levels, which should indicate dilution of urine. The pattern of steroid and creatinine-adjusted steroid levels was also dissimilar in male-exposed males to that in female-exposed males. Males show urinary marking in the presence of both stimulus males and females (cf. Arakawa et al., 2007; Hurst, 1987, 1990a, 1990b; Maruniak et al., 1974), however results of our Experiment 3 suggest that chemistry of males' urine depends on the sex of the stimulus animals. Considering our data and that in previously published work (e.g. Desjardins et al., 1973; Maruniak et al., 1974), polyuria and polydipsia may be induced by exposure to either males or females. Male urination patterns in the presence of stimulus males and females may differ, however, in that we observed active direction of urine toward females, while males in the presence of other males have been observed to deposit urine in multiple droplets around their own housing area, suggestion marking of territory (Arakawa et al., 2007; Desjardins et al., 1973; Maruniak et al., 1974). There is wide consensus that urine chemistry is responsible for males' capacity to accelerate puberty in proximal developing females (the “Vandenbergh effect”; e.g. Beaton et al., 2006; Colby and Vandenbergh, 1974; Cowley and Wise, 1972; Drickamer, 1984; Drickamer and Murphy, 1978; Lombardi and Vandenbergh, 1977; Massey and Vandenbergh, 1981; Price and Vandenbergh, 1992). Urine chemistry has similarly been linked to novel males' capacity to disrupt intrauterine implantation of fertilized ova in previously-inseminated females (the “Bruce effect”, e.g. Brennan and Peele, 2003; deCatanzaro et al., 2006; Dominic, 1965; Marchlewska-Koj, 1981; Parkes and Bruce, 1962; Peele et al., 2003). Both pheromonal phenomena can be mimicked by minute quantities of exogenous estrogens. Bronson (1975) showed that a few injections of less than 0.2 μg/animal of 17βestradiol benzoate induced ovulation in pre-pubertal females to a similar degree as did male exposure. Subsequent research has shown

that estrogens regulate reproductive tract growth in developing females via local growth hormone and IGF-1 activity (Kahlert et al., 2000; Leung et al., 2004; Ogasawara et al., 1983; Sato et al., 2002). Estrogen activity is also well established to set the stage for the expression of female sexual behavior (Pfaff, 1980). Intrauterine implantation can be disrupted in laboratory mice by doses of 17βestradiol as low as 37 ng/day subcutaneously on days 1–5 of gestation (deCatanzaro et al., 1991, 2001). Notably, nasal estradiol administration of as little as 140 ng/day on days 1–3 of gestation similarly disrupts implantation (deCatanzaro et al., 2001, 2006). Exogenous androgens can also disrupt implantation, albeit at higher doses (deCatanzaro et al., 1991, 2001; Harper, 1969). Given low molecular weight (e.g. 272.4 Da for 17β-estradiol), androgens and estrogens could have systemic action subsequent to nasal and transdermal absorption. Data from the present study and previous studies (Beaton and deCatanzaro, 2005; deCatanzaro et al., 2006; Muir et al., 2001) clearly indicate the presence of unconjugated estradiol and testosterone in male urine. Accordingly, female exposure to males' excreted estrogens may help to explain such pheromonal phenomena. Male urinary testosterone could also contribute, most likely via aromatization to estradiol. Previous investigation of the Bruce effect has indicated that the quantities of steroids delivered to inseminated females approach values sufficient to account for this effect (deCatanzaro et al., 2006). The same may be true of the Vandenbergh effect, notwithstanding some evidence suggesting that male preputial gland constituents also contribute to this effect (Ma et al.,1999; Novotny et al.,1999). Polydipsia and polyuria on the part of novel males would serve to increase delivery of steroids and any other urinary constituents that alter female physiology and behavior. The present data also indicate that creatinine was dynamic in relation to conditions, being more abundant in the urine of isolated than female-exposed males. Correction of urinary measures for creatinine has been a longstanding practice, intended to adjust for variation in fluid intake and hydration in various species, based on the assumption that rate of creatinine excretion is fairly constant (e.g. Boeniger et al., 1993; Erb et al., 1970; Muir et al., 2001; Munro et al., 1991). This practice has been criticized by a number of other researchers as unnecessary or misleading (e.g. Boeniger et al., 1993; Hakim et al., 1994; Hall Moran et al., 2001; Miro et al., 2004). Certainly data from the current study suggest that creatinine itself can be dynamic with social and housing conditions in laboratory mice, in interaction with changes in fluid consumption and socially-induced urination. We suggest that future studies of urinary steroid dynamics should present direct analyses of creatinine and both unadjusted and adjusted steroid levels. Acknowledgments This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada awarded to D. deCatanzaro. We greatly appreciate the assistance of Tawnya Pancharovski, Jordan Shaw, and Adam Guzzo. References Arakawa, H., Arakawa, K., Blanchard, D.C., Blanchard, R.J., 2007. Scent marking behavior in male C57BL/6J mice: sexual and developmental determination. Behav. Brain Res. 182, 73–79. Beaton, E.A., deCatanzaro, D., 2005. Novel males' capacity to disrupt early pregnancy in mice (Mus musculus) is attenuated via a chronic reduction of males' urinary 17β-estradiol. Psychoneuroendocrinology 30, 688–697. Beaton, E.A., Khan, A., deCatanzaro, D., 2006. Urinary sex steroids during sexual development in female mice and in proximate novel males. Horm. Metab. Res. 38, 501–506. Bingel, A.S., 1972. Estrous cyclicity in mice housed in the presence or absence of males. Proc. Soc. Exp. Biol. Med. 139, 515–517. Boeniger, M.F., Lowry, L.K., Rosenberg, J., 1993. Interpretation of urine results used to assess chemical exposure with emphasis on creatinine adjustment: a review. Am. Ind. Hyg. Assoc. J. 54, 615–627.

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