Swift-Gallant et al. Biology of Sex Differences (2015) 6:31 DOI 10.1186/s13293-015-0050-x RESEARCH Open Access Removal of reproductive suppression reveals latent sex differences in brain steroid hormone receptors in naked mole-rats, Heterocephalus glaber Ashlyn Swift-Gallant1, Kaiguo Mo1, Deane E. Peragine1, D. Ashley Monks1,2 and Melissa M. Holmes1,2,3* Abstract Background: Naked mole-rats are eusocial mammals, living in large colonies with a single breeding female and 1–3 breeding males. Breeders are socially dominant, and only the breeders exhibit traditional sex differences in circulating gonadal steroid hormones and reproductive behaviors. Non-reproductive subordinates also fail to show sex differences in overall body size, external genital morphology, and non-reproductive behaviors. However, subordinates can transition to breeding status if removed from their colony and housed with an opposite-sex conspecific, suggesting the presence of latent sex differences. Here, we assessed the expression of steroid hormone receptor and aromatase messenger RNA (mRNA) in the brains of males and females as they transitioned in social and reproductive status. Methods: We compared in-colony subordinates to opposite-sex subordinate pairs that were removed from their colony for either 1 day, 1 week, 1 month, or until they became breeders (i.e., produced a litter). Diencephalic tissue was collected and mRNA of androgen receptor (Ar), estrogen receptor alpha (Esr1), progesterone receptor (Pgr), and aromatase (Cyp19a1) was measured using qPCR. Testosterone, 17β-estradiol, and progesterone from serum were also measured. Results: As early as 1 week post-removal, males exhibited increased diencephalic Ar mRNA and circulating testosterone, whereas females had increased Cyp19a1 mRNA in the diencephalon. At 1 month post-removal, females exhibited increased 17β-estradiol and progesterone. The largest changes in steroid hormone receptors were observed in breeders. Breeding females had a threefold increase in Cyp19a1 and fivefold increases in Esr1 and Pgr, whereas breeding males had reduced Pgr and increased Ar. Conclusions: These data demonstrate that sex differences in circulating gonadal steroids and hypothalamic gene expression emerge weeks to months after subordinate animals are removed from reproductive suppression in their home colony. Keywords: Androgen receptor, Aromatase, Estradiol, Estrogen receptor, Eusocial, Naked mole-rat, Progesterone receptor, Reproductive suppression, Sex difference, Testosterone * Correspondence: [email protected] 1Department of Psychology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada 2Department of Cell and Systems Biology, University of Toronto, 100 St. George Street, Toronto, ON M5S 3G3, Canada Full list of author information is available at the end of the article © 2015 Swift-Gallant et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Swift-Gallant et al. Biology of Sex Differences (2015) 6:31 Page 2 of 9 Background Collectively, these data reveal a complex interplay Sexual differentiation of the vertebrate brain and behav- between sex and social status in neuroendocrine func- ior is mediated in large part by gonadal steroid hor- tion in these highly social mammals. To investigate how mones acting in early development. In mammals, an apparent reduction or lack of neural sex differences sexually differentiated hormone production begins at the can be associated with typical sex differences in gonadal earliest stages of gonadal development and produces function seen in breeders, the present study evaluated marked sexual dimorphism in brain and genitals by early the time course of changes in gene expression of gonadal post-natal life (for review, see [1, 2]). steroid hormone receptors and aromatase in brain, as Naked mole-rats (Heterocephalus glaber) do not well as associated circulating hormones. Thus, we quan- appear to fit this framework of sexual differentiation: tified diencephalic levels of messenger RNA (mRNA) most individuals fail to show traditional sex differences expression of androgen receptor (Ar), estrogen receptor in neuroanatomy or behavior, although these features alpha (Esr1), progesterone receptor (Pgr), and aromatase vary based on reproductive and social status [3–6]. (Cyp19a1) and measured circulating T, 17β-estradiol Naked mole-rats are eusocial rodents, living in large (E2), and P, as animals transitioned from subordinate to colonies with a strict reproductive and social hierarchy: breeding status. We hypothesized that sex differences in a single female and 1–3 males reproduce and are socially steroid hormone receptors would be associated with the dominant, while others in the colony are subordinate differences in circulating gonadal steroids that emerge in and usually remain reproductively suppressed for the breeders, specifically predicting that females would show duration of their life [7–9]. The social status within a increases in Esr1 and Pgr while males would show colony is remarkably stable; however, subordinates can increases in Ar. achieve breeder status after loss of a breeder due to death or removal from the colony. In their natural envir- onment, subordinate naked mole-rats can—though rare- Methods ly—leave their natal colony to find an opposite sex Animals and housing partner and establish their own colony [10, 11]. In Sixty-two animals from 11 colonies were used in this ex- captivity, subordinates only undergo sexual maturation if periment. Opposite-sex pairs of animals were established they are separated from their colony [5, 12, 13] or if the either 1 day (24 h), 1 week (7 days), or 1 month (30 days) established breeders die or are removed [14, 15]. prior to tissue collection (6 pairs per time point). Regardless of the specific circumstances in which Breeders were animals that were paired until a litter was subordinates become breeders, they only perform sexual produced (n = 5 pairs) and were collected 30 days after behaviors and show mammalian typical sex differences birth of their first litter. In naked mole-rats, gestation is in gonadal steroid hormones as they transition to between 65 and 74 days long, and lactation ceases at breeding status. Subordinate females have lower urinary approximately 28 days post-partum [5, 23]. Taken with and plasma progesterone (P) levels as well as plasma the fact that none of the breeding females were pregnant luteinizing hormone (LH) concentrations compared to at the time of tissue collection, it seems unlikely that breeder females [16–18]. Within 3 days of separation their values reported below were affected by either preg- from their natal colony, subordinate females show nancy or lactation. Some animals were mis-sexed, result- increases in urinary P levels, and within 7 days, they ing in same-sex pairs. Same-sex pairs were excluded from develop a perforated vagina [16]. Similarly, male subor- analyses; therefore, the final numbers were five opposite- dinates have lower urinary testosterone (T) compared to sex pairs in the 1-day group, 1-week group, and breeder male breeders in the colony [14, 15, 19], and urinary T group and six opposite-sex pairs in the 1-month group. In levels and plasma LH increase when subordinate males addition, seven male and five female subordinate animals are removed from their colony [20]. were selected from the colonies immediately prior to Subordinates and breeders also differ in the neural tissue collection. structures associated with sexual behaviors. However, The colonies were housed in polycarbonate cages where sex differences in the nervous system are found in connected by tubing, as previously described [24]. most mammals, status differences are more prominent The paired animals were housed in single polycar- in the naked mole-rat. For example, breeders, regardless bonate cages (L 43 × W 22 × H 21 cm). All animals of sex, have larger regional volume in various reproduc- were kept on a 12:12-light/dark cycle at 28–30 °C tively relevant hypothalamic structures [4], as well as and had ad libitum access to a diet of sweet potato increased motoneuron soma size in Onuf’s nucleus [3]. and wet protein mash (Harlan Laboratories, Inc). All Furthermore, androgen receptor (AR) and oxytocin procedures were approved by the University Animal immunoreactivity are influenced by social status more Care Committee and adhered to institutional and than sex [6, 21, 22]. federal guidelines. Swift-Gallant et al. Biology of Sex Differences (2015) 6:31 Page 3 of 9 Tissue collection P was measured using an ELISA kit from Cayman The animals were weighed prior to being overdosed with Chemical (Cat. No. 582601) according to the manufac- Avertin (40 mg/100 g). The bloods were collected from turer’sprotocol.Theserumwasdiluted10×withan the trunk, and the brains were extracted. The brains assay buffer provided. The assay sensitivity is 10 pg/ were bisected at midline in the sagittal plane, frozen in mL, and the intra-assay coefficient of variance