Influence of gender and the endocrine environment on the distribution of androgen receptors in the lacrimal gland

Androgens are known to regulate both the structure and function of lacrimal tissue in a variety of species. To explore the endocrine basis for this hormone action, the following study was designed to: (1) determine the cellular distribution of androgen receptors in the lacrimal gland; and (2) examine the influence of gender and the endocrine environment on the glandular content of these binding sites. Lacrimal glands were obtained from intact, castrated, hypophysectomized, diabetic or sham-operated male or female adult rats, mice or hamsters, as well as from orchiectomized rats exposed to placebo compounds or physiological levels of testosterone. The cellular of androgen receptors was evaluated by utilizing an immunoperoxidase protocol, in which a purified rabbit polyclonal antibody to the rat androgen receptor was used as the first antibody. Our findings with lacrimal glands showed that: (1) androgen receptors are located almost exclusively in nuclei of epithelial cells; (2) the cellular distribution or intranuclear density of these binding sites is far more extensive in glands of males, as compared to females; (3) orchiectomy or hypophysectomy, but not sham-surgery or diabetes, lead to a dramatic reduction in the immunocytochemical expression of androgen receptors; and (4) testosterone administration to orchiectomized rats induces a marked increase in androgen receptor content, relative to that in placebo-exposed glands. Our results also reveal that a 10 kb androgen receptor mRNA exists in the rat lacrimal gland. Overall, these findings demonstrate that gender and the endocrine system may significantly influence the distribution of androgen binding sites in rat lacrimal tissue. Moreover, our results show that androgens up-regulate their own lacrimal gland receptors.     

Hormonal influence on the secretory immune system of the eye: endocrine impact on the lacrimal gland accumulation and secretion of IgA and IgG

The objective of the current investigation was to explore the processes underlying the androgen control of tear IgA and to determine whether hormone exposure also modifies tear IgG content. In addition, studies evaluated the impact of diabetes on the androgen regulation of secretory immunity in the eye. Tears and lacrimal glands were collected from age-matched, adult male rats, which had undergone hypophysectomy, selective ablation of the anterior pituitary, streptozotocin-induced diabetes, sham-surgery and/or orchiectomy and had been exposed to vehicle or physiological amounts of testosterone for varying periods of time. Our findings demonstrated that testosterone administration selectively increased the accumulation of IgA, but not IgG, in tears and lacrimal glands of orchiectomized rats. This hormone effect was associated with a 2-fold enhancement of the IgA transfer from lacrimal tissue to tears; IgA movement was against a gradient. In contrast, androgen exposure had no significant influence on the lacrimal gland/tear transfer of IgG, which was down a 90-fold gradient. Testosterone action on the lacrimal gland appeared to involve an increase in IgA production, but not a consistent alteration in the total number of IgA-containing cells. Similarly, androgen exposure had no impact on the population of IgG-containing lymphocytes in lacrimal tissue. Of interest, ablation of the anterior or entire pituitary in orchiectomized rats, which procedure inhibits testosterone-induced stimulation of tear IgA levels, significantly reduced the total number of IgA-containing cells in the lacrimal gland. Induction of diabetes by streptozotocin injection to orchiectomized rats resulted in diminished tear IgA content and decreased numbers of lacrimal IgA-positive lymphocytes, but did not prevent the testosterone-associated rise in IgA antibody content. In summary, our findings demonstrate that androgens increase the lacrimal gland production and secretion of IgA, but not IgG.     

Androgen regulation of secretory component synthesis by lacrimal gland acinar cells in vitro

This study sought to determine whether androgens directly stimulate the production of secretory component (SC) by acinar cells from the rat lacrimal gland. Homogeneous populations of acrinar cells were isolated from lacrimal tissues by serial enzymatic digestion and Ficoll gradient centrifugation and then cultured on reconstituted basement membranes in supplemented, serum-free medium. Acinar cell exposure in vitro to dihydrotestosterone (DHT) resulted in a significant increase in cellular SC output. This hormone action was dose dependent and androgen specific. Testosterone, but not 17 beta-estradiol, progesterone, dexamethasone, or aldosterone, also induced a considerable elevation in acinar cell SC production. The effect of testosterone may not require intracellular enzymatic conversion to DHT. The impact of androgens on SC output was associated with enhanced cellular synthesis and secretion and did not involve variations in acinar cell viability or density. Moreover, the SC response to DHT occurred irrespective of whether lacrimal gland acinar cells were obtained from young adult male or female rats. In contrast, the androgen-related rise in SC production was significantly reduced in acinar cells isolated from tissue of orchiectomized and hypophysectomized rats. In summary, these findings demonstrate that androgens directly increase the synthesis of SC by lacrimal gland acinar cells in vitro. This effect, however, may be significantly altered by prior changes in the endocrine environment of acinar cells in vivo.     

Gender- and androgen-related influence on the expression of proto-oncogene and apoptotic factor mRNAs in lacrimal glands of autoimmune and non-autoimmune mice

Our previous studies have shown that the mRNA levels of c-myb, c-myc, bcl-2 and p53 are higher, and partial Fas antigen (i.e. exons 1-2) lower, in lacrimal tissues of female, as compared to male, MRL/lpr mice, which are a model of Sj?gren's syndrome. We have also found that this gender-related difference in bcl-2 and c-myb expression appears to be due to the influence of androgens. To extend these findings, we sought to determine: first, whether these gender- and/or hormone-associated variations in mRNA content are unique to MRL/lpr mice, or are also present in lacrimal glands of other murine strains, including autoimmune NZB/NZW F1 (F1) and non-obese diabetic (NOD), as well as non-autoimmune C3H/HeJ (C3H) and BALB/c, mice; and second, whether the levels of these apoptotic factor mRNAs are altered in lacrimal tissues of mice (i.e. testicular feminized (Tfm) with dysfunctional androgen receptors, as compared to glandular amounts in their 'normal' controls (i.e. Tabby). Lacrimal tissues were obtained from adult mice, which were either untreated or treated with placebo or testosterone for 21 days. Glands were processed for the analysis of proto-oncogene mRNAs by RT-PCR (at exponential phase of amplification) and data were standardized to the corresponding levels of beta-actin mRNA. Our results demonstrate that Fas antigen, Fas ligand, c-myb, c-myc, bcl-2, Bax and p53 mRNAs are present in lacrimal tissues of F1, NOD, C3H, BALB/c, Tabby and Tfm mice. The relative levels of Fas antigen mRNA are consistently higher in glands of males, whereas amounts of bcl-2 mRNA are greater in tissues of F1, C3H and BALB/c females. Testosterone administration induced a significant increase in the lacrimal gland content of Bax mRNA, but a striking decrease in the lacrimal tissue level of bcl-2 mRNA in F1 and C3H mice. Lacrimal glands of Tfm mice contained elevated amounts of bcl-2 mRNA, as compared to values in tissues of their Tabby controls. In summary, our findings show that fundamental gender-related differences exist in the expression of genes associated with programmed cell death in lacrimal glands of autoimmune and normal mice. In addition, some of these differences may be due, at least in part, to the effect of androgens.     

大鼠泪腺睾酮受体直空负压ABC法光镜观察与免疫金探针超微结构定位

动物实验发现睾酮能改善干眼病动物模型干眼症状,促进泪腺分泌,但作用机理不明,本研究探讨泪腺细胞中是否存在睾酮受体。雌雄各8只大鼠的泪腺分别制成石蜡切片和超薄切片,睾酮分别采用ABC法及免疫金标记,通过真空负压ABC法光镜观察及免疫金探针超微结构定位进行细胞睾酮受体检查。结果：光镜下泪腺导管上皮细胞的胞浆及胞核中出现免疫染色阳性,泪腺上皮细胞则很少见到;电镜下泪腺细胞胞浆及核中可见金颗粒,对照组则染色阴性。结论 泪腺细胞存在着睾酮受体,睾酮通过受体对泪腺发生作用。     

Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production

Males are less susceptible than females to experimental autoimmune encephalomyelitis and many other autoimmune diseases. Gender differences in cytokine production have been observed in splenocytes of experimental autoimmune encephalomyelitis mice stimulated with myelin proteins and may underlie gender differences in susceptibility. As these differences should not be limited to responses specific for myelin proteins, gender differences in cytokine production upon stimulation with Ab to CD3 were examined, and the mechanisms were delineated. Splenocytes from male mice stimulated with Ab to CD3 produced more IL-10 and IL-4 and less IL-12 than those from female mice. Furthermore, splenocytes from dihydrotestosterone (DHT)-treated female mice produced more IL-10 and less IL-12 than those from placebo-treated female mice, whereas there was no difference in IL-4. IL-12 knockout mice were then used to determine whether changes in IL-10 production were mediated directly by testosterone vs indirectly by changes in IL-12. The results of these experiments favored the first hypothesis, because DHT treatment of female IL-12 knockout mice increased IL-10 production. To begin to delineate the mechanism by which DHT may be acting, the cellular source of IL-10 was determined. At both the RNA and protein levels, IL-10 was produced primarily by CD4+ T lymphocytes. CD4+ T lymphocytes were then shown to express the androgen receptor, raising the possibility that testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production. In vitro experiments demonstrated increased IL-10 production following treatment of CD4+ T lymphocytes with DHT. Thus, testosterone can act directly via androgen receptors on CD4+ T lymphocytes to increase IL-10 gene expression.     

Cell-specific expression of kidney androgen-regulated protein messenger RNA is under multihormonal control

Kidney androgen-regulated protein (KAP) gene expression is under androgenic control in the epithelial cells of the proximal convoluted tubule in the mouse kidney. In Tfm/Y androgen receptor-deficient mice, KAP mRNA was detected by in situ hybridization in a subpopulation of these cells only in the S3 segment of the proximal tubules in the outer medulla. Treatment of Tfm/Y animals with testosterone caused a partial induction of KAP mRNA levels, while dihydrotestosterone had no effect. These data suggested that the androgen receptor-independent induction of KAP gene expression in these animals was mediated by an estrogenic metabolite of testosterone, since dihydrotestosterone cannot be aromatized to an estrogenic form. Estrogen treatment of Tfm/Y mice caused an increase in KAP gene expression similar to that observed with testosterone. However, ovariectomy of normal female mice did not eliminate KAP gene expression in the S3 cells and, in fact, resulted in a slight increase. Adrenalectomy in combination with castration had no effect on KAP mRNA levels in S3 cells. However, hypophysectomy alone completely eliminated this cell-specific component of KAP gene expression. These results indicate that KAP gene expression is subject to cell-specific regulation in different segments of the proximal tubule and that this regulation is mediated by hormones of both gonadal and pituitary origin.     

Modulation of Leishmania donovani infection and cell viability by testosterone in bone marrow-derived macrophages: signaling via surface binding sites

Androgens can increase susceptibility toward numerous parasitic infections as well as modulate apoptosis of immune cells. According to the current view, androgens mediate immune cell activities not only through classical intracellular androgen receptors (AR), but also through membrane receptors on the cell surface. Here, using murine bone marrow-derived macrophages (BMMs), we examined the influence of testosterone on Leishmania donovani infection and cell viability in vitro as well as the possible mechanisms. Our data demonstrated that testosterone directly increased intramacrophage infection by L. donovani. In addition, testosterone decreased cell viability by way of apoptosis, accompanied by increased Fas, FasL, and Caspase-8 expression. However, these effects of testosterone could not be associated with the classical AR in BMMs since AR was not detectable using different experimental techniques. Instead, it was found that testosterone could bind to the surface of BMMs by the use of an impermeable testosterone-BSA-FITC in confocal laser scanning microscopy and flow cytometry. Collectively, our data indicated that the influence of testosterone on L. donovani infection and viability of BMMs was mediated through the binding sites of testosterone on cell surfaces, which provided a novel mode of direct action of testosterone on AR-free BMMs.     

Specificity of androgen resistance inMus caroli kidney

Androgen controls the expression of beta-glucuronidase and several other proteins in the kidney of the standard laboratory mouse, Mus musculus. Other species within the genus Mus exhibit a variety of response patterns for kidney beta-glucuronidase and other markers of androgen action. We have investigated the mechanism of androgen action in M. caroli, a Mus species that does not produce beta-glucuronidase in response to testosterone. The failure of testosterone to induce beta-glucuronidase in M. caroli females cannot be overcome by treatment with dihydrotestosterone, with pharmacological doses of testosterone propionate or dihydrotestosterone propionate, or with a variety of potent androgen analogues. All of these compounds induce kidney beta-glucuronidase in M. musculus females and kidney ornithine decarboxylase, submandibular gland renin, and submandibular gland epidermal growth factor in both M. caroli and M. musculus females. Furthermore, kidney androgen receptor proteins from M. caroli and M. musculus animals have the same sedimentation characteristics on sucrose density gradients. These data indicate that androgen resistance in M. caroli is not due to deficient 5 alpha-reductase or aberrant hormone metabolism producing suboptimal levels of functional androgen and is not caused by a defective androgen receptor. They suggest that the resistance of beta-glucuronidase in M. caroli kidney to induction by androgen occurs at the level of the beta-glucuronidase gene.     

Gene expression alterations by conditional knockout of androgen receptor in adult Sertoli cells of Utp14b jsd/jsd (jsd) mice

Spermatogenesis is dependent primarily on testosterone action on the Sertoli cells, but the molecular mechanisms have not been identified. Attempts to identify testosterone-regulated target genes in Sertoli cells have used microarray analysis of gene expression in mice lacking the androgen receptor (AR) in Sertoli cells (SCARKO) and wild-type mice, but the analyses have been complicated both by alteration of germ cell composition of the testis when pubertal or adult mice were used and by differences in Sertoli-cell gene expression from the expression in adults when prepubertal mice were used. To overcome these limitations and identify AR-regulated genes in adult Sertoli cells, we compared gene expression in adult jsd (Utp14b jsd/jsd, juvenile spermatogonial depletion) mouse testes and with that in SCARKO-jsd mouse testes, since their cellular compositions are essentially identical, consisting of only type A spermatogonia and somatic cells. Microarray analysis identified 157 genes as downregulated and 197 genes as upregulated in the SCARKO-jsd mice compared to jsd mice. Some of the AR-regulated genes identified in the previous studies, including Rhox5, Drd4, and Fhod3, were also AR regulated in the jsd testes, but others, such as proteases and components of junctional complexes, were not AR regulated in our model. Surprisingly, a set of germ cell–specific genes preferentially expressed in differentiated spermatogonia and meiotic cells, including Meig1, Sycp3, and Ddx4, were all upregulated about 2-fold in SCARKO-jsd testes. AR-regulated genes in Sertoli cells must therefore be involved in the regulation of spermatogonial differentiation, although there was no significant differentiation to spermatocytes in SCARKO-jsd mice. Further gene ontogeny analysis revealed sets of genes whose changes in expression may be involved in the dislocation of Sertoli cell nuclei in SCARKO-jsd testes.     

Endocrine, neural, and immune control of secretory component output by lacrimal gland acinar cells.

Androgens regulate the synthesis and secretion of secretory component (SC), the IgA antibody receptor, by acinar cells from the lacrimal gland. However, this hormone action may be susceptible to significant modification by other agents from the endocrine, nervous, or immune systems. To investigate the nature of this neuroimmunoendocrine interaction, the present study examined the impact of hormones, neurotransmitters, and lymphokines on basal and androgen-induced SC production by lacrimal gland acinar cells in vitro. Our results demonstrated that vasoactive intestinal peptide, the beta-adrenergic agonist, isoproterenol, PGE2, IL-1 alpha, IL-1 beta, and TNF-alpha significantly increased media SC levels in control or androgen-containing cell cultures. In contrast, the cholinergic agonist, carbachol, significantly decreased cellular SC output. These effects may be mediated through the agents' known capacity to alter intracellular cAMP levels. In support of this hypothesis, exposure of acinar cells to stimulators or analogues of cAMP resulted in a significant enhancement of SC production. Thus, these findings indicate that SC output in lacrimal tissue may be modulated by interactions between the endocrine, nervous and immune systems.     

High abundance of testosterone and salivary androgen-binding protein in the lateral nasal gland of male mice

To better understand androgen function in the mammalian nose, we have determined the levels of testosterone (T) in the olfactory mucosa (OM, which harbors the olfactory receptor neurons) and the lateral nasal gland (LNG, which is the largest anterior nasal gland) of C57BL/6 mice. The results indicated that, in adult male mice, T levels in the LNG were substantially higher than those in the OM and other non-reproductive or non-endocrine tissues examined, including liver, kidney, and brain. Furthermore, in the LNG, the high T levels were accompanied by high levels of salivary androgen-binding protein (sABP) and low microsomal T-hydroxylase activities. The high abundance of T and sABP in the LNG suggests not only that the LNG is a storage site for androgen, but also the possibility that unusually high T levels may occur in other organs that have abundant expression of sABP but low expression of steroid-metabolizing enzymes. Our findings suggest a critical need to determine androgen levels in various organs, as well as to establish the functional significance of an unusually high T level in the LNG, a gland known for its secretion of biologically active molecules, such as odorant binding proteins and immunoglobulin A, to the nasal cavity.     

Primary Rat Lacrimal Cells Undergo Acinar-like Morphogenesis on Reconstituted Basement Membrane and Express Secretory Component under Androgen Stimulation

Single cells or small cell clusters, isolated from the rat lacrimal gland, were incubated on reconstituted basement membrane (matrigel) in a well-defined serum-free medium. During the first days of culture, cells reassociated and reorganized in structures resembling acini. These multicellular structures, maintained in culture for 2 weeks, consisted of well-polarized cuboidal cells surrounding a central lumen and exhibiting apically located microvilli. Myoepithelial cells were observed at the periphery of the acinar structures. Both in the native lacrimal and in the cultured aggregates, epithelial cells displayed strong immunoreactivity for cytokeratin 8, while myoepithelial cells were immunoreactive for vimentin and α-smooth muscle isoactin. These data indicate that the cultured aggregates closely mimic thein vivoarchitecture of lacrimal glands both by morphology and immunohistochemistry. We further demonstrated the presence of an intact androgen receptor and the ability of the cultured aggregates to respond to androgens with increased secretion of the secretory component. Comparable androgen responses were observed in lacrimal gland cultures of 5-week-old male and female rats. In conclusion, we report a morphologically and functionally differentiated culture system of primary rat lacrimal cells, in which androgen-regulated gene expression was observed. This culture model provides a unique experimental paradigm for studying the effects of hormones, cytokines, and growth factors on the morphogenesis, growth, and functional differentiation of lacrimal glands.     

Targeted activation of androgen receptor signaling in the periosteum improves bone fracture repair

Low testosterone level is an independent predictor of osteoporotic fracture in elderly men as well as increased fracture risk in men undergoing androgen deprivation. Androgens and androgen receptor (AR) actions are essential for bone development and homeostasis but their linkage to fracture repair remains unclear. Here we found that AR is highly expressed in the periosteum cells and is co-localized with a mesenchymal progenitor cell marker, paired-related homeobox protein 1 (Prrx1), during bone fracture repair. Mice lacking the AR gene in the periosteum expressing Prrx1-cre (AR^-/Y;Prrx1::Cre) but not in the chondrocytes (AR^-/Y;Col-2::Cre) exhibits reduced callus size and new bone volume. Gene expression data analysis revealed that the expression of several collagens, integrins and cell adhesion molecules were downregulated in periosteum-derived progenitor cells (PDCs) from AR^-/Y;Prrx1::Cre mice. Mechanistically, androgens-AR signaling activates the AR/ARA55/FAK complex and induces the collagen-integrin α2β1 gene expression that is required for promoting the AR-mediated PDCs migration. Using mouse cortical-defect and femoral graft transplantation models, we proved that elimination of AR in periosteum of host mice impairs fracture healing, regardless of AR existence of transplanted donor graft. While testosterone implanted scaffolds failed to complete callus bridging across the fracture gap in AR^-/Y;Prrx1::Cre mice, cell-based transplantation using DPCs re-expressing AR could lead to rescue bone repair. In conclusion, targeting androgen/AR axis in the periosteum may provide a novel therapy approach to improve fracture healing.Subject terms: Bone development, Metabolic bone disease     

Androgen regulation of stage-dependent cyclin D2 expression in Sertoli cells suggests a role in modulating androgen action on spermatogenesis

Regulation of spermatogenesis involves stage-dependent androgen action on Sertoli cells, but the pathways involved are unclear. We assessed if cyclin D2 could play a role. In rats, Sertoli cell nuclear, stage-dependent immunoexpression of cyclin D2 switched on after Day 10 and persisted through Day 35, but disappeared by adulthood. However, ethane dimethane sulfonate (EDS)-induced testosterone withdrawal in adult rats for 6 days induced stage-dependent cyclin D2 immunoexpression in Sertoli cells, with highest expression at stages IX-XII and nondetectable at stages VI-VIII (opposite that for androgen receptor [AR] immunoexpression). In EDS-treated rats, a single injection of testosterone but not of estrogen reversed this change in 4 h, and testosterone administration from the time of EDS treatment prevented expression of cyclin D2 in Sertoli cells. The EDS-induced changes in cyclin D2 immunoexpression were matched by changes in expression of Ccnd2 (cyclin D2) mRNA in isolated stage-dissected tubules. Treatment of adult rats with flutamide induced stage-dependent cyclin D2 immunoexpression in Sertoli cells within 18 h, and confocal microscopy revealed that immunoexpression of AR and cyclin D2 were mutually exclusive within individual seminiferous tubules in these animals. Sertoli cell-selective ablation of the AR in mice using Cre/loxP technology also resulted in stage-dependent Sertoli cell cyclin D2 immunoexpression. Downstream from cyclin D2 action is retinoblastoma 1 (RB1), a tumor suppressor protein, immunoexpression of which paralleled stage-dependent AR expression in Sertoli cells; RB1 stage specificity disappeared after EDS treatment. These results point to a non-cell cycle role for cyclin D2 and RB1 in mature Sertoli cells in the stage-dependent mechanisms regulated by AR expression and androgen action.     

Androgen stimulates endothelial cell proliferation via an androgen receptor/VEGF/cyclin A-mediated mechanism

Growing evidences support that androgen displays beneficial effects on cardiovascular functions although the mechanism of androgen actions remains to be elucidated. Modulation of endothelial cell growth and function is a potential mechanism of androgen actions. We demonstrated in the present study that androgens [dihydrotestosterone (DHT) and testosterone], but not 17β-estradiol, produced a time- and dose-dependent induction of cell proliferation in primary human aortic endothelial cells (HAECs) as evident by increases in viable cell number and DNA biosynthesis. Real-time qRT-PCR analysis showed that DHT induced androgen receptor (AR), cyclin A, cyclin D1, and vascular endothelial growth factor (VEGF) gene expression in a dose- and time-dependent manner. The addition of casodex, a specific AR antagonist, or transfection of a specific AR siRNA blocked DHT-induced cell proliferation and target gene expression, indicating that the DHT effects are mediated via AR. Moreover, coadministration of SU5416 to block VEGF receptors, or transfection of a specific VEGF-A siRNA to knockdown VEGF expression, produced a dose-dependent blockade of DHT induction of cell proliferation and cyclin A gene expression. Interestingly, roscovitine, a selective cyclin-dependent kinase inhibitor, also blocked the DHT stimulation of cell proliferation with a selective inhibition of DHT-induced VEGF-A expression. These results indicate that androgens acting on AR stimulate cell proliferation through upregulation of VEGF-A, cyclin A, and cyclin D1 in HAECs, which may be beneficial to cardiovascular functions since endothelial cell proliferation could assist the repair of endothelial injury/damage in cardiovascular system.     

Sex- and strain-specific expression and vomeronasal activity of mouse ESP family peptides

Male mice secrete exocrine-gland-secreting peptide 1 (ESP1) from the extraorbital lacrimal gland into tear fluid [1]. Other mice detect ESP1 through sensory neurons in the vomeronasal organ (VNO), a secondary olfactory system that senses pheromonal information, including sex, strain, and species. ESP1 is now known to be a member of a multigene family that encodes peptides of various lengths. We herein performed genomic and expression analyses of the ESP family. The ESP family consists of 38 members in mice and 10 members in rat but is absent from the human genome, suggesting rapid molecular evolution. In addition to the male-specific ESP1, we discovered one, which we designated ESP36, that, in adult BALB/c mice, is expressed only in the female extraorbital lacrimal gland. The sexually dimorphic expression is ensured by the release of testosterone after puberty. However, we observed dramatic differences in the expression levels of ESPs between strains. Finally, all ESPs elicited an electrical response in the vomeronasal epithelium but not in the main olfactory epithelium. Multielectrode recording of VNO activity demonstrated that ESP1 induces action potentials in vomeronasal neurons, leading to an increase in the spike firing rate, and that ESP1 is recognized by narrowly tuned vomeronasal sensory neurons. Sexual dimorphism and strain differences of ESPs and their reception in the VNO suggest that the ESP family can convey information about sex and individual identity via the vomeronasal system. The chemosensation of this nonvolatile peptide family by direct contact appears to be one of strategies for sociosexual communication in rodent species.