Activational effects of estradiol and dihydrotestosterone on social recognition and the arginine-vasopressin immunoreactive system in male mice lacking a functional aromatase gene

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Abstract

In rodents, parts of the arginine-vasopressin (AVP) neuronal system are sexually dimorphic with males having more AVP-immunoreactive cells/fibers than females. This neuropeptide neuronal system is highly sensitive to steroids and has been proposed to play an important role in the processing of olfactory cues critical to the establishment of a social memory. We demonstrate here that gonadally intact male aromatase knockout (ArKO) mice, which cannot aromatize androgens into estrogens due to a targeted mutation in the aromatase gene, showed severe deficits in social recognition as well as a reduced AVP-immunoreactivity in several brain regions. To determine whether this reduction is due to a lack of organizational or activational effects of estrogens, we assessed social recognition abilities and AVP-immunoreactivity in male ArKO and wild-type (WT) mice when treated with estradiol benzoate (EB) in association with dihydrotestosterone propionate (DHTP) in adulthood. Adult treatment with EB and DHTP restored social recognition abilities in castrated ArKO males since they showed normal female-oriented ultrasonic vocalizations and were able to recognize an unfamiliar female using a habituation–dishabituation paradigm. Furthermore, adult treatment also restored AVP-immunoreactivity in the lateral septum of ArKO males to levels observed in intact WT males. These results suggest that social recognition in adulthood and stimulation of AVP expression in the adult mouse forebrain depend predominantly on the estrogenic metabolite of testosterone. Furthermore, our results are in line with the idea that the organization of the AVP system may depend on androgen or sex chromosomes rather than estrogens.

Introduction

The ability to recognize a conspecific is fundamental for all social relationships, including parent–offspring recognition, mate recognition, and dominant-subordinate hierarchies. Rodent species, including mice, use primarily olfactory cues to recognize conspecifics (Brown, 1979). The neuropeptide arginine-vasopressin (AVP) has been proposed to play an important role in the processing of olfactory cues critical to the establishment of a social memory (reviewed in Bielsky and Young, 2004). AVP is produced in discrete regions of the brain and is released centrally in the brain as well as peripherally, where it acts as an anti-diuretic hormone. Centrally, AVP projections to the lateral septum are primarily coming from the bed nucleus of the stria terminalis (BNST) and the medial amygdala (Me) in the rat (De Vries and Buijs, 1983).

In rats and mice, as well as in several non-mammalian species, parts of the arginine-vasopressin (AVP) neuronal system are sexually dimorphic and sensitive to sex steroids (Bakker et al., 2006, De Vries and Boyle, 1998, De Vries and Miller, 1998, De Vries and Panzica, 2006). For instance, the BNST and the Me contain more AVP-immunoreactive (-ir) in males than in females. Furthermore, the AVP-ir projections from these nuclei to the lateral septum (LS) are denser in males than in females. These sex differences do not depend on differences in circulating hormone levels in adulthood, since they persist in males and females which are treated with similar levels of testosterone (De Vries and Al Shamma, 1990, De Vries et al., 1994a, De Vries et al., 1994b) or estradiol (Bakker et al., 2006), but are primarily due to differences in hormone levels during development. Treatment of newborn female rats with testosterone induces a male-typical AVP distribution (Han and De Vries, 2003). These masculinizing effects of testosterone on the AVP system may be mediated predominantly by its aromatized metabolite since neonatal treatment of newborn female rats with estradiol benzoate (EB) or with EB and DHTP increased the number of AVP mRNA containing cells in the BNST more than neonatal treatment with DHTP alone (Han and De Vries, 2003). By contrast, in mice, these sex differences may not depend on the presence of estrogens during early development since female alpha-fetoprotein knockout (AFP-KO) mice which are exposed to excessive levels of estrogens prenatally since they lack the protective actions of AFP, did not show a masculinized pattern of AVP expression (Bakker et al., 2006).

Sex differences in steroid responsiveness of AVP-expressing cells have also been reported in adulthood (De Vries et al., 1994b). Treatment of 3 month old rats with estradiol (E2) significantly stimulated AVP mRNA expression in both sexes, but significantly more so in males (De Vries et al., 1994b). DHT treatment by itself did not clearly stimulate AVP mRNA expression, but when given in combination with E2, it significantly increased the number of cells over that of animals treated with E2 alone. However, this increase was seen in males only. These studies in rats thus suggest that brain aromatization may be important in controlling the role of steroid hormones in the hypothalamic/limbic vasopressin system. Indeed, we recently showed that male mice which carry a mutation in the Cyp19 gene encoding the aromatase enzyme (aromatase knockout or ArKO; Honda et al., 1998) and as a result cannot convert androgens into estrogens, had fewer AVP-immunoreactive (ir) structures in the LS, BNST and Me than wild-type males (Plumari et al., 2002). Since only gonadally intact mice were studied in that particular study, it could not be determined whether the reduction in AVP expression in ArKO mice was due to the absence of any organizational or activational effects of estradiol on the AVP system.

Therefore in the present study, we determined the expression of AVP in the LS, BNST and Me of male ArKO mice which were treated with EB and dihydrotestosterone propionate (DHTP) in adulthood. We recently observed that this steroid hormone treatment was successful in stimulating male copulatory behaviors in male ArKO mice (Bakker et al., 2004) emphasizing the importance of estradiol signaling to the activational, as opposed to the organizational, effects of steroids on brain mechanisms controlling male-typical behaviors.

In addition, we determined whether this reduction in AVP expression in male ArKO mice is associated with deficits in social recognition. Site-specific injections of AVP into the LS of male rats resulted in a facilitation of social recognition by prolonging the time over which the memory was held (Engelmann and Landgraf, 1994). Previous results obtained in male ArKO mice have indicated that they do not show a conditioned place preference when estrous female stimuli are used (Pierman et al., 2006b). The absence of a conditioned place preference is most likely not due to any deficits in olfactory recognition per se, since male ArKO mice are perfectly capable to discriminate between different types of urine odors (Pierman et al., 2006a, Wesson et al., 2006). However, it may indicate certain deficits in social recognition. Therefore, we determined social discrimination in gonadally intact male ArKO mice using two different tests. First we used the classic social recognition test as reviewed in Bielsky and Young (2004) and second we used the production of ultrasonic vocalizations as a measure of olfactory recognition. Male mice emit 70 kHz ultrasonic vocalizations under several conditions, mainly during courtship and copulation (Sales, 1972, Whitney et al., 1973). Although females have the capacity to emit these calls, and do so under certain circumstances (Maggio and Whitney, 1985), females rarely call in male–female pairings (Whitney et al., 1973). As a result, male mice produce more vocalizations than females and the stimulation of male calling is highly specific to females or their odors (Dizinno et al., 1978, Nyby et al., 1977). Finally, we determined whether adult treatment with EB and DHTP would correct any deficits in their social recognition.

Section snippets

Animals

Aromatase knockout mice were generated by targeted disruption of exons 1 and 2 of the Cyp 19 gene (Honda et al., 1998). Heterozygous (HET) males and females of the C57Bl/6j strain were bred to generated wild-type (WT), HET and homozygous-null (ArKO) offspring. Mice were genotyped by PCR analysis of tail DNA (for more detailed description see Bakker et al., 2002). All breeding and genotyping were performed at the Center for Cellular and Molecular Neurobiology, University of Liège, Belgium. Food

Social recognition test

Gonadally intact WT males spent less time investigating the stimulus female following successive presentations as she becomes familiar (habituation). However, their investigation time increased again when a new female was presented on the last trial (dishabituation; Fig. 1A). By contrast, gonadally intact ArKO males did not show a clear habituation response when presented with the same female repeatedly. Overall, they continued to investigate the stimulus female and when presented with the same

Discussion

The present study shows that estradiol has strong activational effects on socio-sexual recognition in mice. Gonadally intact ArKO males produced ultrasonic vocalizations when presented with an anesthetized conspecific but unlike WT males, their vocalizations were not directed preferentially toward the female stimulus. These results are consistent with previous studies showing that ArKO males did not investigate female odors preferentially in a Y maze (Bakker et al., 2002). Moreover, results of

Acknowledgments

This work was supported by the National Institutes of Health Grant (HD 044897) and by the Belgian “Fonds National de la Recherche Scientifique (FNRS)” to Sylvie Pierman (FNRS Research Fellow) and Dr. Julie Bakker (FNRS Research Associate; FNRS 1.5.104.06) and by COFIN 2006 (2006072719_003 GCP), the University of Torino (GCP and CVP) and Fondazione CRT (GCP) to Pr. Giancarlo Panzica.

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      The regulatory effects of T on AVP and social recognition are considered in part to be through its conversion into estrogen (Gabor et al., 2012). Gonadally intact male aromatase knockout (ArKO) mice show reduced AVP-ir cells in the MeA and BNST and less AVP fibers in the LS, as well as severe deficits in social recognition due to the inability to encode aromatase for converting T to E2 through aromatization (Pierman et al., 2008; Plumari et al., 2002). Compared with the control males, male mice with lower estrogen levels in the MeA displayed a decreased preference for female urine (Bergan et al., 2014).

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    These authors contributed equally to the presented work.

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