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The maternal hormone in the male brain: Sexually dimorphic distribution of prolactin signalling in the mouse brain

  • Hugo Salais-López,

    Roles Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Unitat Predepartamental de Medicina, Facultat de Ciències de la Salut, Universitat Jaume I, Castelló de la Plana, Spain

  • Carmen Agustín-Pavón,

    Roles Conceptualization, Funding acquisition, Investigation, Writing – original draft, Writing – review & editing

    Affiliations Unitat Predepartamental de Medicina, Facultat de Ciències de la Salut, Universitat Jaume I, Castelló de la Plana, Spain, Departament de Biologia Cel·lular i de Biologia Funcional, Facultat de Ciències Biològiques, Universitat de València, València, Spain

  • Enrique Lanuza,

    Roles Conceptualization, Funding acquisition, Investigation, Supervision, Writing – review & editing

    Affiliation Departament de Biologia Cel·lular i de Biologia Funcional, Facultat de Ciències Biològiques, Universitat de València, València, Spain

  • Fernando Martínez-García

    Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Supervision, Writing – original draft, Writing – review & editing

    Affiliation Unitat Predepartamental de Medicina, Facultat de Ciències de la Salut, Universitat Jaume I, Castelló de la Plana, Spain


Research of the central actions of prolactin is highly focused on females, but this hormone has also documented roles in male physiology and behaviour. Here, we provide the first description of the pattern of prolactin-derived signalling in the male mouse brain, employing the immunostaining of phosphorylated signal transducer and activator of transcription 5 (pSTAT5) after exogenous prolactin administration. Next, we explore possible sexually dimorphic differences by comparing pSTAT5 immunoreactivity in prolactin-supplemented males and females. We also assess the role of testosterone in the regulation of central prolactin signalling in males by comparing intact with castrated prolactin-supplemented males. Prolactin-supplemented males displayed a widespread pattern of pSTAT5 immunoreactivity, restricted to brain centres showing expression of the prolactin receptor. Immunoreactivity for pSTAT5 was present in several nuclei of the preoptic, anterior and tuberal hypothalamus, as well as in the septofimbrial nucleus or posterodorsal medial amygdala of the telencephalon. Conversely, non-supplemented control males were virtually devoid of pSTAT5-immunoreactivity, suggesting that central prolactin actions in males are limited to situations concurrent with substantial hypophyseal prolactin release (e.g. stress or mating). Furthermore, comparison of prolactin-supplemented males and females revealed a significant, female-biased sexual dimorphism, supporting the view that prolactin has a preeminent role in female physiology and behaviour. Finally, in males, castration significantly reduced pSTAT5 immunoreactivity in some structures, including the paraventricular and ventromedial hypothalamic nuclei and the septofimbrial region, thus indicating a region-specific regulatory role of testosterone over central prolactin signalling.


Prolactin (PRL) is a polypeptide hormone produced at the adenohypophysis, best-known for its role in the development of the mammary gland and milk production [1]. To date, PRL has been attributed more than 300 different biological actions [2] in reproduction, homeostasis, angiogenesis or immunity, among others [3]. To fulfil this array of functions, PRL has different target tissues, including the mammary gland, the uterus and other peripheral organs [3] and, importantly, the brain [4,5]. In the brain, PRL is mainly involved in the regulation of a number of behaviours and physiological processes related to female reproduction and lactation [6]. Indeed, PRL has been described as the “maternal hormone”, a pleiotropic hormone responsible for adapting every important aspect of female physiology and behaviour to the demands of motherhood [7].

The key role of prolactin in maternal physiology has traditionally biased neuroendocrinological studies towards its functions in the brain of females, whereas the actions of this hormone in the male brain have drawn less attention. For instance, the distribution of the PRL receptor (PRLR) has been reported for the female [8,9] and only recently studied in the brain of male rodents [10]. Using a genetically modified animal in which a reporter (τGFP) is expressed under the control of the PRLR promoter, the authors of this study have shown that the expression of PRLR is similar in males and females with quantitative differences in only two nuclei, the anteroventral periventricular nucleus (AVPe) and the medial preoptic nucleus (MPO). According to these findings, it is suggested that prolactin may have many equivalent functions in both sexes.

One of the main reasons for the aforementioned bias in functional studies of PRL relates to the fact that male rodent models display low levels of circulating PRL under standard conditions [11], reflecting a limited access and functionality of PRL in the brain. Still, several studies do report acute rises in systemic PRL secretion in males associated to certain physiological conditions, such as the stress response [12] or sexual behaviour [13]. Regarding the latter, PRL is known to be involved in the control of male copulatory behaviour in rats [14], mice [15], other rodent models [16] and humans [17]. Thus, PRL is released acutely with ejaculation [13] and it is proposed to intervene in the satiation following copulation leading to the refractory period [18]. In fact, chronic hyperprolactinaemia suppresses copulatory behaviour in animal models[19] and yields sexual dysfunction and other side-effects in men [20]. The inhibitory role of PRL on sexual function is proposed to have an important central component [21,22], but the exact regions and mechanisms through which PRL operates in the male brain are still poorly understood.

In this light, the main aim of this work is to provide a comprehensive description of the distribution of cells responsive to PRL in the brain of male mice. To do so, we have supplemented males with a standard dose of exogenous PRL and revealed PRL-responsive cells in the brain by means of the immunohistochemical detection of the phosphorylated form of signal transducer and activator of transcription 5 (pSTAT5). Phosphorylation of STAT5 is a key event in the Jak/STAT pathway, the major signalling cascade of the PRL receptor [3]. Thus, pSTAT5 immunodetection can be considered a reliable method to assess central PRL responsiveness [4,5]. Furthermore, the use of pSTAT5 immunohistochemmistry in PRL-supplemented animals provides a functional scope complementary to the study of PRLR expression, revealing those cells that are not just expressing PRL receptors or specific elements of their signalling cascade, but those that are actually fully responsive to the hormone.

The second goal of this work is to compare the pattern of central PRL-derived signalling between male and female mice, in search for intersexual differences. For this purpose, we compared pSTAT5 patterns and levels between PRL-supplemented males and a sample of ovariectomised, steroid-primed females treated with an equivalent dose of PRL.

In accordance with the involvement of PRL in reproductive physiology and behaviour, gonadal steroids are important regulators of PRL function in the brain. In female rodents, estradiol and progesterone have been shown to target most PRL-sensitive neurons in the brain [23] and to regulate PRL signalling at different levels [2426]. On the other hand, testosterone has been documented to inhibit PRL receptor expression in the pituitary gland of male rats and pituitary PRL release in male rats and mice [2729]. Beyond these findings, to our knowledge there is currently no available evidence supporting a regulatory role of testosterone in brain PRL signalling. Hence, in this work we also explore the putative contribution of testosterone in shaping the patterns of PRL responsiveness in the male brain. To this end, we analyse the levels of pSTAT5-ir in the main PRL-responsive regions of the male mouse brain after permanent testosterone withdrawal through castration, as compared to intact male mice, both supplemented with PRL.

By characterizing the patterns of PRL signalling in the male brain, assessing its sexual dimorphism and clarifying their dependence on testosterone, we intend to shed light on the roles of PRL in male behaviour and physiology, as well as on the neural substrates for PRL-dependent, male-specific behaviours.

Material and methods


For the present study, we used 15 male and 6 female mice of the CD1 strain (Charles River Laboratories, France), aging between 8 and 24 weeks. These animals were housed in polypropylene plastic cages under controlled temperature (24 ± 2 °C) and lighting conditions (12h:12h; lights ON at 8 am), with ad libitum access to food and water. Intact males were housed individually (to avoid inter-male aggression) whereas castrated males and females were group-housed (4 to 6 animals per group, to avoid isolation-derived stress). Animals were treated throughout according to the European Union Council Directive 2010/63/EU (6106/1/10 REV1) and procedures were approved by the Committee of Ethics on Animal Experimentation of the University of Valencia (protocol number: 2015/VSC/PEA/00055), where the experiments were performed.

Experimental design

Male mice were randomly assigned to three experimental groups: (1) Male Control (n = 3), which were not supplemented with exogenous PRL; (2) Male+PRL (n = 6) and (3) Castrated+PRL (n = 6). Animals of Male Control and Male+PRL groups were left gonadally intact, whereas males from Castrated+PRL group underwent orchidectomy. Following the procedure by Brown and collaborators [5,30], Male+PRL and Castrated+PRL groups received an exogenous PRL supplementation 45 minutes prior to perfusion.

In order to compare central PRL sensitivity between males and females, we also included in our study a group of ovariectomised, steroid-primed and PRL-supplemented females, which belonged to a sample of a previously published set of experiments [4]. The steroid-replacement schedule conducted on these females intended to emulate the proestrus-estrus phase of the estrous cycle [31]. Given the considerable variability of pSTAT5-ir in the brain of freely-cycling females, likely due to gonadal steroid influence on PRL signalling [4], this group provided a more stable comparison than freely-cycling females and a more accurate comparison than estrous cycle screening.

Data from these four groups of animals (Male Control; Male+PRL; Castrated+PRL and Female+PRL) allowed us to: a) characterize the pattern of PRL-derived signalling in the male brain by analysing the distribution of pSTAT5-ir in group Male+PRL; b) analyse the effect of PRL supplementation on pSTAT5-ir distribution by comparing the Male Control with Male+PRL groups; c) explore sexual dimorphism in the central sensitivity for PRL by comparing Male+PRL with Female+PRL groups; and finally, d) assess the modulatory role of testosterone on PRL signalling in the male brain through the comparison of the Male+PRL and the Castrated+PRL groups.

Ovariectomy and orchidectomy

Males of Castrated+PRL group were orchidectomised at approximately 12 weeks of age. Briefly, animals were deeply anaesthesised with i.p. ketamine (Imalgene 500, Merial, Toulouse, France, 75mg/kg) and medetomidine (Domtor 1mg/ml, Esteve, Barcelona, Spain, 1 mg/kg) and surgery was performed via a single midline incision on the scrotal sac. After surgery, i.p. atipamezol hydrochloride (Antisedan, Pfizer, New York, USA, 1 mg/kg) was administered to reverse anaesthesia and s.c. butorphanol tartrate 1% (Torbugesic, Pfizer, New York, USA, 20 μl) was regularly administered through the postsurgical period for pain control. Castrated males had 2–3 weeks of recovery under undisturbed housing conditions before the beginning of the experiments.

Experimental females underwent ovariectomy 9–10 weeks after birth under identical pharmacological conditions as explained for males [4]. Surgery was conducted through two incisions on both sides of the back of the animal. Ovariectomized females had at least seven days of recovery after surgery.

Hormone treatments

Experimental females underwent a steroid replacement schedule [4] consisting of a sustained treatment with 17-β estradiol and an acute treatment with progesterone. In accordance with the experimental induction of estrous [31], estradiol was administered on a slow-release profile during 7 days, by means of the subcutaneous placement of silastic tubing implants (Dow Corning Corporation, Midland, MI, USA) filled with 20 μg/ml β-estradiol (Sigma, St Louis, MO, USA) diluted in sunflower oil. Silastic tubing had an inner diameter of 1.67 mm and an outer diameter of 2.41 mm, and implants were cut to a length of 20 mm. Implants were inserted subcutaneously on the lumbar region under isoflurane anaesthesia (Isoflo, Esteve Veterinaria, Barcelona, Spain). Animals were also administered a subcutaneous dose of 20 μl butorphanol tartrate 1% (Torbugesic, Pfizer, New York) during implant placement surgery, for pain control. By contrast, progesterone (Sigma, St Louis, MO, USA) was administered to females acutely in a 500 μg subcutaneous injection, diluted in sunflower oil, in the morning of the seventh day of estradiol treatment, specifically 3 hours and 45 minutes prior to perfusion. In order to make males and females directly comparable for the analysis of sexual dimorphism, males of the Male+PRL group received vehicle treatment parallel to that of females (implants with sunflower oil, plus s.c. oil injection 3h 45 minutes prior to perfusion).

Regarding PRL supplementation, except for the Male Control group (which received vehicle injection), all experimental groups were administered an acute 5 mg/kg i.p. dose of ovine PRL (Sigma, St Louis, MO, USA) 45 minutes before perfusion. The dose and timing of PRL administration rendered homogenous, supraphysiological circulating levels of PRL and ensured that the peak of STAT5 phosphorylation in response to a PRL challenge coincided with perfusion [5]. This is intended to reveal the maximal potential response patterns among experimental animals and also to allow direct comparison between experimental groups, regardless of sex (male vs female) or physiological condition (intact vs castrated males).

Tissue collection and histological processing

Animals received an overdose of sodium pentobarbital (Vetoquinol, Madrid, Spain) and were transcardially perfused with 4% paraformaldehyde in 0.1M phosphate buffer (PB), pH 7.4. Brains were carefully extracted and post-fixed overnight through immersion in the same fixative, then cryoprotected by immersion in 30% sucrose in 0.01 M PB until they sank (2–3 days). Using a freezing microtome (Microm HM-450, Walldorf, Germany) brains were sectioned in five parallel series of 40 μm thick coronal sections. Series were stored in PB-30% sucrose at -20°C until their use.

Immunohistochemistry for pSTAT5

Immunohistochemistry was conducted in free-floating sections under light shaking at room temperature (25°C) unless otherwise stated. Immunohistochemistry protocol was adapted from Brown et al. [5,30]. Tissue sections were thoroughly rinsed between stages for at least three 10-min washes in TRIS-buffered saline, 0.05M, pH 7.6 (TBS). After thawing, sections underwent an initial antigen retrieval step, consisting in two sequential 6-minute incubations in 0.01 M TRIS buffer (TB), pH 10 at 85°C, and brought quickly to room temperature in between. Tissue was then incubated in: a) 1% hydrogen peroxide (H2O2) for 30 minutes, for endogenous peroxidase inhibition; b) 2% BSA, 2% goat serum and 0.3% Triton X-100 in TBS for 1h, in order to block unspecific labelling; c) rabbit monoclonal anti-pSTAT5 primary antibody (Phospho-Stat5 Tyr694 (D47E7), Catalog number #4322, Cell Signaling Technology, Danvers, MA) diluted 1:500 in TBS plus Triton X-100 0.1% for 72 h at 4°C; d) biotinylated goat anti-rabbit IgG (Vector Laboratories, Peterborough, UK; RRID AB_2313606) 1:200 in TBS for 90 minutes; and e) avidin-biotin-peroxidase complex (ABC Elite kit; Vector Laboratories; RRID AB_2336819) in TBS for 90 minutes. Peroxidase label was developed using 0.005% 3–3’-diaminobenzidine (Sigma) and 0.01% H2O2 in TB pH 7.6 for about 15 minutes, obtaining thereby a brown nuclear staining. Sections were rinsed in TB and mounted onto gelatinized slides, dehydrated in graded ethanol, cleared with xylene and coverslipped with Entellan.

The specificity of the anti-pSTAT5 primary antibody was demonstrated by means of an antibody competition assay (S1 Fig).

Analysis of pSTAT5 immunoreactivity

The patterns of pSTAT5-ir distribution were mapped for the brain of intact males (Male+PRL) and females (Female+PRL) and illustrated semiquantitatively in camera lucida drawings of selected brain sections (Fig 1). In addition, a quantitative assessment of the density of cells showing pSTAT5 immunoreactivity (pSTAT5-ir) in representative brain sites was performed for PRL-treated females and males, both intact and castrated. To do so, we first selected frames of the chosen nuclei using the stereotaxic atlas of Paxinos and Franklin (2004). Then, we obtained photomicrographs of these frames in both hemispheres using a digital camera (Leica DFC495) attached to a microscope Leitz DMRB (Leica AG, Germany). Thereafter, images were processed and analysed using ImageJ. Briefly, we subtracted background light and converted the RGB colour image to greyscale by selecting the green channel. Then, we binarised the greyscale image setting the 75% of the mode of the histogram as a threshold, thus including every pixel below this threshold as positively labelled. Using the ImageJ commands “fill holes”, “open” (3 iterations) and “watershed”, we filtered small particles (noise or fragmented nuclei) and separated fused objects. The resulting particles were additionally filtered by area (larger than 70 μm2, corresponding to an approximate diameter of 9.4 μm) and finally counted automatically. We calculated the mean (interhemispheric) density of pSTAT5-imunoreactive cell nuclei for each specimen by dividing the counts for both hemispheres by the area of both frames.

Fig 1. Mapping of pSTAT5 immunoreactivity and prolactin receptor expression in the brains of male and female mice.

Semi-schematic camera lucida drawings of coronal sections of the mouse brain showing the distribution patterns of pSTAT5-ir in female and male mice (left side) and the pattern of PRLR expression in the brain of an adult male mouse specimen (right side), as determined by PRLR expression data obtained from the Allen Brain Institute (2004 Allen Institute for Brain Science. Allen Mouse Brain Atlas. Available from: experiment 72340223, In the left side, pink dots represent pSTAT5 expression exclusive of ovariectomized, steroid-primed females, whereas dark blue dots encode overlapping expression of pSTAT5-ir in both the female and male specimens. Black dots in the right illustrate PRLR expression in the male mouse brain. Each dot represents approximately 4 cell nuclei labelled for pSTAT5 or equivalent number of cells expressing the PRLR. Shaded areas indicate the counting frames designed for the AVPe/VMPO region (Fig 1A), the Pa (Fig 1E), and the Arc, VMHvl, VMHc and VMHdm, DM and MePD (all frames in Fig 1F), as part of the quantitative analysis performed in this work (see below). Approximate distance to bregma is indicated for each section.

Statistical analysis

We performed statistical analysis of the resulting data on the SPSS software package. After checking for normality (Kolmogorov-Smirnov test with Lilliefors’ correction) and homogeneity of variances (Levene's test), we first compared levels of pSTAT5-ir density between intact males (Males + PRL) and ovariectomised, steroid-primed females (Female+PRL), in search for dimorphic differences. Then, we checked for the effect of orchidectomy on male pSTAT5-ir levels by comparing intact (Male+PRL) and castrated (Castrated+PRL) males. In both cases, statistical comparisons were ran independently for each of the analysed brain regions. Samples fulfilling the criteria for a parametric analysis were subject to an independent t-test, whereas the samples of the remaining nuclei were subject to a non-parametric Mann-Whitney test. For each statistical test, we applied a significance level of 0.05.


Patterns of pSTAT5 immunoreactivity in the male mouse brain: effect of prolactin supplementation

Immunohistochemistry for pSTAT5 produced a defined staining in the examined brain tissue, which was restricted to the cell nucleus (see Fig 2), as shown in our previous work [4]. Labelling was suppressed by pre-incubation of the antibody with the phosphorylated peptide used for immunization, but not when the antibody was incubated with the equivalent, non-phosphorylated peptide (S1 Fig). Therefore, we safely assume that, in our immunohistochemical procedure, the antibody is detecting specifically pSTAT5. For the following anatomical descriptions, we adhere to the neuroanatomical terminology proposed by Paxinos and Franklin [32] (see also list of abbreviations above).

Fig 2. Representative examples of pSTAT5 immunoreactivity in the brain of females, intact males and castrated male mice.

Photomicrographs illustrating pSTAT5 labelling in representative brain sections in an ovariectomized, steroid-primed female supplemented with PRL (leftmost column), an intact, PRL-supplemented male (left-center column), a castrated, PRL supplemented male (right-center column) and a intact control male lacking PRL supplementation (rightmost column). Sections correspond to the caudal septum (A-D), the posterior medial amygdaloid region (E-H), the preoptic hypothalamus (I-L), the paraventricular hypothalamic region (M-P) and the tuberal hypothalamus (Q-T). The approximate distance to bregma is indicated in each section. Scale bars correspond to 100 μm.

Fig 1 shows the distribution of pSTAT5-ir observed in PRL-supplemented males (blue dots on the left side of the brain), as well as the the distribution of cells expressing PRL receptor (PRLR) in the male mouse brain (Fig 1, black dots on the right side of the brain), as reported by the Allen Brain Institute (Allen Mouse Brain Atlas, experiments 1268 and 72340223, S2 Fig). Fig 2 shows photomicrographs illustrating pSTAT5-ir in selected brain nuclei for all of our experimental groups.

Males supplemented with PRL (Male+PRL) showed moderate-to-dense pSTAT5-ir in several nuclei of the hypothalamus and cerebral hemispheres (Fig 2, left-center column), as well as in the choroid plexus (not shown). In the telencephalon, pSTAT5-ir was mainly observed in the septum, specifically in the area comprising the septofimbrial nucleus (SFi) and the triangular septal nucleus (TS) (Fig 1B and 1C). In this region, the subfornical organ (SFO) also showed some immunoreactive cells. In addition, the posterodorsal nucleus of the medial amygdala (MePD) displayed scarce pSTAT5-ir, with few immunolabelled cells restricted to the upper corner of the nucleus, in close contact to the intramygdaloid BST (BSTia, see Figs 1F and 2F). Importantly, all these brain nuclei display PRLR mRNA expression in the male mouse (Fig 2 right side).

In the hypothalamus, pSTAT5-ir was present in the preoptic, anterior and tuberal regions. In the preoptic hypothalamus (Figs 1A–1D and 2J), pSTAT5-ir cells were concentrated in the juxtaventricular nuclei, namely the vascular organ of the lamina terminalis (VOLT) and the anteroventral periventricular (AVPe), the ventromedial preoptic (VMPO) and the Pe nuclei. In addition, the adjoining MPA displayed a few immunolabelled cell nuclei.

In the anterior hypothalamus, pSTAT5-ir was once again restricted to juxtaventricular structures, the paraventricular nucleus (Pa) and the Pe (Figs 1E and 2N), as well as the retrochiasmatic area (RCh, Fig 1E). In the Pa, labelling was restricted to the medial aspect of the nucleus, mainly to the ventral (PaV), medial magnocellular (PaMM) and medial parvocellular (PaMP) subdivisions, with very few labelling observed in the lateral aspect of the nucleus (PaLM) or the subparaventricular nucleus (SPa). In the tuberal hypothalamus (Figs 1F and 2R), the arcuate nucleus (Arc) displayed abundant pSTAT5-ir, with some immunolabelled cells displaced into the median eminence (ME). In some males, the dorsomedial nucleus (DM) displayed few scattered immunolabelled cells. Finally, pSTAT5-ir appeared in the ventromedial hypothalamic nucleus (VMH), with higher levels in the ventrolateral subdivision (VMHvl) and sparse labelling in the rest (VMHc and VMHdm).

In contrast to the Male+PRL group, control (intact) males not supplemented with exogenous PRL (Male Control, Fig 2, rightmost column), were virtually devoid of pSTAT5-ir, except for some labelled cells in the ventral aspect of the arcuate nucleus (Arc, Fig 2T) and the anterior aspect of the periventricular nucleus (Pe, not shown).

Comparative analysis of pSTAT5-ir the male and female mouse brain

In comparison to males, ovariectomised, steroid-primed females supplemented with PRL (Female+PRL) showed a more extensive distribution of pSTAT5-ir (Fig 1, Fig 2 middle-right column), which matches previous descriptions conducted on freely-cycling female mice [4]. To summarize, labelling in our experimental females was widespread in the basal telencephalon and hypothalamus, but was also found within thalamic, midbrain and brainstem structures. To allow a direct comparison between sexes, Fig 2 shows the distribution of pSTAT5-ir in equivalent brain sections of male and female mice. In addition, in the semi schematic drawings of brain sections depicted in Fig 1, pSTAT5-ir labelling that was common to males and females is shown as blue spots, whereas labelling exclusively present in females is illustrated as red spots. Importantly, there was not a single brain site where labelling was exclusively found in males. In other words, after administration of equivalent doses of exogenous PRL, males show pSTAT5-ir in a reduced number of brain centres, whereas females show immunoreactive cells in the same nuclei, plus a set of additional brain centres. This represents a clear case of sexual dimorphism in favour of females.

We also checked for quantitative sexual dimorphism in PRL-derived signalling, by analysing the density of pSTAT5-ir in the main nuclei showing pSTAT5-ir in PRL-supplemented intact males and steroid-primed ovariectomised females(AVPe/VMPO, Pa, Arc, DM, VMHvl, the VMHc, MePD and SFi). These results are summarized in Fig 3. Females showed a general trend towards higher levels of pSTAT5-ir than males. This effect reached statistical significance in the Arc (t(10) = 3.043, p = 0.012), the DM (U = 32.0; p = 0.026) and the MePD (U = 30.0; p = 0.004), although the AVPe/VMPO also showed an almost significant trend towards higher pSTAT5-ir density in females (t(9) = 2.183; p = 0.057).

Fig 3. Quantitative analysis of pSTAT5 immunoreactivity in selected brain regions of female and male mice.

Assessment of pSTAT5-ir density (pSTAT5-positive cell nuclei/mm2) in the major brain regions with expression of pSTAT5-ir in both male and female mice. Bar histograms show mean interhemispheric pSTAT5-ir density ± SEM in gonadally-intact, vehicle-treated males (Male+PRL; n = 6; black) and ovariectomized females treated with estradiol and progesterone (Female+PRL; n = 6; grey). Counting frames for each of the analyzed nuclei are included in Fig 1. Statistical analysis was applied independently to each brain region (independent t-test for parametric data or Mann-Whitney test for non-parametric data, see Results). *P ≤ 0.05; **P ≤ 0.01; (*) P ≤ 0.06.

Effect of testosterone withdrawal in prolactin-derived signalling in the male mouse brain

Finally, we explored the putative role of testosterone in the regulation of PRL-derived signalling in the male brain. In qualitative terms, castrated and intact male mice displayed similar patterns of pSTAT5-ir, but castrated males showed apparently lower levels of pSTAT5-ir. Quantitative analysis of the labelling in both groups (using the same methodology and exploring the same subset of nuclei analysed in the previous section) allowed comparing pSTAT5-ir between gonadally intact and castrated males by means of independent t-tests for the Arc and AVPe and non-parametric Mann-Whitney tests for the rest of the analysed regions. The results of this statistical comparison reveal a significant effect of testosterone withdrawal in diminishing the levels of pSTAT5-ir in the Pa (U = 33.00; p = 0.004), in both the ventrolateral and central/dorsomedial VMH (U = 30.00, p = 0.015 for both frames) and in the SFi (U = 26.00, p = 0. 05). In the remaining analysed nuclei, the density of pSTAT5-ir cells was similar in Male+PRL and Castrated+PRL animals, as no statistical differences were found (p>0.1) (Fig 4).

Fig 4. Effect of testosterone withdrawal in pSTAT5 immunoreactivity of the male mouse brain.

Assessment of pSTAT5-ir density (pSTAT5-positive cell nuclei/mm2) in gonadally-intact male mice and castrated male mice within the major brain regions showing pSTAT5 expression in the male mouse brain. Bar histograms show mean interhemispheric pSTAT5-ir density ± SEM in intact, PRL-supplemented males (Males+PRL; n = 6; black) and castrated, PRL-supplemented males (Castrated+PRL; n = 6; white). Counting frames for each of the analyzed nuclei are enclosed in Fig 2. Statistical analysis was applied independently to each brain region (independent t-test for parametric data or Mann-Whitney test for non-parametric data). *P ≤ 0.05; **P ≤ 0.01.


The present study examined, for the first time, the responsiveness of the brain of males to PRL. Specifically, we analysed the distribution and steroid regulation of PRL-derived signalling in the male mouse brain, by means of the immunohistochemical detection of pSTAT5. We also compared the pattern of pSTAT5 immunoreactivity in the brain of males and females, to assess its sexual dimorphism. In this section, we will first analyse the distribution of pSTAT5 in the brain of male mice, with or without exogenous PRL administration, and compare it to the pattern of expression of the PRLR, as shown in in situ hybridisation material provided by the Allen Brain Institute (see Fig 1 and S2 Fig) and by other works [10]. Then, we will comment on the observed sexually dimorphic differences in central sensitivity to PRL. Next, we will discuss the role of testosterone as a putative regulator of central PRL signalling and on the mutual regulatory relationship of PRL and testosterone. We will conclude by reviewing how our findings relate to the neuroanatomical substrate of some putative actions of PRL in the brain of males.

Prolactin signalling in the male mouse brain, prolactin supplementation and prolactin receptor expression

In PRL-supplemented male mice (Male+PRL group), brain regions positively labelled for pSTAT5 were located mainly in the hypothalamus (the AVPe and VMPO, MPA, Pe, Pa, Arc, DM and VMH) and, to a lesser extent, in the telencephalon (SFi in the septum and MePD in the amygdala), with no additional pSTAT5-ir detected in any of the remaining brain divisions (Fig 1). Importantly, this pattern of pSTAT5-ir was highly dependent on systemic supplementation with a high dose of exogenous PRL, as male controls which were not supplemented with exogenous PRL (Male Control group) were virtually devoid of pSTAT5-ir, except for some faintly labelled cells in the ventral Arc (Fig 2T) and in the anterior portion of the periventricular hypothalamus (not shown). These results suggest, in the first place, that the patterns of pSTAT5-ir we report in this work are specifically produced by PRL, whereas other endocrine agents signalling through the Jak/STAT pathway, such as growth hormone [33], do not contribute substantially to STAT5 phosphorylation in the studied cases. This is further supported by the fact that pSTAT5 appears only in centres that show expression of PRLR in the brain of male mice (Fig 1, S2 Fig, [10]), whereas the distribution of growth hormone receptor does not fit the pattern of pSTAT5-ir [33,34]. In addition, the reduced pSTAT5-ir found in males not supplemented with exogenous PRL (Male Control group) indicates very low levels of PRL signalling in basal conditions, what fits the reduced circulating levels reported for the hormone for males [11,35]. In fact, the dorsal aspect of the Arc (presumed location of the TIDA neurons as revealed by tyrosine hydroxylase immunohistochemistry, [12]), displays scarce pSTAT5-ir in Male Control mice (Fig 2T).

Altogether, the male mouse brain shows little responsiveness to PRL under basal conditions, but this responsiveness would increase significantly with higher levels of the hormone reaching the brain. Hence, PRL would influence the brain of males especially under some physiological conditions in which there is substantially increased hypophyseal or extra-hypophyseal PRL release, for instance during the dark period of the day [35], after mating [36] or as part of the stress response [12,37].

Sexually dimorphic prolactin-derived signalling in the mouse brain

In comparison to males, female mice displayed a more extensive distribution of pSTAT5-ir, mainly in the hypothalamus and basal telencephalon, but also in different nuclei of the thalamus, midbrain and brainstem. Patterns of pSTAT5-ir in males were always a fraction of those of female mice, with no single brain structure labelled in males but not in females. Furthermore, a number of nuclei with pSTAT5 expression in both sexes displayed significantly higher pSTAT5-ir density in females than in males (the AVPe/VMPO, Arc, DM and MePD), but none of the analysed nuclei showed higher levels of pSTAT5-ir in males (Fig 3). In sum, our study reveals a clear, female-biased sexual dimorphism in PRL-derived signalling in the mouse brain upon exogenous PRL administration. This dimorphic responsiveness to PRL is observed in most of the nodes of the Sociosexual Behavioural Network (SBN), the core system for the integration of social and reproductive behaviour [38], thus supporting one of the defining features of this functional system (sexual dimorphism).

Although it is well known that gonadal steroids, specifically estradiol, promote hypophyseal PRL release [3], the gonadal steroid replacement performed on our experimental females is not likely to explain the higher (dimorphic) pSTAT5-ir levels observed in females. First, the exogenous administration of high doses of PRL homogenises the circulating levels of the hormone in males and females. Moreover, some brain centres show no dimorphic pSTAT5 immunostaining, further demonstrating that estradiol-induced hypophyseal PRL release is not responsible of the intersexual differences observed.

Two of the nuclei showing higher levels of pSTAT5-ir upon PRL administration in females, as compared to males, are the MPO and AVPe/VMPO. This finding fits the sexually dimorphic expression of the PRLR-l reported by Kokay et al [10] for these nuclei. However, the remaining nuclei showing female-biased pSTAT5-ir after PRL administration are reported to express equivalent levels of the PRLR-l in males and females [10]. This important mismatch between expression and activation of the PRLR-l suggests that, in most brain centres, dimorphic responsiveness to PRL is not due to intersexual differences in receptor expression, but to a sexually dimorphic functional regulation of PRLR signalling. This scenario might be the result of signalling pathways other than Jak/STAT (e.g. MAP kinase [3]) being recruited in the male brain. Alternatively, males and females might display differential expression/activation of regulatory elements downstream the PRLR-l (e.g. Jak, STAT5, SOCS, CIS, etc, [3,39,40]). Anyhow, our findings reflect important differences in the functional regulation of central PRL actions in males and females. Moreover, these data stress the importance of analysing hormone signalling rather than restricting the analysis to receptor expression to better understand the role of PRL in the regulation of physiology and behaviour.

Prolactin and testosterone: Mutual neuroendocrine regulation

One of the main outcomes of the present work is the identification of a putative regulatory role of testosterone in PRL signalling in the male mouse brain. Orchidectomy and consequent testosterone withdrawal led to a significant decrease in pSTAT5-ir density in the SFi, the Pa and both the central/dorsomedial and lateral VMH (Fig 4). This indicates a positive regulatory role of testosterone on PRL signalling in those brain regions. The actual (direct or indirect) mechanism of this process is not clear. Even though testosterone has documented inhibitory effects on hypophyseal PRL release in male rats [28] and mice [29], the effect of castration on pSTAT5-ir cannot be attributed to differences in circulating PRL, since intact and castrated males were supplemented with equivalent levels of exogenous PRL. In addition, this effect is not likely explained by a facilitated access of systemic PRL to the brain, as our results reveal that the positive effect of castration on pSTAT5-ir is not generalised but rather specific and restricted to discrete brain sites. Therefore, our results suggest that testosterone might upregulate PRLR expression or perhaps signalling downstream the PRLR in specific neural populations. Further research is required to elucidate the specific action of testosterone on PRL signalling in male mice.

An additional question of interest is the identity of the actual biological modulator of PRL signalling we report here. Testosterone can be locally metabolized into two different neuroactive steroids: it can be reduced to dihydrotestosterone [41], which binds to androgen receptors, or aromatised to estradiol [42] that then binds to oestrogen receptors. Our results make it very unlikely that this regulation occurs through the aromatisation of testosterone to estradiol. Although all the nuclei where PRL signalling is affected by castration express at least one form of oestrogen receptor [43], none of them displays aromatase activity in the male mouse brain [44]. Conversely, all of the nuclei where PRL-derived signalling decreased with castration express androgen receptors, with the Pa and VMH showing very high levels [44]. Hence, evidence suggests that the effect of testosterone on PRL signalling that we report here is likely mediated by androgen rather than oestrogen receptors.

In addition to the reported regulation of testosterone on central PRL signalling, our data, together with published evidence, suggest a reciprocal regulatory action of PRL on the hypothalamus-pituitary-gonadal (HPG) axis and ultimately on testosterone release. According to the literature, PRL is thought to upregulate the HPG axis and testosterone secretion in males, since PRL infusions stimulate the increase in serum testosterone in male rats, whereas immunological disruption of endogenous prolactin eliminates this effect [15,45]. This regulatory action of PRL on the HPG axis likely occurs through the kisspeptin system, which regulates gonadotropin-releasing hormone (GnRH) expressing neurons in the brain [46] and thereby activates the HPG axis. Therefore, the expression of PRL receptors by kisspeptin neurons [47] allows for a direct role of PRL on HPG axis control and testosterone secretion. In this context, our results provide support for the hypothesis that the brain kisspeptin system is subject to PRL regulation in male mice, too, since our sample of male mice displayed substantial pSTAT5-ir in the hypothalamic nuclei containing kisspeptin-positive neurons [48], the AVPe and the Arc (Fig 2J and 2R). Nevertheless, this possibility requires experimental proof by assessing the coexpression of kisspeptin and pSTAT5 immunoreactivity in the aforementioned nuclei.

Prolactin signalling in the male brain: Functional implications

Our findings confirm that the male mouse brain is indeed responsive to PRL, implying that PRL likely exerts certain actions in the male mouse brain, provided sufficient levels of the hormone. The nature of these actions is mostly unknown, but the multiple functional studies on central PRL in female mice provide a good comparative framework to contextualize our findings.

In this respect, the most prominent (but poorly understood) function of PRL in males is the regulation of sexual behaviour [3,17]. It has been shown that central action of PRL after mating suppresses sexual behaviour as part of the satiatory mechanisms operating after ejaculation [18,21]. Similar inhibitory action of PRL onto sexual behaviour occurs under chronic hyperprolactinaemia [4952]. Prolactin has been proposed to do so by modulating the function of the major dopaminergic circuits in the brain, closely involved in the motor component of male sexual function [17, 53]. In support of this, acute elevations of circulating PRL after copulation lead to significant decreases in dopaminergic activity in the target sites of the major dopaminergic pathways [18], including the striatum [22,53] and the MPA region [54]. Several studies have shown that, indeed, PRL is capable of modulating the dopaminergic activity of these pathways at several levels [5357]. However, according to our findings, none of the nuclei originating these dopaminergic pathways [18] display PRL-derived signalling neither in female nor in male mice, even after injection of exogenous PRL (Figs 1 and 2). Furthermore, in situ hybridisation in the mouse brain does not show any expression of PRLR mRNA within these pathways, neither in their sites of origin (substantia nigra or ventral tegmental area), nor in their projection sites (caudatus putamen or nucleus accumbens) (S2 Fig), although certain studies did find evidence of the receptor expression in the rat [58,59]. Hence, this suggests that the proposed action of PRL over these dopaminergic systems to regulate male sexual behaviour should rather be indirect. In this vein, our study evidences a testosterone-dependent action of PRL on two nuclei of the male brain that have also been involved in male sexual behaviour regulation: the VMH and the Pa (Figs 2 and 4). The VMH is sexually dimorphic [60], known to promote the expression of copulatory behaviour in females [61] and to inhibit mounting in male rats [62], whereas the Pa contains magno- and parvocellular oxytocin (OXT) neurons involved in the peripheral and central regulation of male sexual behaviour, respectively [6366].

The interaction between OXT and PRL at the level of the Pa can also have a role in the regulation of parental behaviours. There is solid evidence indicating that PRL [67] and OXT are fundamental in the control of maternal behaviour [68,69] and it is likely that they have an equivalent role in males. In this line, PRL facilitates pup-sensitisation in male rats [70] in a similar fashion as in females [67] and has further roles in other experimental models for paternal behaviour [71]. Tachikawa and collaborators [72] showed that paternal behaviour can be induced in male mice (which are usually infanticidal) by postcopulatory cohabitation with a maternal female, a process apparently dependent on OXT release in the nucleus accumbens (Acb) [73,74]. In turn, Dolen and collaborators [75] demonstrated that the OXT innervation of the Acb originates in the Pa (see also [76]), which, according to our findings, is responsive to PRL in males (Fig 2M). Therefore, PRL might participate in the regulation of parental behaviour in male mice, too, by acting on Pa OXT neurons. This hypothesis requires further study.

Our results indicate a modulatory role of PRL onto the Arc and Pe nuclei of males (2R-T), both of which contain the dopaminergic neuron populations responsible for the inhibitory feedback control of hypophyseal PRL release [77]: the tuberoinfundibular (TIDA) and tuberohypophyseal (THDA) neurons in the dorsomedial and rostral Arc [78], and the periventricular hypophyseal neurons (PHDA) in the Pe [79]. In the Arc, acute rises in circulating PRL have been shown to promote a general increase in STAT5 phosphorylation but, interestingly, also to decrease STAT5 signalling specifically in the TIDA neurons, suggesting a downregulation of the negative feedback exerted by TIDA neurons over pituitary PRL secretion [11]. However, the reported rise in systemic PRL was originated by an acute stress, and thus downregulation of the TIDA inhibitory tone could have occurred as part of the physiological stress response and not as a direct consequence of the increased PRL input.

Prolactin is both a central agent in acute stress response [12] and a significant element in stress regulation. Thus, central inhibition of the expression of PRLR by i.c.v. antisense blocking results in an enhanced activation of HPA axis in male and female rats [80], demonstrating a downregulation of the stress response by PRL. The presence of pSTAT5-ir in the Pa of males (in levels equivalent to females, see Figs 2M, 2N and 3) suggests that this modulatory effect of PRL on the reactivity of the HPA axis would probably take place through PRL action on corticotropin-releasing factor (CRF) or vasopressin (AVP) cells [81]. In fact, PRL is a well-documented regulator of the function of magno- and parvocellular vasopressinergic neurosecretory cells at the level of the hypothalamic Pa [8284]. Although effects of PRL on these parvocellular AVP cells has been proven to be mediated by ERK/MAPK pathway [84], our data in males and females suggest that the Jak/STAT5 signalling cascade can also be involved. This would imply a non-dimorphic regulatory role of PRL on the stress response (in addition to its dimorphic role on anxiety), which requires further research.


This work characterizes the distribution of PRL responsiveness in the male mouse brain and its dependence on circulating PRL levels, sexual dimorphism and testosterone regulation. Male mice display specific patterns of PRL-derived signalling only in the presence of high levels of circulating PRL. These patterns comprise mainly hypothalamic nuclei and some telencephalic sites, and are also more limited in extension and density than the equivalent patterns in female mice, evidencing a clear sexual dimorphism in favour of females. Furthermore, PRL-derived signalling in the male brain is regionally dependent on testosterone input, indicating a regulation of PRL action by testosterone, which might in fact be reciprocal [15,45]. This work confirms that PRL exerts central actions in males, suggesting a possible role of the “maternal hormone” in the regulation of male sexual behaviour, PRL release feedback control or in the regulation of stress response, among others.

Supporting information

S1 Fig. Characterization of antibody specificity.

Immunostaining for pSTAT5 was performed using a monoclonal rabbit anti-pSTAT5 primary antibody provided by Cell Signaling Technology (Phospho-Stat5 Tyr694 (D47E7), Catalog number #4322, Danvers, MA). We validated the specificity of this antibody by performing an antibody competition assay in complete series of brain sections from previously studied (pregnant) female mice (Salais-López et al., 2017) known to display high levels of pSTAT5 immunoreactivity. Sections underwent pSTAT5 immunohistochemistry using a primary antibody solution that had been preincubated overnight with the immunogenic peptide against which this antibody was raised (synthetic peptide corresponding to residues surrounding Tyr694 of human STAT5a protein, aminoacid sequence LAKAVDGyVKPQIKQ, where the phospho-tyrosine is indicated in lower case) (left column in the figure). We added this peptide to the primary antibody incubation solution at saturating concentrations (20 times higher than the molar equivalent of the antibody concentration used in the incubation solution). In order to check that this antibody specifically binds the phosphorylated form of STAT5, we included an additional control, consistent in preincubating the antibody with the non-phosphorylated version of the immunogenic peptide (aminoacid sequence LAKAVDGYVKPQIKQ, where the central tyrosine is not phosphorylated) (right column in the Fig). Both peptides were synthesized by the Peptide and Protein Chemistry Lab (Príncipe Felipe Research Center, CSIC, Valencia, Spain). Preincubation with the phosphorylated immunogenic peptide virtually abolished all pSTAT5-ir in the examined tissue (A, D and G, except for some faintly labelled cells in the arcuate nucleus, see D), as compared to sections preincubated with the non-phosphorylated peptide (B, E and H) or to non-preincubated controls (C, F and I). Altogether, this indicates that this primary antibody specifically binds to the phosphorylated form of pSTAT5, thus validating the employed primary antibody.


S2 Fig. Expression of the prolactin receptor mRNA in the male mouse brain.

Photomicrographs showing brain tissue of an adult male mouse specimen, processed for ISH of the PRLR obtained from the Allen Mouse Brain Atlas (2004 Allen Institute for Brain Science. Allen Mouse Brain Atlas. Available from:, experiment 72340223 ( Riboprobe RP_051101_02_B09, NCBI Accession NM_178921.2. For details on the procedure and use of controls, see the “Allen Mouse Brain Atlas Technical White Paper: In Situ Hybridization Data Production” ( Prolactin receptor labelling appears as dark granules accumulated within the cell body. Plate 1 shows representative regions of the telencephalon, midbrain and hindbrain expressing the PRL: a) septum; b) the medial posterior BST; c) the anterior aspect of the central amygdala; d) the medial posterior amygdaloid region; e) the periaqueductal grey; and f) the parabrachial nucleus. Plate 2 includes representative diencephalic sites: g) the anterior periventricular region of the preoptic hypothalamus; h) the medial preoptic region of the preoptic hypothalamus; i) the paraventricular hypothalamic nucleus; j) the caudatus putamen, globus pallidus and reticular thalamic nucleus (arrowheads point at labelling in this nucleus); k) the tuberal region of the hypothalamus; and l) the posterior intralaminar thalamic nucleus, substantia nigra and VTA. Distance to Bregma enclosed in each section. Scale bars represent 250 μm.


S1 File. Raw data for pSTAT5 analysis.

Raw data of pSTAT5-ir density used for the statistical comparison of PRL-derived signalling among: (A) intact, PRL-supplmented males and ovariectomized, steroid-primed and PRL-supplemented females and (B) intact, PRL-supplemented males and castrated, PRL-supplemented males.



The authors are indebted to The Allen Brain Institute for providing open access to their invaluable material and permission to use and publish its data.


  1. 1. Riddle O, Bates R, Dykshorn S. The preparation, identification and assay of prolactin—a hormone of anterior pituitary. Am J Physiol. 1933;(105):191–216.
  2. 2. Bole-feysot C, Goffin V, Edery M, Binart N, Kelly P a. Prolactin (PRL) and Its Receptor: Actions, Signal PRL Receptor Knockout Mice. Endocr Rev. 1998;19(February):225–68.
  3. 3. Freeman ME, Kanyicska B, Lerant A, Nagy G. Prolactin: structure, function, and regulation of secretion. Physiol Rev [Internet]. 2000 Oct [cited 2015 Sep 16];80(4):1523–631. Available from: pmid:11015620
  4. 4. Salais-López H, Lanuza E, Agustín-Pavón C, Martínez-García F. Tuning the brain for motherhood: prolactin-like central signalling in virgin, pregnant, and lactating female mice. Brain Struct Funct [Internet]. 2017 Mar 25 [cited 2017 Aug 28];222(2):895–921. Available from: pmid:27344140
  5. 5. Brown RSE, Kokay IC, Herbison AE, Grattan DR. Distribution of prolactin-responsive neurons in the mouse forebrain. J Comp Neurol. 2010;518:92–102. pmid:19882722
  6. 6. Grattan DR, Pi XJ, Andrews ZB, Augustine R a, Kokay IC, Summerfield MR, et al. Prolactin receptors in the brain during pregnancy and lactation: implications for behavior. Horm Behav. 2001;40:115–24. pmid:11534971
  7. 7. Grattan DR, Kokay IC. Prolactin: A pleiotropic neuroendocrine hormone. J Neuroendocrinol. 2008;20:752–63. pmid:18601698
  8. 8. Bakowska JC, Morrell JI. Atlas of the neurons that express mRNA for the long form of the prolactin receptor in the forebrain of the female rat. J Comp Neurol. 1997;386(April):161–77.
  9. 9. Bakowska JC, Morrell JI. The distribution of mRNA for the short form of the prolactin receptor in the forebrain of the female rat. Mol Brain Res. 2003;116:50–8. pmid:12941460
  10. 10. Kokay IC, Wyatt A, Phillipps HR, Aoki M, Ectors F, Boehm U, et al. Analysis of prolactin receptor expression in the murine brain using a novel prolactin receptor reporter mouse. J Neuroendocrinol. 2018 Sep;30(9):e12634. pmid:30040149
  11. 11. Guillou A, Romanò N, Steyn F, Abitbol K, Le Tissier P, Bonnefont X, et al. Assessment of lactotroph axis functionality in mice: longitudinal monitoring of PRL secretion by ultrasensitive-ELISA. Endocrinology [Internet]. 2015;(February):en.2014-1571. Available from:
  12. 12. Kirk SE, Xie TY, Steyn FJ, Grattan DR, Bunn SJ. Restraint stress increases prolactin-mediated phosphorylation of signal transducer and activator of transcription 5 in the hypothalamus and adrenal cortex in the male mouse. J Neuroendocrinol [Internet]. 2017 Apr 20 [cited 2017 May 2]; Available from:
  13. 13. Bronson FH, Desjardins C. Endocrine Responses to Sexual Arousal in Male Mice. Endocrinology [Internet]. 1982 Oct [cited 2017 Jul 18];111(4):1286–91. Available from: pmid:6811257
  14. 14. Drago F, Pellegrini-Quarantotti B, Scapagnini U, Gessa GL. Short-term endogenous hyperprolactinaemia and sexual behavior of male rats. Physiol Behav [Internet]. 1981 Feb [cited 2016 Jun 28];26(2):277–9. Available from: pmid:7232533
  15. 15. Bartke A, Morgan WW, Clayton RN, Banerji TK, Brodie AM, Parkening TA, et al. Neuroendocrine studies in hyperprolactinaemic male mice. J Endocrinol [Internet]. 1987 Feb [cited 2016 Jun 16];112(2):215–20. Available from: pmid:3102665
  16. 16. Shrenker P, Bartke A. Effects of hyperprolactinaemia on male sexual behaviour in the golden hamster and mouse. J Endocrinol [Internet]. 1987 Feb [cited 2016 Jun 16];112(2):221–8. Available from: pmid:3819637
  17. 17. Krüger THC, Hartmann U, Schedlowski M. Prolactinergic and dopaminergic mechanisms underlying sexual arousal and orgasm in humans. World J Urol [Internet]. 2005 Jul 12 [cited 2017 Jul 25];23(2):130–8. Available from: pmid:15889301
  18. 18. Krüger THC, Haake P, Hartmann U, Schedlowski M, Exton MS. Orgasm-induced prolactin secretion: feedback control of sexual drive? Neurosci Biobehav Rev [Internet]. 2002 Jan [cited 2017 Jan 21];26(1):31–44. Available from: pmid:11835982
  19. 19. Cruz-Casallas PE, Nasello AG, Hucke EE, Felicio LF. Dual modulation of male sexual behavior in rats by central prolactin: relationship with in vivo striatal dopaminergic activity. Psychoneuroendocrinology [Internet]. 1999 Oct [cited 2016 Jun 10];24(7):681–93. Available from: pmid:10451905
  20. 20. Capozzi A, Scambia G, Pontecorvi A, Lello S. Hyperprolactinemia: pathophysiology and therapeutic approach. Gynecol Endocrinol [Internet]. 2015 Jul [cited 2016 May 22];31(7):506–10. Available from: pmid:26291795
  21. 21. Rehman J, Christ G, Alyskewycz M, Kerr E, Melman A. Experimental hyperprolactinemia in a rat model: alteration in centrally mediated neuroerectile mechanisms. Int J Impot Res [Internet]. 2000 Feb [cited 2017 Jul 25];12(1):23–32. Available from: pmid:10982309
  22. 22. Kalra PS, Simpkins JW, Luttge WG, Kalra SP. Effects on male sex behavior and preoptic dopamine neurons of hyperprolactinemia induced by MtTW15 pituitary tumors. Endocrinology [Internet]. 1983 Dec [cited 2016 Jul 1];113(6):2065–71. Available from: pmid:6641626
  23. 23. Furigo IC, Kim KW, Nagaishi VS, Ramos-Lobo AM, de Alencar A, Pedroso J a. B, et al. Prolactin-sensitive neurons express estrogen receptor-α and depend on sex hormones for normal responsiveness to prolactin. Brain Res [Internet]. 2014;1566:47–59. Available from: pmid:24751572
  24. 24. Neill JD, Freeman ME, Tillson SA. Control of the proestrus surge of prolactin and luteinizing hormone secretion by estrogens in the rat. Endocrinology [Internet]. 1971 Dec [cited 2016 Jul 1];89(6):1448–53. Available from: pmid:5166047
  25. 25. Scully KM, Gleiberman AS, Lindzey J, Lubahn DB, Korach KS, Rosenfeld MG. Role of estrogen receptor-alpha in the anterior pituitary gland. Mol Endocrinol [Internet]. 1997 Jun [cited 2015 Nov 12];11(6):674–81. Available from: pmid:9171231
  26. 26. Anderson GM, Kieser DC, Steyn FJ, Grattan DR. Hypothalamic prolactin receptor messenger ribonucleic acid levels, prolactin signaling, and hyperprolactinemic inhibition of pulsatile luteinizing hormone secretion are dependent on estradiol. Endocrinology [Internet]. 2008 Apr [cited 2015 Dec 17];149(4):1562–70. Available from: pmid:18162529
  27. 27. Tong Y, Simard J, Labrie C, Zhao HF, Labrie F, Pelletier G. Inhibitory effect of androgen on estrogen-induced prolactin messenger ribonucleic acid accumulation in the male rat anterior pituitary gland. Endocrinology [Internet]. 1989 Oct [cited 2016 Jun 21];125(4):1821–8. Available from: pmid:2791967
  28. 28. Gill-Sharma MK. Prolactin and male fertility: the long and short feedback regulation. Int J Endocrinol [Internet]. 2009 Jan [cited 2016 Jun 21];2009:687259. Available from: pmid:20011060
  29. 29. O’Hara L, Curley M, Tedim Ferreira M, Cruickshanks L, Milne L, Smith LB. Pituitary androgen receptor signalling regulates prolactin but not gonadotrophins in the male mouse. PLoS One [Internet]. 2015 Jan [cited 2016 Feb 25];10(3):e0121657. Available from: pmid:25799562
  30. 30. Brown RSE, Herbison a E, Grattan DR. Differential changes in responses of hypothalamic and brainstem neuronal populations to prolactin during lactation in the mouse. Biol Reprod. 2011;84(December 2010):826–36. pmid:21178171
  31. 31. Rissman EF, Early AH, Taylor JA, Korach KS, Lubahn DB. Estrogen receptors are essential for female sexual receptivity. Endocrinology [Internet]. 1997 Jan [cited 2016 May 9];138(1):507–10. Available from: pmid:8977441
  32. 32. Paxinos G, Franklin KBJ. The Mouse Brain in Stereotaxic Coordinates [Internet]. Academic Press; 2004 [cited 2015 Mar 3]. Available from:
  33. 33. Furigo IC, Metzger M, Teixeira PDS, Soares CRJ, Donato J. Distribution of growth hormone-responsive cells in the mouse brain. Brain Struct Funct [Internet]. 2017 Apr 12 [cited 2016 May 27]; Available from:
  34. 34. Burton KA, Kabigting EB, Clifton DK, Steiner RA. Growth hormone receptor messenger ribonucleic acid distribution in the adult male rat brain and its colocalization in hypothalamic somatostatin neurons. Endocrinology [Internet]. 1992 Aug [cited 2015 Mar 3];131(2):958–63. Available from: pmid:1353444
  35. 35. Sinha YN, Salocks CB, Wickes MA, Vanderlaan WP. Serum and pituitary concentrations of prolactin and growth hormone in mice during a twenty-four hour period. Endocrinology [Internet]. 1977 Mar [cited 2015 Mar 4];100(3):786–91. Available from: pmid:45572
  36. 36. Kamel F, Wright WW, Mock EJ, Frankel AI. The influence of mating and related stimuli on plasma levels of luteinizing hormone, follicle stimulating hormone, prolactin, and testosterone in the male rat. Endocrinology [Internet]. 1977 Aug [cited 2016 Jun 10];101(2):421–9. Available from: pmid:885115
  37. 37. Torner L. Actions of Prolactin in the Brain: From Physiological Adaptations to Stress and Neurogenesis to Psychopathology. Front Endocrinol (Lausanne) [Internet]. 2016 Jan [cited 2016 Jun 21];7:25. Available from:
  38. 38. Newman SW. The medial extended amygdala in male reproductive behavior. A node in the mammalian social behavior network. Ann N Y Acad Sci. 1999;877:242–57. pmid:10415653
  39. 39. Pezet A, Favre H, Kelly PA, Edery M. Inhibition and restoration of prolactin signal transduction by suppressors of cytokine signaling. J Biol Chem [Internet]. 1999 Aug 27 [cited 2017 Jan 3];274(35):24497–502. Available from: pmid:10455112
  40. 40. Masuhara M, Sakamoto H, Matsumoto A, Suzuki R, Yasukawa H, Mitsui K, et al. Cloning and characterization of novel CIS family genes. Biochem Biophys Res Commun [Internet]. 1997 Oct 20 [cited 2017 Jan 3];239(2):439–46. Available from: pmid:9344848
  41. 41. Melcangi RC, Giatti S, Garcia-Segura LM. Levels and actions of neuroactive steroids in the nervous system under physiological and pathological conditions: Sex-specific features. Neurosci Biobehav Rev. 2015;
  42. 42. Naftolin F, Ryan KJ, Davies IJ, Reddy V V, Flores F, Petro Z, et al. The formation of estrogens by central neuroendocrine tissues. Recent Prog Horm Res [Internet]. 1975 [cited 2016 Jun 16];31:295–319. Available from: pmid:812160
  43. 43. Mitra SW, Hoskin E, Yudkovitz J, Pear L, Wilkinson H a., Hayashi S, et al. Immunolocalization of estrogen receptor α in the mouse brain: Comparison with estrogen receptor β. Endocrinology. 2003;144(5):2055–67. pmid:12697714
  44. 44. Stanić D, Dubois S, Chua HK, Tonge B, Rinehart N, Horne MK, et al. Characterization of aromatase expression in the adult male and female mouse brain. I. Coexistence with oestrogen receptors α and β, and androgen receptors. PLoS One [Internet]. 2014 Jan [cited 2015 Dec 29];9(3):e90451. Available from: pmid:24646567
  45. 45. Chandrashekar V, Bartke A. Influence of endogenous prolactin on the luteinizing hormone stimulation of testicular steroidogenesis and the role of prolactin in adult male rats. Steroids [Internet]. 1988 [cited 2016 Jul 7];51(5–6):559–76. Available from: pmid:3242177
  46. 46. Dungan HM, Clifton DK, Steiner RA. Minireview: kisspeptin neurons as central processors in the regulation of gonadotropin-releasing hormone secretion. Endocrinology [Internet]. 2006 Mar [cited 2016 Aug 3];147(3):1154–8. Available from: pmid:16373418
  47. 47. Kokay IC, Petersen SL, Grattan DR. Identification of prolactin-sensitive GABA and kisspeptin neurons in regions of the rat hypothalamus involved in the control of fertility. Endocrinology [Internet]. 2011 Feb [cited 2016 Jun 13];152(2):526–35. Available from: pmid:21177834
  48. 48. Semaan SJ, Tolson KP, Kauffman AS. The development of kisspeptin circuits in the Mammalian brain. Adv Exp Med Biol [Internet]. 2013 [cited 2016 Jun 13];784:221–52. Available from: pmid:23550009
  49. 49. Drago F, Lissandrello CO. The “low-dose” concept and the paradoxical effects of prolactin on grooming and sexual behavior. Eur J Pharmacol. 2000;405(1–3):131–7. pmid:11033320
  50. 50. Hernandez ME, Soto-Cid A, Rojas F, Pascual LI, Aranda-Abreu GE, Toledo R, et al. Prostate response to prolactin in sexually active male rats. Reprod Biol Endocrinol [Internet]. 2006 [cited 2016 Jun 15];4:28. Available from: pmid:16707016
  51. 51. Saito TR, Terada M, Oláh M, Nagy GM. The Role of Prolactin in the Regulation of Male Copulatory Behavior. In: Nagy , György M., Toth BE, editor. Prolactin [Internet]. InTech; 2013 [cited 2016 Jun 15]. Available from:
  52. 52. Hull EM, Lorrain DS, Du J, Matuszewich L, Lumley LA, Putnam SK, et al. Hormone-neurotransmitter interactions in the control of sexual behavior. Behav Brain Res [Internet]. 1999 Nov 1 [cited 2017 Jul 25];105(1):105–16. Available from: pmid:10553694
  53. 53. Hernández ML, Fernández-Ruiz JJ, Navarro M, de Miguel R, Cebeira M, Vaticón L, et al. Modifications of mesolimbic and nigrostriatal dopaminergic activities after intracerebroventricular administration of prolactin. J Neural Transm Gen Sect [Internet]. 1994 [cited 2017 May 2];96(1):63–79. Available from: pmid:7857592
  54. 54. Lookingland KJ, Moore KE. Effects of estradiol and prolactin on incertohypothalamic dopaminergic neurons in the male rat. Brain Res [Internet]. 1984 Dec 3 [cited 2017 May 2];323(1):83–91. Available from: pmid:6525510
  55. 55. Chen YF, Ramirez VD. Prolactin stimulates dopamine release from male but not from female rat striatal tissue superfused in vitro. Endocrinology [Internet]. 1982 Nov [cited 2016 Jun 15];111(5):1740–2. Available from: pmid:7128535
  56. 56. Laping NJ, Dluzen DE, Ramirez VD. Prolactin stimulates dopamine release from the rat corpus striatum in the absence of extra-cellular calcium. Neurosci Lett [Internet]. 1991 Dec 16 [cited 2016 Jun 15];134(1):1–4. Available from: pmid:1815141
  57. 57. Gonzalez-Mora JL, Guadalupe T, Mas M. In vivo voltammetry study of the modulatory action of prolactin on the mesolimbic dopaminergic system. Brain Res Bull [Internet]. 1990 Nov [cited 2018 Sep 11];25(5):729–33. Available from: pmid:2289161
  58. 58. Pi X, Voogt JL, Grattan DR. Detection of prolactin receptor mRNA in the corpus striatum and substantia nigra of the rat. J Neurosci Res [Internet]. 2002 Feb 15 [cited 2018 Sep 11];67(4):551–8. Available from: pmid:11835322
  59. 59. Muccioli G, Ghè C, Di Carlo R. Distribution and Characterization of Prolactin Binding Sites in the Male and Female Rat Brain: Effects of Hypophysectomy and Ovariectomy. Neuroendocrinology [Internet]. 1991 Jan [cited 2018 Sep 11];53(1):47–53. Available from: pmid:2046861
  60. 60. Dugger BN, Morris JA, Jordan CL, Breedlove SM. Androgen receptors are required for full masculinization of the ventromedial hypothalamus (VMH) in rats. Horm Behav [Internet]. 2007 Feb [cited 2016 Jul 13];51(2):195–201. Available from: pmid:17123532
  61. 61. Kow LM, Pfaff DW. Mapping of neural and signal transduction pathways for lordosis in the search for estrogen actions on the central nervous system. Behav Brain Res [Internet]. 1998 May [cited 2016 Jul 13];92(2):169–80. Available from: pmid:9638959
  62. 62. Christensen LW, Nance DM, Gorski RA. Effects of hypothalamic and preoptic lesions on reproductive behavior in male rats. Brain Res Bull [Internet]. 1977 Jan [cited 2016 Jul 13];2(2):137–41. Available from: pmid:880486
  63. 63. Waldherr M, Neumann ID. Centrally released oxytocin mediates mating-induced anxiolysis in male rats. Proc Natl Acad Sci U S A [Internet]. 2007 Oct 16 [cited 2016 Oct 21];104(42):16681–4. Available from: pmid:17925443
  64. 64. Nishitani S, Moriya T, Kondo Y, Sakuma Y, Shinohara K. Induction of Fos immunoreactivity in oxytocin neurons in the paraventricular nucleus after female odor exposure in male rats: effects of sexual experience. Cell Mol Neurobiol [Internet]. 2004 Apr [cited 2016 Oct 21];24(2):283–91. Available from: pmid:15176441
  65. 65. Veening JG, de Jong TR, Waldinger MD, Korte SM, Olivier B. The role of oxytocin in male and female reproductive behavior. Eur J Pharmacol [Internet]. 2015 Apr 15 [cited 2016 Oct 24];753:209–28. Available from: pmid:25088178
  66. 66. Gil M, Bhatt R, Picotte KB, Hull EM. Oxytocin in the medial preoptic area facilitates male sexual behavior in the rat. Horm Behav [Internet]. 2011 Apr [cited 2016 Oct 21];59(4):435–43. Available from:
  67. 67. Bridges RS, Ronsheim PM. Prolactin (PRL) regulation of maternal behavior in rats: bromocriptine treatment delays and PRL promotes the rapid onset of behavior. Endocrinology [Internet]. 1990 Feb [cited 2015 Mar 3];126(2):837–48. Available from: pmid:2298174
  68. 68. Bosch OJ. Maternal aggression in rodents: brain oxytocin and vasopressin mediate pup defence. Philos Trans R Soc Lond B Biol Sci [Internet]. 2013;368:20130085. Available from: pmid:24167315
  69. 69. Bosch OJ, Neumann ID. Both oxytocin and vasopressin are mediators of maternal care and aggression in rodents: From central release to sites of action. Horm Behav [Internet]. 2012;61(3):293–303. Available from: pmid:22100184
  70. 70. Sakaguchi K, Tanaka M, Ohkubo T, Doh-ura K, Fujikawa T, Sudo S, et al. Induction of brain prolactin receptor long-form mRNA expression and maternal behavior in pup-contacted male rats: promotion by prolactin administration and suppression by female contact. Neuroendocrinology [Internet]. 1996 Jun [cited 2016 Jul 7];63(6):559–68. Available from: pmid:8793898
  71. 71. Saltzman W, Ziegler TE. Functional significance of hormonal changes in mammalian fathers. J Neuroendocrinol [Internet]. 2014 Oct [cited 2016 Jul 7];26(10):685–96. Available from: pmid:25039657
  72. 72. Tachikawa KS, Yoshihara Y, Kuroda KO. Behavioral transition from attack to parenting in male mice: a crucial role of the vomeronasal system. J Neurosci [Internet]. 2013 Mar 20 [cited 2016 Jul 7];33(12):5120–6. Available from: pmid:23516278
  73. 73. Jin D, Liu H-X, Hirai H, Torashima T, Nagai T, Lopatina O, et al. CD38 is critical for social behaviour by regulating oxytocin secretion. Nature [Internet]. 2007 Mar 1 [cited 2016 Sep 12];446(7131):41–5. Available from: pmid:17287729
  74. 74. Akther S, Korshnova N, Zhong J, Liang M, Cherepanov SM, Lopatina O, et al. CD38 in the nucleus accumbens and oxytocin are related to paternal behavior in mice. Mol Brain [Internet]. 2013 [cited 2016 Sep 12];6:41. Available from: pmid:24059452
  75. 75. Dölen G, Darvishzadeh A, Huang KW, Malenka RC. Social reward requires coordinated activity of nucleus accumbens oxytocin and serotonin. Nature [Internet]. 2013 Sep 12 [cited 2018 Feb 6];501(7466):179–84. Available from: pmid:24025838
  76. 76. Otero-García M, Agustín-Pavón C, Lanuza E, Martínez-García F. Distribution of oxytocin and co-localization with arginine vasopressin in the brain of mice. Brain Struct Funct [Internet]. 2016 Sep [cited 2016 Oct 18];221(7):3445–73. Available from: pmid:26388166
  77. 77. DeMaria JE, Lerant AA, Freeman ME. Prolactin activates all three populations of hypothalamic neuroendocrine dopaminergic neurons in ovariectomized rats. Brain Res [Internet]. 1999 Aug 7 [cited 2016 Jun 6];837(1–2):236–41. Available from: pmid:10434008
  78. 78. Björklund A, Moore RY, Nobin A, Stenevi U. The organization of tubero-hypophyseal and reticulo-infundibular catecholamine neuron systems in the rat brain. Brain Res [Internet]. 1973 Mar 15 [cited 2016 Jun 6];51:171–91. Available from: pmid:4706009
  79. 79. Goudreau JL, Lindley SE, Lookingland KJ, Moore KE. Evidence that hypothalamic periventricular dopamine neurons innervate the intermediate lobe of the rat pituitary. Neuroendocrinology [Internet]. 1992 Jul [cited 2016 Jun 6];56(1):100–5. Available from: pmid:1322505
  80. 80. Torner L, Toschi N, Pohlinger A, Landgraf R, Neumann ID. Anxiolytic and anti-stress effects of brain prolactin: improved efficacy of antisense targeting of the prolactin receptor by molecular modeling. J Neurosci [Internet]. 2001 May 1 [cited 2016 Aug 3];21(9):3207–14. Available from: pmid:11312305
  81. 81. Aguilera G, Subburaju S, Young S, Chen J. The parvocellular vasopressinergic system and responsiveness of the hypothalamic pituitary adrenal axis during chronic stress. Prog Brain Res [Internet]. 2008 Jan [cited 2016 Sep 13];170:29–39. Available from: pmid:18655869
  82. 82. Donner N, Neumann ID. Effects of chronic intracerebral prolactin on the oxytocinergic and vasopressinergic system of virgin ovariectomized rats. Neuroendocrinology [Internet]. 2009 Jan [cited 2016 Sep 13];90(3):315–22. Available from: pmid:19546517
  83. 83. Vega C, Moreno-Carranza B, Zamorano M, Quintanar-Stéphano A, Méndez I, Thebault S, et al. Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei. Am J Physiol Regul Integr Comp Physiol [Internet]. 2010 Dec [cited 2016 Sep 13];299(6):R1701–8. Available from: pmid:20943859
  84. 84. Blume A, Torner L, Liu Y, Subburaju S, Aguilera G, Neumann ID. Prolactin activates mitogen-activated protein kinase signaling and corticotropin releasing hormone transcription in rat hypothalamic neurons. Endocrinology [Internet]. 2009 Apr [cited 2016 Sep 13];150(4):1841–9. Available from: pmid:19022892