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BOR - Papers in Press, published online ahead of print August 10, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.043794
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BIOLOGY OF REPRODUCTION 73, 1182–1190 (2005)
DOI: 10.1095/biolreprod.105.043794
© 2005 by the Society for the Study of Reproduction, Inc.

Research Article

Impaired Male Sexual Behavior in Activin Receptor Type II Knockout Mice1

Xiaoping Ma 3, Andrea Reyna 4, Shailaja K. Mani 4, Martin M. Matzuk 345  , and T. Rajendra Kumar 2 346  

Departments of Pathology,3 Molecular and Cellular Biology,4 and Molecular and Human Genetics,5 Baylor College of Medicine, Houston, Texas 77030 Department of Molecular and Integrative Physiology,6 University of Kansas Medical Center, Kansas City, Kansas 66160

ABSTRACT

Integration of multiple hormonal and neuronal signaling pathways in the medial preoptic area (mPOA) is required for elicitation of male sexual behavior in most vertebrates. Perturbation of nitric oxide synthase (NOS) activity in the mPOA causes significant defects in male sexual behavior. Although activins and their signaling components are highly expressed throughout the brain, including the mPOA, their functional significance in the central nervous system (CNS) is unknown. Here, we demonstrate a neurophysiologic role for activin signaling in male reproductive behavior. Adult activin receptor type II null (Acvr2–/–) male mice display multiple reproductive behavioral deficits, including delayed initiation of copulation, reduced mount, and intromission frequencies, and increased mount, intromission, and ejaculation latencies. These behavioral defects in the adult mice are independent of gonadotropin-releasing hormone (GnRH) homeostasis or mating-induced changes in luteinizing hormone (LH) and testosterone levels. The impairment in behavior can be correlated to the nitric oxide content in the CNS because Acvr2–/– males have decreased NOS activity in the mPOA but not the rest of the hypothalamus or cortex. Olfactory acuity tests confirmed that Acvr2–/– mice have no defects in general odor or pheromone recognition. In addition, motor functions are not impaired and the mutants demonstrate normal neuromuscular coordination and balance. Furthermore, the penile histology in mutant mice appears normal, with no significant differences in the expression of penile differentiation marker genes compared with controls, suggesting the observed behavioral phenotypes are not due to structural defects in the penis. Our studies identify a previously unrecognized role of activin signaling in male sexual behavior and suggest that activins and/or related family members are upstream regulators of NOS activity within the mPOA of the forebrain.

activin, androgen receptor, behavior, gonadotropin-releasing hormone, hypothalamus, male sexual behavior, male sexual function, NOS, steroids, testosterone

INTRODUCTION

Activins are potent autocrine and endocrine stimulators of follicle stimulating hormone (FSH) secretion from the pituitary gonadotropes [1–3]. The components of their signaling pathway are expressed throughout the reproductive axis and in multiple tissues, including the brain [3]. Activin signaling occurs through two type II Ser/Thr kinase receptors (ACVR2 and ACVR2B) and a single type1 receptor (ALK4 or ACVR1B), leading to ligand-induced dimerization of a type II and type I receptor, resulting in phosphorylation of the type I receptor in target cells and phosphorylation of either SMAD2 or SMAD3 proteins [4–7]. Whereas the roles of activins and ACVR2 in pituitary FSH homeostasis, perinatal and craniofacial development, and testis physiology are well-documented [8–12], the in vivo functions of activins and their receptors in the central nervous system are relatively unexplored.

We previously reported that 25% of Acvr2–/–mice die at birth due to mandible and other craniofacial abnormalities, the remaining are viable and demonstrate gonadal defects secondary to suppressed FSH [8, 10]. These include reduced testis size [8], Sertoli and germ cell number [13], and germ cell carrying capacity of the Sertoli cells [12, 13]. Activin ßA and ßB double knockout mice die at birth secondary to craniofacial defects [9–11]. Interestingly, Acvr2–/–males display delayed puberty and infertility with reduced penetrance, independent of GnRH and FSH signaling pathways [14].

The medial preoptic area (mPOA) is a critical integrative site for the converging neuronal signaling pathways that specify male sexual behavior in a variety of species, including rodents. Perturbations in dopaminergic pathways, aromatase levels, and signaling by N-methyl-D-aspartate receptors in the mPOA lead to abnormal male sexual behavior in rodents [15–20]. More important, nitric oxide (NO), a gaseous neurotransmitter molecule has been shown to be an important mediator of dopamine (DA) release in the mPOA [21–24]. NO is a by-product generated during the biochemical conversion of L-arginine to L-citrulline, a reaction catalyzed by NOS [25–28]. The neuronal isoform of nitric oxide synthase (NOS; NOS1) has been shown to affect male sexual behavior as demonstrated by the phenotypic analysis of Nos1 mutant mice [29]. Although many downstream factors are regulated by changes in NOS activity, very little is known about upstream regulators that activate signaling cascades leading to changes in NOS within the mPOA of the brain.

In the brain, ACVR2 is expressed in regions with afferent inputs to the hypothalamus, both in the forebrain, including the mPOA, and within the brain stem [30–32]. Here, we demonstrate that ACVR2 signaling is a critical mediator of male sexual behavior and provide evidence that male reproductive behavioral defects in Acvr2 knockout mice can be correlated with decreased NOS activity in the mPOA.

MATERIALS and METHODS

Mice

All mice used in these studies were of mixed strain (C57BL/6 x 129SvEv); mice were generated and maintained as a breeder colony at Baylor College of Medicine, Houston, TX. Adult heterozygous (Acvr2a +/tm1Zuk) and homozygous mutant (Acvr2a tm1Zuk/tm1Zuk) male mice were used in this study. These mice are referred to as Acvr2+/– and Acvr2–/–, respectively, throughout the text. Wild-type and mutant Acvr2 alleles were identified by Southern blot analysis of tail DNA as described [8]. Mice were kept under standard laboratory conditions and maintained as per the NIH guidelines and approved animal protocols of the Institutional Animal Care and Use Committee of Baylor College of Medicine.

Behavioral Tests

All animals were maintained on a 12L:12D reversed light cycle with lights-off at 1200 h. Food and water were available ad libitum. Eight-week-old Acvr2+/– and Acvr2–/–littermates (n = 14) were tested for male-typical sexual behavior in the presence of sexually receptive stimulus females. Stimulus females consisted of age-matched, ovariectomized wild-type female littermates; primed with 0.5 µg estradiol benzoate (EB) followed 48 h later with 100 µg of progesterone (P). Both EB and P were dissolved in sesame oil and administered s.c. All behavioral tests were conducted 6 h after P administration under red-light illumination during the dark phase of the reversed light cycle. The observers were blind to the genotypes of the experimental mice. The tests were videotaped as well as scored manually. Intromission was scored when vaginal penetration was achieved by a male during mount, accompanied by pelvic thrusting. Behavioral ejaculations were marked when an intromission with longer lasting thrusting, accompanied by characteristic pelvic motor patterns.

Male mice were habituated to an empty clear Plexiglas testing cage 60 min before the introduction of the female. The testing cage was placed on a mirror stand, with the mirror at an inclined angle of 45o to allow ventral viewing. Male sexual behavioral tests were carried out for 30 min after the introduction of the female or until the male ejaculated (whichever occurred first). If the male intromitted within 30 min, a further 60 min was given for him to ejaculate. The male was considered sexually inactive when he failed to intromit within the 30-min testing period. The females were checked for the presence of vaginal plugs immediately after apparent behavioral ejaculation. Male mice were tested four times (with an interval of 6–7 days) and the behavioral parameters were measured as defined [33].

For each male, the latency to mount, intromit and ejaculate after the introduction of the stimulus female and within the test period were recorded. The number of mounts (frequency), intromissions (frequency), and ejaculations (frequency) were also noted. Numbers of thrusts per intromission and ejaculation latency (the time from first mount to ejaculation) were recorded for mice that exhibited these behaviors. The observers were blind to the mouse genotype. The data are represented as mean ± SEM.

Recognition of odor of a hidden food object (extrasharp cheddar cheese) was tested as described [34]. Mice were individually placed in a cage that contained cheese hidden under the bedding. The latency to smell and uncover the food was scored in each case and recorded.

Olfactory preference test was used to evaluate the pheromone odor preference of mice [35]. All mice were tested in a clean polystyrene test arena (50 x 45 x 25 cm) under two different conditions. In the first condition, the olfactory stimulus consisted of three small containers (10 x 10 x 6 cm) containing clean, male, or estrous bedding. The containers were randomly placed, two in the frontal corners and one in the middle section of the arena. Each mouse was allowed to choose between containers with clean, male, or estrous bedding. The time spent by each mouse investigating each bowl was recorded in a 10-min test. A second test under the same condition was performed after 4 days. A week later, the mice were tested a second time and allowed to choose between clean, anestrous, and estrous bedding. As before, the time spent by each mouse investigating each bowl was recorded in a 10-min test. A second test under the same condition was performed after 4 days.

The clean bedding consisted of 50 g of Tek Fresh paper bedding (Harlan Tekland, Indianapolis, IN). The anestrous bedding was collected from 6 ovariectomized and oil-injected female mice, individually housed for 5 days in cages containing 50 g of Tek fresh paper bedding. The estrous bedding (Tek Fresh paper bedding) was collected from the cages of six estrous stimulus female mice, ovariectomized, EB-primed for 48 h followed by P for 6 h before the olfactory test. The male bedding was collected from six adult C57 Bl/6J mice, housed individually in cages containing Tek Fresh paper bedding for 5 days. The observers were blind to the type of bedding in each container.

Motor functions were assessed to determine neuromuscular abnormalities using the vertical pole test and hanging wire test for motor coordination and balance. In the first method, each mouse (n = 6 mice) was placed on a taped metal rod held horizontally [34]. The rod was then slowly moved to a 45° position and eventually to an upright vertical position. The time taken for each mouse to fall off into the cage was recorded. In the second method [36], each mouse was placed on a taped metal wire cage lid ~25 cm above the cage and the lid tapped gently for a few seconds. Subsequently, the metal wire was held in an inverted position and the time taken for each mouse to fall off the lid into the cage was recorded.

NOS Enzyme Assays

Adult Acvr2+/– and Acvr2–/–male mice were isofluorane-anesthetized, decapitated, and the brain regions surgically removed on ice. The hypothalamus and preoptic area (POA) were excised following the mouse brain atlas [37]. Approximately 300-µm slices were cut at a standard rostrocaudal level, beginning approximately 2 mm anterior to optic chiasm and ending 1 mm anterior to the mammillary bodies using a McIlwain tissue chopper. The first four slices were taken, laid out on a cold glass plate, and the mPOA microdissected out using the landmarks based on the atlas [37]. The following five slices containing the anterior, lateral, ventromedial, paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus were microdissected based on the landmarks in the atlas and designated as the rest of the hypothalamus (HYP). A sample of frontal cortex was also removed. The tissue samples were snap frozen on dry ice and stored at –80°C until further use. NOS activity in the mPOA, cortex, and the rest of the hypothalamus extracts was assayed using a NOSdetect assay kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions using arginine (Amersham Biosciences, Piscataway, NJ). The enzyme activity was measured by the conversion of arginine to [3H] citrulline, which was separated by an ion exchange spin column as the flow-through fraction and the radioactivity counted. Rat brain cerebellum extract was used as a positive control. Total protein content of tissues was estimated by a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Tissue extracts were used in duplicate and the data are represented as mean ± standard error of the mean (SEM). The NOS assay was performed twice.

Hormone Assays

Blood was collected from adult male mice by closed cardiac puncture under isofluorane anesthesia, serum separated, and stored at –20°C for estimation of basal levels of hormones. In another independent set of experiments, Acvr2+/– and Acvr2–/–male mice were allowed to mate with wild-type or Acvr2+/– females during an interval of 30 min and thereafter the males were exsanguinated under anesthesia, serum separated and used for testosterone and LH assays. The testosterone content in unextracted serum samples was analyzed by a solid-phase radioimmunoassay (RIA). The sensitivity of the assay is 0.1 ng/ml. Serum estradiol was quantified by a third generation ultrasensitive liquid-phase assay. The sensitivity of the assay is 0.6 pg/ml. Both of the steroid assay kits were purchased from Diagnostic Systems Laboratories, Webster, TX. Serum LH content was estimated using a two-site sandwich immunoassay assay at the Ligand and Hormone assay core, University of Virginia, Charlottesville, using the mouse LH standards. The assay range is 0.08–37 ng/ml. The detailed description of the LH assay method has been reported [38].

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction Assays

Total RNA was isolated from tissues by Trizol method, spectrophotometrically quantified, and 1 µg of total RNA was used for cDNA synthesis by standard methods [12]. For each polymerase chain reaction (PCR), the linear range of amplification of the product representing an optimal cycle number was determined and used for subsequent comparisons between different genotypes. PCR amplifications were done on cDNA samples using mouse primers specific for Inhba, Inhbb, Acvr2, Alk4, Smad2, Smad3, NOS1, Ar, Arm, Shh, Ptc, Hoxd13, and Bmp4 as listed in Table 1. Primers were also designed to amplify cylcophilin A (CypA) and were used as an internal control for all PCR reactions. The amplified products were separated on 1.5% agarose gels, visualized by ethidium bromide staining, and photographed by negative contrast imaging. The intensities of the bands were recorded using densitometry (Molecular Dynamics, Inc., Sunnyvale, CA).


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  		TABLE 1. List of PCR primers used for RT-PCR assays

Histological Analysis

Adult Acvr2+/– and Acvr2–/–male mice were transcardially perfused with ice-cold 4% paraformaldehyde in PBS (pH 7.4) and the brains were immersed in cold PBS at 4°C. External genitalia were collected into formalin and fixed at room temperature for at least 24 h. The tissues were processed through graded alcohol series, paraffin-embedded, and either 30-µm (coronal sections of the brain) and/or 5-µm (external genitalia) sections were cut. External genitalia sections were stained with periodic acid-Schiff reagent and hematoxylin or hematoxylin-eosin, whereas Nissl staining of whole-brain sections was performed. Multiple sections of the brain, including the hypothalamus and mPOA, and external genitalia were analyzed from mice of each genotype. The specific objective of penile histology was to determine whether there were any structural abnormalities in the penile tissue.

Statistical Analysis

The data were analyzed by ANOVA or Student t-test using Prism statistical program (Graphpad Software, San Diego, CA). A P value <0.05 was considered significant.

RESULTS

Reproductive Behavioral Defects in Acvr 2–/– Mice

To further characterize the male reproductive defects in the absence of ACVR2 signaling, we used naive Acvr2+/– and Acvr2–/–adult mice and quantitatively analyzed their male sexual behavior. The mice were subjected to a total of four behavioral tests. The frequencies of mounts, intromissions, and ejaculations were all dramatically reduced in Acvr2–/–males compared with the Acvr2+/– controls, even after the fourth behavioral test (Fig. 1, A–C). In addition, the Acvr2–/–males demonstrated prolonged mount and intromission latencies within the time period tested (Fig. 1, D and E). Acvr2–/–males that did not intromit within the 30-min test period by the fourth trial were considered sexually inactive. Acvr2–/–males that showed intromission within 30 min were given an additional 60 min to allow them to ejaculate in the presence of a stimulus female and ejaculation latencies scored. Acvr2–/–males that demonstrated intromission and visible longer lasting thrusts accompanied by pelvic motor patterns characteristic of behavioral ejaculations often did not deposit visible vaginal plugs in the stimulus female mice (2/14 females tested showed plugs). Furthermore, there was no enhancement in the sexual behavior of mutant males, despite repeated behavioral testing, suggesting a role for ACVR2 in sexual behavior of mice.


Figure 1
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  		FIG. 1. Impaired sexual behavior in Acvr2–/–male mice. Adult Male and female Acvr2+/– and Acvr2–/–mice were obtained from our breeding colony at Baylor College of Medicine. In each of the tests indicated (A–E), as described in Materials and Methods, severe behavioral defects were observed in Acvr2–/–males compared with the control littermates (P < 0.001)

Absence of ACVR2 Signaling Does Not Affect LH and Testosterone Levels

To test whether the reproductive behavioral defects of Acvr2–/–male mice were due to a gonadal defect that affects testosterone production, we assayed serum testosterone levels by a radioimmunoassay. We found no difference in serum testosterone levels (1.5 ± 0.2 ng/ml in Acvr2+/– versus 1.3 ± 0.4 ng/ml in Acvr2–/– males; n = 9; P > 0.05). Furthermore, testosterone levels in Acvr2–/–males exposed to females were not significantly different from those in Acvr2+/– mice similarly exposed to females (Fig. 2). Consistent with the testosterone data, serum LH levels in intact or males exposed to females were also the same between Acvr2–/–and Acvr2+/– male mice [14] (Fig. 2). Together, these results suggest that a deficiency in normal circulating or mating-induced serum LH and testosterone was not responsible for the behavioral defects observed in Acvr2–/–male mice.


Figure 2
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  		FIG. 2. Mating-induced changes in serum testosterone and LH. Male Acvr2+/– and Acvr2–/–mice were allowed to mate with heterozygous or wild-type female mice during half an hour interval of time. Male mice were then exsanguinated, the serum separated and used for quantifying the serum levels of testosterone and LH using separate immunoassays. Note that the mating-induced levels of serum testosterone and LH in Acvr2–/–male mice are not significantly different (P > 0.05; n = 5–6 mice) when compared with those in Acvr2+/– male mice. Values are mean ± SEM. The high SEM values are typical for a testosterone assay using adult serum samples

Acvr2–/– Male Mice Have Reduced NOS Activity in the mPOA

Reverse transcription-PCR analyses indicated that Acvr2 and its signaling components are expressed in the mPOA of adult mice (Fig. 3). While it is well known that pharmacological manipulations of neuronal NOS activity in the mPOA impair male sexual behavior [21, 30, 39], the role of ACVR2 signaling in these behaviors is unknown. To determine whether the behavioral defects in Acvr2–/–male mice were due to alterations in NOS, we tested the activity of NOS in the mPOA, cortex, and the rest of the hypothalamus. Tissue homogenates of the mPOA, cortex, and the rest of the hypothalamus from Acvr2–/–and Acvr2+/– males were analyzed for their NOS activity quantitated by determining the amount of [3H]-citrulline produced by the enzymatic conversion of [3H]-arginine. The values were normalized to the amount of protein input. Approximately, 45% reduction in NOS activity was observed in the mPOA of Acvr2–/–males compared with Acvr2+/– males, while the activity remained unchanged in the cortex and the rest of the hypothalamus (Fig. 4). Although there was a difference in NOS activity, steady-state levels of Nos1 mRNA remained unchanged in the mPOA of Acvr2–/–mice (data not shown). The reduction in NOS activity was not due to anatomical difference in neuron population in the mPOA because histological analysis did not reveal any major differences in cell number and cytoarchitecture of cell grouping and location in the mPOA of Acvr2–/–male mice (data not shown). Furthermore, a similar reduction in NOS activity in the mPOA was observed both in pubertal and postpubertal mice, indicating that delayed puberty is not a factor that would account for developmental differences in NOS activity between controls and mutants. These results suggest that absence of signaling through activin receptor type II in vivo leads to a region-specific decrease in NOS activity in the mPOA.


Figure 3
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  		FIG. 3. Expression of ACVR2 signaling components in the mPOA. Medial preoptic area was surgically isolated from two adult male mice and immediately processed for total RNA isolation using the Trizol reagent (Invitrogen, Carlsbad, CA). The cDNA samples were later amplified using specific primers, the products separated on 1.5% agarose gels, visualized by ethidium bromide staining, and photographed using negative-contrast imaging. Note that, except ßB, all the activin signaling components are expressed in the mPOA. Cyclophilin A (CypA) expression was used as the control for RNA input


Figure 4
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  		FIG. 4. NOS activity in the mPOA, cortex, and the rest of the hypothalamus. Cortex, mPOA, and the rest of the hypothalamus were isolated from adult male Acvr2+/– and Acvr2–/–mice and the NOS enzyme activity was assayed by measuring the conversion of [3H]-arginine to [3H]- citrulline, as described in Materials and Methods. Note that NOS activity is significantly reduced in the mPOA (*P < 0.001), but not the hypothalamus or cortex (P > 0.05) of Acvr2–/– compared with Acvr2+/– male mice

Olfactory Acuity Is Not Decreased in Acvr2–/– Male Mice

To test whether Acvr2–/–male mice have impaired olfactory cues that would explain the sexual behavior defects, control and Acvr2–/–male mice were subjected to olfactory acuity test. Both groups of mice located an individual hidden object by recognizing its odor and the latency to recognize the odor of these hidden objects was similar; the mutants responded better although not statistically significantly (Fig. 5A). Furthermore, when exposed to clean, male, or anestrous and estrous bedding, controls and mutants similarly recognized reproductively relevant odors (Fig. 5B). Together, these results indicate that general odor and pheromone recognition is not impaired in mutant mice.


Figure 5
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  		FIG. 5. Odor recognition and motor coordination tests. A) Recognition of a hidden food object by odor: Adult male Acvr2+/– and Acvr2–/–mice (4–5) were individually placed in a cage and allowed to recognize the smell of the hidden food (cheddar cheese). The latency was scored in each case. Acvr2–/–mice recognize the odor better than the control Acvr2+/– mice (P > 0.05), indicating that there is no defect in sensory function of olfaction. B) Control and mutants were also tested for their ability to investigate reproductively relevant odors from bedding exposed to different pheromones. In these tests, mutants are comparable with controls in pheromone recognition. Values are mean ± SEM (n = 6); P > 0.05. C and D) Test of motor coordination function by wire-hang test. Individual mice were placed on a taped wire held in an inverted position and the latency to fall off into the cage was scored in each case. Note that there is no difference in wire-hang latency (P > 0.05) between Acvr2+/– and Acvr2–/– mice, thus confirming that the motor coordination of Acvr2–/– mice is not affected

Motor Coordination Is Not Impaired in Acvr2–/– Male Mice

To determine whether the consequence of ACVR2 deletion is specific to male sexual behavior, the mice were subjected to another behavior test unrelated to reproductive behaviors. Motor coordination and balance were tested in the wild-type and mutant mice using the vertical-pole test and the hanging-wire test. In the former, the forepaw strength was assessed qualitatively by the ability of the control and mutant mice to climb a rod held at 45° and gradually placed in a vertical position. In the second quantitative test, latency of the control and Acvr2–/–male mice to fall off a hanging wire was scored during a period of 1 min (Fig. 5, C and D). In both these tests, Acvr2–/–male mice performed the tasks comparable with the control mice. Collectively, these behavioral studies indicate that Acvr2–/–male mice demonstrate no motor deficits.

Androgen Receptor and Aromatase Expression Are Not Affected in the mPOA of Acvr2–/– Male Mice

It is well known that steroid hormones regulate Nos1 expression in brain and peripheral tissues and could have effects on behavioral outcomes. Defects in the activity of aromatase or NOS1 in the mPOA [40] or increases in estrogen levels, as observed in ER{alpha}-knockout mice, can cause disruption of male reproductive behavior [40, 41]. Furthermore, male sexual behavioral defects have been reported in aromatase-knockout mice [24, 42]. To test whether Acvr2–/–male mice demonstrate changes in androgen receptor or aromatase gene expression in the mPOA, we performed reverse transcription (RT)-PCR analyses on RNA isolated from mPOA. This analysis identified no changes in androgen receptor and aromatase expression in the mPOA of Acvr2–/–male mice (Fig. 6A). Additionally, serum RIA analyses indicated that Acvr2–/–male mice have estradiol levels comparable with those in control males (Fig. 6B). Collectively, these data demonstrate that a decrease in NOS activity in the mPOA of Acvr2–/–male mice is not correlated to changes in expression of androgen receptor and aromatase or serum estrogen levels.


Figure 6
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  		FIG. 6. Expression of androgen receptor and aromatase in the mPOA and serum estradiol content. A) Total RNA isolated from the mPOA was used to amplify androgen receptor (Ar), aromatase (Cyp19 or Arm), and cyclophilin A (CypA). Note that there were no differences in expression of androgen receptor and aromatase in the mPOA of Acvr2–/– male mice compared with that in Acvr2+/– males. B) Male Acvr2+/– and Acvr2–/–mice were exsanguinated, the serum separated, and used for quantifying the serum levels of estradiol using an ultrasensitive liquid phase RIA. Note that the levels of serum estradiol in Acvr2–/–male mice are not significantly different (P > 0.05; n = 10 mice) when compared with those in Acvr2+/– male mice. Values are mean ± SEM

Acvr2–/– Male Mice Demonstrate Normal Penis Histology and Express Penile Differentiation Marker Genes

Structural defects in penis may cause ejaculatory abnormalities. To determine whether the penis is structurally abnormal, we performed histological analysis and identified that there are no defects in Acvr2–/–mutant mice compared with the heterozygous control mice. The vascular endothelium, smooth muscle bundles, and the general cellular architecture of the penile tissue appear normal in sections obtained from mutants (Fig. 7, A and B). Although activin-signaling components are normally expressed in external genitalia (Fig. 7C), Nos1 mRNA levels were undetectable by a semiquantitative RT-PCR assay (not shown). Furthermore, marker genes that specify normal differentiation program of the penis are all expressed in the mutants and did not significantly differ from the heterozygous control mice (Fig. 8). These data indicate that the observed behavioral defects in the Acvr2–/–mice are unlikely due to developmental or structural abnormalities of the penis.


Figure 7
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  		FIG. 7. Histology of the penis and expression of the ACVR2 signaling components. External genitalia were obtained from adult Acvr2+/– and Acvr2–/– mice and fixed in formalin for at least 12 hours. Paraffin-embedded 5-µm sections were stained with hematoxylin/periodic acid-Schiff reagent and observed with a Zeiss microscope. Note that there are no histological differences in the external genitalia from Acvr2+/– (A) and Acvr2–/– (B) mice. SM, Smooth muscle; N, nerve; arrows indicate endothelium. Both photographs were taken at x10 magnification. C) RT-PCR analysis indicates that expression of the ACVR2 signaling pathway components is not affected in the external genitalia from Acvr2–/– mice


Figure 8
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  		FIG. 8. Expression of penile differentiation markers. Total RNA was isolated from external genitalia tissue from adult Acvr2+/– and Acvr2–/–mice and cDNAs synthesized as described in Materials and Methods section. Semiquantitative RT-PCR assays were performed using primers specific for sonic hedgehog (Shh), patched (Ptc), homeobox gene d-13 (Hoxd13), and bone morphogenetic protein-4 (Bmp4). Cyclophilin A (CypA) expression was used as the control for RNA input. Note that expression of the penile differentiation markers is not affected in Acvr2–/–mutant mice

DISCUSSION

The medial preoptic area in the forebrain is a central integrative site for male sexual behavior [20, 43]. Studies to date have identified a role for several factors, including NOS1, testosterone, dopamine, estrogens, and aromatase in the modulation of male sexual behavior. While signaling proteins, such as activins and their signaling components, are abundantly expressed within mPOA of the forebrain [3, 30], their physiological role in the central nervous system has been relatively unexplored. We have analyzed the consequences of abolishing ACVR2 signaling in male reproductive behavior. Male mice lacking ACVR2 demonstrated severe reproductive behavioral abnormalities. These phenotypes perhaps reflect the gender-specific differential regulation of activins and their signaling components [3, 7].

Correlating with the behavioral defects, we found that NOS activity was suppressed in the mPOA but not the rest of the hypothalamus or cortex of Acvr2–/–male mice. These observations are in accordance with the published findings demonstrating the involvement of NOS activity in the copulatory behavior in male rats. Intracranial administration of L-arginine, an activator of NOS enzymes, into the mPOA enhanced male copulatory behavior, and this enhancement was inhibited by inhibitors of neuronal NOS [44]. Male sexual behavior defects were also observed in neuronal NOS and endothelial NOS knockout mice [29, 45, 46]. Our studies corroborate these earlier reports and also suggest that activin signaling is important for NOS activity in the mPOA but not in other regions of the brain. It is possible that, while ACVR2 is widely expressed in multiple areas of the fore brain, signaling components that orchestrate NOS activity downstream of ACVR2 signaling are mPOA specific. Our current studies do not address which of the NOS (neuronal, inducible, and endothelial) is predominantly regulated in the mPOA downstream of ACVR2 signaling and studies are in progress to determine the isoforms involved.

Although our data indicate that ACVR2 signaling plays an activational role in male sexual behavior, we cannot rule out other organizational defects during development of the mPOA. This issue is relevant because the organizational role of activins in neuroepithelial development during early embryogenesis has been demonstrated. Notably, activin or activin-like signal patterns the domains of the optic vesicle into retinal pigmented epithelium and promotes expression of many specific genes [47, 48]. However, normal GnRH homeostasis in the hypothalamus and the lack of defects in gonadal steroid levels in activin receptor II mutants argues against a developmental or organizational defect resulting in the observed behavioral defects. Given that the perinatal deficits leading to delayed puberty could lead to a slowing of developmental match between steroid hormones and brain development in the Acvr2–/–mice and consequent failure of masculinization, future studies will examine neonatal testosterone levels in mutant mice. Furthermore, detailed mapping studies involving expression of NOS enzymes and their activity will also identify whether specific hypothalamic nuclei, including the paraventricular nucleus, are affected in Acvr2–/–mice.

ACVR2 is highly expressed in olfactory bulbs and also in the amygdala [30, 49]. A disruption in the processing and transmission of chemosensory information from the amygdala to the mPOA may lead to some of the above behavioral defects in Acvr2–/–mice. Two olfactory systems are known to be important in rodents, the main and accessory olfactory system. It was originally assumed that the main olfactory system detected volatile odors generated from food or prey [50, 51], whereas the accessory olfactory system detected large molecular weight nonvolatile odors that influence reproductive behaviors and neuroendocrine functions [52, 53]. More important, volatile urinary odors from opposite-sex conspecifics have been demonstrated to signal the presence of potential mates and motivate approach behaviors [54]. Studies suggest that both the systems interact with each other in mediating social responses to chemical signals from conspecifics of various species [55, 56]. Thus, chemosensory ability mediated by both olfactory systems was evaluated by tests of odor recognition and odor preference.

One possible causative factor for the observed defects in male sexual behavior could be an impairment of odor recognition in Acvr2–/–mice. Impaired olfactory acuity leading to defective information processing in amygdala and signal transmission to mPOA could be responsible for the disruption in sexual responsiveness. However, our findings based on olfactory cue recognition of a hidden food object and reproductively relevant odors indicate that this information processing does not appear to have been affected in the Acvr2–/– mice.

Steroid hormones regulate masculine sexual behavior by their interactions with neurotransmitters, opioids, excitatory amino acids, neuropeptides, prostaglandins, and other peptide hormones in the mPOA [15–21, 23, 57]. Testosterone also affects male sexual behavior by modulating NOS1 activity in the mPOA. Blockade of the androgen receptors by direct intracranial implantation of hydroxyflutamide has been demonstrated to inhibit male sexual behavior in rats [58, 59]. Castration reduced Nos1 levels in the mPOA and testosterone supplementation restored NOS1 immunoreactivity to normal levels [57, 60]. In Acvr2–/–males, expression of androgen receptor in the mPOA and the serum testosterone levels are unaffected, suggesting that the observed decrease in NOS activity in mPOA of these mice is not due to altered testosterone levels. Our observation that testosterone levels are unaffected in Acvr2–/–mice is also supported by normal hypothalamic GnRH content and its receptor expression and LH synthesis and secretion from the pituitary [14], which are regulated by testosterone levels. These data indicate nonpituitary and GnRH-independent roles of ACVR2 for the observed male reproductive defects. It is noteworthy that mPOA NO affects GnRH release in rats [61], but our data clearly suggest that both GnRH and steroids are not affected despite a decrease in NOS activity in the mPOA in Acvr2–/–mice. This can be explained by species-specific differences or factors downstream of NO, critical for GnRH release, not being affected in Acvr2–/–mice, resulting in normal GnRH and consequently normal steroid levels. It will be important in the future to determine whether changes in local concentration of testosterone within the mPOA are critical for alterations in NOS activity that were observed in the Acvr2–/–mice.

Dopamine plays a major role in androgen/NOS1-dependent male sexual behavior. It has also been suggested that dopamine release but not synthesis is regulated by testosterone, which in turn affects nNOS activity in the mPOA [15–19, 21–23, 57, 62–64]. It is possible that perturbation of the ACVR2-signaling pathway could affect dopamine release from mPOA in a testosterone-independent process leading to the observed behavioral defects. Our preliminary studies suggest that at least the mPOA content of dopamine and its metabolites is unaffected in these male mutant mice (unpublished).

Other factors that are known to affect male sexual behavior include estrogen signaling and aromatase [40, 42]. Both aromatase and estrogen receptors are expressed in mPOA and mice lacking estrogen receptor {alpha} and aromatase demonstrate male reproductive behavioral defects [24, 40, 41, 65, 66]. Our data indicate that aromatase gene expression is not affected in mPOA of Acvr2–/–mice and their serum estrogen levels are comparable with those in control male mice. However, further immunolocalization studies are required to confirm whether there are changes in the distribution of estrogen receptor and aromatase-expressing neurons and whether there are changes in local aromatase activity within the mPOA of Acvr2–/–mice.

Abnormal penis development and morphology may cause defects in ejaculation [67–69] in Acvr2–/–mice. Histological analysis of external genitalia of Acvr2–/–male mice indicated normal architecture similar to that in Acvr2+/– mice, suggesting that the male sexual behavioral defects were not due to anatomical defects of the external genitalia. Furthermore, expression of the marker genes critical for penile differentiation is not affected in the mutant external genitalia. An important feature of penile morphology that might affect ejaculatory potential involves the ability of the penis to be fully retracted from the surrounding sheath and the presence of androgen-sensitive cutaneous spines on the glans penis. These structures may be stimulated in the course of intromission, thereby providing genital sensory inputs to the spinal cord, resulting in ejaculation [70, 71]. Although we have not quantified these spines, the similarity in serum testosterone levels between controls and mutants suggests that spine number in activin receptor II mutants may be comparable with that in the control mice. Neuronal NOS is also expressed peripherally in the male external genitalia at very low levels and alterations in penile endothelial but not neuronal NOS can cause ejaculatory defects in mice [45, 72]. It is not known whether some of the male sexual behavior defects in Acvr2–/–mice are due to changes locally in penile NOS activity.

In conclusion, our results suggest that activin receptor II signaling is one of the upstream pathways within the mPOA essential for male sexual behavior. Future studies will determine the signaling pathways involved in the activin-mediated regulation of NOS in the mPOA. Our future studies will also focus on identifying the factors within mPOA that are affected by reduced NOS levels, leading to male reproductive behavioral defects in the absence of ACVR2 signaling.

ACKNOWLEDGMENTS

We thank Ms. Shirley Baker for help with manuscript formatting and Mr. Julio Agno for help with genotyping of mice.

FOOTNOTES

1 Supported in part by The Moran Foundation (T.R.K.), Department of Pathology, Baylor College of Medicine (T.R.K.), National Institutes of Health grants MH57442 and MH63954 (S.K.M.), and HD32067 (M.M.M.) and the National Institutes of Health Specialized Cooperative Centers Program in Reproduction Research (HD07495). The Ligand and Hormone Assay Core at University of Charlottesville, Virginia is supported by an NIH grant (U-54-HD28–394) to the Center for Cellular and Molecular Studies in Reproduction. Back

2 Correspondence. FAX: 913 588 0455; tkumar{at}kumc.edu Back

Received: 12 May 2005.

First decision: 2 June 2005.

Accepted: 8 August 2005.

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