The roles of testicular nuclear receptor 4 (TR4) in male fertility-priapism and sexual behavior defects in TR4 knockout mice.

Abstract

Successful reproductive efforts require the establishment of a situation favorable for reproduction that requires integration of both behavior and internal physiological events. TR4 nuclear receptor is known to be involved in male fertility via controlling spermatogenesis, yet its roles in regulating other biological events related to reproduction have not been completely revealed.
Male TR4 knockout (TR4 -/-) and wild type mice were used for the sexual behavior and penile dysfunction studies. Mice were sacrificed for histological examination and corresponding genes profiles were analyzed by quantitative RT-PCR. Reporter gene assays were performed.
We describe an unexpected finding of priapism in TR4 -/- mice. As a transcriptional factor, we demonstrated that TR4 transcriptionally modulates a key enzyme regulating penis erection and neuronal nitric oxide synthese NOS (nNOS). Thereby, elimination of TR4 results in nNOS reduction in both mRNA and protein levels, consequently may lead to erectile dysfunction. In addition, male TR4 -/- mice display defects in sexual and social behavior, with increased fear or anxiety, as well as reduced mounting, intromission, and ejaculation. Reduction of ER alpha, ER beta, and oxytocin in the hypothalamus may contribute to defects in sexual behavior and stress response.
Together, these results provide in vivo evidence of important TR4 roles in penile physiology, as well as in male sexual behavior. In conjunction with previous finding, TR4 represents a key factor that controls male fertility via regulating behavior and internal physiological events.

Full text

Page 1

RESEARCHOpen Access
The roles of testicular nuclear receptor 4 (TR4) in
male fertility-priapism and sexual behavior
defects in TR4 knockout mice
Loretta L Collins1†, Yi-Fen Lee1†, Huei-Ju Ting1, Wen-Jye Lin1, Ning-Chun Liu1, Charles K Meshul2, Hideo Uno3,
Bo-Ying Bao1,4, Yen-Ta Chen1,5and Chawnshang Chang1,4*
Abstract
Background: Successful reproductive efforts require the establishment of a situation favorable for reproduction
that requires integration of both behavior and internal physiological events. TR4 nuclear receptor is known to be
involved in male fertility via controlling spermatogenesis, yet its roles in regulating other biological events related
to reproduction have not been completely revealed.
Methods: Male TR4 knockout (TR4-/-) and wild type mice were used for the sexual behavior and penile
dysfunction studies. Mice were sacrificed for histological examination and corresponding genes profiles were
analyzed by quantitative RT-PCR. Reporter gene assays were performed.
Results: We describe an unexpected finding of priapism in TR4-/- mice. As a transcriptional factor, we
demonstrated that TR4 transcriptionally modulates a key enzyme regulating penis erection and neuronal nitric
oxide synthese NOS (nNOS). Thereby, elimination of TR4 results in nNOS reduction in both mRNA and protein
levels, consequently may lead to erectile dysfunction. In addition, male TR4-/- mice display defects in sexual and
social behavior, with increased fear or anxiety, as well as reduced mounting, intromission, and ejaculation.
Reduction of ER alpha, ER beta, and oxytocin in the hypothalamus may contribute to defects in sexual behavior
and stress response.
Conclusions: Together, these results provide in vivo evidence of important TR4 roles in penile physiology, as well
as in male sexual behavior. In conjunction with previous finding, TR4 represents a key factor that controls male
fertility via regulating behavior and internal physiological events.
Keywords: TR4, priapism, sexual behavior
Background
Members of the nuclear receptor superfamily are known
to play important roles in differentiation, development,
homeostasis, and metabolism, as well as in disease
development and progression [1]. As a member of the
nuclear receptor superfamily with several known regula-
tory targets [2], Testicular nuclear receptor 4 (TR4) may
affect many signaling pathways and thus have a major
impact on physiological functions [2]. Creation of
mammalian gene knockout models has become a suc-
cessful strategy to study the physiological roles of
orphan receptors in vivo [3,4]. As a nuclear receptor
with wide tissue distribution [5-10] and a relative mys-
tery with respect to physiological function, TR4 was a
good candidate for analysis using a knockout mouse
model. A complex set of phenotypic abnormalities were
found to exist in the TR4 knockout (TR4-/-) mouse,
including significant growth retardation [11], defects in
female reproductive function [12] and maternal behavior
[11], impaired cerebella function [13,14], and reduced
myelination [15]. In addition, recent studies found that
TR4 might be a master regulator controlling glucose
* Correspondence: chang@urmc.rochester.edu
† Contributed equally
1George Whipple Lab for Cancer Research, Departments of Pathology,
Urology, Radiation Oncology, and The Cancer Center, University of Rochester
Medical Center, Rochester, NY 14642, USA
Full list of author information is available at the end of the article
Collins et al. Reproductive Biology and Endocrinology 2011, 9:138
http://www.rbej.com/content/9/1/138
© 2011 Collins et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Page 2

and lipid metabolism [16-18] as well as foam cell forma-
tion [19].
Our previous study revealed that TR4 expresses pre-
dominantly in the testis in a stage-dependent manner
and deletion of TR4 in male mice reduced sperm pro-
duction and disrupted spermatogenesis, thereby
impaired male fertility [20]. Successful reproductive
efforts require the establishment of a situation favorable
for reproduction that requires integration of both beha-
vior and internal physiological events. Following con-
tinue studies of TR4’s roles in male reproduction, we
describe an unexpected finding of priapism in some
TR4-/-mice. Penile priapism, or persistent erection, is
characterized by trapped blood within the corpus caver-
nosa, which leads to reduced tissue oxygenation,
increased blood viscosity, disruption of tissue elasticity,
fibrosis, and finally irreversible failure to achive erection
[21,22]. It is a significant urologic emergency that can
lead to irreversible loss of erectile function if not
promptly resolved. It has been linked to sickle cell dis-
ease as well as the use of vasoactive drugs, yet its mole-
cular mechanism remains unclear. The etiology of this
disorder is obscure. The cyclic nucleotide second mes-
senger cGMP generated by activated guanylyl cyclase in
penile smooth muscle cells regulates penile erection.
These includes activation of guanylyl cyclase requires
nitric oxide (NO), release in the penis upon sexual sti-
mulation from neuronal and endothelial sources con-
taining NO synthase (NOS), and respectively termed
nNOS and eNOS. The crucial role of NOS in mediating
erectile function was further supported by the finding
that the mice lacking the gene for eNOS, nNOS, or
both have a tendency for priapic activity [23]. To our
surprise, in the mice with TR4 knockout [11], penile
priapism was one of the most striking phenotypes
observed.
In characterizing the fertility defects, in addition to
partial penile priapism among TR4-/-males, we also
found that TR4-/-mice displayed delayed sexual matur-
ity, reduced sperm production [20], priapism, and
abnormal sexual behavior, all of which contribute to sig-
nificant reduction of TR4-/-male fertility. Furthermore,
the expressions of nNOS, enzyme involved in produc-
tion of the erectile mediator NO, were reduced in penis
tissue from TR4-knockout mice, where a transcriptional
regulation of nNOS by TR4 was also presented.
Methods
Experimental animals and genotyping
TR4-/-mice were produced as described [11], and
housed in the vivarium facility of the University of
Rochester Medical Center. The animals were provided a
standard diet with constant access to food and water,
and exposed to a 12-hour light/dark cycle. All
experimental protocols were approved by the University
Committee on Animal Resources and the office of
EnvironmentalHealth
implementation.
Genotyping was carried out as described [11]. Briefly,
genomic DNA was isolated from mouse tail samples and
used as template for polymerase chain reaction (PCR). Pri-
mers for amplification of the wildtype and targeted alleles
are TR4-107 (wildtype, forward): 5’-GGAGACACACTG-
CACATGTTCGAATAC-3’, TR4-111 (wildtype, reverse):
5’-CACAGCTCATTTCTCTGCTCACTTACTC-3’, Neo-
3a (targeted allele, forward): 5’-GCAGCGCATCGCCTTC-
TATC-3’, and TR4-34 (targeted allele, reverse): 5’-
TGCAAGCATACTTCTTGTTCC-3’.
and Safetypriorto
Tissue preparation, histology, and immunostaining
Mice were anesthetized with an overdose of pentobarbi-
tal and perfused through the left ventricle with 20 ml
saline (pH 7.3), followed by 20 ml of 4% paraformalde-
hyde or 10% neutral buffered formalin. Tissues were
removed and post-fixed by submersion in 10% formalin.
Alternatively, fresh tissues were fixed by direct submer-
sion, in 10% neutral buffered formalin, prior to proces-
sing. Tissues were processed for embedding in paraffin
using an RHS Tissue Processing System (Hacker Instru-
ments & Industries), or processed manually. Penis tis-
sues from 4.5-7.5 mo. old mice were cut in 5 μm
sections, deparaffinized, and stained with Accustain
Masson Trichrome (Sigma-Aldrich) reagents, according
to the manufacturer’s instructions. Additionally, penis
sections were stained using a polyclonal antibody against
nNOS (BD Transduction Laboratories) at a 1:200 dilu-
tion, a polyclonal antibody against S100 (DAKO) at a
1:500 dilution, or a polyclonal antibody against TR4
(Santa Cruz) at a dilution of 1:200, followed by the
appropriate biotinylated secondary antibody (1:1000
dilution), VECTASTAIN Elite ABC reagent (Vector
Laboratories) and DAB substrate (Vector Laboratories).
Hematoxylin was used as a nuclear counterstain follow-
ing immunostaining.
RT-PCR and Western Blot analysis of gene expression
For RT-PCR analysis, total RNA was isolated from penis
tissue using TRIzol®Reagent (Invitrogen). First strand
cDNA synthesis was achieved using the Superscript™II
RNase H Reverse Transcriptase kit (Invitrogen).
Real time quantitative RT-PCR was carried out, using
SYBR Green PCR MasterMix, on the iCycler iQ™ PCR
cycler and detection system (Bio-Rad Laboratories).
Analysis of data obtained and calculation of relative
gene expression was performed using the 2-ΔΔCT
method [24]. For each gene analyzed, the expression
level of the TR4+/+was set at 1 and relative expression
in TR4-/-samples was calculated.
Collins et al. Reproductive Biology and Endocrinology 2011, 9:138
http://www.rbej.com/content/9/1/138
Page 2 of 9

Page 3

For Western blot analysis, total protein was isolated
from penis tissue using TRIzol®Reagent (Invitrogen). A
total of 75 μg protein per sample was separated on
either a 6% or a 10% SDS-PAGE gel. After transfer to
PVDF membrane, the blots were probed with either a
polyclonal antibody against nNOS (BD Transduction
Laboratories) at a 1:1000 dilution, or an antibody against
b-actin, followed by the appropriate alkaline phospha-
tase-conjugated secondary antibody at a 1:2000 dilution.
Signals were detected using a chromogenic substrate.
Transient transfection/reporter gene assay
The mouse nNOS promoter plasmid pEx2 [25] was pro-
vided by Dr. Ted Dawson (Johns Hopkins University).
CV1 cells were grown in Dulbecco’s Modified Eagle’s
Medium (DMEM) with 10% fetal bovine serum, at 37°C,
and with 5% CO2. Cells were cultured in 24-well plates
(Corning) for 24 h. Transient transfection was carried
out as described previously [26]. The ß-gal activity in
whole cell lysate was measured with a luminometer
using a chemiluminescence-based detection system
(Reaction Buffer, Galacto-Star, TROPIX) according to
the manufacturer’s protocol.
Electrophoretic mobility shift assay (EMSA)
TR4 proteins were synthesized in rabbit reticulocyte
lysate (Promega), according to the manufacturer’s
instructions. The oligonucleotide probe (nNOS-NHR, bp
-192 to -211, 5’-CTGGTCAACCTTGACTTCCT-3’) was
end-labeled with [g]32P in a T4 polynucleotide kinase
reaction (New England Biolabs). The EMSA assay was
performed as described previously [26].
Continuous mating and male sexual behavior analysis
For the continuous mating study, 5 TR4+/+and 5 TR4-/-
males at 5 months of age were each paired with an adult
TR4+/+female for 4 months. The number of litters pro-
duced and the number of pups per litter were recorded. For
male sexual behavior analysis, ovariectomized female mice
of strain ICR, at 8-9 weeks of age, were injected with 12 μg
estradiol valerate one day before mating and with 500 μg
progesterone 4-7 hours before mating. At the time of mat-
ing, one primed female was paired with a 7-8 month-old
TR4+/+or TR4-/-mouse for either 90 min or 6 h, beginning
between 18:00-22:00. Mating behavior was videotaped and
subsequently scored for latency to, and number of mounts,
intromissions, and ejaculations [27,28]. After the 6 h taped
mating trial, experimental pairs remained together until the
following morning. Female mice were then visually exam-
ined for the presence of a vaginal plug.
Statistics
For sexual behavior data, means and standard deviations
for behavior latencies and mean number of behavior
displays, with associated ranges, are shown. Extremely
high variation in both latencies and numbers of specific
behavioral displays was observed both within and
between groups. Differences in numbers of mice of a
particular genotype displaying a particular behavior were
analyzed by one-tailed, independent sample t-tests,
assuming equal variances, after coding for the presence
(scored as 1) or absence (scored as 0) of the behavior.
Results
TR4-/-males exhibit penile priapism
A physical defect observed among TR4-/-male mice,
which may affect sexual function, is partial priapism, or
persistent, partial penile erection. This condition is
observed in a substantial number, but not all of the
TR4-/-males, and increases in prevalence with age (Fig-
ure 1A and 1B). Once priapism was observed in a
mouse, normal penile resheathing was not subsequently
observed, suggesting persistence of priapism thereafter.
As shown in Figure. 2, we observed more densely
packed collagenous and elastic fibers in the corpus
cavernosa from TR4-/-mice with priapism, resulting in
smaller cavernous sinuses, compared to TR4+/+or
TR4-/-without priapism (Figure 2Cvs 2A or 2B). Our
previous studies on characterization of TR4-/-mice
found that heterozygote mice (TR4+/-) are indistinguish-
able with TR4+/+mice [11], and this remains true in
this current study that TR4+/-, like TR4+/+, have no
obvious defects. Therefore, we used TR4+/+as a control
group in following studies. TR4 Comparing histological
sections of penis tissue from a TR4+/+mouse (no priap-
ism), a TR4-/-mouse without priapism, and a TR4-/-
mouse with priapism (Figure 2D-L), we found more red
blood cells within the corpus cavernosa (CC), and cor-
pus spongiosum(CS) (H & I vs G) and dorsal vein (DV)
(K & L vs J) in TR4-/-tissue than in TR4+/+tissue.
Regulation of nNOS transcriptional activation by TR4
To uncover TR4 roles in controlling penile activity, TR4
expression profiles were determined by the immunos-
taining of cross sectioned penis tissue. We observed
TR4 expression in smooth muscle surrounding the
penile vasculature (Additional File 1 Figure S1), a region
where nNOS is also expressed [29]. A neuronal marker,
S100, was used to confirm the dorsal nerve bundles in
all sections. The penile erection requires NO released in
the penis upon sexual stimulation. The synthetic enzyme
for production of NO, a signaling molecule that is
known to play a role in regulation of penile erection
[30,31], was assayed in penis tissue. Through immunos-
taining for nNOS, we confirmed the colocalization of
TR4 and nNOS in smooth muscle surrounding the
penile vasculature, but also found significant variation in
expression of nNOS, even within the same tissue
Collins et al. Reproductive Biology and Endocrinology 2011, 9:138
http://www.rbej.com/content/9/1/138
Page 3 of 9

Page 4

(Additional File 1 Figure S1). To further quantify nNOS
expression, quantitative real time RT-PCR with RNA
from 7-month-old male TR4-/-mice was used, and
nNOS was found to be reduced by more than 65% rela-
tive to TR4+/+levels (Figure 3A). In addition to nNOS,
we also found a significant reduction of mRNA expres-
sion of eNOS gene, another important NOS in regulat-
ing erection in TR4-/-(data not shown). Western blot
analysis showed a clear reduction in the amount of
nNOS protein present in TR4-/-mouse penis tissue (Fig-
ure 3B). As a transcriptional factor, TR4 often regulates
gene expression via binding to TR4 responsive element
(TR4RE). Via sequence searching, a putative TR4RE was
found in nNOS promoter. A reporter assay demon-
strated that transfection of TR4 could enhance nNOS
reporter gene expression (Figure 3C). The nNOS exon 2
contains a nuclear hormone receptor response element
(NHR) through which the nuclear receptor SF-1 was
found to bind and modulate nNOS transcription [25].
Using the EMSA assay, we demonstrated that TR4 pro-
tein can bind to the mouse nNOS NHR sequence (Fig-
ure 3D lane 2, TR4/nNOS-NHR), and this specific TR4-
NHR complex can be super-shifted by addition of a
TR4-specific antibody (lane 3, TR4/nNOS-NHR/Ab). In
summary, we identified TR4 as a key modulator to con-
trol the penile function via a transcriptional regulation
of nNOS expression, and loss of TR4 resulted in
reduced nNOS expression that might contribute to
penile priapism. TR4 represents a novel regulator that
modulates nNOS gene expression to regulate penile
function.
Reduced fertility and aberrant sexual behavior in male
TR4-/-mice
Five adult TR4+/+males were mated with sexually
mature female mice, producing a total of 22 litters. An
average of 4.4 litters were produced per TR4+/+mouse
in 4 months (Table 1). In contrast, only one of the 5
TR4-/-males produced offspring, with a total of two lit-
ters born during the 4 month period (Table 1). Not only
were significantly fewer TR4-/-males able to produce
offspring, the number of litters generated by the known
fertile TR4-/-male was approximately half that of the
TR4+/+average (Table 1).
The initial male sexual behavior analysis that was car-
ried out (Table 2 Trial 1) demonstrated defects in
TR4-/-sexual behavior, as well as social behavior. It was
found that naïve TR4-/-mice do not show defined sex-
ual behavior within 90 minutes of the initial pairing
with a primed female mouse. Out of 9 TR4+/+mice
tested for sexual behavior, all 9 showed mounting beha-
vior, 5 displayed intromission, and two achieved ejacula-
tion, whereas none of the same behaviors were observed
among the 11 TR4-/-mice tested (Table 2). Interestingly,
the TR4-/-mice displayed fear or anxiety in the presence
of a hormonally primed female, with a delay in showing
interaction with the potential mate. In the most severe
cases, the TR4-/-male spent the entire test session
actively avoiding his cage mate.
Given extended pairing time, up to 6 h (Trial 2),
TR4-/-mice did begin to display sexual behavior. Again,
proportionally fewer TR4-/-than TR4+/+mice displayed
each defined sexual behavior, with increased latencies to






Age
Total
TR4-/-
TR4-/- with
priapism
% with
priapism
6-12 wks 276 22.2
13-24 wks 197 36.8
7-12 mo.17 1164.7
A
TR4+/+ TR4-/-
B
Figure 1 TR4-/-males display priapism. A. TR4+/+male (left), and TR4-/-male (right) showing priapism; both mice were 7 mo. old. B. Number
and percentage of TR4-/-mice showing priapism at various ages.
Collins et al. Reproductive Biology and Endocrinology 2011, 9:138
http://www.rbej.com/content/9/1/138
Page 4 of 9

Page 5

each behavior in TR4-/-males (Table 2). After the video-
taped 6 h pairing, male and female mice were left
paired, and female mice were examined for vaginal
plugs the following morning. Vaginal plugs were discov-
ered in each of the female mice paired with TR4+/+
males, whereas none of the females paired with TR4-/-
males had plugs (Table 2).
Expression levels of several genes known to affect
behavior, particularly sexual behavior, were determined
via semi-quantitative RT-PCR and quantitative real time




A
TR4+/+
TR4-/- without
priapism
C
B
TR4-/- with
priapism
D
E F
J
G
I
H
L K
DA
DV
DA
DV
DV
DA
DV
DV
DV
U
CS
U
U
CC
CC
CC
CS
CS
N
N
N
CS
CS
CS
U U
U
Figure 2 Histological staining of penis sections from adult TR4+/+mice, and TR4-/-mice both with and without priapism. Penile cross
sections are compared after Accustain Masson Trichrome (Sigma-Aldrich) staining. Each of panels G-I show a region of the corpus cavernosum
from the sample indicated. DV, dorsal vein; DA, dorsal artery; CC, corpus cavernosum; CS, corpus spongiosum; N, nerve bundle; U, urethra. Scale is
the same for all images in each row. For images A-C, scale bar = 250 μm; D-F, scale bar = 50 μm; G-I, scale bar = 50 μm; J-L, scale bar = 50 μm.
Collins et al. Reproductive Biology and Endocrinology 2011, 9:138
http://www.rbej.com/content/9/1/138
Page 5 of 9

Page 6

PCR, using hypothalamic tissue from TR4-/-and TR4+/+
mice. RT-PCR results demonstrate that hypothalamic
expression of estrogen receptor (ER) alpha (a), ER beta
(b), and oxytocin is reduced in TR4-/-mice (Additional
File 2 Figure S2). No differences in levels of the andro-
gen receptor (AR) or vasopressin (VP) were apparent.
After quantitative real time PCR analysis, it was clear
that the levels of ERa, ERb, and oxytocin were reduced
by approximately 50-60% in the hypothalamic region of
TR4-/-mice compared to TR4+/+(Additional File 2 Fig-
ure S2). More detailed mechanism dissection as to how
TR4 regulates the expression of those genes involved in
the sexual behavior and consequently affects reproduc-
tive function need further investigation.
Discussion
In addition to the inability of the TR4-/-mice with
priapism to retract the penis back within the sheath
(Figure 1A), histological evidence of priapism was
observed (Figure 2). Trapped blood within the corpus
cavernosum of the penis leads to pathology resulting
from reduced tissue oxygenation and, without treatment,
could result in permanent erectile dysfunction
[21,22,32]. Compared with TR4+/+mice, both TR4-/-





nNOS
0
0.2
0.4
0.6
0.8
1
1.2
Relative Gene Expression







B


A

nNOS
nNOSnNOS
β β β β-actin
β β β β-actin
TR4+/+TR4-/-
TR4+/+TR4-/-
?actin
TR4+/+ TR4-/-




TR4+/+ TR4-/-
nNOS
?actin

0.2 0.4 0.6 0.8
(?g)
D


TR4 -
+ +
anti-TR4 antibody
- -
+
TR4/nNOS-NHR
TR4/nNOS-NHR/Ab
n.s
free probe
mouse nNOS CTGGT CAACCTTGA CTTCCT
-211 -192
NHR
Lane 1 2 3
C
Figure 3 Expression of nNOS is reduced in the TR4-/-penis, and TR4 binds and activates the nNOS promoter. A. Qualitative RT-PCR
(upper panel) and real time RT-PCR quantitation (lower panel) of nNOS gene expression in the penis. Expression data were obtained, in
triplicate, from each of two TR4+/+and two TR4-/-mice. b-actin levels were determined as a control for template amount in PCR reactions. B.
Western blot analysis of penis samples from TR4+/+and TR4-/-mice (with priapism) demonstrates reduced nNOS protein expression in TR4-/-
tissue. Samples from each of 3 TR4+/+and 3 TR4-/-mice were analyzed, and a representative blot is shown. C. Transactivation assay for TR4
regulation of nNOS expression. TR4 can activate the nNOS exon 2 promoter and control basal nNOS transcription. pEx2 (0.2 μg) and increasing
amounts of TR4 were cotransfected into CV-1 cells. Promoter activity of each sample was normalized to the average activity of the pEx2
promoter when transfected alone. Results are the mean ± SEM from at least three experiments performed in duplicate. ***P < 0.01 d. By EMSA
assay, TR4 is shown to bind to the nNOS-NHR probe (TR4/nNOS-NHR). The presence of TR4 in the complex was confirmed by supershift (TR4/
nNOS-NHR/Ab) using a TR4-specific antibody. n.s., nonspecific binding.
Table 1 Fertility rates of 5 month old TR4+/+and TR4-/-
males
TR4 genotypeLitters
totalper male
Offspring
total per litter
+/+ (N = 5)
-/- (N = 5)
22
2
4.4
NA
168
13
7.6
6.5
Continuous mating with TR4+/+females for 4 month NA, not applicable since
only one male produced offspring in the TR4-/-group.
Collins et al. Reproductive Biology and Endocrinology 2011, 9:138
http://www.rbej.com/content/9/1/138
Page 6 of 9

Page 7

animals with and without priapism have increased pre-
sence of red blood cells observed in penile cross section,
with the largest amount present in sections of penis tis-
sue from TR4-/-mice with priapism (Figure 2L). Histol-
ogy also revealed more densely packed tissue in the
TR4-/-mice with priapism, compared with TR4+/+mice
and TR4-/-mice without priapism, suggesting loss of
elasticity of the normally flexible penile structures (e.g.
corpus cavernosum, corpus spongiosum) (Figure 2C).
The neurotransmitter NO is a known mediator of
erectile function that promotes vasodilation and the
inflow of blood, resulting in penile tumescence [33].
nNOS is an enzyme responsible for production of NO
in the penis [34], and expression of both nNOS mRNA
and protein were reduced in the TR4-/-mouse penis
(Figure 3A and 3B). It was also found that TR4 is able
to bind the NHR of exon 2 (Figure 3D), as well as
upregulate nNOS gene expression in a reporter gene
assay (Figure 3C). Immunostaining of penis tissue for
TR4 and nNOS resulted in localization of both pro-
teins in smooth muscle surrounding penile vasculature,
further implicating TR4 in the regulation of vascular
function via nNOS. As reported by Champion that
NOS mutant mice: eNOS-/-and eNOS-/-, nNOS-/-dis-
played priapism where the expression of phosphodies-
terase type 5 expression (PDE5A) was reduced, and
transfection of eNOS normalized PDE5A expression
and corrected priapic activity and there is less impact
on loss of nNOS [23]. Therefore, in addition to nNOS,
we also checked the expression of eNOS and PED5A
in TR4-/-penis, and found no significant difference
from TR4+/+(data not shown). These results suggest
that erectile function may be negatively affected by the
loss of TR4 function in TR4-/-mice, with or without
priapism.
In addition to the possible loss of erectile function in
TR4-/-mice via reduced nNOS expression and/or result-
ing from the development of priapism, as well as the
detrimental effects of loss of TR4 on male germ cell
development [20], abnormal sexual behavior is a signifi-
cant contributor to the reduced fertility of male TR4-/-
mice. For each sexual behavior demonstrated by TR4-/-
mice, the latency to behavioral display was increased
compared to TR4+/+(Table 2). These results indicate
that TR4-/-mice still retain sexual motivation, yet take
longer to become acclimated to the pairing situation
and begin showing sexual behaviors. Furthermore,
TR4-/-males are less successful in achieving ejaculation.
In fact, a large proportion of TR4-/-males tested for 6 h
did not even achieve intromission. From observation of
the TR4-/-mice during the mating trials, it became evi-
dent that they had difficulty in maintaining the appro-
priate mounting position through which intromission
and ejaculation could be achieved.
TR4 ubiquitously expresses in almost all the tissues we
examined, with high abundance in testis, and that leads
to our first discovery of TR4 roles in male fertility via
controlling spermatogenesis [20]. This current study
further extends TR4’s role as a master regulator in male
fertility via controlling both sexual/social behavior and
internal physiological events. This discovery points to
the development of novel strategies for male fertility
therapy that specifically target TR4. We have identified
that polyunsaturated fatty acid could potentiate TR4
activity to control the lipid metabolism [19], and it will
be of great interest to identify TR4 upstream modulators
Table 2 Male sexual behavior
BehaviorTRIAL, 90 minTRIAL, 6h
TR4+/+(N = 9)TR4-/-(N = 11)TR4+/+(N = 4)TR4-/-(N = 6)
Mounts
No. of mice
Mean no. of mounts
Range
Latency to behavior++
Intromissions
No. of mice
Mean no. of mounts
Range
Latency to behavior++
Ejaculation
No. of mice
Latency to behavior++
Plug Present
9/9+
0/11*
0
4/4+
5/6
33.8
0-583-7011-24
21.5 ± 20.0136.8 ± 111.1*
5/9 0/11*
0
4/4
14
11-17
2/6
7.2
0-340-98
44.3 ± 32.1 89.8 ± 89.5
2/90/11* 4/41/6*
91
0/6
71.2 ± 58.6
4/4
+Number of mice showing behavior/number of mice tested;++Mean ± standard deviation among animals showing behavior; Latencies are reported in minutes;
*P < 0.05
Collins et al. Reproductive Biology and Endocrinology 2011, 9:138
http://www.rbej.com/content/9/1/138
Page 7 of 9

Page 8

that can promote TR4 activity in male reproductive
organs.
Conclusions
Collectively, the data reported here describe severe
reproductive malfunctions in TR4-/-male mice, with sig-
nificant effects stemming from abnormal sexual beha-
vior, as TR4-/-mice rarely achieve intromission or
ejaculation. In TR4-/-mice with priapism, erectile func-
tion may eventually be lost due to loss of collagenous
tissue elasticity in the corpora cavernosa and corpus
spongiosum, and account for the lack of intromission or
ejaculation. In TR4-/-males with or without priapism,
disruptions of signaling pathways involving nNOS (Fig-
ures 3 and Additional File 1 Figure S1) may also affect
erectile function. These in vivo data demonstrate that
TR4 plays an important part in various aspects of repro-
duction and behavior, and future studies to more finely
dissect the roles of TR4 in penile physiology and in
brain/behavioral function may lead to identification of
additional TR4 target genes, greater knowledge of the
involvement of the receptor in various signaling path-
ways, or the discovery of new physiological ligand for
TR4.
Additional material
Additional file 1: Supplemental Figure S1. Localization of both TR4
and nNOS to vascular smooth muscle in the mouse penis. Penis sections
from adult TR4+/+and TR4-/-(both with and without priapism) mice were
stained for neuronal nitric oxide synthase (nNOS), the neuronal marker
S100, or TR4 protein, as indicated. Immunoreactivity for both nNOS
(upper panels) and TR4 (lower panels) was found in the smooth muscle
surrounding veins (DV, dorsal vein) and arteries (DA, dorsal artery), as
well as venules (V) and arterioles (A), of the penis. TR4 immunoreactivity
is shown in an arteriole of the corpus cavernosum of a TR4+/+mouse,
whereas no immunoreactivity was observed in the same structures from
TR4-/-mice. S100 immunoreactivity was observed in dorsal nerve bundles
in all sections probed for the protein. CC, corpus cavernosum; CS, corpus
spongiosum.
Additional file 2: Supplemental Figure S2. RT-PCR and real time PCR
analysis of sexual behavior/function-related gene expressions in the
hypothalamus. A. Qualitative RT-PCR analysis of AR, ERa, ERb, VP, and OT
mRNA expression. b-actin levels were determined as a control for
template amount in PCR reactions. B. Real time PCR quantitation of
those genes expressing in the hypothalami of TR4+/+and TR4-/-mice.
Relative gene expression levels are shown. For both RT-PCR and real-time
RT-PCR analyses, triplicate data from each of three TR4+/+and three
TR4-/-mice were obtained.
Abbreviations
TR4: testicular orphan nuclear receptor 4; NOS: nitric oxide synthase; RT-PCR:
reverse transcription-polymerase chain reaction; NHR: nuclear hormone
receptor response element.
Acknowledgements
This work was supported by NIH (DK063212 and CA127548)
Author details
1George Whipple Lab for Cancer Research, Departments of Pathology,
Urology, Radiation Oncology, and The Cancer Center, University of Rochester
Medical Center, Rochester, NY 14642, USA.2Research Services, V.A. Medical
Center and Department of Behavioral Neuroscience, Oregon Health &
Science University, Portland, OR 97239, USA.3Wisconsin Regional Primate
Research Center, University of Wisconsin, Madison, WI 53708, USA.4Sex
Hormone Research Center and School of Pharmacy, China Medical School,
China.5Department of Urology, Chang Gung University, Kaohsiung 833,
Taiwan.
Authors’ contributions
LC, YFL carried out the TR4-/-mice sexual behavior experiments and
characterized penile priapsim phenotype and wrote the manuscript. HJT and
BYB carried out eNOS and nNOS expression in gene and protein levels, WJL
and YTC participated in sexual behavior experiments. NCL carried out nNOS
reporter gene assay. CKM, HU and CC participated in its design and
coordination and helped to draft the manuscript. All authors read and
approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 27 June 2011 Accepted: 13 October 2011
Published: 13 October 2011
References
1.Evans RM: The steroid and thyroid hormone receptor superfamily. Science
1988, 240:889-895.
2.Lee YF, Lee HJ, Chang C: Recent advances in the TR2 and TR4 orphan
receptors of the nuclear receptor superfamily. Journal of Steroid
Biochemistry & Molecular Biology 2002, 81:291-308.
3.Qiu Y, Pereira FA, DeMayo FJ, Lydon JP, Tsai SY, Tsai MJ: Null mutation of
mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal
ganglion, axonal projection, and arborization. Genes Dev 1997,
11:1925-1937.
4.Shyr C, Collins LL, Mu X, Platt KA, Chang C: Spermatogenesis and testis
development are normal in mice lacking testicular orphan nuclear
receptor 2. Mol Cell Biol 2002, 22:4661-4666.
5.Young WJ, Smith SM, Chang C: Induction of the intronic enhancer of the
human ciliary neurotrophic factor receptor (CNTFRa) gene by the TR4
orphan receptor. J Biol Chem 1997, 272:3109-3116.
6.Young WJ, Lee YF, Smith SM, Chang C: A bidirectional regulation between
the TR2/TR4 orphan receptors (TR2/TR4) and the ciliary neurotrophic
factor (CNTF) signaling pathway. J Biol Chem 1998, 273:20877-20885.
7.Lee YF, Young WJ, Burbach JPH, Chang C: Negative feedback control of
the retinoid-retinoic acid/retinoid × receptor pathway by the human
TR4 orphan receptor, a member of the steroid receptor superfamily. J
Biol Chem 1998, 273:13437-13443.
8.Lee CH, Chinpaisal C, Wei LN: A novel nuclear receptor
heterodimerization pathway mediated by orphan receptors TR2 and
TR4. Journal of Biological Chemistry 1998, 273:25209-25215.
9.Chang C, Da Silva SL, Ideta R, Lee YF, Yeh S, Burbach JPH: Human and rat
TR4 orphan receptors specify a subclass of the steroid receptor
superfamily. Proceedings of the National Academy of Sciences USA 1994,
1994:6040-6044.
10.van Schaick HSA, Rosmalen JGM, Lopes da Silva S, Chang C, Burbach JPH:
Expression of the orphan receptor TR4 during brain development of the
rat. Molecular Brain Research 2000, 77:104-110.
11.Collins LL, Lee YF, Heinlein CA, Liu NC, Chen YT, Shyr CR, Meshul CK,
Uno H, Platt KA, Chang C: Growth retardation and abnormal maternal
behavior in mice lacking testicular orphan nuclear receptor 4. Proc Natl
Acad Sci USA 2004, 101:15058-15063.
12.Chen LM, Wang RS, Lee YF, Liu NC, Chang YJ, Wu CC, Xie S, Hung YC,
Chang C: Subfertility with defective folliculogenesis in female mice
lacking testicular orphan nuclear receptor 4. Mol Endocrinol 2008,
22:858-867.
13.Chen YT, Collins LL, Uno H, Chang C: Deficits in motor coordination with
aberrant cerebellar development in mice lacking testicular orphan
nuclear receptor 4. Molecular & Cellular Biology 2005, 25:2722-2732.
Collins et al. Reproductive Biology and Endocrinology 2011, 9:138
http://www.rbej.com/content/9/1/138
Page 8 of 9

Page 9

14. Chen YT, Collins LL, Chang SS, Chang C: The roles of testicular orphan
nuclear receptor 4 (TR4) in cerebellar development. Cerebellum 2008,
7:9-17.
Zhang Y, Chen YT, Xie S, Wang L, Lee YF, Chang SS, Chang C: Loss of
testicular orphan receptor 4 impairs normal myelination in mouse
forebrain. Mol Endocrinol 2007, 21:908-920.
Liu NC, Lin WJ, Kim E, Collins LL, Lin HY, Yu IC, Sparks JD, Chen LM, Lee YF,
Chang C: Loss of TR4 orphan nuclear receptor reduces
phosphoenolpyruvate carboxykinase-mediated gluconeogenesis.
Diabetes 2007, 56:2901-2909.
Kim E, Liu NC, Yu IC, Lin HY, Lee YF, Sparks JD, Chen LM, Chang C:
Metformin inhibits nuclear receptor TR4-mediated hepatic stearoyl-CoA
desaturase 1 gene expression with altered insulin sensitivity. Diabetes
60:1493-1503.
Kang HS, Okamoto K, Kim YS, Takeda Y, Bortner CD, Dang H, Wada T,
Xie W, Yang XP, Liao G, Jetten AM: Nuclear orphan receptor TAK1/TR4-
deficient mice are protected against obesity-linked inflammation,
hepatic steatosis, and insulin resistance. Diabetes 60:177-188.
Xie S, Lee YF, Kim E, Chen LM, Ni J, Fang LY, Liu S, Lin SJ, Abe J, Berk B,
et al: TR4 nuclear receptor functions as a fatty acid sensor to modulate
CD36 expression and foam cell formation. Proceedings of the National
Academy of Sciences of the United States of America 2009, 106:13353-13358.
Mu X, Lee Y, Liu N, Chen Y, Kim E, Shyr C, Chang C: Targeted inactivation
of Testicular nuclear Orphan Receptor 4 delays and disrupts late meiotic
prophase and subsequent meiotic divisions of spermatogenesis. Mol Cell
Biochem 2004, 24:5887-5899.
Hauri D, Spycher M, Bruhlmann W: Erection and priapism: A new
physiopathological concept. Urol Int 1983, 88:138-145.
Panteleo-Gandais M, Chalbaud R, Chacon O, Plaza N: Priapism: evaluation
and treatment. Urology 1984, 24:345-346.
Champion HC, Bivalacqua TJ, Takimoto E, Kass DA, Burnett AL:
Phosphodiesterase-5A dysregulation in penile erectile tissue is a
mechanism of priapism. Proceedings of the National Academy of Sciences of
the United States of America 2005, 102:1661-1666.
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using
real-time quantitative PCR and the 2-DDCTmethod. Methods 2001,
25:402-408.
Wei X, Sasaki M, Huang H, Dawson VL, Dawson TM: The orphan nuclear
receptor, Steroidogenic Factor 1, regulates neuronal Nitric Oxide
Synthase gene expression in pituitary gonadotropes. Mol Endocrinol 2002,
16:2828-2839.
Lee YF, Pan HJ, Burbach PH, Morkin E, Chang C: Identification of direct
repeat 4 as a positive regulatory element for the human TR4 orphan
receptor: a modulator for the thyroid hormone target genes. J Biol Chem
1997, 272:12215-12220.
Kriegsfeld LJ, Demas GE, Huang PL, Burnett AL, Nelson RJ: Ejaculatory
abnormalities in mice lacking the gene for endothelial nitric oxide
synthase (eNOS-/-). Physiol Behav 1999, 67:561-566.
Ogawa S, Chan J, Chester AE, Gustafsson J, Korach KS, Pfaff DW: Survival of
reproductive behaviors in estrogen receptor b gene-deficient (bERKO)
male and female mice. Proc Natl Acad Sci USA 1999, 96:12887-12892.
Segal SS, Brett SE, Sessa WC: Codistribution of NOS and caveolin
throughout peripheral vasculature and skeletal muscle of hamsters. Am
J Physiol 1999, 277:H1167-H1177.
Rampin O, Guiliano F: Brain control of penile erection. World J Urol 2001,
19:1-8.
Steers WD: Neural pathways and central sites involved in penile erection:
neuroanatomy and clinical implications. Neurosci Biobehav Rev 2000,
24:507-516.
Winter CC: Priapism. Urological Surgery 1978, 28:163.
Burnett AL: Nitric oxide in the penis: physiology and pathology. J Urol
1995, 157:320-324.
Yun HY, Dawson VL, Dawson TM: Neurobiology of nitric oxide. Crit Rev
Neurobiol 1996, 10:291-316.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
doi:10.1186/1477-7827-9-138
Cite this article as: Collins et al.: The roles of testicular nuclear receptor
4 (TR4) in male fertility-priapism and sexual behavior defects in TR4
knockout mice. Reproductive Biology and Endocrinology 2011 9:138.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Collins et al. Reproductive Biology and Endocrinology 2011, 9:138
http://www.rbej.com/content/9/1/138
Page 9 of 9