ORIGINAL
RESEARCH
Cryptorchidism in Mice with an Androgen Receptor
Ablation in Gubernaculum Testis
Elena M. Kaftanovskaya, Zaohua Huang, Agustin M. Barbara, Karel De Gendt,
Guido Verhoeven, Ivan P. Gorlov, and Alexander I. Agoulnik
Department of Human and Molecular Genetics (E.M.K., Z.H., A.M.B., A.I.A.), Herbert Wertheim College
of Medicine, Florida International University, Miami, Florida 33199; Laboratory for Experimental
Medicine and Endocrinology (K.D.G., G.V.), Katholieke Universiteit Leuven, 3000 Leuven, Belgium;
Department of Genitourinary Medical Oncology (I.P.G.), University of Texas M. D. Anderson Cancer
Center, Houston, Texas 77030; and Department of Obstetrics and Gynecology (A.I.A.), Baylor College of
Medicine, Houston, Texas 77030
Androgens play a critical role in the development of the male reproductive system, including the
positioning of the gonads. It is not clear, however, which developmental processes are influenced
by androgens and what are the target tissues and cells mediating androgen signaling during
testicular descent. Using a Cre-loxP approach, we have produced male mice (GU-ARKO) with
conditional inactivation of the androgen receptor (Ar) gene in the gubernacular ligament connecting the epididymis to the caudal abdominal wall. The GU-ARKO males had normal testosterone levels but developed cryptorchidism with the testes located in a suprascrotal position. Although initially subfertile, the GU-ARKO males became sterile with age. We have shown that
during development, the mutant gubernaculum failed to undergo eversion, a process giving rise
to the processus vaginalis, a peritoneal outpouching inside the scrotum. As a result, the cremasteric sac did not form properly, and the testes remained in the low abdominal position. Abnormal
development of the cremaster muscles in the GU-ARKO males suggested the participation of
androgens in myogenic differentiation; however, males with conditional AR inactivation in the
striated or smooth muscle cells had a normal testicular descent. Gene expression analysis showed
that AR deficiency in GU-ARKO males led to the misexpression of genes involved in muscle
differentiation, cell signaling, and extracellular space remodeling. We therefore conclude that AR
signaling in gubernacular cells is required for gubernaculum eversion and outgrowth. The GUARKO mice provide a valuable model of isolated cryptorchidism, one of the most common birth
defects in newborn boys. (Molecular Endocrinology 26: 598 – 607, 2012)
n most mammals, the male gonads descend from their
original embryonic intraabdominal position into the scrotum. Cryptorchidism, or hidden testis, defined as the absence of one or both testes from the scrotum, is the most
common birth defect, affecting 1– 4% of all newborn
boys with an even higher incidence (up to 30%) in prematurely born boys (1, 2). In the majority of cases, cryptorchidism is presented as an isolated abnormality and the
most common diagnosis is unilateral cryptorchidism in
which one testis is located caudal to the external inguinal
I
rings. Infertility and an increased risk of germ cell cancer
in adulthood are strongly linked to cryptorchidism, and it
has been proposed that all three conditions are the result
of common testicular dysgenesis syndrome (3); however,
recent reviews suggest that there is little evidence of
shared causes between these disorders (4, 5).
Testicular descent, the movement of the testis during
development, can be generally divided into two phases (6,
7). The transabdominal phase occurs in humans between
wk 10 and 15 of gestation and in mice between embryonic
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in U.S.A.
Copyright © 2012 by The Endocrine Society
doi: 10.1210/me.2011-1283 Received October 14, 2011. Accepted January 9, 2012.
First Published Online February 9, 2012
Abbreviations: ACTA1, ␣1-actin; AR, androgen receptor; E, embryonic day; GU-ARKO,
male mice with conditional inactivation of the Ar gene in the gubernacular ligament
connecting the epididymis to the caudal abdominal wall; HAS, hyaluronan synthase; IHC,
immunohistochemistry; INSL3, insulin-like 3; P, postnatal day; qRT-PCR, quantitative
RT-PCR.
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Mol Endocrinol, April 2012, 26(4):598 – 607
days (E) 14.5 and E16.5. This stage is characterized by the
descent of the gonad from the original pararenal location
into a low abdominal position. Experimental data derived
from transgenic mouse models indicate that this phase of
testis descent is androgen independent and controlled by
the peptide hormone insulin-like 3 (INSL3), produced in
testicular Leydig cells (8, 9). The second, inguinoscrotal
phase of testicular descent is believed to be androgen dependent; it is characterized by the movement of the testes
from an intraabdominal position, through the inguinal
canal, and across the pubic region to the scrotum. In
humans, inguinoscrotal descent is finalized before birth,
and in mice, the testes and epididymides move inside the
scrotum within the first 2 wk of neonatal development (2,
10). Although the morphological changes taking place
during testicular descent have been described (6, 11), we
know remarkably little about the cellular targets of hormones, the local cell signaling pathways, and the auto- or
paracrine differentiating stimuli activated by androgens
during testicular descent.
Understanding the mechanisms of testicular maldescent
in androgen receptor (AR)-compromised cryptorchidism is
hampered by an absence of comparable animal models.
Complete ablation of the AR causes testicular feminization
(Tfm) in male mice, characterized by the presence of hypoplastic gonads located in a low abdominal position (12).
The Tfm males have female external genitalia, do not develop a scrotum, and have a low level of testosterone and
therefore are not suitable for the analysis of inguinoscrotal
testicular descent. In this study, a conditional deletion of Ar
using a Cre/loxP approach allowed us to study the significance of AR signaling in the masculinization of different
organs. Notably, none of the reported testis- or epididymis-specific AR mutants exhibited cryptorchidism, suggesting that androgen signaling in other organs was essential for testicular descent (13–18). The objective of the
present study was to demonstrate that the functional
AR in the caudal genital ligament (gubernaculum) is
necessary for normal testicular descent. We describe
here the mouse model of cryptorchidism caused by an
AR deficiency in the gubernaculum (GU-ARKO). Such
males failed to form the processus vaginalis and the
testes were located in a suprascrotal position. The developmental abnormalities in GU-ARKO males and
possible local signaling mechanisms involved in mediating hormonal stimuli in the gubernaculum during testicular descent were analyzed.
Materials and Methods
Production of mice with conditional deletion of AR
All animal studies were approved by the Institutional Animal
Care and Use Committees at the Baylor College of Medicine and
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Florida International University. The mice with Cre-mediated
deletion of AR were produced by crossing females with the
floxed Ar allele Artm1.1Verh/Artm1.1Verh (Arfl/fl) with transgenic
males hemizygous for Tg(Rarb-cre)1Bhr (19), Tg(ACTA1cre)79Jme/J (23), and Tg(Tagln-cre)1Her/J (24). The Rarb-cre
mice were kindly provided by Dr. Richard Behringer (University
of Texas M. D. Anderson Cancer Center, Houston, TX); the last
two strains were from The Jackson Laboratory (Bar Harbor,
ME). Genotyping was performed as previously described for
each mutant using DNA isolated from the ear clips. At least
three mice with the same genotype were analyzed in each
experiment.
Hormone levels
The free testosterone level was determined in testicular homogenates of postnatal day (P) 3 testes and in adult male serum.
The testes were isolated, minced on ice with a mechanical homogenizer in 150 ␮l of PBS, and sonicated for 90 sec to ensure
cell disruption. After centrifugation (16,000 ⫻ g for 1 min) to
remove cell debris, the tissue homogenate was stored at ⫺80 C
until analysis. Five wild-type and six GU-ARKO males were
used in this analysis. The blood from adult males was drawn by
cardiocentesis from six Tg(Rarb-cre), Arfl/Y cryptorchid and
seven Arfl/Y wild-type males euthanized at 4 months of age. The
serum was collected after centrifugation at 3000 ⫻ g for 15 min.
The testosterone and LH levels were determined in the University of Virginia Center for Research in Reproduction Ligand
Assay and Analysis Core (University of Virginia, Charlottesville,
VA) using mouse RIA and two-site sandwich immunoassay,
respectively.
Analysis of transgenic Cre expression using
LacZ staining
To evaluate expression of the Cre transgene, we crossed females
homozygous for ROSA26-LacZ reporter (Gtrosa26tm1Sor) (34)
with Tg(Rarb-cre)1Bhr. The bottom parts of the double-transgenic
Tg(Rarb-cre), Rosa26-LacZ, and wild-type Rosa26-LacZ control
animals at PI were frozen in Tissue-Tek optimum cutting temperature medium, sectioned at 12–15 ␮m, and stained by using a
␤-galactosidase kit (Cell Signaling Technology, Inc., Danvers, MA)
and counterstained with eosin.
Histology and immunohistochemistry (IHC)
The mouse organs were collected, fixed, and embedded in paraffin, and 7-␮m frontal and sagittal sections were cut. The IHC was
performed using the following antibodies: AR (Santa Cruz Biotechnology, Santa Cruz, CA,); desmin (Sigma-Aldrich, St. Louis, MO);
smooth muscle actin (Sigma-Aldrich); and Ki67 (Thermo Scientific, Fremont, CA). For negative controls, normal rabbit IgG or
mouse IgG (Vector Laboratories, Burlingame, CA) was used at
appropriate primary antibody dilutions. Detection was performed
using a Vectastain ABC (avidin-biotin-peroxidase) kit (Vector
Laboratories) as recommended. The color was developed with
diaminobenzidine as chromogen. Samples were counterstained
with Harris hematoxylin. The analysis of cell proliferation in
processus vaginalis walls of the P3 males was performed in three
GU-ARKO and four wild-type littermates in at least 10 viewpoints by counting the percentage of Ki67-positive cells. Stained
slides were examined with a Carl Zeiss Axio A1 microscope
(New York, NY), and images were captured by an AxioCam
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Mol Endocrinol, April 2012, 26(4):598 – 607
Western immunoblotting
FIG. 1. Expression analysis of the Cre transgene in a Tg(Rarb-cre), Gtrosa26tm1Sor
gubernaculum at P1. The expression of Cre recombinase leads to an activation of
␤-galactosidase activity of the ROSA26 allele and blue staining. A, No staining was
detected in control Gtrosa26tm1Sor animals at P1 in gubernaculum (gub) or other
organs. B, Strong expression of Cre is detected in the mesenchymal cells of the
gubernacular bulb (gb), epididymis (ep), vas deferens (vd), and testicular Leydig cells
(t). cm, Differentiating cremaster muscles; wa, abdominal wall. C, Higher
magnification of gubernacular bulb, gb. Scale bar, 100 ␮m.
MRc5 CCD camera (Carl Zeiss). For immunofluorescence detection, sections were incubated with a goat anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Invitrogen,
Carlsbad, CA) to produce green fluorescence and a goat antirabbit Alexa Fluor 555 to produce red fluorescence. Fluorescent
images were captured using an Olympus BX61 motorized fluorescence microscope (Center Valley, PA). Due to a large size in
some cases (indicated in the figure legends), the presented images were manually consolidated from several images of the
same area.
RNA isolation and real-time quantitative RT-PCR
Total RNA was isolated from mouse tissues using the
RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. cDNA was synthesized using an oligo(deoxythymidine) primer and RETROscript kit (Ambion, Austin,
TX). A Q-PCR SybrGreen real-time assay on an Eppendorf
Mastercycler ep realplex instrument (Eppendorf, Westbury,
NY) was used for the real-time quantitative RT-PCR (qRTPCR). Gapdh expression was used for normalization of SybrGreen data. The relative fold change in mRNA level was calculated by the comparative cycle threshold (2–⌬⌬Ct) method. All
primer sequences are available upon request.
Expression microarray analysis
Gene expression profiles were analyzed using the Illumina
MouseRefseq-8 Expression BeadChip platform (Illumina, San
Diego, CA). The cremasteric sac samples from four 4-month-old
cryptorchid Tg(Rarb-cre), Arfl/Y males and four wild-type
Arfl/Y males derived from the same litters were used for RNA
isolation. The array experiments were performed in the Microarray Core Laboratory at the University of Texas Health
Science Center (Houston, TX), using standard Illumina protocols. Data were analyzed using BeadStudio software (Illumina).
Ingenuity IPA 8.5 Pathway Analysis software (Ingenuity Systems, Mountain View, CA) was used to identify pathways enriched by the genes differentially expressed in wild-type vs. cryptorchid tissues.
SDS-PAGE and Western immunoblotting were
performed using protein extracts isolated from the
cremasteric sacs of five GU-ARKO and five wild-type
(Arfl/Y) 6-wk-old littermates. After 4 –15% gradient
SDS-PAGE, the proteins were electrotransferred to a
nitrocellulose membrane (Invitrogen). Mouse monoclonal ␤-tubulin antibody (Millipore, Carlsbad, CA)
and rabbit anti-smooth muscle actin antibody (Abcam, Cambridge, MA) were used as primary antibodies with the appropriate horseradish peroxidase-conjugated secondary antibody (Promega, Madison, WI).
Supersignal West Pico chemiluminescence kit
(Thermo Scientific) was used to detect target proteins.
Statistical analysis
Student t test for two groups and one-way
ANOVA for multiple group comparisons were
used to assess significance of differences. Differences were expressed as mean ⫾SEM; P ⬍ 0.05
was considered significant. All analyses were performed using the GraphPad Software package (GraphPad
Software, La Jolla, CA).
Results
Deletion of the AR in the gubernaculum
causes cryptorchidism
To generate a conditional deletion of the Ar floxed
allele in the gubernaculum, we used a Tg(Rarb-cre) transgene. Cre in Tg(Rarb-cre) transgene is driven by the retinoic acid receptor 2 promoter and is expressed in the
mesonephric mesenchyme and its derivatives, including
different cellular components of the gubernaculum (9,
19). The expression of the transgene was previously detected in E14.5 gubernaculum and in adult cremasteric
sacs (9, 19). To establish the pattern of Cre expression in
the gubernaculum of newborn males, we analyzed male
mice with Tg(Rarb-cre) and a ROSA26-LacZ reporter
transgene. The expression of ␤-galactosidase (LacZ) in
ROSA26 mice is activated upon the Cre-mediated excision of the floxed stop cassette; and thus, only the cells in
which the Cre transgene is expressed and their progenitor
cells are stained in a LacZ assay. Cre activity was detected
in various gubernacular cells of newborn Tg(Rarb-cre),
ROSA26 males (Fig. 1). The strongest expression was
detected in the mesenchymal cells of the gubernacular
bulb and the epithelial cells of the gubernacular ligament.
Much lower, if any, expression was seen in the muscle
cells of the abdominal wall or processus vaginalis, suggesting a possible different cellular ontogeny of the muscle cells. Some LacZ activity was also detected in the
epididymis, vas deferens, and Leydig cells of the testis. We
then mated the homozygous Arfl/fl females with Tg(Rarb-
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FIG. 2. Bilateral cryptorchidism in GU-ARKO [Arfl/Y, Tg(Rarb-cre)]
mice. Aa, The adult wild-type and GU-ARKO males. The mammary
gland nipples are well developed in mutant males. Ab and Ac, In
euthanized males the testes can be manually pushed into the scrotum
in wild-type (Ab) but not in GU-ARKO males (Ac). Note the absence of
pigmented scrotal skin in GU-ARKO males (Ac). Ae, Mutant testes are
located in a suprascrotal position, protruding through the abdominal
wall, whereas in wild-type Arfl/Y littermates (Ad), the testes were fully
descended into the scrotum in the 2-month-old animals shown. Note
the poorly differentiated inguinal scrotum channel, scrotum sac
(dashed brackets), and the reduced testis and vas deferens size in
GU-ARKO mice (Af and Ag). B, Spermatogenesis in 2- (Ba and Bb) and
6-month-old (Bc and Bd) wild-type and GU-ARKO males. Note the
abnormal seminiferous tubules in older mutants. Depletion of sperm in
a mutant epididymis (Be vs. Bf). Abnormal development of the
cremasteric sacs from GU-ARKO 1-month-old males (Bc vs. Bf). Bg and
Bh, The mutant cremasteric sac is smaller and contains poorly
organized muscle cells. Bc and Bf were consolidated from two images
of the same section. a, Anus; pg, preputial glands; cm, cremasteric
muscle; cs, cremasteric sac; ep, epididymis; gc, gubernacular cord; p,
penis; sm, epididymal smooth muscle; sp, sperm; sv, seminal vesicles; t,
testis; vd, vas deferens. *, Seminiferous tubule lumen. Scale bar, Ba,
Bb, Bc, Bd, Be, Bf, 100 ␮m; Bg and Bh, 500 ␮m.
cre) males to obtain F1 males with a conditional inactivation of AR, restricted by Cre expression.
All Arfl/Y, Tg(Rarb-cre) males had an external male
phenotype (Fig. 2A). However, their testes were located
inside the abdominal cavity, cranial to the inguinal canal
601
in a suprascrotal position. The cryptorchid testes were
protruding into the abdominal wall and were located below a thin muscle layer under the skin (Fig. 2Ae). Whereas
in anesthetized wild-type males the temporarily retracted
testes could be easily pushed into the scrotum manually
(Fig. 2Ab), it was not possible to do in mutants (Fig. 2Ac).
Thus, the conditional inactivation of Ar in the gubernaculum (GU-ARKO) caused cryptorchidism. Contrary to
the female external phenotype in Tfm males (12), all parts
of the male reproductive system (testis, epididymis, seminal vesicles, vas deferens, prostate, and penis) were developed in the GU-ARKO mice. The comparison of total
body size and seminal vesicle weight in 10- to 12-wk-old
males did not reveal significant differences between the
GU-ARKO and wild-type (Arfl/Y) littermates (Table 1).
The testis and epididymis weight in mutant males was
reduced to 50% of that of the wild-type males (Table 1
and Fig. 2Af and 2Ag). The GU-ARKO males also had
well-developed mammary nipples, suggesting the inactivation of AR by Cre expression in these organs and hence
the importance of AR in nipple development (Fig. 2Aa).
The fertility of the GU-ARKO males was analyzed in
crosses with wild-type females. When the young 6- to
12-wk-old males were used, few successful matings were
recorded. The older, 4- to 12-month-old, GU-ARKO
males were completely infertile. The spermatozoa number decreased significantly in the epididymal semen of the
10- to 12-wk-old GU-ARKO males (Table 1) without an
appreciable increase in the abnormal motility or shape
(data not shown). The histological examination of the
2-month-old GU-ARKO testis did not reveal noticeable
differences with the wild-type males (Fig. 2Ba and 2Bb).
In older animals, the degeneration of spermatogenesis
was found with increased vacuolization of Sertoli cells,
characteristic for a cryptorchid testis. There was a reduced number of sperm in the mutant epididymis (Fig.
2Bc–f). The size of the adult cremasteric sac was significantly reduced (Table 1); it was more collagenous and had
visibly less muscle tissue in appearance (Fig. 2Bg and
2Bh).
No differences in serum testosterone (38.6 ⫾9.1 vs.
41.7 ⫾10.1 ng/dl) or LH (0.3 ⫾0.1 vs. 0.3 ⫾0.1 ng/ml)
concentrations between 4-month-old wild-type and GU-
TABLE 1. Reproductive organ weights and sperm count in 10- to 12-wk-old wild-type and GU-ARKO male mice
Wild type
GU-ARKO
Body weight
(g)
27.4 ⫾ 1.0 (8)
24.3 ⫾ 1.3 (7)
Testis
(mg)
211 ⫾ 7 (8)
105 ⫾ 4 (7)a
Epididymis
(mg)
81.5 ⫾ 6.4 (8)
34.3 ⫾ 7.8b
Seminal vesicles
(mg)
303.5 ⫾ 32.8 (8)
276 ⫾ 32.2 (7)
Values are mean ⫾ SEM; number in parentheses is the number of animals analyzed; mln, millions.
a
P ⬍ 0.001.
b
P ⬍ 0.01.
Cremasteric sac
(mg)
53.1 ⫾ 3.3 (8)
29.1 ⫾ 3.2 (7)a
Sperm count
(mln/ml)
14.3 ⫾ 3.3 (5)
0.9 ⫾ 0.4 (5)b
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Cryptorchidism in Gubernacular AR Ablation
ARKO males were found. A reduction of AR was also
detected in adult cauda epididymis and vas deferens (data
not shown), in agreement with the expression of Cre in
these organs (Fig. 1).
Development of testicular maldescent in
GU-ARKO mice
To analyze the effect of AR deficiency in GU-ARKO
mice, we compared the testicular descent in mutant and
wild-type littermates at P1, P3, and P12 (Fig. 3). The
morphological differences between the two groups were
already clearly visible in newborn males. In wild-type
males, the processus vaginalis began to form through the
outgrowth and invagination of the gubernacular bulb
into the caudal region (10, 20). At that stage, the epididymis, connected to the body wall through the mesenchymal gubernacular cord, began to move into the forming
inguinal sac. The inverted rims of the gubernacular bulb
FIG. 3. Failure of gubernacular invagination in GU-ARKO male mice.
The lines show the extent of processus vaginalis. bl, Bladder; gb,
gubernacular bulb; pg, preputial gland; pv, processus vaginalis; sc,
scrotum; wa, abdominal wall; other abbreviations are as in Fig. 2. The
presented images were consolidated from two to six images from the
same section. Scale bar, 500 ␮m.
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formed the walls of the processus vaginalis and a dense
population of AR mesenchymal cells was present in the
outgrowing gubernacular bulb (Fig. 4Aa). In mutant GUARKO males, the formation of the processus vaginalis
was delayed, and the epididymis and testis were located
intraabdominally under the bladder (Fig. 3, 4A). At P3,
the wild-type processus vaginalis was significantly increased with the epididymis fully located inside of it. The
processus vaginalis of GU-ARKO littermates was only
marginally increased in size; both epididymis and testis
were only slightly caudally repositioned (Fig. 3). No significant difference in cell proliferation levels in the walls
of the forming cremasteric sacs in P3 GU-ARKO and
wild-type males interrogated with the Ki67 antibody was
found (27.7 ⫾3.4 vs. 32.5 ⫾2.2%).
By P12, the distal end of the wild-type processus vaginalis reached the end of the scrotum; the epididymis and
the testis were located inside the developed scrotum. In
GU-ARKO males, the processus vaginalis reached about
half of the distance achieved by their wild-type counterpart, and only the cauda epididymis was located inside of
it. The mutant testis remained in the abdominal cavity.
Part of the preputial glands moved into the scrotal area,
further preventing gubernacular invagination. We did not
detect any remains of the mammary tissues in mutant
males, which were previously suggested as the reason for
testis maldescent in AR deficiency (21). Interestingly, the
GU-ARKO gubernacular bulb still grew significantly in
size, but inward (Fig. 4, Ad and Bb).
The pattern of AR ablation in the GU-ARKO animals
was confirmed by IHC analysis (Fig. 4). In wild-type newborn males, strong AR expression was detected in the
mesenchymal cells of the gubernacular bulb, peritoneal
epithelium, and walls of the processus vaginalis (Fig. 4A).
The GU-ARKO males had a sharply reduced number of
AR-positive cells in all parts of the P1 gubernaculum (Fig.
4A), with few cells showing staining, perhaps due to a
mosaic expression of Cre. The significant reduction of
AR-positive cells was also visible in stromal cells of the
cauda epididymis (Fig. 4Ac and 4Ad). Histological sections revealed less organized muscle cells in the walls of
the GU-ARKO processus vaginalis and intense staining
for desmin in the mutant gubernacular bulb in P12 males
(Fig. 4B). The muscle cells of the cremasteric sac did not
show significant staining for AR (Fig. 4B).
We then analyzed the level of free intratesticular testosterone in P3 testes from GU-ARKO and wild-type male siblings (Fig. 5A). No difference was detected between the two
groups. Furthermore, the expression of Stra8, P450c17,
Hsd17b6, and Hsd17b3 genes involved in testosterone production did not change in GU-ARKO neonatal testes when
compared with wild-type littermates (Fig. 5B). The qRT-
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gubernaculum. The skeletal muscle ␣1actin, Acta1, was significantly down-regulated in GU-ARKO gubernacula. No
significant differences were detected by
qRT-PCR in the expression of smooth
muscle actin, Acta2, or INSL3 receptor
Rxfp2 in mutant tissues, previously suggested to be a mediator of AR signaling
in abnormal gubernacular development
in LH-deficient mice (22).
Deletion of AR in smooth or
striated muscle cells in the
gubernaculum does not affect
FIG. 4. Immunohistological localization of AR in wild-type and GU-ARKO. A, IHC for AR in the
wild-type and GU-ARKO males indicates an ablation of Ar expression in the gubernacular bulb
testicular descent
(gb, dotted line) of newborn (P1) and 12-d-old (P12) wild-type and GU-ARKO processus vaginalis
The invagination of the gubernaculittermates. Inward growth of gubernaculum in GU-ARKO mutant (Ab and Ad). An asterisk shows
lar bulb gives rise to the processus vagithe development of the hypocellular region in the mutant gubernacular bulb. Aa was consolidated
from two images, Ac, from four images, and Ad, from six images from the same section. B,
nalis and the cremasteric sac (20). The
Immunofluorescence analysis of AR (red), muscle cell marker desmin (green), and nuclei (blue
muscle cells forming the cremasteric
fluorescent) staining showing loss of AR immunostaining in gubernaculum and in processus vaginalis of
sac differentiate at the outer rims of the
P3 GU-ARKO males. Bc and Bd, High magnification of processus vaginalis walls. Bc, Nuclei of the
muscle cells (arrowheads) are negative for AR staining, whereas mesenchymal cells (arrows) are positive
gubernacular bulb. The question then
for AR in wild-type processus vaginalis. Bd, Mutant processus vaginalis is negative for AR apart from the
arises of whether androgen signaling
few cells with weak AR expression (arrows). Note the strong AR staining in epididymal epithelium in A
(brown) and B (red). Scale bar, A, Ba, and Bb, 100 ␮m; Bc and Bd, 10 ␮m.
directly affects the differentiation of
cremaster muscle cells and whether the
PCR analysis of Ar expression in P3 gubernaculum showed inactivation of AR in muscle cells will disrupt testicular
a significant down-regulation (Fig. 5C). The expression of descent. Using the same approach as described above, we
Pax7, a muscle stem cell marker, was up-regulated, suggest- produced mice with an Arfl/Y, Tg(ACTA1-cre) and
ing a delay in muscle cell differentiation in the GU-ARKO Arfl/Y, Tg(Tagln-cre) genotype. The expression of Cre in
these mice was controlled by ACTA1, skeletal muscle
␣1-actin, gene promoter specific for striated muscles (23),
or transgelin (Tagln, smooth muscle protein 22-␣) gene
promoter, specific for smooth muscle cells (24). Both
Acta1 and Tagln genes and the Cre-transgenes were expressed in the gubernacular ligament (25). Indeed, the
analysis of genomic DNA from Arfl/Y, Tg(ACTA1-cre)
and Arfl/Y, Tg(Tagln-cre) adult cremaster muscles indicated a deletion of the Ar floxed allele (Fig. 6). The inten-
FIG. 5. Intratesticular free testosterone concentration and qRT-PCR
analysis of gene expression in neonatal GU-ARKO males. A, Intratesticular
free testosterone concentration in wild-type (n ⫽ 5) and GU-ARKO (n ⫽
6) P3 testes. No significant difference was detected. B, Gene expression in
P3 wild-type and GU-ARKO testis. No statistically significant differences
were detected (n ⫽ 5 in each group). C, Gene expression in P3 wild-type
and GU-ARKO gubernacula revealed significant down-regulation of Ar
and Acta1 genes and up-regulation of Pax7. *, P ⬍ 0.05 (n ⫽ 3 in each
group). Gapdh gene expression was used for normalization.
FIG. 6. Deletion of the Arfl allele in the cremasteric sacs of mice
containing different Cre transgenes. The upper PCR band identifies a
nonrecombinant Arfl allele, and the lower band is a deleted Ar allele
(Arko).
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Mol Endocrinol, April 2012, 26(4):598 – 607
sity of the PCR band corresponding to the deleted Ar
allele was lower in the smooth muscle-specific Cre than
that in Arfl/Y, Tg(Rarb-cre) or Arfl/Y, Tg(ACTA1-cre),
confirming that the adult cremaster muscle contains
mainly striated muscle cells (26). The anatomical examination of males at 1 month of age did not reveal any
difference in size or position of the testes or any appreciable differences in cremasteric sac development in the mutant animals when compared with the wild-type controls.
The testes were fully descended by that time in both mutants. Thus, an ablation of androgen signaling in muscle
cells does not affect gubernaculum development or testicular descent.
Hoxa10, Hoxa11, or Arid5b genes, mutations of which
cause cryptorchidism in mice (28).
We analyzed the expression of smooth muscle actin
in the cremasteric sacs of P3 and adult GU-ARKO
males and their wild-type littermates (Fig. 7). In neonatal gubernacular an increased staining for ACTA2
was detected, perhaps due to a failure of gubernacular
inversion. An increased expression of this marker was
detected in adult mutant scremasteric sacs by IHC and
Western blot hybridization, suggesting a shift toward
smooth muscle myogenic differentiation in GU-ARKO
cremasteric sacs.
Altered extracellular signaling and the shift
from striated to smooth muscle composition
are the main outcomes of AR deletion in
the gubernaculum
To evaluate the consequences of AR ablation on gene
expression in the GU-ARKO gubernacula, we performed a
whole-genome expression analysis of the mutant and wildtype cremasteric sacs in 4-month-old males. Total RNA was
analyzed using the Illumina Expression BeadChip platform.
Both the qRT-PCR (data not shown) and array data indicated a significant down-regulation of Ar expression in the
mutant samples, suggesting an efficient ablation of the gene
in the cremasteric sac. The full list of the genes misexpressed
in the mutant can be found in Supplemental Table 1, published on The Endocrine Society’s Journals Online web site
at http://mend.endojournals.org. In total, 120 genes with
more than a 2-fold difference (P ⬍ 0.01) were misexpressed
in the mutants, of which 63 were down-regulated. Of all
8852 genes detected on the array with P ⬍ 0.01 significance, 982 genes were known to be a direct or secondary
targets of androgen signaling (27). Such genes were significantly overrepresented among the genes with at least a
2-fold difference in expression in the GU-ARKO cremasteric sacs (27 of 120, P ⫽ 0.0002) (Supplemental Table 1).
The functional analysis of the GU-ARKO misexpressed
probe set was performed using Ingenuity IPA 8.5 Pathway
Analysis software. The Gene Ontology molecular functions
and Gene Ontology cellular components were mainly related to the extracellular matrix composition and space, signaling, and receptor binding. Among them, hyaluronan synthases, HAS1 and HAS2, were down-regulated, whereas the
expression of renin was significantly up-regulated in adult
mutant organs. The markers of smooth muscle cells
(ACTA2, ACTG2, MYH11, MYLK, TAGLN) were significantly up-regulated, whereas the skeletal muscle heavy
polypeptide 4 myosin (MYH4) was down-regulated. No
differences were found in the expression of the Rxfp2,
Discussion
The majority of human testicular maldescent cases are
caused by abnormalities during the inguinoscrotal phase,
when the testes move from a low abdominal position into
FIG. 7. Increased expression of smooth muscle actin in a P3
gubernaculum and in a 6-wk-old GU-ARKO cremasteric sac. A, The
ACTA2-positive cells in inverted wild-type noninverted mutant
gubernaculum (gb, arrowheads). Scale bar, 100 ␮m. B, IHC showing
an increase in staining for smooth muscle actin (dark brown,
arrowheads) in a GU-ARKO cremasteric sac. The wild-type image was
consolidated from two images of the same section. ep, Epididymis; gb,
gubernaculum; vd, vas deferens. Scale bar, 500 ␮m. C, Western blot
analysis of two wild-type and two GU-ARKO cremasteric sacs using
smooth muscle actin (ACTA2) antibody. ␤-Tubulin was used as the
loading control.
Mol Endocrinol, April 2012, 26(4):598 – 607
the scrotum. It has been suggested that androgen signaling plays a critical role at this stage by controlling the
differentiation of the gubernaculum, processus vaginalis,
and cremasteric sac. Indeed, prenatal and early neonatal
exposure to antiandrogenic and estrogenic compounds
causes cryptorchidism (21, 29, 30). Similarly, complete
androgen resistance due to mutations in AR causes male
testicular feminization syndrome, with an external female
phenotype and small hypoplastic testes located in a low
abdominal position (12). The use of a Cre/loxP conditional approach in mice allowed the targeting of Ar in
specific organs or cells. It has been previously (16) shown
that the selective inactivation of Ar in different testicular
cells might result in infertility; however, it did not cause
cryptorchidism, suggesting an importance of AR signaling in organs other than the testis during testicular descent. Here we present a mouse model that allows the
study of mechanisms of AR signaling in inguinoscrotal
testis descent and the consequences of cryptorchidism on
germ cell development. We have shown that inactivation
of AR in cells derived from the mesonephric mesenchyme,
which includes different cells of the gubernaculum, leads
to an abnormal cremasteric sac development and testicular maldescent. Thus, the gubernaculum is an essential
target of androgen signaling in testicular descent.
The analysis of Rarb-cre in ROSA26 mice showed Cre
expression in fetal Leydig cells. Consequently, Ar was
deleted in neonatal GU-ARKO Leydig cells, yielding the
question of whether such mutants had abnormal testosterone production. Previously, mice with a Leydig cell
specific knockout of the Ar gene were produced using cre
inserted into anti-Műllerian hormone receptor 2 gene
(Amhr2-cre) (15, 16). Such mice had hypotestosteronemia caused by reduced expression of several key steroidogenic enzymes, including 17␤-hydroxysteroid dehydrogenase-3, 3␤-hydroxysteroid dehydrogenase-6, and
P450c17. It was later demonstrated that the Amhr2-cre
transgene expression is not limited to Leydig cells; it is
also expressed in both fetal and adult Sertoli cells (31).
Thus, a combination of AR deletions in different testicular components might be a reason for hypotestosteronemia in Amhr2-cre, Arfl/Y males. As shown here, the GUARKO mutants had normal levels of testosterone in both
neonatal testis and adult serum, indicating that the deletion of AR in Leydig cells does not, in fact, affect testosterone production. The deletion of AR in GU-ARKO
males was also detected in stromal cells in the cauda epididymis and vas deferens. This might suggest there is a
common origin of all these cells from mesonephric mesenchymal progenitors; however, a more detailed cell fate
and spatiotemporal analysis of Rarb-cre transgene expression is needed.
mend.endojournals.org
605
The cryptorchid animals produced viable sperm and
were able to sire pups until 3 months of age. The analysis of testis sections in older males revealed the appearance of abnormal seminiferous tubules with arrested spermatogenesis and vacuolization of the Sertoli
cells. The epididymal sperm count was significantly
decreased in mutant males, suggesting the causative
role of an abnormal testis position in cryptorchidisminduced infertility. There might be an additional contribution to infertility in GU-ARKO males due to abnormal function of the epididymis and vas deferens, in
which AR deletion in stromal cells was detected.
The most crucial step affected by AR deficiency appeared to be the failure of the gubernaculum to undergo
invagination, leading to the formation of the processus
vaginalis. Two mechanisms of AR-compromised cryptorchidism have been previously proposed. First, it was
suggested that the persistence of mammary gland tissues
in males treated with antiandrogens might prevent the
gubernacular outgrowth (21). However, we did not find
any mammary tissues in the scrotal area of GU-ARKO
males that would interfere with an eversion of the gubernaculum. Second, the stimulatory role of AR signaling
was proposed in the genitofemoral nerve release of calcitonin gene-related peptide, providing a chemotactic gradient to guide gubernacular cell migration (2). However,
the genetic targeting of Calca did not cause cryptorchidism (32). Although we did not analyze the expression of
the Rarb-cre transgene and the deletion of AR in GUARKO neural tissues, our data indicate that the deletion
of Ar in gubernacular cells, derived from the mesonephric
mesenchyme rather than an indirect effect of androgens
through neurotransmitters (33), was essential in the inguinoscrotal maldescent in GU-ARKO males.
We have established that the genes regulated by androgens, or participating in androgen receptor signaling, were
statistically overrepresented among misregulated genes in
the adult GU-ARKO cremasteric sacs. Importantly, an expression analysis revealed a significant number of genes involved in cell signaling, extracellular space, and matrix composition. The extracellular matrix plays a crucial role in
regulating tissue development and function, mainly through
the specific arrangement of macromolecules such as collagens, proteoglycans, glycosaminoglycans, and glycoproteins. Hyaluronan, the glycosaminoglycan, is one of the
main components in the adult gubernaculum. The analysis
revealed a decreased expression of hyaluronan synthases,
HAS1 and HAS2, in the GU-ARKO cremasteric sac, suggesting a transcriptional down-regulation of these genes. In
the newborn gubernaculum, the AR-positive mesenchymal
cells were concentrated in the gubernacular bulb. The secretion of hyaluronan and other extracellular molecules by
606
Kaftanovskaya et al.
Cryptorchidism in Gubernacular AR Ablation
these cells may be important for the formation of increased
hypocellularity in the scrotal region of the gubernacular
bulb floor.
Gene expression analysis also revealed altered expression in a number of muscle-specific markers. The cremaster muscle is mainly composed of striated muscle bundles
with some smooth muscle dispersed between them (26).
We have shown that in neonatal GU-ARKO gubernaculum, the expression of the early muscle developmental
marker Pax7 was increased, whereas the expression of
skeletal muscle actin was decreased, suggesting the inhibition of normal muscle maturation in mutants. The adult
GU-ARKO cremasteric sacs showed an increase in expression of smooth muscle markers and a decreased expression of striated muscle markers. This was consistent
with the morphological changes observed in cryptorchid
animals, in which the striated cremaster muscle organization was noticeably abnormal, and the size of the cremasteric sac was significantly reduced. The selective ablation of Ar in smooth or striated muscle cells using a Cre/
loxP approach did not interfere with the testis descent or
cremaster muscle development, suggesting that the androgen stimuli did not affect the differentiation or function of muscle cells directly. We therefore suggest that the
deficiency of AR and the absence of AR-induced paracrine signals from the gubernacular cells on the myoblast
may be responsible for the abnormal muscle differentiation. Notably, an altered expression of muscle related
genes have been previously detected in a rat strain with
inherited cryptorchidism (25) and in rats treated with
antiandrogen flutamide (30). We also observed an abnormal myogenesis in INSL3/RXFP2-deficient gubernacula
(9). Previously, using the same Cre transgene, we have
shown that the conditional ablation of Notch1 or
␤-catenin genes (7) as well as an Rxfp2 deletion led to a
significant reduction of AR-positive cells in the gubernacular bulb and subsequent failure of the gubernaculum
to undergo invagination. Thus, the presence and normal
function of AR-positive cells in the gubernaculum appear
to be necessary for the normal formation of the processus
vaginalis and testicular descent.
In summary, Cre/loxP gene targeting allowed us to
produce mice with an AR deficiency in the gubernacular
ligament (GU-ARKO). Such males developed all male reproductive organs, had normal hormonal milieu, but exhibited low intraabdominal cryptorchidism, demonstrating that the gubernaculum is a target organ for hormonal
signaling during testis descent. A significant number of
genes involved in extracellular signaling, matrix composition, and muscle differentiation were misregulated in
the GU-ARKO cremasteric sac, a derivative of the gubernacular bulb. Importantly, an ablation of Ar in the guber-
Mol Endocrinol, April 2012, 26(4):598 – 607
nacular smooth or striated muscle cells using specific Cre
transgenes did not affect testis descent, suggesting an indirect effect of AR signaling on myogenesis of the cremaster muscle and cremasteric sac. The GU-ARKO mouse
provides an important in vivo model of AR-compromised
cryptorchidism. Further analysis of this model might reveal the mechanisms of testicular descent and cryptorchidism-induced infertility.
Acknowledgments
We thank Drs. Richard Behringer and Marvin Meistrich (University of Texas M. D. Anderson Cancer Center, Houston, TX)
for providing Tg(Rarb-cre) and Ar-floxed mice; The University
of Texas Health Science Center at Houston Microarray Core
Laboratory for microarray analysis, The University of Virginia
Center for Research in Reproduction Ligand Assay and Analysis
Core, supported by Eunice Kennedy Shriver National Institute
of Child Health and Human Development/National Institutes of
Health (Specialized Cooperative Centers Program in Reproduction and Infertility Research) Grant U54-HD28934, for hormone detection. We also thank Drs. Gen Yamada (Kumamoto
University, Kumamoto, Japan), Irina Agoulnik, and Lydia Ferguson (Florida International University, Miami, FL) for insightful discussions; and Anne Truong, Rhea Pereira, and Giselle
Neukirchner for technical assistance.
Address all correspondence and requests for reprints to:
Alexander I. Agoulnik, Department of Human Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, 11200 SW 8th Street, AHCI 419B, Miami,
Florida 33199. E-mail: [email protected].
This work was supported by the Eunice Kennedy Shriver
National Institute of Child Health and Human Development/
National Institute of Health Grant R01HD37067 (to A.I.A.).
Disclosure Summary: The authors have nothing to disclose.
References
1. Hack WW, Meijer RW, Bos SD, Haasnoot K 2003 A new clinical
classification for undescended testis. Scand J Urol Nephrol 37:
43– 47
2. Hutson JM, Balic A, Nation T, Southwell B 2010 Cryptorchidism.
Semin Pediatr Surg 19:215–224
3. Skakkebaek NE, Rajpert-De Meyts E, Main KM 2001 Testicular
dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 16:972–978
4. Thorup J, McLachlan R, Cortes D, Nation TR, Balic A, Southwell
BR, Hutson JM 2010 What is new in cryptorchidism and hypospadias—a critical review on the testicular dysgenesis hypothesis. J Pediatr Surg 45:2074 –2086
5. Akre O, Richiardi L 2009 Does a testicular dysgenesis syndrome
exist? Hum Reprod 24:2053–2060
6. Gier HT, Marion GB 1969 Development of mammalian testes and
genital ducts. Biol Reprod 1(Suppl 1):1–23
7. Hutson JM 1985 A biphasic model for the hormonal control of
testicular descent. Lancet 2:419 – 421
Mol Endocrinol, April 2012, 26(4):598 – 607
8. Bogatcheva NV, Agoulnik AI 2005 INSL3/LGR8 role in testicular
descent and cryptorchidism. Reprod Biomed Online 10:49 –54
9. Kaftanovskaya EM, Feng S, Huang Z, Tan Y, Barbara AM, Kaur S,
Truong A, Gorlov IP, Agoulnik AI 2011 Suppression of Insulinlike3 receptor reveals the role of ␤-catenin and Notch signaling in
gubernaculum development. Mol Endocrinol 25:170 –183
10. Hadziselimovic F, Herzog B 1993 The development and descent of
the epididymis. Eur J Pediatr 152(Suppl 2):S6 –S9
11. Barteczko KJ, Jacob MI 2000 The testicular descent in human.
Origin, development and fate of the gubernaculum Hunteri, processus vaginalis peritonei, and gonadal ligaments. Adv Anat Embryol Cell Biol 156:III-X, 1–98
12. Lyon MF, Hawkes SG 1970 X-linked gene for testicular feminization in the mouse. Nature 227:1217–1219
13. Walters KA, Simanainen U, Handelsman DJ 2010 Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. Hum Reprod Update 16:543–558
14. Welsh M, Saunders PT, Atanassova N, Sharpe RM, Smith LB 2009
Androgen action via testicular peritubular myoid cells is essential
for male fertility. FASEB J 23:4218 – 4230
15. Xu Q, Lin HY, Yeh SD, Yu IC, Wang RS, Chen YT, Zhang C,
Altuwaijri S, Chen LM, Chuang KH, Chiang HS, Yeh S, Chang C
2007 Infertility with defective spermatogenesis and steroidogenesis
in male mice lacking androgen receptor in Leydig cells. Endocrine
32:96 –106
16. Tsai MY, Yeh SD, Wang RS, Yeh S, Zhang C, Lin HY, Tzeng CR,
Chang C 2006 Differential effects of spermatogenesis and fertility
in mice lacking androgen receptor in individual testis cells. Proc
Natl Acad Sci USA 103:18975–18980
17. Zhang C, Yeh S, Chen YT, Wu CC, Chuang KH, Lin HY, Wang RS,
Chang YJ, Mendis-Handagama C, Hu L, Lardy H, Chang C 2006
Oligozoospermia with normal fertility in male mice lacking the
androgen receptor in testis peritubular myoid cells. Proc Natl Acad
Sci USA 103:17718 –17723
18. De Gendt K, Swinnen JV, Saunders PT, Schoonjans L, Dewerchin
M, Devos A, Tan K, Atanassova N, Claessens F, Lécureuil C, Heyns
W, Carmeliet P, Guillou F, Sharpe RM, Verhoeven G 2004 A Sertoli
cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc Natl Acad Sci USA 101:1327–1332
19. Kobayashi A, Kwan KM, Carroll TJ, McMahon AP, Mendelsohn
CL, Behringer RR 2005 Distinct and sequential tissue-specific activities of the LIM-class homeobox gene Lim1 for tubular morphogenesis during kidney development. Development 132:2809 –2823
20. van der Schoot P 1996 Towards a rational terminology in the study
of the gubernaculum testis: arguments in support of the notion that
the cremasteric sac should be considered the gubernaculum in postnatal rats and other mammals. J Anat 189(Pt 1):97–108
21. Nation T, Balic A, Buraundi S, Farmer P, Newgreen D, Southwell
mend.endojournals.org
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
607
B, Hutson J 2009 The antiandrogen flutamide perturbs inguinoscrotal testicular descent in the rat and suggests a link with mammary development. J Pediatr Surg 44:2330 –2334
Yuan FP, Li X, Lin J, Schwabe C, Büllesbach EE, Rao CV, Lei ZM
2010 The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice. Reproduction
139:759 –769
Miniou P, Tiziano D, Frugier T, Roblot N, Le Meur M, Melki J
1999 Gene targeting restricted to mouse striated muscle lineage.
Nucleic Acids Res 27:e27
Holtwick R, Gotthardt M, Skryabin B, Steinmetz M, Potthast R,
Zetsche B, Hammer RE, Herz J, Kuhn M 2002 Smooth muscleselective deletion of guanylyl cyclase-A prevents the acute but not
chronic effects of ANP on blood pressure. Proc Natl Acad Sci USA
99:7142–7147
Barthold JS, McCahan SM, Singh AV, Knudsen TB, Si X, Campion
L, Akins RE 2008 Altered expression of muscle- and cytoskeletonrelated genes in a rat strain with inherited cryptorchidism. J Androl
29:352–366
Kayalioglu G, Altay B, Uyaroglu FG, Bademkiran F, Uludag B,
Ertekin C 2008 Morphology and innervation of the human cremaster muscle in relation to its function. Anat Rec (Hoboken) 291:790 –
796
Nikitin A, Egorov S, Daraselia N, Mazo I 2003 Pathway studio—
the analysis and navigation of molecular networks. Bioinformatics
19:2155–2157
Klonisch T, Fowler PA, Hombach-Klonisch S 2004 Molecular and
genetic regulation of testis descent and external genitalia development. Dev Biol 270:1–18
Bay K, Main KM, Toppari J, Skakkebæk NE 2011 Testicular descent: INSL3, testosterone, genes and the intrauterine milieu. Nat
Rev Urol 8:187–196
Sanders N, Buraundi S, Balic A, Southwell BR, Hutson JM 2011
Cremaster muscle myogenesis in the tip of the rat gubernaculum
supports active gubernacular elongation during inguinoscrotal testicular descent. J Urol 186:1606 –1613
Tanwar PS, Kaneko-Tarui T, Zhang L, Rani P, Taketo MM, Teixeira J 2010 Constitutive WNT/␤-catenin signaling in murine Sertoli
cells disrupts their differentiation and ability to support spermatogenesis. Biol Reprod 82:422– 432
Lu JT, Son YJ, Lee J, Jetton TL, Shiota M, Moscoso L, Niswender
KD, Loewy AD, Magnuson MA, Sanes JR, Emeson RB 1999 Mice
lacking ␣-calcitonin gene-related peptide exhibit normal cardiovascular regulation and neuromuscular development. Mol Cell Neurosci 14:99 –120
Momose Y, Goh DW, Hutson JM 1993 Calcitonin gene-related
peptide stimulates motility of the gubernaculum via cyclic adenosine monophosphate. J Urol 150:571–573
Soriano P 1999 Generalized lacZ expression with the ROSA26 Cre
reporter strain. Nat Genet 21:70 –71