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From the * Research Centre for Reproductive Health
and Discipline of Obstetrics and Gynaecology,
Discipline of Anatomy, and
Discipline of Pharmacology, University of
Adelaide, Australia.
| Correspondence to: Prof Sarah Robertson, Discipline of Obstetrics and Gynaecology, University of Adelaide, Adelaide, SA 5005 Australia (e-mail: sarah.robertson{at}adelaide.edu.au). |
| Received for publication August 11, 2008; accepted for publication February 16, 2009. |
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Abstract |
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Key words: Penis, erectile dysfunction, sexual behavior, cytokine, skin, mouse model
However, neonatal and adult testosterone administration does not rescue the infertility phenotype of Tgfb1 null mutant male mice (Ingman and Robertson, 2007). This infertility is not due to defects in spermatogenesis because sperm recovered from the epididymis of Tgfb1 null mutant males can fertilize oocytes in vitro, with a normal rate of development to the blastocyst stage. Male reproductive tract tissues including the testis, penis, and seminal vesicle glands are of normal weight and gross morphology. Closer analysis of the behavior of Tgfb1 null mutant males when caged with receptive females reveals that the mice show the expected outward signs of sexual interest in the females, including anogenital investigation and mounting behavior. Although brief intromission behavior is observed in some mice, sustained intromission and ejaculation do not occur, and Tgfb1 null mutant male mice continue to mount females long after control males have successfully mated. Electrical stimulation of penile function leads to erectile activity and ejaculation of motile sperm; however, this stimulus is not physiologic. Based on these observations, we considered the possibility that TGFB1 deficiency causes erectile dysfunction.
Erectile function in males is dependent on central and peripheral neural signaling and a process of concerted smooth muscle contraction and relaxation to obtain erection and ejaculation. A diverse range of central signaling molecules is involved in the cascade of events required for copulation, including dopamine, norepinephrine, serotonin, and acetylcholine (Andersson, 2000). Peripherally, erection is mediated by production of nitric oxide (NO) by penile NO synthase (NOS) enzyme isoforms, causing cGMP production and consequently relaxation of the corpus cavernosa smooth muscle (Burnett, 1995). Noradrenaline, acetylcholine, and dopamine have also been implicated (Andersson, 2001).
The studies presented here explore the physiologic basis for sexual dysfunction in Tgfb1 null mutant males, bred on a Prkdcscid mutant background to allow their survival to reproductive age (Diebold et al, 1995). Our findings indicate that altered structural integrity of penile skin and penile fibrosis contribute to the infertility caused by deficiency in TGFB1.
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Materials and Methods |
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The genotype of each mouse was determined by polymerase chain reaction (PCR) of tail DNA as described previously (Ingman et al, 2006a). Briefly, the PCR detected the intact and disrupted Tgfb1 gene, using the forward primer 5'-GAGAAGAACTGCTGTGTGCG-3' together with reverse primer 5'-GTGTCCAGGCTCCAAATATAGG-3' to detect the intact Tgfb1 gene or 5'-CTCGTCCTGCAGTTCATTCA-3' to detect the mutant Tgfb1 gene containing a neomycin resistance gene inserted into exon 6.
Analysis of Penis Surface
For assessment of the penis surface, mice were sacrificed and the penis was
promptly inflated with fixative via a needle inserted into the main artery at
the base of the penis. The puncture site was immediately sealed with suture
thread prior to excision of the penis. The erect penis samples were then
processed and coated with carbon gold using standard techniques to allow
analysis by scanning electron microscopy (SEM) (using a Philips XL20 scanning
electron microscope [FEI, Hillsboro, Oregon]). Penile spines were evaluated
using analySIS (Olympus Soft Imaging Solutions, Münster, Germany) at the
Adelaide Microscopy Centre.
A loosely attached superficial material, presumed to be shed keratinized epithelial cells on the surface of the penis, was evident during SEM assessment of some tissue. Protrusion of penile spines through this excess material was scored using a semiquantitative scale. Four values in a scale of 0% to 100% of spines protruding through the sloughed epithelial cells were used—0%–25%, 25%–50%, 50%–75%, and 75%–100% protrusion—to span heavy to negligible covering.
Spines covering the surface of the penile skin were counted in 3 sections per mouse: the top, mid, and base sections, which each totaled one-third of the length of the penis. The lengths of an average of 14 fully protruding spines (range, 8–21) were measured in a defined mean area of 292 nm2 (range, 119–510 nm2). The same plane of view was used for each sample so that spines measured from a distance further away owing to curvature of the tissue were excluded.
Histologic Analysis of the Penis
Adult male Tgfb1+/+ and
Tgfb1–/– mice were killed at 10 weeks, 1.5
hours after injection with 1 mg bromodeoxyuridine (BrdU; Sigma-Aldrich, St
Louis, Missouri). The penis was dissected at the joint and either fixed
overnight in 4% paraformaldehyde, followed by phosphate-buffered saline (PBS)
wash and paraffin embedding or frozen in optimal cutting temperature compound
and stored at –80°C prior to staining. Five-µm sections were
mounted on glass slides and stained for histologic analysis using Masson
trichrome staining or immunohistochemical staining using antibodies reactive
with BrdU (to detect proliferating cells), vasoactive intestinal peptide
(VIP), neuronal NOS (nNOS), smooth muscle actin (SMA), endothelial cells,
macrophages, or collagen I.
Skin thickness was quantified in hematoxylin-stained longitudinal sections. Whole sections were scanned using a NanoZoomer (Hamamatsu, Herrsching am Ammersee, Germany), and the midpoint between the base and tip was determined. The thickness of the combined epidermis and dermis was measured on both sides of the penis at the midpoint using NDP view software (Hamamatsu), and values were averaged to give a skin thickness value for each mouse.
Masson trichrome staining was used to evaluate the general histology of the penis. Briefly, dewaxed sections were stained for 20 minutes in Weigert hematoxylin, red cytoplasmic stain (2 parts 1% Xylidine ponceau, 1 part acid fuchsin in 1% acetic acid) for 1 minute and methyl blue for 3 minutes, with washing steps in between. After dehydrating in ethanol, the slides were cleared in Safsolvent (Labchem, New South Wales, Australia) and mounted on coverslips.
Nerve fibers and nNOS were detected by immunohistochemistry using antibodies specific for VIP and nNOS, respectively. Endogenous peroxidase was blocked by treatment for 15 minutes with 3% hydrogen peroxide in 50% methanol. Nonspecific binding was blocked by incubation for 30 minutes with 10% normal mouse serum/10% normal goat serum at 37°C. The primary rabbit polyclonal antibodies (both from Chemicon, Temecula, California) were used at dilutions of 1:1000 (VIP) and 1:1000 (nNOS) incubated overnight at 4°C. Following washing with PBS, the slides were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, California) at a 1:2000 dilution for 40 minutes at room temperature. After washing, the slides were incubated with ABC Elite (Vector Laboratories) according to the manufacturer's instructions, and 3,3'-diaminobenzidine (DAB) staining followed. The slides were counterstained with hematoxylin and mounted.
SMA and endothelial cells were detected as previously described (Godfrey et al, 1988; Palese et al, 2003). Five-µm frozen sections were mounted on glass slides and fixed in ice-cold acetone for SMA staining or fixed in 96% ice-cold ethanol for endothelial cell staining. The slides were blocked with 2% bovine serum albumin for 30 minutes at room temperature and incubated with alkaline phosphatase–conjugated anti-SMA (Sigma-Aldrich) at a 1:30 dilution for 1 hour or endothelial cell–reactive MTS-12 (neat hybridoma supernatant) (kind gift of Richard Boyd, Monash University, Victoria, Australia) with 10% normal mouse serum at 4°C overnight. After washing with PBS, the sections probed for SMA were incubated with Fast Red TR/naphthol AS-MX substrate (Sigma-Aldrich) for 10 minutes, rinsed in PBS, and counterstained with hematoxylin. After dehydrating, aqueous mounting medium (Cell Marque Corporation, Rocklin, California) and coverslips were applied. The sections probed with MTS-12 were incubated with horseradish peroxidase (HRP)–conjugated anti-rat antibody (Dako, Carpinteria, California) at a 1:100 dilution with 10% normal mouse serum for 2 hours at 4°C. After washing with PBS, sections were incubated with DAB, counterstained with hematoxylin, dehydrated, and mounted as above.
Macrophages were detected by F4/80 antibody staining. After dewaxing and rehydrating of the paraffin-embedded sections, the slides were incubated with F4/80 antibody (Caltag Laboratories, Burlingame, California) at a 1:50 dilution at 4°C overnight. After washing in PBS, the slides were incubated with biotinylated rabbit anti-rat IgG (Vector Laboratories) at 1:100 for 40 minutes at room temperature. After washing, the slides were incubated with ABC Elite (Vector Laboratories), followed by standard immunohistochemical steps as described above. Epithelial proliferation was measured by BrdU incorporation. A commercial BrdU detection kit (BD Pharmingen, San Diego, California) was used according to the manufacturer's instructions. Tissues were counterstained with hematoxylin. Manually counted BrdU-positive cells were expressed as a percentage of the total number of epithelial nuclei. The number of proliferating cells was quantified separately in epithelium beneath spines and adjacent to spines.
Collagen I was detected by immunofluorescent microscopy similar to that described previously (Ingman et al, 2006b). Briefly, 5-µm frozen sections mounted on glass slides were blocked for 10 minutes with 10% normal goat serum in PBS and incubated with a 1:40 dilution of rabbit anti-mouse polyclonal collagen I antibody (Chemicon) for 1 hour at room temperature. Following PBS washing, the slides were incubated with a 1:200 dilution of fluorescein isothiocyanate (FITC)–conjugated swine anti-rabbit IgG (Dako). After further PBS washing, Vectashield mounting medium with 4',6-diamidino-2-phenylindole (Vector Laboratories) and coverslips were applied. Collagen immunofluorescence was detected with a confocal microscope (Nikon C1 confocal scanning head, Nikon TE2000E; Nikon Corporation, Tokyo, Japan). Mean fluorescence intensity was calculated in ImageJ software (Rasband).
Western Blot
A Western blot for inducible NOS (iNOS) was performed as described
previously (Marin et al,
1999). Briefly, penis tissue was homogenized in protein extraction
buffer (0.075 M Tris-HCl [pH 6.8], 10% glycerol, 2.3% sodium dodecyl sulfate,
5% β-mercaptoethanol), boiled for 5 minutes, and centrifuged at 13 000
x g for 5 minutes. Protein was quantified using the Bradford
assay (Bio-Rad Laboratories Pty Ltd, Gladesville, New South Wales, Australia),
and 70 µg was mixed with 5 x Laemmli buffer followed by gel
electrophoresis on a 10% acrylamide gel that included prestained molecular
weight markers. Proteins were transferred onto polyvinylidene difluoride
membranes (Immobilon-P; Millipore, North Ryde, New South Wales, Australia).
Membranes were blocked in 10 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween-20
(TBST) containing 4% nonfat milk for 1 hour at room temperature, followed by
incubation with rabbit anti-iNOS antibody (BD Transduction Laboratories,
Lexington, Kentucky) diluted 1:1000 in 4% milk for 2 hours at room
temperature. Membranes were washed 6 times for 5 minutes each in TBST followed
by incubation with a 1:5000 dilution of HRP-linked anti-rabbit IgG (Millipore)
for 1 hour at room temperature. Following washing in TBST, enhanced
chemiluminescence detection was performed according to the manufacturer's
instructions (GE Healthcare Life Sciences, Rydalmere, New South Wales,
Australia). Single bands were detected at 150 kd. The films were scanned and
quantitated using an ImageQuant ECL Capture transilluminator and ImageQuant
Capture software (GE Healthcare Life Sciences).
Behavioral Studies
To investigate the effect of Tgfb1 null mutation on noncontact
erectile behavior, males were placed individually in clear-walled cages and
observed for 1 hour in the afternoon, between 1300 hours and 1600 hours. The
number of genital grooming events and incidence of erections were recorded
under these conditions. A genital grooming event was recorded each time the
mouse bent over on the hind legs to lick the genitals in the absence of an
erection. An erection was counted when the mouse bent over to groom the
genitals as the penis emerged.
To investigate the effect of sildenafil citrate on mating competence, sexual behavior was evaluated in sexually inexperienced, virgin Tgfb1–/– and Tgfb1+/± males of 8 to 10 weeks, as described previously (Ingman and Robertson, 2007). Briefly, 4-week-old B10.BR females (ARC, Perth, Australia) were induced into estrus by treatment with follicle-stimulating hormone (Folligon; 5 IU IP at 1000 hours on day 1) followed by human chorionic gonadotropin (Chorulon; 5 IU IP at 1000 hours on day 3). Male mice were injected with sildenafil citrate (400 µg/kg) 30 minutes prior to addition of a single superovulated female to the male's cage at 2200 hours on day 3 of the superovulation protocol. The dose of sildenafil citrate used was 2-fold greater than the dose required to enhance intracavernosal pressure by electrical stimulation in anaesthetized mice (Mizusawa et al, 2001). Behavior was recorded with a video camera for the following 2 hours under red light. The presence of a plug or sperm-positive vaginal smear the following morning indicated a mating event. Superovulation and thus sexual receptivity in the female was confirmed by the presence of ovulated oocytes in the oviduct. If the female failed to ovulate, data was excluded and the experiment repeated using a new female the following evening.
Male sexual behavior was quantified by blinded analysis of the video tape footage. Parameters of sexual behavior were quantified for each male, including 1) the amount of time spent in anogenital investigation during the first 10 minutes after introduction of a female, 2) latency (the time interval between introduction of the female and first mount), 3) the number and duration of mounting events, 4) the number and duration of intromission events (defined as mounting with thrusting), and 5) the occurrence of ejaculation. Each behavior was identified according to characteristic features as previously described (McGill, 1962).
Statistical Analysis
When Shapiro-Wilk tests showed a parameter to be not normally distributed,
a nonparametric analysis was performed. When normal distribution was evident,
a parametric analysis was performed. Nonparametric analyses were performed
using the Mann-Whitney U test (2-tailed significance values), and
parametric analyses were performed by Student's t test. Statistical
significance was assumed at P < .05, and all tests were analyzed
using SPSS version 12 (SPSS Inc, Chicago, Illinois).
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Results |
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Further analysis was conducted to determine whether the build-up of superficial epithelium was caused by increased epithelial cell proliferation. Representative images of BrdU-positive cells in Tgfb1+/+ and Tgfb1–/– penises are shown (Figure 3A and B, respectively). BrdU-positive cells were observed in the basal epithelium adjacent to and beneath spines but were not present in surface epithelial sheets. No differences in the number of BrdU-positive cells were seen between genotypes, irrespective of their location either beneath or adjacent to spines (Figure 3C). In addition, when the 2 locations were combined, no significant difference was evident between genotypes (data not shown). Similarly, TGFB deficiency was not associated with any change in the relative proportion of epithelial cells incorporating BrdU in other epithelial tissues, including thigh skin or crypts of the small intestine (data not shown).
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Erectile tissue in Tgfb1 replete and null mice was highly innervated, with VIP staining detected in the smooth muscle of the corpus cavernosum and ganglions (Figure 7A and B). These nerves were positively stained for nNOS (Figure 7C and D). The abundance of iNOS detected by Western blot was not altered in protein extract of penis from Tgfb1 null mutant mice (Figure 8A and B). Macrophages, detected by F4/80 staining, were located within the erectile tissue and in the dermal tissue beneath the penile spines of both Tgfb1+/+ and Tgfb1–/– mice (Figure 8C and D, respectively). There was no overt difference between genotypes in the relative density or location of staining for VIP, nNOS, or F4/80.
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A genital grooming event was recorded each time the mice bent over on their hind legs, in the absence of an erection, to lick the genital area. A similar number of Tgfb1+/+ (89%) and Tgfb1–/– (83%) mice performed at least 1 genital grooming act, with the number of grooming acts ranging from 0 to 12. However, a smaller proportion of Tgfb1–/– mice (56%) displayed more than 1 grooming act compared with the Tgfb1+/+ mice (89%; Figure 9C). A significant difference between genotypes was also observed when grooming was expressed on a per hour basis. Tgfb1–/– mice (2.4 ± 0.6 grooming events/hour) performed genital grooming less frequently than Tgfb1+/+ mice (6.4 ± 1.3 grooming events/hour) (P = .007; Figure 9D).
Effect of Sildenafil Citrate on Copulatory Behavior in Tgfb1 Null Mutant Mice
To enhance NO-mediated smooth muscle relaxation and improve erectile
function in Tgfb1 null mutant males, mice were injected with
sildenafil citrate (Pfizer, Sandwich, United Kingdom) prior to exposure to a
superovulated receptive female. The dose of sildenafil citrate used was double
that required to enhance intracavernous pressure by electrical stimulation in
anesthetized mice (Mizusawa et al,
2001). As reported previously, intromission duration and frequency
were reduced in Tgfb1–/– mice, and ejaculation
did not occur (Ingman and Robertson,
2007). Sildenafil citrate did not affect the ability of
Tgfb1+/+ males to mount, intromit, or copulate and did not
improve these parameters in Tgfb1–/– mice
(Table).
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Discussion |
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Reduced Erectile Capacity in Tgfb1 Null Mutant Mice
We previously noted that Tgfb1 null mutant male mice show reduced
intromission frequency when housed with receptive females, and ejaculation
behavior was completely absent (Ingman and
Robertson, 2007). Observation of the erectile response within the
context of behavior to a responsive female is not feasible; therefore, in the
current study, we assessed noncontact spontaneous erectile activity to assess
erectile function. Tgfb1 null mutant mice exhibited a reduction in
both genital grooming activity and the number of noncontact spontaneous
erections. This suggests that reduced sexual function in Tgfb1 null
mutant males might be partly due to diminished erectile activity, although it
should be noted that site-specific lesions in the brain have been seen to
affect noncontact erections differently than copulatory activities
(Liu et al, 1998). Measurement
of corpus spongiosum pressure during erectile activity would be required to
quantify this response (Soukhova-O'Hare et
al, 2007).
Our results suggest that reduced erectile activity might be due to penile fibrosis leading to reduced tissue compliance because increased collagen I was observed in the penis of Tgfb1 null mutant mice compared with wild-type controls. Increased collagen deposition in conjunction with reduced smooth muscle and endothelial cells is consistent with a diagnosis of vasculogenic erectile dysfunction (Gonzalez-Cadavid and Rajfer, 2004); however, we did not observe vascular changes in the penises of Tgfb1 null mutant mice.
TGFB1 is known to increase collagen synthesis by human corpus cavernosum smooth muscle cells (Moreland et al, 1995) and cause penile fibrosis in a rat model of Peyronie disease (El-Sakka et al, 1997; Bivalacqua et al, 2000), contrasting our observation of an increased level of collagen in the absence of TGFB1. In the rat model, TGFB1 treatment causes increased levels of iNOS in the penis, presumably through an inflammatory mechanism, because macrophages are key cells that express iNOS and can mediate vasodilation. We did not observe any evidence of increased iNOS expression or inflammation in tissue from Tgfb1 null mutant mice, and it is therefore likely that the increased collagen deposition in the absence of TGFB1 occurs via a different mechanism than the increased fibrosis observed with increased TGFB1 in the rat model of Peyronie disease.
TGFB1 exerts a diverse array of effects on reproductive tissues, and the
mechanism by which TGFB1 deficiency leads to increased collagen remains
unclear. Erectile dysfunction is not reported in other cytokine-deficient
mouse models (Ingman and Robertson,
2008), apart from mice deficient in colony-stimulating factor 1
(CSF1). These CSF1-deficient mice have reduced mating ability, although this
is secondary to testosterone synthesis
(Cohen et al, 1996). However,
deficiency in the proinflammatory cytokine tumor necrosis factor 
(TNFA) causes an increased frequency of spontaneous noncontact erections and
increased relaxation of the corpus cavernosal smooth muscle in vitro
(Carneiro et al, 2009).
Tnfa null mutation does not cause an overt fertility phenotype
(Taniguchi et al, 1997). This
subclinical result suggests that there may be other cytokines involved in the
fine tuning of the erectile response. The balance between proinflammatory
cytokines such as TNFA and anti-inflammatory cytokines such as TGFB1 might be
of critical significance in regulating the sensitivity of the penis to an
erectile stimulus. Amplification of the erectile response was attempted with
sildenafil citrate, which increases NOS-mediated smooth muscle relaxation.
This was unsuccessful in improving intromission or ejaculation behavior in
Tgfb1 null mutant mice. However, continuous long-term administration
of sildenafil citrate (45 days) is necessary to reduce collagen deposition in
age-related erectile dysfunction in rats
(Ferrini et al, 2007), and
future studies would be required to investigate this in Tgfb1 null
mutant mice.
We cannot exclude the possibility that reduced sexual function in Tgfb1 null mutant mice might also be the result of impaired central signaling mechanisms within the brain that stimulate erectile function. Defects in neuronal innervation of the brain (Farkas et al, 2003), dopamine production (Hull et al, 2004), or central lesions (Liu et al, 1997) are each reported to reduce the incidence of erectile activity.
Abnormal Penile Skin in Tgfb1 Null Mutant Mice
SEM revealed the presence of superficial keratinized epithelial cells
covering the surface of the Tgfb1 null mutant penis. Penile spines
were visible protruding through the covering, although the penile spines were
almost completely obscured in some Tgfb1 null mutant mice. The
superficial epithelial cells could not be attributed to excessive epithelial
cell proliferation, despite several in vitro and in vivo studies showing the
antiproliferative effects of TGFB1 on keratinocytes
(Moses et al, 1991;
Glick et al, 1993;
Wang et al, 1997;
Koch et al, 2000;
Pasonen-Seppanen et al, 2003).
A reduction in sloughing or shedding of keratinized skin tissue in
Tgfb1 null mutant mice, perhaps as a secondary consequence of reduced
genital grooming, might contribute to the abnormal build-up of this material.
However, the additional observation of reduced skin thickness, particularly
the diminished dermal layer, suggests that the surface changes might originate
deeper in the dermis and epidermis. This reduced thickness of the penis skin
is consistent with previous reports of reduced epidermal and dermal thickness
in abdominal skin in the absence of this cytokine
(Koch et al, 2000). Although
the molecular mechanisms linking TGFB1 deficiency with altered skin structure
remain to be defined, this cytokine is synthesized in the dermis and epidermis
of normal skin (Ghahary et al,
1995) and has key roles in regulating extracellular matrix and
adhesion molecule synthesis in skin fibroblasts and keratinocytes
(Kahari et al, 1991;
Jeong and Kim, 2004).
Altered Penile Spines in Tgfb1 Null Mutant Mice
The role of penile spines in copulation and ejaculation of rodent species
is poorly understood, and there is no consensus view on their requirement for
copulation in other mammals (Aronson and
Cooper, 1967; O'Hanlon and
Sachs, 1986; Dixson,
1991). It seems reasonable to speculate that impaired protrusion
of spines owing to excess superficial tissue on the penis surface would
interfere with the anchoring function of penile spines in the Tgfb1
null mutant mice, perhaps resulting in failure to gain adequate attachment to
the female to facilitate sustained intromission. The modest effect of cytokine
deficiency on spine number at the penis tip in TGFB-deficient males could
exacerbate any impaired attachment. Because spine development is androgen
dependent (Murakami, 1987),
this altered distribution pattern might be due to reduced testosterone in
Tgfb1 null mutant mice; however, importantly, any testosterone effect
did not extend to reduced spine size.
The topography of sensory nerve endings adjacent to penile spines in the superficial dermis of the penis is consistent with a sensory function (Johnson and Halata, 1991); in primates, removal of spines extends the duration of intromission required for ejaculation and causes aborted intromission events (Dixson, 1991). In Tgfb1 null mutant mice, impaired male-female anchoring could interfere with a sensory feedback response required for continued copulation. Conversely, reduced penile skin thickness might lead to avoidance of female contact or grooming because of amplified sensation or pain. This situation is likely to be exacerbated by low testosterone in Tgfb1 null mutant mice (Ingman and Robertson, 2007), which is associated with elevated penile tactile sensitivity (Burris et al, 1991). Although the superficial epithelial tissue might be expected to cause irritation to the penile skin, no signs of inflammation were observed, and macrophages are similarly abundant in both wild-type and Tgfb1 null mutant tissue.
Conclusion
These findings shed light on the role of TGFB1 in penile functioning and
male reproductive behavior. In the absence of this cytokine, spontaneous
erections occur at a reduced frequency, and sildenafil citrate treatment does
not improve copulatory behavior. Altered structural integrity of the penile
skin associated with a thinner dermis and build-up of superficial keratinized
epithelial cells results in reduced penile spine protrusion, which potentially
impacts the ability of mice to intromit and ejaculate. Changes in collagen I
deposition might impair tissue compliance and lead to reduced erectile
capacity. Thus, we suggest 2 mechanisms by which the Tgfb1 null
mutation might influence mating competence: 1) altered skin sensory stimulus
or pain and 2) erectile dysfunction. These effects may contribute, together
with the previously described reduction in testosterone, to explain the
profound infertility and inability of the Tgfb1 null mutant mice to
copulate successfully. Further studies are required to investigate the
relative contribution of these 2 mechanisms to the infertility phenotype.
Our observations in mice may be relevant to some forms of erectile dysfunction in men when altered plasma TGFB1 levels are associated with erectile dysfunction and changes to the vascular system (Ryu et al, 2004). Furthermore, because tactile responsiveness of the penile skin is an important determinant of erectile function and ejaculation in men, the Tgfb1 null mutant mouse may have utility in modeling sexual dysfunction linked with anomalous sensitivity owing to aging, vascular pathophysiology, or conditions such as diabetes (Rowland et al, 1993; Morrissette et al, 1999).
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Acknowledgments |
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Footnotes |
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These authors contributed equally to this research. 
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2 and Mann-Whitney U test,
respectively. *P < .05.