of castration and testosterone replacement on veno-occlusion during penile
erection in the rat
Yu-Tian DAI1, Vivienne Stopper2, Ronald Lewis1, Thomas Mills1,2
of Surgery, Urology Section, 2 Department of Physiology and
Endocrinology Medical College of Georgia, Augusta, GA 30912-3000, USA
Asian J Androl 1999 Jun; 1: 53-59
Keywords: castration; testosterone; cavernous sinus; penile erection; nitric oxide; androgens
AbstractAim: To determine if androgens directly regulate veno-occlusion or if androgens act indirectly to maintain the penile structures which control outflow. Methods: Using CASTRATE and TESTO rats, measurement was made of mean arterial pressure (MAP), intracavernosal pressure (CCP), and intracavernosal flow (CCF) during erection resulting from stimulation of the autonomic innervation of the penis. CCP and CCF were also measured during saline infusion into the cavernosal sinuses before and after treatment with sodium nitroprusside (SNP, a nitric oxide donor drug) to fully relax cavernosal smooth muscle. Penile tissue was also collected to measure the content of actin and proline and hydroxyproline to determine if brief withdrawal of androgenic support led to changes in the number of smooth muscle cells or the collagen content of the tissue. Results: Infusion of saline into the cavernosal sinuses demonstrated that veno-occlusion was defective in CASTRATE rats while veno-occlusion was fully functional in TESTO animals. Furthermore, veno-occlusion could be induced in CASTRATE rats if they were first treated with SNP. This observation suggests that failure of veno-occlusion in the CASTRATE rats is due to a deficiency in the production of NO resulting in a reduction in the degree of relaxation of the penile smooth muscle. The measurements of smooth muscle actin and proline and hydroxyproline content of collagen showed that both were unaffected by castration and that the basic structure of the penis did not degenerate after one week without androgenic support. Conclusion: These results can be interpreted to mean that androgens control the veno-occlusive mechanism indirectly via a NO dependent mechanism and not by maintaining the structures of the penis which are essential to veno-occlusion.
1 IntroductionDilation of the arterioles which carry blood into the cavernous sinuses is essential to the mammalian erectile response. Of equal importance, however, is veno-occlusion, the pressure dependent reduction in the rate at which blood flows out of the sinuses. It is the combination of the increased inflow and the decreased outflow which leads to the high intracavernosal pressures characteristic of penile erection. Studies from this and other laboratories have demonstrated that the erectile response in rats is regulated, in part, by androgens with the hormone acting to control the synthesis and actions of nitric oxide, the principle arteriolar and cavernous smooth muscle relaxing neurotransmitter in erection[2-7]. Despite the importance of veno-occlusion to the erectile response, however, the need for androgenic support of this process has not been well studied although we have previously speculated that testosterone may play a role. In our prior report, veno-occlusion appeared to be compromised in castrated animals as demonstrated by the need for a significantly higher rate of saline infusion into the cavernous sinuses to achieve veno-occlusive pressures in the castrated animals receiving no hormone replacement (CASTRATE) than in castrated animals which were implanted with a testosterone pellet (TESTO). In this experiment, however, no attempt was made to fully relax cavernosal smooth muscle prior to the saline infusion. More recently, we employed a laser Doppler flow meter to demonstrate that, during electrically induced erection, veno-occlusion occurred in TESTO rats but not in CASTRATE animals. If, however, the nitric oxide (NO) donor drug sodium nitroprusside (SNP) was first injected, veno-occlusion did occur in CASTRATE rats. Based on this observation, we suggested that veno-occlusion may be an androgen-dependent process and mediated by NO. The present study was undertaken to confirm that veno-occlusion was an androgen-dependent process by measuring the intracavernosal pressure during infusion of saline into the intracavernosal sinuses at rates adequate to cause veno-occlusion. Furthermore, these measurements were made before and then again after complete relaxation of all penile smooth muscle by the administration of a NO donor drug. The studies also investigated the possibility that differences in veno-occlusion in CASTRATE and TESTO rats were due to differences in the structure of the penis specifically the actin content as a measure of the number of smooth muscle cells and the content of collagen, the protein which is responsible for the compliance of the tunica albuginea.
2 Materials and methods
Holtzman rats (Harlan Sprague Dawley, Inc. Indianapolis, IN, USA), 6 to
8 months of age, were used in these studies. Animals were castrated under
ether anesthesia and implanted subcutaneously with a testosterone (Steraloids,
Inc, Wilton, NH, USA) pellet (3 mm diameter, weight 3-5 mg, 1:1, testosterone:
cholesterol-group designation: TESTO) or a control pellet of cholesterol
only (group designation: CASTRATE). The animals were used in the studies
6 to 8 days after castration. We previously reported that one week of
castration led to a significant decline in the magnitude of the erectile response and that
prolonging this post-castration period to as long as 7 weeks did not lead
to a further decline in the erectile response. For all surgical
procedures, rats were anesthetized with intramuscular ketamine (Ketaset,
Fort Dodge Labs, Fort Dodge, IA, USA, 87 mg/kg body weight) plus xylazine
(Sadazine, Fort Dodge Labs, Fort Dodge, IA, USA, 13 mg/kg body weight)
with supplemental ketamine as needed. All procedures were performed in
accordance with the Guiding Principles in the Care and Use of Animals
approved by the American Physiological Society.
2.2 Measurement of the intracavernosal pressure (CCP)
The procedure used to measure the intracavernosal pressure (CCP) has been previously described from this laboratory[4,8,10,11]. Briefly, in this method, the animals are anesthetized, the left carotid artery is cannulated for continuous monitoring of mean arterial blood pressure (MAP). Once this cannula was in place, the right corpus cavernosum was cannulated by insertion of a 30 gauge needle attached to PE 50 tubing which had been drawn to a fine tip and connected to a pressure transducer to permit continuous monitoring of CCP (Statham P23AC transducers and Grass 7D polygraph from AstroMed, Grass Instrument Division, Braintree, MA, USA). The left corpus cavernosum was cannulated with a 30 gauge needle attached to pump for the infusion of saline at rates ranging from 50 to 1000 L/min into the cavernous sinuses. This cannula was also used for the delivery of 1-2 L of saline containing sodium nitroprusside (SNP, Sigma-Aldrich, St Louis, MO, USA).
2.3 Estimation of the intracavernosal pressure at which veno-occlusion occurs.
A laser Doppler flow meter (Periflex PF3, PeriMed Stockholm, Sweden) was employed to measure the movement of blood in the cavernous sinuses during erection. The FC 302 laser Doppler flow detection probe was positioned on the left corpus cavernosum at the base of the penis. The proper positioning of the probe was confirmed during ganglionic stimulation when the turbulence caused by partial veno-occlusion could be heard (audible output of the flow meter) as pulses marking the flow spurts during systole. To estimate the intracavernosal pressure (CCP) at which veno-occlusion takes place, the intracavernosal pressure (CCP) and intracavernosal blood flow (CCF) were continuously monitored before and during erection resulting from ganglionic stimulation at 5 V. Figure 1 shows a diagrammatic representation of the CCP and CCF during induced erection. To estimate the CCP at veno-occlusion, the CCP was determined when CCF was maximal (line A, Figure 1), when CCF had declined by 50% (line B) and at the minimal CCF during veno-occlusion (line C). Based on these measurements, we selected 55 mmHg (7.3 kPa), the CCP approximately half way between points B and C, for use in the subsequent saline infusion studies.
Figure 1. A representative tracing of the intracavernosal blood
pressure (CCP) and intracavernosal blood flow (CCF) during erection induced
by electrical stimulation of the major pelvic ganglion (horizontal black
bar). Measurements were made in 6 TESTO rats to determine the CCP values
at maximal CCF (A), at half
maximal CCF (B) and at minimal CCF (C). Measured average values (meanSEM)
were A=(71) mmHg (1.00.2) kPa, B=464 mmHg (6.10.5) kPa,
C=(595) mmHg (7.80.6) kPa and based on these results, the value
of 55 mmHg (7.3 kPa) was selected as the point at which veno-occlusion
Determination of the saline infusion rate required for veno-occlusion
anesthesized rat was placed on a heating pad and the left corpus cavernosus
was cannulated for continuoum measurement of CCP. The right corpus cavernosum
received a cannula made from PE 50 tubing attached to a 28 gauge needle
connected to a variable speed syringe pump for infusion of saline into
the cavernous sinuses. The infusion rate was increased step-wise (50-800
L/min in 50 or 100 L/min steps) until an infusion rate was reached
at which CCP rose to 55 mmHg (7.3 kPa) or higher within 3 min indicating
veno-occlusion. Once the infusion rate needed for veno-occlusion had been
determined for each animal, sodium nitroprusside (SNP-8 g/kg body weight)
was injected in 1-2 L saline directly into the cavernous sinuses to
achieve complete relaxation of all smooth muscle and the step-wise saline
infusion procedure was repeated.
Determination of the smooth muscle actin content of penile tissue collected
from CASTRATE and TESTO rats.
whole penis was collected from CASTRATE and TESTO rats and rapidly cleaned
including the removal of the dorsal vein and the corpus spongiosum. The
distal portion was discarded and the remainder was cut into four pieces
(left and right crus, left and right shaft) for quick freezing in liquid
nitrogen and storage at -80. For actin, the left crus and left shaft
were weighed and extracted in TRIS buffer containing 2% SDS using a polytron
homogenizer. Following centrifugation to remove particulate matter, an
aliquot was taken for determination of the total protein content using
the BioRad DC protein assay and the remaining samples were stored at -20.
For Western analysis,
10 g of total protein from each sample was loaded on to 12% polyacrylamide
Ready Gels (Bio-Rad Laboratories, Hercules, CA, USA) and the proteins
were separated by electrophoresis with standard molecular weight markers,
a negative control of liver protein and a positive control for actin
prepared from rat aorta. The separated proteins were transferred to a nitrocellulose membrane and
actin was detected on the membrane by immunochemical staining using
a mouse monoclonal first antibody followed by the biotinylated second
antibody. Color was developed using the AEC stain (Fisher Scientific,
Inc, Pittsburgh, PA, USA). The stained membranes were scanned and density
of the actin staining quantified with an IS-1000 Digital Imaging System
(Alpha Innotech Corp, San Leandro CA, USA). The amount of actin in each
sample was determined relative to the standard prepared from rat aorta
Determination of the proline and hydroxyproline content of penile tissues collected
from CASTRATE and TESTO rats.
The right crus and right shaft from each penis (CASTRATE and TESTO animals) were weighed, homogenized in cold 5% perchloric acid (PCA) with a polytron homogenizer and the precipitated proteins collected by centrifugation. The protein pellet was washed several times with cold PCA and the final pellet was reconstituted in 4 mL of 6 mol/L HCl. The proteins were hydrolyzed for 24 h at 110 and then dried under a nitrogen stream. The resulting residue was reconstituted in acetonitrile: water (1:1) and derivatives of the amino acids prepared using PITC. The content of proline and hydroxyproline was determined by high performance liquid chromatography (Model 110A, Beckman Instrument Co, Fullerton, CA, USA) using an Econosphere C18 5 micron column and the resulting quantities expressed as nmol/mg tissue.
In our prior reports[4,8], circulating levels of testosterone were approximately 1 g/L in castrated rats receiving a single pellet of 50% testosterone (TESTO) while in castrated control rats (CASTRATE), levels were less than 25 ng/L. In the present study, blood was not collected for testosterone measurement but, at necropsy, the sizes of the seminal vesicles, ventral prostate, the dorso-lateral prostate were grossly examined for size and apparent secretory activity. Since these are androgen sensitive tissues, their appearance served to confirm the treatment group to which each animal belonged (ie, CASTRATE or TESTO).
determine the intracavernosal pressure at veno-occlusion, measurements
were made of CCP at maximal, minimal and half maximal flow (CCF) rates
(Figure 1). Based on measurements made in 6 TESTO rats, veno-occlusion
must occur when CCP is greater than CCP at maximum inflow of 71 mmHg
(1.00.2 kPa) and greater than the half maximal flow rate (B) of 464
mmHg (6.10.5 kPa) but less than 595 mmHg (7.80.6 kPa) at C. For
use in the intracavernosal saline infusion studies, the assumption was
made that veno-occlusion occurred by the time CCP reached 55 mmHg (7.3
the value of 55 mmHg (7.3 kPa) as the pressure at which veno-occlusion
occurs, saline was infused in a step-wise fashion into the cavernous sinuses
until veno-occlusion occurred. Figure 2
showed the perfusion rate mL/(minkg) which was required for veno-occlusion
and confirms our prior report that a significantly greater rate of saline
infusion is required to achieve veno-occlusion in CASTRATE than in TESTO
rats. After measuring these infusion rates, each rat was injected (intra cavernous
injection) with sodium nitroprusside (SNP) to cause complete relaxation
of all cavernosal and arteriolar smooth muscle in the penis and the step-wise
saline infusion protocol was repeated. After injection of this NO donor
drug, the infusion rates required for veno-occlusion were significantly
reduced in both the CASTRATE and TESTO rats and there was no significant
difference between the rates required for veno-occlusion in the two treatment
Figure 2. Effects of castration and testosterone replacement
on the rate of saline infusion (mLmin-1kg-1
body weight) required to activate the
veno-occlusive mechanism in the rat penis. Infusion rates were determined
before and after intra-cavernosal injection of sodium nitroprusside (SNP).
Note that before SNP, the required infusion rate for veno-occlusion in
CASTRATE rats is significantly higher than the rate in TESTO rats (*).
After SNP, the rate in both
groups is significantly reduced but there is no difference in the rates
between CASTRATE and TESTO animals. Bars represents the meanSEM of 7 TESTO rats or 10 CASTRATE animals.
The results in Figure 3 showed that there was no difference in the actin content of either the shaft or the crus of the penis in CASTRATE and TESTO animals when measured by immunochemical blotting. Similarly, HPLC measurement made in hydrolyzed penile tissue samples revealed no difference between TESTO and CASTRATE rats in the proline or hydroxyproline (Figure 4) content of either the shaft or crus.
Figure 3. Western analysis of the content of actin of the
shaft and crus portion of penis collected from TESTO and CASTRATE rats.
Each bar represents
the SEM of individual measurements made in duplicate on tissues from
4 CASTRATE and 5 TESTO rats. Statistical analysis reveals that there is
no significant difference between CASTRATE and TESTO rats in either shaft
or crus samples.
is no significant difference in either amino acid between CASTRATE and TESTO
in shaft and crus. There is, however, significantly more proline than
hydroxyproline present in both tissue locations.
from this and other laboratories have shown that the erectile response
in the rat is dependent on testosterone[4,7,11,13-15]. After
a period of castration lasting 1 week, there is about a 50% decrease in
the magnitude of the erectile response when expressed as the intracavernosal
pressure relative to mean arterial pressure. If the castrated animals
were treated with testosterone[4,13,15] or dihydrotestosterone
the erectile response was fully maintained. The mechanism by which androgens
maintain erection has been widely investigated and many laboratories have
shown that androgens act to support the synthesis of NO[4,5,7,16].
Androgens have also been reported to reduce the sensitivity of the erectile
tissue to the constricting effects of sympathetic stimulation,
may act via a non-NO dependent pathway or act on central
erectile mechanisms[18,19]. The full erectile response requires
the activation of two processes: 1) relaxation of the smooth muscle in
the cavernosal arterioles to permit greater blood flow into the cavernous
sinuses. 2) relaxation of the smooth muscle in the cavernous sinuses so
that as blood flows in, the sinuses fill and expand against the tunica
albuginea. This sinusoidal expansion compresses and partially, or fully,
occludes the venous system which carries blood out of the sinuses and
the intracavernosal blood pressure rises under the force of the central
arterial pressure. We recently reported measurements of the inflow rates
during erection which showed that inflow is about twice as great in TESTO
rats as in CASTRATE animals demonstrating that the relaxation of the arteriolar
smooth muscle is under androgenic regulation.
The present study was undertaken to determine if the rate of outflow is
under androgenic regulation was well. Erectile dysfunction due to defects
in the veno-occlusive mechanisms is normally diagnosed by saline infusion
directly into the cavernous sinuses at increasing rates until veno-occlusion
is achieved and the flow required to maintain veno-occlusive pressure
is determined. If the flow to maintain is high, a venous
leak or fistula through which blood is diverted out of the cavernous tissue,
may be suspected. Veno-occlusive disease can also result from inadequately
or excessively compliant tunica albuginea or from a failure of the cavernous
sinuses to expand fully due to a reduced number of smooth muscle cells
or to a deficiency in their capacity to respond to the vaso-dilatory neurotransmitters
or reduced synthesis and release of the neurotransmitters[21,22].
Using a similar saline infusion technique, we have previously reported
that approximately twice as high a saline infusion rate was required to
cause veno-occlusion in CASTRATE than
in TESTO rats and we suggested a veno-occlusive disorder in the CASTRATE
animals. However, in our prior studies, the cavernosal smooth
muscle cells were not stimulated to relax prior to saline infusion; incomplete
relaxation of these cells could limit the degree of expansion of the cavernous
sinuses against the tunica albuginea and thereby prevent full veno-occlusion.
The studies of Hatzichristou and coworkers have stressed the importance of
injection of vasodilator drugs into the cavernous sinuses prior to attempting
to measure the pressure required for veno-occlusion. More recently, we
investigated veno-occlusion using a laser Doppler flow meter to follow
intracavernosal blood flow. Our studies showed that during electrically
induced erection, veno-occlusion occurred in the TESTO rats but not in
the CASTRATE animals. Veno-occlusion could, however, be induced in
the CASTRATE rats if they were first treated with SNP prior to saline
infusion. In this prior study, we did not establish how the additional
NO permitted veno-occlusion to occur. The NO could act directly on the
arterioles to permit greater blood inflow and/or NO could cause a greater
reduction in cavernosal smooth muscle tone and permit expansion of the
cavernous sinuses and veno-occlusion. The results presented in Figure 2
confirm our prior
report that a greater rate of saline infusion into the cavernous sinuses
was required in CASTRATE than in TESTO rats to achieve high intracavernosal
pressure. This figure also shows that after treatment with SNP, there
was no difference in the saline flow rate required to achieve veno-occlusion
in the CASTRATE and TESTO rats. Thus, the difference in veno-occlusion
in the CASTRATE and TESTO animals disappears when the NO donor drug was
used and this suggests that a deficiency in NO production or action is
the underlying basis for the failure of veno-occlusion following castration.
The target of the NO could be the arteriolar smooth muscle, the cavernosal
smooth muscle or both.
Next, we sought to determine if differences in the physical properties of the penis such as the compliance of the tunica albuginea contribute to the difference in veno-occlusion between TESTO and CASTRATE animals. If the tunica becomes less compliant, the loss of elasticity could interfere with one of the mechanisms of veno-occlusions. If the cavernosal smooth muscle is decreased or less reactive to neurotransmitters, the tissue would be less expansive and thereby reduce the compression of the outflow venous system and reduce veno-occlusion. Measurements were made of the total collagen content of the penile tissue by determining the amount of proline and hydroxyproline (Figure 4) in hydrolyzed proteins collected for CASTRATE and TESTO animals. These two amino acids constitute a significant portion of the collagen molecules which are present in the tunica albuginea and the trabecular matrix that makes up the framework of the erectile tissue. The failure to detect any difference in the quantities of either amino acid suggests that the collagen content is not changed after one week of castration and there is likely no important difference in the compliance of the tunica albuginea after this brief time period. Our studies were also designed to determine if differences in the veno-occlusive response in CASTRATE and TESTO rats were due to differences in the expression of actin protein in the cavernosal smooth muscle cells. Measurement of the quantity of this protein in the penis (Figure 3) shows no difference between the treatment groups indicating that an androgen dependent maintenance of the cavernosal smooth muscle population is likely not an essential part of veno-occlusion under these experimental conditions.
Thus, based on the results of this study, two conclusions appear to be warranted: First, androgens control the veno-occlusive mechanism during erection indirectly, by regulating the tone of the arteriolar and/or cavernosal smooth muscle. This action appears to be mediated by NO. Secondly, the major structural components of the penis essential to veno-occlusion are unaffected by a short period (one week) of castration even though the erectile response is markedly reduced over the same time period.
Andersson KE, Wagner G. Physiology of penile erection. Physiol Rev 1995;
Correspondence to Thomas M Mills, PhD, Professor.
Fax +1-706-721 7299
Received 1999-03-08 Accepted 1999-05-08