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From the Departments of * Biomedical Sciences and
Biology/CBR/RCMI, Tuskegee University,
Tuskegee, Alabama; and the Department of
Anatomy, Physiology, and Pharmacology, Auburn University, Auburn,
Alabama.
| Correspondence to: H. O. Goyal, Department of Biomedical Science, School of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088 (e-mail: goyalho{at}tuskegee.edu). |
| Received for publication April 28, 2004; accepted for publication July 16, 2004. |
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Abstract |
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Key words: Estrogen, DES, fertility, testosterone, bulbocavernosus
Considering the possible deleterious effects of estrogenic compounds on human health, our long-range goal is to understand how estrogens mediate pathophysiology of male reproduction. In this endeavor, we previously reported that adult rats treated neonatally with DES at a dose of 10 µg/pup/d (equal to a total dose of 3-4 mg/kg) on alternate days from postnatal days 2-12 were infertile, and the loss of fertility was associated with abnormal penile morphology, including replacement of cavernous spaces and associated smooth muscle cells by fat cells in the corpora cavernosa penis (Goyal et al, 2004a). Realizing the novelty of these findings and their possible effect in male human health, we first sought to determine the dose-dependent effects of neonatal DES exposure on penile morphology. In addition, we determined the effects on descent of testes, release of preputial sheath, and weights of bulbocavernosus and levator ani muscles because these parameters can affect function, development, or both of the penis and were not included in our previous study.
Second, we sought to compare the dose-dependent effects of DES with those of another potent estrogenic compound such as estradiol valerate (EV) because it has a uterotrophic effect and binding affinity for estrogen receptors similar to those for DES. Moreover, because both humans and wildlife are constantly exposed to a mixture of environmental estrogenic compounds, it will be significant to determine whether penile abnormalities resulting from the DES treatment can also be caused by another estrogen.
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Materials and Methods |
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Timed-pregnant female rats were housed individually. Within 24 hours of delivery, the litter size was adjusted to 8 pups/litter, with as many as 8 males, if possible. Pups (5-8 males/group, all pups within a group littermates, 2 groups each for controls and the 10- and 1-µg DES and EV treatments) received subcutaneous injections of 25 µL of olive oil containing DES or EV (Sigma, St Louis, Mo) at a dose of 10 µg, 1 µg, 100 ng, 10 ng, or 1 ng/pup/d on alternate days from postnatal days 2-12. The maximal 10-µg dose was selected on the basis of our previous study, in which it caused 100% infertility in male rats at adulthood; the loss of fertility was associated with the loss of cavernous spaces and accumulation of fat cells in the body of the penis (Goyal et al, 2004a). The control pups received olive oil only. Animals were weaned at 22 days and regularly observed for development until necropsied at 150 days.
Descent of Testes
Testicular descent was observed every other day from day 22 to day 38 and
thereafter every third or fourth day (every Friday and Tuesday of the week).
Testes were characterized as fully descended when they were palpated in the
scrotum while holding the animal in a supine position. The formation of a
scrotal bulge usually marked the descent in most animals.
Release of Preputial Sheath
The preputial sheath was examined every third or fourth day, beginning from
day 38 to 119, 1 day prior to the fertility trial. While holding the animal in
a supine position, the prepuce was gently pushed proximally (toward the
abdominal wall) and was characterized as fully released when it completely
retracted from the glans penis up to its transition with the shaft of the
penis. More frequent examination of the sheath was avoided because of the fear
of causing undue stress to the animal or accelerating the release process.
Fertility
Six to seven 120-day-old male rats from each of the 10 µg, 1 µg, 100
ng, 10 ng, and control groups were transferred to mating cages floored with a
mesh grid and cohabited with untreated, 60-70-day-old females (1:1) for 12
days. Cages were checked twice daily for the presence of copulatory plugs. The
plug-positive females were separated and evaluated for the presence of sperm
in vaginal washings. These females were killed on day 15 of pregnancy, and
those without plugs were killed on day 15 after the end of cohabitation. The
uterus was removed and examined for the number of implantation sites and live
fetuses. In addition, both ovaries were removed and the number of corpora
lutea was counted. Data were analyzed to determine the pre- and
postimplantation loss, as described previously
(Goyal et al, 2004a).
Body Weight and Organ Weights
All animals were weighed and terminated at 150 days. The testis, caput and
corpus of the epididymis, cauda of the epididymis, and seminal vesicle
(including coagulating gland) of both sides were weighed, and the relative
weights (weight of the organ per 100 g body weight) were calculated. Testes
and epididymides were trimmed of fat prior to recording their weights. In
addition, the caudal epididymal fat pad, located between the cauda epididymis
and the distal extremity of the testis, was removed and weighed. The rationale
was to determine whether DES, EV, or both had an effect on body fat because
estrogens have been shown to decrease the weight of parametrial fat pads
(Naaz et al, 2003). The caudal
epididymal fat pad was selected for convenience because it can be easily and
cleanly isolated.
Penis and Penile Skeletal Muscles
The penis was grossly examined for its length, diameter, and weight. The
prepuce was retracted or incised in animals in which it was still partially
attached to the glans, and the entire body of the penis was exposed by making
a dorsal skin incision that extended through the body wall up to the ischial
arch, the point of origin of the root of the penis. The stretched length was
measured from the tip of the glans penis to the midpoint of the ischial arch
and the diameter from the middle of the body of the penis with a caliper
(calibrations up to 0.1 mm). After removing the free loose connective tissue,
the entire penis was weighed. Sections (3-5 mm long) from the middle of the
body were processed for histological examination and histochemical
demonstration of fat (n 
5 per group, except n = 4 for the 10-µg EV
group) as described previously from our laboratory
(Goyal et al, 2004b). Briefly,
for histological examination, paraffin sections (5 µm thick) were stained
with hematoxylin and eosin (H&E), and for fat demonstration,
formaldehyde-fixed tissues were stained en block for 8 hours with 1% osmium
tetroxide dissolved in 2.5% potassium dichromate solution, then processed for
paraffin embedding. Undeparaffinized sections (5 µm thick) were examined by
light microscopy, and the adjacent serial sections were stained with H&E
to allow for examination of histological details. In addition, for controls
and the 10- and 1-µg DES and EV groups (n = 3-4 per group), epoxy sections
(1 µm thick) of glutaraldehyde-fixed tissues were stained with toluidine
blue, and frozen sections of formaldehyde-fixed tissues were stained with
Sudan Black. Also, to evaluate the development of the os penis, 1 penis from
each group was radiographed with a cabinet radiographic system (Faxitron
series, Hewlett-Packard, McMinnville, Ore) as described previously by our
laboratory (Goyal et al,
2004b).
The penile skeletal muscles consisted of 3 pairs of muscles: ischicavernosus, bulbocavernosus, and levator ani. The latter 2 were included in this study because they can be isolated easily. The bulbocavernosus is a massive muscle covering the ventro-lateral surface of the bulb of the penis. Conversely, the levator ani muscle is in the form of a circular, thin band that surrounds the anus and terminates at the bulbocavernosus muscle. Both muscles were isolated by freeing the attached connective tissue and fat and then weighed separately.
Digital images of histopathological and histochemical, as well as gross specimens of the penis, were captured with a Leitz Orthoplan microscope (Vashaw Scientific, Inc, Norcross, Ga) and the Kodak Microscopy Documentation System 290 (Eastman Kodak Company, Rochester, NY) and were assembled with the use of Adobe Photoshop 7.0.
Hormone Measurements
One blood sample was collected from the heart of each animal before
necropsy, and plasma was frozen at -20°C until assayed. Luteinizing
hormone (LH) was measured with the use of materials obtained through National
Hormone and Peptide Program (NHPP), National Institute of Diabetes and
Digestive and Kidney Diseases (NIDDK), and Dr A. F. Parlow (antibodies:
NIDDK-anti-rLH-S-11; reference standards: NIDDK-rLH-RP-3; tracers:
NIDDK-rLH-I-10). The sensitivity of the assay was 0.3 ng/mL. Testosterone (T)
was measured by a Coat-a-Count testosterone radioimmunoassay (Diagnostic
Products Corporation, Los Angeles, Calif) according to the manufacturer's
protocol. The sensitivity of the assay was 0.2 ng/mL. All samples were
quantified in a single assay, and the intra-assay coefficient of variation was
6% and 7% for LH and T, respectively.
Statistics
Statistical analyses were performed with Sigma Stat statistical software
(Jandel Scientific, Chicago, Ill). Analysis of variance was performed on body
weight, hormones, penile length, and penile weight. In addition, to account
for the litter effect, penile data from the 10- and 1-µg DES and EV
treatments (n = 2 litters for each treatment) and controls (n = 2 litters)
were grouped by individual litter and analyzed by analysis of variance with
litter as the experimental unit. Treatment groups with means significantly
different (P < .05) from controls were identified by Dunnett's
test. When data were not distributed normally, or heterogeneity of variance
was identified, analyses were performed on transformed data or ranked
data.
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Results |
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Organ Weight
Testis, Epididymis, and Seminal Vesicle—
Although the focus of the study was to determine dose-dependent effects of
DES and EV on the penis and associated skeletal muscles, effects on the
absolute and relative weights of the testis, epididymis, and seminal vesicle
are included here for the sake of comparison and validity because estrogenic
effects on these organs are well established in the literature, including from
our laboratory (Goyal et al,
2001,
2003). Generally, effects were
similar between the DES and EV groups; both caused reductions in weights in a
more or less dose-dependent manner, with higher reductions in the 100-ng and
higher dose groups and minimal to no reductions in the 10-ng and lower dose
groups (Table 1). Among organs,
the reduction was more pronounced in the seminal vesicle than in other organs
(eg, 65% vs 20% in testes in the 1-µg EV group).
Caudal Epididymal Fat Pad— The mean weight of the caudal epididymal fat pad in control animals was 338 mg, ranging from 237 to 460 mg among animals (Figure 1). However, it was significantly (P < .05) reduced to 50% of controls in the 10- and 100-ng DES groups and to less than 10% of controls in the 10-µg DES group. Similar significant reductions in weight were observed in the 100- to 10-µg EV groups (Figure 1).
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Descent of Testes
Testicular descent occurred at 24 days in controls (n = 10), 1- and 10-ng
DES groups (n = 7 per group), and 1- to 100-ng EV groups (n = 7 per group).
Conversely, it was delayed by 3 days in the 100-ng DES group (n = 7), 7-10
days in the 1-µg DES (n = 13) and EV (n = 14 per group) groups, 22-32 days
in the 10-µg DES group (mean age of descent, 47.8 days; n = 16), and 39-67
days in the 10-µg EV group (mean age of descent, 77.0 days; n = 8).
Release of Preputial Sheath
The preputial sheath was completely separate from the glans penis at 47-50
days of age in controls (n = 10) and the 1- and 10-ng DES and EV groups (n = 7
per group). Conversely, it was delayed by 3 days in 2 males and 20 days in 1
male of the 100-ng EV group (n = 7); delayed by 3, 6, or 27 days,
respectively, in 1 male each, and partially attached until day 119 in 4 males
of the 100-ng DES group (n = 7); and partially attached until day 119 in all
males of the 1- and 10-µg EV (n = 11 and 5, respectively) and DES (n = 13
and 16, respectively) groups.
Penile Measurements
Penile measurements, including weight, length, and diameter, were
significantly (P < .05) reduced in a dose-dependent manner in the
10- and 1-µg DES and EV groups (Figure
2), although the percent decrease was higher for weight than
length (eg, 41% vs 19% in the 1-µg EV group). Neither measurement was
significantly different from that of controls in the 100-ng or less DES and EV
groups, except the diameter in the 100-ng DES group
(Figure 2). However, within the
100-ng group, 4 of 7 males had lower penile weight (174-205 vs 244-267 mg in
the remaining 3) and shorter penile length (33-34 vs 40-41 mm), and all 4 of
these males failed to sire pups (see details under "Fertility").
Similarly, the penis in 1 of 7 males in the 100-ng EV group was also lighter
(234 vs 282-318 mg in 6 males) and shorter (34 vs 39-41 mm), and this male
also did not sire pups. An analysis of data with Litter as the statistical
experimental unit, to account for any litter effects (2 litters each for
controls and the 10- and 1-µg DES and EV treatments), also showed
significant reductions (P < .05) for all penile measurements as a
result of treatments (Table
2).
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Penile Skeletal Muscles
The weight of the bulbocavernosus muscle was significantly (P <
.05) decreased to 50% of controls in the 1-µg and to less than 20% of
controls in the 10-µg DES and EV groups
(Figure 3). Conversely,
significant weight reduction in the levator ani muscle was found only in the
10-µg DES and EV groups. Furthermore, the percent weight reduction was
comparatively much lower for the levator ani than the bulbocavernosus in both
the 1- and 10-µg DES and EV groups
(Figure 3).
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Penile Gross Morphology
The rodent penis, including the rat, has 2 distinguishing gross,
morphological features: os penis and right angle between the body and the
glans penis. Both features were adversely altered in the 10- and 1-µg DES
and EV groups, especially so in the 10-µg groups
(Figure 4). The os penis
comprised 2 parts: proximal and distal. A radiographic examination revealed
that not only were both parts of the os penis malformed, underdeveloped, and
undercalcified, but the distal part (closer to the tip of the glans penis) did
not even develop in the 10-µg groups. Except for somewhat less
calcification of the distal part of the os penis in the 100-ng groups, the
penile gross morphology appeared unaltered in the remaining lower dose groups
(Figure 4).
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Penile Histopathology and Histochemistry
The body of the penis in most mammalian species, including rodents,
comprises 3 erectile bodies: 2 corpora cavernosa that are located dorsolateral
to the urethra and a corpus spongiosus that surrounds the urethra. The corpora
cavernosa penis in rats, as in most other species with a vascular penis,
consist of a network of endothelial-lined cavernous spaces (similar to
sinusoids) that are surrounded by smooth muscle cells and separated by dense
bundles of collagen fibers, which also form a part of the tunica albuginea
that surrounds the corpora cavernosa. Both DES and EV in the 1- and 10-µg
groups caused similar, dramatic histological changes in the corpora cavernosa
penis. An examination of H&E-stained paraffin sections revealed that
cavernous spaces and the surrounding smooth muscle cells were altogether
absent and were replaced by cells that appeared to be empty
(Figures 5A through D), which
were confirmed as fat cells in paraffin sections stained with osmium
tetroxide, frozen sections stained with Sudan Black, and epoxy sections
stained with toluidine blue (Figures 5E
through H). Only few, if any, collagen fibers were present between
fat cells, and the tunica albuginea was reduced to a thin band of collagen
fibers. It should be noted that, despite the absence of cavernous spaces,
arterioles and capillaries (the latter have smaller and regular diameter, in
contrast to irregular and wider diameter of the cavernous spaces) were
invariably present in the corpora cavernosa
(Figure 5D).
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The histology of the corpora cavernosa in the 10- and 1-ng DES and EV groups was unaltered. However, it showed a differential response among animals in the 100-ng groups, especially in the DES group. Although cavernous spaces and smooth muscle cells were clearly visible in 3 of 7 males in the DES group and 6 of 7 males in the EV group, they were altogether absent and replaced by fat cells in the remaining males of both groups. Even those males that had cavernous spaces had apparently more fat cells, especially in the DES group, compared with controls (Figures 5I and J).
Unlike the corpora cavernosa, there was no apparent loss of cavernous spaces or smooth muscle cells or aggregation of fat cells in the corpus spongiosus, regardless of the dose or the estrogenic compound (not shown).
Reproductive Hormones
The mean concentration of plasma testosterone in the 1- or 10-ng DES and EV
groups was not significantly (P < .05) different from that of
controls (2.78 ng/mL), but it was significantly reduced in the 100-ng and
higher dose DES and EV groups (Figure
6). Plasma LH level was not significantly different between
controls and any of the DES and EV groups, except in the 10-ng and 1-µg EV
groups.
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Fertility
Although 6 of 7 males in the control group, 7 of 7 males in the 10-ng DES
group, and 5 of 6 males in the 10-ng EV group sired pups, none of the females
mated with the 10- or 1-µg DES and EV groups (n = 6 per group), had pups,
or had copulatory plugs (Table
3). In the 100-ng groups, 4 of 7 males in the DES group and 2 of 6
males in the EV group did not sire pups. Also, none of the females that mated
with these 6 males had copulatory plugs. A comparison of reproductive data
between those who sired and those who did not sire in the 100-ng DES group
revealed that all animals of the latter group had lighter and shorter penes
and lower testosterone levels than those in the former, although there were no
apparent differences in the body weight and organ weights between the 2 groups
(Table 4).
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Discussion |
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Hence, neonatal DES or EV exposure at a cumulative dose of 600 ng or higher per rat can result in permanent morphological changes in the rat penis, which are associated with lower fertility. This information is clinically valuable considering that more than 2 million men in the world are now in their 40s and 50s and were exposed to DES in utero when their mothers were treated with DES for preventing miscarriage (Shawn, 2000). According to one study, DES males born to a cohort of women at the Boston Lying-in Hospital were exposed to a total median dose of 12 200 mg (equal to 200 mg/kg, on the basis of a weight of 60 kg for a pregnant woman; Heinonen, 1973) in contrast to a total dose of 0.03-0.04 mg/kg received by male pups of the 100-ng dose groups of the present study. Although some might argue against the appropriateness of direct comparisons of exposure between neonatal rats and human embryos, male external genitalia are as undifferentiated in rats at birth as in the first trimester of pregnancy in humans (Williams-Ashman and Reddi, 1991; George and Wilson, 1994; Klonisch et al, 2004). Moreover, daughters exposed to DES during the first 7 weeks of their mothers' pregnancy had a much higher chance of developing vaginal adenocarcinoma than those who were exposed after 16 weeks (Shawn, 2000). In light of these observations, it is prudent to find out whether the incidence of erectile dysfunction is higher in DES-exposed men than in the general population. To our knowledge, such information is not available, although fertility in a small cohort of the DES men was found to be normal (Wilcox et al, 1995).
A notable finding of this study, as well as that of our previous studies (Goyal et al, 2004a,b), was the replacement of cavernous spaces and smooth muscle cells with fat cells in the corpora cavernosa penis in rats treated neonatally with DES. Neonatal EV exposure also resulted in similar histopathological changes, reinforcing estrogen-related adverse effects in the penis. Furthermore, both DES and EV induced structural alterations in the corpora cavernosa, but not in the adjoining corpus spongiosus, underscoring the specificity of estrogenic effects within the body of the penis. In light of this information, it is reasonable to predict that cavernous spaces are not likely to be replaced by fat cells in the glans penis because the latter is anatomically an extension of the corpus spongiosus (Dyce et al, 2002). Whether this prediction is true or not remains to be determined, but the glans penis was reduced in size in this study. However, this reduction probably resulted from the underdeveloped os penis, which is a part of the glans penis but is developmentally an extension of the distal part of the corpora cavernosa (Murakami and Mizuno, 1984).
The understanding of how neonatal estrogen exposure selectively transforms the corpora cavernosa could lie in unraveling the mechanisms of early differentiation of corpora cavernosa because estrogen-induced effects were already visible at 18 days of age, the earliest age at which we examined penises from rats treated neonatally with DES in an earlier study (Goyal et al, 2004b). Observations by other authors that cavernous spaces, smooth muscle cells, and os penis are absent in the rat penis at birth and start to differentiate from undifferentiated mesenchymal cells at 2-7 days of age (Murakami 1986, 1987a) suggest that the structural changes that we observed in the corpora cavernosa might have resulted from estrogen-induced alterations in the differentiation of mesenchymal cells. If this hypothesis has some validity, then estrogen exposure during a critical period of differentiation only should result in permanent structural changes in the penis. Although such a study is in progress in our laboratory, it is worth noting that penile morphology did not change in rats treated with DES at adulthood, although they did not sire pups, similar to those treated with DES neonatally (Goyal et al, 2004a). Furthermore, testosterone substitution at adulthood restored fertility in rats that received DES at adulthood but not in rats that received DES neonatally, thus implying that the loss of fertility was probably because of deficits in sexual behavior (central effect) in the former and abnormal penile morphology in the latter (peripheral effect).
In addition to the above histopathological changes, both DES and EV neonatal exposure reduced the weight, length, and diameter of the penis and delayed the release of the preputial sheath and the descent of testes. Interestingly, all these changes, except testicular descent, were directly related with the inability of males to sire pups. The best correlation came from observations in the 100-ng DES group, in which 4 of 7 males, which were unable to sire pups, had lighter and shorter penes and delayed preputial sheath release than the remaining 3 males, which sired. Similar observations were recorded in 1 of 7 males of the 100-ng EV group, which also did not sire. The delayed separation of preputial sheath, dose-dependent reductions in the length of the penis, or both were reported in rats treated neonatally with estrogen (Zanida et al, 1979; Putz et al, 2001). Similarly, phalluses in alligators exposed to excessive estrogenic contaminants in Lake Apopka, Florida, were significantly smaller than those in Lake Woodruff, a control lake in the area (Guillette et al, 1994, 1996).
Another important finding of the study was the dose-dependent reductions in weights of the penile skeletal muscles. More importantly, the reduction between 2 muscles was differential. For example, the bulbocavernosus muscle was decreased by 80% and 50%, respectively, in the 10- and 1-µg DES groups, in contrast to 40% and 10% reductions, respectively, for the levator ani muscle, implying a more severe effect of estrogen in the former muscle. To our knowledge, similar differential response to neonatal estrogen treatment has not been demonstrated in these 2 penile muscles previously, although it is known that both muscles are androgen dependent (Breedlove and Arnold, 1983; Mansouri et al, 2003), innervated by dimorphic spinal nucleus, and reduced in size by antiandrogens (Breedlove, 1992). In addition, their contraction augments erection of the penis (Sachs, 1982; Benson, 1994). Whether the decreased size of both muscles directly contributed in the loss of fertility cannot be assessed in this study because, in the absence of cavernous spaces and smooth muscle cells in the corpora cavernosa, it is difficult for the penis to become erect, or at least attain sufficient erection to enable penetration. However, a recent study reported much thicker ischicavernosus and bulbocavernosus muscles in potent than impotent men (Hsu et al, 2004).
The dose-dependent reductions in organ weights, including the testis,
epididymis, and seminal vesicle, resulting from neonatal estrogen exposure
were essentially similar to those reported earlier from our laboratory
(Goyal et al, 2003), as well
as from other laboratories (Sharpe et al,
1998; Atanassova et al,
2000; Putz et al,
2001). However, new information from this study that, to our
knowledge, has not been reported previously is the effect of neonatal estrogen
exposure on the caudal epididymal fat pad, which was decreased by 90% in the
10-µg and by 30%-50% in the 1-µg DES and EV groups. These decreases were
similar to those observed in the seminal vesicle (eg, 90% and 30% in the 10-
and 1-µg DES groups, respectively) and thus rank the caudal epididymal fat
pad at par, in terms of estrogen sensitivity, with the seminal vesicle, which
is by far one of the most sensitive reproductive organs to estrogen exposure
(Robaire et al, 1987;
Goyal et al, 2001;
Putz et al, 2001). Whether
similar levels of reductions occurred in other body fat pads was not studied
presently; however, parametrial fat pads decreased in weight in adult female
mice treated with 17ß-estradiol or the phytoestrogen genistein
(Naaz et al, 2003). Conversely, epididymal, perirenal, and inguinal fat pads were more than 2-fold
heavier in estrogen receptor-
(ER) knockout mice than in wild
male mice (Cooke et al,
2001).
An important question that remains to be answered is what hormonal and
molecular mechanisms brought about the observed penile changes, especially the
replacement of cavernous spaces by fat cells in the corpora cavernosa. Could
they result from a direct effect of estrogens, an indirect effect via
estrogen-induced decreased T on mesenchymal cells during their
differentiation, or both? Although a detailed discussion on this question is
beyond the scope of this study, some pertinent data are worth noting. Estrogen
receptors have been identified in mesenchymal cells in 1-day-old rat penis
(Jesmin et al, 2002).
ER expression was enhanced in fibroblastlike cells of the corpora
cavernosa at 18 days of age in rats treated neonatally with DES
(Goyal et al, 2004b), implying
a role for ER in estrogen-induced penile abnormalities. Additional
support for a similar role of this receptor in reproduction comes from
observations that ER knockout mice of both sexes are infertile
(Eddy et al, 1996) and that the
prostate (Prins et al, 2001)
and female reproductive tract (Couse et al,
2001) of ER knockout mice are resistant to DES-induced
developmental abnormalities.
Alternatively, androgen receptors are present in the genital tubercle of the fetal rat (Murakami, 1987b); they are seen ubiquitously in endothelial cells of cavernous spaces, smooth muscle cells, and fibroblastlike cells throughout the body of the rat penis at prepuberty, puberty, and adulthood (Goyal et al, 2004b); and their concentration reaches a peak level at a time when the rat penis is differentiating or developing at, or before, puberty (Rajfer et al, 1980; Takane et al, 1990; Goyal et al, 2004b). All these observations point to androgen action in differentiation of the penis. In this context, it is noteworthy that the simultaneous administration of testosterone with DES mitigated most of the male reproductive tract abnormalities in rats treated neonatally with DES alone (Rivas et al, 2003). Although it remains to be seen whether similar supplementation with testosterone will prevent DES-induced penile abnormalities, plasma T level was significantly lower in the 100-ng and higher DES and EV groups in this study. Similarly, previous authors (Rivas et al, 2003), as well as our laboratory (Goyal et al, 2004b), reported marked reduction in plasma T in prepubertal, pubertal, or both ages of rats treated neonatally with DES.
In this study, we describe for the first time neonatal EV exposure, similar to DES, inducing permanent penile abnormalities, including reductions in the length, weight, and diameter of the penis and histopathological changes characterized by the replacement of cavernous spaces and smooth muscle cells with fat cells in the corpora cavernosa penis. These penile abnormalities are associated with a decreased size of penile skeletal muscle cells and lower fertility and can be caused by a dose that is as low as 100 ng/rat/d, given subcutaneously on alternate days from postnatal days 2-12 (cumulative dose of 600 ng, or 0.03-0.04 mg/kg body weight).
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Acknowledgments |
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Footnotes |
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