Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats
The prostate is an exocrine reproductive gland that participates in ejaculation and it is prone to diseases, including cancer. Aim: In the present study, we assessed the longterm effects of copulation on the development of precancerous lesions in rats, and compared them with testosteroneinduced pr...
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| Zitieren: | Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats / D. Herrera-Covarrubias, M.B. Tecamachaltzi-Silvaran, M. Barradas-Moctezuma, J.B. Rosales-Raya, J. Manzo, L.I. García, G.E. Aranda-Abreu, N. Ismail, G.A. Coria-Avila, M.E. Hernández // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 73–79. — Бібліогр.: 40 назв. — англ. |
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irk-123456789-1379992018-06-18T03:06:03Z Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats Herrera-Covarrubias, D. Tecamachaltzi-Silvaran, M.B. Barradas-Moctezuma, M. Rosales-Raya, J.B. Manzo, J. García, L.I. Aranda-Abreu, G.E. Ismail, N. Coria-Avila, G.A. Hernández, M.E. Original contributions The prostate is an exocrine reproductive gland that participates in ejaculation and it is prone to diseases, including cancer. Aim: In the present study, we assessed the longterm effects of copulation on the development of precancerous lesions in rats, and compared them with testosteroneinduced prostatic lesions. Materials and Methods: One group of Wistar males was given 10 copulatory sessions to one ejaculation with ovariectomized, hormoneprimed females. Sessions occurred twice per week for a total of ten trials. A second group was exposed to females during the same trials, but physical contact was prevented. In addition, each group received a subcutaneous implant in the back either filled with testosterone propionate (T, 100 mg/kg) or empty. This resulted in four subgroups: 1) Control + No sex, 2) Control + Sex, 3) T + No sex and 4) T + Sex. Two days after the 10th trial all the males were sacrificed for prostate histo logy (H&E) and hormone analysis (testosterone and prolactin). Results: Males from the group Control + No sex expressed normal histo logy. However, those in the groups Control + Sex and T + No sex expressed metaplasia and dysplasia in both the dorsolateral and ventral portions of the prostate, respectively. Interestingly, males from the group T + Sex expressed dysplasia in the dorsolateral prostate only, but not in the ventral prostate. Conclusions: These results indicate that constant copulation may facilitate the development of prostatic lesions in males with normal levels of testosterone. However, copulation induces less lesions in the ventral prostate of males treated with testosterone. 2016 Article Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats / D. Herrera-Covarrubias, M.B. Tecamachaltzi-Silvaran, M. Barradas-Moctezuma, J.B. Rosales-Raya, J. Manzo, L.I. García, G.E. Aranda-Abreu, N. Ismail, G.A. Coria-Avila, M.E. Hernández // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 73–79. — Бібліогр.: 40 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137999 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Herrera-Covarrubias, D. Tecamachaltzi-Silvaran, M.B. Barradas-Moctezuma, M. Rosales-Raya, J.B. Manzo, J. García, L.I. Aranda-Abreu, G.E. Ismail, N. Coria-Avila, G.A. Hernández, M.E. Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats Experimental Oncology |
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The prostate is an exocrine reproductive gland that participates in ejaculation and it is prone to diseases, including cancer. Aim: In the present study, we assessed the longterm effects of copulation on the development of precancerous lesions in rats, and compared them with testosteroneinduced prostatic lesions. Materials and Methods: One group of Wistar males was given 10 copulatory sessions to one ejaculation with ovariectomized, hormoneprimed females. Sessions occurred twice per week for a total of ten trials. A second group was exposed to females during the same trials, but physical contact was prevented. In addition, each group received a subcutaneous implant in the back either filled with testosterone propionate (T, 100 mg/kg) or empty. This resulted in four subgroups: 1) Control + No sex, 2) Control + Sex, 3) T + No sex and 4) T + Sex. Two days after the 10th trial all the males were sacrificed for prostate histo logy (H&E) and hormone analysis (testosterone and prolactin). Results: Males from the group Control + No sex expressed normal histo logy. However, those in the groups Control + Sex and T + No sex expressed metaplasia and dysplasia in both the dorsolateral and ventral portions of the prostate, respectively. Interestingly, males from the group T + Sex expressed dysplasia in the dorsolateral prostate only, but not in the ventral prostate. Conclusions: These results indicate that constant copulation may facilitate the development of prostatic lesions in males with normal levels of testosterone. However, copulation induces less lesions in the ventral prostate of males treated with testosterone. |
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Article |
| author |
Herrera-Covarrubias, D. Tecamachaltzi-Silvaran, M.B. Barradas-Moctezuma, M. Rosales-Raya, J.B. Manzo, J. García, L.I. Aranda-Abreu, G.E. Ismail, N. Coria-Avila, G.A. Hernández, M.E. |
| author_facet |
Herrera-Covarrubias, D. Tecamachaltzi-Silvaran, M.B. Barradas-Moctezuma, M. Rosales-Raya, J.B. Manzo, J. García, L.I. Aranda-Abreu, G.E. Ismail, N. Coria-Avila, G.A. Hernández, M.E. |
| author_sort |
Herrera-Covarrubias, D. |
| title |
Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats |
| title_short |
Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats |
| title_full |
Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats |
| title_fullStr |
Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats |
| title_full_unstemmed |
Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats |
| title_sort |
effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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2016 |
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Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/137999 |
| citation_txt |
Effect of copulation on potentially precancerous prostate lesions, serum testosterone and prolactin levels in rats / D. Herrera-Covarrubias, M.B. Tecamachaltzi-Silvaran, M. Barradas-Moctezuma, J.B. Rosales-Raya, J. Manzo, L.I. García, G.E. Aranda-Abreu, N. Ismail, G.A. Coria-Avila, M.E. Hernández // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 73–79. — Бібліогр.: 40 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T02:46:53Z |
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2025-07-10T02:46:53Z |
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| fulltext |
Experimental Oncology 38, 73–79, 2016 (June) 73
EFFECT OF COPULATION ON POTENTIALLY PRECANCEROUS
PROSTATE LESIONS, SERUM TESTOSTERONE AND PROLACTIN
LEVELS IN RATS
D. Herrera-Covarrubias1, M.B. Tecamachaltzi-Silvaran2, M. Barradas-Moctezuma2, J.B. Rosales-Raya3,
J. Manzo4, L.I. García4, G.E. Aranda-Abreu4, N. Ismail1, G.A. Coria-Avila4,*, M.E. Hernández4
1School of Psychology, University of Ottawa, Ottawa, ON K1N 6N5, Canada
2Doctorate in Brain Research, University of Veracruz, Xalapa, Veracruz 91190, Mexico
3Veterinary clinical laboratory, Xalapa, Veracruz 91190, Mexico
4Center for Brain Research, University of Veracruz, Xalapa, Veracruz 91190, Mexico
The prostate is an exocrine reproductive gland that participates in ejaculation and it is prone to diseases, including cancer. Aim: In the pre
sent study, we assessed the longterm effects of copulation on the development of precancerous lesions in rats, and compared them with
testosteroneinduced prostatic lesions. Materials and Methods: One group of Wistar males was given 10 copulatory sessions to one ejacu
lation with ovariectomized, hormoneprimed females. Sessions occurred twice per week for a total of ten trials. A second group was exposed
to females during the same trials, but physical contact was prevented. In addition, each group received a subcutaneous implant in the back
either filled with testosterone propionate (T, 100 mg/kg) or empty. This resulted in four subgroups: 1) Control + No sex, 2) Control + Sex,
3) T + No sex and 4) T + Sex. Two days after the 10th trial all the males were sacrificed for prostate histo logy (H&E) and hormone analy
sis (testosterone and prolactin). Results: Males from the group Control + No sex expressed normal histo logy. However, those in the groups
Control + Sex and T + No sex expressed metaplasia and dysplasia in both the dorsolateral and ventral portions of the prostate, respec
tively. Interestingly, males from the group T + Sex expressed dysplasia in the dorsolateral prostate only, but not in the ventral prostate.
Conclusions: These results indicate that constant copulation may facilitate the development of prostatic lesions in males with normal levels
of testosterone. However, copulation induces less lesions in the ventral prostate of males treated with testosterone.
Key Words: prostate, cancer, sexual behavior, ejaculation, dysplasia, testosterone.
Prostate cancer (CaP) is among all types of tumors
in men the second most common worldwide and
every year up to 7% of all the new cases may result
in death [1]. The probability to develop CaP increases
with age (> 65) [2], race (> African-ancestry) [3], diet
(> high-fat products) [4], obesity (body mass index
> 25) [5], smoking [6], and alterations in the level
of hormones such as testosterone [7, 8] and prolactin
(PRL) [9, 10]. Consequently, efforts are made world-
wide to understand its etiology and other possible
contributing factors. For instance, there is no clear
evidence about the role of sexual behavior in the de-
velopment of precancerous and cancerous prostatic
lesions, although some studies in the past argued
a probable link between copulation and CaP [11–14].
For example, in the 1960’s it was observed that ever-
married men expressed higher mortality rates of CaP
than single men in the United States [13]. In other
studies, the number of cases of CaP was positively
correlated with the number of sexual partners [15]
and with high coital frequency [16]. Michalek et al. [17]
described a probable effect of celibacy on the pre-
vention of CaP. They analyzed more than 1000 death
certificates of Catholic clergymen in New York from
1965 to 1977 and found fewer cases of CaP as com-
pared to a matched control group of the same age.
Although mortality was only 1% less in the celibate
group the authors reported significant differences.
However, other studies have shown either no effect
of copulation on CaP or a positive effect. Kaplan [12]
reported that mortality ratios of putative celibate
priests resembled that of a population with a high al-
cohol and caloric intake but a relatively low exposure
to tobacco. The study did not discard the possibility
that celibacy had a protective effect for CaP, but ar-
gued that the evidence to suggest so was too weak.
More recent evidence from a study with controls and
cohorts argued that copulation protects the prostate
from CaP [14]. Such studies provide ambiguous
evidence to support any hypothesis about the role
of copulation on prostate health.
As mentioned above, precancerous or cancerous
lesions in the prostate have also been related to abnor-
mal levels of testosterone and PRL [7–10]. Copulation
increases the levels of serum testosterone [18, 19] and
PRL [20, 21] in both humans and laboratory rats. In ad-
dition, ejaculation increases the levels of androgen
receptors (AR) and mRNA for AR in the prostate [22].
Testosterone can induce cell division and spontaneous
mutations that result in more and abnormal cells within
the gland [7, 8]. Similarly, PRL can activate molecular
pathways (i.e. Jak2-Stat5a/b) that may result in tumor-
igenesis and progression to CaP [23]. Indeed, it has
been shown that rats that receive systemic treatment
with either testosterone or PRL during 4 weeks or more
Submitted: February 04, 2016.
*Correspondence: Phone: +52-228-8418900 EXT. 13609;
Fax: +52-228-8418900 EXT. 13611;
E-mail: gcoria@uv.mx
Abbreviations used: AR — androgen receptor; BHP — benign hy-
perplasia; CaP — prostate cancer; DLP — dorsolateral prostate;
N:C — nucleus : cytoplasm; PRL — prolactin; PRLR — prolactin recep-
tor; s.c. — subcutaneously; T — testoste rone; VP — ventral prostate.
Exp Oncol 2016
38, 2, 73–79
ORIgINAL CONTRIbUTIONS
74 Experimental Oncology 38, 73–79, 2016 (June)
will express precancerous lesions of the prostate (i.e.
dysplasia) [9]. Accordingly, copulation per se induces
short-term endocrine changes that might function
as a long-term risk factor for the development of pre-
cancerous lesions. However, this has not been shown
experimentally in an animal model. Thus, the aim of the
present study was to assess the effects of repeated
copulation to ejaculation on the development of pre-
cancerous prostatic lesions (i.e. dys plasia) in adult
male rats. Our first hypothesis stated that constant
copulation during 5 weeks would induce histological
lesions comparable to those observed in males ex-
posed to exogenous systemic testosterone during
the same period. In addition, we hypothe sized that the
baseline blood levels of testosterone and PRL would
be higher in male rats allowed to copulate constantly.
MATERIALS AND METHODS
Animals. We used 20 Wistar males and 10 females
(Rattus norvegicus albinus). They were purchased
from a certified laboratory animal supplier in Mexico
(Circulo ADN) and were 8 weeks old and sexually na-
ïve at the start of the study. They were group-housed
in large plexiglas cages (50 × 30 × 20 cm), and kept
in a colony room at the Center for Brain Research,
University of Veracruz, Mexico, in a 12–12 h reverse
Light-Dark cycle (lights off at 8:00 hrs). Water and
commercial feed (Rismart rat chow) were provided
ad libitum. All the experimental procedures were car-
ried out according to the Official Mexican Norm NOM-
062-ZOO-1999 for use and care of laboratory animals.
Surgery and hormone treatments. One week
before the start of copulatory trials all the males were
randomly organized in two main groups, depending
on the treatment they received at 8 weeks of age.
The testosterone group (T) was implanted subcutane-
ously (s.c.) in the back with a silastic tube (Dow Corning
Corp 25 mm length, 1.57 mm I.D. × 3.18 mm O.D.), con-
taining solid testosterone propionate (Sigma-Aldrich).
This resulted in approximately 100 mg/kg of body weight
as previously used in other studies [9, 24]. A second
group served as control and was exposed to the same
procedures, but the silastic tube was empty. Surgical
implantation was done under inhaled halothane anes-
thesia, and took less than 3 min for each rat. After confir-
mation of deep anesthesia we performed a 10 mm skin
incision on the lower back. A surgical probe was inserted
s.c. and it was moved rostrally to separate the skin from
the muscle. The silastic tube was slided in and pushed
up to be placed at the upper back, between the two
scapulae. The lower back incision was sutured and the
rat was allowed to fully recover before it was placed
back into its home cage. Early studies showed that
these silastic capsules release testosterone at a rate of
~30 µg/day/cm [25]. Both groups (T and Control) were
further divided in two subgroups, depending on whether
or not they were allowed to engage in sexual behavior
to ejaculation. This resulted in four subgroups: 1) T +
Sex (n = 5), 2) T + No sex (n = 5), 3) Control + Sex (n = 5),
and 4) Control + No sex (n = 5).
The stimulus females were ovariectomized (OVX)
and primed fully with s.c. injections of estradiol benzo-
ate (10 µg) 48 h and progesterone (500 µg) 4 h before
each copulatory trial. For ovariectomy, females were
anesthetized with a mixture of ketamine hydrochloride
(50 mg/ml) and xylazine hydrochloride (4 mg/ml),
mixed at a ratio of 4:3, respectively, injected intra-
peritoneally in a volume of 1 ml/kg of body weight.
Anesthetized females were then OVX bilaterally via
a lumbar incision. Post-surgical treatment for females
included 3 days of s.c. injections of flunixin meglumine
(2.5 mg/kg) for analgesia, and enrofloxacin (5 mg/kg)
every 24 h to prevent post-surgical bacterial infections.
All females were given a week of post-surgical recovery
before they were used as sexual partners.
Sexual behavior. One week after the surgical im-
plantation of the silastic tubes (T or Control), the males
started the first copulatory trial. Thus, the subgroups
T + Sex and Control + Sex were allowed to copulate
twice a week with the OVX sexually receptive female
for a total of 10 trials (5 weeks of sexual activity).
Subgroups T + No sex and Control + No sex were
also exposed to a sexually receptive female behind
a wiremesh that prevented copulation, but allowed
visual, olfactory and acoustic stimulation. Each copu-
latory trial occurred in squared chambers (39 × 21 ×
21 cm) in which males were allowed to interact freely
with the female until the first ejaculation was observed.
Males were gently removed from the chamber one
minute after ejaculation or after 60 min in case that
ejaculation did not occur. The number of males that
ejaculated in each trial and the ejaculation latency
were recorded.
Prostate samples and histology. Two days after
the 10th copulatory session the rats were anesthetized
with sodium pentobarbital (35 mg/kg intraperitoneally).
Then 3 ml of blood were obtained by cardiac puncture
for hormone analysis (see Hormone measurements for
further details). Anesthetized rats were then sacrificed
with an overdose of sodium pentobarbital (120 mg/kg).
An abdominal incision was performed and the acces-
sory sexual organs were carefully removed and placed
into a container with 0.9% saline solution. The prostate
was identified under a dissecting microscope (MEJI,
EMZ-TR) and the gland was further divided into ventral
(VP) and dorsolateral (DLP) prostate. The VP and DLP
were soaked in formol 10% during 24 h, then dehy-
drated in alcohol 70% and 80% (1 h each), and 95%
(3 × 2 h each), and ethanol 100% overnight, plus two
more changes (1 h each), the following day. Then xylene
(3 × 1 h each) was added, always with constant shaking.
Tissue was embedded in paraffin wax 2 × 2 h each),
sliced (5 µm thick) with a microtome (RM 2125RT Leica),
mounted on slides in a bath at 52 °C (containing pork
skin-based gelatin 2.5 mg/100 ml) and then processed
for hematoxylin and eosin (H&E) dye technique as fol-
lows: 1 h at 57 °C, deparaffinization in xylene (3 × 5 min
each), rehydration in alcohol/xylene (1:1) 5 min, ethanol
96% 3 min, hematoxylin (10 min), water (30 s), acid al-
cohol (quick immersion), water (10 s), lithium carbonate
Experimental Oncology 38, 73–79, 2016 (June) 75
(30 s), water (10 s), eosin (4 quick immersions), dehy-
dration in ethanol 96% (3 min), ethanol 100% (2 min),
ethanol/xylene 1:1 (2 min), and xylene (5 min). Then
Permount was added and slides were coverslipped, air
dried, and observed under a light microscope (Olym-
pus Ax70). Photomicrographs were taken at 40× and
analyzed by the same experimenters. As in the previous
study [9], we assessed prostate histology from normal
to abnormal by taking into consideration the following
histological features: 1) epithelium form (cubic in DLP,
columnar in VP, vs hyperplasia, metaplasia, dysplasia,
cancer in situ), 2) epithelium size (even vs anisocytosis),
3) epithelium papillae (scarce vs plenty), 4) interstice
space (even vs compressed), 5) interstice content (col-
lagen vs mononuclear), 6) nucleus size (even vs aniso-
kariosis), 7) nucleus location (basal cell polarity vs non
basal), 8) nucleus to cytoplasm ratio N:C (1:2 vs 1:1),
9) myoepithelium (euplasia vs proplasia), 10) pat-
tern observed at 4× (tubular vs cribiform), 11) lumen
content (amorphous vs granular), and 12) chromatin
(heterochromatin vs euchromatin). A blind score was
performed by researchers and veterinarians for each
prostate sample. Accordingly, the experimenters did not
know to what subgroup of rats belonged each analyzed
gland at the moment of diagnosis.
Hormones measurement. The hormones tes-
tosterone and PRL were measured in blood serum
exclusively two days after the 10th copulatory session.
Namely, on the same day the prostate was obtained.
Blood was collected in vacutainer tubes containing
no anticoagulant and incubated in upright position
at room temperature for 30 min to allow clotting. Tubes
were centrifuged for 15 min at 1000 r.p.m. Supernatant
was aspirated at room temperature and serum was
kept in 500 µl aliquots and frozen at −20 °C during
few days until processing. We used an enzyme-linked
immunosorbent assay (ELISA) and commercial kits
for testosterone (ALPCO 11-TESHU-E01) and PRL
(ALPCO 55-PRLRT-E01) and instructions were fol-
lowed as indicated by the supplier. The assays were
read in an IMARK microplate reader with the software
microplate manager from Bio-Rad.
Variables and statistical analysis. 1) Behavior:
The ejaculation latency (minutes) was analyzed with
a two-way (hormone treatment x session) ANOVA. Sig-
nificant differences were followed by a Fisher LSD
posthoc test. The proportion of ejaculators for each
session was also analyzed with a 2 × 2 contingency
table (one-tailed Fisher exact probability test), con-
sidering the number of males that did (or did not)
ejaculate. 2) Histology: We described 12 histological
features in each male (See section Prostate samples
and histo logy). 3) Hormones: Levels of testosterone
and PRL (ng/mL) were analyzed with a non-parametric
Kruskal — Wallis test, followed by Mann — Whitney
tests to compare individual differences. All statisti-
cal analyses were performed using GraphPad Prism
version 6.00 for Mac, GraphPad Software, La Jolla
California USA, www.graphpad.com and the alpha
level was set at p < 0.05.
RESULTS
Sexual behavior. With regard to the ejaculation
latency the ANOVA failed to detect an interaction be-
tween hormone treatment × session F(9,72) = 0.46,
p < 0.89, or a main effect of treatment F(1,8) = 0.02, p <
0.88, but detected a main effect of session F(9,72) =
2.03, p < 0.04. The posthoc analysis, however, did not
detect specific differences between sessions (Fig. 1).
The analysis of the number of ejaculators showed
significant differences at session 1 (ejaculation was
observed in 2 control vs 8 testosterone-treated males,
p < 0.01), and at session 10 (ejaculation occurred
in 10 control males vs 6 testosterone-treated, p <
0.04), but not at sessions 2–9. These data indicated
that rats from both groups reduced their ejaculation
latency thro ughout the sessions. In addition, testos-
terone-treated males were more likely than controls
to ejaculate at session 1, but less likely at session
10 (Fig. 2).
0
10
20
30
40
50
60
70
1 2 3 4 5 6 7 8 9 10
M
ea
n
+/
–S
EM
, m
in
Ejaculation latency
T
Control
Fig. 1. The ejaculation latency (minutes) of male rats during
10 copulatory trials (5 weeks). Testosterone males received
a subcutaneous implant in the back, filled with 100 mg/kg of tes-
tosterone propionate. Control males received an empty implant
0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10
%
Copulatory sessions
T
Control
*
Fig. 2. Shows the proportion of males from Fig. 1 that ejaculated
in each session. *Significant differences were detected between
control and testosterone males at session 1 and 10
Prostate lesions induced by copulation or testoster-
one. In gene ral, the group Control + No sex expressed
a normal (cubic) epithelium in the DLP, even in size
(normal), with scarce papillae (normal). The interstitial
space was even in size (normal) and contained collagen
(normal). Interestingly, in 3 out of 5 rats the cell nuclei
in the DLP were not homogeneous in size (anisokaryo-
sis), and expressed a relation N:C of 1:1 (normal > 1:2).
Nevertheless, the nucleus had basal cell polarity (nor-
mal), the myoepithelium was euplastic (normal), with
76 Experimental Oncology 38, 73–79, 2016 (June)
Fig. 3. DLP (1) and VP (2) of rats divided in four subgroups: A — Control + No sex, B — Control + Sex, C — T + No sex, D — T +
Sex. A1 — normal cubic epitheium (DLP), A2 — normal columnar epithelium, B1 — metaplasia, B2 — dysplasia, C1 — dysplasia,
C2 — dysplasia, D1 — dysplasia, D2 — columnar
Experimental Oncology 38, 73–79, 2016 (June) 77
tubular pattern (normal), amorphous content (normal),
and there was heterochromatin, which is a less ac-
tive form of chromatin (Fig. 3, A1). In the same group
(Control + No sex) 3/5 animals expressed a columnar
epithelium (normal) in the VP, although some cases
of anisocytosis were observed in 3/5 animals, and also
expressed scarce papillae (normal). The interstitial
space was even in size and contained colagen (normal).
Nuclei were homogeneous in size (normal), with a basal
cell polarity (normal), and a N:C of 1:2, and there was
heterochromatin. The myoepithelium was euplasic and
the pattern observed at 4× was tubular (Fig. 3, A2). Al-
though males from this subgroup (Control + No sex) had
in general normal histological features, none of them
expressed 100% of normal histology. For example,
some males with normal epithelium’s form expressed
what is considered abnormal epithelium’s size (aniso-
cytosis) or abnormal nucleus size (anisokaryosis),
or modified N:C ratio (1:1). This suggests that even
in a sample of putative healthy rats there is variability
in the histological features of the prostate.
Rats from the subgroups Control + Sex, T + Sex
and T + No sex displayed more histological abnormali-
ties in both the DLP and VP. Such differences and the
number of males with normal or abnormal features can
be observed in Fig. 3, B, C, D, and 4, a, b.
0
20
40
60
80
100
Control + No sex Control + Sex T + No sex T + Sex
0
20
40
60
80
100
Control + No sex Control + Sex T + No sex T + Sex
Normal Metaplasia Dysplasia
%
%
b
a
Fig. 4. Effect of constant copulation on the proportion of males
with normal or abnormal epithelium in (a) the DLP and (b) in VP
For instance, in the DLP the Control + Sex males
displayed more cases of abnormal epithelium (must
be cubic in DLP and columnar in VP), or more severely
affected with dysplasia (considered precancerous).
There were also cases of anisocytosis, compressed
interstice, anisokaryosis, apolarity of the nucleus, pro-
plasia of myoepithelium (enlarged), cribriform pattern,
granular content in lumen, and euchromatin (more ac-
tive form of chromatin). In the T + Sex and T + No sex
subgroups there were more cases of epithelium dys-
plasia in the DLP. Interestingly, fewer cases of dysplasia
were observed in the VP of T + Sex animals (Fig. 4, b).
Serum levels of testosterone and PRL. With re-
gard to testosterone, the Kruskal — Wallis test revealed
significant differences between groups H(4,19) = 8.2,
p < 0.05. The Mann — Whitney test indicated individual
differences between the subgroups Control + No sex
(Median = 0.38 ng/ml) vs Control + Sex (Median =
2.05 ng/ml) U = 2, p < 0.05, and between Control +
Sex (Median = 2.05 ng/ml) vs T + No sex (Median =
0.21 ng/ml) U = 1, p < 0.05 (Fig. 5). With regard to PRL,
the analysis failed to detect significant differences be-
tween groups: Control + No sex (Median = 20.86 ng/ml),
Control + Sex (Median = 32.15 ng/ml), T + No sex (Me-
dian = 100.8 ng/ml) and T + Sex (Median = 21.22 ng/ml)
H(4,18) = 3.02, p = 0.37.
0
1
2
3
4
5
Control + No sex Control + Sex T + No sex T + Sex
M
ed
ia
ne
+
/–
in
te
rq
ua
rti
le
, n
g/
m
l
a
b
a
a,b
Fig. 5. Serum baseline levels of testosterone after 10 trials
of copulation (5 weeks). Different letters indicate significant
differences between bars
DISCUSSION
The results of the present study indicate that in rats
repeated copulation induced histological alterations
in both the DLP and VP (e.g. metaplasia and dysplasia).
In the DLP the lesions were milder than those observed
in sexually naïve males that received exogenous
testosterone, but in the VP the effect of copulation
and testosterone were comparable. Copulation plus
exogenous testosterone had additional negative ef-
fects in the DLP as compared to testosterone without
copulation, but in the VP such combination decreased
the cases of dysplasia (see Fig. 4, a, b). The baseline
levels of serum testosterone were higher in males
with constant copulation, but not in males with s.c.
implants filled with testosterone. We speculate that
testosterone was increased during the first week
of implant, but decreased subsequently via negative
feedback mechanisms, resulting in low levels after
6 weeks. Such argument is possibly supported by the
proportion of ejaculators observed in sessions 1 and
10 (see Fig. 2). That is, more testosterone-treated
males ejaculated at session 1 as compared to control
males, but the opposite outcome occurred by the
10th session. The results also indicate that the levels
of serum PRL were not affected by either copulation
or testosterone implants. Thus, we argue that prostate
78 Experimental Oncology 38, 73–79, 2016 (June)
lesions induced by constant copulation (5 weeks) are
mainly due to the effects of increased testosterone.
Specific effects of copulation on prostate le-
sions. After 10 copulatory sessions 60% of males from
the Control + Sex subgroup were affected. Two of them
expressed in the DLP an epithelium with metaplasia
(Fig. 3, B1) that is the reversible replacement of one
differentiated cell type with another mature differenti-
ated cell type. Those rats also expressed anisocytosis
(unequal abnormal size of epithelium cells) and plenty
of papillae, which resulted in reduced interstices. In ad-
dition, cells started to express abnormal nuclear shape
(anisokaryosis), which is one of the key diagnostic tools
used in identifying cancerous cells [26, 27]. Further-
more, the nuclei were no longer polar, the N:C ratio
was abnormal (1:1) and there was a cribriform pattern.
Anysokaryosis, apolarity and alterations in N:C indicate
changes in chromosome organization, which in turn can
affect gene expression [28] that eventually may result
in dysplasia (observed in one male). In the subgroups T
+ No sex and T + Sex 80% of the rats expressed dysplasia
in the DLP (Fig. 3, C1, D1), which refers to a precancerous
lesion characterized by the presence of immature and
abnormal epithelial cells with abnormal function. These
changes indicate that in the DLP s.c. implants of testos-
terone (100 mg/kg) for periods as short as 5 weeks are
more aggressive than the effects of copulation during the
same period. Further experiments are required to confirm
that testosterone is the only responsible of copulation-
induced prostatic lesions. In addition, it is important to fi-
gure out if a long resting period after constant copulation
would result in a total recovery of the prostate.
Effects of copulation and testosterone on DLP
and VP. As mentioned above, copulation affected the
DLP and the combination of copulation plus exogenous
testosterone resulted in more alterations. Males from
the T + Sex subgroup expressed more cases of aniso-
cytosis, proplastic myoepithelium and apolar nuclei than
males from the T + No sex subgroup. However, this was
not observed in the VP. As a matter of fact, copulation
plus exogenous testosterone resulted in fewer cases
of dysplasia in the VP (see Fig. 4, b). Evidence indicates
that ejaculation induces in the VP an acute increase
in the levels of both AR and AR-mRNA [22]. However,
it is not known whether such increase is maintained after
5 weeks of constant copulation in rats. One speculation
is that the opposite occurs after several copulatory tri-
als, namely that AR may be decreased and therefore
VP may be less sensitive to exogenous testosterone.
Like in rats, the human prostate is also heterogeneous
with regard to its embryologic origin and histology [29].
Its three zones (periurethral, central and peripheral)
contain different concentration of glands, conducts,
and AR distribution as observed in cases of CaP [30].
Such heterogeneity may account for the different ef-
fects of copulation on prostatic lesions in humans.
Copulation affected both portions of the prostate
(DLP and VP) of control males, but prevented dysplasia
in the VP of those males that received exogenous tes-
tosterone. Accordingly, the frequency of copulation plus
the le vels of serum testosterone may help to account
the probability of prostatic lesions in VP of rats or in ho-
mologous prostatic areas in other species. For example,
age 65+ is a risk factor to develop CaP in humans. At that
age the blood levels of testosterone are decreased
by more than 50% in some men [31–34]. Human
prostatic areas homologous of rat VP should be more
sensitivity to copulation-induced testosterone, which
would result in higher risk of CaP in low-testosterone
men. By contrast, the same homologous areas of men
with higher levels of testosterone should be benefited
by copulation. Indeed, recent evidence from a study
with controls and cohorts for CaP showed a protective
effect of copulation, which also correlated with higher
levels of serum testosterone in the controls [14]. Some
of the mechanisms to explain the effect of copulation
on DLP and VP may involve the AR directly [35]. It re-
mains to be shown whether the AR gene may mutate
or be amplified by repeated copulation so that the
AR may respond to lower levels of androgens, or per-
haps to other hormones (i.e. PRL) [10, 36–38].
CONCLUSIONS
Either constant copulation or exogenous testoste-
rone resulted in histological alterations of the two pros-
tatic portions (DLP, VP) in rats. However, the combination
of copulation plus exogenous testosterone affected
mainly the DLP, and to a much less extent the VP. This sug-
gests a bimodal effect of copulation in the VP, depending
on the levels of serum testosterone. Accordingly, the
celibacy theory may be supported in males with normal
or low levels of testosterone, but would not be supported
in males with higher-than-normal levels of testosterone.
Further research is needed for understanding the specific
role of copulation at long-term on the levels of AR in the
VP and the development and maintenance of prostatic
diseases, including cancer [39, 40].
ACkNOwLEDgMENTS
This study was supported by Consejo Nacional
de Ciencia y Tecnología (CONACYT) from Mexico, with
a postdoctoral fellowship to DHC (No. 250561), and
to GACA (No. CB-167773). The authors want to give
special thanks to Rodrigo Ramírez-Rodríguez for
helping with animals and samples handling.
CONFLICTS OF INTEREST
The authors declare that they have no conflict
of interest.
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