The male reproductive tract is consisted of the testis, excurrent ductules, and
accessory glands, including prostate and seminal vesicle. The epididymis is a part
of excurrent ductules, which the testis is connected via efferent ductules (Robaire et al., 2006). The epididymis has a
tubular structure, which the lumen is located inside and is surrounded by a layer of
epithelial cells (Robaire et al., 2006).
The epididymis is anatomically divided into four parts, including initial segment,
caput (head region) epididymis, corpus (body region) epididymis, and cauda (tail
region) epididymis, which are characterized by the presence and different frequency
of specific cell types and different histochemical existence of enzymatic molecules
(Robaire et al., 2006). The epididymal
cell types present within the epithelial layer are principal, apical, narrow, clear,
basal, and halo cells (Robaire et al.,
2006). Even though the principal cell is found along the entire epididymal
ductules, the restricted localization of other cell types is commonly observed
(Robaire et al., 2006). For example,
the apical and narrow cells are mainly found in the initial segment (Sun & Flickinger, 1980), while the
clear cells only exist in the caput, corpus, and cauda epididymis (Abou-Haïla & Fain-Maurel, 1984).
In addition, a number of researches have demonstrated that these cell types have
distinctive morphologies, functions, and localization relative to other cell types
(Robaire et al., 2006). Thus, it is
generally considered that each epididymal region plays different roles on control
and regulation of epididymal functions.The epididymis is the site in which immature spermatozoa produced from the testis
become mature and acquire motility and fertilizing capacity (Robaire et al., 2006). The physiological functions of
epididymis include transport and maturation of spermatozoa and storage and
protection of spermatozoa (Robaire et al.,
2006). The regulation of water movement into and out of the epididymal
lumen is a way to form the adequate luminal microenvironment for sperm maturation
(Robaire et al., 2006). Even though
most of testicular fluid is reabsorbed in the efferent ductules (Clulow et al., 1994), luminal water
reabsorption at significant level occurs in the epididymis, especially in the caput
epididymis (Tao et al., 2000). Other
scientific evidences have also shown that not only fluid reabsorption from the
epididymal lumen but also water secretion into the lumen are equally important to
regulate the luminal composition of the epididymis and the fluidity of the
epididymal luminal content (Wong, 1998).
Thus, the controlled transepithelial water movement is important to regulate sperm
maturation of epididymal function.Aquaporins (AQPs) are water channel molecules that are chiefly localized at plasma
membrane in various cell types, including testis, efferent ductules, and epididymis
in the male reproductive tract (Verkman &
Mitra, 2000; Da Silva et al.,
2006). AQPs have 6 membrane-spanning domains in structure and contribute
to water movement across plasma membrane (Verkman
& Mitra, 2000; Ribeiro et al.,
2021). The AQPs have at least 13 isoforms in mammalian cells, and
expression and localization of different types of AQPs in the male reproductive
tract have been extensively examined (Da Silva et
al., 2006; Ribeiro et al.,
2021). AQPs are also capable of transporting ammonia or hydrogen peroxide,
depending on types of AQPs (Da Silva et al.,
2006; Ribeiro et al., 2021).
There are at least 8 types of AQPs expressing in the epididymis, and specific
expression of different AQP types has been detected along the epididymis (Ribeiro et al., 2021). For examples,
expression of AQP1 is found in smooth muscle and endothelial cells adjacent to
epididymal epithelial cells (Brown et al.,
1993), and AQP9 is exclusively localized at the apical microvilli of
principal cells throughout the entire epididymal region (Pastor-Soler et al., 2001). The expression of AQPs in the
male reproductive tract is regulated by various factors, including androgen and
estrogen (Badran & Hermo, 2002; Pastor-Soler et al., 2002; Oliveira et al., 2005). Because expressional
alterations of AQPs in male reproductive tract are associated with the reduction of
reproductive capability (Huang et al.,
2006), it is relatively reasonable to consider that exogenous compound
affecting male fertility could influence expression of AQPs in the male reproductive
tract.Nandrolone decanoate (ND) is an androgenic-anabolic steroidal compound which is
frequently administrated to treat several clinical symptoms, such as muscle wasting
accompanied with human immunodeficiency virus infection and anemia involved in
chronic renal failure (Busardò et al.,
2015). Uncontrolled and unregulated usage of ND is strongly associated
with the reduction of male fertility, probably due to expressional alteration of
steroidogenic enzymes in the testis in part (Min
& Lee, 2018). In addition, chronic administration of ND results in
a decrease of epididymal weight (Mirkhani et al.,
2005), even though the effect of ND on the epididymal has not determined
in detail. Non-clinical use of ND among non-professional people and bodybuilders
appears to be increased (de Souza & Hallak,
2011). But, despite deleterious undesired outcomes on male fertility by
the misuse of ND, the effect of ND on the epididymis has been rarely studied.The present research was focused to determine the effect of ND treatment at different
doses for short or long period on expression of Aqp1 and
Aqp9 in the rat epididymis. The segmental expressional
modulation of Aqp1 and Aqp9 by ND treatment was
examined by using a quantitative real-time polymerase chain reaction (PCR)
analysis.
MATERIALS AND METHODS
Experimental animals, nandrolone decanoate treatment, and tissue
collection
A total of 34 male Sprague Dawley rats at 40 days of age were purchased from
Samtako (Osan, Korea) and individually caged during an entire experimental
period. The animals were allowed ad libitum access to food and
water and kept in controlled conditions. The ND, 50 mg/mL concentration in a
stock solution, was obtained from Organon Korea (Seoul, Korea), and diluted into
adequate concentrations with peanut oil for ND treatment.The equal number of animals at 50 days of age were divided into two different
treatment groups, 2 week-ND treatment and 12 week-ND treatment groups. The
animals in each experimental group were further randomly assigned into one of
three subgroups, including control (peanut oil treatment, n=5), low dose-ND
treatment (2 mg of ND/kg body weight/week, n=6), or high dose-ND treatment (10
mg of ND/kg body weight/week, n=6). The first subcutaneous ND injection was
started at 50 days of age, and the injection amount of peanut oil or ND was
adjusted every week by measuring body weight of animal.After the last injection was allowed at the day of 2nd or 12th week, the animal
was euthanized by over-exposure to CO2 in a closed chamber. The
reproductive tract, including testis and epididymis, was pulled out through an
incision made on the scrotum. The epididymis was rapidly separated from the
testis and efferent ductules in cold phosphate-buffered saline (PBS) solution.
The epididymis transferred into a fresh PBS solution was further dissected out
into initial segment, caput epididymis, corpus epididymis, and caudal
epididymis. The epididymal segments were quickly frozen in liquid nitrogen and
stored in −80°C until further use.
Total RNA isolation and quantitative real-time polymerase chain reaction
(PCR)
Total RNA from the frozen tissue was extracted with Trizol reagent (Molecular
Research Center, Cincinnati, OH, USA). Shortly, the tissue was rapidly
homogenized in Trizol solution, and chloroform and isopropanol were sequentially
supplied to precipitate total RNA. The concentration of total RNA re-suspended
in DEPC-dH2O was measured by NanoDrop Lite spectrophotometer (Thermo
Scientific, Massachusetts, MA, USA). The quality of total RNA was determined by
1.2% agarose gel electrophoresis.One microgram of total RNA was used to generate the first strand complementary
DNA (cDNA) strand by utilizing iScripTM Reverse transcription
Supermix for reverse transcription (RT)-qPCR (Bio-Rad Laboratories, Hercules,
CA, USA). A total volume of the mixture with total RNA was adjusted with
nuclease-free dH2O to 20 μL. The RT reaction was carried out
at 25°C for 5 min, 46°C for 20 min, and 95°C for 1 min as
instructed in the protocol.The quantitative real-time PCR was performed with a mixture of 1 μL of
cDNA, 7 μL of iQTM SYBR® Green Supermix
(Bio-Rad Laboratories), 10 pmol of each oligonucleotide primer, and
nuclease-free dH2O to make a total volume of 25 μL. The
information of oligonucleotide primer used for real-time PCR is shown in Table 1. As an internal control for PCR
analysis, cyclophilin A (Ppia) was used. The PCR was executed
in a pre-denaturation step at 95°C for 5 min, cycles of a denaturation
step at 95°C for 30 sec, an annealing step at Tm for 30 sec,
and an extension step at 72°C for 30 sec in PTC-200 Chromo 4 real-time
system (Bio-Rad Laboratories). At the end of each PCR, an extra extension step
at 72°C for 10 min was added. The PCR product was fractured in 1.2%
agarose gel electrophoresis to check the size.
The levels of Aqp1 and Aqp9 transcripts in each
experimental groups were adjusted to that of Ppia transcript
level. The relative ratios of Aqp1 and Aqp9
expression levels among experimental groups were obtained from
2-ΔΔCt method (Livak & Schmittgen, 2001). Independently triplicated or
quadruplicated RT reactions and PCRs were performed to acquire a mean and
standard error of an experimental groups, and the data were presented in the
relative ratio of Aqp1 or Aqp9 transcript
level against that of control group. The one-way ANOVA was employed to
determined the existence of statistical differences at transcript level of
Aqp1 or Aqp9 among different experimental
groups. If a significance was detected, a post-hoc test, Duncan’s test,
was followed. Less than 0.05 level of probability was considered to be
statistically different.
RESULTS
Expressional changes of Aqp1 and Aqp9 in
the rat epididymal initial segment by nan-drolone decanoate (ND) treatment for 2
weeks or 12 weeks
The transcript level of Aqp1 in the initial segment was not
significantly changed by ND treatment for 2 weeks (Fig. 1A). The similar finding was observed from
Aqp9 transcript level treated with ND for 2 weeks (Fig. 1B). However, a significant decrease of
Aqp1 expression level was detected in the initial segment
by ND treatment at the low dose for 12 weeks (Fig.
1C). The level of Aqp1 transcript at the high
dose-treated group of ND for 12 weeks was also significantly lower than that of
control group, even though there was no statistical difference between the low
and high dose-treated groups of ND (Fig.
1C). The level of Aqp9 transcript was not significantly
changed by ND treatment at the low dose for 12 weeks, while the high dose
treatment of ND for 12 weeks resulted in a decrease of Aqp9
expression in the initial segment (Fig.
1D).
Fig. 1.
Expression changes of Aqp1 and Aqp9
in the initial segment of rat epididymis after ND treatment for 2 weeks
or 12 weeks at different doses.
The animals were treated with ND for 2 weeks (A and B) or 12 weeks (C and
D) at 0 (control), 2 (low dose), or 10 (high dose) mg/kg body
weight/week. The levels of Aqp1 (A and C) and
Aqp9 (B and D) transcripts were normalized to that
of Ppia. a,b Different letters on bars
indicate statistical differences among experimental groups at
p<0.05. Aqp1, aquaporin 1;
Aqp9, aquaporin 9; Ppia,
cyclophilin A; ND, nandrolone decanoate.
Expression changes of Aqp1 and Aqp9
in the initial segment of rat epididymis after ND treatment for 2 weeks
or 12 weeks at different doses.
The animals were treated with ND for 2 weeks (A and B) or 12 weeks (C and
D) at 0 (control), 2 (low dose), or 10 (high dose) mg/kg body
weight/week. The levels of Aqp1 (A and C) and
Aqp9 (B and D) transcripts were normalized to that
of Ppia. a,b Different letters on bars
indicate statistical differences among experimental groups at
p<0.05. Aqp1, aquaporin 1;
Aqp9, aquaporin 9; Ppia,
cyclophilin A; ND, nandrolone decanoate.
Expressional changes of Aqp1 and Aqp9 in
the rat caput epididymis by nandrolone decanoate (ND) treatment for 2 weeks or
12 weeks
The expression of Aqp1 in the caput epididymis with ND treatment
for 2 week was not significantly changed, regardless the dose of treatment
(Fig. 2A). However, a significant
decrease of Aqp9 transcript level was detected in the caput
epididymis with ND treatment at the low dose for 2 weeks, while there was no
significant change of Aqp9 transcript level at the high dose ND
treated-group for 2 weeks (Fig. 2B).
Fig. 2.
Expression changes of Aqp1 and Aqp9
in the rat caput epididymis after ND treatment for 2 weeks or 12 weeks
at different doses.
The animals were treated with ND for 2 weeks (A and B) or 12 weeks (C and
D) at 0 (control), 2 (low dose), or 10 (high dose) mg/kg body
weight/week. The levels of Aqp1 (A and C) and
Aqp9 (B and D) transcripts were normalized to that
of Ppia. a,b Different letters on bars
indicate statistical differences among experimental groups at
p<0.05. Aqp1, aquaporin 1;
Aqp9, aquaporin 9; Ppia,
cyclophilin A; ND, nandrolone decanoate.
Expression changes of Aqp1 and Aqp9
in the rat caput epididymis after ND treatment for 2 weeks or 12 weeks
at different doses.
The animals were treated with ND for 2 weeks (A and B) or 12 weeks (C and
D) at 0 (control), 2 (low dose), or 10 (high dose) mg/kg body
weight/week. The levels of Aqp1 (A and C) and
Aqp9 (B and D) transcripts were normalized to that
of Ppia. a,b Different letters on bars
indicate statistical differences among experimental groups at
p<0.05. Aqp1, aquaporin 1;
Aqp9, aquaporin 9; Ppia,
cyclophilin A; ND, nandrolone decanoate.The expression patterns of Aqp1 and Aqp9 in the
caput epididymis by ND treatment for 12 weeks (Fig. 2C and D) were quietly similar with those of ND treatment for 2
weeks (Fig. 2A and B). That is, the ND
treatment for 2 weeks didn’t give an influence on the level of
Aqp1 transcript at any dose (Fig. 2C). But, the low dose treatment of ND for 12 weeks induced a
reduction of Aqp9 expression in the caput epididymis, even
though there was no significant expression change of Aqp9
observed with the high dose ND treatment for 12 weeks (Fig. 2D).
Expressional changes of Aqp1 and Aqp9 in
the rat corpus epididymis by nandrolone decanoate (ND) treatment for 2 weeks or
12 weeks
Fig. 3 shows the changes of
Aqp1 and Aqp9 transcript levels in the rat
corpus epididymis treated with ND for 2 or 12 weeks. The ND treatment at the low
dose for 2 weeks resulted in a significant increase of Aqp1
transcript level in the corpus epididymis (Fig.
3A). A further increase of Aqp1 transcript amount
was observed in the corpus epididymis exposed to the high dose of ND for 2 weeks
(Fig. 3A). However, expression of
Aqp9 was significantly decreased by ND treatment at the low
dose for 2 weeks (Fig. 3B). The ND
treatment at the high dose for 2 weeks caused an additional reduction of
Aqp9 transcript level in the corpus epididymis (Fig. 3B).
Fig. 3.
Expression changes of Aqp1 and Aqp9
in the rat corpus epididymis after ND treatment for 2 weeks or 12 weeks
at different doses.
The animals were treated with ND for 2 weeks (A and B) or 12 weeks (C and
D) at 0 (control), 2 (low dose), or 10 (high dose) mg/kg body
weight/week. The levels of Aqp1 (A and C) and
Aqp9 (B and D) transcripts were normalized to that
of Ppia. a–c Different letters on
bars indicate statistical differences among experimental groups at
p<0.05. Aqp1, aquaporin 1;
Aqp9, aquaporin 9; Ppia,
cyclophilin A; ND, nandrolone decanoate.
Expression changes of Aqp1 and Aqp9
in the rat corpus epididymis after ND treatment for 2 weeks or 12 weeks
at different doses.
The animals were treated with ND for 2 weeks (A and B) or 12 weeks (C and
D) at 0 (control), 2 (low dose), or 10 (high dose) mg/kg body
weight/week. The levels of Aqp1 (A and C) and
Aqp9 (B and D) transcripts were normalized to that
of Ppia. a–c Different letters on
bars indicate statistical differences among experimental groups at
p<0.05. Aqp1, aquaporin 1;
Aqp9, aquaporin 9; Ppia,
cyclophilin A; ND, nandrolone decanoate.The level of Aqp1 in the corpus epididymis was significantly
decreased by the low dose treatment of ND for 12 weeks (Fig. 3C). But, the Aqp1 transcript level in
the corpus epididymis of the high dose ND treatment groups for 12 weeks was
statistically higher than those of control and low dose ND treatment groups
(Fig. 3C). Expression of
Aqp9 in the corpus epididymis by ND treatment for 12 weeks
was significantly decreased at both doses (Fig.
3D).
Expression changes of Aqp1 and Aqp9
in the rat cauda epididymis after ND treatment for 2 weeks or 12 weeks
at different doses.
The animals were treated with ND for 2 weeks (A and B) or 12 weeks (C and
D) at 0 (control), 2 (low dose), or 10 (high dose) mg/kg body
weight/week. The levels of Aqp1 (A and C) and
Aqp9 (B and D) transcripts were normalized to that
of Ppia. a–c Different letters on
bars indicate statistical differences among experimental groups at
p<0.05. Aqp1, aquaporin 1;
Aqp9, aquaporin 9; Ppia,
cyclophilin A; ND, nandrolone decanoate.
Expressional changes of Aqp1 and Aqp9 in
the rat cauda epididymis by nandrolone decanoate (ND) treatment for 2 weeks or
12 weeks
In the cauda epididymis, ND treatment for 2 weeks resulted in significant
decreases of Aqp1 transcript levels at both doses (Fig. 4A). However, ND treatment at the low
dose for 2 weeks didn’t influence on the level of Aqp9
transcript, even though the high dose treatment of ND for 2 weeks caused a
significant increase of Aqp9 expression in the cauda epididymis
(Fig. 4B).
Fig. 4.
Expression changes of Aqp1 and Aqp9
in the rat cauda epididymis after ND treatment for 2 weeks or 12 weeks
at different doses.
The animals were treated with ND for 2 weeks (A and B) or 12 weeks (C and
D) at 0 (control), 2 (low dose), or 10 (high dose) mg/kg body
weight/week. The levels of Aqp1 (A and C) and
Aqp9 (B and D) transcripts were normalized to that
of Ppia. a–c Different letters on
bars indicate statistical differences among experimental groups at
p<0.05. Aqp1, aquaporin 1;
Aqp9, aquaporin 9; Ppia,
cyclophilin A; ND, nandrolone decanoate.
Expression of Aqp1 in the cauda epididymis was significantly
decreased by the low dose treatment of ND for 12 weeks (Fig. 4C). The level of Aqp1 transcript in
the caudal epididymis treated with the high dose of ND for 12 weeks was
significantly lower than that of control group, but higher than that of the low
dose ND treatment group (Fig. 4C). The low
dose ND treatment for 12 weeks resulted in a significant increase of
Aqp9 transcript level in the cauda epididymis (Fig. 4D). An additional increase of
Aqp9 expression in the cauda epididymis was observed with
the high dose ND treatment for 12 weeks (Fig.
4D).
DISCUSSION
The present research examined expression changes of Aqp1 and
Aqp9 in each epididymal segment by ND treatment at two doses
for 2 or 12 weeks. The current findings are summarized as follows: 1) expression of
Aqp1 and Aqp9 is differentially modulated
among different epididymal segments by ND treatment, even at same dose for same
period; 2) ND treatments at different doses usually result in similar outcomes on
expression of Aqp1 or Aqp9 within a given
epididymal segment; and 3) change patterns of Aqp1 and
Aqp9 expression by ND treatment in an epididymal segment are
not always same, even at same doses and for same period.Hormonal regulation of Aqps expression in the excurrent duct of male
reproductive tract has been demonstrated from other researches. Expression of
Aqp9 is modulated by estrogen and 5α-dihydrotestosterone
in the efferent ductules, but only by 5α-dihydrotestosterone in the initial
segment (Oliveira et al., 2005). The
nonsteroidal antiandrogen flutamide results in a decrease of Aqp9
expression in the epididymis (Pastor-Soler et al.,
2002). The exposure to a steroidal estrogen receptor antagonist,
ICI182,780, leads to a decrease of Aqp1 expression in the monkey
caput epididymis (Shayu et al., 2005). In
addition, 3-beta-diol, an androgenic metabolite, could influence on the regulation
of Aqp9 expression in the efferent ductules (Picciarelli-Lima et al., 2006). Moreover, Badran & Hermo (2002) have shown that
expression of Aqp9 in the initial segment and cauda epididymis
appears to be regulated by testicular factor(s), not by androgen, suggesting
differential regulation of specific Aqps at different epididymal
segments. Thus, these observations indicate that expressional regulation of
Aqps in the efferent ductules and epididymis is under control
of various testicular and/or nontesticular factors. Also, expression of specific
Aqp type in the epididymis could be regulated by
segment-specific manner(s). Indeed, the present research have also shown
differential expression patterns of Aqp1 and Aqp9
in different epididymal regions by same dose and/or duration treatment of ND. For
example, the ND treatment for 2 weeks has not affected expression of
Aqp1 and Aqp9 in the initial segment, while an
increased level of Aqp1 transcript and a decreased level of
Aqp9 transcript have been observed with same treatment in the
corpus epididymis. Also, in the caput epididymis, even though the low dose
treatments of ND for 2 and 12 weeks result in decreased levels of
Aqp9 transcript, the high-dose ND treatments for same periods
have not influenced on Aqp9 expression level. Together with the
other’s findings, the current research data suggest that expression of
Aqp1 and Aqp9 along the epididymis by the
exposure to ND is differentially regulated in segment-specific and/or dose and
treatment duration-dependent manners.Such differential regulation of Aqp1 and Aqp9 by ND
could be due to different distribution of androgen receptor (AR) concentration along
the epididymis. Pujol & Bayard
(1979) have shown that the caput epididymis has the highest AR
concentration and the corpus epididymis has the lowest AR amount. Because ND has a
similar affinity to AR comparable with testosterone (Bergink et al., 1985), ND could exert its biological effect
via AR in a target tissue. Thus, it was expected that the caput epididymis would be
more responsive than the corpus epididymis to ND exposure. However, more dramatic
changes of Aqp1 and Aqp9 expression by ND
treatment have been observed in the corpus epididymis, not in the caput epididymis.
In addition, significant changes of transcript levels in the caput epididymis have
been just detected in Aqp9, not Aqp1, exposed to
the low dose of ND. A clear answer to such ambiguous data could not be provided at
this moment. An additional investigation of the regulatory effect of ND on
Aqp1 and Aqp9 expression in the epididymis is
suggested.It is also suggested that expressional change of Aqp1 and
Aqp9 by ND treatment in the epididymis could be not direct
action of ND but indirect effect accompanied with systemic response to ND. The
exposure to ND frequently accomplishes aberrant expression of steroidogenic enzymes
and abnormal histology in the testis (Svechnikov
et al., 2010; Min & Lee,
2018). In fact, a previous research has demonstrated that ND
administration in a same experimental condition with the present study results in
significant reduction of several testicular steroidogenic enzymes, including
steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage, and
cytochrome P450 17α-hydroxylase, accompanied with a drastic decrease of serum
testosterone concentration (Min & Lee,
2018). Because functions of the epididymis are largely influenced by
testosterone (Robaire et al., 2006), a
decrease of testicular testosterone synthesis by ND exposure could give an impact on
gene expression in the epididymis to result in functional abnormality. However, even
though this suggestion seems to be supported by dose-related decreases of
Aqp9 transcript levels in the corpus epididymis by 2 week-ND
treatment, other data don’t seem to fit to the presumption.The present research shows that expressional regulation of Aqp1 and
Aqp9 in the epididymis by ND is quite complicate. The existence
of complex regulatory mechanism of Aqp1 and Aqp9
in the epididymis by hormonal factor has also been proposed from others (Badran & Hermo, 2002; Oliveira et al., 2005; Squillacioti et al., 2021). Considering the main function of
the epididymis and physiological role of AQPs in the epididymis, it is suggested
that the aberrant expression of Aqp1 and Aqp9 in
the epididymis by ND treatment would cause inappropriate development of mature
spermatozoa, thus leading into ND-induced infertility. In addition, a possible
segment-specific regulation of Aqp1 and Aqp9
expression in the epididymis by ND administration is suggested from the current
study, even though detailed molecular mechanism(s) would be determined from further
researches.
Authors: N Pastor-Soler; C Bagnis; I Sabolic; R Tyszkowski; M McKee; A Van Hoek; S Breton; D Brown Journal: Biol Reprod Date: 2001-08 Impact factor: 4.285
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