| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the * Department of Biochemistry and
Hybridoma Laboratory, National Institute for
Research in Reproductive Health, Mumbai, India.
| Correspondence to: Dr Vijaya P Raghavan, Department of Biochemistry, National Institute for Research in Reproductive Health (ICMR), J.M. Street, Parel – 400 012, Mumbai, India (e-mail: vijuraghavan{at}hotmail.com). |
| Received for publication December 11, 2006; accepted for publication May 7, 2007. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
90 % of that
observed in intact animals. Ethylene dimethane sulfonate (EDS) administration
completely abolished HOXBES2 expression in the epididymis, and supplementation
with DHT or DHT + estradiol for 10 days re-established HOXBES2 expression to
near normalcy. However, in the estradiol alone-supplemented EDS-treated group,
HOXBES2 remained undetected. The unaltered HOXBES2 expression following
efferent duct ligation suggested that HOXBES2 is not critically dependent on
testicular factors. During postnatal development, protein expression in the
epididymis begins to appear from day 40 and 50 and increased from day 60
onward, coinciding with the mature levels of circulating androgens and the
well-differentiated epididymis. Thus, the data obtained from this study
suggests that HOXBES2 expression could be regulated by androgens, and its
expression level is closely associated with the postnatal development of the
epididymis.
Key words: Castration, EDS treatment, DHT supplementation, efferent duct ligation, postnatal development
Several studies have earlier demonstrated that many of the individual
processes that contribute to the creation of an optimal microenvironment
within the epididymis are regulated by circulating luminal androgens and to
some extent by other testicular factors that originate from the testis itself
including basic fibroblast growth factor, androgen-binding protein, retinoids,
etc (Ezer and Robaire, 2002,
2003). Androgen-regulated
regionalized gene expression is complex in the epididymis and has been
discussed in a recent review
(Orgebin-Crist, 1996).
Segment-specific down-regulation of gene expression following withdrawal of
androgens or testicular factors is a hallmark characteristic of the epididymis
(Cornwall et al, 2001).
Androgens such as testosterone (T), 5
-reduced T, and
dihydrotestosterone (DHT) exert their effects by binding and subsequently
activating the androgen receptor (AR) protein with the help of a carrier
molecule, androgen-binding protein (ABP). It is less clear whether the
metabolites of T—namely estradiol—formed by the action of
aromatase or the major metabolites of DHT—androstan-3,
3-diol, and 17ß-diol—also have any significant role to play
in epididymal differentiation or maturation. DHT, the most abundant and active
form of androgen in the epididymis (Hinton
et al, 1998), is produced by the reducing action of 5
reductase and is indispensable for forward progressive and vibrant motility,
capacitation (Holland et al,
1992), sperm-egg interaction
(Lakoski et al, 1988), zona
pellucida (ZP) binding and penetration
(Boue et al, 1994), vitellous
fusion and penetration (Saling,
1982), and fertilizing ability
(Amann et al, 1993) of the
spermatozoa. The functional role of androgens in the synthesis and secretion
of several epididymal proteins such as CD52
(Kirchhoff et al, 2000) and
transcription factors such as ets-like factor PEA3
(Brooks, 1987) and reproductive
homeobox on chromosome X 5 protein (previously known as placenta and embryonic
expression protein; Lindsay and Wilkinson,
1996) was widely demonstrated earlier using the castrated rat
model and DHT supplementation studies
(Jones et al, 1980;
Holland and Orgebin-Crist,
1988; Ghyselinck et al,
1989; Gould and Young,
1990). Further, the withdrawal of androgen by castration or by
chemical suppression leads to involution of epididymal epithelium, gene
down-regulation, and resultant arrest of sperm maturation. It has been
suggested that incomplete sperm maturation is responsible for total failure of
sperm binding to the ZP in unsuccessful in vitro fertilization treatment in
humans (Bedford and Kim, 1993).
In fact, a high percentage of male infertility in humans is believed to
originate from malfunction of the epididymis
(Lunde et al, 1990).
We previously demonstrated the presence of HOXB2 homeodomain containing epididymis-specific sperm protein (HOXBES2) in the cytoplasm of the principal epithelial cells in a region-specific manner showing maximal expression in the distal segments of the rat epididymis (Prabagaran et al, 2007). Indirect immunofluorescence localized the protein to the acrosome, midpiece, and equatorial segment of rat caudal and ejaculated human and monkey spermatozoa, respectively. The aim of the present study was first to determine whether androgens and testicular factors are required to maintain the regional expression patterns of HOXBES2 protein in the adult rat epididymis and second to study how changes in HOXBES2 expression correlate with specific morphologic and biochemical changes that have been reported during the development of the epididymis.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Tissue Samples
Experimental rats were anesthetized with ether, and the tissues were
dissected out (testis, prostate, seminal vesicle, and epididymis). The
epididymis was divided into initial segment, proximal and distal caput,
proximal and distal corpus, and proximal and distal cauda
(Robaire and Viger, 1995) and
processed under sterile conditions for immunohistochemistry, Western blot
analysis, and reverse transcriptase polymerase chain reaction (RT-PCR).
Castration and DHT Supplementation
Three-month-old male rats weighing approximately 300 g were divided into 4
groups of 6 animals each. Group I rats were sham operated (control); the
testes and epididymides were manipulated similar to castration except for the
excision of testes. Group II rats were bilaterally castrated; following
exposure of the scrotal contents, the testicular vascular supplies were
ligated without compromising epididymal blood supplies and the testes were
separated from the epididymides and associated fat pads and then excised. The
epididymides and fat pads were returned, caput first, into the tunica
vaginalis and the incisions sutured. Group III rats were sham operated; this
vehicle-treated control group received 100 µL of sesame seed oil
subcutaneously daily postsham operation for 2 weeks. Group IV rats were
bilaterally castrated and received supplementations of 250 µg DHT/kg body
weight in sesame oil for 7 days in a delayed T repletion regimen that began on
day 7 postcastration. Groups I and II were sacrificed on day 7 postoperation.
Groups III and IV were sacrificed on day 15. Blood was collected from all
groups. From Groups I and II, blood was collected on days 0 and 7 before
sacrifice; blood was collected on days 7 and 14 following castration for
Groups III and IV. Serum T levels were measured in blood samples collected
prior to castration or sham operation. The serum samples were stored at
–20°C until they were assayed for circulatory levels of T determined
by radioimmunoassay (RIA) as described earlier
(Gupta et al, 1975;
Corker and Davidson, 1978). At
the time of sacrifice, epididymides, ventral prostrates, and seminal vesicles
were removed, trimmed from associated fat and connective tissues, and weighed.
The epididymides were dissected into initial segment, proximal and distal
caput, proximal and distal corpus, and proximal and distal cauda
(Teerds et al, 1988), fixed in
Bouin fixative, and subsequently processed for histology and
immunohistochemical analyses.
Chemical Castration and Hormone Supplementation
To study the androgen-dependent expression of HOXBES2 protein in the
epididymis, 3-month-old male rats were administered a single dose of the
Leydig cell–specific toxicant, ethylene dimethane sulfonate (EDS), which
is reported to cause transient infertility. A single dose of EDS was
sufficient to destroy a mature Leydig cell population and to ensure that no
new Leydig cells were formed during the first 10 days of treatment
(Girotti et al, 1992). The
rats were divided into 7 groups of 5 animals each (groups I–VII). Groups
I, II, and III served as controls and were administered intraperitoneally the
vehicle alone (DMSO-H2O, 1:3 vol/vol), sesame oil, and saline,
respectively. Group IV received a single intraperitoneal injection of EDS (75
mg/kg body weight) dissolved in DMSO-H2O (1:3 wt/vol) on day 0.
Group V was administered EDS and in addition received 250 µg DHT/kg body
weight dissolved in sesame oil daily for 10 days. Group VI was EDS treated and
received DHT and 100 µg of ß-estradiol (Sigma-Aldrich, St Louis, Mo)
in saline subcutaneously for 10 days. The EDS-treated rats in group VII were
administered 100 µg of ß-estradiol alone subcutaneously for 10
days.
Blood samples were collected on days 0, 5, and 10 from the controls (groups I–III), EDS-treated (group IV), and hormone-supplemented groups (groups V–VII) to measure circulating levels of T. Serum T levels obtained from untreated control animals or animals prior to treatment were considered basal T levels. The control and experimental groups were sacrificed on day 10, and testes, epididymides, and prostates were removed and processed for immunohistochemistry and Western blot analyses.
Unilateral Efferent Duct Ligation
To determine whether factor(s) of testicular origin are required for the
regionalized expression of HOXBES2 protein in the epididymis, unilateral
efferent duct ligation (EDL) studies were performed on 3-month-old male rats.
Efferent ductules of the right testes were ligated at their junctions with the
extratesticular rete testes without compromising the testicular or epididymal
blood supplies (Hermo et al,
1992b). As controls, the left testes were manipulated similarly to
the right testes, but the efferent ductules were not ligated. Each testis was
returned to its tunica vaginalis, and incisions were sutured. The rats were
sacrificed on days 10 and 15 postligation. The durations of the experiment
were scheduled to simulate the time scales previously followed for EDS
treatment and castration studies, respectively. Five rats per time point of
EDL treatment were used in this study. Ligated and control epididymides were
dissected out and processed for immunohistochemistry and Western blot
analyses.
Developmental Study
To determine the influence of T, if any, on the interrelationship between
the expression pattern of HOXBES2 protein and the postnatal development of the
rat epididymis, normally developing pups
(Hermo et al, 1992a) were
included in the study. The number of animals used at each postnatal age was
optimized to obtain sufficient tissue for RNA isolation, immunohistochemistry,
and Western blot. The animals (n) were sacrificed on postnatal days 5(15),
10(12), 15(10), 20(7), 30(7), 40(7), 50(6), 55(6), 60(6), 70(6), 80(6), 90(6),
100(6), 120(6), and 200(6). Epididymal tissues obtained from each postnatal
age were divided into 3 groups to analyze samples in triplicate. The body
weights of all animals used, as well as the testicular and epididymal weights,
were within the ranges reported earlier
(Scheer and Robaire, 1980;
Hermo et al, 1992a;
Hermo et al, 1998). The time
points were chosen to almost coincide with known developmental events:
postnatal days 0 to 15 represent the proliferative phase in which
undifferentiated cells undergo mitotic activity, and days 16 to 44 represent
the period of differentiation when the blood-epididymal barrier is formed and
the columnar cells differentiate into principal cells, followed by a period of
expansion (days 44 to 91) during which sperm enter the epididymis and are
stored in the lumen of the cauda epididymis
(Cyr et al, 1993). In
addition, postnatal days 20 and 40 reflect the period before and after the
rise in serum androgens; on day 42 specific 5 reductase enzyme activity
is at its peak. Postnatal days 49 and 56 are marked by the first appearance of
spermatozoa in the caput and cauda epididymis, respectively. By day 77,
specific 5 reductase enzyme activity declines rapidly. Finally,
postnatal days 90 to 200 were used to represent the adult animal
(Viger and Robaire, 1992).
RNA Extraction
Total RNA was extracted from immature (15-day) and mature (60-day)
epididymal tissues using the single-step acid guanidium
thiocyanate–phenol-chloroform extraction method as described by
Chomozynski and Sacchi (1987);
quantitation and purity were determined by absorbance at 260 and 280 nm
(Smartspec; BioRad, Hercules, Calif). The integrity of the isolated RNA was
confirmed by electrophoresis on a 1% agarose (Amersham Biosciences,
Piscataway, NJ), 2.2 M formaldehyde gel using formaldehyde-containing buffer,
stained with 0.1 mg/mL ethidium bromide (Sigma-Aldrich) and visualized under
UV (Lehrach et al, 1977).
RT-PCR
Five micrograms each of total RNA extracted from immature and mature rat
epididymides were subjected to RT-PCR analysis by a single-step RT-PCR system
(Roche Diagnostics, Mannheim, Germany) using Hoxbes2 gene-specific
primers, forward primer 5'-CGGCACGAGGACTGCCGG-3' and reverse
primer-5'-GGGTTCTCTCGACAGCCCC-3', which encompass the 581-bp
fragment including the conserved region showing homology to Hoxb2
(Prabagaran et al, 2007). The
PCR conditions used were as follows: initial denaturation at 94°C for 2
minutes; 35 cycles of denaturation at 94°C for 45 seconds, annealing at
55°C for 30 seconds, and extension at 60°C for 45 seconds; and a final
extension at 72°C for 7 minutes. Amplification of a 580 bp ß-actin
housekeeping gene served as the internal control.
Immunohistochemistry
The immunohistochemical analysis of the HOXBES2 protein in the epididymis
followed by image analysis of immunoreactivity was performed based on the
protocols published earlier by our laboratory
(Prabagaran et al, 2007). In
brief, the epididymal tissue sections were immunostained with a goat
polyclonal HOXB2 antibody diluted 1:25 in blocking buffer for 1 hour at room
temperature. Controls were incubated with nonimmune goat serum at a dilution
similar to that used for the primary antibody. All other protocols were
followed according to the manufacturer's instructions (Santa Cruz
Biotechnology, Santa Cruz, Calif). Finally, the sections were dehydrated
through an alcohol gradation (10%–100%), cleaned in xylene, mounted in
p-xylene-bis (pyridinium bromide) Permount, and observed
under a microscope (Leitz, Oberkochen, Germany). Immunoreactivity to the HOXB2
antibody was analyzed using Biovis Image Plus 2.0 software (Expert Vision
Labs, Mumbai, India) based on the intensity of the color reactions observed in
the epididymal tissue sections.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western Blots
For determination of the expression pattern of HOXBES2 protein in the
epididymides of control and experimental rats, the epididymal segments were
dissected, minced, and incubated in 10 mL of 0.1 M phosphate-buffered saline
(pH 7.4) containing 0.1 M phenylmethylsulphonyl fluoride for 30 minutes at
room temperature to release the sperm, followed by extraction of protein as
described earlier (Hu et al,
2003). The protein concentrations of the extracts were estimated
by the method of Folin-Lowry (Lowry et al,
1951). Aliquots of 40 µg protein/lane of each tissue extract
were analyzed on a 10% sodium dodecyl sulfate polyacrylamide gel
(Laemmli, 1970) followed by
Western blot analysis (Towbin et al,
1979). For identification of the immunoreactive protein, the blot
was incubated with an affinity-purified monospecific goat polyclonal primary
antibody (HOXB2 [P-20], SC-17165; Santa Cruz-Biotechnology) generated by
epitope mapping within an internal region of the human HOXB2 protein. This was
followed by incubation with a 1:1000 diluted horseradish peroxidase
(HRP)–conjugated rabbit anti-goat secondary antibody (Bangalore Genei,
Bangalore, India). For developmental expression of AR in the epididymis,
another blot was probed using a rat monoclonal primary antibody to AR (Upstate
Inc, Chicago, Ill) at a dilution of 1:2000. This was followed by incubation
with a 1:2000 diluted HRP-conjugated rabbit anti-rat secondary antibody. The
peroxidase activity was detected using enhanced chemiluminescence (Amersham
Biosciences). The blots were stripped and reprobed with a ß-actin
monoclonal antibody to confirm equal loading of protein samples on the
gel.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
90%
of that observed in the sham-operated animals and further declined to 5% 1
week postcastration. Circulating T levels following castration decreased
within 5 days when compared with levels in the sham-operated controls (data
not shown). Since bilateral orchiectomy lowers blood T concentrations to
undetectable levels, the dramatic decrease in HOXBES2 levels following
castration suggests that T may play a role in the regulation of
Hoxbes2 gene expression. This was further shown when DHT
supplementation to rats 1 week postcastration resulted in restoration of
HOXBES2 expression in the epididymis to levels 50% to 70% of that observed in
the sham-operated animals (Figure
2). The specificity of the immunoreaction was confirmed by
immunolocalizing the sham-operated and castrated epididymal tissue sections
independently using normal goat serum at a dilution similar to the primary
antibody or with secondary antibody alone.
|
|
|
|
|
|
|
|
|
Postnatal Expression of HOXBES2 in the Epididymis
Developmental changes in the levels of immunoreactive HOXBES2 protein were
evaluated by immunohistochemistry and Western blot analyses. In 5-day
postnatal rats, there was no immunostaining for HOXBES2 in the epithelial
cells of the epididymis along its entire length. In the epididymis, a lumen
was already present at day 5 but without any apparent content. Similarly, no
staining was seen on postnatal days 10, 15, 20, 30, and 40. By postnatal day
50, principal cells with complete structural differentiation and the first
appearance of peroxidase staining for the HOXBES2 protein were observed in the
proximal regions of the epididymis. Postnatal days 55 and 60 were
characterized by the presence of spermatozoa, and HOXBES2 staining increased
gradually with a dramatic increase in the number of stained principal
epithelial cells. The intensity of the immunoperoxidase reaction products
appeared similar to that observed in adults by postnatal day 70 onward,
concomitant with markedly elevated circulating androgen levels. Although the
epithelial cells were stained at 200 days, most of the immunoreactivity was
localized in the lumen (Figure
10).
|
In the epididymal tissue sections immunoreacted with normal goat serum or secondary antibody alone (negative control), there was no immunoreactivity in the epithelium, intertubular space, and spermatozoa. In tissues treated with an anti-HOXB2 antibody, HOXBES2 localization was observed in the apical region of the principal epithelial cells. The spread of staining from the apical region on day 50 to the entire cytoplasm from 55 days onward was demonstrated by image analysis (Figure 11). The differences in the localization of the HOXBES2 protein in the epididymis of different age groups were comprehensively complemented by Western blot analysis (Figure 12). Equal loading of the protein samples in all lanes was confirmed by stripping and reprobing the blot using a monoclonal antibody to ß-actin as an internal control. Comparison of the developmental profile of AR and HOXBES2 expression by Western blot analysis indicated that AR was present at all stages of epididymal development with a slight elevation in the later stages of development (Figure 13).
|
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Following castration, which eliminates both circulating androgens and
testicular factors, T levels decreased to <10 % of baseline values within
24 hours (data not shown), whereas HOXBES2 protein levels decreased gradually
to 90% of the control levels by day 5 postcastration and disappeared
completely by day 7. In the current study, the critical dependence of HOXBES2
expression on circulating T was demonstrated by a delayed T repletion regimen
in which the castrated rats were injected daily with DHT beginning day 7
postcastration. The DHT supplementation restored the expression level of
HOXBES2 gradually to approximately 70% of that measured in the sham-operated
rats and maintained the circulating T to levels similar to those reported for
caput fluid in the rat (Turner et al,
1994). Therefore, it could be inferred that the concentration of
DHT used in this study was sufficient to restore HOXBES2 expression in the
castrated epididymis. Nevertheless, the gradual retention in luminal
morphology from caput to cauda and the progressive reappearance of HOXBES2
expression in principal epithelial cells following castration and DHT
supplementation could be explained by the fact that the delivery of exogenous
DHT through the general circulation may affect regionalized Hoxbes2
gene expression differently than T reaching the caput from the testis. Viger
and Robaire (1992) reported
that the expression of 5 reductase can be maintained by normal plasma T
concentrations in the corpus and cauda but not in the initial segment, where
it requires supraphysiologic concentrations of plasma T. Presumably the genes
that respond to a decline in the expression in all segments of the epididymis
by 1 week following orchiectomy are regulated by circulating androgens and not
testicular factors because the latter have been implicated in the regulation
of gene expression in the proximal epididymis but not in the distal segments
(Hinton et al, 1998).
Altogether, the ablation of HOXBES2 expression within 1 week following
castration and its re-establishment by DHT supplementation in caput, corpus,
and followed by cauda epididymis indicated that the expression of the protein
is under the control of circulating androgens.
The EDS-treated rat model (Jackson,
1973) was used to establish the interrelationship between the
intratesticular distribution of T and its influence on the expression of
HOXBES2 in the adjacent epididymis. EDS treatment of adult male rats destroyed
the T-secreting mature Leydig cell populations completely, and that resulted
in the subsequent depletion of T in the epididymis
(Kerr et al, 1985;
Molenaar et al, 1985;
Jackson et al, 1986;
Molenaar et al, 1986;
Morris et al, 1986).
Immunohistochemical analysis indicated a significant decrease in the
expression of the HOXBES2 protein in different segments of the epididymis
following EDS treatment. Similar studies have previously shown that exposure
of epididymal epithelial cells and sperm to EDS results in a significant
decline in the secretion of proteins in the range of 34 to 38 kDa and decrease
in progressive motility and velocity of spermatozoa
(Klinefelter et al, 1992).
Klinefelter et al (1994)
reported that both castration and EDS treatment significantly compromise the
fertilizing ability of sperm from the proximal cauda after 4 days of exposure.
Most Leydig cells exhibit degenerative changes 12 hours after treatment, and
all Leydig cells showed gross degenerative changes after 24 and 48 hours;
these changes disappear completely by 4 and 14 days
(Morris et al, 1986). In the
current study, the absence of mature Leydig cells during the first 10 days
following EDS treatment indicated that the development of new Leydig cells
takes a longer time under this regimen. It was also suggested that the
advanced precursor cells, capable of rapid proliferation into Leydig cells,
are killed by EDS or inhibited from differentiation; the complete repopulation
of Leydig cells was established approximately 49 days following a single dose
of EDS. These changes were accompanied by a decrease in the levels of serum T
and epididymal AR. When EDS-treated rats were subjected to an immediate T
repletion regimen with DHT supplementation, HOXBES2 expression was restored
gradually, concordant with the increase in the levels of T. Results similar to
this have been reported for proteins such as 80-kDa human sperm antigen
(Khobarekar et al, 2007) and
lipocalin-type prostaglandin D synthase
(Zhu et al, 2004) in the
castrated and EDS-treated rat epididymis. In the case of DHT and estradiol
combined supplementation, HOXBES2 expression was comparatively higher than
that observed in the DHT alone–supplemented group. This could be
attributed to the response from estrogen receptors (ER) and
ß (ERß) in the efferent ducts and epididymis
(Meistrich et al, 1975;
Fisher et al, 1997;
Hess et al, 2002). Given that
the postnatal epididymis contains receptors for ERß
(Atanassova et al, 2001) and
that the major circulating steroid during postnatal development is
3-diol (Moger, 1977), a
putative ligand for ERß, an argument can be proposed in favor of a role
for estrogen in the expression of HOXBES2. Earlier reports indicated that the
combined treatment of rats with estradiol (0.02 µg) and androgens
maintained normal motility and transport of spermatozoa
(Bandopadhyaha et al, 1974). Li
et al (2003) reported that the
expression of proteinase inhibitors such as cystatin E1 and E2 were
up-regulated by estrogens in the mouse epididymis. However, HOXBES2 expression
was not restored in the estradiol alone–administered EDS-treated rats.
This observation was similar to the report by Gupta et al
(1991) on the expression of
certain glycolytic enzymes in the rhesus monkey epididymis. These data may
possibly explain the differential response in epididymal gene expression to
androgens and estrogens in different mammalian species. On the other hand, it
emphasizes the importance of the androgen:estrogen balance in epididymal
function. Disturbance in this balance, particularly lowering the androgen and
simultaneously elevating the estrogen, could result in epididymal
abnormalities described in the present study. The fact that these
abnormalities are associated with changes in the expression of both AR and
ER reinforces the close functional relationship between androgen and
estrogen in the maintenance of HOXBES2 expression in the epididymis. Further,
the duality of epididymal response to the 2 sex steroids with respect to
HOXBES2 expression suggests that these 2 hormones could exert their action in
concert at the physiologic level as regulators of epididymal secretory
function.
Testicular factors are known to influence the expression of several proteins in the epididymis (Hinton et al, 1998; Hermo et al, 2000). In this study, the efferent ductules of 1 of the testes were ligated to determine whether the testicular factors are necessary to maintain HOXBES2 expression in the epididymis. EDL, which prevents the flow of testicular fluid into the epididymis, did not elicit any effect on epididymal HOXBES2 expression. Therefore, it could be inferred that the expression of HOXBES2 is dependent mainly on circulating and luminal androgens. Similar results have been reported for the androgen-dependent, tight-junction epididymal glycoprotein cadherins (Cyr et al, 1995), epididymal protein B/C (Brooks and Higgins, 1980), and proenkephalin (Garrett et al, 1990).
The gradual and significant increase in HOXBES2 expression was observed
during postnatal development of the rat epididymis. Epididymal proteins such
as acidic epididymal glycoprotein (Charest
et al, 1989) and protein SP
(Faye et al, 1980) are also
known to exhibit similar postnatal expression patterns. This is in tandem with
the acquisition of hormonal maturation of the epididymis with increases in
age. As early as 2 weeks after birth, the epididymis consists of narrow cells
in the initial segment and clear cells in the remainder of the epididymis.
After 3 weeks, the epididymis acquires detectable activity of the enzyme
5 reductase, although the testicular production of T still remains low.
Nevertheless, the immunoreactivity for HOXBES2 was not observed in the
epididymis during that time point. By day 39, the principal cells of the
epididymis attain adult-like structural features
(Hermo et al, 1992a) along
with high levels of luminal androgens
(Scheer and Robaire, 1980).
The initiation of HOXBES2 expression between day 40 and 50 coincides with the
complete differentiation of principal cells from columnar cells and their
further differentiation to principal cells and apical cells. The fact that the
HOXBES2 protein is barely detectable in the epididymis at 10 days and between
postnatal days 21 and 40, despite high concentrations of androgens and AR mRNA
(Charest et al, 1989),
indicates that the differentiating principal cells may simply be unable to
express Hoxbes2 mRNA, which in turn supports our earlier observation
that the localization of the HOXBES2 protein is mainly restricted to the
principal cells that appear only on day 30 onward.
The increase in the number of HOXBES2-positive cells and the intensity of
HOXBES2 immunostaining observed between postnatal days 60 and 70 onward
coincides with the increasing T levels in the epididymis to mature levels (21
ng/g tissue) and a concomitant rise in AR protein levels. The Hoxbes2
transcript (581-bp RT-PCR–amplified product) could not be detected in
epididymal RNA on day 15 but was present in 60-day-old epididymis, which is in
agreement with the results obtained by immunohistochemistry and Western blot
analyses. Calandra et al (1974)
reported that the AR protein in rat epididymis is not detectable until 20
days. A parallel study that we carried out to compare the ontogeny of AR to
HOXBES2 expression among different age groups indicated that AR was present at
all stages with a slight elevation in its expression during the later stages
of epididymal development. Although our observation was contrary to earlier
reports, several studies have suggested that the expression of AR is essential
for the early development and differentiation of the male reproductive tract,
particularly the epididymis from the wolffian duct. Moreover, the
developmental increase in the levels of HOXBES2 is of functional importance
because it occurs simultaneously with the first appearance of sperm in the
epididymis and immediately after the significant rise in levels of ABP and
5 reductase. The dramatic increase in HOXBES2 expression following this
period could be due to the appearance of critical factors regulating the
expression of the transcript for HOXBES2. Several factors can be proposed to
address the maturation-dependent regulation of the HOXBES2 protein. Garrett et
al (1990) suggested that sperm
may be an essential factor for the expression of proenkephalin mRNA that
appears on day 49 in the proximal initial segment and day 56 in the corpus and
cauda regions coinciding with that of adult ABP expression. However, this
cannot be the case for HOXBES2 because the expression of this protein occurs
even in the absence of testicular factors as demonstrated by unilateral EDL
studies. Further, the expression of the HOXBES2 protein was found to be
maximal in the corpus and caudal regions of the epididymis. These data
reiterated our earlier findings that the HOXBES2 protein is maximally
expressed in the distal regions of the epididymis from where the protein is
transferred to the transiting testicular spermatozoa
(Prabagaran et al, 2007). In
addition, in silico analysis of the 1657-bp Hoxbes2 gene (GenBank
accession number DQ399532) indicated the presence of a putative functional
androgen response element in the 3' untranslated region at positions
1578 to 1592 bp. This observation suggests that the Hoxbes2 gene
could be regulated directly by the circulating and luminal androgens. Taken
together, this finding concurs with the observations from orchiectomized,
EDS-treated, and EDL adult rats, revealing the dependence on T or its
metabolites for HOXBES2 expression in the epididymal principal cells.
Finally, the outcome of our study suggests that HOXBES2 expression is regulated by androgens and the developmental status of the epididymis and attains considerable significance in the realm of epididymal sperm maturation. Being a member of the homeobox gene cluster as a conserved HOXB2 homeodomain-containing protein, the androgen regulation of the HOXBES2 protein indicates that the Hoxbes2 gene might be involved in a cascade of androgen-regulated events in the epididymis. Therefore, future studies would be directed to further delineate the physiologic role of this androgen-dependent, developmentally regulated, and conserved HOXBES2 homeoprotein in sperm function.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Atanassova N, McKinnell C, Williams K, Turner KJ, Fisher JS,
Saunders PTK, Millar MR, Sharpe RM. Age-, cell- and region-specific
immunoexpression of estrogen receptor (but not estrogen receptor
ß) during postnatal development of the epididymis and vas deferens of the
rat and disruption of this pattern by neonatal treatment with
diethylstilbestrol. Endocrinology. 2001; 142: 2874
-2886.
Bandopadhyaha GP, Das RP, Roy S. Effect of estradiol-17-beta on spermatozoa of castrated rats. J Popul Res. 1974; 1: 44 -50.[Medline]
Bedford JM, Kim HH. Sperm-egg binding patterns and oocyte cytology
in retrospective analysis of fertilization failure in vitro. Hum
Reprod. 1993;8: 453
-463.
Berube B, Lefievre L, Coutu L, Sullivan R. Regulation of epididymal synthesis of P26h, a hamster sperm protein. J Androl. 1996; 11: 104 -110.
Boue F, Berube B, De Lamirande E, Gagnon C, Sullivan R. Human sperm-zona pellucida interaction is inhibited by an antiserum against a hamster sperm protein. Biol Reprod. 1994; 51: 577 -587.[Abstract]
Brooks DE. Androgen-regulated epididymal secretory proteins associated with post-testicular sperm development. Ann N Y Acad Sci. 1987;513: 179 -194.[Medline]
Brooks DE, Higgins SJ. Characterization and androgen dependence of
proteins associated with luminal fluid spermatozoa in the rat epididymis.
J Reprod Fertil. 1980; 59: 363
-375.
Calandra RS, Podesta EJ, Rivarola MA, Blaquier JA. Tissue androgens and androphilic proteins in rat epididymis during sexual development. Steroids. 1974;24: 507 -518.[CrossRef][Medline]
Charest NJ, Petresj P, Ordronneau P, Joseph DR, Wilson EM, French
FS. Developmental expression of an androgen regulated epididymal protein.
Endocrinology. 1989; 125: 942
-947.
Chomozynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate–phenol-chloroform extraction. Anal Biochem. 1987;162: 156 -159.[Medline]
Corker CS, Davidson DW. A radioimmunoassay for testosterone in various biological fluids without chromatography. J Steroid Biochem. 1978;9: 373 -374.[CrossRef][Medline]
Cornwall GA, Collis R, Xiao QR, Hsia N, Hann SR. B-Myc, a proximal
caput epididymal protein, is dependent on androgens and testicular factors for
expression. Biol Reprod. 2001; 64: 1600
-1607.
Cornwall GA, Hann SR. Specialized gene expression in the
epididymis. J Androl. 1995; 16: 379
-383.
Cyr DG, Hermo L, Robaire B. Developmental changes in epithelial
cadherin messenger ribonucleic acid and immunocytochemical localization of
epithelial cadherin during postnatal epididymal development in the rat.
Endocrinology. 1993; 132: 1115
-1124.
Cyr DG, Robaire B, Hermo L. Structure turnover of junctional complexes between principal cells of the rat epididymis. Microsc Res Tech. 1995;30: 54 -66.[CrossRef][Medline]
Ezer N, Robaire B. Androgenic regulation of the structure and functions of the epididymis. In: Robaire B, Hinton BT, eds. The Epididymis: From Molecules to Clinical Practice. New York, NY: Kluwer Academic/Plenum Publishers; 2002.
Ezer N, Robaire B. Gene expression is differentially regulated in
the epididymis after orchidectomy. Endocrinology. 2003; 144: 975
-988.
Faye JC, Duquet L, Mazura M, Bayard F. Purification, radioimmunoassay and immunohistochemical localization of a glycoprotein produced by the rat epididymis. Biol Reprod. 1980; 23: 423 -432.[Abstract]
Fisher JS, Millar MR, Majdic G, Saunders PT, Fraser HM, Sharpe RM.
Immunolocalization of estrogen receptor-alpha within the testis and excurrent
ducts of the rat and marmoset monkey from perinatal life to adulthood.
J Endocrinol. 1997; 153: 485
-495.
Garrett JE, Garrett SH, Douglass J. A spermatozoa-associated factor
regulates proenkephalin gene expression in the rat epididymis. Mol
Endocrinol. 1990;4: 108
-118.
Ghyselinck NB, Jimenez C, Courty Y, Dufaure JP. Androgen dependent
messenger RNA(s) related to secretory proteins in the mouse epididymis.
J Reprod Fertil. 1989; 85: 631
-639.
Girotti M, Jones R, Emery DC, Chia W, Hall L. Structure and expression of the rat epididymal secretory protein I gene. An androgen regulated member of the lipocalin superfamily with a rare splice donor site. Biochem J. 1992; 281: 203 -210.[Medline]
Gould K, Young WC. Acquisition of fertilizing capacity by chimpanzee sperm. Folia Primatol. 1990; 54: 857 -865.
Gupta D, Zarzycki J, Rager K. Plasma testosterone and dihydrotestosterone in male rats during sexual maturation and following orchidectomy and experimental bilateral cryptorchidism. Steroids. 1975;25: 33 -42.[CrossRef][Medline]
Gupta G, Srivastava A, Setty BS. Androgen-estrogen synergy in the regulation of energy metabolism in epididymis and vas deferens of rhesus monkey. Endocr Res. 1991; 17: 383 -394.[Medline]
Hermo L, Adamali HI, Andonian S. Immunolocalization of CA II and H+ V-ATPase in epithelial cells of the mouse and rat epididymis. J Androl. 2000;21: 376 -391.[Abstract]
Hermo L, Barin K, Oko R. Androgen binding protein secretion and
endocytosis by principal cells in the adult rat epididymis and during
postnatal development. J Androl. 1998; 19: 527
-541.
Hermo L, Barin K, Robaire B. Structural differentiation of the epithelial cells of the testicular excurrent duct system of rats during postnatal development. Anat Rec. 1992a; 233: 205 -228.[CrossRef][Medline]
Hermo L, Morales C, Oko R. Immunocytochemical localization of sulfated glycoprotein-1 (SGP-1) and identification of its transcripts in epithelial cells of the extra testicular duct system of the rat. Anat Rec. 1992b; 232: 401 -422.[CrossRef][Medline]
Hess RA, Zhou Q, Nie R. The role of estrogens in the endocrine and paracrine regulation of the efferent ductules, epididymis and vas deferens. In: Robaire B, Hinton BT, eds. The Epididymis: From Molecules to Clinical Practice. New York, NY: Kluwer Academic/Plenum Publishers; 2002.
Hinton BT, Lan ZJ, Rudolph DB, Labus JC, Lye RJ. Testicular regulation of epididymal gene expression. J Reprod Fertil. 1998;53: 47 -57.
Holland MK, Orgebin-Crist MC. Characterization and hormonal regulation of protein synthesis by the murine epididymis. Biol Reprod. 1988;38: 487 -496.[Abstract]
Holland MK, Vreeburg JTM, Orgebin-Crist MC. Testicular regulation
of epididymal protein secretion. J Androl. 1992; 13: 266
-273.
Hu Y, Zhou Z, Xu C, Shang Q, Zhang YD, Zhang YL. Androgen down
regulated and region specific expression of germ cell nuclear factor in mouse
epididymis. Endocrinology. 2003; 144: 1612
-1619.
Jackson H. Comparative effects of some antispermatogenic chemicals. In: Segal SJ, Crozier Crofman RPA, Cordliffe PG, eds. The Regulation of Mammalian Reproduction. Springfield, Ill: Thomas;1973.
Jackson NC, Jackson H, Shanks JH, Dixon JS, Lendon RG. Study using
in-vivo binding of 125I-labelled hcG, light and electron microscopy
of the repopulation of rat Leydig cells after destruction due to
administration of ethylene-1,2-dimethanesulphonate. J Reprod
Fertil. 1986;76: 1
-10.
Jervis KM, Robaire B. Dynamic changes in gene expression along the
rat epididymis. Biol Reprod. 2001; 65: 696
-703.
Jones R, Brown CR, Von Glos KI, Parker MG. Hormonal regulation of protein synthesis in the rat epididymis: characterization of androgen dependent and testicular fluid dependent proteins. J Biochem. 1980;188: 667 -676.
Kerr JB, Donachie K, Rommerts FFG. Selective destruction and regeneration of rat Leydig cells in vivo. Cell Tissue Res. 1985;242: 145 -156.[Medline]
Khobarekar BG, Vernekar VJ, Prabagaran E, Raghavan VP, Bandivdekar
AH. Studies on the expression of 80kDa human sperm antigen (80kDaHSA) in rat
testis and epididymis. J Histochem Cytochem. 2007; 55: 753
-762.
Kirchhoff C, Carballada R, Harms B, Kascheike I. CD52 mRNA is modulated by androgens and temperature in epididymal cell cultures. Mol Reprod Dev. 2000; 56: 26 -33.[CrossRef][Medline]
Klinefelter GR, Laskey JW, Perreault SD, Ferrell J, Jeffay S,
Suarez J, Roberts N. The ethane dimethane sulfonate-induced decrease in the
fertilizing ability of cauda epididymal sperm is independent of the testis.
J Androl. 1994;15: 318
-327.
Klinefelter GR, Roberts NL, Suarez JD. Direct effects of ethane
dimethane sulphonate on epididymal function in adult rats. An in vitro
demonstration. J Androl. 1992; 13: 409
-421.
Laemmli UK. Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227: 680 -685.[CrossRef][Medline]
Lakoski KA, Carron CP, Cabot CL, Saling PM. Epididymal maturation and the acrosome reaction in mouse sperm: response to zona pellucida develops coincident with modification of M42 antigen. Biol Reprod. 1988;38: 221 -233.[Abstract]
Lehrach H, Diamond D, Wozney JM, Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977; 16: 4743 -4751.[CrossRef][Medline]
Li Y, Friel PJ, McLean DJ, Griswold MD. Cystatin E1 and E2, new
members of male reproductive tract subgroup within cystatin type 2 family.
Biol Reprod. 2003; 69: 489
-500.
Lindsay S, Wilkinson MF. An androgen-regulated homeobox gene is expressed in rat testis and epididymis. Biol Reprod. 1996; 55: 975 -983.[Abstract]
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurements
with the Folin phenol reagent. J Biol Chem. 1951; 193: 265
-275.
Lunde T, Christiansen E, Purvis E. Epididymis. Anatomy function and pathology [in Norwegian]. Tidsskr Nor Laegeforen. 1990; 110: 3119 -3123.[Medline]
Meistrich ML, Hughes TH, Bruce WR. Alteration of epididymal sperm transport and maturation in mice by estrogen and testosterone. Nature. 1975;258: 145 -147.[CrossRef][Medline]
Moger WH. Serum 5-anrostane,-3, 17ß-diol,
androsterone and testosterone concentrations in the male rat. Influence of age
and gonadotropin stimulation. Endocrinology. 1977; 100: 1027
-1032.
Molenaar R, de Rooij DG, Rommerts FF, Reuvers PJ, van der Molen HJ. Specific destruction of Leydig cells in mature rats after in vivo administration of ethane dimethyl sulfonate. Biol Reprod. 1985;33: 1213 -1222.[Abstract]
Molenaar R, de Rooij DG, Rommerts FF, van der Molen HJ.
Repopulation of Leydig cells in mature rats after selective destruction of the
existent Leydig cells with ethylene dimethane sulfonate is dependent on
luteinizing hormone and not follicle-stimulating hormone.
Endocrinology. 1986; 118: 2546
-2554.
Morris ID, Philips DM, Bardin W. Ethylene dimethane sulfonate
destroys Leydig cells in the rat testis.
Endocrinology. 1986; 118: 709
-719.
Orgebin-Crist MC. Androgens and epididymal function. In: Bhasin S, Wang C, Swerdloff RS, eds. Pharmacology, Biology and Clinical Application of Androgens: Status and Future Prospects. New York, NY: John Wiley & Sons; 1996.
Orgebin-Crist MC. Studies on the function of the epididymis.
Biol Reprod. 1969; 1: 155
-175.
Prabagaran E, Bandivdekar AH, Dighe V, Raghavan VP. HOXBES2: a
novel epididymal HOXB2 homeoprotein and its domain-specific association with
spermatozoa. Biol Reprod. 2007; 76: 314
-326.
Robaire B, Hermo L. Efferent ducts epididymis and vas deferens: structure, functions and their regulation. In: Knobil E, Neill J, eds. The Physiology of Reproduction. New York, NY: Raven Press;1998.
Robaire B, Viger RS. Regulation of epididymal epithelial cell functions. Biol Reprod. 1995; 52: 226 -236.[Abstract]
Saling P. Development of the ability to bind to zona pellucidae during epididymal maturation: reversible immobilization of mouse spermatozoa by lanthanum. Biol Reprod. 1982; 26: 429 -436.[Abstract]
Scheer H, Robaire B. Steroid delta4-5-reductase
and 3-hydroxysteroid dehydrogenase in the rat epididymis during
development. Endocrinology. 1980; 107: 948
-953.
Teerds KJ, De Rooij DG, Rommerts FF, Wensing CJ. The regulation of
the proliferation and differentiation of rat Leydig cell precursor cells after
EDS administration or daily HCG treatment. J Androl. 1988; 9: 343
-351.
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some
applications. Proc Natl Acad Sci U S A. 1979; 76: 4350
-4354.
Turner TT, Avery EA, Sawchuk TJ. Assessment of protein synthesis and secretion by rat seminiferous and epididymal tubules in vivo. Int J Androl. 1994; 17: 205 -213.[Medline]
Viger RS, Robaire B. Expression of 4-ene steroid 5-reductase
messenger ribonucleic acid in the rat epididymis and postnatal development.
Endocrinology. 1992; 131: 1534
-1540.
Xu W, Ensurd KM, Hamilton DW. The 26kDa protein recognized on rat cauda epididymal sperm by monoclonal antibody 4e9 has internal peptide sequence that is identical to the secreted form epididymal protein E. Mol Reprod Dev. 1997; 46: 377 -382.[CrossRef][Medline]
Zhu H, Ma H, Ni H, Ma XH, Mills N, Yang ZM. L-prostaglandin D synthase expression and regulation in mouse testis and epididymis during sexual maturation and testosterone treatment after castration. Endocrine. 2004; 24: 39 -45.[CrossRef][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
