The formation of the biological microenvironment for sperm maturation and storage in
the epididymis occurs via various specific events. One of the most important events
is cell–cell communication among the principal, basal, and clear cells of the
epididymal epithelium to create a specialized environment [1-3]. In addition, the
blood–epididymis barrier (BEB) is comprised of tight junctions (TJs) between
neighboring epithelial cells, which also supports the creation and maintenance of
proper physiological conditions for the maturation and storage of spermatozoa [4-6]. In
male reproductive tracts, the distribution of various tight junctional proteins is
well reported in the testis and the epididymis, especially. In the testis, the
blood–testis barrier (BTB) is located close to the base of the seminiferous
tubule between adjacent Sertoli cells [7,8]. In the epididymis, however, the BEB is
formed at apical surfaces of the epididymal duct between epithelial cells [9,10].
The BTB and BEB show positional differences but have the same function, which is to
regulate the movement of electrolytes, ions, water, nutrients, hormones, and
molecules, and the permeability of the paracellular transport pathway. The BTB and
BEB also maintain the immunologically privileged adluminal compartment, which is a
necessary component of the functioning barrier of the testis and epididymis [11-16].TJs are protein complexes composed of several peripheral membranes and integral
transmembrane proteins. Zonula occludens-1 (ZO-1) was first discovered as a TJ
component in epithelia located on the intracellular side of the plasma membrane
[17]. Additionally, two more proteins,
ZO-2 and ZO-3, were confirmed as members of the ZO protein family [18,19].
These ZO proteins play a critical role in forming bridges between trans-membrane
associated proteins and the actin cytoskeleton [20,21]. In contrast, occludin was
first confirmed as the integral transmembrane protein and has four transmembrane
domains with intracellular N- and C-termini [22]. In addition, claudins (Cldns) are also known as TJ transmembrane
proteins, comprising at least 27 members in mammals [23,24]. Both occludin and Cldns
interact with various intracellular proteins of TJs, including ZO-1, ZO-2, and ZO-3
[25,26]. TJ complexes that form a variety of proteins can create a physical,
physiological, and immunological barrier.Recent studies have reported that hormones can regulate the expression levels and
localization patterns of TJ proteins in the testis. For example, gonadotropin
hormones (GH), follicle stimulating hormone (FSH), and luteinizing hormone (LH)
regulate the formation of the pituitary gland and are necessary for TJ formation in
rat testes because GH plays a critical role in the development and differentiation
of Sertoli cells where TJs are formed [27,28]. Androgens can upregulate
Cldn3 and Cldn11 in Sertoli cells [13,29], and the administration of flutamide, an
androgen receptor antagonist, disrupts Cldn11 protein in rat testes [30]. Additionally, in the epididymis, we
reported that the extracellular signal-regulated kinase (ERK) signaling pathway is a
key player in controlling the expression and localization of TJ proteins, such as
ZO-2, ZO-3, occludin, and Cldn1, 3, and 4 in TJs of the mouse epididymis [31]; however, we know less about the functional
roles of TJs in the epididymis than in the testis. Moreover, there has been no study
on the expression and localization of TJ proteins and changes in TJ proteins during
postnatal development of the epididymis in goats. Hence, in this present study, we
characterized the distribution and localization patterns of TJ proteins in the
immature and mature epididymides of goats using immunofluorescence and western
blotting analyses.
MATERIALS AND METHODS
Antibodies
All information of primary and secondary antibodies used for immunofluorescence
staining and western blotting is described in Tables 1 and 2,
respectively.
Epididymides were collected using a surgical castration procedure from immature
(3-month-old, n = 4), and mature (14-month-old, n = 5) black goats with an
average body weight of 9.95 ± 0.66 kg and 28.04 ± 1.42 kg,
respectively, at the National Institute of Animal Science. All animal
experiments used here were performed under regulation and permission of the
Institutional Animal Care and Use Committee (IACUC) of the National Institute of
Animal Science (NIAS, Approval No. NIAS 2018-290).
Tissue fixation, embedding and sectioning
Following castration, the epididymides were briefly cleaned in a cold
phosphate-buffered saline (PBS) buffer to remove blood. The fixation of the
epididymides was achieved by immersion in 4% paraformaldehyde solution (PFA) for
48–72 hr at 4°C, and the fixative was replaced with fresh solution
every 24 hr. The samples were rinsed with PBS five times for 30 minutes each at
room temperature (RT). To prevent ice crystal formation during freezing tissue
samples, the epididymal tissues were immersed into 30% sucrose solution in PBS
for at least 48–72 hr (until tissue sinks). The 30% sucrose solution was
replaced with fresh one every 24 hr. The tissues were completely embedded in
Tissue Tek compound (Sakura Finetek, Torrance, CA, USA) for 20 minutes, and
freeze at −20°C to −80°C. 10 µm thick tissue
sections were carefully cut and collected using a Leica cryostat (Leica
Biosystems, Buffalo Grove, IL, USA). The cryostat temperature was between
−18°C and −25°C. The tissue sections were mounted on
gelatin coated slides (Sigma Aldrich, St. Louis, MO, USA), dried for 30 minutes
at RT, and stored at −20°C to −80°C until further
use.
Immunofluorescence staining
The cryostat sections were rehydrated in PBS for 15 min. For epitope retrieval,
the sections were immersed into preheated retrieval solution (pH 9.0,
95°C; DAKO, Carpinteria, CA, USA) for 30 minutes and then cooled to RT.
The sections were then incubated in blocking buffer (1% bovine serum albumin
[BSA] in PBS; Thermo Scientific, Rockford, IL, USA) for 30 minutes at RT to
prevent non-specific binding issue on the target epitopes between primary
antibodies and tissues. The sections were then incubated with primary antibodies
diluted in the diluent buffer (DAKO) overnight at 4°C. Thereafter, the
sections were rinsed 3 times for 20 minutes in PBS and incubated with secondary
antibodies diluted in the diluent buffer for 90 minutes at RT. For double
immunostaining, primary and secondary antibody incubation steps were repeated
without the second block step. After this period, the sections were rinsed 3
times for 20 minutes in PBS and then placed in anti-fade mounting media
containing 4′,6-diamidino-2-phenylindole (DAPI; Vecta Labs, Burlingame,
CA, USA) for 5 minutes at RT to counterstain the nuclei. For staining on
negative controls, sections were incubated with the antibody diluent and a
non-immune serum (Thermo Scientific) of the same isotype and at the same
concentration as the primary antibodies. Confocal microscope (LSM800; ZEISS,
Oberkochen, Germany) was used to acquire the images that analyzed by Zen Blue
(ZEISS, Oberkochen, Germany).
Quantitative analysis of tight junction proteins by
immunofluorescence
Epididymis tissues were double-immunostained with ZO-1 and ZO-2, ZO-3, occludin,
or Cldn3. A LSM800 confocal microscope was used to acquire images. All images
used for quantification were obtained in the same conditions. For comparisons of
levels of TJ proteins between groups, the epididymides of three immature and
mature animals was conducted and three cryostat sections from each group was
labeled and analyzed. The quantification of the expression level of ZO-1
colocalization with each target protein in TJs was calculated by using the Image
J software (NIH, Bethesda, MD, USA), as described earlier [31].
Protein extraction and immunoblotting
The epididymides were homogenized on ice for 30 minutes in RIPA buffer (Thermo
Scientific) additive with complete protease inhibitors (Roche, Penzberg,
Germany). The homogenized sample was centrifuged at 18,000×g for 20
minutes at 4°C, and then the supernatant were collected. Protein
concentration was determined by the bicinchoninic acid (BCA) Protein Assay Kit
(Thermo Scientific). Protein samples were diluted with 4x lithium dodecyl
sulfate (LDS) Sample Buffer (Bio-Rad Laboratories, Hercules, CA, USA) in the
presence of 4% b-mercaptoethanol. Samples were heated at 99°C for 10
minutes and cooled on ice before loading on gels. Proteins were separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel
electrophoresis and then wet-blotted onto polyvinylidene difluoride (PVDF)
membranes for 90 minutes at 90 V. Blots were blocked for 60 minutes with 5%
non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at RT.
Primary antibodies diluted in TBS were applied overnight at 4°C. After
washing in TBST, secondary antibodies were applied for 60 minutes at RT.
Antibody binding was visualized by Chemiluminescence reagent (Perkin Elmer Life
Sciences) and were detected with iBright FL1500 (Invitrogen).
Statistical analysis
We have carried out a western blotting with the triplicated samples each group.
The mean value of each target protein was normalized to the value of
β-actin. Data are presented as mean ± standard error of the mean.
Statistical comparison was evaluated by Student’s
t-tests.
RESULTS
Expression and localization of ZO-1, ZO-2, and ZO-3 in immature and mature
goat epididymides
To confirm that ZO-1 is expressed in the goat epididymis, the epididymides at
postnatal month (PNM) 3, and 14 were stained with a ZO-1 monoclonal antibody.
ZO-1 was strictly expressed in the apical region (where TJs are present) of the
epididymal tubules (Figs. 1A and 1B, white arrowheads). The expression level
of ZO-1 was higher in the PNM3 than in the PNM14 epididymis. Quantification of
the average pixel intensity of ZO-1 immunostaining in TJs showed a drastic
decrease in the adult epididymis (Fig. 1C).
The same results were observed by western blot analysis (Fig. 1D).
Fig. 1.
Immunohistochemical expression and localization of zona occludens
(ZO)-1 in the goat epididymis.
The epididymis was immunostained for ZO-1 (green). (A) Epididymis at
postnatal month (PNM) 3. (B) Epididymis at PNM14. Insets indicate ZO-1
labeling with DAPI (Bar = 20 µm). (C) Quantification of ZO-1
labeling in TJs confirmed the decrease in ZO-1 expression in the mature
epididymides compared to that in the immature ones. (D) Western blot
analysis was applied to investigate ZO-1 expression in the epididymis at
PNM3, and PNM14. *p < 0.05, **p
< 0.001.
Immunohistochemical expression and localization of zona occludens
(ZO)-1 in the goat epididymis.
The epididymis was immunostained for ZO-1 (green). (A) Epididymis at
postnatal month (PNM) 3. (B) Epididymis at PNM14. Insets indicate ZO-1
labeling with DAPI (Bar = 20 µm). (C) Quantification of ZO-1
labeling in TJs confirmed the decrease in ZO-1 expression in the mature
epididymides compared to that in the immature ones. (D) Western blot
analysis was applied to investigate ZO-1 expression in the epididymis at
PNM3, and PNM14. *p < 0.05, **p
< 0.001.Next, we investigated whether the other two ZO proteins, ZO-2 and ZO-3, were
present in the TJs of the goat epididymis. Double labeling with ZO-1 and ZO-2
showed that ZO-2 was co-localized with ZO-1 in TJs of the epididymal tubules
(Figs. 2A and 2B, yellow arrowheads). ZO-2 expression pattern was similar
to that of ZO-1 (Figs. 2C and 2D, white arrowheads). ZO-2 labeling was
brighter in the PNM3 epididymides compared to that at PNM14. Quantification of
the average pixel intensity of ZO-2-associated immunostaining in TJs revealed a
drastic decrease in the mature epididymis (Fig.
2E).
Fig. 2.
Immunohistochemical expression and localization of zona occludens
(ZO)-2 in the goat epididymis.
The epididymis was immunostained for ZO-1 (green) and ZO-2 (red). (A, C)
Epididymis at postnatal month (PNM) 3. (B, D) Epididymis at PNM14.
Yellow and white arrowheads indicate an overlap in ZO-1 and ZO-2
staining and ZO-2 single staining, respectively. Insets indicate ZO-1
single staining (Bar = 20 µm). (E) Quantification of ZO-2
labeling in regions co-localized with ZO-1 in TJs revealed a reduction
in the ZO-2 expression level in the mature epididymides compared to that
in the immature ones. (F) Western blot analysis was applied to
investigate ZO-2 expression in the epididymis at PNM3 and PNM14.
*p < 0.05, **p <
0.001.
Immunohistochemical expression and localization of zona occludens
(ZO)-2 in the goat epididymis.
The epididymis was immunostained for ZO-1 (green) and ZO-2 (red). (A, C)
Epididymis at postnatal month (PNM) 3. (B, D) Epididymis at PNM14.
Yellow and white arrowheads indicate an overlap in ZO-1 and ZO-2
staining and ZO-2 single staining, respectively. Insets indicate ZO-1
single staining (Bar = 20 µm). (E) Quantification of ZO-2
labeling in regions co-localized with ZO-1 in TJs revealed a reduction
in the ZO-2 expression level in the mature epididymides compared to that
in the immature ones. (F) Western blot analysis was applied to
investigate ZO-2 expression in the epididymis at PNM3 and PNM14.
*p < 0.05, **p <
0.001.Western blotting verified the decrease in ZO-2 in PNM14 epididymides (Fig. 2F). ZO-3 also colabeled with ZO-1 in
TJs in the goat epididymis (Figs. 3A and
3B, yellow arrowheads). Further, its
expression was significantly decreased at PNM14 compared to that in PNM3
epididymides (Figs. 3C and 3D, white arrowheads). The quantification of
ZO-3 expression was confirmed by immunofluorescence (Fig. 3E) and western blotting (Fig. 3F). These results indicated that all three ZO proteins were
maintained at high expression levels during immature stages, but were
drastically reduced in the mature epididymides.
Fig. 3.
Immunohistochemical expression and localization of ZO-3 in the goat
epididymis.
The epididymis was immunostained for zona occludens (ZO)-1 (green) and
ZO-3 (red). (A, C) Epididymis at postnatal month (PNM) 3. (B, D)
Epididymis at PNM14. Yellow and white arrowheads indicate in overlap of
ZO-1 and ZO-3 staining and ZO-3 single staining, respectively. Insets
indicate ZO-1 single staining (Bar = 20 µm). (E) Quantification
of ZO-3 labeling in regions co-localized with ZO-1 in TJs revealed a
reduction in the ZO-3 expression level in the mature epididymides
compared to that in the immature ones. (F) Western blot analysis was
applied to investigate ZO-3 expression in the epididymis at PNM3 and
PNM14. *p < 0.05, **p <
0.001.
Immunohistochemical expression and localization of ZO-3 in the goat
epididymis.
The epididymis was immunostained for zona occludens (ZO)-1 (green) and
ZO-3 (red). (A, C) Epididymis at postnatal month (PNM) 3. (B, D)
Epididymis at PNM14. Yellow and white arrowheads indicate in overlap of
ZO-1 and ZO-3 staining and ZO-3 single staining, respectively. Insets
indicate ZO-1 single staining (Bar = 20 µm). (E) Quantification
of ZO-3 labeling in regions co-localized with ZO-1 in TJs revealed a
reduction in the ZO-3 expression level in the mature epididymides
compared to that in the immature ones. (F) Western blot analysis was
applied to investigate ZO-3 expression in the epididymis at PNM3 and
PNM14. *p < 0.05, **p <
0.001.
Expression and localization of occludin in immature and mature goat
epididymides
To determine whether occludin, a transmembrane protein, could be localized to the
TJs of the goat epididymis, double-staining was performed with ZO-1 and
occludin. Occludin protein colocalized with ZO-1 in TJs (Figs. 4A and 4B, yellow
arrowheads). Occludin was also significantly decreased in the PNM14 epididymides
compared to that of PNM3 (Figs. 4C and
4D, white arrowheads). The
quantification of occludin expression was confirmed by immunofluorescence (Fig. 4E) and western blotting (Fig. 4F). This result indicated that occludin
was also significantly decreased in the mature epididymis, similar to the three
ZO proteins, known as peripheral membrane proteins.
Fig. 4.
Immunohistochemical expression and localization of occludin in the
goat epididymis.
The epididymis was immunostained for zona occludens (ZO)-1 (green) and
occludin (red). (A, C) Epididymis at postnatal month (PNM) 3. (B, D)
Epididymis at PNM14. Yellow and white arrowheads indicate an overlap in
ZO-1 and occludin staining and occludin single staining, respectively.
Insets indicate ZO-1 single staining (Bar = 20 µm). (E)
Quantification of occludin labeling in regions co-localized with ZO-1 in
TJs revealed a reduction in the occludin expression level in the mature
epididymides compared to that in the immature ones. (F) Western blot
analysis was applied to investigate the occludin expression in the
epididymis at PNM3 and PNM14. *p < 0.05,
**p < 0.001.
Immunohistochemical expression and localization of occludin in the
goat epididymis.
The epididymis was immunostained for zona occludens (ZO)-1 (green) and
occludin (red). (A, C) Epididymis at postnatal month (PNM) 3. (B, D)
Epididymis at PNM14. Yellow and white arrowheads indicate an overlap in
ZO-1 and occludin staining and occludin single staining, respectively.
Insets indicate ZO-1 single staining (Bar = 20 µm). (E)
Quantification of occludin labeling in regions co-localized with ZO-1 in
TJs revealed a reduction in the occludin expression level in the mature
epididymides compared to that in the immature ones. (F) Western blot
analysis was applied to investigate the occludin expression in the
epididymis at PNM3 and PNM14. *p < 0.05,
**p < 0.001.
Changes in claudin3 in immature and mature goat epididymides
To determine whether Cldn3, a transmembrane protein, could be expressed in the
TJs of the goat epididymis, double-labeling was performed using ZO-1 and Cldn3
antibodies. Interestingly, the localization and expression of Cldn3 tended to be
age-dependent. At PNM3, Cldn3 was expressed and localized to the TJs (Figs. 5A and 5C; yellow arrowheads) and basolateral membranes (Figs. 5A and 5C; white arrowheads). At PNM14, Cldn3 was significantly decreased
in TJs where ZO-1 was localized (Figs. 5B
and 5D; yellow arrowheads) and also showed
low expression levels in the basolateral membranes (Figs. 5B and 5D; white
arrowheads). Quantification of the average pixel intensity of Cldn3-associated
immunostaining from TJs revealed a significantly reduced signal at PNM14
compared to that at PNM3 (Fig. 5E). The
total expression level of Cldn3 was examined by western blotting (Fig. 5F). These results indicate that Cldn3
is expressed at and localized to the basolateral membrane and TJs. Moreover, the
changes in Cldn3 expression patterns appear to be age-dependent.
Fig. 5.
Immunohistochemical expression and localization of claudins3 (Cldn3)
in the goat epididymis.
The epididymis was immunostained for zona occludens [ZO]-1 (green) and
Cldn3 (red). (A, C) Epididymis at postnatal month (PNM) 3. (B, D)
Epididymis at PNM14. Yellow and white arrowheads indicate an overlap in
ZO-1 and Cldn3 staining and Cldn3 single staining, respectively. Insets
indicate ZO-1 single staining (Bars = 20 µm). (E) Quantification
of Cldn3 labeling in regions co-localized with ZO-1 in TJs revealed a
reduction in the Cldn3 expression level in the mature epididymides
compared to that in the immature ones. (F) Western blot analysis was
applied to investigate Cldn3 expression in the epididymis at PNM3 and
PNM14. *p < 0.05, **p <
0.001.
Immunohistochemical expression and localization of claudins3 (Cldn3)
in the goat epididymis.
The epididymis was immunostained for zona occludens [ZO]-1 (green) and
Cldn3 (red). (A, C) Epididymis at postnatal month (PNM) 3. (B, D)
Epididymis at PNM14. Yellow and white arrowheads indicate an overlap in
ZO-1 and Cldn3 staining and Cldn3 single staining, respectively. Insets
indicate ZO-1 single staining (Bars = 20 µm). (E) Quantification
of Cldn3 labeling in regions co-localized with ZO-1 in TJs revealed a
reduction in the Cldn3 expression level in the mature epididymides
compared to that in the immature ones. (F) Western blot analysis was
applied to investigate Cldn3 expression in the epididymis at PNM3 and
PNM14. *p < 0.05, **p <
0.001.
DISCUSSION
Incomplete spermatozoa can obtain abilities such as progressive motility and
fertilization capacity through the final maturation step in the epididymis. The
maturation events of spermatozoa in the epididymis depend on a unique luminal
environment that is provided and maintained by the formation of the BEB, which is
formed by TJs composed of several proteins, including transmembrane and peripheral
proteins. These specialized TJ proteins function in forming the seal between
epithelial cells, which is essential for sperm maturation as well as sperm
protection from the immune system. However, there is still limited information on
the regulation mechanism of TJ proteins that are key factors for BEB formation.
Here, we determined the expression and localization patterns of TJ proteins,
including peripheral membrane proteins, three ZOs (ZO-1, ZO-2, and ZO-3), and the
transmembrane proteins, occludin and Cldn3, in the immature and mature goat
epididymis.
Distribution of tight junction proteins in the goat epididymis
ZO proteins comprise a family of TJ-associated proteins that have a functional
role in linking transmembrane proteins to the actin-based cytoskeleton [21]. In the present study, all three ZOs
(ZO-1, ZO-2, and ZO-3) were present and localized along the apical regions of
adjacent epithelial cells of both the immature and mature epididymis. These
results agree with those of a previous study showing that all three ZOs were
present in the TJs of the adult epididymis of mice [31]. In addition, we suggest that in the goat epididymis,
their expression levels are different between the immature and mature stages.
For example, all three ZO proteins were detected at significantly lower levels
in the mature epididymides than in the immature ones (Figs. 1, 2, and 3). Previous studies have demonstrated that
ZO-1/ZO-2 and ZO-1/ZO-3 complexes are present, whereas ZO-2 is not associated
with ZO-3 [21]. Moreover, we previously
demonstrated the protein expression compensatory relationship between ZO-2 and
ZO-3 without affecting ZO-1 expression levels in the epididymis of mice [31]. However, in the present study, we did
not observe any compensation between ZO-2 and ZO-3 during goat epididymis
development. These unexpected findings suggest the possibilities that other TJ
proteins could contribute to BEB formation in the mature epididymis instead of
ZO proteins or that specific relationships among these three ZO proteins might
create an optimal system in this organ. Further studies are needed to clarify
the reason underlying the reduction of all three ZO proteins in the mature
epididymis.We also observed that occludin and Cldn3 proteins, transmembrane proteins
associated with ZO-1, ZO-2, and ZO-3 were localized and expressed in TJs. The
localization and expression of a few types of transmembrane proteins in the
epididymis have been previously reported in various species, including pigs
[32], canines [33], rabbits [34],
mice [31], and rats [35]. Occludin expression in TJs appears
from embryonic day 18.5 in the mouse epididymis and its expression is maintained
until adulthood [36]. Various Cldns were
also observed in the epididymis, including Cldn3, which is expressed in the TJs
and basolateral membranes in mice [31],
rats [37], and bats [38]. In agreement with these previous
studies, we found that occludin was present in TJs and that Cldn3 was expressed
in both the basolateral membranes and TJs in the goat epididymis. Moreover, our
results indicated much higher expression of occludin and Cldn3 in the immature
epididymis, as high levels of ZO proteins were also observed. This might be
because the high expression of ZO proteins in the immature epididymis can
increase the chance of an association with occludin and Cldn3. Otherwise, the
reduction of ZO proteins could result in a decreased chance of binding to other
transmembrane TJ proteins, such as occludin and Cldn3. All TJ proteins examined
in this study were expressed at significantly higher levels in the immature
epididymis than in the mature epididymis. These findings suggest that some
factors regulating the expression of TJ proteins are likely to be present during
epididymis development. Additional studies investigating the expression and
localization patterns of other TJ proteins are needed to elucidate these
unexpected issues in the goat epididymis.
Regulation of tight junction proteins in the goat epididymis
Testicular luminal factors (TLFs) might be important in regulating changes in the
distribution of TJ proteins. TLFs synthesized and secreted from the testis,
include hormones, growth factors, and other luminal nutrients, which are
directly in contact with epididymal epithelial cells that contribute to the
formation of TJs and play an important role in the development of the epididymis
[1, 39-41]. According to a previous study, the secretion of testicular luminal
fluid might occur only in the normal mature testes of many species [42]. Therefore, it is possible that the
epithelial cells exposed to TLFs during puberty might contribute to changes in
the levels of TJ proteins in the mature epididymis. We previously reported that
the epithelium is not perfectly differentiated until PNM2 [43] and PNM3 (unpublished data). Thus, the undifferentiated
epithelium has a stronger TJ association between epithelial cells, and those
that are differentiated by contact with TLFs could reduce their TJ association
at the epididymal epithelium in goats. The reduction of these TJ proteins might
modify the epididymal luminal contents that make up a unique environment in the
adult epididymis for sperm maturation and storage. In addition, among TLFs, an
androgen hormone known to be the most important key factor is responsible for
regulating and maintaining epididymal structure and function. Although not well
understood, an association exists between TJs and androgens. Several studies
have shown that androgens regulate TJ molecules including Cldn3, Cldn11,
occludin, and ZO-1 [44-46] and are crucial for the regulation of
every aspect of spermatogenesis via formation of the BTB [47,48]. However,
direct links between androgens and TJ proteins in the epididymis have not been
well documented. Therefore, more research on the effect of androgens on BEB
formation is needed to confirm this assumption. Moreover, estrogen, another
steroid hormone, is a good candidate factor that controls the expression and
localization of TJ proteins. Estrogens are known as the most important female
hormone, but in males, estrogens are critical to maintain testicular and
epididymal function [49,50]. Without estrogens, male animals are
infertile owing to failed fluid reabsorption, which is necessary for sperm
concentration maintenance in the efferent ductules of the epididymis [51]. Earlier studies also provided some
evidence that estrogens negatively control BTB function. Exposure to estrogens
or diethylstilbestrol (a synthetic nonsteroidal estrogen) delays BTB formation
and induces spermatogenesis failure in rats [27,52]. Moreover, bisphenol A
(an estrogenic environmental toxicant) treatment induces disruption of the TJ
barrier by changing the expression and localization of transmembrane proteins at
the BTB [53]. Therefore, it is possible
that an increased exposure to estrogens in the adult epididymis might decrease
the expression levels of TJ proteins in the BEB.Based on the present results, ZO-1, ZO-2, ZO-3, occludin, and Cldns are expressed
and localized at the TJs of immature and mature epididymides of goats. Moreover,
all TJ proteins were expressed at much higher levels in the immature epididymis
than in the mature epididymis. This finding implies that undifferentiated
epithelial cells might form a stronger BEB through high expression levels of TJ
proteins, and then, the expression of TJ proteins decreases with the
differentiation of epithelial cells in the goat epididymis. Our findings could
contribute to a better understanding of the organization of TJ proteins in the
BEB during the development of the goat epididymis. Although we could not
directly explain the regulatory mechanism of TJ proteins in the BEB, our
findings further the understanding of the reproductive physiological
characteristics of the goat epididymis.
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.