Protein
profiles in various epididymal segments of normal and castrated rats
Premendu
P. Mathur1,2, Aileen Marshall2,
C.Y. Cheng2
1School
of Life Sciences, Pondicherry University, Pondicherry-605
014, India
2The Population Council, Center for Biomedical Research,1230
York Avenue, New York, NY 10021, USA
Asian
J Androl 2000
Mar;
2: 57-64
Keywords:
epididymis;
proteins; sperm maturation; orchiectomy; castration; rats
Abstract
Aim:
Epididymal
proteins are known to play an important role in the maturation
of
spermatozoa. We ought to determine if there are regional differences in
androgen-dependent epididymal proteins. Methods:
A group of adult rats was castrated and epididymides were removed
three days following castration. The epididymides were dissected into caput,
corpus and cauda segments, homogenized, and proteins were
fractionated by anion exchange HPLC. Proteins in selected fractions were
resolved by SDS-PAGE and visualized by silver staining. Results:
It was observed that the levels of multiple proteins drastically reduced
in
the various regions of epididymis of the orchiectomized rats.
Conclusion:
The epididymal proteins appear to be useful markers to study androgenic action
in
the epididymis.
1
Introduction
The
mammalian epididymis plays an important role in male reproduction by providing
a microenvironment for sperm maturation and storage. It is well established
that a functional epididymis is dependent upon androgens and several testicular
factors produced by the testis[1]. In addition, post-testicular
maturation of spermatozoa requires the specialized epididymal environment
which is regulated by selective secretory and absorptive functions[2].
It was, therefore, postulated that sperm maturation is dependent on the
interactions between spermatozoa and specific epididymal proteins during
their transit through the epididymal lumen. The mammalian
epididymis and its role in sperm maturation have been studied extensively
in many species[3-5].
Epididymal
proteins have been the subject of many studies in view of their interactions
with spermatozoa and their possible role in the maturation of male gamete.
It has been shown that rat epididymal epithelium synthesizes and secretes
a large number of androgen-dependent proteins[6-9]. The synthesis
and secretion of prfoteins in the different regions of the human epididymis
were studied
in vitro[10].
Androgen dependence of specific proteins in the epididymis
of adult rhesus
monkeys was also reported[11]. It has also been shown that there
are
changes in protein secretory patterns during the development of rat epididymis[12]. Conspicuous
changes in the protein profile of secretory proteins in various segments
of the epididymis were noted following castration and subsequent administration
of high doses of testosterone propionate[13].
Since epididymal functions are under androgenic control we sought
to identify changes in the proteins in the caput,
corpus and cauda epididymides in rats following castration.
2
Materials and methods
2.1
Biochemicals
Citric
acid, silver nitrate, gelatin, bovine serum albumin (fraction V), ethylenediaminetetracetic
acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF) were obtained from
Sigma (St. Louis, MO). Tris(hy-droxymethyl)aminome thane (Tris),
sodium chloride, sodium phosphate, hydrochloric acid, isopropanol and
sodium hydroxide were from Aldrich (Milwaukee, WI). N,N,N,N-tetramethylethylenediamine
(TEMED), 2-mercaptoethanol, sodium dodecyl sulphate (SDS) and high and
low molecular weight markers were obtained from Bio Rad Laboratories (Richmond,
CA). Ammonium persulfate and glycerol were purchased from Bethesda Research
Laboratories (Gaithersburg, MD). Methanol, acetonitrile, acetic acid and
ammonium hydroxide were from J.T. Baker, Inc. (Phillipsburg, NJ).
Acryliquid-40 (40% w/v acrylamide solution) was purchased from International
Biotechnologies Inc. (New Haven, CT). Formaldehyde (37% v/v) was
obtained from Fisher Scientific Company (Fair
Lawn, NJ). Uniflo 0.2 m filters were from Schleicher & Schuell
(Keene, NH).
2.2
Animals
Sprague-Dawley
rats between 60 and 70 days of age were used for the preparation
of tissue cytosols. Rats were divided into two groups of fifty rats
each. The animals were anaesthetized with MetophaneTM (methoxyflurane)
from Pittman-Moore Inc. (Mundelein, IL). One group of animals
was orchiectomized by ligating the tip of caput
epididymis along with the blood vessels before removal of the
testes through tunica albuginea. The other group of animals was sham operated.
Three days after surgery the epididymides were removed and dissected into
three compartments: caput, corpus and cauda.
2.3
Preparation of cytosols
The
three regional compartments of epididymides were homogenized
individually using Brinkman Polytron homogenizer (Brinkman Instruments
Co., Westbury, NY) in TG buffer (10 mmol/L Tris, 1 mmol/L PMSF, 1 mmol/L
EDTA, pH 7.4 at
22 containing 10% glycerol, v/v) in an ice bath using an organ to buffer
ratio of 1:10. The homogenates were centrifuged at 42000g
at 4 for one
hour and the supernatants
were used as cytosols. Samples were stored at -20 until used.
2.4
Fractionation of epididymal proteins by anion exchange HPLC
After
dialysing against solvent A (20 mmol/L Tris pH 7.4) the samples
were filtered through a 0.2 m
filter. Approximately 10 mg total protein for each sample was loaded
onto Mono Q HR 5/5550 minimum i.d. (particle size 10 m)
from Pharmacia Amersham
Biotech at a flow rate of 1 mL/min. Proteins were eluted using a
linear salt gradient from 0-80% solvent B (20 mmol/L Tris,
600 mmol/L NaCl; pH 7.4) for 30 min. Fractions of 1 mL each
were collected. The eluants were monitored by UV absorbance at
280 nm. An aliquot of each fraction was withdrawn for analysis by SDS-PAGE.
2.5
Analytical SDS-PAGE
Analytical
PAGE in the presence of SDS was performed using the method
of Laemmli[14]. The resolving gel was made up of 12.5% T
(total acrylamide
concentration and 2.6% cross linker using methylene-bis(acrylamide;
% CBIS) with a stacking gel of 5% T and 15% N,N-diallyltartamide
(%CDATD). Aliquots of the HPLC fractions were denatured
in SDS sample buffer[15] and resolved by
SDS-PAGE. After electrophoresis, the gels were stained with silver nitrate
according to the procedure of Wray et al[16].
2.6
General Methods
Protein
estimation was performed by Coomassie blue dye binding assay of Bradford[17]
as modified by Macart & Gerbaut[18]
using
bovine serum albumin as a standard. 3
Results
3.1
Analysis of epididymal proteins in normal rats by SDS-PAGE after
anion-exchange HPLC
When
about 10 mg total protein from either caput (Figure
1A), corpus (Figure
1C), or cauda (Figure 1E) epididymides
were resolved by anion-exchange HPLC,
multiple protein peaks with similar patterns were noted when the eluents
were monitored by UV absorbance at 280 nm. Aliquots of samples
(15 L) from selected fractions were then withdrawn
and resolved by SDS-PAGE as shown in
Figure 1B, 1D and 1F corresponding to samples
from extracts of caput, corpus and cauda epididymis, respectively.
SDS polyacrylamide gels have shown that the three segments of the
epididymis from rats displayed a complex profile of proteins which displayed similarities
in all the three regions of the epididymis. At least 25 different groups
of proteins can be identified in each region. In corpus and cauda segments
28 and 32 different proteins could be identified, respectively. The concentrations
of various proteins were higher in caput and cauda regions as compared
to those in the corpus region of epididymis. There was an apparent
reduction of proteins in the cauda epididymis such as proteins
1, 2, 4-8 when compared to those observed in the caput epididymis.
The amounts of all the proteins were less in corpus region
as compared to those observed in the caput region of epididymis.
Figure
1 (A-F): Fractionation of
rat epididymal cytosol derived from the caput (1A), corpus
(1C) and cauda (1E) epididymis of a normal rat by anion exchange HPLC
using a preparative Mono Q column as described in Material and Me-thods.
The protein profile was allowed to return to the baseline,
and a gradient of 0-80% solvent
B (20 mmol/L Tris and 600 mmol/L NaCl; pH 7.4 at 22) was applied to
the column over 45 min at a flow rate of 1 mL/min.
Fractions (1 mL each) were collected. The effluent
was monitored by the UV absorbance at 280 nm. Roman numerals
indicate the peak numbers. Inj. indicates where the
sample was loaded; grad. indicates where the gradient was started.
An aliquot (15 L) from each fraction obtained from
the anion exchange HPLC fractionation was resolved by SDS-PAGE, and proteins were
visualized by silver nitrate. Figure 1B, 1D, and 1F represent the profile
of proteins
obtained from caput, corpus and cauda epididymis, respectively. The
S lane has molecular weight markers consisting of 0.3 g each
of myosin (Mr 200 000), -galactosidase (Mr 1 165 000), phosphorylase
b (Mr 97 000), bovine serum albumin (Mr 68 000),
and ovalbumin (Mr 45 000). The numbers across top of the gels
correspond to the fraction numbers shown in Figure 1A, 1C and
1E, respectively. D refers to the dye front.
3.2
Analysis of epididymal proteins in castrated rats by SDS-PAGE after
anion-exchange HPLC
Figure
2A, 2C and 2E are the chromatograms corresponding to the anion-exchange
HPLC fractionation of 10 mg protein obtained from extracts of the caput,
corpus, and cauda epididymides from castrated rats, respectively. Selected aliquots
(15 L) of fractions from these runs were resolved by SDS-PAGE
and the gels were
silver stained and are shown in Figure
2B, 2D, and 2F corresponding to the caput, corpus and cauda
epididymides, respectively. The concentrations of the proteins
numbered 1-7, 17, 20-22 reduced in the caput epididymis of castrated
rats as compared to those of the normal rats indicating that
these proteins are regulated by androgens (Figure
2B). In the corpus epididymis the levels of proteins
numbered 1-9, 24 and 25 reduced significantly in castrated rats as compared
to those of the normal rats (Figure
2D). The concentrations of all the proteins numbered
4-27 were found to be decreased in the cauda epididymis of castrated rats
as compared to those of the normal rats (Figure
2F ).
Figure
2 (A-F): Fractionation of
rat epididymal cytosol derived from the caput (2A), corpus (2C) and cauda
(2E) epididymis of a castrated rat by anion exchange
HPLC
using a preparative Mono Q column as described in Material and Methods.
The protein profile was allowed
to return to the baseline, and a gradient of 0-80% solvent B (20 mmol/L
Tris and 600 mmol/L NaCl; pH 7.4 at 22)
was applied to the column over 45 min at a flow rate of 1 mL/min. Fractions
(1 mL each) were collected. The
effluent was monitored by the UV absorbance at 280 nm. Roman numerals indicate
the peak numbers. Inj. indicates where the sample was loaded; grad. indicates
where the gradient was started. An aliquot (15 L)
from each fraction obtained from the anion exchange HPLC fractionation
was resolved by SDS-PAGE, and proteins were visualized by silver nitrate.
Figure 2B, 2D, and 2F represent the profile of
proteins obtained from caput, corpus and cauda epididymis, respectively.
The S lane has molecular weight
markers consisting of 0.3 g
each of myosin (Mr 200 000), -galactosidase
(Mr 1 165 000), phosphorylase b (Mr 97 000), bovine
serum albumin (Mr 68 000), and ovalbumin (Mr 45 000). The numbers across
top of gels correspond to the fraction numbers shown in Figure 2A, 2C
and 2E, respectively. D refers
to the dye front.
4
Conclusions
Using
one-and two-dimensional gel electrophoresis and silver staining
many polypeptides were identified in the luminal fluid of epididymis
and regional differences of the secreted polypeptides were
noted in different segments of rat epididymis[19,20,13,21].
In most of these studies the regionality of protein synthesis
and secretion have been demonstrated to be androgen dependent. It is likely that
some of the secreted proteins get reabsorbed by the epididymal epithelium
under the influence of androgens. Several proteins have already
been shown to be reabsorbed
by the epididymal epithelium[22,23]. It is, therefore, significant
to study the changes in the protein profiles in the secretions
along with the epididymal tissue. In the present study we prepared
cytosols of caput, corpus and cauda epididymis along with their
secretory products and resolved the proteins so that the first
dimension of charge separation was achieved by anion exchange HPLC
and the second dimension of size separation was achieved by SDS-PAGE.
We observed that the caput region followed
by caudal region of the epididymis has high concentrations
of many proteins. However, the concentrations of proteins less than
45 kDa are higher in the caput region while the higher molecular weight
proteins (45 kDa) are more abundant in the cauda epididymis.
The corpus region of epididymis
has less concentration of all the proteins. The number of proteins identified
increased from caput to cauda epididymis. We also sought to identify
if the profiles
of these proteins in various epididymal regions change following castration.
Most of the proteins were observed in all the three epididymal regions
of normal rats (Figure 1B, 1D,
1E). In the caput region (Figure 1B
and 2B) proteins 1-9
(30 kDa), 17 (60 kDa) and 20-22 (70-80 kDa) were decreased in castrated rats
suggesting their androgen-dependence. In the caput epididymis of rabbit
a large group of low molecular weight proteins (30 kDa) were
shown to be androgen dependent[24]. The pattern
of proteins in the corpus epididymis also showed some
changes following castration (Figure
1D and 2D). The proteins numbered
1-9 (40
kDa), 18 (80 kDa) and 24-25 (90-95 kDa) decreased in the corpus segment
showing that these proteins are regulated by androgens. In the cauda epididymis all the proteins numbered
4-27 decreased in the castrated rats (
Figure 1F, 2F) showing
that most of the tissue proteins in the cauda segment are under androgenic
regulation. The levels of 20-30 kDa proteins reduce in all
the three segments of epididymis.
In murine epididymis the maintenance of synthesis and secretion of 24
kDa protein have been shown to be androgen-dependent[25]. In
all the segments of
epididymis especially in the caput and cauda portions the 45-55 kDa proteins were
also reduced following castration. In monkeys several epididymal proteins
(15-40 kDa) have been shown to be reduced in castrated animals[11].
In summary, we have identified several proteins in caput, corpus
and cauda segments of rat epididymis which are reduced following
castration indicating their androgen dependence. Identification
of some of the proteins will help evaluating them as marker
proteins for studying testicular regulation of epididymis.
Acknowledgements
PPM
was a recipient of the Biotechnology Career Award of the Rockefeller Foundation,
New York, U.S.A.
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Correspondence
to: Dr. P P Mathur, Professor, now in School of Life Sciences,
Pondicherry University, Pondicherry-605 014, India.
Tel: +91-413-655212 Fax: +91-413-655211
e-mail:
ppmathur@yahoo.com
Received
1999-12-21 Accepted 2000-02-20
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