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Effect of testosterone on epididymal proteins in castrated rhesus monkeys.

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American Journal of Primatology 4:73-80 (1983)
Effect of Testosterone on Epididymal Proteins in
Castrated Rhesus Monkeys
M Z HAIDER, M.H. QAZI. A KHANUM, AND M ARSLAN
Department of B~olugrccrlScwnces, Quazd-~-AzamWnweixlty, Islumcrbad, Pakistan
The effect of testosterone on regulation of epididymal protein synthesis has
been investigated in castrated rhesus monkeys (Mucaca mulatta). The proteins in the treated monkeys were characterized using polyacrylamide gel
electrophoresis (under nondenaturing and denaturing conditions) and electrofocusing. At least four distinct proteins have been shown to be synthesized by the monkey epididymis under testosterone influence. Two of these
proteins were detected following two days of testosterone treatment while
the other two proteins were detected after a six-day treatment period. None
of these proteins was detectable in monkeys treated with estradiol for six
days. Electrofocusing of epididymal cytosol proteins from untreated and
testosterone-treated and castrated monkeys also confirmed the presence of
four androgen-dependent proteins in this species. The isoelectric points of
these proteins were shown to range between 5.8 and 6.4. The molecular
weights of these proteins were found to vary between 47,500 and 66,000.
The in vitro incorporation of 3H-labeled amino acids was markedly greater
in the androgen-primed epididymis as compared with the control tissue.
Key words: inacaca mulatta, rhesus monkey, epididymis, protein, androgen
INTRODUCTION
Mammalian spermatozoa are known to undergo maturational changes as they pass
through the epididymis [Hamilton, 19751. A number of studies indicate that epididymal secretions which induce maturational changes in spermatozoa are largely
androgen-dependent (Hamilton, 1972; Orgebin-Grist & Davies, 1974; Prasad & Rajalakshmi, 1976; Cameo & Blaquier, 1976; Brooks, 1979; Echeverria et al, 1982).
Available evidence also indicates that the epididymis synthesizes and secretes specific androgen-dependent proteins, some of which coat the sperm surface (Barker &
Amann, 1970; Amann et al, 1973; Huang & Johnson, 1975; Koskimies & Kormano,
1975; Garberi et al, 1979; Brooks & Higgins, 1980). These have been variously
characterized as glycoproteins (Bedford, 1963), acidic proteins (Lea et al, 1978), and
“forward motility proteins” (Brandt et al, 1978). Since most of these studies have
Received June 26, 1982; revision accepted October 18, 1982
Address reprint requests to Dr. M. Arslan, Department of Biological Sciences, Quaid-i-Azam University,
Islamabad, Pakistan.
0275-2565/83/0401-0073$02.500 1983 Alan R. Liss, Inc.
74
Haider e l a1
been carried out in rats or bulls, only limited information is available about specific
epididymal, androgen-dependent proteins in primates [Hinton & Setchell, 1981;
Jones, 1978; Prasad & Rajalakshmi, 1976; Tezon & Blaquier, 1981). Studies were,
therefore, undertaken to characterize some of the androgen-dependent, epididymal
proteins in the rhesus monkey. It is hoped that the information obtained will
enhance our knowledge about the physiology of the epididymis in primates.
METHODS
Animals
The rhesus monkeys (Macaca mulatta) used in the present study were 30-36
months old and weighed 3.0 to 3.5 kg. These animals were castrated 1 year earlier,
but the epididymides were left intact. The epididymis of one side was removed on
day 1 of the experiment to serve as the initial control. Testosterone or estradiol
treatment was initiated the same day. One monkey received only a single injection
of testosterone propionate (10 mg), and the surviving epididymis was removed on
day 3 (48 hr later). Three animals were given injections of 10 mg of testosterone
propionate daily for a period of six days (days 1-6) and the epididymides were
removed on day 7. One animal received 10 mg of estradiol benzoate daily for a period
of six days. On removal, the epididymides were placed in a chilled petri dish, freed
of adhering tissue, and immersed in ice-cold 50 mM-Tris Buffer (pH 7.4) containing
1.5 mM-EDTA.
Preparation of Cytosol
After weighing, the tissues were minced with fine scissors and homogenized in
three volumes of Tris-EDTA buffer in a glass homogenizer. The homogenate was
subsequently centrifuged at 45,OOOg for 90 min to obtain the cytosol fraction. Aliquots were removed for the determination of protein content according to the method
of Lowry et a1 [1951] using bovine serum albumin as the standard. Remaining
supernatant was stored at -20°C until used for analysis.
Electrophoresis
Nondenaturing gels. Polyacrylamide gel electrophoresis (PAGE) was performed
essentially by the method of Raymond & Weintraub (1959).
Denaturing gels. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) was performed as described by Weber & Osborn (1969).The same conditions
were used for molecular weight determination, and the control and treated samples
were run in parallel with standard protein markers (Pharmacia Fine Chemicals,
Uppsala, Sweden).
Electrofocusing
Analytical electrofocusing in polyacrylamide gel was performed using Ampholine
PAG-plates (pH range 3.5-9.5, LKB-Produkter, Bromma, Sweden). Aliquots containing 30-40 pg protein were applied to the gel using 0.5 x 1.0 cm filter paper chips.
One M o-phosphoric acid (85%) was used as anode solution, while the cathode
solution was 1 M sodium hydroxide. Electrofocusing was carried out a t 200 V for 4
hr followed by 400 V for 2 hr. The gel plates were fixed in 500 ml of a solution
containing 57.5 g trichloroacetic acid and 17.25 g sulphosalicylic acid and stained
with Coomassie brilliant blue for 10 min at 60°C. Destaining was carried out
through several changes of the destaining solution. The destained gel plate was
immersed overnight in the preserving solution (40 ml of glycerol in 400 ml of
destaining solution). The gel plate was dried using a hot air blower and preserved i n
plastic sheets. The gel plates were scanned by using an E-C model densitometer
Testosterone on Epididymal Proteins
75
equipped with a Kipp-Zonen integrating recorder. The pH gradient along the gel
was determined by measuring pH in each 1 mm segment of the gel. This was used
to obtain the isoelectric point (PI)of each specific protein band.
In Vitro Synthesis of Proteins
Minced epididymal tissue from castrated control animals and those receiving
androgen treatment for six days were incubated in 10 ml of freshly prepared Eagle’s
medium with 200 pCi of 3H-labeled amino acid mixture (1mCiim1, Radiochemical
Centre, Amersham, England) in HEPES buffer in a Dubnoff metabolic shaker at
33°C for 180 min. After completion of the incubation, the tissues were immediately
transferred to ice and were washed with three successive changes of fresh Eagle’s
medium to remove free radioactivity. The tissue was then homogenized in 5 ml of
sucrose medium (0.7 M-sucrose containing 6 mM MgC12, 0.5 M-Tris-HC1buffer, pH
7.5,50 mM KC1, and 0.2% Triton X-100).The homogenate was centrifuged at 15,OOOg
to obtain the post-nuclear supernatant. An equal volume of 10% ice-cold trichloroacetic acid (TCAI was added slowly with continuous shaking to obtain a precipitate
which was collected by centrifugation at 15000 g. The precipitate was washed thrice
with cold 5% TCA. Finally, the washed TCA-precipitated protein pellet was dissolved in 0.8 ml NCS tissue solubilizer (AmershadSearle, Arlington Heights, IL)
and used for counting in a scintillation cocktail consisting of 0.5% Permablend
(Packard Instrument Go., Downers Grove, IL) in toluene. Radioactivity
was measured with a Packard PL Tri-Carb model liquid scintillation counter. Specific activity was calculated and expressed as c p d m g protein.
RESULTS
Data relating to the weight of the epididymis and the total protein content of the
soluble fraction before and after different treatments are given in Table I. The
epididymal weight obtained a t the end of testosterone treatment was approximately
twice that of the weight of the contralateral epididymis removed immediately before
initiation of treatment. There was also an increase in total protein concentration
per g epididymal tissue in testosterone-treated castrate monkeys. Estradiol treatment for a period of six days failed to stimulate the weight of the epididymis,
although a slight increase in total protein content per g tissue was observed.
The PAGE of epididymal cytosol proteins from control and treated animals is shown
in Figure 1. Using the nomenclature of Cameo & Blaquier (1976), the major band
having the same mobility as that of serum albumin has been designated as Band A.
Under nondenaturing conditions, the electrophoretic profiles showed a t least two
distinct prealbumin bands (B and D) in the epididymis primed with testosterone for
two days. In addition to these, two new bands (C and E) in the prealbumin region
were detected in the epididymides of animals treated with testosterone for six days. The Rf values of these bands were: B, 0.58; C, 0.62; D, 0.67; and
E, 0.74. None of these four prealbumin bands were observed following administration of estradiol for six days t o castrated monkeys.
Analysis of cytosol proteins from untreated and androgen- stimulated epididymides
by SDS-PAGE revealed a pattern which was essentially similar to that obtained
under the nondenaturing conditions. In this system, relative mobilities of the bands
B-E were B: 0.23; C, 0.26; D, 0.32; and E, 0.35 (Fig. 21, and the molecular weights,
determined by the procedure already described, were 66,000, 63,000, 52,500, and
47,500, respectively.
Further evidence for the presence of androgen-dependent epididymal proteins was
obtained by electrofocusing the cytosol € o m untreated and hormone-treated castrated monkeys (Fig. 3). With testosterone treatment for six days, four protein bands
76
Haider et a1
TABLE 1. Epididymal Weight and Total Protein Concentration (Soluble Fraction) in Castrated
Monkeys Treated With Testosterone and Estradiol (10 rnglday)
Epididymal protein
Epididymal
weight
concentration
Duration of
treatment
(mg)
(mgig tissue)
Animal
no.
Treatment
(days)
Pretreatment Post-treatment Pretreatment Post-treatment
812
813
816
817
Testosterone
Testosterone
Testosterone
Estradiol
290
310
210
275
2
6
6
6
4.60
2.77
3.05
2.01
2.02
1.45
1.71
1.55
456
511
445
186
- A
- B
- c
- D
- E
BPB
a
3.
b
b
C
C
Fig. 1. (a) Electrophoresis under nondenaturing conditions, of epididymal cytosol fraction from untreated
castrated rhesus monkey. CD) Castrated nionkey treated with testosterone for 2 days. (c)Castrated monkey
treated with testosterone for 6 days. The positions of albumin (A) aud the prealbumin bands (B-E) are
indicated.
in the treated animals with PI values ranging between 5.8-6.4 were detected. None
of these proteins was focused in material obtained from untreated castrates or those
administered with estradiol.
When epididymal tissue from untreated and treated castrated monkeys was incubated with a n 3H-amino acid mixture, the incorporation of the radiolabel in the
cytosol proteins was markedly greater in the androgen-primed epididymis. The
extent of incorporation of the radiolabel expressed as c p d m g was 1.5 x lo5 in the
control epididymis and 34 x lo5 in the androgen-primed epididymis.
Testosterone on Epididymal Proteins
--A
-
-C
- c
-B
-D
-E
-
A
B
-
77
68,000
- D
- E -
- BPB
a
-
11,500
b
Fig. 2. (a) Electrophoresis under denaturing conditions, of epididymal cytosol fraction from untreated
castrated rhesus monkey, ib) Castrated monkey treated with testosterone for 6 days. Molecular weight ( X
as a function of the migation distance is indicated by the scale at the side of the gels.
DISCIJSSION
Several previous studies carried out in castrated rats have demonstrated maximal
increase in protein content of the epididymis 24 hr after the administration of a
single dose of testosterone (Rajalakshmi & Prasad, 1976). Our data also demonstrate
a similar rise in protein content of the epididymis in castrated monkeys 48 hr after
the injection of testosterone. It has been observed that these changes are specifically
related to testosterone, since estradiol treatment for six days neither increased the
gross weight of the epididymis nor increased the protein content of the tissue to the
same extent as in the testosterone-primed epididymis. Further evidence that the
synthesis of specific proteins in the epididymis is regulated by testosterone is provided by the electrophoretic and electrofocusing studies. It has been observed that
four protein species in the prealbumin area which are absent in castrate epididymis,
are invariably associated with the epididymis of treated castrated animals. Previous
investigations which characterize androgen-regulated epididymal proteins in the rat
have also demonstrated the presence of four major bands in the prealbumin area
(Cameo & Blaquier, 1976). Brooks & Higgins (1980) have studied the androgen
dependence of proteins associated with luminal fluid in the rat epididymis. These
authors were also able to demonstrate four major prealbumin bands (B-E) in the
epididymal fluid obtained from the cauda. These proteins were shown to be of
epididymal origin and regulated by androgens. Previous evidence also suggests that
bands B and C present in the epididymal cytosol of normal rats, may have testicular
Haider et a1
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Fig. 3. (a) Densitometer scan of the epididymal cytosol proteins after electrofncusing from untreated
castrated monkey. cb) Castrated monkey treated with testosterone for 2 days. (c)Castrated monkey treated
with testosterone for 6 days. id) Castrated monkey treated with estradiol for 6 days. - ) absorbance;
(* - -- m i pH-qadient.
as well as epididymal origin (Cameo & Blaquier, 1976; Brooks & Higgins, 1980).
However, in rhesus monkeys, since the epididymal tissue was analyzed one-two
years after castration, the two bands seem to be clearly associated with the epididymis, although a regional synthesis of the different androgen-dependent epididyma1 proteins has yet to be demonstrated. Investigations using radioactive methionine
in rats have shown that two of these proteins (bands B and C) are synthesized in the
initial segment and the caput, and one protein (band D) is produced in the caput and
the cauda (Jones et al, 1980).
The pattern of prealbumin proteins in the epididymal cytosol from untreated and
testosterone-treated castrated monkeys on denaturing gels (SDS-PAGE)was essentially similar to that seen on the nondenatwing gels. This indicates that these
proteins lack disulphide linkages and most probably do not have a subunit structure.
Testosterone on Epididymal Proteins
79
Brooks & Higgins (1980) have also reported a lack of interchain disulphide linkages
in androgen-dependent epididymal proteins in the rat. The approximate molecular
weights of the androgen-dependent epididymal proteins characterized in the present
study varied between 47,500 and 66,000. These values are considerably higher than
those reported for corresponding epididymal proteins in the rat (Cameo & Blaquier,
1976; Brooks & Higgins, 1980; Jones et al, 1980). Jones et a1 (1980) have described
three proteins from the caput with molecular weights ranging from 18,500-32,000
and one protein of caudal origin with a molecular weight of 47,000.
As judged by electrofocusing, the androgen-dependent proteins reported in this
study lie in the acidic region with the PI ranging from 5.8-6.4. The PI’Sfor androgenspecific proteins in the rat epididymis have previously been reported and are shown
to vary between 4.2 and 5.9 (Jones et al, 1980).
The in vitro incorporation of radiolabeled amino acids in the minced epididymal
tissue from androgen-primed castrated monkeys was shown t o be several times
greater than in the epididymal tissue of untreated castrated animals. Jones et a1
(1980) have also demonstrated a 2-%fold increase in total incorporation of 35Smethionine in epididymal tissue f?om castrated rats following testosterone treatment. In contrast, Brooks & Higgins (1980) have noted little quantitative difyerence
between tissues from normal or castrated rats, although increased labeling of a
caput protein (molecular weight 16,000) could be demonstrated under the effect of
androgen. Cameo & Blaquier (1976) have also shown that one major protein @and
C, molecular weight 23,100) is synthesized in vitro by the rat epididymal tissue.
CONCLUSIONS
1. This study shows that the epididymis of the rhesus monkey, like that of the rat,
synthesizes specific proteins in response to androgens.
2. As judged by their electrophoretic mobility, molecular weights, and PI values,
these proteins are apparently not identical with those described in the rat.
3. The four major androgen dependent bands revealed in the castrate monkey
epididymis with testosterone replacement therapy may not represent the total
number of epididymal proteins whose synthesis is regulated by androgens, since
effective production of certain proteins by the epididymis may require a high
level of intraluminal testosterone.
ACKNOWLEDGMENTS
This work was supported in part by the Small Supplies Programme of Special
Programme of Research in Human Reproduction, World Health Organization. The
assistance of Ms. T. Rana and Ms. S. Amin is gratefully acknowledged.
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