journal of Andrology, Vol. 17, No.

1, January/February Copyright u American Society of Andrology

1996

Distribution and Tissue Expression of Semenogelin I and II in Man as Demonstrated In Situ Hybridization and Immunocytochemistry
ANDERS BJARTELL,* JOHAN MALM,t AKE LUNDWALL,f AND HANS LILJAt From the Departments of *Urology Department of Urology, University jClinical Hospital, CHRISTINA MOLLER, MATS

by

GUNNARSSON,*

Chemistry, and Pathology, Lund, University of Lund,

University Sweden.

Hospital,

Malm#{246};and

the

ABSTRACT: Semenogelin I and II (Sgl, Sgll) are two separate gene products of chromosome 20 with extensive (-80%) identity in primary structure. They are mainly responsible for Immediate gel formation of freshly ejaculated semen. Degradation of Sgl and Sgll is due to the proteolytic action of prostate-specific antigen (PSA); it results within 5-15 minutes in liquefaction of semen and release of progressively motile spermatozoa. By means of cDNA cloning and Northern blots, Sgl and SglI transcripts have previously been shown to be abundant in human seminal vesicles, but Sgll alone is suggested to be expressed at low levels in the epididymis. To characterize the expression and tissue distribution of Sgl and Sgll in greater detail, we produced monoclonal Immunoglobulin Gs (lgGs) for immunocytochemistry (ICC) and specific [MS]-, digoxigenin-, or alkaline phosphatase-labeled 30-mer antisense probes to Sgl and Sgll for in situ hybridization (ISH). Immunocytochemical staining for both Sgl and Sgll, and ISH detection of both Sgl and Sgll transcripts, were demonstrated in the cytoplasm of seminal vesicle epithelium. ISH showed Sgll alone to be expressed in the epithelium of the epididymal

cauda. Neither ICC nor ISH yielded any evidence of Sgl or Sgll expression In caput or corpus epithelium or In any stromal cells of the epididymis. Consistent with our previous findings using polyclonal lgG, monoclonal anti-Sgl/SgIl lgGs identified epitopes on the posterior head, midpiece, and tall of ejaculated spermatozoa. Spermatozoa in the epididymal cauda were also immunoreactive, but those in the caput or corpus region of the epididymis as well as those in the testis were negative. As shown by ICC, neither Sgl nor Sgll were expressed In the testis, the prostate, the female genital tract, or other normal human tissue specimens. Although the significance of Sg attachment to epididymal and ejaculated spermatozoa remains to be established, monoclonal anti-Sg lgG might prove useful in establishing the origin of seminal vesicle tissue components in prostate core biopsies or other biopsy specimens. Key words: Semen, spermatozoa, fertilization, Southern blot, oIlgodeoxynucleotide probes. J Androl 1 996;1 7:17-26

he ejaculatory mixing of secretions from the seminal vesicles and the prostate results in immediate formation of a gel in which semenogelin I and II (SgI and SgII) constitute the major gel proteins (Lilja and Laurell, 1984, 1985). Progressively motile spermatozoa are released when the gel dissolves (i.e., the ejaculate liquefies) as a result of proteolytic degradation of the gel proteins (Lilja and Laurell, 1984; McGee and Herr, 1987). This degradation is due to the action of prostate-specific antigen (PSA) (Lilja, 1985; Lilja et al, 1987; McGee and Herr, 1987; Lee et al, 1989), a most abundant prostate-

Supported by the Swedish Medical Research Council (grant project nos. 1 3X-7903 and 8660), the Faculty of Medicine at Lund University, the Research Fund and the Cancer Research Fund at Malm#{246} General Hospital, the Swedish Society of Medicine (SLS), the Foundation for Urology Research in Malm#{246}, the Magnus Bergwall Foundation, the Foundation of Crafoord, and the Fundacion Frederico S.A. Correspondence to: Dr. Anders Bjartell, Department of Urology, University tember Hospital, 19, 1995. Malm#{246}S-205 July 02 Malm#{246}, Sweden. 5, 1995;

Received

for publication

accepted

for publication

Sep-

derived serine protease in the seminal fluid, with extensive structural similarity to the glandular kallikreins but with an enzyme action similar to that of chymotrypsin (Watt et al, 1986; Akiyama et al, 1987; Lundwall and Lilja, 1987; Schaller et a!, 1987; Lilja et a!, 1989; Christensson et al, 1990). The -52-kDa SgI and -70-kDa SgII are predominant secretory proteins from the seminal vesicles (Lilja et al, 1984, Lilja and Laurel, 1985). These proteins have many properties in common with the seminal vesicle-specific antigen (SVSA) described by Herr et a! (1986). Therefore it is likely that the monoclonal antibody MHS-5 used to identify SVSA may detect SgI and SgII. SgI is a singlechain, non-glycosylated protein of 439 amino acids (Lilja et a!, 1989). The structure of SgII is very similar (-80% identity in primary structure), but it contains 559 amino acids (Lilja and Lundwall, 1992). SgI is suggested to be exclusively expressed in the seminal vesicles (Lilja et a!, 1989). The seminal vesicles also constitute the major secretory origin of SgII, although SgII mRNA is also detected at low levels in the epididymis (Lilja and Lundwall,
17

18
1992). Epididymal expression of immunoreactivity epithelium of SgII is further to SgI/SgII supported localized in addition of the par-

Journal of Andrology Tissue Specimens

For ISH and immunocytochemistry

January/Februaiy 1996

by the detection to the secretory

of the epididymis,

to the SgI/SgII-immunostained cells in the epithelium the seminal vesicles (Lilja Ct al, 1989). Moreover, tissue-specific expression of these proteins may be

imens glands

were obtained were removed

at surgery.

(ICC) studies, Seminal vesicles

from urinary bladder. the epididymis

tissue

spec-

and

prostate

at cystoprostatectomy

10 patients Specimens were taken cancer in 10 from two regional disof were im-

(age 60-74 years) with cancer of the from the testis, deferent ducts, and at orchidectomy patients (age patients were

ticularly important, because specific immunostaining for both SgI/SgII and SVSA has been localized to the posterior part of the head, midpiece, and tail of ejaculated

as surgical treatment of prostatic 72-81). In addition, the epididymides divided longitudinally to study the

spermatozoa

(Herr et al, 1986; Lilja et al, 1989). Previous studies have been limited to the use of polyclonal rabbit immunoglobulin G (IgG) against a 52-amino-acid fragment of SgI (Lilja et al, 1989). This antibody cross-reacts
with SgII due to the extensive structural similarity be-

tribution of SgI/SgII the Helsinki Declaration

epitopes in this organ. The provisions regarding the use of human tissues

strictly
Within

observed.
10 minutes in Bouin

after

collection,

all

specimens

were

mersion-fixed

s fixative

or in 4% formaldehyde

in phos-

tween
acterize detail,

SgI and

SgII (Lilja and the tissue expression particularly in the male

Lundwall, 1992). To charof SgI and SgII in greater genital tract, we generated

phate-buffered affin. Sections

chrome-alum-coated or Vectabond-coated

saline were

(PBS), dehydrated, and embedded in parcut at 3-tim thickness and mounted on (subbed) slides for ICC (Huang et al, 1983),
or 3-aminopropyltriethoxysilane (APES)-

and characterized Sgl and SgII for

produced to either antisense

monoclonal

antibodies

against
studies, probes

purified
and we specific

immunocytochemical oligodeoxynucleotide transcripts for use

hybridization

SgI or SgII (ISH)

in detailed

in situ

coated slides (Rentrop fixed paraffin-embedded partment of Pathology matoxylin-eosin-stained

histopathologically

et al, 1986) for ISH. Archival formalinspecimens were obtained from the Deat the University Hospital, Malm#{246}. Hesections from all tissue specimens were
from in 0.05 four volunteer donors phosphate,
seminal

studies.

Fresh ejaculated

normal. spermatozoa twice

with pH plasma

Materials and Methods

Reagents
The SgI fragment as earlier described
Biotinylated goat

normal spermatograms utes and then washed 7.4, and 0.15 M NaC1
components. The

were centrifuged

at 800
M sodium

x g for 10 min-

to remove loosely bound spermatozoa were recovered

by

centrifugation

at 800 encompassing residues 85-136 was purified (Lija and Jeppsson, 1985; Lilja et al, 1989).
anti-rabbit

x g for 10 minutes after each washing cycle, smeared on untreated slides, and stored at - 70#{176}C. ICC, the spermatozoa For were dried overnight at room temperature, fixed in acetone for

IgG

and

biotinylated

horse

anti-

10 minutes,

and air-dried.

mouse

IgG, normal

(NSS) were obtained trup, Denmark). Diaminobenzidine (DAB) and Fast Red, as well as Vectabond coating solution for microscopic slides, were supplied by Vector Laboratories (Burlingame, California). Acetanhydride and triethanolamine were purchased from BDH Ltd. (Poole, UK), formamide and Triton X- 100 from Merck (Darmstadt, Germany), Mount-Quick Aqueous from Daido Sangyo Company, Ltd. (Japan), Pertex from Histolabs (Gothenburg, Sweden), Ilford K-S autoradiography emulsion from Ilford Scientific X-AR5 York), many). Products film from (Cheshire, Eastman UK), Kodak Dektol Company D- 19 developer (Rochester, and New

din-horseradish sheep serum

mouse peroxidase

and normal (SA-HRP)

rabbit IgGl, streptaviconjugate, and normal from Dakopatts A/S (Glos-

Antibodies
Using previously described methods, monoclonal antibodies were

raised in Balb/c mice against SgI purified from human semen collected in alkaline buffer containing urea to prevent degradation et al, of the 1988). semenogelins The purified (Borrebaeck and Eylar, 1981; Laurell

judged
phoresis

from

sodium

SgI was dodecyl sulfate

the only

essentially homogenous as polyacrylamide gel electrocontaminant being minor of with approximately the same amount

(SDS/PAGE),

amounts of SgII. The mice were immunized 15 ig purified SgI and given a booster with

and Age-Fix from Nylon hybridization (no. 4020) SJ 1304) were from

Agfa-Gaevert AG (Leverkusen, Gerfilters (Hybond N), radionucleotides

antigen
with

1 and NS 1 myeloma

after

3 weeks. Before fusion of spleen cells (l0) cells, the mice were given a booster of-V 100

[a-35S)dATP

and [y-32P]ATP,
Amersham

and a 5-end

labeling
(Buckingtailing phosby Boeh-

kit (no.

International

hamshire, UK). The digoxigenin (DIG) oligonucleotide kit (no. 1417 231) and Ft,, fragments of anti-DIG alkaline
phatase (AP) conjugate and proteinase K were supplied

g antigen i.p. on three consecutive days. A solid-phase radioimmunoassay using microtitration wells coated with a mixture of SgI and SgII as antigen (1 big/well) was used to identify positive clones, as previously described (Laurell et al, 1988). Positive
clones and were injected subcloned i.p. into three times by limited Balb/c dilution, mice. expanded, The antipristine-primed

ringer Mannheim GmBH (Mannheim, Germany). Chromospin10 gel-exclusion columns were from Clontech Laboratories Inc. (Palo Alto, California), and T4 polynucleotide kinase was from Pharmacia (Uppsala, Sweden). All other reagents were of reagent grade and purchased from Sigma Chemical Company (St. Louis,
Missouri).

bodies were isolated pharose Fast Flow,

the antibodies were wich enzyme-linked 1985).

by ion-exchange using standard

determined immunosorbent

chromatography on Q-Seprocedures. The isotypes of

with a double-antibody assay (ELISA) (Sugo sandet a!,

Bartell et al

Tissue Expression of Semenogelin I and!!

(ICC)
and polyclonal rabbit IgGs against SgI/SgII

19

Immunocytochemistry
Murine monoclonal

Southern
conjugate and DAB (Elias et a!,

Blot

were 1989).
ferent

detected
IgGs,

using

a SA-HRP

The specificity by Southern

tions

To compare immunostaining patterns obtained by difand to compare ICC with ISH, 3-tim adjacent secwere mounted on separate slides. SgI/SgII epitopes on the

of each of the SgI and SgII probes was verified blot experiments, demonstrating that each probe recognized only the respective cDNA clone under stringent conditions and produced minimal cross-hybridization signals to the
other cDNA clone. The EcoR 1 inserts coding for the cDNA

ejaculated

spermatozoa

were detected

by monoclonal

antibodies

against SgI/SgII using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique with Fast Red as the chromophore (Cordell et a!, 1984). The monoclonal SgI/SgII IgGs were used at a final concentration of -25 ,zg/ml in ICC, and the previously described affinitypurified polyclonal rabbit IgGs against the 52-amino-acid fragment of SgI (Lilja and Laurel, 1985; Lilja et a1, 1989) were used at a final concentration of 12 g/ml. Purified normal mouse and rabbit IgG 1 were used as replacement for the monoclonal and polyclonal Sg antibodies, respectively, and they served as negative controls in ICC. In another procedure to verify the specificity of the immunostaining reactions, the fragment of SgI encompassing residues 85-136 that was added to the affinity-purified polyclonal antibodies in serial dilutions up to 60 times molar excess (-300 ig/ml) was used.

of SgI (XSGLN-IV) and of SgII (XVSRP-XXI) (Lilja et a!, Lilja and Lundwall, 1992) were electrophoresed in 1% agarose gels, vacuum blotted to Hybond N nylon filters, and hybridized to the radioactive probes. The filters were rinsed and washed in 2x standard saline citrate (SSC) and 0.1% SDS for 30 minutes at 45#{176}C 15 minutes and at 54#{176}C exposed and to clones 1989;
Kodak XAR-5 film for 30 minutes.

In situ Hybridization
AP-conjugated
was based

(ISH)

Oligodeoxynucleotide

Probes

Antisense oligodeoxynucleotides (30-mers) were synthesized on an Applied Biosystems 38 1A DNA synthesizer. The probes were chosen to provide an optimal number of mismatches between SgI and SgII: 5-C CAA TCC CCC ATG AGA TCC ATG TFG GTG TC-3 complementary to nucleotides 1280-1309 of the human SgI transcript (Lilja et al, 1989) and 5-T ATG TGA CTC ACT GGA TTC CTG TTT GTA TC-3 complementary to nucleotides 1628-1657 of the human SgII transcript (Lilja and Lundwall, 1992). Both probes were searched for similarities with human mammalian nucleotide sequences in GenBank#{174} (release 71.0) using the FASTA#{174}program. In addition, an oh-

godeoxythymidine
and used
by PAGE as a positive before use

(oligo-dT)
control (Sambrook

probe
in ISH.

(30-mer)

was synthesized
were purified

The probes et a!, 1989).

Probe Labeling
Oligodeoxynucleotides
labeling kit for tailing

were
reaction

labeled
with

with

DIG

using

a 3-end

DIG-il

-dUTP

and dTTP,

catalyzed by terminal deoxytransferase (TdT) according to the manufacturers protocol. Oligodeoxynucleotides complementary to SgI and SgII transcripts, outlined as above, containing 5-terminal amino groups, were also conjugated to alkaline phosphatase (AP) and supplied ready for use by R&D Systems Europe Ltd. (Abingdon, Oxon, UK). Radioactive ISH probes were labeled with [a-35S]dATP to a specific activity of> 1.5 x l0 counts per minute (cpm)4ig probe, using a labeling kit including TdT as the catalyzing enzyme. For Southern blots, oligodeoxynucleotides were 5-end labeled with [y-32P]ATP and T4 polynucleotide kinase to a specific activity of >1 08 cpm4tg probe.

protocol for AP-conjugated probes on methods described by Kiyama et a! (1991) and recently published in detail (Bjartell et a!, 1993). DIG-labeled Probes-The use of DIG-labeled probes in nonradioactive ISH is based on methods developed by Baldino and Lewis (1989). We used a protocol similar to that used for the AP-conjugated probes (see above) but somewhat modified, mainly in the detection procedure. After prehybridization for 1 hour, probes (20-200 ng/ml) were diluted in hybridization buffer (4 x SSC, 10% dextran sulfate, 1 x Denhardts solution [0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin], 400 ig/ml freshly denatured sonicated salmon sperm DNA, and 30% deionized formamide), and hybridized at 37#{176}C overnight (14-18 hours). After hybridization, the tissue sections were washed at a stringency of approximately 10#{176}C below the melting temperature, Tm (Fitzpatrick-McElligott et a!, 1988), equilibrated at room temperature with 1 x SSC for 10 minutes, buffer 1(0.1 M Tris-HC1, pH 7.5, containing 0.14 M NaC1) for 30 minutes, and pretreated with 0.3% Triton X- 100 and 2% normal sheep serum (NSS) in Buffer I for 10 minutes to reduce nonspecific binding of the anti-DIG AP conjugate. F,b fragments of anti-DIG AP conjugate were diluted 1:500 in Buffer I, 0.03% Triton X-100, and 1% NSS and added to the tissue sections for 3 hours. Slides were rinsed and equilibrated in 0.1 M Tris-HCI, pH 9.5, 0.1 M NaCl, and 0.05 M MgCl2 for 2 x 5 minutes. Further, the slides were incubated with the substrate mixture (5-bromo-4-chloro3-indolylphosphate/nitroblue tetrazolium, [BCIP/NBT], containing 0.001 M levamisol) (Leary eta!, 1983). The color reaction was stopped by addition of 10 mM ethylenediaminetetraacetic acid (EDTA), and coverslips were mounted in Mount-Quick
Probes-The Aqueous@. Radioactive

S Probes-Radiolabeled S oligodeoxynucleotide probes were hybridized to tissue sections using a protocol based on the methods developed by Young et a! (1986). Tissue sections were deparaffinized and digested with proteinase K (20 ig/ml) at 37#{176}C 25 minutes. for After 30 seconds in 0.2 M glycine
(in 0.02 M Tris-HC1, pH 7.5), the sections were dehydrated and

dried. Each slide was covered with 1 07 cpm probe per ml hybridization buffer (50% formamide, 4 x SSC, 10% dextran sulfate, 1 x Denhardts, 1% sarcosyl, 2.4 mg/ml Na2HPO4, 400 ig/ ml salmon sperm DNA, and 0.2 M dithiothreitol [DTT]) and hybridized at 37#{176}C 14-18 hours. After the washing procedure, for

20

Journal of Andrology

Januar//February

1996

MrX i94-. 67-

Mabi

Mab 25

3...

43-. 30-

201412 3 1 2 3

FIG. 1. Western blots of Sgl and Sgll using monoclonal antibodies. Partially liquefied human seminal plasma (corresponding to approximately 0.05 iI of seminal plasma, lane 1), intact SgIl (0.6 , lane 2), and intact Sgl (0.6 g, lane 3) were reduced and run on SOS/PAGE. After transfer to a nylon membrane, the semenogelins were visualized with monoclonal anti-Sgl/SgIl lgG (Mab 1 left; Mab 25, rIght). The positions of molecular mass markers

are indicated (far left).

the sections

air-dried, dipped once in 42#{176}C emulsion, diluted 1:1 in DEPC-water, dried for 4 hours, and exposed for 5-14 days in light-sealed boxes at 20#{176}C. Sections were developed for 2 minutes in D- 19 solution, fixed for 3 minutes in AgeFix, rinsed in water, counterstained for 45 seconds in Meyers hematoxylin, dehydrated, and mounted in Pertex. Al! sections were analyzed using an Olympus-CX microscope with brightfield and darkfield illumination.

were

dehydrated,

Ilford K-S autoradiography

-

Results
Characterization Oligonucleotide
During several ing, tion the positive nine different of antibodies.

of Monoclonal Probes
clones were

Sg Antibodies

and
to SgI, subclon-

production

of monoclonal identified,

antibodies and after

hybridomas were used for the producAll monoclonal antibodies (i.e., Mabs

Procedures

to Verify the Specificity

of ISH Reactions
hybridiza-

A DIG-labeled ohigo-dT tion control in all tissues.

probe served as the positive

1, 5, 8, 9, 17A, 22, and 25) were of isotype IgGlK. All clones reacted with both intact SgI and intact SgH. Western blots showed both Mab 1 and Mab 25 to react with intact ments bodies SgI as well as with intact of either (Fig. 1). Some stained SgI more intensely blots, but there were with different fragments the sensitivity SgII, but also with of the monoclonal than SgII and vice fragantiversa

In tissue specimens containing prostate glands, an AP-conjugated or a 35S-labeled antisense probe complementary to nucleotides 528-557 of the PSA transcript (Bjartell et a!, 1993) also served as a positive hybridization control. Four different procedures served as negative controls: 1) hybridization in the absense of any probe; 2) competition with increasing concentrations of an unlabeled probe; 3) hybridization with an unrelated antisense probe, complementary to the PSA transcript (not used in prostate tissues); and 4) RNAse pretreatment of the tissue sections (0.! mg/ml in 2 x SSC, 10 mg MgCl2 for 1 hour at 37#{176}C) before hybridization with either the antisense probe for SgI or that complementary to the SgII transcript.

in western reactivity To a dot blot monoclonal

also differences in the of SgI and SgII. different antibodies,

compare

of the

experiment
antibodies

was

performed
<0.1 However, for SgII

(Table

1). Most
Se-

recognized

ng of intact

menogelin,
of Mab that

SgI as well as SgII. 5 was >20 times higher 1 7A >80 times

the sensitivity than for SgI, and for SgII.

of Mab

higher

for SgI than

Bjartell et al
Table

Tissue Expression of Semenogelin land I!

showing the sensitivity human Sgl
Semenogelin I

21
Table 2. Relative abundance of epithelial cells manifesting specific immunostaining for Sg!ISgll in paraffin sections of male genital organs and smears of freshly ejaculated spermatozoa Mabs 1, 5, 8, 9, 17A, 22, and 25
+ + +

1. Dot blot results

of monoclonal
Semenogelin 0.1 <0.1 <0.1 <0.1 8 <0.1 0.3 II

antibodies

raised against

Mab 1 Mab5 Mab 8 Mab 9 Mabl7A Mab 22 Mab 25

<0.1 2 <0.1 <0.1 <0.1 <0.1 <0.1

Tissue Seminal Epididymis caput vesicle

n 10 10 10 10 10 10 10 6

Polyclonal
+ + +

lgG

Different amounts (1,000, 200, 40, or Sgll were applied to lmmobilone incubated with the different monoclonal reactions were visualized with rabbit phosphatase. The minimum amount detection signal is indicated for each

8, 2, 0.3, and 0.1 ng) of intact Sgl membranes. The membranes were antibodies (4 ig/ml), and positive anti-mouse lgG coupled to alkaline (ng) of Sgl or Sgll to produce a antibody.

corpus
caudal Testis Prostate gland Deferent duct Ejaculated spermatozoa

0 0 0 0 0
+ + +

0 0 0 0 0
+ + +

By

Southern of SgI

blot,

cross-hybridization

of SgII the was similarly

cDNA deter-

with
tion mined

the SgI probe cDNA

to be <0.5%.

was <0.5%, and with SgII probe

cross-hybridiza-

Mabs 1, 5, 8, 9, 17A, 22, and 25 are the different monoclonal lgGs raised against purified Sgl and Sgll, and the affinity-purified polyclonal lgG was raised against a 52-amino-acid fragment of Sgl. Further details concerning the monoclonal antibodies are given in Table 1 and In the text. n, number of tissues/smears examined; + + +, large numbers of cells; 0, no detectable immunoreactive cells or spermatozoa.

Immunocytochemistiy
An tested tration identical (Mabs of -25 the seven

(ICC)
pattern and anti-SgII and was generated concenof The by IgGs

immunostaining

spermatozoa
identical identical clonal IgG

(Fig. 2C). This

staining

pattern

was not only

but also poly-

different jg/ml

anti-SgI (Table

monoclonal

1, 5, 8, 9, 17A,

25), at a final

for the seven different to that obtained with against the 52-amino-acid

monoclonal IgGs, the affinity-purified fragment

2). All monoclonal

antibodies cytoplasm vesicles.

of SgI (not

showed an the majority highest crypts amined glandular stromal

intense immunostaining in the of epithelial cells in the seminal

density of positive cells of the glandular epithelium, contained epithelium cells of the abundant

was found deep in the and all specimens excells in the no staining of the (Fig. 2A). The difthe polyclonal of SgI all imvesicle of the ep-

shown). With none of the anti-SgIISgII IgG monoclonal antibodies tested was immunostaining detected in the germinal epithelium, interstitial cells, or in the stromal cells
of the (Table which testis, 2). gave the deferent ducts, or the prostate gland

immunoreactive

but manifested seminal vesicles

Monoclonal anti-SgI/SgII
immunoreactivity imens pancreas, tum, row, any (urinary

IgG preparations
the most intense

coded used normal

small

Mab

1 and Mab
of tissue liver, bone detected colon,

9,
the

ferent monoclonal IgG against the munostained by using and thin epididymis Using large the

anti-SgIJSgII 52-amino-acid same epithelial tissue

IgGs and fragment cells, sections

immunostaining

IgGs

tested,
in different

were

for screening
human intestine,

of Sg
specspleen, recmarin

as demonstrated of seminal sections antibodies

adjacent specimens. slides with different

bladder,

urethra,

kidney,

esophagus,

stomach,

longitudinal monoclonal

ididymis,

all the

against

lymph node, brain cortex,

SgI and SgII strongly immunostained the epithelium, but only in the caudal region where the spermatozoa are stored in wide secretions in contrast ducts. The spermatozoa contained in the luminal were also immunostained to the caput and corpus in the caudal region, regions, where the were into

No specific

heart, ovary, immunostaining

skin, skeletal muscle, uterus, uterine tube,

for SgI/SgII was

and breast).

of these tissues. In the negative ICC controls, all tissue sections were devoid of immunostaining (Fig. 2D,E). The 5 2-aminoacid fragment of SgI, added in up to 60 times molar excess, completely seminal abolished vesicles, caudal the immunostaining epididymis, and on sections ejaculated of sper-

luminal secretions as well as the glandular epithelium left unstained (Fig. 2B). Dissection of the epididymis

specific
results, ymis

regions
showing

before
SgI/SgII

fixation

and

ICC yielded
in the region.

identical
epidid-

matozoa,
the

using

immunostaining

52-amino-acid

the polyclonal SgI/SgII SgI fragment.

IgGs raised

against

to be confined to the caudal The APAAP technique showed

freshly

ejaculated

sper-

In situ Hybridization
Strong seminal
antisense

(ISH)
demonstrated in the and nonradioactive
SgI and SgII transcripts

matozoa to be immunostained by all the different monoclonal IgGs. The SgI/SgII epitopes were identified on the posterior part of the head, the midpiece, and tail of the

hybridization signals were vesicles by both radioactive

probes to the respective

22

Journal of Andrology

January/February 1996

FIG. 2. Immunocytochemlcal demonstration of Sgl and Sgll In paraffin sections of human seminal vesicles and epididymis. (A), Monoclonal antiSgl/Sgll lgGs (Mab 1) Immunostaln cells, predominantly deep in the crypts of the secretory epithelium. (B), Low-magnification micrograph showing Sg Immunoreactlvity (Mab 9) restricted to the caudal region (arrows) In a longitudinal section of whole epididymis. (C), Freshly ejaculated spermatozoa were immunostained using a monoclonal anti-Sgl/Sgll lgG preparation (Mab 25). APAAP technique with Fast Red as a chromophore shows immunostaining on the postacrosomal part of the sperm head, on the tail, and the most intense staining on the midpiece region. (D), In accord with findings

Bjartell et at

Tissue Expression of Semenogelin land!!

23

FIG. 3. In situ hybridization (ISH) and immunocytochemistry (ICC) on paraffin sections of human seminal vesicles. (A), Using an AP-conjugated 30-mer antisense probe specific for the Sgl transcript, strong hybridization signals were detected In epithellal cells. Note the perlnuclear staining pattern. (B), An adjacent section hybridized with a similar probe specific for SglI transcripts, hybridized to the same epithelial cells, but with less Intense signals. (C), An Irrelevant AP-conjugated probe (PSA) generates no hybridization signals at all. Note the Intracellular lipofuscln granules, which are typical of eplthelial cells of the seminal vesicles. (D), Digoxigenin labeling of the Sgl probe generates a similar staining hybridization pattern, and on an adjacent section (E), the same epithellal cells Immunoreact with the polyclonal Sg-lgG preparation. (F), Radiolabeling (MS) of the Sgl-speclflc 30-mer antisense probe also hybridized to the seminal vesicie epithelum, but with a much more diffuse pattern. BCIP-NBT served as the chromophore In ISH, and ICC was performed with the SA-HRP technique and DAB staining. Bars: A, B, C, and F = 25 pm; D and E = 100 pm.

4-

obtained using the monoclonal lgG preparations, the polyclonal lgG demonstrated immunoreactivity in the epithellal cells and the lumen content in the cauda region of epididymis. (E), Addition of the 52-amino-acid Sgl fragment (10 pg/mI diluted antibody preparation, -40 x molar excess) prior to application on the tissue sections completely prevents the Immunoreaction. Bouin fixation. Bars: A, B, D, E 100 pm; C 10 pm.

24

Journal of Andrology

January/February 1996

FIG. 4. Non-radioactive ISH of the human epididymis with DIG-labeled oligodeoxynucleotide probes. the Sgl transcript does not hybridize to the epithelial cells, in contrast to the well-detectable hybridization (A). Bars 25 m.

(B), The 30-mer antisense probe specific for signals obtained with the Sgli specific probe

(Fig.

3). A perinuclear

staining

pattern

was

detected

in a

subpopulation of epithelial stained cells being present

The signals were consistently tisense probe for radioactive probes

cells, the highest density of in the basal part of the crypts.

more intense with the anthat for SgII. to demonstrate The nonin detail

antisense hybridization prostate Thin

probes tissue

for signals sections. tissue

SgI or SgII in the testis,

generated any specific the deferent ducts, or

adjacent

sections

SgI than with could be used

epididymis were also used to compare of ISH obtained with different probes,

of seminal vesicles and staining patterns

and to evaluate the

that epithelial cells expressing conjugated probes showed in the seminal vesicles than

SgI also expressed SgII. APa better signal-to-noise ratio did DIG-labeled probes. The

oligo-dT sue cells

control hybridization omission

probe (both
for the

labeled epithelial
technique. with

with DIG hybridized and stromal cells)

Negative with controls, antisense unlabeled

to most tisas a positive

including (PSA), and probe, probe

correlation of staining generated by ICC to staining reactions produced by ISH. Thus, cells immunostained for SgI/SgII were found to be identical with the cells expressing the SgI and SgII transcripts detected by ISH (Fig. 3D,E).

irrelevant competition

of probe,

Discussion
The purpose of the present study using ICC and ISH was to further investigate the production and distribution of Sgl and SgII in different normal human tissue specimens,
particularly in the seminal vesicles and the epididymis. Tissues from the male genital tract, obtained at surgery, were prepared and fixed under conditions optimized to preserve (Bjartell mRNA et al, for 1993, ISH BjOrk studies, et al, as 1994). recently Archival described tissue

RNAse digestion of tissue sections prior to hybridization, all supported the specificity of the hybridization signals

(Fig.

3). probe generated

The SgII-specific
in epithelial Sgl probe

hybridization
whereas signals

signals
the were

cells of the caudal did not (Fig. 4). No

epididymis, hybridization

detected in the lumina of tissue sections that still contained luminal spermatozoa after the processing of the slides. ISH with DIG-labeled probes was superior to that with the AP probes because they generated less background staining in epididymal tissue. None of the labeled

specimens were also used for ICC because the monoclonal and polyclonal anti-Sg IgGs worked well on routinely fixed

Bjartell et al

Tissue Expression of Semenoge!in land II

be expressed

25
in the same epithelial cells. In the epididymis, we identified hybridization signals specific to the SgII transcript, whereas the SgI transcript could not be detected. The expression of SgII was confined to the epithelium of the cauda region. No hybridization signals for the SgI or SgII transcripts were detected on spermatozoa present in the lumina of the caput, the corpus, or cauda epididymis or testis, indicating that immunoreactive SgIJ SgII on spermatozoa may represent attached proteins deriving from the cauda epididymis secretion. Whether it really mRNA represents present SgII in the found on the spermatozoa needs to

tissues, facilitating an extensive investigation of the occurrence of immunoreactivity for SgI/SgII in a multiplicity of organs. Monoclonal antibodies against SgI or against SgII have not previously been available. Nine different monoclonal antibodies recognized both SgI and SgII. It is likely that this cross-reactivity is due to the approximately 80% structural similarity between SgI and SgII. The epitopes were not studied in detail, but dot blot and Western blot
experiments suggested that the antibodies recognized at

least

five different

epitope

structures

(Fig.

1).

In this study, all SgI/SgII yielded identical results with polyclonal anti-Sg IgGs. In (Lilja et al, 1989), we also SgII within the epididymis tivity didymis, ulation. completely present ifested to be confined to the where the spermatozoa The caput and corpus unstained. in the lumina Sg immunoreactivity, of the

monoclonal antibodies tested the previously characterized contrast to previous studies examined the distribution of and found the immunoreacepithelium of the cauda epiejacwere manregion are collected before regions of epididymis the specimens only spermatozoa also tissue but

be clarified.
in the deferent ther supports

We were

unable

to identify

epididymis,

any signals of SgI SgI and SgII mRNA

ducts, prostate, both previous

or testis, a result that furand present immunocyto-

chemical findings. SgI and SgII have been shown to be responsible for the immediate gel formation of freshly ejaculated semen (Liii a
and in the Laurell, 1984, region 1985). of the findings The function and of SgII the produced presence of

Interestingly,

cauda semenogelin
established.

epididymis may

epitopes
These

on the spermatozoa

in the cauda

remain to be be important for an un-

of the epididymis. In contrast, Evans and Herr (1986) were unable to detect any SVSA epitopes in the epididymis from with their MHS-5 monoclonal IgG, but it is unclear

derstanding of normal gel formation function, although SgI and SgII may
tions. In this investigation

and

normal

sperm
func-

also have other the findings ofimmunoreactive

their report of the epididymis SgI/SgII described

ulated spermatozoa, studies of previous

whether they examined in detail. All monoclonal

here also immunostained

different parts IgGs against

freshly ejac-

SgI/SgII in a multiplicity of human tissues verified that tissue expression of semenogelins is predominantly confined to the seminal vesicles and the cauda epididymis, and thus the antibodies may be used biopsies specimens. as specific from

a finding
using

in accordance

with

those
IgGs

markers

polycional

anti-SgI/SgII

(Lilja et al, 1989), and with findings regarding the SVSA antigen as studied by Herr et al (1986). Because neither the polyclonal antibodies against SgI/ SgII nor the monoclonal anti-SgIJSgII IgGs described here are specific for either SgI or SgII, we used ISH to demonstrate possible ed probes specific to design showed transcripts in tissue oligodeoxynucleotide for SgI and cross-hybridization sections. probes to be It proved specific to <0.5%. The

of seminal vesicle tissue in core gland or for cauda epididymis

the prostate

a flWflI SW VWIflIWI 5

the respective

mRNA

SgII, and

the construct-

We thank Elise Nilsson and Vinka Filinic at the Department of Pathology, Birgitta Frohm and Ingrid Wigheden and Sanna Hulkko at the Department of Clinical Chemistry, Lund University, University Hospital, Malm#{246}, Sweden, for their expert technical assistance.

non-radioactive ISH methods to the radioactive procedure alize the perinuclear staining
the specific cells (Figs.

were found to be superior in that they distinctly visu-

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