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Regular Article |
a Departments of Obstetrics and Gynecology,
b Clinical Chemistry,
c Urology, Helsinki University Central Hospital, 00290 Helsinki, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, in vitro fertilization, male reproductive tract, seminal vesicles, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Semenogelin I is encoded by a 1.8-kilobase (kb) mRNA, which is abundant in seminal vesicles but absent in epididymis, prostate, and testis [7]. Semenogelin II is expressed as a 2.4-kb transcript both in the epididymis and the seminal vesicles [8]. Semenogelin I and semenogelin II have 78% amino acid sequence identity [8]. The 71- and 76-kDa proteins referred to as semenogelin II have an identical polypeptide backbone that differs only with respect to glycosylation [8]. Semenogelin I and semenogelin II are encoded by 2 homologous genes located 11.5 kb apart in the q12–q13.1 region on chromosome 20 [9].
The purpose of this study was to examine the relationships between semenogelin and semen parameters, biochemical parameters of seminal plasma, and the fertilizing capacity of sperm. In addition, the expression of semenogelin and its mRNA was studied in male and female reproductive tissues.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study was approved by the Ethical Comittee/Institutional Review Board of the Department of Obstetrics and Gynecology, Helsinki University Central Hospital. All the participants had given an informed consent to the use of the tissues or seminal plasma for research after all of the necessary specimens had been taken for clinical purposes. Tissue specimens of the prostate, seminal vesicle, and ampulla of the vas deferens were obtained from 5 men, 52–65 yr of age, undergoing radical prostatectomy for local prostate cancer [10]. Testis and epididymis were from a man 78 yr of age undergoing orchiectomy for advanced prostate cancer. Serum samples were collected from 2 healthy men and 2 healthy women 20–60 yr of age. Three RNA samples from urinary bladder cancer patients were kindly provided by Dr. Kristina Hotakainen (Helsinki University Central Hospital). Amniotic fluid (n = 10) was obtained from the specimens examined for routine prenatal diagnosis of chromosome abnormalities at 15–17 wk of gestation. Endometrium from various phases of the menstrual cycle (n = 22), first trimester decidua (n = 9), placenta, and breast (n = 2) tissues were obtained from specimens removed for clinical reasons. Seminal plasma was collected from male partners of infertile couples (n = 96) participating in an in vitro fertilization program. To prevent degradation of semenogelin I, an aliquot was taken from the ejaculate of a normal healthy volunteer immediately after ejaculation and diluted 6-fold in 4 M urea containing 40 mM Tris, pH 9.6, 25 mM EDTA, and 30 mM dithiothreitol [11]. Purified intact semenogelin I and semenogelin II were kindly provided by Dr. Johan Malm (Lund University, University Hospital, Malmö, Sweden). All the liquid samples were stored at -20°C until studied.
Monoclonal Antibodies
Mice were immunized with the protein fraction of seminal plasma containing glycodelin-S and other proteins [12]. Three antibody clones (F91-11E6, F91-1D6, and F92-10G7) that did not react with highly purified glycodelin detected 7- to 15-kDa peptides from liquefied seminal plasma in Western immunoblots. These antibodies were advanced to production in mouse ascitic fluid or in vitro cell culture medium and purified with Protein G-sepharose (HiTrap Protein G; Pharmacia Biotech, Uppsala, Sweden).
Isolation of Seminal Vesicle mRNA and Construction of the cDNA Library
RNA was purified from the seminal vesicle obtained from a man (54 yr old) undergoing radical prostatectomy for prostate cancer. Total RNA was extracted using the guanidine thiocyanate method (RNA Isolation Kit; Stratagene, La Jolla, CA), and the mRNA fraction was purified using an mRNA Purification Kit (Pharmacia Biotech). A cDNA library was constructed according to the manufacturer's instructions using ZAP Express cDNA Synthesis Kit and ZAP Express cDNA Gigapack III Gold Cloning Kit (Stratagene).
Isolation and Analysis of cDNA Clones
The plaques from an amplified cDNA library were screened following instructions for the picoBlue Immunoscreening Kit (Stratagene), using monoclonal antibody (MAb) F91-11E6. Bound antibody was detected using alkaline phosphatase-conjugated polyclonal anti-mouse antibody (D0314; Dako, Glostrup, Denmark) and nitroblue tetrazolium/5-bromocresyl-3-indolylphosphate (Boehringer-Mannheim, Mannheim, Germany) as a substrate. After tertiary screening in which all of the plaques were positive, in vivo excision of the pBK-CMV phagemid vector from the ZAP express vector was done according to the instructions (Stratagene). DNA was isolated from 4 pBK-CMV clones (Wizard Minipreps, DNA Purification System; Promega, Madison, WI) and sequenced using the DNA Sequencing Kit and ABI PRISM 310 Genetic Analyzer (PerkinElmer, Foster City, CA).
Northern Blot Analysis
Poly(A) RNA (3–5 µg) was subjected to electrophoresis and transferred to a Hybond-N nylon membrane (Amersham, Buckinghamshire, U.K.). The filter was hybridized overnight at 68°C using Express Hyb hybridization solution (Clontech, Palo Alto, CA) and 32P-labeled semenogelin I cDNA cloned as described above. Labeling of the EcoRI fragment was perfomed with a rediprime DNA-labeling system (Amersham). For autoradiography, the filter was exposed to Hyperfilm-MP film (Amersham) for 10 min. A glyceraldehyde phosphate dehydrogenase cDNA probe was used to quantify the amount and control the quality of loaded RNA. A dot blot of poly(A) RNA from 50 different human tissues using Human RNA Master Blot (7770-1; Clontech) was done according to the manufaturer's instructions.
Reverse Transcription Polymerase Chain Reaction
Urinary bladder RNA was extracted by Phase Lock Gel Heavy (5 Prime
3 Prime, Inc., Boulder, CO), and total RNA was extracted from 22 endometrial and 9 decidual samples and male reproductive tissues using the guanidine thiocyanate method [13]. RNA (2 µg) was reverse transcribed to cDNA using Moloney murine leukemia virus reverse transcriptase according to the manufacturer's instructions (Gibco, Gaithersburg, MD; Omniscript RT kit; Qiagen, Valencia, CA). For the semenogelin I polymerase chain reaction (PCR), the sense primer was 5'-TCAAAAGGTCATTTTCACAG-3' and the antisense primer was 5'-CTTTACTTGAATGTTCCTCT-3'. Touchdown PCR (for details, see [10]) for semenogelin I amplification was performed using Dynazyme or Taq DNA polymerase (Finnzymes, Espoo, Finland; Qiagen; respectively), using 1–5 µl of the cDNA reaction as a template. For a positive control, we used cDNA from seminal vesicle. Actin and/or glycodelin PCR was used to control the quality of the cDNAs. For every sample, a negative control PCR was performed using a cDNA reaction mixture without the reverse transcriptase enzyme.
In Vitro Production of Semenogelin I
Escherichia coli was transfected with semenogelin I cDNA in the pBK-CMV phagemid vector (Stratagene). Colonies were grown in Luria broth overnight. Isopropyl-1-thiol-ß-D-galactopyranoside (1 mM) was added, and after 6 h of incubation the cells were collected by centrifugation (15 min at 3000 x g) and stored at -20°C until tested by Western immunoblotting. In vitro transcription/translation of semenogelin I was done using the TNT Coupled Reticulosyte Lysate Systems (Promega) as described in the manufacturers instructions, except that nonradioactive instead of radioactive methionine was used. For Western blotting, l0 µl (of 50 µl) was used per well.
Purification of Semenogelin and Its Fragments
MAb F91-11E6 was conjugated with CNBr-activated Sepharose 4B (Pharmacia Biotech). Urea-treated ejaculate or liquefied pooled seminal plasma was diluted 4-fold in Tris buffer (50 mM Tris HCl, pH 7.7, containing 9 g/L NaCl and 0.5 g/L NaN3) and filtered (Millex HV, 0.45 µm, Millipore, Molsheim, France). After filtration, Triton X-100 was added to a final concentration of 0.1%. The seminal plasma was applied on the MAb-Sepharose column, washed with Tris buffer, 1 M NaCl containing 1% isopropanol, and finally with 10 mM CH3COONH4, pH 5. Elution was performed with 0.1% trifluoroacetic acid. The protein concentrations of purified semenogelin and its fragments were measured by the BioRad protein assay (BioRad Laboratories, Richmond, CA) using BSA as a standard.
Immunohistochemical Staining
Formalin-fixed (10% phosphate-buffered neutral formalin containing 4% formaldehyde) and paraffin-embedded prostate (n = 5), seminal vesicle (n = 5), ampulla of the vas deferens (n = 5), testis (n = 1), epididymis (n = 1), endometrium (n = 2), first trimester decidua (n = 1), placenta (n = 1), and breast (n = 2) tissue specimens were cut in 5-µm sections and immunostained using MAbs against semenogelin and a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) [14].
SDS-PAGE, Western Immunoblot, and Silver Staining
SDS-PAGE of reduced (with ß-mercaptoethanol) samples was performed with a NOVEX X Cell II Mini-Cell system (Novex, San Diego, CA) in a 4–20% gradient gel (Novex). Immunoblotting was performed according to the method of Towbin et al. [15]. After transferring the proteins to Immobilon-P Transfer Membrane (Millipore, Bedford, MA), the membrane was incubated in 1% BSA (Sigma, St. Louis, MO) in Tris buffer (BSA/Tris) overnight at 4°C and then with MAb diluted in BSA/Tris for 2 h at room temperature. After washing with Tris buffer, the membrane was incubated for 1 h at room temperature in 200-fold diluted peroxidase-conjugated anti-mouse antibody (P260; Dako) in BSA/Tris, washed in Tris buffer without NaN3, and stained using 3,3'-diaminobenzidine tetrahydrochloride (0.3 g/L) (Fluka, Buchs, Swizerland) as a substrate. Silver staining was done to identify the proteins transferred to the Immobilon-P Transfer membrane according to the method of Kovarik et al. [16].
Immunofluorometric Assay for Semenogelin
MAb F91-1D6 was labeled with Delfia Eu-labeling reagent (Eu-chelate of isothiocyanatobenzyl-diethylenetriaminetetraacetic acid; Wallac Oy, Turku, Finland) by incubating with a 50-fold molar excess of Eu-chelate in 0.1 M NaHCO3, pH 9.3. After overnight incubation at room temperature, the labeled MAb was purified on a 1 x 50-cm column of Sephacryl S-200 (Pharmacia Biotech) by elution with Tris buffer. For solid-phase coating, MAb F92-10G7 (4 µg/200 µl Tris buffer) was adsorbed onto polystyrene microtiter wells (Eflab Oy, Helsinki, Finland) by overnight incubation at room temperature. After incubation, 250 µl BSA/Tris was added to each well for 2 h at room temperature to minimize nonspecific binding. In the assay, 25 µl of the sample and 200 µl of the assay buffer (0.05 M Tris(hydroxymethyl)aminomethane·HCl, pH 7.7, containing 0.5% BSA, 0.05% bovine 
-globulin, 0.2% diethylenetriaminepentaacetic acid, and 0.01% (w/v) Tween 20) were added in duplicate to microtiter wells and incubated overnight at 4°C. The wells were washed twice with washing solution (Tris buffer containing 0.05% Tween 20), and 50 ng Eu-labeled MAb in 200 µl of assay buffer was added and incubated for 1 h at room temperature. The wells were washed 4 times, and 200 µl of enhancement solution (Wallac) was added. The wells were shaken gently for 5 min, and fluorescence was measured with a 1234 Delfia Research Fluorometer (Wallac). Affinity-purified semenogelin from pooled liquefied seminal plasma was used for estimation of the molar concentrations of the calibrators consisting of diluted seminal plasma.
Correlation of Semenogelin Levels with Sperm Parameters and Fertilization In Vitro
After assessment of sperm parameters according to the World Health Organization guidelines [17], aliquots of seminal plasma (n = 96) were stored at -20°C. Twenty-five of the samples were oligozoospermic (sperm concentration < 20 x 106 sperm/ml) and 21 were asthenozoospermic (<50% spermatozoa with forward progression); 9 samples were both oligozoospermic and asthenozoospermic. In vitro fertilization (IVF) was done as described previously [18]. Semenogelin was measured by immunofluorometric assay (IFMA), and total PSA was determined using a commercial assay (Prostatus PSA Free/Total PSA; Wallac). Statistical analyses were done using StatView 4.1 for Macintosh (Abacus Concepts, Berkeley, CA). The Mann-Whitney U-test was used for comparison of different groups, and the Spearman rank correlation was employed for determining linear relationships between 2 variables.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Positive clones were selected immunologically by MAb F91-11E6 from the human seminal vesicle cDNA library, and about 400 base pairs (bp) from both ends were sequenced. Every screening gave several positive clones. All sequenced clones were identical to semenogelin I [7].
Western Immunoblot
Two of the MAbs (F91-11E6 and F92-10G7) recognized the 5- to 15-kDa proteins corresponding to semenogelin fragments in liquefied ejaculates, and they also recognized the full-length 52-kDa semenogelin I in seminal plasma, fragmentation of which was prevented before liquefaction (Fig. 1). Although urea treatment was performed as soon as possible after ejaculation, part of the semenogelin was fragmented (Fig. 1, lane 2). Affinity-purified semenogelin from urea-treated seminal plasma displayed the same 30- to 52-kDa bands in the immunoblots (MAb F91-11E6 or F92-10G7) and silver stains (not shown). Immunoblotting revealed no bands corresponding to full-length (71 and 76 kDa) semenogelin II in urea-treated seminal plasma or affinity-purified semenogelin. The third antibody (MAb F91-1D6) also recognized semenogelin and its fragments, but the reaction was very weak.
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The specificity of antibodies was also tested with recombinant semenogelin I. Antibodies F91-11E6 and F92-10G7 reacted strongly with full-length semenogelin I and with semenogelin I fragments produced in E. coli. However, these antibodies did not react with seminogelin I produced in reticulisyte lysate systems. Antibody F91-1D6 reacted weakly with semenogelin produced in E. coli but reacted strongly with semenogelin I produced in reticulosyte lysate (results not shown). Purified intact semenogelins I and II reacted with all the MAbs (results not shown).
Reverse Transcription PCR
With the semenogelin I primers, the amplified products from seminal vesicle and the ampullary part of the vas deferens cDNA were 360 bp, which corresponds to the size predicted from the semenogelin I sequence (results not shown). Prostate, testis, epididymis, urinary bladder, and all endometrial and decidual samples were negative for semenogelin. We were able to amplify glycodelin and/or actin cDNA from the same samples, suggesting that the negative results obtained were not artifacts of the technique.
Localization of Semenogelin
Semenogelin was detected by immunohistochemical staining in glandular epithelial cells of the seminal vesicles and the ampullary part of the vas deferens (Fig. 2). No staining was observed in the prostate, epididymis, testis, endometrium, decidua, placenta, or breast tissue. Northern blot analysis confirmed the presence of semenogelin I mRNA in seminal vesicles and ampulla of the vas deferens (Fig. 3), but the prostate, epididymis, and testis were negative. In the filter (human RNA master blot) containing mRNA from 50 different human tissues (including mammary gland, ovary, placenta, and uterus), only prostate and urinary bladder contained semenogelin I mRNA.
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IFMA
A typical standard curve for semenogelin is shown in Figure 4. The detection limit was <1 nM, the intra-assay variation was 6.9–7.5%, and interassay variation was 11–13% at semenogelin concentrations of 5.5–14.0 nM. Similar concentrations were observed in urea-treated seminal plasma (consisting of fragmented and native semenogelin) and liquefied seminal plasma (fragmented semenogelin) from the same ejaculate. The assay also detected intact semenogelin I and semenogelin II. However, dilution curves of urea-treated semenogelin, intact semenogelin I, or semenogelin II were not parallel with that of liquefied seminal plasma, which suggests that the reactivity of the intact and fragmented semenogelin with the MAbs is not identical. As measured by IFMA, none of the serum or amniotic fluid samples contained detectable amounts of semenogelin.
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Correlation of Semenogelin Levels with Sperm Parameters and Fertilization In Vitro
The semenogelin concentration (mean ± SD) in liquefied seminal plasma was 153 ± 61 µM (range, 20–350 µM). The semenogelin concentration was correlated positively with the total protein concentration and negatively with the PSA concentration but was not correlated with the in vitro fertilization rate, volume of the ejaculate, glycodelin concentration, sperm concentration, or percentage of sperm with forward progression (Table 1 and Fig. 5). In addition to the negative correlation with semenogelin, PSA was correlated with the sperm concentration (P = 0.04; 
= 0.22). In spite of the fact that the PSA level was lower in the oligozoospermic group (348 ± 171 mg/L) than in the group with normal sperm concentration (503 ± 253 mg/L, P = 0.005), no significant differences in semenogelin concentration were found between the oligozoospermic group and the group with normal sperm concentration or between the asthenozoospermic group and the group with normal sperm progression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The antibodies were found suitable for immunoaffinity purification of semenogelin and its fragments, immunohistochemical staining, immunoblotting, and development of an IFMA. The immunogen used for immunization was prepared from liquified seminal plasma, which mainly contains fragmented semenogelins. Thus, it is not surprising that the antibodies recognized epitopes occurring both on fragments and on intact semenogelin. Our assay detected both intact semenogelins I and II and the fragments. The use of fragments for calibration is logical when measuring liquefied samples.
We confirmed previous observations [7, 19] that semenogelin I protein and the mRNA are present in the seminal vesicles and ampullary part of the vas deferens but not in the prostate, epididymis, testis, urinary bladder, and the female tissues tested. In the commercial dot blot containing mRNA from 50 different tissue samples, we detected a signal for the prostate and the urinary bladder. However, in keeping with previous studies [7, 19] semenogelin is not expressed in the prostate as shown also here by immunohistochemical staining, Northern blot analysis, and reverse transcription PCR (RT-PCR), or in bladder, as indicated by RT-PCR. Therefore it is possible that the prostate and the urinary bladder samples used for the commercial filter may have been contaminated with seminal vesicle tissue. Many seminal plasma proteins have also been found at high levels in the female reproductive tract [20], but in accordance with previous studies [19] semenogelin was not detected in ovary, placenta, endometrium, mammary gland, or amniotic fluid.
PSA is an enzyme that degrades the seminal plasma clot formed by semenogelin I, semenogelin II, and fibronectin. It is also quite specific for the male reproductive tract, although low levels of PSA have been found in amniotic fluid and female mammary gland [21–23]. Proper processing of semenogelin by PSA is considered important for fertilization [5]. Therefore, we measured semenogelin and PSA in seminal fluid and found a negative correlation between them. PSA cleaves semenogelin into specific fragments, which appear to be detected by the monoclonal IFMA employed in this study. Thus, our assay detected similar amounts of semenogelin in seminal plasma irrespective of whether the PSA activity had been inhibited or not.
Both nonproteolyzed and fragmented semenogelin inhibit sperm motility [24, 25]. Therefore, we studied whether there is any correlation between semenogelin levels and the sperm parameters or their fertilization capacity. Semenogelin concentration was not correlated with any of the sperm parameters measured, and there was no correlation between semenogelin concentration and the fertilization rate in vitro. Given that semenogelin is one of the major proteins in seminal plasma [2], our observation of a positive correlation between semenogelin and the total protein concentration was not surprising.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by grants from the Helsinki University Central Hospital Research Funds, the Federation of Finnish Life and Pension Insurance Companies, the Finnish Cancer Foundation, the Academy of Finland, and the University of Helsinki. 
2 Correspondence: Riitta Koistinen, Biomedicum Helsinki, Department of Obstetrics and Gynecology, Helsinki University Central Hospital, P.O. Box 700 (Haartmaninkatu 8), 00029 HUS (Helsinki), Finland. FAX: 358 9 471 71731; riitta.koistinen{at}hus.fi
Accepted: October 10, 2001.
Received: May 11, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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