Expression of a Uteroglobin-Like Protein in Human Prostate

0022-534 7/88/1401-0176$02.00/0 Vol. 140, July

THE JOURNAL OF UROLOGY

Copyright © 1988 by The Williams & Wilkins Co.

Printed in U.S.A.

EXPRESSION OF A UTEROGLOBIN-LIKE PROTEIN IN HUMAN PROSTATE MICHAEL J. MANYAK,* TADAHIRO KIKUKAWA

AND

ANIL B. MUKHERJEE

From the Experimental Phototherapy Section, Radiation Oncology Branch, National Cancer Institute, and Section on Developmental Genetics, Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland

ABSTRACT

Phospholipase A2 (PLA 2 ) is a key enzyme that initiates the arachidonic acid cascade responsible for the synthesis of prostaglandins and leukotrienes, compounds well known for their inflammatory properties. Inhibition of this enzyme may modulate prostaglandin and leukotriene tissue levels. Uteroglobin is a potent PLA 2 inhibitor found in rabbit uterus, prostate, seminal vesicle, and tracheobronchial tree. Tissue from ten human patients undergoing prostatectomy was examined for presence of a uteroglobin-like protein. Seven patients underwent transurethral resection and three had an open prostatectomy. Preoperative diagnosis in nine of the 10 patients was benign prostatic hypertrophy. One suspected, poorly differentiated, adenocarcinoma was confirmed and one unsuspected, well differentiated, adenocarcinoma was discovered. Specimens were submitted for Western blot, electron microscopy with immunogold staining, radioimmunoassay, and immunofluorescence. Six patients had evidence ofuteroglobin-like protein, three with high levels (2:1000 pg.jmg. protein), two with moderate levels (75 to 250 pg.), one with a low level (:575 pg.). Uteroglobin-like protein was present in all three patients who underwent open prostatectomy and in three of the seven patients with transurethral resections. The uteroglobin-like protein level was 2.5 to five times greater in both prostatic utricle specimens. All four assays corroborated these results. Because rabbit uteroglobin coats sperm and masks spermatic antigenicity in the rabbit female genital tract, this report of biochemical and immunological evidence for uteroglobin-like protein in the human prostate may have implications for human male fertility. (J. Ural., 140:176-182, 1988) Inflammation and immunity are closely related phenomena and, in vertebrate species, most inflammatory processes have an immunological component. 1 Although both inflammation and immunity are essential elements of host defense, these reactions must be specific because unrestrained inflammatory response can be injurious to previously uninvolved tissues. In all vertebrates, including humans, a vast, moist, epithelial layer lines the passages that communicate with the external environment (such as respiratory, gastrointestinal, and genitourinary tracts). The epithelia of these tracts constantly encounter antigens which can elicit injurious inflammatory responses, yet inflammation generally does not occur in these organs. Therefore, it would seem reasonable that endogenous factors are responsible for controlling inflammatory response in normal epithelial cells. Eicosanoids (prostaglandins, leukotrienes) have been suggested as mediators of many inflammatory processes. 2 Phospholipase A2 (PLA 2 ) is a key enzyme which controls the level of available arachidonic acid, the substrate required for prostaglandin and leukotriene synthesis. Inhibition of this enzyme, resulting in a lower level of tissue eicosanoids, has been suggested as a novel means to control inflammation. 3 - 7 Stress of inflammatory reactions may trigger corticosteroid secretion which modulates inflammation. 8- 10 During the past decade, it has been shown that antiinflammatory steroid hormones may exert their effect through a class of PLA2 inhibitory proteins called lipocortins. 7 • 11 - 13 Consequently, endogenous PLA2 inhibitors may be responsible for regulating the inflammatory processes in the moist epithelial lining of various organs. Accepted for publication March 7, 1988. * Requests for reprints: Radiation Oncology Branch, Experimental Phototherapy Section, Bldg. 10, Rm. B3-B69, 9000 Rockville Pike, Bethesda, MD 20892. Presented at the Surgical Forum, Reproductive Biology and Related Endocrinology, American College of Surgeons 73rd Clinical Congress, Oct. 12, 1987.

Uteroglobin (UTG) 14 or blastokinin15 is a potent PLA2 inhibitor genetically distinct from the lipocortins that is dependent upon steroids for synthesis and expression. 16 UTG was first discovered in the gravid rabbit uterus but subsequently has been found in several extrauterine tissues. The gene for this protein is now known to be under multihormonal control. For example, UTG is regulated by estradiol in the rabbit oviduct while in the uterus it is regulated by progesterone. 11-21 Corticosteroids appear to regulate the effect of UTG in the respiratory tract. 22 • 23 The hormonal control of this protein in the rabbit prostate, seminal vesicle, and epididymis is unclear. Although the presence of UTG has been conclusively proven in the rabbit23 • 24 its existence in human tissues is controversial.25 However, the recent detection of a UTG-like protein in the human uterus 26 and tracheobronchial washings 27 has been accomplished along with its biological and immunological characterization. 28 In addition, an inverse relationship between this UTG-like protein and leukotriene C. in the tracheobronchial lavage of children with acute respiratory illness has been demonstrated. 29 The purpose of the present investigation was to determine whether a uteroglobin-like protein is detectable in human prostatic tissues. To our knowledge, this is the first report on the biochemical and immunological evidence for the presence of a protein in human prostate very similar to rabbit uteroglobin. MATERIALS AND METHODS

Prostatic tissues were obtained after informed consent from 10 patients, five black and five white, aged 58 to 95, undergoing prostatectomy (table 1). In this series, seven patients underwent transurethral resection of the prostate (TURP) while three had open prostatectomy. The preoperative diagnosis was benign prostate hypertrophy in nine patients; one patient (#3) had a previous TURP of a prostatic carcinoma. Six patients presented with urinary retention, three patients had sympto-

176

EXPRESSI(Yi~ OF UTEROGLOBIN-LIKE PROTEIN IN HiJMAN PROSTATE T~ble 1. LEVELS OF PROST ATIC UTEROGLOBltJ·UKE PROTEIN (pg/mg Pm!ein) IN 10 PATIENTS CORRELATED WITH THE UROLOGIC HISTORY, SURGICAL PROCEDURE PERFORMED, SPECIMEN LOCATION, AND HISTOPATHOLOGY No

Age 73

TURP HP= UTI

=

Surgery

Specimen Location

Un nary retention, proteus UTI

Open

Lett lobe Right lobe Ulricle

4,014

833 904

75

Obstructive symptoms

TURP

Lateral lobe Lateral lobe

4,680 2,399

71

Carcinoma 1Oyears on estrogen

TURP

Lateral lobe Lateral lobe

124 76

73

Urinary retention

Open

Left lobe Right lobe

83

10

Protein Level

GU History

Urinary retention

TURP

52 11

Lateral lobe Lateral lobe

1,006

None None

Pathology Glandular+ Fibromuscular HP chronic inflammation

Fibromuscular HP

Poorly differentiated adenocarcinoma Glandular+ F1bromuscular HP, chronic prostatitis infarcts; squamous metaplasia Nodularfibromuscular HP

60 Glandular + Fibromuscular HP: acute + chronic inflammation

74

Urinary retention

TURP

Lateral lobe Lateral lobe

77

Urinary retention

Open

Left lobe Right lobe Utricle

95

Urinary retention, klebsiella/pseudomonas UTI

TURP

Lateral lobe Lateral lobe

None None

Glandular + Fibromuscular HP: cystitis cystica et glandularis

58

Obstructive symptoms

TURP

Lateral lobe Lateral lobe

None None

Nodular HP

68

Gross hemaluria: TURP 10yearsago

TURP

Laterral lobe Lateral lobe

None None

Nodular HP

= Transurethral

109 99 258

Well ditt. adenocarcinoma glandular HP

Resection of Prostate

lnlechon

matic obstructive complaints, and one patient had gross hematmia from an enlarged prostate. Of the six patients in urinary retention, four had recent indwelling catheter placement with negative urine cultures while the two patients with long-standing indwelling catheters had positive urine cultures treated with preoperative intravenous antibiotics. Nine of the 10 patients received intravenous antibiotics at surgery. The patient with preoperatively diagnosed prostatic carcinoma also was receiving an oral long-acting estrogen preparation (chlorotrianisene). Two specimens totalling approximately one gram were obtained from each transurethral resection; specimens from open prostatectomies included a wedge resection from each lobe and, when identified, from the prostatic utricle as welL Part of each tissue specimen was placed immediately in 0,25 mM phenylmethylsulfonyl fluoride (PMSF) to prevent protein degradation and was transported to the laboratory at 4C, The remainder of the tissue was fixed in glutaraldehyde and sent for routine histopathology, The specimens were homogenized by a Polytron homogenizer at 4C in three volumes of 50 mM Tris-HCl buffer (pH containing 1.5 mM EDTA, 100 mM NaCl, and 0.25 mM PMSF. The homogenates were centrifuged at 800 g for 10 minutes at 4C and the supematants recentrifuged at 15000 g for 20 minutes at 4C. All samples were lyophilized and stored at -80C until used for immunoprecipitation, SDS PAGE, and Western blotting, Total protein concentrations were determined by the method of Lowry et al. 30 using bovine serum albumin (BSA) as a standard. 1. I mmunoprecipitation of uteroglobin-like protein. Lyophilized samples containing UTG-like protein were reconstituted in PBS at a concentration of 0,8 µ,g. protein/ml. and then centrifuged at 200 g for five minutes. The supernatants were incubated with one mg. of goat anti-UTG antiserum at room temperature for one hour followed by incubation with protein A sepharose (20 µl./ml.) at room temperature for 30 minutes, The samples were then centrifuged at 200 g for five minutes and the precipitates were washed three times with 20 mL of PBS. These washed precipitates were solubilized with 100 µl. SDS sample buffer (20 mM Tris, 9% SDS, 3% /3-mercaptoethanol, and 50% glycerol, pH 8,8), 2, SDS - PAGE. Fifty microliter samples were applied to each lane for electrophoresis using a 10 to 20% SDS gradient polyacrylamide gel according to the method described by Laem-

177

mlL 31 Electrophoresis was carried out at a constant voltage (200 V) for five hours. Gels were fixed in methanol: acetic acid: water (45: 10: 45) for 30 minutes, stained with 0,1% Coomassie Brilliant Blue in 40% ethanol and 10% acetic acid for 30 minutes and destained overnight in methanol: acetic acid: water (20: 10: 70). Molecular weight markers (BioRad, Rockville Centre, NY) used were: phosphorylase B (98 K), bovine serum albumin (68 K), ovalbumin (43 K), carbonic anhydrase (31 K), soybean trypsin inhibitor (20 K), and lysozyme (14.3 K), 3. Purification of anti- UTG antibody by affinity chromatography. UTG was purified from HCG-primed rabbit uterine flushings as previously described, 32 ' 33 Using pure UTG, affinity chromatography was performed with minor modifications of a published method, 34 The procedure is as follows: Pure UTG (10 mg.) was conjugated to three gm, of CNBractivated sepharose-4B (Pharmacia, Piscataway, NJ), This material was then packed into a column (0.5 x 10 cm,) and washed with bicarbonate buffer (0,1 M sodium bicarbonate, 0,5 M NaCl, 0.01 M sodium azide, pH 8,3) ten times the bed volume at 4C The crude anti-UTG antiserum was applied to the column and then washed with bicarbonate buffer until no 280 nm absorbing material could be detected, The column was then washed with the eluting buffer (0.5 M acetic acid, 0.5 M NaCl, pH 2.3) to obtain the pure monospecific antibody to UTG. The column was reactivated for another use by washing with 10 bed volumes of bicarbonate buffer after no more protein could be eluted. After several cycles of purification, the samples were pooled, lyophilized, and reconstituted with PBS in preparation for Western blotting. 4. Western blotting of UTG-like protein. UTG-like protein was electrophoretically transferred to nitrocellulose paper (0.22 m) by the method described by Bumette 35 with minor modifications, The transfers were carried out in a transblot apparatus (BioRad) at 60 V for two hours using a 25 mM tris, 192 mM glycine, 20% methanol buffer (pH 8.3). Non-specific binding was blocked on the nitrocellulose paper by incubation with the same buffer containing 3% bovine albumin (Sigma, St, Louis, MO). This was washed with 0.1% BSA in PBS and then overnight with 1:200 dilution of affinity purified goat antirabbit antibody, The nitrocellulose paper was then washed five times with 150 ml BSA-PBS solution and incubated with 125I-protein A (final concentration 106 cpm/100 ml.) at room temperature for one hour. After washing with cold PBS, the paper was air dried and exposed to Kodak X-Omat AR X ray film (EastmanKodak, Rochester, NY) with intensifying screens for one to two days at -70C. Labeled protein molecular weight markers (BioRad) used were: ovalbumin (43 K), a-chymotrypsin (25.7 K), jJ-lactalbumin (18.4 K), lysozyme (14.3 K), and bovine trypsin inhibitor (6.2 K)o 125 I-UTG was also used as a standard marker. 5. Radioimmunoassay (RIA) of UTG-like protein. RIA of UTG-like protein was performed by using the method of Mayol and Longenecker with minor modifications. 36 Samples and standards were diluted in a one ml. solution containing one mg,/mL BSA, 0.1% NaN3, 0.01 M EDTA, and 0.5% normal goat serum to which a 1:10,000 dilution of goat UTG-antibody was added. After overnight incubation at 4C, 1251-UTG prepared by the Chloramine-T method was added (15 X 10 3 cpm/tube). These mixtures were incubated at 37C for two hours followed by four hours of incubation at 4C. The reaction was stopped by adding 200 µl. of antigoat serum raised in a burro and the mixtures were incubated for 12 hours at 4C. Samples were centrifuged at 1500 g for 30 minutes at 4C and the supernatants were aspirated and discarded. The pellet containing the bound fraction was counted in a Micromedic gamma counter equipped with a logit data reduction program. This assay has been validated using human serum and human uterine and tracheobronchial washings as well as progesterone treated rabbit uterine washings, Negative controls were prepared from rabbit

178

MANYAK, KIKUKAWA AND MUKHERJEE

liver extracts and skeletal muscle homogenates. Proteins with similar molecular weights to UTG, including a-fetoprotein, myosin light chain, SP 1 protein, human atrial natriuretic peptide, and protein peaks from a Sephadex G-100 column, did not cross react in this assay. Inter-assay variations were less than 5%. 6. Immunofluorescence. Biopsy specimens reserved for immunofluorescence were immediately fixed after sectioning in 2.5% glutaraldehyde. The specimens were embedded in paraffin, cut in 0.025 mm. sections, and placed on clean glass slides. One side stained with hematoxilin and eosin was used for histopathological assessment and its counterpart was used for immunofluorescence according to a previously described method. 33 After removal of paraffin by xylene, the tissue samples on the slides were passed through a series of ethanol concentrations (100-30%) and then PBS (pH 7.4). Excess PBS was shaken from the slides and a few drops of 1:100 dilution of affinity purified anti-UTG antibody were added to the tissue sections before incubation for 30 minutes at 37C. A positive control (progesterone primed rabbit uterus) and a negative control (rabbit liver) were added to each batch of tissues. In addition, a second slide with sections of human prostate was stained with UTG-antibody preabsorbed with an excess of UTG prior to its use. After the incubation period the slides were washed with PBS at 4C for one hour. Next, the sections were covered with FITC conjugated rabbit antigoat antibody (1:10), incubated for 45 minutes at 37C, washed for one hour with cold PBS with constant agitation, and rinsed with bicarbonate buffer (pH 9.5). Excess buffer was shaken off the slides and they were mounted in 50% glycerol/bicarbonate buffer, coverslipped, and examined with a Zeiss photomicroscope with epifluorescence. Photomicrographs were taken using Kodak Extachrome-400 film.

2. A well-defined low molecular weight protein (lane 3) corresponding to the rabbit UTG standard (lane 1) is present. Three other high molecular weight bands were also visualized. Further evidence of the staining specificity for rabbit UTG-antibody was established when no bands were visible on similar blots stained with affinity purified antibody preabsorbed with pure rabbit UTG. Indirect immunofluorescence with UTG-antibody was used in glutaraldehyde-fixed human prostate tissue to determine the localization of UTG-like protein to a particular cell type (fig. 3). High levels of immunofluorescence, which were seen in epithelial cells of the human prostatic acini, corresponded to high levels of UTG-like immunoreactivity in the RIA. UTG-like protein was found in all three patients who underwent an open prostatectomy but in only three of the seven who had transurethral resection. In the two cases where a specimen of the pro static utricle was confirmed by histopathology, UTGlike protein levels were 2.5 to five times greater than those from the lateral lobes. There were moderate to low levels of UTGlike protein in both specimens from patients with prostatic

1

2

3

RESULTS

Radioimmunoassay demonstrated the presence of UTG-like immunoreactivity in six of the 10 patients (table 1). The amount of UTG-like immunoreactivity varied: three patients had high levels (>1000 pg./mg. protein), two had moderate levels (75-250 pg./mg.), and one had a low level (<75 pg./mg.). Similarity of the two antigens is strongly suggested by the parallel nature of standard curves generated using various dilutions of pure rabbit uteroglobin and human prostate extracts (fig. 1). Additional characterization of this immunoreactivity by SDS-PAGE and Western blot techniques is presented in figure

43K-

25.7K -

100 . _ . . Standard Uteroglobin 0--0 Prostatic UTG-Like Protein

80

18.4K 14.3K 6.2K-

0

~ 60 ~

'

40

20

15.63

31.25

62.5

125

250

500

1000

UTEROGLOBIN (Pg/tube)

FIG. 1. Standard curves of rabbit uteroglobin and human uteroglobin-like protein. Parallel nature of these curves suggests similarity of antigens.

FIG. 2. Western blots of human prostatic extracts. Lane 1: Molecular standards. Lane 2: Monomeric rabbit uteroglobin standard after disulfide bond reduction of the polymer. Lane 3: Human prostatic extract. Of three immunoreactive bands detected, predominant band has molecular weight of 45 KD.

E:ZCPElESSION OF UTEROGLOBIN--LIKE PROTEII\f II"J I-IDf.11Ai\f PROST;AT'E

Fm. 3. Immunofluorescence from prostatic lateral lobes of patient 2 (table 1) localizing high degree of immunoreactivity to uteroglobin in acinar epithelial cells.

adenocarcinoma, one well-differentiated #7) and one poorly differentiated (patient #3). All three patients with high levels of immunoreactivity had glandular and fibromuscular hyperplasia. The patient with low UTG-like protein levels (patient #4) had histopathological evidence of significant longstanding inflammatory prostatic disease. However, one patient with an open prostatectomy had high levels of the in the presence of chronic inflammation #1) TUR specimens in two of four patients without detectable levels of UTG-like also had evidence of chronic 1m,w..uuicacu•cy processes 1n,ac1oPn1rq#6, DISCUSSION

Since the discovery of UTG in gravid rabbit uterine secretions, much information has been obtained about its structure and function. 16, 37 - 41 UTG also has been found in several rabbit organs besides the uterus: oviduct, lung, gastrointestinal tract, and male genital tract. 21 , 42 - 47 UTG has been localized in rabbit epididymal epithelium but the same study did not show UTGlike immunoreactivity in rabbit testis, prostate, seminal vesicle, and ductus deferens.4 8 However, rabbit prostatic and seminal vesicle localization of UTG has been reported by others. 24 ' 47 The secretory granule of rat ventral prostate cells also have manifested immunocytochemical cross reactivity with UTG antisera.49 In addition, there is homology between UTG and the C 2 chain of the androgen-induced rat prostatic steroid binding protein. 50 Immunocytochemical studies strongly suggest that UTG synthesis occurs in epithelial cells of the organs producing UTG. 51 - 56 For instance, high UTG levels have been

179

located m secretory of both rabbit endometrial nonciliated cells and bronchiolar Clara cells.4 9 Immunofluorescence of the human prostate revealed UTG-like protein thelial cells. Furthermore, immunogold electron ,,,.,,Q,•rm,» has confirmed this localization in epithelial cells (data not shown). Although there have been conflicting reports of UTG-like protein in human endometrium, 25 recent studies have demon·· strated a human endometrial protein cross-l'eactive to monospecific anti-UTG antibody. 26 • 28 This protein, which has the same molecular weight as UTG, also has been found in human neonatal tracheobronchial washings in our laboratory. 27 To our knowledge, this is the first time a protein in the human prostate with similar molecular weight and immunoreactivity to rabbit UTG has been reported. The Western blots of the prostatic tissue extracts (fig. 2, lane 3) revealed three other protein bands with higher molecular weight than the small molecular weight band corresponding to the rabbit UTG. Rabbit UTG has been reported to be a substrate for transglutaminase 57 which crosslinks the protein to higher molecular weight forms. Therefore, it is possible that these higher molecular weight bands represent polymers of human UTG-like protein. Alternatively, the UTG-like protein may be a higher molecular weight form and the low molecular weight bands may represent degradation products. The presence of UTG-like protein was detected in all three patients who had transabdominal surgical removal of the prostate but in only three of the seven patients undergoing prostatic transurethral resection. Several explanations could be offered for this discrepancy. Because no RIA levels were detected in the last 3 TURP specimens, the loss of activity in the stock PMSF preservative could have resulted in UTG-like protein degradation by tissue hydrolases. Secondly, UTG-like protein destruction may have been caused by extensive electrocautery during TURP. Transabdominally removed intact tissue specimens would not be subject to protein denaturation by electrocautery. Of course, there may be true absence of the UTG-like protein in those specimens not expressing UTG-like activity. UTG-like protein levels were obtained from the prostatic utricle in two specimens from patients who had transabdominal prostatic resection. This prostatic structure, embryologically homologous to the uterus where uteroglobin was first discovpossessed UTG-like protein levels 2.5 to five times greater than levels in the lateral lobes of the same specimens. Progesterone receptor mediated androgenic stimulation of rabbit en dometrial UTG 19 suggests that high levels of UTG-like protein may be present in the human prostatic utricle. Although no function has been proven for the utricle, the ducts of simple racemose glands distinct from the prostate are collected in this structure, 58 Utricular UTG-iike secretion could add to the seminal fluid when the 1JUJ.D()C1:trv·er;rro:s1 muscle contracts Prostatic adenocarcinoma was in two specimens that ""'"°"QQc,r1 low to moderate protein levels, There was essentially no difference in UTG-like protein levels between the preoperatively known, transurethrally resected, poorly differentiated adenocarcinoma (patient ,~3) and the unsuspected, transabdominally removed, well differentiated malignancy (patient #7). The significance of UTG-like protein levels in the presence of prostatic adenocarcinoma is unknown. The loss of certain protein expression caused by the rapid neoplastic cellular proliferation in many tumor types may not occur with prostatic carcinoma because of its generally slow growth characteristics. Correlation of UTG-like protein levels, degree of expression, relationship to differentiation, and potential clinical significance for both prostatic hypertrophy and adenocarcinoma is the focus of another study. Diffuse chronic prostatitis was present in one transabdominally resected specimen (patient #4) in which low levels of UTG-like protein were detected. Conversely, high UTG-like

180

MANYAK, KIKUKA WA AND MUKHERJEE

protein levels were found in a patient (#1) with a previous urinary tract infection and chronic inflammation who underwent transabdominal prostatic resection. Although the number of patients in this study is too few to ascertain a correlation between UTG-like protein expression and inflammation, the duration and extent of prostatic inflammatory processes may be related to the degree of UTG-like protein expression. Tracheobronchial washings in children with respiratory tract infections show low UTG-like protein levels and high leukotriene C4 levels, whereas the reverse is true for normal children. 29 This inverse relationship between UTG-like protein and leukotriene C4 may be present in other organs. The intent of this study was to determine whether a prostatic UTG-like protein exists in humans. Although a human prostatic UTG-like protein was found, its clinical significance and physiological function have not yet been demonstrated. It has been proposed that UTG may protect the conceptus from maternal immune and inflammatory response during nidation. 59 In rabbits, UTG has been shown to protect blastocysts from the maternal immunological response, suggesting a role as mediator of pregnancy-related local immunosuppressive action.88·60 Although there has been no proposed explanation for extra-uterine UTG an immunosuppressive response also has been demonstrated for epididymal sperm coated with UTG. 61 Spermatozoa have well established antigenic properties in both animal models and in man. 62-64 However, the female reproductive tract usually does not respond immunologically to ejaculated spermatozoa despite being capable of mounting an immunological response to antigens, 61 implying that local immunosuppression is present. The immunosuppressive activity of seminal fluid is suggested by several studies.65-67 Furthermore, an immunosuppressive response was not found in the highly antigenic rabbit epididymal sperm but was evident once the sperm were coated with UTG. 61 The maximal antigen-masking effect of UTG in ejaculated sperm requires transgultaminase (TG )88 which is found in two forms within the human prostate. 68 TG appears to share a complex relationship with UTG because the immunosuppressive response is ablated by both anti UTG and anti TG antibodies. 83' 69 If the human prostatic UTG-like protein proves to have similar function to rabbit UTG, it may contribute to male fertility by coating sperm, masking spermatic antigenicity in the female genital tract, and allowing conception to occur without mounting an inflammatory response. The preliminary finding of higher levels of UTG-like protein in the human utricle, which is adjacent to the spermatic ejaculatory ducts in the urethra, may not be coincidental. The in vitro PLA2 inhibitory activity of UTG may be related to its presence in the male genital tract. 70 The activation of PLA2cleaves arachidonic acid from phospholipids consequently providing the substrate for synthesis of prostaglandins and leukotrienes, many of which are directly related to inflammation. 71. 72 Prostaglandin and leukotriene production must be strictly controlled in certain organ systems, such as the tracheobronchial tree, where a balance exists between the response to inhaled antigens and the powerful bronchoconstrictive activity associated with these eicosanoids. 72' 73 The high levels of UTG-like protein present in human neonatal tracheobronchial washings27 suggest that this protein is instrumental in maintaining that homeostasis_ This is further supported by the report of an inverse relationship between UTG-like protein and leukotriene C4 levels in tracheobronchial washings of children with respiratory tract infections. 29 A similar form of control may exist for the regulation of seminal prostaglandin levels. Minor amounts of the PGE and PGF prostaglandin series have been found in human prostatic tissue while seminal fluid contains many prostaglandins in large amounts in addition to leukotriene C4.74' 75 Although prostaglandin E 2 (PGE2) has both immunosuppressive 75 and inflammatory76 properties, most prostaglandins in semen have been linked with stimulation of sperm motility or with uterine con-

tractility. However, the complex relationship between prostaglandin levels and male fertility suggests that a balance between various classes of these eicosanoids may be necessary for normal sperm density and motility. 74 In ejaculates with abnormal sperm motility, the seminal concentration of 19-hydroxy-PGE is lower and the levels of 19-hydroxy-PGF are higher. 74 The in vitro addition of physiological amounts of 19-hydroxy-PGF to semen samples with normal motility result in decreased sperm activity and reduced ability to penetrate cervical mucus. 74 In these respects, expression of UTG-like protein in the prostate may contribute to male fertility by modulation of prostaglandin levels. The existence of UTG-like protein in the human prostate provokes many unanswered questions. In light of the known properties of rabbit UTG, the possible relationships of prostatic UTG-like protein to both infertility and inflammation are intriguing. Differences in UTG-like protein expression in normal, benign hypertrophic, and neoplastic prostatic tissue are unknown. Hormonal stimulus of UTG production in other organ systems provides speculation about androgenic influence upon UTG synthesis in the male genital tract. Explanation of the transglutaminase-UTG interrelationship becomes more important with the discovery of UTG-like protein in human prostate. Thus, further characterization of human prostatic UTG-like protein may contribute to our fundamental knowledge of reproductive and immunological physiology. Acknowledgment. The authors would like to thank Drs. Harry C. Miller and Said A. Karmi, Department of Urology, George Washington University Medical Center, Washington, D.C. for their assistance in obtaining prostatic specimens. REFERENCES 1. Oppenheim, J. J. and Potter, M.: Immunity and inflammation. In:

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