Pathogenic Consequences in Semen Quality of an Autoimmune Response against the Prostate Gland: From Animal Models to Human Disease This information is current as of October 2, 2021. Ruben D. Motrich, Mariana Maccioni, Andres A. Ponce, Gerardo A. Gatti, Juan P. Mackern Oberti and Virginia E. Rivero J Immunol 2006; 177:957-967; ;

doi: 10.4049/jimmunol.177.2.957 Downloaded from http://www.jimmunol.org/content/177/2/957

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Pathogenic Consequences in Semen Quality of an Autoimmune Response against the Prostate Gland: From Animal Models to Human Disease1

Ruben D. Motrich,* Mariana Maccioni,* Andres A. Ponce,† Gerardo A. Gatti,* Juan P. Mackern Oberti,* and Virginia E. Rivero2*

We have recently proposed an autoimmune etiology in ϳ35% of chronic nonbacterial prostatitis patients, the most frequent form of prostatitis observed, because they exhibit IFN-␥-secreting lymphocytes specific to prostate Ags. Interestingly, this particular group of patients, but not the rest of chronic nonbacterial prostatitis patients, also presented striking abnormalities in their semen quality. In this work, we use an experimental animal model of autoimmune prostatitis on Wistar rats developed in our laboratory to investigate when, where, and how sperm cells from autoimmune prostatitis individuals are being damaged. As in patients, a

marked reduction in sperm concentration, almost null sperm motility and viability, and an increased percentage of apoptotic Downloaded from spermatozoa were detected in samples from animals with the disease. Prostate-specific autoantibodies as well as elevated levels of NO, TNF-␣, and IFN-␥ were also detected in their seminal plasma. In contrast, epididymal spermatozoa remain intact, indicating that sperm damage occurs at the moment of joining of prostate secretion to sperm cells during ejaculation. These results were further supported by experiments in which mixture of normal sperm cells with autoimmune seminal plasma were performed. We hypothesize that sperm damage in experimental autoimmune prostatitis can be the consequence of an inflammatory milieu,

originally produced by an autoimmune response in the prostate; a diminished prostate functionality, evidenced by reduced levels http://www.jimmunol.org/ of citric acid in semen or by both mechanisms simultaneously. Once more, we suggest that autoimmunity to prostate may have consequences on fertility. The Journal of Immunology, 2006, 177: 957–967.

he past decade has seen a resurgence of interest and ex- amelioration of symptoms rather than on a cure (7). One reason for citing new research on chronic prostatitis and related syn- the lack of significant progress toward the understanding of its T dromes (1). One important reason for this enthusiasm is etiology, pathophysiology, and therefore the design of rationale the recognition that chronic prostatitis syndromes represent an im- therapies is the absence of adequate animal models in which to portant worldwide health care problem (2). Chronic bacterial pros- study the disease process. tatitis is characterized by uropathogenic infections of the prostate Recently, we have analyzed a group of patients with CNBP to by guest on October 2, 2021 gland that respond well to antimicrobial treatment (3). In contrast, search for the presence of a possible autoimmune response to pros- chronic nonbacterial prostatitis (CNBP)3 (now classified as Na- tate Ags. We demonstrated the existence of IFN-␥-secreting lym- tional Institutes of Health category III), which accounts for 90– phocytes that proliferate in response to known human prostate Ags 95% of prostatitis cases, is of unknown etiology and is character- such as prostate-specific Ag and prostatic cancer phosphatase ized by the presence of a mixture of pain, urinary, and ejaculatory (PAP) in 34% of patients with CNBP (8). Only this group of pa- symptoms with no uniformly effective therapy (4–6). A rationale tients showed significantly elevated levels of cytokines such as treatment is urgently required, because until now the goal therapy IFN-␥, IL-1, and TNF-␣ in their seminal plasma, arguing for a for these patients has not had good results, and it has focused on local inflammation of noninfectious cause. The relationship between chronic prostatitis and infertility has been controversial for many years with some reports arguing for a *Centro de Investigaciones en Bioquı´mica Clı´nica e Inmunologı´a, Departamento de positive relationship between chronic prostatitis and altered semen Bioquı´mica Clı´nica, Facultad de Ciencias Quı´micas. Universidad Nacional de Co´r- doba, Co´rdoba. Argentina; and †Instituto de Fisiologı´a Humana, Facultad de Ciencias quality (9, 10), whereas others showed no alterations (11, 12). One Me´dicas, Universidad Nacional de Co´rdoba, Co´rdoba, Argentina possible explanation of such contradictory findings could be that Received for publication December 5, 2005. Accepted for publication April 25, 2006. CNBP comprises an heterogeneous group of patients and, as we The costs of publication of this article were defrayed in part by the payment of page and others have described (8, 13, 14), that the presence of auto- charges. This article must therefore be hereby marked advertisement in accordance immune response against prostate Ags is detected only in a subset with 18 U.S.C. Section 1734 solely to indicate this fact. of patients with CNBP. We have also reported major abnormalities 1 This work was supported by Agencia Nacional de Promocio´n Cientı´fica y Tecno- in some of the ejaculate parameters only in CNBP patients who lo´gica Grant PICT 2003 5/13624, Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de Argentina (CONICET) Grant PEI 6547, and Fundacio´n Antorchas 2002 presented signs of an autoimmune response against the prostate Grant 14264. R.M. and J.P.M. are Ph.D. fellows of CONICET. G.G. is a Ph.D. fellow gland (15). When the comparative analysis of sperm parameters of Fondo para la Investigacio´n Cientifica y Tecnolo´gica, and M.M. and V.R. are members of the Researcher Career of CONICET. was done considering all patients enrolled (CNBP patients with 2 Address correspondence and reprint requests to Dr. Virginia E. Rivero, Haya de la and without an autoimmune response against prostate), nonsignificant Torre y Medina Allende, Ciudad Universitaria, Co´rdoba, 5000 Argentina. E-mail differences were found. These findings allowed us to assume that address: [email protected] these significant differences could have been masked in the other men- 3 Abbreviations used in this paper: CNBP, chronic nonbacterial prostatitis; PAP, pros- tioned studies. Therefore, we hypothesize that the autoimmune re- tatic acid phosphatase; EAP, experimental autoimmune prostatitis; PSBP, prostate steroid-binding protein; SI, stimulation index; HOS, hypoosmotic swelling; PI, pro- sponse to prostate Ags and the inflammatory environment elicited pidium iodide; ROS, reactive oxygen species. could have detrimental consequences on sperm quality.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 958 AUTOIMMUNITY AGAINST PROSTATE AND ITS CONSEQUENCES

From the results obtained in chronic autoimmune prostatitis pa- PAP, purified from human seminal plasma, was purchased from Sigma- tients, some questions arose such as when, where, and how the Aldrich. Recombinant rat TNF-␣ was purchased from BD Pharmingen, and ␥ sperm cells are being damaged and which are the putative medi- recombinant rat IFN- was purchased from BD Biosciences. ators and pathogenic mechanisms involved. Histology Animal models of many human diseases have been studied for many years and have provided a great deal of information about Prostate glands collected 7 days after last immunization (day 42) were mechanisms involved in the autoimmune response. Our laboratory fixed in 4% formalin solution and processed for conventional histology. A prostatitis score was done in a blinded manner and computed for individual has developed over the past decade a rat and a mouse model of glands by summing the grade of each section and dividing the total number experimental autoimmune prostatitis (EAP) that is characterized of sections examined (usually 4). The degree of inflammation was assessed by an organ specific mononuclear infiltration and a cellular and using a scale of 0–3: Ϫ, no inflammation; ϩ, mild, but definite perivascular humoral autoimmune response against prostate Ags (16–19). Pros- cuffing with mononuclear cells; ϩϩ, moderate perivascular cuffing with ϩϩϩ tate steroid-binding protein (PSBP) has been described as the ma- mononuclear cells; , marked perivascular cuffing, hemorrhage, and numerous mononuclear cells in the parenchyma. jor autoantigen involved in the autoimmune response (19, 20). Although very little is known about the immunology of human T cell proliferation assay disease, most of the features already reported on it, are shared with those of our animal model. The type of infiltration characterized by Single cell suspensions were prepared in HBSS (Sigma-Aldrich) from ␥ pooled draining lymph nodes of individual rats. Lymph node cell viability intra-acinar mononuclear cells and the presence of IFN- -secreting was assessed by trypan blue exclusion. Cells were finally resuspended in T cell response against prostate Ags are the main aspects shared by DMEM supplemented with Glutamax, sodium pyruvate, nonessential human and animal diseases. In contrast, the Ab response, which amino acids, HEPES buffer, penicillin/streptomycin, 50 mM 2-ME, and reaches important levels in the animal model, is not detected in the 10% FCS (Techgen) and were distributed at a concentration of 1,5 ϫ 105 Downloaded from human disease. cells/well in a 0.1-ml volume into flat-bottom 96-well microtiter plates (BD Biosciences). Syngeneic naive spleen cells irradiated at 1500 rad served as Moreover, as further characteristics are being described in hu- APCs. These cells were prepulsed for2hat37°C with prostatic Ags: man patients, the validity of EAP as an animal model of the disease prostate extract at 130 ␮g/ml; or purified PSBP at 20 ␮g/ml; or PAP at 20 is being corroborated. In the present work, we used the animal ␮g/ml. APCs were then washed twice and added to 0.1 ml of responder 5 model to respond some unanswered questions and to advance on cells at 4 ϫ 10 cells/well final cell concentration. All cell combinations

were set up in quadruplicate. Plates were incubated for 4 days at 37°C in http://www.jimmunol.org/ the underlying mechanisms and pathogenic consequences of auto- water-saturated 7.5% CO2 atmosphere and pulsed for the final 18 h with 1 immunity against prostate Ags. ␮Ci of methyl-[3H]TdR. Labeled material was automatically harvested and counted in a ␤ plate scanner (Pharmacia). The response was expressed as stimulation index (SI) and as ⌬cpm. Stimulation indexes were calculated from Materials and Methods cpm incorporated in Ag-pulsed cultures/cpm incorporated in cultures with nonpulsed APC. Threshold value for positivity for each Ag assayed (prostate Animals and immunization extract, PSBP, and PAP) was set up above the mean SI plus 3 SDs of the SI ⌬ Sexually mature Wistar rats were bred and maintained under specific observed in the control group. cpm was calculated from cpm incorporated in pathogen-free conditions in the vivarium of the Center of Immunology and Ag-pulsed cultures minus cpm incorporated in cultures with nonpulsed APC. Biochemical Chemistry Research of the National University of Cordoba by guest on October 2, 2021 (Cordoba, Argentina). Fifty-five 2-mo-old male rats were included in our Abs against prostate Ags in serum and in semen study. Animals were maintained in a 16-h light, 8-h dark cycle at 20 Ϯ 2°C, with food and water ad libitum. The experimental protocol and all the Abs against PE, PAP, or PSBP were titrated by conventional ELISA in ␮ performed tests were carefully reviewed and approved by the institutional multiwell plates (Costar) precoated with 50 l/well prostate extract at 100 ␮ ␮ ␮ review board. g/ml, PSBP at 20 g/ml, or PAP at 5 g/ml in 0.05 M carbonate buffer, Animal immunization was achieved by the injection of saline solution pH 9.6. After overnight incubation at 4°C, microwells were washed twice plus CFA (Sigma-Aldrich; group I; n ϭ 17), 1 mg of prostate extract plus and blocked with 3% BSA (Sigma-Aldrich) in PBS for2hat37°C, rinsed ␮ CFA (group II, n ϭ 20) or 120 ␮g of purified PSBP plus CFA (group III; once with PBS-Tween 20 at 0.05%, and then filled with 50 l of serum n ϭ 18), on days 0, 20, and 35. Animals received four intradermal injec- (obtained after cardiac puncture) or seminal plasma dilutions (1/50 or 1/6 tions in different places: right footpad; left footpad; base of the tail; and respectively) for1hat37°C. To detect specific IgG, plates were again shoulders. washed and incubated with HRP-conjugated goat anti-rat IgG (Sigma- Aldrich) for1hat37°C. The reaction was developed with o-phenylene-

diamine-H2O2. OD was measured at 490 nm in a Bio-Rad Plate Reader. Ags and reagents Serum reactivity was expressed as a binding index representing the ratio between the OD given by the test serum and the OD of a preimmune serum Prostate extracts were prepared from Wistar rat prostate glands. Pooled at the same dilution. Minimal threshold value for positivity was set up at glands from 50 rats were homogenized in 0.01 M PBS, pH 7.2, with pro- 2. Semen reactivity was expressed as OD. tease inhibitors in an Ultra-Turrax homogenizer. The homogenate was cen- trifuged at 100,000 ϫ g for 30 min, and the supernatant was used as Ag. Semen collection Protein concentration was determined using the Folin phenol reagent. The preparations were aliquoted and kept frozen at Ϫ20°C. Semen samples were obtained by electroejaculation as described previ- PSBP was purified following the procedure described previously by ously by Ponce et al. (22). Briefly, nonanesthetized rats were placed in a Chen et al. (21). Briefly, ventral prostates were removed in a sterile manner box that covered the cephalic half of the body while the rest laid on a from Wistar male rats (body weight, 250–300 g) and homogenized in 20 metallic grid support. After the genital area was cleaned, the prepuce was mM Tris-HCl using an Ultra-Turrax homogenizer. The cytosolic fraction retracted, and the penis was introduced into an Eppendorf plastic tube. The was obtained after 60 min of centrifugation at 100,000 ϫ g and 4°C. The rectal probe was lubricated, and the bronze bipolar electrode was inserted resultant supernatant was applied onto a Mono-Q FPLC column. Proteins into the rectum to a depth of 20–30 mm and held by the technician. The were eluted with a linear 0–60% NaCl gradient in 20 mM Tris-HCl. Pro- alternative current (220 V, sinusoidal wave, 50 cycles/s) was applied for 5

tein concentration of each fraction was evaluated by measuring its OD280. of every 10 s. The current was controlled by a rheostat and was calculated Each fraction was run under nondenaturing conditions in 15% polyacryl- from an oscilloscope reading of the voltage drop across a resister placed in amide gels (SDS-PAGE); then, the gels were stained with Coomassie blue. the circuit. A series of one to five pulses was normally given at the 6- to Fractions containing two bands of 18 and 20 kDa (corresponding to both 6.8-V setting. Immediately after electrostimulation, a white viscous plug subunits of PSBP) were also run under denaturing conditions to verify their was obtained. Immediately after, a series of 1–10 pulses was given at identity. The purity of the preparation was higher than 95%. Western blot- 13–15 V, and the remaining portion of the ejaculate was collected and ting using a rabbit polyclonal PSBP antiserum or a mouse mAb recogniz- diluted with 150 ␮l of Tyrode’s buffer medium without BSA (pH 7.4, 280 ing the C3 polypeptide (kindly given by Dr. P. Bjork, Pharmacia and Up- mOsm/kg) to avoid coagulation. Functional parameters of spermatozoa john, Uppsala, Sweden) was performed. were determined within 3–10 min by subjective evaluation in fresh semen The Journal of Immunology 959 following standard methods in our laboratory. Seminal plasma was ob- 5 ␮l of annexin V-FITC solution and 5 ␮l of propidium iodide (PI) solu- tained by centrifuging semen at 300 ϫ g for 7 min at room temperature and tion. Samples were incubated in polypropylene tubes for 15 min in the kept at Ϫ80°C until use. dark, and then 400 ␮l of binding buffer were added. Samples were analyzed within 1 h. The samples were analyzed using a FACS Star Plus-Flow Fructose, citric acid, and ␣-glucosidase levels in semen cytometer (Becton Dickinson Immunochemistry Systems) equipped with standard optics. An argon ion laser (INNOVA 90; Coherent) tuned at 488 Citrate, fructose, and ␣-glucosidase seminal levels were measured as tests nm and running at 200 mW was used as light source. From each cell, of function of the prostate, seminal vesicles, and epididymis, respectively. forward light scatter (FSC), orthogonal light scatter (SSC), A-FITC fluo- Neutral ␣-glucosidase, fructose, and citric acid were determined according rescence, and PI fluorescence were evaluated using CellQuest version 3.3 to the World Health Organization semen analysis manual (23). Briefly, for (BD Biosciences). A gate was applied in the FSC-SSC dot plot to restrict determining neutral ␣-glucosidase, 4-nitrophenyl-␣-D-glucopyranoside the analysis to spermatozoa. For the gated cells, the percentages of annexin was converted into 4-nitrophenol and ␣-D-glucopyranoside, and 4-nitro- V-negative or -positive (AϪ or Aϩ), and PI-negative or -positive (PIϪ or phenol was photometrically measured. The acid isoenzyme was inhibited PIϩ), as well as double-positive cells were evaluated, based on quadrants by addition of 1% SDS and phosphate buffer, pH 6.8, so that only the determined from single-stained and unstained control samples. neutral isoform was measured. Fructose was determined according to the hexokinase method, and citric acid was measured with the UV method Analysis of sperm motility in mixture experiments using the citrate lyase-catalyzed reaction. The working medium for analysis of sperm kinetics was modified Krebs- IFN-␥ and TNF-␣ in seminal plasma Ringer bicarbonate solution containing 94.6 mM NaCl, 4.78 mM KCl, 1.71 ␣ ␥ mM CaCl2, 1.19 mM KH2PO4, 1.19 mM MgSO4, 25.07 mM NaHCO2, 21.58 TNF- and IFN- quantities present in seminal plasma were determined by mM sodium lactate, 0.5 mM sodium pyruvate, 5.56 mM glucose, 4.0 mg/ml solid phase sandwich ELISA protocol according to the manufacturer’s in- BSA, 50 ␮g/ml streptomycin sulfate, and 75 ␮g/ml potassium penicillin). structions (BD Biosciences). Briefly, 96-well plates were coated with pri- They were equilibrated overnight to pH 7.4 at 37°C in a 5% CO atmosphere mary anti-TNF-␣ or anti- IFN-␥ capture Abs and blocked with PBS-5% 2

and kept at 37°C all through the protocol. Sperms were collected from Downloaded from BSA. Seminal plasma samples were incubated overnight at 4°C followed normal nontreated adult male Wistar rats by electroejaculation as described by the addition of biotinylated anti-TNF-␣-detecting mAb or biotinylated ␥ in Semen collection and analyzed immediately after collection. Sperm sus- anti-IFN- -detecting mAb. Plates were developed by adding avidin per- pensions (40 ␮l) were placed on grease-free slides warmed at 37°C and oxidase and its substrate (TMB), and the OD was measured at 492 nm in ϫ 2 ␣ ␥ covered with 18- 18-mm coverslips to achieve a chamber depth of a microplate reader (Bio-Rad). The amounts of TNF- or IFN- were 20–50 ␮m, which does not disturb sperm movements. The slides were extrapolated from the standard curve, which was generated in 1/2 dilutions. rapidly prepared and transferred to a heated plate of an inverted Olympus Results are expressed in picograms per milliliter. IX 70 phase contrast microscope. The videomicroscopy system consisted Measurement of NO levels in seminal plasma of a charged-coupled device video camera, a time lapse video recorder, and http://www.jimmunol.org/ a Panasonic monitor (Matsushita) connected to the microscope as previ- Nitrite concentration in seminal plasma was measured by using the Griess ously described (25, 26). For each slide, the motility of sperms in no fewer reagent. Briefly, 0.10 ml of seminal plasma was mixed with 0.2 ml of than 10 fields were recorded for 10 min and analyzed in each experiment. Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine di- Recordings were conducted at videotape speed of one image every 0.016 hydrochloride in 2% phosphoric acid) and incubated for 15 min at room s (continuous running). During the video frame-to-frame playback, cell temperature in the dark. OD was measured at 540 nm, and nitrite concen- tracks (previously selected at random in the pause mode) were drawn by tration was calculated using a calibration curve. Results are expressed as hand on an acetate sheet attached to the monitor screen during 3 s. Then, micromols per milliliter. the tracks were scanned and transferred into a computer, and the per- centage of sperm motility of each sample was calculated and analyzed Epididymal sperm obtainment with SigmaScanPro image analysis software (SPSS). by guest on October 2, 2021

Animals were killed by CO2 asphyxiation followed by cervical dislocation. Statistics The epididymis was removed, trimmed of fat, and clamped at the corpus-cauda junction. Rat spermatozoa were obtained by chopping cauda epididymidis Statistical analysis was performed with the Wilcoxon paired test, paired t with a sharp pair of scissors, allowed to disperse, and homogenized in 5 ml of test, one-way ANOVA (followed by Dunnet’s test when the ANOVA test yielded statistical differences ( p Ͻ 0.05) among experimental groups), and Ham’s F-12 medium for 30 min at 37°C in a 5% CO2-saturated atmosphere. Fisher’s exact test as appropriate. p Ͻ0.05 was considered statistically Assays to determine sperm concentration, motility, viability, and significant. sperm response to the hypoosmotic swelling (HOS) test Results Ϯ Sperm concentration and motility were assessed at 23 2°C in a Makler Autoimmune response can be detected systemically and locally counting chamber (Sefi Medical Instruments) under an inverted micro- scope at ϫ200 magnification. The results are expressed as the percentage in rats bearing experimental autoimmune prostatitis of motile cells (progressive plus nonprogressive spermatozoa). No fewer Autoimmune prostatitis in animals immunized with either prostate than 200 gametes were examined. extract or PSBP was evidenced by different methods (Fig. 1). As The viability parameter was evaluated by supravital staining with Hoechst 33258 (Calbiochem). Using the appropriate UV fluorescence op- expected, histopathological studies revealed lesions of varying in- tics (Axiolab; Zeiss), spermatozoa showing brightly fluorescence nuclei tensity in the prostate gland of almost all experimental animals were scored as dead, and sperm cells that excluded the Hoechst 33258 and irrespective of whether the immunizing Ag was prostate extract were not fluorescent were scored as viable. The viability of 200–250 cells (group II) or PSBP (group III) (Fig. 1A). Observed lesions were was assessed. The HOS test evaluates whether an intact membrane is biochemical and mainly characterized by moderate perivascular cuffing, mononu- functionally active. The procedure used for the HOS test was similar to the clear infiltration accompanied by hemorrhage, and edema. Histo- one described by Jeyendran et al. (24). The sperm suspension was mixed logical alterations were not detected either in prostate samples with a hypoosmotic solution (100 mOsm/L; Fiske Osmometer G-52), pH from animals of group I or in seminal vesicles, testis, and ep- 7.4, for 45 min at 37°C. Evaluations were made by phase contrast micros- ididymis samples from animals of the three groups under study copy at a magnification of ϫ400; 200 cells or more were observed, and the percentage of spermatozoa that showed tail swelling was determined by (Fig. 1A). dividing the number of reacted cells (ϫ100) by the total number of sper- A positive proliferative response against prostate Ags was also de- matozoa counted in the same area. tected only in draining LN cells from prostate extract- and PSBP- Assessment of apoptosis by annexin V staining immunized animals (Fig. 1B). Indeed, lymph nodes cells from control animals of group I exhibited low cpm values in response to prostate Staining for annexin V was performed using an apoptosis detection kit II Ags (group I: ⌬cpm, 118.32 Ϯ 109.76 against prostate extract; ⌬cpm, (BD Pharmingen) containing annexin V conjugated with FITC. Sperm Ϯ ⌬ Ϯ samples were resuspended in binding buffer supplemented with 4 mg/ml 177.25 111.89 against PSBP; and cpm, 165.50 136.95 against BSA (fraction V; A-4503; Sigma-Aldrich) at a concentration of 10 ϫ 106 PAP). On the contrary, autoimmune animals from groups II and III spermatozoa/ml. From this solution, an aliquot of 100 ␮l was stained with presented high cpm values and therefore positive stimulation indexes: 960 AUTOIMMUNITY AGAINST PROSTATE AND ITS CONSEQUENCES Downloaded from http://www.jimmunol.org/

FIGURE 1. Systemic and local autoimmune response in experimental autoimmune prostatitis. A, Histological changes in prostate sections of immunized animals. Prostate glands, seminal vesicles, testes, and epididymis collected 7 days after last immunization (day 42) were fixed and processed for conven- tional histology (n ϭ 5 animals per group). A prostatitis score was computed for individual glands by summing the grade of each section and dividing the total number of sections examined. B, In vitro proliferative response of inguinal and lumbar draining lymph node cells from immunized rats. Five animals per group were killed 7 days after the last immunization. Samples from each animal were processed individually. Proliferation was determined by uptake of [3H]TdR and expressed as a proliferation index calculated from cpm incorporated in Ag-pulsed APC cultures/cpm incorporated in cultures with nonpulsed APC. The threshold value for positivity was set up above the mean SI plus 3 SDs observed in the control group. The cpm incorporated in by guest on October 2, 2021 nonpulsed APC cultures were 230.50 Ϯ 98.48 cpm for animals from group I, 1250.45 Ϯ 435.78 cpm for animals from group II, and 650.45 Ϯ 267.44 for ,p Ͻ 0.05 vs control group. C, Serum IgG response against prostate Ags in immunized animals. IgG against prostate extract, PSBP ,ء .animals from group III and PAP Ags was studied by ELISA in serum samples obtained on day 42 (n ϭ 5 animals per group). Serum reactivity was expressed as a binding index representing the ratio between the OD given by the test serum and the OD of a preimmune serum at the same dilution. Minimal threshold value for positivity p Ͻ 0.05 vs control group. D, Seminal plasma IgG response against prostate Ags in immunized animals. IgG against PSBP was studied by ELISA ,ء .was 2 .p Ͻ 0.05 vs control group ,ء .in seminal plasma samples obtained on day 42. Five animals per group were assayed individually group II: ⌬cpm, 3141.66 Ϯ 722.26 against prostate extract; ⌬cpm, from control and autoimmune rats as a reflection of the prostate 3865 Ϯ 877.70 against PSBP; and ⌬cpm, 2244.66 Ϯ 1148.09 against environment. PAP; and group III: ⌬cpm, 1493.33 Ϯ 239.89 against prostate extract; High levels of NO were detected in seminal plasma from ⌬cpm, 2858.33 Ϯ 631.86 against PSBP; and ⌬cpm, 1361.66 Ϯ autoimmune rats (groups II and III) compared with the levels 504.51 against PAP. observed in the control group (group I; p Ͻ 0.05; Fig. 2A). Autoantibodies against prostate Ags were also detected in cir- Moreover, higher levels of TNF-␣ were also detected in seminal culating blood (Fig. 1C) and as immune complex depositions in the plasma from autoimmune rats of groups II and III when com- prostate gland (16). To investigate whether these autoantibodies pared with the levels observed in rats from group I, although generated against prostate Ags could somehow be part of the pros- only with a significant increase in samples from group II ( p Ͻ tatic fluid and thus appear in the ejaculate, we searched for its 0.05). When the levels of IFN-␥ were evaluated, similar data were presence in seminal plasma from animals under study. Indeed, obtained (Fig. 2C), evidencing significantly increased levels of this PSBP-specific IgG could be locally detected in seminal plasma of cytokine in seminal plasma of autoimmune animals ( p Ͻ 0.05). These animals from groups II and III. results suggest that an inflammatory process is taking place in the Taken together, our results clearly indicate that male rats immu- prostate in prostate extract- and PSBP-immunized animals due to an nized with prostate Ags develop inflammatory lesions in prostatic autoimmune response against prostate Ags. tissue and produce both a cellular and humoral specific autoimmune response, which can be detected systemically and locally. Prostate biochemical markers are altered in seminal plasma High levels of nitric oxide, TNF-␣, and IFN-␥ are detected in from autoimmune rats seminal plasma from autoimmune rats Citric acid and zinc are classical biochemical markers of prostate To analyze the presence of local proinflammatory mediators, the gland function, as well as fructose for seminal vesicles and neutral levels of NO, TNF-␣, and IFN-␥ were studied in seminal plasma ␣-glucosidase for epididymis (23). These markers are usually assayed The Journal of Immunology 961

FIGURE 2. NO, TNF-␣, IFN-␥, and bio- chemical markers of seminal vesicle, epidid- ymis, and prostate levels in seminal plasma samples from rats bearing autoimmune pros- Downloaded from tatitis. NO levels (A) and TNF-␣ (B) and IFN-␥ (C) quantities were determined by measuring the nitrite (Griess reagent) or cy- tokine quantities by a solid phase sandwich ELISA protocol in seminal samples obtained by electroejaculation of immunized animals. ␣-Glucosidase, fructose (D), and citric acid http://www.jimmunol.org/ (E) levels were determined in seminal plasma samples of animals from different groups. In all cases, samples were processed individually, and five animals per group .p Ͻ 0.05 vs control group ,ء .were analyzed by guest on October 2, 2021

as sensors of the functionality of the respective glands in human dis- These results corroborate our previous data on the specificity of eases, and many reports show that an inflammatory response can the autoimmune response in this model (19), confirming that only cause impairment of the functions of these glands. Because the exis- the prostate gland is affected. In other words, the immunization tence of abnormalities in these marker levels has not been demon- procedure generates an immune response that preserves the normal strated in our model, we investigated whether the inflammatory pro- function of seminal vesicles and epididymis but induces an inflam- cess that is taking place in the prostate of autoimmune animals could matory state that to some extent alters the prostate gland function. affect some biochemical functional markers. For that reason, we mea- sured citric acid, fructose, and neutral ␣-glucosidase levels in seminal Sperm quality parameters are altered in semen from animals plasma. No differences were found in the levels of fructose and neutral with autoimmune prostatitis whereas epididymal spermatozoa ␣-glucosidase when measured in the different groups under study, remain intact showing that the immunization performed in groups II and III does We previously demonstrated that chronic prostatitis patients who not affect the seminal vesicle and epididymal function (Fig. 2D). In evidenced autoimmune response to prostate Ags have altered contrast, the levels of citric acid, which mirrors prostate gland func- sperm quality (15). To evaluate whether this also occurs in the tionality, were significantly decreased in samples from autoimmune experimental autoimmune model, semen samples from animals un- rats from groups II and III (Fig. 2E), indicating an altered function of der study were obtained by electroejaculation, and different pa- the gland in autoimmune animals. rameters such as sperm concentration, total sperm motility, sperm 962 AUTOIMMUNITY AGAINST PROSTATE AND ITS CONSEQUENCES viability, and the HOS test were analyzed. For a complete over- zation) as well as in a later stage (after the third immunization) of view of the sperm quality during the development of the prostate- the autoimmune response against prostate ( p Ͻ 0.01). Finally, the specific autoimmune response, the tests were performed after the HOS test, which analyzes whether sperm plasma membrane is in- second immunization (on day 27; data not shown) and after the tact and also whether it is biochemically and functionally active, third immunization (on day 42; data shown in Fig. 3) of the ex- was performed. Very low levels of reactive spermatozoa were de- perimental protocol. In general, all these evaluated parameters tected in both groups of autoimmune animals (groups II and III) com- were impaired in the autoimmune animals, and they worsened as pared with the control group (Fig. 3D, p Ͻ 0.05). Our results suggest long as the autoimmune response had progressed and become es- that the stronger the autoimmune response against prostate the worse tablished. For example, group II (animals immunized with prostate are the alterations observed in semen. Certainly, animals from group extract that develop a florid autoimmune prostatitis) was the first to II, which showed the most severe infiltration and lesions and highest show a decrease in sperm concentration on day 27, but, as the seminal TNF-␣ and IFN-␥ levels, evidenced the most striking alter- immune response progressed (day 42), both groups of autoimmune ations in semen quality. animals (groups II and III) demonstrated a marked reduction in To investigate which is the sperm death process taking place, sperm concentration compared with control animals ( p Ͻ 0.01). sperm apoptosis in ejaculate samples was studied. The Annexin At the same time, sperm motility and viability in semen samples V-PI assay, which measures early and late apoptosis, was per- from animals under study were assayed. Almost null sperm mo- formed in semen samples obtained on day 42 of the experimental tility and viability levels were detected in both autoimmune animal protocol. The annexin V-PI assay identified four categories of cells groups (groups II and III) when compared with the control group (Fig. 3E). Higher levels of apoptotic spermatozoa were detected in (Fig. 3, B and C). These huge alterations in sperm motility and semen from both autoimmune animal groups (groups II and III) viability were seen in an earlier stage (after the second immuni- compared with the control group ( p Ͻ 0.05). The frequency of Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Semen quality in samples from rats with autoimmune prostatitis. Sperm concentration (A), sperm motility (B), sperm viability (C), HOS (D), and apoptosis by annexin V/PI staining (E) were conducted in semen samples obtained by electroejacu- lation of immunized animals. Different pa- rameters of spermatozoa were determined immediately after sperm obtainment by eval- uation following standard methods. In all cases, samples were processed individually, ,ء .and five animals per group were analyzed p Ͻ 0.01 vs control group. The Journal of Immunology 963 early apoptotic cells (Aϩ/PIϪ) was significantly higher in group Animals were sacrificed on days 27 and 42, and the same sperm III than in group II. However, seminal spermatozoa from group II analyses previously performed in semen samples were done in showed significantly higher percentages of late apoptotic, early epididymal sperm. Epididymal sperm concentration, motility, and necrotic (Aϩ/PIϩ), and necrotic (AϪ/PIϩ) spermatozoa than viability revealed no differences between autoimmune animals and those in group III ( p Ͻ 0.05) (Fig. 3E). control animals either on day 27 (data not shown) or on day 42 Physiologically, spermatozoa genesis and differentiation occur (Fig. 4, A–C). In accordance with these results, when the HOS in the testis. In the last steps, spermatozoa mature and are stored in assay was performed, no alterations were observed, revealing nor- the epididymis. The most mature spermatozoa are kept in the cau- mal epididymal sperm cell membrane integrity and functionality in dal region of the epididymis until the ejaculation, when they join both autoimmune groups compared with the control group (Fig. the sex accessory gland fluids to form the semen/ejaculate. Re- 4D). As shown in Fig. 4E, when epididymal sperm apoptosis was garding this fact, we wished to investigate where the sperm alter- analyzed, no increase in the percentage of apoptotic or necrotic ations observed in semen originated. There were two possibilities: spermatozoa was found in samples from both autoimmune animal 1) that these alterations were the result of epididymal damage as a groups (groups II and III) when compared with the control group. result of a satellite effect of the prostate inflammation (although we These findings evidenced that the striking alterations observed had not previously detected any histological alteration in epidid- in semen originated after sperm genesis in testis and its storage in ymis or autoantibodies against epididymal or testicular Ags, (data epididymis, suggesting that the inflammation in the prostate, the not shown)); 2) that these alterations were a result of damage orig- consequence of an autoimmune response against prostate Ags, inated during the encounter of sperm with the prostate fluids dur- could be involved in sperm cell damage. The kinetics of this dam- ing ejaculation. To answer these questions, we also analyzed ep- age appeared to be quick, and its detrimental effect could seriously ididymal sperm quality parameters in immunized animals. compromise the seminal quality of an individual. Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Epididymal sperm function- ality in rats with autoimmune prostatitis. Sperm concentration (A), sperm motility (B), sperm viability (C), HOS (D), and apoptosis by annexin V staining (E) were conducted in epididymal sperm samples of immunized an- imals. Different parameters of spermatozoa were determined immediately after epididy- mal sperm obtainment by evaluation follow- ing standard methods. In all cases, samples were processed individually, and five ani- p Ͻ 0.01 vs ,ء .mals per group were analyzed control group. 964 AUTOIMMUNITY AGAINST PROSTATE AND ITS CONSEQUENCES

Sperm alterations observed in autoimmune rats occur in a short observed, indicating that the immediate contact between sperm time and are caused at least in part by cytokines locally cells and an inflammatory seminal plasma can reproduce the de- produced by the autoimmune response tected abnormalities. To investigate the mechanisms involved in The results presented here supported the hypothesis that the alter- the observed alterations, we performed experiments adding ␣ ations observed in the sperm cells occurred once the prostate se- rTNF- to normal sperm at the concentrations detected in seminal cretions, loaded with the inflammatory mediators produced as a plasma from autoimmune rats. The addition of recombinant ␣ consequence of the autoimmune response, meet the spermatozoa TNF- was able to significantly diminish the sperm motility and during ejaculation. However, one can wonder what mechanism is increase sperm apoptosis of normal spermatozoa (Fig. 6, A and B). ␣ involved in the production of these alterations and whether the Moreover, when TNF- -neutralizing Abs were added, this effect ␥ damage could occur in such a short time. was partially blocked (Fig. 6A). Because IFN- is a crucial medi- To answer this question, the time course of sperm motility was ator of an inflammatory process, and its presence had been de- evaluated in semen samples from different groups. As shown in tected in seminal plasma samples from autoimmune CNBP pa- Fig. 5A, sperm motility diminished slowly in semen samples from tients (8) and autoimmune animals (Fig. 2C), its direct effect on ␥ animals of group I, showing a sperm motility of 60% immediately sperm motility was also investigated. The addition of rIFN- was after ejaculation with a slow drop after 8–9 min. However, marked able to significantly reduce the sperm motility and enhance sperm loss of sperm motility was observed in semen samples from auto- apoptosis of normal spermatozoa (Fig. 6, C and D). Moreover, ␥ immune animals (groups II and III); this effect was more drastic in when IFN- - neutralizing Abs were added, this effect was partially samples from group II. This loss in motility was evident both im- blocked (Fig. 6C). These results allow us to affirm that an inflammatory environment, mediately after sperm collection and during the time course of the Downloaded from analysis. the result of an autoimmune response directed against the prostate To assay whether some metabolite present in seminal plasma gland, can seriously affect the sperm, having a negative impact in the from autoimmune rats could produce these alterations, mixture ex- semen quality of an individual and compromising his fertility. periments were performed. As can be seen in Fig. 5B, when sperm cells from normal nontreated animals were mixed with seminal plasma from autoimmune rats, a marked drop in sperm motility Discussion http://www.jimmunol.org/ was observed. However, when seminal plasma from animals of Witebsky et al. (27) proposed rationales for the autoimmune basis group I (control group) was added to normal sperm cells, the same of clinical disease. They were consciously modeled on Koch’s slow drop on sperm motility seen in animals from group I was postulates and required an autoimmune response to be recognized by guest on October 2, 2021

FIGURE 5. Effect of the addition of seminal plasma from autoimmune animals on normal spermatozoa. A, Sperm kinetics analysis of samples from autoimmune rats during the 10 min after ejaculation. B, Effects of autoimmune seminal plasma (SP) on the motility of p Ͻ 0.01 vs control group. In ,ء .normal spermatozoa all cases, samples were processed individually, and five animals per group were analyzed. The Journal of Immunology 965

FIGURE 6. Effect of the addition of TNF-␣ and IFN-␥ on normal spermato-

zoa. Sperm kinetics analysis of normal Downloaded from spermatozoa during the 10 min after ejaculation in the presence of inflam- matory cytokines. A, Effects of recom- binant TNF-␣ on sperm motility in nor- mal spermatozoa. B, Effects of recombinant TNF-␣ on apoptosis marker expression in normal spermato- http://www.jimmunol.org/ zoa. C, Effects of recombinant IFN-␥ on sperm motility of normal spermatozoa. D, Effects of recombinant IFN-␥ on ap- optosis marker expression of normal p Ͻ 0.01 vs control ,ء .spermatozoa group. Values are representative of at least two experiments performed. SP, Seminal plasma. by guest on October 2, 2021

as either autoantibody- or cell-mediated immunity, the correspond- pathology. Indeed, animal models of numerous human diseases ing Ag to be identified, and an analogous autoimmune response to have been studied for many years and have provided a great deal be induced in an experimental animal. In addition, the immunized of information about mechanisms involved in autoimmune re- animal must also develop a similar disease. Some years later, Rose sponses (29). and Bona (28) proposed novel criteria to consider a disease as an The etiology of the most frequent form of chronic prostatitis, autoimmune disease, based on new information gained from the CNBP (National Institutes of Health category III), has proved elusive use of molecular biology and hybridoma techniques; these include despite years of investigation (7). Recently, we have proposed that direct evidence of the transfer of a pathogenic Ab or pathogenic T CNBP is a heterogeneous syndrome and that the etiology of this dis- cells, indirect evidence based on the reproduction of the autoim- ease, in some cases, involves an autoimmune response against pros- mune disease in experimental animals, and circumstantial evidence tate components. This hypothesis is based on the fact that a significant from clinical clues. These authors emphasized the importance of percentage of these patients exhibited IFN-␥-secreting lymphocytes the animal models to corroborate the autoimmune nature of a given against prostate Ags such as PAP, prostate-specific Ag, and other Ags 966 AUTOIMMUNITY AGAINST PROSTATE AND ITS CONSEQUENCES present in prostate homogenates and seminal plasma (8, 13, 14, 30). Our findings suggest that mediators present in seminal plasma High levels of proinflammatory cytokines and striking abnormalities from autoimmune animals were responsible for these alterations. in semen parameters were also described in CNBP that exhibited this In fact, when seminal plasma from autoimmune rats was added to autoimmune component (15). We consider that Witebsky’s postulates normal ejaculated spermatozoa, reduction of sperm motility was can be readily applied in this particular group of patients, arguing for evident, suggesting that humoral mediator(s) present in seminal an autoimmune etiology of their disease: they have cellular autoim- plasma from autoimmune animals was/were responsible for the mune response against prostate Ags; and the disease can be repro- observed effects. ROS may be involved in the effects observed, and duced in rodents. Indeed, our animal model of autoimmune prostatitis indeed, high levels of NO were detected in the seminal plasma of shows almost all the previously mentioned characteristics of human autoimmune rats. Other putative candidates to produce these ef- chronic autoimmune prostatitis: specific lymphocytes against prostate fects are TNF-␣ and IFN-␥; certainly, seminal plasma from auto- Ags; the presence of Th1 cytokines in the lymphocyte supernatants; immune animals contained high levels of these cytokines. When and the type of infiltration and histological lesions seen in the gland rTNF-␣ or rIFN-␥ were added to normal seminal plasma and then (observed in both the animal models and the autoimmune prostatitis to normal sperm cells, they were able to mirror the observed ef- patients; Refs. 8, 18, and 31). In this work we demonstrate that many fects, diminishing motility and enhancing apoptosis. In concor- of the abnormalities seen in the semen of chronic autoimmune pros- dance, their effects could be blocked, at least in part, by neutral- tatitis patients are also reproduced in the animal model: markers of izing Abs. However, the decrease in sperm quality observed after local inflammation (increased proinflammatory cytokines in seminal recombinant cytokine addition (at doses measured in seminal plasma); reduction of biochemical markers of prostate gland; and plasma from EAP animals) does not reach the levels observed marked alterations of sperm quality, abnormalities detected in both

when total EAP seminal plasma was added. When both cytokines Downloaded from the animal model and in this subset of CNBP patients that evidenced were put together simultaneously, at doses measured in seminal an autoimmune response to prostate gland. plasma from EAP animals, neither additive nor synergic effects were The use of our animal model of autoimmune prostatitis also observed compared with the single addition (data not shown). There- allowed us to address questions otherwise difficult to answer in the fore, TNF-␣, IFN-␥, NO, and probably other proinflammatory medi- human patients. Among them, it was important to determine when, ators play a role in the induction of the damage described. where, and how sperm cells from autoimmune prostatitis individ- ␣ ␣

TNF- exerts its effects via two TNF- -specific membrane- http://www.jimmunol.org/ uals were being damaged. Two possibilities were evaluated: either bound receptors, TNFR1 and TNFR2, which are coexpressed in these alterations were a result of an epididymal damage as a con- most tissues (37, 38). TNFR1 activation is associated with the sequence of a satellite effect of the prostate inflammation; or they activation of kinases and NF-␬B and the induction of apoptosis were the result of a rapid damage originated during the joining of (38). Spermatozoa may be exposed to abnormal levels of TNF-␣ in sperm cells with the prostate fluids at the moment of ejaculation. the male reproductive tract or during their passage into the female Our findings demonstrate that only ejaculated spermatozoa are be- reproductive tract in pathological conditions (39, 40). Some re- ing damaged and that apoptosis is involved. searchers have demonstrated that exposing human spermatozoa to Numerous studies have now shown the presence of nuclear pathological concentrations of TNF-␣ and IFN-␥ can result in a DNA strand breaks and apoptosis in human ejaculated spermato- significant loss of their functional and genomic integrity (41). Ap- by guest on October 2, 2021 zoa from infertile patients and the abnormal persistence of apo- optosis may be a potential mechanism for the occurrence of TNF-␣ ptotic marker proteins (32). Why human spermatozoa possess toxic effects. Upon engagement with its receptor, TNF-␣ activates these abnormalities is still not clear (33). Two processes that have ␣ been linked to the presence of DNA strand breaks in spermatozoa the TNF- receptor-associated factor, TNFR-associated death do- are anomalies in apoptosis during spermatogenesis or abnormali- main, and receptor-interacting protein kinase 1. In turn, caspase-2 ties after spermatogenesis. and caspase-8 become activated with the end result of effector Many recent studies indicate that oxygen-derived free radicals caspase-3 activation followed by cell death (42). Infliximab, a hu- ␣ induce apoptosis to spermatozoa. The excessive generation of manized mAb that blocks specifically TNF- , reverses the toxic ␣ these reactive oxygen species (ROS; superoxide, hydroxyl, NO, effects induced by TNF- in human spermatozoa (43). These toxic ␣ peroxide, peroxynitrile) by spermatozoa and by contaminating leu- effects that TNF- exerts on spermatozoa could possibly be me- ␣ kocytes associated with genitourinary tract inflammation has been diated by ROS because TNF- has the potential to stimulate sper- identified in idiopathic male infertility (34). Henkel et al. (35) re- matozoa to generate ROS. In turn, ROS-related sperm membrane ported that DNA fragmentation was strongly correlated with ROS peroxidation may occur (44). production. However, oxidative stress is the result of elevated It has been reported that sperm cells from human, rabbit, and pig ␥ ROS, depressed total antioxidant capacity, or both. Mammalian species express IFN- receptors, which seem to develop during spermatozoan membranes are rich in polyunsaturated fatty acids, spermatogenesis in the testes (45). Moreover, signaling members making them very susceptible to oxygen-induced damage, which is of the IFN-␥ pathway like the JAK/STAT proteins, TYK 2, mediated by lipid peroxidation. In a normal situation, the antiox- STAT1, and STAT4, are present and active in human sperm (46). idant mechanisms present in the reproductive tissues and their se- Dimitrov et al. (47) studied the effects of recombinant cytokines on cretions are likely to quench these ROS and protect against oxi- spontaneous and ionophore-induced acrosome reaction and dem- dative damage to gonadal cells and mature spermatozoa. The onstrated that spermatozoa that underwent capacitation in medium prostate gland contribute with its secretion to the antioxidant ca- with rIFN-␥ showed a significant increase in spontaneous and in- pacity present in semen (34), and significant oxidative stress in the duced acrosome reaction compared with the control. They postu- semen or prostatic fluid from patients with chronic noninfectious lated that some cases of infertility might result from a defective prostatitis has been reported, comparable with levels reported in acrosome reaction caused by products of activated lymphocytes men with recognized infertility (36). In concordance with these pre- and macrophages that are released into the male and female re- vious reports, we evidenced elevated levels of NO in semen from productive tracts. Indeed, increased levels of IFN-␥ have been re- animals with autoimmune prostatitis. This finding clearly shows that ported in seminal plasma of infertile men and CNBP patients (8, spermatozoa from these animals are exposed to an oxidative stress 15, 48). In the present work, we demonstrate the detrimental effect that could be a cause of the many alterations observed in semen. of IFN-␥ in sperm motility and viability, evidencing its capability The Journal of Immunology 967 of inducing sperm apoptosis. Further work is needed to elucidate 19. Maccioni, M., V. E. Rivero, and C. M. Riera. 1998. Prostatein (or rat prostatic the mechanisms involved in these phenomena. steroid binding protein) is a major autoantigen in experimental autoimmune pros- tatitis. Clin. Exp. Immunol. 112: 159–165. Thus, sperm damage in experimental autoimmune prostatitis can 20. Rivero, V., C. Carnaud, and C. M. Riera. 2002. Prostatein or steroid binding be the consequence of several causes: an inflammatory milieu, protein (PSBP) induces experimental autoimmuneprostatitis (EAP) in NOD mice. Clin. Immunol. 105: 176–184. originally produced by an autoimmune response in the prostate 21. Chen, C., K. Schilling, R. A. Hiipakka, I. Y. Huang, and S. Liao. 1982. Prostate gland, characterized by an enhancement of ROS levels and proin- ␣ protein: isolation and characterization of the polypeptide components and cho- flammatory cytokines in seminal plasma; and a diminished anti- lesterol binding. J. Biol. Chem. 257: 116–121. 22. Ponce, A. A., R. E. Carrascosa, V. A. Aires, M. Fiol de Cuneo, R. D. Ruiz, oxidant capacity caused by an impairment of prostate function; or M. F. Ponzio, and J. L. Lacuara. 1998. Activity of Chinchilla laniger spermato- both mechanisms simultaneously. zoa collected by electroejaculation and cryopreserved. Theriogenology 50: Our results argue in favor of the importance of prostate function- 1239–1249. 23. World Health Organization. 1999. WHO Laboratory Manual for the Examination ality and suggest that besides the clinical manifestations in chronic of Human Semen and Sperm-Cervical Mucus Interaction, 4th Ed. Cambridge prostatitis patients (pelvic pain, irritative voiding symptoms, effects on Univ. Press, Cambridge, U.K. 24. Jeyendran, R. S., W. J. Holmgren, P. Bielfeld, and A. C. Wentz. 1992. Fertilizing sexual function), a group of them who have an autoimmune response capacity of various populations of spermatozoa within an ejaculate. J. Assist. against prostate could also have diminished fertility. Reprod. Genet. 9: 32–35. Utilization of animal models of autoimmune prostatitis will pro- 25. Marı´n, C. I., J. C. Calamera, and R. A. Rovasio. 1995. Development of an ob- jective, semiautomated method for assessment of sperm motility. Andrologia 27: vide important approaches, which may help to identify the patho- 115–119. genic mechanisms and also possible therapies for human disease. 26. Rovasio, R. A., L. C. Giojalas, and C. I. Marı´n. 1994. Three seconds is an efficient tracking time to distinguish different mouse sperm motility patterns. Commun. Biol. 12: 313–320. Acknowledgments 27. Witebsky, E., N. R. Rose, K. Terplan, J. R. Paine, and R. W. Egan. 1957. Chronic Downloaded from We thank Paula Icely for technical assistance and Drs. Laura Giojalas and thyroiditis and autoimmunization. JAMA. 164: 1439–1447. 28. Rose, N. R., and C. Bona. 1993. Defining criteria for autoimmune diseases Hector Guidobaldi from Centro de Biologı´a Molecular y Celular, Facultad (Witebsky’s postulates revisited). Immunol. Today 14: 426–430. de Ciencias Exactas, Fı´sicas y Naturales, Universidad Nacional de Co´r- 29. Furukawa, F., and T. Yoshimasu. 2005. Animal models of spontaneous and drug- doba, for assistance in the sperm motility experiments. induced cutaneous lupus erythematosus. Autoimmun. Rev. 4: 345–350. 30. Ponniah, S., I. Arah, and R. B. Alexander. 2000. 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