Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print May 11, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.041079
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/4/688    most recent
biolreprod.105.041079v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Knee, R. A.
Right arrow Articles by Jones, R. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Knee, R. A.
Right arrow Articles by Jones, R. C.
Agricola
Right arrow Articles by Knee, R. A.
Right arrow Articles by Jones, R. C.
BIOLOGY OF REPRODUCTION 73, 688–694 (2005)
DOI: 10.1095/biolreprod.105.041079
© 2005 by the Society for the Study of Reproduction, Inc.

Transport of IgG across the Blood-Luminal Barrier of the Male Reproductive Tract of the Rat and the Effect of Estradiol Administration on Reabsorption of Fluid and IgG by the Epididymal Ducts1

Rebecca A. Knee 3, Danica K. Hickey 4, Kenneth W. Beagley 4, and Russell C. Jones 2, 3

Discipline of Biological Sciences,3 Faculty of Science and Information Technology, Discipline of Immunology and Microbiology,4 Faculty of Health, University of Newcastle, Callaghan, New South Wales, Australia 2308


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In rats immunized systemically with tetanus toxoid the concentration of specific anti-tetanus-toxoid-specific IgG in fluid from the rete testis and cauda epididymidis were respectively 0.6% and 1.4% the concentration in blood serum. The extratesticular duct system reabsorbed 97% of the IgG and 99% of the fluid leaving the rete, but estradiol administration affected the site of reabsorption. In untreated rats, the ductuli efferentes reabsorbed 94% of the IgG and 96% of the fluid leaving the rete, whereas estradiol-treated rats reabsorbed 83% of the IgG and 86% of the fluid, and the ductus epididymidis fully compensated for these different effects of estradiol on the ductuli efferentes. The concentrations of IgG in secretions of the seminal vesicles and prostate gland were lower (0.1% and 0.3% respectively of the titers in blood serum) than in fluids from the extratesticular ducts, and were not affected by the administration of estradiol. RT-PCR showed that Fcgrt (neonatal Fc receptor, also known as FcRn) is expressed in the reproductive ducts, where IgG is probably transported across epithelium, being particularly strong in the ductuli efferentes (where most IgG was reabsorbed) and distal caput epididymidis. It is concluded that IgG enters the rete testis and is concentrated only 2.5-fold along the extratesticular duct system, unlike spermatozoa, which are concentrated 95-fold. Further, the ductus epididymidis can recognize and compensate for changes in function of the ductuli efferentes.

ductuli efferentes, epididymis, estradiol, immunology, male reproductive tract, Wolffian duct


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Current interests in male immunocontraception and controlling sexually transmitted diseases require an understanding of the potential to deliver antibody to the male reproductive tract. However, there is some doubt whether enough antibodies can be delivered to the tract to effect an immune response sufficient to control conception or an infectious disease. For example, although there are reports of immunocontraception against sperm and epididymal antigens in males [1–4], there is only one report describing contraception in 100% of animals, and that was probably attributable to the immunization's causing tissue inflammation [5].

Although it is well established that there are barriers to the movement of solute between blood and the lumen of the seminiferous tubules [6–8], there is evidence that IgG antibodies can enter the rete testis [9, 10]. It has also been shown that IgG can enter from blood into the interductular compartment of the epididymis [11]. However, there are structural barriers to its transport into the lumen [12], and it is not resolved whether, or how much, IgG can enter the ductuli efferentes or ductus epididymidis through the duct mucosa [11, 13–15], or whether fluid reabsorption by the ducts will concentrate antibodies that enter the rete testis [16]. IgG has been detected in fluid from the distal end of the ductus epididymidis [17], and the accessory glands probably contribute some IgG to semen [18].

This report describes a study of rats immunized with a nonreproductive antigen, tetanus toxoid, which was used because it is highly immunogenic and its antibodies would not interact with reproductive antigens. Comparisons are made within rats of the specific IgG in fluids sampled from the lumen of the rete testis, proximal ductus epididymidis, distal ductus epididymidis, seminal vesicles, and prostate glands. The sampling sites were chosen in order to distinguish between the functions the of major units of the reproductive system: the ductuli efferentes, ductus epididymidis, and the main accessory glands of reproduction. Determinations of the total protein content in the luminal fluids from the extratesticular ducts are included in this report for direct comparison with the concentration of specific IgG, and because in earlier reports on the rat there is confounding between the roles of the ductuli efferentes and the proximal ductus epididymidis in determining the concentration of intraluminal protein [19, 20]. This study confirms that IgG enters the rete testis [9, 10], and shows that most is reabsorbed mainly by the ductuli efferentes, although some is concentrated in the lumen as a consequence of fluid reabsorption by the ducts. The effect of administering estradiol on the concentration of specific IgG in the extratesticular ducts was examined, because the treatment is known to perturb fluid reabsorption in the ductuli efferentes [21– 23] and so may affect the concentration of IgG in the duct system. Importantly, this work showed that the ductus epididymidis must recognize and compensate for a reduced function of the ductuli. Expression of the neonatal Fc receptor (Fcgrt, also known as FcRn) in the reproductive system was also examined in this study because it is recognized to have a role in IgG-mediated immune surveillance in adult epithelium [24–33] and so may provide the mechanism for transporting IgG across the reproductive epithelium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The studies were carried out on outbred male Wistar rats (5 to 10 mo old and weighing 400–500 g, supplied by The Animal Facility, University of Newcastle) with the approval of the Institute's Animal Ethics Committee, and using methods of anesthesia and surgery that were described previously [16]. Rats were killed by an overdose of anesthetic or by carbon dioxide asphyxiation.

Immunization

Rats were immunized subcutaneously. The primary immunization was with 40 µg of tetanus toxoid (a gift from CSL, Melbourne, Australia) in Freund's complete adjuvant (Sigma Chemical Co., St. Louis, MO), and this was followed 4 and 6 wk later by booster immunizations with 25 µg tetanus toxoid in Freund's incomplete adjuvant. Nonimmunized rats received the adjuvants containing no antigen. Antibody titers of IgG in systemic blood were determined by ELISA to confirm that high titers were achieved in the immunized animals before sampling reproductive fluids.

Administration of Estradiol

For 7 days before collecton of the reproductive fluids, the rats received daily subcutaneous injections of peanut oil (Meadow Lea Foods Ltd., NSW, Australia) containing 12.5% v/v benzyl benzoate (Ajax Chemical Co. Ltd., NSW, Australia) and 0 or 400 mg of 17ß-estradiol 3-benzoate.

Collection of Reproductive Fluids

Collection of luminal fluids from the extratesticular ducts has been described [16]. Samples were collected from the rete testis by micropuncture, fluid leaving the ductuli efferentes was collected into a microcannula in the proximal ductus epididymidis (proximal zone 1a [34]), and fluid from the distal cauda epididymidis was collected by retrograde flushing the duct with water saturated oil. Samples were transferred to 10 µl microcaps (Drummond Scientific Co., Broomall, PA) and centrifuged at 10 000 x g (5°C; 15 min for samples leaving the testis and proximal ductus epididymidis and 30 min for fluid from the cauda epididymidis) to separate sperm from epididymal plasma. The concentration of sperm in the samples was determined using a hemocytometer [16]. Prostatic fluid was collected from the penis during direct electrical stimulation of the ventral prostate glands (Harvard Universal kymograph; Harvard Apparatus IMS Ltd., Edenbridge, Kent, United Kingdom). The glands were freed from supporting tissue and electrodes held on either side. The stimulus was applied at a pulse width of 1 ms and frequency of 30 to 60 cycles s–1. The voltage was increased gradually from 0 to 12 volts until a sample was provided. The sample was collected from the penis into a hematocrit tube. Initially, a small amount of fluid was delivered slowly, then fluid was delivered faster (like an ejaculation) to make up a volume of 5–10 µl. The fluid was slightly opaque and viscous, and contained no contamination from other body fluids. Samples of secretions from the seminal vesicles were collected by micropuncturing the large ducts where the secretions accumulated. The secretions from the accessory glands were centrifuged (15 min at 10 000 x g at 5°C) to remove any particulate matter that may have been present. Samples of the reproductive fluids were diluted in 4 mg ml–1 phenylmethylsulfonyl fluoride in borate-buffered saline (BBS: 25 mM sodium borate and 75 mM sodium chloride, pH 8.3) and frozen to –20°C until assays were performed.

Determination of Protein and Tetanus-Toxoid-Specific IgG in Reproductive Fluids

Protein was determined using Coomassie blue [35]. Anti-tetanus-toxoid-specific IgG was determined by ELISPOT assays [36] using a Bio-dot Microfiltration apparatus (Bio-Rad Laboratories Pty. Ptd., Sydney, Australia) to support a rehydrated nitrocellulose membrane (Hypobond C; Bio-Rad). Sample wells were blotted with tetanus toxoid (0.25 mg ml–1) in BBS, and control wells were coated with BBS only. The membrane was incubated overnight at 4°C and then washed (7-times) with Tris-buffered saline (TBS, pH 7.5). The membrane was blocked with 1% (w/v) BSA (BSA-TBS) and incubated for 2.5 h at 37°C, washed with TBS-Tween (TBS containing 0.05% v v–1Tween-20: Sigma-Aldrich, St Louis. MO). Samples were diluted 2-fold in BSA-TBS-Tween, applied to the membrane in duplicate, and incubated for 2 h at 37°C. Each membrane contained samples of standard serum (pooled serum collected from animals with a high titer of tetanus-toxoid-specific IgG) and reproductive fluid from unimmunized animals as well as the samples to be assayed from the immunized animals. The wells were washed with TBS-Tween, loaded with biotinylated goat anti-rat IgG (0.5 mg ml–1 diluted 1:3000 in 1% BSA; Southern Biotechnology Associates Inc., Birmingham, AL) and incubated overnight at 4°C. After washing with TBS-Tween they were incubated in strepavidin-horseradish peroxidase (Amersham, Sydney, Australia) diluted 1:3000 in BSA-TBS-Tween for 2 h at 37°C and washed in TBS-Tween. The membrane was washed in TBS and the color reaction developed for 5 min in a solution containing 10 ml 0.06% w/v diaminobenzadine (Sigma-Aldrich Pty. Ltd., Castle Hill, Australia) in TBS, 1 ml of 3% hydrogen peroxide and 0.1 ml of 3% w/v nickel chloride metal enhancer solution. The reactants were washed away with deionized water and the membrane dried. Volume densities of each immunospot were determined using the analytical software package Multianalyst and a GM 700 Densitometer (Bio-Rad). ELISA units (EU) were assigned to the dilution series of the standard serum (see above) starting with 1000 Elisa Units and decreasing by half for each 2-fold dilution to produce an exponential standard curve for the plot of volume density against EU ml–1.

Determination of Fcgrt mRNA by RT-PCR

Reproductive tissues were removed from animals immediately following euthanasia. Samples from the following regions were extracted for PCR, using the reagents and procedures in a commercial kit (Invitrogen, Mulgrave, Victoria, Australia): testis and ductuli efferentes; Zones 1a (initial segment), 2 (distal caput epididymidis), and 6 (cauda epididymidis) of the epididymis [34]; vas deferens; seminal vesicles; and ventral prostate glands. mRNA was extracted by homogenizing (Tissue Tearor; Biospec Products, Bartlesville, OK) at 4°C in 1 ml TRIzol Reagent (Invitrogen). The resulting pellets were resuspended in 100 µl of diethyl pyrocarbonate (DEPC) water and the concentration of mRNA determined from 260/280 nm absorbances using a Biospec Mini Spectrophotometer (Shimadzu, Rydalmere, NSW, Australia).

For reverse transcription, mRNA (200 µg mRNA in 5 µl of DEPC water) was added to 11 µl DEPC water, heated to 65°C for 5 min, then quenched on ice. A mix of 9 µl of the following (from Invitrogen) was added to the mRNA: 1 µl of 10 µM dNTP, 5µl of 5x Reverse Transcriptase buffer, 2 µl of 0.5 mg/ml Random Primers, 0.5µl of 40 U/µl RNase Out, and 0.5 µl of 200 U/µl M-MLV Reverse Transcriptase. The mixture was incubated (37°C for 60 min, then 95°C for 5 min), quenched on ice, then amplified by real-time PCR. The quality of the cDNA was verified before real-time PCR by amplifying the cDNA by conventional PCR using beta-actin primers and separating the product by electrophoresis on a 2% agarose gel.

Real-time PCR reactions were set up in PCR tubes with a final reaction volume of 25 µl using flanking nucleotide primers and Sybergreen with ROX reference dye (Invitrogen). Primer sequences were designed using Primer Express 2.0 software (PE Applied Biosystems Inc., Foster City, CA). The sequences for rat beta-actin primers were: forward 5'CGC CGT TCC GAA ATT GC 3', reverse 5' GCC GCC GGG TTT TAT AGG 3' (Geneworks Pty Ltd, Hindmarsh, SA, Australia). The rat Fcgrt primers were: forward 5' CGC CCG CCA GAT CTT CT 3', reverse 5'CCA CTA CCA CCA GCA ATA AAC CA 3' (Geneworks Pty. Ltd., Hindmarsh, SA, Australia). The relative increase in reporter fluorescent dye emission was monitored in real time during PCR amplification using the sequence detection system and software (ABI PRISM 7000 sequence detection system and software; PE Applied Biosystems). Each 25 µl reaction volume contained 2 µl of cDNA, 0.5 µl of sense and antisense primers, 0.25 µl of 100X Sybergreen dye, and 12.5 µl of Platinum qPCR supermix-UDG containing ROX reference dye (Invitrogen). Beta-actin RNA was used as an internal standard to control for variability in amplification because of differences in initial RNA concentrations. The level of the Fcgrt RNA relative to beta-actin RNA was calculated using the following formula: relative RNA expression = 2–(Ct of FcRn – Ct of beta-actin) x 1010, where Ct is on the threshold cycle value [37]. Because the level of Fcgrt RNA was considerably lower than the level of beta-actin RNA, all values were arbitrarily multiplied by 1010 [37].

Calculation of Parameters

Daily sperm production (DSP; sperm day–1) was calculated as:

where F1a = flow rate into the cannula that collects fluid leaving the ductuli efferentes (µl h–1) and [sperm] = concentration of sperm in the cannula that collects fluid leaving the ductuli efferentes (sperm µl–1).

Fluid output from the testis (TFO; µl h–1) was calculated as

where [sperm]RT = sperm concentration in RTF (sperm µl–1).

The net rate of fluid reabsorbed by the ductuli efferentes (RRT-1a; µl h–1) was calculated as

The percentage of fluid entering the ductuli efferentes that was reabsorbed was calculated from the ratio of RRT-1a and TFO.

The percentage of fluid reabsorbed between the efferent ducts and cauda epididymidis (% R1a-CE) was calculated as

where [sperm]CE = sperm concentration in fluid from the distal cauda epididymidis.

The rate of flow of fluid at the distal cauda epididymidis (FCE; µl h–1) was estimated as

The flow of solute at a site (SF; units h–1) was calculated from the flow of fluid at the site (F) and solute concentration ([S]), as:

The net amount of solute reabsorbed between two sites was estimated as:

where SFprox and SFdist refer to solute flow at the proximal and distal sites respectively.

Experimental Design and Analyses

Comparisons between samples of the reproductive fluids were made within animals. Differences between means were determined by analyses of variance after transforming absolute values to logs and proportions to angles. The variance between treatments and sampling sites was partitioned into individual degrees of freedom using polynomial coefficients. The standard errors shown in the figures and tables are from the variance between animals calculated from untransformed data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Testicular Output and Fluid Reabsorption

Figure 1 shows that administration of estradiol had no effect on mean testis mass, but reduced the mass of the seminal vesicles. It also reduced estimates of daily sperm production (from 28.33 ± 3.28 x 106 sperm day–1 testis–1 for untreated animals to 18.34 ± 4.54 x 106 sperm day–1 testis–1 for the treated group; P < 0.05; n = 9). Table 1 shows that the mean concentration of sperm was about the same in samples of fluid from the rete testis of untreated and treated rats. The concentration of sperm increased along the extratesticular duct system because of net reabsorption of 99% of the fluid leaving the testis. However, the reabsorption occurred in different parts of the duct system of untreated and estradiol-treated rats. Whereas the efferent ducts reabsorbed 96% of the fluid leaving the testis of untreated rats, they only reabsorbed 86% in estradiol-treated rats (P < 0.001). This resulted in the rate of flow of fluid into the cannula in the proximal ductus epididyidis of estradiol-treated rats being more than 1.8-fold higher than that of untreated rats. However, there was compensation by the ductus epididymidis for the reduced reabsorption by the ductuli efferentes of estradiol-treated rats. The ductus reabsorbed more fluid in estradiol-treated (90%) than in untreated rats (66%, P < 0.001) resulting in the mean concentration of sperm in the distal ductus epididymidis being much the same for both groups.



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 1. The effect of treating rats with 17ß-estradiol 3-benzoate for 7 days on the mass of the testes and seminal vesicles. Unshaded columns are untreated controls and shaded columns are estradiol-treated animals. Means ± SEM from nine rats. *Significantly different from untreated rats (P < 0.05)


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effects of estradiol on the concentration of sperm in luminal fluids from the rete testis and proximal and distal ductus epididymidis (DE), flow of fluid into each site, and reabsorption of fluid between sites.a

Secretion and Reabsorption of Protein

Table 2 shows that the mean concentrations of protein in fluid leaving the testis of untreated and treated rats was only 1.4% and 1.6% of the concentrations in blood (50.4 ± 7.0 µg µl–1 and 63.0 ± 2.8 µg µl–1 respectively). The concentration increased more than 2.5-fold in the efferent ducts (P < 0.001) even though there was a net reabsorption of 68–78% of the protein entering the ductuli. Although there was not a significant difference in the rates of protein reabsorption in the untreated and estradiol-treated groups, the concentration of protein in the proximal epididymis was greater in the former (P < 0.05). This is probably because more fluid was reabsorbed in the efferent ducts of this group. Protein was secreted into the ductus epididymidis of both untreated and estradiol-treated rats (P < 0.01).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Effects of estradiol on the concentration of protein in samples of luminal fluid from the rete testis and proximal and distal ductus epi didymidis (DE), and estimates of protein reabsorption between sites.a

Anti-Tetanus-Toxoid-Specific IgG in Reproductive Fluids

The mean titers of anti-tetanus-toxoid-specific IgG in rete testis fluid were much the same for untreated and estradiol-treated rats (Table 3) and only 0.6% and 0.9% of the titers in blood (17 639 x 105 ± 7 260 x 105 EU ml–1 and 11 319 x 105 ± 1372 x 105 EU ml–1 respectively). Also, there was no significant effect of estradiol treatment on titers of IgG in luminal fluids from the proximal and distal ductus epididymidis. Because of an increase in titers of about 1.5-fold in the ductuli efferentes and 1.5-fold in the ductus epididymidis, the titers of IgG in the distal ductus epididymidis were 1.4% and 2.0% of the titers in blood serum for the untreated and treated rats respectively. The difference in rates of flow of IgG into and net reabsorption from the ductus epididymidis in treated compared with untreated rats (Table 3) reflects the effect of treatment on the rate of fluid reabsorption (described above) by the efferent ducts and ductus epididymidis, and differences in the fluid output from the testis (which did not reach statistical significance).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Effect of treating rats with oestradiol for 7 days on the concentration of anti-tetanus toxoid specific IgG and its reabsorption from the reproductive ducts.a

There was no significant effect of the estradiol treatment on the titers of tetanus-toxoid-specific IgG in fluids collected from the prostate glands or seminal vesicles of rats. The mean titers were much the same for fluid from each type of gland (Table 4), and significantly lower (P < 0.05) than the titers of samples from the rete testis and ductus epididymidis (Table 3).


View this table:
[in this window]
[in a new window]
  		TABLE 4. Concentration of anti-tetanus toxoid specific IgG in serum and fluids secreted from the prostate gland and seminal vesicles of rats.a

Fcgrt mRNA in Reproductive Tract

Figure 2 shows that there was little expression of Fcgrt in the testis and low expression in the accessory glands of reproduction. In the extratesticular ducts there was strong expression in the ductuli efferentes, but the expression varied in the derivatives of the mesonephric duct. In the ductus epididymidis, there was no expression in Zone 1a (initial segment proper), strong expression in Zone 2 (caput epididymidis) and no expression in Zone 6 (cauda epididymidis). There was only low expression in the vas deferens.



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 2. Expression of rat Fcgrt mRNA relative to beta-actin in tissue samples from the male reproductive tract of the rat. Means ± SEM from two rats. The control samples were water and the mean for these was used to correct other values. IS, Zone 2, and CE are respectively zones 1a (initial segment), 2 (distal caput), and 6 (cauda) of the epididymis [34]. DE, Ductuli efferentes; VD, vas deferens; SV, seminal vesicles


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The considerable variation in fluid output from the testes of untreated rats in this study was caused by high flow rates in two rats. We have noted similar responses in other studies, including on mice, wallabies, and elephants (R.C. Jones, unpublished results) and the Japanese quail [38]. The effect is not because of the methods used in this study; for example, the connection between the efferent ducts and testis was severed in studies on elephants [39], and the flow of "rete testis fluid" varied from barely detectable to a spurt, initially reaching about 30 mm high then subsiding within a minute. It is unlikely that the effect would be detected when changes in testis mass are determined following efferent duct ligation [7, 40, 41] because those studies assess changes over longer periods than studies involving cannulation or severing the efferent ducts from the testis. In our experience, changes in flow rate are not associated with changes in the concentration of spermatozoa. Further, exclusion of the two rats from the statistical analyses in the present study did not affect the interpretations described above.

The finding in this study that the administration of estradiol reduces fluid reabsorption by the ductuli efferentes is consistent with our earlier work [21]. The effect is dramatic, approximately doubling the flow of fluid into the cannula in the proximal ductus epididymidis. The effect is also consistent among animals. The finding is not obviously consistent with work on the Esr1 knockout mouse [22] or the effects of an ESR1 antiestrogen in the mouse [42] or rat [43]. However, the methodology of the studies is quite different, and the response to estradiol administration that is described in our work may be because systemic estrogen can reduce expression of Esr1 [43], or its effect may be via Esr2 and not Esr1 [43], or via an effect on androgen regulation (because the estradiol treatment did reduce daily sperm production and the mass to the seminal vesicles: see Results and [21]).

A major finding of this study is that the ductuli efferentes and ductus epididymidis work in tandem in regulating the concentration of sperm in the lumen of the extratesticular ducts. This study confirmed that the ductuli reabsorb 96% of the fluid leaving the testis of untreated rats [16]. However, it also showed that when this reabsorption was reduced by estradiol treatment, the ductus epididymidis fully compensated for the increased flow that it received by increasing fluid reabsorption and delivering sperm to the distal ductus epididymidis at the same concentration as in untreated rats.

Our finding that estrogen treatment does not affect the concentration of sperm in the distal cauda epididymidis does not directly disagree with earlier work on the rat [44] and mouse [45] that showed that estrogen administration increased the rate of sperm transport through the epididymis. That work used a much lower dose of estrogen than the present study, a dose that did not affect daily sperm production. Also, the earlier studies determined the number of sperm in homogenates and not the concentration of sperm in the lumen, so they do not provide direct evidence that the estrogen treatment affected the concentration of sperm in the lumen of the cauda epididymidis. Consequently, the increased rate of sperm transport through the duct system that was observed by others could be a result of estrogen acting on the musculature of the ductus epididymidis and not be an effect of reducing fluid reabsorption by the ductuli efferentes.

This is the first report to examine how the extratesticular duct system handles intraluminal IgG and to relate this to secretions of the accessory glands of reproduction. The report is in agreement with earlier work showing that IgG can enter the rete testis, but only to achieve luminal titers that are less than 1% in blood serum [10, 46, 47]. By showing that enough IgG enters the rete testis to account for the concentrations in more distal regions of the duct system, this report is in agreement with work showing that there is no significant proluminal transport of IgG into the lumen of the ductus epididymidis [13, 48], but in disagreement with an interpretation [17] that "IgG is secreted in the epididymis and does not come from the testis." This report also shows that most IgG entering the rete testis must be reabsorbed along the duct system. This may protect against autoimmunity, because spermatogenesis commences well after immunological tolerance to self has been established [49]. The work is significant in demonstrating that there are physiological mechanisms as well as structural barriers regulating autoimmunity [8, 12]. The demonstration that Fcgrt is expressed in the extratesticular ducts is consistent with the findings of reabsorption of specific IgG mainly by the ductuli efferentes, but also by the ductus epididymidis. It is interesting to note that Fcgrt is not expressed equally along the ductus epididymidis, but this could be because the ductus is regionally specialized for functions other than the transport of IgG.

It is considered that our determinations of IgG in the reproductive fluids of the rat are representative of normal physiological conditions. There was no evidence that the use of Freund's adjuvant caused experimental autoimmune orchitis, which it does in some animals [50, 51], and which is the probable cause of infertility in guinea pigs following immunization with rPH-20 in Freund's complete adjuvant [5]. Also, the level of specific IgG in rete testis fluid is in agreement with values, relative to blood, of total IgG in rete testis fluid from animals receiving no experimental immunization [10].

The transport and maintenance of some IgG in the extratesticular ducts indicates that the system may provide some protection against sexually transmitted and other infections by the humoral as well as the cell-mediated arm of the immune system [49, 52]. However, it is doubtful that the levels of IgG that are present in the epididymal fluid and accessory gland secretions are sufficient to achieve immunocontraception in the rat following immunization with a reproductive antigen. Nevertheless, this does not exclude the chance that a vaccine can achieve some reduction in fertility in other species, such as the hamster [53], because there are interspecies differences in response to immunization with reproductive antigens. Indeed, there is a need to examine the entry of other classes of antibody into the lumen of the reproductive ducts. For example, IgA and IgM as well as IgG have been found in human and boar semen [54, 55], and in humans, prostatic fluid contains IgG [56] and the prostate gland secretes IgA [57–60]. It is possible that sufficient IgA may be secreted into accessory gland fluids for this purpose so that it is worthwhile examining mucosal as well as systemic immunization to raise the immune response.


   ACKNOWLEDGMENTS
 
We appreciate discussions with John Clulow during this study.


   FOOTNOTES
 
1 Supported by the World Health Organization (Project 98187), and the CRC for Pest Animal Control, Australia. Back

2 Correspondence. FAX: 61 2 4921 6923; Russell.Jones{at}newcastle.edu.au Back

Received: 21 February 2005.

First decision: 21 March 2005.

Accepted: 28 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ramarao CS, Myles DG, White JM, Primakoff P. Initial evaluation of fertilin as an immunocontraceptive antigen and molecular cloning of the cynomolgus monkey fertilin beta subunit. Molec Reprod Dev 1996 43:70-75
  2. Primakoff P, Woolman-Gamer L, Tung KSK, Myles DA. Mechanism of infertility in male guinea pig immunized with sperm PH-20. Biol Reprod 1997 56:1142-1146[Abstract]
  3. Ellerman DA, Brantua VS, Martinez SP, Cohen DJ, Conesa D, Cuasnicu PS. Potential contraceptive use of epididymal proteins: immunization of male rats with epididymal protein DE inhibits sperm fusion ability. Biol Reprod 1998 59:1029-1103[Abstract/Free Full Text]
  4. Bérubé B, Sullivan R. Inhibition of in vivo fertilization by active immunization of male hamsters against a 26 kDa sperm glycoprotein. Biol Reprod 1994 51:1255-1263[Abstract]
  5. Tung KSK, Primakoff P, Woolman-Gamer L, Myles DG. Mechanism of infertility in male guinea pig immunized with sperm PH-20. Biol Reprod 1997 56:1133-1141[Abstract]
  6. Nicander L. An electron microscopical study of cell contacts in the seminiferous tubules of some mammals. Z Zellforschung 1967 83:375-397[CrossRef]
  7. Setchell BP, Voglmayr JK, Waites GMH. A blood-testis barrier restricting passage from blood into rete testis fluid but not lymph. J Physiol 1969 200:73-85
  8. Dym M, Fawcett DW. The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol Reprod 1970 3:308-326[Abstract]
  9. Koskimies AI, Kormano M, Lahti A. A difference in the immunoglobulin content of seminiferous tubule fluid and rete testis fluid of the rat. J Reprod Fertil 1971 27:463-465
  10. Johnson MH, Setchell BP. Protein and immunoglobulin content of rete testis fluid of rams. J Reprod Fertil 1968 17:403-406
  11. Pöllänen P, Cooper TG, Kokk K, Saari T, Setchell BP. Microvascular permeability to the F(ab')2 fragment of IgG in the male rat reproductive tract at puberty. J Reprod Immunol 1997 32:221-240[CrossRef][Medline]
  12. Suzuki F, Nagano T. Regional differentiation of cell junctions in the excurrent duct epithelium of the rat testis as revealed by freeze-fracture. Anat Rec 1978 191:503-520[CrossRef][Medline]
  13. Wong PY, Tsang AY, Fu WO, Lau HK. Restricted entry of an anti-rat epididymal protein IgG into the rat epididymis. Int J Androl 1983 6:275-282[Medline]
  14. Pöllänen P, Cooper TG. Immunology of the testis excurrent ducts. J Reprod Immunol 1994 26:167-216[CrossRef][Medline]
  15. Cooper TG, Yeung CH, Bergmann M. Protein transport to the epididymal lumen. Cell Tissue Res 1987 248:527-530[CrossRef][Medline]
  16. Clulow J, Jones RC, Hansen LA. Micropuncture and cannulation studies of fluid composition and transport in the ductuli efferentes testis of the rat: comparisons with the homologous metanephric proximal tubule. Exp Physiol 1994 79:915-928[Abstract]
  17. Weininger RB, Fisher S, Rifkin J, Bedford JM. Experimental studies on the passage of specific IgG to the lumen of the rabbit epididymis. J Reprod Fertil 1982 66:251-258
  18. Bronson RA, Cooper GW, Rosenfeld D. Correlation between regional specificity of antisperm antibodies to the spermatozoan surface and complement mediated sperm immobilization. Am J Reprod Immunol 1982 2:162-168
  19. Turner TT. Resorption versus secretion in the rat epididymis. J Reprod Fertil 1984 72:509-514
  20. Wong PYD, Tsang AYF, Lee WM. Origin of the luminal fluid proteins of the rat epididymis. Int J Androl 1981 4:331-341[Medline]
  21. Hansen LA, Clulow J, Jones RC. Perturbation of fluid reabsorption in the ductuli efferentes testis of the rat by testosterone propionate, 17ß-oestradiol 3-benzoate, flutamide and tamoxifen. Int J Androl 1997 20:265-273[CrossRef][Medline]
  22. Hess RA, Bunick D, Lee KH, Bahr J, Taylor JA, Korach KS, Lubahn DB. A role for oestrogens in the male reproductive system. Nature 1997 390:509-512[CrossRef][Medline]
  23. Hess RA. Estrogen in the adult male reproductive tract: a review. Reprod Biol Endocrinol 2003 1:52-66[CrossRef][Medline]
  24. Blumberg RS, Koss T, Story CM, Barisani D, Polischuk J, Lipin A, Pablo L, Green R, Simister NE. A major histocompatibility complex class I-related Fc receptor for IgG on rat hepatocytes. J Clin Invest 1995 95:2397-2402
  25. Dickinson BL, Badizadegan K, Wu Z, Ahouse JC, Zhu X, Simister NE, Blumberg RS, Lencer WI. Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. J Clin Invest 1999 104:903-911[Medline]
  26. Israel EJ, Taylor S, Wu Z, Mizoguchi E, Blumberg RS, Bhan A, Simister NE. Expression of the neonatal Fc receptor, FcRn, on human intestinal epithelial cells. Immunology 1997 92:60-74[CrossRef][Medline]
  27. Leach JL, Sedmak DD, Osborne JM, Rahill B, Lairmore MD, Anderson CL. Isolation from human placenta of the IgG transporter, FcRn, and localization to the syncytiotrophoblast: implications for maternal-fetal antibody transport. J Immunol 1996 157:3317-3322[Abstract]
  28. Spiekermann GM, Finn PW, Ward ES, Dumont J, Dickinson BL, Blumberg RS, Lencer WI. Receptor-mediated immunoglobulin G transport across mucosal barriers in adult life: functional expression of FcRn in the mammalian lung. J Exp Med 2002 196:303-310[Abstract/Free Full Text]
  29. Zhu X, Meng G, Dickinson BL, Li X, Mizoguchi E, Miao L, Wang Y, Robert C, Wu B, Smith PD. MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells. J Immunol 2001 166:3266-3276[Abstract/Free Full Text]
  30. Claypool SM, Dickinson BL, Yoshida M, Lencer WI, Blumberg RS. Functional reconstitution of human FcRn in MDCK cells requires co-expressed human beta 2-microglobin. J Biol Chem 2002 277:28038-28050[Abstract/Free Full Text]
  31. McCarthy KM, Yoong Y, Simister NE. Bidirectional transcytosis of IgG by the rat neonatal Fc receptor expressed in a rat kidney cell line: a system to study protein transport across epithelia. J Cell Sci 2000 113:1277-1285[Abstract]
  32. Praetor A, Ellinger I, Hunziker W. Intracellular traffic of the MHC class I-like IgG Fc receptor, FcRn, expressed in epithelial MDCK cells. J Cell Sci 1999 112:2291-2299[Abstract]
  33. Abrahamson DR, Powers A, Rodewald R. Intestinal absorption of immune complexes by neonatal rats: a route of antigen transfer from mother to young. Science 1979 206:567-569[Abstract/Free Full Text]
  34. Reid BL, Cleland KW. The structure and function of the epididymis. I. The histology of the rat epididymis. Aust J Zool 1957 5:223-246[CrossRef]
  35. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 1976 72:248-254[CrossRef][Medline]
  36. Monroe D. The solid-phase enzyme-linked immunospot assay: current and potential applications. Biotechniques 1985;222–229
  37. Xia D, Sanders A, Shah M, Bickerstaff A, Orosz C. Real-time polymerase chain reaction analysis reveals an evolution of cytokine mRNA production in allograft acceptor mice. Transplantation 2001 72:907-914[CrossRef][Medline]
  38. Clulow J, Jones RC. Composition of luminal fluid secreted by the seminiferous tubules, and after reabsorption by the extratesticular ducts of the Japanese quail, Coturnix coturnix japonica. Biol Reprod 2004 71:1508-1516[Abstract/Free Full Text]
  39. Jones RC. Luminal composition and maturation of spermatozoa in the genital ducts of the African elephant, Loxodonta africana. J Reprod Fertil 1980 60:87-93
  40. Jegou B, Le Gac F, Irby DC, de Kretser DM. Studies on seminiferous tubule fluid production in the adult rat: effect of hypophysectomy and treatment with FSH, LH and testosterone. Int J Androl 1983 6:249-260[Medline]
  41. Smith G. The effects of ligation of the vasa efferentia and vasectomy on testicular function in the adult rat. J Endocrinol 1962 23:285-299
  42. Cho HW, Nie R, Carnes K, Zhou Q, Sharief NA, Hess RA. The antiestrogen ICI 182,780 induces early effects on the adult male mouse reproductive tract and long-term decreased fertility without testicular atrophy. Reprod Biol Endocrinol 2003 1:57[CrossRef][Medline]
  43. Oliveira CA, Nie R, Carnes K, Franca LR, Prins GS, Saunders PT, Hess RA. The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules. Reprod Biol Endocrinol 2003 1:75[CrossRef][Medline]
  44. Goyal HO, Braden TD, Mansour M, Williams CS, Kamaleldin A, Srivastava KK. Diethylstilbestrol-treated adult rats with altered epididymal sperm numbers and sperm motility parameters, but without alterations in sperm production and sperm morphology. Biol Reprod 2001 64:927-934[Abstract/Free Full Text]
  45. Meistrich ML, Hughes TH, Bruce WR. Alteration of epididymal sperm transport and maturation in mice by oestrogen and testosterone. Nature 1975 258:145-147[CrossRef][Medline]
  46. Pomering M, Jones RC, Holland MK, Beagley KW. Restricted entry of IgG into male and female rabbit reproductive ducts following immunization with recombinant rabbit PH-20. Am J Reprod Immunol 2002 47:174-182
  47. Johnson MH. The distribution of imunoglobulin and spermatozoal autoantigen in the genital tract of the male guinea pig: its relationship to autoallergic orchitis. Fertil Steril 1972 23:383-392[Medline]
  48. Yeung CH, Bergman M, Cooper TG. Non-specific uptake of IgG by rat epididymal tubules in vitro. Int J Androl 1991 14:364-373[Medline]
  49. Beagley KW, Wu ZL, Pomering M, Jones RC. Immune responses in the epididymis: implications for immunocontraception. J Reprod Fertil Suppl 1998 53:235-245[Medline]
  50. Teuscher C, Hickey WF, Grafer CM, Tung KS. A common immunoregulatory locus controls susceptibility to actively induced experimental allergic encephalomyelitis and experimental allergic orchitis in BALB/c mice. J Immunol 1998 160:2751-2756[Abstract/Free Full Text]
  51. Pelletier R-M, Nemirovsky MS, Calvert R, Hugon JS. Effects of immunization with Freund's complete adjuvant and isologous spermatozoa on the seminiferous epithelium and blood-testis barrier in the guinea pig. Anat Rec 1981 199:197-211[CrossRef][Medline]
  52. Serre V, Robaire B. Interactions of the immune system and epididymis. In: Robaire B, Hinton BT (eds.), The epididymis from molecules to clinical practice. A comprehensive survey of the efferent ducts, the epididymis and the vas deferens. New York: Kluwer Academic Press/ Plenum Press Publishers; 2002:219–231
  53. Gaudreault C, Montfort L, Sullivan R. Effect of immunization of hamsters against recombinant P26h on fertility rates. Reproduction 2002 123:307-313[Abstract]
  54. Shibahara H, Tsunoda T, Taneichi A, Hirano Y, Ohno A, Takamizawa S, Yamaguchi C, Tsunoda H, Sato I. Diversity of antisperm antibodies bound to sperm surface in male immunological infertility. Am J Reprod Immunol 2002 47:146-150
  55. Kaiser TJ, Christopher-Hennings J, Nelson EA. Measurement of immunoglobulin G, A and M concentrations in boar seminal plasma. Theriogenology 2000 54:1171-1184[CrossRef][Medline]
  56. Rümke P. The origin of immunologlobulins in semen. Clin Exp Immunol 1974 17:287-297[Medline]
  57. Hickey DK, Jones RC, Bao S, Blake AE, Berry LJ, Beagley KW. Intranasal immunization with C. muridarum major outer membrane protein (MOMP) and cholera toxin elicits local production of neutralizing IgA in the prostate. Vaccine 2004 22:4306-4315[CrossRef][Medline]
  58. Parr MB, Parr EL. Immunohistochemical localization of secretory component and immunoglobulin A in the urogenital tract of the male rodent. J Reprod Fertil 1989 85:115-124
  59. Sirigu P, Perra MT, Turno F. Immunohistochemical study of secretory IgA in the human male reproductive tract. Andrologia 1995 27:335-339[Medline]
  60. Stern JE, Gardner S, Quirk D, Wira CR. Secretory immune system of the male reproductive tract: effects of dihydrotestosterone and estradiol on IgA and secretory component levels. J Reprod Immunol 1992 22:73-85[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/4/688    most recent
biolreprod.105.041079v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Knee, R. A.
Right arrow Articles by Jones, R. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Knee, R. A.
Right arrow Articles by Jones, R. C.
Agricola
Right arrow Articles by Knee, R. A.
Right arrow Articles by Jones, R. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS