Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print June 22, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.040238
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/5/872    most recent
biolreprod.105.040238v1
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 Li, Y.
Right arrow Articles by Griswold, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Y.
Right arrow Articles by Griswold, M. D.
Agricola
Right arrow Articles by Li, Y.
Right arrow Articles by Griswold, M. D.
BIOLOGY OF REPRODUCTION 73, 872–880 (2005)
DOI: 10.1095/biolreprod.105.040238
© 2005 by the Society for the Study of Reproduction, Inc.

Immunolocalization and Regulation of Cystatin 12 in Mouse Testis and Epididymis1

Ying Li , Crystal A. Putnam-Lawson , Heather Knapp-Hoch , Patrick J. Friel , Debra Mitchell , Randy Hively , and Michael D. Griswold 2 

Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In previous studies, we identified a new member of the male reproductive tract subgroup within family 2 cystatins, termed cystatin 12 (Cst12, previously known as Cst TE-1 or Cres3). The mouse Cst12 mRNA was primarily localized to the Sertoli cells in the testis and to the epithelial cells of the proximal caput region of the epididymis. In this report, studies were carried out to characterize the cystatin 12 (CST12) protein in mouse testis and epididymis. A recombinant His-CST12 fusion protein was expressed in E. coli and purified to generate an anti-CST12 polyclonal antibody. Western blot analysis showed little or no cross-reaction between the anti-CST12 antibody and several other known male reproductive tract cystatins. Immunohistochemistry revealed that CST12 protein was predominantly localized to the cytoplasm of Sertoli cells in the seminiferous epithelium in a stage-dependent manner. All stages showed high levels of expression except stages VII and VIII, in which very limited expression of CST12 was observed. In the epididymis, CST12 was highly expressed in the cytoplasm of the epithelial cells in the proximal caput and secreted into the lumen. The mouse CST12 protein was also detected in other regions of the epididymis; however, the localization varied greatly along the epididymal tubules. Indirect immunofluorescence showed that CST12 protein was localized to the cytoplasmic droplets in both testicular and epididymal spermatozoa. These observations suggest that CST12 protein may play a specialized role during spermatogenesis and sperm maturation. Northern blot analyses demonstrated that Cst12 transcript levels in the epididymis decreased after castration, and testosterone propionate (T) treatment further repressed the expression of this gene. However, 17-beta estradiol (E) administration maintained the expression of Cst12 mRNA after castration, whereas treatment with both T and E failed to maintain Cst12 mRNA levels in epididymis. These results suggest that androgen and estrogen, probably with other testicular factors, are involved in the regulation of this gene.

epididymis, estradiol, sperm, testis, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cystatin superfamily is composed of structurally related cysteine proteinase inhibitors subdivided into at least three families: stefins (familiy 1), cystatins (family 2), and kininogens (family 3) (for reviews see [1–3]). Family 2 cystatins are small secretory proteins (13–14 kDa) and form the largest family that includes CST3 (also known as CST C), CST S, CST SA, CST SN, CST D [2] and more recently CST6 (also known as CST E/M) and CST7 (also known as CST F) [4, 5]. Male reproductive tract cystatins are recently identified genes that share similarity with the family 2 cystatins but differ in their expression pattern and altered conserved motifs (for a review see [6]). This subgroup contains at least seven genes, including Cst8 (previously known as Cres) [7, 8], Cst9 (previously known as testatin) [9], Cst SC (8030411F24Rik) [10], Cst12 (previously known as Cst TE-1 or Cres3) [10, 11], Cst 11 (also named Cst E1 or Cres2 in mouse [11–13]), Cst E2 (9230104L09Rik) [12] and Cst13 (previously known as Cst T) [14]. Although the sequence similarity between male reproductive tract cystatin proteins (CST) and other known cystatins is low (<30%), male reproductive tract cystatins share the greatest similarity with family 2 cystatins. All the known or predicted proteins possess four highly conserved cysteine residues at the C-terminus and are located on mouse chromosome 2, which shows a linkage conservation with human chromosome 20, where family 2 cystatins have been previously mapped [15]. Each of these cystatin genes (Cst) has identical exon/intron junction locations as in Cst3. These observations indicate that the male reproductive tract cystatins are members of a subgroup within family 2 cystatins.

However, male reproductive tract cystatins lack some or all of the conserved regions, including the N-terminal glycine, a glutamine-valine-glycine (QXVXG) motif in the first hairpin loop, and the proline-tryptophan (PW) motif in the second hairpin loop thought to be critical for binding to a cysteine proteinase of the papain family [16]. This suggests that the specificity of the members in this subgroup may be quite different from the typical family 2 cystatins. Recently, it was reported that, in in vitro assays, recombinant CST8 did not inhibit the cysteine proteinase papain or cathepsin B; instead, it inhibited the serine proteinase prohormone convertase 2 (PC2), a proteinase involved in prohormone processing in the neuroendocrine system [17]. Moreover, unlike the ubiquitous expression pattern of most family 2 cystatins, the members in this subgroup are predominantly expressed in the male reproductive tract and exhibit tissue- and cell-specificity, with some of them exhibiting a stage-specific expression in testis [6]. These data indicate that male reproductive tract cystatins may have evolved from the archetypal cystatins to perform tissue- and cell-specific functions.

The biological function of male reproductive tract cystatins is not understood. However, it has been suggested that CST8 may perform a role in regulation of intra-acrosomal protein processing or may be involved in fertilization and the gonadotrope-mediated control of reproduction [18– 20]. The expression of the Cst9 gene is upregulated in male gonads but downregulated in female gonads immediately after initiation of sex differentiation, suggesting roles in the early developmental cascade of the testis [9, 21]. CST 11, the human ortholog of mouse CST 11 (also referred to as CST E1 or CRES2), was recently shown to exhibit antimicrobial activity [13], suggesting a functional relationship with the defensins and the possibility that subgroup members may be multifunctional.

In previous studies, we identified a member of male reproductive tract cystatins, Cst12 (previously described as Cst TE-1, for cystatin-related gene highly expressed in testis and epididymis) [10]. The deduced amino acid sequence of mouse CST12 lacks all the short conserved motifs thought to be critical for the cysteine proteinase inhibitory activity. Cst12 transcripts are localized to the Sertoli cells in the testis without apparent stage-dependent expression and to the epithelial cells of the proximal caput epididymis, suggesting that CST12 may play a specialized role in the testis and epididymis [10]. In this study, we analyzed CST12 protein in the testis and epididymis and the hormonal regulation of Cst12 mRNA in epididymis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

All mice used in these studies were adult BL/6-129 male mice (4–6 mo old). They were generated and maintained as a breeder colony at the Washington State University (Pullman, WA). Animals were housed under a 12L:12D cycle in a standard animal facility with free access to food and water. All animal procedures were conducted according to the guidelines stated in the United States Public Health Service's Guide for the Care and Use of Laboratory Animals.

Expression of a Recombinant His-CST12 Fusion Protein in Bacteria and Production of Polyclonal Antiserum

A CST12 fusion protein with a 6x histidine-tag (His-CST12) was synthesized using the QIAexpress bacterial expression system from Qiagen (Valencia, CA). Briefly, a polymerase chain reaction (PCR) using forward primer 5'-CGAGATCGACAAGAATGAAGAGGAG-3' and reverse primer 5'-CACTTAAGTCTGCACACAGGTGCTG-3,' was performed to produce a mouse Cst12 cDNA without the predicted signal peptide sequence and the first three amino acid residues. The PCR product was inserted into the pQE-30 vector digested with SmaI to generate a 6x His-CST12 coding sequence. A His-CST12 fusion protein was expressed in E. coli M15 (pREP4) and purified using Ni-NTA affinity chromatography following the manufacturer's recommendation (Qiagen). Sequence of purified recombinant His-CST12 fusion protein was confirmed by amino acid sequencing.

To raise polyclonal antibody, a New Zealand rabbit was injected subcutaneously with 1 mg of purified His-CST12 fusion protein in 1.5 ml Freund complete adjuvant. The rabbit was boosted with 0.5 mg fusion protein in Freund incomplete adjuvant 28 days later. An affinity-purified CST12 polyclonal antibody (mainly IgG), as well as IgG from preimmune serum, was generated by using a protein A Sepharose column (Pierce, Rockford, IL) following the instructions of the manufacturer.

Western Blot Analysis

Purified recombinant His-CST12, His-CST SC, His-CST E2 and His-CST13 protein was separated by 15% SDS-PAGE followed by transfer to PolyVinyliDine Fluoride (PVDF) membrane (0.22 µm). The membrane was blocked in 1x PBS with 5% nonfat milk for 1 h at room temperature and incubated with affinity-purified CST12 polyclonal antibody (1:400) overnight at 4°C. Control blots were incubated with IgG purified from preimmune serum (1:400) or an affinity-purified CST12 antibody that has been previously incubated with the His-CST12 protein. After incubation, the membranes were rinsed three times with 1x PBS and incubated with horseradish peroxidase conjugated goat anti-rabbit IgG for 1 h at room temperature, followed by three rinses with 1x PBS. Crossreactive proteins were visualized by chemiluminescence (ECL; Amersham, Piscataway, NJ). To obtain the protein-loading control, the membrane, after Western blot analysis, was incubated in stripping buffer containing 65 mM Tris.HCl, pH 6.7, 100 mM beta-mercaptoethanol, 2% SDS at 50°C for 1 h, and stained in 40% methanol, 7% acetic acid and 0.025% Coomassie blue R-250 for 2 min and then destained in 40% methanol, 7% acetic acid until the protein bands were resolved.

Immunohistochemistry

Testes and epididymides were removed from 129 3- to 4-mo-old mice and fixed in freshly prepared 4% (w/v) paraformaldehyde (PFA)/0.25% (v/v) glutaraldehyde (GA) overnight at 4°C. The tissues were embedded in paraffin wax and the sections (6 µm) were mounted on Probe-on-Plus microscope slides (Fisher Scientific Co., Pittsburgh, PA). After deparaffinization in xylene, the sections were exposed to 3% hydrogen peroxide in methanol for 10 min to block any endogenous peroxide activity from the tissue. Following rehydration in decreasing concentrations of ethanol, sections were boiled in 0.01 M sodium citrate, pH 6.0, for 12 min and cooled to room temperature slowly to expose the antigenic epitopes on the sections. For immunohistological staining, the Zymed LAB-SA system (Zymed Laboratory Inc., South San Francisco, CA) was used following the protocol the manufacturer provided with modifications. Briefly, 10% goat serum was used as blocking solution and the affinity-purified CST12 polyclonal antibody was diluted to 1:500 in 10% goat serum. Tissue sections and antibody were incubated together for 2 h at room temperature in a humidified chamber. The slides were then rinsed with PBS three times and incubated with diluted biotinylated secondary antibody for 45 min, followed by the application of streptavidin-horseradish peroxidase. The AEC substrate kit (Zymed) was applied until red color developed, and then sections were rinsed with water to stop the chromogen-substrate reaction. The slides were then counterstained with hematoxylin for 45 seconds and mounted with GVA mounting solution (Zymed). Control sections were incubated with IgG purified from preimmune serum (1:500) or an affinity-purified CST12 antibody that was incubated previously with the His-CST12 protein. Sections were observed with a Nikon Microphot-FX (Meridian Instrument Company Inc., Kent, WA) microscope. Photomicrographs were taken with an Olympus OLY-200 digital camera (Olympus America Inc.) using Olympus MagnaFire Camera Imaging and Control version 1.0 (Olympus America).

Sperm Preparation and Indirect Immunofluorescence

A pure population of testicular spermatozoa was obtained by ligating the efferent duct proximal to the testis for 18–20 h and cutting near the site of ligation. Spermatozoon and rete testis fluid was gently extruded into 500 µl of Hanks medium (Invitrogen Corp. Carlsbad, CA) prewarmed to 37°C and washed with the same medium once before resuspending into Hanks medium at a concentration of 5 x 105/ml. The spermatozoa were then smeared onto Superfrost Plus slides and air dried.

Mouse epididymal spermatozoa were obtained by mincing caput, corpus, or cauda epididymides with scissors into approximately 5 ml of prewarmed Hanks medium. The sperm were washed with same medium once and diluted to approximately 5 x 105/ml and air dried onto slides.

Testicular and epididymal spermatozoa were fixed and permeabilized with 95% methanol:5% glacial acetic acid for 30 min at –20°C. After fixation, spermatozoa were washed with PBS containing 0.1% Tween 20 (PBS-T) and incubated with 10% goat serum at 37°C for 1 h in a humidified chamber to block nonspecific binding. The slides were then incubated with a polyclonal rabbit anti-mouse CST12 antibody (1:400 in 10% goat serum) overnight at 4°C followed by 3 x 5 min washes in PBS-T. The spermatozoa were incubated in a dark container with a fluorescein-conjugated goat anti-rabbit IgG (1:100; Vector Laboratories, Inc., Burlingame, CA) at room temperature for 1 h. The slides were then washed in PBS-T three times and in PBS (pH 8.5) once. Control samples were incubated with IgG purified from preimmune serum (1:400), or secondary antibody alone (1:100). Coverslips with mounting solution containing propodium iodide (Vector) were used to cover the slides. The slides were then examined using a Nikon Microphot-FX microscope equipped with epifluorescence.

Castration, Hormone Replacement and Unilateral Efferent Duct Ligation

Orchidectomies and unilateral efferent duct ligations were done under an i.p. injection of 0.1 mg/kg ketamine-0.05 mg/kg xylene anesthesia and all ligations and sutures were done using nonabsorbable braided #6-0 silk (Surgical Specialist Corp., Reading, PA). Adult mice were castrated at Day 0 and testosterone and/or estrogen replacements were performed at the time of castration. Experimental mice (n = 3 per group) received a daily subcutaneous injection of 3 mg of testosterone propionate (T; Sigma Chemical Co., St. Louis, MO) and/or 17ß-estradiol (E; Sigma) dissolved in 100µl sterile sesame seed oil as a vehicle [22]. Control mice (n = 3 per group) received daily injections of 100µl vehicle only. For sham operation (n = 3 per group), testis and epididymis were manipulated and returned into the tunica vaginalis and animals received 100 µl vehicle every day after surgery. Mice were sacrificed on Day 4, Day 7 and Day 14 after surgery. Plasma T/E levels were determined by radioimmunoassay (RIA) at the Center for Reproductive Biology RIA Core laboratory at Washington State University, using kits obtained from Diagnostic Systems Laboratories, Inc. (Webster, TX). Unilateral efferent ductule ligation was performed by ligating the junction between the left testicular rete testis and the epididymis without compromising testicular or epididymal blood supply [23]. As a control, the right testis was manipulated similarly to the left, but its efferent ductules were not ligated. Epididymides were removed on Days 1, 2, 3, 7 and 10 postligation (n = 3 per group) and put in Trizol for RNA extraction. All the experiments were performed at least twice.

Northern Blot Analysis and Statistical Analysis

Epididymides from experimental animals were homogenized in Trizol reagent (Invitrogen Corp.) and RNA was extracted following the manufacturer's recommendations. Seven micrograms of total RNA isolated from epididymides were fractionated on a 1.0% agarose/formaldehyde gel and transferred to Hybond N membranes (Amersham). Northern blot analysis was carried out as previously described [12] with the following modifications. Mouse Cst12 cDNA (540 bp) was radiolabeled using the Rad Prime DNA Labeling Kit (Invitrogen). The blots were hybridized in a hybridization solution consisting of 1% BSA, 7% SDS, 1 mM EDTA, 0.5 M sodium phosphate, pH 7.2 and 1 x 106 cpm/ml of corresponding cDNA probe overnight at 65°C. After hybridization, the blots were exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA) for 16–48 h. The signals were analyzed using a Molecular Dynamics PhosphorImager 445 SI, ImageQuant software (Molecular Dynamics) and Microsoft Excel (Microsoft, Richmond, WA). The levels of Cst12 mRNA were measured in all mice; the bars in the figures indicate mean ± SEM for all hybridizations of experiments. Differences between groups were statistically analyzed by either Student t-test or one-way ANOVA followed by a Student-Newman-Keul test. The mean differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Protein Purification and Generation of Polyclonal Antibody against CST12 Protein

The purity of His-CST12 protein was determined by 15% SDS-PAGE followed by Coomassie Blue R-250 staining. Western blot analysis was performed to examine cross-reaction between CST12 antibody and several known cystatins sharing similarity with CST12 protein. As shown in Figure 1, the purified His-CST12 protein was more than 95% pure and anti-CST12 antibody recognized this band, which was about 17.5 kDa. No cross-reaction between CST12 antibody and CST SC or CST E2 protein was detected. Cross-reactivity between anti-CST12 antibody and CST13 protein was noticed.



View larger version (64K):
[in this window]
[in a new window]
  		FIG. 1. Western blot analysis showing cross-reactivity between the anti-CST12 antibody and purified recombinant His-cystatin proteins. His-CST12 protein, His-CST E2 protein, His-CST13 and His-CST SC protein produced and purified using the system described in Materials and Methods were separated by 15% SDS-PAGE, transferred to PVDF membrane, and incubated with anti-CST12 antibody (A). The same blot was stripped and incubated with preimmune serum (B). After Western blot analysis, the blot was stripped and stained with Coomassie blue as a protein-loading control (C)

Immunolocalization of CST12 Protein in Adult Mouse Testis

To examine the localization of CST12 protein in testis, cross sections of testis were examined using anti-CST12 antibody and visualized by the technique described in Materials and Methods. As shown in Figure 2, intense CST12 staining in the testis was localized to Sertoli cells and displayed a stage-dependent pattern. The limited immunostaining was found in stages VII–VIII of seminiferous tubules (Fig. 2A). Higher magnification (Fig. 2B) revealed that CST12 protein was highly expressed in the cytoplasm of the Sertoli cells in stages II–III of seminiferous tubules; similar expression level and localization were observed in other stages, but not in stages VII–VIII. The protein level decreased dramatically in the cytoplasm of Sertoli cells in stage VII seminiferous epithelium. Therefore, the signal in Sertoli cells decreased dramatically during spermiation and increased immediately after spermiation. Weak peroxidase reaction was also observed in some pachytene spermatocytes and in elongating and elongated spermatids. No detectable peroxidase reaction was observed in spermatogonia or interstitial tissues (Fig. 2B). No specific immunostaining was detected on the cross section incubated with preimmune serum (Fig. 2C) or on the CST12 antibody preincubated with CST12 protein (data not shown).



View larger version (119K):
[in this window]
[in a new window]
  		FIG. 2. Localization of CST12 protein within the testis. A) Cross section of the mouse testis was incubated with the anti-CST12 antibody followed by incubation with a secondary antibody conjugated to peroxidase. B) High magnification of seminiferous tubules showed the CST12 protein expression and localization in different seminiferous tubules. An intense immunoperoxidase reaction is present within the cytoplasm of Sertoli cells (large bold arrow) and the basal region of these cells (open arrow) at stage II–III of the seminiferous tubule. Intense staining was observed in the Sertoli cell cytoplasm encapsulating the heads of elongating step-14 spermatids (small bold arrowhead). Some nuclei of pachytene spermatocytes (small bold arrow) and cytoplasm of some elongating spermatids (*) were weakly reactive. Spermatogonia (small open arrowhead) were not immunoperoxidase reactive. The immunostaining became very weak in the stage VII seminiferous epithelium. A weak reaction was observed in the basal region of Sertoli cells (large open arrowhead), in pachytene spermatocytes (small bold arrow), and in the heads of the elongated spermatids (large bold arrowhead) lining the lumen. Int: interstitial tissues. C) Cross section of the mouse testis was incubated with preimmune antibody as negative control. Magnification A, C x100; B x 400

Immunolocalization of CST12 Protein in Adult Mouse Epididymis

In our previous studies, we demonstrated by Northern blot analysis and in situ hybridization that Cst12 mRNA was restricted to the epithelial cells of the proximal caput region of the epididymis (initial segment) [10]. To examine the localization of CST12 protein in epididymis, longitudinal sections of the mouse epididymis were incubated with an anti-CST12 antibody and visualized for CST12 protein by immunoperoxidase staining. As shown in Figure 3, the intracellular localization of the CST12 protein in the caput epididymis closely parallels the localization of the Cst12 mRNA. Specifically, the CST12 protein was detected at high levels in the epithelial cells of the initial segment of caput epididymis (Caput I). A higher magnification of the Caput I showed CST12 protein was localized to the cytoplasm of all the epithelial cells with a higher level in the apical region of the cells and secreted to the lumen. No obvious staining was detected in the nuclei of epithelial cells. In the next immediate region, Caput II, only some, not all, of the epithelial cells were immunostained. The staining was random and some of the principal cells and basal cells were stained and other principal cells and basal cells were not stained. The level of immunostaining was significantly less than that observed in Caput I. In Caput III, the immunostaining was barely detectable in the epithelial cells but present in the lumen. In the neck region of Caput IV and the very proximal corpus (Pcorpus) epididymis, CST12 protein was primarily localized to the lumen at very high levels, with little staining found in the epithelial lining. In the corpus, the localization of CST12 protein changed among the epididymal tubules. In some tubules, CST12 protein was localized to the apical region of principal cells and some of the basal cells with only slight staining in the lumen (Corpus I). In other tubules, CST12 protein was mainly localized in the lumen with very little present in epithelial cells (Corpus II). Similar to the corpus region, the localization of CST12 protein also varied among the epididymal tubules in cauda epididymis. However, the immunostaining was much weaker in the cauda region (Cauda) than in other regions of epididymis. The CST12 protein was also detected in the cytoplasm of epithelial cells in the vas deferens. Epididymis incubated with preimmune serum (Neg), secondary antibody only, or CST12 antibody that was preincubated with CST12 protein showed no immunostaining (data not shown), demonstrating the high specificity of the CST12 antibody.



View larger version (148K):
[in this window]
[in a new window]
  		FIG. 3. Immunolocalization of CST12 protein in the mouse epididymis. Longitudinal section of the mouse epididymis was incubated with the CST12 antibody followed by incubation with a secondary antibody conjugated to peroxidase. Caput shows the CST12 protein distribution in different regions of caput, and higher magnification shows the localization of CST12 in caput epididymal tubule (Caput I, Caput II and Caput IV). Corpus I and Corpus II exhibit different localization of CST12 protein in two areas of corpus epididymis. CST12 localization in cauda and in vas deferens (Cauda, Vas deferens) is also shown in this figure. In Caput I, an intense reaction is seen in the cytoplasm of all epithelial cells. In Caput II, immunostaining is present throughout the cytoplasm of some principal cells (large arrow) and within some basal cells (big arrowhead) whereas others (small arrow, principal cells; small arrowhead, basal cells) are not immunoreactive. In Corpus I, an intense immunoreaction is observed in the apical region of some principal cells (arrow) and basal cells (arrowhead). Longitudinal section of caput epididymis was incubated with preimmune antibody as negative control (Neg). Magnification x200, except Caput and Neg x20

Localization of CST12 Protein in Mouse Spermatozoa

CST12 protein was detected in the testis and epididymis, suggesting this protein may interact with spermatozoa during transit from testis to the cauda epididymis. Indirect immunofluorescence analysis was carried out to determine whether CST12 protein was associated with testicular or epididymal spermatozoa. Mouse spermatozoa obtained from rete testis and four regions of epididymis were fixed, permeated and incubated with polyclonal rabbit anti-mouse CST12 antibody followed by a fluorescein-conjugate goat-anti-rabbit secondary antibody. As shown in Figure 4A, a green fluorescent signal representing CST12 protein was detected in the cytoplasmic droplets of the testicular spermatozoa located near the sperm neck along the midpiece of the sperm tail. Figure 4B shows that in corpus and cauda spermatozoa, the immunofluorescence was located in the cytoplasmic droplets that migrated to the distal region near the annulus, at the midpiece-principal piece juncture. Cell counts showed that 77% of testicular sperm, 34% of caput (Caput I, II and III; see Fig. 3 "Caput"), 33% of corpus, and 27% of cauda epididymal sperm were positive. About 61% of sperm from the neck region between Caput IV and Pcorpus were immunostained, which is consistent with the results shown in Figure 3. Very weak fluorescent signal was observed in the acrosomal cap region and negative controls, indicating sperm autofluorescence. Sperm nuclei were stained red by propodium iodide in the mounting solution.



View larger version (66K):
[in this window]
[in a new window]
  		FIG. 4. Localization of CST12 protein in spermatozoa by indirect immunofluorescence analysis. A) The localization of CST12 in testicular sperm. B) The localization of CST12 in corpus and cauda epididymal sperm. C) Epididymal sperm incubated with preimmune serum as negative control. D and F are images of A and C taken under light microscopy. E is an image of B photographed using a wide yellow filter showing sperm nuclei stained by propodium iodide. Magnification x400

Regulation of Cst12 Gene Expression

The effect of castration on Cst12 mRNA level was determined using Northern blot analysis (Fig. 5). Total RNA extracted from the epididymides at 4 and 7 days postcastration was hybridized with the 32P-radiolabelled Cst12 cDNA probes. High levels of transcripts were detected in the epididymis of intact animals. Cst12 transcripts decreased by 46% of sham level on Day 7 postcastration. Serum testosterone levels were 1.6–27 ng/ml on Day 0 and were undetectable (<0.05 ng/ml) by Day 7 postcastration. To determine whether exogenous testosterone can restore Cst12 expression in the epididymides of castrated mice, T was administered to mice at the time of castration in a T maintenance paradigm. The daily administration of T for 7 days did not result in any recovery of Cst12 gene expression. Instead, T treatment further repressed the expression of this gene in the epididymis to 24% of sham level and 52% of castrated level following castration. This suggests that hormones other than T or other testicular factors upregulate Cst12 gene expression. Epididymal retinoic acid-binding protein (ERabp), which has been reported to be upregulated by testosterone in epididymis [24], was used as a control to confirm the epididymal response to T treatment.



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 5. Effect of immediate T replacement after castration (T maintenance) on Cst12 mRNA level. A) Northern blot analysis of Cst12 mRNA from epididymides collected from castrated T-injected (C + T), castrated vehicle-injected (C) and sham-operated mice (S) 4 days and 7 days after surgery. Each group contained three animals (n = 3), and the experiment was performed four times. B) Comparison of Cst12 mRNA levels. The results present in Figure 5A were quantified by densitometry and normalized to ribosomal protein S2 message (S2). Epididymal retinoic acid-binding protein message (Erabp) was used as a control to confirm T regulation. The levels of Cst12 mRNA were measured in all mice, and the bars in this figure indicate mean ± SEM for all hybridizations of experiments. The mean differences between groups were considered significant at P < 0.05 by one-way ANOVA followed by a Student-Newman-Keuls test, as indicated: *S vs. C; #C vs. C + T

To test for possible contributions of testicular factors to the regulation of gene expression, unilateral efferent duct ligation was used to prevent the flow of testicular fluid into one epididymis. After ligation, Cst12 mRNA levels in the isolated epididymis decreased dramatically. As shown in Figure 6, on Days 7 and 10 postligation, the Cst12 mRNA in the ligated epididymis was reduced to 15% of that in the nonligated epididymis, and to about 22% of that in the nonligated epididymis on Day 1 postligation (near-sham level, the mRNA level of CST12 in the nonligated epididymis on Day 1 postligation is close to the sham level shown in Fig. 5).



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 6. Northern blot analysis depicting the effect of efferent ductule ligation on Cst12 expression. Unilateral efferent duct ligation was performed as described in Materials and Methods. Epididymides were collected 1 day (1), 2 days (2), 3 days (3), 7 days (7) and 10 days (10) after surgeries. Seven micrograms of total RNA from epididymis with efferent duct ligation (L) or without efferent duct ligation (N) were analyzed by Northern hybridization. Each group contained three animals (n = 3) and the experiment was performed twice. A) The blots were hybridized to Cst12 probe, stripped, and hybridized to ribosomal protein S2 (S2) cDNA probe to normalize the amount of RNA loaded. B) Comparison of Cst12 mRNA levels. The results present in A were quantified by densitometry and normalized to ribosomal protein S2 message (S2). The levels of Cst12 mRNA were measured in all mice and the bars in this figure indicate mean ± SEM for all hybridizations of experiments. * Significant at P < 0.05 by a Student t-test, N vs. L; ns, not significantly different

Testicular factors include a variety of molecules found in the seminiferous tubular fluid, such as luminal androgens [25], androgen-binding proteins [26], estrogen [27], growth factors [28], and probably unknown molecules as well. To determine whether estrogen regulates the expression of Cst12 expression, we treated the castrated mice with E or E + T for 1 and 2 wk. As shown in Figure 7, Cst12 mRNA levels decreased to 43% of intact levels after 7 days of castration, but E treatment for 1 wk resulted in a full recovery of Cst12 mRNA levels. E + T treatment for 1 wk, however, failed to recover the Cst12 mRNA level. Castration for two weeks failed to change Cst12 mRNA levels significantly when compared to intact levels. However, in castrated mice, E treatment alone for 14 days increased Cst12 mRNA levels to 180% of those in sham-operated mice. E + T administration for 14 days failed to recover the Cst12 mRNA levels. The RIA analysis showed that circulating levels of T and E were undetectable in mice by Day 7 postcastration and were at least 10- to 20-fold higher in treated mice than those in sham-operated animals. In situ hybridization showed similar regulation of Cst12 mRNA by T and/or E treatment in epididymis, and no regional changes in Cst12 mRNA signal were observed after treatment (data not shown).



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 7. Effects of E and E + T maintenance after castration on Cst12 expression. A) Seven micrograms of total RNA from epididymides after 7 (7D) and 14 days (14D) postcastration were analyzed as described in Materials and Methods. Each group contained three animals (n = 3) and the experiment was performed twice. B) Comparison of Cst12 mRNA levels. The results present in Figure 7A were quantified by densitometry and normalized to ribosomal protein S2 (S2) message. The levels of Cst12 mRNA were measured in all mice, and the bars in this figure indicate mean ± SEM for all hybridizations of experiments. Differences between groups were statistically analyzed by one-way ANOVA followed by a Student-Newman-Keuls test. S, sham-operated epididymis; C, castrated epididymis; E, castrated + E administration; ET, castrated + E and T administration; * significantly (P < 0.05) different from S; ns, not significantly different from S; # significantly different from C; nc, not significantly different from C; ;p0 significantly different from E


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the previous studies, we identified a new member of male reproductive tract cystatins, Cst12, using differential display-reverse transcription PCR techniques. It has been shown that Cst12 is highly expressed in testis and epididymis, with very low expression in ovary and prostate. Our study also showed that Cst12 is predominantly expressed in the Sertoli cells and in the epithelial cells of the proximal caput epididymis, suggesting that CST12 plays a specialized role in the testis and epididymis [10]. In this report, we used a prokaryotic expression system to produce a His-tagged CST12 protein, and subsequently generated a polyclonal antibody. The cross-reactivity between the anti-CST12 antibody and other known cystatins was expected to be minimal, because the similarity among male reproductive tract cystatins and other known cystatins is low (<16–30% identical). Because this antibody was against the full-length CST12 protein, not a short peptide, Western blot analysis was carried out to examine the possible cross-reaction between this antibody and other members of the male reproductive tract cystatin subgroup, including CST SC protein, which is the only known cystatin that shares high identity (50.3%) with CST12 protein. A cross-reaction was noticed between the anti-CST12 antibody and His-CST13 protein. However, the localization patterns of CST12 and CST13 in the testis and epididymis as determined by immunohistochemistry do not significantly overlap (Li and Griswold, unpublished observations), suggesting that this cross-reaction does not affect the specificity of this anti-CST12 antibody. Moreover, controls without primary antibody, with preimmune serum, without secondary antibody, and with primary antibody incubated with corresponding protein were always performed to confirm the specificity of this anti-CST12 antibody.

Sertoli cells produce specific products to form a unique and essential environment in the adluminal compartment that is necessary for germ cell survival and development [29]. Sertoli cells synthesize and secrete many different proteinases and proteinase inhibitors [30], and one of the functions ascribed to this class of proteins involves extensive tissue remodeling that occurs when tight junctions must be traversed or when spermatozoa are released from the seminiferous epithelium [31]. However, the CST12 protein lacks all of the conserved regions thought to be critical for binding to a cysteine proteinase of the papain family [16]. This suggests that the specificity of this protein may be quite different from the typical family 2 cystatins. Recently, it was reported that in in vitro assays, recombinant CST8 did not inhibit the cysteine proteinase papain or cathepsin B. Instead, it inhibited the serine proteinase prohormone convertase 2 (PC2), a proteinase involved in prohormone processing in the neuroendocrine system [17]. This raises a possibility that CST12 may also function as a proteinase inhibitor but not as a cysteine proteinase inhibitor. Immunohistochemistry reveals that CST12 protein was highly expressed in the Sertoli cells and showed a stage-dependent pattern in seminiferous tubules. However, no difference in Cst12 mRNA levels among seminiferous tubules was detected by in situ hybridization analysis [10, 11], suggesting a posttranscriptional or translational regulation of the Cst12 gene. This observation indicates that CST12 protein synthesis is under strict regulation in Sertoli cells and CST12 plays a specialized role during spermatogenesis. No or very little Cst12 mRNA signal was detected in developing germ cells in the seminiferous tubules using in situ hybridization [10]. However, weak immunostaining was observed in nuclei of some pachytene spermatocytes, cytoplasm of elongating spermatids and elongated spermatids. It is hard to determine if the protein was synthesized in the developing germ cells or acquired from Sertoli cells by germ cells during germ cell-Sertoli cell interaction.

Because spermatozoa entering epididymis are, for the most part, synthetically inactive, the process of sperm maturation resulting in progressive motility and fertility is thought to involve the interaction of spermatozoa with proteins that are synthesized and secreted by the epididymal epithelium [32]. The localization of CST12 protein in the caput epididymis closely parallels the localization of the Cst12 mRNA [10]. The CST12 protein was highly expressed in the initial segment of epididymis and decreased dramatically in the next immediate region, suggesting that CST12 protein synthesis had significantly decreased. The intense staining in the neck region indicates that the secreted CST12 protein accumulated in and plays a special role related to the function of this region. The concentration and localization of CST12 protein varies in different regions of the epididymis, suggesting that CST12 can be reabsorbed and released by epithelial cells to control the protein interaction with spermatozoa during passage through the epididymis. These observations indicate that CST12 protein plays a highly specialized role during sperm maturation.

The presence of CST12 protein in testis and epididymis prompted us to examine whether CST12 protein was associated with testicular or epididymal sperm. In the present study our analysis using indirect immunofluorescence demonstrated that CST12 protein is present in the cytoplasmic droplets that locate along the midpiece near the neck of testicular spermatozoa and migrate from neck to midpiece-principal piece junction (annulus) during proximal epididymal transport. We speculate that Sertoli cells synthesize and secrete CST12 protein, which can be picked up by germ cells during germ cell-Sertoli cell interaction and can also be adsorbed from the lumen by testicular spermatozoa. Also, spermatozoa in the epididymis can release and reabsorb this protein secreted by epididymal epithelium during their epididymal transit. The role of the cytoplasmic droplet, which remains on the majority of sperm in the epididymis [33], is still unclear. However, several glycolytic enzymes have been localized to the cytoplasmic droplet, suggesting a relationship to lysosomal activity [34, 35]. The identification of active P450 aromatase in the cytoplasmic droplet of several species has led to the implication of estrogens in the regulation of sperm maturation in the epididymis [36]. Recently, Cooper and Yeung suggested a role for cytoplasmic droplets in sperm volume regulation which is crucial for fertility [33]. The percentage of CST12-positive sperm was much higher in the caput-corpus neck region than any other region of epididymis, suggesting CST12 protein performs a role specifically related to the function of this neck region during sperm maturation.

Several proteinase inhibitors or putative proteinase inhibitors have been identified in the epididymis [37–43], including several male reproductive tract cystatins, such as CST 11 {also named CST E1 or CRES2 in mouse, [11– 13]}, CST E2 [12], and CST8 [19]. The synthesis and localization of CST 11 and CST E2 proteins in vivo is unclear. CST8 protein is exclusively synthesized by the proximal caput epithelium in the epididymis and secreted into the lumen [8]. It is mainly localized in the sperm acrosomal cap and released after acrosome reaction. As described in this report, CST12 protein is secreted by the epithelial cells in the initial segment and localized in the cytoplasmic droplets of spermatozoa. Although both CST12 and CST8 are synthesized by the epithelial cells of the proximal caput epididymis and secreted into the lumen, these male reproductive tract cystatins localized to different regions of the spermatozoa, suggesting they interact with unique, corresponding proteinases or targeting proteins and play very specialized roles in sperm maturation, sperm protection, and/or fertilization.

Normal epididymal function depends on both circulating androgens and testicular factors [44–47]. Our study showed that castration for 4 and 7 days decreased Cst12 mRNA levels and that exogenous T administration further repressed Cst12 expression. Moreover, T treatment of castrated animals reduced the Cst12 mRNA (24% of sham level at Day 7 postcastration) to a similar level to that of efferent ductile ligated mice (22% of near-sham level at Day 7 postligation). These results suggest that circulating androgens do downregulate the expression of Cst12, but this downregulation may be the result of clearing of remaining testicular factors from initial segment of caput epididymis induced by androgen-dependent epididymal contractions [48].

Estrogen is one of the testicular factors that is present at high concentration in rete testis fluid [41, 49]. It has been established that estrogen receptors are present in the caput epididymis of mice [50], and estrogen plays an important role in the regulation of epididymal function [51, 52]. Our results showed that Cst12 transcript levels were restored by E administration and that E + T treatment failed to restore Cst12 mRNA. This suggests that E up-regulates the expression of Cst12 whereas T represses the expression. However, Hsia et al. did not notice restoration of Cst12 mRNA by E in castrated animals and the loss of estrogen receptor alpha (ER{alpha}) gene did not affect Cst12 mRNA levels [11]. This contrasting result is probably attributable to the following: 1) Three milligrams of E was administered in our study, resulting in at least a 10-fold increase of circulating E level, whereas only 300 ng (10,000 times less) of E was used in Hsia's report, and no E level was indicated. The high dose of exogenous E may be necessary to increase the E level in testicular or epididymal fluid. 2) Disruption of ER{alpha} (ERKO) in the male alters both morphology and physiology of the male reproductive tract, especially that of testis and efferent ductile [53, 54]. This alteration affects the components and concentration of circulating hormones and testicular factors, which may influence Cst12 mRNA levels in epididymis. In addition, ER{alpha} is present in the caput epididymis of mice. In the ERKO mouse, the epididymal epithelial cells may not respond as normal cells. Because we only examined the Cst12 gene expression in epididymis after 1 and 2 wk postcastration and hormone treatment, the upregulation by E treatment may not be due to direct involvement of E in regulation of this gene. Other testicular factors may also participate in this gene regulation in epididymis. Further experiments should be done to interpret the mechanisms involved in the modulation of Cst12 by testosterone and estrogen. Our previous study demonstrated that Cst E2 mRNA is estrogen-responsive in epididymis, whereas Cst11 is more dependent on other testicular factors [12]. These observations indicate that although these three male reproductive tract cystatins share similar localization in the epididymis, the differences in their regulation by hormones and testicular factors suggest they may play unique and specialized roles.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Drs. Chris Small and Chris Hostetler for critical revision of this manuscript. The authors thank Rong Nie for preparation of the tissue sections. The authors also thank Lizhong Yang for assistance in preparing for the figures.


   FOOTNOTES
 
1 Supported by National Institute of Child Health and Human Development HD 10808. Back

2 Correspondence: Michael D. Griswold, 531 Fulmer Hall, School of Molecular Biosciences, Washington State University, Pullman, WA 99164. FAX: 509 335 9688; griswold{at}mail.wsu.edu Back

Received: 21 January 2005.

First decision: 9 February 2005.

Accepted: 22 June 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Barrett AJ. The cystatins: a diverse superfamily of cysteine peptidase inhibitors. Biomed Biochim Acta 1986 45:1363-1374[Medline]
  2. Henskens YM, Veerman EC, Nieuw Amerongen AV. Cystatins in health and disease. Biol Chem Hoppe Seyler 1996 377:71-86[Medline]
  3. Turk V, Bode W. The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett 1991 285:213-219[CrossRef][Medline]
  4. Ni J, Abrahamson M, Zhang M, Fernandez MA, Grubb A, Su J, Yu GL, Li Y, Parmelee D, Xing L, Coleman TA, Gentz S, Thotakura R, Nguyen N, Hesselberg M, Gentz R. Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins. J Biol Chem 1997 272:10853-10858[Abstract/Free Full Text]
  5. Ni J, Fernandez MA, Danielsson L, Chillakuru RA, Zhang J, Grubb A, Su J, Gentz R, Abrahamson M. Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor. J Biol Chem 1998 273:24797-24804[Abstract/Free Full Text]
  6. Cornwall GA, Hsia N. A new subgroup of the family 2 cystatins. Mol Cell Endocrinol 2003 200:1-8[CrossRef][Medline]
  7. Cornwall GA, Orgebin-Crist MC, Hann SR. The CRES gene: a unique testis-regulated gene related to the cystatin family is highly restricted in its expression to the proximal region of the mouse epididymis. Mol Endocrinol 1992 6:1653-1664[Abstract]
  8. Cornwall GA, Hann SR. Transient appearance of CRES protein during spermatogenesis and caput epididymal sperm maturation. Mol Reprod Dev 1995 41:37-46[CrossRef][Medline]
  9. Tohonen V, Osterlund C, Nordqvist K. Testatin: a cystatin-related gene expressed during early testis development. Proc Natl Acad Sci U S A 1998 95:14208-14213[Abstract/Free Full Text]
  10. Li Y, Friel PJ, Robinson MO, McLean DJ, Griswold MD. Identification and characterization of testis- and epididymis-specific genes: cystatin SC and cystatin TE-1. Biol Reprod 2002 67:1872-1880[Abstract/Free Full Text]
  11. Hsia N, Cornwall GA. Cres2 and Cres3: new members of the cystatin-related epididymal spermatogenic subgroup of family 2 cystatins. Endocrinology 2003 144:909-915[Abstract/Free Full Text]
  12. Li Y, Friel PJ, McLean DJ, Griswold MD. Cystatin E1 and E2, new members of male reproductive tract subgroup within cystatin type 2 family. Biol Reprod 2003 69:489-500[Abstract/Free Full Text]
  13. Hamil KG, Liu Q, Sivashanmugam P, Yenugu S, Soundararajan R, Grossman G, Richardson RT, Zhang YL, O'Rand MG, Petrusz P, French FS, Hall SH. Cystatin 11: a new member of the cystatin type 2 family. Endocrinology 2002 143:2787-2796[Abstract/Free Full Text]
  14. Shoemaker K, Holloway JL, Whitmore TE, Maurer M, Feldhaus AL. Molecular cloning, chromosome mapping and characterization of a testis- specific cystatin-like cDNA, cystatin T. Gene 2000 245:103-108[CrossRef][Medline]
  15. Thiesse M, Millar SJ, Dickinson DP. The human type 2 cystatin gene family consists of eight to nine members, with at least seven genes clustered at a single locus on human chromosome 20. DNA Cell Biol 1994 13:97-116[Medline]
  16. Bode W, Engh R, Musil D, Thiele U, Huber R, Karshikov A, Brzin J, Kos J, Turk V. The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J 1988 7:2593-2599[Medline]
  17. Cornwall GA, Cameron A, Lindberg I, Hardy DM, Cormier N, Hsia N. The cystatin-related epididymal spermatogenic protein inhibits the serine protease prohormone convertase 2. Endocrinology 2003 144:901-908[Abstract/Free Full Text]
  18. Sutton HG, Fusco A, Cornwall GA. Cystatin-related epididymal spermatogenic protein colocalizes with luteinizing hormone-beta protein in mouse anterior pituitary gonadotropes. Endocrinology 1999 140:2721-2732[Abstract/Free Full Text]
  19. Syntin P, Cornwall GA. Immunolocalization of CRES (cystatin-related epididymal spermatogenic) protein in the acrosomes of mouse spermatozoa. Biol Reprod 1999 60:1542-1552[Abstract/Free Full Text]
  20. Wassler M, Syntin P, Sutton-Walsh HG, Hsia N, Hardy DM, Cornwall GA. Identification and characterization of cystatin-related epididymal spermatogenic protein in human spermatozoa: localization in the equatorial segment. Biol Reprod 2002 67:795-803[Abstract/Free Full Text]
  21. Kanno Y, Tamura M, Chuma S, Sakura T, Machida T, Nakatsuji N. A cystatin-related gene, testatin/cresp, shows male-specific expression in germ and somatic cells from the initial stage of murine gonadal sex-differentiation. Int J Dev Biol 1999 43:777-784[Medline]
  22. Walker JE, Jones R, Moore A, Hamilton DW, Hall L. Analysis of major androgen-regulated cDNA clones from the rat epididymis. Mol Cell Endocrinol 1990 74:61-68[CrossRef][Medline]
  23. Girotti M, Jones R, Emery DC, Chia W, Hall L. Structure and expression of the rat epididymal secretory protein I gene. An androgen-regulated member of the lipocalin superfamily with a rare splice donor site. Biochem J 1992 281:pt 1203-210
  24. Lareyre JJ, Zheng WL, Zhao GQ, Kasper S, Newcomer ME, Matusik RJ, Ong DE, Orgebin-Crist MC. Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid. Endocrinology 1998 139:2971-2981[Abstract/Free Full Text]
  25. Turner TT, Jones CE, Howards SS, Ewing LL, Zegeye B, Gunsalus GL. On the androgen microenvironment of maturing spermatozoa. Endocrinology 1984 115:1925-1932[Abstract]
  26. Veeramachaneni DN, Amann RP. Endocytosis of androgen-binding protein, clusterin, and transferrin in the efferent ducts and epididymis of the ram. J Androl 1991 12:288-294[Abstract/Free Full Text]
  27. 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]
  28. Lan ZJ, Labus JC, Hinton BT. Regulation of gamma-glutamyl transpeptidase catalytic activity and protein level in the initial segment of the rat epididymis by testicular factors: role of basic fibroblast growth factor. Biol Reprod 1998 58:197-206[Abstract/Free Full Text]
  29. Griswold MD. Protein secretions of Sertoli cells: general considerations. In: Russell LD, Griswold MD (eds.), The Sertoli Cell, 1st ed. Clearwater, FL: Cache River Press; 1993:195–200
  30. Charron M, Wright WW. Proteinases and proteinase inhibitors. In: Skinner MK, Griswold MD (eds.), Sertoli Cell Biology, 1st ed. San Diego, CA: Elsevier Academic Press; 2005:121–152
  31. Fritz IB, Tung PS, Ailenberg M. Proteinases and antiproteinases in the seminiferous tubule. In: Russell L, Griswold MD (eds.), The Sertoli Cell, 1st ed. Clearwater, FL: Cache River Press; 1993:217–235
  32. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd ed. New York: Raven Press; 1994:189–317
  33. Cooper TG, Yeung CH. Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet. Microsc Res Tech 2003 61:28-38[CrossRef][Medline]
  34. Dingle JT, Dott HM. Lysosomal enzymes in bull and ram semen. Biochem J 1969 111:35P
  35. Harrison RA, White IG. Glycolytic enzymes in the spermatozoa and cytoplasmic droplets of bull, boar and ram, and their leakage after shock. J Reprod Fertil 1972 30:105-115
  36. Janulis L, Hess RA, Bunick D, Nitta H, Janssen S, Asawa Y, Bahr JM. Mouse epididymal sperm contain active P450 aromatase which decreases as sperm traverse the epididymis. J Androl 1996 17:111-116[Abstract/Free Full Text]
  37. Metayer S, Dacheux F, Dacheux JL, Gatti JL. Comparison, characterization, and identification of proteases and protease inhibitors in epididymal fluids of domestic mammals. Matrix metalloproteinases are major fluid gelatinases. Biol Reprod 2002 66:1219-1229[Abstract/Free Full Text]
  38. Uhrin P, Dewerchin M, Hilpert M, Chrenek P, Schofer C, Zechmeister-Machhart M, Kronke G, Vales A, Carmeliet P, Binder BR, Geiger M. Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility. J Clin Invest 2000 106:1531-1539[Medline]
  39. Nothnick WB, Soloway PD, Curry TE Jr. Pattern of messenger ribonucleic acid expression of tissue inhibitors of metalloproteinases (TIMPs) during testicular maturation in male mice lacking a functional TIMP-1 gene. Biol Reprod 1998 59:364-370[Abstract/Free Full Text]
  40. Richardson RT, Sivashanmugam P, Hall SH, Hamil KG, Moore PA, Ruben SM, French FS, O'Rand M. Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis. Gene 2001 270:93-102[CrossRef][Medline]
  41. Free MJ, Jaffe RA. Collection of rete testis fluid from rats without previous efferent duct ligation. Biol Reprod 1979 20:269-278[Abstract]
  42. Wilson CL, Heppner KJ, Rudolph LA, Matrisian LM. The metalloproteinase matrilysin is preferentially expressed by epithelial cells in a tissue-restricted pattern in the mouse. Mol Biol Cell 1995 6:851-869[Abstract]
  43. Howard L, Maciewicz RA, Blobel CP. Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28. Biochem J 2000 348: Pt 1 21-27
  44. Robaire B, Viger RS. Regulation of epididymal epithelial cell functions. Biol Reprod 1995 52:226-236[Abstract]
  45. Turner TT, Miller DW, Avery EA. Protein synthesis and secretion by the rat caput epididymidis in vivo: influence of the luminal microenvironment. Biol Reprod 1995 52:1012-1019[Abstract]
  46. Hermo L, Barin K, Oko R. Androgen binding protein secretion and endocytosis by principal cells in the adult rat epididymis and during postnatal development. J Androl 1998 19:527-541[Abstract/Free Full Text]
  47. Hinton BT, Lan ZJ, Rudolph DB, Labus JC, Lye RJ. Testicular regulation of epididymal gene expression. J Reprod Fertil Suppl 1998 53:47-57[Medline]
  48. Hib J, Ponzio RO. Effect of efferent duct ligation, gonadectomy and testosterone replacement on epididymal contractility in the rat. J Reprod Fertil 1977 50:327-329
  49. Ganjam VK, Amann RP. Steroids in fluids and sperm entering and leaving the bovine epididymis, epididymal tissue, and accessory sex gland secretions. Endocrinology 1976 99:1618-1630[Abstract]
  50. Zhou Q, Nie R, Prins GS, Saunders PT, Katzenellenbogen BS, Hess RA. Localization of androgen and estrogen receptors in adult male mouse reproductive tract. J Androl 2002 23:870-881[Abstract/Free Full Text]
  51. Hess RA. Oestrogen in fluid transport in efferent ducts of the male reproductive tract. Rev Reprod 2000 5:84-92[Abstract]
  52. Hess RA, Bunick D, Lubahn DB, Zhou Q, Bouma J. Morphologic changes in efferent ductules and epididymis in estrogen receptor-alpha knockout mice. J Androl 2000 21:107-121[Abstract]
  53. Eddy EM, Washburn TF, Bunch DO, Goulding EH, Gladen BC, Lubahn DB, Korach KS. Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility. Endocrinology 1996 137:4796-4805[Abstract]
  54. Lee KH, Hess RA, Bahr JM, Lubahn DB, Taylor J, Bunick D. Estrogen receptor alpha has a functional role in the mouse rete testis and efferent ductules. Biol Reprod 2000 63:1873-1880[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/5/872    most recent
biolreprod.105.040238v1
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 Li, Y.
Right arrow Articles by Griswold, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Y.
Right arrow Articles by Griswold, M. D.
Agricola
Right arrow Articles by Li, Y.
Right arrow Articles by Griswold, M. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS