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Biology of Reproduction 67, 133-139 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Rabbit Epididymal Secretory Proteins. I. Characterization and Hormonal Regulation1

Brett Nixona,b, Russell C. Jones2,a, Lyall A. Hansena, and Michael K. Hollandb

a Department of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia b Pest Animal Control Cooperative Research Centre, CSIRO Sustainable Ecosystems, Canberra, ACT 2601, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Analyses of samples of luminal fluid from the rete testis, distal efferent ducts, and epididymal regions 2–5 and 8 revealed that 91% of the fluid leaving the testis is reabsorbed by the efferent ducts, 79% of the remainder is reabsorbed proximal to epididymal regions 4 and 5, and there is a net secretion of fluid into the duct caudally. There is a net reabsorption by the efferent ducts of 73% of the protein leaving the testis and then a net secretion along the epididymis. SDS-PAGE of the luminal fluids indicated that four new protein bands that were not present in blood appeared in the efferent ducts, 5 in epididymal regions 1–5, 6 in regions 6 and 7, and one in region 8. Two bands in samples from the efferent ducts were absent caudally, and one band present in region 7 was absent in region 8. The rates of incorporation of 35S-methionine into minced duct in vitro varied among regions when expressed per milligram of wet weight of tissue (region 2–5 > region 7 > region 6 > region 1 > region 8 > ductuli efferentes), and orchidectomy had little effect on the rates. Incorporation into four proteins that were secreted in vitro (Mr 38 000, 20 000, 15 000, and 13 000) was reduced or abolished by orchidectomy and restored by testosterone therapy. The secretion of three proteins (Mr 52 000, 23 000, and 22 000) was reduced or abolished by orchidectomy and not restored by testosterone therapy. SDS-PAGE of detergent extracts of sperm indicated that five proteins were lost and nine were gained during epididymal transit. Seven of the proteins gained were about the same molecular weight as proteins secreted by the epididymis (Mr 94 000, 52 000, 38 000, 36 000, 22 000, 20 000, and 13 000) and were analyzed using N-terminal amino acid microsequencing.

epididymis, gamete biology, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian sperm are not capable of fertilizing an ovum when they leave the testis and must undergo remodelling of the plasma membrane in the epididymis to achieve this capacity (reviewed in [1]). The epididymis also performs other functions, such as protecting sperm from oxidants, proteases, and the immune system [2]. These processes appear to be mediated by proteins secreted by the epididymal mucosa under the influence of androgens [3, 4]. This report provides a basis for identifying the main epididymal proteins to determine their roles in sperm maturation and storage and assess whether some may be targeted to achieve immunocontraception [5]. The rabbit has been studied because it is a pest in Australia, where there is a program to control populations using virus-vectored immunocontraception [6].

Most workers [7–13] have focused their attention on proteins secreted by the caput epididymidis of the rabbit, where important aspects of sperm maturation seem to occur [14, 15] and a number of epididymal proteins associate with sperm [10–12, 16]. However, we examined all epididymal proteins that may interact with sperm because any of these could be targeted for immunocontraception. To better understand the dynamics of protein secretion and absorption in the rabbit epididymis, we examined the natural occurrence of the luminal proteins on sperm and in the luminal plasma and the incorporation of 35S-methionine in epididymal ducts in vitro. Seven proteins that appear to be secreted by the epididymal mucosa and interact with sperm were examined by amino acid microsequencing.


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

The studies were carried out with the approval of the Institutions' Animal Care and Ethics Committees. Mature male New Zealand White rabbits (3.5–4 kg) were housed under a lighting regime of 16L:8D and were supplied with food and water ad libitum. Bilateral orchidectomy was carried out under fluothane anesthesia, and the epididymides were returned to the scrotum. Implants of testosterone (Innovative Research Corporation of America, Toledo, OH) were administered 14 days after orchidectomy, and the epididymides were recovered 14 days later. We calculated that the implant would maintain a concentration of 2–5 ng/ml of testosterone in systemic blood.

Collection and Processing of Luminal Fluids

Luminal fluids were collected from the reproductive ducts of 6 rabbits anesthetized with sodium pentobarbital. Samples were collected by micropuncture of the rete testis and distal efferent ducts [17] and by flushing the ductus epididymidis (regions 4 and 5 inclusive and regions 7 and 8 [18]) with water-saturated paraffin oil [19]. The efferent ducts were sampled towards the distal end of the coni vasculosa because the flow of fluid into the ductus epididymidis was too low for sufficient volumes of luminal fluid to be sampled there by micropuncture or microcannulation. Sperm and epididymal plasma were separated by centrifugation (12 000 x g, 15 min) in glass Microcaps (Drummond Scientific Co., Philadelphia, PA), and spermatocrits were determined. Protein concentration in plasma was determined using the BCA protein assay (Pierce, Rockford, IL). Sperm concentrations were determined with a hemocytometer, and these values (or spermatocrits) were used to calculate fluid reabsorption along the epididymis and to assess the significance of changes in protein concentration [17].

Spermatozoa from the rete testis and region 8 of the epididymis were washed in 1 ml Krebs Ringer phosphate (KRP: 119 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 15.5 mM Na2HPO4, 1.3 mM CaCl2, 10 mM glucose) and concentrated by centrifugation (500 x g, 5 min). Proteins were extracted [20] for 30 min at 0°C in extraction buffer (0.5 ml PBS, 0.1% Triton X-100, 5 mM EDTA, protease inhibitors, 0.5 mg/ml pepstain, 0.5 mg/ml leupeptain, 5 mM benzamide, and 1 mM PMSF) and separated by centrifugation (2000 x g, 12 min).

Incorporation of 35S-Methionine

Incorporation of 35S-methionine into minced ducts [21] was determined for the efferent ducts and for regions 1, 2–5, 6, 7, and 8 of the epididymis [18]. The study was a randomized block design (replicated twice) comparing animals that were untreated, bilaterally orchidectomized, and bilaterally orchidectomized and implanted with testosterone. Samples (30 mg) were incubated (6 h at 33°C) in siliconized glass vials containing 1 ml KRP supplemented with 0.56 mg penicillin G, 0.94 mg streptomycin sulfate, 0.1 mM of all essential amino acids except L-methionine (Amersham International, Buckinghamshire, U.K.), and 50 µCi 35S-methionine (Tran 35S-label; ICN Radiochemicals, Costa Mesa, CA). Subsequently, the tissue and supernatant (interpreted as containing secreted proteins) were separated by centrifugation (10 000 x g, 10 min). Tissue was homogenized on ice in buffer (0.25 M sucrose, 1.5 mM MgCl2, 0.1 mM L-methionine, 10 mM Tris-HCl, pH 7.4) using a glass homogenizer fitted with a teflon pestle, and both medium and homogenized tissue were centrifuged (100 000 x g, 30 min, 2°C). Labeled proteins were recovered by precipitation with 10% trichloroacetic acid (TCA), washed several times with ether:ethanol (1:1 v/v), and resuspended in 50 µl solubilizing buffer (130 mM Tris-HCl, pH 6.8, 20% v/v glycerol, 4% w/v SDS, 0.005% w/v bromophenol blue, 5% v/v ß-mercaptoethanol). Incorporated 35S-methionine was determined on aliquots (10 µl) that were acid precipitated and collected onto glass microfiber filters (GF/A Whatman International Ltd., Maidstone, U.K.). Filters were washed sequentially with 5% TCA (containing 0.1% mM L-methionine) and 95% ethanol and were counted in scintillation liquid using a liquid scintillation spectrophotometer.

A study of the kinetics of labeled protein release during incubation (2 animals) confirmed that protein was released in a biphasic pattern as described for the rat [22]: an initial lag period for the first 1–2 h followed by a rapid linear increase for the remaining 4 h.

Electrophoresis and Fluorography

Proteins were denatured with SDS and separated using a 10–20% linear polyacrylamide resolving gel (SE 250 Mighty Small II; Hoefer Scientific Instruments, San Francisco, CA). Two gels per set of samples from each animal were loaded with either 0.5 µg unlabeled protein or 4000 cpm of radiolabeled proteins per well in solubilizing buffer and electrophoresed at 100 V/gel. Unlabeled proteins were stained with silver reagent [23], whereas gels containing labeled proteins were impregnated with 20% 2,5-diphenyloxazole in acetic acid and exposed (-70°C, 14 days) to hypersensitized autoradiographic film (Hyperfilm-B max; Amersham). Individual proteins were identified as nominal molecular weights (Mr) using calibration proteins (Pharmacia, Uppsala, Sweden).

N-Terminal Amino Acid Sequence Analysis

Samples from epididymal region 8 were electrophoresed, electrotransferred (250 mA, 1 h) onto Polyscreen polyvinylidene fluoride transfer membranes (NEN Research Products, Boston, MA) using a Mini Trans-Blot Cell (Bio-Rad Inc., Richmond, CA), and stained with Coomassie G-250 (BDH Chemicals Ltd., Poole, England). Protein bands were excised for N-terminal amino acid sequencing using automatic Edman degradation in a Procise Sequencer (Applied Biosystems Inc., Foster City, CA). Trypsin digests were carried out on proteins with an N-amino terminal block [24], and the resulting peptide fragments were sequenced. Searches for amino acid sequence homologies were performed on databases at the National Center for Biotechnology Information (Bethesda, MD) using the BLAST search program and at EMBL (Heidelberg, Germany) using the FASTA and BLITZ alignment programs.

Statistics

Values are presented as mean ± SEM; the SEM was calculated from the variance between animals. Significance of differences was determined based on ANOVA results.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Changes in Concentrations of Sperm and Protein along the Epididymis

Table 1 shows that the efferent ducts reabsorb most of the fluid (91%) leaving the testis. Most of the remainder (79%) is reabsorbed before regions 4 and 5 of the epididymis so that the residual fluid remaining in regions 4 and 5 represents less than 2.5% of the amount that leaves the testis. There was a net secretion of fluid between regions 5 and 8.


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  		TABLE 1. Concentration of sperm and protein in luminal fluids from the rabbit epididymis. Values are mean ± SEM from six animals

There was a net reabsorption in the efferent ducts of 73% of the protein leaving the testis. Between the efferent ducts and regions 4 and 5 there was a net increase in luminal protein of 68% and a further net increase of 45% between regions 4 and 5 and region 8.

Sperm Proteins

There was an apparent loss and gain of a number of proteins as sperm passed through the epididymis (Fig. 1). Five bands were detected in extracts of rete testis sperm (Mr 93 000, 61 000, 35 000, 33 000, 27 000) that were not detected in extracts of sperm from region 8. Nine bands were not detected in rete testis sperm but were detected in sperm from region 8 (Mr 94 000, 88 000, 63 000, 52 000, 38 000, 36 000, 22 000, 20 000, 13 000). Further, there was an increase in staining intensity of 7 proteins associated with epididymal transit (Mr 91 000, 28 000, 26 000, 24 000, 16 000, 15 000, 10 000).



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  		FIG. 1. Electrophoretic profile of detergent extractable sperm proteins from the rete testis (RT) and region 8 (8) of the epididymis. Each well was loaded with 0.5 µg of protein and stained with silver reagent. The numerical values correspond to the molecular weight markers of the standard (Mr x 10-3; left side) and the major proteins that were apparently gained by sperm during epididymal transit (right side). Proteins lost and gained by sperm during epididymal transit are denoted by unshaded and shaded arrowheads, respectively. Proteins that increased in staining intensity during epididymal transit are denoted by a grey arrowhead

Proteins in Luminal Fluids (Collections In Vivo)

Sixteen major proteins were identified in luminal fluid that did not coincide with proteins of the same molecular weight in either blood plasma or rete testis fluid (Fig. 2). Of these proteins, four were thought to have originated in the efferent ducts (Mr 50 000, 46 000, 31 000, 30 000), five from between the efferent ducts and regions 4 and 5 of the epididymis (Mr 63 000, 52 000, 26 000, 22 000, 20 000), 6 from between regions 4 and 5 and region 7 (Mr 94 000, 88 000, 42 000, 38 000, 24 000, 13 000), and 1 from between regions 7 and 8 (Mr 36 000) of the epididymis (Table 2). In addition, one protein that was present in the efferent ducts (Mr 46 000) was absent more distally, and a second protein (Mr 24 000) present in region 7 was absent in region 8 (Fig. 2 and Table 2).



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  		FIG. 2. Electrophoretic profile of epididymal luminal fluids. Blood plasma (B) and micropuncture samples of luminal fluids from the rete testis (RT), distal efferent ducts (ED), regions 4 and 5 (4–5), region 7 (7), and region 8 (8) of the rabbit epididymis were resolved by one-dimensional SDS-PAGE. Each well was loaded with 0.5 µg protein, and the gel was stained with silver reagent. The numerical values correspond to the molecular weight markers of the standard (Mr x 10-3; left side) and the proteins that were selected for further investigation (right side). Proteins interpreted as originating in a particular region are designated by an arrowhead. In the case of proteins for which amino acid sequence was obtained, the arrowheads are shaded. Proteins of Mr 46 000 and 24 000, which were absent in luminal fluids distal to their site of secretion, are indicated by an asterisk


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  		TABLE 2. Properties of the major rabbit epididymal secretory proteins identified in the present study

Incorporation of 35S-Methionine In Vitro: Effects of Orchidectomy

Bilateral orchidectomy reduced the masses of the efferent ducts and all regions of the epididymis (P <= 0.05), and the masses were restored by testosterone therapy in all regions (P <= 0.05) except the efferent ducts and regions 1 and 2–5 of the epididymis. The androgen status of animals had little influence on the overall rates of incorporation of 35S-methionine into different regions of the duct system when the rates were expressed per milligram of wet weight of tissue. The ranking of rates was regions 2–5 > region 7 > region 6 > region 1 > region 8 > ductuli efferentes. Only 10–14% of the incorporated 35S-methionine was secreted by the epididymal samples, and only 6% was secreted by the efferent ducts. This proportion was reduced by 10–25% after orchidectomy, but the original proportion was restored by testosterone therapy. In all regions, orchidectomy caused a significant reduction (P <= 0.05) in incorporation of radiolabel as expressed per epididymal region (Table 3). Testosterone therapy restored the rate of incorporation in all regions except regions 1 and regions 2–5 of the epididymis (P <= 0.05).


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  		TABLE 3. Effect of orchidectomy and orchidectomy followed by testosterone replacement therapy on protein synthesis and secretion per milligram total epididymal region. Minced samples (30 mg) of rabbit epididymal tissue were incubated with 50 µCi 35S-methionine for 6 h in a shaking water bath at 33°C. The incorporation of 35S-methionine is expressed as the amount isolated from tissue and the amount released (secreted) into the incubation medium. Values represent the mean ± SEM from 2 animals

For intact animals, fluorographs (Fig. 3) showed that the major labeled tissue proteins (Mr 94 000, 67 000, 46 000, 25 000, 15 000) were common to all regions of the epididymis, whereas there was considerable variation between regions in secreted proteins (i.e., present in the incubation medium). Eighteen major proteins were identified in the incubation medium (Table 2). The most proximal regions of the epididymis where these were detected were the efferent ducts for seven proteins (Mr 94 000, 63 000, 50 000, 46 000, 42 000, 36 000, 26 000), region 1 for one protein (Mr 23 000), between regions 2 and 5 for four proteins (Mr 120 000, 115 000, 38 000, 20 000), region 6 for three proteins (Mr 88 000, 52 000, 22 000), and region 7 for three proteins (Mr 24 000, 15 000, 13 000) (Fig. 3 and Table 2). Most of the labeled proteins could be related by molecular weight to those identified in the micropuncture samples of epididymal luminal fluid (Table 2). However, not all proteins that were present in gels of native fluids were also present in gels of the incubation medium in the incorporation studies in vitro.



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  		FIG. 3. Fluorographs of SDS-polyacrylamide gels demonstrating the effects of orchidectomy and orchidectomy followed by exogenous administration of testosterone on the incorporation of 35S-methionine by epididymal tissue from the efferent ducts and region 1 (A), epididymal regions 2–5 and region 6 (B), and epididymal regions 7 and 8 (C). Minced tissue samples were incubated in vitro in incubation medium for 6 h at 33°C. Gels were run from homogenates of the tissue and incubation medium. I, Intact animals; C, animals castrated for 14 days; T, animals that were castrated for 14 days prior to supplementation with testosterone for 14 days. The numerical values correspond to the molecular weight markers of the standard (Mr x 10-3; left side) and major epididymal proteins for which amino acid sequence was obtained (right side). {blacktriangleright}, Secretion of protein inhibited by androgen; , secretion of protein stimulated by androgen; , secretion of protein stimulated by testicular factors other than an-drogen.

Consistent with its quantitative effects on label incorporation, orchidectomy had little effect on the profile of labeled proteins in tissue (Fig. 3), except that it inhibited the synthesis of at least one protein (Mr 25 000) in all regions of the epididymis. Testosterone therapy restored the synthesis of this protein. In contrast, the secretion of seven of the major proteins (Mr 52 000, 38 000, 23 000, 22 000, 20 000, 15 000, 13 000) was suppressed by orchidectomy but restored by testosterone therapy (except for the proteins of Mr 52 000, 23 000, 22 000). Further, orchidectomy stimulated secretion of three proteins in the efferent ducts and region 1 of the epididymis, and testosterone therapy reduced their secretion compared to intact animals.

Identification of Major Epididymal Proteins

Table 4 shows partial amino acid sequences and homologies of 7 major proteins in gels of luminal fluid from region 8 of the epididymis. These proteins had the same molecular weight as did proteins that were secreted by the epididymis (Table 2) and associated with sperm during epididymal transit (Mr 94 000, 52 000, 38 000, 36 000, 22 000, 20 000, 13 000). One protein (Mr 52 000) showed no homology with any other previously sequenced proteins.


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  		TABLE 4. Partial amino acid sequence and apparent homology of 7 proteins in luminal plasma from region 8 of the rabbit epididymis. These proteins have the same Mr as proteins that associated with sperm as they passed through the epididymis


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In earlier studies, regional variation in the occurrence of luminal proteins [8, 10, 11] and in the incorporation in vitro of radiolabel into protein [7, 12, 13, 25] has been demonstrated along the rabbit epididymis. However, this study is the first to demonstrate in the rabbit the natural occurrence of proteins along the extratesticular duct system and to relate this occurrence to protein synthesis and secretion in vitro. Table 2 summarizes the occurrence and regulation of epididymal proteins that were detected by the 2 methods and their relationship to epididymal proteins described in previous reports. Regional variation in the occurrence of proteins along the epididymis is mainly due to the secretion of novel proteins in different regions. The majority of new proteins are secreted in regions 2–7 of the epididymis, where the rate of protein synthesis is greatest. These studies are in agreement with reports on the mouse and rat [26, 27] in showing that some labeled proteins were released by minced duct from all regions of the epididymis but were secreted in vivo only in certain epididymal regions. This difference is probably due to differences between autoradiography and silver staining in sensitivity for detecting proteins. Although the procedures involved in in vitro incubation may affect protein synthesis and secretion, the effect must be minor because the findings using the two methods were fairly consistent.

The results of this study support the findings of Brooks and Higgins [21] in showing that the reduction in epididymal mass following orchidectomy was not associated with a concomitant reduction in the rate of protein synthesis per gram of tissue. However, the secretion of four proteins (Mr 38 000, 20 000, 15 000, 13 000) was controlled by androgen, their secretion being reduced or abolished by withdrawal of androgen and restored by replacement therapy. Further, the secretion of three proteins (Mr 52 000, 23 000, 22 000) was reduced or abolished by orchidectomy and not restored by testosterone therapy. Two of these proteins (Mr 54 000, 20 000) appear to correspond to two of the four proteins (Mr 60 000, 54 000, 43 000, 20 000) that Jones et al. [7] found to be secreted by the initial segments (regions 2–5), which are dependent on factors in luminal fluid from the testis [7, 12, 13, 25, 28]. The finding that the secretion of a protein (Mr 22 000) in region 7 is also dependent on luminal fluid is unusual but consistent with our findings for the tammar wallaby (Macropus eugeneii) that there is lumicrine regulation of protein secretion caudal to the initial segments [29]. In addition to the proteins mentioned above, two other proteins (Mr 36 000, 24 000) were secreted in increased amounts in response to androgen withdrawal, an effect consistent with previous work on the rabbit [13].

The comparison of membrane extracts of sperm from the rete testis and region 8 of the epididymis confirms that there are changes in sperm membranes during epididymal transit (reviewed in [1]). There is an apparent loss of five proteins (Mr 93 000, 61 000, 35 000, 33 000, 27 000), a gain of nine (Mr 94 000, 88 000, 63 000, 52 000, 38 000, 36 000, 22 000, 20 000, 13 000), and an increase in the staining intensity of several low molecular weight proteins. Although these modifications may have been mediated by alterations of preexisting sperm proteins, 7 of the new proteins were about the same molecular weight as major epididymal secretory proteins (Mr 94 000, 52 000, 38 000, 36 000, 22 000, 20 000, 13 000). The partial amino acid sequencing of these proteins indicated that all except the Mr 52 000 protein belong to classes that have been previously identified. The Mr 13 000 protein was identified as a homologue of a family of epididymal proteins that includes human epididymal protein HE1 [30, 31]. Members of this family are highly conserved among mammalian species [31], but their function is unknown.

The Mr 20 000 protein was identified as rabbit epididymal protein 20 (EP20 [32]), a protein that shares significant (86%) amino acid sequence homology with human zyxin 2, a component of adhesion plaques [33]. Zyxin is capable of acting as a specific protein-binding interface and mediating adhesion-stimulated changes in gene expression [34].

The Mr 22 000 protein was identified as BE-20 [35], a rabbit homologue of the human HE4 protein [30]. Structural homology between BE-20 and extracellular proteinase inhibitors of the four-disulfide core family indicates that the protein may be involved in protection and stabilization of the sperm acrosome [35]. A similar function has also been postulated for the Mr 36 000 and 94 000 proteins, which were identified as the small and large subunits (respectively) of the rabbit acrosome stabilizing factor (ASF). ASF is a 260-kDa glycoprotein composed of four individual subunits, two each of 38 kDa and 92 kDa [36, 37]. It coats sperm and can reversibly decapacitate them and block the acrosome reaction [38, 39].

The Mr 38 000 protein is homolgous with two rat testicular cDNAs, Odf2 and KTT4, which putatively encode outer dense fiber proteins [40, 41]. Expression of Odf2 and KTT4 mRNA is reported to be predominantly associated with the development of the sperm tail during spermatogenesis in the rat testis. Given that these proteins are expressed in different tissues in the rabbit and rat, they may play different roles.


   ACKNOWLEDGMENTS
 
The authors thank Mrs. Hannah Clarke for her excellent technical assistance and Mrs. Jill McGovern for help with N-terminal amino acid sequencing of the proteins.


   FOOTNOTES
 
First decision: 24 May 1999.

1 This work was supported by grants from the Australian Research Council, Research Management Committee, University of Newcastle, and the Vertebrate Biocontrol Cooperative Research Centre, Canberra, Australia. B.N. was supported by an Australian Postgraduate Award and a Vertebrate Biocontrol Cooperative Research Centre Student Award. Back

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

Accepted: February 22, 2002.

Received: April 8, 1999.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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