Analysis of Epididymal Protein Synthesis and Secretion

Summary

Here, we report the immunofluorescence localization of dynamin to illustrate the protocols for the detection of proteins in paraffin-embedded mouse epididymal sections and those of an immortalized epididymal cell line (mECap18). We also describe the protocols for the isolation of secretory proteins from both epididymal fluid and conditioned cell media.

Abstract

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation and storage of spermatozoa. Such complexity arises due to the combined secretory and absorptive activity of the lining epithelial cells. Here, we describe the techniques for the analysis of epididymal protein synthesis and secretion by focusing on the model protein family of dynamin (DNM) mechanoenzymes; large GTPases that have the potential to regulate bi-directional membrane trafficking events. For the study of protein expression in epididymal tissue, we describe robust methodology for immunofluorescence labeling of target proteins in paraffin-embedded sections and the subsequent detection of the spatial distribution of these proteins via immunofluorescence microscopy. We also describe optimized methodology for the isolation and characterization of exosome like vesicles, known as epididymosomes, which are secreted into the epididymal lumen to participate in intercellular communication with maturing sperm cells. As a complementary approach, we also describe the immunofluorescence detection of target proteins in an SV40-immortalized mouse caput epididymal epithelial (mECap18) cell line. Moreover, we discuss the utility of the mECap18 cell line as a suitable in vitro model with which to explore the regulation of epididymal secretory activity. For this purpose, we describe the culturing requirements for the maintenance of the mECap18 cell line and the use of selective pharmacological inhibition regimens that are capable of influencing their secretory protein profile. The latter are readily assessed via harvesting of conditioned culture medium, concentration of secreted proteins via trichloroacetic acid/acetone precipitation and their subsequent analysis via SDS-PAGE and immunoblotting. We contend that these combined methods are suitable for the analysis of alternative epididymal protein targets as a prelude to determining their functional role in sperm maturation and/or storage.

Introduction

The spermatozoa of all mammalian species acquire the potential to display forward progressive motility and to fertilize an ovum during their prolonged descent through the epididymis, a highly specialized region of the male extra-testicular duct system, which may take 7 - 14 days to navigate (depending on the species)¹. Due to the extreme condensation of the paternal chromatin and the shedding of the majority of cytoplasm that accompanies the cytodifferentiation of spermatozoa within the testes, their subsequent functional maturation is driven exclusively by their interaction with the epididymal microenvironment. This milieu is, in turn, created by the secretory and absorptive activity of the lining epididymal soma and displays an exceptional level of segment-segment variation¹. Thus, the most active segments in terms of protein synthesis and secretion are those located in the proximal portion of the epididymis (namely, the caput and corpus)². This activity mirrors the functional profile of spermatozoa, with the cells first beginning to display hallmarks of functional competence (i.e., progressive motility and the ability to bind to acid-solubilized zona glycoproteins) following their passage through the caput epididymis³. These functional attributes continue to develop before reaching optimal levels as the sperm reach the distal epididymal segment (cauda), wherein they are stored in a quiescent state in readiness for ejaculation. The formation and maintenance of this sperm storage reservoir is also intimately tied to the lining epithelium, which in the cauda is dominated by strong absorptive activity^4,5. Although anatomical differences have been reported^6,7,8, such regionalized division of labor appears to be a characteristic of the epididymis that is shared among the majority of mammalian species studied to date, including our own^9,10. Indeed, from a clinical perspective, it is known that epididymal dysfunction makes an important contribution to the etiology of male factor infertility¹¹, thus highlighting the importance of understanding the regulation of this specialized tissue.

It is therefore regrettable that our understanding of epididymal physiology, and the mechanisms that regulate the sequential phases of sperm maturation and storage within this tissue, remain to be fully resolved. Among the contributing factors, limiting advances in epididymal research are the overall complexity of this tissue and knowledge of the mechanisms that exert regulatory control over its luminal microenvironment. Anatomically, we know that beyond the distinction of caput, corpus and cauda segments, the epididymis can be further subdivided into several zones (Figure 1A), each separated by septa¹² and characterized by discrete profiles of gene/protein expression^{13,14,15,16,17,18}. Indeed, on the basis of detailed transcriptional profiling of segmental gene expression in the epididymis, as many as 6 and 9 distinct epididymal zones have been reported in the mouse and rat models, respectively^19,20. Such complexity presumably reflects the composition of the epididymal soma, a pseudostratified epithelium comprising numerous different cell types; each differing with respect to their abundance, distribution and secretory/absorptive activities along the length of the tract. Thus, principal cells are by far the most abundant epididymal cell type constituting upwards of 80% of all epithelial cells. Accordingly, principal cells are responsible for the bulk of epididymal protein biosynthesis and secretion⁵. In contrast, the clear cell population, which rank as the second most abundant cell type within the epididymal soma, are primarily involved in selective absorption of luminal components and the acidification of this microenvironment⁵. Adding another tier of complexity, androgens and other lumicrine factors of testicular origin exert differential control over each of these epididymal cell types depending on their positioning along the tract.

Despite the limitations imposed by such complexity, significant inroads continue to be made into resolving the mechanistic basis of epididymal function. A key to these studies has been the application of advanced mass spectrometry strategies to establish broad scale inventories of the epididymal proteome, in tandem with detailed analyses of individual proteins selected from among these initial surveys. An illustration of this approach is our recent characterization of the DNM family of mechanoenzymes in the mouse model²¹. Our initial interest in DNM was fueled by its dual action in the coupling of exo- and endocytotic processes. Building on these observations, we were able to demonstrate that the three canonical isoforms of DNM (DNM1 - DNM3) are highly expressed in the mouse epididymis and appropriately positioned to fulfill regulatory roles in protein secretion and absorption²¹. Moreover, we were able to clearly differentiate each DNM isoform on the basis of their cellular and sub-cellular localization, thus suggesting that they possess complementary, as opposed to redundant, activity within the epididymal epithelium²¹.

Here, we describe the experimental methodology employed for the study of DNM expression in the mouse epididymis with the hope that this information will find wider application in the characterization of alternative epididymal proteins and thus contribute to our understanding of the function of this important element of the male reproductive tract. Specifically, we describe the development of robust methodology for immunofluorescence labeling of target proteins in paraffin-embedded epididymal sections and the subsequent detection of the spatial distribution of these proteins via immunofluorescence microscopy. We further document our recently optimized protocols²² for the isolation and characterization of epididymosomes; small exosome-like vesicles that constitute key elements of the epididymal secretory profile and appear to hold a prominent role in promoting sperm maturation²³. As a complementary approach, we also describe the immunofluorescence detection of target proteins in an immortalized mouse caput epididymal epithelial (mECap18) cell line and the use of this resource as a model with which to explore the regulation of epididymal secretory activity in vitro.

Protocol

All experimental procedures involving animal tissue collection were approved by the University of Newcastle's Animal Care and Ethics Committee.

1. Immunofluorescence Staining of the Paraffin-embedded Epididymal Sections (Figures 1 and 2)

1. Immediately after the euthanasia of adult mice via CO₂ inhalation (Swiss mice, over 8 weeks old), carefully dissect the epididymis (using surgical scissors and tweezers) free of overlying connective tissue and fat and immerse in Bouin’s fixative solution (> ten times volume/tissue weight) for overnight fixation.
2. Wash the tissue with 70% ethanol with 2× changes daily for 2 days and then dehydrate through graded ethanol (70%, 95% and 100%) in preparation for infiltration and embedding into a paraffin block.
3. Section the paraffin blocks at a thickness of 4-6 µm and mount on the slides in preparation for immunofluorescence staining.
4. In a fume hood, dewax the epididymal paraffin sections by adding a sufficient amount of xylene to the slide jar to completely immerse the tissue section (3× 5 min each time).
5. Rehydrate the tissue sections by the immersion in graded ethanol solutions diluted in purified H₂O (100% ethanol 5 min, 100% ethanol 5 min, 90% ethanol 1 min, 80% ethanol 1 min, 70% ethanol 1 min, and 50% ethanol 1 min).
6. Wash the sections in a slide jar once for 5 min with sufficient phosphate buffered saline (PBS) to completely immerse the entire tissue section (follow these directions for all subsequent washes).
7. Decant appropriate antigen retrieval solution (i.e., 10 mmol/L sodium citrate, 50 mmol/L Tris pH 10.5 or alternative antigen retrieval solution(s), depending on the antigen to be detected) into a slide rack and microwave until boiling. Immerse the slides into this solution and subject the tissue sections to heat-induced antigen retrieval conditions optimized for individual antibodies (see Table 1).
   Caution: Ensure that the slides are fully immersed in antigen retrieval solution during the antigen retrieval process.
8. Remove the slide container from the microwave and cool to room temperature.
9. Rinse the slides with PBS and use a liquid-repellent slide marker pen to trace around the tissue section.
10. Place the slides in a humidified container (created by a moistened tissue at the base of the container), and apply blocking solution (3% BSA/PBS, previously filtered through a 0.45 µm filter) for 1 h at 37 °C.
11. Rinse the slides once with PBS.
12. Incubate the sections with appropriate primary antibody diluted to an experimentally optimized concentration in filtered 1% BSA/PBS at 4 °C overnight (1:60 for anti-DNM1, DNM2 and DNM3 antibodies; 1:100 for anti-ATP6V1B1 antibody, see Table of Materials for antibody details).
    Note: To distinguish specific from non-specific antibody binding, it is necessary to include stringent negative (i.e., secondary antibody only, primary antibody preabsorbed against immunizing peptide) and positive controls²⁴.
13. Rewarm the slides by placing at room temperature for 30 min.
14. Wash the slides 3× with PBS on a shaking platform (60 rpm) for 10 min each.
15. Incubate the sections with appropriate secondary antibody diluted in 1% BSA/PBS (filtered through a 0.45 µm filter) at 37 °C for 1 h (1:400 dilution for all secondary antibodies, see Table of Materials for antibody details).
    CAUTION: Keep the slide container in the dark from this step onwards. For dual labeling, choose a compatible combination of secondary antibodies (i.e., secondary antibodies must have been raised in different species).
16. Wash the slides 3× with PBS on a shaking platform (60 rpm) for 10 min each.
17. Counterstain the sections with propidium iodide (PI, 7.48 μmol/L) or 4΄,6-diamidino-2-phenylindole (DAPI, 4.37 μmol/L) for 2 min at room temperature to label the cell nucleus.
18. Wash the slides twice with PBS on a shaking platform (60 rpm) for 5 min each.
19. Mount the sections with 10% Mowiol 4-88 prepared in a solution of 30% glycerol in 0.2 mol/L Tris (pH 8.5) and 2.5% 1, 4-diazabicyclo-(2.2.2)-octane.
20. Seal the coverslip with nail varnish and store the slides at 4 °C for future observation.
    CAUTION: It is recommended to perform the imaging of the slides as soon as practical after the preparation to avoid excessive loss of fluorescence.

2  Isolation of Epididymosomes from the Mouse Caput Epididymis (Figure 3)

1. Immediately after the euthanasia of adult mice via CO₂ inhalation (Swiss mice over 8 weeks old), perfuse their vasculature with PBS (pre-warmed to 37 °C) to minimize the blood contamination of epididymal tissue.
   CAUTION: Blood plasma contains diverse populations of exosomes, which are of similar size to epididymosomes²⁵. The efficacy of blood clearance from epididymal tissue can be accessed via the inspection of the initial segment, a highly vascularized epididymal segment located proximal to the caput segment (i.e., zone 1 in Figure 1A)
2. Carefully dissect the epididymis free of overlying fat and connective tissue, and rinse with modified Biggers, Whitten, and Whittingham medium (BWW; pH 7.4, osmolality of 300 mmol/kg water^26,27) to reduce any potential for surface blood contamination.
3. Blot the epididymal tissue to remove excess media, dissect the caput epididymis (i.e., zones 2-5 in Figure 1A) and transfer to a fresh Petri dish (35 × 10 mm) containing BWW medium. Ensure that the amount of medium is sufficient for the final recovery.
   Note: For 6 caput epididymides, it is recommended to use 1.1 mL of the medium to allow for a recovery of ~900 µL, which is then evenly split and applied atop of 2 pre-prepared gradients (see step 2.9).
4. Make a number of small incisions into the caput tissue with a razor blade. Do not mince the tissue and thus avoid contaminating the sample with excessive cytosolic contents. Incubate the plate containing the tissue with mild agitation at 37 °C for 30 min to release the luminal contents.
5. Filter the resultant suspension through 70 µm membranes to remove the cellular debris.
6. Collect the filtrate and subject this to successive centrifugation steps at 4 °C with increasing velocity in order to eliminate cellular debris (i.e., 500 × g, 2,000 × g, 4,000 × g, 8,000 × g, 5 min each; 17,000 × g for 20 min, and finally 17,000 × g for an additional 10 min or until no pellet is formed after centrifugation).
   CAUTION: It is important to assess the color of the pellet after the initial 500 × g centrifugation step to ensure minimal blood contamination is present. Discard any samples in which this pellet displays pink coloration.
7. Prepare discontinuous iodixanol gradients (comprising 40%, 20%, 10%, 5% layers) by diluting a density gradient medium (comprising 60% (w/v) aqueous iodixanol) with a solution of 0.25 mol/L sucrose and 10 mmol/L Tris (pH 7.5).
8. Prepare the gradient in an ultracentrifuge tube (11 × 35 mm), with each fraction of 450 µL (Figure 3). Visually inspect the gradient after the application of each fraction to ensure that the interfaces are successfully formed between each layer prior to loading the epididymal fluid sample. Prepare each gradient fresh on the day of use, however, the epididymal luminal fluid sample can be preserved at 4 °C for up to 2 h prior to loading.
9. Carefully add 450 µL of epididymal luminal fluid suspension (corresponding to the material collected from the caput of 3 epididymides) atop of a single gradient.
10. Ultracentrifuge the gradients at 160,000 × g at 4 °C for 18 h.
    CAUTION: Since this centrifugation is conducted at very high speed, all ultracentrifuge tubes must be paired and balanced precisely. Check the tubes to ensure that they are free of any visible damage that could compromise their integrity.
11. Gently remove 12 equal fractions (each consisting of 185 µL) starting from the uppermost layer and progressing toward the bottom of the gradient. Pool the equivalent fractions recovered from each gradient if applicable (up to two gradients).
    Note: Mouse epididymosomes are most highly enriched in fractions 9 - 11²², see Figure 4 and Discussion.
12. After the recovery and pooling of fractions 9 – 11, dilute into 2 mL of PBS, and ultracentrifuge the samples at 100,000 × g at 4 °C for 3 h (13 × 56 mm tube) to pellet the epididymosomes.
    CAUTION: Since the epididymosome pellet can be difficult to see, ensure that the orientation of the tubes is noted as they are placed into the rotor and mark the tube to indicate the expectant position of the epididymosome pellet. Ensure that each tube contains a sufficient volume (i.e., exceeding 50% of its total capacity) to preclude the risk of tube collapse.
13. Carefully aspirate and discard the supernatant without disturbing the epididymosome pellet.
14. Assess the epididymosome purity (Figure 4).
15. Resuspend the epididymosome pellet into desired medium according to the downstream application(s). For instance, BWW medium is generally used for the experiments involving co-incubation with spermatozoa or alternatively an appropriate lysis buffer in preparation for the resolution of the epididymal proteome via SDS-PAGE.

3. Immunofluorescence Staining of mECap18 Cells

1. Preparation of sterile coverslips (to be conducted in a cell culture hood)
   1. Soak the coverslips (12 × 12 mm) in 70% ethanol for 10 min and disinfect by drying under high temperature above an ethanol lamp.
   2. Cool the coverslip for 10 s before transferring to a 12 well plate.
   3. Apply sterile poly-L-lysine solution to cover the coverslip and settle for 10 min at room temperature.
   4. Discard the poly-L-lysine solution and rinse the coverslip with sterile H₂O or appropriate medium.
2. Preparation mECap18 cells
   1. Passage the aliquots of 2 × 10⁵ mECap18 cells in each well of the 12 well plate containing the coverslips.
   2. Culture the cells with mECap18 cell medium (DMEM supplemented with 1% L-glutamine, 1% sodium pyruvate, 1% penicillin/streptomycin, and 50 μmol/L 5α-androstan-17β-ol-3-oneC-IIIN) containing 10% fetal calf serum (FBS) in a 37 °C incubator under an atmosphere 5% CO₂ overnight.
   3. Once the cells adhere to the coverslip, discard the medium and rinse the cells twice with PBS.
   4. Add a sufficient amount of 4% paraformaldehyde (PFA) diluted in PBS to immerse the entire coverslip and fix the cells at room temperature for 15 min.
   5. Discard the PFA solution and rinse the coverslips twice in PBS.
3. Immunofluorescence staining
   1. Permeabilize mECap18 cells by immersion in 0.1% Triton X-100 in PBS for 10 min.
   2. Rinse the coverslips with PBS.
   3. Block mECap18 cells with 3% BSA and proceed with immunolabeling of cells utilizing equivalent protocols to those described for epididymal tissue sections.

4. Isolation of Proteins from Conditioned Cell Culture Medium

1. Collection of conditioned cell culture medium
   1. Passage aliquots of 4 × 10⁵ mECap18 cells in each well of 6 well plate with mECap18 cell medium supplemented with 10% FBS for 24 h.
   2. Wash mECap18 cells three times with mECap18 cell medium (prepared without FBS) to remove residual FBS and any associated protein contaminants.
   3. Add 1.5 mL of mECap18 cell medium (prepared without FBS) to each well and incubate with mECap18 cells for 12 h in a 37°C incubator under 5% CO₂.
      Note: mECap18 cells at this step can be assessed for different target antigens according to experimental design.
   4. After 12 h incubation, collect the cell medium and centrifuge at 2,000 × g for 10 min to remove all cellular debris.
      Note: The duration of incubation is able to be altered in accordance with experimental design/endpoint assessment and in consideration of the cell’s tolerance to applied treatment(s). It is recommended to tailor the timing of incubation based on specific experimental regimens to ensure that optimal results are achieved.
   5. Assess mECap18 cell viability via the application of a standard trypan blue exclusion assay²⁸. Discard all material in which cell viability has declined below 90% to eliminate bias introduced by proteins released from dead or moribund cells.
   6. Isolate proteins from cell medium as follows or preserve medium at -80 °C.
2. Protein isolation (to be conducted in a fume hood)
   1. Add 20% volume of chilled 100% trichloroacetic acid to 80% volume of conditioned cell medium to precipitate the proteins released from the cultured mECap18 cells. Incubate at 4 °C overnight with constant mixing.
   2. After the incubation, pellet precipitated protein by centrifugation (17, 000 × g, 4 °C for 10 min).
      Note: Due to the limited quantity of protein expected to be secreted into the medium, it is possible that the pellet will not be easily visualized after centrifugation. It is therefore imperative to correctly orientate the tube prior to the centrifugation and take care not to disturb the expectant pellet location during removal of the supernatant.
   3. Discard the supernatant and wash the pellet twice with chilled acetone prior to the re-centrifugation (17, 000 × g, 4 °C for 10 min).
   4. Carefully remove and discard the supernatant before air-drying any residual acetone within a fume hood.
   5. Resuspend the protein pellet in an appropriate extraction buffer in preparation for endpoint analysis to detect complete secretory protein profiles and/or individual target proteins (e.g., SDS-PAGE, immunoblotting).

Results

Figure 1 and Figure 2 show representative results of immunofluorescence localization of DNM in the mouse caput epididymis. Each of the three DNM isoforms investigated display distinct localization profiles. Thus, DNM1 is characterized by relatively modest diffuse labeling of the epididymal cells throughout the initial segment and caput epididymis (Figure 2A). By contrast, the DNM2 is...

Discussion

These studies incorporated the use of Bouin's fixed epididymal tissue that had been subjected to paraffin embedding and standard sectioning protocols. Bouin's fixative solution comprises a mixture of formaldehyde, picric acid and acetic acid, with each component having a specific and complementary function. Thus, formaldehyde reacts with primary amines to form protein cross-links, picric acid slowly penetrates the tissue forming salts and hence coagulation of basic proteins and conversely, acetic acid rapidly pen...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dynamin 1 antibody	Abcam	ab108458	Host species: Rabbit, Isotype: IgG, Class: polyclonal
Dynamin 2 antibody	Santa Cruz	sc-6400	Host species: Goat, Isotype: IgG, Class: polyclonal
Dynamin 3 antibody	Proteintech	14737-1-AP	Host species: Rabbit, Isotype: IgG, Class: polyclonal
ATP6V1B1 antibody	Santa Cruz	sc-21206	Host species: Goat, Isotype: IgG, Class: polyclonal
CD9 antibody	BD Pharmingen	553758	Host species: Rat, Isotype: IgG, Class: monoclonal
Flotillin-1 antibody	Sigma	F1180	Host species: Rabbit, Isotype: IgG, Class: polyclonal
ALOX15 antibody	Abcam	ab80221	Host species: Rabbit, Isotype: IgG, Class: polyclonal
TUBB antibody	Santa Cruz	sc-5274	Host species: Mouse, Isotype: IgG, Class: monoclonal
PSMD7 antibody	Abcam	ab11436	Host species: Rabbit, Isotype: IgG, Class: polyclonal
Anti Rabbit Alexa Fluor 488	Thermo	A11008	Host species: Goat, Isotype: IgG, Class: polyclonal
Anti Goat Alexa Fluor 488	Thermo	A11055	Host species: Donkey, Isotype: IgG, Class: polyclonal
Anti Goat Alexa Fluor 594	Thermo	A11058	Host species: Donkey, Isotype: IgG, Class: polyclonal
Anti Rat Alexa Fluor 594	Thermo	A11007	Host species: Goat, Isotype: IgG, Class: polyclonal
Anti Rabbit HRP	Millipore	DC03L	Host species: Goat, Isotype: IgG, Class: polyclonal
Anti Rat HRP	Millipore	DC01L	Host species: Goat, Isotype: IgG, Class: polyclonal
Anti Mouse HRP	Santa Cruz	sc-2005	Host species: Goat, Isotype: IgG, Class: polyclonal
4', 6-diamidino-2-phenylindole (DAPI)	Sigma	D9564
propidium iodide (PI)	Sigma	P4170
Mowiol 4-88	Calbiochem	475904
Bovine serum albumin (BSA)	Sigma	A7906
fetal bovine serum (FBS)	Bovogen	SFBS-F
DMEM	Thermo	11960-044
L-glutamine	Thermo	25030-081
penicillin/streptomycin	Thermo	15140-122
5α-androstan-17β-ol-3-oneC-IIIN	Sigma	A8380
sodium pyruvate	Thermo	11360-070
Trypsin-ethylenediaminetetraacetic acid (EDTA)	Sigma	T4049
Paraformaldehyde (PFA)	EMS	15710
Xylene	VWR Chemicals	1330-20-7
Ethanol	VWR Chemicals	64-17-5
Phosphate buffered saline (PBS)	Sigma	P4417
Sodium citrate	Sigma	S1804
Tris	Astral	0497-5KG
Glycerol	Sigma	G5516
1, 4-diazabicyclo-(2.2.2)-octane	Sigma	D2522
Poly-L-gysine	Sigma	P4832
Triton X-100	Sigma	78787
Trypan blue	Sigma	T6146
Trichloroacetic acid	Sigma	T9159
Acetone	Ajax Finechem	A6-2.5 L GL
Sucrose	Sigma	S0389
Poly (vinyl alcohol)	Sigma	P8136
D-Glucose	Ajax Finechem	783-500G
OptiPrep Density Gradient Medium	Sigma	D1556
Fluorescence microscopy	Zeiss	Zeiss Axio Imager A1
Ultracentrifuge	BECKMAN COULTER	Optima Max-XP
Microcentrifuges	Eppendorf	5424R
Incubator	Heracell	150
Large Orbital Shaker	Ratek	OM7
Microwave	LG	MS3840SR /00
Lab pH Meter	MeterLab	PHM220
Liquid-repellent slide marker	Daido Sangyo	Mini
Coverslip	Thermo	586
6 well plate	CELLSTAR	657160
12 well plate	CELLSTAR	665180
Slide	Mikro-Glass	SF41296PLMK
0.45 µm filter	Millox-HV	SLHV033RS
Kimwipes Dustfree Paper	KIMTECH	34155
Ultracentrifuge tube (2.2 ml, 11 × 35 mm)	BECKMAN COULTER	347356
Ultracentrifuge tube (3.2 ml, 13 × 56 mm)	BECKMAN COULTER	362305
Cell strainer 70 µm Nylon	FALCON	352350
Petri dish 35 × 10 mm with cams	SARSTED	82.1135.500
Slide jar	TRAJAN	#23 319 00