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Developmental Biology

Analysis of Epididymal Protein Synthesis and Secretion

Published: August 25th, 2018

DOI:

10.3791/58308

1Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, University of Newcastle, 2Hunter Medical Research Institute, 3Department of Physiology, Turku Center for Disease Modeling, Institute of Biomedicine, University of Turku, 4School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle

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.

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.

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....

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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 Boui.......

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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.......

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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.......

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The authors would like to acknowledge the National Health and Medical Research Council of Australia Project Grant APP1103176 for the support of this work.

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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

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