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....

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 Boui.......

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

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