Analysis of Epididymal Protein Synthesis and Secretion

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.

Limitations to intergenerational inheritance: subchronic paternal stress preconception does not influence offspring anxiety

“…The SV40-immortalized mouse caput epididymal epithelial (mECap18) cell line was provided as a kind gift from Dr. Petra Sipila (Turku University, Turku, Finland). mECap18 cells were cultured in DMEM (4.5 g/L glucose) supplemented with 100 μM sodium pyruvate, 200 μM L-glutamate, 100 U/mL penicillin, 10 μg/mL streptomycin, and 10% v/v heat inactivated fetal bovine serum with 50 nM 5α-androstan-17β-ol3-one with passaging as required 52 . Cells were plated and allowed to grow for 72 h prior to treatment with 100 nM 4-pregnen-11β, 21-diol-3, 20-dione 21-acetate (Q1570-000, Steraloids Inc, Rhode Island, USA) for up to 10 days with media replacement occurring at intervals of 48 h. Following treatment, cells were harvested at days 3, 5, 7 and 10 prior to performing RNA extraction.…”

A Kinase Anchor Protein 4 Is Vulnerable to Oxidative Adduction in Male Germ Cells

“…The resulting suspensions were next placed atop a 27% Percoll density gradient and subjected to centrifugation at 400 × g for 15 min at room temperature (RT). The pellet, consisting of an enriched population of >95% corpus spermatozoa was resuspended in fresh BWW and then re-centrifuged at 400 × g for 2 min at RT to again pellet the cells and to allow for the removal of excess Percoll (Zhou et al, 2018). These cell preparations were then pooled with those of the cauda epididymis and assessed for viability and motility as previously described (Zhou et al, 2018), prior to being allocated to appropriate treatment groups in preparation for further analysis.…”

Polarized epithelium-sperm co-culture system reveals stimulatory factors for the secretion of mouse epididymal quiescin sulfhydryl oxidase 1

“…These protein IDs were converted into UniProt gene IDs for future categorization and molecular function annotation studies. Protein IDs that reached the above-mentioned requirements were first categorized into three major subgroups, (1) sperm-origin, (2) epithelium/luminal fluid-origin, and (3) uncategorized, based on published literature [ 2 , 4 , 7 , 25 , 26 , 27 , 28 ].…”