Proteomic analysis of murine testes lipid droplets

Abstract

Testicular Leydig cells contain abundant cytoplasmic lipid droplets (LDs) as a cholesteryl-ester store for releasing cholesterols as the precursor substrate for testosterone biosynthesis. Here, we identified the protein composition of testicular LDs purified from adult mice by using mass spectrometry and immunodetection. Among 337 proteins identified, 144 were previously detected in LD proteomes; 44 were confirmed by microscopy. Testicular LDs contained multiple Rab GTPases, chaperones, and proteins involved in glucuronidation, ubiquination and transport, many known to modulate LD formation and LD-related cellular functions. In particular, testicular LDs contained many members of both the perilipin family and classical lipase/esterase superfamily assembled predominately in adipocyte LDs. Thus, testicular LDs might be regulated similar to adipocyte LDs. Remarkably, testicular LDs contained a large number of classical enzymes for biosynthesis and metabolism of cholesterol and hormonal steroids, so steroidogenic reactions might occur on testicular LDs or the steroidogenic enzymes and products could be transferred through testicular LDs. These characteristics differ from the LDs in most other types of cells, so testicular LDs could be an active organelle functionally involved in steroidogenesis.

Figure 1. Testicular lipid droplets (LDs) staining

A. Hematoxylin-eosin staining of mouse testicular tissue. The asterisk marks the interstitium between the seminiferous tubules in panel a. The amplified images of boxed area are in panel b. B. LDs stained with Nile Red in frozen testis sections. Nuclei were stained with Hoechst 33258. Panel a and b, Nild Red stained LDs. Panel c and d, the merged images. C. LDs purified from mice testes were spread on slides and stained with Lipid-TOX Deep Red.

Figure 2. Lipid and protein patterns in testicular LDs

A. Thin-layer chromatography analysis of total lipids extracted from LD preparations of mice testis, brown adipose tissue (BAT), and Chinese hamster ovary (CHO) cells. TG, triacylglycerols; CE, cholesteryl esters; EL, ether lipid. B. Silver-stained SDS-PAGE gels of protein extracts of different testicular lipid droplet preparations. C. Coomassie Blue-stained SDS-PAGE gels of proteins extracted from fractions of testicular LD, total membrane (TM), cytosol (Cyto), and post-nuclear supernatant (PNS). For the whole proteome, the lane running the testicular LD proteins was excised into 23 gel slices and underwent mass spectrometry.

Figure 3. Properties of murine testicular LD proteins

A. Protein categories of mouse testis LDs. All proteins identified by 2D-LC MS/MS were sorted by subcellular distributions and known functions based on literature or NCBI online sources. B. Network of function-related LD proteins. Lines in different colors represent functional association in various types of evidence. Red, fusion evidence; green, neighborhood evidence; blue, co-occurrence evidence; purple, experiment evidence; yellow, text-mining evidence; black, co-expression evidence.

Figure 4. Confirmation of LD proteins by immunodetection

A. The fractions of LD, total membrane (TM), cytosol (Cyto), and post-nuclear supernatant (PNS) were prepared from mice testes. An equal amount of proteins extracted from different fractions was separated by SDS-PAGE and underwent immunoblotting with the primary antibodies indicated. A representative silver-stained gel showed equivalent protein loading. Plin variants Plin1a~1d were detected on a full-length blot. The blots of proteins were derived from the sample or different samples that were processed in parallel, and the corresponding full-length blots are shown in Supplementary Figure S1. Arom, aromatase; Cav, caveolin; Plin, perilipin; HSL, hormone-sensitive lipase; ATGL, adipose triglyceride lipase. B. Immunofluorescent staining of Plin1 (a,c) Plin2 (e), and 3β-hydroxysteroid dehydrogenase (HSD3B1) (g) in sections of mouse testis. The merged images are shown in panel b,d,f and h.