Proteomic analysis of murine testes lipid droplets

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.

Purification of the LDs from mice testis. For each individual preparation, 20 testes obtained from 10-week-old C57BL/6 mice were used. LDs were purified by the protocol we developed recently 10 . Manipulations were performed at 4 °C or on ice, if required. After removal of blood vessels and connective tissues, 20 testes were grouped and homogenized by use of a Dounce glass homogenizer containing 10 ml buffer A (250 mM sucrose, 0.2 mM phenylmethylsulfonyl fluoride, 25 mM tricine, pH 7.6) by 20 strokes with a loose-fitting pestle and 40 strokes with a tight-fitting pestle. The homogenate was disrupted for 15 min at 750 psi in a nitrogen bomb chamber and cleaned by centrifugation at 3000 × g. The post-nuclear supernatant was transferred to a SW40 tube, then buffer B (20 mM HEPES, pH 7.4, 100 mM KCl and 2 mM MgCl 2 ) was loaded on top of the supernatant. After centrifugation at 38,000 × g for 1 h, a white LD layer appeared on the top of the tube. The membrane was pelleted at the bottom, and the infranatant was the cytosolic fraction. All 3 fractions were collected. The LD fraction was transferred to a new tube and centrifuged for 4 min at 14,000 × g. After removal of the underlying liquid, LDs were washed 3 times, each with 200 μ l buffer B and centrifuged at 14,000 × g for 4 min. The LD fraction on the top was collected.
Protein in-gel digestion and mass spectrometry analysis. Manipulations were performed as we reported recently 11 . Protein components in the LD preparation were precipitated with 100% acetone. Proteins were separated by 10% SDS-PAGE followed by Coomassie Blue or silver staining. For the total proteome, a full lane of Coomassie Blue-stained gel was cut into 23 slices from high to low molecular weight. Each slice was further cut into smaller pieces, destained, washed, dehydrated and vacuum-dried. Proteins in slices were reduced with 10 mM dithiothreitol for 1 h at 56 °C and alkylated with 55 mM iodoacetamide for 45 min. Gel slices were washed with 25 mM ammonium bicarbonate, acetonitrile and vacuum-dried. For in-gel digestion, slices were incubated with 10 ng/μ l trypsin in 25 mM ammonium bicarbonate solution. The digestion reaction proceeded at 37 °C overnight and was stopped by adding 5% formic acid to adjust pH to < 4.0. After two extractions with 60% acetonitrile, the tryptic peptide mixture was vacuum-dried and dissolved in 0.1% formic acid. Peptide extracts were purified on a C18 trap column and analyzed by use of a 2D-HPLC system coupled to a linear ion-trap mass spectrometer (Thermo Fisher Scientific, MA).
Immunoblotting. Proteins from the LD preparation were extracted with acetone, separated by 10% SDS-PAGE, and underwent immunoblotting analysis with primary antibodies, then horseradish peroxidase-conjugated lgG. The blots were developed with enhanced chemiluminescence detection reagents (Applygen Technologies, Beijing).

Histology and immunofluorescence.
Mice testes were fixed with 4% paraformaldehyde and embedded in paraffin and cut. For routine histology, sections were stained with hematoxylin-eosin. For immunofluorescence staining, sections were incubated for 10 min with 3% H 2 O 2 to eliminate endogenous peroxidase activity and underwent antigen retrieval with 0.3% sodium citrate and phosphate buffered saline, pH 7.4, for 15 min at 72 °C. Sections were blocked with 1% defatted albumin and immunostained with primary antibody, then FITC-labeled lgG. Signals were observed under a Nikon Eclipse 50i fluorescence microscope. LD staining. LDs in frozen testicular sections were stained with Nile Red. Nuclei were stained with Hoechst 33258. For in vitro staining, LDs purified from testicular tissue were spread on glass slides, dried, and stained with Lipid-TOX Deep Red. Fluorescent signals were viewed under an Olympus FV1000 confocal microscope.
Thin-layer chromatography. LDs were purified from brown adipose tissue and testes of mice and from cultured Chinese hamster ovary (CHO) cells. Total lipids in different LD preparations were extracted in chloroform and acetone (1:2, v/v) and centrifuged at 14,000 × g for 10 min. The organic phase was collected and dried under nitrogen gas. Lipid extracts were dissolved in chloroform and loaded on silica gel Scientific RepoRts | 5:12070 | DOi: 10.1038/srep12070 plates for analysis. Neutral lipids were separated on plates in a hexane:diethyl ether:acetic acid (80:20:1, v/v/v) solvent system and visualized by the iodine vapor method.
Data mining and bioinformatics. To obtain reliable results, we performed at least two biological replicates of proteomic analysis and results were combined for further analysis. The online database used to sort the proteomic table was http://genome.ucsc.edu/cgi-bin/hgNear. Protein associations were revealed by the Website program String (http://string-db.org/).

Results
Testicular LD staining. Interstitial cells were located in the interstitium between the seminiferous tubules of mouse testicular tissue (Fig. 1A, panel a and b). Numerous small, concrete LDs stained with Nile Red were observed in interstitial Leydig cells rather than in the cells located within the seminiferous tubules (Fig. 1B). Lipid-TOX staining showed that the LDs prepared for proteomic analysis were morphologically intact, with a diameter of about 1 μ m, despite the presence of a few large droplets (Fig. 1C).
Lipid and protein patterns of testicular LDs. Thin-layer chromatography revealed that mice testicular LDs consisted of a fairly equivalent amount of cholesteryl esters and triacylglycerols and a small amount of ether lipid, similar to steroidogenic CHO cells; by contrast, adipose LDs contained a large amount of triacylglycerols but few cholesteryl esters and ether lipid ( Fig. 2A). Equal amounts of protein extracted from different compartments were separated by SDS-PAGE. Silver staining revealed that the proteins in different LD preparations showed a highly consistent band pattern in gels (Fig. 2B), which indicated the reliability of the LD purification. In contrast, the protein band pattern of LD fractions differed from that of total membrane, cytosol, and post-nuclear supernatant fractions (Fig. 2C).
Proteomic analysis of testicular LD proteins. For the whole proteome of testicular LDs, the lane running testicular LD protein was excised into 23 gel slices (Fig. 2C). After in-gel digestion, tryptic peptides underwent mass spectrometry analysis. Only proteins with at least two unique peptides were accepted for identification. A total of 337 proteins were identified; at least 144 (42.7% of total) were previously reported in LD proteomes of other mammalian cells or tissues and 44 were previously confirmed in LDs by microscopy. Each identified protein and its encoding gene were searched in the UniProt and NCBI databases and PubMed. The 337 proteins were classified into 16 groups by known or putative functional annotation for identified proteins ( Fig. 3 and Table 1).
Group 1 proteins represented vimentin 12 and stomatin 13 and particularly Plin1, Plin2/ADRP, Plin3/ Tip47, and Plin4/S3-12, 4 classical LD proteins belonging to the perilipin family of 5 LD proteins (Plin1~5) conserved in their first ~100 amino-terminal residues 9 . Plin1 binds and links vimentin to LDs, then vimentin filaments wrap the LDs tightly in a cage-like spherical structure surrounded by multiple ER tubules, thus facilitating LD formation 12 . Plin2~4 widely express and localize at LDs and non-LD compartments, but Plin1 expresses exclusively in adipose and steroidogenic cells and localizes only at the LD surface 9 . Plin1~4 provide a barrier and protect LDs against access by HSL and adipose triglyceride lipase (ATGL), but native Plin1 is more protective than Plin2~4 14,15 . Interestingly, testicular LDs contained 4 variants of Plin1, termed Plin1a, 1b, 1c, and 1d, which share conserved N-terminal 198 residues and 11-mer regions. This was the first identification of Plin1d protein in the tissue (Table 1).
Group 2 included 7 lipases/esterases/thioesterases, which cover almost all currently known cellular lipases/esterases. HSL 14 , ATGL and its co-lipase CGI-58 represent more than 95% of the lipolytic activity in adipocytes 16 , with the remaining hydrolase activity contributed by triacylglycerol hydrolase/    Table 1. Proteins associated with testicular lipid droplets (LDs). A total of 337 proteins were identified from murine testicular LDs by mass spectrometry; 144 identified proteins had been previously detected in LD proteomic studies and are labeled with "#" and citations to annotate the tissue or cell source of the LDs. A total of 44 proteins had been previously confirmed in LDs by microscopy and are labeled with "&". a Comparison with the reference data involved manual inspection of the GI number and then the standard names of proteins identified in the present and previous proteomic studies. b The expectation value is a statistical term that allows for comparison of the reliability of results. Protein identifications were based on both the expectation value (< 10 −4 ) and the quality of MS/MS spectra of peptide fragments (> 3) identified.
Low expectation values correspond to confident identifications. carboxylesterase 3 17,18 and monoglyceride lipase 19,20 . LD-associated hydrolase (C2orf43 protein) is a cholesteryl ester hydrolase that normally localizes to the ER but is translocated to LDs on lipid loading 21,22 . ATGL expresses specifically in adipose tissue 23 , but HSL expresses primarily in both adipose and steroidogenic tissues. Group 3 proteins represented 22 enzymes involved in the metabolism of fatty acid and glycerolipids and as well as phospholipids and sterols. Five were previously observed in LDs by microscopy. Fatty acid transport protein 1 binds diacylglycerol acyltransferase 2 and colocalizes to the ER-LD interface to facilitate glycerolipid biosynthesis and LD expansion 24 . Long-chain acyl-CoA synthetase Acsl1 25 and Acsl3 25,26 , along with glycerol-3-phosphate O-acyltransferase (Gpat4), are normally localized in the ER microdomain but effectively translocated to nascent LDs to facilitate LD biosynthesis on lipid loading 25,26 . Acsl4 and fatty aldehyde dehydrogenase were morphologically localized in yeast LDs 27 and proteomically detected in LDs of CHO cells 28 , adipocytes 29 and mouse muscle 30 . Carnitine O-palmitoyltransferase 2, very-long-chain acyl-CoA dehydrogenase, and mitochondrial trifunctional enzyme subunit α were detected from mouse muscle LDs 30 . Fatty acid synthase was detected from LDs of granulosa steroidogenic cells from rat ovary 20 . Many proteins in this group are known to specifically or highly express in testes (Table 1).
Group 4 proteins represented 11 phospholipid metabolic enzymes; 3 were previously physiologically confirmed in LDs. Phospholipase B is highly expressed in testis and activated by sterol removal in murine sperm membrane, which localizes at the LD surface and hydrolyzes glycerophospholipids to facilitate the LD structure 31 . Cytosolic phospholipase A2 (cPLA2) is activated by extracellular stimuli-hydrolyzed arachidonic acids from the sn-2 position of glycerophospholipids; in turn, released arachidonic acids induce the translocation of cPLA2 to the ER and LD interface to regulate lipid synthesis and nascent LD formation 32,33 . Phosphocholine cytidylyltransferase binds to growing LDs and then catalyzes phospholipid synthesis and promotes LD expansion 34,35 . Phosphatidylglycerophosphate synthase 1 and phosphoinositide Scientific RepoRts | 5:12070 | DOi: 10.1038/srep12070 lipid phosphatase are highly expressed in testes, and phospholipase DDHD1 is required for spermatogenesis. The proteins in this group also participate in glycerolipid and sterol metabolism.
Group 5 contained 19 proteins that participate in biosynthesis and metabolism of cholesterol, retinol, and hormonal steroids; 6 were previously observed in LDs by microscopy and another 7 were previously detected in LD proteomes. Short-chain dehydrogenase/reductase 3 and retinol dehydrogenase 10 are reciprocally activated and on acyl ester biosynthesis, are translocated from the ER to LDs [36][37][38] .
Group 7 and 8 proteins included 29 enzymes involved in the metabolism of carbohydrate and tricarboxylic acid cycle. NADH-cytochrome b5 reductase was verified in LDs by microscopy 45 . Glutamate dehydrogenase, malate dehydrogenase, succinate dehydrogenase, lactate dehydrogenase, pyruvate kinase 2/3, and citrate synthase were previously reported in LD proteomes 11,20,51 . The identification of 17 other metabolic enzymes in testicular LDs is novel, which might reflect the close relationship between LDs and mitochondria in testicular cells 52 .
Group 9 proteins represented 28 small GTPases; 27 were previously reported in LD proteomes. In cells loaded with fatty acids, Rab5a 53 , Rab11a 53 , Arl2 GTPase Elmod2 54 , and Rab18 53,55 can localize to both the ER and LDs, where Rab18 recruits unknown effectors and microtubules to facilitate membrane trafficking and lipid exchange 53,55 . Testicular LDs might serve as a dock for various small GTPases for mediating Rab signaling.
Group 10 listed 30 protein chaperones; 18 were previously reported in LD proteomes. We previously showed that heat shock protein 70 (Hsp70) can translocate to adipocyte LDs on heat stimulation 56 . Spermatid-specific Hsp70, Hsp70.2 (Hspa2), T-complex protein 11, and protein disulfide-isomerase A3 (PDI3a) are testis-specific and play roles in spermatogenesis. PDI is a component of microsomal triacylglycerol transfer protein complex. T-complex protein 1 contains 8 distinct subunits to form a unique chaperone for escorting actin, tubulin, and numerous other proteins. In Leydig cells, the intermediate filaments of the cytoskeletons may bind to LDs 52 .
Group 12 contained 43 transport proteins; 16 were proteomically reported 11,30,40,58 and 5 were microscopically confirmed in LDs 59,60 . Coatomer protein complex I (COPI) and clathrin adaptor complex mediate intra-Golgi transport and retrograde transport from the Golgi to ER. Arf1/COPI complexes localize between the ER and LDs for targeting the triacylglycerol synthesis enzyme Gpat4 to the LD surface and bud 60-nm nanodroplets from the LDs. In cells loaded with fatty acids, both COPI and COPII (Sec23) structures tend to localize to discrete foci surrounding LDs to create a membrane bridge for transporting ATGL and Plin2 to nascent LDs 60 .
Group 13 contained 26 proteins involved in nucleotide-catabolic processes, such as ion transport, transcription, translation, and cell signaling. Nine proteins were detected by previous LD proteomes. Some proteins might not easily fit into this single category because of the divergence of protein functions. MAPK/ERK kinase 2 is colocalized with cPLA2 in LDs, then rapidly activates cPLA2 for releasing arachidonate from LDs 33 ; it is required for testosterone synthesis in Leydig cells. ATP synthase subunit α and sodium pump subunit α 1 and α 4 are expressed abundantly in testis and regulate spermatogenesis.
Group 14~16 included cytoskeletal proteins, testis-specific and miscellaneous proteins. Only 11 of these 81 proteins were previously reported in LD proteomes. The identification of albumin in the present and previous LD proteomes should represent a contamination because of its abundance in serum. The identification of testis-specific proteins could be due to the contamination or the difficult separation of these protein components from testicular LDs. For example, GAPDH2 and A-kinase anchor protein 3 and 4 participate in spermatogenesis, which can bind the cytoskeletal fibrous sheath and thus might be co-purified with LD-associated cytoskeletons. Also, these testis-specific or spermatogenesis-related proteins might exist in cellular subcomplex structures that associate with testicular LDs 52 .

Confirmation of testicular LD protein identification by immunoblotting and immunofluores-
cence. Some of the identified testicular LD proteins were confirmed by immunoblotting by using marker proteins corresponding to different cellular compartments (Fig. 4A). Four members of the perilipin family, Plin1~4, including the 4 variants of Plin1, Plin1a, 1b, 1c and 1d, were detected only in the LD fraction. This was the first immunodetection of Plin1d in tissue (Fig. 4A). Plin5 signal was not detectable in testicular LD extracts (data not shown), which is consistent with its low level of expression in non-oxidative tissues. ATGL and CGI-58 appeared only in the LD fraction; HSL and 3β -HSD1 were highly enriched in the LD fraction but also detectable in the membrane and cytosol compartments (Fig. 4A). Caveolin-1 and -3, caveolae marker proteins, were not identified in the testicular LD proteome (Table 1) but were immunodetected in the LD fraction or other cellular compartments (Fig. 4A). Aromatase, a cyp19 enzyme that converts androgen to estrogen in seminiferous epithelium, was marginally detected in the testicular LD fraction but appeared mainly in the membrane fraction (Fig. 4A). Lysosome protein Lamp-1, ER protein p62, and cytoplasmic enzyme GAPDH were not detected in the LD fraction. The ER chaperone GRP78 and mitochondrial protein Tim 23 were detected predominately in the membrane and post-nuclear supernatant fractions, but a small amount appeared in the LD fraction (Fig. 4A). Clearly, the isolated LD fraction of mice testes was largely free of other organelle contamination, although a small amount of the ER and mitochondria components might be introduced, likely because of their abundance or general interactions with LDs 61 . Furthermore, immunofluorescent signals of Plin1 appeared strongly in the interstitium of mice testis (Fig. 4B, panel a and c), and the fluorescent signal pattern was consistent with that of interstitial LDs stained with Nile Red (Fig. 1B, panel a and b). Immunofluorescent signals were weaker for Plin2 and 3β -HSD1 than Plin1 but still detectable in interstitial locations (Fig. 4B, panel e and g). The immunofluorescent signal for 17β -HSD11 was not detected (data not shown).

Discussion
We report the first proteomic analysis of LDs purified from adult mice testes. Testicular LDs contained 337 proteins; 144 were previously detected in LD proteomes and 44 were verified by microscopy. From the functions of identified proteins, testicular LDs showed several characteristics different from the LDs of most other cell types. Testicular LDs may be unique, biologically active cellular organelles that might have functional roles in the biosynthesis of hormonal steroids.
First, testicular LDs featured most Plin family and lipase/esterase superfamily proteins and various enzymes for biosynthesis and metabolism of glycerolipids and phospholipids. The classical LD proteins, Plin1~4 and 4 variants of Plin1, are crucial for regulating LD formation 9 . During LD expansion in differentiating adipocytes, nascent small LDs are coated with Plin3 and Plin4, medial-size LDs require both Plin2 and Plin1, and finally, Plin1 replaces Plin2 as a major coat of large LDs in mature adipocytes 62 . We previously revealed that Plin2 is degraded by the proteasome with the induction of Plin1 63 and if Plin1 is null for replacing Plin2, LD growth and adipocyte differentiation are retarded 64 . Different Plins target different types of LDs and have unique functions to govern triacylglyceride-cholesterol ester balance 15 . Plin1a and Plin1b favor triacylglyceride-rich LDs 15 , Plin1c and Plin4 prefer cholesteryl ester-rich LDs, but Plin2 and Plin3 show less specific localization to LDs 15 . Plin1 expresses exclusively in adipose and steroidogenic cells 9 . Thin-layer chromatography revealed that the LD of adipocytes was triacylglyceride-rich, so it associates mainly with Plin1a and Plin1b. In contrast, the testicular LD had a relatively equivalent proportion of triacylglycerides and cholesteryl esters. The accumulation of triacylglycerides promotes and stabilizes storage of cholesteryl esters within Leydig cells 5 . Likely, the coats of Plin1~4, including Plin1a~1d, could cooperatively manipulate the appropriate balance of cholesteryl ester-triacylglycerides in steroidogenic cells of testes.
Also, testicular LDs contained most of the known lipases/esterases/phospholipases and enzymes of glycerolipid and phospholipid metabolism. HSL and ATGL represent ~95% of the lipolytic activity in adipocytes 16 and the remaining activity is contributed by triacylglycerol hydrolase 17,18 and monoglyceride lipase 19 . We and others previously revealed that Plin1 phosphorylation induces the translocation of HSL from the cytosol to LDs 14,65 and also indirectly activates ATGL by unsequestering the ATGL coactivator CGI-58, hence conferring a full lipolytic reaction in adipocytes. HSL is stimulated by catecholamine, thyroxine, and glucocorticoid 66 , and in testes, HSL is activated by chorionic gonadotropin. Inactivation of ATGL causes diacylglyceride accumulation in testes 23 , but HSL ablation disables spermatogenesis and causes male infertility 67 . Despite these crucial roles of lipases, the control of lipolysis and even the catalog of lipases (except HSL) are largely unknown in testes. Although lipases can act on broad lipid substrates (e.g., glycerolipids in adipocytes), in Leydig cells, they predominately hydrolyze cholesterol esters to cholesterols for steroidogenesis 68 . Unlike testicular LDs, the LDs in other types of cells including adipocytes were not found to contain so many lipases/esterases and enzymes for glycerolipid and phospholipid metabolism. Likely, testicular LDs need to be accurately modulated by these different enzymes, to facilitate the biosynthesis and hydrolysis of cholesteryl esters and thereby ensure cholesterol supply for steroidogenesis in testes.
The second unique feature is that testicular LDs contained a large number of steroidogenic enzymes such as lanosterol synthase and demethylase, various hydroxysteroid and retinol dehydrogenases, and various glucuronidation enzymes. Currently, steroidogenic enzymes are known to locate in the ER and mitochondrial membranes and in the adjacent cytoplasm, where they catalyze different reactions, their substrates and products being shuttled between these compartments 47,69 . The enzymes identified in testicular LDs, such as short-chain dehydrogenase 3 37,38 , retinol dehydrogenase 10 36 , 17β -HSD11 41,42 , 3β -HSD1 39,40 , and NAD(P)H steroid dehydrogenase-like protein 43,44 , another 3β -HSD, can translocate from the ER membrane to the LD surface on acyl ester biosynthesis. The substrates, products and metabolites of steroidogenic reactions are mostly insoluble and cannot distribute and move freely in the cytoplasm but instead could be chaperoned and escorted by hydrophobic LDs. Thus, considering that testicular LDs are spatially close to the ER and mitochondria and contain so many steroidogenic Scientific RepoRts | 5:12070 | DOi: 10.1038/srep12070 enzymes at the oil-water interface, the present data suggests that testicular LDs could be a new compartment for carrying out steroidogenic reactions, more than just a simple pool of cholesterol substrates. At least, testicular LDs could be a chaperone vehicle to facilitate the biosynthesis of hormonal steroids, by transferring insoluble intermediate substrates and products between the mitochondria and the adjacent cytoplasm.
Third, testicular LDs contained large numbers of proteins involved in cellular signaling, chaperon, ubiquination, transport, cytoskeleton and spermatogenesis. Proteins in the GTPase superfamily and Rab GTPase subfamily were particularly abundant. Rab18 53,55 can recruit microtubules and localize between the ER and LDs to facilitate membrane trafficking and lipid exchange 53,55 . Ubxd8 and p97/VCP colocalize at the ER-LD interface and promote LD expansion by binding ATGL and inhibiting ATGL-mediated LD lipolysis 49 . Similarly, the vesicle transporters COPI and COPII are membrane bridges between the ER and LDs to deliver and modulate ATGL, Plin2 and Plin3 levels at nascent LDs 60 . Because many of these proteins may exist in large multicomponent complexes, their simultaneous identification from testicular LDs was not surprising. For example, Aup1 localizes to the ER and LDs and contributes to the formation of LDs that may temporarily store misfolded ER proteins under certain conditions 48,57 . Actually, Aup1 is a component of the Hrd1-Sel1l ER quality-control complex and physiologically associates with a hundred other proteins 48 . In comparison, testicular LDs contained at least dozens of Aup1-associated proteins 48 , such as Ubxd8, Ubxd2, p97/VCP, Atad3a, Sel1l, Ruvb-like 1, stomatin, ribophorin I, T-complex proteins, epoxide hydrolase, atlastin-3, Acsl3, pyruvate kinase 2/3, and PDI3a. In addition, testicular LDs contained many cytoskeletal proteins, which might not be simply considered contamination. In steroidogenic cells, the LDs and mitochondria are known to tightly attach to the cytoskeleton and intermediate filaments that are thought to mediate transport of cholesterol 70 . An example is vimentin filaments, which bind Plin1 and wrap LDs 12 . Vimentin ablation results in defective steroidogenesis in adrenocortical and granulosa cells 69 . Overall, these findings suggest that testicular LDs could participate initially in cellular signaling, chaperon, ubiquination, transport, cytoskeleton and spermatogenesis.
In summary, testicular LDs could be considered active cellular organelles participating in the regulation of multiple testicular functions. Plins and lipases/esterases/phospholipases could govern accurate control of the biosynthesis and hydrolysis of cholesteryl esters, thus ensuring appropriate cholesteryl ester-triacylglyceride balance and cholesterol supply for steroidogenesis. Notably, the association with various kinds of steroidogenic enzymes suggests that steroidogenic reactions might occur in testicular LDs or the steroidogenic enzymes and products could be transferred through testicular LDs. Because little was known about testicular LD proteins, the investigation of the roles of testicular LDs has been largely restricted to morphological observations. The present finding uncovers the full set of testicular LD proteins, for further examination of the functional roles of testicular LDs and their proteins in steroidogenesis and spermatogenesis in testes.