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Endocrinology, doi:10.1210/en.2003-1332
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Endocrinology Vol. 145, No. 3 1419-1426
Copyright © 2004 by The Endocrine Society

A Functional Cytochrome P450 Lanosterol 14{alpha}-Demethylase CYP51 Enzyme in the Acrosome: Transport through the Golgi and Synthesis of Meiosis-Activating Sterols

M. Cotman, D. Jezek, K. Fon Tacer, R. Frangez and D. Rozman

Laboratory for Genetics (M.C.) and Institute of Physiology, Pharmacology, and Toxicology (R.F.), Veterinary Faculty, and Medical Center for Molecular Biology (K.F.T., D.R.), Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Solovenia; and Institute of Histology and Embryology (D.J.), School of Medicine, University of Zagreb, Zagreb HR-100000, Croatia

Address all correspondence and requests for reprints to: Professor Damjana Rozman, Ph.D., Medical Center for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia. E-mail: damjana.rozman{at}mf.uni-lj.si.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mammalian lanosterol 14{alpha}-demethylase (CYP51) is a microsomal cytochrome P450 that demethylates lanosterol to FF-MAS, an oocyte meiosis-activating sterol and late intermediate of cholesterol biosynthesis. Herein we report CYP51 unequivocally localized to acrosomal membranes of male germ cells in mouse, bull, and ram, in which it synthesizes FF-MAS in the presence of the acrosomal form of nicotinamide adenine dinucleotide phosphate reduced-P450 reductase. In the mouse, CYP51 (53 kDa) resides in endoplasmic reticulum (ER) and Golgi during all phases of acrosome development, indicating an intracellular transport from ERs through the Golgi to the acrosome. CYP51 (50 kDa) also resides on acrosomal membranes of bull- and ram-ejaculated sperm. In mouse liver, a 53-kDa CYP51 is no longer detected in trans Golgi, suggesting retrieval back to the ER and no further transport to other organelles. Glycosylated high-molecular-mass CYP51-immunoreactive proteins in acrosomal membranes of bull and ram and Golgi-enriched fractions of mouse liver indicate that mammalian CYP51s are subjected to posttranslational modifications in the Golgi. In conclusion, CYP51 is the first cytochrome P450 enzyme to be detected on acrosomal membranes. It exhibits a unique, cell-type-specific intracellular transport that is in agreement with its cell-type-specific physiological role: production of cholesterol in the liver and sterols with signaling properties in sperm. Demethylation of lanosterol to FF-MAS by the acrosomal lanosterol 14{alpha}-demethylase enzyme complex demonstrates for the first time the ability of ejaculate sperm to synthesize meiosis-activating sterols.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENZYMES OF THE cytochrome P450 superfamily are generally located in membranes of the endoplasmic reticulum (microsomal P450s) or inner mitochondrial membranes (1). Lanosterol 14{alpha}-demethylase (CYP51) is a microsomal cytochrome P450 that in the presence of nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase, a ubiquitous P450 redox partner and a microsomal electron transferring protein, NADPH and molecular oxygen, removes the 14{alpha}-methyl group from lanosterol to produce 4,4-dimethyl-5{alpha}-cholesta-8,14,24-diene-3ß-ol or follicular fluid (FF) meiosis-activating sterol (MAS). FF-MAS and testis MAS (T-MAS), the product of the subsequent sterol {Delta}14-reductase reaction, accumulate in female and male gonads, respectively, whereas in the liver they represent barely detectable intermediates in the cholesterol biosynthesis (2, 3). MASs stimulate the reinitiation of meiosis in mouse oocyte in vitro (4) and are believed to have important roles in fertilization (5). Our earlier data show a stage-specific expression of CYP51 mRNA (6) and protein in rat and mouse testis. The highest level of CYP51 protein was observed in step 3–19 spermatids with large amounts of the protein in cytoplasm/residual bodies of step 19 spermatids of the rat. Using classical immunohistochemistry, CYP51 was localized not only to the cytoplasm of male germ cells in which endoplasmic reticulum resides but also to the acrosomal region of round and elongating spermatids (7).

The acrosome is a secretory vesicle containing a number of hydrolytic enzymes that help the sperm to penetrate the egg coat. The organelle seems to be derived from the Golgi apparatus (8, 9, 10). The acrosome of mature sperm has a sac-like shape and is surrounded by inner and outer acrosomal membranes. The formation and evolution of the acrosomal system takes place during four distinct phases of spermiogenesis. In the first Golgi phase, proacrosomal granules are formed from trans-Golgi segments and accumulate in the medullary region. In the second cap phase, the spherical acrosomal granule grows and flattens over the surface of the nucleus. In the third acrosomal phase, the dense acrosomic granule applies itself to the inner acrosomal membrane and becomes hemispherical. At the final maturation phase, acrosome is condensed, several antigens are modified (8), and many proteins are shed, together with organelles, in the residual body. In early stages of spermiogenesis, many Golgi resident proteins are present in the acrosome. For example, in round spermatids, giantin, ß-COP, Golgin-97, and Golgin-95 are localized on Golgi apparatus and membranes surrounding the acrosome (11). In elongating spermatids these proteins are no longer found in the acrosome, suggesting that they may have been retrieved into the Golgi.

Herein we report the first cytochrome P450 enzyme with the acrosomal localization and functional activity. The acrosomal form of NADPH-P450 reductase is also described for the first time. Demonstration that ejaculate sperm can synthesize MAS in situ is of particular importance in better understanding the physiological role of meiosis signaling sterols mammalian reproduction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
All animal experimentation described has been conducted in accordance with accepted standards of humane animal care. In accordance with the Amsterdam protocol on animal protection and welfare, animal experiments were kept to a minimum and performed by competent authorized persons in accordance with the license for the authorization of experiments on animals for scientific and research purposes enacted by Veterinary Administration of the Republic of Slovenia on the basis of Slovenian Protection of Animals Act (98/99) and instructions on the conditions for the authorization of experiments on animals for scientific and research purposes (40/85).

Preparation of mouse liver microsomes and Golgi fractions
Subcellular fractions and protein extracts were prepared from 10 sexually mature CBA male mice, following modified protocols for microsomes (6) and Golgi fractions (12). Livers were washed three times with 0.25 M sucrose, minced with razor blades, diluted with assay buffer C [100 mM KPO4 (pH 7.4), 1 mM EDTA, 4 mM MgCl2 in 0.25 M sucrose] and homogenized by 10 strokes with a hand homogenizer on ice. Cell debris was removed by centrifugation for 10 min at 10,000 x g and 4 C. Microsomal proteins were prepared by spinning the supernatant at 100,000 x g and 4 C for 90 min. The microsomal pellet was resuspended in buffer D [100 mM KPO4 (pH 7.4), 0.1 mM EDTA, 20% glycerol]. For further isolation of Golgi fractions, the pellet was resuspended in 8 ml 1.22 M sucrose, and the suspension was placed on the bottom of a centrifuge tube. Sucrose gradient composed of 8 ml 1.15 sucrose, 8 ml 0.86 M sucrose, 7 ml 0.6 M sucrose, and 7 ml 0.25 M sucrose was overlaid and centrifuged at 82,000 x g for 3 h at 4 C in SW28 rotor (Beckman, Palo Alto, CA). Fractions between 0.25 and 0.6 M sucrose (G3), 0.6 and 0.85 M sucrose (G2), and 0.86 and 1.15 M sucrose (G1) were collected separately, diluted to a final 0.25 M sucrose concentration, and centrifuged at 105,000 x g for 90 min. Pellets were resuspended in buffer D.

Preparation of mouse germ cell microsomes
Germ cells from 10 sexually mature CBA male mice were prepared as described previously (6). Decapsulated testes were minced with an array of scaled razor blades and treated with DNase and trypsin. This procedure destroys Leydig and Sertoli cells, but germ cells remain intact. Germ cells were pelleted, washed, and suspended in 5 ml assay buffer [100 mM KPO4 (pH 7.4), 0.1 mM EDTA, 20% glycerol] and hand homogenized on ice by at least 70 strokes. After centrifugation at 1500 x g for 10 min at 4 C, the supernatant was centrifuged at 100,000 x g for 1 h. The pellet was resuspended in assay buffer.

Isolation of acrosomal membranes from bull and ram sperm
Isolation of sufficient amounts of acrosomal membrane proteins from mouse sperm is practically impossible. Consequently we isolated acrosomal membrane from the bull and ram sperm. Semen was collected from seven Simmentall bulls and 15 rams (Jezerska-Solèavska breed). Acrosomal membranes from bull and ram spermatozoa were isolated by a modified protocol of Zahler and Doak (13). Thirty milliliters of semen were diluted with 2 volumes of PBS buffer containing 2.77 mM fructose. Each 15 ml of the diluted semen were laid on 20 ml of 1.3 M sucrose/0.9% NaCl and centrifuged at 1500 x g 30 min at room temperature. Pellets were resuspended in 108 ml of 0.9% NaCl/5 mM HEPES (pH 7.0). Aliquots of 18 ml of sperm suspension were laid on 20 ml of 1.3 M sucrose/0.9% NaCl and centrifuged 20 min at 20,000 x g in a Beckman SW 28 rotor at 4 C. Pellets were resuspended in 60 ml of 0.9% NaCl/5 mM HEPES (pH 7.0) and hand homogenized in a glass-Teflon homogenizer by 100 strokes. Ten milliliters of homogenate were placed on a sucrose gradient consisting of 14 ml of 1.75 M sucrose/0.9% NaCl and 14 ml of 1.3 M sucrose/0.9% NaCl and centrifuged for 3 h at 100,000 x g in a Beckman SW 28 rotor at 4 C. Acrosomal membranes in band of the 1.3 M/1.75 M sucrose interface were collected, diluted with an equal volume of 0.9% NaCl/5 mM HEPES, and centrifuged for 30 min at 100,000 x g in Beckman 70 Ti rotor at 4 C. Final pellet was resuspended in buffer D.

Immunoblot analysis
Proteins were separated on 8% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred by the Sammy Dry electroblotting apparatus (Schleicher & Schuell, Dassel, Germany) to Optitran BA-S 83 nitrocellulose membranes using three discontinuous transfer buffers at 2.5 mA/cm2. The membrane was incubated for 1 h in 10 ml 5% enhanced chemiluminescence blocking agent (Amersham, London, UK) in Tris-buffered saline (TBS)-T buffer [20 mM Tris (pH 7.4), 300 mM NaCl, 0.1% Tween 20]. The blocking agent was decanted and membranes incubated in 10 ml of primary antibody solution in TBS-T buffer: rabbit polyclonal antibodies against human CYP51 diluted 1:300 and rabbit polyclonal antibodies against rat NADPH-P450 reductase diluted 1:1000. After three washings with 10 ml of TBS-T buffer (first washing for 15 min and twice for 5 min), membranes were incubated in 10 ml of secondary antibody (rabbit IgG horseradish peroxidase-linked whole antibody) diluted 1:2000 in TBS-T buffer for 1 h. After incubation, membranes were washed three times in TBS-T buffer (once for 15 min and twice for 5 min). Proteins were detected by the enhanced chemiluminescence Western blotting kit (Amersham) using autoradiography as described by the manufacturer.

Immunoprecipitation of CYP51 protein and glycosylation test
Five hundred microliters of organelle membranes were solubilized in 20 µl of 10% SDS, 5 µl of 1 mM phenylmethylsulfonyl fluoride, 50 µl of 20% Triton X-100, 1.5 M NaCl, and 0.3 M Tris-HCl (pH 7.5); placed on a shaker; and incubated at 25 C for 15 min. After centrifugation (2 min, 1000 x g) the supernatant was collected, added, and incubated 2 h at room temperature with 20 µl of anti-CYP51 antibodies. Then 100 µl protein-A Sepharose were added, incubated 1 h at room temperature, and centrifuged 15 sec at full speed. Beads were washed three times with 1 ml 20 mM Tris-HCl (pH 7.5), 20 mM EDTA, 0.5% Triton x 100 0.25% SDS, and 150 mM NaCl. After the last washing, proteins were eluted from beads by 30 µl 125 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% ß-mercaptoethanol, and 0.01% bromphenol blue; incubated for 2 min at 95 C; and analyzed on SDS-PAGE. Glycosylated proteins were detected according to the Immuno-blot kit protocol (Bio-Rad Laboratories, Hercules, CA).

CYP51 enzyme activity
Mouse liver, bull sperm, and ram sperm 1500-g proteins were prepared according to earlier published method (6). The CYP51 activity assay (14) and HPLC separation of sterols (15) were performed exactly as described. Twenty-five nanomoles of cold lanosterol (Steraloids) and 0.5–0.8 x 106 cpm of 3H-24,25-dihydrolanosterol tracer (American Radiolabeled Chemicals, Inc.) were added into each activity assay.

Colloidal gold labeling and electron microscopy
Mature CD1 mouse, housed in groups of three, were subjected to a cervical dislocation. Their testes were immediately dissected and fixed using 2% paraformaldehyde in a PBS (pH 7.4) for 1.5–2 h at room temperature. After washings in PBS (twice for 5 min), the samples were dehydrated in a series of graded ethanols and embedded in Unycril resin with UV polymerization for 3 d at 4 C. Ultrathin sections were placed on nickel grids and incubated with a normal goat serum in concentration 1:10 for 1 h at room temperature to prevent nonspecific signals. Specimens were incubated on droplet of 25 µl rabbit polyclonal antihuman CYP51 antibodies in titer 1:20 or antirat NADPH P450 reductase antibodies in titer 1:100 overnight at 4 C. After incubation, specimens were washed on droplets of 50 µl PBS buffer. The appropriate secondary antibody (goat-antirabbit IgG) (Sigma, St. Louis, MO) bearing 10 nm gold particles was used to visualize the location of antigen-antibody complex. Specimens were incubated on 25 µl droplets of secondary antibody in titer 1:100 for 2 h. The procedure was finished by a flash of distilled water. Sections of the mouse testis were examined with a 902A transmission electron microscope (Zeiss, Gottingen, Germany). For the negative control, the process of immunogold labeling was similar, but instead of anti-CYP51 antibodies the preabsorbed anti-CYP51 antibodies with 20-fold excess of CYP51 protein in concentration 1:20 were applied. Instead of anti-NADPH P450 reductase, the normal rabbit serum in concentration 1:100 was applied.

Double immunolabeling
Mouse tissues were obtained from mature male 4-month CBA mice. Tissues were processed as described (7) and blocked in TBS containing 17% native swine serum and 17% native goat serum (Dako, Copenhagen, Denmark) both diluted 1:5 in TBS. Sections were incubated with primary antibodies (antihuman CYP51, 1:20 or anti Golgi 58 protein, 1:50) overnight at 4 C, washed twice in TBS (5 min), and incubated with tetramethylrhodamine isomer R conjugated swine antirabbit IgG (Dako) in dilution 1:30 and with fluorescin isothiocyanate isomer 1-conjugated F(ab)2 fragment of goat antimouse IgG (Dako) for 30 min at room temperature and washed in TBS four times for 5 min. Sections were coverslipped using gelatin medium. Photomicrographs were taken by a multispectral laser scanning confocal microscope (Leica, Québec, Canada), using an argon laser excitation beam of wavelength 488 nm and a helium-neon laser excitation beam with wavelength 543 nm. For the negative control, normal rabbit (1:20) or normal mouse (1: 30) sera were applied.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunodetection of CYP51 protein in mouse liver and testis
CYP51 is considered to be a microsomal cytochrome P450 enzyme. Due to its evolutionary conservation (over 91% amino acid identity in different mammals), the polyclonal antihuman CYP51 antibody recognizes well all mammalian CYP51 enzymes. Using this primary antibody, fluorescent dye-labeled secondary antibodies and confocal microscopy the CYP51 protein was detected in mouse hepatocytes (Fig. 1AGo) and Leydig cells of the testis in sexually mature male mice (Fig. 1BGo). It should be noted that the expression of CYP51 in hepatocytes of normally fed mice is higher than in Leydig cells, which was confirmed also by comparative Western analysis (not shown). Besides Leydig cells, also haploid male germ cells of the testis (spermatids) express CYP51 (Fig. 1CGo). Expression of the protein is observed in round spermatids (two round spermatids in Golgi-cap phase are framed) and elongated spermatids (double arrow). Only background levels of CYP51 are observed in spermatogonia and spermatocytes. The specificity of staining was checked by replacement of primary antibody with nonimmune serum (Fig. 1DGo).



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  		FIG. 1. Immunodetection of CYP51 in mouse liver and testis with immunofluorescent double labeling. Panels A–C and G–J, Anti-CYP51 antibody. Panel E, Anti-Golgi 59-kDa antibody. Panel A, Hepatocytes. Panel B, Leydig cells. Panel C, Mouse testis; double arrow, elongate spermatid; arrow, Leydig cell; frame, round spermatids studied further in panels E–G; opposite arrows, lamina propria with basal membrane. Panel D, Negative control with normal rabbit serum. Panels E–G, Magnification of round spermatids in Golgi phase from panel C. Panel E, Staining of the Golgi by anti-Golgi 59. Panel F, Scheme of organelle position. Panel G, Staining of the ER, Golgi, and acrosome by anti-CYP51. Panel H, Round spermatid in acrosomal phase; arrows, acrosome and Golgi. Panel I, Elongate spermatid in maturation phase; arrows, acrosome. Panel J, Spermatozoa in epididymis, labeling of the acrosome (bar, panels A, B, G, H, I, and J, 10 µm; panels C and D, 50 µm). Panel K, Time scale and subcellular localization of CYP51 protein in different phases of spermatogenesis in the mouse; N, nucleus; A, acrosome; GA, Golgi apparatus; red dot, CYP51.

 
The subcellular location of the CYP51 protein in round spermatids of the Golgi-cap phase was studied further by double labeling using two primary antibodies: anti-CYP51 and a 58-kDa anti-Golgi protein. The 58-kDa Golgi protein is localized only on Golgi compartments and is a good marker for the Golgi apparatus, whereas it does not stain the acrosome. The geometry and organelles of the two round spermatids from Fig. 1CGo are shown in Fig. 1FGo. The acrosome (A) caps the nucleus (N), whereas Golgi stacks (GA) are laid on the top of the acrosome. Only Golgi stacks are stained by the anti-Golgi 58 kDa (green color, Fig. 1EGo). The CYP51 signal (red color, Fig. 1GGo) covers not only the Golgi stacks but also the acrosome capping the nucleus as well as the endoplasmic reticulum. These results indicate that CYP51 likely migrates from smooth endoplasmic reticulum to the Golgi apparatus and then with the acrosomal vesicles to membranes of the newly formed acrosome. In the mouse, approximately 6 d is necessary for the acrosome to be shaped by the Golgi apparatus, and CYP51 is retained on the Golgi during this period. Figure 1Go, H and I, show the CYP51 protein also in acrosomes of later spermatids (acrosomal and maturation phases) and acrosomes of the mature sperm in the epididymis (Fig. 1JGo). A cumulative picture of CYP51 subcellular localization together with the duration of the distinct spermatid maturation phases in the mouse is shown in Fig. 1KGo. Spermatids of the Golgi-cap and acrosomal phases contain CYP51 in three organelles: endoplasmic reticulum (ER), Golgi, and the acrosome. After the majority of cytoplasm is removed as the residual body, spermatids enter the maturation phase during which the CYP51 protein is detected on acrosome only. Localization of CYP51 protein on the acrosome of the mature testicular and epididymal spermatozoa indicates that after the complete formation of the acrosome CYP51 protein persists on this organelle for several weeks.

CYP51 and NADPH cytochrome P450 reductase in mouse spermatids
Confocal microscopy studies (Fig. 1Go) showed the acrosomal location of the CYP51 protein in the mouse. Acrosome is a secretory Golgi-derived vesicle of male germ cells, composed of inner and outer acrosomal membranes and the acrosomal matrix. The matrix is similar to the lysosomal matrix, including soluble proteins with mostly lytic activities, whereas little is known about membrane proteins of the acrosome. Using immunogold electron microscopy, we studied the location of CYP51 and the NADPH cytochrome P450 reductase in different compartments of the acrosome. In the round spermatid, CYP51 enzyme resides primarily on the outer membrane of the acrosome (Fig. 2AGo). Some gold particles were detected also on inner membrane but not in the lumen. A similar picture was observed for the elongated spermatid (Fig. 2BGo). As mentioned earlier, NADPH P450 reductase is a required redox partner in all microsomal cytochrome P450 reactions, including 14{alpha}-demethylation of lanosterol to FF-MAS by CYP51. Figure 2Go, C and D, shows that NADPH P450 reductase also resides primarily on the outer acrosomal membranes of round and elongating spermatids. Gold particles in the area surrounding the round spermatid (Fig. 2CGo) or elongating spermatid (Fig. 2BGo) represent the CYP51 or NADPH-P450 reductase signal, respectively, in ER and Golgi apparatus of Sertoli cells, which are nests for male germ cell development in the testis. Figure 2Go, E and F, shows the negative control-preabsorption of the anti-CYP51 antibody by a 20-fold excess of the CYP51 protein. Colocalization of CYP51 and NADPH P450 reductase on outer mouse acrosomal membranes was the first indication of the potential lanosterol 14{alpha}-demethylase activity in mammalian sperm.



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  		FIG. 2. Immunogold electron microscopy of CYP51 and NADPH P450 reductase in mouse testis. Panel A, Round spermatid, anti-CYP51. Panel B, Elongate spermatid, anti-CYP51. Panel C, Round spermatid, anti-P450 reductase. Panel D, Elongate spermatid, anti-P450 reductase. Panels E and F, Negative control, preabsorption of anti-CYP51 antibodies by the human CYP51 protein. A, Acrosome, N, nucleus, arrows, acrosomal membranes. Bar, 0.1 µm.

 
Different isoforms of the CYP51 protein in acrosomal membranes of ram and bull spermatozoa
High expression of the CYP51 protein was detected in spermatozoa in the lumen of mouse epididymis (Fig. 1JGo). However, as noted earlier, several weeks pass before the epididymal sperm became spermatozoa of the ejaculate. To determine whether the CYP51 protein is present also in acrosomal membranes of ejaculate sperm, spermatozoa were isolated from bull and ram semen. Big mammals have been chosen to allow collection of enough semen material with high sperm concentration, which was practically impossible to achieve with the mouse. The isolated acrosomal membranes were subjected to Western analysis by the antihuman CYP51 antibody (Fig. 3Go). For the ram, isolates were from three batches of semen from different animals, and four immunoblot analyses have been performed with similar results in all cases. The 50-kDa immunoreactive protein corresponds to the size of the ram acrosomal CYP51, which is of the same size as in ram liver (not shown) but smaller, compared with the 53-kDa human CYP51 (Fig. 3AGo, lane C). Surprisingly, in addition to that, several high-molecular-mass (HMM)-CYP51 complexes have been detected in acrosomal membranes (Fig. 3AGo, lanes 1–3). Most prominent bands were of approximately 65 and 70 kDa. A very similar picture has been obtained with bull acrosomal membranes. Bull CYP51 is also of 50 kDa, and HMM-CYP51 immunoreactive complexes of 60 and 70 kDa are detected (Fig. 3BGo, lane 1). To determine whether HMM complexes are modified forms of the CYP51 protein, samples were immunoprecipitated by the antihuman CYP51 antibody and subjected to Coomassie-stained PAGE gels. The major immunoprecipitated proteins are of 50 and 70 kDa, both in ram (Fig. 3AGo, lane 4) and bull (Fig. 3BGo, lane 2), with weaker signals at approximately 60 kDa. Part of the approximately 50-kDa signal likely corresponds to immunoprecipitated immunoglobulins. In the bull, the three CYP51-immunoreactive proteins that are seen on the Western analysis (Fig. 3BGo, lane 1) seem to be glycosylated (lane 3). The most highly glycosylated CYP51-immunoreactive form in the ram is of approximately 60 kDa (Fig. 3AGo, lane 5).



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  		FIG. 3. Immunoblot analysis by antihuman CYP51 antibody, immunoprecipitation, and glycosylation of CYP51 on acrosomal membranes of ram (A) and bull (B) ejaculate sperm. C, In Escherichia coli overexpressed human CYP51 A; 1–3, immunoblot analysis of different isolations of ram acrosomal membranes; 4, Coomassie-stained immunoprecipitate of 2; 5, detection of glycosylated proteins from 4. B, 1, immunoblot analysis of bull acrosomal membranes; 2, Coomassie-stained immunoprecipitate of 1; 3, detection of glycosylated proteins from 2.

 
Acrosomal membranes of bull and ram spermatozoa contain a functional lanosterol 14{alpha}-demethylase enzyme complex
Immunogold electron microscopy studies in the mouse showed both CYP51 and NADPH P450 reductase proteins on acrosomal membranes of mouse spermatids, raising a question whether the cytochrome P450 reaction cycle can be performed in this organelle. Isolated acrosomal membranes have been used as a source of both proteins and the NADPH regenerating system has been added together with the cold and radiolabeled CYP51 substrates (lanosterol and 3H-24,25-dihydrolanosterol). Inhibitors of further metabolic steps toward cholesterol (AY 9944 and NaCN) have also been added. As shown in Fig. 4Go, CYP51 retains enzymatic activity on acrosomal membranes of the ejaculate sperm. The 3H-24,25-dihydrolanosterol (Fig. 4AGo peaks 1a and 1b) in the control mouse liver microsome sample is converted to 3H-24,25-dihydro-FF-MAS (peak 2, marked by arrow). In addition to that, three metabolites between FF-MAS and the final product cholesterol are detected (peaks 3–5). Addition of ketoconazole (Fig. 4BGo) or absence of NADPH (Fig. 4CGo) inhibits the CYP51-catalyzed formation of FF-MAS and further metabolites toward cholesterol. Bull ejaculate sperm homogenate (Fig. 4DGo) and the isolated acrosomal fraction (Fig. 4EGo), as well as ram-ejaculated sperm homogenate (Fig. 4FGo), all show the presence of 3H-24,25-dihydro-FF-MAS, which proves the CYP51 enzymatic activity. Detection of later metabolites (peaks 3–5) indicates that spermatozoa likely contain several enzymes of the cholesterol biosynthesis pathway. Because we did not succeed in blocking completely the further metabolism of 3H-24,25-dihydro-FF-MAS, CYP51 enzymatic activity has been calculated as pmol of metabolized 3H-24,25-dihydrolanosterol per milligram of spermatozoal or acrosomal protein per minute. Activities of different preparations were in the range of 17–42 pmol/mg·min.



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  		FIG. 4. Reverse-phase HPLC chromatograms of 24,25-[3H]dihydrolanosterol (peak 1a; peak 1b is a contaminant of the commercial ARC substrate) and its metabolites. A, Mouse liver homogenate, incubation in the presence of 5 µM ketoconazole (B) or the absence of NADPH (C). D, Bull sperm homogenate. E, Bull acrosomal membranes. F, Ram sperm homogenate. Peak 2, FF-MAS (arrow); peaks 3–5, metabolites of FF-MAS. T-MAS coelutes with peak 1a.

 
CYP51 protein in Golgi-enriched fractions of the mouse liver
Western analysis of ram and bull acrosomal membranes (Fig. 3Go) showed that different HMM-CYP51 immunoreactive complexes exist in these membranes. It was proven recently that acrosomal membranes are derived from Golgi membranes (8), suggesting that all acrosomal proteins were already subjected to Golgi modifications. To investigate whether HMM-CYP51 immunoreactive complexes are a special feature Golgi modifications in male germ cells or they represent a more general phenomena, we isolated different Golgi fractions (G1, cis-Golgi; G2, medial-Golgi; and G3, trans-Golgi) from hepatocytes of normally fed mice. Golgi fractions were subjected to immunoblot analysis with the antihuman CYP51 antibody (Fig. 5Go). A strong signal of approximately 53 kDa corresponding to the mouse CYP51 (lane 1) was detected on the cis-Golgi (lane 2), together with a weaker signal of 70 kDa. On medial-Golgi (lane 3), the 53-kDa band becomes weaker and disappears completely in the trans-Golgi fraction (lane 4). In contrast to that, the intensity of the 70-kDa CYP51 HMM-immunoreactive complex increases from cis through medial to the trans Golgi (lanes 2–4). Immunoprecipitated cis- (lane 5) and trans- (lane 7) Golgi fractions were subjected to glycosylation test. The strongest glycosylation band has been observed again for approximately 60–65 kDa and 70-kDa proteins. As mentioned earlier, the majority of the 50-kDa band likely represents glycosylated immunoglobulins. These results indicate that HMM-CYP51 immunoreactive complexes are likely not a special feature of acrosomal membranes of male germ cells but are found also in Golgi-enriched fractions of somatic cells. The absence of the 53-kDa CYP51 protein in trans-Golgi fraction suggests that the protein is likely retrieved to the ER after passing the cis-Golgi.



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  		FIG. 5. A, Immunoblot analysis by antihuman CYP51, immunoprecipitation, and glycosylation of CYP51 on microsomes and Golgi fractions of the mouse liver. C, In E. coli overexpressed human CYP51; 1, mouse liver microsomes; 2, cis-, 3, med-; 4, trans-Golgi fractions; 5, immunoprecipitate of 2; 6, detection of glycosylated proteins from 5; 7, immunoprecipitate of 4; 8, detection of glycosylated proteins from 7.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Meiosis activating sterols FF-MAS and T-MAS, postlanosterol intermediates of cholesterol biosynthesis and products of CYP51 and sterol {Delta}14-reductase reactions, are able to reinitiate meiosis in oocytes in vitro in the mouse (4) and rat (16). A clear role of FF-MAS as universal oocyte meiosis-inducing substance is not yet resolved due to some contradictory results, such as nonsynchronized timing of resumption of meiosis before FF-MAS peak (17) and limited influence of CYP51 enzyme inhibition on resumption of meiosis (18, 19). The gonadotropin-induced resumption of meiosis is also not yet fully understood, especially in relation with overproduction/accumulation of MAS. It was shown that progestins alter cholesterol synthesis and increase concentration of MAS by inhibition of C-4 sterol methyl oxidase and sterol {Delta}24-reductase, both being post-MAS enzymes (20). Gonadotropin induction of the CYP51 activity in ovaries is confirmatory to the involvement of MAS (21). Expression of ovarian CYP51 depends on gonadotropins in LH (growing follicles) and FSH (corpus luteum) phases, and it seems that the ovarian CYP51 activity is enough for accumulating MAS (22).

High expression of CYP51 mRNA and CYP51 protein in male germ cells in several mammals, including human, suggests an important role of CYP51 protein in male germ cell development. Initially, the presence of CYP51 protein in spermatids indicated its major role in production of cholesterol that accumulates in plasma membranes of these cells during sexual maturation (23, 24, 25, 26). However, high expression of CYP51 and other cholesterogenic enzymes that are responsible for the production of MASs, together with low expression of enzymes that convert MAS toward cholesterol, indicate that production of MAS might be another physiologically important role in mammalian male germ cell maturation (2). The capacity to perform the lanosterol 14{alpha}-demethylase reaction seem to be an evolutionarily conserved trait of mammalian spermatozoa. The entire lanosterol 14{alpha}-demethylase enzyme complex (CYP51 and P450 reductase) is detected in acrosomal membranes of round and elongated spermatids of the mouse, and conversion of lanosterol to FF-MAS is observed by the ejaculated bull and ovine spermatozoa or acrosomal membranes. It was reported previously that the highest concentration of MAS is not in ovaries but in the testis (>30 ppm or µg/ml) and ejaculate (<0.5 ppm) (5). We show here for the first time that MAS can be synthesized by the lanosterol 14{alpha}-demethylase enzyme complex in ejaculated sperm in situ. The MAS-specific receptor has not yet been found. Therefore, the target-cells in which MAS-induced second meiotic division would take place can only be speculated. The autoradiographic localization shows specific binding of FF-MAS to oolemma (oocyte plasma membrane), zona pellucida, and cumulus cells. This suggests the existence of a plasma membrane-associated FF-MAS receptor (27), whereas other studies proposed a G protein-coupled receptor mechanism that might be involved in MAS-signaling (28). It is thus possible to speculate that MAS actively contributes to sperm-oocyte signaling during the fertilization process.

To the best of our knowledge, lanosterol 14{alpha}-demethylase is the first cytochrome P450 localized in acrosomal membranes and the acrosomal form of NADPH cytochrome P450 reductase has also not been described before. It is interesting to note that besides CYP51, acrosomal membranes contain other functional enzymes of the cholesterol biosynthesis pathway because sterol metabolites between FF-MAS and cholesterol have been detected (Fig. 4Go). The aromatase (CYP19) is another cytochrome P450 residing in mammalian male germ cells; however, it has not been localized to the acrosome. CYP19 converts irreversibly androgens to estrogens. Various studies have shown the aromatase expression in the cytoplasm of round and elongating spermatids and flagella of late spermatids and spermatozoa of the epididymis but not on acrosomal membranes (29). As a putative marker of human sperm motility (30), CYP19 mRNA, protein and enzymatic activity have been detected in the ejaculated human spermatozoa (29). It was postulated that the ability of testis to convert androgens into estrogens is related to the presence of a microsomal enzymatic complex composed of the glycoprotein CYP19 and the NADPH P450 reductase (31). It thus seems possible that two forms of the NADPH P450 reductase reside in male germ cells: the microsomal, supporting CYP19 catalyzed conversion of androgens to estrogens, and the acrosomal, supporting conversion of lanosterol to meiosis-activating sterols.

Microsomal preparations of purified spermatozoa contain two isoforms (49 and 53 kDa) of the CYP19 protein (30). The 53 kDa is a glycosylated form of the 49-kDa aromatase. Glycosylation was reported also for the CYP19 isolated from human placenta and is not essential for enzymatic activity (32). Western analysis of CYP51 from acrosomal fractions of ram and bull ejaculate (Fig. 3Go) as well as from Golgi fractions of liver hepatocytes (Fig. 5Go) show HMM isoforms of the CYP51 protein. Besides the regular size of the protein (53 kDa in mouse and 50 kDa in bull and ram), 60–70 kDa immunoreactive complexes have been observed. The 60- to 70-kDa proteins represent glycosylated forms of HMM-CYP51. N-glycosylation would result in 14 oligosaccharides, which would contribute 2.5–3 kDa to the size of the protein. An approximately 3-kDa difference between glycosylated and nonglycosylated proteins was described for the fusion CYP51 protein (33) that was expressed in Saccharomyces cerevisiae for other glycosylated cytochromes P450 (34, 35) as well as for proteins from other families (36, 37). The difference between CYP51 and the HMM-CYP51 complexes is up to 20 kDa, which cannot be explained by glycosylation only. A possibility would be ubiquitination, similarly as described for CYP3A isoforms and CYP2E1 (38). However, the ubiquitination reaction on the HMM-CYP51 complexes was negative (not shown). Besides glycosylation, other posttranslational modifications seem to take place during CYP51 transport through the Golgi. HMM protein complexes with a similar size difference (up to 20 kDa) and nonidentified mechanism of formation have been described recently for transcription factors TCR{zeta} (37), and NFIC (36). It is currently unknown whether HMM-CYP51 proteins are labeled for degradation or still have a functional role. Experiments with 24-h starved mice show redistribution of the protein and indicate that the quantity of HMM-CYP51 complexes diminishes on starvation (not shown). This is in accordance with most recent studies of the C-3 sterol dehydrogenase (Nsdhl), a cholesterogenic enzyme involved in the conversion of lanosterol into cholesterol, in which depletion of fatty acids from culture medium also caused the intracellular redistribution of the protein (39).

CYP51 is believed to be a resident protein of the smooth endoplasmic reticulum; however, our data clearly show that the 53-kDa CYP51 protein in mouse hepatocytes traffics at least to the cis-Golgi compartment (see Fig. 5Go). Recycling of CYP51 protein from ER through cis-Golgi has been described also for S. cerevisiae CYP51-green fluorescent protein. When the fusion protein was expressed from a single copy vector, it remained in the ER, whereas the multicopy vector expressed protein recycled through the Golgi (33). If a protein has a retention signal in the linker region of its transmembrane sequence, it becomes a resident ER protein. An example from the CYP superfamily is CYP2C2 that forms homooligomers due to the signal anchor sequence (40). In the case that a retrieval-retention signal is lacking, proteins are transported through the Golgi and secretory vesicles to other directions (41). The N terminus of the human CYP51 protein does not contain any recognizable retention signal; however, the C terminus contains a potential di-arginine retrieval retention signal KRRS. Our results propose a retention-retrieval mechanism for the microsomal form of CYP51 (Fig. 6AGo). In round spermatids, in which all cytoplasmic organelles are still present (42), CYP51 resides on ER, Golgi, and the acrosomal vesicles, suggesting that the protein traffics from its site of synthesis to the final location on acrosomal membranes (Fig. 6BGo). The proposed transport of the CYP51 protein is in agreement with formation of acrosomal vesicles, which secede from the trans-Golgi network (8).



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  		FIG. 6. Proposed scheme of the intracellular transport of CYP51 in liver (A) and male germ cells (B). N, Nucleus; A, acrosome; GA, Golgi apparatus.

 
In conclusion, the acrosomal lanosterol 14{alpha}-demethylase enzyme complex that is composed of CYP51 and NADPH P450 reductase is the first functional cytochrome P450 complex detected in acrosomal membranes. Its ability to convert lanosterol to FF-MAS shows that MASs are produced by mammalian sperm in situ, which has important implications for better understanding mammalian reproduction.


   Acknowledgments
 
We thank Dr. Mogens Baltsen and Dr. Anne-Grete Byskov (Rikhospitalitet, Univeristy of Copenhagen) for sterol standards and Dr. Ljerka Banek (School of Medicine, University of Zagreb) for support to the project. We also thank Dr. M. R. Waterman (Vanderbilt University School of Medicine, Nashville, TN) for manuscript corrections and helpful suggestions.


   Footnotes
 
This work was supported by the SLO-CRO bilateral collaborative grant of Ministries of Science of Slovenia and Croatia; Grant J1-3438 of Ministry of Science, Education, and Sports (MESS) of Slovenia; Fogarty International Research Collaboration Award/NIH Grant 1 RO3 TW01174-01; and International Centre for Genetic Engineering and Biotechnology Grant CRP/SLO00-01. M.C. and K.F.T. were supported by graduate fellowships from MESS.

Abbreviations: CYP51, Mammalian lanosterol 14{alpha}-demethylase; ER, endoplasmic reticulum; FF, follicular fluid; HMM, high molecular mass; MAS, meiosis-activating sterol; NADPH, nicotinamide adenine dinucleotide phosphate reduced; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TBS-T, Tris, 0.2% Tween 20; T-MAS, testis MAS.

Received October 6, 2003.

Accepted for publication November 13, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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