463 Lanosterol 14-demethylase (CYP51), NADPH–cytochrome P450 reductase and squalene synthase in spermatogenesis: late spermatids of the rat express proteins needed to synthesize follicular fluid meiosis activating sterol G Majdicˇ, M Parvinen1, A Bellamine2, H J Harwood Jr3, WWKu3, M R Waterman2 and D Rozman4 Veterinary Faculty, Clinic of Reproduction, Cesta v Mestni log 47a, 1000 Ljubljana, Slovenia 1Institute of Biomedicine, Department of Anatomy, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland 2Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232–0146, USA 3Pfizer Central Research, Department of Metabolic Diseases, Box No. 0438, Eastern Point Road, Groton, Connecticut 06340, USA 4Institute of Biochemistry, Medical Center for Molecular Biology, Medical Faculty University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia (Requests for offprints should be addressed to D Rozman; Email: [email protected]) (G Majdicˇ is now at Department of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas 75235–8857, USA) Abstract Lanosterol 14-demethylase (CYP51) is a cytochrome detected in step 3–19 spermatids, with large amounts in P450 enzyme involved primarily in cholesterol biosynthe- the cytoplasm/residual bodies of step 19 spermatids, where sis. CYP51 in the presence of NADPH–cytochrome P450 P450 reductase was also observed. Squalene synthase was reductase converts lanosterol to follicular fluid meiosis immunodetected in step 2–15 spermatids of the rat, activating sterol (FF-MAS), an intermediate of cholesterol indicating that squalene synthase and CYP51 proteins are biosynthesis which accumulates in gonads and has an not equally expressed in same stages of spermatogenesis. additional function as oocyte meiosis-activating substance. Discordant expression of cholesterogenic genes may be a This work shows for the first time that cholesterogenic more general mechanism leading to transient accumu- enzymes are highly expressed only in distinct stages of lation of pathway intermediates in spermatogenesis. This spermatogenesis. CYP51, NADPH–P450 reductase (the study provides the first evidence that step 19 spermatids electron transferring enzyme needed for CYP51 activity) and residual bodies of the rat testis have the capacity to and squalene synthase (an enzyme preceding CYP51 in produce MAS sterols in situ. the pathway) proteins have been studied. CYP51 was Journal of Endocrinology (2000) 166, 463–474 Introduction through the action of squalene synthase (Fig. 1A). CYP51 removes the 14-methyl group from lanosterol, forming Lanosterol 14-demethylase (CYP51) is a member of the 4,4,-dimethyl 5-cholesta, 8,14,24-diene-3-ol (Fig. 1B), diverse cytochrome P450 superfamily and is involved in also known as follicular fluid meiosis activating sterol cholesterol biosynthesis in mammals (Nelson 1999). (FF-MAS). At least six enzymatic steps after CYP51 are CYP51 resides in the endoplasmic reticulum (Trzaskos et needed for the production of cholesterol from its precursors al. 1986) and oxidatively demethylates substrates in the (Fig. 1C). presence of NADPH, molecular oxygen and the micro- Cholesterol biosynthesis and its coordinate regulation by somal electron transferring enzyme, NADPH–cytochrome the sterol regulating element binding protein (SREBP)- P450 reductase (Fischer et al. 1991, Shyadehi et al. 1996). dependent pathway is well characterized in the liver Lanosterol, the major endogenous substrate for mammalian (Goldstein & Brown 1990, Brown & Goldstein 1998), but CYP51, is synthesized from squalene through sequential very little is known about this process in the male gonad, epoxidation and cyclization reactions that are catalyzed by especially in germ cells. Human sperm lysates were squalene epoxidase and oxidosqualene cyclase (Fig. 1). shown to have the capacity to synthesize cholesterol from Squalene, which is the first pathway intermediate com- [14C]acetate in vitro, yet the activity seems to be too low to mitted to the formation of cholesterol, is produced directly contribute significantly to the free cholesterol content of from the branch-point substrate, farnesyl pyrophosphate, the sperm (Gunasegaram et al. 1995, Cross 1998). In Journal of Endocrinology (2000) 166, 463–474 Online version via http://www.endocrinology.org 0022–0795/00/0166–463 2000 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 10/02/2021 12:58:36PM via free access 464 G MAJDIC{ and others · Lanosterol 14-demethylase (CYP51) in spermatogenesis Figure 1 Enzymatic conversion of farnesyl pyrophosphate to cholesterol. (A) Conversion of farnesyl pyrophosphate to lanosterol. Farnesyl pyrophosphate is formed from acetate through a multistep pathway that includes the key intermediates mevalonate and isopentenyl pyrophosphate (not shown). (B) Lanosterol 14-demethylase (CYP51) reaction. The methyl group at position 14 is subjected to three consecutive cytochrome P450 oxidation cycles, the intermediates of the reaction being the alcohol and aldehyde. The carbon at position 14 is removed as formic acid during the third oxidation. In each cytochrome P450 cycle, a pair of electrons is transferred from NADPH to NADPH–cytochrome P450 reductase and on the cytochrome P450 enzyme, CYP51. There is no catalytic activity in the absence of NADPH–cytochrome P450 reductase. (C) Conversion of FF-MAS to cholesterol. At least six enyzmes after CYP51 participate in cholesterol biosynthesis. The precise order of the last three reactions might be tissue-specific and remains a matter of discussion. T-MAS is another sterol having in vitro meiosis activating activity. contrast to the majority of tissues in which cholesterol oocytes in vitro and are believed to have important, but not is the main product, male and female gonads over- yet fully understood, roles in fertilization (Byskov et al. produce two late intermediates of the cholesterol bio- 1995, 1999, Grondahl et al. 1998). synthetic pathway, follicular fluid meiosis activating sterol The first mammalian CYP51 gene was characterized (FF-MAS; 4,4-demethyl-5-cholesta-8,14,24-triene-3- in 1994 (Aoyama et al. 1994) and extensively studied ol) and testis meiosis activating sterol (T-MAS; 4,4- since then (Aoyama et al. 1996, Rozman et al. 1996a,b, dimethyl-5-cholesta-8,24-diene-3-ol) (Byskov et al. Strömstedt et al. 1996, Noshiro et al. 1997). The gene 1995, 1999). Both tissue-isolated FF-MAS (Byskov et al. shows remarkable conservation between species: for 1995), the product of lanosterol 14-demethylase example, the amino acid homology between the human (CYP51) and chemically synthesized FF-MAS (Grondahl and the rat genes is 93% (Strömstedt et al. 1996). CYP51 et al. 1998), and T-MAS, the product of the sterol mRNA is expressed in all mammalian tissues, the expres- 14-reductase, stimulate reinitiation of meiosis in mouse sion being an order of magnitude greater in male germ Journal of Endocrinology (2000) 166, 463–474 www.endocrinology.org Downloaded from Bioscientifica.com at 10/02/2021 12:58:36PM via free access Lanosterol 14-demethylase (CYP51) in spermatogenesis · G MAJDIC{ and others 465 cells of the testis compared with any other tissue/cell type Lysozyme was added to a final concentration of (Strömstedt et al. 1998). Testis is also the organ in which 0·5 mg/ml. An equal volume of ice-cold 0·1 mM EDTA, the greatest concentrations of accumulating MAS sterols pH 8, was slowly added and the mixture stirred for have been detected (Byskov et al. 1999). Thus, it seems 30 min. The spheroplasts were collected by centrifugation likely that overexpression of CYP51 mRNA influences at 2000–5000 g for 15 min and resuspended at 5 ml/g accumulation of MAS sterols in the testis. In situ hybridiz- in potassium phosphate buffer pH 7·4 containing ation studies show a stage-specific expression of CYP51 30% glycerol, 0·1 mM dithiothreitol (DTT), 0·1mM mRNA in rat (Strömstedt et al. 1998) and mouse EDTA, 0·1 mM phenylmethylsulphonyl fluoride (PMSF), (Rozman et al. 1999) testis, with the greatest amounts in 1 mg/ml DNase and 10 mM lanosterol. Spheroplasts were postmeiotic germ cells, the spermatids. The earliest detec- homogenized using a Teflon homogenizer. Triton X-114 tion of CYP51 mRNA is in round spermatids of steps 3–5. (Boehringer Mannheim) was added to a final concen- Thereafter, expression increases, reaching the highest level tration of 0·7% and the mixture was stirred for 20 min in elongating spermatids of steps 7–14. CYP51 mRNA (Halkier et al. 1996). The P450-rich phase was separated expressed in germ cells is translated into the protein, as by centrifugation at 100 000 g for 30 min and diluted 1:10 increased CYP51 activity is observed in germ cells of in potassium phosphate buffer pH 7·4 containing 20% sexually mature rats (Strömstedt et al. 1998). glycerol, 0·1 mM PMSF, 0·4% Triton X-100 and 10 mM This study investigated which cell types of the testis lanosterol. The diluted P450 suspension was applied to a have the greatest potential to synthesize MAS sterols Ni2+ nickel nitrilotriacetic acid (NTA) affinity column in situ, by describing immunolocalization of lanosterol (Qiagen). The column was washed with 50 mM potass- 14-demethylase and NADPH–cytochrome P450 re- ium phosphate buffer pH 7·4 containing 500 mM NaCl, ductase – two enzymes needed to produce FF-MAS in the 20% glycerol, 50 mM glycine, 0·4% Triton X-100 and rat testis. By also monitoring squalene synthase, we have 10 mM lanosterol. The human CYP51 was eluted with been able to describe the stage-specific expression of the same buffer containing 40 mM histidine instead of post-farnesyl pyrophosphate enzymes of cholesterol bio- glycine. The CYP51 protein was >90% pure by SDS- synthesis in testicular germ cells, giving insights into the PAGE and silver staining. Antibodies against the human molecular basis by which accumulation of intermediates in CYP51 were raised by injecting New Zealand rabbits cholesterol biosynthesis can occur. with 0·5mgNi2+NTA-eluted protein mixed with one volume of Freund’s complete adjuvant (Sigma). The same amount of protein was used for a booster 4 weeks later, as Materials and Methods a mixture with Freund’s incomplete adjuvant (Sigma).