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-Demethylase (CYP51) and the Synthesis of Oocyte Meiosis-Activating Sterols in Postmeiotic Germ Cells of Male Rats1
Department of Biochemistry, Vanderbilt University School of Medicine (M.S., M.R.W.), Nashville, Tennessee 37232-0146; the Institute of Medical Biochemistry, University of Oslo (T.B.H., K.T.), N-0317 Oslo, Norway; Department of Anatomy, University of Turku (M.P.), Kiinamyllynkatu 10, FIN-20520 Turku, Finland; and Institute of Biochemistry (D.R.), Medical Center for Molecular Biology, Medical Faculty, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
Address all correspondence and requests for reprints to: Dr. Damjana Rozman, Institute of Biochemistry, Medical Center for Molecular Biology, Medical Faculty University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. E-mail: rozman{at}ibmi.mf.uni-lj.si
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-demethylase (P45014DM) that is
involved in the postsqualene part of cholesterol biosynthesis. This
enzyme removes the 14-methyl group from lanosterol and
24,25-dihydrolanosterol producing intermediates in cholesterol
biosynthesis, the oocyte meiosis-activating sterols FF-MAS and MAS-412.
Human and rat CYP51 messenger RNAs (mRNAs) are expressed in all
tissues, with highest levels in the testis due to the presence of an
additional shorter CYP51 transcript in this tissue. In
situ hybridization shows the highest CYP51 mRNA levels in
seminiferous tubules, with only background levels in Leydig cells. The
rat testis-specific CYP51 mRNA arises from the use of an upstream
polyadenylation site and is restricted to germ cells, being most
abundant in elongating spermatids in stages VII–XIV, whereas somatic
CYP51 transcripts are present in all cells. In contrast, the mRNA
levels of squalene synthase are maximal in round spermatids, and no
germ cell-specific transcript is observed. The rat male germ
cell-specific CYP51 transcript is translated in vitro to
two proteins of approximately 55 and 53.5 kDa. CYP51 activity is higher
in protein extracts of testes and germ cells of sexually mature rats
than in prepubertal animals, in which postmeiotic germ cells are not
yet present. This shows increased capacity for the production of MAS
sterols by male germ cells that have already completed meiosis,
suggesting that they serve a role different from meiosis activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-demethylation in
animals, plants, and fungi (1, 2, 3). In animals, lanosterol
14-demethylase (P45014DM) participates in cholesterol biosynthesis,
using lanosterol and 24,25-dihydrolanosterol as substrates (4). Fungal
(5, 6, 7, 8, 9) and plant (10, 11) P45014DM enzymes are involved in the
synthesis of ergosterol and sitosterol, respectively. The mammalian
(rat and human) P45014DM orthologs (12, 13, 14) show 93% amino acid
identity (the high conservation is characteristic of housekeeping
genes) and are 35–42% identical to lower eukaryotic and plant
P45014DM enzymes. Of more than 480 genes within the cytochrome P450
(CYP) superfamily (15), CYP51 is the only enzyme expressed in different
phyla and is believed to be one of the oldest eukaryotic CYP genes,
providing an essential function in these organisms (16). Northern
analysis shows that human CYP51 messenger RNA (mRNA) is expressed in
all tissues, with particularly high levels in testis (14). In addition
to the functional CYP51 gene, intronless CYP51 pseudogenes were found
in human and rat (17, 18). Processed pseudogenes arise from reverse
transcription of mRNA in germ cells followed by their random
incorporation into the genome (19). Only genes highly expressed in germ
cells have such intronless gene copies, and discovery of
CYP51-processed pseudogenes suggested that the high level of CYP51
expression in testis includes expression in germ cells.
The early steps in cholesterol biosynthesis from acetate lead to the
production of other important molecules, including dolichol,
ubiquinone, heme A, isopentyl adenine, and farnesylated proteins (20).
Squalene synthase and subsequent steps have been believed to be
committed to cholesterol biosynthesis. Recently, however, it has been
found that intermediates of the postsqualene part of cholesterol
biosynthesis can serve other roles. The sterols synthesized from
lanosterol by lanosterol 14-demethylase (FF-MAS) followed by sterol
14-reductase (T-MAS) are able to reinitiate meiosis in vitro
in mouse oocytes (21) (Fig. 1
).
Meiosis-activating ability is also observed with the synthetic sterols
MAS-412 and MAS-414, which are produced by the same two enzymes from
24,25-dihydrolanosterol (21).
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-demethylase
mRNA levels in rat testis include the appearance of a translatable germ
cell-specific CYP51 transcript. This transcript is highly expressed in
postmeiotic male germ cells, resulting in moderate elevation of the
P45014DM activity and thus in increased capacity for synthesis of
oocyte meiosis-activating sterols (MAS), whose function in male germ
cells has yet to be established. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Screening of a rat testis cDNA library
A rat testis 
ZAP cDNA library from 6-week-old animals
(Stratagene, La Jolla, CA) was screened with the
32P-labeled rat CYP51 cDNA probe (27). Positive plaques
were purified, and the plasmids were rescued as recommended by the
manufacturer (Qiagen, Chatsworth, CA). Both ends of the inserts were
analyzed by cycle sequencing.
In vitro translation
The rat testis-specific 1856-bp CYP51 cDNA isolated from the rat
testis cDNA library, as described above, was subcloned into the
pBluescript vector and used as a template for the coupled in
vitro transcription/translation by the TNT Coupled Reticulocyte
Lysate System (Promega, Madison, WI). Template (450 ng) was transcribed
by T7 RNA polymerase and translated in the presence of
[35S]methionine according to the instructions.
Radiolabeled proteins were resolved on a 8.5% SDS-polyacrylamide gel
and detected by autoradiography.
In situ hybridization
Rat testes of adult (>100-day-old) animals were rinsed in PBS,
pH 7.4, and immersion fixed overnight in 4% paraformaldehyde in PBS,
pH 7.4. The tissue was dehydrated in ethanol and xylene solutions,
embedded in paraffin, cut into 6-µm thick sections, and hybridized
with rat CYP51 probes (28). The CYP51 cDNA fragment was cloned into
pDIRECT vector (Clontech, Palo Alto, CA), and sense and antisense
riboprobes were prepared after linearization by in vitro
transcription with T3 and T7 RNA polymerases in the presence of
[35S]UTP.
Preparation of rat testis and germ cell protein extracts
After decapsulation of the testis from Sprague-Dawley rats
(Harlan), the tissue was homogenized on ice by 50 strokes with a
glass-Teflon hand-held homogenizer in assay buffer (100 mM
K3PO4, 0.1 m dithiothreitol, 0.1
mM EDTA, and 20% glycerol). Cell debris was removed by
centrifugation for 15 min at 1500 x g and 4 C. The
protein content of the supernatant was measured by the
Bradford method (Bio-Rad, Richmond, CA). Whole testicular protein
extracts were prepared from independent groups of animals: two 52- to
56-day-old sexually mature rats (three experiments) and five 21- to
23-day-old prepubertal rats (two experiments). Germ cells from animals
of the same ages were prepared by mechanical treatment and
trypsinization as previously described (29). Briefly, decapsulated
testes were thoroughly minced with an array of sealed razor blades and
treated with deoxyribonuclease and trypsin. This procedure destroys
most of Sertoli and Leydig cells while germ cells remain intact (29).
Germ cells were pelleted, washed, suspended in assay buffer, and
hand-homogenized on ice by 100 strokes. The 1500 x g
supernatant was prepared, and the protein content was measured.
Contamination with testicular somatic cells was determined by
morphological examination of air-dried smears stained with periodic
acid-Schiff-hematoxylin (29). The germ cell protein extracts were
prepared from independent groups of animals: two 52- to 56-day-old
sexually mature rats (two experiments) and five 21- to 23-day-old
prepubertal rats (one experiment).
Activities of P45014DM and of the two marker enzymes, lactate
dehydrogenase (LDH) and creatine kinase (CK)
The P45014DM assay and HPLC separation of sterols were performed
exactly as previously described (14). Each 0.5-ml reaction
contained 3–8 mg testis or germ cell total protein, 1.6 x
105 cpm pure [3H]24,25-dihydrolanosterol, 25
nmol of the mixture of cold lanosterol and 24,25-dihydrolanosterol in
an equal volume of Triton WR 1339 (0.4 mg/assay), and the inhibitors
NaCN (0.5 mM) and AY9944 (0.1 µm) to block the further
metabolism of MAS-412. Ketoconazole (20 µM, final
concentration) was added in control reactions to block the
CYP51-mediated de-methylation of lanosterol. CK and the LDH
activities were determined by colorimetric kits (no. 520 and 500,
respectively, Sigma Chemical Co., St. Louis, MO). An increase in CK
activity is characteristic of immature male germ cells, whereas an
increase in LDH activity marks the presence of postmeiotic germ cells.
The average value and the SEM of three to seven
measurements were calculated using the Microsoft Excel program.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, lane 8), as previously observed in
humans (14). In addition to the three somatic transcripts, ranging in
size from 2.5–3.1 kb (Fig. 2) previously detected by others in rat
liver (13), a smaller transcript (
2.0 kb) was present in testis
only. Note that although the testis-specific transcript and the
smallest somatic transcript were highly expressed in testis, expression
of the two larger somatic CYP51 transcripts was clearly lower than that
in liver. The identity of the shorter transcript was investigated by
screening a rat testicular cDNA library and sequencing the
3'-untranslated region of the seven isolated clones. Six of these
clones contain 3'-untranslated sequences, none extending beyond
nucleotide 1856 of the 2383-bp rat CYP51 cDNA reported by Sloane (13).
Three of these have polyadenylated tails, all in different positions
(Fig. 3), resulting in transcripts with
significantly shorter (>500 bp) 3'-untranslated regions compared with
clones isolated from rat liver (12, 13). None of the 5'-ends of these
clones exceeded the published rat liver cDNA sequence (not shown). When
translated in vitro, two proteins arose from the 1856-bp rat
testicular CYP51 cDNA, with the expected sizes of about 55 and 53.5 kDa
(Fig. 4). Two translation start sites
with the appropriate Kozak sequence were present in the rat and human
CYP51 mRNAs (13, 14). This suggests that two P45014DM proteins, one
with 17 fewer amino acids at the N-terminus, can be synthesized from
the same CYP51 cDNA template that represents the testis-specific CYP51
mRNA transcript seen in lane 8 of Fig. 2.
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). Only background levels of CYP51 mRNA
were detected in somatic Leydig cells, whereas distinct signals were
observed in these cells with a CYP17 (17-hydroxylase) probe
(positive control; not shown). CYP51 mRNA was detected in H-540 Leydig
cell tumor by Northern analysis (Fig. 6A, lane 5), which may indicate higher levels of expression of CYP51 in
Leydig cell tumor compared with levels in normal Leydig cells. Note
that the intensity of the CYP51 signal varied greatly between different
seminiferous tubule cross-sections (Fig. 5), suggesting expression in a
stage-specific manner. As the seminiferous epithelium consists of
somatic peritubular and Sertoli cells as well as germ cells, we
performed Northern analysis using RNA from different testicular cell
fractions. Figure 6A shows that somatic cell types (Sertoli cells,
peritubular cells, and Leydig tumor H-540 cells) contained only the
three somatic CYP51 transcripts (lanes 1–5), which were present at
relatively low levels. The smaller, testis-specific transcript was
restricted to germ cells and was first detected in the pachytene stage
primary spermatocytes, being stronger in round spermatids from
32-day-old animals (lanes 6 and 7). At this age no elongating
spermatids were yet present. In 44-day-old rats, elongating spermatids
were present and showed the strongest signal (lane 10). The intensity
of the three somatic transcripts varied between different cell
fractions. In all somatic cells (lanes 1–5), the largest transcript
(3.1 kb) dominated, whereas the smaller somatic transcript (2.5 kb)
dominated in germ cells from 32-day-old rats (lanes 6 and 7). In
elongating spermatids from 44 day old rats (lane 10), all three somatic
transcripts were significantly reduced compared with those at earlier
stages of germ cell development.
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with a probe for rat squalene synthase. Figure 6B
shows that the mRNA for this enzyme is also highly expressed in germ
cells (lanes 6–10). At least three transcripts are present in all cell
types and are all stronger in germ cells, but there is no germ
cell-specific squalene synthase mRNA. Increased levels of squalene
synthase mRNA are already present in pachytene spermatocytes from
32-day-old animals, where the testis-specific CYP51 mRNA is not yet
present. The mRNA levels of the squalene synthase peak in round
spermatids of 44-day-old animals and are diminished in elongating
spermatids. Thus, squalene synthase mRNA peaks earlier than the CYP51
mRNA during spermatogenesis.
Expression of CYP51 mRNA at different stages of rat
spermatogenesis
Spermatogenesis in rat seminiferous tubules can be divided into 14
different stages of cellular associations, which can be identified in
specially stained microscopic sections (30). The stages are more
difficult to identify in fresh tissue, but Parvinen (31, 32) has
developed a transillumination technique by which fresh rat seminiferous
tubules can be sectioned into 10 different stages: I, II–III, IV–V,
VI, VIIab, VIIcd, VIII, IX–XI, XII, and XIII–XIV. To further localize
CYP51 in rat testicular germ cells, we performed in situ
hybridization on sections of staged pieces of seminiferous tubules.
Using the same rat CYP51 antisense riboprobe as that in Fig. 5, only a
weak signal was detected in stage I (Fig. 7A) and in stages II and III (same as
stage I; not shown). Stronger signals restricted to the round
spermatids were present in stages IV, V (same as stage VI; not shown),
and VI (Fig. 7B). In stage VII and onward, the signal was very strong,
and in stages VIIab (Fig. 7, C and E) and VIII (same as stage VII; not
shown), it was restricted to late round and early elongating
spermatids. The brightfield in situ hybridization of stage
VIIab (Fig. 7E) shows that there was no signal in most mature
elongating spermatids that face the lumen. In stages IX–X, XI (same as
stages XIII–XIV; not shown), and XIII–XIV (Fig. 7D), the signal was
more dispersed, but continued all the way into the innermost cell layer
facing the lumen, which were the most mature germ cells. In the next
stage, stage I, the CYP51 signal was reduced again to background levels
(Fig. 7A). In summary, a high level of CYP51 mRNA is restricted to
round and early elongating spermatids of stages IV–XIV, as
schematically shown in Fig. 7G.
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-demethylase activity was measured in protein
extracts of the whole testes and isolated germ cells prepared by
trypsinization of decapsulated testes of sexually mature (52- to
56-day-old) and prepubertal (22- to 23-day-old) rats. The periodic
acid-Schiff-stained smears show only premeiotic germ cells
(spermatocytes) in preparations from prepubertal animals (Fig. 8A). No Sertoli cells were observed,
whereas Leydig cells were present at less than 1% of the total cell
population. Preparations of germ cells from the older rats contain
premeiotic spermatocytes as well as postmeiotic round and elongating
spermatids, whereas the extent of contaminating somatic cells of the
testis was similar to that in younger rats (Fig. 8B). To confirm the
quality of protein preparations, control experiments measuring the
activities of CK and LDH were performed. CK activity was higher in
samples containing a higher percentage of immature spermatozoa (33, 34). In contrast, LDH activity was higher in samples containing
postmeiotic germ cells due to the presence of a germ cell-specific
protein isoform that is expressed in these cells (35). The
protein extracts from whole testis or germ cell preparations of
sexually mature rats and prepubertal rats exhibited the expected
activities for the CK and LDH marker enzymes; CK activity was higher in
extracts from prepubertal rats, whereas LDH activity was higher in
protein extracts prepared from sexually mature animals (Table 1). The CYP51 activity followed the
pattern of LDH. In all measurements, a reproducible, approximately 20%
higher P45014DM activity was observed in the protein extracts from
testis of sexually mature rats compared with that from prepubertal
animals. P45014DM activity was also detected in germ cell
protein extracts of sexually mature rats, but was below the limit of
accurate quantification in germ cells of prepubertal rats (Table 1).
These results suggest that the increase in CYP51 activity in testis of
sexually mature rats is due to the increased activity in postmeiotic
germ cells. The appearance of
4,4-dimethyl-5-cholesta-8,14-dien-3ß-ol (MAS-412) (21), which was
produced from 24,25-dihydrolanosterol by P45014DM, was completely
abolished in the presence of the inhibitor ketoconazole or in the
absence of the NADPH-regenerating system (not shown); both are
characteristics of a P45014DM-dependent reaction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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24–25 reduced
analogs, MAS-412 and MAS-414. Sterols FF-MAS and MAS-412 are the
products of lanosterol 14-demethylase and substrates for the sterol
14-reductase, which produces T-MAS and MAS-414. Until the present study
nothing was known about the expression of enzymes that can produce MAS
sterols in male germ cells. Herein we describe that the expression of
CYP51 mRNA and the activity of MAS-producing P45014DM are elevated in
postmeiotic germ cells during spermatogenesis, indicating that MAS
sterols may serve different roles in germ cells of males and
females.
The higher lanosterol 14-demethylase activity in testis and germ
cells of sexually mature rats compared with that in prepubertal animals
follows the pattern of the germ cell-specific LDH expression. The CYP51
activity is reproducibly (20%) higher in protein extracts from
testis of sexually mature rats than in extracts from prepubertal
animals. This suggests that the increased expression of CYP51 in
postmeiotic germ cells, as evidenced from the expression of the
testis-specific transcript, leads to increased P45014DM activity and
elevated synthesis of MAS sterols in male germ cells that have already
completed meiosis. There is no obvious reason why the increased
P45014DM-mediated MAS sterol production in nondividing postmeiotic germ
cells would be associated with increased cholesterol biosynthesis. As
cholesterol is an important membrane constituent, one might expect that
the rapid proliferation of the premeiotic spermatogonia in the testis
would require higher endogenous cholesterol production than postmeiotic
germ cells. If P45014DM-mediated MAS sterol production is important for
meiosis activation during spermatogenesis, MAS sterols would need to be
transported to premeiotic male germ cells. A MAS-specific receptor has
not yet been found. The widely expressed orphan nuclear receptor LXR
can bind FF-MAS, albeit with lower affinity than some other
3ß-hydroxy sterols (38), and is most likely not a specific receptor
for FF-MAS. Thus, the increased production of MAS sterols in
postmeiotic male germ cells may suggest a role for these sterols
different from meiosis activation during spermatogenesis.
There is a large discrepancy between the CYP51 mRNA levels in germ cells and the observed enzymatic activities. The expression of the germ cell-specific CYP51 mRNA is at least an order of magnitude higher than expression of somatic CYP51 transcripts in the testis, but the P45014DM activity is higher in whole testis than in germ cells. On the other hand, P45014DM activity is clearly higher in postmeiotic germ cells or testis containing such cells than in similar samples containing only premeiotic germ cells. Transcription ceases after stage VIII in spermiogenesis, and stable transcripts are required to provide templates for the synthesis of proteins that are needed in later steps of sperm maturation. For example, the mouse protamine 2 gene is transcribed in the round spermatids, yet the mRNA needs to be stable for about 7 days until its delayed translation occurs in the elongating spermatids (39, 40). The temporal profile of CYP51 mRNA during spermatogenesis and the detection of T-MAS in human spermatozoa (Baltsen and Byskov, personal communication) suggest that large amounts of the germ cell-specific CYP51 mRNA are needed to assure sufficient template for P45014DM protein synthesis during the period of germ cell maturation after mRNA synthesis has stopped. Perhaps the germ cell-specific CYP51 transcript is more stable than somatic CYP51 transcripts, and its in vivo translation is delayed as with the protamine-2 gene. Alternatively, less efficient translation, as described for the testis-specific form of the rat farnesyl pyrophosphate synthase gene (41), could be the reason for the high level of testis-specific CYP51 mRNA. In either case, increases in levels of CYP51 mRNA significantly greater than increases in P45014DM activity would be expected. Under any circumstances, P45014DM activity is elevated in postmeiotic germ cells.
The germ cell-specific CYP51 transcript arises by utilization of an
upstream polyadenylation site. The three polyadenylated CYP51 clones
from the rat testicular cDNA library end at nucleotides 1816, 1820, and
1856, and they all correspond to the approximately 2.0-kb germ
cell-specific CYP51 mRNA transcript (Fig. 2, lane 8). These clones
probably arose by using variant cleavage sites within the more upstream
polyadenylation site (42). The presence of shorter, testis-specific
transcripts has been reported for other genes, some of which also arise
from the utilization of upstream polyadenylation sites. Frequently, no
consensus polyadenylation signal is present, as observed for the rat
testis-specific CYP51 transcript, although variant signals may be
detected (43, 44, 45, 46, 47). One such variant signal, AAUGAA, is present at
nucleotides 1799–1804 in the rat CYP51 noncoding sequence, 11
nucleotides upstream from the most 5'-polyadenylation cleavage site
(Fig. 3). In the case of CREM (cAMP-responsive element modulator), a
switch from the most 3' to a more 5' polyadenylation site occurs in the
testis during development (48) at the same time that levels of CREM
transcript increase dramatically. This increase is due to the removal
of 10 AUUUA mRNA destabilizer elements of the longer transcript (49).
Such destabilizer elements are not present in the longer transcript of
the rat CYP51 3'-untranslated region, but other, as yet unidentified,
sequences may affect mRNA stability.
In conclusion, we demonstrate for the first time that postmeiotic male germ cells contain P45014DM and have the capacity to synthesize MAS sterols, probably above the levels needed for cholesterol biosynthesis. Whether these sterols act as signaling molecules to activate meiosis in spermatogenesis (50, 51) or have as yet unknown functions in male germ cell maturation remains to be elucidated. As the onset of meiosis in gonads differs between sexes (50), it would not be surprising if MAS sterols would have different functions in ovaries and testis. Further studies of P45014DM and other enzymes of the postsqualene part of cholesterol biosynthesis as well as studies of potential MAS signaling pathways will help to clarify this point.
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Acknowledgments |
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Footnotes |
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2 Present address: Laboratory of Reproductive Biology, Section 5712,
Center for Women, Children and Reproduction, Rigshospitalet,
University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen,
Denmark.
3 Present address: Andrology Laboratory Department of Gynecology and
Obstetrics, National Hospital, University of Oslo, N-0027 Oslo,
Norway.
Received September 3, 1997.
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 B. LINDENTHAL, A. L. HOLLERAN, T. A. ALDAGHLAS, B. RUAN, G. J. SCHROEPFER Jr, W. K. WILSON, and J. K. KELLEHER Progestins block cholesterol synthesis to produce meiosis-activating sterols FASEB J, March 1, 2001; 15(3): 775 - 784. [Abstract] [Full Text] [PDF]
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 K. M. Vaknin, S. Lazar, M. Popliker, and A. Tsafriri Role of Meiosis-Activating Sterols in Rat Oocyte Maturation: Effects of Specific Inhibitors and Changes in the Expression of Lanosterol 14{{alpha}}-Demethylase During the Preovulatory Period Biol Reprod, January 1, 2001; 64(1): 299 - 309. [Abstract] [Full Text]
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D. Rozman, M. Fink, G. M. Fimia, P. Sassone-Corsi, and M. R. Waterman Cyclic Adenosine 3',5'-Monophosphate(cAMP)/cAMP-Responsive Element Modulator (CREM)-Dependent Regulation of Cholesterogenic Lanosterol 14{alpha}-Demethylase (CYP51) in Spermatids Mol. Endocrinol., November 1, 1999; 13(11): 1951 - 1962. [Abstract] [Full Text]
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D. Rozman and M. R. Waterman Lanosterol 14alpha -Demethylase (CYP51) and Spermatogenesis Drug Metab. Dispos., December 1, 1998; 26(12): 1199 - 1201. [Abstract] [Full Text]
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