1
Author’s institutions: 1Center for Animal Genomics, Veterinary Faculty, University of
Ljubljana, Gerbičeva 60, SI-1000 Ljubljana, Slovenia
2
University of Zagreb, School of Medicine, Dept. Histology and Embryology, Šalata 3, 10000
Zagreb, Croatia
5
3
Centre for Functional genomics and Bio-Chips, Institute of Biochemistry, Medical Faculty,
University of Ljubljana, Zaloška 4, SI-1000 Ljubljana, Slovenia
Title: Initiation of steroidogenesis precedes expression of cholesterolgenic enzymes in the
fetal mouse testes
10
Authors: 1Büdefeld T., 2Jezek D., 3Rozman D., and 1Majdic G.
Short title: Steroidogenesis and cholesterol production in the fetal mouse testis
15
Key words: Mouse, testis, fetal, human, 3beta-hydroxysteroid dehydrogenase, StAR, cyp51,
NADPH cytochrome P450 reductase, immunocytochemistry
Corresponding author: Gregor Majdic
Center for Animal Genomics
20
Veterinary Faculty, University of Ljubljana
Gerbičeva 60, SI-1000 Ljubljana
Slovenia
Phone: +386 1 4779210, Fax: +386 1 2832243
Email: gregor.majdic@vf.uni-lj.si
25
2
Abstract
Sexual differentiation is a carefully regulated process that ultimately results in a development
of the male or female phenotype. Proper development of the male phenotype is dependent
upon the action of testosterone, produced by Leydig cells, and antimullerian hormone,
5
produced by Sertoli cells. Leydig cells start to produce testosterone around day 12.5 in the
fetal mouse testis, and continue to produce high levels of testosterone throughout the
gestation. In the present study we examined whether expression of two enzymes, lanosterol
14α-demethylase (cyp51) and cytochrome P450 NADPH reductase, involved in the
cholesterol production, occurs simultaneously with proteins required for the production of
10
steroid hormones. Immunocytochemical staining with antibodies against lanosterol 14αdemethylase , cytochrome P450 NADPH reductase, steroidogenic acute regulatory protein
(StAR) and 3beta-hydroxysteroid dehydrogenase I (3β-HSD I) was used to determine the
ontogeny of expression of these four proteins. As expected, 3β-HSD I and StAR proteins
were detected on day 12.5 p.c., while expression of cyp51 and NADPH cytochrome P450
15
reductase appeared one day later, on day 13.5. Thereafter, the expression of all four proteins
remained strong throughout the gestation. In human fetal testes, all four proteins were
expressed in the Leydig cells at all ages studied, although very young samples from the time
of initial Leydig cell development were not examined as they were not available. Results of
this study suggest that initial steps of steroid hormone production in murine Leydig cells are
20
dependent on exogenously derived cholesterol. Later on (from day 13.5 onwards), murine and
human Leydig cells are able to synthesize cholesterol and are therefore not dependent on
exogenous cholesterol resources.
3
Introduction
Mammalian gonads develop as genital ridges on the ventral side of the mesonephros and these
subsequently develop into testis or ovary, depending on the presence or absence of Y
chromosome and Sry gene (George & Wilson, 1994; Ross & Capel, 2005). Shortly after
5
differentiation, testes start to produce hormones necessary for the proper development of male
secondary sexual organs. Antimullerian hormone (AMH), produced by Sertoli cells, is
responsible for the regression of the mullerian ducts that would otherwise develop into
oviducts, uterus and upper part of vagina (Josso et al., 2001) while steroid hormone
testosterone is responsible for proper development and masculinization of secondary male
10
sexual organs (George & Wilson, 1994). Female sexual organs develop in the absence of
hormones as ovaries remain hormonally inactive throughout the fetal life and the female
reproductive tract develops also in the complete absence of gonads (George & Wilson, 1994).
Sertoli cells are the first cells that develop in the fetal testis, followed shortly by Leydig cells.
Leydig cells appear in the interstitial tissue around day 12.5 p.c. in mice (Habert et al., 2001).
15
The exact origin of these cells is not yet clear and they might derive either from mesenchymal
like stem cells (perycites and vascular smooth muscle cells) or stem cells from neural crest
(Buehr et al., 1993; Davidoff et al., 2004). Early markers of differentiating Sertoli and
Leydig cells are expression of AMH and steroidogenic enzymes, respectively. Testosterone
synthesis from cholesterol requires four steroidogenic enzymes, of which 3ß-hydroxisteroid
20
dehydrogenase/5-4 isomerase (3ß-HSD) is thought to be constitutively expressed in Leydig
cells and is therefore useful marker for these cells. Greco and Payne (1994) studied the
expression of 3ß-HSD I, P450scc, P450c17 and P450arom mRNA using RT-PCR in fetal
testes and ovaries from C57BL6/J mice. They reported the expression of P450scc, 3β-HSD I
and P450c17 mRNA on day 13.5 p.c. but there are no other reports about early expression of
25
proteins involved in production of steroid hormones in early fetal mouse gonads, although
4
Gondos (1980) reported that testosterone production in the fetal mouse testes starts around
day 12.5 – 13.0 p.c.
The initial steps in the biosynhesis of steroid hormones represent a transport of cholesterol to
the outer mitochondrial membrane, followed by translocation of cholesterol across the outer
5
mitochondrial membrane and intermembrane space to reach cytochrome P450scc, which
resides on the inner mitochondrial membrane. StAR (steroidogenic acute regulatory protein)
is a phosphoprotein that is obligatory for this transport and it is thought that StAR connects
both mitochondrial membranes, forming a hydrophobic tunnel through which cholesterol
could cross hydrophilic intermembrane space (Clark et al., 1994; Stocco, 2001). In situ
10
hybridization studies have shown the presence of StAR mRNA in the fetal mouse gonads as
early as day 10.5 p.c. in both sexes. During subsequent development, StAR mRNA
expression persisted in the fetal testis but was absent from the fetal ovaries (Clark et al.,
1995).
15
Cholesterol is an obligatory precursor for all steroid hormones and has to be either
synthesized by the steroidogenic cells or delivered to them by blood or surrounding cells
(Azhar et al., 2003). Lanosterol 14-demethylase (cyp51) is a member of the cytochrome
P450 superfamily involved in early steps of cholesterol biosynthesis. As cholesterol is an
integral part of cell membranes and is involved in many other functions in the cells, cyp51 is
20
present in most, if not all, mammalian cells (Choudhary et al., 2003). However, its
expression is stronger in steroidogenic tissues such as gonads and adrenal glands in adult
animals (Stromstedt et al., 1996; Majdic et al., 2000), but there are no reports about the cyp51
expression, or expression of any of the other enzymes involved in cholesterologenesis, during
early stages of fetal gonadal development. NADPH cytochrome P450 reductase is another
5
enzyme required for cholesterol production, as it is necessary partner in several enzymatic
reactions during transformation of acetate to cholesterol.
In the present study, immunoexpression of cyp51, NADPH cytochrome P450 reductase, StAR
5
and 3β-HSD I proteins was studied in murine gonads during fetal development with the aim
to determine whether cholesterol is produced endogenously or derived exogenously during
initial steps of steroid hormone production.
6
Materials and Methods
Animals and tissue recovery
C57BL/6 mice were bred in standard conditions. Females were paired with males and
checked every morning for the presence of copulatory plug. The morning on the day when
5
plug was found was designated as day 0.5 p.c. Pregnant females were euthanized by CO2
followed by cervical dislocation on different days of pregnancy. Fetuses were dissected and
either whole fetuses or fetal gonads were fixed in Bouins’ solution overnight (whole fetuses)
or 3-4 hours (isolated gonads) and subsequently processed into paraffin wax using standard
procedures.
10
All animal experiments were done according to ethical principles and in
accordance with EU directive (86/609/EEC). Animal experiments were approved by the
Veterinary commission of Slovenia.
Sex determination
Mice fetuses 11.5, 12.0 and 12.5 days old were genotyped to determine sex. At the time of
15
dissection, amnions were carefully removed and digested in the thermostatic shaker in 200 l
of PCR DNA buffer (Promega, Madison, WI, USA) containing 0.15 mg of Proteinase K
(Sigma, Taufkirchen, Germany) at 55 ºC overnight. Three microliters of lysate were used for
PCR reaction containing primers for the Sry gene as described before (Luo et al., 1994).
20
Immunocytochemistry
Sections 7 µm thick were mounted on the slides coated with 3-aminopropyl triethoxysilane
(TESPA, Sigma) and dried overnight at 42ºC. Before incubation with the primary antibody,
sections were dewaxed in xylene, rehydrated in graded ethanols, washed in water and 0.01M
PBS containing 0.05% Tween 20 (Svanova Biothech AB, Uppsala, Sweden) . Endogenous
25
peroxidases were blocked by incubating the sections for 20 minutes in 1% H2O2 in PBS-
7
Tween 20 at room temperature, followed by 5 minute wash in PBS-Tween 20. For StAR
immunostaining, antigen retrieval was performed in 0.01M Na-citrate pH 6.0 (Sigma) before
incubation with primary antiserum. Sections were boiled in the microwave for 20 minutes
and left undisturbed for additional 20 minutes, followed by a 5 minute wash in PBS-Tween
5
20. All sections were blocked in normal goat serum diluted 1:5 in PBS-Tween 20 (Chemicon
Temecula, CA, USA) for 30min. Rabbit polyclonal antibodies against the human CYP51
protein (gift from Mike Waterman, Vanderbilt University, Nashville, TN, USA), rabbit
polyclonal antibodies against NADPH cytochrome P450 reductase (Abcam, Cambridge, UK),
rabbit polyclonal antibodies against 3ß-HSD I (gift from Ian Mason, University of Edinburgh,
10
Edinburgh, Scotland) and rabbit polyclonal antibodies against StAR (gift from Dale Hales,
Northwestern University, Chicago, IL, USA) were used at dilutions of 1:50, 1:500, 1:1000
and 1:200, respectively (Doody et al., 1990; Ronen-Fuhrmann et al., 1998; Majdic et al.,
2000). Sections were incubated with antibodies diluted in PBS-Tween 20 containing normal
goat serum (5:1 v/v) overnight at 4ºC (for all four antisera). The following day sections were
15
washed twice in PBS-Tween 20 (5 minutes each wash), incubated for 30 minutes with 1:100
dilution of goat anti-rabbit IgG (Dako, Glostrup Denmark) in PBS-Tween 20 and washed
again in PBS-Tween 20 twice for 5 minutes. For detection of bound antibodies, sections were
incubated with a 1:500 dilution of rabbit peroxidase-antiperoxidase complex (Jackson
Immunochemicals, West Grove, PA, USA) in 0.05M Tris, pH 7.4 for 30min and then washed
20
twice in PBS-Tween 20 (5 minutes each).
Color reaction product was developed by
incubating sections in a solution of 0.05% (w/v) 3, 3’-triaminobenzendine tetrahydrochloride
(Sigma) in 0.05M Tris-HCl, pH 7.4 and 0.01% hydrogen peroxide. After 5-30 min, sections
were washed twice in distilled water, counterstained with hematoxyline, dehydrated in graded
ethanols, cleared in xylene and coversliped using Pertex mounting medium (Medite,
25
Burgdorf, Germany). Specificity of the antisera was evaluated by using normal rabbit serum
8
instead of primary antisera.
microscope.
5
Photomicrographs were taken on Nikon microphot FXA
9
Results
Mouse gonads
3β-hydroxysteroid dehydrogenase I (3β-HSD I)
No immunopositive cells were detected in the fetal gonads on days 11.5 p.c and 12.0 p.c.. On
5
day 12.5 p.c., several immunopositive cells were found in the fetal testes (Fig. 1a) while no
immunoexpression was found in the fetal ovary (not shown). Immunoexpression of 3β-HSD I
remained strong in the testes throughout the gestation (Fig. 1c – 13.5 p.c., 1e – 18.5 p.c.).
Steroidogenic acute regulatory protein (StAR)
10
No StAR immunoexpression was detected at 11.5 p.c. and 12.0 p.c., and few scattered
positive cells were found on day 12.5 p.c (Fig. 1b) in the mouse fetal testis. On day 13.5 p.c.,
expression increased and several strongly immunopositive cells were found in the fetal testes
(Fig. 1d) while no immunopositive cells were detected in the fetal ovary at 12.5 or 13.5 p.c.
(not shown). Similar to 3β-HSD I, StAR immunoexpression in the testis remained strong
15
throughout the fetal period (Fig. 1f, 18.5 p.c.).
Lanosterol 14α-demethylase (cyp51)
Cyp51 protein was absent in the fetal gonads on days 11.5 p.c. to 12.5 p.c. (Fig 2a) but
appeared in the fetal testis on day 13.5 (Fig 2c). Thereafter, immunoexpression remained
20
strong in the fetal Leydig cells (Fig 2e) and was absent in the fetal ovaries (not shown).
NADPH cytochrome P450 reductase
NADPH cytochrome P450 reductase protein was not detected in the fetal gonads on days 11.5
p.c., 12.0 p.c. and 12.5 p.c. (Fig 2b) but appeared in the fetal testis on day 13.5 (Fig 2d).
10
Thereafter, immunoexpression remained strong in the fetal Leydig cells (Fig 2f) and was
absent in fetal ovaries (not shown).
11
Discussion
Sexual differentiation is a carefully regulated process that requires precise temporal and
spatial development of different cell types in the developing gonads (George & Wilson, 1994;
Ross & Capel, 2005). Hormones produced by the fetal testis are essential for the
5
development of all male secondary sexual organs. Therefore, proper development and
differentiation of testicular cells is crucial for the normal development of the male phenotype.
In contrast, the fetal development of the female phenotype is largely hormone independent as
even in the absence of ovaries, mullerian ducts develop into the oviducts, uterus and upper
portion of vagina (George & Wilson, 1994). In the present study, immunoexpression of four
10
different proteins involved in the production of steroid hormones or their precursor,
cholesterol, was studied during gonadal development in the mouse fetuses with the aim to
establish whether the newly developed Leydig cells posses enzymes for steroidogenesis and
production of cholesterol or are dependent on exogenously derived cholesterol for the
production of testosterone.
15
Cholesterol is an essential part of the cell membranes, has important roles as a signaling
molecule during the embryonic development and is a precursor for the steroid hormone
synthesis. Requirement for cholesterol during the embryonic development is clearly
demonstrated by embryonic lethality of a knockout mice model lacking squalene synthase,
20
enzyme involved in the production of cholesterol (Tozawa et al., 1999). Most cells in the
mammalian body have the ability to synthesize cholesterol from its precursor acetate.
However, exogenously derived cholesterol is equally important for the proper development
and function of the mammalian body and carefully regulated balance between exo- and
endogenously derived cholesterol is essential for proper function of cells (Azhar et al., 2003).
25
Cyp51 or lanosterol 14α-demethylase is involved in late steps of cholesterol synthesis
12
(Stromstedt et al., 1996). Cyp51 transforms lanosterol into 4,4,-dimethyl 5-cholesta,
8,14,24-diene-3-ol, also called follicular fluid meiosis activating sterol (FF-MAS) as some
studies suggested that this sterol acts as a meiosis activating substance at least in vitro
(Byskov et al., 1997; Byskov et al., 1999). Cyp51 is strongly expressed in the adult gonads
5
and adrenal glands. Interestingly, very strong expression of Cyp51 was detected in
postmeiotic germ cells, suggesting an important role of this enzyme in germ cells during
spermatogenesis (Stromstedt et al., 1998; Cotman et al., 2004). This increased expression of
Cyp51 could be connected with the remodeling of cell membranes in the developing
spermatids, although accumulation of Cyp51 product T-MAS (testes meiosis activating
10
substance) suggest additional roles for this sterol, possibly as a signaling molecule. NADPH
cytochrome P450 reductase is an electron transferring enzyme, required for the activity of
several enzymes involved in cholesterol production, including cyp51. The presence of
NADPH cytochrome P450 reductase is thought to be a rate limiting step in several enzymatic
reactions during cholesterol synthesis and the activity of this enzyme is absolutely necessary
15
for the production of cholesterol. In the fetal gonads, we found immunoexpression of cyp51
together with expression of NADPH cytochrome P450 reductase on day 13.5 p.c. in the fetal
testis and this expression persisted throughout fetal development. In the fetal testes, cyp51
and NADPH cytochrome P450 reductase were present in the interstitial, presumably Leydig
cells, suggesting that de novo cholesterol biosynthesis is at least partially utilized for
20
steroidogenesis in the fetal Leydig cells. Interestingly, the expression of both StAR and 3βHSD I proteins (as shown in this study) and the production of testosterone (Gondos, 1980)
starts earlier, already on day 12.5 p.c. suggesting that initial steps of testosterone production
in the fetal Leydig cells is dependent on exogenously derived cholesterol, what would be in
agreement with previous studies, suggesting that plasma lipoproteins are the main source of
25
cholesterol in steroidogenic cells (Andersen & Dietschy, 1978; Hou et al., 1990; Azhar et al.,
13
1998). However, in our study we only used immunocytochemistry to determine the presence
of the cyp51 and NADPH cytochrome P450 reductase proteins. As immunocytochemistry
has a limited detection potential, it cannot be excluded at the present that both proteins were
expressed at low levels also at an earlier age, but below detection limits of
5
immunocytochemistry. One possibility to get a more conclusive answer would be to examine
the mRNA expression using quantitative RT PCR, although the presence of the mRNA does
not necessarily indicate the presence of bioactive proteins. The only conclusive answer could
therefore be derived from measuring enzyme activity directly, but this would be almost
impossible to conduct precisely, as specific dissection of fetal gonads (with exclusion of all
10
surrounding tissue including nearby adrenal cells) on day 12.5 p.c. is almost impossible
without microscopic dissector, but preparation of tissue for such dissection would interfere
with enzyme activity. As antibodies against both NADPH cytochrome P450 reductase and
cyp51 were shown before by western blot and immunocytochemistry (REFERENCE) to
readily detect the respective proteins, this study nevertheless suggest that the significant
15
amounts of NADPH cytochrome P450 reductase and cyp 51 proteins in the fetal mouse testis
are only present from day 13.5 p.c. onwards.
Both StAR and 3β-HSD I proteins were detected in our study at 12.5 p.c., immediately after
the formation of testicular cords and Sertoli cell differentiation (reviewed in Swain & Lovell20
Badge, 1999). This is interesting, as in the rat fetuses, Leydig cells develop about 1 - 2 days
later then Sertoli cells and several studies suggested that Sertoli cells regulate Leydig cell
differentiation (Lejeune et al., 1992; Racine et al., 1998). The present study together with
previous report of testosterone production in the fetal mouse testis on day 12.5 p.c. ( Gondos,
1980) implicate that delay between Leydig and Sertoli cells appearance in the developing
25
testis is much shorter in mice than in rat. The fetal mouse testis starts to produce testosterone
14
and antimullerian hormone almost simultaneously, unlike in rats where there is at least one
day difference in the onset of expression of AMH and steroidogenic enzymes (Tran et al.,
1987; Munsterberg & Lovell-Badge, 1991; Majdic et al., 1998), suggesting that Sertoli cell
regulation of Leydig cell development in mice must be much quicker process than in rat.
5
In conclusion, in the present study we demonstrated early expression of two proteins required
for the production of steroid hormones, StAR and 3β-HSD I, suggesting almost simultaneous
development of Sertoli and Leydig cells in the fetal mice testes. As expression of cyp51 and
NADPH cytochrome P450 reductase appeared only one day later, these results suggest that
10
early steroid hormone production is mostly dependent on exogenously derived cholesterol.
15
Acknowledgement
The authors are grateful to Mike Waterman, Ian Mason and Dale Hales for their generous
gifts of the antibodies. This work was done with support from Slovenian Ministry of Higher
education and science research programme P4-0053. Tomaz Büdefeld is supported by the
5
fellowship from foundation Stein.
16
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21
Figure legends:
Figure 1: No 3β-HSD I and StAR immunopositive cells were detected in fetal testes on day 12.0
p.c. Few 3β-HSD I (a) and StAR (b) immunopositive cells (arrows) were detected in fetal
5
testes on day 12.5 p.c.. Expression of both proteins increased one day later (c – 3β-HSD I, d –
StAR; day 13.5 p.c.) and remained strong throughout gestation (e – 3β-HSD I, f – StAR; day
18.5 p.c.). Insert - sections incubated with normal rabbit serum. Bar = 50 µm.
Figure 2: No cyp51 (a) or NADPH cytochrome P450 reductase (b) immunopositive cells
10
were detected in fetal testes on day 12.0 p.c. First immunopositive cells appeared on day 13.5
(c – cyp51, d – NADPH cytochrome P450 reductase) and thereafter, strong
immunoexpression persisted throughout the development (e – cyp51, f – NADPH cytochrome
P450 reductase; day 18.5 p.c.). Insert – sections incubated with normal rabbit serum. Bar =
50 µm.
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