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Department of Developmental and Molecular Biology (P.E.C.,
J.W.P.), Department of Obstetrics and Gynecology (J.W.P.), Albert
Einstein College of Medicine, Bronx, New York 10461,
Population Council and The Rockefeller University (M.P.H.),
New York, New York 10021
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase-lyase, with associated reductions in the activity of
all these steroidogenic enzymes, as well as in 17ß-hydroxysteroid
dehydrogenase. The CSF-1-deficient males also have reduced serum LH and
disruption of the normal testosterone negative feedback response of the
hypothalamus, as demonstrated by the failure to increase LH secretion
in castrated males and their lack of response to exogenous
testosterone. However, these males are responsive to GnRH and LH
treatment. These studies have identified a novel role for CSF-1 in the
development and/or regulation of the male
hypothalamic-pituitary-gonadal axis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The absence of CSF-1 in csfmop/csfmop male mice results in 90% lower serum testosterone (T) concentrations than wild type males (10) and is associated with low epididymal sperm numbers and reduced libido (10). In the testis, CSF-1 regulates the resident population of testicular macrophages (TMs) (12), which form an intimate structural relationship with neighboring steroidogenic Leydig cells (13, 14, 15). In csfmop/csfmop males, TM numbers are severely depleted throughout development (12) and this, together with previous reports of a regulatory role for TMs on Leydig cell function (for review see 16 , suggests that CSF-1 might act in a paracrine fashion, through TMs, to regulate steroidogenesis. However, because CSF-1, by acting through microglia, is involved in the development of functional neuronal processing in the brain (17), CSF-1 might also act at the level of the hypothalamus and pituitary to modulate the release of GnRH and/or the gonadotropins, LH and FSH, such that the LH-mediated regulation of Leydig cell function is disrupted in csfmop/csfmop males. The present studies were aimed at defining the molecular basis of the fertility defects in csfmop/csfmop males and demonstrate that this growth factor plays an essential role in the development and functioning of the hypothalamic-pituitary-gonadal axis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The 86.3% reduction in basal T
synthesis by Leydig cells derived from
csfmop/csfmop males is
similar to the 
90% lower serum T observed in these animals (10),
suggesting that the reduction in serum T is due directly to lowered
testicular steroidogenic capacity. Similar T synthesis results were
obtained from incubations of whole testis in vitro (data not
shown).
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-hydroxylase
lyase (P45017), and 17ß-hydroxysteroid dehydrogenase
(17ßHSD) activities are all significantly reduced in isolated Leydig
cells from csfmop/csfmop
males compared with +/csfmop Leydig cells (Table 2). P450scc catalyzes the
conversion of cholesterol to pregnenolone in the mitochondria. Its
activity in csfmop/csfmop
Leydig cells is reduced to 18.1% of that found in heterozygote Leydig
cells. The activity of 3ßHSD, which is responsible for the conversion
of pregnenolone to progesterone, is reduced to 66.3% of normal.
P45017 is a dual-activity enzyme responsible for the
conversion of progesterone to 17-hydroxyprogesterone through its
hydroxylase activity and the subsequent formation of androstenedione
through its lyase activity. The hydroxylase activity of
P45017 is reduced to 51.3%, while its lyase activity is
80.5% of normal levels (Table 2). 17ßHSD, which completes the
pathway by converting androstenedione to T, is also reduced in
csfmop/csfmop Leydig
cells to 40.6% of that seen in +/csfmop Leydig
cells (Table 2).
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were measured, either in whole testis or in
isolated Leydig cells, or both. Anti-P450scc antibody
detected a single band of 52 kDa in testis, Leydig cells, ovary, and
adrenal extracts but not in spleen (Fig. 1, A and B). This band corresponds to
that detected in a commercial control sample of purified bovine
mitochondria (see Materials and Methods; Fig. 1A). The
concentrations of P450scc in whole testicular extracts of
csfmop/csfmop males, when
normalized to glyceraldehyde phosphodehydrogenase (GAPDH) protein
concentration in three separate Western blots, was very significantly
reduced (P < 0.0005, Mann Whitney test, n = 9) to
16.7% of wild type concentrations (Fig. 1A). These data were confirmed
by examination of an isolated Leydig cell protein extract in which the
level of P450scc enzyme was 14.7% of that found in
heterozygote Leydig cells (Fig. 1B). Interestingly, however, mRNA
expression of P450scc, as determined by ribonuclease
protection assay (RPA) and normalized to GAPDH mRNA expression, was
similar in the testes of
csfmop/csfmop and
+/csfmop males (Fig. 1C), indicating a
posttranscriptional effect on the testicular content of this
enzyme.
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, A and B). The specificity of the
anti-3ßHSD antibody was confirmed using protein extracts from COS-1
cells expressing either isoform I or VI, both of which are expressed in
testicular Leydig cells, while only isoform I is found in the adrenal
gland (18). Both isoforms migrate at similar rates on 10%
polyacrylamide gels, having only 2 kDa difference in molecular mass
(18), and both are detected by the antibody. This band migrates at the
same rate as that detected in the steroidogenic tissues, but no
comigratory band was detected in the spleen samples. The 3ßHSD
concentration in whole testis extracts from
csfmop/csfmop males,
determined by densitometric analysis of four separate Western blots, is
43.1% of the +/csfmop level (Fig. 2A), a figure
that is statistically significant (P < 0.05, Mann
Whitney test, n = 12). In an extract of Leydig cells from
csfmop/csfmop males,
anti-3ßHSD antibody Western blotting detects only 14.8% the amount
of 3ßHSD detected in a Leydig cell extract from
+/csfmop males (Fig. 2B). Northern blot analysis
of total RNA from +/csfmop and
csfmop/csfmop testes detects a mRNA transcript
of 1.6 kb whose concentration does not differ between genotypes (Fig. 2C). A similar band is detected in RNA isolated from ovaries and
adrenal glands, but not spleen (Fig. 2C). These results were confirmed
by a RPA using a probe specific to nucleotides 502–839 of the
published mRNA sequence (data not shown).
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protein is also reduced in
whole testis extracts from
csfmop/csfmop males to
90% of normal (Fig. 3A) which, although
being only a 10% reduction, is statistically significant
(P < 0.05, Mann Whitney test for the means of two
Western blots, n = 6). The antibody against P45017
detects a band of 52 kDa in protein extracts from testis, Leydig cell,
and ovary, but not in adrenal or splenic samples. In testis and to a
lesser extent in spleen, the antibody also detects a major
cross-reactive band of around 49 kDa. This band is not present in
isolated Leydig cell extracts. The P45017 protein level
in two isolated Leydig cell extracts from
csfmop/csfmop males is
reduced to 44.7% of that seen in heterozygote Leydig cells (Fig. 3B).
However, P45017 mRNA concentrations are normal in total
RNA extracts from
csfmop/csfmop whole
testis (Fig. 3C). Expression of the 1.7-kb P45017
message is also detected in ovarian total RNA but not in adrenal and
spleen RNA (Fig. 3C). The normal expression of P45017
mRNA in csfmop/csfmop
testes was confirmed by RPA (data not shown).
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, A and B).
Furthermore, serum corticosterone concentrations in
csfmop/csfmop and
+/csfmop males were also not
significantly different (Fig. 4C).
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) and are limited to a small
portion of the cytoplasmic volume. In
csfmop/csfmop males,
however, the membranous whorls occupy the majority of the Leydig cell
cytoplasm, often occluding and displacing the nucleus (Fig. 5B). The
increased size of the whorls observed in
csfmop/csfmop Leydig
cells appears to result from unravelling of the whorls and/or dilation
of the intermembrane spaces. The membranes of the mitochondria also
appeared to be disrupted, with the occasional loss of cisternal
structures.
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). As expected, LH stimulated T synthesis by Leydig
cells taken from heterozygote males by 13-fold. Similarly, LH
stimulated basal T synthesis in
csfmop/csfmop Leydig
cells to a level that, although still significantly lower than control
values, was factorially greater (65-fold) as a result of the lower
basal T concentration. Similar responses were obtained in
vivo by treating mice with a single dose of the LH analog, human
chorionic gonadotropin (hCG), and measuring serum T concentrations
1 h later. In confirmation of previous data (10), basal serum T
concentrations were reduced by 90% in
csfmop/csfmop males.
Heterozygous males responded to hCG injection with an 11-fold increase
in circulating concentrations of T (Table 3). Circulating T levels rose even
more dramatically (147-fold) in
csfmop/csfmop
males to a concentration equivalent to that of hCG-stimulated control
males (Table 3). Together, these results, confirmed by analysis of
LH-stimulated T synthesis by whole testes in vitro (data not
shown), demonstrate that the steroidogenic ability of
csfmop/csfmop males can
be enhanced greatly by gonadotropin treatment both in vitro
and in vivo, indicating that a sufficient number of LH
receptors are present to elicit steroidogenic responses comparable to
that of wild type males.
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). However,
the level of stimulation in
csfmop/csfmop Leydig
cells is far greater than that of +/csfmop
Leydig cells due to the differences in basal T synthesis between
genotypes.
Hypothalamic-Pituitary Function in
csfmop/csfmop
Males
The steroidogenic failure in
csfmop/csfmop males might
be due to low circulating LH concentrations. For this reason, serum LH
concentrations were measured by RIA. Basal serum LH in
csfmop/csfmop males is
90% lower than that of heterozygote males (Table 4). Treatment with the potent GnRH
agonist, histerilin, at a dose shown to cause maximal stimulation (data
not shown), increases serum LH by 35-fold in
csfmop/csfmop males,
compared with a 5-fold increase in +/csfmop
males (Table 4). Despite this large stimulation, however,
histerilin-treated LH levels remain significantly lower in
csfmop/csfmop males than
in heterozygote controls. Interestingly, at a dose of 1 ng histerilin
per g body weight,
csfmop/csfmop males
displayed a 10-fold increase in serum LH. However, this dose was not
sufficient to stimulate LH secretion in +/csfmop
males, whose serum LH concentrations remained at basal levels. Thus at
this dose, the serum LH concentrations were comparable between
genotypes.
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To investigate the negative feedback sensitivity of
csfmop/csfmop males,
heterozygote and homozygote mutant mice were subjected to either
bilateral orchidectomy or sham surgery, and serum samples were obtained
5 days later. Serum LH concentrations in bilaterally orchidectomized
+/csfmop males was 1.8-fold higher than in
control males, as would be expected by release of negative feedback
(Table 4), while sham-operated males showed no such stimulation (not
shown). In contrast, bilateral orchidectomy had no effect on the serum
LH concentrations in
csfmop/csfmop males,
indicating a loss of feedback sensitivity in these males. Furthermore,
treatment with physiological concentrations of T postpubertally results
in elevated, although not significant (P < 0.06),
basal LH concentrations as compared with that of untreated
csfmop/csfmop males.
These effects of both castration and T treatment contradict the
expectation that low serum T would increase, while high serum T would
decrease, circulating LH concentrations via the classic negative
feedback regulatory system. Taken together, these studies suggest that
the pituitaries of
csfmop/csfmop males are
able to respond to GnRH by increasing gonadotropin output to a higher
level, but that the hypothalamic-pituitary response to circulating
androgen is disrupted in these mice.
Effect of CSF-1 on Reproductive Function in
csfmop/csfmop
Males
To investigate the effect of postnatal restoration of serum CSF-1
concentrations on male reproductive function,
csfmop/csfmop males were
treated with human recombinant CSF-1 (hrCSF-1) from day 2 of life
(C-csfmop/csfmop). This
regimen has been shown in previous studies to restore circulating
concentrations of CSF-1 (7). Leydig cells obtained from CSF-1-treated
csfmop/csfmop males
produce significantly more T ( 2.3-fold) than those from
untreated csfmop/csfmop
Leydig cells (Table 1), but their T synthesis is not restored to wild
type levels, in line with the failure of hrCSF-1 treatment to restore
circulating T concentrations in these mice (10). LH treatment of
C-csfmop/csfmop Leydig
cells results in stimulation of T synthesis to the same level of
LH-stimulated heterozygote Leydig cells (Table 1), while 22R-CHOL
treatment produces a level of T synthesis that is only 62.7% of the
level of 22R-CHOL-stimulated +/csfmop Leydig
cells, despite being 27-fold stimulated from basal levels (Table 1).
Interestingly, however, Leydig cells isolated from CSF-1-treated
C-csfmop/csfmop males
contain increased, and often almost normal, protein levels for
P450scc (74.2% of wild type level; Fig. 1B), 3ßHSD
(93.9% of the +/csfmop level; Fig. 2B) and
P45017 (68.5% of wild type levels; Fig. 3B). The
recovery of steroidogenic enzyme proteins in
C-csfmop/csfmop Leydig
cells and testis is associated with the restoration of apparently
normal Leydig cell ultrastructure in these mice (Fig. 5C).
To investigate the effect of CSF-1 treatment on hypothalamic-pituitary function in csfmop/csfmop males, serum LH concentrations were measured as described in Materials and Methods. While basal LH concentrations in untreated csfmop/csfmop males is 7.4% of the normal circulating concentration, CSF-1 treatment results in a significant increase (2.5-fold) in LH concentrations to 18.2% of normal, consistent with the increased basal synthesis of T by Leydig cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The hypothalamus is the primary regulator of sexual function, liberating GnRH in a pulsatile fashion which, in turn, stimulates release of LH from the pituitary (26). The hypothalamus is rich in mononuclear phagocytic-lineage microglial cells (27) that express the CSF-1R and respond in culture to CSF-1 (17). Although hypothalamic populations of microglia have not been studied in csfmop/csfmop mice, in the retina the acquisition of these cells is delayed during development (7). Furthermore, adult csfmop/csfmop mice have intracortical processing problems associated with deficits in both excitatory and inhibitory neuronal firing, even though at a gross morphological level, anatomical abnormalities could not be detected in the cortical regions of the brain (17). In culture, CSF-1 is a trophic factor for embryonic neurons derived from many areas of the brain including the hypothalamus, provided that microglia are present in the cultures (17). Together, these data suggest that CSF-1 acting through the microglia plays an important developmental role in the establishment of brain function including hypothalamic function. However, these actions in the hypothalamus and pituitary must be fairly specific since adrenal steroidogenesis is unaffected, suggesting normal secretion of CRF by the parvocellular neurons and the subsequently normal release of ACTH from anterior pituitary corticotrophs.
While it is reasonable to conclude that the reduced steroidogenic
capacity of csfmop/csfmop
Leydig cells is due to the low circulating LH in adult mice, there are
a number of paradoxical observations that suggest there might also be a
local requirement for CSF-1 in the testis. Our data show that all the
steroidogenic enzyme activities are reduced, with P450scc
being the most depressed, consistent with the reduction in protein
concentration observed. However, the mRNA concentrations for these
enzymes are unaffected, suggesting that the deficit is
posttranscriptional. This is contrary to expectation for a deficiency
of LH, since this gonadotropin has been shown to regulate
steroidogenic enzyme mRNA expression (28, 29). Furthermore, while
the activity of P45017 is most affected by LH (30, 31),
in the csfmop/csfmop
mouse this enzyme is the least affected, whereas P450scc
whose activity is relatively less modulated by LH (31), is the most
affected by the csfmop mutation. Although,
experimentally, the consequence of maintaining LH concentrations at
10% in a pulsatile manner in wild type mice throughout life is
unknown, the data suggest that there may also be local influences of
CSF-1 in the testis interacting with the systemic regulation by LH.
Interestingly, despite its statistical significance, the reduction in
serum FSH in
csfmop/csfmop males is
not sufficient to cause any severe effects on the gross testicular
morphology and seminiferous tubule cytology.
There is a large body of literature reporting both anatomical and
physiological interactions between Leydig cells and TMs. The idea that
TMs and Leydig cells are functionally coupled arose from the
characterization of their unique structural relationship, together with
the observations that vasectomy causes simultaneous destruction of both
cell types (32), while pharmacological depletion of either cell type
has severe consequences on the performance of the other cell type
(33, 34, 35, 36). The appearance of adult Leydig cells during puberty occurs in
an environment in which the TM population is already established (12, 15, 16, 37, 38) and is prevented when TMs are pharmacologically ablated
before puberty (36). In adult males, the removal of TMs results in
disruption of Leydig cell steroidogenesis (for review see 16 . In
addition, TM-conditioned media, as well as cytokines that can be
macrophage-derived (including tumor necrosis factor- and
interleukin-1), alter the steroidogenic function of Leydig cells
in vitro (39, 40, 41, 42, 43). We have shown that CSF-1 is the major
regulator of TMs, the only testicular cell type that expresses CSF-1R
(10), and that this macrophage population is severely depleted in
csfmop/csfmop mice
throughout development. This depletion of macrophages in the CSF-1
nullizygous testis, therefore, is likely to have significant
consequences for the functioning of Leydig cells. Consistent with this
is the disruption of Leydig cell morphology in the mutant.
Interestingly, an increase in abundance and/or disruption of the Leydig cell membranous whorls has also been described in other testicular perturbation models, such as in experimentally cryptorchid rats (44) and rats with experimentally induced damage to the seminiferous tubules (45), which are also deficient in steroidogenesis (46, 47). The functional significance of these whorls remains unknown, but previous studies in wild type mice have demonstrated that the whorls are contiguous with the smooth endoplasmic reticulum (SER), suggesting they are regional modifications of their system (48). Since three of the testosterone-biosynthetic enzymes are intimately associated with the SER, the membrane disruptions seen in the csfmop/csfmop males might result in destabilization of these enzymes with the consequent reduction in protein content without significant effects on mRNA concentration. Interestingly, P450scc, the enzyme that is the most affected by the csfmop mutation, resides in the mitochondria and is therefore unaffected by the disruption of the endoplasmic reticular system. However, our preliminary ultrastructural analysis indicates that there might be similar, albeit more subtle, disruptions of the mitochondria in csfmop/csfmop Leydig cells. This idea that enzyme activities are perturbed as a result of physical disruption of Leydig cell architecture is supported by the observation that treatment of the nullizygous mice from birth with CSF-1 corrects both the TM population and Leydig cell morphology and restores steroidogenic enzyme protein levels to near wild type concentrations without completely correcting the steroidogenic deficiencies. It is interesting to note that, despite the remarkable ultrastructural defects in the Leydig cells of csfmop/csfmop males, they are still capable of responding acutely to hCG/LH treatment both in vitro and in vivo, suggesting, perhaps, that the total surface area of SER is not reduced in these cells, despite the obvious membrane disruption.
Androgen biosynthesis by testicular Leydig cells is required for normal
reproductive function in adult males, as well as for the processes of
sexual differentiation and puberty. During sexual differentiation, a
surge of T secretion by Leydig cells of the fetal testis masculinizes
the hypothalamic-pituitary axis (49). However, disrupting pituitary
secretion of LH and FSH by a targeted mutation of their common
-chain does not affect gonadal development until after birth (50)
despite the requirement for T during pituitary development, suggesting
that neonatal and, perhaps early postnatal, Leydig cell T synthesis
occurs independently of gonadotropins. During puberty, another surge of
T secretion is required to initiate changes in the hypothalamus and
pituitary, resulting in an alteration in the pattern of GnRH release
from the hypothalamus (49). This resetting of the GnRH system at
puberty results in maturation of the classic T-mediated negative
feedback loop from the testis to the hypothalamic-pituitary axis and
the subsequent onset of fecundity (49, 51, 52). Disruptions in T
biosynthesis by Leydig cells as a consequence of the absence of TMs in
csfmop/csfmop males
during development might have a significant impact on the establishment
of this feedback loop, in a manner observed in the nullizygous mice.
Furthermore, Gaytan et al. (35) suggested that TMs produce a
factor that directly affects hypothalamic-pituitary development. These
observations, together with the physiological interactions between
Leydig cells and TMs, suggest that the phenotype of
csfmop/csfmop males might
result from a complex mix of the actions of CSF-1 in the hypothalamus,
pituitary, and testis during development.
All the reproductive defects described in this paper can be ascribed to the lack of CSF-1. However, restoration of circulating CSF-1 during the postnatal period only partially restores many of these deficits. This may be due to a requirement for embryonic or local synthesis of CSF-1 or, because CSF-1 is synthesized in a variety of forms (including cell surface, secreted, and secreted proteoglycanated forms), there may be a requirement for a form other than the soluble human recombinant form administered. Indeed, evidence has been obtained for both local and humoral actions of CSF-1 in regulating other mononuclear phagocytic populations (7).
These studies in the csfmop/csfmop mouse model provide compelling evidence that CSF-1 plays a fundamental role in the establishment and functioning of the hypothalamic-pituitary-gonadal axis, through its action on macrophages or microglia. This unique role for CSF-1/macrophage interactions in male reproductive function will have a significant impact on the understanding of the development of classic endocrine feedback systems and the involvement of immune cells in these processes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Bilateral orchidectomies were performed under methoxyflurane anesthesia (Metofane, Pittman-Moore Inc., Mundelein, IL). Sham-operated animals received similar surgical treatment without testicular excision. The mice were allowed to recover for 5 days before blood sampling.
Hormone Treatments and RIAs
To assess the effects of hCG treatment in vivo,
heterozygote and homozygote mutant males were injected with 5 IU hCG
per 10 g body weight (ip), and blood samples were obtained 1
h later for serum T measurement. For GnRH agonist experiments,
histerilin was administered subcutaneously (at doses of 1, 50, or 100
ng per gram body weight in 0.9% saline solution containing 5%
mannitol), 3 h prior to blood sampling. Sera from mice injected
with histerilin or vehicle alone were analyzed by RIA for LH. Animals
undergoing T treatment were given an subcutaneous Silastic implant
containing T in arachis oil (0.6 mg/ml) from 10 weeks of age
(T-csfmop/csfmop).
Implants were constructed as previously described (53).
T concentrations were assessed by RIA using a commercially available kit (DSL Inc., Webster, TX). Inter- and intraassay coefficients of variance were 38.8% and 3.0%, respectively. LH RIAs were performed as previously described (54). Inter- and intraassay coefficients of variance were 26.9% and 6.7%, respectively.
Serum corticosterone concentrations were measured using a commercially available kit (Diagnostic Products Corp., Los Angeles, CA).
Leydig Cell Isolation and Treatment
Leydig cells were isolated as previously described (55) and
subjected to 3 h of incubation at 34 C in the presence or absence
of either LH (100 ng/ml) or 22R-hydroxycholesterol (22R-CHOL: 5
µM). For each isolation procedure, eight to ten
age-matched males were used. For each tube, 100,000 cells were
incubated in a total volume of 0.5 ml, and incubations were performed
in quadruplet. Media T content was assessed by RIA.
Western Blotting
Soluble protein extracts from 50,000 Leydig cells or transfected
COS-1 cells or from testis, adrenals, ovaries, and spleen samples were
used for Western blotting as previously described (56). Steroidogenic
enzyme proteins were detected using the Boehringer Mannheim
chemiluminescence Western blotting kit (Indianapolis, IN) and
antibodies raised in rabbits against bovine adrenal
P450scc, human placental 3ß-HSD, and porcine testicular
P45017. Densitometric analyses were performed using
ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Whole testis
Western blot densitometry represents the means (± SD) of
at least three separate Western blots (and, therefore, at least nine
different testis samples for each genotype). The quantification was
shown to be linear within the range of protein concentrations used
(data not shown), and all protein concentrations were normalized to
GAPDH protein levels on the same blots. Additional controls were used
for each specific antibody; for P450scc and
P45017, samples of purified bovine mitochondria or
purified sheep microsomes (Oxygene Dallas Corp., Dallas, TX) were run
at the same time as the testis samples, while for 3ß-HSD, cell
extracts were prepared from COS-1 cells transfected with
cytomegalovirus-driven expression vectors containing the entire cDNA
for 3ß-HSD isoforms I or VI (see below). Each antibody showed
specificity for its respective enzyme, as determined by antigen
localization in other steroidogenic tissues (adrenal and ovary) and the
absence of antigen in nonsteroidogenic tissue (spleen). The antibodies
detected proteins of 52 kDa for P450scc, 46 kDa for
3ß-HSD, 52 kDa for P45017, and 36 kDa for GAPDH.
All of the primary antibodies used were generous gifts from various
sources: anti-P450scc and anti-P45017 (both
from Dr. Dale Buchanan Hales, Department of Physiology and Biophysics,
University of Illinois, Chicago, IL), anti-3ßHSD (Dr. Ian Mason,
Department of Biochemistry and Obstetrics and Gynecology, Southwestern
Medical School, Dallas, TX), and anti-GAPDH (Dr. Y. G. Yeung,
Department of Developmental and Molecular Biology, Albert Einstein
College of Medicine, Bronx, NY).
Transient Expression of 3ßHSD Isoforms
Expression vectors containing the cytomegalovirus-driven
full-length cDNA for 3ßHSD isoform I and 3ßHSD isoform VI
were generously provided by Dr Anita Payne (Department of Obstetrics
and Gynecology, Stanford University, Stanford, CA). Transient
expression in COS-1 cells was obtained using the
diethylaminoethyl-dextran method as described (57). Seventy-two hours
after transfection, the cells were harvested, and soluble protein
extracts were prepared as described above.
Measurement of Steroidogenic Enzyme Activity in Freshly Isolated
Leydig Cells
With the exception of P450scc, steroidogenic enzyme
activities were measured by incubation of purified Leydig cells with
radiolabeled substrates and separation of products by TLC as described
by O’Shaughnessy and Payne (58). Reaction mixture (0.5 ml) was
prepared in Leydig cell medium that contained 1 µM
substrate (1 µCi, 1 µM) in Leydig cell culture medium.
The reaction mixture was maintained at pH 7.2. Reactions were initiated
by adding to the reaction mixture an aliquot of 0.1 x
106 preincubated intact Leydig cell suspension, using
endogenous cofactors. The reaction mixtures, conducted in triplicate,
were maintained at 34°C in a shaking water bath for 30 min. With this
incubation time, the conversion of substrates to products was linear
with respect to cell number and time of incubation. Reactions were
terminated by adding ice-cold ethyl acetate, and steroids were rapidly
extracted. The organic layer was dried under nitrogen. The steroid
residues were chromatographed on TLC plates, and radioactivity was
measured with a scanning radiometer (System 200/AC3000, Bioscan, Inc.,
Washington, DC).
Activity of P450scc was determined by measuring the conversion of side-chain-labeled [26,27-3H]25-hydroxycholesterol to [3H]4-hydroxyl-4-methyl-pentanoic acid as previously described (59). 25-Hydroxycholesterol, rather than cholesterol, was used as substrate to measure the P450scc reaction, since it does not depend on active transfer to the inner mitochondrial membrane and has a high aqueous solubility. The reaction was performed in 0.5 ml of medium containing 0.1 x 106 cells and 1 µM substrate for 30 min. Blank incubations were performed with medium containing BSA (1.5 mg/ml) in place of cell suspension. At the end of the incubation, 0.1 ml 1 M NaOH was added followed by 0.5 ml phosphate buffer adjusted to pH 13. Nonmetabolized substrate was removed by extracting twice with chloroform. An aliquot was counted in a liquid scintillation counter. Counts from the blank incubation were subtracted from the counts in the test system.
[26,27-3H]25-hydroxycholesterol (specific activity, 81.9
Ci/mmol), [7-3H(N)]-pregnenolone (specific activity, 25
Ci/mmol), [1,2-3H(N)]-17-hydroxyprogesterone (specific
activity, 48.7 Ci/mmol),
[1ß,2ß-3H(N)]-androstenedione (specific activity,
45.3 Ci/mmol), and [1,2,6,7-3H(N)]-testosterone (specific
activity, 101 Ci/mmol) were purchased from Dupont-New England Nuclear
(Boston, MA). [1,2,6,7-3H(N)]-Progesterone (specific
activity, 92 Ci/mmol) was purchased from Amersham International PLC
(Amersham, England). 25-Hydroxycholesterol, pregnenolone, progesterone,
17-hydroxyprogesterone, androstenedione, and T were purchased from
Sigma Chemical Co. (St. Louis, MO). TLC plates (Baker-flex/UV254) were
obtained from J.T. Baker, Inc. (Phillipsburg, NJ).
Northern Blot Analysis
Probes for Northern blots were obtained by RT-PCR using pairs of
oligonucleotide primers designed from the published sequences of genes
encoding the following steroidogenic enzymes: mouse
P45017, 5'-CCA GAC GTG GTC ATA TGC ATG CCA-3' and 5'-GAT
GAG CGT AGA CAG ATC TCG GGA-3' (60); and mouse 3ß-HSD, 5'-TGG TCT GAT
CCA TAC CCA TAC AGC-3' and 5'-TGG TGC GGG GTG TCA TCT GAG ATG-3' (61).
Aliquots of 20 µg total mRNA, isolated as previously described (62, 63), were separated by agarose gel electrophoresis and transferred to
nitrocellulose membrane using routine methods. After a 3-h incubation
in prehybridization buffer at 45 C, [32P]-labeled probes
were allowed to hybridize to the blots overnight at 42 C, before high
stringency washing at 55 C and exposure to autoradiographic film for
appropriate lengths of time.
RPAs
For RPA, [32P]-labeled antisense riboprobes were
prepared from cDNA templates cloned into the pCRII vector (InVitrogen
Corp., San Diego, CA). The templates were prepared by RT-PCR using
pairs of oligonucleotide primers designed from the published sequences
of genes encoding the following steroidogenic enzymes: rat
P450scc, 5'-CCG CTT TGC CTT TGA GTC CAT-3' and 5'-ACA CCC
AGA ACT TCT ACT GGG-3' (64); mouse P45017 (see above);
and mouse 3ß-HSD (see above). Aliquots of 20 µg total RNA were
combined with approximately 105 cpm labeled riboprobe and
analyzed by RPA using standard techniques. Consistent mRNA loading was
ensured by the simultaneous hybridization to
[32P]-labeled antisense probe transcribed from the
pTRI-GAPDH-mouse antisense control template (Ambion, Austin, TX), which
contains a fragment of the mouse glyceraldehyde 3-phosphate
dehydrogenase housekeeping gene. Protected RNA fragments were separated
on a denaturing 6% PAGE/8 M urea gel and visualized by
exposing the dehydrated gel to Hyperfilm (Amersham Life Sciences Inc.,
Arlington Heights, IL) for appropriate periods of time.
Electron Microscopy
Male +/csfmop,
csfmop/csfmop,
C-csfmop/csfmop mice were
given a single dose of heparin (130 mU per g body weight ip) and
anesthetized 15 min later. Mice were perfused transcardially for 30 min
with fixative (1.3% sym-collidine, 1.2% acrolein, 25% glutaraldehyde
in 45 µM HCl) before removal of both testes. The testes
were stored in fresh fixative at 4 C until processing by conventional
methods.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
|---|
This work was funded by the Population Council (M.P.H.) and by grants from the NIH: R29-HD32588 (to M.P.H.) and HD/AI 30280 (to J.W.P.), The Albert Einstein Core Cancer Grant P30-CA13330 (to J.W.P.), and Chiron Corporation (to J.W.P.). J.W.P. is a Monique Weill-Caulier Scholar.
Received for publication November 19, 1996. Revision received June 9, 1997. Accepted for publication July 24, 1997.
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