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Department of Obstetrics, Gynecology, and Reproductive Sciences (N.A.C., S.H.M.), the Metabolic Research Unit (S.H.M.), and the Department of Pediatrics (E.S., L.J.S.), University of California, San Francisco, California 94143; and Universidad de La Laguna (E.S.), Laguna, Spain
Address all correspondence and requests for reprints to: Synthia H. Mellon, Ph.D., Department of Obstetrics and Gynecology, University of California, Box 0556, San Francisco, California 94143-0556.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric
acidA (GABAA) and
N-methyl-D-aspartate receptors rather than
through classical nuclear steroid hormone receptors. Steroid hormones
are known to exist not only as free compounds, but also as sulfated
derivatives. Pharmacological studies indicate that unconjugated and
sulfated steroids, such as pregnenolone and pregnenolone sulfate, may
have opposite effects on GABAA receptors. Thus,
pregnenolone acts as a potent positive allosteric modulator of
-aminobutyric acid action at GABAA receptors, whereas
pregnenolone sulfate acts as a potent negative modulator. Recent
experiments also suggest that dehydroepiandrosterone and
dehydroepiandrosterone sulfate may have distinct effects on growth of
neurites from embryonic neocortical neurons in vitro.
Thus, regulation of steroid sulfation may have profound behavioral and
morphological effects on the nervous system. We, therefore, studied the
developmental expression of the enzyme steroid sulfatase (STS), which
converts sulfated steroids to free steroids. By in situ
hybridization, STS messenger RNA was expressed in the embryonic mouse
cortex, hindbrain, and thalamus during the last third of gestation. The
sites of expression of STS were similar to those of P450c17, suggesting
that these two enzymes may have concerted actions in similar functional
processes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxy-DHEAS in the
liver and other tissues, but it must undergo the sulfatase reaction in
the placenta to be metabolized to estriol, the main estrogen of the
placenta. Diminished or absent placental sulfatase activity reduces the
pool of free DHEA available for placental conversion to estrogen,
resulting in prolonged gestation and failure of induction of labor. Previous studies have demonstrated STS activity in a variety of tissues other than the placenta, including the brain (7), and in virtually all tissues of the rhesus monkey (8), mouse macrophages (9), serum (10), human leukocytes (11), and testes (12). In general, high levels of STS activity have been found in tissues in which the biological activity of DHEA or its metabolites regulates cellular function.
We demonstrated that P450c17, the enzyme necessary for the synthesis of DHEA from pregnenolone, is transiently expressed in the neocortical subplate during embryogenesis (13). Furthermore, we recently demonstrated that DHEA and DHEAS affect the development of embryonic neocortical neurons in vitro (14). We further showed that these two steroids have different biological effects. Thus, regulation of the interconversion of these two steroids may play an important role in the development of the nervous system in vivo. We, therefore, studied the expression of STS in the developing rodent to characterize the distribution of this enzyme and thus determine the areas in the brain where sulfate hydrolysis may regulate neurosteroid activity. The rat and mouse STS have recently been cloned (15, 16), and we generated riboprobes from the mouse STS complementary DNA (cDNA) to study the expression of this enzyme by in situ hybridization. This report shows distribution of mouse STS during late embryogenesis. Our results show that the cortex, the hindbrain, and, interestingly, the thalamus, which is involved in supporting cortical differentiation during development (17), are sites in the central nervous system where STS is expressed during the last third of gestation. The sites of expression of STS are similar to those of P450c17, suggesting that these two enzymes may have concerted actions in similar functional processes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ribonuclease (RNase) protection assays
Mouse steroid sulfohydrolase (mSTS) cDNA fragments were
subcloned into pSKII+ (Stratagene, La Jolla, CA) for the
synthesis of RNA probes. A 447-bp HindIII/BamHI
fragment of mouse STS cDNA (nucleotides 1289–1736) was used to prepare
a 507-base mSTS probe. RNA was extracted from tissues using a Trizol
messenger RNA (mRNA) extraction kit (Life Technologies, Grand Island,
NY), and analyzed by RNase protection assays. Total RNA was analyzed
from the following sources: E18.5 central nervous system tissues and
E18.5 intestine (200 µg), E18.5 lung (150 µg), E18.5 skin (60
µg), and adult mouse cortex (160 µg). RNA was combined with
0.5 x 106 cpm 32P-labeled riboprobe;
precipitated with ethanol; resuspended in 80% formamide, 400
mM piperazine-N-N'-bis[ethanesulfonic acid]
(pH 6.4), and 1 mM EDTA; boiled for 5 min; and incubated
overnight at 42 C. Samples were diluted 10-fold with 10 mM
Tris-HCl (pH 7.9), 300 mM NaCl, and 5 mM EDTA;
treated with RNase A (30 µg/ml); incubated for 30 min at 37 C;
treated with 0.25 µg/ml proteinase K and 0.5% SDS for an additional
15 min at 37 C; extracted once with phenol/chloroform; and precipitated
with ethanol. Protected 32P-labeled fragments were
separated by electrophoresis on 6% polyacrylamide-7.5 M
urea gels.
In situ hybridization
Mouse STS cDNA has 63% nucleotide identity with human STS and
75% nucleotide identity with rat STS (16). Furthermore, mouse STS does
not share homology with other sulfatases, such as iduronate sulfate
(mouse) and chondroitin sulfate (goat), because these sequences only
have 36% nucleotide identity with the mSTS. A 447-bp
HindIII-BamHI (nucleotides 1289–1736) cDNA
fragment of mSTS cDNA was subcloned into PSKII (Stratagene) for
35S-labeled sense and antisense RNA probe synthesis.
In situ hybridization was performed as previously described
(18). Briefly, slides were fixed in 4% paraformaldehyde in PBS for 20
min at room temperature, and sections were treated with 1 µg/ml
proteinase K in 100 mM Tris-HCl, pH 8, containing 50
mM EDTA and acetylated with acetic anhydride in 0.1
M triethanolamine buffer for 10 min at room temperature.
Slides were hybridized overnight at 52 C with 106 cpm probe
in buffer containing 50% formamide, 5 x SSC (1 x SSC
= 0.15 M NaCl and 0.015 M sodium citrate), 10
mM ß-mercaptoethanol, 10% dextran sulfate, 2 x
Denhardt’s solution (0.02% polyvinylpyrrolidone, 0.02% Ficoll, and
0.02% BSA), 250 µg/ml yeast transfer RNA, and 500 µg/ml salmon
sperm DNA. Nonhybridized probe was washed from the slides with 2
x SSC containing 10 mM ß-mercaptoethanol for 30 min at
room temperature. Sections were then treated with 20 µg/ml RNase A at
room temperature for 40 min, followed by two washes for 30 min each
time at 55 C in 50% formamide-2 x SSC containing 1
mM EDTA and 10 mM ß-mercaptoethanol and a
wash for 2 min at room temperature in 0.2 x SCC. Slides were
dipped in Ilford K5 nuclear emulsion (Warrington, PA), dried, and
stored at 4 C until developed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
On E18.5, we detected mSTS mRNA expression in the cortex and hindbrain,
but we did not detect this mRNA in the basal ganglia or diencephalon of
the mouse embryo. Using reverse transcription-PCR, we detected mSTS
mRNA in the mouse diencephalon (not shown), suggesting that this mRNA
was expressed at extremely low levels in this region.
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In the adult rat, we detected STS mRNA in the cortex (using 160 µg total RNA). As the signal in the cortex was robust, we used less RNA from other regions of the adult brain (thalamus, hypothalamus, hindbrain, and cerebellum; 25–90 µg) and detected no STS mRNA in these regions.
In situ hybridization
We detected STS mRNA in cartilage primordia and in the liver on
E14.5 (Fig. 2A). In the liver, STS mRNA
was observed in restricted areas (Fig. 2E). In cartilage, expression of
mSTS mRNA was first observed at the base of the skull and in the
cartilage of the ribs and vertebrae (E14.5). Later in ontogeny
(E16.5–E18.5), expression of mSTS mRNA was observed in the cartilage
of the temporal and occipital regions of the head as well as in the
facial region, the eye, and the ear (Fig. 2, B, C, F, and G). Figure 2D
shows a higher magnification of the vertebral cartilage of the rib.
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). As
expected, mSTS mRNA was strongly expressed in the skin of E16.5 embryos
(Fig. 2I).
In the central nervous system, a hybridization signal was first
observed on E16.5. At this time, STS mRNA was found in the thalamus
(Fig. 3C). By E18.5, STS mRNA was found
in the cortex, hippocampus, thalamus, cerebellum, and spinal cord (not
shown), and expression in these regions persisted 9 days after birth
(Fig. 3B). Expression of STS mRNA in the cortex and cerebellum was
diffuse, suggesting that mSTS mRNA may be expressed in cells not
restricted to anatomical nuclei. By contrast, expression of mSTS mRNA
in the thalamus was highly selective and was found in a small number of
cells whose distribution suggested that its expression was restricted
to a particular anatomical domain of the anterior thalamus (Fig. 3D).
Hybridization signals for mSTS mRNA persisted in 9-day-old neonatal
mouse brains (Fig. 3, B and D) in the (subicular area) CA1 region of
the hippocampus and in the anterior thalamus.
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) and in dorsal root ganglia (Fig. 2D) as well
as in the inner neural layer of the retina (Fig. 2G).
To ensure the specificity of the hybridization signal, we performed
in situ hybridizations on sister sections of postnatal day 9
mice brains using a sense RNA probe (Fig. 3A). The sense probe did not
show any hybridization signal in the thalamus. There was a weak signal
in the dentate gyrus and in Ammon’s horn, which probably represents
nonspecific RNA binding, but no signal was observed in the subicular
area, suggesting that the hybridization signals observed using the STS
antisense RNA probe were specific.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to the known roles of STS in the placenta and skin, our results emphasize other possible roles for STS in the function of other organs during embryogenesis. In the liver the signal is clustered in restricted areas resembling blood islands, suggesting that STS may be active in the hematopoiesis signaling processes. Spleen expression of mSTS is very high, and this strongly suggests a role for mSTS in lymphocyte production. Sulfatase activity, recently found in endothelial cells and monocytes, has been shown to be an important regulator of the inflammatory response, as the efficiency of this response relies on the ability of leukocytes, platelets, and/or endothelial cells to degrade the subendothelial basement membrane, which is considered to be an important barrier to the entry of leukocytes into inflammatory sites (19). Sulfatase activity in endothelial cells, but not in monocytes, could be induced by tumor necrosis factor, interleukin-1, and interleukin-8 (19).
Expression of STS mRNA in the developing cartilage early during embryogenesis coincides with the appearance of ossification centers (E14–E14.5), suggesting that this enzyme may participate in the differentiation of the skeletal system. Consistent with our observation of mSTS mRNA expression in developing ossification centers, STS may continue to play an important role in the maintenance of bones throughout life. The conversion of estrone sulfate to estradiol plays an important role in modulating bone turnover. Estrone sulfatase activity was detected in the MG63 human osteoblast cell line (20), suggesting that development of osteoporosis in postmenopausal women may be due to a reduction in the activity of the enzymes responsible for local estrogen production.
Expression of mSTS mRNA in the skin only appeared on E16.5, concomitant with the formation of the multilayered structure of the epidermis and with a peak in expression of cholesterol sulfatase, another sulfatase involved in epidermal differentiation (21).
In the central and peripheral nervous systems, the expression of mSTS mRNA may be related to the expression of steroidogenic enzymes involved in neurosteroid synthesis. We have previously shown the expression of P450c17 in restricted areas of the developing nervous system (13), particularly in the neocortex, and demonstrated a role for both DHEA and DHEAS in neocortical neuron differentiation. Although DHEA promotes axonal growth, DHEAS promotes dendrite growth and cell clustering (14). The expression of STS in the neocortex, hippocampus, and thalamus may regulate the ratio of DHEA/DHEAS. Furthermore, as the thalamus has been shown to influence the survival of the cortical subplate (17), where we detected P450c17-expressing neurons, the ratio of these two steroids may play crucial roles in controlling axonal vs. dendritic growth of cortico-thalamic fibers during neocortex organization. The afferent and efferent connections of the cerebral neocortex develop simultaneously toward the end of embryogenesis. At this stage, the neocortex comprises two main cell-dense layers: the thicker and more superficial cortical plate (future layers 2–6) and the thinner underlying subplate. Many early thalamocortical projections temporarily innervate the subplate before leaving it to locate their ultimate targets in the overlying cortical plate. The subplate then disappears. In addition, subplate neurons have been shown to prime thalamic connections in the cortex (22, 23). Hence, we propose that thalamic conversion of DHEA to DHEAS in the thalamus may be a regulatory mechanism controlling axonal growth of thalamic fibers during neocortex cytoarchitectural organization. Furthermore, as DHEA has been shown to modulate GABAA and N-methyl-D-aspartate receptor activities (24, 25, 26, 27, 28, 29), STS activity in the thalamus might be considered a regulatory process of the thalamic neurosecretion.
STS expression most likely persists beyond embryonic development into adulthood. In earlier studies assaying STS activity, investigators detected abundant quantities of STS activity in the central nuclei of the thalamus of two adult males, aged 44 and 63 yr, who died of lymphatic leukemia and pulmonary embolus, respectively (30). The thalamus had the greatest amount of STS activity, which was about twice as much activity as any other region per g tissue. The hippocampus and hypothalamus had lesser, but still substantial, amounts of activity. Many other regions of the brain contained less activity. Thus, the thalamus may continue to be a central region for coordinating the relative abundance of sulfated and free steroids in the brain throughout life.
In addition to possible roles in axonal and dendritic growth during embryogenesis, others have proposed a role for DHEAS in memory in adult humans and rodents (31, 32, 33, 34, 35). Chemical inhibition of STS activity in vivo was associated with changes in memory in adult rats (31, 36). Thus, regulation of STS activity in the thalamus and hippocampus may have implications for learning and/or memory enhancement throughout life.
The distribution of STS mRNA in other regions of the central and peripheral nervous systems is compatible with a concerted action of both STS and P450c17 in similar processes during embryogenesis. The concept of functional regulation of some neurosteroids by their sulfation or sulfate hydrolysis, suggested by our previous data (14) and others (reviewed in Ref.37) is thus supported by the localization and ontogeny of mSTS mRNA.
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Footnotes |
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2 Supported in part by funds from the Phillipe Foundation and the
Singer Polignac Foundation.
Received May 14, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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