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Experimental Studies |
Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and reprint requests to: Meera S. Ramayya, M.D., M.S., Children’s Hospital and Medical Center, 4800 Sand Point Way N.E., P. O. Box C5371, Seattle, Washington 98105.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit of
the glycoprotein hormones (9, 10), the ß-gene promoter of luteinizing
hormone (11, 12), DAX-1 (13), and steroidogenic acute regulatory
protein (StAR) (14). In situ hybridization studies in mouse
embryos demonstrated that SF-1 was expressed in the urogenital sinus
and the developing adrenal cortex, gonads, and diencephalon (15).
Targeted disruption of the SF-1 gene in mice established its essential
role in the organogenesis of these tissues (10, 16, 17, 18, 19, 20, 21). Thus, these
mice had agenesis of their adrenal glands, gonads, and ventromedial
nucleus of the hypothalamus, resulting in complete congenital adrenal
insufficiency and male-to-female sex reversal.
To determine the role of SF-1 in the human, we previously cloned and
sequenced the human SF-1 (hSF-1) complementary DNA (cDNA) by
heterologous probing of a 
gt11 fetal adrenal cDNA library using a
PCR-amplified 32P-labeled mouse SF-1 cDNA probe that did
not include the region coding for the zinc finger domain (22). Sequence
analysis revealed an entire open reading frame, most of the
5'-untranslated region, and the complete 3'-untranslated region,
including the poly-A signal and tail. The derived amino acid sequence
showed a high degree of homology (>95%) was found to the bovine and
murine sequences. The zinc fingers, the FTZ-1 box, and the AF2 domains
showed 100% conservation of the derived amino acid residues with a
lesser degree of homology in the ligand binding/dimerization domains
(regions I and II) (22).
In this study, we defined the sites of hSF-1 messenger RNA (mRNA) expression in human tissues by both Northern blot and in situ hybridization analyses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Restriction enzymes, the rapid DNA ligation kit, Pwo DNA polymerase, and PCR nucleotide mix were purchased from Boehringer Mannheim Co. (Indianapolis, IN). Multiple tissue Northern blots and ExpressHyb hybridization solution were purchased from Clontech (Palo Alto, CA). Cloning vector pBluescript II KS ±, expression vector pBK-CMV, and competent cells XLI-Blue MRF’ were purchased from Stratagene (La Jolla, CA) and Sephadex G-50 spin columns from Lofstrand Laboratories (Gaithersburg, MD).
Methods
Northern blot analyses. The hSF-1 cDNA was cloned and sequenced as previously described (22). Prehybridization and hybridization were carried out in ExpressHyb hybridization solution, according to the manufacturer’s protocol. The stringency of the washing conditions used in the Northern blot experiments is as follows: Low-stringency wash was done at room temperature x 30 min with 3 changes of wash solution containing sodium chloride 300 mmol/L, sodium citrate 300 mmol/L (2 x SSC), and 0.1% sodium dodecyl sulfate. High-stringency wash was done at 50 C x 40 min with 2 changes of wash solution containing 0.2x SSC, 0.1% SDS.
The probe was prepared by PCR amplification of hSF-1 cDNA using Pwo DNA polymerase and PCR nucleotide mix in Perkin-Elmer Gene Amp PCR system 9600 (Perkin-Elmer Corp., Foster City, CA). The primers used for the PCR amplification of the probe were: forward primer, 5'-GCAGAAGAAGGCACAGATTC-3'; and reverse primer, 5'-TCACCAGGATGTGGTTATTC-3'.
The probe was radiolabeled with Klenow DNA polymerase-directed incorporation of 32P deoxycytidine 5'-triphosphate, primed by random deoxynucleotide monophosphate hexamers. The probe was purified by removal of unincorporated 32P-labeled deoxycytidine 5'-triphosphate by Sephadex-G-50 spin column.
In Situ hybridization. The template for the sense probe was generated by Xho-1 digestion and that for the antisense probe by Xba-1 digestion of pBluescript SKII ± containing the 3.109-kb hSF-1 fragment. These cDNA template fragments were gel purified for in vitro transcription to generate the sense and antisense RNA probes. The sense probe template spanned the entire hSF-1 cDNA. The antisense probe template was located in the 3' untranslated region and spanned nucleotides 2355–3109 of the hSF-1 cDNA.
Human tissues (liver, pituitary, ovary, and testes) included fresh-frozen necropsy specimens from young adults (having died suddenly as a result of trauma; obtained through the National Disease Research Interchange, Philadelphia, PA) and fresh-frozen surgical specimens from patients undergoing nephrectomy at the NIH Clinical Center (spleen, kidney, and adrenal). Patients gave informed consent to the disposition of their surgically removed tissues under a protocol approved by the NIH Clinical Center Institutional Review Board. Aside from the diagnosis of presumed renal tumor, the patients were free of endocrine or renal disease and were 43–48 yr old. Rhesus monkey ovaries were obtained from healthy animals undergoing ovariectomy for a protocol approved by the NICHD Animal Committee. Two to five independent specimens were evaluated for each tissue type. Tissue was snap-frozen and stored at -70 C until sectioning. Sections of 10-µm thickness were cut at -15 C and thaw-mounted onto poly-L-lysine-coated slides and stored at -70 C until use.
35S-labeled RNA probes were synthesized to SA 
2 x 108 dpm/µg, as previously described (23). Equal
concentrations of labeled probe (106 cpm/100 µL) were applied to all
sections, which were then hybridized and washed as previously described
(23). Sense and antisense probes were used in parallel experiments to
evaluate nonspecific signal level. Sections were exposed to NTB2
nuclear emulsion for 2 weeks, developed, and counterstained with
hematoxylin and eosin for microscopic evaluation and photography.
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Results |
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A hSF-1 expression plasmid, containing our hSF-1 cDNA, produced functional hSF-1 that, as expected, stimulated a StAR promoter-luciferase reporter construct, 5-fold, in HeLa cells (data not shown). The human StAR promoter-luciferase reporter construct was kindly donated by Dr. Jerome F. Strauss III, of the University of Pennsylvania.
The expression of hSF-1 mRNA in human tissues was examined both by Northern blot and in situ hybridization analyses using a hSF-1 cDNA and a cRNA probe, respectively.
Northern blot analysis of multiple peripheral tissues revealed a single
message size of approximately 3.5 kb in the steroidogenic tissues,
i.e. adrenal cortex, ovary, and testis (Fig. 1
, A and B). However, a nonsteroidogenic tissue, the
spleen, also showed strong expression of the hSF-1 message with a
signal intensity less than that of the adrenal cortex but higher than
or similar to that of the gonads. Also, several lymphoid tissues,
including the lymph nodes, thymus, appendix, and peripheral blood
leukocytes, showed an hSF-1 transcript of a signal intensity less than
that of the spleen (Fig. 1D). In keeping with these data, human
lymphoid and myeloid cancer cell lines, a lymphoid B cell line
(Burkitt’s lymphoma), the myeloid cell lines HL-60 (promyelocytic
leukemia) and K-562 (chronic myelogenous leukemia), also expressed
hSF-1 mRNA (Fig. 1E). In the spleen, the main transcript, as well as
two additional transcripts of 4.4 kb and 8 kb, were noted. The myeloid
and lymphoid cancer cell lines, several tissues of the immune system,
skeletal muscle, and pancreas showed the 4.4-kb transcript in addition
to the main transcript. However, the heart and adult liver showed only
a single 4.4-kb transcript (Fig. 1C).
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). A weak signal was detected after an 8-week
exposure (data not shown).
Northern blot analysis of several human central nervous system (CNS)
components, including the spinal cord, revealed widespread presence of
an hSF-1 message of 3.5–4 kb. An additional mRNA signal of 4.4 kb also
was widely expressed. The hSF-1 mRNA concentration was highest in the
caudate and subthalamic nuclei and also in the thalamus. hSF-1 mRNA
also was present in the hippocampus, corpus callosum, and cerebral
cortex (Fig. 2, A and B).
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, A–D). No
hybridization was detected in the kidney or liver (data not shown).
SF-1 mRNA localization was investigated in both human and rhesus monkey
ovary sections, and findings were identical in both species. In the
ovary, hSF-1 mRNA was abundant in granulosa and theca cells at all
stages of follicular development (Fig. 4, C and D),
except for primordial follicles. hSF-1 message also was present in
corpora lutea (data not shown). Testicular SF-1 mRNA was most abundant
in clusters of large round cells with abundant eosinophilic cytoplasm
containing fine granular basophilic stippling around large, pale blue
nuclei (Fig. 4, A and B). These cells have the typical
appearance of steroidogenically active Leydig cells and are localized
in clusters in the interstitial compartment, as are Leydig cells (Fig. 4A); thus it is concluded that SF-1 mRNA is concentrated in Leydig
cells. SF-1 hybrid signal also is specifically detected in a diffuse
distribution within the seminiferous tubules (Fig. 4, A and B). Given
this diffuse pattern, it cannot be determined whether the grains are
localized over Sertoli cells or spermatocytes or both cell types, and
thus, it is concluded simply that SF-1 mRNA is present in the germinal
epithelium of the human testis. In the spleen, hSF-1 mRNA expression
had a reticular pattern, and the signal was concentrated in the red
pulp, primarily within reticuloendothelial cells (Fig. 5, A–D).
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Discussion |
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SF-1 in the human was expressed clearly in the adrenal cortex, the ovary, and the testis. The adrenal cortex had higher hSF-1 mRNA intensity than the gonads. Expression of hSF-1 also was seen in the adrenal medulla by Northern blot analysis. However, this expression probably reflects the presence of hSF-1 in adrenal cortical rests within the medullary tissue. Histological studies have shown the presence of islands of adrenal cortical tissue within this portion of the gland (25), and this was confirmed by our in situ hybridization studies showing hSF-1 mRNA expression in steroidogenic but not chromaffin cells.
In situ hybridization studies of normal architecture adrenal
gland showed similar distribution of SF-1 mRNA signal in all the three
zones of the adrenal cortex. However, within each cortical zone, the
signal distribution was heterogeneous (Fig. 3, C and D). Sesano
et al. (26), using immunohistochemistry, also demonstrated
the heterogenous distribution of the signal of Ad4BP, the bovine
homolog of SF-1, within each of the three cortical zones of normal,
neoplastic, and atrophied human adrenal glands. Interestingly, in our
in situ hybridization studies of a normal nodular variant of
the adrenal gland, we detected a very high SF-1 gene expression in the
proliferative nodules (Fig. 3, A and B). These data suggest that, in
addition to regulation of steroidogenesis, SF-1 also may have a role in
regulating the growth and proliferation of adult adrenal cortical
cells.
Parallel examinations of rhesus and human ovary for SF-1 mRNA localization, by in situ hybridization studies, revealed an identical distribution in the two species. These studies demonstrated that hSF-1 mRNA was abundant in granulosa cells at all stages of follicular development, except for primordial follicles, and also was present in corpora lutea. Both the theca interna and externa cells surrounding the graafian follicles also expressed hSF-1 mRNA. In addition, a thecoma seen in a section of Rhesus monkey ovary also showed strong expression of hSF-1 mRNA, suggesting active steroidogenesis in this tissue. hSF-1 mRNA, however, also was seen in atretic follicles, which normally are not steroidogenic. Takayama et al. (27) recently correlated the expression of steroidogenic enzymes to the expression of immunoreactive Ad4BP in the human ovary. Although there was such a correlation, Ad4BP expression also was seen in preantral and atretic follicles and degenerating corpora lutea, where no steroidogenesis was detected. These data collectively suggest a broader role for hSF-1, beyond steroidogenesis, perhaps in differentiation and/or apoptosis processes of the ovary. Data from studies in the rat with in vitro cultured granulosa cells are compatible with this process (28).
In the adult testis, hSF-1 expression was seen in both the interstitial cells and the inner border of the seminiferous tubules, suggesting expression in the steroidogenic Leydig cells and the germinal epithelium. Previous studies in the embryonic mouse testis and postnatal day-15 rat Sertoli cells also show expression of SF-1 mRNA in the seminiferous tubules of the mouse and the Sertoli cells of the rat (15). In immunohistochemical studies in the rat testis, using Ad4BP antiserum, immunoreactivity was detected in the cells surrounding the seminiferous tubules but not within the tubules (3). This discrepancy is probably a species-specific difference or could be explained as incongruous expression of hSF-1 mRNA and protein. Our results suggest that, in the human testis, in addition to steroidogenesis, hSF-1 also may have a role in the function of spermatogenesis.
Northern blot analysis of the human placenta did not reveal hSF-1 message after a 16-h exposure; however, a weak signal was noted after 8 weeks of exposure (data not shown). SF-1 message expression in the bovine (6) and human placenta (29) was reported previously using the highly sensitive RT-PCR technique. The apparently low expression of hSF-1 in human placenta suggests that it may not have a major role in placental steroidogenesis. This view is supported by previous studies that demonstrated expression of SF-1 mRNA in BeWo human choriocarcinoma cells only by the highly sensitive RT-PCR technique (14), nonexpression of the StAR gene in the placenta (30), expression of P450 side-chain-cleavage enzyme in the placenta of SF-1- deficient mice (21), use of SFRE-deficient aromatase promoter 1.1 for placental aromatase gene transcription (31), and regulation of placental p450scc gene transcription by a 55-kd protein that is expressed in the placenta but not in the adrenal cortex (32). The data from these studies, along with our own data, suggest that alternative pathways of steroid metabolism or functional homologues of hSF-1 may be operational in human placental steroidogenesis.
We detected a widespread expression of hSF-1 mRNA in components of the human brain, by Northern blot analysis. In addition to the main message of 3.5–4 kb, also seen in peripheral tissues, a message of 4.4 kb also was detected. The hSF-1 mRNA concentration was highest in the caudate and subthalamic nuclei; furthermore, hSF-1 mRNA was detected in the amygdala, hippocampus, corpus callosum, and brain cortex. In contrast to our findings in the human, SF-1 expression was found to be quite limited in animal brains. In the cow brain, the Ad4BP message was detected only by RT-PCR (6), whereas in the mouse brain, SF-1 expression was restricted to the hypothalamus and pituitary (10, 15, 16, 20). The widespread expression of SF-1 mRNA in the human brain, as opposed to the restricted expression in the hypothalamus and pituitary in mice, suggests a broader role for this transcription factor in the human. It is tempting to speculate that in the human, SF-1 may have a role in the regulation of neurosteroid biosynthesis and/or in the development of the CNS. At the present time, we can only speculate about the additional message found in the brain. This could be an alternatively spliced or polyadenylated product of the hSF-1 gene, or an altogether different, but highly homologous, molecule.
The unexpected detection of hSF-1 transcripts in the spleen demonstrates that the role of this transcription factor may not be limited to steroidogenic tissues and the brain in the human. The Northern blot analysis of the spleen revealed that the signal strength in this organ was second only to that of the adrenal cortex and greater than or equal to that of the gonads. The hSF-1 mRNA also was detected in the myeloid K-562, and HL-60 and Burkitt’s lymphoma cells, which are related to the hematopoetic/immune system. The in situ hybridization study showed that hSF-1 transcripts were concentrated selectively in the reticuloendothelial network of the spleen. In cows, Ad4BP was not detected in the spleen by either Northern blot analysis or by RT-PCR (6). Additionally, SF-1 gene-knockout studies in the mouse did not report agenesis or dysgenesis of the spleen or any obvious immune dysfunction (19). The strong hSF-1 mRNA expression and the two additional transcripts of 4.4 kb and 8 kb seen in the spleen, together with the additional 4.4-kb transcript seen in the rest of the immune tissues and cancer cell lines, indicate that this transcription factor may have a significant role in human immune cell maturation or function.
We conclude that in the human, SF-1 may have a more comprehensive role than in previously studied animals. Its unexpected widespread expression in the brain and its strong expression in the spleen are indicative of such a broader role. It is, therefore, tempting to speculate that in the human, SF-1 may have a fundamental role in neurosteroid biosynthesis and/or brain development, as well as in hematopoetic/immune cell proliferation and/or differentiation.
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Acknowledgments |
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Received September 17, 1996.
Accepted February 20, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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