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a Westmead Institute for Cancer Research, University of Sydney, Westmead Hospital, Westmead, New South Wales 2145, Australia
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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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Studies from this laboratory, aimed at the identification of progestin-responsive genes, have shown that SOX4, a member of the 
RY-related HMG-b
(SOX) gene family which is related to the mammalian testis-determining gene SRY, was expressed in T-47D cells [9], a human hormone-responsive breast cancer cell line. SOX4 transcription was directly induced by progestins, and this was accompanied by an increase in SOX-specific transcriptional activity. E2 decreased SOX4 mRNA expression in these cells. SOX4 mRNA was also expressed in the normal human breast [9], suggesting that SOX4 may play a developmental role in normal steroid hormone-responsive tissues in the adult.
SOX4 belongs to a family of transcriptional regulators that all contain an HMG-box DNA-binding domain and interact with DNA in a sequence-specific fashion [10]. Like all other SOX proteins analyzed, SOX4 binds to the DNA motif A/TA/TCAAA/TG (the most frequently occurring nucleotides appear in bold) and induces bending of the DNA, thus facilitating protein-protein or protein-DNA interaction; this may form the basis of the transactivating property of the SOX genes [11–17]. SOX4 transactivates the human CD2 gene [18], and the region mediating its transactivating properties has been mapped to the carboxyl terminal of the gene [14]. Many SOX genes have now been identified in the human and other species, and they show diverse patterns of expression during embryogenesis, suggestive of a role in cell fate determination in a wide range of developmental processes. Indeed, many SOX genes play important roles in development, specifically of neural tissues, central and peripheral nervous systems, and skeletal tissue during embryogenesis [10].
The murine Sox4 gene was first isolated as the Sry-related gene a4 [19]. It is expressed in the ovary, testis, and thymus of adult mice and in mouse T and pre-B lymphocytic cell lines [14]. During embryogenesis, Sox4 is essential for normal cardiac development. Sox4 null mice die during embryogenesis because of failure in endocardial ridge development, which prevents proper formation of semilunar valves and results in circulatory failure [20]. Furthermore, introduction of bone marrow cells from these animals into an irradiated host results in a block in B-cell lineage progression at the stage of pro-B-lymphocyte expansion [20]. This indicates that Sox4 is involved in developmental processes in the adult animal as well as during embryogenesis.
Sox4 is expressed in the steroid hormone-responsive ovary and testis, but it is not known whether it is also expressed in other hormone-responsive tissues such as the uterus and mammary gland. Studies in our laboratory have shown expression of SOX4 mRNA in the normal human breast and steroidal regulation of SOX4 mRNA expression in breast cancer cells [9], but there is no information on hormonal regulation of Sox4 in hormone-responsive tissues in the mouse under physiological conditions in which endogenous hormone levels fluctuate. This study was undertaken to examine Sox4 expression and regulation in the mouse female reproductive system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals used were of analytical grade and obtained from either Astral Scientific (Gymea, NSW, Australia) or Sigma Chemical Co. (St. Louis, MO). P4, E2, and TRI Reagent were obtained from Sigma Chemical Co.; [
-32P]dCTP (10 Ci/mmol), [
-32P]ATP (5 Ci/mmol), and the Megaprime DNA labeling kit were from Amersham Australia (North Ryde, Sydney, Australia). [-32P]UTP (3000 Ci/mmol) was from Bresatec (Thebarton, SA, Australia), the Ambion MAXIscript and RPA II kits from Ambion Inc. (Austin, TX), and the poly dA-dT tract poly(A)+ RNA isolation kit from Promega (Madison, WI). The IMMULITE P4 kit was from Bio-Mediq DPC Pty. Ltd. (Victoria, Australia).
Animals
Female BALB/c mice were maintained under standard temperature-controlled conditions with 12L:12D cycles. Animals were handled and ovariectomies performed according to the guidelines of the Animal Research Act, 1985, and the National Health & Medical Research Council Australian Code of Practice for Care and Use of Animals for Scientific Purposes, 6th edition, 1997.
Sox4 mRNA levels were determined during the estrous cycle using two experimental designs, and cycle stages were determined by vaginal smear cytology [21]. In the first experiment, 12 mice that had gone through 2 cycles were killed for RNA analysis at various stages of the third cycle. In the second experiment, 8 mice were isolated from males for 3 wk and then reintroduced into the presence of male mice to synchronize their cycles. Two mice were killed on each of the 4 following consecutive days, and cycle stage was determined. There was no difference in results obtained with the two experimental designs.
Ovariectomy and Hormone Treatment of Mice
Bilateral ovariectomies were performed 12 days before the start of hormonal treatments and according to standard techniques. Hormones were dissolved in ethanol, then diluted in mineral oil to final concentrations of 10 µg/µl and 10 ng/µl for P4 and E2, respectively. Hormone treatment was administered by daily s.c. injections of 0.1 ml of mineral oil containing no hormone (C), 1 mg of P4 (P), 1 µg of E2 (E), or 1 µg E2 + 1 mg P4 (E+P); and the mice were killed at various time points. Animals received either a single injection of vehicle or hormone, alone or in combination, 6 h (C6, P6, E6, E6+P6) or 14 h (C14, P14, E14, and E14+P14) before they were killed, or multiple injections every 24 h (C, P, E, and E+P with a number designating the time in hours between first injection and killing, i.e., C72 and E72+P48). Tissues (inguinal mammary glands, uterus, thymus, ovaries, and liver), collected immediately after the mice were killed, were snap-frozen in liquid nitrogen for subsequent RNA isolation.
To assess serum levels of P4, blood was collected from the mice at the time they were killed, and serum was analyzed using the IMMULITE P4 kit. Where required, the serum was diluted for the values to fit within the range of detection of the assay. Human serum purchased with the kit and routinely used for dilution and analysis of human serum was tested for its suitability with mouse serum: a mouse serum sample known to contain high levels of P4 was analyzed at various dilutions, and as the dilution pattern paralleled the standard curve, the human serum was deemed to be suitable for use to dilute the mouse serum where required. Serum P4 levels were all below 10 ng/ml in the cycling mice and below 3 ng/ml in the ovariectomized mice. At 6 h after P4 injection, levels reached an average of 98.4 ng/ml (± 51.5 SEM), and by 14 h levels had decreased to an average of 9.4 ng/ml (± 14.4 SEM). Uterine weights, which correlate with serum E2 levels [22], were 35.0 ± 6.0 (mean mg ± SEM) and 31.4 ± 7.7, respectively, in E and E+P mice that received E2 and 21.9 ± 7.8 and 20.1 ± 4.7, respectively, in the C and P mice that received no E2 treatment.
Hybridization Probes
A partial Sox4 cDNA (nucleotides 1108–1887; [14]), showing little identity to other Sox genes with sequences in the data bases, was isolated from a pGEMT-cloned fragment (nucleotides 1108–2026) previously amplified from mouse genomic DNA using polymerase chain reaction and specific oligonucleotide primers msoxf1 (nucleotides 1108–1125) 5'-AGAAGGGCGACAAGGTCG-3' and msoxr1 (nucleotides 2026–2008) 5'-CTCCTCTCCTGCCTCTTGG-3', labeled by random priming and used as a Sox4-specific probe. The glyceraldehyde phosphate dehydrogenase (GAPDH) probe, a 1.1-kbp (kilobase pair) of rat cDNA fragment cloned in pBluescribe, was a gift from G. Brown (CSIRO, North Ryde, NSW, Australia).
A pGEMT clone containing a Sox4 cDNA fragment (nucleotides 1108–1887) was linearized at the SmaI site (nucleotide 1549) and used as a template for riboprobe transcription using SP6 polymerase and the MAXIscript kit. This generated a 388-base riboprobe containing 50 bases of vector sequence and 338 bases of antisense Sox4-specific RNA. For the Sox4 riboprobe, the reaction mixture contained 1 µg of linearized template, 0.3 nmol of UTP, and 0.017 nmol of [-32P]UTP to produce a riboprobe of specific activity 2.4 x 108 cpm/µg. T4 gene 32 protein (2.5 µg), a single-strand DNA binding protein (SSBP), was also added to facilitate generation of full-length transcript from the Sox4 guanine/cytosine (GC)-rich template (65% GC-rich). The full-length transcript was purified from a polyacrylamide gel as described in the kit protocol, the activity of the eluted riboprobe was determined by liquid scintillation counting, and an amount equivalent to 2 fmol of RNA was used per hybridization reaction.
The mouse ß-actin template provided with the kit was used to generate an actin-specific riboprobe. The reaction was as above except that the SSBP was omitted, and 0.3 nmol of UTP and 0.017 nmol of [-32P]UTP were used to generate a riboprobe of specific activity 1 x 107 cpm/µg.
RNA Isolation and Analysis
Frozen tissues were weighed, pulverized, resuspended in TRI reagent (1 ml/50 mg tissue), and aspirated several times through a 21-gauge needle; and RNA was isolated using TRI reagent according to the manufacturer's instructions. Where indicated, poly(A)+ RNA was isolated using the poly dA-dT tract kit and the manufacturer's instructions.
Sox4 mRNA expression was analyzed by Northern blot hybridization or ribonuclease protection assay (RPA). For Northern blot, RNA was separated on 2.2 M formaldehyde/1% agarose gels, transferred to nylon membranes (Zeta Probe GT; Bio-Rad, Cambridge, MA) by vacuum blotting, and UV cross-linked to the membrane. Filters were prehybridized at 50°C for 1–2 h in standard hybridization solution containing 50% formamide, radiolabeled probe was added (106 cpm/ml), and hybridization was carried out for 16–20 h at 50°C. Membranes were washed twice at ambient temperature for 15 min in single-strength SSC, 0.1% SDS and for 15 min in 0.25-strength SSC, 0.1% SDS; and at 65°C for 15 min in 0.1-strength SSC, 0.1% SDS. Membranes were exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) screen and subsequently to an x-ray film at -70°C. Sox4 transcripts were quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics) or by scanning densitometry. The filters were reprobed for GAPDH mRNA or 18S ribosomal RNA, and levels were measured as above. Sox4 mRNA values were normalized against either GAPDH or 18S levels and expressed as arbitrary units (AU).
RPA analysis was performed using the AMBION kit and the streamline method as outlined in the manufacturer's instructions. Hybridization was carried out for 16 h at 45°C. Protected RNA fragments were separated on a denaturing polyacrylamide gel; and the gel was transferred to 3M paper, dried, and exposed to a PhosphorImager screen. Protected fragments were quantified as described for the Northern blots; Sox4 values were normalized against ß-actin values and expressed as AU.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine whether Sox4 mRNA was expressed in the uterus and mammary glands of mice, total RNA was extracted from the uterus, and poly(A)+ RNA was extracted from mammary glands. Total RNA was extracted also from thymus and ovaries, known Sox4-positive tissues, and from the liver, a known negative tissue [12, 23]. An mRNA species of 5.0 kilobases (kb), consistent with the reported size of Sox4 transcript [12], was detected by Northern blot hybridization in the uterus and mammary glands (Fig. 1). Sox4 mRNA in the uterus was present at levels comparable to those in the ovary and thymus but was less abundant in the mammary glands. A less abundant mRNA species, migrating ahead of the 4.8-kb 28S ribosomal band, was detected in the uterus and thymus after longer exposure (not shown). This species could represent the 3.9-kb species that has been reported in testis and brain [23]. No signal was detected in liver RNA.
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Effect of Estrous Cycle, Developmental Stage, and Ovariectomy on Sox4 mRNA Expression in Mouse Tissues
To investigate whether Sox4 mRNA expression was modulated in physiological circumstances in which ovarian hormones are modulated, Sox4 mRNA levels were measured in the uterus of mice during the estrous cycle and in 4-wk-old pubertal or ovariectomized adult mice in which ovarian hormones are absent (Fig. 2).
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The vaginal smear cytology used to estimate the estrous cycle stages reflects daily changes in the secretion of ovarian hormones [21, 24]. E2 levels increase from baseline values throughout the first day of the cycle (estrus and metestrus1) and are maintained at a moderate level until the afternoon of Day 2 (metestrus2), when levels steadily increase through the diestrous stage to reach peak values by the morning of proestrus (Day 4, 5, or 6, depending on cycle length) before rapidly returning to baseline by estrus. P4 levels show a small peak at metestrus2 as a result of the autonomous activity of the corpus luteum of the previous ovulation, but the levels decline rapidly on Day 3 as the corpus luteum regresses. The major rise of P4 occurs late at proestrus, with levels peaking in the night preceding estrus, and then abruptly declining to baseline levels by the morning of estrus.
Sox4 mRNA was detected at all stages of the cycle, with levels being maximal at estrus (100%), reduced at metestrus1 (80%) and metestrus2 (34%), and then diminishing to minimal levels at diestrus (16%) and proestrus (19%). Ovariectomy reduced Sox4 mRNA expression 2-fold when compared to levels detected in estrous mice. Pubertal mice, before initiation of ovarian function, showed Sox4 mRNA expression comparable to that found in proestrous mice.
Total RNA isolated from the ovary and the thymus, and poly(A)+ RNA from the mammary glands from some of the mice above, were analyzed for Sox4 mRNA expression (Fig. 3). Sox4 mRNA was expressed throughout the cycle in the ovaries, with maximal levels at metestrus1 and minimal levels at proestrus. However, the changes in level during the cycle were not marked, and the difference between maximal and minimal levels was no more than 2-fold (Fig. 3A). Sox4 mRNA was expressed at very low abundance in the mammary gland (11-day exposure was required compared to 2 days with the other tissues) and remained relatively constant throughout the estrous cycle (Fig. 3B). Sox4 expression was also measured by RPA in total RNA from the mammary glands of a proestrous mouse, an estrous mouse, an ovariectomized mouse, and two 4-wk-old mice. The lowest levels of Sox4 mRNA were detected in the ovariectomized mouse, and the levels were essentially equivalent in the 4-wk-old mice and proestrous and estrous mice. However, absolute levels of Sox4 mRNA in the mammary gland were too close to the limit of detection of the assay for any apparent difference to be significant. Sox4 mRNA was detected at similar levels in the thymus throughout the cycle, and ovariectomy did not lead to any changes in expression (Fig. 3C).
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Effects of P4 and E2 on Sox4 mRNA Expression in Mouse Uterus
The modulation of Sox4 mRNA expression in the uterus under varying physiological conditions in which ovarian hormones fluctuate suggested a role for these hormones in this process. To further investigate this possibility, the contribution of P4 or E2, alone or in combination, was determined on modulation of Sox4 mRNA expression in the mouse uterus. Sox4 mRNA was analyzed in tissues harvested at various time points after ovariectomized mice received s.c. injections of oil, P, E, or E+P as outlined in the methods. Total RNA from the uterus after single injection (6 h, 14 h) was analyzed by RPA (Fig. 4A) and after multiple injections (38 h, 48 h, 72 h) by Northern blot hybridization (Fig. 4B).
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E2 treatment resulted in a 6-fold decrease in Sox4 mRNA compared to the oil-injected control after 6-h and 14-h treatment (Fig. 4C). This effect was diminished after longer treatment times. P4 treatment resulted in a 2-fold decrease in Sox4 mRNA levels compared to the oil-treated control at all time points. Combined E+P treatment had different effects depending on the length of treatment. At 6 h, the effect of the combined hormones was the same as that of E2 alone. At 14 h the effect was intermediate between each individual treatment, and after longer treatments, the Sox4 mRNA levels were higher than with the individual treatments.
Effects of P4 and E2 on Sox4 mRNA Expression in Mouse Mammary Glands and Thymus
Sox4 mRNA expression was measured by RPA and Northern blot hybridization, respectively, in total RNA from the mammary glands and thymus from some of the above mice (Fig. 5). There were no effects of any of the treatments on Sox4 mRNA expression in the mammary glands or the thymus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sox4 mRNA expression was modulated in the adult uterus at various stages of the estrous cycle, suggesting a role for ovarian hormones in the control of Sox4 expression. Low levels of Sox4 mRNA were observed in proestrus and diestrus, stages in which serum E2 levels are increasing and P4 levels are low. Maximal levels were observed at estrus and metestrus1, when serum E2 levels are low, and immediately after maximal serum P4 levels during the preceding night. These results suggest a role for E2 in down-regulation of Sox4 mRNA expression and a role for P4 in its induction at estrus. This interpretation is consistent with the observation that progestins directly induced, and E2 down-regulated, SOX4 mRNA expression in breast cancer cells [9].
A role for ovarian hormones in the control of Sox4 expression in the uterus was suggested also by the observation that hormonal ablation by ovariectomy resulted in levels of Sox4 mRNA intermediate between those found at estrus and proestrus, suggesting that ovarian hormones play a role in the decrease as well as the induction of Sox4 gene expression in the uterus.
In the uterus, E2 treatment of ovariectomized mice led to a 6-fold decrease in Sox4 mRNA. This was comparable with the decrease in Sox4 mRNA observed at proestrus. P4, when administered together with E2, partially abrogated the E2 effect after prolonged treatment. The delay in the P4 effect may be explained by the fact that E2 is known to up-regulate the P4 receptor and is required for P4 action in the ovariectomized uterus where P4 receptor (PR) content is low [3]. If the serum P4 peak on the night preceding estrus plays a role in the induction of Sox4 mRNA expression in the cycling mice, exposure to E2 in the days preceding estrus would have resulted in PR induction and hence optimal P4 effects in the uterus.
P4 alone did not induce Sox4 mRNA expression in ovariectomized mice, and a decrease in Sox4 mRNA levels was observed. This is despite observations in the estrous mice described above, implying a role for P4 in induction of Sox4 mRNA expression, and previous studies demonstrating direct PR-mediated induction of SOX4 mRNA expression in breast cancer cells [9]. The simplest explanation for the modest effects of P4 alone is that PR levels were low in these animals, which did not receive E2 priming, and this needs to be examined in more detail in future experiments.
Modulation of Sox4 mRNA expression in the mouse mammary gland was not observed, either during the estrous cycle or upon hormone administration to ovariectomized animals. In view of the low abundance of Sox4 mRNA in this tissue, and the fact that PR and ER, the mediators of hormonal effects, are expressed in only a small proportion of the mammary epithelial cell population [25, 26], analysis of RNA isolated from whole tissue may not be sufficiently sensitive to detect changes if they occur. It will be important to analyze cell-specific expression of Sox4 in the mammary gland by immunohistochemistry. Furthermore, the most striking hormonal effects in the mammary gland occur in pubertal development and pregnancy rather than during the estrous cycle, and analysis of Sox4 expression at these stages form the basis of ongoing studies in our laboratory.
The demonstration that Sox4 mRNA expression is modulated in the uterus of cycling mice suggests a role for Sox4 in the reproductive system. The functions of members of the SOX gene family are not well elucidated, but target genes for some of the SOX proteins implicate SOX genes in transcriptional control in a tissue-specific manner. The human SOX4 has been shown to transactivate the CD2 enhancer in T cells [18], and SOX9 has been implicated in the regulation of type II collagen in a chondrocyte-specific manner [27]. An emerging feature of SOX proteins is their cooperative interactions with other transcription factors, more specifically the POU proteins, and this may form the basis of their complex tissue-specific roles [28, 29].
In summary, this study identified expression of Sox4 mRNA in the mouse female reproductive tissues and modulation of that expression in the uterus under different physiological conditions in which the hormonal milieu fluctuates. The results suggest that Sox4 expression is under ovarian control in the mouse uterus and suggest a putative role for Sox4 in the female reproductive system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 61 2 9845 8319; sybille{at}hemonc.wh.su.edu.au
Accepted: March 16, 1999.
Received: January 6, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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