The nuclear receptor SF-1 mediates sexually dimorphic expression of Mullerian Inhibiting Substance, in vivo

Mullerian Inhibiting Substance (MIS) functions to promote regression of the Mullerian duct during male development. Maintaining the sexually dimorphic pattern of MIS expression is essential for proper mammalian reproductive tract development. Here, we show that the intricate spatial and temporal pattern of MIS expression is directed by a remarkably small proximal promoter of only 180 base pairs in length. Expression of the MIS-human growth hormone transgene (MIS/GH) is restricted to Sertoli cells in embryonic testis and to granulosa cells of postnatal ovary, consistent with the known MIS expression pattern. The proximal MIS promoter is therefore sufficient to direct the initiation and the maintenance of MIS gene expression in both sexes. Moreover, in vivo MIS promoter activity requires an intact binding site for the orphan nuclear receptor SF-1. Taken together, these data strongly suggest that SF-1 directly activates MIS in embryonic and postnatal gonads. Consistent with the proposed role of SF-1 in mammalian sex-determination, our study provides physiological evidence that a SF-1 binding site is essential for gene activation of an embryonic testis-specific marker.     

Sex determination in dioecious Silene latifolia. Effects of the Y chromosome and the parasitic smut fungus (Ustilago violacea) on gene expression during flower development

We have embarked on a molecular cloning approach to the investigation of sex determination in Silene latifolia Poiret, a dioecious plant species with morphologically distinguishable sex chromosomes. One of our key objectives was to define a range of genes that are up-regulated in male plants in response to Y chromosome sex-determination genes. Here we present the characterization of eight male-specific cDNA sequences and classify these according to their expression dynamics to provide a range of molecular markers for dioecious male flower development. Genetically female S. latifolia plants undergo a partial sex reversal in response to infection by the parasitic smut fungus Ustilago violacea. This phenomenon has been exploited in these studies; male-specific cDNAs have been further categorized as inducible or noninducible in female plants by smut fungus infection. Analysis of the organ-specific expression of male-specific probes in male and female flowers has also identified a gene that is regulated in a sex-specific manner in nonreproductive floral tissues common to both male and female plants. This observation provides, to our knowledge, the first molecular marker for dominant effect of the Y chromosome in nonreproductive floral organs.     

Photodynamic therapy for rheumatoid arthritis?

Songbirds have emerged as extremely important animals for investigating sex steroid hormone effects on the central nervous system. The masculinizing effects of exogenous estrogen on the neural circuits controlling song in female zebra finches are well documented. There is evidence that estrogens are necessary for the full activation of singing behavior in several species. These kinds of studies have led us and others to investigate the mechanisms whereby estrogens are made available to the brains of songbirds during development and adulthood. In this article, I review results of some of these studies examining the estrogen synthetic enzyme aromatase and its expression and activity in brain and in other tissues of songbirds. I will discuss some results and thoughts we have about the interactions of aromatase with the two remaining androgen-metabolizing enzymes in the avian brain, 5alpha-reductase, the enzyme that converts T into the active androgen 5alpha-dihydrotestosterone (DHT); and 5beta-reductase, the enzyme that converts T into the inactive 5beta-DHT. Finally, I will consider some ideas raised by these studies concerning potential sources of the androgen substrate for brain aromatization as well as some possible new functions that aromatase might be playing in the songbird telencephalon.     

SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene

The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.     

Mutations in SOX9, the gene responsible for Campomelic dysplasia and autosomal sex reversal

Campomelic dysplasia (CD) is a skeletal malformation syndrome frequently accompanied by 46,XY sex reversal. A mutation-screening strategy using SSCP was employed to identify mutations in SOX9, the chromosome 17q24 gene responsible for CD and autosomal sex reversal in man. We have screened seven CD patients with no cytologically detectable chromosomal aberrations and two CD patients with chromosome 17 rearrangements for mutations in the entire open reading frame of SOX9. Five different mutations have been identified in six CD patients: two missense mutations in the SOX9 putative DNA binding domain (high mobility group, or HMG, box); three frameshift mutations and a splice-acceptor mutation. An identical frameshift mutation is found in two unrelated 46,XY patients, one exhibiting a male phenotype and the other displaying a female phenotype (XY sex reversal). All mutations found affect a single allele, which is consistent with a dominant mode of inheritance. No mutations were found in the SOX9 open reading frame of two patients with chromosome 17q rearrangements, suggesting that the translocations affect SOX9 expression. These findings are consistent with the hypothesis that CD results from haploinsufficiency of SOX9.     

Mammalian sex determination: from gonads to brain

In mammals, sex is determined by the Y chromosome, which encodes a testis-determining factor (TDF). This factor causes the undifferentiated embryonic gonads to develop as testes rather than ovaries. The testes subsequently produce the male sex hormones that are responsible for all male sexual characteristics. In 1990, the sex-determining gene, TDF, was identified and termed SRY in humans (Sry in mice). It encodes a protein containing a high mobility group (HMG) motif, which confers the ability to bind and to bend DNA. Genetic evidence supporting SRY as TDF came from the observation of a male phenotype in XX mice transgenic for a small genomic fragment containing Sry, and from the study of XY sex-reversed individuals who harbor de novo mutations in the SRY coding sequence. Other non-Y-linked genes involved in sex determination were subsequently found by genetic analysis of XY sex-reversed patients not explained by mutations in SRY. These genes are WT1, SF1, DAX1, and SOX9. A regulatory cascade hypothesis for mammalian sex determination, proposing that SRY represses a negative regulator of male development, was recently supported by observation of mice that expressed a DAX1 transgene and developed as XY sex-reversed females. The role of some sex-determining genes, such as DAX1 and SF1, in the development of the entire reproductive axis, a functionally integrated endocrine axis, leads to a new concept. Normal sexual development may result from the functional and developmental integration of a number of different genes that play roles in sex determination, sexual differentiation, and sexual behavior.     

Steroid binding and metabolism in the luteinizing hormone-releasing hormone-producing neuronal cell line GT1-1

LHRH synthesis and release are modulated in vivo by gonadal steroids. Although immunocytochemical and autoradiographic studies failed to detect appreciable amounts of estrogen or androgen receptor in LHRH-producing neurons, the recent finding that the promoter region of the LHRH gene contains several steroid hormone-responsive elements indicates a possible direct effect of sex steroids on these specialized neurons. The immortalized LHRH-producing neuronal cell line, GT1, which became recently available, may allow the study of LHRH dynamics. The presence of specific binding sites for estrogen and androgens as well as the presence of the two major enzymatic pathways involved in modulation of androgen action (the 5 alpha-reductase/3 alpha-hydroxysteroid dehydrogenase and the aromatase) have been studied in the GT1-1 clone. High affinity, low capacity binding sites for [3H]estradiol (Kd, 0.11 nM; binding capacity, 6.2 fmol/mg protein) and for a ligand of the androgen receptor, [3H]R1881 (Kd, 0.054 nM; binding capacity, 9.58 fmol/mg protein), have been identified in this cell line. A 2-fold induction of androgen-binding sites has been observed after 3 days of treatment of GT1-1 cells with estradiol (1 microM), indicating that the estradiol binding is probably linked to a functional estrogen receptor. Aromatase and 5 alpha-reductase/3 alpha-hydroxysteroid dehydrogenase activities have been also tested in GT1-1 cells. Under the culture conditions adopted, no detectable aromatization of [1 beta 3H]delta 4-androstenedione to estrone was observed using the tritiated water method. On the other hand, GT1-1 cells efficiently converted testosterone into dihydrotestosterone and subsequently into 5 alpha-androstan-3 alpha,17 beta-diol. In conclusion, GT1-1 cells possess several elements of the machinery through which sex steroids may influence LHRH dynamics.