Sdmg1 is a conserved transmembrane protein associated with germ cell sex determination and germline-soma interactions in mice.

In mammals, the supporting cell lineage in an embryonic gonad communicates the sex-determining decision to various sexually dimorphic cell types in the developing embryo, including the germ cells. However, the molecular nature of the sex-determining signals that pass from the supporting cells to the germ cells is not well understood. We have identified a conserved transmembrane protein, Sdmg1, owing to its male-specific expression in mouse embryonic gonads. Sdmg1 is expressed in the Sertoli cells of embryonic testes from 12.5 dpc, and in granulosa cells of growing follicles in adult ovaries. In Sertoli cells, Sdmg1 is localised to endosomes, and knock-down of Sdmg1 in Sertoli cell lines causes mis-localisation of the secretory SNARE Stx2 and defects in membrane trafficking. Upregulation of Sdmg1 appears to be part of a larger programme of changes to membrane trafficking pathways in embryonic Sertoli cells, and perturbing secretion in male embryonic gonads in organ culture causes male-to-female germ cell sex reversal. These data suggest that changes that occur in the cell biology of embryonic Sertoli cells may facilitate the communication of male sex-determining decisions to the germ cells during embryonic development.

A novel molecular assay to discriminate transcriptional effects caused by xenoestrogens.

A phenotypic definition of the term estrogen has become increasingly problematic due to the multiple modes of estrogen action which can now be defined by differing nuclear and membrane receptors for the classic ligand, 17beta-estradiol, and by the multiple signalling pathways that are consequently addressed. This has led to the term xenoestrogen being largely determined by whatever assay system is used for its definition. Here we describe a novel and simple matrix for a transfection system using MBA-MD231 and MCF-7 breast cancer cells as hosts. This matrix is able to vary the type of nuclear estrogen receptor used, and by varying the promoter-reporter construct between one using a classic estrogen response element (ERE) enhancer, and one using an enhancer element derived from the bovine oxytocin gene promoter binding an orphan nuclear receptor, direct classical effects can be neatly discriminated from non-classical and non-genomic actions of test substances. This assay matrix has been used to examine a selection of phytoestrogens and xenobiotics, thereby providing new information on the mechanism of action of some of these substances in breast cancer cells.

Differentiation-specific action of orphan nuclear receptor NR5A1 (SF-1): transcriptional regulation in luteinizing bovine theca cells.

BACKGROUND: The orphan nuclear receptor NR5A1 (steroidogenic factor-1, SF-1) is a master regulator of tissue-specific gene expression in reproductive and steroidogenic tissues. Two activating functions, AF-1 and AF-2, have been described to function in a cooperative manner to recruit transcriptional coactivators to the promoter regions of NR5A1-controlled genes. METHODS: The role of the NR5A1 activating functions AF-1 and AF-2 was studied in primary bovine theca cells. Bovine theca cells were infected with recombinant adenovirus vectors over-expressing wild-type NR5A1 or NR5A1 mutants, in which one of the activating functions of this orphan nuclear receptor had been impaired. Under different culture conditions, theca cell-specific transcript levels were measured by reverse transcription and real-time PCR. RESULTS: Under culture conditions optimized for cell growth, transcriptional up-regulation of CYP11A1 (P450 side chain-cleavage enzyme) and INSL3 (Insulin-like factor 3, Relaxin-like factor (RLF)) was found to be dependent on the presence of NR5A1 carrying an intact AF-2. Under conditions inducing luteal differentiation of theca cells, CYP11A1 and STAR (Steroidogenic acute regulatory protein) were up-regulated by the action of luteinizing hormone (LH), whereas the differentiation-specific up-regulation of INSL3 was suppressed by LH in luteinizing theca cells. Inhibition of insulin- or IGF1- (insulin-like growth factor I) dependent signal transduction by the RAF1 kinase inhibitor GW5074 and the mitogen-activated protein kinase kinase inhibitor PD98059 resulted in the finding that RAF1 kinase inhibition was able to counteract the LH-dependent regulation of NR5A1-controlled genes, whereas inhibition of the mitogen-activated protein kinase (MAP kinase) pathway did not have any significant effect. CONCLUSION: The regulation of the three NR5A1-controlled genes CYPA11, STAR, and INSL3 in luteinizing theca cells apparently is not dependent on NR5A1 activating functions AF-1 or AF-2. Activation of AF-1 here even appears to have an impairing effect on NR5A1 transcriptional activity, implying that up-regulation of NR5A1-controlled genes uses a different pathway. Our results might be explained by the possible existence of an interconnection between the RAF1 kinase and the cyclic AMP-protein kinase A pathway. Such a non-classical regulatory pathway might play an important role in the control of gene expression in reproductive and steroidogenic tissues.

Expression of androgen and estrogen receptors in sertoli cells: studies using the mouse SK11 cell line.

Sertoli cells (Sc) play a major role in the establishment and maintenance of spermatogenesis. In the adult testis, Sc contain androgen receptor (AR) and estrogen receptor (ER)-beta but exhibit a loss of steroid responsiveness when maintained in primary culture. In the present study, we demonstrated that a transformed murine cell line (SK11) has retained a Sc phenotype and remains steroid responsive. SK11 cells expressed mRNAs found in Sc (aromatase, sulfated glycoprotein-1, sulfated glycoprotein-2, GATA-1, Sry-type high-mobility-group box transcription factor-9, testatin, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) including those for AR and ERbeta but not ERalpha. AR and ERbeta were immunolocalized to cell nuclei, and their ability to activate gene expression was investigated using transient transfections with reporter constructs containing either 3xERE or pem-androgen-responsive element promoters. Expression of the 3xERE reporter was induced after incubation with 17beta-estradiol (E2), 5alpha-androstane-3-beta, 17beta-diol (3betaAdiol), or testosterone (T); up-regulation of the pem-androgen-responsive element reporter was detected only in the presence of T or dihydrotestosterone. Activation of the ERE reporter did not occur after targeted knockdown of ERbeta mRNA. Expression of AR and ERbeta mRNAs was increased after incubation of cells with T or E2, respectively. In conclusion, we have demonstrated that the SK11 Sc cell line contains functional AR and ERbeta and that treatment of the cells with their respective steroids results in an increase in the amount of their mRNAs. Our results suggest that E2 or 3betaAdiol acting via ERbeta might modulate Sc function in vivo and that SK11 cells provide a useful model that can be used to complement studies using Sc selective gene ablation.

Promyelocytic leukaemia zinc finger protein (PLZF) is a glucocorticoid- and progesterone-induced transcription factor in human endometrial stromal cells and myometrial smooth muscle cells.

The promyelocytic leukaemia zinc finger (PLZF) protein belongs to the family of Krüppel-like zinc finger proteins. It is a transcriptional repressor involved in cell cycle control and has been implicated in limb development, differentiation of myeloid cells, and spermatogenesis. Little is known about the regulation of PLZF expression. In search for mediators of progesterone signalling in the female reproductive tract, we discovered induction of PLZF mRNA in primary cultures of human endometrial stromal cells and myometrial smooth muscle cells (SMC) in response to progesterone. Surprisingly, dexamethasone was a more potent inducer of PLZF expression than progesterone and elicited a sustained up-regulation of PLZF mRNA levels within 2 h. Immunofluorescence showed localization of PLZF to the nuclei of dexamethasone-treated SMC. In uterine biopsies, nuclear staining for PLZF was found in myometrial cells and endometrial stromal cells of the secretory phase. The transcriptional start site of the PLZF gene was located to position -5801 in SMC. Transfected promoter constructs containing up to 4.1 kb of 5'-flanking DNA were not induced by activated glucocorticoid or progesterone receptor. In contrast, co-transfection of c-jun and c-fos expression vectors resulted in stimulation of reporter gene activity, indicating an involvement of AP-1 transcription factors in PLZF expression.

SPEER--a new family of testis-specific genes from the mouse.

Differential cloning revealed a partial mRNA sequence expressed in the mouse testis, which on further molecular characterization proved to be a member of a new family of 14 transcribed genes. Six of the genes appear to be expressed pseudogenes. The remainder indicate an open reading frame of approximately 200-220 amino acids encoding proteins with a very high proportion of alpha helical secondary structure, comprising approximately 15% glutamate residues. Because of this property, the family has been named SPErm-associated glutamate (E)-Rich protein (SPEER). Three members were chosen for more detailed characterization: SPEER-1 (pseudogene), SPEER-2, and SPEER-4D. All three are expressed tissue specifically in the testis of mice, with only very weak expression evident in the rat testis but in no other species tested. Using reverse transcription-polymerase chain reaction (RT-PCR), all three transcripts can be detected also in the epididymis, presumably due to the presence of spermatozoa. All three transcripts are expressed to high levels in haploid germ cells at the spermatocyte-spermatid transition. SPEER-1 mRNA is present in the cytoplasm as a sense transcript, SPEER-2 appears to be made mostly as an antisense transcript, whereas SPEER-4D appears to be localized within a subcellular compartment as a conventional sense transcript. Codon usage analysis suggests that all but the pseudogenes can be expressed as protein, confirmed for SPEER-2 and SPEER-4D by in vitro transcription/translation. An antibody raised against a peptide region of SPEER-4D, which probably cross-reacts with other SPEER members, immunohistochemically stains the nuclei of early round spermatids. While there are no true homologies to other proteins in the genome databases, some motifs are present that suggest a relationship to nuclear matrix proteins, implying that the SPEER family is a new group of haploid sperm-specific nuclear factors.