The mouse insulin-like growth factor 2 (Igf2) locus is a complex genomic region that produces multiple transcripts from alternative promoters. Expression at this locus is regulated by parental imprinting. However, despite the existence of putative imprinting control elements in the Igf2 upstream region, imprinted transcriptional repression is abolished by null mutations at the linked H19 locus. To clarify the extent to which the Igf2 upstream region contains autonomous imprinting control elements we have performed functional and comparative analyses of the region in the mouse and human. Here we report the existence of multiple, overlapping imprinted (maternally repressed) sense and antisense transcripts that are associated with a tandem repeat in the mouse Igf2 upstream region. Regions f lanking the repeat exhibit tissue-specific parental allelic methylation patterns, suggesting the existence of tissue-specific control elements in the upstream region. Studies in H19 null mice indicate that both parental allelic methylation and monoallelic expression of the upstream transcripts depends on an intact H19 gene acting in cis. The homologous region in human IGF2 is structurally conserved, with the significant exception that it does not contain a tandem repeat. Our results support the proposal that tandem repeats act to target methylation to imprinted genetic loci.

high, a transgene expressed at high levels, caused adrenal aplasia in newborn mice. Analysis of fetal adrenal development with Sf1/Cre high -mediated β-catenin inactivation showed decreased proliferation in presumptive adrenocortical precursor cells. By contrast, the Sf1/Cre low transgene effected a lesser degree of β-catenin inactivation that did not affect all adrenocortical cells, permitting adrenal survival to reveal age-dependent degeneration of the cortex. These results define crucial roles for β-catenin -presumably as part of the Wnt canonical signaling pathway -in both embryonic development of the adrenal cortex and in maintenance of the adult organ.

Background: Germline-speci®c differential DNA methylation that persists through fertilization and embryonic development is thought to be thè imprint' distinguishing the parental alleles of imprinted genes. If such methylation is to work as the imprinting mechanism, however, it has to be reprogrammed following each passage through the germline. Previous studies on maternally methylated genes have shown that their methylation imprints are ®rst erased in primordial germ cells (PGCs) and then re-established during oocyte growth.

The orphan nuclear receptor Ad4BP/SF-1 (adrenal 4 binding protein/steroidogenic factor 1) is essential for the proper development and function of reproductive and steroidogenic tissues. Although the expression of Ad4BP/SF-1 is specific for those tissues, the mechanisms underlying this tissue-specific expression remain unknown. In this study, we used transgenic mouse assays to examine the regulation of the tissue-specific expression of Ad4BP/SF-1. An investigation of the entire Ad4BP/SF-1 gene locus revealed a fetal adrenal enhancer (FAdE) in intron 4 containing highly conserved binding sites for Pbx-Prep, Pbx-Hox, and Ad4BP/ SF-1. Transgenic assays revealed that the Ad4 sites, together with Ad4BP/SF-1, develop an autoregulatory loop and thereby maintain transcription, while the Pbx/Prep and Pbx/Hox sites initiate transcription prior to the establishment of the autoregulatory loop. Indeed, a limited number of Hox family members were found to be expressed in the adrenal primordia. Whether a true fetal-type adrenal cortex is present in mice remained controversial, and this argument was complicated by the postnatal development of the so-called X zone. Using transgenic mice with lacZ driven by the FAdE, we clearly identified a fetal adrenal cortex in mice, and the X zone is the fetal adrenal cells accumulated at the juxtamedullary region after birth.

The nuclear receptor Ad4BP/SF-1 is essential for development of the adrenal cortex and the gonads, which derive from a common adrenogonadal primordium. The adrenal cortex subsequently forms morphologically distinct compartments: the inner (fetal) and outer (definitive or adult) zones. Despite considerable effort, the mechanisms that mediate the differential development of the adrenal and gonadal primordia and the fetal and adult adrenal cortices remain incompletely understood. We previously identified a fetal adrenal-specific enhancer (FAdE) in the Ad4BP/SF-1 locus that directs transgene expression to the fetal adrenal cortex and demonstrated that this enhancer is autoregulated by Ad4BP/SF-1. We now combine the FAdE with the Cre/loxP system to trace cell lineages in which the FAdE was active at some stage in development. These lineage-tracing studies establish definitively that the adult cortex derives from precursor cells in the fetal cortex in which the FAdE was activated before the organization into two distinct zones. The potential of these fetal adrenocortical cells to enter the pathway that eventuates in cells of the adult cortex disappeared by embryonic day 14.5. Thus, these studies demonstrate a direct link between the fetal and adult cortices involving a transition that must occur before a specific stage of development.

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The nuclear receptor steroidogenic factor 1 (SF-1 [officially designated NR5A1]) is essential for fetal gonadal development, but its roles in postnatal ovarian function are less well defined. Herein, we have extended our analyses of knockout (KO) mice with markedly decreased SF-1 expression in granulosa cells. As described, these SF-1 KO mice had hypoplastic ovaries that contained a decreased number of follicles and lacked corpora lutea. In the present study, we showed that SF-1 KO mice exhibited abnormal estrous cycles, were infertile, and released significantly fewer oocytes in response to a standard superovulation regimen. Moreover, they had blunted induction of plasma estradiol in response to gonadotropins. The granulosa cell-specific SF-1 KO also significantly affected ovarian expression of putative SF-1 target genes. Consistent with their decreased follicle number, these mice had reduced ovarian expression of anti-müllerian hormone (Amh), which correlates with the reserve pool of ovarian follicles, as well as decreased gonadotropin-induced ovarian expression of aromatase (Cyp19a1) and cyclin D2 (Ccnd2). In contrast, perhaps because of their abnormal cyclicity, SF-1 KO ovaries had higher basal expression of inhibin-alpha. They also had decreased immunoreactivity for genes related to proliferation (Ccnd2 and Mki67 [also known as Ki67]) and increased expression of Cdkn1b, also known as p27, which inhibits cyclin-dependent kinases, arguing for a role of SF-1 in granulosa cell proliferation. These findings demonstrate that SF-1 has a key role in female reproduction via essential actions in granulosa cells.

Adrenal 4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) (Nr5a1) is a nuclear receptor essential for reproductive tissue development and endocrine regulation. This factor is expressed in steroidogenic tissues (e.g. adrenal glands and gonads), and expression of this factor is tightly regulated in a tissue and cell type-specific manner. Our previous studies have identified tissue and cell type-specific enhancers in the introns of the Ad4BP/SF-1 gene in fetal adrenal glands, ventromedial hypothalamus, and pituitary gonadotrope. Characterization of the enhancers had provided new insights into tissue and cell development. However, these studies have failed to identify any gonad-specific enhancer. Here, we identified a fetal Leydig cell-specific enhancer in the upstream region of the mouse Ad4BP/SF-1 gene using transgenic mouse assays. Alignment of the upstream regions among vertebrate animal species demonstrated that the enhancer consisted of three conserved regions, whereby the most highly conserved region contained an Ad4BP/SF-1 binding sequence and an E-box. Mutation of each sequence abolished the enhancer activity and led to a loss of reporter gene expression. These results suggested that Ad4BP/SF-1 gene expression in the fetal Leydig cell is regulated by a yet unidentified E-box binding protein(s) and by an autoregulatory loop formed by Ad4BP/SF-1. Although fetal Leydig cells have been thought to play crucial roles for masculinization of various fetal tissues through androgen production, other functions have remained elusive. Our identification of a fetal Leydig cell-specific enhancer in the Ad4BP/SF-1 gene would be a powerful tool to address these gaps in the knowledge base.