CD9-positive cells in the intermediate lobe migrate into the anterior lobe to supply endocrine cells.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL), and secretes important hormones for growth, sexual development, metabolism, and reproduction. In the marginal cell layer (MCL) facing Rathke's cleft between the IL and AL, cluster of differentiation (CD) 9-, CD81-, S100ß-, and SOX2-quadruple positive (CD9/CD81/S100ß/SOX2-positive) cells in the adult IL are settled as tissue-resident stem/progenitor cells supplying hormone-producing cells to the AL. However, it is unclear how CD9/CD81/S100ß/SOX2-positive cells in the IL-side MCL migrate into the AL across Rathke's cleft. In the present study, we performed chimeric pituitary tissue culture using S100ß/GFP-transgenic rats and Wistar rats, and traced the footprint of S100ß/GFP-expressing cells. We detected IL-side S100ß/GFP-expressing cells in the AL tissue, demonstrating that these cells migrate from the IL to the AL. However, the cells failed to migrate in the opposite direction. Consistently, scanning electron microscopic analysis revealed well-developed cytoplasmic protrusions in the IL-side MCL, but not in the AL-side MCL, suggesting that IL-side CD9/CD81/S100ß/SOX2-positive cells had higher migratory activity. We also searched for a specific marker for IL-side CD9/CD81/S100ß/SOX2-positive cells and identified tetraspanin 1 (TSPAN1) from microarray analysis. Downregulation of Tspan1 by specific siRNA impaired cell migration and significantly reduced expression of snail family transcriptional repressor 2 (Slug), a marker of epithelial-mesenchymal transition (EMT). Therefore, CD9/CD81/S100ß/SOX2-positive cells in the IL-side MCL can be stem/progenitor cells that provide stem/progenitor cells to the AL-side MCL via SLUG-mediated EMT and cell migration.

Di(n-butyl) phthalate induces vimentin filaments disruption in rat sertoli cells: a possible relation with spermatogenic cell apoptosis.

Phthalate esters have been extensively used as a plasticizer of synthetic polymers. Previous studies have revealed that some phthalate esters including di(n-butyl) phthalate (DBP) induce spermatogenic cell apoptosis, although its mechanism is not yet clear. The present study describes that disruption of Sertoli cell vimentin filaments by DBP administration may relate to spermatogenic cell apoptosis. The present histopathological study revealed that a single oral administration of 500 mg/kg DBP caused progressive detachment and displacement of spermatogenic cells away from the seminiferous epithelium and sloughing of them into the lumen. Degenerative spermatogenic cells characterized by chromatin condensation were frequently observed in DBP-treated rats. Ultrastructurally, the degenerative spermatogenic cells were separated from their neighbours, and a collapse of Sertoli cell vimentin filaments was recognized in DBP-treated rats. Sertoli cell cultures showed the increased number and size of vacuoles in their cytoplasm. In agreement with the in vivo experiment, vimentin filaments clearly showed a gradual collapse in DBP-exposed Sertoli cells in vitro. These in vivo and in vitro experiments indicate that DBP-induced collapse of Sertoli cell vimentin filaments may lead to detachment of spermatogenic cells, and then detached cells may undergo apoptosis because of loss of the support and nurture provided by Sertoli cells.

Comparative uterine gene expression analysis after dioxin and estradiol administration.

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many organisms. TCDD exposure is known to be associated with abnormal development, hepatotoxicity and endocrine effects. It has also been reported to have antiestrogenic activity in addition to estrogenic activity. In order to clarify the effects of TCDD in the uterus, we evaluated the patterns of gene expression after TCDD and estradiol administration. Of the 10 000 arrayed genes, only a few were affected by both estradiol and TCDD. Although the subset of genes that responded to estrogen was also activated by TCDD, the response to TCDD was more limited than that observed in response to estradiol. Therefore, according to our analysis of gene expression patterns, TCDD had partial and weak estrogenic activity in the uterus.

Effects of 3,3',4,4',5-pentachlorobiphenyl, a coplanar polychlorinated biphenyl congener, on cultured neonatal mouse testis.

3,3',4,4',5-Pentachlorobiphenyl (PCB126), a congener with a planar configuration, has been established to have relatively strong toxicities similar to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via aryl hydrocarbon receptors. We investigated the effects of this coplanar PCB on mammalian early spermatogenesis and steroidogenesis in a mouse neonatal testicular organ culture system. Testes collected from newborn mice were subjected to organ culture in medium containing 0, 10, 100 or 1000 nM PCB126. Histochemical analysis revealed that the BrdU-labeling indices of both spermatogenic cells and Sertoli cells were unchanged in all testis specimens exposed to the coplanar PCB. CYP1A1 and steroidogenic enzymes (P450scc, P450c17, 3beta-HSD and 17beta-HSD) mRNA levels were determined by semiquantitative RT-PCR. The CYP1A1 mRNA level in cultured testis was significantly increased by PCB126 in a dose-dependent manner. Although mRNA levels of 3beta-HSD and 17beta-HSD were unchanged, the P450scc mRNA level was significantly down-regulated by PCB126 in a dose-dependent manner. In contrast, the P450c17 mRNA level was significantly higher in 1000 nM PCB126-exposed testis than in control testis. These results suggest that the coplanar PCB does not alter the proliferative activity of spermatogenic cells and Sertoli cells in neonatal testis, but that it directly affects the expression of steroidogenic enzyme genes.

Maternal exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the development of reproductive organs of male rats: dose-dependent increase of mRNA levels of 5alpha-reductase type 2 in contrast to decrease of androgen receptor in the pubertal ventral prostate.

To assess the health risks associated with exposure to 2,3,7,8-tetrachlorodebenzo-p-dioxin (TCDD), we studied the effects of a relatively low dose of TCDD on the male reproductive system of rats, using the experimental protocol of T. A. Mably et al. (1992, Toxicol. Appl. Pharmacol. 114, 97-107, 108-117, 118-126), and searched for the most sensitive and reliable among several indices of TCDD toxicity. Pregnant Holtzman rats were given a single oral dose of 0, 12.5, 50, 200, or 800 ng TCDD/kg body weight on gestational day (GD) 15, and male offspring were sacrificed on postnatal day (PND) 49 or 120. GC-MS analysis of the abdominal fat tissue and testis clearly showed increased amounts of TCDD in these offspring. However, there was no TCDD effect on body weight of offspring. There were no changes on testicular or epididymal weights by TCDD administration, even at the 800-ng/kg dose in rats sacrificed on either PND 49 or 120. In addition, TCDD administration resulted in no changes in daily sperm production or sperm reserve at any of the doses used. However, the weight of the urogenital complex, including the ventral prostate, was significantly reduced at doses of 200 and 800 ng TCDD/kg in rats sacrificed on PND 120. Moreover, the anogenital distance (AGD) of male rats sacrificed on PND 120 showed a significant decrease in the groups receiving doses greater than 50 ng TCDD/kg. TCDD administration resulted in no apparent dose-dependent changes in levels of either serum testosterone or luteinizing hormone. Interestingly, reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that, in the ventral prostates of the PND 49 group, TCDD administration resulted in both a dose-dependent increase in 5alpha-reductase type 2 (5alphaR-II) mRNA level and a dose-dependent decrease in androgen receptor (AR) mRNA level. These results suggest that low-dose TCDD administration had a greater effect on the development of the external genital organs and ventral prostate than on development of the testis and other internal genital organs. Moreover, it is highly suggested that the decrease in the size of the ventral prostate by maternal TCDD exposure might be due to decreased responsiveness of the prostate to androgen due to an insufficient expression level of androgen receptor during puberty.

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis.

In the prespermatogenesis period, male germ cells (gonocytes) begin to reproliferate and move to the basement membrane of the seminiferous tubule. Although these two events-reproliferation and relocation-are important for establishment of spermatogenesis, they have not been greatly analyzed both in a mechanical and in an endocrine or paracrine aspect. In this study, the relationship between reproliferation and relocation of gonocytes was examined, using the thymidine analog bromodeoxyuridine (BrdU) labeling method and transmission electron microscopy (TEM). BrdU was injected into the fetuses [day 13.5 post coitus (dpc) to 18.5 dpc] and pups [day 0. 5 post partum (dpp) to 6.5 dpp] of C57BL/6J mice. Two hours later, BrdU positive gonocytes were examined immunohistochemically and these data were analyzed. TEM and LM observation was carried out as well. Gonocytes began to relocate on the basement membrane from 18.5 dpc (1.4%) while BrdU-labeled gonocytes were first detected on 1.5 dpp (13.6%). Relocated BrdU-negative gonocytes were recognized from 18.5 dpc (1.4%), and relocated BrdU-labeled gonocytes were recognized from 1.5 dpp (8.4%). On the other hand, non-relocated BrdU-labeled gonocytes were detected from 1.5 dpp (5.2%). Gonocyte relocation began 2 days earlier than reproliferation during the late fetal period. After birth, the two events occurred at random. These results indicate that the reproliferation of the gonocyte does not correlate with relocation. The two events may be regulated by different mechanisms.

[Health risk assessment of endocrine disrupting chemicals].

To assess possible health effects of endocrine disrupting chemicals(EDCs), standardized procedures for the health risk assessment of various chemicals could be employed. The procedures are comprised from 4 components: risk identification, exposure assessment, dose-response relationship and risk characterization. However, it should be born in mind that exposure assessment is particularly important to select candidate chemicals from 67 chemicals that were enlisted as suspected EDCs in the report "SPEED'98" of Environment Agency, Japan. At the same time, one should investigate low-dose effects, non-threshold mechanism and combined exposure to various EDCs since the issue of EDCs brings up a new paradigm in the filed of toxicology.

The spectrum of apoptotic defects and clinical manifestations, including systemic lupus erythematosus, in humans with CD95 (Fas/APO-1) mutations.

OBJECTIVE: To determine the clinical spectrum of disease in humans with mutations in the CD95 (Fas/ APO-1) receptor and to obtain mechanistic insight into the different clinical phenotypes observed. METHODS: Clinical information for each of the index cases, first-degree relatives, and any family members reported to have Canale-Smith syndrome (or another autoimmune disease) was gathered by direct interview, chart review, and verification of data by the physician or pathologist concerned. Apoptosis of activated T or B lymphocytes was induced by agonistic anti-CD95 antibodies and quantified by a cell death assay (propidium iodide staining in the subdiploid peak) or cell viability assay (alamar blue or 3H-thymidine incorporation). RESULTS: Evaluation of an additional 8 probands with novel heterozygous CD95 mutations revealed hypergammaglobulinemia and immune-mediated cytopenias in all patients, as well as urticarial rash, oral ulceration, lymphopenia, and peripheral neuropathy in some individuals. One patient (P4) had systemic lupus erythematosus (SLE) characterized by a World Health Organization class V lupus nephropathy, a recurrent, reversible multifocal central nervous system disorder, high-titer antiphospholipid autoantibodies, and autoimmune cytopenias. In the P4 pedigree, the father had reduced T and B cell apoptosis associated with a CD95 mutation, whereas an independent B cell apoptotic defect was demonstrated in maternal family members who did not have a CD95 mutation. Three cases of B cell lymphoma occurred in carriers of the CD95 mutation. CONCLUSIONS: CD95 mutations are associated with loss of regulation of B lymphocytes, which predisposes to systemic autoimmunity including SLE. The P4 family provides a model of the complex genetic and functional interactions that are required for the development of a lupus-like syndrome.

Dynamic expression pattern of Ca(2+)/calmodulin-dependent protein kinase II gene in the central nervous system of Drosophila throughout development.

Calcium/calmodulin-dependent protein kinase II (CaM KII) is thought to be involved in the majority of the neuronal functions mediated by intracellular free Ca(2+), and has been implicated in long-term potentiation, learning, and memory. In this work, we have examined in detail the RNA expression pattern for the Drosophila CaM KII gene by in situ hybridization, during embryonic, larval, pupal, and adult stages. Our results indicate that expression of CaM KII was homogeneous in early embryos, but that during development the gene transcription rapidly became restricted to neuroblasts and their progeny in the nervous system. This predominant expression in the nervous system is maintained during late embryogenesis and post-embryonic development. A signal compartmentalization appeared in the larval central nervous system, where the CaM KII expression became progressively concentrated in the anterior ganglia. In the adult brain, a specific expression was more abundant in a subset of neurons around the central brain, particularly the mushroom bodies and the central complex, structures that play an important role in learning and memory.

Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.

Ovalbumin was detected in developing chicken eggs. The large majority of these ovalbumin molecules was found to be in a heat-stable form reminiscent of S-ovalbumin. About 83 and 90% of the ovalbumin population was in a heat-stable form in day 14 or stage 40 amniotic fluid and day 18 or stage 44 egg yolk, respectively, whereas ovalbumin in newly deposited eggs was in the heat-unstable, native form. Purified preparations of stable ovalbumin from egg white and amniotic fluid showed a less ordered configuration than native ovalbumin, as analyzed by circular dichroism and differential scanning calorimetry. In addition, mass spectrometric analysis exhibited distinct size microheterogeneity between the stable and native forms of ovalbumin. Immunohisotochemical study revealed that ovalbumin was present in the central nervous system and other embryonic organs. These results indicated that egg white ovalbumin migrates into the developing embryo while changing its higher order structure.

Apoptosis in the effector phase of autoimmune diabetes, multiple sclerosis and thyroiditis.

The immune system is unusual in two respects. It produces billions of new cells daily that traffic throughout the body and cells within the system proliferate rapidly following exposure to an infectious agent. Both of these attributes require that cell production be regulated by cell death. Human diseases characterized by accelerated cell death leading to immunodeficiency disorders or by reduced cell death leading to systemic autoimmune diseases have been identified. In certain autoimmune diseases, the immune system directs its powerful cytotoxic effector mechanisms against specialized cells such as oligodendrocytes in multiple sclerosis, the beta cells of the pancreas in diabetes mellitus and thyrocytes in Hashimoto's thyroiditis. In this review, we examine the cytotoxic effector pathways implicated in cell death in organ specific autoimmune disorders.

Identification and characterization of a Drosophila homologue of the yeast UBC9 and hus5 genes.

The yeast UBC9 and hus5 gene products have been identified as putative E2 members of the ubiquitin-conjugating enzyme (UBC) family and have been shown to play an essential role in cell cycle progression. We have identified a Drosophila Ubc9/Hus5 homologue (termed dUBC9) in an attempt to identify proteins that interact with the amino-terminal transcriptional repression domain of the Groucho corepressor by use of the yeast two-hybrid system. The predicted dUBC9 protein consists of 159 amino acids and shows 85, 68, and 54% amino acid sequence identities with human UBC9 homologue, Schizosaccharomyces pombe Hus5, and Saccharomyces cerevisiae Ubc9 proteins, respectively. Expression of dUBC9 cDNA complements a temperature-sensitive ubc9-1 mutation of S. cerevisiae to fully restore normal growth, indicating that the dUBC9 protein can act as a substitute for the yeast Ubc9 protein. The dUBC9 transcripts were about 1.2 kb and were detected at all stages of Drosophila development and in ovaries and Schneider cells. However, an increased level was observed in early embryos and ovaries. The dUBC9 gene is present as a single copy in the genome and localized in segment 21C-D on the left arm of the second chromosome.

Characterization of domains in mice of calnexin-t, a putative molecular chaperone required in sperm fertility, with use of glutathione S-transferase-fusion proteins.

Calnexin-t (calmegin) is a male germ cell-specific variant of calnexin, a membrane bound-molecular chaperone in the endoplasmic reticulum (ER). Although it is temporally expressed during spermatogenesis, it has recently been shown to be highly involved in sperm fertility. To investigate the biochemical states of calnexin-t during spermatogenesis, we produced a series of glutathione S-transferase-fusion proteins with several specific coding domains of calnexin-t. Immunostaining and 45Ca2+ overlay assays clearly showed that the internal proline-rich repeat region has Ca2+-binding ability and contains an epitope recognized by monoclonal antibody 1C9. Western blot analysis of protein extracts from the testes of 10-, 18-, 26-, and 60-day-old mice revealed only a single 101-kDa protein during testicular development by 1C9. Anti-C, a cytoplasmic domain-specific antibody generated by immunization with recombinant protein, produced the same results, indicating that the 101-kDa form of calnexin-t is prevalent at all stages of spermatogenesis expressing calnexin-t. In paraffin sections of mouse testis, Anti-C stained spermatocytes and spermatids intensely, whereas 1C9 stained spermatocytes only slightly but spermatids intensely, suggesting that the affinity of 1C9 for its epitope is lower in pachytene spermatocytes than in spermatids. Acid phosphatase treatment of the 101-kDa form generated a 93-kDa band that in turn could be recovered to the 101-kDa form by incubation with HeLa cell S100 fraction, indicating that the 101-kDa form is a phosphorylated type of calnexin-t. The sites of phosphorylation were shown to be restricted to the cytoplasmic domain. Our results suggest that the structure of the ER luminal domain of calnexin-t is likely to differ in middle pachytene versus haploid germ cell phases. In addition, the cytoplasmic domain of calnexin-t was shown to be highly phosphorylated immediately after protein synthesis and constitutively phosphorylated during spermatogenesis.

Multivariate analysis of mandible in the Ryukyu wild pig (Sus scrofa riukiuanus).

We measured adult mandibles of Ryukyu wild pig (Sus scrofa riukiuanus) from Tokunoshima Island and compared the osteometrical data with those from six Nansei Islands. The mandibles in Tokunoshima Island were larger than those from Amami-Oshima and Okinawa Islands in some measurements. We concluded that the size cline was not statistically recognized among populations. In the principal component analysis, the size cline was also denied, and the separation could be made among island populations in female. It is suggested that the populations in Tokunoshima and Okinawa Islands may be different from those in Amami-Oshima, Kakeroma, Ishigaki and Iriomote Islands in skull proportion.

Histological changes in mouse nipple tissue during the reproductive cycle.

To obtain detailed information about the histological changes occurring in the mouse nipple during the reproductive cycle, we examined and quantified the S-phase of cell by immunohistochemical staining with bromodeoxyuridine (BrdU), and analysed histologically the subepithelial fibrous elements. The nipple markedly increased in size dramatically on days 15-18 of pregnancy. The densities of cells in the epidermis and dermis were very high during the early stages of pregnancy but low during lactation. In the epithelium of the lactiferous sinus, the densities of cells did not differ significantly among stages. The BrdU antibody labeling revealed a number of BrdU-positive cells in the basal layer of the epidermis and epithelium of the lactiferous sinus. The ratios of BrdU-positive cells to total cells in the epidermis and the epithelium of the lactiferous sinus were highest on day 15 and day 10 of pregnancy, respectively. After lactation, however, the ratios were similar to those in the virgin stage. No significant differences were detected in the dermis among all stages. The number of collagen and elastic fibers increased during lactation. These results indicate that cells in the epidermis and lactiferous sinus proliferated actively from day 10 to day 15 of pregnancy. The observation that cellular proliferation in the epithelial system of the nipple was stimulated at the early stage of pregnancy, while the dermis has two growth phases, with cellular proliferation during pregnancy and an increase in extracellular matrix during lactation, suggests that these two phenomena might be regulated by different factors.

A histometrical study on the long bones of raccoon dogs, Nyctereutes procyonoides and badgers, Meles meles.

To obtain the data required for identification of skeletal remains excavated from archaeological sites, histometrical observations were made in the cross sections of the mid-shaft of humerus, radius, femur and tibia of raccoon dogs (Nyctereutes procyonoides) and badgers (Meles meles) captured in Kagoshima Prefecture. There were interspecific differences between both animals in the breadth, the depth and the area of medullary cavity at the mid-shaft of the bones, all measurements were greater in male than in female bones. The thickness and the area of compact bones in male raccoon dogs were larger than those of female. No differences in histological structure could be detected among the bones, but an interspecific difference was found in the shape of osteons; round and constant-sized osteons consisting of 3 to 5 lamellae in raccoon dogs, while round or elliptic osteons varying in size from 3 to 8 lamellae in badgers. The ratios, the osteon areas per unit compact bone areas, were higher in all the bones of raccoon dogs. The short diameters of osteons and the ratios were greater in males in both animals. In females, the short diameter of osteons was smaller, and the number of osteons was larger. The results revealed interspecific differences between both animals and sexual dimorphism in each species.

Morphology and morphometry of skulls of raccoon dogs, Nyctereutes procyonoides and badgers, Meles meles.

In order to obtain the basic data to identify the skeletal remains from the archaeological sites, morphological and morphometrical studies were carried out on skulls of living raccoon dogs (35 males and 45 females) and badgers (16 males and 8 females) from Kagoshima Prefecture. Macroscopically, the sexual differences were observed in badgers for the parts of the zygomatic process of the temporal bone and the occipital squama, but were not in raccoon dogs. Among 24 cranial measurements, significant sexual differences were found in five measurement items in raccoon dogs, while 12 items in badgers. Mandibles showed significant sexual differences in both species. Raccoon dogs had significantly larger values than badgers in most of the items concerning length of cranium and most mandibular measurements. The discrimination efficiencies of discriminant formulae between both sexes were lower in raccoon dogs, but higher in badgers, and the efficiencies between both species were obtained 100%. In the regression formulae for estimating skull length, some formulae showed high coefficients of determination in both species. These observations represented interspecific and sexual differences in the skulls of raccoon dogs and badgers.

Histological and morphometrical studies on the rat nipple during the reproductive cycle.

Histological changes in the rat nipple during the reproductive cycle were observed. In virgin and the first half (days 5 and 10) of pregnancy, the nipple had a dull conical shape and the germinative layer of epidermis, thicker than that of the skin surrounding the nipple, deeply ingrew into the dermis in the basal region. From the second half (days 15 and 20) of pregnancy to the post-weaning period, the nipple appeared columnar in shape and many wrinkles were observed in the nipple wall especially during the lactating period. Collagen fibers longitudinally running in the nipple wall mainly comprised the dermis of the nipple and became loose during lactation. Small numbers of elastic fibers running parallel with smooth muscles were also observed in the nipple wall, and these increased in number and thickness from the second half of pregnancy, and most became frizzy structures during lactation. Around the lactiferous sinus, smooth muscle cells were arranged longitudinally but a few muscle cells were seen in a concentric layer, but during the lactating period the sinus was distended and many epithelial folds were observed. Morphometrical analysis indicated that the length of the nipple increased from the second half of pregnancy and reached the maximum on day 15 of lactation, approximately 3.7 times that in the virgin period. The outer diameter of the nipple and thickness of the nipple wall during lactation also reached approximately twice that in virgin. The size of the nipple decreased gradually after weaning. These observations suggest that the histological changes in the rat nipple during the reproductive cycle were mainly characterized by hyperplasia of the epidermis and hypertrophy of connective tissue in the dermis from the second half of pregnancy.

Comparison of the nuclear DNA stability against freezing-thawing and high temperature treatments between spermatozoa and somatic cells.

Thermostability of sperm genome against freezing-thawing and high temperature treatments was assessed by comparing the degradation patterns of genomic DNAs from epididymal sperms and somatic tissues. Golden hamster liver, kidney, epididymal sperm, and testis were frozen and thawed repeatedly, or incubated in a hot water bath. Genomic DNAs were isolated and then separated by agarose gel electrophoresis. It was revealed that the size of sperm genomic DNA was hardly changed after freezing-thawing treatment, however, the DNA sizes of the other three tissues were gradually reduced with an increasing number of freezing-thawing cycles. In contrast, high temperature treatment appears to damage not only the genomic DNAs of somatic cells but also those of spermatozoa.

The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain.

Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.