Morc1 reestablishes H3K9me3 heterochromatin on piRNA-targeted transposons in gonocytes.

To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltransferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified.

Partial male-to-female reprogramming of mouse fetal testis by Sertoli cell ablation.

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions: near the testicular surface epithelia and around its adjacent mesonephros. The surface FOXL2-positive cells, together with ectopic expression of Lgr5 and Gng13 (markers of ovarian cords), were derived from the testis epithelia/subepithelia, whereas another FOXL2-positive population was the 3ßHSD-negative stroma near the mesonephros. In addition to high expression of Fgfr1/Fgfr2 and heparan sulfate proteoglycan (a reservoir for FGF ligand) in these two sites, exogenous FGF9 additives repressed DT-dependent Foxl2 upregulation in Tg testes. These findings imply retention of Foxl2 inducibility in the surface epithelia and peri-mesonephric stroma of the testicular parenchyma, in which certain paracrine signals, including FGF9 derived from fetal Sertoli cells, repress feminization in these two sites of the early fetal testis.

Identification and characterization of dystrophin-locus-derived testis-specific protein: A testis-specific gene within the intronic region of the rat dystrophin gene.

The mammalian X chromosome exhibits enrichment in genes associated with germ cell development. Previously, we generated a rat model of Becker muscular dystrophy (BMD) characterized by an in-frame mutation in the dystrophin gene, situated on the X chromosome and responsible for encoding a protein crucial for muscle integrity. Male BMD rats are infertile owing to the absence of normal spermatids in the epididymis. Within the seminiferous tubules of BMD rats, elongated spermatids displayed abnormal morphology. To elucidate the cause of infertility, we identified a putative gene containing an open reading frame situated in the intronic region between exons 6 and 7 of the dystrophin gene, specifically deleted in male BMD rats. This identified gene, along with its encoded protein, exhibited specific detection within the testes, exclusively localized in round to elongated spermatids during spermiogenesis. Consequently, we designated the encoded protein as dystrophin-locus-derived testis-specific protein (DTSP). Given the absence of DTSP in the testes of BMD rats, we hypothesized that the loss of DTSP contributes to the infertility observed in male BMD rats.

Gonadal Sex Differentiation and Ovarian Organogenesis along the Cortical-Medullary Axis in Mammals.

In most mammals, the sex of the gonads is based on the fate of the supporting cell lineages, which arises from the proliferation of coelomic epithelium (CE) that surfaces on the bipotential genital ridge in both XY and XX embryos. Recent genetic studies and single-cell transcriptome analyses in mice have revealed the cellular and molecular events in the two-wave proliferation of the CE that produce the supporting cells. This proliferation contributes to the formation of the primary sex cords in the medullary region of both the testis and the ovary at the early phase of gonadal sex differentiation, as well as to that of the secondary sex cords in the cortical region of the ovary at the perinatal stage. To support gametogenesis, the testis forms seminiferous tubules in the medullary region, whereas the ovary forms follicles mainly in the cortical region. The medullary region in the ovary exhibits morphological and functional diversity among mammalian species that ranges from ovary-like to testis-like characteristics. This review focuses on the mechanism of gonadal sex differentiation along the cortical-medullary axis and compares the features of the cortical and medullary regions of the ovary in mammalian species.

Sox17 is essential for proper formation of the marginal zone of extraembryonic endoderm adjacent to a developing mouse placental disk.

In mouse conceptus, two yolk-sac membranes, the parietal endoderm (PE) and visceral endoderm (VE), are involved in protecting and nourishing early-somite-stage embryos prior to the establishment of placental circulation. Both PE and VE membranes are tightly anchored to the marginal edge of the developing placental disk, in which the extraembryonic endoderm (marginal zone endoderm: ME) shows the typical flat epithelial morphology intermediate between those of PE and VE in vivo. However, the molecular characteristics and functions of the ME in mouse placentation remain unclear. Here, we show that SOX17, not SOX7, is continuously expressed in the ME cells, whereas both SOX17 and SOX7 are coexpressed in PE cells, by at least 10.5 days postconception. The Sox17-null conceptus, but not the Sox7-null one, showed the ectopic appearance of squamous VE-like epithelial cells in the presumptive ME region, together with reduced cell density and aberrant morphology of PE cells. Such aberrant ME formation in the Sox17-null extraembryonic endoderm was not rescued by the chimeric embryo replaced with the wild-type gut endoderm by the injection of wild-type ES cells into the Sox17-null blastocyst, suggesting the cell autonomous defects in the extraembryonic endoderm of Sox17-null concepti. These findings provide direct evidence of the crucial roles of SOX17 in proper formation and maintenance of the ME region, highlighting a novel entry point to understand the in vivo VE-to-PE transition in the marginal edge of developing placenta.

Regionally distinct patterns of STAT3 phosphorylation in the seminiferous epithelia of mouse testes.

In mouse testes, Sertoli cells support the continuous process of spermatogenesis, which is dependent on seminiferous epithelial cycles along the longitudinal axis of the seminiferous tubule. Sertoli cell function is modulated partly by local cytokines and/or growth factors derived from adjacent tissues such as blood vessels, macrophages, rete testis, etc. However, the spatial activation patterns by local signals in vivo remain unclear. In this study, we focused on Signal Transducers and Activators of Transcription (STAT) signaling in Sertoli cells, because STAT is a major crucial cytokine transducer for somatic cyst cell regulation in Drosophila testis niches. In mouse testes, STAT3 was ubiquitously expressed in Sertoli cells throughout the seminiferous tubules. Phosphorylated STAT3 (p-STAT3) was predominantly observed in the Sertoli cells within the valve-like structure adjacent to the rete testis (i.e., the Sertoli valve [SV]) in the terminal segment of the proximal seminiferous tubules. In the distal seminiferous tubules with active spermatogenesis, most Sertoli cells were negative for anti-p-STAT3 staining. Albeit rarely, a small patch of several p-STAT3-positive Sertoli cells was detected frequently in seminiferous epithelial cycle stages I-VI. Such p-STAT3-positive ratios in the convoluted seminiferous epithelia were significantly increased in germ cell-less testes than in the wild-type testes, but with considerably lower ratios than in the SV region. These findings imply that regionally distinct patterns of STAT3 phosphorylation in the Sertoli cells depend on either location or spermatogenic activity in normal healthy testes in vivo, highlighting a novel entry point to understanding STAT signaling in mammalian spermatogenesis.

CRISPR/Cas9-mediated knock-in of the murine Y chromosomal Sry gene.

Mammalian zygote-mediated genome editing via the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system is widely used to generate genome-modified animals. This system allows for the production of loss-of-function mutations in various Y chromosome genes, including Sry, in mice. Here, we report the establishment of a CRISPR-Cas9-mediated knock-in line of Flag-tag sequences into the Sry locus at the C-terminal coding end of the Y chromosome (YSry-flag). In the F1 and successive generations, all male pups carrying the YSry-flag chromosome had normal testis differentiation and proper spermatogenesis at maturity, enabling complete fertility and the production of viable offspring. To our knowledge, this study is the first to produce a stable Sry knock-in line at the C-terminal region, highlighting a novel approach for examining the significance of amino acid changes at the naive Sry locus in mammals.

Heterogeneity in sexual bipotentiality and plasticity of granulosa cells in developing mouse ovaries.

In mammalian sex determination, SRY directly upregulates the expression of SOX9, the master regulatory transcription factor in Sertoli cell differentiation, leading to testis formation. Without SRY action, the bipotential gonadal cells become pre-granulosa cells, which results in ovarian follicle development. When, where and how pre-granulosa cells are determined to differentiate into developing ovaries, however, remains unclear. By monitoring SRY-dependent SOX9 inducibility (SDSI) in an Sry-inducible mouse system, we were able to identify spatiotemporal changes in the sexual bipotentiality/plasticity of ovarian somatic cells throughout life. The early pre-granulosa cells maintain the SDSI until 11.5 d.p.c., after which most pre-granulosa cells rapidly lose this ability by 12.0 d.p.c. Unexpectedly, we found a subpopulation of the pre-granulosa cells near the mesonephric tissue that continuously retains SDSI throughout fetal and early postnatal stages. After birth, these SDSI-positive pre-granulosa cells contribute to the initial round of folliculogenesis by the secondary follicle stage. In experimental sex reversal of 13.5-d.p.c. ovaries grafted into adult male nude mice, the differentiated granulosa cells re-acquire the SDSI before other signs of masculinization. Our data provide direct evidence of an unexpectedly high sexual heterogeneity of granulosa cells in developing mouse ovaries in a stage- and region-specific manner. Discovery of such sexually bipotential granulosa cells provides a novel entry point to the understanding of masculinization in various cases of XX disorders of sexual development in mammalian ovaries.

Visceral adipose tissue-derived serine proteinase inhibitor inhibits apoptosis of endothelial cells as a ligand for the cell-surface GRP78/voltage-dependent anion channel complex.

RATIONALE: Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified from visceral adipose tissues of genetically obese rats. OBJECTIVE: The role of vaspin in the diabetic vascular complications remains elusive, and we investigated the effects of vaspin on the vascular function under the diabetic milieu. METHODS AND RESULTS: Adenovirus carrying the full length of the vaspin gene (Vaspin-Ad) ameliorated intimal proliferation of balloon-injured carotid arteries in diabetic Wistar rats. The expression of Ccl2, Pdgfb, and Pdgfrb genes was significantly reduced by the treatment of Vaspin-Ad. In cuff-injured femoral arteries, the intimal proliferation was ameliorated in vaspin transgenic (Vaspin Tg) mice. The application of recombinant vaspin and Vaspin-Ad promoted the proliferation and inhibited the apoptosis of human aortic endothelial cells. Adenovirus expressing vaspin with calmodulin and streptavidin-binding peptides was applied to human aortic endothelial cells, subjected to tandem tag purification and liquid chromatography-tandem mass spectrometry, and we identified GRP78 (78-kDa glucose-regulated protein) as an interacting molecule. The complex formation of vaspin, GRP78, and voltage-dependent anion channel on the plasma membrane was confirmed by the immunoprecipitation studies using aortas of Vaspin Tg mice. The binding assay using (125)I-vaspin in human aortic endothelial cells revealed high-affinity binding (dissociation constant = 0.565×10(-9) m) by the treatment of 5 µM thapsigargin, which recruited GRP78 from the endoplasmic reticulum to plasma membrane by inducing endoplasmic reticulum stress. In human aortic endothelial cells, vaspin induced phosphorylation of Akt and inhibited the kringle 5-induced Ca(2+) influx and subsequent apoptosis. CONCLUSIONS: Vaspin is a novel ligand for the cell-surface GRP78/voltage-dependent anion channel complex in endothelial cells and promotes proliferation, inhibits apoptosis, and protects vascular injuries in diabetes mellitus.

External genitalia phenotypes of a Mab21l1-null mouse model for cerebellar, ocular, craniofacial, and genital (COFG) syndrome.

The cerebellar, ocular, craniofacial, and genital (COFG) syndrome is a human genetic disease that is caused by MAB21L1 mutations. A COFG mouse model with Mab21l1-null mutation causes severe microphthalmia and fontanelle dysosteogenesis, similar to the symptoms in human patients. One of the typical symptoms is scrotal agenesis in male infants, while male Mab21l1-null mice show hypoplastic preputial glands, a rodent-specific derivative of the cranial scrotal fold. However, it is still unclear where and how MAB21Ll acts in the external genitalia in both mice and humans. Here we show that, at the neonatal stage, MAB21L1 expression in the external genitalia was restricted to two mesenchymal cell populations-underneath the scrotal and labial skin and around the preputial and clitoral glands (PG/CG). Morphometric analyses of the Mab21l1-/- pups revealed a significant reduction in the external size of the scrotum, vulva, and CG, as well as PG. In the periglandular region around PG and CG, the periglandular mesenchymal cells showed a drastic reduction in both cell density and immunoreactive signals for several extracellular matrix proteins (e.g., collagen I, fibronectin, and proteoglycans), together with their reduced Ki67-positive cell proliferation index. In the Mab21l1-/- PG/CG, together with reduced vascularization, the glandular epithelia displayed atrophy with discontinuous basal lamina along the basal surface and defective glycogen accumulation in their cytoplasm. Under a 5-day organ culture of the isolated PG, the Mab21l1-/- explants showed poor outgrowth and retention of the glandular structure in vitro. However, the addition of exogenous Matrigel could partially rescue such tissue-autonomous phenotypes, showing glandular morphology similar to that of the wild-type explants. These findings suggest that MAB21L1+ mesenchymal cells play a crucial role in providing nutrient ECM support for glandular outgrowth and morphogenesis in the peripheral external genitalia.

Impact of gallbladder hypoplasia on hilar hepatic ducts in biliary atresia.

BACKGROUND: Biliary atresia (BA) is an intractable disease of unknown cause that develops in the neonatal period. It causes jaundice and liver damage due to the destruction of extrahepatic biliary tracts,. We have found that heterozygous knockout mice of the SRY related HMG-box 17 (Sox17) gene, a master regulator of stem/progenitor cells in the gallbladder wall, exhibit a condition like BA. However, the precise contribution of hypoplastic gallbladder wall to the pathogenesis of hepatobiliary disease in Sox17 heterozygous embryos and human BA remains unclear. METHODS: We employed cholangiography and histological analyses in the mouse BA model. Furthermore, we conducted a retrospective analysis of human BA. RESULTS: We show that gallbladder wall hypoplasia causes abnormal multiple connections between the hilar hepatic bile ducts and the gallbladder-cystic duct in Sox17 heterozygous embryos. These multiple hilar extrahepatic ducts fuse with the developing intrahepatic duct walls and pull them out of the liver parenchyma, resulting in abnormal intrahepatic duct network and severe cholestasis. In human BA with gallbladder wall hypoplasia (i.e., abnormally reduced expression of SOX17), we also identify a strong association between reduced gallbladder width (a morphometric parameter indicating gallbladder wall hypoplasia) and severe liver injury at the time of the Kasai surgery, like the Sox17-mutant mouse model. CONCLUSIONS: Together with the close correlation between gallbladder wall hypoplasia and liver damage in both mouse and human cases, these findings provide an insight into the critical role of SOX17-positive gallbladder walls in establishing functional bile duct networks in the hepatic hilus of neonates.

Biliary atresia (BA) is a disease in newborns that causes a serious liver condition due to damage to the bile ducts (the pathways that carry bile juice). Although reduced function of a key gene called Sox17, which is essential for forming the gallbladder wall, has been observed in some BA cases, the link between gallbladder issues and liver damage is unknown. This study has shown how damage spreads through the bile ducts in the liver around the time of birth when there are problems in the gallbladder wall due to reduced SOX17 function. The findings indicate that proper growth of the gallbladder wall during this critical period is essential for forming a normal network of bile ducts in the developing liver. This discovery is promising for early diagnosis and better treatment of BA in newborns.

FGF signaling directs a center-to-pole expansion of tubulogenesis in mouse testis differentiation.

In mouse embryogenesis, Sry is transiently activated in a center-to-pole wavelike manner along the anteroposterior (AP) axis of developing XY gonads. However, the mechanism and significance of the center-to-pole expansion of testis initiation pathways downstream of Sry expression remain unclear. Here we demonstrate that FGF9 can act as a diffusible conductor for a poleward expansion of tubulogenic programs at early phases of testis differentiation. In XY genital ridge cultures of anterior, middle and posterior segments at 11.0-11.25 days post-coitum, male-specific activation of Sry and its target gene, Sox9, was still observed in both anterior and posterior pole segments despite their isolation from the central domain. However, high-level Sox9 expression was not maintained, resulting in the failure of testis cord organization in most pole segments. A reconstruction experiment using ROSA:lacZ middle segments showed rescue of the tubulogenic defect in the poles without any appreciable contribution of lacZ-positive gonadal parenchyma cells. A partition culture assay also showed a possible contribution of soluble/diffusible factors secreted from the gonadal center domain to proper tubulogenesis in the poles. Among various signaling factors, Fgf9 expression was significantly lower in both anterior and posterior pole segments than in the central domain. The supportive role of the central domain could be substituted by exogenous FGF9 supply, whereas reduction of Wnt4 activity did not rescue the tubulogenesis defect in the pole segments. These observations imply that center-to-pole FGF9 diffusion directs a poleward expansion of testiculogenic programs along the AP axis of developing XY gonads.

Overexpression of KLF15 transcription factor in adipocytes of mice results in down-regulation of SCD1 protein expression in adipocytes and consequent enhancement of glucose-induced insulin secretion.

Krüppel-like factor 15 (KLF15), a member of the Krüppel-like factor family of transcription factors, has been found to play diverse roles in adipocytes in vitro. However, little is known of the function of KLF15 in adipocytes in vivo. We have now found that the expression of KLF15 in adipose tissue is down-regulated in obese mice, and we therefore generated adipose tissue-specific KLF15 transgenic (aP2-KLF15 Tg) mice to investigate the possible contribution of KLF15 to various pathological conditions associated with obesity in vivo. The aP2-KLF15 Tg mice manifest insulin resistance and are resistant to the development of obesity induced by maintenance on a high fat diet. However, they also exhibit improved glucose tolerance as a result of enhanced insulin secretion. Furthermore, this enhancement of insulin secretion was shown to result from down-regulation of the expression of stearoyl-CoA desaturase 1 (SCD1) in white adipose tissue and a consequent reduced level of oxidative stress. This is supported by the findings that restoration of SCD1 expression in white adipose tissue of aP2-KLF15 Tg mice exhibited increased oxidative stress in white adipose tissue and reduced insulin secretion with hyperglycemia. Our data thus provide an example of cross-talk between white adipose tissue and pancreatic ß cells mediated through modulation of oxidative stress.

Brd2 is required for cell cycle exit and neuronal differentiation through the E2F1 pathway in mouse neuroepithelial cells.

To understand genetic programs controlling mammalian central nervous system (CNS) development, we have identified one transgene-inserted mutation, which showed embryonic lethality during neurulation. Determination of the transgene integration site and rescue experiments revealed that the Brd2 gene, whose products specifically bind acetylated histone H4 and can mediate transcription, was the cause of this mutation. Expression studies with specific markers demonstrated that cell cycle progression was accelerated and neuronal differentiation as well as cell cycle exit were impaired in Brd2-deficient neruoepithelial cells. To investigate whether Brd2 regulates neuronal differentiation through a E2F1 transcriptional factor, which directly binds Brd2 and controls genes expression for cell cycle progression and exit, we analyzed Brd2;E2F1 double mutant phenotypes and, consequently found that abnormalities in neuronal differentiation and cell cycle progression due to Brd2-deficiency were restored by removing the E2F1 gene. These findings suggest that Brd2 is required for cell cycle exit and neuronal differentiation of neuroepithelial cells through the E2F1 pathway during mouse CNS development.

Evidence for almost complete sex-reversal in bovine freemartin gonads: formation of seminiferous tubule-like structures and transdifferentiation into typical testicular cell types.

During mammalian sex determination of XY fetuses, SRY induces SOX9 in Sertoli cells, resulting in formation of testes with seminiferous tubules, interstitial Leydig cells and peritubular myoid cells. Meanwhile XX fetuses without SRY develop ovaries. In cattle, most XX heifers born with a male twin, so-called freemartins, develop nonfunctioning ovaries and genitalia with an intersex phenotype. Interestingly, freemartins sometimes develop highly masculinized gonads with seminiferous tubule-like structures despite the absence of SRY. However, in these cases, the degree of masculinization in each gonadal somatic cell type is unclear. Here, we report a rare case of a freemartin Japanese black calf with almost complete XX sexreversal. Gross anatomical analysis of this calf revealed the presence of a pair of small testis-like gonads with rudimentary epididymides, in addition to highly masculinized genitalia including a pampiniform plexus, scrotum and vesicular gland. Histological and immunohistochemical analyses of these masculinized gonads revealed well-defined seminiferous tubule-like structures throughout the whole gonadal parenchyma. In epithelia of these tubules, SOX9-positive supporting cells (i.e., Sertoli cells) were found to be arranged regularly along the bases of tubules, and they were also positive for GDNF, one of the major factors for spermatogenesis. 3ß-HSD-positive cells (i.e., Leydig cells) and SMA-positive peritubular myoid cells were also identified around tubules. Therefore, for the first time, we found the transdifferentiation of ovarian somatic cells into all testicular somatic cell types in the XX freemartin gonads. These data strongly support the idea of a high sexual plasticity in the ovarian somatic cells of mammalian gonads.

Clinical effects of wasabi extract containing 6-MSITC on myalgic encephalomyelitis/chronic fatigue syndrome: an open-label trial.

BACKGROUND: Wasabi (Eutrema japonicum) is a common pungent spice used in Japan. 6-Methylsulfinylhexyl isothiocyanate (6-MSITC) found in the rhizome of wasabi has been shown to have anti-inflammatory and antioxidant effects, as well as improve neuroinflammation and memory. Therefore, we hypothesized that these effects would be beneficial for treating myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The present study was conducted to investigate the effectiveness of wasabi extract containing 6-MSITC on ME/CFS in an open-label trial. METHODS: Fifteen patients (3 males, 12 females, 20-58 years old) were orally administered wasabi extract (9.6 mg of 6-MSITC/day) for 12 weeks. The following parameters and test results were compared pre- and post-treatment: performance status (PS), self-rating questionnaires, pressure pain threshold (PPT) on the occiput, Trail Making test-A (TMT-A), and hemodynamic patterns determined by an active standing test. RESULTS: After treatment with 6-MSITC, PS improved significantly (p = 0.001). Although the scores on the 11-item Chalder Fatigue scale (CFS-11) and numerical rating scale (NRS) of fatigue did not show significant changes, subjective symptoms improved significantly, including headache frequency (4.1 to 3.0 times/week, p = 0.001) and myalgia (4.1 to 2.4 times/week, p = 0.019), NRS brain fog scores (5.7 to 4.5, p = 0.011), difficulty finding appropriate words (4.8 to 3.7, p = 0.015), photophobia (4.8 to 3.5, p = 0.008), and the Profile of Mood Status vigor score (46.9 to 50.0, p = 0.045). The PPT of the right occiput (17.3 to 21.3 kPa, p = 0.01) and TMT-A scores (53.0 to 38.1 s, p = 0.007) also changed, suggesting reduced pain sensitivity, and improved cognitive function, respectively. Orthostatic patterns determined by a standing test did not show remarkable changes. There were no serious adverse reactions. CONCLUSION: This study suggests that 6-MSITC improves PS as well as subjective symptoms such as pain and cognitive dysfunction, and psychological vitality of patients with ME/CFS. It also improved cognitive performance and increased pain thresholds in these patients. 6-MSITC may be a promising therapeutic option especially for improving cognitive dysfunction associated with ME/CFS.

SOX17-positive rete testis epithelium is required for Sertoli valve formation and normal spermiogenesis in the male mouse.

Seminiferous tubules (STs) in the mammalian testes are connected to the rete testis (RT) via a Sertoli valve (SV). Spermatozoa produced in the STs are released into the tubular luminal fluid and passively transported through the SV into the RT. However, the physiological functions of the RT and SV remain unclear. Here, we identified the expression of Sox17 in RT epithelia. The SV valve was disrupted before puberty in RT-specific Sox17 conditional knockout (Sox17-cKO) male mice. This induced a backflow of RT fluid into the STs, which caused aberrant detachment of immature spermatids. RT of Sox17-cKO mice had reduced expression levels of various growth factor genes, which presumably support SV formation. When transplanted next to the Sox17+ RT, Sertoli cells of Sox17-cKO mice reconstructed the SV and supported proper spermiogenesis in the STs. This study highlights the novel and unexpected modulatory roles of the RT in SV valve formation and spermatogenesis in mouse testes, as a downstream action of Sox17.

Role of hepatic STAT3 in brain-insulin action on hepatic glucose production.

STAT3 regulates glucose homeostasis by suppressing the expression of gluconeogenic genes in the liver. The mechanism by which hepatic STAT3 is regulated by nutritional or hormonal status has remained unknown, however. Here, we show that an increase in the plasma insulin concentration, achieved either by glucose administration or by intravenous insulin infusion, stimulates tyrosine phosphorylation of STAT3 in the liver. This effect of insulin was mediated by the hormone's effects in the brain, and the increase in hepatic IL-6 induced by the brain-insulin action is essential for the activation of STAT3. The inhibition of hepatic glucose production and of expression of gluconeogenic genes induced by intracerebral ventricular insulin infusion was impaired in mice with liver-specific STAT3 deficiency or in mice with IL-6 deficiency. These results thus indicate that IL-6-STAT3 signaling in the liver contributes to insulin action in the brain, leading to the suppression of hepatic glucose production.

FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets.

White adipocytes are unique in that they contain large unilocular lipid droplets that occupy most of the cytoplasm. To identify genes involved in the maintenance of mature adipocytes, we expressed dominant-negative PPARgamma in 3T3-L1 cells and performed a microarray screen. The fat-specific protein of 27 kDa (FSP27) was strongly downregulated in this context. FSP27 expression correlated with induction of differentiation in cultured preadipocytes, and the protein localized to lipid droplets in murine white adipocytes in vivo. Ablation of FSP27 in mice resulted in the formation of multilocular lipid droplets in these cells. Furthermore, FSP27-deficient mice were protected from diet-induced obesity and insulin resistance and displayed an increased metabolic rate due to increased mitochondrial biogenesis in white adipose tissue (WAT). Depletion of FSP27 by siRNA in murine cultured white adipocytes resulted in the formation of numerous small lipid droplets, increased lipolysis, and decreased triacylglycerol storage, while expression of FSP27 in COS cells promoted the formation of large lipid droplets. Our results suggest that FSP27 contributes to efficient energy storage in WAT by promoting the formation of unilocular lipid droplets, thereby restricting lipolysis. In addition, we found that the nature of lipid accumulation in WAT appears to be associated with maintenance of energy balance and insulin sensitivity.

Role of STAT-3 in regulation of hepatic gluconeogenic genes and carbohydrate metabolism in vivo.

The transcription factor, signal transducer and activator of transcription-3 (STAT-3) contributes to various physiological processes. Here we show that mice with liver-specific deficiency in STAT-3, achieved using the Cre-loxP system, show insulin resistance associated with increased hepatic expression of gluconeogenic genes. Restoration of hepatic STAT-3 expression in these mice, using adenovirus-mediated gene transfer, corrected the metabolic abnormalities and the alterations in hepatic expression of gluconeogenic genes. Overexpression of STAT-3 in cultured hepatocytes inhibited gluconeogenic gene expression independently of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), an upstream regulator of gluconeogenic genes. Liver-specific expression of a constitutively active form of STAT-3, achieved by infection with an adenovirus vector, markedly reduced blood glucose, plasma insulin concentrations and hepatic gluconeogenic gene expression in diabetic mice. Hepatic STAT-3 signaling is thus essential for normal glucose homeostasis and may provide new therapeutic targets for diabetes mellitus.