Although di-n-butyl phthalate (DBP) induces germ cell apoptosis, the underlying mechanism is not yet clear in quail. In this study, prepubertal quails were given a single dose of 500mg kg-1 DBP by gavage and were then killed 3, 6 and 24h after treatment. There was a significant reduction in intratesticular testosterone (ITT) concentrations and testicular steroidogenic enzyme mRNA expression and a significant increase in germ cell apoptosis in DBP-treated compared with control quails at all time points. Maximum apoptosis was detected 6h after treatment and the maximum reduction in testosterone concentrations was at 3h. To investigate whether DBP suppressed testicular steroidogenesis by affecting the hypothalamic-pituitary-testicular axis, we analysed pituitary LH subunit β (Lhb) mRNA expression and serum LH concentrations. At all time points, pituitary Lhb expression and serum LH concentrations were significantly decreased following DBP treatment. The present observations suggest the possibility that DBP blocked LH secretion from the hypothalamus and/or pituitary, thereby decreasing LH stimulation of Leydig cells and reducing ITT concentrations. DBP-induced decreases in ITT concentrations may cause changes to the physical structure of Sertoli cells, which, in turn, may induce germ cell apoptosis.

In mammalian testes, undifferentiated spermatogonia (Aundiff) undergo differentiation in response to retinoic acid (RA), while their progenitor states are partially maintained by fibroblast growth factors (FGFs). Sertoli valve (SV) is a region located at the terminal end of seminiferous tubule (ST) adjacent to the rete testis (RT), where the high density of Aundiff is constitutively maintained with the absence of active spermatogenesis. However, the molecular and cellular characteristics of SV epithelia still remain unclear. In this study, we first identified the region-specific AKT phosphorylation in the SV Sertoli cells and demonstrated non-cell autonomous specialization of Sertoli cells in the SV region by performing a Sertoli cell ablation/replacement experiment. The expression of Fgf9 was detected in the RT epithelia, while the exogenous administration of FGF9 caused ectopic AKT phosphorylation in the Sertoli cells of convoluted ST. Furthermore, we revealed the SV region-specific expression of Cyp26a1, which encodes an RA-degrading enzyme, and demonstrated that the increased RA levels in the SV region disrupt its pool of Aundiff by inducing their differentiation. Taken together, RT-derived FGFs and low levels of RA signaling contribute to the non-cell-autonomous regionalization of the SV epithelia and its local maintenance of Aundiff in the SV region.

The postnatal testicular development and actin distribution in the seminiferous epithelium were examined by light microscopy, using the testes of the Habu (Trimeresurus flavoviridis; snake) from 0-year-old to 3-year-old. At 0-year-old (about 1 month after birth), the testis was quite small in size, and the seminiferous epithelium was composed of only Sertoli cells and large spermatogonia. Actin immunoreactivity was observed in the peritubular myoid cells, but could not be detected in the seminiferous epithelium. At 1-year-old (about 10 months after birth), the testicular size increased to a great degree. In the seminiferous epithelium, spermatocytes newly appeared. Actin could still not be detected in the seminiferous epithelium. At 2-year-old (about 1 year and 10 months after birth), the testes continued to develop in size. In the seminiferous epithelium, elongate spermatids and round spermatids were frequently seen, in addition to Sertoli cells, spermatogonia and spermatocytes. Thus, active spermatogenesis was clearly recognized at this age. Moreover, the actin distribution in the seminiferous epithelium was observed at the site between Sertoli cells and spermatids, as well as that at adult stage. The immunoreactivity of actin in the peritubular myoid cells gradually increased from 0-year-old to 2-year-old. Conclusively, it seems likely that spermatogenesis in the Habu initiates at 2-year-old, accompanying with the appearance of actin in the seminiferous epithelium.

Sialic acids (Sia) are terminal components of glycoconjugates that are involved in molecular and cellular interactions in the olfactory system. Diverse glycoconjugates are expressed in the salamander olfactory projection; however, their sialylation and the linkage of Sia to underlying sugars remain largely unknown. The present study aimed to determine the expression of Sia linked to galactose (Gal)-N-acetylglucosamine and N-acetylgalactosamine (GalNAc) in the olfactory bulbs of three species of salamanders using lectin binding. Abundant distribution of sialoglycoconjugates was observed in the salamander olfactory bulb by lectins, Sambucus sieboldiana (SSA) and Maackia amurensis (MAM). Moreover, SSA and MAM showed heterogeneous bindings in the primary olfactory projection of Cynops pyrrhogaster and C. orientalis. Lectin reactivities obviously decreased in all layers of the olfactory bulb after sialidase digestion, indicating selective binding to sialoglycoconjugates. Next, we examined the expression of the subterminal sugar residues, Gal and GalNAc, after terminal Sia removal. Desialylation in the olfactory bulb enhanced the reactivity of Jacalin and Vicia villosa (VVA) lectins that recognize Gal and GalNAc respectively. Together with the binding of SSA and MAM, Sia linked to Gal and GalNAc might be a major component of sialoglycoconjugates in the salamander olfactory projection.

Organic cation transporters (OCTs) are poly-specific carriers for endogenous and exogenous cationic compounds. These are widely distributed in the nervous system and mediate neuronal activities. As antineoplastic cationic drugs accumulate in the dorsal root ganglion (DRG), OCT function has been studied mainly in cultured DRG neurons. However, the histological distribution of OCTs in the DRG is unclear. This study investigated the localization of OCT2 (a member of OCTs) in mouse DRG neurons and determined their histochemical properties. OCT2 expression was found in about 20% of DRG neurons, which were small to medium size. OCT2-expressing neurons were labeled with markers for peptidergic nociceptive (substance P or calcitonin gene-related peptide) and tactile/proprioceptive (neurofilament 200 or tropomyosin receptor kinase B or C) neurons. OCT2 was also expressed in cholinergic DRG neurons identified by choline acetyltransferase promoter-derived Cre expression. In the spinal dorsal horn, OCT2 was distributed in superficial to deep laminae. OCT2 immunoreactivity was punctate in appearance and localized in the nerve terminals of sensory afferents with labeling of neurochemical markers. Our findings suggest that OCT2 as a low-affinity, high-capacity carrier may take up substrates including cationic neurotransmitters and drugs from the extracellular space around cell bodies in DRG neurons.

Biliary atresia (BA) is a rare neonatal disease characterized by inflammation and obstruction of the extrahepatic bile ducts (EHBDs). The Sox17-haploinsufficient (Sox17+/- ) mouse is an animal model of BA that encompasses bile duct injury and subsequent BA-like inflammation by the neonatal stage. Most Sox17+/- neonates die soon after birth, but some Sox17+/- pups reach adulthood and have a normal life span, unlike human BA. However, the phenotype and BA-derived scars in the hepatobiliary organs of surviving Sox17+/- mice are unknown. Here, we examined the phenotypes of the hepatobiliary organs in post-weaning and young adult Sox17+/- mice. The results confirmed the significant reduction in liver weight, together with peripheral calcinosis and aberrant vasculature in the hepatic lobule, in surviving Sox17+/- mice as compared with their wild-type (WT) littermates. Such hepatic phenotypes may be sequelae of hepatobiliary damage at the fetal and neonatal stages, a notion supported by the slight, but significant, increases in the levels of serum markers of liver damage in adult Sox17+/- mice. The surviving Sox17+/- mice had a shorter gallbladder in which ectopic hepatic ducts were more frequent compared to WT mice. Also, the surviving Sox17+/- mice showed neither obstruction of the EHBDs nor atrophy or inflammation of hepatocytes or the intrahepatic ducts. These data suggest that some Sox17+/- pups with BA naturally escape lethality and recover from fetal hepatobiliary damages during the perinatal period, highlighting the usefulness of the in vivo model in understanding the hepatobiliary healing processes after surgical restoration of bile flow in human BA.

The distribution of actin filaments was examined in the seminiferous epithelium of the Habu (Trimeresurus flavoviridis; snake), by transmission electron microscopy and fluorescence histochemistry. By transmission electron microscopy, actin filaments were clearly found only at the site between Sertoli cell and spermatid without a lattice-like structure. Fluorescence histochemistry showed a weak labelling of actin filaments in the seminiferous epithelium, whereas these findings seem to be common among reptiles, they are different from those in mammals. Additionally, the bundles of actin filaments adjacent to the plasma membrane of Sertoli cells, appeared in other reptiles, were not observed in the Habu.

In most of mammalian embryos, gonadal sex differentiation occurs inside the maternal uterus before birth. In several fetal ovarian grafting experiments using male host mice, an experimental switch from the maternal intrauterine to male-host environment gradually induces partial masculinization of the grafted ovaries even under the wild-type genotype. However, either host-derived factors causing or molecular basis underlying this masculinization of the fetal ovaries are not clear. Here, we demonstrate that ectopic appearance of SOX9-positive Sertoli cell-like cells in grafted ovaries was mediated by the testosterone derived from the male host. Neither Sox8 nor Amh activity in the ovarian tissues is essential for such ectopic appearance of SOX9-positive cells. The transcriptome analyses of the grafted ovaries during this masculinization process showed early downregulation of pro-ovarian genes such as Irx3, Nr0b1/Dax1, Emx2, and Fez1/Lzts1 by days 7-10 post-transplantation, and subsequent upregulation of several pro-testis genes, such as Bhlhe40, Egr1/2, Nr4a2, and Zc3h12c by day 20, leading to a partial sex reversal with altered expression profiles in one-third of the total numbers of the sex-dimorphic pre-granulosa and Sertoli cell-specific genes at 12.5 dpc. Our data imply that the paternal testosterone exposure is partially responsible for the sex-reversal expression profiles of certain pro-ovarian and pro-testis genes in the fetal ovaries in a temporally dependent manner.

Diverse glycoconjugates are expressed in the vertebrate olfactory bulb and serve as guidance cues for axons of nasal receptor neurons. Although the involvement of glycoconjugates in the segregation of the olfactory pathway has been suggested, it is poorly understood in salamanders. In this study, lectin histochemistry was used to determine glycoconjugate distribution in the olfactory bulb of the Chinese fire-bellied newt (Cynops orientalis). Succinylated wheat germ agglutinin (sWGA), Ricinus communis agglutinin-I and Lens culinaris agglutinin showed different bindings in the nerve fibre layer or glomerular layer, or both, between the main and accessory olfactory bulbs. We then investigated the lectin-binding pattern after the removal of terminal sialic acids using neuraminidase. Desialylation resulted in a change in the binding reactivities with seven lectins. Wheat germ agglutinin, sWGA, soybean agglutinin (SBA) and peanut agglutinin showed different degrees of binding between the main and accessory olfactory bulbs. In addition, SBA showed a heterogeneous labelling of glomeruli in the rostral region of the main olfactory bulb. Our results suggest that terminal sialic acids mask the heterogeneity of glycoconjugates in the olfactory bulb of C. orientalis.

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.

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.

The gallbladder is the hepatobiliary organ for storing and secreting bile fluid, and is a synapomorphy of extant vertebrates. However, this organ has been frequently lost in several lineages of birds and mammals, including rodents. Although it is known as the traditional problem, the differences in development between animals with and without gallbladders are not well understood. To address this research gap, we compared the anatomy and development of the hepatobiliary systems in mice (gallbladder is present) and rats (gallbladder is absent). Anatomically, almost all parts of the hepatobiliary system of rats are topographically the same as those of mice, but rats have lost the gallbladder and cystic duct completely. During morphogenesis, the gallbladder–cystic duct domain (Gb–Cd domain) and its primordium, the biliary bud, do not develop in the rat. In the early stages, SOX17, a master regulator of gallbladder formation, is positive in the murine biliary bud epithelium, as seen in other vertebrates with a gallbladder, but there is no SOX17‐positive domain in the rat hepatobiliary primordia. These findings suggest that the evolutionary loss of the Gb–Cd domain should be translated simply as the absence of a biliary bud at an early stage, which may correlate with alterations in regulatory genes, such as Sox17, in the rat. A SOX17‐positive biliary bud is clearly definable as a developmental module that may be involved in the frequent loss of gallbladder in mammals.

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.

The gallbladder excretes cytotoxic bile acids into the duodenum through the cystic duct and common bile duct system. Sox17 haploinsufficiency causes biliary atresia-like phenotypes and hepatitis in late organogenesis mouse embryos, but the molecular and cellular mechanisms underlying this remain unclear. In this study, transcriptomic analyses revealed the early onset of cholecystitis in Sox17+/- embryos, together with the appearance of ectopic cystic duct-like epithelia in their gallbladders. The embryonic hepatitis showed positive correlations with the severity of cholecystitis in individual Sox17+/- embryos. Embryonic hepatitis could be induced by conditional deletion of Sox17 in the primordial gallbladder epithelia but not in fetal liver hepatoblasts. The Sox17+/- gallbladder also showed a drastic reduction in sonic hedgehog expression, leading to aberrant smooth muscle formation and defective contraction of the fetal gallbladder. The defective gallbladder contraction positively correlated with the severity of embryonic hepatitis in Sox17+/- embryos, suggesting a potential contribution of embryonic cholecystitis and fetal gallbladder contraction in the early pathogenesis of congenital biliary atresia.

USP9X (ubiquitin-specific peptidase 9, X chromosome) is the mammalian orthologue of Drosophila deubiquitinase fat facets that was previously shown to regulate the maintenance of the germ cell lineage partially through stabilizing Vasa, one of the widely conserved factors crucial for gametogenesis. Here, we demonstrate that USP9X is expressed in the gonocytes and spermatogonia in mouse testes from newborn to adult stages. By using Vasa-Cre mice, germ cell-specific conditional deletion of Usp9x from the embryonic stage showed no abnormality in the developing testes by 1 week and no appreciable defects in the undifferentiated and differentiating spermatogonia at postnatal and adult stages. Interestingly, after 2 weeks, Usp9x-null spermatogenic cells underwent apoptotic cell death at the early spermatocyte stage, and then, caused subsequent aberrant spermiogenesis, which resulted in a complete infertility of Usp9x conditional knockout male mice. These data provide the first evidence of the crucial role of the spermatogonial USP9X during transition from the mitotic to meiotic phases and/or maintenance of early meiotic phase in Usp9x conditional knockout testes.

In mouse ovaries, the first wave of folliculogenesis perinatally starts near the medullary region, which directs the initial round of follicular growth soon after birth. At the same time, cortical primordial follicles start forming in the ovarian surface region, and then some are cyclically recruited for the second and subsequent rounds of follicular growth. Recent studies suggest different dynamics between the first and subsequent waves of follicular growth in postnatal ovaries. However, the phenotypic differences between these phases remain unclear. Here, we show direct evidence that XO female mice, a murine model for Turner Syndrome, lack the first wave of folliculogenesis. Our histopathological analyses of XX and XO littermates revealed a lack of anti-Müllerian hormone (AMH)-positive primary follicles in the XO ovaries by 4 days post partum (dpp). This loss of first follicles was also confirmed by histological bioassay for SRY-dependent SOX9 inducibility, a specific marker for the first follicular granulosa cells. In contrast, cortical primordial follicles formed properly in XO ovaries, and some of them formed primary and secondary follicles in the subcortical region by 7 dpp. They rapidly developed into late antral follicles, showing similarities to XX littermate ovaries by 21 dpp. These results suggest distinct X-monosomy effects between the first and subsequent waves of follicular growth, highlighting the high susceptibility to elimination of XO oocytes in the first wave of mammalian folliculogenesis.

In mouse testes, spermatogonial stem cells (SSCs), a subpopulation of GFR alpha 1 (GDNF family receptor-alpha 1)-positive spermatogonia, are widely distributed along the convoluted seminiferous tubules. The proliferation and differentiation of the SSCs are regulated in part by local expression of GDNF (glial cell-derived neurotorphic factor), one of major niche factors for SSCs. However, the in vivo dynamics of the GDNF-stimulated GFR alpha 1-positive spermatogonia remains unclear. Here, we developed a simple method for transplanting Dil-labeled and GDNF-soaked beads into the mouse testicular interstitium. By using this method, we examined the dynamics of GFRO-positive spermatogonia in the tubular walls close to the transplanted GDNF-soaked beads. The bead-derived GDNF signals were able to induce the stratified aggregate formation of GFR alpha 1-positive undifferentiated spermatogonia by day 3 post-transplantation. Each aggregate consisted of tightly compacted Asingle and marginal A(paired)-A(aligned) GFR alpha 1-positive spermatogonia and was surrounded by A(aligned) GFR alpha 1-negative spermatogonia at more advanced stages. These data not only provide in vivo evidence for the inductive roles of GDNF in forming a rapid aggregation of GFR alpha 1-positive spermatogonia but also indicate the usefulness of this in vivo assay system of various growth factors for the stem/progenitor spermatogonia in mammalian spermatogenesis. (C) 2016 Elsevier Inc. All rights reserved.

In early embryogenesis, the posteroventral foregut endoderm gives rise to the budding endodermal organs including the liver, ventral pancreas and gallbladder during early somitogenesis. Despite the detailed fate maps of the liver and pancreatic progenitors in the mouse foregut endoderm, the exact location of the gallbladder progenitors remains unclear. In this study, we performed a DiI fate-mapping analysis using whole-embryo cultures of mouse early somite-stage embryos. Here, we show that the majority of gallbladder progenitors in 9-11-somite-stage embryos are located in the lateral-most domain of the foregut endoderm at the first intersomite junction level along the anteroposterior axis. This definition of their location highlights a novel entry point to understanding of the molecular mechanisms of initial specification of the gallbladder.