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Urodele amphibian newts have unique biological properties in male gametogenesis, in addition to their extreme regenerative capacity. Male newts are able to regenerate new testes even after reaching sexual maturity and can possess multiple testes. Notably, these animals maintain primordial germ cell-like cells in a tissue adjacent to the testis. Spermatogenesis proceeds while synchronizing in a region-specific manner in the testis. However, the newt species that have been used most commonly require 2-3 years to achieve sexual maturity, and spermatogenesis in these species shows seasonality. These traits have restricted the use of newts for studies on testicular development and spermatogenesis, and testis development in newts remains poorly characterized. Recently, the Iberian ribbed newt Pleurodeles waltl has been established as an emerging model organism. P. waltl reaches sexual maturity more quick after birth than do other newts and is capable of breeding year-round. Thus, P. waltl is expected to serve as an appealing experimental model for studying the mechanisms of male gametogenesis in the urodeles. In the present study, we use P. waltl to describe the entire developmental process of the newt testis from primordial gonad to maturity. Notably, the mature testes show synchronized progression of spermatogenesis along the anteroposterior axis. Additionally, we demonstrate that the process of spermatogenesis in P. waltl proceeds irrespective of day length. Our results show that P. waltl newts are a suitable model for investigating the process of testicular development. We also expect that these results will be useful for the maintenance of P. waltl bioresources.

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.

USP9X (ubiquitin-specific peptidase 9, X chromosome) is the mammalian orthologue ofDrosophiladeubiquitinase 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 usingVasa-Cremice, germ cell-specific conditional deletion ofUsp9xfrom 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 ofUsp9xconditional 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 inUsp9xconditional knockout testes.

In mouse testes, spermatogonial stem cells (SSCs), a subpopulation of GFRα1 (GDNF family receptor-α1)-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 GFRα1-positive spermatogonia remains unclear. Here, we developed a simple method for transplanting DiI-labeled and GDNF-soaked beads into the mouse testicular interstitium. By using this method, we examined the dynamics of GFRα1-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 GFRα1-positive undifferentiated spermatogonia by day 3 post-transplantation. Each aggregate consisted of tightly compacted Asingle and marginal Apaired-Aaligned GFRα1-positive spermatogonia and was surrounded by Aaligned GFRα1-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 GFRα1-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.

To explore possible roles of heparanase in cancer-host crosstalk, we examined whether heparanase influences expression of inflammatory chemokines in colorectal cancer cells. Murine colorectal carcinoma cells incubated with heparanase upregulated MCP-1, KC, and RANTES genes and released MCP-1 and KC proteins. Heparanase-dependent production of IL-8 was detected in two human colorectal carcinoma cell lines. Addition of a heparanase inhibitor Heparastatin (SF4) did not influence MCP-1 production, while both latent and mature forms of heparanase augmented MCP-1 release, suggesting that heparanase catalytic activity was dispensable for MCP-1 production. In contrast, addition of heparin to the medium suppressed MCP-1 release in a dose-dependent manner. Similarly, targeted suppression of Ext1 by RNAi significantly suppressed cell surface expression of heparan sulfate and MCP-1 production in colon 26 cells. Taken together, it is concluded that colon 26 cells transduce the heparanase-mediated signal through heparan sulfate binding. We propose a novel function for heparanase independent of its endoglycosidase activity, namely as a stimulant for chemokine production.

The sodium pump (Na,K-ATPase) in animal cells is vital for actively maintaining ATP hydrolysis-powered Na- and K+ electrochemical gradients across the cell membrane. These ion gradients drive co-And countertransport and are critical for establishing the membrane potential. It has been an enigma how Na,K-ATPase discriminates between Na- and K+, despite the pumped ion on each side being at a lower concentration than the other ion. Recent crystal structures of analogs of the intermediate conformations E2-Pi-2K+ and Na-bound E1-P-ADP suggest that the dimensions of the respective binding sites in Na,KATPase are crucial in determining its selectivity. Here, we found that the selectivity at each membrane face is pH-dependent and that this dependence is unique for each face. Most notable was a strong increase in the specific affinity for K+ at the extracellular face (i.e. E2 conformation) as thepHis lowered from 7.5 to 5.We also observed a smaller increase in affinity for K+ on the cytoplasmic side (E1 conformation), which reduced the selectivity for Na-. Theoretical analysis of the pKa values of ion-coordinating acidic amino acid residues suggested that the face-specific pH dependences and Na-/K+ selectivities may arise from the protonation or ionization of key residues. The increase in K+ selectivity at low pH on the cytoplasmic face, for instance, appeared to be associated with Asp808 protonation. We conclude that changes in the ionization state of coordinating residues in Na,K-ATPase could contribute to altering face-specific ion selectivity.

Great variations in the size, shape, color, feather structure and behavior are observed among fowl breeds. Because many types of domestic fowls have been bred for various purposes, they are ideal to assess the relationship between brain morphology and avian biology. However, little is known about changes in brain shape that may have occurred during fowl domestication. We analyzed the brains of red jungle fowl and domestic fowl to clarify differences in the brain shape between these breeds, as well as the shape changes associated with size enlargement using three-dimensional geometric morphometrics. Principal component and multivariate regression analyses showed that ventrodorsal bending, anteroposterior elongation and width reduction were significantly correlated with brain size. According to the size-dependent analysis, the red jungle fowl brain has an intermediate shape between the brain of young broilers and that of large domestic fowl and adult broilers. After the size effect is removed, geometric morphometric analyses show that the brain of red jungle fowl is different from that of domestic fowl, with large round cerebral hemispheres. Significant correlations exist between the skull length and brain volume among fowl, while the brain volume relative to the skull length is distinctly larger in red jungle fowl compared with domestic fowl. The distinct brain shape and increased relative brain size of red jungle fowl may be driven by the presence of large, rounded cerebral hemispheres.

Cell ablation technology is useful for studying specific cell lineages in a developing organ in vivo. Herein, we established a novel anti-Müllerian hormone (AMH)-toxin receptor-mediated cell knockout (Treck) mouse line, in which the diphtheria toxin (DT) receptor was specifically activated in Sertoli and granulosa cells in postnatal testes and ovaries respectively. In the postnatal testes of Amh-Treck transgenic (Tg) male mice, DT injection induced a specific loss of the Sertoli cells in a dose-dependent manner, as well as the specific degeneration of granulosa cells in the primary and secondary follicles caused by DT injection in Tg females. In the testes with depletion of Sertoli cell, germ cells appeared to survive for only several days after DT treatment and rapidly underwent cell degeneration, which led to the accumulation of a large amount of cell debris within the seminiferous tubules by day 10 after DT treatment. Transplantation of exogenous healthy Sertoli cells following DT treatment rescued the germ cell loss in the transplantation sites of the seminiferous epithelia, leading to a partial recovery of the spermatogenesis. These results provide not only in vivo evidence of the crucial role of Sertoli cells in the maintenance of germ cells, but also show that the Amh-Treck Tg line is a useful in vivo model of the function of the supporting cell lineage in developing mammalian gonads.

Heparanase cleaves macromolecular heparin in the secretory granules of connective tissue-type mast cells. We investigated roles of the cleavage under a microenvironment mimicking where the mast cells physiologically reside. A connective tissue-type mast cell line MST and mouse peritoneal cell-derived mast cells stored macromolecular heparin in the secretory granules. The cells expressing heparanase stored fragmented heparin (~10 kDa) due to heparanase-dependent cleavage of the heparin. We produced an artificial collagen-based extracellular matrix and placed the live cells or glycosaminoglycans purified from the cells in the matrix to measure the release of sulfated macromolecules into the medium. The sulfate-radiolabelled molecules from the degranulating heparanase-expressing cells and the purified glycosaminoglycans showed significantly greater release into the medium than those derived from mock cells, which was not the case in suspension culture. The mast cell granular enzyme chymase, but not β-hexosaminidase, showed significantly greater release from the degranulating heparanase-expressing cells than from mock cells. Purified chymase mixed with fragmented heparin derived from heparanase-expressing cells showed greater release from collagen gels than the enzyme alone or mixed with macromolecular heparin derived from mock cells. We propose that the cleavage of macromolecular heparin by heparanase accelerates the release of heparin and chymase from extracellular matrices.

Background: The spermatogonial transplantation experiment can be used as an unequivocal detection assay of spermatogenic stem cells (SSCs) in both a qualitative and quantitative manner, based on their regenerative capacity. In this study, the proliferative patterns and kinetics of donor-derived GFRα1-positive spermatogonia containing potential SSCs were examined during early colonization following spermatogonial transplantation.Results: Donor-derived GFRα1-positive cells frequently formed several aggregates of Aal(aligned)/morula-like structures in a single spermatogenic cell patch before and on day 14 post-transplant, indicating a possible involvement in the formation of a stable spermatogenic colony at 21 days post-transplant. The appearance of these Aal/morula-like aggregates is positively correlated with regional, high-level expression of immunoreactive GDNF signals, a ligand for GFRα1, associated with colony expansion. Conclusions: These data raise the hypothesis that regional GDNF signals regulate the balance between donor-derived Aal-like cell aggregates and their differentiation in each small patch, which subsequently leads to further selection of survival colonies at later stages. Developmental Dynamics 241:1374–1384, 2012. © 2012 Wiley Periodicals, Inc.

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.

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.

In early-organogenesis-stage mouse embryos, the posteroventral foregut endoderm adjacent to the heart tube gives rise to liver, ventral pancreas and gallbladder. Hepatic and pancreatic primordia become specified in the posterior segment of the ventral foregut endoderm at early somite stages. The mechanisms for demarcating gallbladder and bile duct primordium, however, are poorly understood. Here, we demonstrate that the gallbladder and bile duct progenitors are specified in the paired lateral endoderm domains outside the heart field at almost the same timing as hepatic and pancreatic induction. In the anterior definitive endoderm, Sox17 reactivation occurs in a certain population within the most lateral domains posterolateral to the anterior intestinal portal (AIP) lip on both the left and right sides. During foregut formation, the paired Sox17-positive domains expand ventromedially to merge in the midline of the AIP lip and become localized between the liver and pancreatic primordia. In Sox17-null embryos, these lateral domains are missing, resulting in a complete loss of the gallbladder/bile-duct structure. Chimera analyses revealed that Sox17-null endoderm cells in the posteroventral foregut do not display any gallbladder/bile-duct molecular characters. Our findings show that Sox17 functions cell-autonomously to specify gallbladder/bile-duct in the mouse embryo.

During mouse gastrulation, primordial germ cells (PGCs) become clustered at the base of the allantois and move caudally into the hindgut endoderm before entering the genital ridges. The precise roles of endoderm tissues in PGC migration, however, remain unclear. By using Sox17 mutants with a specific endoderm deficiency, we provide direct evidence for the crucial role of hindgut expansion in directing proper PGC migration. In Sox17-null embryos, PGCs normally colonize in the allantois and then a small front-row population of PGCs moves properly into the most posterior gut endoderm. Defective hindgut expansion, however, causes the failure of further lateral PGC movement, resulting in the immobilization of PGCs in the hindgut entrance at the later stages. In contrast, the majority of the remaining PGCs moves into the visceral endoderm layer, but relocate outside of the embryonic gut domain. This leads to a scattering of PGCs in the extraembryonic yolk sac endoderm. This aberrant migration of Sox17-null PGCs can be rescued by the supply of wildtype hindgut cells in chimeric embryos. Therefore, these data indicate that hindgut morphogenic movement is crucial for directing PGC movement toward the embryonic gut side, but not for their relocation from the mesoderm into the endoderm.

In mammals, the Y-linked sex-determining gene Srycell-autonomously promotes Sertoli cell differentiation from bipotential supporting cell precursors through SRY-box containing gene 9 (Sox9),leading to testis formation. Without Sry action, the supporting cells differentiate into granulosa cells, resulting in ovarian development. However,how Sry acts spatiotemporally to switch supporting cells from the female to the male pathway is poorly understood. We created a novel transgenic mouse line bearing an inducible Sry transgene under the control of the Hsp70.3 promoter. Analysis of these mice demonstrated that the ability of Sry to induce testis development is limited to approximately 11.0-11.25 dpc, corresponding to a time window of only 6 hours after the normal onset of Sry expression in XY gonads. If Sry was activated after 11.3 dpc, Sox9 activation was not maintained, resulting in ovarian development. This time window is delimited by the ability to engage the high-FGF9/low-WNT4 signaling states required for Sertoli cell establishment and cord organization. Our results indicate the overarching importance of Sry action in the initial 6-hour phase for the female-to-male switching of FGF9/WNT4 signaling patterns.

Knock-in embryonic stem (ES) cells, in which GFP or lacZ was expressed from the endogenous mouse vasa homolog ( Mvh ), which is specifically expressed in differentiating germ cells, were used to visualize germ cell production during in vitro differentiation. The appearance of MVH-positive germ cells depended on embryoid body formation and was greatly enhanced by the inductive effects of bone morphogenic protein 4-producing cells. The ES-derived MVH-positive cells could participate in spermatogenesis when transplanted into reconstituted testicular tubules, demonstrating that ES cells can produce functional germ cells in vitro . In vitro germ cell differentiation provides a paradigm for studying the molecular basis of germ line establishment, as well as for developing new approaches to reproductive engineering.

Germ cell-specific ATP-dependent RNA helicase, the product of the mouse vasa homolog (Mvh), has been shown to play an essential role in the development of the male germ cell. In male Mvh knockout mice, premeiotic germ cells arrest at the zygotene stage. To investigate the role of MVH protein in the progression of meiosis, we searched for genes encoding partners that interact with MVH in testicular germ cells. Using the yeast two-hybrid system, we found that MVH interacts with mouse RanBPM, a Ran-GTP binding protein involved in microtubule nucleation. RanBPM is predominantly expressed in the testis, especially in maturating spermatocytes. Within the cell, RanBPM and MVH are closely associated with perinuclear RNA-protein complexes and chromatoid bodies. The interaction of MVH with RanBPM points to a functional relationship between translational regulation and the microtubule nucleation during meiosis. Mol. Reprod. Dev. 66: 1-7, 2004.

In invertebrate species such as flies and nematodes, germline stem cells are maintained in a niche environment, which is restricted to the terminal end of the tubular structure in the gonads. In mice, spermatogonial stem cells (SSCs), a subpopulation of Asingle GFRα1 (glial cell line-derived neurotrophic factor [GDNF] family receptor-α1)-positive spermatogonia, are widely distributed along the longitudinal axis in the convoluted seminiferous tubules, preferentially juxtaposed to the interstitial vasculature. However, whether this area is the only SSC niche is not known. In this study, we identified a valve-like terminal segment of the seminiferous tubules, the Sertoli valve (SV), adjacent to the rete testis as another niche for GFRα1-positive spermatogonia in hamsters. Here, we show that the SV epithelium is composed of the modified Sertoli cells that are still capable of proliferation and missing most spermatogenic activities in the adult stage. The SV epithelium constitutively expresses GDNF, a major niche factor for SSCs, and supports the stable proliferation and selective maintenance of an Asingle subpopulation of GFRα1-positive spermatogonia in hamsters. The SV region of hamster seminiferous tubules has features that are similar to the stem cell niche in invertebrate gonads. Therefore, we propose that the SV may be a novel niche for Asingle GFRá1-positive spermatogonia potentially including a SSC population, at the terminal segments of the seminiferous tubules in hamsters. Stem Cells 2015;33:2811–2824

Many vasa homologue genes to Drosophila vasa have been isolated in various animal species. They provide specific molecular probes to analyze the establishment and the differentiation of germ cell lineage. In mammals, the expression of VASA protein becomes detectable in PGCs at the late migrating stage. Interestingly, during spermatogenesis the intracellular localization of VASA protein is closely associated with the chromatoid body.

Hsc70t is a member of the Hsp70 family of genes and is constitutively expressed after meiosis in mouse spermatogenesis. Immunohistochemistry and in situ hybridization techniques were used to examine the precise localization of the Hsc70t product during the various stages of spermatogenesis. A rabbit antiserum raised againstthe mouse Hsc70t-lacZ fusion protein detected the Hsc70t protein in the late spermatid-enriched fraction after two-dimensional Western blot analyses. On histological sections, the protein appears in the cytoplasm of spermatids as they progress from step 9 to the final step of spermatogenesis. An antisense RNA probe generated from the 3' untranslated region of Hsc70t cDNA detected Hsc70t mRNA in late round spermatids from step 7 onward with the signal disappearing in spermatids at step 15. Thus, Hsc70t mRNA first appears after meiosis in haploid cells but is not translated effectively until these cells progress to the transcriptionally inactive stage which coincides with chromatin condensation. These results establish that the synthesis of Hsc70t protein is under strict translational control.

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.

Although di(n-butyl) phthalate (DBP), a suspected endocrine disruptor, induces testicular atrophy in prepubertal male rats, whether it exerts estrogenic activity in vivo remains a matter of debate. In the present study, we explored the estrogenic potency of DBP using 3-week-old male rats, and then examined the relationship between estrogen-induced spermatogenic cell apoptosis and testicular steroidogenesis. Daily exposure to DBP for 7 days caused testicular atrophy due to loss of spermatogenic cells, whereas testicular steroidogenesis was almost the same with the control values. A single exposure of DBP decreased testicular steroidogenesis in addition to decreasing the level of serum LH at 3 h after DBP treatment, with an extremely high incidence of apoptotic spermatogenic cells at 6 h after administration. To elucidate the estrogenic activity of DBP, we carried out an inhibition study using pure antiestrogen ICI 182,780 (ICI) in a model of spermatogenic cell apoptosis induced by DBP or estradial-3-benzoate (EB). Although both the DBP- and EB-treated groups showed a significant increase in spermatogenic cell apoptosis, ICI pretreatment significantly decreased the number of apoptotic spermatogenic cells in these two groups. In contrast, testicular steroidogenesis and serum FSH were significantly reduced in all the treated groups, even in the DBP+ICI and EB+ICI groups. Taken together, these findings led us to conclude that estrogenic compounds such as DBP and EB induce spermatogenic cell apoptosis in prepubertal rats, probably by activating estrogen receptors in testis, and that reduction in testicular steroidogenic function induced by estrogenic compounds is not associated with spermatogenic cell apoptosis.

Morphological alterations in seminiferous tubules caused by single administration of di(n-butyl) phthalate (DBP) in 3-week-old rats were investigated throughout the first wave of spermatogenesis. Single administration of DBP (500 mg/kg) showed progressive detachment and displacement of spermatogenic cells and disappearance of tubular lumen at 3 h after treatment, and then showed thin seminiferous epithelia and wide tubular lumen at day 1 (D1). At D1, quite significant numbers of apoptotic spermatogenic cells were detected, and then they gradually decreased in accordance with the passage of time. In contrast, the testes revealed lower weight gain, even after completion of first wave of spermatogenesis in the DBP-treated group, compared to the control. In order to clarify whether spermatogenic cells differentiate into mature spermatids in the DBP-treated rats, immunohistochemical staining for Hsc 70t, a specific marker for elongate spermatids, was carried out. As a result, the decrease in mature spermatids in the DBP-treated testes, compared to the control, was demonstrated. For example, at D20 (41-day-old) after treatment, the most advanced spermatids in the tubules from rats in the DBP-treated groups were steps 2–4, while those of the control were steps 12–13. Moreover, in some tubules, pachytene spermatocytes were the most advanced spermatogenic cell. At D30 (51-day-old) after treatment, maturation of spermatogenic cells in the DBP-treated rats proceeded further, and the most advanced spermatids in tubules were steps 8–9, while those of the control were steps 15–19. These results lead us to the postulation that a single administration of DBP to prepubertal rats delays maturation of spermatogenic cells, even after completion of first wave of spermatogenesis.

Sox7, -17 and -18 constitute the Sox subgroup F (SoxF) of HMG box transcription factor genes, which all are co-expressed in developing vascular endothelial cells in mice. Here we characterized cardiovascular phenotypes of Sox17/Sox18-double and Sox17-single null embryos during early-somite stages. Whole-mount PECAM staining demonstrated the aberrant heart looping, enlarged cardinal vein and mild defects in anterior dorsal aorta formation in Sox17 single-null embryos. The Sox17/Sox18 double-null embryos showed more severe defects in formation of anterior dorsal aorta and head/cervical microvasculature, and in some cases, aberrant differentiation of endocardial cells and defective fusion of the endocardial tube. However, the posterior dorsal aorta and allantoic microvasculature was properly formed in all of the Sox17/Sox18 double-null embryos. The anomalies in both anterior dorsal aorta and head/cervical vasculature corresponded with the weak Sox7 expression sites. This suggests the region-specific redundant activities of three SoxF members along the anteroposterior axis of embryonic vascular network.

The role of phagocytosis in eliminating apoptotic spermatogenic cells caused by mono(2-ethylhexyl) phthalate (MEHP) was studied. Twenty-one-day-old C57Bl/6N male mice were given a single dose of 800 mg/kg MEHP in corn oil by oral gavage and sacrificed at 1, 3, 5, 7 and 9 days after initial exposure. At the same time, the role of phagocytosis in MEHP related apoptosis was examined using microinjection of annexin V into the seminiferous tubules of living mice. Results showed that mice treated with MEHP had a lower rate of testis weight gain (lower regression line) and a significant TUNEL-positive spermatogenic cell number compared to control. However, this incident was reversible, and the number of TUNEL-positive cells returned to normal after 9 days. Mice microinjected with annexin V and later treated with MEHP showed a large amount of TUNEL-positive cells compared to mice treated with MEHP only. This clearly proves that phagocytosis plays an efficient and highly important role in eliminating dead cells in the injured testis of mice treated with MEHP.

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility. Here we show that Cnot7(-/-) males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor, is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7(-/-) mice. Transplantation of spermatogonial stem cells from male Cnot7(-/-) mice to seminiferous tubules of Kit mutant mice (Kit(W/W-v)) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7(-/-) condition. The testicular phenotypes of Cnot7(-/-) mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb). We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.

Background and Aims In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo. Methodology and Principal Findings Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules. Conclusion/Significance Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.

In mouse sex differentiation, SRY promotes Sertoli cell differentiation via SOX9 action, resulting in testis formation. SRY/SOX9 also initiates various testis-specific morphogenic events including glycogenesis in pre-Sertoli cells, suggesting the importance of glucose storage for certain SRY/SOX9-downstream events in gonadal sex determination. However, it remains unclear which cell types and what molecular/cellular events require sex-dimorphic high-energy metabolic rate. Here we show that the establishment of SOX9 activation itself is a metabolically active process with sex-dimorphic high-energy requirements in gonadal sex differentiation. The glucose-deprivation and metabolic rescue experiments using genital ridge cultures of the XY/XX-wildtype and XX/Sry transgenic embryos demonstrated that, among the various somatic cell types, pre-Sertoli cells are the most sensitive to glucose starvation despite the differences between XX/Sry and XY genotypes. Moreover, our data showed that, in developing pre-Sertoli cells, the high-glucose metabolic state is required for the establishment of SOX9 expression through an ECM (extracellular matrix)-mediated feed-forward pathway. In contrast, the expression of SRY, SF1/Ad4Bp, GATA4 and WT1, as well as initiation of early SOX9 expression, is properly maintained in the glucose-deprived condition. Therefore, our results imply the metabolic importance of the high-glucose condition for the establishment of SOX9 activation in testis differentiation.

The gonad as well as the reproductive tracts, kidney, and adrenal cortex are derived from the intermediate mesoderm. In addition, the intermediate mesoderm forms the mesonephros. Although the mesonephros is the source of certain testicular cell types, its contribution to gonad formation through expression of growth factors is largely unknown. Here, we examined the expression profiles of FGF9 in the developing mesonephros of chick embryos at sexually indifferent stages, and found that the expression domain is adjacent to the gonadal primordium. Moreover, FGFR3 (FGF receptor 3) showed a strong expression in the gonadal primordium. Next, we examined the functions of FGF signal during gonadal development with misexpressed FGF9. Interestingly, misexpression of FGF9 led to gonadal expansion through stimulation of cell proliferation. In contrast, treatment with a chemical inhibitor for FGFR decreased cell proliferation and resulted in reduction of the gonadal size. Simultaneously, the treatment resulted in reduction of gonadal marker gene expression. Our study demonstrated that FGF expressed in the developing mesonephros is involved in the development of the gonad at the sexually indifferent stages through stimulation of gonadal cell proliferation and gonadal marker gene expression.