Comparative morphological study of skeletal muscle weight among the red jungle fowl (Gallus gallus) and various fowl breeds (Gallus domesticus).

We examined the weight distribution of skeletal muscles of the red jungle fowl, then compared these values with those of domesticated populations to determine how muscle distribution has changed by selecting breeding. Sonia, Fayoumi, and Rhode Island Red were selected for comparison from livestock breeds, while Japanese Shamo and Thai fighting cocks were selected from cockfighting groups. Principal component analysis was applied using body size-free data. The mass distribution of muscles clearly differed between the wild, livestock, and cockfighting groups, demonstrating that muscle distribution has changed after selecting breeding, coupled with functional demands of each group. The red jungle fowl, which has the ability to fly, could be clearly distinguished from the flightless domesticated populations due to differences in flight pectoral muscle size. The cervical muscles in the wild population were smaller than in the domesticated groups; these do not contribute to flight. The gluteal muscles were larger in the fighting cock group, functionally coupled to their traditionally preferred upright posture. Wild bird populations typically exhibit reduced weight of their hind limbs, associated with flight, but as the red jungle fowl displays largely terrestrial behavior, these muscles are similar in arrangement and relative size to those of the livestock groups. We showed that the mass distribution pattern of skeletal muscles expresses selecting breeding strategy and clearly reflects the specific traits for each group.

Photoperiod-independent testicular development in the model newt Pleurodeles waltl.

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.

Multiple mutations in RNA polymerase ß-subunit gene (rpoB) in Streptomyces incarnatus NRRL8089 enhance production of antiviral antibiotic sinefungin: modeling rif cluster region by density functional theory.

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) ß-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

Mechanism of the E2 to E1 transition in Ca2+ pump revealed by crystal structures of gating residue mutants.

Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a) pumps two Ca2+ per ATP hydrolyzed from the cytoplasm and two or three protons in the opposite direction. In the E2 state, after transferring Ca2+ into the lumen of sarcoplasmic reticulum, all of the acidic residues that coordinate Ca2+ are thought to be protonated, including the gating residue Glu309. Therefore a Glu309Gln substitution is not expected to significantly perturb the structure. Here we report crystal structures of the Glu309Gln and Glu309Ala mutants of SERCA1a under E2 conditions. The Glu309Gln mutant exhibits, unexpectedly, large structural rearrangements in both the cytoplasmic and transmembrane domains, apparently uncoupling them. However, the structure definitely represents E2 and, together with the help of quantum chemical calculations, allows us to postulate a mechanism for the E2 → E1 transition triggered by deprotonation of Glu309.

Embryonic cholecystitis and defective gallbladder contraction in the Sox17-haploinsufficient mouse model of biliary atresia.

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.

Distinct pH dependencies of Na+/K+ selectivity at the two faces of Na,K-ATPase.

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,K-ATPase 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 the pH is 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.

Morphological variation in brain through domestication of fowl.

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.

Spermatogonial deubiquitinase USP9X is essential for proper spermatogenesis in mice.

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.

Defects in the first wave of folliculogenesis in mouse XO ovaries.

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.

IL-17A-producing CD30(+) Vδ1 T cells drive inflammation-induced cancer progression.

Although it has been suspected that inflammation is associated with increased tumor metastasis, the exact type of immune response required to initiate cancer progression and metastasis remains unknown. In this study, by using an in vivo tumor progression model in which low tumorigenic cancer cells acquire malignant metastatic phenotype after exposure to inflammation, we found that IL-17A is a critical cue for escalating cancer cell malignancy. We further demonstrated that the length of exposure to an inflammatory microenvironment could be associated with acquiring greater tumorigenicity and that IL-17A was critical for amplifying such local inflammation, as observed in the production of IL-1ß and neutrophil infiltration following the cross-talk between cancer and host stromal cells. We further determined that γδT cells expressing Vδ1 semi-invariant TCR initiate cancer-promoting inflammation by producing IL-17A in an MyD88/IL-23-dependent manner. Finally, we identified CD30 as a key molecule in the inflammatory function of Vδ1T cells and the blockade of this pathway targeted this cancer immune-escalation process. Collectively, these results reveal the importance of IL-17A-producing CD30(+) Vδ1T cells in triggering inflammation and orchestrating a microenvironment leading to cancer progression.

Heparanase augments inflammatory chemokine production from colorectal carcinoma cell lines.

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.

In vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay.

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.

Sox17 haploinsufficiency results in perinatal biliary atresia and hepatitis in C57BL/6 background mice.

Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute 'embryonic hepatitis', leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17(+/-) B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17(+/-) gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.

A Niche for GFRα1-Positive Spermatogonia in the Terminal Segments of the Seminiferous Tubules in Hamster Testes.

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.

Identification and characterization of the zosA gene involved in copper uptake in Bacillus subtilis 168.

DL-Penicillamine, a copper-specific metal chelator, remarkably suppressed the growth of Bacillus subtilis 168 when added to a synthetic medium under Cu(2+) limitation. DNA microarray and screening of 2,602 knockout mutants showed that the zosA gene was de-repressed in the presence of 0.1% dl-penicillamine, and that the zosA mutant was sensitive to dl-penicillamine medium. The zosA mutant delayed the growth under Cu-limitation even without the chelator, and the sensitivity to dl-penicillamine was reversed by induction using 0.3 mM IPTG and the Pspac promoter inserted directly upstream of the zosA gene. Furthermore, the zosA mutant showed elevated tolerance of excessive Cu(2+) but not of excessive Zn(2+) added to LB and synthetic media. Homology modeling of the ZosA protein suggested that the protein can fold itself into essential domains for constituting a metal transporting ATPase. Our study suggests that zosA is a candidate gene involved in copper uptake.

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.

Heparanase-mediated cleavage of macromolecular heparin accelerates release of granular components of mast cells from extracellular matrices.

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 ß-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.

A novel Amh-Treck transgenic mouse line allows toxin-dependent loss of supporting cells in gonads.

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.

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.

Oligo-astheno-teratozoospermia in mice lacking Cnot7, a regulator of retinoid X receptor beta.

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.