Removal of serum factors by charcoal treatment promotes adipogenesis via a MAPK-dependent pathway

Human TSPY is a candidate oncogene and is supposed to function as a proliferation factor during spermatogenesis. It is the only mammalian protein-coding gene known to be organized as a tandem repeat gene family. It is expressed at highest level in spermatogonia and to a lower amount in primary spermatocytes. To characterize the human TSPY promoter we used the luciferase reporter system in a mouse spermatogonia derived cell line (GC-1spg) and in a GC-4spc cell line, that harbour prophase spermatocytes of the preleptotene and early pachytene stage. We isolated a 1303 bp fragment of the 5′-flanking region of exon 1 that shows significant promoter activity in GC-1spg and reduced activity in GC-4spc cells. In order to gain further insight into the organization of the TSPY-promoter, stepwise truncations of the putative promoter sequence were performed. The resulting fragments were cloned into the pGL3-vector and analysed for reporter gene activity in the murine germ cell lines GC-1spg and GC-4spc, leading to the characterization of a core promoter (−159 to −1), an enhancing region (−673 to −364) and a silencing region (−1262 to −669). Database research for cis-active elements yielded two putative SOX-like binding sites in the enhancing region and reporter gene activity was drastically reduced when three nucleotides of the AACAAT SOX core sequence were mutated. Our findings strongly suggest that testis-specific expression of human TSPY is mediated by Sox proteins. (Mol Cell Biochem 276: 159–167, 2005)     

Molecular cloning of a novel mouse testis-specific spermatogenic cell apoptosis inhibitor gene mTSARG7 as a candidate oncogene

A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and.ll introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFPtagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(-)/mTSARG7 plasmid was constructed and introduced into GC- 1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.     

Promoter elements required for developmental expression of the maize Adh1 gene in transgenic rice.

To define the regions of the maize alcohol dehydrogenase 1 (Adh1) promoter that confer tissue-specific expression, a series of 5' promoter deletions and substitution mutations were linked to the Escherichia coli beta-glucuronidase A (uidA) reporter gene and introduced into rice plants. A region between -140 and -99 not only conferred anaerobically inducible expression in the roots of transgenic plants but was also required for expression in the root cap, embryo, and in endosperm under aerobic conditions. GC-rich (GC-1, GC-2, and GC-3) or GT-rich (GT-1 and GT-2) sequence motifs in this region were necessary for expression in these tissues, as they were in anaerobic expression. Expression in the root cap under aerobic conditions required all the GC- and GT-rich motifs. The GT-1, GC-1, GC-2, and GC-3 motifs, and to a lesser extent the GT-2 motif, were also required for anaerobic responsiveness in rice roots. All elements except the GC-3 motif were needed for endosperm-specific expression. The GC-2 motif and perhaps the GT-1 motif appeared to be the only elements required for high-level expression in the embryos of rice seeds. Promoter regions important for shoot-, embryo-, and pollen-specific expression were proximal to -99, and nucleotides required for shoot-specific expression occurred between positions -72 and -43. Pollen-specific expression required a sequence element outside the promoter region, between +54 and +106 of the untranslated leader, as well as a silencer element in the promoter between -72 and -43.     

Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m6A deposition to activate the PI3K/AKT signaling

Melatonin is a key neuroendocrine hormone that promotes spermatogenesis and sperm motility, but the underlying mechanisms remains poorly understood. In this study, we aimed to investigate the possible roles of m⁶A (N⁶--methyl-adenosine) in mediating melatonin-regulated spermatogonia activity alterations. In this study, mouse-derived GC-1 spermatogonia (spg) cell line was used as the in vitro cellular model. The viability, proliferation rates and apoptosis of spermatogonia were detected via CCK-8, Edu staining and flow cytometry respectively. Total m⁶A level was quantitated by dot blot, while mRNA and proteins contents in spermatogonia were measured by qRT-PCR and western blot respectively. Differentially expressed mRNAs were characterized by deep RNA sequencing method. Results showed that melatonin significantly promoted viability and proliferation rate while inhibited apoptosis in the GC-1 spg cells. The total m⁶A levels in GC-1 spg cells were also greatly increased by melatonin treatment, accompanied by remarkable expressional elevation of the m⁶A writer KIAA1429. Moreover, the regulation of GC-1 spg cell viability, proliferation and apoptosis by melatonin were greatly abrogated by KIAA1429 silencing but effectively strengthened by KIAA1429 overexpression. In addition, KIAA1429 overexpression regulates multiple biological process and signaling pathways in spermatogonia such as the PI3K/AKT signaling. The PI3K inhibitor LY294002 effectively mitigated the regulation of spermatogonia activity by KIAA1429 overexpression under melatonin treatment. Taken together, melatonin promotes spermatogonia activity via enhancing KIAA1429 expression and m⁶A RNA methylation to activate the downstream PI3K/AKT signaling pathway.     

Zebrafish pou5f1/pou2, homolog of mammalian Oct4, functions in the endoderm specification cascade

pou5f1, also known as Oct4, is required to establish the pluripotent cell population necessary for embryogenesis in mouse. Additional roles during development, including endoderm formation, have been proposed. In zebrafish, the zygotic pou5f1/pou2 mutant spiel ohne grenzen (spg) shows neural plate patterning defects and reduced endoderm at the tailbud stage. To investigate the function of maternal and early zygotic pou5f1 expression, we rescued zygotic spg(m793) mutants by injecting pou5f1 mRNA at the one-cell stage and raised them into fertile homozygous spg(m793) adults that mate to produce maternal-zygotic spg (MZspg) mutant embryos. Although neurectoderm, mesoderm, and germ cells develop in MZspg mutants, gastrulation is delayed and proceeds abnormally. Further, MZspg mutants do not maintain expression of sox32/casanova, express little or no sox17, and fail to develop endodermal tissue. Constitutively active Nodal receptor TARAM-A or sox32 overexpression induces ubiquitous sox17 expression in wild-type embryos, but not in MZspg mutants. Overexpression of a Pou5f1-VP16 activator fusion protein can rescue gastrulation and endodermal tissues in MZspg mutants. We propose that pou5f1 plays an activating role in zebrafish endodermal development, where it maintains sox32 expression during gastrulation and acts with sox32 to induce sox17 expression in endodermal precursor cells.     

The alpha4 isoform of the Na,K-ATPase is expressed in the germ cells of the testes.

In addition to the three isoforms of the catalytic subunit of the Na, K-ATPase originally identified (alpha1, alpha2, and alpha3), a fourth alpha polypeptide (alpha4) has recently been found in mammalian cells. This novel alpha-subunit of the Na,K-ATPase is selectively expressed in male gonadal tissues. In the testes, alpha4 is functionally active and comprises approximately half of the Na, K-ATPase activity of the organ. At present, the pattern of expression of the alpha4 polypeptide within the cells of the male gonad is unknown. By in situ hybridization, immunocytochemistry, and the ouabain inhibition profile of Na,K-ATPase activity, we show that the alpha4-subunit is expressed in the germ cells of rat testes. The highest amounts of the isoform are found in spermatozoa, where it constitutes two thirds of the Na,K-ATPase activity of the gametes. The other Na pump present in the cells is the ubiquitously expressed alpha1 polypeptide. The characteristic localization of alpha4 in the gonad is further supported by the drastic reduction of the polypeptide in mice that are infertile as a consequence of arrest in maturation of the germ cells. In addition, GC-1spg cells, a murine cell line derived from testis spermatogonia, also contain the Na, K-ATPase alpha4 polypeptide. However, the level of expression of the isoform in these cells is much lower than in the spermatozoa, a fact that may depend on the limited ability of the GC-1spg cells to differentiate in vitro. The particular expression of the Na,K-ATPase alpha4 isoform we encounter and the specific enzymatic properties of the polypeptide suggests its importance for ionic homeostasis of the germ cells of the testes.     

Heparin-like dextran derivatives as well as glycosaminoglycans inhibit the enzymatic activity of human cathepsin G

6-Phosphofructo-2-kinase catalyzes the synthesis and degradation of fructose 2,6-bisphosphate, activator of phosphofructokinase-1 and inhibitor of fructose 1,6-bisphosphatase. These properties confer to this bifunctional enzyme a key role in the control of glycolysis and gluconeogenesis. Several mammalian isozymes generated by alternative splicing from four genes, designated pfkfb1–4, have been identified. The results presented in this study demonstrate the expression of the pfkfb3 gene in C2C12 cells and its downregulation during myogenic cell differentiation. We also show that the decrease of ubiquitous 6-phosphofructo-2-kinase isozyme levels, product of pfkfb3 gene, is due to its enhanced degradation through the ubiquitin-proteasome proteolytic pathway.     

spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories.