Prmt7 regulates epiboly and gastrulation cell movements by facilitating syntenin.

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation. Despite of its fundamental function, little is known about the molecular mechanisms that control this coordinated cell movement. In this study, we investigated protein arginine methyltransferase 7 (Prmt7) morphants with an epibolic delay defect in zebrafish. The ratio of morphants with epiboly delay phenotypes increased as the dose of the injected morpholino (MO) increased. Here, syntenin transcripts are maternally deposited and ubiquitously expressed from the oocyte period to the early larva stage. Furthermore, we demonstrated that Prmt7 modulates epibolic movements of the enveloping layer by regulating F-actin organization. These defects can be partially rescued by re-expression of Prmt7 or syntenin protein. Analysis of the earliest cellular defects suggested a role of Prmt7 in the autonomous vegetal expansion of the yolk syncytial layer and the rearrangement of the actin cytoskeleton in extra-embryonic tissues. By a combination of knockdown studies and rescue experiments in zebrafish, we showed that epiboly relies on the molecular networking of Prmt7 by facilitating syntenin, which acts as a regulator for cytoskeleton. This study identifies the important function of the Prmt7 for the progression of zebrafish epiboly and establishes its key role in directional cell movements during early development.

Protein Arginine Methyltransferase 6 (Prmt6) Is Essential for Early Zebrafish Development through the Direct Suppression of gadd45αa Stress Sensor Gene.

Histone lysine methylation is important in early zebrafish development; however, the role of histone arginine methylation in this process remains unclear. H3R2me2a, generated by protein arginine methyltransferase 6 (Prmt6), is a repressive mark. To explore the role of Prmt6 and H3R2me2a during zebrafish embryogenesis, we identified the maternal characteristic of prmt6 and designed two prmt6-specific morpholino-oligos (MOs) to study its importance in early development, application of which led to early epiboly defects and significantly reduced the level of H3R2me2a marks. prmt6 mRNA could rescue the epiboly defects and the H3R2me2a reduction in the prmt6 morphants. Functionally, microarray data demonstrated that growth arrest and DNA damage-inducible, α, a (gadd45αa) was a significantly up-regulated gene in MO-treated embryos, the activity of which was linked to the activation of the p38/JNK pathway and apoptosis. Importantly, gadd45αa MO and p38/JNK inhibitors could partially rescue the defect of prmt6 morphants, the downstream targets of Prmt6, and the apoptosis ratios of the prmt6 morphants. Moreover, the results of ChIP quantitative real time PCR and luciferase reporter assay indicated that gadd45αa is a repressive target of Prmt6. Taken together, these results suggest that maternal Prmt6 is essential to early zebrafish development by directly repressing gadd45αa.

GABA exists as a negative regulator of cell proliferation in spermatogonial stem cells. [corrected].

γ-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA is also found in many peripheral tissues, where it has important functions during development. Here, we identified the existence of the GABA system in spermatogonial stem cells (SSCs) and found that GABA negatively regulates SSC proliferation. First, we demonstrated that GABA and its synthesizing enzymes were abundant in the testes 6 days postpartum (dpp), suggesting that GABA signaling regulates SSCs function in vivo. In order to directly examine the effect of GABA on SSC proliferation, we then established an in vitro culture system for long-term expansion of SSCs. We showed that GABAA receptor subunits, including α1, α5, ß1, ß2, ß3 and γ3, the synthesizing enzyme GAD67, and the transporter GAT-1, are expressed in SSCs. Using phosphorylated histone H3 (pH3) staining, we demonstrated that GABA or the GABAAR-specific agonist muscimol reduced the proliferation of SSCs. This GABA regulation of SSC proliferation was shown to be independent of apoptosis using the TUNEL assay. These results suggest that GABA acts as a negative regulator of SSC proliferation to maintain the homeostasis of spermatogenesis in the testes.

Erratum to: GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells.

[This corrects the article DOI: 10.2478/s11658-013-0081-4.].

Splice blocking of zygotic sox31 leads to developmental arrest shortly after Mid-Blastula Transition and induces apoptosis in zebrafish.

Here we report that splice blocking morpholinos (Sb MO) against zebrafish sox31 elicit developmental arrest, likely through creating a series of dominant negative splicing variants. Embryos injected with the Sb MO develop normally before the Mid-Blastula Transition (MBT); however, they do not initiate epiboly. Microarray analysis of mRNAs collected at the dome stage revealed that the Sb MO impairs activation of a large set of zygotic genes and reduces degradation of maternal mRNA during MBT. Furthermore, an apoptotic response occurs in Sb morphants at about 6hpf. SoxB1 family genes including sox31 thus play an essential role for early embryos traversing the transitional stage.

Differential response of multiple zebrafish hepatic F-box protein genes to 17alpha-ethinylestradiol treatment.

Estrogens are accumulating in environment and their effects on a variety of reproductive processes and tumorigenesis were reported by previous study, but the mechanism of estrogen promoting neoplasia was still not clear. F-box protein (FBP) is the component of E3 ubiquitin ligase which takes part in a variety of key biological processes. In this study, using mature male zebrafish, which are more sensitive to estrogen treatment, we examined influence of 17alpha-ethinylestradiol (EE2) exposure on the expression of a series of hepatic FBP genes, which take part in a variety of biological processes, including tumorigenesis. The influence of EE2 on the expression of hepatic mRNA concentrations of FBP genes were quantified based on the expression of the optimal internal control gene in male zebrafish after 7-day exposure to EE2, from a low-dose concentration (1 ng/L) to environmentally relevant concentrations (10, 100 ng/L). Our results showed that EE2 exposure reduced the expression of fbxl14a, fbxl14b, fbxo25 and beta-TRCP2b, but enchanced the expression of skp2. While the alterations in fbxl2, fbxw7, fbxo9, beta-TRCP2a, fbxl18 and fbxo45 mRNA levels were not observed after EE2 exposure. Thus, our results showed that the expression of hepatic FBP genes exhibited differentially in male zebrafish exposed EE2. The changes of the expression level of FBP genes induced by EE2 may be an important clue to elucidate the correlations of estrogen and hepatic tumors.

Sox31 is involved in central nervous system anteroposterior regionalization through regulating the organizer activity in zebrafish.

Sox superfamily proteins are DNA-binding transcriptional factors that contain highly conserved high-mobility group (HMG) box and take part in various development process. Sox31 is a maternal factor supplied in the oocyte and starts its zygotic expression during mid-blastula transition (MBT). From gastrulation stage, it mainly resides in neural tissue. Ectopically expression of Sox31 mRNA leads to cyclopia, fusion eyes, or totally loss of anterior head structure, in accompany with severe notochord defects. Molecular markers indicate that forebrain tissue reduces sharply while the posterior neural tissue expands anteriorly. In addition, organizer specification is also suppressed. Oppositely, an antisense morpholino designed functionally knockdown Sox31 causes typically dorsalized phenotype and reversed central nervous system (CNS) anteroposterior (AP) patterning. Gain of function with chimeric construct, where Sox31 HMG DNA binding domain is fused to a transcription activation domain (VP16) or transcription suppression domain (EnR), suggests that Sox31 acts as a transcriptional suppressor in vivo. The expression of Bozozok (Dharma), a direct target gene of pre-MBT Wnt/ß-catenin signal, is suppressed by Sox31. Thus, to unveil the relationship between Sox31 and ß-catenin-related transcriptional activity, we designed Top/Fop luciferase assay in HEK293T cells, and found that Sox31 could indeed suppress Tcf/Lef-dependent transcriptional activity without influencing the stability of ß-catenin. Moreover, post-MBT Wnt signal was reduced in Sox31 morphants corresponding to the suppressed hindbrain structure, while phenotypic defects caused by excessive Sox31 could be rescued by Wnt antagonist dkk1. Taken together, Sox31 functions as an essential CNS AP patterning determinant and coordinates the CNS AP patterning process with organizer specification.

The protective effects of alpha-ketoacids against oxidative stress on rat spermatozoa in vitro.

The aim of this study was to determine the effects of antioxidants, including alpha-ketoacids (alpha-ketoglutarate and pyruvate), lactate and glutamate/malate combination, against oxidative stress on rat spermatozoa. Our results showed that H(2)O(2) (250 micromol L(-1))-induced damages, such as impaired motility, adenosine triphosphate (ATP) depletion, inhibition of sperm protein phosphorylation, reduced acrosome reaction and decreased viability, could be significantly prevented by incubation of the spermatozoa with alpha-ketoglutarate (4 mmol L(-1)) or pyruvate (4 mmol L(-1)). Without exogenous H(2)O(2) in the medium, the addition of pyruvate (4 mmol L(-1)) significantly increased the superoxide anion (O(2)(-).) level in sperm suspension (P < or = 0.01), whereas the addition of alpha-ketoglutarate (4 mmol L(-1)) and lactate (4 mmol L(-1)) significantly enhanced tyrosine-phosphorylated proteins with the size of 95 kDa (P < or = 0.04). At the same time, alpha-ketoglutarate, pyruvate, lactate, glutamate and malate supplemented in media can be used as important energy sources and supply ATP for sperm motility. In conclusion, the present results show that alpha-ketoacids could be effective antioxidants for protecting rat spermatozoa from H(2)O(2) attack and could be effective components to improve the antioxidant capacity of Biggers, Whitten and Whittingham media.

Dynamic regulation of glutamate decarboxylase 67 gene expression by alternative promoters and splicing during rat testis maturation.

Glutamate decarboxylase produces GABA, the main inhibitory neurotransmitter in adult mammalian brain. Two homologous forms of GAD encoded by separate genes have been identified in mammalian brain, with molecular weight of 67 kDa (GAD67) and 65 kDa (GAD65). Here, we studied the transcriptional regulation of GAD67. Three transcript variants (GAD67A, GAD67B, and GAD67C) transcribed from distinct categories of transcriptional start sites were identified. RT-PCR revealed these transcripts have distinct tissues distributions. Though GAD67A and GAD67B were co-expressed in brain and many nonneural tissues, in heart, only GAD67A was expressed. GAD67C was specifically expressed in testis. These transcripts also showed distinct developmental expression patterns during testis maturation. GAD67A was expressed at all age points examined. GAD67B was only detected at postnatal day 1 and day 5, while GAD67C was expressed from postnatal day 30. Characterizing the genome sequence upstream of transcriptional start sites of these transcripts revealed the presence of TATA-less promoters. Potential promoter activities were analyzed by coupling these promoter sequences to the open reading frame of a luciferase reporter gene in transient expression experiments. Moreover, our results showed GAD67 gene expression was also regulated by alternative splicing in postnatal day 1 and day 5 testis. The above results suggested GAD67 gene expression was dynamically regulated by alternative promoters and splicing during postnatal rat testis maturation.

Dynamic regulation of glutamic acid decarboxylase 65 gene expression in rat testis.

Glutamate decarboxylase 65 (GAD65) produces gamma-aminobutyric acid, the main inhibitory neurotransmitter in adult mammalian brain. Previous experiments, performed in brain, showed that GAD65 gene possesses two TATA-less promoters, although the significance is unknown. Here, by rapid amplification of cDNA ends method, two distinct GAD65 mRNA isoforms transcribed from two independent clusters of transcription start sites were identified in post-natal rat testis. RT-PCR results revealed that the two mRNA isoforms had distinct expression patterns during post-natal testis maturation, suggesting that GAD65 gene expression was regulated by alternative promoters at the transcription level. By using GAD65-specific antibodies, western blotting analysis showed that the 58-kDa GAD65, N-terminal 69 amino acids truncated form of full-length GAD65 protein, was developmentally expressed during post-natal testis maturation, suggesting that GAD65 gene expression in testis may also be regulated by post-translational processing. Confocal immunofluorescence microscopy revealed that GAD65 protein was presented in Leydig cells of Day 1 testis, primary spermatocytes and spermatids of postnatal of Day 90 testis. The above results suggested that GAD65 gene expression is dynamically regulated at multiple levels during post-natal testis maturation.

Utilization of an intron located polyadenlyation site resulted in four novel glutamate decarboxylase transcripts.

Glutamate decarboxylase (GAD) is the rate-limiting enzyme in the synthesis of gamma-aminobutyric acid (GABA), the most important inhibitory neurotransmitter in central nervous system (CNS). Two homologous forms of GAD encoded by separate genes have been identified in mammalian brain, with molecular weight of 65 kDa (GAD65) and 67 kDa (GAD67). In the present study, four novel GAD67 transcripts produced by alternative splicing and polyadenlyation were cloned from rat testis. These novel GAD67 transcripts were widely expressed in non-neuronal tissues. During rat testis maturation, their expression level showed a time dependent change. These transcripts were predicted to synthesis of GAD proteins truncated of the binding site for pyridoxal phosphate, an essential cofactor, therefore cannot function as a decarboxylase. Thus, post-transcriptional processing mechanism as alternative splicing and polyadenlyation may play a crucial role in regulating rat GAD67 gene expression.

Identification and expression of GABAC receptor in rat testis and spermatozoa.

Our previous studies showed that gamma-aminobutyric acid (GABA)A and GABAB receptors are involved in rat sperm acrosome reaction induced by progesterone or GABA. Here, we report the presence of GABAC receptor in rat testis and spermatozoa. Full-length complementary DNA encoding the rho1, rho2 and rho3 subunits of GABAC receptor were cloned from rat testis; their sequences are identical to those of rat GABAC receptor in retina. Reverse transcription-polymerase chain reaction analysis showed that during the development of rat testis, the transcript levels of the rho1 and rho2 subunits showed little change, while the expression of rho3 was gradually up-regulated. Immunofluorescence analysis using an anti-rho1 antibody revealed that GABAC receptor exists on the elongated spermatid and sperm. Using a chlortetracycline assay, we found that N(4)-chloroacetylcytosine arabinoside, a GABAC receptor agonist, triggered rat sperm acrosome reaction; whereas (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid, a GABAC receptor antagonist, inhibited the ability of N(4)-chloroacetylcytosine arabinoside to induce acrosome reaction. These results suggested that GABAC receptors are also involved in rat sperm acrosome reaction.

Identification of WSB1 gene as an important regulator in the development of zebrafish embryo during midblastula transition.

To uncover novel genes potentially involved in embryo development, especially at the midblastula transition (MBT) phase in the developing embryo of zebrafish, Affymetrix zebrafish GeneChip microarray analysis was carried out on the expression of 14,900 gene transcripts. The results of the analysis showed that 360 genes were clearly up-regulated and 119 genes were markedly down-regulated. Many of these genes were involved in transcription factor activity, nucleic acid binding, and cell growth. The present study showed that significant changes in transcript abundance occurred during the MBT phase. The expression of eight of these 479 genes was identified by reverse transcription-polymerase chain reaction analysis, confirming the microarray results. The WSB1 gene, found to be down-regulated by the microarray and reverse transcription-polymerase chain reaction analyses, was selected for further study. Sequence analysis of the WSB1 gene showed that it encodes a protein with 75% identity to the corresponding active human orthologs. In addition, WSB1 gene expression was detected at a higher level at 2 h post fertilization and at a lower level at 4 h post fertilization, consistent with the chip results. Overexpression of the WSB1 gene can result in the formation of abnormalities in embryos, as determined by fluorescence-activated cell sorting. The present study showed unequivocally that the occurrence of WSB1 expression is an important event during the MBT phase in the development of zebrafish embryos.

A strategy for constructing and verifying short hairpin RNA expression vectors.

The application of RNA interference (RNAi) to study gene function is now commonplace in a variety of biological systems. Producing short hairpin RNA (shRNA) by DNA vectors is one popular strategy for RNAi applications. Here, we describe a one-step PCR method, termed reverse PCR, for constructing shRNA expression vectors. Characteristically, the pair of primers binds to circular plasmid in a back-to-back manner. The anchored primers provide the templates of shRNA sense strand and antisense strand locating to the two separate ends of PCR segment, which will benefit the PCR amplification and subsequent cloning by avoiding premature formation of a hairpin configuration. Finally, the establishment of a circular vector is achieved by self-ligation of the single PCR product. In addition, our results indicated that the hairpin loop including a single restriction site is resistant to digestion, while inclusion of twin restriction sites in the loop leads to activity, creating an optimal strategy for verifying sequences of shRNA template.

Transactivated minimal E1b promoter is capable of driving the expression of short hairpin RNA.

The strategy that transcribes short hairpin RNAs (shRNAs) by RNA polymerase II promoters is expected to present flexible approaches for regulating the patterns of shRNA expression. The capacity of generating shRNA by a modified adenovirus RNA polymerase II E1b promoter was studied. This 49bp promoter consists of a TATA-box and an initiation element. It is demonstrated that this modified E1b promoter is capable of driving shRNA transcription and causing either long-term suppression against the target gene in response to the transactivation of constitutively expressed Gal4-VP16 fusion protein or inducible suppression given that the expression of Gal4-VP16 is subject to a dexamethasone inducer.

Identification and characterization of a novel splice variant of beta3 subunit of GABA(A) receptor in rat testis and spermatozoa.

Gamma-aminobutyric acid type A (GABA(A)) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of beta3 subunit of GABA(A) receptor, designated as beta3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the beta3 subunit of neuronal GABA(A) receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The beta3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the beta3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of beta3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the beta3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the beta3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of beta3 subunit of GABA(A) receptor, designated as beta3t, is found in germ cells of rat testis and sperm. The inability of the beta3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus.

Identification of GABABR2 in rat testis and sperm.

GABA is capable of mimicking and potentiating the action of progesterone in initiating the acrosome reaction (AR) of mammalian sperm. The GABA-initiated AR is mediated by GABA(A)R; whereas GABA(B)R1 protein found in rat testis and sperm tends to modify this process. Moreover, the occurrence of GABA(B)R2, a subunit essential for the formation of a functionally active GABA (B)R, in rat testis and sperm has not been established. In the present study, rat testis and sperm were analyzed for the presence of GABA(B)R2 transcript and protein by RT-PCR, Northern blot, Western blot and an indirect immunofluorescence technique. Northern blot shows that the transcript of testis GABA(B)R2 is shorter (~3.0 Kb) than that of the brain (~5.6 Kb). The full length testis GABA(B)R2 cDNA was prepared by RACE-PCR and found to be shorter by 2.2 Kb in the segment at the extreme terminus of 3'UTR of rat brain GABA(B)R2 but, the sequences corresponding to the open reading frame and 5'-UTR of rat testis GABA(B)R2 were found to be identical to those of rat brain. GABA(B)R2 protein isolated from rat epididymal sperm was slighter larger than those of rat testis and brain. It was principally localized in the acrosome region of the head of rat sperm by an indirect immunofluorescence technique. The present results establish that GABA(B)R2 protein is produced in rat testis and sperm and may play a role in GABA triggering of AR.